Modulation of CD4(+) T Cell-Dependent Specific Cytotoxic CD8(+) T Cells Differentiation and Proliferation by the Timing of Increase in the Pathogen Load

Background. Following infection with viruses, bacteria or protozoan parasites, naive antigen-specific CD8(+) T cells undergo a process of differentiation and proliferation to generate effector cells. Recent evidences suggest that the timing of generation of specific effector CD8(+) T cells varies widely according to different pathogens. We hypothesized that the timing of increase in the pathogen load could be a critical parameter governing this process. Methodology/Principal Findings. Using increasing doses of the protozoan parasite Trypanosoma cruzi to infect C57BL/6 mice, we observed a significant acceleration in the timing of parasitemia without an increase in mouse susceptibility. in contrast, in CD8 deficient mice, we observed an inverse relationship between the parasite inoculum and the timing of death. These results suggest that in normal mice CD8(+) T cells became protective earlier, following the accelerated development of parasitemia. the evaluation of specific cytotoxic responses in vivo to three distinct epitopes revealed that increasing the parasite inoculum hastened the expansion of specific CD8(+) cytotoxic T cells following infection. the differentiation and expansion of T. cruzi-specific CD8(+) cytotoxic T cells is in fact dependent on parasite multiplication, as radiation-attenuated parasites were unable to activate these cells. We also observed that, in contrast to most pathogens, the activation process of T. cruzi-specific CD8(+) cytotoxic T cells was dependent on MHC class II restricted CD4(+) T cells. Conclusions/Significance. Our results are compatible with our initial hypothesis that the timing of increase in the pathogen load can be a critical parameter governing the kinetics of CD4(+) T cell-dependent expansion of pathogen-specific CD8(+) cytotoxic T cells.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Millennium Institute for Vaccine Development and TechnologyConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Biofis Carlos Chagas Filho, Ilha Fundao, Ctr Ciencias Saude, BR-21941 Rio de Janeiro, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, CINTERGEN, São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilMillennium Institute for Vaccine Development and Technology: CNPq - 420067/2005-1Web of Scienc

ATP activates a Reactive Oxygen Species-dependent oxidative stress response and secretion of pro-inflammatory cytokines in macrophages

Secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, usually requires two signals. The first, due to microbial products such as lipopolysaccharide, initiates transcription of the cytokine genes and accumulation of the precursor proteins. Cleavage and secretion of the cytokines is mediated by caspase-1, in association with an inflammasome containing Nalp3, which can be activated by binding of extracellular ATP to purinergic receptors. We show that treatment of macrophages with ATP results in production of reactive oxygen species (ROS), which stimulate the phosphatidylinositol 3-kinase (PI3K) pathway and subsequent Akt and ERK1/2 activation. ROS exerts its effect through glutathionylation of PTEN (phosphatase and tensin homologue deleted from chromosome 10), whose inactivation would shift the equilibrium in favor of PI3K. ATP-dependent ROS production and PI3K activation also stimulate transcription of genes required for an oxidative stress response. In parallel, ATP-mediated ROS-dependent PI3K is required for activation of caspase-1 and secretion of IL-1β and IL-18. Thus, an increase in ROS levels in ATP-treated macrophages results in activation of a single pathway that promotes both adaptation to subsequent exposure to oxidants or inflammation, and processing and secretion of proinflammatory cytokines

The role of P2 receptors in controlling infections by intracellular pathogens

A growing number of studies have demonstrated the importance of ATPe-signalling via P2 receptors as an important component of the inflammatory response to infection. More recent studies have shown that ATPe can also have a direct effect on infection by intracellular pathogens, by modulating membrane trafficking in cells that contain vacuoles that harbour intracellular pathogens, such as mycobacteria and chlamydiae. A conserved mechanism appears to be involved in controlling infection by both of these pathogens, as a role for phospholipase D in inducing fusion between lysosomes and the vacuoles has been demonstrated. Other P2-dependent mechanisms are most likely operative in the cases of pathogens, such as Leishmania, which survive in an acidic phagolysosomal-like compartment. ATPe may function as a ‘danger signal–that alerts the immune system to the presence of intracellular pathogens that damage the host cell, while different intracellular pathogens have evolved enzymes or other mechanisms to inhibit ATPe-mediated signalling, which should, thus, be viewed as virulence factors for these pathogens

"Endemol Turkey launches drama unit" - C21 Media

"Endemol Turkey has set up a drama unit and has appointed an executive from one of the country’s biggest free-to-air broadcasters to head it. Hulya Vural, who was head of drama at ATV and previously held the same post at Turkey’s Star network, started in the newly created head of drama role at Endemol Turkey in January. The company now aims to launch a drama project in the next 12 months, as it aims to cash in on the country’s booming scripted market, Endemol Turkey MD Ansi Elagoz told C21. “..

Specific cytotoxicity in C57BL/6 mice challenged with irradiated or non-irradiated trypomastigotes of <i>T. cruzi.</i>

<p>Groups of C57BL/6 mice were challenged or not i.p. with 10³ or 10⁴ irradiated or non-irradiated bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. A) Fifteen days after challenge, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of 4 mice±SD per group. B) Fifteen days after challenge, IFN-γ producing spleen cells specific to the peptide VNHRFTLV were estimated by the ELISPOT assay. The results represent the mean number of SFC per 10⁶ splenocytes±SD (n = 4). Asterisks denote statistically significant differences (<i>P</i><0.05) when we compared mice challenged with irradiated or non-irradiated trypomastigotes of <i>T. cruzi</i>. Results are representative of two independent experiments.</p

Kinetics of CD8⁺ T-cell mediated immune responses specific for sub-dominant epitopes in C57BL/6 mice.

<p>Groups of C57BL/6 mice were challenged or not i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. At the indicated days, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide TsKb-18 or TsKb-20 was determined as described in the Methods Section. The results represent the mean of 4 mice±SD per group. Asterisks denote statistically significant differences when we compared <i>T. cruzi</i> challenged with control mice (<i>P</i><0.05). ND = Not done. Results are representative of two or more independent experiments.</p

Trypomastigote-induced parasitemia in C57BL/6 mice challenged with different doses of trypomastigotes of <i>T. cruzi.</i>

<p>C57BL/6 mice were infected i.p. with 10²,10³, 10⁴ or 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Parasitemia was followed daily from days 0 to 14 after challenge. The results represent the mean of 5–6 mice±SD. At the peak of infection, the parasitemia of mice infected with each different dose was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) 10²×10³, Non-Significant (NS); ii) 10²×10⁴, <i>P</i><0.01; iii) 10²×10⁵, <i>P</i><0.01; iv) 10³×10⁴, <i>P</i><0.05; v) 10³×10⁵, NS; vi) 10⁴×10⁵, NS. Results are representative of two independent experiments.</p

Specific cytotoxicity in WT or genetically deficient mice challenged with <i>T. cruzi.</i>

<p>Groups of WT C57BL/6 (n = 4), WT 129 mice (n = 4), MHC-II KO (n = 4), perforin KO (n = 8), CD4 KO (n = 4), IL-12 KO (n = 4), and IFN-I receptor KO (n = 4) were challenged or not i.p. with 10⁵ bloodstream trypomastigotes of the Y strain of <i>T. cruzi</i>. Ten days after challenge, the <i>in vivo</i> cytotoxic activity against target cells coated with peptide VNHRFTLV was determined. The results represent the mean of the above indicated number of mice±SD per group. The <i>in vivo</i> cytotoxicity was compared by One-way Anova and Tukey HSD tests. The results of the comparisons were as follows: i) WT C57BL/6×MHC-II KO (<i>P</i><0.01); ii) WT C57BL/6×Perforin KO (<i>P</i><0.01); iii) WT C57BL/6×CD4 KO (<i>P</i><0.01); iv) WT C57BL/6×IL-12 KO (NS); v) WT 129×IFN-I receptor KO (NS). Results are representative of two or more independent experiments.</p