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Find video protocols related to scientific articles indexed in Pubmed.
Proteomic white adipose tissue analysis of obese mice fed with a high-fat diet and treated with oral angiotensin-(1-7).
Peptides
PUBLISHED: 08-04-2014
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Angiotensin-(1-7) has been described as a new potential therapeutic tool for the treatment and prevention of metabolic disorders by regulating several pathways in visceral white adipose tissue (vWAT). The aim of this study was to access the proteins differentially regulated by Ang-(1-7) using proteomic analysis of visceral adipose tissue. Male mice were divided into three groups and fed for 60 days, with each group receiving one of the following diets: standard diet+HP?CD (ST), high fat diet+HP?CD (HFD) and high fat diet+Ang-(1-7)/HP?CD (HFD+Ang-(1-7)). Body weight, fat weight and food intake were measured. At the end of treatment, Ang-(1-7) induced a decrease in body and fat weight. Differential proteomic analysis using two-dimensional electrophoresis (2-DE) combined with mass spectrometry were performed. Results of protein mapping of mesenteric adipose tissue using 2-DE revealed the presence of about 450 spots in each gel (n=3/treatment) with great reproducibility (>70%). Image analysis and further statistical analysis allowed the detection and identification of eight proteins whose expression was modulated in response to HFD when compared to ST. Among these, two proteins showed a sensitive response to Ang-(1-7) treatment (eno1 and aldehyde dehydrogenase). In addition, three proteins were expressed statistically different between HFD+Ang-(1-7) and HFD groups, and four proteins were modulated compared to standard diet. In conclusion, comparative proteomic analysis of a mice model of diet-induced obesity allowed us to outline possible pathways involved in the response to Ang-(1-7), suggesting that Ang-(1-7) may be a useful tool for the treatment of metabolic disorders.
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Identification of virulence factors in leishmania infantum strains by a proteomic approach.
J. Proteome Res.
PUBLISHED: 03-24-2014
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Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.
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Aberrant patterns of X chromosome inactivation in a new line of human embryonic stem cells established in physiological oxygen concentrations.
Stem Cell Rev
PUBLISHED: 03-18-2014
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One of the differences between murine and human embryonic stem cells (ESCs) is the epigenetic state of the X chromosomes in female lines. Murine ESCs (mESCs) present two transcriptionally active Xs that will undergo the dosage compensation process of XCI upon differentiation, whereas most human ESCs (hESCs) spontaneously inactivate one X while keeping their pluripotency. Whether this reflects differences in embryonic development of mice and humans, or distinct culture requirements for the two kinds of pluripotent cells is not known. Recently it has been shown that hESCs established in physiological oxygen levels are in a stable pre-XCI state equivalent to that of mESCs, suggesting that culture in low oxygen concentration is enough to preserve that epigenetic state of the X chromosomes. Here we describe the establishment of two new lines of hESCs under physiological oxygen level and the characterization of the XCI state in the 46,XX line BR-5. We show that a fraction of undifferentiated cells present XIST RNA accumulation and single H3K27me foci, characteristic of the inactive X. Moreover, analysis of allele specific gene expression suggests that pluripotent BR-5 cells present completely skewed XCI. Our data indicate that physiological levels of oxygen are not sufficient for the stabilization of the pre-XCI state in hESCs.
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Proteomic analysis of Trypanosoma cruzi response to ionizing radiation stress.
PLoS ONE
PUBLISHED: 01-01-2014
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Trypanosoma cruzi, the causative agent of Chagas disease, is extremely resistant to ionizing radiation, enduring up to 1.5 kGy of gamma rays. Ionizing radiation can damage the DNA molecule both directly, resulting in double-strand breaks, and indirectly, as a consequence of reactive oxygen species production. After a dose of 500 Gy of gamma rays, the parasite genome is fragmented, but the chromosomal bands are restored within 48 hours. Under such conditions, cell growth arrests for up to 120 hours and the parasites resume normal growth after this period. To better understand the parasite response to ionizing radiation, we analyzed the proteome of irradiated (4, 24, and 96 hours after irradiation) and non-irradiated T. cruzi using two-dimensional differential gel electrophoresis followed by mass spectrometry for protein identification. A total of 543 spots were found to be differentially expressed, from which 215 were identified. These identified protein spots represent different isoforms of only 53 proteins. We observed a tendency for overexpression of proteins with molecular weights below predicted, indicating that these may be processed, yielding shorter polypeptides. The presence of shorter protein isoforms after irradiation suggests the occurrence of post-translational modifications and/or processing in response to gamma radiation stress. Our results also indicate that active translation is essential for the recovery of parasites from ionizing radiation damage. This study therefore reveals the peculiar response of T. cruzi to ionizing radiation, raising questions about how this organism can change its protein expression to survive such a harmful stress.
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Crotalus durissus collilineatus Venom Induces TNF- ? and IL-10 Production in Human Peripheral Blood Mononuclear Cells.
ISRN Inflamm
PUBLISHED: 01-01-2014
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Snake venom has been the subject of numerous studies in an attempt to find properties and biological effects that may be beneficial to man. In this study we evaluated in vitro the effects of Crotalus durissus terrificus (Cdt) and Crotalus durissus collilineatus (Cdc) venom in human peripheral blood mononuclear cells (PBMCs). At 24?h, a significant decrease of viable cells was observed in cells stimulated with the Cdc venom at 0.0005?mg/mL and 0.005?mg/mL compared to the negative control. At 48?h, a significant decrease of viable cells was observed only in cells stimulated with Cdc venom at 0.005?mg/mL. A significant increase of TNF- ? and IL-10 was detected 48 hours after culture of PBMC with Cdc, but not with Cdt venom. The expression of CD69 and PD1 (programmed death-1), activation and regulatory cell markers, on CD8+ and CD8- T cells did not change in the presence of Cdt and Cdc venom. Our results suggest the presence of proinflammatory and anti-inflammatory components in the Cdc venom. Further analysis should be done to identify those Cdc venom components as it has been done for the Cdt venom by other authors, indicating that modulatory components are found in the venom of different species of Crotalus snakes.
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Proteomic changes in Bacteroides fragilis exposed to subinhibitory concentration of piperacillin/tazobactam.
Anaerobe
PUBLISHED: 04-12-2013
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Bacteroides fragilis is the anaerobe most frequently isolated from clinical specimens and piperacillin/tazobactam is among the drugs that can be used to treat polymicrobial infections in which this bacteria is often involved. During antibiotic therapy, inhibitory concentrations of antibiotics are always followed by subinhibitory concentrations which can generate phenotypic changes in bacteria. So, in this study we aimed to evaluate changes in the proteomic profile of B. fragilis grown in a sub-MIC of PTZ, using 2-D electrophoresis followed by matrix-assisted laser desorption/ionization time-of-flight/time of-flight. Analysis of the 2-DE gels showed 18 spots with significantly different volume percentages between experimental conditions and 12 were successfully identified by MS/MS. Two proteins with decreased abundance in sub-MIC condition were involved in the glycolysis (glyceraldehyde-3-phosphate dehydrogenase and triose phosphate isomerase), others two involved in amino acid metabolism (Oxoacyl-(acyl-carrier protein) synthase II and dihydrodipicolinate reductase), and finally, one protein involved in fatty acid metabolism (UDP-N-acetylglucosamine acyltransferase). Among the proteins with increased abundance, we founded three ATP synthase (alpha, beta, and alpha type V), which could be involved in antibiotic bacterial resistance by efflux pump, one protein involved in glycolysis (enolase), and one involved in protein degradation (aminoacyl-histidine dipeptidase). In conclusion, our data show overall changes in the proteome of B. fragilis conducted by sub-MIC of PTZ, whose consequences on bacterial physiology deserve further investigation.
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Down-regulation of CTLA-4 by HIV-1 Nef protein.
PLoS ONE
PUBLISHED: 01-23-2013
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HIV-1 Nef protein down-regulates several cell surface receptors through its interference with the cell sorting and trafficking machinery. Here we demonstrate for the first time the ability of Nef to down-regulate cell surface expression of the negative immune modulator CTLA-4. Down-regulation of CTLA-4 required the Nef motifs DD175, EE155 and LL165, all known to be involved in vesicle trafficking. Disruption of the lysosomal functions by pH-neutralizing agents prevented CTLA-4 down-regulation by Nef, demonstrating the implication of the endosomal/lysosomal compartments in this process. Confocal microscopy experiments visualized the co-localization between Nef and CTLA-4 in the early and recycling endosomes but not at the cell surface. Overall, our results provide a novel mechanism by which HIV-1 Nef interferes with the surface expression of the negative regulator of T cell activation CTLA-4. Down-regulation of CTLA-4 may contribute to the mechanisms by which HIV-1 sustains T cell activation, a critical step in viral replication and dissemination.
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Inflammatory and Immunological parameters in adults with Down syndrome.
Immun Ageing
PUBLISHED: 04-16-2011
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The increase in life expectancy within the general population has resulted in an increasing number of elderly adults, including patients with Down syndrome (DS), with a current life expectancy of about 50 years. We evaluate the parameters of humoral and cellular immune response, the quantitative expression of the regulator of calcineurin1 gene (RCAN1) and the production of cytokines. The study group consisted of adults DS (n = 24) and a control group with intellectual disability without Down syndrome (ID) (n = 21) and living in a similar environmental background. It was evaluated serology, immunophenotyping, the quantitative gene expression of RCAN1 and the production of cytokines.
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Unique features of memory T cells in HIV elite controllers: a systems biology perspective.
Curr Opin HIV AIDS
PUBLISHED: 03-25-2011
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Elite controllers constitute a rare group of HIV-infected individuals who control HIV replication and maintain normal CD4 cell counts without antiretroviral therapy (ART). The mechanisms involved in the control of infection are poorly understood. This review will focus on the identification of signaling pathways upregulated or downregulated in different memory T-cell subsets in elite controllers by using systems biology approaches. Features of memory T cells in simian immunodeficiency virus (SIV) natural hosts will be also highlighted. Finally, we will discuss how these approaches will guide the development of new vaccines and therapeutic interventions.
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Myocardial gene and protein expression profiles after autoimmune injury in Chagas disease cardiomyopathy.
Autoimmun Rev
PUBLISHED: 09-12-2010
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One third of the 16 million of individuals infected by the protozoan Trypanosoma cruzi in Latin America eventually develop chronic Chagas disease cardiomyopathy (CCC), an inflammatory dilated cardiomyopathy with shorter survival than non-inflammatory cardiomyopathies. The presence of a T cell-rich mononuclear inflammatory infiltrate and the relative scarcity of parasites in the heart suggested that chronic inflammation secondary to the autoimmune recognition of cardiac proteins could be a major pathogenetic mechanism. Sera from CCC patients crossreactively recognize cardiac myosin and T. cruzi protein B13. T cell clones elicited from peripheral blood with T. cruzi B13 protein or its peptides could crossreactively recognize epitopes from cardiac myosin heavy chain. Likewise, CD4+ T cell clones infiltrating CCC myocardium crossreactively recognize cardiac myosin and T. cruzi protein B13, and intralesional T cell lines produce the inflammatory cytokines IFN-? and TNF-?. Conversely, IFN-?-induced genes and chemokines were found to be upregulated in CCC heart samples, and IFN-? is able to induce cardiomyocyte expression of atrial natriuretic factor, a key member of the hypertrophy/heart failure signature. Proteomic analysis of CCC heart tissue showed reduced expression of the energy metabolism enzymes. It can be hypothesized that cytokine-induced modulation of cardiomyocyte gene/protein expression may be a novel disease mechanism in CCC, in addition to direct inflammatory damage.
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A vaccine encoding conserved promiscuous HIV CD4 epitopes induces broad T cell responses in mice transgenic to multiple common HLA class II molecules.
PLoS ONE
PUBLISHED: 03-18-2010
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Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, "promiscuous" (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.
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Programmed death-1-induced interleukin-10 production by monocytes impairs CD4+ T cell activation during HIV infection.
Nat. Med.
PUBLISHED: 01-22-2010
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Viral replication and microbial translocation from the gut to the blood during HIV infection lead to hyperimmune activation, which contributes to the decline in CD4+ T cell numbers during HIV infection. Programmed death-1 (PD-1) and interleukin-10 (IL-10) are both upregulated during HIV infection. Blocking interactions between PD-1 and programmed death ligand-1 (PD-L1) and between IL-10 and IL-10 receptor (IL-10R) results in viral clearance and improves T cell function in animal models of chronic viral infections. Here we show that high amounts of microbial products and inflammatory cytokines in the plasma of HIV-infected subjects lead to upregulation of PD-1 expression on monocytes that correlates with high plasma concentrations of IL-10. Triggering of PD-1 expressed on monocytes by PD-L1 expressed on various cell types induced IL-10 production and led to reversible CD4+ T cell dysfunction. We describe a new function for PD-1 whereby microbial products inhibit T cell expansion and function by upregulating PD-1 levels and IL-10 production by monocytes after binding of PD-1 by PD-L1.
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Proteomic analysis of Escherichia coli with experimentally induced resistance to piperacillin/tazobactam.
Res. Microbiol.
PUBLISHED: 01-12-2010
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The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with host-pathogen interactions. The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of Escherichia coli (minimal inhibitory concentration [MIC] = 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922 (MIC = 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis, pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E. coli resistance to piperacillin/tazobactam.
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Peripheral blood CCR4+CCR6+ and CXCR3+CCR6+CD4+ T cells are highly permissive to HIV-1 infection.
J. Immunol.
PUBLISHED: 12-30-2009
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There is limited knowledge on the identity of primary CD4(+) T cell subsets selectively targeted by HIV-1 in vivo. In this study, we established a link between HIV permissiveness, phenotype/homing potential, and lineage commitment in primary CD4(+) T cells. CCR4(+)CCR6(+), CCR4(+)CCR6(-), CXCR3(+)CCR6(+), and CXCR3(+)CCR6(-) T cells expressed cytokines and transcription factors specific for Th17, Th2, Th1Th17, and Th1 lineages, respectively. CCR4(+)CCR6(+) and CXCR3(+)CCR6(+) T cells expressed the HIV coreceptors CCR5 and CXCR4 and were permissive to R5 and X4 HIV replication. CCR4(+)CCR6(-) T cells expressed CXCR4 but not CCR5 and were permissive to X4 HIV only. CXCR3(+)CCR6(-) T cells expressed CCR5 and CXCR4 but were relatively resistant to R5 and X4 HIV in vitro. Total CCR6(+) T cells compared with CCR6(-) T cells harbored higher levels of integrated HIV DNA in treatment-naive HIV-infected subjects. The frequency of total CCR6(+) T cells and those of CCR4(+)CCR6(+) and CXCR3(+)CCR6(+) T cells were diminished in chronically infected HIV-positive subjects, despite viral-suppressive therapy. A high-throughput analysis of cytokine profiles identified CXCR3(+)CCR6(+) T cells as a major source of TNF-alpha and CCL20 and demonstrated a decreased TNF-alpha/IL-10 ratio in CXCR3(+)CCR6(-) T cells. Finally, CCR4(+)CCR6(+) and CXCR3(+)CCR6(+) T cells exhibited gut- and lymph node-homing potential. Thus, we identified CCR4(+)CCR6(+) and CXCR3(+)CCR6(+) T cells as highly permissive to HIV replication, with potential to infiltrate and recruit more CCR6(+) T cells into anatomic sites of viral replication. It is necessary that new therapeutic strategies against HIV interfere with viral replication/persistence in discrete CCR6(+) T cell subsets.
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Immunological and non-immunological effects of cytokines and chemokines in the pathogenesis of chronic Chagas disease cardiomyopathy.
Mem. Inst. Oswaldo Cruz
PUBLISHED: 04-20-2009
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The pathogenesis of Chagas disease cardiomyopathy (CCC) is not well understood. Since studies show that myocarditis is more frequent during the advanced stages of the disease, and the prognosis of CCC is worse than that of other dilated cardiomyopathies of non-inflammatory aetiology, which suggest that the inflammatory infiltrate plays a major role in myocardial damage. In the last decade, increasing evidence has shown that inflammatory cytokines and chemokines play a role in the generation of the inflammatory infiltrate and tissue damage. CCC patients have an increased peripheral production of the inflammatory Th1 cytokines IFN-gamma and TNF-alpha when compared to patients with the asymptomatic/indeterminate form. Moreover, Th1-T cells are the main producers of IFN-gamma and TNF-alpha and are frequently found in CCC myocardial inflammatory infiltrate. Over the past several years, our group has collected evidence that shows several cytokines and chemokines produced in the CCC myocardium may also have a non-immunological pathogenic effect via modulation of gene and protein expression in cardiomyocytes and other myocardial cell types. Furthermore, genetic polymorphisms of cytokine, chemokine and innate immune response genes have been associated with disease progression. We will review the molecular and immunological mechanisms of myocardial damage in human CCC in light of recent findings.
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Presumptive germ cells derived from mouse pluripotent somatic cell hybrids.
Differentiation
PUBLISHED: 03-25-2009
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The fusion of embryonic stem (ES) cells with differentiated somatic cells is an approach that reverses a somatic cell nucleus to a state of pluripotency. The resulting ES-somatic cell hybrids (ES-SCH) retain most of the properties of ES cells: differentiate into multiple cell types and have the ability to produce embryoid bodies (EB) and chimeras. However, it is still unknown whether ES-SCH will be able to complete the differentiation into germ cells (GC) in vitro similar to ES cells. Here, we show that near diploid ES-SCH, obtained by the fusion of mouse ES and spleen cells, were able to differentiate in vitro into presumptive GC. Differentiation of ES-SCH was induced through EB formation and by the addition of retinoic acid. Presumptive GC obtained reacted positively with anti-EMA, Vasa, Fragilis and Dazl antibodies and expressed GC-specific genes, such as Vasa, Stella, Dazl, Piwil 2, Tex14, Bmp8b, Tdrd1 and Rnf17. Fluorescent in situ hybridization analysis indicates chromosome reduction in the GC-like cells. Expression of meiotic and postmeiotic GC-specific genes such as Haprin, Acrosin, Scyp1, Scyp3 and Stra-8 were also detected. Transmission electron microscopy confirmed ES-SCH differentiation into presumptive GC. The presence of several autosomes and the X chromosome originated from the "somatic" partner did not prevent ES-SCH differentiation towards presumptive GC. Overall our study suggests an interesting in vitro model, which allows the study GC differentiation in reprogrammed somatic cells.
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Cellular and molecular mechanisms of memory T-cell survival.
Expert Rev Vaccines
PUBLISHED: 03-03-2009
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Long-term maintenance of the memory T-cell response is the hallmark of immune protection and, hence, constitutes one of the most important objectives of vaccine-development strategies. Persistent memory T cells, developed after vaccination or microbial infections, ensure the generation of an antimicrobial response upon re-exposure to the pathogen through rapid clonal proliferation and activation of effector functions. However, in the context of many pathogen infections, these memory T cells fail to persist and die. In this review, we will highlight recent exciting findings in studies of memory T cells, their generation, their lineage relationships and their survival pathways; indeed, survival of memory T cells and maintenance of their functionality are key features of the immune response in its quest to control disease progression and in the development of vaccines to persistent microbial infections.
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JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.