Elevated frequencies of 6-thioguanine-resistant lymphocytes in multiple sclerosis patients treated with cyclophosphamide: a prospective study

An autoradiographic assay for 6-thioguanine-resistant (TGr) lymphocytes was used to determine the frequency of in vivo derived variant T lymphocytes in peripheral blood from multiple sclerosis (MS) patients treated with monthly intravenous infusions of 750 mg/m2 of cyclophosphamide (CP). To analyze the time-course of response to CP, the MS patients were studied prospectively. Samples were obtained from the patients before the beginning of CP therapy, 4-5 times during the course of treatment, and, finally, 2 or 3 months after the completion of therapy. 2 weeks after the first CP infusion, the variant frequencies (Vfs) of the MS patients were significantly increased (p less than 0.05) above their pre-treatment values, but by 4 weeks following the first CP infusion the Vfs had fallen to normal or near-normal levels. After subsequent treatments, the frequencies of variant TGr cells were again higher than pre-treatment Vfs. However, within 7-13 weeks after the cessation of CP therapy, the Vfs of all subjects had returned to normal levels. The transient nature of the response indicates rapid in vivo selection against CP-induced TGr mutant cells. The mean pre-treatment Vf of the 4 MS patients who were cigarette smokers was 6.56 X 10(-6) which was significantly greater (p less than 0.05) than the mean Vf (1.52 X 10(-6) of the 4 MS patients who were non-smokers. The mean Vf from 8 assays of healthy non-smokers was 1.92 X 10(-6).     

An inhibitor of arachidonic acid metabolism stimulates luteinizing hormone (LH) release from cultured pituitary cells

5, 8, 11, 14 eicosatetraynoic acid ("ETYA", Roche 3-1428) is a competitive inhibitor of arachidonic acid metabolism. It effectively inhibits the action of both the lipoxygenases and the fatty acid cyclooxygenases both of which utilize arachidonic acid as a substrate. In the present work, we have shown that ETYA stimulates luteinizing hormone (LH) release from cultured pituitary cells (ED50 = 10 micrograms/ml). Stimulation is not due to contaminants present in the preparation, since highly purified ETYA (characterized by GC-MS) stimulates release, while contaminants removed by silicic acid chromatography do not. In addition, neither oxidized solutions of ETYA nor arachidonic acid itself stimulate LH release. ETYA stimulated release is dose dependent and is inhibited by ions which antagonize Ca2+ action. The observation that neither indomethecin (10, 100 micrograms/ml) nor meclofenamate (1.0, 10 micrograms/ml) stimulate LH release suggests that the effect of ETYA cannot be explained by an action on cyclooxygenase. The action of ETYA may be mediated either via an effect on lipoxygenase or through some nonspecific action (such as altered membrane fluidity).     

MAP2 IHC detection: a marker of antigenicity in CNS tissues

Immunohistochemistry (IHC) is used to detect antibody-specific antigens in tissues; the results depend on the ability of the primary antibodies to bind to their antigens. Therefore, results depend on the quality of preservation of the specimen. Many investigators have overcome the deleterious effects of over-fixation on the binding of primary antibodies to specimen antigens using IHC, but if the specimen is under-fixed or fixation is delayed, false negative results could be obtained despite certified laboratory practices. Microtubule-associated protein 2 (MAP2) is an abundant microtubule-associate protein that participates in the outgrowth of neuronal processes and synaptic plasticity; it is localized primarily in cell bodies and dendrites of neurons. MAP2 immunolabeling has been reported to be absent in areas of the entorhinal cortex and hippocampus of Alzheimer’s disease brains that were co-localized with the dense-core type of amyloid plaques. It was hypothesized that the lack of MAP2 immunolabeling in these structures was due to the degradation of the MAP2 antigen by the neuronal proteases that were released as the neurons lysed leading to the formation of these plaques. Because MAP2 is sensitive to proteolysis, we hypothesized that changes in MAP2 immunolabeling may be correlated with the degree of fixation of central nervous system (CNS) tissues. We detected normal MAP2 immunolabeling in fixed rat brain tissues, but MAP2 immunolabeling was decreased or lost in unfixed and delayed-fixed rat brain tissues. By contrast, two ubiquitous CNS-specific markers, myelin basic protein and glial fibrillary acidic protein, were unaffected by the degree of fixation in the same tissues. Our observations suggest that preservation of various CNS-specific antigens differs with the degree of fixation and that the lack of MAP2 immunolabeling in the rat brain may indicate inadequate tissue fixation. We recommend applying MAP2 IHC for all CNS tissues as a pre-screen to assess the quality of the tissue preservation and to avoid potentially false negative IHC results.     

Bradykinin stimulation of inositol polyphosphate production in porcine aortic endothelial cells

Bradykinin stimulation of inositol polyphosphate production was followed using [3H]inositol-labeled porcine aortic endothelial cells grown in culture. Bradykinin stimulated a significant increase in inositol trisphosphate (IP3) production within 15 s. This increase reached a maximum value of 5-fold above control at 30 s and returned toward baseline by 90 s. Production of inositol bisphosphate increased with time reaching 4-fold by 60 s. Bradykinin stimulated the production of IP3 and inositol biphosphate in a dose-dependent manner with an EC50 of 9 X 10(-9) M. Labeled pools of phosphatidylinositol-4,5-bisphosphate (PIPP) decreased by 50% within 30 s, corresponding to the rise in IP3, while labeled lysophosphatidylinositol pools increased 3-fold by 60 s. Pertussis toxin, a protein which ribosylates GTP-binding proteins, did not inhibit bradykinin-stimulated inositol polyphosphate production. Incubation of labeled cells in the absence of extracellular Ca2+ also did not affect bradykinin-stimulated inositol polyphosphate production. Further, A23187, a Ca2+ ionophore, failed to stimulate PIPP metabolism. Finally, Ca2+ influx into cell monolayers occurred with a time course which paralleled rather than preceded the increase in IP3 levels. These data suggest that bradykinin stimulates phospholipase C metabolism of PIPP to IP3 by a mechanism which does not contain a pertussis toxin sensitive GTP-binding protein. Also, this receptor-linked phospholipase C activity does not appear to be activated by extracellular Ca2+ influx. The results support the proposal that IP3 production initiates Ca2+ mobilization and suggest that the calcium-dependent step in arachidonate release is distal to IP3 production.     

Osteogenesis imperfecta: the audiological phenotype lacks correlation with the genotype

Background

Neurofibromatosis 1 (NF1), a common autosomal dominant disorder, was shown in one study to be associated with a 15-year decrease in life expectancy. However, data on mortality in NF1 are limited. Our aim was to evaluate mortality in a large retrospective cohort of NF1 patients seen in France between 1980 and 2006.

Methods

Consecutive NF1 patients referred to the National French Referral Center for Neurofibromatoses were included. The standardized mortality ratio (SMR) with its 95% confidence interval (CI) was calculated as the ratio of observed over expected numbers of deaths. We studied factors associated with death and causes of death.

Results

Between 1980 and 2006, 1895 NF1 patients were seen. Median follow-up was 6.8 years (range, 0.4-20.6). Vital status was available for 1226 (65%) patients, of whom 1159 (94.5%) survived and 67 (5.5%) died. Overall mortality was significantly increased in the NF1 cohort (SMR, 2.02; CI, 1.6-2.6; P < 10^-4). The excess mortality occurred among patients aged 10 to 20 years (SMR, 5.2; CI, 2.6-9.3; P < 10^-4) and 20 to 40 years (SMR, 4.1; 2.8-5.8; P < 10^-4). Significant excess mortality was found in both males and females. In the 10-20 year age group, females had a significant increase in mortality compared to males (SMR, 12.6; CI, 5.7-23.9; and SMR, 1.8; CI, 0.2-6.4; respectively). The cause of death was available for 58 (86.6%) patients; malignant nerve sheath tumor was the main cause of death (60%).

Conclusions

We found significantly increased SMRs indicating excess mortality in NF1 patients compared to the general population. The definitive diagnosis of NF1 in all patients is a strength of our study, and the high rate of death related to malignant transformation is consistent with previous work. The retrospective design and hospital-based recruitment are limitations of our study. Mortality was significantly increased in NF1 patients aged 10 to 40 years and tended to be higher in females than in males.     

Prostaglandin mediation of potassium effects on renin release

The effects of varying potassium concentrations on renin release from rabbit renal cortical slices were investigated. High potassium concentrations inhibited and low concentrations stimulated renin release, although the magnitude of the effect was greater with reduced concentrations. The fatty acid cyclooxygenase inhibitor Δ^5,8,11,14-eicosatetraynoic acid abolished the stimulatory effects of low potassium, suggesting a prostaglandin dependent effect of potassium on renin release.     

An evaluation of the mouse sperm morphology test and other sperm tests in nonhuman mammals. A report of the U.S. Environmental Protection Agency Gene-Tox Program

The literature on the mouse sperm morphology test and on other sperm tests in nonhuman mammals was reviewed (a) to evaluate the relationship of these tests to chemically induced spermatogenic dysfunction, germ-cell mutagenicity, and carcinogenicity, and (b) to make an interspecies comparison to chemicals. A total of 71 papers were reviewed. The mouse sperm morphology test was used to assess the effects of 154 of the 182 chemical agents covered. 4 other murine sperm tests were also used: the induction of acrosomal abnormalities (4 agents), reduction in sperm counts, (6 agents), motility (5 agents), and F1 sperm morphology (7 agents)). In addition, sperm tests for the spermatogenic effects of 35 agents were done in 9 nonmurine mammalian species; these included analyses for sperm count, motility, and morphology, using a large variety of study designs. For the mouse sperm morphology test, 41 agents were judged by the reviewing committee to be positive inducers of sperm-head shape abnormalities, 103 were negative, and 10 were inconclusive. To evaluate the relationship between changes in sperm morphology and germ cell mutagenicity, the effects of 41 agents on mouse sperm shape were compared to available data from 3 different mammalian germ-cell mutational tests (specific locus, heritable translocation, and dominant lethal). The mouse sperm morphology test was found to be highly sensitive to germ-cell mutagens; 100% of the known mutagens were correctly identified as positives in the sperm morphology test. Data are insufficient at present to access the rate of false positives. Although it is biologically unclear why one might expect changes in sperm morphology to be related to carcinogenesis, we found that (a) a positive response in the mouse sperm morphology test is highly specific for carcinogenic potential (100% for the agents surveyed), and (b) overall, only 50% of carcinogens were positive in the test (i.e., sensitivity approximately equal to 50%). Since many carcinogens do not produce abnormally shaped sperm even at lethal doses, negative findings with the sperm test cannot be used to classify agents as noncarcinogens. We conclude that the mouse sperm morphology test has potential use for identifying chemicals that induce spermatogenic dysfunction and perhaps heritable mutations. Insufficient numbers of chemicals agents have been studied by the other sperm tests to permit similar comparisons. A comparison of 25 chemicals tested with sperm counts, motility, and morphology in at least 2 species (including man, mouse and 9 other mammals) demonstrated good agreement in response among species. With further study, interspecies comparisons of chemically induced sperm changes may be useful for predicting and evaluating human effects.