Efeito de medicamentos indicados para a prevenção da mastite bovina no período seco sobre a função fagocítica in vitro de leucócitos do leite de caprinos

O uso de medicamentos antimamíticos específicos para vacas no período seco é indicado para prevenção de infecções na lactação seguinte. Não obstante, a ação das células envolvidas no período de secagem tem fundamental importância para a involução da glândula mamária e seu restabelecimento para a lactação subseqüente. A indisponibilidade de tais medicamentos para uso em cabras tem resultado na extrapolação do uso de produtos recomendados para vacas sem que se considerem as particularidades e diferenças anátomo-fisiológicas entre as espécies bovina e caprina. O presente estudo teve por objetivo avaliar a influência de cinco antimamíticos específicos para vacas secas sobre a função dos fagócitos provenientes de leite caprino. Para tal, fez-se o isolamento de células somáticas de 20 amostras de leite provenientes de 10 cabras lactantes, sem antecedentes de tratamento de mamite nos últimos 30 dias, sob condições higiênico-sanitárias de colheita e com resultados negativos ao cultivo microbiológico do leite. As células aderidas a lamínulas de vidro foram confrontadas com formulações contendo princípios ativos disponíveis no mercado como Gentamicina (M1), Cefalônio Anidro (M2), Ampicilina (M3), Cloxacilina Benzatínica (M4) e Cefapirina Benzatínica (M5). Avaliou-se, por microscopia, a fagocitose de partículas de Zymosan. As médias dos índices de fagocitose das células submetidas ao tratamento com M2 (15,12% ± 16,22), M3 (6,02% ± 7,96), M4 (4,54% ± 5,45) e M5 (2,47% ± 4,64) foram menores (p<0,001) que a média dos índices de fagocitose do grupo controle (40,67% ± 19,68). A média dos índices de fagocitose das células submetidas ao tratamento com M2 foi maior (p<0,05) que as médias dos tratamentos com M3, M4 e M5 enquanto estas foram estatisticamente iguais entre si. As amostras celulares submetidas ao medicamento M1 exibiram adesão insuficiente ou ausente às lamínulas, inviabilizando a avaliação da fagocitose por meio da técnica utilizada. Os resultados obtidos permitem concluir que os medicamentos estudados exerceram influência negativa sobre os fagócitos do leite, porém, esta interferência sobre as funções das células somáticas não pode por si só determinar o insucesso da terapia proposta para o período seco, pois deve ser considerada, outrossim, a eficácia do princípio ativo sobre o patógeno causador do processo infeccioso.

Efeito da inclusão de diferentes fontes lipídicas e óleo mineral na dieta sobre a digestibilidade dos nutrientes e os níveis plasmáticos de gordura em equinos

Objetivou-se avaliar os efeitos da inclusão de gordura de origem animal ou vegetal e de óleo mineral sobre a aceitabilidade, a digestibilidade dos nutrientes e as concentrações plasmáticas de triglicérides e colesterol em equinos. Utilizaram-se quatro potros de 13 a 16 meses de idade recebendo dieta contendo feno de gramínea e concentrado, em delineamento quadrado latino, analisado por contrastes ortogonais. A aceitabilidade não foi influenciada pela quantidade nem pelo tipo de óleo adicionado às dietas. Os menores valores de digestibilidade de matéria seca (MS), matéria orgânica (MO) e extrato etéreo (EE) foram observados para as dietas acrescidas de óleo mineral (58,90; 60,29 e 32,02%) em comparação à dieta controle, cujos valores foram de 62,58; 64,41 e 77,72%. O coeficiente de digestibilidade do EE obtido com a dieta com óleo mineral foi menor (32,02%) que o obtido para as dietas com gordura animal (90,26%) e gordura vegetal (86,47%). A dieta com óleo mineral reduziu a concentração de HDL-C (68,75 mg/dL) em relação à dieta controle (76,00 mg/dL). A adição de fontes lipídicas e óleo mineral não influencia na aceitabilidade da dieta por equinos. O óleo vegetal não se diferencia da gordura animal quanto à digestibilidade dos nutrientes, no entanto, essas fontes de lipídios afetam a digestibilidade do extrato etéreo. A adição de óleo mineral reduz os níveis plasmáticos de HDL-C, ao passo que a adição de gordura animal e gordura vegetal não altera as concentrações plasmáticas de colesterol.

Efeito do processamento na atividade antioxidante do grão de amaranto (Amaranthus cruentus L. BRS-Alegria)

As características nutricionais, funcionais e agrícolas do amaranto são responsáveis pelo aumento do interesse por este alimento nas últimas décadas. O grão pode ser cozido, estourado, torrado, extrusado ou moído para ser consumido. Foi avaliado o efeito destes processamentos na atividade antioxidante do grão de amaranto, através das determinações do teor de fenólicos totais e da atividade antioxidante in vitro por dois métodos: inibição da oxidação lipídica pelo sistema \03B2-caroteno/ácido linoléico e índice de atividade antioxidante pelo aparelho Rancimat®. Os processamentos reduziram em média o teor de fenólicos totais do grão de amaranto de 31,7 para 22,0 mg de equivalentes de ácido gálico/g de resíduo seco. Observou-se que o extrato obtido por etanol do grão torrado foi o único a apresentar menor índice de atividade antioxidante (IAA) em relação ao grão cru (1,3 v 1,7). Os processos de extrusão, torração e explosão não alteraram a capacidade de inibição da oxidação lipídica (IOL) do amaranto (55 por cento). Já o cozimento aumentou o IOL (79 por cento), o que pode ter ocorrido devido ao maior tempo de processamento sob alta temperatura (100ºC/10min). Os métodos mais comuns de processamento do grão de amaranto ocasionaram redução do teor de fenólicos totais, no entanto a atividade antioxidante do estourado e do extrusado, avaliada pelos dois métodos, foi semelhante ao do grão cru. O grão de amaranto tanto cru como processado apresenta potencial antioxidante. Polifenóis, antocianinas, flavonóides, tocoferóis, vitamina C e compostos gerados na reação de Maillard podem estar relacionados à atividade antioxidante deste grão

Efeito da aplicação de fertilização nitrogenada e lodo de esgoto nas características da madeira juvenil de árvores de Eucalyptus grandis

This work evaluated the wood anatomical and physical characteristics of 24 months-old Eucalyptus grandis trees, planted in 3x2m spacing and fertilized with nitrogen (6, 12, 18 month old) and sewage sludge (planting, 8 month old). For each treatment 10 eucalypts trees were cut according to the distribution of basal area. Wood samples were collected in different percentages of the total height to analyze the anatomical (vessels and fibers) and physical (wood density) characteristics. The results showed that the wood apparent density and wood basic density of the eucalypt trees in the nitrogen and sewage sludge were larger in comparison to the control. Radial profiles of wood apparent density, were similar in the three treatments, presenting the exected characteristics of juvenile wood of 24 months-old eucalypt trees. Fiber and vessel dimensions were not affected by fertilization.

Unexpected Diversity of Cellular Immune Responses against Nef and Vif in HIV-1-Infected Patients Who Spontaneously Control Viral Replication

Background: HIV-1-infected individuals who spontaneously control viral replication represent an example of successful containment of the AIDS virus. Understanding the anti-viral immune responses in these individuals may help in vaccine design. However, immune responses against HIV-1 are normally analyzed using HIV-1 consensus B 15-mers that overlap by 11 amino acids. Unfortunately, this method may underestimate the real breadth of the cellular immune responses against the autologous sequence of the infecting virus. Methodology and Principal Findings: Here we compared cellular immune responses against nef and vif-encoded consensus B 15-mer peptides to responses against HLA class I-predicted minimal optimal epitopes from consensus B and autologous sequences in six patients who have controlled HIV-1 replication. Interestingly, our analysis revealed that three of our patients had broader cellular immune responses against HLA class I-predicted minimal optimal epitopes from either autologous viruses or from the HIV-1 consensus B sequence, when compared to responses against the 15-mer HIV-1 type B consensus peptides. Conclusion and Significance: This suggests that the cellular immune responses against HIV-1 in controller patients may be broader than we had previously anticipated.

Real-Time PCR in HIV/Trypanosoma cruzi Coinfection with and without Chagas Disease Reactivation: Association with HIV Viral Load and CD4(+) Level

Background: Reactivation of chronic Chagas disease, which occurs in approximately 20% of patients coinfected with HIV/Trypanosoma cruzi (T. cruzi), is commonly characterized by severe meningoencephalitis and myocarditis. The use of quantitative molecular tests to monitor Chagas disease reactivation was analyzed. Methodology: Polymerase chain reaction (PCR) of kDNA sequences, competitive (C-) PCR and real-time quantitative (q) PCR were compared with blood cultures and xenodiagnosis in samples from 91 patients (57 patients with chronic Chagas disease and 34 with HIV/T. cruzi coinfection), of whom 5 had reactivation of Chagas disease and 29 did not. Principal Findings: qRT-PCR showed significant differences between groups; the highest parasitemia was observed in patients infected with HIV/T. cruzi with Chagas disease reactivation (median 1428.90 T. cruzi/mL), followed by patients with HIV/T. cruzi infection without reactivation (median 1.57 T. cruzi/mL) and patients with Chagas disease without HIV (median 0.00 T. cruzi/mL). Spearman's correlation coefficient showed that xenodiagnosis was correlated with blood culture, C-PCR and qRT-PCR. A stronger Spearman correlation index was found between C-PCR and qRT-PCR, the number of parasites and the HIV viral load, expressed as the number of CD4(+) cells or the CD4(+)/CD8(+) ratio. Conclusions: qRT-PCR distinguished the groups of HIV/T. cruzi coinfected patients with and without reactivation. Therefore, this new method of qRT-PCR is proposed as a tool for prospective studies to analyze the importance of parasitemia (persistent and/or increased) as a criterion for recommending pre-emptive therapy in patients with chronic Chagas disease with HIV infection or immunosuppression. As seen in this study, an increase in HIV viral load and decreases in the number of CD4(+) cells/mm(3) and the CD4(+)/CD8(+) ratio were identified as cofactors for increased parasitemia that can be used to target the introduction of early, pre-emptive therapy.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

Viral mutation and its influence in the time evolution of the immunizations

In this paper, we study the behavior of immune memory against antigenic mutation. Using a dynamic model proposed by one of the authors in a previous study (A. de Castro [Phys. J. Appl. Phys. 33, 147 (2006) and Simul. Mod. Pract. Theory. 15, 831 (2007)]), we have performed simulations of several inoculations, where in each virtual sample the viral population undergoes mutations. Our results suggest that the sustainability of the immunizations is dependent on viral variability and that the memory lifetimes are not random, what contradicts what was suggested by Tarlinton et al. [Curr. Opin. Immunol. 20, 162 (2008)]. We show that what may cause an apparent random behavior of the immune memory is the antigenic variability.

Detection of Human Bocavirus mRNA in Respiratory Secretions Correlates with High Viral Load and Concurrent Diarrhea

Human bocavirus (HBoV) is a parvovirus recently identified in association with acute respiratory infections (ARI). Despite its worldwide occurrence, little is known on the pathogenesis of HBoV infections. In addition, few systematic studies of HBoV in ARI have been conducted in Latin America. Therefore, in order to test whether active viral replication of human bocavirus is associated with respiratory diseases and to understand the clinical impact of this virus in patients with these diseases, we performed a 3-year retrospective hospital-based study of HBoV in outpatients and inpatients with symptoms of Acute Respiratory Infections (ARI) in Brazil. Nasopharyngeal aspirates (NPAs) from 1015 patients with respiratory symptoms were tested for HBoV DNA by PCR. All samples positive for HBoV were tested by PCR for all other respiratory viruses, had HBoV viral loads determined by quantitative real time PCR and, when possible, were tested by RT-PCR for HBoV VP1 mRNA, as evidence of active viral replication. HBoV was detected in 4.8% of patients, with annual rates of 10.0%, 3.0% and 3.0% in 2005, 2006 and 2007, respectively. The range of respiratory symptoms was similar between HBoV-positive and HBoV-negative ARI patients. However, a higher rate of diarrhea was observed in HBoV-positive patients. High HBoV viral loads (> 10(8) copies/mL) and diarrhea were significantly more frequent in patients with exclusive infection by HBoV and in patients with detection of HBoV VP1 mRNA than in patients with viral co-infection, detected in 72.9% of patients with HBoV. In summary, our data demonstrated that active HBoV replication was detected in a small percentage of patients with ARI and was correlated with concurrent diarrhea and lack of other viral co-infections.

Complete Genome Viral Phylogenies Suggests the Concerted Evolution of Regulatory Cores and Accessory Satellites

We consider the concerted evolution of viral genomes in four families of DNA viruses. Given the high rate of horizontal gene transfer among viruses and their hosts, it is an open question as to how representative particular genes are of the evolutionary history of the complete genome. To address the concerted evolution of viral genes, we compared genomic evolution across four distinct, extant viral families. For all four viral families we constructed DNA-dependent DNA polymerase-based (DdDp) phylogenies and in addition, whole genome sequence, as quantitative descriptions of inter-genome relationships. We found that the history of the polymerase gene was highly predictive of the history of the genome as a whole, which we explain in terms of repeated, co-divergence events of the core DdDp gene accompanied by a number of satellite, accessory genetic loci. We also found that the rate of gene gain in baculovirus and poxviruses proceeds significantly more quickly than the rate of gene loss and that there is convergent acquisition of satellite functions promoting contextual adaptation when distinct viral families infect related hosts. The congruence of the genome and polymerase trees suggests that a large set of viral genes, including polymerase, derive from a phylogenetically conserved core of genes of host origin, secondarily reinforced by gene acquisition from common hosts or co-infecting viruses within the host. A single viral genome can be thought of as a mutualistic network, with the core genes acting as an effective host and the satellite genes as effective symbionts. Larger virus genomes show a greater departure from linkage equilibrium between core and satellites functions.

From sabotage to camouflage: viral evasion of cytotoxic T lymphocyte and natural killer cell-mediated immunity

The outcome of a virus infection is strongly influenced by interactions between host immune defences and virus 'anti-defence' mechanisms. For many viruses, their continued survival depends on, the speed of their attach: their capacity to replicate and transmit to uninfected hosts prior to their elimination by an effective immune response. In contrast, the success of persistent viruses lies in their capacity for immunological subterfuge: the evasion of host defence mechanisms by either mutation (covered elsewhere in this issue, by Gould and Bangham, pp. 321-328) or interference with the action of host cellular proteins that are important components of the immune response. This review will focus on the strategies employed by persistent viruses against two formidable host defences against virus infection: the CD8+ cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

The murine cytomegalovirus chemokine homolog, m131/129, is a determinant of viral pathogenicity

Chemokines are important mediators of the early inflammatory response to infection and modify a wide range of host immune responses. Functional homologs of cellular chemokines have been identified in a number of herpesviruses, suggesting that the subversion of the host chemokine response contributes to the pathogenesis of these viruses. Transcriptional and reverse transcription-PCR analyses demonstrated that the murine cytomegalovirus (MCMV) chemokine homolog, m131, was spliced at the 3' end to the adjacent downstream open reading frame, m129, resulting in a predicted product of 31 kDa, which is significantly larger than most known chemokines. The in vivo impact of m131/129 was investigated by comparing the replication of MCMV mutants having m131/129 deleted (Delta m131/129) with that of wild-type (wt) MCMV. Our studies demonstrate that both wt and Delta m131/129 viruses replicated to equivalent levels during the first 2 to 3 days following in vivo infection. However, histological studies demonstrated that the early inflammatory response elicited by Delta m131/129 was reduced compared with that of wt MCMV. Furthermore, the Delta m131/129 mutants failed to establish a high-titer infection in the salivary glands, These results suggest that m131/129 possesses proinflammatory properties in vivo and is important for the dissemination of MCMV to or infection of the salivary gland. Notably, the Delta m131/129 mutants were cleared more rapidly from the spleen and liver during acute infection compared with wt MCMV. The accelerated clearance of the mutants was dependent on NK cells and cells of the CD4(+) CD8(+) phenotype. These data suggest that m131/129 may also contribute to virus mechanisms of immune system evasion during early infection, possibly through the interference of NK cells and T cells.

Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.