RESUMEN
Plasma fibronectin levels in sickle cell anaemia and apparently healthy Nigerians were investigated to determine any correlation with disease severity. A cheaper in-house plasma fibronectin assay was also developed that could be adapted for use in Africa and elsewhere. Plasma fibronectin assay was concurrently carried out using the newly developed inhibition enzyme-linked immunosorbent assay (ELISA) and a commercial competitive binding ELISA. The in-house assay compared favourably with that of the commercial kit. The mean plasma fibronectin levels in sickle cell anaemia subjects were significantly lower than that of control subjects (P < 0.001). Plasma fibronectin concentration could therefore be useful in assessing the severity of sickle cell anaemia.
Asunto(s)
Anemia de Células Falciformes/sangre , Fibronectinas/sangre , Adolescente , Adulto , Unión Competitiva , Biomarcadores/sangre , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Nigeria , Sensibilidad y Especificidad , Índice de Severidad de la EnfermedadRESUMEN
Histological studies have demonstrated vascular damage in all types of allograft rejection. It is likely that donor endothelium suffers the major and the first insult by the recipient's immune system since, in vivo, capillary endothelium expresses human lymphocyte antigen (HLA) class I and class II antigens. The present study was designed to examine whether injury to donor endothelial cells (ECs) by recipient peripheral blood mononuclear cells (PBMCs) can be demonstrated in vitro, and whether there is a relationship between the in vitro findings and the clinical outcome of renal allografts. Twenty renal transplant recipients were included in this study, and all patients were followed up for 6 months. PBMCs were isolated from the renal transplant recipients on three occasions; in the first 24-h post-transplantation, at the beginning of the second week, and in the third week post-transplantation. Additional samples were taken at the time of any acute rejection episode. These patients received renal allografts from 15 local cadaveric donors whose ECs were isolated. Donor-specific ECs and the corresponding renal transplant recipients' PBMCs and sera were employed in proliferation and cytotoxicity assays. Our results show that donor-specific ECs consistently induced a highly significant degree of recipient lymphocyte proliferative response (p < 0.05). However, no significant correlation between acute graft rejection and the degree of donor-specific EC-induced recipient lymphocyte proliferation was found. In contrast, there was a significant correlation between lymphocyte-induced EC cytolytic effects and acute renal graft rejection (p < 0.05). When conducted in larger studies, such information can have important implications in clinical transplantation.
Asunto(s)
Endotelio Vascular/inmunología , Rechazo de Injerto/inmunología , Trasplante de Riñón/inmunología , Linfocitos/inmunología , Técnicas de Cocultivo , Citotoxicidad Inmunológica , Antígenos de Histocompatibilidad Clase II/análisis , Antígenos de Histocompatibilidad Clase II/inmunología , Humanos , Interferón gamma/farmacología , Activación de Linfocitos , Donantes de TejidosRESUMEN
OBJECTIVE: To determine the relationship between the mechanism of apoptosis and intracellular killing of Mycobacterium bovis BCG. Apoptosis, "programmed cell death"--a physiologically beneficial and distinct form of cell death. SETTING: In vitro study was carried out in murine macrophage cell line J774 that was infected with Mycobacterium bovis BCG in different set of conditions. Percentage of surviving BCG and apoptotic cells was determined. METHODS: IFN-g and/or LPS-activated and non-activated J774 mouse macrophage cells were infected with BCG in a ratio of 1:5. The morphology of the host cells was studied after 4 hours, 24 hours and 48 hours of infection in cytospins stained with Jenner-Giemsa. Surviving bacteria were counted by incorporation of radiolabelled-uridine after cell lysis. RESULTS: Both in the activated and non-activted J774 cultures some cells undergo apoptosis. In cells activated with IFN-g or LPS without BCG, less than 10% of cells were found to be apoptotic. More apoptosis was seen when LPS-activated cells were infected with BCG. In the cells activated with IFN-g or LPS-activated cells the percentage of apoptotic cells was much higher than in non-activated cells or cells activated with either INF-g or LPS alone. After 24 hours culture, without BCG, about 15% of the cells were found to be apoptotic and with BCG infection this increased to 23% (p < 0.01). The level further increased after 48 hours of infection. BCG growth inhibition was observed in both non-activated J774 cells and cells activated with LPS, INF-g or both and was sustained to 48 hours of co-culture. CONCLUSION: It is evident that BCG-infected J774 cells undergo apoptosis in the presence of a high concentration of RNI and/or ROI. During this process the cells shrink considerably in volume with the removal of water that may concentrate toxic products in the cell. The increased concentration of toxic species and the disorganisation of the phagocytic vacuoles may account for the enhanced stasis and/or death of the intracellular micro-organisms. We conclude that host cell apoptosis may arrest the growth and account for the death of the intracellular mycobacterial pathogen.
Asunto(s)
Apoptosis , Macrófagos/fisiología , Mycobacterium bovis , Animales , Células Cultivadas , RatonesRESUMEN
Bacterial infections are the major determinants of fatality in severe protein-energy malnutrition (PEM). Unfortunately, these infections are difficult to diagnose clinically. C-reactive protein (CRP) levels were determined in 17 infected and 10 non-infected Nigerian children with severe PEM and compared with age/sex-matched apparently healthy controls. The aim was to study the response of this acute phase protein to bacterial infections as well as to assess its value in the diagnosis of infections in severe PEM. C3 complement protein levels were also determined in the same group of subjects. The major organisms isolated in samples from these subjects were S. aureus and the coliforms. Mean CRP level in the non-infected children with severe PEM was 13.8 +/- 6.21 mg/l and rose to 159.83 +/- 124.07 mg/l in the presence of infection. The mean value in healthy non-infected controls was 2.01 +/- 0.96 mg/l. The difference in the mean CRP levels between the infected and non-infected PEM children was statistically significant at p < 0.01. The mean difference between the non-infected and the control subjects was not significant. Using a diagnostic level of 20.00 mg/l of CRP gave a sensitivity of 85.0% and a specificity of 80.0%. This CRP level is a useful index of bacterial infections in severe PEM. C3 complement protein was low in the non-infected malnourished group, but rose significantly in the presence of infection to values similar to that of the healthy controls. C3 protein thus behaves as an acute phase reactant in the presence of infection in severe PEM, and does not appear to be consumed, probably due to a deficiency in the early components of the complement cascade. This suggests a role for C3 measurement in the monitoring of bacterial infections in severe PEM.
Asunto(s)
Infecciones Bacterianas/sangre , Proteína C-Reactiva/análisis , Complemento C3/análisis , Desnutrición Proteico-Calórica/sangre , Infecciones Bacterianas/complicaciones , Preescolar , Países en Desarrollo , Femenino , Humanos , Lactante , Masculino , Monitoreo Fisiológico , Nigeria , Desnutrición Proteico-Calórica/complicaciones , Valores de Referencia , Sensibilidad y Especificidad , Albúmina Sérica/metabolismo , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To explore the possibility that an analysis of antibody specificity to separated components of mycobacteria in a group of tuberculous patients may reveal a combination of target antigens whose antibodies could form the basis of a useful serodiagnostic test. DESIGN: Immunoblots of 1-dimensional (SDS-PAGE) and 2-dimensional (isoelectric focusing/SDS-PAGE) separation of antigenic extracts of Mycobacterium tuberculosis H37Rv (MTSE) and M. bovis bacille Calmette-Guérin (BCG) (MBSE) with 52 tuberculous and 59 BCG-vaccinated control human sera were analyzed for band and spot reactivity patterns that are indicative of infection with M. tuberculosis. RESULTS: Reactivity to antigens banding in the 10-18 kDa, 37-43 kDa and 70-90 kDa regions allowed a good discrimination between patients and normal subjects. Patients' sera reacting with antigens in the 22-30 and 70-88 kDa regions differentiated responses to MTSE and MBSE. In 2-D immunoblotting, patients' sera only reacted with antigens separating at approximately pI 6.5/26-28 kDa, pI 4.8/38 kDa and pI 6.5/70-79 kDa position and the responses were specific for M. tuberculosis (MTSE). CONCLUSION: These results provide evidence that a combination of these M. tuberculosis antigens may be a useful basis for developing a diagnostic antibody test. Additionally, they may help to define antigens, and host antibody responses that are specific to one but not the other of the two closely related species.
Asunto(s)
Antígenos Bacterianos/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis/diagnóstico , Anticuerpos Antibacterianos/sangre , Especificidad de Anticuerpos , Western Blotting , Electroforesis en Gel Bidimensional , Electroforesis en Gel de Poliacrilamida , Humanos , Sueros Inmunes/inmunología , Immunoblotting , Focalización Isoeléctrica , Mycobacterium bovis/inmunología , Tuberculosis/inmunologíaRESUMEN
OBJECTIVE: To evaluate and compare the lymphoproliferative response of human peripheral blood mononuclear cells (PBMC) to fractionated soluble extracts of Mycobacterium tuberculosis H37Rv (MTSE) and M. bovis bacille Calmette-Guérin (BCG) (MBSE), and thereby determine responses that correlate to infection, and to contrast antibody and T-cell responses. DESIGN: Membrane blots of SDS-PAGE fractionated M. tuberculosis H37Rv and M. bovis BCG were employed for antibody immunoblotting and T-cell proliferative responses using sera and PBMC from seven tuberculous and seven BCG vaccinated control subjects. RESULTS: The profiles of responses contrasted rather interestingly, with antibody and T-cells responding more to higher and lower molecular weight fractions respectively. T-cells responding to antigens in the 59-88 kDa region discriminated between tuberculous and BCG vaccinated controls (P < 0.05) even though the differences were more toward the 70-75 kDa fractions within the region in question. Responses to smaller molecular weight fractions of both MTSE and MBSE were high in direct contrast to antibody responses. Additionally, responses to MBSE in these regions were generally higher than for MTSE in vaccinated controls. The reverse was the case with tuberculous subjects where responses to MTSE were generally higher, though not sufficiently significant in enough of the tuberculous subjects to be considered discriminatory. CONCLUSION: T-cell proliferative responses to mycobacterial antigens in the 59-88 kDa region, and particularly antigens in the 70-75 kDa region, can be an indication of infection with M. tuberculosis, as well as the basis for discriminating between active disease and vaccination with BCG.
Asunto(s)
Antígenos Bacterianos/inmunología , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T/inmunología , Tuberculosis/inmunología , Antígenos Bacterianos/química , Western Blotting , Técnicas de Cultivo de Célula , División Celular/inmunología , Células Clonales/inmunología , Humanos , Activación de Linfocitos , Peso MolecularRESUMEN
Fibronectin-binding protein (FnBp) antigens are a prominent secretory protein of short term culture supernatants of M. tuberculosis and M. bovis (BCG) and is conserved within the genus Mycobacterium. The 30/31 kDa antigen of M. tuberculosis is one of the major secretory molecules and is probably routinely recognised by the host immune system in the early stage of tuberculosis infection. Serum immune complexes, prepared from TB patients and normals, were analysed for the presence of FnBp by ELISA using an anti-30/31 kDa (FnBp) monoclonal antibody (CF8) and by western blotting using Fibronectin-HRP. A significant difference was seen between normals and TB patients (p < 0.05). This test was found to have a specificity of 80% and a positive predictive value of 73%. This is a preliminary finding and the test needs to be evaluated further for its performance on a larger number of confirmed TB patients and controls.
Asunto(s)
Adhesinas Bacterianas , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas Bacterianas , Proteínas Portadoras , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/diagnóstico , Anticuerpos Monoclonales , Complejo Antígeno-Anticuerpo/análisis , Complejo Antígeno-Anticuerpo/inmunología , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Humanos , Sensibilidad y Especificidad , Tuberculosis Pulmonar/inmunologíaRESUMEN
Several studies have addressed the possible importance of anti-epithelial cell antibodies in kidney transplantation using the A549 cell line as an in vitro model. In this paper we report our results using for the first time an enzyme-linked immunosorbent assay (ELISA) to detect the anti-A549 cell antibodies. Sera from 129 kidney transplant patients were tested for IgM anti-epithelial cell antibodies directed against the A549 cell line prior to transplantation; only three sera were positive (2.3%). 101 of these patients were then followed-up post-transplantation; sera were collected routinely at 2, 6 and 12 weeks and at the time of rejection episodes. All samples were also tested for cytomegalovirus (CMV) IgM antibodies. Sixteen patients developed anti-A549 IgM antibodies, and there was no correlation with acute graft rejection. Anti-epithelial antibodies showed no binding to sections of normal kidney or biopsies of rejected kidneys. Eleven patients were positive for anti-CMV IgM antibodies. In nine cases both IgM anti-A549 and IgM anti-CMV antibodies were found, which was a highly significant association (p < 0.001). Analysis of A549 cellular proteins by immunoblotting gave evidence for the presence of CMV polypeptides in the cell lysate. Electron-microscopic examination of A549 cell preparations revealed intracellular particles which were compatible in size with CMV. Polymerase chain reaction analysis confirmed the presence of a specific CMV DNA sequence in A549 cells of several batches from different sources. Our data strongly suggest that the A549 cell line used in several published reports is infected with CMV and that in the majority of cases the anti-A549 'anti-epithelial' antibodies found in renal transplant patients are anti-CMV antibodies.
Asunto(s)
Especificidad de Anticuerpos , Trasplante de Riñón/inmunología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Artritis Reumatoide/inmunología , Secuencia de Bases , Niño , Citomegalovirus/genética , Citomegalovirus/aislamiento & purificación , Citomegalovirus/ultraestructura , Epitelio/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/farmacología , Inmunoglobulina M/sangre , Neoplasias Pulmonares/inmunología , Lupus Eritematoso Sistémico/inmunología , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Estudios Prospectivos , Factor Reumatoide/sangre , Células Tumorales Cultivadas , Proteínas Virales/análisisRESUMEN
The concept of a common mucosal immune system in man was tested by examining the concurrent presence of specific-secretory IgA (SIgA) antibodies in human milk and saliva from three groups of subjects: 64 Sri Lankan women living in Sri Lanka; 20 immigrant Asian women living in Birmingham (median duration of residence in the United Kingdom five years); and 75 Caucasian women living in Birmingham (controls). Enzyme linked immunosorbent assays (ELISA) were developed to detect enterotoxigenic Escherichia coli (ETEC) colonisation factor/1 (CFA/1) specific SIgA antibodies in milk and saliva. ETEC CFA/1 specific SIgA antibody activity was detectable in milk (37.5% and 25%) and saliva (42.1% and 35%) of Sri Lankan and immigrant Asian women, respectively, but not in any of the Caucasian controls. Eighty five point two per cent of subjects who were positive had specific antibodies detectable in both milk and saliva; 5% of all Sri Lankan women and 10% of all immigrant Asian women had detectable antibody only in saliva. These observations lend further strong support to the idea that a common mucosal immune system exists in man. The continuing presence of specific SIgA antibodies in Asian immigrants to previously encountered antigens suggests that there may be an 'immunological memory' in the human secretory immune system.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Proteínas Fimbrias , Inmunoglobulina A Secretora/inmunología , Leche Humana/inmunología , Saliva/inmunología , Adolescente , Adulto , Asia/etnología , Inglaterra , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Sri LankaRESUMEN
The concept of an enteromammary link in secretory IgA (SIgA) antibody production was tested by hypothesising that specific SIgA antibody profiles in human milk might be an epidemiological marker for enteropathogens in a community. Milk from three subject groups was studied: 64 Sri Lankan women living in poor suburbs of Colombo, 20 Asian immigrant women domiciled in Birmingham, for a median period of five years (range 14 days-16 years), and 75 white women living in Birmingham. An enzyme linked immunosorbent assay (ELISA) was developed for the detection and measurement of SIgA antibodies to a panel of 14 crude O and 10 pure lipopolysaccharide antigens of diarrhoeagenic Escherichia coli strains well known to be endemic in the Indian subcontinent. The number of Sri Lankan and Asian immigrant women with SIgA antibodies to all 14 diarrhoeagenic E coli antigens (except O127 in Asian women) was significantly higher than in the white controls. The amount of E coli O antigen specific SIgA antibody activity as a percentage of total SIgA also gave significantly higher median values in Sri Lankan (6%) and in Asian immigrant (4%) women than in white controls (0.7%). SIgA antibodies were highly O serogroup specific and showed excellent concordance between crude O and the corresponding purified lipopolysaccharide antigens. These results suggest that milk antibody profiles represent an epidemiological marker of exposure to enteral pathogens. The continuing specific milk antibody response in Asian women who have been domiciled in the United Kingdom for many years may indicate 'memory' in the human secretory immune system.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Inmunoglobulina A Secretora/análisis , Enfermedades Intestinales/inmunología , Leche Humana/inmunología , Sistema del Grupo Sanguíneo ABO , Adolescente , Adulto , Asia/etnología , Inglaterra/epidemiología , Escherichia coli/inmunología , Femenino , Humanos , Enfermedades Intestinales/sangre , Enfermedades Intestinales/epidemiología , Sri Lanka/etnologíaRESUMEN
AIMS: To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. METHODS: Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. RESULTS: Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine. CONCLUSION: It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.
Asunto(s)
Anticuerpos Antibacterianos/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Neisseria meningitidis/inmunología , Polisacáridos Bacterianos/inmunología , Especificidad de Anticuerpos , Humanos , Polilisina , Reproducibilidad de los Resultados , Albúmina SéricaRESUMEN
PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.
Asunto(s)
Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/estadística & datos numéricos , Secuencia de Bases , Cartilla de ADN/genética , ADN Bacteriano/genética , Errores Diagnósticos , Humanos , Laboratorios , Datos de Secuencia Molecular , Mycobacterium bovis/genética , Mycobacterium bovis/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Control de Calidad , Reproducibilidad de los Resultados , Saliva/microbiología , Sensibilidad y Especificidad , Esputo/microbiología , Tuberculosis/diagnóstico , Microbiología del AguaRESUMEN
Venom-insoluble adsorbents were employed to absorb out the cross-reacting antibodies from monovalent polyclonal antivenoms. The absorbed antivenoms were tested by enzyme-linked immunosorbent assay against homologous and heterologous venoms and showed species-specificity throughout a range of venom concentrations. The same absorbed antisera were used in immunoblots under non-reducing conditions as probes to reveal species-specific antigens. In all cases studied this was achieved. The range of mol. wts of specific antigens was between 20,000 and 120,000, approximately. Venoms added to human serum experimentally were specifically detected by their homologous absorbed antivenom antibodies. The work here described could be important in the development of diagnostic assays for envenomings involving snakes from the Bothrops and Lachesis genera.
Asunto(s)
Antígenos/análisis , Venenos de Crotálidos/inmunología , Adsorción , Especificidad de Anticuerpos , Antivenenos/análisis , Antivenenos/inmunología , Western Blotting , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunoglobulinas/análisis , Peso Molecular , Especificidad de la EspecieRESUMEN
The enzyme-linked immunoabsorbent assay (ELISA) was used to determine the specific streptococcal antibodies in the sera of patients with post-streptococcal acute glomerulonephritis. Attempts to use this same technique to determine the immunoglobulin content of immune complexes (IC) after immunochemical dissociation proved futile. It became necessary to perform an inhibition assay before performing the ELISA. This modification involved the determination of the antigen concentration necessary to produce 50 per cent inhibition of the dissassociated antibody. The antigen inhibitors, M-types 41 and 55 cell membranes, were found to inhibit optimally at 12.5 ug/ml and were used to absorb dissassociated immune complexes diluted 1/00. The absorbed solution was then used in the ELISA test system. While specific streptococcal antibodies to M-55 were lower than those to M-41 (p<0.001) in PSAGN patients and those in controls (p<0.001), in the IC controls, specific antibodies to both inhibitors were of the same order. In PSAGN, however, the antibody levels were increased when compared with controls. Interestingly enough, specific streptococcal antibodies were demonstrated in IC of resolved GN cases, chronic GN and in those with intermittent proteinuria (AU)
Asunto(s)
Humanos , Glomerulonefritis/inmunología , Infecciones Estreptocócicas/inmunología , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
Four-day-old chickens infected orally with a spectinomycin-resistant (Spcr) mutant of a highly invasive avian Salmonella typhimurium strain excreted salmonellae in the feces for at least 10 weeks. When these chickens were reinfected at this time with a nalidixic acid-resistant (Nalr) mutant of the same strain, they excreted this mutant in significantly smaller numbers (P less than 0.01) than did a previously uninfected control group. The Nalr mutant had a shorter survival rate in the tissues of the immunized chickens than in tissues of the control birds. The Spcr mutant stimulated strong IgG, IgA, and IgM responses in serum, small-intestinal contents, and bile. These were detected by enzyme-linked immunosorbent assay (ELISA) against antigens of crude whole bacterial cell protein sonicate, lipopolysaccharide, flagella, and outer-membrane proteins. There was some evidence of an anamnestic response with IgA in bile following reinfection with the Salmonella. The peak response of antibody-producing cells from the spleens of infected chickens, assayed by solid-phase ELISA, occurred at 3 weeks postinoculation. A strong delayed hypersensitivity reaction, detected by foot-pad swelling after inoculation with either whole-cell or outer-membrane proteins, was observed between 2 and 5 weeks after infection with the Spcr mutant. The data indicate that outer-membrane proteins are major immunogens for both humoral and cell-mediated arms of the immune system.
Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Pollos , Enfermedades de las Aves de Corral/inmunología , Salmonelosis Animal/inmunología , Salmonella typhimurium/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Células Productoras de Anticuerpos/inmunología , Bilis/inmunología , Ciego/microbiología , Heces/microbiología , Hipersensibilidad Tardía , Inmunidad Celular , Inmunoglobulinas/biosíntesis , Inmunoglobulinas/sangre , Intestinos/inmunología , Enfermedades de las Aves de Corral/microbiología , Recurrencia , Salmonelosis Animal/microbiología , Salmonella typhimurium/aislamiento & purificación , Organismos Libres de Patógenos EspecíficosRESUMEN
The gene encoding a 23 kilodalton protein antigen has been cloned from Mycobacterium tuberculosis by screening of a recombinant DNA library with monoclonal antibodies. The product of the gene has been identified as the superoxide dismutase (SOD) of M. tuberculosis on the basis of sequence comparison and by expression of the recombinant protein in a functionally active form. The derived amino acid sequence of M. tuberculosis SOD reveals a close similarity to manganese-containing SODs from other organisms, in spite of the fact that previous studies using the purified enzyme have identified iron as the preferred metal ion ligand. SOD is present in the extracellular fluid of logarithmic-phase cultures of M. tuberculosis, but the structural gene is not preceded by a signal peptide sequence. Insertion of the M. tuberculosis SOD gene into a novel shuttle vector demonstrated the mycobacteria but is ineffective in Escherichia coli.
Asunto(s)
Antígenos Bacterianos/genética , Mycobacterium tuberculosis/genética , Superóxido Dismutasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Western Blotting , Clonación Molecular , ADN Bacteriano , Electroforesis en Gel de Poliacrilamida , Vectores Genéticos , Inmunoensayo , Datos de Secuencia Molecular , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Homología de Secuencia de Ácido Nucleico , Superóxido Dismutasa/inmunología , Superóxido Dismutasa/metabolismoRESUMEN
IS986 of Mycobacterium tuberculosis belongs to the IS3-like family of insertion sequences, and it has previously been shown to be present in multiple copies in the chromosome of M. tuberculosis. In this study we investigated the value of a IS986-based DNA probe in the diagnosis and epidemiology of tuberculosis. IS986 was found only in species belonging to the M. tuberculosis complex. Independent isolates of M. tuberculosis complex strains showed a very high degree of polymorphism of restriction fragments which contained IS986 DNA. In contrast, Mycobacterium bovis BCG vaccine strains as well as clinical isolates of M. bovis BCG contained one copy of IS986, which was present at the same location in the chromosome. Different M. tuberculosis isolates from a recent M. tuberculosis outbreak showed an identical banding pattern. We concluded that IS986 is an extremely suitable tool for the diagnosis and epidemiology of tuberculosis.
Asunto(s)
Elementos Transponibles de ADN , Mycobacterium tuberculosis/genética , Tuberculosis/diagnóstico , Animales , Secuencia de Bases , Sondas de ADN , Reordenamiento Génico , Cobayas , Humanos , Datos de Secuencia Molecular , Mycobacterium bovis/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Tuberculosis/epidemiologíaRESUMEN
A repetitive element (IS986), previously isolated from Mycobacterium tuberculosis and shown to detect multiple restriction fragment-length polymorphisms (RFLPs), has been sequenced. It consists of a potential insertion sequence of 1358bp, with 30-bp inverted repeat ends. IS986 has four potentially significant open reading frames (ORFs): ORFa1, ORFa2 and ORFb on one strand and ORFc on the complementary strand. The sequences of the potential translated products identify IS986 as a member of the IS3 family, with an apparent frameshift between ORFa1 and ORFa2. IS986 has potential as a highly specific probe for detection and typing of M. tuberculosis, as well as for transposon mutagenesis of mycobacteria. The sequence of IS986 is virtually identical to that of another recently described element, IS6110 (Thierry et al., 1990).
Asunto(s)
Elementos Transponibles de ADN , Mycobacterium tuberculosis/genética , Secuencias Repetitivas de Ácidos Nucleicos , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Polimorfismo de Longitud del Fragmento de Restricción , Biosíntesis de Proteínas , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
Antigenic cross-reactivity between venoms of the genus Bothrops has been shown to be an extensive problem. However, some venom components are species-specific. In this study we have produced species-specific antivenoms against some members of the genus Bothrops. Monospecific rabbit antivenoms (IgG) were absorbed on venom affinity adsorbents. The species-specificity was tested by ELISA assays and immunoblots. The results of both assays showed complete species-specificity in some cases and highly increased species-specificity in others. These reagents can be used to determine the envenomating species in snake bite patients as an aid to improved serotherapy.
Asunto(s)
Antivenenos/análisis , Venenos de Crotálidos/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Inmunoglobulina G/análisis , Brasil , Reacciones Cruzadas/inmunología , Immunoblotting , Especificidad de la EspecieRESUMEN
Antigenic cross-reactivity between venoms of the genus Bothrops has been shown to be an extensive problem. However, some venom components are species-specific. In this study we have produced species-specific antivenoms against some members of the genus Bothrops. Monospecific rabbit antivenoms (IgG) were absorbed on venom affinity adsorbents. The species-specificity was tested by ELISA assays and immunoblots. The results of both assays showed complete species-specificity in some cases and highly increased species-specificity in others. These reagents can be used to determine the envenomating species in snake bite patients as an aid to improved serotherapy