Corticotropin-releasing Hormone Stimulation Research Articles

A new pharmacological strategy against treatment-resistant depression

Major depressive disorder affects more than 50 million people in the world. However, 50% of patients don't respond to two or more drugs or psychotherapeutic treatments, named treatment-resistant depression (TRD). In this work, we propose a new augmentation treatment against TRD based on combining Fluoxetine (FLX) and the N-terminal fragment Galanin, GAL(1–15). In Wistar Kyoto (WKY) rats, akin to endogenous depression genetically, we evaluate GAL(1–15)’s impact on FLX-induced behaviours on tests measuring despair and anhedonia. We explored GALR2 involvement using the antagonist M871 and an in vivo model with siRNA 5-HT1A knockdown. Also, the 5-HT1AR was analyzed by autoradiography binding in several brain regions. We analyze the corticosterone levels and a dexamethasone-suppressed corticotropin-releasing hormone stimulation to study the HPA axis regulation. Our results shows that only the combination of FLX + GAL(1–15) induced antidepressant effects in the WKY animals in Behavioural tests related to despair. This combination also reduced corticosterone levels in the WKY animals and modulated the functional characteristics of the serotoninergic receptor 5-HT1A in the prefrontal cortex. These novel results suggest combining GAL(1–15) with FLX is a new potentiation strategy in TRD cases. It shows the innovative potential of the interactions between the galaninergic and serotonergic systems to find new strategies and drugs against resistant depression.

Difficult Diagnosis and Management of Concealed Cushing.

Adrenocorticotropic Hormone (ACTH)-secreting tumors account for 5- 10% of Cushing syndrome cases and are often difficult to diagnose and treat. A 44-year-old man presented with arterial hypertension and weight gain. On the physical examination, he exhibited central obesity, abdominal striae rubrae, and facial plethora. Due to the clinical suspicion of Cushing syndrome, the Nugent test and Liddle-1 test were performed, which showed a lack of cortisol suppression. ACTH levels were also high (138 pg/mL), so pituitary MRI and dynamic tests were performed, including the Corticotropin-releasing Hormone (CRH) stimulation test and Liddle-2. MRI showed a 3 mm pituitary microadenoma, but hormonal testing suggested ectopic ACTH production. Chest CT detected a 10-mm nodule in the upper lobe of the right lung, suspicious for a carcinoid tumor. However, the nodule did not exhibit any enhancement on 68-Gallium-DOTATOC PET-CT, and further, 18-FDG PET-CT was inconclusive. In addition, the nodule was deemed non-biopsiable due to its location. Meanwhile, the patient developed osteoporosis, resulting in two vertebral fractures and one rib fracture, which was treated with zoledronate. Furthermore, the patient developed acute aortic insufficiency. During bioprosthetic valve replacement, the thoracic surgeon performed wedge resection of the right upper lung lobe. The histological examination of the lesion revealed a typical lung carcinoid (1.2x0.9 cm, pT1bNXR0, Ki671%, ACTH positive in 95% of neoplastic elements). ACTH levels dropped to 4 pg/mL on the fourth postoperative day. ACTH-secreting tumors are particularly challenging diseases. A comprehensive hormonal and instrumental valuation is often required, necessitating a multidisciplinary approach.

Characterizing Adrenal Insuffciency in a Rat Model of Prader-Willi Syndrome: Insights into Hypothalamic-Pituitary-Adrenal Axis Dysregulation

Prader-Willi Syndrome (PWS) is a complex genetic disorder with distinct developmental stages. In the first stage, PWS manifests as failure to thrive, hypotonia and feeding diffculty in infants. This is followed by hyperphagia, hypogonadism, and behavioral challenges which characterize the later stage of this disease. Hypothalamic-pituitary dysregulation is believed to be responsible for many aspects of the PWS phenotype, potentially contributing to several of the hallmark characteristics of this disorder. Changes in the ability of the central nervous system to respond to stressors by integrating and responding to insults to the homeostatic state are well documented in PWS, and adrenal insuffciency may further complicate endocrine dysfunction in PWS. The inability of PWS patients to respond to stress may be due to alterations at any or all three levels of the hypothalamo-pituitary-adrenal (HPA) axis. In this study, we sought to investigate the hypothesis that adrenal insuffciency in Magel2-deficient rats, a model of PWS, is at least in part due to loss of pituitary secretory capacity, since disruption of Magel2 expression, as is observed in PWS patients, is known to affect vesicular traffcking and secretion. To address this hypothesis, anterior pituitaries collected from Magel2-deficient and wild type rats were dispersed and aliquoted. The following day, cells were treated with either 0.1, 1.0, 10, 100, or 1000 nM of corticotrophin-releasing hormone (CRH). Following a 60 minute incubation, media were collected for the assessment of adrenocorticotropin (ACTH) by radioimmunoassay. We also collected pituitary cells for measurement of intracellular content of ACTH. Our results showed that cells from Magel2-deficient rats had decreased basal ACTH secretion, but remained responsive to CRH stimulation, albeit to a lesser degree than cells isolated from wild type animals. This suggests a pituitary deficiency in the response to hypothalamic factors (e.g. CRH) that normally control ACTH release. It is also possible that the other two levels of the HPA axis are altered in PWS. We now are investigating potential alterations in CRH prohormone processing or release (i.e. hypothalmic deficiencies) and responsiveness of the adrenal gland itself to ACTH administration. Furthermore, we have investigated circulating levels of cortisol in stressed and non-stressed animals to determine if the Magel2 rat model recapitulates the the aberrant stress response observed in PWS patients. Through this investigation of Magel2’s potential contributions to adrenal insuffciency, we hope to advance the understanding of PWS pathophysiology and lay the groundwork for developing new treatment strategies. This work has been paid for through a grant from the Foundation for Prader-Willi Research. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The diagnostic yield of inferior petrosal sinus sampling in Cushing syndrome in the era of ovine CRH shortage

PurposeThe ovine corticotropin-releasing hormone (oCRH) stimulation test has been routinely used in the diagnostic work-up of ACTH-dependent Cushing syndrome (CS). With oCRH currently being out-of-stock in Europe, we aimed at evaluating the diagnostic performance of inferior petrosal sinus sampling (IPSS) without oCRH stimulation.MethodsWe compared the values of 40 patients with ACTH-dependent CS and negative MRI findings in whom ACTH was measured before and after oCRH stimulation.ResultsThe ratio of central-to-peripheral ACTH measurement (IPS:P) before the combined 3, 5, and 10 min of oCRH stimulation yielded diminished sensitivity (85% vs. 97%), alongside markedly decreased specificity (57% vs. 71%), as well as reduced positive and negative predictive values (90% vs. 94% and 44% vs. 83%), respectively.ConclusionsWith the current drug shortages in Europe, ACTH measurements without oCRH stimulation in IPSS cannot be recommended. Thus, we call for desmopressin or the commercially available human CRH as a potential alternative in the confirmation of ACTH excess by IPSS in equivocal MRI findings.

P-61 From Cushing to Nelson Syndrome: Diagnostic challenges and Surgical Dilemma

Abstract Introduction Diagnosis of the origin of ACTH-dependent Cushing's syndrome (CS) can be difficult in some cases, especially when MRI of the pituitary gland does not reveal an adenoma. Inferior petrosal (IPSS) or cavernous sinus sampling (CSS) with CRH stimulation helps confirm the central origin in most cases. CSS also helps lateralize the tumor within the pituitary gland with a sensitivity of 80%. Because transsphenoidal pituitary surgery is successful in the majority of MRI-negative cases, bilateral adrenalectomy is performed less frequently. Even with advanced radiologic studies, the origin may not be detected. Adrenalectomy may be an option in these cases. However, loss of feedback inhibition of the hypothalamic-pituitary-adrenal axis; can lead to aggressive transformation of an existing ACTH-secreting pituitary adenoma into Nelson's syndrome within 1-5 years. Clinical Case A 46-year-old female patient complaining of hot flashes and unable to lose weight was evaluated for CS and referred to the Department of Neurosurgery for a pituitary adenoma (Table 1). Since the adenoma was <6mm CSS with CRH stimulation was performed. The results did not support central origin (Table 2). Ectopic ACTH secretion was considered, but without finding the origin of the tumor on radiological examination, and treatment with ketoconazole and metyrapone was started for CS. However the patient then discontinued outpatient follow-up. The patient presented to our clinic 6 years later with a new pituitary MRI showing tumor growth (Fig.1b). She complained of fatigue and obesity. She was depressed and anxious. The skin on her face, hands and hump was dark and patchy (Fig.1c). During these years, she consulted several endocrinologists. MRI was performed annually. Brain MRI had showed a fronto-basal extraaxial mass lesion. The patient underwent microsurgical resection and pathology revealed a microcystic meningioma (WHO grade I). 3 months later bilateral nodular hyperplasia of adrenal gland was detected; she underwent bilateral laparoscopic adrenalectomy (no neoplasia). ACTH gradually increased up to 418pg/ml; the diagnosis of Nelson's syndrome was established. The patient underwent endoscopic transsphenoidal adenomectomy. The ACTH level was found to be 12pg/ml on the 2nd postoperative day. Pathological examination revealed a ACTH secreting pituitary adenoma (Ki-67:7%). CS patients suffer from emotional lability due to the effects of elevated cortisol and to the physical changes of their body. Proximal muscle weakness and obesity causes fatigue symptoms. The diagnosis is often delayed until complications occur. Differential diagnosis could be difficult and challenging. Patient's emotional lability could adversely affect treatment/follow-up compliance. The presented case had meningioma resection which could be delayed until remission from CD and bilateral adrenalectomy which could be avoided by endoscopic pituitary surgery. This case is a good example of the need for pituitary centers of excellence.Figure.Fig.1.a. MRI scan of the pituitary gland at first admission shows (a) a lesion extending posteriorly inferior to the right half of the pituitary gland, hyperintense at T2-weighted, iso-hypointense at T1-w, with heterogeneous contrast enhancement consistent with pituitary microadenoma (4mm*2mm) Fig.1.b. Progression in size of the adenoma (6mm*4mm) Fig.1.c. Darkening of the skin due to hypersecretion of ACTH. Table 1.Cavernous sinus sampling with corticotropin-releasing hormone stimulation Table 2.Laboratory tests in different times

OR2803 Corticotroph Fate Weighted Cell Differentiation Paradigm For Human Induced Pluripotent Stem Cells

Abstract Disclosure: M.E. Moore: None. K.G. Chen: None. K. Park: None. D. Asuzu: None. N. Ramavenkat: None. S. Walbridge: None. D. Mullaney: None. M. Arhin: None. D. Maric: None. D. Mandal: None. P. Chittiboina: None. Introduction: The vertebrate anterior pituitary gland is a highly conserved ‘master’ organ that controls hormonal milieu in real-time. Pituitary adenomas and their treatments (surgery, chemotherapy or radiation) can result in pituitary organ failure, specifically hypocortisolism, a challenging condition to manage. Here, we created an optimized paradigm (anterior pituitary corticotroph or APcor) to preferentially drive human induced pluripotent stem cells (hiPSCs) towards a pituitary corticotroph fate. Methods: We directed U112I hiPSCs towards terminally differentiated cell types using a combination of rho-associated kinase inhibitor (Y-27632), bone morphogenic protein 4 (BMP4), transforming growth factor β inhibitor (SB431542), sonic hedgehog (SHH), fibroblast growth factor 8b (FGF8b), fibroblast growth factor 10 (FGF10) and leukemia inhibitory factor (LIF). Markers for pluripotent stem cells, cranial placode (CP), Rathke’s Pouch (RP), anterior pituitary progenitors and mature cell types were assessed at 10-day intervals using quantitative qRT-PCR and multiplex immunocytochemistry (mICC) or immunohistochemistry (IHC). ACTH activity was measured using ELISA (MD Bio). Results: We tested initial differentiation conditions and found optimal CP initiation in presence of SB431542. We observed decrease in embryonic stem cell markers OCT4 and NANOG by day 10, with concomitant increase in CP/RP markers SIX1, PAX6 and SIX6 using mICC/IHC and qRT-PCR. Continuation of differentiation paradigm with LIF showed strong expression of anterior pituitary progenitor markers PITX1, TBX19 and POU1F1 at day 20. pRT-PCR analysis at this time point confirms differentiation towards pituitary cell types without needing enrichment for SIX1. At day 30, POMC expression was observed by IHC in 10-15% of cells as measured by quantitative analysis (Qupath 0.2.0), indicating mature corticotrophs. Corticotroph cell fate was confirmed with corticotropin releasing hormone (CRH) stimulation which led to up to 40x-fold increase in ACTH expression detected by ELISA (D30 w/CRH vs Pluripotent w/CRH). Conclusions: We found with the APcor paradigm, we can reliably drive towards corticotroph cell fate. We successfully differentiated hiPSCs into mature functioning corticotrophs with ability to recapitulate pituitary organ function. Our method is amenable to 3D organoid formation and pre-clinical implantation in animal models of hypophysectomy as a novel hormone replacement therapy. Presentation: Sunday, June 18, 2023

Enhancing Cushing's disease diagnosis: exploring the impact of desmopressin on ACTH gradient during BIPSS.

The differential diagnosis between Cushing's disease (CD) and ectopic ACTH syndrome (EAS) is complex, and bilateral inferior petrosal sinus sampling (BIPSS) is considered the gold-standard test. However, BIPSS with corticotropin-releasing hormone (CRH) stimulation is rarely available. This retrospective cohort study aimed to assess the accuracy of the inferior petrosal sinus to peripheral ACTH gradient (IPS:P) before and after desmopressin stimulation for the differential diagnosis of ACTH-dependent Cushing's syndrome (CS), applying different cutoff values. A total of 50 patients (48 with CD and 2 with EAS) who underwent BIPSS were included in this study. The sensitivity and specificity of IPS:P in BIPSS before and after desmopressin stimulation were evaluated. Various cutoff values for IPS:P were examined to determine their diagnostic accuracy. Using the traditional IPS:P cutoff, the sensitivity was 85.1% before stimulation, 89.6% after stimulation, and a combined sensitivity of 91.7%. Applying cutoff values of IPS:P >1.4 before and >2.8 after stimulation, the sensitivity was 87.2% and 89.6%, respectively, with a combined sensitivity of 91.7%. Receiver operating characteristic (ROC) curve analysis determined optimal cutoff values of 1.2 before stimulation and 1.57 after stimulation, resulting in a sensitivity of 93.6% and 93.8%, respectively, with a combined sensitivity of 97.9%. Specificity remained at 100% throughout all analyses. Among the 43 patients who responded positively to stimulation, 42 (97.7%) did so within the first three minutes, and all 43 (100%) did so within the first five minutes. None of the assessed clinical variables predicted the ACTH response to stimulation in BIPSS with statistical significance. ACTH stimulation with desmopressin during BIPSS improves the accuracy of IPS:P, making it a valuable tool for investigating ACTH-dependent Cushing's syndrome. Considering the low risk of complications, we recommend the use of desmopressin stimulation during BIPSS for the differential diagnosis of ACTH-dependent CS.

Ovine CRH Stimulation and 8 mg Dexamethasone Suppression Tests in 323 Patients With ACTH-Dependent Cushing's Syndrome.

Determining the etiology of adrenocorticotropin (ACTH)-dependent Cushing's syndrome (CS) is often difficult. The gold standard test, inferior petrosal sinus sampling (IPSS), is expensive and not widely available. Evaluate the performance of the corticotropin-releasing hormone stimulation test (CRH-ST) and the 8 mg high-dose dexamethasone suppression test (HDDST) in distinguishing Cushing's disease (CD) from ectopic ACTH syndrome (EAS). Retrospective review in a tertiary referral center. A total of 323 patients with CD or EAS (n = 78) confirmed by pathology or biochemical cure (n = 15) in 96% underwent CRH-ST and HDDST performed between 1986 and 2019. We calculated test sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value, and diagnostic accuracy (DA) for the diagnosis of CD, and determined optimal response criteria for each test, alone and in combination. The CRH-ST performed better than the HDDST (DA 91%, 95% CI 87-94% vs 75%, 95% CI 69-79%). Optimal response criteria were a ≥40% increase of ACTH and/or cortisol during the CRH test and a ≥69% suppression of cortisol during the HDDST. A ≥40% cortisol increase during the CRH test was the most specific measure, PPV 99%. Seventy-four percent of subjects had concordant positive CRH test and HDDST results, yielding Se 93%, Sp 98%, DA 95%, and PPV 99%, with a pretest likelihood of 85%. A proposed algorithm diagnosed 64% of patients with CD with near perfect accuracy (99%), obviating the need for IPSS. CRH is a valuable tool to correctly diagnose the etiology of ACTH-dependent CS. Its current worldwide unavailability impedes optimal management of these patients.

Acute iv CRH administration significantly increases serum active ghrelin in postmenopausal PCOS women compared to postmenopausal controls.

In women with Polycystic Ovarian Syndrome (PCOS), an increased risk of disordered eating has been described. There is growing interest regarding a possible interconnection between psychological states and increased appetite in women with PCOS. Acute stress is characterized by increased Corticotropin Releasing Hormone (CRH) secretion. The aim was to estimate the ghrelin concentrations during CRH test. Twenty postmenopausal women with PCOS and twenty age- and BMI- matched postmenopausal control women were recruited at Aretaieion University Hospital. In the morning (9 am) all subjects had anthropometric measurements (weight, height, waist circumference) and a fasting sample for hormonal measurements. An intravenous (iv) CRH stimulation test conducted over 1 min. Serum active ghrelin levels were measured at 0, 15, 30, 60, 90, 120 min after iv CRH administration. The postmenopausal PCOS group had a higher waist circumference compared to postmenopausal controls. Active ghrelin concentrations increased significantly from 0 to 15 min, to 30 min, to 60 min, to 90 min and then decreased to 120 min. However, within the postmenopausal control group there were no significant changes in serum active ghrelin levels. Serum active ghrelin concentrations were significantly greater in the postmenopausal control group at 0, 15 and 120 min compared to the postmenopausal PCOS group. At 90 min active ghrelin concentrations were significantly greater in the postmenopausal PCOS group. Delta Area Under the Curve of active ghrelin (ΔAUCghr) was significantly greater in the postmenopausal PCOS group compared to controls. In postmenopausal PCOS, but not in postmenopausal controls, iv CRH administration induces increased serum active ghrelin secretion, suggesting a possible anti-stress adaptive mechanism. An increase in serum active ghrelin may induce hunger as a side-effect of this presumed adaptive mechanism.

Bilateral inferior petrosal sinus sampling with human CRH stimulation in ACTH-dependent Cushing's syndrome: results from a retrospective multicenter study.

Bilateral inferior petrosal sinus sampling (BIPSS) is regarded as gold standard to differentiate between Cushing´s disease (CD) and ectopic Cushing's syndrome (ECS). However, published data e.g. on the diagnostic value of additional prolactin analysis is controversial. Thus, we evaluated the diagnostic performance of BIPSS with and without prolactin in a multicenter study. Retrospective study in 5 European reference centers. Patients with overt adrenocorticotropin (ACTH)-dependent Cushing's syndrome at the time of BIPSS with human corticotropin-releasing hormone stimulation were eligible. Cut-offs for the inferior petrosal sinus (IPS) to peripheral (P) ACTH ratio and the normalized ACTH:prolactin IPS:P ratio were calculated via receiver operator characteristics analyses (reference: CD). 156 patients with BIPSS were identified. Of these, 120 patients (92 (77%) females; 106 (88%) CD, 14 (12%) ECS) had either histopathologically confirmed tumors or biochemical remission and/or adrenal insufficiency after surgery; only this subgroup was analyzed by ROC analysis. The optimal cut-offs for the ACTH IPS:P ratio were ≥1.9 at baseline (sensitivity 82.1% (95%CI 73.2-88.6), specificity 85.7% (95%CI 56.2-97.5), AUC 0.86) and ≥2.1 at 5 minutes post-CRH (sensitivity 91.3% (95%CI 83.6-95.7), specificity 92.9%(95%CI 64.1-99.6), AUC 0.96). A subgroup underwent additional prolactin analysis. An optimal cut-off of ≥1.4 was calculated for the normalized ACTH:prolactin IPS:P ratio (sensitivity 96.0% (95%CI 77.7-99.9), specificity 100% (95%CI 56.1-100), AUC 0.99). Our study confirms the high accuracy of BIPSS in the differential diagnosis of ACTH-dependent Cushing's syndrome and suggests that the simultaneous measurement of prolactin might further improve the diagnostic performance of this test.

Pituitary hypoadrenocorticism and hypothyroidism after immunochemotherapy followed by salvage surgery for lung cancer: a case report

BackgroundImmune checkpoint inhibitors (ICIs) have been shown to prolong the survival of patients with non-small cell lung cancer (NSCLC) and have allowed complete resection for advanced lung cancer. However, immune-related adverse events (irAEs) have been recognized as concerning side effects of ICIs.Case presentationA 62-year-old man visited our hospital because of fever, dyspnea, and anorexia. A tumor was found in the right hilum of the lung. It compressed the left atrium and was also thought to be invading the esophagus and a vertebral body. A bronchoscopic biopsy revealed squamous cell carcinoma of the lung (cT4N2M0-IIIB). We thought that a complete resection was impossible because of the N2 status of the tumor and because it had invaded several organs. Radiotherapy was thought to be contraindicated because of the patient’s marked emphysema. Therefore, we administered 4 courses of pembrolizumab plus carboplatin plus nab-paclitaxel immunochemotherapy.After immunochemotherapy, the tumor was downstaged to ycT2bN0M0-IIA and was determined to be acceptable for salvage surgery. A right lower lobectomy and systematic dissection of the mediastinal lymph nodes were performed. The histopathological examination of the resected specimen found that the proportion of the remaining tumor cells was 5%, indicating achievement of a major pathologic response.On postoperative day 79, the patient visited the emergency room because of anorexia. Blood tests showed hyponatremia, hypoglycemia, and eosinophilia. The serum thyroid hormone and thyroid-stimulating hormone levels were low and high, respectively. A corticotropin-releasing hormone stimulation test revealed levels of adrenocorticotropic hormone and cortisol far below the normal ranges. We speculated that the patient had developed pituitary hypoadrenocorticism and hypothyroidism as irAEs associated with ICI treatment. We administered hydrocortisone and levothyroxine, with improvement in the patient’s appetite and normalization of the patient’s serum sodium level. The patient has been receiving ongoing supplementation with oral hydrocortisone and levothyroxine and is doing well 11 months after surgery.ConclusionsThe increasing numbers of patients treated with perioperative ICIs might lead to increasing numbers of patients who develop perioperative irAEs. Careful attention should be paid to the possible development of irAEs during the perioperative management of patients undergoing surgery for lung cancer.

The addition of corticotropin-releasing hormone to 2-day low dose dexamethasone suppression test provides additional case detection.

PURPOSE: The diagnostic value of adding a Corticotropin-Releasing Hormone (CRH) Stimulation Test to the 2-day Low Dose Dexamethasone Suppression Test (Dex-CRH Test) has been debated in the literature. We identified 65 patients with Cushing disease (CD) and 42 patients in whom a diagnosis of Cushing disease could not be confirmed (NCD) after a minimum follow-up of 14 months who underwent the Dex-CRH test. The female sex ratio, median (range) age, and BMI were similar between the two groups. The follow-up for patients with CD and NCD was 74 (4-233) and 52 (14-146) months, respectively. Among 65 patients with CD, 5 (7.7%) had a cortisol level ≤1.4 µg/dl after LDDST but were appropriately classified as CD with a cortisol level >1.4 µg/dL at 15-min post CRH stimulation. In contrast, 3/42 patients (7.1%) in NCD had an abnormal Dex-CRH test. In only one of three patients, the LDDST was marginally normal (cortisol was 1.4 µg/dL and increased to 3.1 µg/dL 15-min post CRH). A cortisol cutoff value of >1.4 µg/dL during the Dex-CRH test provided a sensitivity of 100%, specificity of 93%, and diagnostic accuracy of 97% to diagnose CD. When patients without a Dex level were excluded (n = 74), the sensitivity did not change, but the specificity and accuracy of the Dex-CRH test increased to 97 and 99%, respectively. The Dex-CRH Test provided additional case detection in 5/65 (7.7%) patients with CD. It resulted in one false-positive case compared to LDDST. Measurement of dexamethasone improved diagnostic accuracy of the test.

Subcutaneous transplantation of human embryonic stem cells-derived pituitary organoids.

The pituitary gland, regulating various hormones, is central in the endocrine system. As spontaneous recovery from hypopituitarism is rare, and exogenous-hormone substitution is clumsy, pituitary replacement via regenerative medicine, using pluripotent stem cells, is desirable. We have developed a differentiation method that in mice yields pituitary organoids (POs) derived from human embryonic stem cells (hESC). Efficacy of these POs, transplanted subcutaneously into hypopituitary mice, in reversing hypopituitarism was studied. hESC-derived POs were transplanted into inguinal subcutaneous white adipose tissue (ISWAT) and beneath dorsal skin, a relatively avascular region (AR), of hypophysectomized severe combined immunodeficient (SCID) mice. Pituitary function was evaluated thereafter for ¾ 6mo, assaying basal plasma ACTH and ACTH response to corticotropin-releasing hormone (CRH) stimulation. Histopathologic examination of organoids 150d after transplantation assessed engraftment. Some mice received an inhibitor of vascular endothelial growth factor (VEGF) to permit assessment of how angiogenesis contributed to subcutaneous engraftment. During follow-up, both basal and CRH-stimulated plasma ACTH levels were significantly higher in the ISWAT group (p < 0.001 - 0.05 and 0.001 - 0.005, respectively) than in a sham-operated group. ACTH secretion also was higher in the ISWAT group than in the AR group. Histopathologic study found ACTH-producing human pituitary-cell clusters in both groups of allografts, which had acquired a microvasculature. POs qPCR showed expression of angiogenetic factors. Plasma ACTH levels decreased with VEGF-inhibitor administration. Subcutaneous transplantation of hESC-derived POs into hypopituitary SCID mice efficaciously renders recipients ACTH-sufficient.

Adrenal gland involvement in 11-ketotestosterone production analyzed using LC-MS/MS.

11-ketotestosterone (11KT), which is derived by the bioconversion of testosterone via 11β-hydroxytestosterone (11OHT), is a potent agonist of the human androgen receptor. The adrenal gland is considered an important organ in 11KT production because CYP11B1, which catalyzes testosterone to 11OHT, is expressed in the adrenal glands. The present study aimed to demonstrate adrenal gland involvement in 11KT production in prepubertal children, a topic which has not yet been addressed by any previous studies. Three, retrospective, observational studies were performed. Study 1 enrolled patients aged 8 months to 7 years with severe Kawasaki disease (KD) who were treated with mPSL pulse. Studies 2 and 3 included patients who had received a corticotropin-releasing hormone (CRH) stimulation test and adrenocorticotropic hormone (ACTH) stimulation test, respectively. Samples were collected before and after treatment or drug administration, and serum 11KT, 11OHT, and other 11-oxygenated androgens were measured by LC-MS/MS. Steroid hormone values before and after medication were analyzed using the Wilcoxon signed rank test. Studies 1, 2, and 3 included twenty patients with severe KD, eight patients with a CRH stimulation test, and eight patients with an ACTH stimulation test, respectively. Study 1 demonstrated that the median (IQR) 11KT level was significantly higher before, than after, mPSL pulse (0.39 (0.28-0.47) nmol/L versus 0.064 (0.012-0.075) nmol/L; P < 0.001). Studies 2 and 3 indicated no significant difference in the median 11KT value before and after the CRH or ACTH stimulation test while the 11OHT value was significantly higher after the test. In conclusion, the mediation of 11KT production by ACTH demonstrated the importance of the adrenal glands in the synthesis of this androgen in prepubertal children.

Sensitivity of Different ACTH and Cortisol Concentration Values in Corticotropin-Releasing Hormone Based Tests in Cushing’s Disease

ABSTRACT Purpose In Cushing’s disease (CD) patients, the aim of the present study is to confirm sensitivity of several ACTH and cortisol concentration values in different time points, during corticotropin-releasing hormone (CRH) stimulation test and during CRH stimulation following dexamethasone suppression (DEX-CRH) test. Methods We retrospectively analyzed cortisol and ACTH concentration increment during CRH and DEX-CRH tests in 23 patients with confirmed CD. Cortisol and ACTH concentrations were determined immediately before, 15 min and 30 min after CRH stimulation. We evaluated the sensitivity of different cutoff values including those reported in previous studies, in the diagnosis of CD. Results During DEX–CRH test, 15 min serum cortisol concentration of 1.4 μg/dl (38 nmol/L) had a sensitivity of 90.9%, and serum cortisol concentration ≥1.27 μg/dl (35 nmol/L) had a sensitivity of 100%. For plasma ACTH, sensitivity of 100% was obtained using ACTH ≥3.5pmol/L (16 pg/ml) at 30 min. During CRH test, 35% increase from baseline in ACTH concentration had a sensitivity of 72.7%. Twenty percent increase in cortisol 30 minutes after stimulation yielded a sensitivity of 85.7%. The best sensitivity of ACTH and cortisol increment was obtained 15 min after stimulation, using 19% and 9% increase, respectively (sensitivity of 100% and 92.8%, respectively). Conclusion During CRH and DEX-CRH tests, the study findings agree with the good sensitivity of ACTH and cortisol cutoff values suggested in previous studies; yet, other cutoff values may give a higher diagnostic sensitivity.

The hypothalamic-pituitary-adrenal axis response to ovine corticotropin-releasing-hormone stimulation tests in healthy and hospitalized foals.

The hypothalamic-pituitary-adrenocortical axis (HPAA) response to sepsis can be impaired in critical illness. Corticotropin-releasing hormone (CRH) stimulation test might assess HPAA function in foals. To evaluate plasma cortisol, ACTH, arginine vasopressin (AVP), and endogenous CRH (eCRH) response to different doses of ovine CRH (oCRH). Healthy (n=14) and hospitalized (n=15) foals <7 days of age. In this prospective randomized study, oCRH (0.1, 0.3, and 1μg/kg) was administered intravenously and blood samples were collected before, 15, 30, 60, and 90 minutes after administration of oCRH to determine plasma hormone concentrations. The hormonal response was evaluated as the difference (Delta; μg/dL or pg/mL) or percent change between baseline hormone concentration and each time point after oCRH stimulation. Cortisol concentrations increased from baseline at 15 minutes with 0.1 and 0.3μg/kg and at 30 and 60 minutes from baseline with 1μg/kg oCRH (P < .05) in healthy and hospitalized foals. ACTH concentrations increased from baseline at 15 minutes with 0.1μg/kg and at 30 minutes with 1μg/kg oCRH (P < .05) in hospitalized foals. Delta cortisol 0 - 30, ACTH 0 - 30, and eCRH 0 - 30 was higher for the 1μg/kg compared with 0.1μg/kg oCRH in healthy foals (P < .05). Delta ACTH 0 - 15 and eCRH 0 - 30 was higher for the 1μg/kg compared with the lower doses of oCRH in hospitalized foals (P < .05). Cortisol, ACTH, and eCRH concentrations increased in response to administration of all doses of oCRH. One microgram per kilogram of oCRH appears to be optimal for the assessment of HPAA in healthy and hospitalized foals.

A Cistus incanus Extract Blocks Psychological Stress Signaling and Reduces Neurogenic Inflammation and Signs of Aging in Skin, as Shown in In-Vitro Models and a Randomized Clinical Trial

Psychological stress exerts its effects mainly through the release of corticotropin releasing hormone (CRH), which activates inflammatory pathways in skin (inter alia), resulting in redness, extracellular matrix degradation, loss of skin elasticity and firmness, and the appearance of wrinkles—namely, accelerated skin aging. In order to propose a solution to this neurogenic aging phenomenon, we report here on studies using a myricitrin-rich extract of Cistus incanus, a Mediterranean shrub used in traditional medicine for the treatment of inflammatory and other diseases. These studies include a CRH receptor (CRH-R1) blocking assay; in vitro inflammatory cytokine reduction under CRH stimulation, and ex vivo NF-kB inhibition; and a double-blind clinical trial performed on highly stressed panelists, evaluating skin inflammation and wrinkling (active formulation vs. placebo control, applied split-face following a computer-generated randomization scheme; 36 subjects recruited and randomized, 30 analyzed; no adverse effects recorded; EMA/INFARMED registration #118505, internally funded). The results show that this extract can effectively block the CRH-R1 receptor, preventing NF-κB activation and the production of related pro-inflammatory cytokines. In a clinical setting, this same extract delivered significant anti-inflammatory and anti-aging effects. Taken together, these results demonstrate the value of this extract as a cosmetic active to counter neurogenic inflammation and skin aging.

Relevance of inferior petrosal sinus sampling in the diagnosis of Cushing's syndrome: a case report.

Non-specific signs and symptoms of Cushing's syndrome (CS) can pose a diagnostic challenge. We report the case of a man referred to the service of endocrinology for suspected CS. Hypercortisolism was confirmed on CS screening tests, whereas diagnostic tests confirmed the presence of adrenocorticotropin (ACTH) -dependent CS. The corticotropin-releasing hormone stimulation (CRH) test was performed to determine whether CS hadan endogenous or ectopic origin. Since the CRH and the magnetic resonance imaging (MRI) test were negative, IPSS was performed and suggested that CS was originated in the pituitary glands. Transsphenoidal pituitary resection was carried out. Histopathological analysis confirmed the cortitotrope origin of the tumor. The etiological study and differential diagnosis of CS are complex processes that involve a variety of biochemical and imaging tests. It is important that a sequence of biochemical screening and diagnostic tests is performed, along with studies for establishing the location of the lesion, to determine whether CS has an adrenal, pituitary or ectopic origin. Despite its good diagnostic performance, the results of biochemical tests may not be conclusive, especially in ACTH-dependent CS. In the case reported, the inconclusive results obtained in the CRH test rendered an invasive procedure (IPSS) necessary, which ultimately confirmed diagnosis.

ODP301 Corticotroph Macroadenomas with Extensions to Third Ventricle: Four Case Reports

Abstract Background Pathophysiology of corticotroph macroadenomas has not been elucidated. In this study, we present four cases of adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas with extensions to the third ventricle (TV) and investigate their clinical and pathological characteristics. Case 1 A male in his seventies went to a hospital because of a fall. Upon performing computed tomography, a tumor was incidentally noted. Pituitary MRI revealed a macroadenoma with extension to TV. He presented with Cushingoid features (CF). His basal ACTH level was 112 pg/mL, and urinary free cortisol (UFC) was 129.1 μg/day. Cortisol was not suppressed in the 0.5 mg dexamethasone suppression test (0.5 mg DST). ACTH level increased in the corticotropin-releasing hormone (CRH) stimulation test and the desmopressin (DDAVP) stimulation test. He was diagnosed with Cushing's disease (CD), and endoscopic transsphenoidal surgery (ETSS) was performed. Pathological findings showed densely granulated corticotroph adenoma (DGCA). Somatostatin receptor type 5 (SSTR5) was negative, and ubiquitin-specific protease 8 (USP8) was not mutated. Case 2 A female in her fifties had a visual field defect and pigmentation of the face. MRI revealed a macroadenoma with extension to TV. She presented with CF. Her basal ACTH level was 57.9 pg/mL, and UFC was 259.7 μg/day. Cortisol was not suppressed in the 0.5 mg DST. ACTH level increased in the CRH test and the DDAVP test. She was diagnosed with CD. Pathological findings showed DGCA. SSTR5 was moderate positive, and USP8 was suspected to be mutated. Case 3 A female in her fifties had a throbbing headache. MRI showed a macroadenoma with extension to TV. She did not present with CF. Her basal ACTH level was 71.8 pg/mL, and UFC was 134.3 μg/day. Cortisol was not suppressed in the 0.5 mg DST. ACTH showed response to the CRH test and the DDAVP test. She was diagnosed with subclinical Cushing's disease (SCD) and sparsely granulated corticotroph adenoma (SGCA). SSTR5 was slightly positive, and USP8 was not mutated. Case 4 A male in his fifties had general malaise and polyuria. MRI showed a macroadenoma with extension to TV. He did not present with CF. His basal ACTH level was 179 pg/mL, and UFC was 187.4 μg/day. Cortisol was not suppressed in the 0.5 mg DST. ACTH did not show response to the CRH test and the DDAVP test. He was diagnosed with SCD, and the tumor had oncocytic changes. SSTR5 was negative, and USP8 was not mutated. Conclusion The findings from these case reports suggest that phenotypes of CD and SCD are associated with the differences between SGCA and DGCA. Moreover, it is also suggested that USP8 mutations correlate with SSTR5 expression in macroadenomas that extend to the TV. Presentation: No date and time listed

PSAT043 Liraglutide Ameliorates Symptoms of Cushing Syndrome in a Pediatric Patient

Abstract Background Cushing Syndrome (CS) is a rare disease characterized by hypercortisolism. Pituitary adenomas resulting in ACTH-dependent disease may be difficult to visualize radiologically, resulting in a prolonged diagnostic course and delay in definitive treatment. Adjuvant medical treatment with pharmacologic agents, including steroidogenesis inhibitors and adrenolytics, can be utilized for symptom management. However, hypercortisolemia may be refractory to medical therapy making it necessary to consider symptomatic treatments. We present a case of a pediatric patient with Cushing syndrome in whom treatment with liraglutide, a GLP-1 agonist approved for the treatment of hyperglycemia and obesity, resulted in improvement of symptoms of hypercortisolemia. Case A 13 year old Caucasian female presented with a 2 year history of linear growth arrest, accelerated weight gain and delayed puberty. Her physical examination was remarkable for overt signs of hypercortisolemia. Initial laboratory evaluation showed normal thyroid function, hyperglycemia with HbA1c 7.2% (normal &amp;lt; 5.7%), dyslipidemia and elevated midnight salivary cortisol 0.898 (normal &amp;lt; 0.112 mcg/mL). Hypercortisolemia was confirmed when morning serum cortisol was 25.6 mcg/dL after 1 mg overnight dexamethasone suppression test (DST). This along with an elevated morning ACTH level, 72.8 (7.2- 63.3 pg/mL), confirmed a diagnosis of ACTH-dependent Cushing syndrome. Magnetic Resonance Imaging of the pituitary gland with contrast showed a 4 mm non- enhancing lesion consistent with a Rathke's cleft cyst which was unlikely to be the source of excess ACTH. Results from high dose (8 mg overnight) DST, Corticotropin Releasing Hormone (CRH) stimulation test, and Inferior Petrosal Sinus Sampling for ACTH post CRH stimulation were partially inconsistent with pituitary disease. Further workup for ectopic source(s) of ACTH showed normal imaging of the neck, chest, abdomen and pelvis. A DOTATATE PET scan failed to identify an ACTH source. Adrenolytic adjuvant treatment with mitotane was started but was discontinued due to intolerable gastrointestinal effects. Treatment was changed to ketoconazole, however, hypercortisolemia remained refractory to treatment as evidenced by continued elevation of 24- hour urine free cortisol levels. Metformin was initiated for hyperglycemia treatment but was not tolerated due to persistent nausea and vomiting. The patient was switched to liraglutide and within 3 months she had 10% weight loss, normalization of fasting blood glucose levels and reduction of HbA1c to 5.7%. Additionally, the patient's energy level and baseline hidradenitis suppurativa improved. Conclusion In some pediatric patients with CS, definitive treatment of hypercortisolemia may be delayed or not feasible due to a lack of biochemical or radiological evidence showing the specific etiology for CS. Medical treatment with pharmacologic agents can be utilized for symptom management, however, hypercortisolemia may persist and even be recalcitrant to therapy. In this setting, clinicians should consider using liraglutide to ameliorate some of the cardinal symptoms of CS in pediatric patients. Presentation: Saturday, June 11, 2022 1:00 p.m. - 3:00 p.m.