RESUMO
In order to gain more information about local humoral immune responses to HPV infection, we quantified IgG, IgM, secretory-IgA (S-IgA), and total-IgA by ELISA, and lysozyme and lactoferrin by TR-IFMA, in cervical and cervicovaginal secretions of 40 healthy women and 28 high-risk HPV infected patients (11 were HPV16+). IgG, total-IgA, and S-IgA concentrations in cervicovaginal secretions (p < 0.0001) and high IgG and total-IgA concentrations (p < 0.001 and p < 0.01, respectively) in endocervical secretions were significantly higher in HPV+ patients than in the healthy group. Since the S-IgA/total-IgA ratio was significantly lower in cervicovaginal (7.5%) and endocervical secretions (36.5%) in HPV+ women compared to the control group (p < 0.003 and p < 0.001, respectively), HPV could be responsible for an increase in local production of non-secretory IgA (monomeric and dimeric forms). IgG and total-IgA concentrations in cervicovaginal and endocervical secretions fell in the same general percentage range in both HPV16+ and HPV+ groups (80% and 15%, respectively). However, the S-IgA/total-IgA ratio was much lower in HPV16+ than in HPV+ women, in both cervicovaginal secretions (3.4%) (p < 0.003) and in endocervical secretions (23.3%) (p < 0.001). Innate immunity proteins and local S-IgA response could not stop the spread of HPV infection in spite of high lysozyme and lactoferrin concentrations. HPV16+ disturbed the local humoral immune system, which could partly explain its low clearance.
Assuntos
Anticorpos Antivirais/análise , Colo do Útero/imunologia , Infecções por Papillomavirus/imunologia , Vagina/imunologia , Adolescente , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Isotipos de Imunoglobulinas/análise , Lactoferrina/sangue , Pessoa de Meia-Idade , Muramidase/sangue , Albumina Sérica/análiseRESUMO
Apolipoprotein E (apo E) is postulated to be a major lipid carrier protein in the brain involved in brain development and repair. Multiple sclerosis (MS) is a major demyelinating disease characterized by destruction of myelin and marked alteration of myelin cholesterol and lipid metabolism. We have determined serum and cerebrospinal fluid (CSF) apo E concentrations using an original time-resolved immunofluorometric assay and calculated intrathecal apo E concentration. Apo E concentrations were determined in 13 control subjects and 129 neurological patients: 34 definite MS patients, 25 with Guillain-Barré syndrome (GBS), 32 with amyotrophic lateral sclerosis (ALS) and 38 with other neurological diseases. Seven clinical parameters (sex, age, age at MS onset, duration of the disease, course, clinical status and disability score) were considered in MS patients. Significant (P < 0.01) decrease in CSF apo E was observed in MS, linked to a decrease in intrathecal apo E. The decreased CSF apo E concentration in MS patients occur independent of the apo E genotype. Apo E is considered as a neurotrophic factor in the brain. Any decrease in intrathecal apo E synthesis would thus contribute to progression of neurological diseases, such as MS.
Assuntos
Apolipoproteínas E/sangue , Esclerose Múltipla/sangue , Adulto , Idoso , Apolipoproteínas E/líquido cefalorraquidiano , Apolipoproteínas E/genética , Feminino , Fluorometria , Humanos , Técnicas Imunológicas , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Concentração Osmolar , Polimorfismo Genético/genéticaRESUMO
The role of the mucosal immune response against Cryptosporidium has been suggested by studies on the therapeutic effects of hyperimmune colostrum. In order to study the intestinal response to this infection, we have developed a sandwich-type time-resolved immunofluorometric assay for the determination of anti-Cryptosporidium coproantibodies. This assay has the inherent sensitivity of an immunoassay without the problems due to background responses from other biological compounds, and is thus suitable for faecal samples. The intra-assay coefficients of variation (5.1%, 4.6%, and 5.8% for immunoglobulins (Ig) A, M and G respectively), inter-assay coefficients of variation (9.4%; 10.5% and 12.2% for IgA, IgM and IgG, respectively) and specificity (100% for all 3 isotypes) were all satisfactory. Using this assay to study 12 patients with the acquired immune deficiency syndrome (AIDS) who were infected with cryptosporidiosis, we found a marked elevation of anti-Cryptosporidium IgA and IgM coproantibody titres relative to 18 healthy control values, but no correlation with the gravity of the infection in terms of oocyst shedding. These results suggest that a non-protective mucosal immune response develops to Cryptosporidium in AIDS patients.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Anticorpos Antiprotozoários/análise , Criptosporidiose/diagnóstico , Cryptosporidium/imunologia , Fezes/química , Fluorimunoensaio/métodos , Adulto , Animais , Antígenos de Protozoários/imunologia , Criptosporidiose/complicações , Feminino , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Masculino , Pessoa de Meia-IdadeRESUMO
A technique to specifically quantify monomeric IgA and total IgA in colostrum has been developed using a modified one-dimensional immunoelectrophoretic assay. This method employed electrophoresis in antibody-containing polyacrylamide-agarose gel in the presence of a gel barrier which blocks polymeric IgA. The addition of PEG (polyethylene-glycol 6000) to the anodic gel increased the sharpness of the peaks, the height of which was proportional to the antigen concentration. This method proved to be sufficiently simple, precise, reproducible (CV less than 3%) and linear (from 20-300 mg/l) to measure the monomeric IgA: total IgA ratio rapidly (14 +/- 4.5% for 20 samples in duplicate). Immunoelectrodiffusion studies confirmed that human colostral and serum IgA standards could be used to determine directly monomeric IgA, total IgA and polymeric IgA levels (by difference) rather than to apply correction factors to estimate these IgA levels.
Assuntos
Imunoglobulina A/análise , Biopolímeros , Colostro/imunologia , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunodifusão/métodos , Imunoeletroforese Bidimensional , GravidezRESUMO
The molecular composition of fecal IgA is poorly documented, although it is of theoretical and practical importance to determine the different forms of IgA in faeces. Two main molecular forms were isolated by successive steps of ion-exchange chromatography and gel-filtration. The first consisted of secretory IgA dimers dissociating into slightly lower molecular mass forms under the influence of the electric field during electrophoresis. The other contained cleaved-IgA complexed with alpha 1-antitrypsin, that is considered to be a serum origin marker. These results confirm that secretory IgA are relatively resistant to digestive enzymes in vivo, and suggest that alpha 1-antitrypsin-bound fragments originate from serum IgA monomers. Analysis of the proportions of these forms may be of value in the investigation of gut diseases.
Assuntos
Fezes/química , Imunoglobulina A/análise , Cromatografia em Gel , Cromatografia por Troca Iônica , Eletroforese em Gel de Poliacrilamida , Humanos , Imunoglobulina A/química , Substâncias Macromoleculares , Peso MolecularRESUMO
Time-resolved fluorometric assay is based on lanthanide fluorescence. This time-resolved fluorescence has a narrow-band emission line whose wavelength differs from that of emission-pulsed light and has a long decay-time. These characteristics make it possible to avoid background interference from sample constituents (protein, light-scattering particles, etc). Europium and its chelates are the most commonly used lanthanides. The europium-labelling of antigens or antibodies is followed by immunoassay. In the final step, fluorescence is measured, after enhancement, as counts per second. This assay has several advantages, including a wide working range, high sensitivity and good practicability. The method has widespread applications in the field of immunoassays in both clinical and research laboratories. The use of non-radioactive europium-labelled probes and the development of simultaneous multiple tests are possible future orientations.
Assuntos
Fluorometria/métodos , Európio , Imunoensaio de Fluorescência por Polarização/métodos , Humanos , Laboratórios , Fatores de TempoRESUMO
The detection of occult blood in the stools is the only simple screening method for colorectal cancer. The aim of this study was to compare the results obtained with the new Hemolex kit (Orion diagnostica, Fumouze France) with those given by three gaiac tests--Hemoccult (Smithkline diagnostics), Hemofec (Boehringer Mannheim) and Hemopreuve (Fumouze) of 165 stools from patients without special diet. Seventy-one patients with at least two positive gaiac tests or a positive Hemolex test underwent colonoscopy followed, if negative, by fibroscopy: 28 had lesions of the lower digestive tract and five of the upper digestive tract. Sensitivity, specificity and negative and positive predictive value were of 70, 98, 91 and 92% respectively for Hemolex; 82, 74, 94 and 44% for Hemoccult; 94, 67, 98 and 42% for Hemopreuve and 91, 73, 97 and 46% for Hemofec. The results obtained in this study confirm the value of the Hemolex test for the detection of human occult blood in the stools whereas the gaiac tests used are influenced by dietary components (unless restricted), explaining their poor positive predictive value. In conclusion, due to their good negative predictive values, the authors recommend that screening for colorectal tumours should be based on the use of two or three gaiac tests which should be confirmed, when positive, by an immunological test for human hemoglobin.
Assuntos
Testes de Fixação do Látex/métodos , Sangue Oculto , Colonoscopia , Neoplasias Colorretais/diagnóstico , Tecnologia de Fibra Óptica , Humanos , Sensibilidade e EspecificidadeRESUMO
The concentrations of seven proteins [albumin (Alb), immunoglobulins (IgG, A, M), lactoferrin (Lf), lysozyme (Lzm) and beta 2-microglobulin (beta 2 m)] were measured in the tears and serum of 10 patients with acquired immune deficiency syndrome (AIDS) and in 13 normal subjects. In the AIDS group, alteration of the blood-lacrimal barrier was revealed by the high Alb clearance. In this group also, the significantly high IgG, IgA, IgM and beta 2 m concentrations indicated a systemic and local immune response, with passive transfer of these proteins from serum to tears. However, Lf and Lzm concentrations were normal, showing that the secretory activity of the lacrimal gland remained unchanged.
Assuntos
Síndrome da Imunodeficiência Adquirida/sangue , Imunodifusão , Proteínas/análise , Lágrimas/análise , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Olho/imunologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Aparelho Lacrimal/imunologia , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos TestesAssuntos
Apolipoproteínas E/biossíntese , Esclerose Múltipla/metabolismo , Adulto , Idoso , Apolipoproteínas E/líquido cefalorraquidiano , Estudos de Casos e Controles , Estudos de Avaliação como Assunto , Feminino , Fluorimunoensaio , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla/líquido cefalorraquidiano , Sensibilidade e EspecificidadeRESUMO
The infectious diarrhea in AIDS is principaly due to Cryptosporidium. The study of the inflammatory and humoral immunity proteins reveal a high exudative enteropathy associated with an IgA, IgG and IgM intestinal immune response. However, this barrier of defence is not sufficient to eradicate the infectious agent.
Assuntos
Síndrome da Imunodeficiência Adquirida/complicações , Criptosporidiose/complicações , Infecções Oportunistas/complicações , Síndrome da Imunodeficiência Adquirida/imunologia , Adulto , Formação de Anticorpos , Criptosporidiose/imunologia , Diarreia/etiologia , Feminino , Humanos , Imunoglobulina A/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Intestinos/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
A time-resolved immunofluorometric assay (TR-IFMA) was used for the measurement of glycated C3. The very high sensitivity of this technique allowed the direct measurement of glycated and non-glycated proteins (especially C3) in chromatography eluates. C3 glycation in vitro after incubation with 20 mmol/l glucose was always less than 3.5% by day 5. As determined with the TR-IFMA, the means +/- standard deviations of glycated C3 were 0.20% +/- 0.04 for non-diabetic subjects and 0.88% +/- 0.06 for insulin-dependent diabetic patients. The low percentages of glycated C3 in both our in vitro and in vivo studies show that this protein is subject to only moderate rates of glycation.
Assuntos
Complemento C3/metabolismo , Diabetes Mellitus Tipo 1/sangue , Adolescente , Adulto , Complexo Antígeno-Anticorpo/metabolismo , Feminino , Fluorimunoensaio , Glicosilação , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-IdadeRESUMO
In order to evaluate a commercial immunoglobulin G (IgG) antibody capture enzyme-linked immunoassay (ELISA) (Wellcozyme HIV1 + 2 Gacelisa; Murex Diagnostics Limited, Dartford, United Kingdom) for the detection of antibodies to human immunodeficiency virus (HIV) in vaginal secretion samples (VS) from HIV-seropositive and -seronegative women, serum samples (S) and VS were obtained from 129 African women living in the Central African Republic, a country of high HIV prevalence. Sera were tested for HIV by routine second-generation ELISA with confirmatory Western blot (immunoblot) (WB). By the Gacelisa IgG immuno-capture assay, 45 VS were positive and 84 were negative, whereas by WB, 44 VS were confirmed positive and 85 were confirmed negative. Considering WB as a reference, the IgG immunocapture assay in VS was 97.7% sensitive (43 of 44 positive samples) and 97.6% specific (83 of 85 negative samples). Of 42 HIV-seropositive women, 41 (97.6%) had S and VS that both were HIV positive (S+ VS+), and of 87 HIV-seronegative women, 83 (95.4%) had S and VS that both were HIV negative (S- VS-). Five women had discordant results for S and VS. One (S+ VS-) possibly had a false-negative VS result. Two (S- VS+) had similar indeterminate patterns for S and VS in WB. Two (S- VS+) had a typical HIV-positive pattern on WB of VS, whereas S results in WB were indeterminate in one case and negative in the other case; for both women, detection of prostatic acid-phosphatase was positive in VS, strongly suggesting recent sexual intercourse with an HIV-positive man. Because all HIV-infected men have detectable IgG antibodies to HIV in the seminal fluid, an HIV-seronegative rape victim with HIV-positive VS (S- VS+) should receive short-term antiviral therapy to prevent possible HIV transmission.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Anticorpos Anti-HIV/análise , Sêmen/imunologia , Vagina/imunologia , Adolescente , Adulto , República Centro-Africana , Coito , Feminino , Anticorpos Anti-HIV/sangue , Infecções por HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/transmissão , HIV-1/imunologia , HIV-2/imunologia , Humanos , Imunoglobulina G/análise , Masculino , Nonoxinol/administração & dosagem , Estupro , Zidovudina/administração & dosagemRESUMO
The intestinal clearance of alpha 1-antiproteinase, monomeric IgA and IgG, and the daily fecal output of polymeric IgA and IgM were investigated in patients with inflammatory bowel diseases (inactive and active Crohn's disease, ulcerative colitis) and in a control group. The intestinal clearance of alpha 1-antiproteinase was significantly increased in all patients with inflammatory bowel diseases (p less than 0.01), irrespective of the grade of the disease. In contrast, increases in intestinal clearances of monomeric IgA and IgG were more closely related to the severity of the intestinal lesions. The associate determination of these three quantities should therefore be of interest for monitoring the degree of intestinal bowel inflammation. Faecal output of polymeric IgA was significantly increased in active intestinal disease (p less than 0.01), whereas faecal IgM levels were not. The determination of the faecal output of polymeric IgA should contribute to the assessment of the activity of inflammatory bowel diseases, and may provide insight into the activation of the mucosal immune system.
Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Imunoglobulinas/metabolismo , alfa 1-Antitripsina/metabolismo , Adolescente , Adulto , Idoso , Criança , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Fezes/química , Feminino , Humanos , Imunoglobulina A/metabolismo , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Masculino , Pessoa de Meia-Idade , PolímerosRESUMO
Oropharyngeal candidiasis (OPC), mainly caused by Candida albicans, is commonly observed in HIV-infected patients. Secreted aspartic proteinases (Saps) are virulent agents involved in adherence to the mucosal surface and in tissue invasion. The immune secretory response to these agents was investigated in 15 HIV-infected patients, during oral yeast colonization and episodes of oropharyngeal candidiasis (OPC), in a 1-year longitudinal study. We developed an avidin-biotin-amplified immunofluorometric assay for the detection of specific immunoglobulins G, A, and M against somatic, Sap2 and Sap6 antigens. We report increases in anti-somatic, anti-Sap2, and anti-Sap6 salivary antibodies in patients with OPC. Over the 1-year period, not only OPC episodes but also variations in yeast colonization levels were correlated with variations in salivary anti-Sap6 antibody levels. Our results show the ability of HIV-infected patients to produce high levels of salivary antibodies; however, these antibodies were not efficient in limiting candidal infection, probably because of cellular cooperation deficiency and the enhanced virulence of the infecting strain.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Anticorpos Antifúngicos/análise , Ácido Aspártico Endopeptidases/imunologia , Candida albicans , Candidíase/imunologia , Proteínas Fúngicas , Infecções por HIV/imunologia , Mucosa Bucal/imunologia , Saliva/microbiologia , Infecções Oportunistas Relacionadas com a AIDS/microbiologia , Adulto , Antígenos de Fungos , Avidina , Biotina , Candida albicans/imunologia , Candidíase/microbiologia , Candidíase Bucal/imunologia , Candidíase Bucal/microbiologia , Feminino , Imunofluorescência , Infecções por HIV/complicações , Humanos , Imunoglobulina A Secretora/análise , Imunoglobulina G/análise , Imunoglobulina M/análise , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Doenças Faríngeas/imunologia , Doenças Faríngeas/microbiologia , Saliva/imunologiaRESUMO
The aim of this study was to explore anti-Candida albicans systemic and mucosal humoral responses against Candida virulence antigens such as somatic antigen and secreted aspartic proteases (Saps) in HIV-infected patients with oral candidiasis. Twenty-eight subjects were included in the study: 11 HIV-positive patients without oral candidiasis (group A), 6 HIV-positive patients with oral candidiasis (group B) and 11 HIV-negative healthy controls (group C). Total IgA, IgG and IgM concentrations and antibodies to C. albicans (somatic antigen, Sap1, Sap6) were measured in serum and saliva. We developed a time-resolved immunofluorometric assay with biotin and europium-labeled streptavidin for this purpose. Salivary total IgA, IgG and IgM concentrations were higher in group B. IgA, IgG and IgM anti-C. albicans antibodies (against somatic antigen, Sap1, Sap6) were higher in saliva and serum from patients from group B compared with patients from group A and controls. Our results suggest that, in oral candidiasis, HIV-infected patients have a high mucosal response, specifically directed against C. albicans virulence antigens, such as somatic antigen, Sap1 and Sap6.
Assuntos
Infecções Oportunistas Relacionadas com a AIDS/imunologia , Anticorpos Antifúngicos/sangue , Anticorpos Antifúngicos/metabolismo , Candida albicans/imunologia , Candidíase Bucal/complicações , Candidíase Bucal/imunologia , Saliva/imunologia , Adulto , Antígenos de Fungos , Ácido Aspártico Endopeptidases/imunologia , Candida albicans/enzimologia , Candida albicans/patogenicidade , Estudos de Casos e Controles , Feminino , Infecções por HIV/imunologia , Humanos , Imunidade nas Mucosas , Imunoglobulina A/sangue , Imunoglobulina A Secretora/metabolismo , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Imunoglobulina M/sangue , Imunoglobulina M/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
Total (t), monomeric (m) and polymeric (p) immunoglobulin A (IgA) levels were simultaneously measured in mixed faecal material collected over three consecutive days using a modified one-dimensional immunoelectrophoretic assay, which is a precise and reproducible method. Faecal t-, m- and p-IgA levels were statistically higher in 32 diarrhoeic patients than in a group of 16 healthy subjects (respectively 2.2, 2.6 and 2.0 times the normal). The values were also significantly different (P less than 0.001) when results were expressed as output per day in order to estimate the secreting capacity of gut. Moreover, the m-IgA/t-IgA percentages were significantly higher in faeces from diarrhoeic patients (42.3%) than in normal faeces (26.5%). These results confirm the importance of faecal IgA determination in estimating the local production of the IgA in various gastrointestinal tract disorders.
Assuntos
Diarreia/imunologia , Fezes/análise , Imunoglobulina A/análise , Polímeros/análise , Adulto , Idoso , Anticorpos Monoclonais/análise , Doença Crônica , Feminino , Humanos , Imunodifusão , Masculino , Pessoa de Meia-Idade , Proteínas/análiseRESUMO
Enhanced serum IgA concentrations are common in alcoholic liver cirrhosis, but functional differences between IgA subclasses and their relation with interleukin-6 (IL-6) have not been described. Distinct immunoregulatory mechanisms may exist that selectively affect one subclass. This possibility prompted us to investigate the distribution of IgA1 and IgA2 subclasses in the serum of 25 heavy alcohol drinkers (alcohol: 80 to 200 g per day) without clinical disorders, in comparison with 35 patients affected by alcoholic liver cirrhosis, 29 viral hepatitis patients and 33 social drinkers as a control group. Mean (+/- SD) IgA2 concentration (0.56 +/- 0.31 g/l) was significantly increased (p < 0.01) in heavy alcohol drinkers, with an IgA2/IgA1 ratio of 0.33 +/- 0.12, while the mean total IgA concentration was similar to the control group. Mean IgA1 and IgA2 concentrations were significantly increased (p < 0.001) in alcoholic liver cirrhosis patients (6.13 +/- 4.52 g/l and 1.83 +/- 1.93 g/l respectively, with an IgA2/IgA1 ratio of 0.32 +/- 0.19) and viral hepatitis patients (3.66 +/- 2.59 g/l and 0.69 +/- 0.67 g/l respectively, with an IgA2/IgA1 ratio of 0.21 +/- 0.14) High serum IL-6 concentrations (34 +/- 33 ng/l) were correlated with elevated IgA1 and IgA2 concentrations only in patients with alcoholic liver cirrhosis. IgA2 subclass and IgA2/IgA1 ratio could therefore be used as markers of chronic alcohol abuse directly related to the extent and duration of the alcohol abuse and the effectiveness of alcohol withdrawal.
Assuntos
Alcoolismo/imunologia , Imunoglobulina A/sangue , Adulto , Consumo de Bebidas Alcoólicas/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Feminino , Hepatite Viral Humana/imunologia , Humanos , Interleucina-6/sangue , Cirrose Hepática Alcoólica/imunologia , Masculino , Pessoa de Meia-IdadeRESUMO
Elevation of serum interleukin-1 beta (IL-1 beta) levels, and to a lesser degree tumor necrosis factor alpha levels, was found in cachectic human immunodeficiency virus (HIV)-infected African patients without concurrent opportunistic infection or neoplasia (HIV wasting syndrome). A heterogeneous pattern of elevations of cytokine levels, including mild elevations of IL-1 beta and pronounced elevations of IL-6 levels, was found in other cachectic states.