Green Synthesis of Copper Oxide Nanoparticles using Cucumis Sativus (Cucumber) Extracts and their Bio-Physical and Biochemical Characterization for Cosmetic and Dermatologic Applications.

BACKGROUND & OBJECTIVE: Nanoparticles are used in cosmetic and dermatologic products, due to better skin penetration properties. Incorporation of natural products exhibiting medicinal properties in nano-preparations could significantly improve the efficacy of these products and improve the quality of life without the side effects of synthetic formulations. METHODS: We here report the green synthesis of Copper Oxide nanoparticles, using Cucumber extract, and their detailed bio-physical and bio-chemical characterization. RESULTS: These Copper Oxide-Cucumber nanoparticles exhibit significant anti-bacterial and anti-fungal properties, Ultra Violet-radiation protection ability and reactive-oxygen species inhibition properties. Importantly, these nanoparticles do not exhibit significant cellular toxicity and, when incorporated in skin cream, exhibit skin rejuvenating properties. CONCLUSION: Our findings have implications for nanoparticle-based cosmetics and dermatologic applications.

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.

Inherent Immunogenicity or Lack Thereof of Pluripotent Stem Cells: Implications for Cell Replacement Therapy.

Donor-specific induced pluripotent stem cells (iPSCs) offer opportunities for personalized cell replacement therapeutic approaches due to their unlimited self-renewal potential and ability to differentiate into different somatic cells. A significant progress has been made toward generating iPSC lines that are free of integrating viral vectors, development of xeno-free culture conditions, and differentiation of pluripotent stem cells (PSCs) into functional somatic cell lineages. Since donor-specific iPSC lines are genetically identical to the individual, they are expected to be immunologically matched and these iPSC lines and their cellular derivatives are not expected to be immunologically rejected. However, studies in mouse models, utilizing rejection of teratomas as a model, have claimed that syngenic iPSC lines, especially the iPSC lines derived with integrating viral vectors, could be inherently immunogenic. This manuscript reviews current understanding of inherent immunogenicity of PSC lines, especially that of the human iPSC lines and their cellular derivatives, and strategies to overcome it.

Derivation of Human Induced Pluripotent Stem Cell (iPSC) Lines and Mechanism of Pluripotency: Historical Perspective and Recent Advances.

Derivation of human embryonic stem cell (hES) lines in 1998 was not only a major technological breakthrough in the field of regenerative medicine; it also triggered a passionate debate about the ethical issues associated with the utilization of human embryos for derivation of hESC lines. Successful derivation of induced pluripotent stem cell (iPS) lines from human somatic cells with defined reprogramming factors by Shinya Yamanaka`s group in 2007 was another breakthrough that generated enormous excitement and hope for the development of donor-specific personalized cell replacement therapies (CRT) without the ethical dilemma associated with it. As we approach twentieth anniversary of derivation of hESC lines and the tenth anniversary of isolation of donor-specific iPSC lines, this manuscript summarizes the key advances in pluripotent stem cell (PSC) research field that led to derivation of human iPSC lines, different methodologies for derivation iPSC lines and characterization of the mechanism of reprogramming. We will also review progress towards generating donor-specific somatic cell lineages from iPSC lines, especially the functional immune cell lineages, and progress towards advancing these findings to the clinic. Finally, we will discuss the challenges, such as genome instability and inherent immunogenicity of hPSC lines that need to be addressed to develop safe and effective iPSC-based CRT.

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.

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.

Suppression of inducible CD4 regulatory cells by MHC class I-restricted human tumor epitope specific TCR engineered multifunctional CD4 T cells.

Regulatory T cells (Treg) can interfere with the generation and function of anti-tumor immune effectors. Accordingly, ways that could block Treg function would be useful in cancer immunotherapy. We have previously shown that incorporation of CD4+CD25-ve T cells in an in vitro cytolytic T lymphocyte (CTL) generation assay leads to generation of induced regulatory T cells (iTregs), and that these iTreg block the generation of productive CTL response (Chattopadhyay et al., 2006). We here show that human CD4 T cells engineered to express MHC class I-restricted human melanoma associated epitope, MART-127-35, specific T cell receptor (TCR), that can simultaneously exhibit helper as well as cytolytic effector functions (Chhabra et al., 2008, Ray et al., 2010), can interfere with the generation of inducible Treg, block iTreg-mediated suppression, and allow the activation and expansion of MART-127-35 specific CTL responses, in vitro. We also show that mitigation of Treg generation by TCR engineered CD4 T cells is not mediated by a soluble factor and may involve "licensing/conditioning" of the dendritic cells (DC). Our data offer novel insights on the biology of MHC class I restricted TCReng CD4 T cells and have translational implications.

Death receptor-independent activation-induced cell death in human melanoma antigen-specific MHC class I-restricted TCR-engineered CD4 T cells.

Engaging CD4 T cells in antitumor immunity has been quite challenging, especially in an Ag-specific manner, because most human solid tumors usually do not express MHC class II molecules. We have recently shown that human CD4 T cells engineered to express a human melanoma-associated antigenic epitope, MART-127-35, specific MHC class I-restricted transgenic TCR function as polyfunctional effectors that can exhibit a helper as well as cytolytic effector function, in an epitope-specific and MHC class I-restricted manner (Chhabra et al. 2008. J. Immunol. 181: 1063-1070; Ray et al. 2010. Clin. Immunol. 136: 338-347). TCR-engineered (TCReng) CD4 T cells therefore have translational potential, and clinical trials with MHC class I TCReng CD4 T cells are under way. In this study, we show that although TCReng CD4 T cells could be useful in cancer immunotherapy, they are also susceptible to epitope-specific activation-induced cell death (AICD). We also show that the AICD in TCReng CD4 T cells is a death receptor-independent process and that JNK and p53 play critical roles in this process as pharmacological inhibitors targeting JNK activation and p-53-mediated transcription-independent mitochondria-centric death cascade rescued a significant fraction of TCReng CD4 T cells from undergoing AICD without affecting their effector function. Our data offer novel insights toward AICD in TCReng CD4 T cells and identify several potential targets to interfere with this process.

Analyses of T cell-mediated immune response to a human melanoma-associated antigen by the young and the elderly.

Elderly cancer patients are often excluded from immune-based clinical trials and therapies based on the belief that they respond poorly to tumor antigens. Using melanoma as a model and melanoma related Mart-127-35 epitope specific T cell receptor (TCR) engineered T cells as a tool we compared the T cell responses from young and elderly to the Mart-127-35 epitope, ex vivo. We also compared the natural Treg (nTreg) activities and the expression of a number of genes associated with immune response by quantitative real-time reverse Transcription Polymerase Chain Reaction (qRTPCR) in formalin fixed primary melanomas, in situ. We detected a significant difference in CD8(+) T cell response to Flu antigen (influenza matrix peptide Flu MP58-66), but the responses of the two cohorts to melanoma antigen were comparable. nTreg activities in the elderly was significantly compromised. The qPCR analyses of tissues from elderly patients revealed lower levels of Fox-P3 expression but comparable levels of expression of IL-2, IFNγ, TNFα, IL-4, IL-10, IDO, and TGFß. These findings indicate that elderly patients might be capable of responding to tumor antigens, and need not be excluded from immune-based therapies or clinical trials.

TCR-engineered, customized, antitumor T cells for cancer immunotherapy: advantages and limitations.

The clinical outcome of the traditional adoptive cancer immunotherapy approaches involving the administration of donor-derived immune effectors, expanded ex vivo, has not met expectations. This could be attributed, in part, to the lack of sufficient high-avidity antitumor T-cell precursors in most cancer patients, poor immunogenicity of cancer cells, and the technological limitations to generate a sufficiently large number of tumor antigen-specific T cells. In addition, the host immune regulatory mechanisms and immune homeostasis mechanisms, such as activation-induced cell death (AICD), could further limit the clinical efficacy of the adoptively administered antitumor T cells. Since generation of a sufficiently large number of potent antitumor immune effectors for adoptive administration is critical for the clinical success of this approach, recent advances towards generating customized donor-specific antitumor-effector T cells by engrafting human peripheral blood-derived T cells with a tumor-associated antigen-specific transgenic T-cell receptor (TCR) are quite interesting. This manuscript provides a brief overview of the TCR engineering-based cancer immunotherapy approach, its advantages, and the current limitations.

MHC-I-restricted melanoma antigen specific TCR-engineered human CD4+ T cells exhibit multifunctional effector and helper responses, in vitro.

MHC class I-restricted human melanoma epitope MART-1(27-35) specific TCR-engineered CD4+CD25- T cells synthesize Th1 type cytokines and exhibit cytolytic effector function upon cognate stimulation. A detailed characterization of such TCR-engineered CD4+CD25- T cells now reveals that they are multifunctional. For example, they undergo multiple rounds of division, synthesize cytokines (IFN-gamma, TNF-alpha, IL-2, and MIP1ss), lyse target cells, and "help" the expansion of the MART-1(27-35) specific CD8+ T cells when stimulated by the MART-1(27-35) peptide pulsed DC. Multiparametric analyses reveal that a single TCR-engineered CD4+ T cell can perform as many as five different functions. Nearly 100% MART-1(27-35) specific TCR expressing CD4+ T cells can be generated through retroviral vector-based transduction and one round of in vitro stimulation by the peptide pulsed DC. MHC class I-restricted tumor epitope specific TCR transduced CD4+ T cells, therefore, could be useful in immunotherapeutic strategies for melanoma or other human malignancies.

Mitochondria-centric activation induced cell death of cytolytic T lymphocytes and its implications for cancer immunotherapy.

Premature death of the adoptively transferred cytolytic T lymphocytes (CTL) by means of activation induced cell death (AICD) represents one of the major constraints in devising an effective anti-cancer immune intervention strategy. Understanding the mechanism of AICD is, therefore, critical for developing methods to interfere with this death process. Although the existing paradigm on AICD centers around the initiation of the cascade of events originating from the engagement of death receptors leading to the activation of effector caspases and eventually resulting in cell death, recent findings have questioned the universal role of caspases as the cell death executioners. We here review our current understanding of the contribution of caspase-dependent and caspase-independent death executioners in AICD of T cells. We will also discuss the involvement of mitochondria-centric death pathway in AICD of human tumor associated antigen-specific primary CTL and its implications in cancer immunotherapy.

Obstacles to and opportunities for more effective peptide-based therapeutic immunization in human melanoma.

Melanoma cells can play a number of tricks to evade the host immune response. They can make themselves invisible to cells of the immune system poised to attack them, elaborate molecules that are frankly immunosuppressive, and can create a microenvironment that is hostile to cells of the immune system. Efforts are underway to institute measures that would make tumor cells more susceptible to immune attack, but these efforts have not been all that successful so far. This contribution reviews the history and the rationale of cancer vaccines, the major obstacles to peptide-based immunization, and a discussion on how to surmount them. Also included are the roles played by peripheral tolerance, low-affinity T-cell receptors, T-cell ignorance, activation-induced cell death, exhaustion of T cells and regulation of the immune response, helpless cytolytic T lymphocytes, and evasion by tumor cells.

MHC class I TCR engineered anti-tumor CD4 T cells: implications for cancer immunotherapy.

T cell immunity is critical for a protective immune response against cancers. Traditionally, this function has been ascribed to CD8 T lymphocytes with cytotoxic activity, which are restricted by MHC class I molecules. The lack of direct cytolytic effector function on part of CD4 T cells, which are MHC class II restricted, coupled with the MHC class II negative nature of most human cancers have been the main reasons for CD8 centered cancer immunotherapy approaches, so far. However, recent findings showing that CD4 T cells play an essential role towards the generation of a productive CD8 response and that the CD4 T cells can also play a direct role in anti-tumor immunity have resulted in growing enthusiasm towards engaging CD4 T cells in cancer immunotherapy. We here discuss the current approaches used for immune based cancer therapy, role of natural MHC class II-restricted CD4 T cells in tumor immunity, factors limiting the engagement of natural CD4 T cells in cancer immunotherapy protocols alongside CD8 T cells, and recent advances in TCR engineering approach to address these limitations. We will also discuss the significance of the MHC class I directed anti-tumor CD4 T cells in tumor immunity.

CD4+CD25- T cells transduced to express MHC class I-restricted epitope-specific TCR synthesize Th1 cytokines and exhibit MHC class I-restricted cytolytic effector function in a human melanoma model.

Cytolytic T cell-centric active specific and adoptive immunotherapeutic approaches might benefit from the simultaneous engagement of CD4(+) T cells. Considering the difficulties in simultaneously engaging CD4(+) and CD8(+) T cells in tumor immunotherapy, especially in an Ag-specific manner, redirecting CD4(+) T cells to MHC class I-restricted epitopes through engineered expression of MHC class I-restricted epitope-specific TCRs in CD4(+) T cells has emerged as a strategic consideration. Such TCR-engineered CD4(+) T cells have been shown to be capable of synthesizing cytokines as well as lysing target cells. We have conducted a critical examination of functional characteristics of CD4(+) T cells engineered to express the alpha- and beta-chains of a high functional avidity TCR specific for the melanoma epitope, MART-1(27-35), as a prototypic human tumor Ag system. We found that unpolarized CD4(+)CD25(-) T cells engineered to express the MART-1(27-35) TCR selectively synthesize Th1 cytokines and exhibit a potent Ag-specific lytic granule exocytosis-mediated cytolytic effector function of comparable efficacy to that of CD8(+) CTL. Such TCR engineered CD4(+) T cells, therefore, might be useful in clinical immunotherapy.

Presence of low dose of fludarabine in cultures blocks regulatory T cell expansion and maintains tumor-specific cytotoxic T lymphocyte activity generated with peripheral blood lymphocytes.

BACKGROUND: For tumor vaccine-based immunotherapy of cancer, the expansion of tumor antigen-specific cytotoxic T lymphocytes (CTL) in the patients by blocking induced regulatory T (Treg) cells is the most important objective now. Fludarabine (FLU), a known anticancer drug, has been shown to downregulate Treg cells in vivo in chronic leukemia patients. Melanoma tumor antigen Mart-1(27-35)-specific CD8+ CTLs generated in vitrowith total peripheral blood lymphocytes (PBL) lose their activity within 14-21 days with concomitant expansion of Treg cells. When CD4+ cells are removed from PBL and CTL are generated with purified CD8+ cells, the CTL survive and maintain their activity for a significantly longer period. METHODS: We used a low dose of FLU in the cultures in Mart-1-specific CTL generation assays with total PBL. Blood samples were taken from HLA-A2-positive melanoma patients and normal donors. Autologous matured dendritic cells pulsed with Mart-1(27-35) peptide were used to generate CTL responses using purified CD8+ cells or total PBL. RESULTS: The presence of FLU in the cultures with PBL helped to generate a significantly higher number of antigen-specific CTLs as detected by Mart-1 HLA-A2 tetramer staining. Specificity of such CTLs was determined by IFN-gamma secretion or by cytotoxicity against the target cells bearing the specific antigen. The presence of FLU stopped the expansion of IL-10-producing CD4+ Treg cells in the cultures with PBL. Analyses of expanded CD4+ cells isolated from PBL in vitro cocultures with FLU showed a Th1 type of function. Those cells secreted higher amounts of IFN-gamma and very low levels of IL-10, or no IL-10 at all, upon restimulation. CONCLUSION: The observations of the study are as important for adaptive immunotherapy of cancer as they are for vaccine-based approaches.

Silencing of endogenous IL-10 in human dendritic cells leads to the generation of an improved CTL response against human melanoma associated antigenic epitope, MART-1 27-35.

Dendritic cells (DC) present antigenic epitopes to and activate T cells. They also polarize the ensuing T cell response to Th1 or Th2 type response, depending on their cytokine production profile. For example, IL-12 producing DC generate Th1 type T cell response whereas IL-10 producing DC is usually tolerogenic. Different strategies--such as the use of cytokines and anti-cytokine antibodies, dominant negative forms of protein, anti-sense RNA etc.--have been employed to influence the cytokine synthetic profile of DC as well as to make DC more immunogenic. Utilizing GFP expressing recombinant adenoviruses in association with lipid-mediated transfection of siRNA, we have silenced the endogenous IL-10 gene in DC. We show that IL-10 gene silenced DC produces more IL-12 and also generates a better cytolytic T cell response against the human melanoma associated epitope, MART-1(27-35), in vitro. We also show that the GFP expressing adenoviral vector can be used to optimize the parameters for siRNA delivery in primary cells and show that RNA interference methodology can efficiently knock down virus encoded genes transcribed at very high multiplicity of infection in DC.

Inhibition of c-Jun N-terminal kinase rescues influenza epitope-specific human cytolytic T lymphocytes from activation-induced cell death.

Cytolytic T lymphocytes (CTL) play an important role in defense against viral infections. Following clonal expansion and effector functions, a vast majority of the antigen-specific CTL undergoes programmed cell death to maintain homeostasis. We have shown earlier that melanoma epitope-specific CTL are quite sensitive to activation-induced cell death (AICD) even on the secondary encounter of the antigen. Excessive sensitivity of viral antigen-specific CTL to AICD, however, would be counterproductive. It might be argued that although CTL for a "self" epitope might be more prone to AICD for maintaining self-tolerance, viral antigen-specific CTL are likely to be less sensitive to AICD. We show here that influenza matrix protein-derived MP(58-66) epitope-specific CTL, activated in vitro and bearing a memory phenotype, are just as sensitive to AICD. The AICD in these CTL is not blocked by the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethylketone or by soluble Ig-Fc chimeras of the death receptors [Fas, TNF receptor (TNF-R), TRAIL-RI, TRAIL-RII]. However, the MP(58-66)-specific CTL can be rescued from AICD by the c-jun-N-terminal kinase (JNK) inhibitor SP600125. These results have implications for immunotherapeutic intervention in rescuing viral epitope-specific CTL from AICD.

Activation-induced cell death of human melanoma specific cytotoxic T lymphocytes is mediated by apoptosis-inducing factor.

Activation-induced cell death (AICD) of T cells can be an impediment towards achieving a robust and long-lived cytolytic T lymphocyte (CTL) response from active specific immunization or after adoptive cell transfer in cancer immunotherapy. The mechanism of AICD in primary CTL, however, remains poorly understood. It is widely believed that AICD is driven by signals from death receptors (DR) and that the cell death takes place in a caspase-dependent manner, although it has been shown that AICD of T cells can be induced by internal triggers and that death takes place in a caspase-independent manner. We show here that AICD in human melanoma epitope-specific primary CTL involves selective mitochondrio-nuclear translocation of the apoptosis inducing factor (AIF) without cytochrome c release, caspase-3 and caspase-8 activation, and results from large-scale DNA fragmentation. The c-jun-N terminal kinase (JNK) inhibitor, SP600125, blocks the mitochondrio-nuclear translocation of AIF and prevents AICD in these CTL. These findings suggest that the AICD in human melanoma epitope specific primary CTL is mediated by mitochondrial AIF release and JNK is involved in regulation of this death process.

Effect of CD4+CD25+ and CD4+CD25- T regulatory cells on the generation of cytolytic T cell response to a self but human tumor-associated epitope in vitro.

CD4+ T cells naturally expressing CD25 molecules (natural T regulatory cells (Tregs)) have a role in maintaining self tolerance and in regulating responses to infectious agents, transplantation Ags, and tumor Ags. CD4+ Tregs induced from CD4+CD25- precursors (induced Tregs) also regulate immune responses in the periphery. However, which of these Tregs is a major impediment in generating antitumor CTL responses is not clear. We show that although the CD4+CD25+ subsets isolated from peripheral blood-derived lymphocytes do suppress the proliferation of CD4+CD25- effector T cells, they do not suppress the activation and expansion of the self but melanoma-associated, melanoma Ag-reactive T cell 1 (MART-1)27-35-specific CD8+ T cells stimulated by the respective peptide-loaded matured dendritic cells in vitro. The CD4+CD25- counterparts, in contrast, lead to the generation of CD25+ glucocorticoid-inducible TNFR+-Forkhead/winged helix transcription factor+ populations and efficiently suppress the activation and expansion of the MART-127-35 epitope-specific CTLs. Our data suggest that when CTL precursors are optimally stimulated, natural Tregs are not a formidable constraint toward generating a robust antitumor CTL response, but induced Tregs could be.