A peptide binding motif for I-Eg7, the MHC class II molecule that protects E alpha-transgenic nonobese diabetic mice from autoimmune diabetes.

The nonobese diabetic (NOD) mouse, a model of spontaneous insulin-dependent diabetes mellitus (IDDM), fails to express surface MHC class II I-Eg7 molecules due to a deletion in the E alpha gene promoter. E alpha-transgenic NOD mice express the E alpha E beta g7 dimer and fail to develop either insulitis or IDDM. A number of hypotheses have been proposed to explain the mechanisms of protection, most of which require peptide binding to I-Eg7. To define the requirements for peptide binding to I-Eg7, we first identified an I-Eg7-restricted T cell epitope corresponding to the sequence 4-13 of Mycobacterium tuberculosis 65-kDa heat shock protein (hsp). Single amino acid substitutions at individual positions revealed a motif for peptide binding to I-Eg7 characterized by two primary anchors at relative position (p) 1 and 4, and two secondary anchors at p6 and p9. This motif is present in eight of nine hsp peptides that bind to I-Eg7 with high affinity. The I-Eg7 binding motif displays a unique p4 anchor compared with the other known I-E motifs, and major differences are found between I-Eg7 and I-Ag7 binding motifs. Analysis of peptide binding to I-Eg7 and I-Ag7 molecules as well as proliferative responses of draining lymph node cells from hsp-primed NOD and E alpha-transgenic NOD mice to overlapping hsp peptides revealed that the two MHC molecules bind different peptides. Of 80 hsp peptides tested, none bind with high affinity to both MHC molecules, arguing against some of the mechanisms hypothesized to explain protection from IDDM in E alpha-transgenic NOD mice.     

Phoretic Behaviour of Bromeliad Annelids (<Emphasis Type="Italic">Dero</Emphasis>) and Ostracods (<Emphasis Type="Italic">Elpidium</Emphasis>) using Frogs and Lizards as Dispersal Vectors

The phoretic behaviour of ostracods (Elpidium bromeliarum) andannelids (Dero superterrenus) that inhabit tank bromeliads was studied. Our previous field observations had shown that bromeliad ostracods can be found attached to the skin of amphibians and reptiles that move among bromeliads, probably allowing the ostracods to colonise new tanks. In this paper, we present the first record of bromeliad annelids found attached to frogs moving among bromeliads in the field. We have also enlarged the database on bromeliad ostracods engaged in phoretic association with terrestrial vertebrates in three locations in southeastern Brazil. In our laboratory experiments bromeliad annelids show a strong significant tendency to climb onto papers that had been in contact with frog skin when compared with control papers, indicating a kind of chemically oriented behaviour. Bromeliad ostracods, on the other hand, attached themselves to treated and untreated papers with same frequency. When brought into contact with various species of frogs and lizards, the bromeliad annelids and ostracods both presented preference to attach themselves to frogs, but the annelids showed a stronger preference to attach to frogs and to avoid attachment to lizards. Another experiment demonstrated that bromeliad annelids are much more prone to dehydration than are ostracods. We suggest that the chemically oriented behaviour presented by bromeliad annelids toward frogs could diminish the risk of death by dehydration during the transport among bromeliads due to the moist characteristic of frog skins.     

Hyaluronan chain conformation and dynamics

An overview of the present state of research in the field of hyaluronan chain conformational aspects is presented. The relationship between structure and dynamics are illustrated for a series of hyaluronan oligomers. Conformational characteristics of hyaluronan chains are discussed, together with the dynamic chain patterns, evaluated by using a theoretical approach to diffusive polymer dynamics. The dependence of correlation times and NMR relaxation parameters from the chain dimension are investigated. Topological features and dimensional properties are related to the structural determinants by using classical computational methods of molecular mechanics and Monte Carlo simulation.     

Early Th1 response in unprimed nonobese diabetic mice to the tyrosine phosphatase-like insulinoma-associated protein 2, an autoantigen in type 1 diabetes

The insulinoma-associated protein 2 (IA-2) is a phosphatase-like autoantigen inducing T and B cell responses associated with human insulin-dependent diabetes mellitus (IDDM). We now report that T cell responses to IA-2 can also be detected in the nonobese diabetic (NOD) mouse, a model of human IDDM. Cytokine secretion in response to purified mouse rIA-2, characterized by high IFN-gamma and relatively low IL-10 and IL-6 secretion, was elicited in spleen cells from unprimed NOD mice. Conversely, no response to IA-2 was induced in spleen cells from BALB/c, C57BL/6, or Biozzi AB/H mice that express, like NOD, the I-A(g7) class II molecule, but are not susceptible to spontaneous IDDM. The IA-2-induced IFN-gamma response in NOD spleen cells could already be detected at 3 wk and peaked at 8 wk of age, whereas the IL-10 secretion was maximal at 4 wk of age and then waned. IA-2-dependent IFN-gamma secretion was induced in CD4(+) cells from spleen as well as pancreatic and mesenteric lymph nodes. It required Ag presentation by I-A(g7) molecules and engagement of the CD4 coreceptor. Interestingly, cytokines were produced in the absence of cell proliferation and IL-2 secretion. The biological relevance of the response to IA-2 is indicated by the enhanced IDDM following a single injection of the recombinant protein emulsified in IFA into 18-day-old NOD mice. In addition, IFN-gamma production in response to IA-2 and IDDM acceleration could be induced by IL-12 administration to 12-day-old NOD mice. These results identify IA-2 as an early T cell-inducing autoantigen in the NOD mouse and indicate a role for the IA-2-induced Th1 cell response in IDDM pathogenesis.     

Frequencies of virus-specific CD4(+) and CD8(+) T lymphocytes secreting gamma interferon after acute natural rotavirus infection in children and adults

Human rotavirus-specific CD4(+) and CD8(+) T-cell responses in peripheral blood lymphocytes were studied using a flow cytometric assay that detects the intracellular accumulation of cytokines after short-term in vitro antigen stimulation. The frequencies of virus-specific T cells that secrete gamma interferon and interleukin-13 (IL-13) were determined in adults and children during the acute or convalescent phase of rotavirus-induced diarrhea, in asymptomatically infected adults and laboratory workers who worked with human stool samples containing rotavirus, and in healthy adults. Significantly higher frequencies of rotavirus-specific interferon gamma-secreting CD8(+) and CD4(+) T cells, but not IL-13-secreting T cells, were detected in symptomatically infected adults and exposed laboratory workers than in healthy adults and children with acute rotavirus diarrhea. The levels of rotavirus-specific T cells returned to levels found in healthy adults by 32 days after the onset of rotavirus diarrhea in most adult subjects. Children with rotavirus diarrhea had undetectable or very low levels of CD4(+) and CD8(+) T cells that secrete gamma interferon. Adult cytomegalovirus-seropositive individuals had frequencies of cytomegalovirus-specific T cells that secrete gamma interferon that were approximately 20 times the level of rotavirus-specific T cells. This result suggests that rotavirus is a relatively poor inducer of circulating memory T cells that secrete gamma interferon. The frequencies of gamma interferon-secreting CD4(+) and CD8(+) T cells and the frequencies of IL-13-secreting CD4(+) T cells responding to the T-cell superantigen staphylococcal enterotoxin B (SEB) were lower in children than in adults. In both adults and children, the frequencies of CD4(+) cells secreting gamma interferon in response to SEB were higher than the frequencies of cells secreting IL-13.     

Assessment of neuropathic pain in leprosy patients with relapse or treatment failure by infrared thermography: A cross-sectional study

BackgroundNeuropathic pain (NP) is one of the main complications of leprosy, and its management is challenging. Infrared thermography (IRT) has been shown to be effective in the evaluation of peripheral autonomic function resulting from microcirculation flow changes in painful syndromes. This study used IRT to map the skin temperature on the hands and feet of leprosy patients with NP.Methodology/Principal findingsThis cross-sectional study included 20 controls and 55 leprosy patients, distributed into 29 with NP (PWP) and 26 without NP (PNP). Thermal images of the hands and feet were captured with infrared camera and clinical evaluations were performed. Electroneuromyography (ENMG) was used as a complementary neurological exam. Instruments used for the NP diagnosis were visual analog pain scale (VAS), Douleur Neuropathic en 4 questions (DN4), and simplified neurological assessment protocol. The prevalence of NP was 52.7%. Pain intensity showed that 93.1% of patients with NP had moderate/severe pain. The most frequent DN4 items in individuals with NP were numbness (86.2%), tingling (86.2%) and electric shocks (82.7%). Reactional episodes type 1 were statistically significant in the PWP group. Approximately 81.3% of patients showed a predominance of multiple mononeuropathy in ENMG, 79.6% had sensory loss, and 81.4% showed some degree of disability. The average temperature in the patients’ hands and feet was slightly lower than in the controls, but without a significant difference. Compared to controls, all patients showed significant temperature asymmetry in almost all points assessed on the hands, except for two palmar points and one dorsal point. In the feet, there was significant asymmetry in all points, indicating a greater involvement of the lower limbs.ConclusionIRT confirmed the asymmetric pattern of leprosy neuropathy, indicating a change in the function of the autonomic nervous system, and proving to be a useful method in the approach of pain.     

microRNA-induced translational control of antiviral immunity by the cap-binding protein 4EHP

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Pentoxifylline Reverses Chronic Experimental Chagasic Cardiomyopathy in Association with Repositioning of Abnormal CD8+ T-Cell Response

BackgroundChronic chagasic cardiomyopathy (CCC), the main clinical sign of Chagas disease, is associated with systemic CD8⁺ T-cell abnormalities and CD8-enriched myocarditis occurring in an inflammatory milieu. Pentoxifylline (PTX), a phosphodiesterase inhibitor, has immunoregulatory and cardioprotective properties. Here, we tested PTX effects on CD8⁺ T-cell abnormalities and cardiac alterations using a model of experimental Chagas’ heart disease.Conclusions/SignificancePTX therapy ameliorates critical aspects of CCC and repositioned CD8⁺ T-cell response towards homeostasis, reinforcing that immunological abnormalities are crucially linked, as cause or effect, to CCC. Therefore, PTX emerges as a candidate to treat the non-beneficial immune deregulation associated with chronic Chagas'' heart disease and to improve prognosis.     

Bone marrow mesenchymal stem cells increase motility of prostate cancer cells via production of stromal cell‐derived factor‐1α

Prostate cancer frequently metastasizes to the bone, and the interaction between cancer cells and bone microenvironment has proven to be crucial in the establishment of new metastases. Bone marrow mesenchymal stem cells (BM‐MSCs) secrete various cytokines that can regulate the behaviour of neighbouring cell. However, little is known about the role of BM‐MSCs in influencing the migration and the invasion of prostate cancer cells. We hypothesize that the stromal cell‐derived factor‐1α released by BM‐MSCs may play a pivotal role in these processes. To study the interaction between factors secreted by BM‐MSCs and prostate cancer cells we established an in vitro model of transwell co‐culture of BM‐MSCs and prostate cancer cells DU145. Using this model, we have shown that BM‐MSCs produce soluble factors which increase the motility of prostate cancer cells DU145. Neutralization of stromal cell‐derived factor‐1α (SDF1α) via a blocking antibody significantly limits the chemoattractive effect of bone marrow MSCs. Moreover, soluble factors produced by BM‐MSCs greatly activate prosurvival kinases, namely AKT and ERK 1/2. We provide further evidence that SDF1α is involved in the interaction between prostate cancer cells and BM‐MSCs. Such interaction may play an important role in the migration and the invasion of prostate cancer cells within bone.     

Regulation of Stress-Inducible Phosphoprotein 1 Nuclear Retention by Protein Inhibitor of Activated STAT PIAS1

Stress-inducible phosphoprotein 1 (STI1), a cochaperone for Hsp90, has been shown to regulate multiple pathways in astrocytes, but its contributions to cellular stress responses are not fully understood. We show that in response to irradiation-mediated DNA damage stress STI1 accumulates in the nucleus of astrocytes. Also, STI1 haploinsufficiency decreases astrocyte survival after irradiation. Using yeast two-hybrid screenings we identified several nuclear proteins as STI1 interactors. Overexpression of one of these interactors, PIAS1, seems to be specifically involved in STI1 nuclear retention and in directing STI1 and Hsp90 to specific sub-nuclear regions. PIAS1 and STI1 co-immunoprecipitate and PIAS1 can function as an E3 SUMO ligase for STI. Using mass spectrometry we identified five SUMOylation sites in STI1. A STI1 mutant lacking these five sites is not SUMOylated, but still accumulates in the nucleus in response to increased expression of PIAS1, suggesting the possibility that a direct interaction with PIAS1 could be responsible for STI1 nuclear retention. To test this possibility, we mapped the interaction sites between PIAS1 and STI1 using yeast-two hybrid assays and surface plasmon resonance and found that a large domain in the N-terminal region of STI1 interacts with high affinity with amino acids 450–480 of PIAS1. Knockdown of PIAS1 in astrocytes impairs the accumulation of nuclear STI1 in response to irradiation. Moreover, a PIAS1 mutant lacking the STI1 binding site is unable to increase STI1 nuclear retention. Interestingly, in human glioblastoma multiforme PIAS1 expression is increased and we found a significant correlation between increased PIAS1 expression and STI1 nuclear localization. These experiments provide evidence that direct interaction between STI1 and PIAS1 is involved in the accumulation of nuclear STI1. This retention mechanism could facilitate nuclear chaperone activity.Stress-inducible phosphoprotein I (STI1)¹ is a conserved cochaperone protein that assists Hsp90 in managing client proteins, by mediating the transfer of proteins between Hsp70 and Hsp90 (1–3). STI1 contains several tetratricopeptide-repeat domains (TRP) that can serve as interaction modules with Hsp90 and Hsp70 (4). STI1 helps to drive the sequential steps involved in the Hsp90 chaperone machinery (5) and regulates the ATPase activity of Hsp90 (6, 7). STI1 is also secreted by distinct cells (8–12), using a noncanonical mechanism involving extracellular vesicles (11). Secreted STI1 can activate multiple signaling pathways in distinct cell types (8–10, 13–18).Elimination of STI1 in yeast sensitizes cells to Hsp90 inhibitors, but it is not by itself lethal (19). STI1 can also be eliminated in C. elegans, although it results in decreased life span (20). In contrast, STI1 mutant mice do not survive E10.5 and present several morphological defects, owing to decreased levels of several Hsp90-client proteins (21). Mouse embryonic fibroblasts obtained from STI1-deficient embryos also fail to thrive and present increased levels of the DNA damage marker γ-H2AX, suggestive of increased cellular stress (21). Hence, in mammals STI1 seems to play additional roles in cellular survival that are not yet fully understood.STI1 is abundantly expressed in the cytoplasm of cells, but can also be found in the Golgi (22), in vesicles and in multivesicular bodies (11). Moreover, this cochaperone has been shown to shuttle between the cytoplasm and the nucleus in cell lines (23). Cellular stress, arrest in G1/S phase of the cell cycle and phosphorylation are factors that seem to regulate STI1 nuclear localization (23, 24). Presumably nuclear STI1 can regulate chaperone activity, but whether it can interact with nuclear proteins is unknown.Previous experiments using cell lines have shown that knockdown of STI1 increases susceptibility of cells to irradiation (25). Whether changes in STI1 levels in primary differentiated cells, such as astrocytes, may affect their response to irradiation stress is unknown. This is of interest, as astrocytes, which can give rise to distinct tumor cells, are highly radioresistant (26). Indeed, astrocytes have a noncanonical DNA damage response (DDR) to irradiation (26). Here we show that STI1 undergoes nuclear translocation in astrocytes after γ-radiation-induced DNA damage. Moreover, astrocytes haploinsufficient for STI1 are more susceptible to cell death induced by irradiation. To understand potential mechanisms involved with STI1 nuclear retention, we have performed yeast-two hybrid screenings to identify STI1 nuclear partners. We identified protein inhibitor of activated STAT (PIAS1) as a direct interactor of STI1 and provide evidence that it acts as a small ubiquitin-like modifier (SUMO) E3 ligase for STI1. We show this interaction is involved with STI1 nuclear retention after irradiation. Interestingly, tissue microarray analysis demonstrated that higher PIAS1 levels are found in glioblastoma multiforme (GBM) when compared with non-neoplastic tissue. Furthermore, we uncovered a positive relationship between increased PIAS1 expression in GBMs and augmented STI1 nuclear localization. Our results reveal a novel mechanism by which increased expression of PIAS1, as observed in GBM, can increase the retention of nuclear STI1, a critical regulator of the chaperone machinery.