IL-4 regulates Bim expression and promotes B cell maturation in synergy with BAFF conferring resistance to cell death at negative selection checkpoints.

IL-4 plays an essential role in the activation of mature B cells, but less is known about the role of IL-4 in B cell maturation and tolerance checkpoints. In this study, we analyzed the effect of IL-4 on in vitro B cell maturation, from immature to transitional stages, and its influence on BCR-mediated negative selection. Starting either from purified CD19(+)IgM(-) B cell precursors, or sorted bone marrow immature (B220(low)IgM(low)CD23(-)) and transitional (B220(int)IgM(high)CD23(-)) B cells from C57BL/6 mice, we compared the maturation effects of IL-4 and BAFF. We found that IL-4 stimulated the generation of CD23(+) transitional B cells from CD23(-) B cells, and this effect was comparable to BAFF. IL-4 showed a unique protective effect against anti-IgM apoptotic signals on transitional B cell checkpoint, not observed with BAFF. IL-4 and BAFF strongly synergized to promote B cell maturation, and IL-4 also rendered it refractory to BCR-mediated cell death. IL-4 blocked upregulation of proapoptotic Bim protein levels induced by BCR crosslinking, suggesting that diminished levels of intracellular Bim promote protection to BCR-induced cell death. Evidence was obtained indicating that downmodulation of Bim by IL-4 occurred in a posttranscriptional manner. Consistent with data obtained in vitro, IL-4 in vivo was able to inhibit Bim upregulation and prevent cell death. These results contribute to the understanding of the role of IL-4 in B lymphocyte physiology, unveiling a previously undescribed activity of this cytokine on the maturation of B cells, which could have important implications on the breaking of B cell central tolerance in autoimmunity.

The introduction of anti-HTLV testing of blood donations and the risk of transfusion-transmitted HTLV, UK: 2002-2006.

The objectives of the study were to describe the introduction of testing blood donations for antibodies to human T-cell lymphotropic virus (anti-HTLV) and to determine the risk of HTLV potentially infectious donations entering the UK blood supply. The rationale for testing was based on (i) evidence of transmission through transfusion in the UK, (ii) the serious nature of HTLV I-associated morbidity and (iii) evidence of infection in UK blood donors. From mid-2002, all blood donations made at UK blood centres were tested in pooled samples using Abbott-Murex HTLV I/II GE 80/81 enzyme immunoassay (EIA). Surveillance data were used to calculate the incidence and prevalence of anti-HTLV and derive estimates of risk. Between August 2002 and December 2006, 106 donations were confirmed positive for anti-HTLV (95 anti-HTLV I and 11 anti-HTLV II). Prevalence was 10-fold higher among donations from new donors than repeat (4.0 and 0.42 per 100 000 donations), and only one repeat donor had evidence of seroconversion. The risk of an HTLV I potentially infectious donation entering the UK blood supply was estimated at 0.11 per million donations (95% confidence interval 0.06 to 0.18). The current very low observed incidence and prevalence among blood donors reflect the very low estimated risk of an HTLV I-positive donation entering the UK blood supply. A change in either the epidemiology of HTLV in UK blood donors or the length of the window period of the test should prompt further review of the risk and a reassessment of anti-HTLV testing in the UK.

Improvement in the performance of HIV screening kits.

SUMMARY: The aim of this study was to assess the performance of HIV screening kits introduced over a 12-year period. HIV kits used by the National Blood Service (NBS) were assessed in the context of other HIV kits employed by diagnostic and reference laboratories. Thirty-three HIV screening kits were assessed and 13 had the potential to be used by the NBS. Specimens applied to NBS evaluations included 2000 HIV-negative specimens collected from blood donors, 200 HIV-positive specimens and 21 seroconversion panels, with larger numbers applied to the latter two categories prior to implementation of Communauté Européennes (CE) marking. The 33 HIV kits gave repeat reactive rates, based on HIV-negative specimens, of between 0% and 0.8% (and between 0% and 0.2% for kits relevant to the NBS). When examined for diagnostic sensitivity, the 33 kits gave sensitivities between 99.78% and 100%. Kits relevant to NBS gave sensitivities of 100% except one kit, which failed to detect one anti-HIV-2-positive specimen. Twenty-six kits were compared for detection of primary HIV infection. Of these, the 10 combined HIV antigen/antibody kits examined were more sensitive than other formats and have been exclusively adopted by NBS where operational considerations allow. Their added seroconversion sensitivity makes them the screening method of choice for populations at increased risk, e.g. in sexually transmitted infection (STI) clinics. The regular review of evaluation results has demonstrated a continuing improvement over time in the performance of HIV screening kits and contributed to advances in blood safety.

Results of an Aboriginal community-based renal disease management program incorporating point of care testing for urine albumin:creatinine ratio.

INTRODUCTION: There has been a significant increase in the burden of renal disease among Aboriginal Australians over the past 15 years. Urine albumin:creatinine ratio (ACR) is a well-established marker of microalbuminuria and can be conveniently performed on the DCA 2000 point-of-care testing (POCT) analyser (Bayer Australia; Melbourne, VIC, Australia) with an on-site result available in 7 min. The application of the urine ACR POCT for renal disease risk assessment was pioneered by our group in the Umoona Kidney Project. This article describes the results of the management arm of the Umoona Kidney Project, which used point-of-care urine ACR testing for the first time within a management framework to monitor albuminuria in patients at highest risk of renal disease. The article also examines the analytical quality of POCT results and overall community acceptance of the Umoona Kidney Project. METHODS: Adults clinically assessed by Flinders Medical Centre renal specialists as being at greatest risk for renal disease were offered the ACE inhibitor (ACEI) perindopril on a voluntary basis. Selected renal markers, including POCT urine ACR (conducted on-site by Umoona's Aboriginal health worker team), plasma electrolytes, urea, creatinine, calculated glomerular filtration rate and blood pressure were measured six monthly. Regular quality control testing was undertaken to monitor the analytical performance of the POCT analyser. A culturally appropriate questionnaire was designed and implemented to assess community satisfaction with the project. RESULTS: In all, 231 patient management consultations were conducted over a two year period, with over 70% of patients having four or more (up to a maximum of eight) consultations; 35 patients (mean age 49.2 [+/-2.3] years, 54% males) participated voluntarily in the management arm. All were overtly hypertensive, hypertensive with other risk factors or had diabetes. The renal status of these patients was followed for a mean of 63 +/- 4.5 weeks. In total, 111 POCT urine ACR tests were performed for patient management (mean 3.2 tests per patient). There was no significant difference in POCT urine ACR in the study period with a median (and inter-quartile range) of 5.7 mg/mmol (1.2-15.2) pre-ACEI and 4.3 mg/mmol (1.3-16.7) post-ACEI treatment (p = 0.50, Wilcoxon signed ranks test). The calculated glomerular filtration rate altered from 110 to 118 mL/min (p = 0.019, paired t-test). There was no change in the group plasma potassium, urea and creatinine. Collectively these results indicate a stabilisation in renal function among the management group. Blood pressure (both lying and standing) fell significantly in the study period. The imprecision for urine ACR quality control POCT conducted during the management program was within nationally and internationally accepted precision goals for urine albumin, creatinine and ACR. Fifty community members completed the satisfaction questionnaire. Three-quarters of respondents felt there were no cultural barriers in providing a urine sample for urine ACR POCT. CONCLUSIONS: The management arm of the Umoona Kidney Project was effective in stabilising the renal function and improving the blood pressure of community members identified to be at greatest risk of kidney disease. POCT urine ACR testing can be utilised, not only for community risk assessment, but also for patient management. The Umoona Kidney Project was well accepted by the health service and community members.

HOW PHYSICIAN LEADERS CAN SUCCESSFULLY RESOLVE TEAM CONFLICTS.

Examine how physician leaders can become more knowledgeable about assessing the nature of health care team conflicts and intervening with teams to improve their collaboration skills.

Cytomegalovirus and blood transfusion.

The problem of transfusion-transmitted cytomegalovirus (CMV) infection differs from that for other transfusion-transmitted infections in that only patients who are immunocompromised require CMV-free blood or components. The virus is cell-associated and transmission appears to be due to reactivation of latent virus in white blood cells. As a herpes virus, CMV can be responsible for primary infections, reactivations or reinfections in humans. The use of restriction endonuclease techniques is sometimes necessary to pinpoint the origin of infections. Serological studies have shown that CMV infection is worldwide, but seropositivity rates vary widely being highest in underdeveloped countries, rising both with age and lower socio-economic status. Provision of CMV seronegative blood therefore involves considerable administrative as well as laboratory effort and planning, especially if panels of previously tested, seronegative donors are organized. Serious complications of transfusion-transmitted CMV infection (which can occasionally prove fatal) are only seen with immuno-suppressed patients (commonly low birth weight infants or transplant recipients). Prevention or amelioration of CMV infection with appropriate patients can be attempted by reducing the number of white blood cells present in blood or components by filtration or washing, administration of CMV immune globulin or provision of blood found by serological screening to be CMV-seronegative.

Post-transfusion NANBH in the light of a test for anti-HCV.

The incidence of post-transfusion hepatitis (PTH) varies over an order of magnitude in different parts of the world. For example, prospective studies from Spain and the UK reveal rates of PTH of approximately 10 and 0.5% respectively. Similarly the association of a history of transfusion in patients with chronic liver disease varies widely; in Japan, with high rates of PTH, the association appears obvious whereas in the UK less obvious. These factors must be taken into account when assessing the cost-effectiveness of pre-transfusion screening for anti-HCV. A useful approach to assessing the value of screening donors for anti-HCV is to study prospectively the correlation of anti-HCV and PTH. In carefully selected cases of PTH, the correlation of anti-HCV and PTH in donor-recipient sets of samples may be very high. However, the predictive value of 'first-generation' assays for anti-HCV in routine studies of unselected cases of PTH may be less than 20% in countries with low rates of transfusion-transmitted non-A, non-B hepatitis (NANBH). The anti-HCV screening tests and supplementary assays are continually evolving. More recent assays incorporate structural as well as non-structural antigens in both types of ELISA used for screening and in the supplementary tests such as the recombinant based immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS)

Islet allograft rejection can be mediated by CD4+, alloantigen experienced, direct pathway T cells of TH1 and TH2 cytokine phenotype.

BACKGROUND: It is widely believed that Thl cells that secrete interferon-gamma are primarily involved in the rejection of allografts whereas Th2 cells [interleukin(IL) 4 and IL-10] are thought to be protective of this process. However, the exact role and specificity of these helper T lymphocytes in mediating allograft damage is presently unknown. METHODS: Th0, Th1, and Th2 cell lines specific for the class II MHC molecule H2IAb were adoptively transferred into T cell deficient, syngeneic, diabetic mice before transplantation of fully allogeneic C57BL/10 (H2b) or (CBKxBALB/c)F1 (H2k/d+Kb) islet grafts. T cells were 5-(and-6-)-carboxyfluorescein diacetate succinimidyl ester- (CFSE) labeled to allow detection, immunohistochemistry was performed, and IL-4 transcripts within the rejected islet grafts were quantified by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Adoptive transfer (IV) of Th0-, Th1-, and Th2 IAb-specific T cells resulted in rejection of H2b islet allografts. CFSE-labeling demonstrated that these T cells were able to home to the graft site. CD4+ T cells and CD11b+ macrophages were present within the graft after adoptive transfer of both Thl and Th2 cells. Interestingly, CD8+ T cells and B cells were absent from these rejecting grafts. Even when Th2 cells were introduced directly at the graft site, prompt rejection was still observed despite the presence of increased IL-4 mRNA expression within the islet allografts. CONCLUSIONS: Th2 and Th0 alloreactive CD4+ T helper cells can reject islet grafts with similar efficiency to Th1 cells. These results suggest that deviation of the immune response from a Th1 to Th2 phenotype will not be sufficient to allow successful engraftment of allogeneic organs or tissues.

The visualization of T cell responses.

Transplanting allogeneic grafts is still significantly hampered by the rejection process, despite the use of powerful immunosuppressive agents. The T cell is recognized as playing a central role in the process of rejection, and it is believed that graft tolerance will ultimately be achieved by immunological manipulation of this cell (1, 2). As immunologists strive to define the role of the T cell in the fundamental processes of immunity and tolerance, new methods are emerging that will facilitate visualization of the T cells directly involved in the rejection response (3, 4). This overview addresses the visualization of T cell responses as made possible by these technological developments.

Suppression mediated by anergic CD4+ T cells requires stimulation by MHC-peptide complexes and can be induced in the presence of costimulation.

BACKGROUND: In this study, we have investigated the mechanisms involved in both the induction of suppressive anergy, the stability of the anergy induced, and the possible mechanisms by which the response of immunocompetent CD4+ T cells are suppressed. METHODS: We used immobilized anti-CD3 monoclonal antibody (mAb) to induce anergy in T helper (Th) 1 and Th0 cells reactive with MHC class II molecule H2 I-Ab. RESULTS: We observed that suppressive anergy was induced independently of costimulation in Th0 but not Th1 cells. Although the anergic and suppressive states of Th0 cells were stable in the presence of exogenous interleukin-2, this was not the case for Th1 cells. No evidence for linked epitope suppression was observed for any of the I-Ab reactive cells investigated. Neither anergy nor suppression was observed in Th0 cells upon restimulation with anti-CD3 in the presence of syngeneic antigen-presenting cells (APCs). However, anergy but not suppression was observed in co-cultures restimulated with anti-T-cell antigen receptor (TCR) mAbs/syngeneic APCs and suppression could be restored by the addition of I-Ab+ APCs. CONCLUSIONS: Overall, these data suggested that the MHC-peptide complex recognized by the Th0 cells was required for suppression of the response of immunocompetent cells. We propose that suppression is mediated either by down-modulation of the MHC-peptide complex recognized by the anergic T cells or that a molecule specific to the MHC-peptide/TCR interaction facilitates negative regulation by APC:T or T:T interactions.

HBV DNA levels and transmission of hepatitis B by health care workers.

BACKGROUND: Laboratory-based study funded by the Research and Development Division of the Department of Health to inform the decision making on guidelines for the conduct of exposure prone procedures (EPPs) by health care workers who are hepatitis B carriers. OBJECTIVES: Define the quantity and nature of hepatitis B virus (HBV) DNA in hepatitis carriers whose serum does not contain hepatitis B e antigen (HBeAg) and in surgeons previously cleared to conduct EPPs who have transmitted HBV to their patients. STUDY DESIGN: Cross-sectional survey using HBV DNA quantification, genotyping and sequencing comparing transmitting surgeons and asymptomatic carriers. RESULTS: HBV DNA could be detected and quantified in 64.5% (136 of 211) of carriers whose serum did not contain HBeAg with a median level 3.6 log(10) copies/ml (range of 5.7 log(10) copies). Pre-core mutation appeared not to affect the HBV DNA level, however, all surgeons carried codon 28 variants and transmitted these variants to their patients. The lowest HBV DNA level in a transmitting surgeon was 4 x 10(4) copies/ml. CONCLUSIONS: Pre-core mutations are common in carriers whose serum does not contain HBeAg and do not specifically identify carriers whose HBV DNA levels are high. It was possible to define a level of virus above which transmission of hepatitis B during conduct of EPPs could not be excluded.

Safety aspects of cord blood banking.

The safety of cord blood for transplantation depends upon a considered approach to donor selection, testing and processing of donations. It should be undertaken within a total quality system and good manufacturing practice facilities. Protocols should be developed based upon risk assessment and cost efficiency. In this context the retesting of donors for HIV is considered and the risk of a serological window period HIV transmission by cord blood illustrated to be minimal compared to the risks of transplant procedures or of not having a donor.

Challenges in transfusion microbiology.

PIP: Transfusion microbiology must minimize the risks of transfusion transmitted infection (TTI) in a cost-effective and efficient manner. The wide range of transmissible microbial agents exhibits different relative risks in different circumstances; therefore, the same safety rules are not always applicable. TTIs can also exist as asymptomatic diseases in their hosts, so donors must be screened for high-risk behavior. Then blood must be screened by detecting antibodies to infectious agents. Since antibody reactivity is difficult to confirm, and cross-reactive or nonspecific effects are possible, a reactive sample can be tested by a range of assays, each based on a different immunoassay principle, or by a supplementary assay. Despite the advanced nature of these tests, problems of indeterminate donors remain, and a compromise must be made between the sensitivity and specificity of screening assays; maximum sensitivity protects transfusion recipients, but specificity prevents wasting blood or making false reports about donor health. An ideal screening assay would have maximum sensitivity, optimal specificity, simplicity, objectivity, standard format, rapid processing time, safety of reagents, and economy. Assay users must chose the best combination of available characteristics for their particular situation. For example, a French study found that 30% of seronegative, but HIV-infectious, donors are anti-HBc positive; this test would be too specific, however, in a country where hepatitis B is common. Combined assays are being used to screen for anti-HIV-1 and -2 and anti-human T-cell lymphotropic virus-I and -II; this should save time and money. Screening donors selectively (for example, Latin American migrants to North America for Trypanosoma cruzi) may protect previously unexposed populations. In some countries, transmission of HIV via seronegative blood is a problem, and HIV antigen screening and confirmation of reactivity by neutralization should occur as soon as reagents become less expensive. As test methodology continues to move through rapid developmental "generations," the development of sensitive techniques to detect nucleic acids holds promise. Several problems will remain to be solved including cost cross-contamination and carry-over in automated sampling systems, and operational and clerical errors. While there are methods to inactivate viruses in plasma pools, inactivation must incur a minimal loss of biological activity. All of these challenges are exacerbated in developing countries by a lack of resources. As physicians become knowledgeable about when to use transfusion and when to use an alternative method, such as blood salvage, refusing transfusion will be more risky than contracting a TTI.^ieng

Post transfusion septicaemia 1980-1989: importance of donor arm cleansing.

AIMS: To determine the prevalence of Pseudomonas fluorescens on the arms of blood donors, and to elucidate one possible cause for its predominance (60% of cases during 1980-89) in exogenous post transfusion septicaemia (PTS). METHODS: Skin swabs were taken from the arms of 782 blood donors and cultured on to heated blood agar. After incubation, Oxidase reagent and the Gram stain were used to select non-fermentative Gram negative rods, which were then subcultured and identified using the Analytical Profile System (API) 20 NE system. RESULTS: Non-fermentative Gram negative rods were found on the arms of 11.7% of donors, Pseudomonas spp on 1.0%, and Ps fluorescens on the arms of 0.3% of donors. CONCLUSIONS: This evidence emphasises the absolute requirement for efficient skin cleansing of blood donors' arms to minimise the risk of exogenous PTS.

A sensitive single reverse passive haemagglutination test for detecting both HBsAg and anti-HBs.

A trial of a modified reverse passive haemagglutination test for HBsAg using a 0.1% cell suspension instead of the recommended 1% showed an approximately eight-fold increase in detection sensitivity. The test can be performed within 30 minutes and lends itself to mass screening techniques. Confirmation tests can be done using the 0.1% method. In addition, the same serological plates and cells used for HBsAg screening can then be used to screen for high-titre anti-HBs. This makes the overall screening for both HBsAg and high-titre anti-HBs donors cheap and convenient.

Diagnostic reagents for hepatitis C virus.

The development of diagnostic methods for hepatitis C virus is presented. Special attention is paid to the selection of antigenic markers, the type of assay selected and the interpretation of results. A few of the pitfalls and ambiguities of various assays are discussed and possible future methods are described.

A comparison of four enzyme immunoassays for the simultaneous detection of HIV-1- and HIV-2-specific antibody.

The performance of four HIV 1 and 2 combined assays has been compared with current type-specific assays using three panels of sera. The first panel comprised single samples from 19 HIV-1-infected persons; the second panel comprised 19 sera from 16 HIV-2-infected persons. Samples from both these panels were titrated across interpolated end points of detectability. The third panel comprised sera from 5200 consecutive blood donors. The four combined assays, manufactured by Abbott Laboratories, Behring Laboratories, Diagnostics Pasteur and Wellcome Diagnostics Laboratories detected all anti-HIV-1 sera at high dilution; the immunometric assay from Wellcome was particularly proficient. All assays were broadly similar in their ability to detect sera from recently infected persons. The same assays were also effective in detecting anti-HIV 2 in sera both from seropositive individuals and from a single recently-infected person, though none was as sensitive as an inhouse competitive EIA. When used for donor screening the repeat reactive rates for donors negative for HIV 1 and 2 antibodies ranged between 1.80% for Elavia Mixt from Pasteur, 0.27% Abbott combined and 0.15% for the Wellcome combined assays.