1 Inflammmatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leucocyte infiltration and upregulation of proinflammatory cytokines. In this study, we have investigated the protective effects of curcumin, an anti-inflammatory and antioxidant food derivative, on 2,4,6-trinitrobenzene sulphonic acid-induced colitis in mice, a model for IBD. 2 Intestinal lesions (judged by macroscopic and histological score) were associated with neutrophil infiltration (measured as increase in myeloperoxidase activity in the mucosa), increased serine protease activity (may be involved in the degradation of colonic tissue) and high levels of malondialdehyde (an indicator of lipid peroxidation). 3 Dose -response studies revealed that pretreatment of mice with curcumin (50 mg kg À1 daily i.g. for 10 days) significantly ameliorated the appearance of diarrhoea and the disruption of colonic architecture. Higher doses (100 and 300 mg kg À1 ) had comparable effects. 4 In curcumin-pretreated mice, there was a significant reduction in the degree of both neutrophil infiltration (measured as decrease in myeloperoxidase activity) and lipid peroxidation (measured as decrease in malondialdehyde activity) in the inflamed colon as well as decreased serine protease activity. 5 Curcumin also reduced the levels of nitric oxide (NO) and O 2 À associated with the favourable expression of Th1 and Th2 cytokines and inducible NO synthase. Consistent with these observations, nuclear factor-kB activation in colonic mucosa was suppressed in the curcumin-treated mice. 6 These findings suggest that curcumin or diferuloylmethane, a major component of the food flavour turmeric, exerts beneficial effects in experimental colitis and may, therefore, be useful in the treatment of IBD.

The virulence of Leishmania donovani in mammals depends at least in part on cysteine proteases because they play a key role in CD4+ T cell differentiation. A 6-fold increase in NO production was observed with 0.5 μM chicken cystatin, a natural cysteine protease inhibitor, in IFN-γ-activated macrophages. In a 45-day BALB/c mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by cystatin in synergistic activation with a suboptimal dose of IFN-γ. In contrast to the case with promastigotes, cystatin and IFN-γ inhibited the growth of amastigotes in macrophages. Although in vitro cystatin treatment of macrophages did not induce any NO generation, significantly enhanced amounts of NO were generated by macrophages of cystatin-treated animals. Their splenocytes secreted soluble factors required for the induction of NO biosynthesis, and the increased NO production was paralleled by a concomitant increase in antileishmanial activity. Moreover, splenocyte supernatants treated with anti-IFN-γ or anti-TNF-α Abs suppressed inducible NO generation, whereas i.v. administration of these anticytokine Abs along with combined therapy reversed protection against infection. mRNA expression and flow cytometric analysis of infected spleen cells suggested that cystatin and IFN-γ treatment, in addition to greatly reducing parasite numbers, resulted in reduced levels of IL-4 but increased levels of IL-12 and inducible NO synthase. Not only was this treatment curative when administered 15 days postinfection, but it also imparted resistance to reinfection. These studies provide a promising alternative for protection against leishmaniasis with a switch of CD4+ differentiation from Th2 to Th1, indicative of long-term resistance.

No abstract

A1122 is a T7-related bacteriophage infecting most isolates of

The bacteriophage vB_YecM-R1-37 (R1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage R1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of R1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the R1-37 capsid consists of hexameric capsomers arranged on a T‫72؍‬ lattice similar to the bacteriophage KZ. The tail of R1-37 has a contractile sheath. We conclude that phage R1-37 is a representative of a novel phage type that carries the dU-containing genome in a KZ-like head. Bacteriophages, the viruses that infect bacteria, are the most abundant organisms on Earth, and it is estimated that for each microbial isolate at least 10 different phages exist (19,35). Present knowledge indicates that phages are extremely diverse in nature (7a). Studies on bacteriophages have escalated, since they are excellent targets for genomic and evolutionary research and as models for systems biology studies; in addition, they are important vehicles in horizontal gene transfer. Phages are used as tools in bacterial genetics, and phage gene products are used as tools in molecular biology. Furthermore, their potential as therapeutic agents during the increasing emergence of antibiotic resistance is being reexamined (45,46). In summary, a thorough knowledge of the bacteriophage and its biology is considered essential to all phage research.We have isolated several Yersinia enterocolitica-specific bacteriophages that use different parts of lipopolysaccharide (LPS) as receptors and used them to study the molecular biology and genetics of LPS biosynthesis (2,23,24,33,34,(46)(47)(48). The bacteriophage vB_YecM-R1-37 (R1-37) was isolated from sewage based on its ability to infect Y. enterocolitica strain YeO3-R1, an O-polysaccharide (O-PS)-lacking Y. enterocolitica serotype O:3 strain (44, 48). The host range of R1-37, as well as genetic and structural data, showed that the LPS outer core (OC) hexasaccharide of Y. enterocolitica O:3 is the phage receptor (23,37,38,48).Electron microscopy and analysis of its genome indicated that R1-37 is an exceptionally large-tailed phage with an estimated genome size of 270 kb (23). Structural studies on large bacteriophages with contractile tails are currently limited to the Pse...

No abstract

The incidence of oropharyngeal squamous cell carcinoma (OPSCC) has increased over the past decades in many western countries. This trend is mainly attributed to the human papillomavirus (HPV). Cancer-related actions of immunological defense systems are being intensively researched. Human toll-like receptors (TLRs) are a family of pattern recognition receptors that participate in the immunological defense against pathogens, but their actions are also linked to cancer. The expression of TLRs in cervical epithelium alters both during the clearance of HPV infection and the HPV-induced neoplasia, but the expression of TLRs has not been studied in OPSCC. Thirty-five paraffin-embedded, formalin-fixed, squamous cell carcinoma tissue specimens were analyzed for TLRs 2, 3, 4, 5, 7, and 9 and HPV and p16 statuses. The TLR 9 expression was lower in HPV-positive tumors compared with HPV-negative tumors. TLR 7 was expressed in all cancer specimens, but elevated expression was evident in HPV and/or p16-positive tumors. The majority of p16-positive tumors did not express TLR 5, whereas its expression was stronger in p16-negative tumors. The results of in vitro analysis of five human OPSCC cell lines and one human oral tongue squamous cell carcinoma cell line agree with the in vivo trends: low levels of TLR 5 and high levels of TLR 7 in p16-positive OPSCC. Overall, TLR 7 and 9 expression patterns are demonstrated here to relate to the HPV status in vivo and TLR 5 and 7 expression patterns to the p16 status in vivo and in vitro.

Active targeting of CpG-containing oligodeoxynucleotide (CpG-ODN) to macrophages was studied by incorporating it in mannose-coated liposomes, using visceral leishmaniasis as the model macrophage disease. Mannosylated liposomal CpG-ODN was more effective than liposomal or free CpG-ODN in inhibiting amastigote multiplication within macrophages. Moreover, in a 60-day mouse model of visceral leishmaniasis, complete elimination of spleen parasite burden was achieved by mannosylated liposomal CpG-ODN, compared to 62% and 81% parasite suppression by free and liposomal ODN, respectively, at a similar dose. Although in vitro exposure of CpG-ODN did not induce marked nitric oxide (NO) generation by macrophages, considerably enhanced amount of NO was generated by macrophages of CpG-ODN-treated animals. Their splenocytes secreted soluble factors required for the induction of NO generation, and the increased NO generation was paralleled by an increase in antileishmanial activity. Inducible NO generation was suppressed by treating splenocyte supernatants with anti-IFN-+ or anti-IL-12 antibodies, whereas in vivo administration of these anti-cytokine Ab along with CpG-ODN reversed protection against infection. CpG-ODN treatment resulted in reduced levels of IL-4, but increased levels of IFN-+ , IL-12 and inducible NO synthase in infected spleen cells, which was magnified by encapsulation in mannose-coated liposomes. This targeted treatment was not only curative, but it also imparted resistance to reinfection. These results represent a general approach for intracellular targeting of CpG-ODN, which effectively enhances its therapeutic potential in redirecting curative Th1 responses in Th2-driven disorders.