Component 3 Research Articles

Associations between fruit and vegetable variety and low-grade inflammation in Portuguese adolescents from LabMed Physical Activity Study.

The dietary guidelines for the consumption of a variety of fruits and vegetables have been recognized as an important factor for achieving healthy eating patterns to reduce the risk of chronic disease throughout the lifespan. Our aim is to assess the association between fruit and vegetable variety and low-grade inflammation in adolescents. This cross-sectional analysis was conducted with 412 adolescents (ages 14.4±1.7years; 52% girls). The consumption of a variety of fruits and vegetables was assessed with a food-frequency questionnaire, considering the number of individual/category of fruit or vegetable intake at least once month, and categorized into tertiles. Blood samples were collected to determine C-reactive protein (CRP), interleukin-6 (IL-6), complement component 3 (C3), and 4 (C4). We created categories of lower or higher (inflammatory state) for each biomarker, considering sex- and age-adjusted median values. Then, we computed an overall inflammatory score, by adding all points awarded wherein one point was assigned if biomarker was higher or zero if lower, and created categories of 0-1 or 2-4 biomarkers above the median. The odds ratio (OR) and 95% interval confidence (95% CI) were calculated from binary logistic regression to estimate the magnitude of association between fruit and vegetable variety and inflammatory biomarkers. Adolescents with a greater variety of vegetable consumption (≥13 categories/month) had lower odds of having a higher CRP (OR 0.31, 95% CI 0.15-0.64, p trend=0.004) when compared to those with lower variety consumption (≤6 categories/month), independent of vegetable quantity intake. However, a greater variety of fruit consumption (≥12 categories/month) had higher odds of having a higher IL-6 (OR 4.41, 95% CI 1.67-11.71, p trend=0.012), C3 (OR 3.30, 95% CI 1.23-8.86, p trend=0.047), and inflammatory score (OR 4.90, 95% CI 1.62-14.86, p trend=0.017), when compared to those with lower variety consumption (≤9 categories/month), independent of fruit quantity intake, only for girls. The consumption of a variety of vegetables is inversely associated with lower CRP. This finding supports the current dietary guidelines regarding the consumption of a variety of vegetables. The role of fruit variety in low-grade inflammation should be further studied.

The effects of dietary leucine on the growth performances, body composition, metabolic abilities and innate immune responses in black carp Mylopharyngodon piceus

The present study was focused on the growth, body composition, metabolic abilities and innate immune responses in juvenile black carp Mylopharyngodon piceus fed with six levels of dietary leucine (7.3, 12.4, 16.2, 21.9, 28.3 and 34.5 g kg−1) for 9 weeks. Results showed that the highest weight gain (WG) and the lowest feed conversion ratio (FCR) was obtained at 23.5 and 23.9 g kg−1 dietary leucine using second-order polynomial model, respectively. Adequate dietary leucine content (21.9 and 28.3 g kg−1) could significantly up-regulate the expression levels of neuropeptide Y (NPY) and ghrelin (GRL) in the brain of black carp juveniles. The protein efficiency ratio (PER), feed efficiency ratio (FER) and protein deposition ratio (PDR) were also significantly increased by adequate dietary leucine content (21.9 and 28.3 g kg−1) (p < 0.05). Adequate dietary leucine content (21.9 and 28.3 g kg−1) could significantly up-regulate the activities of metabolic enzymes, such as α amylase, trypsin, chymotrypsin and elastase in the liver of Black carp (p < 0.05). However, the activities of alanine transaminase (ALT), aspartate aminotransferase (AST) and leucine aminopeptidase (LAP) were significantly reduced in the fish serum by adequate dietary leucine content (21.9 and 28.3 g kg−1) compared with leucine-deficient diet (7.3 and 12.4 g kg−1). In addition, 21.9 and 28.3 g kg−1 dietary leucine could significantly increase complement component 3 (C3) and C4 contents, lysozyme (LYZ) activities in the serum compared with the leucine-deficient diet (7.3 and 12.4 g kg−1) (p < 0.05). Furthermore, optimal dietary leucine could also significantly up-regulate the mRNA expression levels of LYZ, interferon α (IFN-α), hepcidin (HEPC), natural resistance-associated macrophage protein (NRAMP), C3 and C9 in the blood of juvenile black carp compared with the leucine-deficient diets (7.3 and 12.4 g kg−1) (p < 0.05). In conclusion, these results suggest that adequate dietary leucine (21.9 and 28.3 g kg−1) could increase growth performances, improve metabolic abilities and then enhance non-specific immunities in black carp juveniles.

Effects of partial replacement of fish meal by yeast hydrolysate on complement system and stress resistance in juvenile Jian carp (Cyprinus carpio var. Jian)

A 10-week feeding trial was carried out to investigate the effects of dietary fish meal replacement by yeast hydrolysate (YH) on growth performance, complement system and stress resistance of juvenile Jian carp (Cyprinus carpio var. Jian) (initial average weight 19.44 ± 0.06 g). In the study, there were five groups: one control group was fed with a basal diet (YH0), and four treatment groups were fed with dietary fish meal replaced by 1% YH (YH1), 3% (YH3), 5% (YH5) and 7% (YH7), respectively. Each group had four replicates. At the end of feeding trial, twelve fish from each group (three fish per replicate) were randomly selected for assessing the growth and immunity. Meanwhile, 20 fish per replicate were injected by Aeromonas hydrophila. The results showed that (1) Replacement levels of YH significantly affected the growth of the fish with the highest values of weight gain (WG) occurred in fish fed YH3 diet. However, no significant difference in feed conversion ratios (FCR) was observed among all groups. (2) Pre-stressed plasma lysozyme activity, total protein and albumin contents and complement component 3 (C3) and complement component 4 (C4) levels of fish fed YH3 diet were significantly higher than those of fish fed YH0 diet. However, post-stressed immune parameters of fish in all groups were significantly lower. (3) There was a trend that the expression levels of the complement-related genes (c1r/s-A, c4-1, c3-H1, c5-1, fb/c2-A, mbl-2 and masp) initially increased and then decreased except mbl-2 and masp, with the maximum values observed in fish fed YH3 diet. Before stress, the expression levels of the inflammation-related genes (alp, il-1β and tnf-α) in the hepatopancreas and spleen of fish fed YH1 diet and YH7 diet were significant higher than that of fish fed YH0 diet. After stress, no significant difference in the expression levels of those genes was observed among all groups. These results indicated that FM replacement by YH could improve growth performance, enhance innate immunity, and activate complement via the alternative complement pathway (ACP) and the classical complement pathway (CCP).

Efficacy of Spirulina platensis diet supplements on disease resistance and immune-related gene expression in Cyprinus carpio L. exposed to herbicide atrazine

The present study evaluated the immunotoxicological effects of the herbicide atrazine (ATZ) at sub-lethal concentrations and the potential ameliorative influence of Spirulina platensis (SP) over a sub-chronic exposure period on Cyprinus carpio L., also known as common carp. Common carp was sampled after a 40-days exposure to ATZ (428 μg/L) and SP (1%), individually or in combination to assess the non-specific immune response, changes in mRNA expression of immune-related genes [lysozyme (LYZ), immunoglobulin M (IgM), and complement component 3 (C3)] in the spleen, and inflammatory cytokines (interleukins IL–1ß and IL–10) in the head kidney using real-time PCR. Additionally, disease resistance to Aeromonas sobria was evaluated. The results revealed that ATZ exposure caused a significant decline in most of the hematological variables, lymphocyte viability, and lysozyme and bactericidal activity. Moreover, ATZ increased the susceptibility to disease, reflected by a significantly lower post-challenge survival rate of the carp. ATZ may induce dysregulated expression of immune-related genes leading to downregulation of mRNA levels of IgM and LYZ in the spleen. However, expression of C3 remained unaffected. Of the cytokine-related genes examined, IL–1B was up-regulated in the head kidney. In contrast, the expression of IL–10 gene was down-regulated in the ATZ-exposed group. The SP supplementation resulted in a significant improvement in most indices; however, these values did not match with that of the controls. These results may conclude that ATZ affects both innate and adaptive immune responses through the negative transcriptional effect on genes involved in immunity and also due to the inflammation of the immune organs. In addition, dietary supplements with SP could be useful for modulation of the immunity in response to ATZ exposure, thereby presenting a promising feed additive for carps in aquaculture.

Increase in complement iC3b is associated with anti-inflammatory cytokine expression during late pregnancy in mice.

Immunological tolerance between fetal allograft and mother is crucial for pregnancy establishment and maintenance; however, these mechanisms particularly those during the latter part of pregnancy have not been definitively elucidated. The aim of this study was to examine the presence and potential function of innate immunity characteristic to the middle to late pregnancy. We first characterized up-regulated proteins in decidua from day 11 pregnant (P11) mice using 2D-PAGE, followed by MALDI-TOF/MS analysis. These analyses identified increased complement component 3 (C3) and its derivatives in P11 decidua. We then found that in the decidual tissues, C3 mRNA increased on P15 and remained high on P19. C3 is converted to C3b and then iC3b by complement component factor I (Cfi) and complement receptor 1-like protein (Crry), both of which were present in P19 placentas. In addition, iC3b proteins and its receptor CR3 (Cd11b/Cd18) in decidual and placental tissues increased toward the latter phase of pregnancy. Moreover, CR3 subunit CD11b protein was predominantly localized to spongiotrophoblast layer in the P19 placenta. Because iC3b is known to induce anti-inflammatory cytokine production, the analysis was extended to examine changes in pro- and anti-inflammatory cytokines, Il12, Il10, and Tgfb1. Il12 expression decreased in P15 and P19 placenta, while high mRNA expression of Il10 and Tgfb1 was found in P19 placental tissues. Furthermore, placental Il10 and Tgfb1 mRNAs were down-regulated when pregnant mice were treated with an anti-C3 antibody, detecting C3, C3b and iC3b. These results indicated that C3 derivatives, in particular, iC3b and its receptor CR3 were up-regulated at the fetal-maternal interface, and suggest that iC3b may regulate the placental expression of anti-inflammatory cytokines, IL10 and TGFB1, during the latter phase of pregnancy.

Complement Component C3 Variant (R102G) and the Risk of Neovascular Age-Related Macular Degeneration in a Tunisian Population.

Purpose To explore the association between the polymorphism (S/F) p.R102G in the complement component 3 (C3) gene and age-related macular degeneration (AMD) in a Tunisian population. Methods The molecular study was performed by polymerase chain reaction using sequence-specific primers (PCR-SSP) in 207 control subjects free of any eye disease (fundus normal) and 145 patients with exudative AMD. The CH50 activity and quantification of C3 and C4 have been made by technical home method and nephelometry, respectively. Results The prevalence of C3 GG genotype polymorphism was significantly higher in AMD patients compared to controls (OR: 2.41, IC 95% [1.90-3.05], p = 0.0007). However, no correlation was found between this allelic variant and the type of neovascularization. Similarly, there is no association between this polymorphism and the presence of functional and/or quantitative hypocomplementemia. Conclusions The C3 GG genotype of the gene could be a susceptibility factor for AMD in the Tunisian population. However, it does not seem to influence the clinical profile of the disease.

Daily Moderate Exercise Is Beneficial and Social Stress Is Detrimental to Disease Pathology in Murine Lupus Nephritis.

Daily moderate exercise (DME) and stress management are underemphasized in the care of patients with lupus nephritis (LN) due to a poor comprehensive understanding of their potential roles in controlling the inflammatory response. To investigate these effects on murine LN, disease progression was monitored with either DME or social disruption stress (SDR) induction in NZM2410/J mice, which spontaneously develop severe, early-onset LN. SDR of previously established social hierarchies was performed daily for 6 days and DME consisted of treadmill walking (8.5 m/min for 45 min/day). SDR significantly enhanced kidney disease when compared to age-matched, randomly selected control counterparts, as measured by histopathological analysis of H&E staining and immunohistochemistry for complement component 3 (C3) and IgG complex deposition. Conversely, while 88% of non-exercised mice displayed significant renal damage by 43 weeks of age, this was reduced to 45% with exercise. DME also reduced histopathology in kidney tissue and significantly decreased deposits of C3 and IgG complexes. Further examination of renal infiltrates revealed a macrophage-mediated inflammatory response that was significantly induced with SDR and suppressed with DME, which also correlated with expression of inflammatory mediators. Specifically, SDR induced IL-6, TNF-α, IL-1β, and MCP-1, while DME suppressed IL-6, TNF-α, IL-10, CXCL1, and anti-dsDNA autoantibodies. These data demonstrate that psychological stressors and DME have significant, but opposing effects on the chronic inflammation associated with LN; thus identifying and characterizing stress reduction and a daily regimen of physical activity as potential adjunct therapies to complement pharmacological intervention in the management of autoimmune disorders, including LN.

Metastasis: Breaching barriers.

Boire et al. describe a mechanism by which cancer cells can survive and grow in the leptomeninges of the brain by upregulating complement component 3 (C3) to disrupt blood–cerebrospinal fluid barrier function, allowing entry of growth-promoting factors.

Expression of a novel complement C3 gene in the razor clam Sinonovacula constricta and its role in innate immune response and hemolysis

Complement component 3 (C3) is a core component of the complement system, and directly participates in immune regulation and immune defense. Isoforms of C3 have been reported in several species of vertebrate, but invertebrates, and more specifically clams, have been less well studied. An isoform of C3, named ScC3-2, was identified in Sinonovacula constricta (Chinese razor clam). ScC3-2 included eight conserved regions, a thioester bond and two predicted junction sites (α-β and α-γ). The gene was expressed in the liver, gill, foot, hemolymph, mantle, gonad and siphon tissues. The gene was significantly upregulated in umbo larvae, suggesting that initial larval immunity may develop in umbo larvae. Moreover, the ScC3-2 mRNA expression patterns after challenge with Vibrio parahemolyticus and Micrococcus lysodeikticus exhibited an obvious upregulation at 8 h in the hemolymph and at 4 h in the liver, respectively. Furthermore, ScC3-2 showed effective membrane rupture of heterologous rabbit erythrocytes. The ScC3-2 protein was located on the surface of the cells during the process of hemolysis. After a comparative analysis, we suggest that the major structure and function of ScC3 and ScC3-2 are analogous. Our findings suggest that ScC3-2 plays an important immune function, and an intricate complement response may exist in S. constricta.

Metastatic cells feed off a complement.

Cancer Cancer patients with metastases in the cerebrospinal fluid (CSF) have a poor prognosis. How cancer cells survive in the CSF has been an enigma because this microenvironment is devoid of mitogens and nutrients required for cell growth. Studying mice and patient samples, Boire et al. show that cancer cells metastasizing to the CSF overexpress a protein called complement component 3 (C3). C3 activates a specific receptor in the choroid plexus epithelium, a barrier system in the brain that prevents cells and molecules in the blood from entering the CSF. This activation disrupts the blood-CSF barrier, allowing circulating growth factors into the CSF. A drug that blocks this activation suppressed metastasis to the CSF in mice. Cell 168 , 1101 (2017).

Complement Component 3 Adapts the Cerebrospinal Fluid for Leptomeningeal Metastasis

We molecularly dissected leptomeningeal metastasis, or spread of cancer to the cerebrospinal fluid (CSF), which is a frequent and fatal condition mediated by unknown mechanisms. We selected lung and breast cancer cell lines for the ability to infiltrate and grow in CSF, a remarkably acellular, mitogen-poor metastasis microenvironment. Complement component 3 (C3) was upregulated in four leptomeningeal metastatic models and proved necessary for cancer growth within the leptomeningeal space. In human disease, cancer cells within the CSF produced C3 in correlation with clinical course. C3 expression in primary tumors was predictive of leptomeningeal relapse. Mechanistically, we found that cancer-cell-derived C3 activates the C3a receptor in the choroid plexus epithelium to disrupt the blood-CSF barrier. This effect allows plasma components, including amphiregulin, and other mitogens toenter the CSF and promote cancer cell growth. Pharmacologic interference with C3 signaling proved therapeutically beneficial in suppressing leptomeningeal metastasis in these preclinical models.

Hyperuricemia as an independent risk factor for major cardiovascular events: a 10-year cohort study from Southern Italy.

To date, whether hyperuricemia may represent a marker or an independent risk factor for cardiovascular disease remains unclear. This study aimed at assessing the role of hyperuricemia in the onset of major cardiovascular events (MACE). Baseline clinical data were collected through a 1998/1999 longitudinal survey as part of the larger Valle dell'Irno Prevenzione Project. Ten years later, MACE incidence was evaluated. A total of 1175 patients (50% men, aged 25-74 years) completed the study. At least one MACE was reported by 135 patients, whose mean uric acid values were significantly higher compared with patients without events (6.0 ± 4.8 and 4.6 ± 4.0 mg/dl, respectively; P < 0.01). Patients with uric acid values of at least 6 mg/dl (prevalence of 14.6%) had significantly lower levels of high-density lipoprotein cholesterol and increased values of BMI, blood pressure (BP), cholesterol, triglycerides, white blood cells, complement component 3 (C3) and creatinine. After subgrouping patients in tertiles and considering the first one as reference [odds ratio (OR): 1], the OR (95% confidence interval) was 1.44 (0.7-2.9) in the second and 2.2 (1.3-3.5) in the third tertile, respectively. Confounder-adjusted stepwise linear regression revealed uric acid, age, creatinine, glucose and systolic BP as independent predictors of MACE. Diastolic BP and creatinine were independently correlated with uric acid in the entire population, diastolic BP only in men and BMI, creatinine, age and C3 in women. Hyperuricemia was shown to be a strong independent risk factor for MACE and should be included in cardiovascular prevention strategies. Whether hypouricemic drugs can decrease cardiovascular disease risk warrants further studies.

Surface complement C3 fragments and cellular binding of microparticles in patients with SLE

ObjectivesTo examine microparticles (MPs) from patients with SLE and healthy controls (HCs) by determining the cellular origin of the MPs, quantifying attached fragments of complement component 3 (C3) and assessing...

Complement component 3 (C3) as a biomarker for insulin resistance after bariatric surgery

ObjectivesSince complement system has been recently associated with metabolic and cardiovascular diseases, and closely related to insulin resistance, we investigated the association of plasma complement factor 3 (C3) and factor 4 (C4) with insulin sensibility and weight loss after bariatric surgery. MethodsSerum levels of C3, C4, total cholesterol, triacylglycerol, HDL-cholesterol, LDL-cholesterol and homeostatic model assessment (HOMA-IR) measurements were assessed in morbidly obese patients before and after bariatric surgery, including a 6-month follow-up period, as well as a comparison with a lean group. ResultsWeight loss decreased body mass index (BMI), serum triacylglycerol, and increased serum HDL-cholesterol and insulin sensitivity, as expected. C3 and C4 were significantly higher in obese individuals when compared to lean subjects (p<0.001). In addition, C3 and C4 positively correlated with BMI and HOMA-IR, however, only C3 were significantly decreased 6months after surgery. ConclusionC3 was strongly associated with insulin sensitivity after bariatric surgery,

Redox dysregulation, immuno-inflammatory alterations and genetic variants of BDNF and MMP-9 in schizophrenia: Pathophysiological and phenotypic implications

BackgroundAlthough a clear mechanism underlying the pathophysiology of schizophrenia (SZ) remains elusive, oxidative stress, inflammatory syndrome and immune activation have become an attractive hypothesis for explaining the pathophysiology of SZ. Data from prior studies on the role of matrix metalloproteinase 9 (MMP-9) and brain-derived neurotrophic factor (BDNF) single nucleotide polymorphisms (SNPs) in SZ are contradictory. We aimed to investigate whether oxidative stress, inflammatory and immune activation markers as well as MMP-9 levels may be implicated in SZ pathogenesis. The association of MMP-9 and BDNF SNPs with the clinical expression of SZ was examined. Subjects and methodsNinety-four subjects were recruited, including 44 SZ patients and 50 healthy controls. Serum levels of thiobarbituric acid reactive substances (TBARS), protein carbonyl content (PCC), nitrite, C-reactive protein (CRP), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), Beta-2 microglobulin (Β2M), complement component 3 (C3), C4 and MMP-9 were measured. The MMP-9 −1562C>T and BDNF196G>A SNPs were genotyped using polymerase chain reaction-restriction fragment length polymorphism assay. Psychopathology was assessed using the positive and negative syndrome scale (PANSS). ResultsSZ patients showed significantly higher TBARS, PCC, nitrite, CRP, IL-6, TNF-α, Β2M, C3 and MMP-9 levels than controls. In distinguishing SZ patients from healthy controls, CRP and MMP-9 yielded similar discriminatory performance, and both perform better than IL-6, Β2M, C3, nitrite, TBARS, PCC, TNF-α and C4. The MMP-9 −1562C>T SNP genotypes distribution didn't differ significantly between controls and SZ patients. As compared to controls, SZ patients harbor a significantly higher frequency of the BDNF196GG genotype and a lower frequency of the BDNF196GA/AA genotype. Patients carrying the MMP-9 −1562CC or BDNF196GG genotype revealed a significantly higher PANSS than those carrying MMP-9 −1562CT/TT or BDNF196GA/AA genotype. Male gender and the MMP-9 −1562CC genotype were identified as independent predictive factors for higher PANSS. ConclusionsRedox dysregulation and alterations in the immuno-inflammatory pathways are major culprits in the pathogenesis of SZ. MMP-9 and BDNF SNPs are associated with the clinical phenotype of SZ and, thus, may be a useful marker predicting the phenotypic expression and prognosis of SZ patients.

Serum Proteomic Profiling of Obsessive-Compulsive Disorder, Washing Subtype: A Preliminary Study.

Introduction:Obsessive-Compulsive Disorder (OCD) is a disabling mental condition that its proteomic profiling is not yet investigated. Proteomics is a valuable tool to discover biomarker approaches. It can be helpful to detect protein expression changes in complex disorders such as OCD.Methods:Here, by the application of 2D gel electrophoresis (2DE), a pilot study of serum proteome profile of females with washing subtype of OCD was performed. Serum samples were obtained from females with washing subtype of OCD. Following the protein extraction from the serum with acetone perception, the samples were subjected to 2DE for separation based on pI and molecular weight (MW) with triple replications. Finally, the protein spots were visualized using Coomassie blue staining method and analyzed by Progenesis SameSpots software. Furthermore, protein-protein interaction (PPI) network analysis was handled by the application of Cytoscape software.Results:The results suggested that 41 matched spots demonstrated significant expression alterations among which 5 proteins including immunoglobulin heavy constant alpha-1 (IGHA1), apolipoprotein A-4 (APOA4), haptoglobin (HP), protein α-1-antitrypsin (SERPINA1), and component 3 (C3) were identified by database query. Additionally, PPI network analysis indicated the central role of SERPINA1 and C3 in the network integrity. However, albumin (ALB), amyloid precursor protein (APP), and protein α-1-antitrypsin (APOA1) proteins were important in OCD PPI network as well. The identified proteins were related to 3 processes: acute-phase response, hydrogen peroxide catabolic process, and regulation of triglyceride metabolic process.Conclusion:It was concluded that these proteins may have a fundamental role in OCD pathogenesis. Moreover, the dysregulation of inflammatory and antioxidant systems in OCD risk was suggested by the current study. However, evaluation of bigger sample sizes and application of mass spectrometry are essential requirements to confirm this preliminary evaluation.

Effect of a Single Dose of Vitamin D3 on Postprandial Arterial Stiffness and Inflammation in Vitamin D-Deficient Women.

Cholecalciferol (vitamin D3) improves vascular function and inflammation, potentially providing an explanation for the proposed cardiovascular protection of vitamin D. We investigated whether cholecalciferol supplementation reduces postprandial arterial dysfunction and inflammation. Randomized, 1:1, double-blind trial. Diabetes and Vascular Center, Franciscus Gasthuis, Rotterdam, The Netherlands. Twenty-four healthy, premenopausal, overweight or obese, vitamin D-deficient women. A single high (300,000 IU) or low dose (75,000 IU) of cholecalciferol. The effect of low- and high-dose cholecalciferol on postprandial leukocyte activation markers, pulse wave velocity (PWV), and augmentation index (AIx) during an oral fat loading test, expressed as area under the curve (AUC). High- and low-dose supplementation increased vitamin D by 163% ± 134% (P < 0.001) and 66% ± 59% (P < 0.001), respectively. Monocyte CD11b-AUC slightly increased after low but not high dose (6% ± 2%, P = 0.012, and 4% ± 1%, P = 0.339, respectively). There were no significant effects on postprandial PWV or AIx by high- or low-dose vitamin D. Fasting complement component 3 (C3) levels decreased by 5.9% (P = 0.004) in the high-dose group and by 4.0% (P = 0.018) in the low-dose group. A single dose of vitamin D does not seem to reduce arterial stiffness and leukocyte activation in overweight, vitamin D-deficient women. Vitamin D may decrease fasting C3. Possibly, higher vitamin D concentrations may be needed to decrease inflammation and improve vascular function in overweight or obese vitamin D-deficient women.

C3 Modulates RBC Antigen to Negatively Regulate Antibody Response

Background:Red blood cell (RBC) transfusion can result in the development of alloantibodies that can make it difficult to find compatible RBCs for future transfusions and increase the risk of hemolytic transfusion reactions. Despite the consequences of RBC alloimmunization, the factors that regulate this process remain relatively unknown. Recent studies suggest that complement deposition on an antigen surface can significantly enhance the immune response to foreign antigen. As many anti-RBC alloantibodies fix complement and RBCs otherwise lack known adjuvants, early antibody-mediated complement deposition may serve as a key regulator that enhances antibody production. To test this, we employed the KEL RBC model system, which employs RBCs that transgenically express the human KEL antigen specifically on RBCs (KEL RBCs). Using this system, we examined the immunological consequence of KEL RBC exposure following transfusion into C57BL/6 wild-type (WT) or complement component 3 (C3) knockout (KO) recipients.Methods: KEL RBCs were transfused into WT or C3 KO recipients, followed by serum collection on days 3, 5, 7, 14, and 21 post-transfusion. Antibody development in WT or C3 KO recipients was examined by flow crossmatch, where serum was incubated with KEL RBCs followed by antibody detection with fluorescently-tagged secondary anti-IgM and anti-IgG antibodies using flow cytometry. To determine the impact of complement deposition on the level of detectable antigen on the RBC surface, RBCs were labeled with the lipophilic dye, DiI, prior to transfusion and then sampled 1, 2, 3, 5, 7 and 9 days post-transfusion. The level of detectable KEL antigen, complement deposition, KEL RBC survival and antibody bound to the RBC surface was measured by flow cytometry. To examine the effect of complement deposition on the level of KEL protein in the RBC membrane post-transfusion, RBCs stroma was isolated at various time points post transfusion, followed by western blot analysis for the KEL protein.Results: While KEL RBCs induced robust anti-KEL antibody formation and C3 deposition in WT recipients, similar exposure to KEL RBCs in C3 KO recipients actually resulted in an unexpected increase in IgM and IgG anti-KEL antibodies when compared to WT recipients. To determine the consequence of C3 deposition, we examined the potential impact of antibody engagement and complement fixation on KEL antigen levels. Consistent with a potential role for complement in directly impacting KEL antigen availability to the immune system, KEL RBCs transferred into WT recipients experienced a decrease in the level of detectable KEL antigen over time that paralleled the development of anti-KEL antibodies and C3 deposition. In contrast, C3 KO recipients failed to experience the same degree of KEL antigen reduction despite the development of significant anti-KEL antibodies over this same time period. Western blot analysis of RBCs post-transfusion revealed that loss of detectable KEL antigen on the RBC surface paralleled a complete lack of detectable KEL antigen in RBC membranes, indicating that C3 may actually facilitate the removal of KEL from the RBC surface.Conclusion: These results suggest an unexpected role for C3 in negatively regulating antibody responses following RBC transfusion. The impact of C3 on the developing alloantibody response strongly suggests that C3-mediated loss of antigen over time likely reduces antigen availability to the immune system, thereby facilitating the inhibition of antibody production over time. These results not only provide novel insight into potential impact of antigen modulation on the development of an immune response to a RBC alloantigen, but also suggest a completely unexpected role for complement in negatively regulating alloantibody production. In doing so, these results suggest that unique differences in complement activity and overall activation following RBC alloantigen exposure between individuals may represent a previously unrecognized factor that influences alloantibody formation following RBC transfusion. DisclosuresNo relevant conflicts of interest to declare.

Three Cases of Patients with Complement Regulatory Factor Genetic Mutations and Acquired Thrombotic Thrombocytopenic Purpura (TTP)

Acquired TTP is a thrombotic microangiopathy (TMA) associated with deficiency of the vonWillebrandfactor cleaving protein ADAMTS13. Atypical hemolytic uremic syndrome (aHUS) is a TMA with a genetic predisposition todysregulationin the alternative complement pathway. Causes ofaHUSinclude mutations in CD46, complement factor I (CFI), complement factor B, complement component 3 (C3), complement factor H-related 5 (CFHR5), andthrombomodulin(THBD) or secondary to Complement factor H (CFH) autoantibodies.Treatment of these two TMAs differs. Whereas therapeutic plasma exchange (TPE), steroids, rituximab, cyclophosphamide and/or vincristine are used in TTP,aHUSrequires controlling unrestrained complement activation with eculizumab, an antibody preventing cleavage of C5.Testing for genetic predisposition to complementdysregulationis not commonly considered in TTP. To our knowledge, only one case report has described such a genetic association (Ref: PMID# 22409250). Here we describe 3 new cases with concurrent TTP,aHUSand genetic changes in complement genes.Case 1: 24y.o. African American (AA) man with XXY syndrome presented with bloody diarrhea, diffuse abdominal pain and renal dysfunction and a presumptive diagnosis ofaHUS. He was treated with eculizumab prior to transfer to our institution. On arrival to us, ADAMTS13 was low and inhibitor elevated. He initially responded to TPE but renal failure progressed andaHUS gene testing was ordered revealing a CFHR1-CFHR3 homologous deletion. Eculizumab was restarted but increased ADAMTS13 inhibitor required continued TPE, rituximab and vincristine; eculizumab was held until ADAMTS13 inhibitor was no longer detectable. At 8 months he continues on eculizumab with undetectable ADAMTS13 inhibitor and normal renal function, platelets and LDH (Figure).Case 2: 35y.o. AA woman presented with complaints of slurred speech, left-sided numbness, and thrombocytopenia, but normal renal function. Testing revealed ADAMTS13 <5%, presence of inhibitor (2.2 BU; normal <0.4 BU) and normal complement levels. TPE and steroids were initiated, with rituximab and vincristine added due to inhibitor boosting after initial response. Despite continued treatment, clinical/laboratory parameters did not improve, C3 and C4 levels declined, andaHUS genetic mutation analysis was ordered. This revealed heterozygous missense variants in exon 11 (c.1246A>C, plle416Leu) and exon 7 (c.1135G>C,p.Val379Leu). Once platelets recovered, we treated her with one dose of eculizumab; however, subsequently her plateletsdropped as she had not yet cleared the inhibitor. We then resumed plasma exchange and added eculizumab to her treatment at a later point (figure). Her laboratory parameters have normalized and she is on eculizumab maintenance.Case 3: 67y.o. AA woman presented with thrombocytopenia, microangiopathic hemolytic anemia, and left arm weakness. Renal function was mildly affected, with GFR ~ 30 mL/min/1.73m2. ADAMTS13 was <5% and inhibitor titer was 1.1 BU. TPE and steroids were initiated on admission, followed by rituximab, but due to lack of response after 14 days, she was given eculizumab. She had an immediate rise in platelet count after one dose and a sustained rise after the second dose. Her CBC and hemolysis parameters normalized after 4 doses. She continued treatment with eculizumab for a total of 6 months after which she opted to stop the drug. Within a month of discontinuing treatment, she developed a deep venous thrombosis and received anticoagulation for 6 months. She is now 2 years out of stopping eculizumab and has a normal CBC and LDH. Subsequent gene testing revealed the same CFHR1-CFHR3 homologous deletion identified in case 1. (Figure)Conclusion:We found treatment of TTP first allowed control of complementdysregulation, while treatment ofaHUSfirst did not allow control of TTP. This suggests that low ADAMTS13 levels may be the initiating factor in uncontrolled complement activation inaHUS. These cases suggest the need to test forgermlinemutations leading to abnormal complement activation in patients with refractory TTP, as they may have a dual diagnosis of both TTP andaHUSas our patients did. We therefore suggest treating the underlying TTP first, monitoring the ADAMTS13 activity and inhibitor levels to assess the clearance of the inhibitor, and initiating treatment with eculizumab once the inhibitor has cleared. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Serum biomarkers and changes in clinical/MRI evidence of golimumab-treated patients with ankylosing spondylitis: results of the randomized, placebo-controlled GO-RAISE study.

BackgroundIn the present study, we evaluated relationships between serum biomarkers and clinical/magnetic resonance imaging (MRI) findings in golimumab-treated patients with ankylosing spondylitis.MethodsIn the GO-RAISE study, 356 patients with ankylosing spondylitis randomly received either placebo (n = 78) or golimumab 50 mg or 100 mg (n = 278) injections every 4 weeks through week 24 (placebo-controlled); patients continuing GO-RAISE received golimumab through week 252. Up to 139/125 patients had sera collected for biomarkers/serial spine MRI scans (sagittal plane, 1.5-T scanner). Two blinded readers employed modified ankylosing spondylitis spine magnetic resonance imaging score for activity (ASspiMRI-a) and ankylosing spondylitis spine magnetic resonance imaging score for chronicity. Spearman correlations (r s) were assessed between serum biomarkers (n = 73) and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI), C-reactive-protein (CRP)-based Ankylosing Spondylitis Disease Activity Score (ASDAS), modified Stokes Ankylosing Spondylitis Spine Score (mSASSS), and ASspiMRI scores. Serum biomarkers predicting postbaseline spinal fatty lesion development and inflammation were analyzed by logistic regression.ResultsSignificant, moderately strong correlations were observed between baseline inflammatory markers interleukin (IL)-6, intracellular adhesion molecule-1, complement component 3 (C3), CRP, haptoglobin, and serum amyloid-P and baseline ASDAS (r s = 0.39–0.66, p ≤ 0.01). Only baseline leptin significantly correlated with ASDAS improvement at week 104 (r s = 0.55, p = 0.040), and only baseline IL-6 significantly predicted mSASSS week 104 change (β = 0.236, SE = 0.073, p = 0.002, model R 2 = 0.093). By logistic regression, baseline leptin, C3, and tissue inhibitor of metalloproteinase (TIMP)-1 correlated with new fatty lesions per spinal MRI at week 14 and week 104 (both p < 0.01). Changes in serum C3 levels at week 4 (r s = 0.55, p = 0.001) and week 14 (r s = 0.49, p = 0.040) significantly correlated with BASDAI improvement at week 14. Baseline IL-6 and TIMP-1 (r s = −0.63, −0.67; p < 0.05) and reductions at week 4 in IL-6 (r s = 0.61, p < 0.05) and C3 (r s = 0.72; p < 0.05) significantly correlated with week 14 ASspiMRI-a improvement.ConclusionsExtensive serum biomarker multiparametric analyses in golimumab-treated patients with ankylosing spondylitis demonstrated few correlations with disease activity or MRI changes; IL-6 weakly correlated with radiographic progression.Trial registrationClinicalTrials.gov identifier: NCT00265083. Registered on 12 December 2005.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1200-1) contains supplementary material, which is available to authorized users.