T cell depletion of human bone marrow. Comparison of Campath-1 plus complement, anti-T cell ricin A chain immunotoxin, and soybean agglutinin alone or in combination with sheep erythrocytes or immunomagnetic beads.

The aim of this study was to compare the extent of in vitro T cell depletion and recovery of hematopoietic progenitor cells achieved with five methods of T cell depletion. Bone marrow samples from the same source were treated with monoclonal antibody Campath-1 (CP1) and human complement, XomaZyme-H65 (anti-T cell ricin A chain immunotoxin), or soybean agglutinin (SBA) alone or in combination with sheep erythrocytes (EAET) or a cocktail of immunomagnetic beads (B) directly coated with anti-CD2, anti-CD3, or anti-CD8 monoclonal antibodies. Residual T cells were enumerated by limiting dilution analysis, EAET rosetting, and proliferative responses to phytohemagglutinin. The results of this study demonstrated the following reductions in BM T cells as detected by limiting dilution analysis (mean % control): SBA+B (99.9%), SBA+EAET (99.8%), CP1+C' (99.4%), anti-T cell ricin A chain immunotoxin (99.0%), and SBA alone (94.2%). Neither PHA response nor enumeration of residual EAET rosettes provided discriminating differences in the degree of T cell depletion by treatment method when T cell reductions exceeded 99.0% by LDA. These results demonstrate the ability of CP1+C', XomaZyme-H65, and SBA plus sheep erythrocyte or magnetic bead depletion to achieve a greater than 99% reduction of BM T cells and the importance of limiting dilution analysis in defining differences in T cell numbers when depletion exceeded 99%.

Optimal conditions for in vitro T cell depletion of human bone marrow by Campath-1a plus complement as demonstrated by limiting dilution analysis.

Quantitations of residual T cells by limiting dilution analysis (LDA), immunofluorescence analysis, sheep red cell rosetting, and proliferative responses to phytohemagglutinin were done to identify treatment conditions that maximized the ex vivo T cell depletion (TCD) of human bone marrow (BM) with the rat monoclonal antibody Campath-1 (CP1) and complement (C'). Different treatment approaches achieved levels of TCD varying from 0.4 to 2.6 log10. However, under optimal treatment conditions, a mean (+/- SEM) log10 TCD of 2.60 +/- 0.12 was demonstrated by LDA. Concentrations of CP1 ranging from 5 micrograms to 300 micrograms/10(7) cells/ml achieved equally effective TCD as determined by LDA. An inverse relationship between the concentration of BM cells/ml and the extent of TCD was observed. Additional C' treatment did not increase TCD as detectable by LDA. Mean recoveries of CFU-GM (day 7), CFU-GM (day 14), CFU-GEMM, and BFU-E growth following CP1 + C' were 51, 43, 42, and 45% respectively. These results demonstrate the importance of cell concentration and treatment conditions for maximizing the depletion of BM T cells with CP1 + C'.

[Inducing high-specificity anti-cortisol antibodies for use in RIA (radioimmunoassay)]. / Inducción de anticuerpos anticortisol con alta especificidad para uso en RIA.

A method for inducing highly specific cortisol antibodies for measuring plasmatic cortisol by RIA is described. The method is fast, direct (without extraction or separation), with high specificity, due to the high quality of the antibody produced in our laboratory. Reliability was demonstrated by: the sensitivity of the method, with a pattern curve of 50 pg of cortisol; the precision tests showed a variation rate intranalysis of < 9.7% and internalysis of < 17.6%; accuracy was > 87%; and specificity was demonstrated by antiserum characterization with other steroids. Measurings of cortisol in plasma of normal subjects could detect the circadian cycle pattern as well as normal supression with dexametasone. These findings support that our method allows fast measurement of plasmatic cortisol with accuracy and sensitivity, reducing operative cost.

Clonable T lymphocytes in T cell-depleted bone marrow transplants correlate with development of graft-v-host disease.

Early clinical trials using T lymphocyte-depleted human marrow for transplantation have reported that such grafts reduce, to varying degrees, both the incidence and the severity of graft-v-host disease (GVHD). However, to date, no clear estimates have been made as to what degree of T cell depletion is necessary to prevent GVHD in every case. To address this problem, we used a limiting dilution assay (LDA) to quantitate residual clonable T lymphocytes in human T cell-depleted bone marrow in 31 HLA-identical transplants for leukemia. The number of phytohemagglutinin -interleukin 2-responsive T lymphocytes determined by LDA and expressed as T cell per kilogram recipient weight was found to correlate with the subsequent development of GVHD: no patients who received less than 1 X 10(5) T cell per kilogram developed GVHD (N = 24). Of the seven patients who received 1 X 10(5) to 4.4 X 10(5) T cell per kilogram, four patients developed grade I or II skin GVHD. This study thus provides a quantitative estimate of the number of T lymphocytes necessary to initiate clinically detectable GVHD in an HLA-identical host.

Contaminación Enterobacteriana del Guarapo de una Fábrica de Cochabamba, en Fermentación Normal y Fermentación Flemosa / Guarapo Enterobacteriana Pollution in the Normal and Fermentation Fermentation Phlegmy of a Factory Cochabamba

El guarapo es una bebida alcohólica que sigue la vía de fermentación y un desvío del mismo resulta en guarapo de consistencia flemosa. La presencia de enterobacterias (entéricas fecales y la E. coli) son indicadores de contaminación y tienen relación con los grupos tifoide-paratifoide. El objetivo de la investigación fue determinar la contaminación enterobacteriana presente en el guarapo en fermentación normal y en fermentación de consistencia flemosa en muestras recolectadas en una fábrica de la provincia Cercado-Cochabamba. Se realizó un estudio de tipo transversal y descriptivo tomando en cuenta 9 muestras de guarapo de consistencia flemosa y una muestra de guarapo en fermentación normal tomada al azar, durante el tiempo de estudio establecido. Se encontró 100% de coliformes fecales en las muestras dos, tres y cinco, existiendo contaminación de gravedad entre 20.000 a 2.640.000 UFC/ml sobrepasando el parámetro normal < 10 UFC/ml, causado posiblemente por el escaso control de calidad y saneamiento.

The cane juice is an alcoholic beverage that follows the route of fermentation and diverted the same results in consistency guarapo phlegmy. The presence of Enterobacteriaceae (enteric coliforms and E. coli) are indicators of pollution and are linked to the typhoid groups - paratyphoid.The research objective was to determine the contamination present in the juice enterobacteriana in normal fermentation and fermentation phlegmy consistency in samples collected from a factory in the province Cercado - Cochabamba.A study of cross-sectional and descriptive sample taking into account 9 phlegmy consistency of juice and a sample of juice in normal fermentation chosen at random during the study period established. We found 100% of fecal coliform in the samples two, three and five, there contamination of gravity between 20,000 to 2,640,000 CFU / ml exceeding the normal parameter < 10 CFU / ml, possibly caused by the low quality control and sanitation.