Serum Complement Activity Research Articles

#2314 Circulating immune complexes concentration and complement activation in sensitized kidney transplant recipients

Abstract Background and Aims Chronic antibody mediated rejection of the kidney transplant is the most common cause of graft loss in the late posttransplant period. Crucial in this process seems to be the role of classical pathway complement activation due to the formation of immune complexes. The formation of immune complexes is a benign process of a normally functioning immune system. In kidney transplant recipients (KTRs), the production of donor specific IgG/IgM antibodies (DSAs) against donor antigens (MHC molecules, ABO blood group antigens) leads to the attachment of immune complexes to cell membranes and subsequent tissue damage through complement activation. The attachment of C1q complement fragment to the immune complexes holds an important part in this process. This study aims to determine the levels of immune complexes as well as classical pathway complement activation in sensitized kidney transplant recipients. Method In this cross-sectional study 3 groups of KTRs were compared as to the levels of immunocomplexes concentration and classical pathway complement activation. Fourteen KTRs with preformed or de novo donor specific antibodies were compared to 28 kidney transplant recipients with no DSAs and to 22 subjects with no history of chronic kidney disease (controls). We used MicroVue Complement CIC-C1q EIA that is designed to detect immunocomplexes that bind to C1q in human serum or plasma. In order to estimate total classical pathway complement activation in serum, we used MicroVue Complement CH50 Eq EIA. Results Kidney function (eGFR) of KTRs with and without DSAs was similar (53.1 ± vs. 60.8 ± ml/min/1.73 m2, p = 0.54) but lower in comparison to controls (106.4 ml/min/1.73 m2, p < 0.001). We found a significantly higher concentration of C1q-immunocomplexes in the serum of KTRs with donor specific antibodies, in comparison to controls, as well as to the group of kidney transplant recipients with no DSAs (6.78 ± 2.7 vs. 4.83 ± 1.9 vs. 5.04 ± 1.2 μgEq/ml respectively, p = 0.0078). Furthermore, there was no significant difference of C1q-immunocomplexes between kidney transplant recipients with no DSAs and controls. Finally, a significant reduction of CH50 levels was observed in KTRs with DSAs as compared to controls and KTRs with no DSAs (39.2 ± 14.9 vs. 70.6 ± 34.3 vs. 67.9 ± 39.9 U/ml respectively, p = 0.003), which suggests higher levels of classical pathway complement activation. CH50 levels in KTRs with DSAs, who were further subcategorized in those with preformed and those with de novo DSAs were similar (39.68 ± 15.6 vs 39.08 ± 16.04 U/ml). Conclusion Kidney transplant recipients with donor specific antibodies have increased serum levels of immune complexes and increased classical pathway complement activation.

#933 Serum and kidney complement and severity of ANCA associated vasculitis in the kidney

Abstract Background and Aims Kidney involvement in ANCA associated vasculitis (AAV) is commonly described as pauci-immune based on histological characteristics. However, we now know that the complement system is activated and contributes to the pathogenesis of AAV – with Avacopan (C5a inhibitor) demonstrating superior efficacy in maintenance of remission over Prednisolone in the ADVOCATE trial. Our aim was to evaluate if there is an association between kidney disease severity and the presence of serum and histological complement activation markers. Method Retrospective cohort study of 59 patients with AAV diagnosed between November 2015 and May 2019. Complement activation markers were serum complements (C3 and C4) levels and presence of complement (C3, C1q and C4) in glomerular capillary walls in immunofluorescence with intensity of 2+ and 3+. Kidney disease severity markers were peak serum creatinine value at presentation, anti-MPO or anti-PR3 levels, and number and proportion of glomeruli with crescents on kidney biopsy. Associations were evaluated using Spearman correlation or Mann Whitney U test as appropriate Results Among 59 patients with biopsy-proven AAV in the kidney, the median (25th percentile, 75th percentile) age was 68.8 (60.7, 73.4) years, serum creatinine was 263 (156, 560) µmol/L, and anti-MPO and anti-PR3 levels were 100.0 (81.5, 199.2) and 128.8 (22.9, 200) in 41 and 7 patients, respectively. respectively. The number and proportion of glomeruli with crescents were 6 (3, 12) and 33.3% (12.0%, 42.6%), respectively. Serum C3 and C4 were performed in 41 patients and the median levels were 1.00 (0.83, 1.14) and 0.24 (0.18, 0.31), respectively. Serum C3 level did not correlate with serum creatinine, anti-MPO or anti-PR3 titers, and number and proportion of glomeruli with crescents. Serum C4 level was negatively correlated with anti-MPO titer (34 patients; r = −0.393, p = 0.02) but did not correlate with serum creatinine or number or proportion of crescents. Complement in the glomerular capillary wall was present in 8 patients (13.6%): C3, C1q and C4 were present in 8, 3 and 1 patients, respectively. Serum creatinine [median 299 (IQR 133, 998) µmol/L versus 261 (156, 359) µmol/L, p = 0.39], anti-MPO titer [100 (88.3, 200) IU/ml versus 100 (79.4, 198) IU/ml, p = 0.74], number of crescents [14 (4, 21) versus 5 (3, 12), p = 0.90] and proportion of crescents [38.9% (12.5%, 42.3%) versus 31.3% (11.1%, 44.3%), p = 0.59] were not different in those with and without complement in the kidney biopsy. Conclusion The presence of complement activation markers was not associated with kidney disease severity in AAV. However, the detection of clinically significant associations may have been limited by our cohort size.

Escherichia coli isolated from pyometra and cystitis in the same animal exhibit a wide phenotypic similarity.

Pyometra and cystitis caused by Escherichia coli are common diseases identified in canine or feline females. The origin of pyometra infection remains uncertain, and effective prevention strategies for this disease are still unknown. This study aimed to provide a phenotypic characterisation, including antimicrobial resistance and virulence profiles, of Endometrial Pathogenic (EnPEC) and Uropathogenic (UPEC) E. coli strains isolated simultaneously from the same animal. Sixteen E. coli strains, from eight different animals, were analysed in this study. The antimicrobial susceptibility profile of EnPEC and UPEC strains was determined using the disc diffusion method, which showed a similar susceptibility profile among strains (EnPEC and UPEC) from the same animal. The virulence profile of the strains was assessed through biofilm formation, as well as serum resistance abilities. EnPEC and UPEC strains from the same animal exhibited slight variations in their virulence and antimicrobial resistance capabilities. Overall, most of the strain pairs showed a high similarity in their ability to establish biofilms and survive in serum complement activity. Overall, strains of E. coli isolated from both pyometra and cystitis in the same animal, despite presenting distinct clinical diseases, exhibit a wide phenotypic similarity, suggesting a common origin for the strains.

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

Low C4A copy numbers and higher HERV gene insertion contributes to increased risk of SLE, with absence of association with disease phenotype and disease activity.

Low copy numbers (CNs) of C4 genes are associated with systemic autoimmune disorders and affects autoantibody diversity and disease subgroups. The primary objective of this study was to characterize diversity of complement (C4) and C4-Human Endogenous Retrovirus (HERV) gene copy numbers in SLE. We also sought to assess the association of C4 and C4-HERV CNs with serum complement levels, autoantibodies, disease phenotypes and activity. Finally, we checked the association of C4 and HERV CNs with specific HLA alleles. Genomic DNA from 70 SLE and 90 healthy controls of south Indian Tamil origin were included. Demographic, clinical and serological data was collected in a predetermined proforma. CNs of C4A and C4B genes and the frequency of insertion of 6.4kb HERV within C4 gene (C4AL, C4BL) was determined using droplet digital polymerase chain reaction (ddPCR). A four digit high resolution HLA genotyping was done using next generation sequencing. In our cohort, the total C4 gene copies ranged from 2 to 6. Compared to controls, presence of two or less copies of C4A gene was associated with SLE risk (p = 0.005; OR = 2.79; 95% CI = 1.29-6.22). Higher frequency of HERV insertion in C4A than in C4B increases such risk (p = 0.000; OR = 12.67; 95% CI = 2.80-115.3). AL-AL-AL-BS genotype was significantly higher in controls than SLE (9%vs1%, p = 0.04; OR = 0.15, 95% CI = 0.00-0.16). Distribution of HLA alleles was not different in SLE compared to controls as well as in SLE subjects with ≤ 2 copies and > 2 copies of C4A, but HLA allele distribution was diverse in subjects with C4B ≤ 2 copies and > 2 copies. Finally, there was no correlation between the C4 and the C4-HERV diversity and complement levels, autoantibodies, disease phenotypes and activity. In conclusion, our data show that, low C4A copy number and higher insertion of HERV-K in C4A increases the risk for SLE. C4 and C4-HERV CNs did not correlate with serum complements, autoantibodies, disease phenotypes and activity in SLE. Further validation in a larger homogenous SLE cohort is needed.

Differentiated pattern of complement system activation between MOG-IgG-associated disease and AQP4-IgG-positive neuromyelitis optica spectrum disorder.

Myelin oligodendrocyte glycoprotein antibody (MOG) immunoglobulin G (IgG)-associated disease (MOGAD) has clinical and pathophysiological features that are similar to but distinct from those of aquaporin-4 antibody (AQP4-IgG)-positive neuromyelitis optica spectrum disorders (AQP4-NMOSD). MOG-IgG and AQP4-IgG, mostly of the IgG1 subtype, can both activate the complement system. Therefore, we investigated whether the levels of serum complement components, regulators, and activation products differ between MOGAD and AQP4-NMOSD, and if complement analytes can be utilized to differentiate between these diseases. The sera of patients with MOGAD (from during an attack and remission; N=19 and N=9, respectively) and AQP4-NMOSD (N=35 and N=17), and healthy controls (N=38) were analyzed for C1q-binding circulating immune complex (CIC-C1q), C1 inhibitor (C1-INH), factor H (FH), C3, iC3b, and soluble terminal complement complex (sC5b-9). In attack samples, the levels of C1-INH, FH, and iC3b were higher in the MOGAD group than in the NMOSD group (all, p<0.001), while the level of sC5b-9 was increased only in the NMOSD group. In MOGAD, there were no differences in the concentrations of complement analytes based on disease status. However, within AQP4-NMOSD, remission samples indicated a higher C1-INH level than attack samples (p=0.003). Notably, AQP4-NMOSD patients on medications during attack showed lower levels of iC3b (p<0.001) and higher levels of C3 (p=0.008), C1-INH (p=0.004), and sC5b-9 (p<0.001) compared to those not on medication. Among patients not on medication at the time of attack sampling, serum MOG-IgG cell-based assay (CBA) score had a positive correlation with iC3b and C1-INH levels (rho=0.764 and p=0.010, and rho=0.629 and p=0.049, respectively), and AQP4-IgG CBA score had a positive correlation with C1-INH level (rho=0.836, p=0.003). This study indicates a higher prominence of complement pathway activation and subsequent C3 degradation in MOGAD compared to AQP4-NMOSD. On the other hand, the production of terminal complement complexes (TCC) was found to be more substantial in AQP4-NMOSD than in MOGAD. These findings suggest a strong regulation of the complement system, implying its potential involvement in the pathogenesis of MOGAD through mechanisms that extend beyond TCC formation.

Dissociation Phenomenon of Erythrocyte Agglutination and Its Application to Assay of Functional Activity of the Complement System in Clinical Laboratory.

The objective of the study was to validate the dissociation phenomenon of erythrocyte agglutination which is based on erythrocyte fragments and to apply it in the functional activity assay of the complement system. The dissociation-agglutination effect of erythrocyte fragments was validated by detecting the number of free erythrocytes after the action of erythrocyte fragments on agglutinated erythrocytes. The number of free erythrocytes produced after hemolysis of agglutinated erythrocytes caused by complements and complement activators(CAs) was detected by auto hematology analyzer and the results were indicated by mean hemoglobin concentration of erythrocytes (MCHC). We optimized the test conditions and validated the inter-batch stability, explored the resolution of the assay method, and assayed for the total complement activity (AC) and the CAs activated complement activity (ACA) in serum from patients and healthy individual groups. Erythrocyte fragments have a dissociative effect on agglutinated erythrocytes. The auto hematology analyzer was able to detect AC and ACA, where AC showed an inverse correlation with MCHC, and ACA demonstrated a positive correlation with MCHC. The inter-batch CV of AC, ACA, and ACA/AC was found to be 5%, 9%, and 11.7%, respectively, with good stability. The study found that serum samples from acute phase reaction patients showed significant differences in ACA compared with healthy individuals, with a p value of 0.018; serum samples from patients with nephrotic syndrome showed significant differences in AC, ACA, and ACA/AC compared with healthy individuals, with p values of 0.014, 0.002, and 0.041, respectively. Erythrocyte fragments have dissociation-agglutination effect. The complement system immunological functional detection method, based on this effect, has potential clinical application value due to its sensitivity and accuracy.

Effects of whey-enriched drinking water on fattening merino lamb growth, hemogram, inflammation, oxidant and antioxidant parameters

This study aimed to evaluate the oxidative stress and inflammation status of ad libitum whey consumption as drinking water on fatting Merino lambs (n=24). Lambs were randomly divided into two groups and received ad libitum freshwater or whey enriched water and had clover as roughage and lamb grower feed as concentrate daily. Blood samples were collected before the trial (T1), on the 15th (T2) and 30th (T3) days and analyses were conducted on hemogram, serum antioxidant enzymes (Superoxide dismutase, SOD; Glutathione peroxidase, GSH-Px) inflammation response (C-Reactive Protein, CRP), complement activation, and chemokine marker (complement component 4, C4), lymphocyte activation factor (Interleukin 1 beta, IL-1 β), systemic inflammation cytokine (Tumor necrosis factor-alpha, TNF-α), lipid peroxidation marker (Malondialdehyde, MDA). The rate of hematocrit was found to be higher in the experimental group in T3 (35.55 ± 10.54) compared to the control group (44.50 ± 2.58). The platelet amounts were found to be higher in the whey group in T2 and T3 compared to the control group (p&lt;0.05). In conclusion, with the use of whey protein-enriched drinking water, no significant change was observed in the amounts of TNF-α, SOD, MDA, IL-1β, C4, CRP under these study conditions.

Frass from Black Soldier Fly Larvae, Hermetia illucens, as a Possible Functional Dietary Ingredient in Channel Catfish Feed

Frass–the by-product of the larva meal industry–was recently shown to be a sustainable ingredient for use in diets of several fish species. Additionally, because of its possible immunomodulatory activity and anti-microbial properties, frass may have great potential as an organic method of pathogen control in aquaculture. Five diets containing 0, 5, 10, 20 and 30% frass from black soldier fly larvae, Hermetia illucens, were fed to channel catfish (5.24 ± 0.04 g) in quadruplicate aquaria to apparent satiation twice daily. At the end of the 10-week feeding trial, blood samples were collected from all groups to measure hematological and immune parameters, and to determine the effects of dietary frass on resistance to Flavobacterium covae infection. Hematological parameters (red blood cell count, hemoglobin, and hematocrit)—but not white blood cell count—were improved with the inclusion of frass. Serum glucose levels were significantly lower in fish on the diet with frass than fish on the diet without frass. Fish fed the highest dietary levels of frass (30%) had a significantly higher serum cholesterol level than fish on the control diet. Serum complement activity was significantly higher in fish on diets containing frass at levels of 10% and 20%. No significant differences were observed in other measure serum components including albumin, alkaline phosphatase, alanine aminotransferase, amylase, calcium, phosphorus, potassium, total protein, globulin, thyroxine and lysozyme activity. Even though overall mortality was low (0–17%), fish on the diets containing frass at levels 20% or more showed significantly higher survival than that of control fish or fish on lower levels of dietary frass. The use of frass in the catfish diet may prove beneficial by improving hematological parameters, and select serum immune effectors, and the overall resistance of juvenile channel catfish against F. covae infection.

O043 Evaluation of minimal factor H therapy administered to kidneys during ex vivo normothermic perfusion as a treatment to improve ischaemia reperfusion injury

Abstract Introduction Complement activation is a key mechanism of Ischaemia reperfusion injury, with the alternative pathway driving damage in particular. The main regulator of the alternative pathway is factor H. We hypothesised that homodimeric mini-factor H (HDM-FH; PMID:29588430) may protect transplanted kidneys from complement mediated damage when administered during normothermic perfusion machine perfusion. Methods Kidneys were retrieved from female white landrace pigs following an optimised protocol. One kidney from each pair was randomised to receive 5mg of HDM-FH (∼8μg/mL). Kidneys were perfused at 37°C with autologous blood for 6 hours. HDM-FH binding was measured using ELISA and immunofluorescence. Complement activation was measured by quantifying Bb deposition in tissue. The ability of HDM-FH to inhibit complement in serum was measured using haemolytic assays. Results 4mgs of HDM-FH bound from perfusate during perfusion, with &amp;lt;10% lost in urine suggesting saturation was achieved. HDM-FH binding within the kidneys was confirmed using immunofluorescence. HDM-FH localised to glomeruli with deposition increasing over the time of the perfusion. Alternative pathway activation was reduced in kidneys receiving HDM-FH as demonstrated by reduced Bb deposition. Fibrinogen deposition was also reduced and found to colocalise with C3 in glomeruli. HDM-FH inhibits complement activity in serum in a dose-dependent manor. Conclusion Infusion with HDM-FH prior to simulated kidney transplant conditions reduced complement activation and other secondary negative outcomes to the organ. Therefore, organ perfusion with HDM-FH is highly likely to help prolong graft survival after transplant and this will be assessed in future studies.

Regulation of protein corona on liposomes using albumin-binding peptide for targeted tumor therapy.

Nanocarriers entering the body are usually coated by plasma protein, leading to a protein "corona" easily recognized by tissues and cells. Adjusting the composition of protein coronas may be an efficient way to change the properties and behavior of nanoparticles in vivo. In this study, we modified doxorubicin-loaded liposomes (Lip/DOX) with an albumin-binding domain (ABD) to prepare nanoparticles (ABD-Lip/DOX) that can specifically bind to albumin and form albumin-based protein coronas in vivo for targeted tumor therapy. The prepared liposomes were spherical with a particle size of about 100 nm. After incubating the liposomes with rat serum, the albumin content was eight times higher on ABD-Lip than on control liposomes. ABD-Lip significantly inhibited adsorption of IgG and complement activation in rat serum in vitro, while corona-coated ABD-Lip was internalized to a significantly greater extent than corona-coated control liposomes. In addition, ABD-Lip showed longer blood circulation time, higher tumor accumulation and greater antitumor efficacy than control liposomes in mice bearing 4 T1 tumors, while both liposome formulations showed similar biocompatibility. These results confirm that adjusting the component of protein coronas around nanoparticles can improve their therapeutic efficacy.

Complement Heat Tolerance as a Marker of Protein Fragility and Its Clinical Significance.

This study aimed to establish a complement tolerance test (CTT) as a marker of protein fragility and discuss its clinical significance. Total complement activity (TCA) of serum was measured using a self-hemolysis colorimetric method. Human O-erythrocytes and rabbit anti-human O-erythrocyte antibodies were used to replace sheep erythrocytes and the corresponding hemolysin for the hemolysis test, respectively. The antigen-antibody specific binding activated the classical pathway of complement, generating a membrane attack complex, and the red blood cells rupture. A CTT was established to measure complement heat tolerance according to the sensitivity of complement proteins to temperature, which was calculated according to differences in TCA at different temperatures. The smaller the CTT the stronger the complement resistance to heat. The method was applied to the detection of diabetic patients and healthy controls. The mean value of CTT (mean) = 0.063 ± 0.003 with a coefficient of variation of 4.8% for the same specimen tested for complementary thermal resistance on 5 consecutive days, which is a good stability of the assay. Application of CTT on samples from patients with different ages revealed significantly higher mean CTT values for elderly patients (≥60-years old) relative to those for younger patients (20-40-years old) (p < 0.05). In addition, the mean CTT values for diabetic patients were significantly higher than those for healthy patients (p < 0.001). We successfully established a method that uses complement thermal resistance as a marker of protein fragility, with the results demonstrating the ability of the CTT identify age- and disease-related variations in patient samples and its potential efficacy for clinical application.

Edwardsiella piscicida Ail1: An outer membrane protein required for host infection

Edwardsiella piscicida is a significant pathogenic microorganism, and can lead to systematic infections in multifold hosts, including teleost, amphibians, and mammals. Ail is a surface protein involved in virulence. However, the underlining mechanism is still to be investigated in E. piscicida. This study examined the functions and immunoprotective properties of an Ail molecule from E. piscicida. Ail1 possessed a conserved Ail/Lom superfamily domain and was exposed to the bacterial surface. Mutation of ail1 significantly reduced the survival rate under acid and oxidizing environment, motility, biofilm formation, cellular infection, tissue dissemination, and causing mortality in infected fish. The loss abilities of TX01Δail1 were rescued by overexpression of the ail1 gene. Recombinant Ail1 (rAil1) could interact with peripheral blood leukocytes from Japanese flounder and affect E. piscicida infection in vitro. Vaccine trial showed that rAil1 was used as a potential subunit vaccine and could induce effective immune protection against E. piscicida infection. Fish vaccinated with rAil1 produced the specific antibody against rAil1 in serum, which increased the bactericidal activity of serum complement. In addition, rAil1-vaccinated fish displayed the higher ROS and acid phosphatase activity of macrophages. The above findings indicated that Ail1 played a pivotal role in the virulence of E. piscicida and may be explored as a subunit vaccine in aquaculture.

Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis

Complement component 5 (C5), an important molecule in the complement cascade, blockade by antibodies shows clinical efficacy in treating complement-mediated disorders. However, insufficient blockading induced by single-nucleotide polymorphisms in the C5 protein or frequent development of "breakthrough" intravascular hemolysis in patients with paroxysmal nocturnal hemoglobinuria treated with eculizumab have been reported. Herein, we developed a lipid nanoparticle (LNP)-formulated siRNA targeting C5 that was efficiently delivered to the liver and silenced C5 expression. We identified a potent C5-siRNA with an invitro IC50 of 420 pM and invivo ED50 of 0.017mg/kg following a single administration. Single or repeated administrations of the LNP-formulated C5-siRNA allowed robust and durable suppression of liver C5 expression in mice. Complement C5 silencing ameliorated C5b-dependent anti-acetylcholine receptor antibody-induced myasthenia gravis and C5a-dependent collagen-induced arthritis symptoms. Similarly, in nonhuman primates, a single administration of C5-siRNA/LNP-induced dose-dependent plasma C5 suppression and concomitantly inhibited serum complement activity; complement activity recovered to the pre-treatment levels at 65days post administration, thus indicating that the complement activity can be controlled for a specific period. Our findings provide the foundation for further developing C5-siRNA delivered via LNPs as a potential therapeutic for complement-mediated diseases.

A novel C1qDC (PoC1qDC) with a collagen domain in Paralichthys olivaceus mediates complement activation and against bacterial infection

Complement C1q domain containing protein (C1qDC) is a vital recognition molecule and has an important effect on immunity. The C1qDCs exhibit opsonic activity in fish, while the mechanisms of C1qDCs in activation complement still remain unclear. This study explored immunological characteristics of a C1qDC from Japanese flounder (Paralichthys olivaceus) (PoC1qDC). PoC1qDC consists of 296 amino acid residues, possessing a collagen domain and a C1q domain. According to our results, PoC1qDC was expressed in 9 diverse tissue samples and showed up-regulation after bacterial challenge. Recombinant PoC1qDC (rPoC1qDC) activated normal serum bactericidal and hemolytic activities by interaction with Japanese flounder IgM, but not enhanced the complement activity of C3-depeleted serum. rPoC1qDC was significantly bound to various bacterial species and agglutination activity against Edwardsiella piscicida and Streptococcus iniae. Furthermore, rPoC1qDC showed direct interaction with peripheral blood leucocytes while enhancing phagocytic and chemotactic activity. When PoC1qDC was overexpressed in Japanese flounder before E. piscicida infection, bacterial replication was significantly inhibited in fish tissues. Consistently, when PoC1qDC expression in Japanese flounder was knocked down, bacterial replication was significantly enhanced. The above findings first suggested the role of PoC1qDC in teleost in mediating complement activation by interaction with IgM, which can positively influence bacterial infection.

P17.10: Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation. This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development [Project No. PJ015607 (HK and KO)], Rural Development Administration, Republic of Korea. This research was partly supported by Hallym University Research Fund (HL).

Immunomodulatory Effect of Vitamin C and E on Non-Specific Immune Parameters in fishes: A Review

Aquaculture has been fastest growing industry for several decades that include fish and shellfish production. Culture of fishes in narrow space or uncontrolled production becomes the reason of various infectious diseases in fishes. So, prevention from diseases and increasing immunity is important for healthy production. There are many ways in which immunity may be enhanced like use of antibiotics and vaccines in fishes. The use of antibiotics is not very successful and vaccination is an expensive method, so there is need of eco-friendly and less expensive method. Immunostimulants are chemical compounds that activate the innate or non-specific immune system of fishes by activating the cells of immune system. Several vitamins like vitamin E (Alpha-Tocopherol) and vitamin C (Ascorbic Acid) act as an immunostimulant. Vitamin E is a fat soluble compound that enhances the growth, reproduction and survival by increasing the non-specific immunity. It also acts as an antioxidant by controlling the production of free-radicals. Vitamin C is also essential for fish growth, health and survival. It enhances the phagocytosis, serum haemolytic activity, complement activity and proliferation of immune cells. In past few years, stabled beneficial effects of immunostimulants in fishes promoted their application for disease management in aquaculture practice. As we have taken up similar study on the effects of vitamins as natural immunostimulants on Asian, catfish Clarias batrachus, so, this review is an attempt to throw light on the role of Vitamin C and Vitamin E as an immunostimulants in fish culture.

Complement component 3 haplotypes influence serum complement activity and milk production traits in Chinese Holstein cattle.

Complement component 3 (C3) is the key molecule of the three pathways of complement activation (alternative, classical, and lectin pathways), which are involved in phagocytosis, inflammation, and immunoregulation processes to destroy infectious microorganisms. In this study, three novel single-nucleotide polymorphisms (SNPs) (g.-1293C>G located in the 5'-flanking region, g.56T>C in exon I, and g.7017C>T in exon XII) of the C3 gene were detected using created restriction site polymerase chain reaction, restriction fragment length polymorphism, and DNA sequencing in 952 cattle from three Chinese breeds. The genotypes and haplotypes were analyzed to investigate the polymorphisms and their possible implications, with particular investigative focus on their associations with serum C3 level, complement hemolytic activity (CH50 and ACH50), and milk production traits. The g.56T>C SNP in exon I affected the serum ACH50 (P<0.01) and the milk somatic cell score (SCS) (P<0.05), and the g.7017C>T SNP in exon XII significantly affected the serum ACH50 values (P<0.01). Moreover, statistical analyses revealed that individuals with genotypic combination CCC/GCC showed significantly lower SCS and the lowest C3 concentration in serum compared with cows with CCC/GTT (P = 0.0007) and CTT/CTT (P = 0.0021); the individuals with CCC/CCT had significantly higher ACH50 values than those with CCC/CTC (P = 0.0008) and CTC/GTC (P = 0.001); cows with CCT/CTT had higher values of CH50 and 305-day milk yield (P>0.05). The C3 expression levels were significantly increased in lung and mammary tissues (P<0.05), while significantly decreased in heart, spleen, liver, and kidney tissues in mastitis cows compared with those in healthy animals (P<0.01), respectively. Bacterial counts of serum antibacterial activities were also completed to verify the effect of SNPs on resistance to mastitis pathogens. Genetically resistant cows (CCC/GCC) had serum with noticeably higher antibacterial activity against S. aureus and E. coli in vitro than the genetically susceptible CCC/GTT cows (P<0.05). Results from this study imply that the C3 gene plays a role in resistance to bacterial infection and that it can be used as a molecular marker for complement activity and traits related to milk production.

Effects of Biotronic® Top3, a feed additive containing organic acids, cinnamaldehyde and a permeabilizing complex on growth, digestive enzyme activities, immunity, antioxidant system and gene expression of barramundi (Lates calcarifer)

The Biotronic® Top3 (OCAP: organic acids, cinnamaldehyde, and permeabilising complex) is used for improving microbial community in monogastric animals. This research was aimed to test the five dosages of OCAP (0%: control, 0.05%: OCAP0.05, 0.10%: OCAP0.1, 0.15%: OCAP0.15%, and 0.20%: OCAP0.2) in barramundi (Lates calcarifer) (8.3 ± 0.3 g) for eight weeks. The results showed a linear relation between OCAP level and growth rate, feed conversion ratio, protease, lipase, lysozyme in serum and mucus, alternative complement activity (ACH50) in serum, catalase, glutathione peroxidase, and malondialdehyde levels (P < 0.05). Accordingly, the OCAP0.2 treatment had the best performance in the abovementioned parameters. While there was no significant difference in expression of heat shock proteins (HSP70, HSP90) and tumour necrosis factor (TNF-a) among groups, interleukins (IL-1β, IL-8) were significantly lower in the OCAP0.15 and OCAP0.2 group than others. The expression of IGF-1 followed the growth rate trend. Generally, feeding fish with the OCAP0.2 diet improved growth rate, digestive enzymes activities, immune response, antioxidant system, and gene expressions in barramundi. As the linear relation was observed, supplementing more levels of OCAP to reach a plateau is recommended.

Germline rare variants of lectin pathway genes predispose to asymptomatic SARS-CoV-2 infection in elderly individuals

PurposeEmerging evidence suggest that infection-dependent hyperactivation of complement system (CS) may worsen COVID-19 outcome. We investigated the role of predicted high impact rare variants — referred as qualifying variants (QVs) — of CS genes in predisposing asymptomatic COVID-19 in elderly individuals, known to be more susceptible to severe disease. MethodsExploiting exome sequencing data and 56 CS genes, we performed a gene-based collapsing test between 164 asymptomatic subjects (aged ≥60 years) and 56,885 European individuals from the Genome Aggregation Database. We replicated this test comparing the same asymptomatic individuals with 147 hospitalized patients with COVID-19. ResultsWe found an enrichment of QVs in 3 genes (MASP1, COLEC11, and COLEC10), which belong to the lectin pathway, in the asymptomatic cohort. Analyses of complement activity in serum showed decreased activity of lectin pathway in asymptomatic individuals with QVs. Finally, we found allelic variants associated with asymptomatic COVID-19 phenotype and with a decreased expression of MASP1, COLEC11, and COLEC10 in lung tissue. ConclusionThis study suggests that genetic rare variants can protect from severe COVID-19 by mitigating the activity of lectin pathway and prothrombin. The genetic data obtained through ES of 786 asymptomatic and 147 hospitalized individuals are publicly available at http://espocovid.ceinge.unina.it/