Antinociception induced by atorvastatin in different pain models.

Atorvastatin is a statin that inhibits the 3-hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase. Several landmark clinical trials have demonstrated the beneficial effects of statin therapy for primary and secondary prevention of cardiovascular disease. It is assumed that the beneficial effects of statin therapy are entirely due to cholesterol reduction. Statins have an additional activity (pleiotropic effect) that has been associated to their anti-inflammatory effects. The aim of the present study was to assess the antinociceptive activity of atorvastatin in five animal pain models. The daily administration of 3-100mg/kg of atorvastatin by oral gavage induced a significant dose-dependent antinociception in the writhing, tail-flick, orofacial formalin and formalin hind paw tests. However, this antinociceptive activity of atorvastatin was detectable only at high concentrations in the hot plate assay. The data obtained in the present study demonstrates the effect of atorvastatin to reduce nociception and inflammation in different animal pain models.

In vivo determination of genotoxicity induced by metals from orthodontic appliances using micronucleus and comet assays.

Orthodontic appliances are usually made of stainless steel, which contains metals such as nickel, chromium and iron that have been associated with DNA damage. The aim of the present study was to determine the genetic toxicity associated with orthodontic fixed appliances in twenty healthy patients (16 +/- 2.5 years) undergoing orthodontic treatment (fixed appliances - basic composition: stainless steel alloy), using the micronucleus (MN) and comet (CA) assays in buccal cells. Primary DNA damage level, as assessed by the CA, was low either before the beginning (1.5 +/- 1.05 damage index - DI) or 10 days after the placement of the orthodontic appliance (2.5 +/- 3.08 DI) and did not change significantly between these time points (p= 0.0913). Conversely, there was a significant increase in MN frequency 30 days after the beginning of the treatment (p= 0.0236). In this study, the MN assay was shown to be more sensitive than the CA. Other investigations are necessary in order to assess the genotoxic potential of orthodontic fixed appliances associated with long-term studies concerning these effects in orthodontic patients.

[Latin-American Dialysis and Kidney Transplantation Registry: data on the treatment of end-stage renal disease in Latin America]. / Registro latino-americano di dialisi e trapianto: dati relativi al trattamento dell'insufficienza renale cronica terminale nell'America Latina.

Latin America, a region composed of a series of neighboring countries that share their history, Latin ancestry and language (Spanish or Portuguese), includes Mexico, Central America, the Spanish Caribbean islands, and South America. The Latin-American Dialysis and Kidney Transplantation Registry, which has been operative since 1991, collects data from 20 countries (Argentina, Brazil, Bolivia, Chile, Colombia, Costa Rica, Cuba, Ecuador, El Salvador, Guatemala, Honduras, Mexico, Nicaragua, Panama, Paraguay, Peru, Puerto Rico, Dominican Republic, Venezuela and Uruguay), where 97% of Latin Americans live. The prevalence of renal replacement therapy (RRT) has increased from 119 patients per million (pmp) in 1991 to 478.2 in 2005 (147,158 patients [57%] on chronic hemodialysis, 58,251 [23%] on peritoneal dialysis and 52,565 [20%] living with a functioning kidney graft). The incidence rate also increased from 27.8 pmp in 1992 to 167 in 2005. The increment in prevalence and incidence occurred in all Latin- American countries. The transplantation rate increased from 3,7 pmp in 1987 to 15 pmp in 2005 (7,968 kidney transplants performed this year, the cumulative number being 98,415). Access to RRT was available for every patient diagnosed with end-stage renal disease only in Argentina, Brazil, Chile, Cuba, Puerto Rico, Venezuela and Uruguay. In Latin America, the incidence and prevalence of RRT increased year by year. Only in some countries is access to RRT available to 100% of diagnosed patients. Detection and prevention programs for chronic kidney disease are needed in the region. Meanwhile, access to RRT has to be improved for everybody who needs it.

Alimentação de leitoas pré-púberes com dietas contendo zearalenona / Feeding of pre-pubertal gilts with diets containing zearalenone

O desempenho, o peso de alguns órgãos e a morfologia vulvar de leitoas pré-púberes, alimentadas por 28 dias com dietas contendo zearalenona, foram avaliados. O delineamento experimental utilizado foi inteiramente ao acaso, com dois tratamentos, dieta controle (DC) e dieta controle + 2mg kg-1 de zearalenona (DZ), e seis repetições cada. Não houve diferença (P>0,05) entre os tratamentos para consumo médio diário de ração (1,24 x 1,19kg), ganho médio diário de peso (0,68 x 0,71kg), conversão alimentar (1,86 x 1,71) e peso vivo (PV); (30,9 x 30,4kg). A zearalenona não alterou (P>0,05) os pesos absoluto e relativo do coração (137 x 141g e 0,45 x 0,45 por cento PV), fígado (699 x 699g e 2,31 x 2,26 por centoPV), rins (47 x 49g e 0,15 x 0,16 por centoPV) e baço (166 x 171g e 0,55 x 0,55 por centoPV). Houve aumento (P<0,05) no comprimento (17 x 27cm) e no peso (23 x 157g e 0,07 x 0,51 por centoPV) do trato reprodutivo das leitoas do grupo DZ. O volume vulvar ao final do período foi 820 por cento maior (P<0,05) nos animais alimentados com zearalenona (941 x 8658mm³/kgPV0,6). Os resultados indicam que em suínos a zearalenona e seus metabólitos possuem atividade estrogênica, mas não interferem no desempenho dos animais.

The performance, the weights of some organs, and the vulvae morphology in pre-pubertal gilts fed diets containing zearalenone were evaluated during 28 days. The experimental design was completely randomized with two treatments (control diet, ZD - control diet + 2mg kg-1 of zearalenone) and six replications of each were done. No differences (P>0.05) between treatments for daily feed intake (1.24 x 1.19kg), average daily gain (0.68 x 0.71kg), feed conversion ratio (1.86 x 1.71), and live weight (30.9 x 30.4kg) were observed. Zearalenone did not change (P>0.05) the absolute and relative weights of heart (137 x 141g and 0.45 x 0.45 percentBW), liver (699 x 699g and 2.31 x 2.26 percentBW), kidneys (47 x 49g and 0.15 x 0.16 percentBW), and spleen (166 x 171g and 0.55 x 0.55 percent BW). However, zearalenone increased (P<0.05) the length (17 x 27cm) and weight (23 x 157g and 0.07 x 0.51 percentBW) of the reproductive tract. The final vulvae volume was 820 percent larger (P<0.05) in gilts fed diets containing zearalenone than those fed control diet (941 x 8658mm³/kgBW0.6). Results suggested that zearalenone and its metabolites have an estrogenic activity in pigs without changing the animal performance.

Administration of eliprodil during ethanol withdrawal in the neonatal rat attenuates ethanol-induced learning deficits.

RATIONALE: Prenatal exposure to alcohol can disrupt brain development, leading to a variety of behavioral alterations, including learning deficits. We have postulated that some central nervous system damage may be due to N-methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity that occurs during ethanol withdrawal. Consistent with this hypothesis, we previously demonstrated that administration of MK-801, an NMDA receptor antagonist, during ethanol withdrawal attenuates ethanol-related learning deficits using an animal model of fetal alcohol effects. However, MK-801 binds to the phencyclidine site, which affects all NMDA receptor subtypes and can cause adverse side effects and toxicity. Eliprodil is a more selective NMDA receptor antagonist that acts at the polyamine modulatory site of NMDA receptors. OBJECTIVES: The purpose of this study was to determine if administration of eliprodil during ethanol withdrawal would reduce the severity of learning deficits associated with developmental alcohol exposure. METHODS: Male rat pups were randomly assigned to ethanol-exposed or control treatments. On postnatal day (PD) 6, during a period of brain development similar to that of the mid-third trimester in humans, subjects were exposed to 6.0 g/kg ethanol or isocaloric maltose solutions via oral gavage. Twenty-four hours after the end of the ethanol treatment, during ethanol withdrawal, all subjects received an intraperitoneal injection of one of three doses of eliprodil (5, 10, or 25 mg/kg) or vehicle. On PD 40, all subjects were tested on a serial spatial discrimination reversal learning task. RESULTS: Ethanol-exposed subjects treated with vehicle committed a significantly greater number of errors compared to controls. Administration of eliprodil during ethanol withdrawal significantly decreased the number of errors in the ethanol-exposed groups, but had no significant effect on the performance of controls. CONCLUSION: These data support the hypothesis that NMDA receptor-mediated excitotoxicity during ethanol withdrawal contributes to fetal alcohol effects.

Single-cell analyses reveal two defects in peptide-specific activation of naive T cells from aged mice.

Confocal fluorescent microscopy was used to study redistribution of membrane-associated proteins in naive T cells from young and old mice from a transgenic stock whose T cells express a TCR specific for a peptide derived from pigeon cytochrome C. About 50% of the T cells from young mice that formed conjugates with peptide-pulsed APC were found to form complexes, at the site of binding to the APC, containing CD3epsilon, linker for activation of T cells (LAT), and Zap-70 in a central area and c-Cbl, p95(vav), Grb-2, PLC gamma, Fyn, and Lck distributed more uniformly across the interface area. Two-color staining showed that those cells that were able to relocalize c-Cbl, LAT, CD3epsilon, or PLC gamma typically relocalized all four of these components of the activation complex. About 75% of conjugates that rearranged LAT, c-Cbl, or PLC gamma also exhibited cytoplasmic NF-AT migration to the T cell nucleus. Aging had two effects. First, it led to a diminution of approximately 2-fold in the proportion of T cell/APC conjugates that could relocalize any of the nine tested proteins to the immune synapse. Second, aging diminished by approximately 2-fold the frequency of cytoplasmic NF-AT migration among cells that could generate immune synapses containing LAT, c-Cbl, or PLC gamma. Thus naive CD4 T cells from old mice exhibit at least two separable defects in the earliest stages of activation induced by peptide/MHC complexes.

Age-dependent alterations in the assembly of signal transduction complexes at the site of T cell/APC interaction.

TCR interaction with peptide-MHC complexes triggers migration of protein kinases, actin-binding proteins, and other accessory molecules to the T cell/APC synapse. We used confocal immunofluorescence methods to show that the adapter protein LAT (linker for activation of T cells) and the guanine nucleotide exchange factor Vav also move to the APC interface in mouse CD4 T cells conjugated to anti-CD3 hybridoma cells, and in TCR-transgenic CD4 cells conjugated to APC bearing agonist (but not closely related nonagonist) peptides. The proportion of CD4+ T cells able to relocalize LAT or Vav, or to relocate cytoplasmic NT-AT (NF-ATc) from cytoplasm to nucleus, declines about 2-fold in aged mice. The decline in LAT relocalization is accompanied by a similar decline in tyrosine phosphorylation of LAT in CD4 cells stimulated by CD3/CD4 cross-linking. Two-color experiments show that LAT redistribution is strongly associated with relocalization of both NF-ATc and protein kinase C-theta among individual cells. LAT migration to the immunological synapse depends on actin polymerization as well as on activity of Src family kinases, but aging leads to only a small change in the percentage of CD4 cells that redistribute F-actin to the site of APC contact. These results suggest that defects in the ability of T cells from aged donors to move kinase substrates and coupling factors, including LAT and Vav, into the T cell/APC contact region may contribute to the decline with age in NF-ATc-dependent gene expression, and thus to defects in T cell clonal expansion.

Endotoxin-triggered haematological interactions in Fusobacterium necrophorum infections.

The haematological mechanisms in the course of liver abscess formation were evaluated. They were examined by employing viable cells of Fusobacterium necrophorum subsp. necrophorum and Fusobacterium necrophorum subsp. funduliforme in comparison with their endotoxins. Whole cell infection with F.n. necrophorum led to neutrophilia and to a concomitant monocytosis in parallel with those responses induced by the in vivo injection of its endotoxin. Viable infection with F.n. funduliforme was characterized by a sustained endotoxin-related monocytosis against neutropenia. The stimulatory impact of endotoxin on monocytes when released from a viable F.n. funduliforme infection suggested an inherently peculiar mechanism which differed from the induction of both neutrophilia and monocytosis when F.n. funduliforme endotoxin was administered alone. The neutrophilic inducing capacity of the F.n. necrophorum endotoxin was equally illustrated by its positive chemotactic effect on polymorphonuclear neutrophils in vitro. The data presented here emphasize the virulence of F.n. necrophorum viewed in reference to changes in leucocyte trafficking and as complemented by a relatively high endotoxin content.

Chemical composition of endotoxins produced by Fusobacterium necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme.

The endotoxins from two recently-classified subspecies of Fusobacterium, namely F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme, were compared. Chemical analysis of the isolated endotoxins revealed that they were clearly different. Distinct levels of polysaccharides were demonstrated. The endotoxins isolated were devoid of heptose and 3-deoxy-D-manno-octulosonate (KDO). The endotoxins of F. n. necrophorum and F. n. funduliforme contained lipid A in a ratio of 4:1 which may account for the variations in their virulence.

Increased Zap-70 association with CD3zeta in CD4 T cells from old mice.

Aging diminishes the amount of phosphotyrosine in the CD3zeta chains of resting and activated mouse CD4 T cells by about threefold and might therefore be expected to a corresponding decline in Zap-70 association with CD3zeta and in Zap-70 kinase function in CD3zeta complexes. We show here that aging leads, unexpectedly, to an approximately twofold increase in the amount of Zap-70 associated with CD3zeta in resting CD4 T cells. There is, however, no effect of age on total intracellular Zap-70 content. Cross-linking CD3 to CD4 leads to an increase of only 50% in the functional activity of Zap-70 in CD3zeta complexes from freshly isolated CD4 T cells of young donors. Compared to Jurkat and HT-2 cells, fresh T cells show both higher baseline levels and lower induced levels of Zap-70 function in CD3zeta complexes. CD4 T cells from old mice have baseline levels of Zap-70 activity similar to those seen in activated T cells from young mice, and these levels do not increase after CD3/CD4 cross-linking. Tyrosine-specific phosphorylation of Zap-70 is also higher at rest in old T cells than in young T cells and inducible only in cells from young donors. These data suggest that age-related defects in T cell activation are not likely to be attributable simply to a decline in Zap-70 association with CD3zeta or to diminished Zap-70 phosphorylation. The increase with age in CD3zeta-Zap association, despite the loss with age in CD3zeta tyrosine phosphorylation, suggests that the pattern of tyrosine phosphate groups among CD3zeta ITAM groups may be different in T cells from young and old donors or that access to ITAM regions within CD3zeta may be blocked by inter- or intramolecular steric hindrance in young CD4 T cells.

Differential tyrosine phosphorylation of zeta chain dimers in mouse CD4 T lymphocytes: effect of age.

Antibody to the zeta chain of the CD3/TCR signal transduction complex precipitates a series of tyrosine-phosphorylated proteins that can be discriminated by electrophoresis in nonreducing polyacrylamide gels. Stimulation of resting mouse splenic CD4 T cells by cross-linking CD3 to CD4 leads to increases in tyrosine phosphorylation of five such proteins, of which three are likely to be dimeric forms of zeta as judged by behavior on two-dimensional gels. Two other phosphoproteins, of MW 38 and 55 kDa, also coprecipitate with the CD3zeta complex. The level of induced phosphorylation of each of these five stimulus-responsive phosphoproteins declines with donor age, T cells from 18-month-old mice being almost wholly nonresponsive. Resting T cells have only a single major form of tyrosine-phosphorylated zetazeta. Phosphorylation of this rapidly migrating species is not influenced by activation, but is about threefold lower in resting T cells from 12- to 18-month-old mice than in cells from 6-month-old animals. Thus the phosphorylation of the CD3-zeta chain of CD4 T cells from aged mice exhibits abnormalities both in the resting state and within the first 5 min of the activation process.

Structural domains of interleukin-2 receptor beta critical for signal transduction: kinase association and nuclear complex-formation.

The structural domains of interleukin-2 receptor beta (IL-2R beta) were examined, characterizing the protein domains, associated phosphoproteins and nuclear complexes of IL-2-induced signal transduction. A series of IL-2R beta cytoplasmic deletion mutants were constructed and transfected into a murine pre-B-cell line, Ba/F3. The proliferative response of characterized clones was determined. A minimal linear cytoplasmic sequence required for proliferation and a sequence motif (PQPLXP) needed along with Box1-Box2 for IL-2-induced proliferation were identified. Anti-phosphotyrosine Western-blot analysis of a stimulated biologically active clone showed several IL-2-induced tyrosylphosphorylated proteins with molecular masses ranging from 45 to 116 kDa. In vitro kinase studies of biologically active clone-receptor complexes showed a 116 kDa protein (p116) to be the major tyrosine-phosphorylated component. The presence of the p116 kinase in the receptor complex correlates with IL-2-induced proliferation. An IL-2-inducible p116 kinase has recently been characterized as a Jak kinase family member and named Jak3. Nuclear complexes were formed with the GRR oligomer only when the IL-2R beta mutant supported proliferation. This led us to conclude that Box1-Box2 and PQPLXP motifs associate with Jak3 and that this association is an essential element in the IL-2 signal-transduction pathway culminating in the formation of a nuclear complex.

Pervanadate simulates the effects of interleukin-2 (IL-2) in human T cells and provides evidence for the activation of two distinct tyrosine kinase pathways by IL-2.

Pervanadate has been shown to rapidly increase the level of tyrosine phosphorylation in intact cells. Because one of the most rapidly detectable events following treatment of human T cells with interleukin-2 (IL-2) is tyrosine kinase activation, we were interested to determine whether pervanadate could act to induce IL-2-associated events. We show here that pervanadate does act to induce IL-2 signal transduction pathways as determined by induction of mitogenesis and interferon gamma production in normal human T cells and the factor independent T cell line YT. Analysis of signal transduction events shows that pervanadate induces the activity of the src family of tyrosine kinases lck and fyn and the tyrosine phosphorylation of a major IL-2 responsive protein of 97 kDa. Pervanadate does not, however, induce the activity of tyrosine kinases associated with the IL-2 receptor or the phosphorylation of a major IL-2 responsive protein of 116 kDa (Jak-3). Together these data suggest that src family kinase activation is a down stream event following IL-2 stimulation and is not directly associated with the activation of the IL-2 receptor-associated tyrosine kinase. The data also imply that tyrosine phosphorylation of p116/Jak-3 is strictly associated with activation of tyrosine kinases associated with the IL-2 receptor. With the use of pervanadate as a tool, we have established a dissociation of src family kinases with IL-2 receptor activation and imply the involvement of two distinct tyrosine kinase pathways, a receptor-associated pathway closely coupled with Jak-3 phosphorylation and a downstream pathway involving src family kinase activation.

Characterization of a tyrosine kinase activity associated with the high-affinity interleukin 2 receptor complex.

The IL-2 receptor complex is minimally composed of two genetically unrelated subunits of relative molecular masses 55 and 75 kDa respectively. Structural information deduced from the cDNA sequences of either subunit have not revealed significant information as to the basis of the mechanisms of IL-2 receptor signal transduction. Nevertheless, IL-2 stimulates the activation of one or more tyrosine kinases requiring the functional participation of the p75 member of the receptor complex. Here we have developed the methods to isolate the receptor complex with an associated tyrosine protein kinase. Extracts of membrane glycoproteins from activated normal human T lymphocytes and cell lines demonstrated catalytic activation of tyrosine kinase activity when stimulated with IL-2. Purification of the receptor complex with biotinylated IL-2 revealed the presence of two dominant phosphotyrosyl-proteins of approximate molecular masses 58 and 97 kDa. Denaturation gel electrophoresis followed by renaturation of proteins associated with the IL-2 receptor complex demonstrated that the 97 kDa protein had catalytic autophosphorylation activity. The results indicate that the 58 and 97 kDa phosphotyrosyl-proteins can be found to co-precipitate with the IL-2 receptor complex and that the 97 kDa protein was demonstrated to have protein kinase activity. The association of such kinases with receptors devoid of catalytic structure may represent a unique paradigm of growth-factor receptor mechanisms.

Regulation of the interleukin 2 receptor complex tyrosine kinase activity in vitro.

Interleukin 2 (IL-2) has been shown to stimulate tyrosine phosphorylation of a number of proteins requiring only the p75 beta chain of the IL-2 receptor. Unlike the receptors for epidermal growth factor, insulin, and other growth factors, the p55-alpha and p75-beta chains of the IL-2 receptor have no tyrosine protein kinase domain suggesting that the IL-2 receptor complex activates protein kinases by a unique mechanism. The activation of tyrosine kinases by IL-2 in situ was studied and using a novel methodology has shown tyrosine kinase activity associated with the purified IL-2R complex in vitro. IL-2 stimulated the in situ tyrosine phosphorylation of 97 kDa and 58 kDa proteins which bound to poly(Glu,Tyr)4:1, a substrate for tyrosine protein kinases, suggesting these proteins had characteristics found in almost all tyrosine kinases. IL-2 was found to stimulate tyrosine protein kinase activity in receptor extracts partially purified from human T lymphocytes and the YT cell line. Biotinylated IL-2 was used to precipitate the high-affinity-receptor complex and phosphoproteins associated with it. The data indicated that the 97-kDa and 58-kDa phosphotyrosyl proteins were tightly associated with the IL-2 receptor complex. These proteins were phosphorylated on tyrosine residues by IL-2 stimulation of intact cells and ligand treatment of in vitro receptor extracts. Furthermore, the 97-kDa and 58-kDa proteins were found in streptavidin-agarose/biotinylated IL-2 purified receptor preparations and showed high affinity for tyrosine kinase substrate support matrixes. The experiments suggest that these two proteins are potential candidates for tyrosine kinases involved in the IL-2R complex signal transduction process.