Vimal Patel

Abstract The novel coronavirus disease (COVID-19) outbreak, caused by SARS-CoV-2, represents the greatest medical challenge in decades. We provide a comprehensive review of the clinical course of COVID-19, its comorbidities, and mechanistic considerations for future therapies. While COVID-19 primarily affects the lungs, causing interstitial pneumonitis and severe acute respiratory distress syndrome (ARDS), it also affects multiple organs, particularly the cardiovascular system. Risk of severe infection and mortality increase with advancing age and male sex. Mortality is increased by comorbidities: cardiovascular disease, hypertension, diabetes, chronic pulmonary disease, and cancer. The most common complications include arrhythmia (atrial fibrillation, ventricular tachyarrhythmia, and ventricular fibrillation), cardiac injury [elevated highly sensitive troponin I (hs-cTnI) and creatine kinase (CK) levels], fulminant myocarditis, heart failure, pulmonary embolism, and disseminated intravascular coagulation (DIC). Mechanistically, SARS-CoV-2, following proteolytic cleavage of its S protein by a serine protease, binds to the transmembrane angiotensin-converting enzyme 2 (ACE2) —a homologue of ACE—to enter type 2 pneumocytes, macrophages, perivascular pericytes, and cardiomyocytes. This may lead to myocardial dysfunction and damage, endothelial dysfunction, microvascular dysfunction, plaque instability, and myocardial infarction (MI). While ACE2 is essential for viral invasion, there is no evidence that ACE inhibitors or angiotensin receptor blockers (ARBs) worsen prognosis. Hence, patients should not discontinue their use. Moreover, renin–angiotensin–aldosterone system (RAAS) inhibitors might be beneficial in COVID-19. Initial immune and inflammatory responses induce a severe cytokine storm [interleukin (IL)-6, IL-7, IL-22, IL-17, etc.] during the rapid progression phase of COVID-19. Early evaluation and continued monitoring of cardiac damage (cTnI and NT-proBNP) and coagulation (D-dimer) after hospitalization may identify patients with cardiac injury and predict COVID-19 complications. Preventive measures (social distancing and social isolation) also increase cardiovascular risk. Cardiovascular considerations of therapies currently used, including remdesivir, chloroquine, hydroxychloroquine, tocilizumab, ribavirin, interferons, and lopinavir/ritonavir, as well as experimental therapies, such as human recombinant ACE2 (rhACE2), are discussed.

Pulmonary cavities, the hallmark of tuberculosis (TB), are characterized by high mycobacterial load and perpetuate the spread of M. tuberculosis. The mechanism of matrix destruction resulting in cavitation is not well defined. Neutrophils are emerging as key mediators of TB immunopathology and their influx are associated with poor outcomes. We investigated neutrophil-dependent mechanisms involved in TB-associated matrix destruction using a cellular model, a cohort of 108 patients, and in separate patient lung biopsies. Neutrophil-derived NF-kB-dependent matrix metalloproteinase-8 (MMP-8) secretion was up-regulated in TB and caused matrix destruction both in vitro and in respiratory samples of TB patients. Collagen destruction induced by TB infection was abolished by doxycycline, a licensed MMP inhibitor. Neutrophil extracellular traps (NETs) contain MMP-8 and are increased in samples from TB patients. Neutrophils lined the circumference of human pulmonary TB cavities and sputum MMP-8 concentrations reflected TB radiological and clinical disease severity. AMPK, a central regulator of catabolism, drove neutrophil MMP-8 secretion and neutrophils from AMPK-deficient patients secrete lower MMP-8 concentrations. AMPK-expressing neutrophils are present in human TB lung biopsies with phospho-AMPK detected in nuclei. These data demonstrate that neutrophil-derived MMP-8 has a key role in the immunopathology of TB and is a potential target for host-directed therapy in this infectious disease.

Cyclooxygenase-2 (Cox-2), an inducible form of the enzyme that catalyzes the first step in the synthesis of prostanoids, has been shown to be overexpressed in a wide range of tumors and possesses proangiogenic and antiapoptotic properties. To understand the molecular mechanism of Cox-2 action we used adenovirus-mediated transfer of rat Cox-2 cDNA into renal rat mesangial cells and determined the differential gene expression using cDNA microarrays. One of the several genes that were highly up-regulated by over expressed Cox-2 was MDR1. MDR1 or P-glycoprotein (P-gp), the product of the MDR1 gene, is implicated as the primary cause of multidrug resistance (MDR) in tumors where it acts as an efflux pump for chemotherapeutic agents. It is also expressed in normal tissues of the liver and kidney where it functions to actively transport lipophilic xenobiotics. Reverse transcriptase-PCR analysis confirmed the results of the microarray, showing increased mRNA levels for MDR1 in Cox-2 overexpressing cells. This increase in mRNA translated to an increase in MDR1 protein expression, which was dose-dependent on Cox-2 expression. Furthermore, using rhodamine 123 efflux assay we observed a significant increase in P-gp activity in Cox-2 overexpressing renal mesangial cells. The specific Cox-2 inhibitor NS398 was able to block the Cox-2-mediated increase in MDR1 expression and activity, suggesting that Cox-2 products may be implicated in this response. These results prove the existence of a causal link between Cox-2 and P-gp activity, which would have implications for kidney function and multidrug resistance in tumors where Cox-2 is overexpressed. Cyclooxygenase-2 (Cox-2), an inducible form of the enzyme that catalyzes the first step in the synthesis of prostanoids, has been shown to be overexpressed in a wide range of tumors and possesses proangiogenic and antiapoptotic properties. To understand the molecular mechanism of Cox-2 action we used adenovirus-mediated transfer of rat Cox-2 cDNA into renal rat mesangial cells and determined the differential gene expression using cDNA microarrays. One of the several genes that were highly up-regulated by over expressed Cox-2 was MDR1. MDR1 or P-glycoprotein (P-gp), the product of the MDR1 gene, is implicated as the primary cause of multidrug resistance (MDR) in tumors where it acts as an efflux pump for chemotherapeutic agents. It is also expressed in normal tissues of the liver and kidney where it functions to actively transport lipophilic xenobiotics. Reverse transcriptase-PCR analysis confirmed the results of the microarray, showing increased mRNA levels for MDR1 in Cox-2 overexpressing cells. This increase in mRNA translated to an increase in MDR1 protein expression, which was dose-dependent on Cox-2 expression. Furthermore, using rhodamine 123 efflux assay we observed a significant increase in P-gp activity in Cox-2 overexpressing renal mesangial cells. The specific Cox-2 inhibitor NS398 was able to block the Cox-2-mediated increase in MDR1 expression and activity, suggesting that Cox-2 products may be implicated in this response. These results prove the existence of a causal link between Cox-2 and P-gp activity, which would have implications for kidney function and multidrug resistance in tumors where Cox-2 is overexpressed. cyclooxygenase nonsteroidal antiinflammatory drugs multidrug resistance P-glycoprotein rat mesangial cells multiplicity of infection reverse transcriptase green fluorescent protein Cyclooxygenase (Cox),1also known as prostaglandin endoperoxide synthase, is the key enzyme required for the conversion of arachidonic acid to prostaglandins (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1854) Google Scholar). There are two isoforms of the enzyme that have been identified, Cox-1 and Cox-2. In most tissues, the Cox-1 enzyme is produced constitutively, whereas Cox-2 is highly inducible by growth factors and cytokines (e.g. at sites of inflammation) (1Smith W.L. Garavito R.M. DeWitt D.L. J. Biol. Chem. 1996; 271: 33157-33160Abstract Full Text Full Text PDF PubMed Scopus (1854) Google Scholar, 2Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2225) Google Scholar). Traditional nonsteroidal antiinflammatory drugs (NSAIDs) inhibit both enzymes leading to their antiinflammatory, analgesic, and antipyretic effects. However, a new class of Cox-2 selective inhibitors (COXIBs) preferentially inhibit the Cox-2 enzyme thereby reducing side effects such as gastrointestinal ulceration and bleeding and platelet dysfunction caused by inhibiting Cox-1. These specific Cox-2 inhibitors have emerged as important therapeutic tools for treatment of pain and arthritis (3Brune K. Zeilhofer H.U. Hinz B. Emery P. Cyclo-oxygenase inhibitors: new insights. Health Press, Oxford, UK1999Google Scholar). There is compelling evidence linking Cox-2 and carcinogenesis, where selective Cox-2 inhibitors have been shown to have strong chemopreventative actions against colon carcinogenesis in rats, inhibiting tumors to a greater degree than conventional NSAIDs (4Kawamori T. Rao C.V. Seibert K. Reddy B.S. Cancer Res. 1998; 58: 409-412PubMed Google Scholar). Cox-2-derived prostaglandins were found to modulate the production of angiogenic factors by colon cancer cells thereby inducing new blood vessel formation to sustain tumor growth (5Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. Dubois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar). Also, overexpression of Cox-2 either in epithelial cells or in PC12 cells has been shown to lead to resistance to apoptosis, which would lead to a dysregulation in cell death and cell growth (6Tsujii M. Dubois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar, 7McGinty A. Chang Y.W. Sorokin A. Bokemeyer D. Dunn M.J. J. Biol. Chem. 2000; 275: 12095-12101Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Apart from the colon, elevated Cox-2 levels have been observed in cancer of the lung, breast, gastric, and prostate. Multidrug resistance (MDR) has been a major obstacle to successful cancer chemotherapy. One important mechanism of MDR involves the multidrug transporter, P-glycoprotein (P-gp), which confers upon cancer cells the ability to resist lethal doses of certain cytotoxic drugs by actively pumping the drugs out of the cells and thus reducing their cytotoxicity (8Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (412) Google Scholar, 9Johnstone R.W. Ruefli A.A. Smyth M.J. Trends Biochem. Sci. 2000; 25: 1-6Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). P-gp belongs to the ATP-binding cassette family of transporter molecules that require hydrolysis of ATP to run the transport mechanism. The substrates of P-gp may be endogenous (steroid hormones, cytokines) or xenobiotics (cytostatic drugs). A number of studies have demonstrated a negative correlation between P-gp expression levels and chemosensitivity or survival in a range of human malignancies. P-gp is encoded by a group of related genes termed MDR; only MDR1 is known to confer the drug resistance, and its overexpression in cancer cells has been a therapeutic target to circumvent the resistance. Only recently has the role of P-gp expressed in normal tissues as a determinant of drug pharmacokinetics and pharmacodynamics been examined. P-gp is present in organ systems that influence drug absorption (intestine), distribution to the site of action (central nervous system and leukocytes), and elimination (liver and kidney), as well as several other tissues. In the kidney, P-gp is present in the brush border membrane of the proximal tubule (9Johnstone R.W. Ruefli A.A. Smyth M.J. Trends Biochem. Sci. 2000; 25: 1-6Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 11Ernest S. Rajaraman S. Megyesi J. Bello-Reuss E.N. Nephron. 1997; 77: 284-289Crossref PubMed Scopus (88) Google Scholar), a site compatible with a role in xenobiotic secretion. It is also expressed in the mesangium, the thick ascending limb of Henle's loop, and the collecting duct (11Ernest S. Rajaraman S. Megyesi J. Bello-Reuss E.N. Nephron. 1997; 77: 284-289Crossref PubMed Scopus (88) Google Scholar), locations that are not traditionally associated with drug excretion. The purpose of this study was to determine the mechanism of the biological actions of Cox-2 within RMC. We employed cDNA expression microarrays for this purpose and observed an increase in P-gp gene expression within cells with enforced expression of Cox-2. This translated to increased protein expression and functional activity of P-gp. This is the first report that has found a direct link between Cox-2 expression and P-gp, and further analysis into the mechanism of this relationship would help us to understand the nature of multidrug resistance and possible strategies to block this effect. Unless otherwise stated all chemicals were obtained from Sigma or Invitrogen. MDR1 antibody was obtained from RDI (Flanders, NJ). Cox-2 antibody was obtained from Santa Cruz (La Jolla, CA). Rat glomerular mesangial cells were cultured as described previously reported (12Foschi M. Chari S. Dunn M.J. Sorokin A. EMBO J. 1997; 16: 6439-6451Crossref PubMed Scopus (142) Google Scholar, 13Simonson M.S. Dunn M.J. Methods Enzymol. 1990; 187: 544-553Crossref PubMed Scopus (37) Google Scholar). Briefly both kidneys from male Sprague-Dawley rats (120–170 g) were removed and decapsulated under sterile conditions. Cortical tissue was cut away from the medulla and minced in isolation buffer solution containing 5 mmKCl, 2 mm CaCl2, 130 mm NaCl, 10 mm glucose, 20 mm sucrose, and 10 mm Tris, pH 7.4. Glomeruli were isolated by sequential sieving and collected on a 50-μm sieve. After incubation with 0.1% collagenase in isolation buffer solution for 30 min at 37 C° to remove epithelial cells and obtain glomerular cores consisting mostly of mesangium and capillary loops, the glomeruli suspension was centrifuged at 2200 r.p.m. for 7 min at room temperature. Isolated glomeruli were plated in RPMI 1640 medium supplemented with 17% fetal bovine serum, 100 units/ml penicillin, 100 units/ml streptomycin, 5 μg/ml each insulin and transferrin, and 5 ng/ml selenite. The flasks were incubated at 37 C° and 5% CO2 in a humidified atmosphere in a CO2-controlled incubator for 3–6 weeks, and the medium was changed every third day. RMC were used between passages 5 and 24. The recombinant adenovirus vector AdCox-2, expressing COX-2, was constructed from replication-deficient adenovirus type 5(Ad5) with deletions in the E1 and E3 genes, Addl327, and a plasmid containing Ad5 sequences from 22 to 5790 with a deletion of the E1 region from bp 342 to 3523, a polycloning site under control of the cytomegalovirus promoter, the COX-2 cDNA, and the simian virus 40 polyadenylation signal. Rat mesangial cells were incubated with serum-free media for 24 h prior to infection with AdCox-2, Ad5-based green fluorescent protein expression vector (AdGFP) or a wild type Ad5-based virus containing no transgene at a varying multiplicity of infection (m.o.i.) for 1 h. The cells remained in serum-free conditions for 48 h after infection before experiments were performed. AdCox2 or AdGFP-infected rat mesangial cells were grown for 48 h in serum-free media. mRNA was isolated from two 100-mm diameter plates of each condition using TRIzol reagent. 32P-labeled cDNA probes were prepared and hybridized to the nylon Atlas Rat Stress cDNA expression array (Clontech) as instructed by the manufacturer. Membranes were exposed to Kodak BioMax MS x-ray film with a BioMax MS intensifying screen at −70 °C for 24 h. MDR1 gene expression was determined by RT-PCR analysis using the Advantage RT-for-PCR kit (Clontech). Total RNA was prepared, and RT-PCR was performed as instructed by the manufacturer using rat MDR1 and β-actin primers (Clontech). Western blot analysis was carried out as described previously (14Bokemeyer D. Guglielmi K.E. McGinty A. Sorokin A. Lianos E.A. Dunn M.J. J. Clin. Invest. 1997; 100: 582-588Crossref PubMed Scopus (75) Google Scholar). Cells were lysed in 50 mmHEPES, pH 7.5, containing 150 mm NaCl, 1.5 mmMgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mmphenylmethylsulfonyl fluoride, and 200 μm orthovanadate. Samples standardized for protein (BCA, Pierce) were separated by SDS-polyacrylamide gel electrophoresis using 7% acrylamide (Criterion Gels, Bio-Rad, Hercules, CA). Protein was transferred to nitrocellulose and probed with the appropriate polyclonal antibody. Rat mesangial cells were washed once with prewarmed HEPES-buffered solution (37 °C) consisting of 125 mm NaCl, 5 mm KCl, 1 mmMgSO4, 1.36 mm Na2HPO4, 10 mm sodium acetate, 5 mm HEPES, 1.8 mm CaCl2, and 8 mm glucose titrated to pH 7.4. The cells were incubated for 1 h at 37 °C in the same solution containing 1 μm rhodamine 123 (R123). The R123 solution was removed from the extracellular medium, and cells were detached from the cell culture flask by incubation in a trypsin-containing, Ca2+-free, phosphate-buffered solution with 1 μm R123. Trypsin digestion was stopped by addition of cold HEPES-buffered solution supplemented with newborn bovine serum, 1 μm R123. Cell aliquots were placed in 12 × 75-mm polystyrene Falcon tubes (Becton Dickinson, Lincoln Park, NJ) and centrifuged at 1000 × g for 5 min at 4 °C. The supernatant was removed by aspiration, 2 ml of the cold incubation solution was added to the tube, and aliquots were kept at 4 °C. Loading was measured at 0 °C. The efflux of R123 was initiated by incubating the cells at 25 °C. In some experiments, the cells were resuspended in incubation medium containing 100 μm of the P-gp blockers (verapamil and cyclosporin). Samples were kept at 4 °C until assayed. Fluorescence of R123 was collected through a 530/30 nm bandpass on a FACscalibur flow cytometer (Becton-Dickinson). After gating for live cells 10,000 cells were recorded for each sample and processed by Cell Quest software (Becton-Dickinson). Our first objective was to confirm our ability to introduce the transgene of interest into rat mesangial cells with the use of adenovirus infection. Fig. 1 Ashows GFP-infected cells at 100 m.o.i. Under the fluorescence microscope 90% of the cells show fluorescence; however, more sensitive flow cytometry analysis revealed 100% transgene expression by the cells. Fig. 1 B (middle panel) shows a Western blot of Cox-2 expression in rat mesangial cells infected with GFP and Cox-2 adenovirus. The figure shows that we are able to increase the expression of Cox-2 within the cells using this system and that basal levels of Cox-2 expression (AdGFP-treated) were negligible 48 h after infection. To identify the differential gene expression as a result of Cox-2 overexpression, AtlasTM rat stress cDNA expression arrays (Clontech) were screened with32P-labeled cDNA generated from mRNA from RMC infected with AdCox-2 or AdGFP. A difference of more than 3-fold was considered significant. Signals were normalized to signals from housekeeping genes (data not shown). Of genes that were up-regulated in RMC MDR1 or P-gp showed the most significant increase, and because of the roles of Cox-2 and P-gp in cancer we investigated this correlation further. To confirm the results of the cDNA microarray we analyzed the samples using RT-PCR with primers specific for rat P-gp and actin for control purposes. Fig. 1 C shows the result of this experiment and confirmed the data that we obtained with the cDNA arrays by showing increased expression of the MDR1 gene. It was necessary to investigate whether this increased gene expression resulted in an increased translation to protein. RMC were infected with AdGFP or AdCox-2, and P-gp and Cox-2 expression were analyzed by Western immunoblot. Fig. 1 B shows the results of this initial experiment where we observed a basal level of P-gp expression, which increased in RMC overexpressing Cox-2. The P-gp protein is 170 kDa but is hyperglycosylated and as such appears as a diffuse band on the Western immunoblot. Using a new preparation of RMC and AdCox-2 we were able to reproduce these results, and we subsequently found that the P-gp protein expression was dependent on Cox-2 levels as shown in Fig. 2. This relationship was studied further using an assay to measure specific P-gp activity. The assay measures P-gp-mediated transport by the decrease in the intracellular fluorescence of rhodamine 123 (R123, a P-pg substrate) using flow cytometry. Intracellular rhodamine 123 was measured at time 0 (load) and after 2 h of efflux in RMC infected with AdCox-2 and wild type adenovirus. AdGFP was not used due to fluorescence from GFP interfering with R123 measurements. Fig.3 A shows the results of this experiment with control RMC (top panel) effluxing R123 consistent with the basal protein expression observed in Fig.2 B. Efflux was measured by the number of cells in the M1 region of the plot. In the RMC infected with AdCox-2 (Fig3 A, bottom panels) there was an increase in R123 efflux from the cells indicating greater P-gp activity. This experiment was carried out several times, and each time the increase in R123 efflux in Cox-2 expressing RMC over control levels was measured. The average percentage increase in R123 efflux from the Cox-2 overexpressing RMC was 75% ± 10 (p < 0.0001). To confirm the specificity of the R123 efflux from the RMC we employed two P-gp blockers, verapamil and cyclosporin A. The R123 efflux assays were performed in the presence of these inhibitors, and the results are shown in Fig. 3 B. The inhibitors at concentrations of 100 μm were able to significantly reduce the efflux of R123 from the RMC thus confirming that the increase in efflux form Cox-2 overexpressing RMC is due to an increase in P-gp expression and activity. The effect of the specific Cox-2 inhibitor NS398 on the Cox-2-mediated MDR1 activity and expression was assessed. Fig.4, A and B show the results of R123 efflux assays measuring MDR1 activity in RMC infected with the Cox-2 adenovirus and treated with increasing concentrations of NS398. The histograms in figure 4A show a dose-dependent decrease in R123 efflux as measured by the number of cells in the M1 region of the plot. Fig. 4 B gives the percentage values for the efflux in all treatments. They confirm that we see a greater efflux in Cox-2 overexpressing cells (39%versus 14%) and that with the treatment of NS398 the efflux in Cox-2 overexpressing cells is reduced to control levels (14%). Our next objective was to see if the reduced MDR1 activity with NS398 treatments was due to reduced MDR1 expression. Fig. 4 C shows a representative Western blot of RMC infected with control and Cox-2 adenovirus plus or minus NS398 (25 μm). We again confirm that Cox-2 overexpressing RMC have increased MDR1 expression. We also observed that the addition of NS398 blocked the up-regulation of the MDR1 protein, consistent with the R123 efflux assay showing reduced MDR1 activity. These additional results show that the Cox-2-mediated MDR1 response is dependent on Cox-2 activity. We have shown that the overexpression of Cox-2 leads to increased P-gp expression and activity in RMC, and this effect is dependent on Cox-2 activity. The products of Cox-2 activity that may be implicated in this response are varied. They include prostaglandin endoperoxide H2 (PGH2), PGD2, PDE2, PGF2α, PGI2 (prostacyclin) or TxA2 (thromboxane A2). P-glycoprotein is the product of the MDR gene and has been of great interest due to its role in multidrug resistance in a variety of cancers (8Sharom F.J. J. Membr. Biol. 1997; 160: 161-175Crossref PubMed Scopus (412) Google Scholar, 9Johnstone R.W. Ruefli A.A. Smyth M.J. Trends Biochem. Sci. 2000; 25: 1-6Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar, 15Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3564) Google Scholar). It has been shown to be overexpressed in cancer cells that actively extrude chemotherapeutic agents (15Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3564) Google Scholar). Aberrant up-regulation of Cox-2 expression is also increasingly implicated in the pathogenesis of epithelial cell carcinomas such as human colorectal adenocarcinomas and colorectal tumors (16Prescott S.M. Fitzpatrick F.A. Biochim. Biophys. Acta. 2000; 1470: M69-M78PubMed Google Scholar). Previous studies in rat medullary interstitial cells have shown that Cox-2 overexpression leads to a number of effects that could be associated with tumorigenesis: increased adhesion to extracellular matrix proteins, inhibition of butyrate-induced apoptosis, decreased expression of both E-cadherin and transforming growth factor β2 receptor, and stimulation of Bcl-2 protein expression (6Tsujii M. Dubois R.N. Cell. 1995; 83: 493-501Abstract Full Text PDF PubMed Scopus (2137) Google Scholar). Cox-2-expressing cells also produce high levels of angiogenic factors that stimulate angiogenesis (5Tsujii M. Kawano S. Tsuji S. Sawaoka H. Hori M. Dubois R.N. Cell. 1998; 93: 705-716Abstract Full Text Full Text PDF PubMed Scopus (2214) Google Scholar). The results we have shown suggest another possible role of Cox-2 in carcinogenesis by promoting multidrug resistance. Immunohistochemical analyses of human breast tumor specimens revealed a strong correlation between expression of Cox-2 and P-gp. In drug-resistant cell lines that overexpress P-gp there was also significant expression of Cox-2 expression (17Ratnasinghe D. Daschner P.J. Anver M.R. Kasprzak B.H. Taylor P.R. Yeh G.C. Tangrea J.A. Anticancer Res. 2001; 21: 2141-2147PubMed Google Scholar). Studies indicate that the use of NSAIDs may enhance the antitumor activity of cancer chemotherapeutic agents and reduce the risk of many cancers. The best known function of NSAIDs is to block Cox and stop the conversion of arachidonic acid to prostaglandins. We have shown the first report of direct causal relationship between Cox-2 expression and P-gp regulation. These results may give us an indication as to the nature of multidrug resistance in tumors where Cox-2 is overexpressed. MDR1 is also expressed in normal human tissues including the kidney, liver, pancreas and colon (10Thiebaut F. Tsuruo T. Hamada H. Gottesman M.M. Pastan I. Willingham M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7735-7738Crossref PubMed Scopus (2570) Google Scholar). MDR1 expression has been shown to be up-regulated by many extracellular stimuli including UV irradiation, heat shock, and growth factors, and by a number of drugs. In the kidney it is localized at sites compatible with its role in xenobiotic secretion. Human mesangial cells express P-pg in culture where it is localized to the plasma membrane (18Bello-Reuss E. Ernest S. Am. J. Physiol. 1994; 267: C1351-C1358Crossref PubMed Google Scholar) and functionally transports xenobiotics. Our results would have implications as to how up-regulated Cox-2 activity may mediate these effects in the kidney, notably the excretion of drugs by the kidney via P-gp. P-gp has also been implicated in a role in augmenting cell survival of a number of cell systems (19Pallis M. Russell N. Blood. 2000; 95: 2897-2904Crossref PubMed Google Scholar, 20Thevenod F. Friedmann J.M. Katsen A.D. Hauser I.A. J. Biol. Chem. 2000; 275: 1887-1896Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). Up-regulation of P-gp via nuclear factor-κB (NF-κB) activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis, which was blocked by the MDR1 inhibitors such as PSC833, cyclosporin A, or verapamil (19Pallis M. Russell N. Blood. 2000; 95: 2897-2904Crossref PubMed Google Scholar). It is also possible that MDR1 is capable of preventing transformation by pumping out xenobiotic carcinogens. Cox-2 and its products are also implicated in cell survival. Treatment of human colon cancer cells (HCA7) with SC-58125, a highly selective Cox-2 inhibitor results in inhibition of growth and increased apoptosis that is reversed by PGE2 (21Sheng H. Shao J. Morrow J.D. Beauchamp R.D. Dubois R.N. Cancer Res. 1998; 58: 362-366PubMed Google Scholar). Cox-2 promotes resistance to trophic withdrawal apoptosis in PC12 cells by stimulation of dynein light chain expression and inhibition of neuronal nitric-oxide synthase activity (22Chang Y.W. Jakobi R. McGinty A. Foschi M. Dunn M.J. Sorokin A. Mol. Cell. Biol. 2000; 20: 8571-8579Crossref PubMed Scopus (81) Google Scholar). It has been established that MDR1 can translocate a wide variety of short-chain lipid molecules, including ceramides, across the plasma membrane to the cell surface (23van Helvoort A. Smith A.J. Sprong H. Fritzsche I. Schinkel A.H. Borst P. van Meer G. Cell. 1996; 87: 507-517Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). It was also shown that TNFα-induced ceramide generation and apoptosis was restored by MDR1 inhibitors in TNFα-resistant leukemia cells (24Bezombes C. Maestre N. Laurent G. Levade T. Bettaieb A. Jaffrezou J.P. FASEB J. 1998; 12: 101-109PubMed Google Scholar). Apoptosis can be triggered by ceramide generation, and Cox-2 antiapoptotic activity is sometimes assigned to prevention of ceramide formation due to arachidonic acid depletion (25Cao Y. Pearman A.T. Zimmerman G.A. McIntyre T.M. Prescott S.M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11280-11285Crossref PubMed Scopus (384) Google Scholar). Our results suggest that one of the mechanisms of Cox-2 antiapoptotic action is mediated via P-gp. To date there has been no evidence of Cox-2-mediated P-glycoprotein expression. We hope that the uncovering of this link and further studies will help to devise strategies to combat multidrug resistance in tumors overexpressing the Cox-2 protein. We thank Dr. Adiba Ishaque and Dr. Jeffrey Woodliff for assistance with flow cytometry experiments and Chris Roberts for technical assistance.

Abstract Tumor neovasculature is a potential but, until very recently, unexplored target for boron neutron capture therapy (BNCT) of cancer. In the present report, we describe the construction of a vascular endothelial growth factor (VEGF)–containing bioconjugate that potentially could be used to target up-regulated VEGF receptors (VEGFR), which are overexpressed on tumor neovasculature. A fifth-generation polyamidoamine dendrimer containing 128 reactive amino groups was reacted with 105 to 110 decaborate molecules to produce a macromolecule with 1,050 to 1,100 boron atoms per dendrimer. This was conjugated to thiol groups of VEGF at a 4:1 molar ratio using the heterobifunctional reagent sulfo-LC-SPDP. In addition, the boronated dendrimer was tagged with a near-IR Cy5 dye to allow for near-IR fluorescent imaging of the bioconjugate in vitro and in vivo. As would be predicted, the resulting VEGF-BD/Cy5 bioconjugate was not cytotoxic to HEK293 cells engineered to express 2.5 × 106 VEGFR-2 per cell. Furthermore, it showed binding and activation of VEGFR-2 comparable with that of native VEGF. Internalization of VEGF-BD/Cy5 by PAE cells expressing 2.5 × 105 VEGFR-2 per cell was inhibited by excess VEGF, indicating a VEGFR-2-mediated mechanism of uptake. Near-IR fluorescent imaging of 4T1 mouse breast carcinoma revealed selective accumulation of VEGF-BD/Cy5, but not BD/Cy5, particularly at the tumor periphery where angiogenesis was most active. Accumulation of VEGF-BD/Cy5 in 4T1 breast carcinoma was diminished in mice pretreated with a toxin-VEGF fusion protein that selectively killed VEGFR-2-overexpressing endothelial cells. Our data lay the groundwork for future studies using the VEGF-BD/Cy5 bioconjugate as a targeting agent for BNCT of tumor neovasculature.

Current models of autoimmunity suggest that delayed clearance of apoptotic cells leads to the presentation of apoptotic antigens in the context of inflammatory signals, with resultant autoimmunity. These models implicitly assume that, in contrast to early apoptotic cells (that retain membrane integrity), late apoptotic cells (with compromised membranes) act like necrotic cells (which also lack intact membranes), possibly because of the release of proinflammatory intracellular contents. We showed previously that early apoptotic and necrotic cells induce distinct mitogen-activated protein kinase modules in macrophages with which they interact. Exposure to apoptotic cells led to nearly complete inhibition of both basal and macrophage colony-stimulating factor-induced ERK1/2 by macrophages. In contrast, necrotic cells induced ERK1/2. We show here that apoptotic cells also strongly induced both c-Jun N-terminal kinase and p38, whereas necrotic cells had no detectable effect on c-Jun N-terminal kinase and p38. We also compared the signaling events induced in macrophages by exposure to early apoptotic cells, late apoptotic cells, and necrotic cells. The signaling events induced by late apoptotic cells were identical to and just as potent as those induced by early apoptotic cells. Thus, apoptotic cells are functionally equivalent throughout the cell death process, irrespective of membrane integrity. Moreover, the effects of both early and late apoptotic cells on signaling were dominant over those of necrotic cells. These data show that apoptotic cells do not become proinflammatory upon the loss of membrane integrity and are inconsistent with the notion that delayed clearance alone can lead to autoimmunity.

Developing tissue engineering scaffolds with immobilized growth factors requires facile and reliable methods for the covalent attachment of functionally active proteins. We describe here a new approach to immobilize recombinant proteins based on expression of the protein of interest with a 15-aa long fusion tag (Cys-tag), which avails a free sulfhydryl group for site-specific conjugation. To validate this approach, we conjugated a single-chain vascular endothelial growth factor expressed with an N-terminal Cys-tag (scVEGF) to fibronectin (FN) using a common thiol-directed bi-functional cross-linking agent. We found that the FN-scVEGF conjugate retains VEGF activity similar to that of free scVEGF when used as a soluble ligand. Cells expressing VEGF receptor VEGFR-2 grown on plates coated with FN-scVEGF displayed morphological phenotypes similar to those observed for cells grown on FN in the presence of equivalent amounts of free scVEGF. In addition, 293/KDR cell growth stimulation was observed in the same concentration range with either immobilized or free scVEGF. The effects of immobilized scVEGF, and soluble scVEGF were blocked by NVP-AAD777-NX, a VEGF receptor tyrosine kinase inhibitor. These data indicate that site-specific immobilization via Cys-tag provides a facile and reliable method for permanent deposition of functionally active growth factors on synthetic or protein scaffolds with applications for advanced tissue engineering.

The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of “self” versus “other” considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The purpose of physiological cell death is the noninflammatory clearance of cells that have become inappropriate or nonfunctional. Consistent with this function, the recognition of apoptotic cells by professional phagocytes, including macrophages and dendritic cells, triggers a set of potent anti-inflammatory responses manifest on multiple levels. The immediate-early inhibition of proinflammatory cytokine gene transcription in the phagocyte is a proximate consequence of recognition of the apoptotic corpse, independent of subsequent engulfment and soluble factor involvement. Here, we show that recognition is linked to a characteristic signature of responses, including MAPK signaling events and the ablation of proinflammatory transcription and cytokine secretion. Specific recognition and response occurs without regard to the origin (species, tissue type, or suicidal stimulus) of the apoptotic cell and does not involve Toll-like receptor signaling. These features mark this as an innate immunity fundamentally distinct from the discrimination of “self” versus “other” considered to be the hallmark of conventional immunity. This profound unconventional innate immune discrimination of effete from live cells is as ubiquitous as apoptotic cell death itself, manifest by professional and nonprofessional phagocytes and nonphagocytic cell types alike. Innate apoptotic immunity provides an intrinsic anti-inflammatory circuit that attenuates proinflammatory responses dynamically and may act systemically as a powerful physiological regulator of immunity. The process of physiological cell death assures the elimination of functionally inappropriate cells in a manner that does not elicit inflammation (1.Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-256Crossref PubMed Scopus (12819) Google Scholar, 2.Savill J. Dransfield I. Gregory C. Haslett C. Nat. Rev. Immunol. 2002; 2: 965-975Crossref PubMed Scopus (1325) Google Scholar). Professional phagocytes, including resident macrophages and dendritic cells, participate in the efficient clearance of apoptotic corpses in vivo (3.Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1347) Google Scholar, 4.Surh C.D. Sprent J. Nature. 1994; 372: 100-103Crossref PubMed Scopus (934) Google Scholar, 5.Huang F.-P. Platt N. Wykes M. Major J.R. Powell T.J. Jenkins C.D. MacPherson G.G. J. Exp. Med. 2000; 191: 435-444Crossref PubMed Scopus (779) Google Scholar, 6.Wood W. Turmaine M. Weber R. Camp V. Maki R.A. McKercher S.R. Martin P. Development. 2000; 127: 5245-5252Crossref PubMed Google Scholar). Studies of neutrophil death and resulting phagocytosis by macrophages provided the first experimental evidence that a variety of cytokines and chemokines associated with inflammation, including interleukin (IL) 4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.4The abbreviations used are: IL, interleukin; EGF, epidermal growth factor; PI, propidium iodide; PS, phosphatidylserine; LPS, lipopolysaccharide; RLU, relative light units; CFDA, 5,6-carboxyfluorescein diacetate succinimidyl ester; PBS, phosphate-buffered saline; CMTMR, 5-(and 6)-(((4-chloromethyl)benzoyl)amino) tetramethyl rhodamine; TNFα, tumor necrosis factor α; PMA, phorbol 12-myristate 13-acetate; NF-κB, nuclear factor κB; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; ERK1/2, extracellular signal-regulated kinase 1 and 2; TGRβ, transforming growth factor β; TLR, toll-like receptor.-6 and IL-8, are not secreted from phagocytes that engulf apoptotic targets (3.Savill J.S. Wyllie A.H. Henson J.E. Walport M.J. Henson P.M. Haslett C. J. Clin. Invest. 1989; 83: 865-875Crossref PubMed Scopus (1347) Google Scholar, 7.Meagher L.C. Savill J.S. Baker A. Fuller R.W. Haslett C. J. Leukocyte Biol. 1992; 52: 269-272Crossref PubMed Scopus (247) Google Scholar, 8.Hughes J. Liu Y. Damme J.V. Savill J. J. Immunol. 1997; 158: 4389-4397PubMed Google Scholar, 9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). More significantly, the lack of inflammatory cytokine release reflects an affirmative inhibitory response. For example, whereas stimulation of macrophages via the Toll-like receptor (TLR) 4 signaling complex (11.Hoshino K. Takeuchi O. Kawai T. Sanjo H. Ogawa T. Takeda Y. Takeda K. Akira S. J. Immunol. 1999; 162: 3749-3752Crossref PubMed Google Scholar) upon engagement with bacterial lipopolysaccharide (LPS) triggers significant cytokine secretion, the additional ingestion of apoptotic cells attenuates this response potently (9.Fadok V.A. Bratton D.L. Konowal A. Freed P.W. Westcott J.Y. Henson P.M. J. Clin. Invest. 1998; 101: 890-898Crossref PubMed Scopus (2552) Google Scholar, 10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 12.Voll R.E. Herrmann M. Roth E.A. Stach C. Kalden J.R. Girkontaite I. Nature. 1997; 390: 350-351Crossref PubMed Scopus (1505) Google Scholar). The ability of apoptotic cells to be cleared in a noninflammatory manner by professional phagocytes, such as macrophages, is a consequence of their specific expression of determinants for recognition and modulation of proinflammatory responses. The acquisition of these apoptotic determinants represents a gain of function and is common to all physiological cell deaths, without regard to suicidal stimulus (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Cells that die necrotically also are recognized by professional phagocytes; in contrast, however, necrotic corpses do not down-regulate inflammatory responses. Discrimination between apoptotic and necrotic corpses occurs on the level of binding and not engulfment and involves distinct and noncompeting mechanisms of recognition (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The modulatory activity of the apoptotic corpse is manifest as an immediate-early inhibition of macrophage proinflammatory cytokine gene transcription and is exerted directly upon binding to the macrophage, independent of subsequent engulfment and soluble factor involvement (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Apoptotic cells target the proinflammatory transcriptional machinery of macrophages, with which they interact through a novel regulatory pathway. Inhibition appears to involve sequestration of critical transcriptional co-activator molecules without effect on proximal signaling events induced by inflammatory receptors, including innate immune receptors of the TLR family (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The ubiquity of apoptotic cells in all tissues throughout organismal life prompted us to ask whether this innate discrimination of apoptotic cells is limited to professional phagocytes. Certainly, normal homeostatic cell turnover in vivo, and especially in solid tissues and intact cellular strata, results in apoptotic cells that are in immediate contact with their neighbors independent of (and before the arrival of) mobile phagocytes (6.Wood W. Turmaine M. Weber R. Camp V. Maki R.A. McKercher S.R. Martin P. Development. 2000; 127: 5245-5252Crossref PubMed Google Scholar, 14.Saunders Jr., J.W. Science. 1966; 154: 604-612Crossref PubMed Scopus (720) Google Scholar, 15.Wyllie A.H. Kerr J.F.R. Currie A.R. Int. Rev. Cytol. 1980; 68: 251-305Crossref PubMed Scopus (6720) Google Scholar, 16.Parnaik R. Raff M.C. Scholes J. Curr. Biol. 2000; 10: 857-860Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 17.Monks J. Rosner D. Geske F.J. Lehman L. Hanson L. Neville M.C. Fadok V.A. Cell Death Differ. 2005; 12: 107-114Crossref PubMed Scopus (178) Google Scholar). We asked whether nonprofessional phagocytes discriminate and respond specifically to apoptotic cells in an anti-inflammatory manner. Here we describe studies that reveal that they do. Remarkably, this profound innate immune function is manifest fully and ubiquitously among professional and nonprofessional phagocytes and even nonphagocytic cell types. Cells and Death Induction—RAW 264.7 murine macrophages, DO11.10 murine T hybridoma cells, Jurkat human acute T leukemia cells, Ramos (RA-1) human Burkitt's B lymphoma cells, and PLB-985 human myelomonoblastic leukemia cells (generously provided by Dr. Peter Henson, National Jewish Medical and Research Center) were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in RPMI 1640 medium (Mediatech, Herndon, VA) supplemented with heat-inactivated fetal bovine serum (10% v/v), 2 mm l-glutamine, and 50 μm 2-mercaptoethanol. HeLa human cervical carcinoma cells and 293T human transformed kidney epithelial cells were grown in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10% (v/v); HyClone Laboratories, Logan, UT) and 2 mm l-glutamine. Chinese hamster ovary cells were grown in α-minimal essential medium (Invitrogen) supplemented only with fetal bovine serum (10%, v/v). Human umbilical vein endothelial cells were grown in supplemented endothelial growth medium (Cambrex Bio Science, East Rutherford, NJ) on gelatin-coated plates. Immortalized murine 3T3 fibroblast cell lines were derived from mouse embryo fibroblasts following the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar). Briefly, the embryo fibroblasts were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in Dulbecco's modified Eagle's medium with 4.5 g/liter glucose (Mediatech) supplemented with fetal bovine serum (10%, v/v; HyClone Laboratories), 2 mml-glutamine, and 50 μm 2-mercaptoethanol, replating at 3 × 105/60-mm diameter dish every 3 days. Immortalized cell lines were established from cells that grew from cultures that had become senescent. Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitors actinomycin D (200 ng/ml, 12 h) or cycloheximide (1 μg/ml, 12 h) (19.Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar). Cells were killed pathologically (necrotic death) by incubation in phosphate-buffered saline (PBS) at 55 °C for 20 min (until trypan blue uptake indicated compromise of membrane integrity) (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). In all cases, target cells (viable, apoptotic, and necrotic) were washed twice and resuspended in the medium of the responder cells to be tested. In some experiments, apoptotic and viable target cells were fixed by incubation with formaldehyde (125 mm in PBS, 25 °C, 20 min; Polysciences, Inc., Warrington, PA) or were cycled through three rounds of freezing and thawing. Target preparations then were washed twice and resuspended in the medium of the responder cells to be tested. Plasma membrane vesicles were prepared from HeLa cells following the approach of Baumann et al. (20.Baumann N.A. Vidugiriene J. Machamer C.E. Menon A.K. J. Biol. Chem. 2000; 275: 7378-7389Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Monolayers of cells, either untreated or induced to die with actinomycin D (and still adherent), were stimulated to vesiculate by incubation at 37 °C in Vesiculation Buffer (10 mm Hepes (pH 7.4), 150 mm NaCl, 2 mm CaCl2, 2 mm dithiothreitol, and 25 mm formaldehyde). Supernatants were collected after ∼2.5 h (when abundant small membrane vesicles were apparent in the culture fluid). Nonadherent cells were removed (1,000 × g for 10 min), and vesicles were pelleted from the cleared supernatant by centrifugation (30,000 × g, 60 min, 4 °C). Cytofluorimetric analysis indicated that vesicles were ∼0.8 μm in diameter and free of contaminating intact cells. Phagocytosis Assay and Other Cytofluorimetric Analyses—Phagocytosis was assessed as previously described for macrophages (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Target cells were labeled green with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μm; Molecular Probes, Inc., Eugene, OR) and were then induced to undergo apoptotic cell death, killed pathologically by heat treatment, or left untreated. Phagocytes (or cells being tested for phagocytic activity) were labeled red with 5-(and 6)-(((4-chloromethyl)benzoyl)-amino) tetramethyl rhodamine (CMTMR; 10 μm; Molecular Probes). In all cases, cells were labeled on the day preceding the experiment and cultured in serum-containing medium overnight to eliminate unbound label. Labeled phagocytes were co-cultured with the apoptotic, necrotic, or viable target cells for 30 min at 37 °C. Cells were harvested with PBS supplemented with 0.4 mm Na2EDTA and analyzed cytofluorimetrically on a FACSCaliber instrument (BD Biosciences). Cytofluorimetric data were processed with WinMDI software (Joe Trotter, Scripps Research Institute, La Jolla, CA). Cells that were both CMTMR-positive (Exλ = 488 nm, Emλ = 610 ± 15 nm) and CFDA-positive (Exλ = 488 nm; Emλ = 530 ± 15 nm) and that had scatter properties of the phagocyte population represented phagocytes that had engulfed targets. Engulfment is calculated as the fraction of double-positive phagocytes (all CMTMR-positive cells that also are CFDA-positive). Most targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis, although they could be enumerated under static microscopic examination (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar). The accessibility of phosphatidylserine was revealed by the binding of fluorescein isothiocyanate-conjugated annexin V (Pharmingen; San Diego, CA; Exλ = 488 nm, Emλ = 525 nm). Cells were harvested and washed twice with cold PBS. Cells were resuspended in 100 μl of binding buffer (10 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm CaCl2) and incubated with 5 μl of fluorescein isothiocyanate-conjugated annexin V for 15 min in the dark at 25 °C. After incubation, 400 μl of binding buffer was added per sample, and cells were analyzed cytofluorimetrically. Propidium iodide (PI) was employed to assess plasma membrane integrity. PI was added to cells at 1 μg/ml immediately before cytofluorimetric analysis (Exλ = 488 nm, Emλ = 610 nm). Transfections and Luciferase Assays—Apoptotic modulation of specific transcription (e.g. dependent on nuclear factor κB (NF-κB) or the IL-8 promoter) was assessed in various cell types following transfection of relevant transcriptional reporter constructs, using a dual luciferase strategy, as described previously (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The efficiencies of transfection were measured in parallel with farnesylated green fluorescent protein as a transfection marker (21.Harvey K.J. Lukovic D. Ucker D.S. Cytometry. 2001; 43: 273-278Crossref PubMed Scopus (27) Google Scholar). Cells were co-transfected with pNF-κB-Luc, a plasmid containing the firefly (Photinus pyralis) luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif (Clontech), together with pRL-SV40, a Renilla (sea pansy; R. reniformis) luciferase control vector, the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI). RAW 264.7 macrophages (5.0 × 106 cells/60-mm diameter dish) and HeLa, 293T, and Chinese hamster ovary cells, at ∼75% confluence, were transfected using Effectene Transfection Reagent (Qiagen, Valencia, CA). 3T3 cells were transfected using the MEF1 Nucleofector Kit (AMAXA Biosystems; Gaithersburg, MD), with MEF Nucleofector Solution 1 and a machine setting of A-23. Jurkat, Ramos RA-1, and human umbilical vein endothelial cells were transfected using GenePORTER2 Transfection Reagent (Gene Therapy Systems, San Diego, CA). The next day, the cells were replated in 24-well plates (1.0 × 105 cells/well) and incubated without or with the indicated target cells (at a target cell/macrophage ratio of 10:1) and/or a proinflammatory stimulus in a final volume of 2 ml. The proinflammatory stimuli used included LPS (100 ng/ml; Escherichia coli O111:B4; Sigma), tumor necrosis factor-α (TNFα; 10 ng/ml; R&D Systems; Minneapolis, MN), IL-1β (5 ng/ml; R&D Systems), and phorbol 12-myristate 13-acetate (PMA; 1.3 ng/ml; EMD Biosciences, San Diego, CA) alone or with ionomycin (200 ng/ml; Molecular Probes). Cell extracts were prepared after further incubation as indicated, and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) in an FB12 luminometer (Zylux; Oak Ridge, TN). Each condition was repeated in triplicate wells, and the luciferase activities in cells from each well were determined independently. Within any experiment, Renilla luciferase activities among samples varied less than 6%. The firefly luciferase activity in each sample was normalized with respect to the internal Renilla luciferase activity, and the relative level of normalized firefly luciferase activity compared with the activity in an untreated population was taken as a measure of specific (e.g. NF-κB-dependent) transcriptional activity. Stably transfected reporter cells were generated by transfection of 293T cells with another NF-κB-Luc reporter construct, 4×NF-κB(HIV)tkluc (22.Wissink S. van de Stolpe A. Caldenhoven E. Koenderman L. van der Saag P.T. Immunobiology. 1997; 198: 50-64Crossref PubMed Scopus (40) Google Scholar), and an unlinked vector conferring hygromycin resistance. Cells resistant to hygromycin (50 μg/ml) were selected, cloned at limiting dilution, and tested for NF-κB-dependent responsiveness. Extracts were prepared and analyzed as above, except that the luciferase assay system (Promega) was used. Data with one clone, B2, are described here. Quantification of Cytokine Release—Cytokine production was assessed following incubation of responder cells with target cells. Where indicated, proinflammatory stimuli were added simultaneously with the addition of targets. Culture supernatants were withdrawn from wells at the indicated times and frozen at –20 °C until analysis. Secreted cytokines were quantified by ELISA, using matched pair cytokine-specific capture and biotinylated reporter antibodies for murine IL-6 (eBiosciences; San Diego, CA) or human IL-8 (BIOSOURCE, Camarillo, CA). The reporter reactions were developed with horseradish peroxidase-conjugated streptavidin (R&D Systems) and measured spectrophotometrically at 450 nm (corrected for turbidity at 550 nm; Microplate Autoreader model EL311; Bio-Tek Instruments, Winooski, VT). Cellular Extract Preparation and Immunoblot Analysis—Activation of Akt and inhibition of ERK1/2 were assessed in 3T3 cells cultured overnight in serum-free medium and left unstimulated or stimulated for 15 min with a 5-fold excess of apoptotic DO11.10 cells (the apoptotic cells, which had been cultured under serum-free conditions, were centrifuged briefly onto the adherent 3T3 cells to initiate the interaction) and/or subsequent stimulation with epidermal growth factor (EGF; 10 nm; Calbiochem). After washing, cell extracts were prepared from the adherent 3T3 cells. Cells were lysed in lysis buffer (150 mm NaCl, 50 mm HEPES (pH 7.5), 1.5 mm MgCl2, 1 mm EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 200 μm orthovanadate). Lysates were centrifuged at 10,000 × g for 10 min at 4 °C, and the supernatants were stored at –70 °C. Protein samples (20 μg each, determined by the bicinchoninic acid protein assay; Pierce) were boiled in 5× sample buffer, run on 12% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA). Blots were blocked with 5% dry milk in 150 mm PBS, 20 mm Tris HCl, pH 7.5, before probing with a phospho-Akt(Thr308)-specific rabbit anti-serum (Cell Signaling, Beverly, MA) or an affinity-purified phospho-ERK1/2 (Thr183/Tyr185)-specific polyclonal rabbit IgG (Promega, Madison, WI). Following incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase, immunoreactive bands were visualized by the luminol reaction (ECLplus; Amersham Biosciences). Equivalent loading of protein samples was monitored by Ponceau S staining (0.25% (w/v) (Sigma) in 0.1% acetic acid; 5 min) of blotted proteins. Specific Recognition and Response to Apoptotic Cells Is Not Limited to Macrophages—The anti-inflammatory response triggered in macrophages by their specific recognition of apoptotic cells is exerted on the level of cytokine gene transcription (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Although key transcriptional activators of cytokine gene expression, such as NF-κB (23.Shakhov A.N. Collart M.A. Vassalli P. Nedospasov S.A. Jongeneel C.V. J. Exp. Med. 1990; 171: 35-47Crossref PubMed Scopus (733) Google Scholar, 24.Collart M.A. Baeuerle P. Vassalli P. Mol. Cell. Biol. 1990; 10: 1498-1506Crossref PubMed Google Scholar), are not the molecular targets of apoptotic modulation, modulation is evident on the level of NF-κB-dependent transcription, and an NF-κB-dependent transcriptional reporter serves as a sensitive, reliable, and convenient readout for the modulatory effect exerted by apoptotic targets (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). The experiment in Fig. 1A exemplifies this analysis. We transiently transfected RAW 264.7 macrophages with pNF-κB-Luc, a plasmid containing the firefly luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif. Macrophages were co-transfected with a constitutive (NF-κB-independent) Renilla luciferase control vector, which served as an internal normalization control for transfection efficiency and cell viability. Following transfection, macrophages were incubated with different target cell populations and/or bacterial LPS, a potent proinflammatory agonist. Firefly and Renilla luciferase activities then were measured. As indicated by these luciferase reporters, LPS-activated NF-κB-dependent transcription (but not global transcription) in macrophages is inhibited specifically following their interaction with apoptotic cells; necrotic and viable cells do not exert this effect (13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). This response is elicited by apoptotic cells generally, regardless of species, cell type, or suicidal stimulus. Here, murine DO11.10 T cells and human HeLa epithelial carcinoma cells, triggered to die with different suicidal stimuli (inhibitors of translation and transcription, respectively), were equally effective at triggering modulation in these murine macrophages. As a first test of the ability of nonprofessional phagocytes to recognize and respond to apoptotic cells, we examined the responsiveness of HeLa cells, the same cells used as targets in Fig. 1A, utilizing the identical transcriptional reporter strategy. The transfected HeLa cells were incubated with apoptotic, necrotic, or viable populations of target cells and/or the inflammatory cytokine TNFα as a stimulus of an NF-κB-dependent transcriptional response. Significantly, HeLa cells do not die in response to TNFα unless the NF-κB-dependent response is attenuated (e.g. by an inhibitor of macromolecular synthesis) (25.Beg A.A. Baltimore D. Science. 1996; 274: 782-784Crossref PubMed Scopus (2933) Google Scholar, 26.Wang C.-Y. Mayo M.W. Baldwin A.S.J. Science. 1996; 274: 784-787Crossref PubMed Scopus (2509) Google Scholar, 27.Harvey K.J. Lukovic D. Ucker D.S. J. Cell Biol. 2000; 148: 59-72Crossref PubMed Scopus (83) Google Scholar). Just as with LPS-stimulated macrophage responsiveness, robust TNFα-activated NF-κB-dependent transcription in HeLa cells was inhibited specifically and profoundly following the interaction of those cells with apoptotic, but not necrotic or viable, targets (Fig. 1B). It is notable that the ranges of absolute values of NF-κB-dependent luciferase activities were quite different in HeLa cells and macrophages at comparably early times (as much as 300-fold; see Fig. 1), reflecting differences in the efficiencies of transfection and transgene expression. Still, expressed relative to basal luciferase levels, these data present a consistent pattern of modulation and reveal an identical response to apoptotic targets in different responder cell populations. Again, this selective response to apoptotic cells occurred without species restriction. Most dramatically, HeLa cells even were able to recognize and respond specifically to homotypic apoptotic cells. The Characteristic Repertoire of Anti-inflammatory Responses Elicited Specifically upon Apoptotic Cell Recognition Is Evident in Murine Fibroblasts—To begin a more comprehensive exploration of the recognition and response to apoptotic targets by nonprofessional phagocytes, we examined nontransformed murine fibroblasts. Immortalized murine embryo fibroblast cells, derived by the 3T3 protocol of Todaro and Green (18.Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (2003) Google Scholar), were highly phagocytic for dead cells (Fig. 2A; for consistency, we used DO11.10 cells as targets in this and the following experiments). Apoptotic and necrotic cell targets were engulfed rapidly and to equal extents by 3T3 fibroblasts, whereas viable cells were not ingested (Fig. 2A; we take the low level of engulfment of “viable” cells to reflect the small fraction of dead and dying apoptotic cells present in any cell culture). These 3T3 fibroblasts secrete the inflammatory cytokine IL-6 in response to a variety of proinflammatory stimuli, including IL-1β, TNFα, and, to a lesser degree, bacterial LPS (Fig. 2B and data not shown) (28.Kurt-Jones E.A. Sandor F. Ortiz Y. Bowen G.N. Counter S.L. Wang T.C. Finberg R.W. J. Endotoxin Res. 2004; 10: 419-424Crossref PubMed Google Scholar). We characterized the release of IL-6 from fibroblasts following their interaction with target cells as one indication of inflammatory responsiveness; IL-6 secretion in macrophages reflects inflammatory responsiveness generally (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163) Google Scholar, 13.Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). IL-1β-stimulated IL-6 secretion was potently attenuated when fibroblasts interacted with apoptotic targets, but not with necrotic or viable targets (Fig. 2B). Apoptotic target cell contact was necessary for this response, since supernatants from apoptotic cell cultures could not substitute for the target cells themselves to modulate IL-6 secretion (data not shown). The ability of apoptotic cells to block IL-6 secretion by IL-1β-stimulated fibroblasts parallels their ability to abrogate the secretion of IL-6 and other inflammatory cytokines by LPS-stimulated macrophages (10.Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (163

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR+/+, PSR+/-, and PSR-/- fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells. The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated, PSR+/+, PSR+/-, and PSR-/- fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells. Physiological cell death is a process whose purpose is the elimination of functionally inappropriate cells in a manner that does not elicit inflammation. The ability of apoptotic corpses to be cleared in a noninflammatory manner by phagocytes is a consequence of their specific expression of determinants for recognition and modulation of pro-inflammatory responses. The acquisition of these apoptotic determinants is a gain-of-function common to all physiological cell deaths, without regard to suicidal stimulus, and conserved widely across species (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar, 2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar).Numerous cellular alterations associated with apoptotic cell death have been described, including plasma membrane reorganization associated with blebbing (3Hogue M.J. J. Exp. Med. 1919; 30: 617-647Crossref PubMed Scopus (24) Google Scholar), shrinkage, and the loss of membrane phospholipid asymmetry (4Kerr J.F.R. Wyllie A.H. Currie A.R. Br. J. Cancer. 1972; 26: 239-256Crossref PubMed Scopus (12723) Google Scholar, 5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). In particular, phosphatidylserine (PS), 4The abbreviations used are: PS, phosphatidylserine; CFDA, 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester; CMTMR, 5 (and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; GFP, green fluorescent protein; GST, glutathione S-transferase; MFG-E8, milk fat globule factor-E8; PSR, presumptive PS-specific receptor; RT, reverse transcriptase; IL-1β, interleukin 1β; IL-6, interleukin 6; TGFβ, transforming growth factor-β; TNFα, tumor necrosis factor-α; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; mAb, monoclonal antibody; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; CRIB, Cdc42/Rac interactive binding. 4The abbreviations used are: PS, phosphatidylserine; CFDA, 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester; CMTMR, 5 (and 6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; GFP, green fluorescent protein; GST, glutathione S-transferase; MFG-E8, milk fat globule factor-E8; PSR, presumptive PS-specific receptor; RT, reverse transcriptase; IL-1β, interleukin 1β; IL-6, interleukin 6; TGFβ, transforming growth factor-β; TNFα, tumor necrosis factor-α; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; mAb, monoclonal antibody; MEF, mouse embryo fibroblast; PBS, phosphate-buffered saline; CRIB, Cdc42/Rac interactive binding. an anionic phospholipid normally cloistered in the inner leaflet of the plasma membrane, is externalized during physiological cell death (5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar). It still remains to be determined what specific molecular events are responsible for the recognition of the effete cell.The view that externalized PS serves as a ligand for macrophage recognition of apoptotic cells followed from studies demonstrating that similar changes target aged erythrocytes for clearance (6Schroit A.J. Madsen J.W. Tanaka Y. J. Biol. Chem. 1985; 260: 5131-5138Abstract Full Text PDF PubMed Google Scholar, 7McEvoy L. Williamson P. Schlegel R.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 3311-3315Crossref PubMed Scopus (191) Google Scholar) and gained support from observations that phospho-l-serine and PS vesicles could inhibit partially the interaction of dying nucleated cells with macrophages (5Fadok V.A. Voelker D.R. Campbell P.A. Cohen J.J. Bratton D.L. Henson P.M. J. Immunol. 1992; 148: 2207-2216PubMed Google Scholar, 8Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (126) Google Scholar, 9Fadok V.A. Warner M.L. Bratton D.L. Henson P.M. J. Immunol. 1998; 161: 6250-6257PubMed Google Scholar).A presumptive cell surface PS-specific receptor (PSR) was identified molecularly following a screen for monoclonal antibodies whose binding to human macrophages was inhibited by PS-containing liposomes (10Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekowitz R.A.B. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar). The product of that screen, mAb 217, bound to cell surface determinants on macrophages and other cell types, notably excluding lymphoid cells. Significantly, mAb 217 triggered macrophages to release the anti-inflammatory cytokine TGFβ, further suggesting that mAb 217 engaged an apoptotic-like recognition mechanism (10Fadok V.A. Bratton D.L. Rose D.M. Pearson A. Ezekowitz R.A.B. Henson P.M. Nature. 2000; 405: 85-90Crossref PubMed Scopus (1247) Google Scholar).Controversy regarding this presumptive receptor arose, however, when PSR was observed to localize to the nucleus in mammalian cells (11Cui P. Qin B. Liu N. Pan G. Pei D. Exp. Cell Res. 2004; 293: 154-163Crossref PubMed Scopus (95) Google Scholar) as well as in Hydra (12Cikala M. Alexandrova O. David C.N. Pröschel M. Stiening B. Cramer P. Böttger A. BMC Cell Biol. 2004; 5: 26Crossref PubMed Scopus (81) Google Scholar). The role of PSR has been further clouded by the disparate results of three groups of investigators who independently generated mice with targeted disruptions of the PSR locus (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar, 14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar, 15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar). While homozygous PSR disruptions result in perinatal lethality in each case, different effects on the phagocytosis of apoptotic cells have been reported for the three PSR deficiencies.Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) described a disruption beginning upstream of exon 1 (which includes the translational start site of the PSR gene product) and extending through exon 2, in a mixed 129 × C57BL/6 background, which leads to severe lung defects as well as less penetrant brain aberrations. Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar) generated a disruption, also starting upstream of exon 1 and extending into exon 3, in a chimeric 129 × C57BL/6 background, which results in defective erythroid differentiation and severe anemia. The disruption generated by Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) in a pure C57BL/6 background is limited to exons 1 and 2 and leads to growth retardation, defects in embryonic lung, kidney, gut, and erythroid development, and, at low frequency, aberrant eye and brain development. Hemizygous PSR deficiency has no phenotype in any of these cases.An increased number of apoptotic cells (with digested genomic DNA, identified by TUNEL (terminal deoxyribonucleotidyltransferase end labeling) staining) was observed by Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) in the affected tissues of their PSR-deficient mice and attributed to the diminished engulfment of dead cells. Interestingly, Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar) also suggested that phagocytosis of apoptotic cells by macrophages was diminished during embryogenesis, based on immunohistochemical of staining fetal liver and thymus sections their PSR-/- of mice. Their data are confounded, however, by an apparent reduction in the number of tissue-resident macrophages (stained with F4/80 antibody; Ref. 16Austyn J.M. Gordon S. Eur. J. Immunol. 1981; 11: 805-815Crossref PubMed Scopus (1278) Google Scholar), hinting at another possible developmental impairment related to PSR deficiency. Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) further explored engulfment in vitro with PSR-deficient macrophages generated by adoptive transfer of PSR-/- fetal liver into wild-type hosts. They reported a 50% overall reduction and a complete absence of PS-inhibitable engulfment by PSR-deficient elicited macrophages. In light of the observations of Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar), it is important to note that the differentiation and activation states of these PSR-/- and PSR+/+ macrophages were not compared; previous work has suggested that PS-inhibitable engulfment pertains particularly to activated macrophages (8Pradhan D. Krahling S. Williamson P. Schlegel R.A. Mol. Biol. Cell. 1997; 8: 767-778Crossref PubMed Scopus (126) Google Scholar, 9Fadok V.A. Warner M.L. Bratton D.L. Henson P.M. J. Immunol. 1998; 161: 6250-6257PubMed Google Scholar). In marked contrast to the results of Li et al. (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar) and Kunisaki et al. (14Kunisaki Y. Masuko S. Noda M. Inayoshi A. Sanui T. Harada M. Sasazuki T. Fukui Y. Blood. 2004; 103: 3362-3364Crossref PubMed Scopus (96) Google Scholar), extensive histological analysis of numerous tissues by Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) revealed no defects in the phagocytosis of apoptotic (TUNEL+) cells. Their in vitro phagocytosis studies with fetal liver-derived macrophages (differentiated in culture) also showed no engulfment defect of PSR-deficient macrophages. Of great significance, Böse et al. (15Böse J. Gruber A.D. Helming L. Schiebe S. Wegener I. Hafner M. Beales M. Köntgen F. Lengeling A. J. Biol. 2004; 3: 15.1-15.18Crossref Google Scholar) reported that the ability of apoptotic cells to trigger an antiinflammatory response in engulfing macrophages (both the inhibition of secretion of pro-inflammatory cytokines, such as TNFα, and the induction of anti-inflammatory cytokines, such as TGFβ and IL-10) was unimpaired in the absence of PSR expression.Our studies of the process of apoptotic cell clearance have demonstrated that the ability of apoptotic corpses to be engulfed specifically and in a non-inflammatory manner by macrophages and other phagocytes is a consequence of a process of specific recognition and modulation of pro-inflammatory phagocyte responses (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar). The modulatory effect of the apoptotic corpse is manifest as an immediate-early inhibition of pro-inflammatory cytokine gene transcription within the responding phagocyte with which it interacts, and is exerted upon binding, independent of subsequent engulfment or soluble factor involvement (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Cells that die pathologically (that is, necrotic corpses) also are recognized by phagocytes but do not down-regulate inflammatory responses. The recognition of these two classes of native dying cells occurs via distinct and non-competing mechanisms (1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar).Importantly, non-professional phagocytes are fully capable of noninflammatory recognition and clearance of apoptotic cells. 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. Indeed, we have found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including non-phagocytic lymphocytes, and reflects an innate immunity that discriminates live from effete cells without regard to self (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. In particular, the ability of fibroblastic cells to respond to apoptotic corpses permits mouse embryo fibroblasts established from animals with targeted disruptions of genes of interest (including essential genes) to be used in the evaluation of genetic contributions to apoptotic recognition and response without the generation or selection of particular cell populations.We have applied this analysis to embryo fibroblasts that harbor a disruption of the PSR locus. Our data, which demonstrate that PSR-deficient cells are fully competent to recognize apoptotic cells, definitively exclude a general role for PSR in apoptotic recognition and inflammatory modulation.EXPERIMENTAL PROCEDURESCells and Death Induction—Immortalized murine fibroblast cell lines were derived from mouse embryo fibroblasts (MEFs) following the 3T3 protocol of Todaro and Green (17Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1993) Google Scholar). Briefly, MEFs were cultured at 37 °C in a humidified, 5% (v/v) CO2 atmosphere in Dulbecco’s modified Eagle’s medium with 4.5 g/liter glucose (Mediatech, Herndon, VA) supplemented with fetal bovine serum (10% v/v; HyClone Laboratories, Logan, UT), 2mml-glutamine, and 50 μm 2-mercaptoethanol, replating at 3 × 105/60-mm diameter dish every 3 days. Immortalized cell lines were established from cells that grew from cultures that had become senescent.NIH 3T3 cells were maintained similarly. 293T human transformed kidney epithelial cells were grown in the same medium without 2-mercaptoethanol, and DO11.10 murine T hybridoma and CEM human T lymphoblastoid cells were grown in RPMI 1640 medium (Mediatech) supplemented with heat inactivated fetal bovine serum (10% v/v), 2 mml-glutamine, and 50 μm 2-mercaptoethanol.Physiological cell death (apoptosis) was induced by treatment of target cells with the macromolecular synthesis inhibitor actinomycin D (200 ng/ml, 12 h; Ref. 18Chang S.H. Cvetanovic M. Harvey K.J. Komoriya A. Packard B.Z. Ucker D.S. Exp. Cell Res. 2002; 277: 15-30Crossref PubMed Scopus (18) Google Scholar) or with UV-B irradiation (20 mJ/cm2). Cells were killed pathologically (necrotic death) by incubation at 55 °C for 20 min (until trypan blue uptake indicated compromise of membrane integrity; Ref. 1Cocco R.E. Ucker D.S. Mol. Biol. Cell. 2001; 12: 919-930Crossref PubMed Scopus (162) Google Scholar). In all cases, target cells (viable, apoptotic, and necrotic) were washed twice in PBS or complete medium before addition to experimental cultures.Reverse Transcriptase (RT)-PCR Analysis—PSR expression on the level of transcripts was evaluated by RT-PCR analysis. Total cellular RNA was isolated using TRIzol reagent (Invitrogen). cDNA synthesis and PCR were performed sequentially using the SuperScript One-Step RT-PCR with Platinum Taq kit (Invitrogen). For the PSR-specific reaction, the forward primer was 5′-CAAGACGGTAAGAGGGAGACC-3′ (nucleotides 1086-1106, within exon 4), and the reverse primer was 5′-GTCACCTGGAGGAGCTGCG-3′ (complementary to nucleotides 1402-1384, within exon 6), yielding a product of 276 bp. As a control for RNA integrity, transcripts of constitutively expressed glyceraldehyde-3-phosphate dehydrogenase were assessed in parallel, using forward (5′-CCATGGAGAAGGCTGGGG-3′) and reverse (5′-CAAAGTTGTCATGGATGACC-3′) primers, generating a 188-bp product from 5′ end of the mRNA.Phagocytosis Assay—Phagocytosis by 3T3 fibroblasts was assessed as previously described for macrophages (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). Target cells were labeled green with 5 (and 6)-carboxyfluorescein diacetate succinimidyl ester (CFDA; 0.2 μm; Molecular Probes, Eugene, OR) and were then induced to undergo cell death, killed by heat treatment, or left untreated. 3T3 cells were labeled red with 5 (and 6)-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine (CMTMR; 10 μm; Molecular Probes). In all cases, cells were labeled on the day preceding the experiment and cultured in serum-containing medium overnight to eliminate unbound label. Labeled 3T3 cells were co-cultured with the apoptotic, necrotic, or viable target cells for 30 min at 37 °C. Cells were harvested with PBS supplemented with 0.4 mm Na2EDTA and analyzed cytofluorimetrically on a FacsCaliber instrument (BD Biosciences). Cells with 3T3-like scatter properties that were both CMTMR-positive (λEx = 488 nm, λEm = 610 nm ± 15 nm) and CFDA-positive (λEx = 488 nm; λEm = 530 ± 15 nm) represented 3T3 cells that had engulfed targets. Engulfment is calculated as the fraction of double-positive 3T3 fibroblasts (all CMTMR-positive cells that also are CFDA-positive). Targets that are bound but not engulfed are disrupted and do not remain adherent during the analysis.Phagocytosis by transfected 293T cells was assayed similarly, employing a green fluorescent protein (GFP) marker expressed from a bicistronic vector to label the phagocytes green in place of CFDA. 293T cells were transfected with pIRES-2-PSR, pCX-β5 (which includes the same cytomegalovirus promoter/internal ribosome entry site (IRES) structure), or empty pIRES-2 vector (19Albert M.L. Kim J.I. Birge R.B. Nat. Cell Biol. 2000; 2: 899-905Crossref PubMed Scopus (324) Google Scholar). Target cells were labeled red with PKH26-GL red fluorescent cell linker kit (Sigma). 48 h after transfection, the 293T cells were co-cultured with labeled apoptotic cells for 2 h at 37 °C and analyzed as above.Transfections and Luciferase Assays—Apoptoticmodulation of NFκB-dependent transcription was assessed using a dual luciferase strategy, as described previously (2Cvetanovic M. Ucker D.S. J. Immunol. 2004; 172: 880-889Crossref PubMed Scopus (158) Google Scholar). We found that routine transfection protocols for 3T3 cells triggered high levels of cell death and NFκB activation (“transfection stress”). A transfection protocol that was reasonably efficient (∼40% viable cell transfection, as measured in parallel with farnesylated GFP as a transfection marker; Ref. 20Harvey K.J. Lukovic D. Ucker D.S. Cytometry. 2001; 43: 273-278Crossref PubMed Scopus (27) Google Scholar) and minimally stressful (i.e. low spontaneous NFκB activation) was selected.3T3 cells were transfected using the MEF1 Nucleofector Kit (AMAXA Biosystems, Gaithersburg, MD), with “MEF Nucleofector Solution 1” and a machine setting of “A-23.” 2 × 106 3T3 fibroblasts were transfected with 4.5 μg of pNFκB-Luc, a plasmid containing the firefly (Photinus pyralis) luciferase gene, the expression of which is driven by a basal transcriptional promoter linked to four copies of the κB motif (Clontech Laboratories; Palo Alto, CA), together with 0.5 μg of pRL-SV40, a Renilla (sea pansy; Renilla reniformis) luciferase control vector, the constant expression of which is dependent on the SV40 early enhancer/promoter region (Promega, Madison, WI). Transfected cells were cultured overnight in 100-mm diameter dishes and replated the following day into 24-well plates at 1 × 105 cells/2 ml/well. After culturing a further 24 h, transfected cells were incubated without or with the indicated target cells (at a target cell:fibroblast ratio of 10:1) and/or IL-1β (5 ng/ml; R&D Systems; Minneapolis, MN) or TNFα (10 ng/ml; R&D Systems) for 12 h.Cell extracts were prepared, and luciferase activities were measured by the Dual Luciferase Reporter Assay System (Promega) in an FB12 Luminometer (Zylux, Oak Ridge, TN). Each condition was repeated in triplicate wells, and the luciferase activities in cells from each well were determined independently. The firefly luciferase activity in each sample was normalized with respect to the internal Renilla luciferase activity, and the relative level of normalized firefly luciferase activity compared with the activity in an untreated population was taken as a measure of NFκB-dependent transcriptional activity.For other studies involving transfection, routine methods were employed. NIH 3T3 cells were transfected with Lipofectamine 2000 Transfection Reagent (Qiagen, Valencia, CA), and 293T cells were transfected using Effectene Transfection Reagent (Qiagen).Intracellular Immunostaining—5 × 104 NIH 3T3 cells were plated on poly-d-lysine-coated cover slips and transfected with epitope-tagged PSR constructs (either N-terminal HA-PSR in pRK or C-terminal PSR-V5-His in pcDNA3.1). 24 h post-transfection, the cells were fixed with paraformaldehyde (5% in PBS). The cells then were permeabilized with Triton X-100 (0.1% in PBS) for 5 min and incubated for 1 h with anti-HA (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-V5 (Invitrogen) antibodies (diluted in PBS + 0.1% gelatin). After four washes (5 min each) in PBS + 0.1% gelatin, the cells were stained with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody and rhodamine-conjugated phalloidin (Molecular Probes). After several further washes, the coverslips were dried and mounted with mounting medium (ProLong Antifade Kit; Molecular Probes). The cells were visualized by epifluorescence microscopy.Cellular Extract Preparation and Immunoblot Analysis—PSR expression was assessed in unmanipulated 3T3 cells by immunoblotting. Activation of Akt and inhibition of extracellular signal-regulated kinases 1 and 2 (ERK1/2) were assessed in 3T3 cells cultured overnight in serum-free medium and left unstimulated or stimulated for 15 min with a 5-fold excess of apoptotic DO11.10 cells (the apoptotic cells, which had been cultured under serum-free conditions, were centrifuged briefly onto the adherent 3T3 cells to initiate the interaction) and/or subsequent stimulation with epidermal growth factor (10 nm; Calbiochem). After washing, cell extracts were prepared from the adherent 3T3 cells. Cells were lysed in lysis buffer 1 (150 mm NaCl, 50 mm HEPES (pH 7.5), 1.5 mm MgCl2,1mm EGTA, 10% glycerol, 1% Triton X-100, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 200 μm orthovanadate). Lysates were centrifuged at 10,000 × g for 10 min at 4 °C and the supernatants stored at -70 °C.Protein samples (20 μg each, determined by the bicinchoninic acid protein assay; Pierce) were boiled in 5× sample buffer, run on 12% SDS-polyacrylamide gels, and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA). Blots were blocked with 5% dry milk in PBS before probing with a phospho-Akt(Thr308)-specific rabbit antiserum (Cell Signaling, Beverly,MA), an affinity-Purified phospho-ERK1/2 (Thr183/Tyr185)-specific rabbit antibody (Promega), or an affininty-purified PSR-specific rabbit antibody (specific for amino acids 363-381; Abcam, Cambridge, MA). Following incubation with an anti-rabbit secondary antibody conjugated to horseradish peroxidase, immunoreactive bands were visualized by the luminol reaction (ECLplus; Amersham Biosciences). Equivalent loading of protein samples was monitored by Ponceau S staining (0.25% (w/v; Sigma) in 0.1% acetic acid; 5 min) of blotted proteins.Rac1 Pull-down Assay—The level of GTP-bound Rac1 was determined by the GST-PAK CRIB “pull-down” assay as described previously (21Akakura S. Singh S. Spataro M. Akakura R. Kim J.I. Albert M.L. Birge R.B. Exp. Cell Res. 2004; 292: 403-416Crossref PubMed Scopus (177) Google Scholar). In brief, 5 × 105 3T3 fibroblasts were plated for 2 h on 60-mm diameter dishes that had been coated previously with MFG-E8 or bovine serum albumin (10 μg/ml). The cells then were lysed for 10 min in lysis buffer 2 (50 mm Tris (pH 7.2), 1% (v/v) Triton X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 500 mm NaCl, and 10 mm MgCl2, plus protease inhibitors). 500 μg of each cell lysate was incubated for 1 h with glutathione-agarose beads coated with a bacterially expressed fusion protein of glutathione S-transferase (GST) with the GTPase binding domain (CRIB domain; amino acid residue 56-272) of the human PAK kinase, a downstream effector molecule for Rac1, which specifically binds the activated, GTP-loaded form of Rac1. The beads then were washed four times in wash buffer (50 mm Tris (pH 7.2), 1% (v/v) Triton X-100, 150 mm NaCl, and 10 mm MgCl2, plus protease inhibitors). Active Rac1, precipitating with beads, and total Rac1, in the starting lysates, were quantified by densitometry following immunoblotting with a Rac1 antibody (Upstate Biotechnology, Waltham, MA).RESULTSImmortalized 3T3 Cell Lines from PSR+/+, PSR+/-, and PSR-/- Mouse Embryo Fibroblasts—The role of PSR in the non-inflammatory clearance of apoptotic cells remains unresolved. Taking advantage of the ability of non-professional phagocytes to recognize, engulf, and respond to apoptotic cells specifically, 5M. Cvetanovic, J. E. Mitchell, V. Patel, B. S. Avner, Y. Su, P. T. van der Saag, P. L. Witte, S. Fiore, J. S. Levine, and D. S. Ucker, submitted for publication. we established immortalized 3T3 fibroblast cells lines from PSR+/+, PSR+/-, and PSR-/- mouse embryo fibroblasts to test the role of PSR in these processes. The immortalized cell lines were derived from (129 × C57BL/6) embryo fibroblasts taken at day E14.5, prior to manifestations of lethality associated with PSR deficiency (13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar), following the 3T3 protocol of Todaro and Green (17Todaro G.J. Green H. J. Cell Biol. 1963; 17: 299-313Crossref PubMed Scopus (1993) Google Scholar). We chose to examine cells with the targeted PSR locus of Li et al. (Fig. 1A; Ref. 13Li M.O. Sarkisian M.R. Mehal W.Z. Rakic P. Flavell R.A. Science. 2003; 302: 1560-1563Crossref PubMed Scopus (335) Google Scholar), because the best genetic evidence implicating PSR in apoptotic clearance is derived from studies of mice harboring that disruption.PSR expression in these cells was evaluated by RT-PCR analysis of PSR transcripts (Fig. 1B) and by immunoblotting with a PSR-specific antibody (Fig. 1C). These tests confirmed both the presence of PSR expression in wild-type cells and in PSR+/- heterozygotes, at roughly equivalent levels, and its absence in PSR-/- fibroblasts. These results a

Background. We reported that rapamycin impairs recovery after acute renal failure (ARF) in rats. The objective of this study was to determine if recovery will eventually occur after ARF despite continued rapamycin treatment. Methods. ARF was induced in rats by renal artery occlusion. Glomerular filtration rate (GFR), morphology, and tubular cell proliferation were assessed either 2, 4, 6, or 7 days later. Rats were treated daily with rapamycin or vehicle throughout the study. Cultured mouse proximal tubular (MPT) cells were used to compare the antiproliferative effects of rapamycin after exposure for 1 and 7 days. Results. Two days after ARF, GFR was reduced severely but comparably in vehicle and rapamycin rats. In controls, GFR began to increase after day 2 and was normal by day 6. In rapamycin rats, GFR did begin to improve until after day 4 and reached normal values by day 7. In controls, many proliferating tubular cells were present in outer medulla on day 2, after which proliferation progressively decreased. By contrast, in rapamycin rats, proliferating cells were sparse on day 2, but then increased substantially through days 4 and 6. Cultured MPT cells exposed to rapamycin for 7 days were ∼10-fold more resistant to the antiproliferative effects of rapamycin than cells exposed for 1 day. Conclusions. Rapamycin delays but does not prevent renal recovery after ARF. MPT cells become resistant to rapamycin after prolonged exposure. We speculate that the ultimate recovery of renal function after ARF is due to the development of acquired tubular cell resistance to rapamycin.

Previous studies using verapamil as an adjunct therapy to anti-seizure medications have used doses ranging from 120 to 240 mg per day. However, despite showing promising results, there was an increased incidence of side effects. The aim of this study is to assess the efficacy and tolerability of low dose verapamil (20 mg p.o. tid) as adjunct therapy to patient’s anti-seizure medications irrespective of the type or etiology of the epilepsy. In an open-label pilot study we enrolled 20 adult patients with history of epilepsy who continued to have a minimum of 2 seizures a month despite being on or having tried maximum tolerated doses of 3 or more standard antiepileptic drugs under the supervision of an epileptologist. 10 of the 19 patients (53%) who continued in the study had >50% reduction in seizure frequency. 2 of the patients (10%) had <50% seizure reduction. The remaining 7 patients (37%) had no reduction in their seizures. There was no discontinuation due to adverse events. P-Glycoprotein is a prototypical drug transporter that has been strongly implicated in drug resistance in epilepsy. Verapamil at a relatively low dose was well tolerated compared to previous studies which used up to 240 mg per day and seems to have contributed to a statistically significant improvement in seizure control in patients with medically refractory epilepsy, especially in patients with Lennox-Gastaut syndrome. A randomized double blind controlled study at this low dose with larger sample size may be more informative.

Atypical antipsychotic agents, such as clozapine, are used to treat schizophrenia and other psychiatric disorders by a mechanism that is believed to involve modulating the immune system. Multiple sclerosis is an immune-mediated neurological disease, and recently, clozapine was shown to reduce disease severity in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). However, the mode of action by which clozapine reduces disease in this model is poorly understood.Because the mode of action by which clozapine reduces neuroinflammation is poorly understood, we used the EAE model to elucidate the in vivo and in vitro effects of clozapine.In this study, we report that clozapine treatment reduced the infiltration of peripheral immune cells into the central nervous system (CNS) and that this correlated with reduced expression of the chemokines CCL2 and CCL5 transcripts in the brain and spinal cord. We assessed to what extent immune cell populations were affected by clozapine treatment and we found that clozapine targets the expression of chemokines by macrophages and primary microglia. Furthermore, in addition to decreasing CNS infiltration by reducing chemokine expression, we found that clozapine directly inhibits chemokine-induced migration of immune cells. This direct target on the immune cells was not mediated by a change in receptor expression on the immune cell surface but by decreasing downstream signaling via these receptors leading to a reduced migration.Taken together, our study indicates that clozapine protects against EAE by two different mechanisms; first, by reducing the chemoattractant proteins in the CNS; and second, by direct targeting the migration potential of peripheral immune cells.

We examined the role of AMP-activated protein kinase (AMPK) in modulating the viability of cultured kidney proximal tubular cells subjected to metabolic stress induced by either dextrose deprivation, inhibition of glycolysis, or inhibition of mitochondrial respiration. We used BU.MPT cells, a conditionally immortalized kidney epithelial cell line derived from the proximal tubules of transgenic mice bearing a temperature-sensitive mutation of the simian virus 40 large-tumor antigen. All three forms of metabolic stress increased the phosphorylation and activity of AMPK. Activation of AMPK led to changes in the phosphorylation of two downstream targets of AMPK, acetyl coenzyme A carboxylase and p70 S6 kinase. Inhibition of AMPK, either pharmacologically with compound C (CC) or by gene silencing, significantly increased the amount of apoptosis in response to all three forms of metabolic stress. Although the amount of apoptosis was directly related to the severity of ATP depletion, inhibition of AMPK had no effect on cellular ATP levels. Notably, metabolic stress increased the phosphorylation and activity of Akt. Furthermore, inhibition of AMPK, with CC or gene silencing, abrogated the ability of metabolic stress to activate Akt. The augmentation of apoptosis induced by inhibition of AMPK was comparable to that induced by inhibition of Akt. We conclude that activation of AMPK following acute metabolic stress plays a major role in promoting the viability of cultured proximal tubular cells. Protection by AMPK appears to be due not to AMPK-mediated conservation of cell energy stores, but rather, at least in part, to AMPK-mediated activation of Akt.

To determine whether opioids during the first 24 postoperative hours were significantly altered when receiving intravenous (IV) acetaminophen during that time compared with those receiving placebo (normal saline). One hundred forty patients undergoing any type of craniotomy were randomly assigned to receive either 1 g of IV acetaminophen or placebo upon surgical closure, and every 6 hours thereafter, up to 18 hours postoperatively. Analgesic requirements for the first 24 postoperative hours were recorded. Time to rescue medications in the postanesthesia care unit (PACU)/intensive care unit (ICU), amount of rescue medication, ICU and hospital lengths of stay, number of successful neurological examinations, sedation, delirium, satisfaction, and visual analog scale pain scores were also recorded. Compared with the placebo group, more patients in the IV acetaminophen group (10/66 [15.2%] vs. 4/65 [6.2%] in the placebo group) did not require opioids within the first 24 postoperative hours, but this did not reach significance (odds ratio, −9.0%, 95% confidence interval −20.5% to 1.8%; P = 0.166). Both groups had similar times to rescue medications, amounts of rescue medications, ICU and hospital lengths of stay, numbers of successful neurological examinations, sedation, delirium, satisfaction scores, visual analog scale pain scores, and temperatures within the first 24 postoperative hours. The opioid requirements within the first 24 postoperative hours were similar in the placebo and acetaminophen groups. This study is informative for the design and planning of future studies investigating the management of postoperative pain in patients undergoing craniotomies.

Administration of a goitrogen (methimazole) and a low iodine diet to rats over a two-week period resulted in hypothyroidism and thyroid hyperplasia compared with controls (control: total serum thyroxine (T4) 66 +/- 4 nmol/l, thyroid weight 5 +/- 1 mg/100 g body weight; experimental: T4 undetectable, thyroid weight 27 +/- 4 mg/100 g body weight after 2 weeks of treatment; mean +/- S.D., n = 10). Immunohistochemistry carried out using a specific endothelial cell marker, CD31, and morphometric analysis (point counting of immunopositive cells) revealed that the progression of goitre in the rat thyroid is accompanied by an increase in capillary endothelial cell growth (neovascularisation). Fibroblast growth factor-2 (FGF-2) immunohistochemistry revealed widespread staining for the protein in the follicular cells of control glands. Less intense staining was found in the stroma and follicular cell nuclei. During hyperplasia and subsequent neovascularisation there was a progressive increase in the FGF-2 immunoreactivity at all locations during the two-week treatment period. Thrombospondin-1 (TSP1) immunoreactivity in the control rat thyroid was found in the stroma and in the endothelial cells, while weak follicular cell staining was also present. In the goitrous rat thyroid the TSP immunoreactivity was present after 1 week of treatment in the endothelial cells and most follicular cells, whilst stroma localisation was weak. After week 2 of treatment the endothelial cell and stromal localisation was no longer apparent, although a follicular localisation was still present. Transforming growth factor-beta 1 (TGF beta 1) immunoreactivity was present in the cytoplasm of a minority of the follicular cells in control rat thyroids, while their nuclei were unstained. In the goitrous rat thyroid an increase intensity of staining for TGF beta 1 was seen in all follicular cells, many of which now also demonstrated immuno-positive nuclei, within one week of goitrogen administration. These results show that in the hyperplastic thyroid increases in FGF-2 and TGF beta 1, and decreases in TSP1 accompany angiogenesis. These factors may interact in an autocrine/paracrine relationship to stimulate the neovascularisation that occurs during goitre formation.

Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway. Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin αIIbβ3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin αIIbβ3 in resting platelets and in human embryonal kidney 293 cells expressing αIIbβ3. The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin αIIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to αIIbβ3 during platelet adhesion decreased integrin-associated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Acα in 293 cells decreased αIIbβ3-mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated αIIbβ3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac α-depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2Acα expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac α can negatively regulate integrin αIIbβ3 signaling by suppressing the ERK1/2 signaling pathway. Integrin cytoplasmic tails are devoid of any intrinsic catalytic activity. Nevertheless, integrins can transmit bidirectional signals across the plasma membrane of a cell and regulate several cellular processes, such as, adhesion, migration, and apoptosis. In the context of the major platelet integrin αIIbβ3, emerging evidence indicates that cytoplasmic tails act as a molecular scaffold for intracellular enzymes and for both cytoskeletal and adaptor proteins and can either positively or negatively regulate signaling (1Shattil S.J. Newman P.J. Blood. 2004; 104: 1606-1615Crossref PubMed Scopus (448) Google Scholar). For example, during an agonist-mediated inside-out signaling process, talin interacts with the integrin β3 tail and induces integrin αIIbβ3 activation (2Tadokoro S. Shattil S.J. Eto K. Tai V. Liddington R.C. de Pereda J.M. Ginsberg M.H. Calderwood D.A. Science. 2003; 302: 103-106Crossref PubMed Scopus (983) Google Scholar), whereas calcium and integrin-binding protein 1 binds to the αIIb tail and negatively regulates αIIbβ3 activation (3Yuan W. Leisner T.M. McFadden A.W. Wang Z. Larson M.K. Clark S. Boudignon-Proudhon C. Lam S.C. Parise L.V. J. Cell Biol. 2006; 172: 169-175Crossref PubMed Scopus (64) Google Scholar). Subsequent binding of fibrinogen to the activated αIIbβ3 integrin initiates an outside-in signaling process that regulates platelet function. Outside-in signaling can be mediated by intricate interplay of a set of proteins that associate constitutively with the integrin and by others that either associate or dissociate with the integrin in response to fibrinogen binding. For instance, c-Src associates constitutively to the β3 tail (4Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar). Fibrinogen binding to αIIbβ3 induces association of protein tyrosine phosphatase 1B, spleen tyrosine kinase, and protein kinase Cβ to the β3 tail (4Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar, 5Arias-Salgado E.G. Haj F. Dubois C. Moran B. Kasirer-Friede A. Furie B.C. Furie B. Neel B.G. Shattil S.J. J. Cell Biol. 2005; 170: 837-845Crossref PubMed Scopus (103) Google Scholar, 6Buensuceso C.S. Obergfell A. Soriani A. Eto K. Kiosses W.B. Arias-Salgado E.G. Kawakami T. Shattil S.J. J. Biol. Chem. 2005; 280: 644-653Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) and calcium and integrin-binding protein 1 to the αIIb tail (7Naik U.P. Naik M.U. Blood. 2003; 102: 1355-1362Crossref PubMed Scopus (64) Google Scholar) and causes the dissociation of C terminus Src kinase from the β3 tail (4Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar) and the catalytic subunit of protein phosphatase 1 (PP1c) 6The abbreviations used are: PP1c, catalytic subunit of protein phosphatase 1; PAR4AP, protease-activated receptor 4-activating peptide; VASP, vasodilator-associated phosphoprotein; ERK, extracellular signal-regulated kinase; PP2Ac, catalytic subunit of protein phosphatase 2A; siRNA, short interference RNA; BSA, bovine serum albumin; VWF, von Willebrand factor; WT, wild type. from the αIIb tail (8Vijayan K.V. Liu Y. Li T.T. Bray P.F. J. Biol. Chem. 2004; 279: 33039-33042Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Reversible phosphorylation of multiple effector proteins that are downstream of the integrin signaling pathway is one of the mechanisms by which these αIIbβ3-associated proteins can initiate and/or transduce signals. The phosphorylation status of most signaling proteins is determined by a fine balance between the activities of kinases and phosphatases. Thus far, among the reported αIIbβ3-associated signaling molecules, kinases have outnumbered phosphatases. Consequently, in contrast to integrin-associated kinases, a role for phosphatases in platelet signaling, with rare exception (5Arias-Salgado E.G. Haj F. Dubois C. Moran B. Kasirer-Friede A. Furie B.C. Furie B. Neel B.G. Shattil S.J. J. Cell Biol. 2005; 170: 837-845Crossref PubMed Scopus (103) Google Scholar), is not well understood. Protein phosphatase 2A (PP2A) is a ubiquitously expressed Ser/Thr phosphatase and is implicated in β1 integrin function in cell types other than platelets (9Ivaska J. Nissinen L. Immonen N. Eriksson J.E. Kahari V.M. Heino J. Mol. Cell. Biol. 2002; 22: 1352-1359Crossref PubMed Scopus (154) Google Scholar). The PP2A holoenzyme consists of a ∼36-kDa catalytic subunit C (PP2Ac) and a ∼65-kDa structural subunit A (PP2Aa) that together form an AC core dimer (PP2Aac). The A subunit in the core dimer links multiple regulatory B subunits in a fashion that determines the substrate specificity, the subcellular location, and the catalytic activity of the phosphatase (10Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1548) Google Scholar). Blockade by generic Ser/Thr phosphatase inhibitors like okadaic acid and calyculin A impair agonist-induced platelet aggregation, secretion (11Nishikawa M. Toyoda H. Saito M. Morita K. Tawara I. Deguchi K. Kuno T. Shima H. Nagao M. Shirakawa S. Cell Signal. 1994; 6: 59-71Crossref PubMed Scopus (26) Google Scholar, 12Hoyt C.H. Lerea K.M. Biochemistry. 1995; 34: 9565-9570Crossref PubMed Scopus (20) Google Scholar, 13Higashihara M. Takahata K. Kurokawa K. Ikebe M. FEBS Lett. 1992; 307: 206-210Crossref PubMed Scopus (27) Google Scholar), and αIIbβ3 outside-in signaling functions such as adhesion and spreading to immobilized fibrinogen. This act may be independent of β3 Thr753 phosphorylation status (14Lerea K.M. Cordero K.P. Sakariassen K.S. Kirk R.I. Fried V.A. J. Biol. Chem. 1999; 274: 1914-1919Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 15Lerea K.M. Venjara A.Y. Olson S.C. Kelly M.R. Biochim. Biophys. Acta. 2006; 1773: 185-191Crossref PubMed Scopus (13) Google Scholar); however, okadaic acid and calyculin A can inhibit multiple Ser/Thr phosphatases such as PP1, PP2A, and PP4 (16McCluskey A. Sim A.T. Sakoff J.A. J. Med. Chem. 2002; 45: 1151-1175Crossref PubMed Scopus (215) Google Scholar). Therefore, these agents are unable to specifically elucidate the role of PP2A in integrin αIIbβ3 signaling and function. In this study, we show that a pool of the catalytic subunit of PP2A constitutively associates with integrin αIIbβ3. By using a genetic (gene knockdown and/or gene overexpression) approach in two distinct model systems, such as the 293 and the primary murine megakaryocytes, PP2Acα was identified to negatively regulate αIIbβ3 adhesiveness to immobilized and soluble fibrinogen. PP2Acα can negatively regulate αIIbβ3 signaling by repressing the ERK1/2 activation pathway. Immunoprecipitation and Western Blotting—Blood was drawn in an acid/citrate/dextrose anticoagulant from normal, healthy, fasting donors. Each donor signed an informed consent approved by the Institutional Review Board of Baylor College of Medicine, Houston, TX. Washed platelets were prepared as previously described (17Vijayan K.V. Liu Y. Dong J.F. Bray P.F. J. Biol. Chem. 2003; 278: 3860-3867Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Using either 750 μg/ml 1% Triton X-100 or Igepal CA-630, lysates were obtained from either resting platelets or 293 cells expressing αIIbβ3 or the various mutants. The lysates were immunoprecipitated using anti-αIIb (Sew-8, gift from Dr. Newman, Blood Research Institute, Milwaukee, WI), anti-PP2Ac (Santa Cruz Biotechnology, Santa Cruz, CA), or rabbit IgG (Pierce) using Protein A-Sepharose beads (Amersham Biosciences). Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, probed with monoclonal antibodies to αIIb (132.1), PP2Ac (Upstate Biotechnology/Millipore, Billerica, MA), or PP2Cc (Alexis Biochemicals, San Diego, CA), and developed using the ECL system (Amersham Biosciences). Interaction of PP2Ac with the Integrin αIIb Subunit—Truncation of the cytoplasmic domain of integrin αIIb at residue 989 was generated according to the manufacturer's protocol for the QuikChange™ site-directed mutagenesis kit (Stratagene, La Jolla, CA). Integrin β3 with truncation of the cytoplasmic domain at residue 716 was kindly provided by Dr. Michael H. Kroll (Baylor College of Medicine, Houston, TX). These constructs were sequenced to confirm the presence of the desired truncations. 293 cells were transiently transfected with wild-type αIIb and β3 or with truncated integrin tails using Lipofectamine (Invitrogen) for 48 h. Cells were lysed in 1% Triton X-100, and lysates were immunoprecipitated with anti-PP2Ac antibody and immunoblotted with anti-αIIb antibody. In an alternate approach, biotinylated peptides corresponding to residues 985–995 of the integrin αIIb (LAMWKVGFFKR) and a control with scrambled sequence (LWKRVAGPFKM) were synthesized at the Baylor College of Medicine Protein Sequencing Core Facility, Houston, TX. We mixed 25–50 μg/ml peptide with 1 μg/ml purified PP2A or 750 μg/ml platelet lysate, and the mixtures were precipitated using streptavidin-agarose beads. The beads were washed three times, and bound proteins were eluted using SDS sample buffer. Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose, and probed with anti-PP2Ac antibody, and signals were then detected using ECL. siRNA Construct, Transfection, and Adhesion—The following SMARTpool siRNA reagents, purchased from Dharmacon (Thermo Fisher Scientific, Lafayette, CO), were used in this study: 1) catalog # M-003598 targeting human PP2A catalytic subunit, α isoforms (NM_002715); 2) catalog # M-04065700 targeting murine PP2A catalytic subunit, α isoforms (NM_019411); and 3) catalog # D-001206-13-05 a nonspecific control pool with no sequence homology to any human or mouse sequence. According to manufacturer's instructions, we used siImporter (Upstate Biotechnology) to transfect 293 αIIbβ3 cells with 100 nm siRNA. After 48 h, the cells were used for Western blotting or adhesion experiments. For the adhesion studies, 1 × 105 cells, suspended in Tyrode's buffer containing 1.8 mm CaCl2 and 0.49 mm MgCl2, were incubated with either 5% BSA (control), 12.5 μg/ml fibrinogen (Enzyme Research Laboratories Inc., South Bend, IN), or 10 μg/ml VWF (gift from Dr. Jing-fei Dong, Baylor College of Medicine)-coated wells for varying time points. During certain experiments, the cells were pretreated with either control DMSO, 10 μm U0126 (ERK1/2 inhibitor), or 10 μm SB203580 (p38 inhibitor). Unbound cells were washed, and the adherent cells were quantified by assaying for acid phosphatase activity at 405 nm. The number of bound cells was obtained using a standard curve for absorbance versus cell number. Percent adhesion was calculated as the number of bound cells divided by the total number of cells added per well multiplied by 100. Specific fibrinogen binding was calculated after subtracting values obtained for BSA-coated wells. In some experiments, 293 αIIbβ3 cells were transiently transfected using Lipofectamine with cDNA for HA-tagged PP2Acα or the control vector (gift from Dr. A. Verin, University of Chicago, Chicago, IL). After 48 h, cells were analyzed for Western blotting and adhesion as described above. In some experiments, fibrinogenor BSA-coated dishes were incubated with 2 × 108 platelets for 30 min at 37 °C. The fibrinogen-bound platelets and the non-adherent platelets from BSA-coated plates were lysed in a phosphate-free buffer, and αIIb was immunoprecipitated. The integrin αIIb-associated PP2Ac activity assay was quantified using a PP2A phosphatase assay kit (Upstate Biotechnology). Using a malachite green assay, αIIb immunoprecipitates were evaluated for PP2Ac activity by dephosphorylation of the phosphopeptide K-Rp-I-R-R. Megakaryocyte Culture, Transfection, and Flow Cytometry—Megakaryocytes were obtained from the bone marrow cultures of BALB/c mice as described previously (18Shiraga M. Ritchie A. Aidoudi S. Baron V. Wilcox D. White G. Ybarrondo B. Murphy G. Leavitt A. Shattil S. J. Cell Biol. 1999; 147: 1419-1430Crossref PubMed Scopus (78) Google Scholar). At day 5, megakaryocytes were transfected with 100 nm control siRNA or PP2Acα siRNA by using a transfecting agent from Mirus (Mirus Bio Corp., Madison, WI) for 48 h at 37 °C. On day 7, a portion of the differentiated megakaryocytes was used to assess the expression of PP2Acα. We mixed 50 μl of the megakaryocyte suspension with 2.5 mm agonist PAR4AP (AYPGKF, Protein Sequencing Core Facility, Baylor College of Medicine). Next, a non-blocking anti-αIIb antibody, 7-amino-actinomycin D (BD Bioscience, San Jose, CA) and Alexa 488-conjugated fibrinogen (20 μg/ml final concentration, Invitrogen) were added in the presence or absence of 10 mm EDTA at room temperature for 15–20 min. Alexa-fibrinogen binding was measured using an EPICS-XL flow cytometer (Beckman Coulter, Miami, FL). The FL1 expression was evaluated from the gated population of only large megakaryocytes (size) that expressed αIIbβ3 (FL2) and were viable (defined as negative for 7-amino-actinomycin D in the FL3 parameter). In resting platelets, a pool of the catalytic subunit of protein phosphatase 1 (PP1c) constitutively associates with αIIbβ3 complex (8Vijayan K.V. Liu Y. Li T.T. Bray P.F. J. Biol. Chem. 2004; 279: 33039-33042Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Studies from other cell types have revealed that PP2Ac can associate with integrin β1 (19Pankov R. Cukierman E. Clark K. Matsumoto K. Hahn C. Poulin B. Yamada K.M. J. Biol. Chem. 2003; 278: 18671-18681Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Because the primary amino acid sequence within the catalytic subunits of PP1 and PP2A are closely related, we considered whether the catalytic subunit of PP2A (PP2Ac) could associate with αIIbβ3 complex in platelets. During co-immunoprecipitation assays with lysates from resting human platelets, we detected the presence of PP2Ac in the αIIb immunoprecipitate. Conversely, in a reciprocal co-immunoprecipitation assay, αIIb was observed in the PP2Ac immunoprecipitate (Fig. 1A). Furthermore, the association of PP2Ac with integrin αIIbβ3 could be recapitulated in human embryonal kidney cell line 293 expressing αIIbβ3. Thus, suggesting the phosphatase-integrin association is intrinsic to integrin αIIbβ3 (Fig. 1B). In contrast to PP2Ac, the catalytic subunit of a structurally distinct Ser/Thr phosphatase, protein phosphatase 2C (PP2Cc) was not detected in the αIIb immunoprecipitate, indicating the PP2Ac-αIIbβ3 interaction is specific (Fig. 1C). Although the phosphatase-integrin association may appear modest, its consequence on cellular function is not (see Fig. 4). PP2Acα and PP2Acβ are the two ubiquitously expressed isoforms of PP2Ac and share roughly 97% similarity in the primary amino acid sequence. The PP2Ac antibody used in these studies recognizes both isoforms. Thus, these studies indicate a specific and constitutive interaction of the catalytic subunit of PP2A with the resting integrin αIIbβ3. To identify whether αIIb or the β3 cytoplasmic domains support PP2Ac interaction, 293 cells were transiently transfected with wild-type αIIbβ3 or with αIIb and β3 cytoplasmic domain truncation mutants. The association of PP2Ac was then assessed by co-immunoprecipitation assays. Integrin αIIb co-immunoprecipitated with PP2Ac in 293 cells transiently expressing the wild-type (WT) αIIbβ3 (Fig. 2A). Furthermore, cells expressing the WT αIIb along with the β3 cytoplasmic truncation mutant (β3Δ716) also supported the interaction of αIIb with PP2Ac. In contrast, αIIb failed to associate with PP2Ac in 293 cells that expressed 1) no integrin αIIbβ3, 2) αIIb cytoplasmic truncation mutant (αIIbΔ989) along with the WT β3, and 3) αIIb (αIIbΔ989) and β3 (β3Δ716) cytoplasmic truncation mutants. The apparent increased αIIb association with PP2Ac in β3Δ716-expressing cells, seen in Fig. 2, was not consistently reproducible and may be due to an increased amount of immunoprecipitated PP2Ac. The inability of αIIbΔ989 mutant to support PP2Ac association was not due to a lack of αIIb expression (Fig. 2A, 293 cell lysates). These studies suggest that the cytoplasmic domain of integrin αIIb, but not β3, supports the interaction of PP2Ac. To ascertain whether PP2A could directly associate with the integrin, we examined the interaction of purified PP2A enzyme and purified αIIb cytoplasmic peptide. Because PP1c (a PP2Ac-related phosphatase) interacts with the αIIb cytoplasmic tail, containing a PP1c binding motif, 989KVXF992, we considered whether the membrane proximal, as opposed to membrane distal, residues of the αIIb subunit could also support PP2Ac interaction. Purified PP2A enzyme and PP2Ac from the resting platelet lysates bound specifically to a biotinylated αIIb peptide containing the residues 985–995 of the integrin αIIb, but not to a control peptide with scrambled sequence (Fig. 2B). These studies suggest that the αIIb membrane proximal region containing the KVGFFKR sequence can support the direct interaction of PP2Ac. Next, we examined if αIIbβ3 activation and ligand engagement regulates PP2Ac-αIIbβ3 association or αIIbβ3-associated PP2A activity. The association of PP2Ac with the integrin was evaluated following platelet adhesion to immobilized fibrinogen, an αIIbβ3-mediated event. The association of PP2Ac with αIIbβ3 was maintained regardless of whether the platelets were held in suspension over the BSA substrate or adhered to immobilized fibrinogen (Fig. 3A). Densitometric quantification revealed that a comparable (p = 0.642) amount of PP2Ac, associated with the integrin immunoprecipitates, was obtained from platelets that either adhered to fibrinogen or suspended over BSA (Fig. 3B). Similarly, a stable association of PP2Ac with the integrin was also observed during soluble fibrinogen binding induced by Mn+2 (data not shown). Next, the activity of PP2Ac associated with αIIbβ3 was quantified in the αIIb immunoprecipitates. Platelets that adhered to fibrinogen exhibited ∼45% decreased (p = 0.02) αIIbβ3-associated PP2Ac activity compared with platelets that were maintained in suspension over the BSA substrate (Fig. 3C). Consistent with the decreased αIIbβ3-associated PP2Ac activity in fibrinogen-adhered platelets, we observed an increased Ser157 phosphorylation of vasodilator-associated phosphoprotein (VASP), a PP2Ac substrate in fibrinogen adhered platelets (Fig. 3D). By densitometry, when compared with platelets suspended over BSA, fibrinogenadhered platelets exhibited a ∼2-fold increase of VASP phosphorylation (Fig. 3E). Thus, decreased integrin-associated PP2Ac activity in fibrinogen-adhered platelets correlated with the increased phosphorylation of PP2Ac substrate VASP in platelets. Collectively, these results indicate that the integrinfibrinogen engagement may not significantly disrupt the association of a pool of phosphatase with the integrin, but rather decreases the phosphatase activity of PP2Ac associated with the αIIbβ3. To explore a functional role for PP2Ac in integrin αIIbβ3 signaling, we overexpressed a HA-tagged PP2Acα in 293 αIIbβ3 cells and evaluated adhesion to immobilized fibrinogen. PP2Acα was chosen because of the reported 10-fold abundance over PP2Acβ in most tissues (21Khew-Goodall Y. Hemmings B.A. FEBS Lett. 1988; 238: 265-268Crossref PubMed Scopus (79) Google Scholar). Immunoblotting with anti-HA antibody confirmed the overexpression of PP2Acα (Fig. 4A). Compared with the vector control-treated cells, PP2Acα overexpression significantly decreased the adhesion of αIIbβ3 cells to fibrinogen (Fig. 4B). The αIIbβ3-specific blocking antibody, 10E5, inhibited the adhesion of 293 cells. Thus, indicating that the adhesion was primarily mediated through αIIbβ3. Comparable levels of integrin αIIb expression were observed by densitometry of αIIb immunoblots in control vector and PP2Acα HA-overexpressed cells (54.59 ± 7.9 versus 55.10 ± 8.02, respectively). To further verify these findings, in complementary studies, we used short interference RNA (siRNA) to knock down the expression of endogenous PP2Acα in 293 cells expressing αIIbβ3. Knockdown was maximal (∼50–60%) and specific for PP2Ac, because PP1c and actin protein levels were comparable between the control and PP2Ac siRNA-treated cells (Fig. 4C). Compared with the control siRNA-treated cells, PP2Acα knockdown significantly increased the adhesion of αIIbβ3 cells to fibrinogen (Fig. 4D). To further determine whether the increased adhesiveness exhibited by PP2Acα-depleted cells was specific to immobilized fibrinogen, we studied adhesion to immobilized VWF. PP2Acα knockdown significantly increased the adhesion of αIIbβ3 cells to VWF (Fig. 4E). This suggests the differential adhesion due to PP2Acα depletion is not ligand-specific. The mean fluorescence intensity for αIIbβ3 expression was 266.12 ± 55 and 321.3 ± 68 for control and PP2Acα siRNA-treated 293 cells, respectively (p = 0.89). Thus, integrin expression levels may not account for the observed difference in adhesion. Taken together, these results indicate that PP2Acα negatively regulates αIIbβ3 outside-in signaling function in 293 cells. To investigate a potential mechanism by which PP2Acα can negatively regulate cell adhesion, we explored the effect of depleting endogenous PP2Acα in 293 cells on p38 and ERK (PP2Acα effectors) signaling pathways. These pathways are implicated in the modulation of cellular cytoskeletal reorganization and cell adhesiveness (22Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar). The siRNA-mediated depletion of PP2Acα resulted in an increased activation of p38 and ERK1/2 signaling (Fig. 5, A and C). Using densitometry, the comparison of control siRNA-treated cells and PP2Acα-depleted cells exhibited a ∼2-fold (p = 0.046) increase for p38 activation and a ∼2.5-fold (p = 0.0058) increase in ERK1/2 activation (Fig. 5, B and D). Next, we ascertained whether the increased adhesiveness of PP2Acα-depleted cells was due to increased ERK1/2 or p38 signaling. Compared with control siRNA-treated cells, PP2Acα depletion significantly (p = 0.0001) increased adhesion. This increase was abolished (p = 0.373) by the ERK1/2 inhibitor (U0126) (Fig. 5E). In contrast, the p38 inhibitor, SB203580, failed to repress the increased adhesiveness of PP2Acα-depleted cells (Fig. 5E). Similar results were obtained with another p38 inhibitor (SB202190) (not shown). These studies indicate that PP2Acα may suppress αIIbβ3 adhesiveness, in part, by down-regulating ERK1/2 activation pathway. Next, we evaluated if genetic manipulation of PP2Acα negatively regulated αIIbβ3 signaling in an additional model system that has more direct relevance to platelet biology. It is not feasible to manipulate gene expression in platelets, because they are anucleate and mice lacking PP2Acα die around embryonic day 6.5, thus, precluding the study of platelets from PP2Acα null mice (23Gotz J. Probst A. Ehler E. Hemmings B. Kues W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12370-12375Crossref PubMed Scopus (184) Google Scholar). In recent years megakaryocytes, from which platelets are derived, have emerged as an attractive system for studying integrin inside-out signaling. They express αIIbβ3, are activated by agonists, and are amenable to genetic manipulation (3Yuan W. Leisner T.M. McFadden A.W. Wang Z. Larson M.K. Clark S. Boudignon-Proudhon C. Lam S.C. Parise L.V. J. Cell Biol. 2006; 172: 169-175Crossref PubMed Scopus (64) Google Scholar, 18Shiraga M. Ritchie A. Aidoudi S. Baron V. Wilcox D. White G. Ybarrondo B. Murphy G. Leavitt A. Shattil S. J. Cell Biol. 1999; 147: 1419-1430Crossref PubMed Scopus (78) Google Scholar). Therefore, we chose to study the role of PP2Acα in murine megakaryocytes, which is a physiologically relevant model comparable to platelets. In concurrence with the previously published reports using this model system, we noticed increased fibrinogen binding in protease-activated receptor 4 activating peptide (PAR4AP)-stimulated murine megakaryocytes compared with the unstimulated megakaryocytes. Addition of EDTA, a divalent cation chelator, decreased fibrinogen binding in PAR4AP-treated megakaryocytes to the level of the unstimulated megakaryocytes. This indicates that the increased fibrinogen binding in response to PAR4AP is specific to integrin activation (Fig. 6A). To elucidate the role of PP2Acα in agonist-induced αIIbβ3 fibrinogen binding, we used murine siRNAs to knock down PP2Acα expression in megakaryocytes. Knockdown was maximal (∼40–50%) and specific for PP2Ac, because PP1c and actin protein levels were comparable between the control and PP2Acα siRNA-treated megakaryocytes (Fig. 6B). The residual PP2Ac signal could represent incomplete PP2Acα knockdown or expression of PP2Acβ. Knockdown of PP2Acα significantly increased binding of soluble fibrinogen in response to PAR4AP (Fig. 6C) compared with the megakaryocytes treated with control siRNA (Fig. 6C). An increased trend that did not reach statistical significance was also noted for MnCl2-induced fibrinogen binding in PP2Acα-depleted megakaryocytes. This suggests that PP2Acα may also negatively regulate αIIbβ3 outside-in signaling in megakaryocytes (data not shown). Surface expression of αIIbβ3 was not different between the control (mean fluorescence intensity, 94.67 ± 16) and PP2Acα (mean fluorescence intensity, 89.33 ± 13) siRNA-treated megakaryocytes and could not account for the observed difference in fibrinogen binding. These results suggest that PP2Acα negatively regulates integrin αIIbβ3 inside-out signaling in a murine megakaryocyte model system. Signal transduction by kinases and phosphatases control integrin αIIbβ3 adhesiveness and activation events. However, a specific role for PP2A in integrin αIIbβ3 signaling is unclear. In this work, we show that a pool of PP2Ac associates constitutively with the integrin αIIbβ3 in resting platelets. Studies in 293 model systems revealed that PP2Acα can negatively regulate integrin αIIbβ3 adhesiveness, in part, via the suppression of ERK1/2, but not the p38 signaling pathway. Furthermore, PP2Acα negatively regulated PAR4AP-induced integrin αIIbβ3 inside-out signaling in a murine megakaryocyte model system. Co-immunoprecipitation assays revealed a close proximal association of a pool of PP2Ac with integrin αIIbβ3. Additional studies, using integrin tail truncation mutants and integrin αIIb cytoplasmic peptides, have indicated that the αIIb membrane proximal region containing KVGFFKR is sufficient to mediate a direct interaction with PP2Ac (Fig. 2). This observation is consistent with a previous study that showed an association of the inhibitor for PP2A (I1PP2A) with the membrane proximal GFFKR motif of integrin α3Aβ1 (24Mutz D. Weise C. Mechai N. Hofmann W. Horstkorte R. Bruning G. Danker K. J. Neurosci. Res. 2006; 84: 1759-1770Crossref PubMed Scopus (6) Google Scholar). The membrane proximal region of αIIb can host the binding of calcium and integrin-binding protein 1, PP1c, and ICIn (8Vijayan K.V. Liu Y. Li T.T. Bray P.F. J. Biol. Chem. 2004; 279: 33039-33042Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 25Barry W.T. Boudignon-Proudhon C. Shock D.D. McFadden A. Weiss J.M. Sondek J. Parise L.V. J. Biol. Chem. 2002; 277: 28877-28883Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 26Larkin D. Murphy D. Reilly D.F. Cahill M. Sattler E. Harriott P. Cahill D.J. Moran N. J. Biol. Chem. 2004; 279: 27286-27293Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar); therefore, it is conceivable that only a subpopulation of αIIbβ3 may harbor PP2Ac. Because GFFKR sequence is conserved in other α subunits, it is likely that PP2Ac association may not be limited to αIIb subunits. Despite the similar binding motifs for PP1c and PP2Ac on integrin αIIb, integrin engagement resulted in a different effect for the two phosphatases. PP1c dissociated from the integrin complex (8Vijayan K.V. Liu Y. Li T.T. Bray P.F. J. Biol. Chem. 2004; 279: 33039-33042Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar), whereas a great extent of PP2Ac remained associated with the integrin (Fig. 3A). This suggests that integrin engagement may differentially regulate the two phosphatases in platelets. Interestingly, fibrinogen binding during platelet adhesion to αIIbβ3 repressed the αIIbβ3-associated PP2Ac activity (Fig. 3B). It is conceivable that the decreased PP2Ac activity, following fibrinogen binding, may be due to an increased Tyr307 phosphorylation of PP2Ac that is mediated in part by αIIbβ3-associated Src. In fact, fibrinogen binding to αIIbβ3 during platelet adhesion resulted in an increased αIIbβ3-associated Src activity (4Obergfell A. Eto K. Mocsai A. Buensuceso C. Moores S.L. Brugge J.S. Lowell C.A. Shattil S.J. J. Cell Biol. 2002; 157: 265-275Crossref PubMed Scopus (353) Google Scholar). Also, phosphorylation of Tyr307 residue, within the catalytic subunit of PP2A, by the tyrosine kinases PP60Src or PP56Lck resulted in a reduction of the PP2A activity (27Chen J. Martin B.L. Brautigan D.L. Science. 1992; 257: 1261-1264Crossref PubMed Scopus (539) Google Scholar). Decreased αIIbβ3-associated PP2A activity also correlates with the increased αIIbβ3-associated Ser/Thr kinase protein kinase C activity following fibrinogen binding (6Buensuceso C.S. Obergfell A. Soriani A. Eto K. Kiosses W.B. Arias-Salgado E.G. Kawakami T. Shattil S.J. J. Biol. Chem. 2005; 280: 644-653Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar), implying αIIbβ3-associated Ser/Thr kinase and phosphatase activity are tightly controlled. It is difficult to ascertain a specific role for integrin-associated PP2Ac in functional assays obtained from cells expressing either point mutations or deletions of the KVGFFKR region in αIIb, because multiple proteins dock in this region. Moreover, deletion of GFFKR sequence in αIIb can lead to integrin activation via disruption of a salt bridge between the αIIb and β3 subunits (28Hughes P.E. Diaz-Gonzalez F. Leong L. Wu C. McDonald J.A. Shattil S.J. Ginsberg M.H. J. Biol. Chem. 1996; 271: 6571-6574Abstract Full Text Full Text PDF PubMed Scopus (510) Google Scholar). Therefore, in this study, we analyzed αIIbβ3 adhesive function in cells that are depleted of PP2Acα by an siRNA approach. Although this approach can decipher a specific functional role for PP2Acα independent of other KVGFFKR-binding proteins, we cannot stringently rule out the functional contribution of PP2Acα that is also present in other subcellular locations. Nevertheless, our studies indicated that PP2Acα knockdown in 293 αIIbβ3 model cells resulted in an increased αIIbβ3 adhesion to immobilized fibrinogen and VWF. These results are in contrast to an earlier study, wherein platelets treated with PP1/PP2A inhibitor calyculin A produced a decreased adhesive phenotype to immobilized fibrinogen (14Lerea K.M. Cordero K.P. Sakariassen K.S. Kirk R.I. Fried V.A. J. Biol. Chem. 1999; 274: 1914-1919Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This discrepancy could be due to inhibition of multiple phosphatases other than PP2A in calyculin A-treated platelets or could merely reflect the differences between platelets and cell lines. The siRNA approach we have undertaken provides us an opportunity to evaluate, more specifically, a role for PP2Ac independent of PP1c, because PP1c expression was not decreased in PP2Ac knockdown cells (Fig. 4C). Because platelets are anucleate and PP2Acα null mice are embryonically lethal, we employed murine megakaryocytes as a comparable model to study platelet αIIbβ3 inside-out signaling process. We observed increased fibrinogen binding in PAR4AP-treated megakaryocytes that were treated with PP2Acα siRNA (Fig. 6). Taken together with the results obtained from the 293 cell model system, these observations strengthen the conclusion that PP2Acα negatively regulates integrin signaling. How could integrin-associated PP2Ac exert a negative regulation of integrin signaling? The association of PP2Ac with the αIIbβ3 complex does not directly regulate the integrin affinity, because the basal fibrinogen binding in PP2Acα-depleted megakaryocytes was not statistically increased (Fig. 6C). Furthermore, the association of PP2Ac with the αIIbβ3 complex does not regulate Thr753 phosphorylation of integrin β3, because we failed to observe β3 Thr753 phosphorylation in PP2Acα-depleted 293 cells (data not shown). Perhaps, multiple substrates for PP2Ac may exist in the focal adhesion protein complexes organized by αIIb andβ3 cytoplasmic tails. Suppression of the phosphorylation or activation of these proteins within the complex by the αIIbβ3-associated PP2Acα is likely to participate in limiting integrin activation and function. Indeed, we noticed that PP2Acα repressed the activation of its effectors p38 and ERK1/2 (Fig. 5). Although activation of ERK1/2 is more intensely studied as a regulator of gene expression and cell proliferation, this pathway can also regulate cellular functions. For example, cell adhesion and spreading are inhibited by dominant-negative ERK (22Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar) and promoted by ERK activation (29Zhu X. Assoian R.K. Mol. Biol. Cell. 1995; 6: 273-282Crossref PubMed Scopus (394) Google Scholar). ERK and p38 are required for platelet spreading on fibrinogen (30Mazharian A. Roger S. Berrou E. Adam F. Kauskot A. Nurden P. Jandrot-Perrus M. Bryckaert M. J. Biol. Chem. 2007; 282: 5478-5487Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cell migration is inhibited by blocking p38 activation (31Hedges J.C. Dechert M.A. Yamboliev I.A. Martin J.L. Hickey E. Weber L.A. Gerthoffer W.T. J. Biol. Chem. 1999; 274: 24211-24219Abstract Full Text Full Text PDF PubMed Scopus (350) Google Scholar). We observed that PP2Acα-depleted cells exhibited increased ERK1/2 and p38 activation and increased adhesion to fibrinogen and VWF. Inhibition of ERK1/2, but not p38 signaling, abolished the increased adhesion of PP2Acα-depleted cells (Fig. 5E). Other investigators have reported that pharmacological inhibition of PP2Ac leads to increased migration of endothelial and carcinoma cells (32Young M.R. Kolesiak K. Meisinger J. Int. J. Cancer. 2002; 100: 276-282Crossref PubMed Scopus (36) Google Scholar, 33Young M.R. Liu S.W. Meisinger J. Int. J. Cancer. 2003; 103: 38-44Crossref PubMed Scopus (25) Google Scholar). Thus, the enhanced ERK1/2 and p38 signaling in PP2Acα-depleted cells would be predicted to exhibit greater adhesive and migratory properties. Moreover, at the molecular level, evidence exists that ERK1/2 signaling can regulate focal adhesion complexes and integrin activation. Formation of peripheral actin microspikes are blocked by inhibiting ERK activation (34Brunton V.G. Fincham V.J. McLean G.W. Winder S.J. Paraskeva C. Marshall J.F. Frame M.C. Neoplasia. 2001; 3: 215-226Crossref PubMed Scopus (25) Google Scholar). After integrin engagement, active ERK is targeted to the newly forming focal adhesion via receptor for activated protein kinase C 1 (35Vomastek T. Iwanicki M.P. Schaeffer H.J. Tarcsafalvi A. Parsons J.T. Weber M.J. Mol. Cell. Biol. 2007; 27: 8296-8305Crossref PubMed Scopus (66) Google Scholar). Interestingly, receptor for activated protein kinase C 1 also interacts with PP2A (36Kiely P.A. O'Gorman D. Luong K. Ron D. O'Connor R. Mol. Cell. Biol. 2006; 26: 4041-4051Crossref PubMed Scopus (85) Google Scholar); therefore, it may provide the scaffold for cross-talk between PP2A and ERK signaling. Finally, the p38 and ERK signaling pathways are reported to play a critical role in the activation of integrin αIIbβ3 when induced by agonists like VWF and thrombin (20Li Z. Zhang G. Feil R. Han J. Du X. Blood. 2006; 107: 965-972Crossref PubMed Scopus (133) Google Scholar). Thus, suppression of ERK1/2 signaling by PP2A could constitute a potential mechanism for limiting integrin function. In summary, our understanding of the role for PP2A in integrin signaling is fairly limited based on the use of pharmacological inhibitors. Using a genetic approach, our data indicate that PP2Acα negatively regulates αIIbβ3 signaling. Given that platelets are derived from megakaryocytes, this finding could be directly relevant to platelets. Moreover, such functional interactions might also extend to other α integrin subunits, thereby providing additional regulatory mechanisms to control integrin activation. We thank Drs. Leslie Parise and Jun Liu (University of North Carolina, Chapel Hill, NC) for providing the necessary training to isolate, culture, transfect, and analyze murine megakaryocytes and Dr. Paul Bray (Jefferson Medical College, Philadelphia, PA) for scientific advice during the initial phase of this study.

Functional outcomes after traumatic brain injury (TBI) vary widely across patients with apparently similar injuries. This variability hinders prognosis, therapy, and clinical innovation. Recently, single nucleotide polymorphism (SNPs) that influence outcome after TBI have been identified. These discoveries create opportunities to personalize therapy and stratify clinical trials. Both of these changes would propel clinical innovation in the field. This review focuses on one of most well-characterized of these SNPs, the Val66Met SNP in the brain-derived neurotrophic factor (BDNF) gene. This SNP influences neurological function in healthy subjects as well as TBI patients and patients with similar acute insults to the central nervous system. A host of other patient-specific factors including ethnicity, age, gender, injury severity, and post-injury time point modulate this influence. These interactions confound efforts to define a simple relationship between this SNP and TBI outcomes. The opportunities and challenges associated with personalizing TBI therapy around this SNP and other similar SNPs are discussed in light of these results.

Summary Objective Using the electronic medical record ( EMR ) to capture structured clinical data at the point of care would be a practical way to support quality improvement and practice‐based research in epilepsy. Methods We describe our stepwise process for building structured clinical documentation support tools in the EMR that define best practices in epilepsy, and we describe how we incorporated these toolkits into our clinical workflow. Results These tools write notes and capture hundreds of fields of data including several score tests: Generalized Anxiety Disorder‐7 items, Neurological Disorders Depression Inventory for Epilepsy, Epworth Sleepiness Scale, Quality of Life in Epilepsy–10 items, Montreal Cognitive Assessment/Short Test of Mental Status, and Medical Research Council Prognostic Index. The tools summarize brain imaging, blood laboratory, and electroencephalography results, and document neuromodulation treatments. The tools provide Best Practices Advisories and other clinical decision support when appropriate. The tools prompt enrollment in a DNA biobanking study. We have thus far enrolled 231 patients for initial visits and are starting our first annual follow‐up visits and provide a brief description of our cohort. Significance We are sharing these EMR tools and captured data with other epilepsy clinics as part of a Neurology Practice Based Research Network, and are using the tools to conduct pragmatic trials using subgroup‐based adaptive designs.

ABBREVIATIONS BBB = blood-brain barrier; DHA = docosahexaenoic acid; EPA = eicosapentaenoic acid; GCS = Glasgow Coma Scale; ICP = intracranial pressure; O3FA = omega-3 polyunsaturated fatty acid; TBI = traumatic brain injury.

Macrophages from nonobese diabetic (NOD) mice, which spontaneously develop type I diabetes, share a defect in elicited cytokine production with macrophages from multiple diverse strains of systemic lupus erythematosus (SLE)-prone mice. We have previously shown that, in SLE-prone mice, this defect is triggered by exposure to apoptotic cells. We report in this work that macrophages from prediseased NOD mice also respond abnormally to apoptotic cells, mimicking closely the apoptotic cell-dependent abnormality that we have observed in multiple SLE-prone strains. This defect is characterized by the underexpression of IL-1 beta and multiple other cytokines. In the presence of apoptotic cells or FBS, elicited expression of IL-1 beta by NOD macrophages is markedly reduced compared with that by macrophages from control mice, including three strains of mice that develop type II (nonautoimmune) diabetes. Given the increasing role of apoptotic cells in tolerance and autoimmunity, a macrophage defect triggered by apoptotic cells has broad potential to upset the balance between tolerance and immunity. The concordance of this defect among so many diverse autoimmune-prone strains suggests that the genetic basis for this abnormality may constitute a permissive background for autoimmunity.

Random conjugation of therapeutic or diagnostic payloads to targeting proteins generates functionally heterogeneous products. Conjugation of payloads to an adapter that binds to a peptide tag engineered into a targeting protein provides an alternative strategy. To progress into clinical development, an adapter/docking tag system should include humanized components and be stable in circulation. We describe here an adapter/docking tag system based on mutated fragments of human RNase I that spontaneously bind to each other and form a conjugate with a disulfide bond between complimentary cysteine residues. This self-assembled “dock and lock” system utilizes the previously described fusion C-tag, a 1−15 aa fragment of human RNase I with the R4C amino acid substitution, and a newly engineered adapter protein (Ad-C), a 21−127-aa fragment of human RNase I with the V118C substitution. Two vastly different C-tagged recombinant proteins, human vascular endothelial growth factor (VEGF) and a 254-aa long N-terminal fragment of anthrax lethal factor (LFn), retain functional activities after spontaneous conjugation of Ad-C to N-terminal or C-terminal C-tag, respectively. Ad-C modified with pegylated phospolipid and inserted into the lipid membrane of drug-loaded liposomes (Doxil) retained the ability to conjugate C-tagged proteins, yielding targeted liposomes decorated with functionally active proteins. To further optimize the system, we engineered an adapter with an additional cysteine residue at position 88 for site-specific modification, conjugated it to C-tagged VEGF, and labeled with a near-infrared fluorescent dye Cy5.5, yielding a unique functionally active probe for in vivo molecular imaging. We expect that this self-assembled “dock and lock” system will provide new opportunities for using functionally active proteins for biomedical purposes.

During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses. During apoptosis, cells acquire new activities that enable them to modulate the fate and function of interacting phagocytes, particularly macrophages (mϕ). Although the best known of these activities is anti-inflammatory, apoptotic targets also influence mϕ survival and proliferation by modulating proximal signaling events, such as MAPK modules and Akt. We asked whether modulation of these same signaling events extends to epithelial cells, a minimally phagocytic cell type. We used BU.MPT cells, a mouse kidney epithelial cell line, as our primary model, but we also evaluated several epithelial cell lines of distinct tissue origins. Like mϕ, mouse kidney epithelial cells recognized apoptotic and necrotic targets through distinct non-competing receptors, albeit with lower binding capacity and markedly reduced phagocytosis. Also, modulation of inflammatory activity and MAPK-dependent signaling by apoptotic and necrotic targets was indistinguishable in kidney epithelial cells and mϕ. In contrast, modulation of Akt-dependent signaling differed dramatically between kidney epithelial cells and mϕ. In kidney epithelial cells, modulation of Akt was linked to target cell recognition, independently of phagocytosis, whereas in mϕ, modulation was linked to phagocytosis. Moreover, recognition of apoptotic and necrotic targets by kidney epithelial cells elicited opposite responses; apoptotic targets inhibited whereas necrotic targets stimulated Akt activity. These data confirm that nonprofessional phagocytes recognize and respond to dying cells, albeit in a manner partially distinct from mϕ. By acting as sentinels of environmental change, apoptotic and necrotic targets may permit neighboring viable cells, especially non-migratory epithelial cells, to monitor and adapt to local stresses.

Virtually all cells in the body have the capacity to recognize and respond to dead cells. Viable cells discriminate apo from nec targets via distinct cell surface receptors. Engagement of these receptors induces "recognition-dependent" signaling events in viable responding cells that differ for apo vs. nec targets. Although "engulfment-dependent" signaling events also contribute to the response by viable cells, these events do not differ for apo vs. nec targets. While many signaling events are conserved across diverse cell lineages, other signaling events, especially those involving Akt, demonstrate lineage-specific variation. Whereas apo targets activate Akt in MPhi, they inhibit Akt in kidney epithelial cells. Differences in the responses to dead targets by viable migratory cells, such as MPhi, and viable fixed cells, such as kidney epithelial cells, permit cell-specific adaptations to local environmental change or stress. We propose that dead cells (apo and nec) act as sentinels to alert nearby viable cells to local environmental change or stress.

Scientific knowledge of omega-3 fatty acids (FAs) has grown in the last decade to a greater understanding of their mechanisms of action and their potential therapeutic effects. Omega-3 FAs have shown therapeutic potential with respect to hyperlipidemia, depression, attention-deficit hyperactivity disorder, and mild cognitive impairment. Laboratory evidence and clinical interest have grown such that omega-3 FAs have now assumed a role in concussion management. This has coincided with recent research that has also helped to increase the scientific understanding of cerebral concussion; although concussion or mild traumatic brain injury was assumed to be a malfunctioning brain without anatomical damage, we now know that there is the potential for damage and dysfunction at the cellular and microstructural levels. Specifically, with concussion abnormal metabolism of glucose may occur and intracellular mitochondrial dysfunction can persist for several days. In this article, we discuss the role of omega-3 FAs, particularly docosahexaenoic acid, in concussion management.

Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses. Apoptosis plays an indispensable role in the maintenance and development of tissues. We have shown that receptor-mediated recognition of apoptotic target cells by viable kidney proximal tubular epithelial cells (PTECs) inhibits the proliferation and survival of PTECs. Here, we examined the effect of apoptotic targets on PTEC cell growth (cell size during G1 phase of the cell cycle). Using a cell culture model, we show that apoptotic cells potently activate AMP-activated protein kinase (AMPK), a highly sensitive sensor of intracellular energy stores. AMPK activation leads to decreased activity of its downstream target, ribosomal protein p70 S6 kinase (p70S6K), and concomitant inhibition of cell growth. Importantly, these events occur without detectable change in intracellular levels of AMP, ADP, or ATP. Inhibition of AMPK, either pharmacologically by compound C or molecularly by shRNA, diminishes the effects of apoptotic targets and largely restores p70S6K activity and cell size to normal levels. Apoptotic targets also inhibit Akt, a second signaling pathway regulating cell growth. Expression of a constitutively active Akt construct partially relieved cell growth inhibition but was less effective than inhibition of AMPK. Inhibition of cell growth by apoptotic targets is dependent on physical interaction between apoptotic targets and PTECs but independent of phagocytosis. We conclude that receptor-mediated recognition of apoptotic targets mimics the effects of intracellular energy depletion, activating AMPK and inhibiting cell growth. By acting as sentinels of environmental change, apoptotic death may enable nearby viable cells, especially nonmigratory epithelial cells, to monitor and adapt to local stresses.

Four new compounds (2-5) structurally related to the microtubule-stabilizing agent (-)-zampanolide (1) have been isolated from the Tongan marine sponge Cacospongia mycofijiensis. Three of these new structures, zampanolides B-D (2-4), exhibit nanomolar cytotoxicity toward the HL-60 cell line, are antimitotic, and induce in vitro tubulin polymerization at levels comparable to 1. Zampanolide E (5), saturated at C-8/C-9, was significantly less potent and does not stabilize purified tubulin, even at 10-fold higher concentrations. The structural differences across these compounds reveal a plasticity of the zampanolide pharmacophore. While unsaturation is required at Δ8, the configuration of this alkene and those of Δ4 and Δ4' have little effect on tubulin polymerization. The first natural co-occurrence of 1 and (-)-dactylolide (6) from the same sponge extract is also noted.

From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells, and expression was increased by agents that elevate cAMP. Nitric oxide synthase (NOS) 3, the endothelial form of NOS, was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen fibroblast growth factor (FGF)-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was, however, able to stimulate nitric oxide release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120 kDa) and immunolocalized to the cytosol and nucleus. Thyrotropin (TSH) did not regulate FGFR1, but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor, but neither TSH nor 3,5,3'-triiodothyronine had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells, but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.

Growing evidence exists for a new role for apoptotic cell recognition and clearance in immune homeostasis. Apoptotic cells at all stages, irrespective of membrane integrity, elicit a signature set of signaling events in responding phagocytes, both professional and non-professional. These signaling events are initiated by receptor-mediated recognition of apoptotic determinants, independently of species, cell type, or apoptotic stimulus. We propose that the ability of phagocytes to respond to apoptotic targets with a characteristic set of signaling events comprises a second distinct dimension of innate immunity, as opposed to the traditional innate discrimination of self vs. non-self. We further propose that a loss or abnormality of the signaling events elicited by apoptotic cells, as distinct from the actual clearance of those cells, may predispose to autoimmunity.

This study looks at the changes in injuries after the implementation of a new rule by the International Boxing Association (AIBA) to remove head guards from its competitions.A cross-sectional observational study performed prospectively. This brief report examines the removal of head guards in 2 different ways. The first was to examine the stoppages due to blows to the head by comparing World Series Boxing (WSB), without head guards, to other AIBA competitions with head guards. Secondly, we examined the last 3 world championships: 2009 and 2011 (with head guards) and 2013 (without head guards).World Series Boxing and AIBA world championship boxing.Boxers from WSB and AIBA world championships.The information was recorded by ringside medical physicians.Stoppages per 10 000 rounds; stoppages per 1000 hours.Both studies show that the number of stoppages due to head blows was significantly decreased without head guards. The studies also showed that there was a notable increase in cuts.Removing head guards may reduce the already small risk of acute brain injury in amateur boxing.

The leaf homogenate of Psychotria insularum is widely used in Samoan traditional medicine to treat inflammation associated with fever, body aches, swellings, wounds, elephantiasis, incontinence, skin infections, vomiting, respiratory infections, and abdominal distress. However, the bioactive components and underlying mechanisms of action are unknown. We used chemical genomic analyses in the model organism Saccharomyces cerevisiae (baker's yeast) to identify and characterize an iron homeostasis mechanism of action in the traditional medicine as an unfractionated entity to emulate its traditional use. Bioactivity-guided fractionation of the homogenate identified two flavonol glycosides, rutin and nicotiflorin, each binding iron in an ion-dependent molecular networking metabolomics analysis. Translating results to mammalian immune cells and traditional application, the iron chelator activity of the P. insularum homogenate or rutin decreased proinflammatory and enhanced anti-inflammatory cytokine responses in immune cells. Together, the synergistic power of combining traditional knowledge with chemical genomics, metabolomics, and bioassay-guided fractionation provided molecular insight into a relatively understudied Samoan traditional medicine and developed methodology to advance ethnobotany.

ABSTRACT In the course of Bacillus anthracis infection, B. anthracis lethal factor (LF) and edema factor bind to a protective antigen (PA) associated with cellular receptors ANTXR1 (TEM8) or ANTXR2 (CMG2), followed by internalization of the complex via receptor-mediated endocytosis. A new group of potential antianthrax drugs, β-cyclodextrins, has recently been described. A member of this group, per-6-(3-aminopropylthio)-β-cyclodextrin (AmPrβCD), was shown to inhibit the toxicity of LF in vitro and in vivo. In order to determine which steps in lethal factor trafficking are inhibited by AmPrβCD, we developed two targeted fluorescent tracers based on LFn, a catalytically inactive fragment of LF: (i) LFn site specifically labeled with the fluorescent dye AlexaFluor-594 (LFn-Al), and (ii) LFn-decorated liposomes loaded with the fluorescent dye 8-hydroxypyrene-1,3,6-trisulfonic acid (LFn-Lip). Both tracers retained high affinity to PA/ANTXR complexes and were readily internalized via receptor-mediated endocytosis. Using fluorescent microscopy, we found that AmPrβCD inhibits receptor-mediated cell uptake but not the binding of LFn-Al to PA/ANTXR complexes, suggesting that AmPrβCD works outside the cell. Moreover, AmPrβCD and LFn-Al synergistically protect RAW 264.7 cells from PA-mediated LF toxicity, confirming that AmPrβCD did not affect the binding of LFn-Al to receptor-associated PA. In contrast, AmPrβCD did not inhibit PA-mediated internalization of LFn-Lip, suggesting that multiplexing of LFn on the liposomal surface overcomes the inhibiting effects of AmPrβCD. Notably, internalized LFn-Al and LFn-Lip protected cells that overexpressed anthrax receptor TEM8 from PA-induced, LF-independent toxicity, suggesting an independent mechanism for PA inhibition inside the cell. These data suggest the potential for the use of β-cyclodextrins in combination with LFn-Lip loaded with antianthrax drugs against intracellular targets.

Concussion has a long and interesting history spanning at least the 5 millennia of written medical record and closely mirrors the development of surgery and neurosurgery. Not surprisingly, much of the past and present experimental head injury and concussion work has been performed within neurosurgically driven laboratories or by several surgically oriented neurologists. This historical review chronicles the key aspects of neurosurgical involvement in sports concussion as related to the diagnosis, treatment, mitigation, and prevention of injury using the example of American football. In addition, we briefly trace the developments that led to our current understanding of the biomechanical and neurophysiological basis of concussion. ABBREVIATIONS: AFCA, American Football Coaches Association CTE, chronic traumatic encephalopathy EEG, electroencephalography LOC, loss of consciousness RTP, return to play

Internal jugular vein (IJV) compression has been shown to reduce axonal injury in pre-clinical traumatic brain injury (TBI) models and clinical concussion studies. However, this novel approach to prophylactically mitigating TBI through venous congestion raises concerns of increasing the propensity for hemorrhage and hemorrhagic propagation. This study aims to test the safety of IJV compression in a large animal controlled cortical impact (CCI) injury model and the resultant effects on hemorrhage. Twelve swine were randomized to placement of a bilateral IJV compression collar (CCI+collar) or control/no collar (CCI) prior to CCI injury. A histological grading of the extent of hemorrhage, both subarachnoid (SAH) and intraparenchymal (IPH), was conducted in a blinded manner by two neuropathologists. Other various measures of TBI histology were also analyzed including: β-amyloid precursor protein (β-APP) expression, presence of degenerating neurons, extent of cerebral edema, and inflammatory infiltrates. Euthanized 5 h after injury, the CCI+collar animals exhibited a significant reduction in total SAH (p = 0.024-0.026) and IPH scores (p = 0.03-0.05) compared with the CCI animals. There was no statistically significant difference in scoring for the other markers of TBI (β-APP, neuronal degeneration, cerebral edema, or inflammatory infiltration). In conclusion, IJV compression was shown to reduce hemorrhage (SAH and IPH) in the porcine CCI model when applied prior to injury. These results suggest the role of IJV compression for mitigation of not only axonal, but also hemorrhagic injury following TBI.

High-affinity interactions of two fragments of human RNase I (1−15-aa Hu-tag and 21−125-aa HuS adapter protein) can be used for assembly of targeting drug delivery complexes. In this approach, a targeting protein is expressed as a fusion protein with a 15-aa Hu-tag, while HuS is conjugated to a drug (or a drug carrier) creating a “payload” module, which is then bound noncovalently to the Hu-tag of the targeting protein. Although this approach eliminates chemical modifications of targeting proteins, the payload modules are still constructed by random cross-linking of drugs or drug carriers to an adapter protein that might lead to functional heterogeneity of the complexes. To avoid this problem, we engineered an adapter protein HuS(N88C) with an unpaired cysteine in position 88 that can be directly modified without interference with activity of assembled targeting complexes. HuS(N88C) binds Hu-tagged annexin V with KD of 50 ± 6 nM, which is comparable to that of wild-type HuS. To demonstrate the utility of HuS(N88C) for developing uniform payload modules, we constructed a HuS(N88C)−lipid conjugate and inserted it into preformed liposomes loaded with a fluorescent dye. Targeting proteins, Hu-tagged vascular endothelial growth factor or Hu-tagged annexin V, were docked to liposomes decorated with HuS, and the assembled complexes delivered liposomes selectively to target cells.

Macrophages (mphi) from prediseased mice of all the major murine models of spontaneous autoimmunity have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. We show here that mphi from prediseased mice of the same models of spontaneous autoimmunity share a serum-dependent defect in the activity of Rho, a cytoplasmic G protein and cytoskeletal regulator. Affected strains include those developing lupus (BXSB, LG, MRL/l+, MRL/lpr, NZBWF1) and autoimmune diabetes (nonobese diabetic). No similar defect in Rho activity occurred in seven control strains. In the presence of serum, Rho activity in mphi from all autoimmune-prone strains was reduced to less than 10% of that in control mice. In contrast, under serum-free conditions, Rho activity was completely normal in autoimmune-prone mphi. The activities of Ras, another cytoplasmic G protein, and Rac and Cdc42, two additional G protein regulators of the cytoskeleton, were regulated normally in autoimmune-prone strains. Serum-dependent dysregulation of Rho was associated with multiple abnormalities, including increased adhesion to various surfaces, a more spread dendritic morphology, and an altered actin cytoskeletal organization. Our results suggest that mphi from multiple, genetically diverse, autoimmune-prone strains share a mutation or allelic difference affecting signal transduction within a specific Rho-regulatory pathway.

Integrin αIIbβ3 signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing αIIbβ3 to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 αIIbβ3 cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 αIIbβ3 cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 αIIbβ3 cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 αIIbβ3 cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate αIIbβ3 adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune αIIbβ3 outside-in signaling. Integrin αIIbβ3 signaling mediated by kinases and phosphatases participate in hemostasis and thrombosis, in part, by supporting stable platelet adhesion. Our previous studies indicate that the genetic manipulation of PP2Acα (α isoform of the catalytic subunit of protein phosphatase 2A) negatively regulate the adhesion of human embryonal kidney 293 cells expressing αIIbβ3 to fibrinogen. Here, we demonstrated that small interference RNA (siRNA) mediated knockdown of PP2Acα in 293 αIIbβ3 cells led to the dephosphorylation of Src Tyr-529, phosphorylation of Src Tyr-418 and an increased Src kinase activity. Conversely, overexpression of PP2Acα decreased the basal Src activity. Pharmacological inhibition of PP2Ac in human platelets or PP2Acα knockdown in primary murine megakaryocytes resulted in Src activation. PP2Acα-depleted 293 αIIbβ3 cells did not alter the serine (Ser) phosphorylation of Src but enhanced the Ser-50 phosphorylation of protein tyrosine phosphatase 1B (PTP-1B) with a concomitant increase in the PTP-1B activity. Src activation in the PP2Acα-depleted 293 αIIbβ3 cells was abolished by siRNA mediated knockdown of PTP-1B. Pharmacological inhibition of Src or knockdown of Src, PTP-1B blocked the enhanced activation of extracellular signal-regulated kinase (ERK1/2) and the increased adhesiveness of PP2Acα-depleted 293 αIIbβ3 cells to fibrinogen, respectively. Thus, inactivation of PP2Acα promotes hyperphosphorylation of PTP-1B Ser-50, elevates PTP-1B activity, which dephosphorylates Src Tyr-529 to activate Src and its downstream ERK1/2 signaling pathways that regulate αIIbβ3 adhesion. Moreover, these studies extend the notion that a cross-talk between Ser/Thr and Tyr phosphatases can fine-tune αIIbβ3 outside-in signaling.

Case series identifying chronic traumatic encephalopathy (CTE) in former athletes and military veterans have been, and continue to be, reported. Recently, there have also been substantial descriptions of the neuropathological similarities and differences between CTE and other neurodegenerative diseases such as Alzheimer disease (AD). Finally, there is growing evidence that neurodegenerative diseases, including Parkinson disease (PD), afflict those with a history of head trauma and blast exposure at higher rates than the general population.1-4 Despite the strong supporting evidence, there is still debate about the relationship between neurotrauma and neurodegeneration, particularly CTE.5-7 In this work, we provide a brief summary of the origin of CTE as it relates to dementia pugilistica (DP) while discussing common clinical presentations of CTE and neuropathological features required for diagnosis. We highlight the role of injury severity, namely concussive or subconcussive injury, in the risk of development of CTE. Additionally, we discuss other risk factors for CTE and elaborate on the relationship between CTE and other neurodegenerative diseases such as AD. Finally, we suggest possible mechanisms of mitigating CTE going forward on the basis of current knowledge and potential changes to playing styles and rules. CTE: AN EXTENSION OF DP? Chronic effects after an extensive history of traumatic brain injury (TBI) were first recognized in a cohort of boxers by Martland8 in the 1920s and referred to as “punch drunk” syndrome. This condition, DP, was later expanded on in numerous other studies that detailed more cases and characterized gross and microscopic pathological findings.9-11 Although DP is a form of traumatic encephalopathy, it is believed to represent a more severe form of traumatic encephalopathy and, by definition, is restricted to boxers. DP is characterized, according to Corsellis et al,10 as a tetrad of gross and microscopic findings: (1) abnormalities (cavum) of the septum pellucidum, (2) scarring of the cerebellum and other brain regions, (3) degeneration of the substantia nigra, and (4) the regional presence of neurofibrillary tangles (NFTs).12,13 Notably, modern CTE, defined initially by Omalu et al,14 is not represented by gross neuroanatomical changes, which is distinct from DP.15-33 CTE SYMPTOMOLOGY AND PRESENTATION CTE can be defined as a progressive neurodegenerative syndrome caused by single, episodic, or usually repetitive blunt force impacts to the head and transfer of acceleration-deceleration and rotational forces to the brain. CTE presents clinically after a prolonged latent period as a composite syndrome of mood disorders, neuropsychiatric symptoms, and cognitive impairment (Figure 1). Typically, a latency period of 6 to 12 years follows the end of a playing career in a contact sport. Thereafter, symptoms consisting of difficulties with personal, business, and financial affairs ensue, with an evolution to depression, often alcohol and substance abuse, and progressive disorganization in activities of life. CTE sufferers may go on to develop mild cognitive impairment and dementia, and many lives end by suicide. Frequently reported CTE symptoms generally fall into 4 broad classifications: behavior, cognition, mood, and motor.22 Behavioral changes reported include those related to heightened aggression, increased impulsive and often risky behaviors, and impaired judgment.34,35 These behavioral changes collectively may occasionally lead to or be associated with a tendency toward violent and explosive outbursts.24 Cognitive changes include short-term memory loss and learning impairments.23 In fact, our prior studies indicate that former professional football players may have a greater risk of severe memory impairment and an increased incidence of mild cognitive impairment and depression compared with the general population.36,37 Common mood changes associated with CTE include depression, apathy, and irritability, potentially leading to an increased risk of suicide. This heightened suicidal risk is in stark contrast to other tauopathies such as AD.15,16,18,38,39 Finally, rare cases of CTE have been associated with motor deficits, possibly a tribute to degeneration within the substantia nigra and loss of dopaminergic neurons.32,40,41 Reported deficits center on decreased reaction time, disordered eye movements, and frequent falls.15,32,33,40,41FIGURE 1: Chronic traumatic encephalopathy timeline. The disease usually presents clinically after a prolonged latent period as a composite syndrome of mood disorders and neuropsychiatric and cognitive impairment.Most patients with CTE have exhibited only some of these symptoms, and consequently, 2 broad classifications of CTE presentation have been described.25 The first, occurring at a younger age of onset, involves primarily mood and behavioral changes.25 These behavioral changes may be attributable to the pathology in the midbrain and pontine dopaminergic, noradrenergic, and serotonergic neurotransmitter alterations (see neuropathology description below). The second, occurring at an older age of onset, manifests itself primarily with cognitive deficits.25 These 2 presentation paradigms demonstrate the continuum of CTE, a disease that has variable latency between neurotrauma exposure and disease onset. Case series have outlined the rarity, range, and continuum of neurobehavioral and cognitive symptoms.6,13-22,26,29-31 GROSS, MICROSCOPIC, AND IMAGING CHARACTERIZATION OF CTE The emerging picture for CTE diagnosis in retired amateur or professional contact sport athletes has been defined in a recent National Institutes of Health report and by research groups.15,26,42 Postmortem brain tissue has revealed the presence of topographically distributed NFTs and neuritic threads with or without the presence of diffuse amyloid plaques (Figure 2 and Table 1). In the pure form, there is usually absence of classic or neuritic amyloid plaques and pathognomonic changes of other types of tauopathies such as progressive supranuclear palsy, corticobasal degeneration, postencephalitic parkinsonism, Guam Parkinson dementia, Lewy bodies, or Lewy neurites. CTE has been a neuropathological diagnosis and is defined most generally as a tauopathy.FIGURE 2: Tau immunohistochemical staining showing (A) a normal control and (B) a chronic traumatic encephalopathy specimen, with characteristic tau protein deposits within the neurofibrillary tangles and neuritic threads.TABLE 1: Neuropathology of Chronic Traumatic EncephalopathyaBecause numerous other neurodegenerative diseases are characterized as tauopathies and, in the case of AD and PD, are associated with neurotrauma, it becomes critically important to delineate between tauopathies. Both clinical presentation and neuropathological abnormalities can be used in this regard. Compared with CTE, AD exhibits more generalized cerebral atrophy and the consistent presence of diffuse and neuritic amyloid plaques. However, diffuse and neuritic plaques are found in an estimated 40% to 50% and 25% to 30% of CTE cases, respectively.33 Focusing on tau, a defining feature of both CTE and AD, reveals differences in not only distribution but also density. NFTs in CTE are generally perivascular and more superficial within the cortex, being most prominent in layers II and III. In AD, in addition to laminar neuritic tau changes in the neutrophil, NFTs are found predominantly in not only layers III but also deeper layers of the cortex, layers V and VI.43 Other differences in NFT distribution can be seen within the hippocampus and parahippocampal gyrus. In CTE, NFTs are seen throughout the Ammon horn (CA1-CA4), whereas NFTs are most heavily focused within CA1 for AD. However, although NFTs can be seen in the locus ceruleus and raphe nucleus in AD, in a number of young individuals without amyloid plaques, these areas can be profoundly affected (Figure 3). Furthermore, in CTE, NFTs are seen in the substantia nigra, an unusual finding in strictly AD cases.43 It is also characteristic to find NFTs throughout the entire amygdala, pontine tegmentum (outside the locus ceruleus and raphe nucleus), and thalamus (particularly within the pulvinar) in CTE.44FIGURE 3: Chronic traumatic encephalopathy in a 41-year-old former professional football player. A, low-power view of the pontine tegmentum with paired helical filament-1 (PHF-1) tau staining of the locus ceruleus (LC) and the raphe nuclei (RN). B, high-power view of the LC PHF-1–positive neurofibrillary tangles. C, high-power view of the RN PHF-1–positive neurofibrillary tangles.Attempts have been made to develop standardized scoring/staging systems for CTE, akin to Braak staging for AD, with leading groups in the CTE field proposing unique classification schemes.15,26,45 The cellular pathophysiology for CTE remains unclear but under active investigation by our group and others.46,47Figure 4 shows possible mechanisms whereby TBI leads to formation of tau oligomers in the neuronal soma. Additional mechanisms that occur after TBI and are potential targets for preventing CTE include endoplasmic reticulum dysfunction, elevation in tau kinases compared with phosphatases, glutamate excitotoxicity, and microglial activation.46 Furthermore, the recent development of imaging technologies recognizing tau deposits and tau distribution in former athletes suspected of having CTE may prove invaluable in tracking disease development and progression in an in vivo study rather than the traditional postmortem analysis. Studies at the University of California Los Angeles using F18DDNP positron emission tomography have shown a characteristic binding density and distribution not seen in other neurodegenerative syndromes (Figure 5).48 Although this radionucleotide labels both tau and amyloid-β (Aβ), the latter may be seen in 10% to 15% of CTE cases, and CTE appears to be in distinct patterns.48,49FIGURE 4: Factors contributing to the pathology of chronic traumatic encephalopathy. Traumatic brain injury (TBI) can lead to diffuse traumatic axonal injury and the shearing of axons. This results in the disruption of tau binding to tubulin, leading to hyperphosphorylation of tau and the formation of tau oligomers in the neuronal soma. Eventually, neurofibrillary tangles form and secreted into the extracellular milieu or spread to other neurons via transsynaptic propagation. Additionally, TBI can cause a rapid blood pressure spike, resulting in disruption of the blood-brain barrier (BBB). This leads to an inflammatory cascade and thus microglia and astrocyte activation.FIGURE 5: Coronal and transaxial 2-(1-{6-[(2-[fluorine-18]fluoroethyl)(methyl)amino]-2-naphthyl}-ethylidene)malononitrile (FDDNP) positron emission tomography scan results for National Football League (NFL) players and a control. The players' scans show consistently high signals in the amygdala and subcortical regions and a range of cortical binding from extensive to limited, whereas the control subject shows limited binding in these regions. Red and yellow areas indicate high-FDDNP binding signals.SUMMARY OF REPORTED CTE CASES CTE, originally described by Omalu and colleagues20 in 2005, has been characterized extensively in recent years with cases in professional football players (both the National Football League and Canadian Football League),17,19,26,50 professional wrestlers (World Wrestling Entertainment),16,26 military veterans,14,27 and ice hockey players.26 In fact, recent reports have described and summarized CTE-related neuropathological findings over the course of the past decade alone, including a few cases in athletes who played exclusively in high school or college football.14-17,19,20,22,27,50 Importantly, these cases may increase because we are just beginning to more clearly understand the clinical problem. TBI remains a prominent problem across the globe, with upward of 3.8 million independent events occurring each year in the United States alone.51 Maroon et al52 recently published an excellent systemic review of all CTE cases. ROLE OF CONCUSSION AND SUBCONCUSSIVE INJURY Although the risk of developing CTE has historically been discussed in the context of concussive injury and extensive neurotrauma exposure, emerging evidence indicates that a history of diagnosed or major concussions is not a requirement; instead, repetitive subconcussive injury may play a prominent role in CTE development.53 This finding is based on a lack of documented concussive injury in numerous individuals diagnosed with CTE, although a lack of self-reporting by athletes was common, and concussion without loss of consciousness was not recognized and treated as seriously as it is today. Perhaps the most alarming evidence related to subconcussive injury and possible predilection to neurodegenerative disease is based on (1) documented rates and severity of impacts in football linemen, a position in which retired athletes are commonly diagnosed with CTE49; (2) demonstration of neuroimaging and cognitive changes in those without a history of documented concussions54,55; and (3) laboratory evidence indicating cellular and ultrastructural alterations without changes in levels of alertness or behavior.56 EVOLVING RISK FACTORS FOR CTE: ROLE OF GENOTYPE AND ENVIRONMENT Despite the large number of people exposed to concussive and subconcussive injury, CTE appears problematic for only a small subset of the population exposed to neurotrauma. Other variables may be involved as risk factors for CTE. Identification of these variables remains in its early stage, with speculation that genetics and lifestyle may be implicated.25 Just as in other forms of neurodegeneration such as AD, it has been postulated that the role of the apolipoprotein E (ApoE) ε4 (ApoE4) allele may be a susceptibility factor for the development of CTE; however, this has yet to be borne out. Thus far, no definitive genetic marker has emerged to define CTE risk.52 ApoE is a ligand for low-density lipoprotein receptors and mediates the receptor binding of ApoE-containing lipoproteins to the low-density lipoprotein receptor for cellular uptake and intracellular cholesterol metabolism. In the central nervous system, ApoE is synthesized and secreted primarily by astrocytes and microglia, and its importance is underscored by the absence of most other plasma apolipoproteins in the brain. It is the primary cholesterol transporter in the brain, where it is proposed to function as a ligand directing the delivery of lipids for neuronal repair and remodeling after injury.15,25 In our original cohort, all CTE-positive cases exhibited either an E3/E3 or E3/E4 genotype, with the majority (71%) having the E3/E3 genotype.15 Overrepresentation of the ApoE3 allele has been reported in patients with tangle-only dementia,25,26 whereas the ApoE4 allele has been associated with an increase in the risk of AD, with ApoE4/E4 homozygotes demonstrating a 19-fold increased risk of the development of AD. Those with a CTE diagnosis carry a similar rate of ApoE4 homozygosity as the AD population. In the AD population, it is the greatest risk factor associated with sporadic development of the disease.25,57 The question of whether ApoE4 may be a risk factor for CTE development is consistent with preclinical work in which ApoE4 polymorphism correlated with increased amyloid-β (Aβ) deposition, microgliosis, tau phosphorylation, and neuronal loss after neurotrauma.58,59 The role that lifestyle and environment play in the risk of CTE development remains less investigated and is poorly understood at this time. Our group has shown that administration of anabolic steroids, either before or after neurotrauma, had no effect on amyloid precursor protein expression in the corticospinal tracts after impact-acceleration injury in a rodent model. However, we did not assess tau hyperphosphorylation, the development of NFTs, or damage in other brain regions.60 OVERLAP WITH OTHER NEURODEGENERATIVE DISEASES Over the past few years as autopsy-based CTE diagnoses have become more common in those with histories of neurotrauma, a number of studies have documented the effects of prior TBI, including subconcussion, in the risk of developing other neurodegenerative diseases.1-4,43,53 Perhaps the most well documented of these include AD and PD. Previously, it was felt that AD and CTE were distinct entities, failing to exist within the same patient. Emerging evidence demonstrates that this may not be the case and that patients may exhibit clinical and neuropathological features consistent with both AD and CTE (Figure 6).25 These case presentations raise further questions for future investigation. Does the presence of CTE accelerate the development of AD? Can CTE and AD coexist within a patient? Does CTE exhibit a progressive or nonprogressive course with aging? It is entirely possible that CTE may exhibit age-dependent characteristics that act synergistically with AD development. These questions are clearly difficult to answer in a clinical population, but significant insight has been provided through preclinical investigation. The development of a preclinical model of CTE has led to the observation that it is possible to recreate both biochemical and behavioral deficits consistent with CTE. Likewise, application of neurotrauma in preclinical AD models has shown that repetitive neurotrauma accelerates the rate of AD, CTE, and PD development.35,61,62FIGURE 6: Patients with chronic traumatic encephalopathy (CTE) may exhibit clinical and neuropathological features consistent with other neurodegenerative diseases. AD, Alzheimer disease; PD, Parkinson disease.ADVANCING CTE EPIDEMIOLOGY THROUGH LONGITUDINAL STUDIES Although numerous case reports and case series involving CTE patients have been reported, the precise incidence and scope of the disease remain unclear and cannot be answered without prospective, long-term studies.6,52 Undertaking this research will be costly and time consuming, much like the Framingham Heart Study, but has the potential not only to contribute to elucidation of CTE epidemiology but also to provide insight into disease progression through serial clinical and neuroimaging assessments, which would occur before eventual neuropathological analysis at the time of death. Likewise, such a study would ideally be initiated in demographically matched groups of exposed and unexposed patients, allowing chronological monitoring and tracking of symptoms. In these regards, disease progression can be mapped while eliminating recall bias from retrospective reporting of cognitive impairment, dementia, and other neurodegenerative diseases in retired players.3,36,63-65 The first longitudinal prospective study has been initiated for mixed martial arts fighters and uses functional magnetic resonance imaging to track changes.38 PREVENTION AND MITIGATION OF CTE Currently, there are no established treatments for CTE. Therefore, reducing the risk for CTE development becomes the goal, and limiting the exposure to concussive and subconcussive head impacts is an obvious approach. Widespread acceptance of CTE by professional medical and research personnel, as well as the lay public, has led to extensive discussion of this topic. Rule changes in sports with a high incidence of concussion, along with proper enforcement, may reduce the frequency and severity of concussion. Pop Warner Football, a youth football league, has endorsed the prevention of cranial collisions and in 2012 added regulations to limit the amount of contact allowed in practice.50,66 The National Football League, National Collegiate Athletic Association, and National Federation of High Schools also have taken steps to limit cranial collisions through tougher enforcement of targeting penalties and changes in the length of time permitted for organized team activities and contact practicies.67 Although each of these steps may reduce the number of cranial impacts sustained, particularly among linemen, who sustain impacts on every play, these changes may not go far enough to address the number and severity of impacts. Therefore, the possibility remains that other rule changes may need to be implemented, one of which involves elimination of the 3-point stance along the offensive and defensive lines.53 Having linemen start in a more upright, squatting position not only would reduce cranial involvement in collisions but also would decelerate linemen at the point of contact, thereby reducing both the frequency and magnitude of cranial impacts.53 Another option for reducing the frequency of cranial impacts in football is slowing the tempo and number of plays in the game. Acute concussion management via education, culture change, and proper medical surveillance should also minimize detrimental exposures to repetitive TBI. Beyond rule changes, further improvements in technology may substantially reduce the incidence of concussion. Efforts aimed at preventing TBI have continued to focus on extracranial protection in the form of improved helmet technology. Although helmets are effective in preventing the infrequent penetration or fracture of the skull, they have little ability to limit the high-velocity acceleration-deceleration mechanisms that lead to concussive injury.68 Whether headgear can prevent or reduce the cumulative effects of repetitive head trauma is unknown. Efforts to reduce intracranial slosh phenomenon also have potential. Published preclinical studies found that internal jugular vein compression produced an 80% reduction in the number of torn brain fibers in a standard laboratory rodent concussion model.69,70 It is believed that mitigating slosh by increasing blood volume within venous structures within the skull will significantly reduce the propensity for differential motion between the skull and its contents. Likewise, pharmacological approaches to preventing CTE through modulation of membrane integrity or reduction in cellular stress and subsequent tauopathy may be on the horizon.71,72 Potential molecular targets for such intervention are shown in Figure 4. For the beneficial effects of any proposed rule changes, preventive technologies, and therapeutics to be fully assessed in the context of CTE development, prospective studies must be conducted. These studies must incorporate advanced imaging to track neurometabolic, microscopic neurostructural, and neurofunctional changes. CONCLUSION Approximately 150 cases of CTE are reported in the literature, if one accounts for publication of duplicative cases.52 Because thus far only case reports and retrospective selected series have been published, long-term and longitudinal studies are needed for clarification. The ability to perform in vivo imaging will allow us to follow living subjects to track the progression of tau protein deposition and to learn whether it is dynamic, reversible, or treatable, which is an exciting scientific frontier. There are many remaining uncertainties about CTE (Table 2), and although the true incidence and prevalence are unknown, the senior author (J.E.B.) believes that only a small minority of former contact sport athletes will develop the full clinical and neuropathological syndrome. The potential for confounders, environmental factors, role of comorbidities, neurodegenerative syndrome overlap, natural history, and numerous other factors and their impact are still presently unknown. Tau proteinopathy and the effects of the 6 different isoforms of tau remain to be elucidated. And as always, the genetic influence will no doubt end up being a major contributing factor as to which individuals are at risk if they sustain multiple concussions, concussion with loss of consciousness, and subconcussive impacts, as well as total lifetime exposure to TBI. The importance of age at onset of exposure and whether the human brain is more vulnerable at a specific age also are unclear.TABLE 2: Uncertainties of Chronic Traumatic EncephalopathyThe cases of CTE reported thus far have been primarily in those athletes from prior decades who participated in contact sports during the last 4 decades. As a result of the reforms that occurred in 2010 in football, ice hockey, and other sports to address the issues related to head contact, the playing styles and management of these CTE individuals were under what would now be considered outdated protocols. The limitation of head-to-head contact in practice; the outlawing of egregious head-to-head hits in games; the improvement in education, coaching techniques, and reporting of concussions; and the advances in helmets, biosensors, and other protective equipment make us optimistic that the incidence of CTE may be significantly reduced in the future (Table 3).TABLE 3: Elimination of Chronic Traumatic EncephalopathyaWorldwide, TBI, particularly “mild” TBI, remains an important topic in both lay and scientific communities after the emergence of CTE as a possible consequence of repetitive concussive or subconcussive neurotrauma. Although the epidemiology of CTE remains unclear and is unlikely to be resolved in the near future because of the inherent challenges, it is clear that CTE is a burgeoning problem affecting athletes and military veterans alike. Similarly, emerging evidence indicates that the consequences of TBI, particularly with repetitive injury, are not limited to increased risk for CTE, AD, and PD independently but also may predispose to the development of these diseases concurrently. How these conditions develop and potentially coalesce after neurotrauma remains unknown but is a point of interest going forward. Future studies addressing the relationship between neurotrauma factors (severity, number, interinjury interval, age at time of injury, etc) and development of CTE, AD, PD, and CTE-AD are needed to provide further insight into disease pathophysiology. By increasing our understanding of disease pathophysiology, we can better implement rule changes and preventive strategies that can mitigate or prevent the development and progression of CTE. Emerging in vivo diagnosis may provide clarification of many aspects of CTE.49 Disclosure The authors have no personal, financial, or institutional interest in any of the drugs, materials, or devices described in this article.

Cyclooxygenase-2 (COX-2) is constitutively expressed in restricted subpopulations of kidney cells, where it presumably acts as an antiapoptotic factor. In conditions that are characterized by inflammation, COX-2 expression also has been described in glomerular mesangial cells (GMC), where COX-2 is not expressed constitutively. It was shown previously that adenovirus-mediated gene transfer of COX-2 into rat GMC led to increased expression and activity of multidrug resistance protein 1 (MDR-1), a membrane transporter that functions as an efflux pump for chemotherapeutic drugs, including Adriamycin (ADR). In ADR nephrotoxicity, a pathologic change in glomeruli could be partially explained by ADR-mediated changes in GMC. Here it is demonstrated that ADR (also known as doxorubicin; 1 microg/ml) induced apoptosis in 15.3 +/- 2.2% of GMC, whereas after adenovirus-mediated COX-2 expression, only 6.6 +/- 0.4% of ADR-treated cells underwent apoptosis. This protective effect was nullified by treatment with NS398, specific COX-2 inhibitor. ADR efflux is greater in COX-2-overexpressing cells, when compared with control, which is attributed to the increased MDR-1 expression. Addition of PSC833, the specific MDR-1 inhibitor, completely abolished the protective effect of COX-2 overexpression and increased the level of apoptosis in GMC that were exposed to ADR. These data suggest that COX-2 protects GMC from ADR-mediated apoptosis via transcriptional upregulation of MDR-1 and that induced COX-2 expression would lessen ADR nephrotoxicity.

The authors trace the Oxford, England, roots of World War II (WWII)-related advances in head injury management, the biomechanics of concussion and brain injury, and postwar delineation of pathological findings in severe concussion and diffuse brain injury in man. The prominent figure in these developments was the charismatic and innovative Harvey Cushing-trained neurosurgeon Sir Hugh Cairns. Cairns, who was to closely emulate Cushing's surgical and scholarly approach, is credited with saving thousands of lives during WWII by introducing and implementing innovative programs such as helmets for motorcyclists, mobile neurosurgical units near battle zones, and the military usage of penicillin. In addition, he inspired and taught a generation of neurosurgeons, neurologists, and neurological nurses in the care of brain and spinal cord injuries at Oxford's Military Hospital for Head Injuries. During this time Cairns also trained the first full-time female neurosurgeon. Pivotal in supporting animal research demonstrating the critical role of acceleration in the causation of concussion, Cairns recruited the physicist Hylas Holbourn, whose research implicated rotary acceleration and shear strains as particularly damaging. Cairns' work in military medicine and head injury remain highly influential in efforts to mitigate and manage brain injury.

A cute death resulting from head trauma is a rare but catastrophic and unfortunate occurrence in contact sports, particularly American football.Although the incidence of acute death is lower now than it was decades before, it nonetheless continues to occur and is associated with several important characteristics.][23][24][25][26][27] Despite the warnings and notoriety of "second-impact syndrome" (SIS), this entity is a poorly understood, but often ascribed, phenomenon and one that has often led to plaintiffs' attorneys and their experts accusing medical personnel, athletic trainers, and coaches of ignoring or missing the warning and prior impacts, which subsequently are blamed for the second, or fatal, blow.Our experience, however, indicates that rather than attributing head injury to the poorly documented and ill-defined SIS, it is quite common and understandable that the fatal head blow is often in reality the first, horrific one.Thus, the majority of these occurrences are most appropriately termed "firstimpact syndrome.

ObjectiveWe describe our experience with routinely capturing and analyzing Mediterranean diet data via structured clinical documentation support tools built into the electronic medical record and describe adherence to the Mediterranean diet in patients at risk for either stroke or dementia in a US neurology clinical practice.Patients and MethodsThe Mediterranean diet is associated with a reduced risk of stroke and dementia. The Department of Neurology at NorthShore University HealthSystem routinely evaluates patients at initial and annual outpatient visits using structured clinical documentation support (SCDS) tools built into the electronic medical record (EMR). For patient evaluations in our Vascular Neurology and Brain Health subspecialty clinics, SCDS tools in the EMR include the validated 14-item questionnaire for Mediterranean diet adherence (PREvención con DIeta MEDiterránea [PREDIMED]) that autoscores, auto-interprets, writes to the progress note, and electronically captures data. Our study population includes patients seen at these clinics from July 1, 2015, through November 29, 2017.ResultsAt their initial office visit, 25.5% (95/373) of Brain Health patients scored 10 or more points (“strongly adherent”) on the PREDIMED (median, 8; range, 0-14) whereas 6.7% (55/829) of Vascular Neurology patients achieved a score of 10 or more points (median, 6; range, 0-12). By contrast, 34.7% (2586/7447) of individuals in the original PREDIMED cohort were strongly adherent to the Mediterranean diet.ConclusionPREDIMED scores can be electronically captured to tailor nutrition interventions by assessing baseline adherence at the time of their initial neurology clinic visit. Patients in our Midwestern US clinics were weakly adherent to the Mediterranean diet. This suggests a major opportunity for nutrition intervention and education in US neurology clinical practices, toward preserving and improving brain health.

Abstract Macrophages (mφ) from prediseased mice of the major murine models of lupus have an identical defect in cytokine expression that is triggered by serum and/or apoptotic cells. It is striking that cytokine expression in the absence of serum and apoptotic cells is equivalent to that of nonautoimmune mice. Here, we show that mφ from prediseased lupus-prone MRL/MpJ (MRL/+) or MRL/MpJ-Tnfrsf6lpr (MRL/lpr) mice also have reversible abnormalities in morphology, cytoskeletal organization, and adhesive properties. In the presence of serum, MRL mφ adhered in increased numbers to a variety of extracellular matrix proteins compared with mφ from two nonautoimmune strains. However, in the absence of serum, adhesion by MRL mφ was similar to that of nonautoimmune mφ. Increased adhesion by MRL mφ was also observed in the presence of apoptotic, but not necrotic, cells. The morphology and actin-staining pattern of adherent MRL mφ were consistent with reduced activity of Rho, a cytoskeletal regulator. Indeed, MRL mφ cultured in the presence of serum had markedly decreased levels of active Rho compared with nonautoimmune mφ. It is remarkable that when cultured in the absence of serum, MRL mφ displayed normal Rho activity and cytoskeletal morphology. Addition of a Rho inhibitor to normal mφ reproduced the morphologic and cytoskeletal abnormalities observed in MRL mφ. Taken together, our findings support the hypothesis that mφ from MRL and other systemic lupus erythematosus-prone mice have an apoptotic, cell-dependent, autoimmune phenotype that affects a broad range of mφ functions, including cytokine gene expression and Rho-dependent cytoskeletal regulation.

Endoscopic transsphenoidal surgery has become the most commonly performed surgical procedure for pituitary tumor removal. As such, there are many patient-oriented educational materials on the technique available online for members of the public who desire to learn more about the surgery. It has been recommended that educational resources be written to the national average reading level, which in the United States is between sixth and seventh grade. This study assesses the reading level of the educational materials currently available online for endoscopic transsphenoidal surgery and determines whether these resources are written at a suitable comprehension level for most readers.Sixteen patient educational resources describing endoscopic transsphenoidal surgery were identified online and assessed using 4 standard readability assessments.Patient educational resources written for endoscopic transsphenoidal surgery are written far above the recommended reading level of sixth grade.The online educational resources written for patients about endoscopic transsphenoidal surgery are above the recommended reading level for patient education materials. Further revisions to simplify these resources on endoscopic transsphenoidal surgery are needed to ensure that most patients can comprehend this important material and make informed decisions about their health care.

Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular β-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses. Apoptosis allows for the removal of damaged, aged, and/or excess cells without harm to surrounding tissue. To accomplish this, cells undergoing apoptosis acquire new activities that enable them to modulate the fate and function of nearby cells. We have shown that receptor-mediated recognition of apoptotic versus necrotic target cells by viable kidney proximal tubular epithelial cells (PTEC) modulates the activity of several signaling pathways critically involved in regulation of proliferation and survival. Generally, apoptotic and necrotic targets have opposite effects with apoptotic targets inhibiting and necrotic targets stimulating the activity of these pathways. Here we explore the consequences of these signaling differences. We show that recognition of apoptotic targets induces a profound decrease in PTEC viability through increased responder cell death and decreased proliferation. In contrast, necrotic targets promote viability through decreased death and increased proliferation. Both target types mediate their effects through a network of Akt-dependent and -independent events. Apoptotic targets modulate Akt-dependent viability in part through a reduction in cellular β-catenin and decreased inactivation of Bad. In contrast, Akt-independent modulation of viability occurs through activation of caspase-8, suggesting that death receptor-dependent pathways are involved. Apoptotic targets also activate p38, which partially protects responders from target-induced death. The response of epithelial cells varies depending on their tissue origin. Some cell lines, like PTEC, demonstrate decreased viability, whereas others (e.g. breast-derived) show increased viability. By acting as sentinels of environmental change, apoptotic targets allow neighboring cells, especially non-migratory epithelial cells, to monitor and potentially adapt to local stresses.

The purpose of this study was to determine whether AMPK influences the survival of primary cultures of mouse proximal tubular (MPT) cells subjected to metabolic stress. Previous studies, using an immortalized MPT cell line, suggest that AMPK is activated during metabolic stress, and ameliorates stress-induced apoptosis of these cells. Primary MPT cells were cultured from AMPK knockout (KO) mice lacking either the α1 or the α2 isoform of the catalytic domain of AMPK. MPT cells were subjected to ATP depletion using antimycin A. Surprisingly, there was no difference in the amount of death induced by metabolic stress of MPT cells from either type of AMPK KO mice compared to its WT control. Moreover, inhibition of the activity of the α1 isoform in primary MPT cells from α2-/- mice (pharmacologically, via compound C) or inhibition of the α2 isoform in primary MPT cells from α1-/- mice (molecularly, via knockdown) both decreased cell viability equivalently in response to metabolic stress. The explanation for this unexpected result appears to be an adaptive increase in expression of the non-deleted α-isoform. As a consequence, total α-domain expression (i.e. α1 + α2), is comparable in kidney cortex and in cultured MPT cells derived from either type of KO mouse versus its WT control. Importantly, each α-isoform appears able to compensate fully for the absence of the other, with respect to both the phosphorylation of downstream targets of AMPK and the amelioration of stress-induced cell death. These findings not only confirm the importance of AMPK as a pro-survival kinase in MPT cells during metabolic stress, but also show, for the first time, that each of the two α-isoforms can substitute for the other in MPT cells from AMPK KO mice with regard to amelioration of stress-induced loss of cell viability.

Internal jugular vein (IJV) compression before blast injury will lead to reduced risk of traumatic hearing injury following exposure to a blast injury.IJV compression and its effects on not only intracranial, but also intracochlear pressure may potentiate blast induced hearing injury, therefore, precluding its use as a prophylactic therapy for blast induced traumatic brain injury.Twenty Sprague Dawley rats were exposed to a 17.9 ± 0.4 PSI (195.8 dB SPL) right sided shock wave in which 10 had application of a custom IJV compression collar before injury. All rodents received baseline and post blast injury otoacoustic emission (OAE) and auditory brainstem response (ABR) testing followed by cochlear histology.IJV compression was shown to significantly reduce ABR and OAE threshold shifts in comparison to the non-intervention group by: 14.9 ± 4.8 dB (right ear ABR 0.5 kHz Day 1 post blast, p = 0.01), 13.1 ± 4.9 dB (right ear ABR 4 kHz Day 1 post blast, p = 0.04), 16.5 ± 4.5 dB (right ear ABR click Day 1 post blast, p = 0.003), 12.1 ± 4.6 dB (right ear ABR click Day 6 post blast, p = 0.04), and 14.0 ± 3.2 dB (both ears OAE 3.2-10 kHz, p < 0.0001). Also, those animals with collar application had a greater number of total hair cells per mm from 70 to 100% distance from the cochlear apex following blast injury in comparison to those without intervention (blast: 211.8 ± 27.5 versus blast+collar: 355.5 ± 39.5 [p = 0.0002]).This study supports the use of IJV compression in a pre-clinical model as a new prophylactic mechanism to combat blast induced hearing injury.

There is limited research from Australasian EDs describing the demographic make-up, injury severity and impact of alcohol in patients requiring computed tomography (CT) for suspected traumatic brain injury (TBI). The present study aims to review the frequency and presenting patterns of patients who consume alcohol prior to presenting with suspected TBI.Retrospective observational study of patients referred for head CT to exclude TBI from a major referral centre and regional ED in New Zealand, between 1 September 2018 and 31 August 2019. Comparison groups were defined as 'alcohol involved' or 'no alcohol involved'.97/425 (22.8% [95% CI 18.3-27.4]) of included TBI presentations involved alcohol. 73/97 (75.3% [95% CI 58.6-93.5]) were male and 41/97 (42.3% [95% CI 29.3-55.2]) were aged 18-30 years. The alcohol group were more likely to report assault as the injury mechanism (19.6% [95% CI 10.8-28.4] vs 5.2% [95% CI 2.7-7.7], P < 0.05) and have Glasgow Coma Scale scores reflecting more moderate (13.5% [95% CI 5.9-21.1] vs 3.5% [95% CI 1.5-5.6]) and severe (5.6% [95% CI 0.7-10.5] vs 3.2% [95% CI 1.2-5.2] TBI. Presentation times post-injury were delayed compared to the no alcohol group (3.4 h [interquartile range 1.9-14.8] vs 2.8 h [interquartile range 1.8-6.6], P < 0.05).One quarter of patients with suspected TBI had consumed alcohol prior to their injury. Predominantly, those affected were young males who reported higher rates of assault; however, alcohol use was recorded in all age groups and sex. Alcohol-affected patients presented later, potentially delaying time to diagnosis. The present study supports the call for public health interventions that aim to reduce alcohol misuse.

We set out to determine whether adding medium chain triglyceride (MCT) oil as a dietary supplement to standard diet in adult subjects with intractable epilepsy in a U.S. neurology clinical practice was associated with a reduction in number of seizures. We secondarily aimed to determine whether subjects experienced any side effects and whether there was a presence of urinary ketones while using MCT oil as a dietary supplement.Adult patients with intractable epilepsy were recruited at standard of care clinical visits with their epileptologist. Once enrolled, subjects were instructed to supplement their diet with MCT oil as tolerated twice daily for three months (including a 1-2 week titration period, followed by a 1-2 week tapering off window) while keeping a seizure diary to record total number of seizures, presence of urinary ketones, and any side effects.Our data although limited by small sample size, shows that there is an estimated 42% reduction (p < 0.0001) in the rate of seizures. The MCT oil supplementation was well tolerated by most subjects except for minor GI side effects like nausea and loose stools. Most subjects developed ketones in their urine at some point during the trial.MCT oil supplementation reduced seizure frequency in study participants. The reported side effects included mild nausea, stomachache, loose stools. A placebo-controlled study will be more informative.

Traumatic brain injury is a common ED presentation. CT-head utilisation is escalating, exacerbating resource pressure in the ED. The biomarker S100B could assist clinicians with CT-head decisions by excluding intracranial pathology. Diagnostic performance of S100B was assessed in patients meeting National Institute of Health and Clinical Excellence Head Injury Guideline (NICE HIG) criteria for CT-head within 6 and 24 hours of injury.This multicentre prospective observational study included adult patients presenting to the ED with head injuries between May 2020 and June 2021. Informed consent was obtained from patients meeting NICE HIG CT-head criteria. A venous blood sample was collected and serum was tested for S100B using a Cobas Elecsys-S100 module; >0.1 µg/mL was the threshold used to indicate a positive test. Intracranial pathology reported on CT-head scan by the duty radiologist was used as the reference standard to review diagnostic performance.This study included 265 patients of whom 35 (13.2%) had positive CT-head findings. Within 6 hours of injury, sensitivity of S100B was 93.8% (95% CI 69.8% to 99.8%) and specificity was 30.8% (22.6% to 40.0%). Negative predictive value (NPV) was 97.3% (95% CI 84.2% to 99.6%) and area under the curve (AUC) was 0.73 (95% CI 0.61 to 0.85; p=0.003). Within 24 hours of injury, sensitivity was 82.9% (95% CI 66.4% to 93.44%) and specificity was 43.0% (95% CI 36.6% to 49.7%). NPV was 94.29% (95% CI 88.7% to 97.2%) and AUC was 0.65 (95% CI 0.56 to 0.74; p=0.046). Theoretically, use of S100B as a rule-out test would have reduced CT-head scans by 27.1% (95% CI 18.9% to 36.8%) within 6 hours and 37.4% (95% CI 32.0% to 47.2%) within 24 hours. The risk of missing a significant injury with this approach would have been 0.75% (95% CI 0.0% to 2.2%) within 6 hours and 2.3% (95% CI 0.5% to 4.1%) within 24 hours.Within 6 hours of injury, S100B performed well as a diagnostic test to exclude significant intracranial pathology in low-risk patients presenting with head injury. In theory, if used in addition to NICE HIGs, CT-head rates could reduce by one-quarter with a potential miss rate of <1%.

A new peloruside congener, peloruside E (5), has been isolated in sub-milligram quantities from a specimen of the New Zealand marine sponge Mycale hentscheli. The structure of 5 differs from the parent compound peloruside A (1) by replacement of the C-10 gem-dimethyl moiety with a monomethyl substituent and represents the first structural deviation in the pelorusane scaffold. Peloruside E (5) is potently antiproliferative (HL-60, IC50 90 nM, cf. 1, 19 nM) and polymerizes purified tubulin, albeit at a rate lower than that of 1.

Hypothesis: Internal jugular vein (IJV) compression influences not only intracranial but also intracochlear physiology and has demonstrated preclinical effectiveness in reducing acute audiological injury in a rodent blast model. However, the long-term effects in this model are unknown. Background: Blast wave-induced audiological injury from an improvised explosive device is a leading cause of morbidity among service members in theater but there are limitations to the current protective measures. Methods: For this study, we exposed 20 Sprague Dawley rats to a 16.8 ± 0.3 PSI (195.3 dB SPL) right-sided shock wave in which 10 had application of a custom IJV compression collar in place at the time of injury. Results: IJV compression at the time of injury was shown acutely to significantly reduce the incidence of tympanic membrane rupture and the initial temporary threshold shift on otoacoustic emissions in both the right and left ears of animals who had collar application immediately after and 7 days post injury. At 28 days from injury, collared animals demonstrated a return to baseline of otoacoustic emission values while the noncollared animals had persistent threshold shifts, signifying the presence of a permanent threshold shift only in those animals without collar application. IJV compression was also found to significantly reduce hair cell loss at the base of the cochlea secondary to mechanical trauma from the blast wind. Conclusion: Previously observed acute protective effects of IJV compression are sustained at chronic time points. IJV compression can potentially be used to reduce long-term permanent morbidity from blast-induced audiological trauma.

Intracerebral hemorrhage (ICH) with or without intraventricular hemorrhage (IVH) is a highly morbid disease process due to the mass effect and secondary injury that occurs upon the surrounding brain. Historically, surgical evacuation has failed to demonstrate improved outcomes in comparison to standard medical therapy likely due to the significant brain trauma when accessing the clot. Recent minimally invasive techniques have proposed a way to improve outcomes by reducing this injury. We report here a 62-year-old male with ICH and IVH with acute neurological deterioration due to hydrocephalus was found to have no improvement following external ventricular drainage. A repeat non-contrasted computed tomography (CT) head was obtained which demonstrated the worsening mass effect from peri-hematoma edema. Surgical intervention was employed that uses a variety of techniques (endoscopic and exoscopic visualization, stereotactic trans-sulcal approach and side cutting aspiration, and intraventricular thrombolytic therapy) to reduce cerebral trauma while effectively removing both ICH and IVH. The surgical intervention reduces the mass effect and associated secondary injury, lessens the likelihood of shunt placement and length of stay, and improves long-term morbidity. We conclude that the effectiveness of surgical management of ICH could potentially be improved by employing a multifaceted approach to address the different characteristics of the hemorrhagic stroke.

Macrophages (mϕ) from pre-diseased mice of the major murine inbred models of spontaneous autoimmunity (AI), including multiple lupus-prone strains and the type I diabetes-prone NOD (non-obese diabetic) strain, have identical apoptotic target-dependent abnormalities. This characteristic feature of mϕ from AI-prone mice suggests that abnormal signaling events induced within mϕ following their interaction with apoptotic targets may predispose to AI. Such signaling abnormalities would affect predominantly the processing and presentation of self-antigen (i.e., derived from apoptotic targets), while sparing the processing and presentation of foreign antigen (i.e., derived from non-apoptotic sources). Here, we used DNA microarrays to test the hypothesis that mϕ from AI-prone mice (MRL/MpJ [MRL/+] or MRL/MpJ-Tnfrsf6 lpr [MRL/lpr]) differentially express multiple genes in comparison to non-AI mϕ (BALB/c), but do so in a largely apoptotic cell-dependent manner. Mϕ were stimulated with lipopolysaccharide, a potent innate stimulus, in the presence or absence of serum (an experimental surrogate for apoptotic targets). In accord with our hypothesis, the number of genes differentially expressed by MRL mϕ was significantly increased in the presence vs. the absence of serum, the apoptotic target surrogate (n = 401 vs. n = 201). Notably, for genes differentially expressed by MRL mϕ in the presence of serum, serum-free culture normalized their expression to a level statistically indistinguishable from that by non-AI mϕ. Comparisons of mϕ from AI-prone NOD and non-AI C57BL/6 mice corroborated these findings. Together, these data support the hypothesis that mϕ from MRL and other AI-prone mice are characterized by a conditional abnormality elicited by serum lipids or apoptotic targets.

In Traditional Chinese Medicine, Qi, the life force, is said to have impeded or abnormal flow through the body when disease is present. Restoration of Qi flow is said to regain balance between Yin, Yang and Qi, resulting in the restoration of normal health. A number of therapies have evolved which attempt to restore the normal function of meridians, the channels in the body within which Qi flows. The best known is acupuncture which involves the insertion of needles into pressure points along blocked or affected meridians to restore Qi flow. Acupressure is believed by its advocates to achieve the same goal by applying pressure instead of needles.

<h3>Introduction</h3> Myocardial abnormalities have been identified in hypertrophic cardiomyopathy(HCM) gene mutation carriers without hypertrophy(G+LVH-). Some of these changes may be mutation-related but whether they can predict gene carriage in relatives of HCM probands is unknown. We developed a method for tracking trabecular and mitral valve(MV) development in embryonic mouse hearts using high-resolution episcopic microscopy(HREM). We used these insights to instruct on human cardiac morphology by cardiovascular magnetic resonance(CMR) hypothesising that a combination of cardiac abnormalities could predict gene carriage in HCM before the appearance of LVH. <h3>Method</h3> MOUSE DEVELOPMENT-63 Wild-type hearts were examined from the time of ventricular septation till just before birth. Trabeculae ware charted by box-counting fractal analysis. MV volumes were calculated from 3D volumetric reconstructions of HREM datasets. HUMAN MORPHOLOGY-74 G+LVH- sarcomere mutation carriers (29 ± 13 yr [SD] 51% M) were identified in 12 US-centres(HCMNet n35) and UCL (n39). Subjects underwent CMR and fractal analysis. Results were compared with 111 overt HCM patients(G+LVH+ n71;G-LVH+ n40) and 136 matched controls(36 ± 16 yr 63% M). We analysed a single-centre (UCL) G+LVH- case-control cohort to identify factors associated with gene carriage evaluating anterior MV leaflets (AMVL), wall thickness, clefts, trabeculae and other variables. We validated associations in the multicenter HCMNet and combined parameters into a model predicting gene carriage. <h3>Results</h3> In developing mice MV volumes trebled between stages E14.5 to 18.5 and a fractal atlas tracked trabecular development revealing a basal drop in LV trabecular complexity(E14.5–18.5 p &lt; 0.0001 Figure 1). Contrasting the UCL case-control populations 5 differences were borne out in the validation cohort (Figure 2). These were: I) longer AMVL(22 ± 3 vs 20 ± 3 mm p &lt; 0.0001); II) increased maximal-apical trabecular complexity(1.242 ± 0.07 vs 1.196 ± 0.05 p &lt; 0.0001); III) increased maximal-septal systolic wall thickness(13 ± 3 vs 12 ± 2 mm p = 0.02); IV) lower indexed-end-systolic LV volume(23 ± 6 vs 26 ± 7 mls/m²p = 0.005); and V) presence of clefts(35 vs 7% p &lt; 0.0001). Conditional logistic regression provided a model containing these parameters which predicted gene carriage with a high level of accuracy (78%). <h3>Conclusion</h3> The normal pattern of cardiac trabecular and MV development may be studied in mouse using HREM. Similar approaches applied to CMR in humans reveal cardiac structural abnormalities in HCM gene mutation carriers even in the absence of LVH. These abnormalities are an early phenotype of sarcomere mutations and a CMR imaging pentad exhibits promising potential for predicting gene carriage in HCM.

Introduction The integration of clinical oncology pharmacists into multidisciplinary healthcare teams is not well-described in the community practice setting. This study aims to analyze the clinical and financial impact of a remote-based clinical oncology pharmacist in four community oncology practices within The US Oncology Network. Methods Oncology-trained clinical pharmacists electronically reviewed chemotherapy orders for clinical optimization and financial stewardship within four community oncology practices. Each pharmacist was appointed at 0.5 full-time equivalents per practice. Financial, clinical, and workload metrics were tracked to monitor the impact of pharmacist engagement. Results Over 12 months, 5716 order reviews were completed with an intervention rate of 57%. The most common interventions identified by the pharmacists were interventions with clinical impact on the patient (36%), followed by dose rounding (35%) and therapeutic interchange (30%). Overall, interventions improved the cumulative practice margins by $1,455,033 and reduced total medication costs by $5,962,551. The average program return on investment was 415% (range 100–915%). Conclusion Community oncology practices seek to provide high-value care in a lean, resource-constrained model. An oncology clinical pharmacist is a cost-effective and clinically invaluable care team member in community oncology practice. Pharmacists in this setting identified opportunities to improve medication safety and regimen optimization and demonstrated a significant tremendous financial impact on small-scale budgets in community oncology.

The use of brain-computer interface (BCI) technologies is a rapidly developing area of interest in neurosurgery. Studies investigating the use of BCIs have primarily focused on surface electrodes; however, subcortically implanted microchips may provide novel, non-pharmacological approaches for managing nervous system disorders. Accessing subcortical regions without disrupting surrounding tissues remains a significant challenge. Accordingly, we conducted an observational, proof-of-concept study to evaluate the use of a modified BrainPath® device to implant subcortical microchips in a rodent model. All animals survived the procedure and lived with a microchip in situ for one or three months with no apparent neurological effects. Histological examination revealed that implantation of a small chip was associated with some tissue damage, tract formation, and surrounding reactive changes. Gliosis and hemosiderin-laden macrophages were evident surrounding tract marks after one month and focal cystic cavitation consistent with infarcted tissue was evident after three months. While a more extensive cellular proliferation response was apparent one month after implantation of a large microchip, there was no significant collagen deposition suggestive of fibrosis. This study is the first demonstration of subcortical microchip implantation using a novel neurosurgical technique that may facilitate the development of BCI technologies for treatment of neurological disorders.

Dr. Sindelar is Senior Neurosurgery Resident, Dr. Patel is Clinician Researcher, and Dr. Bailes is Chairman, Department of Neurosurgery, and Co-Director, NorthShore Neurological Institute, 2650 Ridge Ave, 3rd Floor Kellogg Bldg, Evanston, IL 60201; E-mail: [email protected]. The authors, faculty, and staff in a position to control the content of this CME activity, and their spouses/life partners (if any), have disclosed that they have no financial relationships with, or financial interests in, any commercial organizations related to this CME activity.Category: General NeurosurgeryLippincott Continuing Medical Education Institute, Inc. is accredited by the Accreditation Council for Continuing Medical Education to provide continuing medical education for physicians.Lippincott Continuing Medical Education Institute, Inc. designates this enduring material for a maximum of 2.0 AMA PRA Category 1 Credits™. Physicians should claim only the credit commensurate with the extent of their participation in the activity. To earn CME credit, you must read the CME article and complete the quiz and evaluation on the enclosed form, answering at least 70% of the quiz questions correctly. This activity expires on April 29, 2018.

Introduction The use of CT head scanning for traumatic brain injury (TBI) is a vital diagnostic tool, guided by risk stratification tools. This study aims to review the use of CT head scans and adherence to guidelines for TBI in two New Zealand emergency departments (EDs). Methods Retrospective observational study of patients referred for head CT from EDs to exclude a significant intracranial injury between 1st September 2018 and 31st August 2019. Clinical data were collected regarding presenting patterns, identification of injuries on CT scan and adherence to National Institute of Clinical Excellence (NICE) CT head guidelines. Results Out of 425 included cases, 41 (10%) patients had an intracranial injury seen on their CT head scan. Patients who reported loss (32% vs 20%, p &lt; 0.05) or possible loss of consciousness (34% vs 22%, p &lt; 0.05) and had a Glasgow Coma Score (GCS) &lt;13 (17% vs 8%, p &lt; 0.05) or focal neurology (10% vs 3%, p &lt; 0.05) were more likely to have an intracranial injury on CT. Interestingly, 17 (41%) patients with CT diagnosed injuries had a GCS 15 and no focal neurology. NICE guidelines were adhered to in 364 (86%) of CT requests. In the 14% of cases that did not meet guideline criteria, all CT head scans were negative. Conclusion CT head scans are a valuable tool in TBI, and guidelines successfully identify those with significant intracranial injuries. However, the rate of significant injury for the total population requiring head CT remains low, with over 90% of head CTs in the population normal, despite high guideline compliance, perhaps identifying a role for novel objective tests in ED guidelines internationally.

Abstract Integrin αIIbβ3 activation is critical for platelet physiology and is controlled by agonists- induced signal transduction through kinases and phosphatases. Compared to kinases, a role of phosphatases in platelet integrin αIIbβ3 signaling and activation is less understood. We have previously demonstrated that the catalytic subunit of protein phosphatase 1 (PP1c) associates constitutively with the integrin αIIbβ3 and regulates myosin light chain phosphorylation. Platelets express three isoforms of PP1c namely PP1cα, PP1cβ/δ and PP1cγ1, however their role in platelet physiology is unclear. In this study, we explored a role for the PP1cγ in integrin αIIbβ3 activation using the PP1cγ null mice that lacks both PP1cγ1 and PP1cγ2 splice variants. Deletion of PP1cγ did not alter the expression of PP1cα and PP1cβ/δ in platelets. Compared to the wild type mice, PP1cγ null mice had significantly (P=0.03) more platelets but not other blood cells and increased megakaryocytes in an in vitro bone marrow culture system, suggesting that PP1cγ may regulate platelet production. Compared to the wild type platelets, PP1cγ null platelets exhibited a ∼50% decreased activation (P=0.007) of integrin αIIbβ3 in response to low doses of thrombin as measured by the binding of soluble Alexa-flour 488-conjugated fibrinogen or Jon/A, an antibody to high affinity integrin αIIbβ3. Consistent with this observation, PP1cγ null platelets showed decreased aggregation in response to thrombin or protease activated receptor activating peptide. Soluble fibrinogen binding and aggregation to ADP, collagen and cross linked collagen -related peptide was not different between the PP1cγ null and wild type platelets. Surface expression of integrin αIIbβ3 was comparable in the PP1cγ null platelets indicating that the decreased activation of αIIbβ3 in PP1cγ platelets was not due to decreased integrin expression. These studies suggest that PP1cγ is required for thrombin-induced αIIbβ3 inside-out signaling process. In contrast, αIIbβ3 outside-in signaling process was not altered in PP1cγ null mice as revealed by normal adhesion to immobilized fibrinogen. Intravital microscopy revealed that the thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly (P=0.03) delayed in PP1cγ null mice compared to wild type mice. These studies illustrates that PP1cγ is an essential component of thrombin signaling that is downstream of protease activated receptors and promotes thrombus formation in part by supporting soluble fibrinogen binding and platelet aggregation.

April 19, 2016April 5, 2016Free AccessQuality Improvement and Practice Based Research inNeuro-Oncology Using the Electronic Medical Record (P4.245)Ryan Merrell, Nina Martinez, Susan Stasinos, James Mureson, Shaun Walters, Laura Amelse, Inna Glatman, Magdalena Dudek, Vimal Patel, Roberta Frigerio, and Demetrius MaraganoreAuthors Info & AffiliationsApril 5, 2016 issue86 (16_supplement)https://doi.org/10.1212/WNL.86.16_supplement.P4.245 Letters to the Editor

1) to develop a structured clinical documentation support (SCDS) toolkit within the electronic medical record (EMR) that navigates care, writes notes, and captures data at office visits; 2) To share the toolkit with Neuro-oncologists using the Epic EMR platform, as part of a Neurology Practice Based Research Network (NPBRN). We developed a seven-stage process for quality improvement and practice based research in Neurology using the EMR: 1) SCDS toolkits (custom navigators, electronic forms, smart data elements); 2) enrollment reports; 3) data quality reports; 4) descriptive cohort reports; 5) quality improvement dashboards; 6) clinical decision support; 7) sharing of toolkits and data (NPBRN). We enrolled subjects in a germline DNA biobank. We met every two weeks for three months to develop content: define the cohort, select outcome measures, delineate factors known to modify disease progression. We assigned tasks to the care team, and mapped data elements to the progress note. We then met every two weeks for three more months with programmer analysts to build and test the SCDS toolkit. Autoscored and interpreted tests included the Generalized Anxiety Disorder 7-item, Center for Epidemiological Studies Depression, Karnofsky Performance Scale, MD Anderson Symptom Inventory, and Short Test of Mental Status. Our toolkit captures ~800 fields of data per office visit. We have used the toolkits ~200 times to assess brain tumor patients (initially, six monthly if malignant, annually if benign). We have enrolled 129 brain tumor patients in our biobank: 53 “benign”, 36 “malignant primary”, and 40 “metastatic”. We will present screenshots of our SCDS toolkit, descriptive data, and pairwise correlations and principle component analyses for score test measures. The EMR can be effectively structured to standardize Neuro-oncology office visits, capture data, and support multicenter quality improvement and practice based research initiatives.

Abstract Piperlongumine (PL) from piper longum induces cancerous, but not normal cells to undergo reactive oxygen species (ROS)-mediated apoptosis and blocks the activation of the transcription factor Stat3 in breast cancer cells. Paradoxically, this ROS-inducing agent also inhibits platelet activation and aggregation induced by collagen, which is reported to induce ROS production in platelets. Several types of ROS are known to activate platelets. We test the hypothesis that PL inhibits platelet reactivity to collagen by regulating a non-transcriptional activity of Stat3 that we have recently characterized. Consistent with previous reports, we found that PL dose-dependently blocked collagen-induced platelet activation, aggregation, and thrombus formation with a maximal inhibition at 100 mM. PL was equally effective in blocking platelet aggregation induced by a collagen-related peptide, suggesting that it primarily targets the collagen-receptor GP VI. Several lines of evidence suggest that this inhibitory effect of PL on collagen-induced platelet reactivity is mediated through blocking JAK2-Stat3 signaling. First, PL blocked the tyrosine phosphorylation of JAK2 and Stat3 in collagen-stimulated platelets. This inhibitory effect was significantly reduced in platelets that were pretreated with the Stat3 inhibitor STA21. Second, the JAK2 inhibitor AG490 blocked collagen-induced platelet aggregation and thrombus formation. Third, PL did not inhibit the tyrosine phosphorylation of Syk, the key kinase for collagen-GP VI signaling in platelets. However, it was effective in blocking PLCγ2 phosphorylation, a regulatory profile that is identical to that of Stat3 inhibitors to block collagen-induced platelet activation. Consistent with a report on cancer cells, PL at 100 mM induced platelets to release ROS detected by two specific probes and by a mass spectrometric measurement of platelet free glutathione (GSH). This effect was additive to that of collagen. Quenching oxidants by 20 µM of GSH (cell non-permeable) and 0.5 mM of L-Cysteine (cell permeable) did not block the inhibitory effects of PL. Neither was Apocynin, an inhibitor to reduced nicotinamide adenine dinucleotide phosphate, which blocks collagen-induced ROS production in platelets. The PL treated platelets were viable and responsive to other platelet agonists, suggesting that PL did not induce platelets to undergo apoptosis. Finally, two PL analogs that are active (MZ370) and inactive (MZ306) in inhibiting the growth of breast cancer cells were synthesized and found to be equally active in blocking collagen-induced platelet activation. In summary, we made several observations that define a mechanism for PL to inhibit collagen-induced platelet reactivity and provide new information regarding differential effects of ROS on platelet reactivity to collagen. First, we demonstrate that the inhibitory activity of PL on collagen-induced platelet reactivity was primarily mediated by blocking the activation of JAK2 and Stat3, but not a direct inhibition of collagen-GP IV signaling. Second, both PL and collagen induced ROS release in platelets, but PL’s inhibitory activity was not affected by ROS. The results suggest that PL and collagen induce the release of different oxidants that have differential effects on platelets. Studying these differential effects may uncover new mechanisms of regulating platelet functions by oxidants. Disclosures No relevant conflicts of interest to declare.

Abstract Ultra-large von Willebrand factor (ULVWF) multimers secreted from the Weibel-Palade bodies (WPBs) of endothelial cells plays a crucial role in the development of thrombotic microangiopathies. Secretion of WPB contents is regulated, in part, by the phosphorylation of vesicle trafficking proteins that constitutes the endothelial exocytotic machinery. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate the exocytosis of WPB contents, a role for protein phosphatases in regulating endothelial exocytosis is currently undefined. In this study, we show that inhibition of serine/threonine protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus (FK506) or a cell-permeable PP2B autoinhibitory (AI) peptide promotes the secretion of hyper-adhesive ULVWF from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs induce the tethering of adhesive platelets. In support of a role for PP2B in VWF secretion, we demonstrated that the catalytic subunit of PP2B expressed as a GST fusion protein interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, was significantly increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis and secretion of VWF. Furthermore, plasma VWF antigen levels were also enhanced in CsA-treated mice, and siRNA-mediated knockdown of PP2Bb in HUVECs significantly enhanced VWF secretion. These observations suggest that CsA promotes VWF release, at least in part by inhibition of PP2B activity. These observations are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans, as well as the association between prolonged exposure to CsA and thrombotic microangiopathy in a subset of exposed patients.

Abstract Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase that regulates a variety of cellular functions. In the context of the platelets, we have previously shown that a pool of the catalytic subunit of PP2A (PP2Ac) associates constitutively with the resting αIIbβ3 and negatively regulates αIIbβ3 signaling. However, the mechanism by which PP2Ac controls αIIbβ3 adhesive function is incompletely understood. In this study, we demonstrated that PP2Ac expressed as a GST fusion protein interacts with the tyrosine kinase Src. Activation of Src is essential to initiate αIIbβ3 outside-in signaling. Small interference RNA mediated knockdown of endogenous PP2Acα expression in 293 cells overexpressing αIIbβ3 (293-αIIbβ3) and murine megakaryocytes, resulted in the activation of Src, as evidenced by the dephosphorylation of Src Tyr-529 and phosphorylation of Src Tyr-418. In contrast to PP2Acα, knockdown of the catalytic subunit of protein phosphatase 1 (PP1cα) did not activate Src, indicating that the regulation of Src activity by PP2Ac is specific. Dephosphorylation of Src Tyr-529 was not observed in PP2Aca depleted 293 cells treated with sulfanamido-benzbromarone compound, a selective protein tyrosine phosphatase 1 (PTP-1) inhibitor. These results suggest that inhibition of PP2Ac may activate a tyrosine phosphatase, capable of dephosphorylating Src Tyr-529. Activation of Src in PP2Ac depleted 293-αIIbβ3 cells had functional consequences for integrin αIIbβ3. PP2Ac depleted 293-αIIbβ3 cells exhibited ~2 fold increased adhesion to immobilized fibrinogen. Inhibition of Src kinase with a pharmacological agent PP2 but not by PP3 an inactive analogue of PP2, abolished the increased adhesiveness of PP2Acα–depleted 293 cells to fibrinogen. Finally, the increased activation of extracellular signal-regulated kinase (ERK1/2) in PP2Acα-depleted cells that we previously demonstrated was also blocked by Src inhibitor PP2 but not by PP3. These results indicate that both Src and ERK1/2 are activated in response to PP2Ac inhibition, with activation of Src being upstream of ERK1/2. These studies illustrate that inhibition of PP2Ac promotes αIIbβ3 adhesiveness by activating Src, and imply that the control of αIIbβ3 adhesive function can be further fine-tuned by a cross talk between the serine/threonine phosphatase PP2A and the tyrosine kinase Src.

Abstract Background: Traumatic brain injury (TBI) is a common Emergency Department (ED) presentation. CT-head utilisation is escalating, exacerbating resource pressure in ED. The biomarker S100B could assist clinicians with CT-head decisions by excluding intracranial pathology. Diagnostic performance of S100B was assessed in patients meeting NICE CT-head criteria within 6 and 24-hours of injury. Methods: This multi-centre prospective observational study included adult patients presenting to ED with mild TBI who were able to give their own informed consent and had a CT-head scan performed as part of standard care. Patients were recruited using consecutive sampling methods. Informed consent was obtained prior to blood sampling. Serum was tested for S100B using a Cobas Elecsys S100 module; &gt;0.1ug/ml is the validated threshold indicating a positive test. Diagnostic performance was assessed using ROC analysis as well as sensitivity, specificity, NPV and PPV calculations. Results: There were 265 included patients, and 35 (13.2%) had a positive CT-head. The sensitivity of S100B when performed within 24 hours of injury was 82.9% (95%CI 66.4-93.44) and specificity was 43.0% (95%CI 36.6–49.7%). NPV was 94.29% (95%CI 88.7–97.2). Within 6 hours sensitivity was 93.8% (95%CI 69.8–99.8%) and specificity was 30.8% (22.6–40.0%). NPV was 97.3% (95%CI 84.2–99.6%). Theoretically, use of S100B as a rule out test would have reduced CT scans by 37.4% within 24 hours and 27.1% within 6-hours. The risk of missing a significant injury with this approach would have been 2.3% within 24 hours and 0.75% within 6-hours. Conclusion Within 6-hours of injury, S100B performed well as a diagnostic test to exclude significant intracranial pathology in patients presenting with mild head injuries, with low risk of missed injury. In theory, if used in addition to NICE CT-head guidelines, CT-head request rates could reduce by one quarter and one fifth of patients could be discharged earlier from ED. What is already known on this topic S100B has shown promise diagnostically as an objective marker that can rule out intracranial injuries in head injured patients. S100B has a validated platform that could potentially be used to reduce CT head demand in ED. However, evidence testing the added value of biomarkers to existing clinical guidelines are limited. What this study adds This study demonstrates that S100B could theoretically reduce CT-head demand and enable earlier ED discharge in low-risk patients meeting NICE CT-head criteria. The included population were GCS 15, had no post traumatic amnesia and were able to consent for themselves. This low-risk population have a low yield of traumatic CT abnormalities and could be a suitable population to target biomarker use. How this study might affect research, practice or policy S100B has the potential to reduce CT-head demand, thereby benefitting EDs by reducing overcrowding, resource burden and healthcare costs; and from the patients’ perspective, enable shorter waits, earlier intervention and earlier discharge. Further research should focus on optimising the performance of S100B within the NICE CT-head guidelines in low-risk patients, and review real-time economic benefits.

Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance. Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their increased proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of myocardial infarction prevented cardiac remodeling and heart failure. Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair and prevent the onset of heart failure after MI.

Thoracic aortic aneurysm (TAA) involves extracellular matrix (ECM) remodeling of the aortic wall, leading to reduced biomechanical support with risk of aortic dissection and rupture. Elusive pathophysiology of initiation and progression of TAA has hindered efforts to develop pharmacological therapeutic interventions to delay or prevent progressive aortic dilatation. Activation of the renin-angiotensin system, and resultant Angiotensin (Ang) II synthesis, is critically involved in the onset and progression of TAA. The current study investigated the effects of Angiotensin (Ang) 1-7 on a murine model of TAA. 8-10 weeks old apolipoprotein-deficient mice (ApoEKO) were infused with Ang II (1.44 mg/kg/day) and treated with Ang 1-7 (0.576 mg/kg/day). Echocardiography and histological analyses revealed increased thoracic aortic dilatation, extracellular matrix (ECM) remodeling, perivascular fibrosis, and inflammation in Ang II-treated mice. Infusion of Ang 1-7 led to suppression of Ang II-induced dilatation, remodeling, and inflammation in the thoracic aorta. The immunofluorescence imaging showed reduced α-SMA fluorescence (contractile marker) in vascular smooth muscle cells (VSMCs) of the aortic media indicating phenotypic switching. In response to Ang II, the thoracic VSMCs from ApoEKO mice exhibited phenotypic switching indicated by reduced contractile (Acta2, Cnn1, Myh11) and increased synthetic (Il-6, Mmp2, Mmp9, Col1a1, Col3a1) gene expression. Ki67 staining and flow cytometry analysis showed VSMCs hyperproliferation with Ang II treatment. Ang 1-7 preserved the contractile phenotype of VSMCs and attenuated hyperproliferation. The mitochondrial structure was assessed using MitoTracker Red staining which showed Ang II-induced excessive mitochondrial fission. DHE and MitoSOX Red staining were used to assess the reactive oxygen species (ROS). Ang II-treated thoracic VSMCs demonstrated elevated cellular and mitochondrial ROS generation. Ang 1-7 mitigated Ang II-induced mitochondrial fission and oxidative stress. Ang 1-7 prevented Ang II-induced vascular remodeling and the development of TAA. Enhancing Ang 1-7 actions may provide a novel therapeutic strategy to prevent or delay the progression of TAA.

Abstract Exocytosis of Weibel Palade bodies (WPBs) containing ultra-large Von Willebrand factor (ULVWF) plays an essential role in the processes of inflammation and thrombosis. The exocytosis of ULVWF involves the fusion of WPBs to the endothelial cell plasma membrane via vesicular trafficking families of proteins. Reversible post-translational modification, including the phosphorylation of tyrosine (Tyr) and/or serine/threonine (Ser/Thr) residues or S-nitrosylation of cysteine residues on one or more vesicular trafficking proteins tightly regulates the exocytotic process. The phosphorylation of any trafficking protein is determined by the interplay between protein kinases and protein phosphatases and any alteration in the activity of either enzyme can affect exocytosis. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate exocytosis in endothelial cells, there is scant information about any possible role for the regulation of protein phosphatases in this process. Inhibition of protein phosphatase 2B (PP2B) also called calcineurin by the immunosuppressive drug, cyclosporine (CsA), is associated with thrombotic microangiopathy in a subset of transplant patients. Calcineurin is the target of a cyclophilin-CsA complex. We detected both calcineurin and cyclophilin A in resting human umbilical vein endothelial cells (HUVECs). Because CsA is a potent inhibitor of PP2B, we hypothesized that CsA induces ULVWF secretion and thrombotic events at least in part, by facilitating the phosphorylation of vesicle trafficking proteins that promote the exocytosis of ULVWF from endothelial cells. In vitro, we found that nanomolar concentrations of CsA or a cell permeable PP2B auto inhibitory peptide stimulated the release of ULVWF from HUVECs. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) viability assays revealed that PP2B inhibitors did not damage the HUVECs. In vivo, plasma levels of VWF were increased after intraperitoneal injection of CsA. These studies suggest that PP2B negatively regulate ULVWF exocytosis. Microscopic studies revealed that the ULVWF strings secreted by HUVECs treated with CsA or PP2B auto-inhibitory peptide induced the adhesion of platelets. CsA-treated HUVECs exhibited an increased serine phosphorylation of Munc18c, a vesicle trafficking associated protein that promotes granule exocytosis. These findings support a mechanism whereby inhibition of PP2B by CsA enhances cell secretion, including endothelial cell secretion of ULVWF by facilitating Munc18c phosphorylation.

Preoperative embolization of highly vascular tumors of the posterior fossa can decrease morbidity and operative blood loss. No clear consensus exists for the embolization agent of choice for optimal devascularization of these tumors. The purpose of this study was to assess effectiveness of microsphere embolization in reducing tumor hypervascularity before surgical resection.We retrospectively reviewed medical records of patients with hypervascular posterior fossa tumors who were treated at a single institution from 2009 to 2016.Four of 9 patients with hypervascular posterior fossa tumors underwent embolization with 300–500 μm microspheres before surgical resection. Patients selected for embolization had large tumors with large feeding vessels evident on brain magnetic resonance imaging. Surgical resection was performed within 24 hours of embolization in all 4 patients. Mean (SD) patient age was 42.5 years (18.4), and mean (SD) tumor size was 4.3 cm (1.4) in greatest dimension. All patients presented with symptoms related to mass effect. Gross total tumor resection was achieved in all patients. There were no intraoperative complications related to the embolization or craniotomy; mean (SD) blood loss was 350 mL (208).Preoperative embolization with microspheres can effectively reduce vascularity of the hypervascular posterior fossa tumor bed. This technique helped achieve complete resection, particularly for patients with recurrence after previous resection.

Background: The success in the management of haemophilia in the last two decades has been predominantly due to the availability of sufficient quantities of safe anti-haemophilic factor concentrates. Unfortunately, the prohibitive cost of these products has prevented this benefit from being available to the vast majority (~80%) of haemophiliacs living in the developing world. Methods: With this background, the article aims at demonstrating the development and characterization of a safe, economically viable, highly pure and efficient plasma-derived Factor-VIII/von Willebrand Factor complex concentrate (pdF-VIII/vWF) prepared from freshly frozen recovered plasma collected from approved blood banks across India, which is comparable to other commercially available products. This complex was subjected to solvent detergent and dry heat treatment for virus inactivation and further, characterized by various physicochemical and functional assays along with other marketed products. Results: The indigenously manufactured pdF-VIII/vWF drug product yielded ~150 IU of F-VIII/L of plasma with specific activity of 60±20 IU F-VIII/mg of total protein and functional vWF demonstrating F-VIIIC : F-VIII:Ag ratio ≈1 which is also optimum in managing vWF deficient patients, in addition to its ability to treat F-VIII deficient population. Identity of pdF-VIII/vWF was confirmed by Western blot. The high order secondary structure was confirmed using Far UV Circular Dichroism (CD) in accordance with different marketed products. pdF-VIII/vWF was proved to be stable for 24 months at 5±3 ℃. Conclusions: The present study is a milestone as it demonstrates highly pure, safe, low-cost and stable pdF-VIII/vWF complex that will be able to cater to the clinical demands of both Haemophilia A and vWF deficient patients in India as well as other developing countries of similar origin.

Abstract Introduction/Objective Ordering pre-operative type and screen specimens before the day of surgery is very important to prevent delays in provision of blood products due to unexpected alloantibody findings, unexpected ABO discrepancies, or need for a repeat ABO-RH testing due to lack of historical testing. However, the important pre- operative type and screen was not always ordered prior to the day of surgery by the clinician. Due to this observation, after discussion and collaboration with the Blood Utilization Committee (BUC), an educational and mentoring intervention for the pre-operative clinicians was undertaken. Methods Education and mentoring of clinicians involved in the pre-operative clinics, surgical services, and new incoming physicians on an ongoing basis was undertaken through the BUC since March 2018. As part of quality assurance, to track the results of this widespread educational effort, the daily OR lists from January 2017 to December 2019 were examined with the medical record to determine if a pre-operative type and screen was ordered prior to the day of surgery. The established Maximum Surgery Blood order Schedule (MSBOS) for the facility was used to determine if a pre-operative type and screen would be indicated for the listed procedure. Results As we started reinforced pre type and screen should be ordered there has been significant increase in percent of compliance rate average from 76% (2017), 87% (2018), and 95.6% (2019). By the T-test, there was a statistically significant change in the monthly ordering rates (p-value &amp;lt; 0.05). The VAMC’s preferred threshold value of at least 95% with pre-operative type and screen prior to the day of procedure was achieved in the 2019 calendar year with no operating room delays. Conclusion Appropriately performing the pre-operative type and screen prior to the day of the elective surgical procedure would reduce the risk of delays due to unexpected findings or lack of a historical ABO-RH type. Educational interventions help tremendously in ensuring this critical patient care practice is followed. The value of collaborating and educating clinicians cannot be understated as it is both cheap, simple, and highly effective.

Loosemore, Michael P. MBBS, PhD; Butler, Charles F. MD, PhD; Khadri, Abdelhamid MD; McDonagh, David MD; Patel, Vimal PhD; Bailes, Julian E. MD Author Information

The role of the presumptive phosphatidylserine receptor (PSR) in the recognition and engulfment of apoptotic cells, and the antiinflammatory response they exert, has been of great interest. Genetic deficiency of PSR in the mouse is lethal perinatally, and results to date have been ambiguous with regard to the phagocytic and inflammatory phenotypes associated with that deficiency. Recently, we found that the specific functional recognition of apoptotic cells is a ubiquitous property of virtually all cell types, including mouse embryo fibroblasts, and reflects an innate immunity that discriminates live from effete cells. Taking advantage of this property of fibroblasts, we generated PSR / , PSR / , and PSR / fibroblast cell lines to examine definitively the involvement of PSR in apoptotic recognition and inflammatory modulation. Our data demonstrate that PSR-deficient cells are fully competent to recognize, engulf, and respond to apoptotic cells. Signal transduction in the responder cells, including the activation of Akt and Rac1, is unimpaired in the absence of PSR. We confirm as well that PSR is localized predominantly to the nucleus. However, it does not play a role in pro-inflammatory transcription or in the anti-inflammatory modulation of that transcriptional response triggered by apoptotic cells. We conclude that PSR is not involved generally in either specific innate recognition or engulfment of apoptotic cells.

Abstract Objective: The use of CT head scanning for traumatic brain injury (TBI) is a vital diagnostic tool, guided by risk stratification tools. This study aims to review the use of CT head scans for TBI in two Australasian Emergency Departments (ED) in New Zealand. Methods: Retrospective observational design of patients referred for head CT from ED to exclude a significant intracranial injury between 1st September 2018 and 31st August 2019. Clinical data were collected regarding presenting patterns, identification of injuries on CT scan and adherence to CT guidelines. Results: Out of 425 cases reviewed, a clinically significant injury was identified in 41 (10%) patients. Patients who reported loss (32% vs 20% p &lt; 0.05) or possible loss of consciousness (34% vs 22% p &lt; 0.05) and had GCS &lt; 13 (17% vs 8%, p &lt; 0.05) or focal neurology (10% vs 3%, p &lt; 0.05) were more likely to have a significantly intracranial injury on CT. Interestingly, 17 (41%) patients with significant injury were GCS 15 with no focal neurology. NICE guidelines were adhered to in 364 (86%) patients. In the 14% of cases that did not meet guideline criteria, all CT head scans were negative. Conclusion: CT head scans are a valuable tool in TBI and guidelines successfully identify those with significant intracranial injuries. However, the rate of significant injury for the total population requiring head CT remains low, with over 90% of head CTs in the population normal, despite high guideline compliance perhaps identifying a role for novel objective tests in ED guidelines internationally.

BACKGROUND Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance. METHODS AND RESULTS Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of myocardial infarction prevented cardiac remodeling and heart failure. Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. CONCLUSION Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair after MI. Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance. Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of myocardial infarction prevented cardiac remodeling and heart failure. Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair after MI.

Abstract: Variable Compression Ratio (VCR) engine test can be used to determine the effect of Compression Ratio (CR) on the performance and emissions of the engine. The combustion situation, when provided with a pressure transducer. The performance frequency parameters like efficiencies, power, and specific fuel consumption are determined. The combustion phenomenon is also observed through this work, we can find the optimum compression ratio for which the best performance is possible. In order to find out the optimum compression ratio, experiments were carried out on a single-cylinder four-stroke variable compression ratio engine. Tests were carried out at compression ratios of 18, 17, and 16 at different loads the performance characteristics of the engine like Brake power (BP), Thermal Efficiency, Brake Specific Fuel Consumption (BSFC). A variable compression ratio engine is able to operate at different compression ratios, depending on particular vehicle performance needs. The VCR engine is optimized for the full range of driving conditions, as acceleration, speed, and load. Keywords: Performance, Compression ratio, Load, Break Power, William Line’s Method, Emission, Thermal Efficiency, Diesel Engine, Fuel Consumption