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Most patients who became critically ill following infection with COVID-19 develop severe acute respiratory syndrome (SARS) attributed to a maladaptive or inadequate immune response. The complement system is an important component of the innate immune system that is involved in the opsonization of viruses but also in triggering further immune cell responses. Complement activation was seen in plasma adsorber material that clogged during the treatment of critically ill patients with COVID-19. Apart from the lung, the kidney is the second most common organ affected by COVID-19. Using immunohistochemistry for complement factors C1q, MASP-2, C3c, C3d, C4d, and C5b-9 we investigated the involvement of the complement system in six kidney biopsies with acute kidney failure in different clinical settings and three kidneys from autopsy material of patients with COVID-19. Renal tissue was analyzed for signs of renal injury by detection of thrombus formation using CD61, endothelial cell rarefaction using the marker E-26 transformation specific-related gene (ERG-) and proliferation using proliferating cell nuclear antigen (PCNA)-staining. SARS-CoV-2 was detected by in situ hybridization and immunohistochemistry. Biopsies from patients with hemolytic uremic syndrome (HUS, n = 5), severe acute tubular injury (ATI, n = 7), zero biopsies with disseminated intravascular coagulation (DIC, n = 7) and 1 year protocol biopsies from renal transplants (Ctrl, n = 7) served as controls. In the material clogging plasma adsorbers used for extracorporeal therapy of patients with COVID-19 C3 was the dominant protein but collectin 11 and MASP-2 were also identified. SARS-CoV-2 was sporadically present in varying numbers in some biopsies from patients with COVID-19. The highest frequency of CD61-positive platelets was found in peritubular capillaries and arteries of COVID-19 infected renal specimens as compared to all controls. Apart from COVID-19 specimens, MASP-2 was detected in glomeruli with DIC and ATI. In contrast, the classical pathway (i.e. C1q) was hardly seen in COVID-19 biopsies. Both C3 cleavage products C3c and C3d were strongly detected in renal arteries but also occurs in glomerular capillaries of COVID-19 biopsies, while tubular C3d was stronger than C3c in biopsies from COVID-19 patients. The membrane attack complex C5b-9, demonstrating terminal pathway activation, was predominantly deposited in COVID-19 biopsies in peritubular capillaries, renal arterioles, and tubular basement membrane with similar or even higher frequency compared to controls. In conclusion, various complement pathways were activated in COVID-19 kidneys, the lectin pathway mainly in peritubular capillaries and in part the classical pathway in renal arteries whereas the alternative pathway seem to be crucial for tubular complement activation. Therefore, activation of the complement system might be involved in the worsening of renal injury. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury.

Background:Despite serum albumin levels being predictive for clinical outcome in ANCA-associated renal vasculitis, implications providing a direct link between low serum albumin levels and intrarenal lesions remain elusive.Objectives:We here aimed to systematically assess the clinical relevance of low albumin levels and scrutinize clinicopathological correlations to expand our current knowledge.Methods:We here retrospectively enrolled biopsy-proven cases of ANCA-associated renal vasculitis between 2015 till 2020 in a single-center observational study. Survival-curve analyses on long-term renal survival and short-term clinical recovery were performed. Correlative analyses between serum albumin levels, laboratory parameters, proteinuria levels, and histopathological lesions including tubulointerstitial immune cell infiltrates, lesions analogous to the Banff score and intrarenal complement deposition of C3c and C4d were performed.Results:We here show that hypoalbuminemia below the median of 2.4 g/dL impairs long-term renal survival especially in MPO-positive ANCA-associated renal vasculitis (p = 0.006, HR: 5.0, 95% CI: 1.2-21.5), while short-term clinical recovery particularly in critically ill patients is negatively affected by low serum albumin levels (p=0.0082, HR: 3.6, 95% CI: 1.1-11.7). Low albumin levels are associated with the urinary marker of tubular damage in the total cohort (β = -0.5, p = 0.01), PR3-ANCA (β = -0.5, p = 0.04) and MPO-ANCA (p = 0.002, β = -0.8), but not in critically ill patients (β = -0.5, p = 0.07) implying hypoalbuminemia to occur by extrarenal causation in the state of critical illness. We identified plasmacytic infiltrates to correlate with low albumin levels in the total cohort (β = -0.6, p = 0.004) and in the subgroup of critically ill patients (β = -0.7, p = 0.005). Banff-scored interstitial inflammation (i) was further observed to inversely correlate with albumin levels (β = -0.7, p = 0.02). Intrarenal C4d deposition showed a significant correlation with low serum albumin levels in the glomerular tuft (β = -0.4, p = 0.04) and in interstitial arteries (β = -0.7, p = 0.01).Conclusion:In conclusion, long-term renal outcome is significantly affected by low serum albumin levels especially in the setting of MPO-ANCA seropositivity. By contrast, short-term recovery was predicted by low albumin levels in critically ill patients with ANCA-associated renal vasculitis. We here provide evidence that low levels of serum albumin might directly affect tubulointerstitial inflammation as reflected by plasmacytic immune-cell infiltration, and intrarenal C4d complement deposition in ANCA-associated renal vasculitis.REFERENCES:[1] Berden, A. E., F. Ferrario, E. C. Hagen, D. R. Jayne, J. C. Jennette, K. Joh, I. Neumann, L. H. Noel, C. D. Pusey, R. Waldherr, J. A. Bruijn, and I. M. Bajema. “Histopathologic Classification of Anca-Associated Glomerulonephritis.” J Am Soc Nephrol 21, no. 10 (2010): 1628-36.[2] Flossmann, O., A. Berden, K. de Groot, C. Hagen, L. Harper, C. Heijl, P. Hoglund, D. Jayne, R. Luqmani, A. Mahr, C. Mukhtyar, C. Pusey, N. Rasmussen, C. Stegeman, M. Walsh, K. Westman, and Group European Vasculitis Study. “Long-Term Patient Survival in Anca-Associated Vasculitis.” Ann Rheum Dis 70, no. 3 (2011): 488-94.[3] Weidner, S., S. Geuss, S. Hafezi-Rachti, A. Wonka, and H. D. Rupprecht. “Anca-Associated Vasculitis with Renal Involvement: An Outcome Analysis.” Nephrol Dial Transplant 19, no. 6 (2004): 1403-11.[4] Hakroush, S., D. Tampe, P. Strobel, P. Korsten, and B. Tampe. “Comparative Histological Subtyping of Immune Cell Infiltrates in Mpo-Anca and Pr3-Anca Glomerulonephritis.” Front Immunol 12 (2021): 737708.[5] Ge, Y., G. Yang, X. Yu, B. Sun, B. Zhang, Y. Yuan, M. Zeng, N. Wang, H. Mao, and C. Xing. “Outcome Predictors of Biopsy-Proven Myeloperoxidase-Anti-Neutrophil Cytoplasmic Antibody-Associated Glomerulonephritis.” Front Immunol 11 (2020): 607261.Figure 1.Correlative analyses of serum albumin levels and histopathological lesions. (A) Heatmaps reflect the mean values of Spearman’s ρ, circle size represents significance level in the stepwise multivariable linear regression.Acknowledgements:NIL.Disclosure of Interests:None declared.

S. pneumoniae (SP) serotypes 15B and 15C are frequent causative agents of invasive pneumococcal disease (IPD), otitis media, and nasopharyngeal colonization in the post PCV13 era. The principal difference between serotypes 15B and 15C is the presence of an O-acetyl group on the pentasaccharide repeating unit of 15B polysaccharide. It remains unclear if antibodies against SP15B polysaccharide demonstrate functional cross reaction with SP15C strains. We compared functional activity of polyclonal rabbit anti-capsular 15B sera against SP15B and SP15C isolates. Using flow cytometry we measured complement factors C3c and C4d deposition on SP15B and 15C in the presence of polyclonal rabbit anti capsular 15B sera. We measured the binding of C3c, common to all complement pathways, and C4d, specific to classical pathway, on SP serotypes 15B and 15C when co-incubated with polyclonal rabbit anti capsular 15B sera and antibody depleted complement. Both the proportion of bacteria with complement deposition and the fluorescence intensity were measured. We also measured agglutination as the increase in forward and side scatter. Polyclonal rabbit anti-capsular 15B sera activated classical pathway resulting in deposition of C4d and C3c at high intensity on all SP15B cells but only achieved limited deposition and intensity of C4 with SP15C. Similarly, polyclonal rabbit anti-capsular 15B sera induced agglutination of SP15B strains in a dose dependent manner and limited agglutination of SP15C. Anti-capsular 15B sera induce limited C4d and C3c deposition, and minimal agglutination with SP15C strains, reflecting lower classical pathway activation in contrast to high C4d and C3c deposition and agglutination of SP15B. These observations support limited functional cross reactivity of anti-15B to SP15C strains and are consistent with the reduction in opsonophagocytic killing of SP15C reported following immunization with vaccines containing 15B polysaccharide. •Antibodies against SP serotype 15B activated both classical and alternative pathway of complement activation on the surface of SP15B.•15B antibodies showed relatively less complement binding and agglutination with 15C compared with 15B.•Study suggest antibodies against SP15B may not confer protection against SP 15C by complement dependent killing.

Background:Anti-tumor necrosis factor-α (anti-TNF) therapy is often part of the treatment strategy for rheumatic diseases (RD) and inflammatory bowel disease (IBD). An association between anti-TNF agents and the development of lupus erythematosus (LE)-like syndrome has been established and some patients may have a definitive diagnosis of systemic lupus erythematosus (SLE), requiring appropriate treatment. To date, no studies have been conducted to analyze follow-up between rheumatic and IBD patients who have developed LE-like syndrome induced by anti-TNF agents.Objectives:To assess clinical and serological markers of LE-like syndrome induced by anti-TNF therapy, and to characterize its development, in patients with RD and IBD receiving treatment with anti-TNF.Methods:In this monocentric descriptive study, with a minimum 6-month follow-up and conducted at a tertiary university hospital (Rheumatology Department), we reviewed the clinical and serological parameters of 8 patients with RD and 11 patients with IBD, all diagnosed with LE-like syndrome induced by anti-TNF therapy. Statistical analysis, using student’s t test, was performed to compare differences in anti-double-stranded DNA (anti-dsDNA) levels between patients with positive and negative anti-histone antibodies (anti-H).Results:Nineteen patients were included, mostly female (n=14). The mean age at the diagnosis of LE-like syndrome was 45.42 (±16.77) years. Ten IBD and 2 RD patients received infliximab. At the time of symptoms onset, anti-TNF agent was discontinued. Fifteen (78.9%) patients developed polyarthritis and 3 (15.8%) polyarthralgias. Three (15.8%) patients had constitutional symptoms (such as fatigue or fever), 2 (10.5%) patients developed skin rash, 2 (10.5%) patients had cytopenia (1 with anemia and 1 with anemia and leukopenia; both in RD group), 2 (10.5%) patients developed serositis (pleural effusion) and 1 (5.3%) patient had vasculitis-like skin lesions in the hands; 1 (5.3%) patient developed venous thrombosis associated with antiphospholipid antibodies positivity. At the time of LE-like syndrome diagnosis, all patients (n=100, 19%) tested positive for antinuclear antibodies (ANA), and almost all patients (n=14, 73.7%) tested positive (≥100 international units/L) for anti-dsDNA. Except for 4 (21.1%) patients (3 in IBD group and 1 in RD group) who had low complement (C3c/C4), complement levels were mostly normal. The relevant data are detailed in Table 1. Of note, positive anti-H were accompanied by low serum levels of anti-dsDNA antibodies, p<0.001 (Table 2).Seven (36.8%), all IBD patients, needed immunosuppressive therapy (methotrexate, azathioprine or hydroxychloroquine) to resolve their LE-like syndrome symptoms (in 3 patients, anti-dsDNA were negative only after at least 1 year of treatment) and continued to receive immunosuppressants during follow-up. In 11 (57.9%) cases, including all RD patients, discontinuation of anti-TNF therapy (and sometimes using low dose of glucocorticoids) was sufficient for symptoms resolution and antibody negativity. Five patients (26.3%) switched to another anti-TNF without experiencing relapse of symptoms.Conclusion:Studies show that SLE-like syndrome frequency has been increasing and recognition of the condition is very important as anti-TNF therapy is commonly used. This study, although limited by its descriptive nature and small sample size, revealed differences between the RD and IBD groups regarding the specific anti-TNF agents used and the necessity of immunosuppressive therapy, more prevalent in the IBD group. On the other hand, most patients improved after discontinuing therapy and those who tolerated the switch to another anti-TNF-α agent had no SLE recurrence. Of interest, positive anti-H was associated with absence or low levels of anti-dsDNA, suggesting that this clinical entity is an independent disease and different from classical drug-induced lupus with positive anti-histone antibodies. Further studies are required to establish definite differences between IBD and RD groups and determine the impact of definitive SLE diagnosis secondary to anti-TNF therapy.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of Interests:None declared.

Hidradenitis suppurativa (HS) is a chronic auto-inflammatory skin disease with a complex and multifactorial pathogenesis involving both the innate and adaptive immune system. Despite limited evidence for local complement activation, conflicting results have been published on the role of systemic complement activation in HS. It was hypothesized that complement was consumed in highly inflamed HS skin, trapping complement from the circulation. Therefore, the aim of this study was to evaluate this local complement deposition in HS skin lesions using routine and commonly used complement antibodies.Direct immunofluorescence for C1q, C3c, C4d, C5b-9, and properdin was performed on frozen tissue sections of 19 HS patients and 6 controls. C5a receptor 1 (C5aR1) was visualized using immunohistochemistry. Overall, we found no significant local complement deposition in HS patients versus controls regarding C1q, C3c, C4d, C5b-9, or properdin on either vessels or immune cells. C5aR1 expression was exclusively found on immune cells, predominantly neutrophilic granulocytes, but not significantly different relatively to the total infiltrate in HS lesions compared with controls. In conclusion, despite not being able to confirm local complement depositions of C1q, C3c, C4d, or properdin using highly sensitive and widely accepted techniques, the increased presence of C5aR1 positive immune cells in HS suggests the importance of complement in the pathogenesis of HS and supports emerging therapies targeting this pathway.

The link between clinical recovery and immune restoration after severe COVID-19 remains poorly defined. Although most survivors experience symptomatic improvement, persistent symptoms have been hypothesized to reflect ongoing immune dysregulation. This prospective cohort study followed 93 unvaccinated adults with RT-PCR-confirmed moderate-to-critical COVID-19 at 3, 6, and 12 months post-discharge. Clinical assessments used structured interviews to evaluate the persistent symptoms. Peripheral blood analyses were used to measure lymphocyte subsets, immunoglobulins, and complement components. Clinical recovery was substantial; fatigue prevalence declined from 70.9% to 24.7% and dyspnea prevalence from 81.7% to 25.8% by 12 months ( < 0.001 for both). However, immune recovery exhibited divergent patterns. Activated T cells (CD3 HLA-DR ) decreased significantly (from 20% to 13%; < 0.001), complement C3c levels paradoxically increased from 1.23 to 1.35 g/L ( < 0.001), and serum IgA increased by 32% ( = 0.003). NK cells remained stable overall but were persistently reduced in a subset (~25%) of patients, particularly among those with fatigue and dyspnea. Critical illness was associated with slower T-cell resolution, prolonged IgM elevation, and increased complement activity. One year after hospitalization, most patients achieved substantial clinical improvement, but immune reconstitution lagged behind. These findings highlight the dissociation between clinical and immunological recovery and suggest that persistent immune dysregulation may be associated with long COVID manifestations. Incorporating immune monitoring into post-COVID care may help identify patients at risk of prolonged sequelae and guide targeted therapeutic strategies.

Introduction: The environment of the infection site affects bacterial growth and antibiotic activity. When bacterial growth and antibiotic activity are studied in body fluids, samples of multiple subjects are usually pooled, averaging out potentially relevant differences in composition. The ascitic fluid (AF) environment is frequently associated with spontaneous bacterial peritonitis (SBP) in cirrhotic patients. In this study, bacterial growth and ceftriaxone activity were evaluated in individual AF using an in vitro model of SBP, reflecting the environment and pharmacokinetics at the infection site. Methods: AF was obtained from nine cirrhotic patients with non-infected ascites. Growth of nine bacterial strains (three Escherichia coli , four Staphylococcus aureus , one Enterococcus faecalis , and one Klebsiella pneumoniae ) in individual AF was assessed and correlated with biomarkers including potential risk factors for SBP. Ceftriaxone time-kill experiments, in which the pharmacokinetic profile observed in AF following a 1 g intravenous infusion was replicated, were performed with two  E. coli and two S. aureus isolates with minimum inhibitory concentrations around the ceftriaxone resistance breakpoint. Results: Significant correlations were found between bacterial growth and AF levels of protein (Spearman’s rank correlation coefficient ρ = −0.35), albumin (ρ = −0.31), and complement C3c (ρ = −0.28), and serum levels of bilirubin (ρ = 0.39) and aspartate aminotransferase (ρ = 0.25). Ceftriaxone was active in AF, even against resistant isolates, generally resulting in ≥2 log reductions in bacterial count within 24 h. Conclusion: Ascites patients may be predisposed to or protected against SBP based on the antimicrobial capacity of their AF. Ceftriaxone at clinical AF concentrations is active in the AF environment.

Objective: Complement deposition is prevalent in kidney biopsies of patients with arterial hypertension and hypertensive nephropathy, but an association of hypertension and complement deposition or involvement of complement in the pathogenesis of hypertensive nephropathy has not been shown to date. Methods: In this study, we analyzed complement C1q and C3c deposition in a rat model of overload and hypertension by subtotal nephrectomy (SNX) and in archival human renal biopsies from 217 patients with known hypertension and 91 control patients with no history of hypertension using semiquantitative scoring of C1q and C3c immunohistochemistry and correlation with parameters of renal function. To address whether complement was only passively deposited or actively expressed by renal cells, C1q and C3 mRNA expression were additionally analyzed. Results: Glomerular C1q and C3c complement deposition were significantly higher in kidneys of hypertensive SNX rats and hypertensive compared to nonhypertensive patients. Mean arterial blood pressure (BP) in SNX rats correlated well with the amount of glomerular C1q and C3c deposition and with left ventricular weight, as an indirect parameter of high BP. Quantitative mRNA analysis showed that C3 was not only deposited but also actively produced by glomerular cells of hypertensive SNX rats and in human renal biopsies. Of note, in patients CKD-stage correlated significantly with the intensity of glomerular C3c staining, but not with that of C1q. Conclusion: Renal complement deposition correlated with experimental hypertension as well as the presence of hypertension in a variety of renal diseases. To answer the question, if and how exactly renal complement is causative for the pathogenesis of arterial hypertension in men, further studies are needed.

Propionibacterium acnes is thought to be a causative agent of sarcoidosis. Patients with sarcoidosis have circulating immune complexes. We attempted to detect P. acnes-derived immune complexes in sarcoid lesions. We evaluated formalin-fixed and paraffin-embedded lymph node samples from 38 sarcoidosis patients and 90 non-sarcoidosis patients (27 patients with necrotizing lymphadenitis, 28 patients with reactive lymphadenitis, 16 patients with colon cancer, 19 patients with gastric cancer) by immunohistochemistry using anti-human immunoglobulins (IgG, IgA, and IgM) and complement (C1q and C3c) antibodies, and a P. acnes-specific monoclonal antibody (PAB antibody) that reacts with the membrane-bound lipoteichoic acid of P. acnes. Small round bodies (SRBs) bound to IgA, IgM, or IgG were detected in sinus macrophages, in 32 (84%), 32 (84%), or 11 (29%) sarcoid samples, respectively, and in 19 (21%), 26 (29%), or no (0%) control samples, respectively. Some of these insoluble immune complexes (IICs) also bound to C1q and C3c. We developed a microwave treatment followed by brief trypsin digestion (MT treatment) to detect PAB-reactive SRBs bound to immunoglobulins (IIC-forming P. acnes). MT treatment revealed abundant IIC-forming P. acnes in most (89%) of the sarcoid samples and sparse distribution in some (20%) of the control samples with lymphadenitis, but no IIC-forming P. acnes was detected in control samples without inflammation. IIC-forming P. acnes were mostly bound to both IgA and IgM. The PAB-reactive antigen and immunoglobulins were both located at the peripheral rim of the IIC-forming P. acnes. Conventional electron microscopy identified many SRBs (0.5-2.0 μm diameter) in sinus macrophages of sarcoid lymph nodes with many IIC-forming P. acnes, some of which were in phagolysosomes with a degraded and lamellar appearance. P. acnes-derived IICs in sinus macrophages were frequent and abundant in sarcoid lymph nodes, suggesting a potential etiologic link between sarcoidosis and this commensal bacterium.

Studies conducted in animal models have suggested that membrane complement regulatory proteins play an important role in the pathophysiology of coronary artery disease (CAD). In this study, a total of 100 individuals, with stable CAD and 100 healthy controls, both groups predominantly male, were recruited. We evaluated the plasma levels of complement regulatory proteins (Cregs) CD35, CD46, CD55, and CD59 and their surface expression on granulocytes, lymphocytes, and monocytes by flow cytometry. The mRNA expression of these Cregs in total leukocytes was determined by quantitative PCR. The soluble forms of Cregs, C3c, Mannose binding protein-associated serine protease 2 (MASP-2), Platelet activating factor-acetyl hydrolase (PAF-AH), and inflammatory cytokines were quantified by ELISA. High plasma levels of C3c, indicative of complement activation, in addition to significantly low levels of Cregs, were observed in CAD patients. A significantly lower expression of CD46 and CD55 on the surface of lymphocytes, monocytes, and granulocytes and higher surface expression of CD35 and CD59 on granulocytes ( < 0.0001) was seen in CAD patients as compared to healthy donors. The high expression of CD59 on granulocytes positively correlated with the severity of disease and may serve as a potential marker of disease progression in CAD.