Role of cell-cell contact in the preservation of differentiation and response to thyrotrophin in cultured porcine thyroid cells

Cultured porcine thyroid cells did not reassociate into functional follicles in the presence of TSH unless the initial seeding density was adequate. At 0.2 X 10(6) cells/35 mm diameter culture dish the cells rapidly formed a monolayer even in the presence of TSH (128 microunits./ml), and radioiodide uptake was not significantly increased compared with that in control cells. Seeding densities of 1-3 X 10(6) cells/dish resulted in cultures which responded to TSH with follicular development and increased radioiodide uptake. A cell-free membrane fraction of thyroid homogenate restored the ability of cultures seeded at low densities to respond to TSH with development of follicular morphology and increased radioiodide uptake. Delaying the addition of TSH by 48 h markedly reduced the stimulation of follicular development and radioiodide uptake of cultures. Addition of membrane fractions, or an alkali-soluble fraction of membranes, at zero time improved the responses to TSH added after a 48-h delay. It was concluded that maintenance of differentiation and of TSH-responsiveness in cultured thyroid cells was influenced by cell-cell contact.     

Smoking and coffee intake following alcohol withdrawal in alcoholic inpatients

The aim of this study was to assess alcoholic inpatients' smoking and coffee intake variation following withdrawal. Only moderate smokers (less than 30 cigarettes/day) showed a significant increase of cigarette consumption after alcohol withdrawal. However, their urinary cotinine level did not vary, suggesting a behavioral, and not biological, compensation through smoking following alcohol withdrawal. Heavy smokers (30 cigarettes/day or more) showed no significant clinical or biological variation of smoking behavior. Coffee consumption increased after alcohol withdrawal in all patients, irrespective of smoking habits.     

Structure-activity studies of potassium channel opening in pinacidil-type cyanoguanidines, nitroethenediamines, thioureas, and ureas.

Potassium channel opening activity for pinacidil-type cyanoguanidines, nitroethenediamines, thioureas, and ureas, has been assessed through simultaneous measurement of spontaneous contractile activity and stimulation of 86Rb+ efflux from rat portal veins loaded with 86Rb+. The good correlation between these two effects suggests that the vasodilator activity of the compounds is directly attributable to an increased opening of potassium channels. The resulting quantitative in vitro data has been used to analyze the structure-activity relationships for potassium channel opening, allowing the biological activity to be rationalized in terms of a pharmacophore involving a hydrogen-bond-acceptor element, a hydrogen-bond-donor element, and a lipophilic binding group. A model for the binding of pinacidil-related compounds to their potassium channel receptor has been developed, and compounds designed to test this model have been synthesized and tested. Prototropic equilibria are implicated as playing a fundamental role in determining the hydrogen-bonding ability of the compounds, and conformational changes in the receptor are invoked to explain disparities in the chiral recognition of lipophilic groups in different compounds.     

Validation in the sheep of an ultrasound velocity dilution technique for haemodialysis graft flow

BACKGROUND: A simple, rapid, inexpensive method for measuring the flow in a patient's vascular access would permit routine monitoring during haemodialysis, and hence provide information of access graft deterioration sufficiently early to increase the success of minimally invasive remedial procedures. This paper reports the validation of such a method in animals. METHODS: A PTFE graft was implanted in sheep between the carotid artery and the jugular vein. While the sheep was under general anaesthesia and on an haemodialysis circuit, ultrasound velocity in its blood was perturbed by the injection of a 5-10 ml bolus of isotonic NaCl. The pump tubing flow was measured by a transit-time blood flow meter. This flow was combined with the areas of perturbation generated by the injection before and after mixing in the access flow to estimate graft flow. The calculated graft flow was compared to flow measured directly by a transit-time probe on the same carotid artery. RESULTS: Over a 10-fold range, 120-1260 ml/min, graft flow measured by ultrasound velocity dilution agreed well with graft flow measured directly with a scatter of 76 ml/min about the regression line. CONCLUSION: Ultrasound velocity dilution provides a method for measuring flow in the graft accurate enough for clinical evaluation of patients on dialysis.      

Intraosseous infusions by prehospital personnel in critically ill pediatric patients

A program to instruct ground and aeromedical prehospital emergency medical system providers in the intraosseous infusion technique was developed and implemented. Paramedics and flight nurses received training through lectures and performance of the procedure in several animal models. The records of attempts on 15 patients who subsequently received intraosseous infusions were then reviewed. An intraosseous infusion was successful in 12 of 15 attempts (80%), and all needles were placed in less than 30 seconds. Drugs administered included phenobarbitol, phenytoin, atropine, epinephrine, sodium bicarbonate, isoproterenol, and pancuronium. Observed complications were limited to minimal subcutaneous infiltration in three cases and slow infusion in another. No serious sequelae were noted, but most patients did not survive and the ability of this study to detect sequelae may be limited. These data suggest that intraosseous infusion is a safe and reliable technique in the prehospital setting. Research is needed to study this technique in more detail.     

Combination of ABO blood group incompatibility and glucose-6-phosphate dehydrogenase deficiency: effect on hemolysis and neonatal hyperbilirubinemia

The incidence (%) of hyperbilirubinemia (serum bilirubin ≥257 μmol/l) was similar in neonates with a combination of ABO incompatibility and glucose-6-phosphate dehydrogenase (G-6-PD) deficiency (45%), with ABO incompatibility (54%) or G-6-PD deficiency (37%), alone (ns). Carboxyhemoglobin values, corrected for inspired CO, were similarly elevated in all three groups (0.87 ± 0.32%, 0.82 ± 0.29%, 0.76 ± 0.18%, respectively, ns), but correlated with bilirubin only in those with ABO incompatibility alone. ABO-incompatible/G-6-PD-deficient neonates, compared with those with either condition alone, are not at increased risk for hemolysis or hyperbilirubinemia.     

Characterization of HLA DR3/DQ2 transgenic mice: a potential humanized animal model for autoimmune disease studies

Linkage studies indicate close associations of certain HLA alleles with autoimmune diseases. To better understand how specific HLA alleles are related to disease pathogenesis, we have generated an HLA DR3/DQ2 transgenic mouse utilizing a 550-kb yeast artificial chromosome (YAC) construct containing the complete DRalpha, DRbeta1, DRbeta3, DQalpha, and DQbeta regions. The transgenic mouse (4D1/C2D) in an I-Abeta(o) background appears healthy with no signs of autoimmune diseases. Lymphoid tissues as well as CD4(+) T cells develop normally. Characterization of the transgene expression demonstrates that approximately 90% of B cells express high levels of DR3 and 50-70% of B cells express DQ2. CD11c(+) dendritic cells express high levels of DR and DQ. Approximately 12-18% of resting T cells are positive for DR expression, and further up-regulation to 40-50% expression is seen upon activation with anti-CD3/anti-CD28 mAb. These results suggest that the transgenic construct confers a high fidelity to the normal human temporal and spatial expression profile. Analysis of T cell receptor repertoire in transgenic mice confirms that DR3/DQ2 are able to mediate thymic selection. Furthermore, transgenic mice respond to a DR3-restricted antigen, demonstrating antigen processing and presentation by antigen-presenting cells (APC). Purified T cells from ovalbumin (OVA)-immunized 4D1 mice respond to human APC co-cultured with OVA, suggesting appropriate antigen/DR3 or DQ2 recognition by murine T cells. Immunoglobulin isotype switching is also observed, indicating functional T-B cognate interactions. Thus, the DR3/DQ2 transgenic mouse has normal lymphoid development and functionality that are mediated by HLA transgenes and can be used to investigate HLA-associated immunological questions.     

New insights into water transport and edema in the central nervous system from phenotype analysis of aquaporin-4 null mice

Aquaporin-4 (AQP4) is the major water channel in the CNS. Its expression at fluid-tissue barriers (blood-brain and brain-cerebrospinal fluid barriers) throughout the brain and spinal cord suggests a role in water transport under normal and pathological conditions. Phenotype studies of transgenic mice lacking AQP4 have provided evidence for a role of AQP4 in cerebral water balance and neural signal transduction. Primary cultures of astrocytes from AQP4-null mice have greatly reduced osmotic water permeability compared with wild-type astrocytes, indicating that AQP4 is the principal water channel in these cells. AQP4-null mice have reduced brain swelling and improved neurological outcome following water intoxication and focal cerebral ischemia, establishing a role of AQP4 in the development of cytotoxic (cellular) cerebral edema. In contrast, brain swelling and clinical outcome are worse in AQP4-null mice in models of vasogenic (fluid leak) edema caused by freeze-injury and brain tumor, probably due to impaired AQP4-dependent brain water clearance. AQP4-null mice also have markedly reduced acoustic brainstem response potentials and significantly increased seizure threshold in response to chemical convulsants, implicating AQP4 in modulation of neural signal transduction. Pharmacological modulation of AQP4 function may thus provide a novel therapeutic strategy for the treatment of stroke, tumor-associated edema, epilepsy, traumatic brain injury, and other disorders of the CNS associated with altered brain water balance.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.