Down regulation of RANTES in pleural site is associated with inhibition of antigen specific response in tuberculosis.

Tuberculosis is the most common infectious reason for death and a major cause of pleural effusion globally. To understand the role of chemokines in trafficking of cells during TB pleurisy, we studied the responses to MTB, Ag85A in cells from pleural fluids and peripheral blood. Patients with TB pleural effusions, malignant effusions and asymptomatic healthy controls were enrolled. High expression (p < 0.05) of IP-10, MCP-1, MIG, IL-8, IFN-γ and IL-23 were observed in pleural fluids of TB patients compared to their plasma where expression of RANTES was significantly higher (p < 0.05). On specific stimulation of PFMCs with Ag85A, expression of RANTES was significantly lower in TB compared to NTB patients. We also observed increased expression of T regs and PD1 on CD8+T cells in PFMC of TB patients. Though some of the inflammatory chemokine/cytokines were up-regulated in pleura of TB patients, antigenic stimulation failed to induce them indicating poor antigenic responses at the site. Low expression of RANTES might be a reason for decreased trafficking of cells to the site and dissemination of infection into pleural site. The pattern of RANTES expression in pleural fluid vs serum is interesting. The observations necessitate further studies to investigate the levels of RANTES for its potential biological relevance in TB immunity and its use as a biomarker for diagnosis of pleural TB.

Glutathione-redox balance regulates c-rel-driven IL-12 production in macrophages: possible implications in antituberculosis immunotherapy.

The glutathione-redox balance, expressed as the ratio of intracellular reduced glutathione (GSH) and oxidized glutathione, plays an important role in regulating cellular immune responses. In the current study, we demonstrate that alteration of glutathione-redox balance in macrophages by GSH donors like cell-permeable glutathione ethyl ester reduced or N-acetyl-L-cysteine (NAC) can differentially regulate production of IL-12 cytokine in macrophages. A low concentration of NAC increased IL-12 p40/p70 production, whereas at high concentration, IL-12 production was inhibited due to increased calmodulin expression that binds and sequesters c-rel in the cytoplasm. Although NAC treatment increased the IkappaBalpha phosphorylation, it failed to increase TNF-alpha levels due to enhanced expression of suppressor of cytokine signaling 1, which specifically prevented nuclear translocation of p65 NF-kappaB. We demonstrate that NAC at 3 mM concentration could increase bacillus Calmette-Guérin-induced IFN-gamma production by PBMCs from patients with active tuberculosis and shifts the anti-bacillus Calmette-Guérin immune response toward the protective Th1 type. Our results indicate that redox balance of glutathione plays a critical role in regulating IL-12 induction in native macrophages, and NAC can be used in tailoring macrophages to induce enhanced Th1 response that may be helpful to control tuberculosis and other pathophysiological disorders.

Anti-B7-1/B7-2 antibody elicits innate-effector responses in macrophages through NF-kappaB-dependent pathway.

Blocking T cell co-stimulatory signals by anti-B7-1/B7-2 mAb is an attractive approach to treat autoimmune diseases. However, anti-B7-1/B7-2 mAb treatment is known to exacerbate autoimmune diseases through mechanisms not fully understood. Tumor necrosis factor alpha (TNF-alpha) and reactive oxygen species (ROS) also play important roles in determining the clinical outcome of autoimmune diseases. In this study, we demonstrate that the anti-B7-1 and the anti-B7-2 mAbs activate macrophages for higher induction of TNF-alpha and other effector responses such as bacterial cytotoxicity and production of ROS. Nuclear factor-kappaB (NF-kappaB) was found to be increased with anti-B7-1/B7-2 mAb treatment. Inhibition of NF-kappaB activity by over-expression of phosphorylation-defective I-kappaB alpha in anti-B7-1/B7-2 mAb-treated macrophages decreased TNF-alpha production. These data indicate that anti-B7-1 and anti-B7-2 mAbs can trigger innate-effector responses in macrophages by activating NF-kappaB-signaling pathway. Our results suggest that the B7 molecules are not only essential for induction of adaptive immune responses but also play roles in activation of innate immune responses.

Hydrogen peroxide inhibits IL-12 p40 induction in macrophages by inhibiting c-rel translocation to the nucleus through activation of calmodulin protein.

Although the antimicrobial activity of reactive oxygen species (ROSs) is well defined, the role of ROSs in regulating the immune response of the body is not well understood. We now provide evidence that hydrogen peroxide (H2O2), a major component of ROSs, inhibits interleukin-12 (IL-12) p40 and IL-12 p70 induction in murine macrophages and catalase pretreatment prevents H2O2-mediated down-regulation of IL-12. Endogenous accumulation of H2O2/ROSs in macrophages treated with alloxan resulted in IL-12 p40 inhibition. Although nuclear expression of both p50 and p65 NF-kappaB increased on H2O2 exposure, nuclear c-rel level was inhibited. Overexpression of c-rel restored IL-12 p40 on stimulation with lipopolysaccharide plus IFN-gamma during H2O2 treatment. H2O2 did not inhibit c-rel induction in cytosol; however, it prevented the transport of c-rel from cytosol to the nucleus. H2O2 activated calmodulin (CaM) protein in the cytosol, which subsequently sequestered c-rel in the cytosol preventing its transport to the nucleus. The CaM inhibitor trifIuoperazine increased both nuclear c-rel and IL-12 p40 levels in H2O2-treated macrophages, emphasizing a role of CaM in these processes. H2O2/ROSs thus down-regulate IL-12 induction in macrophages by a novel pathway inhibiting c-rel translocation to the nucleus through activation of CaM protein.

Use of fluorescent amplified fragment length polymorphism for molecular epidemiology of leptospirosis in India.

Nineteen isolates of leptospires recovered from patients during three epidemics that occurred at different places and different times in the Andaman Islands and eight isolates from sporadic cases were characterized using serological and molecular genetic techniques. Group sera and monoclonal antibodies were used for antigenic characterization, whereas fluorescent amplified fragment length polymorphism (FAFLP) was used for genotyping. Of the 27 isolates, 19 were identified as belonging to serogroup Grippotyphosa, 3 belonged to serogroup Australis, 2 belonged to serogroup Icterohaemorrhagiae, and 1 each belonged to serogroups Hebdomadis, Canicola, and Sejroe. Analysis of FAFLP data grouped these 27 isolates into two main clusters of genotypes. One of the clusters, populated by 19 isolates, included 16 outbreak isolates. Seven of these 19 isolates belonged to serovar Ratnapura, 10 belonged to serovar Valbuzzi, and 1 each belonged to serovar Grippotyphosa and serovar Saxkoebing. Of the 27 patients from whom isolates were obtained, 9 had severe illness, and 6 of these 9 patients had pulmonary involvement, 1 had pulmonary and hepatorenal involvement, and the remaining 2 had hepatorenal involvement alone. Two patients out of the nine severe cases died subsequently. The isolates from sporadic cases showed great genetic diversity and were also diverse antigenically. Perhaps the strains belonging to a dominant genotype (the outbreak-associated cluster) possessed epidemic potential and higher virulence with a greater predilection to cause pulmonary complications than strains belonging to other genetic backgrounds.

Microevolution between paired antral and paired antrum and corpus Helicobacter pylori isolates recovered from individual patients.

Sequence variations located at the signal sequence and mid-region within the vacA gene, the 3'-end of the cagA gene, the indel motifs at the 3'-end of the cag pathogenicity island and the regions upstream of the vacA and ribA genes were determined by PCR in 19 paired antral or antrum and corpus Helicobacter pylori isolates obtained at the same endoscopic session, and three antral pairs taken sequentially. Random amplification of polymorphic DNA (RAPD)-PCR and fluorescent amplified fragment length polymorphism (FAFLP)-PCR fingerprinting were applied to these paired clinical isolates. The FAFLP-PCR profiles generated were phylogenetically analysed. For the 22 paired isolates there were no differences within pairs at five of the genetic loci studied. However, six pairs of isolates (27%), of which four were antrum and corpus pairs, showed differences in the numbers of repeats located at the 3'-end of the cagA gene. RAPD-PCR fingerprinting showed that 16 (73%) pairs, nine of which were antrum and corpus pairs, possessed identical profiles, while six (27%) displayed distinctly different profiles, indicating mixed infections. Three of the six pairs showing differences at the 3'-end of the cagA gene yielded identical RAPD-PCR fingerprints. FAFLP-PCR fingerprinting and phylogenetic analysis revealed that all 16 pairs that displayed identical RAPD-PCR profiles had highly similar, but not identical, fingerprints, demonstrating that these pairs were ancestrally related but had undergone minor genomic alterations. Two antrum and corpus pairs of isolates, within the latter group, were isolates obtained from two siblings from the same family. This analysis demonstrated that each sibling was colonized by ancestrally related strains that exhibited differences in vacA genotype characteristics.

Fine-structure molecular typing of Irish Helicobacter pylori isolates and their genetic relatedness to strains from four different continents.

Genotyping of 74 Irish Helicobacter pylori isolates was performed at four different loci (vacA signal sequence and mid-region, insertion-deletion polymorphisms at the 3' end of the cag pathogenicity island, and cagA). The predominant vacA alleles and insertion-deletion motifs suggest an ancestral relationship between Irish isolates and either specific East Asian or Northern European strains. In addition, fluorescent amplified fragment length polymorphism-PCR genotyping and phylogenetic analysis of 32 representative Irish H. pylori isolates and 22 isolates from four different continents demonstrated that the Irish H. pylori isolates examined were weakly clonal and showed some association with both European and Asian isolates. These three genotyping techniques show that Irish H. pylori isolates have distinctive features that may have evolved in this insular European population.