Complement Reaction Research Articles

Bioinformation Analysis of Differential Expression Proteins in Different Processes of COVID-19.

COVID-19 is a highly infectious respiratory disease whose progression has been associated with multiple factors. From SARS-CoV-2 infection to death, biomarkers capable of predicting different disease processes are needed to help us further understand the molecular progression of COVID-19 disease. The aim is to find differentially expressed proteins that are associated with the progression of COVID-19 disease or can be potential biomarkers, and to provide a reference for further understanding of the molecular mechanisms of COVID-19 occurrence, progression, and treatment. Data-independent Acquisition (DIA) proteomics to obtain sample protein expression data, using R language screening differentially expressed proteins. Gene Ontology and Kyoto Encyclopedia for Genes and Genomes analysis was performed on differential proteins and protein-protein interaction (PPI) network was constructed to screen key proteins. A total of 47 differentially expressed proteins were obtained from COVID-19 incubation patients and healthy population (L/H), mainly enriched in platelet-related functions, and complement and coagulation cascade reaction pathways, such as platelet degranulation and platelet aggregation. A total of 42 differential proteins were obtained in clinical and latent phase patients (C/L), also mainly enriched in platelet-related functions and in complement and coagulation cascade reactions, platelet activation pathways. A total of 10 differential proteins were screened in recovery and clinical phase patients (R/C), mostly immune-related proteins. The differentially expressed proteins in different stages of COVID-19 are mostly closely associated with coagulation, and key differential proteins, such as FGA, FGB, FGG, ACTB, PFN1, VCL, SERPZNCL, APOC3, LTF, and DEFA1, have the potential to be used as early diagnostic markers.

Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells.

Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells. Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source. Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants. TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion. TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE. TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology.

Mechanism of Qiwei Guibao Granules in treatment of premature ovarian failure based on proteomics

In this study, the underlying mechanism of Qiwei Guibao Granules(QWGB) in the treatment of premature ovarian fai-lure(POF) was explored by the proteomics technique. Firstly, the POF model was induced in mice by intragastric administration of Tripterygium wilfordii glycosides solution at 50 mg·kg~(-1) for 14 days. Ten days prior to the end of the modeling, the estrous cycle of mice was observed every day to evaluate the success of modeling. From the 1st day after modeling, the POF model mice were treated with QWGB by gavage every day and the treatment lasted four weeks. On the 2nd day after the end of the experiment, blood was collected from the eyeballs and the serum was separated by centrifugation. The ovaries and uterus were collected and the adipose tissues were carefully stripped. The organ indexes of the ovaries and uterus of each group were calculated. The serum estrogen(E_2) level of mice in each group was detected by ELISA. Protein samples were extracted from ovarian tissues of mice, and the differential proteins before and after QWGB intervention and before and after modeling were analyzed by quantitative proteomics using tandem mass tags(TMT). As revealed by the analysis of differential proteins, QWGB could regulate 26 differentially expressed proteins related to the POF model induced by T. wilfordii glycosides, including S100A4, STAR, adrenodoxin oxidoreductase, XAF1, and PBXIP1. GO enrichment results showed that the 26 differential proteins were mainly enriched in biological processes and cellular components. The results of KEGG enrichment showed that those differential proteins were involved in signaling pathways such as completion and coalescence cascades, focal adhesion, arginine biosynthesis, and terpenoid backbone biosynthesis. The complement and coalescence cascades signaling pathway was presumably the target pathway of QWGB in the treatment of POF. In this study, the proteomics technique was used to screen the differential proteins of QWGB in the treatment of POF in mice induced by T. wilfordii glycosides, and they were mainly involved in immune regulation, apoptosis regulation, complement and coagulation cascade reactions, cholesterol metabolism, and steroid hormone production, which may be the main mechanisms of QWGB in the treatment of POF.

Rituximab Induces an Inflammatory Cytokine Response with Increased IP-10 (CXCR10) in Patients with Infusion Reactions

Most patients initiating anti-CD20 monoclonal antibody (mAb) therapy for CLL have a disruptive infusion reaction that can cause serious complications and be fatal. A better understanding of the risk factors and etiology of infusion reactions is required to improve preventative measures and management. We report a study to test the hypothesis that IV rituximab induces an inflammatory cytokines response with differences in individual cytokine concentrations correlating with infusion reactions. We designed a clinical trial (NCT03788291) for initial treatment of patients with progressive CLL that provided clinical data and blood specimens from 37 patients treated with 50 mg of IV rituximab infused at 25 mg/h. Blood specimens were collected immediately prior to initiation of rituximab (baseline), one hour later (1h), at the end of the infusion (post-infusion) and at 48 h after initiation of therapy (48h). Infusion reactions (CTCAE grade ≥2 events) were managed by interrupting the infusion and initiating symptomatic treatment. We measured circulating CLL cell counts and CD20 levels, and serum rituximab concentration, complement levels, and cytokine concentrations of IFN-γ, IL-10, IL-18, IL-2, IL-4, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α using standard methods. Twenty-four (65%) patients had 25 infusion reactions (all CTCAE grade 2) at a median of 78 minutes (range 35-128) after initiation of rituximab therapy (median dose 32 mg, range 15-50). All patients completed the 50 mg rituximab infusion (median time 150 minutes, range 119-195). Patient and CLL characteristics including baseline CLL cell counts did not predict the risk of infusion reactions. Median CLL counts at 1h decreased to 14.7% of baseline with no further changes post-infusion and no significant difference between patients with or without infusion reactions (60x109/L vs. 45x109/L, p=0.61). Median CLL cell membrane CD20 levels (x103 molecules/cell) decreased from 4.77 at baseline to 3.10 at 1h (p=0.002) and 1.59 post-infusion (p<0.0001 vs. baseline and 1h) with no significant differences between patients with or without an infusion reaction. The median estimated number of circulating CLL cell membrane CD20 molecules (x1012 molecules/L) decreased from 325.8 at baseline to 34.1 at 1h (p<0.0001) and 14.5 post-infusion (p<0.0001 vs. baseline, p<0.03 vs. 1h) with no significant differences between patients with or without an infusion reaction. There was no correlation between risk of infusion reaction and serum levels of rituximab or serum complement. Thirty-five (95%) patients had a ≥4 fold increase in serum concentration of ≥1 cytokine with resolution of the cytokine response at 48h. All patients with an infusion reaction had significantly higher median post-infusion serum concentrations of IP-10, IL-6 and IL-8. All 17 patients with IP-10 serum concentrations above the limit of detection (40,000 pg/ml) post-infusion had an infusion reaction, and all 24 patients with an infusion reaction had a ≥4 fold increase in serum IP-10 concentrations. Of 13 patients without an infusion reaction, the highest measured serum IP-10 concentration was 22,013 pg/ml and only 3 (23%) had ≥4 fold increases in serum IP-10 concentrations. Slow infusion of a reduced dose of rituximab induced a cytokine response in 95% of patients and therapy disrupting infusion reactions occurred at a comparable rate (65%) to standard rituximab therapy. Infusion reactions were associated with increased levels of IP-10, IL-6 and IL-8. IP-10 levels were increased by ≥4 fold in all patients with an infusion reaction compared to 23% without an infusion reaction. These data suggest that rituximab infusion reactions are caused by inflammatory cytokines produced by cytotoxic effector cells activated by rituximab independent of the pre-treatment amount of circulating CLL cells or CD20 levels, or efficacy of rituximab in reducing the circulating CLL cell count. Infusion reactions occurred after at least 35 minutes of therapy concurrent with a rapid decrease in circulating CLL cells. This observation suggests that cytokine release could be a result of activation of effector cells responsible for CLL cell elimination. Our data provides novel insights into the potential mechanism of infusion reactions and could form the basis of further research to determine methods to control this clinically important and therapy limiting complication.

The impact of human complement on the clinical outcome of malaria infection

The tropical disease malaria remains a major cause of global morbidity. Once transmitted to the human by a blood-feeding mosquito, the unicellular malaria parasite comes into contact with the complement system and continues to interact with human complement during its intraerythrocytic replication cycles. In the course of infection, both the classical and the alternative pathway of complement are activated, leading to parasite opsonization and lysis as well as the induction of complement-binding antibodies. While complement activity can be linked to the severity of malaria, it remains to date unclear, whether human complement is beneficial for protective immunity or if extensive complement reactions may rather enhance pathogenesis. In addition, the parasite has evolved molecular strategies to circumvent attack by human complement and has even developed means to utilize complement factors as mediators of host cell infection. In this review, we highlight current knowledge on the role of human complement for the progression of malaria infection. We discuss the various types of interactions between malaria parasites and complement factors with regard to immunity and infection outcome and set a special emphasis on the dual role of complement in the context of parasite fitness.

Characterization of hypersensitivity reactions to polysulfone hemodialysis membranes

BackgroundIn recent years, cases have been reported in which unexpected systemic hypersensitivity reactions occurred in patients dialyzed with polysulfone- or polyethersulfone-biocompatible membranes in the absence of other risk factors. The pathomechanisms involved in these reactions are largely unknown. ObjectiveTo characterize hypersensitivity reactions to polysulfone hemodialysis using clinical and laboratory data and to identify biomarkers suitable for endotype identification and diagnosis. MethodsWe prospectively collected data from 29 patients with suspected hypersensitivity reactions to polysulfone hemodialysis membranes. Clinical laboratory parameters such as tryptase, blood cell counts, and complement levels were recorded. Acute samples were obtained from 18 cases for the ex vivo assessment of basophil activation by flow cytometry analysis of CD63, CD203, and FcεRI cell membrane expression. Serum cytokines and anaphylatoxin concentrations were evaluated in 16 cases by Luminex and cytometric bead array analysis. ResultsTryptase was elevated during the acute reaction in 4 cases. Evidence of basophil activation was obtained in 10 patients. Complement activation was found in only 2 cases. However, C5a serum levels tended to increase during the acute reaction in those patients with hypoxemia. Significantly higher serum levels of interleukin-6 were observed during the acute reactions to polysulfone hemodialysis (P = .0103). ConclusionBased on biomarker analysis, various endotypes were identified, including type I-like (with the involvement of mast cells or basophils), complement, and cytokine (interleukin-6) release-related reactions, with some patients showing mixed reactions. Further research is needed to unravel the exact mechanisms involved in the activation of these cellular and molecular pathways.

Oxygen, pH, Lactate, and Metabolism-How Old Knowledge and New Insights Might Be Combined for New Wound Treatment.

Over time, we have come to recognize a very complex network of physiological changes enabling wound healing. An immunological process enables the body to distinguish damaged cells and begin a cleaning mechanism by separating damaged proteins and cells with matrix metalloproteinases, a complement reaction, and free radicals. A wide variety of cell functions help to rebuild new tissue, dependent on energy provision and oxygen supply. Like in an optimized “bio-reactor,” disturbance can lead to prolonged healing. One of the earliest investigated local factors is the pH of wounds, studied in close relation to the local perfusion, oxygen tension, and lactate concentration. Granulation tissue with the wrong pH can hinder fibroblast and keratinocyte division and proliferation, as well as skin graft takes. Methods for influencing the pH have been tested, such as occlusion and acidification by the topical application of acidic media. In most trials, this has not changed the wound’s pH to an acidic one, but it has reduced the strong alkalinity of deeper or chronic wounds. Energy provision is essential for all repair processes. New insights into the metabolism of cells have changed the definition of lactate from a waste product to an indispensable energy provider in normoxic and hypoxic conditions. Neovascularization depends on oxygen provision and lactate, signaling hypoxic conditions even under normoxic conditions. An appropriate pH is necessary for successful skin grafting; hypoxia can change the pH of wounds. This review describes the close interconnections between the local lactate levels, metabolism, healing mechanisms, and pH. Furthermore, it analyzes and evaluates the different possible ways to support metabolism, such as lactate enhancement and pH adjustment. The aim of wound treatment must be the optimization of all these components. Therefore, the role of lactate and its influence on wound healing in acute and chronic wounds will be assessed.

Complement C5 inhibition protects against hemolytic anemia and acute kidney injury in anthrax peptidoglycan-induced sepsis in baboons

Late-stage anthrax infections are characterized by dysregulated immune responses and hematogenous spread of Bacillus anthracis, leading to extreme bacteremia, sepsis, multiple organ failure, and, ultimately, death. Despite the bacterium being nonhemolytic, some fulminant anthrax patients develop a secondary atypical hemolytic uremic syndrome (aHUS) through unknown mechanisms. We recapitulated the pathology in baboons challenged with cell wall peptidoglycan (PGN), a polymeric, pathogen-associated molecular pattern responsible for the hemostatic dysregulation in anthrax sepsis. Similar to aHUS anthrax patients, PGN induces an initial hematocrit elevation followed by progressive hemolytic anemia and associated renal failure. Etiologically, PGN induces erythrolysis through direct excessive activation of all three complement pathways. Blunting terminal complement activation with a C5 neutralizing peptide prevented the progressive deposition of membrane attack complexes on red blood cells (RBC) and subsequent intravascular hemolysis, heme cytotoxicity, and acute kidney injury. Importantly, C5 neutralization did not prevent immune recognition of PGN and shifted the systemic inflammatory responses, consistent with improved survival in sepsis. Whereas PGN-induced hemostatic dysregulation was unchanged, C5 inhibition augmented fibrinolysis and improved the thromboischemic resolution. Overall, our study identifies PGN-driven complement activation as the pathologic mechanism underlying hemolytic anemia in anthrax and likely other gram-positive infections in which PGN is abundantly represented. Neutralization of terminal complement reactions reduces the hemolytic uremic pathology induced by PGN and could alleviate heme cytotoxicity and its associated kidney failure in gram-positive infections.

Drug Packaging Management Based on the Effect of Medical Images on the Intracellular Polysaccharide Synthesis and Antivertigo Activity of Phalaenopsis.

Many clinically important drugs come directly or indirectly from higher plants. People are increasingly aware of the role of the human immune system in maintaining good health. Diseases related to physical dysfunction, such as vertigo, have attracted increasing attention from medical researchers and clinicians. In this paper, some compounds isolated and identified from medicinal fomes showed promising antivertigo properties. Medical images were used to classify and synthesize polysaccharides in the management of drug subpackages of Cladosporium intracellular polysaccharides. The scientific explanation of how these compounds work in animal and human systems is increasing exponentially. Studies have found that all of these compounds can enhance the innate and adaptive immune responses of the host and activate various immune cells that are important for maintaining homeostasis, such as host cells and chemical messengers, triggering complement and acute phase reactions. The antivertigo compounds derived from the intracellular polysaccharides of Phellinus mucronatus had an activity interference of 35% without drug subpackage. Although the antivertigo activity of many intracellular polysaccharides from Fovea xylostella can reach 86%, only a few of them have been proved to have antivertigo activity. In addition, they can be considered as multicytokine inducers that can induce the expression of various immune-regulatory cytokines and cytokine receptor genes. Lymphocytes that control antibody production and cell-mediated cytotoxicity are also stimulated.

Multiplex gene analysis reveals T-cell and antibody-mediated rejection-specific upregulation of complement in renal transplants

In renal transplantation, complement is involved in ischemia reperfusion injury, graft rejection and dysfunction. However, it is still unclear how induction of complement and its activation are initiated. Using allograft biopsies of a well-characterized cohort of 28 renal transplant patients with no rejection (Ctrl), delayed graft function (DGF), acute T-cell-mediated (TCMR) or antibody-mediated rejection (ABMR) we analyzed differences in complement reaction. For that mRNA was isolated from FFPE sections, quantified with a multiplex gene expression panel and correlated with transplant conditions and follow-up of patients. Additionally, inflammatory cells were quantified by multiplex immunohistochemistry. In allograft biopsies with TCMR and ABMR gene expression of C1QB was 2-4 fold elevated compared to Ctrl. In TCMR biopsies, mRNA counts of several complement-related genes including C1S, C3, CFB and complement regulators CFH, CR1 and SERPING1 were significantly increased compared to Ctrl. Interestingly, expression levels of about 75% of the analyzed complement related genes correlated with cold ischemia time (CIT) and markers of inflammation. In conclusion, this study suggest an important role of complement in transplant pathology which seems to be at least in part triggered by CIT. Multiplex mRNA analysis might be a useful method to refine diagnosis and explore new pathways involved in rejection.

Exploration of Serum Marker Proteins in Mice Induced by Babesia microti Infection Using a Quantitative Proteomic Approach.

Babesia microti is a protozoan that mainly parasitizes rodent and human erythrocytes. B. microti infection can result in changes in the expression levels of various proteins in the host serum. To explore the mechanism underlying the regulation of serum proteins by the host during B. microti infection, this study used a data-independent acquisition (DIA) quantitative proteomic approach to perform comprehensive quantitative proteomic analysis on the serum of B. microti-infected mice. We identified and analysed 333 serum proteins during the infectious stage and recovery stage within 30days of infection by B. microti in mice. Through quantitative analysis, we found 57 proteins differentially expressed in the infection stage and 69 proteins differentially expressed in the recovery stage. Bioinformatics analysis revealed that these differentially expressed proteins were mainly concentrated in organelles, cell parts, and extracellular regions that are mainly involved in immune system, metabolic, and cellular processes. Additionally, the differentially expressed proteins mainly had catalytic activity. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis showed that many of the differentially expressed proteins participate in the complement and coagulation cascade reaction, including complement C3, complement FP, and coagulation factor XII. The results of this study can provide more information for the selection of biomarkers for the early clinical monitoring of babesiosis and help in the treatment of babesiosis.

Factor H-related protein 1: a complement regulatory protein and guardian of necrotic-type surfaces.

Factor H-related protein 1 (FHR-1) is a member of the factor H protein family, which is involved in regulating innate immune complement reactions. Genetic modification of the encoding gene, CFHR1 on human chromosome 1, is involved in diseases such as age-related macular degeneration, C3 glomerulopathy and atypical haemolytic uraemic syndrome, indicating an important role for FHR-1 in human health. Recent research data demonstrate that FHR-1 levels increase in IgA nephropathy and anti-neutrophilic cytoplasmic autoantibodies (ANCA) vasculitis and that FHR-1 induces strong inflammation in monocytes on necrotic-type surfaces, suggesting a complement-independent role. These new results increase our knowledge about the role of this complement protein in pathology and provide a new therapeutic target, particularly in the context of inflammatory diseases induced by necrosis. This review summarizes current knowledge about FHR-1 and discusses its role in complement reactions and inflammation. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

The Population of Fusarium oxysporum f. sp. lactucae in California and Arizona.

Fusarium wilt of lettuce, caused by Fusarium oxysporum f. sp. lactucae, is now found in all major lettuce producing regions in California and Arizona. The population structure of F. oxysporum f. sp. lactucae in California and Arizona was characterized based on somatic compatibility and sequences of the translation elongation factor 1-α gene (EF-1α) and rDNA intergenic spacer region (IGS). In this study, 170 isolates were tested for somatic compatibility based on heterokaryon formation, using complementary nitrate nonutilizing (nit) mutants. Five subgroups (A to E) of somatic compatibility group 0300 were identified. Isolates associated with the same subgroup had a strong complementation reaction, whereas reactions between isolates of different subgroups were weak or delayed. An isolate from the first known infestation of Fusarium wilt of lettuce in California was associated with subgroup A, which predominated among isolates in our collection. Isolates representative of each subgroup were confirmed to be associated with race 1, based on the reaction of differential lettuce cultivars. It is possible that somatic compatibility subgroups B to E of F. oxysporum f. sp. lactucae were derived from subgroup A, as a consequence of somatic mutations affecting compatibility. If so, subgroups of F. oxysporum f. sp. lactucae may represent an intermediate step in divergence that will lead to clearly separable compatibility groups. Sequences of EF-1α and IGS were both identical for 58 isolates of F. oxysporum f. sp. lactucae that represented all somatic compatibility subgroups and locations from which isolates were obtained, indicating that subgroups were derived from the same clonal lineage (VCG 0300).

The changes of immune-related molecules within the ileal mucosa of piglets infected with porcine circovirus type 2.

BackgroundEnteritis is one of the most frequently reported symptoms in piglets infected with porcine circovirus type 2 (PCV2), but the immunopathogenesis has not been reported.ObjectivesThis study examined the effect of a PCV2 infection on the intestinal mucosal immune function through morphological observations and immune-related molecular detection.MethodsMorphological changes within the ileum of piglets during a PCV2 infection were observed. The expression of the related-molecules was analyzed using a gene chip. The immunocyte subsets were analyzed by flow cytometry. The secretory immunoglobulin A (SIgA) content was analyzed by enzyme-linked immunosorbent assay.ResultsThe PCV2 infection caused ileal villus damage, intestinal epithelial cells exfoliation, and an increase in lymphocytes in the lamina propria at 21 days post-infection. Differentially expressed genes occurred in the defense response, inflammatory response, and the complement and coagulation cascade reactions. Most of them were downregulated significantly at the induction site and upregulated at the effector site. The genes associated with SIgA production were downregulated significantly at the induction site. In contrast, the expression of the Toll-like receptor-related genes was upregulated significantly at the effector site. The frequencies of dendritic cells, B cells, and CD8+T cells were upregulated at the 2 sites. The SIgA content decreased significantly in the ileal mucosa.ConclusionsPCV2 infections can cause damage to the ileum that is associated with changes in immune-related gene expression, immune-related cell subsets, and SIgA production. These findings elucidated the molecular changes in the ileum after a PCV2 infection from the perspective of intestinal mucosal immunity, which provides insights into a further study for PCV2-induced enteritis.

Preanalytical classical and alternative complement pathway activity loss.

IntroductionComplement functional analyses provide insight into the integrity of the entire complement reaction cascade. These tests are suitable for investigating suspected complement deficiencies. Falsely reduced test outcomes may result from preanalytical instabilities of individual complement components. To generate rationale for this or potential alternative practices, this study aimed to extend the knowledge on the preanalytical stability of widely used tests to screen the complement system. We assessed the influence of time, temperature and EDTA on classical (CH50) and alternative pathway (AP50) functional assay test results.Materials and methodsWe used nephelometric (C3d) and immunofixation (C3c) techniques to support the investigation of the preanalytical phase of basic complement system activity tests. Quantitative determination of classical and alternative pathway function was performed with a haemolytic activity assay and a C5b-9 neo-epitope ELISA-based assay respectively. Blood of five healthy volunteers was sampled and complement components allowed to degrade under different conditions.ResultsCH50 and AP50 remain stable for approximately one week in serum samples incubated on ice. CH50 activity decreased almost twice as fast in EDTA plasma compared to serum at room temperature. AP50 activity contrastingly, decreased twice as slow in EDTA plasma compared to serum at room temperature.ConclusionSerum on ice remains the preferred specimen for functional complement analyses. In the absence of serum transported on ice, serum kept at room temperature (not exceeding 24h) is suitable for classical and alternative pathway analyses. For alternative pathway analyses specifically, the C3-stabilising effect of EDTA allows for the extended use of EDTA plasma (not over 4 days). In these conditions, at least 85% of baseline complement activity remains.

Cutting Edge: Role of MASP-3 in the Physiological Activation of Factor D of the Alternative Complement Pathway.

The complement system, a part of the innate immune system, can be activated via three different pathways. In the alternative pathway, a factor D (FD) plays essential roles in both the initiation and the amplification loop and circulates as an active form. Mannose-binding lectin-associated serine proteases (MASPs) are key enzymes of the lectin pathway, and MASP-1 and/or MASP-3 are reported to be involved in the activation of FD. In the current study, we generated mice monospecifically deficient for MASP-1 or MASP-3 and found that the sera of the MASP-1-deficient mice lacked lectin pathway activity, but those of the MASP-3-deficient mice lacked alternative pathway activity with a zymogen FD. Furthermore, the results indicate that MASP-3 but not MASP-1 activates the zymogen FD under physiological conditions and MASP-3 circulates predominantly as an active form. Therefore, our study illustrates that, in mice, MASP-3 orchestrates the overall complement reaction through the activation of FD.

Protection of Baculovirus Vectors Expressing Complement Regulatory Proteins against Serum Complement Attack.

Baculovirus vectors (BVs) enable safe and efficient gene delivery to mammalian cells and are useful in a wide range of applications, including gene therapy and in vivo analysis of gene functions. We previously developed BVs expressing malaria sporozoite surface proteins for targeting liver cells or hepatocytes. However, BVs are known to be very vulnerable to complement attack and efforts to overcome their inactivation based on complement are important. In this study, BVs expressing complement regulatory proteins (CRPs) on the surfaces of virions were developed to inhibit complement reactions. Decay accelerating factor (DAF; CD55)-type BVs exhibited significantly higher complement resistance than control BVs without any CRPs in HepG2 cells transduction, although the transduction efficacy of DAF-type BV was low. In contrast, CD46-DAF-CD59 fusion type BVs showed significantly higher transduction efficacy and complement resistance than both control and DAF-type BVs. DAF-type and CD46-DAF-CD59 type BVs repressed formation of the membrane attack complex, a terminal product of complement reaction cascades, induced by BVs. These results suggest that the CD46-DAF-CD59 fusion construct confers complement protection ability superior to that of the DAF construct in gene delivery under complement active serum.

Evaluation of Cytotoxicity for Immunity Rejection of US11, hDAF

Xenotransplantation is proposed as a solution to the problem of organ shortage. However, transplantation of xenogeneic organs induces an antigen-antibody reaction in α-1,3-gal structure that are not present in humans and primates, and thus complement is also activated and organs die within minutes or hours. In this study, we used FasL gene, which is involved in the immune response of NK cell, and US11, which suppresses MHC Class I cell membrane surface expression, to inhibit cell mediated rejection in the interspecific immunity rejection, and also hDAF(CD55) was introduced to confirm the response to C3 complement. These genes were tranfeced into Korean native pig fetal fibroblasts using pCAGGS vector. And cytotoxicity of NK cell and human complement was confirmed in each cell line. The US11 inhibited the cytotoxicity of NK cell and, in addition, the simultaneous expression of US11 and Fas ligand showed excellent suppress to T-lymphocyte cytotoxicity, hDAF showed weak resistance to cytotoxicity of natural killer cell but not in CD8+ CTLs. Cytotoxicity study with human complement showed that hDAF was effective for reducing complement reaction. In this studies have demonstrated that each gene is effective in reducing immune rejection.

Differential gene expression in Anopheles stephensi following infection with drug-resistant Plasmodium yoelii

BackgroundThe transmission of drug-resistant parasites by the mosquito may be influenced by the altered biological fitness of drug-resistant parasites and different immune reactions or metabolic change in the mosquito. At this point, little is known about the variations in mosquito immunity and metabolism when mosquitoes are infected with drug-resistant parasites. To understand the differential gene expression in Anopheles following infection with drug-resistant Plasmodium, we conducted a genome-wide transcriptomic profiling analysis of Anopheles stephensi following feeding on mice with drug-resistant or drug-sensitive P. yoelii, observed changes in gene expression profiles and identified transcripts affected by the drug-resistant parasite.ResultsTo study the impact of drug-resistant Plasmodium infections on An. stephensi gene transcription, we analyzed the three major transition stages of Plasmodium infections: at 24 h and 13 and 19 days after blood-feeding. Six cDNA libraries (R-As24h, R-As13d, R-As19d,S-As24h, S-As13dand S-As19d) were constructed, and RNA sequencing was subsequently performed. In total, approximately 50.1 million raw reads, 47.9 million clean reads and 7.18G clean bases were obtained. Following differentially expressed gene (DEG) analysis, GO enrichment analysis of DEGs, and functional classification by KEGG, we showed that the variations in gene expression in An. stephensi infected by the drug-resistant P. yoelii NSM occurred mainly at 13 days after blood meal during sporozoite migration through the hemolymph. The differentially expressed genes included those functioning in some important immune reaction and iron metabolism pathways, such as pattern recognition receptors, regulators of the JNK pathway, components of the phagosome pathway, regulators of the melanization response, activators of complement reactions, insulin signaling cascade members, oxidative stress and detoxification proteins.ConclusionsOur study shows that drug-resistant P. yoelii NSM has an impact on the transcript abundance levels of An.stephensi mostly at 13 days after blood meal during sporozoite migration through the hemolymph and that most differentially expressed genes were downregulated. Our results highlight the need for a better understanding of selective pressures from these differentially expressed genes of the drug-resistant Plasmodium in the mosquito and the different transmission patterns of drug-resistant Plasmodium by Anopheles.

Phylogenetic Analysis of Antibiotic Resistance Genes and Virulence Genes of Klebsiella species in silico

A total of twelve isolates were screened for virulence and antibiotic resistance genes associated with Klebsiella pneumoniae infections. Virulence and antibiotic resistance genes were detected by in silico PCR amplification. Iron uptake protein entB was detected in 66.67% (n=8) of the isolates while no isolate was found to harbour chelating agent irp2. Iron uptake system kfu, involved in purulent tissue infections and capsule formation, was identified in 25% (n=3) of the isolates. Regulator of mucoid phenotype A, rmpA was not found in any of the isolates. The wabG gene, responsible for urinary tract infections was found in seven K. pneumoniae strains. Five uge positive strains might play role in the pathogenicity of K. pneumoniae infections. About 83.33% of the isolates were positive for type 1 fimbriae fimH1 while no type 3 fimbriae mrkD gene was found. Complement reaction blocked by plasmid traT gene was not observed in Klebsiella species while eight isolates harboured outer membrane lipoprotein, ycfM which protects Klebsiella species from antibiotics. Antibiotic resistance genes blaTEM and blaSHV were detected in 33.33% (n=4) and 66.67% (n=8) of the isolates while 25% isolates carried both blaTEM and blaSHV genes. Genotype 1 carried fimH1 and ycfM genes while all the virulence genes studied were present in genotype 2 and 3. The blaSHV gene was detected in all the genotypes while blaTEM gene was found in only genotype 1 and 3. The findings of this study would be helpful to predict virulence gene associated with Klebsiella infections. This data also helps us to choose antibiotics for treating Klebsiella infections. By assessing the genotypic distribution of antibiotic resistance gene, correct antibiotic can be used to treat the infection. This could help reduce emergence of antibiotic resistance since it is known that incorrect choice of antibiotics contributes to antibiotic resistance.Dhaka Univ. J. Pharm. Sci. 16(1): 119-127, 2017 (June)