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Background: This study examined the development of chronic pain, a cardinal symptom of rheumatoid arthritis (RA), in mice with antigen- and collagen-induced arthritis (ACIA). Since the role of CD8+ T cells in arthritis is controversial, we investigated the consequences of CD8-depletion on arthritis development and opioid modulation of pain in this novel model of chronic autoimmune arthritis.Methods: Disease severity in control and CD8-depleted animals was determined by histological assessment of knee-joint sections and measurement of autoantibody formation. Pain was evaluated by measuring mechanical allodynia and thermal hyperalgesia in von Frey and Hargreaves tests, respectively. The production and release of endogenous opioids and inflammatory cytokines was assessed in immunoassays.Results: In ACIA, mice display persistent mechanical allodynia and thermal hyperalgesia for more than 2 months after induction of arthritis. The blockade of peripheral opioid receptors with naloxone-methiodide (NLXM) transiently increased thermal hyperalgesia, indicating that endogenous opioid peptides were released in the arthritic joint to inhibit pain. CD8+ T cell depletion did not affect autoantibody formation or severity of joint inflammation, but serum levels of the pro-inflammatory cytokines TNFα and IL-17 were increased. The release of opioid peptides from explanted arthritic knee cells and the NLXM effect were significantly reduced in the absence of CD8+ T cells.Conclusions: We have successfully modeled the development of chronic pain, a hallmark of RA, in ACIA. Furthermore, we detected a yet unknown protective role of CD8+ T cells in chronic ACIA since pro-inflammatory cytokines rose and opioid peptide release decreased in the absence of these cells. [ABSTRACT FROM AUTHOR]

Background: Assembly of recombinant capsid proteins into virus-like particles (VLPs) still represents an interesting challenge in virus-based nanotechnologies. The structure of VLPs has gained importance for the development and design of new adjuvants and antigen carriers. The potential of Tobacco etch virus capsid protein (TEV CP) as adjuvant has not been evaluated to date. Findings: Two constructs for TEV CP expression in Escherichia coli were generated: a wild-type version (TEV-CP) and a C-terminal hexahistidine (His)-tagged version (His-TEV-CP). Although both versions were expressed in the soluble fraction of E. coli lysates, only His-TEV-CP self-assembled into micrometric flexuous filamentous VLPs. In addition, the His-tag enabled high yields and facilitated purification of TEV VLPs. These TEV VLPs elicited broader IgG2- specific antibody response against a novel porcine reproductive and respiratory syndrome virus (PRRSV) protein when compared to the potent IgG1 response induced by the protein alone. Conclusions: His-TEV CP was purified by immobilized metal affinity chromatography and assembled into VLPs, some of them reaching 2-μm length. TEV VLPs administered along with PRRSV chimeric protein changed the IgG2/IgG1 ratio against the chimeric protein, suggesting that TEV CP can modulate the immune response against a soluble antigen. [ABSTRACT FROM AUTHOR]

Background: The subcommissural organ (SCO) is a highly conserved brain gland present throughout the vertebrate phylum; it secretes glycoproteins into the cerebrospinal fluid (CSF), where they aggregate to form Reissner's fiber (RF). SCO-spondin is the major constituent protein of RF. Evidence exists that the SCO also secretes proteins that remain soluble in the CSF. The aims of the present investigation were: (i) to identify and partially characterize the SCO-secretory compounds present in the SCO gland itself and in the RF of the Sprague-Dawley rat and nonhydrocephalic hyh mouse, and in the CSF of rat; (ii) to make a comparative analysis of the proteins present in these three compartments; (iii) to identify the proteins secreted by the SCO into the CSF at different developmental periods. Methods: The proteins of the SCO secreted into the CSF were studied (i) by injecting specific antibodies into ventricular CSF in vivo; (ii) by immunoblots of SCO, RF and CSF samples, using specific antibodies against the SCO secretory proteins (AFRU and anti-P15). In addition, the glycosylated nature of SCO-compounds was analysed by concanavalin A and wheat germ agglutinin binding. To analyse RF-glycoproteins, RF was extracted from the central canal of juvenile rats and mice; to investigate the CSF-soluble proteins secreted by the SCO, CSF samples were collected from the cisterna magna of rats at different stages of development (from E18 to PN30). Results: Five glycoproteins were identified in the rat SCO with apparent molecular weights of 630, 450, 390, 320 and 200 kDa. With the exception of the 200-kDa compound, all other compounds present in the rat SCO were also present in the mouse SCO. The 630 and 390 kDa compounds of the rat SCO have affinity for concanavalin A but not for wheat germ agglutinin, suggesting that they correspond to precursor forms. Four of the AFRU-immunoreactive compounds present in the SCO (630, 450, 390, 320 kDa) were absent from the RF and CSF. These may be precursor and/or partially processed forms. Two other compounds (200, 63 kDa) were present in SCO, RF and CSF and may be processed forms. The presence of these proteins in both, RF and CSF suggests a steadystate RF/CSF equilibrium for these compounds. Eight AFRU-immunoreactive bands were consistently found in CSF samples from rats at E18, E20 and PN1. Only four of these compounds were detected in the cisternal CSF of PN30 rats. The 200 kDa compound appears to be a key compound in rats since it was consistently found in all samples of SCO, RF and embryonic and juvenile CSF. Conclusion: It is concluded that (i) during the late embryonic life, the rat SCO secretes compounds that remain soluble in the CSF and reach the subarachnoid space; (ii) during postnatal life, there is a reduction in the number and concentration of CSF-soluble proteins secreted by the SCO. The molecular structure and functional significance of these proteins remain to be elucidated. The possibility they are involved in brain development has been discussed. [ABSTRACT FROM AUTHOR]

An abstract of the research "Molecular mechanisms underlying neuroepithelial/ependymal denudation in the hydrocephalic hyh mutant: spatial and temporal expression of alpha-SNAP and N-cadherin" is presented.

An abstract of the research "Long-lasting hydrocephalus in hyh mutant mice: gain and loss of a brain surviving hydrocephalus" is presented.

Background Hyh mutant mice suffer a congenital hydrocephalus triggered by ependyma denudation [1]. The ventricular surface in non hydrocephalic newborn mice is lined by the immature ependyma, which is characterized for being vimentin (-) and S100ß (-), at variance in the adult animals the mature ependyma expresses vimentin and S100ß [2]. On the other hand, in the hydrocephalic mice the ependyma begins to denudate on the 12th day of gestation, and at PN8 only some areas of lateral ventricle are still endowed with ependyma. In parallel, astroglia starts to cover the denuded surface forming a new cell layer, the glial scar, which lines the damaged ventricular surface. We have studied the permeability to horseradish peroxidase (HRP) of these four regions at the ventricular wills: mature ependyma, and denuded areas with or without glial scar. Materials and methods Control and hydrocephalic hyh mice (Jackson Lab., USA) at 3rd and 30th day of post-natal life were injected into a lateral ventricle with 3% HRP. 15 min after the injection the animals were sacrificed under anesthesia. HRP was detected by immunocitochemistry with specific antibodies. Inmunocitochemistry for PCNA (to label proliferating cells) and GFAP, S100ß and vimentin was used Results In non-hydrocephalic mice the immature ependymal layer was impermeable to HRP, whereas the mature ependyma was permeable. In hydrocephalic animal the areas where the ependyma had detached and the glial scar had not yet form were permeable to HRP, diffusing through the parenchyma. The glial scar was recognized for being GFAP positive and surprisingly, vimentin positive. When this barrier was fully developed at PN-30, it was apparently impermeable. However, the presence in the neuropile of cells labelled with HRP might indicates that some HRP has passaged through the glial scar. In adult hydrocephalic animals, there are zones where the ependyma is not denuded. This ependyma and the neighbouring glial scar appear impermeable to HRP. However, the HRP labelling of subventricular structures in these levels suggest that some tracer has passed through. Conclusion The different permeability properties between mature and immature ependymal layers suggest that differences exist in cell adhesion features and permeability. In hydrocephalic mice, denudated areas devoid of glial scar are very permeable to HRP. Thus, ependymal denudation implies the loss of CSF-parenchyma barrier, which could influence the CNS development. In adult hidrocephalic mice there are ependimal patches that do not detach. This particular ependyma, as the glial layer lining the denuded area, prevents partially or completely the passage of HRP. The HRP labelling of subventricular structures in this two regions could be an indication that some HRP has passed through the non-detached ependyma and through the glial sheath by an as yet unknown mechanism. This suggests that these ependymal areas could correspond to an would be a tight ependyma, and that such an ependyma would have the same barrier properties as those of the glial scar. What actually are these barrier properties are being further investigated in our laboratory. specific ependyma population that in the normal animal would be a tight ependyma, and that such an ependyma would have the same barrier properties as those of the glial scar. What actually are these barrier properties are being further investigated in our laboratory. [ABSTRACT FROM AUTHOR]