Philadelphia-negative myeloproliferative neoplasms as disorders marked by cytokine modulation

Background: Cytokines are key immune mediators in physiological and disease processes, whose increased levels have been associated with the physiopathology of hematopoietic malignancies, such as myeloproliferative neoplasms. Methods: This study examined the plasma cytokine profiles of patients with essential thrombocythemia, primary myelofibrosis, polycythemia vera and of healthy subjects, and analyzed correlations with JAK2 V617F status and clinical-hematological parameters. Results: The proinflammatory cytokine levels were increased in myeloproliferative neoplasm patients, and the presence of the JAK2 V617F mutation was associated with high IP-10 levels in primary myelofibrosis patients. Conclusions: Essential thrombocythemia, primary myelofibrosis, and polycythemia vera patients exhibited different patterns of cytokine production, as revealed by cytokine network correlations. Together, these findings suggest that augmented cytokine levels are associated with the physiopathology of myeloproliferative neoplasms.

GM-CSF Priming Drives Bone Marrow-Derived Macrophages to a Pro-Inflammatory Pattern and Downmodulates PGE(2) in Response to TLR2 Ligands

In response to pathogen recognition by Toll-like receptors (TLRs) on their cell surface, macrophages release lipid mediators and cytokines that are widely distributed throughout the body and play essential roles in host responses. Granulocyte macrophage colony-stimulating factor (GM-CSF) is important for the immune response during infections to improve the clearance of microorganisms. In this study, we examined the release of mediators in response to TLR2 ligands by bone marrow-derived macrophages (BMDMs) primed with GM-CSF. We demonstrated that when stimulated with TLR2 ligands, non-primed BMDMs preferentially produced PGE(2) in greater amounts than LTB4. However, GM-CSF priming shifted the release of lipid mediators by BMDMs, resulting in a significant decrease of PGE(2) production in response to the same stimuli. The decrease of PGE(2) production from primed BMDMs was accompanied by a decrease in PGE-synthase mRNA expression and an increase in TNF-alpha and nitric oxide (NO) production. Moreover, some GM-CSF effects were potentiated by the addition of IFN-gamma. Using a variety of TLR2 ligands, we established that PGE(2) release by GM-CSF-primed BMDMs was dependent on TLR2 co-receptors (TLR1, TLR6), CD14, MyD88 and the nuclear translocation of NF kappa B but was not dependent on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) activation. Indeed, GM-CSF priming enhanced TLR2, TLR4 and MyD88 mRNA expression and phospho-I kappa B alpha formation. These findings demonstrate that GM-CSF drives BMDMs to present a profile relevant to the host during infections.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)FAPESP - Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Pesquisa (CNPq)CNPq - Conselho Nacional de Pesquis

Combined immunization using DNA-Sm14 and DNA-Hsp65 increases CD8+ memory T cells, reduces chronic pathology and decreases egg viability during Schistosoma mansoni infection

Abstract\ud \ud Background\ud Schistosomiasis is one of the most important neglected diseases found in developing countries and affects 249 million people worldwide. The development of an efficient vaccination strategy is essential for the control of this disease. Previous work showed partial protection induced by DNA-Sm14 against Schistosoma mansoni infection, whereas DNA-Hsp65 showed immunostimulatory properties against infectious diseases, autoimmune diseases, cancer and antifibrotic properties in an egg-induced granuloma model.\ud \ud \ud Methods\ud C57BL/6 mice received 4 doses of DNA-Sm14 (100 μg/dose) and DNA-Hsp65 (100 μg/dose), simultaneously administrated, or DNA-Sm14 alone, once a week, during four weeks. Three groups were included: 1- Control (no immunization); 2- DNA-Sm14; 3- DNA-Sm14/DNA-Hsp65. Two weeks following last immunization, animals were challenged subcutaneously with 30 cercariae. Fifteen, 48 and 69 days after infection splenocytes were collected to evaluate the number of CD8+ memory T cells (CD44highCD62low) using flow cytometry. Forty-eight days after challenge adult worms were collected by portal veins perfusion and intestines were collected to analyze the intestinal egg viability. Histological, immunohistochemical and soluble quantification of collagen and α-SMA accumulation were performed on the liver.\ud \ud \ud Results\ud In the current work, we tested a new vaccination strategy using DNA-Sm14 with DNA-Hsp65 to potentiate the protection against schistosomiasis. Combined vaccination increased the number of CD8+ memory T cells and decreased egg viability on the intestinal wall of infected mice. In addition, simultaneous vaccination with DNA-Sm14/DNA-Hsp65 reduced collagen and α-SMA accumulation during the chronic phase of granuloma formation.\ud \ud \ud Conclusion\ud Simultaneous vaccination with DNA-Sm14/DNA-Hsp65 showed an immunostimulatory potential and antifibrotic property that is associated with the reduction of tissue damage on Schistosoma mansoni experimental infection.We are grateful to Elaine Medeiros Floriano, Olinda Mara Brigoto and Fabiana\ud Rossetto de Morais for their technical assistance. This study was supported\ud by São Paulo Research Foundation (FAPESP, grant# 2009/01501-8 and 2009/\ud 07169-5) and Conselho Nacional de Desenvolvimento Científico e\ud Tecnológico (CNPq)

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination732175184FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO – FAPESPsem informaçã

The synergy between structural stability and DNA-binding controls the antibody production in EPC/DOTAP/DOPE liposomes and DOTAP/DOPE lipoplexes

We present a comparative study of the physico-chemical properties, in vitro cytotoxicity and in vivo antibody production of surface-complexed DNA in EPC/DOTAP/DOPE (50/25/25% molar) liposomes and DOTAP/DOPE (50/50% molar) lipoplexes. The study aims to correlate the biological behavior and structural properties of the lipid carriers. We used DNA-hsp65, whose naked action as a gene vaccine against tuberculosis has already been demonstrated. Additionally, surface-complexed DNA-hsp65 in EPC/DOTAP/DOPE (50/25/25% molar) liposomes was effective as a single-dose tuberculosis vaccine. The results obtained showed that the EPC inclusion stabilized the DOTAP/DOPE structure, producing higher melting temperature and lower zeta potential despite a close mean hydrodynamic diameter. Resemblances in morphologies were identified in both structures, although a higher fraction of loaded DNA was not electrostatically bound in EPC/DOTAP/DOPE. EPC also induced a striking reduction in cytotoxicity, similar to naked DNA-hsp65. The proper immune response lead to a polarized antibody production of the IgG2a isotype, even for the cytotoxic DOTAP/DOPE. However, the antibody production was detected at 15 and 30 days for DOTAP/DOPE and EPC/DOTAP/DOPE, respectively. Therefore, the in vivo antibody production neither correlates with the in vitro cytotoxicity, nor with the structural stability alone. The synergistic effect of the structural stability and DNA electrostatic binding upon the surface of structures account for the immunological effects. By adjusting the composition to generate proper packing and cationic lipid/DNA interaction, we allow for the optimization of liposome formulations for required immunization or gene therapy. In a specific manner, our results contribute to studies on the tuberculosis therapy and vaccination. (C) 2009 Elsevier B.V. All rights reserved.Fundacao de Auxilio a Pesquisa de Sao Paulo (FAPESP

The influence of dehydroepiandrosterone on effector functions of neutrophils

Dehydroepiandrosterone (DHEA) is a steroid hormone secreted by the adrenal glands, gonads and brain. It is a precursor to sex hormones and also is known to have immune modulatory activity. However, little is known about the relationship between DHEA and neutrophils and thus our study evaluates the influence of DHEA in the effector functions of neutrophils. Human neutrophils were treated in vitro with DHEA and further infected with Salmonella enterica serovar Typhimurium. The treatment of neutrophils with 0.01 μM of DHEA increased the phagocytosis of Salmonella independent of TLR4 as the treatment did not modulate the TLR4 expression. Additionally, DHEA caused a decrease in ROS (reactive oxygen species) production and did not influence the formation of the neutrophil extracellular trap (NET). Steroid treated neutrophils, infected or stimulated with LPS (lipopolysaccharide), showed reduced production of IL-8, compared to untreated cells. Also, the protein levels of p-NFκB were decreased in neutrophils treated with DHEA, and this reduction could explain the reduced levels of IL-8. These results led us to conclude that the steroid hormone DHEA has important modulatory functions in neutrophils

Philadelphia-negative myeloproliferative neoplasms as disorders marked by cytokine modulation

Background: Cytokines are key immune mediators in physiological and disease processes, whose increased levels have been associated with the physiopathology of hematopoietic malignancies, such as myeloproliferative neoplasms. Methods: This study examined the plasma cytokine profiles of patients with essential thrombocythemia, primary myelofibrosis, polycythemia vera and of healthy subjects, and analyzed correlations with JAK2 V617F status and clinical-hematological parameters. Results: The proinflammatory cytokine levels were increased in myeloproliferative neoplasm patients, and the presence of the JAK2 V617F mutation was associated with high IP-10 levels in primary myelofibrosis patients. Conclusions: Essential thrombocythemia, primary myelofibrosis, and polycythemia vera patients exhibited different patterns of cytokine production, as revealed by cytokine network correlations. Together, these findings suggest that augmented cytokine levels are associated with the physiopathology of myeloproliferative neoplasms. Keywords: Ph-negative myeloproliferative neoplasms, Inflammation, Plasma cytokines, JAK2 V617

IFN-γ potentiates the GM-CSF priming effects on BMDM.

<p>BMDM WT cells were preincubated for 24 h with GM-CSF or GM-CSF plus IFN-γ (500 UI/mL) and then stimulated with AraLAM (5000 ng/mL), LM (5000 ng/mL), Malp2 (300 ng/mL), Pam₃-CSK₄ (5000 ng/mL) or LPS (500 ng/mL) for 24 h <i>in vitro</i>. The supernatants were analyzed for the production of NO (Nitrite) (<b>A</b>), TNF-α (<b>B</b>), PGE₂ (<b>C</b>) and IL-10 (<b>D</b>). Medium alone was used as a negative control. ELISA was used to measure TNF-α and PGE₂, and the Griess Method was used to measure Nitrite production in the supernatants. Results are the average ± S.E.M from mice cells (<i>n</i> = 6 per group) from two independent experiments; *, <i>p</i><0.05 compared with unstimulated cells; <b>^#</b>, <i>p<0.05</i> compared with GM-CSF priming alone.</p

GM-CSF-modified lipid mediators released by BMDM in response to bacterial TLRs-ligands.

<p>BMDM from WT mice were primed (or not) with GM-CSF for 24 h and then incubated with AraLAM (5000 ng/mL), LM (5000 ng/mL), Malp2 (300 ng/mL), Pam₃-CSK₄ (5000 ng/mL) or LPS (500 ng/mL) for 24 h. PGE₂ (<b>A</b>) and LTB₄ (<b>B</b>) release were measured in the supernatant by ELISA. Medium alone was used as a negative control. Results are the average ± S.E.M from mice cells (<i>n</i> = 6 per group), from two independent experiments; *, <i>p</i><<i>0.05</i> compared with unstimulated cells; <b>^#</b>, <i>p<0.05</i> compared with non-priming GM-CSF cells.</p

Effect of GM-CSF priming on NO production by BMDM in response to bacterial stimulus^a.

a<p>BMDM from WT (C57Bl/6) or deficient mice (^−/−) for TLR2, TLR1, TLR6, TLR4, CD14 and MyD88 primed with GM-CSF, were incubated with AraLAM (5000 ng/mL), LM (5000 ng/mL), Malp2 (300 ng/mL), Pam₃-CSK₄ (5000 ng/mL) or LPS (500 ng/mL) for 24 h <i>in vitro</i>. NO (Nitrite) was measured in the supernatants by Griess Method. Percentage of inhibition of NO production in deficient mice compared to WT.</p>b<p><i>p<0.05</i>, significantly versus WT production. Data are mean ± S.D, <i>n</i> = 6 mice per genotype from two independent experiments.</p>c<p>Negative values corresponds an increment in correlation with WT production.</p