Activation Of Alternative Complement Pathway Research Articles

Targeted complement inhibition using bispecific antibodies that bind local antigens and endogenous complement regulators

Complement activation protects against infection but also contributes to pathological mechanisms in a range of clinical conditions such as autoimmune diseases and transplant rejection. Complement-inhibitory drugs, either approved or in development, usually act systemically, thereby increasing the risk for infections. We therefore envisioned a novel class of bispecific antibodies (bsAbs) which are capable of site-directed complement inhibition by bringing endogenous complement regulators in the vicinity of defined cell surface antigens. Here, we analyzed a comprehensive set of obligate bsAbs designed to crosslink a specific target with either complement regulator factor H (FH) or C4b-binding protein (C4BP). The bsAbs were assessed for their capacity to inhibit complement activation and cell lysis in an antigen-targeted manner. We observed that the bsAbs inhibited classical, lectin, and alternative pathway complement activation in which sufficient endogenous serum FH and C4BP could be recruited to achieve local inhibition. Importantly, the bsAbs effectively protected antigen-positive liposomes, erythrocytes, and human leukocytes from complement-mediated lysis. In conclusion, localized complement inhibition by bsAbs capable of recruiting endogenous human complement regulators (such as FH or C4BP) to cell surfaces potentially provides a novel therapeutic approach for the targeted treatment of complement-mediated diseases.

Synergistic benefits of dietary silymarin and selenium on growth, immune functions, antioxidants, and gut/liver health of Thinlip mullet (Liza ramada) juveniles

Abstract This study investigates the synergistic impact of silymarin (SI) levels combined with inorganic selenium (sodium selenite: Se) on growth, feed utilization, biochemical parameters, antioxidants, innate immunity, intestinal and liver histology, and gene expression of Thinlip mullet (Liza ramada) juveniles. The experimental design involved thinlip mullets initially weighing 3.5±0.13 g, distributed in a completely randomized design with 30 fish per hapa (0.5 × 0.5 × 1 m), and conducted in triplicate over 60 days. Seven experimental diets were employed, including a control (without SI and Se supplementation), a negative control (with only Se supplementation), and four treatments with varying levels of silymarin (250, 450, 650, 850 mg/kg) alongside selenium (0.5 mg/kg diet). The growth performance results highlighted significant enhancements in final body weight, weight gain, and specific growth rate, particularly in the SI 850 mg/kg + Se treatment. Survival rates, feed intake, and feed conversion ratios showed positive trends across the SI-Se supplemented groups. Biochemical profiles of serum exhibited that the control diet-induced elevated concentrations of glucose, cholesterol, Alanine aminotransferase, Aspartate aminotransferase, and urea, while Se or SI supplementation significantly mitigated these levels, with the lowest concentrations observed in the SI-Se supplemented groups. Moreover, SI supplementation increased serum protein content. Antioxidant enzyme activities, represented by superoxide dismutase (SOD), catalase (CAT), and catalase (GPx), demonstrated notable improvements in the SI-Se fortified groups, with significantly elevated GPx activity compared to the Se-supplemented and control groups. Immune system responses, including lysozyme, bactericidal, Nitro-blue Tetrazolium (NBT%), and serum alternative complement pathway (ACH50) activities, were highest in the SI-Se augmented groups. SI and Se in L. ramada reduce liver pro-inflammatory gene expression (il-1β, hepcidin) vs. control group. Histological examinations of the intestine and liver depicted structural enhancements, especially at moderate and high levels of SI with Se supplementation. The results indicate improved intestinal villi morphology and hepatic architecture, supporting the positive influence of dietary treatments on the health of thinlip mullet juveniles. In conclusion, the combined supplementation of SI at 850 mg/kg diet and Se at 0.5 mg/kg diet positively influenced the growth, biochemical profiles, antioxidant status, immune responses, gene expression, and histological integrity of Thinlip mullet juveniles, providing valuable insights for optimizing aquafeed formulations.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration.

Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear. We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing. The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (∼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells. Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.

High-dose systemic adeno-associated virus vector administration causes liver and sinusoidal endothelial cell injury

We analyzed retrospective data from toxicology studies involving administration of high doses of adeno-associated virus expressing different therapeutic transgenes to 21 cynomolgus and 15 rhesus macaques. We also conducted prospective studies to investigate acute toxicity following high-dose systemic administration of enhanced green fluorescent protein-expressing adeno-associated virus to 10 rhesus macaques. Toxicity was characterized by transaminitis, thrombocytopenia, and alternative complement pathway activation that peaked on post-administration day 3. Although most animals recovered, some developed ascites, generalized edema, hyperbilirubinemia, and/or coagulopathy that prompted unscheduled euthanasia. Study endpoint livers from animals that recovered and from unscheduled necropsies of those that succumbed to toxicity were analyzed via hypothesis-driven histopathological and unbiased single-nucleus RNA sequencing. All liver cell types expressed high transgene transcript levels at early unscheduled timepoints that subsequently decreased. Thrombocytopenia coincided with sinusoidal platelet microthrombi and sinusoidal endothelial injury identified via immunohistology and single-nucleus RNA sequencing. Acute toxicity, sinusoidal injury, and liver platelet sequestration were similarly observed with therapeutic transgenes and enhanced green fluorescent protein at doses ≥1×1014 GC/kg, suggesting it was the consequence of high-dose systemic adeno-associated virus administration, not green fluorescent protein toxicity. These findings highlight a potential toxic effect of high-dose intravenous adeno-associated virus on nonhuman primate liver microvasculature.

Inhibition of the Alternative Complement Pathway May Cause Secretion of Factor B, Enabling an Early Detection of Pancreatic Cancer.

This study aims to elucidate the cellular mechanisms behind the secretion of complement factor B (CFB), known for its dual roles as an early biomarker for pancreatic ductal adenocarcinoma (PDAC) and as the initial substrate for the alternative complement pathway (ACP). Using parallel reaction monitoring analysis, we confirmed a consistent ∼2-fold increase in CFB expression in PDAC patients compared with that in both healthy donors (HD) and chronic pancreatitis (CP) patients. Elevated ACP activity was observed in CP and other benign conditions compared with that in HD and PDAC patients, suggesting a functional link between ACP and PDAC. Protein-protein interaction analyses involving key complement proteins and their regulatory factors were conducted using blood samples from PDAC patients and cultured cell lines. Our findings revealed a complex control system governing the ACP and its regulatory factors, including Kirsten rat sarcoma viral oncogene homolog (KRAS) mutation, adrenomedullin (AM), and complement factor H (CFH). Particularly, AM emerged as a crucial player in CFB secretion, activating CFH and promoting its predominant binding to C3b over CFB. Mechanistically, our data suggest that the KRAS mutation stimulates AM expression, enhancing CFH activity in the fluid phase through binding. This heightened AM-CFH interaction conferred greater affinity for C3b over CFB, potentially suppressing the ACP cascade. This sequence of events likely culminated in the preferential release of ductal CFB into plasma during the early stages of PDAC. (Data set ID PXD047043.).

Complement System Deficiencies in Elite Athletes.

Although regular physical activity improves immune competency and reduces the prevalence of inflammatory diseases, strenuous training in elite athletes is associated with an increased susceptibility to infectious complications. Therefore, the objective of our study was to assess the routinely examined parameters of the complement system in elite athletes. The study was carried out in a cohort of elite athletes (n = 134) and healthy control subjects (n = 110). In all subjects, besides a routine laboratory check-up, serum concentrations of the C3 and C4 complement components, mannose-binding lectin (MBL), as well as activation of all three complement pathways were determined. Compared to healthy controls, lower C3 and C4 complement component concentrations were observed in elite athletes (0.96 ± 0.1 vs. 1.08 ± 0.2mg/L, and 0.18 ± 0.1 vs. 0.25 ± 0.1mg/L, respectively, p < 0.05); with much higher frequency rates of C3 and C4 deficiencies in athletes (31.3 vs. 14.5%, and 6 vs. 0%, p < 0.05). Simultaneously, athletes had much higher frequency rates of deficiencies of activation of classical and alternative complement pathways; while, deficiency of activation of the lectin pathway was similar in both cohorts. We confirmed a high frequency of defects in the complement system in elite athletes. Lower concentrations of C3 and C4 complement components, with high frequencies of deficiencies of the classical and alternative complement activation pathways were the most prevalent disorder of the complement system in elite athletes. Further studies are needed to uncover the functional impacts of these observations upon the susceptibility to infectious diseases.

The spectrum of thrombotic microangiopathy related to monoclonal gammopathy.

Recent studies showed a high prevalence of monoclonal gammopathy (MG) in patients with thrombotic microangiopathy (TMA) aged over 50years and suggested that complement dysregulation is pivotal for the disease to develop. Here, we studied this premise in seven patients with TMA and coexisting MG. Patients with TMA on kidney biopsy and/or peripheral blood were recruited from the prospective COMPETE cohort (NCT04745195) and Limburg Renal Registry. Patients were screened for complement dysregulation, including genetics/factor H autoantibodies (FHAA) and functional ex vivo testing on microvascular endothelial cells. Seven (8%) out of 84 patients with TMA presented with a coexisting MG. MG clustered in patients aged over 50years (n/N=6/32, 19%). C4 and/or C3 levels were low in three patients, while four patients presented with normal complement levels. None of the patients carried rare variants in complement genes. Massive ex vivo C5b9 formation on the endothelium was noted in one patient; purified IgG from this patient caused massive ex vivo C5b9 formation via the alternative pathway of complement activation, pointing to complement dysregulation in the fluid phase. Kidney biopsies from other nephropathies linked to MG rarely exhibited concurrent TMA (n/N=1/27, 4%). MG clustered in patients with TMA aged over 50 years. TMA and coexisting MG represents a heterogeneous disease spectrum, including a small subset of patients who may present with complement dysregulation.

Advances in the treatment of IgA nephropathy with biological agents.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerular disease, and the "four-hit" theory represents its currently accepted pathogenic mechanism. Mucosal immunity triggered by infections in the respiratory tract, intestines, or other areas leads to antigen presentation, T cell stimulation, B cell maturation, and the production of IgA-producing plasma cells. The proteins B-lymphocyte stimulator (BLyS) and aproliferation-inducing ligand (APRIL) are involved in this process, and alternative complement and lectin pathway activation are also part of the pathogenic mechanism. Kidney DiseaseImproving Global Outcomes guidelines indicate that a specific effective treatment for IgAN is lacking, with renin-angiotensin-aldosterone system inhibitors being the primary therapy. Recent research shows that biological agents can significantly reduce proteinuria, stabilize the estimated glomerular filtration rate, and reverse some pathological changes, such as endocapillary proliferation and crescent formation. There are four main categories of biological agents used to treat IgA nephropathy, specifically anti-CD20 monoclonal antibodies, anti-BLyS or APRIL monoclonal antibodies, monoclonal antibodies targeting both BLyS and APRIL (telitacicept and atacicept), and monoclonal antibodies inhibiting complement system activation (narsoplimab and eculizumab). However, further research on the dosages, treatment duration, long-term efficacy, and safety of these biological agents is required.

Prospective Clinical and Biomarker Validation of the ASTCT Consensus Definition for Transplant-Associated Thrombotic Microangiopathy (TA-TMA)

Introduction: Transplantation-associated thrombotic microangiography (TA-TMA) is a disorder that causes severe complications post-hematopoietic cell transplantation (HCT). Diagnosing TA-TMA is challenging due to non-standardized criteria. The American Society for Transplantation and Cellular Therapy (ASTCT) has proposed new consensus diagnostic criteria for standard risk (ASTCT-SR) and high risk (ASTCT-HR) but their correlation with older criteria based on microangiopathic hemolytic anemia (MAHA) or diagnostic complement biomarkers used in previous TA-TMA studies, such as levels of the terminal complement activation product sC5b-9 and the alternative complement pathway activation product Factor Ba, is unknown. Using data and samples from an ongoing prospective pediatric cohort study, we compared the two definitions and their associations with biomarkers. Methods: Patients aged 0 to 18 years who underwent first allogeneic HCT 2021-2023 at Texas Children's Hospital were included in the clinical and biomarker study. Those who did not have at least 100-day post-HCT event-free follow-up were excluded. Two different definitions for TA-TMA were applied. Clinical TMA (cTMA) was diagnosed if a patient 1) met 4/4 concurrent criteria of MAHA (schistocytosis, thrombocytopenia, anemia, and elevated lactate dehydrogenase (LDH)) at least twice in 14 days and 2) clinically adjudicated to exclude known mimics or alternative causes of TMA. In contrast, ASTCT-HR TMA was diagnosed if a patient 1) met 4/7 non-concurrent criteria (schistocytosis, thrombocytopenia, anemia, elevated LDH, elevated blood pressure, elevated urinary protein excretion, and elevated sC5b9) at least twice in 14 days and 2) had evidence of LDH &amp;gt;2x normal, proteinuria ≥1 mg/mg, elevated sC5b9, acute graft-versus-host disease, active bacterial/viral infection, or end-organ damage. Cumulative incidence of TA-TMA was assessed by the competing risk method. Overall survival was compared by the Kaplan Meier estimator and log-rank test. Biomarker levels for samples collected at day 15 (+/- 14 days) were compared using the t-test. Results: A total of 32 patients met the inclusion criteria. The median age was 5.7 years (range 1.1-18.8) and most were White (68.8%) and Hispanic (56.2%). Malignant disease was the indication for HCT in 50% of patients. While the classic MAHA-based cTMA definition had an incidence of 12.7%, the newly proposed ASTCT-HR definition doubled it to 28.5% by day-100 (Figure 1). In contrast to patients with concordant TA-TMA diagnosis (+/+) who had significantly worse post-transplant survival (p=0.0047) compared with those with concordant non-TMA (-/-), those reclassified as TA-TMA by the new definition only (-/+) had an unexpectedly good prognosis (100% survival at day 100) despite the lack of TMA-directed therapy. Longitudinal biomarker analysis was conducted on 29 patients. The highest average plasma levels of sC5b-9 and Ba during the first 100 days were observed in patients meeting both cTMA and ASTCT-HR criteria (+/+). When focused on early biomarkers measured around day 15, sC5b-9 (P=0.041) and Ba (P=0.004) were significantly elevated in the doubly positive TMA group (+/+) vs. non-TMA group (-/-) (Figure 2). We did not observe significant differences for ASTCT-HR singly positive (-/+) vs. non-TMA group (-/-). Conclusions: In the current prospective clinical and biomarker study, the recently proposed ASTCT-HR definition was more sensitive but less prognostic than the traditional cTMA definition for TA-TMA. Patients who solely met the ASTCT-HR diagnostic criteria but not the cTMA criteria had benign complement biomarker profiles and excellent survival prognosis without TMA-directed therapy. While clinicians need to be vigilant for this infrequent yet potentially severe complications, it is crucial that diagnostic criteria accurately identify affected patients given the expense and potential complications of treatment. We recommend ongoing validations of the new consensus diagnostic criteria from other prospective cohort studies with larger sample sizes and extended follow-up.

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

Combined Effects of Spirulina platensis and Pediococcus acidilactici on the Growth Performance, Digestive Enzyme Activity, Antioxidative Status, and Immune Genes in Zebrafish

Abstract The combined effects of Spirulina platensis (SP) and Pediococcus acidilactici (PA) on growth performance, digestive enzyme activity, antioxidative status, and immune genes in zebrafish were investigated in the present study. Four experimental diets were designed: control and three test diets mixed with SP at 2.5%, PA at 107 CFU/g, or a combination of 2.5% SP and 107 CFU/g PA. After 56 days, fish treated with PA and SP mixture had higher final weight, weight gain, SGR, and lower FCR than fish fed the control and SP diets (P&lt;0.05). The results also illustrated that fish fed PA, SP, and their mixture had higher (P&lt;0.05) protease and amylase activities than the control. The lipase activity was significantly higher in fish treated with PA or the mixture of PA and SP than in the control (P&lt;0.05). The alternative complement pathway (ACH50) and lysozyme activity in the mucus samples of fish treated with PA or both PA and SP were significantly higher (P&lt;0.05) than in fish treated with the control and SP diets. The total immunoglobulin level in the skin mucus was significantly higher (P&lt;0.05) in fish fed PA than in control. In the body homogenates samples, the lysozyme activity and immunoglobulin levels were markedly higher (P&lt;0.05) in fish treated with the mixture of PA and SP than in the control. The dietary PA and SP diet mixture improved the glutathione peroxidase, superoxide dismutase, and total antioxidative capacity. The expression of IL-1β and IL-8 genes in fish treated with PA or the mixture of PA and SP was significantly higher (P&lt;0.05) than in fish treated with the control or SP diets. Fish treated with PA, SP, or both PA and SP had marked (P&lt;0.05) upregulation of the lysozyme gene expression. In conclusion, the mixture of S. platensis and P. acidilactici is more effective than using each individually for improving the growth performance, digestive enzyme activity, and the immune and antioxidative capacity of zebrafish.

Results of a randomized double-blind placebo-controlled Phase 2 study propose iptacopan as an alternative complement pathway inhibitor for IgA nephropathy

Targeting the alternative complement pathway is an attractive therapeutic strategy given its role in the pathogenesis of immunoglobulin A nephropathy (IgAN). Iptacopan (LNP023) is an oral, proximal alternative complement inhibitor that specifically binds to Factor B. Our randomized, double-blind, parallel-group adaptive Phase 2 study (NCT03373461) enrolled patients with biopsy-confirmed IgAN (within previous three years) with estimated glomerular filtration rates of 30 mL/min/1.73 m2 and over and urine protein 0.75 g/24 hours and over on stable doses of renin angiotensin system inhibitors. Patients were randomized to four iptacopan doses (10, 50, 100, or 200 mg bid) or placebo for either a three-month (Part 1; 46 patients) or a six-month (Part 2; 66 patients) treatment period. The primary analysis evaluated the dose–response relationship of iptacopan versus placebo on 24-hour urine protein-to-creatinine ratio (UPCR) at three months. Other efficacy, safety and biomarker parameters were assessed. Baseline characteristics were generally well-balanced across treatment arms. There was a statistically significant dose-response effect, with 23% reduction in UPCR achieved with iptacopan 200 mg bid (80% confidence interval 8 -34%) at three months. UPCR decreased further through six months in iptacopan 100 and 200 mg arms (from a mean of 1.3 g/g at baseline to 0.8 g/g at six months in the 200 mg arm). A sustained reduction in complement biomarkers levels including plasma Bb, serum Wieslab, and urinary C5b-9 was observed. Iptacopan was well-tolerated, with no reports of deaths, treatment-related serious adverse events or bacterial infections, and led to strong inhibition of alternative complement pathway activity and persistent proteinuria reduction in patients with IgAN. Thus, our findings support further evaluation of iptacopan in the ongoing Phase 3 trial (APPLAUSE-IgAN; NCT04578834).

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Circulating alternative pathway complement cleavage factor Bb is associated with vascular lesions and outcomes in IgA nephropathy.

Complement alternative pathway (AP) activation is linked to immunoglobulin A nephropathy (IgAN) prognosis severity, but Bb fragment's role is unclear. We examined the relationship between serum Bb fragment concentration at IgAN diagnosis with disease activity and outcomes. This retrospective study included 125 biopsy-proven IgAN patients (age 39.9 years, 75% male, eGFR 82mL/min, proteinuria 0.5g/day) enrolled from 1984 to 2010 and followed for a minimum of 18 months. Monitoring continued until the last follow-up, end-stage kdiney disease (ESKD), or death. Serum Bb fragment was measured using ELISA at diagnosis. Oxford classification and global optical score (GOS) were utilized for pathology assessment. Patients were followed for a median of 16 years, 42% developed CKD stage 3 or higher, 19% reached ESKD, and 9% died. Serum Bb fragment concentration negatively correlated with eGFR values at last follow-up and positively with vascular and tubular histopathological indices. In univariate Cox regression analyses, higher Bb fragment concentration was associated with ESKD alongside older age, increased body-mass-index, arterial hypertension, lower eGFR, higher proteinuria, E1, S1, T1-2, GOS, and corticotherapy. Patients with Bb levels≥14.3μg/mL had shorter mean kidney survival time (19.5vs 22.7 years, p=0.07); after adjusting for progression risk factors, the association persisted (HR 4.76, 95% CI 1.56-14.43). Serum Bb fragment concentration at diagnosis may predict long-term IgAN outcomes, potentially due to AP activation at the endothelial surface. Further research is needed to confirm these results and evaluate Bb fragment's role in IgAN management.

Dietary fulvic acid increased growth, stress tolerance and disease resistance against Vibrio harveyi in Asian seabass (Lates calcarifer) juvenile

An 8-week husbandry trial was carried out toexamine the impacts of graded levels of dietary fulvic acid (FA) on growth, stress, and disease resistance of Asian seabass (Lates calcarifer, 38.2 g) juveniles. Five levels of FA were included in a basal diet (470 g kg-1 crude protein, 150 g kg-1 crude lipid) to prepare experimental diets, including: 0 (control), 2.5, 5.0, 10, and 15 g kg-1 diet. Two hundred and twenty-five fish (38.2 ± 0.1 g) were stocked randomly into 15 1000-L circular polyethylene tanks (15 fish tank-1) filled with 800 L disinfected seawater and fed with the experimental feeds three times daily. Each dietary treatment had three replicates. Tanks were supplied with seawater (32.2 °C, 45.0 ppt) in a flow-throw system. At the end of the husbandry trial, fish were challenged with Vibrio harveyi and air exposure stress for one min. Fish fed FA-supplemented diets showed a positive quadratic growth response, and those fed with a 5 g FA kg-1 diet had higher final weight and weight gain percentage than the control. Feed intake in fish fed FA-supplemented diets were higher than in control, showing significant linear and quadratic trends (P < 0.05). The highest and lowest skin mucus lysozyme (LYZ) and serum alternative complement pathway (ACH50) activities were in fish fed 10 and 15 g FA kg-1 diets, respectively, exhibiting a quadratic response to dietary FA. Serum total protein level (TP) in fish fed FA-supplemented diets was higher than in the control group. Medium or high levels (10–15 g kg-1) of FA significantly enhanced the survival rate against Vibrio harveyi, which was associated with increased activities of serum lysozyme and ACH50 as well as liver glutathione peroxidase activity in these groups. Air exposure induced oxidative stress in all treatments and gradually reduced antioxidant enzyme activities associated with increased malondialdehyde content in the liver. Furthermore, air exposure suppressed some immune responses, including serum total immunoglobulin and TP levels. Still, LYZ activity was restored after 24 h, with the highest level found in fish fed 5 g FA kg-1 diet. Based on the broken-line regression analysis, the adequate level of FA in the diet for Asian seabass is 4.4 g kg-1.

Urinary markers of the alternative and lectin complement pathway are increased in IgA vasculitis nephritis.

IgA vasculitis (IgAV) is the most common form of childhood vasculitis. Nephritis (IgAVN) occurs in 50% of patients and 1-2% progress to chronic kidney disease stage 5. The pathophysiology of nephritis remains largely unknown, but recent evidence suggests that the complement system may be involved. The aim of this cross-sectional study was to explore whether there is evidence of alternative and/or lectin complement pathway activation in children with IgAVN. Children with IgAV were recruited and grouped according to proteinuria: IgAVN or IgAV without nephritis (IgAVwoN). Age and sex-matched healthy controls (HCs) were also recruited. Cross-sectional urine and plasma concentrations of complement factor D (CFD), factor B (CFB), and MBL-associated protease 1 (MASP-1) were performed using commercially available enzyme-linked immunoassays. A total of 50 children were included (IgAVN, n=15; IgAVwoN, n=20, HCs, n=15). The mean age was 8.5±3.7 years old, male:female ratio was 1:1. Urinary CFD and CFB concentrations were statistically significantly increased in children with IgAVN (3.5±5.4μg/mmol; 25.9±26.5μg/mmol, respectively) compared to both IgAVwoN (0.4±0.4μg/mmol, P=0.002; 9.2±11.5μg/mmol, P=0.004) and HCs (0.3±0.2μg/mmol, P<0.001; 5.1±6.0μg/mmol, P<0.001). No statistically significant difference was reported for the plasma concentrations of CFD and CFB. Urinary MASP-1 concentrations were statistically significantly increased in IgAVN (116.9±116.7ng/mmol) compared to HCs (41.4±56.1ng/mmol, P=0.006) and plasma MASP-1 concentrations were increased in IgAVwoN (254.2±23.3ng/mL) compared to HCs (233.4±6.6ng/mL, P=0.046). There is evidence of complement pathway products in the urine of children with IgAVN that warrants further investigation.

Comparative characteristics of the complement system in patients with C3-glomerulopathy and atypical hemolytic uremic syndrome of chronic course who suffered an acute episode of thrombotic microangiopathy

To compare changes in the complement system in C3-glomerulopathy (C3-GP) and atypical hemolytic uremic syndrome (aHUS) after the relief of an acute episode of thrombotic microangiopathy. The study included 8 patients diagnosed with C3-GP and 8 with aHUS in remission. The blood levels of the complement system components were determined: C3, C4, C3a, C5a, factor H (CFH), factor B (CFB), membrane-attacking complex (MAC), antibodies to C3b (anti-C3b-AT), the level of hemolytic activity (CH50), the content of factor D (CFD) in the urine. C3 and CH50 levels were within the reference range in both groups, however, in the C3-GP group they were at the lower limit, and C3 level was significantly lower than in the aHUS group: 0.56 [0.44; 0.96] vs 1.37 [1.16; 2.52] (p=0.003). CFB increased level was detected in both groups, but in the C3-GP group it was significantly lower than in the aHUS group - 275.1 [222.1; 356.6] vs 438.7 [323.3; 449.3] (p=0.010). C3a, C5a and MAC levels were increased in both groups, but the maximum was in the C3-GP group, and the MAC level in the C3-GP group was 2 times higher than that in aHUS, and these differences reached statistical significance - 123 555±6686 vs 5603±1294 (p=0.036). CFH and CFD levels was increased in both groups, but their highest values was in the aHUS group. Alternative complement pathway activation signs were present in both groups of patients with complement-mediated nephropathies, regardless the stage of the disease. In C3-GP, alternative complement pathway activation was more pronounced than in aHUS after the relief of an acute episode of thrombotic microangiopathy.

Complement and coagulation crosstalk – Factor H in the spotlight

The immune complement and the coagulation systems are blood-based proteolytic cascades that are activated by pathway-specific triggers, based on protein–protein interactions and enzymatic cleavage reactions. Activation of these systems is finely balanced and controlled through specific regulatory mechanisms. The complement and coagulation systems are generally viewed as distinct, but have common evolutionary origins, and several interactions between these homologous systems have been reported. This complement and coagulation crosstalk can affect activation, amplification and regulatory functions in both systems. In this review, we summarize the literature on coagulation factors contributing to complement alternative pathway activation and regulation and highlight molecular interactions of the complement alternative pathway regulator factor H with several coagulation factors. We propose a mechanism where factor H interactions with coagulation factors may contribute to both complement and coagulation activation and regulation within the haemostatic system and fibrin clot microenvironment and introduce the emerging role of factor H as a modulator of coagulation. Finally, we discuss the potential impact of these protein interactions in diseases associated with factor H dysregulation or deficiency as well as evidence of coagulation dysfunction.

Can a Single Glomerulus Morphology Implicate Successful Therapy?

Recurrent gross hematuria of glomerular origin is frequently encountered in clinical practice, and in absence of specific serological marker, renal biopsy is mandatory to address the definitive diagnosis, and set out an appropriate therapeutic protocol. Technical deficiencies associated with practice of renal biopsy are frequently encountered, as inadequate number of glomeruli or poor immunofluorescence staining of kidney biopsy specimen; however, these deficiencies can be offset by detailed electron microscopy analysis of a single abnormal glom.We present a single middle-aged Libyan woman, with a rare glomerular disease, related to abnormal activation of alternative complement pathway, where renal biopsy report was initially not adequate and lacking immunohistochemistry workup. However, electron microscopy reports a characteristic abnormal glomerular deposit, coupled with clinical and biochemical data that guided our therapeutic protocol.In a middle-aged female who presented with recurrent gross hematuria and nephrotic range proteinuria, we should suspect a glomerular pathology. Further to immunoglobulin A nephropathy or lupus nephritis, particularly in presence of complement abnormalities and negative serology for glomerulopathy-related autoantibodies, dense deposit disease and C3 glomerulonephritis that are rare complement mediated glomerulopathy should be considered as a seronegative lupus nephritis-equivalent, in terms of their membranoproliferative features on light microscopy, and when setting out appropriate therapeutic protocol. Patient and family counseling for C3 glomerulopathy is essential because this type of glomerulopathy has a recurrence rate after kidney transplant.