Mechanism of chemical-induced toxicity. I. Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes

A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death. This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells. In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis. Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds. As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable. The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced. The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS. These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K+ and glutathione concentrations. Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment. These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult.     

Serum zinc levels in sheep with experimental pancreatic abnormalities

Serum zinc levels were measured in sheep before and after total pancreatectomy, pancreatic duct ligation, alloxan treatment, and pancreatic biopsy. Values were elevated after each procedure, but only total pancreatectomy and pancreatic duct ligation caused a significant increase (t = 3.5, p less than 0.05; t = 2.8, p less than 0.05, respectively). These effects were not due to changes in serum proteins. The results are consistent with a model in which the pancreas removes zinc from blood and subsequently eliminates it via the pancreatic duct. Facilitation of intestinal absorption of zinc by pancreatic products and effects of insulin on serum zinc are less significant.     

Risky behavior in teens with cystic fibrosis or sickle cell disease: a multicenter study

OBJECTIVE: To determine the prevalence and age of onset of common risky behaviors such as smoking and sexual activity in teens with cystic fibrosis and those with sickle cell disease and to compare their behaviors with those of adolescents in the general population. DESIGN: Survey. SETTING: All five major pediatric tertiary care centers in North Carolina (study participants with sickle cell disease or cystic fibrosis) and North Carolina public schools (comparison population). PARTICIPANTS: Three hundred twenty-one adolescents with cystic fibrosis or sickle cell disease aged 12 to 19 years (mean age, 15.6 years; 49% female). Demographically matched comparison teens for each group were selected from 2760 in-school adolescents (mean age, 16.0 years; 51% female). MAIN OUTCOMES MEASURES: Prevalence of tobacco and marijuana use, alcohol use, sexual intercourse, sexually transmitted diseases, seat belt use, weapon carrying, and age of onset of these behaviors. RESULTS: Chronically ill teens reported significantly less lifetime and current use of tobacco, marijuana, and alcohol; less sexual intercourse; less weapon carrying, less drunk driving, and more seat belt use than their peers. Nonetheless, 21% of the teens with cystic fibrosis and 30% of those with sickle cell disease had smoked; sexual intercourse was reported by 28% and 51%, respectively. Age of onset of these behaviors was frequently older for the chronically ill teens. CONCLUSION: Teens with cystic fibrosis or sickle cell disease took more potentially damaging health risks than might be expected, although the prevalence was lower than reported by their peers. Future longitudinal studies should examine the relationships between chronic illness, physical and psychosocial maturation, and risky behavior. Screening for psychosocial issues, including risky behaviors, should be incorporated into the routine health care of chronically ill teens.     

Evaluation of factor VIII-rich cryoprecipitate and the plasma fibronectin-rich, heparin-precipitable fraction prepared from single- donor plasma units

A plasma fibronectin-rich component was prepared by heparin-induced 4 degrees C precipitation of fresh or stored (21 days at 4 degrees C), single-donor plasma. The recovery of plasma fibronectin was 45 percent at a concentration of 0.05 mg heparin per ml (7.5 units/ml) and 75 percent at 0.1 mg per ml (15 units/ml). The biologic activity of plasma fibronectin, as assessed by the spreading of Chinese hamster ovary cells or attachment of monocytes to gelatin-coated surfaces, was similar to that of plasma fibronectin concentrates made from fresh or stored plasma. Only 20 to 30 percent of the factor VIII activity in fresh plasma was recovered in cryoprecipitate produced after the heparin-induced precipitate containing fibronectin was removed. Cryoprecipitate prepared from the supernatant plasma that remains after heparin-induced cold precipitation in the presence of CaCl2 (5 mM) contained approximately 50 percent less factor VIII. The relatively low recovery of factor VIII in cryoprecipitate prepared from fibronectin-depleted plasma makes cryoprecipitation an unsuitable method of producing fibronectin-rich and factor VIII-rich components effectively from a single unit of fresh plasma. However, heparin-induced cold precipitation provides an efficient method for preparing plasma fibronectin concentrates from small plasma pools or single units of stored or fresh plasma.     

Low tuberculosis notification in mountainous Vietnam is not due to low case detection: a cross-sectional survey

Background  

Studies show that tuberculosis notification declines with increasing altitude. This can be due to declining incidence or declining case detection. In Vietnam notification rates of new smear-positive tuberculosis in the central mountainous provinces (26/100,000 population) are considerably lower than in Vietnam in general (69/100,000 population). In order to clarify whether this is explained by low incidence or low case detection, we aimed to assess the prevalence of new smear-positive tuberculosis among adults with prolonged cough in three mountainous provinces in central Vietnam.     

Kaposi's sarcoma (KS)-associated herpesvirus-like DNA sequences in peripheral blood mononuclear cells: association with KS and persistence in patients receiving anti-herpesvirus drugs

Herpesvirus-like DNA sequences (KSHV/HHV-8) have recently been described in AIDS-associated Kaposi's sarcoma (KS) lesions. Many questions remain regarding the role of this virus in KS and the therapeutic implications of this finding. In the current study, KSHV/HHV-8 DNA was detected in peripheral blood mononuclear cells (PBMCs) from human immunodeficiency virus (HIV)-infected patients with KS (34/98) more often than in HIV-infected individuals without KS (12/64, P = .03). The detection of KSHV/HHV-8 DNA did not correlate with the CD4 lymphocyte count. Five patients demonstrated KSHV/HHV-8 DNA in their PBMCs during administration of intravenous foscarnet and/or ganciclovir. The continued detection of KSHV/HHV-8 DNA in the PBMCs of patients receiving these anti-herpesvirus drugs has potential implications regarding the virus-cell relationship of KSHV/HHV-8, as well as for the value of these drugs in treating or preventing KS, but additional studies are needed.     

Interleukin-10 is an autocrine growth factor for acquired immunodeficiency syndrome-related B-cell lymphoma [see comments]

Interleukin-10 (IL-10) is an acid-sensitive protein of 35 kD that has pleiotropic effects including inhibition of cytotoxic T-cell response, induction of major histocompatibility complex type II in B lymphocytes, induction of B-cell growth and differentiation, and autocrine growth factor activity in monocytes. We and others have shown that IL-10 is produced spontaneously by blood mononuclear cells from human immunodeficiency virus-seropositive patients. In an attempt to ascertain the potential role of IL-10 in acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma, we evaluated the expression of human IL-10 in both tumor-derived B-cell lines and primary tumor cells. Expression of human IL-10 (hIL-10) mRNA and protein was detected in four of five cell lines examined. An IL-10 antisense oligonucleotide inhibited IL-10 mRNA expression and IL-10 protein production. The proliferation of all B-cell lines was inhibited by an antisense oligonucleotide in a dose-dependent manner that was abrogated by the addition of recombinant hIL-10 protein. No effect of antisense oligonucleotide was observed in the B-cell line not producing hIL-10. Evaluation of primary tumor cells from patients with AIDS-lymphoma cells showed similar production and response to IL-10. These data suggest an autocrine growth mechanism for IL-10 in AIDS-related lymphoma cells and that IL-10 may be important in its pathogenesis.     

Deoxycoformycin-induced immunosuppression in patients with hairy cell leukemia

Immune function in patients with hairy cell leukemia (HCL) was examined serially during treatment with alternating monthly cycles of recombinant interferon alpha-2a and 2'-deoxycoformycin (dCF). At presentation, most patients had normal numbers of T lymphocytes and their cells had normal proliferative responses to mitogens [phytohemagglutinin (PHA) and concanavalin A (Con A)] and alloantigens. Patients had severe monocytopenia, decreased delayed-type hypersensitivity (DTH) reactions, and decreased peripheral blood natural killer (NK) activity. Treatment caused a profound decrease in all lymphocyte subpopulations. T cells were more affected than B cells or NK cells. Numbers of CD4+ and CD8+ lymphocytes decreased to levels less than 200 cells/microliters in all patients during treatment. This decrease in T cell number was associated with a marked decrease in proliferative responsiveness to PHA, Con A, and alloantigens. These abnormalities persisted throughout the 14 months of treatment and have continued for up to 6 months beyond discontinuation of treatment. NK cell activity increased during treatment, but cycled depending on the phase of treatment; highest activities were observed after interferon (IFN)-alpha and lower levels of activity were observed after dCF. DTH responses generally did not improve during therapy. Levels of IgM, IgG, IgA, and IgD did not change during treatment, but IgE levels rose in most patients. All immunosuppressive effects were attributable to dCF since patients receiving IFN-alpha 2a alone did not exhibit these same immunosuppressive effects, and patients receiving dCF alone after IFN failure exhibited similar abnormalities. Despite this severe immunosuppression from dCF, life-threatening opportunistic infections have not been observed in our patient population. Six patients developed localized Herpes zoster infection among 21 patients who had received dCF. Pending the results of long-term follow-up, we recommend that dCF be reserved for patients who have failed splenectomy and IFN therapy.