Pancreastatin secretion by pituitary adenomas and regulation of chromogranin B mRNA expression.

Pancreastatin, a carboxyl-terminal amidated peptide derived from chromogranin (Cg)A, inhibits secretion of insulin and parathyroid hormone. Our recent studies found significant amounts of immunoreactive pancreastatin in all pituitary adenomas except prolactin adenomas. To analyze the effects of pancreastatin on pituitary cell function, 17 cultured pituitary adenomas were examined for immunoreactive pancreastatin and pancreastatin secretion by the tumors. The effects of pancreastatin on pituitary hormone secretion and on pituitary hormone (follicle-stimulating hormone and prolactin), CgA, and CgB mRNA levels were also examined. Immunoreactive pancreastatin and CgA were present diffusely in gonadotroph and null cell adenomas, but only a few prolactin adenoma cells expressed pancreastatin or CgA. When cells were treated with hypothalamic peptides, gonadotroph adenomas were the only group that released increased amounts of pancreastatin in response to gonadotropin-releasing hormone (10(-7) mol/L). Pancreastatin (10(-7) mol/L) treatment did not stimulate pituitary hormone secretion significantly. In situ hybridization analyses showed that gonadotropin-releasing hormone and pancreastatin treatment led to significant increases in CgB and follicle-stimulating hormone mRNAs in gonadotroph adenomas, whereas CgA mRNA levels did not change significantly. These results show that there is a differential distribution of pancreastatin secretion in pituitary adenomas and that the hypothalamic hormone gonadotropin-releasing hormone and the CgA-derived peptide pancreastatin can regulate CgB mRNA in gonadotroph adenomas, suggesting an autocrine effect of pancreastatin on pituitary tumor function.     

Influence of co-culture with established human endometrial epithelial and stromal cell lines on sperm movement characteristics

The effects of co-culture of human spermatozoa with human immortalized endometrial cells - epithelial or stromal - on sperm movement characteristics, including hyperactivation, were studied using computer- assisted sperm analysis (CASA). Epithelial and stromal cell types could be separated following 8-10 days of culture of endometrial cells originating from human biopsies. Both cell types were immortalized by the SV 40 large T antigen. Co-incubation of sperm with epithelial and stromal monolayers enhanced the rate of hyperactivation: 24.9% (P <0.05) and 17.8% (P = 0.05) versus 9.5% as control, respectively, whereas the majority of motility parameters remained unchanged. Conditioned media had no effect upon sperm parameters, including hyperactivation. Co-incubation with either monolayer was able to maintain sperm motility over a longer period than incubation in control medium alone. In four patients whose spermatozoa did not exhibit hyperactivation, co-incubation with epithelial cells, but not conditioned medium, allowed normal rates of hyperactivation (range: 6.9- 15.6%).      

The mechanical efficiency of treadmill running against a horizontal impeding force

1. A method for determining the apparent mechanical efficiency of running against a horizontal impeding force is described. The results of studies on three well trained athletes have been summarized.2. A linear relation was found between metabolic rate and external work rate. The apparent mechanical efficiency was calculated as the inverse of the slope of the linear regression line of metabolic rate on external work rate x 10(2) (percentage).3. The over-all mean for apparent load-running efficiency (LRE) was 36.1%. The differences in LRE between subjects were not statistically significant. No measurable change in LRE occurred with habituation.4. Values are given for E(k), the net energy cost of unloaded horizontal treadmill running per kilogram of bodyweight and per kilometre of distance covered. The over-all mean for E(k) was 0.83 kcal. kg(-1). km(-1).5. The efficiency values obtained are discussed and compared with other values of running efficiency obtained by different methods, and with studies on isolated muscle.     

Ocular inflammatory effects of intravitreally injected tumor necrosis factor-alpha and endotoxin

Intravitreal injection of human recombinant tumor necrosis factor-alpha (TNF) induced inflammation in the rabbit eye characterized by dilation of blood vessels in the iris, disruption of the blood-ocular barriers, infiltration of inflammatory cells into the anterior chamber, and accumulation of prostaglandin E in intraocular fluids. Inflammation first appeared on day 1, increased on day 2, and remained elevated on day 7. The inflammatory eell infiltrate in the anterior segment of the eye was largely monocytic on days 1 and 2; by day 7 large numbers of lymphocytes were also present. TNF-induced ocular inflammation therefore differed from that reported for intravitreally injected endotoxin in terms of time course and the types of inflammatory cells in the aqueous humor. In a series of experiments in which combinations of TNF and endotoxin were used, intravitreal injection of TNF, 24 h after a low dose ofEscherichia coli endotoxin, produced no more inflammation than that produced by TNF following an injection of endotoxin vehicle. However, if TNF was injected 24 h before endotoxin, the resulting inflammation was greater than that observed in animals given TNF followed by endotoxin vehicle.     

Direct Detection of Legionella Species from Bronchoalveolar Lavage and Open Lung Biopsy Specimens: Comparison of LightCycler PCR, In Situ Hybridization, Direct Fluorescence Antigen Detection, and Culture

We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila, and by Warthin-Starry (WS) staining. The results were compared to the "gold standard" method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.     

Microassay for Sindbis virus and interferon activity.

A simple and rapid microplaque assay for Sindbis virus was developed which uses microtiter plates and overlay medium consisting of methylcellulose and specific antibody to Sindbis virus. Discrete plaque formation was consistently observed on baby hamster kidney (BHK-15) cells within 24 h. The assay was reproducible, quantitative, and had about the same sensitivity as the agar overlay technique on chicken embryo cells in 35-mm petri dishes. The microplaque assay could be accurately applied to measuring interferon activity, particularly at low interferon levels. Overall, the microassay methods described here for assay of Sindbis virus yields and interferon activity retain the accuracy of chicken cell macroplaque assays while offering greater simplicity, rapidity, and economy of materials. This assay is also potentially applicable for use with other togaviruses.     

Estrogen-induced hyperplasia and neoplasia in the rat anterior pituitary gland. An immunohistochemical study

Diethylstilbestrol (DES) treatment of weanling F344 female rats resulted in enlarged pituitary glands and diffuse pituitary prolactin (PRL) cell hyperplasia in all animals after 9 and 12 weeks of treatment. Serum PRL was significantly greater than in control rats (P less than 0.001). Immunohistochemical studies showed that most of the pituitary gland cells consisted of PRL cells. Ultrastructural studies showed increased numbers of PRL cells with hyperplasia of the rough endoplasmic reticulum and decreased numbers of secretory granules. There was a decrease in the relative number of growth hormone (GH) and other cell types in the anterior pituitary. Pituitary tumors and normal pituitary glands were dissociated with trypsin and maintained in culture for 3 weeks. The numbers of PRL and GH cells decreased with time in both groups, and there was an increase in the number of fibroblasts. Staining of the culture cells with neuron-specific enolase showed that the anterior pituitary cells were positive for this enzyme, while the fibroblastic cells were negative. When dissociated pituitary cells were cultured in the presence of 10(-9) M DES for 7 days, there was a 42% increase in the number of immunoreactive PRL cells. These results indicate that DES-treated rats provide an excellent model for study of the in vivo and in vitro regulation of pituitary hyperplasia and neoplasia.     

Expression of superoxide dismutase and xanthine oxidase in myometrium, fetal membranes and placenta during normal human pregnancy and parturition

Superoxide, an agent which attenuates the half-life of nitric oxide, is metabolized and synthesized by superoxide dismutase (SOD) and xanthine oxidase, respectively. Over the last few years much work has focused on the role of nitric oxide in human parturition. The aim of this study was to determine whether the onset of human parturition is associated with a change in the expression of copper/zinc superoxide dismutase (Cu/Zn SOD), manganese superoxide dismutase (Mn SOD) or xanthine oxidase within the uterus. Samples of myometrium, placenta, decidua and fetal membranes were obtained from women before and after the onset of labour at term. Immunocytochemistry was used to localize Cu/Zn SOD, Mn SOD and xanthine oxidase and measure SOD enzyme activity. Cu/Zn and Mn SOD-like immunoreactivity was detected in syncytiotrophoblast cells, villous stromal cells and endothelial cells of blood vessels in the placenta. In the myometrium Cu/Zn and Mn SOD were localized to myocytes and endothelial cells and to some vascular smooth muscle cells. In the fetal membranes we observed staining for Cu/Zn SOD and Mn SOD in the amnion, chorion, extravillous trophoblast and decidua. There was no difference in SOD enzyme activity or staining intensity for SOD between different cell types before and during labour. Xanthine oxidase immunoreactivity was identified in each of the tissues examined and again there was no difference in immunostaining in tissues obtained from women delivered before or after the onset of labour. These results show that the pregnant uterus is capable of both synthesizing and degrading superoxide and suggest that superoxide dismutase and xanthine oxidase may play a role in the maintenance of uterine quiescence during pregnancy, but not in the initiation of parturition.      

Analysis of endocrine active and clinically silent corticotropic adenomas by in situ hybridization.

The distribution of pro-opiomelanocortin (POMC) messenger RNA (mRNA) in 7 functional and 17 clinically silent corticotropic adenomas was analyzed by in situ hybridization (ISH) with 35S-labeled oligonucleotide probes using formalin-fixed paraffin-embedded tissue sections cut from blocks that were in storage between 1 to 14 years. All 7 functional adenomas and 4 subtype 1 tumors had detectable POMC mRNA, while 3 of 6 subtype 2 and 1 of 7 subtype 3 silent adenomas contained detectable POMC mRNA. In situ hybridization analysis with an 35S-labeled beta-actin probe showed a positive hybridization signal in 22 of 22 cases, indicating that the absence of detectable POMC mRNA in some adenomas was not due to loss of the mRNAs during processing of the tissues or because of the age of the embedded tissue blocks. Northern hybridization analysis with the oligonucleotide probes in 2 normal pituitaries and an adenoma causing Cushing's disease detected a 1.2-Kb mRNA in all three tissues, indicating that the oligonucleotide probes were very specific. These results indicate that subtype 1 silent adenomas and clinically active adenomas associated with Cushing's disease contain POMC mRNA that is readily detectable by ISH in routinely processed tissue specimens, while only a few of the subtypes 2 and 3 adenomas have POMC mRNA that can be detected in paraffin blocks with the oligonucleotide probes used in this study.