Paul M. Stewart

Introduction: Oral once-daily dual-release hydrocortisone (DR-HC) therapy provides a more physiological cortisol profile than conventional thrice-daily (TID) replacement therapy and has demonstrated improved metabolic profile among patients with adrenal insufficiency (AI). The mechanisms by which this metabolic improvement occurs may be due to less total exposure, changed cortisol time exposure profile, but also modified metabolism of cortisol. Objective: The aim was to study steroid enzyme activities related to corticosteroids during DR-HC and TID. Methods: Patients with primary AI received DR-HC or an equal total daily dose of TID hydrocortisone in a 12-week crossover multi-center study. 24 h urinary collection was performed during both treatment and in 124 healthy controls. Urinary cortisol metabolites were measured using gas chromatography/mass spectrometry providing an index of total cortisol exposure and metabolism. Results: Fifty patients (22 female, mean age 47 years (range 19–71)) and 124 healthy controls [73 females, mean age 48 years (range 20–81)] were included in the study. Total cortisol metabolites (F, E, THF, 5αTHF, THE, cortols, cortolones) were significantly decreased during DR-HC treatment [median: 6380 μg/24h] compared to TID (8825 μg/24 h); PP=0.089). Compared to controls, the urinary THF+5αTHF/THE ratio reflecting 11βHSD1 activity was increased during both DR-HC (PPPPP=0.358). The 5α-reduced metabolite, 5αTHF, was similar in patients compared to control but decreased significantly with DR-HC compared to TID (PPPPP<0.001) indicating that the main driver for this was an increase in 5β-reductase activity during TID and to a lesser extent during DR-HC. Conclusion: The urinary cortisol metabolome shows more striking abnormalities in patients receiving TID compared to DR-HC replacement therapy and maybe a more sensitive marker of “optimal cortisol replacement”. The increased 11βHSD1 activity in patients on TID may account for the deleterious metabolic phenotype reported in patients with AI and its reduced activity during DR-HC may also mediate some of the beneficial effects previously seen with this treatment.

Introduction: Primary adrenal insufficiency (PAI) leads to a drastically reduced production of steroids from the adrenal cortex, but a few patients may keep some residual adrenal steroid secretion that may simplify replacement therapy and prevent adrenal crisis. Irrespectively, the conventional glucocorticoid (GC) replacement therapy, using thrice-daily oral hydrocortisone, does not restore the patients’ physiological cortisol profile. Objective: The primary objective was to study enzyme activity related to GC action, synthesis and degradation in patients with PAI and GC replacement by assessing urinary steroid profile. The secondary aim was to analyze if any residual adrenal function can be detected in patients with long-standing PAI. Methods: Case-control study comparing patients with PAI >6 months after diagnosis, under stable thrice-daily hydrocortisone replacement (20-40mgs/day) with healthy controls. Urinary steroid profile was measured using gas chromatography/mass spectrometry. Activity of enzymes involved in synthesis and metabolism of steroid hormones was evaluated by calculation of ratios (substrates/products). Results: Fifty PAI patients (22 females, mean age 47 years (range 19-71)) were compared with 124 healthy controls (73 females, mean age 48 years (range 20-81). Urinary cortisol (F) (PPP and P respectively). The F/5αTHF (PPP=0.023) ratios were higher in patients suggesting a lower 5α-reductase activity. Similarly, ratios evaluating 5β-reductase, 20αHSD and 20βHSD indicated a lower activity in patients. Androgens, mineralocorticoids and their precursors as well as the GC precursors were drastically decreased in patients, although residual secretion of one or more steroids was detected in 9 patients. Conclusion: The urinary steroid metabolome showed that activity of important enzymes involved in the modulation of corticosteroid action and metabolism was markedly abnormal in PAI patients treated with conventional hydrocortisone therapy. This results in an abnormal cortisol metabolite profile that may contribute to adverse metabolic effects. Production of androgens, mineralocorticoids and GCs is markedly reduced in PAI patients, although 18% of them exhibited residual steroid secretion.

Volume 82, Issue 1, January 2015, Pages 2–11. DOI: 10.1111/cen.12603

Variation in endogenous glucocorticoid (GC) activity during inflammation has been linked to susceptibility to developing rheumatoid arthritis (RA). It has been shown that in patients with RA inflamed synovial tissue can generate active GCs through the expression of the 11β-hydroxysteroid dehydrogenase type 1 enzyme (11β-HSD1), which converts cortisone to cortisol. We examined whether the total body activity of 11β-HSD1 or its expression within synovium was associated with the risk of developing persistent arthritis in patients first presenting with joint inflammation. Blood, urine, and synovial tissue biopsies were obtained from 76 patients at first presentation with arthritis. Total body 11β-HSD1 activity was determined by urinary gas chromatography/mass spectrometry and calculated as the tetrahydrocortisol+allotetrahydrocortisol/tetrahydrocortisone ((THF+alloTHF)/THE) and the cortols:cortolones ratios. Urinary 11β-HSD2 activity was measured as the UFF:UFE ratio. Synovial tissue expression of 11β-HSD1 and 11β-HSD2 was assessed by qPCR. Arthritis severity was assessed by ESR, CRP, and DAS28. Total body 11β-HSD1 activity was significantly lower in patients with arthritis that subsequently resolved than in patients with arthritis that went on to develop into persistent arthritis (Persistent RA, 1.34 (0.013) and Resolvers, 0.96 (0.07), P=0.012). Similar changes were seen in the cortols:cortolones ratio (P=0.0002). There was no difference in renal 11β-HSD2 activity between patients who resolved and those that developed persistent arthritis. Despite the difference in total body 11β-HSD1 activity there was no significant difference between groups in synovial tissue 11β-HSD1 expression. There was no difference in ESR or DAS28 between patients that resolved or went on to persistence although resolvers had a lower level of CRP. These studies demonstrate that a high total body 11β-HSD1 activity during early arthritis is associated with a reduced probability of resolution. The excess 11β-HSD1 may have an articular and/or extra-articular origin. This work raises the possibility that targeting 11β-HSD1 activity in early arthritis could impact on the development of RA.

To review the literature with regard to mortality in patients with hypopituitarism with a focus on the role of growth hormone (GH) deficiency and therapy

Glucocorticoid (GC) excess adversely affects skin integrity, inducing thinning and impaired wound healing. Aged skin, particularly that which has been photo-exposed, shares a similar phenotype. Previously, we demonstrated age-induced expression of the GC-activating enzyme 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) in cultured human dermal fibroblasts (HDFs). Here, we determined 11β-HSD1 levels in human skin biopsies from young and older volunteers and examined the aged 11β-HSD1 KO mouse skin phenotype. 11β-HSD1 activity was elevated in aged human and mouse skin and in PE compared with donor-matched photo-protected human biopsies. Age-induced dermal atrophy with deranged collagen structural organization was prevented in 11β-HSD1 KO mice, which also exhibited increased collagen density. We found that treatment of HDFs with physiological concentrations of cortisol inhibited rate-limiting steps in collagen biosynthesis and processing. Furthermore, topical 11β-HSD1 inhibitor treatment accelerated healing of full-thickness mouse dorsal wounds, with improved healing also observed in aged 11β-HSD1 KO mice. These findings suggest that elevated 11β-HSD1 activity in aging skin leads to increased local GC generation, which may account for adverse changes occurring in the elderly, and 11β-HSD1 inhibitors may be useful in the treatment of age-associated impairments in dermal integrity and wound healing.

Context: Inactivating mutations in the enzyme hexose-6-phosphate dehydrogenase (H6PDH, encoded by H6PD) cause apparent cortisone reductase deficiency (ACRD). H6PDH generates cofactor NADPH for 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1, encoded by HSD11B1) oxo-reductase activity, converting cortisone to cortisol. Inactivating mutations in HSD11B1 cause true cortisone reductase deficiency (CRD). Both ACRD and CRD present with hypothalamic-pituitary-adrenal (HPA) axis activation and adrenal hyperandrogenism.Objective: To describe the clinical, biochemical and molecular characteristics of two additional female children with ACRD and to illustrate the diagnostic value of urinary steroid profiling in identifying and differentiating a total of six ACRD and four CRD cases.Design: Clinical, biochemical and genetic assessment of two female patients presenting during childhood. In addition, results of urinary steroid profiling in a total of ten ACRD/CRD patients were compared to identify distinguishing characteristics.Results: Case 1 was compound heterozygous for R109AfsX3 and a novel P146L missense mutation in H6PD. Case 2 was compound heterozygous for novel nonsense mutations Q325X and Y446X in H6PD. Mutant expression studies confirmed loss of H6PDH activity in both cases. Urinary steroid metabolite profiling by gas chromatography/mass spectrometry suggested ACRD in both cases. In addition, we were able to establish a steroid metabolite signature differentiating ACRD and CRD, providing a basis for genetic diagnosis and future individualised management.Conclusions: Steroid profile analysis of a 24-h urine collection provides a diagnostic method for discriminating between ACRD and CRD. This will provide a useful tool in stratifying unresolved adrenal hyperandrogenism in children with premature adrenarche and adult females with polycystic ovary syndrome (PCOS).

Introduction: Fibroblast-like synoviocytes (FLS) play a central role in defining the stromal environment in inflammatory joint diseases. Despite a growing use of FLS isolated from murine inflammatory models, a detailed characterisation of these cells has not been performed. Methods: In this study, FLS were isolated from inflamed joints of mice expressing both the T cell receptor transgene KRN and the MHC class II molecule Ag7 (K/BxN mice) and their purity in culture determined by immunofluorescence and real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Basal expression of proinflammatory genes was determined by real-time RT-PCR. Secreted interleukin 6 (IL-6) was measured by enzyme-linked immunosorbent assay (ELISA), and its regulation by tumor necrosis factor-alpha (TNF-α and corticosterone (the major glucocorticoid in rodents) measured relative to other mesenchymal cell populations. Results: Purity of FLS culture was identified by positive expression of fibronectin, prolyl 4-hydroxylase, cluster of differentiation 90.2 (CD90.2) and 248 (CD248) in greater than 98% of the population. Cultured FLS were able to migrate and invade through matrigel, a process enhanced in the presence of TNF-α. FLS isolated from K/BxN mice possessed significantly greater basal expression of the inflammatory markers IL-6, chemokine ligand 2 (CCL-2) and vascular cell adhesion molecule 1 (VCAM-1) when compared to FLS isolated from non-inflamed tissue (IL-6, 3.6 fold; CCL-2, 11.2 fold; VCAM-1, 9 fold; P < 0.05). This elevated expression was abrogated in the presence of corticosterone at 100 nmol/l. TNF-α significantly increased expression of all inflammatory markers to a much greater degree in K/BxN FLS relative to other mesenchymal cell lines (K/BxN; IL-6, 40.8 fold; CCL-2, 1343.2 fold; VCAM-1, 17.8 fold; ICAM-1, 13.8 fold; P < 0.05), with secreted IL-6 mirroring these results (K/BxN; con, 169 ± 29.7 versus TNF-α, 923 ± 378.8 pg/ml/1 × 10⁵ cells; P < 0.05). Dose response experiments confirmed effective concentrations between 10 and 100 nmol/l for corticosterone and 1 and 10 ng/ml for TNF-α, whilst inflammatory gene expression in FLS was shown to be stable between passages four and seven. Conclusions: This study has established a well characterised set of key inflammatory genes for in vitro FLS culture, isolated from K/BxN mice and non-inflamed wild-type controls. Their response to both pro- and anti-inflammatory signalling has been assessed and shown to strongly resemble that which is seen in human FLS culture. Additionally, this study provides guidelines for the effective characterisation, duration and treatment of murine FLS culture.

Introduction: Inflammatory arthritis is associated with increased bone resorption and suppressed bone formation. The Wnt antagonist dickkopf-1 (DKK1) is secreted by synovial fibroblasts in response to inflammation and this protein has been proposed to be a master regulator of bone remodelling in inflammatory arthritis. Local glucocorticoid production is also significantly increased during joint inflammation. Therefore, we investigated how locally derived glucocorticoids and inflammatory cytokines regulate DKK1 synthesis in synovial fibroblasts during inflammatory arthritis. Methods: We examined expression and regulation of DKK1 in primary cultures of human synovial fibroblasts isolated from patients with inflammatory arthritis. The effect of TNFα, IL-1β and glucocorticoids on DKK1 mRNA and protein expression was examined by real-time PCR and ELISA. The ability of inflammatory cytokine-induced expression of the glucocorticoid-activating enzyme 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) to sensitise fibroblasts to endogenous glucocorticoids was explored. Global expression of Wnt signalling and target genes in response to TNFα and glucocorticoids was assessed using a custom array. Results: DKK1 expression in human synovial fibroblasts was directly regulated by glucocorticoids but not proinflammatory cytokines. Glucocorticoids, but not TNFα, regulated expression of multiple Wnt agonists and antagonists in favour of inhibition of Wnt signalling. However, TNFα and IL-1β indirectly stimulated DKK1 production through increased expression of 11β-HSD1. Conclusions: These results demonstrate that in rheumatoid arthritis synovial fibroblasts, DKK1 expression is directly regulated by glucocorticoids rather than TNFα. Consequently, the links between synovial inflammation, altered Wnt signalling and bone remodelling are not direct but are dependent on local activation of endogenous glucocorticoids.

Objectives The Clinical Research Training Fellowship (CRTF) allows up to 3 years support for clinically qualified candidates to undertake specialised or further research training in biomedical sciences. CRTFs are perceived as a crucial step in the career development and progression of Clinical Academics but there are no published data to support this notion. We conducted an electronic survey of a large cohort of Medical Research Council (MRC) CRTFs followed for up to 20 years. Design Retrospective analysis of CRTF outcome data held with the MRC, UK. Participants Two cohorts comprising 40 CRFTs awarded by the MRC in the year 1991 and 299 MRC CRTFs who were awarded a fellowship between 1993 and 2003. Results The MRC CRTF scheme built capacity in clinical academia across the UK with 40% of CRTFs progressing to a University professorship. Importantly, the CRTF scheme is also providing NHS consultants who remain research active. Conclusions This is the first analysis of outcome of CRTFs in the UK and provides robust evidence of the importance of this capacity building mode of funding to underpin research excellence at the University–NHS interface.