Postoperative ACTH-stimulated aldosterone predicts biochemical outcome in primary aldosteronism.

OBJECTIVE: Primary aldosteronism (PA) is the most common surgically curable cause of hypertension. Unilateral aldosterone-producing adenoma can be treated with adrenalectomy. Clinical and biochemical outcomes are assessed 6-12 months after adrenalectomy according to primary aldosteronism surgical outcome (PASO) consensus criteria. Earlier prediction of biochemical remission would be desirable as it could reduce cumbersome follow-up visits. We hypothesized that postoperative adrenocorticotropic hormone (ACTH) stimulated plasma aldosterone concentrations (PAC) measured shortly after adrenalectomy can predict PASO outcomes. DESIGN: Retrospective cohort study. METHODS: We analyzed 100 patients of the German Conn's registry who underwent adrenalectomy and postoperative ACTH stimulation tests within the first week after adrenalectomy. Six to twelve months after adrenalectomy we assessed clinical and biochemical outcomes according to PASO criteria. Serum cortisol and PAC were measured by immunoassay at baseline and 30 min after the intravenous ACTH infusion. We used receiver operating characteristics (ROC) curve analysis and matched the parameters to PASO outcomes. RESULTS: Eighty-one percent of patients had complete, 13% partial, and 6% absent biochemical remission. Complete clinical remission was observed in 28%. For a cut-off of 58.5 pg/mL, stimulated PAC could predict partial/absent biochemical remission with a high sensitivity (95%) and reasonable specificity (74%). Stimulated PAC's area under the curve (AUC) (0.89; confidence interval (CI) 0.82-0.96) was significantly higher than other investigated parameters. CONCLUSIONS: Low postoperative ACTH stimulated PAC was predictive of biochemical remission. If confirmed, this approach could reduce follow-up visits to assess biochemical outcome.

Differential central regulatory mineralocorticoidreceptor systems for anxiety and depression - Could KCNJ5 be an interesting target for further investigations in major depression?

The mineralocorticoid receptor (MR) is suggested to play a role in the pathophysiology of depression and anxiety. Main support comes from studies in patients with primary aldosteronism (PA) which suggested different central pathways for depression and anxiety mediated via the MR and gender differences. We investigated 118 patients with PA over 3 years using self-rating questionnaires for anxiety (GAD-7) and depression (PHQD) at baseline and once a year under specific treatment with adrenalectomy (ADX; n = 48) or a MR antagonist (MRA; n = 70). Genotyping for KCNJ5 mutation was performed in resected tumors. At baseline, patients treated by ADX or MRA had comparable scores for anxiety and depression. Females showed a better metabolic profile but higher scores of depression and anxiety, compared to males. Initiation of specific treatment for PA resulted in a better response in depressive symptoms after ADX and of anxiety under MRA treatment. However, GAD-7 and PHQD remained high in women over the three-year follow-up. KCNJ5 mutation, linked to co-secretion of hybrid steroids as 18-oxocortisol and 18-hydroxycortisol, was detected in 10 female and 2 male patients. They tended to have higher GAD and PHQD scores at baseline compared to patients without KNCJ5 mutation, but showed a significant better reduction in symptoms of anxiety during the 3-year follow up compared to patients without this mutation (all p < 0.05). These data support a differentiated regulation of depression and anxiety by the MR. Moreover, genetic mutations such as KCNJ5 could affect the pathophysiology of these disorders by impacting in adrenal steroidogenesis.

Targeted sequencing of candidate gene regions for myelofibrosis in dogs.

BACKGROUND: Myelofibrosis often lacks an identifiable cause in dogs. In humans, most primary myelofibrosis cases develop secondary to driver mutations in JAK2, CALR, or MPL. OBJECTIVES: To determine the prevalence of variants in JAK2, CALR, or MPL candidate regions in dogs with myelofibrosis and in healthy dogs. ANIMALS: Twenty-six dogs with myelofibrosis that underwent bone marrow biopsy between 2010 and 2018 and 25 control dogs matched for age, sex, and breed. METHODS: Cross-sectional study. Amplicon sequencing of JAK2 exons 12 and 14, CALR exon 9, and MPL exon 10 was performed on formalin-fixed, decalcified, paraffin-embedded bone marrow (myelofibrosis) or peripheral blood (control) DNA. Somatic variants were categorized as likely-benign or possibly-pathogenic based on predicted impact on protein function. Within the myelofibrosis group, hematologic variables and survival were compared by variant status (none, likely-benign only, and ≥1 possibly-pathogenic). The effect of age on variant count was analyzed using linear regression. RESULTS: Eighteen of 26 (69%) myelofibrosis cases had somatic variants, including 9 classified as possibly-pathogenic. No somatic variants were detected in controls. Within the myelofibrosis group, hematologic variables and survival did not differ by variant status. The number of somatic variants per myelofibrosis case increased with age (estimate, 0.69; SE, 0.29; P = .03). CONCLUSIONS AND CLINICAL IMPORTANCE: Somatic variants might initiate or perpetuate myelofibrosis in dogs. Our findings suggest the occurrence of clonal hematopoiesis in dogs, with increasing incidence with age, as observed in humans.

Retrospective evaluation of fluid production at the time of thoracostomy tube removal following elective and emergency surgery in dogs (2010-2017): 185 cases.

OBJECTIVE: To report the rate of fluid production at the time of removal of thoracostomy tubes placed intraoperatively and to determine the association of this rate with specific patient factors, surgical factors, or clinical diagnosis. The secondary objective was to determine whether identification of pleural effusion within 2 weeks of thoracostomy tube removal was associated with the same variables. DESIGN: Retrospective study. SETTING: University teaching hospital. ANIMALS: One hundred eighty-five client-owned dogs with thoracostomy tubes placed intraoperatively between January 2010 and March 2017. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Thoracostomy tubes were removed at a median fluid production of 0.09 mL/kg/h (range, 0-7.0 m L/kg/h). Median fluid production at the time of thoracostomy tube removal was significantly higher in dogs with preoperative pleural effusion compared to dogs without preoperative pleural effusion (0.21 vs 0.05 mL/kg/h; P = 0.0001) and in dogs that had a median sternotomy compared to dogs that had a lateral thoracotomy (0.14 vs 0.09 mL/kg/h; P = 0.04). Of the 169 dogs available for follow-up, 12 (7.1%) had pleural effusion within 2 weeks of removal of the thoracostomy tube. Detection of pleural effusion during the follow-up period was significantly associated with the presence of preoperative pleural effusion (P = 0.0019) and the diagnosis (P = 0.01). A greater proportion of dogs with a lung lobe torsion (4/9, 44.4%) and idiopathic chylothorax (2/7, 28.5%) had pleural effusion within 2 weeks compared to other diagnoses. Reintervention was performed in 4.7% of dogs. CONCLUSIONS: Thoracostomy tubes were removed at pleural fluid production rates that frequently exceeded current veterinary guidelines. However, the fluid production rate at the time of thoracostomy tube removal was not associated with the detection of pleural effusion within 2 weeks of thoracostomy tube removal, and the overall need for reintervention following thoracostomy tube removal was low (4.7%).

Characteristics of preoperative steroid profiles and glucose metabolism in patients with primary aldosteronism developing adrenal insufficiency after adrenalectomy.

Treatment of choice in patients with unilateral aldosterone producing adenoma (APA) is adrenalectomy. Following surgery, most patients retain normal adrenal function, while some develop adrenal insufficiency (AI). To facilitate early detection and treatment of AI, we aimed to identify variables measured pre-operatively that are associated with post-operative AI. Variables obtained from 66 patients before and after surgery included anthropometrical data, clinical chemistry, endocrine work-up. LC-MS/MS steroid hormone profiles from tests before surgery (ACTH-stimulation, saline infusion, dexamethasone suppression) were obtained. Based on 78 variables, machine-learning methods were used in model fitting for classification and regression to predict ACTH-stimulated cortisol after surgery. Among the 78 variables, insulin concentration during pre-operative oral glucose tolerance test (OGTT) correlated positively, and dexamethasone suppressed glucocorticoids correlated negatively with ACTH-stimulated cortisol after surgery. Inclusion of LC-MS/MS measurements allowed construction of better models associated with the occurrence of AI in the training data, but did not allow reliable prediction in cross-validation. Our results suggest that glucocorticoid co-secretion (low insulin during pre-operative OGTT and insufficient suppression of glucocorticoids following dexamethasone) are correlated with the development of post-operative AI. Addition of steroid profiles improved the accuracy of prediction, but cross validation revealed lack of reliability in the prediction of AI.

Atherosclerotic Burden and Arterial Stiffness are Not Increased in Patients with Milder Forms of Primary Aldosteronism Compared to Patients with Essential Hypertension.

Patients with primary aldosteronism (PA) are at increased cardiovascular risk, compared to patients with essential hypertension (EH). Cardiovascular damage could depend on PA phenotype, potentially being lower in milder forms of PA. Our aim was to assess atherosclerotic burden and arterial stiffness in 88 prospectively recruited patients, including 44 patients with mild PA and EH respectively. All patients underwent a structured study program, including measurements of ankle-brachial index, oscillometric measurement of central pulse wave velocity (cPWV) and vascular ultrasound examination of the supraaortic arteries, the abdominal aorta, and the femoropopliteal arteries. A plaque score was calculated to estimate atherosclerotic burden for each patient. This is a prospective case-control study set at a tertiary care hospital. Patients with PA and EH matched well for age, gender, blood pressure, BMI, and cardiovascular risk factors such as diabetes mellitus and smoking status. Common carotid intima-media thickness (0.77 vs. 0.75 mm; p=0.997) and cPWV (7.2 vs. 7.1 m/s; p=0.372) were comparable between patients with PA and EH. The atherosclerotic burden, as expressed by the plaque score, did not differ between the two groups (p=0.159). However, after initiation of treatment cPWV was significantly decreased in patients with PA (p=0.017). This study shows that subclinical atherosclerotic burden and arterial stiffness in patients with milder forms of PA is comparable to patients with EH. Nevertheless, specific treatment for PA significantly improved cPWV, which argues for a more liberal use of mineralocorticoid receptor antagonists in patients with arterial hypertension.

Steroid Profiling and Immunohistochemistry for Subtyping and Outcome Prediction in Primary Aldosteronism-a Review.

PURPOSE OF REVIEW: Steroid profiling and immunohistochemistry are both promising new tools used to improve diagnostic accuracy in the work-up of primary aldosteronism (PA) and to predict treatment outcomes. Herein, we review the recent literature and present an outlook to the future of diagnostics and therapeutic decision-making in patients with PA. RECENT FINDING: PA is the most common endocrine cause of arterial hypertension and unilateral forms of the disease are potentially curable by surgical resection of the overactive adrenal. Recent studies have shown that adrenal steroid profiling by liquid chromatography-tandem mass spectrometry (LC-MS/MS) can be helpful for subtyping unilateral and bilateral forms of PA, classifying patients with a unilateral aldosterone-producing adenoma (APA) according to the presence of driver mutations of aldosterone production in APAs, and potentially predicting the outcomes of surgical treatment for unilateral PA. Following adrenalectomy, immunohistochemistry of aldosterone synthase (CYP11B2) in resected adrenals is a new tool to analyze "functional" histopathology and may be an indicator of biochemical outcomes after surgery. Biochemical and clinical outcomes of therapy in PA vary widely among patients. Peripheral venous steroid profiling at baseline could improve diagnostic accuracy and help in surgical decision-making in cases of a suspected APA; results of "functional" histopathology could help determine which patients are likely to need close post-surgical follow-up for persistent aldosteronism.

Adrenal Insufficiency After Unilateral Adrenalectomy in Primary Aldosteronism: Long-Term Outcome and Clinical Impact.

CONTEXT: Primary aldosteronism (PA) represents a secondary form of arterial hypertension that can be cured by surgery. Evidence of adrenal insufficiency (AI) was recently found in patients with PA who had undergone unilateral adrenalectomy (uADX). OBJECTIVE: To study the incidence and long-term outcome of postoperative AI after uADX for PA. DESIGN: Prospective registry study (August 2014 until the end of 2018). SETTING: Tertiary referral center. PATIENTS: One hundred consecutive patients undergoing uADX for PA were included. All patients underwent postoperative ACTH stimulation testing. INTERVENTION: Postoperative ACTH stimulation testing to identify patients with AI. MAIN OUTCOME MEASURES: Incidence of patients with postoperative AI and definition of long-term outcome. RESULTS: Twenty-seven percent of patients developed postoperative AI. Of these, 48% had postoperative ACTH stimulation serum cortisol levels ≤13.5 µg/dL (severe AI); 52% were classified into the group with moderate AI (stimulated serum cortisol levels: 13.5 to 17 µg/dL). Patients with severe AI required significantly longer hydrocortisone replacement therapy than the moderate group (median [25th, 75th percentiles]: 353 [294, 476] days; 95% CI: 284 to 322 days; vs 74 [32, 293] days; 95% CI: 11 to 137 days; P = 0.016). One patient with severe AI was hospitalized for an acute adrenal crisis. With a cumulative follow-up of 14.5 years, this produced an incidence rate of 6.9 adrenal crises per 100 patient-years. CONCLUSION: We suggest performing postoperative ACTH stimulation tests in all patients who undergo uADX for PA.

Cytology and the cell block method in diagnostic characterization of canine lymphadenopathy and in the immunophenotyping of nodal lymphoma.

Minimally invasive techniques used to evaluate canine peripheral lymphadenopathy (PLN), including fine needle aspiration biopsy with cytological evaluation (FNAB-C) and flow cytometry (FC), have benefits and limitations. The cell block (CB) method is an alternate processing technique in which fine needle aspirate biopsy samples are concentrated, fixed, and embedded in paraffin for routine histological processing/staining. Utilizing three observers, we determined the diagnostic value of the CB in evaluating canine PLN across six categories (non-diagnostic, reactive, inflammatory/infectious, probable lymphoma and lymphoma, metastatic neoplasia) and correlated findings to immunophenotypic and clonal antigen receptor rearrangement results in canine nodal lymphoma. Eighty-five paired FNAB-C and CB samples were evaluated from canine patients presenting to the University of Minnesota Veterinary Oncology or Internal Medicine services. Diagnostic quality samples were obtained in 55/85 (65%) CB and 81/85 (95%) FNAB-C samples, respectively, and nodal pathology impacted CB diagnostic yield. Overall percent agreement between diagnostic-quality FNAB-C and CB samples was 86%, but increased to 95% if the categories of lymphoma and probable lymphoma were combined. There was 100% agreement for both the diagnoses of metastatic neoplasia and reactive lymph nodes and 92% agreement for the diagnosis of lymphoma/probable lymphoma. Using immunohistochemistry (IHC), CB samples correctly immunophenotyped 22/23 (96%) cases of B-cell lymphoma, but only 1/6 (17%) cases of T-cell lymphoma. IHC was not completed on nine cases of lymphoproliferative disease because of insufficient cellularity. When the CB method (CBM) yielded diagnostic quality samples there was good to excellent agreement with FNAB-C samples and CB samples were suitable for some IHC tests.

Cortisol Excess in Patients With Primary Aldosteronism Impacts Left Ventricular Hypertrophy.

Context: Primary aldosteronism (PA) represents the most frequent form of endocrine hypertension. Hyperaldosteronism and hypercortisolism both induce excessive left ventricular hypertrophy (LVH) compared with matched essential hypertensives. In recent studies frequent cosecretion of cortisol and aldosterone has been reported in patients with PA. Objective: Our aim was to investigate the impact of cortisol cosecretion on LVH in patients with PA. We determined 24-hour excretion of mineralocorticoids and glucocorticoids by gas chromatography-mass spectrometry and assessed cardiac remodeling using echocardiography initially and 1 year after initiation of treatment of PA. Patients: We included 73 patients from the Munich center of the German Conn's registry: 45 with unilateral aldosterone-producing adenoma and 28 with bilateral adrenal hyperplasia. Results: At the time of diagnosis, 85% of patients with PA showed LVH according to left ventricular mass index [(LVMI); median 62.4 g/m2.7]. LVMI correlated positively with total glucocorticoid excretion (r2 = 0.076, P = 0.018) as well as with tetrahydroaldosterone excretion (r2 = 0.070, P = 0.024). Adrenalectomy led to significantly reduced LVMI in aldosterone-producing adenoma (P < 0.001) whereas mineralocorticoid receptor antagonist therapy in bilateral adrenal patients with hyperplasia reduced LVMI to a lesser degree (P = 0.024). In multivariate analysis, the decrease in LVMI was positively correlated with total glucocorticoid excretion and systolic 24-hour blood pressure, but not with tetrahydroaldosterone excretion. Conclusion: Cortisol excess appears to have an additional impact on cardiac remodeling in patients with PA. Treatment of PA by either adrenalectomy or mineralocorticoid receptor antagonist improves LVMI. This effect was most pronounced in patients with high total glucocorticoid excretion.

Lymphocyte populations in joint tissues from dogs with inflammatory stifle arthritis and associated degenerative cranial cruciate ligament rupture.

OBJECTIVE: To evaluate lymphocyte populations in stifle synovium and synovial fluid of dogs with degenerative cranial cruciate ligament rupture (CCLR). STUDY DESIGN: Prospective clinical study. ANIMALS: Dogs (n=25) with stifle arthritis and CCLR, 7 dogs with arthritis associated with cartilage degeneration (osteoarthritis [OA]), and 12 healthy Beagle dogs with intact CCL. METHODS: Arthritis was graded radiographically in CCLR dogs. After collection of joint tissues, mononuclear cells were isolated and subsequently analyzed using flow cytometry for expression of CD3, CD4, CD8, and CD21. RESULTS: The proportions of CD4(+) T helper lymphocytes, CD8(+) cytotoxic T lymphocytes, and CD3(+) CD4(-) CD8(-) T lymphocytes were increased in synovium from dogs with CCLR compared with synovium from healthy Beagle dogs (P<.05). The proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was increased in dogs with CCLR compared with dogs with OA (P<.05). In dogs with CCLR, the proportion of CD3(+) CD4(-) CD8(-) T lymphocytes in synovial fluid was inversely correlated with radiographic arthritis (S(R) =-0.68, P<.005). CONCLUSION: Lymphocytic inflammation of stifle synovium and synovial fluid is an important feature of the CCLR arthropathy. Lymphocyte populations include T lymphocytes expressing CD4 and CD8, and CD3(+) CD4(-) CD8(-) T lymphocytes. Presence of CD3(+) CD4(-) CD8(-) T lymphocytes was associated with development of stifle synovitis. Further work is needed to fully identify the phenotype of these cells.

Reactive oxygen species mediate mitogenic growth factor signaling pathways in human leiomyoma smooth muscle cells.

Uterine leiomyomas are benign uterine tumors characterized by extracellular matrix remodeling, increased collagen deposition, and increased smooth muscle cell (SMC) proliferation. The reactive oxygen species (ROS) producing NADPH oxidase complex has been shown to be involved in the signaling pathways of several growth factors, cytokines, and vasoactive agents that stimulate proliferation of a variety of cell types. Our objective was to test the hypothesis that ROS derived from NADPH oxidase is a necessary component of the MAP kinase mitogenic pathway activated by platelet derived growth factor (PDGF) and epidermal growth factor (EGF) in leiomyoma SMCs (LSMCs). Primary cell cultures of LSMCs were used as our experimental model. Our results showed that stimulation of these cells with PDGF or EGF caused a marked increase in intracellular ROS production and that the NADPH oxidase inhibitor, DPI, blocks ROS production. In addition, inhibition of ROS production by NADPH oxidase inhibitors blocked, in a dose-dependent manner, the EGF- and PDGF-induced increase in [(3)H]thymidine incorporation by LSMCs. Furthermore, an exogenous source of ROS, hydrogen peroxide, was sufficient to stimulate [(3)H]thymidine incorporation in LSMCs but did not affect COL1A2 and COL3A1 mRNA levels. Inhibition of the NADPH oxidase complex decreased PDGF-induced MAPK1/MAPK3 activation, whereas exogenous hydrogen peroxide induced MAPK1/MAPK3 activation. This article is the first report suggesting the presence of the NADPH oxidase system and its importance in mitogenic signaling pathways in LSMCs. The necessity of NADPH oxidase-derived ROS for EGF and PDGF signaling pathways leading to cell proliferation points to another potential therapeutic target for treatment and/or prevention of uterine leiomyomas.