Autoantibodies to truncated GAD(96-585) antigen stratify risk of early insulin requirement in adult-onset diabetes.

We investigated whether characterisation of full-length (f-)GADA responses could identify early insulin requirement in adult-onset diabetes. In 179 f-GADA positive participants diagnosed with type 2 diabetes, we assessed associations of truncated (t-)GADA positivity, f-GADA IgG subclasses, and f-GADA affinity with early insulin requirement (<5 years), type 1 diabetes genetic risk score (T1D GRS), and C-peptide. t-GADA positivity was lower in f-GADA positive without early insulin in comparison to f-GADA positive type 2 diabetes requiring insulin within 5 years, and type 1 diabetes (75% vs. 91% and 95% respectively, p<0.0001). t-GADA positivity (in those f-GADA positive) identified a group with a higher type 1 diabetes genetic susceptibility (mean T1D GRS 0.248 vs. 0.225, p=0.003), lower C-peptide (1156 pmol/L vs. 4289 pmol/L, p=1x10-7), and increased IA-2A positivity (23% vs. 6%, p=0.03). In survival analysis, t-GADA positivity was associated with early insulin requirement compared with those only positive for f-GADA, independently from age of diagnosis, f-GADA titre and duration of diabetes [adjusted HR 5.7 (95% CI 1.4, 23.5), p=0.017]. The testing of t-GADA in f-GADA positive individuals with type 2 diabetes identifies those who have genetic and clinical characteristics comparable to type 1 diabetes and stratifies those at higher risk of early insulin requirement.

Improved Specificity of Glutamate Decarboxylase 65 Autoantibody Measurement Using Luciferase-Based Immunoprecipitation System Assays.

Autoantibodies to glutamate decarboxylase (GADA) are widely used in the prediction and classification of type 1 diabetes. GADA radiobinding assays (RBAs) using N-terminally truncated antigens offer improved specificity, but radioisotopes limit the high-throughput potential for population screening. Luciferase-based immunoprecipitation system (LIPS) assays are sensitive and specific alternatives to RBAs with the potential to improve risk stratification. The performance of assays using the Nanoluc luciferase (Nluc)-conjugated GAD65 constructs, Nluc-GAD65(96-585) and full length Nluc-GAD65(1-585), were evaluated in 434 well-characterized serum samples from patients with recent-onset type 1 diabetes and first-degree relatives. Nonradioactive, high-throughput LIPS assays are quicker and require less serum than RBAs. Of 171 relatives previously tested single autoantibody positive for autoantibodies to full-length GAD65 by RBA but had not progressed to diabetes, fewer retested positive by LIPS using either truncated (n = 72) or full-length (n = 111) antigen. The Nluc-GAD65(96-585) truncation demonstrated the highest specificity in LIPS assays overall, but in contrast to RBA, N-terminus truncations did not result in a significant increase in disease-specificity compared with the full-length antigen. This suggests that binding of nonspecific antibodies is affected by the conformational changes resulting from addition of the Nluc antigen. Nluc-GAD65(96-585) LIPS assays offer low-blood-volume, high-specificity GADA tests for screening and diagnostics.

A novel, high-performance, low-volume, rapid luciferase immunoprecipitation system (LIPS) assay to detect autoantibodies to zinc transporter 8.

BACKGROUND: Zinc transporter 8 autoantibodies (ZnT8A) are thought to appear close to type 1 diabetes (T1D) onset and can identify high-risk multiple (≥2) autoantibody positive individuals. Radiobinding assays (RBA) are widely used for ZnT8A measurement but have limited sustainability. We sought to develop a novel, high-performance, non-radioactive luciferase immunoprecipitation system (LIPS) assay to replace RBA. METHODS: A custom dual C-terminal ZnT8 (aa268-369; R325/W325) heterodimeric antigen, tagged with a NanoluciferaseTM (Nluc-ZnT8) reporter, and LIPS assay was developed. Assay performance was evaluated by testing sera from new onset T1D (n = 573), healthy schoolchildren (n = 521), and selected first-degree relatives (FDRs) from the Bart's Oxford family study (n = 617; 164 progressed to diabetes). RESULTS: In new-onset T1D, ZnT8A levels by LIPS strongly correlated with RBA (Spearman's r = 0.89; P < 0.0001), and positivity was highly concordant (94.3%). At a high specificity (95%), LIPS and RBA had comparable assay performance [LIPS pROC-AUC(95) 0.032 (95% CI: 0.029-0.036); RBA pROC-AUC(95) 0.031 (95% CI: 0.028-0.034); P = 0.376]. Overall, FDRs found positive by LIPS or RBA had a comparable 20-year diabetes risk (52.6% and 59.7%, respectively), but LIPS positivity further stratified T1D risk in FDRs positive for at least one other islet autoantibody detected by RBA (P = 0.0346). CONCLUSION: This novel, high-performance, cheaper, quicker, higher throughput, low blood volume Nluc-ZnT8 LIPS assay is a safe, non-radioactive alternative to RBA with enhanced sensitivity and ability to discriminate T1D progressors. This method offers an advanced approach to current strategies to screen the general population for T1D risk for immunotherapy trials and to reduce rates of diabetic ketoacidosis at diagnosis.

Ulipristal acetate versus levonorgestrel-releasing intrauterine system for heavy menstrual bleeding (UCON): a randomised controlled phase III trial.

Background: Heavy menstrual bleeding affects one in four women and negatively impacts quality of life. Ulipristal acetate is prescribed to treat symptoms associated with uterine fibroids. We compared the effectiveness of ulipristal acetate and the levonorgestrel-releasing intrauterine system at reducing the burden of heavy menstrual bleeding, irrespective of the presence of fibroids. Methods: This randomised, open-label, parallel group phase III trial enrolled women over 18 years with heavy menstrual bleeding from 10 UK hospitals. Participants were centrally randomised, in a 1:1 ratio, to either three, 12-week treatment cycles of 5 mg ulipristal acetate daily, separated by 4-week treatment-free intervals, or a levonorgestrel-releasing intrauterine system. The primary outcome, analysed by intention-to-treat, was quality of life measured by the Menorrhagia Multi-Attribute Scale at 12 months. Secondary outcomes included menstrual bleeding and liver function. The trial is registered with ISRCTN, 20426843. Findings: Between June 5th, 2015 and February 26th, 2020, 236 women were randomised, either side of a recruitment suspension due to concerns of ulipristal acetate hepatoxicity. Subsequent withdrawal of ulipristal acetate led to early cessation of recruitment but the trial continued in follow-up. The primary outcome substantially improved in both groups, and was 89, (interquartile range [IQR] 65 to 100, n = 53) and 94, (IQR 70 to 100, n = 50; adjusted odds ratio 0.55, 95% confidence interval [CI] 0.26-1.17; p = 0.12) in the ulipristal and levonorgestrel-releasing intrauterine system groups. Rates of amenorrhoea at 12 months were higher in those allocated ulipristal acetate compared to levonorgestrel-releasing intrauterine system (64% versus 25%, adjusted odds ratio 7.12, 95% CI 2.29-22.2). Other outcomes were similar between the two groups and there were no cases of endometrial malignancy or hepatotoxicity due to ulipristal acetate use. Interpretation: Our findings suggested that both treatments improved quality of life. Ulipristal was more effective at inducing amenorrhoea. Ulipristal has been demonstrated to be an effective medical therapeutic option but currently its use has restrictions and requires liver function monitoring. Funding: UK Medical Research Council and National Institute of Health Research EME Programme (12/206/52).

Evaluation and deployment of isotype-specific salivary antibody assays for detecting previous SARS-CoV-2 infection in children and adults.

BACKGROUND: Saliva is easily obtainable non-invasively and potentially suitable for detecting both current and previous SARS-CoV-2 infection, but there is limited evidence on the utility of salivary antibody testing for community surveillance. METHODS: We established 6 ELISAs detecting IgA and IgG antibodies to whole SARS-CoV-2 spike protein, to its receptor binding domain region and to nucleocapsid protein in saliva. We evaluated diagnostic performance, and using paired saliva and serum samples, correlated mucosal and systemic antibody responses. The best-performing assays were field-tested in 20 household outbreaks. RESULTS: We demonstrate in test accuracy (N = 320), spike IgG (ROC AUC: 95.0%, 92.8-97.3%) and spike IgA (ROC AUC: 89.9%, 86.5-93.2%) assays to discriminate best between pre-pandemic and post COVID-19 saliva samples. Specificity was 100% in younger age groups (0-19 years) for spike IgA and IgG. However, sensitivity was low for the best-performing assay (spike IgG: 50.6%, 39.8-61.4%). Using machine learning, diagnostic performance was improved when a combination of tests was used. As expected, salivary IgA was poorly correlated with serum, indicating an oral mucosal response whereas salivary IgG responses were predictive of those in serum. When deployed to household outbreaks, antibody responses were heterogeneous but remained a reliable indicator of recent infection. Intriguingly, unvaccinated children without confirmed infection showed evidence of exposure almost exclusively through specific IgA responses. CONCLUSIONS: Through robust standardisation, evaluation and field-testing, this work provides a platform for further studies investigating SARS-CoV-2 transmission and mucosal immunity with the potential for expanding salivo-surveillance to other respiratory infections in hard-to-reach settings.

If a person has been previously infected with SARS-CoV-2 they will produce specific proteins, called antibodies. These are present in the saliva and blood. Saliva is easier to obtain than blood, so we developed and evaluated six tests that detect SARS-CoV-2 antibodies in saliva in children and adults. Some tests detected antibodies to a particular protein made by SARS-CoV-2 called the spike protein, and these tests worked best. The most accurate results were obtained by using a combination of tests. Similar tests could also be developed to detect other respiratory infections which will enable easier identification of infected individuals.

Islet Autoantibody Standardization Program: interlaboratory comparison of insulin autoantibody assay performance in 2018 and 2020 workshops.

AIMS/HYPOTHESIS: The Islet Autoantibody Standardization Program (IASP) aims to improve the performance of immunoassays measuring autoantibodies in type 1 diabetes and the concordance of results across laboratories. IASP organises international workshops distributing anonymised serum samples to participating laboratories and centralises the collection and analysis of results. In this report, we describe the results of assays measuring IAA submitted to the IASP 2018 and 2020 workshops. METHODS: The IASP distributed uniquely coded sera from individuals with new-onset type 1 diabetes, multiple islet autoantibody-positive individuals, and diabetes-free blood donors in both 2018 and 2020. Serial dilutions of the anti-insulin mouse monoclonal antibody HUI-018 were also included. Sensitivity, specificity, area under the receiver operating characteristic curve (ROC-AUC), partial ROC-AUC at 95% specificity (pAUC95) and concordance of qualitative/quantitative results were compared across assays. RESULTS: Results from 45 IAA assays of seven different formats and from 37 IAA assays of six different formats were submitted to the IASP in 2018 and 2020, respectively. The median ROC-AUC was 0.736 (IQR 0.617-0.803) and 0.790 (IQR 0.730-0.836), while the median pAUC95 was 0.016 (IQR 0.004-0.021) and 0.023 (IQR 0.014-0.026) in the 2018 and 2020 workshops, respectively. Assays largely differed in AUC (IASP 2018 range 0.232-0.874; IASP 2020 range 0.379-0.924) and pAUC95 (IASP 2018 and IASP 2020 range 0-0.032). CONCLUSIONS/INTERPRETATION: Assay formats submitted to this study showed heterogeneous performance. Despite the high variability across laboratories, the in-house radiobinding assay (RBA) remains the gold standard for IAA measurement. However, novel non-radioactive IAA immunoassays showed a good performance and, if further improved, might be considered valid alternatives to RBAs.

Increased levels of anti-BSA antibodies in children with Down syndrome.

Introduction: Autoimmune diabetes occurs more often in the first 2 years of life in children with Down syndrome (DS) compared with the general population. We previously observed increased frequencies of islet autoantibodies, including insulin autoantibodies (IAA), in children with DS. Assays for IAA using 125I-labelled insulin require competition to overcome cross reactivity with antibodies to the cow's milk protein, bovine serum albumin (BSA). 125I-IAA assay results suggested that levels of antibodies to BSA may also be increased in children with DS. The aim of this study therefore was to determine whether the levels of anti-BSA antibodies differed in children with DS compared with controls. Methods: Samples were available from two populations with DS: one from the UK, (UK DS cohort n=106, 58 male, median age 12.5 years) and one from Estonia (Estonian DS cohort: n=121, 65 male, median age 9.75 years). A UK control population was provided by sex and age-matched healthy siblings of probands participating in the Bart's Oxford (BOX) family study of type 1 diabetes. A competitive-displacement radiobinding assay (RBA) and a Dissociation Enhanced Lanthanide Fluoroimmunoassay (DELFIA) were developed to measure and confirm anti-BSA antibody levels. HLA class II genotype was analysed by PCR using sequence specific primers (PCR-SSP). Results: Overall, levels of anti-BSA antibodies were increased in those with DS compared with controls (p<0.0001) but this was not HLA associated. Conclusion: Increased levels of anti-BSA antibodies may reflect a defect in immune maturation or increased gut permeability in children with DS, increasing their risk of developing autoimmunity.

Exocrine Proteins Including Trypsin(ogen) as a Key Biomarker in Type 1 Diabetes.

OBJECTIVE: Proteomic profiling can identify useful biomarkers. Monozygotic (MZ) twins discordant for a condition represent an ideal test population. We aimed to investigate and validate proteomic profiling in twins with type 1 diabetes and in other well-characterized cohorts. RESEARCH DESIGN AND METHODS: A broad, multiplex analysis of 4,068 proteins in serum samples from MZ twins concordant (n = 43) and discordant (n = 27) for type 1 diabetes identified major differences that were subsequently validated by a trypsin(ogen) assay in MZ pairs concordant (n = 39) and discordant (n = 42) for type 1 diabetes, individuals at risk for (n = 195) and with (n = 990) type 1 diabetes, as well as individuals with non-insulin-requiring adult-onset diabetes diagnosed as either autoimmune (n = 96) or type 2 (n = 291). RESULTS: Proteomic analysis identified major differences between exocrine enzyme levels in discordant MZ twin pairs despite a strong correlation between twins, whether concordant or discordant for type 1 diabetes (P < 0.01 for both). In validation experiments, trypsin(ogen) levels were lower in twins with diabetes than in the co-twin without diabetes (P < 0.0001) and healthy control participants (P < 0.0001). In recently diagnosed participants, trypsin(ogen) levels were lower than in control participants across a broad age range. In at-risk relatives, levels <15 ng/mL were associated with an increased risk of progression (uncorrected P = 0.009). Multiple linear regression in recently diagnosed participants showed that trypsin(ogen) levels were associated with insulin dose and diabetic ketoacidosis, while age and BMI were confounders. CONCLUSIONS: Type 1 diabetes is associated with altered exocrine function, even before onset. Twin data suggest roles for genetic and nongenetically determined factors. Exocrine/endocrine interactions are important underinvestigated factors in type 1 diabetes.

Development and evaluation of low-volume tests to detect and characterize antibodies to SARS-CoV-2.

Low-volume antibody assays can be used to track SARS-CoV-2 infection rates in settings where active testing for virus is limited and remote sampling is optimal. We developed 12 ELISAs detecting total or antibody isotypes to SARS-CoV-2 nucleocapsid, spike protein or its receptor binding domain (RBD), 3 anti-RBD isotype specific luciferase immunoprecipitation system (LIPS) assays and a novel Spike-RBD bridging LIPS total-antibody assay. We utilized pre-pandemic (n=984) and confirmed/suspected recent COVID-19 sera taken pre-vaccination rollout in 2020 (n=269). Assays measuring total antibody discriminated best between pre-pandemic and COVID-19 sera and were selected for diagnostic evaluation. In the blind evaluation, two of these assays (Spike Pan ELISA and Spike-RBD Bridging LIPS assay) demonstrated >97% specificity and >92% sensitivity for samples from COVID-19 patients taken >21 days post symptom onset or PCR test. These assays offered better sensitivity for the detection of COVID-19 cases than a commercial assay which requires 100-fold larger serum volumes. This study demonstrates that low-volume in-house antibody assays can provide good diagnostic performance, and highlights the importance of using well-characterized samples and controls for all stages of assay development and evaluation. These cost-effective assays may be particularly useful for seroprevalence studies in low and middle-income countries.

The measurement of autoantibodies to insulin informs diagnosis of diabetes in a childhood population negative for other autoantibodies.

AIMS: Some childhood type 1 diabetes cases are islet autoantibody negative at diagnosis. Potential explanations include misdiagnosis of genetic forms of diabetes or insufficient islet autoantibody testing. Many NHS laboratories offer combinations of three autoantibody markers. We sought to determine the benefit of testing for additional islet autoantibodies, including insulin (IAA) and tetraspanin 7 (TSPAN7A). METHODS: Radiobinding assays (RBAs) were used to test for four islet autoantibodies in children with newly diagnosed type 1 diabetes (n = 486; 54.1% male; median age 10.4 years [range 0.7-18.0]; median duration 1 day [range -183 to 14]). Islet autoantibody negative children were tested for TSPAN7A using a luminescence-based test. Where available, islet cell antibody (ICA) and human leucocyte antigen (HLA) data were considered. RESULTS: Using three autoantibody markers, 21/486 (4.3%) children were autoantibody negative. Testing for IAA classified a further 9/21 (42.9%) children as autoantibody positive. Of the remaining 12 (2.5%) autoantibody negative children, all were TPAN7A negative, seven were ICA negative and one was positive for the protective variant DQB1*0602. One was subsequently diagnosed with Maturity Onset of Diabetes in the Young, but follow-up was not available in all cases. CONCLUSIONS: Using highly sensitive assays, testing for three autoantibodies fails to detect islet autoimmunity in approximately 1/20 children diagnosed with type 1 diabetes. Testing for IAA in children <5 years and GADA in those >10 years was the most effective strategy for detecting islet autoimmunity. The ability to test for all islet autoantibodies should inform clinical decisions and make screening for monogenic diabetes more cost-effective.

Improving the Diagnosis of Endometrial Hyperplasia Using Computerized Analysis and Immunohistochemical Biomarkers.

Endometrial hyperplasia (EH) is a precursor lesion to endometrial carcinoma (EC). Risks for EC include genetic, hormonal and metabolic factors most notably those associated with obesity: rates are rising and there is concern that cases in pre-menopausal women may remain undetected. Making an accurate distinction between benign and pre-malignant disease is both a challenge for the pathologist and important to the gynecologist who wants to deliver the most appropriate care to meet the needs of the patient. Premalignant change may be recognized by histological changes of endometrial hyperplasia (which may occur with or without atypia) and endometrial intraepithelial neoplasia (EIN). In this study we created a tissue resource of EH samples diagnosed between 2004 and 2009 (n = 125) and used this to address key questions: 1. Are the EIN/WHO2014 diagnostic criteria able to consistently identify premalignant endometrium? 2. Can computer aided image analysis inform identification of EIN? 3. Can we improve diagnosis by incorporating analysis of protein expression using immunohistochemistry. Our findings confirmed the inclusion of EIN in diagnostic criteria resulted in a better agreement between expert pathologists compared with the previous WHO94 criteria used for the original diagnosis of our sample set. A computer model based on assessment of stromal:epithelial ratio appeared most accurate in classification of areas of tissue without EIN. From an extensive panel of putative endometrial protein tissue biomarkers a score based on assessment of HAND2, PTEN, and PAX2 was able to identify four clusters one of which appeared to be more likely to be benign. In summary, our study has highlighted new opportunities to improve diagnosis of pre-malignant disease in endometrium and provide a platform for further research on this important topic.

Multiomic Characterization of High-Grade Serous Ovarian Carcinoma Enables High-Resolution Patient Stratification.

PURPOSE: High-grade serous ovarian carcinoma (HGSOC) is the most common ovarian cancer type; most patients experience disease recurrence that accumulates chemoresistance, leading to treatment failure. Genomic and transcriptomic features have been associated with differential outcome and treatment response. However, the relationship between events at the gene sequence, copy number, and gene-expression levels remains poorly defined. EXPERIMENTAL DESIGN: We perform multiomic characterization of a large HGSOC cohort (n = 362) with detailed clinical annotation to interrogate the relationship between patient subgroups defined by specific molecular events. RESULTS: BRCA2-mutant (BRCA2m) and EMSY-overexpressing cases demonstrated prolonged survival [multivariable hazard ratios (HR) 0.40 and 0.51] and significantly higher first- and second-line chemotherapy response rate. CCNE1-gained (CCNE1g) cases demonstrated underrepresentation of FIGO stage IV cases, with shorter survival but no significant difference in treatment response. We demonstrate marked overlap between the TCGA- and Tothill-derived subtypes. IMR/C2 cases displayed higher BRCA1/2m frequency (25.5%, 32.5%) and significantly greater immune cell infiltration, whereas PRO/C5 cases had the highest CCNE1g rate (23.9%, 22.2%) and were uniformly low in immune cell infiltration. The survival benefit for cases with aberrations in homologous recombination repair (HRR) genes was apparent across all transcriptomic subtypes (HR range, 0.48-0.68). There was significant co-occurrence of RB loss and HRR gene aberrations; RB loss was further associated with favorable survival within HRR-aberrant cases (multivariable HR, 0.50). CONCLUSIONS: These data paint a high-resolution picture of the molecular landscape in HGSOC, better defining patients who may benefit most from specific molecular therapeutics and highlighting those for whom novel treatment strategies are needed to improve outcomes.

Young infants exhibit robust functional antibody responses and restrained IFN-γ production to SARS-CoV-2.

Severe COVID-19 appears rare in children. This is unexpected, especially in young infants, who are vulnerable to severe disease caused by other respiratory viruses. We evaluate convalescent immune responses in 4 infants under 3 months old with confirmed COVID-19 who presented with mild febrile illness, alongside their parents, and adult controls recovered from confirmed COVID-19. Although not statistically significant, compared to seropositive adults, infants have high serum levels of IgG and IgA to SARS-CoV-2 spike protein, with a corresponding functional ability to block SARS-CoV-2 cellular entry. Infants also exhibit robust saliva anti-spike IgG and IgA responses. Spike-specific IFN-γ production by infant peripheral blood mononuclear cells appears restrained, but the frequency of spike-specific IFN-γ- and/or TNF-α-producing T cells is comparable between infants and adults. On principal-component analysis, infant immune responses appear distinct from their parents. Robust functional antibody responses alongside restrained IFN-γ production may help protect infants from severe COVID-19.

Pharmacodynamics and pharmacokinetics of a copper intrauterine contraceptive system releasing ulipristal acetate: A randomized proof-of-concept study.

OBJECTIVES: To assess pharmacodynamic and pharmacokinetic outcomes of a novel copper (Cu) intrauterine system (IUS) releasing ulipristal acetate (UPA) in healthy women. STUDY DESIGN: In this single-blinded, randomized proof-of-concept study, ovulatory women received one of three Cu-IUSs releasing low-dose UPA (5, 20 or 40 µg/d) for 12 weeks. The study included a baseline cycle, three 4-week treatment-cycles and 2 recovery cycles. Primary outcomes included effects of the IUS on bleeding profile, ovarian function, and the occurrence of progesterone receptor modulator associated endometrial changes (PAEC). Pharmacokinetics and safety profile were secondary outcomes. We compared outcomes in treatment-cycle 3 with baseline, using generalized linear mixed models with orthogonal contrasts. RESULTS: We randomized 29 women (5 µg/d = 10, 20 µg/d = 10, 40 µg/d = 9). All had a successful IUS insertion; 27 completed the 12-week treatment period. Compared to baseline, the mean number of bleeding-only days at treatment-cycle 3 declined by 16.7% in the 5 µg/d group (3.6 vs 3.0, p = 0.66), 40.5% in the 20 µg/d group (4.2 vs 2.5, p = 0.14), and 77% in the 40 µg/d group (3.9 vs 0.9, p = 0.002). Most women reported reduction in the amount of bleeding: 4/8, 8/10, and 7/9 for the 5 µg/d, 20 µg/d, and 40 µg/d groups, respectively. During IUS use, ovulation occurred in most cycles [5 µg/d: 23/24 (96%), 20 µg/d: 26/30 (87%), 40 µg/d: 22/27 (81%)]. The frequency of PAEC at IUS removal was 1/10 (10%), 1/10 (10%) and 4/9 (44%) in the 5 µg/d, 20 µg/d, and 4 0 µg/d groups, respectively. No serious adverse events occurred. CONCLUSIONS: Reduction in bleeding, low incidence of PAEC, and no serious adverse events are reassuring findings of the novel Cu-UPA-IUS. The 20 µg/d seems the lowest dose promoting a favorable bleeding profile and limiting PAEC. IMPLICATIONS: The preliminary results of this short-term study of a novel copper intrauterine system (IUS) delivering ulipristal acetate showed reduction of bleeding, low incidence of progesterone receptor modulator associated endometrial changes, and absence of serious adverse events. By preventing copper-induced increase in bleeding, this IUS could provide a noncontraceptive benefit, especially for women with low hemoglobin.

The endometrial response to modulation of ligand-progesterone receptor pathways is reversible.

OBJECTIVE: To study the impact of the progesterone receptor modulator (PRM), ulipristal acetate (UPA), on endometrial morphology and function. DESIGN: Cross-sectional. SETTING: University Research Institute. PATIENT(S): Endometrial biopsies from 16 patients with heavy menstrual bleeding with a structurally normal uterus or in association with structural abnormalities identified on radiological imaging (fibroids, adenomyosis or a combination of fibroids and adenomyosis). INTERVENTION(S): Participants received UPA (5 mg once daily) for three 12-week courses, each separated by 4 weeks without treatment. MAIN OUTCOME MEASURE(S): Gene expression by real-time quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and digital image analysis were analyzed to investigate the endometrial impact of modulation of progesterone receptor pathways upon expression of steroid receptors, steroid metabolizing enzymes, cell proliferation, and progesterone-regulated genes in the same patients at 3 time points: before, during, and after discontinuation of PRM treatment. RESULT(S): Ulipristal acetate treatment resulted in increased messenger ribonucleic acid (mRNA) levels of steroid receptors compared with pretreatment secretory endometrium; decreased mRNA levels of 17- and 11-beta-hydroxysteroid dehydrogenases compared with pretreatment proliferative endometrium and pretreatment secretory endometrium; reduced cell proliferation compared with pretreatment proliferative endometrium; and altered mRNA levels of progesterone-regulated genes. A strong consistency between immunohistochemistry-digital image analysis and real-time quantitative reverse transcription polymerase chain reaction results was evident. Alterations in the mRNA levels and endometrial morphology returned to a pretreatment phenotype after the cessation of PRM exposure. CONCLUSION(S): The endometrial impact of the modulation of progesterone receptor pathways with PRM (UPA) treatment is reversible. CLINICAL TRIAL REGISTRATION NUMBER: Ulipristal acetate versus conventional management of heavy menstrual bleeding (UCON) trial (EudraCT 2014-003408-65; REC14/LO/1602).

Investigating the potential impact of post translational modification of auto-antigens by tissue transglutaminase on humoral islet autoimmunity in type 1 diabetes.

BACKGROUND: Post-translational modification (PTM) of antigens plays a role in the pathogenesis of many autoimmune disorders. In coeliac disease (CD), tissue transglutaminase (tTG) deamidates gliadin peptides to activate the immune response against the gut endomysium. CD is six times more prevalent in type 1 diabetes (T1D) patients than in the general population. HYPOTHESIS: tTG also modifies auto-antigens implicated in the pathogenesis of T1D, leading to an autoimmune response to pancreatic ß-cells. METHODS: tTG PTM was investigated in the following auto-antigens, which had been previously shown to have high importance in the development of T1D: glutamic acid decarboxylase isoform 65 (GAD65), full length islet antigen (IA-2), intracellular portion of IA-2 (IA-2ic), and both isoforms of zinc transporter 8 (ZnT8W and ZnT8R), on antibody binding. Radiolabelled antigen was incubated with tTG for 20 h at 37 °C in 100 mM Denver buffer, 3.33 nM CaCl2, at pH 7.3. Antibody binding in 20 mixed samples from the Bart's-Oxford (BOX) cohort was measured by radiobinding assay. RESULTS: Results varied between serum samples. Generally, tTG treatment of ZnT8W, ZnT8R and IA-2ic showed no significant change in antigen: autoantibody binding, while increases in binding were observed with tTG-treated GAD65 and full length IA-2. CONCLUSION: In the case of GAD65, full length IA-2, the strength of antibody: antigen binding increased after incubation with tTG. However, the exact tTG-modification events that occurred requires further elucidation.

Effects of the levonorgestrel intrauterine system on the endometrium after long-term exposure to mifepristone: Secondary outcomes of a randomized controlled trial.

OBJECTIVE: Long-term treatment with progesterone receptor modulators (PRM) is associated with a distinct histological entity termed progesterone receptor modulator associated endometrial changes (PAEC). While accumulating evidence implies that these changes are benign and reversible after cessation of treatment, there are currently no data underpinning their development. Consequently, as a precaution, endometrial shedding is recommended after long-term PRM intake. Avoiding endometrial shedding after treatment with a PRM and prior to the start of a progestin treatment would be beneficial for women in reproductive age to avoid pregnancy and bleeding. However, the endometrial morphology with such a treatment regimen is unknown. The aim of this study was to delineate the endometrial morphology following continuous long-term treatment with the PRM mifepristone and subsequent placement of a levonorgestrel intrauterine system (LNG-IUS) without prior shedding of the endometrium. STUDY DESIGN: This study reports the secondary outcome from a double-blinded randomized controlled trial conducted at Karolinska University Hospital, Sweden, November 2009 to January 2015. Healthy women aged 18-43 years with regular menstrual cycles were included. Eligible women were randomized to receive either 50 mg of mifepristone (n = 29) or a comparator (n = 29), every other day for two months followed by insertion of an LNG-IUS 52 mg. Endometrial biopsies were obtained at baseline and three months after placement of the device. The samples were histologically assessed. The main outcome measure of this sub-study was the endometrial morphology including presence of PAEC three months after LNG-IUS insertion. RESULTS: Nine and eight paired biopsies from the mifepristone and comparator group, respectively, were included in the histological analysis. There were no differences in baseline characteristics between the groups and all baseline endometrial biopsies were physiological. Three months after LNG-IUS placement the endometrial morphology was still benign without PAEC in all samples treated with either mifepristone or comparator. A progestin effect on the endometrium was seen in all samples. CONCLUSIONS: Placement of an LNG-IUS immediately following two months' treatment with the PRM mifepristone, without any prior shedding of the endometrium, may represent a feasible approach in terms of endometrial safety. However, larger studies are needed to confirm our results.

Type 1 diabetes in Africa: an immunogenetic study in the Amhara of North-West Ethiopia.

AIMS/HYPOTHESIS: We aimed to characterise the immunogenic background of insulin-dependent diabetes in a resource-poor rural African community. The study was initiated because reports of low autoantibody prevalence and phenotypic differences from European-origin cases with type 1 diabetes have raised doubts as to the role of autoimmunity in this and similar populations. METHODS: A study of consecutive, unselected cases of recently diagnosed, insulin-dependent diabetes (n = 236, ≤35 years) and control participants (n = 200) was carried out in the ethnic Amhara of rural North-West Ethiopia. We assessed their demographic and socioeconomic characteristics, and measured non-fasting C-peptide, diabetes-associated autoantibodies and HLA-DRB1 alleles. Leveraging genome-wide genotyping, we performed both a principal component analysis and, given the relatively modest sample size, a provisional genome-wide association study. Type 1 diabetes genetic risk scores were calculated to compare their genetic background with known European type 1 diabetes determinants. RESULTS: Patients presented with stunted growth and low BMI, and were insulin sensitive; only 15.3% had diabetes onset at ≤15 years. C-peptide levels were low but not absent. With clinical diabetes onset at ≤15, 16-25 and 26-35 years, 86.1%, 59.7% and 50.0% were autoantibody positive, respectively. Most had autoantibodies to GAD (GADA) as a single antibody; the prevalence of positivity for autoantibodies to IA-2 (IA-2A) and ZnT8 (ZnT8A) was low in all age groups. Principal component analysis showed that the Amhara genomes were distinct from modern European and other African genomes. HLA-DRB1*03:01 (p = 0.0014) and HLA-DRB1*04 (p = 0.0001) were positively associated with this form of diabetes, while HLA-DRB1*15 was protective (p < 0.0001). The mean type 1 diabetes genetic risk score (derived from European data) was higher in patients than control participants (p = 1.60 × 10-7). Interestingly, despite the modest sample size, autoantibody-positive patients revealed evidence of association with SNPs in the well-characterised MHC region, already known to explain half of type 1 diabetes heritability in Europeans. CONCLUSIONS/INTERPRETATION: The majority of patients with insulin-dependent diabetes in rural North-West Ethiopia have the immunogenetic characteristics of autoimmune type 1 diabetes. Phenotypic differences between type 1 diabetes in rural North-West Ethiopia and the industrialised world remain unexplained.

Hierarchical Order of Distinct Autoantibody Spreading and Progression to Type 1 Diabetes in the TEDDY Study.

OBJECTIVE: The first-appearing ß-cell autoantibody has been shown to influence risk of type 1 diabetes (T1D). Here, we assessed the risk of autoantibody spreading to the second-appearing autoantibody and further progression to clinical disease in The Environmental Determinants of Diabetes in the Young (TEDDY) study. RESEARCH DESIGN AND METHODS: Eligible children with increased HLA-DR-DQ genetic risk for T1D were followed quarterly from age 3 months up to 15 years for development of a single first-appearing autoantibody (GAD antibody [GADA], insulin autoantibody [IAA], or insulinoma antigen-2 autoantibody [IA-2A]) and subsequent development of a single second-appearing autoantibody and progression to T1D. Autoantibody positivity was defined as positivity for a specific autoantibody at two consecutive visits confirmed in two laboratories. Zinc transporter 8 autoantibody (ZnT8A) was measured in children who developed another autoantibody. RESULTS: There were 608 children who developed a single first-appearing autoantibody (IAA, n = 282, or GADA, n = 326) with a median follow-up of 12.5 years from birth. The risk of a second-appearing autoantibody was independent of GADA versus IAA as a first-appearing autoantibody (adjusted hazard ratio [HR] 1.12; 95% CI 0.88-1.42; P = 0.36). Second-appearing GADA, IAA, IA-2A, or ZnT8A conferred an increased risk of T1D compared with children who remained positive for a single autoantibody, e.g., IAA or GADA second (adjusted HR 6.44; 95% CI 3.78-10.98), IA-2A second (adjusted HR 16.33; 95% CI 9.10-29.29; P < 0.0001), or ZnT8A second (adjusted HR 5.35; 95% CI 2.61-10.95; P < 0.0001). In children who developed a distinct second autoantibody, IA-2A (adjusted HR 3.08; 95% CI 2.04-4.65; P < 0.0001) conferred a greater risk of progression to T1D as compared with GADA or IAA. Additionally, both a younger initial age at seroconversion and shorter time to the development of the second-appearing autoantibody increased the risk for T1D. CONCLUSIONS: The hierarchical order of distinct autoantibody spreading was independent of the first-appearing autoantibody type and was age-dependent and augmented the risk of progression to T1D.

Slow progressors to type 1 diabetes lose islet autoantibodies over time, have few islet antigen-specific CD8+ T cells and exhibit a distinct CD95hi B cell phenotype.

AIMS/HYPOTHESIS: The aim of this study was to characterise islet autoantibody profiles and immune cell phenotypes in slow progressors to type 1 diabetes. METHODS: Immunological variables were compared across peripheral blood samples obtained from slow progressors to type 1 diabetes, individuals with newly diagnosed or long-standing type 1 diabetes, and healthy individuals. Polychromatic flow cytometry was used to characterise the phenotypic attributes of B and T cells. Islet autoantigen-specific B cells were quantified using an enzyme-linked immunospot (ELISpot) assay and islet autoantigen-specific CD8+ T cells were quantified using peptide-HLA class I tetramers. Radioimmunoassays were used to detect islet autoantibodies. Sera were assayed for various chemokines, cytokines and soluble receptors via ELISAs. RESULTS: Islet autoantibodies were lost over time in slow progressors. Various B cell subsets expressed higher levels of CD95 in slow progressors, especially after polyclonal stimulation, compared with the corresponding B cell subsets in healthy donors (p < 0.05). The phenotypic characteristics of CD4+ and CD8+ T cells were similar in slow progressors and healthy donors. Lower frequencies of CD4+ T cells with a central memory phenotype (CD27int, CD127+, CD95int) were observed in slow progressors compared with healthy donors (mean percentage of total CD4+ T cells was 3.00% in slow progressors vs 4.67% in healthy donors, p < 0.05). Autoreactive B cell responses to proinsulin were detected at higher frequencies in slow progressors compared with healthy donors (median no. of spots was 0 in healthy donors vs 24.34 in slow progressors, p < 0.05) in an ELISpot assay. Islet autoantigen-specific CD8+ T cell responses were largely absent in slow progressors and healthy donors. Serum levels of DcR3, the decoy receptor for CD95L, were elevated in slow progressors compared with healthy donors (median was 1087 pg/ml in slow progressors vs 651 pg/ml in healthy donors, p = 0.06). CONCLUSIONS/INTERPRETATION: In this study, we found that slow progression to type 1 diabetes was associated with a loss of islet autoantibodies and a distinct B cell phenotype, consistent with enhanced apoptotic regulation of peripheral autoreactivity via CD95. These phenotypic changes warrant further studies in larger cohorts to determine their functional implications.