Lipid peroxidation and protein thiol depletion are not involved in the cytotoxicity of N-hydroxy-2-acetylaminofluorene in isolated rat hepatocytes

Freshly isolated rat hepatocytes were used to study the mechanism of cell death induced by N-hydroxy-2-acetylaminofluorene (N-OH-AAF). Exposure to 1.0 mM N-OH-AAF resulted in more than 90% cell death (as measured by LDH leakage) of hepatocytes isolated from male rats within 6 hr. Only 36% of the hepatocytes isolated from female rats died within this period. When inorganic sulfate was omitted from the incubation medium, a 6 hr exposure to 1.0 mM N-OH-AAF resulted in only 40% cell death of male hepatocytes. These findings are in accordance with the sex difference and sulfation dependence of N-OH-AAF hepatotoxicity observed in the rat in vivo. N-OH-AAF decreased glutathione (GSH) in male hepatocytes in a concentration-dependent manner. This GSH consumption was only partly dependent on the presence of inorganic sulfate. No lipid peroxidation was observed during N-OH-AAF exposure; N-OH-AAF even prevented endogenous and diethyl maleate (DEM)-induced lipid peroxidation. No reduction of free protein thiol groups was found after exposure to N-OH-AAF, even after 75% cell death had occurred. A reduction of protein thiols after N-OH-AAF exposure was observed in GSH depleted hepatocytes (obtained by DEM plus vitamin E pretreatment). Under these conditions N-OH-AAF-induced cell death occurred earlier. Therefore, GSH protects against protein thiol depletion by N-OH-AAF in control cells. N-OH-AAF-induced cell death was preceded by a loss of intracellular ATP. It is concluded, therefore, that neither lipid peroxidation nor depletion of protein thiols, but possibly loss of intracellular ATP, is involved in the sulfation-dependent cytotoxic mechanism of N-OH-AAF in isolated rat hepatocytes.     

Ion channel regulation of the dynamical instability of the resting membrane potential in saccular hair cells of the green frog (Rana esculenta)

AIMS: We investigated the ion channel regulation of the resting membrane potential of hair cells with the aim to determine if the resting membrane potential is poised close to instability and thereby a potential cause of the spontaneous afferent spike activity. METHODS: The ionic mechanism and the dynamic properties of the resting membrane potential were examined with the whole-cell patch clamp technique in dissociated saccular hair cells and in a mathematical model including all identified ion channels. RESULTS: In hair cells showing I/V curves with a low membrane conductance flanked by large inward and outward rectifying potassium conductances, the inward rectifier (K(IR)), the delayed outward rectifier (K(V)) and the large conductance, calcium-sensitive, voltage-gated potassium channel (BK(Ca)) were all activated at rest. Under current clamp conditions, the outward current through these channels balanced the inward current through mechano-electrical transduction (MET) and Ca2+ channels. In 45% (22/49) of the cells, the membrane potential fluctuated spontaneously between two voltage levels determined by the voltage extent of the low membrane conductance range. These fluctuations were not influenced by blocking the MET channels but could be reversibly stopped by increasing [K+]o or by blocking of K(IR) channels. Blocking the BK(Ca) channels induced regular voltage oscillations. CONCLUSIONS: Two intrinsic dynamical instabilities of V(m) are present in hair cells. One of these is observed as spontaneous voltage fluctuations by currents through K(IR), K(V) and h-channels in combination with a steady current through MET channels. The other instability shows as regenerative voltage changes involving Ca2+ and K(V) channels. The BK(Ca) channels prevent the spontaneous voltage fluctuations from activating the regenerative system.     

B lymphocyte differentiation in the rat: production and characterization of monoclonal antibodies to B lineage-associated antigens

Three mouse monoclonal antibodies (mAb) directed against rat B lineage antigens were produced. The mAb, designated HIS14 (IgG1), HIS22 (IgM) and HIS24 (IgG2b), were characterized for binding to lymphoid and nonlymphoid tissues by immunoperoxidase staining of frozen sections and by (double-) immunofluorescence staining of single cell suspensions from lymphoid organs. HIS14 recognized a pan B cell determinant: it reacted with virtually all cells of each anatomic B cell compartment and with about 95% of surface (s)Ig+ cells in thoracic duct lymph and in suspensions of spleen and lymph nodes. HIS22 and HIS24 detected B lineage-associated antigens expressed by major subpopulations of B cells. HIS22 predominantly stained the lymphocyte corona, but not (or weakly) the germinal centers and splenic marginal zones, whereas HIS24 reacted with both corona and germinal center and not (or weakly) with marginal zone. In accordance with this, substantial proportions of sIg+ cells in spleen cell suspensions did not express HIS22 or HIS24 determinants (20% and 27%, respectively). In bone marrow the vast majority of cytomplasmic mu+ pre-B cells were HIS14+ and HIS24+, and up to one third also HIS22+, indicating an appearance of the determinants early in B lymphocytopoiesis. The antigens recognized by HIS14, HIS22 and HIS24 are lost during the final stage of B cell differentiation: none of the mAb bound to plasma cells. As far as detectable, neither cells of myeloid and erythroid lineages in bone marrow nor thymocytes were stained by HIS14, HIS22, or HIS24. In suspensions of peripheral lymphoid organs (spleen and lymph nodes) but not in thoracic duct lymph, HIS14 and HIS24 labeled a small proportion (12% and 14%, respectively) of Ig- cells. HIS22 did not bind to Ig- peripheral lymphocytes. Reactivity of HIS14, HIS22 and HIS24 with nonlymphoid tissues was virtually absent; HIS22 stained the high endothelial venules in lymph nodes and Peyer's patches. As determined by immunoblotting, the antigenic determinants on lymph node cells recognized by HIS14, HIS22 and HIS24 were present on molecules with an apparent molecular mass of 205 kDa, 210 (and 175) kDa and 205 kDa, respectively, which is similar to the molecular mass of the B cell form of the rat leukocyte common antigen. In addition, the antigens recognized by HIS14, HIS22 and HIS24 co-capped with the leukocyte common antigen. This suggests that each of the three mAb recognize determinants present on the B cell form of the leukocyte common antigen.     

How can the evaluation of genetic tests be enhanced? Lessons learned from the ACCE framework and evaluating genetic tests in the United Kingdom.

Advances in genetic technology are increasing the availability of genetic tests, not only for rare single gene disorders, but also for common diseases such as breast and colo-rectal cancer. Before there can be widespread uptake of these tests, they must be evaluated to confirm the benefits of their use. But how should genetic tests be evaluated, given the speed at which new tests are emerging? One highly influential approach is the analytic validity, clinical validity, clinical utility and ethical, legal and social issues (ACCE) framework, which has provided a benchmark for the evaluation of genetic tests. The approach has been adopted and adapted by the United Kingdom Genetic Testing Network, with the help of the Public Health Genetics Unit in Cambridge, to evaluate new genetic tests for use in the National Health Service. We discuss a number of conceptual, methodological, and practical issues concerning the evaluation of genetic tests, based on lessons learned from applying the ACCE framework and from the UK experience, and make a number of recommendations to further strengthen the evaluation of genetic tests.     

Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity

Long term B lineage chimeras are used here to study the originof plasma cells in the mouse. Chlmeric mice are constructedby reconstituting lethally irradiated mice with peritoneal cells(PerC) and bone marrow cells from congenic pairs of mice differingIn Igh-C allotype. All conventional B cells in these mice expressthe allotype of the bone marrow donor and nearly all Ly-1 Blineage cells express the allotype of the PerC donor. FACS analysisand immunohistology of these mice shows that virtually all (sig⁺)B cells in peripheral lymphoid organs are derived from the bonemarrow donor. However, despite this overwhelming number of bonemarrow-derived B cells in these animals, immunohistologicalstaining of lymphold organs and gut shows that nearly half ofthe IgM, IgG, and IgA plasma cells derive from the PerC donor.These data demonstrate that the peritoneal cavity contains amajor reservoir of self-replenishing cells that play a significantrole in the mucosal immune response. The possibility that theseare B cells that belong to the Ly-1 B lineage is discussed.     

Cancer risk assessment for health care workers occupationally exposed to cyclophosphamide

In the present study a cancer risk assessment of occupational exposure to cyclophosphamide (CP), a genotoxic carcinogenic antineoplastic agent, was carried out following two approaches based on (1) data from an animal study and (2) data on primary and secondary tumors in CP-treated patients. Data on the urinary excretion of CP in health care workers were used to estimate the uptake of CP, which ranged from 3.6 to 18 g/day. Based on data from an animal study, cancer risks were calculated for a health care worker with a body weight of 70 kg and a working period of 40 years, 200 days a year (linear extrapolation). The lifetime risks (70 years) of urinary bladder cancer in men and leukemias in men and women were found to be nearly the same and ranged from 95 to 600 per million. Based on the patient studies, cancer risks were calculated by multiplication of the 10-year cumulative incidence per gram of CP in patients by the estimated mean total uptake in health care workers over 10 years, 200 days a year. The risk of leukemias in women over 10 years ranged from 17 to 100 per million using the secondary tumor data (linear extrapolation). Comparable results were obtained for the risk of urinary bladder tumors and leukemias in men and women when primary tumor data were used. Thus, on an annual basis, cancer risks obtained from both the animal and the patient study were nearly the same and ranged from about 1.4 to 10 per million. In The Netherlands it is proposed that, for workers, a cancer risk per compound of one extra cancer case per million a year should be striven for (target risk) and that no risk higher than 100 per million a year (prohibitory risk) should be tolerated. From the animal and the patient study it appears that the target risk is exceeded but that the risk is still below the prohibitory risk.     

Bacterial population analysis of human colon and terminal ileum biopsies with 16S rRNA-based fluorescent probes: commensal bacteria live in suspension and have no direct contact with epithelial cells

BACKGROUND: The commensal intestinal microflora has important metabolic and perhaps also immune modulatory functions. Evidence has accumulated that the microflora plays a role in the pathogenesis of inflammatory bowel disease. Therefore, there is a growing interest in the intestinal microflora and its interaction with the host. Presumably, this interaction takes place at the mucus layer. In this study, we investigated the microflora that is present at the mucus layer and addressed the following questions. Does a specific mucus-adherent microflora exist? Is there direct contact between commensal bacteria and epithelial cells? METHODS: Snap-frozen biopsies were taken of 5 colon regions and of the terminal ileum in 9 subjects with a normal colon. Fecal samples were also collected. Bacteria were detected in cryosections with fluorescent in situ hybridization (FISH) with 16S ribosomal (r)RNA-targeted probes for all bacteria and specific probes for the major representatives of anaerobic microflora (bifidobacteria, Bacteroides, clostridia, atopobia) and aerobic microflora (Enterobacteriaceae, enterococci, streptococci, lactobacilli). RESULTS: With this sensitive technique, bacteria were only observed at the luminal side of the intestinal mucus layer. Very few microcolonies were present at the mucus layer, and the composition of the bacterial microflora present in the feces was similar to that at the mucus layer of the terminal ileum and colon regions. CONCLUSIONS: We did not observe direct contact between bacteria and epithelial cells. The equal distribution of bacterial species suggests that intestinal commensal bacteria live in suspension in the lumen and that there is no specific mucus-adherent microflora.     

Paraproteinaemia plus osteolytic lesions in typical hairy-cell leukaemia

Most cases of hairy-cell leukaemia (HCL) involve proliferations of neoplastic B lymphocytes. In rare cases, M-proteins or osteolytic lesions have been documented in patients with HCL. In this study two patients with typical HCL are reported in whom both paraproteinaemia and osteolytic lesions of the femoral neck developed. In one of the patients the production of the M-protein by hairy cells could be established. In the other patient, at autopsy no signs of myeloma were found. The hairy cells from inside the osteolytic lesion had the same immunological phenotype as hairy cells from the peripheral blood, the spleen, and other parts of the bone marrow. These cases once more confirm the B-cell nature of many cases of HCL, and show that hairy cells can have functional capacities usually attributed to much more mature B lymphocytes, i.e. plasma cells.