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Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the central nervous system that results in demyelination of axons, inefficient signal transmission and reduced muscular mobility. Recent findings suggest that B cells play a significant role in disease development and pathology. To further explore this, B cell profiles in peripheral blood from 28 treatment-naive patients with early MS were assessed using flow cytometry and compared to 17 healthy controls. Conventional and algorithm-based analysis revealed a significant increase in MS patients of IgA+ memory B cells (MBC) including CD27+, CD27- and Tbet+ subsets. Screening circulating B cells for markers associated with B cell function revealed a significantly decreased expression of the B cell activation factor receptor (BAFF-R) in MS patients compared to controls. In healthy controls, BAFF-R expression was inversely associated with abundance of differentiated MBC but this was not observed in MS. Instead in MS patients, decreased BAFF-R expression correlated with increased production of proinflammatory TNF following B cell stimulation. Finally, we demonstrated that reactivation of Epstein Barr Virus (EBV) in MS patients was associated with several phenotypic changes amongst MBCs, particularly increased expression of HLA-DR molecules and markers of a T-bet+ differentiation pathway in IgM+ MBCs. Together, these data suggest that the B cell compartment is dysregulated in MS regarding aberrant MBC homeostasis, driven by reduced BAFF-R expression and EBV reactivation. This study adds further insights into the contribution of B cells to the pathological mechanisms of MS, as well as the complex role of BAFF/BAFF-R signalling in MS.

B cells are critical to the development of multiple sclerosis (MS), but the mechanisms by which they contribute to the disease are poorly defined. We hypothesised that the expression of CD32b (FcγRIIb), a receptor for the Fc region of IgG with inhibitory activities in B cells, is lower on B cell subsets from people with clinically isolated syndrome (CIS) or MS. CD32b expression was highest on post-naive IgM+ B cell subsets in healthy controls. For females with MS or CIS, significantly lower CD32b expression was identified on IgM+ B cell subsets, including naive and IgMhi MZ-like B cells, when compared with control females. Lower CD32b expression on these B cell subsets was associated with detectable anti-Epstein Barr Virus viral capsid antigen IgM antibodies, and higher serum levels of B cell activating factor. To investigate the effects of lower CD32b expression, B cells were polyclonally activated in the presence of IgG immune complexes, with or without a CD32b blocking antibody, and the expression of TNF and IL-10 in B cell subsets was assessed. The reduction of TNF but not IL-10 expression in controls mediated by IgG immune complexes was reversed by CD32b blockade in naive and IgMhi MZ-like B cells only. However, no consequence of lower CD32b expression on these cells from females with CIS or MS was detected. Our findings highlight a potential role for naive and marginal zone-like B cells in the immunopathogenesis of MS in females, which requires further investigation.

Altered composition of gut bacteria and changes to the production of their bioactive metabolites, the short-chain fatty acids (SCFAs), have been implicated in the development of multiple sclerosis (MS). However, the immunomodulatory actions of SCFAs and intermediaries in their ability to influence MS pathogenesis are uncertain. In this study, levels of serum SCFAs were correlated with immune cell abundance and phenotype as well as with other relevant serum factors in blood samples taken at first presentation of Clinically Isolated Syndrome (CIS; an early form of MS) or MS and compared to healthy controls. There was a small but significant reduction in propionate levels in the serum of patients with CIS or MS compared with healthy controls. The frequencies of circulating T follicular regulatory cells and T follicular helper cells were significantly positively correlated with serum levels of propionate. Levels of butyrate associated positively with frequencies of IL-10-producing B-cells and negatively with frequencies of class-switched memory B-cells. TNF production by polyclonally-activated B-cells correlated negatively with acetate levels. Levels of serum SCFAs associated with changes in circulating immune cells and biomarkers implicated in the development of MS.

Objectives At the end of a 60‐day course of narrowband UVB phototherapy, administered to individuals with early multiple sclerosis, there were changes in the relative proportions of circulating B‐cell subsets. This study investigated phototherapy‐associated changes to cytokine responses of B cells when exposed to a TLR7 ligand. Methods PBMCs from participants of the PhoCIS (Phototherapy for Clinically Isolated Syndrome) trial taken before (day 1) and after phototherapy for 8 weeks (day 60) were incubated with, or without, the TLR7 ligand, R848, for 18 h. Production of TNF and IL‐10 in seven B‐cell subsets was examined, with cytokine responses in each individual at day 60, adjusted for responses at day 1. Paired PBMCs were from participants administered phototherapy (n = 7) or controls (n = 6). Results At day 60, significantly fewer B cells, particularly marginal zone‐like B cells (CD27+/IgD+), from participants administered phototherapy produced TNF in response to TLR7 stimulation. When responses by seven B‐cell subsets were analysed together using multivariate methods, a phototherapy‐specific signature was observed. An increased responsiveness from day 1 to day 60 in IgM‐only memory B cells (CD27+/IgD−/IgM+) after TLR7 stimulation also predicted slower progression from CIS to MS. Phototherapy was without significant effect on B‐cell IL‐10 production. Conclusions Reduced TNF responses after TLR7 stimulation in marginal zone‐like B cells from participants administered phototherapy suggested treatment‐associated priming effects that were detected upon subsequent polyclonal B‐cell activation. Changes in responsiveness to TLR7 stimulation also suggested that IgM‐only memory B cells may be important in conversion from CIS to MS.

Background: UV radiation exposure has immunosuppressive effects in humans. Low environmental UV radiation exposure is associated with multiple sclerosis risk. Narrowband UVB phototherapy is an established treatment for psoriasis, and is under investigation in people with clinically isolated syndrome (CIS) at risk of developing MS. Objective: To determine whether systemic leukocyte phenotypes in blood are significantly altered by a short course of narrowband UVB phototherapy in people with CIS. Methods: 20 participants with CIS were recruited and half received narrowband UVB phototherapy in 24 sessions over 2 months. Peripheral blood mononuclear cells (PBMC) were collected at baseline, and 1, 2, 3, 6 and 12 months after enrolment. Multicolour flow cytometry was used to identify T cell, B cell, monocyte, dendritic cell, and natural killer cell subsets. The effects of phototherapy on the frequencies of PBMCs were examined using linear regression. Results: Significant differences in the frequencies of PBMC phenotypes were detected between treated and untreated individuals during phototherapy, including in subsets of B cells, monocytes and T cells. No effects were detected after phototherapy treatment ceased. The largest treatment effects were detected in B cell subsets, where phototherapy was associated with significantly higher naïve B cell frequency and significantly lower frequency of switched memory B cells. Conclusions: Narrowband UVB phototherapy may contribute to immune suppression through regulation of systemic leukocyte phenotypes in people with CIS.

Clinically isolated syndrome (CIS) is the earliest clinical episode in multiple sclerosis (MS). Low environmental exposure to UV radiation is implicated in risk of developing MS, and therefore, narrowband UVB phototherapy might delay progression to MS in people with CIS. Twenty individuals with CIS were recruited, and half were randomised to receive 24 sessions of narrowband UVB phototherapy over a period of 8 weeks. Here, the effects of narrowband UVB phototherapy on the frequencies of circulating immune cells and immunoglobulin levels after phototherapy are reported. Peripheral blood samples for all participants were collected at baseline, and 1, 2, 3, 6 and 12 months after enrolment. An extensive panel of leukocyte populations, including subsets of T cells, B cells, monocytes, dendritic cells, and natural killer cells were examined in phototherapy-treated and control participants, and immunoglobulin levels measured in serum. There were significant short-term increases in the frequency of naïve B cells, intermediate monocytes, and fraction III FoxP3+ T regulatory cells, and decreases in switched memory B cells and classical monocytes in phototherapy-treated individuals. Since B cells are increasingly targeted by MS therapies, the effects of narrowband UVB phototherapy in people with MS should be investigated further.

Clinically isolated syndrome (CIS) is a first episode of neurological symptoms that may precede a diagnosis of multiple sclerosis (MS). Therefore, studying individuals with CIS may lead to breakthroughs in understanding the development and pathogenesis of MS. In this study, serum levels of immunoglobulin (Ig)G, IgA, IgM, and IgG1–4 were measured in 20 people with CIS and compared with those in 10 healthy controls (HC) and 8 people with MS. Serum Ig levels in individuals with CIS were compared with (a) the time to their conversion from CIS to MS, (b) serum levels of antibodies to Epstein–Barr virus, (c) frequencies of T regulatory (Treg), T follicular regulatory (Tfr), and B cell subsets, and (d) Treg/Tfr expression of Helios. Serum IgG, IgM, and IgG2 levels were significantly lower in people with CIS than HC, and IgG, IgM, and IgG1 levels were significantly lower in people with CIS than MS. After adjusting for age, sex, and serum 25(OH) vitamin D3 [25(OH)D] levels, CIS was associated with lower serum levels of IgG and IgG2 compared with HC (p = 0.001 and p < 0.001, respectively). People with MS had lower IgG2 levels (p < 0.001) and IgG2 proportions (%IgG; p = 0.007) compared with HC. After adjusting for age, sex, and 25(OH)D, these outcomes remained, in addition to lower serum IgA levels (p = 0.01) and increased IgG3 levels (p = 0.053) in people with MS compared with HC. Furthermore, serum from people with MS had increased proportions of IgG1 and IgG3 (p = 0.03 and p = 0.02, respectively), decreased proportions of IgG2 (p = 0.007), and greater ratios of “upstream” to “downstream” IgG subclasses (p = 0.001) compared with HC. Serum IgG3 proportions (%IgG) from people with CIS correlated with the frequency of plasmablasts in peripheral blood (p = 0.02). Expression of Helios by Treg and Tfr cell subsets from individuals with CIS correlated with levels of serum IgG2 and IgG4. IgG3 levels and proportions of IgG3 (%IgG) in serum at CIS diagnosis were inversely correlated w

Antibody responses have not been fully characterised in chronically HIV/HCV patients receiving antiretroviral therapy (ART). Seventeen HIV/HCV patients receiving ART were followed for a median (range) interval of 597 (186–766) weeks. Prior to ART, HIV/HCV patients had lower levels of antibodies reactive with HCV core and JFH-1, and lower genotype cross-reactive neutralising antibodies (nAb) titres, than HCV patients. Levels of JFH-1 reactive antibody increased on ART, irrespective of CD4+ T-cell counts or changes in serum ALT levels. The appearance of nAb coincided with control of HCV viral replication in five HIV/HCV patients. In other patients, HCV viral loads remained elevated despite nAb responses. Sustained virological responses following HCV therapy were associated with reduced antibody responses to JFH-1 and core but elevated responses to non-structural proteins. We conclude that nAb responses alone may fail to clear HCV, but contribute to control of viral replication in some HIV/HCV patients responding to ART.

Type I interferons (IFNs) exert anti-viral effects through the induction of numerous IFN-stimulated genes and an immunomodulatory effect on innate and adaptive immune responses. This is beneficial in controlling virus infections but prolonged IFN-α activity in persistent virus infections, such as HIV infection, may contribute to immune activation and have a detrimental effect on the function of monocytes and T and B lymphocytes. Activation of monocytes, associated with increased IFN-α activity, contributes to atherosclerotic vascular disease, brain disease and other ‘age-related diseases’ in HIV patients treated with long-term antiretroviral therapy (ART). In HIV patients receiving ART, the anti-viral effects of IFN-α therapy have the potential to contribute to eradication of HIV infection while IFN-α inhibitor therapy is under investigation for the treatment of immune activation. The management of HIV patients receiving ART will be improved by understanding more about the opposing effects of IFN-α on HIV infection and disease and by developing methods to assess IFN-α activity in clinical practice.

Objectives: To examine the relationship between plasma markers of microbial translocation and antibodies to lipopolysaccharide (LPS) and circulating memory B cells in patients with HIV infection. Design: Cross-sectional study in antiretroviral therapy (ART)-naive (n = 23) and ART-treated (n = 27) HIV patients. Methods: Antibodies to LPS and immunoglobulins, assayed in stored serum, and matched memory B-cell counts were correlated with levels of LPS and bacterial 16S ribosome DNA (16S rDNA), assayed in stored plasma. Results: In ART-naive patients, plasma LPS levels correlated inversely with serum levels of IgG and IgA antibodies to LPS (P = 0.03 and 0.006, respectively), serum levels of IgA anti-LPS correlated with total IgA (P < 0.0001) and levels of IgG anti-LPS correlated with IgM+ memory B-cell counts (P = 0.025). In ART-treated patients, plasma LPS levels were not related to levels of LPS antibodies, but were related to CD4+ T-cell and switched memory B-cell counts. There were no correlations with plasma levels of 16S rDNA. Conclusion: Plasma LPS levels were associated with antibody and possibly B-cell responses to LPS in ART-naive HIV patients, whereas they were associated with the degree of immune reconstitution in ART-treated patients.

Objectives: To describe characteristics and management of people with community acquired needle stick injuries (CANSI) attending urban emergency departments; and suggest a guideline to improve assessment, management, and documentation. Methods: A retrospective analysis of cases with CANSI attending emergency departments in two tertiary hospitals between 2001 and 2005 using medical record review with follow up phone and written survey. Results: Thirty-nine cases met the criteria for CANSI. Persons younger than 30 years sustained 48.72% of all injuries. Source serology was available for only five cases (12.82%). Thirty-one of thirty-nine patients (79.49%) were classed as not immune to hepatitis B but only four of these (12.90%) received both hepatitis B vaccination and hepatitis B immunoglobulin. Six patients (15.38%) received HIV prophylaxis; of which two (33.33%) did not receive baseline HIV testing. Of ten patients referred to immunology clinic for follow up only two (20.00%) attended at 6 months. Conclusion: We have identified groups that are at high risk of CANSI, including young males, security workers and cleaners. In the majority of cases protection against hepatitis B was inadequately provided, and a substantial proportion had inadequate baseline assessment and documentation. A guideline is suggested that may be used to improve these deficits. What is known about this topic? Occupationally acquired needle stick injury guidelines are well established, but no guidelines currently exist for community acquired needle stick injuries (CANSI) which may require different risk stratification, assessment and management. Management of CANSI in Emergency Departments has not been well described. What does this paper add? An audit of Emergency Department management of community acquired needle stick injuries demonstrates deficits in risk assessment, documentation and use of post-exposure immunisation and prophylaxis. A guideline is suggested that may be used to improve the

Allogeneic hematopoietic stem cell transplant (HSCT) recipients were assessed to elucidate memory B cell defects underlying their increased susceptibility to infections, particularly by encapsulated bacteria. Circulating IgM memory B cells (CD19+, CD27+, IgM+) and switched memory B cells (CD19+, CD27+, IgM-) were enumerated in allogeneic HSCT recipients (n = 37) and healthy controls (n = 35). T lymphocyte subpopulations and serum levels of immunoglobulins, including IgG subclasses, and antibodies to pneumococcal polysaccharides were also assayed. Allogeneic HSCT recipients were deficient in both switched memory and IgM memory B cells compared to healthy controls (both P < .0001), irrespective of time post-HSCT. Switched memory B cell deficiency correlated with CD4+ T cell deficiency, and both correlated with serum levels of IgG1 (P < .0001), possibly reflecting impaired B cell isotype switching in germinal centres. "Steady-state" serum levels of antibodies to pneumococcal polysaccharides did not correlate with circulating memory B cells. Graft-versus-host disease (GVHD) was associated with lower IgM memory B cell counts and lower serum levels of IgG2, IgG4, IgA, and pneumococcal antibodies. The increased susceptibility of allogeneic HSCT patients to infection may reflect a combination of memory B cell defects, which are most common in patients with a history of GVHD.

OBJECTIVE: To determine if the depletion of IgM memory B cells might contribute to the increased susceptibility of HIV patients to pneumococcal infection, memory B-cell subpopulations were investigated in HIV patients, including patients receiving antiretroviral therapy (ART). METHODS: Blood B cells with the phenotype of IgM memory B cells (CD27, IgM) and switched memory B cells (CD27, IgM) were measured in antiretroviral-treated (n = 32) and untreated (n = 24) HIV patients and non-HIV controls (n = 35). Serum levels of IgG and IgG2 antibodies to pneumococcal polysaccharides, IgG, IgG subclasses, IgM and IgA were also assayed in HIV patients. RESULTS: Switched memory B-cell counts were lower than controls in HIV patients (P < 0.01) irrespective of antiretroviral status and correlated with CD4 T-cell counts (r = 0.56, P = 0.001) in treated patients. In untreated patients, IgM memory B-cell counts correlated with CD4 T-cell counts (r = 0.73, P < 0.0001) reflecting higher values than controls in patients with CD4 T-cell counts greater than 300 cells/μl (P = 0.004) and lower values than controls in patients with CD4 T-cell counts below 300 cells/μl (P = 0.0001). There was no relationship between serum levels of pneumococcal antibodies and IgM or switched memory B cells. CONCLUSION: The depletion of IgM memory B cells in untreated HIV patients with a CD4 T-cell count below 300 cells/μl might be a risk factor for pneumococcal infection. The depletion of switched memory B cells is a complication of HIV infection irrespective of ART and might contribute to impaired IgG antibody responses. Memory B-cell subpopulations might predict the risk of pneumococcal sepsis more accurately than the CD4 T-cell count or pneumococcal antibody levels.

Objectives: HIV-infected patients responding to combination antiretroviral therapy (ART) after experiencing severe immunodeficiency may exhibit persistent immune defects and occasionally experience opportunistic infections (OIs) despite increased CD4 T-cell counts. The investigation of immune defects in such patients was examined in this study. Methods: CD4 effector memory T-cell (T em-cell) function [assessed by blood cytomegalovirus (CMV) interferon-γ (IFN-γ) enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) counts] and B-cell dysregulation [assessed by serum immunoglobulin A (IgA) and IgE levels] were examined in 27 patients with increased CD4 T-cell counts after receiving ART for over 2 years. Two of these patients and one other had developed OIs on ART and are described in detail. Results: Serum levels of IgA and IgE were higher than reference intervals (P<0.001) and CMV IFN-γ ELISPOT counts were lower than those in non-HIV-infected controls (P<0.001) in the HIV-infected patients. Low CMV IFN-γ ELISPOT counts were associated with high IgA levels (r = -0.5, P = 0.01, Spearman's correlation test) and segregated with high IgE levels (P = 0.06, Fisher's test). CMV IFN-γ ELISPOT counts and serum IgA and IgE levels did not change significantly over a median time of 35 (range 8-60) months after the first measurement, whereas CD4 T-cell counts increased. All three patients who experienced OIs had repeatedly low CMV IFN-γ ELISPOT counts and increased serum levels of IgA and/or IgE. Conclusion: Low CD4 T em-cell function and B-cell dysregulation are immune defects that may persist independently of changes in the CD4 T-cell count in HIV-1-infected patients responding to ART and are associated with an increased risk of developing an OI.

OBJECTIVE: To examine the relationships between blood CD4 natural regulatory T (Treg) cells, plasma HIV RNA level, CD4 T-cell count and immune activation in untreated HIV-infected patients and immunodeficient patients beginning antiretroviral therapy (ART), using a novel phenotype to define Treg cells (CD25CD127CD4). Data were compared with established Treg cell markers (FoxP3, CTLA-4 and GITR). METHODS: Twenty-nine untreated HIV-infected patients with CD4 T-cell counts of < 300 or > 400/μl were compared in a cross-sectional study and 12 patients beginning combination ART with < 100 CD4 T cells/μl were followed for 1 year on therapy. Three- and four-colour flow cytometry was used to quantitate proportions of Treg cells. RESULTS: In control donors and patients with high CD4 T-cell counts, 28-89% (median 60%) of CD25CD127CD4 cells were FoxP3, but < 10% expressed GITR or CTLA-4. Immunodeficient patients also had CD4-negative lymphocytes with the phenotype FoxP3CD127. Proportions of CD25CD127 cells and activated (HLA-DR) cells in the CD4 T-cell population were increased in patients with low CD4 T cell counts. The proportion of CD25CD127CD4 T cells correlated positively with plasma HIV RNA level and CD4 T-cell activation, but inversely with CD4 T-cell count. Longitudinal studies of 12 patients receiving ART in two distinct cohorts (Western Australia and Malaysia) showed that the proportion of CD25CD127CD4 cells decreased slightly over time, but remained above levels seen in non-HIV controls. CONCLUSIONS: Proportions of circulating T cells with a regulatory cell phenotype increase with HIV-associated immune activation and remain high after 1 year on ART.

Low-level production of interferon-γ (IFN-γ) marks human immunodeficiency virus (HIV)-induced immunodeficiency and has been ascribed to a bias towards T2 cytokines. This was investigated in two cross-sectional studies of HIV patients who were immunodeficient when they began antiretroviral therapy (ART) and had stable increases in CD4 T-cell counts. Blood leucocytes were assessed unstimulated or after stimulation with cytomegalovirus (CMV), anti-CD3 or mitogen. IFN-γ and interleukin (IL)-5 responses were initially assessed by enzyme-linked immunosorbent spot-forming cell assay (ELISPOT) and enzyme-linked immunosorbent assay (ELISA). We then adopted a sensitive reverse transcription–polymerase chain reaction (RT–PCR) system to assess IFN-γ, IL-5, IL-4 and IL-4δ2 (an inhibitory splice variant of IL-4) mRNA. The results were correlated with putative serological markers of a T1 [lymphocyte activation gene-3 (LAG-3), CD26] or a T2 [CD30, immunoglobulin E (IgE)] cytokine environment. IL-5 production and IgE levels were elevated in patients. IgE levels did not correlate with IFN-γ, but showed an inverse correlation with IL-5 released in culture (P = 0·05). The levels of IL-4, IFN-γ, IL-5 and IL-4δ2 mRNA were correlated after anti-CD3 stimulation, where IL-5 was the best predictor of IFN-γ mRNA (P = 0·006). Weak positive correlations were evident between CD30 and cytokine mRNA levels, whilst IgE correlated inversely with IL-4, IL-4δ2, IL-5 and IFN-γ mRNA levels. These analyses provide no evidence for an inverse relationship between T1 and T2 cytokine responses in HIV patients, but suggest that the elevation of IgE marks low cytokine responses.

We investigated whether polymorphisms in genes associated with HIV disease progression and/or immune activation affect CD4 + T-cell recovery in HIV patients who began combination antiretroviral therapy (ART) with advanced immunodeficiency and achieved stable control of plasma viremia. Patients with CD4 + T-cell counts <300 cells/μL (n = 33) and >400 cells/μL (n = 37) on ART were compared. A multiple case-control logistic regression associated carriage of BAT1(1,2) or interleukin (IL)6-174(2,2) with low CD4 + T-cell counts (P = 0.012). BAT1*2 uniquely marks the central major histocompatibility complex region of a conserved haplotype (HLA-A1,B8,BAT1*2,TNFA-308*2,DR3,DQ2). There was no association between alleles carried at CCR5Δ32, CCR5 59029, CCR5 59353, CCR2+190 (V64I), SDF1 3′UTR, IL1A+4845, IL1B+3953, IL4-589, IL10-592, IL10-R1+536, IL10-R1+1112, IL12B 3′UTR, TNFA-308, or TNFA-1031 and CD4 + T-cell counts. We suggest that immune activation and/or CD4 + T-cell apoptosis in HIV patients on effective ART is influenced by genetic factors.

Extended assessments of memory T-cell responses in HIV patients who have a satisfactory virological response to combination antiretroviral therapy (CART) have been limited by availability of longitudinal samples and of antigens to which most individuals (including HIV-negative controls) have been exposed. Studies of cytomegalovirus (CMV) show that interferon-gamma (IFN-;3) responses never recover completely, but this may be antigen-specific. Here we present responses to Candida and CMV antigens analyzed using a statistical approach that derives overall trends from samples collected at variable time points. Results were considered in relation to putative markers of T-regulatory cells. Blood mononuclear cells collected from seventeen HIV-1 patients (nadir <100 CD4 T cells/mL) 0 138 years after initiation of CART were stimulated with Candida spp lysate, Candida enolase protein or CMV lysate and production of IFN-;3 was assessed by ELISpot assay. CD4 T-cell counts increased fivefold and stabilized within 24 months on CART, following control of plasma viremia. IFN-;3 responses to Candida antigens began low and increased slowly, generating positive slope up to 60 months on CART (Candida enolase p=0.008; Candida lysate p=0.03; mixed-model Wald test). Only two patients displayed a CMV or Candida-specific IFN-;3 response above the median for seronegative controls. Proportions of T cells expressing CD25 or CD57 did not correlate with IFN-;3 responses. Slow reconstitution of IFN-;3 responses to CMV and Candida in previously immunodeficient patients with restored CD4+ T-cell counts on CART suggests a broad and nonresolving defect in memory T-cell responses.

The adverse effects of immune activation on CD4+ T-cell recovery and the relationship between CD4+ T-cell counts and effector T-cell function were examined in HIV-1 patients receiving long-term effective ART. Patients with nadir CD4+ T-cell counts <100/;Cl, > 12 months on ART and >6 months with <50 HIV RNA copies/ml were stratified by current CD4+ T-cell counts and patients from the lowest (n = 15) and highest (n = 12) tertiles were studied. We assessed proliferation (Ki67), activation (HLA-DR, CD38) and replicative senescence (CD57) by flow cytometry and CD4+ T-cell responses to CMV by IFN-;3 ELISpot. Proportions of CD4+ T-cells expressing HLA-DR or CD57 were strong univariate predictors of total (P = 0.0002 and P = 0.002) and naive (P < 0.0001 and P < 0.0001, respectively) CD4+ T-cell counts, suggesting that CD4+ T-cell activation drives the depletion of naive CD4+ T-cells. This was clearest in patients with a small/undetectable thymus. IFN-;3 responses to CMV were similar in patients with low or high CD4+ T-cell counts.

A novel technique for obtaining time-integrated 99Tc concentrations in seawater has been developed, using diffusive gradients in thin films (DGT). The performance of TEVA resin as a binding agent for 99Tc was investigated via laboratory experiments. The accumulated 99Tc activity per unit area of resin-gel was proportional to both the bulk solution activity and the exposure time for deployments of up to 2 weeks. The response of DGT was found to be independent of solution chemistries over the pH range 3-8 and ionic strength range 0.01-1.3 M. Seawater has pH 8 and ionic strength of approx. 0.7 M; therefore, the potential of the technique for field deployment in seawater was demonstrated. Detection limits of 0.05 and 0.025 Bq/L, for 2- and 4-week DGT deployments, respectively, were calculated for 99Tc measurement by liquid scintillation spectrometry. Using quadrupole ICPMS to measure bound 99Tc could reduce these detection limits to 0.125 mBq/L for a 4-week deployment. These detection limits are sufficiently low for monitoring contaminated environments, including the Irish Sea. This method is simpler and faster than other 99Tc analysis methods and represents the only means of obtaining timeintegrated data.

Objectives. To define the level of pathogen-specific immune reconstitution persisting over 3 to 5 years of highly active antiretroviral therapy (HAART) in HIV-infected patients who began therapy with CD4 T-cell counts below 50 cells/μL. Methods. Cytomegalovirus (CMV)-specific T-cell responses were analysed in adult HIV-1-infected patients with nadir CD4 T-cell counts below 50 cells/μL before HAART. CMV-specific CD4 T-cell responses were measured by interferon-γ enzyme-linked immunospot assay (ELISpot assay), lymphoproliferation and interferon-γ levels in cell culture supernatants. Results. CD4 T-cell responses to CMV were low in untreated patients and remained low during the first year on HAART, but increased progressively to levels similar to those found in HIV-seronegative CMV-seropositive controls at 3 years. Responses then declined markedly and at 5 years were lower than controls. This could not be explained by changes in CD4 or CD8 T-cell counts or plasma HIV RNA levels. Interferon-γ and interleukin-5 responses to a mitogen were maintained or elevated. Conclusions. CMV-specific CD4 T-cell responses were found to decline after 3-5 years on HAART and may provide inadequate long-term protection against CMV disease in patients who are severely immunodeficient prior to treatment.

Objective: Some HIV patients treated with highly active antiretroviral therapy (HAART) do not resolve their plasma viraemia or HIV RNA can reappear after a period of virological control. We investigate whether polymorphisms in cytokine genes affect the control of plasma HIV RNA over 5 years on HAART. Design: The study utilized adult HIV-infected patients in Western Australia. Plasma HIV-RNA levels were assessed from commencement of HAART in patients who had a CD4 T-cell count less than 100 cells/μl before HAART and achieved immune reconstitution assessed by CD4 T-cell counts. Results: Control of plasma viraemia could be predicted from carriage of allele 2 at position -889 in the IL1A gene (IL1A-889*2). This was significant when assessed by the proportion of patients with a plasma HIV-RNA level of 400 copies/ml or less (P = 0.002). At 48 months post-HAART, proportions were approximately 0.76, 0.51 and 0.32 for IL1A (1,1), (1,2) and (2,2) patients, respectively. The outcome was independent of the patients' CD4 T-cell counts before or on therapy, drug regimen or age. Polymorphisms in IL6, TNFA, IL1B or IL12B had less significant effects, which became marginal when IL1A was included in the statistical model. IL1A-889 was in linkage disequilibrium with a non-synonymous polymorphism at IL1A+4845. Conclusion: Alleles carried at IL1A-889 or IL1A+4845 may predict the control of HIV replication in previously immunodeficient patients responding to HAART.

Purpose: To compare inhibition of HIV replication, improvements in CD4+ T-cell counts, metabolic parameters, and body shape changes after 2 years of assigned therapy in OzCombo patients. Method: Study participants were those who were recruited into the open-label OzCombo 1 (1996/1997) and OzCombo 2 (1997/1998) trials. Patients in OzCombo 1 were randomized to receive indinavir in combination with zidovudine+lamivudine (AZT+3TC; n = 35), stavudine (d4T)+3TC (n = 34), or d4T+didanosine (ddI) (n = 37). OzCombo 2 patients were randomized to the same nucleoside reverse transcriptase inhibitor (NRTI) backbones with nevirapine (n = 20, 22, 23, respectively). The mean time-weighted changes from baseline in CD4 T-cell count/mL, HIV RNA (log copies/mL plasma), and proportions with detectable viral load (<500 copies plasma HIV RNA/mL) between NRTI arms over 2 years were compared by formal meta-analysis. A cross-sectional study of metabolic and body shape complications was also undertaken. Results: For the comparison of d4T+3TC and d4T+ddI to AZT+3TC, mean differences in time-weighted change from baseline in CD4 T-cell count/wL and log copies HIV RNA/mL adjusted for baseline CD4+ T-cell and HIV RNA counts were: m44 (p = .08) and m14 (p = .56) cells/wL and m0.1 (p = .40) and m0.1 (p = .6) copies/mL. Odds ratios for detectable viral load in the last study quarter were 0.6 (p = .44) and 1.0 (p = .95). The mean percent leg fat was lower in the d4T+3TC and d4T+ddI than the AZT+3TC arm (mean difference 5.1% [p = .07] and 7.6% [p = .02], respectively). Conclusion: For all regimens, virological control and immunological response were maintained over 2 years. Regimens containing d4T and particularly d4T+ddI were significantly associated with increased peripheral fat loss compared with AZT+3TC.

The objective of this study was to evaluate T cell responses in HIV-infected patients after highly active antiretroviral therapy (HAART), using four assays of immune function, and to determine which best reflects the presence of CD4+ T cells able to respond to CMV antigen. Peripheral blood mononuclear cells from 41 HIVinfected patients and 31 healthy HIV-seronegative controls were cultured with mitogen (PMA/Ca2+ ionophore) or antigen (CMV). Production of interferon γ (IFN-γ) determined by ELISpot assay was compared with lymphoproliferation, IFN-γ production assessed by ELISA, and CD69 expression and intracellular IFN-γ assessed by flow cytometry. Cells from patients whose CD4+ T cells counts increased 4-fold or to >200 cells/μl after HAART responded as well as control cells when assessed by IFN-γ production and CD69 expression after mitogenic stimulation, but lymphoproliferation responses were depressed by about 52%. Patients who did not meet these criteria for immune reconstitution had lymphoproliferative responses up to 30-fold lower than control subjects, while intracellular IFN-γ and CD69 expression and ELISpot counts were less than 3-fold lower. Responses to CMV antigen could not be detected by flow cytometry, but were readily detected by ELISpot in CMV-seropositive patients whose CD4+ T cell counts had increased after HAART. This included patients with low responses assessed by lymphoproliferation. Moreover, ELISpot responses measured with fresh and frozen cells were comparable, while lymphoproliferation assays required fresh cells. In conclusion, the ELISpot assay is a sensitive and efficient technique for detecting CMV-specific IFN-γ responses in samples that display poor responses when assessed by lymphoproliferation assays.

Background: To determine if infectious disease events in HIV-infected patients treated with highly active antiretroviral therapy (HAART) are a consequence of the restoration of pathogen-specific immune responses, a single-centre retrospective study of all HIV-infected patients commencing HAART prior to 1 July 1997 was undertaken to determine the incidence, characteristics and time of onset of disease episodes in HAART responders (decrease in plasma HIV RNA of > 1 log10 copies/mL). Methods: Baseline and post-therapy changes in CD4 T-cell counts and HIV RNA were compared in patients with and without disease and delayed-type hypersensitivity responses to mycobacterial antigens were measured in selected patients. Results: Thirty-three of 132 HAART responders (25%) exhibited one or more disease episodes after HAART, related to a pre-existent or subclinical infection by an opportunistic pathogen. Disease episodes were most often related to infections by mycobacteria or herpesviruses but hepatitis C virus (HCV), molluscum contagiosum virus and human papilloma virus were also implicated. They were most common in patients with a baseline CD4 T-cell count of < 50/uL and occurred most often during the first 2 months of therapy and when CD4 T-cell counts were increasing. Mycobacteria- and HCV-related diseases were associated with restoration of pathogen-specific immune responses. Conclusions: We conclude that improved immune function in immunodeficient patients treated with HAART may restore pathogen-specific immune responses and cause inflammation in tissues infected by those pathogens.

Periodontal attachment loss is mediated by overproduction of tumour necrosis factor (TNF) and interleukin (IL)-1, and appears to have a genetic component. The 8.1 major histocompatibility complex (MHC) ancestral haplotype (HLA-A1,B8,TNFA-308(2),DR3) is associated with elevated TNF production and predisposes carriers to several autoimmune/immunopathological disorders, including rapid progression of HIV disease, but not early onset periodontal disease in healthy individuals. Rather a high proportion of subjects with severe periodontal disease carry allele 2 at IL-1A-889 and IL-1B+3953. We predicted that genetic associations may be different or clearer in HIV patients, as they often show elevated production of TNF and IL-1 and periodontal attachment loss. Hence periodontal parameters and IL-1 polymorphisms were assessed in HIV-positive subjects expressing HLA-B8 with or without other markers of the 8.1 haplotype. Of 16 HLA-B8 subjects, 13 demonstrated elevated probing pocket depth and clinical attachment loss. The difference was statistically significant and did not correlate with smoking, age, CD4 T-cell counts, HIV viral load or levels of dental plaque. As TNFA-308 (allele 2) was present in four non-B8 subjects who had minimal attachment loss, it may not mediate the effect of the 8.1 haplotype. Moreover, polymorphisms at IL-1A-889 and IL-1B+3953 did not significantly affect periodontal parameters. Thus a central MHC gene characteristic of the 8.1 haplotype was the clearest determinant of periodontal attachment loss in HIV-infected individuals.xm.

No abstract available

To assess time trends in incidence of AIDS illnesses in Australia, a retrospective cohort of people diagnosed with AIDS from January 1, 1983 to December 31, 1994 in three HIV medicine units in Sydney, Melbourne, and Perth was established. Data on initial and subsequent AIDS illnesses were available for 2580 AIDS cases, or 45% of Australian AIDS notifications over the study period. Males represented 97.2% of the cohort, and HIV exposure category was homosexual contact for 89.9%. Subcohorts were formed by interval of AIDS diagnosis: 1983 through 1987, 1988 through 1990, and 1991 through 1994, with estimation of cumulative risk for each AIDS illness by the Kaplan-Meier method. The cumulative risk declined for Pneumocystis carinii pneumonia (PCP) (p < 0.0001) and for Kaposi's sarcoma (KS) (p < 0.0001); PCP cumulative risk estimates 2 years following AIDS diagnosis were 70% for people diagnosed with AIDS in 1983 through 1987 and 48% in 1991 through 1994, and KS cumulative risk estimates 2 years following AIDS diagnosis were 44% in 1983 through 1987 and 32% in 1991 through 1994. In contrast, cumulative risk increased from 34% to 40% for cytomegalovirus (CMV) disease (p = 0.005), from 47% to 50% for Mycobacterium avium complex (MAC) (p < 0.0001), and from 26% to 33% for esophageal candidiasis (p < 0.0001). Corresponding to this changing spectrum of AIDS illness has been an increase in severity of immunodeficiency at AIDS, with median CD4 cell count declining from 54 cells/mm3 in 1983 through 1987 to 34/mm3 in 1991 through 1994 (p = 0.002).

Objective: To explore trend in cumulative incidence of Kaposi's sarcoma (KS) and the level of immunodeficiency at KS diagnosis among people with AIDS in Australia. Setting: Three hospital-based HIV units. Study population: Retrospective cohort of 2580 people diagnosed with AIDS over the period 1983-1994, representing 45% of cases of AIDS in Australia over this period. Methods: Data including date and CD4 T-lymphocyte count of KS diagnosis was abstracted from medical records. KS occurring as both an initial and subsequent AIDS illness was included. Three subcohorts were defined based on interval of AIDS diagnosis: 1983-1987, 1988-1990, 1991-1994. Cumulative risk estimates for KS development were calculated by the Kaplan-Meier method. Results: KS was diagnosed in 716 people (27.8%), and in 451 (63%) of these as the initial AIDS illness. There was a decline over time in cumulative incidence of KS (P < 0.0005); the cumulative risk of KS at 1 year after AIDS diagnosis declined from 35% for those diagnosed with AIDS during 1983-1987 to 25% for 1991-1994. This decline was not due to a decline in homosexual HIV exposure category, and was independent of CD4 T-lymphocyte count at AIDS. In multivariate analysis independent risk factors for KS development were year of AIDS diagnosis (P = 0.003), male homosexuality (P = 0.003), and CD4 T-lymphocyte count at AIDS greater than 150x106/l (P = 0.02). A decline in median CD4 T-lymphocyte count at KS diagnosis was seen, from 67x106/l in 1984-1987 to 20x106/l for 1991-1994 (P < 0.0005). Conclusion: The decline in incidence and later occurrence of KS suggest several hypotheses, including declining prevalence or reduced virulence of a KS cofactor.

No abstract available

OBJECTIVE: To determine the incidence and nature of occupational exposures to blood and body fluids in health care workers. DESIGN: 332 reports of occupational exposure were analysed and are presented. SETTING: A major teaching hospital. PARTICIPANTS: All staff at Royal Perth Hospital who reported an occupational exposure to blood or body fluids to the Department of Clinical Immunology between 1 January 1990 and 31 August 1991. OUTCOME MEASURES: The rate of reported occupational exposure according to staff category, nature of exposure, HIV status of source patient, activity at the time of exposure and compliance with infection control measures. RESULTS: 332 reports from 323 health care workers were received, giving an overall incidence of 6.1 per 100 full time equivalent (FTE) years. Nursing staff (9.4/100 FTE years) and medical staff (9.0/100 FTE years) reported exposure more frequently than housekeeping staff (2.5/100 FTE years) or paramedical staff (2.3/100 FTE years) (P < 0.001). The rate of exposure to HIV antibody positive patients was only 0.24/100 FTE years. Needlestick or other blood contaminated sharps injuries accounted for 83.4% (277/332) of reports and failure to observe universal precautions for 34.0% of reports. Insertion and operation of parenteral lines (24%) and performing operations (15.4%) were the activities most often associated with occupational exposure. No occupationally acquired infections were observed. Despite the immediate availability of zidovudine, acceptance by health care workers with high risk occupational exposure was low (18.8%). CONCLUSIONS: Occupational exposure to blood and body fluids is common among health care workers but most exposures confer a low risk of blood borne infection. The introduction of an occupational exposure assessment program has many benefits, including optimal management of injuries and acquisition of data on infection control measures, and may protect health care institutions from false claims for compens

OBJECTIVE To determine the incidence of and reasons for late presentation of patients infected with the human immunodeficiency virus (HIV), and the demographic characteristics of these late presenters, who first seek medical attention when they have an illness that defines the acquired immunodeficiency syndrome (AIDS). DESIGN Retrospective analysis of clinical and demographic data relating to 106 sequential AIDS diagnoses in Western Australia. Surviving patients were questioned during routine medical and social work interviews to identify the reasons for late presentation. SETTING Royal Perth Hospital, the sole specialist referral centre for the management of infection with the human immunodeficiency virus (HIV) in Western Australia. PARTICIPANTS All patients presenting or referred to Royal Perth Hospital with an AIDS-defining condition between 1 January 1988 and 1 July 1991. RESULTS Forty-one of 106 (39%) patients presenting with an AIDS-defining condition had known of their HIV status for eight weeks or less (late presenters). The proportion of late presenters decreased from 61% in 1988 to 34% in 1989 (P less than 0.05). Six of the 41 late presenters died during their initial admission compared with only one of the 65 early presenters (P less than 0.02). Sixty-six per cent of late presenters compared with only 35% of early presenters had pneumocystis pneumonia as their AIDS-defining condition (P less than 0.01). There was no significant difference in age at diagnosis of AIDS, martial status, sex or risk factors between the early and late presentation groups. Reasons given for late presentation included lack of knowledge of advances in the treatment of HIV infection, concerns about confidentiality and beliefs that sexual behaviour had been relatively "safe" from risk of HIV infection. CONCLUSIONS While the proportion of patients presenting late is decreasing, late presentation continues to be a problem. Reasons for delayed presentation are not entirely clear, but c