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.

Obstructive Sleep Apnea and Cardiovascular Risk Markers (Fibrinogen & Gamma Glutamyl Transferase) in Obese Males

Aim: To determine and compare gamma glutamyl transferase (GGT) and fibrinogen among obese males with and without obstructive sleep apnea (OSA). Second objective was to investigate correlation between blood pressure and GGT. Methodology: Sixty-four obese males aged 20-45 years with BMI > 25kg/m2 were included by convenience sampling. The study was conducted, after obtaining ethical approval from IRB, at the Department of Physiology, Post Graduate Medical Institute, Lahore from August 2014 to May 2015. Participants having acute or chronic inflammatory conditions were excluded. Participants were screened for OSA by Berlin and STOP BANG questionnaires. Diagnosis of OSA was made by overnight portable pulse oximetry. The participants were divided into two groups. Group I had 32 obese males with OSA. Group II contained 32 obese males without OSA. After an overnight fasting of 10-12 hours blood samples were drawn. Serum fibrinogen and GGT were measured by spectrophotometer. The data was analyzed using SPSS-22. Quantitative variables were compared between the two groups by Mann-Whitney U test. Spearman correlation was used to correlate blood pressure and GGT among the participants. Results: Fibrinogen was significantly raised (p=0.015) in obese males with OSA. Systolic blood pressure (p=0.003), diastolic blood pressure (p=0.001) and mean arterial blood pressure (p<0.001) showed strong positive correlation with GGT in obese males with OSA. Conclusion: Proinflammatory, procoagulant and proatherogenic marker fibrinogen levels were significantly raised in obese otherwise healthy males with OSA. Oxidative stress marker GGT showed strong positive correlation with blood pressure in obese males with OSA. Keywords: Fibrinogen, gamma glutamyl transferase, inflammation, obstructive sleep apnea, oxidative stress

697 Binding of gamma-glutamyl transferase to toll-like receptor-4 allows tissue factor activation in monocytes

Aims Gamma-glutamyl transferase (GGT) is well known to play a key role in the antioxidant processes, however, it also exerts pro-oxidant effects by activating NFkB, a redox-sensitive transcription factor key in the induction of tissue factor (TF) gene expression, the principal initiator of the clotting cascade. GGT may potentially modulate TF expression, an assumption verified by previous studies carried out in human peripheral blood mononuclear cell (PBMC). Quite importantly, TF expression in response to GGT stimulation was independent of its enzymatic properties since those experiments were conducted by using human recombinant (hr)GGT, a wheat germ-derived protein enzymatically inert because of missing post-translational glycosylation site. Thus, GGT may act through a cytokine-like mechanism although the precise determinants of its action and the receptor involved were not defined by those experiments. To assess whether GGT-induced TF stimulation is a consequence of binding to toll-like receptor (TLR)-4 and activation of NFkB, as suggested by results recently obtained in different experimental contexts. Methods PBMCs obtained from healthy donors through a discontinuous Ficoll/Hystopaque density gradient and THP-1 cells, a human monocytic cell line derived from an acute monocytic leukaemia patient, were incubated with hrGGT (0.5 ng/μL for PBMCs and 1 ng/µL for THP-1). LPS-Rs (0.5 ng/µL for PBMCs and 1 ng/µL for THP-1), CLI-095 (3 × 10−6M) and BAY-11-7082 (10−5 M) were used to block TLR-4 receptors, TLR4 signalling and NFkB respectively. TF protein concentration was determined by western blot analysis. TF pro-coagulant activity (PCA) was assessed through the use of Start Max coagulometer and results were expressed in pg/mL after calibration with a standard curve. HEK-Blue hTLR4-positive and HEK-Blue hTLR4-negative cells are used to evaluate the engagement of TLR4 by hrGGT. Results hrGGT increased TF expression in both PBMCs (PCA from 110 ± 70 to 510 ± 43, n = 7, P &lt; 0.01) and THP-1 cells (PCA from 170 ± 64 to 460 ± 80, n = 15, P &lt; 0.001), result confirmed by western blot. In PBMCs GGT-induced TF stimulation was antagonized by LPS-Rs (PCA: −72 ± 17% n = 4, P &lt; 0.01) a TLR-4 antagonist, CLI-095 (PCA: −74 ± 34%, n = 7, P &lt; 0.001) a TLR-4 intracellular antagonist and BAY-11-7082 (PCA: −71 ± 32%, n = 7, P &lt; 0.001), a NF-kB inhibitor. Similar results were obtained in THP-1 cells (LPS-Rs: −76 ± 15%, n = 6, P &lt; 0.01; CLI-095: −100 ± 6,6 %, n = 6, P &lt; 0.01; BAY-11-7082: −100 ± 2,1%, n = 6, P &lt; 0.01). hrGGT activates NFkB in hTLR4-positive HEK cell lines while does not induces effect in TLR4-negative HEK cell lines. Conclusions These data confirm the cytokine-like activity of GGT and its procoagulant effect in PBMCs and THP-1 cells. Furthermore, it is highlighted for the first time the possible role of TLR-4 as the receptor of GGT and NFkB as the involved signal transduction pathway. The GGT-TLR-4 link may provide an explanation to the consistent association between circulating GGT levels and increased risk of acute thrombotic events as well as to the involvement of GGT in the morbid evolution of the silent atherosclerotic plaque in which GGT colocalizes with monocytes and foam cells, the prime sources of TF within the plaque.

Role of C-reactive protein and gamma-glutamyl transferase in the diagnosis of metabolic syndrome

Previous studies have reported that metabolic syndrome (MetS) is associated with an increased risk of major cardiovascular events and levels of C-Reactive protein (CRP) can be considered as markers of MetS and its constituent components. Oxidative stress plays a major role in the development of MetS, and levels of gamma-glutamyl transferase (GGT) change with response to oxidative stress are also associated with MetS, which may be modulated by CRP. This study was conducted to identify the role of GGT and CRP as biomarkers in the diagnosis of MetS, a high-risk factor for cardiovascular diseases. One hundred and fifty patients meeting the diagnostic criteria of MetS and an equal number of controls were included in the study. The cases were selected from pathology and molecular biology laboratories, Karachi, while the controls came from the general population. Anthropometric indices of adiposity and blood pressure were recorded for both cases and controls. Blood samples were taken from all subjects to determine the levels of CRP and GGT. All those cases and control height, weight, hip waist circumference were noted and the comparison of CRP and GGT by applying students' t-test as markers for detection of metabolic syndrome. p-value 0.001 was considered as significant. This study suggests that in patients with metabolic syndrome were found to have raised the basal metabolic rate, C-reactive protein and GGT were synergistically associated with MetS independently of another confounding factor in the general population. Keywords: C-reactive protein (CRP), gama glutamyl transferase (GGT), metabolic syndrome, (Met-S), inflammation, body mass index.