Expression of a soluble IL-10 receptor enhances the therapeutic effects of a papillomavirus-associated antitumor vaccine in a murine model.

The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

Expression of bacterial virulence factors and cytokines during in vitro macrophage infection by enteroinvasive Escherichia coli and Shigella flexneri: a comparative study.

Enteroinvasive Escherichia coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and biochemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.

A Chemiresistor Sensor Based on Azo-Polymer and Graphene for Real-Time Monitoring of Mitochondrial Oxygen Consumption.

In the present study, a chemiresistor sensor based on a poly(Bismarck Brown Y)-reduced graphene oxide nanocomposite was developed to analyze the respiratory capacity of the constituent complexes of the electron transport chain. The sensorial platform was characterized using electrochemical impedance spectroscopy, and oxygen detection was accomplished by measuring the resistive properties of the sensor at fixed AC frequency. The impedance decreased significantly in response to small variations of the O2 concentrations tested up to saturation of the electrolyte solution with molecular oxygen. The resistive response of the sensor at 0.1 Hz was linear over the oxygen concentration range from 1.17 × 10-5 mol L-1 to 1.02 × 10-3 mol L-1, with a detection limit of 3.60 × 10-7 mol L-1. Using the new O2 sensing platform, we monitored gradients in static cultures of adherent cells exposed to graded oxygen both at rest and upon metabolic stimulation. Under high dissolved oxygen conditions, the respiration of resting cells dictated that local O2 was moderately reduced, while cell metabolic stimulation triggered a major redistribution of O2. The usefulness of the developed sensor was demonstrated by continuous monitoring of mitochondrial oxygen consumption in various biologic applications.

The Combined Use of Melatonin and an Indoleamine 2,3-Dioxygenase-1 Inhibitor Enhances Vaccine-Induced Protective Cellular Immunity to HPV16-Associated Tumors.

Immunotherapy has become an important ally in the fight against distinct types of cancer. However, the metabolic plasticity of the tumor environment frequently influences the efficacy of therapeutic procedures, including those based on immunological tools. In this scenario, immunometabolic adjuvants arise as an alternative toward the development of more efficient cancer therapies. Here we demonstrated that the combination of melatonin, a neuroimmunomodulator molecule, and an indoleamine 2,3-dioxygenase (IDO) inhibitor (1-methyl-DL-tryptophan, DL-1MT) improves the efficacy of an immunotherapy (gDE7) targeting human papillomavirus (HPV)-associated tumors. Melatonin or IDO inhibitors (D-1MT and DL-1MT) directly reduced proliferation, migration, adhesion and viability of a tumor cell line (TC-1), capable to express the HPV-16 E6 and E7 oncoproteins, but could not confer in vivo antitumor protection effects. Nonetheless, combination of gDE7 with melatonin or D-1MT or DL-1MT enhanced the antitumor protective immunity of gDE7-based vaccine in mice. Notably, expression of IDO1 in stromal cells and/or immune cells, but not in tumor cells, inhibited the antitumor effects of the gDE7, as demonstrated in IDO1-deficient mice. Finally, co-administration of gDE7, melatonin and DL-1MT further improved the protective antitumor effects and the numbers of circulating E7-specific CD8+ T cells in mice previously transplanted with TC-1 cells. The unprecedented combination of melatonin and IDO inhibitors, as immunometabolic adjuvants, thus, represents a new and promising alternative for improving the efficacy of immunotherapeutic treatments of HPV-associated tumors.

The expanding roles of 1-methyl-tryptophan (1-MT): in addition to inhibiting kynurenine production, 1-MT activates the synthesis of melatonin in skin cells.

Indoleamine 2,3-dioxygenase 1 (IDO1), the rate-limiting enzyme of tryptophan catabolism, has been strongly associated with the progression of malignancy and poor survival in melanoma patients. As a result, IDO1 is a leading target for interventions aimed at restoring melanoma immune surveillance. Here, in a scenario involving the tryptophan catabolism, we report that melatonin biosynthesis is driven by 1-methyl-tryptophan (1-MT), a competitive inhibitor of IDO1, in human fibroblasts, melanocytes and melanoma cells. In addition to melatonin biosynthesis, 1-MT induced the expression of tryptophan hydroxylase, arylalkylamine-N-acetyltransferase and hydroxyindole O-methyltransferase mRNA in fibroblasts and melanocytes. We observed a great variability in the levels of IDO1 mRNA expression and kynurenine release between skin cells and melanoma cell lines in response to interferon-γ, a classical IDO1 inducer. In this setting, melatonin was shown to downregulate kynurenine production. Furthermore, in a condition of low basal activity of IDO1, it was observed that 1-MT, as well melatonin, inhibited the proliferation of human melanoma cells. Taken together, our results suggest that 1-MT may serve as more than just a tool to disrupt tumor immune escape (via the inhibition of IDO1) because it was shown to act directly on the proliferation of human melanoma cells and induce melatonin biosynthesis in the tumor milieu. Moreover, 1-MT-mediated inhibition of IDO occurs in normal skin and melanoma cells, which addresses the possibility that all cells in the skin microenvironment can be targeted by 1-MT. Our findings provide innovative approaches into understanding tumor therapy related to the control of tryptophan metabolism by 1-MT.