Interaction of kisspeptin and the somatotropic axis.

Kisspeptin, a regulator of gonadotropin-releasing hormone, has been hypothesized as an integrator of nutrition and hormones critical to metabolism and the regulation of reproduction. Growth hormone (GH) is necessary for optimal reproduction and recent evidence suggests that its secretion may be influenced by kisspeptin. The objectives of this study were to determine whether the effect of kisspeptin to stimulate GH release is due to an interaction with growth hormone-releasing hormone (GHRH) or somatostatin (SS), or an effect at the hypothalamus. Intravenous injection and infusion of kisspeptin [500 pmol/kg BW (650 ng/kg)/h × 5 h] to cows (n = 5) increased serum concentrations of luteinizing hormone (LH) but not GH. Pretreatment with kisspeptin injection and infusion in cows (n = 5) reduced the stimulatory effect of GHRH (0.05 µg/kg BW) on GH secretion. However, the magnitude of the GH response to GHRH (assessed by incremental AUC) was not affected by kisspeptin. In these same cows, administration of kisspeptin prevented the increase in GH induced by SS infusion (0.5 µg/kg BW/ h × 1.5 h) withdrawal. Peripheral administration of kisspeptin [200 and 1,000 pmol/kg BW (260 and 1,300 ng/kg)] increased serum concentrations of LH but not GH in ewes (n = 8). However, concentrations of GH were stimulated by central kisspeptin treatment [100 and 200 pmol/kg BW (130 and 260 ng/kg)] in ewes. In addition to activating the gonadotropic axis, kisspeptin can activate the somatotropic axis in ruminants. Present data support the concept of a central site of action for this effect.

The incidence of Zolpidem use in suspected DUI drivers in Miami-Dade Florida: a comparative study using immunalysis Zolpidem ELISA KIT and gas chromatography-mass spectrometry screening.

In 1993, Zolpidem (Ambien), a non-benzodiazepine hypnotic agent, was approved for use in the United States for the short-term treatment of insomnia. Zolpidem has a rapid onset of action and short elimination half-life, rendering it ideal as a sleep aid. The objective of this study was to evaluate, and retrospectively compare, the use of the Immunalysis ELISA kit and gas chromatograpy-mass spectrometry (GC-MS) to screen blood/urine specimens for zolpidem. In addition, results for the incidence of zolpidem in suspected DUI drivers in 2007 are compared to previous years' data. The ELISA kit was evaluated for cross-reactivity with zaleplon and zopiclone and zolpidem metabolite I. Urine samples (n = 100) and blood samples (n = 100) were selected from specimens received into the DUI laboratory in 2007 and were screened via the Immunalysis Zolpidem ELISA kit and on GC-MS in full EI scan mode following an alkaline liquid-liquid extraction. Results show 5% of the urine and blood samples screened positive for zolpidem using the ELISA kits, and all 5% confirmed positive for zolpidem using GC-MS. The ELISA kit demonstrated no cross-reactivity to zaleplon or zopiclone at a spiked urine concentration of 1000 ng/mL. Ten cases of suspected DUI drivers in 2007 confirmed positive for zolpidem by ELISA and GC-MS in blood/urine, a higher incidence rate than in the previous years. Because of the low percentage elimination of the parent compound in urine, a screening method for the detection of the main metabolite of zolpidem may be needed for better detection of drug impairment driving due to zolpidem.

Modeling growth factor activity during proinflammatory stress: methodological considerations in assessing cytokine modulation of IGF binding proteins released by cultured bovine kidney epithelial cells.

The present research was conducted to model potential mechanisms through which IGFBPs might be affected by a key proinflammatory response initiating cytokine tumor necrosis factor (TNF-)-alpha. Madin-Darby bovine kidney epithelial (MDBK) cells, known to release IGFBPs in response to several stimuli, were grown under several conditions and challenged with forskolin (F) or recombinant TNF-alpha for 24h. Forskolin increased IGFBP-3 gene expression and media content of BP-3 protein. TNF-alpha increased basal and augmented F-mediated IGFBP-3 gene expression. However, TNF-alpha effects on the measurable media content of IGFBPs were influenced by culture conditions; in the absence of added protease inhibitors (PIs) or sufficient media albumin concentration (high BSA, 1mg/ml), the effect of TNF-alpha was to decrease (P<0.02) measurable IGFBPs. In the presence of PI and high BSA, media IGFBP-3 levels were shown to be increased by TNF-alpha consistent with the gene expression data. Changes in media IGFBP-3 protease activity were examined further to explain the observed effects of TNF-alpha on production and destruction of IGFBPs in media. When recombinant human IGFBP-3 (500 ng/ml) was added to PI-free, low BSA 100 microg/ml) media from TNF-treated MDBK cells, less than 10% of the BP-3 was recognizable by Western blot in 30 min; conversely, inclusion of High BSA and PI in media resulted in attenuation of the protease effect on the IGFBPs. The data suggest that the MDBK model of cellular response to proinflammatory stimulus is affected by culture conditions and that TNF-alpha affects media content of IGFBPs through effects on IGFBP gene expression coupled with degradation of IGFBPs via enhanced proteolytic enzyme release.

Interleukin-1beta and tumor necrosis factor-alpha mediation of endotoxin action on growth hormone.

In humans and sheep, endotoxin (LPS) administration results in increased growth hormone (GH) concentrations. To determine the role of cytokines in the effect of LPS on GH, sheep were challenged with IL-1beta or TNF-alpha. GH data were compared with results with LH, where the major effects of LPS are known to act via the hypothalamus. Intracerebroventricular (icv) administration of IL-1beta or TNF-alpha did not alter plasma concentrations of GH. Endotoxin was then administered intravenously (iv) in combination with icv injection of IL-1 receptor antagonist (IL-1RA), TNF antagonist (sTNF-R1), or saline. Administration of LPS increased GH (P < 0.0001), although coadministration of IL-1ra or sTNF-R1 icv did not alter GH response to LPS. In contrast, plasma concentrations of LH were profoundly inhibited by icv administration of either cytokine (P < 0.03), but the LH response to LPS was not altered by cytokine antagonists. Intravenous administration of either IL-1beta or TNF-alpha increased plasma concentrations of GH (P < 0.0001). Administration of IL-1RA and sTNF-R1 iv prevented LPS-induced increases in GH. Although LH was suppressed by high iv doses of IL-1beta (P = 0.0063), the antagonists did not alter the LH response to LPS. To determine whether LPS might directly activate GH release, confocal microscopy revealed colocalization of CD14, the LPS receptor, with GH and, to a lesser extent, LH and some prolactin (PRL)-containing cells, but not ACTH or TSH. These data are consistent with the effects of LPS on GH secretion originating through peripheral cytokine presentation to the pituitary, as well as a potential to act directly on selective populations of pituitary cells via CD14.

Leptin, tumor necrosis factor-alpha (TNF), and CD14 in ovine adipose tissue and changes in circulating TNF in lean and fat sheep.

Four studies were designed to determine whether 1) tumor necrosis factor-alpha (TNF) and the Lipopolysaccharide (LPS) binding ligand, CD14, are produced by sheep adipose tissue; 2) nutritional reserves and/or short-term fasting affect circulating concentrations of TNF; 3) there is a relationship between TNF and metabolic factors in sheep; and 4) inflammation alters circulating concentrations of leptin. In Exp. 1 and 2, ewes were assigned, based on ultrasonic assessments of last-rib subcutaneous fat measurements to fat (fat thickness > 1 cm; mean = 1.52 +/- 0.03 cm) or thin (fat thickness < 1 cm; mean = 0.25 +/- 0.03 cm) groups. Fat and thin ewes were assigned to fed or fasted groups for a total of four groups (fed-fat; fasted-fat; fed-thin; fasted-thin). Fed-ewes had ad libitum access to feed, and fasted-ewes were prohibited feed 48 h before initiation of sample collection. In Exp. 1, subcutaneous fat samples were collected from just above the last rib for detection of TNF and CD14 mRNA, and immunoreactivity. Tumor necrosis factor-alpha-like immunoreactivity in adipocytes was sparse, more pronounced in cells in fed-ewes than fasted-ewes, and localized to membranes between adjacent cells in nucleated regions. Immunoreactivity for CD14 was minimally observed but present in adipocytes and widely expressed in infiltrating monocytes and epithelial vascular cells. Leptin was detected in adipocytes. In Exp. 2, plasma samples collected every 6 h for 24 h were analyzed for plasma concentrations of TNF. Fat ewes had greater plasma concentrations of TNF than thin ewes (P = 0.039). In Exp. 3, wethers were injected i.v. with interleukin-1beta or TNF. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not affected by treatment (P > 0.39). In Exp. 4, wethers were injected with LPS. Blood samples were collected every 15 min for 8 h following injection. Plasma concentration of leptin was not altered by LPS (P > 0.20). These results provide evidence: 1) of TNF-like immunoreactivity within fat tissue; 2) that elements within fatty tissues have CD14 that may allow adipocyte function to be directly affected by LPS; 3) that plasma concentrations of leptin are not altered by LPS treatment; and 4) that circulating concentrations of TNF are elevated with obesity in sheep.

Estradiol plus progesterone treatment modulates select elements of the proinflammatory cytokine cascade in steers: attenuated nitric oxide and thromboxane B2 production in endotoxemia.

Estradiol plus progesterone (EP) implants have been shown to favorably alter the time course or decrease the severity of many of the clinical manifestations associated with coccidiosis and endotoxemia in calves. This study evaluated the effect of EP treatment on plasma tumor necrosis factor-alpha (TNF), thromboxane (TXB), prostacyclin (PRC), nitrite and nitrate (NO[x]), and cortisol. Holstein steer calves were divided into four groups: control, EP, endotoxin (LPS), and EP + LPS (n = five/group). Estradiol/progesterone pellets (Synovex-S) were implanted subcutaneously when calves reached 20 wk of age. One week after implantation, calves were injected i.v. with endotoxin (i.e., lipopolysaccharide; LPS, 0.6 microgram/kg of BW) or nonpyrogenic saline placebo. Body temperature was measured and blood was collected before injection and at 1, 2, 3, 4, 6, and 8 h thereafter. Plasma concentrations of TNF, cortisol, TXB, PRC, NO[x], were measured. Body temperature increased in both LPS and LPS-EP calves, but had returned to normal by 6 h in the LPS-EP group (P < 0.05). Plasma TNF and cortisol increased after LPS (P < 0.01), but were not differentially affected by EP treatment. Likewise, EP did not affect the magnitude of increase in LPS-induced PRC, but EP decreased the magnitude of increase in TXB (P < 0.05). Plasma NO[x]) levels were increased (P < 0.01) in calves after LPS; treatment with EP attenuated the LPS-associated increase in plasma NO[x] levels. These results suggest that EP exerts specific effects on different components of the proinflammatory cytokine cascade. Although the initiation of responses mediated by TNF, cortisol, and PRC do not seem to be differentially affected by EP, components of the nitric oxide- and TXB-axis responses to LPS are decreased in calves pretreated with EP.

Intrauterine growth of full-term infants: impact of prenatal cocaine exposure.

OBJECTIVE: The objectives of this study were to estimate the effect of prenatal cocaine exposure on fetal growth and gestational age after controlling for exposure to alcohol, tobacco, and marijuana and other covariates; to evaluate whether prenatal cocaine exposure has a disproportionate adverse effect on head circumference compared with overall somatic growth; and to assess whether the effect of prenatal cocaine exposure on fetal growth is mediated by cocaine's suspected effect on gestational age. METHODS: The study population includes 476 neonates participating in the Miami Prenatal Cocaine Study, a longitudinal follow-up of in utero cocaine exposure. The sample, restricted to full-term neonates born to African-American inner-city mothers, included 253 infants exposed prenatally to cocaine (with or without alcohol, tobacco, or marijuana exposure) and 223 non-cocaine-exposed infants, of whom 147 were drug-free and 76 were exposed to varying combinations of alcohol, tobacco, or marijuana. RESULTS: Evidence based on structural equations and multiple regression models supports a hypothesis of cocaine-associated fetal growth deficits (0.63 standard deviation) and an independent mild effect on gestational age (0.33 standard deviation). There was no evidence of a disproportionate adverse effect on birth head circumference once the impact on overall growth was estimated. There was evidence that some but not all of the cocaine effect on fetal growth was direct and some was indirect, acting via an intermediate influence of cocaine on gestational age. CONCLUSIONS: Cocaine-associated growth deficits, symmetrical and partially mediated by gestational age, were observed in this sample of inner-city African-American full-term infants prospectively enrolled at birth. Long-term implications will be the subject of future reports from this longitudinal investigation.

Subsite-specific incidence rate and stage of disease in colorectal cancer by race, gender, and age group in the United States, 1992-1997.

BACKGROUND: Subsite specific incidence rates of colorectal cancer vary considerably by age, gender, and race. This variation may be related not only to distinctions in exposure to genetic and environment factors but also to current strategies of early detection screening. Patterns of stage of disease in anatomic subsite may reflect the effect of screening. This study used the largest aggregation of cancer incidence data in the U.S. to examine subsite specific incidence rates of colorectal cancer and the relation of stage of disease to anatomic subsites by race, gender, and age group. METHODS: Data on the incidence of invasive colorectal cancer were obtained from 28 population-based central cancer registries. Age-specific and age-adjusted rates and stage distributions were analyzed by subsite, race, and gender. RESULTS: The impact of screening can be observed in the percentage of localized disease, which increased from 31.9% among cancers in the proximal colon to 37.0% in the descending colon to 41.5% in the distal colorectum. Within the same subsite, blacks were less likely than whites to receive a diagnosis of localized disease and more likely to receive a diagnosis of distant disease whereas stage distributions were approximately the same for males and females. Blacks were more likely than whites to receive a diagnosis of proximal colon cancer than distal colorectal cancer. The male-to-female rate ratios progressively increased from the proximal colon to the distal colorectum. The ratios of proximal-to-distal colorectal cancer gradually increased with advancing age. CONCLUSIONS: Differentials in stage of disease by subsites indicate a need for a targeted effort at early detection of cancer in the proximal colon. Risk factors and higher risk populations for colorectal cancers in each subsite need to be studied further to guide actions for improving the efficacy of screening.

Predictive medicine: computational techniques in therapeutic decision-making.

The current paradigm for surgery planning for the treatment of cardiovascular disease relies exclusively on diagnostic imaging data to define the present state of the patient, empirical data to evaluate the efficacy of prior treatments for similar patients, and the judgement of the surgeon to decide on a preferred treatment. The individual variability and inherent complexity of human biological systems is such that diagnostic imaging and empirical data alone are insufficient to predict the outcome of a given treatment for an individual patient. We propose a new paradigm of predictive medicine in which the physician utilizes computational tools to construct and evaluate a combined anatomic/physiologic model to predict the outcome of alternative treatment plans for an individual patient. The predictive medicine paradigm is implemented in a software system developed for Simulation-Based Medical Planning. This system provides an integrated set of tools to test hypotheses regarding the effect of alternate treatment plans on blood flow in the cardiovascular system of an individual patient. It combines an Internet-based user interface developed using Java and VRML, image segmentation, geometric solid modeling, automatic finite element mesh generation, computational fluid dynamics, and scientific visualization techniques. This system is applied to the evaluation of alternate, patient-specific treatments for a case of lower extremity occlusive cardiovascular disease.

Estradiol/progesterone implants increase food intake, reduce hyperglycemia and increase insulin resistance in endotoxic steers.

High doses of lipopolysaccharide (LPS) induce transient hyperglycemia, then chronic hypoglycemia and increased insulin resistance. In addition, appetite is reduced, while body temperature and concentrations of cortisol and tumor necrosis factor alpha (TNFalpha) are elevated. Furthermore, concentrations of GH and IGF-I are reduced in cattle. The objectives of this study were to determine whether a gonadal steroid implant (20 mg estrogen and 200 mg progesterone) given to endotoxemic steers would: (1) reduce hyperglycemia, reduce hypoglycemia, reduce insulin resistance, (2) reduce changes in concentrations of GH and IGF-I, (3) reduce inappetence and reduce concentrations of blood urea nitrogen (BUN) and non-esterified fatty acids (NEFA), and (4) reduce fever and concentrations of TNFalpha and cortisol. Holstein steers were assigned within a 2x2 factorial arrangement of treatments as follows (n=5 per group): C/C, no steroid and vehicle; S/C, steroid and vehicle; C/E, no steroid and LPS (1 microg/kg body weight (BW), i.v.); S/E, steroid and endotoxin. Steroid implants were given at 20 weeks of age (day 0) and serial blood samples (15 min) were collected on day 14 for 8 h, with vehicle or LPS injected after 2 h. Intravenous glucose tolerance tests (100 mg/kg BW) were carried out at 6 h and 24 h. Hyperglycemia was 67% lower (P<0.05) in S/E- compared with C/E-treated steers between 30 and 150 min after i.v. injection of LPS. Hypoglycemia developed after 4 h and insulin resistance was greater in S/E- compared with C/E-treated steers (P<0. 05) at 6 and 24 h. Concentrations of IGF-I were restored earlier in steroid-treated steers than in controls. Concentrations of GH were not affected by steroids, but increased 1 h after injection of LPS, then were reduced for 2 h. Appetite was greater (P<0.05) in S/E- (2.1% BW) compared with C/E-treated steers (1.1% BW) (pooled s.e.m.=0.3). Concentrations of NEFA increased after injecting LPS, but concentrations were lower (P<0.05) in S/E- compared with C/E-treated steers. LPS did not affect concentrations of BUN, but concentrations were lower in steroid-treated steers. Steroids did not affect body temperature or concentrations of TNFalpha and cortisol. In summary, gonadal steroids reduce hyperglycemia, reduce inappetence and tissue wasting, but increase insulin resistance. Furthermore, concentrations of IGF-I are restored earlier in steroid-treated than in non-steroid-treated steers injected with LPS. It is concluded that gonadal steroids reduce severity of some endocrine and metabolic parameters associated with endotoxemia. However, it is unlikely that gonadal steroids acted via anti-inflammatory and immunosuppressive actions of glucocorticoids or through reducing concentrations of cytokines.

Cytokine-mediated growth hormone release from cultured ovine pituitary cells.

Previous studies have demonstrated that intravenous lipopolysaccharide (LPS) will increase concentrations of growth hormone (GH). One possible explanation for this may reside in the response of the pituitary to specific cytokines. This study sought to determine the effects of recombinant bovine tumor necrosis factor alpha (TNF), recombinant ovine (ro) interleukin-1alpha (IL-1alpha), roIL-1beta, ro interleukin-2 (IL-2), and ro gamma-interferon (INT) on GH release from cultured sheep pituitary cells. Sheep were sacrificed and pituitary cells cultured in DMEM with 10% fetal bovine serum for 3 days. On day 4, cells were washed and serum-free DMEM added to cells. IL-1alpha and IL-1beta were used at 0.2, 2 and 20 ng/ml and the remaining cytokines at 2, 20 and 200 ng/ml. Neither IL-2 nor INT had effects on basal or on GH-releasing hormone (GRH)-stimulated GH release. TNF inhibited GRH-stimulated GH release (p < 0.05). Both IL-1alpha and IL-1beta stimulated GH release from cultured pituitary cells at all doses tested (p < 0.01). Neither IL-1alpha nor IL-1beta had an effect on GRH-stimulated GH release. IL-1 effects were inhibited by H-89 (p < 0.05; a protein kinase A inhibitor) and by nifedipine (p < 0.05; a calcium channel blocker). Both of these mechanisms are central signal transduction mechanisms mediating GRH-stimulated GH release. IL-1-stimulated GH release is partially inhibited (p < 0.05) by lipoxygenase pathway blockers. Phorbol myristate acetate downregulation of protein kinase C did not alter IL-1-stimulated GH release. IL-1beta increased the content of both GH and GH mRNA in cultured sheep pituitary cells. We conclude that IL-1 produces a strong stimulus to GH release, which is mediated by calcium entry and protein kinase A activation. IL-1 also activates lipoxygenase pathways. This latter pathway as well as calcium entry were shown to mediate LPS stimulation of GH release from cultured pituitary cells. The similarity between IL-1 and LPS signal transduction suggests that LPS may activate pituitary production of IL-1 to produce the stimulus to GH. The lack of inhibitory effects of INT, TNF and IL-2 as opposed to what is seen in the rat may suggest a partial mechanism to explain the different effects of LPS on GH release between sheep and that seen in cattle and rats.

Hormonal modulation of the physiologic responses of calves infected with Eimeria bovis.

OBJECTIVE: To determine whether an estradiol-progesterone (EP) growth implant would have an effect on febrile responses and on the catabolic component of Eimeria bovis infection. ANIMALS: 27 Holstein bull calves. PROCEDURE: Calves were assigned to treatment groups as: control (n = 5), EP implant (EP, n = 5), E bovis-inoculated (coccidia: C, n = 7), pair fed (n = 4), or EP plus E bovis-inoculated coccidia (EP/C, n = 6) groups. Calves were provided subcutaneous EP implants at 8 weeks of age, and were inoculated with 2 x 10(5) oocysts of E bovis at 11 weeks of age. Body weight was measured on postinoculation day (PID) 0, 14, and 28. Rectal temperature and food intake were determined and fecal samples were collected daily from PID 15 to 28. Blood samples were collected on PID 24 for analysis of CD2+, CD4+, and CD8+ antigens and plasma insulin-like growth factor I concentration. Blood samples were collected at 15-minute intervals for measurement of pulsatile growth hormone release. RESULTS: Group-EP/C calves had fever for 2 days versus 5 days for group-C calves (P < 0.05). These calves had diarrhea for fewer days than did their group-C counterparts (P < 0.05). Fibrinogen and glucose values were high in group-C (P < 0.05) but not group-EP/C calves. The latter had positive weight gain from PID 14 to 28, whereas group-C calves had weight loss (P < 0.05). Plasma insulin-like growth factor I concentration was reduced by infection (P < 0.05). EP-treated noninfected calves had increased numbers of CD2+, CD4+, and CD8+ blood mononuclear cells (P < 0.05). CONCLUSIONS: EP has a protective effect in calves infected with E bovis. This may relate to changes in immune function induced by EP. CLINICAL RELEVANCE: Treatment of calves with EP could offer some protection against the often severe wasting and debilitation associated with E bovis infection.

The safety of transdermal nicotine as an aid to smoking cessation in patients with cardiac disease.

BACKGROUND: Transdermal nicotine therapy is widely used to aid smoking cessation, but there is uncertainty about its safety in patients with cardiac disease. METHODS: In a randomized, double-blind, placebo-controlled trial at 10 Veterans Affairs medical centers, we randomly assigned 584 outpatients (of whom 576 were men) with at least one diagnosis of cardiovascular disease to a 10-week course of transdermal nicotine or placebo as an aid to smoking cessation. The subjects were monitored for a total of 14 weeks for the primary end points of the study (death, myocardial infarction, cardiac arrest, and admission to the hospital due to increased severity of angina, arrhythmia, or congestive heart failure); the secondary end points (admission to the hospital for other reasons and outpatient visits necessitated by increased severity of heart disease); any side effects of therapy; and abstinence from smoking. RESULTS: There were 48 primary and 78 secondary end points noted in a total of 95 subjects. At least one of the primary end points was reached by 5.4 percent of the subjects in the nicotine group and 7.9 percent of the subjects in the placebo group (difference, 2.5 percent; 95 percent confidence interval, -1.6 to 6.5 percent; P=0.23). In the nicotine group, 11.9 percent of the subjects had at least one of the secondary end points, as compared with 9.7 percent in the placebo group (difference, 2.2 percent; 95 percent confidence interval, -2.2 to 7.4 percent; P= 0.37). After 14 weeks the rate of abstinence from smoking was 21 percent in the nicotine group, as compared with 9 percent in the placebo group (P=0.001), but after 24 weeks the abstinence rates were not significantly different (14 percent vs. 11 percent, P= 0.67). CONCLUSIONS: Transdermal nicotine does not cause a significant increase in cardiovascular events in high-risk outpatients with cardiac disease. However, the efficacy of transdermal nicotine as an aid to smoking cessation in such patients is limited and may not be sustained over time.

Interaction of cyclic AMP- and calcium-dependent mechanisms in the regulation of growth hormone-releasing hormone-stimulated growth hormone release from ovine pituitary cells.

Growth hormone-releasing hormone (GHRH)-stimulated growth hormone (GH) release from the sheep pituitary is mediated through Ca(2+)-and cyclic AMP-dependent mechanisms. The initial Ca2+ influx is suggested to result from depolarization, whereas a secondary Ca2+ influx is thought to result from second messengers. This study sought to determine whether there was an interaction between these two signal transduction pathways. Sheep pituitary cells were placed in culture for 4 d and were then washed and incubated for 1 hr in serum-free medium before the application of specific antagonises and/or agonists. Both KCl and forskolin stimulated GH release (P < 0.05), but neither produced an effect similar to that of GHRH. The combination of both stimuli, however, mimicked GH release, as seen with a maximal dose of GHRH. Pretreatment with H89 (protein kinase A [PKA] inhibitor) inhibited GHRH, forskolin- and KCl-stimulated GH release (P < 0.001) but had no effect on phorbol myristate acetate (PMA)-stimulated GH release. Verapamil (voltage-dependent Ca2+ channel blocker) inhibited the GHRH effects on GH release (P < 0.0002) but did not influence forskolin or PMA actions. These data suggest that Ca(2+)-dependent pathways converge with cyclic AMP-dependent pathways before or with the activation of PKA. The data also suggest that PKA activation by cyclic AMP alone is insufficient to reproduce either the effects of GHRH stimulation or the combined effects of Ca2+ influx plus PKA activation on GH release. A calmodulin blocker, W7, reduced GHRH-stimulated GH release, a reduction equivalent to the Ca2+ effect on GH release. This suggests that Ca2+ activates calmodulin, which in turn enhances adenylyl cyclase and/or PKA activity to release GH from the sheep pituitary.

Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status.

CD26 is a multifunctional molecule implied to have a variety of roles in the immune response including its activity as a membrane exopeptidase (Dipeptidyl peptidase IV) which cleaves several protein molecules. In order to further define the expression and functional activity of CD26 in the developing thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of DPP IV activity with a fluorochrome-conjugated peptide substrate and surface staining of the T lymphocyte lineage antigens CD4 and CD8. Neonatal and adult murine thymi were examined using the three-color assay and significant differences in DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. Single-positive cells bore higher activity than CD4-/CD8- cells and neonates had higher activity than adults. Thymocytes with characteristics consistent with apoptotic cells expressed higher DPP IV activity. Thus, DPP IV appears to be upregulated both as thymocytes mature and among thymocytes which are undergoing programmed cell death. These results suggest that CD26 is ontogenically controlled during T cell maturation and may play a role in thymic deletion of emerging clones.

Cortisol inhibition of growth hormone-releasing hormone-stimulated growth hormone release from cultured sheep pituitary cells.

Cortisol inhibits growth hormone (GH) release in short-term culture and is stimulatory in long-term cultures of rat and human pituitary cells. This study sought to determine the in vitro effects of cortisol on GH release and the signal transduction pathways mediating the effects of cortisol on GH release from cultured ovine somatotrophs. Pituitary cells were dispersed with collagenase and placed in culture medium for 4 days. The data indicate that cortisol inhibited growth hormone-releasing hormone (GHRH)-stimulated GH release by at least 2 h. In short-term culture GHRH-, forskolin- and dibutyryl cyclic AMP-stimulated GH release were inhibited by cortisol, suggesting an effect distal to the membrane and involving a protein kinase A (PKA)-dependent pathway. GH release initiated by KCl was inhibited by cortisol, but GH release caused by the calcium ionophore A23187 was unaffected. This suggests a possible action of cortisol on the calcium channels. The inhibition by cortisol of the calcium-dependent secretion of GH release appeared to play a smaller role in mediating cortisol inhibition of GH release than that seen with PKA. Attempts to overcome cortisol inhibition of GH release using puromycin, arachidonic acid or pertussis toxin were unsuccessful. Since cortisol inhibition of GH release does not occur via the mechanisms found in other cell types, cortisol inhibition of pituitary cell secretions appears to be cell-specific rather than utilizing a single inhibitory mechanism. The majority of cortisol actions on the somatotroph appear to act at a site distal to the production of cyclic AMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Human T lymphocyte subpopulation and NK cell alterations in persons exposed to cocaine.

Immunophenotypic changes in peripheral blood mononuclear cells were analyzed by flow cytometry in several patient groups positive for cocaine in their urine. Single- and dual-color immunofluorescence staining was performed to examine total numbers of NK, T, and B cells, as well as the coexpression of surface molecules on T cells associated with memory function, helper/inducer capacity, and activation status. In addition, levels of several serum proteins (including immunoglobulins) and other demographic variables were evaluated. Our results show that cocaine-intoxicated patients display reductions in the total percentage of CD4+ T cells and increases in the number of NK cells. Dramatic shifts within certain T cell subpopulations were also observed. In particular, there appeared to be a preferential stimulation of "activated" T cells as indicated by increased levels of class II+ CD4 and CD8 T cells and IL2r+ CD4 T cells. "Memory" CD8+ T cell subpopulations (i.e., CD45RO+) were reduced in the cocaine-positive patients, whereas CD45R+/CD8+ T cells were accordingly increased in the same individuals. In some cases, direct correlation could be established between certain T cell percentages and cocaine levels. None of the serum protein levels measured appeared to be influenced by cocaine. These findings demonstrate that cocaine utilization is associated with variations in levels of certain T cell subpopulations and other immune cells. This may represent a disruption of particular immunologic cell networks which could ultimately influence host resistance to infection and malignancy.

Prenatal compensatory renal growth: documentation with US.

PURPOSE: To determine whether compensatory renal growth (CRG), which occurs in adults, children, and infants after loss of functioning renal tissue, occurs in fetal life. MATERIALS AND METHODS: Prenatal ultrasound scans were used to select 29 patients with a unilateral multicystic dysplastic kidney and four patients with unilateral renal agenesis. Accurate measurements of prenatal renal length were obtained in 21 of these patients; an accurate measurement of postnatal renal length and birth weight was obtained in 27 of these patients. Prenatal and postnatal renal measurements of the contralateral kidney were compared with renal length in 23 matched control patients and previously reported normal renal lengths. RESULTS: The single functioning kidneys in the study patients were significantly longer than those in the control patients before and after birth (P = .001). CONCLUSION: This study provides strong evidence to support the belief that CRG occurs before birth.

Renal abscess in children.

Three cases of renal abscesses in children are described to illustrate the variable presenting features. An additional 23 pediatric cases, reported over the past ten years, were reviewed for clinical features and therapy. Fever, loin pain, and leukocytosis were common presenting features, but less than half of all abscesses were associated with either an abnormal urinalysis or a positive urine culture. The presenting features were sometimes confused with appendicitis, peritonitis, or a Wilms tumor. An organism was identified in 17 cases--Escherichia coli in 9 children and Staphylococcus aureus in 8 children. The majority of E. coli infections occurred in girls and the majority of S. aureus infections occurred in boys. Reflux was documented in 5 patients, and 2 children had a possible extrarenal source of infection. Antibiotics alone produced a cure in 10 children (38%), but 16 children (62%) required a surgical procedure.