Intravenous Endotoxin Challenge in Healthy Humans: An Experimental Platform to Investigate and Modulate Systemic Inflammation.

Activation of inflammatory pathways represents a central mechanism in multiple disease states both acute and chronic. Triggered via either pathogen or tissue damage-associated molecular motifs, common biochemical pathways lead to conserved yet variable physiological and immunological alterations. Dissection and delineation of the determinants and mechanisms underlying phenotypic variance in response is expected to yield novel therapeutic advances. Intravenous (IV) administration of endotoxin (gram-negative bacterial lipopolysaccharide), a specific Toll-like receptor 4 agonist, represents an in vivo model of systemic inflammation in man. National Institutes for Health Clinical Center Reference Endotoxin (CCRE, Escherichia coli O:113:H10:K negative) is employed to reliably and reproducibly generate vascular, hematological, endocrine, immunological and organ-specific functional effects that parallel, to varying degrees, those seen in the early stages of pathological states. Alteration of dose (0.06 - 4 ng/kg) and time-scale of exposure (bolus vs. infusion) allows replication of either acute or chronic inflammation and a range of severity to be elicited, with higher doses (2 - 4 ng/kg) frequently being used to create a 'sepsis-like' state. Established and novel medicinal compounds may additionally be administered prior to or post endotoxin exposure to appreciate their effect on the inflammatory cascade. Despite limitations in scope and generalizability, human IV endotoxin challenge offers a unique platform to gain mechanistic insights into inducible physiological responses and inflammatory pathways. Rationally employed it may aid translation of this knowledge into therapeutic innovations.

ATTIRE: Albumin To prevenT Infection in chronic liveR failurE: study protocol for a single-arm feasibility trial.

INTRODUCTION: Circulating prostaglandin E2 levels are elevated in acutely decompensated cirrhosis and have been shown to contribute to immune suppression. Albumin binds and inactivates this hormone. Human albumin solution could thus be repurposed as an immune restorative drug in these patients.This feasibility study aims to determine whether it is possible and safe to restore serum albumin to >30 g/L and maintain it at this level in patients admitted with acute decompensated cirrhosis using repeated 20% human albumin infusions according to daily serum albumin levels. METHODS AND ANALYSIS: Albumin To prevenT Infection in chronic liveR failurE (ATTIRE) stage 1 is a multicentre, open label dose feasibility trial. Patients with acutely decompensated cirrhosis admitted to hospital with a serum albumin of <30 g/L are eligible, subject to exclusion criteria. Daily intravenous human albumin solution will be infused, according to serum albumin levels, for up to 14 days or discharge in all patients. The primary end point is daily serum albumin levels for the duration of the treatment period and the secondary end point is plasma-induced macrophage dysfunction. The trial will recruit 80 patients. Outcomes will be used to assist with study design for an 866 patient randomised controlled trial at more than 30 sites across the UK. ETHICS AND DISSEMINATION: Research ethics approval was given by the London-Brent research ethics committee (ref: 15/LO/0104). The clinical trials authorisation was issued by the medicines and healthcare products regulatory agency (ref: 20363/0350/001-0001). RESULTS: Will be disseminated through peer reviewed journals and international conferences. Recruitment of the first participant occurred on 26/05/2015. TRIAL REGISTRATION NUMBER: The trial is registered with the European Medicines Agency (EudraCT 2014-002300-24) and has been adopted by the NIHR (ISRCTN 14174793). This manuscript refers to V.4.0 of the protocol; Pre-results.

Feasibility and cost of using mobile phones for capturing drug safety information in peri-urban settlement in Ghana: a prospective cohort study of patients with uncomplicated malaria.

BACKGROUND: The growing need to capture data on health and health events using faster and efficient means to enable prompt evidence-based decision-making is making the use of mobile phones for health an alternative means to capture anti-malarial drug safety data. This paper examined the feasibility and cost of using mobile phones vis-à-vis home visit to monitor adverse events (AEs) related to artemisinin-based combination therapy (ACT) for treatment of uncomplicated malaria in peri-urban Ghana. METHODS: A prospective, observational, cohort study conducted on 4270 patients prescribed ACT in 21 health facilities. The patients were actively followed by telephone or home visit to document AEs associated with anti-malarial drugs. Call duration and travel distances of each visit were recorded. Pre-paid call cards and fuel for motorbike travels were used to determine cost of conducting both follow-ups. Ms-Excel 2010 and STATA 11.2 were used for analysis. RESULTS: Of the 4270 patients recruited, 4124 (96.6 %) were successfully followed up and analyzed. Of these, 1126/4124 (27.3 %) were children under 5 years. Most 3790/4124 (91.9 %) follow-ups were done within 7 days of ACT intake. Overall, follow up by phone (2671/4124-64.8 %) was almost two times the number done by home visits (1453/4124-35.2 %). Duration of telephone calls ranged from 38 s to 53 min, costing between GH¢0.26 (0.20USD) and GH¢41.70 (27.USD). On the average, the calls lasted 3 min 51 s (SD = 3 min, 21 s) costing GH¢2.70 (0.77USD). Distance travelled for home visit ranged from 0.65 to 62 km costing GH¢0.29 (0.20USD) and GH¢279.00 (79.70USD). Thirty-two per cent (1128/4124) of patients reported AEs. In total, 1831 AE were reported, 1016/1831(55.5 %) by telephone and 815/1831 (44.5 %) by home visits. Events such as nausea, dizziness, diarrhoea, and vomiting were commonly reported. CONCLUSION: Majority of patients was successfully followed up by telephone and reported the most AEs. The cost of telephone interviewing was almost two times less than the cost of home visit. Telephone follow up should be considered for monitoring drug adverse events in low resource settings.

Severe alkalosis and hypokalemia with stanozolol misuse.

Stanozolol is a popular androgenic anabolic steroid, used by body builders and athletes for physical performance enhancement. There are few data on its potential adverse effects and no documented cases of it causing severe electrolyte imbalance. Here, we report a patient presenting to a tertiary care emergency department with reduced conscious level, profound hypokalemia, and severe metabolic alkalosis, resulting from stanozolol misuse. This is the first such case reported.

Development of an allogeneic whole-cell tumor vaccine expressing xenogeneic gp100 and its implementation in a phase II clinical trial in canine patients with malignant melanoma.

A xenogeneic melanoma-antigen-enhanced allogeneic tumor cell vaccine (ATCV) is an appealing strategy for anti-cancer immunotherapy due to its relative ease of production, and the theoretical possibility that presentation of a multiplex of antigens along with a xenogeneic antigen would result in cross-reaction between the xenogeneic homologs and self-molecules, breaking tolerance and ultimately resulting in a clinically relevant immune response. In this study, we evaluated the efficacy of such a strategy using a xenogeneic melanoma differentiation antigen, human glycoprotein 100 (hgp100) in the context of a phase II clinical trial utilizing spontaneously arising melanoma in pet dogs. Our results demonstrate that the approach was well tolerated and resulted in an overall response rate (complete and partial response) of 17% and a tumor control rate (complete and partial response and stable disease of >6 weeks duration) of 35%. Dogs that had evidence of tumor control had significantly longer survival times than dogs that did not experience control. Delayed type hypersensitivity (DTH) to 17CM98 canine melanoma cells used in the whole cell vaccine was enhanced by ATCV and correlated with clinical response. In vitro cytotoxicity was enhanced by ATCV, but did not correlate with clinical response. Additionally, anti-hgp100 antibodies were elicited in response to ATCV in the majority of patients tested; however, this also did not correlate with clinical response. This approach, along with further elucidation of the mechanisms of tumor protection after xenogeneic immunization, may allow the development of more rational vaccines. This trial also further demonstrates the utility of spontaneous tumors in companion animals as a valid translational model for the evaluation of novel vaccine therapies.

Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption.

Mechanisms responsible for increased jejunal transport rates observed in tissues treated with orally administered insulin-like growth factor-I (IGF-I) were studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg. kg(-1). day(-1)) or control vehicle was given orogastrically for 4 days. Disaccharidase activity, fructose uptake, and Na+-glucose cotransporter SGLT-1 protein abundance were similar between groups. Oral IGF-I produced greater rates of enterocyte Na+-K+-ATPase activity with no significant differences in Na+-K+-ATPase abundance. Cellular mechanisms responsible for transport changes were studied in Ussing chambers. In control tissues, the presence of IGF-I in mucosal solutions increased basal short-circuit current (I(sc)), potential difference, D-glucose-stimulated I(sc), and Na+-K+-ATPase activity; these changes were abolished by preincubation of tissues with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. The results suggest that the effect of IGF-I on jejunal ion and nutrient transport involves activation of PI 3-kinase and stimulation of Na+-K+-ATPase activity in enterocytes.

Oral IGF-I enhances nutrient and electrolyte absorption in neonatal piglet intestine.

The effect of orally administered insulin-like growth factor-I (IGF-I) on small intestinal structure and function was studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg. kg(-1). day(-1)) or control vehicle was given orogastrically for 4 days. Body weights, jejunal and ileal mucosa wet and dry weights, and serum IGF-I levels were similar in the two groups. Small intestinal villus height and crypt depth and jejunal enterocyte microvillar dimensions were also similar between groups. Oral IGF-I produced higher rates of jejunal ion transport because of increased basal Na+ absorption. Short-circuit current responses to mucosal addition of D-glucose and L-alanine and net transepithelial absorption of 3-O-methylglucose were increased by IGF-I. Carrier-mediated uptake of D-glucose per milligram in everted jejunal sleeves was greater in IGF-I-treated piglets because of a significantly greater maximal rate of uptake. We conclude that rates of net Na+ and Na+-dependent nutrient absorption are enhanced in piglets treated with oral IGF-I, and this effect is independent of changes in mucosal mass or surface area.

Clinical and immunohistochemical characterization of thymic lymphosarcoma in a heifer.

A 2-year-old Holstein heifer with a swollen brisket, jugular vein distention, muffled heart sounds, tachycardia, and free gas bloat was examined. Thymic lymphosarcoma was suspected based on a negative agar gel immunodiffusion test for bovine leukemia virus, presence of atypical lymphocytes in pleural fluid, and detection of a mass in the thoracic inlet. Right-sided cardiac catheterization was performed, and markedly increased jugular venous pressures (41 mm Hg) with a pressure gradient of 29 mm Hg immediately cranial to the heart indicated constriction of the cranial vena cava. Immunohistochemical staining of formalin fixed, paraffin-embedded tissue sections of the tumor using a rabbit antihuman T cell, CD3 polyclonal antibody confirmed that the neoplastic lymphocytes were of thymic origin.

Single dose responses to the gonadotropin-releasing hormone agonist analogue [(imBz 1)-D-His6,Pro9-NEt]GnRH.

The agonist analogue of gonadotropin-releasing hormone (GnRH), [(imBz1)-D-His6,Pro9-NEt]GnRH, has a potency 200 times that of the native hormone in vitro. In single dose studies in man, this analogue resulted in 2- to 4-fold elevation of LH and FSH, and demonstrated a prolonged duration of activity. [(imBz1)-D-His6,Pro9-NEt]GnRH appears to be safe and, as with other analogues of GnRH, may have application to clinical medicine.

Clinical studies with synthetic ovine corticotropin-releasing factor.

Ovine corticotropin-releasing factor (oCRF) stimulates increased plasma immunoreactive adrenocorticotropin (IR-ACTH) and IR-cortisol at threshold, half-maximal, and maximal doses of 0.01-0.03, 0.3-1, and 3-10 micrograms/kg, respectively. Side effects occur with increasing frequency, severity, and duration at doses above 1 microgram/kg. oCRF has a prolonged duration of action, at least in part because of the long circulating half-life of intact oCRF in plasma. Increasing doses of oCRF given in late afternoon progressively diminish the next morning's circadian rise in plasma IR-ACTH in normal subjects, but not in Addisonian patients or subjects receiving metyrapone, indicating that prolonged oCRF-induced hypercortisolemia is the cause. Plasma IR-lipotropins and IR-beta-endorphin rise and fall concomitantly with IR-ACTH after oCRF injection. Arginine vasopressin increases the IR-ACTH response to oCRF fourfold when given simultaneously with oCRF. Cushing's disease patients respond variably, suggesting that oCRF may not be a very useful diagnostic agent in Cushing's syndrome. However, the combination of oCRF with growth hormone-releasing factor, gonadotropin-releasing hormone, and thyrotropin-releasing hormone appears to provide a rapid and useful test of combined anterior pituitary function.

Suppression of plasma testosterone leads to an increase in serum total and high density lipoprotein cholesterol and apoproteins A-I and B.

Men have lower high density lipoprotein (HDL) and higher low density lipoprotein (LDL) levels than women. To dynamically evaluate the role of endogenous testosterone on the lipoprotein profile, eight normal men received a long-acting gonadotropin releasing hormone analog (LHRHA) for 10 weeks by SC injection. Plasma testosterone levels were acutely lowered below 1 ng/ml after 4 weeks of LHRHA treatment and remained depressed at this level for the duration of administration of the analog. There were prompt increases in total cholesterol [baseline vs. peak (milligrams per dl) mean +/- SEM, 177 +/- 18 vs. 208 +/- 22; P less than 0.005], apoprotein B (apo B; 69 +/- 12 vs. 97 +/- 13; P less than 0.05), HDL-cholesterol (23 +/- 2 vs. 33 +/- 2; P less than 0.005), and apo A-I (80 +/- 7 vs. 112 +/- 5; P less than 0.005), but not in apo A-II (40 +/- 3 vs. 40 +/- 4; P = NS) levels. The peaks occurred after 10 weeks of treatment and were followed by a fall in these values after discontinuing LHRHA. These changes were largely prevented in a second study (six men) in which LHRHA was administered together with im testosterone enanthate, which was given every 2 weeks. These results show that suppression of endogenous testosterone leads to increases in HDL and LDL, demonstrating that testosterone has an important effect on lipoprotein metabolism and plays a key role in defining the lipoprotein profile in men.

Gonadotropin and steroid secretory patterns during chronic treatment with a luteinizing hormone-releasing hormone agonist analog in men.

LHRH agonist analogs induce hypogonadism in man but the mechanism is uncertain. To evaluate this, we treated 13 normal men with 50 micrograms/day D-Trp6,Pro9-NEt LHRH (LHRHA) for periods up to 8 weeks and measured (1) patterns of endogenous gonadotropin and testosterone secretion, (2) gonadal response to exogenous human LH infusions, and (3) gonadotropin and testosterone responses to hourly bolus doses of LHRH. Seven men were evaluated with frequent sampling for 12-h periods every 4 week during treatment with LHRHA. Before treatment, all had three to four spikes of LH in 12 h. On the first day of treatment, the response to LHRHA was tested in four of the men, and there were significant increases in LH, FSH, and testosterone. After 4 weeks, all men had dramatic decreases in mean testosterone levels and blunted or absent gonadotropin responses to acute injection of LHRHA. Mean gonadotropin levels at 4 and 8 weeks were variable; values lower than pretreatment basal levels were found in three men, while unchanged or higher values were found in the remaining four. The pulsatile pattern of LH secretion, characteristic among these men before treatment, was lost during LHRHA therapy. Forty-eight-hour constant infusion of human LH in four other analog-treated men resulted in increases in serum testosterone comparable to those in untreated men. Pulsatile administration of native sequence LHRH to two other men during chronic treatment with LHRHA failed to elicit demonstrable responses in serum gonadotropin or testosterone levels. LHRHA produced a qualitative change in the pattern of LH release from the pituitary. Mean basal LH levels varied during treatment, but the normal pulsatile pattern was diminished. The gonadotropin response to pulsatile administration of LHRH was lost during chronic treatment with LHRHA, but the Leydig cell remained responsive to exogenous human LH. Thus, the locus of action of the analog appears to be at the level of the pituitary in man.

Heterogeneity of sperm density profiles following 20-week therapy with high-dose LHRH analog plus testosterone.

Eight normal male volunteers received an LHRH analog, 100 to 500 micrograms daily, for 20 weeks. Testosterone enanthate, 100 mg, was given by injection every second week. Sperm density fell to 5.5 X 10(6)/ml and to 0 in two of the subjects receiving 100 micrograms, but was unchanged in the third. Three of the subjects who received 500 micrograms displayed azoospermia, whereas the other two showed no significant change in sperm density. The reasons for the heterogeneity are not clear. Only one of the three nonresponders had testosterone values that were higher than the other subjects. Gonadotropin levels were similar in responders and nonresponders. It is possible that the response to LHRH analog in man is determined by the extent of the reduction in LH bioactivity.

Synthetic ovine corticotropin-releasing hormone: simultaneous release of proopiolipomelanocortin peptides in man.

The response of plasma proopiolipomelanocortin-derived peptide levels to synthetic ovine corticotropin-releasing hormone (CRH) was studied in six normal men. CRH was given as a 30-sec iv injection of 30 micrograms/kg body weight in the late afternoon, and blood samples were drawn for up to 16 h thereafter. Low levels of immunoreactive (IR)-ACTH, IR-beta-endorphin and IR-lipotropins (LPH) were measured before CRH administration. All subjects had prompt, concomitant, biphasic, and prolonged release of all of these proopiolipomelanocortin-derived peptides. The plasma levels of these IR-peptides rose in all subjects by 5 min after CRH, reached a first peak at 10-15 min, fell until 90 min, rose to a second peak at 2-4 h, and then gradually declined over several hours. The molar concentrations of the IR-peptides closely paralleled one another at all times, especially during the first 90 min after CRH administration. Later, IR-LPH increased slightly more and remained slightly higher than did the other IR-peptides, although the difference was not significant. This observation probably reflects the longer plasma disappearance half-life of IR-LPH. The maximum change (mean +/- SEM) in the concentration of these IR-peptides was similar: IR-ACTH, 18.0 +/- 4.0; IR-LPH, 20.5 +/- 4.0; and IR-beta-endorphin, 16.9 +/- 3.2 fmol/ml. The next morning's circadian rise in IR-peptides was blocked, presumably due to negative feedback inhibition of the hypothalamic-pituitary-adrenal axis by the prolonged high plasma cortisol levels stimulated by CRH the previous evening.

Arginine vasopressin potentiates adrenocorticotropin release induced by ovine corticotropin-releasing factor.

Arginine vasopressin (AVP) stimulates ACTH release in man and acts synergistically with synthetic ovine corticotropin-releasing factor (oCRF) in vitro. This study was designed to examine in man the combined effects of synthetic AVP (10 U intramuscularly) and oCRF (1 micrograms/kg intravenously) on ACTH release. Five normal male volunteers participated in five separate experiments: (a) AVP alone; (b) oCRF alone; (c) AVP followed by oCRF 15 min later; (d) simultaneous AVP and oCRF; and (e) insulin-induced hypoglycemia. Plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol were measured for 4 h after injection of each hormone; basal levels for all subjects were less than or equal to 9 +/- 1.2 pg/ml and 4.9 +/- 0.4 micrograms/dl (mean +/- SE), respectively. AVP and oCRF, when given individually, caused rapid rises in IR-ACTH to similar peak levels of 25 +/- 6.6 and 33 +/- 4.6 pg/ml, respectively. AVP given 15 min before oCRF caused a 2.6-fold potentiation of the oCRF response, with a peak IR-ACTH of 85 +/- 4.6 pg/ml. AVP given at the same time as oCRF produced a fourfold potentiation of the peak IR-ACTH response to 132 +/- 11 pg/ml. These ACTH responses were far greater than those previously observed after 30-fold greater doses of oCRF alone. By way of comparison, insulin-induced hypoglycemia caused a peak IR-ACTH of 169 +/- 20 pg/ml. IR-ACTH returned to base line at 60-90 min after AVP alone, whereas the prolonged effect of oCRF was apparent whether it was given alone or in combination with AVP. The mean peak IR-cortisol responses to AVP, oCRF, and AVP given 15 min before oCRF were similar (16.5 +/- 0.9, 16.4 +/- 2.3, and 18.5 +/- 0.8 micrograms/dl, respectively), but the peak IR-cortisol responses to AVP and oCRF given simultaneously and to insulin-induced hypoglycemia were 1.5 and 1.7 times greater, respectively. IR-cortisol returned to base line within 2-3 h after AVP alone, but remained elevated for at least 4 h after oCRF alone or in combination with AVP. These results indicate that AVP acts synergistically with oCRF to release ACTH in man and suggest that AVP may play a physiologic role in modulating the ACTH response mediated by corticotropin-releasing factor.

Plasma distribution, disappearance half-time, metabolic clearance rate, and degradation of synthetic ovine corticotropin-releasing factor in man.

The plasma distribution, disappearance half-time, MCR, and degradation of corticotropin-releasing factor (CRF) were studied in normal men who received a pulse injection of synthetic ovine CRF (oCRF). Graded iv doses of oCRF produced a linear increase in plasma immunoreactive oCRF (IR-oCRF). The calculated total plasma content of IR-oCRF 2 min after injection represented 41.7 +/- 2.5% (mean +/- SE) of the injected dose. The disappearance of IR-oCRF from plasma was characterized by a biexponential decay curve, with initial distribution and subsequent metabolic t 1/2 values of 6.1 +/- 0.5 and 55 +/- 3.8 min (mean +/- SE), respectively. In two subjects who were studied for 14-16 h after being given the largest dose of oCRF, there was third phase of disappearance, with a t 1/2 of 198 +/- 54 min. The MCR of IR-oCRF was 2.4 +/- 0.2 ml/min . kg (146 +/- 12 l/m2 . day) and was relatively constant over a 3000-fold dose range. The volume of distribution of IR-oCRF was 6.2 +/- 0.6 liters. The plasma IR-oCRF component, examined at increasing intervals after injection, was indistinguishable from the injected oCRF in that its apparent molecular size had not been altered, nor had its biological activity been attenuated. The continued circulation of apparently intact, biologically active oCRF for at least 90 min after injection was associated with sustained release of ACTH into the plasma. Thus, the clearance of oCRF from circulating human plasma is prolonged and appears to be responsible for the sustained release of ACTH that occurs after injection of this hormone-releasing factor.

Combined treatment with an LHRH agonist and testosterone in man. Reversible oligozoospermia without impotence.

We have previously shown that LHRH agonist [D-Trp6,Pro9-NEt]LHRH (LHRHA) results in reversible oligozoospermia when given to normal subjects for up to ten weeks. A fall in plasma testosterone was accompanied by loss of libido and potency. We now report six subjects who were evaluated by semen analysis and hormone profile at two-week intervals during ten-week basal, 20-week treatment, and post-treatment periods lasting at least ten weeks. Treatment consisted of LHRHA (50 microgram subcutaneously daily), and testosterone enanthate (100 mg intramuscularly every two weeks). Sperm density (mean basal 76.7 +/- 8.7 x 10(6)/ml) fell consistently in each subject to a mean nadir of 12.3 +/- 4.5 x 10(6)/ml (p less than 0.001). This is similar to the mean nadir of 11.6 +/- 5.8 x 10(6)/ml achieved when LHRHA was given alone. In each individual subject, sperm density returned to his basal level after cessation of treatment. No consistent changes were seen in sperm motility of morphology, or in semen volume. Libido and potency were maintained in all subjects. An additional three subjects received testosterone enanthate alone in identical dosage for 20 weeks. No change in sperm density was observed. In contrast to treatment with LHRHA alone, combination treatment produces reversible oligozoospermia without attendant change in potency.

Effect of synthetic ovine corticotropin-releasing factor: prolonged duration of action and biphasic response of plasma adrenocorticotropin and cortisol.

The duration of the response to synthetic ovine corticotropin-releasing factor (CRF) was studied in 13 healthy male volunteer subjects. Placebo or CRF (0.3, 3, or 30 micrograms/kg BW) was administered as an iv bolus or, in the case of the largest dose, a 30-sec infusion in single blind fashion in the late afternoon. Basal plasma immunoreactive ACTH (IR-ACTH) and IR-cortisol were 10.8 +/- 7.7 pg/ml and 5.0 +/- 1.8 micrograms/dl (mean +/- SD), respectively. IR-ACTH rose rapidly after CRF, reached an initial peak at 15 min, fell rapidly until 1.5 h after CRF, and then either fell more slowly (after the lowest dose) or rose to a second major peak at 2-3 h before falling back to baseline. After 0.3, 3, and 30 micrograms/kg CRF, IR-ACTH remained elevated for 4, 7, and 8 h, respectively. The effect on plasma IR-cortisol was similar, but more prolonged. The magnitude of both peaks of IR-ACTH, the duration of the response, and the area under the curve all appeared dose dependent. The same was true for IR-cortisol, except that the first peak height was similar after all three doses. The duration of CRF's action is probably due to its long circulating half-life. The biphasic response curve may reflect initial secretion of a readily releasable pool of ACTH, followed by later secretion of a second pool of newly synthesized and/or matured peptide. The next morning's normal circadian rise in both IR-ACTH and IR-cortisol was delayed and diminished after 3 micrograms/kg CRF; there was no increase in IR-ACTH after 30 micrograms/kg CRF, and the IR-cortisol level was diminished. Inhibition of the normal circadian rise may reflect inhibition of ACTH secretion by the sustained high plasma cortisol levels.

Effect of synthetic ovine corticotropin-releasing factor. Dose response of plasma adrenocorticotropin and cortisol.

Synthetic ovine corticotropin-releasing factor (CRF) was administered to normal male volunteer subjects as an intravenous bolus or 30-s infusion. Doses of CRF ranging from 0.001 to 30 micrograms/kg body wt were administered, and plasma immunoreactive (IR)-ACTH and IR-cortisol concentrations were measured. The threshold dose appeared to be 0.01-0.03 micrograms/kg, the half-maximal dose 0.3-1 micrograms/kg, and the maximally effective dose 3-10 micrograms/kg. Basal concentrations of IR-ACTH and IR-cortisol were 14 +/- 7.6 pg/ml (mean +/- SD) and 5.6 +/- 2.2 micrograms/dl, respectively. IR-ACTH rose as early as 2 min after CRF injection, reached peak levels in 10-15 min, and declined slowly thereafter. IR-cortisol rose at 10 min or later and reached peak levels in 30-60 min. At a dose of 30 micrograms/kg, neither IR-ACTH nor IR-cortisol fell from peak levels of 82 +/- 21 pg/ml (mean +/- SE) and 23 +/- 1.4 micrograms/dl, respectively, during the 2-h course of the experiment, indicating that CRF has a sustained effect on ACTH release and/or a prolonged circulating plasma half-life. There was little or no increase in the levels of other anterior pituitary hormones. At doses of 1 microgram/kg and higher, facial flushing, tachycardia, and, in some subjects, a 15-29-mmHg decline in systemic arterial blood pressure were observed, even though blood volume was replaced and the subjects remained supine. These data indicate that synthetic ovine CRF is a very potent and specific ACTH secretagogue in man. Administered with caution until its vasomotor effects are more fully defined, CRF promises to be a safe and very useful investigative, diagnostic, and, possibly, therapeutic agent in man.

Pituitary microadenomas causing Cushing's disease respond to corticotropin-releasing factor.

Corticotropin-releasing factor (CRF) was administered as an iv bolus to two young women with mild Cushing's disease shortly before and one week after successful transsphenoidal microadenomectomy. The dose of CRF (1 microgram/kg body weight) had previously been shown to stimulate increased plasma ACTH and cortisol in normal subjects. In the first patient, prior to surgery, there were brisk increases in ACTH and cortisol that exceeded those observed in normal subjects. ACTH rose by 2 min and reached a peak between 15-30 min. Cortisol increased by 10 min and peaked between 45-60 min. After surgery, at a time when plasma cortisol was maintained at similar levels with exogenous hydrocortisone, there was no plasma ACTH or LH, TSH and prolactin increased after administration of LRH and TRH, and GH increased in response to insulin-induced hypoglycemia. The second patient had higher basal plasma ACTH and cortisol than the first patient. CRF-induced increments in ACTH and cortisol were much less, but the time course was similar and peak levels attained were still higher than those in normal subjects. After surgery, at a time when plasma cortisol was maintained at a much lower level with exogenous hydrocortisone, there was no plasma ACTH or cortisol response. She had mild, transient diabetes insipidus. Basal levels of all other anterior pituitary hormones were normal. These results demonstrate that two microadenomas causing Cushing's disease were responsive to CRF in situ and suggest that CRF may be involved in the etiology and/or the responses to changes in plasma glucocorticoid concentrations observed in patients with Cushing's disease.