Takashi Kishimoto

The neutrophil Mac-1 and gp100MEL-14 adhesion proteins are involved in neutrophil extravasation during inflammation. Both the expression and activity of Mac-1 are greatly increased after neutrophil activation. In contrast, neutrophils shed gp100MEL-14 from the cell surface within 4 minutes after activation with chemotactic factors or phorbol esters, releasing a 96-kilodalton fragment of the antigen into the supernatant. Immunohistology showed that gp100MEL-14 was downregulated on neutrophils that had extravasated into inflamed tissue. The gp100MEL-14 adhesion protein may participate in the binding of unactivated neutrophils to the endothelium; rapid shedding of gp100MEL-14 may prevent extravasation into and damage of normal tissues by activated neutrophils.

Exhausted CD8 T (Tex) cells are a distinct cell lineage that arise during chronic infections and cancers in animal models and humans. Tex cells are characterized by progressive loss of effector functions, high and sustained inhibitory receptor expression, ...Read More

There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany.Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine.The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine.Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

This chapter focuses on the molecular biology of the leukocyte integrins, LFA-1, Mac-1, and p150,95, and on their role in mediating inflammation. Three recent developments have underscored the importance of the leukocyte integrins as adhesion receptors of the immune system: The recognition that the leukocyte integrins are evolutionarily related to other integrins; Identification of intercellular adhesion molecule-1 (ICAM-l), a ligand for LFA-1, which is induced during inflammation, and may regulate leukocyte migration and localization; and discovery and characterization of immunodeficiency patients who are genetically deficient in their expression of the leukocyte integrins. Researchers have found a class of immune-deficient patients who suffer from recurrent, life-threatening bacterial and fungal infections, and who have neutrophils deficient in chemotaxis and phagocytosis. Infected, necrotic lesions in these patients contain few leukocytes, despite the observation that these patients have chronic leukocytosis. The leukocyte integrins are α1β1 heterodimers, in which the α subunit is noncovalently associated with the β subunit. The α subunits of LFA-1, Mac-1, and p150, 95 are 1,80,000, 1,70,000, and 1,50,000 Da, respectively. The α subunits have been shown to be distinct by MAb reactivity, antigen-preclearing studies, and tryptic peptide mapping. In contrast, the β subunit, Mr = 95,000, has been shown to be identical in all three proteins by the same criteria. There is also substantial evidence that other ligands for LFA-1, Mac-1, and p150, 95 exist. Rational strategies must be designed to identify these ligands and to assess their contributions in different phases of the immune response. Multiple ligands may provide quite distinct signals and positional information to leukocytes.

Background Maternal viral infection is associated with increased risk for schizophrenia. It is hypothesized that the maternal immune response to viruses may influence fetal brain development and lead to schizophrenia. Methods To mimic a viral infection, the synthetic double strand RNA polyriboinosinic-polyribocytidilic acid (poly I:C) was administered into pregnant mice. Behavioral evaluations (thigmotaxis, methamphetamine [MAP]-induced hyperactivity, novel-object recognition test [NORT]), sensorimotor gating (prepulse inhibition [PPI]), and biochemical evaluation of the dopaminergic function of the offspring of phosphate-buffered saline (PBS)-treated dams (PBS-mice) and that of poly I:C-treated dams (poly I:C-mice) were examined. Results In juveniles, no difference was found between the poly I:C-mice and PBS-mice. However, in adults, the poly I:C-mice exhibited attenuated thigmotaxis, greater response in MAP-induced (2 mg/kg) hyperlocomotion, deficits in PPI, and cognitive impairment in NORT compared with the PBS-mice. Cognitive impairment in the adult poly I:C-mice could be improved by subchronic administration of clozapine (5.0 mg/kg) but not haloperidol (.1 mg/kg). Increased dopamine (DA) turnover and decreased receptor binding of D2-like receptors, but not D1-like receptors, in the striatum were found in adult poly I:C-mice. Conclusions Prenatal poly I:C administration causes maturation-dependent increased subcortical DA function and cognitive impairment in the offspring, indicating a neurodevelopmental animal model of schizophrenia. Maternal viral infection is associated with increased risk for schizophrenia. It is hypothesized that the maternal immune response to viruses may influence fetal brain development and lead to schizophrenia. To mimic a viral infection, the synthetic double strand RNA polyriboinosinic-polyribocytidilic acid (poly I:C) was administered into pregnant mice. Behavioral evaluations (thigmotaxis, methamphetamine [MAP]-induced hyperactivity, novel-object recognition test [NORT]), sensorimotor gating (prepulse inhibition [PPI]), and biochemical evaluation of the dopaminergic function of the offspring of phosphate-buffered saline (PBS)-treated dams (PBS-mice) and that of poly I:C-treated dams (poly I:C-mice) were examined. In juveniles, no difference was found between the poly I:C-mice and PBS-mice. However, in adults, the poly I:C-mice exhibited attenuated thigmotaxis, greater response in MAP-induced (2 mg/kg) hyperlocomotion, deficits in PPI, and cognitive impairment in NORT compared with the PBS-mice. Cognitive impairment in the adult poly I:C-mice could be improved by subchronic administration of clozapine (5.0 mg/kg) but not haloperidol (.1 mg/kg). Increased dopamine (DA) turnover and decreased receptor binding of D2-like receptors, but not D1-like receptors, in the striatum were found in adult poly I:C-mice. Prenatal poly I:C administration causes maturation-dependent increased subcortical DA function and cognitive impairment in the offspring, indicating a neurodevelopmental animal model of schizophrenia.

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor α precursor (proTGF-α) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-α, cell adhesion receptor L-selectin, interleukin 6 receptor α subunit, β-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene. The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor α precursor (proTGF-α) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-α, cell adhesion receptor L-selectin, interleukin 6 receptor α subunit, β-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.

Leukocyte adhesion deficiency (LAD) is a heritable disease involving deficient expression of three related leukocyte adhesion glycoproteins: LFA-1, Mac-1, and p150,95. These proteins are alpha beta heterodimers containing identical 95,000 dalton beta subunits. Here we demonstrate that the primary defect in LAD is in the beta subunit gene. We identified five distinct beta subunit phenotypes in LAD patients: undetectable beta subunit mRNA and protein precursor; low levels of beta subunit mRNA and precursor; an aberrantly large beta subunit precursor, probably due to an extra glycosylation site; an aberrantly small precursor; and a grossly normal precursor. Mutant beta subunit precursors from LAD patients failed to associate with the LFA-1 alpha subunit. In family studies, inheritance of the aberrant precursors correlates with the known inheritance of the LAD defect.

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.

In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions.Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case-control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants.A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin.Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause of the outbreak.

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8+ T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25+ T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

Antigen-specific immune tolerance has been a long-standing goal for immunotherapy for the treatment of autoimmune diseases and allergies and for the prevention of allograft rejection and anti-drug antibodies directed against biologic therapies. Nanoparticles have emerged as powerful tools to initiate and modulate immune responses due to their inherent capacity to target antigen-presenting cells (APCs) and deliver coordinated signals that can elicit an antigen-specific immune response. A wide range of strategies have been described to create tolerogenic nanoparticles (tNPs) that fall into three broad categories. One strategy includes tNPs that provide antigen alone to harness natural tolerogenic processes and environments, such as presentation of antigen in the absence of costimulatory signals, oral tolerance, the tolerogenic environment of the liver, and apoptotic cell death. A second strategy includes tNPs that carry antigen and simultaneously target tolerogenic receptors, such as pro-tolerogenic cytokine receptors, aryl hydrocarbon receptor, FAS receptor, and the CD22 inhibitory receptor. A third strategy includes tNPs that carry a payload of tolerogenic pharmacological agents that can "lock" APCs into a developmental or metabolic state that favors tolerogenic presentation of antigens. These diverse strategies have led to the development of tNPs that are capable of inducing antigen-specific immunological tolerance, not just immunosuppression, in animal models. These novel tNP technologies herald a promising approach to specifically prevent and treat unwanted immune reactions in humans. The first tNP, SEL-212, a biodegradable synthetic vaccine particle encapsulating rapamycin, has reached the clinic and is currently in Phase 2 clinical trials.

KEYWORDS: Gene therapyadeno-associated virusAAV toxicityhepatotoxicitythrombotic microangiopathyhepatocellular carcinomadorsal root ganglia toxicityAAV capsid engineeringimmune tolerance

Mac-1 (CD 11b/CD18) is a leukocyte adhesion heterodimeric glycoprotein which functions both as a receptor for iC3b (CR3) and in several cell-cell and cell-substrate adhesive interactions. We describe full-length cDNA clones for the alpha subunit of Mac-1. Mac-1 alpha subunit message was detected in blood monocytes and phorbol-12-myristate acetate-induced myeloid cell lines, but not in cells of the T or B lineages, correlating with Mac-1 protein surface expression. The alpha subunit of Mac-1 is a transmembrane protein of 1137 residues with a long extracellular domain (1092 residues) and a 19-amino acid cytoplasmic tail. The extracellular domain contains three putative divalent cation-binding sequences and 19 potential N-glycosylation sites. The amino acid sequence of Mac-1 alpha shows that it is a member of the integrin superfamily; Mac-1 alpha shows 63% identity to the alpha subunit of the leukocyte adhesion glycoprotein p150.95 and 25% to the alpha subunits of the extracellular matrix receptors platelet glycoprotein IIb/IIIa, the fibronectin receptor, and the vitronectin receptor. The Mac-1 alpha subunit putative divalent cation-binding sites and the flanking regions exhibit a high degree of identity both to the p150.95 alpha subunit (87% identity at the amino acid level) and to the rest of the integrin alpha subunits (38%). The alpha subunit of Mac-1, like the p150.95 alpha subunit, contains a domain of 187 amino acids in the extracellular region which is absent in other integrins. This leukocyte or L domain is homologous to the A domains of von Willebrand factor, which in turn are homologous to regions of the C3-binding proteins factor B and C2. These findings draw attention to this region of Mac-1 as a potential binding site for iC3b.

E-selectin was evaluated for its ability to support neutrophil adhesion under conditions of flow. At a wall shear stress of 1.85 dyn/cm2, neutrophils were found to attach to E-selectin expressed on the apical surface of L cell monolayers. The initial intercellular contact was most often evidenced by neutrophils rolling on the monolayer at a mean rate of congruent to 10 microns/s. Anti-E-selectin monoclonal antibody, CL2/6, inhibited this interaction by > 90%. Rolling neutrophils often transiently stopped, but in contrast to the behavior on stimulated endothelial cells, they remained spherical in shape and did not migrate on or beneath the monolayer. A possible contribution of neutrophil L-selectin to this interaction was indicated by the findings that anti-L-selectin monoclonal antibody, DREG-56, inhibited E-selectin-dependent adhesion under flow by > 65%, and there was a highly significant correlation between surface levels of L-selectin and E-selectin-dependent adhesion under flow. E-selectin also appeared to support neutrophil adhesion to IL-1 beta-stimulated endothelial cells under conditions of flow, but it accounted for only congruent to 30% of the level of adherence, in contrast to L-selectin which accounted for > 65%. Thus, both L-selectin and E-selectin can support neutrophil adhesion at wall shear stresses that preclude intercellular adhesion molecule-1-dependent adhesion, and they participate in neutrophil adherence to stimulated endothelial cells under conditions of flow.

Previous studies have suggested that the leukocyte adhesion proteins Mac-1 and p150,95 are stored in a latent intracellular pool in neutrophils, and cellular fractionation studies have shown that Mac-1 is localized primarily in the peroxidase-negative specific granules. To determine the subcellular location of leukocyte adhesion receptors (LAR), we used immunocytochemical techniques on frozen thin sections of human blood leukocytes that had been incubated for peroxidase to mark the peroxidase-positive azurophil granules. To enhance the sensitivity of detection, polyclonal antibodies against immunoaffinity-purified p150,95 were raised in rabbits and absorbed with leukocytes from a patient deficient in this protein. The antiserum reacted with p150,95 and two other antigens with the same beta subunit, Mac-1 and lymphocyte function-associated antigen 1 (LFA-1). In neutrophils, we observed immunogold label for LAR predominantly on the membranes of peroxidase-negative granules, and in smaller amounts on the plasma and perinuclear membranes. In double-label experiments, there was colocalization of LAR with lactoferrin in some of the peroxidase-negative granules. We conclude that the latent pool of LAR resides in the membranes of peroxidase-negative granules. A significant increase in label on the plasma membrane of neutrophils stimulated with PMA is consistent with secretion of LAR to the exterior of the cell during degranulation. While LFA-1 appears very early in neutrophil maturation, it is becoming clear that Mac-1 and p150,95 are upregulated from an intracellular storage pool of peroxidase-negative granules that appear during the myelocyte stage of differentiation. Further studies are indicated to determine the significance of these proteins on the plasma membrane of two other granulocytes, eosinophils and basophils.

Abstract The localization of the adhesion protein L-selectin in human neutrophils was determined by sub-cellular fractionation and immunoelectron microscopy and compared with the localization of Mac-1 (α m β z ) and alkaline phosphatase, the marker for secretory vesicles. L-selectin was found to be localized exclusively on the plasma membrane of unstimulated cells and also of stimulated cells, although markedly diminished. This was in contrast to Mac-1, which was also localized in secretory vesicles and in specific/gelatinase granules as shown previously [Sengeiov, H., et al. J. Clin. Invest. (1993) 92, 1467–1476]. Stimulation of neutrophils with inflammatory mediators such as tumor necrosis factor (TNF), platelet-activating factor (PAF), or f-Met-Leu-Phe (fMLP), induced parallel up-regulation of the surface membrane content of alkaline phosphatase and Mac-1 and down-regulation of L-selectin, as evidenced by flow cytometry. Preimbedding immunoelectron microscopy confirmed that L-selectin was present mainly on tips of microvilli in unstimulated cells and showed that alkaline phosphatase and Mac-1 were randomly distributed on the surface membrane of fMLP-stimulated cells. These studies indicate that the transition of neutrophils from L-selectin-presenting cells to Mac-l-presenting cells induced by inflammatory mediators is mediated by incorporation of secretory vesicle membrane, rich in Mac-1 and devoid of L-selectin, into the plasma membrane. J. Leukoc. Biol. 56: 80–87; 1994.

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.

L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band co-migrated with mitogen- activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab′)2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion. L-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates rolling of leukocytes along endothelial surfaces. In addition to its role in adhesion, an intracellular signaling role for L-selectin has recently been recognized. In particular, cross-linking L-selectin leads to increased cytosolic Ca2+ levels and potentiation of the oxidative burst. As several cell surface glycoproteins have been shown to be linked to tyrosine kinases, we examined the hypothesis that L-selectin may be linked to pathways involving tyrosine phosphorylation in human neutrophils. Ligation of L-selectin by three different antibodies recognizing separate epitopes led to increased tyrosine phosphorylation of several cellular proteins as judged by anti-phosphotyrosine immunoblots of whole cell lysates with prominent bands at 40-42, 55-60, 70-72, and 105-120 kDa. The 42-kDa band co-migrated with mitogen- activated protein (MAP) kinase as determined by immunoblotting with anti-MAP kinase antibody. This effect was specific for L-selectin, because antibodies against CD18, CD45, and CD10 did not increase tyrosine phosphorylation. Phosphorylation was not due to Fc binding, since F(ab′)2 fragments of the anti-L-selectin antibodies were similarly effective, and the response was unaffected by Fc receptor blockade. Cross-linking of L-selectin was not required for enhanced tyrosine phosphorylation, because monovalent Fab fragments also increased tyrosine phosphorylation. The response to L-selectin antibodies was not inhibited by cytochalasin, suggesting that reorganization of the actin cytoskeleton was not required for this response. Sulfatides, sulfated glycolipids which may be natural ligands for L-selectin, also induced a rapid, dose-dependent increase in tyrosine phosphorylation. In addition, sulfatides, but not control glycolipids, resulted in enhanced tyrosine phosphorylation of MAP kinase. Both sulfatides and anti-L-selectin antibodies increased kinase activity of MAP kinase as determined by gel renaturation assay. The tyrosine kinase inhibitor, genistein, blocked the transient increase in intracellular Ca2+ and the oxidative burst induced by sulfatides, suggesting that this tyrosine phosphorylation is functionally important. We conclude that L-selectin is able to transmit intracellular signals, including increased tyrosine phosphorylation and activation of MAP kinase in neutrophils. We speculate that these events may contribute to the activation of neutrophils during adhesion.

Atherosclerosis is increasingly thought to be a chronic inflammatory disease. Inflammation requires transmigration of leukocytes from the circulation to the tissues. Adhesion of leukocytes to endothelial cells is the initial event in an inflammatory response and is mediated by expression of several adhesion molecules. In this study we characterize the contribution of intercellular adhesion molecule-1 (ICAM-1) and L-selectin in patients with different coronary artery disease syndromes. Serum concentrations of cICAM-1 and sL-selectin were measured by enzyme-linked immunosorbent assay in 31 patients with stable angina, 30 patients with unstable angina, 18 patients with acute myocardial infarction and 20 healthy subjects in a control group. All patients underwent coronary angiography. Mean (±SE) cICAM-1 levels were higher (p < 0.05) in patients with stable angina (249 ± 16 ng/ml), unstable angina (260 ± 16 ng/ml), or acute myocardial infarction (261 ± 24 ng/ml) compared with those in subjects in the control group (171 ± 11 ng/ml). In contrast, levels of sL-selectin were lower (p < 0.01) in patients with stable angina (1.2 ± 0.1 μg/ml), unstable angina (1.1 ± 0.6 μg/ml), or acute myocardial infarction (1.1 ± 0.1 μg/ml) compared with those in subjects in the control group (1.8 ± 0.1 μg/ml). No difference was found in cICAM-1 or sL-selectin levels among patients with stable angina, unstable angina, or acute myocardial infarction. No correlation was seen between cICAM-1 or sL-selectin levels and extent (or severity) of coronary artery disease or leukocyte count. L-selectin expression was observed to be depressed in patients with severe angina compared with that in members of the control group. To examine the mechanism of reduction in sL-selectin levels and L-selectin expression on leukocytes, leukocytes from the control group were stimulated in vitro. Stimulation of leukocytes resulted in a rapid downregulation of surface L-selectin expression, measured by flowcytometry, similar to the suppressed expression of L-selectin found on leukocytes from patients with coronary artery disease. In conclusion, altered cICAM-1 and sL-selectin levels in patients with coronary artery disease reflect the presence of a chronic inflammatory process. This inflammatory process results in downregulation of leukocyte expression of L-selectin and thus lower circulating sL-selectin levels.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required.

Tumor lymphangiogenesis is now known to play a causal role in lymph node metastasis, and thus its inhibition would have great significance for the prevention of lymph node metastasis in cancer therapy. VEGF‐C has recently been identified as a key molecule that involved in tumor lymphangiogenesis and lymphatic metastasis. However, the expressional regulation of VEGF‐C is not fully understood. We investigated the role of mTOR, which is a downstream kinase of the phosphatidylinositol 3‐kinase/Akt pathway, and the MAPK family (MEK1/2, p38, and JNK) in the regulation of VEGF‐C and VEGF‐A expression in B13LM cells, a lymphatic metastasis‐prone pancreatic tumor cell line. We also investigated the antilymphangiogenic effect of rapamycin, a specific inhibitor of mTOR in vivo using male BALB/c nu/nu mice. VEGF‐C expression was inhibited by the inhibitors for mTOR, p38, and JNK, but not by the inhibitor for MEK1/2, whereas VEGF‐A expression was inhibited by all four of these inhibitors. The serum starvation‐induced expression of VEGF‐C was inhibited by rapamycin, whereas that of VEGF‐A was incompletely inhibited. The metastatic experiment in vivo demonstrated that the number and the area of lymphatic vessels in the primary tumors were significantly decreased by rapamycin. Finally, the lymph node metastasis was significantly suppressed in rapamycin‐treated mice. Our results suggest that mTOR, p38, and JNK play important roles in VEGF‐C expression, and that rapamycin has an antilymphangiogentic effect and exerts the expected inhibition of lymphatic metastasis. ( Cancer Sci 2007; 98: 726–733)

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

T cells reacting to self-components can promote tissue damage when escaping tolerogenic control mechanisms which may result in autoimmune disease. The current treatments for these disorders are not antigen (Ag) specific and can compromise host immunity through chronic suppression. We have previously demonstrated that co-administration of encapsulated or free Ag with tolerogenic nanoparticles (tNPs) comprised of biodegradable polymers that encapsulate rapamycin are capable of inhibiting Ag-specific transgenic T cell proliferation and inducing Ag-specific regulatory T cells (Tregs). Here, we further show that tNPs can trigger the expansion of endogenous Tregs specific to a target Ag. The proportion of Ag-specific Treg to total Ag-specific T cells remains constant even after subsequent Ag challenge in combination with a potent TLR7/8 agonist or complete Freund's adjuvant. tNP-treated mice do not develop experimental autoimmune encephalomyelitis (EAE) after adoptive transfer of encephalitogenic T cells; furthermore, tNP treatment provided therapeutic protection in relapsing EAE that was transferred to naïve animals. These findings describe a potent therapy to expand Ag-specific Tregs in vivo and suppress T cell-mediated autoimmunity.

Biologic drugs have transformed the standard of care for many diseases. However, many biologics induce the formation of anti-drug antibodies (ADAs), which can compromise their safety and efficacy. Preclinical studies demonstrate that biodegradable nanoparticles-encapsulating rapamycin (ImmTOR), but not free rapamycin, mitigate the immunogenicity of co-administered biologic drugs. Here we report the outcomes from two clinical trials for ImmTOR. In the first ascending dose, open-label study (NCT02464605), pegadricase, an immunogenic, pegylated uricase enzyme derived from Candida utilis, is assessed for safety and tolerability (primary endpoint) as well as activity and immunogenicity (secondary endpoint); in the second single ascending dose Phase 1b trial (NCT02648269) composed of both a double-blind and open-label parts, we evaluate the safety of ImmTOR (primary endpoint) and its ability to prevent the formation of anti-drug antibodies against pegadricase and enhance its pharmacodynamic activity (secondary endpoint) in patients with hyperuricemia. The combination of ImmTOR and pegadricase is well tolerated. ImmTOR inhibits the development of uricase-specific ADAs in a dose-dependent manner, thus enabling sustained enzyme activity and reduction in serum uric acid levels. ImmTOR may thus represent a feasible approach for preventing the formation of ADAs to a broad range of immunogenic biologic therapies.

Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-α. Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-α.

This chapter discusses the key adhesion molecules involved in leukocyte trafficking to sites of inflammation, focuses on mechanisms to regulate adhesion molecule expression and function, and defines some of the steps involved in neutrophil–endothelial cell interactions. The chapter summarizes the progress made in testing anti-adhesion molecule monoclonal antibodies (MAbs) in animal models of inflammatory diseases. Neutrophils are the front line of defense and are rapidly mobilized and recruited to sites of tissue injury or infection. This efficient response requires that neutrophils gain access to virtually any tissue. In addition to traditional random screening of large chemical libraries, it may be possible to rationally design drugs based on known ligand structures, such as small carbohydrates for selectins or peptide-based antagonists. In addition to direct receptor antagonists, drugs that modulate adhesion molecule regulation or expression may also be effective.

L-selectin expression is regulated in part by membrane-proximal cleavage from the cell surface of leukocytes and L-selectin-transfected cells. The downregulation of L-selectin from the surface of neutrophils is speculated to be a process involved in the adhesion cascade leading to neutrophil recruitment to sites of inflammation. We previously reported that L-selectin is cleaved between Lys321 and Ser322 in a region that links the second short consensus repeat (SCR) and the transmembrane domain. We demonstrate that replacing this cleavage domain of L-selectin with the corresponding region of E-selectin prevents L-selectin shedding, as judged by inhibiting the generation of the 68-kD soluble and 6-kD transmembrane cleavage products of L-selectin. Unexpectedly, we found that point mutations of the cleavage site, as well as mutations of multiple conserved amino acids within the cleavage domain, do not significantly affect L-selectin shedding. However, short deletions of four or five amino acids in the L-selectin cleavage domain inhibit L-selectin downregulation. Mutations that appeared to inhibit L-selectin shedding resulted in higher levels of cell surface expression, consistent with a lack of apparent proteolysis from the cell membrane. One deletion mutant, I327 delta N332, retains the native cleavage site yet inhibits L-selectin proteolysis as well. Restoring the amino acids deleted between I327 and N332 with five alanine residues restores L-selectin proteolysis. Thus, the proteolytic processing of L-selectin appears to have a relaxed sequence specificity at the cleavage site, and it may depend on the physical length or other secondary structural characteristics of the cleavage domain.

Leukocyte adhesion deficiency (LAD) is a heritable deficiency of the LFA-1, Mac-1, p150,95 family of leukocyte alpha beta heterodimers (the leukocyte integrins). We have studied the defect in patients who synthesize an aberrantly small form of the beta subunit common to all three proteins. S1 nuclease protection showed the presence of a 90-nucleotide mismatch in RNA from patients and relatives, correlating with inheritance of the disease. Use of the Taq polymerase chain reaction to amplify this region of RNA after first strand cDNA synthesis and sequencing showed an in-frame deletion of 90 nucleotides in the extracellular domain. Thus, this highly conserved region, 63% and 53% identical in amino acid sequence to two other beta subunits of the integrin family, is required for association of the beta subunit with alpha subunits. The 90-nucleotide region corresponds to a single exon present in both the normal and patient genome. The patient DNA has a single G to C substitution in the 5' splice site. This results in the direct joining of nonconsecutive exons in an unusual type of abnormal RNA splicing. A small amount of normally spliced message, detected by S1 nuclease protection and Taq polymerase chain reaction, encodes a normal sized beta subunit which is surface-expressed and accounts for the low levels of leukocyte integrin expression observed in these patients, and hence the moderate phenotype.

The adhesion receptors Mac-1, LFA-1, and p150,95 are cell surface alpha/beta heterodimers that play a key role in leukocyte adhesion processes. The genes for Mac-1, LFA-1, and p150,95 alpha subunits have been located to chromosome 16 by means of Southern blot analysis using a series of somatic cell hybrids. Chromosomal in situ hybridization has demonstrated that the genes for the three alpha subunits map to the short arm of chromosome 16, between bands p11 and p13.1, defining a cluster of genes involved in leukocyte adhesion. The gene encoding the LFA-1/Mac-1/p150,95 beta subunit, and defective in leukocyte adhesion deficiency, has been located on chromosome 21, band q22. The leukocyte adhesion receptor alpha and beta subunits are mapped to chromosomal regions that have been shown to be involved in cytogenetic rearrangements in certain patients with acute myelomonocytic leukemia and the blast phase of chronic myelogenous leukemia, respectively.

Abstract Objective B and T lymphocyte attenuator (BTLA), a coreceptor expressed on lymphocytes, was recently described as an inhibitory coreceptor that negatively regulates lymphocyte activation. The purpose of this study was to investigate the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity. Methods We examined the levels of immunoglobulins and autoantibodies to nuclear antigens and the activation status of T cells in BTLA −/− mice. We also examined histopathologic changes in the organs of BTLA −/− mice. Results We observed that BTLA −/− mice gradually developed hypergammaglobulinemia, antinuclear antibodies, anti‐SSA antibodies, anti–double‐stranded DNA antibodies, and an increased number of activated CD4+ T cells in the periphery with age. Lack of BTLA led to spontaneous development of autoimmune hepatitis–like disease characterized by an elevation in the level of transaminases, interface hepatitis, and spotty necrosis of the liver. BTLA −/− mice also showed inflammatory cell infiltration of multiple organs, including the salivary glands, lungs, and pancreas; these features are similar to those of Sjögren's syndrome, which is a frequent complication of autoimmune hepatitis. Furthermore, the survival rate of BTLA −/− mice was significantly reduced after the age of 7 months. Conclusion Our results indicate that BTLA plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases.

CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates.

Napsin A is a reliable marker for pulmonary adenocarcinoma and is expressed in a subset of ovarian clear cell carcinomas (O-CCCs), endometrial (EM) CCCs, and endometrioid carcinomas (EC). We investigated napsin A levels in O-CCC and EM-CCC and compared these with levels in other nonmucinous ovarian carcinomas and EM-EC, respectively. Napsin A, thyroid transcription factor (TTF)-1, paired box (PAX) 8, and cancer antigen (CA) 125 expression was evaluated in 111 ovarian and uterine carcinoma cases (22 O-CCC, 15 EM-CCC, 13 ovarian EC (O-EC), 39 high-grade serous carcinoma [HGSC], and 22 EM-EC) using immunohistochemistry. Napsin A immunoreactivity was observed in 21 (95.5%) of 22 O-CCC and 10 (66.7%) of 15 EM-CCC cases but was rare in O-EC and EM-EC (7.7% and 4.5%) and undetectable in HGSC cases. Thyroid transcription factor 1 was not expressed in O-CCC but was detected in 1 (6.7%) of 15 EM-CCC, 3 (23.1%) of 13 O-EC, 2 (5.1%) of 39 HGSC, and 1 (4.5%) of 22 EM-EC cases. All 111 cases examined were positive for PAX8, whereas 3 (20.0%) of 15 of EM-CCC and 1 (4.5%) of 22 EM-EC cases were negative for CA125. There were no napsin A/TTF-1 double-positive cases, except for 1 EM-CCC, in which cells had a focal expression pattern. All napsin A- and/or TTF-1-positive cases expressed PAX8 and CA125. In conclusion, napsin A is frequently expressed in O-CCC and EM-CCC, rarely in O-EC and EM-EC, and never in HGSC cases. These findings confirm the importance of using a panel of antibodies that includes napsin A, TTF-1, and PAX8 when evaluating metastatic carcinomas of unknown origin, particularly when gynecologic and pulmonary adenocarcinomas are included in the differential diagnosis.

The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and hypersensitivity reactions for many biologics. The focus of this review is the development of ImmTOR, platform technology designed to prevent the formation of ADAs that can be applied broadly across a wide variety of biologics by inducing immunological tolerance with ImmTOR nanoparticles encapsulating rapamycin. The induction of tolerance is antigen-specific and dependent on the incorporation of rapamycin in nanoparticles and the presence of the antigen at the time of administration of ImmTOR. Evidence for the induction of specific immune tolerance versus general immune suppression is supported by the findings that: 1) ImmTOR induces regulatory T cells specific to the co-administered antigen; 2) tolerance can be transferred by adoptive transfer of splenocytes from treated animals to naïve recipients; 3) the tolerance is durable to subsequent challenge with antigen alone; and 4) animals tolerized to a specific antigen are capable of responding to an unrelated antigen. ImmTOR nanoparticles can be added to new or existing biologics without the need to modify or reformulate the biologic drug. The ability of ImmTOR to mitigate the formation of ADAs has been demonstrated for coagulation factor VIII in a mouse model of hemophilia A, an anti-TNF monoclonal antibody in a mouse model of inflammatory arthritis, pegylated uricase in hyperuricemic mice and in nonhuman primates, acid alpha-glucosidase in a mouse model of Pompe disease, recombinant immunotoxin in a mouse model of mesothelioma, and adeno-associated vectors in a model of repeat dosing of gene therapy vectors in mice and in nonhuman primates. Human proof-of concept for the mitigation of ADAs has been demonstrated with SEL-212, a combination product consisting of ImmTOR + pegadricase, a highly immunogenic enzyme therapy for the treatment of gout. ImmTOR represents a promising approach to preventing the formation of ADAs to a broad range of biologic drugs.

Liver-directed AAV transgene expression is enhanced by ImmTOR nanoparticles.

SEL-212 is a developmental treatment for uncontrolled gout characterized by serum uric acid (sUA) levels ≥ 6 mg/dl despite treatment. It comprises a novel PEGylated uricase (SEL-037; also called pegadricase) co-administered with tolerogenic nanoparticles containing sirolimus (rapamycin) (SEL-110; also called ImmTOR®), which mitigates the formation of anti-drug antibodies (ADAs) against uricase and SEL-037 (PEGylated uricase), thereby enabling sustained sUA control (sUA < 6 mg/dl). The aim of this study was to identify appropriate dosing for SEL-037 and SEL-110 for use in phase 3 clinical trials. This open-label phase 2 study was conducted in adults with symptomatic gout and sUA ≥ 6 mg/dl. Participants received five monthly infusions of SEL-037 (0.2 or 0.4 mg/kg) alone or in combination with three or five monthly infusions of SEL-110 (0.05–0.15 mg/kg). Safety, tolerability, sUA, ADAs, and tophi were monitored for 6 months. A total of 152 adults completed the study. SEL-037 alone resulted in rapid sUA reductions that were not sustained beyond 30 days in most participants due to ADA formation and loss of uricase activity. Levels of ADAs decreased with increasing doses of SEL-110 up to 0.1 mg/kg, with anti-uricase titers < 1080 correlating with sustained sUA control and reductions in tophi. Overall, 66% of evaluable participants achieved sUA control at week 20 following five monthly doses of SEL-037 0.2 mg/kg + SEL-110 0.1–0.15 mg/kg, whereas only 26% achieved sUA control at week 20 when SEL-110 was withdrawn after week 12. Compared to other dose combinations, SEL-037 0.2 mg/kg + SEL-110 0.15 mg/kg achieved the greatest sUA control at week 12 and was well-tolerated with no safety concerns. Results provide continued support for the use of multiple monthly administrations of SEL-037 0.2 mg/kg + SEL-110 0.1-0.15 mg/kg in clinical trials for SEL-212. ClinicalTrials.gov identifier, NCT02959918.

Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity - a particular concern surrounding rAAV treatment.AAV8-MDR3 was re-administered to infant Abcb4-/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment.Co-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma.These outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis.Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration.

The indication for surgical resection of intraductal papillary mucinous neoplasms (IPMNs) is defined by imaging features, such as mural nodules. Although carbohydrate antigen (CA) 19-9 was selected as a parameter for worrisome features, no serum biomarkers were considered when deciding on surgical indications in the latest international consensus guideline. In this study, we assessed whether clinical factors, imaging findings, and serum biomarkers are useful in predicting malignant IPMNs. A total of 234 resected IPMN cases in Chiba University Hospital from July 2005 to December 2021 were retrospectively analyzed. Among the 234 patients with resected IPMNs diagnosed by preoperative imaging, 117 were diagnosed with malignant pathologies (high-grade dysplasia and invasive IPMNs) according to the histological classification. In the multivariate analysis, cyst diameter ≥30 mm; p = 0.035), enhancing mural nodules on multidetector computed tomography (≥5 mm; p = 0.018), and high serum elastase-1 (≥230 ng/dl; p = 0.0007) were identified as independent malignant predictors, while CA19-9 was not. Furthermore, based on the receiver operator characteristic curve analyses, elastase-1 was superior to CA19-9 for predicting malignant IPMNs. Additionally, high serum elastase-1 levels (≥230 ng/dl; p = 0.0093) were identified as independent predictors of malignant IPMNs in patients without mural nodules on multidetector computed tomography (MDCT) in multivariate analysis. The serum elastase-1 level was found to be a potentially useful biomarker for predicting malignant IPMNs.

The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 <i>O</i>-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked <i>O</i>-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2<i>O</i>-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an <i>O</i>-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an <i>O</i>-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.

Four cases of hepatoid adenocarcinoma, three in the stomach, and one in the sigmoid colon, are presented to emphasize venous permeation and mimicry of hepatocellular carcinoma by metastatic liver nodules.Tumour cells in all cases extensively invaded vems, and intravenous tumour thrombi in two cases were grossly observed as anastomosing, worm-like cords up to 10 mm in diameter in the lesser omentum and mesentery in continuity with the primary mucosal lesions. The cytological features and trabecular architecture of the metastatic liver nodules in these subjects mimicked primary hepatocellular carcinoma. In a third case the tumour contained grossly visible bile in a metastatic lung nodule, but there was no evidence of bile production in the primary gastric or metastatic liver lesions. In the fourth case, detailed histopathological study revealed a gastric origin of the hepatoid adenocarcinoma, rather than primary hepatocellular carcinoma metastatic to the stomach, the initial diagnosis.These cases are reported here to draw attention to this rare variant of gastrointestinal adenocarcinoma, its mimicry of hepatocellular carcinoma when metastatic to the liver and other sites, and its propensity for venous permeation.

Antibody blocking studies in the mouse suggest that the MEL-14 antigen is involved in neutrophil-endothelial cell interactions and may be important in neutrophil extravasation to sites of inflammation in vivo. We recently showed that chemotactic factor activation causes a rapid (within minutes) shedding of a large fragment of the MEL-14 antigen from the surface of neutrophils. We report here that chymotrypsin, at low doses (0.1 units/1 × 106 cells), but not trypsin, elastase, or collagenase, causes an activation-independent rapid loss (>90%) of the MEL-14 antigen from the surface of murine neutrophils. Under the same treatment conditions chymotrypsin has no effect on the expression of four other neutrophil surface antigens, including the Mac-1 adhesion protein. Chymotrypsin treatment has no effect on neutrophil adhesion to plastic, migration to C5a, or regulation of the Mac-1 antigen, but causes a greater than 95% reduction in neutrophil binding to high endothelial venules (HEV) in peripheral lymph nodes measured in the ex vivo frozen section HEV binding assay. The level of inhibition of neutrophil adhesion to HEV was comparable to that seen with the MEL-14 antibody. This experimental system allows us for the first time to specifically examine the consequences of removing the MEL-14 antigen from the surface of neutrophils on function in vivo. We show that treatment with chymotrypsin blocks >85% of the ability of neutrophils injected back into the animal to home to the inflamed peritoneum. In similar in vivo experiments the MEL-14 antibody blocks neutrophil homing by 60–70%. These results further support the importance of the MEL-14 antigen in neutrophil extravasation in vivo and indicate that chymotrypsin could be useful in examining the molecular mechanisms involved in extravasation of leukocytes into a variety of diverse tissue sites of inflammation.

Serum levels of recently discovered circulating forms of adhesion molecules, ICAM-1 and L-selectin, were found to be elevated in IDDM patients and in subjects at risk for developing IDDM compared with 100 normal, nondiabetic blood donors. Both adhesion molecules were determined by sandwich ELISA. Serum concentrations of either clCAM-1 or cL-selectin were > 2SD of normal mean in 10 of 14 recent-onset IDDM patients (P < 0.05). Serum levels of clCAM-1 and cL-selectin did not correlate. In first-degree relatives, elevated adhesion molecule levels were observed in the 6 ICA+ individuals and in the ICA- individuals all (n = 14) with a genetic risk of IDDM (sharing HLA-DR3 and/or-DR4 with the diabetic relative) but not in the HLA-DR3- and/or -DR4- relatives (n = 13). We conclude that elevated clCAM-1 and cL-selectin levels occur independently of ICA status and probably reflect ongoing immune processes in recent-onset IDDM patients and first-degree relatives at risk for IDDM.

Adhesion to the vascular endothelium precedes or is a necessary prelude to leukocyte migration into the underlying tissue. Constitutive lymphocyte trafficking through lymphoid organs is controlled by tissue-specific interactions between molecules expressed on the surface of the lymphocyte (homing receptors) and ligands (vascular addressins) expressed on endothelial cells (HEV) within lymphoid tissues. Preliminary evidence suggests that lymphocytes may employ related but distinct interactions in their entry into some chronic sites of inflammation. Other leukocytes, such as neutrophils and monocytes, express molecules related or identical to lymphocyte homing receptors, and these molecules are exquisitely regulated by chemotactic factors and appear to be involved in the homing of these cells to inflamed tissues. In addition, inflammation in vivo induces increased endothelial cell adhesiveness for leukocytes that undoubtedly plays a key role in regulating leukocyte extravasation. Tissue- and inflammation-specific leukocyte/endothelial cell adhesion molecules constitute attractive targets for suppression or manipulation of the early stages of tissue inflammation.

Mouse immunoglobulin epsilon chain gene was cloned from DNA of a hybridoma producing anti-dinitrophenyl IgE, which was constructed by fusing a spleen cell of a BALB/c mouse with a variant clone of MOPC21 myeloma (IgG1 producer). Because a given active heavy chain constant region (CH) gene is linked to a heavy chain joining segment (JH) gene at its 5' side, the expressed C epsilon gene of the hybridoma was cloned from a phage library containing partial Sau3A digests of IgE hybridoma DNA by using a J gene fragment as a probe. Among 6 X 10(5) phages screened, five positive clones were obtained and three of them were identified as C epsilon gene clones by restriction mapping, Southern blot hybridization, R-loop formation, and partial nucleotide sequence determination. The determined nucleotide sequence predicted the amino acid sequence which resembles a part of the CH3 domain of human epsilon chain. The deletion profile of the C epsilon gene in various myelomas expressing different CH genes indicates that the C epsilon gene is located between the C gamma 2a and C alpha genes. The linkage (5'-epsilon-alpha-3') was directly confirmed by molecular cloning of the overlapping chromosomal segments from newborn mouse DNA.

This study retrospectively analyzed the clinical outcomes of endoscopic resection of 26 sporadic (i. e., not associated with polyposis syndrome) nonampullary duodenal lesions representing high-grade dysplasia or intramucosal carcinoma (duodenal HGD/IMC) in 23 patients. No severe complications such as perforation were observed, but three cases of delayed bleeding were seen. The use of endoscopic clips significantly decreased the delayed bleeding rate (0 /19, 0 %) compared with cases in which clips were not used (3 /7, 42.9 %; <i>P</i> = 0.013, χ² test). Eighteen lesions (69.2 %) were removed by en bloc resection. The follow-up period after resection was 25.5 ± 23.3 months. Two lesions (7.7 %) that recurred locally were detected at the first surveillance endoscopy 3 months after resection. These lesions were 22 and 15 mm in size respectively and were resected piecemeal. Endoscopic resection is an effective and safe procedure for treating duodenal HGD/IMC. En bloc resection and prophylactic clip usage are encouraged.

Some follow-up studies of large regenerative nodules (LRNs) and dysplastic nodules (DNs) were reported previously. However, the pre-malignant potentiality of LRNs has remained controversial up to now. No LRNs showed malignant transformation in our previous study. We aimed to evaluate the pre-malignant potentiality of LRNs and DNs with a greater number of cases and longer follow-up periods. From 1982 to 2005, 1,500 consecutive nodular lesions up to 2 cm in diameter were subjected to US guided thin-needle biopsy in cirrhotic patients at Chiba University Hospital. Of these lesions, 68 LRNs in 60 cases and 20 DNs in 22 cases were followed up for more than 6 months without any anti-cancer therapy. The last US examination was in 2010. The total study period was 28 years. We analyzed the histological findings and the clinical data of all cases retrospectively. The outcome of the lesions was examined. The mean follow-up period was 38.9 (16–119) months in LRNs and 31.9 (6–101 months) in DNs. Rate of nodule enlargement was higher in DNs (8/24 nodules, 33 %) than LRNs (11/68 nodules, 16 %), (p = 0.0743, not significant). Rate of malignant transformation was also higher in DNs (10/24 nodules, 42 %) than LRNs (9/68 nodules, 13 %), (p = 0.0040, significant). The rate of disappearance in images was similar between LRNs and DNs. We should recognize LRN as low risk pre-malignant lesions whereas DNs as high risk lesions.

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation.

Ultrasound is noninvasive and useful to evaluate liver disease despite its operator dependency. This pilot study was conducted to quantitatively assess liver fibrosis using ultrasound.Fibrosis extraction ratios (FER) (fiber volume/total volume) of ultrasound and histological images of 8 autopsy specimens were compared. We also compared FER of ultrasound images from clinical patients (n=79) with histological fibrosis stages.In the autopsy study, FER correlation coefficient between histological images and ultrasound images was 0.992. Regarding clinical patients, there was sufficient evidence to indicate differences in the distributions of FER for each fibrosis stage (Kruskal-Wallis test P<0.0001). With FER cut-off to distinguish > or =F2 from F0 and F1 defined as mean plus standard deviation of F0 and F1, sensitivity, specificity, positive predictive value, negative predictive value, and likelihood ratio were 62, 75, 78, 57%, and 2.47, respectively. Regarding HCV cohort (n=44), they were 55, 87, 89, 50%, and 4.14, respectively. Areas under receiver operating characteristic curves were 0.78, 0.79, 0.83 and 0.83 for > or =F1, > or =F2, > or =F3 and =F4, respectively. Regarding HCV cohort, they were 0.74, 0.71, 0.79 for > or =F2, > or =3 and =4, respectively.The FER method has great potential for diagnosing liver fibrosis using ultrasound.

The T-cell receptor for antigen (Ti) was purified from the human tumor cell line HPB-ALL. Amino-terminal sequence analysis of an acid-cleaved peptide of the Tiα chain showed that it is highly homologous to a putative murine α chain recently described. Amino-terminal sequence analysis of the Tiβ chain revealed that it shares 50 percent homology with the Tiβ chain amino acid sequences from two other human T-cell tumors. Nucleotide sequence analysis of a complementary DNA clone encoding the Tiβ chain from the HPB-MLT cell line showed that this chain represents a second human constant region gene segment and suggested that it arises from direct joining of the variable and joining gene segments without any intervening D region sequences.

Current treatments for unwanted antibody responses largely rely on immunosuppressive drugs compromising overall immunity. New approaches to achieve antigen-specific tolerance are desirable to avoid unwanted side effects. Several nanoparticle-based approaches that utilize different mechanisms to tolerize the B or T cell arms of the humoral immune response have shown promise for induction of antigen-specific tolerance, raising the possibility that they could work synergistically if combined. Earlier we showed that Siglec-engaging tolerance-inducing antigenic liposomes (STALs) that display both an antigen (Ag) and glycan ligands of the inhibitory co-receptor CD22 (CD22L) lead to robust antigen-specific B cell tolerance to protein antigens in naive mice. In another approach, administration of free Ag with poly(lactic-co-glycolic acid)-rapamycin nanoparticles (PLGA-R) induced robust antigen-specific tolerance through production of regulatory T cells. Here we illustrate that coadministration of STALs together with PLGA-R to naive mice induced more robust tolerance to multiple antigen challenges than either nanoparticle alone. Moreover, in K/BxN mice that develop spontaneous autoimmune arthritis to the self-antigen glucose-6-phosphate-isomerase (GPI), co-delivery of GPI-LP-CD22L and PLGA-R delayed onset of disease and in some mice prevented the disease indefinitely. The results show synergy between B cell-tolerizing STALs and T cell-tolerizing PLGA-R and the potential to induce tolerance in early stage autoimmune disease.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model.

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.

Metastasisto the liver often occurs in patients during the natural course of pancreatic cancer.Using carcinoma cell lines established from 9 such patients, we examined phenotypes of cell lines to search for correlations with their potential to metastasize to the liver.Anti-asialo GM I -treated nude mice were used.PCI-43, -55, -24 and -6, in this order, had frequent metastases, while PCI-10, -19, -35, -64, and -66 did not.In vitro doubling time, surface expression of sialyl Lewis' (SLe'), VLA-4/6, LFA-I /3, CEA, E-selectin, VCAM-I, NCAM, Mac-I, HLA-ABC/ DR/DQ, ICAM-I /2, production of interleukin-I a, tumor necrosis factor-a, and matrix metalloproteinase, as well as susceptibility to cytotoxicity by natural killer cells, were all examined.Expression of surface SLe' was significantly associated with metastasis; numbers of metastatic colonies of SLea-positive and -negative cell lines were 21.6 k 33.9 and 6.5 -C 14.3 (p < O.Ol), respectively.Moreover, the intensity of surface SLe" expression of each PCI line correlated with the number of metastatic colonies in the liver.When anti-SLe" monoclonal antibody (MAb) was administered, the development of liver metastasis by PCI-43 cells was significantly repressed, as compared with a control MAb.Although a reverse correlation between surface ICAM-I expression and liver metastasis was noted, the speciesrestricted function of ICAM-I makes interpretation difficult.Collective evidence indicates that expression of SLea is an important positive mediator in the hematogenous metastasis of pancreas carcinoma.

Hepatoid carcinoma of the ovary is an ovarian carcinoma that has phenotypic properties in common with hepatocellular carcinomas. However, the extent of the tumor cells’ similarity to and their difference from hepatocytes is largely unknown. In addition, the precursor cell of origin for hepatoid carcinoma of the ovary has not been identified. Three cases of alpha-fetoprotein-producing hepatoid carcinoma of the ovary that were admixed with an adenocarcinoma of common surface epithelial type are reported. The hepatoid carcinomas had a trabecular architecture with canaliculi detected by polyclonal (but not monoclonal) anticarcinoembryonic antigen antibodies. A hepatic phenotype in the hepatoid tumor cells was further supported by the production of albumin mRNA by in situ hybridization. The adenocarcinomas in the three cases were mucinous (Case 1), serous (Case 2), and endometrioid (Case 3), respectively. The cytokeratin (CK) profile in both the hepatoid and adenocarcinomatous components was CK18+/CK19+/CK20±, whereas normal and neoplastic hepatocytes were CK18+/CK19-/CK20-. Although this study supports a hepatic phenotype in ovarian hepatoid carcinoma, the CK profile of hepatoid carcinoma differs from that of normal and neoplastic hepatocytes but resembles that of the associated common epithelial adenocarcinoma. These findings suggest that hepatoid carcinoma of the ovary is probably derived from carcinomas of surface epithelial origin by a process of neometaplasia or transdifferentiation.

A new type of multiple imaging and multiple Fourier transformation system under coherent illumination using microlens arrays has been developed. The optical system is based on geometrical optics instead of convolution or diffraction. As a result, it has the advantage of design flexibility especially in alignment of the duplicate images. The experimental results of the system, which are implemented using planar microlens arrays fabricated by an ion exchange technique, are also discussed.

HistopathologyVolume 53, Issue 4 p. 495-498 Predominance of IgG4+ plasma cells and CD68 positivity in sclerosing angiomatoid nodular transformation (SANT) Y Nagai, Y Nagai Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this authorN Hayama, N Hayama Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 1 T Kishimoto, T Kishimoto Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 2 M Furuya, M Furuya Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 2 Y Takahashi, Y Takahashi Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 3 M Otsuka, M Otsuka Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 4 M Miyazaki, M Miyazaki Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 4 Y Nakatani, Y Nakatani Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 3 Y Nagai, Y Nagai Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this authorN Hayama, N Hayama Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 1 T Kishimoto, T Kishimoto Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 2 M Furuya, M Furuya Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 2 Y Takahashi, Y Takahashi Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 3 M Otsuka, M Otsuka Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 4 M Miyazaki, M Miyazaki Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 4 Y Nakatani, Y Nakatani Department of Pathology, Chiba Medical Centre, 1Chiba University School of Medicine, 2Department of Molecular Pathology and 3Diagnostic Pathology, and 4General Surgery, Chiba University Postgraduate School of Medicine, Chiba, JapanSearch for more papers by this author 3 First published: 24 September 2008 https://doi.org/10.1111/j.1365-2559.2008.03118.xCitations: 40Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume53, Issue4October 2008Pages 495-498 RelatedInformation

After the report of the Cardiac Arrhythmia Suppression Trial, a tabular framework of the Sicilian Gambit has been proposed to display actions of antiarrhythmic drugs on ion channels and receptors and to provide more rational pharmacotherapy of arrhythmias. However, because effects of antiarrhythmic drugs on If have not been thoroughly examined, we used patch clamp techniques to determine the effects of various antiarrhythmic drugs on the HCN (hyperpolarization-activated cyclic nucleotide-gated) channel currents. HCN4 channels, a dominant isoform of HCN channels in the heart, were expressed in HEK293 cells. Amiodarone and bepridil potently inhibited the HCN4 channel current with IC50 values of 4.5 and 4.9 microM, respectively, which were close to their therapeutic concentrations. The inhibitory effects of quinidine, disopyramide, cibenzoline, lidocaine, mexiletine, aprindine, propafenone, flecainide, propranolol, and verapamil on the HCN4 channel current were weak in their therapeutic concentrations, with IC50 values of 78.3, 249, 46.8, 276, 309, 43.7, 14.3, 1700, 50.5, and 44.9 microM, respectively, suggesting that the inhibitory effects on If would be clinically small. D,L-Sotalol hardly affected the HCN4 channel current. Information about the HCN4-channel effects of many antiarrhythmic drugs may be useful for determining the appropriate drug for treatment of various arrhythmias while minimizing adverse effects.

A pivotal role of c-jun N-terminal kinase (JNK) on neuronal apoptosis has been demonstrated in a rodent stroke model. MAP kinase phosphatase 1 (MKP-1) is an archetypal member of the dual-specificity protein phosphatase (DUSP) family, which inactivates mitogen-activated protein kinase (MAPK) including JNK through dephosphorylation. MKP-1, one of immediate early genes in stress conditions, was induced at transcriptional level in hypoxia/re-oxygenation (H/R) in neuroblastoma N1E115 cells, however the activation of JNK was not suppressed in the acute phase of re-oxygenation. Small interference RNA-mediated knock-down of MKP-1 enhanced phospho-JNK and neuronal death that is rescued by JNK inhibitor in H/R. Conversely, conditional over-expression of MKP-1 suppressed phospho-JNK, the expression of proapoptotic genes, and neuronal death in H/R. Further the immunoreactivity of MKP-1 was detected in the neurons and partially co-localized with that of phospho-JNK in the surrounding zone of ischemia in rat MCA-O (middle cerebral artery occlusion) reperfusion model. These findings indicate that over-expression of MKP-1 could suppress neuronal death possibly through regulating JNK signaling in vitro and be a prominent neuroprotective target for the treatment of acute cerebral infarction.

Preoperative assessment of longitudinal extension of cholangiocarcinoma (CCA) is essential for making decisions concerning surgical resection and selecting operative procedures. We evaluated the accuracy of peroral video-cholangioscopy (PVCS) in diagnosing longitudinal extension of CCA.Patients with CCA who underwent preoperative PVCS were considered for this study. We evaluated the accuracy of PVCS in diagnosing longitudinal extension of perihilar cholangiocarcinoma (PCCA) and distal extrahepatic cholangiocarcinoma (DCCA) to the secondary biliary radicles and confluence of the hepatic ducts, respectively, on the hepatic side and to the intrapancreatic common bile duct on the papillary side. Diagnostic accuracy was determined by comparing the results with those of histopathological analyses of surgical specimens.Forty-three consecutive patients were enrolled. The cholangioscope could not be advanced into the hepatic side in eight of the 25 patients with PCCA and in five of the 18 patients with DCCA. The accuracy of PVCS in diagnosing longitudinal extension of CCA on the hepatic and papillary sides was 82.4% and 92.0%, respectively, in patients with PCCA and 92.3% and 100%, respectively, in patients with DCCA. PVCS accurately detected longitudinal extension of CCA to the hepatic and papillary sides that was not detected previously by endoscopic retrograde cholangiography in 20.0% and 11.6% patients, respectively.PVCS proved useful for the preoperative assessment of longitudinal extension of CCA. Therefore, it can aid surgeons in deciding surgical resectability and selecting operative procedures. This, in turn, may impact overall patient prognosis.

HistopathologyVolume 35, Issue 1 p. 90-92 Hepatoid adenocarcinoma of the pancreas T Yano, T Yano Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorH Ishikura, H Ishikura Departments of Pathology,Search for more papers by this authorT Wada, T Wada Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorT Kishimoto, T Kishimoto Departments of Pathology,Search for more papers by this authorS Kondo, S Kondo Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorH Katoh, H Katoh Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorT Yoshiki, T Yoshiki Departments of Pathology,Search for more papers by this author T Yano, T Yano Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorH Ishikura, H Ishikura Departments of Pathology,Search for more papers by this authorT Wada, T Wada Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorT Kishimoto, T Kishimoto Departments of Pathology,Search for more papers by this authorS Kondo, S Kondo Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorH Katoh, H Katoh Surgery, Hokkaido University School of Medicine, Sapporo, JapanSearch for more papers by this authorT Yoshiki, T Yoshiki Departments of Pathology,Search for more papers by this author First published: 10 December 2003 https://doi.org/10.1046/j.1365-2559.1999.0728f.xCitations: 36Read the full textAbout ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article.Citing Literature Volume35, Issue1July 1999Pages 90-92 RelatedInformation

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.

Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.

Abstract Cellular adhesion of sialyl‐Lewis‐a(SLe a )‐positive pancreas carcinoma to endothelial cells (EC) is augmented by activation of EC via up‐regulated E‐selectin expression on EC. Co‐cultivation of pancreas‐carcinoma cells, PCI‐24, with human umbilical‐vein endothelial cells (HUVEC) for 5 hr at the PCI‐to‐HUVEC ratio of 1:10 induced E‐selectin expression on the endothelial‐cell surface, augmenting SLe a ‐positive pancreas‐carcinoma cell attachment with HUVEC. Culture supernatants of 6 tested pancreas‐carcinoma cell lines contained soluble, E‐selectin‐inducing factor(s). The E‐selectin‐inducing effect by the supernatants was blocked by the protein‐kinase‐C inhibitor, H7. Antibodies against SLe a and E‐selectin but not SLe x or ICAM‐I blocked the increased pancreas‐carcinoma‐to‐endothelial attachment. Paraformaldehyde(PFA)‐fixed PCI‐24 cells also induced E‐selectin on vascular endothelial cells upon direct contact with endothelial cells, indicating the presence of a membrane‐bound form. The 6 pancreas‐carcinoma lines all produced IL‐1α mRNA and protein but not IL‐1β or TNF‐α protein and/or mRNA Absorption of IL‐lα from the supernatants by IL‐lα‐specific antibody almost completely abolished E‐selectin‐inducing activity. Anti‐IL‐lα antibody also abolished the E‐selectin‐inducing activity of PFA‐fixed PCI. IL‐1α production by PCI cells was up‐regulated by TNF‐α. These observations suggest that substance(s) produced by pancreas‐carcinoma cells, in this case, IL‐1α, may contribute to pancreas‐carcinoma‐cell colonization in non‐inflamed, distant locations in vivo , by activating vascular endothelial cells.

Gastroenteropancreatic neuroendocrine tumors are uncommon and their tumor biology has not been well elucidated to date. Currently the WHO classification is widely used for the diagnosis and distinction of this tumor entity, which is sometimes cumbersome. Although neuroendocrine tumor markers do exist (ie chromograninA, synaptopyhsin, etc), sensitive and specific markers that accurately predict tumor growth and tumor behavior are still absent. In the present study, we assessed the expression of transcription factors (NeuroD and mASH1) essential for the normal fetal neuronal development in 33 gastroenteropancreatic neuroendocrine tumor patients (12 well-differentiated neuroendocrine tumors, 7 well-differentiated neuroendocrine carcinomas, and 14 poorly differentiated neuroendocrine carcinomas). NeuroD was less expressed in poorly differentiated neuroendocrine carcinoma (small-cell type) compared to well-differentiated neuroendocrine tumor (carcinoid) by reverse transcription–polymerase chain reaction. Immunohistochemical staining revealed that mASH1 was highly (sensitivity of 71%) and specifically (specificity of 95%) expressed in poorly differentiated neuroendocrine carcinoma. High NeuroD expression was seen in all well-differentiated neuroendocrine carcinoma and tumor (carcinoid) patients. Low NeuroD expression was seen in 36% (5 of 14) of poorly differentiated neuroendocrine carcinoma patients, which was associated with significant shorter overall survival. The expression pattern of these transcription factors may represent the biological and pathophysiological difference of gastroenteropancreatic neuroendocrine tumors and may become a new marker for the distinction of gastroenteropancreatic neuroendocrine tumors.

We address the following question: Determine the affine cones over smooth projective varieties which admit an action of a connected algebraic group different from the standard C*-action by scalar matrices and its inverse action. We show in particular that the affine cones over anticanonically embedded smooth del Pezzo surfaces of degree at least 4 possess such an action. A question by Flenner and the third author whether this is also true for cubic surfaces, occurs to be out of reach for our methods. Nevertheless, we provide a general geometric criterion that could be helpful also in this case.

An affine algebraic variety X is called cylindrical if it contains a principal Zariski dense open cylinder U ≃ Z × A1. A polarized projective variety (Y, H) is called cylindrical if it contains a cylinder U = Y \ supp D, where D is an effective Q-divisor on Y such that [D] ∈ Q+[H] in PicQ(Y ). We show that cylindricity of a polarized projective variety is equivalent to that of a certain Veronese affine cone over this variety. This gives a criterion of the existence of a unipotent group action on an affine cone.

In [KPZ11b] we showed that for any del Pezzo surface Y of degree d 4 and for any r 1, the affine cone X = cone r(-K Y ) (Y ) admits an effective G a -action.In particular, the group Aut(X) is infinite-dimensional.In this note we prove that for a del Pezzo surface Y of degree 2, the generalized cones X as above do not admit any nontrivial action of a unipotent affine algebraic group.

Acute ischemic stroke (AIS) is a serious cause of mortality and disability. AIS is a serious cause of mortality and disability. Early diagnosis of atherosclerosis, which is the major cause of AIS, allows therapeutic intervention before the onset, leading to prevention of AIS.Serological identification by cDNA expression cDNA libraries and the protein array method were used for the screening of antigens recognized by serum IgG antibodies in patients with atherosclerosis. Recombinant proteins or synthetic peptides derived from candidate antigens were used as antigens to compare serum IgG levels between healthy donors (HDs) and patients with atherosclerosis-related disease using the amplified luminescent proximity homogeneous assay-linked immunosorbent assay.The first screening using the protein array method identified death-inducer obliterator 1 (DIDO1), forkhead box J2 (FOXJ2), and cleavage and polyadenylation specificity factor (CPSF2) as the target antigens of serum IgG antibodies in patients with AIS. Then, we prepared various antigens including glutathione S-transferase-fused DIDO1 protein as well as peptides of the amino acids 297-311 of DIDO1, 426-440 of FOXJ2, and 607-621 of CPSF2 to examine serum antibody levels. Compared with HDs, a significant increase in antibody levels of the DIDO1 protein and peptide in patients with AIS, transient ischemic attack (TIA), and chronic kidney disease (CKD) but not in those with acute myocardial infarction and diabetes mellitus (DM). Serum anti-FOXJ2 antibody levels were elevated in most patients with atherosclerosis-related diseases, whereas serum anti-CPSF2 antibody levels were associated with AIS, TIA, and DM. Receiver operating characteristic curves showed that serum DIDO1 antibody levels were highly associated with CKD, and correlation analysis revealed that serum anti-FOXJ2 antibody levels were associated with hypertension. A prospective case-control study on ischemic stroke verified that the serum antibody levels of the DIDO1 protein and DIDO1, FOXJ2, and CPSF2 peptides showed significantly higher odds ratios with a risk of AIS in patients with the highest quartile than in those with the lowest quartile, indicating that these antibody markers are useful as risk factors for AIS.Serum antibody levels of DIDO1, FOXJ2, and CPSF2 are useful in predicting the onset of atherosclerosis-related AIS caused by kidney failure, hypertension, and DM, respectively.

Recruitment of polymorphonuclear leukocytes (PMN) through upregulation of cellular adhesion molecules is a proposed mechanism of injury in sepsis and acute respiratory distress syndrome (ARDS). We hypothesized that pretreatment of baboons with a monoclonal antibody to human E- and L-selectin (EL-246) during sepsis would decrease PMN influx into tissues and result in less organ injury during gram-negative sepsis. We studied 14 anesthetized, ventilated adult baboons; six animals received 1 mg/kg of EL-246 before infusion of an LD100 of live Escherichia coli and six received the E. coli infusion without antibody therapy. Two other animals received 1 mg/kg of EL-246 intravenously without an infusion of bacteria. Intermittent measurements were made of circulatory pressures, cardiac output, urine output, arterial blood gases, ventilation:perfusion ratio (VA/Q), and hematologic status. The experiments were ended at 48 h or at the time of death. Tissues were harvested for pathology and biochemical measurements. The E. coli infusions were associated with a hyperdynamic state, pulmonary hypertension, systemic hypotension, decreased urine output (UOP), and metabolic acidosis. The antibody partly blocked PMN migration, but there were few significant physiologic or biochemical differences between the EL-246-treated and untreated animals. In the antibody-treated animals, UOP was decreased, metabolic acidosis was worsened, and median survival time was decreased significantly. We conclude that treatment with an antibody to E- and L-selectin in gram-negative sepsis does not improve gas exchange or protect against lung injury, and is associated with decreased survival time in primates.

The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils.

We report a case of α-fetoprotein (AFP)–producing pulmonary carcinoma with studies for messenger RNA (mRNA) expression of hepatocyte nuclear factor (HNF)-4α, which is a transcription factor that is highly expressed in the process of liver development. The patient was a 64-year-old man with a pulmonary tumor in his left lower lobe. Serum AFP was 673 ng/mL. The microscopic analysis of the surgical specimen revealed a large cell neuroendocrine carcinoma with occasional hepatoid foci. The competitive reverse transcriptase polymerase chain reaction analysis revealed that HNF-4α mRNA was expressed on the order of 102- to 103-fold more abundantly than control pulmonary carcinomas and normal lung tissues. In addition, AFP and HNF-4α expression was restricted in hepatoid foci. Two previously reported cases of AFP-producing pulmonary carcinoma were also positive, whereas all 18 control pulmonary carcinomas were negative for HNF-4α. These findings suggest that aberrant expression of HNF-4α is implicated in the emergence or maintenance of hepatoid foci in AFP-producing pulmonary carcinomas.

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.

Adamantinoma-like Ewing family tumor (EFT) is a rare subset of EFTs showing mixed features of Ewing sarcoma and adamantinoma of the long bones. All currently reported cases of the adamantinoma-like type have been associated with bone. Recently, a unique type of EFT was reported showing complex epithelial differentiation associated with the vagus nerve. Here we describe another unique type of EFT arising in the soft tissue of the neck associated with the vagus nerve. An 11-year-old girl presented to our hospital with a neck tumor on her right side. Surgical resection was performed, and histopathologic examination demonstrated a high-grade malignant neoplasm. The tumor was composed of sheets of small round proliferating cells, basaloid tumor nests with marked squamous differentiation, biphasic growth pattern with epithelioid tumor nests, and spindle cell proliferation. Immunohistochemically, the tumor cells showed diffuse expression of CD99 and FLI-1. In addition, small round cells and basaloid/squamoid components were immunoreactive for AE1/AE3, CAM5.2, cytokeratin 5/6, high–molecular weight keratin, p63, and p40 (ΔNp63). Reverse transcription polymerase chain reaction and direct sequencing analysis revealed that the tumor harbored a t(11;22) translocation, involving EWSR1 and FLI-1, which are characteristic of EFTs. According to these findings, our case has characteristics of both a subset of adamantinoma-like EFT and EFT with complex epithelial differentiation. We suggest that EFT with complex epithelial differentiation is in a common spectrum with the adamantinoma-like type and that adamantinoma-like EFTs can arise in soft tissue, leading to difficulty in differential diagnosis with malignant epithelial tumors.

MRL/Mp-Fas (lpr) (MRL-lpr) mice develop a systemic autoimmune disease and are considered to be a good model for systemic lupus erythematosus in humans. We have recently shown that mice lacking B and T lymphocyte attenuator (BTLA), an inhibitory co-receptor expressed mainly on lymphocytes, on a 129SvEv background spontaneously develop lymphocytic infiltration in multiple organs and an autoimmune hepatitis (AIH)-like disease. In this study, we investigated the role of BTLA in the pathogenesis of autoimmune diseases in MRL-lpr mice. We found that BTLA-deficient (BTLA−/−) MRL-lpr/lpr mice developed severe lymphocytic infiltration in salivary glands, lungs, pancreas, kidneys and joints as compared with BTLA-sufficient (BTLA+/+) MRL-lpr/lpr mice. In addition, although AIH-like disease was not found in BTLA+/+ MRL-lpr/lpr mice, AIH-like disease was exacerbated in BTLA−/− MRL-lpr/lpr mice as compared with that in BTLA−/− 129SvEv mice. These results suggest that BTLA plays a protective role in autoimmune diseases in MRL-lpr mice and that AIH-like disease develops in BTLA−/− mice even in the absence of Fas-dependent signaling.

A major obstacle to enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) for Pompe disease is the development of high titers of anti-rhGAA antibodies in a subset of patients, which often leads to a loss of treatment efficacy. In an effort to induce sustained immune tolerance to rhGAA, we supplemented the rhGAA therapy with a weekly intravenous injection of synthetic vaccine particles carrying rapamycin (SVP-Rapa) during the first 3 weeks of a 12-week course of ERT in GAA-KO mice, and compared this with three intraperitoneal injections of methotrexate (MTX) per week for the first 3 weeks. Empty nanoparticles (NP) were used as negative control for SVP-Rapa. Co-administration of SVP-Rapa with rhGAA resulted in more durable inhibition of anti-rhGAA antibody responses, higher efficacy in glycogen clearance in skeletal muscles, and greater improvement of motor function than mice treated with empty NP or MTX. Body weight loss was observed during the MTX-treatment but not SVP-Rapa-treatment. Our data suggest that co-administration of SVP-Rapa may be an innovative and safe strategy to induce durable immune tolerance to rhGAA during the ERT in patients with Pompe disease, leading to improved clinical outcomes.

Hepatitis C virus (HCV) is frequently associated with various extrahepatic manifestations such as autoimmune features and immune complex deposit diseases. Oral lichen planus (OLP) is one of the representative extrahepatic manifestations of HCV infection. Direct-acting antivirals (DAA) are highly effective and safe for the eradication of HCV. However, there is a lack of information regarding the association between HCV-associated OLP and interferon (IFN)-free DAA therapy. Herein, we present the case of a 60-year-old female who was diagnosed with OLP during routine periodontal treatment by a dentist. The patient was referred for hepatitis C treatment using IFN-free DAA, which resulted in the improvement of the symptoms of OLP. This case represents the safety and efficacy of IFN-free DAAs in patients with HCV-associated OLP. However, long-term follow-up studies are required to elucidate the therapeutic effects of this therapy in these patients.

Lactobacilli are commensal bacteria colonizing the human oral cavity, gastrointestinal and gynecologic tracts. Lactobacilli, especially Lactobacillus paracasei, are widely used in probiotics, and are effective in the treatment of both vaginal candidiasis and diarrhea [ [1] Alvarez-Olmos M.I. Oberhelman R.A. Probiotic agents and infectious diseases: a modern perspective on a traditional therapy. Clin. Infect. Dis. 2001; 32: 1567-1576 Crossref PubMed Scopus (280) Google Scholar ]. On the other hand, rare cases of infective endocarditis due to L. paracasei have been reported [ [2] Franko B. Vaillant M. Recule C. Vautrin E. Brion J.P. Pavese P. Lactobacillus paracasei endocarditis in a consumer of probiotics. Med. Mal. Infect. 2013; 43: 171-173 Crossref PubMed Scopus (22) Google Scholar ]. Herein, we describe a case of L. paracasei endocarditis in a consumer of probiotics with severe bicuspid aortic valve stenosis and diffuse mid-layer fibrosis in the left ventricular (LV) myocardium on computed tomography (CT).

Abstract Some cancers are related to atherosclerotic diseases; therefore, these two types of disease may share some antibody biomarkers in common. To investigate this, a first screening of sera was performed from patients with esophageal squamous cell carcinoma (ESCC) or acute ischemic stroke (AIS) for serological identification of antigens using recombinant cDNA expression cloning (SEREX). The amplified luminescent proximity homogeneous assay‐linked immunosorbent assay (AlphaLISA) method, which incorporates glutathione donor beads and anti‐human IgG acceptor beads, was used to evaluate serum antibody levels. SEREX screening identified low‐density lipoprotein receptor–related protein–associated protein 1 (LRPAP1) as a target antigen of serum IgG antibodies in the sera of patients with ESCC or AIS. Antigens, including recombinant glutathione S‐transferase–fused LRPAP1 protein, were prepared to examine serum antibody levels. AlphaLISA revealed significantly higher antibody levels against the LRPAP1 protein in patients with solid cancers such as ESCC and colorectal carcinoma and some atherosclerosis‐related diseases such as AIS and diabetes mellitus compared with healthy donors. Correlation analysis revealed that the elevated serum antibody levels against LRPAP1 were associated with smoking, a well‐known risk factor for both cancer and atherosclerosis. Serum LRPAP1 antibody is therefore a common marker for the early diagnosis of some cancers and atherosclerotic diseases and may reflect diseases caused by habitual smoking.

We give constructions of completions of the affine 3-space into total spaces of del Pezzo fibrations of every degree other than 7 over the projective line. We show in particular that every del Pezzo surface other than $${\mathbb {P}}^{2}$$ blown-up in one or two points can appear as a closed fiber of a del Pezzo fibration $$\pi :X\rightarrow {\mathbb {P}}^{1}$$ whose total space X is a $${\mathbb {Q}}$$ -factorial threefold with terminal singularities which contains $${\mathbb {A}}^{3}$$ as the complement of the union of a closed fiber of $$\pi $$ and a prime divisor $$B_{h}$$ horizontal for $$\pi $$ . For such completions, we also give a complete description of integral curves that can appear as general fibers of the induced morphism $$\bar{\pi }:B_{h}\rightarrow {\mathbb {P}}^{1}$$ .

L-Selectin, previously known as LEC.CAM-1, LECAM-1, LAM1, and as the MEL-14, Leu-8, TQ1, and DREG-56 antigens, is a leukocyte membrane protein that participates in adhesion to endothelium. We studied its expression on eosinophils using flow cytometry and the MAb Dreg-56 and Leu-8. Unstimulated peripheral blood eosinophils from healthy adults expressed about one third the level of L-selectin as neutrophils (mean +/- SD specific fluorescence: 20.9 +/- 3.2 versus 54.5 +/- 8.4, p = 0.0001, n = 18). After stimulation with A23187, L-selectin expression on eosinophils was rapidly lost. This was temporally correlated with increased expression of Mac-1 (CD11b/CD18); the kinetics on eosinophils and neutrophils were similar. Eosinophil expression of L-selectin decreased modestly after stimulation with platelet activating factor, but was minimally affected by N-formyl-methionyl-leucyl-phenylalanine, leukotriene B4, or C5a compared with their effects on neutrophils. Eosinophils from cord blood of healthy neonates born at term expressed less L-selectin than adult eosinophils (10.4 +/- 3.8 versus 19.4 +/- 2.7, p = 0.0001, n = 9); the relative reduction was the same as on cord blood neutrophils (36.4 +/- 8.2 versus 55.5 +/- 4.8, p = 0.0001, n = 9). Relative to baseline expression, the responses of neonatal and adult cells to stimulation did not differ. We conclude that neonatal eosinophils have abnormalities in L-selectin expression similar to neonatal neutrophils and suggest that decreased expression of L-selectin and a diminished responsiveness to direct stimulation with chemotactic factors are possible mechanisms that may limit the exudation of eosinophils.

Abstract Purpose: Gastrointestinal neuroendocrine carcinoma (NEC) is extremely aggressive, but its pathophysiologic features remain poorly understood. There have been no biologically specific markers for this disease. In this study, distinctive up-regulation of human achaete-scute homologue 1 (hASH1) in gastrointestinal NECs was clarified. Experimental Design: Expression of hASH1 in NECs (n=10), carcinoid tumors (n = 10), other tumors (10adenocarcinomas, 2 squamous cell carcinomas and 1 malignant lymphoma), and the corresponding normal mucosa were investigated by in situ hybridization, reverse transcription-PCR (RT-PCR), real-time RT-PCR, and immunohistochemistry. Results: By in situ hybridization, mild to intense signals of hASH1 mRNA were detected in 9 of 10 NECs, but not in other tumors or normal mucosa, except for focally weak signals in one carcinoid tumor. RT-PCR showed strong expression of hASH1 in a small cell NEC, followed by a moderately differentiated NEC, and a carcinoid tumor, whereas it is undetectable in adenocarcinomas or normal mucosa. By real-time RT-PCR, the amounts of hASH1 mRNA in a small cell NEC were 16,600 times higher than those in adenocarcinomas and 110 times higher than those in a carcinoid tumor. Immunohistochemically, mammalian homologue of hASH1 was positive in 7 of 10 NECs but was negative in the other tumors. Pan-endocrine markers chromogranin A and synaptophysin were positive in almost all carcinoid tumors, in 4 and 7 of the 10 NECs, respectively. Conclusions: These findings revealed that hASH1 is distinctly up-regulated in gastrointestinal NECs. hASH1 may be used as a more sensitive and specific marker than conventional pan-endocrine markers for clinical diagnosis of gastrointestinal NECs.

Aim Hepatoid adenocarcinoma, a putative chemosensitive tumour, is defined as a tumour with aberrant hepatocellular differentiation occurring in extrahepatic organs such as the stomach, usually in the gastrointestinal tract. Differentiation in the hepatocellular direction is usually supported by the production of α-fetoprotein (AFP) and, more recently, albumin (ALB) mRNA. We investigated ALB mRNA to address whether adenocarcinoma with hepatoid morphology, regardless of AFP production, can be diagnosed solely by morphological criteria as a hepatoid adenocarcinoma. Methods We performed in situ hybridisation (ISH) and immunohistochemistry (IH) for ALB mRNA on AFP-negative gastric adenocarcinomas with hepatoid morphology. AFP- positive hepatoid adenocarcinomas and AFP-negative conventional gastric adenocarcinomas were also investigated as positive and negative controls, respectively. Results All three gastric adenocarcinomas with hepatoid morphology with no evidence of AFP production stained positive for ALB mRNA, thus providing evidence of differentiation in the hepatocellular direction. Three of five cases of AFP-positive hepatoid adenocarcinoma of the stomach were positive for ALB mRNA, while 11 cases of AFP-negative conventional gastric adenocarcinoma were negative. Conclusion The present study demonstrates that, irrespective of AFP production, gastric adenocarcinoma with morphological patterns suggestive of hepatoid differentiation should be diagnosed as hepatoid adenocarcinoma with important prognostic implications.

Aims Hepatocyte nuclear factor (HNF)-4α is a developmental regulator of the visceral endoderm, which is expressed in the embryonic gut and later in the adult intestine and colon. However, adult gastric mucosa does not express HNF-4α We investigated the possible involvement of HNF-4α in intestinal metaplasia and the intestinalisa- tion of gastric adenocarcinomas. Methods Thirty-five cases of adenocarcinomas and 46 cases of adjacent non-neoplastic mucosa with (22 lesions) or without (24 lesions) intestinal metaplasia were immunos- tained for HNF-4α The gastric or the intestinal phenotype was also examined using immunohistochemistry for MUC5AC, MUC2, CD10, and gastric-type mucin (GTM). Adenocarcinomas were classified into the gastric type (G- type, 42.9%), the mixed gastric and intestinal type (GI-type, 31.4%), and the intestinal type (I-type, 25.7%). Results The HNF-4α expression was exclusively seen in glandular cells with intestinal metaplasia, which was correlated with MUC2 expression (p<0.05) and inversely correlated with MUC5AC expression (p<0.05). All adenocarcinomas more or less expressed HNF-4α, with an intense expression being seen in the I-type (p<0.01) and in well-differentiated adenocarcinomas (p<0.03). Conclusions HNF-4α expression is associated with the intestinal phenotype of non-neoplastic and neoplastic gastric glandular cells, suggesting a possible involvement in the establishment and/or maintenance of the intestinal phenotype of the gastric mucosa and adenocarcinomas.

Gastrointestinal neuroendocrine carcinomas (NECs) are extremely aggressive and poorly prognostic. We showed previously that human achaete-scute homologue gene 1 (hASH1), a basic helix-loop-helix transcription factor regulated by Notch, was aberrantly expressed in NECs. To date, no effective therapeutic strategies for NECs have been investigated. Notch, Wnt and Hedgehog (Hh) signalings are important for stem cell self-renewal and carcinogenesis in the gastrointestinal epithelium. In this study, we showed that Hh signaling was clearly upregulated in NECs in Gli1-dependent manner. Specific therapeutic effects of cyclopamine on NECs were also demonstrated. RT-PCR showed that among eight frozen samples (three NECs, one carcinoid tumor, three adenocarcinomas and one normal mucosa), the band intensities of Gli1 were the strongest in NECs, moderately strong in a carcinoid tumor, very weak in adenocarcinomas and undetectable in a normal mucosa. In real-time RT-PCR, the expression levels of Gli1 in NECs were 108.4, 28.6 and 16.3 times higher than that in an adenocarcinoma. In immunohistochemistry using 25 paraffin-embedded tissues, all twelve NECs and three of six carcinoid tumors showed positive stainings for Gli1, whereas six of seven adenocarcinomas were negative. In vitro, RT-PCR showed that NEC cell lines expressed Gli1 mRNA significantly. Administration of cyclopamine suppressed cell proliferation and invasion, and induced apoptosis in NECs. In cyclopamine-treated NECs, downregulation of Gli1, Ptch1, Snail and hASH1, and upregulation of E-cadherin were demonstrated at mRNA levels. Such effects were not observed in a Gli1-negative colonic adenocarcinoma cell line or in control alkaloid-treated NECs. Hh signaling may play a crucial role in the pathophysiology of NECs. Blockade of Hh pathway using cyclopamine or its synthetic derivatives might open an effective therapeutic strategy to NECs, not only by suppressing tumor viability but also by altering tumor cell nature.

Insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3) is a valuable marker that distinguishes malignant from benign lesions and predicts prognosis. First, we evaluated IMP3 expression in 77 resected specimens of pancreatic ductal adenocarcinoma (PDAC), intraductal papillary mucinous neoplasm (IPMN), and chronic pancreatitis (CP). Eleven PDAC patients preoperatively underwent endoscopic ultrasound-guided fine needle aspiration (EUS-FNA). Survival analysis of IMP3 and clinicopathological factors was performed. IMP3 and p53 expression was evaluated in another 127 EUS-FNA samples of solid pancreatic masses to compare the diagnostic value of routine and immunohistochemical staining. IMP3 expression was detected in 72.3%, 50%, 20%, and 0% of PDAC, malignant IPMN, benign IPMN, and CP, respectively. Evaluation of IMP3 expression in EUS-FNA specimens coincided with that in resected specimens in 10 of 11. IMP3 expression correlated with tumor differentiation in PDAC samples (p = .006) and with poor prognosis through univariate analysis (p = .045). Tumor differentiation and lymph node metastasis were significantly associated with poor prognosis through multivariate analysis. In EUS-FNA specimens, the sensitivity, specificity, and accuracy of cytohistological analysis were 80.8%, 100%, and 85.0%, respectively. IMP3 and p53 expression were detected in 80.8% and 44.9% of malignant and 0% and 5% of benign lesions. Combined with IMP3 immunostaining, the sensitivity, specificity and accuracy of cytohistological analysis significantly increased to 87.9%, 100%, and 90.8% (p = .016), respectively. Meanwhile, p53 staining had no impact on the results. IMP3 immunohistochemical staining can improve the diagnostic accuracy of EUS-FNA for malignant pancreatic tumors.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1–2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13–14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses.

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human <i>OTC</i> cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTC^Spf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic.

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4+ and CD8+ T cells, and the emergence of a large population of CD4-CD8- (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment.

Abstract Low dose IL-2 therapy and IL-2 molecules engineered to be selective for the high affinity IL-2 receptor have been shown to expand Tregs in vivo, and, in the case of low dose IL-2 therapy, has demonstrated promising therapeutic benefit in autoimmune diseases. One of the potential limitations of IL-2 therapy is the nonselective expansion of pre-existing Treg populations rather than induction of antigen-specific Tregs, as well as potential activation of effector cells. We have recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, to induce selective immune tolerance to co-administered antigens, such as immunogenic biologic drugs. Unlike Treg-selective IL-2 therapy, ImmTOR alone does not increase total Treg numbers. However, here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Treg when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. ImmTOR also showed the potential to increase the therapeutic window of engineered IL-2 molecules by mitigating effector T cell expansion typically observed at higher doses of IL-2 and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, engineered IL-2 molecules showed potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases.

Hepatic fibrosis has recently been evaluated using ultrasonography or magnetic resonance elastography. Although the shear wave velocity (SWV) obtained using point shear wave elastography (pSWE) provides a valuable measure of fibrosis, underlying steatosis may affect its measurement.Using hepatic fibrosis samples, this study evaluated the effect of steatosis on the shear wave velocity of pSWE (Vs) and viscoelastic properties (assessed by dynamic mechanical analysis) of rat liver. Fifty rats with various grades of steatosis and fibrosis underwent open abdominal in vivo Vs measurements using a commercial ultrasound scanner. The mechanical properties of hepatic tissue were also characterized under ex vivo conditions using dynamic mechanical analysis and the Zener model of viscoelasticity.Fibrosis and steatosis progression influenced Vs and elasticity. The SWV computed using the Zener model and Vs showed a substantial correlation (r > 0.8). Fibrosis progression increased SWV. Steatosis was also related to SWV. Steatosis progression obscured the SWV change associated with fibrosis progression.We conclude that steatosis progression affects the evaluation of fibrosis progression. This finding could aid discrimination of non-alcoholic steatohepatitis from non-alcoholic fatty liver disease using SWV.