Relating specific connexin co-expression ratio to connexon composition and gap junction function.

Cardiac connexin 43 (Cx43), Cx40 and Cx45 are co-expressed at distinct ratios in myocytes. This pattern is considered a key factor in regulating the gap junction channels composition, properties and function and remains poorly understood. This work aims to correlate gap junction function with the connexin composition of the channels at accurate ratios Cx43:Cx40 and Cx43:Cx45. Rat liver epithelial cells that endogenously express Cx43 were stably transfected to induce expression of accurate levels of Cx40 or Cx45 that may be present in various areas of the heart (e.g. atria and ventricular conduction system). Induction of Cx40 does not increase the amounts of junctional connexins (Cx43 and Cx40), whereas induction of Cx45 increases the amounts of junctional connexins (Cx43 and Cx45). Interestingly, the non-junctional fraction of Cx43 remains unaffected upon induction of Cx40 and Cx45. Co-immunoprecipitation studies show low level of Cx40/Cx43 heteromerisation and undetectable Cx45/Cx43 heteromerisation. Functional characterisation shows that induction of Cx40 and Cx45 decreases Lucifer Yellow transfer. Electrical coupling is decreased by Cx45 induction, whereas it is decreased at low induction of Cx40 and increased at high induction. These data indicate a fine regulation of the gap junction channel make-up in function of the type and the ratio of co-expressed Cxs that specifically regulates chemical and electrical coupling. This reflects specific gap junction function in regulating impulse propagation in the healthy heart, and a pro-arrhythmic potential of connexin remodelling in the diseased heart.

Sex differences in expression and subcellular localization of heart rhythm determinant proteins.

To evaluate sex differences in protein expression in the heart, we performed Western blot studies on a subset of Heart Rhythm Determinant (HRD) proteins. We examined key components of a variety of types of mechanical and electrical junctions including, connexin43, plakophilin-2, N-cadherin and plakoglobin, ankyrin-2 and actin. We describe novel findings in sex differences in cardiac protein expression and membrane localization. For most proteins examined, sex differences were significantly more pronounced in the membrane compartment than in overall expression. These studies extend our previous findings in microarray studies to demonstrate that sex differences in gene expression are likely to confer distinct functional properties on male and female myocardium.

Failure of surgical decompression for a presumed case of piriformis syndrome. Case report.

Diagnosis of piriformis syndrome is difficult and its precise definition is highly controversial. In this article, the authors present the case of a patient who had clinical features suggestive of piriformis syndrome. During surgery the patient was found to have a rare variation in anatomical structures, in which the peroneal nerve was displaced by the piriformis muscle. Surgical decompression did not alleviate the patient's symptoms.

Who does best on CAPD? A study to identify self-care determinants.

Individuals with end-stage renal failure (ESRF) may be offered two main types of renal replacement therapy--haemodialysis or continuous ambulatory peritoneal dialysis (CAPD). At present approximately 50% of individuals within the United Kingdom who require dialysis are maintained on CAPD whilst the remainder receive haemodialysis (1).

Anti-islet autoantibodies detected by monoclonal antibody 1A2: further studies suggesting a role in the pathogenesis of IDDM.

Insulin-dependent diabetes mellitus (IDDM) is associated with autoantibodies to several pancreatic islet antigens. We have described an assay in which autoantibodies displace a radiolabelled monoclonal anti-islet antibody. Sera from 87% of 429 children at time of diagnosis of IDDM were positive, while sera from control groups had much lower prevalences (1.3-19%). Sera from 41.9% of diabetic subjects remained positive after 20 years duration of IDDM. Sera from 23.6% of parents and 37.9% of non-diabetic siblings were positive. Twenty relatives who subsequently developed IDDM had the same prevalence of the antibodies (85%) as did the patients at time of diagnosis. These findings confirm that the autoantibodies detected by monoclonal antibody (mAb) 1A2 are common at the onset of IDDM and their presence prior to the onset of hyperglycaemia suggests that this method may be useful in screening non-diabetic populations. The high prevalence of antibodies in relatives reduces the efficacy for diabetes prediction, but suggests either that generation of these antibodies is an autosomal dominant trait, or that the antigen detected by these antibodies is cross-reactive with a common environmental antigen. Differentiation between these hypotheses will await the identification of the specific islet-cell antigen detected by mAb 1A2.

Effects of the pure anti-oestrogen ICI 182780 on oestrogen receptors, progesterone receptors and Ki67 antigen in human endometrium in vivo.

The new steroidal pure anti-oestrogen ICI 182780 was studied for the first time in pre-menopausal women. A total of 30 patients requiring hysterectomy for benign gynaecological disease were randomized to ICI 182780, 12 mg/day i.m. (n = 19) or no treatment (n = 11) for 7 days prior to surgery. Immunohistochemical measurements were made in the snap-frozen, resected endometrium for oestrogen receptors (ER), progesterone receptors (PgR) and Ki67, a nuclear antigen whose expression is closely related to proliferation. Five control patients ovulated prior to surgery and, as expected, the secretory endometria had lower Ki67 antigen concentrations than endometria had lower proliferative phase. The endometria from patients treated with ICI 182780 had reduced Ki67 compared with controls. This demonstration of reduced proliferative activity indicates that the pharmacological effectiveness of the treatment was maintained despite increased plasma oestradiol concentrations. In contrast to results from rodents, ICI 182780 did not markedly reduce ER expression, although there was significantly lower ER in the myometrial cells of the treated group. The lack of effect on PgR shows a dissociation between the drug's effect on this oestrogen-dependent protein and its effects on proliferation.

The effects of ICI 182,780, a pure anti-oestrogen, on the hypothalamic-pituitary-gonadal axis and on endometrial proliferation in pre-menopausal women.

ICI 182,780 has shown pure oestrogen antagonism in vitro and in vivo in animals. A total of 17 women with normal menstrual cycles were administered ICI 182,780, 12 mg daily for 7 days in the follicular phase prior to hysterectomy; 11 normal women were used as controls. Of the 17 patients, three (18%) experienced a luteinizing hormone (LH) surge in the treatment group compared with five (45%) in the controls (P = 0.24), and these patients were only included up to the surge. There were no differences in the daily mean plasma LH and follicle stimulating hormone concentrations between the treatment (n = 17) and control (n = 10) groups. The mean plasma oestradiol was higher in the treatment group than controls (P < 0.05) on days 5, 6 and 7. However, there was no increase in endometrial thickness in the treatment group throughout the study. In the control group, endometrial thickness increased during the study and was significantly higher (P < 0.05) on day 7. There was no ultrasonic evidence of ovarian hyperstimulation and no serious adverse events reported. This study shows that treatment for 7 days with ICI 182,780 does not cause ovarian hyperstimulation and has a potent anti-oestrogenic action on the endometrium. We conclude that ICI 182,780 may be a useful compound in the treatment of oestrogen-dependent gynaecological disease.

Interleukin-6 and interleukin-8 production by mononuclear cells of chronic alcoholics during treatment.

Chronic alcohol consumption has been associated with suppression of a number of immune parameters. This study was designed to investigate the relationship between chronic alcohol ingestion and cessation with respect to release of interleukin-6 (IL-6) and interleukin-8 (IL-8) using highly specific and sensitive ELISA assays, as well as a functional assay, natural killer cell cytotoxic activity. ELISAs were developed to determine the amount of IL-6 and IL-8 release by peripheral blood mononuclear cells (PBMCs). Two groups of subjects were recruited: young (18-22 years old), nonalcoholic users (controls) and long-term alcoholics (35-55 years old). Blood samples were collected at time 0 from all subjects and from alcoholics 28 days after treatment had begun and alcohol use had ceased. Then mitogen-stimulated release of cytokines by peripheral blood cells was determined. The abstaining controls, and the alcoholics, after 30 days of abstinence, tended to produce lower amounts of IL-6 and IL-8, although these differences were not statistically significant. Natural killer cell activity was not statistically different between the young groups, yet appeared to increase once alcohol use discontinued. Some of the cells from the controls (abstainers) were incubated with ethanol (EtOH). Its content in sealed wells was measured after the time of incubation of PBMCs. When EtOH was serially diluted in plates, some well-well diffusion was noted, but the maximum concentration of EtOH never fell below 0.3% from an initial concentration of 0.5%, and at no time was the EtOH concentration gradient completely lost, even after 66 hr of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

The combination of a depot gonadotrophin releasing hormone agonist and cyclical hormone replacement therapy for dysfunctional uterine bleeding.

OBJECTIVE: To observe if a combination of a depot GnRH agonist and cyclical hormone replacement therapy decreases menstrual blood loss. DESIGN: An open, observational study comparing the objective assessment of menstrual blood loss before, during and after 3 months treatment. SUBJECTS: 20 women with a subjective complaint of heavy menstrual loss in whom no cause could be discovered. INTERVENTIONS: Each woman received 3 months of depot goserelin (Zoladex) combined with cyclical hormone replacement therapy (Cyclo-Progynova, 1 mg). Menstrual loss and symptoms were assessed before, throughout and after the study. MAIN OUTCOME MEASURES: Changes in objective and subjective assessments of menstrual blood loss and the acceptability of the treatment. RESULTS: The median pretreatment menstrual loss was 68 ml (range 23-397). Only 8 (40%) of the patients had a loss exceeding 80 ml per period. The median blood loss was 30 ml, 16 ml, and 17 ml in the three treatment cycles (P less than 0.001 Wilcoxon rank sum for the third cycle). There was a significant decrease in the median length of menstruation (P less than 0.001) and the number of towels or tampons (P less than 0.01) used per period in the third treatment cycle. There was a significant decrease (P less than 0.005) in the number of women complaining of dysmenorrhoea, premenstrual symptoms, flooding and the passage of clots. Seventeen patients experienced hot flushes. Eighteen of the 20 patients were completely satisfied with the treatment and would have been happy to continue with it for longer than 12 months. CONCLUSIONS: The combination of a depot gonadotrophin releasing hormone agonist and cyclical hormone replacement therapy is a successful and acceptable treatment of dysfunctional uterine bleeding.

Strong association between diabetes and displacement of mouse anti-rat insulinoma cell monoclonal antibody by human serum in vitro.

In an attempt to identify novel pancreatic beta-cell surface antigens, mouse monoclonal antibodies (MoAbs) were raised against rat insulinoma (RIN5F) cells with standard techniques. Several clones were identified whose antibodies bound specifically to RIN5F cells but not to other rat, mouse, and human target cells. Each of these MoAbs was radiolabeled, and the specificity of binding of each MoAb was determined by the ability of excess cold homologous MoAb to displace the labeled MoAb. Six RIN5F cell-specific MoAbs of different epitopic specificities were identified. The relevance of these beta-cell epitopes to human insulin-dependent diabetes (IDDM) was demonstrated by the differential ability of human serums from control and diabetic children to displace the radiolabeled MoAbs from the RIN5F cells. Serums from 333 children without diabetes or a family history of diabetes and from 156 newly diagnosed IDDM patients were tested. Only one IgM MoAb was specifically displaced by the IDDM serums, i.e., 146 of 156, compared to serums from control children, i.e., 10 of 333. With immunofluorescence, the serum component responsible for the displacement of the mouse MoAb was identified as IgG. Most of the positive control serums were from children with active autoimmune thyroiditis. Serums from children with other forms of glucose intolerance did not displace MoAb 1A2. There was no correlation between age and the degree of displacement of 1A2. Thus, the displacement of 1A2 is a specific and sensitive marker of diabetes susceptibility easily applicable to mass screening.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple low-dose streptozotocin-induced diabetes in the mouse: further evidence for involvement of an anti-B cell cytotoxic cellular auto-immune response.

Anti-B cell auto-immunity may play a role in the pathogenesis of diabetes in mice resulting from multiple subdiabetogenic doses of the pancreatic B cell toxin, streptozotocin. In the present study we have investigated the cytotoxic anti-B cell response in these mice. A major role for B lymphocytes, macrophages, or their products in the cytotoxic response originally detected in vitro was eliminated by passing splenocytes from the mice treated with multiple subdiabetogenic doses of streptozotocin over a nylon wool column. The removal of the adherent cells enhanced the cytotoxicity against a rat insulinoma cell line in vitro by that expected due to enrichment of T-lymphocytes by approximately two-fold. The induction of diabetes after multiple subdiabetogenic doses of streptozotocin is strain dependent. Mice of five strains were immunized with rat insulinoma cells, but only splenocytes from the two strains susceptible to multiple subdiabetogenic doses of streptozotocin demonstrated a significant cytotoxic response against the rat insulinoma cells in vitro. Mice pre-immunized with either the rat insulinoma cells or with syngeneic islets labelled in vitro with the hapten trinitrophenol developed hyperglycaemia more rapidly than control mice after multiple subdiabetogenic doses of streptozotocin. In the latter experiment the control mice immunized with complete Freund's adjuvant alone also became hyperglycaemic after a modified multiple subdiabetogenic dose of streptozotocin that did not cause diabetes in non-immunized mice. In mice pre-treated with either adjuvant or cyclophosphamide and then given a modified multiple subdiabetogenic dose of streptozotocin (35 mg/kg X 5 rather than 40 mg/kg) the degree of hyperglycaemia was reduced and there was no protective effect of cyclophosphamide.(ABSTRACT TRUNCATED AT 250 WORDS)

Maintenance of fetal human pancreatic beta cells in tissue culture.

Large quantities of viable human islet tissue (beta cells) are required for transplant and for investigations of the autoimmune basis of Type I diabetes. Fetal pancreas offers a potential advantage over other possible sources of beta cells in that it retains some capacity for growth in vitro. We have cultured a total of 45 human pancreata from fetuses of gestational ages from 18 to 23 weeks. Each pancreas was obtained within minutes after delivery and usually cultured within 30 minutes. Pancreata were dispersed and cultured for up to 32 days. Maintenance and growth of the beta cells was assessed by the content of insulin in extracts of cultured tissue. As has been reported by others, fetal human beta cells survived in vitro for over 4 weeks. In three experiments in which a direct comparison was made, collagenase digestion of the fetal pancreas resulted in a significantly greater loss of insulin content compared to minced tissue cultured without digestion. Storage of three pancreata in medium overnight at 4 degrees C significantly reduced the insulin content of the pancreas compared to pancreata cultured immediately. During culture, the majority of the beta cells (based on insulin content) were found in small, macroscopic clumps attached to the surface of the culture dish, and surrounded by a nearly confluent monolayer of fibroblastoid cells. There was a marked decrease in the insulin content of the tissue during culture, most of it (to less than 25% of the original) occurring over the first 4-6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)