Colony formation by human haemopoietic precursor cells cultured in semi-solid agar in diffusion chambers

A technique which allows colony growth of haematologically normal human bone marrow cells is described. The cells are supported by semi-solid-agar-medium inside modified Millipore diffusion chambers implanted in the peritoneal cavity of irradiated mice. After 9 days incubation colonies containing up to 1000 cells are found in these Agar Diffusion Chambers. All haematologically normal patients studied so far produced colonies, the majority with between 10 and 40 colonies per 2 X 10(5) bone marrow cells inoculated. This culture system therefore provides a convenient and reliable clonal assay for human bone marrow cells which, in contrast to the agar colony assay in vitro, does not require a source of Colony Stimulating Factor (CSF).     

Increased fetal loss in women with heritable thrombophilia

BACKGROUND: A successful outcome of pregnancy requires an efficient uteroplacental vascular system. Since this system may be compromised by disorders of haemostasis associated with a prothrombotic state, we postulated that maternal thrombophilia might be a risk factor for fetal loss. We studied the relation between heritable thrombophilic defects and fetal loss in a cohort of women with factor V Leiden or deficiency of antithrombin, protein C, or protein S. METHODS: We studied 1384 women enrolled in the European Prospective Cohort on Thrombophilia (EPCOT). Of 843 women with thrombophilia 571 had 1524 pregnancies; of 541 control women 395 had 1019 pregnancies. The controls were partners of male members of the EPCOT cohort or acquaintances of cases. We analysed the frequencies of miscarriage (fetal loss at or before 28 weeks of gestation) and stillbirth (fetal loss after 28 weeks of gestation) jointly and separately. FINDINGS: The risk of fetal loss was increased in women with thrombophilia (168/571 vs 93/395; odds ratio 1.35 [95% Cl 1.01-1.82]). The odds ratio was higher for stillbirth than for miscarriage (3.6 [1.4-9.4] vs 1.27 [0.94-1.71]). The highest odds ratio for stillbirth was in women with combined defects (14.3 [2.4-86.0]) compared with 5.2 (1.5-18.1) in antithrombin deficiency, 2.3 (0.6-8.3) in protein-C deficiency, 3.3 (1.0-11.3) in protein-S deficiency, and 2.0 (0.5-7.7) with factor V Leiden. The corresponding odds ratios for miscarriage in these subgroups were 0.8 (0.2-3.6), 1.7 (1.0-2.8), 1.4 (0.9-2.2), 1.2 (0.7-1.9), and 0.9 (0.5-1.5). Significantly more pregnancy terminations had been done in women with thrombophilia than in controls (odds ratio 2.9 [1.8-4.8]); this discrepancy was apparent in nine of 11 participating centres and for all thrombophilia subgroups. INTERPRETATION: Women with familial thrombophilia, especially those with combined defects or antithrombin deficiency, have an increased risk of fetal loss, particularly stillbirth. Our findings have important implications for therapy and provide a rationale for clinical trials of thromboprophylaxis for affected women with recurrent fetal loss.     

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic beta-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K+ channels (KATP) to insulin secretion via depolarization-driven increases in intracellular Ca2+ ([Ca2+]i). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block KATP. A membrane-permeant analog, methyl pyruvate, however, produced a block of KATP, a sustained rise in [Ca2+]i, and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of KATP responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the beta-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.     

Detection of aneuploidy in human and rodent sperm using FISH and applications of sperm assays of genetic damage in heritable risk evaluation

Efficient molecular methods are being developed for detecting various types of cytogenetic genetic damage in sperm, especially numerical aneuploidy for chromosomes involved in trisomies that survive at birth. These methods provide new approaches for identifying potentially detrimental environmental exposures, genetic predisposition, chromosomal rearrangements, and physiologic factors which may increase a man's risk of fathering a genetically defective offspring. Corollary methods are also being developed for detecting sperm aneuploidy in laboratory rodents and these will be used to make inter-species comparisons of mutagen sensitivities and for investigating mechanisms of induction and persistence of aneuploidy. Validated assays for detecting genetic alterations in human and rodent sperm (of which sperm aneuploidy is a first example) permit comparisons of somatic and germinal response to mutagens within individuals, comparisons of human and rodent germinal sensitivity to mutagens, and can be applied in an extended parallelogram model to sperm for assessing heritable risk resulting from paternal mutagen exposures.     

Treatment of patients with pneumonia, chronic obstructive bronchitis and mucoviscidosis with cefpirome

Twenty two patients with inflammatory respiratory tract infection were treated with cefpirome. Among the patients 14 were with severe pneumonia, 4 with exacerbated obstructive chronic purulent bronchitis and 4 with mucoviscidosis. All the patients were subjected to clinical, laboratory and x-ray examinations, electrocardiography, estimation of the external respiration and sputum bacteriological tests. The cefpirome susceptibility was determined by the agar diffusion assay with standard disks from Roussel Uclaf. Cefpirome was administered by slow intravenous infusion in a daily dose of 2 to 4 g every 12 hours depending on the disease severity. After 2 or 3 days of the patient afebrile temperature and normal differential blood count the therapy was discontinued. The favourable time course of the disease was recorded in 12 out of the 14 patients with pneumonia. Recovery and clinical improvement were stated in 64.3 and 21.4 per cent of the cases respectively. In 2 patients the treatment failed. In all the patients with exacerbated severe chronic purulent bronchitis the cefpirome therapy resulted in the disease remission. The clinical effect of the mucoviscidosis treatment was observed in 3 out of the 4 patients. The drug tolerance in the doses used was good.     

Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin

The genome of tomato pseudo-curly top virus (TPCTV), originating from Florida, has been cloned and sequenced. TPCTV is the only geminivirus identified with a vector specificity which falls outside the Cicadellidae (leafhoppers) and Aleyrodidae (whiteflies). Infectivity of the cloned viral genome was demonstrated by Agrobacterium-mediated inoculation of several host species. Progeny virus was transmissible by the treehopper vector of TPCTV, Micrutalis malleifera (Fowler). The genome of TPCTV shows features typical of both subgroups I and III genera of the family Geminiviridae. The coat protein of TPCTV, although distinct from all previously characterized geminiviruses, exhibits features more akin to the leafhopper-transmitted geminiviruses than those transmissible by the whitefly Bemisia tabaci Genn. The relationship of TPCTV to other geminiviruses, particularly beet curly top virus, is discussed in relation to the possible evolutionary origins of this virus.     

Laparotomy for abdominal sepsis in the critically ill

A culture system designed to support the development of individual preantral mouse ovarian follicles has been employed to study follicle growth in the New World marsupial species Monodelphis domestica. Preantral follicles were isolated mechanically and cultured individually in microdrops under oil. Preliminary results indicate that follicle growth was positively correlated to the concentration of follicle-stimulating hormone (FSH) provided, with 1.0-1.5 IU FSH mL-1 producing the best results. Incubation at the body temperature of M. domestica (33 degrees C) was found to be preferable to that at 37 degrees C. The culture system was able to support follicle growth; however, despite follicles exceeding the size when antrum formation occurs in vivo, they remained preantral.     

Mutagenicity of kaempferol in V79 cells: the role of cytochromes P450

The flavonol kaempferol is widely found in the diet and is directly mutagenic in some short-term tests, such as the induction of chromosomal aberrations in eukaryotic cells. The presence of exogenous metabolizing systems enhances its mutagenicity. We have evaluated the role of cytochromes P450 in the induction of chromosomal aberrations by kaempferol in V79 cells. The results obtained suggest that there is a time-dependent biotransformation of kaempferol to quercetin, by cytochromes P450, as assessed by high pressure liquid chromatography. Quercetin seems to contribute to the mutagenicity of kaempferol in the presence of microsomal metabolizing systems. On the other hand, the direct induction of chromosomal aberrations by kaempferol does not seem to depend on the production of reactive oxygen species.     

Cranial MR imaging in Wilson's disease

OBJECTIVE: The purpose of the study was to describe the range of abnormalities seen on cranial MR images of patients with Wilson's disease and correlate the findings with clinical severity, duration of disease, and duration of neurologic signs and symptoms before treatment. In those patients with serial studies, the changes on MR images were compared with the clinical response. SUBJECTS AND METHODS: Twenty-five patients with Wilson's disease underwent MR imaging of the brain using conventional spin-echo sequences (n = 25), phase maps (n = 8), and partially refocused interleaved multiple-echo sequences (n = 5). RESULTS: MR imaging findings were abnormal in 22 patients and normal in three patients. The basal ganglia were interpreted as abnormal in 19 (86%) of 22 patients, involving the putamen in 19 (86%), the thalami in 12 (54%), the caudate head in 10 (45%), and the globus pallidus in nine (41%). We found a predilection for involvement of the outer rim of the putamen and the ventral nuclear mass of the thalami. The claustrum was abnormal in three patients. The midbrain was abnormal in 17 (77%) of these 22 patients, affecting predominantly the tegmentum but also the substantia nigra, red nuclei, inferior tectum, and crura. The pons was abnormal in 18 (82%) of 22 patients, and the cerebellum was abnormal in 11 patients (50%), with involvement of the superior and middle cerebellar peduncles. Atrophy was present in 18 (82%) of 22 patients, and cortical white matter changes were apparent in 13 (59%) of 22 patients. The scan of one untreated patient revealed shortening of the T1 relaxation time in the thalami, which was consistent with the paramagnetic effects of copper. Phase maps and partially refocused interleaved multiple-echo sequences performed in eight and five patients, respectively, and used to reveal a susceptibility change induced by iron or copper showed normal findings. We found a significant inverse relationship between severity, but not extent, of change in signal intensity and the length of untreated disease (p = .030) and the total duration of disease (p = .015). The study group was too small to show a correlation with clinical findings. Changes seen on MR images matched the clinical response to treatment in only two of the seven patients who underwent follow-up studies. CONCLUSION: MR imaging revealed abnormalities in the basal ganglia, cerebral white matter, midbrain, pons, and cerebellum. The paramagnetic effects of copper were detected only in untreated patients. Patients with a longer duration of disease had less severe changes in signal intensity. MR imaging was of limited value in follow-up.     

New model of ErbB-2 over-expression in human mammary luminal epithelial cells

The ErbB-2 receptor has been strongly implicated in the development of breast cancer. To establish a new model system to investigate the role of erbB-2 in tumorigenesis of the breast, the conditionally immortalised human mammary luminal epithelial cell line HB4a was transfected with erbB-2 cDNA. Biological and biochemical characterisation of the resulting cell lines demonstrated that high levels of ErbB-2 expression were sufficient to cause transformation in vitro but did not cause tumours in vivo. Transformation by overexpression of ErbB-2 correlated with ligand-independent tyrosine phosphorylation of ErbB-2 and the adaptor protein Shc. Over-expression of ErbB-2 also resulted in the ligand-independent constitutive association between Shc and another adaptor protein, Grb2, indicating that receptor activation was sufficient to activate downstream signalling pathways. Using the model described, it was found that elevation of ErbB-2 expression levels caused marked quantitative and qualitative alterations in responses to the ligands epidermal growth factor and heregulin. Data indicate a central role for ErbB-2 in mediating the responses induced by these ligands and suggest that these altered ligand-dependent responses play an important role in tumorigenesis in vivo.