Experimental murine mycobacteriosis: evaluation of the functional activity of alveolar macrophages in thalidomide- treated mice

Thalidomide is a selective inhibitor of tumor necrosis factor-alpha (TNF-alpha), a cytokine involved in mycobacterial death mechanisms. We investigated the role of this drug in the functional activity of alveolar macrophages in the presence of infection induced by intranasal inoculation of Mycobacterium avium in thalidomide-treated and untreated adult Swiss mice. Sixty animals were inoculated with 5 x 10(6) M. avium by the respiratory route. Thirty animals received daily thalidomide (30 mg/kg mouse) and 30 received water by gavage up to sacrifice. Ten non-inoculated mice were used as a control group. Lots of animals from each group were evaluated until 6 weeks after inoculation. Infection resulted in an increased total number of inflammatory cells as well as increased activity of pulmonary macrophages. Histologically, intranasal inoculation of bacilli resulted in small mononuclear infiltrates located at the periphery of the organ. Culture of lung fragments revealed the presence of bacilli only at the beginning and at the end of the experimental period. Thalidomide administration did not affect the microbiological or histological features of the infection. Thalidomide-treated and untreated animals showed the same amount of M. avium colonies 3 weeks after infection. Although it did not affect bacillary clearance, thalidomide administration resulted in a decreased percent of spread cells and release of hydrogen peroxide, suggesting that factors other than TNF-alpha play a role in the killing of mycobacteria by alveolar macrophages. Thalidomide administration also reduced the number of spread cells among resident macrophages, suggesting a direct effect of the drug on this phenomenon.

PATHOGENICITY AND IMMUNOGENICITY OF PARACOCCIDIOIDES-BRASILIENSIS ISOLATES IN THE HUMAN-DISEASE AND IN AN EXPERIMENTAL MURINE MODEL

The pathogenicity and immunogenicity of six recently isolated Paracoccidioides brasiliensis samples derived from patients presenting distinct and well defined clinical forms of paracoccidioidomycosis (PCM) were compared as to their virulence, tropism to different organs and ability to induce specific cellular and humoral immune response in susceptible (B10.A) inbred mice. Isolates Pb44 and Pb47 were obtained from acute cases, Pb50 from a chronic severe form, Pb45 from a chronic moderate case and both Pb56 and Pb57 from chronic mild forms of PCM. Pathogenicity and tropism of each fungal sample were evaluated by LD50% estimation, examination of gross lesions on various organs at 2, 4, 12 and 16 weeks post-infection, and by colony-forming unit (CFU) counts in the lungs at week 16 post-infection of mice. Fungal tropism in human PCM and in B10.A mice was always dissociated. A well defined relationship between virulence of the fungal sample and the clinical findings of the correspondent patient was not evident, although a tendency to higher LD50% and less intense paracoccidioidic lesions was observed in mice infected with Pb56 and Pb57. The specific DTH response patterns varied according to the infectant sample, but positive DTH reactions at the beginning of the infection and a tendency to anergy or low DTH responses at week 12 and/or week 16 post-infection were always observed. A correspondence between the DTH response in humans and in mice was noticeable only when the isolates from the most benign cases (Pb56 and Pb57) were considered. The specific antibody patterns in mice and in the correspondent patients were also not analogous. Collectively, these results indicate that an association between the fungal pathogenicity and immunogenicity in the human disease and in susceptible mice was discernible only when isolates obtained from very mild cases (Pb56 and Pb57) were considered.

Ketoconazole in the treatment of experimental murine paracoccidioidomycosis

In a murine model of chronic disseminated paracoccidioidomycosis (strain 18; intravenous route), Ketoconazole (200 mg/kg in 0.2% agar) was given daily by gavage in three different schedules. Continuous treatment from an early stage of infection (day 3) up to week 20 was the most effective protocol, leading to remission of histopathological lesions and of both humoral and cellular anti-P. brasiliensis immune response, and clearance of the fungus in lungs; only 1 treated animal at week 20 showed pulmonary granulomas, although less extensive than control mice. Continuous treatment from early stage up to week 8, followed by a 16 week-period of drug discontinuity, caused remission of lesions in all but 3 treated mice which showed active pulmonary paracoccidioidomycosis similar to controls (14.2% of unresponsiveness to treatment). The continuous Ketoconazole protocol since a late stage of infection (week 4) up to week 20 produced a slower remission of lesions and immune response when compared with the first drug schedule. In this model of paracoccidioidomycosis, Ketoconazole showed no detectable side-effects and was a very effective drug especially in a prolonged administration protocol from an early stage of infection.

Modulação da resposta imune através do consumo de produto probiótico de soja durante o desenvolvimento de câncer de mama murino experimental

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Immunological and parasitological parameters in Schistosoma mansoni-infected mice treated with crude extract from the leaves of Mentha x piperita L.

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

Protection induced in BALB/c mice by the high-molecular-mass (hMM) fraction of Paracoccidioides brasiliensis

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Cell-free antigens from precocious Paracoccidioides brasiliensis culture induce a typical delayed-type hypersensitivity reaction

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Characterization of S3Pvac Anti-Cysticercosis Vaccine Components: Implications for the Development of an Anti-Cestodiasis Vaccine

Background: Cysticercosis and hydatidosis seriously affect human health and are responsible for considerable economic loss in animal husbandry in non-developed and developed countries. S3Pvac and EG95 are the only field trial-tested vaccine candidates against cysticercosis and hydatidosis, respectively. S3Pvac is composed of three peptides (KETc1, GK1 and KETc12), originally identified in a Taenia crassiceps cDNA library. S3Pvac synthetically and recombinantly expressed is effective against experimentally and naturally acquired cysticercosis.Methodology/ Principal Findings: In this study, the homologous sequences of two of the S3Pvac peptides, GK1 and KETc1, were identified and further characterized in Taenia crassiceps WFU, Taenia solium, Taenia saginata, Echinococcus granulosus and Echinococcus multilocularis. Comparisons of the nucleotide and amino acid sequences coding for KETc1 and GK1 revealed significant homologies in these species. The predicted secondary structure of GK1 is almost identical between the species, while some differences were observed in the C terminal region of KETc1 according to 3D modeling. A KETc1 variant with a deletion of three C-terminal amino acids protected to the same extent against experimental murine cysticercosis as the entire peptide. on the contrary, immunization with the truncated GK1 failed to induce protection. Immunolocalization studies revealed the non stage-specificity of the two S3Pvac epitopes and their persistence in the larval tegument of all species and in Taenia adult tapeworms.Conclusions/ Significance: These results indicate that GK1 and KETc1 may be considered candidates to be included in the formulation of a multivalent and multistage vaccine against these cestodiases because of their enhancing effects on other available vaccine candidates.

Immunomodulation and protection induced by DNA-hsp65 vaccination in an animal model of arthritis

We described a prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65- kDa heat shock protein (DNA-hsp65) in experimental murine tuberculosis. However, high homology of the vaccine to the corresponding mammalian hsp60, together with the CpG motifs in the plasmidial vector, could trigger or exacerbate an autoimmune disease. In the present study, we evaluate the potential of DNA- hsp65 vaccination to induce or modulate arthritis in mice genetically selected for acute inflammatory reaction (AIR), either maximal (AIRmax) or minimal (AIRmin). Mice immunized with DNA-hsp65 or injected with the corresponding DNA vector (DNAv) developed no arthritis, whereas pristane injection resulted in arthritis in 62% of AIRmax mice and 7.3% of AIRmin mice. Administered after pristane, DNA- hsp65 downregulated arthritis induction in AIRmax animals. Levels of interleukin (IL)- 12 were significantly lower in mice receiving pristane plus DNA- hsp65 or DNAv than in mice receiving pristane alone. However, when mice previously injected with pristane were inoculated with DNA- hsp65 or DNAv, the protective effect was significantly correlated with lower IL-6 and IL-12 levels and higher IL-10 levels. Our results strongly suggest that DNA-hsp65 has no arthritogenic potential and is actually protective against experimentally induced arthritis in mice.

Immune modulation induced by tuberculosis DNA vaccine protects non-obese diabetic mice from diabetes progression

We have described previously the prophylactic and therapeutic effect of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in experimental murine tuberculosis. However, the high homology of this protein to the corresponding mammalian 60 kDa heat shock protein (Hsp60), together with the CpG motifs in the plasmid vector, could trigger or exacerbate the development of autoimmune diseases. The non-obese diabetic (NOD) mouse develops insulin-dependent diabetes mellitus (IDDM) spontaneously as a consequence of an autoimmune process that leads to destruction of the insulin-producing beta cells of the pancreas. IDDM is characterized by increased T helper 1 (Th1) cell responses toward several autoantigens, including Hsp60, glutamic acid decarboxylase and insulin. In the present study, we evaluated the potential of DNA-HSP65 injection to modulate diabetes in NOD mice. Our results show that DNA-HSP65 or DNA empty vector had no diabetogenic effect and actually protected NOD mice against the development of severe diabetes. However, this effect was more pronounced in DNA-HSP65-injected mice. The protective effect of DNA-HSP65 injection was associated with a clear shift in the cellular infiltration pattern in the pancreas. This change included reduction of CD4(+) and CD8(+) T cells infiltration, appearance of CD25(+) cells influx and an increased staining for interleukin (IL)-10 in the islets. These results show that DNA-HSP65 can protect NOD mice against diabetes and can therefore be considered in the development of new immunotherapeutic strategies.

Efeito de micobactéria ambiental (Mycobacterium avium) na imunidade induzida pela vacina gênica para tuberculose (pVAXhsp65)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Resposta imune induzida pelas peçonhas do bagre Cathorops agassizii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Dermatophyte-host relationship of a murine model of experimental invasive dermatophytosis

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

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.