Immunohistochemical localization of TNF-α and IL-4 in granulomas of immunocompetent and immunosuppressed New Zealand white rabbits infected with Encephalitozoon cuniculi.

Encephalitozoon cuniculi is a fungi-related, obligate, zoonotic, spore-forming intracellular eukaryotic microorganism. This emerging pathogen causes granulomas to form in the brain and kidneys of infected individuals. The objective of the current study was to detect the distribution of TNF-α- and IL-4-positive cells using immunohistochemistry within these granulomas in both infected immunocompetent (group A) and immunosuppressed (group B) New Zealand white rabbits. In the brain, labeled TNF-α immune cells were mainly located in the granuloma peripheries in group B. Granulomas examined in the kidneys of groups A and B were TNF-α positive, but were significantly different (p < 0.001) when compared with the brain. IL-4-producing immune cells in the brain and kidneys were disseminated within granulomas in groups A and B; however, no significant difference (p > 0.05), was observed. IL-4 positive cells were more numerous in brain sections of group B and differed significantly (p < 0.05) when compared with kidneys. Granulomas were not observed in control animals (groups C and D). In conclusion, we identified TNF-α positive cells in both the brain and kidneys of immunocompetent and immunosuppressed animals; IL-4 positive cells were numerous in the brains of immunosuppressed rabbits; however, in terms of percentage were numerous in the brains of immunocompetent rabbits. Immunosuppression appeared to stimulate a change in the cellular phenotype of Th1- to Th2-like granulomas in the brain and kidneys via an unknown mechanism. Expression of pro- and pre-inflammatory cytokines in microsporidian granulomas suggests a mechanism by which E. cuniculi evades the immune response, causing more severe disease. These results increase our understanding of TNF-α and IL-4-positive cells within the E. cuniculi granuloma microenvironment.

WT1 gene silencing by aerosol delivery of PEI-RNAi complexes inhibits B16-F10 lung metastases growth.

The Wilms' tumor gene 1 (WT1) is a universal tumor antigen and consequently a good therapeutic target for the development of gene therapy strategies. Earlier, we reported the in vitro efficacy of WT1 RNAi in the inhibition of B16F10 murine melanoma cell line growth. In this study, we used an aerosol system to deliver WT1 RNAi complexes, polyethyleneimine (PEI)-WT1-1 or PEI-WT1-2, to the lungs of mice with B16F10 lung metastasis. This treatment produced a statistically significant (P=0.020) reduction in the number and size of lung tumor foci, resulting in decreased lung weight and tumor index in treated mice compared with controls. The WT1 RNAi treatment also reduced the number and size of tumor blood vessels, suggesting decreased angiogenesis. Furthermore, the treated lung tissue showed cells in the tumor infiltrations undergoing apoptosis and elevated expression of the proapoptotic genes Bcl-xS and Bax, suggesting an activation of the intrinsic apoptotic pathway. Overall, WT1-1 treatment prolonged the mean survival time of tumor-bearing mice in comparison with the control and WT1-2-treated mice. Our data show that WT1 gene silencing in vivo by aerosol delivery of PEI-WT1 RNAi complexes is an effective therapeutic strategy for the treatment of lung metastases.