Regulation of the proliferative potential of cord blood long-term culture-initiating cells (LTC-IC) by different stromal cell lines: implications for LTC-IC measurement

BACKGROUND AND OBJECTIVE: Long-term culture-initiating cells (LTC-IC) are the best available approximation to an in vitro assay of stem cells in humans although they still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord blood (CB) is rich in hemopoietic progenitor cells, as measured by clonogenic assays and contains stem cells capable of reconstituting the marrow after ablation in clinical transplantation. We evaluated the influence of culture conditions on the in vitro behavior of LTC-IC from CB. DESIGN AND METHODS: LTC-IC were evaluated in long-term cultures, comparing two types of murine stromal cell lines: M2-10B4 and M2-10B4 transfected with cDNAs for human G-CSF and IL-3. RESULTS: Two and five fold higher numbers of terminally differentiated cells were produced during nine weeks of culture of CB mononuclear or CD34+ cells respectively, in cultures containing a M2-10B4 IL-3 G-CSF cell line compared to cultures containing the parental cell line. Likewise, a higher number of colony-forming cells (CFC) were detected in the supernatant of cultures with the transfected cell line. In contrast, the number of CFC generated within the stromal layer, after 5 or 9 weeks of culture, was significantly higher in cultures on M2-10B4 cells than those on M2-10B4 IL-3 G-CSF. INTERPRETATION AND CONCLUSIONS: Our results show that the proliferative capacity of CB LTC-IC can be strongly influenced by culture conditions and that the frequency of LTC-IC estimated using these cell lines as stromal support is not identical.     

Two regions in the CD80 cytoplasmic tail regulate CD80 redistribution and T cell costimulation

CD28 is a major T cell costimulatory molecule, delivering signals distinct from those of the CD3/TCR complex, which regulate cytokine and cytokine receptor expression, cell proliferation, and cell viability. CD28 needs to be cross-linked to initiate signals, yet both of its ligands, CD80 and CD86, are expressed as monomers. Previously, we determined the cytoplasmic tail of CD80 is required for CD28-mediated costimulation and subcellular relocalization of CD80 in lymphocytes. In this study, we report that Reh B cell transfectants expressing CD80 with mutations in the cytoplasmic tail region either at 275-278 (RRNE-->AAAA, CD80/4A) or serine 284 (S-->A, CD80/SA) can bind ligand similar to transfectants expressing wild-type CD80, yet are unable to costimulate T cell proliferation. These mutant CD80 molecules are expressed on the surface of the Reh cells in small clusters or foci indistinguishable from those of wild-type CD80 molecules. However, mutant CD80 molecules unlike wild-type CD80 cannot be readily induced by ligand into caps. Thus, small clusters of CD80 found on APC are insufficient to initiate CD28-mediated signals, and the formation of CD80 caps appears to be a critical factor regulating the initiation of T cell costimulation. A 30-kDa phosphoprotein that associates with the cytoplasmic tail of CD80 in activated cells may play a role in CD80 redistribution and thus CD28-mediated costimulation. These results indicate two distinct regions of the CD80 cytoplasmic tail regulate its costimulatory function, and both regions are required for CD80 function.     

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.     

The behavioral development of rats with prenatal hypoinsulinemia

Influence of the prenatal hypoinsulinaemia (streptozocin diabetes) on the behaviour of the offsprings of Wistar rats was studied from 1 to 20 postnatal days. In experimental pups there were slower weight increment, later eye opening, later development of elementary behavioural acts (grooming elements, rearing, sniffing), and later formation of complex behavioural patterns (investigation of an environment) than in the control offsprings of healthy females.     

The sanatorium-health resort treatment of children with thyroid hyperplasia

A favourable effect of sanatorium treatment on the key body functions was found in children with thyroid hyperplasia consequent to environmental pollution due to Chernobyl accident. It is emphasized that balneofactors in the above children should be used in sparing regimens.     

Contrast-medium-induced ventricular fibrillation: arrhythmogenic mechanisms and the role of antiarrhythmic drugs in dogs

RATIONALE AND OBJECTIVES: Small electrolyte additions to a nonionic contrast medium reduce the risk of ventricular fibrillation (VF) during wedged catheter injection of a contrast medium. The current study was designed to further investigate contrast-medium-induced VF by studying the effect of pretreatment with different antiarrhythmic drugs. METHODS: During a simulated wedged catheter situation, iohexol was injected into the anterior descending branch of the left coronary artery in five open-chest, anesthetized dogs pretreated with lidocaine, propranolol, amiodarone, almokalant, or verapamil. RESULTS: Wedging the catheter for 60 sec did not induce VF. However, all 15 wedged catheter injections with iohexol induced VF within 28 sec (19 +/- 1 [mean +/- standard error of the mean]) despite pretreatment with antiarrhythmic drugs. Prior to VF, conduction was slowed and monophasic action potential duration lengthened in the contrast-medium-perfused myocardium, although no significant changes occurred in the control area. CONCLUSION: The combination of catheter wedging and long-lasting contrast medium injection has a high risk of causing VF. Although adding a small amount of electrolytes to nonionic contrast media can reduce the risk of VF, antiarrhythmic drug therapy may not have a protective effect.     

Inhaled nitric oxide is not a myocardial depressant in a porcine model of heart failure

BACKGROUND: Inhaled nitric oxide has been shown to be a potent and selective pulmonary vasodilator. Reports of increases in left ventricular end-diastolic pressure and episodes of pulmonary edema during the clinical use of inhaled nitric oxide in patients with preexisting left ventricular dysfunction have raised concerns that this agent may have myocardial depressant effects. We therefore undertook a study of the effects of inhaled nitric oxide on myocardial contractility in a porcine model of ventricular failure and pulmonary hypertension. METHODS: After inducing heart failure in 10 pigs by rapid ventricular pacing, hemodynamic measurements and pressure-volume diagrams (by the conductance method) were obtained in six animals at baseline and during administration of inhaled nitric oxide at concentrations of 20 and 40 ppm. Myocardial contractile state was assessed by the end-systolic pressure-volume relationship and preload-recruitable stroke work, whereas diastolic function was measured in terms of the end-diastolic pressure-volume relationship and the pressure decay time constant T. RESULTS: Baseline hemodynamics reflected heart failure and pulmonary hypertension, and inhaled nitric oxide induced significant reductions in mean pulmonary artery pressure and pulmonary vascular resistance. Although left ventricular end-diastolic pressure increased during administration of inhaled nitric oxide, no changes were observed in measures of systolic or diastolic function. CONCLUSIONS: Inhaled nitric oxide reduced pulmonary vascular resistance but did not alter myocardial contractility or diastolic function. Increases in left ventricular end-diastolic pressure during inhaled nitric oxide therapy are therefore not due to myocardial depression and may be related to increases in volume delivery to the left side of the heart resulting from reduced pulmonary vascular resistance.