Marker Of Cell Function Research Articles

7316 A Unique Presentation of Diabetes Mellitus in an 8-Year-Old Male Positive For 3 Different Mutations Related to MODY

Abstract Disclosure: R. Senguttuvan: None. A. Pamfil: None. Background: Maturity-Onset Diabetes of the Young (MODY) is the most common form of inherited non-autoimmune diabetes mellitus, characterized by autosomal dominant inheritance, young age at onset and beta cell dysfunction. There are at least 14 MODY mutations and we present a pediatric case not meeting criteria for type 1[T1DM] or type 2 diabetes mellitus[T2DM], who tested positive for mutations in 3 different genes known to cause MODY. Case: An 8-year-old male, born full term, 2776 grams and 48.3 cm, without history of neonatal hypoglycemia or hyperglycemia, was referred for new onset diabetes mellitus management. While undergoing evaluation for abdominal pain and constipation, he was found to have a fasting glucose of 126 mg/dL (7 mmol/L) and hemoglobin A1C of 6.5%. He endorsed polyuria, but no polydipsia. He had no clinical signs of insulin resistance (e.g., acanthosis nigricans) and a BMI of 23.65 kg/m2, at the 97th percentile. Family history is positive for diabetes mellitus requiring insulin, in father (diagnosed at 11 years) and paternal grandmother and T2DM in paternal aunt. Laboratory evaluation was negative for all 5 antibodies (GAD, IA-2, Anti-islet cell antibody, Zinc Transporter 8 and Insulin antibodies) and revealed an insulin level of 15.9 uIU/mL (RR: <2.0-13.0) and C peptide: 2.3 ng/mL (RR: 1.1-4.4). Lacking diagnostic criteria for T1DM or T2DM, genetic testing was performed. Patient tested heterozygous for mutations in 3 genes known to cause MODY: GCK [(c.350 G>T p. (G117V)], ABCC8 [(c.2270 C>G p.(T757R)], and HNF1B [(c.68A>G p.(K23R)], all variant of uncertain significance. Over an 8-month period, patient's weight decreased from 98 lbs. (44.9 kg) to 90 lbs. (41.1 kg) after meeting with our dietician and implementing healthy dietary changes, and his beta cell function markers improved from diagnosis: Fasting insulin (4.5 uIU/mL), C peptide (1.3 ng/mL), and glucose 119 mg/dL; Hemoglobin A1C remained relatively stable at 6.1%. A kidney ultrasound was normal. No medical intervention were started yet, and patient is being monitored clinically and biochemically every 3-4 months. Discussion: Clinical monitoring over time, genetic testing in the parents, are critical in this patient’s long-term treatment plan and prognosis. To our knowledge, this may be the first case of diabetes mellitus in a pediatric patient, positive for mutations in 3 different genes known to cause MODY. South Texas has one of the highest incidences of T2DM in the USA. In addition to screening for genetic forms of diabetes, in patients with negative T1DM antibodies and atypical phenotype for T2DM, we should consider genetic screening in mildly obese pediatric and young adult patients without significant acanthosis nigricans, who were already diagnosed as T2DM. Presentation: 6/1/2024

ImmCellTyper facilitates systematic mass cytometry data analysis for deep immune profiling.

Mass cytometry is a cutting-edge high-dimensional technology for profiling marker expression at the single-cell level, advancing clinical research in immune monitoring. Nevertheless, the vast data generated by cytometry by time-of-flight (CyTOF) poses a significant analytical challenge. To address this, we describe ImmCellTyper (https://github.com/JingAnyaSun/ImmCellTyper), a novel toolkit for CyTOF data analysis. This framework incorporates BinaryClust, an in-house developed semi-supervised clustering tool that automatically identifies main cell types. BinaryClust outperforms existing clustering tools in accuracy and speed, as shown in benchmarks with two datasets of approximately 4 million cells, matching the precision of manual gating by human experts. Furthermore, ImmCellTyper offers various visualisation and analytical tools, spanning from quality control to differential analysis, tailored to users' specific needs for a comprehensive CyTOF data analysis solution. The workflow includes five key steps: (1) batch effect evaluation and correction, (2) data quality control and pre-processing, (3) main cell lineage characterisation and quantification, (4) in-depth investigation of specific cell types; and (5) differential analysis of cell abundance and functional marker expression across study groups. Overall, ImmCellTyper combines expert biological knowledge in a semi-supervised approach to accurately deconvolute well-defined main cell lineages, while maintaining the potential of unsupervised methods to discover novel cell subsets, thus facilitating high-dimensional immune profiling.

Evaluation of Regulatory B Cells and Serum IL-10 Concentration in Peripheral Blood of Women with Recurrent Pregnancy Loss

Background: Although the role of B cells in normal pregnancy has been recently highlighted, their importance and function are not completely clarified. Until now, some investigations have shown that during pregnancy, regulatory B cells (Breg), a subset of B cells, are one of the key players in immune regulation by both producing IL-10 and cell-cell interactions. Therefore, any decrease in the number or function of these cells may lead to recurrent pregnancy loss (RPL). Thus, the objective of this study was to characterize Breg cell frequency and function in women who suffered from RPL in comparison with healthy non-pregnant and pregnant women (under twenty weeks of gestational age) as controls. Method: In this study, peripheral blood samples of women suffering from RPL (n=8), women with normal pregnancy under 20 weeks of gestational age (n=14), and healthy nonpregnant women (n=10) were collected. The frequency of Breg cells (CD19+CD24hiCD38hi) was measured by flow cytometry. The serum level of the IL-10 cytokine, as a marker of Breg cell function, was measured by ELISA. Results: The Percentage of Breg cells in women who suffered from RPL was significantly lower than that of women who had normal pregnancies (P=0.0016). The percentages of Breg cells in women who suffered from RPL were also significantly lower than in non-pregnant women (P=0.0001). Furthermore, no significant differences were observed in Breg cell percentages between normal pregnant and non-pregnant women. Evaluation of IL-10 concentration in the serum of women who had participated in this study showed no significant differences between the three groups. Conclusion: Based on our results, the number of Breg cells was significantly lower in RPL women than in healthy non-pregnant and normal-pregnant women, which shows the significance of these cells in the maintenance of normal pregnancy. However, we could not detect significant differences in the serum levels of IL-10, bringing to mind the notion that the beneficial and supportive function of these cells during pregnancy might be independent of IL-10 secretion. by these cells. Thus, screening of Breg cells in women with pregnancy complications, especially RPL, could be helpful for predicting a healthy pregnancy.

Simple methodology to score micropattern quality and effectiveness.

Micropatterns (MPs) are widely used as a powerful tool to control cell morphology and phenotype. However, methods for determining the effectiveness of how well cells are controlled by the shape of MPs have been inconsistently used and studies rarely report on this topic, indicating lack of standardization. We introduce an evaluation score that quantitatively assesses the MP fabrication quality and effectiveness, which can be broadly used in conjunction with all currently available MP design types. This score uses four simple and quick steps: (i) scoring MP and (ii) background fabrication quality, (iii) defining the type(s) of MP of interest, and (iv) assigning so-called efficiency descriptors describing cell behavior. These steps are based on visual inspection and quick categorization of various aspects of MP fabrication quality and cell behaviour, presented in illustrations and microscopy image examples intended to serve as a reference "atlas". To illustrate the advantage of using this score, we determined differences in cell morphology and F-actin intensity between scored vs. non-scored cells. These measurements, which could be different in other studies, were chosen because both are understood as markers of cell phenotype and function. We combined intensity-calibrated immunofluorescence microscopy and image-based single cell protein analysis. Importantly, significant differences in cell morphology and cytoskeletal protein content between scored vs. non-scored cells were noted: the unconditional inclusion of all experimental read-outs (i.e., all MP data regardless of MP quality and effectiveness) into the final results significantly misjudged the experimental readouts vs. only including experimental read-outs of quality-controlled and effective MPs, identified by scoring. Specifically, non-scoring underestimated the F-actin intensity per cell and quantitative cellular morphometric descriptors circularity and solidity and overestimated aspect ratio. Scoring improved the precision of cellular readouts, advocating the use of a MP quality and efficiency score as a quantitative decision-supporting tool in deciding whether or not particular MPs should be used for experiments, saving time and money. This simple scoring methodology can be used for improving MP fabrication, comparing results across studies, benefiting basic science studies and potential future clinical use of MPs by introducing standardization.

Polygenic polymorphism is associated with NKG2A repertoire and influences lymphocyte phenotype and function

AbstractCD94/NKG2A is a heterodimeric receptor commonly found on natural killer (NK) and T cells, and its interaction with its ligand HLA-E on adjacent cells leads to inhibitory signaling and cell suppression. We have identified several killer cell lectin–like receptor (KLR)C1 (NKG2A) single nucleotide polymorphisms (SNPs) that are associated with NKG2A expression on NK cells, CD8+ T cells, and Vγ9/Vδ2+ T cells. Additionally, due to strong linkage disequilibrium, polymorphisms in KLRC2 (NKG2C) and KLRK1 (NKG2D) are also associated with NKG2A surface density and frequency. NKG2A surface expression correlates with single-cell NK responsiveness, and NKG2A+ NK cell frequency is associated with total NK repertoire response and inhibitability, making the identification of SNPs responsible for expression and frequency important for predicting the innate immune response. Because HLA-E expression is dependent on HLA class I signal peptides, we analyzed the relationship between peptide abundance and HLA-E expression levels. Our findings revealed a strong association between peptide availability and HLA-E expression. We identified the HLA-C killer immunoglobulin–like receptor ligand epitope as a predictive marker for HLA-ABC expression, with the HLA-C1 epitope associated with high HLA-E expression and the HLA-C2 epitope associated with low HLA-E expression. The relationship between HLA-C epitopes and HLA-E expression was independent of HLA-E allotypes and HLA-B leader peptides. Although HLA-E expression showed no significant influence on NKG2A-mediated NK education, it did affect NK cell inhibition. In summary, these findings underscore the importance of NKG2A SNPs and HLA-C epitopes as predictive markers of NK cell phenotype and function and should be evaluated as prognostic markers for diseases that express high levels of HLA-E.

Reduced anti-Müllerian hormone levels in males with inherited bone marrow failure syndromes.

Fanconi anemia (FA), dyskeratosis congenita-related telomere biology disorders (DC/TBD), and Diamond-Blackfan anemia (DBA) are inherited bone marrow failure syndromes (IBMFS) with high risks of bone marrow failure, leukemia, and solid tumors. Individuals with FA have reduced fertility. Previously, we showed low levels of anti-Müllerian hormone (AMH), a circulating marker of ovarian reserve, in females with IBMFS. In males, AMH may be a direct marker of Sertoli cell function and an indirect marker of spermatogenesis. In this study, we assessed serum AMH levels in pubertal and postpubertal males with FA, DC/TBD, or DBA and compared this with their unaffected male relatives and unrelated healthy male volunteers. Males with FA had significantly lower levels of AMH (median: 5 ng/mL, range: 1.18-6.75) compared with unaffected male relatives (median: 7.31 ng/mL, range: 3.46-18.82, P = 0.03) or healthy male volunteers (median: 7.66 ng/mL, range: 3.3-14.67, P = 0.008). Males with DC/TBD had lower levels of AMH (median: 3.76 ng/mL, range: 0-8.9) compared with unaffected relatives (median: 5.31 ng/mL, range: 1.2-17.77, P = 0.01) or healthy volunteers (median: 5.995 ng/mL, range: 1.57-14.67, P < 0.001). Males with DBA had similar levels of AMH (median: 3.46 ng/mL, range: 2.32-11.85) as unaffected relatives (median: 4.66 ng/mL, range: 0.09-13.51, P = 0.56) and healthy volunteers (median: 5.81 ng/mL, range: 1.57-14.67, P = 0.10). Our findings suggest a defect in the production of AMH in postpubertal males with FA and DC/TBD, similar to that observed in females. These findings warrant confirmation in larger prospective studies.

Role of Anti-Müllerian Hormone in Male Reproduction and Sperm Motility.

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells and is responsible for the regression of Müllerian ducts in the male fetus as part of the sexual differentiation process. Serum AMH concentrations are at their lowest levels in the first days after birth but increase after the first week, likely reflecting active Sertoli cell proliferation. AMH rises rapidly in concentration in boys during the first month, reaching a peak level at ∼6 months of age, and it remains high during childhood, then they will slowly decline during puberty, falling to low levels in adulthood. Serum AMH measurement is used by pediatric endocrinologist as a specific marker of immature Sertoli cell number and function during childhood. After puberty, AMH is released especially by the apical pole of the Sertoli cells toward the lumen of the seminiferous tubules, resulting in higher levels in the seminal plasma than in the serum. Recently, AMH has received increasing attention in research on male fertility-related disorders. This article reviews and summarizes the potential contribution of serum AMH measurement in different male fertility-related disorders.

Tirzepatide Improved Markers of Islet Cell Function (Fasting Glucagon and HOMA2-B) and Insulin Sensitivity (Fasting Insulin and HOMA2-IR) Compared to Semaglutide in People with Type 2 Diabetes

Tirzepatide Improved Markers of Islet Cell Function (Fasting Glucagon and HOMA2-B) and Insulin Sensitivity (Fasting Insulin and HOMA2-IR) Compared to Semaglutide in People with Type 2 Diabetes

Abstract 3423: Spatial-resolved single-cell analysis of the tumor microenvironment in Alaska Native colorectal cancer patients

Abstract Tribal Health Organizations recognize the high rates of colorectal cancer (CRC) among Alaska Native peoples and are undertaking initiatives to address it. The tumor microenvironment (TME) is a complex ecosystem including tumor, stromal, and immune cells. Understanding the cell composition and spatial organization of the TME among Alaska Native patients with CRC will provide new insights into disease progression. We performed spatial profiling on 3 tissue microarrays (TMA) from 37 patients using the Akoya Biosciences’ s PhenoCycler system. Patients with CRC were selected from a nested case-control study which includes 16 patients who died of CRC and 21 patients who lived as long as patients with lethal CRC and matched on age at diagnosis, sex, tumor site and tumor stage. We designed a 40-antibody panel that captured tumor, epithelium, stromal, vascular, and immune cells, as well as cell functional states (e.g., PD1). Initial images were processed using QuPath for image stitching, artifact removal, background subtraction, cell segmentation, and measurement of average intensity of each marker per cell. Cell quality control (QC) was performed to filter out low quality cells based on cell size and log10-transformed sum of intensity. After quantile normalization and arcsinh transformation, cells that passed QC were clustered using the R package ‘Seurat’ and manually annotated based on the cell type and function marker intensities for each cluster. We identified 1.17 million cells and 15 cell types. Those cell types included 3 stromal and vascular cells, 1 epithelium, 1 mixed immune cluster, and 10 different immune cells. We quantified each cell type as a fraction of total cells on a per-patient basis. Among the subset of immune cells, the proportions of macrophages, CD4+T cells, and CD8+T cells were high (21%, 11.2%, and 14.5%, respectively), and the proportion of B cells was low (5.1%). We also identified regulatory T cells, cytotoxic CD8+T cells, and monocytes at 1-3%. We observed differences in the composition of cell clusters by CRC-specific death. The frequency of epithelium was higher among patients with lethal CRC, and the frequency of CD4+T cells and CD8+T cells were higher among patients without lethal CRC. In summary, the overall composition of tumor and immune cells varies between patients with and without CRC-specific death, indicating TME heterogeneity. Further investigation of spatial domains and relationships with clinical molecular features may facilitate discovery of novel predictors of CRC-specific death among Alaska Native peoples. We are clustering cell types into different cellular neighborhoods using spatial information and conducting statistical analysis integrating RNA sequencing data from the same patients. Further, we are generating spatial profiling data for an additional 5 TMAs comprising 60 additional patients and will present results of the combined data analyses. Citation Format: Hang Yin, Diana Redwood, Kimberly Smythe, Daniel Jones, McGarry Houghton, Kevin C. Barry, Amanda L. Koehne, Elizabeth Donato, Cecilia Yeung, Mingang Lin, James J. Tiesinga, Tabitha A. Harrison, Sushma S. Thomas, Li Hsu, Jane C. Figueiredo, Li Li, Timothy K. Thomas, Christopher Li, Ulrike Peters, Jeroen R. Huyghe. Spatial-resolved single-cell analysis of the tumor microenvironment in Alaska Native colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3423.

Comprehensive analysis of lung macrophages and dendritic cells in two murine models of allergic airway inflammation reveals model- and subset-specific accumulation and phenotypic alterations.

Allergic asthma has been mainly attributed to T helper type 2 (Th2) and proinflammatory responses but many cellular processes remain elusive. There is increasing evidence for distinct roles for macrophage and dendritic cell (DC) subsets in allergic airway inflammation (AAI). At the same time, there are various mouse models for allergic asthma that have been of utmost importance in identifying key inflammatory pathways in AAI but that differ in the allergen and/or route of sensitization. It is unclear whether and how the accumulation and activation of specialized macrophage and DC subsets depend on the experimental model chosen for analyses. In our study, we employed high-parameter spectral flow cytometry to comprehensively assess the accumulation and phenotypic alterations of different macrophage- and DC-subsets in the lung in an OVA- and an HDM-mediated mouse model of AAI. We observed subset-specific as well as model-specific characteristics with respect to cell numbers and functional marker expression. Generally, alveolar as opposed to interstitial macrophages showed increased MHCII surface expression in AAI. Between the models, we observed significantly increased numbers of alveolar macrophages, CD103+ DC and CD11b+ DC in HDM-mediated AAI, concurrent with significantly increased airway interleukin-4 but decreased total serum IgE levels. Further, increased expression of CD80 and CD86 on DC was exclusively detected in HDM-mediated AAI. Our study demonstrates a model-specific involvement of macrophage and DC subsets in AAI. It further highlights spectral flow cytometry as a valuable tool for their comprehensive analysis under inflammatory conditions in the lung.

Symptomatic post COVID patients have impaired alveolar capillary membrane function and high VE/VCO2

BackgroundPost COVID-19 syndrome is characterized by several cardiorespiratory symptoms but the origin of patients’ reported symptomatology is still unclear.MethodsConsecutive post COVID-19 patients were included. Patients underwent full clinical evaluation, symptoms dedicated questionnaires, blood tests, echocardiography, thoracic computer tomography (CT), spirometry including alveolar capillary membrane diffusion (DM) and capillary volume (Vcap) assessment by combined carbon dioxide and nitric oxide lung diffusion (DLCO/DLNO) and cardiopulmonary exercise test. We measured surfactant derive protein B (immature form) as blood marker of alveolar cell function.ResultsWe evaluated 204 consecutive post COVID-19 patients (56.5 ± 14.5 years, 89 females) 171 ± 85 days after the end of acute COVID-19 infection. We measured: forced expiratory volume (FEV1) 99 ± 17%pred, FVC 99 ± 17%pred, DLCO 82 ± 19%, DM 47.6 ± 14.8 mL/min/mmHg, Vcap 59 ± 17 mL, residual parenchymal damage at CT 7.2 ± 3.2% of lung tissue, peakVO2 84 ± 18%pred, VE/VCO2 slope 112 [102–123]%pred. Major reported symptoms were: dyspnea 45% of cases, tiredness 60% and fatigability 77%. Low FEV1, Vcap and high VE/VCO2 slope were associated with persistence of dyspnea. Tiredness was associated with high VE/VCO2 slope and low PeakVO2 and FEV1 while fatigability with high VE/VCO2 slope. SPB was fivefold higher in post COVID-19 than in normal subjects, but not associated to any of the referred symptoms. SPB was negatively associated to Vcap.ConclusionsIn patients with post COVID-19, cardiorespiratory symptoms are linked to VE/VCO2 slope. In these patients the alveolar cells are dysregulated as shown by the very high SPB. The Vcap is low likely due to post COVID-19 pulmonary endothelial/vasculature damage but DLCO is only minimally impaired being DM preserved.

Tirzepatide Improved Markers of Islet Cell Function and Insulin Sensitivity in People With T2D (SURPASS-2).

In previous SURPASS studies tirzepatide reduced hemoglobin glycated A1c (HbA1c) and body weight and improved markers of insulin sensitivity and β-cell function to a greater extent than comparators. Explore changes in biomarkers of β-cell function and insulin sensitivity and in efficacy profiles in baseline biomarker quartile analyses with tirzepatide compared to semaglutide. Post hoc analysis of SURPASS-2 phase 3 trial (participants randomly assigned to receive weekly subcutaneous tirzepatide or semaglutide for 40 weeks). Post hoc analysis of 128 sites in 8 countries. A total of 1879 participants with type 2 diabetes. Once-weekly tirzepatide (5, 10, 15 mg) or semaglutide 1 mg. Change in homeostatic model assessment indices for pancreatic β-cell function (HOMA2-B) and for insulin resistance (HOMA2-IR), fasting glucagon, fasting C-peptide, and fasting insulin. At week 40, a greater increase in HOMA2-B was seen with tirzepatide (5, 10, 15 mg) doses (96.9-120.4%) than with semaglutide 1 mg (84.0%) (P < .05). There was a greater reduction in HOMA2-IR with all doses of tirzepatide (15.5%-24.0%) than with semaglutide 1 mg (5.1%) (P < .05). Tirzepatide 10 and 15 mg resulted in a significant reduction in both fasting C-peptide (5.2%-6.0%) and fasting glucagon (53.0%-55.3%) compared with an increase of C-peptide (3.3%) and a reduction of glucagon (47.7%) with semaglutide 1 mg (P < .05). HbA1c and body weight reductions were greater with all tirzepatide doses than semaglutide within each HOMA2-B and HOMA2-IR baseline quartile. In this post hoc analysis, improvements in HbA1c and weight loss were consistent and significantly higher with tirzepatide, regardless of baseline β-cell function and insulin resistance, compared with semaglutide.

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

TMEM219 regulates the transcription factor expression and proliferation of beta cells.

Pancreatic beta cells replenishment is considered the next therapeutic option for type 1 diabetes; while stimulating endogenous beta cells proliferation is the "holy grail" for those patients with exhausted beta cell mass. Here we are demonstrating that the pro-apoptotic receptor TMEM219 is expressed in fetal pancreas, in beta cell precursors and in in vitro embryonic-derived endocrine progenitors. TMEM219 signaling negatively regulates beta cells at early stages and induces Caspase 8-mediated cell death. Pharmacological blockade of TMEM219 further rescued beta cell precursor and proliferation markers, and decreased cell death, both in islets and in in vitro-derived endocrine progenitors, allowing for beta cell preservation. While addressing the upstream controlling TMEM219 expression, we determined the TMEM219 miRNet; indeed, one of those miRNAs, miR-129-2, is highly expressed in human islets, particularly in patients at risk or with established type 1 diabetes. miR-129-2 mimic downregulated TMEM219 expression in islets, in in vitro embryonic-derived endocrine progenitors and in highly proliferating insulinoma-derived cells. Moreover, miR-129-2 inhibitor induced a TMEM219 overexpression in insulinoma-derived cells, which restored cell proliferation and functional markers, thus acting as endogenous regulator of TMEM219 expression. The TMEM219 upstream regulator miR129-2 controls the fate of beta cell precursors and may unleash their regenerative potentials to replenish beta cells in type 1 diabetes.

Investigating the Modulating Effects of Tirzepatide on NAFLD/NASH Progression Using 3D Human Liver Organoids

Background: Nonalcoholic fatty liver disease (NAFLD) is a disease characterized by the accumulation of lipids in the liver that ultimately progresses to nonalcoholic steatohepatitis (NASH) and cirrhosis. Currently, there is no known FDA-approved treatment for NASH. Tirzepatide (brand name Mounjaro), a glucagon-like peptide 1 (GLP-1) agonist, has been hypothesized to have potential effects in reversing NAFLD/NASH progression and restoring liver function. However, this hypothesis remains untested in current literature. The aim of this study is to use 3D human liver organoids (3D-HLOs) as an ex vivo model system to determine the potential effects of tirzepatide on the progression of NAFLD/NASH.&#x0D; &#x0D; Methods: 3D-HLOs were constructed by incorporating 5 major human hepatic cell types isolated from NAFLD livers, including hepatocytes, hepatic stellate cells, liver endothelial cells, cholangiocytes, and Kupffer cells. NAFLD 3D-HLOs were maintained in the hepatocyte medium supplemented with 50mM free fatty acid (FFA) only (control group) or FFA+tirzepatide (100nM, 200nM, or 500 nM) for 14 days. By the end of the treatment, 3D-HLOs were subjected to BODIPY493/503 staining to determine lipid droplet accumulation. Immunofluorescence staining and confocal microscopy analysis were performed to confirm hepatocyte function (ALBUMIN) and fibrosis (COL1A1). qPCR was performed to determine the relative expression markers of hepatocyte function (ALBUMIN, HNF4A), angiogenesis (PECAM-1, VCAM-1, ICAM-1), and fibrosis (ACTA2, COL1A1).&#x0D; &#x0D; Results: BODIPY 493/503 revealed no significant difference in lipid deposition between 3D-HLOs in all treatment groups. Mean immunofluorescence staining intensity for albumin in controls, 100nM, 200nM, and 500nM tirzepatide were 154.4, 181.4, 221.1, and 236.4, respectively (p&gt;0.05). Dose-dependent quadratic regression revealed a strong correlation between dose and albumin fluorescence intensity (R2 = 0.963). qRT-PCR analysis revealed that tirzepatide treatment did not significantly alter transcriptional levels of ALB, HNFA4, ACTA2, PECAM-1, VCAM-1, and ICAM-1 compared to the control group.&#x0D; &#x0D; Conclusion: Although there is no statistically significant difference in liver cell function markers at different tirzepatide dosages in the setting of NAFLD/NASH, the highest dose of tirzepatide improved ALB expression despite ongoing FFA exposure, indicating a potential hyperbolic relationship between tirzepatide concentration and albumin production. Future investigation with altered treatment duration and dosage will elucidate whether there are any direct modulating effects of tirzepatide on NAFLD/NASH progression.

A novel model for predicting diabetes remission after bariatric surgery based on the measurement of C-peptide and creatinine in serum: A pilot study

Background and aimsBariatric surgery is effective for treating type 2 diabetes (T2D) in patients with obesity, although a significant proportion of these patients do not achieve diabetes remission after the surgery even after significant weight loss and metabolic improvement. C-peptide is a valuable marker of beta cell function and insulin secretion, but renal function must be considered when interpreting measurements in patients with T2D. The study aims to investigate the association of serum levels of C-peptide adjusted for creatinine with diabetes remission and glycemic target achievement after bariatric surgery in patients with obesity and T2D. Methods and resultsProspective data from a cohort of 84 patients with obesity and T2D submitted to Roux-en-Y gastric bypass (RYGB) were collected at baseline and at least a 6-month follow up. A multivariate binomial regression model showed that Ln(C-peptide/creatinine) and age were significantly associated with 6-month T2D remission. The area under the curve for the receiver operating characteristic analysis (AUROC) to predict remission was 0.87, and more accurate than the AUROC based on C-peptide levels alone (0.75). The same model was also able to predict achieving an HbA1c target of 7 % (53 mmol/mol) (AUROC 0.96). ConclusionIn conclusion, Ln(C-peptide/creatinine) ratio could be a useful tool in predicting T2D remission and target achievement after RYGB surgery, providing a more accurate reflection of beta cell function in bariatric patients.

BinaryClust2: An Integrated Computational Pipeline for Systematic Mining of Mass Cytometry Data to Assist Deep Immune Profiling in Haematological Research

Introduction: The key role of immune system in modulating treatment efficacy and clinical outcomes in the settings of haematological diseases has been well recognized through the application of flow cytometry technologies over years. Mass cytometry (MC) is a next generation cytometry platform to simultaneously profile over 40 protein markers on single-cell resolution, which facilitates clinical research on immune monitoring and biomarker discovery. Nonetheless, the high-dimensional data generated present a daunting challenge for clinical investigators. Therefore, we describe BinaryClust2 (https://github.com/JingAnyaSun/BinaryClust2), an R-based computational framework integrated with the state-of-the-art algorithms and novel cell lineage characterisation method to assist comprehensive exploration of liquid MC data. Method: Here, we present the implementation of BinaryClust2 to three example MC datasets: In-house dataset from peripheral blood mononuclear cells (PBMCs) of 9 baseline myeloproliferative neoplasm (MPN) patients (~4 million cells), previously published data from 11 MPN patients receiving influenza vaccine (~0.2million cells), and published dataset of 59 covid patients and 23 healthy donors (~2million cells). BinaryClust2 has a streamlined analytical workflow, which comprises the following steps (Figure 1A): Step 1: Quality control, batch effect evaluation and correction Data quality control is a separate step before downstream analysis which includes diagnostic plots and batch effect examination. Algorithms CytofRUV and CytoNorm are available in the pipeline to remove unwanted variations caused by batch effects. Step 2: Semi-supervised identification of main cell types BinaryClust2 adopts a knowledge-based semi-supervised approach to predict main cell types. Users are required to provide a simple marker expression matrix of pre-defined cell types along with fcs files and metadata to construct a SingleCellExperiment (SCE) object, then the embedded algorithm can automatically classify cell populations without manual annotation. Step 3: In-depth interrogation and differential testing Specific population can be further extracted from whole cells and subject to in-depth exploration using unsupervised algorithms for subpopulation discovery. Dimensionality reduction tools UMAP and TSNE, unsupervised clustering methods Phenograph and flowSOM, and various data visualization plotting functions are offered in BinaryClust2. For statistical analysis, multiple study group comparison (n&amp;gt;2) of cell abundance and functional marker expression is supported via Kruskal Wallis test with multiple testing correction and post hoc analysis. Results: The performance of the semi-supervised classification function was tested independently in the MPN and influenza PBMC datasets, 7 main cell lineages were identified with accuracy comparable to manual gating by human experts (Figure 1B): average F-measure reached 0.93 and 0.98 respectively. Moreover, taking manual gating as ground truth reference, BinaryClust2 outperformed the unsupervised approach flowSOM concerning accuracy (F-measure: 0.93 vs. 0.70) and speed (140s vs 339s) in MPN dataset, while remaining equivalent to the well-performing semi-supervised approach LDA in accuracy (F-measure: 0.93 vs. 0.93) but faster in runtime (140s vs 595s), as shown in Table 1. Application to covid-19 dataset by Chevrier et al. achieved reproducible results and additional discoveries. 13 main cell types were characterised, abundance of B cells, Basophils, cDCs, DN T cells, Monocytes, Neutrophils, NK cells, pDCs, CD8 T cells obtained statistical significance (all P&amp;lt;0.05) among study conditions (healthy, mild covid, severe covid). We also grouped markers reflecting functional status of immune cells and found Granzyme B expression was significantly increased in the majority of main immune cells of covid patients. Phenograph was further applied in neutrophils and monocytes and returned 14 and 16 subsets respectively. Conclusion: Overall, BinaryClust2 incorporates expert's prior biological knowledge in a semi-supervised fashion to accurately deconvolute well-defined main cell lineages, while also preserving the potential of unsupervised approaches to discover novel cell subsets and providing a user-friendly toolset to remove the analytical barrier for high-dimensional immune profiling.

Circulating acyl-CoA-binding protein/diazepam-binding inhibitor in gestational diabetes mellitus

BackgroundAcyl-CoA-binding protein (ACBP)/diazepam-binding inhibitor has recently been characterized as an endocrine factor affecting energy balance and lipid metabolism. However, regulation of ACBP in women with gestational diabetes mellitus (GDM) during pregnancy, as well as postpartum, has not been investigated, so far.MethodsACBP was quantified in 74 women with GDM and 74 healthy, gestational age-matched, pregnant controls using an enzyme-linked immunosorbent assay. Furthermore, ACBP was quantified post-partum in 82 women (i.e. 41 women with previous GDM vs. 41 previous control women). ACBP was related to measures of obesity, hypertension, glucose and lipid metabolism, renal function, and inflammation during pregnancy and postpartum.ResultsDuring pregnancy, median [interquartile range] ACBP levels were not significantly different in women with GDM (40.9 [40.0] µg/l) compared to healthy, pregnant controls (29.1 [32.3] µg/l) (p = 0.215). ACBP serum concentrations increased from 30.3 [40.5] µg/l during pregnancy to 59.7 [33.2] µg/l after pregnancy in the entire cohort (p < 0.001). This observed elevation was consistent across both subgroups of women, those with prior GDM and those without. Multivariate analysis revealed that homeostasis model assessment of beta cell function (HOMA2-B) and creatinine positively and independently correlated with serum ACBP after pregnancy, while multivariate analysis during pregnancy showed no significant correlations.ConclusionsCirculating ACBP is not a marker of GDM status, but ACBP is decreased during pregnancy, irrespective of GDM status. Furthermore, ACBP is related to beta cell function and renal markers in women after pregnancy.

Ulipristal acetate versus levonorgestrel-releasing intrauterine system for heavy menstrual bleeding: the UCON randomised controlled trial and mechanism of action study

Background Heavy menstrual bleeding affects one in four women and negatively impacts quality of life. The levonorgestrel-releasing intrauterine system is an effective long-term treatment but is discontinued by many due to unpredictable bleeding, or adverse effects. The selective progesterone receptor modulator ulipristal acetate is used to treat symptomatic fibroids but long-term efficacy for the symptom of heavy menstrual bleeding, irrespective of presence of fibroids, is unknown. Objectives To determine whether ulipristal acetate is more effective at reducing the burden of heavy menstrual bleeding than levonorgestrel-releasing intrauterine system after 12 months of treatment in women with and without fibroids. We investigated mechanism of action of ulipristal acetate in a subset of 20 women. Design Randomised, open-label, parallel group, multicentre trial with embedded mechanistic study. Setting Ten UK hospitals. Participants Women with heavy menstrual bleeding aged 18 and over with no contraindications to levonorgestrel-releasing intrauterine system or ulipristal acetate. Interventions Three 12-week treatment cycles of 5 mg ulipristal acetate daily, separated by 4-week treatment-free intervals, or continuous levonorgestrel-releasing intrauterine system following allocation in a 1 : 1 ratio using a web-based minimisation procedure. Main trial outcome measures Primary outcome was quality-of-life measured by menorrhagia multi-attribute scale at 12 months. Secondary outcomes included menstrual bleeding and patient satisfaction. Impact on fibroid size, endometrial appearance and liver function was also collected. Mechanistic study outcome Cellular markers for endometrial cell structure and function, determined from endometrial biopsies; volume of uterus and fibroids and microcirculation parameters were determined from magnetic resonance images. Results Sample size was increased from 220 to 302 as a result of temporary halt to recruitment due to concerns of ulipristal acetate hepatoxicity. Subsequent withdrawal of ulipristal acetate and the COVID-19 pandemic led to a premature closure of recruitment, with 118 women randomised to each treatment and 103 women completing 12-month menorrhagia multi-attribute scale scores prior to this point. Primary outcome scores substantially improved in both arms, but at 12 months there was no evidence of a difference between those receiving three cycles of ulipristal acetate [median score category: 76–99, interquartile range (51–75 to 100), n = 53] and levonorgestrel-releasing intrauterine system [median score category: 76–99, interquartile range (51–75 to 100), n = 50; adjusted odds ratio 0.55, 95% confidence interval 0.26 to 1.17; p = 0.12]. Rates of amenorrhoea were much higher in those allocated ulipristal acetate compared with the levonorgestrel-releasing intrauterine system (12 months: 64% vs. 25%, adjusted odds ratio 7.12, 95% confidence interval 2.29 to 22.2). There was no evidence of a difference in other participant-reported outcomes. There were no cases of endometrial malignancy and no hepatotoxicity due to ulipristal acetate use. Mechanistic study results Ulipristal acetate produced a reversible reduction in endometrial cell proliferation, as well as reversible alteration of other endometrial cellular markers. Ulipristal acetate did not produce a reduction in the volume of the uterus irrespective of coexisting fibroids, nor an effect on uterine microvascular blood flow. Limitations The urgent safety measures and premature closure of recruitment impacted final sample size. Conclusions We found no evidence of a difference in quality of life between the two treatments, but ulipristal acetate was superior to levonorgestrel-releasing intrauterine system at inducing amenorrhoea. Ulipristal acetate currently has restricted availability due to concerns regarding hepatotoxicity. Future work There is a need to develop new, safe, effective and fertility-sparing medical treatments for heavy menstrual bleeding. The observed acceptability and effectiveness of ulipristal acetate warrants further research into the selective progesterone receptor modulator class of pharmacological agents. Study registration This trial is registered as ISRCTN 20426843.

The Association between 4-Tertiary-Octylphenol, Apoptotic Microparticles, and Carotid Intima-Media Thickness in a Young Taiwanese Population.

As one of the most common alkylphenols, 4-tertiary-octylphenol (4-tOP) is commonly used in many consumer products. Our previous epidemiological study revealed a negative correlation between serum 4-tOP levels and carotid intima-media thickness (CIMT), which serves as a biomarker of arteriosclerosis. We aimed to explore the role of apoptotic microparticles, markers of vascular endothelial cell function, in the 4-tOP and CIMT connection. To investigate this, we enrolled 886 Taiwanese adolescents and young adults (aged 12-30 years) and examined the relationships among serum 4-tOP levels, apoptotic microparticles (CD31+/CD42a-, CD31+/CD42a+), and CIMT. Our results showed negative associations among serum 4-tOP levels, both apoptotic microparticles, and CIMT in multiple linear regression analysis. The odds ratios for CIMT (≥75th percentile) and the natural logarithm of 4-tOP were highest when both CD31+/CD42a- and CD31+/CD42a+ were greater than the 50th percentile. Conversely, the odds ratios were lowest when both CD31+/CD42a- and CD31+/CD42a+ were less than the 50th percentile. In the structural equation model, we demonstrated that serum 4-tOP levels were negatively correlated with CIMT and indirectly and negatively correlated with CIMT through both apoptotic microparticles. In conclusion, our study reported the inverse association between 4-tOP apoptotic microparticles and CIMT in a young Taiwanese population. Further experimental studies are needed to clarify these associations.