Ac2PIM-responsive miR-150 and miR-143 Target Receptor-interacting Protein Kinase 2 and Transforming Growth Factor Beta-activated Kinase 1 to Suppress NOD2-induced Immunomodulators

Specific and coordinated regulation of innate immune receptor-driven signaling networks often determines the net outcome of the immune responses. Here, we investigated the cross-regulation of toll-like receptor (TLR)2 and nucleotide-binding oligomerization domain (NOD)2 pathways mediated by Ac₂PIM, a tetra-acylated form of mycobacterial cell wall component and muramyl dipeptide (MDP), a peptidoglycan derivative respectively. While Ac₂PIM treatment of macrophages compromised their ability to induce NOD2-dependent immunomodulators like cyclooxygenase (COX)-2, suppressor of cytokine signaling (SOCS)-3, and matrix metalloproteinase (MMP)-9, no change in the NOD2-responsive NO, TNF-α, VEGF-A, and IL-12 levels was observed. Further, genome-wide microRNA expression profiling identified Ac₂PIM-responsive miR-150 and miR-143 to target NOD2 signaling adaptors, RIP2 and TAK1, respectively. Interestingly, Ac₂PIM was found to activate the SRC-FAK-PYK2-CREB cascade via TLR2 to recruit CBP/P300 at the promoters of miR-150 and miR-143 and epigenetically induce their expression. Loss-of-function studies utilizing specific miRNA inhibitors establish that Ac₂PIM, via the miRNAs, abrogate NOD2-induced PI3K-PKCδ-MAPK pathway to suppress β-catenin-mediated expression of COX-2, SOCS-3, and MMP-9. Our investigation has thus underscored the negative regulatory role of Ac₂PIM-TLR2 signaling on NOD2 pathway which could broaden our understanding on vaccine potential or adjuvant utilities of Ac₂PIM and/or MDP.     

Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population

Cytotoxic T Lymphocyte antigen 4 (CTLA4) is a potent immunoregulatory molecule that suppresses antitumor response by down-regulating T cell activation. We examined candidate disease-susceptibility single nucleotide polymorphism (SNPs) of CTLA4 at +49A/G, CT60A/G and ?318C/T genes in bladder cancer (BC) patients of North Indian population. Histopathologically confirmed 200 patients of BC and 200 unrelated, healthy controls of similar ethnicity were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) and amplification refractory mutation specific (PCR–ARMS) methods. In present study SNP CTLA4 +49A/G, variant genotype showed 3.74-fold risks for BC. While looking at G allele carrier level, risk for BC was high (OR = 1.54). The risk for BC was also evident in case G allele (OR = 1.58). CTLA4 CT60A/G gene polymorphism variant genotype showed 1.36-fold risks for BC. While at G allele carrier and with variant G allele it showed significantly reduced risk for BC. CTLA4 +49A/G genotype exhibited 1.57-fold risks with smoking in BC patients in homozygous mutant condition. In silico analysis further supports the results of SNP at CTLA4 +49A/G and CTLA4 CT60A/G. None of the above SNPs of CTLA4 demonstrated association with tumor stage/grade for BC severity and progression. BCG immunotherapy had no impact on CTLA4 gene polymorphism revealing no significant association. Haplotype GAC showed high risk for BC while other haplotype AGT showed reduced risk for BC. Our results indicated that genetic variations in CTLA4 gene (+49A/G, CT60A/G) play role in susceptibility to BC. Haplotype GAC showed high risk for BC. An association study utilizing a larger sample size and different ethnicity warrant further investigation through replication and advance techniques.     

Inhibition of 5′-UTR RNA Conformational Switching in HIV-1 Using Antisense PNAs

Background

The genome of retroviruses, including HIV-1, is packaged as two homologous (+) strand RNA molecules, noncovalently associated close to their 5′-end in a region called dimer linkage structure (DLS). Retroviral HIV-1 genomic RNAs dimerize through complex interactions between dimerization initiation sites (DIS) within the (5′-UTR). Dimer formation is prevented by so calledLong Distance Interaction (LDI) conformation, whereas Branched Multiple Hairpin (BMH) conformation leads to spontaneous dimerization.

Methods and Results

We evaluated the role of SL1 (DIS), PolyA Hairpin signal and a long distance U5-AUG interaction by in-vitro dimerization, conformer assay and coupled dimerization and template-switching assays using antisense PNAs. Our data suggests evidence that PNAs targeted against SL1 produced severe inhibitory effect on dimerization and template-switching processes while PNAs targeted against U5 region do not show significant effect on dimerization and template switching, while PNAs targeted against AUG region showed strong inhibition of dimerization and template switching processes.

Conclusions

Our results demonstrate that PNA can be used successfully as an antisense to inhibit dimerization and template switching process in HIV -1 and both of the processes are closely linked to each other. Different PNA oligomers have ability of switching between two thermodynamically stable forms. PNA targeted against DIS and SL1 switch, LDI conformer to more dimerization friendly BMH form. PNAs targeted against PolyA haipin configuration did not show a significant change in dimerization and template switching process. The PNA oligomer directed against the AUG strand of U5-AUG duplex structure also showed a significant reduction in RNA dimerization as well as template- switching efficiency.The antisense PNA oligomers can be used to regulate the shift in the LDI/BMH equilibrium.     

Association of inflammatory chemokine gene CCL2I/D with bladder cancer risk in North Indian population

Chemokine genes have been proposed as good candidate genes for conferring susceptibility to Bladder cancer (BC). We examined the combined effect of multiple alleles of pro inflammatory chemokine genes for determining the risk of BC. We tested association of three gene polymorphisms of CCL2I/D (rs3917887), CCL2A2518G (rs1024611) and CCR2V64I (rs1799864) with BC risk in North Indian population. Genotypes were assessed in hospital-based case-control study comprising of 200?BC patients and 200 healthy controls. Genomic DNA was isolated from blood and genotyping done using PCR-RFLP method. In CCL2I/D polymorphism, the heterozygous genotype (I/D) showed high risk of BC p?相似文献   

Highly aggregated antibody therapeutics can enhance the in vitro innate and late-stage T-cell immune responses

Aggregation of biotherapeutics has the potential to induce an immunogenic response. Here, we show that aggregated therapeutic antibodies, previously generated and determined to contain a variety of attributes (Joubert, M. K., Luo, Q., Nashed-Samuel, Y., Wypych, J., and Narhi, L. O. (2011) J. Biol. Chem. 286, 25118-25133), can enhance the in vitro innate immune response of a population of naive human peripheral blood mononuclear cells. This response depended on the aggregate type, inherent immunogenicity of the monomer, and donor responsiveness, and required a high number of particles, well above that detected in marketed drug products, at least in this in vitro system. We propose a cytokine signature as a potential biomarker of the in vitro peripheral blood mononuclear cell response to aggregates. The cytokines include IL-1β, IL-6, IL-10, MCP-1, MIP-1α, MIP-1β, MMP-2, and TNF-α. IL-6 and IL-10 might have an immunosuppressive effect on the long term immune response. Aggregates made by stirring induced the highest response compared with aggregates made by other methods. Particle size in the 2-10 μm range and the retention of some folded structure were associated with an increased response. The mechanism of aggregate activation at the innate phase was found to occur through specific cell surface receptors (the toll-like receptors TLR-2 and TLR-4, FcγRs, and the complement system). The innate signal was shown to progress to an adaptive T-cell response characterized by T-cell proliferation and secretion of T-cell cytokines. Investigating the ability of aggregates to induce cytokine signatures as biomarkers of immune responses is essential for determining their risk of immunogenicity.     

Evidence for an association between thyroid-stimulating hormone and insulin-like growth factor 1 receptors: a tale of two antigens implicated in Graves' disease

Thyroid-stimulating hormone receptor (TSHR) plays a central role in regulating thyroid function and is targeted by IgGs in Graves' disease (GD-IgG). Whether TSHR is involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO), the orbital manifestation of GD, remains uncertain. TSHR signaling overlaps with that of insulin-like grow factor 1 receptor (IGF-1R). GD-IgG can activate fibroblasts derived from donors with GD to synthesize T cell chemoattractants and hyaluronan, actions mediated through IGF-1R. In this study, we compare levels of IGF-1R and TSHR on the surfaces of TAO and control orbital fibroblasts and thyrocytes and explore the physical and functional relationship between the two receptors. TSHR levels are 11-fold higher on thyrocytes than on TAO or control fibroblasts. In contrast, IGF-1R levels are 3-fold higher on TAO vs control fibroblasts. In pull-down studies using fibroblasts, thyrocytes, and thyroid tissue, Abs directed specifically against either IGF-1Rbeta or TSHR bring both proteins out of solution. Moreover, IGF-1Rbeta and TSHR colocalize to the perinuclear and cytoplasmic compartments in fibroblasts and thyrocytes by confocal microscopy. Examination of orbital tissue from patients with TAO reveals similar colocalization to cell membranes. Treatment of primary thyrocytes with recombinant human TSH results in rapid ERK phosphorylation which can be blocked by an IGF-1R-blocking mAb. Our findings suggest that IGF-1R might mediate some TSH-provoked signaling. Furthermore, they indicate that TSHR levels on orbital fibroblasts are considerably lower than those on thyrocytes and that this receptor associates with IGF-1R in situ and together may comprise a functional complex in thyroid and orbital tissue.     

Molecular Analysis of Chickpea (Cicer arietinum L) Cultivars Using AFLP and STMS Markers

Fifteen AFLP and eighteen STMS primer pairs were employed to reveal genetic diversity and relationship in twenty-one cultivars of chickpea (Cicer arietinum L). Fifteen AFLP primer pairs generated 1804 amplicons, out of which 1732 amplicons (96%) were polymorphic and 600 amplicons (∼33%) were genotype specific. Eighteen polymorphic STMS primer pairs generated 64 amplicons with an average of 3.55 amplicons per primer pair. Polymorphic information content (PIC) varied from 0.52 to 1.0 for STMS markers. The genetic similarity between cultivars varied from 0.30 to 0.85 for AFLP and 0.22 to 0.83 for STMS markers. Dendrogram constructed after combining both AFLP and STMS markers data with Bootstrap analysis, grouped all the cultivars into four clusters. Association of varietal type and flower colour was observed as cultivars E 100Ymu and Nabin (Both Desi type and pink flower) clustered together in the dendrogram.     

A bacterial haloalkane dehalogenase (<Emphasis Type="Italic">dhlA</Emphasis>) gene as conditional negative selection marker for rice callus cells

The dhlA gene of Xanthobacter autotrophicus encodes dehalogenase that hydrolyzes dihaloalkanes such as 1,2-dichloroethane (DCE) into cytotoxic halogenated alcohol and an inorganic halide. As plants do not contain dehalogenase activity, they grow normally in the presence of DCE. We tested the transgenic expression of the bacterial dhlA gene in rice as a conditional negative selection marker. We developed 24 transgenic callus lines containing dhlA gene driven by rice actin-1 promoter, verified the expression of dhlA by Northern blot analysis, and subjected these transgenic lines to DCE treatment. We found that, while untransformed callus (Nipponbare) was unaffected by the DCE treatment, most of the transformed lines displayed symptoms of toxicity, indicating that dhlA is an effective conditional negative selection marker gene for rice in vitro cultures.