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Pitting, the removal of dead parasites from their host erythrocyte, has been studied in patients with severe malaria treated parenterally with quinine or artesunate, and was recently shown to contribute to delayed hemolysis, a frequent adverse event of artesunate. We quantified pitting in 81 travelers treated with oral antimalarial therapy. Pitting rate was high (55.8%) with artemisinin-based combinations, but <10% with the nonartemisinin drugs quinine, mefloquine, and atovaquone-proguanil. This may, in part, explain the slower parasite clearance in patients treated with antimalarial drugs lacking an artemisinin component, as well as the absence of posttreatment hemolysis with these drugs., (© The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Context: Restrictive anorexia nervosa (AN) presents an adaptive appetite regulating profile including mainly high levels of ghrelin. Because this adaptive mechanism is not effective on food intake, other appetite-regulating peptides need to be explored. 26RFa is a hypothalamic neuropeptide that stimulates appetite, gonadotropin release, and bone metabolism., Objective: The objective of the study was to evaluate the circadian levels of 26RFa in AN patients compared with healthy subjects, other eating disorders, and constitutional thinness (CT)., Design and Settings: This was a cross-sectional study performed in an endocrine unit and an academic laboratory., Investigated Subjects: Five groups of age-matched young women were included in the study: 19 restrictive AN, 10 AN with bingeing/purging episodes, 14 with CT, 10 bulimic, and 10 normal-weight controls., Main Outcome Measures: Twelve-point circadian profiles of plasma 26RFa levels were measured in each subject., Results: Significant circadian variations of 26 RFA were noticed in controls with higher values in the morning and abrupt decrease at noon. Twenty-four-hour mean 26RFa levels were significantly increased in restrictive AN and AN with bingeing/purging episodes (P < 0.001), predominantly in the afternoon and evening when compared with controls. Preprandial rises of 26 RFA were noticed in AN patients. Mean 26RFa levels trend to be higher in CT than in controls (P = 0.06) and significantly lower than in AN. The bulimic patients presented a circadian profile of 26RFa similar to that of controls., Conclusion: High levels of circulating 26RFa observed in AN patients might reflect an adaptive mechanism of the organism to promote energy intake and to increase fat stores in response to undernutrition.

26RFa is a hypothalamic RFamide neuropeptide that was identified as the endogenous ligand of the orphan G protein-coupled receptor, GPR103, and that stimulates appetite in mice. Up until now, the mechanism of action of 26RFa in the hypothalamic control of food intake remains unknown. The high density of GPR103 in the arcuate nucleus (Arc) prompted us to investigate, in the present study, the effects of 26RFa on the rat neuropeptide Y (NPY)/proopiomelanocortin (POMC) system. Intracerebroventricular injection of 26RFa stimulated NPY expression and release in the basal hypothalamus, whereas it decreased POMC expression and alpha-MSH release, and these effects were associated with an increase in food intake. A double in situ hybridization procedure indicated that the 26RFa receptor is present in NPY neurons of the Arc, but not in POMC neurons. Central administration of NPY Y1 and Y5 receptor antagonists abolished the inhibitory effects of 26RFa on POMC expression and alpha-MSH release, and reversed 26RFa-induced food consumption. Finally, 26RFa antagonized the effects of leptin on NPY expression and release, POMC expression and alpha-MSH release, and food intake. Altogether, the present data demonstrate for the first time that 26RFa exerts its orexigenic activity by stimulating the release of NPY in the Arc, which in turn inhibits POMC neurons by activating the Y1 and Y5 receptors. It is also suggested that the balance 26RFa/leptin is an important parameter in the maintenance of energy homeostasis.

Context: Neurotensin (NT) modulates corticosteroid secretion from the mammalian adrenal gland., Objective: The objective of this study was to investigate the possible involvement of NT in the control of cortisol secretion in the human adrenal gland., Design: In vitro studies were conducted on cultured human adrenocortical cells., Setting: This study was conducted in a university research laboratory., Patients: Adrenal explants from patients undergoing expanded nephrectomy for kidney cancer were studied., Main Outcome Measure: Cortisol secretion from cultured adrenocortical cells was measured., Results: NT1-11, the N-terminal fragment of NT, dose-dependently inhibited basal and ACTH-stimulated cortisol production by human adrenocortical cells in primary culture. In contrast, NT had no influence on cortisol output at concentrations up to 10(-6) m. HPLC and RT-PCR analyses failed to detect any significant amounts of NT and NT mRNA, respectively, in adrenal extracts. Molecular and pharmacological studies were performed to determine the type of NT receptor involved in the corticostatic effect of NT1-11. RT-PCR analysis revealed the expression of NT receptor type (NTR) 3 mRNA but not NTR1 and NTR2 mRNAs in the human adrenal tissue. However, the pharmacological profile of the adrenal NT1-11 receptor was different from that of NTR3, indicating that this receptor type is not involved in the action of NT1-11 on corticosteroidogenesis., Conclusion: Our results indicate that NT1-11 may act as an endocrine factor to inhibit cortisol secretion through activation of a receptor distinct from the classical NTR1, NTR2, and NTR3.

Objective: Previous work from our laboratory has demonstrated that frog urotensin-II (UII), at a high concentration, inhibits glucose-induced insulin release in the rat pancreas. We have investigated the effect of rat UII and two structural analogs on insulin secretion and searched for the presence of UII-immunoreactivity in rat pancreatic extracts., Methods: The study was performed in the perfused rat pancreas. UII as well as its analogs were synthesized by solid phase methodology. Pancreatic extracts were analyzed for UII by reversed-phase HPLC combined with a sensitive UII RIA., Results: Infusion of synthetic rat UII inhibited glucose-induced insulin release in a dose-dependent manner (IC(50): 0.12 nmol/l). UII (1 nmol/l) also inhibited the insulin responses induced by carbachol, glucagon-like peptide-1, and a calcium channel agonist (BAY K 8644). The inhibitory effect of UII was mimicked by the potent G protein-coupled receptor (GPR14) agonist [3-iodo-Tyr(6)]UII(4-11). In contrast, [Ala(8)]UII(4-11), a UII analog devoid of contractile activity on rat aortic rings, did not affect glucose-induced insulin secretion. Analysis of rat pancreatic extracts revealed the presence of an immunoreactive peptide exhibiting the same retention time as synthetic rat UII., Conclusions: Our results demonstrate that UII is a potent insulinostatic peptide. The observation that UII is actually present in the pancreas suggests that this peptide may play a physiological role in the control of insulin secretion. Concerning the two UII analogs tested, only [3-iodo-Tyr(6)]UII(4-11), reportedly possessing GPR14-mediated contractile activity, mimics the insulinostatic effect of UII. This finding would support the view that UII acts on the pancreatic beta cell through the GPR14 receptor.

Adrenochromaffin cells have been shown to physiologically synthesize and secrete ACTH. We have thus hypothesized that excessive intraadrenal ACTH production may be involved in the pathogenesis of primary adrenal Cushing's syndrome. In this report we describe a case of Cushing's syndrome due to bilateral adrenocortical macronodular hyperplasia associated with suppression of plasma ACTH levels. HPLC analysis of adrenal tissue extracts revealed the presence of a peptide coeluting with bioactive ACTH. Immunohistochemical studies showed that ACTH immunoreactivity was detectable in a subpopulation of steroidogenic cells, but not in chromaffin cells. ACTH-positive cells were also labeled by antibodies against relaxin-like factor, a marker of Leydig cells. The presence of ACTH in the hyperplastic tissue resulted from local expression of the gene encoding the ACTH precursor proopiomelanocortin. Finally, hyperplasia fragments, contrary to normal adrenal cortex explants, appeared to release in vitro measurable amounts of ACTH. In conclusion, this observation shows that Cushing's syndromes associated with suppressed plasma ACTH levels may be dependent upon ACTH produced within adrenocortical tissue. The term ACTH-independent used to designate primary adrenal Cushing's syndrome may therefore be inappropriate in some cases of bilateral macronodular adrenal hyperplasia with hypercortisolism and undetectable plasma ACTH levels.

The biosynthesis of various hypothalamic neuropeptides has been previously reported in anterior pituitary cells but not in intermediate lobe cells. We have recently demonstrated the occurrence of two somatostatin isoforms in the frog brain, namely somatostatin-14 (SS1) and [Pro(2),Met(13)]somatostatin-14 (SS2). In the present study, we demonstrate that the gene encoding the SS2 precursor (PSS2) is actively expressed in the intermediate lobe of the frog pituitary. High concentrations of PSS2 mRNA have been detected by Northern blot analysis and in situ hybridization in the frog pars intermedia but not in the pars distalis or pars nervosa. The distribution of PSS1- and PSS2-derived peptides has been investigated by immunohistochemistry using two antisera directed against SS1 and the sequence 54-66 of PSS2 (PSS2(54-66)), respectively. The SS1 antiserum stained only a network of fibers in the neural lobe and a few nerve processes in the intermediate lobe. In contrast, the PSS2(54-66) antiserum produced intense labeling of melanotrope cells in the pars intermedia. Biochemical characterization of the immunoreactive materials present in pituitary extracts was performed by combining high-performance liquid chromatography analysis and RIA detection. The SS1 RIA revealed the existence of two major immunoreactive peaks that exhibited the same retention times as synthetic SS1 and SS2. The PSS2(54-66) RIA detected a single peak that likely corresponds to the N-flanking peptide of SS2 (PSS2(1-66)). The present study reveals that melanotrope cells of the frog pituitary selectively express the PSS2 gene and fully process PSS2 to generate the mature somatostatin variant SS2. Taken together, these data provide the first evidence that the gene encoding a hypophysiotropic neuropeptide is intensely expressed in the intermediate lobe of the pituitary.

In amphibians, the secretion of alpha-MSH by melanotrope cells is stimulated by TRH and inhibited by NPY. We have previously shown that NPY abrogates the stimulatory effect of TRH on alpha-MSH secretion. The aim of the present study was to characterize the receptor subtypes mediating the action of NPY and to investigate the intracellular mechanisms involved in the inhibitory effect of NPY on basal and TRH-induced alpha-MSH secretion. Y(1) and Y(5) receptor mRNAs were detected by RT-PCR and visualized by in situ hybridization histochemistry in the intermediate lobe of the pituitary. Various NPY analogs inhibited in a dose-dependent manner the spontaneous secretion of alpha-MSH from perifused frog neurointermediate lobes with the following order of potency porcine peptide YY (pPYY) > frog NPY (fNPY) > porcine NPY (pNPY)-2-36) > pNPY-(13-36) > [D-Trp(32)]pNPY > [Leu(31),Pro(34)]pNPY. The stimulatory effect of TRH (10(-8)6 M) on alpha-MSH release was inhibited by fNPY, pPYY, and [Leu(31),Pro(34)]pNPY, but not by pNPY-(13-36) and [D-Trp(32)]pNPY. These data indicate that the inhibitory effect of fNPY on spontaneous alpha-MSH release is preferentially mediated through Y(5) receptors, whereas the suppression of TRH-induced alpha-MSH secretion by fNPY probably involves Y(1) receptors. Pretreatment of neurointermediate lobes with pertussis toxin (PTX; 1 microg/ml; 12 h) did not abolish the inhibitory effect of fNPY on cAMP formation and spontaneous alpha-MSH release, but restored the stimulatory effect of TRH on alpha-MSH secretion, indicating that the adenylyl cyclase pathway is not involved in the action of fNPY on TRH-evoked alpha-MSH secretion. In the majority of melanotrope cells, TRH induces a sustained and biphasic increase in cytosolic Ca(2+) concentration. Preincubation of cultured cells with fNPY (10(-7) M) or omega-conotoxin GVIA (10(-7) M) suppressed the plateau phase of the Ca(2+) response induced by TRH. However, although fNPY abrogated TRH-evoked alpha-MSH secretion, omega-conotoxin did not, showing dissociation between the cytosolic Ca(2+) concentration increase and the secretory response. Collectively, these data indicate that in frog melanotrope cells NPY inhibits spontaneous alpha-MSH release and cAMP formation through activation of a Y(5) receptor coupled to PTX- insensitive G protein, whereas NPY suppresses the stimulatory effect of TRH on alpha-MSH secretion through a Y(1) receptor coupled to a PTX-sensitive G protein-coupled receptor.

The primary structure of neurotensin has been recently determined for the frog Rana ridibunda (Endocrinology 139: 4140-4146, 1998). In the present study, we have investigated the distribution and biochemical characterization of neurotensin-like immunoreactivity in the frog adrenal gland, using an antiserum directed against the conserved C-terminal region of the peptide. Neurotensin-like immunoreactivity was detected in two populations of nerve fibers: numerous varicose fibers coursing between adrenal cells, and a few processes located in the walls of blood vessels irrigating the gland. Reversed-phase HPLC analysis of frog adrenal gland extracts revealed the existence of a major peak of neurotensin-like immunoreactivity that exhibited the same retention time as synthetic frog neurotensin. The possible involvement of neurotensin in the regulation of steroid secretion was studied in vitro using perifused frog adrenal slices. For concentrations ranging from 10(-10) to 10(-5) M, synthetic frog neurotensin increased corticosterone and aldosterone production in a dose-dependent manner (EC50 = 1.2 x 10(-9) M and 5.8 x 10(-10) M, respectively). Repeated administration of neurotensin induced a reproducible stimulation of steroid output without any tachyphylaxis. Prolonged administration (3 h) of frog neurotensin caused a transient increase in corticosterone and aldosterone secretion followed by a decline of corticosteroid secretion. Neurotensin also produced a significant stimulation of corticosteroid secretion from dispersed frog adrenal cells. This study demonstrates that neurotensin is located in nerve processes innervating the adrenal gland of amphibians. The results also show that synthetic frog neurotensin exerts a direct stimulatory effect on corticosteroid output. Taken together, these data support the view that neurotensin, released by nerve fibers, may act as a local regulator of corticosteroid secretion.

Vasoactive intestinal polypeptide (VIP) and galanin were isolated in pure form from the stomach of the European green frog, Rana ridibunda. Frog VIP is identical to the previously characterized VIP from chicken and alligator. The primary structure of frog galanin contains only two amino acid substitutions (asparagine for histidine at position 23 and histidine for tyrosine at position 26) compared with porcine galanin. The data indicate that evolutionary pressure to conserve the amino acid sequence of both peptides during the evolution of amphibia to mammals has been strong. Synthetic frog VIP produced a dose-dependent increase in cAMP concentration in frog anterior pituitary fragments. The potency of the peptide (ED50 = 1.2 x 10(-6) M; mean +/- SE; n = 8) was comparable to that of porcine VIP (EC50 = 1.3 x 10(-6) M), but was approximately 10-fold less than that of frog pituitary adenylate cyclase-activating polypeptide [PACAP-(1-38); ED50 = 1.1 x 10(-7) M] in the same system. The increases in cAMP concentrations produced by maximal doses of PACAP (10(-5) M) and VIP (10(-5) M) were not additive. The data suggest that the effects of both peptides are mediated through a common PACAP-preferring receptor that is pharmacologically different from the mammalian PACAP type I receptor. Synthetic frog galanin also produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 9.3 x 10(-8) M) consistent with a possible role for the peptide as a hypophysiotropic factor in amphibians.

In a previous report, we have shown that frog pituitary adenylate cyclase-activating polypeptide (fPACAP38) is a potent stimulator of corticosteroid secretion by frog adrenal slices in vitro. The aim of the present study was to determine the mode of action of PACAP on the frog adrenal gland. Immunoelectron microscopic labeling revealed that PACAP-like immunoreactivity is present in electron-dense vesicles within nerve endings located in the vicinity of both adrenocortical and chromaffin cells. Exposure of dispersed adrenal cells to fPACAP38 caused stimulation of corticosteroid secretion. Labeling of cultured adrenal cells with [125I]PACAP27 revealed the existence of PACAP-binding sites on both adrenocortical and chromaffin cells. Saturation and competition experiments showed the occurrence of high affinity and selective receptors for fPACAP38 on cultured adrenal cells. fPACAP38 (10(-8)-10(-5) M) provoked a dose-dependent stimulation of cAMP production by frog adrenal slices. Microflurimetric studies demonstrated that fPACAP38 induced a substantial elevation of the intracellular calcium concentration in both adrenocortical and chromaffin cells. The present results indicate that in the frog adrenal gland, PACAP fibers innervate both adrenocortical and chromaffin cells. The data show the presence of PACAP receptors on the two cell types. PACAP exerts a direct stimulatory effect on corticosteroid-producing cells. This effect is probably mediated through stimulation of adenylyl cyclase activity and/or augmentation of intracellular Ca2+. PACAP also increases intracellular Ca2+ in chromaffin cells. These data suggest that PACAP, released locally in the adrenal gland, acts as a neuroendocrine factor, regulating the activity of adrenocortical and chromaffin cells.

Pituitary adenylate cyclase-activating polypeptide (PACAP), a peptide of the glucagon-secretin-vasoactive intestinal polypeptide superfamily, was isolated in pure form from the brain of the European green frog, Rana ridibunda. The primary structure of the peptide indicates that evolutionary pressure to conserve the complete amino acid sequence has been very strong. Frog PACAP comprises 38 amino acid residues and contains only 1 substitution (isoleucine for valine at position 35) compared with human/ovine/rat PACAP. In the presence of the phosphodiesterase inhibitor isobutylmethylxanthine, synthetic ovine PACAP-(1-38) produced a dose-dependent increase in the concentration of cAMP in isolated frog anterior pituitary fragments (ED50 = 2.1 +/- 0.6 x 10(-7) M; mean +/- SE; n = 6). Maximum stimulation (an approximately 8-fold increase in concentration over basal values) was produced by 10(-6) M peptide. The truncated form of PACAP [PACAP-(1-27)] also produced a dose-dependent increase in cAMP in frog anterior pituitary fragments, and the potency of the peptide (ED50 = 5.9 +/- 0.6 x 10(-8) M) was comparable to that of PACAP-(1-38). The data suggest, therefore, that the function as well as the structure of PACAP have been conserved during the evolution of amphibia to mammals.