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Clayton E Mathews

Publications and source records attributed to Clayton E Mathews.

16 recordsLinked to original sources
PubMed2026

A murine model of sepsis induces age- and sex-specific chromatin remodeling in myeloid-derived suppressor cells.

INTRODUCTION: Sepsis survivors frequently develop long-term immune dysfunction, but the epigenetic mechanisms underlying persistent myeloid suppression remain unclear. Myeloid-derived suppressor cells (MDSCs), whose function is shaped by host age and sex, are key contributors to post-sepsis immune dysregulation. METHODS: Here, we present a high-resolution epigenetic map targeting gene promoters of MDSCs after sepsis and daily chronic stress using MAPit-FENGC, a single-molecule assay that simultaneously profiles DNA methylation and chromatin accessibility. In a clinically relevant murine model, including young and older adult male and female mice, splenic MDSCs were isolated for MAPit-FENGC and single-cell RNA sequencing. RESULTS: Unsupervised clustering identified nine promoter classes reflecting chromatin dynamics: age- and sex-dependent sepsis-induced opening (Classes 1-4), persistent closure with varying levels of DNA methylation (Classes 5-7), and constitutive openness post-sepsis (Classes 8, 9). Transcriptomic profiling corroborated these promoter states, linking accessibility with gene expression. CONCLUSIONS: These findings define promoter-level epigenetic classes across a targeted locus panel in splenic CD11b+Gr1+ cells within this murine sepsis model and generate mechanistic hypotheses regarding age- and sex-associated chromatin states.

Animals↗
PubMed2024

Proteome-Scale Tissue Mapping Using Mass Spectrometry Based on Label-Free and Multiplexed Workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput. Since many of these existing challenges are associated with mass spectrometry-based protein identification and quantification, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ∼3500 proteins at a spatial resolution of 50 μm and the highest quantification dynamic range, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provides robust protein quantifications in identifying differentially abundant proteins and spatially covariable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables the identification of protein markers specific to different cell types, but more importantly, it also reveals unknown or hidden protein patterns by spatial coexpression analysis.

Proteome↗
PubMed2024

Proteome-scale tissue mapping using mass spectrometry based on label-free and multiplexed workflows.

Multiplexed bimolecular profiling of tissue microenvironment, or spatial omics, can provide deep insight into cellular compositions and interactions in healthy and diseased tissues. Proteome-scale tissue mapping, which aims to unbiasedly visualize all the proteins in a whole tissue section or region of interest, has attracted significant interest because it holds great potential to directly reveal diagnostic biomarkers and therapeutic targets. While many approaches are available, however, proteome mapping still exhibits significant technical challenges in both protein coverage and analytical throughput. Since many of these existing challenges are associated with mass spectrometry-based protein identification and quantification, we performed a detailed benchmarking study of three protein quantification methods for spatial proteome mapping, including label-free, TMT-MS2, and TMT-MS3. Our study indicates label-free method provided the deepest coverages of ~3500 proteins at a spatial resolution of 50 µm and the highest quantification dynamic range, while TMT-MS2 method holds great benefit in mapping throughput at >125 pixels per day. The evaluation also indicates both label-free and TMT-MS2 provide robust protein quantifications in identifying differentially abundant proteins and spatially co-variable clusters. In the study of pancreatic islet microenvironment, we demonstrated deep proteome mapping not only enables the identification of protein markers specific to different cell types, but more importantly, it also reveals unknown or hidden protein patterns by spatial co-expression analysis.

Journal Article↗
PubMed2006

Nuclear and mitochondrial interaction involving mt-Nd2 leads to increased mitochondrial reactive oxygen species production.

NADH dehydrogenase subunit 2, encoded by the mtDNA, has been associated with resistance to autoimmune type I diabetes (T1D) in a case control study. Recently, we confirmed a role for the mouse ortholog of the protective allele (mt-Nd2(a)) in resistance to T1D using genetic analysis of outcrosses between T1D-resistant ALR and T1D-susceptible NOD mice. We sought to determine the mechanism of disease protection by elucidating whether mt-Nd2(a) affects basal mitochondrial function or mitochondrial function in the presence of oxidative stress. Two lines of reciprocal conplastic mouse strains were generated: one with ALR nuclear DNA and NOD mtDNA (ALR.mt(NOD)) and the reciprocal with NOD nuclear DNA and ALR mtDNA (NOD.mt(ALR)). Basal mitochondrial respiration, transmembrane potential, and electron transport system enzymatic activities showed no difference among the strains. However, ALR.mt(NOD) mitochondria supported by either complex I or complex II substrates produced significantly more reactive oxygen species when compared with both parental strains, NOD.mt(ALR) or C57BL/6 controls. Nitric oxide inhibited respiration to a similar extent for mitochondria from the five strains due to competitive antagonism with molecular oxygen at complex IV. Superoxide and hydrogen peroxide generated by xanthine oxidase did not significantly decrease complex I function. The protein nitrating agents peroxynitrite or nitrogen dioxide radicals significantly decreased complex I function but with no significant difference among the five strains. In summary, mt-Nd2(a) does not confer elevated resistance to oxidative stress; however, it plays a critical role in the control of the mitochondrial reactive oxygen species production.

Alleles↗
PubMed2006

Apoptotic cells induce Mer tyrosine kinase-dependent blockade of NF-kappaB activation in dendritic cells.

Dendritic cells (DCs) play a key role in immune homeostasis and maintenance of self-tolerance. Tolerogenic DCs can be established by an encounter with apoptotic cells (ACs) and subsequent inhibition of maturation and effector functions. The receptor(s) and signaling pathway(s) involved in AC-induced inhibition of DCs have yet to be defined. We demonstrate that pretreatment with apoptotic but not necrotic cells inhibits activation of IkappaB kinase (IKK) and downstream NF-kappaB. Notably, receptor tyrosine kinase Mer (MerTK) binding of ACs is required for mediating this effect. Monocyte-derived DCs lacking MerTK expression (MerTKKD) or treated with blocking MerTK-specific antibodies (Abs) are resistant to AC-induced inhibition and continue to activate NF-kappaB and secrete proinflammatory cytokines. Blocking MerTK activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway prevents AC-induced inhibition. These results demonstrate an essential role for MerTK-mediated regulation of the PI3K/AKT and NF-kappaB pathways in AC-induced inhibition of monocyte-derived DCs.

Animals↗
PubMed2006

HLA-A*0201-restricted T cells from humanized NOD mice recognize autoantigens of potential clinical relevance to type 1 diabetes.

In both humans and NOD mice, particular MHC genes are primary contributors to development of the autoreactive CD4+ and CD8+ T cell responses against pancreatic beta cells that cause type 1 diabetes (T1D). Association studies have suggested, but not proved, that the HLA-A*0201 MHC class I variant is an important contributor to T1D in humans. In this study, we show that transgenic expression in NOD mice of HLA-A*0201, in the absence of murine class I MHC molecules, is sufficient to mediate autoreactive CD8+ T cell responses contributing to T1D development. CD8+ T cells from the transgenic mice are cytotoxic to murine and human HLA-A*0201-positive islet cells. Hence, the murine and human islets must present one or more peptides in common. Islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) is one of several important T1D autoantigens in standard NOD mice. Three IGRP-derived peptides were identified as targets of diabetogenic HLA-A*0201-restricted T cells in our NOD transgenic stock. Collectively, these results indicate the utility of humanized HLA-A*0201-expressing NOD mice in the identification of T cells and autoantigens of potential relevance to human T1D. In particular, the identified antigenic peptides represent promising tools to explore the potential importance of IGRP in the development of human T1D.

Alleles↗
PubMed2006

Modulatory role of DR4- to DQ8-restricted CD4 T-cell responses and type 1 diabetes susceptibility.

This study addressed an important biological question, namely how certain HLA molecules modulate the disease risk conferred by other HLA molecules. The HLA molecules under investigation were HLA-DQ8 and -DR4, the two most prevalent HLA class II alleles found in Caucasian type 1 diabetic patients. A panel of human GAD (hGAD65)-specific CD4 T-cell lines and hybridomas was generated to serve as detection reagents for evaluating the peptide occupancy of DQ8 and DR4. Results indicated that DQ8 and DR4 (0401) were able to bind the same hGAD65 peptides. The coexpression of DR4 (0401) diminished DQ8-restricted T-cell responses. In addition, we also demonstrated that the diminished T-cell response varied according to the specific DRB1*04 alleles. Taken together, this study provides evidence that DR4 is able to modulate DQ8-restricted T-cell responses, possibly by competing for peptides. Given that DQ8 is a primary genetic determinant of type 1 diabetes, the decreased DQ8-restricted CD4 T-cell activity due to peptide competition may be the mechanism explaining the modulation effect of DR4 to type 1 diabetes susceptibility.

Amino Acid Sequence↗
PubMed2005

Mechanisms underlying resistance of pancreatic islets from ALR/Lt mice to cytokine-induced destruction.

Nuclear and mitochondrial genomes combine in ALR/Lt mice to produce systemically elevated defenses against free radical damage, rendering these mice resistant to immune-mediated pancreatic islet destruction. We analyzed the mechanism whereby isolated islets from ALR mice resisted proinflammatory stress mediated by combined cytokines (IL-1beta, TNF-alpha, and IFN-gamma) in vitro. Such damage entails both superoxide and NO radical generation, as well as peroxynitrite, resulting from their combination. In contrast to islets from other mouse strains, ALR islets expressed constitutively higher glutathione reductase, glutathione peroxidase, and higher ratios of reduced to oxidized glutathione. Following incubation with combined cytokines, islets from control strains produced significantly higher levels of hydrogen peroxide and NO than islets from ALR mice. Nitrotyrosine was generated in NOD and C3H/HeJ islets but not by ALR islets. Western blot analysis showed that combined cytokines up-regulated the NF-kappaB inducible NO synthase in NOD-Rag and C3H/HeJ islets but not in ALR islets. This inability of cytokine-treated ALR islets to up-regulate inducible NO synthase and produce NO correlated both with reduced kinetics of IkappaB degradation and with markedly suppressed NF-kappaB p65 nuclear translocation. Hence, ALR/Lt islets resist cytokine-induced diabetogenic stress through enhanced dissipation and/or suppressed formation of reactive oxygen and nitrogen species, impaired IkappaB degradation, and blunted NF-kappaB activation. Nitrotyrosylation of beta cell proteins may generate neoantigens; therefore, resistance of ALR islets to nitrotyrosine formation may, in part, explain why ALR mice are resistant to type 1 diabetes when reconstituted with a NOD immune system.

Active Transport, Cell Nucleus↗
PubMed2005

Proteasome inhibition alters glucose-stimulated (pro)insulin secretion and turnover in pancreatic {beta}-cells.

Metabolic labeling studies were conducted in freshly isolated mouse islets and a beta-cell line (MIN6) to examine the effects of proteasome inhibition on glucose-stimulated (pro)insulin synthesis and secretion. Glucose-stimulated (pro)insulin synthesis, as determined by the incorporation of [(3)H]tyrosine, decreased significantly by 90% in islets and 71% in MIN6 cells pretreated with the proteasome inhibitor lactacystin (10 microM) for 2 h. To follow the fate of newly synthesized (pro)insulin, islets were pulse-labeled with [(3)H]tyrosine (40 microCi) for 20 min and chased +/- lactacystin (10 microM) for up to 4 h. The release of newly synthesized (pro)insulin ([(3)H]tyrosine-labeled) was similar between lactacystin-treated and control islets despite a 51% decrease (p <0.05) in total immunoreactive (pro)insulin secretion by lactacystin-treated islets. The specific radioactivity of [(3)H]tyrosine-labeled (pro)insulin in the extracellular medium of lactacystin-treated islets (0.52 +/- 0.16 cpm/microunits) was 2-fold greater relative to control islets (0.25 +/- 0.06 cpm/microunits). Induction of the unfolded protein response by lactacystin, as evidenced by the up-regulation of endoplasmic reticulum (ER) chaperones (GRP78/BiP, GRP94, protein disulfide isomerase) and induction of the stress-inducible transcription factor C/EBP-homologous protein/GADD153 (CHOP/GADD153), likely contributed to the release of newly synthesized (pro)insulin to relieve ER stress. The present data indicate proteasome inhibition did not prevent, but increased (p <0.05), the intracellular degradation of [(3)H]tyrosine-labeled (pro-)insulin from 8 to 24% in islets. Collectively, these data indicate beta-cells may balance glucose-stimulated (pro)insulin synthesis and secretion with the activity of the proteasome to regulate protein concentrations in the ER.

Acetylcysteine↗
PubMed2005

Major histocompatibility complex-linked diabetes susceptibility in NOD/Lt mice: subcongenic analysis localizes a component of Idd16 at the H2-D end of the diabetogenic H2(g7) complex.

The diabetogenic major histocompatibility complex (MHC) (H2(g7)) of NOD mice comprises contributions from several class II loci collectively designated as Idd1. Introduction of the H2(gx) haplotype from the related but diabetes-resistant cataract Shionogi (CTS) strain demonstrated an additional MHC-linked locus designated Idd16. The NOD-related alloxan resistant (ALR)/Lt strain is also characterized by the H2(gx) haplotype, which does not differ from H2(g7) from the class I H2-K(d) gene distally through the class II and into the class III region. Polymorphisms distal to the heat shock protein 70 locus (Hspa1b) include a rare H2-D(dx) rather than the H2(g7) encoded D(b) allele. Two differential-length NOD.ALR-H2(gx) congenic stocks (D.R1 and D.R2), both containing H2-D(dx), significantly suppressed diabetogenesis. This protection was lost when ALR alleles between the class III region and H2-D were removed in a shorter interval congenic (D.R3). Because no differences were observed in the ALR-derived interval extending 0.41 mB proximal to H2-K in any of these congenic stocks, a component of what was originally designated "Idd16" was sited to an interval shorter than 7.33 mB, distinguishing D.R2 from D.R3. Evidence supporting the candidacy of the ALR/CTS-shared H2-D(dx) MHC class I variant present in both diabetes-resistant stocks, but not the susceptible stock, is discussed.

Aging↗
PubMed2004

ALS/Lt: a new type 2 diabetes mouse model associated with low free radical scavenging potential.

Outbred CD-1 mice carry a spectrum of genetic susceptibilities for obesity and type 2 diabetes. ALS is an inbred strain with low antioxidant defenses produced by inbreeding CD-1 mice, with selection for susceptibility to alloxan, a generator of highly reactive oxygen free radicals and a potent beta-cell toxin. The objective of this study was to determine if the low ability to diffuse free radical stress would contribute to spontaneous type 2 diabetes development in alloxan-untreated males. Indeed, both hyperinsulinemia and impaired glucose tolerance developed spontaneously between 6 and 8 weeks of age in alloxan-untreated males. Further aging was accompanied by increases in body mass, progressively more severe hyperinsulinemia, and development of overt hyperglycemia. Transition from impaired glucose tolerance to overt hyperglycemia correlated with a decreased ratio of reduced to oxidized glutathione. Evidence that the increased oxidative burden elicited the type 2 diabetes syndrome was obtained by the systemic elevation of the antioxidative capacity through daily administration of R-lipoic acid. R-lipoic acid (30 mg/kg) prevented hyperglycemia, reduced insulin levels, and increased free radical scavenging potential. This mouse model with reduced ability to diffuse free radical stress is of obvious interest because free radical-mediated damage is implicated in the pathogenesis and complications of both type 1 and type 2 diabetes.

Aging↗
PubMed2003

Genetic analysis of resistance to Type-1 Diabetes in ALR/Lt mice, a NOD-related strain with defenses against autoimmune-mediated diabetogenic stress.

ALR mice are closely related to type-1 diabetes mellitus (T1DM)-prone NOD mice. The ALR genome confers systemically elevated free radical defenses, dominantly protecting their pancreatic islets from free radical generating toxins, cytotoxic cytokines, and diabetogenic T cells. The ALR major histocompatibility complex (MHC) ( H2(gx) haplotype) is largely, but not completely identical with the NOD H2(g7) haplotype, sharing alleles from H2-K through the class II and distally into the class III region. This same H2(gx) haplotype in the related CTS strain was linked to the Idd16 resistance locus. In the present study, ALR was outcrossed to NOD to fine map the Idd16 locus and establish chromosomal regions carrying other ALR non-MHC-linked resistance loci. To this end, 120 (NODxALR)xNOD backcross progeny females were monitored for T1DM and genetic linkage analysis was performed on all progeny using 88 markers covering all chromosomes. Glucosuria or end-stage insulitis developed in 32 females, while 88 remained both aglucosuria and insulitis free. Three ALR-derived resistance loci segregated. As expected, one mapped to Chromosome 17, with peak linkage mapping just proximal to H2-K. A novel resistance locus mapped to Chr 8. A pairwise scan for interactions detected a significant interaction between the loci on Chr 8 and Chr 17. On Chr 3, resistance segregated with a marker between previously described Idd loci and coinciding with an independently mapped locus conferring a suppressed superoxide burst by ALR neutrophils (Susp). These results indicate that the Idd16 resistance allele, defined originally by linkage to the H2(gx) haplotype of CTS, is immediately proximal to H2-K. Two additional ALR-contributed resistance loci may be ALR-specific and contribute to this strain's ability to dissipate free-radical stress.

Animals↗
PubMed2002

New mouse model to study islet transplantation in insulin-dependent diabetes mellitus.

BACKGROUND: Islet transplantation studies with diabetic rodents frequently use treatment with diabetogens such as alloxan or streptozotocin to render hosts hyperglycemic. These chemicals produce unwanted toxic side effects, which complicate interpretations of damage produced by hyperglycemia versus direct toxin-induced damage. A mouse that spontaneously developed insulin-sensitive diabetes without beta-cell autoimmunity would provide an excellent vehicle for testing beta-cell replacement protocols. The Ins2Akita mutation disrupts normal insulin processing and causes a failure in secretion of mature insulins, which results in the early development of hyperglycemia. This report examines the insulin sensitivity of mice that carry Ins2Akita and their responsiveness to engraftment with syngeneic pancreatic islets. METHODS: Ten-week-old C57BL/6J-Ins2Akita/+ males were given 1 unit of insulin to determine insulin sensitivity. Also, 10-week-old, hyperglycemic B6-Ins2Akita/+ received either 400 islets isolated from syngeneic C57BL/6J males (n=7) or from allogeneic BALB/cJ males (n=5) under the renal capsule. These mice were followed for 8 weeks after engraftment or until remission of euglycemia. Nephrectomy of the graft-containing kidney was performed on mice that remained euglycemic. These mice were then followed for 2 weeks for return of hyperglycemia. RESULTS: B6-Ins2Akita/+ mice are insulin responsive. Insulin treatment of hyperglycemic B6-Ins2Akita/+ males significantly lowered blood glucose values within 1 hr. In addition, B6-Ins2Akita/+ recipients of syngeneic islet grafts reversed their diabetic state in less than 72 hr. These islet-engrafted mice remained normoglycemic until removal of the graft-containing kidney. Removal of the graft resulted in a return to hyperglycemia. Mice that received allogeneic grafts efficiently rejected the graft. CONCLUSIONS: Our data support the hypothesis that B6-Ins2Akita/+ mice are insulin sensitive and provide an excellent model for islet transplantation studies. In addition, the reduced beta-cell mass and the absence of beta-cell autoimmunity, coupled to the fact that these mice also reject allografts, suggest that these mice may be useful for a variety of other applications, including testing functionality of human islets prepared for transplantation and perhaps also for exploring beta-cell restorative therapy using pancreatic islet stem cells.

Animals↗
PubMed2002

Genetic control of neutrophil superoxide production in diabetes-resistant ALR/Lt mice.

The neutrophil oxidative burst reaction differentiates ALR/Lt mice, known for an unusual systemic elevation of antioxidant defenses, from ALS/Lt mice, a related strain known for reduced ability to withstand oxidative stress. Neutrophils from marrow of ALS mice produced a normal neutrophil oxidative burst following phorbol ester stimulation. In contrast, ALR mice exhibited a markedly suppressed superoxide burst. F1 progeny from reciprocal outcrosses between ALR and ALS mice exhibited an intermediate burst level, higher than ALR but significantly lower than ALS. To elucidate the genetic basis for this strain difference, F1 mice were backcrossed to ALS mice, and marrow neutrophils isolated from the progeny were phenotyped for oxidative burst capacity. A genome-wide sweep using polymorphic markers distinguishing the two parental strains was performed to map the trait. A 1:1 phenotypic distribution was observed, and a locus (Suppressor of superoxide production, Susp) controlling this phenotype was mapped to Chromosome 3 near D3Mit241 at 33.1 cM. This locus probably represents an important regulatory element in the overall ALR strain resistance to oxidative stress, since diminished ability to mount a neutrophil burst in backcross segregants correlated with elevated hepatic superoxide dismutase 1 (SOD1) activity, an ALR strain characteristic.

Animals↗