Caracterización funcional de líneas de linfocitos T específicos para la proteína básica de mielina: modulación de la respuesta de citocinas con prostaglandina E2 / Functional characterization of long-term T celllines specific for myelin basic protein: modulation of cytokine response by prostaglandin E2

Presentamos el estudio funcional de 57 líneas de linfocitos T CD4+ específicas para la proteína básica de mielina (MBP) derivadas de la sangre periférica de seis pacientes de EM (tres con EM-RR y tres con EM-SP) y de dos controles sanos. Todas ellas respondieron a la MBP con intensas respuestas proliferativas y variable espectro de citocinas. El análisis de frecuencia de poblaciones funcionales reveló una mayor proporción de células con citocinas intracitoplásmicas en las líneas derivadas de pacientes que en las obtenidas de sujetos normales. En un 60 por ciento de las líneas se observó predominio de células Th1, en un 19 por ciento de Th2 y en un 4 por ciento de Th0. En el resto, no se apreció un patrón de secreción predominante. Las líneas de cada paciente individual tendieron a producir un mismo espectro de citocinas. La adición de prostaglandina E2 (PGE2) provocó un dramático efecto modulador, desviando de manera dosis-dependiente la respuesta funcional hacia un espectro Th2 en las líneas derivadas de pacientes con esclerosis múltiple remitente - recidivante. Las líneas derivadas de enfermos con la forma secundaria progresiva mostraron una mayor resistencia a esta modulación (AU)

Enumeration of CD4(+) T-cells in the peripheral blood of HIV-infected patients: an interlaboratory study of the FACSCount system.

The aim of the present study was to assess the interlaboratory reproducibility of the FACSCount system for the enumeration of peripheral blood (PB) CD4(+) T-cells. In each of the seven participating centers, both previously stained and unstained PB samples (n = 49) were received and either analyzed or stained and then analyzed. Interlaboratory reproducibility was checked in two different groups of centers (n = 3 and n = 4) where the study was performed in parallel. In addition, both the intralaboratory precision and accuracy of this system were analyzed in comparison with results obtained with conventional flow cytometry. Accordingly, upon comparing both methods, a high degree of correlation was observed in the total number of CD3(+) T-cells (coefficient of correlation of 0.9750 +/- 0.0184, slope of the best linear fit: 0. 9214 +/- 0.0311, y-intercept of 12 +/- 47) as well as in the number of CD3(+)/CD4(+) (coefficient of correlation of 0.9794 +/- 0.1457, slope of the best linear fit: 0.9463 +/- 0.0753, y-intercept of -11 +/- 36) and CD3(+)/CD8(+) (coefficient of correlation of 0.9728 +/- 0.0192, slope of the best linear fit: 0.9682 +/- 0.0735, y-intercept of 7 +/- 95) major subsets. In addition, low coefficients of variation (CV) were obtained for replicates, indicating the method's high degree of accuracy. The present study shows that with respect to the interlaboratory reproducibility reported for most techniques used for the enumeration of PB CD4(+) T-cells, the FACSCount system results in data with much lower coefficients of variance (CVs) (mean CV of less than 10%). Upon measuring the impact on results of different variables associated with either sample preparation or data acquisition and analysis, our study clearly shows that data acquisition and analysis does not influence the results by increasing variability since the coefficients of variation obtained for samples prepared in the same laboratory under the same conditions and read in different laboratories with different instruments were identical to those obtained for the replicates of the same samples read in each individual center. In contrast, interlaboratory variability, although low, significantly increased when sample preparation was carried out in different laboratories, suggesting that pipetting still represents the major source of variability in the FACSCount system.

DNA staining changes associated with apoptosis and necrosis in blood lymphocytes of individuals with HIV infection.

We used flow cytometry to quantitate cells that die by apoptosis or necrosis. The method uses low concentrations of two DNA binding dyes that allow one to establish selective regions for live, apoptotic, and necrotic cells in a rat thymocytes model. Quantitative analysis of blood lymphocyte death in individuals with HIV infection by this technique shows the presence of nonviable cells that exhibit a spectrum of changes in staining by DNA binding dyes. These changes range from typical features of cells undergoing programmed cell death or apoptosis to changes observed in cells that die by accidental death or necrosis. The proportion of cells exhibiting these lethal changes increases significantly in patients who progress to AIDS, but, although cells with staining features associated with apoptosis and necrosis were both found to be increased in in vitro-activated cells from AIDS patients, spontaneous in vivo activation preferentially leads to apoptotic changes without a significant increase of cells exhibiting the staining changes associated with necrosis.

Quantification of chicken alpha-fetoprotein: a useful tool in studies of embryo development and pathology.

Chicken alpha-fetoprotein (AFP) from the plasma of 12-day-old chick embryos was purified by electroelution from SDS/PAGE gels, and used to produce polyclonal and monoclonal antibodies. Both reagents were then used to design a sandwich-type enzyme-immunoassay for the quantification of AFP in biological fluids. The assay was used to quantify AFP in the serum and amniotic fluid of chick embryos with abnormalities of the neural tube. Serum AFP was significantly greater in these embryos than in normal ones of similar age. Moreover, substantial amounts of AFP were demonstrated in the amniotic fluid, whereas this protein was undetectable in the amniotic fluid of normal embryos. This method of assay may provide a reliable tool for studies of chick embryogenesis and abnormalities of embryonal differentiation.

Imbalance of the CD4+ subpopulations expressing CD45RA and CD29 antigens in the peripheral blood of adults and children with Down syndrome.

Peripheral blood lymphoid subsets expressing either CD45RA or CD29 antigens, were quantified in 30 children and 59 adults with Down Syndrome and appropriate age-matched controls, by dual immunofluorescence and flow cytometry. Down's patients, both adults and children, displayed a significant decrease of CD4+CD45RA+ cells in comparison with the observed in their age-matched controls, but no difference was found in the CD4+CD29+ subset. These results show clearly the imbalance of these subpopulations in the peripheral blood of individuals with Down syndrome that result in the inversion of the CD45RA/CD29 ratio, due to a major reduction of the CD45RA subset. No obvious difference was found in the CD45RA/CD29 ratio within the CD4 negative cells. Abnormalities of these subpopulations could be indicative of early senescence of the immune system, since age-related changes in Down's persons were in parallel with those observed in normal individuals and the proportion of both subpopulations were roughly similar in Down's children and normal adults.

CD8+CD38+ and CD8+DR+ peripheral blood lymphoid subsets of HIV-infected intravenous drug abusers correlate with CD4+ cell counts and proliferation to mitogens.

Twenty-nine intravenous drug abusers (ivda), with asymptomatic HIV infection at entry, were sequentially studied at 4- to 6-month intervals for variable follow-up periods (mean, 19.6 months). Two of them progressed to AIDS and another one fell into the IV-C2 stage of the CDC classification at the end of the study. CD8+ lymphoid subsets (CD57+, CD38+, and HLA-DR+) were sequentially analyzed in peripheral blood samples along the follow-up. Both absolute number and percentage of cells within these subsets were found significantly increased over those observed in normal controls. Minor changes were appreciated throughout the follow-up. CD8+CD38+ and CD8+DR+ cells increased slightly (P < 0.05), but the CD8+CD57+ subset did not change significantly. In order to determine whether abnormalities in these subsets are associated with immune dysfunction, we looked for correlation between the quantification of CD8+ subpopulations and other parameters of cellular immunity. Percentage of CD8+CD38+ or CD8+DR+ cells inversely correlates with absolute number of CD4+ cells (P < 0.0001), and percentage of CD38+ subset also correlates with the proliferative response to mitogens in lymphoid cultures. Thus, the enumeration of these populations of CD8+ cells may provide some additional information about the immune status of HIV-infected ivda.

Down's syndrome and celiac disease.

The association between Down's syndrome (DS) and autoimmune diseases has long been recognized. However, its relationship to celiac disease (CD) has only recently been reported, and a clear-cut association remains to be fully established. We have studied the prevalence of CD in a random sample of 70 individuals with DS. IgA anti-gliadin antibodies (IgA AGAs) were determined in all and found to be positive in nine (13%). In eight, anti-endomysium antibodies (AEAs) were investigated, and jejunal biopsies were performed. AEAs were positive in two, and three had flat intestinal mucosa. The class I and II human leukocyte antigens of two patients with CD were determined. Results were as follows: A2/B8 B39/DR1 DR3/DQW1 DQW2 in one case and A2 A28/B44 B17/DR4 DR5/DQW3 in the other. This implies a 43% prevalence of CD in DS, which is well above that previously found by us in our population (0.62/1,000 live births). We conclude that the association between DS and CD is not fortuitous and suggest that the determination of such serologic markers as IgA AGA and AEA should be part of health assessment in DS patients.

[Prognostic factors in HIV-infected heroin addicts: a multivariate analysis of nonspecific serological factors in the evolution of the infection]. / Factores pronósticos en heroinómanos infectados por el VIH: análisis multivariable de factores serológicos inespecíficos en la evolución de la infección.

We present a prognostic analysis of the quantifying of serum levels of beta 2 microglobulin, neopterina, IL-2 soluble receptor and three major classes of immunoglobulins, in a group of 68 heroin-addicts infected by the human immune deficiency virus, type I, clinically assessed for a period of at least three years. High levels of any of these unspecific serologic factors were correlated with the illness progression. Survival curves were generated with the categorized variables, showed a significant decrease on the time interval prior to the diagnosis of AIDS, in the patients with these variables assigned on the higher groups, being neopterine and IgA the more predictive factors when the Cox proportional regression model is applied. We conclude that the quantifying of these unspecific serum factors provides a useful information regarding the clinical evolution of heroin-addicts with HIV infection.

Differential expression of lymphocyte function-associated antigen (LFA-1) on peripheral blood leucocytes from individuals with Down's syndrome.

We analysed the expression of lymphocyte function-associated antigen LFA-1 on the cell surface of peripheral blood lymphocytes, monocytes and granulocytes from 20 children with Down's syndrome. No differences in LFA-1 expression was found within monocytes or granulocytes from either normal or Down's syndrome children; however, a clear-cut difference was observed on lymphoid cells. Both normal and Down's syndrome lymphocytes displayed a bimodal pattern of LFA-1 staining by flow cytometry, with a predominance of cells with low expression in normal population, and an increased proportion of lymphocytes with high level of LFA-1 expression in Down's syndrome children. This difference correlates well with the abnormal proportion of T cell subsets and inversion of CD4/CD8 observed in a majority of our cases, and therefore, it could merely reflect the increase of certain T cell subsets normally expressing higher number of LFA-1 molecules. Taken together, our results do not support an abnormally increased expression of leucocytes integrins in trisomy 21 cells, and raise some doubt about the suggested role of the abnormal cellular expression of LFA-1 in the pathogensis of secondary immunodeficiency associated to Down's syndrome.