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Biomedical subjects

M J Torres

Publications and source records attributed to M J Torres.

At least 91 records · Page 5Linked to original sources
PubMed1993

Dual genitotropic human papillomavirus infections in genital warts.

BACKGROUND AND METHODS: We have carried out a prospective study of dual genitotropic human papillomavirus (HPV) infections by means of two different DNA detection methods in biopsy specimens obtained from patients who were examined for genital warts at the STD clinic of the School of Medicine in Seville, between January 1990 and December 1991. RESULTS: 100 patients with a clinical diagnosis of condilomata acuminata were seen during the study period. DNA of the genitotropic HPV 6/11, 16/18 and 31/33/35 was detected by an in situ hybridisation method in 75 (77%) of the 98 evaluable samples; one of the genotypes tested in 59 (61%) samples, and two or more genotypes tested in the remaining 16 (15%) samples. In 21 (98%) of the 23 negative samples by in situ hybridisation, we were able to detect DNA of genital HPV using a polymerase chain reaction amplification method (PCR). Among the 34 samples where PCR was applied we confirmed the presence of two different HPV genotypes in eight samples. CONCLUSIONS: The frequency of dual infections with human genitotropic papillomavirus in genital warts was 8%, although we believe that this rate should be higher as we have not used the PCR method in all of the samples.

Adolescent↗
PubMed1992

Evaluation of a fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae.

This study evaluates a four-hour fluorescent DNA hybridization assay using both known bacterial isolates and clinical specimens. A biotinylated oligonucleotide probe from a sequence of the plasmid-encoded gene cppB was used. Hybrids were detected by addition of a streptavidin-alkaline phosphatase conjugate, followed by incubation for 30 min in a fluorescent substrate for alkaline phosphatase. The level of detection of the fluorescent assay was 0.1 pg of cryptic plasmid DNA or 200 cfu of the plasmid-containing strain NG 34/85 of Neisseria gonorrhoeae. A total of 119 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All Neisseria gonorrhoeae strains, including eight plasmid-free strains, hybridized with the probe. Fluorescence ratios were 2.67 for plasmid-free strains and 3.85 for plasmid-containing strains. Of the heterologous microorganisms tested, only one of six strains of Neisseria cinerea gave a fluorescence ratio above the 2.0 cut-off value for positivity with the probe at a cell density of 1 x 10(4) cfu. The probe was also evaluated using clinical specimens from 100 patients attending a clinic for sexually transmitted diseases. The sensitivity of the assay was 100% while the specificity was 97.5%. Positive and negative predictive values were 91.2% and 100%, respectively. The fluorescent DNA hybridization assay for the detection of Neisseria gonorrhoeae described here thus appears to be a highly specific and sensitive assay.

Base Sequence↗
PubMed1992

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.

Alkaline Phosphatase↗
PubMed1992

[Chlamydia trachomatis detection by DNA-RNA hybridization].

BACKGROUND: A one-chain DNA probe, that complements ribosomal RNA of Chlamydia trachomatis was used as a detection method for this microorganism on clinical samples. We compare the method with the cell culture one. METHODS: A total of 175 samples (cervix swabs) from women seen at the STD center of the Facultad de Medicina de Sevilla were examined by both diagnostic techniques. When the results were different, a third method (ELISA) was also used. RESULTS: Using serial dilutions of a C. trachomatis cell culture as reference pattern, we determine the minimum number of inclusion forming units needed in order to be detected by the probe was 1000. Of all 175 samples, in 24 (14%) cell culture was positive for C. trachomatis, and 26 were positive using the DNA probe test. Sensitivity and specificity for this test were 93% and 95%, respectively. CONCLUSIONS: We believe that the DNA probe test was similar to the cell culture test as screening test in Chlamydia trachomatis infections diagnosis, specially among high risk populations.

Chlamydia Infections↗
PubMed1992

DNA hybridization assay using ATTOPHOS, a fluorescent substrate for alkaline phosphatase.

A fluorometric procedure for the detection of DNA-DNA hybrids is described. The procedure involved the detection of probe-bound alkaline phosphatase with the fluorescent substrate ATTOPHOS. This substrate is converted to ATTOFLUOR by alkaline phosphatase and fluoresces strongly at 550 nm when excited with a wavelength of 440 nm. DNA hybridization assays were performed both with dilutions of purified target plasmid DNA (pSE9 or PBR322) and whole bacterial cells. Streptavidin-alkaline phosphatase conjugates were added to react with bound probe. Fluorometric assays, as well as colorimetric assays, using 5-bromo-4-chloro-3-indolylphosphate + nitroblue tetrazolium for alkaline phosphatase activity were performed. The fluorescence of the substrate was measured at time intervals, and the slope of the regression line calculated. A slope four times greater than that of background was considered positive. One hundred femtograms or 2.2 x 10(4) molecules of homologous DNA were detected with the fluorescent assay as compared with 10,000 femtograms or 2.2 x 10(6) molecules of homologous DNA with the colorimetric assay. Similar results were obtained with whole cells. Approximately 1 x 10(3) homologous cells were detected fluorometrically and 1 x 10(5) cells were detected colorimetrically. Based on these results, we conclude that, in our hands, the DNA hybridization assay described here using ATTOPHOS as the substrate for alkaline phosphatase is a very sensitive assay for the detection of DNA-DNA hybrids.

Alkaline Phosphatase↗
PubMed1991

Evaluation of a DNA probe of plasmid origin for the detection of Neisseria gonorrhoeae in cultures and clinical specimens.

This study evaluates a cryptic plasmid-derived DNA probe in a dot-blot hybridization assay of 4-h duration, using both known bacterial isolates and clinical specimens. The probe, consisting of a 237 bp segment of the plasmid-encoded gene cppB, sequences of which are also found in the chromosome, was labelled with digoxigenin-11-dUTP. The sensitivity of the probe was approximately 25 pg of DNA or 500 cfu of Neisseria gonorrhoeae. A total of 170 reference strains of Neisseria gonorrhoeae and other related bacteria were tested for reactivity with the probe. All N. gonorrhoeae strains, including three plasmid-free strains, hybridized with the probe. Among the heterologous bacterial cultures, only one strain of N. cinerea reacted with the probe when the cell concentration was 5 x 10(6) cfu. The probe was also evaluated in a clinical study. A total of 201 patients visiting the STD clinic at the University Hospital, University of Seville, participated in the study. The sensitivity of the assay was 95% while the specificity was 98%. Positive and negative predictive values were 97% and 98%, respectively. It appears that the plasmid-derived probe used in this study could serve as a useful tool in the rapid and specific detection of Neisseria gonorrhoeae in clinical specimens.

Cloning, Molecular↗
PubMed1989

Cross-reactivity between penicillins and cephalosporins: clinical and immunologic studies.

Nineteen well-characterized penicillin-allergic patients were investigated for their sensitivity to cephalosporins containing potentially cross-reactive side chains. All patients were administered cephamandole parenterally and, if this was tolerated, a course of oral cephaloridine was administered. Only two patients responded to the cephamandole; none of the remaining patients reacted to cephaloridine. Benzylpenicilloyl RAST-inhibition studies with sera from three subjects who had not reacted to the cephalosporins demonstrated that cephamandole linked to proteins was capable of recognizing benzylpenicilloyl-specific IgE antibody. It is concluded that consideration of side chain structures can help to predict possible cross-reactions between penicillins and cephalosporins, but carefully controlled challenge tests are advisable before penicillin-allergic patients are treated with cephalosporins. In relation to cross-reacting potential, in vitro experimental studies are difficult to interpret and may in some circumstances overestimate the risk.

Adult↗
PubMed1981

[Neviform hyperkeratosis of the mammary areola].

A case of neviform hyperkeratosis of the areola is reported. This case may be identified as type III of the Lévy-Franckel classification: not associated to other cutaneous alterations. A review of the literature is made. The authors emphasize the importance of the hormonal factors in the development of these lesions.

Adult↗
PubMedDate not supplied

Different lymphocyte markers and cytokine expression in peripheral blood mononuclear cells in children with acute atopic dermatitis.

BACKGROUND: The pathogenesis of atopic dermatitis (AD) is still not completely understood. AD is characterized by the presence of clinical symptoms of both IgE antibody-mediated immediate hypersensitivity and specific T lymphocyte-mediated delayed hypersensitivity. OBJECTIVE: To evaluate the immunological mechanisms involved in children with acute AD lesions. MATERIAL AND METHODS: Ten children with acute AD lesions and 10 non-atopic controls were studied. Total IgE was measured by immunoassay. T cell marker expression (CD3, CD4, CD8, cutaneous lymphocyte-associated antigen [CLA]) and cytokine production (interferon [IFN]-gamma, interleukin [IL]-13) were analyzed in peripheral blood mononuclear cells by flow cytometry. RESULTS: In children with AD the percentage of CD3+ cells (p = 0.015) increased while that of CD8+ cells (p = 0.023) decreased, with no differences in CLA expression. We found increased IL-13 production in CD3+ cells (p = 0.01) and CD3+CD4+ (p = 0.001) cells with no difference in IFN-gamma. Total IgE was significantly higher in patients with AD (p = 0.01). Comparison of IL-13 production in CD4+ cells categorized by total IgE level showed that IL-13 production was significantly increased in subjects with a higher IgE level. CONCLUSION: Peripheral blood from children with AD showed an increase in IgE levels and a Th2 pattern. There was a correlation between IL-13 production and total IgE levels.

Antigens, CD↗
PubMedDate not supplied

Differences in the immunological responses in drug- and virus-induced cutaneous reactions in children.

The cutaneous symptoms in non-immediate reactions to drugs are not always clinically distinguishable from those induced by viruses, especially during the early phase of the reaction. Moreover, viral infections and drug reactions often coexist and identification of the etiological agent is necessary. Discerning the differences in the immunological response between both may help in the diagnosis. The aim of this study was to determine possible differences in the immunological response in non-immediate cutaneous reactions to drugs versus cutaneous viral-induced diseases in children. Two groups of children were evaluated: one with non-immediate drug-induced cutaneous reactions (DICR) and another with virus-induced cutaneous reactions (VICR). A third group of children taking the same drugs as the DICR group and with no cutaneous disease or viral infections was included as controls. The lymphocyte markers CD3, CD4, CD8, CD16, CD19, CLA, CD25, CD69, CD45RO, CD45RA were determined by flow cytometry. IL-2, IL-4, IL-5, IFN-gamma, TNF-alpha and IL-10 mRNA were measured by RT-PCR. Data were compared by non-parametric and chi(2) statistical analysis. In DICR group (n=8) the diagnosis was established by temporal association, improvement after drug withdrawal, patch testing, and in some cases by controlled administration. All patients in the VICR group (n=10) were diagnosed based on a positive viral serology: the presence of IgM antibodies or seroconversion of IgG antibodies. There were significant differences between the three groups in peripheral lymphocytes expressing the skin homing receptor CLA (P < 0.01), the early activation marker CD69 (P < 0.001), and the memory (CD3+CD45RO+) (P < 0.02) and naive (CD3+CD45RA+) (P < 0.03) T cell subsets. Children with DICR showed a TH1 mRNA cytokine pattern whereas those with VICR showed a TH0 pattern. In lymphocyte subpopulations from children, differences in the immunological response between DICR and VICR can be detected in the expressions of the activation marker CD69 and the cutaneous homing receptor CLA, and in cytokine mRNA profiles.

Adolescent↗
PubMedDate not supplied

Memory to the hapten in non-immediate cutaneous allergic reactions to betalactams resides in a lymphocyte subpopulation expressing both CD45RO and CLA markers.

The cutaneous lymphocyte-associated antigen (CLA) is a homing receptor expressed in a subpopulation of memory T lymphocytes that migrates to the skin and participates in different inflammatory processes. The aim of the study was to compare the T cell response to betalactams in both CLA+ and CLA- memory T cell subsets from subjects with non-immediate allergic reactions to these drugs. Three patients with a non-immediate reaction to penicillins were studied during their acute episodes. Peripheral blood mononuclear cells were isolated by Ficoll density gradient and were used for flow cytometry and lymphocyte transformation test assays. CD3+ cells were purified via high affinity negative selection columns. CD45RA+ and CD45RO+ subpopulations were obtained by magnetic sorting and the memory subpopulation was subdivided into CLA+ and CLA- fractions. These were cultured in triplicate together with feeder cells and different concentrations of amoxicillin and benzylpenicillin. In all cases, the proliferative responses to the drugs were confined to the CD45RO+CLA+ subpopulation. The CD45RO+CLA- subset showed no proliferative response to either drug at any concentration. We have shown that the in vitro memory to penicillins in non-immediate cutaneous allergic reactions to these drugs resides in the CD45RO+CD3+ subset expressing CLA, which enables these T cells to migrate to the skin. These findings may have relevance to understanding the involvement of T cells in allergic reactions to penicillins.

Aged↗
PubMedDate not supplied

Failure to thrive, wasting syndrome, and immunodeficiency in rabies: a hypophyseal/hypothalamic/thymic axis effect of rabies virus.

Studies of rabies virus in several animal models consistently showed hypothalamic infection, hypophyseal infection, dramatic growth impairment (in the form of failure to thrive), wasting syndrome, and immune depletion. Rabies virus infection was studied through routine monoclonal antinucleocapsid antibody immunofluorescence and through a peroxidase-antiperoxidase immunoperoxidase method. The latter was modified to detect the in situ production of growth hormone by uninfected and rabies virus-infected adeno-a-pituicytes (with confirmation of the results both in vivo and in vitro). Infection with rabies virus made the specialized pituicytes produce less growth hormone. Growth before rabies virus infection and its reduction due to infection were investigated in a linear regression model. The fit was statistically significant (P less than .05) in all species studied: mouse, rat, rabbit, cow, and cat. Immune depression was studied in terms of alterations in the immunotopography of the thymus and also the specific T- and B-cell homing areas of the spleen (although spleen data are not presented here). On the basis of these results and a thorough review of wasting syndromes encountered in other diseases, a primary failure to thrive and an ensuing wasting syndrome were described and characterized for rabies, and their origin was assigned to a dysfunction of the hypophyseal/hypothalamic/thymic axis associated with at least (but not necessarily only) one of the centrally controlled growth hormones.

Acquired Immunodeficiency Syndrome↗
PubMedDate not supplied

IgG and IgE antibodies in subjects allergic to penicillins recognize different parts of the penicillin molecule.

BACKGROUND: IgE and IgG antibodies to penicillins can have different specificities. However, this response, including the recognition of the different parts of penicillin, has never been studied in the same subject. OBJECTIVE: Study of the specificity of IgE and IgG antibodies and the relevant parts of the penicillin molecule that contribute to the hapten binding site in sera from human. METHODS: Specific IgE antibodies were determined by RAST and specific IgG antibodies by ELISA. The recognition of the different molecules was studied by inhibition studies. RESULTS: Seven sera with IgG and IgE antibodies to amoxicillin and benzyl penicillin were analyzed. IgE antibodies recognized mainly two different epitopes: in one, the side chain was a relevant part of the epitope, in the other, it was the nuclear portion. IgG antibodies recognized the nuclear portion in all instances. In the same subject, antibodies of different isotype and specificity were found. CONCLUSIONS: Subjects who develop simultaneously IgE and IgG antibodies to penicillins show different specificities. This proves that different populations of antibodies recognize different epitopes.

Antibody Specificity↗
PubMedDate not supplied

[Use of PCR in the epidemiological identification of Campylobacter spp].

BACKGROUND: With arbitrary primer PCR technique it is possible to obtain amplification lane patterns easily and with good reproducibility from the genomic DNA of bacteria studied. There is also no need for prior information regarding the DNA sequence. METHOD: This method implies two cycles of amplification with low stringency, followed by a PCR of high stringency. RESULTS: Using the above mentioned technique, we were able to show that Campylobacter spp from clinical samples could be separated as well as different strains from the same species. CONCLUSIONS: According to our results as well as the ones from different authors applied to other microorganisms, we can assume that the method could be used in any bacterial species for epidemiologic studies purposes.

Base Sequence↗
PubMedDate not supplied

T cell assessment in allergic drug reactions during the acute phase according to the time of occurrence.

Allergic drug reactions can be classified as immediate, accelerated or delayed. This classification usually correlates with the mechanism involved: immediate reactions are IgE mediated and delayed reactions are T cell dependent. We analyzed lymphocyte involvement in patients with these reactions by determining cell subpopulations, activation state and skin homing receptor expression (CLA) in blood and skin. Patients with immediate, accelerated and delayed reactions were evaluated during the acute phase and after resolution. Controls taking drugs were included. Phenotypic immunofluorescence analysis was done by flow cytometry in peripheral blood, and by immunohistochemistry in skin for delayed reactions. Forty-six patients were included, 17 with immediate reactions, 10 accelerated and 19 delayed. At the acute phase CLA was significantly increased in delayed reactions and HLA-DR in all three types of reaction. In the severest delayed reactions, Steven-Johnson/Lyell syndromes, the CD4 subsets were increased in peripheral blood and skin compared to maculopapular exanthemas and urticaria and HLA-DR when compared with urticaria. In maculopapular exanthemas CLA was significantly increased in peripheral blood and skin compared to urticaria and the severe reactions. We found that T-cells are implicated, besides delayed reactions, in immediate and accelerated reactions. In delayed reactions there is a parallelism between results found in skin and peripheral blood with a higher participation of CD4+ cells the more severe the reaction.

Acute-Phase Reaction↗