Immunotherapy against experimental canine visceral leishmaniasis with the saponin enriched-Leishmune((R)) vaccine

In order to assess the immunotherapeutic potential on canine visceral leishmaniasis of the Leishmune (R) vaccine, formulated with an increased adjuvant concentration (1 mg of saponin rather than 0.5 mg), 24 mongrel dogs were infected with Leishmania (L.) chagasi. The enriched-Leishmune (R) vaccine was injected on month 6, 7 and 8 after infection, when animals were seropositive and symptomatic. The control group were injected with a saline solution. Leishmune (R)-treated dogs showed significantly higher levels of anti-FML IgG antibodies (ANOVA; p < 0.0001), a higher and stable IgG2 and a decreasing IgG I response, pointing to a TH1 T cell mediated response. The vaccine had the following effects: it led to more positive delayed type hypersensitivity reactions against Leishmania lysate in vaccinated dogs (75%) than in controls (50%), to a decreased average of CD4+ Leishmania-specific lymphocytes in saline controls (32.13%) that fell outside the 95% confidence interval of the vaccinees (41.62%, CI95% 43.93-49.80) and an increased average of the clinical scores from the saline controls (17.83) that falls outside the 95% confidence interval for the Leishmune (R) immumotherapy-treated dogs (15.75, CI95% 13.97-17.53). All dogs that received the vaccine were clustered, and showed lower clinical scores and normal CD4+ counts, whereas 42% of the untreated dogs showed very diminished CD4+ and higher clinical score. The increase in clinical signs of the saline treated group was correlated with an increase in anti-FML antibodies (p < 0.0001), the parasitological evidence (p = 0.038) and a decrease in Leishinania-specific CD4+ lymphocyte proportions (p = 0.035). These results confirm the immunotherapeutic potential of the enriched-Leishmune (R) vaccine. The vaccine reduced the clinical symptoms and evidence of parasite, modulating the outcome of the infection and the dog's potential infectiosity to phlebotomines. The enriched-Leishmune (R) vaccine was subjected to a safety analysis and found to be well tolerated and safe. (c) 2007 Elsevier Ltd. All rights reserved.

Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages

Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action. In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection. The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection. TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection. A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals. These results suggest that during the early stages of infection with P. brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination. In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death. These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis.

Experimental infections with Paracoccidioides brasiliensis obtained from armadillos: comparison to clinical isolates

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM) that is one of the most prevalent systemic human mycoses in Latin America. Armadillos show a high incidence of PCM infection and could, therefore, be a natural reservoir for this fungus. In this study were compared the virulence profiles of isolates obtained from nine-banded armadillos (Dasypus novemcinctus) (PbT1 and PbT4) and isolates from PCM patients (Pb265 and Bt83). Pathogenicity was evaluated by fungal load and analysis of colony morphology. Immunity against the fungus was tested by delayed type hypersensitivity test (DTH) and antibody quantification by ELISA. The higher virulence of PbT1 and PbT4 was suggested by higher fungal load in spleen and lungs. Armadillo isolates and Bt83 presented a cotton-like surface contrasting with the cerebriform appearance of Pb265. All isolates induced cellular and humoral immune responses in infected BALB/c mice. DTH reactions were similarly induced by the four isolates, however, a great variability was observed in specific antibody levels, being the highest ones induced by Bt83 and PbT4. The present work confirms that armadillos harbor P. brasiliensis, whose multiplication and induced immunity in experimentally infected mice are heterogeneous, resembling the behavior of isolates from human PCM. This study reinforces the possibility that armadillos play an important role in the biological cycle of this pathogen.

Modulatory effects of tuberculosis vaccines on experimental autoimmune encephalomyelitis

Although BCG is the only accepted vaccine against tuberculosis (TB), its protective ability is very limited. Therefore, many new vaccines are being evaluated. Our group has been working on DNAhsp65 - a genetic construction containing the hsp65 gene from Mycobacterium leprae. In previous experimental works, we demonstrated that both DNAhsp65 alone or associated with BCG, in a prime-boost regimen, were effective to control TB. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 alone or associated with BCG in a rat model of multiple sclerosis. Female Lewis rats were immunized with three doses of DNAhsp65 or primed with BCG followed by two DNAhsp65 boosters. The animals were, then, immunized with myelin associated with complete Freund's adjuvant to develop experimental autoimmune encephalomyelitis (EAE). The following parameters were evaluated: weight loss, clinical score, central nervous system (CNS) inflammation and anti-myelin immune response. No deleterious effect was associated with these immunizations schedules. Immunized animals equally lost weight, the clinical scores were similar and CNS inflammation did not increase. Interestingly, both procedures determined decreased inflammation in the brain and lumbar spinal cord. This was concurrent with a modulatory effect over cytokine production by peripheral lymphoid organs. Cell cultures from spleen and lymph nodes in vitro stimulated with myelin produced less IFN-gamma and IL-10, respectively. This phenomenon was more clear in rats immunized with the genetic vaccine alone than with the prime-boost strategy. Together the results suggest that these strategies for TB prophylaxis would not accelerate or aggravate multiple sclerosis, being therefore, safe in this aspect. In addition, they indicate that these vaccination regimens have a potential anti-inflammatory activity that could be better explored in the future.

The inhibitory effect of MK886 on the inflammatory process caused by Paracoccidioidomycosis brasiliensis infection in genetically selected mice

Leukotrienes are classic inflammatory response mediators considered chemotactic agents and microbicidal activity regulators in cells of the innate immune system, playing a protective role against different infectious agents. In this study, we investigated the involvement of leukotrienes in the course of murine paracoccidioidomycosis based on the following immunologic parameters: cell influx, mieloperoxydase activity, NO production, cytokine production, and fungal recovery in lungs of mice selected according to the intensity of their low (AIRmin) and high (AIRmax) acute inflammatory response. Infection by P. brasiliensis induced considerable production of IL-6, IL-10, IFN-gamma and TNF-alpha cytokines, and led to cell recruitment, as well as NO production in lungs at different study periods. In animals treated with MK886, a leukotriene biosynthesis inhibitor, IFN-gamma, IL-6 and TNF-alpha production was lower, while neutrophil influx and NO production decreased. These results may explain the higher fungal load in lungs of animals in which leukotriene synthesis was inhibited, suggesting that leukotrienes have a possible protective role in experimental paracoccidioidomycosis. AIRmax animals had lower fungal load in comparison with AIRmin ones, which can be related to the AIR phenotype regarding neutrophil migration, besides lower production of NO and pro-inflammatory cytokines. Thus, mice presenting AIRmax background are more resistant to infection by P. brasiliensis.

Cytokine production in experimental paracoccidioidomycosis of murine selected lineages for acute inflammatory response (air)

Paracoccidioidomycosis is a systemic human mycosis caused by Paracoccidioides brasiliensis (P. brasiliensis), an imperfect dimorphic fungus whose conidia are its infective form. Mice genetically selected for maximum (AIRmax) and minimum (AIRmin) acute inflammatory response were used as experimental paracoccidioidomycosis models. The animals were intraperitoneally inoculated with P. brasiliensis (strain 18) and killed 6, 12 and 24 hours or 3, 7 and 14 days after infection. In these periods, fragments from their spleen, liver and lung were removed for evaluation of the infection level by fungal cells, assessment of macrophagic activity by peritoneal and splenic macrophages - through the determination of nitric oxide (NO) concentrations and production of pro- and anti-inflammatory cytokines of lung and spleen homogenate supernatants. In the present study, it was observed that AIRmax lineages presented greater control of the infectious process than the AIRmin ones. Regarding NO production, AIRmax animals produced more metabolites in late periods, what may help control the infectious process. Concerning cytokine production, it was observed that the production of INF-gamma, TNF-alpha, IL-1, IL-6, IL-8 and IL-12 were increased in AIRmax lineages in most analyzed organs and periods, thus contributing to the greater resistance exhibited by such lineages against infection, except for IL-4 and IL-10 that showed decreased production in AIRmax lineage, reproducing its suppressive biological effect. From these results, it was observed that the AIRmax lineage was more effective in controlling the infectious process, with an important involvement of the analyzed cytokines. These findings are probably related to the genetically selected factors involved in the acute inflammatory response.

Impact of malnutrition on immunity and infection

Malnutrition may be a consequence of energy deficit or micronutrient deficiency. It is considered the most relevant risk factor for illness and death, particularly in developing countries. In this review we described the magnitude of this problem, as well as its direct effect on the immune system and how it results in higher susceptibility to infections. A special emphasis was given to experimental models used to investigate the relationship between undernutrition and immunity. Malnutrition is obviously a challenge that must be addressed to health authorities and the scientific community.

Recovery from Strongyloides venezuelensis infection in Lewis rats is associated with a strong Th2 response

P>In this study, we investigated the characteristics of the infection and subsequent immunity induced by Strongyloides venezuelensis in Lewis rats. Animals were infected with 4000 L3 of S. venezuelensis and number of eggs per gram of faeces indicated an acute phase around day 8 and a recovery phase around day 32 after infection. A strong Th2 polarization during recovery phase was ascertained by a significant increase in IgG1 and IgE compared with that in the acute period. A shift in the cytokine profile confirmed these findings. A predominant production of IFN-gamma during the acute phase was followed by IL-10 production during recovery. Together these findings show that experimental infection of Lewis rats with S. venezuelensis presents a kinetics of parasite establishment and immunity similar to that described in other models of helminthic infection.

Influence of dietary protein supply on resistance to experimental infections with Haemonchus contortus in Ile de France and Santa Ines lambs

The effect of Haemonchus contortus infection in sheep fed with a moderate and high protein content diet was evaluated in two breeds of sheep. Forty-eight Ile de France and Santa Ines lambs were maintained indoors since birth, in worm-free conditions. The lambs were allocated after weaning in four groups of six animals per breed, which were either infected or remain uninfected and given access to either a moderately or highly metabolizable protein diet. The moderately and highly metabolizable protein diets were calculated to supply 75 and 129 g metabolizable protein per kg of dry matter (MP/kg DM), respectively. The infection consisted of a trickle infection with 300 infective larvae, three times a week, for 12 weeks. Significant differences were observed for mast cell, globule leukocyte and eosinophil counts in the abomasal mucosa of the infected groups compared to the control of both breeds (P < 0.05), regardless of the diet supplied. Significantly higher IgA anti-L5 antibody was detected in the infected Santa Ines groups than in the infected Ile de France groups (P < 0.05). Increased metabolizable protein supply resulted in larger body weight gain and higher packed cell volumes for both breeds (P < 0.05). Both breeds showed an increased ability to withstand the pathophysiological effects of H. contortus infection when given access to the highly metabolizable protein diet. However, increased metabolizable protein supply resulted in reduced worm burdens in Santa Ines lambs but not in the Ile de France lambs (P < 0.05). The present results show that the increase in protein content in growing lamb diets may benefit resistance and resilience to gastrointestinal parasites but that these benefits may vary among breeds. (c) 2005 Elsevier B.V. All rights reserved.

Strongyloides venezuelensis infection susceptibility of seven inbred strains of mice

Foi investigada a susceptibilidade de sete linhagens isogênicas de camundongos à infecção experimental, primária e secundária, por Strongyloides venezuelensis a fim de servir de base para estudos genéticos sobre a resistência. Foram utilizados 12 camundongos machos, com seis semanas de idade, das seguintes linhagens isogênicas: A/J, BALB/c, CBA/J, C3H/Hepos, C57BL/6, DBA/2 e NIH. Os animais foram inoculados, via sub-cutânea, com 2000 larvas infectantes. As contagens médias (± desvio padrão) de parasitas no intestino delgado dos camundongos seis dias após a infecção, em ordem crescente, foram: 28 (± 19) na linhagem NIH; 647 (± 228) na BALB/c; 709 (± 425) na DBA/2; 731 (± 151) na C3H/Hepos, 801 (± 174) na CBA/J; 1024 (± 267) na C57BL/6 e 1313 (± 483) na A/J. Os camundongos C57BL/6 apresentaram as mais elevadas contagens de ovos de S. venezuelensis por grama de fezes (OPG) e os NIH, as mais baixas. Não foram detectados ovos nos exames de fezes e não foram encontrados parasitas no intestino delgado dos animais re-infectados 14 dias após a infecção primária. A linhagem NIH apresentou elevada resistência contra as infecções primárias por S. venezuelensis. Entre as outras seis linhagens, uma das mais susceptíveis foi a linhagem C57BL/6.

Concurrent dengue and malaria in the Amazon region

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

Experimental systemic virulence of oral Candida dubliniensis isolates in comparison with Candida albicans, Candida tropicalis and Candida krusei

There are no previous studies on the comparative virulence of Candida dubliniensis with other non-albicans species. The aim of this study was to compare the virulence and infection kinetics of C. dubliniensis and other species. Candida albicans, C. dubliniensis, Candida tropicalis and Candida krusei (reference strains) were inoculated intravenously in mice. For infection kinetics evaluation, a group of five animals were sacrificed after 6 h, 3, 7, 14 and 21 days. Microbiological evaluations (liver, spleen, kidneys, lungs and brain) and histopathological examination of the kidney were performed. The results of virulence evaluation were analysed using Kaplan-Meier survival analysis (5%). Candida dubliniensis-inoculated mice survived for longer periods compared with those with C. albicans (P = 0.005). No differences were detected in relation to C. tropicalis (P = 0.326) and C. krusei (P = 0.317). Most of the organs were persistently colonised by C. albicans and C. dubliniensis even by day 21. Tendency of C. krusei clearance was observed in all organs. Fungal masses and renal lesions were observed after inoculation of C. albicans, C. dubliniensis and C. tropicalis. Within the limits of the study, data on survival rate and dissemination capacity suggest that C. dubliniensis is less virulent than C. albicans.

Response activity of alveolar macrophages in pulmonary dysfunction caused by Leptospira infection

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

Metodologia de infecção experimental e grau de susceptibilidade do híbrido tambacu e Leporinus macrocephalus Garavello & Britski (Osteichthyes, Anostomidae) a quatro inóculos de trofozoítos de Icthyophthirius multifiliis Fouquet (Protozoa, Ciliophora)

Tropical fishes like Leporinus macrocephalus Garavello & Britski, 1988 (piauçu) and the hybrid tambacu (Piaractus mesopotamicus Holmberg, 1887 pacu male x Colossoma macropomum Cuvier, 1818 tambaqui female) were experimentally infected with four inocula of trophonts of Ichthyophthirius multifiliis Fouquet, 1876 (Protozoa, Ciliophora) and the infection level was evaluated. Each one of twelve experimental units consisted of a tank with 200 liters of water and two specimens of each kind of fish. The following treatments were designed to each tank, in triplicate: 8, 16,32 and 64 trophonts. After 8 days of exposure the fishes were killed and the number of trophonts was counted at a stereoscopic microscope. This assay showed to be feasible at tropical conditions and was able to show that tambacu was more susceptiblc than piauçu.

Infecção experimental por Salmonella enterica subespécie enterica sorotipo Panama e tentativa de transmissão nasonasal em leitões desmamados

O objetivo deste experimento foi produzir uma infecção experimental de Salmonella enterica subespécie. enterica sorotipo Panama e verificar a importância da via nasonasal na transmissão entre leitões desmamados. Foram utilizados seis leitões recém-desmamados, adquiridos de granja livre de Salmonella spp. Utilizaram-se baias isoladoras, que proporcionavam o contato nasonasal e eliminavam a possibilidade de outras vias de transmissão e de contaminação externa. Três grupos foram formados: controle, sentinela e infectado. Não foram encontradas amostras positivas para Salmonella spp. em leitões do grupo-controle e sentinelas, e nos animais infectados foi isolada Salmonella Panama em suabes retais e tecidos necropsiados. Os resultados revelaram não haver a transmissão pela via nasonasal entre leitões desmamados, pois, em nenhum momento, o agente foi isolado dos animais sentinelas