Inhibition of TNF alpha during maturation of dendritic cells results in the development of semi-mature cells: a potential mechanism for the beneficial effects of TNF alpha blockade in rheumatoid arthritis.

BACKGROUND: Dendritic cells orchestrate pivotal immunological processes mediated by the production of cytokines and chemokines. OBJECTIVE: To assess whether neutralisation of tumour necrosis factor alpha (TNF alpha) during maturation of dendritic cells affects their phenotype and behaviour, which might explain the beneficial effects of TNF alpha neutralisation in rheumatoid arthritis. METHODS: Immature and fully matured dendritic cells were cultured from blood monocytes from patients with rheumatoid arthritis and healthy controls following standardised protocols. TNF alpha was neutralised by addition of the p55 soluble TNF alpha receptor, PEGsTNFRI. The effect of TNF alpha neutralisation on the phenotype (CD14, CD16, CD32, CD64, CD80, CD83, CD86, and MHC) of dendritic cells was investigated by flow cytometry. Expression of chemokines (CCL17, CCL18, CCL19, CCL22, CCL3, and CXCL8) and production of IL1 beta and IL6 during dendritic cell differentiation and maturation were examined. RESULTS: Neutralisation of TNF alpha during the differentiation and maturation of dendritic cells did not result in an altered dendritic cell phenotype in the rheumatoid patients or the healthy controls. In contrast, the expression of CCL17, CCL18, CCL19, CCL22, CCL3, and CXCL8 by dendritic cells was significantly reduced when TNF alpha activity was inhibited during lipopolysaccharide triggered dendritic cell maturation. The production of IL1 beta and IL6 by mature dendritic cells was inhibited by PEGsTNFRI. CONCLUSIONS: Inhibition of TNF alpha activity during dendritic cell maturation leads to the development of semi-mature cells. These data suggest a novel pathway by which the neutralisation of TNF alpha might exert its therapeutic effects.

Increased FcgammaRII expression and aberrant tumour necrosis factor alpha production by mature dendritic cells from patients with active rheumatoid arthritis.

OBJECTIVES: To investigate potential differences in phenotype and behaviour of immature (iDC) and mature dendritic cells (mDC) from patients with RA and healthy subjects. METHODS: iDC and mDC were derived from blood monocytes of patients with RA and healthy controls following standardised protocols. FACS was used to analyse expression of FcgammaRI, II, and III and molecules to characterise DC. Discrimination between FcgammaRIIa and FcgammaRIIb was achieved by RT-PCR. Immunohistochemistry was performed on synovial biopsy specimens of three patients with RA and three healthy controls. TNFalpha production by iDC and mDC upon FcgammaR dependent stimulation was compared between patients with RA and controls by ELISA. RESULTS: iDC from patients with active RA but not from patients with inactive RA or healthy controls markedly up regulated FcgammaRII. mDC from patients with active RA also lacked the physiological down regulation of FcgammaRII that occurs upon maturation in both control groups. RT-PCR analysis confirmed the increased expression of FcgammaRII in RA-especially marked for FcgammaRIIb. FcgammaR dependent stimulation of DC using antigen-IgG immune complexes (IC) significantly increased TNFalpha production by DC from healthy subjects, but significantly decreased TNFalpha by DC from patients with RA. Overlapping expression patterns between FcgammaRII and DC-LAMP in the synovial tissue of patients with RA imply that in vivo, also, mature DC express increased levels of FcgammaRIIb. CONCLUSION: The presence and altered characteristics of DC during active RA suggest that DC help to modulate autoimmunity in RA. Further studies should elucidate the role of local factors in altering the function of DC in RA and in increasing expression of FcgammaRII.

High production of proinflammatory and Th1 cytokines by dendritic cells from patients with rheumatoid arthritis, and down regulation upon FcgammaR triggering.

OBJECTIVE: To assess whether DC from RA produce altered cytokine levels and whether this is regulated by triggering of Fc gamma receptors (FcgammaR). METHODS: The production of proinflammatory (TNFalpha, IL1, IL6), Th1 (IL12, IFNgamma), and Th2 (IL10) cytokine profiles of immature DC (iDC) from patients with RA and healthy subjects upon triggering of FcgammaR dependent and independent pathways was investigated. iDC, derived from blood monocytes by standardised protocols, were stimulated with immune complexes (IC) at day 6 for 48 hours and, subsequently, for 2 days with LPS in the presence or absence of IC or IFNgamma, resulting in fully matured DC (mDC). IL1, IL6, TNFalpha, IFNgamma, IL12, and IL10 levels in supernatants were measured by ELISA and RIA. RESULTS: mDC from patients with RA showed a markedly increased production of IL1, IL6, TNFalpha, and IL10 compared with DC from healthy donors. Triggering of FcgammaR decreased the production of proinflammatory cytokines IL1, IL12, and IFNgamma by iDC and mDC in RA and controls. The production of IL6 and TNFalpha decreased in patients with RA, whereas it was increased in controls. Triggering of FcgammaR independent mechanisms using IFNgamma increased the production of proinflammatory and Th1 cytokines, which was more pronounced in RA. CONCLUSION: FcgammaR dependent pathways influence cytokine production by DC. A skewed balance towards proinflammatory and Th1 cytokines in RA can, at least partly, be restored by triggering FcgammaR on DC in RA. Insight into the mechanism which determines the FcgammaR balance might lead to new strategies to abrogate Th1 driven inflammatory processes in RA.

Depression of plasma levels of cytokines and ex-vivo cytokine production in relation to the activity of the pituitary-adrenal axis, in patients undergoing major vascular surgery.

The relation between the immune and neuroendocrine response during surgery was studied. In 18 patients undergoing major vascular surgery, circulating interleukin (IL)-1beta and ex-vivo production of IL-1beta and tumour necrosis factor (TNF)-alpha were lower on day 1 after surgery compared to pre-operation values (-14+/-5%, P<0.05; -62+/-9%, P<0.05; and -31+/-54%, P<0.005, respectively). Circulating IL-1 receptor antagonist (IL-1ra) was higher on the 5th day post-operatively compared to pre-operation values (mean +640%+/-400, P<0.05). In a more detailed study in six patients, the ex-vivo production of IL-1beta and TNF-alpha started to decrease at induction of general anaesthesia and dropped to under 10% of initial values at the end of surgery. Circulating IL-1ra and ex-vivo production of IL-1ra started to increase at the end of surgery and remained elevated up to 6 days post-operatively. Plasma antidiuretic hormone (ADH) and adrenocorticotropic hormone (ACTH) increased during surgery, but cortisol remained unchanged. We demonstrate a depression of circulating pro-inflammatory IL-1beta and an increase of circulating anti-inflammatory IL-1ra during surgical stress. The ex-vivo production of IL-1beta and TNF-alpha was suppressed, indicating a downregulation of the production of these cytokines. This parallelled the hormonal reaction with high ADH and ACTH, but not of cortisol, suggesting that glucocorticoid is not the key-factor in downregulation of production and release of pro-inflammatory cytokines.

A neuropeptide in human semen: oxytocin.

The neuropeptide oxytocin (OT) has been detected in testis and epididymis of several mammals. The peptide affects steroidogenesis and sperm transport in vivo. Effects of OT on sperm motility in vitro seems to be contradictory. As no data are available on the presence of OT in human semen and on the relationship of OT with sperm characteristics, we assessed OT level in semen samples in 3 groups of patients: (I) normozoospermic, (II) astheno-/oligo-/teratozoospermic, and (III) azoospermic subjects. Furthermore, we studied the relationship between the concentration of OT in semen and the sperm characteristics. OT was measured in seminal plasma by radioimmunoassay after extraction. OT semen levels did not differ in control patients (I: 1.72 +/- 0.78 pg/mL; n = 10), patients with poor semen quality (II: 1.66 +/- 0.91 pg/mL; n = 11), or in vasectomized patients (III: 1.28 +/- 0.65 pg/mL; n = 11). No statistically significant relationships between the OT levels and sperm characteristics (density: 0.0693; total sperm count: 0.0845; percentage of motility: 0.1341; morphology: 0.3478) have been found. The neurosecretory peptide oxytocin is present in human seminal plasma of normal as well as of vasectomized subjects. OT is not only derived from the testis; OT levels in poor semen samples are not different from controls. No relationship was found between OT seminal plasma levels and sperm characteristics.

Circulating and ex vivo production of pyrogenic cytokines and interleukin-1 receptor antagonist in 123 patients with fever of unknown origin. The Netherlands Fever of Unknown Origin Study Group.

Circulating and ex vivo production of interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, IL-6, and IL-1 receptor antagonist (ra) and the diagnostic utility of these cytokines were studied in 123 patients with fever of unknown origin (FUO). Diagnoses were infections, 28; neoplasms, 14; noninfectious inflammatory diseases (NIID), 32; miscellaneous diseases, 10; and none made, 39. IL-1beta, IL-6, and IL-1ra concentrations were higher in patients with infections, neoplasms, and NIID than in healthy controls. Patients with infections had higher concentrations of TNF-alpha than controls. The ex vivo production of IL-1beta and IL-1ra in all patients with FUO did not differ from that in controls; however, production of TNF-alpha was lower in patients with neoplasms and NIID, and IL-6 production was lower in patients with neoplasms. Thirty-five patients with fever did not have elevated cytokines. Although some significant differences were found among the diagnostic subgroups, there was wide variation. Thus, measurement of these cytokines does not aid in the diagnosis of FUO.

Chronic stimulation of the hypothalamus-pituitary-adrenal axis in rats by interleukin 1beta: central and peripheral mechanisms.

The authors have studied mechanisms which could be involved in the sustained activation of the hypothalamus-pituitary-adrenal (HPA) axis during continuous infusion of rats with recombinant human interleukin-1beta (IL-1beta). First, the effects of 3 days of intracerebroventricular (i.c.v.) infusion of rats with IL-1 on plasma adrenocorticotropin (ACTH) and corticosterone (B) levels were investigated. Thereafter, changes in plasma ACTH and B levels were followed in rats intraperitoneally (i.p.) infused with IL-1beta after immunoneutralization of corticotropin-releasing hormone (CRH), hypophysectomy (HPX), macrophage depletion using dichloromethylene diphosphonate (Cl2MDP)-containing liposomes, adrenalectomy (ADX) and dexamethasone (DEX) administration, respectively. Infusion of IL-1beta i.c.v., even in doses as low as 0.1 microg/day, induced significant increases in plasma ACTH and B levels. HPX and ADX rats died within 18 h after starting the IL-1beta infusion (0.5 microg/day). Immunoneutralization of CRH significantly decreased and macrophage depletion significantly increased the stimulation of the HPA axis by IL-1 (4.0 microg/day). Administration of high doses of DEX completely abolished the stimulation of the HPA axis by IL-1beta (2.0 microg/day). The present study demonstrates that lower doses of IL-1beta were able to activate the HPA axis when infused i.c.v. compared with i.p. Regarding stimulation of the HPA axis by chronic i.p. infusion of IL-1beta the present study: (1) provides evidence that the CRH system is involved; (2) provides no evidence for a direct stimulatory effect of IL-1beta on the release of B by the adrenal gland which is of sufficient magnitude to resist the stress of chronic i.p. IL-1beta infusion; (3) shows that endogenous macrophage-derived mediators, induced by i.p. IL-1beta infusion, express an overall inhibitory rather than a stimulatory effect on the activity of the HPA axis; (4) demonstrates that exogenous administration of DEX blocks the effect of IL-1beta, which fits well in the concept of an immunoregulatory feedback between IL-1beta and glucocorticoids.

Effects of cytokines on pituitary beta-endorphin and adrenal corticosterone release in vitro.

We investigated the effects of recombinant human IL-1 alpha, -1 beta, -2, -6 and TNF on the in vitro secretion of beta-endorphin-immunoreactivity (beta E-IR) by the rat anterior and neurointermediate lobes (AL and NIL, respectively) and of B by the rat adrenal gland. Isolated AL and NIL cells were incubated for 2 h with cytokines (1 pg/m1(-1) mu g/ml), CRH (5.10(-10) M) or with cytokines in combination with CRH (AL cells), isolated adrenal cells were incubated for 2 h with cytokines, ACTH (25 pg/ml) or with cytokines in combination with ACTH. Furthermore, AL, NIL and adrenal tissue fragments were superfused for 30 or 60 min with cytokines (10 and/or 100 ng/ml). Incubation of AL, NIL and adrenal cells and superfusion of these tissues with cytokines had no significant effect on beta E-IR and B release. However, there are some exceptions: incubation of AL cells with IL-2 increased CRH-induced beta E-IR release, incubation of NIL cells with IL-2 induced an increase of basal beta E-IR release, ACTH-induced B secretion was reduced after co-incubation of adrenal cells with TNF and after prolonged (6 h) superfusion of adrenal tissue with TNF, and finally, prolonged (6 h) superfusion of adrenal fragments with IL-1 beta increased basal B release. Taken together, these data suggest that the acute activation of the pituitary-adrenal axis of rats by administration of cytokines (at least IL-1, IL-6 and TNF) in vivo is not mediated by a direct action of these cytokines at the level of the pituitary and/or adrenal gland.

Acute stimulation of the hypothalamic-pituitary-adrenal axis by IL-1 beta, TNF alpha and IL-6: a dose response study.

We investigated the effects of i.v. and intracerebroventricular (i.c.v) administration of increasing doses of recombinant human IL-1 beta, TNF alpha and IL-6 on plasma corticosterone (B) levels in rats. Rats were equipped with a jugular cannula for repeated blood sampling anda subgroup of rats also received an i.c.v implanted cannula. I.v. administration of IL-1 beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta and IL-6 induced a significant dose-dependent increase in plasma B levels, whereas i.c.v injection of TNF alpha in doses up to 1000 ng/rat was not effective. I.v. pretreatment of rats with anti-CRH antiserum had no significant overall effect on the plasma B response to i.v. administered IL-1 beta (500 and 3000 ng/rat), whereas the plasma B response to i.v. TNF alpha or IL-6 administration (3000 ng/rat) were significantly reduced. I.v. pretreatment of the animals with recombinant human IL-1 receptor antagonist (IL-1ra) significantly blocked the plasma B response to i.v. treatment with IL-1 beta, whereas the TNF alpha- and IL-6-induced increases in plasma B levels were not affected. Our data show that 1) i.v. administration of IL-beta, TNF alpha or IL-6 and i.c.v administration of IL-1 beta or IL-6 dose-dependently stimulate the HPA axis; 2) when given i.v. or i.c.v, IL-1 beta is more powerful than TNF alpha and IL-6 in activating the HPA axis; 3) endogenous CRH is involved in the activation of the HPA axis by acute i.v. administration of TNF alpha and IL-6. It is most likely that in case of i.v. treatment with IL-1 beta a CRH-independent mechanism is involved. This study provides no arguments for the involvement of endogenous IL-1 in TNF alpha- or IL-6-induced activation of the HPA axis.

Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production.

We investigated whether a 6-h endurance run changes cytokine plasma concentrations and lipopolysaccharides (LPS) stimulated ex vivo production of cytokines in a whole blood culture of 19 well-trained athletes. The average distance covered was 65.1 +/- 8.64 (SD) km. At the end of the exercise, the mean plasma concentration of interleukin-1-receptor agonist (IL-1ra), which was 188 pg/ml 24 h before finish, increased to 886 pg/ml (P < 0.0005). The mean plasma interleukin-6 concentration increased from 18.5 +/- 4.2 to 71.5 +/- 33.3 pg/ml (P < 0.0001). The increase of neutrophils correlated with the increase of IL-1ra concentrations (r = 0.58, P < 0.005). We could not detect an effect of exercise on plasma concentrations of interleukin-1 beta (IL-1 beta) or tumor necrosis factor-alpha (TNF-alpha). The ex vivo LPS-stimulated production of IL-1 beta in athletes 24 h before the run was significantly higher than in sedentary controls. Exercise induced a decrease of LPS-stimulated production of IL-1 beta and TNF-alpha, whereas production of IL-1ra was unchanged. These results show that prolonged exercise elicits a selective downregulation of the proinflammatory cytokine production and upregulation of the cytokines IL-1ra and interleukin-6.

Bioavailability and pharmacokinetics of sublingual oxytocin in male volunteers.

The aim of this investigation was to assess the bioavailability and pharmacokinetics of oxytocin in six male subjects after a sublingual dose of 400 int. units (684 micrograms) and after an intravenous dose of 1 int. unit (1.71 micrograms). After intravenous administration, the pharmacokinetic profile could be described with a two-compartment model. The distribution half-life was 0.049 +/- 0.106 h, the elimination half-life was 0.33 +/- 0.23 h, the total body clearance was 67.1 +/- 13.4 L h-1 and the volume of distribution was 33.2 +/- 28.1 L. After sublingual administration, a poor bioavailability with a 10-fold variation between 0.007 and 0.07% was observed. The pharmacokinetic profile could be described with a one-compartment model. The lag time was subject-dependent and ranged between 0.12 and 0.30 h (40% CV). The absorption half-life was 0.45 +/- 0.29 h, and the apparent elimination half-life 0.69 +/ - 0.26 h. This study showed a very poor and interindividual variability in bioavailability. The sublingual route of administration with its 'long' lag time and 'long' absorption half-life would not seem a reliable route for accurate high dosing for immediate prevention of post-partum haemorrhage.

Synergism between IL-1 beta and TNF-alpha on the activity of the pituitary-adrenal axis and on food intake of rats.

We investigated the effects of separate and combined intraperitoneal administration for 3 days of recombinant human interleukin-1 beta (IL-1) and recombinant human tumor necrosis factor-alpha (TNF) on plasma adrenocorticotropic hormone (ACTH) and corticosterone (B) levels, adrenal weight, food intake, and rectal temperature. Rats were equipped with a jugular cannula for daily blood sampling and with an intraperitoneally implanted Alzet osmotic minipump loaded with either saline, IL-1 (2.0 micrograms/day), TNF (0.2, 2.0, or 10.0 micrograms/day), or IL-1 in combination with TNF. Plasma ACTH and B levels and adrenal weight were significantly increased, in a dose-dependent way, by simultaneous infusion of IL-1 and TNF but not by administration of either cytokine alone. Chronic administration of IL-1 alone induced a significant decrease in food intake and a significant elevation of rectal temperature, whereas infusion of only the highest dose of TNF significantly elevated rectal temperature. Coinfusion of IL-1 and TNF induced both effects in a dose-dependent and synergistic way. Our data show that simultaneous infusion of IL-1 and TNF in rats has a synergistic effect on the activity of the pituitary-adrenal axis as well as on food intake and rectal temperature. The existence of two pathways, which act synergistically, may increase the sensitivity of the host to respond to subtle inflammatory stimuli.

Multiple forms of bioactive and immunoreactive adrenocorticotropin in human pituitary and blood of patients with Nelson's syndrome.

A pool of human pituitaries obtained from allegedly healthy subjects (traffic victims) and plasma samples from patients with Nelson's syndrome were analyzed by high performance liquid chromatography, and the corticosteroidogenic bioactivity and ACTH immunoreactivity were measured. Three bioactive forms of ACTH were detected in plasma samples and pituitary extract. The major form (peak III) coeluted with human ACTH-(1-39), showed a bioactive to immunoreactive ratio (B/I ratio) of about 1, and represented about 80% of the total bioactivity in both the plasma samples and the pituitary extract. Peak I, with a B/I ratio greater than 1, represented about 5%, and peak II, with a highly variable B/I ratio, represented about 7% of the bioactivity in both the plasma and pituitary extracts. A fraction with a very low B/I ratio was found to coelute with corticotropin-like intermediate lobe peptide. These data suggest that in Nelson's syndrome, ACTH secretion by the pituitary gland does not differ from that in normal subjects, at least qualitatively.

Major contribution of the basic amino acid lysine at position 11 to the bioactivity of ACTH in purified isolated rat adrenocortical cells.

The contribution of the positive charge of lysine in position 11 of ACTH to the corticosteroidogenicity was tested in a purified isolated rat adrenal cell assay. ACTH analogues with Lys11 or Arg11 had the same bioactivity. Replacement of lysine by norleucine or protection of its positive charge diminished the potency by about a factor of 80. The extent of this reduction has been discussed in relation to the importance of the basic amino acid sequence 15-18 of the peptide. The result suggests a ten times higher contribution of Lys11 to the bioactivity of ACTH than the surmised potency of the individual amino acids present in the sequence 15-18.

Increase of the proportion of corticosteroidogenic activity of ACTH versus immunoreactive ACTH in rat plasma extracts during stress.

The relationship between bioactive and immunoreactive adrenocorticotrophin (ACTH) was studied under various conditions. Rats were stressed by ether and the immunoreactive (I-ACTH) and bioactive (B-ACTH) ACTH levels were compared to the normal values obtained from "handled" rats. The bioactivity was determined by measuring the corticosteroid production in an isolated rat adrenal cell system. Stress exposure enhanced both the I-ACTH and B-ACTH levels. I-ACTH rose from 86.9 +/- 37.9 to 363 +/- 148.7 pg/ml, whereas the B-ACTH levels rose from 34.2 +/- 11.9 to 424.2 +/- 170.9 pg/ml. The bioactive to immunoreactive ratio (B/I ratio) was calculated as 0.47 +/- 0.13 in the control group and rose to the significantly higher value of 1.10 +/- 0.22 in the stressed rats. The conclusion is that under normal circumstances only about 50% of plasma immunoreactive ACTH shows corticosteroidogenic activity and that the B/I ACTH ratio might be a useful tool to estimate the physiological phase of the pituitary releasing activity.

Chronic stimulation of the pituitary-adrenal axis in rats by interleukin-1 beta infusion: in vivo and in vitro studies.

It has been shown that acute administration of interleukin-1 (IL-1) to rats elicits a transitory increase in plasma ACTH and corticosterone (B) levels. To investigate the effects of chronic administration of IL-1 on plasma ACTH and B levels, in the present study rats were equipped with Alzet osmotic minipumps loaded with either IL-1 (delivery rate 0.5, 2.0, or 4.0 micrograms/24 h, ip, for 1 week) or saline. At the end of the treatment the rats were decapitated, the adrenals were weighed, and the in vitro release of beta-endorphin (beta E) by the anterior pituitary and that of B by the adrenal gland were measured. Continuous administration of 2.0 and 4.0 micrograms IL-1/24 h resulted in a persistent increase in plasma ACTH and B concentrations compared to the levels in saline-infused rats, with peak levels on the first day of administration. In addition, adrenal weights of IL-1 rats were significantly higher than those of saline rats. The 4.0-micrograms IL-1/day in vivo treatment induced an increase in spontaneous in vitro secretion of beta E and B, while the in vitro responses of the pituitary (to CRF) and the adrenal (to ACTH) of animals treated in vivo with IL-1 were significantly diminished. IL-1 at a dose of 0.5 microgram failed to affect plasma ACTH and B values, adrenal weight, and in vitro beta E and B secretion. Chronic infusion of rats with 4.0 micrograms IL-1/day induced prolonged fever, whereas at lower doses of IL-1 (2.0 and 0.5 micrograms), temperatures were elevated only on the first 2 days of infusion. IL-1 at doses of 2.0 and 4.0 micrograms/day induced suppression of body weight gain on the first 2 days of the treatment period compared to saline treatment. Plasma norepinephrine and/or epinephrine concentrations were raised only on day 1 of the 2.0- and 4.0-micrograms IL-1 experiments. Thus, the observed effects of IL-1 on the hypothalamo-pituitary-adrenal axis probably do not result merely from stress induced by the treatment. Taken together, our data show the potential of IL-1 to induce a dose-dependent and long term activation of the pituitary-adrenal axis.

The bioactivity of immunoreactive adrenocorticotrophin in human blood is dependent on the secretory state of the pituitary gland.

A highly sensitive bioassay, using preincubated purified isolated rat adrenal cells, has been developed for measuring plasma ACTH. This bioassay enables detection of ACTH in plasma of about 0.9 pmol/l. Bioactive ACTH (B-ACTH) plasma levels were determined during insulin-induced hypoglycaemia in 12 female subjects and the values were compared with immunoreactive ACTH (I-ACTH) levels. The mean (+/- SD) basal B-ACTH level amounted to 1.7 +/- 0.8 pmol/l, the mean I-ACTH to 2.9 +/- 1.4 pmol/l. The highest mean B-ACTH plasma value was found 30 min after insulin injection: 14.7 +/- 15.7 pmol/l (I-ACTH: 12.8 +/- 9.9 pmol/l). By 90 min the B-ACTH level had returned to baseline (1.6 +/- 0.8 pmol/l), whereas the I-ACTH level was still significantly higher (5.4 +/- 2.7 pmol/l) than at time zero. Remarkably, the B-ACTH to I-ACTH ratio (B/I ratio) showed a biphasic profile during the insulin tolerance test, the ratio increasing from 0.60 +/- 0.77 at time zero to 1.08 +/- 0.35 at the ACTH peak, and decreasing after that to a lower value of 0.33 +/- 0.11 at 90 min. From these results it is concluded: (1) in the morning hours a considerable amount of circulating I-ACTH has no steroidogenic activity; (2) the B/I ratio temporarily increases immediately after insulin injection but gradually decreases afterwards to values half the baseline level at 90 min. Whereas this decrease at 90 min can be explained by differences in disappearance rates, the increase of B-ACTH relative to I-ACTH at 30 min indicates that estimations of immunoreactive ACTH reflect the biological activity of newly released ACTH with greater precision than at steady state level. Thus, the B/I-ACTH ratio can be used as a tool for measuring the state of the pituitary releasing activity.

The melanotropin potentiating factor and beta-endorphin do not modulate the alpha-melanotropin-or adrenocorticotropin-induced corticosteroidogenesis in purified isolated rat adrenal cells.

The ability of two pro-opiomelanocortin-derived peptides, the melanotropin potentiating factor (MPF) and beta-endorphin (beta EP), to affect corticosteroid production was studied in purified isolated rat adrenal cells. Addition of MPF or beta EP, in doses from 5 pg to 5 micrograms, alone did not result in a corticosterone production. Furthermore, no effect of MPF or beta EP in doses from 5 pg to as high as 5 micrograms for both peptides upon the ACTH or alpha-MSH-induced corticosteroidogenesis was observed (p greater than 0.1). It is concluded that both MPF and beta EP do not influence the steroidogenic activity in the adrenal gland. Use of these peptides for discrimination of the ACTH/alpha-MSH receptor interactions is suggested.

Coexistence of hypothalamic and pituitary failure after successful pituitary surgery in Cushing's disease?

The type of secondary adrenal failure, which occurs after successful pituitary surgery in Cushing's disease was studied using CRH administration. Eight patients with Cushing's disease were studied in the immediate postoperative period (9-18 days) of successful pituitary surgery. The results in these patients were compared with those in 13 healthy subjects, 7 patients with secondary adrenal failure of the pituitary type and 9 patients with secondary adrenal failure of the hypothalamic type. In all postoperative patients with Cushing's disease, except one, clear ACTH and cortisol responses occurred after CRH, demonstrating hypothalamic adrenal failure in these patients. This demonstration of hypothalamic adrenal failure after neurosurgery strongly argues against a pivotal role for endogenous CRH in the pathogenesis of Cushing's disease in these patients. The mean ACTH response to CRH in the postoperative patients with Cushing's disease was significantly lower than that in patients with adrenal failure of the hypothalamic type. This suggests the presence of pituitary failure in the postadenomectomy patients. In conclusion, this study provides arguments that the secondary adrenal failure after adenomectomy in Cushing's disease is caused by both hypothalamic and pituitary failure.