Enhancement of NETosis by ACE2-cross-reactive anti-SARS-CoV-2 RBD antibodies in patients with COVID-19.

BACKGROUND: High levels of neutrophil extracellular trap (NET) formation or NETosis and autoantibodies are related to poor prognosis and disease severity of COVID-19 patients. Human angiotensin-converting enzyme 2 (ACE2) cross-reactive anti-severe acute respiratory syndrome coronavirus 2 spike protein receptor-binding domain (SARS-CoV-2 RBD) antibodies (CR Abs) have been reported as one of the sources of anti-ACE2 autoantibodies. However, the pathological implications of CR Abs in NET formation remain unknown. METHODS: In this study, we first assessed the presence of CR Abs in the sera of COVID-19 patients with different severity by serological analysis. Sera and purified IgG from CR Abs positive COVID-19 patients as well as a mouse monoclonal Ab (mAb 127) that can recognize both ACE2 and the RBD were tested for their influence on NETosis and the possible mechanisms involved were studied. RESULTS: An association between CR Abs levels and the severity of COVID-19 in 120 patients was found. The CR Abs-positive sera and IgG from severe COVID-19 patients and mAb 127 significantly activated human leukocytes and triggered NETosis, in the presence of RBD. This NETosis, triggered by the coexistence of CR Abs and RBD, activated thrombus-related cells but was abolished when the interaction between CR Abs and ACE2 or Fc receptors was disrupted. We also revealed that CR Abs-induced NETosis was suppressed in the presence of recombinant ACE2 or the Src family kinase inhibitor, dasatinib. Furthermore, we found that COVID-19 vaccination not only reduced COVID-19 severity but also prevented the production of CR Abs after SARS-CoV-2 infection. CONCLUSIONS: Our findings provide possible pathogenic effects of CR Abs in exacerbating COVID-19 by enhancing NETosis, highlighting ACE2 and dasatinib as potential treatments, and supporting the benefit of vaccination in reducing disease severity and CR Abs production in COVID-19 patients.

Folic Acid Ameliorates Renal Injury in Experimental Obstructive Nephropathy: Role of Glycine N-Methyltransferase.

Folic acid exerts both anti-inflammatory and antifibrotic effects. Glycine N-methyltransferase (GNMT), the major folic acid-binding protein in the liver, is a crucial enzyme that regulates the cellular methylation process by maintaining S-adenosylmethionine levels. However, as yet neither the therapeutic effects of folic acid in renal fibrosis nor whether GNMT is involved in these folic acid-associated mechanisms has been investigated. First, the expression of GNMT was examined in human kidneys with or without obstructive nephropathy. Later, wild-type and GNMT knockout (GNMT-/-) mice were subjected to unilateral ureteral obstruction (UUO) and then treated with either folic acid or vehicle for 14 days. Renal tubular injury, inflammation, fibrosis, and autophagy were evaluated by histological analysis and Western blotting. We observed increased expression of GNMT in humans with obstructive nephropathy. Furthermore, UUO significantly increased the expression of GNMT in mice; in addition, it caused renal injury as well as the development of both hydronephrosis and tubular injury. These were all alleviated by folic acid treatment. In contrast, GNMT-/- mice exhibited exacerbated UUO-induced renal injury, but the protective effect of folic acid was not observed in GNMT-/- mice. We propose a novel role for folic acid in the treatment of renal fibrosis, which indicates that GNMT may be a therapeutic target.

Reversal of High-Fat Diet-Induced Non-Alcoholic Fatty Liver Disease by Metformin Combined with PGG, an Inducer of Glycine N-Methyltransferase.

Nonalcoholic fatty liver disease (NAFLD) is a major cause of liver-related morbidities and mortality, and no effective drug treatment currently exists. We aimed to develop a novel treatment strategy to induce the expression of glycine N-methyltransferase (GNMT), which is an important enzyme regulating S-adenosylmethionine metabolism whose expression is downregulated in patients with NAFLD. Because 1,2,3,4,6-pentagalloyl glucose (PGG) is a GNMT inducer, and metformin was shown to upregulate liver mitochondrial GNMT protein expression, the effect of PGG and metformin was evaluated. Biochemical analysis, histopathological examination, immunohistochemical staining, reverse transcription-quantitative PCR (RT-qPCR), Western blotting (WB), proteomic analysis and Seahorse XF Cell Mito Stress Test were performed. The high-fat diet (HFD)-induced NAFLD mice were treated with PGG and metformin. Combination of PGG and metformin nearly completely reversed weight gain, elevation of serum aminotransferases, and hepatic steatosis and steatohepatitis. In addition, the downregulated GNMT expression in liver tissues of HFD-induced NAFLD mice was restored. The GNMT expression was further confirmed by RT-qPCR and WB analysis using both in vitro and in vivo systems. In addition, PGG treatment was shown to increase oxygen consumption rate (OCR) maximum capacity in a dose-dependent manner, and was capable of rescuing the suppression of mitochondrial OCR induced by metformin. Proteomic analysis identified increased expression of glutathione S-transferase mu 4 (GSTM4), heat shock protein 72 (HSP72), pyruvate carboxylase (PYC) and 40S ribosomal protein S28 (RS28) in the metformin plus PGG treatment group. Our findings show that GNMT expression plays an important role in the pathogenesis of NAFLD, and combination of an inducer of GNMT and metformin can be of therapeutic potential for patients with NAFLD.

High prevalence of HIV-1 transmitted drug resistance and factors associated with time to virological failure and viral suppression in Taiwan.

BACKGROUND: Integrase strand transfer inhibitor (InSTI)-based regimens have become the major first-line treatment for HIV-1-infected patients in Taiwan. Transmitted drug resistance (TDR) and several clinical characteristics are associated with time to virological failure or viral suppression; however, these have not been investigated in Taiwan. OBJECTIVES: To determine the impact of several factors on treatment outcomes in HIV-1-infected patients in Taiwan. METHODS: The cohort included 164 HIV-1 treatment-naive patients in Taiwan from 2018 to 2020. Blood specimens were collected to determine the genotypic drug resistance using the Stanford University HIV drug resistance database. Cox proportional hazards models were used to identify factors associated with time to virological failure or viral suppression. RESULTS: The prevalence of TDR in Taiwan was 27.4% and an increasing trend was seen from 2018 to 2020. TDR mutations related to NNRTIs were the most prevalent (21%) while TDR to InSTIs remained at a relatively low level (1.3%). A baseline HIV-1 viral load of ≥100 000 copies/mL was associated with a shorter time to virological failure [multivariate hazard ratio (mHR) 7.84; P = 0.018] and longer time to viral suppression (mHR 0.46; P < 0.001). Time to viral suppression was shorter in patients receiving InSTI-based regimens (mHR 2.18; P = 0.006). Different InSTI-based regimens as initial treatment did not affect the treatment outcomes. CONCLUSIONS: This study found an increasing trend of HIV-1 TDR prevalence from 2018 to 2020 in Taiwan. Baseline HIV-1 viral load and receiving InSTI-based regimens are important factors associated with time to virological failure or viral suppression.

HIV-1 Tat Interacts with a Kaposi's Sarcoma-Associated Herpesvirus Reactivation-Upregulated Antiangiogenic Long Noncoding RNA, LINC00313, and Antagonizes Its Function.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), an AIDS-defining cancer with abnormal angiogenesis. The high incidence of KS in human immunodeficiency virus (HIV)-infected AIDS patients has been ascribed to an interaction between HIV type 1 (HIV-1) and KSHV, focusing on secretory proteins. The HIV-1 secreted protein HIV Tat has been found to synergize with KSHV lytic proteins to induce angiogenesis. However, the impact and underlying mechanisms of HIV Tat in KSHV-infected endothelial cells undergoing viral lytic reactivation remain unclear. Here, we identified LINC00313 as a novel KSHV reactivation-activated long noncoding RNA (lncRNA) that interacts with HIV Tat. We found that LINC00313 overexpression inhibits cell migration, invasion, and tube formation, and this suppressive effect was relieved by HIV Tat. In addition, LINC00313 bound to polycomb repressive complex 2 (PRC2) complex components, and this interaction was disrupted by HIV Tat, suggesting that LINC00313 may mediate transcription repression through recruitment of PRC2 and that HIV Tat alleviates repression through disruption of this association. This notion was further supported by bioinformatics analysis of transcriptome profiles in LINC00313 overexpression combined with HIV Tat treatment. Ingenuity Pathway Analysis (IPA) showed that LINC00313 overexpression negatively regulates cell movement and migration pathways, and enrichment of these pathways was absent in the presence of HIV Tat. Collectively, our results illustrate that an angiogenic repressive lncRNA, LINC00313, which is upregulated during KSHV reactivation, interacts with HIV Tat to promote endothelial cell motility. These results demonstrate that an lncRNA serves as a novel connector in HIV-KSHV interactions.IMPORTANCE KS is a prevalent tumor associated with infections with two distinct viruses, KSHV and HIV. Since KSHV and HIV infect distinct cell types, the virus-virus interaction associated with KS formation has focused on secretory factors. HIV Tat is a well-known RNA binding protein secreted by HIV. Here, we revealed LINC00313, an lncRNA upregulated during KSHV lytic reactivation, as a novel HIV Tat-interacting lncRNA that potentially mediates HIV-KSHV interactions. We found that LINC00313 can repress endothelial cell angiogenesis-related properties potentially by interacting with chromatin remodeling complex PRC2 and downregulation of cell migration-regulating genes. An interaction between HIV Tat and LINC00313 contributed to the dissociation of PRC2 from LINC00313 and the disinhibition of LINC00313-induced repression of cell motility. Given that lncRNAs are emerging as key players in tissue physiology and disease progression, including cancer, the mechanism identified in this study may help decipher the mechanisms underlying KS pathogenesis induced by HIV and KSHV coinfection.

Utilizing an Animal Model to Identify Brain Neurodegeneration-Related Biomarkers in Aging.

Glycine N-methyltransferase (GNMT) regulates S-adenosylmethionine (SAMe), a methyl donor in methylation. Over-expressed SAMe may cause neurogenic capacity reduction and memory impairment. GNMT knockout mice (GNMT-KO) was applied as an experimental model to evaluate its effect on neurons. In this study, proteins from brain tissues were studied using proteomic approaches, Haemotoxylin and Eosin staining, immunohistochemistry, Western blotting, and ingenuity pathway analysis. The expression of Receptor-interacting protein 1(RIPK1) and Caspase 3 were up-regulated and activity-dependent neuroprotective protein (ADNP) was down-regulated in GNMT-KO mice regardless of the age. Besides, proteins related to neuropathology, such as excitatory amino acid transporter 2, calcium/calmodulin-dependent protein kinase type II subunit alpha, and Cu-Zn superoxide dismutase were found only in the group of aged wild-type mice; 4-aminobutyrate amino transferase, limbic system-associated membrane protein, sodium- and chloride-dependent GABA transporter 3 and ProSAAS were found only in the group of young GNMT-KO mice and are related to function of neurons; serum albumin and Rho GDP dissociation inhibitor 1 were found only in the group of aged GNMT-KO mice and are connected to neurodegenerative disorders. With proteomic analyses, a pathway involving Gonadotropin-releasing hormone (GnRH) signal was found to be associated with aging. The GnRH pathway could provide additional information on the mechanism of aging and non-aging related neurodegeneration, and these protein markers may be served in developing future therapeutic treatments to ameliorate aging and prevent diseases.

Amino Acid Deletions in p6Gag Domain of HIV-1 CRF07_BC Ameliorate Galectin-3 Mediated Enhancement in Viral Budding.

HIV-1 CRF07_BC is a recombinant virus with amino acid (a.a.) deletions in p6Gag, which are overlapped with the Alix-binding domain. Galectin-3 (Gal3), a ß-galactose binding lectin, has been reported to interact with Alix and regulate HIV-1 subtype B budding. This study aims to evaluate the role of Gal3 in HIV-1 CRF07_BC infection and the potential effect of a.a. deletions on Gal3-mediated regulation. A total of 38 HIV-1+ injecting drug users (IDUs) were enrolled in the study. Viral characterization and correlation of Gal3 were validated. CRF07_BC containing 7 a.a. deletions and wild-type in the p6Gag (CRF07_BC-7d and -wt) were isolated and infectious clones were generated. Viral growth kinetic and budding assays using Jurkat-CCR5/Jurkat-CCR5-Gal3 cells infected with CRF07_BC were performed. Results indicate that 69.4% (25/38) of the recruited patients were identified as CRF07_BC, and CRF07_BC-7d was predominant. Slow disease progression and significantly higher plasma Gal3 were noted in CRF07_BC patients (p < 0.01). Results revealed that CRF07_BC infection resulted in Gal3 expression, which was induced by Tat. Growth dynamic and budding assays indicated that Gal3 expression in Jurkat-CCR5 cells significantly enhanced CRF07_BC-wt replication and budding (p < 0.05), while the promoting effect was ameliorated in CRF07_BC-7d. Co-immunoprecipitation found that deletions in the p6Gag reduced Gal-3-mediated enhancement of the Alix-Gag interaction.

Chemical Constituents with GNMT-Promoter-Enhancing and NRF2-Reduction Activities from Taiwan Agarwood Excoecaria formosana.

Hepatocellular carcinoma (HCC) is considered to be a silent killer, and was the fourth leading global cause of cancer deaths in 2018. For now, sorafenib is the only approved drug for advanced HCC treatment. The introduction of additional chemopreventive agents and/or adjuvant therapies may be helpful for the treatment of HCC. After screening 3000 methanolic extracts from the Formosan plant extract bank, Excoecaria formosana showed glycine N-methyltransferase (GNMT)-promoter-enhancing and nuclear factor erythroid 2-related factor 2 (NRF2)-suppressing activities. Further, the investigation of the whole plant of E. formosana led to the isolation of a new steroid, 7α-hydroperoxysitosterol-3-O-ß-d-(6-O-palmitoyl)glucopyranoside (1); two new coumarinolignans, excoecoumarin A (2) and excoecoumarin B (3); a new diterpene, excoeterpenol A (4); and 40 known compounds (5-44). Among them, Compounds 38 and 40-44 at a 100 µM concentration showed a 2.97 ± 0.27-, 3.17 ± 1.03-, 2.73 ± 0.23-, 2.63 ± 0.14-, 6.57 ± 0.13-, and 2.62 ± 0.05-fold increase in GNMT promoter activity, respectively. In addition, Compounds 40 and 43 could reduce NRF2 activity, a transcription factor associated with drug resistance, in Huh7 cells with relative activity of 33.1 ± 0.2% and 45.2 ± 2.5%. These results provided the basis for the utilization of Taiwan agarwood for the development of anti-HCC agents.

Tumor suppressor gene glycine N-methyltransferase and its potential in liver disorders and hepatocellular carcinoma.

Glycine N-methyltransferase is a protein with many functions. In addition to catalyzing the production of sarcosine in the one carbon metabolism pathway, it plays a role in the detoxification of environmental carcinogens such as benzo[a]pyrene, aflatoxin B1, and aristocholic acid. There is also increasing evidence suggesting a role of GNMT deficiency in liver carcinogenesis. In this review, we discuss the role of GNMT in the detoxification of xenobiotics and the mechanism of GNMT suppression during liver tumorigenesis. The protective role of GNMT in the liver allows GNMT to not only serve as a marker of liver disease, but also potentially be applied in the treatment of liver disorders and hepatocellular carcinoma. We describe the potential use of GNMT in gene therapy and we introduce the development of a GNMT promoter reporter assay that can be used to screen medicinal drugs and herbal libraries for natural compounds with anti-cancer properties.

Loss of Glycine N-Methyltransferase Associates with Angiopoietin-Like Protein 8 Expression in High Fat-Diet-Fed Mice.

Imbalance of lipid metabolism is a main cause of metabolic syndrome leading to life-threatening metabolic diseases. Angiopoietin-like protein 8 (Angptl8) was recently identified as a liver and adipose tissue-released hormone that is one of the molecules involved in triglyceride metabolism. However, the regulatory mechanism of Angptl8 is largely unknown. A high fat diet (HFD)-fed mouse model, which showed high cholesterol, high triglyceride, and high insulin in the blood, revealed the upregulation of hepatic and plasma Angptl8 and the downregulation of hepatic glycine N-methyltransferase (GNMT). The inverse correlation of hepatic Angptl8 and GNMT expression in the livers of HFD-fed mice was also confirmed in a publicly available microarray dataset. The mechanistic study using primary hepatocytes showed that the Angptl8 expression could be induced by insulin treatment in a dose- and time-dependent manner. Inhibition of PI3K/Akt pathway by the specific inhibitors or the dominant-negative Akt blocked the insulin-induced Angptl8 expression. Moreover, knockout of GNMT promoted the Akt activation as well as the Angptl8 expression. These results suggested that GNMT might be involved in insulin-induced Angptl8 expression in HFD-mediated metabolic syndrome.

Simple Strategy for Rapid and Sensitive Detection of Avian Influenza A H7N9 Virus Based on Intensity-Modulated SPR Biosensor and New Generated Antibody.

In 2013 a new reassortant avian influenza A H7N9 virus emerged in China, causing human infection with high mortality. An accurate and timely diagnosis is crucial for controlling the outbreaks of the disease. We therefore propose a simple strategy for rapidly and sensitively detecting the H7N9 virus using an intensity-modulated surface plasmon resonance (IM-SPR) biosensor integrated with a new generated monoclonal antibody. The novel antibody exhibits significant specificity to recognize H7N9 virus compared with other clinical human influenza isolates (p < 0.01). Experimentally, the detection limit of the proposed approach for H7N9 virus detection is estimated to be 144 copies/mL, which is a 20-fold increase in sensitivity compared with homemade target-captured ELISA using the identical antibody. For the measurement of mimic clinical specimens containing the H7N9 virus mixed with nasal mucosa from flu-like syndrome patients, the detection limit is calculated to be 402 copies/mL, which is better than conventional influenza detection assays; quantitative reverse transcription polymerase chain reaction (qRT-PCR) and rapid influenza diagnostic test (RIDT). Most importantly, the assay time took less than 10 min. Combined, the results of this study indicate that the proposed simple strategy demonstrates high sensitivity and time-saving in H7N9 virus detection. By incorporating a high specific recognizer, the proposed technique has the potential to be used in applications and development of other emerging or re-emerging microbe detection platforms.

Bioactive Triterpenoids from the Leaves and Twigs of Lithocarpus litseifolius and L. corneus.

Phytochemical investigation of the leaves and twigs of Lithocarpus litseifolius and Lithocarpus corneus resulted in the isolation of four new triterpenoids (1: -4: ), three triterpenoids (5: -7: ) isolated from a natural source for the first time, and six known compounds (8: -13: ). In addition, four known triterpenoids (14: -17: ) were isolated from L. corneus. Compound 1: is a 3,4-seco-lupane-type triterpenoid, and compounds 2: -4: are lupane-type triterpenoids in different oxidation states. The structures of all isolated compounds were identified by spectroscopic methods, especially NMR and mass spectrometry data. The absolute configuration of 2: and 3: was confirmed by X-ray single crystallographic analysis. The anti-inflammatory activities of 1: -17: and anti-HIV activities of 2: -17: were evaluated. Among them, 3-epi-betulinic acid (8: ) showed a strong anti-HIV activity comparable to abacavir, a drug used for treating HIV/AIDS. 3,4-seco-4(23),20(29)-lupadiene-3,28-dioic acid (5: ) exhibited potent inhibition of superoxide-anion generation with 86.9 ± 2.8% inhibition at 1 µM.

Human Immunodeficiency Virus Infection Increases the Risk of Incident Autoimmune Hemolytic Anemia: A Population-Based Cohort Study in Taiwan.

Background: Currently, the association between human immunodeficiency virus (HIV) infection and subsequent development of autoimmune hemolytic anemia (AIHA) remains unclear. This nationwide population-based cohort study aimed to determine the association between incident AIHA and HIV infection in Taiwan. Methods: During 2000-2012, we identified people aged ≧15 years living with HIV (PLWH) from the Taiwan Centers for Disease Control HIV Surveillance System. Individuals were considered to be infected with HIV on the basis of positive results of an HIV type 1 Western blot. Age- and sex-matched controls without HIV infection were selected from the Taiwan National Health Insurance Research Database for comparison. All patients were followed until 31 December 2012 and observed for occurrence of AIHA. Results: Of 171468 subjects (19052 PLWH and 152416 controls), 30 (0.02%) had incident AIHA during a mean follow-up of 5.45 years, including 23 PLWH (0.12%) and 7 controls (0.01%). After adjustment for age, sex, and comorbidities, HIV infection was found to be an independent risk factor of incident AIHA (adjusted hazard ratio, 20.9; 95% confidence interval, 8.34-52.3). Moreover, PLWH who were receiving highly active antiretroviral therapy were more likely to develop AIHA than those who were not receiving these drugs (adjusted hazard ratio, 16.2; 95% confidence interval, 3.52-74.2). Conclusions: Our study suggests that HIV infection is an independent risk factor for incident AIHA.

Incidence of autoimmune diseases in a nationwide HIV/AIDS patient cohort in Taiwan, 2000-2012.

OBJECTIVES: It is not known if the incidences of autoimmune diseases are higher in individuals living with HIV infection or AIDS. Our study investigated the incidences of autoimmune diseases among people living with HIV/AIDS (PLWHA) in Taiwan during 2000-2012. METHODS: The Taiwan National Health Insurance Research Database was used to identify PLWHA. The incidence densities of systemic and organ-specific autoimmune diseases were calculated, and age-adjusted, sex-adjusted and period-adjusted standardised incidence rates (SIRs) were obtained by using two million people from the general population as controls. To examine the effects of highly active antiretroviral therapy (HAART) on the incidence of autoimmune diseases, the incidence densities and SIRs of autoimmune diseases were calculated after stratifying PLWHA by HAART status. RESULTS: Of the 20 444 PLWHA identified, the overall mean (SD) age was 30.1 (11.0) years; 67.2% of the subjects received HAART. As compared with the general population, SIRs were higher for incident Sjögren syndrome (SIR=1.64; 95% CI 1.24 to 2.13), psoriasis (SIR=2.05; 95% CI 1.67 to 2.48), systemic lupus erythematosus (SLE) (SIR=2.59; 95% CI 1.53 to 4.09), autoimmune haemolytic anaemia (SIR=35.06; 95% CI 23.1 to 51.02) and uveitis (SIR=2.50; 95% CI 2.05 to 3.02), but were lower for incident ankylosing spondyloarthritis (SIR=0.70; 95% CI 0.48 to 0.99). When the effect of HAART on incident autoimmune diseases was considered, PLWHA who received HAART had higher SIRs for psoriasis, autoimmune haemolytic anaemia and uveitis, but had lower risks of rheumatoid arthritis (RA) and ankylosing spondyloarthritis. In contrast, PLWHA who did not receive HAART had higher SIRs for Sjögren syndrome, psoriasis, RA, SLE, scleroderma, polymyositis, autoimmune haemolytic anaemia and Hashimoto's thyroiditis. CONCLUSIONS: PLWHA had higher risks of incident Sjögren syndrome, psoriasis, SLE, autoimmune haemolytic anaemia and uveitis.

Glycine N-methyltransferase deficiency in female mice impairs insulin signaling and promotes gluconeogenesis by modulating the PI3K/Akt pathway in the liver.

BACKGROUND: Glycine N-methyltransferase (GNMT) is abundantly expressed in the normal liver but is down-regulated in liver cancer tissues. GNMT knockout (Gnmt-/-) mice can spontaneously develop chronic hepatitis, fatty liver, and liver cancer. We previously demonstrated that hepatic GNMT is decreased in high-fat-diet-induced type 2 diabetes mellitus, but its contribution to metabolic syndrome is unclear. Here we show that GNMT modulates key aspects of metabolic syndrome in mice. METHODS: Eleven-week-old Gnmt-/- and wild-type (WT) mice with a C57BL/6 genetic background were used in this study. The metabolic defects of GNMT deficiency were measured by glucose and insulin tolerance tests, lipid homeostasis, gluconeogenesis, and insulin signaling. RESULTS: Gnmt-/- mice, especially females, exhibited glucose intolerance and insulin resistance. However, their body fat and lean mass, food and water intakes, and energy expenditure did not differ from those of WT mice. In addition, glucose-stimulated insulin secretion and insulin-stimulated glucagon secretion were normal in the serum and pancreatic islets of Gnmt-/- mice. Importantly, we found that GNMT deficiency increased lipogenesis and triglycerides in the liver. The elevated triglycerides disrupted the ability of insulin to induce Akt and S6 ribosomal protein phosphorylation, and then triggered insulin resistance and gluconeogenesis in female Gnmt-/- mice. CONCLUSIONS: Our data indicate that hepatic GNMT regulates lipid and glucose homeostasis, and provide insight into the development of insulin resistance through modulating the PI3K/Akt pathway.

Identification of 1,2,3,4,6-Penta-O-galloyl-ß-d-glucopyranoside as a Glycine N-Methyltransferase Enhancer by High-Throughput Screening of Natural Products Inhibits Hepatocellular Carcinoma.

Glycine N-methyltransferase (GNMT) expression is vastly downregulated in hepatocellular carcinomas (HCC). High rates of GNMT knockout mice developed HCC, while overexpression of GNMT prevented aflatoxin-induced carcinogenicity and inhibited liver cancer cell proliferation. Therefore, in this study, we aimed for the identification of a GNMT inducer for HCC therapy. We established a GNMT promoter-driven luciferase reporter assay as a drug screening platform. Screening of 324 pure compounds and 480 crude extracts from Chinese medicinal herbs resulted in the identification of Paeonia lactiflora Pall (PL) extract and the active component 1,2,3,4,6-penta-O-galloyl-ß-d-glucopyranoside (PGG) as a GNMT inducer. Purified PL extract and PGG induced GNMT mRNA and protein expression in Huh7 human hepatoma cells and in xenograft tumors. PGG and PL extract had potent anti-HCC effects both in vitro and in vivo. Furthermore, PGG treatment induced apoptosis in Huh7 cells. Moreover, PGG treatment sensitized Huh7 cells to sorafenib treatment. Therefore, these results indicated that identifying a GNMT enhancer using the GNMT promoter-based assay might be a useful approach to find drugs for HCC. These data also suggested that PGG has therapeutic potential for the treatment of HCC.

Role of glycine N-methyltransferase in the regulation of T-cell responses in experimental autoimmune encephalomyelitis.

Glycine N-methyltransferase (GNMT) is known for its function as a tumor suppressor gene. Since 100% of female Gnmt(-/-) mice developed hepatocellular carcinoma, we hypothesized that Gnmt(-/-) mice may have defective immune surveillance. In this study, we examined the immune modulation of GNMT in T-cell responses using experimental autoimmune encephalomyelitis (EAE). The results showed that EAE severity was reduced significantly in Gnmt(-/-) mice. Pathological examination of the spinal cords revealed that Gnmt(-/-) mice had significantly lower levels of mononuclear cell infiltration and demyelination than the wild-type mice. In addition, quantitative real-time PCR showed that expression levels of proinflammatory cytokines, including interferon (IFN)-γ and interleukin (IL)-17A, were much lower in the spinal cord of Gnmt(-/-) than in that of wild-type mice. Accordingly, myelin oligodendrocyte glycoprotein (MOG)-specific T-cell proliferation and induction of T-helper (Th)1 and Th17 cells were markedly suppressed in MOG(35-55)-induced Gnmt(-/-) mice. Moreover, the number of regulatory T (Treg) cells was increased significantly in these mice. When the T-cell receptor was stimulated, the proliferative capacity and the activation status of mTOR-associated downstream signaling were decreased significantly in Gnmt(-/-) CD4(+) T cells via an IL-2- and CD25-independent manner. Moreover, GNMT deficiency enhanced the differentiation of Treg cells without affecting the differentiation of Th1 and Th17 cells. Furthermore, the severity of EAE in mice adoptive transferred with GNMT-deficient CD4(+) T cells was much milder than in those with wild-type CD4(+) T cells. In summary, our findings suggest that GNMT is involved in the pathogenesis of EAE and plays a crucial role in the regulation of CD4(+) T-cell functions.

The serologic decoy receptor 3 (DcR3) levels are associated with slower disease progression in HIV-1/AIDS patients.

BACKGROUND/PURPOSE: The decoy receptor 3 (DcR3) is a member of the tumor necrosis factor receptor (TNFR) super-family. It counteracts the biological effects of Fas ligands and inhibits apoptosis. The goals of this study were to understand the associations between serologic DcR3 (sDcR3) levels and different human immunodeficiency virus type 1 (HIV-1) subtypes, as well as the AIDS disease progression. METHODS: Serum samples from 61 HIV/AIDS patients, who had been followed up every 6 months for 3 years, were collected. sDcR3 levels were quantified using an enzyme immunoassay (EIA). RESULTS: The sDcR3 levels in patients with HIV-1 subtype B were significantly higher than those in patients infected with subtype CRF01_AE (p < 0.001). In addition, multivariable linear mixed model analysis demonstrated that HIV-1 subtype B and slow disease progression were associated with higher levels of sDcR3, adjusting for potential predictors (p = 0.0008 and 0.0455, respectively). CONCLUSION: HIV-1-infected cells may gain a survival advantage by activating DcR3, which prevents infected cell detection by the host immune system. These data indicate that the sDcR3 level is a biomarker for AIDS disease progression.

Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins.

The severe acute respiratory syndrome coronavirus (SARS-CoV) still carries the potential for reemergence, therefore efforts are being made to create a vaccine as a prophylactic strategy for control and prevention. Antibody-dependent enhancement (ADE) is a mechanism through which dengue viruses, feline coronaviruses, and HIV viruses take advantage of anti-viral humoral immune responses to infect host target cells. Here we describe our observations of SARS-CoV using ADE to enhance the infectivity of a HL-CZ human promonocyte cell line. Quantitative-PCR and immunofluorescence staining results indicate that SARS-CoV is capable of replication in HL-CZ cells, and of displaying virus-induced cytopathic effects and increased levels of TNF-α, IL-4 and IL-6 two days post-infection. According to flow cytometry data, the HL-CZ cells also expressed angiotensin converting enzyme 2 (ACE2, a SARS-CoV receptor) and higher levels of the FcγRII receptor. We found that higher concentrations of anti-sera against SARS-CoV neutralized SARS-CoV infection, while highly diluted anti-sera significantly increased SARS-CoV infection and induced higher levels of apoptosis. Results from infectivity assays indicate that SARS-CoV ADE is primarily mediated by diluted antibodies against envelope spike proteins rather than nucleocapsid proteins. We also generated monoclonal antibodies against SARS-CoV spike proteins and observed that most of them promoted SARS-CoV infection. Combined, our results suggest that antibodies against SARS-CoV spike proteins may trigger ADE effects. The data raise new questions regarding a potential SARS-CoV vaccine, while shedding light on mechanisms involved in SARS pathogenesis.

Role of glycine N-methyltransferase in experimental ulcerative colitis.

BACKGROUND AND AIM: Inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with unclear etiology and mechanism(s). Glycine N-methyltransferase (GNMT) plays a central role in inflammatory diseases such as hepatitis and atherosclerosis. However, little is known about the impact of GNMT and the involved mechanism in the pathogenesis of IBD. In the current study, we investigated the role of GNMT in the mouse model of dextran sulfate sodium (DSS)-induced colitis. METHODS: Protein expression was determined by Western blotting or immunohistochemistry. Histopathology was examined by hematoxylin and eosin staining. Levels of pro-inflammatory cytokines were evaluated by ELISA kits. RESULTS: GNMT was expressed in the epithelium of the colon under normal conditions, and with DSS treatment, its expression was predominant in infiltrated leukocytes of lesions. Mice with genetic deletion of GNMT (GNMT(-/-) ) showed increased susceptibility to DSS induction of colitis, as revealed by the progression of colitis. Additionally, severe colonic inflammation, including increased crypt loss, leukocyte infiltration, and hemorrhage, was greater with DSS treatment in GNMT(-/-) than wild-type mice. Furthermore, the expression of adhesion molecule and inflammatory mediators in the colon was significantly higher with DSS treatment in GNMT(-/-) than wild-type mice. Moreover, loss of GNMT decreased cell apoptosis in colitis lesions with DSS treatment. CONCLUSIONS: Collectively, our findings suggest that GNMT may be a crucial molecule in the pathogenesis of DSS-induced colitis. This finding may provide new information for a potential therapeutic target in treating IBD.