Complement C3 Aggravates Post-epileptic Neuronal Injury Via Activation of TRPV1

Epilepsy is a brain condition characterized by the recurrence of unprovoked seizures.Recent studies have shown that complement component 3(C3)aggravate the neuronal injury in epilepsy.And our previous studies revealed that TRPV1(transient receptor potential vanilloid type 1)is involved in epilepsy.Whether complement C3 regulation of neuronal injury is related to the activation of TRPV1 during epilepsy is not fully understood.We found that in a mouse model of status epilepticus(SE),complement C3 derived from astrocytes was increased and aggravated neuronal injury,and that TRPV 1-knockout rescued neurons from the injury induced by complement C3.Circular RNAs are abundant in the brain,and the reduction of circRad52 caused by complement C3 promoted the expression of TRPV 1 and exacerbated neuronal injury.Mechanistically,disorders of neuron-glia interaction mediated by the C3-TRPV1 signaling pathway may be important for the induction of neuronal injury.This study provides support for the hypothesis that the C3-TRFV1 pathway is involved in the prevention and treatment of neuronal injury and cognitive disorders.

Complement C3a activates osteoclasts by regulating the PI3K/PDK1/SGK3 pathway in patients with multiple myeloma

Objective:Myeloma bone disease(MBD)is the most common complication of multiple myeloma(MM).Our previous study showed that the serum levels of C3/C4 in MM patients were significantly positively correlated with the severity of bone disease.However,the mechanism of C3 a/C4 a in osteoclasts MM patients remains unclear.Methods:The formation and function of osteoclasts were analyzed after adding C3 a/C4 a in vitro.RNA-seq analysis was used to screen the potential pathways affecting osteoclasts,and the results were verified by Western blot,q RT-PCR,and pathway inhibitors.Results:The osteoclast area per view induced by 1μg/m L(mean±SD:50.828±12.984%)and 10μg/m L(53.663±12.685%)of C3 a was significantly increased compared to the control group(0μg/m L)(34.635±8.916%)(P<0.001 and P<0.001,respectively).The relative m RNA expressions of genes,OSCAR/TRAP/RANKL/cathepsin K,induced by 1μg/m L(median:5.041,3.726,1.638,and 4.752,respectively)and 10μg/m L(median:5.140,3.702,2.250,and 5.172,respectively)of C3 a was significantly increased compared to the control group(median:3.137,2.004,0.573,and 2.257,respectively)(1μg/m L P=0.001,P=0.003,P<0.001,and P=0.008,respectively;10μg/m L:P<0.001,P=0.019,P<0.001,and P=0.002,respectively).The absorption areas of the osteoclast resorption pits per view induced by 1μg/m L(mean±SD:51.464±11.983%)and 10μg/m L(50.219±12.067%)of C3 a was also significantly increased(33.845±8.331%)(P<0.001 and P<0.001,respectively)compared to the control.There was no difference between the C4 a and control groups.RNA-seq analysis showed that C3 a promoted the proliferation of osteoclasts using the phosphoinositide 3-kinase(PI3 K)signaling pathway.The relative expressions of PIK3 CA/phosphoinositide dependent kinase-1(PDK1)/serum and glucocorticoid inducible protein kinases(SGK3)genes and PI3 K/PDK1/p-SGK3 protein in the C3 a group were significantly higher than in the control group.The activation role of C3 a in osteoclasts of MM patients was reduced by the SGK inhibitor(EMD638683).Conclusions:C3 a activated osteoclasts by regulating the PI3 K/PDK1/SGK3 pathways in MM patients,which was reduced using a SGK inhibitor.Overall,our results identified potential therapeutic targets and strategies for MBD patients。

EFFECTS OF ACUPUNCTURE ON THE IMMUNOLOGICAL FUNCTIONS IN HEPATITIS B VIRUS CARRIERS

A contrast study on the effects of manual acupuncture and electroacupuncture wasconducted in 60 cases of chronic hepatitis B carriers.The results demonstrated that theimmunological functions,both cellular and humoral,were markedly regulated asevidenced by the negative turnover rates of HBsAg,HBeAg,anti-HBc and HBcAg,as wellas the positive turnover rate of anti-HBe.

Risk of connective—tissue disease in men with testicular or penile prostheses：a preliminary study

AIM: To help clarifying the possibility of connective-tissue diseases in men with penile or testicular prostheses. METHODS: Eight patients underwent inflatable penile prostheses and 15, testicular prostheses consented to the study. Their medical records were reviewed and a follow-up interview and physical and serological examinations were performed. RESULTS: In patients with penile prostheses, there was no abnormal antinuclear antibody (ANA) or IgM elevation. The serum levels of the rheumatoid factor (RF), C4, IgA and IgG were abnormal in one patient, and the levels of erythrocyte sedimentation rate (ESR) and C3, abnormal in two. Four had elevated IgE. In patients with testicular prostheses, there was no abnormal RF, ANA or IgM. The serum levels of ESR and IgA were abnormal in two, and three had abnormal C4, ten abnormal C3, and eleven decreased IgG. All had increased IgE. Men with penile prostheses had higher serum levels of IgG and IgM than those with testicular prostheses (P=0.001, P=0.016, respectively). The rates of abnormal values of IgE and IgG were higher in men with testicular prostheses than in men with penile prostheses (P=0.008, P=0.009, respectively). Physical examination was normal in all patients and nobody had documented symptoms pertinent to connective-tissue diseases. CONCLUSION: Our findings suggest that the risk of connective-tissue diseases is not higher in patients wearing prostheses as the ANA is negative and there is no apparent manifestation suggestive of connective-tissue diseases.

Combined liver-kidney transplantation for rare diseases

Combined liver and kidney transplantation(CLKT)is indicated in patients with failure of both organs,or for the treatment of end-stage chronic kidney disease(ESKD)caused by a genetic defect in the liver.The aim of the present review is to provide the most up-to-date overview of the rare conditions as indications for CLKT.They are major indications for CLKT in children.However,in some of them(e.g.,atypical hemolytic uremic syndrome or primary hyperoxaluria),CLKT may be required in adults as well.Primary hyperoxaluria is divided into three types,of which type 1 and 2 lead to ESKD.CLKT has been proven effective in renal function replacement,at the same time preventing recurrence of the disease.Nephronophthisis is associated with liver fibrosis in 5%of cases and these patients are candidates for CLKT.In alpha 1-antitrypsin deficiency,hereditary C3 deficiency,lecithin cholesterol acyltransferase deficiency and glycogen storage diseases,glomerular or tubulointerstitial disease can lead to chronic kidney disease.Liver transplantation as a part of CLKT corrects underlying genetic and consequent metabolic abnormality.In atypical hemolytic uremic syndrome caused by mutations in the genes for factor H,successful CLKT has been reported in a small number of patients.However,for this indication,CLKT has been largely replaced by eculizumab,an anti-C5 antibody.CLKT has been well established to provide immune protection of the transplanted kidney against donor-specific antibodies against class I HLA,facilitating transplantation in a highly sensitized recipient.

Gene testing for osteonecrosis of the femoral head in systemic lupus erythematosus using targeted next-generation sequencing:A pilot study

BACKGROUND Previous publications indicated that genetic predisposition might play important roles in the onset of osteonecrosis of the femoral head(ONFH)in systemic lupus erythematosus(SLE).Some gene loci such as complement C3d receptor 2(CR2),nitric oxide synthase 3(NOS3),collagen type II alpha 1 chain(COL2A1),protein tyrosine phosphatase non-receptor type 22(PTPN22),and transient receptor potential cation channel subfamily V member 4(TRPV4)were reported to be involved in this process.AIM To investigate whether the risk of ONFH in SLE is associated with single nucleotide variations(SNVs)in these five genes.METHODS SNVs in the CR2,NOS3,COL2A1,PTPN22,and TRPV4 genes were examined by using FastTarget and Illumina Miseq sequencing technologies in 49 cases of SLE with ONFH.Burrows–wheeler aligner was used to align the sequencing reads to hg19,and GATK and Varscan programs were used to perform SNV calling.PolyPhen-2,SIFT,and MutationTaster were used to assess the functional effects of non-synonymous SNVs.RESULTS Six of the 49 patients were confirmed to have low frequency SNVs,including one patient with SNVs in NOS3(exon 6:c.814G>A:p.E272K and exon 7:c.814G>A:p.E272K.),four in COL2A1(rs41263847:exon 29:c.1913C>T:p.T638I,exon 28:c.1706C>T:p.T569I,and rs371445823:exon 8:c.580G>A:p.A194T,exon 7:c.373G>A:p.A125T),and one in CR2(rs45573035:exon 2:c.200C>G:p.T67S).CONCLUSION The onset of ONFH in SLE might be associated with the identified SNVs in NOS3,COL2A1,and CR2.

Ginsenoside modified lipid-coated perfluorocarbon nanodroplets: A novel approach to reduce complement protein adsorption and prolong in vivo circulation

Lipid-coated perfluorocarbon nanodroplets(lp-NDs)hold great promise in bio-medicine as vehicles for drug delivery,molecular imaging and vaccine agents.However,their clinical utility is restricted by limited targeted accumulation,attributed to the innate immune system(IIS),which acts as the initial defense mechanism in humans.This study aimed to optimize lp-ND formulations to mini-mize non-specific clearance by the IIS.Ginsenosides(Gs),the principal components of Panax ginseng,possessing complement inhibition ability,structural similarity to cholesterol,and comparable fat solubi-lity to phospholipids,were used as promising candidate IIS inhibitors.Two different types of ginsenoside-based Ip-NDs(Gs Ip-NDs)were created,and their efficacy in reducing IS recognition was examined.The Gs p-NDs were observed to inhibit the adsorption of C3 in the protein corona(PC)and the generation of SC5b-9.Adding Gs to Ip-NDs reduced complement adsorption and phagocytosis,resulting in a longer blood circulation time in vivo compared to lp-NDs that did not contain Gs.These results suggest that Gs can act as anti-complement and anti-phagocytosis adjuvants,potentially reducing non-specific clear-ance by the IS and improving lifespan.

Development of a C3c-based ELISA method for the determination of anti-complementary potency of Bupleurum polysaccharides

Traditionally, determination of inhibitory potency of complement inhibitors is performed by the hemolytic assay. However, this assay is not applicable to the lectin pathway, thus impeding the understanding of complement inhibitors against the overall function of the complement system. The main objective of our study was to develop a specific enzyme-linked immunosoihent assay (ELISA) as an alternative method to assess the anti-complement activity, particularly against the lectin pathway. By using respective coating substrates against different activation pathways, followed by capturing the stable C3c fragments, our ELBA method can be used to screen complement inhibitors against the classical pathway and the lectin pathway. The inhibitory effect of sununin on the classical pathway, as measured by our hemolytic assay is consistent with previous reports. Further assessment of suramin and Bupleurum polysaccharides against the lectin pathway showed a good reproducibility of the method. Comparison of the lectin pathway IC5is between Ruplearum.smithii var, purviPliam polysaccharides (1.055 ingtmE) and Buplcurann chinense polysaccharides (0.98 ing/mL) showed that similar to the classical and alterative pathway, these two Bupleurum polysaccharides had comparable anti complementary properties against the lectin pathway. The results demonstrate that the described EIASA assay can compensate for he shortcomings of the hemolytic assay in lectin pathway. (C) 2015 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

CD59 prevents human complement-mediated injuries in isolated guinea pig hearts

OBJECTIVE: To assess complement-mediated myocardial injury on isolated guinea pig working hearts and cardioprotective effects of CD59. METHODS: Using a modified Langendorff apparatus, isolated guinea-pig working hearts were perfused with a modified Krebs Henseleit buffer containing 3% heat-inactivated human plasma and zymosan (IPZ) (control) (n = 10), 3% normal human plasma and zymosan (NPZ) (n = 10), or 3% normal human plasma and zymosan and 1.5 microg/ml CD59 (NPZC) (n = 10), respectively. Epicardial electrocardiogram (ECG), cardiac output (CO), coronary arterial flow (CF), maximum left ventricular developed pressure (LVP(max)), maximum left ventricular developed pressure increase rate (+ dp/dt(max)), maximum left ventricular developed pressure decrease rate (- dp/dt(max)) and heart rate (HR) were recorded at 0, 15, 30, 45 and 60 min of treatment. After the experiment, immunohistochemical examination was performed to detect the presence of C3a or C5b-9 in the myocardium of the isolated hearts. RESULTS: Compared the IPZ group, hearts treated with NPZ showed a slight depression on ST segments of epicardial ECG at 15 min, a significant elevation between 30 min to 60 min, a decrease in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max), and an increase in HR at 15 min. The observed alterations in CF, CO, LVP(max), + dp/dt(max) and - dp/dt(max) remained decreased, while the HR remained increased until the end of the protocol. The all above parameters of hearts treated with NPZC were similar to the control group (IPZ) at any given time. Immunohistochemical examination showed positive signals of C3a and C5b-9 in the myocardium of hearts treated with NPZ. C3a was positive in NPZC, and C3a and C5b-9 were negative in IPZ. CONCLUSIONS: Activated human complements directly damage isolated guinea pig working hearts, and CD59 offers a significant protection against the injuries.

Analysis of Graves＇ ophthalmopathy patients＇ tear protein spectrum

Background Graves＇ ophthalmopathy/orbitopathy （GO） patients often suffer ocular surface damages and tear fluid proteins play a significant role in maintaining healthy ocular surfaces,while changes in tear protein components reflect the changes ocular surface abnormalities.In this study proteomics techniques were used to investigate tear protein compositions in GO patients.Methods We carried out a case-control study by comparing tear fluid contents of GO patients with that of healthy subjects.In the first step the tears were subjected to SDS-PAGE electrophoresis and then single protein bands were analyzed by to in-gel trypsin digestion and nano-flow liquid mass spectrometry （LC-MS/MS） using a MS software.Results In tear samples of GO subjects,the protein fractions of inflammation-related protein immunoglobulin kappa chain C region （IgKC） and serum albumin were essentially reduced,whereas a novel isoform of complement component 3 （C3）,which we detected in control subjects,was completely absent in the GO patients＇ tears.Conclusions Reduced protein concentrations of particularly IgKC and complement C3 as well as albumin in the tears of GO patients may contribute to changes in their ocular surfaces via diminished reactive oxygen species （ROS） depletion and adaptive immune responses.The completely absent of C3 in the GO patients＇ tears,may imply that an important inflammatory signaling pathway is affected,which needs further investigation.

Growth of G422 glioma implanted in the mouse brain was affected by the immune ability of the host

Background It is generally accepted that gliomas are the most common primary brain tumors with poor prognosis. We aimed to explore the relationship of the immunity of the central nervous system and the genesis and development of glioma. Methods G422 glioma was implanted in the brain of BALB/c mice （immuno-competent mice）, nude mice （T cell related immuno-deficient） and complement C3 knock-out mice （complement C3 related immunodeficient）. The survival time of the host, growth and histopathology of the tumor, and concentrations of tumor necrosis factor-α （TNF-α） and interferon-γ （INF-γ） in tumor tissues were assessed. Results Tumor spheres were formed in all mice after injection, and glial fibrillary acidic protein （GFAP） positive staining of the cells declared their glioma origin. The longest median survival time of （44.3±6.0） days was found in BALB/c mice, followed by （24.8±5.2） days in nude mice and the shortest （18.6±5.8） days in complement C3 knock-out mice. Accordingly, the growth of the tumor was fastest in complement C3 knock-out mice, followed by the nude mice and slowest in the BALB/c mice. Although the proportions of infiltrating CD68^± lymphocytes in tumor tissues showed no significant difference （P 〉0.05）, TNF-α level in the nude and C3 knock-out mice, （28.11±4.86） μmol/L and （22.87±6.36） μmol/L respectively, were significantly lower （P 〈0.01） than that in the BALB/c mice, which was （230.21±39.17） μmol/L. The INF-γ level was highest in the BALB/c mice （（180.76±29.19） μmol/L）, followed by the nude mice （（113.46±23.76） μmol/L） and then the C3 knock-out mice （（16.84±4.45） μmol/L）. Conclusions The G422 glioma implanted in the brains of mice with different immune ability would be a useful model for studying the relationship of the immune system and tumor in the central nervous system. Furthermore, the T cells and complement C3 compartments of the immune response may affect the growth of implanted tumors and inflammatory factors such as TNF-α and INF-γ.