Molecular forms of gonadotropin-releasing hormone associated peptide (GAP): changes within the rat hypothalamus and release from hypothalamic cells in vitro

We have developed RIAs using antisera directed against the cryptic peptide of the GnRH precursor (termed GnRH-associated peptide, GAP) and have used these together with a GnRH assay to characterize proGnRH-derived peptides in rat hypothalamic extracts. On Sephadex chromatography we have identified three molecular forms of GAP-like immunoreactivity (GAP-LI), in addition to the GnRH decapeptide. The largest of these forms is an 8.0-kilodalton (kD) GAP-LI which appears to be the complete proGnRH peptide. The second is a 6.5-kD GAP-LI, and is similar to the complete cryptic peptide (i.e. proGnRH14-69 or GAP1.56). The third peptide is a 2.5 kD C-terminal fragment of the cryptic peptide, representing a processed form of GAP. In whole hypothalamic extracts from normal rats the 8.0-kD form was the major form, comprising 60-70% of the total GAP-LI. All three forms were present in three distinct areas of the rat hypothalamus, namely median eminence (ME), anterior and mid-hypothalamus. However in the ME the proportion of 8.0-kD GAP-LI was significantly reduced and the proportion of 6.5-kD GAP-LI significantly increased compared to anterior and mid-hypothalamic samples (p less than 0.05). In whole hypothalamic extracts from pregnant and lactating rats the total content of proGnRH-derived peptides was reduced but the relative proportions of these peptides were not significantly changed from normal female rats. However, in postlactating rats, 2 weeks after removal of pups, the total levels of GAP-LI were unchanged compared to normals, but the percentage of 8.0-kD GAP-LI was significantly decreased and the percentage of 2.5-kD GAP-LI significantly increased compared to normals (p less than 0.05), suggesting that proGnRH may undergo additional processing dependent on physiological condition. In fetal and neonatal rats the proportion of the 6.5-kD peptide was increased and that of the 8.0-kD peptide decreased compared to adults, and this change became less significant with increasing age. In ovariectomized rats the proportion of 6.5-kD GAP-LI was increased and that of 8.0-kD GAP-LI decreased; this change was partially reversed with steroid treatment. Both the 6.5 and 2.5-kD forms were released by high K+ stimulation of neonatal hypothalamic cells in culture. These results indicate that there is differential processing of the proGnRH precursor within the hypothalamus and in altered physiological states.     

Epidermal growth factor-induced epithelio-mesenchymal transition in human breast carcinoma cells

PMC42-LA cells display an epithelial phenotype: the cells congregate into pavement epithelial sheets in which E-cadherin and beta-catenin are localized at cell-cell borders. They abundantly express cytokeratins, although 5% to 10% of the cells also express the mesenchymal marker vimentin. Stimulation of PMC42-LA cells with epidermal growth factor (EGF) leads to epithelio-mesenchymal transition-like changes including up-regulation of vimentin and down-regulation of E-cadherin. Vimentin expression is seen in virtually all cells, and this increase is abrogated by treatment of cells with an EGF receptor antagonist. The expression of the mesenchyme-associated extracellular matrix molecules fibronectin and chondroitin sulfate proteoglycan also increase in the presence of EGF. PMC42-LA cells adhere rapidly to collagen I, collagen IV, and laminin-1 substrates and markedly more slowly to fibronectin and vitronectin. EGF increases the speed of cell adhesion to most of these extracellular matrix molecules without altering the order of adhesive preference. EGF also caused a time-dependent increase in the motility of PMC42-LA cells, commensurate with the degree of vimentin staining. The increase in motility was at least partly chemokinetic, because it was evident both with and without chemoattractive stimuli. Although E-cadherin staining at cell-cell junctions disappeared in response to EGF, beta-catenin persisted at the cell periphery. Further analysis revealed that N-cadherin was present at the cell-cell junctions of untreated cells and that expression was increased after EGF treatment. N- and E-cadherin are not usually coexpressed in human carcinoma cell lines but can be coexpressed in embryonic tissues, and this may signify an epithelial cell population prone to epithelio-mesenchymal-like responses.     

In-hospital mortality after transurethral resection of the prostate in Victorian public hospitals

BACKGROUND: The purpose of the present paper was (i) to identify trends in in-hospital mortality after transurethral resection of the prostate (TURP) in Victorian public hospitals; and (ii) to explore associations between in-hospital mortality after TURP and age, adverse events, type of admission (emergency/planned), location of the hospital (metropolitan/rural), teaching status of the hospital and length of stay. METHODS: Trends in in-hospital mortality after TURP and the associations between in-hospital mortality and the aforementioned variables were studied using International Classification of Diseases, 9th revision, Clinical Modification (ICD-9-CM) coded Victorian hospital morbidity data from public hospitals between 1987-88 and 1994-95. Crude and adjusted odds ratios (OR) and 95% confidence intervals (CI) were based on univariate and multivariate logistic regression, respectively. RESULTS: After adjustment for age, comorbidity, and other confounding variables, the trend in mortality reduction over time was highly significant (P for trend < 0.0001, 95% CI for trend: 0.84-0.95). Highly significant associations with mortality were observed for emergency admissions (OR = 1.99, P < 0.0001), presence of adverse events (OR = 2.69, P < 0.0001), length of hospital stay (P for trend < 0.0001, 95% for trend: 1.88-2.15) and age (P for trend < 0.0001; 95% CI for trend: 1.26-1.48). CONCLUSIONS: Routinely collected data from hospitals can provide tentative evidence of improved effectiveness of a surgical treatment, provided analysis takes careful account of potential sources of bias, especially those related to possible changes in case selection over time. These kinds of data should stimulate a joint effort between clinicians, quality assurance experts and epidemiologists to confirm this attribution, and to locate the causative factors.     

Low-risk febrile neutropenia in a medical oncology unit

Background: Febrile neutropenia occurring in patients receiving chemotherapy for solid tumours or lymphoma is usually of short duration, and therefore may have a better outcome compared to patients with acute leukaemia or patients receiving myeloablative chemotherapy. Aims: To review retrospectively the outcomes for febrile neutropenia occurring in patients of the Medical Oncology Unit at our institution, and to identify factors associated with worse outcome, particularly prolonged admission or death. Methods: We reviewed 102 episodes of febrile neutropenia occurring in 85 patients treated between 1992 and 1994. Demographic factors, tumour-related factors and clinical aspects of the episodes were correlated with outcome. Results: The median age was 60 years (range, 18–87), with 56 (55%) episodes occurring in females. Twenty-eight (27%) episodes occurred in patients with lymphoma, with the remaining 74 (73%) occurring in patients with solid tumours. At presentation, the median absolute neutrophil count (ANC) was 0.14×10⁹/L with a median duration of significant neutropenia (ANC<0.5×10 ⁹/L) of three days. The median duration of fever was two days. Twenty-nine (28%) episodes had positive cultures; of these 11 had bacteraemia. Forty-four (43%) episodes were classified as unexplained fevers. The remaining 29 episodes were associated with clinically documented infection but negative cultures. There was a high treatment success rate (81%) with first-line empirical antibiotics. Of 19 treatment failures, 13 were due to the necessity for antibiotic modification; the other six patients died from infection. Factors associated with a worse outcome (including prolonged admission and death) include: diagnosis of lymphoma; increasing number of chemotherapy courses; early onset of neutropenia; pneumonia; severe hypotension; and multiple co-morbidities. Conclusions: Febrile neutropenia in adult patients with solid tumours or lymphoma is associated with a relatively good outcome, possibly due to the short duration of neutropenia. A future prospective study to validate the risk factors indentified in this study would be useful for defining patients at low risk for the adverse outcomes examined, in whom less intensive management for this condition may be possible.     

Abnormal copper metabolism in cultured fibroblasts from patients with Wilson's disease

Skin fibroblasts derived from patients with Wilson's disease have, under certain conditions, elevated concentrations of copper. However, the levels of intracellular copper varied from one experiment to another and the reason for this inconsistency has not yet been determined. 64Cu retention after 24 hours and its release in "chase" experiments was not abnormal, thus distinguishing these fibroblasts from Menkes' syndrome fibroblasts. The data provides evidence that the mutant gene responsible for Wilson's disease is expressed in fibroblasts under certain conditions.     

Does an Unloader Brace Reduce Knee Loading in Normally Aligned Knees?

Background

Unloading knee braces often are used after tibiofemoral articular cartilage repair. However, the experimental basis for their use in patients with normal tibiofemoral alignment such as those undergoing cartilage repair is lacking.

Questions/purposes

The purpose of this study was to investigate the effect of varus and valgus adjustments to one commercially available unloader knee brace on tibiofemoral joint loading and knee muscle activation in populations with normal knee alignment.

Methods

The gait of 20 healthy participants (mean age 28.3 years; body mass index 22.9 kg/m²) was analyzed with varus and valgus knee brace conditions and without a brace. Spatiotemporal variables were calculated as were knee adduction moments and muscle activation during stance. A directed cocontraction ratio was also calculated to investigate the relative change in the activation of muscles with medial (versus lateral) moment arms about the knee. Group differences were investigated using analysis of variance. The numbers available would have provided 85% power to detect a 0.05 increase or decrease in the knee adduction moment (Nm/kg*m) in the braced condition compared with the no brace condition.

Results

With the numbers available, there were no differences between the braced and nonbraced conditions in kinetic or muscle activity parameters. Both varus (directed cocontraction ratio 0.29, SD 0.21, effect size 0.95, p = 0.315) and valgus (directed cocontraction ratio 0.28, SD 0.24, effect size 0.93, p = 0.315) bracing conditions increased the relative activation of muscles with lateral moment arms compared with no brace (directed cocontraction ratio 0.49, SD 0.21).

Conclusions

Results revealed inconsistencies in knee kinetics and muscle activation strategies after varus and valgus bracing conditions. Although in this pilot study the results were not statistically significant, the magnitudes of the observed effect sizes were moderate to large and represent suitable pilot data for future work. Varus bracing increased knee adduction moments as expected; however, they produced a more laterally directed muscular activation profile. Valgus bracing produced a more laterally directed muscular activation profile; however, it increased knee adduction moments.

Clinical Relevance

When evaluating changes in knee kinetics and muscle activation together, this study demonstrated conflicting outcomes and questions the efficacy for the use of unloader bracing for people with normally aligned knees such as those after articular cartilage repair.     

Advancing Pharmacological Treatments for Opioid Use Disorder (ADaPT-OUD): an Implementation Trial in Eight Veterans Health Administration Facilities

BackgroundIdentifying effective strategies to improve access to medication treatments for opioid use disorder (MOUD) is imperative. Within the Veterans Health Administration (VHA), provision of MOUD varies significantly, requiring development and testing of implementation strategies that target facilities with low provision of MOUD.ObjectiveDetermine the effectiveness of external facilitation in increasing the provision of MOUD among VHA facilities with low baseline provision of MOUD compared to matched controls.DesignPre-post, block randomized study designed to compare facility-level outcomes in a stratified sample of eligible facilities. Four blocks (two intervention facilities in each) were defined by median splits of both the ratio of patients with OUD receiving MOUD and number of patients with OUD not currently receiving MOUD (i.e., number of actionable patients). Intervention facilities participated in a 12-month implementation intervention.ParticipantsVHA facilities in the lowest quartile of MOUD provision (35 facilities), eight of which were randomly assigned to participate in the intervention (two per block) with twenty-seven serving as matched controls by block.InterventionExternal facilitation included assessment of local barriers/facilitators, formation of a local implementation team, a site visit for action planning and training/education, cross-facility quarterly calls, monthly coaching calls, and consultation.Main MeasuresPre- to post-change in the facility-level ratio of patients with an OUD diagnosis receiving MOUD compared to control facilities.Key ResultsIntervention facilities significantly increased the ratio of patients with OUD receiving MOUD from an average of 18% at baseline to 30% 1 year later, with an absolute difference of 12% (95% confidence interval [CI]: 6.6%, 17.0%). The difference in differences between intervention and control facilities was 3.0% (95% CI: − 0.2%. 6.7%). The impact of the intervention varied by block, with smaller, less complex facilities more likely to outperform matched controls.ConclusionsIntensive external facilitation improved the adoption of MOUD in most low-performing facilities and may enhance adoption beyond other interventions less tailored to individual facility contexts.