Human Dendritic Cell-Derived Induced Pluripotent Stem Cell Lines Are Not Immunogenic.

Donor-specific induced pluripotent stem cells (iPSC) can be used to generate desired cell types, including naive immune effectors, for the treatment of different diseases. However, a greater understanding of the inherent immunogenicity of human iPSC and their cellular derivatives is needed for the development of safe and effective cell-replacement therapies, given that studies in mouse models claimed that the syngenic mouse iPSC lines can be immunogenic. We report the characterization of the innate and adaptive immune mechanisms in human iPSC lines derived from peripheral blood-derived dendritic cells using a nonintegrating RNA virus, Sendai virus. We show that these iPSC lines express mRNA of TLR molecules and the Ag-presentation pathway intermediates; however, these mRNA are not translated into functional proteins, and these iPSC lines do not induce TLR-mediated inflammatory cytokine responses or inflammasome activation. We also show that these iPSC lines do not activate T cells in an allogenic MLR; however, they express low levels of MHC class I molecules that can efficiently acquire antigenic peptides from their microenvironment and present them to Ag-specific T cells. In addition, we show that these iPSC lines can be efficiently differentiated into hematopoietic stem cell precursors, as well as APC, under appropriate culture conditions. Taken together, our data show that the dedifferentiation of human dendritic cells effectively shuts down their immunogenic pathways and implicates transcriptional and posttranscriptional mechanisms in this process.

Vitamin D modulates human macrophage response to Mycobacterium tuberculosis DNA.

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that infects macrophages where it avoids elimination by interfering with host defense mechanisms, including phago-lysosome fusion. Endosomal Toll-like receptors (TLRs) generate Type I Interferons (IFNs), which are associated with active tuberculosis (TB). We aimed to explore if DNA from different Mtb lineages lead to differences in the inflammatory response of human monocytic/macrophage cells. THP-1 cells which express two inducible reporter constructs for interferons (IFNs) as well as for NF-κB, were stimulated via endosomal delivery of Mtb DNA as a nanocomplex with PEI. DNA from different Mtb phylogenetic lineages elicited differential inflammatory responses in human macrophages. An initial relatively weak IRF-mediated response to DNA from HN878 and H37Rv increased if the cells were pre-treated with Vitamin D (Vit D) for 72 h. RNAseq of THP-1 under different transformation conditions showed that pre-treatment with Vit D upregulated several TLR9 variants, as well as genes involved in inflammatory immune response to infection, immune cell activation, Type I IFN regulation, and regulation of inflammation. Vit D appears to be important in increasing low IRF responses to DNA from certain lineages of Mtb. Variations in the IRF-mediated response to DNA derived from different Mtb genotypes are potentially important in the pathogenesis of tuberculosis since Type I IFN responses are associated with active disease. The role of Vit D in these responses could also translate into future therapeutic approaches.

Activation induced cell death (AICD) of human melanoma antigen-specific TCR engineered CD8 T cells involves JNK, Bim and p53.

OBJECTIVES: Adoptive cancer immunotherapy (ACT) with transgenic T cell receptor (TCR) engineered (TCReng) anti-tumor T cells has produced encouraging results, however, efficacy of these approaches need improvement. Since premature activation induced cell death (AICD) of adoptively administered T cells could be a major impediment, we examined the mechanism(s) underlying AICD in TCReng CD8+ cytolytic T lymphocytes (CTL). METHODS: AICD in human tumor antigen-specific MHC class I restricted TCR engineered CD8+ CTL was induced by exposing them to cognate peptide epitope. RESULTS: We show that TCReng CD8+ human primary CTL undergo AICD even upon encountering their cognate peptide epitope for the very first time. AICD in TCReng CTL is a death-receptor-independent, JNK activation-driven intrinsic processes, in which p53-mediated mitochondria-centric, non-transcription-dependent pathway plays an essential role. Activated JNK modulates mitochondrial membrane integrity in CTL undergoing AICD by directly interacting with Bcl family protein, Bim, and the mitochondrial membrane pore complex, voltage dependent anion channel (VDAC), leading to the release of caspase-independent death executioner, apoptosis inducing factor (AIF), accumulation of single strand DNA breaks and eventually to cell death. CONCLUSIONS: Our findings offer opportunities to interfere with AICD in TCReng CD8+ anti-tumor CTL for sustaining them longer for producing better clinical outcomes.

DNA vaccine for rabies: relevance of the trans-membrane domain of the glycoprotein in generating an antibody response.

Various studies have demonstrated the potential of immunization with DNA vaccines encoding the rabies virus glycoprotein (RV-G) to elicit humoral responses. In the present study, we have designed four constructs using a VR1020 vector, wherein the RV-G ectodomain has been cloned without the signal sequence (SS) and the trans-membrane domain (TD) (rGVR), without the SS but with the TD (rGVRt), with the SS but without the TD (rGVRs) and with the SS and the TD (rGVRst), under the control of a cytomegalovirus (CMV) promoter, and downstream of the tissue plasminogen activator (TPA) signal sequence. In addition, RV-G has been expressed as a His6 tag fusion protein, both in Escherichia coli as well as in baculovirus expression systems. Using a prime-boost strategy, BALB/cJ mice administered with the rGVRt construct either in saline (intramuscularly) or adsorbed onto gold microcarriers (delivered intradermally by gene gun) generated the highest rabies virus neutralizing antibody (RVNA) titers. Inclusion of the SS, in addition to the TD (rGVRst), led to a significant decrease in RVNA titers, compared to the rGVRt construct. The DNA vaccine construct lacking both the SS and the TD domain and the vaccine having only the SS generated lower antibody responses, compared to the rGVRt construct. After priming with DNA vaccine, boosting with both E. coli- as well as baculovirus-expressed rRV-G led to an increase in the RVNA titers. The present results demonstrate that a DNA vaccine encoding the full-length sequence of the ectodomain plus TD of the mature native RV-G is capable of expressing an 'ideal' immunogen to produce RVNA titers.

Update on zona pellucida glycoproteins based contraceptive vaccine.

Zona pellucida (ZP) glycoproteins, due to their critical role in mammalian fertilization, have been proposed as candidate immunogens for development of a contraceptive vaccine. Active immunization studies in a variety of animal species, employing either native or recombinant zona proteins, has established their contraceptive potential. Hence, ZP glycoprotein-based contraceptive vaccines have a very good potential for controlling wild life population. To make it a realistic proposition, additional research inputs are required to develop new potent adjuvants and novel practical strategies for vaccine delivery. The observed ovarian dysfunction, often associated with immunization by ZP glycoproteins, is one of the major obstacles for their application in the control of human population. Ongoing studies to delineate epitopes of ZP glycoproteins that will generate an immune response capable of inhibiting fertility without any untoward effects on ovarian functions will help in determining their feasibility for human use.

Baculovirus expressed C-terminal fragment of bonnet monkey (Macaca radiata) zona pellucida glycoprotein-3 inhibits ZP3-mediated induction of acrosomal exocytosis.

Zona pellucida glycoprotein-3 (ZP3) has been postulated as the primary sperm receptor in various mammalian species including bonnet monkey (Macaca radiata). However, information on the domain responsible for its binding to spermatozoa is inadequate. In the present study, bonnet monkey ZP3 (bmZP3), corresponding to amino acid (aa) residues 223-348 [bmZP3(223-348)] has been cloned and expressed using baculovirus expression system. SDS-PAGE and Western blot analysis of the purified renatured recombinant protein revealed it as a closely spaced doublet of approximately 25 kDa. Lectin-binding studies documented the presence of both O- as well as N-linked glycans. The biotinylated r-bmZP3(223-348) binds to the acrosomal region of the capacitated spermatozoa but fails to bind to the acrosome-reacted spermatozoa as investigated by immunofluorescence studies. In ELISA, nonbiotinylated r-bmZP3(223-348) and baculovirus expressed r-bmZP3, devoid of signal sequence and transmembrane-like domain [r-bmZP3(23-348)] competitively inhibit its binding to the capacitated spermatozoa. Interestingly, binding of biotinylated r-bmZP3(23-348) to the capacitated sperm is also inhibited by nonbiotinylated r-bmZP3(223-348). In contrast to r-bmZP3(23-348), r-bmZP3(223-348) failed to induce acrosomal exocytosis in the capacitated sperm. Interestingly, it competitively inhibits the acrosomal exocytosis induced by r-bmZP3(23-348). These studies, for the first time, identify a domain of ZP3 capable of binding to capacitated spermatozoa and inhibiting ZP3-mediated induction of acrosomal exocytosis furthering our understanding of mammalian fertilization.

Characterization of immune response in mice to plasmid DNA encoding dog zona pellucida glycoprotein-3.

To investigate the immunogenicity of plasmid DNA encoding dog zona pellucida glycoprotein-3 (dZP3), the cDNA corresponding to dZP3, was cloned in mammalian expression vector VR1020 downstream of tissue plasminogen activator signal sequence under cytomegalovirus promoter (VRdZP3). In vitro transfection of COS-1 mammalian cells with VRdZP3 plasmid DNA led to its cytosolic expression. The expressed dZP3 has an apparent molecular weight of 45kDa as compared to calculated molecular weight of 38.4 kDa, suggesting possible glycosylation. Immunization of male BALB/cJ mice with VRdZP3 plasmid DNA in saline, by electroporation or adsorbed onto gold microcarriers (delivered by gene gun) generated antibody response against Escherichia coli expressed recombinant dZP3 (r-dZP3). Administration of r-dZP3 in saline following immunization with plasmid DNA led to boosting of the antibody response. Although mice immunized with gene gun exhibited highest antibody titres, the differences in the antibody titres seen by the three modes of plasmid DNA delivery were not statistically significant (P>0.05). Interestingly, female mice immunized with VRdZP3 plasmid DNA using gene gun also generated antibodies against r-dZP3. A dominant IgG1 isotype response was observed in mice immunized with VRdZP3 plasmid DNA using gene gun as compared to a mixed IgG1-IgG2a isotype response when delivered in saline or by electroporation. Immunization with VRdZP3 plasmid DNA also generated cell mediated immune response. The antibodies generated by VRdZP3 plasmid DNA recognized dog native zona pellucida. These studies for the first time, demonstrate the feasibility of generating an immune response to ZP3 by DNA vaccine and that the antibodies thus generated recognize native zona pellucida.

Impact of anemia on hospitalization time, charges, and mortality in patients with heart failure.

OBJECTIVES: Although anemia is known to influence clinical outcomes in heart failure (HF) patients, little is known about its impact on economic outcomes. A retrospective analysis was performed to determine the impact of hemoglobin (Hb) level on hospital length of stay (LOS), total charges, and hospital mortality in HF patients. METHODS: Claims data were drawn from 21 teaching and nonteaching hospitals for patients hospitalized between October 1, 2000 and September 30, 2001. The impact of Hb on LOS, charges, and hospital mortality was determined using multivariate analyses. Two-stage least squares regression methods were used to assess the potential endogeneity of the economic outcomes (LOS and total charges) and Hb level. RESULTS: Of the 8569 patients in the analysis, 40.2% had Hb < 12 g/dl and 73.8% were > or = 70 years of age. Hemoglobin had significant independent effects on all three outcomes. A 1 g/dl increase in Hb was associated with a 5.1% reduction in LOS (P < 0.001), a 4.3% decrease in charges (P < 0.001), and an 8.7% reduction in mortality risk (P < 0.001). The impact of Hb on all outcomes was greatest in younger HF patients. CONCLUSIONS: This analysis demonstrates that higher Hb is associated with reductions in LOS, charges, and mortality in hospitalized HF patients. Further clinical studies are necessary to validate the cost effectiveness of pharmacologic intervention in anemic HF patients and its impact on patient care.