Whole-school interventions promoting student commitment to school to prevent substance use and violence: a systematic review.

OBJECTIVES: Whole-school interventions that promote student commitment to school are a promising modality to reduce health inequalities through school-level change; however, evidence for the effectiveness of these interventions in improving policy-relevant health outcomes, such as substance use and violence, has not been comprehensively synthesised. STUDY DESIGN: This was a systematic review and meta-analysis. METHODS: We searched 20 databases and a range of other sources to identify randomised trials meeting our intervention definition and reporting substance use and violence outcomes. Extracted effect estimates were meta-analysed using robust variance estimation with random effects, separating effects <1 year from baseline and effects at or more than 1 year from baseline. RESULTS: We included 18 evaluations with varying risk of bias. Pooled effects suggested significant impacts on short-term (odds ratio [OR] = 0.85, 95% confidence interval [CI] 0.76, 0.96) and long-term (OR = 0.79, 95% CI 0.65, 0.98) violence perpetration, short-term (OR = 0.84, 95% CI 0.72, 0.98) and long-term (OR = 0.85, 95% CI 0.73, 0.99) violence victimisation, and short-term (OR = 0.83, 95% CI 0.70, 0.97) and long-term (OR = 0.79, 95% CI 0.62, 0.998) substance use outcomes, with effects relatively stable between short-term and long-term analyses. Stratifying substance use meta-analyses by type (e.g. smoking, alcohol) did not impact results. All meta-analyses had substantial heterogeneity. CONCLUSION: Although diverse in content, interventions appear effective with respect to the review outcomes and as a form of universal prevention. Future research should consider contextual contingencies in intervention effectiveness, given considerable policy and practice interest in these interventions and the need to support schools in effective decision-making as to intervention choice.

Retrospective study found that outpatient care for infants exposed to drugs during pregnancy was sustainable and safe.

AIM: We determined the safety, feasibility and sustainability of an outpatient model of care for infants exposed to intra-uterine drugs. METHODS: This was a retrospective chart review of 774 drug-exposed infants born between 1998 and 2016 at the Royal Hospital for Women, Sydney, Australia. RESULTS: Most (86%) of the mothers used multiple drugs, including opioids (58%). More than three-quarters (78%) of the infants were born full term at a mean gestation of 38 weeks and hospitalised for a median of seven days. This rose to 14 days if they were medicated for neonatal abstinence syndrome (NAS). Most of the NAS patients (83%) were discharged on medication, namely morphine, and the median duration of NAS treatment was 76 (interquartile range 35-120). Three medication errors occurred: two extra doses of phenobarbitone and one infant weaned off morphine faster than prescribed. No infants were rehospitalised for NAS. Four died from sudden infant death syndrome at 2.2-5.8 months after discharge and one drowned at 15 months. None were medicated at the time of death. CONCLUSION: Outpatient care for drug-exposed infants was sustainable and had low complication rates, even for those with NAS. The optimum duration of follow-up and impact on hospital costs should be examined.

The Barts Health NHS Trust COVID-19 cohort: characteristics, outcomes and risk scoring of patients in East London.

BACKGROUND: Barts Health National Health Service Trust (BHNHST) serves a diverse population of 2.5 million people in London, UK. We undertook a health services assessment of factors used to evaluate the risk of severe acute respiratory coronavirus 2 (SARS-CoV-2) infection.METHODS: Patients with confirmed polymerase chain reaction (PCR) test results admitted between 1 March and 1 August 2020 were included, alongwith clinician-diagnosed suspected cases. Prognostic factors from the 4C Mortality score and 4C Deterioration scores were extracted from electronic health records and logistic regression was used to quantify the strength of association with 28-day mortality and clinical deterioration using national death registry linkage.RESULTS: Of 2783 patients, 1621 had a confirmed diagnosis, of whom 61% were male and 54% were from Black and Minority Ethnic groups; 26% died within 28 days of admission. Mortality was strongly associated with older age. The 4C mortality score had good stratification of risk with a calibration slope of 1.14 (95% CI 1.01-1.27). It may have under-estimated mortality risk in those with a high respiratory rate or requiring oxygen.CONCLUSION: Patients in this diverse patient cohort had similar mortality associated with prognostic factors to the 4C score derivation sample, but survival might be poorer in those with respiratory failure.

Are child passenger fatalities and child passenger severe injuries equally affected by child restraint legislation? The case of Chile.

The objective of this study is to determine the contribution of Chile's 2005 child restraint legislation to the reduction of child passenger fatalities and severe injuries. We analysed motor vehicle injury and fatality data from Chile's National Road Safety Commission of the Ministry of Transport from 2000 to 2012 to determine the effect of Chile's 2005 mandatory child restraint legislation. Using interrupted time-series Poisson regression models, we assessed the effect of the law on two dependent variables: (1) number of child fatalities in car and (2) number of children severely injured. The independent variable was the 2005 enactment of Chile's mandatory child restraint legislation. Coefficients from the interrupted time-series Poisson regression models indicate that Chile's enactment of child restraint legislation in 2005 is significantly associated with a total of 35% reduction in child passenger severely injured but only three years after its enactment, and significant associations between this policy and child fatalities were less evident.

Ovine placental eluate immunoglobulins recognise isologous and third party acid-treated trophoblast microvesicle antigens in vitro.

Placental microvesicles were prepared from ovine placentae and immunoglobulins eluted with 0.5 M glycine buffer pH 2.5. The ability of eluate immunoglobulins to re-associate with isologous (self) and third party acidified microvesicles was tested by ELISA. Ovine placental immunoglobulins re-associated with isologous and third party acidified microvesicles suggesting that at least 2 types of antigenic epitopes I and II maybe expressed on the ovine placentae. Type I antigens may be present on placentae of all ovines while type II epitopes may be paternally derived, hence unique to each pregnancy. Analysis by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27 kDa, respectively, together giving a relative molecular weight of 158 kDa. Results suggest that immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the trophoblast, possibly defining a mechanism by which the foetus evades maternal immunological rejection.

Induction of silent myocardial ischemia with mental stress testing: relation to the triggers of ischemia during daily life activities and to ischemic functional severity.

OBJECTIVES: This study examined the relations among the triggers of ischemia during the activities of daily life, mental stress-induced ischemia in the laboratory and functional severity of ischemia on exercise testing. BACKGROUND: Myocardial ischemia is readily induced with exercise testing, but most episodes of ischemia in daily life occur during relatively sedentary activities. Although mental and emotional arousal are known to trigger myocardial ischemia, mental stress testing induces ischemia in only approximately 50% of patients with active coronary disease. It is not known whether such patients are particularly susceptible to nonexertional ischemia during daily activity. METHODS: We studied 45 men (mean age +/- SD 58 +/- 9 years) with coronary artery disease by means of 48-h Holter ambulatory electrocardiography for ST segment analysis and quantification of physical and mental activity with a structured diary system. These data were cross-tabulated with new left ventricular dyssynchrony (detected on two-dimensional echocardiography) induced by two mental stressors and by bicycle exercise. RESULTS: During mental stress testing, 24 patients (53%) (Group I) had a new wall motion abnormality; the other 21 patients (Group II) did not. The average wall motion dyssynchrony score increased from 1.20 +/- 0.29 to 1.34 +/- 0.36 (p = 0.001), but the increase was less than that with exercise stress (1.52 +/- 0.41, p = 0.001). The total duration of ischemia during sedentary activities was greater in Group I (22.9 +/- 24.5 min) than in Group II (3.6 +/- 3.9 min, p = 0.025). Group I had more ischemic events while sedentary (23 of 290 diary entries) than did Group II (8 of 256 diary entries, p = 0.015). The magnitude of dyssynchrony with mental stress and the number of mental stressors capable of triggering ischemia were related to severity of ischemia with exercise. CONCLUSIONS: Patients with ischemia during mental stress testing also have increased ischemia during sedentary activities in daily life. This finding may reflect greater functional severity of coronary artery disease or a propensity toward coronary vasoconstriction while sedentary.

Subcapsular hematoma of the liver after laparoscopic cholecystectomy.

Two female patients underwent an uneventful laparoscopic chloecystectomy (LC) for cholelithiasis. Their past medical history was insignificant. The first patient had diclofenac sodium for her postoperative pain relief. Both patients returned in the early postoperative period with pain in the right hypochondrium. Laboratory investigations revealed elevated leucocytes, C reactive protein (CRP), and deranged liver function tests. A computed tomography (CT) scan showed subcapsular haematoma of liver. CT-guided aspiration of hematoma was done in one case. Both patients improved over a period of time and a follow-up radiological scan showed resolving hematoma. The presentation, diagnostic evaluation, treatment, and possible causes are discussed.

Immunoglobulin G bound to ovine placenta is eluted by surgical cannulation and acid perfusion in situ.

OBJECTIVE: To elute placental bound immunoglobulin G (IgG) in situ. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department, The University of Newcastle Australia and the Department of Biochemistry, University of Nairobi, Kenya. SUBJECTS: Twelve pregnant ewes 10 to 15 days before the onset of natural parturition. RESULTS: Placental eluates were rich in IgG, and IgG2. The relative molecular weight of placental IgG was estimated at 158kDa by gel filtration chromatography. Analysis of eluate by SDS PAGE revealed the heavy and light chains of IgG at 57 and 27kDa respectively together giving a relative molecular weight of 168kDa. CONCLUSION: Placental bound IgG may be crucial in immunology of pregnancy and together with the cognate antigen thereof may be useful as models for the study of maternal-fetal interaction in human pregnancy and in the development of experimental immunotherapy to immunologically compromised pregnancies in humans and livestock.

Human placental immunoglobulins show unique re-association patterns with isologous and third party acid treated trophoblast microvesicles in vitro.

OBJECTIVE: To study re-association pattern of human placental eluate immunoglobulins with acid treated isologous and third party trophoblast derived placental microvesicles. DESIGN: Laboratory based experimentation. SETTING: Biological Sciences Department and Discipline for Reproductive Medicine University of Newcastle, Australia and the Department of Biochemistry, University of Nairobi, Kenya. RESULTS: Placental eluate immunoglobulins re-associated with isologous and third party acidified microvesicles in three distinct patterns. I: eluate immunoglobulins re-associated more strongly with isologous and third party acid treated placental microvesicles, II: eluate immunoglobulins re-associated strongly with isologous but weakly with third party acid treated placental microvesicles, III: eluate immunoglobulins did not show preferential re-association with isologous and third party acid treated placental microvesicles. CONCLUSION: Two types of antigenic epitopes I and II may be expressed on the human placentae. Type I antigens may be present on all human placentae while type II epitopes may be paternally derived hence unique to each pregnancy. Also, immunoglobulins produced to placental microvesicle antigens may be directed to some but not all antigenic epitopes expressed on the human placental trophoblast.

Immunoreactive methionine-enkephalin in cerebrospinal fluid and blood plasma during acute stress in conscious sheep.

The opioid peptide methionine-enkephalin (Met-enkephalin) was measured in plasma and cerebrospinal fluid (CSF) of sheep in which the cisterna magna, carotid artery, and jugular vein were chronically cannulated. Venous blood plasma and CSF were collected before and after stress treatment and in control studies in conscious animals. Plasma and CSF were extracted with octadecylsilica and oxidized, and Met-enkephalin was measured as its Met-sulfoxide derivative by specific RIA. The molecular form of immunoreactive Met-enkephalin was characterized by peptide size exclusion chromatography of an octadecylsilica extract of sheep plasma through Bio-Gel P2, followed by reverse phase liquid chromatography, and was identical to Met-enkephalin and Met-sulfoxide-enkephalin. Insulin-induced hypoglycemia produced an elevation of plasma cortisol and an increase in the plasma concentration of Met-enkephalin. Acute hemorrhage led to an earlier and greater rise in plasma cortisol than that associated with insulin-induced hypoglycemia, but did not increase the concentration of Met-enkephalin in plasma. Neither form of acute stress increased the concentration of Met-enkephalin in CSF. These studies confirm that secretion of Met-enkephalin into blood can be dissociated from stimulation of the pituitary-adrenocortical system. They also show that circulating Met-enkephalin is elevated in conscious sheep during acute hypoglycemic stress, but plasma Met-enkephalin is unlikely to exert effects on the opiate receptors of periaqueductal or spinal nociceptive neurons under these conditions, since it does not enter cerebrospinal fluid in significant amounts.

Acute hemorrhagic stress in conscious sheep elevates immunoreactive beta-endorphin in plasma but not in cerebrospinal fluid.

The effects of acute hemorrhagic stress on the concentrations of immunoreactive beta-endorphin (IR beta EP) in cerebrospinal fluid (CSF) and blood plasma were investigated in conscious sheep in which the cisterna magna, a carotid artery, and a jugular vein were chronically cannulated. Serial samples of CSF and jugular venous blood were collected before and after acute arterial hemorrhage and in control experiments. Basal concentrations of IR beta EP were higher in plasma than in CSF. Plasma concentrations of cortisol and IR beta EP increased within 45 min of the commencement of hemorrhage and returned to near baseline levels within 2.25 h. The concentrations of cortisol and IR beta EP in plasma observed after hemorrhage were significantly different from those observed in controls (analysis of variance). Neither the molar nor the relative changes from initial concentrations of IR beta EP in CSF were significantly different between hemorrhage-stressed and controls by analysis of variance. These results show that hemorrhagic stress in conscious sheep elevates concentrations of IR beta EP in plasma but not in CSF, indicating that pituitary beta EP secreted into blood does not enter CSF in significant amounts.

A corticotropin-releasing hormone type I receptor antagonist delays parturition in sheep.

In sheep, corticotropin-releasing hormone (CRH) can stimulate the fetal release of ACTH to produce a cortisol surge which leads to the onset of parturition. We tested the hypothesis that fetal CRH is a primary factor in the onset of parturition in sheep by using a Type I CRH receptor antagonist, antalarmin, to block the endogenous action of CRH. Pregnant ewes were cannulated at 130-135 days of gestation. Five catheters were placed into the amniotic sac, fetal femoral artery, fetal tarsal vein, maternal jugular vein and carotid artery. After 5 days' recovery, blood samples from maternal and fetal vessels were collected at the following times: a day before the start of infusion, at [-1, 0, 1, 2, 4, 8 and 24]h, on the first day of infusion, and thereafter daily throughout a 10-day infusion. Animals (n=6 per group) received infusions into a fetal vein of either a vehicle comprising 1:1 mixture of ethanol and polyethoxylated castor oil (Cremophor EL) or antalarmin (50 g/L) in the vehicle at a rate of 0.3 mL/h. The plasma samples were assayed for ACTH and cortisol using commercial RIA kits. Fetuses infused with vehicle delivered at a mean gestational age of 141.8 +/- 0.9 days compared with antalarmin-infused sheep at 148.8 +/- 1.6 days (P = 0.0036, unpaired Student's t-test). Fetal ACTH and cortisol did not change in the antalarmin-infused sheep after 3 days' infusion compared to significant increases in vehicle-infused sheep (P=0.004 and P = 0.016 respectively, ANOVA). These data show that CRH receptor antagonism in the fetus can delay the onset of parturition. It supports the hypothesis that hypothalamic CRH drives fetal production of ACTH and is essential for the onset of parturition triggered by a surge in fetal cortisol.

Effect of restriction of placental growth on the concentrations of insulin, glucose and placental lactogen in the plasma of sheep.

The effect of restricting placental growth on maternal glucose, insulin and placental lactogen was investigated in 16 ewes carrying singleton lambs. Uterine caruncles were removed from seven ewes (caruncle ewes) before pregnancy, resulting in reduced placental size and retarded intra-uterine fetal growth. The concentration of insulin in maternal plasma was similar in both control and caruncle ewes. The concentration of glucose was significantly higher in the caruncle than in the control ewes (3.26 +/- 0.15 (S.E.M.) mmol/l, number of observations (n) = 9, vs 2.75 +/- 0.1, n = 9, P less than 0.02, and 3.27 +/- 0.16, n = 7, vs 2.46 +/- 0.11, n = 12, P less than 0.001, for the carotid artery and utero-ovarian vein respectively). The concentration of ovine placental lactogen (oPL) in the utero-ovarian vein was reduced in the caruncle compared with the control ewes (283 +/- 65 micrograms/l, n = 8, and 705 +/- 106 micrograms/l, n = 18, P less than 0.02, respectively). Restriction of placental growth by removal of endometrial caruncles similarly reduced the concentrations of oPL in maternal arterial plasma (231 +/- 54 micrograms/l, n = 9, and 621 +/- 96 micrograms/l, n = 18, P less than 0.002). Production of oPL by the placenta was also reduced by limiting placental growth to 30 +/- 11 micrograms/min, n = 8, compared with 133 +/- 43 micrograms/min, n = 15, P less than 0.05, for the controls. Production of oPL per gram of placenta in the caruncle group, although only 34% of the control value, was not reduced significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of beta-endorphin into the maternal circulation by uteroplacental tissues in response to hypoglycaemic stress.

The placenta has been shown to contain ACTH and beta-endorphin but the roles of these peptides are unknown. To investigate whether they are released into the maternal circulation from the placenta in response to physiological stimuli the effects of hypoglycaemic stress were investigated. Plasma samples were collected from the femoral artery (FA) and uterovarian (UV) vein of nine pregnant sheep before and during hypoglycaemia induced by intravenous insulin (100U). Plasma concentrations of ovine beta-endorphin (o beta-EP) were measured by radioimmunoassay. Concentrations of o beta-EP rose in both vessels by 60 min after insulin. The peak concentrations of o beta-EP (pmol/l) were 122 +/- 29 (mean +/- SEM, n = 8) in the UV and 96 +/- 24 (n = 9) fmol/ml in the FA 60 min after insulin injection. There was no difference between the concentrations of o beta-EP in the vessels before insulin injection but at 60 and 120 min after insulin the concentrations of o beta-EP were significantly higher in the UV than FA (P less than 0.02, analysis of variance). This indicates that the pregnant uterus or placenta can respond to hypoglycaemia by secreting beta-EP into the maternal circulation. It is therefore possible that placental pro-opiomelanocortin (POMC) peptides may have a role in maternal endocrinology and metabolism.

Somatomedin-like activity in cattle: the effect of breed, lactation and time of day.

Somatomedin-like activity (SLA) was measured in plasma samples from cattle using the porcine costal cartilage disk bioassay. In 40-day-old pre-ruminant bull calves, SLA was highest in the plasma of eight Friesians (mean potency 0.97 +/- 0.06 (S.E.M.) units/ml), lowest in five Aberdeen Angus x Friesians (0.78 +/- P < 0.05) and intermediate in six Hereford x Friesians (9.89 +/- 0.09). Plasma levels of SLA were significantly (P < 0.01) lower in eight lactating Friesian (high-yielding) cows (0.53 +/- 0.04) than in five dry cows (0.76 +/- 0.06). Levels of SLA in the plasma of a further eight lactating Hereford x Friesian (low-yielding) cows (0.76 +/- 0.07) were similar to the levels in the plasma of the five dry dairy cows. Large diurnal changes in plasma SLA occurred in lactating Hereford x Friesian and Friesian heifers. In three Hereford x Friesian heifers plasma levels of SLA were lowest at 19.00 h (0.39 +/- 0.07) and highest at 03.00 h (0.77 +/- 0.08). A similar pattern was present in plasma samples from three Friesian heifers; the lowest levels of SLA being at 21.00 h (0.37 +/- 0.07) and the highest at 05.00 h (0.61 +/- 0.13). Throughout the period of 24 h the overall mean levels of SLA were significantly (P < 0.01) higher in the plasma samples from the three Hereford x Friesian heifers (0.6 +/- 0.01) than in the samples from the three Friesian heifers (0.49 +/- 0.02 units/ml).

Somatomedin-like activity in plasma after foetal hypophysectomy or nephrectomy and in experimental intra-uterine growth retardation in sheep.

Plasma samples from pregnant ewes and their foetuses during the last quarter of gestation were assayed for somatomedin-like activity (SLA) using the procine costal cartilage assay. In maternal plasma, the mean potency (compared with pooled serum from six sheep) was 0.84 +/- 0.05 (S.E.M.) units/ml (n = 15). Somatomedin-like activity in the plasma of five control foetuses (0.91 +/- 0.1 units/ml) was similar to the maternal levels and did not change with gestational age. After foetal hypophysectomy the SLA in foetal plasma (0.37 +/- 0.05 units/ml, n = 4) was significantly less than in control animals. In two nephrectomized foetuses, the mean SLA in plasma (0.08 and 0.51 units/ml respectively) was less than in control animals. Retardation of intra-uterine foetal growth was induced by removal of endometrial caruncles before pregnancy in four sheep. The SLA in plasma from these foetuses was 0.38 +/- 0.05 units/ml (P less than 0.01 v. control animals). The results suggest that SLA in the foetus may be important in the regulation of foetal growth, but they also indicate that factors other than growth hormone may be important in the control of SLA in foetal plasma.

Peripheral blood cells from weight-losing cancer patients control the hepatic acute phase response by a primarily interleukin-6 dependent mechanism.

Cancer cachexia is associated with an elevated hepatic acute phase protein response, poor outcome and elevated cytokine production from peripheral blood mononuclear cells (PBMC). This study investigates the mechanism by which PBMC can induce a hepatic acute phase response. Supernatants from the peripheral blood cells of cancer patients induced significantly higher C-reactive protein (CRP) from hepatocytes (198+/-21 ng ml-1) than did supernatants from healthy controls (64+/-20, p<0.005). CRP production in vitro correlated with IL-6 production by PBMC from patients with pancreatic cancer (r=0.76, p<0.0001). This C-reactive protein production was reduced by 84% using neutralising antibody to IL-6 (p<0.001). There was a significant negative correlation between PBMC-induced hepatocyte C-reactive protein production and survival (r=-0.45, p<0.01). PBMC from cancer patients induce the hepatic acute phase response via a primarily IL-6-dependent mechanism.

Reverse haemolytic plaque assay study of corticotropin-releasing hormone and arginine vasopressin interaction in ovine corticotropes.

Abstract Corticotropin-releasing hormone and arginine vasopressin are known to interact in stimulating secretion of adrenocorticotropin-related peptides from corticotropes. However, the mechanism mediating this interaction is uncertain. Recently, evidence has been provided using a reverse haemolytic plaque assay that in rat pituitary cells, arginine vasopressin potentiates the effects of corticotropin-releasing hormone by increasing the percentage of target cells that secrete adrenocorticotropin. To determine whether a similar mechanism also operates in the sheep corticotrope, which is reportedly more sensitive to arginine vasopressin than that of the rat, a reverse haemolytic plaque assay for beta-endorphin secretion was used to study the response of ovine corticotropes to stimulation by increasing doses of corticotropin-releasing hormone or arginine vasopressin (0.1 nM to 10.0 nM) alone or in combination. In the reverse haemolytic plaque assay, beta-endorphin antiserum at 1:50 and complement at 1:10 were found to be optimal dilutions for plaque formation. A concentration-dependent response curve to corticotropin-releasing hormone was obtained with a significant increase in plaque area from basal to reach maximal levels at 1.0 nM. Arginine vasopressin also stimulated an increase in plaque area, however, plaques formed were significantly smaller than those caused by corticotropin-releasing hormone. Since in the reverse haemolytic plaque assay, plaque area is related to the amount of hormone secreted by the cell, results demonstrate that although corticotropin-releasing hormone and arginine vasopressin both stimulate beta-endorphin secretion from ovine corticotropes, corticotropin-releasing hormone is a more potent secretagogue than arginine vasopressin in that it causes the formation of significantly larger plaques. The addition of arginine vasopressin to low concentrations of corticotropin-releasing hormone caused plaque areas to reach maximal levels at 0.1 nM whereas these levels were only attained at 1.0 nM when corticotropin-releasing hormone was used alone. Therefore, arginine vasopressin interacts with corticotropin-releasing hormone to increase corticotrope responses by increasing their secretory response to corticotropin-releasing hormone. These data are consistent with previous work suggesting that arginine vasopressin increases the expression of corticotropin-releasing hormone receptors on the corticotrope cell surface. However, no significant increase in the percentage of plaque-forming cells was seen with either corticotropin-releasing hormone or arginine vasopressin alone or in combination implying that there was no recruitment of previously non-secreting cells.

Large Molecular Weight Immunoreactive Corticotrophin-Releasing Hormone has Bioactivity on Superfused Ovine Pituitary Cells.

Abstract Human placental extracts fractionated with Sephadex G-50 produced three peaks of corticotrophin-releasing hormone immunoreactivity, a large molecular weight peak (M(r)30,000), an intermediate peak (4,758 < M(r) < 10,000) and a low molecular weight peak coeluting with the 41-residue hormone. All three peaks of immunoreactivity stimulated the release of beta-endorphin-like immunoreactivity from ovine pituitary cells superfused in vitro. No response was observed from unstimulated cells superfused in parallel. Gel chromatography indicated that intermediate and small molecular weight forms of human corticotrophin-releasing hormone immunoreactivity remained intact after contact with the ovine pituitary cells, whereas the large molecular weight material dissociated to produce 41-residue hormone immunoreactivity. The secreted beta-endorphin immunoreactivity was shown by gel chromatography to comprise both beta-lipotrophin-like and the 31-residue beta-endorphin-like immunoreactivity. The data show that the intermediate and low molecular weight forms of placental corticotrophin-releasing hormone immunoreactivity are bioactive and suggest that the intermediate form is a hormone precursor, possibly procorticotrophin-releasing hormone(125-196), and the small form is identical to the hypothalamic hormone. The results with the larger molecular weight material indicate that it is likely to be a complex of the mature 41-residue hormone and a binding protein.

Desensitization of superfused isolated ovine anterior pituitary cells to human corticotropin-releasing factor.

Abstract We have employed an in vitro system to study the time-course of beta-endorphin (beta-EP) immunoreactivity release from anterior pituitary cells stimulated with corticotropin-releasing factor (CRF) and whether exposure to CRF desensitizes the cells to subsequent stimulation. Ovine anterior pituitaries were enzymatically disrupted into single cells, mixed with Siegel P2 and superfused in mini-columns with carbogen-gassed medium at 37 degrees C. Superfusate fractions were collected at 5-min intervals and beta-EP immunoreactivity in the eluate was measured by radioimmunoassay. Peaks of beta-EP release that rose significantly above baseline noise were detected using the PULSAR algorithm. Unstimulated cell columns did not display any spontaneous peaks of beta-EP discharge detectable by PULSAR whereas peaks were identified in the output of columns exposed to 1 nM CRF for 100 min. beta-EP release increased after 10 min of stimulation and maximum stimulated output was achieved after 20 min of continuous CRF exposure. Between 20 and 60 min of CRF stimulation the rate of beta-EP release declined progressively but stabilized in the last 40 min of the exposure at a level significantly above controls for baseline secretion. Peak duration did not depend on the inclusion of calcium in the superfusion medium while peak amplitude and area were significantly reduced when cells were denied extracellular calcium. Following a 100-min exposure to 1 nM CRF, pituitary cell columns were given a 30-min rest period then restimulated with either 1 nM CRF or 50 mM KCI for 20 min. The columns given prior exposure to CRF did not mount a response (detectable by PULSAR) to a subsequent dose of 1 nM CRF whereas PULSAR detected a clear response in all members of a control group that had not received prior CRF challenge. Both CRF exposed and control columns responded to 50 mM KCI although the response was significantly attenuated in the cells that had received prior CRF treatment. These results indicate that unstimulated superfused isolated ovine anterior pituitary cells do not possess an inherent rhythmicity of beta-EP release that can be detected by the PULSAR algorithm while treatment of the cells with CRF results in detectable discharge. The rapid response of beta-EP discharge to CRF treatment suggests the presence of intracellular beta-EP stores available for rapid mobilization. Continuous exposure to 1 nM CRF can tonically amplify corticotrope output for the duration of its presence in the environment of the corticotrope, but the maximum rate of release cannot be maintained. An inrush of extracellular calcium is not essential for the corticotrope to mount a detectable response to continuous CRF exposure but the release of a maximum amount of beta-EP relies on calcium entry. Long-term treatment with CRF prevents the corticotrope releasing a detectable peak of beta-EP on subsequent CRF stimulation and therefore CRF exposure leaves a lasting impression on the physiological machinery of the corticotrope. The attenuation of responsiveness to 50 mM KCI after long-term CRF treatment indicates that depletion of beta-EP stores may play a part in corticotrope desensitization although a reduction in CRF receptor number and an alteration in the intracellular mechanisms controlling beta-EP release may also be a factor.