Yersinia pestis Ail recruitment of C4b‐binding protein leads to factor I‐mediated inactivation of covalently and noncovalently bound C4b

The outer membrane protein Ail of Yersinia pestis mediates several virulence functions, including serum resistance. Here, we demonstrate that Ail binds C4b‐binding protein (C4BP), the primary fluid‐phase regulator of the classical and lectin pathways. Non‐covalent binding of C4 and C4b to Ail was also observed. C4BP bound to Ail can act as a cofactor to the serine protease factor I (fI) in the cleavage of fluid‐phase C4b. Employing a panel of C4BP alpha‐chain mutants, we observed that the absence of complement control protein domain 6 and 8 reduced binding to Ail. Immunoblot analysis of normal human serum (NHS)‐treated bacteria revealed minimal C4b alpha’‐chain complexes with bacterial outer membrane targets. Addition of the anti‐C4BP monoclonal antibody MK104 to NHS restored C4b‐alpha’ chain target complexes, suggesting that C4b binds covalently to targets on the Y. pestis surface. C4b bound to Ail noncovalently was also cleaved in a C4BP and fI‐dependent manner, leaving the C4c fragment bound to Ail. MK104 also prevented the cleavage of noncovalently bound C4b. Collectively, these data suggest that when C4BP is bound to Ail, fI can cleave and inactivate C4b that has bound covalently to bacterial surface structures as well as C4b bound noncovalently to Ail.     

Membrane cofactor protein (MCP,CD46) in seminal plasma and on spermatozoa in normal and “sterile” subjects

A sperm protein of molecular mass 43 kDa (the spermatozoa membrane cofactor protein, smMCP) and a seminal plasma protein of 60 kDa (ssMCP) were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by immunoblotting with four monoclonal antibodies (mAb) against membrane cofactor protein (MCP, CD46).These proteins served as factor I cofactors for the cleavage of methylamine-treated C3 (C3ma), the activity of which was blocked by M75, an MCP cofactor-activity-blocking mAb. Thus, these semen proteins are antigenic and functional homologoues of MCP. On SDS-PAGE analysis these MCP migrated as single-band proteins which differed from the two-band forms of MCP expressed on other cells. smMCP was N-glycosylated but not O-glycosylated, while ssMCP was O-glycosylated: after deglycosylation of these proteins bands were detected at 38-40 kDa and 43 kDa on SDS-PAGE, respectively. These semen MCP are therefore, structurally different from the conventional MCP. ssMCP in both normal and “sterile” subject groups was determined by sandwich enzyme-linked immunosorbent assay. Seminal plasma in the two groups contained 250-700 ng/ml ssMCP. The difference between the two groups was marginal, although samples from normal subjects tended to show higher concentrations of ssMCP than samples from “sterile” subjects. No molecular difference was observed with ssMCP and smMCP in the two groups by SDS-PAGE/immunobloblotting analysis. Immunohistochemical analysis suggested that MCP was positive in glandular epithelial cells and the lumen of the prostate, and in most intra-lumen cells of the testis. Using antibody M177, solubilized prostate and testis were analyzed by immunoblotting and compared with other cell MCP The major band of MCP in the testis, but not in the prostate, was of 60 kDa, which aligned with ssMCP. No band of testis or prostate MCP, however, aligned with smMCP. ssMCP may be produced in the testis, while the origin of smMCP remains unknown. We hypothesize that ssMCP is important in the survival of spermatozoa, protecting them against local secretion of immunoglobulin and complement in the female genital tract, and that smMCP, which is expressed on acrosome-reacted spermatozoa, plays an essential role in the interaction of spermatozoa with oocytes.     

Identification of the Complement Regulatory Proteins CD46, CD55, and CD59 in Human Fallopian Tube,Endometrium, and Cervical Mucosa and Secretion

PROBLEM : Complement lytic activity has been demonstrated, and a potential for its activation is present in human cervical and tubal secretions and in the endometrium. This necessitates the presence of regulatory mechanisms for protection of the sperm and the implanting allogeneic conceptus in the female genital tract. Complement regulatory proteins demonstrated on sperm and in seminal fluid have been attributed such a role. It is however likely that additional protection is required for a successful conception and implantation to take place. This lead us to investigate the distribution of the complement regulatory factors in cervical mucus and mucosa, uterine endometrium, and fallopian tube. METHOD : Endometrium and cervical mucosa were obtained from patients undergoing hysterectomy for benign conditions, and specimens were selected from different stages of the menstrual cycle. Fallopian tubes were obtained from patients submitted for sterilization, while cervical mucus was aspirated from volunteers undergoing gynecological examination. Immunohistochemistry was performed on all tissue samples, using monoclonal antibodies to membrane cofactor protein (MCP), decay accelerating factor (DAF), CD59 and complement receptor 1 (CR1). Western blot analysis was performed on cervical mucus under nonreducing conditions. RESULTS : MCP, DAF, and CD59 were found to be expressed in human endometrium and fallopian tube. No variation in expression was detected throughout the menstrual cycle. CR1 was not expressed. Soluble forms of DAF and CD59 were found to be present in cervical mucus. CONCLUSION : The complement regulatory proteins MCP, DAF, and CD59 are expressed throughout the female genital tract, and may thus play an important role in protecting the traversing sperm and implanting blastocyst from complement mediated damage.     

Searching for links between endotoxin exposure and pregnancy loss: CD14 polymorphism in idiopathic recurrent miscarriage

PROBLEM: Lipopolysaccharide (LPS) (endotoxin) is a well-known inducer of abortions in mice. In addition it has been proposed that gut-derived LPS of gram-negative bacteria may play a role in triggering idiopathic recurrent miscarriage (IRM) in humans. CD14 is one of the key molecules that mediates the effects of LPS. Promoter region polymorphism (-159C/T) in the CD14 gene is functionally important by regulating CD14 levels. High-producing CD14 genotype (TT) associates with deleterious effects of gut-derived LPS in hepatic cirrhosis in humans. It is not known whether women with IRM are genetically more prone to suffer from toxic effects of LPS. METHOD OF STUDY: By using polymerase chain reaction we analyzed the CD14 promoter region polymorphism in 38 women with IRM and in 127 normal controls of Finnish origin. RESULTS: There were no significant differences in the CD14 (-159C/T) allele or the genotype frequencies between the IRM women and the controls. However, there was a trend associating the presence of the T allele with increased odds of miscarriage. CONCLUSIONS: Although we were not able to find a statistically significant association between CD14 genotypes and IRM in our relatively small study population, a further study with a larger sample size is warranted to explore the role of high-producing CD14 genotypes in IRM. Also studies highlighting environmental LPS triggers and other intrinsic mediators of LPS signalling are needed to solve the enigmatic role of LPS in IRM in humans.     

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35. More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsCD55 and rsCD35 to regulate human complement activation mediated by the classical and alternative pathways was directly compared. Regulation of the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46 + rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not as effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCD55 and rsCD35, may be a useful therapeutic agent.     

Expression of complement C3, C5, C3aR and C5aR1 genes in resting and activated CD4+ T cells

Complement activation is traditionally thought to occur in the extracellular space. However, it has been suggested that complement proteins are activated and function at additional locations. T cells contain intracellular stores of C3 and C5 that can be cleaved into C3a and C5a and bind to intracellular receptors, which have been shown to be of vital importance for the differentiation and function of these cells. However, whether the origin of the complement proteins located within T cells is derived from endogenous produced complement or from an uptake dependent mechanism is unknown.The presence of intracellular C3 in T cells from normal donors was investigated by fluorescence microscopy and flow cytometry. Moreover, mRNA expression levels of several genes encoding for complement proteins with primary focus on C3, C3aR, C5 and C5aR1 during resting state and upon activation of CD4⁺ T cells were investigated by a quantitative PCR technique. Furthermore, the gene expression level was evaluated at different time points.We confirmed the presence of intracellular C3 protein in normal T-cells. However, we could not see any increase in mRNA levels using any activation strategy tested. On the contrary, we observed a slight increase in C3 and C5aR1 mRNA only in the non-activated T-cells compared to the activated T cells, and a decrease in the activated T-cells at different incubation time points.Our results show that there is a baseline intracellular expression of the complement C3, C5, C3aR and C5aR1 genes in normal CD4⁺ T cells, but that expression is not increased during T-cell activation, but rather down regulated. Thus, the pool of intracellular complement in CD4⁺ T cells may either be due to accumulated complement due low-grade expression or arise from the circulation from an uptake dependent mechanism, but these possibilities are not mutually exclusive.     

Lack of association between polymorphisms in C4b‐binding protein and atypical haemolytic uraemic syndrome in the Spanish population

Dysregulation of the alternative pathway of complement activation, caused by mutations or polymorphisms in the genes encoding factor H, membrane co‐factor protein, factor I or factor B, is associated strongly with predisposition to atypical haemolytic uraemic syndrome (aHUS). C4b‐binding protein (C4BP), a major regulator of the classical pathway of complement activation, also has capacity to regulate the alternative pathway. Interestingly, the C4BP polymorphism p.Arg240His has been associated recently with predisposition to aHUS and the risk allele His240 showed decreased capacity to regulate the alternative pathway. Identification of novel aHUS predisposition factors has important implications for diagnosis and treatment in a significant number of aHUS patients; thus, we sought to replicate these association studies in an independent cohort of aHUS patients. In this study we show that the C4BP His240 allele corresponds to the C4BP*2 allele identified previously by isoelectric focusing in heterozygosis in 1·9–3·7% of unrelated Caucasians. Crucially, we found no differences between 102 unrelated Spanish aHUS patients and 128 healthy age‐matched Spanish controls for the frequency of carriers of the His240 C4BP allele. This did not support an association between the p.Arg240His C4BP polymorphism and predisposition to aHUS in the Spanish population. In a similar study, we also failed to sustain an association between C4BP polymorphisms and predisposition to age‐related macular degeneration, another disorder which is associated strongly with polymorphisms in factor H, and is thought to involve alternative pathway dysregulation.     

Major histocompatibility complex class III (C2, C4, factor B) and C3 gene variants in patients with pulmonary tuberculosis

The complement system is an integral part of the host immune system and plays an immunoregulatory role at the interface of innate and acquired immune responses. Limited data are available on the influence of variations in complement genes in infectious diseases such as pulmonary tuberculosis (PTB). The aim of this study was to investigate the role of genetic variations in complement system components C2, C4, BF, and C3 in PTB (n = 125) compared with healthy controls (n = 125) in the Indian population. The study showed, for the first time, an increased occurrence of null alleles at the C4A, i.e., C4AQ0; an increased frequency of BF*FA and C3*F in patients with PTB compared with healthy individuals, and contributed a risk with odds ratios of 18.16 (95% confidence interval [CI] = 3.0-108.6, p = 0.0004), 2.9 (95% CI = 1.9-4.37, p(c) = 3.15E-06), and 2.26 (95% CI = 1.5-3.3, p(c) = 6.7E-05), respectively. A combinatorial analysis of complement gene variants as risk determinants and their phenotypic effects in various populations may provide unique insights into the genetic basis of susceptibility to PTB.     

血清超敏C反应蛋白、转铁蛋白、补体C3和C4在儿童全身炎性反应综合征中的临床价值

目的 探讨全身炎性反应综合征(SIRS)患儿血清超敏C反应蛋白(hs-CRP)、转铁蛋白(TRf)和补体C3、C4水平的变化及意义.方法 2012年7月至2013年6月本院PICU、NICU和急诊综合病区符合SIRS诊断的患儿93例(SIRS组),对照组为年龄、性别与患儿相匹配的健康体检儿童65例,测定两组儿童血清中hs-CRP、TRf、补体C3和C4的水平.对93例患儿进行SIRS评分,根据评分分成A组(SIRS 2分)37例、B组(SIRS 3分)32例、C组(SIRS 4分)24例,比较3组血清中hs-CRP、TRf、补体C3和C4的水平并统计病死率,与SIRS评分进行相关分析.结果 SIRS组患儿血清hs-CRP水平高于对照组[(33.85±17.76)mg/L比(2.34±1.54) mg/L,P＜0.05].SIRS组血清TRf、补体C3和C4的水平均低于对照组[血清TRf:(1.26±0.48)g/L比(2.81±0.57)g/L,补体C3:(0.48±0.19)g/L比(1.14±0.21)g/L,补体C4:(0.19±0.09)g/L比(0.39±0.10)g/L,均P＜0.05].SIRS患儿血清hs-CRP水平随SIRS评分升高而升高,呈正相关(r=0.863,P＜0.05),血清TRf、补体C3和C4的水平则随SIRS评分的升高而降低,呈负相关(r=-0.834、-0.715、-0.691,均P＜0.05).病死率也随SIRS评分的升高而逐渐升高,呈正相关(r=1.00,P＜0.05).结论 血清中hs-CRP、TRf、补体C3和C4的水平对评估SIRS患儿的病情和预后有重要意义.     

Analysis of plasminogen activator inhibitor-1, integrin beta3, beta fibrinogen, and methylenetetrahydrofolate reductase polymorphisms in Iranian women with recurrent pregnancy loss

Citation
Jeddi‐Tehrani M, Torabi R, Zarnani AH, Mohammadzadeh A, Arefi S, Zeraati H, Akhondi MM, Chamani‐Tabriz L, Idali F, Emami S, Zarei S. Analysis of plasminogen activator inhibitor‐1, integrin beta3, beta fibrinogen and methylenetetrahydrofolate reductase polymorphisms in Iranian women with recurrent pregnancy loss. Am J Reprod Immunol 2011; 66: 149–156 Problem To identify the associations of the plasminogen activator inhibitor‐1 (PAI‐1) ?675 4G/5G, beta fibrinogen (BF) ?455G/A, integrin beta 3 (ITGB3) 1565T/C, and methylenetetrahydrofolate reductase (MTHFR) 677C/T and 1298A/C polymorphisms with recurrent pregnancy loss (RPL). Method of study Polymerase chain reaction and restriction fragment length polymorphism (PCR‐RFLP) were performed to assess the frequency of five candidate genetic risk factors for RPL, and the frequencies of the polymorphisms were calculated and compared between case and control groups. Results The BF ?455G/A, MTHFR 677C/T, and 1298A/C polymorphisms were found to be positively, and ITGB3 1565T/C polymorphism negatively, associated with RPL. Homozygosity but not heterozygosity for PAI‐1 ?675 4G/5G polymorphism was significantly higher in patients with RPL than in the control group. The presence of both mutations of MTHFR genes highly increased the risk of RPL. Conclusion The data highlight the importance of thrombophilia screening in patients with RPL.     

Low maternal serum concentrations of mannose‐binding lectin are associated with the risk of shorter duration of pregnancy and lower birthweight

Chronic inflammation has been implicated as the underlying mechanism responsible for the pathophysiology of preterm labour. Mannose‐binding lectin (MBL) plays a central role in the innate immune response and is thus an important component of the first line of defense. The aim of this study was to investigate whether serum concentrations of MBL correlated with the incidence of preterm birth and low birthweight in a cohort of women with signs of threatened preterm birth. A cohort of 60 patients who presented with regular contractions and/or short cervix (group A) between 24 and 32 weeks of gestation and 20 healthy controls (group B) who had no pregnancy complications and delivered at term were recruited into a prospective study. The following outcomes were recorded: presence of preterm labour and birthweight in all patients. MBL and high sensitivity C‐reactive protein levels were measured in all serum samples. The serum concentrations of MBL were significantly reduced in patients with threatened preterm labour (Group A), compared to the control Group B. Furthermore, infants born to Group A mothers with MBL deficiency (n = 13, MBL ≤100 ng/mL) had significantly lower birthweights, compared to those born to Group A women with normal MBL serum concentrations (P  < .0001). Our small cohort study demonstrated a strong association between MBL deficiency and preterm delivery, and associated low birthweight. MBL deficiency could thus be considered an important risk factor for preterm birth.     

不明原因复发性流产与精子DNA完整性的关系

目的探讨不明原因复发性流产与男方精子DNA完整性的关系。方法精子DNA完整性检测采用精子染色质扩散试验（sperm chromatin dispersion,SCD）,以DNA断裂指数（DNA fragmentation index,DFI）表示。分别对不明原因复发性流产男方与无流产史男方精子进行精液常规分析和DFI分析。结果流产组精子密度、活动率、前向运动率和DFI与生育组比较差异有统计学意义（P〈0.05）。结论不明原因复发性流产可能与精子DNA损伤存在一定关系。     

Complement 4 phenotypes and genotypes in Brazilian patients with classical 21‐hydroxylase deficiency

The aim of this work was to analyse C4 genotypes, C4 protein levels, phenotypes and genotypes in patients with the classical form of 21‐hydroxylase deficiency. Fifty‐four patients from 46 families (36 female, 18 male; mean age 10·8 years) with different clinical manifestations (31 salt‐wasting; 23 simple‐virilizing) were studied. Taq I Southern blotting was used to perform molecular analysis of the C4/CYP21 gene cluster and the genotypes were defined according to gene organization within RCCX modules. Serum C4 isotypes were assayed by enzyme‐linked immunosorbent assay. The results revealed 12 different haplotypes of the C4/CYP21 gene cluster. Total functional activity of the classical pathway (CH50) was reduced in individuals carrying different genotypes because of low C4 concentrations (43% of all patients) to complete or partial C4 allotype deficiency. Thirteen of 54 patients presented recurrent infections affecting the respiratory and/or the urinary tracts, none of them with severe infections. Low C4A or C4B correlated well with RCCX monomodular gene organization, but no association between C4 haplotypes and recurrent infections or autoimmunity was observed. Considering this redundant gene cluster, C4 seems to be a well‐protected gene segment along the evolutionary process.     

A distinct chemokine axis does not account for enrichment of Foxp3+ CD4+ T cells in carcinogen‐induced fibrosarcomas

The frequency of CD4⁺ Foxp3⁺ regulatory T (Treg) cells is often significantly increased in the blood of tumour-bearing mice and people with cancer. Moreover, Treg cell frequencies are often higher in tumours compared with blood and lymphoid organs. We wished to determine whether certain chemokines expressed within the tumour mass selectively recruit Treg cells, thereby contributing to their enrichment within the tumour-infiltrating lymphocyte pool. To achieve this goal, the chemokine profile of carcinogen-induced fibrosarcomas was determined, and the chemokine receptor expression profiles of both CD4⁺ Foxp3⁻ and CD4⁺ Foxp3⁺ T cells were compared. These analyses revealed that the tumours are characterized by expression of inflammatory chemokines (CCL2, CCL5, CCL7, CCL8, CCL12, CXCL9, CXCL10 and CX3CL1), reflected by an enrichment of activated Foxp3⁻ and Foxp3⁺ T cells expressing T helper type 1-associated chemokine receptors. Notably, we found that CXCR3⁺ T cells were significantly enriched in the tumours although curiously we found no evidence that CXCR3 was required for their recruitment. Instead, CXCR3 marks a population of activated Foxp3⁻ and Foxp3⁺ T cells, which use multiple and overlapping ligand receptor pairs to guide their migration to tumours. Collectively, these data indicate that enrichment of Foxp3⁺ cells in tumours characterized by expression of inflammatory chemokines, does not occur via a distinct chemokine axis, thus selective chemokine blockade is unlikely to represent a meaningful therapeutic strategy for preventing Treg cell accumulation in tumours.     

B‐cell activation with CD40L or CpG measures the function of B‐cell subsets and identifies specific defects in immunodeficient patients

Around 65% of primary immunodeficiencies are antibody deficiencies. Functional tests are useful tools to study B‐cell functions in vitro. However, no accepted guidelines for performing and evaluating functional tests have been issued yet. Here, we report our experience on the study of B‐cell functions in infancy and throughout childhood. We show that T‐independent stimulation with CpG measures proliferation and differentiation potential of memory B cells. Switched memory B cells respond better than IgM memory B cells. On the other hand, CD40L, a T‐dependent stimulus, does not induce plasma cell differentiation, but causes proliferation of naïve and memory B cells. During childhood, the production of plasmablasts in response to CpG increases with age mirroring the development of memory B cells. The response to CD40L does not change with age. In patients with selective IgA deficiency (SIgAD), we observed that switched memory B cells are reduced due to the absence of IgA memory B cells. In agreement, IgA plasma cells are not generated in response to CpG. Unexpectedly, B cells from SIgAD patients show a reduced proliferative response to CD40L. Our results demonstrate that functional tests are an important tool to assess the functions of the humoral immune system.     

Spontaneous CD4+ and CD8+ T‐cell responses directed against cancer testis antigens are present in the peripheral blood of testicular cancer patients

Cancer/testis antigen (CTAg) expression is restricted to spermatogenic cells in an immune‐privileged site within the testis. However, these proteins are expressed aberrantly by malignant cells and T‐cell responses against CTAgs develop in many cancer patients. We investigated the prevalence, magnitude and phenotype of CTAg‐specific T cells in the blood of patients with testicular germ cell tumors (TGCTs). CD8⁺ and CD4⁺ T‐cell responses against MAGE‐A family antigens were present in 44% (20/45) of patients’ samples assayed by ex vivo IFN‐γ ELISPOT. The presence of MAGE‐specific CD8⁺ T cells was further determined following short‐term in vitro expansion through the use of pMHC‐I multimers containing known immunogenic peptides. Longitudinal analysis revealed that the frequency of MAGE‐specific T cells decreased by 89% following orchidectomy suggesting that persistence of tumor antigen is required to sustain CTAg‐specific T‐cell immunity. Notably, this decrease correlated with a decline in the global effector/memory T‐cell pool following treatment. Spontaneous T‐cell immunity against CTAg proteins therefore develops in many patients with testicular cancer and may play an important role in the excellent clinical outcome of patients with this tumor subtype.