Key Matrix Proteins Within the Pancreatic Islet Basement Membrane Are Differentially Digested During Human Islet Isolation.

Clinical islet transplantation achieves insulin independence in selected patients, yet current methods for extracting islets from their surrounding pancreatic matrix are suboptimal. The islet basement membrane (BM) influences islet function and survival and is a critical marker of islet integrity following rodent islet isolation. No studies have investigated the impact of islet isolation on BM integrity in human islets, which have a unique duplex structure. To address this, samples were taken from 27 clinical human islet isolations (donor age 41-59, BMI 26-38, cold ischemic time < 10 h). Collagen IV, pan-laminin, perlecan and laminin-α5 in the islet BM were significantly digested by enzyme treatment. In isolated islets, laminin-α5 (found in both layers of the duplex BM) and perlecan were lost entirely, with no restoration evident during culture. Collagen IV and pan-laminin were present in the disorganized BM of isolated islets, yet a significant reduction in pan-laminin was seen during the initial 24 h culture period. Islet cytotoxicity increased during culture. Therefore, the human islet BM is substantially disrupted during the islet isolation procedure. Islet function and survival may be compromised as a consequence of an incomplete islet BM, which has implications for islet survival and transplanted graft longevity.

Expression of apoptotic genes in immature and in vitro matured equine oocytes and cumulus cells.

The gene expression of Bax, Bcl-2, survivin and p53, following in vitro maturation of equine oocytes, was compared in morphologically distinct oocytes and cumulus cells. Cumulus-oocyte complexes (COC) were harvested and divided into two groups: G1 - morphologically healthy cells; and G2 - less viable cells or cells with some degree of atresia. Total RNA was isolated from both immature and in vitro matured COC and real-time reverse transcription polymerase chain reaction (qRT-PCR) was used to quantify gene expression. Our results showed there was significantly higher expression of survivin (P < 0.05) and lower expression of p53 (P < 0.01) in oocytes compared with cumulus cells in G1. No significant difference in gene expression was observed following in vitro maturation or in COC derived from G1 and G2. However, expression of the Bax gene was significantly higher in cumulus cells from G1 (P < 0.02).

Erythropoietin non-viral gene therapy does not affect motility, viability, morphology or concentration of rabbit sperm.

Erythropoietin (EPO) gene therapy can be used for several purposes; however, its effects on reproductive performance are unknown. The aim of this study was to evaluate the toxicological effects of non-viral (EPO) gene transfer on sperm motility, viability, morphology and concentration. Rabbit EPO cDNA was cloned into a pTarget mammalian expression vector. Rabbits were administered with: (1) pTarget/EPO vector, (2) recombinant human EPO (rHuEpo) and (3) saline (control). Both pTarget/EPO and rHuEpo significantly increased (P < 0.05) hematocrit levels 1 week after injection and they remained significantly higher than the control for up to 5 weeks (P < 0.05), showing that both EPO treatments were effective in stimulating the production of red blood cells in rabbits. The EPO gene transfer or rHuEPO administration had no significant effect (P > 0.05) on sperm motility, vigor, viability, concentration or morphology in the testis.

CR1/2 is an important suppressor of Adenovirus-induced innate immune responses and is required for induction of neutralizing antibodies.

Human complement receptors 1 and 2 are well described as important regulators of innate and adaptive immune responses, having pivotal roles in regulating complement activation (CR1) and B-cell maturation/survival. In contrast, the role of the murine homologs of CR1 and CR2 (mCR1/2) have been primarily defined as modulating activation of the adaptive immune system, with very little evidence available about the role of mCR1/2 in regulating the innate immune responses to pathogens. In this paper, we confirm that mCR1/2 plays an important role in regulating both the innate and adaptive immune responses noted after Adenovirus (Ad)-mediated gene transfer. Our results uncovered a novel role of mCR1/2 in downregulating several complement-dependent innate immune responses. We also unveiled the mechanism underlying the complement-dependent induction of neutralizing antibodies to Ad capsids as a CR1/2-dependent phenomenon that correlates with B-cell activation. These results confirm that Ad interactions with the complement system are pivotal in understanding how to maximize the safety or potency of Ad-mediated gene transfer for both gene therapy and vaccine applications.

Naïve donor responses to Schistosoma mansoni soluble egg antigens.

Schistosome infection induces profound Th-biasing and immune suppression. Although much has been examined in mice, few studies have examined responses of naïve humans to schistosome antigens. In this study, we examined the response of naïve human peripheral blood mononuclear cells (nPBMC) to stimulation with Schistosoma mansoni soluble egg antigen (SEA) using a priming in vitro (PIV) assay. We found that SEA induced a pronounced CD4+ T-helper cell response based on cytokine secretion and phenotyping markers. SEA-stimulated nPBMC (SEA cells) at day 7 post-priming and after the first recall consisted predominantly of Th0-like CD4+ T cells. Following the second recall, the majority of donor (10/12) responses were Th2-like. The cell population consisted of approximately 64% CD4+, 17% CD8(+high), 12% CD19+, and 7% CD23+ cells. The CD4+ population also expressed HLA-DR+, CD54+, CD45RO+ and CD25+ whereas the CD19+ cells expressed CD80 and CD86. Following priming, we detected high levels of IL-6, IFN-gamma, IL-12p40, IL-10 and IL-5. Upon restimulation, SEA cells secreted IL-5 and high levels of IL-10, typical of a Th2-like response. The data presented herein shows that the majority of naïve donor dendritic cells, following stimulation with SEA, prime and clonally expand SEA-specific T cells towards a Th2-type response. However, two donors responded with an atypical response, producing IFN-gamma coincident with low levels of IL-10. Whether this differential response was due to HLA or other genes was not determined but is currently under investigation.

Expression of the B-cell and T-cell epitopes of the rabies virus nucleoprotein in Mycobacterium bovis BCG and induction of an humoral response in mice.

Expression vectors containing rabies virus nucleoprotein B-cell and T-cell epitopes in Mycobacterium bovis BCG were constructed. The epitopes were subcloned into the M. leprae 18-kDa gene to ensure correct presentation to the host immune system. Expression of the 18-kDa::B+T epitope fusion protein was driven by either the hsp60 promoter, which is constitutively activated at a high level in M. bovis BCG, or the 18-kDa promoter, which is strongly induced in vivo. Mice were immunised intra-peritoneally with the recombinant BCG cultures and compared to a control group vaccinated with the commercial rabies vaccine Rai-SAD. Both of the expression vectors elicited a higher antibody titre than that of the rabies vaccine, with the highest response shown by M. bovis BCG (pUP203), expression controlled by the 18-kDa promoter. Immunisation with M. bovis BCG (pUP202), expression controlled by the hsp60 promoter, resulted in a continuously increasing antibody titre up to 60 days post immunisation. The mice antibodies were also capable of recognising the whole rabies virus and not only the synthetic peptide epitopes.

Needle fixation, the drug user's perspective: a qualitative study.

AIMS: Changing drug users' injecting behaviour is central to the project of drug services. Information about the idea of "needle fixation" is fragmentary and of uncertain relevance to contemporary injecting drug users. The aim of the study is to describe injecting drug users' ideas about needle fixation. PARTICIPANTS AND DESIGN: Twenty-four participants, some of whom identified themselves as needle fixated, were recruited from four drugs agencies in south Wales. Participants took part in semi-structured interviews, recorded, transcribed and qualitatively analysed using Atlas/ti software. FINDINGS: Participants describe a range of behaviours and experiences which fit with previous ideas of needle fixation, including ritualization, substitution of other drugs, injection of water and associations with deliberate-self-harm and sex. Participants describe high levels of needle aversion and add detail to previous partial descriptions of needle fixation. CONCLUSIONS: Issues conveniently considered together as needle fixation are current among injecting drug users and may be relevant to the inability of some drug users to change from injecting drug use.

The drug treatment of alcohol withdrawal symptoms: a systematic review.

A computer-assisted and cross-reference literature search identified trials of therapy for alcohol withdrawal symptoms. Those with a randomized, double-blind placebo-controlled design were systematically assessed for quality of methodology. Fourteen studies were identified investigating 12 different drugs. The quality of methodological design, even among this highly selected group of published studies, was often poor. Study populations were generally under-defined, most studies excluded severely ill patients, control groups were poorly matched, and the use of additional medication may have confounded results in some studies. Twelve different rating scales were used to assess severity of symptoms. All 12 compounds investigated were reported to be superior to placebo, but this has only been replicated for benzodiazepines and chlormethiazole. Further research using better methods is required to allow comparison of different drugs in the treatment of alcohol withdrawal symptoms. On the evidence available, a long-acting benzodiazepine should be the drug of first choice.

Self detoxication by amphetamine dependent patients: a pilot study.

Fifty current or past amphetamine dependent clients attending a Community Drug and Alcohol Team service took part in structured interviews about their previous attempts to stop using amphetamine. Thirty three had made a total of 47 attempts at self detoxication; 15 had undergone enforced withdrawal and ten had previously sought medical treatment. A total of 86% of subjects described significant withdrawal symptoms on stopping use of amphetamines. Increased use of other drugs was commonly reported as a means of coping with withdrawal, psycho-social techniques being used less systematically. The implications for the provision of attractive, effective treatments are discussed.

Three cases of nalbuphine hydrochloride dependence associated with anabolic steroid use.

Three case reports are presented of nalbuphine hydrochloride dependence meeting DSM IIIR and ICD10 criteria for opioid dependence. Nalbuphine hydrochloride is being obtained from illicit sources and used by those using performance enhancing drugs. In some cases this leads to opioid dependence. There is a potential risks of crossover between the misuse of drugs of performance and the misuse of psychoactive drugs by injection. Further research into the dependence potential of nalbuphine and the extent of the crossover between steroid misuse and other psychoactive drug misuse is required. The legal status of nalbuphine should be reviewed in the light of its availability on the black market.

Compound opioid/paracetamol analgesics: misuse and dependence.

Ten cases of individuals with a history of chronic compound opioid/paracetamol misuse are described. Potentially useful indicators of toxicity are suggested.

Cannabis use in a drug and alcohol clinic population.

Cannabis using behaviour was determined, by structured interview, in 100 consecutive clinic attenders who had used cannabis within 28 days. Subjects were white, 85 were male, mean age was 27.6 years and duration of cannabis use was 12.4 years. The mean consumption of the past week was 10.5 g. Sixty percent smoked daily. The average cannabis cigarette comprised three cigarette papers, the tobacco from three-quarters of a king sized cigarette, and 0.35 g of cannabis resin. Eating cannabis was unusual. Fifty subjects had a conviction for possession of cannabis. Subjects fulfilled criteria for 'heavy' use. The findings are discussed in the context of previous research.