Effect of Supplementation with Omega-3 Polyunsaturated Fatty Acids on Metabolic Modulators in Skeletal Muscle of Rats with an Obesogenic High-Fat Diet.

Previous studies provided evidence of the benefits of omega-3 polyunsaturated fatty acids (ω-3 PUFA) on the cardiovascular system and inflammation. However, its possible effect on skeletal muscle is unknown. This study aimed to evaluate whether ω-3 PUFA reverses the dysregulation of metabolic modulators in the skeletal muscle of rats on a high-fat obesogenic diet. For this purpose, an animal model was developed using male Wistar rats with a high-fat diet (HFD) and subsequently supplemented with ω-3 PUFA. Insulin resistance was assessed, and gene and protein expression of metabolism modulators in skeletal muscle was also calculated using PCR-RT and Western blot. Our results confirmed that in HFD rats, zoometric parameters and insulin resistance were increased compared to SD rats. Furthermore, we demonstrate reduced gene and protein expression of peroxisome proliferator-activated receptors (PPARs) and insulin signaling molecules. After ω-3 PUFA supplementation, we observed that glucose (24.34%), triglycerides (35.78%), and HOMA-IR (40.10%) were reduced, and QUICKI (12.16%) increased compared to HFD rats. Furthermore, in skeletal muscle, we detected increased gene and protein expression of PPAR-α, PPAR-γ, insulin receptor (INSR), insulin receptor substrate 1 (ISR-1), phosphatidylinositol-3-kinase (PI3K), and glucose transporter 4 (GLUT-4). These findings suggest that ω-3 PUFAs decrease insulin resistance of obese skeletal muscle.

Glycosaminoglycans are involved in bacterial adherence to lung cells.

BACKGROUND: Lower respiratory infections are among the top ten causes of death worldwide. Since pathogen to cell adhesion is a crucial step in the infection progress, blocking the interaction between eukaryotic receptors and bacterial ligands may enable the pathogenesis process to be stopped. Cell surface glycosaminoglycans (GAGs) are known to be mediators in the adhesion of diverse bacteria to different cell types, making it of interest to examine their involvement in the attachment of various pathogenic bacteria to lung cells, including epithelial cells and fibroblasts. METHODS: The function of cell surface GAGs in bacterial adhesion was studied by reducing their levels through inhibiting their biosynthesis and enzymatic degradation, as well as in binding competition experiments with various species of GAGs. The participation of the different bacterial adhesins in attachment was evaluated through competition with two peptides, both containing consensus heparin binding sequences. Blocking inhibition assays using anti-syndecans and the enzymatic removal of glypicans were conducted to test their involvement in bacterial adhesion. The importance of the fine structure of GAGs in the interaction with pathogens was investigated in competition experiments with specifically desulfated heparins. RESULTS: The binding of all bacteria tested decreased when GAG levels in cell surface of both lung cells were diminished. Competition experiments with different types of GAGs showed that heparan sulfate chains are the main species involved. Blocking or removal of cell surface proteoglycans evidenced that syndecans play a more important role than glypicans. The binding was partially inhibited by peptides including heparin binding sequences. Desulfated heparins also reduced bacterial adhesion to different extents depending on the bacterium and the sulfated residue, especially in fibroblast cells. CONCLUSIONS: Taken together, these data demonstrate that the GAG chains of the cell surface are involved in the adhesion of bacterial adhesins to lung cells. Heparan sulfate seems to be the main species implicated, and binding is dependent on the sulfation pattern of the molecule. These data could facilitate the development of new anti-infective strategies, enabling the development of new procedures for blocking the interaction between pathogens and lung cells more effectively.

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Transforming and tumorigenic activity of JAK2 by fusion to BCR: molecular mechanisms of action of a novel BCR-JAK2 tyrosine-kinase.

Chromosomal translocations in tumors frequently produce fusion genes coding for chimeric proteins with a key role in oncogenesis. Recent reports described a BCR-JAK2 fusion gene in fatal chronic and acute myeloid leukemia, but the functional behavior of the chimeric protein remains uncharacterized. We used fluorescence in situ hybridization and reverse transcription polymerase chain reaction (RT-PCR) assays to describe a BCR-JAK2 fusion gene from a patient with acute lymphoblastic leukemia. The patient has been in complete remission for six years following treatment and autologous transplantation, and minimal residual disease was monitored by real-time RT-PCR. BCR-JAK2 codes for a protein containing the BCR oligomerization domain fused to the JAK2 tyrosine-kinase domain. In vitro analysis of transfected cells showed that BCR-JAK2 is located in the cytoplasm. Transduction of hematopoietic Ba/F3 cells with retroviral vectors carrying BCR-JAK2 induced IL-3-independent cell growth, constitutive activation of the chimeric protein as well as STAT5 phosphorylation and translocation to the nuclei, where Bcl-xL gene expression was elicited. Primary mouse progenitor cells transduced with BCR-JAK2 also showed increased proliferation and survival. Treatment with the JAK2 inhibitor TG101209 abrogated BCR-JAK2 and STAT5 phosphorylation, decreased Bcl-xL expression and triggered apoptosis of transformed Ba/F3 cells. Therefore, BCR-JAK2 is a novel tyrosine-kinase with transforming activity. It deregulates growth factor-dependent proliferation and cell survival, which can be abrogated by the TG101209 inhibitor. Moreover, transformed Ba/F3 cells developed tumors when injected subcutaneously into nude mice, thus proving the tumorigenic capacity of BCR-JAK2 in vivo. Together these findings suggest that adult and pediatric patients with BCR-ABL-negative leukemia and JAK2 overexpression may benefit from targeted therapies.

Candida albicans beta-glucan exposure is controlled by the fungal CEK1-mediated mitogen-activated protein kinase pathway that modulates immune responses triggered through dectin-1.

Innate immunity to Candida albicans depends upon the recognition of molecular patterns on the fungal cell wall. However, the masking of major components such as beta-glucan seems to be a mechanism that fungi have evolved to avoid immune cell recognition through the dectin-1 receptor. Although the role of C. albicans mitogen-activated protein kinase (MAPK) pathways as virulence determinants has been established previously with animal models, the mechanism involved in this behavior is largely unknown. In this study we demonstrate that a disruption of the C. albicans extracellular signal-regulated kinase (ERK)-like 1 (CEK1)-mediated MAPK pathway causes enhanced cell wall beta-glucan exposure, triggering immune responses more efficiently than the wild type, as measured by dectin-1-mediated specific binding and human dendritic cell (hDC)- and macrophage-mediated phagocytosis, killing, and activation of intracellular signaling pathways. At the molecular level, the disruption of CEK1 resulted in altered spleen tyrosine kinase (Syk), Raf-1, and ERK1/2 activations together with IkappaB degradation on hDCs and increased dectin-1-dependent activator protein 1 (AP-1) activation on transfected cells. In addition, concurring with these altered pathways, we detected increased reactive oxygen species production and cytokine secretion. In conclusion, the CEK1-mediated MAPK pathway is involved in beta-glucan exposure in a fungal pathogen, hence influencing dectin-1-dependent immune cell recognition, thus establishing this fungal intracellular signaling route as a promising novel therapeutic target.

Dynamic redistribution of the activating 2B4/SAP complex at the cytotoxic NK cell immune synapse.

The 2B4 molecule (CD244) has been described as a coreceptor in human NK cell activation. However, the behavior of 2B4 during the cytotoxic NK cell immune synapse (NK-IS) formation remains undetermined. In this study, we demonstrate the redistribution of 2B4 and the signaling adaptor molecule, signaling lymphocyte activation molecule-associated protein (SAP), to the cytotoxic NK-IS upon formation of conjugates between resting NK cells and EBV-infected 721.221 human cells. Confocal microscopy showed that 2B4 localized at the central supramolecular activation cluster, surrounded by a peripheral supramolecular activation cluster containing talin within NK cell and ICAM-1 on target cells. Videomicroscopy studies with 2B4-GFP-transfected NK cells revealed that 2B4 redistributed to cytotoxic NK-IS as soon as the cell contact occurred. Simultaneously, a SAP-GFP also clustered at the contact site, where it remained during the interaction period. The 2B4 molecular clusters remained bound to the target cell even after NK cell detachment. These results underscore the function of 2B4 as an adhesion molecule and suggest a relevant role in the initial binding, scanning of target cells, and formation of cytotoxic NK-IS. Finally, these findings are indicative of an important role of the activating 2B4/signaling lymphocyte activation molecule-associated protein complex during the recognition of EBV-infected cells.

Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class Ia and HLA-E expression: impact on target susceptibility to NK cell subsets.

We compared in an inducible expression system the individual effect of US2, US6 and US11 human cytomegalovirus (HCMV) proteins on HLA-E and HLA class Ia surface expression, assessing in parallel their influence on target susceptibility to NK cell clones. To this end, the RPMI 8866 B lymphoma cell line (HLA-A2, HLA-A3, HLA-B7, HLA-Cw7, HLA-E(R), HLA-E(G)) was stably cotransfected with the ecdysone receptor, together with the US sequences under the control of an ecdysone-inducible promoter. Biosynthesis of viral proteins was turned on by incubating transfectants with Ponasterone A. US6 down-regulated expression of all class I molecules, hampering target resistance to NK cell clones controlled by the CD94/NKG2A, KIR2DL2 and/or CD85j (ILT2 or LIR-1) inhibitory receptors. By contrast, US11 reduced the surface levels of class Ia molecules but preserved HLA-E; this rendered US11(+) cells sensitive to NK clones under the control of KIR2DL2 and/or CD85j, while their resistance to CD94/NKG2A(+)KIR2DL2(-) effector cells was maintained. US2 preserved as well HLA-E expression but selectively targeted class Ia molecules; in fact, HLA-A and HLA-C allotypes were down-modulated whereas HLA-B7 remained unaltered. US2(+) targets became sensitive to KIR2DL2(+) cells but remained resistant to CD94/NKG2A(+)CD85j(+) NK clones. The differential effects of US proteins on HLA class Ia and HLA-E likely reflect the evolutionary adaptation of HCMV to counteract NK-mediated surveillance.

High molecular response rate and clinical correlation in patients with follicular lymphoma treated with cyclophosphamide-vincristine-prednisone plus interferon alpha 2b.

PURPOSE: The role of molecular monitoring of minimal residual disease (MRD) in low-grade non-Hodgkin's lymphoma is controversial. We have performed a prospective study of the molecular behavior of 35 patients with follicular non-Hodgkin's lymphoma who received cyclophosphamide-vincristine-prednisone chemotherapy in conjunction with IFN-alpha 2b. EXPERIMENTAL DESIGN: Bcl-2 and clonal immunoglobulin heavy chain (IgH) gene rearrangements were assayed at diagnosis by PCR in lymph node and bone marrow (BM) and sequentially after treatment. RESULTS: Molecular markers were detected in BM of 29 (83%) patients at diagnosis: Bcl-2 rearrangement in 20 patients (90% major breakpoint and 10% minor cluster) and clonal IgH rearrangement in 9 of 15 patients negative for Bcl-2. Molecular and clinical response was noted in 25 (86%) patients after induction treatment. Progression-free survival at 5 years was 78.1 +/- 8%. A correlation between clinical and molecular response was found in 24 patients with molecular markers in BM at diagnosis and >2 years of follow-up: 94% of patients with undetectable MRD showed continuous clinical remission, whereas 50% of patients who reverted back to positive molecular markers relapsed (P < 0.05). CONCLUSIONS: The rate of molecular response is high in patients treated with cyclophosphamide-vincristine-prednisone and IFN and MRD sequential analysis is useful for disease surveillance.