ChaperISM: improved chaperone binding prediction using position-independent scoring matrices.

MOTIVATION: Understanding the mechanisms of client protein interaction with Hsp70 chaperones is essential to analyze the complex dynamics in the context of normal or dysregulated metabolism. Because Hsp70 can bind millions of proteins, including key molecules involved in processes of stemness, tumorigenesis and survival, in silico prediction of Hsp70 interactions has great value in validating possible new clients. Currently, two algorithms are available to predict binding to DnaK-the bacterial Hsp70-but both are based on amino acid sequence and energy calculations of qualitative information-binders and non-binders. RESULTS: We introduce a new algorithm to identify Hsp70 binding sequences in proteins-ChaperISM-a position-independent scoring matrix trained on either qualitative or quantitative chemiluminescence data previously published, which were obtained from the interaction between DnaK and different ligands. Both versions of ChaperISM, qualitative or quantitative, resulted in an improved performance in comparison to other state-of-the-art chaperone binding predictors. AVAILABILITY AND IMPLEMENTATION: ChaperISM is implemented in Python version 3. The source code of ChaperISM is freely available for download at https://github.com/BioinfLab/ChaperISM. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Immune response of toddlers with history of prematurity.

BACKGROUND: It is not quite well established how immune responses differ in term and preterm infants beyond the first year of life. This study aimed to evaluate aspects of the innate and adaptive immune responses in a group of preterm infants in comparison with their term peers. METHODS: In this cross-sectional study peripheral blood mononuclear cells (PBMC) were isolated from preterm and term children at age three years. Innate immune response was evaluated by the analysis of TLR receptors expression on CD11c+HLADRhigh cells and inflammatory cytokine production after PBMC stimulation with Toll like receptors (TLR) ligands. Adaptive immune response was evaluated by T cells' phenotyping and function after stimulation with polyclonal conventional T cell stimulus. CONCLUSION: We have found that the patterns of innate and adaptive immune responses at 3 years of age were not affected by the fact of the children having being born preterm or at term.

Respiratory syncytial virus induces phosphorylation of mTOR at ser2448 in CD8 T cells from nasal washes of infected infants.

Respiratory syncytial virus (RSV)-specific CD8(+) T cell responses do not protect against reinfection. Activation of mammalian target of rapamycin (mTOR) impairs memory CD8(+) T cell differentiation. Our hypothesis was that RSV inhibits the formation of CD8(+) T cells memory responses through mTOR activation. To explore this, human and mouse T cells were used. RSV induced mTOR phosphorylation at Ser2448 in CD8 T cells. mTOR activation by RSV was completely inhibited using rapamycin. RSV-infected children presented higher mTOR gene expression on nasal washes comparing to children infected with metapneumovirus and rhinovirus. In addition, RSV-infected infants presented a higher frequency of CD8(+) pmTORser2448(+) T cells in nasal washes compared to RSV-negative infants. Rapamycin treatment increased the frequency of mouse CD8 RSV-M282-90 pentamer-positive T cells and the frequency of RSV-specific memory T cells precursors. These data demonstrate that RSV is activating mTOR directly in CD8 T cells, indicating a role for mTOR during the course of RSV infection.

Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo.

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11clow/medB220+NK1.1+. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.

Cancer research in Brazil - stuck in second gear?

This article describes the main issues regarding clinical cancer research in Brazil, including both the opportunities and the hurdles. Scientists and clinicians in this field had the opportunity to talk to regulatory agencies and to the Health Ministry representative at a meeting held in the State of Rio de Janeiro, Brazil, in April 2014. Our conclusions are that we do indeed have opportunities; however, we need to move forward regarding partnerships between academia and industry, increase the availability of funding, and provide easier navigation through the regulatory processes.

An efficient gene transfer system for hematopoietic cell line using transient and stable vectors.

The hematopoietic system represents an interesting model for gene transfer protocols. Here, we have evaluated the efficiency of a gene transfer system using the polycationic compound SuperFect (Qiagen) and the K562 hematopoietic cell line. Transient and stable vectors carrying the enhanced green fluorescent protein (EGFP) reporter gene were employed. The stable vector was constructed based on Epstein-Barr virus sequences such as EBV oriP (origin of replication) and EBNA (EBV nuclear antigen)-1, both for DNA replication. The transfection efficiency of the viable cells was estimated by flow cytometry at approximately 98% for transient and stable vectors. Transiently transfected cells presented optimal EGFP expression until day 2 when fluorescence started to decrease. In contrast, stable transfectants continuously expressed the marker gene product for 10 weeks in the presence of G418. Our results represent an efficient gene transfer method for K562 hematopoietic cells and may be used as an alternative approach for further gene transfer studies involving hematopoietic cells.

Neuroprotective effects of a new skin care formulation following ultraviolet exposure.

BACKGROUND: Chronic ultraviolet (UV) exposure is a major environmental factor involved in extrinsic skin ageing (photo-ageing). Skin nerve fibres are significantly reduced in number following UV irradiation and new skincare compounds with neuroprotective effects are thus highly warranted. OBJECTIVES: We developed a new skincare formulation from a plant extract and evaluated its neuroprotective effects of ex vivo UV irradiation. MATERIALS AND METHODS: The new skincare emulsion was formulated from Echinacea purpurea extract and was enriched with antioxidants (patent no. PROV020110087075). Skin samples were obtained from 20 healthy patients enrolled for plastic surgery and were immediately treated with placebo (SPF 15) or test emulsions. Skin samples were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody, anti-human CD56. Cell damage was quantified by image analysis. RESULTS: UVA and UVB significantly reduced (40-60%) densities of nerve endings in control samples treated with placebo (P < 0.001). Samples treated with test emulsion completely blocked UV-related effects on skin nerve endings. These neuroprotective effects were similarly observed regardless of age or tissue analysed (breast versus abdomen). CONCLUSIONS: Our new skincare formulation obtained from E. purpurea provides important neuroprotective effects of UV irradiation and could be used together with SPFs to prevent chronic deleterious effects of solar exposure.

Spontaneous autoimmunity in mice that carry an IghV partial transgene: a required arginine in VHCDR3.

We describe a unique spontaneous mouse model of autoimmunity, which occurs on a non-autoimmune-prone SWR genetic background. In this model, SWR mice carry an IghV partial transgene (pTg) encoding only the heavy chain variable domain of an antibody directed against chromatin. Autoimmune disease in pTg mice was manifested by some of the features of systemic lupus erythematosus (SLE), including the presence of serum anti-nuclear antibodies, splenomegaly, skin lesions and a moderate degree of kidney pathology, in various combinations among individuals. Autoimmunity was observed in three independent transgenic lines, but not in three control lines carrying a nearly identical pTg, in which a VHCDR3 codon for Arg was replaced by one for Ser to ablate chromatin reactivity. Various features of disease were often but not always accompanied by anti-chromatin antibodies. Unexpectedly, the anti-chromatin antibodies detected in seropositive animals were not encoded by the pTg. These observations strongly implicate a role for the transgene product in disease initiation but not necessarily for end-state pathology, and they raise the possibility that autoreactive B cells may play a previously unappreciated role in initiating the development of systemic autoimmunity.

Effects of ultraviolet radiation on human cutaneous nerve fibres.

UNLABELLED: Due to an increasing number of skin diseases as a result of exposure to ultraviolet (UV) radiation, it is necessary to evaluate the effectiveness of new skin care formulations with broad-spectrum sunscreens. OBJECTIVES: This study aims to assess the status of nerve fibres in healthy human skin, to quantify effects of UV radiation on nerve endings, and to evaluate neuroprotective effects of new skin care formulations against UV exposure damage. METHODS: Samples were obtained from 34 female patients enrolled for plastic surgery and were immediately treated (10 min) with three emulsions: Cream 1, Cream 2 (placebo) and a sunscreen with sun protection factor 15 (SPF15). Control samples and those treated with the cream emulsions were exposed to UVA and UVB for 60 min. Nerve fibres were identified by immunofluorescence using a monoclonal antibody (anti-human CD56/NCAM). Cell damage was assessed by image analysis. RESULTS: Several cellular nervous structures were identified in the skin samples, including free nerve endings. UVA and UVB significantly decreased (40-60%) density of nerve endings in the control samples and those treated with placebo (Cream 2) or SPF15 (all P < 0.001). Cream 1 completely blocked effects of UV radiation on nerve endings (P > 0.05 vs. control). CONCLUSIONS: Quantification of cell damage induced by UV radiation provides useful information for identification of new skin care compounds with neuroprotective properties.

Immune response of toddlers with history of prematurity

Background: It is not quite well established how immune responses differ in term and preterm infants beyond the first year of life. This study aimed to evaluate aspects of the innate and adaptive immune responses in a group of preterm infants in comparison with their term peers. Methods: In this cross-sectional study peripheral blood mononuclear cells (PBMC) were isolated from preterm and term children at age three years. Innate immune response was evaluated by the analysis of TLR receptors expression on CD11c+HLADRhigh cells and inflammatory cytokine production after PBMC stimulation with Toll like receptors (TLR) ligands. Adaptive immune response was evaluated by T cells’ phenotyping and function after stimulation with polyclonal conventional T cell stimulus. Conclusion: We have found that the patterns of innate and adaptive immune responses at 3 years of age were not affected by the fact of the children having being born preterm or at term (AU)

No disponible

Heat shock genes in the willistoni group of Drosophila: induced puffs and proteins.

Seven species of the willistoni group of Drosophila were comparatively analysed for their response to heat shock and anoxia, both at the transcriptional (induced puff) and translational (induced protein) levels. The number of induced puffs varied among the species, but were concentrated at chromosomes 3 and XR. One puff present under control (25 degrees C) conditions was found to increase in size in response to stress in all species. Proteins of 83 and 70 kD were induced in all species in response to heat shock, as well as proteins of 50 and 32 kD, but not reported for other species of Drosophila under similar conditions. The number and size of the small heat shock proteins induced, as expected, showed considerable interspecific variability.

Characteristics of the strong antibody response to mycobacterial Hsp70: a primary, T cell-dependent IgG response with no evidence of natural priming or gamma delta T cell involvement.

Despite its high degree of evolutionary conservation, hsp70 is a surprisingly robust Ag, to such a degree that it is under consideration as a potential substrate in vaccine development. The cellular basis of the strong humoral response, however, is unknown, although it is often hypothesized to derive from restimulation of memory T cells that have been primed by hsp of intestinal flora. In this study, we tested this hypothesis and performed additional studies on the immune response to hsp70 of Mycobacterium tuberculosis. Superficially, the primary Ab response to this protein resembles a T cell-dependent secondary one, constituted almost exclusively by IgG. However, there is no evidence of natural priming, as revealed both by in vitro stimulation experiments and by immunity in germfree mice. Although hsp70 stimulates gammadelta and alphabeta T cells from unprimed mice to proliferate in vitro, gammadelta cells are not required for the strong humoral response, which is indistinguishable in normal and gammadelta T cell-deficient mice. Thus, the unusual immunogenicity of this protein in eliciting a humoral response appears to be due to a strong alphabeta T cell response with no evidence of natural priming or a gammadelta T cell involvement.

Somatic origin of T-cell epitopes within antibody variable regions: significance to monoclonal therapy and genesis of systemic autoimmune disease.

During an immune response, specific antibody variable region genes are diversified by a somatic point mutation process that generates de novo "foreign" V-region sequences. This creates an interesting problem in immune regulation because B cells are highly proficient at self-presenting V-region peptides in the context of class II MHC. Though our studies indicate that the corresponding T-cell repertoire attains a state of tolerance to germline-encoded antibody V-region diversity, it is presently unknown whether the same is true of mutationally generated diversity. On the basis of immunoregulatory considerations, we hypothesize that contact exclusion or tolerance normally precludes T cells from helping B cells via self-presented mutant V-region peptides. The lack of recurrent somatic mutations that create known T-cell epitopes in antibody V regions lends some support to this idea. In contrast, our studies of spontaneously autoreactive B cells in systemic autoimmune disease strongly suggest that precursors of such cells are recruited by T-cell help directed to self-presented mutant idiopeptides. Failures in tolerance or contact exclusion mechanisms may be responsible for this apparently abnormal event. In addition to their importance in immune regulation, somatic mutations or other differences from germline-encoded V-region sequence may be largely responsible for undesirable patient responses to therapeutic monoclonal antibodies. These reactions might be averted or diminished by inducing tolerance in the T-cell repertoire with synthetic peptide correlates of non-germline-encoded V-region sequences in humanized antibodies.

Mycobacterial heat shock protein 70 induces interleukin-10 production: immunomodulation of synovial cell cytokine profile and dendritic cell maturation.

Cytokines are key modulators of the immune responses that take place in the inflamed synovium of arthritis patients. Consequently, substances that can reverse the inflammatory profile of the inflamed joint are potential tools for clinical management of the disease. Mycobacterial heat shock protein 70 (MTBHSP70) has been found to protect rats from experimentally induced arthritis through the induction of interleukin (IL)-10-producing T cells. In this study, we have demonstrated that MTBHSP70 induces IL-10 production in synoviocytes from arthritis patients and peripheral blood monoculear cells (PBMCs) from both patients and healthy controls. IL-10 production was accompanied by a decrease in tumour necrosis factor (TNF)-alpha production by synovial cells. Separation studies showed that the target cells were mainly monocytes. Accordingly, we observed that MTBHSP70 delayed maturation of murine bone marrow-derived dendritic cells. Our results suggest that MTBHSP may act on antigen-presenting cells (APCs) to modulate the cytokine response in arthritis and support an anti-inflammatory role for this protein, suggesting that it may be of therapeutic use in the modulation of arthritis.

Mycobacterium tuberculosis aerogenic rechallenge infections in B cell-deficient mice.

OBJECTIVE: Use gene disrupted mice to examine the possible role of secretory antibody on resistance to re-exposure to pulmonary tuberculosis. DESIGN: Mice deficient in B cells due to targeted gene disruption were infected by aerosol exposure with Mycobacterium tuberculosis. A further set were identically exposed then given isoniazid to clear the infection and establish a state of memory immunity. RESULTS: Control of the aerosol infection and generation of gamma interferon proceeded in a similar manner in both naive and memory immune mice, regardless of B cell deficiency. CONCLUSIONS: The absence of antibody responses did not affect the course of infection, thus confirming the classical literature that antibody plays no significant protective role.