Toll-like receptor-4 deficiency inhibits ultraviolet radiation-induced tumor development by modulation of immune and inflammatory responses.

Ultraviolet (UV) B irradiation of the skin induces acute inflammation, as characterized by erythema, edema, and immunosuppression, and is subsequently linked to the progression of skin cancer. Toll-like receptor 4 (TLR4), a component of innate immunity, has been shown to play an important role in cancer. To elucidate the role of TLR4 in UVB-induced tumor development, TLR4-proficient (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice were exposed to multiple doses of UVB radiation (200 mJ/cm2 ) for 40 weeks. Photocarcinogenesis was retarded in terms of tumor incidence, and tumor latency, in mice deficient in TLR4 compared with TLR4-proficient mice, whereas significantly greater numbers of tumors occurred in TLR4-proficient mice. There was significant upregulation of inflammatory markers like COX-2, PGE2 , S100A8, and S100A9 in the skin of TLR4-proficient mice than the skin of TLR4-deficient mice. Furthermore, we found that TLR4-proficient mice had a significantly higher number of Gr1+CD11b+ myeloid cells CD4+CD25+ regulatory T-cells than TLR4-deficient mice. Furthermore, the levels of interferon (IFN)-γ cytokine was increased and the levels of interleukin (IL)-4, IL-10, and IL-17 cytokines were decreased in serum, skin, and tumor lysates of TLR4-deficient mice in comparison with samples from TLR4-proficient mice. Together, our data indicate that TLR4-mediated inflammation may cause suppression of antitumor responses and trigger the development of UVB-induced skin cancers. Thus, strategies to inhibit TLR4-mediated immune suppression may allow us to develop preventive and therapeutic approaches for the management of UVB-induced cutaneous tumors.

Evaluation of two mutants of Mycobacterium avium subsp. paratuberculosis as candidates for a live attenuated vaccine for Johne's disease.

Control of Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis, has been difficult because of a lack of an effective vaccine. To address this problem we used targeted gene disruption to develop candidate mutants with impaired capacity to survive ex vivo and in vivo to test as a vaccine. We selected relA and pknG, genes known to be important virulence factors in Mycobacterium tuberculosis and Mycobacterium bovis, for initial studies. Deletion mutants were made in a wild type Map (K10) and its recombinant strain expressing the green fluorescent protein (K10-GFP). Comparison of survival in an ex vivo assay revealed deletion of either gene attenuated survival in monocyte-derived macrophages compared to survival of wild-type K10. In contrast, study in calves revealed survival in vivo was mainly affected by deletion of relA. Bacteria were detected in tissues from wild-type and the pknG mutant infected calves by bacterial culture and PCR at three months post infection. No bacteria were detected in tissues from calves infected with the relA mutant (P<0.05). Flow cytometric analysis of the immune response to the wild-type K10-GFP and the mutant strains showed deletion of either gene did not affect their capacity to elicit a strong proliferative response to soluble antigen extract or live Map. Quantitative RT-PCR revealed genes encoding IFN-γ, IL-17, IL-22, T-bet, RORC, and granulysin were up-regulated in PBMC stimulated with live Map three months post infection compared to the response of PBMC pre-infection. A challenge study in kid goats showed deletion of pknG did not interfere with establishment of an infection. As in calves, deletion of relA attenuated survival in vivo. The mutant also elicited an immune response that limited colonization by challenge wild type Map. The findings show the relA mutant is a good candidate for development of a live attenuated vaccine for Johne's disease.