As major urothelial differentiation products, uroplakins are targeted to the apical surface of umbrella cells. Via the sequential actions of Rabs 11, 8, and 27b and their effectors, uroplakin vesicles are transported to a subapical zone above a K20 network and fuse, via a SNARE-mediated and MAL-facilitated step, with the urothelial apical membrane.

Polytope approach of genetic immunization is a promising strategy for the prevention of infectious disease as it is capable of generating effective cell mediated immunity by delivering the T cell epitopes assembled in series. Leishmaniasis is a significant world wide health problem for which no vaccine exists. In this study we have compared immunogenicity and efficacy of three types of DNA vaccines: single antigen Gp63 (Gp63/pcDNA), polytope (Poly/pcDNA) and Polytope fused with hsp70 (Poly/hsp/pcDNA) against visceral leishmaniasis in susceptible BALB/c mice. Mice vaccinated with these plasmids generated strong Th1 immune response as seen by dominating IFN-γ over IL-10 cytokine. Interestingly, cytotoxic responses generated by polytope DNA plasmid fused with hsp70 of Leishmania donovani were significantly higher when compared to polytope and single antigen Gp63 vaccine. Challenge studies revealed that the parasite load in liver and spleen was significantly lower with Poly/hsp/pcDNA vaccination compared to other vaccines. Therefore, our study indicates that polytope DNA vaccine is a feasible, practical and effective approach for visceral leishmaniasis.

Clinical reports indicate that some infected individuals control HIV-1 replication through undefined mechanisms. Our group reported that human protein named X-DING-CD4 holds a potent antiviral activity blocking transcription of HIV-1 LTR through inhibition of NF-κB/DNA binding. Based on observations that transformed HIV-1 resistant CD4+T cells produce higher levels of soluble X-DING-CD4 protein upon their exposure to virus, we hypothesized that resistance to HIV-1 in these cells might be regulated through function of X-DING-CD4 gene. Real time PCR evaluations of X-DING-CD4 mRNA expression confirmed our hypothesis; HIV-1 exposure caused rapid up-regulation of X-DING-CD4 mRNA in resistant but not susceptible cells; and the burst of X-DING-CD4 mRNA expression correlated with restriction of HIV-1 transcription. Subsequently, we examined the activity of X-DING-CD4 gene in monocytes and macrophages from (n=13) HIV-negative donors. The assessment of HIV-1 gag mRNA showed that majority of cells were permissive to virus replication; however, macrophages from four donors were refractory to HIV-1 infection. In response to virus these cells up-regulated the X-DING-CD4 gene expression by 2 to 1000-fold. This data provide evidence that X-DING-CD4 gene contributes to early cellular protection from HIV infection in some individuals and this protection depends solely on the unique genetic regulation of the host.

Independent research groups reported that DING protein homologues isolated from bacterial, plant and human cells demonstrate the anti-HIV-1 activity. This might indicate that diverse organisms utilize a DING-mediated broad-range protective innate immunity response to pathogen invasion, and that this mechanism is effective also against HIV-1. We performed structural analyses and evaluated the anti-HIV-1 activity for four DING protein homologues isolated from different species. Our data show that bacterial PfluDING, plant p38SJ (pDING), human phosphate binding protein (HPBP) and human extracellular DING from CD4 T cells (X-DING-CD4) share high degrees of structure and sequence homology. According to earlier reports on the anti-HIV-1 activity of pDING and X-DING-CD4, other members of this protein family from bacteria and humans were able to block transcription of HIV-1 and replication of virus in cell based assays. The efficacy studies for DING-mediated HIV-1 LTR and HIV-1 replication blocking activity showed that the LTR transcription inhibitory concentration 50 (IC50) values ranged from 0.052–0.449 ng/ml; and the HIV-1 replication IC50 values ranged from 0.075–0.311 ng/ml. Treatment of cells with DING protein alters the interaction between p65-NF-κB and HIV-1 LTR. Our data suggest that DING proteins may be part of an innate immunity defense against pathogen invasion; the conserved structure and activity makes them appealing candidates for development of a novel therapeutics targeting HIV-1 transcription.

HIV-1 restriction factor, the X-DING-CD4 protein blocks transcription of HIV-1 LTR and pathogen induced proinflammatory response and the increased activity of its gene associates with cellular resistance to virus; therefore HIV-1 elite controllers (ECs), who innately restrict viremia, should have significantly higher X-DING-CD4 and reduced proinflammatory mRNA activity than viremic or uninfected individuals. We posited that depending on cell stimulating factor the expression of X-DING-CD4 mRNA in ECs might be autonomous or contingent on interferon signaling. We compared expression of X-DING-CD4, IFN-α and IL-8 mRNA in naïve, PHA- or HIV-1 exposed PBMCs from ECs, HIV progressors and negative controls. We tested correlation between X-DING-CD4 and IFN-α mRNA expression and sensitivity of X-DING-CD4 gene to IFN-α regulation. We also evaluated interactions between innate and proinflammatory genes under different status of T cell activation. We found that X-DING-CD4 and IFN-α mRNAs were significantly up-regulated in ECs, and their expression correlated when cells were stimulated with mitogen but not HIV-1. The X-DING-CD4 gene was more sensitive to HIV-1 than rIFN-α stimulation. The ECs had significantly lower levels of IL-8 mRNA when PBMCs were exposed to the exogenous HIV-1. The two-way ANOVA showed that control of HIV-1 and virus-induced proinflammatory response by ECs stems from interactions between expression of innate immunity and proinflammatory genes, state of cell stimulation and status of virus control. We provide evidence that interaction of multiple host innate immune responses rather than single mechanism regulates unique capacity of ECs to restrict HIV-1.

Onsets of bacterial infections devastate the compromised immune system in AIDS patients. Damaged gut mucosa permits dissemination of bacterial toxins into deeper layers and hyper-activation of the immune system. We reported previously that the un-fractionated supernatants of HIV-resistant CD4+ T cells impeded the NF-κB/DNA binding in macrophages induced by either HIV-1 or lipopolysaccharide (LPS). The active component of this soluble material was identified as X-DING-CD4 (extracellular DING from CD4 T cells). We hypothesized that the anti-inflammatory effect of X-DING-CD4 protein might extend to the non-immune cells, for example endothelial cells, undergoing persistent endotoxin stimulation in the course of the advanced HIV disease. To test this proposition we evaluated the efficiency of NF-κB and Ap-1 binding to IL-8 promoter in LPS activated endothelial cells and control human macrophages exposed to native X-DING-CD4 protein. We found deficiency of NF-κB-but not AP-1-DNA binding in the systems where cells were treated with native soluble X-DING-CD4 protein. The X-DING-CD4 mediated inhibition of IL-8 promoter resulted also in reduction of the soluble IL-8 protein in endothelial cells and human macrophages infected with a subset of enteric bacteria frequently causing diarrhea in a progressive HIV disease. Bacterial endotoxin did not induce the endogenous X-DING-CD4 mRNA activity in human macrophages and transformed CD4+T cells, indicating that the reduction of LPS mediated IL-8 promoter activation was not related to de novo X-DING-CD4 protein synthesis but depended on function of the exogenous X-DING-CD4 protein. This study provides evidence that X-DING-CD4 protein might be developed as a novel biotherapeutic to control the LPS mediated inflammation in the advanced HIV disease.

No abstract

X-DING-CD4 is a novel phosphatase mediating antiviral responses to HIV-1 infection. This protein is constitutively expressed and secreted by HIV-1 resistant CD4+ T cells and its mRNA transcription is up-regulated in PBMCs from HIV-1 elite controllers. The secreted/soluble X-DING-CD4 protein form is of particular importance because it blocks virus transcription when added to HIV-1 susceptible cells. Our research aimed to determine the contribution of this factor to induction of antiviral response in target cells. We found that soluble X-DING-CD4 enters cells by endocytosis and influx of this protein induced transcription of IFN-α and endogenous X-DING-CD4 mRNA in transformed CD4+ T cells and primary macrophages. Treatment of HIV-1 susceptible cells with exogenous X-DING-CD4 caused depletion of phosphorylated p50 and p65 NF-κB subunits and a significant reduction in p50/p65 NF-κB binding to the HIV-1 LTR. Together, these findings indicate a novel antiviral mechanism mediated by the influx of soluble X-DING-CD4, its signaling to promote self-amplification and functional duality as an endogenous innate immunity effector and exogenous factor regulating gene expression in bystander cells.