Activation Of Alternative Complement Pathway Research Articles

Initial investigation on the feasibility of porcine red blood cells from genetically modified pigs as an alternative to human red blood cells for transfusion.

The decline in blood donation rates and the ongoing shortage of blood products pose significant challenges to medical societies. One potential solution is to use porcine red blood cells (pRBCs) from genetically modified pigs as an alternative to human red blood cells (hRBCs). However, adverse immunological reactions remain a significant obstacle to their use. This study aimed to evaluate the compatibility of diverse genetically modified pRBCs with human serum. We acquired human complement-competent serum, complement 7 (C7)-deficient serum, and hRBCs from all ABO blood types. Additionally, we used leftover clinical samples from health checkups for further evaluation. pRBCs were collected from wild-type (WT) and genetically modified pigs: triple knockout (TKO), quadruple KO (QKO), and TKO/hCD55.hCD39 knockin (hCD55.hCD39KI). The extent of C3 deposition on RBCs was measured using flow cytometry after incubation in C7-deficient serum diluted in Ca++-enriched or Ca++-depleted and Mg++-enriched buffers. The binding of immunoglobulin (Ig) M/IgG antibody to RBCs after incubation in ABO-type human serum was evaluated using flow cytometry. Naïve human serum- or sensitized monkey serum-mediated hemolysis was also evaluated. Phagocytosis was assessed by incubating labeled RBCs with the human monocytic cell line THP-1 and measurement by flow cytometry. All three genetic modifications significantly improved the compatibility of pRBCs with human serum relative to that of WT pRBCs. The extent of IgM/IgG binding to genetically modified pRBCs was lower than that of WT pRBCs and similar to that of O-type hRBCs. Total and alternative pathway complement activation in all three genetically modified pRBCs was significantly weaker than that in WT pRBCs and did not differ from that in O-type hRBCs. The extent of serum-mediated hemolysis and phagocytosis of these genetically modified pRBCs was low and similar to that of O-type hRBCs. Sensitized monkey serum-mediated hemolysis in QKO and TKO/hCD55.hCD39KI pRBCs was higher than in O-type hRBCs but lower than in TKO pRBCs. The elimination of porcine carbohydrate antigens in genetically modified pigs significantly enhanced pRBC compatibility with naïve human sera, which was comparable to that of O-type hRBCs. These findings provide valuable insights into the development of pRBCs as potential alternatives to hRBCs.

Combined Effects of Spirulina platensis and Pediococcus acidilactici on the Growth Performance, Digestive Enzyme Activity, Antioxidative Status, and Immune Genes in Zebrafish

Abstract The combined effects of Spirulina platensis (SP) and Pediococcus acidilactici (PA) on growth performance, digestive enzyme activity, antioxidative status, and immune genes in zebrafish were investigated in the present study. Four experimental diets were designed: control and three test diets mixed with SP at 2.5%, PA at 107 CFU/g, or a combination of 2.5% SP and 107 CFU/g PA. After 56 days, fish treated with PA and SP mixture had higher final weight, weight gain, SGR, and lower FCR than fish fed the control and SP diets (P<0.05). The results also illustrated that fish fed PA, SP, and their mixture had higher (P<0.05) protease and amylase activities than the control. The lipase activity was significantly higher in fish treated with PA or the mixture of PA and SP than in the control (P<0.05). The alternative complement pathway (ACH50) and lysozyme activity in the mucus samples of fish treated with PA or both PA and SP were significantly higher (P<0.05) than in fish treated with the control and SP diets. The total immunoglobulin level in the skin mucus was significantly higher (P<0.05) in fish fed PA than in control. In the body homogenates samples, the lysozyme activity and immunoglobulin levels were markedly higher (P<0.05) in fish treated with the mixture of PA and SP than in the control. The dietary PA and SP diet mixture improved the glutathione peroxidase, superoxide dismutase, and total antioxidative capacity. The expression of IL-1β and IL-8 genes in fish treated with PA or the mixture of PA and SP was significantly higher (P<0.05) than in fish treated with the control or SP diets. Fish treated with PA, SP, or both PA and SP had marked (P<0.05) upregulation of the lysozyme gene expression. In conclusion, the mixture of S. platensis and P. acidilactici is more effective than using each individually for improving the growth performance, digestive enzyme activity, and the immune and antioxidative capacity of zebrafish.

Results of a randomized double-blind placebo-controlled Phase 2 study propose iptacopan as an alternative complement pathway inhibitor for IgA nephropathy

Targeting the alternative complement pathway is an attractive therapeutic strategy given its role in the pathogenesis of immunoglobulin A nephropathy (IgAN). Iptacopan (LNP023) is an oral, proximal alternative complement inhibitor that specifically binds to Factor B. Our randomized, double-blind, parallel-group adaptive Phase 2 study (NCT03373461) enrolled patients with biopsy-confirmed IgAN (within previous three years) with estimated glomerular filtration rates of 30 mL/min/1.73 m2 and over and urine protein 0.75 g/24 hours and over on stable doses of renin angiotensin system inhibitors. Patients were randomized to four iptacopan doses (10, 50, 100, or 200 mg bid) or placebo for either a three-month (Part 1; 46 patients) or a six-month (Part 2; 66 patients) treatment period. The primary analysis evaluated the dose–response relationship of iptacopan versus placebo on 24-hour urine protein-to-creatinine ratio (UPCR) at three months. Other efficacy, safety and biomarker parameters were assessed. Baseline characteristics were generally well-balanced across treatment arms. There was a statistically significant dose-response effect, with 23% reduction in UPCR achieved with iptacopan 200 mg bid (80% confidence interval 8 -34%) at three months. UPCR decreased further through six months in iptacopan 100 and 200 mg arms (from a mean of 1.3 g/g at baseline to 0.8 g/g at six months in the 200 mg arm). A sustained reduction in complement biomarkers levels including plasma Bb, serum Wieslab, and urinary C5b-9 was observed. Iptacopan was well-tolerated, with no reports of deaths, treatment-related serious adverse events or bacterial infections, and led to strong inhibition of alternative complement pathway activity and persistent proteinuria reduction in patients with IgAN. Thus, our findings support further evaluation of iptacopan in the ongoing Phase 3 trial (APPLAUSE-IgAN; NCT04578834).

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

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

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 with disease activity and outcomes. This retrospective study included 125 biopsy-proven IgAN patients (age 39.9 years, 75% male, eGFR 82mL/min, proteinuria 0.5g/day) enrolled from 1984 to 2010 and followed for a minimum of 18 months. Monitoring continued until the last follow-up, end-stage kdiney disease (ESKD), or death. Serum Bb fragment was measured using ELISA at diagnosis. Oxford classification and global optical score (GOS) were utilized for pathology assessment. Patients were followed for a median of 16 years, 42% developed CKD stage 3 or higher, 19% reached ESKD, and 9% died. Serum Bb fragment concentration negatively correlated with eGFR values at 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≥14.3μg/mL had shorter mean kidney survival time (19.5vs 22.7 years, p=0.07); after adjusting for progression risk factors, the association persisted (HR 4.76, 95% CI 1.56-14.43). 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.

Dietary fulvic acid increased growth, stress tolerance and disease resistance against Vibrio harveyi in Asian seabass (Lates calcarifer) juvenile

An 8-week husbandry trial was carried out toexamine the impacts of graded levels of dietary fulvic acid (FA) on growth, stress, and disease resistance of Asian seabass (Lates calcarifer, 38.2 g) juveniles. Five levels of FA were included in a basal diet (470 g kg-1 crude protein, 150 g kg-1 crude lipid) to prepare experimental diets, including: 0 (control), 2.5, 5.0, 10, and 15 g kg-1 diet. Two hundred and twenty-five fish (38.2 ± 0.1 g) were stocked randomly into 15 1000-L circular polyethylene tanks (15 fish tank-1) filled with 800 L disinfected seawater and fed with the experimental feeds three times daily. Each dietary treatment had three replicates. Tanks were supplied with seawater (32.2 °C, 45.0 ppt) in a flow-throw system. At the end of the husbandry trial, fish were challenged with Vibrio harveyi and air exposure stress for one min. Fish fed FA-supplemented diets showed a positive quadratic growth response, and those fed with a 5 g FA kg-1 diet had higher final weight and weight gain percentage than the control. Feed intake in fish fed FA-supplemented diets were higher than in control, showing significant linear and quadratic trends (P < 0.05). The highest and lowest skin mucus lysozyme (LYZ) and serum alternative complement pathway (ACH50) activities were in fish fed 10 and 15 g FA kg-1 diets, respectively, exhibiting a quadratic response to dietary FA. Serum total protein level (TP) in fish fed FA-supplemented diets was higher than in the control group. Medium or high levels (10–15 g kg-1) of FA significantly enhanced the survival rate against Vibrio harveyi, which was associated with increased activities of serum lysozyme and ACH50 as well as liver glutathione peroxidase activity in these groups. Air exposure induced oxidative stress in all treatments and gradually reduced antioxidant enzyme activities associated with increased malondialdehyde content in the liver. Furthermore, air exposure suppressed some immune responses, including serum total immunoglobulin and TP levels. Still, LYZ activity was restored after 24 h, with the highest level found in fish fed 5 g FA kg-1 diet. Based on the broken-line regression analysis, the adequate level of FA in the diet for Asian seabass is 4.4 g kg-1.

Urinary markers of the alternative and lectin complement pathway are increased in IgA vasculitis nephritis.

IgA vasculitis (IgAV) is the most common form of childhood vasculitis. Nephritis (IgAVN) occurs in 50% of patients and 1-2% progress to chronic kidney disease stage 5. The pathophysiology of nephritis remains largely unknown, but recent evidence suggests that the complement system may be involved. The aim of this cross-sectional study was to explore whether there is evidence of alternative and/or lectin complement pathway activation in children with IgAVN. Children with IgAV were recruited and grouped according to proteinuria: IgAVN or IgAV without nephritis (IgAVwoN). Age and sex-matched healthy controls (HCs) were also recruited. Cross-sectional urine and plasma concentrations of complement factor D (CFD), factor B (CFB), and MBL-associated protease 1 (MASP-1) were performed using commercially available enzyme-linked immunoassays. A total of 50 children were included (IgAVN, n=15; IgAVwoN, n=20, HCs, n=15). The mean age was 8.5±3.7 years old, male:female ratio was 1:1. Urinary CFD and CFB concentrations were statistically significantly increased in children with IgAVN (3.5±5.4μg/mmol; 25.9±26.5μg/mmol, respectively) compared to both IgAVwoN (0.4±0.4μg/mmol, P=0.002; 9.2±11.5μg/mmol, P=0.004) and HCs (0.3±0.2μg/mmol, P<0.001; 5.1±6.0μg/mmol, P<0.001). No statistically significant difference was reported for the plasma concentrations of CFD and CFB. Urinary MASP-1 concentrations were statistically significantly increased in IgAVN (116.9±116.7ng/mmol) compared to HCs (41.4±56.1ng/mmol, P=0.006) and plasma MASP-1 concentrations were increased in IgAVwoN (254.2±23.3ng/mL) compared to HCs (233.4±6.6ng/mL, P=0.046). There is evidence of complement pathway products in the urine of children with IgAVN that warrants further investigation.

Comparative characteristics of the complement system in patients with C3-glomerulopathy and atypical hemolytic uremic syndrome of chronic course who suffered an acute episode of thrombotic microangiopathy

To compare changes in the complement system in C3-glomerulopathy (C3-GP) and atypical hemolytic uremic syndrome (aHUS) after the relief of an acute episode of thrombotic microangiopathy. The study included 8 patients diagnosed with C3-GP and 8 with aHUS in remission. The blood levels of the complement system components were determined: C3, C4, C3a, C5a, factor H (CFH), factor B (CFB), membrane-attacking complex (MAC), antibodies to C3b (anti-C3b-AT), the level of hemolytic activity (CH50), the content of factor D (CFD) in the urine. C3 and CH50 levels were within the reference range in both groups, however, in the C3-GP group they were at the lower limit, and C3 level was significantly lower than in the aHUS group: 0.56 [0.44; 0.96] vs 1.37 [1.16; 2.52] (p=0.003). CFB increased level was detected in both groups, but in the C3-GP group it was significantly lower than in the aHUS group - 275.1 [222.1; 356.6] vs 438.7 [323.3; 449.3] (p=0.010). C3a, C5a and MAC levels were increased in both groups, but the maximum was in the C3-GP group, and the MAC level in the C3-GP group was 2 times higher than that in aHUS, and these differences reached statistical significance - 123 555±6686 vs 5603±1294 (p=0.036). CFH and CFD levels was increased in both groups, but their highest values was in the aHUS group. Alternative complement pathway activation signs were present in both groups of patients with complement-mediated nephropathies, regardless the stage of the disease. In C3-GP, alternative complement pathway activation was more pronounced than in aHUS after the relief of an acute episode of thrombotic microangiopathy.

Complement and coagulation crosstalk – Factor H in the spotlight

The immune complement and the coagulation systems are blood-based proteolytic cascades that are activated by pathway-specific triggers, based on protein–protein interactions and enzymatic cleavage reactions. Activation of these systems is finely balanced and controlled through specific regulatory mechanisms. The complement and coagulation systems are generally viewed as distinct, but have common evolutionary origins, and several interactions between these homologous systems have been reported. This complement and coagulation crosstalk can affect activation, amplification and regulatory functions in both systems. In this review, we summarize the literature on coagulation factors contributing to complement alternative pathway activation and regulation and highlight molecular interactions of the complement alternative pathway regulator factor H with several coagulation factors. We propose a mechanism where factor H interactions with coagulation factors may contribute to both complement and coagulation activation and regulation within the haemostatic system and fibrin clot microenvironment and introduce the emerging role of factor H as a modulator of coagulation. Finally, we discuss the potential impact of these protein interactions in diseases associated with factor H dysregulation or deficiency as well as evidence of coagulation dysfunction.

Can a Single Glomerulus Morphology Implicate Successful Therapy?

Recurrent gross hematuria of glomerular origin is frequently encountered in clinical practice, and in absence of specific serological marker, renal biopsy is mandatory to address the definitive diagnosis, and set out an appropriate therapeutic protocol. Technical deficiencies associated with practice of renal biopsy are frequently encountered, as inadequate number of glomeruli or poor immunofluorescence staining of kidney biopsy specimen; however, these deficiencies can be offset by detailed electron microscopy analysis of a single abnormal glom.We present a single middle-aged Libyan woman, with a rare glomerular disease, related to abnormal activation of alternative complement pathway, where renal biopsy report was initially not adequate and lacking immunohistochemistry workup. However, electron microscopy reports a characteristic abnormal glomerular deposit, coupled with clinical and biochemical data that guided our therapeutic protocol.In a middle-aged female who presented with recurrent gross hematuria and nephrotic range proteinuria, we should suspect a glomerular pathology. Further to immunoglobulin A nephropathy or lupus nephritis, particularly in presence of complement abnormalities and negative serology for glomerulopathy-related autoantibodies, dense deposit disease and C3 glomerulonephritis that are rare complement mediated glomerulopathy should be considered as a seronegative lupus nephritis-equivalent, in terms of their membranoproliferative features on light microscopy, and when setting out appropriate therapeutic protocol. Patient and family counseling for C3 glomerulopathy is essential because this type of glomerulopathy has a recurrence rate after kidney transplant.

#4463 JAPANESE AND WHITES SHARE SIMILAR IPTACOPAN PHARMACOKINETICS AND PHARMACODYNAMICS

Abstract Background and Aims Iptacopan (LNP023) is a first-in-class, oral, proximal complement inhibitor that specifically binds to Factor B and inhibits the alternative complement pathway (AP). Current Phase III studies of iptacopan focus on diseases associated with AP activation, such as paroxysmal nocturnal hemoglobinuria, C3 glomerulonephritis, IgA nephropathy, and atypical hemolytic uremic syndrome. These studies are enrolling patients across geographical regions and ethnicities, including those from Japan. The aim of this study was to evaluate the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of iptacopan in Japanese patients. Method CLNP023 × 1102 was a randomized, subject-blinded, placebo-controlled, single-dose Phase I study conducted in Japan in healthy Japanese male subjects to assess safety, tolerability, PK, and AP blood biomarkers (Wieslab, Bb) in three dose cohorts, 25, 100, and 400 mg (8 active/2 placebo per cohort). Subjects were dosed on day 1 and observed for 96 hours post-dose. PK and PD data from this study was compared to White data from the similarly designed, previous CLNP023 × 2101 first-in-human study. Results Iptacopan was well tolerated in both Japanese and White subjects. White subjects were on average 15.7 years older and 19.5 kg heavier than Japanese subjects. Iptacopan mean (±SD) Cmax and AUCinf as well as mean (±SD) % change from baseline at 12 hours post-dose for Wieslab and Bb by dose are shown in the table below. All three dose groups manifested a general trend of increased systemic exposure and increased AP biomarker inhibition with increasing dose in both Japanese and White subjects. Conclusion Japanese and White healthy subjects had similar PK and PD results at all dose levels. The slightly higher Cmax and AUCinf in Japanese subjects may be explained in part by the lower average weight of these subjects. This study provides reassurance that there are no clinically meaningful differences in the human pharmacology of iptacopan between these ethnic groups.

#3278 HUMAN PHARMACOKINETICS OF MULTIPLE, STEADY-STATE DOSING OF IPTACOPAN

Abstract Background and Aims Iptacopan (LNP023) is a first-in-class, oral, proximal complement inhibitor that specifically binds to Factor B and inhibits the alternative complement pathway (AP). Current Phase III studies of iptacopan focus on diseases associated with AP activation, such as paroxysmal nocturnal hemoglobinuria, C3 glomerulonephritis, IgA nephropathy, and atypical hemolytic uremic syndrome. Early modeling efforts suggested that an iptacopan concentration of ∼1000 ng/mL would provide near-maximal AP inhibition. The aim of this study was to evaluate the PK of oral iptacopan in healthy subjects to determine the dose that results in a ∼1000-ng/mL mean steady-state concentration over the entire 12-h dosing interval. Method In this blinded, controlled, randomized study, a total of 32 healthy subjects were enrolled into 5 cohorts receiving twice-daily dosing for 14 days: placebo (n = 8) or iptacopan 25, 50, 100, or 200 mg (n = 6 per group). Plasma iptacopan concentration was intensively measured for 72 hours post day 14 dose using a validated LC-MS/MS assay (LLOQ = 1 ng/mL). Results Iptacopan treatment was safe and well tolerated at all dose levels. Iptacopan steady-state concentration-time curves are shown below. Iptacopan was rapidly absorbed, with a median Tmax of approximately 2 hours (Figure 1). The mean half-life was moderately long at 18 to 25 hours. The inter-subject variability (CV%) of iptacopan Cmax and AUCinf was low at approximately 12% to 27%, and both parameters were under-dose proportional with increasing dose. At all time points from 0 to 12 hours, mean iptacopan concentration for the 200-mg dose cohort was &amp;gt;1000 ng/mL. Conclusion Iptacopan was rapidly absorbed, had low inter-subject variability, and a moderately long half-life. Only the 200-mg b.i.d dose produced mean iptacopan concentrations of ≥1000 ng/mL over the dosing interval, and thus is expected to result in near-complete, continuous AP inhibition. These results support the rationale for use of iptacopan 200 mg twice daily as the therapeutic dose in ongoing clinical trials and provide evidence of durable AP inhibition in patients administered oral iptacopan therapy.

#4447 EFFECT OF RENAL IMPAIRMENT ON IPTACOPAN PHARMACOKINETICS

Abstract Background and Aims Iptacopan (LNP023) is a first-in-class, oral, proximal complement inhibitor that specifically binds to Factor B and inhibits the alternative complement pathway (AP). Current Phase III studies of iptacopan focus on diseases associated with AP activation, such as paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulonephritis (C3G), IgA nephropathy (IgAN), and atypical hemolytic uremic syndrome. The liver is the primary route of iptacopan elimination. Based on a previous healthy volunteer pharmacokinetic (PK) study, the contribution of the renal route of iptacopan elimination was measured to be low, approximately 14%. A population PK (PopPK) analysis was done to further quantify the effect of renal impairment on iptacopan PK. Method A PopPK dataset pool was created by pooling across 6 Phase II/III studies in PNH, C3G, and IgAN indications. The pool comprised patient data on the two highest dose levels (100 and 200 mg bid) and included 2439 datapoints in 234 unique patients. Factors (baseline characteristics) that could possibly affect exposure, body weight, age, gender, ethnicity, and eGFR (mL/min/1.73 m2) at baseline were investigated. The range of eGFR was 27.4 to 142.8 mL/min/1.73 m2. Age, ethnicity, and body weight were included as the significant covariates in the final model. Results The median eGFR was 87.5 mL/min/1.73 m2. As shown in the table below, the PopPK model detected a significant (p = 4.3 × 10−11) but modest effect of eGFR on simulated mean AUC0-24. At an eGFR of 34.3, AUC0-24 is expected to increase by only 38%, compared with median eGFR. Changes of eGFR of ≤15% with respect to median eGFR were observed with eGFR range between 59.7 and 132.0 mL/min /1.73 m2. Conclusion Consistent with previously observed primarily hepatic clearance of iptacopan in preclinical and clinical studies, renal impairment down to an eGFR of 34.3 did not have a clinically meaningful effect on iptacopan PK. Currently, iptacopan PK in patients with eGFR &amp;lt;30 mL/min/1.73 m2 is being explored. These results support the use of iptacopan in patients with mild and moderate renal impairment.

#4015 RECOMBINANT HIGH-DENSITY LIPOPROTEIN MODULATES INFLAMMATORY RESPONSE AND RENAL DYSFUNCTION IN A SWINE MODEL OF SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Sepsis is a severe and dysregulated inflammatory disease that often precedes the development of acute kidney injury (AKI) with consequent worsening outcome. Although clinical data demonstrate that high-density lipoprotein (HDL) levels drop in septic patients with a poor prognosis, little is known about the molecular basis of HDL's role in systemic inflammation and renal function. Here we investigate the possible effects of a novel engineered HDL-mimetic (CER-001) in a swine model of lipopolysaccharide (LPS)-induced AKI. Method Sepsis was induced by intravenous infusion of a saline solution containing 300 μg/kg of LPS in a porcine model. The animals were randomized into three groups: LPS (endotoxemic pigs, n = 6), CER20 (endotoxemic pigs treated with a single dose of CER-001 20mg/kg; n = 6), and CER20 × 2 (endotoxemic pigs treated with two doses of CER001 20mg/kg; n = 6). Animals were sacrificed after 24h from the start of experimental procedure. Renal histologic and biochemical changes were analyzed. Endothelial dysfunction biomarkers, circulating pro-inflammatory mediators, LPS and Apolipoprotein A-I (Apo A-I) levels were quantified with ELISA assay. Systemic complement activation was evaluated by Wieslab kit. Results Untreated animals were highly susceptible to LPS challenge and usually succumbed before completing the study protocol (LPS group 16.7%). CER-001 treatment increased the survival rate of endotoxemic pigs, compared to the LPS group (CER20, 50%; CER20 × 2, 66.7%). Furthermore, as shown in Figure 1A, LPS injection led to a time-dependent increase of IL-6 in endotoxemic animals respect to basal condition (T0). CER-001 treatment was able to reverse LPS effects. In particular, the second infusion of CER-001 three hours after the first dose (T3) strongly reduced IL-6 serum levels back to basal level (LPS p &amp;lt;0.05). Similarly, we found high levels of TNF-α and MCP-1 in endotoxemic pigs that were significantly decreased in both CER-001 treatment arms (T24, CER20 × 2 vs LPS group, IL-6, p = 0.0086; TNF-α, p&amp;lt;0.0001; MCP-1, p = 0.0009). In addition, CER-001 treatment ameliorated systemic endothelial dysfunction by reducing VCAM and ICAM serum levels. A significant activation of classical and alternative complement pathway (vs T0 p &amp;lt;0.05) was observed at 1h, 3h and 24h after LPS infusion. CER001 treatment significantly prevented systemic complement activation in both treated groups (vs LPS p &amp;lt;0.05). We also investigated whether CER001 infusions significantly prevented renal tissue damage. Endotoxemic pigs presented oliguric AKI with increased tubulo-interstitial infiltrate, extensive collagen deposition, and glomerular thrombi; CER-001 treatment preserved renal parenchyma, recovered urine output, decreased creatine levels, and reduced the biomarkers of tubular damage, Cystatin C and KIM-1, both in serum and urine samples. Considering that HDL has a very high affinity for LPS, we evaluated the circulating LPS concentration in treated animals. We observed that LPS levels were greatly reduced in treated animals and the effects are more evident after the second infusion of CER-001(Figure 1B). Therefore, we also examined LPS levels in bile samples and we observed a dose-dependent increasing amount of endotoxin in the CER001 treated septic pigs. Conclusion This preclinical data indicates that CER001 treatment prevents systemic inflammation thereby limiting renal damage. The mechanism of action is two-fold consisting of both the scavenging of endotoxin and a direct anti-inflammatory effect of CER-001.

#3053 CORRELATION OF SYSTEMIC AND IN SITU BIOMARKERS OF COMPLEMENT ACTIVATION WITH HISTOPATHOLOGICAL FEATURES AND PROGNOSIS IN C3 GLOMERULOPATHY

Abstract Background and Aims C3 glomerulopathy is a rare complement mediated disease, resulting from complement alternative pathway (AP) activation. In addition to AP activation, there are emerging evidence for terminal pathway (TP) implication in kidney damage. A C3G histopathologic index has recently been proposed to evaluate both activity and chronicity parameters. Only a chronicity index &amp;gt; 4 has been independently validated as prognosis factor for end stage kidney disease. We aimed to determine the impact of local activation of the TP on disease phenotype. Method C3G histopathological index grading were evaluated via central review of 53 kidney biopsies from 43 patients carrying C3G. C5b-9 staining was performed on 53 FFPE kidney sections and quantified (scoring from grade 0 to 3 for C5b-9 deposits in glomeruli and from 0 to 2 in the tubulointerstitial compartment). Clinical data, C3 and sC5b-9 levels at the time of KB were retrospectively collected. Results KB were performed at diagnosis for n = 31. In 22 cases, KB was performed during follow up, under specific treatment (n = 10), without treatment (n = 7), or at disease relapse (n = 5). Twenty-five (47%) were children. Median[Q1-Q3] C3 level at KB was 623 mg/l [236-931]mg/l. Median[Q1-Q3] sC5b-9 level at KB, available in 16, was 374 ng/ml [203-1295]. Median [Q1-Q3] activity and chronicity index were respectively 9[6-12] and 1[0-6]. We confirmed the prognostic impact of chronicity index (p = 0.03) (Figure 1A) without impact of the activity index. Glomerular C5b-9 staining was positive in 47/53 (87%) (grade 1: n = 16 (30%); 2: n = 17 (32%); 3: n = 14 (26%)) and n = 40 (75%) had interstitial C5b-9 staining (grade 1: n = 28 (53%); 2: n = 12 (23%)). Soluble C5b-9 measurement correlated with intensity of C5b-9 glomerular deposit (coef corr = 0.69). As compared to patients with normal C3 level, patients with AP activation had higher proteinuria but kidney function at KB was similar. We found distinct histological features according to C3 plasmatic level (Table 1): patients with low C3 level had higher histological activity index (p = 0.03) and higher glomerular and tubulointerstitial C5b-9 staining (p = 0.001 and p = 0.002 respectively). C5b-9 glomeruli staining allows the identification of 3 groups of KB (1: no or low, 2: medium and 3: high C5b-9 staining) with distinct histological and clinical features. Activity index were higher in groups 2 &amp; 3 (p = 0.05) and chronicity index in groups 1 &amp; 3 (p = 0.003) (Table 2). Renal prognosis was poorer in patients with higher glomerular C5b-9 deposits (Figure 1B). Patients with chronicity index &amp;gt;4 and high C5b-9 staining had the poorest renal survival (Figure 1C). Conclusion Our results suggest that TP activation in glomeruli may have an impact on histological features, contributing therefore to renal prognosis. Deeper exploration of correlation between systemic and in situ complement activation and histological features could allow to better identify patients with worst prognosis or who could benefit from emerging complement inhibition therapeutics.

#5076 MONOCLONAL GAMMOPATHY IN C3 GLOMERULOPATHY: DOES THE BUCKET STOP THERE?

Abstract Background and Aims C3 glomerulopathy (C3G) is a rare diagnosis, characterized by dysregulation of the alternative pathway of complement, classically presenting in children and young adults. Recently, it has been reported the association between the production of a monoclonal protein and the development of C3G, affecting a larger age span. Method We report the case of a 53-year-old male patient, that attended the emergency department due to persistent holocranial headache. He was an IV drug abuser with a history of treated Hepatitis C infection and free lambda light chain monoclonal protein, being studied by the Haematology Department. On admission, he was hypertensive (187/98 mmHg), and labs showed an acute on chronic kidney disease (serum creatinine 2.35 mg/dL from a basal of 1.5 mg/dL) with an active urinary sediment (38 erythrocytes/field, with dysmorphia), a urinary protein/creatinine ratio of 626 mg/g, and C3 consumption (0.52 g/L). The patient was admitted, and a kidney biopsy was performed. Results Further lab results confirmed a serum IgG Lambda monoclonal protein. Autoantibodies screening (ANCAs, ANAs, Anti-GBM), ASLO and Rheumatoid Factor were negative. Cryoglobulinemia was detected, and the cryoprecipitate's immunofixation revealed monoclonal proteins IgG Lambda and IgM Kappa. HCV viral load was negative. Bone marrow biopsy revealed a small population (&amp;lt; 3%) of plasma cells with Lambda light chain restriction. Kidney biopsy's light microscopy showed increased mesangial matrix and cells, endocapillary hypercellularity, and some glomeruli with lobulated appearance. Five out of 15 glomeruli presented fibrocellular crescents. Immunofluorescence showed C3 dominant staining on capillary walls and in the mesangium, with absent IgG and light chain deposits. A presumptive diagnosis of C3 glomerulopathy was made and the patient was empirically started on corticosteroids (3 pulses of intravenous methylprednisolone, followed by oral prednisolone 1mg/Kg/day), which was followed by paraprotein-directed chemotherapy with bortezomib, cyclophosphamide and dexamethasone (VCD). The patient is currently on the third VCD cycle, presenting both renal and haematological responses: recovery of serum creatinine to basal levels (1.54 mg/dL), resolution of haematuria and proteinuria, normalisation of C3 levels. Serum immunofixation is currently negative for monoclonal protein, with normal serum immunoglobulin levels. Electron microscopy revealed electron-dense deposits in the mesangium, and mesangium-capillary transition, supporting the diagnosis of C3 glomerulonephritis. Genetic testing of complement-related genes revealed a rare missense variation of unknown significance (c.4855A&amp;gt;C) on the C3 gene, that has been previously reported in association with C3 glomerulopathy and atypical Haemolytic-Uremic syndrome. Conclusion This case represents a monoclonal gammopathy associated-C3 glomerulonephritis with a favourable response to paraprotein-directed chemotherapy. Although the association between monoclonal gammopathy and C3G rests on epidemiologic findings, experimental evidence suggests that several monoclonal proteins have complement dysregulation features, which may enhance alternate complement pathway activation. Our patient's response is in line with observational data that have shown improved renal outcomes in patients who achieve a haematological response following clone-directed chemotherapy, which further supports the role of monoclonal gammopathy in C3G pathogenesis [1]. Interestingly, the genetic variation identified in our patient might have provided a favourable genetic background for the development of C3G, which is thought to rely upon a complex interaction of triggering events, such as a monoclonal gammopathy, and underlying complement abnormalities, such as genetic variants in complement genes.

Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

Venous thromboembolism (VTE) is a common, multi-causal disease with potentially serious short- and long-term complications. In clinical practice, there is a need for improved plasma biomarker-based tools for VTE diagnosis and risk prediction. Here we show, using proteomics profiling to screen plasma from patients with suspected acute VTE, and several case-control studies for VTE, how Complement Factor H Related 5 protein (CFHR5), a regulator of the alternative pathway of complement activation, is a VTE-associated plasma biomarker. In plasma, higher CFHR5 levels are associated with increased thrombin generation potential and recombinant CFHR5 enhanced platelet activation in vitro. GWAS analysis of ~52,000 participants identifies six loci associated with CFHR5 plasma levels, but Mendelian randomization do not demonstrate causality between CFHR5 and VTE. Our results indicate an important role for the regulation of the alternative pathway of complement activation in VTE and that CFHR5 represents a potential diagnostic and/or risk predictive plasma biomarker.

Multiple Complement Pathway-related Proteins Might Regulate Immunopathogenesis of Major Depressive Disorder

Exacerbated inflammatory pathway has emerged as a predominant etiological construct of major depressive disorder (MDD). Innate immune molecules like complement proteins induce inflammatory responses and also regulate key neurobiological processes. However, there is a dearth of literature on the impact of critical complement proteins in MDD. Herein, plasma profiling of seven complement proteins was carried out to obtain a better insight into the role of the complement pathway in MDD. Plasma levels of C1q, C3, C3b/iC3b, C4, Factor B, Factor H, and properdin were assayed in 22 patients with MDD and 27 healthy controls by multiplex suspension assay. The patients with MDD were diagnosed as per DSM IV-TR. Hamilton Depression Rating Scale (HAM-D), Montgomery Depression Rating Scale and Clinical Global Improvement were used for clinical assessments of the patients. The plasma levels of these complement proteins were also correlated with various clinical scores and phenotypes of MDD. The patients with MDD and healthy controls did not differ in terms of age and gender (p > 0.1). The patients with MDD had a mean duration of illness of around 3 years, with average number of depressive episodes being 6 and the mean HAM-D score was 19. Of the seven complement components, the plasma levels of C1q, Factor B, and Factor H (p ≤ 0.05) were significantly elevated in MDD patients compared to healthy controls. However, the plasma levels of these complement proteins were not found to correlate with the clinical profile of MDD patients. Both Factor B and Factor H are crucial in the induction and regulation of the alternative pathway of complement activation. The alternative pathway also plays a critical role in inflammation. These findings suggest an important role of the alternative complement pathway in immuno-inflammation in MDD.

Leukocytes, microRNA, and complement activity in raw, heat-treated, and frozen colostrum and their dynamics as colostrum transitions to mature milk in dairy cows

The objectives of this study were to evaluate the abundance and viability of leukocytes, the abundance of microRNA, and the activity of the complement pathway in (1) colostrum following heat-treatment or freezing, and (2) colostrum, transition milk, and mature milk. In experiment 1, composite colostrum samples were harvested from individual cows (n = 14) on a commercial dairy farm in NY and split into 3 aliquots using single-use colostrum bags. One aliquot was immediately cooled on ice following harvest (RAW) and stored at 4°C overnight, one was heat-treated for 60 min at 60°C (HT) before being cooled on ice and stored at 4°C overnight, and one was frozen at -20°C overnight (FR). The following morning, all samples were warmed to 40°C before further processing. In experiment 2, cows were sampled in a longitudinal study where composite samples were collected from colostrum (first milking, n = 23), transition milk (3 to 4 d postpartum, n = 13), and mature milk (6 to 7 d postpartum, n = 13). In both experiments colostrum was harvested from the first milking within 8 h of calving and samples were processed within 14 h of collection. Colostral leukocytes were isolated before viability was determined by trypan blue exclusion and manual differential cell counts were performed. Extracellular vesicles were isolated from whey by ultracentrifugation to isolate and quantify microRNA. Activity of the alternative complement pathway was determined in casein-depleted whey by semi-solid phase hemolysis assay. Somatic cell counts were determined for all raw samples. Macrophages and neutrophils made up the greatest proportion of leukocytes in colostrum followed by lymphocytes. Lymphocyte proportion increased as colostrum transitioned to mature milk, but overall somatic cell numbers declined concurrently. Viable cells were not isolated from HT or FR samples. Abundance of microRNA isolated from transition and mature milk was decreased compared with colostrum, did not differ between HT and RAW, but was increased in FR compared with RAW. Alternative complement pathway activity was decreased in HT, but not FR compared with RAW, and was not measurable in transition or mature milk. Postharvest heat-treatment and freezing of colostrum eliminated viable colostral leukocytes and affected microRNA abundance and complement activity. Leukocyte proportions, microRNA abundance, and complement activity changed as colostrum transitioned to mature milk. Although there were clear changes in the colostral components under study in relation to treatment and transition to mature milk, the biological significance of the described treatment effects and temporal changes were not investigated here.

Genetic coding variant in complement factor B (CFB) is associated with increased risk for perianal Crohn’s disease and leads to impaired CFB cleavage and phagocytosis

ObjectivePerianal Crohn’s disease (pCD) occurs in up to 40% of patients with CD and is associated with poor quality of life, limited treatment responses and poorly understood aetiology. We performed...