Epithelial cells are key players in the first line of defense offered by the mucosal immune system against invading pathogens. In the present study we sought to determine whether human corneal epithelial cells expressing Toll-like receptors (TLRs) function as pattern-recognition receptors in the innate immune system and, if so, whether these TLRs act as a first line of defense in ocular mucosal immunity. Incubation of human primary corneal epithelial cells and the human corneal epithelial cell line (HCE-T) with peptidoglycan or LPS did not lead to activation, at the level of DNA transcription, of NF-κB or the secretion of inflammation-associated molecules such as IL-6, IL-8, and human β-defensin-2. However, when incubated with IL-1α to activate NF-κB, the production by these cells of such inflammatory mediators was enhanced. Human corneal epithelial cells were observed to express both TLR2- and TLR4-specific mRNA as well as their corresponding proteins intracellularly, but not at the cell surface. However, even when LPS was artificially introduced into the cytoplasm, it did not lead to the activation of epithelial cells. Taken together, our results demonstrate that the intracellular expression of TLR2 and TLR4 in human corneal epithelial cells fails to elicit innate immune responses and therefore, perhaps purposely, contributes to an immunosilent environment at the ocular mucosal epithelium.

The antitumor activity of cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) depends on its being converted to 5-fluorouracil (5-FUra) by the enzyme thymidine phosphorylase (dThdPase, EC 2.42.4). We prepared mouse anti-human dThdPase monoclonal antibodies to serve as tools for clinical studies with this drug. Partially purified dThdPase obtained form HCT116 human colon cancer cells grown in athymic mice was used as and antigen for the immunization of mice. Six hybridomas were cloned which produced anti-human dThdPase antibodies, as detected by Western blot analysis with human dThdPase. With these antibodies, we developed an ELISA method sensitive enough to measure dThdPase levels, even in tumor tissue samples weighing as little as 10 mg. In addition, one monoclonal antibody was suitable for immunologically staining the enzyme in tumor tissues. Thus, these anti-human dThdPase monoclonal antibodies could be used to measure levels of the enzyme in tumor cells, which is essential for the activation of 5'-dFUrd.

The present study shows that various cytokines such as tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1 alpha), and interferon-gamma (IFN gamma) make tumor cells much more susceptible to the cytostatic 5'-deoxy-5-fluorouridine (5'-dFUrd) than to 5-fluorouracil (5-FUra) and other cytostatics. These three cytokines increased the susceptibility of human cancer cell lines (COLO201, MKN45 and WiDr) but did not affect that of normal fibroblast WI38 cells. The cytokine mixture induced a 50-fold increase in the susceptibility of COLO201 to 5'-dFUrd, whereas a 12-fold increase and a less than 5-fold enhancement in the susceptibility to 5-FUra and other cytostatics, respectively, were observed. The increased susceptibility would be a result of the induction of thymidine phosphorylase (TdR Pase), which is the essential enzyme for the conversion of 5'-dFUrd to 5-FUra. The cytokine mixture increased TdR Pase activity by up to 47 times and greatly induced its mRNA expression in the cancer cell lines. These results suggest that the therapeutic benefit of 5'-dFUrd would be improved by its use in combination with the cytokines.

Interaction of the TCR complex with self- or foreign peptides is a central event in the immune responses. Upon TCR stimulation, a protein-tyrosine kinase (PTK), ZAP-70, is recruited to signaling units of the TCR complex, such as TCRzeta, to play an essential role in T cell activation. Here, we find that mice lacking adaptor proteins Dok-1 and Dok-2 show augmented responses to thymus-dependent, but not thymus-independent, antigens, and that their T cells show elevated responses to TCR stimulation, including the activation of ZAP-70 and subsequent proliferation and cytokine production. Furthermore, the forced expression of Dok-1 or Dok-2 in a CD3(+)CD4(+) T cell clone inhibited the activation of ZAP-70 upon TCR stimulation. Interestingly, the Dok-1 and Dok-2 COOH-terminal moieties bearing the src homology 2 target motifs were dispensable for this negative regulation, even though they are crucial for the known adaptor function of Dok-family proteins. Thus, by an as yet unidentified mechanism, Dok-1 and Dok-2 play an essential role in the negative regulation of TCR signaling. Consistently, all mice lacking these proteins exhibited elevated titers of antibodies to double-stranded DNA and developed lupus-like renal disease.