Increased expression of HSP70 by colon cancer cells is not always associated with access to the dendritic cell cross-presentation pathway

Dendritic cells (DCs) are highly specialized antigen-presenting cells endowed with the unique ability to not only present exogenous antigens upon exposure to MHC II, but also to cross-present these upon exposure to MHC I. This property was exploited to generate the tumor-specific CD8 cytotoxic lymphocyte (CTL) response in DCs-based cancer vaccine protocols. In this context, the source of tumor antigens remains a critical challenge. A crude tumor in the context of danger signals is believed to represent an efficient source of tumor antigens (TAs) for DCs loading. In our previous work, increased DCs cross-presentation of antigens from necrotic gastric carcinoma cells paralleled up-regulation of the heat shock protein hsp70. We studied the expression of hsp70 on primary colon carcinoma cells and its relevance in the cross-priming of anti-tumor CTL by tumor-loaded DCs. Hsp70 was expressed on all three of the tumors studied, but was never detected in the peritumoral normal mucosa (NM). The uptake of the tumor induced a trend towards down-modulation of the monocyte-specific marker CD14, but had no effect on the chemokine receptors CCR4 and CCR7. The IFN-γ enzyme-linked immunospot assay (ELIspot) showed cross-priming of CTL by tumor-loaded but not NM-loaded DCs in four of the six cases studied. The CTL response generated in DC+tumor cultures was directed towards the tumor, but not towards NM, and it was characterized by refractoriness to polyclonal (Ca ionophores, PKC activators) stimuli. Of the three CTL-generating tumors, only one expressed hsp70. This data indicates a tumor-specific expression of hsp70, but does not support its relevance in the DC cross-presentation of TAs.     

Reference proteome of highly purified human Th1 cells reveals strong effects on metabolism and protein ubiquitination upon differentiation

The differentiation of human CD4⁺ T cells into T helper cell subtypes and regulatory T cells is crucial to the immune response. Among subtypes, Th1 cells are dominant, representing approximately 50% of all lymphocytes. Thus far, most global proteomic studies have used only partially purified T helper cell subpopulations and/or have employed artificial protocols for inducing specific T helper cell subtypes and/or used gel‐based approaches. These studies have shed light on molecular details of certain aspects of the proteome; nevertheless a global analysis of high purity primary naïve and Th1 cells by LC‐MS/MS is required to provide a reference dataset for proteome‐based T cell subtype characterization. The utilization of highly purified Th1 cells for a global proteome assessment and the bioinformatic comparison to naïve cells reveals changes in cell metabolism and the ubiquitination pathway upon T cell differentiation. All MS data have been deposited in the ProteomeXchange with identifier PXD001066 ( http://proteomecentral.proteomexchange.org/dataset/PXD001066 ).     

Whole-genome sequence of Wolbachia strain wAlbB, an endosymbiont of tiger mosquito vector Aedes albopictus

Although bacteria of the genus Wolbachia induced significant extended phenotypes to infected hosts, most molecular mechanisms involved are still unknown. To gain insight into the bacterial genetic determinants, we sequenced the whole genome of Wolbachia wAlbB strain, a commensal obligate intracellular of the tiger mosquito Aedes albopictus.     

Regulation of the type IV secretion ATPase TrwD by magnesium: implications for catalytic mechanism of the secretion ATPase superfamily

TrwD, the VirB11 homologue in conjugative plasmid R388, is a member of the large secretion ATPase superfamily, which includes ATPases from bacterial type II and type IV secretion systems, type IV pilus, and archaeal flagellae assembly. Based on structural studies of the VirB11 homologues in Helicobacter pylori and Brucella suis and the archaeal type II secretion ATPase GspE, a unified mechanism for the secretion ATPase superfamily has been proposed. Here, we have found that the ATP turnover of TrwD is down-regulated by physiological concentrations of magnesium. This regulation is exerted by increasing the affinity for ADP, hence delaying product release. Circular dichroism and limited proteolysis analysis indicate that magnesium induces conformational changes in the protein that promote a more rigid, but less active, form of the enzyme. The results shown here provide new insights into the catalytic mechanism of the secretion ATPase superfamily.     

Polyphenols in brewed green tea inhibit prostate tumor xenograft growth by localizing to the tumor and decreasing oxidative stress and angiogenesis

It has been demonstrated in various animal models that the oral administration of green tea (GT) extracts in drinking water can inhibit tumor growth, but the effects of brewed GT on factors promoting tumor growth, including oxidant damage of DNA and protein, angiogenesis and DNA methylation, have not been tested in an animal model. To explore these potential mechanisms, brewed GT was administered instead of drinking water to male severe combined immunodeficiency (SCID) mice with androgen-dependent human LAPC4 prostate cancer cell subcutaneous xenografts. Tumor volume was decreased significantly in mice consuming GT, and tumor size was significantly correlated with GT polyphenol (GTP) content in tumor tissue. There was a significant reduction in hypoxia-inducible factor 1-alpha and vascular endothelial growth factor protein expression. GT consumption significantly reduced oxidative DNA and protein damage in tumor tissue as determined by 8-hydroxydeoxyguanosine/deoxyguanosine ratio and protein carbonyl assay, respectively. Methylation is known to inhibit antioxidative enzymes such as glutathione S-transferase pi to permit reactive oxygen species promotion of tumor growth. GT inhibited tumor 5-cytosine DNA methyltransferase 1 mRNA and protein expression significantly, which may contribute to the inhibition of tumor growth by reactivation of antioxidative enzymes. This study advances our understanding of tumor growth inhibition by brewed GT in an animal model by demonstrating tissue localization of GTPs in correlation with inhibition of tumor growth. Our results suggest that the inhibition of tumor growth is due to GTP-mediated inhibition of oxidative stress and angiogenesis in the LAPC4 xenograft prostate tumor in SCID mice.     

Iron overload,measured as serum ferritin,increases brain damage induced by focal ischemia and early reperfusion

High levels of iron, measured as serum ferritin, are associated to a worse outcome after stroke. However, it is not known whether ischemic damage might increase ferritin levels as an acute phase protein or whether iron overload affects stroke outcome. The objectives are to study the effect of stroke on serum ferritin and the contribution of iron overload to ischemic damage.     

Cellular proliferation, differentiation and apoptosis in polyether-polyurethane sponge implant model in mice

The integration of implanted material to host organism requires spatial and temporal organization of several cellular processes, such as proliferation, differentiation and apoptosis. Despite the clinical relevance of these processes, there is little information regarding the sequence of such events in synthetic matrices. Here, we present a combination of techniques used to characterize the fibrovascular response in subcutaneous polyether-polyurethane sponge implants in mice at days 4, 7, 10 and 14 postimplantation. The AgNOR technique was modified and used as a surrogate marker for proliferating and activated cells invading the implant. The number of AgNOR-stained cells increased progressively from day 4 (606+/-76) to day 14 (2146+/-71) postimplantation. The number of TUNEL-positive (apoptotic index) cells also increased progressively from day 4 (459+/-40) to day 14 (1157+/-119) postimplantation. However, the ratio of TUNEL-labeled/proliferating cells had its highest peak in the early phase of the process remaining stable until day 14. Using Picrosirius staining it was shown that thin collagen increased from day 4, peaking at day 10 and falling markedly at day 14, whereas dense collagen increased progressively during the whole period. These experiments hold potential to investigate not only distinct phases of tissue repair induced by synthetic matrices but also to study underlying mechanisms involved.     

Changes of adolescents' defence mechanisms during the first year of high school education

The goal of this study was to analyse the defence mechanism's changes during adolescent's adaptation to the new schooling terms at the first year in high school. Sample of 266 students, mean age 15, have been included in the research. Kellerman's Life Style Questionnaire (LSQ) was administred for measuring defence mechanisms at the beginning and at the end of the school year. Results show that regression and reactive formation are changing at most; regression raises (p < 0.005) while reactive formation decreases (p < 0.000). Boys used repression, while girls reactive formation more frequently in both measuring. Displacement was 'male" defence in the first measuring; but at repeated measuring this difference in sex vanished. The sources of anxiety can be found partly in new school environment and in adolescent's effort to "solve the problem" with sexual impulses and aggression using different sets of defences.     

Melatonin inhibits cardiolipin peroxidation in mitochondria and prevents the mitochondrial permeability transition and cytochrome c release

Cardiolipin oxidation is emerging as an important factor in mitochondrial dysfunction as well as in the initial phase of the apoptotic process. We have previously shown that exogenously added peroxidized cardiolipin sensitizes mitochondria to Ca²⁺-induced mitochondrial permeability transition (MPT) pore opening and promotes the release of cytochrome c. In this work, the effects of intramitochondrial cardiolipin peroxidation on Ca²⁺-induced MPT and on the cytochrome c release from mitochondria were studied. The effects of melatonin, a compound known to protect the mitochondria from oxidative damage, on both of these processes were also tested. tert-Butylhydroperoxide (t-BuOOH), a lipid-soluble peroxide that promotes lipid peroxidation, was used to induce intramitochondrial cardiolipin peroxidation. Exposure of heart mitochondria to t-BuOOH resulted in the oxidation of cardiolipin, associated with an increased sensitivity of mitochondria to Ca²⁺-induced MPT and with the release of cytochrome c from the mitochondria. All these processes were inhibited by micromolar concentrations of melatonin. It is proposed that melatonin inhibits cardiolipin peroxidation in mitochondria, and this effect seems to be responsible for the protection afforded by this agent against the MPT induction and cytochrome c release. Thus, manipulating the oxidation sensitivity of cardiolipin with melatonin may help to control MPT and cytochrome c release, events associated with cell death, and thus, be used for treatment of those disorders characterized by mitochondrial cardiolipin oxidation and Ca²⁺ overload.