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During viral infections high levels of antibodies can form soluble immune complexes (sICs) with antigen and trigger Fcγ receptors (FcγR) leading to increased immunopathology. Here the authors measure FcγRs activation by sICs and consider how these may lead to excessive immunopathology during severe SARS-CoV-2 infection.

Abstract Aims Heart failure (HF) is a systemic inflammatory disorder with infections being an important cause of morbidity and mortality. We asked if HF patients have a higher susceptibility to infections compared with the general population and if a subtle secondary immunodeficiency facilitates infectious complications. Methods and results In a cohort of 92 patients with HF with reduced ejection fraction, we analysed recirculating lymphocyte subpopulations, serum immunoglobulin levels, and specific antibody titres against pneumococcal antigens. We quantified susceptibility to infections of the respiratory tract with a validated questionnaire and compared it to the general population. Susceptibility to infections of the respiratory tract was comparable in HF patients and the general population. Hypogammaglobulinaemia was present in 16% of HF patients, but anti‐pneumococcal titres showed no evidence of specific secondary antibody deficiency. Relative lymphopaenia in our HF cohort was due to B lymphocytopenia with a relative reduction in naive B‐cells and expansion of memory B‐cells while CD4+ and CD8+ T‐lymphocytes as well as NK‐cell counts were comparable between HF and healthy donors. The intake of the angiotensin receptor neprilysin (CD10) inhibitor (ARNI) sacubitril/valsartan was associated with increased B‐lymphocyte counts, possibly by an increased output of CD10+ transitional B lymphocytes from the bone marrow. Conclusion Despite a reduction of B lymphocytes in HF and mild hypogammaglobulinaemia, patients showed no evidence of secondary immunodeficiency or increased susceptibility to infections. The relevance of B‐cell lymphopenia in HF patients and modulation of B‐cell counts under ARNI treatment remains to be investigated.

Abstract Fc‐gamma receptor (FcγR) activation by soluble IgG immune complexes (sICs) represents a major mechanism of inflammation in certain autoimmune diseases such as systemic lupus erythematosus (SLE). A robust and scalable test system allowing for the detection and quantification of sIC bioactivity is missing. We developed a comprehensive reporter cell panel detecting activation of FcγRs. The reporter cell lines were integrated into an assay that enables the quantification of sIC reactivity via ELISA or a faster detection using flow cytometry. This identified FcγRIIA(H) and FcγRIIIA as the most sIC‐sensitive FcγRs in our test system. Reaching a detection limit in the very low nanomolar range, the assay proved also to be sensitive to sIC stoichiometry and size reproducing for the first time a complete Heidelberger‐Kendall curve in terms of immune receptor activation. Analyzing sera from SLE patients and mouse models of lupus and arthritis proved that sIC‐dependent FcγR activation has predictive capabilities regarding severity of SLE disease. The assay provides a sensitive and scalable tool to evaluate the size, amount, and bioactivity of sICs in all settings.

Storage of packed red blood cells is associated with changes in erythrocytes that over time increasingly impair cellular function and potentially contribute to adverse effects associated with blood transfusion. Exposure of phosphatidylserine at the outer membrane leaflet of erythrocytes and shedding of microvesicles (MVs) during packed red blood cell storage are alterations assumed to increase the risk of prothrombotic events in recipients. Here, we used rotational thromboelastometry to study the coagulation process in blood samples with erythrocytes from stored PRBCs reconstituted with freshly prepared platelet-rich plasma. We explored the influence of following effects on the coagulation process: 1) PRBC storage duration, 2) differences between erythrocytes from stored PRBCs compared to freshly drawn erythrocytes, and 3) the contribution of added MVs. Interestingly, despite of a higher fraction of PS-positive cells, erythrocytes from PRBCs stored for 6 weeks revealed longer clotting times than samples with erythrocytes stored for 2 or 4 weeks. Further, clotting times and clot formation times were considerably increased in samples reconstituted with erythrocytes from stored PRBCs as compared to fresh erythrocytes. Moreover, MVs added to reconstituted samples elicited only comparably small and ambiguous effects on coagulation. Thus, this study provides no evidence for an amplified clotting process from prolonged storage of PRBCs but on the contrary implicates a loss of function, which may be of clinical significance in massive transfusion. Our observations add to the increasing body of evidence viewing erythrocytes as active players in the clotting process.

Abstract Background Cytomegalovirus (CMV) infection is one of the main driving forces of T-cell senescence in the general population, whereas its differential impact in people living with HIV (PLWH) is less well characterized. The study explores the effect of latent CMV infection on T-cell subsets, monocyte/macrophages activation markers, and CRP in PLWH on long-term ART. Methods Cross-sectional cohort study including PLWH on long-term suppressive ART. Individuals of 4 groups (HIV+CMV−, HIV+CMV+, HIV−CMV+, and HIV−CMV−) were matched 1:1:1:1 for age and sex. Immunophenotyping of lymphocyte and T-cell subsets by multicolor flow cytometry was performed in fresh blood samples collected from patients and healthy donors. Results Both, latent CMV and treated HIV infection were associated with an expansion of CD8 T cells, a reduced CD4/CD8 ratio, and with CD8 T-cell activation with a cumulative effect in CMV/HIV-coinfected individuals. CMV was associated with elevated numbers of late effector and terminally differentiated CD8 T-cells. Compared to CMV monoinfection, CMV/HIV coinfection showed to be associated with lower proportion of CD28−CD8+ T cells expressing CD57 suggesting that HIV preferentially expands CD28−CD57−CD8+ T cells and impedes terminal differentiation of CD28−CD8+ T cells. We could not show any association between HIV or CMV infection status and concentration of CRP and CD163. Conclusions CMV infection is associated with phenotypic signs of T-cell senescence, promoting exacerbation and persistence of alterations of the T-cell compartment in PLWH on effective ART, which are associated with adverse clinical outcomes and may be an attractive target for therapeutic interventions.

Abstract Background Cytotoxic Natural Killer (NK) cells are increasingly recognized as a powerful tool to induce targeted cell death in cancer and autoimmune diseases. Still, basic blood NK cell parameters are poorly defined. The aims of this study were 1) to establish reference values of NK cell counts and percentages in healthy adults; 2) to describe these parameters in the prototype autoimmune disease group ANCA-associated vasculitis (AAV); and 3) to investigate whether NK cell counts and percentages may be used as activity biomarkers in the care of AAV patients, as suggested by a preceding study. Methods CD3-(CD56 or 16)+ NK cell counts and percentages were determined in 120 healthy adults. Lymphocyte subset and clinical data from two German vasculitis centers were analyzed retrospectively (in total 407 measurements, including 201/49/157 measurements from 64/16/39 patients with granulomatosis with polyangiitis (GPA), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA), respectively). Results CD3-(CD56 or 16)+ NK cell counts and percentages in healthy adults were highly variable, not Gaussian distributed and independent of age and sex. NK cell percentages ranged from 1.9 to 37.9% of lymphocytes, and were significantly more dispersed in AAV (0.3 to 57.6%), while the median percentage was not different between AAV and healthy donors. In contrast, median NK cell counts were significantly lower in AAV compared to healthy donors. Sub-group analyses revealed that NK cell counts were low independent of AAV entity and disease activity. Azathioprine therapy was associated with significantly lower NK cell counts and percentages compared to non-azathioprine therapies. In 13.6% of azathioprine-treated patients, percentages were

IntroductionAnti-DFS70 antibodies occur in healthy individuals with various medical conditions. Unlike other anti-nuclear autoantibodies (ANA), they are not associated with systemic autoimmune disease in adult patients. To date, only a few studies have addressed the prevalence and/or clinical relevance of anti-DFS70 autoantibodies in children with and without autoimmune disease.MethodsIncluded in this retrospective cross-sectional mono-centric study were 308 pediatric patients with suspected or known autoimmune conditions who had a positive ANA in indirect immune fluorescence (IIF) screening and who were screened for anti-DFS70 antibodies by extractable nuclear antigen antibodies (ENA) immunoblot. Patients were assigned to four different diagnostic categories according to their diagnosis in the corresponding medical record: (a) absence of autoimmune or rheumatic disease (noARD, n = 116); (b) suspected autoimmunity without definitive diagnosis (sAI, n = 48); (c) other rheumatic disease (ORD) (n = 115); and (d) ANA-associated autoimmune disease (AARD, n = 29).ResultsThe prevalence of anti-DFS70 antibodies in the overall cohort was 33.8%. Among children without ARD (46.6%, 54/116), prevalence was significantly higher than among children with ORD (23.7%, 27/115, p = 0.0003) or AARD (17.2%, 5/29, p = 0.0054). Among all of the anti-DFS70 positive patients with AARD, other autoantibodies were found in the ENA immunoblot. In contrast, among anti-DFS70 positive patients with ORD (11.5%, 4/27), sAI (33.3%, 6/18) and noARD (16.7%, 9/54), other autoantibodies infrequently were detected (p = 0.0005). Patients with uveitis rarely were positive for anti-DFS70 antibodies (7.7%, 1/13). No association was found between anti-DFS70 antibodies and a history of allergic conditions (p = 0.51). The concordance between a typical DFS pattern in IIF and the detection of anti-DFS70 antibodies by immunoblot was 59.3%.ConclusionAs with adults, the higher prevalence of anti-DFS70 among children without autoimmune disease confirms the mutual exclusion for this autoantibody in the pathogenesis of ARD. Among ANA-positive children, monospecific anti-DFS70 antibodies may help to discriminate between AARD and not-AARD-related conditions.

Abstract Objective Pantothenate kinase 2‐associated neurodegeneration (PKAN) is a rare neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene. PKAN is associated with iron deposition in the basal ganglia and, occasionally, with the occurrence of misshaped erythrocytes (acanthocytes). The aim of this study was to assess residual activity of PANK2 in erythrocytes of PKAN patients and to correlate these data with the type of PANK2 mutations and the progression of neurodegeneration. Methods Residual PANK2 activities in erythrocytes of 14 PKAN patients and 14 related carriers were assessed by a radiometric assay. Clinical data on neurodegeneration included the Barry–Albright Dystonia Scale (BAD‐Scale) besides further general patient features. A molecular visualization and analysis program was used to rationalize the influence of the PKAN causing mutations on a molecular level. Results Erythrocytes of PKAN patients had markedly reduced or no PANK2 activity. However, patients with at least one allele of the c.1583C > T (T528M) or the c.833G > T (R278L) variant exhibited 12–56% of residual PANK2 activity. In line, molecular modeling indicated only minor effects on enzyme structure for these point mutations. On average, these patients with c.1583C > T or c.833G > T variant had lower BAD scores corresponding to lower symptom severity than patients with other PANK2 point mutations. Interpretation Residual erythrocyte PANK2 activity could be a predictor for the progression of neurodegeneration in PKAN patients. Erythrocytes are an interesting patient‐derived cell system with still underestimated diagnostic potential.

FLT3‐ITD tyrosine kinase inhibitors (TKI) show limited clinical activity in acute myeloid leukemia (AML) due to emerging resistance. TKI resistance is mediated by secondary FLT3‐ITD mutations only in a minority of cases. We hypothesize that the cytokine CCL5 protects AML cells from TKI‐mediated cell death and contributes to treatment resistance. We generated PKC412‐ and sorafenib‐resistant MOLM‐13 cell lines as an in vitro model to study TKI resistance in AML. Increased CCL5 levels were detected in supernatants from PKC412‐resistant cell lines compared to TKI‐sensitive cells. Moreover, CCL5 treatment of TKI‐sensitive cells induced resistance to PKC412. In resistant cell lines with high CCL5 release, we observed a significant downregulation of the CCL5‐receptor CCR5 and CXCR4. In these cell lines, TKI resistance could be partly overcome by addition of the CXCR4‐receptor antagonist plerixafor. Microarray and intracellular flow cytometry analyses revealed increased p‐Akt or p‐Stat5 levels in PKC412‐resistant cell lines releasing high amounts of CCL5. Treatment with the CXCR4 antagonist plerixafor, αCCL5, or CCR5‐targeting siRNA led to a decrease of p‐Akt‐positive cells. Transient transfection of sensitive MOLM‐13 cells with a CCL5‐encoding vector mediated resistance against PKC412 and led to an increase in p‐Akt‐positive and p‐Stat5‐positive cells. Isolated AML blasts from patients treated with PKC412 revealed that CCL5 transcript levels increase significantly at relapse. Taken together, our findings indicate that CCL5 mediates resistance to FLT3‐TKIs in FLT3‐ITD‐mutated AML and could possibly serve as a biomarker to predict drug resistance.

Ascorbic acid (AA; or vitamin C) is an important physiological antioxidant and radical scavenger. Some mammalian species, including homo sapiens, have lost the ability to synthetize AA and depend on its nutritional uptake. Erythrocytes from AA-auxotroph mammals express high amounts of the glucose transporter GLUT1. This isoform enables rapid uptake of glucose as well as dehydroascorbate (DHA), the fully oxidized form of AA. Here, we explored the effects of DHA uptake on the redox metabolism of human erythrocytes. DHA uptake enhanced plasma membrane electron transport (PMET) activity. This process is mediated by DCytb, a membrane bound cytochrome catalyzing extracellular reduction of Fe3+ and ascorbate free radical (AFR), the first oxidized form of AA. DHA uptake also decreased cellular radical oxygen species (ROS) levels. Both effects were massively enhanced in the presence of physiological glucose concentrations. Reduction of DHA to AA largely depleted intracellular glutathione (GSH) and induced the efflux of its oxidized form, GSSG. GSSG efflux could be inhibited by MK-571 (IC50 = 5 μM), indicating involvement of multidrug resistance associated protein (MRP1/4). DHA-dependent GSH depletion and GSSG efflux were completely rescued in the presence of 5 mM glucose and, partially, by 2-deoxy-glucose (2-DG), respectively. These findings indicate that human erythrocytes are physiologically adapted to recycle AA both intracellularly via GLUT1-mediated DHA uptake and reduction and extracellularly via DCytb-mediated AFR reduction. We discuss the possibility that this improved erythrocyte-mediated AA recycling was a prerequisite for the emergence of AA auxotrophy which independently occurred at least twice during mammalian evolution.

Changed dietary habits in Western countries such as reduced fiber intake represent an important lifestyle factor contributing to the increase in inflammatory immune-mediated diseases. The mode of action of beneficial fiber effects is not fully elucidated, but short-chain fatty acids (SCFA) and gut microbiota have been implicated. The aim of this study was to explore the impact of dietary fiber on lupus pathology and to understand underlying mechanisms. Here, we show that in lupus-prone NZB/WF1 mice low fiber intake deteriorates disease progression reflected in accelerated mortality, autoantibody production and immune dysregulation. In contrast to our original assumption, microbiota suppression by antibiotics or direct SCFA feeding did not influence the course of lupus-like disease. Mechanistically, our data rather indicate that in low fiber-fed mice, an increase in white adipose tissue mass, fat-inflammation and a disrupted intestinal homeostasis go along with systemic, low-grade inflammation driving autoimmunity. The links between obesity, intestinal leakage and low-grade inflammation were confirmed in human samples, while adaptive immune activation predominantly correlated with lupus activity. We further propose that an accelerated gastro-intestinal passage along with energy dilution underlies fiber-mediated weight regulation. Thus, our data highlight the often-overlooked effects of dietary fiber on energy homeostasis and obesity prevention. Further, they provide insight into how intricately the pathologies of inflammatory immune-mediated conditions, such as obesity and autoimmunity, might be interlinked, possibly sharing common pathways.

Pantothenate kinase-associated neurodegeneration (PKAN) is a progressive neurodegenerative disease caused by mutations in the pantothenate kinase 2 (PANK2) gene and associated with iron deposition in basal ganglia. Pantothenate kinase isoforms catalyze the first step in coenzyme A (CoA) biosynthesis. Since PANK2 is the only isoform in erythrocytes, these cells are an excellent ex vivo model to study the effect of PANK2 point mutations on expression/stability and activity of the protein as well as on the downstream molecular consequences. PKAN erythrocytes containing the T528M PANK2 mutant had residual enzyme activities but variable PANK2 abundances indicating an impaired regulation of the protein. Patients with G521R/G521R, G521R/G262R, and R264N/L275fs PANK2 mutants had no residual enzyme activity and strongly reduced PANK2 abundance. G521R inactivates the catalytic activity of the enzyme, whereas G262R and the R264N point mutations impair the switch from the inactive to the active conformation of the PANK2 dimer. Metabolites in cytosolic extracts were analyzed by gas chromatography–mass spectrometry and multivariate analytic methods revealing changes in the carboxylate metabolism of erythrocytes from PKAN patients as compared to that of the carrier and healthy control. Assuming low/absent CoA levels in PKAN erythrocytes, changes are consistent with a model of altered citrate channeling where citrate is preferentially converted to α-ketoglutarate and α-hydroxyglutarate instead of being used for de novo acetyl-CoA generation. This finding hints at the importance of carboxylate metabolism in PKAN pathology with potential links to reduced cytoplasmic acetyl-CoA levels in neurons and to aberrant brain iron regulation.

Abstract Background Some rheumatologic disorders may initially manifest with central nervous system (CNS) affection, mimicking the clinical, magnetic resonance imaging, and cerebrospinal fluid findings of multiple sclerosis (MS). The MRZ reaction (MRZR), composed of the three respective antibody indices (AIs) against measles, rubella, and varicella zoster virus, has been found positive frequently in MS patients. However, it is unclear whether the MRZR is helpful to distinguish rheumatologic disorders with CNS involvement (RDwCNS) from MS. Methods The MRZR was evaluated in patients with RDwCNS (n = 23), MS (n = 46; age and sex matched to patients with RDwCNS), and other inflammatory autoimmune neurological diseases affecting the CNS (OIND; n = 48). Both the stringency levels that have been used in previous MRZR studies, MRZR-1 (≥ 1 of 3 AIs positive) and MRZR-2 (≥ 2 of 3 AIs positive), were applied. Results There was no statistically significant difference in the prevalence of positive MRZR between patients with RDwCNS (MRZR-1: 13.0% and MRZR-2: 8.7%, respectively) and OIND (MRZR-1: 22.9% and MRZR-2: 8.3%, respectively). Compared to these two study cohorts, the MS group exhibited significantly higher prevalences of positive MRZR (MRZR-1: 82.6%, MRZR-2: 63.0%; p

Summary: B cell activating factor (BAFF) provides B cells with essential survival signals. It binds to three receptors: BAFFR, TACI, and BCMA that are differentially expressed by B cell subsets. BAFFR is early expressed in circulating B cells and provides key signals for further maturation. Here, we report that highly regulated BAFFR processing events modulate BAFF responses. BAFFR processing is triggered by BAFF binding in B cells co-expressing TACI and it is executed by the metalloproteases ADAM10 and ADAM17. The degree of BAFF oligomerization, the expression of ADAM proteins in different B cell subsets, and the activation status of the cell determine the proteases involved in BAFFR processing. Inhibition of ADAM10 augments BAFF-dependent survival of primary human B cells, whereas inhibition of ADAM17 increases BAFFR expression levels on germinal center B cells. Therefore, BAFF-induced processing of BAFFR regulates BAFF-mediated B cell responses in a TACI-dependent manner. : Smulski et al. report that the B cell survival receptor BAFFR undergoes ligand-induced shedding but only in cells co-expressing a second receptor for BAFF called TACI. BAFFR shedding can be performed by ADAM10 in circulating B cells or by ADAM17 in germinal center B cells and limits BAFF-mediated survival signals. Keywords: B cell, BAFF-receptor, BAFFR, TACI, ADAM10, ADAM17, metalloprotease, processing, ectodomain shedding, germinal center, survival

Background: Organic psychiatric disorders can be caused by immunological disorders, such as autoimmune encephalitis or systemic lupus erythematosus (SLE). SLE can affect most organs, as well as the central nervous system (CNS). In this paper, we describe a patient with an isolated psychiatric syndrome in the context of SLE and discuss the role of antibody detection in the cerebrospinal fluid (CSF).Case presentation: The 22-year-old German male high school graduate presented with obsessive–compulsive and schizophreniform symptoms. He first experienced obsessive–compulsive symptoms at the age of 14. At the age of 19, his obsessive thoughts, hallucinations, diffuse anxiety, depressed mood, severe dizziness, and suicidal ideation became severe and did not respond to neuroleptic or antidepressant treatment. Due to increased antinuclear antibodies (ANAs) with anti-nucleosome specificity in serum and CSF, complement activation, multiple bilateral white matter lesions, and inflammatory CSF alterations, we classified the complex syndrome as an isolated psychiatric variant of SLE. Immunosuppressive treatment with two times high-dose steroids, methotrexate, and hydroxychloroquine led to a slow but convincing improvement.Conclusion: Some patients with psychiatric syndromes and increased ANA titers may suffer from psychiatric variants of SLE, even if the American College of Rheumatology criteria for SLE are not met. Whether the psychiatric symptoms in our patient represent a prodromal stage with the later manifestation of full-blown SLE or a subtype of SLE with isolated CNS involvement remains unclear. Regardless, early diagnosis and initiation of immunosuppressive treatment are essential steps in preventing further disease progression and organ damage. Intrathecal ANAs with extractable nuclear antigen differentiation may be a more sensitive marker of CNS involvement compared with serum analyses alone.

Objective: Rheumatic diseases with involvement of the central nervous system (RDwCNS) may mimic multiple sclerosis (MS). Inversely, up to 60% of MS-patients have antinuclear autoantibodies (ANAs) and may be misdiagnosed as RDwCNS. The detection of antibodies against extractable nuclear antigens (ENA) and oligoclonal bands (OCB) are established valuable diagnostic tools in the differential diagnosis of RDwCNS and MS. The MRZ-reaction (MRZR) is defined by three antibody indices (AIs) against neurotropic viruses and is frequently positive in MS. To investigate the added value of MRZR combined with testing for antibodies against ENAs and OCB detection to distinguish RDwCNS from ANA positive MS.Methods: MRZR was evaluated in RDwCNS (n = 40) and 68 ANA positive MS-patients. Two stringency levels, MRZR-1 and MRZR-2 (at least one respectively two of three AIs positive) were applied. Autoantibody testing included ANA plus ENA and anti-dsDNA antibodies, antiphospholipid antibodies, and anti-neutrophil cytoplasmic antibodies.Results: Most of the RDwCNS patients (n = 32; 80%) suffered from systemic lupus erythematosus. Within the RDwCNS group 20% had a positive MRZR-1 and 8.5% a positive MRZR-2 compared to 80.9 and 60%, respectively within the MS-group (p < 0.0001 for both comparisons). Oligoclonal bands were found in 28.6% of the RDwCNS patients and 94.3% of the MS-patients (p < 0.0001). Conversely, autoantibodies to specific nuclear antigens or phospholipids were found more frequently in RDwCNS. A positive MRZR in conjunction with the absence of ENA autoantibodies distinguished MS from RDwCNS with high specificity (97.5%).Conclusions: We suggest combining MRZR, OCBs, and specific autoantibody diagnostics to differentiate RDwCNS from MS.

Stomatin is an ancient, widely expressed, oligomeric, monotopic membrane protein that is associated with cholesterol-rich membranes/lipid rafts. It is part of the SPFH superfamily including stomatin-like proteins, prohibitins, flotillin/reggie proteins, bacterial HflK/C proteins and erlins. Biochemical features such as palmitoylation, oligomerization, and hydrophobic "hairpin" structure show similarity to caveolins and other integral scaffolding proteins. Recent structure analyses of the conserved PHB/SPFH domain revealed amino acid residues and subdomains that appear essential for the structure and function of stomatin. To test the significance of these residues and domains, we exchanged or deleted them, expressed respective GFP-tagged mutants, and studied their subcellular localization, molecular dynamics and biochemical properties. We show that stomatin is a cholesterol binding protein and that at least two domains are important for the association with cholesterol-rich membranes. The conserved, prominent coiled-coil domain is necessary for oligomerization, while association with cholesterol-rich membranes is also involved in oligomer formation. FRAP analyses indicate that the C-terminus is the dominant entity for lateral mobility and binding site for the cortical actin cytoskeleton.

Erythrocytes are known to shed vesicles in vivo, under various conditions in vitro, and, with impact for transfusion medicine, during storage of red blood cell concentrates (Vsto vesicles). Vsto vesicles of blood transfusions have been shown to deliver glycosylphosphatidylinositol-linked proteins to recipient erythrocytes, to display prothrombotic activity, and to have an inhibitory effect on macrophages. The interaction of Vsto vesicles with and their effect on neutrophilic granulocytes has not yet been studied in detail. Fluorescentlylabeled Vsto and calcium-induced vesicles were preparedin order to study the uptake of labeled vesicular components by neutrophils as compared to the process of phagocytosis of zymosan using flow cytometry and confocal microscopy. The activating effect of Vsto vesicles on neutrophils was addressed by a luminometric assay for stimulated radical oxygen species (ROS) generation. Coincubation of vesicles and neutrophils results in a transfer of vesicular components to the cells. This uptake is different from a phagocytotic process and is enhanced upon interference with the cellular actin cytoskeleton. Preincubation of neutrophils with Vsto vesicles results in an enhanced ROS generation by neutrophils, which is further increased upon fMLP stimulation and during zymosan phagocytosis. The activating effect of Vsto vesicles on neutrophils might be due to the specific accumulation of lysophospholipidsin Vsto vesicles and should be considered as a possible contributor to the pathogenesis of transfusion-related acute lung injury.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a form of Neurodegeneration with Brain Iron Accumulation (NBIA) associated with mutations in the pantothenate kinase 2 gene (PANK2). Pantothenate kinases catalyze the rate-limiting step of coenzyme A synthesis and Pank2 is the only pantothenate kinase isoform in humans that is localized to mitochondria. Acanthocytosis, the occurrence of spiculated erythrocytes, is observed in about 10% of the PKAN patients. Therefore PKAN is also classified together with other rare neurodegenerative diseases like Chorea Acanthocytosis (ChAc) and McLeod syndrome (MLS) into the Neuroacanthocytosis (NA) syndromes. It has not been investigated yet whether acanthocytosis in PKAN is associated with a specific subset of Pank2 mutations. In this study, we analyzed acanthocytosis of a cohort of 25 PKAN patients from the Dominican Republic that are homozygous for the c.680 A>G mutation in the PANK2 gene as compared to control donors that are heterozygous or wild-type with respect to this mutation. 3D modeling of this mutation indicated that the replacement of a tyrosine by a cysteine at position 227 in Pank2 disrupts a polar interaction within the A domain of the enzyme. Mean acanthocyte count was elevated in the cohort of patients, however, acanthocytosis varied among the patients with nearly half of them showing high (>20%) or elevated acanthocytosis and the rest showing mild (6-10%) or no (

Currently very little is known about the differential expression and function of the transcription factor SOX5 during B cell maturation. We identified two new splice variants of SOX5 in human B cells, encoding the known L-SOX5B isoform and a new shorter isoform L-SOX5F. The SOX5 transcripts are highly expressed during late stages of B-cell differentiation, including atypical memory B cells, activated CD21low B cells and germinal center B cells of tonsils. In tonsillar sections SOX5 expression was predominantly polarized to centrocytes within the light zone. After in vitro stimulation, SOX5 expression was down-regulated during proliferation while high expression levels were permissible for plasmablast differentiation. Overexpression of L-SOX5F in human primary B lymphocytes resulted in reduced proliferation, less survival of CD138neg B cells, but comparable numbers of CD138+CD38hi plasmablasts compared to control cells. Thus, our findings describe for the first time a functional role of SOX5 during late B cell development reducing the proliferative capacity and thus potentially affecting the differentiation of B cells during the germinal center response.

Neuroacanthocytosis (NA) refers to a group of heterogenous, rare genetic disorders, namely chorea acanthocytosis (ChAc), McLeod syndrome (MLS), Huntington's disease-like 2 (HDL2) and pantothenate kinase associated neurodegeneration (PKAN), that mainly affect the basal ganglia and are associated with similar neurological symptoms. PKAN is also assigned to a group of rare neurodegenerative diseases, known as NBIA (neurodegeneration with brain iron accumulation), associated with iron accumulation in the basal ganglia and progressive movement disorder. Acanthocytosis, the occurrence of misshaped erythrocytes with thorny protrusions, is frequently observed in ChAc and MLS patients but less prevalent in PKAN (about 10%) and HDL2 patients. The pathological factors that lead to the formation of the acanthocytic red blood cell shape are currently unknown. The aim of this study was to determine whether NA/NBIA acanthocytes differ in their functionality from normal erythrocytes. Several flow-cytometry-based assays were applied to test the physiological responses of the plasma membrane, namely drug-induced endocytosis, phosphatidylserine exposure and calcium uptake upon treatment with lysophosphatidic acid. ChAc red cell samples clearly showed a reduced response in drug-induced endovesiculation, lysophosphatidic acid-induced phosphatidylserine exposure, and calcium uptake. Impaired responses were also observed in acanthocyte-positive NBIA (PKAN) red cells but not in patient cells without shape abnormalities. These data suggest an "acanthocytic state" of the red cell where alterations in functional and interdependent membrane properties arise together with an acanthocytic cell shape. Further elucidation of the aberrant molecular mechanisms that cause this acanthocytic state may possibly help to evaluate the pathological pathways leading to neurodegeneration.

TNFRSF13B/TACI defects have been associated with CVID pathogenesis and/or phenotype, especially the development of benign lymphoproliferation and autoimmunity. Our purpose was to investigate the role of TNFRSF13B/TACI defects in the pathogenesis of two common lymphoproliferative disorders, namely, sarcoidosis and tonsillar hypertrophy (TH). 105 patients (71 with sarcoidosis and 34 with TH, including 19 without infectious causative and 15 due to Haemophilus influenzae) were analyzed for TNFRSF13B/TACI defects. Two out of 19 TH patients without infectious cause (10.5%) and 2 patients with sarcoidosis (2.8%) displayed rare TNFRSF13B/TACI defects (I87N, L69TfsX12, E36L, and R202H, resp.). Both mutations identified in TH patients have been assessed as deleterious for protein function, while the patient with the R202H mutation and sarcoidosis exhibited also sIgG4D. Our study further supports the notion that TNFRSF13B/TACI defects alone do not result in CVID but may be also found frequently in distinct clinical phenotypes, including benign lymphoproliferation and IgG subclass deficiencies.

OBJECTIVE: To assess the impact of immunosuppressive therapy with cyclophosphamide (CYC) and rituximab (RTX) on serum immunoglobulin (Ig) concentrations and B lymphocyte counts in patients with ANCA-associated vasculitides (AAVs). METHODS: Retrospective analysis of Ig concentrations and peripheral B cell counts in 55 AAV patients. RESULTS: CYC treatment resulted in a decrease in Ig levels (median; interquartile range IQR) from IgG 12.8 g/L (8.15-15.45) to 9.17 g/L (8.04-9.90) (p = 0.002), IgM 1.05 g/L (0.70-1.41) to 0.83 g/L (0.60-1.17) (p = 0.046) and IgA 2.58 g/L (1.71-3.48) to 1.58 g/L (1-31-2.39) (p = 0.056) at a median follow-up time of 4 months. IgG remained significantly below the initial value at 14.5 months and 30 months analyses. Subsequent RTX treatment in patients that had previously received CYC resulted in a further decline in Ig levels from pre RTX IgG 9.84 g/L (8.71-11.60) to 7.11 g/L (5.75-8.77; p = 0.007), from pre RTX IgM 0.84 g/L (0.63-1.18) to 0.35 g/L (0.23-0.48; p

Autosomal recessive mutations in the cytolinker protein plectin account for the multisystem disorders epidermolysis bullosa simplex (EBS) associated with muscular dystrophy (EBS-MD), pyloric atresia (EBS-PA), and congenital myasthenia (EBS-CMS). In contrast, a dominant missense mutation leads to the disease EBS-Ogna, manifesting exclusively as skin fragility. We have exploited this trait to study the molecular basis of hemidesmosome failure in EBS-Ogna and to reveal the contribution of plectin to hemidesmosome homeostasis. We generated EBS-Ogna knock-in mice mimicking the human phenotype and show that blistering reflects insufficient protein levels of the hemidesmosome-associated plectin isoform 1a. We found that plectin 1a, in contrast to plectin 1c, the major isoform expressed in epidermal keratinocytes, is proteolytically degraded, supporting the notion that degradation of hemidesmosome-anchored plectin is spatially controlled. Using recombinant proteins, we show that the mutation renders plectin's 190-nm-long coiled-coil rod domain more vulnerable to cleavage by calpains and other proteases activated in the epidermis but not in skeletal muscle. Accordingly, treatment of cultured EBS-Ogna keratinocytes as well as of EBS-Ogna mouse skin with calpain inhibitors resulted in increased plectin 1a protein expression levels. Moreover, we report that plectin's rod domain forms dimeric structures that can further associate laterally into remarkably stable (paracrystalline) polymers. We propose focal self-association of plectin molecules as a novel mechanism contributing to hemidesmosome homeostasis and stabilization.

Stiff person syndrome (SPS) is a rare, neurological disorder characterized by sudden cramps and spasms. High titers of enzyme-inhibiting IgG autoantibodies against the 65 kD isoform of glutamic acid decarboxylase (GAD65) are a hallmark of SPS, implicating an autoimmune component in the pathology of the syndrome. Studying the B cell compartment and the anti-GAD65 B cell response in two monozygotic twins suffering from SPS, who were treated with the B cell-depleting monoclonal anti-CD20 antibody rituximab, we found that the humoral autoimmune response in SPS is composed of a rituximab-sensitive part that is rapidly cleared after treatment, and a rituximab-resistant component, which persists and acts as a reservoir for autoantibodies inhibiting GAD65 enzyme activity. Our data show that these potentially pathogenic anti-GAD65 autoantibodies are secreted by long-lived plasma cells, which may either be persistent or develop from rituximab-resistant memory B lymphocytes. Both subsets represent only a fraction of anti-GAD65 autoantibody secreting cells. Therefore, the identification and targeting of this compartment is a key factor for successful treatment planning of SPS and of similar autoimmune diseases.

The study determines the molecular cause of inflammatory bowel disease by examining the genetic mutations affecting the interleukin-10 receptor. Evidence reveals mutations in genes encoding the IL10R subunit proteins in patients with early-onset enterocolitis, involving hyper-inflammatory immune responses in the intestine.