Mena Farag

The interaction of multiple myeloma (MM) cells with their microenvironment in the bone marrow (BM) provides a protective environment and resistance to therapeutic agents. We hypothesized that disruption of the interaction of MM cells with their BM milieu would lead to their sensitization to therapeutic agents such as bortezomib, melphalan, doxorubicin, and dexamethasone. We report that the CXCR4 inhibitor AMD3100 induces disruption of the interaction of MM cells with the BM reflected by mobilization of MM cells into the circulation in vivo, with kinetics that differed from that of hematopoietic stem cells. AMD3100 enhanced sensitivity of MM cell to multiple therapeutic agents in vitro by disrupting adhesion of MM cells to bone marrow stromal cells (BMSCs). Moreover, AMD3100 increased mobilization of MM cells to the circulation in vivo, increased the ratio of apoptotic circulating MM cells, and enhanced the tumor reduction induced by bortezomib. Mechanistically, AMD3100 significantly inhibited Akt phosphorylation and enhanced poly(ADP-ribose) polymerase (PARP) cleavage as a result of bortezomib, in the presence of BMSCs in coculture. These experiments provide a proof of concept for the use of agents that disrupt interaction with the microenvironment for enhancement of efficacy of cytotoxic agents in cancer therapy.

Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoplasmacytic lymphoma. We demonstrate up-regulated Akt activity in WM, and that Akt down-regulation by Akt knockdown and the inhibitor perifosine leads to significant inhibition of proliferation and induction of apoptosis in WM cells in vitro, but not in normal donor peripheral blood and hematopoietic progenitors. Importantly, down-regulation of Akt induced cytotoxicity of WM cells in the bone marrow microenvironment (BMM) context. Perifosine induced significant reduction in WM tumor growth in vivo in a subcutaneous xenograft model through inhibition of Akt phosphorylation and downstream targets. We also demonstrated that Akt pathway down-regulation inhibited migration and adhesion in vitro and homing of WM tumor cells to the BMM in vivo. Proteomic analysis identified other signaling pathways modulated by perifosine, such as activation of ERK MAPK pathway, which induces survival of tumor cells. Interestingly, MEK inhibitor significantly enhanced perifosine-induced cytotoxicity in WM cells. Using Akt knockdown experiments and specific Akt and PI3K inhibitors, we demonstrated that ERK activation is through a direct effect, rather than feedback activation, of perifosine upstream ERK pathway. These results provide understanding of biological effects of Akt pathway in WM and provide the framework for clinical evaluation of perifosine in WM patients.

Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow at the time of diagnosis, implying continuous homing of WM cells into the marrow. The mechanisms by which trafficking of the malignant cells into the bone marrow has not been previously elucidated. In this study, we show that WM cells express high levels of chemokine and adhesion receptors, including CXCR4 and VLA-4. We showed that CXCR4 was essential for the migration and trans-endothelial migration of WM cells under static and dynamic shear flow conditions, with significant inhibition of migration using CXCR4 knockdown or the CXCR4 inhibitor AMD3100. Similarly, CXCR4 or VLA-4 inhibition led to significant inhibition of adhesion to fibronectin, stromal cells, and endothelial cells. Decreased adhesion of WM cells to stromal cells by AMD3100 led to increased sensitivity of these cells to cytotoxicity by bortezomib. To further investigate the mechanisms of CXCR4-dependent adhesion, we showed that CXCR4 and VLA-4 directly interact in response to SDF-1, we further investigated downstream signaling pathways regulating migration and adhesion in WM. Together, these studies demonstrate that the CXCR4/SDF-1 axis interacts with VLA-4 in regulating migration and adhesion of WM cells in the bone marrow microenvironment.

The nuclear factor-kappaB (NF-kappaB) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-kappaB pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-kappaB activity. We demonstrated that perifosine and bortezomib both targeted NF-kappaB through its recruitment to the promoter of its target gene IkappaB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-kappaB pathway.

Waldenström macroglobulinemia (WM) is an incurable low-grade B-cell lymphoma characterized by high protein turnover. We dissected the biologic role of the proteasome in WM using 2 proteasome inhibitors, NPI-0052 and bortezomib. We found that NPI-0052 inhibited proliferation and induced apoptosis in WM cells, and that the combination of NPI-0052 and bortezomib induced synergistic cytotoxicity in WM cells, leading to inhibition of nuclear translocation of p65NF-kappaB and synergistic induction of caspases-3, -8, and -9 and PARP cleavage. These 2 agents inhibited the canonical and noncanonical NF-kappaB pathways and acted synergistically through their differential effect on Akt activity and on chymotrypsin-like, caspaselike, and trypsinlike activities of the proteasome. We demonstrated that NPI-0052-induced cytotoxicity was completely abrogated in an Akt knockdown cell line, indicating that its major activity is mediated through the Akt pathway. Moreover, we demonstrated that NPI-0052 and bortezomib inhibited migration and adhesion in vitro and homing of WM cells in vivo, and overcame resistance induced by mesenchymal cells or by the addition of interleukin-6 in a coculture in vitro system. Theses studies enhance our understanding of the biologic role of the proteasome pathway in WM, and provide the preclinical basis for clinical trials of combinations of proteasome inhibitors in WM.

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Pharmacological treatments for Tourette syndrome (TS) vary in efficacy between different patients. The evidence base is limited as even high quality controlled studies tend to be of relatively short duration which may lose relevance in clinical usage. Patients are frequently treated with serial agents in the search for efficacy and tolerability. The success of this strategy has not been previously documented. We examined 400 consecutive TS patients seen over a 10-year period, some with a longer prior history in other clinics; 255/400 (64%) were prescribed medication. We present this heterogeneous cohort in terms of the number of drugs they had tried, and as a proxy measure of some benefit of the last drug used, whether it had been prescribed under our supervision for ≥ 5 months. The most commonly prescribed medications were aripiprazole (64%), clonidine (40%), risperidone (30%) and sulpiride (29%) with changes in prescribing practises over the period examined. The number of different drugs tried were one (n = 155), two (n = 69), three (n = 36), four (n = 14), five (n = 15), six (n = 5), seven (n = 2) and eight (n = 1). The data illustrate the difficulty in drug treatment of tics and suggest that even after trials of several agents there is potential benefit in trying further options.

Abstract Waldenstrom macroglobulinemia (WM) is an incurable low-grade lymphoma characterized by bone marrow (BM) involvement of IgM secreting lymphoplasmacytic cells. The induction of unfolded protein response (UPR) genes (“physiologic” UPR) enables cells to differentiate into professional secretory cells capable of production of high amounts of endoplasmic reticulum (ER)–processed proteins, such as immunoglobulins. Ultimately, the initially cytoprotective UPR triggers an apoptotic cascade if ER stress is not corrected, called proapoptotic/terminal UPR. We show that WM cells inherently express the physiologic UPR machinery compared with normal BM cells, and that increased ER stress leads to proapoptotic/terminal UPR in WM cells. We therefore examined tunicamycin, ER stress inducer, for potential antitumor effects in WM. Tunicamycin induced significant cytotoxicity, apoptosis and cell-cycle arrest, and inhibited DNA synthesis in WM cell lines and primary BM CD19+ cells from patients with WM with an inhibitory concentration (IC50) of 0.5 μg/mL to 1 μg/mL, but not in healthy donor cells. Importantly, coculture of WM cells in the context of the BM microenvironment did not inhibit tunicamycin-induced cytotoxicity. Finally, we demonstrate that ER stress inducer synergizes with other agents used in the treatment of WM. These preclinical studies provide a framework for further evaluation of ER stress inducing agents as therapeutic agents in WM.

Indomethacin-responsive headaches encompass a group of disorders which include a subset of the trigeminal autonomic cephalalgias and other paroxysmal, often precipitated primary headaches. Many patients show a rapid therapeutic response to indomethacin, which is limited by intolerability. Etoricoxib and celecoxib, selective inhibitors of cyclo-oxygenase-2 (COX-2), spare gastroduodenal COX-1 activity and are less likely to cause gastrointestinal adverse effects than indomethacin. We report a case series of eight patients, seven who responded to etoricoxib and one patient who responded to celecoxib.

Abstract Purpose: Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia. Experimental Design: We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia. Results: We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G1 cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells. Conclusions: Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)

An investigation into long-term cognitive impairment and Quality of Life (QoL) after severe burns. A proof of principle, cohort design, prospective, observational clinical study. Patients with severe burns (>15% TBSA) admitted to Burns ICU for invasive ventilation were recruited for psychocognitive assessment with a convenience sample of age and sex-matched controls. Participants completed psychological and QoL questionnaires, the Cogstate® electronic battery, Hopkins Verbal Learning, Verbal Fluency and Trail making tasks. 15 patients (11M, 4F; 41 ± 14 years; TBSA 38.4% ± 18.5) and comparators (11M, 4F; 40 ± 13 years) were recruited. Burns patients reported worse QoL (Neuro-QoL Short Form v2, patient 30.1 ± 8.2, control 38.7 ± 3.2, p = 0.0004) and cognitive function (patient composite z-score 0.01, IQR −0.11 to 0.33, control 0.13, IQR 0.47–0.73, p = 0.02). Compared to estimated premorbid FSIQ, patients dropped an equivalent of 8 IQ points (p = 0.002). Cognitive function negatively correlated with burn severity (rBaux score, p = 0.04). QoL strongly correlated with depressive symptoms (Rho = −0.67, p = 0.009) but not cognitive function. Severe burns injuries are associated with a significant, global, cognitive deficit. Patients also report worse QoL, depression and post-traumatic stress. Perceived QoL from cognitive impairment was more closely associated with depression than cognitive impairment.

We report on two patients with secondary cough headache who responded to the cyclo-oxygenase-2 (COX-2) inhibitor etoricoxib and showed an independent temporal course. This case report shows that secondary cough headache can also respond to medical treatment and can respond to a COX-2 inhibitor, not previously reported. As is seen in primary cough headache, the headache disorder can go into natural remission (case 1) while the secondary pathology progresses and conversely, persist once the secondary pathology has resolved (case 2). The course of the headache and that of the secondary pathology do not necessarily correlate. It is, therefore, proposed that any treatment of the secondary pathology is independent to that of the headache. In NSAID-intolerant cases a COX-2 inhibitor can be trialled first line.

Abstract Assisted reproductive technologies (ART) account for 1–6% of births in developed countries. While most children conceived are healthy, increases in birth and genomic imprinting defects have been reported; such abnormal outcomes have been attributed to underlying parental infertility and/or the ART used. Here, we assessed whether paternal genetic and lifestyle factors, that are associated with male infertility and affect the sperm epigenome, can influence ART outcomes. We examined how paternal factors, haploinsufficiency for Dnmt3L, an important co-factor for DNA methylation reactions, and/or diet-induced obesity, in combination with ART (superovulation, in vitro fertilization, embryo culture and embryo transfer), could adversely influence embryo development and DNA methylation patterning in mice. While male mice fed high-fat diets (HFD) gained weight and showed perturbed metabolic health, their sperm DNA methylation was minimally affected by the diet. In contrast, Dnmt3L haploinsufficiency induced a marked loss of DNA methylation in sperm; notably, regions affected were associated with neurodevelopmental pathways and enriched in young retrotransposons, sequences that can have functional consequences in the next generation. Following ART, placental imprinted gene methylation and growth parameters were impacted by one or both paternal factors. For embryos conceived by natural conception, abnormality rates were similar for WT and Dnmt3L+/− fathers. In contrast, paternal Dnmt3L+/− genotype, as compared to WT fathers, resulted in a 3-fold increase in the incidence of morphological abnormalities in embryos generated by ART. Together, the results indicate that embryonic morphological and epigenetic defects associated with ART may be exacerbated in offspring conceived by fathers with sperm epimutations.

Epidemiological measures of the prevalence of fetal alcohol spectrum disorders (FASD) vary greatly in the literature. Irrespective of the methodology, the criteria to define a 'case' are set by the researchers. Hence, estimates of the prevalence of FASD primarily depend on the diagnostic criteria currently available. The problem lies therein - the aforementioned criteria are ill-defined.A critical analysis of the diagnostic criteria from the Institute of Medicine, Hoyme, 4-Digit Diagnostic Code and Canadian guidelines was performed, with particular attention focused on the inconsistencies in specificities of the fetal alcohol syndrome (FAS) facial phenotype.To date, the Canadian guidelines represent the only guidelines that have pushed for a uniform diagnostic capacity through harmonizing the IoM and 4-Digit Diagnostic Code criteria. In the absence of a reliable biochemical marker of effect to confirm maternal drinking during pregnancy, the importance and dependence on diagnostic guidelines for FASD is understated. With the availability of four published guidelines for diagnoses across the spectrum of FASD, there is a need to reach a set standard globally. There are profound implications of relaxed and strict diagnostic approaches on FAS prevalence reporting in the literature.This review exposes the clinical burden of diagnosing the range of FASD with disputing diagnostic criteria. Discrepancies in the criteria pose a danger to the validity of FASD diagnoses with respect to inaccurate estimates of incidence and prevalence. In turn, these discrepancies risk compromising the future healthcare of affected individuals with regards to intervention, counselling and treatment.

This is the first study to ascertain the value of multidisciplinary team (MDT) meetings within an early pregnancy assessment unit (EPAU). Our national telephone survey identified that in the United Kingdom, overall 37% of EPAU utilise regular MDT meetings. Secondary and tertiary hospitals are just as likely to hold regular MDT meetings. The participants in our interview study expressed the principal benefits of regular MDT meetings as communication, education and effective stress management. The perceived additional benefits included improved care quality, better patient experience and enhanced team cohesion. During the meetings, at least, one representative from every tier of staffing was present. The caseload of the MDT meeting comprised ectopic pregnancies and pregnancies of unknown location. We propose a number of research studies, which would build on this study. Such efforts will help enhance the effectiveness of the MDT-based EPAU service.

This study compared the past-year prevalence rates of nonsuicidal self-injury produced by a behavioral checklist assessment and a single-item measure. In order to extend previous research on differences in prevalence rates across research samples, we employed both assessment types within a single sample of young adults (N = 433). Moreover, as an indicator of convergent validity, those rates were analyzed in association with participants' depression and anxiety scores. Findings indicated that the checklist assessment, relative to the single-item measure, produced a substantially higher prevalence rate, thereby providing greater evidence for the validity of its outcomes on the basis of convergence.

Within the past few years, major advances in the preclinical and clinical testing of novel therapeutic agents have occurred in Waldenström's macroglobulinemia (WM). These include agents that target the PI3K/Akt/mTOR pathway, PKC pathways, NF-kB signaling pathway, as well as tyrosine kinases and histone deacetylase inhibitors. In this review, we summarize the current understanding of the clinical development of these agents in WM.

We report a patient who has peripheral demyelination in the form of chronic inflammatory demyelinating polyneuropathy (CIDP) with central demyelination following a relapsing-remitting disease course. The patient developed bilateral sequential optic neuritis predating the diagnosis of CIPD, then developed a profound brainstem syndrome with ataxia, dysarthria, a complex eye movement disorder, visual disturbance and urinary incontinence. Interval imaging fulfilled McDonald criteria for multiple sclerosis (MS) with a right parieto-occipital tumefactive lesion showing contrast enhancement and new lesions in the right temporal white matter and midbrain tegmentum. Oligoclonal bands (OCBs) were matched and serum antibodies against aquaporin-4 (AQP-4) and myelin oligodendrocyte glycoprotein (MOG) were negative. Genetic sequence analysis and deletion/duplication testing revealed variants of uncertain significance with compound heterozygosity for point mutations in two genes, DYNC1H1 and SH3TC2, which are associated with Charcot-Marie-Tooth (CMT) disease though the patient was negative for known CMT mutations. The patient responded poorly to steroids and regular intravenous immunoglobulin (IVIg) but clinically improved following aggressive immunomodulatory therapy with pulsed steroids and plasmapheresis, followed by Rituximab. Combined central and peripheral demyelination (CCPD) is rare. Autoimmune mechanisms are postulated in the pathogenesis. Whether overlap of central and pe- ripheral demyelination is coincidental or caused by a shared epitope in both the peripheral and central nervous systems still remains to be elucidated. There is no clear therapeutic consensus in the treatment of both central and peripheral demyelination, though immunomodulating treatment strategies may minimise disability and improve prognosis.

Advance care planning (ACP) is a useful tool that benefits adult patients, care providers, and surrogate decision makers, through providing opportunities for patients to consider, express, and formalize their beliefs, preferences, and wishes pertaining to decisions regarding future medical care at a time when they retain decision-making capacity. Early and timely consideration of ACP discussions is paramount in Huntington’s disease (HD) given the potential challenges in ascertaining decision-making capacity in the advanced stages of the disease. ACP helps to empower and extend patient autonomy, providing clinicians and surrogate decision makers with reassurance that management is consistent with a patient’s expressed wishes. Regular follow up is vital to establish consistency of decisions and wishes. We outline the framework of the dedicated ACP clinic integrated within our HD service to highlight the importance of a patient-centred and tailored care plan that fulfils the patient’s expressed goals, preferences, and values.

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

<div>Abstract<p><b>Purpose:</b> Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.</p><p><b>Experimental Design:</b> We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.</p><p><b>Results:</b> We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G<sub>1</sub> cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.</p><p><b>Conclusions:</b> Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)</p></div>

<div>Abstract<p><b>Purpose:</b> Waldenstrom macroglobulinemia is a lymphoplasmacytic lymphoma characterized by widespread involvement of the bone marrow. Despite different options of therapy, Waldenstrom macroglobulinemia is still incurable. Src tyrosine kinase has been shown to play a central role in the regulation of a variety of biological processes, such as cell proliferation, migration, adhesion, and survival in solid tumors. We sought to determine whether the protein tyrosine kinase Src regulates adhesion, migration, and survival in Waldenstrom macroglobulinemia.</p><p><b>Experimental Design:</b> We tested the expression of Src tyrosine kinase in Waldenstrom macroglobulinemia and normal cells, and the effect of the specific Src inhibitor AZD0530 on the adhesion, migration, cell cycle, and survival of a Waldenstrom macroglobulinemia cell line and patient samples. Moreover, we tested the effect of AZD0530 on cytoskeletal and cell cycle signaling in Waldenstrom macroglobulinemia.</p><p><b>Results:</b> We show that Src is overexpressed in Waldenstrom macroglobulinemia cells compared with control B cells, and that the use of the Src inhibitor AZD0530 led to significant inhibition of adhesion, migration, and cytoskeletal signaling induced by SDF1. Moreover, inhibition of Src activity induced G<sub>1</sub> cell cycle arrest; however, it had minimal effect on survival of Waldenstrom macroglobulinemia cells, and no significant effect on survival of normal cells.</p><p><b>Conclusions:</b> Taken together, these results delineate the role of Src kinase activity in Waldenstrom macroglobulinemia and provide the framework for future clinical trials using Src inhibitors in combination with other drugs to improve the outcome of patients with Waldenstrom macroglobulinemia. (Clin Cancer Res 2009;15(19):6035–41)</p></div>

Supplementary Data from Src Tyrosine Kinase Regulates Adhesion and Chemotaxis in Waldenstrom Macroglobulinemia

Abstract Introduction Hyposexuality is a well-defined and prevalent side effect of individuals prescribed SSRI medication. However, hypersexuality has never been found in patients taking SSRI and antitypical antidepressant medications. We present two new case reports to the scanty literature found up to now in this area. This condition needs to be differentiated from the hyperactive arousal disorder already described in the sexological literature. This demands further study. With several documented reports of these side effects, failure to examine and develop therapy options for our patients would be a disservice. Objective The objective of this care report is to educate both patients and medical providers of this side effect as well as further investigation to include the pharmacological mechanism involved. Methods Two patients presented to the office complaining of hypersexuality. These patients were then followed for four months before completing analysis. Results Of the two patients presenting on the drug regimen specified, both experienced hypersexuality. Conclusions This case study illustrates that the use of Bupropion and Fluoxetine/Sertraline concomitantly can cause hypersexuality in perimenopausal women. This is an area that needs to be studied more to conclude the mechanism of action. Disclosure No

Abstract Promising blood-based biomarkers of neuropathology have emerged with potential for therapeutic development and disease monitoring. However, these tools will require specialist tertiary services for integration into clinical management. Remote sampling for biomarker assessment could ease the burden of in-person clinical visits for such tests and allow for frequent sampling. Here we evaluated a capillary finger-prick collection for remote quantification of blood neurofilament light (NfL), a common blood-based biomarker evident in various neurological disorders, and other exploratory markers of neuronal injury and neuroinflammation (GFAP, tau). Matched samples from venepuncture and finger-prick were collected and processed into plasma and/or serum to directly compare NfL levels across four different neurological conditions (HD, MS, ALS, PD). Two delayed processing conditions were compared, three- and seven-day delay, simulating ambient shipment. Capillary NfL and GFAP concentrations were equivalent to those in venous blood serum and plasma. Only NfL remained stable after seven-day processing delay. Capillary NfL replicated disease group differences displayed in venous blood. This data supports our finger-prick method for remote collection and quantification of NfL. With the widespread applications for NfL across the spectrum of neurological disorders, this has the potential to transform disease monitoring, prognosis, and therapeutic development within clinical practice and research. Graphical abstract: Figure 1

In a recent study, Wilton and colleagues link activation of the classical complement pathway with corticostriatal synapse loss and cognitive decline in Huntington's disease.1.

Burn injuries are a debilitating cause of morbidity and mortality associated with the long-term impact of psychological factors on quality of life. Accurate assessment of the differential impact of burn sequelae and anxiety is often complicated by the overlap between psychological and somatic symptoms in burns patients. The Beck Anxiety Inventory (BAI) is one validated psychometric tool for anxiety assessment. The primary objective of this study is to investigate whether utilising the BAI as a tool to assess for anxiety in burns patients is biased due to the confounding of symptoms of anxiety with the physical sequelae of a burn injury.This is a single-centre, prospective, cross-sectional study. The study was conducted in accordance with the UK Good Clinical Practice guidelines (CAPP reference number 506). Patients were recruited over a three-month period from November 2016 to February 2017 and were offered a modified BAI questionnaire to complete. Patients were asked to indicate to what degree they attributed each symptom to their physical injury or their psychological state on a visual analogue scale (VAS).50 patients, comprising 33 females (66%) and 17 males (34%), participated in the study with a median age of 33.5 years (range: 20-88). Date of injury spanned May 1991 to January 2017. Percentage of the total body surface area (% TBSA) affected by burn ranged from 1 to 86%. Patients attributed eight of the 21 self-report items within the BAI as being more physical than psychological in origin. The results reveal a statistical significant difference in patient VAS scores between physical (mean: 34.16, 95% CI: 29.04-39.28) and psychological (mean: 61.2, 95% CI: 56.33-66.17) BAI items, with p<0.0001. In addition, patients with a facial burn injury were more likely to report 'face flushed' (Mann-Whitney U Test, Z=-2.11, p<0.05) and patients with a hand burn injury were more likely to report 'hands trembling' (Mann-Whitney U Test, Z=-2.52, p<0.05).This feasibility study found preliminary evidence suggesting that the BAI may, in part, represent misattributed symptoms of cutaneous injury from burns. However, whilst our findings suggest an attribution bias, there is not enough evidence from this data to comment on whether its use should be restricted in burns patients. Further research is needed to formally quantify convergent and divergent validity through structured interviews. In addition, further research using other self-report tools of anxiety in burns patients would be useful to corroborate the prospect of biased and confounded anxiety scores.

Three south-London hospital trusts undertook a feasibility study, comparing data from 93 patients who received the 14-day adhesive ambulatory electrocardiography (ECG) patch Zio XT with retrospective data from 125 patients referred for 24-hour Holter for cryptogenic stroke and transient ischaemic attack following negative 12-lead ECG. As the ECG patch was fitted the same day as the clinical decision for ambulatory ECG monitoring was made, median time to the patient having the monitor fitted was significantly reduced in all three hospital trusts compared with 24-hour Holter being ordered and fitted. Hospital visits reduced by a median of two for patients receiving Zio XT. This project supports that it is feasible to use a patch as part of routine clinical care with a positive impact on care pathways.

Abstract Background. WM is characterized by excess secretion of IgM in the serum. Response to therapy is assessed according to the Consensus panel recommendations with the use of IgM by serum protein electrophoresis (M spike). However, there are many limitations to the use of M-spike level, and new markers are needed. We, and others, have recently demonstrated that the measurement of involved sFLC values accurately identified patients with poor prognostic features of WM. We sought to determine whether involved sFLC level can be used as a reliable and sensitive marker to study response to therapy in WM. Methods. We prospectively studied M-spike and involved sFLC levels in 32 patients with relapse/refractory WM, treated on two phase II clinical trials, single agent perifosine (N=21; given 150mg oral daily for 28 days), or bortezomib-rituximab (N=13; given IV bortezomib 1.6mg/m2 at days 1, 8, 15 q28 days x 6 cycles and rituximab 375mg/m2 at days 1, 8, 15, 22 on cycles 1 and 4). Levels of sFLC and M spike were measured on day 1 of each cycle. In addition, sFLCs were measured weekly for the first month in the bortezomib-rituximab study (N=12). Responses were assessed after 2 cycles of therapy. Time to response was determined from initiation of therapy to the time of initial response. Results. Characteristics in the overall population were not different between the 2 groups treated, and median values were: beta-2 microglobulin (B2M) 2.7mg/L, hemoglobin level 11g/dL, serum IgM 40g/L, M-spike 2.4g/dL, and involved sFLC 111mg/L. The median follow-up was 5 months, and was not significantly different between the 2 trials. The overall response rate (ORR) of the bortezomib/rituximab clinical trial was 85% and the perifosine clinical trial was 33%. The ORR as calculated by the sFLC in the bortezomib-rituximab trial was 100% and in the perifosine trial was 39%. Eleven (34%) patients achieved better responses by sFLC compared to M spike. Of these, 7 were classified as minor response (MR) by M spike but achieved a partial response by sFLC, and 4 were classified as stable disease by M-spike, but achieved MR using involved sFLC. Only 2 patients (6%) had a better response using M spike compared to sFLC. The overall median time to response was 79 and 110 days using M-spike, and 68 and 41 days using sFLC, in bortezomib-rituximab and perifosine trials, respectively. We next found that neither M-spike nor involved sFLC measured prior to therapy could predict response. We then investigated whether sFLC could predict response to therapy using weekly sFLC for the first month. Among the 14 patients studied, 7 showed a decrease in sFLC within the first month, which predicted response by using M spike. On the other hand, the other 7 patients had an sFLC “flare” within the first 4 weeks of therapy, and these patients had a delayed response to therapy using M spike criteria. Conclusion. Involved sFLC is a sensitive marker for monitoring response to therapy in patients with WM. Using sFLC measurement, we showed a higher response rate in patients treated on 2 prospective clinical trials than using M-spike measurement. More importantly, we demonstrated that responses occurred earlier using sFLC compared to M-spike measurement. sFLC measurement could predict response to therapy within the first month of therapy.

Neuropsychiatric symptoms are not uncommon in patients with autoimmune diseases, including inflammatory bowel disease (IBD).1 The link between the two is bidirectional with multiple and additive causes. These include the impact of chronic illness on well-being2 and the effects of medication used to treat IBD.3 We report the neuropsychiatric presentation of a 34-year-old man to the emergency department (ED). His medical history included Crohn disease, in remission for 6 years while taking azathioprine, mesalazine, and the biologic adalimumab. Two months before presenting, he switched from reference adalimumab (Humira) to a biosimilar, Imraldi, with his last dose 17 days before presentation. Two weeks before admission he developed a prodromal illness with lethargy, malaise, and a self-limiting widespread maculopapular rash. He experienced an intense holocranial headache with photosensitivity and vomiting, an unusual taste in his mouth, seeing “multicolored dots on the wall,” and excoriation without pruritis. He described low mood, restless sleep, early awakening, and poor concentration. Preoccupied with losing his family, he experienced emotional lability and panic attacks. Transcripts from calls to a crisis line revealed a perplexed, highly anxious state. He attended his local ED and was discharged on promethazine. That day, he experienced another panic attack and, overwhelmed, took an eight-inch kitchen knife and stabbed himself three times in the abdomen with the intent to die. He expressed regret and denied passivity of control and premeditation, describing derealization, depersonalization, and absence of pain sensation. During his admission, organic causes of psychosis were excluded and he was transferred to a psychiatric facility. He self-discharged the following day with community follow-up and promethazine and zopiclone. One day later, severe anxiety reoccurred, culminating in an episode where he stabbed himself in the chest and neck, cut both wrists, and slid his finger inside the wound “to see where my heart is.” He was admitted to a critical care unit. No psychotic symptoms or thoughts of self-harm were elicited. His flat affect and inability to explain events was striking, despite being orientated and having insight into the events of the preceding weeks. Investigations were unremarkable (Table 1). 3.7 mmol/L (serum 6.0 mmol/L) 3.1 mmol/L (serum 4.6 mmol/L) This was considered an acute stress reaction possibly related to adalimumab switch. Empirical treatment with plasmapheresis was not recommended and adalimumab was withheld. Steroids were avoided in the subsequent Crohn flare given the risk of psychiatric side effects. Multidisciplinary team consensus was for him to commence vedolizumab and mirtazapine. He was discharged and referred to his local mental health team. Encephalitis or temporal lobe seizures were differentials given the prodromal illness and anxiety, panic, depersonalization, and derealization, but were ruled out given normal investigations. While temporal lobe seizures cannot be ruled out completely, the possibility of temporal lobe seizures was excluded on the basis of: (1) atypical clinical features given the prolonged episode with complex, purposeful behavior; (2) unremarkable serial electroencephalograms; and (3) absence of structural abnormalities on magnetic resonance imaging of the head to support the focal onset of temporal lobe seizures. Psychiatric disturbance can be a side effect of tumor necrosis factor α inhibitors such as adalimumab,4 with some incidences in the supplementary information. Intriguingly, our patient's symptoms started in close temporal association with switching reference adalimumab to a biosimilar. Biosimilars are considered to be as safe and effective as, and often represent a less expensive version of, the reference product.5 Studies suggest that the side effect profile of both Humira and its biosimilars was similar and reporting predominantly minor adverse events after treatment with either drug.6-8 However, one study6 recorded two unexpected events: one loss of consciousness and one episode of psychosis, neither thought at the time to be related to treatment with Humira. The co-occurrence of other autoimmune disease–causing psychiatric symptoms should be contemplated when patients with autoimmune disease have psychiatric presentations. Various factors exist explaining the link between autoimmune disease and psychosis, including inflammation, genetic vulnerability, brain-reactive antibodies, predisposing infections9 and the stress of chronic disease.1 Immune dysregulation can also affect the risk of suicidal ideation with elevated levels of the cytokine interleukin 6 found consistently elevated and associated with suicidal ideation, nonfatal suicide attempts, and suicides.10 There are many causes of psychiatric presentations in patients with autoimmune disease, including the disease itself, its management, and the psychosocial impact of chronic disease. Although causation cannot be wholly attributed without symptom recurrence on rechallenging of the drug, the exclusion of other causes and the patient's recovery following Imraldi's suspension makes a compelling case for the recent switch from reference Adalimumab (Humira) being a precipitating factor in the patient's presentation. The authors have no conflicts of interests to declare. The authors confirm that written consent for submission of this case report including associated text has been obtained from the patient in line with Committee on Publication Ethics (COPE) guidance. Appendix S1. A series of case studies or articles referencing psychiatric events following use of TNF-α inhibitors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The enzyme DNA methyltransferase 3-like (DNMT3L) is essential for the acquisition of DNA methylation patterns in germ cells as well as normal germ cell development and has been reported as the first paternal effect gene in mammals. Although Dnmt3L heterozygous males are fertile, they have abnormalities in X chromosome compaction, postmeiotic gene expression and sire an increased number of pups with XO monosomy. To examine the postulated epigenetic basis of the paternal effects, we examined DNA methylation acquisition at intergenic (non-CpG island, non-repeat) sites along chromosome 9 in male germ cells isolated during the prenatal and postnatal periods. At 16.5 dpc (days post coitum), 42% of intergenic loci examined were hypomethylated, by 20–30%, in Dnmt3L heterozygotes as compared to Dnmt3L wildtype mice. By day 1 after birth DNA methylation differences in spermatogonia between the two genotypes had resolved for 67% of the sites; in contrast, 33% of sites showed residual hypomethylation, in the range of 10%, associated with Dnmt3L haploinsufficiency. By postnatal days 4 and 6, germ cell DNA methylation levels were similar in the heterozygous and wildtype mice and remained similar later in spermatogenesis in spermatozoa. The findings suggest that germ cell DNA methylation differences between the genotypes resolve shortly after birth before the spermatogonia begin to divide. To confirm and extend these results, higher resolution studies using Reduced Representation Bisulphite Sequencing (RRBS) are being carried out to examine methylation differences at 3-5 million CpG sites across the genome. The DNA methylation defects described here in prenatal and early postnatal male germ cells of Dnmt3L heterozygous mice are the earliest epigenetic perturbation yet identified for a mammalian paternal effect gene and may influence downstream epigenetic and genetic events that lead to the previously observed abnormalities during spermatogenesis and paternal effects in the offspring. (Supported by CIHR).

Abstract Background: Waldenstrom’s Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy. Insulin-like growth factor 1 (IGF1) is a polypeptide hormone that has been shown to have a proactive role in many cancer cell types, including multiple myeloma and solid tumor cells. We evaluated the role of IGF1 in WM. Methods: WM cell lines (BCWM1 and WSU-WM) and IgM secreting low-grade lymphoma cell lines (MEC1, RL) were used. Bone marrow-derived primary CD19+ cells and bone marrow stromal cells (BMSC) were obtained from patients with WM after informed consent. The small molecule IGF-1R inhibitor II (Calbiochem) and the inhibitory antibody αIR3 (Calbiochem) were used. Cytotoxicity and DNA synthesis were measured by MTT assay and thymidine uptake assay, respectively. Cell signaling and apoptotic pathways were determined by Western Blot. Cell cycle and receptor analysis was obtained through flow cytometry. IGF1 levels were measured by ELISA. Receptor tyrosine kinase activity was evaluated in WM cell lines using the Luminex-microbeads-based assay. Results: We demonstrated that the IGF1 receptor (IGF1R) was expressed on WM primary patients and normal CD19+ control B cells, while IGF1 levels of serum and bone marrow samples in both patient and healthy donor samples were similar, suggesting a possible constitutive activation of the pathway downstream of IGF1R in WM and independent of receptor activity or cytokine levels. Surface IGF1R was expressed on BCWM1 cells, in serum-starved conditions, while, it was internalized with the addition of FBS that includes IGF-1. Finally, we also showed that IGF1 induces the activation of IGF1R as demonstrated by the induction of related tyrosine kinase activity. To demonstrate the protective effect of IGF1 on WM cells, IGF1 was added to serum starved BCWM1 cells, and we found it rescued nearly 100% of the cells from apoptosis in concentrations as low as 25ng/ml. The effect of IGF1R inhibitor on WM cells has been investigated and found that it blocked migration, induced cytotoxicity and decreased cell proliferation; without any effect on healthy donor peripheral blood mononuclear cells. Furthermore, the inhibitor decreased the tyrosine kinase activity of IGF1R, overcoming its initial activation in the presence of IGF1. Conclusion: IGF1 plays a complex role in WM cells with no evidence of constitutive activation of the receptor itself or higher levels of cytokine in WM marrow samples, indicating that activation of this pathway is downstream of IGF1R. Inhibition of IGF1R by a specific small molecule or antibody inhibitor led to a significant decrease in proliferation, survival, and migration in WM cells, but not in control mononuclear cells. These studies provide the framework to investigate the role of IGF1R inhibitors in clinical trials in WM.

Abstract BACKGROUND: Tyrosine phosphorylation is a central event in the regulation of a variety of biological processes such as cell proliferation, migration, adhesion, and survival. Waldenström Macroglobulinemia (WM) is an incurable lymphoplasmacytic lymphoma with limited options of therapy and characterized by widespread involvement of the bone marrow. We sought to determine whether the protein tyrosine kinase Src regulates adhesion and survival in WM. METHODS: The WM cell lines BCWM1, WM-WSU, and IgM secreting lymphoma cells lines MEC-1 and RL were used in these studies. Primary CD19+ WM cells were obtained from the bone marrow of patients after informed consent. AstraZeneca Biopharmaceuticals (London, England) provided the Src inhibitor AZD0503 (AZD). Cytotoxicity and DNA synthesis were measured using MTT assay and [3H]-thymidine uptake, respectively. Apoptosis was measured using flow cytometry with Apo 2.7 staining. Western blotting was performed to determine downstream signaling pathways. Migration was performed using the transwell migration assay. RESULTS: We demonstrated that pSrc is overexpressed in WM cells compared to control B cells. Similarly, phospho-Src protein expression was upregulated in WM cell lines, specifically BCWM.1 but not in WM-WSU. We then showed that pSrc regulates migration and adhesion in response to the chemokine SDF-1, as well as in vivo homing using in vivo flow cytometry. The use of the specific Src inhibitor AZD0530 led to significant inhibition of adhesion and migration in cell lines with pSrc activation, but not in those deficient of Src activation. Similarly, inhibition of Src activity led to significant inhibition of proliferation and survival through inhibition of STAT3, Akt, and ERK/MAPK pathways. The monoclonal antibody rituximab signals through Src kinase, and the combination of AZD0530 and rituximab was synergistic in vitro. CONCLUSION: Taken together, these studies delineate the role of Src kinase activity in WM and provide the framework for future clinical trials using Src inhibitors in combination with rituximab to improve the outcome of patients with WM.

Abstract PURPOSE: Recent advances in understanding of the molecular alterations that occur at the genetic and epigenetic levels in Multiple Myeloma (MM) have led to major leaps in identifying molecular pathways that regulate progression and resistance to therapeutic agents. However, despite great scientific advances at the genomic level, studies to identify signaling pathways deregulated at the functional proteomic level in MM are limited. In this study, we used a rapid and reproducible antibody-based protein microarray technique to screen the functional differences between malignant plasma cells in samples obtained from patients with MM compared to normal plasma cells (NPC) from the bone marrow of healthy volunteers. METHODS: We determined the protein expression level of 512 polypeptides in 12 samples of newly diagnosed patients with MM using high-throughput proteomic analysis with antibody-based protein microarray. Primary CD138+ sorted MM cells were obtained from the bone marrow of patients after informed consent. MM1.S was used in this study. Using immunohistochemistry and immunoblotting were confirmed. Lentivirus was used to knockdown CRIK. Gene expression datasets from the Mayo Clinic (accession number GSE 6477) and the UAMS (accession number GSE 2658) were obtained from the Gene Expression Omnibus for analysis. The Mayo dataset was generated using Affymetrix U133A platform whereas the UAMS dataset was generated using Affymetrix U133plus 2.0 platform. RESULTS: We identified four subgroups of MM using unsupervised clustering analysis. We confirmed overexpression of some of these proteins including CRIK and CDK4 using immunohistochemistry and immunoblotting. Many of these proteins are known to be deregulated in MM, indicating that this technique can accurately identify proteins that are over or under-expressed in MM in a high-throughput fashion. In addition, we identified novel proteins that are not previously known to be differentially expressed in MM, including the small GTPase member of the Rho family, CRIK protein. We then showed using knockdown of CRIK that this novel protein specifically regulates migration and adhesion in MM. There was no effect on survival of MM cells using the CRIK knockdown. Analyzing the GEP data of the 15 NPC, 46 monoclonal gammopathy of undetermined significance (MGUS) or smoldering myeloma (SMM) and 101 MM samples from the Mayo Clinic, there was a significant increase in expression of CIT from NPC to MM. Among the 351 patients entered into TTII trials from UAMS, CIT expression was similar across the different TC class. Using a cut-off normalized expression level of 1.25 (a level above expression in NPC), MM with a high CIT expression (n=81) had a significantly shorter survival than the other patients. CONCLUSION: In this study, we show that MM cells express a high level of CRIK, and that inhibition of this protein leads to significant inhibition of adhesion and migration of MM cells. In addition, CRIK protein expression correlated with CIT gene expression, with high expression in MM samples compared to NPC. In addition, high CIT expression correlated with poor survival in patients with MM.

Introduction and Aim Acute neurological problems are prevalent, accounting for 10–20% of medical admissions, but management is often challenging and require input from different teams (A&amp;E, acute medicine, neurology). There is no standardised training for staff in the management of acute neurological conditions and existing clinical pathways for these conditions are often underutilised with variable impact on patient outcome. The use of e-learning has gained popularity in the last decade, showing that it can be as effective as conventional didacticism [Warnecke et al, 2011]. Here, we detail the development of an e-learning course on management of acute neurological conditions to support use of integrated clinical pathways and to facilitate self-directed learning. Method and Results Using principles of People, Analysis, Content and Technology (PACT), we defined target learners, their learning needs, challenges in assimilating knowledge and the resources required. Topics were derived from three themes: acute neurological emergencies, commonly seen conditions, and ‘lessons from serious incidents’. Each module lasts 10 minutes and is embedded with a pre- and post-course knowledge quiz. In collaboration with the Neurosciences Institute Programme at King’s Health Partners, an academic Health Science Centre, the course will be hosted and supported by the KHP e-learning platform. Summary This e-learning course supports the use of clinical pathways and facilitates clinicians in consoli- dating knowledge. It is flexible, highly transferable, and can be integrated into work-based learning for continuous professional development. kitwu@doctors.org.uk

We present a case of adhesive spinal arachnoiditis, as a complication of a fall with traumatic subarach- noid haemorrhage, in a patient with progressive multiple sclerosis (MS). A 68 year-old woman with MS and spasticity, on Baclofen, Pregabalin and Nabiximols, was admitted following a fall. CT scanning demonstrated traumatic subarachnoid haemorrhage. She was admitted to the ITU initially and managed conservatively. Four months later, while undergoing inpatient rehabilitation, her condition deteriorated over six weeks with worsening paraparesis, neurogenic bladder and bowel dys- function with a C5–6 sensory level. She received steroids and underwent MRI of her neuroaxis. Imaging of her brain showed unchanged demyelinating lesions. Spinal imaging identified distortion with considerable T2 signal change in the cervical and thoracic regions, extending from the C5/6 to the T8/9 level, radiologi- cally consistent with a syrinx and myelopathy. It was felt her deterioration was not due to demyelination but secondary to cervicothoracic adhesions ventral to the cord as sequelae of cerebral haemorrhage. Following neurosurgical review, the patient underwent C4-T1 laminoplasty, adhesionolysis and expansion duraplasty with histopathology showing calcified connective tissue but the clinical outcome has thus far been poor. It is important to consider all the causes of deterioration in patients with progressive MS. menafarag@doctors.org.uk

Chorea can be associated with autoimmune diseases such as antiphospholipid syndrome and has been associated with the isolated presence of antiphospholipid antibodies (aPL). Chorea is a rare neurological manifestation of antiphospholipid syndrome. The pathophysiological mechanisms underlying aPL-related chorea are still debated. One postulated mechanism is aPL or other autoantibody binding to brain-blood vessel endothelium, resulting in endothelial dysfunction secondary to a proinflammatory cascade, with sequalae of inflammation and local microthrombosis. Another postulated mechanism considers immune-mediated attack (aPL or antibasal ganglia antibodies) against specific basal ganglia epitopes. Here, we report a patient with isolated aPL-related chorea that followed a relapsing-remitting course. We highlight the role of brain metabolic imaging with fluorodeoxy glucose positron-emission tomography in the diagnostic workup of chorea and the challenges in the practical management of aPL-related chorea with symptomatic treatments.

Abstract Background. Waldenstrom Macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow (BM). Adhesion of malignant cells to the BM microenvironment induces proliferation and resistance to therapy. We previously showed that SDF-1/CXCR4 axis regulates migration and adhesion of WM cells in vitro indicating a potential role in homing. Homing of malignant cells to the BM niches requires active navigation through the endothelial cell barrier where tumor cells must adhere, roll and transmigrate. We hypothesized that CXCR4 and its interaction with VLA-4 is critical for this 3-step process. Methods. The level of CXCR4 and VLA-4 was determined using flow cytometry and RT-PCR in patient samples and WM cell lines (BCWM.1 and WM-WSU). The CXCR4 inhibitor AMD3100 (10–100uM, Sigma, MO), Gi protein inhibitor pertussis toxin, PTX (10–200ng/ml, Sigma, MO) were used to inhibit CXCR4 signaling. These studies were confirmed using CXCR4 knockdown using shRNA with lentivirus infection. Adhesion assay was done with either plates coated with the VLA-4 ligand fibronectin for adhesion of tumor cells in presence and absence of SDF1 (30nM), or coated with endothelial cells (HUVEC) and BMSCs for cell-cell adhesion experiments. Transmigration assay was performed using the transwell system with either SDF1 in the lower chamber to study migration towards SDF1, or with the upper chamber coated overnight with endothelial cells (HUVEC) and BMSCs to study transmigration. Experiments with HUVEC cells were performed in absence or presence of TNF-alpha that activates the HUVEC cells. Results. WM tumor cells from patients and cell line expressed high surface levels of CXCR4 (mean 70%) and VLA-4 (mean 95%). Adhesion of WM cells to fibronectin was significantly increased with SDF1 compared to BSA control, and AMD3100 20uM inhibited adhesion by 50% compared to control. Similar results were obtained with PTX 200ng/mL and using CXCR4 knockdown in WM cells. Furthermore, WM cells adhesion was greatly enhanced in co-culture with BMSCs and endothelial cells, and was significantly inhibited using AMD3100 and PTX. To further dissect the mechanisms of interaction of CXCR4 and VLA-4, we showed that AMD3100 inhibited surface expression of CXCR4 but not of VLA-4. Immunoprecipitation studies showed that CXCR4 and VLA-4 directly interact in response to SDF-1. Downstream signaling pathways showed that CXCR4 regulates migration and adhesion through the PI3K/Akt and ERK/MAPK pathways. We next studied whether CXCR4 was involved in transmigration through the endothelial barrier. We demonstrated that BCWM.1 cells transmigrated through the barrier of endothelial cells, that was increased when SDF1 was added to the lower chamber, and that AMD3100 inhibited the transmigration of BCWM.1 cells. Studies of the role of CXCR4 in rolling on endothelial cells is ongoing. Conclusion. We showed CXCR4/SDF1 axis regulates migration and adhesion of WM cells to the BM microenvironment indicating a potential role in homing. Moreover, we demonstrate that CXCR4/SDF1 axis is critical for transendothelial migration, a critical step of homing and egression of tumor cells in and out the BM niches.

Abstract Background: The NF-kB pathway has been implicated in tumor survival and growth, and to induce resistance to conventional agents. Because of the natural ability of tumor cells to overcome single agent antitumor activity, we hypothesized that targeting differentially NF-kB pathway with combination of proteasome inhibitor bortezomib and of Akt inhibitor perifosine might lead to synergistic cytotoxicity on WM. Methods: The WM cell line BCWM.1 was used in these studies. Primary CD19+ WM cells were obtained from the bone marrow (BM) of patient after informed consent. Perifosine (P) was provided by Keryx (NY), Bortezomib (B) from Millennium (MA) and Rituximab (R) from Genentech (CA). NF-kB was studied using the Chromatin Immunoprecipitation (ChIP)-based assays, a new technology that allows for the specific analysis of activation/inhibition of one specific gene in the entire genome. IkB gene was used in this study as NF-kB target-activation gene. Quantitative real-time PCR (qPCR) for IkB in the p65NF-kB-DNA immunoprecipitated fragments was assessed and those results were confirmed at the transcriptional level using qPCR and immunoblotting on cytoplasmic/nuclear fractionation of cells. NF-kB activity assay was confirmed using the DNA-binding ELISA-based assay Active Motif kit on nuclear extract. Antibody-dependent cellular cytotoxicity assays (ADCC) was performed in presence of rituximab or human control IgG1. Results: We first showed that perifosine and bortezomib combination [P+B] induced synergistic cytotoxicity and inhibition of proliferation in WM cells. Similar cytotoxicity was observed in primary patient tumor cells and in presence of BM microenvironment, but spared normal hematopoietic cells. We next demonstrated that P and B inhibited p65NF-kB nuclear translocation and downstream IkB target gene using ChIP, and that the combination [P+B] showed additive inhibitory activity. We next sought to further dissect molecular mechanisms of synergy induced by the combination. B 5-10nM inhibited phosphorylation of ERK MAPK pathway and slightly upregulated Akt activity. On the other hand, P 10uM inhibited Akt phosphorylation, but induced activation of the ERK MAPK pathway. Interestingly, while either agent differentially upregulated one of those 2 signaling pathways, their combination [P+B] was able to overcome resistance induced by the other agent. This was demonstrated by significant inhibition of downstream target proteins of Akt, and by a significant decreased phosphorylation of fusion protein GSK3a/b using an in vitro Akt kinase assay. We next sought to examine the combination of R with [B+P], since R is known to inhibit Akt and NF-kB pathways. Pretreatment of WM cells with P, B or the combination [P+B] led to a significant increase in the ADCC cytotoxicity of R compared to R alone (p=0.025). We next confirmed that the combination [P+B+R] inhibited NF-KBp65 function in WM cells, using Active Motif assay and ChIP for IkB. Conclusion: Together these studies provide the framework for clinical studies of combination of perifosine with bortezomib and rituximab in a sequential approach to improve patient outcome in WM.

Nine participants were interviewed about their experiences with unintentionally severe injury during engagement in nonsuicidal self‐injury. Using interpretative phenomenological analysis, we identified four common themes among participants: (a) explosive affect prior to unintentional injury, (b) loss of control during unintentional injury, (c) unfamiliar method, and (d) consequences of unintentional injury. Implications for research and practice are discussed.