Biomarkers Associated With Future Severe Liver Disease in Children With Alpha-1-Antitrypsin Deficiency.

Background and Aims: Children with alpha-1-antitrypsin deficiency (AATD) exhibit a wide range of liver disease outcomes from portal hypertension and transplant to asymptomatic without fibrosis. Individual outcomes cannot be predicted. Liver injury in AATD is caused by the accumulation in hepatocytes of the mutant Z alpha-1-antitrypsin (AAT) protein, especially the toxic, intracellular polymerized conformation. AATD patients have trace Z polymer detectable in serum with unknown significance. Methods: The Childhood Liver Disease Research Network is an NIH consortium for the study of pediatric liver diseases, including AATD. We obtained data and samples with the aim of identifying biomarkers predictive of severe AATD liver disease. Results: We analyzed prospective AATD Childhood Liver Disease Research Network data and serum samples in 251 subjects from 2007 to 2015 for outcomes and Z polymer levels. Fifty-eight of 251 had clinically evident portal hypertension (CEPH) at enrollment, and 10 developed CEPH during follow-up. Higher Z AAT polymer levels were associated with existing CEPH (P = .01). In infants without CEPH, higher polymer levels were associated with future CEPH later in childhood, but total AAT was not predictive. Higher gamma-glutamyl transferase (GGT) in the first few months of life was also significantly associated with future CEPH, and risk-threshold GGT levels can be identified. A model was constructed to identify subjects at high risk of future CEPH by combining clinical GGT and polymer levels (area under the curve of 0.83; 95% confidence interval: 0.656-1.00, P = .019). Conclusion: High circulating Z polymer levels and high GGT early in life are associated with future CEPH in AATD, and the use of predictive cutoffs may assist in future clinical trial design.

Fibrosis, biomarkers and liver biopsy in AAT deficiency and relation to liver Z protein polymer accumulation.

BACKGROUND AND AIMS: The course of adults with ZZ alpha-1-antitrypsin deficiency (AATD) liver disease is unpredictable. The utility of markers, including liver biopsy, is undefined. METHODS: A prospective cohort, including protocol liver biopsies, was enrolled to address these questions. RESULTS: We enrolled 96 homozygous ZZ AATD adults prospectively at three US sites with standardized clinical evaluations, and protocol liver biopsies. Fibrosis was scored using Ishak (stages 0-6). Also, 51% of the 96 subjects had Ishak score >1 fibrosis (49% Ishak 0-1, 36% Ishak 2-3 and 15% ≥4). Elevated aspartate aminotransferase (AST) more than alanine aminotransferase (ALT), high body mass index (BMI), obesity, AST platelet ratio index and elevated serum Z alpha 1 antitrypsin (AAT) polymer levels were associated with increased fibrosis. Steatosis did not correlate to fibrosis. Increased fibrosis was associated with increased mutant Z polymer globular inclusions (p = .002) and increased diffuse cytoplasmic Z polymer on biopsy (p = .0029) in a direct relationship. Increased globule Z polymer was associated with increased serum AST (p = .007) and increased periportal inflammation on histopathology (p = .004), but there was no relationship of Z polymer hepatocellular accumulation with ALT, gamma glutamine transferase, inflammation in other parts of the lobule, necrosis or steatosis. Serum Z polymer levels were directly correlated to hepatic Z protein polymer content. Lung function, smoking and alcohol consumption patterns were not associated with fibrosis. CONCLUSION: In AATD high BMI, obesity and elevated AST are associated with increased fibrosis. Liver biopsy features are correlated to some serum tests. Serum Z AAT polymer levels could be a future biomarker to detect fibrosis early and is directly correlated to liver Z content.

SMIM1 absence is associated with reduced energy expenditure and excess weight.

BACKGROUND: Obesity rates have nearly tripled in the past 50 years, and by 2030 more than 1 billion individuals worldwide are projected to be obese. This creates a significant economic strain due to the associated non-communicable diseases. The root cause is an energy expenditure imbalance, owing to an interplay of lifestyle, environmental, and genetic factors. Obesity has a polygenic genetic architecture; however, single genetic variants with large effect size are etiological in a minority of cases. These variants allowed the discovery of novel genes and biology relevant to weight regulation and ultimately led to the development of novel specific treatments. METHODS: We used a case-control approach to determine metabolic differences between individuals homozygous for a loss-of-function genetic variant in the small integral membrane protein 1 (SMIM1) and the general population, leveraging data from five cohorts. Metabolic characterization of SMIM1-/- individuals was performed using plasma biochemistry, calorimetric chamber, and DXA scan. FINDINGS: We found that individuals homozygous for a loss-of-function genetic variant in SMIM1 gene, underlying the blood group Vel, display excess body weight, dyslipidemia, altered leptin to adiponectin ratio, increased liver enzymes, and lower thyroid hormone levels. This was accompanied by a reduction in resting energy expenditure. CONCLUSION: This research identified a novel genetic predisposition to being overweight or obese. It highlights the need to investigate the genetic causes of obesity to select the most appropriate treatment given the large cost disparity between them. FUNDING: This work was funded by the National Institute of Health Research, British Heart Foundation, and NHS Blood and Transplant.

Cosmic kidney disease: an integrated pan-omic, physiological and morphological study into spaceflight-induced renal dysfunction.

Missions into Deep Space are planned this decade. Yet the health consequences of exposure to microgravity and galactic cosmic radiation (GCR) over years-long missions on indispensable visceral organs such as the kidney are largely unexplored. We performed biomolecular (epigenomic, transcriptomic, proteomic, epiproteomic, metabolomic, metagenomic), clinical chemistry (electrolytes, endocrinology, biochemistry) and morphometry (histology, 3D imaging, miRNA-ISH, tissue weights) analyses using samples and datasets available from 11 spaceflight-exposed mouse and 5 human, 1 simulated microgravity rat and 4 simulated GCR-exposed mouse missions. We found that spaceflight induces: 1) renal transporter dephosphorylation which may indicate astronauts' increased risk of nephrolithiasis is in part a primary renal phenomenon rather than solely a secondary consequence of bone loss; 2) remodelling of the nephron that results in expansion of distal convoluted tubule size but loss of overall tubule density; 3) renal damage and dysfunction when exposed to a Mars roundtrip dose-equivalent of simulated GCR.

Diagnosis and treatment of anti-insulin antibody-mediated labile glycaemia in insulin-treated diabetes.

AIMS: Anti-insulin antibodies in insulin-treated diabetes can derange glycaemia, but are under-recognised. Detection of significant antibodies is complicated by antigenically distinct insulin analogues. We evaluated a pragmatic biochemical approach to identifying actionable antibodies, and assessed its utility in therapeutic decision making. METHODS: Forty people with insulin-treated diabetes and combinations of insulin resistance, nocturnal/matutinal hypoglycaemia, and unexplained ketoacidosis were studied using broad-specificity insulin immunoassays, polyethylene glycol (PEG) precipitation and gel filtration chromatography (GFC) with or without ex vivo insulin preincubation. RESULTS: Twenty-seven people had insulin immunoreactivity (IIR) below 3000 pmol/L that fell less than 50% after PEG precipitation. Insulin binding by antibodies in this group was low and judged insignificant. In 8 people IIR was above 3000 pmol/L and fell by more than 50% after PEG precipitation. GFC demonstrated substantial high molecular weight (HMW) IIR in 7 of these 8. In this group antibodies were judged likely significant. In 2 people immunosuppression was introduced, with a good clinical result in one but only a biochemical response in another. In 6 people adjustment of insulin delivery was subsequently informed by knowledge of underlying antibody. In a final group of 5 participants IIR was below 3000 pmol/L but fell by more than 50% after PEG precipitation. In 4 of these GFC demonstrated low levels of HMW IIR and antibody significance was judged indeterminate. CONCLUSIONS: Anti-insulin antibodies should be considered in insulin-treated diabetes with unexplained glycaemic lability. Combining immunoassays with PEG precipitation can stratify their significance. Antibody depletion may be beneficial, but conservative measures often suffice.

Pregnancy glucagon-like peptide 1 predicts insulin but not glucose concentrations.

AIMS: Incretin hormones glucagon-like peptide 1 (GLP-1) and gastric inhibitory peptide (GIP) cause increased insulin secretion in non-pregnant adults, but their role in pregnancy, where there are additional metabolically-active hormones from the placenta, is less clear. The aim of the present study was to assess if fasting and post-load incretin concentrations were predictive of pregnancy insulin and glucose concentrations. METHODS: Pregnant women (n = 394) with one or more risk factors for gestational diabetes were recruited at 28 weeks for a 75 g oral glucose tolerance test (OGTT). Glucose, insulin, GLP-1 and GIP were measured in the fasting state and 120 min after glucose ingestion. RESULTS: Fasting plasma GLP-1 concentrations were associated with plasma insulin (standardised ß' 0.393 (0.289-0.498), p = 1.3 × 10-12; n = 306), but not with glucose concentrations (p = 0.3). The association with insulin was still evident when adjusting for BMI (ß' 0.271 (0.180-0.362), p = 1.1 × 10-8; n = 297). Likewise, at 120 min the OGTT GLP-1 concentrations were associated with plasma insulin concentrations (ß' 0.216 (0.100-0.331), p = 2.7 × 10-4; n = 306) even after adjusting for BMI (ß' 0.178 (0.061-0.294), p = 2.9 × 10-3; n = 296), but not with glucose (p = 0.9). GIP concentrations were not associated with insulin or glucose concentrations at either time point (all p > 0.2). In pregnancy plasma GLP-1, but not GIP, concentrations appear to be predictive of circulating insulin concentrations, independently of associations with BMIs. CONCLUSIONS: These results suggest that the relationship between insulin and incretins is preserved in pregnancy, but that other factors, such as placental hormones or counter-regulatory hormones, may be more important determinants of glycaemia and gestational diabetes aetiology.

Association of GDF15 levels with body mass index and endocrine status in ß-thalassaemia.

OBJECTIVE: GDF15 has emerged as a stress-induced hormone, acting on the brain to reduce food intake and body weight while affecting neuroendocrine function. Very high GDF15 levels are found in thalassaemia, where growth, energy balance and neuroendocrine function are impaired. We examined the relationships between GDF15 and anthropometric measures and endocrine status in ß-thalassaemia. DESIGN: Cross sectional study. PATIENTS: All ß-thalassaemia patients attending the thalassaemia unit of Colombo North Teaching Hospital for blood transfusions. MEASUREMENTS: Anthropometric data, appetite scores, circulating GDF15, IGF, thyroid and reproductive hormone levels in 103 ß-thalassaemia patients were obtained. RESULTS: GDF15 levels were markedly elevated in thalassaemia patients (24.2-fold with ß-thalassaemia major compared with healthy controls). Among patients with ß-thalassaemia major, the relationship between GDF15 and body mass index (BMI) was curvilinear with all individuals with GDF15 levels above 24,000 pg/mL having a BMI below 20 kg/m2 . After adjustment for BMI, age and Tanner stage, serum IGF1 concentrations correlated negatively with GDF15 in all thalassaemia patients (ß = -.027, p = .02). We found a significant positive relationship between GDF15 and gonadotropin (in both sexes) and testosterone (in males). CONCLUSIONS: GDF15 levels were markedly elevated in patients with ß-thalassaemia and its association with BMI is consistent with the known effect of GDF15 to reduce body weight. The inverse association between GDF15 with IGF1 levels may reflect a neuroendocrine impact of GDF15 or an indirect effect via impaired nutritional state. The positive association with testosterone in males and gonadotropins in both sexes, was surprising and should prompt further GDF15 studies on the hypothalamic pituitary gonadal axis.

A Wars2 mutant mouse shows a sex and diet specific change in fat distribution, reduced food intake and depot-specific upregulation of WAT browning.

Background: Increased waist-to-hip ratio (WHR) is associated with increased mortality and risk of type 2 diabetes and cardiovascular disease. The TBX15-WARS2 locus has consistently been associated with increased WHR. Previous study of the hypomorphic Wars2 V117L/V117L mouse model found phenotypes including severely reduced fat mass, and white adipose tissue (WAT) browning, suggesting Wars2 could be a potential modulator of fat distribution and WAT browning. Methods: To test for differences in browning induction across different adipose depots of Wars2 V117L/V117L mice, we measured multiple browning markers of a 4-month old chow-fed cohort in subcutaneous and visceral WAT and brown adipose tissue (BAT). To explain previously observed fat mass loss, we also tested for the upregulation of plasma mitokines FGF21 and GDF15 and for differences in food intake in the same cohort. Finally, to test for diet-associated differences in fat distribution, we placed Wars2 V117L/V117L mice on low-fat or high-fat diet (LFD, HFD) and assessed their body composition by Echo-MRI and compared terminal adipose depot weights at 6 months of age. Results: The chow-fed Wars2 V117L/V117L mice showed more changes in WAT browning marker gene expression in the subcutaneous inguinal WAT depot (iWAT) than in the visceral gonadal WAT depot (gWAT). These mice also demonstrated reduced food intake and elevated plasma FGF21 and GDF15, and mRNA from heart and BAT. When exposed to HFD, the Wars2 V117L/V117L mice showed resistance to diet-induced obesity and a male and HFD-specific reduction of gWAT: iWAT ratio. Conclusion: Severe reduction of Wars2 gene function causes a systemic phenotype which leads to upregulation of FGF21 and GDF15, resulting in reduced food intake and depot-specific changes in browning and fat mass.

The renal-bone axis in older people living with HIV on stable antiretroviral therapy: A sub-analysis of the GS-US-104-0423 study.

BACKGROUND: Data on low bone mineral density (BMD) in people living with HIV (PLWH) are mainly derived from younger adults; little is known about how antiretroviral therapy (ART) and alterations in the renal-bone axis relate to BMD in older PLWH. METHODS: Cross-sectional study of men > 50 years and post-menopausal women with HIV. Antiretroviral therapy exposure was stratified into four groups based on use of tenofovir disoproxil fumarate (TDF) and protease inhibitors (PI): non-TDF/non-PI, non-TDF/PI, TDF/non-PI, and TDF/PI. Bone mineral density was measured by dual X-ray absorptiometry (DXA). Bone turnover/regulatory markers and renal tubular function were analysed in stored plasma and urine samples. The association of ART exposure and bone/renal biomarkers on BMD was explored using logistic regression models. RESULTS: 247 individuals (median [IQR] age 57 [53, 65] years; 47% female; 13% of Black ethnicity; CD4 count 643 [473, 811] cells/mm3; and 98% with HIV RNA < 200 copies/mL) were included. Bone turnover and renal tubular function differed significantly by ART exposure. In analyses adjusted for demographic and traditional renal/bone risk factors, exposure to TDF and PI was associated with a fourfold greater risk of low BMD at the femoral neck and exposure to TDF and/or PI with a threefold greater risk of low BMD at the lumbar spine. The relationship between ART and low BMD was not altered by further adjustment for bone turnover or renal tubular function markers. CONCLUSIONS: The associations between low BMD and ART exposure (TDF vs. non-TDF and boosted vs. unboosted third agents) were minimally affected by adjustments for bone and kidney biomarkers.

The Common H202D Variant in GDF-15 Does Not Affect Its Bioactivity but Can Significantly Interfere with Measurement of Its Circulating Levels.

BACKGROUND: There is growing interest in the measurement of growth differentiation factor 15 (GDF-15) in a range of disorders associated with cachexia. We undertook studies to determine whether a common histidine (H) to aspartate (D) variant at position 202 in the pro-peptide (position 6 in the mature peptide) interfered with its detection by 3 of the most commonly used immunoassays. METHODS: Three synthetic GDF-15-forms (HH homo-, HD hetero-, and DD-homodimers) were measured after serial dilution using Roche Elecsys®, R&D QuantikineTM ELISA, and MSD R&D DuoSet® immunoassays. GDF-15 concentrations were measured by the Roche and the MSD R&D immunoassays in 173 genotyped participants (61 HH homozygotes, 59 HD heterozygotes, and 53 DD homozygotes). For the comparative statistical analyses of the GDF-15 concentrations, we used non-parametric tests, in particular Bland-Altman difference (bias) plots and Passing-Bablok regression. The bioactivity of the 2 different homodimers was compared in a cell-based assay in HEK293S-SRF-RET/GFRAL cells. RESULTS: The Roche assay detected H- and D-containing peptides similarly but the R&D reagents (Quantikine and DuoSet) consistently underreported GDF-15 concentrations in the presence of the D variant. DD dimers had recoveries of approximately 45% while HD dimers recoveries were 62% to 78%. In human serum samples, the GDF-15 concentrations reported by the R&D assay were a median of 4% lower for HH, a median of 36% lower for HD, and a median of 61% lower for DD compared to the Roche assay. The bioactivities of the HH and DD peptides were indistinguishable. CONCLUSIONS: The D variant of GDF-15 substantially affects its measurement by a commonly used immunoassay, a finding that has clear implications for its interpretation in research and clinical settings.

Exploration of a panel of urine biomarkers of kidney disease in two paediatric cohorts with Type 1 diabetes mellitus of differing duration.

BACKGROUND: The pathogenesis of diabetic kidney disease (DKD) is complex and involves both glomerular and tubular dysfunction. A global assessment of kidney function is necessary to stage DKD, a progressive kidney disease that is likely to begin in childhood. The present study evaluated whether kidney injury biomarkers identified as early DKD biomarkers in adults have any prognostic value in the very early stages of childhood diabetes. METHODS: We measured urine free Retinol-binding protein 4 (UfRBP4), albumin (UAlb), Kidney injury molecule-1 (KIM-1) and the microRNAs miR-155, miR-126 and miR-29b in two cohorts of paediatric T1DM patients without evidence of DKD, but with diabetes of short-duration, ≤ 2.5 years (SD, n = 25) or of long-duration, ≥ 10 years (LD, n = 29); non-diabetic siblings (H, n = 26) were recruited as controls. A p value < 0.05 was considered significant for all results. RESULTS: UfRBP4 and UAlb were not significantly different across the three groups. No differences were found in KIM-1 excretion between any of the three groups. UfRBP4 was correlated with UAlb in all three groups (r 0.49; p < 0.001), whereas KIM-1 showed no correlation with albumin excretion. Among microRNAs, miR-29b was higher in all diabetic children compared with the H control group (p = 0.03), whereas miR-155 and miR-126 were not significantly different. No differences were found between the SD and LD groups for all three microRNAs. No associations were identified between these biomarkers with sex, age, BMI, eGFR, T1DM duration or glycaemic control. CONCLUSIONS: UfRBP4, KIM-1, miR-155, and miR-126 were unaffected by the presence and duration of diabetes, whereas miR-29b showed a modest elevation in diabetics, regardless of duration. These data support the specificity of a panel of urine biomarkers as DKD biomarkers, rather than any relationship to diabetes per se or its duration, and not as early DKD biomarkers in a paediatric setting.

The imprinted Igf2-Igf2r axis is critical for matching placental microvasculature expansion to fetal growth.

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here, we demonstrate, through fetal, endothelial, hematopoietic, and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The angiocrine effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signaling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.

The impact of islet mass, number of transplants, and time between transplants on graft function in a national islet transplant program.

The UK islet allotransplant program is nationally funded to deliver one or two transplants over 12 months to individuals with type 1 diabetes and recurrent severe hypoglycemia. Analyses were undertaken 10 years after program inception to evaluate associations between transplanted mass; single versus two transplants; time between two transplants and graft survival (stimulated C-peptide >50 pmol/L) and function. In total, 84 islet transplant recipients were studied. Uninterrupted graft survival over 12 months was attained in 23 (68%) single and 47 (94%) (p = .002) two transplant recipients (separated by [median (IQR)] 6 (3-8) months). 64% recipients of one or two transplants with uninterrupted function at 12 months sustained graft function at 6 years. Total transplanted mass was associated with Mixed Meal Tolerance Test stimulated C-peptide at 12 months (p < .01). Despite 1.9-fold greater transplanted mass in recipients of two versus one islet infusion (12 218 [9291-15 417] vs. 6442 [5156-7639] IEQ/kg; p < .0001), stimulated C-peptide was not significantly higher. Shorter time between transplants was associated with greater insulin dose reduction at 12 months (beta -0.35; p = .02). Graft survival over the first 12 months was greater in recipients of two versus one islet transplant in the UK program, although function at 1 and 6 years was comparable. Minimizing the interval between 2 islet infusions may maximize cumulative impact on graft function.

Safety of Tenofovir Alafenamide in People With HIV Who Experienced Proximal Renal Tubulopathy on Tenofovir Disoproxil Fumarate.

BACKGROUND: Proximal renal tubulopathy (PRT) is an infrequent complication of tenofovir disoproxil fumarate (TDF). It remains to be established whether tenofovir alafenamide (TAF) can be safely administered to individuals who experienced PRT on TDF. METHODS: Individuals with a history of TDF-associated PRT and current estimated glomerular filtration rate (eGFR) over 30 mL/min/1.73 m2 initiated TAF and were followed for 96 weeks. The primary outcome of interest was recurrent PRT. Secondary outcomes were changes in kidney biomarkers, bone biomarkers, and bone mineral density (BMD). Data were analyzed using multilevel mixed-effects linear regression models. The trial was registered under EudraCT 2016-003345-29. RESULTS: All 31 participants [median age 55 (inter-quartile range 51, 60) years, 97% men, 87% White ethnicity] remained on TAF at week 96, and none developed glycosuria or recurrent PRT. Participants experienced small declines in eGFR-creatinine [-1.9 (95% confidence interval: -3.5 to -0.3) mL/min/1.73 m2/yr; P = 0.024], but not in eGFR-cystatin C [-0.9 (-2.1 to 0.4) mL/min/1.73 m2/yr; P = 0.16]. Ten (32%) and 5 (16%) participants experienced rapid (>5 mL/min/1.73 m2/yr) decline in eGFR-creatinine and eGFR-cystatin C. No significant change in other kidney biomarkers, bone turnover, or BMD was observed (P > 0.2). CONCLUSIONS: In individuals with a history of PRT on TDF, 96 weeks of TAF was not associated with recurrent PRT or adverse effects on renal tubular function, bone turnover, or BMD. These data suggest that TAF is a treatment option for this vulnerable population.

Activation of the hypothalamic-pituitary-adrenal axis by exogenous and endogenous GDF15.

An acute increase in the circulating concentration of glucocorticoid hormones is essential for the survival of severe somatic stresses. Circulating concentrations of GDF15, a hormone that acts in the brain to reduce food intake, are frequently elevated in stressful states. We now report that GDF15 potently activates the hypothalamic-pituitary-adrenal (HPA) axis in mice and rats. A blocking antibody to the GDNF-family receptor α-like receptor completely prevented the corticosterone response to GDF15 administration. In wild-type mice exposed to a range of stressful stimuli, circulating levels of both corticosterone and GDF15 rose acutely. In the case of Escherichia coli or lipopolysaccharide injections, the vigorous proinflammatory cytokine response elicited was sufficient to produce a near-maximal HPA response, regardless of the presence or absence of GDF15. In contrast, the activation of the HPA axis seen in wild-type mice in response to the administration of genotoxic or endoplasmic reticulum toxins, which do not provoke a marked rise in cytokines, was absent in Gdf15-/- mice. In conclusion, consistent with its proposed role as a sentinel hormone, endogenous GDF15 is required for the activation of the protective HPA response to toxins that do not induce a substantial cytokine response. In the context of efforts to develop GDF15 as an antiobesity therapeutic, these findings identify a biomarker of target engagement and a previously unrecognized pharmacodynamic effect, which will require monitoring in human studies.

Increased C-Peptide Immunoreactivity in Insulin Autoimmune Syndrome (Hirata Disease) Due to High Molecular Weight Proinsulin.

BACKGROUND: Determination of C-peptide is important in the investigation of unexplained hyperinsulinemic hypoglycemia because a high C-peptide concentration usually indicates endogenous insulin hypersecretion. Insulin autoimmune syndrome (IAS) denotes hyperinsulinemic hypoglycemia due to insulin-binding antibodies that prolong insulin half-life. C-peptide clearance is considered to be unaffected, and although a marked C-peptide immunoreactivity in hypoglycemic samples has been reported, it has been suspected to be artifactual. High-resolution mass spectrometry enables examination of the basis of C-peptide-immunoreactivity in IAS. METHODS: Precipitation of plasma with polyethylene glycol was followed by C-peptide immunoassay. Plasma peptides extracted by solvent precipitation were characterized by nano-LC-MS/MS and analyzed using an untargeted data-dependent method. Peptides related to proinsulin, in amino acid sequence, were identified using proprietary bioinformatics software and confirmed by repeat LC-MS/MS analysis. Gel filtration chromatography coupled to LC-MS/MS was used to identify proinsulin-related peptides present in IAS immunocomplexes. Results were compared with those from C-peptide immunoassay. RESULTS: Polyethylene glycol precipitation of IAS plasma, but not control plasma, depleted C-peptide immunoreactivity consistent with immunoglobulin-bound C-peptide immunoreactivity. LC-MS/MS detected proinsulin and des 31,32 proinsulin at higher abundance in IAS plasma compared with control plasma. Analysis by gel filtration chromatography coupled to LC-MS/MS demonstrated proinsulin and des 31,32 proinsulin, but no C-peptide, in plasma immunocomplexes. CONCLUSIONS: Antibody binding can enrich proinsulin and des 31,32 proinsulin in IAS immunocomplexes. Proinsulin cross-reactivity in some C-peptide immunoassays can lead to artifactually increased C-peptide results.

Evaluation of PSA and PSA Density in a Multiparametric Magnetic Resonance Imaging-Directed Diagnostic Pathway for Suspected Prostate Cancer: The INNOVATE Trial.

Objectives: To assess the clinical outcomes of mpMRI before biopsy and evaluate the space remaining for novel biomarkers. Methods: The INNOVATE study was set up to evaluate the validity of novel fluidic biomarkers in men with suspected prostate cancer who undergo pre-biopsy mpMRI. We report the characteristics of this clinical cohort, the distribution of clinical serum biomarkers, PSA and PSA density (PSAD), and compare the mpMRI Likert scoring system to the Prostate Imaging-Reporting and Data System v2.1 (PI-RADS) in men undergoing biopsy. Results: 340 men underwent mpMRI to evaluate suspected prostate cancer. 193/340 (57%) men had subsequent MRI-targeted prostate biopsy. Clinically significant prostate cancer (csigPCa), i.e., overall Gleason ≥ 3 + 4 of any length OR maximum cancer core length (MCCL) ≥4 mm of any grade including any 3 + 3, was found in 96/195 (49%) of biopsied patients. Median PSA (and PSAD) was 4.7 (0.20), 8.0 (0.17), and 9.7 (0.31) ng/mL (ng/mL/mL) in mpMRI scored Likert 3,4,5 respectively for men with csigPCa on biopsy. The space for novel biomarkers was shown to be within the group of men with mpMRI scored Likert3 (178/340) and 4 (70/350), in whom an additional of 40% (70/178) men with mpMRI-scored Likert3, and 37% (26/70) Likert4 could have been spared biopsy. PSAD is already considered clinically in this cohort to risk stratify patients for biopsy, despite this 67% (55/82) of men with mpMRI-scored Likert3, and 55% (36/65) Likert4, who underwent prostate biopsy had a PSAD below a clinical threshold of 0.15 (or 0.12 for men aged <50 years). Different thresholds of PSA and PSAD were assessed in mpMRI-scored Likert4 to predict csigPCa on biopsy, to achieve false negative levels of ≤5% the proportion of patients whom who test as above the threshold were unsuitably high at 86 and 92% of patients for PSAD and PSA respectively. When PSA was re tested in a sub cohort of men repeated PSAD showed its poor reproducibility with 43% (41/95) of patients being reclassified. After PI-RADS rescoring of the biopsied lesions, 66% (54/82) of the Likert3 lesions received a different PI-RADS score. Conclusions: The addition of simple biochemical and radiological markers (Likert and PSAD) facilitate the streamlining of the mpMRI-diagnostic pathway for suspected prostate cancer but there remains scope for improvement, in the introduction of novel biomarkers for risk assessment in Likert3 and 4 patients, future application of novel biomarkers tested in a Likert cohort would also require re-optimization around Likert3/PI-RADS2, as well as reproducibility testing.

Pregnancy Serum DLK1 Concentrations Are Associated With Indices of Insulin Resistance and Secretion.

CONTEXT: Delta like noncanonical notch ligand 1 (DLK1) is a paternally expressed imprinted gene that encodes an epidermal growth factor repeat-containing transmembrane protein. A bioactive, truncated DLK1 protein is present in the circulation and has roles in development and metabolism. OBJECTIVE: We sought to investigate links between maternal pregnancy circulating DLK1 concentrations and: (1) maternal and fetal DLK1 genotypes, (2) maternal insulin resistance and secretion, and (3) offspring size at birth. PATIENTS, DESIGN, AND SETTING: We measured third-trimester maternal serum DLK1 concentrations and examined their associations with parentally transmitted fetal and maternal DLK1 genotypes, indices of maternal insulin resistance and secretion derived from 75-g oral glucose tolerance tests performed around week 28 of pregnancy, and offspring size at birth in 613 pregnancies from the Cambridge Baby Growth Study. RESULTS: Maternal DLK1 concentrations were associated with the paternally transmitted fetal DLK1 rs12147008 allele (P = 7.8 × 10-3) but not with maternal rs12147008 genotype (P = 0.4). Maternal DLK1 concentrations were positively associated with maternal prepregnancy body mass index (P = 3.5 × 10-6), and (after adjustment for maternal body mass index) with both maternal fasting insulin resistance (Homeostatic Model Assessment of Insulin Resistance: P = 0.01) and measures of maternal insulin secretion in response to oral glucose (insulinogenic index: P = 1.2 × 10-3; insulin disposition index: P = 0.049). Further positive associations were found with offspring weight (P = 0.02) and head circumference at birth (P = 0.04). CONCLUSION: These results are consistent with a partial paternal or placental origin for the maternal circulating DLK1 which may lead to increased maternal circulating DLK1 concentrations, stimulation of maternal insulin resistance and compensatory hyperinsulinemia during pregnancy, and the promotion of fetal growth.

Dyslipidemia, Insulin Resistance, Ectopic Lipid Accumulation, and Vascular Function in Resistance to Thyroid Hormone ß.

PURPOSE: In resistance to thyroid hormone due to mutations in thyroid hormone receptor ß, peripheral tissues are variably refractory to the action of circulating thyroid hormones. We evaluated parameters contributing to atherosclerotic risk in this disorder. METHODS: We measured low-density lipoprotein cholesterol (LDL-C), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), nonesterified fatty acids (NEFA), intrahepatic lipid (IHL) and intramyocellular lipid (IMCL), Homeostasis-model assessment of insulin resistance (HOMA-IR), augmentation index (AIx) and pulse wave velocity (PWV), flow-mediated dilatation, and carotid intima-media thickness (cIMT) in an unselected, genetically confirmed cohort of adult RTHß patients (n = 27-77) and compared these with measurements in healthy subjects (up to n = 100) and thyrotoxic patients (n = 40). RESULTS: Resistance to thyroid hormone beta (RTHß) patients exhibited higher LDL-C (P = 0.008) and TG (P = 0.002) and lower HDL-C concentrations (P = 0.015 × 10-2) than control subjects, with LDL-C being higher than in thyrotoxic patients with comparable hyperthyroxinemia. Proprotein convertase subtilisin/kexin 9 (P = 0.002) and apolipoprotein B (P = 0.0009) levels were reduced in thyrotoxic patients but not lower in RTHß patients or control subjects. Intrahepatic lipid (P = 0.02 × 10-4), IMCL (P = 0.002), HOMA-IR (P = 0.01 × 10-2), and NEFA (P = 0.04 × 10-6) were significantly higher in RTHß patients than control subjects. Flow-mediated dilatation was increased (P = 0.04) but cIMT (P = 0.71), PWV P = 0.81), and AIx (P = 0.95) were unaltered in RTHß patients. CONCLUSIONS: We have documented mixed dyslipidemia with hepatic and IMCL accumulation in RTHß, suggesting that surveillance for these metabolic abnormalities is warranted. How they combine with enhanced endothelial function and unaltered vessel wall thickness and compliance to determine overall cardiometabolic risk in this disorder remains to be defined.