Molecular mechanisms of the induction of IL-12 and its inhibition by IL- 10

Exogenously added IL-10 rapidly inhibited Staphylococcus aureus- or LPS- induced cytokine mRNA expression in human PBMCs and monocytes, with a maximal effect observed when IL-10 was added from 20 h before until 1 h after the addition of the inducers. Nuclear run-on assays revealed that the inhibition of IL-12 p40, IL-12 p35, and TNF-α was at the gene transcriptional level and that the addition of IL-10 to S. aureus- or LPS-treated PBMCs did not affect mRNA stability. The inhibitory activity of IL-10 was abrogated by cycloheximide (CHX), suggesting the involvement of a newly synthesized protein(s). The addition of CHX at 2 h before S. aureus or LPS also inhibited the accumulation of IL-12 p40 mRNA, but did not inhibit IL-12 p35 and TNF-α mRNA. This finding suggests that p40 transcription is regulated through a de novo synthesized protein factor(s), whereas the addition of CHX at 2 h after S. aureus activation caused superinduction of the IL-12 p40, IL-12 p35, and TNF-α genes. These results indicate that in human monocytes, the mechanism(s) of IL-10 suppression of both IL-12 p40 and IL-12 p35 genes is primarily seen at the transcriptional level, and that the induction of the IL-12 p40 and p35 genes have different requirements for de novo protein synthesis.

Interval between first dose and booster affected antibody production in cattle vaccinated against rabies

In this study, we compared the levels of neutralizing antibodies induced by inactivated rabies vaccine in cattle by using three alternative immunization procedures. Forty-five bovines (breed nelore) were then organized in three groups (A, B and C, with 15 animals/group). Group A received only one vaccine dose at day zero and Group B received the first dose at day zero and then another dose at day 30 (early booster). Group C was also immunized with two doses; however, the booster was postponed until day 180 after the first dose (delayed booster). Blood samples were withdrawn at days zero (before the first dose) and 30, 210, 390, and 540 after the beginning of immunization and the antibody titers were evaluated by mouse neutralization test. The protocol used to immunize Group C (booster at day 180) was clearly more efficient. In this group, antibody levels were higher and also remained higher for longer periods in comparison with the other two groups. These results show that booster timing significantly affected antibody levels. Therefore, programs addressed to control this disease in cattle should consider not only the use of a booster but also its administration time.

Genetic vaccine for tuberculosis (pVAXhsp65) primes neonate mice for a strong immune response at the adult stage

Background: Vaccination of neonates is generally difficult due to the immaturity of the immune system and consequent higher susceptibility to tolerance induction. Genetic immunization has been described as an alternative to trigger a stronger immune response in neonates, including significant Th1 polarization. In this investigation we analysed the potential use of a genetic vaccine containing the heat shock protein (hsp65) from Mycobacterium leprae (pVAXhsp65) against tuberculosis (TB) in neonate mice. Aspects as antigen production, genomic integration and immunogenicity were evaluated. Methods: Hsp65 message and genomic integration were evaluated by RT-PCR and Southern blot, respectively. Immunogenicity of pVAXhsp65 alone or combined with BCG was analysed by specific induction of antibodies and cytokines, both quantified by ELISA. Results: This DNA vaccine was transcribed by muscular cells of neonate mice without integration into the cellular genome. Even though this vaccine was not strongly immunogenic when entirely administered (three doses) during early animal's life, it was not tolerogenic. In addition, pVAXhsp65 and BCG were equally able to prime newborn mice for a strong and mixed immune response (Th1 + Th2) to pVAXhsp65 boosters administered later, at the adult life. Conclusion: These results suggest that pVAXhsp65 can be safely used as a priming stimulus in neonate animals in prime-boost similar strategies to control TB. However, priming with BCG or pVAXhsp65, directed the ensuing immune response triggered by an heterologous or homologous booster, to a mixed Th1/Th2 pattern of response. Measures as introduction of IL-12 or GM-CSF genes in the vaccine construct or even IL-4 neutralization, are probably required to increase the priming towards Th1 polarization to ensure control of tuberculosis infection. © 2007 Pelizon et al; licensee BioMed Central Ltd.

Produção deficiente de citocinas Th1 em camundongos BALB/c jovens

O objetivo do presente estudo foi comparar a produção de IFN-γ, IL-12 e IL-4 entre camundongos jovens (5, 12 e 19 dias de idade) e adultos (30 dias de idade). As avaliações foram feitas por estimulação, in vitro, de células esplênicas com Concanavalina A (ConA) , Staphylococcus aureus (S. aureus) e lipopolissacarídeo (LPS). Diferentes concentrações de cada estímulo foram testadas e os sobrenadantes das culturas foram coletados após 48 horas de incubação e as concentrações de IFN-γ, IL-12 e IL-4 determinadas por ELISA. Células de camundongos jovens e adultos produziram níveis igualmente elevados de IFN-γ após estímulo com ConA. Somente animais adultos produziram IFN-γ em resposta ao estímulo com S. aureus. Em culturas estimuladas com LPS, a produção desta citocina foi baixa e similar nos animais jovens e significativamente elevada nos animais adultos. Somente células de animais adultos estimuladas com S. aureus foram capazes de produzir IL-12. O único estímulo capaz de induzir níveis detectáveis de IL-4 foi ConA, sendo que estes níveis foram mais elevados nos animais com 12 e 19 dias de idade em comparação com animais neonatos e adultos. A diminuição das doses ótimas dos estímulos não mudou o perfil de produção de cada citocina nos animais jovens. Estes resultados permitem concluir que a idade afeta a produção de citocinas: ocorre maior produção de IL-4 em camundongos jovens e maior produção de IL-12 e IFN-γ em animais adultos. Estas informações são importantes devido ao papel destas citocinas na polarização das respostas imunes nos sentidos Th1 e Th2. Palavras-chave: camundongo; citocina; interferon-gama; interleucina-4; interleucina-12.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs. © 2009 Ishikawa et al; licensee BioMed Central Ltd.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Background: Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods: In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results: DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion: The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases. © 2009 Santos et al; licensee BioMed Central Ltd.

Vascularização arterial e venosa do linfonodo poplíteo em cães

Poplíteo lympho nodes in the dogs is placed, to the right and the left in popliteo an appointed space fossa, in distal part of the muscles femoral biceps, laterally and semi-tendinous, medially, projecting in the height of the face volume of the joint to femoro tibial e femoro patellar (joint of the knee). In this study twenty and five dogs, males and females adult, originating the Araçatuba municipal kennel had been used whose captures had been effected by the animal sanitary defense of this city. The arterial vessels destined to this structure always derive, of both the sides, the femoral artery distal volume and vary of 10 the 1, more frequently of 2 (7 times, 28%) to the right and of 6 the 2, equally more frequently of 2 (8 times, 32%). So soon as one has still to right 5 and 6 branches (3 times, 12%), 1, 7 and 10 (1 time, 4%). Relatively to the veins derived from this lympho nodes, always converges to the lateral safena vein, these vessels oscillates between 9 and 2, more frequently of 3 (6 times, 24%), to the right and of 12 the 2, more frequently of 3 (10 times, 40%) to the left. Thus, others deriving branches of popliteo lympho nodes right and that if they insert in the above-mentioned vein are in number of 2 and 5 (5 times, 20%), 4 and 6 (2 times, 8%) and 8 and 9 (1 time, 4%). In spite of, to the left side it is examined in number de 2 branches (6 times, 24%), 4 (4 times, 16%), 6 (3 times, 12%) and finishing 9 and 12 branches (1 time, 4%). The size of popliteo lympho node in seropositive dogs for Visceral Leishmaniasis can meets enters 7,8 × 3,8 × 6,1 a 50,0 × 20,7 × 28,5, in mm, being average 26,18 × 10,5 × 15,97 mm for right and 26,98 × 11,14 × 15,25 mm for left (concerning the measures dorsoventral, latero-lateral and cranial-caudal, respectively).

Sistema de reprodução, parentesco e tamanho efetivo em sementes de polinização livre de populações fragmentadas de Copaifera langsdorffii Desf. por análise de locos microssatélites

Anthropogenic changes in the reproductive population density can affect the mating system and result in an increase in selfing and correlated matings. This study investigated the mating system in small fragmented populations of the insect pollinated tropical tree species Copaifera langsdorffii, using microsatellite loci and the mixed and correlated mating models. Open-pollinated seeds were collected from 15 seed-trees located in a small forest fragment (4.8 ha), denominated Bosque and from 14 other seed-trees located in other small forest fragments of the north-western region of São Paulo State. No significant differences were observed between the seed-trees from Bosque (tm=0.933±0.028) and other fragments (tm=0.971±0.032), although these estimates were significantly different from 1.0, suggesting that selfing was occurring. Differences between multilocus and unilocus outcrossing rate were significantly high in both seed-trees of Bosque (tm -ts=0.478±0.05) and other forest fragments (tm -ts=0.475±0.018), suggesting a spatial genetic structure in those stands. The results also showed high rates of correlated mating in the samples, indicating that a good part of the offspring were full-sibs. As a consequence of selfing, mating among relatives and correlated matings, the coancestry within families was equally high in the seed-trees of Bosque ((Θ=0.237) and in the seed-trees of the other forest fragments (Θ=0.241) and the effective population size was lower than expected in panmitic populations (Ne<4). The results were discussed, focusing on the sample size of seed-trees to collect seeds for genetic conservation and enviromental reforestation.

Variação e parâmetros genéticos em teste de progênies de polinização livre de Peltophorum dubium (Sprengel) taubert em Luiz Antonio -SP

Intensive deforestation and forest fragmentation of the Brazilian forest biomes has contributed to increase the number of trees species under the risk of extinction. Peltophorum dubium (Sprengel) Taubert is one of these species. Some of its populations are being conserved ex situ by the Instituto Florestal de São Paulo through provenance and progeny tests. The aim of this study was to investigate the genetic variation and to estimate genetic parameters at age 24 years in a progeny test of P. dubium established in Luiz Antônio, São Paulo State, Brazil. The trial was established in a random block design, with six replications, five plants per plot and 18 open-pollinated progenies. Measured were: diameter at breast height (DBH), height and stem form. The genetic parameters heritability, genetic variation and effective population size were estimated. Significant genetic differences were observed only for DBH. This trait also presented a high coefficient of genetic variation (CV g=4.8%) and heritability, especially among progeny means (h m 2=0.6607). This indicates that DBH is the most indicated trait for selection in the population. The effective population size conserved ex situ in the test was estimated to be 38.9. Concerning genetic conservation, although the effective population size in the test is small, the values of the genetic variation and of the heritability indicate that the ex situ population has sufficient genetic variation and potential to respond to changes promoted by natural and artificial selection.

Hepatoencephalopathy syndrome due to Cassia occidentalis (Leguminosae, Caesalpinioideae) seed ingestion in horses

Cassia occidentalis is a bush from the Leguminosae family, subfamily Caesalpinoideae, and is a toxic plant of veterinary interest due to the occasional contamination of animal rations. This report describes the clinical and histopathological findings of an outbreak of C.occidentalis poisoning in horses. Twenty mares were poisoned after consuming ground corn contaminated with 8% of C.occidentalis seeds. Of the 20 animals affected, 12 died: 8 mares were found dead, 2 died 6h after the onset of clinical signs compatible with hepatic encephalopathy and the 2 other animals were subjected to euthanasia 12h after the onset of the clinical signs. The remaining 8 mares presented with mild depression and decreased appetite, but improved with treatment and no clinical sequelae were observed. In 6 animals that underwent a necropsy, an enhanced hepatic lobular pattern was noted and within the large intestine, a large number of seeds were consistently observed. Hepatocellular pericentrolobular necrosis and cerebral oedema were the main histological findings. In one mare, there was mild multifocal semimembranosus rhabdomyocytic necrosis and haemorrhage. Seeds collected from intestinal contents and sifted from the culpable feedstuff were planted. Examination of the leaves, flowers, fruits and seeds of the resultant plants identified C.occidentalis. Horses poisoned by C.occidentalis seeds demonstrate clinical signs associated with hepatoencephalopathy and frequently die suddenly. Lesions primarily involve the liver and secondarily, the central nervous system. Cassia occidentalis poisoning should be considered a differential diagnosis in horses with hepatoencephalopathy and special caution should be taken with horse rations to avoid contamination with seeds of this toxic plant. © 2012 EVJ Ltd.

Previous contact with Strongyloides venezuelensis contributed to prevent insulitis in MLD-STZ diabetes

Epidemiological and experimental studies support the idea that helminth infections can induce a protective effect against the development of autoimmune and allergic diseases. In this study we characterized the immune response induced by Strongyloides venezuelensis infection in C57BL/6 mice and then evaluated the effect of a previous contact with this helminth in the outcome of type 1 diabetes. Animals were initially infected with 2000 L3 larvae from S. venezuelensis and euthanized 22. days later. An acute phase, identified by a high amount of eggs per gram of feces, was established between days 7 and 9 post-infection. Recovery from infection was associated with a Th2 polarized response characterized by a significant level of serum IgG1 specific antibodies and also a significant production of IL-5 and IL-10 by spleen cells stimulated with S. venezuelensis soluble antigen. Immunization with soluble S. venezuelensis antigen associated with complete Freund's adjuvant followed by infection with S. venezuelensis protected mice from diabetes development induced by streptozotocin. Protection was characterized by a higher body weight gain, lower glycemic levels, much less severe insulitis and preserved insulin production. Together, these results indicate that S. venezuelensis contributed to protect C57BL/6 mice against experimental diabetes induced by streptozotocin. © 2013 Elsevier Inc.

Persistent inflammation in the CNS during chronic EAE despite local absence of IL-17 production

Experimental autoimmune encephalomyelitis (EAE) is an artificially induced demyelination of the central nervous system (CNS) that resembles multiple sclerosis in its clinical, histopathological, and immunological features. Activated Th1 and Th17 cells are thought to be the main immunological players during EAE development. This study was designed to evaluate peripheral and local contribution of IL-17 to acute and chronic EAE stages. C57BL/6 mice were immunized with MOG plus complete Freund's adjuvant followed by pertussis toxin. Mice presented an initial acute phase characterized by accentuated weight loss and high clinical score, followed by a partial recovery when the animals reached normal body weight and smaller clinical scores. Spleen cells stimulated with MOG produced significantly higher levels of IFN-γ during the acute period whereas similar IL-17 levels were produced during both disease stages. CNS-infiltrating cells stimulated with MOG produced similar amounts of IFN-γ but, IL-17 was produced only at the acute phase of EAE. The percentage of Foxp3+ Treg cells, at the spleen and CNS, was elevated during both phases. The degree of inflammation was similar at both disease stages. Partial clinical recovery observed during chronic EAE was associated with no IL-17 production and presence of Foxp3+ Treg cells in the CNS. © 2013 Sofia Fernanda Gonçalves Zorzella-Pezavento et al.

Differential arthritogenicity of Staphylococcus aureus strains isolated from biological samples

Background: Staphylococcus aureus is the most common agent of septic arthritis that is a severe, rapidly progressive and destructive joint disease. Superantigens produced by S. aureus are considered the major arthritogenic factors. In this study, we compared the arthritogenic potential of five superantigen-producing staphylococcal strains.Methods: Male C57BL/6 mice were intravenously infected with ATCC 19095 SEC+, N315 ST5 TSST-1+, S-70 TSST-1+, ATCC 51650 TSST-1+ and ATCC 13565 SEA+ strains. Clinical parameters as body weight, arthritis incidence and clinical score were daily evaluated. Joint histopathological analysis and spleen cytokine production were evaluated at the 14th day after infection.Results: Weight loss was observed in all infected mice. ATCC 19095 SEC+, N315 ST5 TSST-1+ and S-70 TSST-1+ were arthritogenic, being the highest scores observed in ATCC 19095 SEC+ infected mice. Intermediate and lower clinical scores were observed in N315 ST5 TSST-1+ and S-70 TSST-1+ infected mice, respectively. The ATCC 13565 SEA+ strain caused death of 85% of the animals after 48 h. Arthritis triggered by the ATCC 19095 SEC+ strain was characterized by accentuated synovial hyperplasia, inflammation, pannus formation, cartilage destruction and bone erosion. Similar joint alterations were found in N315 ST5 TSST-1+ infected mice, however they were strikingly more discrete. Only minor synovial proliferation and inflammation were triggered by the S-70 TSST-1+ strain. The lowest levels of TNF-α, IL-6 and IL-17 production in response to S. aureus stimulation were found in cultures from mice infected with the less arthritogenic strains (S-70 TSST-1+ and ATCC 51650 TSST-1+). The highest production of IL-17 was detected in mice infected with the most arthritogenic strains (ATCC 19095 SEC+ and N315 ST5 TSST-1+).Conclusions: Together these results demonstrated that S. aureus strains, isolated from biological samples, were able to induce a typical septic arthritis in mice. These results also suggest that the variable arthritogenicity of these strains was, at least in part, related to their differential ability to induce IL-17 production. © 2013 Colavite-Machado et al.; licensee BioMed Central Ltd.

Intervalos de chuva na eficiência de herbicidas aplicados em pós-emergência

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Anatomia e aspectos ultra-estruturais de pulvinos de leguminosas de cerrado

Pós-graduação em Ciências Biológicas (Botânica) - IBB