DDT induces DNA damage in blood cells. Studies in vitro and in women chronically exposed to this insecticide.

In this study, DDT-induced DNA damage on blood cells was analyzed both in vitro and in vivo. Peripheral blood mononuclear cells (PBMC) were isolated from healthy donors and incubated in the presence of three different concentrations (40, 80, and 100 microg/mL) of p,p'-DDT, p,p'-DDE, and p,p'-DDD at three different treatment times (24, 48, and 72 h). Then, DNA damage was assessed by the single-cell electrophoresis assay (comet assay) as well as by flow cytometry detection of hypodiploid cells (DNA content assay). All compounds induced significant DNA damage as shown by the comet assay. Accordingly, cells exposed to DDT, DDE, and DDD showed a significant increase in the percentage of hypodiploid cells compared with untreated PBMC. In agreement with the in vitro data, a significant correlation between blood levels of DDT, DDD, and DDE and DNA damage (comet assay) was found in women with different amounts of environmental exposure. This association remained significant after controlling for nutritional status, smoking habits, alcohol ingestion, and reported exposure to other pesticides. Although the precise biological importance remains to be explained, our results strongly suggest that DDT and its metabolites are able to induce DNA damage in PBMC both in vitro and in vivo.

DDT induces apoptosis in human mononuclear cells in vitro and is associated with increased apoptosis in exposed children.

The aim of the present work was to investigate whether DDT and its metabolites are able to induce apoptosis of human peripheral blood mononuclear cells (PBMC) both in vitro and in vivo. Cells isolated from healthy individuals were incubated in the presence of increasing concentrations of p'p-DDT, p'p-DDE, or p'p-DDD (0-150 microg/mL) for different intervals. Apoptosis was then determined by flow cytometry (DNA cell content analysis) and fluorescence microscopy (Hoechst staining). A significant level of apoptosis was induced by DDT, DDD, and DDE at 80 microg/mL compared to controls, reaching a maximum effect at 100 microg/mL. We began to detect apoptosis at 12h, with a maximum effect at 24h of incubation. These results were confirmed using the TUNEL assay in cells treated with the three compounds tested as well as with o'p-DDT at 100 microg/mL and 24h of incubation. Our data demonstrate that DDT and its metabolites are able to induce apoptosis of human PBMC in vitro. Therefore, we performed a preliminary study in children exposed to this insecticide. When compared to a control population, the exposed children had higher levels of DDT, DDD, and DDE in blood and also had a higher frequency of apoptosis. In the exposed children, a weak positive association was found between the frequency of apoptosis and the exposure to DDT and DDE. Our results showed that more studies are needed in people exposed to DDT, as apoptosis may cause serious public health effects such as immunosuppression.

Expression of CD64 as a potential marker of neonatal sepsis.

The aim of this study was to identify a novel immunological indicator useful for the early diagnosis (through a rapid and single determination) of neonatal sepsis (NS). Peripheral blood samples were taken from 63 neonates, who were classified into four groups: proven NS (n = 17); clinical NS (n = 14); disease without infection (n = 17); and healthy newborns (n = 15). Neutrophil expression of CD64, CD43, CD44, CD50, CD62L and Mac-1, and plasma levels of interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha (TNF-alpha) and soluble L-selectin (sCD62L), were determined. Expression of CD64 was significantly enhanced in the group with proven sepsis and clinical NS compared to newborns without infection (p < 0.05). Eight newborns with proven or clinical sepsis, but only one with disease without infection, showed an increased percentage of CD64+ cells (diagnostic specificity = 96.8%). No significant differences were found in the expression of the other leucocyte differentiation antigens studied. As previously described, TNF-alpha and IL-6 levels were significantly elevated in newborns with proven or clinical sepsis compared to neonates without infection (p < 0.05). Our results suggest that, through a single determination, the enhanced expression of CD64 is a highly specific indicator of NS, although its diagnostic sensitivity is low (25.8%). In contrast, we found that plasma levels of IL-1beta and sCD62L, as well as the expression of Mac-1, CD43, CD44, CD50, and CD62L, do not appear to be useful for the diagnosis of NS.

Lack of involvement of P-glycoprotein (P-gp) in pemphigus patients with poor response to steroid therapy.

A small but significant fraction of pemphigus patients do not show an adequate response to steroid therapy. P-glycoprotein (or P-gp) is a cell membrane efflux pump that expels drugs, including glucocorticoids, from the cytosol to the extracellular medium. An increased expression and/or function of P-glycoprotein in lymphoid cells could decrease the intracellular concentration of glucocorticoids, diminishing its therapeutic effects. The aim of this work was to assess the expression and activity of P-glycoprotein in the mononuclear cells (MNC) from pemphigus patients with good and poor response to steroid therapy. We studied 20 patients with pemphigus vulgaris, eight of them classified as poor responders and 12 as good responders to steroid therapy. The expression and activity of P-glycoprotein by MNC were quantified by flow cytometry, and P-glycoprotein mRNA levels were determined by a semi-quantitative RT-PCR technique. We found that the expression of P-glycoprotein at both mRNA and protein levels was similar in pemphigus patients with good and poor response to steroid therapy. Similar results were obtained regarding P-glycoprotein activity. P-glycoprotein does not seem to be involved in the poor response to steroid treatment seen in some pemphigus patients. It is important to investigate additional mechanisms that could account for the glucocorticoid resistance seen in some pemphigus patients.

Comparative and prospective study of different immune parameters in healthy subjects at risk for tuberculosis and in tuberculosis patients.

It has not been fully elucidated which of the components of the immune response against Mycobacterium tuberculosis is indicative of resistance or susceptibility. The aim of this study was to identify an immune parameter that could be indicative of either resistance or susceptibility to M. tuberculosis infection. We prospectively studied (three determinations, at months 0, 8, and 12) 15 patients with chronic pulmonary tuberculosis and 42 healthy individuals with a recent and frequent contact with tuberculosis patients. Peripheral blood mononuclear cells were stimulated with a whole-protein extract or the 30-kDa antigen of M. tuberculosis for 6 days, and several immune parameters were determined. No consistent differences between tuberculosis patients and healthy controls were detected in most immune parameters studied, including the expression of different activation antigens, cytokine secretion, lymphocyte proliferation, and nitric oxide production. However, the synthesis of tumor necrosis factor alpha, the intracellular detection of gamma interferon, and the apoptosis of monocytes under certain culture conditions tended to show clear-cut differences in cells from patients and controls (P < 0.05 in all cases for most determinations). Nevertheless, when results were analyzed on an individual basis, it was evident that a significant degree of overlapping of values from patients and controls occurred for all parameters studied. We conclude that although the immune parameters tested do not allow the identification of individuals susceptible to M. tuberculosis, the specificity and sensitivity of some of them could be improved through future studies.

Efecto in vivo del diclofenaco potásico sobre la expresión de selectina-L por leucocitos polimorfonucleares / In vivo effect of diclofenac potasium on the expression of L-selectina by human normal polymorphonuclear leukocytes

La selectina-L (CD62L) juegan un papel importante en la interacción inicial leucocito-endotelio, fenómeno clave en la extravasación de células del torrente sanguíneo hacia focos inflamatorios. Recientemente se ha demostrado que ciertos antiinflamatorios no esteroideos (AINE) inducen in vitro una disminución en la expresión de CD62L e inhiben la interacción de leucocitos polimorfonucleares (PMN) y células endoteliales. En el presente trabajo exploramos in vivo el efecto de diclofenaco potásico sobre expresión de CD62L por PMN. Encontramos que el diclofenaco K, a la dosis que se admistra usualmente, induce una disminución rápida y significativa en la expresión de selectina-L por PMNs de sangre periférica de individuos sanos. Este efecto tendió a desaparecer 24 horas después de la suspensión del AINE. No se observó un incremento significativo en la concentración de CD62L soluble en suero a las 48 horas del inicio de la administración de diclofenaco K. Nuestros datos indican que el efecto in vitro del diclofenaco K sobre la expresión de selectina-L por leucocitos PMN ocurre también in vivo; además, y apoyan fuertemente la hipótesis de que el diclofenaco K ejerce su efecto antiinflamatorio, al menos parcialmente, a través de inducir una disminución en el expresión de CD62L por PMN. Es necesario llevar a cabo mediciones seriadas de CD62L soluble para corroborar si efectivamente la administración de diclofenaco K no afecta los niveles sérico de esta molécula