Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Impact of dietary betaine and conjugated linoleic acid on insulin sensitivity, protein and fat metabolism of obese pigs.

To determine possible mechanisms of action that might explain the nutrient partitioning effect of betaine and conjugated linoleic acid (CLA) in Iberian pigs and to address potential adverse effects, twenty gilts were restrictively fed from 20 to 50 kg BW Control, 0.5% betaine, 1% CLA or 0.5% betaine + 1% CLA diets. Serum hormones and metabolites profile were determined at 30 kg BW and an oral glucose test was performed before slaughter. Pigs were slaughtered at 50 kg BW and livers were obtained for chemical and histological analysis. Decreased serum urea in pigs fed betaine and betaine + CLA diets (11%; P = 0.0001) indicated a more efficient N utilization. The increase in serum triacylglycerol (58% and 28%, respectively; P = 0.0098) indicated that CLA and betaine + CLA could have reduced adipose tissue triacylglycerol synthesis from preformed fatty acids. Serum glucose, low-density lipoprotein (LDL) cholesterol and non-esterified fatty acids were unaffected. CLA and betaine + CLA altered serum lipids profile, although liver of pigs fed CLA diet presented no histopathological changes and triglyceride content was not different from Control pigs. Compared with controls, serum growth hormone decreased (20% to 23%; P = 0.0209) for all treatments. Although serum insulin increased in CLA, and especially in betaine + CLA pigs (28% and 83%; P = 0.0001), indices of insulin resistance were unaffected. In conclusion, CLA, and especially betaine + CLA, induced changes in biochemical parameters and hormones that may partially explain a nutrient partitioning effect in young pigs. Nevertheless, they exhibited weak, although detrimental, effects on blood lipids. Moreover, although livers were chemically and histologically normal, pigs fed CLA diet challenged with a glucose load had higher serum glucose than controls.

Mechanisms of hydrogen peroxide-induced vasoconstriction in the isolated perfused rat kidney.

The vasoconstrictor effect of hydrogen peroxide (H(2)O(2)) on isolated perfused rat kidney was investigated. H(2)O(2) induced vasoconstriction in the isolated rat kidney in a concentration-dependent manner. The vasoconstrictor effects of H(2)O(2) were completely inhibited by 1200 U/ml catalase. Endothelium-removal potentiated the renal response to H(2)O(2). The H(2)O(2) dose-response curve was not significantly modified by administration of the NO inhibitor L-NAME (10(-4) mol/l), whereas it was increased by the non-specific inhibitor of K+-channels, tetraethylammonium (3.10(-3) mol/l). Separately, removal of extracellular Ca(2+), administration of a mixture of calcium desensitizing agents (nitroprusside, papaverine, and diazoxide), and administration of a protein kinase C (PKC) inhibitor (chelerythrine, 10(-5) mol/l) each significantly attenuated the vasoconstrictor response to H(2)O(2), which was virtually suppressed when they were performed together. The pressor response to H(2)O(2) was not affected by: dimethyl sulfoxide (7.10(-5) mol/l) plus mannitol (3.10(-5) mol/l); intracellular Ca(2+) chelation using BAPTA (10(-5) mol/l); calcium store depletion after repeated doses of phenylephrine (10(-5) g/g kidney); or the presence of indomethacin (10(-5) mol/l), ODYA (2.10(-6) mol/l) or genistein (10(-5) mol/l). We conclude that the vasoconstrictor response to H(2)O(2) in the rat renal vasculature comprises the following components: 1) extracellular calcium influx, 2) activation of PKC, and 3) stimulation of pathways leading to sensitization of contractile elements to calcium. Moreover, a reduced pressor responsiveness to H(2)O(2) in female kidneys was observed.

Spanish multicenter study of the epidemiology and mechanisms of amoxicillin-clavulanate resistance in Escherichia coli.

We conducted a prospective multicenter study in Spain to characterize the mechanisms of resistance to amoxicillin-clavulanate (AMC) in Escherichia coli. Up to 44 AMC-resistant E. coli isolates (MIC ≥ 32/16 μg/ml) were collected at each of the seven participant hospitals. Resistance mechanisms were characterized by PCR and sequencing. Molecular epidemiology was studied by pulsed-field gel electrophoresis (PFGE) and by multilocus sequence typing. Overall AMC resistance was 9.3%. The resistance mechanisms detected in the 257 AMC-resistant isolates were OXA-1 production (26.1%), hyperproduction of penicillinase (22.6%), production of plasmidic AmpC (19.5%), hyperproduction of chromosomic AmpC (18.3%), and production of inhibitor-resistant TEM (IRT) (17.5%). The IRTs identified were TEM-40 (33.3%), TEM-30 (28.9%), TEM-33 (11.1%), TEM-32 (4.4%), TEM-34 (4.4%), TEM-35 (2.2%), TEM-54 (2.2%), TEM-76 (2.2%), TEM-79 (2.2%), and the new TEM-185 (8.8%). By PFGE, a high degree of genetic diversity was observed although two well-defined clusters were detected in the OXA-1-producing isolates: the C1 cluster consisting of 19 phylogroup A/sequence type 88 [ST88] isolates and the C2 cluster consisting of 19 phylogroup B2/ST131 isolates (16 of them producing CTX-M-15). Each of the clusters was detected in six different hospitals. In total, 21.8% of the isolates were serotype O25b/phylogroup B2 (O25b/B2). AMC resistance in E. coli is widespread in Spain at the hospital and community levels. A high prevalence of OXA-1 was found. Although resistant isolates were genetically diverse, clonality was linked to OXA-1-producing isolates of the STs 88 and 131. Dissemination of IRTs was frequent, and the epidemic O25b/B2/ST131 clone carried many different mechanisms of AMC resistance.

Mechanisms of Photoaging and Cutaneous Photocarcinogenesis, and Photoprotective Strategies with Phytochemicals.

Photoaging and photocarcinogenesis are primarily due to solar ultraviolet (UV) radiation, which alters DNA, cellular antioxidant balance, signal transduction pathways, immunology, and the extracellular matrix (ECM). The DNA alterations include UV radiation induced thymine-thymine dimers and loss of tumor suppressor gene p53. UV radiation reduces cellular antioxidant status by generating reactive oxygen species (ROS), and the resultant oxidative stress alters signal transduction pathways such as the mitogen-activated protein kinase (MAPK), the nuclear factor-kappa beta (NF-κB)/p65, the janus kinase (JAK), signal transduction and activation of transcription (STAT) and the nuclear factor erythroid 2-related factor 2 (Nrf2). UV radiation induces pro-inflammatory genes and causes immunosuppression by depleting the number and activity of the epidermal Langerhans cells. Further, UV radiation remodels the ECM by increasing matrixmetalloproteinases (MMP) and reducing structural collagen and elastin. The photoprotective strategies to prevent/treat photoaging and photocarcinogenesis include oral or topical agents that act as sunscreens or counteract the effects of UV radiation on DNA, cellular antioxidant balance, signal transduction pathways, immunology and the ECM. Many of these agents are phytochemical derivatives and include polyphenols and non-polyphenols. The flavonoids are polyphenols and include catechins, isoflavones, proanthocyanidins, and anthocyanins, whereas the non-flavonoids comprise mono phenolic acids and stilbenes. The natural sources of polyphenols include tea, cocoa, grape/wine, soy, pomegranate, and Polypodium leucotomos. The non-phenolic phytochemicals include carotenoids, caffeine and sulphoraphance (SFN). In addition, there are other phytochemical derivatives or whole extracts such as baicalin, flavangenol, raspberry extract, and Photomorphe umbellata with photoprotective activity against UVB radiation, and thereby carcinogenesis.