Complement activation in the plasma and placentas of women with different subsets of antiphospholipid syndrome.

PROBLEM: As antiphospholipid antibody-positive women with adverse pregnancy outcomes have higher plasma complement activation product levels, and the placentas of women with antiphospholipid syndrome (APS) exhibit C4d complement component deposition, complement activation involvement has been hypothesized in APS pregnancy complications. METHOD OF STUDY: Plasma levels of C5a and C5b-9 complement components of 43 APS non-pregnant patients and 17 pregnant APS women were measured using enzyme-linked immunosorbent assay. The results were compared with those of 16 healthy non-pregnant women and eight healthy pregnant women, respectively. Placenta samples of five APS patients at high risk of pregnancy complications and of five healthy controls were subjected to immunoblotting analysis with specific antibodies to C5b-9 and CD46, CD55, CD59 complement regulators. RESULTS: The mean plasma C5a and C5b-9 levels were significantly higher in the non-pregnant APS patients with previous thrombosis ± pregnancy morbidity (P = .0001 and P = .0034, respectively) and in the pregnant APS women with adverse outcomes (P = .0093 for both). Similarly, C5b-9 amounts were significantly higher in the adverse pregnancy outcome placenta (P = .0115) than in those associated to a favorable outcome. The mean CD46, CD55 and CD59 amounts were, instead, lower, although not always significantly, in the placentas of all the high-risk APS women with respect to the control placentas. CONCLUSION: Data analysis demonstrated that there was significant complement activation in the more severe subset of APS patients and in only the adverse pregnancy outcome APS women. Further studies will clarify whether the lower CD46, CD55, and CD59 expressions in the APS placentas are limited to only high-risk APS patients.

Clinical features and new diagnostic criteria for the syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis.

AIM: The syndrome of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) is a common inflammatory disease that presents with periodic fever. We aimed to establish more specific diagnostic criteria for PFAPA based on the clinical characteristics of PFAPA patients in our directory. METHOD: The clinical, laboratory, genetic, and family history details of 257 Japanese PFAPA patients treated at our and other affiliated hospitals between April 2000 and April 2018 were analyzed along with quantitative measurements of the number of CD64 molecules on neutrophils, and the levels of serum inflammatory cytokines. The sensitivity and specificity of the criteria were calculated for several diseases. RESULTS: Because recurrent fevers were crucial findings, they were defined as the required criterion. Tonsillitis/pharyngitis with white moss were important accompanying signs. Other symptoms associated with febrile episodes were cervical lymphadenitis with tenderness, aphthous stomatitis, sore throat, vomiting, and headache but not cough. A total of 159 (62%) patients had a family history of recurrent fevers, indicating autosomal dominant inheritance. C-reactive protein levels were extremely elevated during febrile attacks but normal in attack-free periods. Serum immunoglobulin D levels were high in 72 of the 199 tested patients. Oral glucocorticoid and cimetidine were extremely effective in all and 51.6% of the patients, respectively. We defined the above as supportive criteria. These criteria were sensitive and specific enough to distinguish PFAPA from other recurrent fever diseases. Raised serum interferon-γ levels and remarkable CD64 expression on neutrophils during flare-ups were recognized, indicating they contributed to diagnosis. CONCLUSION: Our new criteria are useful for diagnosing PFAPA.

Defective gene expression of the membrane complement inhibitor CD46 in patients with progressive immunoglobulin A nephropathy.

BACKGROUND: Complement is thought to play a role in immunoglobulin A nephropathy (IgAN), though the activating mechanisms are unknown. This study focused on the gene expression of CD46 and CD55, two key molecules for regulating C3 convertase activity of lectin and alternative complement pathways at a cellular level. METHODS: The transcriptional expression in peripheral white blood cells (WBCs) of CD46 and CD55 was investigated in 157 patients enrolled by the Validation of the Oxford Classification of IgAN group, looking for correlations with clinical and pathology features and estimated glomerular filtration rate (eGFR) modifications from renal biopsy to sampling. Patients had a previous median follow-up of 6.4 (interquartile range 2.8-10.7) years and were divided into progressors and non-progressors according to the median value of their velocity of loss of renal function per year (-0.41 mL/min/1.73 m2/year). RESULTS: CD46 and CD55 messenger RNA (mRNA) expression in WBCs was not correlated with eGFR values or proteinuria at sampling. CD46 mRNA was significantly correlated with eGFR decline rate as a continuous outcome variable (P = 0.014). A significant difference was found in CD46 gene expression between progressors and non-progressors (P = 0.013). CD46 and CD55 mRNA levels were significantly correlated (P < 0.01), although no difference between progressors and non-progressors was found for CD55 mRNA values. The prediction of progression was increased when CD46 and CD55 mRNA expressions were added to clinical data at renal biopsy (eGFR, proteinuria and mean arterial blood pressure) and Oxford MEST-C (mesangial hypercellularity, endocapillary hypercellularity, segmental glomerulosclerosis, tubular atrophy/interstitial fibrosis, presence of any crescents) score. CONCLUSIONS: Patients with progressive IgAN showed lower expression of mRNA encoding for the complement inhibitory protein CD46, which may implicate a defective regulation of C3 convertase with uncontrolled complement activation.

A pilot study of the effect of ezetimibe for postprandial hyperlipidemia.

This study aimed to explore the feasible effect of ezetimibe for postprandial hyperlipidemia (PPHP).Sixty participants were included in this study. Of these, 30 subjects in the intervention group received ezetimibe, while the remaining 30 participants in the control group did not undergo ezetimibe. All patients in intervention group were treated for a total of 2 weeks. Primary endpoints consisted of serum levels of total cholesterol (Total-C), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG). Secondary endpoints included apoB-48, remnant lipoprotein cholesterol (RLP-C), blood glucose, insulin, hemoglobin A1c (HbA1c), and monocyte chemotactic protein (MCP). All outcomes were measured before and after 2-week treatment.After 2-week treatment, participants in the intervention group did not show better outcomes in primary endpoints of Total-C, LDL-C, HDL-C, and TG; and secondary endpoints of apoB-48, RLP-C, blood glucose, insulin, HbA1c, and MCP, compared with subjects in the control group.The results of this study showed that ezetimibe may be not efficacious for participants with PPHP after 2-week treatment.

Increased systemic microbial translocation is associated with depression during early pregnancy.

Plasma level of microbial translocation is a marker of mucosal permeability. Increased mucosal permeability ignites elevated microbial translocation and as a consequence of systemic inflammation. Pregnant women with depression have higher levels of inflammatory markers relative to pregnant women without depression, however, no studies have reported whether systemic microbial translocation will change in depressed women during pregnancy. In this study, we examined the plasma LPS level of depressed women during pregnancy. The results showed that the plasma LPS level was significantly increased in depressed mothers during their 8-12 weeks gestation compared to healthy controls. Compared to 8-12 weeks gestation, the plasma LPS levels were significantly decreased at 24-28 weeks gestation and 6-8 weeks postpartum in both depressed subjects and healthy controls. Furthermore, the plasma levels of pro-inflammatory cytokines (TNF-α and MCP/CCL2) associated with microbial translocation were significantly increased in depressed subjects during 8-12 weeks gestation compared to healthy controls. These results indicate that the level of microbial translocation is increased in depressed women during early pregnancy.

Role of CD46 Polymorphisms in the Occurrence of Disease in Young Chinese Men With Human Adenovirus Type 55 Infection.

OBJECTIVE: Human adenovirus type 55 (HAdV-55) has recently caused multiple outbreaks. This study examined polymorphisms in CD46 to determine their involvement in HAdV-55 infection. METHODS: A total of 214 study subjects infected with HAdV-55 were included in our study. The study subjects were divided into those with silent infections (n=91), minor infections (n=85), and severe infections (n=38). Ten single nucleotide polymorphisms (SNPs) from CD46 were examined. RESULTS: Compared with the AA genotype, the TT genotype at rs2724385 (CD46, A/T) was associated with a protective effect against disease occurrence, with an odds ratio (95% confidence interval) of 0.20 (0.04-0.97) (P=0.038). There were no significant differences between the patients with minor and severe infection and those who had silent HAdV-55 infection in the other CD46 SNPs. We next compared the polymorphisms of these genes according to disease severity in HAdV-55-infected patients with clinical symptoms. The results showed that there were no significant differences between minor infections and severe infections. CONCLUSIONS: Our results suggested that the CD46 SNP at rs2724385 is associated with the occurrence of disease in HAdV-55-infected patients. A much larger number of samples is required to understand the role of CD46 polymorphisms in the occurrence and progression of infection by HAdV-55. (Disaster Med Public Health Preparedness. 2018;12:427-430).

Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus.

IFN-γ-producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4+ T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN-γ production. Further, CD46-mediated signals also induce co-expression of immunosuppressive IL-10 in Th1 cells and transition into a (self)-regulating and contracting phase. In consequence, C3 or CD46-deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46-regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP-9-mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP-9 activity normalized release of soluble CD46 and restored Th1 contraction in patients' T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46-cleaving proteases could be a novel avenue to modulate Th1 responses.

Soluble forms of CD46 are detected in Bos taurus plasma and neutralize BVDV, the bovine pestivirus.

The pestivirus bovine viral diarrhea virus (BVDV) is known to bind to the CD46 molecule, which subsequently promotes entry of the virus. Mapping of the BVD-virion-binding site has shown that two peptides, 66EQIV69 and 82GQVLAL87, located on antiparallel beta sheets in the most distal complement control protein module (CCP1), provide the attachment platform. In the present study, we reveal new CD46-encoding transcripts that are predicted to encode CCP1-containing soluble forms. Further, we show that the serum of most adult cattle contains soluble CD46 (sCD46) and that a recombinant soluble isoform neutralizes BVDV infectivity in an in vitro assay. We have then established an ELISA for determination of plasma sCD46 in a large cohort of animals. Overall, serum sCD46 amounts to 8±18ng/mL (mean±SD, n=440), with a IC [95-105] ranging from 6,4 to 9,8ng/mL and extreme values ​​between 0 and 178ng/mL. We found that sCD46 is not detectable in fetal and neonatal sera and that its plasma concentration increases progressively up to adulthood. We also detected high- and low-sCD46 performers and show that this phenotype does not depend of environment. As modern rearing techniques make it possible to disseminate genetically-determined phenotypes very quickly in a population, a large-scale study examining whether high-sCD46 animals provide epidemiological protection against BVDV infection and transmission should be undertaken.

Evaluation of the markers of inflammation in the umbilical cord blood of newborns of mothers with thrombophilia.

OBJECTIVE: To answer the question as to whether the markers of thrombophilia in pregnant women, whose pregnancies ended in success, are reflected in the level of inflammation in the blood of the umbilical cord of the newborn. MATERIALS AND METHODS: Umbilical cord blood and placenta after childbirth were secured from 16 patients with inherited (n = 7), acquired (n = 9) thrombophilia, and control group (n = 20). The concentrations of cytokines IL1ß, IL10, TNFα, C5a anaphylatoxin, and granzyme A were assessed. decay accelerating factor (DAF) and membrane cofactor protein (MCP) levels were determined in the placentas, and the incidence of thrombotic changes was evaluated. RESULTS: Higher levels of anaphylatoxin C5a (P = 0.041), TNFα (P = 0.016), and IL1ß (P = 0.037) were observed in the study group compared to the control group. In the study group, C5a levels correlated with the levels of TNFα (P = 0.018) and IL1ß (P = 0.012). Higher levels of DAF and MCP proteins in study group were found in the control group (P < 0.001). In placentas from study group, there was a more frequent occurrence of incidences of thrombotic changes. CONCLUSION: The observed, increased levels of pro-inflammatory factors in the cord blood of newborns of mothers with thrombophilia may result from a reaction of the prothrombotic and pro-inflammatory markers of thrombophilia present in maternal blood.

Does dysregulated complement activation contribute to haemolytic uraemic syndrome secondary to Streptococcus pneumoniae?

We describe two patients with haemolytic uraemic syndrome (HUS) associated with invasive Streptococcus pneumoniae infection. Both patients had transiently reduced serum concentrations of complement C3. One had reduced expression of CD46 and never recovered renal function. No constitutive defect in regulation of the alternative pathway of complement activation was demonstrated in the second patient but there was an apparent improvement in her condition after administration of eculizumab. The most widely accepted mechanism for pneumococcal HUS is endothelial cell damage by pre-formed antibodies against the Thomsen-Friedenreich antigen. This explanation does not bear rigorous scrutiny. We postulate that transiently dysregulated complement activation may play a role in the pathogenesis of pneumococcal disease. We further postulate that the mechanism could be enhanced binding of factor H to the neuraminidase-altered surface of endothelial cells or reduced binding of factor H to the endothelial cell surface mediated by competitive binding of factor H by pneumococcal surface protein C (pspC).

Use of complement regulators, CD35, CD46, CD55, and CD59, on leukocytes as markers for diagnosis of viral and bacterial infections.

Several complement regulatory proteins exist on self-cells to prevent damage by the serum complement system. In the present study, we aimed to perform quantitative analysis of membrane-bound complement regulators, CR1 (CD35), MCP (CD46), DAF (CD55), and MIRL (CD59), on peripheral blood neutrophils, monocytes, and lymphocytes from healthy controls (n=36) and febrile patients diagnosed with either bacterial (n=21) or viral (n=26) infections. Our results show that: (a) increased CD35 and CD55 levels on neutrophils and monocytes present potent markers of bacterial infection, (b) increased expression of CD46 on monocytes is an indicator of viral infection, and (c) increased CD59 expression on neutrophils and monocytes is a general infection marker. Additionally, CD19-positive B-lymphocytes represent practically the only lymphocyte population capable of expressing CD35. We further developed two novel clinical flow cytometric markers (indices), specifically, clinical mononucleosis (CM)-INDEX (incorporating CD35, CD55, and CD59 expression on lymphocytes) and clinical bacterial infection (CBI)-INDEX (incorporating CD35 and CD55 expression on neutrophils and lymphocytes), for the effective detection of viral mononucleosis and bacterial infection, respectively. In summary, bacterial and viral infections induce different expression patterns of membrane-bound complement regulators in human leukocytes, which may be effectively exploited in clinical differential diagnosis.

Expression of human CD46 has no effect on porcine circovirus type 2 infection and shedding in the experimental pig model.

Xenotransplantation of tissues from transgenic pigs with desired genetic modifications such as CD46 expression help minimize xenograft rejections. However, CD46 is a known receptor for some viruses. In this study, pigs transgenic for human CD46 (CD46-TG) and appropriate non-transgenic (non-TG) control pigs were utilized to determine possible differences in the level of replication and shedding of porcine circovirus type 2 (PCV2). Non-TG and CD46-TG were blocked by transgenic status and randomly divided into three groups: Non-TG negative controls (n = 3), non-TG-PCV2 (n = 10; PCV2a = 5, PCV2b = 5), and CD46-TG-PCV2 (n = 6; PCV2a = 3, PCV2b = 3). Blood, oral, nasal and fecal swabs were collected at regular intervals from the day of arrival until 70 days post inoculation (DPI). All samples were tested by quantitative real-time PCR for the presence of PCV2 DNA and serum was tested for presence of PCV2 antibodies by ELISA. Overall, the main effects "transgenic status" and "PCV2 subtype" had no influence on degree of PCV2 viremia and shedding or the anti-PCV2 humoral immune response in CD46-TG-PCV2 pigs compared to non-TG-PCV2 pigs. Differences in PCV2 concentrations between non-TG-PCV2 and CD46-TG-PCV2 pigs were minimal and limited to DPI 35 in sera, DPI 7 in fecal swabs and DPI 5 in nasal swabs when CD46-TG-PCV2 pigs had significantly higher concentrations of PCV2 DNA. At DPI 1, CD46-TG-PCV2 pigs had significantly lower concentrations of PCV2 DNA in oral swabs. Under the study conditions, the presence of human CD46 in transgenic pigs had no effect on PCV2 infection in otherwise healthy pigs capable of a normal immune response.

Expression of complement regulatory proteins: CD46, CD55, and CD59 and response to rituximab in patients with CD20+ non-Hodgkin's lymphoma.

Rituximab is an anti-CD20 humanized monoclonal antibody widely used in the treatment of B-cell non-Hodgkin's lymphomas (NHLs). Its mechanism of action is related with complement function-complement mediated cytotoxicity. CD46, CD55, and CD59 are complement regulatory proteins. The aim of this study was to analyze expression of complement inhibitors CD46, CD55, and CD59 in patients with CD20(+) NHLs treated with rituximab combined with chemotherapy. A total of 27 patients with CD20(+) NHLs were evaluated (13 females and 14 males). The median age of patients was 56 years. All patients were examined before treatment with rituximab. Expression of CD46, CD55, and CD59 was determined by two-color flow cytometry. A total of 15 patients achieved complete response (CR), 5 patients achieved partial response, and 7 patients had no or minimal response (NR) after rituximab therapy. We observed that expression of CD46 and CD59 were higher in patients with CR than in group with NR. Expression of CD55 and CD59 were higher in patients with bulky disease. In conclusion level of expression of CD46, CD55, and CD59 could be clinically helpful to predict the response to rituximab therapy.

Evaluation of a possible inflammatory response after appendectomy for non-perforated appendicitis in children.

BACKGROUND: The inflammatory process in the post-appendectomy period is not well characterized. In a pilot study, we prospectively followed the kinetics of different inflammatory mediators before and after appendectomy in children, and compared the results of the groups open appendectomy (OA) and laparoscopic appendectomy (LA). MATERIAL AND METHODS: Levels of sP-selectin, tPA, MCP-1, IL-6, IL-8, sVCAM-1, and sCD40L were measured before appendectomy and on the next three consecutive days in the serum of 25 children (16 males and 9 females) aged 7 - 16 years (mean 12.6+/-2.47 years) with non-perforated acute appendicitis. RESULTS: LA and OA were performed in 16 and 9 patients respectively. None of the markers of inflammation differed significantly by surgical approach at any point of observation. However, sP-selectin, MCP-1 and sVCAM-1 levels were found to have significantly different postoperative kinetics with a trend towards higher values in the laparoscopic group compared to the open appendectomy group (p=0.034, p=0.016 and p=0.025, respectively). CONCLUSIONS: The cytokines sP-selectin, MCP-1 and sVCAM-1 may play a role in the possible post-appendectomy cytokine activation after non-perforated appendicitis. Since this phenomenon is more evident after LA than after OA, the contribution of the different LA procedures has to be further investigated.

Detailed immunophenotypic characterization of different major and minor subsets of peripheral blood cells in patients with paroxysmal nocturnal hemoglobinuria.

BACKGROUND: Paroxysmal nocturnal hemoglobinuria (PNH) is characterized by a deficient expression of glycosylphosphatidylinositol-anchored proteins (GPI-APs), due to somatic mutations of the phosphatidylinositolglycan complementation Class A (PIG-A) gene. STUDY DESIGN AND METHODS: In this study, the expression of a high number of GPI-APs on different subsets of peripheral blood (PB) cells from 14 PNH patients and their potential association with underlying genetic abnormalities has been analyzed. RESULTS: This study confirms the existence of variable patterns of expression of different GPI-APs on both major and minor PB-cell subsets from PNH patients. The size of the PNH clone within PB neutrophils and monocytes was systematically higher than that of other cell populations. Genetic changes were detected in the PIG-A gene in 5 of 13 cases analyzed. Interestingly, the reactivity for many GPI-APs was significantly higher on different subsets of normal PB cells from PNH patients than those observed on healthy volunteers. CONCLUSION: The best combination of markers for the diagnostic screening of PNH would include evaluation of CD14 on monocytes and of CD16 on neutrophils, although further analysis of CD55 and CD59 expression may contain additional clinically useful information. Clear association between the genetic changes detected in the PIG-A gene in 5 of 13 cases analyzed, and the phenotypic profile of PNH cells has not been found. Additionally, an abnormally higher expression of several GPI-APs among normal residual cells from PNH patients in comparison to healthy donors was observed, suggesting that factors other than the PIG-A mutation could determine the phenotypic profile of PB cells in PNH.

Identification of MCP/CD46 analogue on bovine erythrocytes using the new monoclonal antibody IVA-520.

MCP/CD46 is a widely distributed C3b/C4b binding regulatory glycoprotein of the complement system that has been identified on all human peripheral blood cells except erythrocytes. In this paper, we describe the identification of bovine CD46 on all blood cells, including erythrocytes, with the newly prepared monoclonal antibody IVA-520. This antibody cross-reacts with human and pig cells. Furthermore, the molecule identified by IVA-520 functionally behaves as the MCP molecule, showing cofactor activity for the factor I-mediated cleavage of bovine C3 complement factor.

Levels of expression of complement regulatory proteins CD46, CD55 and CD59 on resting and activated human peripheral blood leucocytes.

The cell surface complement regulatory (CReg) proteins CD46, CD55 and CD59 are widely expressed on human lymphoid and non-lymphoid cells. This study aimed to compare systematically levels of CReg expression by different leucocyte subsets and to determine whether levels were increased following activation in vitro. Levels of each CReg protein were similar on freshly isolated monocytes and all major lymphocyte subsets, except that CD4(+) cells expressed significantly less CD46 than CD8(+) cells (P < 0.05) while the reverse was observed for CD55 (P < 0.02). CD56(+) cells, predominantly natural killer cells, expressed significantly lower levels of CD59 than T cells (P < 0.02). CD45RO(+) cells had higher levels of surface CD46 and CD59, but lower levels of CD55, than CD45RO(-) cells (P < 0.02); CD25(+) cells also expressed significantly less CD55 than CD25(-) cells (P < 0.002). Neutrophils expressed higher levels of CD59, but lower levels of CD55, than monocytes. Following activation with phytohaemagglutinin, CD46 was up-regulated on all leucocyte subsets with the exception of CD56(+) cells. Both CD55 and CD59 were also markedly up-regulated on monocytes, and CD55 expression was greater on CD8(+) than CD4(+) cells following activation (P < 0.02). Lipopolysaccharide treatment did not significantly alter B-cell expression of CReg proteins whereas CD55 and CD59, but not CD46, were significantly up-regulated on monocytes (P < 0.02). These observations that CReg proteins are up-regulated on certain activated leucocyte subsets indicate that levels would be increased following immune responses in vivo. This could enhance both protection against local complement activation at inflammatory sites and also the immunoregulatory properties of these leucocytes.

Quantitative expression analysis of HHV-6 cell receptor CD46 on cells of human cord blood, peripheral blood and G-CSF mobilised leukapheresis cells.

Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection.