Defects in B-lymphopoiesis and B-cell maturation underlie prolonged B-cell depletion in ANCA-associated vasculitis.

OBJECTIVES: B-cell depletion time after rituximab (RTX) treatment is prolonged in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) compared with other autoimmune diseases. We investigated central and peripheral B-cell development to identify the causes for the defect in B-cell reconstitution after RTX therapy. METHODS: We recruited 91 patients with AAV and performed deep phenotyping of the peripheral and bone marrow B-cell compartment by spectral flow and mass cytometry. B-cell development was studied by in vitro modelling and the role of BAFF receptor by quantitative PCR, western blot analysis and in vitro assays. RESULTS: Treatment-naïve patients with AAV showed low transitional B-cell numbers, suggesting impaired B-lymphopoiesis. We analysed bone marrow of treatment-naïve and RTX-treated patients with AAV and found reduced B-lymphoid precursors. In vitro modelling of B-lymphopoiesis from AAV haematopoietic stem cells showed intact, but slower and reduced immature B-cell development. In a subgroup of patients, after RTX treatment, the presence of transitional B cells did not translate in replenishment of naïve B cells, suggesting an impairment in peripheral B-cell maturation. We found low BAFF-receptor expression on B cells of RTX-treated patients with AAV, resulting in reduced survival in response to BAFF in vitro. CONCLUSIONS: Prolonged depletion of B cells in patients with AAV after RTX therapy indicates a B-cell defect that is unmasked by RTX treatment. Our data indicate that impaired bone marrow B-lymphopoiesis results in a delayed recovery of peripheral B cells that may be further aggravated by a survival defect of B cells. Our findings contribute to the understanding of AAV pathogenesis and may have clinical implications regarding RTX retreatment schedules and immunomonitoring after RTX therapy.

Nonpermissive bone marrow environment impairs early B-cell development in common variable immunodeficiency.

Common variable immunodeficiency (CVID) is a disease characterized by increased susceptibility to infections, hypogammaglobulinemia, and immune dysregulation. Although CVID is thought to be a disorder of the peripheral B-cell compartment, in 25% of patients, early B-cell development in the bone marrow is impaired. Because poor B-cell reconstitution after hematopoietic stem cell transplantation has been observed, we hypothesized that in some patients the bone marrow environment is not permissive to B-cell development. Studying the differentiation dynamics of bone marrow-derived CD34+ cells into immature B cells in vitro allowed us to distinguish patients with B-cell intrinsic defects and patients with a nonpermissive bone marrow environment. In the former, immature B cells did not develop and in the latter CD34+ cells differentiated into immature cells in vitro, but less efficiently in vivo. In a further group of patients, the uncommitted precursors were unable to support the constant development of B cells in vitro, indicating a possible low frequency or exhaustion of the precursor population. Hematopoietic stem cell transplantation would result in normal B-cell repopulation in case of intrinsic B-cell defect, but in defective B-cell repopulation in a nonpermissive environment. Our study points to the importance of the bone marrow niche in the pathogenesis of CVID.

Abatacept modulates CD80 and CD86 expression and memory formation in human B-cells.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) limits T-cell activation and is expressed on T-regulatory cells. Human CTLA-4 deficiency results in severe immune dysregulation. Abatacept (CTLA-4 Ig) is approved for the treatment of rheumatoid arthritis (RA) and its mechanism of action is attributed to effects on T-cells. It is known that CTLA-4 modulates the expression of its ligands CD80 and CD86 on antigen presenting cells (APC) by transendocytosis. As B-cells express CD80/CD86 and function as APC, we hypothesize that B-cells are a direct target of abatacept. OBJECTIVES: To investigate direct effects of abatacept on human B-lymphocytes in vitro and in RA patients. METHODS: The effect of abatacept on healthy donor B-cells' phenotype, activation and CD80/CD86 expression was studied in vitro. Nine abatacept-treated RA patients were studied. Seven of these were followed up to 24 months, and two up to 12 months only and treatment response, immunoglobulins, ACPA, RF concentrations, B-cell phenotype and ACPA-specific switched memory B-cell frequency were assessed. RESULTS: B-cell development was unaffected by abatacept. Abatacept treatment resulted in a dose-dependent decrease of CD80/CD86 expression on B-cells in vitro, which was due to dynamin-dependent internalization. RA patients treated with abatacept showed a progressive decrease in plasmablasts and serum IgG. While ACPA-titers only moderately declined, the frequency of ACPA-specific switched memory B-cells significantly decreased. CONCLUSIONS: Abatacept directly targets B-cells by reducing CD80/CD86 expression. Impairment of antigen presentation and T-cell activation may result in altered B-cell selection, providing a new therapeutic mechanism and a base for abatacept use in B-cell mediated autoimmunity.

Disturbed B-lymphocyte selection in autoimmune lymphoproliferative syndrome.

Fas is a transmembrane receptor involved in the maintenance of tolerance and immune homeostasis. In murine models, it has been shown to be essential for deletion of autoreactive B cells in the germinal center. The role of Fas in human B-cell selection and in development of autoimmunity in patients carrying FAS mutations is unclear. We analyzed patients with either a somatic FAS mutation or a germline FAS mutation and somatic loss-of-heterozygosity, which allows comparing the fate of B cells with impaired vs normal Fas signaling within the same individual. Class-switched memory B cells showed: accumulation of FAS-mutated B cells; failure to enrich single V, D, J genes and single V-D, D-J gene combinations of the B-cell receptor variable region; increased frequency of variable regions with higher content of positively charged amino acids; and longer CDR3 and maintenance of polyreactive specificities. Importantly, Fas-deficient switched memory B cells showed increased rates of somatic hypermutation. Our data uncover a defect in B-cell selection in patients with FAS mutations, which has implications for the understanding of the pathogenesis of autoimmunity and lymphomagenesis of autoimmune lymphoproliferative syndrome.

Impact of tofacitinib treatment on human B-cells in vitro and in vivo.

B-cells are pivotal to the pathogenesis of rheumatoid arthritis and tofacitinib, a JAK inhibitor, is effective and safe in its treatment. Tofacitinib interferes with signal transduction via cytokine receptors using the common γ-chain. Despite extensive data on T-lymphocytes, the impact of tofacitinib on B-lymphocytes is poorly understood. In this study we assessed the effect of tofacitinib on B-lymphocyte differentiation and function. Tofacitinib treatment strongly impaired in vitro plasmablast development, immunoglobulin secretion and induction of B-cell fate determining transcription factors, Blimp-1, Xbp-1, and IRF-4, in naïve B-cells. Interestingly, class switch and activation-induced cytidine deaminase (AICDA) induction was only slightly reduced in activated naïve B-cells. The effect of tofacitinib on plasmablast formation, immunoglobulin secretion and proliferation was less profound, when peripheral blood B-cells, including not only naïve but also memory B-cells, were stimulated. In line with these in vitro results, the relative distribution of B-cell populations remained stable in tofacitinib treated patients. Nevertheless, a temporary increase in absolute B-cell numbers was observed 6-8 weeks after start of treatment. In addition, B-cells isolated from tofacitinib treated patients responded rapidly to in vitro activation. We demonstrate that tofacitinib has a direct impact on human naïve B-lymphocytes, independently from its effect on T-lymphocytes, by impairing their development into plasmablasts and immunoglobulin secretion. The major effect of tofacitinib on naïve B-lymphocyte development points to the potential inability of tofacitinib-treated patients to respond to novel antigens, and suggests planning vaccination strategies prior to tofacitinib treatment.

Non-apoptotic FAS signaling controls mTOR activation and extrafollicular maturation in human B cells.

Defective FAS (CD95/Apo-1/TNFRSF6) signaling causes autoimmune lymphoproliferative syndrome (ALPS). Hypergammaglobulinemia is a common feature in ALPS with FAS mutations (ALPS-FAS), but paradoxically, fewer conventional memory cells differentiate from FAS-expressing germinal center (GC) B cells. Resistance to FAS-induced apoptosis does not explain this phenotype. We tested the hypothesis that defective non-apoptotic FAS signaling may contribute to impaired B cell differentiation in ALPS. We analyzed secondary lymphoid organs of patients with ALPS-FAS and found low numbers of memory B cells, fewer GC B cells, and an expanded extrafollicular (EF) B cell response. Enhanced mTOR activity has been shown to favor EF versus GC fate decision, and we found enhanced PI3K/mTOR and BCR signaling in ALPS-FAS splenic B cells. Modeling initial T-dependent B cell activation with CD40L in vitro, we showed that FAS competent cells with transient FAS ligation showed specifically decreased mTOR axis activation without apoptosis. Mechanistically, transient FAS engagement with involvement of caspase-8 induced nuclear exclusion of PTEN, leading to mTOR inhibition. In addition, FASL-dependent PTEN nuclear exclusion and mTOR modulation were defective in patients with ALPS-FAS. In the early phase of activation, FAS stimulation promoted expression of genes related to GC initiation at the expense of processes related to the EF response. Hence, our data suggest that non-apoptotic FAS signaling acts as molecular switch between EF versus GC fate decisions via regulation of the mTOR axis and transcription. The defect of this modulatory circuit may explain the observed hypergammaglobulinemia and low memory B cell numbers in ALPS.

BJ-PI2, a non-hemorrhagic metalloproteinase from Bothrops jararaca snake venom.

BACKGROUND: Envenoming by Bothrops jararaca can result in local pain, edema, hemorrhage and necrosis, partially mediated by snake venom metalloproteinases (SVMPs). Here, we describe the characterization of BJ-PI2, a P-I class SVMP from B. jararaca venom, and its local tissue actions. METHODS: BJ-PI2 was purified by a combination of gel filtration, anion-exchange chromatography and reverse phase HPLC, and identified by mass spectrometry. Clotting and fibrin(ogen)olytic activities were assayed using conventional methods. Hemorrhagic activity and changes in vascular permeability were examined in rat dorsal skin. Myonecrosis and inflammatory activity were examined in mouse gastrocnemius muscle. RESULTS: BJ-PI2 was a 23.08kDa single-chain polypeptide. Tryptic fragments showed highest homology with SVMP insularinase A from Bothrops insularis, but also with B. jararaca SVMP bothrojaractivase; less similarity was observed with B. jararaca SVMPs BJ-PI and jararafibrases II and IV. BJ-PI2 did not clot fibrinogen or rat citrated plasma but had α- and ß-fibrinogenolytic activity (inhibited by EDTA and 1,10-phenanthroline but not by PMSF) and attenuated coagulation after plasma recalcification. BJ-PI2 had fibrinolytic activity. BJ-PI2 increased the vascular permeability of rat dorsal skin (inhibited by 1,10-phenanthroline). BJ-PI2 was not hemorrhagic or myonecrotic but caused migration of inflammatory cells. In contrast, venom was strongly hemorrhagic and myonecrotic but caused less infiltration of inflammatory cells. CONCLUSIONS: BJ-PI2 is a non-hemorrhagic, non-myonecrotic, non-coagulant P-I class SVMP that may enhance vascular permeability and inflammatory cell migration in vivo. GENERAL SIGNIFICANCE: BJ-PI2 contributes to enhanced vascular permeability and inflammatory cell migration after envenoming, but not to venom-induced hemorrhage and necrosis.

JAK inhibitors differentially modulate B cell activation, maturation and function: A comparative analysis of five JAK inhibitors in an in-vitro B cell differentiation model and in patients with rheumatoid arthritis.

Background: Janus kinase (JAK) inhibitors have been approved for the treatment of several immune-mediated diseases (IMIDs) including rheumatoid arthritis (RA) and psoriatic arthritis and are in clinical trials for numerous other IMIDs. However, detailed studies investigating the effects of different JAK inhibitors on B cells are missing. Within this study, we therefore aimed to characterize the effect of JAK inhibition on the B cell compartment. Methods: To this end, we investigated the B cell compartment under JAK inhibition and compared the specific effects of the different JAK inhibitors tofacitinib (pan-JAK), baricitinib (JAK1/2), ruxolitinib (JAK1/2), upadacitinib (JAK1/2) as well as filgotinib (selective JAK1) on in-vitro B cell activation, proliferation, and class switch recombination and involved pathways. Results: While B cell phenotyping of RA patients showed an increase in marginal zone (MZ) B cells under JAK inhibition, comparison with healthy donors revealed that the relative frequency of MZ B cells was still lower compared to healthy controls. In an in-vitro model of T-cell-independent B cell activation we observed that JAK1/2 and selective JAK1 inhibitor treatment led to a dose-dependent decrease of total B cell numbers. We detected an altered B cell differentiation with a significant increase in MZ-like B cells and an increase in plasmablast differentiation in the first days of culture, most pronounced with the pan-JAK inhibitor tofacitinib, although there was no increase in immunoglobulin secretion in-vitro. Notably, we further observed a profound reduction of switched memory B cell formation, especially with JAK1/2 inhibition. JAK inhibitor treatment led to a dose-dependent reduction of STAT3 expression and phosphorylation as well as STAT3 target gene expression and modulated the secretion of pro- and anti-inflammatory cytokines by B cells. Conclusion: JAK inhibition has a major effect on B cell activation and differentiation, with differential outcomes between JAK inhibitors hinting towards distinct and unique effects on B cell homeostasis.

Sleep behaviour differs in women and men with psoriatic arthritis and axial spondyloarthritis with impact on quality of life and depressive symptoms.

OBJECTIVES: Axial spondyloarthritis (axSpA) and psoriatic arthritis (PsA) may have a profound impact on sleep and health-related quality of life. The aim of this study was to assess sleep quality and quality of life and determine associated factors in patients treated with spondyloarthritides (SpA). METHODS: Cross-sectional questionnaire-based assessment of sleep behaviour, quality of life, functional impairment and depression (Regensburg Insomnia Scale, WHO Quality of Life questionnaire, Funktionsfragebogen Hannover questionnaire, Beck Depression Inventory II, Patient health questionnaire 9) and retrospective medical chart analysis of a monocentric cohort of 330 patients with SpA (n=168 PsA and n=162 axSpA). RESULTS: 46.6% of patients with SpA demonstrated abnormal sleep behaviour. Linear regression models showed HLA-B27 positivity, Bath Ankylosing Spondylitis Disease Activity Index, depressive symptoms, functional capacity and disease duration to be predictive of insomnia symptoms in axSpA, respectively, depressive symptoms, female sex and Disease Activity Score 28 in patients with PsA. Patients with unrestful sleep had a significantly reduced health-related quality of life (p<0.001) as well as significantly more depressive symptoms (p<0.001). Satisfaction with health was rated significantly lower (p<0.001), indicating poor sleep as a burden on general well-being.In particular, female patients had a significantly worse sleep quality with a prolonged sleep latency (p=0.009), increased sleep disturbances (p=0.014) and unrestful sleep (p<0.001) as well as a reduced physical and mental health-related quality of life (p=0.015, p<0.001) and more depressive symptoms (p=0.015). CONCLUSION: Despite treatment, many patients with SpA demonstrate abnormal sleep behaviour with symptoms of insomnia and a reduced quality of life with significant differences between male and female patients. An interdisciplinary and holistic approach may be needed to address unmet needs.

Respiratory tract infections and risk factors for infection in a cohort of 330 patients with axial spondyloarthritis or psoriatic arthritis.

Respiratory tract infections (RTIs) are the most common infections in patients with rheumatic diseases under immunosuppressive treatment and may contribute to morbidity and mortality as well as increased healthcare costs. However, to date only limited data on infection risk in spondyloarthritis (SpA) patients are available. In this study we assessed the occurrence of respiratory tract infections in a monocentric real-world cohort consisting of 330 patients (168 psoriatic arthritis and 162 axial spondyloarthritis patients) and determined factors associated with increased infection risk. Out of 330 SpA patients, 89.3% had suffered from ≥ 1 upper respiratory tract infection (URTI) and 31.1% from ≥ 1 lower respiratory tract infection (LRTI) within the last two years. The most common URTIs were rhinitis and laryngitis/pharyngitis with 87.3% and 36.1%, respectively. Bronchitis constituted the most common LRTI, reported in 29.7% of patients. In a multivariate binomial logistic regression model occurrence of LRTI was associated with chronic lung disease (OR 17.44, p=0.006), glucocorticoid therapy (OR 9.24, p=0.012), previous history of severe airway infections (OR 6.82, p=0.013), and number of previous biological therapies (OR 1.72, p=0.017), whereas HLA B27 positivity was negatively associated (OR 0.29, p=0.025). Female patients reported significantly more LRTIs than male patients (p=0.006) and had a higher rate of antibiotic therapy (p=0.009). There were no significant differences between axSpA and PsA patients regarding infection frequency or antibiotic use. 45.4% of patients had required antibiotics for respiratory tract infections. Antibiotic therapy was associated with smoking (OR 3.40, p=0.008), biological therapy (OR 3.38, p=0.004), sleep quality (OR 1.13, p<0.001) and age (OR 0.96, p=0.030). Hypogammaglobulinemia (IgG<7g/l) was rare (3.4%) in this SpA cohort despite continuous immunomodulatory treatment. Awareness of these risk factors will assist physicians to identify patients with an increased infection risk, who will benefit from additional preventive measures, such as vaccination and smoking cessation or adjustment of DMARD therapy.

High antibody levels and reduced cellular response in children up to one year after SARS-CoV-2 infection.

The COVID-19 course and immunity differ in children and adults. We analyzed immune response dynamics in 28 families up to 12 months after mild or asymptomatic infection. Unlike adults, the initial response is plasmablast-driven in children. Four months after infection, children show an enhanced specific antibody response and lower but detectable spike 1 protein (S1)-specific B and T cell responses than their parents. While specific antibodies decline, neutralizing antibody activity and breadth increase in both groups. The frequencies of S1-specific B and T cell responses remain stable. However, in children, one year after infection, an increase in the S1-specific IgA class switch and the expression of CD27 on S1-specific B cells and T cell maturation are observed. These results, together with the enhanced neutralizing potential and breadth of the specific antibodies, suggest a progressive maturation of the S1-specific immune response. Hence, the immune response in children persists over 12 months but dynamically changes in quality, with progressive neutralizing, breadth, and memory maturation. This implies a benefit for booster vaccination in children to consolidate memory formation.

TRAIL-R1 and TRAIL-R2 Mediate TRAIL-Dependent Apoptosis in Activated Primary Human B Lymphocytes.

The maintenance of B cell homeostasis requires a tight control of B cell generation, survival, activation, and maturation. In lymphocytes upon activation, increased sensitivity to apoptotic signals helps controlling differentiation and proliferation. The death receptor Fas is important in this context because genetic Fas mutations in humans lead to an autoimmune lymphoproliferative syndrome that is similar to lymphoproliferation observed in Fas-deficient mice. In contrast, the physiological role of TNF-related apoptosis-inducing ligand receptors (TRAIL-Rs) in humans has been poorly studied so far. Indeed, most studies have focused on tumor cell lines and on mouse models whose results are difficult to transpose to primary human B cells. In the present work, the expression of apoptosis-inducing TRAIL-R1 and TRAIL-R2 and of the decoy receptors TRAIL-R3 and TRAIL-R4 was systematically studied in all developmental stages of peripheral B cells isolated from the blood and secondary lymphoid organs. Expression of TRAIL-Rs is modulated along development, with highest levels observed in germinal center B cells. In addition, T-dependent and T-independent signals elicited induction of TRAIL-Rs with distinct kinetics, which differed among B cell subpopulations: switched memory cells rapidly upregulated TRAIL-R1 and -2 upon activation while naïve B cells only reached similar expression levels at later time points in culture. Increased expression of TRAIL-R1 and -2 coincided with a caspase-3-dependent sensitivity to TRAIL-induced apoptosis in activated B cells but not in freshly isolated resting B cells. Finally, both TRAIL-R1 and TRAIL-R2 could signal actively and both contributed to TRAIL-induced apoptosis. In conclusion, this study provides a systematic analysis of the expression of TRAIL-Rs in human primary B cells and of their capacity to signal and induce apoptosis. This dataset forms a basis to further study and understand the dysregulation of TRAIL-Rs and TRAIL expression observed in autoimmune diseases. Additionally, it will be important to foresee potential bystander immunomodulation when TRAIL-R agonists are used in cancer treatment.

Synthesis and pharmacological evaluations of sildenafil analogues for treatment of erectile dysfunction.

The 5-[2-ethoxy-5-(4-methylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7 H-pyrazolo[4,3-d]pyrimidin-7-one, sildenafil, is a cGMP-specific phosphodiesterase-5 (PDE5) inhibitor used for penile erectile dysfunction. In the search for more potent and selective PDE5 inhibitors, new sildenafil analogues (6a-v), characterized by the presence on the sulfonyl group in the 5' position of novel N-4-substituted piperazines or ethylenediamine moiety, were prepared by traditional and microwave-assisted synthesis and tested in rabbit isolated aorta and corpus cavernosum. Similarly to sildenafil, several analogues showed IC50 values in the nanomolar range. In the in vitro studies, all the tested compounds caused concentration-dependent relaxations in both rabbit isolated aorta and corpus cavernosum. All sildenafil analogues potentiated the nitric oxide-dependent vasodilation in endothelium-intact rabbit aorta. Compound 6f exhibited great pEC50 value in corpus cavernosum, and compounds 6r and 6u in isolated aorta were found as potent as sildenafil for inhibiting PDE5. Because several analogues were significantly more lipophilic than sildenafil, these compounds may offer a new lead for development of new sildenafil analogues.

Pharmacological characterization of a novel phosphodiesterase type 5 (PDE5) inhibitor lodenafil carbonate on human and rabbit corpus cavernosum.

Nitrergic nerves and endothelial cells release nitric oxide (NO) in the corpus cavernosum, a key mediator that stimulates soluble guanylyl cyclase to increase cGMP levels causing penile erection. Phosphodiesterase 5 (PDE5) inhibitors, such as sildenafil, prolong the NO effects by inhibiting cGMP breakdown. Here, we report a novel PDE5 inhibitor, lodenafil carbonate, (Bis-(2-{4-[4-ethoxy-3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-benzenesulfonyl]piperazin-1-yl}-ethyl)carbonate) that is a dimer of lodenafil. We therefore aimed to compare the effects of sildenafil, lodenafil and lodenafil carbonate on in vitro human and rabbit cavernosal relaxations, activity of crude PDE extracts from human platelets, as well as stability and metabolic studies in rat, dog and human plasma. Pharmacokinetic evaluations after intravenous and oral administration were performed in male beagles. Functional experiments were conducted using organ bath techniques. Pharmacokinetics was studied in beagles by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), following oral or intravascular administration. All PDE5 inhibitors tested concentration-dependently relaxed (0.001-100 microM) phenylephrine-precontracted rabbit and human corpus cavernosum. The cavernosal relaxations evoked by either acetylcholine (0.01-100 microM) or electrical field stimulation (EFS, 1-20 Hz) were markedly potentiated by sildenafil, lodenafil and lodenafil carbonate. Lodenafil carbonate was more potent to inhibit the cGMP hydrolysis in PDE extracts compared with lodenafil and sildenafil. Following intravascular and single oral administration of lodenafil carbonate, only lodenafil and norlodenafil were detected in vivo. These results indicate that lodenafil carbonate works as a prodrug, being lodenafil the active moiety of lodenafil carbonate.

Simultaneous quantification of GMP, AMP, cyclic GMP and cyclic AMP by liquid chromatography coupled to tandem mass spectrometry.

Phosphodiesterases are drug targets for treating various diseases. Inhibition of these can increase cAMP and cGMP levels, which can affect a variety of physiological responses. Here we report a new method for determining PDE activity by combining high-performance liquid chromatography and tandem mass spectrometry. Characteristic peaks of the substrates, cGMP or cAMP and products, GMP or AMP, were identified in positive-ion electrospray ionization using multiple reaction monitoring. The method can be applied to determine activity of PDE inhibitors. Our results showed that this new method was fast, sensitive and highly reproducible.

Hepatotoxicity in HIV-infected children and adolescents on antiretroviral therapy.

CONTEXT AND OBJECTIVE: Adverse drug reactions are a significant problem in patients on antiretroviral therapy (ART). We determined liver enzyme elevation frequencies in HIV-infected children and adolescents receiving ART, and their association with risk factors. DESIGN AND SETTING: Cross-sectional study, at the Pediatrics Immunodeficiency Division, University Hospital, Universidade Estadual de Campinas. METHODS: Medical records of 152 children and adolescents (54.6% male; median age 7.48 years) were analyzed, with a mean of 2.6 liver enzyme determinations per patient. Clinically, patients were classified in categories N (6), A (29), B (78) and C (39). Serum levels of aspartate aminotransferase and alanine aminotransferase were evaluated. Hepatotoxicity was scored as grade 1 (1.1-4.9 times upper limit of normality, ULN), grade 2 (5.0-9.9 times ULN), grade 3 (10.0-15.0 times ULN) and grade 4 (> 15.0 times ULN). To assess hepatotoxicity risk factors, odds ratios (OR) and adjusted odds ratios (aOR) for age, gender, TCD4+ cell count, viral load and medication usage were calculated. RESULTS: We observed grade 1 hepatotoxicity in 19.7 % (30/152) patients. No cases of grade 2, 3 or 4 were detected. There was a significant association between hepatotoxicity and use of sulfonamides (OR, 3.61; 95% confidence interval (CI), 1.50-8.70; aOR, 3.58; 95% CI, 1.44-8.85) and antituberculous agents (OR, 9.23; 95% CI, 1.60-53.08; aOR, 9.05; 95% CI, 1.48-55.25). No toxicity was associated with ART. CONCLUSIONS: One fifth of patients experienced mild hepatotoxicity, attributed to antituberculous agents and sulfonamides. Our results suggest that ART was well tolerated.

B cell repopulation kinetics after rituximab treatment in ANCA-associated vasculitides compared to rheumatoid arthritis, and connective tissue diseases: a longitudinal observational study on 120 patients.

BACKGROUND: B cell depletion with rituximab (RTX) is approved for treatment of rheumatoid arthritis (RA) and ANCA-associated vasculitides (AAV). Recently, RTX has been shown to be effective in AAV maintenance therapy, but an optimal RTX treatment schedule is unknown and the time to B cell repopulation after RTX has not been studied. METHODS: Retrospective single-center analysis of B cell repopulation in patients with AAV, RA or connective tissue disease (CTD) treated with RTX. RESULTS: Beginning B cell repopulation within the first year after RTX treatment was observed in 93% of RA and 88% of CTD patients. Only 10% of patients with granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) and no patient with eosinophilic granulomatosis with polyangiitis (EGPA) showed B cell repopulation within this time. Median time of B cell depletion was 26 months in GPA/MPA, and 21 months in EGPA compared to 9 months in RA, and 8 months in CTD (p < 0.0001). In 25 AAV-patients B cell depletion lasted for at least 44 months. There was a significant decline in serum immunoglobulin concentrations in GPA/MPA patients, but not in patients with RA or CTD. Significantly more GPA/MPA patients developed hygogammaglobulinemia (IgG <7 g/L) compared to patients with RA or CTD. CONCLUSIONS: In contrast to RA and CTD, in AAV RTX induces long-lasting depletion of B cells that is associated with decreased antibody production. This observation points toward potential defects in the B cell compartment in AAV that are unmasked by immunosuppressive treatment and has important implications for the design of maintenance treatment schedules using RTX.

Hetero-oligomerization between the TNF receptor superfamily members CD40, Fas and TRAILR2 modulate CD40 signalling.

TNF receptor superfamily members (TNFRSF) such as CD40, Fas and TRAIL receptor 2 (TRAILR2) participate to the adaptive immune response by eliciting survival, proliferation, differentiation and/or cell death signals. The balance between these signals determines the fate of the immune response. It was previously reported that these receptors are able to self-assemble in the absence of ligand through their extracellular regions. However, the role of this oligomerization is not well understood, and none of the proposed hypotheses take into account potential hetero-association of receptors. Using CD40 as bait in a flow cytometry Förster resonance energy transfer assay, TNFRSF members with known functions in B cells were probed for interactions. Both Fas and TRAILR2 associated with CD40. Immunoprecipitation experiments confirmed the interaction of CD40 with Fas at the endogenous levels in a BJAB B-cell lymphoma cell line deficient for TRAILR2. TRAILR2-expressing BJAB cells displayed a robust CD40-TRAILR2 interaction at the expense of the CD40-Fas interaction. The same results were obtained by proximity ligation assay, using TRAILR2-positive and -negative BJAB cells and primary human B cells. Expression of the extracellular domains of Fas or TRAILR2 with a glycolipid membrane anchor specifically reduced the intrinsic signalling pathway of CD40 in 293T cells. Conversely, BJAB cells lacking endogenous Fas or TRAILR2 showed an increased NF-κB response to CD40L. Finally, upregulation of TRAILR2 in primary human B cells correlated with reduced NF-κB activation and reduced proliferation in response to CD40L. Altogether, these data reveal that selective interactions between different TNFRSF members may modulate ligand-induced responses upstream signalling events.

Inhibition of human platelet aggregation by eosinophils.

AIMS: The relationship between the activity of eosinophils and platelets has been observed in recent decades by many scientists. These observations include increased numbers of eosinophils associated with platelet disorders, including changes in the coagulation cascade and platelet aggregation. Based on these observations, the interaction between eosinophils and platelets in platelet aggregation was analyze. MAIN METHODS: Human platelets were incubated with eosinophil cytosolic fraction, promyelocytic human HL-60 clone 15 cell lineage, and eosinophil cationic protein (ECP). Platelet rich plasma (PRP) aggregation was induced by adenosine diphosphate, platelet activating factor, arachidonic acid, and collagen, and washed platelets (WP) were activated by thrombin. KEY FINDINGS: Aggregation induced by all agonists was dose dependently inhibited by eosinophil cytosolic fraction. This inhibition was only partially reversed by previous incubation of the eosinophils with l-Nitro-Arginine-Methyl-Ester (l-NAME). Previous incubation with indomethacin did not prevent the cytosolic fraction induced inhibition. The separation of eosinophil cytosolic fraction by gel filtration on Sephadex G-75 showed that the inhibitory activity was concentrated in the lower molecular weight fraction. HL-60 clone 15 cells differentiated into eosinophils for 5 and 7 day were able to inhibit platelet aggregation. The ECP protein inhibited the platelet aggregation on PRP and WP. This inhibition was more evident in WP, and the citotoxicity MTT assay proved the viability of tested platelets, showing that the observed inhibition by the ECP protein does not occur simply by cell death. SIGNIFICANCE: Our results indicate that eosinophils play a fundamental role in platelet aggregation inhibition.