Immune Response and Partial Protection against Heterologous Foot-and-Mouth Disease Virus Induced by Dendrimer Peptides in Cattle.

Synthetic peptides mimicking protective B- and T-cell epitopes are good candidates for safer, more effective FMD vaccines. Nevertheless, previous studies of immunization with linear peptides showed that they failed to induce solid protection in cattle. Dendrimeric peptides displaying two or four copies of a peptide corresponding to the B-cell epitope VP1 [136-154] of type O FMDV (O/UKG/11/2001) linked through thioether bonds to a single copy of the T-cell epitope 3A [21-35] (termed B2T and B4T, resp.) afforded protection in vaccinated pigs. In this work, we show that dendrimeric peptides B2T and B4T can elicit specific humoral responses in cattle and confer partial protection against the challenge with a heterologous type O virus (O1/Campos/Bra/58). This protective response correlated with the induction of specific T-cells as well as with an anamnestic antibody response upon virus challenge, as shown by the detection of virus-specific antibody-secreting cells (ASC) in lymphoid tissues distal from the inoculation point.

Relationship between dental occlusion and functional dependence in elderly Caucasians.

OBJECTIVE: To determine whether a relationship exists between the number of functional masticatory units (FMUs) and the level of functional dependence of elderly. SUBJECTS AND METHODS: The study group comprised 502 elderly Caucasians living in nursing homes in north-west Spain and Portugal. The number of FMUs was counted on direct visual inspection. The degree of dependence was assessed using the Barthel index. The results were validated in a group of 156 elderly. Statistical analysis of the results was performed using a generalised linear model (GLM), a logistic GLM, a ROC-GLM curve and a confusion matrix. RESULTS: The number of FMUs significantly affected the Barthel index score (explained deviance = 27.5%). The number of FMUs was significantly associated with a lower probability of dependence, both for women (explained deviance = 31%) and for men (explained deviance = 33%). The model based on FMUs showed a good discriminatory capacity for dependence (AUC = 0.84 in women and 0.82 in men). The predictive capacity of the dependence model based on FMUs was very high (sensitivity = 0.9 in women and 0.8 in men). CONCLUSIONS: In institutionalised elderly Caucasians, the number of FMUs is significantly associated with the Barthel index score and could be a predictive factor for dependence.

Inhibition of herpes virus infection in oligodendrocyte cultured cells by valproic acid.

Valproic acid (VPA) is a small fatty acid used for treatment of different neurologic diseases such as epilepsy, migraines or bipolar disorders. VPA modulates different processes of cell metabolism that can lead to alterations in susceptibility of several cell types to the infection of Human Immunodeficiency Virus (HIV), Epstein-Barr virus (EBV), as well as to exert an inhibitory effect on the replication of different enveloped viruses in cultured cells. Taken these data into account and the fact that HSV-1 has been involved in some neuropathies, we have characterized the effect of VPA on this herpesvirus infection of the differentiation/maturation-inducible human oligodendrocyte cell line HOG, which resulted more susceptible to VPA inhibition of virus growth after cell differentiation. In these cells, the role of VPA in virus entry was tackled. Incubation with VPA induced a slight but reproducible inhibition in the virus particles uptake mainly observed when the drug was added in the adsorption or early upon infection. In addition, transcription and expression of viral proteins were significantly downregulated in the presence of VPA. Remarkably, when the infective viral production was assessed, VPA dramatically blocked the detection of infectious HSV-1 particles. Herein, our results indicate that VPA treatment of HOG cells significantly reduces the effect of HSV-1 infection, virus entry and productivity without affecting cellular viability.

Gene expression profiles in the cerebellum of transgenic mice over expressing the human FMR1 gene with CGG repeats in the normal range.

Modifications in the GABA pathway are considered to be responsible for motor alterations in animal models for fragile X-associated tremor ataxia syndrome. We analyzed the expression profile in the cerebellum in a transgenic mouse model that over expresses the human FMR1 gene with CGG repeats in the normal range. We used the "GeneChip Mouse Gene 1.0 ST Array" from Affymetrix analyzing 28,853 well-described and -characterized genes. Based on data from the comparative analysis of the expression profile, we detected a significant gradient with a P value <0.1 and changes in expression equal to or greater than 1.5 times compared to the control mouse genes. There were significant changes in the expression of 104 genes, among which 72% had decreased and 28% had increased expression. With the exception of GabarapL2, no changes in expression of genes from the GABA pathway were observed, which may explain the absence of an altered motor phenotype in these mice. These results further support the view that toxic effects in fragile X-associated tremor ataxia syndrome are due to expansion of CGG repeats rather than increased mRNA levels, since in the transgenic mice the FMR1 mRNA levels were increased 20-100 times compared with those of control littermates.

Genomic and antigenic characterization of viruses from the 1993 Italian foot-and-mouth disease outbreak.

The origin and evolution of the type O foot-and-mouth disease viruses (FMDV) that caused the outbreak occurrence in Italy in 1993, the first episode of the disease in the EU after adoption of a non-vaccination policy in 1991, have been studied by the analysis of sequences encoding three main antigenic sites on the viral capsid proteins. The phylogenetic tree derived from sequences spanning the carboxyterminal end of VP1 showed that these Italian viruses were grouped in the ME-SA topotype, closely related to viruses that circulated previously in the Middle East. The analysis of the nucleotide sequences in VP1, VP2 and VP3 showed a co-circulation during the epizootic of genetic variants, including viruses with amino acid replacements in VP3. For some of the isolates analyzed, values of fixation of nucleotide substitutions per year were observed in the three regions analyzed, ranging from 1.5 to 5.1 x 10(-2). The use of a panel of new monoclonal antibodies raised against an isolate from this outbreak, as well as monoclonal antibodies to FMDV O1-Switzerland 1965, showed differences in the reactivity pattern among some of the Italian isolates analyzed, which were consistent with the co-circulation of antigenic variants. These results support the potential for FMDV diversification in a limited period of time and under epidemiological conditions in which no vaccination campaigns were being implemented.

Myeloperoxidase release after allergen-specific conjunctival challenge.

BACKGROUND: Allergen-specific conjunctival challenge is a fruitful and complete tool in evaluating pathophysiological phenomena of allergic inflammation. After challenge, a significant neutrophil infiltrate occurred in allergic subjects. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether allergen-specific conjunctival challenge (ASCC) is able to elicit MPO release. We also investigated the possible role of immunotherapy (IT) in the release of MPO. METHOD: The groups studied included Dactylis glomerata-sensitive adult atopic patients suffering from seasonal allergic rhinoconjunctivitis, and healthy adult nonatopic volunteer controls. One group of allergic patients received no specific hyposensitization (not-IT allergic group). A second group of allergic patients had been immunotherapy-treated with Dactylis glomerata extract for the preceding three years and continued to receive a maintenance dose within the highest potency of the extract (IT-allergic group). ASCC with Dactylis glomerata was performed outside the pollen season in all subjects. Myeloperoxidase was assayed by MPO-enzyme immunoassay method. RESULTS: Thirty minutes after challenge, myeloperoxidase levels in the non-immunotherapy allergic patients were significantly higher compared than in the healthy group (p<0.001). The levels of myeloperoxidase released in the immunotherapy allergic group were significantly lower than those in the nonimmunotherapy allergic group (p<0.001) and higher than those in nonallergic subjects (p<0.001). CONCLUSION: These results indicate that after ASCC there is a release of MPO. Our study suggests that immunotherapy actively modifies the release of MPO after ASCC.

¿Es el neutrófilo una célula reguladora del eosinófilo en los procesos alérgicos mediados por la IgE? / Is the neutrophil leukocyte an eosinophil­ regulating cell in IgE- mediated allergic processes?

Aunque desde hace años se ha implicado al eosinófilo en la fisiopatología de los procesos alérgicos mediados por la lgE todavía no se conocen lo mecanismo exactos que atraen a esta células al órgano de choque del proceso atópico. Por una parte se habla de la propia presencia de lo tres receptores de la lgE (Fcel R, FccRII y galectina 3) en los eosinófilos de los pacientes alérgicos. pero si exite es mínima. Y por la otra no e ha podido activar a esos eosinófilo mediante un mecanismo mediado por la IgE. Pero el neutrófilo. que es una célula cuya participación en la respuesta alérgica es cada día más evidente, posee también los tres receptores de la IgE y. al contrario de lo que sucede en los eosinófilo . puede activarse a través de un mecanismo dependiente de la lgE como nuestro grupo ha demostrado en reiterada ocasiones. En este artículo comentamos como la activación mediada por la lgE de los neutrófilos puede modular la respuesta de los eosinófilos en los procesos atópicos (AU)

Even though the eosinophil has been implicated in the pathophysiology of IgE­mediated allergic processes for many a year. the precise mechanism that attract the e cells to the target organ of the aiopic proces are a yet unknown. Sorne author invoke the presence of the three lgE receptor (Fcalk. FccRII and gallectin 3) on the eosinophils of allergic patient ; yet, if so. such a presence is minimal. Furthermore. it ha not been posible to activate those eosinophil through an IgE­mediated mechanim. However, the neutrophil, a cell who e participation in the allergic repone i increasingly evident, al o possese the three IgE receptor and, to the contrary of what happen with the eosinophil. it can be activated through an IgE­mediated rnechanism, a our group has repeatedly hown. We here di cus how IgE­mediated neutrophil activation may modulate the repone of eosinophil in atopic procese (AU)

Allergen-dependent CD14 modulation and apoptosis in monocytes from allergic patients.

BACKGROUND: CD14 is a most important monocyte surface molecule. Recently, it has been reported that there is an important relationship between CD14 and immunoglobulin E, and that regulation of CD14 expression is an effector mechanism mediating apoptosis of monocytes. OBJECTIVE: The present study was designed to determine whether specific allergens were able to modulate CD14 expression and apoptosis by monocytes from allergic patients or whether specific immunotherapy (IT) might affect these processes. METHODS: One group of adult allergic asthmatic patients had received IT for the previous 3 years. Another similar group was not treated with IT. We challenged peripheral blood monocytes from both groups of asthmatic patients in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. The cells were also challenged with allergen to which the patients were not sensitive. Monocytes from normal subjects were also challenged with allergens. Expression of CD14 on the monocyte surface was analyzed by flow cytometry, and soluble CD14 (sCD14) in culture supernatant by enzyme-linked immunosorbent assay. The three groups of subjects were challenged with allergens, and apoptosis was analyzed by flow cytometry. RESULTS: When monocytes from non-IT-treated asthmatic patients were cultivated with the allergens to which the patients were sensitive, a significant up-regulation on the monocyte surface was observed compared with results from the healthy group (P < 0.003) and from the IT asthmatic group (P < 0.003). A significantly higher sCD14 level was observed in the culture supernatant of the monocytes from the IT asthmatic group were observed compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (P < 0.001). A significantly higher apoptosis level was observed in monocytes from the IT asthmatic group compared with those from the healthy group (P < 0.001) and those from the non-IT asthmatic group (<0.001). CONCLUSIONS: We present evidence that the expression of CD14 on the surface of monocytes and the apoptosis of the same cells can be modulated by an allergen-dependent mechanism. These processes can be affected by IT.

Stable expression of antisense RNAs targeted to the 5' non-coding region confers heterotypic inhibition to foot-and-mouth disease virus infection.

The antiviral potential of transcripts targeted to the non-coding regions (NCRs) of foot-and-mouth disease virus (FMDV) RNA have been studied during transient and constitutive expression in susceptible BHK-21 cells. Transient expression of antisense transcripts corresponding to the 5' and 3'NCRs, alone or in combination, confers specific inhibition of homologous (serotype C) virus infection in BHK-21 cells. Constitutive expression of antisense 5'NCR transcripts (5'AS) exerted higher levels of inhibition to homologous and heterologous (serotypes O, A, Asia, SAT 1, SAT 2 and SAT 3) FMDV infection, as estimated by a 10-fold reduction in virus titre in the supernatants from infected clones and by a plaque reduction assay. These inhibitions were also observed, albeit to a lesser extent, in clones stably expressing antisense 3'NCR transcripts. The antiviral response was specific for FMDV, as the picornavirus encephalomyocarditis virus was not inhibited in any of the transformed cell lines. In all cases, a correlation was found between the level of transcript expression and the extent of virus inhibition. The potential to efficiently inhibit FMDV, including isolates representing the seven serotypes, by expressing interfering 5'AS transcripts opens the possibility of developing transgenic animals with a reduced susceptibility to FMDV.

Respiratory burst in neutrophils from asthmatic patients.

BACKGROUND: The role of oxygen radicals has been implicated in disease processes of asthma. We have previously shown that specific allergens were able to activate respiratory burst by neutrophils from allergic patients sensitized to allergens of the same type as those which produce clinical allergy. OBJECTIVES: In this study, we attempted to evaluate the production of respiratory burst by an anti-IgE Ab in neutrophils from asthmatic allergic patients (with and without immunotherapy treatment) and in neutrophils from healthy subjects. METHOD: Neutrophils were stimulated by 10 microg/mL of anti-IgE Ab for 15 min at 37 degrees C. The production of respiratory burst from neutrophils was assayed by luminol-amplified chemiluminescence method. RESULTS: The respiratory burst was significantly higher in neutrophils from non-IT-asthmatic patients than in neutrophils from both healthy (p < 0.001) and IT-asthmatic (p < 0.001) groups. The IT-asthmatic group presented levels of respiratory burst approximately equal to those from non-allergic subjects (p=0.426). CONCLUSIONS: We conclude that neutrophils obtained from allergic asthmatic patients have an increased propensity to generate respiratory bursts, in comparison with neutrophils from healthy subjects. Immunotherapy actively modifies the respiratory burst by neutrophils from allergic asthmatic patients.

Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties.

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.

Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells.

The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.

Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation.

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA-NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K gamma gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kgamma. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.

IgE-dependent release of myeloperoxidase by neutrophils from allergic patients.

BACKGROUND: The three forms of IgE receptor: the heterotrimeric high-affinity receptor for IgE (FcepsilonRI), the low-affinity receptor for IgE (FcepsilonRII/CD23) and the Mac-2/IgE-binding protein (epsilonBP), have previously been found on human neutrophils. We have previously shown that specific allergens are able to activate functional responses by neutrophils from allergic patients sensitized to those allergens. Neutrophils are present in the sites of allergic inflammation. The primary (azurophilic) granules of neutrophils contain a variety of enzymes that might potentiate inflammation, such as myeloperoxidase (MPO). It is not known whether specific allergens are able to elicit MPO release by neutrophils from allergic patients. METHODS: Neutrophils were challenged in vitro with the specific allergen that produced clinical symptoms in asthmatic patients. Also, the cells were challenged with allergens that the patients were not sensitive to. Neutrophils from normal subjects were also challenged with allergens. RESULTS: The in vitro challenge of neutrophils with allergens that the patients were sensitive to elicited a release of MPO by these cells. The in vitro activation of neutrophils was highly allergen-specific, in such a way that allergens other than those accounting for clinical symptoms did not evoke MPO release, and allergens were ineffective on neutrophils from healthy donors. CONCLUSION: An IgE-dependent mechanism might promote MPO release by neutrophils at allergic sites.

Age-related changes in membrane lipid composition, fluidity and respiratory burst in rat peritoneal neutrophils.

The O2*(-) production has been studied in rat peritoneal neutrophils of different age (3, 12 and 24 months), in order to analyse whether the neutrophil respiratory burst is modified with increasing age. To stimulate NADPH oxidase, the enzyme responsible for the respiratory burst, two stimuli that act in different way have been used: phorbol myristate acetate (PMA) and N-formyl-methionyl-leucyl-phenylalanine (N-FMLP). Production of O2*(-) decreased with age in neutrophils stimulated with N-FMLP (about 40%), but not in the stimulated with PMA. No difference in NADPH oxidase activity was found with age. The NADPH is supplied to the respiratory burst mainly by the pentose phosphate shunt. A progressive and significant decrease in the two most important enzymes of this route, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was detected as a function of age; in spite of this reduction, the NADPH produced by cells from old animals seems not limiting for the O2*(-) production. The N-FMLP-induced decrease in the O2*(-) production may be related to the age-dependent increase in the membrane fluidity observed. A decline in the cholesterol/phospholipid ratio and a rise in the total polyunsaturated fatty acids content were found, that correlated well with the increase in the membrane fluidity. The decrease (50%) of phosphatidylinositols in the 24-month-old animals may be also related to the age-impairment in the respiratory burst found after stimulation with N-FMLP. These studies suggest that the age-related alterations in neutrophil may result in diminished neutrophil function and increased susceptibility to infection in the ageing.

Elevated secretion of myeloperoxidase by neutrophils from asthmatic patients: the effect of immunotherapy.

BACKGROUND: There is increasing evidence of neutrophil participation in asthma and the allergic process. After activation, neutrophils release myeloperoxidase (MPO) together with other granule enzymes. OBJECTIVES: In this study we attempted to evaluate the release of MPO in vitro by neutrophils from asthmatic patients and the relationship between neutrophil degranulation and lung function, measured as FEV(1), of the patients. We also investigated the possible role of immunotherapy in the release of MPO by neutrophils. METHODS: Neutrophils were stimulated with formyl-methionyl-leucyl-phenylalanine for 45 minutes at 37 degrees C. MPO released from neutrophils was assayed by using an MPO enzyme immunoassay. RESULTS: Neutrophils released statistically significantly higher MPO levels in the asthmatic patients not receiving immunotherapy than in the healthy group. A significant inverse correlation was observed in the asthmatic group not receiving immunotherapy between MPO secretion and lung function, measured as FEV(1), of the patients. Neutrophils of the asthmatic group receiving immunotherapy released significantly less MPO than did those of the asthmatic group not receiving immunotherapy, with MPO levels equal to those from nonallergic subjects. CONCLUSIONS: We conclude that neutrophils obtained from allergic asthmatic patients have an increased propensity to release MPO. The experiments described here provide evidence that there is a significant inverse relationship between levels of MPO released by neutrophils from allergic patients and lung function, as assessed by FEV(1). Our study suggests that immunotherapy actively modifies the release of MPO in vitro by neutrophils from allergic asthmatic patients.