"Spontaneous" FRA16B is a hot spot for sister chromatid exchanges.

Six heterozygous carriers of a fragile site at 16q22 were available for the current study. We demonstrated that the observed fragile site was a "BrdUrd-sensitive" fra(16)(q22) or FRA16B, capable of spontaneous expression in some individuals (= "spontaneous" FRA16B). Significant differences either in spontaneous or in ethylmethane sulfonate (EMS)-induced sister chromatid exchange (SCE) frequencies were found between the fragile 16q22 site, whether expressed or not, and its homologous normal site. These data complement our previous findings on FRAXA and provide additional arguments indicating that fragility and SCE are variable cytogenetic expressions of the same DNA structural alteration.

No significant increase in spontaneous and ethyl methane sulfonate-induced sister chromatid exchanges at the Xq27.3 fragile site.

The present study was performed to determine if the fra(X)(q27.3) site is inherently a high sister chromatid exchange (SCE) site independent of fragility. Therefore, we studied baseline and ethyl methane sulfonate (EMS)-induced SCEs in peripheral blood lymphocytes from 10 retarded fragile-X male patients and eight retarded nonfragile-X male controls. The distributions of SCE scores per chromosome within each experimental condition showed significant interindividual variability in response to EMS treatment in the fragile-X group. Each fragile-X patient was therefore compared with a matched control. No significant differences were found in the distribution of SCE scores per chromosome. In addition, at the Xq27 site, whatever the degree of expressed fragility, no significant differences between fragile-X and control groups were evident in the spontaneous or EMS-induced SCEs. The fra(X)(q27.3) site therefore is not a hot spot for spontaneous or EMS-induced SCEs. Because fluorodeoxyuridine (FUdR) treatment has been shown to induce SCE at this site, our results indicate that the Xq27.3 site must be structurally different from other nonfragile SCE sites.

The probability of dizygosity of phenotypically concordant twins.

A basic element in the determination of the zygosity of a twin pair is the proportion of genotypically concordant pairs among the dizygotic pairs. Two methods to derive this proportion are in common use: the first method requires a laborious enumeration of parental genotypic mating types, and the second method relies on a set of formulas, one for each of the possible combinations of genotypes of two full sibs. In this paper the relation between both methods is uncovered. The set of formulas of the second method is reduced to a single general formula, of which the connection with the ITO method (Li and Sacks 1954) is indicated. By applying both methods in turn to an example concerning the MNS blood group system (Fisher 1951), Fisher's way of performing the calculations according to the first method is unraveled, and the preferability of the second method is made clear. Next, formulas are derived for the probability of genotypic or phenotypic concordance of dizygotic twins when direct information on the genotype or phenotype of one of the parents is available. The case of an X-linked locus is also considered. To facilitate applications, tables are given.

Enhancement of experimental allergic encephalomyelitis in mice by antibodies against IFN-gamma.

Acute experimental allergic encephalomyelitis (EAE) was induced in C57BL/6J and SJL/J mice by injection of isologous spinal cord homogenate given in conjunction with Bordetella pertussis and Freund's adjuvant. SJL/J mice showed a highly aggressive and 100% lethal form of the disease; C57BL/6J mice were much less susceptible as they had low morbidity rates (20 to 40%), low disease scores, and mostly no mortality. Treatment of these low susceptibility mice with neutralizing mAb against IFN-gamma caused an increase in morbidity rates as well as significant mortality (up to 80%). Similar antibody treatment did not affect the course of the disease in the high susceptibility SJL/J mice. However, treatment of these mice with IFN-gamma resulted in reduced morbidity and mortality. A similar but less pronounced inhibition of the disease in SJL/J mice could be obtained by administration of IFN-alpha/beta or by acute infection with lactate dehydrogenase virus. The results indicate that endogenous as well as exogenous IFN can exert a down-regulating effect on the development of EAE. They also indicate that endogenous IFN-gamma is produced during the development of EAE and plays a disease-limiting role.

Non-uniform distribution of sister chromatid exchanges in human lymphocytes.

To test whether sister chromatid exchange (SCE) scores on human chromosomes have a uniform distribution, simulated SCE scores were generated and compared with observed scores using log-linear models. The analysis was performed at the level of the chromosome groups. Using this method we first tested whether the number of SCEs was distributed uniformly, i.e. proportional to the relative length of the chromosomes. Refinements of this hypothesis were made by considering a variable region around a first SCE to be inert for other SCEs and by making the occurrence of an SCE on a chromosome dependent on the occurrence of another SCE on the same chromosome. In further analyses it was tested whether the number of SCEs was proportional to the number of G bands on a chromosome, or to the DNA content of the chromosomes. None of the tested hypotheses fitted the observed data, establishing the non-uniform distribution of these events.

Interferon production by cultured peripheral leucocytes of MS patients.

Peripheral blood leucocytes from multiple sclerosis (MS) patients and from normal individuals were tested for their interferon (IFN) producing capacity after stimulation in vitro with various lectins and viruses. The lectins, Con A, PHA and PWM, induced IFN-gamma. In a kinetic study, the response to Con A revealed itself as an all or none event: the number of responding cultures increased with increasing mitogen dose, but the IFN yield in responding cultures did not differ significantly between dose levels. Thus, any patient or donor could easily be rated as a responder or non-responder. About 1/2 of the MS patients were found to be non-responders if Con A or PHA were used as stimuli. Ninety per cent of the normal donors on the other hand were responders. With PWM as a stimulus 100% of both the MS patients and normal donor groups were found to be responders. Also, with PWM very small doses were sufficient to obtain a 100% response rate among tested cultures, and IFN production persisted for 5 days, while with Con A or PHA it was arrested after 2-3 days. The results indicate that the MS associated lesion is not the absence of functional impairment of all IFN-gamma producing cells, but in only a fraction of them or in an accessory cell population required for the response to Con A and PHA but not to PWM. Newcastle disease virus (NDV) and vesicular stomatitis virus (VSV) both induced IFN-alpha. With NDV as the inducer response rates were 100% and yields were high irrespective of whether the cells were derived from patients or control donors. In contrast, with VSV as the inducer lower response rates were found in cultures from MS patients than in those from controls.

Statistical evaluation of sister chromatid exchanges.

Log-linear models are fitted to sister chromatid exchange (SCE) scores in order to test the significance of the differences in SCE scores observed between individuals or between experimental treatments. The analysis is performed at the level of chromosome groups. In each single test all measurements from all chromosome groups, both from the control and from the experimental sets, are utilized. By proceeding in this way full use is made of all the available information on the SCE scores at the level of chromosome groups and the shortcomings of the classical Student-t and chi-square tests are avoided.

Statistical evaluation of sister chromatid exchanges: refined method.

The statistical problems in evaluating the results of sister chromatid exchange (SCE) scores can be solved by fitting log-linear models to the number of SCE scores (2). In each single test all measurements from all chromosome groups, both from the control and from the experimental sets are utilized. Previously, we analyzed for each chromosome group the number of metaphases with specified total SCE score for the group. We have refined this method to an analysis of the total number of chromosomes with specified SCE score over all metaphases. The results show that the refined method uses more information and can lead to conclusions which are at variance with the conclusions of the first method. By proceeding in this way, better use is made of all available information on the SCE scores and the shortcomings of the classical Student's t-test and chi-square (X2) tests are avoided.

Interferon production and natural killer (NK) activity in leukocyte cultures from multiple sclerosis patients.

Peripheral blood leukocyte (PBL) cultures from only 37% of MS patients produced detectable HuIFN-gamma in response to ConA as opposed to 85% of the cultures derived from normal blood donors. However, the yields in patient-derived cultures that were responsive, were not lower than those in cultures from controls. Production of HuIFN-alpha after stimulation with Sendai virus was not aberrant in cells taken from MS patients. The difference in HuIFN-gamma response rate between MS and normal donor-derived cells was more pronounced when DR2+ carriers were compared amongst each other than when DR2-k carriers were compared. Among the MS patients, the failure of PBLs to produce HuIFN-gamma in response to ConA was not correlated with age, sex, disease duration and type of disease. However, positive correlations were found with current disability indices and past disease progression rates. Unstimulated NK-activities of MS patient-derived PBLs were not different from those of normal donor-derived cells. the degree of augmentation of the activity by stimulation with ConA and interferon-alpha was also normal. Within the MS patients group, but not in the control group, there was a trend for DR2+ carriers to have lower spontaneous and stimulated NK-activities than DR2- individuals.

Changes in some lipid variables in obese women during the early days of fasting.

Adult obese human subjects with a normal or slightly disturbed oral glucose tolerance test, were submitted to a 7-day fast. Initial serum triglyceride levels were inversely related to the Intralipid fractional removal rate, reflecting the close dependency of triglyceridemia on clearance efficiency; the correlation was less significant on the 5th day of fasting. The direction of the change in triglycerides during fasting was clearly related to the prefast triglyceride levels, the groups of patients with a lower level showing an increase, the group with a higher level showing a decrease. Changes in triglyceridemia were inversely related to fractional removal rate; changes in lipid clearing from plasma, however, could not explain the direction of changes in triglyceridemia in all subjects investigated. The group of patients responding to fasting with a decrease in triglyceridemia had a lower mean initial serum cholesterol value and their serum cholesterol levels showed a less prolonged increase during fasting than the other groups of patients.

Beta-blockers: once or three times a day?

In a double-blind, crossover trial 16 hypertensive patients were treated, in random order, with placebo, metoprolol 300 mg in a single daily dose, or metoprolol 300 mg/day in three doses. Both therapeutic regimens produced detectable plasma metoprolol concentrations and appreciable beta-blockade, estimated from exercise tachycardia, throughout the day. Fluctuations throughout the day in plasma drug concentrations and degree of beta-blockade were insignificant on the thrice-daily regimen, but they varied considerably on the single-dose regimen. Both therapeutic regimens also significantly lowered blood pressure throughout the day. Although the thrice-daily regimen again tended to produce a stronger and less fluctuating hypotensive action, the differences in hypotensive effect between the two regimens were not statistically significant. A single-dose of 300 mg of metoprolol can therefore be recommended if the only aim is to reduce blood pressure but not if a steady degree of beta-blockade is needed.

Adipose tissue cellularity in patients with amyotrophic lateral sclerosis.

The subcutaneous mean fat-cell volumes as measured in 20 patients suffering from amyotrophic lateral sclerosis (ALS) were definitely larger than those measured in a control group. In contrast with the control subjects, the mean fat-cell volume in patients with ALS appeared to be independent of body weight. The noted disturbances in carbohydrate-insulin metabolism in patients with ALS and the increase in serum triglycerides as observed in some patients may be related to the enlarged fat cells. The possibility that the enlargement of the fat-cells may, at least partially, have a neurogenic basis cannot be ignored. The hypothesis is put forward that in patients with ALS there is not only a lesion of the anterior horn, but also an involvement of sympathic structures responsible for the innervation of adipose tissue vessels. This involvement may lead to an inhibition of the lipolysis, and consequently to an enlargemnt of the fat cell. Interaction between the interference with metabolic and neurogenic factors may be at play.

Secretion rates of LH and FSH during infusion of LH-FSH/RH in normal women and in patients with secondary amenorrhea: suggestive evidence for two pools of LH and FSH.

Normal women in the early follicular phase and in the luteal phase of the cycle, and patients with secondary amenorrhea received on consecutive days a rapid intravenous injection (50 mug) and a two or four-hour infusion (25 mug/h) of synthetic LH-FSH/RH. The responses of LH and FSH were evaluated by the measured plasma concentrations, as well as by the calculated pituitary secretion rates and by the amounts of hormone released. To estimate these pituitary secretion rates of LH and FSH, a simplified mathematical model is proposed. During an infusion of LH-FSH/RH the secretion rates of both LH and FSH increased in the three groups of women in a biphasic way with a dip after 1 to 2h of infusion, suggesting that besides the pool mobilized by a rapid intravenous injection of LH-FSH/RH there is a second pool of (stored) gonadotropins. For LH the increase above baseline concentrations was higher in group II (luteal phase) than in group I (follicular phase) or in group III (amenorrhea) and this both after a bolus injection and during infusion of LH-FSH/RH. For FSH a similar pattern of response prevailed during an infusion of LH-FSH/RH. After a bolus administration, however, the FSH release was relatively higher in group III (amenorrhea) than in both groups of normal women in which the increases were about the same. The latter finding suggests that the first pool of FSH is released by a different mechanism than the second pool.

Variations in the capacity of the steroid binding beta globulin (SBbetaG) in plasma from normal young twins.

The capacity of human plasma to bind specifically dihydrotestosterone (DHTBC) has been determined in normal monozygotic and dizygotic twins less than 20 years old. The total variation in DHTBC was analysed by stepwise multiple regression, combined with variance analysis, applied to the pair sums in DHTBC, body height and body weight, as well as to age. A significant part of the variation in DHTBC was explained. Due to the strong interwining between body weight, body height and age in growing children it was impossible to make out which one of the latter variable contributed most to the negative correlation which was observed. Previous studies in adults did, however, show that only body weight and not body height or age correlated negatively with DHTBC. By doing the same calculations for the within-pair differences in DHTBC, body height and body weight it was hoped to delineate the within-family part of the environmental variation in DHTBC. Body weight and body height did, however, not offer any significant explanation as to this part of the variation in DHTBC. The upper limit of heritability (h2) for DHTBC was estimated by comparing intraclass-correlation coefficients in monozygotic and dizygotic twins. According to the method used to calculate the latter intraclass-correlation coefficients h2 estimates for DHTBC of 0.774 and 0.960 were arrived at. However, due to the rather small number of dizygotic twins available the confidence limits of these estimates were rather large.