Splenectomy at early stage of autoimmune arthritis delayed inflammatory response and reduced joint deterioration in mice.

The spleen plays a role in innate and adaptive immunity, and autoimmune diseases like rheumatoid arthritis (RA). We investigated the effect of splenectomy in early and moderate stages of autoimmune arthritis in a mouse model. To induce recombinant human G1-induced arthritis (GIA), BALB/c mice were immunized intraperitoneally three times in 4-week intervals with the rhG1 antigen. Mice were splenectomized on day 7 (SPE1) or day 35 (SPE2) after the initiation of immunization; tested for clinical severity, joint radiological and histological changes, serum levels of inflammatory cytokines and autoantibodies, and rhG1-specific immune responses; and compared to those in control mice with spleen left intact. Circulating Tregs and T-helper subset ratios in the spleen and inguinal lymph nodes (LNs) were also examined using flow cytometry. The onset of severe inflammatory response was significantly delayed in SPE1 and SPE2 groups compared to control mice at early stages of GIA, which was associated with increased circulating Tregs. After the third immunization, as disease progressed, the severity scores were robustly increased in all mice. Nevertheless, in splenectomized mice, we observed reduced joint deterioration and cartilage damage, more Th2 cells in LNs, and reduced levels of pro-inflammatory cytokines and autoantibodies in their sera. Mesenteric LN cells of splenectomized mice exhibited weaker response in vitro against the rhG1 antigen compared to control mice spleen. In conclusion, splenectomy in the early stages of GIA delayed the inflammatory response, suggesting a protective effect against the development and progression of severe destructive arthritis.

Altered Levels of Natural Autoantibodies against Heat Shock Proteins in Pregnant Women with Hashimoto's Thyroiditis.

The function of natural autoantibodies (nAAbs) in maintaining immunological tolerance has been comprehensively explained; however, their function in pregnant patients dealing with autoimmune diseases has not been thoroughly investigated. As Hashimoto's thyroiditis (HT) is the predominant organ-specific autoimmune condition of women of childbearing age, this study's objective was to evaluate IgM and IgG nAAbs targeting mitochondrial citrate synthase (CS) and heat shock proteins (Hsp60 and Hsp70) in women diagnosed with HT who were pregnant (HTP). Serum samples collected from HTP and healthy pregnant (HP) women in the first and third trimesters were tested using in-house-developed enzyme-linked immunosorbent assays (ELISAs). Our findings indicate the stability of nAAbs against CS and Hsps throughout the pregnancies of both healthy women and those with HT. However, during both trimesters, HTP patients displayed elevated levels of IgM isotype nAAbs against Hsp60 and Hsp70 compared to HP women, suggesting a regulatory role of IgM nAAbs during the pregnancies of patients with HT. Nonetheless, levels of IgG isotype nAAbs against Hsps were lower solely in the third trimester among HTP patients, resulting in a higher IgM/IgG ratio, which indicates their importance in alterations of the nAAb network during pregnancy in patients with HT.

Natural Autoantibodies in Biologic-Treated Rheumatoid Arthritis and Ankylosing Spondylitis Patients: Associations with Vascular Pathophysiology.

Cardiovascular (CV) morbidity and mortality have been associated with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Natural autoantibodies (nAAb) are involved in innate immunity, as well as autoimmunity, inflammation, and atherosclerosis. There have not been any studies assessing the effects of biologics on nAAbs in RA and AS, also in relation to vascular pathophysiology. Fifty-three anti-TNF-treated RA and AS patients were included in a 12-month follow-up study. Anti-citrate synthase (CS) and anti-topoisomerase I fragment 4 (TOPO-F4) IgM and IgG levels were determined by ELISA. Ultrasonography was performed to assess brachial artery flow-mediated vasodilation (FMD), common carotid intima-media thickness (ccIMT), and arterial pulse-wave velocity (PWV). Other variables were also evaluated at baseline and 6 and 12 months after treatment initiation. Anti-TNF therapy improved FMD in RA and PWV in AS and stabilized ccIMT. TNF inhibition increased anti-CS IgM and IgG, and possibly also anti-TOPO-F4 IgG levels. Various correlation analyses revealed that nAAbs might be independently involved in autoimmunity as well as changes in inflammation and vascular pathology over time in biologic-treated patients (p < 0.05). We also found associations between anti-TOPO-F4 IgG and anti-Hsp60 IgG (p < 0.05). Baseline nAAb levels or nAAb level changes might determine changes in CRP, disease activity, FMD, PWV, and ccIMT over time (p < 0.05). The interplay between arthritis and inflammatory atherosclerosis, as well as the effects of anti-TNF biologics on these pathologies, might independently involve nAAbs.

Complexity of the Immune Response Elicited by Different COVID-19 Vaccines, in the Light of Natural Autoantibodies and Immunomodulatory Therapies.

Despite the abundance of data on the COVID-19 vaccine-induced immune activation, the impact of natural autoantibodies (nAAbs) on these processes is less well defined. Therefore, we investigated potential connections between vaccine efficacy and nAAb levels. We were also interested in the impact of immunomodulatory therapies on vaccine efficacy. Clinical residual samples were used for the assessment of the COVID-19 vaccine-elicited immune response (IR) (n=255), as well as for the investigation of the immunization-associated expansion of the nAAb pool (n=185). In order to study the potential interaction between immunomodulatory therapies and the vaccine-induced IR, untreated, healthy individuals and patients receiving anti-TNFα or anti-IL-17 therapies were compared (n total =45). In-house ELISAs (anticitrate synthase, anti-HSP60 and-70) and commercial ELISAs (anti-SARS-CoV-2 ELISAs IgG, IgA, NeutraLISA and IFN-γ release assay 'IGRA') were applied. We found significant differences in the IR given to different vaccines. Moreover, nAAb levels showed plasticity in response to anti-COVID-19 immunization. We conclude that our findings may support the theorem about the non-specific beneficial 'side effects' of vaccination, including the broadening of the nAAb repertoire. Considering immunomodulation, we suggest that anti-TNFα and anti-IL17 treatments may interfere negatively with MALT-associated IR, manifested as decreased IgA titers; however, the modest sample numbers of the herein presented model might be a limiting factor of reaching a more comprehensive conclusion.

An Optimized Flow Cytometric Method to Demonstrate the Differentiation Stage-Dependent Ca2+ Flux Responses of Peripheral Human B Cells.

Calcium (Ca2+) flux acts as a central signaling pathway in B cells, and its alterations are associated with autoimmune dysregulation and B-cell malignancies. We standardized a flow-cytometry-based method using various stimuli to investigate the Ca2+ flux characteristics of circulating human B lymphocytes from healthy individuals. We found that different activating agents trigger distinct Ca2+ flux responses and that B-cell subsets show specific developmental-stage dependent Ca2+ flux response patterns. Naive B cells responded with a more substantial Ca2+ flux to B cell receptor (BCR) stimulation than memory B cells. Non-switched memory cells responded to anti-IgD stimulation with a naive-like Ca2+ flux pattern, whereas their anti-IgM response was memory-like. Peripheral antibody-secreting cells retained their IgG responsivity but showed reduced Ca2+ responses upon activation, indicating their loss of dependence on Ca2+ signaling. Ca2+ flux is a relevant functional test for B cells, and its alterations could provide insight into pathological B-cell activation development.

Different Kinetics of Serum ADAMTS13, GDF-15, and Neutrophil Gelatinase-Associated Lipocalin in the Early Phase of Aneurysmal Subarachnoid Hemorrhage.

Growth differentiation factor 15 (GDF-15), neutrophil gelatinase-associated lipocalin (NGAL), and ADAMTS13 have previously been implicated in the pathophysiological processes of SAH. In the present study, we aim to examine their role in the early period of SAH and their relationship to primary and secondary outcomes. Serum samples were collected at five time periods after SAH (at 24 h (D1), at 72 h (D3), at 120 h (D5), at 168 h (D7) and at 216 h (D9), post-admission) and) were measured by using MILLIPLEX Map Human Cardiovascular Disease (CVD) Magnetic Bead Panel 2. We included 150 patients with SAH and 30 healthy controls. GDF-15 levels at D1 to D9 were significantly associated with a 3-month unfavorable outcome. Based on the ROC analysis, in patients with a good clinical grade at admission (WFNS I-III), the GDF-15 value measured at time point D3 predicted a 3-month unfavorable outcome (cut-off value: 3.97 ng/mL, AUC:0.833, 95%CI: 0.728-0.938, sensitivity:73.7%, specificity:82.6%, p < 0.001). Univariate binary logistic regression analysis showed that serum NGAL levels at D1-D5 and ADAMTS13 levels at D7-D9 were associated with MVS following SAH. GDF-15 is an early indicator of a poor 3-month functional outcome even in patients with mild clinical conditions at admission.

Following Natural Autoantibodies: Further Immunoserological Evidence Regarding Their Silent Plasticity and Engagement in Immune Activation.

Contradictory reports are available on vaccine-associated hyperstimulation of the immune system, provoking the formation of pathological autoantibodies. Despite being interconnected within the same network, the role of the quieter, yet important non-pathological and natural autoantibodies (nAAbs) is less defined. We hypothesize that upon a prompt immunological trigger, physiological nAAbs also exhibit a moderate plasticity. We investigated their inducibility through aged and recent antigenic triggers. Anti-viral antibodies (anti-MMR n = 1739 and anti-SARS-CoV-2 IgG n = 330) and nAAbs (anti-citrate synthase IgG, IgM n = 1739) were measured by in-house and commercial ELISAs using Croatian (Osijek) anonymous samples with documented vaccination backgrounds. The results were subsequently compared for statistical evaluation. Interestingly, the IgM isotype nAAb showed a statistically significant connection with anti-MMR IgG seropositivity (p < 0.001 in all cases), while IgG isotype nAAb levels were elevated in association with anti-SARS CoV-2 specific seropositivity (p = 0.019) and in heterogeneous vaccine regimen recipients (unvaccinated controls vector/mRNA vaccines p = 0.002). Increasing evidence supports the interplay between immune activation and the dynamic expansion of nAAbs. Consequently, further questions may emerge regarding the ability of nAAbs silently shaping the effectiveness of immunization. We suggest re-evaluating the impact of nAAbs on the complex functioning of the immunological network.

Natural and Pathological Autoantibodies Show Age-Related Changes in a Spontaneous Autoimmune Mouse (NZB) Model.

The natural autoantibody (natAAb) network is thought to play a role in immune regulation. These IgM antibodies react with evolutionary conserved antigens; however, they do not lead to pathological tissue destruction as opposed to pathological autoantibodies (pathAAb). The exact relation between the natAAbs and pathAAbs is still not completely understood; therefore, in the present study, we set out to measure nat- and pathAAb levels against three conserved antigens in a spontaneous autoimmune disease model: the NZB mouse strain which develops autoimmune hemolytic anemia (AIHA) from six months of age. There was an age dependent increase in the natAAb levels in the serum against Hsp60, Hsp70, and the mitochondrial citrate synthase until 6-9 months of age, followed by a gradual decrease. The pathological autoantibodies appeared after six months of age, which corresponded with the appearance of the autoimmune disease. The changes in nat/pathAAb levels were coupled with decreasing B1- and increasing plasma cell and memory B cell percentages. Based on this, we propose that there is a switch from natAAbs towards pathAAbs in aged NZB mice.

Splenectomy modulates the immune response but does not prevent joint inflammation in a mouse model of RA.

The spleen is the largest secondary lymphoid organ which is involved in the development of B cells and also in systemic (auto)immune responses. Using the recombinant human G1 domain-induced arthritis (GIA) model in splenectomized and control BALB/c mice, we investigated the role of the spleen in the induction and pathogenesis of autoimmune arthritis. Splenectomized mice developed GIA with a similar clinical picture to the control group. However, we observed significant alterations in the humoral and cellular immune responses in splenectomized mice. In the sera of the splenectomized mice, we found lower pro-inflammatory cytokine and anti-rhG1 IgM levels, but higher IL-4, anti-rhG1 IgG1 and anti-CCP and RF antibodies. The arthritis induction in the splenectomized group was associated with a significant expansion of activated helper T cells and an increase in the proportion of the circulating B1 and marginal zone B cell subsets. Importantly, immunization of the splenectomized mice with rhG1 induced the formation of germinal centers in the inguinal- and mesenteric lymph nodes (i/mLNs) which showed an active immune response to rhG1. Finally, both B and T cells from the mLNs of the splenectomized mice showed decreased intracellular Ca2+ signaling than those of the control group. Collectively, these findings indicate that the presence of the spleen is not critical for the induction of GIA, and in its absence the autoimmune arthritis is most likely promoted through the compensatory activity of the i/mLNs. However, our data implies the immunological role of the spleen in arthritis which could be further assessed in human RA.

Repository of intra- and inter-run variations of quantitative autoantibody assays: a European multicenter study.

OBJECTIVES: No reference data are available on repositories to measure precision of autoantibody assays. The scope of this study was to document inter- and intra-run variations of quantitative autoantibody assays based on a real-world large international data set. METHODS: Members of the European Autoimmunity Standardisation Initiative (EASI) group collected the data of intra- and inter-run variability obtained with assays quantifying 15 different autoantibodies in voluntary participating laboratories from their country. We analyzed the impact on the assay performances of the type of immunoassay, the number of measurements used to calculate the coefficient of variation (CVs), the nature and the autoantibody level of the internal quality control (IQC). RESULTS: Data were obtained from 64 laboratories from 15 European countries between February and October 2021. We analyzed 686 and 1,331 values of intra- and inter-run CVs, respectively. Both CVs were significantly dependent on: the method of immunoassay, the level of IQC with higher imprecision observed when the antibody levels were lower than 2-fold the threshold for positivity, and the nature of the IQC with commercial IQCs having lower CVs than patients-derived IQCs. Our analyses also show that the type of autoantibody has low impact on the assay' performances and that 15 measurements are sufficient to establish reliable intra- and inter-run variations. CONCLUSIONS: This study provides for the first time an international repository yielding values of intra- and inter-run variation for quantitative autoantibody assays. These data could be useful for ISO 15189 accreditation requirements and will allow clinical diagnostic laboratories to assure quality of patient results.

Ligation of TLR Homologue CD180 of B Cells Activates the PI3K/Akt/mTOR Pathway in Systemic Sclerosis and Induces a Pathological Shift in the Expression of BAFF Receptors.

The phosphatidylinositol-3-kinase (PI3K)/Akt and the mammalian target of rapamycin (mTOR) pathways are known to play a key role in B-cell activation and fibrosis in systemic sclerosis (SSc). Receptors of B-cell activator factor (BAFF) utilize these pathways, which can be influenced by Toll-like receptors (TLRs), as TLRs can alter the expression of BAFF-binding receptors. Our results show that B-cell stimulation via TLR homologue CD180 phosphorylates Akt in diffuse cutaneous SSc (dcSSc) to a lower extent than in healthy controls (HCs). We found basal downregulated BAFF receptor (BAFF-R) and enhanced transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI) expression in dcSSc B cells, which might enhance the formation of autoantibody-secreting plasma cells. Moreover, this pathological shift was observed in naive B cells, emphasizing the importance of their increase in SSc. Additionally, we measured higher serum levels of autoantibodies to BAFF in dcSSc patients, suggesting that an imbalance in the complex system of BAFF/anti-BAFF autoantibodies/BAFF-binding receptors may contribute to the development of SSc. Anti-CD180 antibody treatment had opposite effects on the expression of BAFF-R and TACI in HC B cells, resulting in similar levels as observed in SSc B cells without stimulation, which argues against the usefulness of such therapy in SSc.

Biomarker Associations in Delayed Cerebral Ischemia after Aneurysmal Subarachnoid Hemorrhage.

The prognosis for patients with aneurysmal subarachnoid hemorrhage (aSAH) is heavily influenced by the development of delayed cerebral ischemia (DCI), but the adequate and effective therapy of DCI to this day has not been resolved. Multiplex serum biomarker studies may help to understand the pathophysiological processes underlying DCI. Samples were collected from patients with aSAH at two time points: (1) 24 h (Day 1) and (2) 5−7 days after ictus. Serum concentrations of eotaxin, FGF-2, FLT-3L, CX3CL1, Il-1b, IL-4, IP-10, MCP3, and MIP-1b were determined using a customized MILLIPLEX Human Cytokine/Chemokine/Growth Factor Panel A multiplex assay. The functional outcome was defined by the modified Rankin scale (favorable: 0−2, unfavorable: 3−6) measured on the 30th day after aSAH. One-hundred and twelve patients with aSAH were included in this study. The median level of CX3CL1 and MCP-3 measured on Days 5−7 were significantly higher in patients with DCI compared with those without DCI (CX3CL1: with DCI: 110.5 pg/mL, IQR: 82−201 vs. without DCI: 82.6, 58−119, p = 0.036; and MCP-3: with DCI: 22 pg/mL (0−32) vs. without DCI: 0 (0−11), p < 0.001). IP-10, MCP-3, and MIP-1b also showed significant associations with the functional outcome after aSAH. MCP-3 and CX3CL1 may play a role in the pathophysiology of DCI.

Increased level of LIGHT/TNFSF14 is associated with survival in aneurysmal subarachnoid hemorrhage.

OBJECTIVES: Multiple cytokines have been implicated in aneurysmal subarachnoid hemorrhage (aSAH), but tumor necrosis factor superfamily 14 (LIGHT/TNFSF14) and oncostatin-M (OSM) have not been previously explored. AIMS OF THE STUDY: The primary objective of this study was to examine the relationship between TNFSF14 and OSM levels and survival. Our secondary goal was to investigate a potential association between these markers and the incidence of delayed cerebral ischemia (DCI). MATERIALS & METHODS: We consecutively recruited 60 patients with a clinical diagnosis of aSAH. LIGHT/TNFSF14 and OSM serum concentrations were determined by ELISA. The primary endpoint was survival at Day 30, while development of DCI was assessed as secondary outcome. RESULTS: Patients had significantly higher levels of both markers than the control group (median of LIGHT: 18.1 pg/ml vs. 7 pg/ml; p = 0.01; median of OSM: 10.3 pg/ml vs. 2.8 pg/ml, p < 0.001). Significantly lower serum level of LIGHT/TNFSF14 was found in nonsurviving patients (n = 9) compared with survivors (n = 51; p = 0.011). Based on ROC analysis, serum LIGHT/TNFSF14 with a cutoff value of >7.95 pg/ml predicted 30-day survival with a sensitivity of 71% and specificity of 78% (Area: 0.763; 95% CI: 0.604-0.921, p = 0.013). In addition, it was also a predictor of DCI with a sensitivity of 72.7% and a specificity of 62.5% (AUC: 0.702; 95% CI: 0.555-0.849, p = 0.018). Based on binary logistic regression analysis, LIGHT/TNFSF14 was found to be independently associated with 30-day mortality, but not with DCI. CONCLUSION: In this cohort, a higher serum level of LIGHT/TNFSF14 was associated with increased survival of patients with aSAH.

Analysis of PI3K Pathway Associated Molecules Reveals Dysregulated Innate and Adaptive Functions of B Cells in Early Diffuse Cutaneous Systemic Sclerosis.

B cell activation is an early event in the development of systemic sclerosis (SSc). The classical activation of B cells downstream of the B-cell receptor (BCR) involves the phosphatidylinositol-3 kinase (PI3K) pathway that integrates the effects of multiple co-stimulatory receptors. Our analysis of PI3K pathway associated molecules in peripheral blood B cells of early diffuse cutaneous SSc (dcSSc) patients showed altered mRNA expression of Toll-like receptor (TLR) homolog CD180, TLR4, complement component 3, IL-4 receptor and secreted phosphoprotein 1 (SPP1). Parallel to this, we found elevated basal SPP1 secretion in dcSSc B cells, but, with BCR + IL-4 receptor co-stimulation, we could not induce further secretion. CD180 stimulation alone resulted in NF-κB activation in more B cells than CD180 + BCR co-stimulation both in dcSSc and healthy control (HC), but the co-engagement increased the phosphorylation of NF-κB only in dcSSc B cells. Additionally, in contrast with HC B cells, the lower basal production of IL-10 by dcSSc B cells could not be elevated with CD180 stimulation. Furthermore, activation via CD180 increased the percentage of CD86+ switched memory (CD27+IgD-) B cells in dcSSc compared to HC. Our results suggest that alternative B cell activation and CD180 dysfunction cause imbalance of regulatory mechanisms in dcSSc B cells.

Fatty Acid-Binding Protein 3 and CXC-Chemokine Ligand 16 are Associated with Unfavorable Outcome in Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: Aneurysmal subarachnoid hemorrhage (aSAH) is associated with activation of the inflammatory cascade contributing to unfavorable outcome and secondary complications, such as delayed cerebral ischemia (DCI). Both fatty acid-binding protein 3 (FABP3) and CXC-chemokine ligand 16 (CXCL-16) have been linked to vascular inflammation and cellular death. The authors aimed to assess the 30-day prognostic value of serum levels of FABP3 and CXCL-16 and explore their associations with DCI in aSAH patients. METHODS: A total of 60 patients with aSAH were prospectively enrolled. Sampling for markers was done at 24 hours after the index event. FABP3 and CXCL-16 serum concentrations were determined by MilliPlex multiplex immunoassay method. The primary endpoint was unfavorable outcome at Day 30 based on the modified Rankin Scale. RESULTS: Both FABP3 and CXCL-16 levels were significantly elevated in patients with unfavorable outcome compared to those with favorable outcome after aSAH (FABP3: 2133 pg/mL, IQR: 1053-4567 vs. 3773, 3295-13116; p<0.003 and CXCL-16: 384 pg/mL, 313-502 vs. 498, 456-62, p<0.001). The area under the curve (AUC) for serum CXCL-16 levels as a predictor of unfavorable outcome at Day 30 was 0.747 (95% CI =0.622-0.871; p<0.001). Based on binary logistic regression analysis, serum CXCL-16 with a cut-off level >446.7 ng/L independently predicted Day 30 unfavorable outcome with a sensitivity of 81% and a specificity of 62%. Neither CXCL-16 nor FABP3 showed a significant correlation with DCI. CONCLUSION: Early FABP3 and CXCL-16 levels are significantly associated with poor 30-day outcome in patients with aSAH.

Optimization of Lyophilized Hyperacute Serum (HAS) as a Regenerative Therapeutic in Osteoarthritis.

Hyperacute serum (HAS) is a blood derivative product that promotes the proliferation of various cell types and controls inflammation in vitro. The aim of this study is to investigate the regenerative potential of different formulations of HAS, including lyophilized and hyaluronic acid combined versions, to obtain a stable and standardized therapeutic in osteoarthritis (OA), which may be able to overcome the variability limitations of platelet-rich plasma (PRP). Primary human osteoarthritic chondrocytes were used for testing cellular viability and gene expression of OA-related genes. Moreover, a co-culture of human explants of cartilage, bone and synovium under inflammatory conditions was used for investigating the inflammatory control capacities of the different therapeutics. In this study, one formulation of lyophilized HAS achieved the high cell viability rates of liquid HAS and PRP. Gene expression analysis showed that HAS induced higher Col1a1 expression than PRP. Cytokine quantification from supernatant fluids revealed that HAS treatment of inflamed co-cultures significantly reduced levels of IL-5, IL-15, IL-2, TNFα, IL-7 and IL-12. To conclude, lyophilized HAS is a stable and standardized therapeutic with high potential in joint regeneration.

Regulatory T cells are less sensitive to glucocorticoid hormone induced apoptosis than CD4+ T cells.

Earlier we have reported that thymic regulatory T cells (Treg) are resistant to in vivo glucocorticoid hormone (GC)-induced apoptosis, while the most GC-sensitive DP thymocytes died through the activation of mitochondrial apoptotic pathway. Here we analyzed the apoptosis-inducing effect of high dose (10-6 M) in vitro dexamethasone (DX) treatment in mouse thymic- and splenic Tregs and CD4+ T cells. Activation of both extrinsic and intrinsic apoptotic pathways started after 2 h of DX treatment in CD4 SP thymocytes and was 3 × higher than in CD4+ splenocytes, while in Treg cells, weak activation of the extrinsic apoptotic pathway started only after 3 h. We also investigated the expression of 21 apoptosis-related molecules using a protein array and found higher level of both pro-and anti-apoptotic molecules in Tregs compared to CD4+ T cells. 4 h in vitro DX treatment induced upregulation of most apoptosis-related molecules both in Tregs and CD4+ T cells, except for the decrease of Bcl-2 expression in CD4+ T cells. We found high basal cytosolic Ca2+ levels in untreated Treg cells, which further increased after DX treatment, while the specific TCR-induced Ca2+ signal was lower in Tregs than in CD4+ T cells. Our results suggest that in the background of the relative apoptosis resistance of Treg cells to GCs might be their high basal cytosolic Ca2+ level and upregulated Bcl-2 expression. In contrast, downregulation of Bcl-2 expression in CD4+ T cells can explain their higher, DX-induced apoptosis sensitivity.

Ameliorated Autoimmune Arthritis and Impaired B Cell Receptor-Mediated Ca2+ Influx in Nkx2-3 Knock-out Mice.

B cells play a crucial role in the pathogenesis of rheumatoid arthritis. In Nkx2-3-deficient mice (Nkx2-3-/-) the spleen's histological structure is fundamentally changed; therefore, B cell homeostasis is seriously disturbed. Based on this, we were curious, whether autoimmune arthritis could be induced in Nkx2-3-/- mice and how B cell activation and function were affected. We induced arthritis with immunization of recombinant human proteoglycan aggrecan G1 domain in Nkx2-3-/- and control BALB/c mice. We followed the clinical picture, characterized the radiological changes, the immune response, and intracellular Ca2+ signaling of B cells. Incidence of the autoimmune arthritis was lower, and the disease severity was milder in Nkx2-3-/- mice than in control BALB/c mice. The radiological changes were in line with the clinical picture. In Nkx2-3-/- mice, we measured decreased antigen-induced proliferation and cytokine production in spleen cell cultures; in the sera, we found less anti-CCP-IgG2a, IL-17 and IFNγ, but more IL-1ß, IL-4 and IL-6. B cells isolated from the lymph nodes of Nkx2-3-/- mice showed decreased intracellular Ca2+ signaling compared to those isolated from BALB/c mice. Our findings show that the transcription factor Nkx2-3 might regulate the development of autoimmune arthritis most likely through modifying B cell activation.

Toll-Like Receptor Mediated Activation of Natural Autoantibody Producing B Cell Subpopulations in an Autoimmune Disease Model.

Altered expression and function of the Toll-like receptor (TLR) homologue CD180 molecule in B cells have been associated with autoimmune disorders. In this study, we report decreased expression of CD180 at protein and mRNA levels in peripheral blood B cells of diffuse cutaneous systemic sclerosis (dcSSc) patients. To analyze the effect of CD180 stimulation, together with CpG (TLR9 ligand) treatment, on the phenotype defined by CD19/CD27/IgD/CD24/CD38 staining, and function (CD69 and CD180 expression, cytokine and antibody secretion) of B cell subpopulations, we used tonsillar B cells. After stimulation, we found reduced expression of CD180 protein and mRNA in total B cells, and CD180 protein in B cell subpopulations. The frequency of CD180+ cells was the highest in the CD19+CD27+IgD+ non-switched (NS) B cell subset, and they showed the strongest activation after anti-CD180 stimulation. Furthermore, B cell activation via CD180 induced IL-6 and natural autoantibody secretion. Treatment with the combination of anti-CD180 antibody and CpG resulted in increased IL-6 and IL-10 secretion and natural autoantibody production of B cells. Our results support the role of CD180 in the induction of natural autoantibody production, possibly by NS B cells, and suggest an imbalance between the pathologic and natural autoantibody production in SSc patients.

Subacute Elevation of Plasma Level of Caspase-Cleaved Cytokeratin-18 is Associated with Hemorrhagic Transformation and Functional Outcome in Ischemic Stroke.

BACKGROUND: Caspase-cleaved cytokeratin-18 (CCCK-18) is an apoptosis marker. Here, we analyzed the relationship between plasma level of CCCK-18 in the acute and subacute stage of ischemic stroke and early and late functional outcome. Besides, correlation among CCCK-18 and complications, such as hemorrhagic transformation (HT) were also explored. METHODS: Plasma concentration of CCCK-18 was investigated in 54 patients at admission and poststroke 72 hours. HT was evaluated by CT scans on 24 poststroke hours. Outcome measures were assessed by modified Rankin scale at hospital discharge and 6-month later. Receiver operating characteristics (ROC) analysis was used to determine the best cut-off values of CCCK-18 as a predictor of unfavorable functional outcome. RESULTS: Significantly elevated CCCK-18 level was observed at 72 hours after onset of stroke, in nonsurviving compared to surviving patients (331 ± 191 ng/L versus 251 ± 164 ng/L, P = .01). Based on ROC analysis, the cut-off value of plasma CCCK-18 levels >223 ng/L at 72 poststroke hours predicted 6-month unfavorable stroke outcome with a sensitivity of 84.4% and a specificity of 77.3% (area under the curve: .851, 95% confidence interval = .745-.955, P < .001). The rate of complications such as HT and in-hospital infection was significantly higher in patients presented with a plasma CCCK-18 level above the cut-off value. CONCLUSIONS: The association between high serum CCCK-18 levels and unfavorable early and late stroke outcome in an unselected study population was first described here. Besides, the apoptosis marker CCCK-18 might be a predictor of further complication such as HT and in-hospital infection.