Anzac Research Institute

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that is familial in 10% of cases. We have identified a missense mutation in the gene encoding fused in sarcoma (FUS) in a British kindred, linked to ALS6. In a survey of 197 familial ALS index cases, we identified two further missense mutations in eight families. Postmortem analysis of three cases with FUS mutations showed FUS-immunoreactive cytoplasmic inclusions and predominantly lower motor neuron degeneration. Cellular expression studies revealed aberrant localization of mutant FUS protein. FUS is involved in the regulation of transcription and RNA splicing and transport, and it has functional homology to another ALS gene, TARDBP, which suggests that a common mechanism may underlie motor neuron degeneration.

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

UNLABELLED: The safety and bone antiresorptive effect of a single subcutaneous dose of AMG 162, a human monoclonal antibody to RANKL, was investigated in 49 postmenopausal women. AMG 162 is a potent antiresorptive agent for diseases such as osteoporosis. INTRODUCTION: RANKL is an essential osteoclastic differentiation and activation factor. MATERIALS AND METHODS: The bone antiresorptive activity and safety of AMG 162, a fully human monoclonal antibody to RANKL, were evaluated in postmenopausal women in this randomized, double-blind, placebo-controlled, single-dose, dose escalation study. Six cohorts of eight to nine women were randomly assigned to receive a single subcutaneous injection of either AMG 162 or placebo (3:1 ratio). AMG 162 doses were 0.01, 0.03, 0.1, 0.3, 1.0, and 3.0 mg/kg. Subjects were followed up to 6 months in all cohorts and 9 months in the three highest dose cohorts. Second morning void urinary N-telopeptide/creatinine (NTX; Osteomark), serum NTX, and serum bone-specific alkaline phosphatase (BALP, Ostase) were assessed as bone turnover markers. RESULTS AND CONCLUSIONS: Forty-nine women were enrolled. A single subcutaneous dose of AMG 162 resulted in a dose-dependent, rapid (within 12 h), profound (up to 84%), and sustained (up to 6 months) decrease in urinary NTX. At 6 months, there was a mean change from baseline of -81% in the 3.0 mg/kg AMG 162 group compared with -10% in the placebo group; serum NTX changes were -56% and 2%, respectively. BALP levels did not decrease remarkably until after 1 month, indicating that the effect of AMG 162 is primarily antiresorptive. Intact parathyroid hormone (PTH) levels increased up to approximately 3-fold after 4 days in the 3.0 mg/kg dose group, but returned toward baseline with follow-up. Albumin-adjusted serum calcium did not decrease >10% on average in any group, and no subject had values below 2 mmol/liter. AMG 162 was well tolerated. No related serious adverse events occurred. No clinically meaningful laboratory changes, other than those described above, were observed. In summary, a single subcutaneous dose of AMG 162 resulted in a dose-dependent rapid and sustained decrease from baseline in bone turnover and could be an effective and convenient treatment for osteoporosis.

Globally, cardiovascular disease will continue causing most human deaths for the foreseeable future. The consistent gender gap in life span of approximately 5.6 yr in all advanced economies must derive from gender differences in age-specific cardiovascular death rates, which rise steeply in parallel for both genders but 5-10 yr earlier in men. The lack of inflection point at modal age of menopause, contrasting with unequivocally estrogen-dependent biological markers like breast cancer or bone density, makes estrogen protection of premenopausal women an unlikely explanation. Limited human data suggest that testosterone exposure does not shorten life span in either gender, and oral estrogen treatment increases risk of cardiovascular death in men as it does in women. Alternatively, androgen exposure in early life (perinatal androgen imprinting) may predispose males to earlier onset of atherosclerosis. Following the recent reevaluation of the estrogen-protection orthodoxy, empirical research has flourished into the role of androgens in the progression of cardiovascular disease, highlighting the need to better understand androgen receptor (AR) coregulators, nongenomic androgen effects, tissue-specific metabolic activation of androgens, and androgen sensitivity. Novel therapeutic targets may arise from understanding how androgens enhance early plaque formation and cause vasodilatation via nongenomic androgen effects on vascular smooth muscle, and how tissue-specific variations in androgen effects are modulated by AR coregulators as well as metabolic activation of testosterone to amplify (via 5alpha-reductase to form dihydrotestosterone acting on AR) or diversify (via aromatization to estradiol acting upon estrogen receptor alpha/beta) the biological effects of testosterone on the vasculature. Observational studies show that blood testosterone concentrations are consistently lower among men with cardiovascular disease, suggesting a possible preventive role for testosterone therapy, which requires critical evaluation by further prospective studies. Short-term interventional studies show that testosterone produces a modest but consistent improvement in cardiac ischemia over placebo, comparable to the effects of existing antianginal drugs. By contrast, testosterone therapy has no beneficial effects in peripheral arterial disease but has not been evaluated in cerebrovascular disease. Erectile dysfunction is most frequently caused by pelvic arterial insufficiency due to atherosclerosis, and its sentinel relationship to generalized atherosclerosis is insufficiently appreciated. The commonality of risk factor patterns and mechanisms (including endothelial dysfunction) suggests that the efficacy of antiatherogenic therapy is an important challenge with the potential to enhance men's motivation for prevention and treatment of cardiovascular diseases.

Elite athletic competitions have separate male and female events due to men's physical advantages in strength, speed, and endurance so that a protected female category with objective entry criteria is required. Prior to puberty, there is no sex difference in circulating testosterone concentrations or athletic performance, but from puberty onward a clear sex difference in athletic performance emerges as circulating testosterone concentrations rise in men because testes produce 30 times more testosterone than before puberty with circulating testosterone exceeding 15-fold that of women at any age. There is a wide sex difference in circulating testosterone concentrations and a reproducible dose-response relationship between circulating testosterone and muscle mass and strength as well as circulating hemoglobin in both men and women. These dichotomies largely account for the sex differences in muscle mass and strength and circulating hemoglobin levels that result in at least an 8% to 12% ergogenic advantage in men. Suppression of elevated circulating testosterone of hyperandrogenic athletes results in negative effects on performance, which are reversed when suppression ceases. Based on the nonoverlapping, bimodal distribution of circulating testosterone concentration (measured by liquid chromatography-mass spectrometry)-and making an allowance for women with mild hyperandrogenism, notably women with polycystic ovary syndrome (who are overrepresented in elite athletics)-the appropriate eligibility criterion for female athletic events should be a circulating testosterone of <5.0 nmol/L. This would include all women other than those with untreated hyperandrogenic disorders of sexual development and noncompliant male-to-female transgender as well as testosterone-treated female-to-male transgender or androgen dopers.

With the ageing population in most countries, disorders of bone and mineral metabolism are becoming increasingly relevant to every day clinical practice. Consequently, the interest in, and the need for effective measures to be used in the screening, diagnosis and follow-up of such pathologies has markedly grown. Together with clinical and imaging techniques, biochemical tests play an important role in the assessment and differential diagnosis of metabolic bone disease. In recent years, the isolation and characterisation of cellular and extracellular components of the skeletal matrix have resulted in the development of molecular markers that are considered to reflect either bone formation or bone resorption. These biochemical indices are non-invasive, comparatively inexpensive and, when applied and interpreted correctly, helpful tools in the diagnostic and therapeutic assessment of metabolic bone disease. Part I of this article provides an overview of the basic biochemistry of bone markers, and sources of non-specific variability. Part II (to be published in a subsequent issue of this journal) will review the current evidence regarding the clinical use of biochemical markers of bone remodelling in metabolic and metastatic bone disease.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with a lifetime risk of one in 350 people and an unmet need for disease-modifying therapies. We conducted a cross-ancestry genome-wide association study (GWAS) including 29,612 patients with ALS and 122,656 controls, which identified 15 risk loci. When combined with 8,953 individuals with whole-genome sequencing (6,538 patients, 2,415 controls) and a large cortex-derived expression quantitative trait locus (eQTL) dataset (MetaBrain), analyses revealed locus-specific genetic architectures in which we prioritized genes either through rare variants, short tandem repeats or regulatory effects. ALS-associated risk loci were shared with multiple traits within the neurodegenerative spectrum but with distinct enrichment patterns across brain regions and cell types. Of the environmental and lifestyle risk factors obtained from the literature, Mendelian randomization analyses indicated a causal role for high cholesterol levels. The combination of all ALS-associated signals reveals a role for perturbations in vesicle-mediated transport and autophagy and provides evidence for cell-autonomous disease initiation in glutamatergic neurons.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most lethal malignancies. Neovascularization during tumorigenesis supplies oxygen and nutrients to proliferative tumor cells, and serves as a conduit for migration. Targeting oncogenes involved in angiogenesis is needed to treat organ-confined and locally advanced ESCC. Although the phospholipase C epsilon-1 (PLCE1) gene was originally identified as a susceptibility gene for ESCC, how PLCE1 is involved in ESCC is unclear. METHODS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry were used to measure the methylation status of the PLCE1 promoter region. To validate the underlying mechanism for PLCE1 in constitutive activation of the NF-κB signaling pathway, we performed studies using in vitro and in vivo assays and samples from 368 formalin-fixed esophageal cancer tissues and 215 normal tissues with IHC using tissue microarrays and the Cancer Genome Atlas dataset. RESULTS: We report that hypomethylation-associated up-regulation of PLCE1 expression was correlated with tumor angiogenesis and poor prognosis in ESCC cohorts. PLCE1 can activate NF-κB through phosphoinositide-phospholipase C-ε (PI-PLCε) signaling pathway. Furthermore, PLCE1 can bind p65 and IκBα proteins, promoting IκBα-S32 and p65-S536 phosphorylation. Consequently, phosphorylated IκBα promotes nuclear translocation of p50/p65 and p65, as a transcription factor, can bind vascular endothelial growth factor-C and bcl-2 promoters, enhancing angiogenesis and inhibiting apoptosis in vitro. Moreover, xenograft tumors in nude mice proved that PLCE1 can induce angiogenesis, inhibit apoptosis, and increase tumor aggressiveness via the NF-κB signaling pathway in vivo. CONCLUSIONS: Our findings not only provide evidence that hypomethylation-induced PLCE1 confers angiogenesis and proliferation in ESCC by activating PI-PLCε-NF-κB signaling pathway and VEGF-C/Bcl-2 expression, but also suggest that modulation of PLCE1 by epigenetic modification or a selective inhibitor may be a promising therapeutic approach for the treatment of ESCC.

Familial amyotrophic lateral sclerosis (FALS) is an inherited neurodegenerative disorder of the motor neurons. While 10-15% of cases are caused by mutations in the copper/zinc superoxide-dismutase-1 (SOD-1) gene, the dying-forward hypothesis, in which corticomotoneurons induce anterograde excitotoxic motoneuron degeneration, has been proposed as a potential mechanism. The present study applied novel threshold tracking transcranial magnetic stimulation techniques to investigate the mechanisms underlying neurodegeneration in FALS. Studies were undertaken in 14 asymptomatic and 3 pre-symptomatic SOD-1 mutation carriers, followed longitudinally for up to 3-years. The pre-symptomatic subjects were asymptomatic at the time of their initial study but developed symptoms during the follow-up period. Results were compared to 7 SOD-1 FALS patients, 50 sporadic ALS patients and 55 normal controls. Short-interval intracortical inhibition (SICI) was significantly reduced in SOD-1 FALS (-1.2 +/- 0.6%) and sporadic ALS patients (-0.7 +/- 0.3%) compared to asymptomatic SOD-1 mutation carriers (9.8 +/- 1.5%, P<0.00001) and normal controls (8.5 +/- 1.0%, P<0.00001). SICI reduction was accompanied by increases in intracortical facilitation, motor evoked potential amplitudes and the slope of the magnetic stimulus-response curve. In two pre-symptomatic SOD-1 mutation carriers SICI was completely absent (SICI patient 1, -3.2%; patients 2, -1.3%), while in one subject there was a 32% reduction in SICI prior to symptom onset. These three individuals subsequently developed clinical features of ALS. Simultaneous investigation of central and peripheral excitability has established that cortical hyperexcitability develops in clinically affected SOD-1 FALS patients, similar to that seen in sporadic ALS patients, thereby suggesting that a similar pathophysiological process in evident in both familial and sporadic ALS patients. In addition, the present study has established that cortical hyperexcitability precedes the development of clinical symptoms in pre-symptomatic carriers of the SOD1 mutation, thereby suggesting that cortical hyperexcitability underlies neurodegeneration in FALS.

OBJECTIVE: To study the acceptor specificity for human ABCG1 (hABCG1)-mediated cholesterol efflux. METHODS AND RESULTS: Cells overexpressing hABCG1 were created in Chinese Hamster Ovary (CHO-K1) cells and characterized in terms of lipid composition. hABCG1 expressed in these cells formed homodimers and was mostly present intracellularly. Cholesterol efflux from hABCG1 cells to HDL2 and HDL3 was increased but not to lipid-free apolipoproteins. A range of phospholipid containing acceptors apart from high-density lipoprotein (HDL) subclasses were also efficient in mediating ABCG1-dependent export of cholesterol. Importantly, a buoyant phospholipid-containing fraction generated from incubation of lipid-free apoA-I with macrophages was nearly as efficient as HDL2. The capacity of acceptors to induce ABCG1-mediated efflux was strongly correlated with their total phospholipid content, suggesting that acceptor phospholipids drive ABCG1-mediated efflux. Most importantly, acceptors for ABCG1-mediated cholesterol export could be generated from incubation of cells with lipid-free apoA-I through the action of ABCA1 alone. CONCLUSIONS: These results indicate a synergistic relationship between ABCA1 and ABCG1 in peripheral tissues, where ABCA1 lipidates any lipid-poor/free apoA-I to generate nascent or pre-beta-HDL. These particles in turn may serve as substrates for ABCG1-mediated cholesterol export.

OBJECTIVE: To report the levels and trends of prevalence, deaths, and disability-adjusted life years (DALYs) due to musculoskeletal disorders, categorized as low back pain, neck pain, osteoarthritis (OA), rheumatoid arthritis (RA), gout, and other musculoskeletal disorders, across 195 countries and territories from 1990 to 2017 according to age, sex, and Sociodemographic Index (SDI; a composite of sociodemographic factors). METHODS: Data were obtained from the Global Burden of Disease (GBD) Study 2017. The fatal and nonfatal burdens of musculoskeletal disorders were estimated using the Cause of Death Ensemble model and Bayesian meta-regression tool, respectively. Estimates were provided for all musculoskeletal disorders and the corresponding 6 categories at global, regional, and national levels from 1990 to 2017. Counts and age-standardized rates per 100,000 population along with 95% uncertainty intervals (95% UIs) were reported for prevalence, deaths, and DALYs. RESULTS: Globally, there were ~1.3 billion prevalent cases (95% UI 1.2 billion, 1.4 billion), 121.3 thousand deaths (95% UI 105.6 thousand, 126.2 thousand), and 138.7 million DALYs (95% UI 101.9 million, 182.6 million) due to musculoskeletal disorders in 2017. Age-standardized prevalence, death, and DALY rates per 100,000 population were 16,276.2 (95% UI 15,495.5, 17,145.8), 1.6 (95% UI 1.4, 1.6), and 1,720 (95% UI 1,264.4, 2,259.2), respectively. Age-standardized prevalence (-1.6% [95% UI -2.4, -0.8]) and DALY rates (-3.5% [95% UI -4.7, -2.3]) decreased slightly from 1990. The global point prevalence rate of musculoskeletal disorders in 2017 was higher in women than in men and increased with age up to the oldest age group. Globally, the proportion of prevalent cases according to category of musculoskeletal disorders in 2017 was greatest for low back pain (36.8%), followed by other musculoskeletal disorders (21.5%), OA (19.3%), neck pain (18.4%), gout (2.6%), and RA (1.3%). These proportions did not change appreciably compared with 1990. The burden due to musculoskeletal conditions was higher in developed countries. The countries with the highest age-standardized prevalence rates of musculoskeletal disorders in 2017 were Switzerland (23,346.0 [95% UI 22,392.6, 24,329.8]), Chile (23,007.9 [95% UI 21,746.5, 24,165.8]), and Denmark (22,166.1 [95% UI 20,817.2, 23,542.1]). The greatest increases from 1990 were found in Chile (10.8% [95% UI 6.6, 15.4]), Benin (8.8% [95% UI 6.7, 11.1]), and El Salvador (8.5% [95% UI 5.5, 11.9]). CONCLUSION: There is a large burden of musculoskeletal disorders globally, with some notable inter-country variation. Some countries have twice the burden of other countries. Increasing population awareness regarding risk factors, consequences, and evidence-informed treatment strategies for musculoskeletal disorders with a focus on the older female population in developed countries is needed, particularly for low back and neck pain and OA, which contribute a large burden among this cohort.

Amyotrophic lateral sclerosis (ALS) is a devastating and universally fatal neurodegenerative disease. Mutations in two related RNA-binding proteins, TDP-43 and FUS, that harbor prion-like domains, cause some forms of ALS. There are at least 213 human proteins harboring RNA recognition motifs, including FUS and TDP-43, raising the possibility that additional RNA-binding proteins might contribute to ALS pathogenesis. We performed a systematic survey of these proteins to find additional candidates similar to TDP-43 and FUS, followed by bioinformatics to predict prion-like domains in a subset of them. We sequenced one of these genes, TAF15, in patients with ALS and identified missense variants, which were absent in a large number of healthy controls. These disease-associated variants of TAF15 caused formation of cytoplasmic foci when expressed in primary cultures of spinal cord neurons. Very similar to TDP-43 and FUS, TAF15 aggregated in vitro and conferred neurodegeneration in Drosophila, with the ALS-linked variants having a more severe effect than wild type. Immunohistochemistry of postmortem spinal cord tissue revealed mislocalization of TAF15 in motor neurons of patients with ALS. We propose that aggregation-prone RNA-binding proteins might contribute very broadly to ALS pathogenesis and the genes identified in our yeast functional screen, coupled with prion-like domain prediction analysis, now provide a powerful resource to facilitate ALS disease gene discovery.

between cells and biomaterial scaffolds is called focal adhesion. To understand the factors that influence cell adhesive ability is a key in the development and application of new tissue engineering scaffold. Cell attachment is a complex process, affected by numerous aspects, such as cell behavior, material surface properties, and environmental factors. Material surface properties comprise the hydrophobicity, charge, roughness, softness and chemical composition of the biomaterial surface itself.

CLICK HERE FOR AN UPDATE ON THE REQUIREMENT FOR MASS SPECTROMETRY SEX STEROIDS ASSAYS The accurate measurement of hormones is the pivot of modern endocrinology. Although tracing its roots to ancient writings, the modern scientific discipline came into existence after a series of mid-20th century Nobel Prize-winning achievements in identifying and measuring hormones. Arguably the most decisive step was the invention of immunoassay in 1959 (1), originally for peptide hormones but extended a decade later to nonimmunogenic steroids (2). This armed the nascent discipline with revolutionary impact through the ability to measure virtually every hormone at orders of magnitude lower than had ever been possible before with earlier cumbersome, insensitive, whole-animal bioassays. Yet the pioneers of steroid immunoassay understood its limitations as that decade's delay was consumed in finding the additional workarounds required to create valid formats for immunoassay of small, subimmunogenic molecules like steroids. These essentials comprised solvent extraction, chromatography, and structurally authentic tracers, a triplet of validity criteria. Employing these required skills originally confined steroid measurement to manual methods in specialized laboratories; however, in the 1980–1990s, the steeply increasing demand for steroid measurement in the clinic and laboratory drove assay simplification to adapt steroid immunoassays into popular one-step kits and multiplex assay platform formats. This commodification resulted in “direct” steroid immunoassays, which bypassed all the original triplet of validity criteria (most fundamentally extraction), sacrificing accuracy and specificity for throughput speed and lower cost. Although problems with validity (analytical specificity, accuracy) of direct immunoassays were soon identified and were reported over 25 years ago (3, 4), the fundamental significance of violating these validity criteria resurfaced prominently in the last decade as the accuracy of direct hormone assays came under more stringent scrutiny—and as a solution, affordable and accessible mass spectrometry (MS) steroid assays came into view. The method-specific bias and analytical nonspecificity of direct T immunoassays are worst at the low circulating T levels in children, women, and men with pathological, functional, or experimentally induced T deficiency (5–7) where such direct immunoassays have been famously likened to random number generation (8). By modern standards, putative findings using such suboptimal methods would require verification by more specific MS-based assays. This renders direct T immunoassays methodologically inadequate for high-quality clinical reproductive medicine research. Not surprisingly, aiming to measure circulating (picomolar) estradiol concentrations 10- to 100-fold lower than circulating (nanomolar) T levels that render direct T immunoassays unreliable is clearly problematic. Thus, direct estradiol immunoassays also lack accuracy in children (9), men (10, 11), and postmenopausal (3, 12–15) or aromatase inhibitor-treated (16) women who all share comparably low circulating estradiol concentrations. It is beyond doubt now that direct immunoassays of sex steroids are methodologically inadequate for high-quality clinical research. MS-based steroid measurements have been available for decades, indeed as long as immunoassays (17). Although they always remained the reference method for steroid specificity and structure, MS-based steroid assays were always too expensive and unavailable for routine use in the laboratory to support clinical research because they required highly skilled operators using complex, difficult-to-access equipment. Hence, while remaining the reference method for steroids, MS was too insensitive, expensive, and unavailable for routine clinical research use. All of this changed over the last decade with the advent of bench-top mass spectrometers with sensitivity matching the best immunoassays while retaining reference level specificity and introducing multianalyte capability. Not only does the availability of MS-based steroid assays resolve the problems of direct steroid immunoassays, but multianalyte profiling also ushers in a new era of snapshot profiling of the overlooked importance of steroid pathway fluxes that will broaden the interpretation of clinical studies beyond the standard restricted single analyte-by-analyte analysis. In this context, it is salient that the nonspecificity and single analyte limitations of steroid immunoassays always rendered them inadequate for antidoping testing where MS-based profiling has been standard for decades. Where an elite athlete's pursuit of his or her profession could be ended by a positive antidoping test, detection of banned steroids had to be undertaken at the highest standards of scientific rigor to withstand the most vigorous medico-legal challenge. It is time that clinical research and practice operates under equally high standards that have now become feasible. Recognizing the limitations of direct steroid immunoassays, The Endocrine Society commissioned two Position Papers written by experienced immunoassayists on the challenges to T (18) and estradiol (19) measurement and supported a renewed effort to standardize T assays (20). Although all assays require rigorous quality control to minimize random errors, additional methodological limitations of steroid immunoassay technology constituting systematic errors are impervious to such performance-based standardization. For example, the method-specific bias of immunoassays, a function of antibody epitope, allows for cross-reactivity in all steroid assays with steroid precursors, metabolites, and/or conjugates as well as matrix effects for unextracted direct assays. These limitations are aggravated by regular changes in proprietary assay reagents, especially, but not only, primary antibodies causing changed assay cross-reactivity profiles. Typically, validation of such modified assays via correlation of new with old assays (rather than with the reference method) allows for subtle, gradual “drift” in assay performance from reference standards. The net effect of method-specific bias is that steroid immunoassays do not constitute durable, definitive chemical measurements, independent of method, like hemoglobin, cholesterol, urea, and creatinine for which measurements are comparable in studies separated widely in time, place, and/or laboratory. Such long-term assay “drift” and inaccuracy is troublesome in epidemiology (19) and in evaluating temporal trends in population-based studies (21, 22) where the noncomparability of different steroid immunoassays over time hinders interpretation. In addition, method-specific bias is incompatible with research studies that promulgate clinical decision-making thresholds (23). The wider ramifications of the limitations of direct steroid immunoassays and challenges arising from them are well discussed in The Endocrine Society's Position Papers (18, 19). Similarly, derived sex steroid measures (“free,” “bioavailable”) are often calculated rather than actually measured (24). However, such calculational shortcuts use inaccurate formulae for “free” (25–27) or “bioavailable” (28, 29) measurements with wide deviations from measured values due to mistaken stoichiometry and arbitrary binding affinity constants in the formulae. Furthermore, such questionable calculations purport to be hormonal measurements, yet they have no reproducibility measures like measured hormone variables. Such unreliable calculations are misleading if absolute measurements are projected, inferred, or adopted for clinical use outside the study, with the potential for confusion and misunderstanding, rather than the enlightenment intended by high-quality clinical research. It is timely to recognize that for high-impact clinical research, the steroid immunoassay era is gradually drawing to a close, and for direct immunoassays it is effectively over. Over recent years an increasing number of manuscripts submitted to the JCEM have been rejected, largely for their reliance on direct steroid immunoassays as major endpoints. Because the JCEM remains a leader in publishing clinical endocrinology research, reflected in consistently high bibliometrics (total citations, Eigenfactor score), the Journal is taking the next step (24) in setting acceptable assay standards for the field in upgrading its submission requirements for publication of studies using sex steroid measurements. Effective January 1, 2015, manuscripts reporting sex steroid assays as important endpoints must use MS-based assays including reporting or citing their methods with sufficient detail to allow them to be reproduced together with standard quality control, specificity, and reproducibility metrics. Some limited editorial discretion may be exercised for rare circumstances, such as where a study reports hormone measurements made long ago and where samples no longer exist; however, that would not extend to the sort of high-quality, well-designed clinical studies that the JCEM expects to publish where it is expected that extra serum samples would be stored for key endpoints. It is anticipated that this requirement for MS-based assays will extend to adrenal steroids and vitamin D in the near future. In introducing the new standard, the Journal makes provision for a realistic timescale for the switchover. This allows for changes in planning or conduct of clinical research to comply with the new requirement. Although resolving the problems of direct sex steroid immunoassays is at hand, it is natural to expect that convenience of a familiar, old but failing technology may foster rationalization and resistance to necessary progressive change. When Endocrinology recently instituted the requirement for more rigorous reporting of antibody specificity, a few months were allowed for authors to come into compliance (30). When a consortium of general medical journals required a momentous change to combat publication bias by requiring advance registration of all clinical trials before they commenced patient intake (31), a grace period of 12 months was allowed for studies in progress to become compliant (32). Hence, the JCEM is adopting a generous grace period to allow ample time for investigators to ensure that future submissions for which sex steroid assays are an important endpoint have suitably rigorous assay formats. The best clinical research will always feature standards that are rising, usually gradually but sometimes more abruptly. Ultimately, the need for the Journal to foster the highest standards in clinical research and practice will drive us—or we will be driven out of that top echelon by failing to do what is necessary and timely. Disclosure Summary: The authors have nothing to declare. mass spectrometry.

Polycystic ovary syndrome (PCOS) affects 5-10% of women of reproductive age, causing a range of reproductive, metabolic and endocrine defects including anovulation, infertility, hyperandrogenism, obesity, hyperinsulinism, and an increased risk of type 2 diabetes and cardiovascular disease. Hyperandrogenism is the most consistent feature of PCOS, but its etiology remains unknown, and ethical and logistic constraints limit definitive experimentation in humans to determine mechanisms involved. In this study, we provide the first comprehensive characterization of reproductive, endocrine, and metabolic PCOS traits in 4 distinct murine models of hyperandrogenism, comprising prenatal dihydrotestosterone (DHT, potent nonaromatizable androgen) treatment during days 16-18 of gestation, or long-term treatment (90 days from 21 days of age) with DHT, dehydroepiandrosterone (DHEA), or letrozole (aromatase inhibitor). Prenatal DHT-treated mature mice exhibited irregular estrous cycles, oligo-ovulation, reduced preantral follicle health, hepatic steatosis, and adipocyte hypertrophy, but lacked overall changes in body-fat composition. Long-term DHT treatment induced polycystic ovaries displaying unhealthy antral follicles (degenerate oocyte and/or > 10% pyknotic granulosa cells), as well as anovulation and acyclicity in mature (16-week-old) females. Long-term DHT also increased body and fat pad weights and induced adipocyte hypertrophy and hypercholesterolemia. Long-term letrozole-treated mice exhibited absent or irregular cycles, oligo-ovulation, polycystic ovaries containing hemorrhagic cysts atypical of PCOS, and displayed no metabolic features of PCOS. Long-term dehydroepiandrosterone treatment produced no PCOS features in mature mice. Our findings reveal that long-term DHT treatment replicated a breadth of ovarian, endocrine, and metabolic features of human PCOS and provides the best mouse model for experimental studies of PCOS pathogenesis.

The liver has an established ability to induce tolerance. Recent evidence indicates that this unique property might be related to its distinctive architecture allowing T cells to be activated in situ independently of lymphoid tissues. Unlike lymph node-activated T cells, liver-activated T cells are short-lived, a mechanism that might contribute to the "liver tolerance effect." Although the potential role of hepatocytes as tolerogenic antigen-presenting cells has been demonstrated, the question as to whether these cells are able to interact with CD8(+) T cells in physiological settings remains controversial. Contradicting the immunological dogma stating that naïve T lymphocytes are prevented from interacting with parenchymal cells within non-lymphoid organs by an impenetrable endothelial barrier, we show here that the unique morphology of the liver sinusoidal endothelial cell (LSEC) permits interactions between lymphocytes and hepatocytes. Using electron microscopy, we demonstrate that liver resident lymphocytes as well as circulating naïve CD8(+) T cells make direct contact with hepatocytes through cytoplasmic extensions penetrating the endothelial fenestrations that perforate the LSECs. Furthermore, the expression of molecules required for primary T cell activation, MHC class I and ICAM-1, is polarized on hepatocytes to the perisinusoidal cell membrane, thus maximizing the opportunity for interactions with circulating lymphocytes. In conclusion, this study has identified, at the ultrastructural level, a unique type of interaction between naïve T lymphocytes and liver parenchymal cells in vivo. These results hold implications for the pathogenesis of viral hepatitis in which hepatocytes may represent the main antigen-presenting cell, and for the development of immune tolerance as lymphocytes pass through the liver.

BACKGROUND: Participation in cardiac rehabilitation (CR) benefits patients with coronary heart disease (CHD), yet worldwide only some 15–30% of those eligible attend. To improve understanding of the reasons for poor participation we undertook a systematic review and meta-synthesis of the qualitative literature. METHODS: Qualitative studies identifying patient barriers and enablers to attendance at CR were identified by searching multiple electronic databases, reference lists, relevant conference lists, grey literature, and keyword searching of the Internet (1990–2010). Studies were selected if they included patients with CHD and reviewed experience or understanding about CR. Meta-synthesis was used to review the papers and to synthesize the data. RESULTS: From 1165 papers, 34 unique studies were included after screening. These included 1213 patients from eight countries. Study methodology included interviews (n = 25), focus groups (n = 5), and mixed-methods (n = 4). Key reasons for not attending CR were physical barriers, such as lack of transport, or financial cost, and personal barriers, such as embarrassment about participation, or misunderstanding the reasons for onset of CHD or the purpose of CR. CONCLUSIONS: There is a vast amount of qualitative research which investigates patients’ reasons for non-attendance at CR. Key issues include system-level and patient-level barriers, which are potentially modifiable. Future research would best be directed at investigating strategies to overcome these barriers.

levels represents an opportunity to rescue female reproductive function in mammals.

Polycystic ovary syndrome (PCOS) is the most frequent female endocrine disorder, affecting 5%-10% of women, causing infertility due to dysfunctional follicular maturation and ovulation, distinctive multicystic ovaries and hyperandrogenism, together with metabolic abnormalities including obesity, hyperinsulinism, an increased risk of type 2 diabetes, and cardiovascular disease. The etiology of PCOS is unclear, and decisive clinical studies are limited by ethical and logistic constraints. Consequently treatment is palliative rather than curative and focuses on symptomatic approaches. Hence, a suitable animal model could provide a valuable means with which to study the pathogenesis of the characteristic reproductive and metabolic abnormalities and thereby identify novel and more effective treatments. So far there is no consensus on the best experimental animal model, which should ideally reproduce the key features associated with human PCOS. The prenatally androgenized rhesus monkey displays many characteristics of the human condition, including hyperandrogenism, anovulation, polycystic ovaries, increased adiposity, and insulin insensitivity. However, the high cost of nonhuman primate studies limits the practical utility of these large-animal models. Rodent models, on the other hand, are inexpensive, provide well-characterized and stable genetic backgrounds readily accessible for targeted genetic manipulation, and shorter reproductive life spans and generation times. Recent rodent models display both reproductive and metabolic disturbances associated with human PCOS. This review aimed to evaluate the rodent models reported to identify the advantages and disadvantages of the distinct rodent models used to investigate this complex endocrine disorder.