Augmentation of differentiation and gap junction function by kaempferol in partially differentiated colon cancer cells

Kaempferol induces differentiation in partially differentiated colon cancer cells which express low levels of connexin43 protein and connexin43 mRNA (KNC cells). Differentiation was observed as changes in cell morphology and the activity of alkaline phosphatase. Increased differentiation in kaempferol-treated KNC cells correlated with restoration of gap junctional intercellular communication (GJIC), increased levels of connexin43 protein and its phosphorylation status. Phosphorylation (activation) of Stat3 and Erk was also reduced by kaempferol. An inhibitor of Stat3 phosphorylation also induced morphological changes in KNC cells similar to those in kaempferol-treated cells, suggesting that kaempferol-induced differentiation may be mediated by inhibition of Stat3 phosphorylation. These effects were not observed in HCT116 cells, a poorly differentiated colon cancer cell line deficient in expression of connexin43 mRNA and connexin43 protein. In conclusion, kaempferol might function as an anticancer agent by re-establishing GJIC through enhancement of the expression and phosphorylation of connexin43 protein in a tumorigenic colon cancer cell line that already expresses connexin43 mRNA via a Stat3-dependent mechanism. In contrast, kaempferol had no effect in a tumorigenic colon cancer cell line that did not express connexin43 mRNA and was deficient in GJIC.     

Toll-like receptor 2 ligands inhibit TH2 responses to mite allergen

BACKGROUND: There is intense interest in the interaction between microbial compounds and allergy. Although Toll-like receptor (TLR)-2 ligands derived from Gram-positive bacteria alter allergic sensitization in animal models, it is not clear what effect TLR2 ligands have on allergen-specific T-cell memory in human beings. OBJECTIVE: To determine whether in vitro exposure to TLR2 ligands modifies the immune response to house dust mite allergen (HDM). METHODS: Blood mononuclear cells were obtained from individuals both allergic (n = 23) and not allergic (n = 22) to HDM, and stimulated with HDM in the presence or absence of TLR2 ligands. RESULTS: In subjects allergic to HDM, IL-5 and IL-13 responses to HDM were inhibited by heat-killed Staphylococcus aureus, staphylococcal lipoteichoic acid, and the synthetic lipoprotein Pam3CSK4 (P < .005; all stimuli). Although the whole staphylococcal bacteria increased IFN-gamma responses, the purified TLR2 ligands lipoteichoic acid and Pam3CSK4 inhibited HDM-specific IFN-gamma synthesis. In contrast, TLR2 ligands had minimal effects on responses to HDM in subjects without allergy. TLR2 ligands induced upregulation of HLA-DR expression but did not inhibit antigen uptake or processing by antigen-presenting cells. CONCLUSION: Toll-like receptor 2 ligands inhibit allergen-specific T(H)2 responses in sensitized individuals. This effect appears to be mediated by the actions of TLR2 ligands on antigen-presenting cells, and at least for the purified TLR2 ligands does not involve the induction of a strong T(H)1 immune response. CLINICAL IMPLICATIONS: These findings provide an impetus for further preclinical studies examining the potential use of TLR2 ligands in allergic disease.     

TLR3 and RIG-I gene variants: associations with functional effects on receptor expression and responses to measles virus and vaccine in vaccinated infants

Measles virus causes severe morbidity and mortality, despite the availability of measles vaccines. Successful defence against viral pathogens requires early recognition of virus-specific patterns by innate receptors like Toll-like receptor (TLR)3 and the RNA helicase, retinoic acid inducible gene-I (RIG-I). Genetic differences in these receptors may influence the primary immune responses to measles and the efficacy of measles vaccine. In 1-year-old Australian infants after their first measles vaccine dose, we investigated functional effects of TLR3 and RIG-I polymorphisms on intracellular protein expression using flow cytometry, cytokine responses to receptor ligands and measles lysate, and post-vaccination measles IgG levels. We found that TLR3 Leu412Phe was significantly associated with IFN-α/β response after stimulation with TLR3 ligand, poly(I:C) (P=0.024). Downregulation of TLR3 protein expression in NK cells after poly(I:C) was also associated with this variant (P=0.011). In contrast, measles-specific expression, cytokine responses and antibody responses were not associated with TLR3 polymorphisms. No associations were found with RIG-I variants. These results suggest that a TLR3 polymorphism has functional effects on receptor expression and cytokine response. However, this did not translate to an effect on specific responses to measles virus or vaccine. We found no evidence that RIG-I polymorphisms were involved in measles immune responses.     

Inhibition of gap junctional intercellular communication by perfluorinated fatty acids is dependent on the chain length of the fluorinated tail

Perfluorinated fatty acids (PFFAs), such as perfluorooctanoic acid (PFOA) and perfluorodecanoic acid (PFDA), are known peroxisome proliferators and hepatocarcinogens. A causal link between an increase in the oxidative stress by peroxisomes and tumor promotion has been proposed to explain the hepatocarcinogenicity of PFOA and PFDA. However, the down-regulation of gap junctional intercellular communication (GJIC) has also been linked to the tumor-promoting properties of many carcinogens. Therefore, the effect of PFFAs on GJIC in WB-rat liver epithelial cells was determined. The chain length of the PFFAs tested for an effect on GJIC ranged from 2 to 10, 16 and 18 carbons. Carbon lengths of 7 to 10 inhibited GJIC in a dose–response fashion, whereas carbon lengths of 2 to 5, 16 and 18 did not appreciably inhibit GJIC. Inhibition occurred within 15 min and was reversible, with total recovery from inhibition occurring within 30 min after the removal of the compound from the growth medium. This short time of inhibition suggests that GJIC was modified at the post-translational level. Also, this short time period was not long enough for peroxisome proliferation. The post-translational modification of the gap junction proteins was not a consequence of altered phosphorylation as determined by Western blot analysis. Perfluorooctanesulfonic acid also inhibited GJIC in a dose–response fashion similar to PFDA, indicating that the determining factor of inhibition was probably the fluorinated tail, which required 7–10 carbons. Our results suggest that PFFAs could potentially act as hepatocarcinogens at the level of gap junctions in addition to or instead of through peroxisome proliferation.Int. J. Cancer 78:491–495, 1998. © 1998 Wiley-Liss, Inc.     

Hydrogen peroxide inhibits gap junctional intercellular communication in glutathione sufficient but not glutathione deficient cells

Cell to cell communication via gap junctions is essential in the maintenance of the homeostatic balance of multicellular organisms. Aberrant intercellular gap junctional communication (GJIC) has been implicated in tumor promotion, neuropathy and teratogenesis. Oxidative stress has also been implicated in similar pathologies such as cancer. We report a potential link between oxidative stress and GJIC. Hydrogen peroxide, a known tumor promoter, inhibited GJIC in WB-F344 rat liver epithelial cells with an I50 value of 200 microM. Inhibition of GJIC by H2O2 was reversible as indicated by the complete recovery of GJIC with the removal of H2O2 via a change of fresh media. Free radical scavengers, such as t-butyl alcohol, propylgallate, and Trolox, did not prevent the inhibition of GJIC by H2O2, which indicated that the effects of H2O2 on GJIC was probably not a consequence of aqueous free radical damage. The depletion of intracellular GSH reversed the inhibitory effect of H2O2 on GJIC. The treatment of glutathione- sufficient cells with H2O2 resulted in the hyperphosphorylation of connexin43, which is the basic subunit of the hexameric gap junction protein, as determined by Western blot analysis. TPA, a well-known tumor promoter, also inhibits GJIC via hyperphosphorylation of GJIC, which is a result of protein kinase-C activation. However, H2O2 also induced hyperphosphorylation in GSH-deficient cells that had normal rates of GJIC. Therefore, the mechanism of GJIC inhibition must be different from the TPA-pathway and involves GSH.      

Inhibition of gap junctional intercellular communication by perfluorinated compounds in rat liver and dolphin kidney epithelial cell lines in vitro and Sprague-Dawley rats in vivo.

Gap junctional intercellular communication (GJIC) is the major pathway of intercellular signal transduction, and is thus important for normal cell growth and function. Recent studies have revealed a global distribution of some perfluorinated organic compounds, especially perfluorooctane sulfonic acid (PFOS) in the environment. Because other perfluoroalkanes had been shown to inhibit GJIC, the effects of PFOS and related sulfonated fluorochemicals on GJIC were studied using a rat liver epithelial cell line (WB-F344) and a dolphin kidney epithelial cell line (CDK). In vivo effects on GJIC were studied in Sprague-Dawley rats orally exposed to PFOS for 3 days or 3 weeks. Effects on GJIC were measured using the scrape loading dye technique. PFOS, perfluorooctane sulfonamide (PFOSA), and perfluorohexane sulfonic acid (PFHA) were found to inhibit GJIC in a dose-dependent fashion, and this inhibition occurred rapidly and was reversible. Perfluorobutane sulfonic acid (PFBS) showed no significant effects on GJIC within the concentration range tested. A structure activity relationship was established among all 4 tested compounds, indicating that the inhibitory effect was determined by the length of fluorinated tail and not by the nature of the functional group. The results of the studies of the 2 cell lines and the in vivo exposure were comparable, suggesting that the inhibitory effects of the selected perfluorinated compounds on GJIC were neither species- nor tissue-specific and can occur both in vitro and in vivo.     

Dendritic cell immaturity during infancy restricts the capacity to express vaccine-specific T-cell memory

The capacity of the immune system in infants to develop stable T-cell memory in response to vaccination is attenuated, and the mechanism(s) underlying this developmental deficiency in humans is poorly understood. The present study focuses on the capacity for expression of in vitro recall responses to tetanus and diphtheria antigens in lymphocytes from 12-month-old infants vaccinated during the first 6 months of life. We demonstrate that supplementation of infant lymphocytes with "matured" dendritic cells (DC) cultured from autologous CD14+ precursors unmasks previously covert cellular immunity in the form of Th2-skewed cytokine production. Supplementation of adult lymphocytes with comparable prematured autologous DC also boosted vaccine-specific T-cell memory expression, but in contrast to the case for the infants, these cytokine responses were heavily Th1 skewed. Compared to adults, infants had significantly fewer circulating myeloid DC (P < 0.0001) and plasmacytoid DC (P < 0.0001) as a proportion of peripheral blood mononuclear cells. These findings suggest that deficiencies in the numbers of antigen-presenting cells and their functional competence at 12 months of age limit the capacity to express effector memory responses and are potentially a key factor in reduced vaccine responsiveness in infants.     

IXth International Conference on AIDS and STDs in Africa (ICASA), Kampala,Uganda - December 10-14, 1995

This article summarizes the results of scientific investigations presented at the 9th International Conference on AIDS and Sexually Transmitted Diseases in Africa held December 10-14, 1995 in Kampala, Uganda. The studies indicated that AIDS epidemic is starting to level off in urban areas, with rates among women, especially those who are pregnant, continuing to climb steadily in the outlying semiurban areas. In addition, a high prevalence and sizable rate of new infections are occurring among militaries. Furthermore, the economic tendency to put a price on services appears potentially harmful for the campaigns against AIDS in Africa, especially among the economically disadvantaged. In Uganda, this condition has led to a significant reduction in the number of clients visiting the counseling and testing centers (-8.3% between 1993 and 1994, from 45,484 to 41,688 clients, with more of a pronounced fall-off among men (-12.6%) as well as among persons without primary education (-44.3%)). According to private sources, the Ugandan AIDS prevention program targeted at truckers has not yet been fully extended throughout the Ugandan society. Finally, it is indicated that the economic cost of AIDS for families is much more burdensome, contrary to the findings of the World Bank Study in Tanzania which confirmed that families do not suffer economically as a result of AIDS.