Immune micro-environment analysis and drug screening for ovarian endometriosis.

BACKGROUND: Patients of ovary endometriosis have an abnormal immune micro-environment, leading to endometrial tissue that from retrograde menstruation evade immune surveillance and subsequently develop into ectopic lesions. OBJECTIVE: This study aims to elucidate the crucial immune cells and molecular pathways that are associated with an aberrant immune micro-environment of endometriosis. METHOD: In this study, we identified differentially expressed genes between ovarian ectopic endometrial tissue (OVE) and eutopic endometrial tissue from patients with endometriosis (PE) and non-endometriosis patients (CON) by analyzing the mRNA sequencing data. Additionally, we used WGCNA(Weighted Gene Co-expression Network Analysis) to screen for key genes related to immune cell infiltration and compared the sub-types of infiltrating immune cells using CIBERSORT(cell-type identification by estimating relative subsets of RNA transcript). Subsequently, we conducted a single-cell analysis on the identified key genes. Furthermore, we analyzed potential drugs suitable for ovarian endometriosis treatment using pRRophertic. RESULTS: Seven key genes associated with immune cell infiltration were screened out. The expression of these genes in OVE was significantly lower than that in PE and CON. These key genes were mainly enriched in the NK cell-mediated cytotoxicity pathway, especially for CD16 + CD56dim NK. Moreover, NK cells infiltration in ovarian endometriosis was significantly reduced compared with PE and CON, while M2 macrophage shown the opposite. Results of the single-cell analysis showed that the expression of the seven key genes in NK cells and monocyte-macrophages in OVE was significantly lower than that in PE or CON. Additionally, we identified potential drugs suitable for ovarian endometriosis treatment. CONCLUSION: The decreased infiltration of NK cells and increased infiltration of M2 macrophages contribute to the evasion of immune surveillance against endometrial tissue, promoting the progression of OVE. Therefore, potential strategies for the treatment of OVE include increasing NK cell activation and decreasing M2 macrophage polarization.

Endometriosis of the skeletal muscular system (ESMS): a systematic review.

BACKGROUND: Extrapelvic endometriosis occurring at skeletal muscle and joint sites is not rare and is prone to delayed diagnosis and inappropriate treatment. Herein, endometriosis of the skeletal muscular system (ESMS) is systematically reviewed to facilitate early diagnosis and treatment. METHODS: Literature on ESMS published before March 2022 was retrieved from the Ovid Medline and Web of Science databases, and the major clinical data were extracted for descriptive analysis. RESULTS: A total of 62 studies (78 ESMS cases) met these requirements. The ESMS included the abdominal muscles (50.7%), pelvic floor muscles (11.6%), lower limb muscles (11.6%), hip muscles (8.7%), lumbar muscles (7.2%), joints (5.8%), upper limb muscles (2.9%), and shoulder-neck muscles (1.4%). The age was 34.0 ± 7.2 years (range 17-49 years). Approximately 63.8% of patients had at least one previous pelvic surgery, and 76.8% of local symptoms were related to the menstrual cycle. The course of disease was 29.6 ± 25.4 months (range 0.5-96 months). Only 30.3% of the patients sought initial medical advice from gynecologists, while 69.7% sought initial medical advice from a nongynecological physician. Twenty-seven patients underwent fine-needle aspiration (FNA) under ultrasound or CT monitoring, and only 44.4% (12/27) were confirmed to have endometriosis by FNA tissue pathology. Approximately 47.4% (37/78) of the patients had a normal pelvic cavity appearance. Surgical resection was performed in 92.3% (72/78) of the patients, of whom 88.9% (64/72) underwent complete resection of the lesion (negative surgical margin) and 20.8% (15/72) received postoperative hormone therapy. At 16.7 months of follow-up, 83.3%, 13.8%, 2.9%, and four patients had complete response, partial response, recurrence, and permanent function impairment, respectively. CONCLUSION: Endometriosis can occur at almost any site in the musculoskeletal system. For women of reproductive age with catamenial pain or a mass in the musculoskeletal system, endometriosis should be suspected. Fine-needle aspiration can easily lead to missed diagnoses. Surgical resection for negative margins is the main treatment, and permanent impairment of function may occur in a few patients due to delayed diagnosis. Vascular lymphatic metastasis is the most likely mechanism of pathogenesis.

Immune micro-environment and drug analysis of peritoneal endometriosis based on epithelial-mesenchymal transition classification.

Background: Epithelial-mesenchymal transition (EMT) is a complex event that drives polar epithelial cells transform from adherent cells to motile mesenchymal cells, in which are involved immune cells and stroma cells. EMT plays crucial roles in migration and invasion of endometriosis. The interaction of endometrial implants with the surrounding peritoneal micro-environment probably affects the development of peritoneal endometriosis. To date, very few studies have been carried out on peritoneal endometriosis sub-type classification and micro-environment analysis based on EMT. The purpose of this study is to investigate the potential application of EMT-based classification in precise diagnosis and treatment of peritoneal endometriosis. Method: Based on EMT hallmark genes, 76 peritoneal endometriosis samples were classified into two clusters by consistent cluster classification. EMT scores, which calculated by Z score of 8 epithelial cell marker genes and 8 mesenchymal cell marker genes, were compared in two clusters. Then, immune scores and the abundances of corresponding immune cells, stroma scores and the abundances of corresponding stroma cells were analyzed by the "xCell" package. Futhermore, a diagnostic model was constructed based on 9 diagnostic markers which related to immune score and stroma score by Lasso-Logistic regression analysis. Finally, based on EMT classification, a total of 8 targeted drugs against two clusters were screened out by drug susceptibility analysis via "pRRophetic" package. Results: Hallmark epithelial-mesenchymal transition was the mainly enriched pathway of differentially expressed genes between peritoneal endometriosis tissues and endometrium tissues. Compared with cluster 2, EMT score and the abundances of most infiltrating stroma cell were significantly higher, while the abundances of most infiltrating immune cells were dramatically less. The diagnostic model could accurately distinguish cluster 1 from cluster 2. Pathway analysis showed drug candidates targeting cluster 1 mainly act on the IGF-1 signaling pathway, and drug candidates targeting cluster 2 mainly block the EGFR signaling pathway. Conclusion: In peritoneal endometriosis, EMT was probably promoted by stroma cell infiltration and inhibited by immune cell infiltration. Besides, our study highlighted the potential uses of the EMT classification in the precise diagnosis and treatment of peritoneal endometriosis.

HOXA10 Regulates the Synthesis of Cholesterol in Endometrial Stromal Cells.

Background: The expression of homeobox A10 (HOXA10) in endometrial stromal cells is regulated by steroid hormones, especially by estrogen. As a precursor molecule of estrogen, abnormal cholesterol metabolism is significantly positively correlated with endometriosis. The purpose of this study was to explore the regulation of HOXA10 on cholesterol synthesis in endometrial stromal cells. Method: mRNA expression data of eutopic endometrial stromal cell (ESC) and ovarian endometriotic cysts stromal cell (OESC) were download from the Gene Expression Omnibus (GEO) databases. Overexpression and silence of HOXA10 were conducted in cultured ESC and subjected to mRNA sequencing. The differentially expressed genes (DEGs) were selected by analyzing the sequencing data. Weighted gene co-expression network analysis (WGCNA) was applied to identify the key genes associated with HOXA10. The methylation rate of HOXA10 CpGs and the correlation between HOXA10 expression and the methylation in eutopic endometrial tissue (EU) and ovarian cyst (OC) were analyzed. Results: HOXA10 in ESC was significantly higher expressed than that in OESC. Six key genes (HMGCR, MSMO1, ACAT2, HMGCS1, EBP, and SQLE), which were regulated by HOXA10, were identified from the salmon4 module by WGCNA. All these key genes were enriched in cholesterol synthesis. Moreover, the expression of HOXA10 was negatively related to its CpGs methylation rate. Conclusion: In this study, six key genes that were regulated by HOXA10 were selected, and all of them were enriched in cholesterol synthesis. This finding provided a new insight into the metabolic mechanism of cholesterol in ESC. It also provided a potential treatment strategy for cholesterol metabolism maladjustment in patients with ovarian endometriosis.

SARS-CoV-2 delta (B.1.617.2) spike protein adjuvanted with Alum-3M-052 enhances antibody production and neutralization ability.

Background: Optimizing adjuvant is one of the critical methods to improve the vaccine. 3M-052, a novel TLR7/8 agonist which was designed for slow dissemination at the injection site, has a potential as adjuvant, but its performance as a vaccine adjuvant for SARS-CoV-2 (B.1.617.2) spike protein has not been studied. The present study aimed to evaluate the effect of Alum-3M-052 as an adjuvant to improve mice serum antibody titers and pseudovirus neutralization efficiency. Method: Female Balb/c mice were immunized 3 times at day 0, 7 and 21 intramuscularly with SARS-CoV-2 (B.1.617.2) spike protein and adjuvant (Alum or Alum-3M-052). Mice serum was collected weekly since day 7. Antibody titers of mice serum anti-SARS-CoV-2 (B.1.617.2) IgG and IgM were detected by ELISA. Inhibition rates of mice serum blocking SARS-CoV-2 (B.1.617.2) spike protein binding to ACE2 were detected by SARS-CoV-2 (B.1.617.2) Inhibitor Screening Kit. Neutralization efficiencies of mice serum against both SARS-CoV-2 (BA.2.12.1) pseudovirus and SARS-CoV-2 (B.1.617.2) pseudovirus were detected by pseudovirus neutralizing assay. Result: Serum of mice immunized by SARS-CoV-2 (B.1.617.2) spike protein adjuvanted with Alum-3M-052 had highest antibody titers and higher neutralization efficiency against both SARS-CoV-2 (BA.2.12.1) pseudovirus and SARS-CoV-2 (B.1.617.2) pseudovirus. Besides, neutralization efficiency of anti-SARS-CoV-2 (B.1.617.2) spike protein antibody against SARS-CoV-2 (BA.2.12.1) pseudovirus was lower than that of SARS-CoV-2 (B.1.617.2) pseudovirus. Conclusion: Alum-3M-052 rapidly increased the titer of anti-SARS-CoV-2 (B.1.617.2) spike protein neutralizing antibodies and enhanced the neutralization ability against pseudoviruses and variants. This study provided evidence for the application of Alum-3M-052 as an adjuvant in COVID-19 vaccines production.

Identification of Immune-Related Key Genes in Ovarian Cancer Based on WGCNA.

Background: Ovarian cancer (OV) is a fatal gynecologic malignancy and has poor survival rate in women over the age of forty. In our study, we aimed to identify genes related to immune microenvironment regulations and explore genes associated with OV prognosis. Methods: The RNA-seq data of GDC TCGA Ovarian Cancer cohort of 376 patients was retrieved from website. Weighted gene co-expression network analysis (WGCNA) and ESTIMATE algorithm were applied to identify the key genes associated with the immune scores. The correlation between key genes and 22 immune cell types were estimated by using CIBERSORT algorithms. Results: WGCNA showed that the pink module was most correlated with the immune score. Seven of 14 key genes (FCRL3, IFNG, KCNA3, LY9, PLA2G2D, THEMIS, and TRAT1) were significantly associated with the OS of OV patients. Correlation analysis showed our key genes positively related to M1 macrophages, CD8 T cells, plasma cells, regulatory T (Treg) cells and activated memory CD4 T cells, and negatively related to naive CD4 T cells, M0 macrophages, activated dendritic cells (DCs) and memory B cells. Kaplan-Meier survival analysis showed that lower abundances of neutrophils and higher abundances of M1 macrophages, plasma cells, T cells gamma delta (γδT) cells and follicular helper T (Tfh) cells predicted better OV prognosis. Conclusion: Forteen key genes related to the immune infiltrating of OV were identified, and seven of them were significantly related to prognosis. These key genes have potential roles in tumor infiltrating immune cells differentiation and proliferation. This study provided potential prognostic markers and immunotherapy targets for OV.

Identification of Autophagy-Related Prognostic Signature and Analysis of Immune Cell Infiltration in Low-Grade Gliomas.

Autophagy plays an important role in cancer. Many studies have demonstrated that autophagy-related genes (ARGs) can act as a prognostic signature for some cancers, but little has been known in low-grade gliomas (LGG). In our study, we aimed to establish a prognostical model based on ARGs and find prognostic risk-related key genes in LGG. In the present study, a prognostic signature was constructed based on a total of 8 ARGs (MAPK8IP1, EEF2, GRID2, BIRC5, DLC1, NAMPT, GRID1, and TP73). It was revealed that the higher the risk score, the worse was the prognosis. Time-dependent ROC analysis showed that the risk score could precisely predict the prognosis of LGG patients. Additionally, four key genes (TGFß2, SERPING1, SERPINE1, and TIMP1) were identified and found significantly associated with OS of LGG patients. Besides, they were also discovered to be strongly related to six types of immune cells which infiltrated in LGG tumor. Taken together, the present study demonstrated the promising potential of the ARG risk score formula as an independent factor for LGG prediction. It also provided the autophagy-related signature of prognosis and potential therapeutic targets for the treatment of LGG.

DNA methylation markers in the diagnosis and prognosis of common leukemias.

The ability to identify a specific type of leukemia using minimally invasive biopsies holds great promise to improve the diagnosis, treatment selection, and prognosis prediction of patients. Using genome-wide methylation profiling and machine learning methods, we investigated the utility of CpG methylation status to differentiate blood from patients with acute lymphocytic leukemia (ALL) or acute myelogenous leukemia (AML) from normal blood. We established a CpG methylation panel that can distinguish ALL and AML blood from normal blood as well as ALL blood from AML blood with high sensitivity and specificity. We then developed a methylation-based survival classifier with 23 CpGs for ALL and 20 CpGs for AML that could successfully divide patients into high-risk and low-risk groups, with significant differences in clinical outcome in each leukemia type. Together, these findings demonstrate that methylation profiles can be highly sensitive and specific in the accurate diagnosis of ALL and AML, with implications for the prediction of prognosis and treatment selection.

Impaired lipid metabolism by age-dependent DNA methylation alterations accelerates aging.

Epigenetic alterations and metabolic dysfunction are two hallmarks of aging. However, the mechanism of how their interaction regulates aging, particularly in mammals, remains largely unknown. Here we show ELOVL fatty acid elongase 2 (Elovl2), a gene whose epigenetic alterations are most highly correlated with age prediction, contributes to aging by regulating lipid metabolism. Impaired Elovl2 function disturbs lipid synthesis with increased endoplasmic reticulum stress and mitochondrial dysfunction, leading to key accelerated aging phenotypes. Restoration of mitochondrial activity can rescue age-related macular degeneration (AMD) phenotypes induced by Elovl2 deficiency in human retinal pigmental epithelial (RPE) cells. We revealed an epigenetic-metabolism axis contributing to aging and potentially to antiaging therapy.

Histone demethylase JMJD6 regulates cellular migration and proliferation in adipose-derived mesenchymal stem cells.

BACKGROUND: Adipose-derived mesenchymal stem cells (ADSCs) have been extensively explored as a promising therapeutic agent due to their differentiation, proliferation and migration abilities. The epigenetic mechanisms that regulate the fate of mesenchymal stem cells (MSCs) have been described in detail. However, the epigenetic modulation of ADSCs proliferation and migration is poorly understood. METHODS: The present study examined histone demethylases roles and expression by RT-PCR, as well as through siRNA screening and ChIP-qPCR assay. Cellular proliferation and migration assays were employed in shRNA-mediated JMJD6 knockdown and control ADSCs. PDE1C inhibition studies were conducted to confirm its role in JMJD6-mediated epigenetic regulation of ADSCs. RESULTS: The data demonstrate that the histone demethylase JMJD6 plays a critical role in regulating the proliferation and migration of ADSCs by removing H4R3me2a at the promoter regions of PDEC1 and suppressing PDEC1 expression. Importantly, the depletion of JMJD6 in ADSCs significantly increased cellular proliferation and motility, which was associated with increases in PDE1C expression and decreases in the levels of both cAMP and cGMP. The increase in proliferation and migration was reversed by treatment with a PDE1C inhibitor, suggesting that JMJD6 attenuates the proliferation and migration of ADSCs as an epigenetic regulator and PDE1C partially contributes to the JMJD6-mediated regulation. CONCLUSIONS: Taken together, our results indicate for the first time that JMJD6 plays an important role in the regulation of ADSCs proliferation and migration through the modulation of PDE1C expression.

Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma.

An effective blood-based method for the diagnosis and prognosis of hepatocellular carcinoma (HCC) has not yet been developed. Circulating tumour DNA (ctDNA) carrying cancer-specific genetic and epigenetic aberrations may enable a noninvasive 'liquid biopsy' for diagnosis and monitoring of cancer. Here, we identified an HCC-specific methylation marker panel by comparing HCC tissue and normal blood leukocytes and showed that methylation profiles of HCC tumour DNA and matched plasma ctDNA are highly correlated. Using cfDNA samples from a large cohort of 1,098 HCC patients and 835 normal controls, we constructed a diagnostic prediction model that showed high diagnostic specificity and sensitivity (P < 0.001) and was highly correlated with tumour burden, treatment response, and stage. Additionally, we constructed a prognostic prediction model that effectively predicted prognosis and survival (P < 0.001). Together, these findings demonstrate in a large clinical cohort the utility of ctDNA methylation markers in the diagnosis, surveillance, and prognosis of HCC.

[Identification of novel compound heterozygous mutations of USH2A gene in a family with Usher syndrome type II].

OBJECTIVE: To identify potential mutations in a Chinese family with Usher syndrome type II. METHODS: Genomic DNA was obtained from two affected and four unaffected members of the family and subjected to amplification of the entire coding sequence and splicing sites of USH2A gene. Mutation detection was conducted by direct sequencing of the PCR products. A total of 100 normal unrelated individuals were used as controls. RESULTS: The patients were identified to be a compound heterozygote for two mutations: c.8272G>T (p.E2758X) in exon 42 from his mother and c.12376-12378ACT>TAA(p.T4126X) in exon 63 of the USH2A gene from his father. Both mutations were not found in either of the two unaffected family members or 100 unrelated controls, and had completely co-segregated with the disease phenotype in the family. Neither mutation has been reported in the HGMD database. CONCLUSION: The novel compound heterozygous mutations c.8272G>T and c.12376-12378ACT>TAA within the USH2A gene may be responsible for the disease. This result may provide new clues for molecular diagnosis of this disease.

[Genetic diagnosis for a Chinese Han family with hereditary multiple osteochondromas].

OBJECTIVE: To identify the genetic cause for a Chinese Han family affected with hereditary multiple osteochondromas. METHODS: Two patients, five unaffected relatives of the family and 100 unrelated healthy controls were collected. The coding sequences and intron/exon boundaries of EXT1 gene were amplified with polymerase chain reaction (PCR) and sequenced. RESULTS: A heterozygous c.600G>A (p.Trp200X) mutation in exon 1 of the EXT1 gene was detected in the patients. The same mutation was not found in unaffected family members and 100 healthy controls. CONCLUSION: The hereditary multiple osteochondromas in the family is caused by a nonsense mutation (p.Trp200X) in the EXT1 gene.

Genetic analysis of the forkhead transcriptional factor 2 gene in three Chinese families with blepharophimosis syndrome.

PURPOSE: Clinically, blepharophimosis syndrome (BPES) has been divided into two subsets according to the association of ocular malformation with (type I) or without (type II) premature ovarian failure (POF). BPES is ascribed to mutations in the forkhead transcriptional factor 2 (FOXL2) gene. This study aimed at identifying clinical features and mutations within the FOXL2 gene in three Chinese families with BPES. METHODS: A clinical and molecular genetic investigation was performed in affected and unaffected members from three Chinese families with BPES. Genomic DNA was prepared from leucocytes of peripheral venous blood, the entire coding region of FOXL2 were amplified with PCR, and direct DNA sequencing of the PCR products was performed for mutations in FOXL2. RESULTS: Three mutations in FOXL2 were found in three families, including c.672_701dup30, c.663_692dup30, and c.475dupC. Of the three, the c.475dupC (p.His159fs) was novel in family C and resulted in a frameshift mutation to generate a truncated protein owing to a premature stop codon at codon 238. The new duplication mutation was associated with BPES type II. The c.672_701dup30 (p.Ala224_Ala234dup10) and the c.663_692dup30 (p.Ala221_Ala231dup10) were detected in family A and family B, respectively, leading to expansions of the polyalanine (poly-Ala) tract that is frequently the hot spot of mutations within FOXL2. CONCLUSIONS: Our results expand the spectrum of FOXL2 mutations, and further indicate the association of a novel duplication mutation leading to a truncated protein with BPES type II. The other two known mutations may support the previous hypothesis regarding expansions of the polyalanine tract associated with BPES type II as a mutational hot spot in FOXL2.

[Clinical investigation of a Chinese family with hypotrichosis simplex of the scalp and mutational analysis of CDSN gene].

OBJECTIVE: To analyze clinical symptoms and disease-causing mutations of corneodesmosin (CDSN) gene in a Chinese family affected with hypotrichosis simplex of the scalp and to establish a method for prenatal diagnosis. METHODS: Family survey and clinical examinations were carried out to determine the inheritance pattern. Three patients and 7 unaffected relatives from the family, in addition with 100 unrelated healthy controls were recruited. Genomic DNA from peripheral blood leukocytes was extracted. Five pairs of primers were designed based on the CDSN gene sequence. Exons and flanking regions of the CDSN gene were amplified using polymerase chain reaction (PCR). Potential mutations were analyzed through direct sequencing and comparison by BLAST. RESULTS: The type of alopecia of the family was diagnosed as hypotrichosis simplex of the scalp with an autosomal dominant inheritance pattern. A nonsense mutation (C717G) in cDNA sequence of the CDSN gene was identified in all three patients of the family, which resulted in a premature stop codon (Y239X). The same mutation was not found among healthy members of the family and 100 healthy controls. CONCLUSION: A Chinese family was diagnosed with hypotrichosis simplex of the scalp, which was caused by a novel nonsense mutation (Y239X) in the CDSN gene.

[Mutation analysis of FGFR3 gene in a family featuring hereditary dwarfism].

OBJECTIVE: To investigate the clinical symptoms and potential mutation in FGFR3 gene for a family featuring hereditary dwarfism in order to attain diagnosis and provide prenatal diagnosis. METHODS: Five patients and two unaffected relatives from the family, in addition with 100 healthy controls, were recruited. Genome DNA was extracted. Exons 10 and 13 of the FGFR3 gene were amplified using polymerase chain reaction (PCR). PCR products were sequenced in both directions. RESULTS: All patients had similar features including short stature, short limbs, lumbar hyperlordosis but normal craniofacial features. A heterozygous mutation G1620T (N540K) was identified in the cDNA from all patients but not in the unaffected relatives and 100 control subjects. A heterozygous G380R mutation was excluded. CONCLUSION: The hereditary dwarfism featured by this family has been caused by hypochondroplasia (HCH) due to a N540K mutation in the FGFR3 gene.

T lymphocyte responses against hepatitis B virus-related hepatocellular carcinoma induced by adenovirus vaccine encoding HBx.

HBx is an oncogenic tumor-associated antigen and is dominantly expressed in hepatitis and hepatoma tissues, the induction of active cellular responses against HBx should be a promising approach for the treatment of hepatitis B virus-related hepatocellular carcinoma. The present study was designed to test whether a replication-defective adenovirus vaccine expressing HBx antigen could be effectively used in the immunotherapy of hepatocellular carcinoma. To validate the possibility, we developed a novel HBx-positive hepatocellular carcinoma in mice by inoculated the pcDNA-HBx transfected Hepa1-6 cells subcutaneously into the right flank of mice. We found that immunotherapy with Ad-HBx was effective at both protective and therapeutic antitumor immunity in the hepatoma models in immune-competent mice. Histological examination revealed that Ad-HBx treatment led to significantly increased induction of apoptosis, tumor necrosis, and elevated CD8+ lymphocyte infiltration. In addition, the induction efficacy of the CTL response is dramatically enhanced by immunotherapy. Cytokine analysis confirmed that the antitumor efficacy of Ad-HBx may mostly result from cellular immunity. Our findings may prove useful in development of adenovirus vaccine based on HBx antigen to the treatment of HBV-associated hepatocellular carcinoma.