Treatment outcomes in a cohort of patients with mucosal-predominant pemphigus vulgaris.

BACKGROUND: Pemphigus vulgaris (PV) is a rare autoimmune blistering condition. Treatment typically combines corticosteroids with another immunosuppressive agent, such as azathioprine, mycophenolate mofetil (MMF) or rituximab. AIM: This study aims to compare these second agents for their clinical efficacy and steroid-sparing effect. METHODS: This was a single-centre, retrospective observational cohort study of 21 patients with oral PV over a 6-year period, 18 of whom were newly diagnosed. Of the latter, the first 13 were initially given azathioprine, progressing to MMF and then rituximab on treatment failure, while the next five patients started directly on MMF. RESULTS: Of the 13 newly diagnosed patients, 2/13 were intolerant of azathioprine, and only 1/11 was controlled, with a median time to treatment failure (MTTF) of 254 days. MMF was given to 17 patients, either de novo (5) or after azathioprine (12), and was significantly more effective, controlling activity in 4/17 patients, and for a significantly longer time (MTTF 395 days, P = 0.019). All 13 patients failing MMF received rituximab, seven required a second dose, and three, a third dose. All patients responded, with 11/13 able to cease steroids. Control was maintained for a similar time to MMF (MTTF 364 days, P = NS). Rituximab also had the best steroid-sparing effect followed by MMF, then azathioprine. Side-effects were common with azathioprine, while the other two agents were well tolerated. CONCLUSION: Rituximab was the most effective of the three immunosuppressives for PV, although repeat dosing was frequently required. These observations have significant implications for the choice of drugs for this condition.

Vancomycin-associated drug reaction with eosinophilia and systemic symptoms syndrome.

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome is a rare but potentially life-threatening multi-system disorder characterised by the delayed onset of fever, rash and internal organ involvement following the administration of a drug. We report three definite cases of vancomycin-associated DRESS syndrome occurring and review the literature regarding this syndrome.

Circulating phenotypic B-1 cells are decreased in common variable immunodeficiency and correlate with immunoglobulin M levels.

B-1 cells are innate-like lymphocytes characterized by spontaneous production of 'natural' polyspecific antibodies, often of self-specificity, and thought to be responsible for tissue homeostasis, mucosal protection, maintaining resting serum immunoglobulin (Ig)M levels and for early immunoglobulin production following infection. Although defined most clearly in mice, a human B-1 cell counterpart, defined by the phenotype CD19 or 20(+) CD27(+) CD43(+) CD69 or 70(-) , has been proposed recently, facilitating a study of their role in human humoral immunodeficiencies, such as common variable immunodeficiency (CVID). This study examined circulating B-1 cells in 27 CVID patients in comparison to age-matched controls (n = 28). Phenotypic putative B-1 cell proportions varied widely, but there was an overall 60-70% decrease in CVID (0·039 ± 0·033% of lymphocytes, mean ± standard deviation) compared with controls (0·110 ± 0·159% of lymphocytes, P = 0·0012). This decrease was, however, explained largely by concomitant loss of total CD27(+) memory B cells characteristic of CVID, although those with higher memory B cell proportions appeared to show a true decrease. No age-related effects were apparent in B-1 cell proportions. However, among CVID patients, there was a strong positive correlation between the B-1 cell proportion and serum IgM levels, a relationship that was not evident for IgA, nor was there a relationship between memory B cell proportions and serum IgM. Patients with CVID have fewer circulating putative phenotypic B-1 cells, which largely reflected the overall decrease in memory B cells. However, B-1 cell proportions correlated with resting serum IgM levels, suggesting a possible role in IgM deficiency in CVID.

Prevalence of paraproteinaemia in older Australians.

BACKGROUND: Estimates of the prevalence of paraproteinaemia vary, from 1% in persons aged over 25 years to 10% in those aged over 80 years, although there are limited data from well-defined populations. We sought to determine the prevalence of paraproteinaemia in Australians aged 50 years and over, and to determine risks factors for its presence. METHODS: We performed a population-based, cross-sectional study using data and serum collected in the Blue Mountains Eye Study. Serum samples from 2933 patients were analysed by capillary zone electrophoresis and, where indicated, immunosubtraction, which allowed for both quantitation and isotype detection. RESULTS: A paraprotein was detected in 134 of the 2933 samples, giving an overall prevalence of 4.6% (95% confidence interval, 3.8-5.3%). The presence of a paraprotein was strongly age-related (P(trend) = 0.001), with a prevalence of 2.8% in persons aged 50-59 years, rising steadily to 9.1% in those aged 80 years and over. The prevalence was significantly higher in men (5.9%) compared with women (4.0%) (P= 0.03). CONCLUSION: We conclude that approximately one in 20 Australians aged 50 years or over harbours a paraprotein, a prevalence that appears higher than from similar cohorts in other countries.

Invariant natural killer (iNK) T cell deficiency in patients with common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a B cell immunodeficiency disorder characterized frequently by failure of memory B cell development and antibody secretion. A unifying cellular pathogenesis for CVID has not been forthcoming, but given the immunoregulatory role of invariant NK (iNK) T cells and their absence in several other immunodeficiencies, we quantified these cells in the blood of 58 CVID patients. There was a marked decrease in the proportion of iNK T cells in CVID patients compared with controls. This was particularly notable in those with low isotype-switched memory B cells, but subset analysis demonstrated no difference when stratified by specific clinical features. We propose that the decreased proportion of iNK T cells in CVID might be linked to the failure of memory B cell generation, which may contribute to reduced antibody production in these patients.

Decrease in phenotypic regulatory T cells in subsets of patients with common variable immunodeficiency.

Common variable immunodeficiencies (CVID) are a heterogeneous group of antibody deficiency disorders complicated by autoimmune, lymphoproliferative and/or granulomatous manifestations, suggesting variations in immunoregulation. We sought to quantify regulatory CD4 T cells (T(reg) cells) in the blood of CVID patients and to correlate the frequency with clinical manifestations and classification subgroups. Blood samples from 99 CVID patients in Freiburg, London and Sydney, who had been phenotyped clinically and stratified according to their memory B cell phenotype (Freiburg and Paris classification schemes), were analysed for the proportion of T(reg) cells, defined either as CD25(+)/forkhead box P3 (FoxP3)(+), CD25(+)/CD127(low)/FoxP3(+) or CD25(+)/CD127(low) CD4(+) T cells, and results compared with 49 healthy controls. Irrespective of the phenotype used to define them, there was a significant decrease in the T(reg) cell proportion in patients with granulomatous disease and immune cytopenias. This allowed the definition of a subgroup of CVID patients with abnormally low T(reg) cells, which had a higher rate of these two manifestations as well as autoimmune disease in general. There was also a significant reduction in the proportion of T(reg) cells in the Freiburg group Ia compared with other CVID patients and controls, but there were no differences between the Paris groups. The reduction in T(reg) cells in subsets of CVID patients may be relevant to their clinical manifestations, and may contribute to our understanding of the pathogenesis of CVID complications.

Polymorphisms within the CTLA4 gene are associated with infant atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is one of the most common childhood disorders. It can have a significant impact on the physical and psychological well-being of affected individuals. Although environmental triggers are important, AD also has a strong genetic component. Identifying genes associated with AD may help to understand better the basis of this disorder and its relationship with other allergic disorders such as asthma. OBJECTIVES: Polymorphisms in the gene encoding the inhibitory CTLA4 receptor, an important regulator of T cells, are associated with asthma as well as autoimmune disorders. We have now tested whether polymorphisms in the CTLA4 gene are also associated with early childhood AD. METHODS: A family-based cohort of 112 children and their parents was recruited from Western Sydney, Australia. All children were seen by a paediatric dermatologist and presented with AD within the first 3 years of life. Using the transmission disequilibrium test, individual and haplotypic associations with the +49 and CT60 polymorphisms in exon 1 and the 3' nontranslated DNA of the CTLA4 gene were tested. RESULTS: Single tests of association revealed significant association of the +49(A) [P = 0.037, odds ratio (OR) 1.59, 95% confidence interval (CI) 1-2.55] and borderline significance of the CT60(A) alleles (P = 0.055, OR 1.51, 95% CI 1-2.38). Significant association of the +49(A)/CT60(A) haplotype was detected (P = 0.002, OR 1.78, 95% CI 1.2-2.65). CONCLUSIONS: Polymorphisms within the gene encoding CTLA4 were associated with early onset infant AD. This is in agreement with findings from asthmatic cohorts, suggesting that the +49(A)/CT60(A) haplotype is a genetic risk factor common to asthma and AD.

The -590C/T and -34C/T interleukin-4 promoter polymorphisms are not associated with atopic eczema in childhood.

Susceptibility to the development of asthma and other atopic diseases is known to have a genetic component. To date, several studies have linked chromosome 5q31 to asthma and atopy in human beings. This region harbors a cluster of cytokine and growth factor genes, IL-4 presenting as a prime atopy candidate gene, inasmuch as it plays a pivotal role in the atopy pathway. Our approach was to identify polymorphisms within the promoter regions of IL-4 and test their association with atopic eczema. Polymorphisms were typed in a cohort of 76 small nuclear families and 25 triads with childhood atopic eczema. The genotypes were used to test for linkage in the presence of association with atopic eczema. A new polymorphism, -34C/T, was identified and studied with a known polymorphism, -590C/T. On its own, each polymorphism showed no association with atopic eczema. The 2 polymorphisms were used to generate haplotypes, and a significant result was found for the -590C/-34C haplotype. However, after Bonferroni correction for multiple testing, the association became nonsignificant. Neither polymorphism predisposes to early-onset atopic eczema by itself, but suggestive linkage was found for the -590C/-34C haplotype in this study.

In vitro HIV-specific CTL activity from HIV-seropositive individuals is augmented by interleukin-12 (IL-12).

IL-12 production is reduced in HIV infection, and recombinant human IL-12 (rhIL-12) augments in vitro HIV-specific proliferative responses in PBMC from HIV-seropositive individuals. To determine whether rhIL12 could also augment HIV-specific CTL responses we studied 41 HIV-seropositive individuals. Recombinant hIL-12 increased the detectable in vitro HIV-specific CD8 CTL activity of PBMC taken from HIV-seropositive individuals with CD4 counts >500 cells/microl and from some individuals with lower CD4 counts. IL-12 increased cell recovery in cultures of PBMC from HIV-seropositive individuals with CD4 counts >500 cells/microl and also increased the precursor CTL frequency. However, the increase in HIV-specific CTL activity was not due to IL-2 or IFN-gamma production or an increase in the number of cells with surface markers characteristic of CTL effector cells. This study demonstrates that rhIL-12 augments in vitro HIV-specific CTL activity and provides evidence to justify further investigation within clinical trials of this cytokine in HIV infection.

Carboxyfluorescein succinimidyl ester-based proliferative assays for assessment of T cell function in the diagnostic laboratory.

Immune deficiency diseases are often accompanied by abnormalities in one or both arms of the specific immune system. Impairment can often be detected as a decrease in the number of T or B lymphocytes or their products in the circulation, but questions are often asked as to the functional capabilities of T lymphocytes in patients with recurrent infections. Function of T cells has traditionally been measured by their uptake of [3H]- thymidine following stimulation with antigen or mitogen in vitro. However, the ability of carboxyfluorescein succinimidyl ester (CFSE) to label lymphocytes intracellularly and track their mitotic activity by progressive two-fold reduction in fluorescence intensity prompted an alternative methodology based on flow cytometry, an approach which has the advantage of allowing specific gating on particular T cell subsets and simultaneous assessment of activation markers. This method was therefore evaluated for T cell responses to mitogen and antigen. Phytohaemagglutinin-induced blast transformation of CFSE-labelled T cells was reflected by an increase in forward and orthogonal light scatter and a progressive two-fold decrease in CFSE fluorescence intensity. These changes allowed the derivation of various measures of mitotic activity, which correlated well with [3H]-thymidine uptake. Patients with T cell functional deficiencies showed impairment in their responses by both assays, whereas the CFSE-based assay demonstrated that impaired blastogenesis was not simply due to depressed T cell numbers. Concomitant measurement of the activation markers CD69 and CD25 showed that CD69 was rapidly expressed on non-mitotic cells and that this expression was progressively diluted with subsequent rounds of cell division. In contrast, CD25 expression was unaffected by cell cycle, but was expressed in proportion to the PHA dose. Antigen-specific responsiveness to Candida was also assessed using a CFSE-based assay. Initial gating on the relatively minor population of T cells that underwent blast transformation demonstrated progressive twofold dilutions of CFSE intensity in responsive cells. These normal Candida responses, found in patients who had recovered from Candida infection, contrasted with those who had not been infected with Candida or who had chronic recurrent infection, in whom neither blast transformation nor significant mitosis could be detected. Again, there was good correlation with [3H]-thymidine uptake. The CFSE-based assays are equivalent to traditional measures of mitogen- and antigen-specific T cell responsiveness in the diagnostic laboratory and have significant advantages in terms of decreased labour intensiveness, avoidance of radioactivity, the ability to gate on a specific population of lymphocytes and the concomitant measurement of activation markers.

Diagnostic value of classical and atypical antineutrophil cytoplasmic antibody (ANCA) immunofluorescence patterns.

BACKGROUND: The "classical" antineutrophil cytoplasmic antibody (C-ANCA) pattern seen on indirect immunofluorescence (IIF) is characterised by granular cytoplasmic staining showing central or interlobular accentuation, and is strongly associated with antiproteinase-3 antibodies (PR3-ANCA) and Wegener's granulomatosis. However, many laboratories report C-ANCA in the presence of any cytoplasmic IIF staining, regardless of pattern, which risks reducing the diagnostic value of this pattern. AIMS: To classify different cytoplasmic ANCA patterns and thus determine whether stringent application of the classical criteria for C-ANCA would produce better correlation between C-ANCA and (1) PR3-ANCA enzyme linked immunosorbent assay (ELISA) results; (2) a diagnosis of systemic vasculitis (including Wegener's granulomatosis). METHODS: 72 sera with cytoplasmic IIF collected over a two year period were analysed by IIF and a commercial PR3-ANCA ELISA kit. RESULTS: Three IIF patterns were defined: "classical/true" C-ANCA as described above (n = 27 (37.5%)); "flat" ANCA with homogeneous cytoplasmic staining (n = 21 (29%)); and "atypical" ANCA which included all other cytoplasmic patterns (n = 24 (33.5%)). Twenty five of the 27 true C-ANCA sera (92.5%) contained PR3-ANCA (p < 0.0001), but none of the 21 with flat ANCA and only one of the 24 with atypical ANCA. From clinical data on 23 of the 27 true C-ANCA positive patients, 20 (87%) had evidence of Wegener's granulomatosis or systemic vasculitis (p < 0.0001 v the other two patterns). However, none of 19 sera with flat ANCA and clinical data had evidence of systemic vasculitis. CONCLUSIONS: Restricting the term "c-ANCA" to the "classical" description of central/interlobular accentuation on IIF, will improve its correlation with PR3-ANCA positivity and a diagnosis of systemic vasculitis.

Measurement of Candida-specific blastogenesis: comparison of carboxyfluorescein succinimidyl ester labelling of T cells, thymidine incorporation, and CD69 expression.

Measurement of the T cell blastogenic response to Candida may be useful in the evaluation of patients with suspected immunodeficiency. The classic blastogenesis assay is based on uptake of [3H]thymidine by peripheral blood lymphocytes stimulated with Candida antigens for 5 days. An alternative approach involves staining peripheral blood lymphocytes with the intracellular fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) and measuring mitotic activity by the successive twofold reductions in fluorescent intensity using flow cytometry (FCM). The two approaches were compared in 16 subjects who demonstrated various proliferative responses to Candida. FCM-derived indices all involved initial gating on CD3+ T cells and included 1) blastic transformation as measured by changes in light scatter, 2) cell division, measured by CFSE fluorescence, and 3) CD69 expression. A good correlation was found between [3H]thymidine uptake and CFSE-derived indices, irrespective of the analysis algorithm used to interpret CFSE division profiles. Furthermore, significant T cell proliferation occurred only in subjects who had had one or more symptomatic episodes of vaginal candidiasis whereas controls with no such history, and patients with chronic vaginal infection, showed minimal proliferation. The increase in proportion of CD69+ T cells in culture also correlated with the blastogenic response to Candida, but less well than mitotic indices. CFSE-derived indices of T cell blastogenesis to Candida are equivalent to [3H]thymidine-based assays and may allow useful laboratory distinction between subjects who have been exposed to and recovered from vaginal Candida infection, who have a strong proliferative response, from those with no exposure or chronic infection who demonstrate a poor response.

Influences on the lifespan of B cell subpopulations defined by different phenotypes.

The turnover of mature and immature B cells defined by a range of cell surface markers was investigated by feeding normal or bcl-2-transgenic (bcl-2-Tg) mice 5'-bromo-2-deoxyuridine (BrdUrd) for up to 6 weeks. In peripheral lymphoid tissue, B cells accumulated BrdUrd with a 50% labeling time of 4.3 weeks and a pattern of uptake indicative of the presence of both long-lived and short-lived cells. These two kinetic populations could be resolved into immature B220lo/heat-stable antigen (HSA)hi cells which labeled rapidly, and B220hiHSAlo cells which were uniformly long-lived with a half-life of about 6 weeks. During loading and pulse-chase experiments, BrdUrd uptake by cells within the mature B220hiHSAlo population clearly followed an exponential kinetic pattern, suggesting that their loss was governed by stochastic processes. Using other surface markers, the long-lived population could also be defined by high expression of IgD, representing cells in the follicular mantle zone of the spleen, and by the phenotype IgMhiIgDloHSAlo which most likely represented marginal zone memory B cells. CD23 expression on B cells did not differentiate well between long and short-lived cells. Only about half of newly labeled B cells appearing in the spleen progressed to the long-lived compartment, a proportion which was not altered significantly in bcl-2-Tg mice. The most likely explanation was that a combination of both positive and negative selection was operating at this site which was mediated by pathways not regulated by bcl-2. On the other hand, overexpression of bcl-2 did result in a two- to threefold increase in the rate of appearance of newly labeled B cells in the spleen, consistent with a possible role for this protein during early selection events within the bone marrow. Selection processes appeared to be very active in young mice during the shaping of the B cell repertoire, since B cells from 6-week-old non-Ig mice displayed a rapid rate of turnover irrespective of their surface phenotype, and a significant population of long-lived cells did not become evident until the mice had reached about 12 weeks of age.

B-cell activation versus tolerance--the central role of immunoglobulin receptor engagement and T-cell help.

The critical variables responsible for the decision between activation and tolerance in the B-cell compartment were studied in the well-characterised hen egg lysozyme:antilysozyme transgenic model. Mature B-cells exposed to a low antigenic signal, which resulted in a receptor occupancy of between 5% and 25%, remained 'indifferent' (i.e. were neither activated nor tolerant), had a normal lifespan and recirculated through B-cell follicles. When receptor occupancy reached a critical threshold of approximately 50%, the B-cells became activated and moved to the outer T-cell zones where they died. This threshold appeared to be lower in the case of immature B-cells. On addition of T-cell help, however, the B-cells were rescued, proliferated and secreted antibody. Thus the outcome of the interaction between B-cells and antigen (self or foreign) is determined largely by the degree of antigen receptor engagement and availability of T-cell help.

B cell life span: a review.

Debate has surrounded the subject of B cell life span since it was first measured in mice in the early 1970s. In the 25 years which have passed since then, it has become increasingly apparent that the methods employed to measure rates of B cell turnover, such as [3H]-thymidine labelling, cell transfer or cell ablation, brought about significant disruptions to normal physiology which in themselves might have affected B cell turnover. More recently the use of bromodeoxyuridine has overcome many of these methodological difficulties and has allowed rates of B cell renewal to be measured within B cell subpopulations defined by multiparameter flow cytometry. Such studies have largely resolved the issue, concluding that about 85% of peripheral B cells are phenotypically mature and display first-order exponential kinetics defined by a half-life of 5-6 weeks, whilst the remainder are short-lived with a life span of several days. This review examines both traditional and recent methods and discusses the influence of age, self-tolerance and randomness in the overall shaping of a kinetically stable mature B cell population.

Managing HIV. Antiretroviral therapy.

Initiating or changing antiretroviral therapy requires specialist knowledge of indications, side effects and drug interactions. This brief guide highlights the key points for monitoring antiretroviral therapy.