Complement Split Product Research Articles

Study of Serum Complement 3 Split Product iC3b /C3 Ratio in a Cohort of Systemic Lupus Erythematosus Patients

Abstract Background Systemic lupus erythematosus (SLE) is a chronic autoimmune systemic disease that causes widespread multi organ inflammation. The exact cause of SLE remains unclear; however, genetic, hormonal, and environmental factors contribute to the development of the disease. Aim of the Work The aim of this study is to measure the ratio of complement split product iC3b to C3 and its relation to disease activity and lupus nephritis in a cohort of SLE patients. Patients and Methods This study is a Cross sectional case control study conducted in internal medicine and Rheumatology unit, Ain Shams university hospital and Rheumatology unit, Maadi military hospital. Our study included. Results The present study shows that female were (76%) in active group of SLE patients and (60%) in inactive group while male were (24%) and (40%) respectively. with no significant difference. The mean of Disease Duration was (5.66 ± 1.97) with a range from 3 and 10 years in active group of SLE patients and (5.08 ± 1.54) with a range from 3 and 9 years in inactive group. with no significant difference. Regarding clinical manifestations of SLE the present study shows that active group of SLE patients had statistically highly significant increase in incidence of lupus nephritis, arthritis, serositis, oral ulcers, malar rash (p < 0.001) with significant increase in incidence of alopecia and neurological manifestations (p < 0.05). The current study shows that active group of SLE patients had statistically significantly higher mean ESR, anti-DNA titre, 24hr urinary proteins, serum creatinine, serum urea, and GFR levels (p = 0.000) than inactive group, Also active group had significant leucopenia, anemia (p = 0.000, 0.005 respectively) and C3 was significantly more consumed than inactive group (p < 0.05) Conclusion The ratio of complement split product iC3b to C3 concentrations correlates with the extent of SLE disease activity more than C3 alone. Furthermore, the iC3b:C3 ratio may discriminate between patients with lupus nephritis and patients without lupus nephritis. Detection of SLE disease activity and lupus nephritis by iC3b/C3 ratio help to make a proper decision in management to induce remission.

Abstract C103: Preclinical and clinical safety and tolerability evaluation of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP) for treatment of advanced non-small cell lung cancer (NSCLC)

Abstract Glutathione S-transferase Pi (GSTP) has an important role in detoxification and anti-oxidative damage responses. Additionally, GSTP has a chaperone function that regulates key oncogenic pathways such as the KRAS and JNK pathways (E Laborde, 2010). Since alternative or redundant pathways could compensate for the incomplete inhibition of a single kinase, targeting multiple pathways could lead to more effective therapy. NBF-006 is a novel drug product containing a GSTP siRNA encapsulated within a proprietary lipid nanoparticle (LNP). It has been designed to deliver siRNA to tumors of the lung and common sites of metastatic spread (liver and marrow). As GSTP downregulation leads to inhibition of KRAS signaling through modulation of both MAPK and PI3K pathways, NBF-006 is being developed to treat KRAS mutant tumors. Preclinical studies have demonstrated activity across KRAS mutations, as well as efficacy in orthotopic non-small cell lung cancer (NSCLC) models. Immune toxicities have been recognized as safety concerns associated with LNP therapeutics. The safety assessment of NBF-006 was conducted both preclinically and clinically. In vitro preclinical toxicity studies included hemolysis in hRBC samples, cytokine release in hPBMCs, and TLR activation in human THP1 cells. GLP-compliant general toxicity studies were conducted in both mice and cynomolgus monkeys, IV administration, once a week, for 5 weeks. Complement splits were measured in the monkey study to assess any immune-related responses. Safety pharmacology parameters were also conducted in monkeys. The mouse was slightly more sensitive than monkeys to NBF-006, and the highest non-seriously toxic dose level in mice was 20 mg/kg/week, which yielded a good safety margin to proceed for a Phase I clinical trial. The highest dose level tested in monkeys was of 18 mg/kg/week and was well tolerated by the monkeys. Immunological assessments were conducted during Phase 1 dose escalation and expansion in patients with NSCLC, pancreatic cancer, or colorectal cancer. NBF-006 was administered by IV infusion over 70 minutes, once a week, for 4 weeks followed by a 2-week rest period. Clinical symptoms and reactions were monitored, and lab tests were conducted according to the protocol, including CBC, complement split products (Bb, C3a, C5a, and CH50), cytokines (IL-6, IL-1β, IFNg, and TNFα), and anti-drug antibodies against the LNPs. Dose-dependent increases in C3a and Bb of 1.2 and 1.6 mg/kg were observed following NBF-006 administration at doses ranging from 0.15 to 1.6 mg/kg. The increases were transient at either 10 minutes or 1 hour after the end of the infusion. Due to lack of corresponding increases in C5a or decreases in CH50, transient elevations of C3a and Bb were not considered clinically significant. In summary, NBF-006 was well tolerated in both animal species and patients: 20 mg/kg/week was the non-seriously toxic dose level in mice; the highest dose level of 18 mg/kg/week tested in monkeys was the NOAEL; and 1.6 mg/kg/week was the highest dose level evaluated in humans. Citation Format: Jian Liu, Cima Cina, Douglas Kornbrust, Sonya Zabludoff. Preclinical and clinical safety and tolerability evaluation of NBF-006, a novel siRNA inhibitor of glutathione-s-transferase P (GSTP) encapsulated in a lipid nanoparticle (LNP) for treatment of advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C103.

Detailed Analysis of Simultaneous Renal and Liver Allografts in the Presence of DSA.

We reevaluated allograft biopsies from simultaneous liver-kidney transplant recipients who had both allografts biopsied within 60 d of one another and within 30 d of DSA being positive in serum (positive: mean florescence intensity ≥5000). Routine histology, C4d staining, and specialized immunohistochemistry for Kupffer cells (KCs; CD163) and a C4d receptor immunoglobulin-like transcript-4 were carried out in 4 patients with 6 paired biopsies. Overt antibody-mediated rejection was found in 3 of 4 renal and liver allografts. One patient had biopsy-confirmed renal and liver allograft antibody-mediated rejection despite serum clearance of DSA. All biopsies showed KC hypertrophy (minimal: 1; mild: 2; moderate: 1; severe: 2) and cytoplasmic C4d KC staining was easily detected in 2 biopsies from 2 patients; minimal and negative in 2 biopsies each. Implications of which are discussed. Control 1-y protocol liver allograft biopsies from DSA- recipients showed neither KC hypertrophy nor KC C4d staining (n = 6). Partial renal allograft protection by a liver allograft from the same donor may be partially mediated by phagocytosis/elimination of antibody and complement split products by KCs, as shown decades ago in controlled sensitized experimental animal experiments.

Exploration of complement split products in plasma and urine as biomarkers of kidney graft rejection

IntroductionThe complement system, consisting of more than thirty different soluble and cell-bound proteins, exerts essential functions both in the innate and adaptive immune systems and is believed to be an important contributor to allograft injury in kidney transplantation. The anaphylatoxins C3a and C5a are powerful chemoattractants, recruiting immune effector cells toward the site of complement activation and enhance T-cell response, while C3dg binding to CR2 on B-cells, enhances B-cell immunity at several stages of the B-cell differentiation.Complement split products in plasma and urine could reflect ongoing inflammation and tissue injury. We, therefore, investigated if complement split products increase in plasma and urine in kidney transplant recipients with rejection. MethodIn this case-control feasibility study, complement factors C3a, C3dg, C4a, and C5a were measured in plasma and C3dg and sC5b-9 associated C9 neoantigen in urine in 15 kidney transplant recipients with rejection (cases) and 15 kidney transplant recipients without (controls). The groups were matched on the type of transplantation and the time from transplantation to sampling. The complement split products were compared (i) between cases and controls and (ii) within the rejection group over time, comparing the measurements at rejection with measurements where the kidney transplant recipients were clinically stable. Possible moderators were explored, and results adjusted accordingly. P values < 0.05 were considered significant.Plasma C3dg was analyzed by immune-electrophoresis, plasma C3a, plasma C4a, and plasma C5a by flow cytometry, and urine C3dg and urine C9neo by ELISA. ResultsIn plasma, there were no significant differences between the rejection and the control group. However, steroids and pretransplant C3dg levels significantly influenced C3dg.Within the rejection group, plasma C3a and C3dg were significantly higher at the time of rejection compared to the stable phase (p < 0.01).In urine, C3dg/creatinine and C9 neoantigen/creatinine ratios were not different between the rejection and the control group. Urine C3dg/creatinine and urine C9 neoantigen/creatinine ratios correlated to urine albumin and significantly increased after the transplantation (p < 0.001). ConclusionThis study shows increased plasma C3a and C3dg in kidney transplant recipients, primarily with T cell mediated rejection. This finding suggests that consecutive measurements of C3a and C3dg in plasma could be applicable to monitor alloreactivity in kidney transplant recipients. Urine complement split products are unsuitable as rejection biomarkers since the permeability of the glomerular filtration barrier strongly influences them.Prospective longitudinal studies on plasma C3a and C3dg dynamics will be needed to validate present findings.

Modulation of Neutrophil Activity by Soluble Complement Cleavage Products—An In-Depth Analysis

The cellular and fluid phase-innate immune responses of many diseases predominantly involve activated neutrophil granulocytes and complement factors. However, a comparative systematic analysis of the early impact of key soluble complement cleavage products, including anaphylatoxins, on neutrophil granulocyte function is lacking. Neutrophil activity was monitored by flow cytometry regarding cellular (electro-)physiology, cellular activity, and changes in the surface expression of activation markers. The study revealed no major effects induced by C3a or C4a on neutrophil functions. By contrast, exposure to C5a or C5a des-Arg stimulated neutrophil activity as reflected in changes in membrane potential, intracellular pH, glucose uptake, and cellular size. Similarly, C5a and C5a des-Arg but no other monitored complement cleavage product enhanced phagocytosis and reactive oxygen species generation. C5a and C5a des-Arg also altered the neutrophil surface expression of several complement receptors and neutrophil activation markers, including C5aR1, CD62L, CD10, and CD11b, among others. In addition, a detailed characterization of the C5a-induced effects was performed with a time resolution of seconds. The multiparametric response of neutrophils was further analyzed by a principal component analysis, revealing CD11b, CD10, and CD16 to be key surrogates of the C5a-induced effects. Overall, we provide a comprehensive insight into the very early interactions of neutrophil granulocytes with activated complement split products and the resulting neutrophil activity. The results provide a basis for a better and, importantly, time-resolved and multiparametric understanding of neutrophil-related (patho-)physiologies.

Functional variant rs12614 in CFB confers a low risk of IgA nephropathy by attenuating complement alternative pathway activation in Han Chinese.

Activation of the alternative pathway (AP) of complement is thought to play an important role in Immunoglobin A nephropathy (IgAN). Our previous study showed that rs4151657 within the complement factor B (CFB) gene increased the risk of IgAN. The protein encoded by the CFB gene is an initial factor that promotes AP activation. The aim of this study was to investigate whether other variants of CFB confer susceptibility to IgAN and elucidate their potential roles in AP activation. A total of 1,350 patients with IgAN and 1,420 healthy controls were enrolled and five tag single-nucleotide polymorphisms were selected for genotyping. The levels of key AP components, such as CFB, complement factor H and complement split product C3a, were measured by enzyme-linked immunosorbent assay. Molecular docking and molecular dynamic simulation were carried out to characterize the mutation of residues in the protein structure and the dynamic properties of wide type and mutation models of CFB protein. The allele-specific effect on CFB expression and its binding affinity to C3b were investigated through cell transfection and surface plasmon resonance analysis, respectively. We found that rs12614 significantly reduced the risk of IgAN (OR = 0.69, 95% CI = 0.52–0.91, P = 0.009), and the rs12614-T (R32W mutation) was correlated with lower CFB levels, higher serum C3 level, and less mesangial C3 deposition in patients with IgAN. The structural model showed that the R32W mutation reduced the structural stability of CFB protein. Furthermore, in vitro study revealed that rs12614-T decreased the expression of CFB and reduced its binding affinity to C3b by four-fold compared with rs12614-C. In conclusion, the rs12614-T in CFB was associated with low risk of IgAN probably by attenuating AP activation.

The Role of Complement Split-products as Biomarkers for Acute Antibody-mediated Rejection of Kidney Allografts.

Background.Acute antibody-mediated rejection (AMR) is mediated by the activation of the classical complement system in addition to noncomplement-dependent inflammatory pathways. Complement fixation by donor-specific antibodies leads to cleavage of the complement proteins C4, C3, and C5 to produce multiple complement split-products (CSP) and the end-effector membrane attack complex, C5b-9. In this study, we investigate CSP as potential biomarkers for AMR.Methods.In an Institutional Review Board–approved, prospective, controlled study, CSP levels were measured in blood and urine samples from consecutive kidney transplant recipients with biopsy-proven AMR (n = 10), acute cellular rejection (ACR) (n = 5), or no rejection (n = 5). After obtaining informed consent, samples were collected at the time of biopsy (day 0) and days 15 (end of rejection treatment) and 30 postbiopsy for AMR and ACR patients. ELISA was used to measure C5a, C4d, and soluble C5b-9 concentrations in blood and urine, in addition to factor Bb (Bb) concentration in blood only. Kidney transplant histopathology was evaluated using the Banff 2013 classification. Rejection treatment and follow-up were performed per standard of care.Results.Blood and urine CSP levels adjusted to urine creatinine were not elevated in AMR compared to no rejection and ACR arms. There was significant variability in CSP concentration within each of the study groups.Conclusion.Our study does not support the utility of CSP as surrogate biomarkers of AMR; however, it is limited by the small sample size and larger studies may be warranted.

POS-041 BCX9930, AN ORAL FACTOR D INHIBITOR, SUPPRESSES COMPLEMENT ALTERNATIVE PATHWAY ACTIVITY IN PATIENTS WITH COMPLEMENT 3 GLOMERULOPATHY

POS-041 BCX9930, AN ORAL FACTOR D INHIBITOR, SUPPRESSES COMPLEMENT ALTERNATIVE PATHWAY ACTIVITY IN PATIENTS WITH COMPLEMENT 3 GLOMERULOPATHY

CD4+ and CD8+CD60+ T cells in PBMC of IgE+ allergic adults express IL-4 or IFN alpha but not IFN gamma, and some express receptors for complement split products (CSP) C3a and C5a

We previously reported that two distinct T cell subsets (CD4+ and CD8+CD60+) and IL-4, IFN alpha, and IFN gamma, but not IL-13, were required for human ragweed specific memory IgE responses. Nevertheless, the exact subsets producing these cytokines and their numbers are unknown, as are the numbers of C3a+ or C5a+ receptor T cells expressing IL-4.

Distributions of T cell subsets expressing receptors for complement split products (CSP) C3a or C5a in blood of serum IgE+ aeroallergen allergic adults

Previous studies demonstrated that CD4+ T cells and IL-4 are required for human and rodent IgE responses. We demonstrated that two distinct T cell subsets (CD4+, CD8+CD60+) and 6 cytokines (including IL-4, IFN alpha, IFN gamma, but not IL-13) are required for human ragweed specific memory IgE responses. However, little is known about events upstream of T cell participation leading to IgE production. Antigen/allergen cleaves complement, but it is unknown if/which T cells express receptors for CSP.

Efficacy and Safety of Vilobelimab (IFX-1), a Novel Monoclonal Anti-C5a Antibody, in Patients With Early Severe Sepsis or Septic Shock-A Randomized, Placebo-Controlled, Double-Blind, Multicenter, Phase IIa Trial (SCIENS Study).

This phase II clinical trial was conducted to study safety, tolerability, pharmacokinetics, and pharmacodynamics of vilobelimab, a recombinant monoclonal antibody against C5a, in patients with severe sepsis or septic shock. Multicenter, randomized, and placebo-controlled study. Eleven multidisciplinary ICUs across Germany. Adult patients with severe sepsis or septic shock and with early onset of infection-associated organ dysfunction. Patients were randomly assigned in a ratio of 2:1 to three subsequent dosing cohorts for IV vilobelimab or placebo receiving either 2 × 2 mg/kg (0 and 12 hr), 2 × 4 mg/kg (0 and 24 hr), and 3 × 4 mg/kg (0, 24, and 72 hr). Co-primary endpoints were pharmacodynamics (assessed by C5a concentrations), pharmacokinetics (assessed by vilobelimab concentrations), and safety of vilobelimab. Preliminary efficacy was evaluated by secondary objectives. Seventy-two patients were randomized (16 patients for each vilobelimab dosing cohort and eight patients for each placebo dosing cohort). Vilobelimab application was associated with dosing dependent decrease in C5a compared with baseline (p < 0.001). Duration of C5a decrease increased with more frequent dosing. Membrane attack complex lysis capacity measured by 50% hemolytic complement was not affected. Vilobelimab was well tolerated with similar safety findings in all dose cohorts. No vilobelimab-specific adverse events emerged. For vilobelimab-treated patients, investigators attributed less treatment-emergent adverse events as related compared with placebo. Dosing cohorts 2 and 3 had the highest ICU-free and ventilator-free days. There was no difference in mortality, vasopressor-free days, or renal replacement therapy-free days between the groups. Administration of vilobelimab in patients with severe sepsis and septic shock selectively neutralizes C5a in a dose-dependent manner without blocking formation of the membrane attack complex and without resulting in detected safety issues. The data warrant further investigation of C5a inhibition in sepsis.

Platelet bound complement split product (PC4d) is a marker of platelet activation and arterial vascular events in Systemic Lupus Erythematosus

Platelet-bound complement activation products (PC4d) are associated with thrombosis in Systemic Lupus Erythematosus (SLE). This study investigated the effect of PC4d on platelet function, as a mechanistic link to arterial thrombosis. In a cohort of 150 SLE patients, 13 events had occurred within five years of enrollment. Patients with arterial events had higher PC4d levels (13.6 [4.4–24.0] vs. 4.0 [2.5–8.3] net MFI), with PC4d 10 being the optimal cutoff for event detection. The association of arterial events with PC4d remained significant after adjusting for antiphospholipid status, smoking, and prednisone use (p = 0.045). PC4d levels correlated with lower platelet counts (r = −0.26, p = 0.002), larger platelet volumes (r = 0.22, p = 0.009) and increased platelet aggregation: the adenosine diphosphate (ADP) concentration to achieve 50% maximal aggregation (EC50) was lower in patients with PC4d 10 compared with PC4d < 10 (1.6 vs. 3.7, p = 0.038, respectively). These results suggest that PC4d may be a mechanistic marker for vascular disease in SLE.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

<h3>Purpose</h3> Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. <h3>Methods</h3> All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. <h3>Results</h3> 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. <h3>Conclusion</h3> Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Local and Systemic Concentrations of Pattern Recognition Receptors of the Lectin Pathway of Complement in a Cohort of Patients With Interstitial Lung Diseases.

BackgroundThe role of the lectin pathway of complement in the pathogenesis of interstitial lung diseases (ILDs) is largely unknown. Pattern recognition receptors (PRR) of the lectin pathway are involved in the clearance of apoptotic cells either via activation of the complement system or as direct opsonins. As recent findings suggest a role of apoptosis in the development of pulmonary fibrosis, the influence of plasma lectins has lately been considered in various ILDs, but data on local concentrations in the lungs are lacking. This study investigated the role of mannose-binding lectin (MBL), ficolin-2 and ficolin-3 in ILD patients with a focus on idiopathic pulmonary fibrosis (IPF) and sarcoidosis.MethodsA case control study was conducted involving 80 patients with different forms of ILD as well as 40 control patients undergoing routine flexible bronchoscopy with bronchoalveolar lavage (BAL). Plasma and BAL fluid (BALF) levels of MBL, ficolin-2 and ficolin-3 as well as complement split products C4d and C5a (only in BALF) were measured by enzyme-linked immunosorbent assays. Eight single-nucleotide polymorphisms (SNPs) of MBL and ficolin-2 were determined by genotyping and tested for their association with ILDs.ResultsWe included 35, 35, 10, and 40 patients with sarcoidosis, idiopathic pulmonary fibrosis (IPF), other ILD, and a control group, respectively. BALF but not plasma levels of the three PRR were significantly elevated in sarcoidosis patients compared to a control group without ILD (MBL: median 66.8 vs. 24.6 ng/ml, p = 0.02, ficolin-2: 140 vs. 58.8 ng/ml, p = 0.01, ficolin-3: 2523 vs. 1180 ng/ml, p = 0.02), whereas the frequency of the investigated SNPs was similar. In line, complement split products were markedly elevated in BALF of sarcoidosis patients (C4d, median 97.4 vs. 0 ng/ml, p = 0.10; C5a, 23.9 vs. 9.1 ng/ml, p = 0.01). There was a weak positive correlation of BALF ficolin-3 with serum neopterin, a marker of sarcoidosis activity. In IPF patients, we observed numerically higher MBL plasma and BALF levels (plasma, median 1511 vs. 879 ng/ml, p = 0.44; BALF, 37.5 vs. 24.6 ng/ml, p = 0.7) as well as lower ficolin-2 plasma levels (plasma 1111 vs. 1647 ng/ml, p = 0.11). Ficolin-2 plasma levels were inversely correlated with the forced vital capacity (r = 0.55, p = 0.1).ConclusionThis is the first study to simultaneously assess systemic and local lectin pathway protein levels in ILD patients. Our data suggest an involvement of PRR of the lectin pathway in the pathogenesis of sarcoidosis given the significantly higher BALF levels compared to a control group. Additional analyses in a larger patient cohort are required to confirm or refute a potential effect of local and/or systemic ficolin-2 levels in IPF patients.

Complement split product C3c in saliva as biomarker for periodontitis and response to periodontal treatment.

Background and ObjectiveThe complement system is engaged in inflammatory reactions both in the periodontal pockets and in the periodontium itself, where it can mediate tissue destruction. The aim of this study was, first, to compare salivary levels of the total complement system protein C3 and its split product, fluid‐phase C3c in patients with periodontitis and periodontally healthy controls. Next, to determine if C3 and C3c levels had biomarker potential in diagnosing and monitoring periodontitis and its treatment. We hypothesized that salivary levels of total C3 and the split product C3c associated with the severity of periodontitis and reflected decreased inflammatory activity after periodontal treatment.MethodsAt baseline, stimulated saliva samples were collected from patients with periodontitis (n = 18) and periodontally healthy controls (n = 15). Subsequently, non‐surgical periodontal treatment was performed in the patients, and saliva sampling from patients was repeated two‐, six‐, and twelve weeks post‐treatment (NCT02913248 at clinicaltrials.gov).The patients were grouped as good and poor responders to treatment according to the achieved reduction in bleeding on probing (BOP).Salivary levels of C3 and C3c were quantified using sandwich ELISA.ResultsPatients with periodontitis had higher baseline levels of both total C3 and the split product C3c in saliva than did periodontally healthy controls (P < .0001). Receiver operating curve (ROC) analyses discriminated patients with periodontitis from controls based on both C3 (AUC (area under curve) = 0.91, P < .001) and C3c levels (AUC = 0.84, P < .001) in saliva. Periodontal treatment improved all clinical parameters (P < .01). Good responders (n = 10) had lower baseline levels of C3c than poor responders (n = 8), (P < .05), and baseline levels of C3c discriminated between good and poor responders (AUC = 0.80, P < .05).ConclusionIn conclusion, patients with periodontitis had higher salivary levels of C3c, and the C3c levels were predictive of reductions in BOP, that is, the poor responders. This suggests that salivary C3c levels possess potential to serve as a biomarker predicting the clinical response to non‐surgical periodontal treatment.

New Insights in the Pathogenesis and Therapy of Cold Agglutinin-Mediated Autoimmune Hemolytic Anemia.

Autoimmune hemolytic anemias mediated by cold agglutinins can be divided into cold agglutinin disease (CAD), which is a well-defined clinicopathologic entity and a clonal lymphoproliferative disorder, and secondary cold agglutinin syndrome (CAS), in which a similar picture of cold-hemolytic anemia occurs secondary to another distinct clinical disease. Thus, the pathogenesis in CAD is quite different from that of polyclonal autoimmune diseases such as warm-antibody AIHA. In both CAD and CAS, hemolysis is mediated by the classical complement pathway and therefore can result in generation of anaphylotoxins, such as complement split product 3a (C3a) and, to some extent, C5a. On the other hand, infection and inflammation can act as triggers and drivers of hemolysis, exemplified by exacerbation of CAD in situations with acute phase reaction and the role of specific infections (particularly Mycoplasma pneumoniae and Epstein-Barr virus) as causes of CAS. In this review, the putative mechanisms behind these phenomena will be explained along with other recent achievements in the understanding of pathogenesis in these disorders. Therapeutic approaches have been directed against the clonal lymphoproliferation in CAD or the underlying disease in CAS. Currently, novel targeted treatments, in particular complement-directed therapies, are also being rapidly developed and will be reviewed.

Sarcolemmal Staining by Anti-C4d/C3d in Cardiac Transplant Endomyocardial Biopsies and Associated Serum HLA Antibodies and Pathologic Rejection

Immunologic evidence of complement fixation in endomyocardial biopsies (EMB) is a feature used to assess pathologic antibody-mediated rejection in cardiac transplants. Uncommonly, sarcolemmal staining (SS) of myocytes is observed. The significance of this is unclear and under-investigated. This study aims to temporally correlate SS with changes in HLA antibodies and pathologic rejection. All EMBs that had SS by anti-C4d and/or C3d on immunofluorescence or immunohistochemistry (Figure 1) were identified via the laboratory information system between 10/1/2016 and 10/1/2019. The HLA antibodies and all other EMB results since time of transplant for each case were reviewed and temporally correlated to the biopsy that showed SS. 28 patients (mean: 31 yrs; range: 6-73 yrs) with 31 SS events were identified. Associated features are summarized in Table 1. Of note, 29/31 events of SS had HLA antibodies measured within a 2 week period before/after the time of biopsy. A temporally associated expansion of HLA Class I/II antibodies was noted in 12 (39%) cases. 21 (68%) cases had prior donor specific antibodies before EMB showing SS, with less immediate temporal association. Sarcolemmal staining of complement split products in EMBs was associated with a concurrent expansion in strength and/or breadth of HLA antibodies in 39% of cases. This is a novel observation that may partially explain the appearance of SS by immunologic assays. Further investigation is required to fully elucidate the significance of SS in EMB.

Identification of susceptibility locus shared by IgA nephropathy and inflammatory bowel disease in a Chinese Han population.

Genome-wide association studies (GWAS) had discovered several genetic risk loci for IgA nephropathy (IgAN), where the susceptibility genes of CARD9 and HORMAD2 for IgAN were also implicated in inflammatory bowel disease (IBD), suggesting a shared genetic etiology of these two diseases. The aim of this study is to explore the common susceptibility loci between IgAN and IBD and provide evidences to elucidate the shared pathogenesis between these two autoimmune diseases. Nineteen single-nucleotide polymorphisms (SNPs) associated with IBD in Asian populations were selected through the National Human Genome Research Institute (NHGRI) GWAS Catalog, and 2078 IgAN patients and 2085 healthy individuals of Chinese Han ancestry were included in the two-stage case-control association study. Serum levels of complement factor B (CFB) and complement split product C3a were detected by enzyme-linked immunosorbent assay (ELISA). One significant shared association at rs4151657 (OR = 1.28, 95%CI = 1.13-1.45, P = 1.42 × 10-4) was discovered between these two diseases, which implicated CFB as a susceptibility gene for IgAN. Genotype-phenotype correlation analysis found significant association of the rs4151657-C allele with decreased serum C3 levels. In addition, the rs4151657-C allele was also associated with higher CFB levels and C3a levels, which suggested a certain degree of systemic complement activation in IgAN patients with the rs4151657-CT or CC genotypes. Our study identified one risk locus (CFB) shared by IgAN and IBD, and genetic variants of CFB may affect complement activation and associate with the predisposition to IgAN.

Neuropilin-1 Acts as a Receptor for Complement Split Products.

Complement split products (CSPs), such as the fragments C4d and C3d, which are generated as a consequence of complement regulatory processes, are established markers for disease activity in autoimmunity or antibody-mediated graft rejection. Since immunoglobulin-like transcript 4 (ILT4) was previously shown to interact with soluble CSPs, but not with CSPs covalently-bound to target surfaces following classical complement activation, the present study aimed to identify novel cellular receptors interacting with covalently-deposited CSPs. By applying an unbiased screening approach using a cDNA mammalian expression library generated from human monocyte-derived dendritic cells and probed with recombinant human C4d, we identified neuropilin-1 (NRP1) as a novel receptor for C4d, C3d, and iC3b. NRP1, a highly conserved type 1 transmembrane protein, plays important roles in the development of the nervous and cardiovascular system as well as in tumorigenesis through interaction with its established binding partners, such as vascular endothelial growth factor (VEGF) and semaphorin 3A (Sema3A). NRP1 is also expressed on immune cells and serves as a marker for murine Tregs. Although NRP1 contains domains homologous to ones found in some complement proteins, it has not been linked to the complement system. We demonstrate that binding of C4d to NRP1 expressing cells was dose-dependent and saturable, and had a KD value of 0.71 μM. Importantly, and in contrast to ILT4, NRP1 interacted with CSPs that were covalently bound to target surfaces in the course of complement activation, therefore representing a classical complement receptor. The binding site of CSPs was mapped to the b1 domain of the coagulation factor V/VIII homology domain of NRP1. Taken together, our results demonstrate a novel role for NRP1 as a receptor for CSPs deposited on surfaces during complement activation. Further work is required to elucidate the functional consequences of the NRP1-CSP interactions in immunity.

Case report: a peculiar glomerulopathy in a patient suffering from nephrotic syndrome

BackgroundPodocyte infolding glomerulopathy (PIG) is a rare histopathologic finding with global infolding of the podocytes into the glomerular basement membrane (GBM), accompanied by microstructures underneath. Described in 2002 for the first time, PIG was proposed as a new pathological entity in 2008 based on the largest case series so far. Yet all of the described cases derive from Asian countries. We report a case from Germany fulfilling the diagnostic criteria of PIG. Considering the scarcity of data on this entity especially in Western countries, collecting cases like ours and multicentric meta-analyses will be crucial to obtain a better understanding of PIG, its causes, clinical course and potential treatment options.Case presentationA 56-year-old Caucasian woman with a history of rheumatoid arthritis (RA), no other comorbidities and no known renal disease was admitted to the hospital with acute kidney injury (AKI) and nephrotic syndrome. Physical examination was unremarkable except for anasarca. Renal ultrasound revealed no abnormalities. Laboratory and urine analyses were consistent with the nephrotic syndrome and renal failure. Serological studies regarding ANA, ANCA, anti-PLA2R autoantibodies, complement, virus infections, immunofixation and quantitative light chain analysis were unremarkable. A renal biopsy was performed. Light microscopic examination showed flattened tubular epithelium consistent with acute tubular damage, no infiltrates and unremarkable glomeruli except diffuse and global holes in the GBM (Fig. 1a) and negative staining for immunoglobulin heavy-chains, light-chains and complement split products. Electron microscopy revealed a rare correlate for these holes: global peculiar infolding of podocyte cytoplasm into the GBM. Most of these infoldings were accompanied by condensation of the GBM underneath. No such condensation or electron dense deposits were found without these infoldings or outside the GBM.ConclusionHere we report the first case of PIG outside of Asia. Since there are only few reports about this specific finding, we feel there is a need to share information in an attempt to accumulate knowledge about this possible new entity and potential treatment options.