GRANULYSIN PEPTIDE AND GENE POLYMORPHISM IN THE PATHOGENESIS OF HASHIMOTO THYROIDITIS.

Background: Hashimoto thyroiditis (HT) is an autoimmune disease and the most common cause of hypothyroidism. The widespread lymphocyte infiltration in the thyroid gland and intolerance of the body against its thyroid antigens leads to the destruction of thyroid cells and impaired thyroid function. Granulysin (GNLY) is a cytolytic antimicrobial peptide that has been associated with a wide range of diseases such as various infections, cancer, transplantation, and skin problems. However, there are a few studies investigating the relationship between HT and granulysin. Aim: Our study aims to investigate whether granulysin levels and GNLY gene polymorphism contribute to the damaged immune response leading to HT. Material and Methods: 100 unrelated patients diagnosed with HT and 140 healthy individuals were included in our study. Frequencies of GNLY rs10180391 and rs7908 gene polymorphisms were determined using PCR- RFLP method and serum granulysin levels were determined using ELISA. Results: There is no statistical significance between patient and control groups in terms of genotype and allele frequencies of GNLY gene polymorphisms and serum levels of granulysin. Conclusion: In conclusion, granulysin and GNLY gene polymorphisms do not appear to relate to HT disease.

A new nucleic acid-based agent inhibits cytotoxic T lymphocyte-mediated immune disorders.

BACKGROUND: Stevens-Johnson syndrome (SJS), toxic epidermal necrolysis (TEN), and graft-versus-host disease (GVHD) are distinct immune reactions elicited by drugs or allogeneic antigens; however, they share a pathomechanism with the activation of cytotoxic T lymphocytes (CTLs). CTLs produce cytotoxic proteins, cytokines, chemokines, or immune alarmins, such as granulysin (GNLY), leading to the extensive tissue damage and systemic inflammation seen in patients with SJS/TEN or GVHD. Currently, there is no effective therapeutic agent specific for CTL-mediated immune disorders. OBJECTIVES: By targeting GNLY(+) CTLs, we aimed to develop a nucleic acid-based agent consisting of an anti-CD8 aptamer with GNLY small interfering RNA (siRNA). METHODS: We performed systematic evolution of ligands using exponential enrichment to select and identify effective anti-CD8 aptamers. We developed an aptamer-siRNA chimera using a "sticky bridge" method by conjugating the aptamer with siRNA. We analyzed the inhibitory effects of the aptamer-siRNA chimera on CTL responses in patients with SJS/TEN or GVHD. RESULTS: We identified a novel DNA aptamer (CD8AP17s) targeting CTLs. This aptamer could be specifically internalized into human CTLs. We generated the CD8AP17s aptamer-GNLY siRNA chimera, which showed a greater than 79% inhibitory effect on the production of GNLY by drug/alloantigen-activated T cells. The CD8AP17s aptamer-GNLY siRNA chimera decreased cytotoxicity in in vitro models of both SJS/TEN (elicited by drug-specific antigen) and GVHD (elicited by allogeneic antigens). CONCLUSIONS: Our results identified a new nucleic acid-based agent (CD8 aptamer-GNLY siRNA chimera) that can significantly inhibit CTL-mediated drug hypersensitivity, such as that seen in patients with SJS/TEN, as well as the alloreactivity seen in patients with GVHD. This study provides a novel therapeutic strategy for CTL-mediated immune disorders.

Implications of accumulation of clonally expanded and senescent CD4+GNLY+ T cells in immunological non-responders of HIV-1 infection.

Increased CD4+GNLY+ T cells have been confirmed to be inversely associated with CD4+ T cell count in immunological non-responders (INRs), however, the underlying mechanisms are unknown. This study aimed to elucidate the characteristics of CD4+GNLY+ T cells and their relationship with immune restoration. Single-cell RNA sequencing, single-cell TCR sequencing, and flow cytometry were used to analyze the frequency, phenotypes, and function of CD4+GNLY+ T cells. Moreover, Enzyme linked immunosorbent assay was performed to detect plasma cytokines production in patients. CD4+GNLY+ T cells were found to be highly clonally expanded, characterized by higher levels of cytotoxicity, senescence, P24, and HIV-1 DNA than CD4+GNLY- T cells. Additionally, the frequency of CD4+GNLY+ T cells increased after ART, and further increased in INRs, and were positively associated with the antiretroviral therapy duration in INR. Furthermore, increased IL-15 levels in INRs positively correlated with the frequency and senescence of CD4+GNLY+ T cells, suggesting that CD4+GNLY+ T cells may provide new insights for understanding the poor immune reconstitution of INRs. In conclusion, increased, highly clonally expanded, and senescent CD4+GNLY+ T cells may contribute to poor immune reconstitution in HIV-1 infection.

Identification of AP002498.1 and LINC01871 as prognostic biomarkers and therapeutic targets for distant metastasis of colorectal adenocarcinoma.

BACKGROUND: Increasing evidence suggests that lncRNA (Long non-coding RNA, lncRNA)-mediated ceRNA (competing endogenous RNA, ceRNA) networks are involved in the occurrence and progression of colorectal cancer (CRC). However, the roles of the lncRNA-miRNA-mRNA ceRNA network in distant metastasis of CRC are still unclear. METHODS: In this study, we constructed a specific ceRNA network to identify potential biomarkers and therapeutic targets for distant metastasis of CRC. Specifically, RNA-Seq data from The Cancer Genome Atlas (TCGA) were used to screen for differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) related to metastasis. After validation and selection by qRT-PCR and univariate and multivariate analysis of the metastasis- and prognosis-related lncRNAs, the regulated microRNAs (miRNAs) and coexpressed mRNAs were used to construct a ceRNA network for distant metastasis of CRC. RESULTS: Two key distant metastasis-related DElncRNAs, AP002498.1 and LINC01871, were identified by univariate and multivariate analysis in combination with analyses of clinical data and expression levels. Furthermore, lncRNA-associated ceRNA subnetworks were constructed from the predicted miRNAs and 13 coexpressed DEmRNAs (SERPINA1, ITLN1, REG4, L1TD1, IGFALS, MUC5B, CIITA, CXCL9, CXCL10, GBP4, GNLY, IDO1, and NOS2). The AP002498.1- and LINC01871-associated ceRNA subnetworks regulated the expression of the target genes SERPINA1 and MUC5B and GNLY, respectively, through the associated miRNAs. CONCLUSION: The DElncRNA AP002498.1 and the LINC01871/miR-4644 and miR-185-5p/GNLY axes were identified as being closely associated with distant metastasis and could represent independent prognostic biomarkers or therapeutic targets in colorectal adenocarcinoma.

A Missense Variant in Granulysin is Associated with the Efficacy of Pegylated-Interferon-Alpha Therapy in Chinese Patients with HBeAg-Positive Chronic Hepatitis B.

PURPOSE: Granulysin (GNLY) is a cytotoxic granule that has been reported to have various antimicrobial activities. We evaluated the association between a missense variant in GNLY (rs11127) and treatment efficacy of pegylated interferon-alpha (PegIFNα) or nucleos(t)ide analogs (NUCs) in patients with chronic hepatitis B (CHB). PATIENTS AND METHODS: We included a total of 1823 patients with hepatitis B e antigen (HBeAg)-positive CHB (954 patients treated with PegIFNα and 869 patients treated with NUCs) in four Phase IV multicenter randomized controlled trials. The association of the GNLY rs11127 genotype with the combined response (CR), defined as HBeAg seroconversion and hepatitis B virus (HBV) DNA level <2000 IU/mL was evaluated. A polygenic score (PGS) was constructed to evaluate the cumulative effect of multiple single-nucleotide polymorphisms (SNPs), including rs11127 and several other SNPs, STAT4 rs7574865, CFB rs12614, and CD55 rs28371597, which were reported to be associated with CR. RESULTS: GNLY rs11127 was significantly associated with CR in patients treated with PegIFNα. The CR rate in patients with the rs11127 CC genotype was higher than that with the CT or TT genotype (40.98% vs 30.34% or 27.09%, P = 0.003). Furthermore, a PGS integrating GNLY rs11127 and three other SNPs was significantly associated with CR in PegIFNα-treated patients (P < 0.001). However, no significant correlation was found between GNLY rs11127 and CR in NUCs-treated patients. CONCLUSION: GNLY rs11127 is an independent biomarker for predicting the response to PegIFNα therapy in HBeAg-positive CHB patients. Furthermore, the PGS, including GNLY rs11127, provides new insights for individualized treatment in clinical practice.

Probing infectious disease by single-cell RNA sequencing: Progresses and perspectives.

The increasing application of single-cell RNA sequencing (scRNA-seq) technology in life science and biomedical research has significantly increased our understanding of the cellular heterogeneities in immunology, oncology and developmental biology. This review will summarize the development of various scRNA-seq technologies; primarily discussing the application of scRNA-seq on infectious diseases, and exploring the current development, challenges, and potential applications of scRNA-seq technology in the future.

Potential biomarkers for the early diagnosis of colorectal adenocarcinoma - transcriptomic analysis of four clinical stages.

BACKGROUNDS: Colorectal cancer is the third most common cancer in economically developed countries. Molecular studies and, in particular, gene expression have contributed to advances in the diagnosis and treatment of many cancers. Genes can be molecular and therapeutic markers, but because of the large molecular diversity in colorectal cancer the knowledge is not yet fully established. Probably one of the most crucial processes during early cancer development is inflammation. The inflammatory response in the tumor is an important indicator of molecular etiology and later of cancer progression. OBJECTIVE: The aim of this work is to identify potential biomarkers for early stage of colorectal adenocarcinoma in patients' bowel tissues using transcriptomic analysis. METHODS: Expression of the inflammatory response genes of colorectal cancer at all clinical stages (I-IV) and control of the bowel were evaluated by oligonucleotide microarrays. RESULTS: Based on statistical analysis many differentially expressed genes were selected. LCK (LCK Proto-Oncogene, Src Family Tyrosine Kinase), GNLY (granulysin), SLC6A6 (Solute-Carrier Family 6 Member 6) and LAMP2 (Lysosomal Associated Membrane Protein 2) were specific for the early stage of the disease. These genes had the properties of the good biomarkers. CONCLUSIONS: The expression of LCK, GNLY, SLC6A6 and LAMP2 genes could be valuable potential diagnostic biomarkers of the early stage of colorectal adenocarcinoma.

Expression of genes associated with cholesterol and lipid metabolism identified as a novel pathway in the early pathogenesis of Mycobacterium avium subspecies paratuberculosis-infection in cattle.

Johne's disease (JD) is a chronic disease affecting ruminants and other species caused by the pathogenic mycobacterium, Mycobacterium avium subsp. paratuberculosis (MAP). MAP has developed a multitude of mechanisms to persist within the host, and these in turn are counteracted by the host through various immune pathways. Identifying and characterising the different strategies employed by MAP to alter the host immune system in its favour, and thereby persist intracellularly, could hold the key to developing strategies to fight this disease. In this study we analysed a subset of bovine microarray data derived from early time points after experimental infection with MAP. A specifically developed integrated approach was used to identify and validate host genes involved in cholesterol homeostasis (24DHCR, LDLR, SCD-1), calcium homeostasis and anti-bacterial defence mechanisms, (CD38, GIMAP6) which were downregulated in response to MAP exposure. A trend for upregulation of granulysin gene expression in MAP-exposed cattle in comparison to unexposed cattle was also observed. From these analyses, a model of potential pathogen-host interactions involving these novel pathways was developed which indicates an important role for host lipids in mycobacterial survival and persistence.