Plasma Kallikrein Cleaved H-kininogen: An End-Point Marker for Contact Activation in vitro and ex vivo.

Objectives: The contact system consists of coagulation factor XII (FXII), prekallikrein, and H-kininogen (HK) and plays important roles in many diseases. Plasma kallikrein (PKa) cleaved HK (cHK) is a marker of contact activation. Presently, we developed a specific and precise enzyme-linked immunosorbent assay (ELISA) for determination of cHK in vitro and ex vivo. Methods: Cleaved HK specific mouse monoclonal antibodies (mAbs) were generated using a peptide corresponding to the PKa cleavage site on HK as immunogen. ELISA, surface plasmon resonance analysis, and immunoprecipitation established the specificity of the antibody, which subsequently was used in a sandwich ELISA. The analytical imprecision and the concentration of cHK in a reference population and in women receiving oral contraceptives (OC) were determined. cHK was assessed in vitro in plasma exposed to polytetrafluoroethylene, silicone, and glass tubes. Results: The selected mAb showed excellent specificity towards cHK. The intra-assay and inter-assay CV of the ELISA were 3.6 and 6.0%, respectively. The reference population (60 women, 60 men) displayed a median cHK plasma concentration of 1.38 µg/mL and a reference interval of 0.82 - 2.56 µg/mL. Women receiving OC had significantly higher concentrations, p < 0.001. cHK was significantly elevated in plasma exposed to polytetrafluoroethylene, p = 0.001, and glass, p < 0.0001. Conclusion: The ELISA showed excellent precision and specificity. cHK assessment ex vivo demonstrated ongoing contact activation in healthy individuals, augmented by OC. The cHK antibody and the ELISA could be promising tools in contact activation related diseases and in vitro investigations of the plasma compatibility of blood contacting biomaterials.

Monitoring process-related impurities in biologics-host cell protein analysis.

During biologics development, manufacturers must demonstrate clearance of host cell impurities and contaminants to ensure drug purity, manufacturing process consistency, and patient safety. Host cell proteins (HCPs) are a major class of process-related impurities and require monitoring and documentation of their presence through development and manufacturing. Even in residual amounts, they are known to affect product quality and efficacy as well as patient safety. HCP analysis using enzyme-linked immunosorbent assay (HCP-ELISA) is the standard technique, due to its simple handling, short analysis time, and high sensitivity for protein impurities. Liquid chromatography mass spectrometry (LC-MS) is an orthogonal method for HCP analysis and is increasingly included in regulatory documentation. LC-MS offers advantages where HCP-ELISA has drawbacks, e.g., the ability to identify and quantify individual HCPs. This article summarizes the available knowledge about monitoring HCPs in biologics and presents the newest trends in HCP analysis with current state-of-the-art HCP measurement tools. Through case studies, we present examples of HCP control strategies that have been used in regulatory license applications, using an MS-based coverage analysis and HCP-ELISA and LC-MS for HCP quantification. This provides novel insight into the rapid evolving strategy of HCP analysis. Improvements in technologies to evaluate HCP-ELISA suitability and the implementation of orthogonal LC-MS methods for HCP analysis are important to rationally manipulate, engineer, and select suitable cell lines and downstream processing steps to limit problematic HCPs.

Hemodialysis leads to plasma depletion of lectin complement pathway initiator molecule ficolin-2.

INTRODUCTION: This study aimed to investigate changes in complement system-related molecules in patients undergoing hemodialysis. METHODS: Patients >18 years of age on maintenance hemodialysis were included. Using enzyme-linked immunosorbent assays (ELISA) methods complement related molecules ficolin-1, ficolin-2, ficolin-3 mannose-binding lectin, long pentraxin 3, complement activation products C3c, and complement activation potentials were measured before and after a single hemodialysis treatment. All patients were dialyzed with synthetic high flux filters >1.6 m2 , respectively, Polyamix and Polysulfone, and the Kt/V was maintained >1.3. FINDINGS: Three hundred and four patients were included. There was a modest decrease in plasma level of ficolin-1 (p < 0.001). Ficolin-2 was virtually depleted with median 3.9 (interquartile range [IQR]: 2.6-6.1, range 0.3-13.5) µg/ml before dialysis to median 0.0 (IQR: 0.0-0.5, range 0.0-5.5) µg/ml after dialysis (p < 0.001). No significant difference before and after hemodialysis was seen for mannose-binding lectin and long pentraxin 3 (p > 0.05). In a random subgroup of 160 patients ficolin-2-binding, ficolin-3-mediated lectin pathway capacity and classical pathway capacity were significantly decreased due to hemodialysis. The complement capacity of the alternative pathway was increased after hemodialysis (p = 0.0101), while mannose-binding lectin-mediated lectin pathway capacity was unaltered (p = 0.79). There was an increase in the complement activation product C3c (p < 0.0001), while the concentration of total C4 and C3 did not change (p > 0.158). Multivariate Cox proportional hazard analyses showed an increased risk for all-cause mortality with increasing ficolin-2 (p = 0.002) after hemodialysis. DISCUSSION: Plasma ficolin-2 was virtually depleted from the circulation after hemodialysis. However, elevated plasma ficolin-2 levels after hemodialysis was independently associated with increased mortality.

Prediction of survival in amyotrophic lateral sclerosis: a nationwide, Danish cohort study.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with great heterogeneity. Biological prognostic markers are needed for the patients to plan future supportive treatment, palliative treatment, and end-of-life decisions. In addition, prognostic markers are greatly needed for the randomization in clinical trials. OBJECTIVE: This study aimed to test the ALS Functional Rating Scale-Revised (ALSFRS-R) progression rate (ΔFS) as a prognostic marker of survival in a Danish ALS cohort. METHODS: The ALSFRS-R score at test date in association with duration of symptoms, from the onset of symptoms until test date, (defined as ΔFS') was calculated for 90 Danish patients diagnosed with either probable or definite sporadic ALS. Median survival time was then estimated from the onset of symptoms until primary endpoint (either death or tracheostomy). ΔFS' was subjected to survival analysis using Cox proportional hazards modelling, log-rank test, and Kaplan-Meier survival analysis. RESULTS AND CONCLUSIONS: Both ΔFS' and age was found to be strong predictors of survival of the Danish ALS cohort. Both variables are easily obtained at the time of diagnosis and could be used by clinicians and ALS patients to plan future supportive and palliative treatment. Furthermore, ΔFS', is a simple, prognostic marker that predicts survival in the early phase of disease as well as at later stages of the disease.

Complement Profiles in Patients with Amyotrophic Lateral Sclerosis: A Prospective Observational Cohort Study.

BACKGROUND: The complement system has been suggested to be involved in the pathophysiology of amyotrophic lateral sclerosis (ALS), a progressive motor neuron disease. In the present study, we compared levels of selected complement markers to clinical outcome in ALS patients. METHODS: This observational, explorative cohort study included 92 ALS patients, 61 neurological controls (NCs) admitted for suspected aneurysmal subarachnoid haemorrhage, and 96 neurologically healthy controls (NHCs). Peripheral blood and cerebrospinal fluid (CSF) were obtained for the measurement of ficolin-1, -2, and -3; collectin-11, MBL, MASP-3, MAP-1, C4, C3, PTX-3, and complement activation products C4c, C3bc, and sC5b-9. We recorded clinical outcomes of ALS patients for 24 to 48 months after inclusion in order to analyse the effects of the complement markers on survival time. RESULTS: Compared with both control groups, ALS patients exhibited increased collectin-11, C4 and sC5b-9 in plasma, as well as increased ficolin-3 in CSF. Ficolin-2 was significantly decreased in plasma of the ALS patients compared with NHCs, but not with NCs. The concentration of collectin-11, C3 and C3bc correlated negatively with the revised ALS functional rating scale (ALSFRS-R). No association was found between levels of complement markers and survival as estimated by hazard ratios. CONCLUSION: ALS patients exhibit aberrant expression of selected mediators of the lectin complement pathway as well as increased activation of the terminal complement pathway, corroborating the notion that the complement system might be involved in the pathophysiology of ALS.

Amyotrophic lateral sclerosis and the innate immune system: protocol for establishing a biobank and statistical analysis plan.

INTRODUCTION: Amyotrophic lateral sclerosis (ALS) is a devastating, progressive disease that causes degeneration of the motor neurons leading to paresis of the bulbar and the skeletal musculature. The pathogenesis of ALS remains unknown. We will test the hypothesis that the complement system is involved in the pathophysiology of ALS. This protocol article describes our efforts to establish a national Danish ALS biobank. The primary aim is to obtain biological material from patients with ALS for the current study as well as for future studies. METHODS AND ANALYSIS: We intend to establish an observational ALS biobank; some of the material from this biobank will be used for a prospective, observational case-control study. The participants are patients with ALS, neurologically healthy controls and non-ALS neurological controls. Each participant consents to be interviewed and to donate blood and cerebrospinal fluid to the biobank. Analysis of the complement system will be carried out on the three groups of patients and compared. ETHICS AND DISSEMINATION: The project has been approved by the Committees on Health Research Ethics in the Capital Region of Denmark (Approval number H-16017145) and the Danish Data Protection Agency (file number 2012-58-0004). All results will be published in peer-reviewed, medical journals and presented at scientific conferences. TRIAL REGISTRATION NUMBER: NCT02869048.

Local complement activation is associated with primary graft dysfunction after lung transplantation.

BACKGROUNDThe complement system plays a key role in host defense but is activated by ischemia/reperfusion injury (IRI). Primary graft dysfunction (PGD) is a form of acute lung injury occurring predominantly due to IRI, which worsens survival after lung transplantation (LTx). Local complement activation is associated with acute lung injury, but whether it is more reflective of allograft injury compared with systemic activation remains unclear. We proposed that local complement activation would help identify those who develop PGD after LTx. We also aimed to identify which complement activation pathways are associated with PGD.METHODSWe performed a multicenter cohort study at the University of Pennsylvania and Washington University School of Medicine. Bronchoalveolar lavage (BAL) and plasma specimens were obtained from recipients within 24 hours after LTx. PGD was scored based on the consensus definition. Complement activation products and components of each arm of the complement cascade were measured using ELISA.RESULTSIn both cohorts, sC4d and sC5b-9 levels were increased in BAL of subjects with PGD compared with those without PGD. Subjects with PGD also had higher C1q, C2, C4, and C4b, compared with subjects without PGD, suggesting classical and lectin pathway involvement. Ba levels were higher in subjects with PGD, suggesting alternative pathway activation. Among lectin pathway-specific components, MBL and FCN-3 had a moderate-to-strong correlation with the terminal complement complex in the BAL but not in the plasma.CONCLUSIONComplement activation fragments are detected in the BAL within 24 hours after LTx. Components of all 3 pathways are locally increased in subjects with PGD. Our findings create a precedent for investigating complement-targeted therapeutics to mitigate PGD.FUNDINGThis research was supported by the NIH, American Lung Association, Children's Discovery Institute, Robert Wood Johnson Foundation, Cystic Fibrosis Foundation, Barnes-Jewish Hospital Foundation, Danish Heart Foundation, Danish Research Foundation of Independent Research, Svend Andersen Research Foundation, and Novo Nordisk Research Foundation.

Complement Activation Is Associated With Mortality in Patients With Necrotizing Soft-Tissue Infections-A Prospective Observational Study.

Aim: We assessed whether different complement factors and complement activation products were associated with poor outcome in patients with necrotizing soft-tissue infection (NSTI). Methods: We conducted a prospective, observational study in an intensive care unit where treatment of NSTI is centralized at a national level. In 135 NSTI patients and 65 control patients, admission levels of MASP-1, MASP-2, MASP-3, C4, C3, complement activation products C4c, C3bc, and terminal complement complex (TCC) were assessed. Results: The 90-day mortality was 23%. In a Cox regression model adjusted for sex, and SAPS II, a higher than median MASP-1 (HR 0.378, CI 95% [0.164-0.872], p = 0.0226) and C4 (HR 0.162, 95% CI [0.060-0.438], p = 0.0003), C4c/C4 ratio (HR 2.290 95% CI [1.078-4.867], p = 0.0312), C3bc (HR 2.664 95% CI [1.195-5.938], p = 0.0166), and C3bc/C3 ratio (HR 4.041 95% CI [1.673-9.758], p = 0.0019) were associated with 90-day mortality, while MASP-2, C4c, C3, and TCC were not. C4 had the highest ROC-AUC (0.748, [95% CI 0.649-0.847]), which was comparable to the AUC for SOFA score (0.753, [95% CI 0.649-0.857]), and SAPS II (0.862 [95% CI 0.795-0.929]). Conclusion: In adjusted analyses, high admission levels of the C4c/C4 ratio, C3bc, and the C3bc/C3 ratio were significantly associated with a higher risk of death after 90 days while high admission levels of MASP-1 and C4 were associated with lower risk. In this cohort, these variables are better predictors of mortality in NSTI than C-reactive protein and Procalcitonin. C4's ability to predict mortality was comparable to the well-established scoring systems SAPS score II and SOFA on day 1.

Circulating Ficolin-2 and Ficolin-3 Form Heterocomplexes.

The complement system constitutes an important part of the innate immune system. The collectins and the ficolins are soluble pattern recognition molecules that contribute to complement activation via the lectin pathway. During previous experiments with ficolin-2 and ficolin-3, we have observed that the molecules may interact. We therefore hypothesized the existence of stable ficolin-2/-3 heterocomplexes. We could demonstrate ficolin-2/-3 heterocomplexes in normal human serum and plasma by ELISA using Abs specific for ficolin-2 and ficolin-3. The formation of heteromeric protein complexes were validated by coimmunoprecipitation and Western blot analysis. When recombinant ficolin-2 and recombinant ficolin-3 were mixed, no complexes were formed. However, when coexpressing ficolin-2 and ficolin-3 in Chinese hamster ovary cells, we could detect ficolin-2/-3 heterocomplexes in the supernatant. Furthermore, we measured concentration of the ficolin-2/-3 heterocomplexes in arbitrary units in 94 healthy individuals. We also established the relationship between the concentrations of ficolin-2, ficolin-3, and the ficolin-2/-3 heterocomplexes. We observed that the concentration of the ficolin-2/-3 heterocomplex correlated significantly with ficolin-2 (ρ: 0.24, p < 0.018) and ficolin-3 concentrations (ρ: 0.46, p < 0.0001). In conclusion, we describe a novel protein complex between ficolin-2 and ficolin-3 present in serum and plasma, which might be of additional biological relevance apart from the native ficolin-2 and ficolin-3 molecules.

A novel approach to evaluate ELISA antibody coverage of host cell proteins-combining ELISA-based immunocapture and mass spectrometry.

Monitoring host cell proteins (HCPs) is one of the most important analytical requirements in production of recombinant biopharmaceuticals to ensure product purity and patient safety. Enzyme-linked immunosorbent assay (ELISA) is the standard method for monitoring HCP clearance. It is important to validate that the critical reagent of an ELISA, the HCP antibody, covers a broad spectrum of the HCPs potentially present in the purified drug substance. Current coverage methods for assessing HCP antibody coverage are based on 2D-Western blot or immunoaffinity-purification combined with 2D gel electrophoresis and have several limitations. In the present study, we present a novel coverage method combining ELISA-based immunocapture with protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS): ELISA-MS. ELISA-MS is used to accurately determine HCP coverage of an early process sample by three commercially available anti-Escherichia coli HCP antibodies, evading the limitations of current methods for coverage analysis, and taking advantage of the benefits of MS analysis. The results obtained comprise a list of individual HCPs covered by each HCP antibody. The novel method shows high sensitivity, high reproducibility, and enables tight control of nonspecific binding through inclusion of a species-specific isotype control antibody. We propose that ELISA-MS will be a valuable supplement to existing coverage methods or even a replacement. ELISA-MS will increase the possibility of selecting the best HCP ELISA, thus improving HCP surveillance and resulting in a final HCP profile with the lowest achievable risk. Overall, this will be beneficial to both the pharmaceutical industry and patient safety.

Complement related pattern recognition molecules as markers of short-term mortality in intensive care patients.

OBJECTIVES: To evaluate the complement related pattern recognition molecules (PRMs) PTX3, MBL, CL-11, ficolin-2 and -3, along with the established marker CRP, to predict 28-day mortality and disease severity of sepsis in patients admitted to the intensive care unit (ICU). METHODS: In a single-center, prospective, observational study 547 patients were included over a period of 18 months. Blood samples were obtained at admission to the ICU and the following 4 days. RESULTS: PTX3 baseline levels were significantly higher in non-survivors compared to survivors, whereas MBL and ficolin-2 levels were significantly lower in non-survivors compared to survivors. A PTX3 level above the median was independently associated with 28-day mortality in the adjusted analysis including age, sex, chronic disease and immunosuppression (HR 1.87, 95% CI [1.41-2.48], p < 0.0001), while a MBL level above the median was associated with increased chance of survival (HR 0.75, 95% CI [0.57-0.98], p = 0.034). Ficolin-2 was only borderline significant (HR 0.79, 95% CI [0.60-1.03], p = 0.084). In a ROC analysis PTX3 was superior to CRP in predicting septic shock. CONCLUSIONS: PTX3, MBL and CRP levels were independently associated with 28-day mortality in ICU patients. PTX3 was a better marker of septic shock compared to CRP.

Decreased Ficolin-3-mediated Complement Lectin Pathway Activation and Alternative Pathway Amplification During Bacterial Infections in Patients With Type 2 Diabetes Mellitus.

Bacterial infections are frequent and severe in patients with diabetes mellitus. Whether diabetes per se induces functional alterations in the complement system hampering activation during infection is unknown. We investigated key elements of the complement system during bacterial infections in patients with type 2 diabetes mellitus (T2DM) and compared them to non-diabetic (ND) individuals. Using a prospective design, we included 197 T2DM, and 196 ND subjects, all with clinical diagnosis of acute community-acquired bacterial infections. Functional activities of the ficolin-3-mediated lectin (F3-LP), mannose binding lectin-mediated lectin- (MBL-LP), classical (CP), and alternative pathways (AP), as well as concentrations of complement activation products C4d and sC5b-9 were determined. Functional in vitro activities of F3-LP and AP were significantly higher in T2DM than in ND subjects, (median 64% vs. 45%, p = 0.0354 and 75 vs. 28%, p = 0.0013, respectively), indicating a decreased in vivo activation and lack of consumption of F3-LP and AP in T2DM patients, whereas no difference in functional capacities of CP and MBL-LP were observed between T2DM and ND subjects. Diminished F3-LP and AP activation was most pronounced in diabetic patients with urinary tract infections with positive microbiological culture results for Escherichia coli bacteria. In the T2DM group 3-months mortality significantly associated with diminished F3-LP and AP, but not with CP activation. Concentrations of C4d and sC5b-9 were significantly lower in the T2DM than in ND patients. In conclusion, we found impaired F3-LP activation and lack of AP amplification during bacterial infections in patients with type 2 diabetes, compared to non-diabetic subjects, suggesting a diminished complement mediated protection to bacterial infections in T2DM.

Alpha-cyclodextrin inhibits cholesterol crystal-induced complement-mediated inflammation: A potential new compound for treatment of atherosclerosis.

BACKGROUND AND AIMS: Cholesterol crystal (CC)-induced inflammation is a critical step in the development of atherosclerosis. CCs activate the complement system and induce an inflammatory response resulting in phagocytosis of the CCs, production of reactive oxygen species (ROS) and release of cytokines. The cyclodextrin 2-hydroxypropyl-ß-cyclodextrin has been found to reduce CC-induced complement activation and induce regression of established atherosclerotic plaques in a mouse model of atherosclerosis, thus inhibition of complement with cyclodextrins is a potential new strategy for treatment of inflammation during atherosclerosis. We hypothesized that other cyclodextrins, like α-cyclodextrin, may have related functions. METHODS: The effect of cyclodextrins on CC-induced complement activation, phagocytosis, and production of ROS from granulocytes and monocytes was investigated by flow cytometry and ELISA. RESULTS: We showed that α-cyclodextrin strongly inhibited CC-induced complement activation by inhibiting binding of the pattern recognition molecules C1q (via IgM) and ficolin-2. The reduced CC-induced complement activation mediated by α-cyclodextrin resulted in reduced phagocytosis and reduced ROS production in monocytes and granulocytes. Alpha-cyclodextrin was the most effective inhibitor of CC-induced complement activation, with the reduction in deposition of complement activation products being significantly different from the reduction induced by 2-hydroxypropyl-ß-cyclodextrin. We also found that α-cyclodextrin was able to dissolve CCs. CONCLUSIONS: This study identified α-cyclodextrin as a potential candidate in the search for therapeutics to prevent CC-induced inflammation in atherosclerosis.

Chimeric Proteins Containing MAP-1 and Functional Domains of C4b-Binding Protein Reveal Strong Complement Inhibitory Capacities.

The complement system is a tightly regulated network of proteins involved in defense against pathogens, inflammatory processes, and coordination of the innate and adaptive immune responses. Dysregulation of the complement cascade is associated with many inflammatory disorders. Thus, inhibition of the complement system has emerged as an option for treatment of a range of different inflammatory diseases. MAP-1 is a pattern recognition molecule (PRM)-associated inhibitor of the lectin pathway of the complement system, whereas C4b-binding protein (C4BP) regulates both the classical and lectin pathways. In this study we generated chimeric proteins consisting of MAP-1 and the first five domains of human C4BP (C4BP1-5) in order to develop a targeted inhibitor acting at different levels of the complement cascade. Two different constructs were designed and expressed in CHO cells where MAP-1 was fused with C4BP1-5 in either the C- or N-terminus. The functionality of the chimeric proteins was assessed using different in vitro complement activation assays. Both chimeric proteins displayed the characteristic Ca2+-dependent dimerization and binding to PRMs of native MAP-1, as well as the co-factor activity of native C4BP. In ELISA-based complement activation assays they could effectively inhibit the lectin and classical pathways. Notably, MAP-1:C4BP1-5 was five times more effective than rMAP-1 and rC4BP1-5 applied at the same time, emphasizing the advantage of a single inhibitor containing both functional domains. The MAP-1/C4BP chimeras exert unique complement inhibitory properties and represent a novel therapeutic approach targeting both upstream and central complement activation.

Amyotrophic lateral sclerosis: The complement and inflammatory hypothesis.

Amyotrophic lateral sclerosis (ALS) is a devastating, neurodegenerative motor neuron disease. The aetiology of ALS remains an enigma which hinders the design of an effective treatment to prevent, postpone, or reverse the pathophysiological changes occurring during the aggressive progression of this disease. During the last decade, basic research within the innate immune system, and in particular the complement system, has revealed new, important roles of the innate immune system during development, homeostasis, and ageing within as well as outside the central nervous system. Several lines of evidence indicate that aberrant activation of the complement system locally in the central nervous system as well as systemically may be involved in the pathophysiology of ALS. This exciting new knowledge could point towards the innate immune system as a potential target of medical intervention in ALS. Recently, the historic perception of ALS as a central neurodegenerative disease has been challenged due to the significant amount of evidence of a dying-back mechanism causing the selective destruction of the motor neurons, indicating that disease onset occurs outside the borders of the blood-brain-barrier. This review addresses the function of the innate immune system during ALS. We emphasize the role of the complement system and specifically suggest the involvement of ficolin-3 from the lectin pathway in the pathophysiology of ALS.

Mannose-binding lectin genotypes and outcome in end-stage renal disease: a prospective cohort study.

Background: Patients with end-stage renal disease (ESRD) have high morbidity and mortality rates, with cardiovascular diseases and infections being the major causes of death. Mannose-binding lectin (MBL) has been suggested to play a protective role in this regard. The aim of this study was to investigate a possible clinical association of MBL genotypes (MBL2) with outcome among patients on dialysis or with a functioning graft. Methods: A total of 98 patients with ESRD accepted for living-donor renal transplantation or on the waiting list for transplantation were included and prospectively followed for an average of 9 years (range 7.5-9.9). Medical records were evaluated regarding transplantation status, diabetes mellitus, vascular parameters and infections for all the patients. Cox regression models and logistic regression analysis were used for statistical analyses. The cohort was divided into two groups according to the MBL2 genotype (normal A/A versus variant A/O or O/O). Results: We found no evidence for an association between the MBL2 genotype and all-cause mortality, cardiovascular events or bacterial infections (pneumonia, urinary tract infection, fistula infection or other infections). Conclusion: In this cohort, the MBL2 genotype did not seem to be associated with any long-term clinical effects in ESRD patients on dialysis or with a functioning graft.

Associations of Plasma Nitrite, L-Arginine and Asymmetric Dimethylarginine With Morbidity and Mortality in Patients With Necrotizing Soft Tissue Infections.

BACKGROUND: The nitric oxide system could play an important role in the pathophysiology related to necrotizing soft tissue infection (NSTI). Accordingly, we investigated the association between plasma nitrite level at admission and the presence of septic shock in patients with NSTI. We also evaluated the association between nitrite, asymmetric dimethylarginine (ADMA), L-arginine, L-arginine/ADMA ratio, and outcome. METHODS: We analyzed plasma from 141 NSTI patients taken upon hospital admission. The severity of NSTI was assessed by the presence of septic shock, Simplified Acute Physiology Score (SAPS) II, Sepsis-Related Organ Failure Assessment (SOFA) score, use of renal replacement therapy (RRT), amputation, and 28-day mortality. RESULTS: No difference in nitrite levels was found between patients with and without septic shock (median 0.82 µmol/L [interquartile range (IQR) 0.41-1.21] vs. 0.87 µmol/L (0.62-1.24), P = 0.25). ADMA level was higher in patients in need of RRT (0.64 µmol/L (IQR 0.47-0.90) vs. (0.52 µmol/L (0.34-0.70), P = 0.028), and ADMA levels correlated positively with SAPS II (rho = 0.32, P = 0.0002) and SOFA scores (rho = 0.22, P = 0.01). In a logistic regression analysis, an L-arginine/ADMA ratio below 101.59 was independently associated with 28-day mortality, odds ratio 6.03 (95% confidence interval, 1.41-25.84), P = 0.016. None of the other analyses indicated differences in the NO system based on differences in disease severity. CONCLUSIONS: In patients with NSTI, we found no difference in baseline nitrite levels according to septic shock. High baseline ADMA level was associated with the use of RRT and patients with a low baseline L-arginine/ADMA ratio were at higher risk of dying within 28 days after hospital admission.

Prognostic value of lectin pathway molecules and complement proteins in ascitic fluid and blood in patients with liver cirrhosis.

BACKGROUND AND AIM: Patients with liver cirrhosis and ascites have a poor prognosis with increased risk of infection related death, as advanced stages of cirrhosis are associated with immunodeficiency. We aimed to investigate immunologically active molecules in ascitic fluid and blood and their potential association to survival. MATERIALS AND METHODS: In an exploratory pilot study; blood and ascitic fluid from 34 patients with liver cirrhosis of different etiology were analyzed for pattern recognition molecules (ficolin-1, ficolin-2, ficolin-3 and MBL) and complement proteins (C4 and C3). An observational follow-up study (minimum 17 months) was conducted to assess the association to all-cause mortality or liver transplantation. RESULTS: Ficolin-1, ficolin-2, MBL, C4 and C3 in ascitic fluid and ficolin-1, C4 and C3 in blood were significantly (p = .001-.027) lower in patients with Child-Pugh stage C (n = 16, 47%) compared to Child-Pugh stage B cirrhosis (n = 18, 53%). In multivariate COX-regression analysis low levels of ficolin-1(p = .036) and C3 (p = .025) in ascitic fluid and C4(p = .005) and C3 (p = .032) in serum were associated with all-cause mortality or liver transplantation independent of Child-Pugh score. CONCLUSION: Levels of lectin-complement pathway molecules in ascitic fluid and blood are lower in patients with more advanced stage of cirrhosis. Low C4 and C3 in serum and C3 and ficolin-1 in ascitic fluid are risk factors for all-cause mortality or liver transplantation independently of liver function in patients with cirrhosis and ascites.

Cyclodextrin Reduces Cholesterol Crystal-Induced Inflammation by Modulating Complement Activation.

Cholesterol crystals (CC) are abundant in atherosclerotic plaques and promote inflammatory responses via the complement system and inflammasome activation. Cyclic oligosaccharide 2-hydroxypropyl-ß-cyclodextrin (BCD) is a compound that solubilizes lipophilic substances. Recently we have shown that BCD has an anti-inflammatory effect on CC via suppression of the inflammasome and liver X receptor activation. The putative effects of BCD on CC-induced complement activation remain unknown. In this study, we found that BCD bound to CC and reduced deposition of Igs, pattern recognition molecules, and complement factors on CC in human plasma. Furthermore, BCD decreased complement activation as measured by terminal complement complex and lowered the expression of complement receptors on monocytes in whole blood in response to CC exposure. In line with this, BCD also reduced reactive oxygen species formation caused by CC in whole blood. Furthermore, BCD attenuated the CC-induced proinflammatory cytokine responses (e.g., IL-1α, MIP-1α, TNF, IL-6, and IL-8) as well as regulated a range of CC-induced genes in human PBMC. BCD also regulated complement-related genes in human carotid plaques treated ex vivo. Formation of terminal complement complex on other complement-activating structures such as monosodium urate crystals and zymosan was not affected by BCD. These data demonstrate that BCD inhibits CC-induced inflammatory responses, which may be explained by BCD-mediated attenuation of complement activation. Thus, these findings support the potential for using BCD in treatment of atherosclerosis.

C-Reactive Protein Binds to Cholesterol Crystals and Co-Localizes with the Terminal Complement Complex in Human Atherosclerotic Plaques.

Inflammation is a part of the initial process leading to atherosclerosis and cholesterol crystals (CC), found in atherosclerotic plaques, which are known to induce complement activation. The pentraxins C-reactive protein (CRP), long pentraxin 3 (PTX3), and serum amyloid P component (SAP) are serum proteins associated with increased risk of cardiovascular events and these proteins have been shown to interact with the complement system. Whether the pentraxins binds to CC and mediate downstream complement-dependent inflammatory processes remains unknown. Binding of CRP, PTX3, and SAP to CC was investigated in vitro by flow cytometry and fluorescence microscopy. CRP, PTX3, and SAP bound to CC in a concentration-dependent manner. CRP and PTX3 interacted with the complement pattern recognition molecule C1q on CC by increasing the binding of both purified C1q and C1q in plasma. However, CRP was the strongest mediator of C1q binding and also the pentraxin that most potently elevated C1q-mediated complement activation. In a phagocytic assay using whole blood, we confirmed that phagocytosis of CC is complement dependent and initiated by C1q-mediated activation. The pathophysiological relevance of the in vitro observations was examined in vivo in human atherosclerotic plaques. CRP, PTX3, and SAP were all found in atherosclerotic plaques and were located mainly in the cholesterol-rich necrotic core, but co-localization with the terminal C5b-9 complement complex was only found for CRP. In conclusion, this study identifies CRP as a strong C1q recruiter and complement facilitator on CC, which may be highly relevant for the development of atherosclerosis.