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Syndecan-1 is a heparan sulfate proteoglycan (HSPG) commonly upregulated in AIDS-related B lymphoid malignancies. Tat is the main HIV-1 transactivating factor that has a major role in the pathogenesis of AIDS-related lymphomas (ARL) by engaging heparan sulfate proteoglycans (HSPGs), chemokine receptors and integrins at the lymphoid cell (LC) surface. Here B-lymphoid Namalwa cell clones that do not express or overexpress syndecan-1 (EV-Ncs and SYN-Ncs, respectively) were compared for their responsiveness with Tat: in the absence of syndecan-1, Tat induces a limited EV-Nc migration via C-X-C motif chemokine receptor 4 (CXCR4), G-proteins and Rac. Syndecan-1 overexpression increases SYN-Nc responsiveness to Tat and makes this response independent from CXCR4 and G-protein and dependent instead on pp60src phosphorylation. Tat-induced SYN-Nc migration and pp60src phosphorylation require the engagement of alpha sub(v) beta sub(3) integrin and consequent pp125FAK phosphorylation. This complex set of Tat-driven activations is orchestrated by the direct interaction of syndecan-1 with pp60src and its simultaneous coupling with alpha sub(v) beta sub(3). The Tat/syndecan-1/ alpha sub(v) beta sub(3 ) interplay is retained in vivo and is shared also by other syndecan-1 super(+) B-LCs, including BJAB cells, whose responsiveness to Tat is inhibited by syndecan-1 knockdown. In conclusion, overexpression of syndecan-1 confers to B-LCs an increased capacity to migrate in response to Tat, owing to a switch from a CXCR4/G-protein/Rac to a syndecan-1/ alpha sub(v) beta sub(3)/pp60src/pp125FAK signal transduction pathway that depends on the formation of a complex in which syndecan-1 interacts with Tat via its HS-chains, with alpha sub(v) beta sub(3) via its core protein ectodomain and with pp60src via its intracellular tail. These findings have implications in ARL progression and may help in identifying new therapeutical targets for the treatment of AIDS-associated neoplasia.

Decitabine, which reverses hypermethylation of the p15 super(INK4B) gene in vitro, has been used to relieve cytopenias and blast excess in over 50% of patients with high-risk myelodysplastic syndrome (MDS). In this study, heme oxygenase-1 (HO-1) was overexpressed in MDS cell line SKM-1, which was closely related to resistance to decitabine-induced apoptosis. We aimed to further investigate the role of HO-1 in apoptosis induced by low-dose decitabine in SKM-1 cells. Upregulation of HO-1 by transfecting it into SKM-1 cells with lentivirus vector promoted cell proliferation and protected them against apoptosis. In contrast, downregulation of HO-1 enhanced decitabine-induced apoptosis but reduced accumulation of the S phase in cell cycle. To explore the mechanism, the expressions of cell cycle-related proteins were detected after the cells were treated by decitabine in each group. p15 super(INK4B) and CDK4 were overexpressed in SKM-1 cells in which HO-1 was inhibited, and the expression-depending apoptosis was related to the caspase-3 pathway. Even though HO-1 was silenced, the apoptotic rate never increased as the caspase-3 pathway was blocked. It is well known that p15 super(INK4B) dominantly regulates the S phase of the cell cycle. p15 super(INK4B) was herein demethylated more evidently in the group of SKM-1 cells in which HO-1 was downregulated, as well as in the mononuclear cells of patients suffering from MDS. In the case of poor prognosis, the mRNA level of HO-1 was raised. In conclusion, overexpression of HO-1 indicated resistance to demethylation of p15 super(INK4B) induced by decitabine.

Cell-based therapy using mesenchymal stem cells (MSC) has emerged as a promising approach to regenerating failing hearts. However, studies need to be done to optimise and enhance the beneficial effects of mesenchymal stem cell therapy. Here we show that genetic silencing of Plasma Membrane Calcium ATP-ase 4 (PMCA4) in MSC produces a protective effect when co-cultured with cardiomyocytes in response to hypoxia. Using the lentivirus system we have generated MSC with stable  integration of PMCA4 shRNA (MSC-PMCA4KD). PMCA4 expression was reduced by more than 75% in these cells (p<0.01). To test the protective effect of MSC-PMCA4KD we co-cultured these cells with neonatal rat cardiomyocytes and cultured them in hypoxic conditions (95% N2, 5% CO2, 1% O2) for 24 hours. We found that cardiomyocytes co-cultured with MSC-PMCA4KD displayed higher survival and less apoptosis, as indicated by a significantly lower level of caspase 3/7 activity (n=3, p<0.05) compared to cells co-cultured with control MSC. We also found that MSC-PMCA4KD expressed and secreted higher levels of sFRP2 (secreted frizzled related protein 2) compared to control, as indicated by qPCR, Western blot and ELISA analyses (n=3, p<0.05). sFRP2 is a potent inhibitor of the Wnt signalling pathway and may be responsible for the protective effect of MSC-PMCA4KD. In conclusion, our data show that PMCA4 genetic silencing increases the protective effect of MSC against hypoxic stimulus. Thus, PMCA4 may be a target to enhance the therapeutic effects of MSC.

ObjectiveTo reprogramme the induced pluripotent stem (iPS) cells from Human umbilical cord mesenchymal cells (HuMSCs) and induce the iPS cells into germ cells by BMP4.MethodsOCT4, SOX2, Klf4,c-myc, Nanog, Lin28 were transfected into HuMSCs with lentivirus to reprogram HuMSCs into iPS cells. Morphological observation, Alkaline Phosphatase staining, karyotype analysis, RT-PCR, immunofluorescence staining, tumour formation in vivo and embryniod body formation in vitro were performed to examine the pluripotency of the iPS cell lines. Then we induced one of the iPS cells lines into germ cells by BMP4. Gene expression was measured by qRT-PCR at days 0, 3, 7, 10 and 14. Early-stage germ specific protein VASA and meiosis specific protein SYCP3 were assesssed by immunofluorescence staining.ResultWe obtained two iPS cell lines completely reprogrammed, HuMSC-iPS1 and HuMSC-iPS2. HuMSC-iPS1 expresses germ cell markers at undifferentiated state. BMP4 can upregulate germ cell markers at different time points highly while the spontaneous differentiation just upregulate DPPA3, DAZL and VASA modestly at day 3. However, all of these genes were downregulated at day 14. VASA and SYCP3 immunofluorescence staining indicates there is a high VASA expression in BMP4 induced group in contrast to low expression in the spontaneous group at day 7. Meanwhile, there is a modest SYCP3 fluorescence in BMP4 induced group in contrast to no immunofluoresence in the spontaneous group.ConclusionThis system can reprogram HuMSCs into iPS cells effectively. The MSC- iPS1 can differentiate into early germ cells spontaneously while the germ cells induced by BMP4 can enter meiosis.

Ciliary neurotrophic factor (CNTF) acts as a potent neuroprotective agent in multiple retinal degeneration animal models. Recently, CNTF has been evaluated in clinical trials for the inherited degenerative disease retinitis pigmentosa (RP) and for dry age-related macular degeneration (AMD). Despite its potential as a broad-spectrum therapeutic treatment for blinding diseases, the target cells of exogenous CNTF and its mechanism of action remain poorly understood. We have shown previously that constitutive expression of CNTF prevents photoreceptor death but alters the retinal transcriptome and suppresses visual function. Here, we use a lentivirus to deliver the same secreted human CNTF used in clinical trials to a mouse model of RP. We found that low levels of CNTF halt photoreceptor death, improve photoreceptor morphology, and correct opsin mislocalization. However, we did not detect corresponding improvement of retinal function as measured by the electroretinogram. Disruption of the cytokine receptor gp130 gene in Muller glia reduces CNTF-dependent photoreceptor survival and prevents phosphorylation of STAT3 and ERK in Muller glia and the rest of the retina. Targeted deletion of gp130 in rods also demolishes neuroprotection by CNTF and prevents further activation of Muller glia. Moreover, CNTF elevates the expression of LIF and endothelin 2, thus positively promoting Muller and photoreceptor interactions. We propose that exogenous CNTF initially targets Muller glia, and subsequently induces cytokines acting through gp130 in photoreceptors to promote neuronal survival. These results elucidate a cellular mechanism for exogenous CNTF-triggered neuroprotection and provide insight into the complex cellular responses induced by CNTF in diseased retinas.

IntroductionBASHH guidance recommends proactively educating HIV-infected patients regarding the availability of post-exposure prophylaxis (PEP). Existing evidence suggests PEP awareness is low amongst HIV-infected cohorts, particularly amongst heterosexuals, older patients and those with long-standing HIV diagnoses. We reviewed our educational provision by assessing current awareness in our cohort.AimsTo establish current PEP knowledge, and patient factors influencing that knowledge.MethodsAll HIV outpatients were prospectively assessed via questionnaire between 3/7/14-3/1/15. Following data collation PEP aware and PEP unaware patients were compared using chi-squared and Mann-Whitney testing with significance defined as p < 0.05.Results155 patients responded, 148 were Caucasian; 118 identified as men who have sex with men. 117 (75.5%) were PEP aware of which 108 knew how to access PEP if required. 109 (70.3%) had an undetectable HIV viral load (<20 copies/mL). Attaining an undetectable viral load did not significantly affect awareness (83/117 v 26/38, p = 0.768). Patients who were currently sexually active were not significantly more aware (77/117 v 19/38, p = 0.082) but those reporting contact with HIV-negative partners were (50/117 v 7/38, p = 0.007). Median time since diagnosis was significantly less in those aware of PEP (7.88 years v 11.33 years, p = 0.006). Age, gender and ethnicity did not significantly affect awareness.ConclusionPEP awareness was prevalent and distributed evenly across all demographics. Awareness was significantly higher in those reporting HIV-negative partners, a group in which PEP awareness is especially important. Patients with long-standing diagnoses were shown to have poorer awareness and should be a target group for PEP education.

BackgroundAcutely deranged liver function tests (LFTs) in HIV positive pregnant women present challenges in balancing pregnancy-related conditions, antiretroviral (ARVs) toxicities and prevention of mother to child transmission (MTCT). A 34 year old HIV positive lady with a history of poor engagement in care, psychosis, cognitive impairment and recent nevirapine resistance was admitted at 26 weeks gestation under mental health legislation due to cognitive impairment and self-neglect.MethodShe was commenced on darunavir/ritonavir 600 mg bd, truvada and raltegravir but three weeks later, at 29 weeks gestation, she developed rapidly progressive hepatic transaminitis. Abdominal ultrasound scan was normal and tests for viral hepatitis negative. Pre-eclampsia was excluded, leaving three working diagnoses: drug-induced hepatitis, obstetric cholestasis or acute fatty liver of pregnancy. ARVs were stopped but transaminases continued to rise (ALT 614 and AST 716 U/L). Clotting screen and platelet count remained normal but the patient began to complain of epigastric pain. HIV viral load had risen to 241 copies/ml. In view of deteriorating maternal health and the increasing risk of MTCT (HIV viral load expected to rise), the baby was delivered at 31 weeks' gestation by semi-elective caesarean after a course of antenatal steroids. The baby received antiviral prophylaxis in the form of abacavir, lamivudine and zidovudine; HIV RNA was undetectable at three months (MTCT extremely unlikely). Nine days after delivery the patient's LFTs normalised.ConclusionDarunavir-induced hepatitis typically presents with increased AST and ALT. In this case, LFTs only started to improve following delivery of the baby, suggesting a pregnancy related cause.

Background/introductionAmphetamine (AM) use is associated with HIV infection among MSM. There are various toxic effects of AM, cardiotoxicity being one of them.Aim(s)/objectivesTo present a case of report of cardiomyopathy secondary to AM misuse in a patient with well-controlled HIV.Case reportA 51 year old HIV positive MSM was admitted to hospital with dyspnoea, orthopnoea and decreased exercise tolerance. He was HIV positive since 1990 and this is stable on ARVs. CD4 count pre-admission was 514 with undetectable viral load. He used 25-30 grams of AM per week over a period of 20 years and had multiple casual unprotected MSM partners. On admission, the patient was tachycardic and hypoxic. Chest X-Ray on admission showed cardiomegaly and bi-basal opacification. Echocardiogram demonstrated severe left and right ventricular dysfunction, at a level requiring cardiac transplant. ECG showed prolonged QT interval. The patient was diagnosed with toxic dilated cardiomyopathy secondary to long term AM abuse. UK guidelines for Heart transplantation in adults deem chronic viral infection and ongoing substance misuse as relative contraindications to transplant. He was consequently commenced on medication for cardiac failure and received benzodiazepine as inpatient for managing withdrawal symptoms. On discharge, psychiatry follow-up was organised for support to help reduction of AM. At follow up, the patient reported reduced AM use by quarter, but felt he could never abstain.Discussion/conclusionAM related cardiac fatalities are caused by acute myocardial necrosis, ventricular rupture, cardiomyopathy or arrhythmia. Evidence is mostly derived from case-reports. Patients using AM should be fully counselled regarding possible toxic effects.

Background/introductionCHIVA standards recommend all adolescents and young people living with HIV have an individualised care plan to transition them to adult services over time, as is appropriate to their age, developmental stage and social circumstances. Within the UK, adolescents living with HIV acquire the infection either via vertical transmission or sexual acquisition. These 2 groups differ in terms of medical, social and psychological needs, with the former group historically doing less well in terms of adherence and prognosis compared to the latter group.Aim(s)/objectivesTo understand and characterise patients under the age of 25 attending for HIV care in a provincial UK adult HIV clinic, and identify care needs.MethodsCase note review of all HIV positive patients attending care under the age of 25.ResultsOf 39 patients (29 male, 10 female), mode of transmission was 27(69%) sexual, 11(28%) vertical, and 1 unknown. The vertically-acquired cohort have lower CD4 counts (64% vs 93% CD4 >350), more resistance mutations (including triple class resistance) and lower rates of viral suppression (45% vs 90%) compared to the sexually-acquired cohort. Retention in care is also lower, (72% vs 92% attending in the last year). STI rates are high overall but higher in the sexual transmission cohort, 75% vs 55%.Discussion/conclusionThe under 25 HIV clinic cohort comprises 2 distinct groups: a vertically -acquired cohort with poorer outcomes, who consistently require more support and motivation to remain engaged in care; and a sexually-acquired cohort who adhere to HAART, but have higher rates of STIs and would benefit from support involving motivational interviewing and health promotion.

Background/introductionBetween 2012 and 2013 the rate of gonorrhoea in Southend increased significantly from 24.6 to 42.4 per 100,000. A multidisciplinary Incident Management Team was established in June 2014 to assess the situation and implement appropriate control measures. However, the number of cases had already begun to fall. A retrospective case review was initiated.Aim(s)/objectivesTo identify factors that contributed to the increase and subsequent decrease in diagnoses.MethodsEnhanced questionnaires were completed for each case diagnosed between October 2012 and March 2014. Antibiotic resistance profiles were provided by the local laboratory. Previous STI and HIV test history was extracted from the Genitourinary Medicine Clinic Activity Dataset (GUMCADv2).ResultsProvisional results show that enhanced forms were completed for 160 cases. Majority of cases were of white ethnicity (64%) and born in the UK (87%). Cases were aged between 15 to 63 years (median 28 years), 62% were male and 60% heterosexual. Most cases had 1 or 2 partners in the preceding 3 months and attended because of symptoms (40%). However, approximately 30 cases had been referred from a level 2 service - some of which had negative results when re-tested. The majority of cases were treated with first line therapy and had a test of cure undertaken.Discussion/conclusionFull details of the epidemiology, presentation and diagnosis of the cases will be presented - including a comparison with the cases diagnosed before and after the increase, the use of social network techniques and an analysis against the auditable outcome measures in the BASHH guidelines.