C4d, rather than C3d and C5b-9, is associated with graft loss in recurrent IgA deposition after kidney transplantation.

INTRODUCTION: Recurrent IgA deposition is common after kidney transplantation. However, it is difficult to define whether IgA deposition is innocuous or contributes to organ damage. Next, although complement is known to be involved in the pathogenesis of IgA nephropathy (IgAN), its involvement has not been studied systematically in kidney transplant recipients (KTR). METHODS: KTR with biopsy-proven native IgAN who underwent kidney biopsy after transplantation between 1995 and 2020 were included. Recurrent IgA deposition was defined as IgA deposit in the glomerulus. Staining of complement factors C4d, C3d, and C5b-9 were quantitatively evaluated using ImageScope. RESULTS: Sixty-seven KTR (85% male, 46±13 years old, 12 [6-24] months after transplantation, 58% with indication biopsy) were included in the analyses. Of them, 25 (37%) had recurrent IgA deposition. There were no clinical differences between KTR with and without recurrent IgA deposition. C3d and C5b-9 were always present in biopsies with IgA deposition, while C4d was present in 48% of the biopsies. During a median follow-up of 9.6 [4.8-14] years, 18 (27%) KTR developed death-censored graft failure. Recurrent IgA deposition was not associated with graft failure. Of the evaluated complement factors, only C4d staining was associated with graft failure in KTR with recurrent IgA deposition (Hazard ratio = 2.55, 95% confidence interval = 1.07-6.03, p = 0.034). CONCLUSIONS: Recurrent IgA deposition was not associated with graft failure in itself. C4d, when present, is strongly associated with graft loss in KTR with recurrent IgA deposition, suggesting a pathogenic role for the lectin pathway in recurrent IgAN.

Three-Year Clinical Outcomes of the First South Asian Prospective Longitudinal Observational IgA Nephropathy Cohort.

INTRODUCTION: Glomerular Research And Clinical Experiments-IgA Nephropathy in Indians (GRACE-IgANI) is the first prospective South Asian IgA nephropathy (IgAN) cohort with prespecified objectives, protocolized longitudinal follow-up, and extensive biosample collection. The baseline risk scores predicted high risk of kidney disease progression. METHODS: A total of 195 of 201 patients (97%) completed 3-year follow-up in September 2020. All patients received optimized supportive care, and those at high risk of progression were offered systemic corticosteroids. RESULTS: A total of 76 patients (76 of 193, 39.4%) had rapid progression in 3 years (≥5 ml/min per 1.73 m2 decline in estimated glomerular filtration rate [eGFR] per year). A total of 72 patients (72 of 195, 36.9%) experienced the composite outcome (CO), defined as ≥50% fall in eGFR, eGFR < 15 ml/min per 1.73 m2, commenced kidney replacement therapy or death, in 3 years. At each scheduled follow-up, achievement of proteinuria level < 1 g/d significantly delayed the time to the CO. The receiver operating characteristic curve of average annual decline in eGFR ≥ 5 ml/min per 1.73 m2 had 86% sensitivity and 89% specificity for CO in 3 years and had good discrimination from 1 year onwards (area under the curve 0.8, SE 0.04, 95% CI 0.7-0.9, P < 0.0001). The significant predictors of CO by Cox proportional-hazards model were as follows: baseline MEST-T2 score (hazard ratio [HR] 3.3, 95% CI 1.7-6.5, P < 0.001), along with 24-hour urine protein level ≥ 1 g/d (HR 2.1, 95% CI 1.1-3.9, P = 0.02), eGFR < 60 ml/min per 1.73 m2 (HR 2.9, 95% CI 1.1-7.6, P = 0.03), and rate of eGFR decline ≥ 5 ml/min per 1.73 m2/yr (HR 2.7, 95% CI 1.6-4.8, P < 0.001) all measured at 6 months. Mortality was 11 of 195 (5.6%). CONCLUSION: We identified longitudinal clinical variables measured at 6 months and ≥5 ml/min per 1.73 m2 annual fall in eGFR after kidney biopsy as important predictors for composite outcome in addition to baseline histology.

Pentraxin-3-mediated complement activation in a swine model of renal ischemia/reperfusion injury.

Pentraxins are a family of evolutionarily conserved pattern recognition molecules with pivotal roles in innate immunity and inflammation, such as opsonization of pathogens during bacterial and viral infections. In particular, the long Pentraxin 3 (PTX3) has been shown to regulate several aspects of vascular and tissue inflammation during solid organ transplantation. Our study investigated the role of PTX3 as possible modulator of Complement activation in a swine model of renal ischemia/reperfusion (I/R) injury. We demonstrated that I/R injury induced early PTX3 deposits at peritubular and glomerular capillary levels. Confocal laser scanning microscopy revealed PTX3 deposits co-localizing with CD31+ endothelial cells. In addition, PTX3 was associated with infiltrating macrophages (CD163), dendritic cells (SWC3a) and myofibroblasts (FSP1). In particular, we demonstrated a significant PTX3-mediated activation of classical (C1q-mediated) and lectin (MBL-mediated) pathways of Complement. Interestingly, PTX3 deposits co-localized with activation of the terminal Complement complex (C5b-9) on endothelial cells, indicating that PTX3-mediated Complement activation occurred mainly at the renal vascular level. In conclusion, these data indicate that PTX3 might be a potential therapeutic target to prevent Complement-induced I/R injury.

Epidemiology, baseline characteristics and risk of progression in the first South-Asian prospective longitudinal observational IgA nephropathy cohort.

INTRODUCTION: Glomerular Research And Clinical Experiments-IgA Nephropathy in Indians (GRACE-IgANI) is the first prospective South Asian IgAN cohort with protocolized follow-up and extensive biosample collection. Here we report the baseline clinical, biochemical, and histopathologic characteristics of GRACE IgANI and calculate baseline risk of progression for the cohort. METHODS: 201 incident adults with kidney biopsy-proven primary IgAN were recruited into GRACE-IgANI between March 2015 and September 2017. As of April 30, 2020, the cohort had completed a median follow-up of 30 months (interquartile range [IQR] 16-39). RESULTS: The commonest clinical presentation in GRACE IgANI was hypertension, with or without proteinuria, and nephrotic-range proteinuria was present in 34%, despite <10 months of lead time to kidney biopsy. The GRACE-IgANI kidney biopsy data demonstrated a disproportionate absence of active glomerular lesions and overrepresentation of segmental sclerosing lesions and tubulointerstitial fibrosis at presentation, often coexistent with relatively well-preserved estimated glomerular filtration rate (eGFR) and low levels of proteinuria, especially in males. Baseline risk of progression was calculated for each evaluable patient using 2 different risk prediction tools. The median 5-year absolute risk of end-stage kidney disease (ESKD) was 19.8% (IQR 2.7-57.4) and median 5-year risk of progression to the combined endpoint of 50% decline in eGFR or ESKD was 35.5% using the 2 tools. CONCLUSIONS: The predicted risk of progression in this cohort was considerable. Over the next 5 years, we will dissect the pathogenic pathways that underlie this severe South Asian IgAN phenotype.

Brain death-induced lung injury is complement dependent, with a primary role for the classical/lectin pathway.

In brain-dead donors immunological activation occurs, which deteriorates donor lung quality. Whether the complement system is activated and which pathways are herein involved, remain unknown. We aimed to investigate whether brain death (BD)-induced lung injury is complement dependent and dissected the contribution of the complement activation pathways. BD was induced and sustained for 3 hours in wild-type (WT) and complement deficient mice. C3-/- mice represented total complement deficiency, C4-/- mice represented deficiency of the classical and lectin pathway, and factor properdin (P)-/- mice represented alternative pathway deficiency. Systemic and local complement levels, histological lung injury, and pulmonary inflammation were assessed. Systemic and local complement levels were reduced in C3-/- mice. In addition, histological lung injury and inflammation were attenuated, as corroborated by influx of neutrophils and gene expressions of interleukin (IL)-6, IL-8-like KC, TNF-α, E-selectin, and MCP-1. In C4-/- mice, complement was reduced on both systemic and local levels and histological lung injury and inflammatory status were ameliorated. In P-/- mice, histological lung injury was attenuated, though systemic and local complement levels, IL-6 and KC gene expressions, and neutrophil influx were not affected. We demonstrated that BD-induced lung injury is complement dependent, with a primary role for the classical/lectin activation pathway.

Properdin Pattern Recognition on Proximal Tubular Cells Is Heparan Sulfate/Syndecan-1 but Not C3b Dependent and Can Be Blocked by Tick Protein Salp20.

Introduction: Proteinuria contributes to progression of renal damage, partly by complement activation on proximal tubular epithelial cells. By pattern recognition, properdin has shown to bind to heparan sulfate proteoglycans on tubular epithelium and can initiate the alternative complement pathway (AP). Properdin however, also binds to C3b(Bb) and properdin binding to tubular cells might be influenced by the presence of C3b(Bb) on tubular cells and/or by variability in properdin proteins in vitro. In this study we carefully evaluated the specificity of the properdin - heparan sulfate interaction and whether this interaction could be exploited in order to block alternative complement activation. Methods: Binding of various properdin preparations to proximal tubular epithelial cells (PTEC) and subsequent AP activation was determined in the presence or absence of C3 inhibitor Compstatin and properdin inhibitor Salp20. Heparan sulfate proteoglycan dependency of the pattern recognition of properdin was evaluated on PTEC knocked down for syndecan-1 by shRNA technology. Solid phase binding assays were used to evaluate the effectivity of heparin(oids) and recombinant Salp20 to block the pattern recognition of properdin. Results: Binding of serum-derived and recombinant properdin preparations to PTECs could be dose-dependently inhibited (P < 0.01) and competed off (P < 0.01) by recombinant Salp20 (IC50: ~125 ng/ml) but not by Compstatin. Subsequent properdin-mediated AP activation on PTECs could be inhibited by Compstatin (P < 0.01) and blocked by recombinant Salp20 (P < 0.05). Syndecan-1 deficiency in PTECs resulted in a ~75% reduction of properdin binding (P = 0.057). In solid-phase binding assays, properdin binding to C3b could be dose-dependently inhibited by recombinant Salp20> heparin(oid) > C3b. Discussion: In this study we showed that all properdin preparations recognize heparan sulfate/syndecan-1 on PTECs with and without Compstatin C3 blocking conditions. In contrast to Compstatin, recombinant Salp20 prevents heparan sulfate pattern recognition by properdin on PTECs. Both complement inhibitors prevented properdin-mediated C3 activation. Binding of properdin to C3b could also be blocked by heparin(oids) and recombinant Salp20. This work indicates that properdin serves as a docking station for AP activation on PTECs and a Salp20 analog or heparinoids may be viable inhibitors in properdin mediated AP activation.

Arteriolar C4d in IgA Nephropathy: A Cohort Study.

RATIONALE & OBJECTIVE: Glomerular C4d (C4dG) as an indicator of the lectin pathway of complement activation in immunoglobulin A nephropathy (IgAN) has been associated with more severe kidney damage. Recent studies have suggested that vascular lesions in IgAN biopsy specimens with complement deposition are also associated with disease progression. We aimed to study the clinical significance of arteriolar C4d (C4dA) in IgAN kidney biopsy tissue. STUDY DESIGN: Retrospective cohort study. SETTING & PARTICIPANTS: Kidney biopsy specimens from 126 adults with IgAN diagnosed by Oxford classification criteria were stained using immunohistochemistry and classified according to C4dG and C4dA deposition. Additionally, vascular lesions including acute and chronic microangiopathy, arteriolar hyalinosis, and arterial intima fibrosis were characterized. PREDICTOR: C4dA. OUTCOME: Progressive kidney disease, defined as a decline in estimated glomerular filtration rate by≥50% or occurrence of kidney failure. ANALYTICAL APPROACH: The association of C4dA and C4dG with baseline clinical and histologic characteristics, as well as progressive kidney disease, were assessed with survival analysis using multivariable Cox regression analysis. RESULTS: C4dA was identified in 21 (17%) patients and was associated with mean arterial pressure, arterial intima fibrosis, and chronic microangiopathy. C4dA was also significantly associated with C4dG and both were associated with progressive kidney disease. In regression analysis, C4dA remained significantly associated with progressive kidney disease after adjusting for other significant predictors, including baseline estimated glomerular filtration rate, mean arterial pressure, and the presence of crescents. LIMITATIONS: Findings based on the retrospective evaluation of a single center's experience, limited number of events, a small number of patients with a broad range of kidney disease stages, and use of immunohistochemistry rather than immunofluorescence to detect C4d. CONCLUSIONS: C4dA is a potential biomarker for disease progression in IgAN. It should be further investigated in larger cohorts to determine the value of C4dA in improving prediction of IgAN disease progression.

Administration of Intravenous Iron Formulations Induces Complement Activation in-vivo.

Background: Intravenous (IV) iron is widely used to treat anemia in chronic kidney disease patients. Previously, iron formulations were shown to induce immune activation in-vitro. The current study aimed to investigate the effect of IV iron on complement activation in-vivo, and whether this subsequently induces inflammation and/or oxidative stress. Methods: Two distinct patient groups were included: 51 non-dialysis and 32 dialysis patients. The non-dialysis group received iron sucrose or ferric carboxymaltose, based on physicians' choice. Plasma samples were collected prior to and 1 h after completion of IV iron infusion. The dialysis group received iron sucrose exclusively. Plasma samples were collected at the start and end of two consecutive hemodialysis sessions, one with and one without IV iron. Finally, plasma levels of MBL, C1q, properdin, factor D, sC5b-9, MPO, PTX3 were assessed by ELISA. Results: In the non-dialysis group, sC5b-9 levels significantly increased after IV iron by 32%, while levels of factor D and MBL significantly dropped. Subgroup analysis demonstrated that iron sucrose induced complement activation whereas ferric carboxymaltose did not. In the dialysis group, levels of sC5b-9 significantly increased by 46% during the dialysis session with IV iron, while factor D levels significantly fell. Furthermore, the relative decrease in factor D by IV iron correlated significantly with the relative increase in sC5b-9 by IV iron. MPO levels rose significantly during the dialysis session with IV iron, but not in the session without iron. Moreover, the relative increase in MPO and sC5b-9 by IV iron correlated significantly. PTX3 levels were not affected by IV iron. Conclusions: Iron sucrose but not ferric carboxymaltose, results in complement activation possibly via the lectin and alternative pathway partially mediating oxidative stress but not inflammation.

Intradialytic Complement Activation Precedes the Development of Cardiovascular Events in Hemodialysis Patients.

Background: Hemodialysis (HD) is a life-saving treatment for patients with end stage renal disease. However, HD patients have markedly increased rates of cardiovascular morbidity and mortality. Previously, a link between the complement system and cardiovascular events (CV-events) has been reported. In HD, systemic complement activation occurs due to blood-to-membrane interaction. We hypothesize that HD-induced complement activation together with inflammation and thrombosis are involved in the development of CV-events in these patients. Methods: HD patients were followed for the occurrence of CV-events during a maximum follow-up of 45 months. Plasma samples were collected from 55 patients at different time points during one HD session prior to follow-up. Plasma levels of mannose-binding lectin, properdin and C3d/C3 ratios were assessed by ELISA. In addition, levels of von Willebrand factor, TNF-α and IL-6/IL-10 ratios were determined. An ex-vivo model of HD was used to assess the effect of complement inhibition. Results: During median follow-up of 32 months, 17 participants developed CV-events. In the CV-event group, the C3d/C3-ratio sharply increased 30 min after the start of the HD session, while in the event-free group the ratio did not increase. In accordance, HD patients that developed a CV-event also had a sustained higher IL-6/IL-10-ratio during the first 60 min of the HD session, followed by a greater rise in TNF-α levels and von Willebrand factor at the end of the session. In the ex-vivo HD model, we found that complement activation contributed to the induction of TNF-α levels, IL-6/IL-10-ratio and levels of von Willebrand factor. Conclusions: In conclusion, these findings suggest that early intradialytic complement activation predominantly occurred in HD patients who develop a CV-event during follow-up. In addition, in these patients complement activation was accompanied by a pro-inflammatory and pro-thrombotic response. Experimental complement inhibition revealed that this reaction is secondary to complement activation. Therefore, our data suggests that HD-induced complement, inflammation and coagulation are involved in the increased CV risk of HD patients.

C1-Inhibitor Treatment Decreases Renal Injury in an Established Brain-Dead Rat Model.

BACKGROUND: Kidneys derived from brain-dead (BD) donors have lower graft survival rates compared with kidneys from living donors. Complement activation plays an important role in brain death. The aim of our study was therefore to investigate the effect of C1-inhibitor (C1-INH) on BD-induced renal injury. METHODS: Brain death was induced in rats by inflating a subdurally placed balloon catheter. Thirty minutes after BD, rats were treated with saline, low-dose or high-dose C1-INH. Sham-operated rats served as controls. After 4 hours of brain death, renal function, injury, inflammation, and complement activation were assessed. RESULTS: High-dose C1-INH treatment of BD donors resulted in significantly lower renal gene expression and serum levels of IL-6. Treatment with C1-INH also improved renal function and reduced renal injury, reflected by the significantly lower kidney injury marker 1 gene expression and lower serum levels of lactate dehydrogenase and creatinine. Furthermore, C1-INH effectively reduced complement activation by brain death and significantly increased functional levels. However, C1-INH treatment did not prevent renal cellular influx. CONCLUSIONS: Targeting complement activation after the induction of brain death reduced renal inflammation and improved renal function before transplantation. Therefore, strategies targeting complement activation in human BD donors might clinically improve donor organ viability and renal allograft survival.

Distinct in vitro Complement Activation by Various Intravenous Iron Preparations.

BACKGROUND: Intravenous (IV) iron preparations are widely used in the treatment of anemia in patients undergoing hemodialysis (HD). All IV iron preparations carry a risk of causing hypersensitivity reactions. However, the pathophysiological mechanism is poorly understood. We hypothesize that a relevant number of these reactions are mediated by complement activation, resulting in a pseudo-anaphylactic clinical picture known as complement activation-related pseudo allergy (CARPA). METHODS: First, the in-vitro complement-activating capacity was determined for 5 commonly used IV iron preparations using functional complement assays for the 3 pathways. Additionally, the preparations were tested in an ex-vivo model using the whole blood of healthy volunteers and HD patients. Lastly, in-vivo complement activation was tested for one preparation in HD patients. RESULTS: In the in-vitro assays, iron dextran, and ferric carboxymaltose caused complement activation, which was only possible under alternative pathway conditions. Iron sucrose may interact with complement proteins, but did not activate complement in-vitro. In the ex-vivo assay, iron dextran significantly induced complement activation in the blood of healthy volunteers and HD patients. Furthermore, in the ex-vivo assay, ferric carboxymaltose and iron sucrose only caused significant complement activation in the blood of HD patients. No in-vitro or ex-vivo complement activation was found for ferumoxytol and iron isomaltoside. IV iron therapy with ferric carboxymaltose in HD patients did not lead to significant in-vivo complement activation. CONCLUSION: This study provides evidence that iron dextran and ferric carboxymaltose have complement-activating capacities in-vitro, and hypersensitivity reactions to these drugs could be CARPA-mediated.

Monomeric C-reactive protein inhibits renal cell-directed complement activation mediated by properdin.

Previous studies have shown that complement activation on renal tubular cells is involved in the induction of interstitial fibrosis and cellular injury. Evidence suggests that the tubular cell damage is initiated by the alternative pathway (AP) of complement with properdin having an instrumental role. Properdin is a positive regulator of the AP, which can bind necrotic cells as well as viable proximal tubular epithelial cells (PTECs), inducing complement activation. Various studies have indicated that in the circulation there is an unidentified inhibitor of properdin. We investigated the ability of C-reactive protein (CRP), both in its monomeric (mCRP) and pentameric (pCRP) form, to inhibit AP activation and injury in vitro on renal tubular cells by fluorescent microscopy, ELISA, and flow cytometry. We demonstrated that preincubation of properdin with normal human serum inhibits properdin binding to viable PTECs. We identified mCRP as a factor able to bind to properdin in solution, thereby inhibiting its binding to PTECs. In contrast, pCRP exhibited no such binding and inhibitory effect. Furthermore, mCRP was able to inhibit properdin-directed C3 and C5b-9 deposition on viable PTECs. The inhibitory ability of mCRP was not unique for viable cells but also demonstrated for binding to necrotic Jurkat cells, a target for properdin binding and complement activation. In summary, mCRP is an inhibitor of properdin in both binding to necrotic cells and viable renal cells, regulating complement activation on the cell surface. We propose that mCRP limits amplification of tissue injury by controlling properdin-directed complement activation by damaged tissue and cells.

Mutations in Complement Factor H Impair Alternative Pathway Regulation on Mouse Glomerular Endothelial Cells in Vitro.

Complement factor H (FH) inhibits complement activation and interacts with glomerular endothelium via its complement control protein domains 19 and 20, which also recognize heparan sulfate (HS). Abnormalities in FH are associated with the renal diseases atypical hemolytic uremic syndrome and dense deposit disease and the ocular disease age-related macular degeneration. Although FH systemically controls complement activation, clinical phenotypes selectively manifest in kidneys and eyes, suggesting the presence of tissue-specific determinants of disease development. Recent results imply the importance of tissue-specifically expressed, sulfated glycosaminoglycans (GAGs), like HS, in determining FH binding to and activity on host tissues. Therefore, we investigated which GAGs mediate human FH and recombinant human FH complement control proteins domains 19 and 20 (FH19-20) binding to mouse glomerular endothelial cells (mGEnCs) in ELISA. Furthermore, we evaluated the functional defects of FH19-20 mutants during complement activation by measuring C3b deposition on mGEnCs using flow cytometry. FH and FH19-20 bound dose-dependently to mGEnCs and TNF-α treatment increased binding of both proteins, whereas heparinase digestion and competition with heparin/HS inhibited binding. Furthermore, 2-O-, and 6-O-, but not N-desulfation of heparin, significantly increased the inhibitory effect on FH19-20 binding to mGEnCs. Compared with wild type FH19-20, atypical hemolytic uremic syndrome-associated mutants were less able to compete with FH in normal human serum during complement activation on mGEnCs, confirming their potential glomerular pathogenicity. In conclusion, our study shows that FH and FH19-20 binding to glomerular endothelial cells is differentially mediated by HS but not other GAGs. Furthermore, we describe a novel, patient serum-independent competition assay for pathogenicity screening of FH19-20 mutants.

Zinc supplementation inhibits complement activation in age-related macular degeneration.

UNLABELLED: Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. AMD is a multifactorial disorder but complement-mediated inflammation at the level of the retina plays a pivotal role. Oral zinc supplementation can reduce the progression of AMD but the precise mechanism of this protective effect is as yet unclear. We investigated whether zinc supplementation directly affects the degree of complement activation in AMD and whether there is a relation between serum complement catabolism during zinc administration and the complement factor H (CFH) gene or the Age-Related Maculopathy susceptibility 2 (ARMS2) genotype. In this open-label clinical study, 72 randomly selected AMD patients in various stages of AMD received a daily supplement of 50 mg zinc sulphate and 1 mg cupric sulphate for three months. Serum complement catabolism-defined as the C3d/C3 ratio-was measured at baseline, throughout the three months of supplementation and after discontinuation of zinc administration. Additionally, downstream inhibition of complement catabolism was evaluated by measurement of anaphylatoxin C5a. Furthermore, we investigated the effect of zinc on complement activation in vitro. AMD patients with high levels of complement catabolism at baseline exhibited a steeper decline in serum complement activation (p<0.001) during the three month zinc supplementation period compared to patients with low complement levels. There was no significant association of change in complement catabolism and CFH and ARMS2 genotype. In vitro zinc sulphate directly inhibits complement catabolism in hemolytic assays and membrane attack complex (MAC) deposition on RPE cells. This study provides evidence that daily administration of 50 mg zinc sulphate can inhibit complement catabolism in AMD patients with increased complement activation. This could explain part of the mechanism by which zinc slows AMD progression. TRIAL REGISTRATION: The Netherlands National Trial Register NTR2605.

Impact of the common genetic associations of age-related macular degeneration upon systemic complement component C3d levels.

Age-related macular degeneration (AMD) is a common condition that leads to severe vision loss and dysregulation of the complement system is thought to be associated with the disease. To investigate associations of polymorphisms in AMD susceptibility genes with systemic complement activation, 2655 individuals were genotyped for 32 single nucleotide polymorphisms (SNPs) in or near 23 AMD associated risk genes. Component 3 (C3) and its catabolic fragment C3d were measured in serum and AMD staging was performed using multimodal imaging. The C3d/C3 ratio was calculated and associations with environmental factors, SNPs and various haplotypes of complement factor H (CFH) genes and complement factor B (CFB) genes were analyzed. Linear models were built to measure the influence of genetic variants on the C3d/C3 ratio. The study cohort included 1387 patients with AMD and 1268 controls. Higher C3d/C3 ratios were found for current smoker (p = 0.002), higher age (p = 1.56 × 10(-7)), AMD phenotype (p = 1.15 × 10(-11)) and the two SNPs in the C3 gene rs6795735 (p = 0.04) and rs2230199 (p = 0.04). Lower C3d/C3 ratios were found for diabetes (p = 2.87 × 10(-6)), higher body mass index (p = 1.00 × 10(-13)), the SNPs rs1410996 (p = 0.0001), rs800292 (p = 0.003), rs12144939 (p = 4.60 × 10(-6)) in CFH, rs4151667 (p = 1.01 × 10(-5)) in CFB and individual haplotypes in CFH and CFB. The linear model revealed a corrected R-square of 0.063 including age, smoking status, gender, and genetic polymorphisms explaining 6.3% of the C3d/C3 ratio. After adding the AMD status the corrected R-square was 0.067. In conclusion, none of the evaluated genetic polymorphisms showed an association with increased systemic complement activation apart from two SNPs in the C3 gene. Major genetic and non-genetic factors for AMD were not associated with systemic complement activation.

New insights into the pathogenesis of IgA nephropathy.

IgA nephropathy (IgAN) is the most common diagnosis amongst primary glomerular diseases in most countries where renal biopsies are regularly performed. Only a fraction of these patients is at high risk of losing glomerular filtration rate (GFR) in particular those with high grade proteinuria, uncontrolled hypertension or already impaired GFR at diagnosis, and those with renal scars in the renal biopsy. Genetic modifiers of IgAN onset and/or course are emerging. Spontaneous animal models of IgAN are problematic given considerable species differences between the rodent and human IgA system. However, new transgenic models help to better understand the pathogenesis. A key pathogenetic role appears to be played by underglycated IgA1 as well as autoantibodies to these IgA glycoforms and IgA receptors such as CD89 and transferrin receptor 1. Once IgA and/or IgA-containing immune complexes are deposited or formed in the mesangium, secondary effector mechanisms become important including complement activation, release of mesangial growth factors (in particular platelet-derived growth factor), and finally non-IgAN-specific events that culminate in glomerular and subsequently renal tubulointerstitial scaring. Here, we review these processes and describe potential novel therapeutic targets in IgAN.

Allergy is a protective factor against age-related macular degeneration.

PURPOSE: To investigate the role of allergy on AMD. METHODS: Age-related macular degeneration staging was performed for 3585 individuals (1878 from Cologne, Germany, and 1707 from Nijmegen, The Netherlands). Interviewer-assisted questionnaires were evaluated for the factors smoking, use of corticosteroids, and history of allergy, including causative allergens. Serum complement component C3d and C3 levels were measured and the C3d:C3 ratio was calculated. Associations of allergy with AMD/late AMD were assessed by logistic regression analysis; C3d:C3 ratio was compared between groups. RESULTS: The discovery cohort from Cologne included 864 AMD patients and 1014 controls; 495 patients had late AMD. Positive history of allergy showed strong protective effects on the phenotype AMD (OR 0.52; P = 3.42 × 10(-9)) and late AMD (OR 0.32; P = 2.57 × 10(-13)). Subclassification in allergy-provoking agents showed significant protective effects in all groups. After adjustment for age, sex, smoking, and corticosteroid use, protective effects for AMD (OR 0.75; P = 0.018) and late AMD (OR 0.49; P = 2.87 × 10(-5)) were confirmed. Although the C3d:C3 ratio was higher in AMD/late AMD patients (both P < 0.001), there was no association with allergy in AMD (P = 0.22). The protective effect of allergy on AMD was confirmed in the replication cohort from Nijmegen (P = 0.002 for AMD; P = 0.0001 for late AMD). CONCLUSIONS: Allergy has a protective effect on the development of AMD independent of the provoking allergen, which cannot be explained by complement activation. Further investigations are necessary to elucidate the molecular mechanisms underlying the protective effect of allergy on AMD.

Reversible differentiation of pro- and anti-inflammatory macrophages.

Macrophages (Mφ) represent dynamic cell populations that develop according to the nature of environmental signals. It has been demonstrated that human Mφ can be polarized in vitro into pro-inflammatory (Mφ1) and anti-inflammatory cells (Mφ2) by the lineage-determining factors GM-CSF and M-CSF, respectively. Here we show that polarized Mφ1 and Mφ2 are not an end stage of differentiation but are able to reversibly undergo functional re-differentiation into anti-inflammatory and pro-inflammatory Mφ. GM-CSF-driven Mφ1 exposed to M-CSF for an additional 6 days obtained a Mφ2-like phenotype, inhibited the production of pro-inflammatory cytokine IL-6 and TNF-α, and exhibited a reduced T cell stimulatory capacity. Vice versa, Mφ2 exposed to GM-CSF exhibited a Mφ1-like phenotype with significant lower production of anti-inflammatory cytokine IL-10 and a higher T cell stimulatory activity, and a decreased capacity for phagocytosis of early apoptotic cells. Our data suggest that polarized macrophages are flexible in modulating their immune functions upon environmental changes, i.e., steady-state versus inflammatory conditions. These observations are important for our understanding of the regulatory role of macrophages in tissue homeostasis and disease pathogenesis.

Modulation of activated leukocyte cell adhesion molecule-mediated invasion triggers an innate immune gene response in melanoma.

Activated leukocyte cell adhesion molecule (ALCAM/CD166) is a progression marker of a variety of cancers, including melanoma, and is a marker for mesenchymal stem cells. ALCAM expression triggers matrix metalloproteinase activity and correlates with the transition between superficial melanoma growth and deep dermal invasion in vivo. We previously showed that manipulating ALCAM functionality could both decrease and increase melanoma invasion, depending on the manner by which ALCAM function was altered. How ALCAM exerts these opposing invasive phenotypes remained elusive. In the present study, we analyzed differences in melanoma cell gene expression in two- and three-dimensional cultures as function of ALCAM-mediated adhesion. We identified a cluster of genes highly responsive to ALCAM functionality and relevant for melanoma invasion. This cluster is characterized by known invasion-related genes similar to L1 neuronal cell adhesion molecule and showed a remarkable induction of several innate immune genes. Unexpectedly, we identified major variations in the expression of genes related to an immunological response when modulating ALCAM function, including complement factors C1r and C1s. The expression and function of these proteinases were confirmed in protein assays and in vivo. Together, our results demonstrate a link between ALCAM functionality and the immune transcriptome, and support the assumption that ALCAM-ALCAM interactions could function as a cell signaling complex to promote melanoma tumor invasion.

Low Mannose-Binding Lectin (MBL) genotype is associated with future cardiovascular events in type 2 diabetic South Asians. A prospective cohort study.

BACKGROUND: South Asians have a high burden of type 2 diabetes and vascular complications. Vascular inflammation is considered central in the pathophysiology of atherosclerosis, and the complement system is thought to play an important role. Mannose-Binding Lectin (MBL), which activates the lectin pathway of complement activation, has been introduced as a risk marker of vascular damage. The present study explores the association of MBL levels, genotype and cardiovascular events in type 2 diabetic South Asians. METHODS: We conducted a prospective observational study. A cohort consisting of 168 type 2 diabetic South Asians was followed for a median duration of 7.66 years. At baseline, MBL levels and genotype were determined. The association with future cardiovascular events was assessed by Cox proportional hazard regression. RESULTS: During follow-up, 31 cardiovascular events occurred in 22 subjects (11 men, 11 women). The O/O genotype was significantly associated with the occurrence of cardiovascular events (hazard ratio 3.42, 95%CI 1.24-9.49, P = 0.018). However, log MBL levels were not associated with the occurrence of cardiovascular events (hazard ratio 0.93, 95% CI 0.50-1.73). CONCLUSIONS: In type 2 diabetic South Asians, the O/O MBL genotype is associated with cardiovascular events, although single serum MBL levels are not.