Clinical relevance of serum lipids in the carcinogenesis of oral squamous cell carcinoma.

BACKGROUND: Dyslipidaemia is associated with cancers. However, the specific expression of serum lipids in oral potentially malignant disorders (OPMD) and oral squamous cell carcinoma (OSCC) remains unclear, and it remains unknown whether serum lipids are associated with the development of OPMD and OSCC. This study investigated the serum lipid profiles of OPMD and OSCC patients, and the association of serum lipids with the occurrence of OPMD and OSCC. METHODS: A total of 532 patients were recruited from the Affiliated Hospital of Stomatology, Nanjing Medical University. Serum lipid parameters including total cholesterol (TC), triglycerides (TGs), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), apolipoprotein A (Apo-A), apolipoprotein B (Apo-B), and lipoprotein (a) (Lpa) were analysed, and clinicopathological data were collected for further analysis. Furthermore, a regression model was used to evaluate the relationship between serum lipids and the occurrence of OSCC and OPMD. RESULTS: After adjusting for age and sex, no significant differences were observed in serum lipid or body mass index (BMI) between OSCC patients and controls (P > 0.05). HDL-C, Apo-A, and Apo-B levels were lower in OSCC patients than in OPMD patients (P < 0.05); HDL-C and Apo-A levels were higher in OPMD patients than in controls (P < 0.05). Furthermore, female OSCC patients had higher Apo-A and BMI values than males. The HDL-C level was lower in patients under 60 years of age than in elders (P < 0.05); and age was related to a higher risk of developing OSCC. Female patients with OPMD had higher TC, HDL-C, and Apo-A levels than males (P < 0.05); OPMD patients over 60 years of age had higher HDL-C than youngers (P < 0.05), whereas the LDL-C level was lower in elders (P < 0.05). The HDL-C and BMI values of the patients with oral leukoplakia (OLK) with dysplasia were more elevated than those of the oral lichen planus group, and the LDL-C, and Apo-A levels in patients with OLK with dysplasia were decreased (P < 0.05). Sex, high HDL-C and Apo-A values were associated with the development of OPMD. CONCLUSION: Serum lipids exhibited certain differences according to the occurrence and development of OSCC; high levels of HDL-C and Apo-A might be markers for predicting OPMD.

Clinical and pathological studies of eight cases of lipidized fibrous histiocytoma.

Lipidized fibrous histiocytoma (FH) is a rare type of FH. The present study aimed to describe the clinical and pathological features of lipidized FH. A total of eight patients diagnosed with lipidized FH were retrospectively reviewed in the present study. The cohort included three male and five female patients (male to female ratio, 1.7:1) with a mean age of 48 years (range, 38-62 years). In total, four tumors were located on the buttock, three on the lower leg and one on the forearm. Histological, lipidized FH showed a wide spectrum. Some cases included prominent stromal hyalinization and hyalinized vessels with scant lipid-laden histiocytes. Other cases exhibited the prominent lipid-laden histiocytes and scant stromal hyalinization. Overall, lipidized FH must be differentiated from other benign and malignant tumors, taking into account the therapeutic and prognostic differences between these different entities.

Effects of bone morphogenetic protein 4 on TGF-ß1-induced cell proliferation, apoptosis, activation and differentiation in mouse lung fibroblasts via ERK/p38 MAPK signaling pathway.

Fibroblasts, in particular myofibroblasts, are the critical effector cells in idiopathic pulmonary fibrosis (IPF), a deadly lung disease characterized by abnormal lung remodeling and the formation of "fibroblastic foci". Aberrant activation of TGF-ß1 is frequently encountered and promotes fibroblast proliferation, activation, and differentiation in pulmonary fibrosis. Hence, the inhibition of TGF-ß1-induced lung fibroblast activation holds promise as a therapeutic strategy for IPF. The present study aimed to investigate the potential effect and underlying mechanisms of bone morphogenetic protein 4 (BMP4) on TGF-ß1-induced proliferation, apoptosis, activation and myofibroblast differentiation of adult lung fibroblasts. Here, we demonstrated that BMP4 expression was significantly decreased in TGF-ß1-stimulated mouse primary lung fibroblasts (PLFs). BMP4 inhibited proliferation and apoptosis resistance of TGF-ß1-stimulated mouse PLFs. BMP4 suppressed TGF-ß1-induced fibroblast activation and differentiation in mouse PLFs. We also found that BMP4 inhibited TGF-ß1-induced ERK and p38 MAPK phosphorylation. Our findings indicate that BMP4 exerts its anti-fibrotic effects by regulating fibroblast proliferation, apoptosis, activation and differentiation via the inhibition of the ERK/p38 MAPK signaling pathway, and thus has a potential for the treatment of pulmonary fibrosis.

Gastritis cystica profunda: clinical and pathologic study of seven cases and review of literature.

Gastritis cystica profunda (GCP) is a rare lesion characterized by hyperplasia and cystic dilatation of the gastric glands in the submucosal layer. Here we report seven cases of GCP. The patients are 5 women and 2 men with a mean age of 62 (range, 42-82) years at the time of diagnosis. The patients presented with abdominal distension, sour regurgitation, and heartburn. One case had the previous gastric surgery and the other six cases had no special history. The lesions were located in the fundus (4/7), corpus (1/7), cardia (1/7), and antrum (1/7). Endoscopic analysis revealed pedunculated polyps, or a dome-shaped polyp. Histologically, all cases showed dilated tubular glands, mainly located in the submucosa, among the muscularis mucosa, and occasionally in the lamina propria. The glands were lined by bland single columnar epithelium with infolding features in some areas. Mitotic activity and marked cellular atypia were not present. The stroma in some cases was mildly edematous with infiltrated lymphocytes and plasma cells. There was no epithelial dysplasia in the overlying mucosa. Immunohistochemically, the Ki-67 index was < 1%. P53 immunostaining was generally characterized as wild type in all cases. Based on the morphology of the glands and the cells and the possible mechanism of hyperplasia and cystic dilatation of the gastric glands, it is easy to differentiate GCP from a well-differentiated adenocarcinoma.

Elevated Expression of Gamma-Glutamyl Hydrolase Is Associated With Poor Prognosis and Altered Immune Signature in Uterine Corpus Endometrial Carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is a common malignant tumor of the female reproductive system with poor prognosis in advanced, recurrent, and metastatic cases. Identification of reliable molecular markers will help in the development of clinical strategies for early detection, diagnosis, and intervention. Gamma-glutamyl hydrolase (GGH) is a key enzyme in folate metabolism pathway. High expression of GGH is associated with severe clinicopathological features and poor prognosis of several cancers. High GGH expression is also related to cell resistance to antifolate drugs such as methotrexate. In this study we focused on the prognostic value of immunohistochemical GGH expression level in UCEC tissue and RNA-seq data from The Cancer Genome Atlas to establish associations with clinical features and outcomes. Further, we conducted comprehensive bioinformatics analyses to identify and functionally annotate differentially expressed genes (DEGs) associated with UCEC upregulation and assessed the effects of upregulation on immune infiltration. Both GGH mRNA and protein expression levels were elevated in tumor tissues, and higher expression was significantly associated with advanced clinicopathological features and poor prognosis by univariate analysis. Further multivariate analysis identified elevated GGH expression as an independent risk factor for poor outcome. Nomograms including GGH expression yielded a c-index for disease-specific survival prediction of 0.884 (95% confidence interval: 0.861-0.907). A total of 520 DEGs (111 upregulated and 409 downregulated) were identified between high and low GGH expression groups. Analysis using Gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, Gene set enrichment analysis, and protein‒protein interaction indicated significant associations of altered GGH expression with cell proliferation, immune response, and the occurrence and development of UCEC tumors. Finally, GGH expression level was associated with high Th2 cell and low natural killer CD56bright cell infiltration. Collectively, these findings indicate that GGH drives UCEC progression and could be a useful biomarker for survival prediction as well as a therapeutic target.

Berberine Relieves Metabolic Syndrome in Mice by Inhibiting Liver Inflammation Caused by a High-Fat Diet and Potential Association With Gut Microbiota.

Whether berberine mediates its anti-inflammatory and blood sugar and lipid-lowering effects solely by adjusting the structure of the gut microbiota or by first directly regulating the expression of host pro-inflammatory proteins and activation of macrophages and subsequently acting on gut microbiota, is currently unclear. To clarify the mechanism of berberine-mediated regulation of metabolism, we constructed an obese mouse model using SPF-grade C57BL/6J male mice and conducted a systematic study of liver tissue pathology, inflammatory factor expression, and gut microbiota structure. We screened the gut microbiota targets of berberine and showed that the molecular mechanism of berberine-mediated treatment of metabolic syndrome involves the regulation of gut microbiota structure and the expression of inflammatory factors. Our results revealed that a high-fat diet (HFD) significantly changed mice gut microbiota, thereby probably increasing the level of toxins in the intestine, and triggered the host inflammatory response. The HFD also reduced the proportion of short-chain fatty acid (SCFA)-producing genes, thereby hindering mucosal immunity and cell nutrition, and increased the host inflammatory response and liver fat metabolism disorders. Further, berberine could improve the chronic HFD-induced inflammatory metabolic syndrome to some extent and effectively improved the metabolism of high-fat foods in mice, which correlated with the gut microbiota composition. Taken together, our study may improve our understanding of host-microbe interactions during the treatment of metabolic diseases and provide useful insights into the action mechanism of berberine.

Radiation-induced second primary squamous cell carcinoma of the oral cavity after radiotherapy for nasopharyngeal carcinoma.

BACKGROUND: The increasing occurrence of radiation-induced second primary squamous cell carcinoma of the oral cavity (RISCCO) after radiotherapy for nasopharyngeal carcinoma (NPC) has become a noteworthy complication that can influence long-term survival. This study aimed to analyze the associations of clinicopathologic characteristics with prognostic factors among patients who developed RISCCO after radiotherapy for NPC. METHODS: A total of 41,446 NPC patients admitted to Sun Yat-sen University Cancer Center (SYSUCC) between August 1989 and January 2019 were reviewed. Among these patients, 88 RISCCO patients who satisfied the inclusion criteria were included in the study. RESULTS: During our study, the incidence of RISCCO after radiotherapy was 0.21% (88/41,446) among NPC patients at SYSUCC. The latency period ranged from 1.0 to 34.0 years (median, 9.0 years), and the latency of RISCCO was notably shorter for patients who received intensity-modulated radiation therapy than that for patients who received conventional radiotherapy using cobalt-60 or 6-MV X-rays (median, 4.0 years vs. 11.0 years, P = 0.013). The 1-, 3-, and 5-year overall survival (OS) rates for the entire cohort of 88 patients were 79.0%, 46.6%, and 35.2%, respectively. The 5-year OS rate for the 79 patients who received treatment was 45.7%, and the 5-year OS rate for the 9 patients who refused treatment was 0%. T classification and surgery were identified as independent prognostic factors associated with a high OS rate. CONCLUSIONS: Surgery as the first-choice treatment may improve survival and prognosis. A long-term follow-up is needed for early detection of RISCCO in NPC patients.

Vitamin D receptor genetic polymorphisms are associated with oral lichen planus susceptibility in a Chinese Han population.

BACKGROUND: Vitamin D receptor (VDR) is involved in multiple immune-mediated disorders including oral lichen planus (OLP). This study investigated the association between VDR gene polymorphisms and the risk of OLP. METHODS: In total, 177 OLP patients and 207 healthy participants were recruited from the Affiliated Hospital of Stomatology, Nanjing Medical University. Eight single nucleotide polymorphisms (SNPs: rs731236, rs739837, rs757343, rs2107301, rs2239185, rs7975232, rs11574129 and rs11568820) in the VDR gene were selected and genotyped. RESULTS: The results showed that OLP risk was increased in subjects with the rs2239185 TT genotype (Recessive model: adjusted Odd ratio(OR) = 2.68, 95% Confidence interval(CI) = 1.28-5.62, P = 0.009) and rs7975232 CC genotype (Recessive model: adjusted OR = 2.25, 95% CI = 1.10-4.58, P = 0.026). Moreover, rs2239185 and rs7975232 (P < 0.01) showed significant cumulative effects on OLP risk.Haplotype analysis showed that the CC haplotype (rs2239185-rs7975232) was associated with an increased risk of OLP (OR = 3.11, 95% CI = 1.42-6.83, P = 0.005), compared with the AC haplotype. CONCLUSION: The rs2239185 and rs7975232 variants of VDR may influence OLP susceptibility, and VDR gene polymorphisms may be candidate susceptibility regions for OLP in a Chinese Han population.

Systematic genetic analyses of genome-wide association study data reveal an association between the key nucleosome remodeling and deacetylase complex and bipolar disorder development.

BACKGROUND: Genome-wide association studies (GWASs) are used to identify genetic variants for association with bipolar disorder (BD) risk; however, each GWAS can only reveal a small fraction of this association. This study systematically analyzed multiple GWAS data sets to provide further insights into potential causal BD processes by integrating the results of Psychiatric Genomics Consortium Phase I (PGC-I) for BD with core human pathways and functional networks. METHODS: The i-Gsea4GwasV2 program was used to analyze data from the PGC-I GWAS for BD (the pathways came from Reactome), as well as the nominally significant pathways. We established a gene network of the significant pathways and performed a gene set analysis for each gene cluster of the Enhancing Neuroimaging Genetics through Meta-Analysis (ENIGMA) GWAS data for the volumes of the intracranial region and seven subcortical regions. RESULTS: A total of 30 of 1816 Reactome pathways were identified and showed associations with BD risk. We further revealed 22 interconnected functional and topologically interacting clusters (Clusters 0-21) that were associated with BD risk. Moreover, we obtained brain transcriptome data from BrainSpan and found significant associations between common variants of the genes in Cluster 1 with the hippocampus (HIP; P = .026; family-wise error [FWE] correction) and amygdala (AMY; P = .016; FEW correction) in Cluster 8 with HIP (P = .022; FWE correction). The genes in Cluster 1 were enriched for the transcriptional co-expression profile in the prenatal AMY, and core genes (CDH4, MTA2, RBBP4, and HDAC2) were identified to be involved in regulating early brain development. CONCLUSION: This study demonstrated that the HIP and AMY play a central role in neurodevelopment and BD risk.

A transcriptomic study of myogenic differentiation under the overexpression of PPARγ by RNA-Seq.

To study the cellular and molecular function of peroxisome proliferator-activated receptor γ (PPARγ) in skeletal muscle differentiation, we have generated inducible gain-of-function to overexpress PPARγ in C2C12 myoblasts. In order to identify PPARγ targets, RNA sequencing (RNA-seq) was used to evaluate and quantify the transcriptomes and expression patterns during myogenic differentiation under the overexpression of PPARγ. The formation of myotubes and the expression of muscle-specific myogenic genes such as MyoD and MyoG may be inhibited by PPARγ overexpression. Multiple genes and pathways were significantly involved in this process, including 11 genes such as Fndc9 and Slc14a1 with fundamental change of regulation modes, 9 genes of which were validated by the data of qRT-PCR. Our studies demonstrate that PPARγ would play critical roles on myoblasts differentiation, mediating crosstalk among several pathways and transcription factors. Our data is available in the Gene Expression Omnibus (GEO) database with the accession number as GSE99399.

A systematic analysis of the association studies between CASP8 D302H polymorphisms and breast cancer risk.

Caspase 8 (CASP8) is a regulator of apoptosis, whose genetic variation has been reported to be associated with the risk of various cancers. Especially, the single-nucleotide polymorphism (SNP) rs1045485, which generates the substitution D302H in CASP8, is likely to be associated with breast cancer. Several previous studies have reported the association of CASP8 D302H polymorphism with breast cancer; however, the results are inconsistent. To validate the association between CASP8 D302H polymorphism and breast cancer risk, we performed an updated meta-analysis of 18 studies including 27,807 cases and 32,332 controls. We tested the overall association between this SNP and breast cancer susceptibility and stratified subgroups based on countries where cases are from. We confirmed a significant correlation between CASP8 D302H polymorphism and the reduced breast cancer susceptibility in population from UK, Germany and Poland, but no significant association was observed in other countries, such as Finland or USA. Our findings indicate the relationship of SNP CASP8 D302H and breast cancer would not be universal but only be sensitive in some particular European countries. The genetic difference for diverse countries may be useful in individual and precision medicine or health.

Comprehensive tissue-specific gene set enrichment analysis and transcription factor analysis of breast cancer by integrating 14 gene expression datasets.

Breast cancer is the most commonly diagnosed malignancy in women. Several key genes and pathways have been proven to correlate with breast cancer pathology. This study sought to explore the differences in key transcription factors (TFs), transcriptional regulation networks and dysregulated pathways in different tissues in breast cancer. We employed 14 breast cancer datasets from NCBI-GEO and performed an integrated analysis in three different tissues including breast, blood and saliva. The results showed that there were eight genes (CEBPD, EGR1, EGR2, EGR3, FOS, FOSB, ID1 and NFIL3) down-regulated in breast tissue but up-regulated in blood tissue. Furthermore, we identified several unreported tissue-specific TFs that may contribute to breast cancer, including ATOH8, DMRT2, TBX15 and ZNF367. The dysregulation of these TFs damaged lipid metabolism, development, cell adhesion, proliferation, differentiation and metastasis processes. Among these pathways, the breast tissue showed the most serious impairment and the blood tissue showed a relatively moderate damage, whereas the saliva tissue was almost unaffected. This study could be helpful for future biomarker discovery, drug design, and therapeutic and predictive applications in breast cancers.

A comprehensive transcriptomic analysis of differentiating embryonic stem cells in response to the overexpression of Mesogenin 1.

The mutation of somitogenesis protein Mesogenin 1 (Msgn1) has been widely used to study the direct link between somitogenesis and the development of an embryo. Several studies have used gene expression profiling of somitogenesis to identify the key genes in the process, but few have focused on the pathways involved and the coexpression patterns of associated pathways. Here we employed time-course microarray datasets of differentiating embryonic stem cells by overexpressing the transcription factor Msgn1 from the public database library of Gene Expression Omnibus (GEO). Then we applied gene set enrichment analysis (GSEA) to the datasets and performed candidate transcription factors selection. As a result, several significantly regulated pathways and transcription factors (TFs), as well as some of the specific signaling pathways, were identified during somitogenesis under Msgn1 overexpression, most of which had not been reported previously. Finally, significant core genes such as Hes1 and Notch1 as well as some of the TFs such as PPARs and FOXs were identified to construct coexpression networks of related pathways, the expression patterns of which had been validated by our following quantitative real-time PCR (qRT-PCR). The results of our study may help us better understand the molecular mechanisms of somitogenesis in mice at the genome-wide level.

A comprehensive meta-analysis of genetic associations between five key SNPs and colorectal cancer risk.

Genome-wide association studies (GWAS) on colorectal cancer (CRC) have identified dozens of single nucleotide polymorphisms (SNPs) in more than 19 independent loci associated with CRC. Due to the heterogeneity of the studied subjects and the contrary results, it is challenging to verify the certainty of the association between these loci and CRC.We conducted a critical review of the published studies of SNPs associated with CRC. Five most frequently reported SNPs, which are rs6983267/8q24.21, rs4939827/18q21.1, rs10795668/10p14, rs4444235/14q22.2 and rs4779584/ 15q13.3, were selected for the current study from the qualified studies. Then meta-analyses based on larger sample sizes with average of 33,000 CRC cases and 34,000 controls were performed to assess the association between SNPs and CRC risk. Heterogeneity among studies and publication bias were assessed by the χ2-based Q statistic test Begg's funnel plot or Egger's test, respectively.Our meta-analysis confirmed significant associations of the five SNPs with CRC risk under different genetic models. Two risk variants at rs6983267 {Odds Ratio (OR) 1.388, 95% Confidence Interval (CI) 1.180-1.8633} and rs10795668 (OR 1.323, 95% CI 1.062-1.648) had the highest ORs in homogeneous model. While ORs of the other three variants at rs4939827 {OR 1.298, 95% CI 1.135-1.483}, rs4779584 (OR 1.261, 95% CI 1.146-1.386) and rs4444235 (OR 1.160, 95% CI 1.106-1.216) were also statistically significant. Sensitivity analyses and publication bias assessment indicated the robust stability and reliability of the results.

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-ß receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.

Insertion sequence ISRP10 inactivation of the oprD gene in imipenem-resistant Pseudomonas aeruginosa clinical isolates.

Carbapenem resistance mechanisms were investigated in 32 imipenem-resistant Pseudomonas aeruginosa clinical isolates recovered from hospitalised children. Sequence analysis revealed that 31 of the isolates had an insertion sequence element ISRP10 disrupting the porin gene oprD, demonstrating that ISRP10 inactivation of oprD conferred imipenem resistance in the majority of the isolates. Multilocus sequence typing (MLST) was used to discriminate the isolates. In total, 11 sequence types (STs) were identified including 3 novel STs, and 68.3% (28/41) of the tested strains were characterised as clone ST253. In combination with random amplified polymorphic DNA (RAPD) analysis, the imipenem-resistant isolates displayed a relatively high degree of genetic variability and were unlikely associated with nosocomial infections.

Ubiquitination and regulation of Smad7 in the TGF-ß1/Smad signaling of aristolochic acid nephropathy.

Aristolochic acid I (AAI) affects TGF-ß1/Smad signaling, which causes AA nephropathy (AAN), but the mechanisms are not fully understood. We aimed to clarify whether Arkadia and UCH37 participate in TGF-ß1/Smad signaling via Smad7, and the regulatory mechanisms of Smad7. One side, mice and cultured mouse renal tubular epithelial cells (RTECs) were treated with various AAI doses and concentrations, respectively; on the other side, RTECs were transfected with small interfering RNA (siRNA) expression vectors against Arkadia and UCH37 and then treated with 10 µg/ml AAI. And then detect the mRNA and protein levels of Smad7, UCH37, Arkadia and any other relative factors by RT-PCR and Western blotting. In kidney tissues and RTECs, the mRNA and protein levels of Smad7 decreased with increasing AAI doses concentrations by real-time PCR and Western blotting, whereas those of Arkadia, UCH37, Smad2, Smad3 and TßRI increased. Cells transfected with the Arkadia siRNA expression vector showed reduced mRNA and protein levels of vimentin, α-SMA, Smad2, Smad3 and TßRI after AAI treatment, while those of CK18 and Smad7 increased compared with those of untransfected RTECs. Conversely, cells transfected with the UCH37 siRNA expression vector showed the opposite effect on analyzed signaling molecules after AAI treatment. Arkadia and UCH37 participate in TGF-ß1/Smad signaling-mediated renal fibrosis, and Smad7 blocks TGF-ß1 signaling by inhibiting Smad2/Smad3 phosphorylation and enhancing the degradation of TßRI.

Limitation of immune tolerance-inducing thymic epithelial cell development by Spi-B-mediated negative feedback regulation.

Medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator AIRE and various tissue-specific antigens (TSAs) are critical for preventing the onset of autoimmunity and may attenuate tumor immunity. However, molecular mechanisms controlling mTEC development remain elusive. Here, we describe the roles of the transcription factor Spi-B in mTEC development. Spi-B is rapidly up-regulated by receptor activator of NF-κB ligand (RANKL) cytokine signaling, which triggers mTEC differentiation, and in turn up-regulates CD80, CD86, some TSAs, and the natural inhibitor of RANKL signaling, osteoprotegerin (OPG). Spi-B-mediated OPG expression limits mTEC development in neonates but not in embryos, suggesting developmental stage-specific negative feedback regulation. OPG-mediated negative regulation attenuates cellularity of thymic regulatory T cells and tumor development in vivo. Hence, these data suggest that this negative RANKL-Spi-B-OPG feedback mechanism finely tunes mTEC development and function and may optimize the trade-off between prevention of autoimmunity and induction of antitumor immunity.

DNA methylation profile of Aire-deficient mouse medullary thymic epithelial cells.

BACKGROUND: Medullary thymic epithelial cells (mTECs) are characterized by ectopic expression of self-antigens during the establishment of central tolerance. The autoimmune regulator (Aire), which is specifically expressed in mTECs, is responsible for the expression of a large repertoire of tissue-restricted antigens (TRAs) and plays a role in the development of mTECs. However, Aire-deficient mTECs still express TRAs. Moreover, a subset of mTECs, which are considered to be at a stage of terminal differentiation, exists in the Aire-deficient thymus. The phenotype of a specific cell type in a multicellular organism is governed by the epigenetic regulation system. DNA methylation modification is an important component of this system. Every cell or tissue type displays a DNA methylation profile, consisting of tissue-dependent and differentially methylated regions (T-DMRs), and this profile is involved in cell-type-specific genome usage. The aim of this study was to examine the DNA methylation profile of mTECs by using Aire-deficient mTECs as a model. RESULTS: We identified the T-DMRs of mTECs (mTEC-T-DMRs) via genome-wide DNA methylation analysis of Aire(-/-) mTECs by comparison with the liver, brain, thymus, and embryonic stem cells. The hypomethylated mTEC-T-DMRs in Aire(-/-) mTECs were associated with mTEC-specific genes, including Aire, CD80, and Trp63, as well as other genes involved in the RANK signaling pathway. While these mTEC-T-DMRs were also hypomethylated in Aire(+/+) mTECs, they were hypermethylated in control thymic stromal cells. We compared the pattern of DNA methylation levels at a total of 55 mTEC-T-DMRs and adjacent regions and found that the DNA methylation status was similar for Aire(+/+) and Aire(-/-) mTECs but distinct from that of athymic cells and tissues. CONCLUSIONS: These results indicate a unique DNA methylation profile that is independent of Aire in mTECs. This profile is distinct from other cell types in the thymic microenvironment and is indicated to be involved in the differentiation of the mTEC lineage.

Distinct subcellular localization of three isoforms of insulinoma-associated protein 2ß in neuroendocrine tissues.

AIMS: Insulinoma-associated protein 2ß (IA-2ß) is considered to play a significant role in regulated secretion. Recent studies have shown that the mouse brain expresses three major isoforms of IA-2ß, named IA-2ß60, IA-2ß64, and IA-2ß71. In this study, we analyzed the tissue-, cell- and organelle-specific distributions of IA-2ß isoforms in mice. MAIN METHODS: To localize IA-2ß expression in mouse tissues and cells, western blot and immunohistochemical analyses were carried out. The subcellular distribution of IA-2ß isoforms was assessed by sedimentation of mouse brain homogenates in a discontinuous sucrose density gradient. KEY FINDINGS: IA-2ß60 was abundant in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, and pituitary, and in the muscular and mucosal layers of the digestive organs. In contrast, the expression of IA-2ß64 and IA-2ß71 was restricted to the cerebrum, cerebellum, medulla oblongata, and pituitary, and the muscular layers of the digestive organs. Immunohistochemical analysis of mouse pancreatic islets revealed that pancreatic beta cells expressed IA-2ß60 exclusively, whereas alpha and delta cells expressed all three isoforms. By the sedimentation of mouse brain homogenates, it was shown that IA-2ß64 and IA-2ß71 were co-localized with IA-2 on secretory granules, but were absent from synaptic vesicles (SVs). On the other hand, IA-2ß60 was co-localized with synaptophisin on SVs, but was absent from secretory granules. SIGNIFICANCE: The tissue-, cell- and organelle-specific distributions of IA-2ß isoforms suggest that IA-2ß60 has a role in secretion from SVs, whereas IA-2ß64 and IA-2ß71 are involved in secretion from secretory granules.