Mast cell stabilization with sodium cromoglycate modulates pulmonary vessel wall remodeling during four-day hypoxia in rats.

AIM OF THE STUDY: In rats, the environment with low content of oxygen induces hypoxic pulmonary hypertension. Remodeling of pulmonary resistance arteries is particularly triggered by the mast cell degranulation products, e.g., rodent-like interstitial collagenase (matrix metalloproteinase 13). Administration of sodium cromoglycate leads to stabilization of mast cell granules, and thus to the modified remodeling process. MATERIALS AND METHODS: During four-day hypoxia, we treated rats with sodium cromoglycate. Pulmonary vascular remodeling was assessed as well as counts of periarterial pulmonary mast cells, both total and matrix metalloproteinase 13-positive ones. RESULTS: Four-day hypoxia induced remodeling of both resistance arteries and large conduit arteries. We have found increase in the tunica media thickness of resistance arteries. Tunica adventitia thickness of both resistance arteries and large conduit arteries with a diameter of over 300 µm increased as well; the latter ones revealed increase in the number of vasa vasorum in their walls. Mast cell stabilization suppressed hypoxic pulmonary vascular remodeling in resistance pulmonary arteries. Four-day hypoxia led to changes in distribution of toluidine blue-detected and MMP-13 positive periarterial mast cells; this redistribution was also influenced by the administration of sodium cromoglycate. CONCLUSIONS: The number of pulmonary periarterial mast cells seemingly decreases during hypoxia due to their degranulation, which disables their identification. Large conduit arteries do not affect final blood pressure in the pulmonary vascular bed; however, their structure changes substantially under hypoxia. Such remodeling changes are not mediated by mast cell products only since they have occurred in spite of stabilization of mast cell granules.

Genetic analysis of the CYP21A2 gene in neonatal dried blood spots from children with transiently elevated 17-hydroxyprogesterone.

BACKGROUND: Neonatal screening for congenital adrenal hyperplasia (CAH) identifies a certain proportion of newborns with transient moderate elevation of 17-hydroxyprogesterone (17-OHP). These children require regular follow-up until normalization of their 17-OHP levels. We investigated the possibility of reducing the individuals' recall rates by using genetic methods on their original neonatal dried blood spots. PATIENTS AND METHODS: We analysed neonatal dried blood spots from 753 subjects with transiently elevated levels of 17-OHP. The CYP21A2 gene was sequenced to detect point mutations, and the presence of CYP21A2 was further confirmed by two methods utilizing the difference between CYP21A2 and its CYP21A1P pseudogene in the sequence of exon 3 (8-bp deletion). The accuracy of the methods was verified using samples from 70 subjects with known CYP21A2 mutations and 181 healthy children. RESULT: Among the 701 successfully sequenced samples from subjects with transiently elevated 17-OHP, 670 (95%) had no point mutations or novel variants in the CYP21A2 gene. We found no individuals carrying genotypes consistent with the diagnosis of CAH (i.e. homozygotes or compound heterozygotes for point mutations, large deletions or rearrangements). However, 21 heterozygous carriers of known point mutations that cause the classic and nonclassic forms of CAH were identified. Additionally, we detected eight heterozygous and two homozygous point variants with unknown functional significance. CONCLUSION: Although CAH caused by 21-hydroxylase deficiency could be genetically excluded with a reasonable degree of confidence in 95% of the genotyped subjects that had transiently elevated 17-OHP, the performance of the tests was suboptimal when performed using dried blood spots and time-consuming in comparison with the current practice of repeated measurements of 17-OHP. The introduction of this method into clinical practice seems to be impractical at this stage.

Lessons learned from 5 years of newborn screening for congenital adrenal hyperplasia in the Czech Republic: 17-hydroxyprogesterone, genotypes, and screening performance.

The aims were to summarize the experience and to determine the performance metrics of newborn screening (NBS) for congenital adrenal hyperplasia (CAH) in the Czech Republic. 17-Hydroxyprogesterone (17OHP) was measured in NBS samples prospectively in 545,026 newborns and retrospectively in 31 CAH patients born outside the study period. A total of 2,811 screened newborns had abnormal 17OHP; CAH was confirmed in 46 probands. One patient with a severe-moderate genotype of CAH had 17OHP below the cut-off and was diagnosed clinically. This corresponds to a screening sensitivity of 98% and a false positive rate (FPR) of 0.51%. The median of 17OHP in the most severe genotypes was 484 nmol/L (n = 21); in severe/moderate, 321 nmol/L (n = 30); in moderate, 61 nmol/L (n = 20); and in mild genotypes, 31 nmol/L (n = 7). NBS is efficient to detect severe CAH but may fail to detect milder variants. However, the FPR is too high but could be improved by application of a second tier test.

Novel presentation of the c.1856A > G (p.Asp619Gly) TSHR gene-activating variant: relapsing hyperthyroidism in three subsequent generations manifesting in early childhood and an in vitro functional study.

BACKGROUND: Familial non-autoimmune hyperthyroidism is a rare disease caused by germline activating variants in the thyroid-stimulating hormone receptor (TSHR) gene. The c.1856A > G (p.Asp619Gly) pathogenic variant has been described in cases of toxic adenoma but never before, to our knowledge, in a case of familial non-autoimmune hyperthyroidism. PATIENT FINDINGS: A 3-year-old boy was admitted for acute gastroenteritis presenting with goiter and tall stature. Laboratory findings revealed peripheral hyperthyroidism and negativity for thyroid autoantibodies. Antithyroid drug treatment was effective, but relapses occurred shortly after attempts to decrease the drug dose. As the boy's father and paternal grandmother also experienced relapsing hyperthyroidism manifesting in early childhood, genetic testing of TSHR was indicated. The c.1856A > G (p.Asp619Gly) pathogenic variant was found in all three affected family members. Functional in vitro characterization of the variant verified that it enhances constitutional activation of the receptor, leading to increased production of cyclic adenosine monophosphate. Total thyroidectomy was indicated in the boy due to an unsatisfactory prognosis. Due to persistent positive thyroglobulin serum concentration, a diagnostic radioiodine scan was performed approximately 2 years later. Residual thyroid tissue was revealed; therefore, radioiodine ablative therapy was performed. Despite adequate thyroxine substitution over a long period of follow-up, TSH remained suppressed. CONCLUSIONS: Unlike Graves' disease, familial non-autoimmune hyperthyroidism cases present with antithyroid drug-dependence. Not ultrasound but positive thyroglobulin serum concentration indicated residual thyroid tissue. Early detection of residual thyroid tissue and radioiodine ablation prevented the subject from experiencing relapsing hyperthyroidism and undergoing unnecessary repeated surgery. Life-long hormone substitution should be adjusted to free thyroxine rather than TSH serum concentrations.

Functional Analyses of HNF1A-MODY Variants Refine the Interpretation of Identified Sequence Variants.

CONTEXT: While rare variants of the hepatocyte nuclear factor-1 alpha (HNF1A) gene can cause maturity-onset diabetes of the young (HNF1A-MODY), other variants can be risk factors for the development of type 2 diabetes. As has been suggested by the American College of Medical Genetics (ACMG) guidelines for variant interpretation, functional studies provide strong evidence to classify a variant as pathogenic. OBJECTIVE: We hypothesized that a functional evaluation can improve the interpretation of the HNF1A variants in our Czech MODY Registry. DESIGN, SETTINGS, AND PARTICIPANTS: We studied 17 HNF1A variants that were identified in 48 individuals (33 female/15 male) from 20 Czech families with diabetes, using bioinformatics in silico tools and functional protein analyses (transactivation, protein expression, DNA binding, and nuclear localization). RESULTS: Of the 17 variants, 12 variants (p.Lys120Glu, p.Gln130Glu, p.Arg131Pro, p.Leu139Pro, p.Met154Ile, p.Gln170Ter, p.Glu187SerfsTer40, p.Phe215SerfsTer18, p.Gly253Arg, p.Leu383ArgfsTer3, p.Gly437Val, and p.Thr563HisfsTer85) exhibited significantly reduced transcriptional activity or DNA binding (< 40%) and were classified as (likely) pathogenic, 2/17 variants were (likely) benign and 3/17 remained of uncertain significance. Functional analyses allowed for the reclassification of 10/17 variants (59%). Diabetes treatment was improved in 20/29 (69%) carriers of (likely) pathogenic HNF1A variants. CONCLUSION: Functional evaluation of the HNF1A variants is necessary to better predict the pathogenic effects and to improve the diagnostic interpretation and treatment, particularly in cases where the cosegregation or family history data are not available or where the phenotype is more diverse and overlaps with other types of diabetes.

HIV Drug Efavirenz Inhibits CYP21A2 Activity with Possible Clinical Implications.

BACKGROUND: The HIV drugs lopinavir and ritonavir have recently been reported to cause transient adrenal insufficiency in preterm newborns. We, therefore, considered HIV drugs as a cause of transiently elevated 17-hydroxyprogesterone (17OHP) levels in a neonatal screening test for congenital adrenal hyperplasia in a preterm girl exposed to zidovudine, efavirenz, tenofovir, and emtricitabine. OBJECTIVE: So far, HIV drugs have not been tested for their effect on steroidogenesis and the steroidogenic enzyme activity of CYP21A2 specifically in an in vitro system. METHODS: We tested the effect of efavirenz, tenofovir, emtricitabine, and zidovudine on steroidogenesis of human adrenal H295R cells. Cells were treated with the drugs at different concentrations including concentrations in therapeutic use. The effect on CYP21A2 activity was assessed by testing the conversion of radiolabeled 17OHP to 11-deoxycortisol. Cell viability was tested by an MTT assay. In addition, recombinant human CYP21A2 protein was used to assess direct drug effects on CYP21A2 activity. RESULTS: We observed significantly decreased CYP21A2 activity in both in vitro testing systems after treatment with efavirenz at therapeutic concentrations. Moreover, efavirenz affected cell viability. By contrast, the other test drugs did not affect steroidogenesis. Follow-up of our patient revealed elevated 17OHP and androgen levels during the first weeks of life, but values normalized spontaneously. Genetic testing for CYP21A2 mutations was negative. Thus, it remains unsettled whether the transient 17OHP elevation in this baby was due to a drug effect. CONCLUSION: The HIV drug efavirenz inhibits CYP21A2 activity in vitro through direct interaction with enzyme catalysis at therapeutic concentrations. This may have clinical implications for HIV treatment in children and adults. However, so far, clinical data are scarce, and further studies are needed to be able to draw clinical conclusions.

Effectiveness and safety of the tri-iodothyronine analogue Triac in children and adults with MCT8 deficiency: an international, single-arm, open-label, phase 2 trial.

BACKGROUND: Deficiency of the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) causes severe intellectual and motor disability and high serum tri-iodothyronine (T3) concentrations (Allan-Herndon-Dudley syndrome). This chronic thyrotoxicosis leads to progressive deterioration in bodyweight, tachycardia, and muscle wasting, predisposing affected individuals to substantial morbidity and mortality. Treatment that safely alleviates peripheral thyrotoxicosis and reverses cerebral hypothyroidism is not yet available. We aimed to investigate the effects of treatment with the T3 analogue Triac (3,3',5-tri-iodothyroacetic acid, or tiratricol), in patients with MCT8 deficiency. METHODS: In this investigator-initiated, multicentre, open-label, single-arm, phase 2, pragmatic trial, we investigated the effectiveness and safety of oral Triac in male paediatric and adult patients with MCT8 deficiency in eight countries in Europe and one site in South Africa. Triac was administered in a predefined escalating dose schedule-after the initial dose of once-daily 350 µg Triac, the daily dose was increased progressively in 350 µg increments, with the goal of attaining serum total T3 concentrations within the target range of 1·4-2·5 nmol/L. We assessed changes in several clinical and biochemical signs of hyperthyroidism between baseline and 12 months of treatment. The prespecified primary endpoint was the change in serum T3 concentrations from baseline to month 12. The co-primary endpoints were changes in concentrations of serum thyroid-stimulating hormone (TSH), free and total thyroxine (T4), and total reverse T3 from baseline to month 12. These analyses were done in patients who received at least one dose of Triac and had at least one post-baseline evaluation of serum throid function. This trial is registered with ClinicalTrials.gov, number NCT02060474. FINDINGS: Between Oct 15, 2014, and June 1, 2017, we screened 50 patients, all of whom were eligible. Of these patients, four (8%) patients decided not to participate because of travel commitments. 46 (92%) patients were therefore enrolled in the trial to receive Triac (median age 7·1 years [range 0·8-66·8]). 45 (98%) participants received Triac and had at least one follow-up measurement of thyroid function and thus were included in the analyses of the primary endpoints. Of these 45 patients, five did not complete the trial (two patients withdrew [travel burden, severe pre-existing comorbidity], one was lost to follow-up, one developed of Graves disease, and one died of sepsis). Patients required a mean dose of 38.3 µg/kg of bodyweight (range 6·4-84·3) to attain T3 concentrations within the target range. Serum T3 concentration decreased from 4·97 nmol/L (SD 1·55) at baseline to 1·82 nmol/L (0·69) at month 12 (mean decrease 3·15 nmol/L, 95% CI 2·68-3·62; p<0·0001), while serum TSH concentrations decreased from 2·91 mU/L (SD 1·68) to 1·02 mU/L (1·14; mean decrease 1·89 mU/L, 1·39-2·39; p<0·0001) and serum free T4 concentrations decreased from 9·5 pmol/L (SD 2·5) to 3·4 (1·6; mean decrease 6·1 pmol/L (5·4-6·8; p<0·0001). Additionally, serum total T4 concentrations decreased by 31·6 nmol/L (28·0-35·2; p<0·0001) and reverse T3 by 0·08 nmol/L (0·05-0·10; p<0·0001). Seven treatment-related adverse events (transiently increased perspiration or irritability) occurred in six (13%) patients. 26 serious adverse events that were considered unrelated to treatment occurred in 18 (39%) patients (mostly hospital admissions because of infections). One patient died from pulmonary sepsis leading to multi-organ failure, which was unrelated to Triac treatment. INTERPRETATION: Key features of peripheral thyrotoxicosis were alleviated in paediatric and adult patients with MCT8 deficiency who were treated with Triac. Triac seems a reasonable treatment strategy to ameliorate the consequences of untreated peripheral thyrotoxicosis in patients with MCT8 deficiency. FUNDING: Dutch Scientific Organization, Sherman Foundation, NeMO Foundation, Wellcome Trust, UK National Institute for Health Research Cambridge Biomedical Centre, Toulouse University Hospital, and Una Vita Rara ONLUS.

Anticancer and antiproliferative activity of natural brassinosteroids.

Brassinosteroids (BRs) are steroid plant hormones that are essential for many plant growth and developmental processes, including cell expansion, vascular differentiation and stress responses. Up to now the inhibitory effects of BRs on cell division of mammalian cells are unknown. To determine basic anticancer structure-activity relationships of natural BRs on human cells, several normal and cancer cell lines have been used. Several of the tested BRs were found to have high cytotoxic activity. Therefore, in our next series of experiments, we tested the effects of the most promising and readily available BR analogues with interesting anticancer properties, 28-homocastasterone (1) and 24-epibrassinolide (2), on the viability, proliferation, and cycling of hormone-sensitive/insensitive (MCF-7/MDA-MB-468) breast and (LNCaP/DU-145) prostate cancer cell lines to determine whether the discovered cytotoxic activity of BRs could be, at least partially, related to brassinosteroid-nuclear receptor interactions. Both BRs inhibited cell growth in a dose-dependent manner in the cancer cell lines. Flow cytometry analysis showed that BR treatment arrested MCF-7, MDA-MB-468 and LNCaP cells in G(1) phase of the cell cycle and induced apoptosis in MDA-MB-468, LNCaP, and slightly in the DU-145 cells. Our results provide the first evidence that natural BRs can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells. Therefore, these plant hormones are promising leads for potential anticancer drugs.

CYP17A1 inhibitor abiraterone, an anti-prostate cancer drug, also inhibits the 21-hydroxylase activity of CYP21A2.

Abiraterone is an inhibitor of CYP17A1 which is used for the treatment of castration resistant prostate cancer. Abiraterone is known to inhibit several drug metabolizing cytochrome P450 enzymes including CYP1A2, CYP2D6, CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, but its effects on steroid metabolizing P450 enzymes are not clear. In preliminary results, we had observed inhibition of CYP21A2 by 1µM abiraterone. Here we are reporting the effect of abiraterone on activities of CYP21A2 in human adrenal cells as well as with purified recombinant CYP21A2. Cells were treated with varying concentrations of abiraterone for 24h and CYP21A2 activity was measured using [3H] 17-hydroxyprogesterone as substrate. Whole steroid profile changes were determined by gas chromatography-mass spectrometry. Binding of abiraterone to purified CYP21A2 protein was measured spectroscopically. Computational docking was used to study the binding and interaction of abiraterone with CYP21A2. Abiraterone caused significant reduction in CYP21A2 activity in assays with cells and an inhibition of CYP21A2 activity was also observed in experiments using recombinant purified proteins. Abiraterone binds to CYP21A2 with an estimated Kd of 6.3µM. These inhibitory effects of abiraterone are at clinically used concentrations. A loss of CYP21A2 activity in combination with reduction of CYP17A1 activities by abiraterone could result in lower cortisol levels and may require monitoring for any potential adverse effects.

Androgen production in pediatric adrenocortical tumors may occur via both the classic and/or the alternative backdoor pathway.

Children with adrenocortical tumors (ACTs) often present with virilization due to high tumoral androgen production, with dihydrotestosterone (DHT) as most potent androgen. Recent work revealed two pathways for DHT biosynthesis, the classic and the backdoor pathway. Usage of alternate routes for DHT production has been reported in castration-resistant prostate cancer, CAH and PCOS. To assess whether the backdoor pathway may contribute to the virilization of pediatric ACTs, we investigated seven children suffering from androgen producing tumors using steroid profiling and immunohistochemical expression studies. All cases produced large amounts of androgens of the classic and/or backdoor pathway. Variable expression of steroid enzymes was observed in carcinomas and adenomas. We found no discriminative pattern. This suggests that enhanced androgen production in pediatric ACTs is the result of deregulated steroidogenesis through multiple steroid pathways. Thus future treatments of ACTs targeting androgen overproduction should consider these novel steroid production pathways.