Noradrenergic and opioidergic alterations in neuropathy in different rat strains

The Fischer 344 (F344) rat strain differs from the Lewis strain in the response to neuropathic pain. Recently, we found that F344 rats totally recover from mechanical allodynia induced by chronic constriction injury (CCI) of the sciatic nerve 28 days after surgery whereas Lewis rats are initiating their recovery at this time point. Thus, the use of this neuropathic pain model in these different rat strains constitutes a good strategy to identify possible target genes involved in the development of neuropathic pain. Since differences between Lewis and F344 rats in their response to pain stimuli in acute pain models have been related to differences in the endogenous opioid and noradrenergic systems, we aimed to determine the levels of expression of key genes of both systems in the spinal cord and dorsal root ganglia (DRG) of both strains 28 days after CCI surgery. Real time RT-PCR revealed minimal changes in gene expression in the spinal cord after CCI despite the strain considered, but marked changes in DRG were observed. A significant upregulation of prodynorphin gene expression occurred only in injured DRG of F344 rats, the most resistant strain to neuropathic pain. In addition, we found a significant downregulation of tyrosine hydroxylase and proenkephalin gene expression levels in both strains whereas δ-opioid receptor was found to be significantly downregulated only in injured DRG of Lewis rats although the same trend was observed in F344 rats. The data strongly suggest that dynorphins could be involved in strain differences concerning CCI resistance.     

Oligonucleotide Ligation Assay for Detecting Mutations in the Human Immunodeficiency Virus Type 1 pol Gene That Are Associated with Resistance to Zidovudine, Didanosine, and Lamivudine

This report describes the detection of mutations in the pol gene of human immunodeficiency virus type 1 associated with resistance to zidovudine, didanosine, and lamivudine by genotyping by an oligonucleotide ligation assay specific codons in the pol gene amplified by PCR. Our studies demonstrate the sensitivity, simplicity, and specificity of this genotyping system.     

Engineering porcine arteries: effects of scaffold modification

Techniques have been developed to culture bovine or porcine vascular cells on polyglycolic acid (PGA) scaffolds to form engineered vessels. Previously, it was shown that smooth muscle cells (SMCs) that were in close proximity to PGA remnants after 8 weeks of culture had lower expression of SMC markers of differentiation and were more mitotic compared with SMCs that were distant from polymer residuals. Modifications of PGA were explored as a means to minimize residual polymer fragments after culture. To hasten degradation, polymer was treated with heat, NaOH, or gamma-irradiation. Differential scanning calorimetry, mass and tensile strength degradation, and inherent viscosity were used to assess polymer characteristics. When polymer was maintained in aqueous conditions, tensile strength of treated PGA degraded to zero within 3 weeks for each treatment. Engineered vessel constructs cultured on NaOH and gamma-treated polymer displayed smooth muscle alpha-actin throughout the vessel wall. Scaffold treatment impacted graft morphology, cellular differentiation, and mechanical integrity.     

Essential role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity

Defects in death receptor-mediated apoptosis have been linked to cancer and autoimmune disease in humans. The in vivo role of caspase 8, a component of this pathway, has eluded analysis in postnatal tissues because of the lack of an appropriate animal model. Targeted disruption of caspase 8 is lethal in utero. We generated mice with a targeted caspase 8 mutation that is restricted to the T-cell lineage. Despite normal thymocyte development in the absence of caspase 8, we observed a marked decrease in the number of peripheral T-cells and impaired T-cell response ex vivo to activation stimuli. caspase 8 ablation protected thymocytes and activated T-cells from CD95 ligand but not anti-CD3-induced apoptosis, or apoptosis activated by agents that are known to act through the mitochondria. caspase 8 mutant mice were unable to mount an immune response to viral infection, indicating that caspase 8 deletion in T-cells leads to immunodeficiency. These findings identify an essential, cell-stage-specific role for caspase 8 in T-cell homeostasis and T-cell-mediated immunity. This is consistent with the recent identification of caspase 8 mutations in human immunodeficiency.     

Neutrophils CD11b and fibroblasts PGE(2) are elevated in Alzheimer's disease

To evaluate whether inflammation-like mechanisms present in the brain of Alzheimer's disease (AD) patients are reflected in the periphery, the expression of CD11b in peripheral blood neutrophils and the expression and activity of inflammatory markers in cultured skin fibroblasts were examined. We found significantly higher levels of CD11b in neutrophils from sporadic AD patients than in controls and this elevation was positively correlated with disease severity and progression rate of mental decline. Cultured skin fibroblasts from familial (FAD) and sporadic AD patients and from controls were immunopositive for both isoforms of cyclooxygenase with no differences between groups. In unstimulated culture, the production of prostaglandin-E2 in the medium was significantly higher in fibroblasts from sporadic AD and FAD patients than in controls, and this elevation was reverted by the addition of 25 microM of ibuprofen. Our findings provide further evidence of the presence of inflammatory and immuno-related markers in the periphery of AD patients and support those studies indicating the beneficial effects of anti-inflammatory therapy in AD.     

Improved overall survival in dendritic cell vaccination-induced immunoreactive subgroup of advanced melanoma patients

Background  

We present our experience of therapeutic vaccination using dendritic cells (DC) pulsed with autologous tumor antigens in patients with advanced melanoma.     

Upregulation of TGF-beta, FOXP3, and CD4+CD25+ regulatory T cells correlates with more rapid parasite growth in human malaria infection

Understanding the regulation of immune responses is central for control of autoimmune and infectious disease. In murine models of autoimmunity and chronic inflammatory disease, potent regulatory T lymphocytes have recently been characterized. Despite an explosion of interest in these cells, their relevance to human disease has been uncertain. In a longitudinal study of malaria sporozoite infection via the natural route, we provide evidence that regulatory T cells have modifying effects on blood-stage infection in vivo in humans. Cells with the characteristics of regulatory T cells are rapidly induced following blood-stage infection and are associated with a burst of TGF-beta production, decreased proinflammatory cytokine production, and decreased antigen-specific immune responses. Both the production of TGF-beta and the presence of CD4+CD25+FOXP3+ regulatory T cells are associated with higher rates of parasite growth in vivo. P. falciparum-mediated induction of regulatory T cells may represent a parasite-specific virulence factor.     

Copolymer effects on microglia and T cells in the central nervous system of humanized mice

The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85-99-specific T cell receptor and HLA-DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85-99 and treated with these copolymers had reduced expression of HLA-DR, and thus reduced capacity to present MBP85-99 and activate transgenic T cells. In vitro microglia up-regulated empty HLA-DR2 upon activation with GM-CSF with or without LPS or IFN-gamma, but not with IL-4 or IL-10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK-treated mice differentially expressed pro- and anti-inflammatory molecules during MBP85-99-induced EAE. Interestingly, microglia expressed the full-length gammabeta and alphabeta subunits of the tetrameric adaptor protein complexes AP-1 and AP-2 respectively, but after treatment with GM-CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85-99 was composed of equal numbers of hVbeta2+ MPB85-99-specific transgenic and hVbeta2- endogenous T cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hVbeta2- endogenous T cells.