RESUMO
The aim of the present study was to (1) investigate the relationship between late-onset Alzheimer's disease (AD) and DNA methylation levels in six of the top seven AD-associated genes identified through a meta-analysis of recent genome wide association studies, APOE, BIN1, PICALM, CR1, CLU, and ABCA7, in blood, and (2) examine its applicability to the diagnosis of AD. We examined methylation differences at CpG island shores in the six genes using Sanger sequencing, and one of two groups of 48 AD patients and 48 elderly controls was used for a test or replication analysis. We found that methylation levels in three out of the six genes, CR1, CLU, and PICALM, were significantly lower in AD subjects. The combination of CLU methylation levels and the APOE genotype classified AD patients with AUC = 0.84 and 0.80 in the test and replication analyses, respectively. Our study implicates methylation differences at the CpG island shores of AD-associated genes in the onset of AD and suggests their diagnostic value.
Assuntos
Doença de Alzheimer/genética , Clusterina , Ilhas de CpG , Metilação de DNA , Proteínas Monoméricas de Montagem de Clatrina , Receptores de Complemento 3b , Idoso , Doença de Alzheimer/diagnóstico , Biomarcadores/sangue , Clusterina/sangue , Clusterina/genética , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Proteínas Monoméricas de Montagem de Clatrina/sangue , Proteínas Monoméricas de Montagem de Clatrina/genética , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/genéticaRESUMO
The CR1 gene has been widely studied in Alzheimer's disease (AD), since its first association with the disease in 2009. Even after 11 years of this discovery, the role of this gene in AD has not yet been fully elucidated and the association of its variants was not validated in Latin American populations. We genotyped five CR1 single nucleotide polymorphisms (SNPs rs6656401, rs3849266, rs2274567, rs4844610, and rs12034383) in up to 162 AD patients and 137 controls through PCR-SSP and iPLEX MassARRAY Platform (Sequenom), and measured soluble CR1 (sCR1) levels in plasma of 40 AD patients and 39 controls with an enzyme-linked immunosorbent assay (ELISA). Homozygosity for haplotype rs3849266*C_rs2274567*A (CA/CA genotype) was associated with susceptibility to AD (OR = 2.94, p = 0.018). Patients presented higher sCR1 levels in plasma than controls (p = 0.038). Furthermore, patients that carry the rs2274567*G allele (p.1208Arg) presented higher sCR1 levels than A/A (p.1208His/His) homozygotes (p = 0.036). This is the first study to validate the association of CR1 polymorphisms with late-onset Alzheimer's disease, as well as to evaluate sCR1 levels in a Latin American population. SNPs present in the regulatory and coding regions of this gene may be playing a key role in the observed association, probably by interfering in Aß plaques clearance. Inhibition may be due to the increase in local sCR1 levels observed in patients, which may result from polymorphisms leading to larger isoforms of CR1 and/or structural alterations of the protein that makes it less functional, as well as increased vesiculation of the molecules.
Assuntos
Doença de Alzheimer/genética , Polimorfismo de Nucleotídeo Único , Receptores de Complemento 3b/genética , Doença de Alzheimer/sangue , Haplótipos , Homozigoto , Humanos , América Latina , Receptores de Complemento 3b/sangueRESUMO
Pemphigus foliaceus is an autoimmune disease that is sporadic around the world but endemic in Brazil, where it is known as fogo selvagem (FS). Characterized by autoantibodies against the desmosomal cadherin desmoglein 1, FS causes painful erosions, and crusts that may be widespread. The recognition of antigens, including exposed sugar moieties, activates the complement system. Complement receptor 1 (CR1, CD35), which is responsible for the Knops blood group on erythrocytes (York and McCoy antigens), is also expressed by antigen-presenting cells. This regulates the complement system by removing opsonized antigens, blocking the final steps of the complement cascade. Membrane-bound CR1 also fosters antigen presentation to B cells, whereas soluble CR1 has anti-inflammatory properties. CR1 gene polymorphisms have been associated with susceptibility to complex diseases. In order to investigate the association of CR1 polymorphisms with FS susceptibility, we developed a multiplex sequence-specific assay to haplotype eleven polymorphisms in up to 367 FS patients and 242 controls from an endemic area and 289 from a non-endemic area. We also measured soluble CR1 (sCR1) in the serum of 53 FS patients and 27 controls and mRNA levels in the peripheral blood mononuclear cells of 63 genotyped controls. The haplotypes CR1*3B2B (with the York antigen-encoded by p.1408Met) and CR1*3A2A (with p.1208Arg) were associated with protection against FS (OR = 0.57, P = 0.027, and OR = 0.46, P = 0.014, respectively). In contrast, the CR1*1 haplotype (with the McCoy antigen - encoded by p.1590Glu) was associated with FS susceptibility (OR = 4.97, P < 0.001). Heterozygote rs12034383*A/G individuals presented higher mRNA expression than homozygotes with the G allele (P = 0.04). The lowest sCR1 levels occurred in patients with active disease before treatment (P = 0.036). Patients in remission had higher levels of sCR1 than did healthy controls (P = 0.013). Among those under treatment, patients with localized lesions also presented higher sCR1 levels than those with generalized lesions (P = 0.0073). In conclusion, the Knops blood group seems to modulate susceptibility to the disease. Furthermore, corticosteroid treatment might increase sCR1 serum levels, and higher levels may play an anti-inflammatory role in patients with FS, limiting the distribution of lesions. Based on these results, we suggest CR1 as a potential new therapeutic target for the treatment of FS.
Assuntos
Pênfigo/sangue , Pênfigo/etiologia , Polimorfismo Genético , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Alelos , Biomarcadores , Estudos de Casos e Controles , Suscetibilidade a Doenças , Feminino , Loci Gênicos , Predisposição Genética para Doença , Haplótipos , Humanos , Masculino , Pessoa de Meia-Idade , Pênfigo/diagnóstico , Filogenia , RNA Mensageiro/genética , Adulto JovemRESUMO
Severe malarial anaemia (SMA) is the most common life-threatening complication of Plasmodium falciparum infection in African children. SMA is characterised by haemolysis and inadequate erythropoiesis, and is associated with dysregulated inflammatory responses and reduced complement regulatory protein levels (including CD35). However, a deeper mechanistic understanding of the pathogenesis requires improved animal models. In this comparative study of two closely related macaque species, we interrogated potential causal factors for their differential and temporal relationships to onset of SMA. We found that rhesus macaques inoculated with blood-stage Plasmodium coatneyi developed SMA within 2 weeks, with no other severe outcomes, whereas infected cynomolgus macaques experienced only mild/ moderate anaemia. The abrupt drop in haematocrit in rhesus was accompanied by consumption of haptoglobin (haemolysis) and poor reticulocyte production. Rhesus developed a greater inflammatory response than cynomolgus macaques, and had lower baseline levels of CD35 on red blood cells (RBCs) leading to a significant reduction in the proportion of CD35+ RBCs during infection. Overall, severe anaemia in rhesus macaques infected with P. coatneyi has similar features to SMA in children. Our comparisons are consistent with an association of low baseline CD35 levels on RBCs and of early inflammatory responses with the pathogenesis of SMA.
Assuntos
Anemia/sangue , Anemia/parasitologia , Eritrócitos/metabolismo , Malária/sangue , Plasmodium/metabolismo , Receptores de Complemento 3b/sangue , Anemia/patologia , Animais , Eritrócitos/parasitologia , Eritrócitos/patologia , Feminino , Inflamação/sangue , Inflamação/parasitologia , Inflamação/patologia , Macaca fascicularis , Macaca mulatta , Malária/parasitologia , Malária/patologia , Especificidade da EspécieRESUMO
BACKGROUND The aim of this study was to determine the relationship between the expression of CD35 and CD64 from white blood cells (neutrophil, monocytes, and lymphocytes) and acute infectious diseases in children. MATERIAL AND METHODS The blood samples were collected from 104 children with infections (42 viral infections and 62 bacterial infections). Blood samples were stained with CD45-PC5, CD35-FITC, and CD64-PE, and the fluorescence intensities were measured by flow cytometer, and then the ratio of CD35 to CD64 was calculated. RESULTS The ratio of CD64/CD35 on neutrophils (NCD35/NCD64) was significantly different between the bacterial group, the virus group, and the healthy control group. According to receiver operating characteristic (ROC) analysis, a cutoff value of 7.256 (sensitivity: 90.0%, specificity: 93.7%) was determined for the NCD35/NCD64 ratio. CONCLUSIONS This study shows that NCD35/NCD64 is helpful in the differential diagnosis of acute viral infection and bacterial infection in children.
Assuntos
Infecções Bacterianas/diagnóstico , Neutrófilos/imunologia , Receptores de Complemento 3b/imunologia , Receptores de IgG/imunologia , Viroses/diagnóstico , Infecções Bacterianas/sangue , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Biomarcadores/sangue , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Citometria de Fluxo/métodos , Humanos , Contagem de Leucócitos/métodos , Leucócitos/metabolismo , Linfócitos/metabolismo , Masculino , Monócitos/metabolismo , Neutrófilos/citologia , Neutrófilos/metabolismo , Receptores de Complemento 3b/sangue , Receptores de IgG/sangue , Viroses/sangue , Viroses/imunologia , Viroses/microbiologiaRESUMO
INTRODUCTION: Phagocytes such as granulocytes and monocytes are fundamental players in the innate immune system. Activation of these cells can be quantified by the measurement of activation marker expression using flow cytometry. Analysis of receptor expression on inflammatory cells facilitates the diagnosis of inflammatory diseases and can be used to determine the extent of inflammation. However, several major limitations of this analysis precludes application of inflammation monitoring in clinical practice. Fast and automated analysis would minimalize ex vivo manipulation and allow reproducible processing. The aim of this study was to evaluate a fully automated "load & go" flow cytometer for analyzing activation of granulocytes and monocytes in a clinically applicable setting. METHODS: Blood samples were obtained from 10 anonymous and healthy volunteers between the age of 18 and 65â¯years. Granulocyte and monocyte activation was determined by the use of the markers CD35, CD11b and CD10 measured in the automated AQUIOS CL® "load & go" flow cytometer. This machine is able to pierce the tube caps, add antibodies, lyse and measure the sample within 20â¯min after vena puncture. Reproducibility tests were performed to test the stability of activation marker expression on phagocytes. The expression of activation markers was measured at different time points after blood drawing to analyze the effect of bench time on granulocyte and monocyte activation. RESULTS: The duplicate experiments demonstrate a high reproducibility of the measurements of the activation state of phagocytes. Healthy controls showed a very homogenous expression of activation markers at Tâ¯=â¯0 (immediately after vena puncture). Activation markers on neutrophils were already significantly increased after 1â¯h (Tâ¯=â¯1) depicted as means (95%Cl) CD35: 2.2× (1.5×-2.5×) pâ¯=â¯.028, CD11b: 2.5× (1.7×-3.1×) pâ¯=â¯.023, CD10: 2.5× (2.1×-2.7×) pâ¯=â¯.009) and a further increase in activation markers was observed after 2 and 3â¯h. Monocytes also showed a increase in activation markers in 1â¯h (mean (95%Cl) CD35: 1.8× (1.3×-2.2×) pâ¯=â¯.058, CD11b: 2.13× (1.6×-2.4×) pâ¯=â¯.025) and also a further significant increase in 2 and 3â¯h was observed. CONCLUSION: This study showed that bench time of one hour already leads to a significant upregulation and bigger variance in activation markers of granulocytes and monocytes. In addition, it is likely that automated flow cytometry reduces intra-assay variability in the analysis of activation markers on inflammatory cells. Therefore, we found that it is of utmost importance to perform immune activation analysis as fast as possible to prevent drawing wrong conclusions. Automated flow cytometry is able to reduce this analysis from 2â¯h to only 15-20â¯min without the need of dedicated personnel and in a point-of-care context. This now allows fast and automated inflammation monitoring in blood samples obtained from a variety of patient groups. FUND: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Assuntos
Antígeno CD11b/sangue , Citometria de Fluxo , Imunofenotipagem/métodos , Leucócitos/metabolismo , Neprilisina/sangue , Sistemas Automatizados de Assistência Junto ao Leito , Testes Imediatos , Receptores de Complemento 3b/sangue , Adolescente , Adulto , Idoso , Automação Laboratorial , Biomarcadores/sangue , Feminino , Citometria de Fluxo/instrumentação , Voluntários Saudáveis , Humanos , Imunofenotipagem/instrumentação , Leucócitos/imunologia , Masculino , Pessoa de Meia-Idade , Monócitos/imunologia , Monócitos/metabolismo , Ativação de Neutrófilo , Neutrófilos/imunologia , Neutrófilos/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Fenótipo , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de Tempo , Fluxo de Trabalho , Adulto JovemRESUMO
The complement receptor 1 (CR1) gene was shown to be involved in Alzheimer's disease (AD). We previously showed that AD is associated with low density of the long CR1 isoform, CR1*2 (S). Here, we correlated phenotype data (CR1 density per erythrocyte (CR1/E), blood soluble CR1 (sCR1)) with genetic data (density/length polymorphisms) in AD patients and healthy controls. CR1/E was enumerated using flow cytometry, while sCR1 was quantified by ELISA. CR1 polymorphisms were assessed using restriction fragment length polymorphism (RFLP), pyrosequencing, and high-resolution melting PCR. In AD patients carrying the H allele (HindIII polymorphism) or the Q allele (Q981H polymorphism), CR1/E was significantly lower when compared with controls carrying the same alleles (p < 0.01), contrary to sCR1, which was significantly higher (p < 0.001). Using multivariate analysis, a reduction of 6.68 units in density was associated with an increase of 1% in methylation of CR1 (estimate -6.68; 95% confidence intervals (CIs) -12.37, -0.99; p = 0.02). Our data show that, in addition to inherited genetic factors, low density of CR1/E is also acquired. The involvement of CR1 in the pathogenesis of AD might be linked to insufficient clearance of amyloid deposits. These findings may open perspectives for new therapeutic strategies in AD.
Assuntos
Doença de Alzheimer/genética , Eritrócitos/patologia , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/genética , Idoso , Idoso de 80 Anos ou mais , Alelos , Sítios de Ligação/genética , Estudos de Coortes , Eritrócitos/química , Feminino , Genótipo , Humanos , Masculino , Metilação , Análise Multivariada , Placa Amiloide/patologia , Polimorfismo de Fragmento de Restrição , Isoformas de Proteínas/sangue , Isoformas de Proteínas/genética , Fatores de RiscoRESUMO
Alzheimer's disease (AD) is the most common type of dementia, with a prevalence that is rising every year. AD is associated with type 2 diabetes mellitus (T2DM) and insulin resistance, and is therefore sometimes called "type 3 diabetes mellitus". The aim of this study was to examine whether the variants of some candidate genes involved in the development of AD, namely BIN1 (rs744373), CLU (rs11136000), CR1 (rs3818361), and PICALM (rs3851179), are related to several disorders of glucose metabolism-gestational diabetes (GDM), T2DM and impaired glucose tolerance (IGT). Our study included 550 women with former GDM and 717 control women, 392 patients with T2DM and 180 non-diabetic controls, and 117 patients with IGT and 630 controls with normal glucose tolerance. Genotyping analysis was performed using specially-designed TaqMan assays. No significant associations of the genetic variants rs744373 in BIN1, rs11136000 in CLU, or rs3818361 in CR1 were found with GDM, T2DM or IGT, but rs3851179 in PICALM was associated with an increased risk of GDM. The frequency of the AD risk-associated C allele was significantly higher in the GDM group compared to controls: OR 1.21; 95% CI (1.03-1.44). This finding was not apparent in T2DM and IGT; conversely, the C allele of the PICALM SNP was protective for IGT: OR 0.67; 95% CI (0.51-0.89). This study demonstrates an association between PICALM rs3851179 and GDM as well as IGT. However, elucidation of the possible role of this gene in the pathogenesis of GDM requires further independent studies.
Assuntos
Doença de Alzheimer/genética , Diabetes Gestacional/genética , Intolerância à Glucose/genética , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Adaptadoras de Transdução de Sinal/sangue , Proteínas Adaptadoras de Transdução de Sinal/genética , Adulto , Idoso , Alelos , Doença de Alzheimer/complicações , Clusterina/sangue , Clusterina/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Gestacional/metabolismo , Feminino , Frequência do Gene , Estudos de Associação Genética/métodos , Predisposição Genética para Doença , Variação Genética , Intolerância à Glucose/metabolismo , Humanos , Pessoa de Meia-Idade , Proteínas Monoméricas de Montagem de Clatrina/sangue , Proteínas Nucleares/sangue , Proteínas Nucleares/genética , Razão de Chances , Polimorfismo de Nucleotídeo Único/genética , Gravidez , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/genética , Fatores de Risco , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética , População Branca/genéticaRESUMO
OBJECTIVE: The delicate balance which exists between complement activation and its regulation is altered in HIV infection and pregnancy disorders such as pre-eclampsia. Therefore, the purpose of this study was to investigate the expression of complement regulatory (Creg) proteins (CD35 and CD55) in HIV associated normal and pre-eclamptic pregnancies. STUDY DESIGN: The total study population (n=100) consisted of normotensive pregnant (n=50) and pre-eclamptic (n=50) women. These groups were equally sub-stratified into HIV infected and uninfected groups (n=25 per group). Standard haematological tests were conducted. Flow cytometric analysis of isolated neutrophils were performed using fluorescein isothiocyanate-conjugated anti-CD35 and phycoerythrin-cyanine 5 conjugated anti-CD55. RESULTS: HELLP syndrome characteristics of increased lactate dehydrogenase enzymes levels, low platelet counts, cell morphological abnormalities (red cell fragmentation) and anaemia were observed in 40% of the HIV infected pre-eclamptic group. Red cell fragmentation inclusive of burr cells and schistocytes were also noted. Activated partial thromboplastin time and fibrinogen differed significantly between the HIV uninfected pre-eclamptic compared to the HIV infected pre-eclamptic groups (p<0.01). Irrespective of HIV status, the mean fluorescence intensity of CD35 and CD55 were significantly higher in the pre-eclamptic compared to the normotensive pregnant (p=0.0001; p=0.0001 respectively) groups. In the pre-eclamptic groups, the expression of both CD35 and CD55 did not significantly differ between HIV infected and uninfected women (p=0.486; p=0.767 respectively). CONCLUSIONS: This study demonstrates an up-regulation of complement regulatory proteins, CD35 and CD55 in HIV associated pre-eclamptic compared to normotensive pregnancy. This elevation of the Creg proteins is an adaptive immune response to the high complement-mediated cell lysis that occurs in HIV infection and further aggravated by the complement activated state of pre-eclampsia.
Assuntos
Antígenos CD55/sangue , Infecções por HIV/sangue , Pré-Eclâmpsia/sangue , Complicações Infecciosas na Gravidez/sangue , Receptores de Complemento 3b/sangue , Adulto , Feminino , Citometria de Fluxo , Infecções por HIV/complicações , Humanos , Neutrófilos/metabolismo , Gravidez , Regulação para Cima , Adulto JovemRESUMO
Chronic activation of the complement system and induced inflammation are associated with neuropathology in Alzheimer's disease (AD). Recent large genome wide association studies (GWAS) have identified single nucleotide polymorphisms (SNPs) in the C3b/C4b receptor (CR1 or CD35) that are associated with late onset AD. Here, anti-CR1 antibodies (Abs) directed against different epitopes of the receptor, were used to localize CR1 in brain, and relative binding affinities of the CR1 ligands, C1q and C3b, were assessed by ELISA. Most Abs tested stained red blood cells in blood vessels but showed no staining in brain parenchyma. However, two monoclonal anti-CR1 Abs labeled astrocytes in all of the cases tested, and this reactivity was preabsorbed by purified recombinant human CR1. Human brain-derived astrocyte cultures were also reactive with both mAbs. The amount of astrocyte staining varied among the samples, but no consistent difference was conferred by diagnosis or the GWAS-identified SNPs rs4844609 or rs6656401. Plasma levels of soluble CR1 did not correlate with diagnosis but a slight increase was observed with rs4844609 and rs6656401 SNP. There was also a modest but statistically significant increase in relative binding activity of C1q to CR1 with the rs4844609 SNP compared to CR1 without the SNP, and of C3b to CR1 in the CR1 genotypes containing the rs6656401 SNP (also associated with the larger isoform of CR1) regardless of clinical diagnosis. These results suggest that it is unlikely that astrocyte CR1 expression levels or C1q or C3b binding activity are the cause of the GWAS identified association of CR1 variants with AD. Further careful functional studies are needed to determine if the variant-dictated number of CR1 expressed on red blood cells contributes to the role of this receptor in the progression of AD, or if another mechanism is involved.
Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Regulação da Expressão Gênica , Polimorfismo de Nucleotídeo Único , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/sangue , Astrócitos/metabolismo , Encéfalo/patologia , Complemento C1q/metabolismo , Complemento C3b/metabolismo , Eritrócitos/metabolismo , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Transporte Proteico , Receptores de Complemento 3b/sangueRESUMO
The study was carried out to analyze rate of expression of antigens CD35 and CD21 in norm and under different forms of B-cell lymphoproliferative diseases. The level of average intensity of fluorescence of antigens CD35, CD21 and CD200 is compared for different groups of patients with B-cell lymphoproliferative diseases. The established patterns of expression of antigens CD35 and CD21 under B-cell lymphoproliferative diseases permit considering expression of the given markers as a characteristic of differential diagnostic.
Assuntos
Biomarcadores Tumorais/sangue , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B , Linfoma de Células B , Proteínas de Neoplasias/sangue , Receptores de Complemento 3b/sangue , Receptores de Complemento 3d/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Diagnóstico Diferencial , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/diagnóstico , Linfoma de Células B/sangue , Linfoma de Células B/diagnóstico , Masculino , Pessoa de Meia-IdadeRESUMO
BACKGROUND: In clinical practice it is often troublesome to discriminate bacterial etiologies from viral etiologies in pediatric lower respiratory tract infections (LRTIs). The aim of this study was to develop an accurate analytic method to improve diagnostic determination for bacterial and viral etiologies in pediatric LRTIs. METHODS: A total of 45 children with confirmed bacterial LRTIs and 51 children with viral LRTIs were finally included after assessment of the children visiting the emergency department with a suspected infection and identification of pathogens. C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), CD35, and CD64 were assessed and then the areas under receiver operating characteristic (ROC) curves (AUC) of PCT, IL-6, CD35, and CD64 in combination with CRP were compared to the AUC of CRP alone in all subjects. RESULTS: The levels of CRP, PCT, IL-6, CD45, and CD64 observed in children with bacterial LRTIs were statistically higher than for viral infections. The AUC of CRP combined with CD53 (0.963, 95% confidence interval (CI) 0.921-1.002) or CD64 (0.952, 95% CI 0.907-0.998) or CD35/CD64 (0.971, 95% CI 0.932-1.004) increased compared with that of the single biomarker. CONCLUSIONS: The combined analysis improved diagnostic accuracy in children with bacterial and viral LRTIs.
Assuntos
Infecções Bacterianas/diagnóstico , Infecções Respiratórias/diagnóstico , Viroses/diagnóstico , Infecções Bacterianas/microbiologia , Biomarcadores/sangue , Proteína C-Reativa/análise , Calcitonina/sangue , Criança , Pré-Escolar , Feminino , Humanos , Interleucina-6/sangue , Masculino , Receptores de Complemento 3b/sangue , Infecções Respiratórias/microbiologia , Infecções Respiratórias/virologia , Sensibilidade e Especificidade , Viroses/virologiaRESUMO
The long complement receptor type 1 (CR1) isoform, CR1*2 (S), has been identified as being associated with Alzheimer's disease (AD) risk. We aimed to analyze the phenotypic structural and expression aspects (length and density) of CR1 in erythrocytes of 135 Caucasian subjects (100 AD and 35 controls). CR1 length polymorphism was assessed at protein and gene levels using Western blot and high-resolution melting, respectively. CR1 sites on erythrocytes were enumerated by flow cytometry. CR1 gene analysis, spotting the rs6656401 and rs3818361 polymorphisms, was performed by pyrosequencing. The CR1 density was significantly lower in AD patients expressing the CR1*2 isoform compared with the controls (p = 0.001), demonstrating lower expression of CR1 in CR1*2 carriers. Our data suggested the existence of silent CR1 alleles. Finally, rs6656401 and rs3818361 were strongly associated with CR1 length polymorphism (p < 0.0001). These observations indicate that AD susceptibility is associated with the long CR1 isoform (CR1*2), albeit at a lower density, suggesting that AD results from insufficient clearance of plaque deposits rather than increased inflammation.
Assuntos
Doença de Alzheimer/genética , Estudos de Associação Genética , Receptores de Complemento 3b/química , Receptores de Complemento 3b/genética , Alelos , Eritrócitos/metabolismo , Expressão Gênica , Predisposição Genética para Doença/genética , Heterozigoto , Humanos , Fenótipo , Polimorfismo Genético , Estudos Prospectivos , Isoformas de Proteínas , Receptores de Complemento 3b/sangue , RiscoRESUMO
Malaria is responsible for close to 1 million deaths each year, mostly among African children. Red blood cells (RBCs) of children with severe malarial anemia show loss of complement regulatory proteins such as complement receptor 1 (CR1). We carried out this study to identify socio-economic, environmental, and biological factors associated with the loss of RBC CR1. A cross-sectional study was conducted in a malaria holoendemic area of western Kenya. Twelve socioeconomic, environmental, and biological factors were examined for a relationship with RBC CR1 level using bivariate linear regression followed by creation of a multivariate linear regression model. A significant positive relationship between RBC CR1 level and use of mosquito countermeasures was found. However, there was no evidence of a significant relationship between RBC CR1 level and malaria infection or parasitemia level. Reducing mosquito exposure may aid in the prevention of severe malarial anemia by reducing the number of infections and thus preserving RBC CR1.
Assuntos
Eritrócitos/metabolismo , Insetos Vetores , Malária/epidemiologia , Controle de Mosquitos , Receptores de Complemento 3b/sangue , Animais , Feminino , Humanos , Quênia/epidemiologia , Malária/transmissão , MasculinoRESUMO
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.
Assuntos
Infecções Bacterianas/diagnóstico , Antígenos CD55/sangue , Antígenos CD59/sangue , Mononucleose Infecciosa/diagnóstico , Leucócitos/metabolismo , Proteína Cofatora de Membrana/sangue , Receptores de Complemento 3b/sangue , Adulto , Idoso , Infecções Bacterianas/sangue , Biomarcadores/sangue , Proteínas Inativadoras do Complemento/análise , Diagnóstico Diferencial , Citometria de Fluxo , Humanos , Mononucleose Infecciosa/sangue , Linfócitos/metabolismo , Pessoa de Meia-Idade , Monócitos/metabolismo , Neutrófilos/metabolismo , Sensibilidade e Especificidade , Adulto JovemRESUMO
We investigate the distribution of the membrane protein complement receptor 1 (CR1/CD35) in human erythrocyte membrane ghosts using scanning near-field optical microscopy. Recent studies have demonstrated that levels of Aß peptide, associated with Alzheimers disease (AD) and present in brain and peripheral blood, vary significantly when bound by complement C3b-dependent adherence to CR1. It is unknown why patients with AD have a markedly impaired ability to bind Aß to CR1 via this mechanism, but one possibility is a defect in the localization and/or conformation of CR1 in the membrane. Scanning near-field optical microscopy does not require the harsh preparation of electron microscopy techniques and may therefore be better suited for measuring membrane protein distributions. The clustering phenomenon of CR1 identified in electron microscopy studies is confirmed and quantified. The standard deviation of the inter-cluster spacing of CR1 is constant (79 ± 9 nm) across erythrocytes with between 61 and 124 clusters per membrane ghost.
Assuntos
Eritrócitos/química , Eritrócitos/metabolismo , Receptores de Complemento 3b/sangue , Adulto , Análise por Conglomerados , Membrana Eritrocítica/química , Membrana Eritrocítica/metabolismo , Humanos , Masculino , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura/métodosRESUMO
OBJECTIVES: The expression level of CD64 on neutrophils can be used to differentiate between an infection and a disease flare in rheumatoid arthritis (RA) patients. However, the CD64 expression is elevated by both bacteria and viruses, so it cannot be used to distinguish the type of infection. We herein investigated the results of a simultaneous quantitative analysis of the expression of CD64 and CD35 on neutrophils to determine whether these molecules can be used to distinguish between bacterial and viral infections in RA patients. METHODS: We collected blood from 22 RA patients with pathogen-proven infections (15 bacterial and 7 viral infections). Blood samples were stained with QuantiBRITE CD64PE/CD45PerCP and CD35PE, and the mean fluorescence intensities were assessed by a flow cytometer. The mean numbers of molecules were calculated using QuantiBrite PE beads. RESULTS: We calculated the ratio of CD64 to the CD35 level (CD35/CD64), and used a cut-off value of 2.8 for the CD35/CD64 ratio. At this value, the sensitivity for diagnosing a bacterial infection was 87%, and the specificity was 86%. CONCLUSIONS: Simultaneous quantitative analysis of CD64 and CD35 expression on neutrophils might be useful to distinguish between bacterial and viral infections in RA patients.
Assuntos
Artrite Reumatoide/patologia , Infecções Bacterianas/diagnóstico , Neutrófilos/imunologia , Receptores de Complemento 3b/sangue , Receptores de IgG/sangue , Viroses/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Artrite Reumatoide/complicações , Artrite Reumatoide/imunologia , Infecções Bacterianas/complicações , Infecções Bacterianas/imunologia , Biomarcadores/sangue , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Viroses/complicações , Viroses/imunologiaRESUMO
Complement Receptor 1 (CR1) is a key complement regulatory protein (CRP) involved in the clearance of immune complexes. Earlier, we reported a marked decline of leukocyte CR1 (L-CR1) transcript and protein in patients with active systemic lupus erythematosus (SLE) and suggested L-CR1 transcript as a putative non-invasive disease marker for SLE. This follow-up study involving 18 patients with active SLE was conducted for further confirmation of the relationship between L-CR1 and SLE. Blood samples from the patients were collected on day 1 of the diagnosis (0 month) and at different time intervals (3 and 6 months) for analysis of L-CR1 transcript and L-CR1 protein by semi-quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR) and western blotting respectively. Within 6 months, 15 patients entered remission. On day 1, the mean values of L-CR1 transcript (8.42 ± 3.53) and L-CR1 protein (4683 ± 1094) in the SLE patients were 6 times and 12 times lower than the normal controls (n = 103). At the end of month 6, these values increased by 4.5 and 6.5 times respectively for CR1 transcript (37.86 ± 8.52) and protein (30,265 ± 8614). Simultaneously, the SLE Disease Activity Index (SLEDAI) scores decreased by 4.8 times (4.47 ± 3.32) as compared with the scores obtained on day 1 (21.45 ± 5.67). Moreover, CR1 values correlated negatively with the SLEDAI scores. Levels of L-CR1 protein and transcript remained low in the three patients who did not enter remission. All of the above results suggested that an increase in the levels of L-CR1 related to good prognosis. Since the levels of L-CR1 protein is influenced by variables like proteolytic cleavage and secretion from leukocytes, the values of L-CR1 transcript on day 1 and subsequent follow-up points may bring a better insight into the state of the disease activity. An extended follow-up study is needed to confirm the significance of L-CR1 as a prognostic marker for SLE.
Assuntos
Lúpus Eritematoso Sistêmico/sangue , Lúpus Eritematoso Sistêmico/genética , Receptores de Complemento 3b/sangue , Receptores de Complemento 3b/genética , Adulto , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Seguimentos , Humanos , Leucócitos/metabolismo , Lúpus Eritematoso Sistêmico/fisiopatologia , Masculino , Prognóstico , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Adulto JovemAssuntos
Integrina alfaXbeta2/sangue , Antígeno de Macrófago 1/sangue , Receptores de Complemento 3b/sangue , Receptores de Complemento 3d/sangue , Biomarcadores/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Citometria de Fluxo/métodos , Humanos , Integrina alfaXbeta2/química , Integrina alfaXbeta2/fisiologia , Síndrome da Aderência Leucocítica Deficitária/diagnóstico , Lúpus Eritematoso Sistêmico/diagnóstico , Antígeno de Macrófago 1/química , Antígeno de Macrófago 1/fisiologia , Fagocitose , Receptores de Complemento 3b/química , Receptores de Complemento 3b/fisiologia , Receptores de Complemento 3d/química , Receptores de Complemento 3d/fisiologia , Valores de Referência , Manejo de EspécimesRESUMO
Complement receptor 1 (CR1) on human erythrocytes (Es) and complement factor H (CFH) on rodent platelets perform immune adherence, which is a function that allows the processing of immune complexes (ICs) bearing C3 by the mononuclear phagocyte system. Similar immune adherence occurs in the glomerular podocyte by CR1 in humans and CFH in rodents. As a model for human IC processing, we studied transgenic mice lacking CFH systemically but with human CR1 on Es. These CR1(hu)Tg/CFH(-/-) mice spontaneously developed proliferative glomerulonephritis, which was accelerated in a chronic serum sickness model by active immunization with heterologous apoferritin. ICs containing Ag, IgG and C3 bound to Es in CR1(hu)Tg/CFH(-/-) mice. In this setting, there was increased IC deposition in glomeruli, attributable to the presence of CR1 on Es, together with the absence of CFH on platelets and podocytes. In the absence of plasma CFH, the accumulated ICs activated complement, which led to spontaneous and chronic serum sickness-induced proliferative glomerulonephritis. These findings illustrate the complexities of complement-dependent IC processing by blood cells and in the glomerulus, and the importance of CFH as a plasma complement regulator.