Impact of Strongyloides stercoralis infection on complement activation in Type 2 diabetes mellitus: Insights from a clinical and anthelmintic intervention study.

BACKGROUND: Numerous studies indicate a potential protective role of helminths in diabetes mellitus (DM) progression. The complement system, vital for host defense, plays a crucial role in tissue homeostasis and immune surveillance. Dysregulated complement activation is implicated in diabetic complications. We aimed to investigate the influence of the helminth, Strongyloides stercoralis (Ss) on complement activation in individuals with type 2 DM (T2D). METHODOLOGY: We assessed circulating levels of complement proteins (C1q, C2, C3, C4, C4b, C5, C5a, and MBL (Lectin)) and their regulatory components (Factor B, Factor D, Factor H, and Factor I) in individuals with T2D with (n = 60) or without concomitant Ss infection (n = 58). Additionally, we evaluated the impact of anthelmintic therapy on these parameters after 6 months in Ss-infected individuals (n = 60). RESULTS: Ss+DM+ individuals demonstrated reduced levels of complement proteins (C1q, C4b, MBL (Lectin), C3, C5a, and C3b/iC3b) and complement regulatory proteins (Factor B and Factor D) compared to Ss-DM+ individuals. Following anthelmintic therapy, there was a partial reversal of these levels in Ss+DM+ individuals. CONCLUSION: Our findings indicate that Ss infection reduces complement activation, potentially mitigating inflammatory processes in individuals with T2D. The study underscores the complex interplay between helminth infections, complement regulation, and diabetes mellitus, offering insights into potential therapeutic avenues.

Oral Iptacopan Monotherapy in Paroxysmal Nocturnal Hemoglobinuria.

BACKGROUND: Persistent hemolytic anemia and a lack of oral treatments are challenges for patients with paroxysmal nocturnal hemoglobinuria who have received anti-C5 therapy or have not received complement inhibitors. Iptacopan, a first-in-class oral factor B inhibitor, has been shown to improve hemoglobin levels in these patients. METHODS: In two phase 3 trials, we assessed iptacopan monotherapy over a 24-week period in patients with hemoglobin levels of less than 10 g per deciliter. In the first, anti-C5-treated patients were randomly assigned to switch to iptacopan or to continue anti-C5 therapy. In the second, single-group trial, patients who had not received complement inhibitors and who had lactate dehydrogenase (LDH) levels more than 1.5 times the upper limit of the normal range received iptacopan. The two primary end points in the first trial were an increase in the hemoglobin level of at least 2 g per deciliter from baseline and a hemoglobin level of at least 12 g per deciliter, each without red-cell transfusion; the primary end point for the second trial was an increase in hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. RESULTS: In the first trial, 51 of the 60 patients who received iptacopan had an increase in the hemoglobin level of at least 2 g per deciliter from baseline, and 42 had a hemoglobin level of at least 12 g per deciliter, each without transfusion; none of the 35 anti-C5-treated patients attained the end-point levels. In the second trial, 31 of 33 patients had an increase in the hemoglobin level of at least 2 g per deciliter from baseline without red-cell transfusion. In the first trial, 59 of the 62 patients who received iptacopan and 14 of the 35 anti-C5-treated patients did not require or receive transfusion; in the second trial, no patients required or received transfusion. Treatment with iptacopan increased hemoglobin levels, reduced fatigue, reduced reticulocyte and bilirubin levels, and resulted in mean LDH levels that were less than 1.5 times the upper limit of the normal range. Headache was the most frequent adverse event with iptacopan. CONCLUSIONS: Iptacopan treatment improved hematologic and clinical outcomes in anti-C5-treated patients with persistent anemia - in whom iptacopan showed superiority to anti-C5 therapy - and in patients who had not received complement inhibitors. (Funded by Novartis; APPLY-PNH ClinicalTrials.gov number, NCT04558918; APPOINT-PNH ClinicalTrials.gov number, NCT04820530.).

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

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

CRISPR Manipulation of Age-Related Macular Degeneration Haplotypes in the Complement System: Potential Future Therapeutic Applications/Avenues.

Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss among the elderly in the developed world. Whilst AMD is a multifactorial disease, the involvement of the complement system in its pathology is well documented, with single-nucleotide polymorphisms (SNPs) in different complement genes representing an increased risk factor. With several complement inhibitors explored in clinical trials showing limited success, patients with AMD are still without a reliable treatment option. This indicates that there is still a gap of knowledge in the functional implications and manipulation of the complement system in AMD, hindering the progress towards translational treatments. Since the discovery of the CRISPR/Cas system and its development into a powerful genome engineering tool, the field of molecular biology has been revolutionised. Genetic variants in the complement system have long been associated with an increased risk of AMD, and a variety of haplotypes have been identified to be predisposing/protective, with variation in complement genes believed to be the trigger for dysregulation of the cascade leading to inflammation. AMD-haplotypes (SNPs) alter specific aspects of the activation and regulation of the complement cascade, providing valuable insights into the pathogenic mechanisms of AMD with important diagnostic and therapeutic implications. The effect of targeting these AMD-related SNPs on the regulation of the complement cascade has been poorly explored, and the CRISPR/Cas system provides an ideal tool with which to explore this avenue. Current research concentrates on the association events of specific AMD-related SNPs in complement genes without looking into the effect of targeting these SNPs and therefore influencing the complement system in AMD pathogenesis. This review will explore the current understanding of manipulating the complement system in AMD pathogenesis utilising the genomic manipulation powers of the CRISPR/Cas systems. A number of AMD-related SNPs in different complement factor genes will be explored, with a particular emphasis on factor H (CFH), factor B (CFB), and complement C3 (C3).

A new alternative: inhibiting complement activation in patients with IgA nephropathy.

Mesangial complement C3 deposits, reflecting alternative and possibly lectin pathway activation, are characteristic in biopsies of patients with IgA nephropathy (IgAN). A recent randomized controlled trial tested the efficacy and safety of iptacopan, a factor B inhibitor, in patients with IgAN. Iptacopan dose-dependently reduced proteinuria, and there was a pronounced decrease of urinary C5b-9. This offers the perspective of "personalizing" therapy, which would be a unique feature of this novel approach to IgAN. A phase III clinical trial (APPLAUSE-IgAN) is ongoing.

Inhibition of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) by microbially derived natural compounds: a computational study.

The majority of the world population (around 25%) has latent Mycobacterium tuberculosis (Mtb) infection, among which only 5-10% of individuals develop active tuberculosis (TB), and 90-95% continue to have latent tuberculosis infection. This makes it the biggest global health concern. It has been reported that the resuscitation-promoting factor B (RpfB) is an exciting potential target for tuberculosis drug discovery due to its significant role in the reactivation of latent TB infection to an active infection. Several attempts have been made to investigate potential inhibitors against RpfB utilizing in-silico approaches. The present study also utilized a computational approach to investigate microbially derived natural compounds against the Mtb RpfB protein which is a very cost-effective This evaluation used structure-based virtual screening (SBVS), drug-likeness profiling, molecular docking, molecular dynamics simulation, and free-binding energy calculations. Six potential natural compounds, viz. Cyclizidine I, Boremexin C, Xenocoumacin 2, PM-94128, Cutinostatin B, and (+)1-O-demethylvariecolorquinone A were selected, which displayed a potential binding affinity between -52.39 and -60.87 Kcal/mol MMGBSA score and docking energy between -7.307 Kcal/mol to -6.972 Kcal/mol. All the complexes showed acceptable stability (<2.7 Å RMSD) during 100 ns MD simulation time except the RpfB protein-xenocoumacin 2 complex. This result exhibited that the selected compounds have high efficiency in inhibiting the Mtb RpfB and can be taken into account for additional in vitro and in vivo experimental validation.Communicated by Ramaswamy H. Sarma.

Major histocompatibility complex complement (MHC) Bf alleles show trans species evolution between man and chimpanzee.

HLA and disease studies by using single allele statistics have been fruitless during the last 40 years for explaining association pathogenesis of the associated diseases.Other approaches are necessary to untangle this puzzle. We aim to revisit complement alleleism in humans and primates for both studying MHC and disease association to complotypes and extended MHC haplotypes in order to also explain the positive directional selection of maintaining immune response genes (complement, MHC adaptive and MHC non-specific genes) that keeps these three type of genes together in a short chromosome stretch (MHC) for million years. These genes may be linked to conjointly avoid microbes attack and autoimmunity. In the present paper, it is obtained a new Bf chimpanzee allele, provisionaly named Patr-Bf*A:01,that differs from other Bf alleles by having CTG at eleventh codon of exon 2 in order to start the newly suggested methodology and explain functional and evolutionary MHC obscure aspects. Exons 1 to 6 of Ba fragment of Bf gene were obtained from chimpanzee. This new chimpanzee Factor B allele (Patr-Bf*A:01) is to be identical to a infrequent human Bf allele (SNP rs641153); it stresses the strong evolutive pressure upon certain alleles that are trans specific. It also may apply to MHC extended haplotipes which may conjointly act to start an adequate immune response. It is the first time that a complement MHC class III allele is described to undergo trans species evolution,in contrast to class I and class II alleles which had already been reported . Allelism of complement factors are again proposed for studying MHC complement genes, complotypes, and extended MHC haplotypes which may be more informative that single MHC marker studies.

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

BACKGROUND: Complement alternative pathway (AP) activation is linked to immunoglobulin A nephropathy (IgAN) prognosis severity, but Bb fragment's role is unclear. We examined the relationship between serum Bb fragment concentration at IgAN diagnosis and disease activity and outcomes. METHODS: This retrospective study included 125 biopsy-proven IgAN patients [age 39.9 years, 75% male, estimated glomerular filtration rate (eGFR) 82 ml/min, proteinuria 0.5 g/day] enrolled from 1984 to 2010 and followed for a minimum of 18 months. Monitoring continued until the last follow-up, end-stage kidney disease (ESKD) or death. Serum Bb fragment was measured using an enzyme-linked immunosorbent assay at diagnosis. Oxford classification and global optical score (GOS) were utilized for pathology assessment. RESULTS: Patients were followed for a median of 16 years; 42% developed chronic kidney disease stage ≥3, 19% reached ESKD and 9% died. Serum Bb fragment concentration negatively correlated with eGFR values at the last follow-up and positively with vascular and tubular histopathological indices. In univariate Cox regression analyses, higher Bb fragment concentration was associated with ESKD alongside older age, increased body mass index, arterial hypertension, lower eGFR, higher proteinuria, E1, S1, T1-2, GOS and corticotherapy. Patients with Bb levels ≥14.3 µg/ml had shorter mean kidney survival time (19.5 versus 22.7 years, P = .07); after adjusting for progression risk factors, the association persisted [hazard ratio 4.76 (95% confidence interval 1.56-14.43)]. CONCLUSIONS: Serum Bb fragment concentration at diagnosis may predict long-term IgAN outcomes, potentially due to AP activation at the endothelial surface. Further research is needed to confirm these results and evaluate Bb fragment's role in IgAN management.

Temporal properties of the speed-accuracy trade-off for arm-pointing movements in various directions around the body.

Human body movements are based on the intrinsic trade-off between speed and accuracy. Fitts's law (1954) shows that the time required for movement is represented by a simple logarithmic equation and is applicable to a variety of movements. However, few studies have determined the role of the direction in modulating the performance of upper limb movements and the effects of the interactions between direction and distance and between direction and target size. This study examined the variations in temporal properties of the speed-accuracy trade-off in arm-pointing movements that directly manipulate objects according to the direction, distance, and target size. Participants performed pointing movements to the targets with 3 different sizes presented at 15 locations (5 directions and 3 distances) on a horizontal plane. Movement time (MT) for each trial in each condition was obtained. Subsequently, Mackenzie's model (1992), MT = a + b log2(D/W +1), where D and W represent the distance and width of the target, respectively, was fitted. The slope factor b, a fitted parameter in the equation, was calculated and evaluated according to the changes in the direction, distance, and target size. The results showed that MTs exhibited anisotropy in the hemifield, being the smallest in the right-forward direction. Additionally, the slope factor b, as a function of distance, was smaller in the rightward direction than in the forward and left-forward directions. These results suggest that the degree of difficulty of upper limb movements expands heterogeneously in various directions around the body.

Cross-Cultural Adaptation and Validation of the Revised Brief Diabetes Knowledge Test (DKT2) in Individuals With Type 2 Diabetes Mellitus and Their Caregivers.

PURPOSE: The purpose of the study was to develop an Italian version of the Revised Brief Diabetes Knowledge Test (DKT2), providing a cultural and linguistic validation supported by psychometrics and hypotheses testing. METHODS: This multimethods study was divided into 4 phases: (a) cultural-linguistic validation, with a translation and back-translation process; (b) confirmatory factor analysis (CFA) considering the original scale's structure (knowledge and insulin-specific knowledge); (c) criterion validity via hypotheses testing; and (d) cross-group measurement invariance. The internal consistency reliability was assessed by the Kuder-Richardson Formula 20 (KR-20) of the overall scale. RESULTS: A total of 251 patients and 251 caregivers were enrolled. The CFA showed good goodness of fit for both patients and caregivers. The tested hypotheses supported criterion validity in both groups. Reliability was adequate: All KR-20 values in both groups and domains were higher than 0.60. The mean percentage of knowledge score on DKT2 was lower for patients than caregivers. CONCLUSION: The DKT2 is a valid and reliable scale to assess overall knowledge of diabetes, considering its role in promoting appropriate self-care behaviors in patients with type 2 diabetes mellitus. The Italian version of DKT2 demonstrated reliability and validity, and it can be used by researchers and diabetes care and education specialists to assess a patient's or population's overall knowledge of diabetes.

The Bouma law accounts for crowding in 50 observers.

Crowding is the failure to recognize an object due to surrounding clutter. Our visual crowding survey measured 13 crowding distances (or "critical spacings") twice in each of 50 observers. The survey includes three eccentricities (0, 5, and 10 deg), four cardinal meridians, two orientations (radial and tangential), and two fonts (Sloan and Pelli). The survey also tested foveal acuity, twice. Remarkably, fitting a two-parameter model-the well-known Bouma law, where crowding distance grows linearly with eccentricity-explains 82% of the variance for all 13 × 50 measured log crowding distances, cross-validated. An enhanced Bouma law, with factors for meridian, crowding orientation, target kind, and observer, explains 94% of the variance, again cross-validated. These additional factors reveal several asymmetries, consistent with previous reports, which can be expressed as crowding-distance ratios: 0.62 horizontal:vertical, 0.79 lower:upper, 0.78 right:left, 0.55 tangential:radial, and 0.78 Sloan-font:Pelli-font. Across our observers, peripheral crowding is independent of foveal crowding and acuity. Evaluation of the Bouma factor, b (the slope of the Bouma law), as a biomarker of visual health would be easier if there were a way to compare results across crowding studies that use different methods. We define a standardized Bouma factor b' that corrects for differences from Bouma's 25 choice alternatives, 75% threshold criterion, and linearly symmetric flanker placement. For radial crowding on the right meridian, the standardized Bouma factor b' is 0.24 for this study, 0.35 for Bouma (1970), and 0.30 for the geometric mean across five representative modern studies, including this one, showing good agreement across labs, including Bouma's. Simulations, confirmed by data, show that peeking can skew estimates of crowding (e.g., greatly decreasing the mean or doubling the SD of log b). Using gaze tracking to prevent peeking, individual differences are robust, as evidenced by the much larger 0.08 SD of log b across observers than the mere 0.03 test-retest SD of log b measured in half an hour. The ease of measurement of crowding enhances its promise as a biomarker for dyslexia and visual health.

Proteomic Analysis of Pediatric Hemophagocytic Lymphohistiocytosis: a Comparative Study with Healthy Controls, Sepsis, Critical Ill, and Active Epstein-Barr virus Infection to Identify Altered Pathways and Candidate Biomarkers.

Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome characterized by excessive activation of the immune system, along with uncontrolled proliferation of activated macrophages and lymphocytes. The clinical features of HLH often overlap with the clinical features of other severe inflammatory conditions such as sepsis, hindering accurate and timely diagnosis. In this study, we performed a data-independent acquisition mass spectrometry-based plasma proteomic analysis of 33 pediatric patients with HLH compared with four control groups: 39 healthy children, 43 children with sepsis, 39 children hospitalized in the pediatric intensive care unit without confirmed infections, and 21 children with acute Epstein-Barr virus infection. Proteomic comparisons between the HLH group and each of the control groups showed that HLH was characterized by alterations in complement and coagulation cascades, neutrophil extracellular trap formation, and platelet activation pathways. We identified eight differentially expressed proteins in patients with HLH, including plastin-2 (LCP1), vascular cell adhesion protein 1, fibrinogen beta chain, fibrinogen gamma chain, serum amyloid A-4 protein, extracellular matrix protein 1, apolipoprotein A-I, and albumin. LCP1 emerged as a candidate diagnostic marker for HLH with an area under the curve (AUC) of 0.97 in the original cohort and an AUC of 0.90 (sensitivity = 0.83 and specificity = 1.0) in the validation cohort. Complement C1q subcomponent subunit B was associated with disease severity in patients with HLH. Based on comparisons with multiple control groups, this study provides a proteomic profile and candidate biomarkers of HLH, offering researchers novel information to improve the understanding of this condition.

Liver factor B silencing to cure C3 glomerulopathy: Evidence from a mouse model of complement dysregulation.

Uncontrolled activation of the alternative pathway (AP) of complement, due to genetic and/or acquired defects, plays a primary pathogenetic role in C3 glomerulopathy (C3G), a rare and heterogeneous disease characterised by predominant C3 fragment deposition within the glomerulus, as well as glomerular damage. There are currently no approved disease-specific treatments for C3G, but new drugs that directly counteract AP dysregulation, targeting components of the pathway, have opened promising new perspectives for managing the disease. Complement factor B (FB), which is primarily synthesised by hepatocytes, is a key component of the AP, as it drives the central amplification loop of the complement system. In this study we used a GalNAc (N-Acetylgalactosamine)-conjugated siRNA to selectively target and suppress liver FB expression in two mouse models characterised by the complete (Cfh-/- mice) or partial (Cfh+/-) loss of function of complement factor H (FH). Homozygous deletion of FH induced a severe C3G phenotype, with strong dysregulation of the AP of complement, glomerular C3 deposition and almost complete C3 consumption. Mice with a heterozygous deletion of FH had intermediate C3 levels and exhibited slower disease progression, resembling human C3G more closely. Here we showed that FB siRNA treatment did not improve serum C3 levels, nor limit glomerular C3 deposition in Cfh-/- mice, while it did normalise circulating C3 levels, reduce glomerular C3 deposits, and limit mesangial electron-dense deposits in Cfh+/- mice. The present data provide important insights into the potential benefits and limitations of FB-targeted inhibition strategies and suggest RNA interference-mediated FB silencing in the liver as a possible therapeutic approach for treating C3G patients with FH haploinsufficiency.

Quantification of the zymogenicity and the substrate-induced activity enhancement of complement factor D.

Complement factor D (FD) is a serine protease present predominantly in the active form in circulation. It is synthesized as a zymogen (pro-FD), but it is continuously converted to FD by circulating active MASP-3. FD is a unique, self-inhibited protease. It has an extremely low activity toward free factor B (FB), while it is a highly efficient enzyme toward FB complexed with C3b (C3bB). The structural basis of this phenomenon is known; however, the rate enhancement was not yet quantified. It has also been unknown whether pro-FD has any enzymatic activity. In this study, we aimed to measure the activity of human FD and pro-FD toward uncomplexed FB and C3bB in order to quantitatively characterize the substrate-induced activity enhancement and zymogenicity of FD. Pro-FD was stabilized in the proenzyme form by replacing Arg25 (precursor numbering) with Gln (pro-FD-R/Q). Activated MASP-1 and MASP-3 catalytic fragments were also included in the study for comparison. We found that the complex formation with C3b enhanced the cleavage rate of FB by FD approximately 20 million-fold. C3bB was also a better substrate for MASP-1, approximately 100-fold, than free FB, showing that binding to C3b renders the scissile Arg-Lys bond in FB to become more accessible for proteolysis. Though easily measurable, this cleavage by MASP-1 is not relevant physiologically. Our approach provides quantitative data for the two-step mechanism characterized by the enhanced susceptibility of FB for cleavage upon complex formation with C3b and the substrate-induced activity enhancement of FD upon its binding to C3bB. Earlier MASP-3 was also implicated as a potential FB activator; however, MASP-3 does not cleave C3bB (or FB) at an appreciable rate. Finally, pro-FD cleaves C3bB at a rate that could be physiologically significant. The zymogenicity of FD is approximately 800, i.e., the cleavage rate of C3bB by pro-FD-R/Q was found to be approximately 800-fold lower than that by FD. Moreover, pro-FD-R/Q at approximately 50-fold of the physiological FD concentration could restore half-maximal AP activity of FD-depleted human serum on zymosan. The observed zymogen activity of pro-FD might be relevant in MASP-3 deficiency cases or during therapeutic MASP-3 inhibition.

Absorption, Distribution, Metabolism, and Excretion of [14C]iptacopan in Healthy Male Volunteers and in In Vivo and In Vitro Studies.

Iptacopan (LNP023) is an oral, small-molecule, first-in-class, highly potent proximal complement inhibitor that specifically binds factor B and inhibits the alternative complement pathway. Iptacopan is currently in development as a targeted treatment of paroxysmal nocturnal hemoglobinuria and multiple other complement-mediated diseases. In this study, the absorption, distribution, metabolism, and excretion (ADME) of iptacopan was characterized in six healthy volunteers after a single 100 mg oral dose of [14C]iptacopan. This was supplemented with an in vivo rat ADME study and metabolite exposure comparisons between human, rat, and dog, in addition to in vitro assays, to better understand the clearance pathways and enzymes involved in the metabolism of iptacopan. The fraction of [14C]iptacopan absorbed was estimated to be about 71%, with a time to maximum concentration of 1.5 hours and elimination half-life from plasma of 12.3 hours. Following a single dose of [14C]iptacopan, 71.5% of the radioactivity was recovered in feces and 24.8% in urine. [14C]iptacopan was primarily eliminated by hepatic metabolism. The main biotransformation pathways were oxidative metabolism via CYP2C8, with M2 being the major oxidative metabolite, and acyl glucuronidation via UGT1A1. The two acyl glucuronide metabolites in human plasma, M8 and M9, each accounted for ≤ 10% of the total circulating drug-related material; systemic exposure was also observed in toxicology studies in rat and dog, suggesting a low risk associated with these metabolites. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan. SIGNIFICANCE STATEMENT: We characterized the pharmacokinetics, excretion, metabolism and elimination of [14C]iptacopan (an oral, selective small-molecule inhibitor of factor B) in healthy human subjects. [14C]iptacopan was primarily eliminated by metabolism. The primary biotransformation pathways were oxidative metabolism via CYP2C8 and acyl glucuronidation via UGT1A1. Direct secretion of iptacopan into urine and potentially bile represented additional elimination mechanisms. Binding of iptacopan to its target, factor B, in the bloodstream led to a concentration-dependent blood:plasma distribution and plasma protein binding of [14C]iptacopan.

Complement proteins in serum astrocyte-derived exosomes are associated with mild cognitive impairment in type 1 diabetes mellitus patients.

BACKGROUND: The complement system plays a crucial role in cognitive impairment. The aim of this study is to investigate the correlation between the complement proteins levels in serum astrocyte-derived exosomes (ADEs) and mild cognitive impairment (MCI) in type 1 diabetes mellitus (T1DM) patients. METHODS: In this cross-sectional study, the patients with immune-mediated T1DM were enrolled. Healthy subjects matched for age and sex with T1DM patients were selected as controls. The cognitive function was evaluated by a Beijing version of the Montreal Cognitive Assessment (MoCA) questionnaire. The complement proteins including C5b-9, C3b and Factor B in serum ADEs were measured by ELISA kits. RESULTS: This study recruited 55 subjects immune-mediated T1DM patients without dementia, including 31 T1DM patients with MCI, 24 T1DM patients without MCI. 33 healthy subjects were enrolled as controls. The results showed higher complement proteins including C5b-9, C3b and Factor B levels in ADEs from T1DM patients with MCI than those in the controls (P < 0.001, P < 0.001, P = 0.006) and T1DM patients without MCI (P = 0.02, P = 0.02, P = 0.03). The C5b-9 levels in ADEs were independently associated with MCI in T1DM patients(OR: 1.20, 95% CI: 1.00-1.44, P = 0.04). The C5b-9 levels in ADEs were significantly correlated with global cognitive scores (ß = -0.360, P＜0.001) and visuo-executive (ß = -0.132, P＜0.001), language(ß = -0.036, P = 0.026) and delayed recall score (ß = -0.090,P = 0.007). There was no correlation between the C5b-9 levels in ADEs and the fasting glucose, HbA1c, fasting c-peptide and GAD65 antibody in T1DM patients. Furthermore, the C5b-9, C3b and Factor B levels in ADEs exhibited a fair combined diagnostic value for MCI, with an area under the curve of 0.76 (95% CI: 0.63-0.88, P = 0.001). CONCLUSION: The elevated C5b-9 levels in ADEswere significantly associated with theMCI in T1DM patients. The C5b-9 in ADEs may be used as a marker of MCI in T1DM patients.

Heat-inactivated Factor B inhibits alternative pathway fluid-phase activation and convertase formation on endothelial cell-secreted ultra-large von Willebrand factor strings.

Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.

Regulatory Role of Anti-Sigma Factor RsbW in Clostridioides difficile Stress Response, Persistence, and Infection.

The anaerobic pathogen Clostridioides difficile, which is a primary cause of antibiotic-associated diarrhea, faces a variety of stresses in the environment and in the mammalian gut. To cope with these stresses, alternative sigma factor B (σB) is employed to modulate gene transcription, and σB is regulated by an anti-sigma factor, RsbW. To understand the role of RsbW in C. difficile physiology, a rsbW mutant (ΔrsbW), in which σB is assumed to be "always on," was generated. ΔrsbW did not show fitness defects in the absence of stress but tolerated acidic environments and detoxified reactive oxygen and nitrogen species better compared to the parental strain. ΔrsbW was defective in spore and biofilm formation, but it displayed increased adhesion to human gut epithelia and was less virulent in a Galleria mellonella infection model. A transcriptomic analysis to understand the unique phenotype of ΔrsbW showed changes in expression of genes associated with stress responses, virulence, sporulation, phage, and several σB-controlled regulators, including the pleiotropic regulator sinRR'. While these profiles were distinct to ΔrsbW, changes in some σB-controlled stress-associated genes were similar to those reported in the absence of σB. Further analysis of ΔrsbW showed unexpected lower intracellular levels of σB, suggesting an additional post-translational control mechanism for σB in the absence of stress. Our study provides insight into the regulatory role of RsbW and the complexity of regulatory networks mediating stress responses in C. difficile. IMPORTANCE Pathogens like Clostridioides difficile face a range of stresses in the environment and within the host. Alternative transcriptional factors like sigma factor B (σB) enable the bacterium to respond quickly to different stresses. Anti-sigma factors like RsbW control sigma factors and therefore the activation of genes via these pathways. Some of these transcriptional control systems provide C. difficile with the ability to tolerate and detoxify harmful compounds. Here, we investigate the role of RsbW in C. difficile physiology. We demonstrate distinctive phenotypes for a rsbW mutant in growth, persistence, and virulence and suggest alternate σB control mechanisms in C. difficile. Understanding C. difficile responses to external stress is key to designing better strategies to combat this highly resilient bacterial pathogen.