Plasma C3 Research Articles

Using genetics to explore complement C5 as a druggable protein in periodontitis.

An excessively activated or dysregulated complement system has been proven to be a vital contributor to the pathogenesis of periodontitis. It has been previously hypothesized that inhibiting the activity of complement component C5 by targeting the C5a receptor is a powerful candidate for treating periodontitis. Here, we apply the drug target instrumental variable (IV) approach to investigate the therapeutic effect of genetically proxied inhibition of C5 on periodontitis. In our primary analysis, we used 26 independent 'cis' single nucleotide polymorphisms as IVs from the vicinity of the encoding locus of C5 that are associated with plasma C5 levels. In a secondary analysis, we assess the validity of our primary findings, exploring the involvement of alternative downstream biomarkers, interleukin 17 (IL-17), interleukin 1β (IL-1β), and tumor necrosis factor (TNF). Summary statistics of plasma levels (C5, IL-17, IL-1β, and TNF) were obtained from a genome-wide association study (GWAS) of 35,559 European descent individuals. We extracted association statistics from a GWAS of 17,353 clinical periodontitis cases and 28,210 European controls. Wald ratios were combined using inverse-variance weighted meta-analysis. In our primary approach, inhibiting C5 reduced the risk of periodontitis (Odds ratio 0.89 per 1 standard deviation reduction in C5; 95% confidence Interval 0.80-0.98, p value=0.022). Our secondary analysis suggests an involvement of IL-17 within the potential causal pathway, but was inconclusive for other biomarkers. The findings from our study suggest that C5 inhibition may reduce the risk of periodontitis, prioritizing C5 inhibitors as a potential adjunctive therapeutic intervention in this disease.

Reversal of insulin resistance in people with obesity by lifestyle-induced weight loss does not impact the proportion of circulating 12α-hydroxylated bile acids.

Bile acids (BAs) are implicated in the pathogenesis of several metabolic syndrome-related diseases, including insulin resistance (IR) and type 2 diabetes (T2D). It has been reported that IR and T2D are associated with an increased ratio of 12α/non-12α-hydroxylated BAs in the circulating BA pool. It is, however, unknown whether the improvement of insulin sensitivity inversely affects BA composition in humans. Therefore, we assessed whether lifestyle-induced weight loss induces changes in BA metabolism in people with obesity, with or without T2D, and if these changes are associated with metabolic parameters. Individual BAs and C4 were quantified by ultra-high-performance liquid chromatography-tandem mass spectrometry in plasma samples collected from two cohorts of people with obesity (OB) and with T2D and obesity (T2D), before and after a lifestyle intervention. Lifestyle-induced weight loss improved glycaemic control in both cohorts, with plasma BA concentrations not affected by the lifestyle interventions. The ratio of 12α/non-12α-hydroxylated BAs remained unchanged in OB (p = .178) and even slightly increased upon intervention in T2D (p = .0147). Plasma C4 levels were unaffected in OB participants (p = .20) but significantly reduced in T2D after intervention (p = .0003). There were no significant correlations between the ratio of 12α/non-12α-hydroxylated BAs and glucose, insulin, or homeostatic model assessment-IR, nor in plasma triglycerides, low-density lipoprotein cholesterol, lipoprotein (a) in the T2D cohort. Lifestyle-induced weight loss did improve glycaemic control but did not affect BA concentrations. Improvements in insulin sensitivity were not associated with changes in BA parameters in people with obesity, with or without T2D.

Mannan-Binding Lectin Is Associated with Inflammation and Kidney Damage in a Mouse Model of Type 2 Diabetes.

Autoreactivity of the complement system may escalate the development of diabetic nephropathy. We used the BTBR OB mouse model of type 2 diabetes to investigate the role of the complement factor mannan-binding lectin (MBL) in diabetic nephropathy. Female BTBR OB mice (n = 30) and BTBR non-diabetic WT mice (n = 30) were included. Plasma samples (weeks 12 and 21) and urine samples (week 19) were analyzed for MBL, C3, C3-fragments, SAA3, and markers for renal function. Renal tissue sections were analyzed for fibrosis, inflammation, and complement deposition. The renal cortex was analyzed for gene expression (complement, inflammation, and fibrosis), and isolated glomerular cells were investigated for MBL protein. Human vascular endothelial cells cultured under normo- and hyperglycemic conditions were analyzed by flow cytometry. We found that the OB mice had elevated plasma and urine concentrations of MBL-C (p < 0.0001 and p < 0.001, respectively) and higher plasma C3 levels (p < 0.001) compared to WT mice. Renal cryosections from OB mice showed increased MBL-C and C4 deposition in the glomeruli and increased macrophage infiltration (p = 0.002). Isolated glomeruli revealed significantly higher MBL protein levels (p < 0.001) compared to the OB and WT mice, and no renal MBL expression was detected. We report that chronic inflammation plays an important role in the development of DN through the binding of MBL to hyperglycemia-exposed renal cells.

Partial liver resection alters the bile salt-FGF19 axis in patients with perihilar cholangiocarcinoma: Implications for liver regeneration.

Extended liver resection is the only treatment option for perihilar cholangiocarcinoma (pCCA). Bile salts and the gut hormone FGF19, both promoters of liver regeneration (LR), have not been investigated in patients undergoing resection for pCCA. We aimed to evaluate the bile salt-FGF19 axis perioperatively in pCCA and study its effects on LR. Plasma bile salts, FGF19, and C4 (bile salt synthesis marker) were assessed in patients with pCCA and controls (colorectal liver metastases), before and after resection on postoperative days (PODs) 1, 3, and 7. Hepatic bile salts were determined in intraoperative liver biopsies. Partial liver resection in pCCA elicited a sharp decline in bile salt and FGF19 plasma levels on POD 1 and remained low thereafter, unlike in controls, where bile salts rose gradually. Preoperatively, suppressed C4 in pCCA normalized postoperatively to levels similar to those in the controls. The remnant liver volume and postoperative bilirubin levels were negatively associated with postoperative C4 levels. Furthermore, patients who developed postoperative liver failure had nearly undetectable C4 levels on POD 7. Hepatic bile salts strongly predicted hyperbilirubinemia on POD 7 in both groups. Finally, postoperative bile salt levels on day 7 were an independent predictor of LR. Partial liver resection alters the bile salt-FGF19 axis, but its derailment is unrelated to LR in pCCA. Postoperative monitoring of circulating bile salts and their production may be useful for monitoring LR.

#1940 Plasma complement activation profile in kidney transplant recipients with IgAN supports the importance of the complement alternative pathway in IgAN

Abstract Background and Aims The alternative complement and lectin pathways are key players in IgA nephropathy (IgAN). However, the precise dynamics of complement activation in IgAN remain poorly understood. Non-invasive complement biomarkers are urgently needed in view of the evolving landscape of complement-targeting therapies under evaluation in IgAN. Method We analyzed plasma complement activation biomarkers in a cohort of 87 kidney transplant recipients (KTR) with IgAN and 30 controls consisting of autosomal dominant polycystic kidney disease (ADPKD) KTR (NCT05234463, DC-2013-1990). Patients were excluded if 1) they had a biological inflammatory syndrome at the time of sampling or 2) they had experienced a cellular/humoral rejection in the year prior to or after sampling. We simultaneously measured a large panel of complement activation and regulation fragments (C3a, C5a, C4a, sC5b-9, Ba, Bb) and intact proteins (C3, C4, FI, FH, properdin) in plasma using multiplex ELISA (Quidel Ortho®, A900), nephelometry and in-house ELISA. Results There was no consumption of C3 and C4 proteins in IgAN patients. However, C3a and Bb concentrations were elevated in IgAN patients compared to ADPKD patients (161 [98-227] vs. 102 [66-172] ng/mL, p = 0.007; 3.0 [2.6-4.0] vs. 2.5 [2.0-2.8] µg/mL, p &amp;lt; 0.0001). Overall, 70/87 IgAN patients (80.5%) had elevated Bb levels. The plasma concentration of C5a was elevated in IgAN patients compared to ADPKD patients (6.3 [3.8-9.2] vs. 4.9 [2.8-6.3] ng/mL, p = 0.0274), with values within the reference range in 92.0% of IgAN patients. There was no difference in plasma levels of sC5b-9 between the two groups (p = 0.186). The combined analysis of C3a, C5a and sC5b-9 allowed us to distinguish 4 clusters of patients (Fig. 1). A total of 48 patients (55.2%) show evidence of C3 convertase activity in plasma (clusters 3 and 4), including 16 patients (18.4%) with the highest plasma concentrations of sC5b-9, C3a and Bb. In contrast, 15 patients (17.2%, cluster 1) have no (or very low) complement activation. The last group (n = 32, 36.8%, cluster 2) shows an isolated increase in sC5b-9 without evidence of plasma C3 convertase activity. Proteinuria was significantly increased in groups 3 and 4, which had the highest C3a concentrations, compared to groups 1 and 2 (p = 0.0051). Conclusion This study provides insights into the mechanisms of complement activation in IgAN, with a pivotal role for the activity and regulation of the alternative C3 pathway convertase. In addition, the combined analysis of complement biomarkers identified, for the first time, clusters of patients who may benefit from complement targeting therapies.

Abstract WMP115: Rapid Vascular Complement Activation: A Key Driver of Blood-Brain Barrier Disruption in Ischemic Stroke With Acute Hyperglycemia

Background: Acute hyperglycemia, which occurs in over 40% of ischemic stroke patients irrespective of pre-existing diabetes, exacerbates stroke injury. Understanding its mechanisms is vital for innovative treatments. Here we investigate the role of complement in hyperglycemia-exacerbated damage after stroke. Method: Male C57/BL6 mice (10-11 weeks) underwent 30-min suture-induced middle cerebral artery occlusion followed by reperfusion, resembling clinical thrombectomy. Acute hyperglycemia was induced 10 min pre-stroke via glucose injection. Mice were sacrificed at 4.5h and 24h post-stroke to analyze brain swelling, blood-brain barrier (BBB) leakage and hemorrhagic transformation (HT). Mortality, neurological deficits, and motor-sensory functions were assessed at 14 days post-stroke. Complement activation was visualized via C3d immunostaining. Complement C3's role was examined using knock-out mice and the targeted complement inhibitor CR2-Crry fusion protein. Result: Hyperglycemia rapidly worsens BBB leakage (p&lt;0.0001), brain swelling (p&lt;0.05), and HT (p&lt;0.0001) at 4.5 hr after stroke. Hyperglycemic stroke mice exhibit higher mortality (100% vs 25%, p=0.0008), body weight loss, and behavior impairment at the sub-acute phase compared to normoglycemic mice. Notably, acute hyperglycemia rapidly increased plasma C3 levels at 1hr after stroke (p&lt;0.01), accompanied by rapid and time-dependent activation of C3 in ischemic brain vessels, as marked by increased vascular C3d; and this co-localized with autoantibody (IgM/IgG) vessel buildup and vessel leakage. Additionally, C3d levels positively correlated with brain swelling and HT (p&lt;0.01). Vascular C3d and leakage into parenchyma were markedly reduced in the absence of reperfusion. Blocking complement via C3 knock-out or post-reperfusion systemic injection of C3 inhibitor CR2-Crry mitigates hyperglycemia's detrimental effects, including BBB leakage. Conclusion: Rapid vascular activation of complement C3 is a significant driver of hyperglycemic-exacerbated pathology in experimental stroke. Inhibiting C3 activation could be a potential therapeutic approach to improve hyperglycemic stroke outcome.

De-stabilizing innate immunity in COVID-19: effects of its own positive feedback and erratic viraemia on the alternative pathway of complement.

Complement provides powerful, fast responses in the human circulation to SARS-CoV-2 (COVID-19 virus) infection of the lower respiratory tract. COVID-19 effects were investigated in a revised human in silico Mass Action model of complement's alternative pathway (AP) responses. Bursts of newly circulating virions increased the fission of Complement protein C3 into C3a and C3b via stimulation of the lectin pathway or inhibited complement factor H. Viral reproduction sub-models incorporated smoothly exponential or step-wise exponential growth. Starting complement protein concentrations were drawn randomly from published normal male or female ranges and each infection model run for 10 days. C3 and factor B (FB) syntheses driven by Lectin Pathway stimulation led to declining plasma C3 and increasing FB concentrations. The C3-convertase concentration, a driver of viral elimination, could match viral growth over three orders of magnitude but near-complete exhaustion of circulating C3 was more prevalent with step-wise than with 'smooth' increases in viral stimulation. C3 exhaustion could be prolonged. Type 2 Diabetes and hypertension led to greatly increased peak C3-convertase concentrations, as did short-term variability of COVID-19 viraemia, pulmonary capillary clotting and secondary acidosis. Positive feedback in the AP greatly extends its response range at the expense of stability.

Surgery for Crohn’s Disease Is Associated With a Dysbiotic Microbiome and Metabolome: Results From Two Prospective Cohorts

Background and AimsCrohn’s disease is associated with alterations in the gut microbiome and metabolome described as dysbiosis. We characterized the microbial and metabolic consequences of ileal resection, the most common Crohn’s disease surgery. MethodsPatients with and without intestinal resection were identified from the Diet to Induce Remission in Crohn’s Disease and Study of a Prospective Adult Research Cohort with Inflammatory Bowel Disease studies. Stool samples were analyzed with shotgun metagenomics sequencing. Fecal butyrate was measured with 1H nuclear magnetic resonance spectroscopy. Fecal bile acids and plasma 7α-hydroxy-4-cholesten-3-one (C4) was measured with mass spectrometry. ResultsIntestinal resection was associated with reduced alpha diversity and altered beta diversity with increased Proteobacteria and reduced Bacteroidetes and Firmicutes. Surgery was associated with higher representation of genes in the KEGG pathway for ABC transporters and reduction in genes related to bacterial metabolism. Surgery was associated with reduced concentration of the But gene but this did not translate to reduced fecal butyrate concentration. Surgery was associated with decreased abundance of bai operon genes, with increased plasma C4 concentration, increased primary bile acids and reduced secondary bile acids, including isoLCA. Additionally, E lenta, A equalofaciens and G pamelaeae were lower in abundance among patients with prior surgery in both cohorts. ConclusionsIn two different populations, prior surgery in Crohn’s disease is associated with altered fecal microbiome. Patients who had undergone ileal resection had reduction in the potentially beneficial bacteria E lenta and related actinobacteria as well as secondary bile acids, including isoLCA, suggesting that these could be biomarkers of patients at higher risk for disease progression.

Renin and renin blockade have no role in complement activity

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 ⁰C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

Complement alternative pathway determines disease susceptibility and severity in antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis

Activation of the alternative pathway (AP) of complement is involved in the pathogenesis of antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), although the underlying molecular mechanisms are unclear. To gain insight into the role of the AP, common gene variants in CFH/CFHR1-5, CFB, C3 and MCP, and longitudinal determinations of plasma C3, C4, FH, FHR-1, FHR-2, FHR-5, FB, properdin and sC5b-9 levels were analyzed in a Spanish AAV cohort consisting of 102 patients; 54 with active AAV (active cohort) and 48 in remission not receiving immunosuppressants or dialysis therapy (remission cohort). The validation cohort consisted of 100 patients with ANCA-associated glomerulonephritis. Here, we demonstrated that common genetic variants in complement components of the AP are associated with disease susceptibility (CFB32Q/W) or severity of kidney damage in AAV (CFH-H1, CFHH2 and ΔCFHR3/1). Plasma levels of complement components were significantly different between active and remission cohorts. In longitudinal observations, a high degree of AP activation at diagnosis was associated with worse disease outcome, while high basal FHR-1 levels and lower FH/FHR-1 ratios determined severe forms of kidney associated AAV. These genetic and plasmatic findings were confirmed in the validation cohort. Additionally, autoantibodies against FH and C3 convertase were identified in one and five active patients, respectively. Thus, our study identified key genetic and plasma components of the AP that determine disease susceptibility, prognosis, and severity in AAV. Our data also suggests that balance between FH and FHR-1 is critical and supports FHR-1 as a novel AP-specific therapeutic target in AAV.

Prospects and Pitfalls of Plasma Complement C4 in Schizophrenia: Building a Better Biomarker

Complex brain disorders like schizophrenia may have multifactorial origins related to mis-timed heritable and environmental factors interacting during neurodevelopment. Infections, inflammation, and autoimmune diseases are over-represented in schizophrenia leading to immune system-centered hypotheses. Complement component C4 is genetically and neurobiologically associated with schizophrenia, and its dual activity peripherally and in the brain makes it an exceptional target for biomarker development. Studies to evaluate the biomarker potential of plasma or serum C4 in schizophrenia do so to understand how peripheral C4 might reflect central nervous system-derived neuroinflammation, synapse pruning, and other mechanisms. This effort, however, has produced mostly conflicting results, with peripheral C4 sometimes elevated, reduced, or unchanged between comparison groups. We undertook a pilot biomarker development study to systematically identify sociodemographic, genetic, and immune-related variables (autoimmune, infection-related, gastrointestinal, inflammatory), which may be associated with plasma C4 levels in schizophrenia (SCH; n = 335) and/or in nonpsychiatric comparison subjects (NCs; n = 233). As with previously inconclusive studies, we detected no differences in plasma C4 levels between SCH and NCs. In contrast, levels of general inflammation, C-reactive protein (CRP), were significantly elevated in SCH compared to NCs (ANOVA, F = 20.74, p < 0.0001), suggestive that plasma C4 and CRP may reflect different sources or causes of inflammation. In multivariate regressions of C4 gene copy number variants, plasma C4 levels were correlated only for C4A (not C4B, C4L, C4S) and only in NCs (R Coeff = 0.39, CI = 0.01–0.77, R² = 0.18, p < 0.01; not SCH). Other variables associated with plasma C4 levels only in NCs included sex, double-stranded DNA IgG, tissue-transglutaminase (TTG) IgG, and cytomegalovirus IgG. Toxoplasma gondii IgG was the only variable significantly correlated with plasma C4 in SCH but not in NCs. Many variables were associated with plasma C4 in both groups (body mass index, race, CRP, N-methyl-D-aspartate receptor (NMDAR) NR2 subunit IgG, TTG IgA, lipopolysaccharide-binding protein (LBP), and soluble CD14 (sCD14). While the direction of most C4 associations was positive, autoimmune markers tended to be inverse, and associated with reduced plasma C4 levels. When NMDAR-NR2 autoantibody-positive individuals were removed, plasma C4 was elevated in SCH versus NCs (ANOVA, F = 5.16, p < 0.02). Our study was exploratory and confirmation of the many variables associated with peripheral C4 requires replication. Our preliminary results point toward autoimmune factors and exposure to the pathogen, T. gondii, as possibly significant contributors to variability of total C4 protein levels in plasma of individuals with schizophrenia.

Effects of dietary ginger (Zingiber officinale) extract and curcumin on growth, hematology, immunity, and antioxidant status in rainbow trout (Oncorhynchus mykiss)

The use of dietary ginger (Zingiber officinale) and curcumin as immunostimulants is a promising method in fish culture. This study evaluated the impacts of dietary ginger extract and curcumin on growth, hematology, immune and antioxidant status, and expression of some growth and immune related genes in rainbow trout (Oncorhynchus mykiss) fingerlings. Fish (7.5 ± 0.1 g) were randomly distributed into 12 fiberglass tanks (30 fish per tank), divided into four treatments in triplicate and received no supplementation (control) and diets supplemented with 0.5% ginger extract (ginger treatment), 0.5% curcumin (curcumin treatment), and 0.5% ginger extract plus 0.5% curcumin (mix treatment). The growth of fish and muscle crude protein content were highest in the mix treatment. Higher protein efficiency ratio, hemoglobin, and plasma ALP activity were observed in fish fed supplemented diets than the control. Red blood cells (RBC) counts and hematocrit value were higher in the ginger treatment, then curcumin and mix treatments and lower in the control. White blood cells (WBC) counts and plasma lysozyme, ACH50, catalase, and SOD activities were highest in fish fed the mix diet. Moreover, plasma C4 value and GPx activity were significantly higher and triglyceride value was lower in the curcumin and mix treatments compared to the control and ginger treatments. The highest plasma C3 and total immunoglobulin (Ig) values were found in fish fed curcumin and mix diets, but those were not significantly different from the ginger treatment. Growth hormone (GH) and lysozyme relative expression levels were highest in fish fed the mix diet and lowest in the control. Thus, the combination of 0.5% ginger extract and 0.5% curcumin (mix diet) to improve growth, body composition, hematology, immune responses, and antioxidant status in rainbow trout can be recommended.

Effects of fucoidan on growth performance, immunity, antioxidant ability, digestive enzyme activity, and hepatic morphology in juvenile common carp (Cyprinus carpio)

Fucoidan with its excellent biological activities such as growth promotion, antioxidant and strong immunity, is widely used in animal production. The present study was conducted to investigate the influences of feeding fucoidan on growth performance, biochemical indices, immunity, the antibacterial ability of plasma, the digestive enzyme activity of the intestine, antioxidant capacity, and the histological structure of liver in juvenile common carp. Five experimental diets added with 0 (Diet 1), 500 (Diet 2), 1,000 (Diet 3), 1,500 (Diet 4), and 2,000 (Diet 5) mg/kg fucoidan were fed to triplicate groups of 30 fish (35.83 ± 0.24 g) respectively for 8 weeks. The results showed that fish fed diets with a fucoidan supplementation of 1,666.67–1,757 mg/kg might have the best growth performance (p&amp;lt; 0.05). The levels of plasma total protein (TP) and albumin (ALB) in Diet 3, Diet 4, and Diet 5 were higher than those in Diet 1 and Diet 2 (p&amp;lt; 0.05). Moreover, the contents of plasma C3, LYZ, and IgM; the antibacterial ability of serum; and the activity of SOD, CAT, POD, and GPX in the liver, and ACP, AKP, LPS, AMS, and TRY in the intestine significantly improved; the contents of LPO and MDA in the liver were notably decreased in diets with fucoidan supplement (p&amp;lt; 0.05). Furthermore, the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the contents of total bilirubin (TB) and glucose (Glu) in Diet 5 were the highest among the groups. Meanwhile, proinflammatory factors (plasma IL-6 and IL-1β) had a higher expression, but anti-inflammatory factors (plasma IL-1) had a lower expression in Diet 5 (p &amp;gt; 0.05). It indicated that a higher dose (2,000 mg/kg) of fucoidan may induce inflammation and metabolic disorders. Interestingly, histological results of liver also indicated that dietary fucoidan intake in certain amounts (500–1,500 mg/kg) could ameliorate hepatic morphology, but the high dosage (2,000 mg/kg) probably damaged the liver. To the best of our knowledge, this is the first study on the application of fucoidan as a functional additive to juvenile common carp. The results of the present study can be used to guide the application of fucoidan in healthy aquaculture and can further reveal the effect and mechanism of fucoidan on the nutritional physiology of aquatic animals.

An integrated analysis of fecal microbiome and metabolomic features distinguish non-cirrhotic NASH from healthy control populations.

There is great interest in identifying microbiome features as reliable noninvasive diagnostic and/or prognostic biomarkers for non-cirrhotic NASH fibrosis. Several cross-sectional studies have reported gut microbiome features associated with advanced NASH fibrosis and cirrhosis, where the most prominent features are associated with cirrhosis. However, no large, prospectively collected data exist establishing microbiome features that discern non-cirrhotic NASH fibrosis, integrate the fecal metabolome as disease biomarkers, and are unconfounded by BMI and age. Results from shotgun metagenomic sequencing performed on fecal samples prospectively collected from 279 US patients with biopsy-proven NASH (F1-F3 fibrosis) enrolled in the REGENERATE I303 study were compared to those from 3 healthy control cohorts and integrated with the absolute quantification of fecal bile acids. Microbiota beta-diversity was different, and BMI- and age-adjusted logistic regression identified 12 NASH-associated species. Random forest prediction models resulted in an AUC of 0.75-0.81 in a receiver operator characteristic analysis. In addition, specific fecal bile acids were significantly lower in NASH and correlated with plasma C4 levels. Microbial gene abundance analysis revealed 127 genes increased in controls, many involving protein synthesis, whereas 362 genes were increased in NASH many involving bacterial environmental responses (false discovery rate < 0.01). Finally, we provide evidence that fecal bile acid levels may be a better discriminator of non-cirrhotic NASH versus health than either plasma bile acids or gut microbiome features. These results may have value as a set of baseline characteristics of non-cirrhotic NASH against which therapeutic interventions to prevent cirrhosis can be compared and microbiome-based diagnostic biomarkers identified.

Complement system changes in blood in Parkinson's disease and progressive Supranuclear Palsy/Corticobasal Syndrome

Parkinson's Disease (PD) is diagnosed clinically, and early PD is often challenging to differentiate from atypical parkinsonian disorders such as the Four-repeat (4R-) Tauopathies Progressive Supranuclear Palsy and Corticobasal Syndrome. Diagnostic biomarkers are needed, and proteomic studies have suggested that the plasma complement system is altered in PD, but validation studies are lacking.In this study, plasma from 148 individuals (PD, 4R-Tauopathies, and healthy controls (HC)) were used to quantify 12 complement proteins with immunoassays, and CH50 classical pathway complement activity was quantified in sera from further 78 individuals (PD and HC).Complement factors C1q and C3 in plasma were lower in individuals with 4R-Tauopathies (ANOVA, p = 0.0041, p = 0.0057 respectively) compared to both PD and HC. None of the complement proteins were altered between PD and HC, however a few proteins correlated with clinical parameters within the PD group. Notably, levels of C3 correlated with non-motor symptoms in female patients. Classical pathway complement activity was not altered in PD serum, but did correlate with mental fatigue.In conclusion, individuals with 4R-Tauopathies showed lower plasma C1q and C3 compared PD and HC. Neither complement levels nor CH50 activity were significantly altered in PD versus HC but may associate with PD symptom severity.

Relief of CoA sequestration and restoration of mitochondrial function in a mouse model of propionic acidemia.

Propionic acidemia (PA, OMIM 606054) is a devastating inborn error of metabolism arising from mutations that reduce the activity of the mitochondrial enzyme propionyl-CoA carboxylase (PCC). The defects in PCC reduce the concentrations of nonesterified coenzyme A (CoASH), thus compromising mitochondrial function and disrupting intermediary metabolism. Here, we use a hypomorphic PA mouse model to test the effectiveness of BBP-671 in correcting the metabolic imbalances in PA. BBP-671 is a high-affinity allosteric pantothenate kinase activator that counteracts feedback inhibition of the enzyme to increase the intracellular concentration of CoA. Liver CoASH and acetyl-CoA are depressed in PA mice and BBP-671 treatment normalizes the cellular concentrations of these two key cofactors. Hepatic propionyl-CoA is also reduced by BBP-671 leading to an improved intracellular C3:C2-CoA ratio. Elevated plasma C3:C2-carnitine ratio and methylcitrate, hallmark biomarkers of PA, are significantly reduced by BBP-671. The large elevations of malate and α-ketoglutarate in the urine of PA mice are biomarkers for compromised tricarboxylic acid cycle activity and BBP-671 therapy reduces the amounts of both metabolites. Furthermore, the low survival of PA mice is restored to normal by BBP-671. These data show that BBP-671 relieves CoA sequestration, improves mitochondrial function, reduces plasma PA biomarkers, and extends the lifespan of PA mice, providing the preclinical foundation for the therapeutic potential of BBP-671.

Effect of replacement of fishmeal by soybean products with attractants supplementation on the growth performance, feed utilization, body composition, plasma physiological responses and hepatic antioxidant abilities of juvenile golden pompano Trachinotus ovatus

A feeding experiment was carried out to develop a practical diet with low fishmeal for juvenile golden pompano (Trachinotus ovatus) by substituting fishmeal protein with soybean products mixture. Six isocaloric (17.6 KJ g−1) diets were designed by replacing 0 (FM100), 5 (FM95), 10 (FM90), 15 (FM85), 20 (FM80), and 25% (FM75) of the fishmeal with soybean products mixture (SPM) (soybean meal and soybean protein concentrate). The experiment diets were supplemented with 1.5%, 3%, 4.5%, 6% and 7.5% squid paste as attractants or palatability enhancer. The experimental fish (6.9 g) were cultured in sea cages (1.0 m × 1.0 m × 1.5 m) with 25 fish in each cage. Fish were fed the corresponding experimental diets to satiation twice daily for 8 weeks. At the end of the feeding experiment, no differences were found in the final weight and weight gain (WG) of fish fed FM100, FM85, FM80, and FM75 diets (P &gt; 0.05). The WG was significantly (P &lt; 0.05) better in the fish-fed FM95 diet compared to FM100 and FM75 diets. FCR in diet FM95 and FM90 was significantly (P &lt; 0.05) than that of FM80 and FM75, but these values were not significantly different from those in other treatments. Although the condition factor value in diets FM90, FM85, and FM80 was significantly (P &lt; 0.05) higher than that of FM100, these values were not significantly different from the rest of the other groups. There was no statistical difference in SGR, FR, VSI, and survival rates among different dietary treatments. The plasma AST activity was significantly lower in fish fed FM90 diet compared to fish fed with control, FM85, FM80, and FM75 (P &lt; 0.05). There was no significant difference in plasma AST among all dietary treatments except FM95 and FM90. In contrast, the plasma AKP activity showed the opposite trend. Compared with the control, the fish-fed FM80 and FM75 diets had significantly decreased plasma cholesterol, triglyceride total protein, albumin, and globulin level (P &lt; 0.05) while significantly increasing plasma COR levels (P &lt; 0.05). There was no significant difference in ALT, Urea and glucose values among all treatments. No statistical difference was observed among the FM100, FM80, and FM75 treatments for those of plasma LYZ activity, C3, and C4 levels. The hepatic T-AOC and SOD activities were significantly increased in fish fed the FM90 diet (P &lt; 0.05) compared to those in fish fed with FM100 diet. According to the current experiment, it can be concluded that SPM supplemented with SP could partially substitute fishmeal (25%) for juvenile golden pompano without any adverse symptoms.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies

BackgroundIdiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels...

Elevated CSF and plasma complement proteins in genetic frontotemporal dementia: results from the GENFI study

BackgroundNeuroinflammation is emerging as an important pathological process in frontotemporal dementia (FTD), but biomarkers are lacking. We aimed to determine the value of complement proteins, which are key components of innate immunity, as biomarkers in cerebrospinal fluid (CSF) and plasma of presymptomatic and symptomatic genetic FTD mutation carriers.MethodsWe measured the complement proteins C1q and C3b in CSF by ELISAs in 224 presymptomatic and symptomatic GRN, C9orf72 or MAPT mutation carriers and non-carriers participating in the Genetic Frontotemporal Dementia Initiative (GENFI), a multicentre cohort study. Next, we used multiplex immunoassays to measure a panel of 14 complement proteins in plasma of 431 GENFI participants. We correlated complement protein levels with corresponding clinical and neuroimaging data, neurofilament light chain (NfL) and glial fibrillary acidic protein (GFAP).ResultsCSF C1q and C3b, as well as plasma C2 and C3, were elevated in symptomatic mutation carriers compared to presymptomatic carriers and non-carriers. In genetic subgroup analyses, these differences remained statistically significant for C9orf72 mutation carriers. In presymptomatic carriers, several complement proteins correlated negatively with grey matter volume of FTD-related regions and positively with NfL and GFAP. In symptomatic carriers, correlations were additionally observed with disease duration and with Mini Mental State Examination and Clinical Dementia Rating scale® plus NACC Frontotemporal lobar degeneration sum of boxes scores.ConclusionsElevated levels of CSF C1q and C3b, as well as plasma C2 and C3, demonstrate the presence of complement activation in the symptomatic stage of genetic FTD. Intriguingly, correlations with several disease measures in presymptomatic carriers suggest that complement protein levels might increase before symptom onset. Although the overlap between groups precludes their use as diagnostic markers, further research is needed to determine their potential to monitor dysregulation of the complement system in FTD.

Abstract 018: B Cells Induce Hypertension, AT1AA, And Complement Activation In Preeclampsia Or In Response To A Prior Covid-19 Infection During Pregnancy

Preeclampsia (PE), new onset hypertension (HTN) during pregnancy, is associated with placental ischemia and chronic inflammation that includes increased CD4+ T cells, B cells secreting agonistic autoantibodies against the angiotensin II type 1 receptor (AT1AA), and activation of the complement system. Previous studies have shown AT1-AA is produced in patients with COVID-19 infection. Interestingly, having had COVID-19 during pregnancy is associated with increased incidence of developing a PE phenotype during pregnancy. We have previously shown an important role for B cell depletion or AT1AA inhibition to attenuate HTN in rat models of PE. Collectively, this data suggests B cells contribute to PE development and that B cells may increase incidence of PE in patients with a history (Hx) of COVID-19 during pregnancy through production of the AT1AA. We hypothesize B cells from PE or CV Hx PE patients produce AT1AA resulting in HTN and complement activation in pregnancy. Placental B cells were isolated from normal pregnant (NP), PE, normotensive (NT) CV Hx, or PE CV Hx patients at delivery. B cells were transferred i.p. into pregnant athymic rats at gestation (GD) 12. On GD18, carotid catheters were inserted. On GD19, blood pressure was measured and tissues collected. PE B cell recipients had increased Mean Arterial Pressure (MAP) (115±3 mmHg n=6) compared to NP B cell recipients (97±4 mmHg n=6 p&lt;0.05). PE B cell recipients had increased AT1AA (20±2 ΔBPM n=4) compared to NP B cell recipients (6±1 ΔBPM n=4 p&lt;0.05). PE B cell recipients had increased markers of complement activation such as reduced plasma C4 (1302±169 μg/mL n=4) and C3 (516±45 μg/mL n=4) compared to recipients of NP B cells (2348±338 μg/mL n=4 p&lt;0.05) and (790±66 μg/mL n=4 p&lt;0.05) respectively. CV Hx PE B cell recipients had elevated MAP (108±3 mmHg n=4) compared to CV Hx NT B cell recipients (101±7 mmHg n=4) and increased AT1AA (24±3 ΔBPM n=3) compared to CV Hx NT B cell Recipients (4±1 ΔBPM n=4 p&lt;0.05). Collectively, this study demonstrates an important role for B cells to cause HTN during pregnancy; and indicates that B cells contribute to a higher incidence of PE in women with a Hx of CV infection during pregnancy possibly by secreting AT1-AA.