Proteomic Analysis in Esophageal Eosinophilia Reveals Differential Galectin-3 Expression and S-Nitrosylation.

BACKGROUND AIMS: Esophageal eosinophilia (EE) can be caused by gastroesophageal reflux disease (GERD), proton-pump inhibitor-responsive EE (PPI-REE) or eosinophilic esophagitis (EoE). This study quantified protein expression and S-nitrosylation (SNO) post-translational modifications in EE to elucidate potential disease biomarkers. METHODS: Proximal and distal esophageal (DE) biopsy proteins in patients with EE and in controls were assayed for protein content and fluorescence-labeled with and without ascorbate treatment. Protein SNO was determined, and selected protein spots were identified by matrix-assisted laser desorption ionization time-of-flight/mass spectrometry. Western blot and ingenuity pathway analysis were performed. RESULTS: Ninety-one of 648 proteins showed differential expression. There were significantly altered levels of abundance for 11 proximal and 14 DE proteins. Hierarchal clustering revealed differential SNO in inflamed tissues, indicating reactive nitrogen/oxygen species involvement. Galectin-3 was upregulated in both proximal (p < 0.04) and distal (p < 0.004) esophageal EE biopsies compared to controls. In distal EE samples, galectin-3 was significantly S-nitrosylated (p < 0.004). Principal component analysis revealed sample group discrimination distally. CONCLUSION: Proteomic analysis in EE esophageal mucosa revealed a distinct abundance and nitrosylation profile, most prominently in distal biopsies. Galectin-3 was upregulated in expression and SNO, which may indicate its potential role in mucosal inflammation. These results call for more studies to be performed to investigate the role of galectin-3 in GERD, PPI-REE and EoE.

Preclinical development of the green tea catechin, epigallocatechin gallate, as an HIV-1 therapy.

BACKGROUND: Previously, we presented evidence that at physiologic concentrations the green tea catechin, epigallocatechin gallate (EGCG), inhibited attachment of HIV-1 glycoprotein 120 to the CD4 molecule on T cells, but the downstream effects of EGCG on HIV-1 infectivity were not determined. OBJECTIVE: To evaluate the inhibition of HIV-1 infectivity by EGCG and begin preclinical development of EGCG as a possible therapy. METHODS: PBMCs, CD4(+) T cells, and macrophages were isolated from blood of HIV-1-uninfected donors. HIV-1 infectivity was assessed by an HIV-1 p24 ELISA. Cell survival was assessed by cell viability by Trypan blue exclusion assay, cell growth by thymidine incorporation, and apoptosis by flow-cytometric analysis of annexin-V binding. RESULTS: Epigallocatechin gallate inhibited HIV-1 infectivity on human CD4(+) T cells and macrophages in a dose-dependent manner. At a physiologic concentration of 6 mumol/L, EGCG significantly inhibited HIV-1 p24 antigen production across a broad spectrum of both HIV-1 clinical isolates and laboratory-adapted subtypes (B [P < .001], C, D, and G [P < .01]). The specificity of the EGCG-induced inhibition was substantiated by the failure of EGCG derivatives lacking galloyl and/or pyrogallol side groups to alter HIV-1 p24 levels. EGCG-induced inhibition of HV-1 infectivity was not a result of cytotoxicity, cell growth inhibition, or apoptosis. CONCLUSION: We conclude that by preventing the attachment of HIV-1-glycoprotein 120 to the CD4 molecule, EGCG inhibits HIV-1 infectivity. Because this inhibition can be achieved at physiologic concentrations, the natural anti-HIV agent EGCG is a candidate as an alternative therapy in HIV-1 therapy.

Epigallocatechin gallate, the main polyphenol in green tea, binds to the T-cell receptor, CD4: Potential for HIV-1 therapy.

BACKGROUND: The green tea flavonoid, epigallocatechin gallate (EGCG), has been proposed to have an anti-HIV-1 effect by preventing the binding of HIV-1 glycoprotein (gp) 120 to the CD4 molecule on T cells. OBJECTIVE: To demonstrate that EGCG binds to the CD4 molecule at the gp120 attachment site and inhibits gp120 binding at physiologically relevant levels, thus establishing EGCG as a potential therapeutic treatment for HIV-1 infection. METHODS: Nuclear magnetic resonance spectroscopy was used to examine the binding of EGCG and control, (-)-catechin, to CD4-IgG2 (PRO 542). Gp120 binding to human CD4+ T cells was analyzed by flow cytometry. RESULTS: Addition of CD4 to EGCG produced a linear decrease in nuclear magnetic resonance signal intensity from EGCG but not from the control, (-)-catechin. In saturation transfer difference experiments, addition of 5.8 micromol/L CD4 to 310 micromol/L EGCG produced strong saturation at the aromatic rings of EGCG, but identical concentrations of (-)-catechin produced much smaller effects, implying EGCG/CD4 binding strong enough to reduce gp120/CD4 binding substantially. Molecular modeling studies suggested a binding site for EGCG in the D1 domain of CD4, the pocket that binds gp120. Physiologically relevant concentrations of EGCG (0.2 micromol/L) inhibited binding of gp120 to isolated human CD4+ T cells. CONCLUSION: We have demonstrated clear evidence of high-affinity binding of EGCG to the CD4 molecule with a Kd of approximately 10 nmol/L and inhibition of gp120 binding to human CD4+ T cells. CLINICAL IMPLICATIONS: Epigallocatechin gallate has potential use as adjunctive therapy in HIV-1 infection.