Detection of reactive oxygen species (ROS) and apoptosis in human fragmented embryos

In human in-vitro fertilization (IVF)-embryo transfer, the in-vitro culture environment differs from in-vivo conditions in that the oxygen concentration is higher, and in such conditions the mouse embryos show a higher concentration of reactive oxygen species (ROS) in simple culture media. ROS are believed to cause damage to cell membranes and DNA fragmentation in somatic cells. This study was conducted to ascertain the level of H2O2 concentration within embryos and the morphological features of cell damage induced by H2O2. A total of 62 human oocytes and embryos (31 fragmented, 15 non-fragmented embryos, 16 unfertilized oocytes) was obtained from the IVF-embryo transfer programme. The relative intensity of H2O2 concentrations within embryos was measured using 2',7'-dichlorodihydrofluorescein diacetate by Quanti cell 500 fluorescence imaging and DNA fragmentation was observed with transmission electron microscopy and an in-situ apoptosis detection kit. The H2O2 concentrations were significantly higher in fragmented embryos (72.21 +/- 9.62, mean +/- SEM) compared to non-fragmented embryos (31.30 +/- 3.50, P < 0.05) and unfertilized oocytes (30.75 +/- 2.67, P < 0.05). Apoptosis was observed only in fragmented embryos, and was absent in non-fragmented embryos. Electron microscopic findings confirmed apoptotic bodies and cytoplasmic condensation in the fragmented blastomeres. We conclude that there is a direct relationship between increased H2O2 concentration and apoptosis, and that further studies should be undertaken to confirm these findings.      

A 17-year-old with rash, fever, myalgias, and nephritis

Patients on hemodialysis are at increased risk for developing active tuberculosis (TB) after primary infection. Although this increased risk is well documented, the prevalence of TB infection, as indicated by a positive tuberculin skin test (TST), is not well described. End-stage renal disease is also known to be a risk factor for skin test anergy, but the rate of anergy in hemodialysis patients is unclear. We sought to identify rates of anergy and TST positivity in patients at four hemodialysis units in St Louis, Missouri, from June 1996 through August 1996. Data obtained from patients and medical records included age, years on hemodialysis, medical history, and basic laboratory data. Patients without a history of TB or a positive TST had a TST with Tubersol, as well as candida and tetanus controls, placed by the Mantoux method. Tests were read 48 hours later. Of the patients enrolled at these units, 307 of 331 (93%) were evaluated. Patients had a mean age of 58 years (range, 19 to 91 years) and had been on hemodialysis for a mean of 3.7 years (range, 1 week to 18.7 years). Blacks made up 81% of the population. A history of a positive TST was obtained from 24 patients (8%), and an additional seven (2%) had a history of active TB. Of the 276 patients tested, 93 did not respond to either control antigen, but five of these patients had a positive TST, leaving 88 (32%) anergic. Anergy was related to age, immunosuppressive drug use, and the reagents used, but not to urea reduction ratio. Positive TSTs were found in 17 of 188 of nonanergic patients (9%) (6% of all tested patients). Overall, 48 of 307 patients (16%) had a positive TST or history of TB. TB or a positive TST was associated with liver disease and peptic ulcer disease, but not socioeconomic status. All 17 newly identified TST-positive patients received chest radiographs. No new cases of active TB were found. Only two of 17 of these patients (12%) were started on isoniazid (INH) prophylaxis. We identified high rates of TST positivity and anergy in the hemodialysis patients tested. Hemodialysis patients should receive regular TST screening, and INH prophylaxis needs to be more strongly encouraged. Studies are ongoing to define the rate of TST conversion over time.     

Racial/Ethnic differences in smoking, drinking, and illicit drug use among American high school seniors, 1976-89.

BACKGROUND. This paper reports racial/ethnic differences in the use of licit and illicit drugs by high school seniors in the United States. METHODS. The study uses questionnaire data from annual, nationally representative surveys of seniors from 1976 through 1989. Combined sample sizes were 57,620 for 1976-79; 75,772 for 1980-84; and 73,527 for 1985-89. RESULTS. Native American had the highest prevalence rates for cigarettes, alcohol, and most illicit drugs; White students had the next highest rates for most drugs. Asian Americans had the lowest prevalence rates, and Black students had levels nearly as low except for marijuana. Prevalence rates for the Hispanic groups were mostly in the intermediate ranges except for relatively high cocaine use among the males. Trend patterns for most forms of drug use were similar across subgroups, although cigarette use declined more sharply for Black than White seniors, resulting in greater Black-White differences in recent years. CONCLUSIONS. This study, other school-based studies, and general population surveys all show relatively low levels of drug use by most non-White youth, especially Black Americans and Asian Americans. Multivariate analyses indicate that such subgroup differences in high school seniors' drug use are not primarily attributable to family composition, parents' education, region, or urban-rural distinctions.     

Human macrophage maturation and heterogeneity: analysis with a newly generated set of monoclonal antibodies to differentiation antigens

We have analyzed the expression of late differentiation antigens during terminal in vitro maturation of human macrophages (M phi) from blood monocytes (MO) in comparison to their distribution among mature M phi residing in various tissue sites. By immunizing mice with M phi derived from blood MO by culture on hydrophobic Teflon foils, monoclonal antibodies (mAbs) were developed (MAX.1, MAX.2, MAX.3, MAX.11) that reacted with lineage-restricted differentiation antigens. These antigens were expressed exclusively on M phi or were markedly increased after in vitro differentiation. The only overlap to another hemopoietic cell lineage was observed with MAX.3, which is shared by platelets and megakaryocytes. In the course of M phi maturation in vitro, the MAX.1 and MAX.3 antigens are detected within the cytoplasm two days before they appear on the cell surface. In contrast, the MAX.11 antigen is expressed simultaneously in the cytoplasm and at the cell surface, is found in varying degrees on a minor portion of blood MO and U937 cells, and is expressed rapidly at high density during early M phi differentiation in vitro. Among conventional mAbs that do not react with MO we found those against the transferrin (TF)-receptor, the BA-2, and the PCA1 antigen to label M phi. M phi matured in vivo and isolated from body fluids were positive with some but not all MAX mAbs. Distinctive patterns were observed with pulmonary M phi, exudate M phi from pleural and peritoneal effusions, synovial fluids, and early lactation milk. M phi from the alveolar space, for example, constantly expressed the MAX.2 antigen but not the MAX.3 antigen. Pleural effusion M phi, however, did not react with the MAX.1 mAb, but in most cases, it did react with the MAX.3 mAb. The detection of novel differentiation antigens, all expressed on monocyte-derived M phi but differently expressed on site-specific M phi in situ, underlines the remarkable heterogeneity among human M phi. The expression of these antigens is flexible because those MAX antigens that were not expressed in situ could be induced if cells from distinct tissue sites were cultured in vitro for several days. MAX mAbs may be of potential value to study both the sequential stages of maturation within the M phi lineage as well as differential developments induced by various culture conditions in parallel to environmental factors in vivo.     

Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.     

内皮下脂蛋白滞留——动脉粥样硬化的始动过程

以动脉粥样硬化为基础的心血管疾病是人类健康面临的严重挑战.人们对动脉粥样硬化发生、发展进行漫长和不懈的探索.至今,其机制与过程仍不十分清楚.