Simultaneous in situ hybridisation of native mRNA and immunoglobulin detection by conventional immunofluorescence in paraffin wax embedded sections.

AIMS: The development of a technique for simultaneous in situ hybridisation for native mRNA and conventional immunofluorescence for cytoplasmic antigens in routine pathology specimens. METHODS: Cocktails of synthetic deoxyoligonucleotides coding for immunoglobulin J chain and kappa light chain were 3' end labelled enzymatically with digoxigenin using terminal deoxynucleotidyl transferase. Native mRNA sequences were "unmasked" using proteolytic digestion with proteinase K and hybrid detection was achieved with an alkaline phosphatase labelled anti-digoxigenin antibody. Alkaline phosphatase was visualised with Fast red/naphthol AS-MX phosphate. Fluorescein isothiocyanate (FITC) conjugated anti-isotype antibodies were used simultaneously at the detection stage to identify the isotype production by individual plasma cells in endoscopic duodenal biopsy specimens. RESULTS: The IgA plasma cells of the lamina propria were identified by immunofluorescence and hybrids were detected in the anticipated plasma cell population by Fast red visualisation. The reaction product was visible in bright field or ultraviolet illumination which allowed FITC and Fast red labels to be visualised together under ultraviolet light at 490 nm. Dual labelled cells were clearly visible. Morphology was well preserved throughout. CONCLUSIONS: This technique permits the demonstration of specific mRNA species in cells expressing immunoglobulin. It combines all the advantages of non-radioactive synthetic oligonucleotide probes and conventional immunofluorescence techniques in routine formol-saline fixed and paraffin wax embedded sections with good retention of morphology.     

Preincubation of human granulosa cells with gonadotrophin prevents the cloprostenol-induced inhibition of progesterone production.

Human granulosa cells, from women undergoing ovum collection for in-vitro fertilization (IVF), will luteinize in vitro and provide a model for investigating the antigonadotrophic action of a prostaglandin F2 alpha (PGF2 alpha) analogue, cloprostenol, on granulosa-derived luteal cells. The granulosa cells were cultured in a defined medium and exposed to treatments during a preincubation period of 0 to 3 days and a final incubation with low density lipoprotein (LDL) from days 3 to 4. In the absence of human chorionic gonadotrophin (HCG), progesterone production was low, whereas exposure to HCG in the final incubation resulted in a 10-fold increase in progesterone concentrations. The inclusion of cloprostenol with HCG in the final incubation significantly (P less than 0.05) inhibited HCG-stimulated progesterone production. Exposure to HCG during the preincubation prevented the antigonadotrophic action of cloprostenol in the final incubation. The antigonadotrophic action of cloprostenol was retained when the granulosa cells were exposed to cloprostenol during the preincubation. Omission of LDL from the final incubation lowered the production of progesterone but the pattern of responses to HCG and cloprostenol were similar. Prevention of the antigonadotrophic action of cloprostenol after exposure to HCG may be a mechanism through which chorionic gonadotrophin can prevent regression of the corpus luteum in early pregnancy. Cloprostenol does not appear to inhibit LDL-stimulated steroidogenesis in human granulosa cells.     

Digital replantation teaching model in rats

Replant surgery is a complex procedure that requires advanced microsurgical skills and is usually performed as an emergency operation, lasting many hours. For these reasons, teaching replantation is difficult. Although teaching models exist, they are often too general or complicated for routine use and do not simulate the stages and the pitfalls of human replant surgery. We have designed a model that is simple and imitates human replant surgery. After reviewing the rat anatomy, students dissect and replant a rat hind limb that has been sharply amputated by the instructor. They follow the same principles of "real" surgery like debridement, minimizing ischemia time, and stable fixation before anatomosis of vessels. After marking the structures, bony fixation followed by vessel and nerve anastomosis are performed. Muscle is reattached to the skin and limb vascularity evaluated. After we designed this model, plastic surgery residents performed the technique on 10 rats. An 80% limb viability rate was achieved. This model is simple to perform, simulates all the relevant structures and pitfalls of human surgery, and the rats are relatively cheap and can be used for other parallel projects.     

Auditing paediatric diabetes care and the impact of a specialist nurse trained in paediatric diabetes

AIMS: To define outcome measures for auditing the clinical care of children and adolescents with insulin dependent diabetes mellitus (IDDM) and to assess the benefit of appointing a dedicated paediatric trained diabetes specialist nurse (PDSN). METHODS: Retrospective analysis of medical notes and hospital records. Glycaemic control, growth, weight gain, microvascular complications, school absence, and the proportion of children undergoing an annual clinical review and diabetes education session were assessed. The effect of the appointment of a PDSN on the frequency of hospital admission, length of inpatient stay, and outpatient attendance was evaluated. RESULTS: Children with IDDM were of normal height and grew well for three years after diagnosis, but grew suboptimally thereafter. Weight gain was above average every year after diagnosis. Glycaemic control was poor at all ages with only 16% of children having an acceptable glycated haemoglobin. Eighty five per cent of patients underwent a formal annual clinical review, of whom 16% had background retinopathy and 20% microalbuminuria in one or more samples. After appointing the PDSN the median length of hospital stay for newly diagnosed patients decreased from five days to one day, with 10 of 24 children not admitted. None of the latter was admitted during the next year. There was no evidence of the PDSN affecting the frequency of readmission or length of stay of children with established IDDM. Non-attendance at the outpatient clinic was reduced from a median of 19 to 10%. CONCLUSIONS: Outcome measures for evaluating the care of children with IDDM can be defined and evaluated. Specialist nursing support markedly reduces the length of hospital stay of newly diagnosed patients without sacrificing the quality of care.     

Serum leptin concentrations in relation to pubertal development

OBJECTIVES: The amount of adipose tissue influences pubertal development and fertility in girls. A candidate for mediating this is the hormone leptin, derived from adipocytes. This work was carried out to determine whether the leptin concentration in serum is regulated during pubertal development. SUBJECTS AND METHODS: Serum concentrations of leptin were determined by radioimmunoassay in a sample of 252 healthy children representing all pubertal stages. RESULTS: Serum leptin concentrations correlated directly with age (r = 0.53), body mass index (BMI) (r = 0.71), and weight for height SD score (r = 0.44) in girls and with BMI (r = 0.33) and weight for height SD score in boys (r = 0.36). Leptin concentrations increased with pubertal development in girls, resulting in significantly higher concentrations at pubertal stages 4 and 5 than at the prepubertal stage, whereas there was no change in the boys. CONCLUSIONS: Serum leptin concentrations increased during pubertal development in the girls, but remained constant in the boys. Whether the increase in serum leptin concentrations in girls is of importance for, or a consequence of, pubertal development is still to be determined.     

Proliferative lesions of oviduct and uterus in CD-1 mice exposed prenatally to tamoxifen

Tamoxifen (TAM) is widely used as adjuvant breast cancer therapy after surgery and as a chemopreventive agent in women of child-bearing age. However, TAM therapy has been shown to result in an increased incidence of endometrial carcinoma in women. The present study was designed to investigate the effects of TAM (5 mg/kg and 7.5 mg/kg body wt) given i.g. to pregnant CD-1 mice (1x/day, days 12 through 18 of gestation) on their female offspring. Progressive proliferative hyperplasia of the oviduct was frequently seen in TAM-exposed offspring, reaching 100% incidence by 52 weeks in both treatment groups. These females also developed progressive proliferative uterine lesions, including moderate/severe cystic endometrial hyperplasia (34-50%) and polypoid adenomas (27-30%) between 53 and 78 weeks. Deciduomas (15%) occurred at young ages (12 and 24 weeks) while leiomyomas (14%), a malignant leiomyosarcoma, and ovarian granulosa cell tumors (14%), were found between 72 and 78 weeks. Our findings thus suggest a strong association between transplacental TAM and reproductive tract abnormalities in female CD-1 mice.      

Median nerve compression in Proteus syndrome

Proteus syndrome is a multi–organ disorder, a prime feature of which is localized gigantism, usually clinically obvious. Symptoms secondary to hypertrophy of nerves has not been previously recognized as a part of the syndrome. Accepted: 16 May 1997