OIM and related animal models of osteogenesis imperfecta

Osteogenesis imperfecta (OI) is characterized by fragile bones, skeletal deformity, and growth retardation. This heritable disorder of connective tissue is the result of mutations affecting the COL1A1 and COL1A2 genes of type I collagen. Progress in OI research has been limited because of dependence on human fibroblast and osteoblast specimens and the absence of a naturally occurring animal model for this genetic disorder. Recent technology in molecular biology has led to the development of transgenic models of OI based on site directed mutagenesis of type I collagen genes. OIM is a naturally occurring model which incorporates both the phenotypic and biochemical defects of moderate to severe osteogenesis imperfecta. This powerful tool permits the development of models based on different type I collagen mutations. The collagen type I mutation in OIM is a C propeptide deletion which impairs the production of normal pro-alpha2(I). Tissues in OIM contain only [pro-alpha1(I)]3 homotrimer. Thus, although several animal models are now available for research in osteogenesis imperfecta few are viable or fully mimic human disease disorders. OIM duplicates the phenotype and biochemistry of human disease and has a normal life span.     

Evaluation of endothelial cell migration with a novel in vitro assay system

In this study we introduce a novel in vitro 'oil-drop' assay system for the measurement of endothelial cell (EC) migration, based on the original concept of the Teflon fence assay (Pratt et al., 1984; Am. J. Pathol. 117: 349–354). An aliquot of 15–20,000 human umbilical vein EC (HUVEC) is pipetted through a layer of mineral oil. The cells readily attach, spread and migrate on the surface of a matrix-coated tissue culture dish as a confluent circular monolayer. Migration is measured as the net increase in the total area covered at 24 hours. We have used this system to quantify EC migration on matrices composed of a mixture of type I collagen and either von Willebrand factor (vWF) or fibronectin (FN) in the presence or absence of tumor necrosis factor (TNF). Plating efficiency on both vWF/collagen and FN/collagen, measured by counting cells after attachment and spreading, is about 80%. With this method, migration on vWF/collagen was about 6.4 mm² and 5.3 mm² for TNF-treated and untreated HUVEC, respectively. HUVEC migration on FN/collagen was slightly greater – 6.4 mm² and 6.5 mm² with and without TNF treatment, respectively. During the 24 hour time period, HUVEC numbers increased 30–40% on vWF/collagen, and 60–80% on FN/collagen, with increased proliferation observed with TNF- treatment. EC proliferation could be completely inhibited by 2 mM hydroxyurea. This assay system has proven useful in our studies to quantify cell migration and proliferation.     

Clinical,hematologic, and immunologic effects of interleukin-10 in humans

We conducted a double-blind, placebo-controlled study to investigate the safety, pharmacokinetics, and immunological properties of interleukin-10 (IL-10) administration in healthy humans. Volunteers received a single intravenous bolus injection of recombinant human IL-10 (1, 10, or 25g/kg) or placebo. Cytokine production in whole blood and peripheral blood mononuclear cells (PBMC) was assessed before and 3, 6, 24, and 48 hr after the injection. Peak serum concentrations of IL-10 (15±1.1, 208±20.1, and 505±22.3 ng/ml) occurred after 2–5 min for 1, 10, and 25g/kg IL-10, respectively. The terminal-phase half-life was 3.18 hr. A transient leukocytosis (24–63% above baseline) was observed 6 hr after injection, which coincided with a dose-dependent decrease (12–24%) in neutrophil superoxide generation. There was a marked inhibition (60–95%) of endotoxin-induced IL-6 production from whole blood in each group receiving IL-10. Production of IL-8 in endotoxin-stimulated blood was reduced in the 10g/kg group. In PBMC stimulated with phytohemagglutinin and phorbol ester, there was a decrease (72–87%) in interferon- (IFN) production 6 hr after IL-10 with a return to pre-IL-10 levels after 24 hr. This reduction was only partially associated with a decrease in the number of CD2-bearing cells. We conclude that IL-10 administration into humans is without significant side effects, and a single injection reducesex vivo production of IL-6, IL-8, and IFN.     

The effects of stress and caffeine on hypertensives

Eighteen male hypertensives on diuretic medication between the ages of 37 and 60 were studied in a double-blind, randomized, crossover design under three conditions: 200 mg of caffeine and mental arithmetic; placebo and mental arithmetic; and 200 mg of caffeine alone. Systolic and diastolic blood pressure, heart rate, and skin conductance were recorded. During rest, caffeine compared to placebo increased blood pressure by 8/6 mm Hg, but had no effect on heart rate or skin conductance. During mental arithmetic, the combined effect of mental stress and caffeine led to a further increase of 17/7 mm Hg, reaching a pressure level of 163/100 mm Hg. Heart rate and skin conductance were increased above their prior caffeine levels. There were no significant differences between the blood pressure response to mental arithmetic with caffeine and that response to mental arithmetic with a placebo, which may have been due to the fact that the hypertensives were already responding at ceiling level during the mental stressor.     

Epidemiology of group C rotavirus infection in Western New York women of childbearing age.

Umbilical cord serum samples (380), an average of 10 per month for 3 years (1990 to 1992), were tested by indirect immunofluorescence assay for group C rotavirus immunoglobulin G. Thirty percent were positive, suggesting that approximately one-third of women of childbearing age in western New York have experienced group C rotavirus infection.     

The risk of myocardial infarction after quitting smoking in men under 55 years of age

We assessed the effect of quitting cigarette smoking on the incidence of nonfatal myocardial infarction in men under the age of 55 in a case-control study of 1873 men with first episodes of myocardial infarction and 2775 controls. For "current" smokers (men who had smoked in the previous year) as compared with those who had never smoked, the estimated relative risk of myocardial infarction, adjusted for age, was 2.9 (95 per cent confidence interval, 2.4 to 3.4). Among exsmokers (those who had last smoked at least one year previously), the relative-risk estimate declined to a value close to unity for those who had abstained for at least two years; the estimate was 2.0 (1.1 to 3.8) for men who had abstained for 12 to 23 months, and the estimates were about 1.0 for men who had abstained for longer intervals. The results were unchanged by allowance for multiple potential confounding factors. A similar pattern was apparent among exsmokers who had smoked heavily for many years; among those predisposed to a myocardial infarction because of family history, hypertension, or other risk factors; and among those with no apparent predisposition. The results suggest that the risk of myocardial infarction in cigarette smokers decreases within a few years of quitting to a level similar to that in men who have never smoked.     

African trypanosome antigens recognized during the course of infection in N''dama and Zebu cattle.

The humoral immune responses to Trypanosoma brucei infection were examined in N'dama and in Zebu, two breeds of cattle recognized for their differing susceptibility to trypanosomiasis. Regardless of the clinical course, animals of both breeds produced antibodies to nonsurface trypanosome antigen(s) detectable by both immunodiffusion and immune fluorescence. As a new approach to assessment of the humoral response to trypanosome infection, protein antigens responded to were isolated by immune precipitation, and their molecular weights were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This allowed the detection of differences in the immune response which correlated with the clinical course of the disease. All cattle of both breeds which exhibited a capacity to control the disease recognized at least one of three specific antigens: protein of 110,000, 150,000, and 300,000 daltons. The N'dama, which proved less susceptible to the disease, generally responded to more of the three identified trypanosome protein antigens than did the Zebu. Animals which died of trypanosomiasis failed to produce detectable antibodies to any of the three specific proteins, although they sometimes exhibited antibodies to another trypanosome antigen.     

Diastolic Blood Pressure and Heart Rate Biofeedback Training During Orthostatic Stress

Forty male college student volunteers were asked either to increase their diastolic pressure or their heart rate while sitting and during orthostatic stress (going from a sitting to a standing position), and half of them were also given second- to-second visual feedback for the target variable. Systolic blood pressure was also continuously recorded. Comparisons were made between baseline and voluntary control conditions, and test trials were included to examine immediate carry-over effects. With voluntary control instructions, substantial increases in tonic levels were obtained for the three cardiovascular variables in both sitting and postural change conditions. In general, the increases were significantly greater for feedback than for no-feedback conditions. Phasic effects of feedback were also observed during postural change conditions: the blood pressure troughs and the heart rate peak occurred earlier with feedback than for instructions only. Immediate transfer effects were obtained in feedback conditions only. The results were discussed in terms of concomitant effects of somatic and cognitive activities. The distinctive feedback effects on the time course of the responses suggest that a precise analysis of response patterns is needed in selecting appropriate feedback methods in the management of orthostatic hypotension.     

Detection of N-myc gene amplification by fluorescence in situ hybridization. Diagnostic utility for neuroblastoma.

We assessed fluorescence in situ hybridization (FISH) as an alternative to Southern blot analysis for determination of N-myc gene amplification in neuroblastoma. In the 44 pediatric solid tumor cell lines examined (20 neuroblastomas), the mean number of N-myc copies determined by FISH correlated closely with Southern blot results. There was wide intercellular variability in gene copy number in tumors that had evidence of amplification; however, tumors judged to be non-amplified completely lacked any cells with high N-myc copy number. FISH provided reliable estimates of N-myc amplification in 12 clinical samples even when the percentage of tumor was low. The other advantages of FISH over Southern blot analysis were speed and technical simplicity, ability to discern heterogeneous gene amplification among tumor cells in the same specimen, and capacity to determine the source of the amplified N-myc signal, whether extrachromosomal double-minute chromosomes, expanded intrachromosomal regions, or chromosome 2 aneuploidy. We conclude that FISH would refine the analysis of N-myc amplification in neuroblastoma and thus improve the assignment of patients to prognostic groups based on this unfavorable risk factor.