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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.

Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation., GRAPHICAL ABSTRACT

The “Adopt a Bacterium” project is based on the use of social network as a tool in Microbiology undergraduate education, improving student learning and encouraging students to participate in collaborative learning. The approach involves active participation of both students and teachers, emphasizing knowledge exchange, based on widely used social media. Students were organized in groups and asked to adopt a specific bacterial genus and, subsequently, submit posts about “adopted genus”. The formative assessment is based on posting information on Facebook®, and the summative assessment involves presentation of seminars about the adopted theme. To evaluate the project, students filled out three anonymous and voluntary surveys. Most of the students enjoyed the activities and positively evaluated the experience. A large amount of students declared a change in their attitude towards the way they processed information, especially regarding the use of scientific sources. Finally, we evaluated knowledge retention six months after the end of the course and students were able to recall relevant Microbiology concepts. Our results suggest that the “Adopt a Bacterium” project represents a useful strategy in Microbiology learning and may be applied to other academic fields.

Considering the great lethality and sequels caused by meningitis, rapid diagnosis and prompt treatment initiation have a great impact on patient outcome. Here, we developed a multiplex-PCR for simultaneous detection of the four most prevalent bacterial pathogens directly in CSF samples. The multiplex-PCR was designed to detect the following genes: fbsA (Streptococcus agalactiae), lytA (Streptococcus pneumoniae), crtA (Neisseria meningitidis), p6 (Haemophilus influenzae), and 16S rRNA (any bacterial agent). The multiplex-PCR showed a DNA detection limit of 1 pg/μL. Among 447 CSF samples tested, 40 were multiplex-PCR positive, in which 27 and 13 had positive and negative bacterial culture, respectively. Our multiplex-PCR is fast, reliable, and easily implementable into a laboratory routine for bacterial meningitis confirmation, especially for patients who previously started antimicrobial therapy. Our molecular approach can substantially improve clinical diagnosis and epidemiological measures of meningitis disease burden.

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.

The activity of the enzyme indoleamine 2,3 dioxygenase-1 (IDO) has been associated with the maintenance of the immunosuppressive microenvironment in many types of cancers. IDO catalyzes the degradation of tryptophan via the kynurenine pathway, which impacts on the effector activity of immune cells. The role of IDO in immune tolerance observed in human papillomaviruses-associated tumors is poor understood. Here, we targeted IDO to improve the efficacy of a therapeutic protein vaccine against HPV-16-associated tumors. The combination of IDO inhibitors with immunotherapy showed evidence that can support a new antitumor therapeutic approach. In the murine model C57BL/6, different biologic parameters were evaluated in order to determine the activation of the protective immunity obtained by the combined immunotherapy. We observed that isoforms of 1-methyl tryptophan, combined with our therapeutic vaccine, led to an activation of cytotoxic CD8 (+) T cells in mice grafted with tumor cells expressing the HPV-16 oncoproteins. In addition, the combination of melatonin enhanced the efficacy of the immunotherapeutic approach, resulting in the complete eradication of tumor masses. Altogether, our results indicate that blocking the immunosuppressive mechanisms mediated by IDO may improve the antitumor response induced by immunotherapies. Citation Format: Ana Carolina Ramos Moreno, Bruna F. M. M. Porchia, Patrícia da Cruz Souza, Roberta Liberato Pagni, Rafael Pegoraro, Luís Carlos de Souza Ferreira. Targeting indoleamine 2,3 dioxygenase to improve the efficacy of a therapeutic vaccine against human papillomaviruses-associated tumors [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B55.

We examined the effects of hypertonic saline (HS) on inflammatory, metabolic variables, and bacterial translocation (BT) in rats submitted to intestinal obstruction and ischemia (IO). Male Wistar rats were submitted to IO and treated, 2 h thereafter, with lactated Ringer's (LR) (4 mL/kg per 5 min, i.v.) or HS (7.5% NaCl, 4 mL/kg per 5 min, i.v.). Twenty-four hours after IO, rats were also submitted to enterectomy/enteroanastomosis to resection of necrotized small bowel. Leukocyte-endothelial interactions were investigated by intravital microscopy and the expression of P-selectin and intercellular adhesion molecule 1 by immunohistochemistry. Bacterial cultures of mesenteric lymph nodes, liver, spleen, and blood were used to evaluate BT. Levels of chemokines (cytokine-induced neutrophil chemoattractants 1 and 2), insulin, and corticosterone were determined by enzyme-linked immunosorbent assay. Intestinal histology, serum urea and creatinine levels, and hepatic enzymes activities were performed to evaluate local and remote damage. Relative to IO and LR-treated rats, which exhibited increases in the number of rolling (1.5-fold), adhered (3.5-fold) and migrated (9.0-fold) leukocytes, and increased expression of P-selectin (3-fold) and intercellular adhesion molecule 1 (3-fold) on mesenteric microcirculation, treatment with HS followed by enterectomy reduced leukocyte-endothelial interactions and expression of both adhesion molecules to values attained in sham rats. Serum chemokines were normalized after treatment with both solutions followed by enterectomy. Hypertonic saline-treated rats demonstrated a significant reduction in BT to 50% in liver and spleen samples and bacteremia (14%), compared with 82% of BT in liver and spleen samples of IO and LR-treated rats and bacteremia (57%). Local intestinal damage was attenuated, and renal and hepatic function preserved by treatment with HS followed by enterectomy. Survival rate increased to 86% up to 15 days. Data presented suggest that HS solution followed by enterectomy reduces mesenteric microcirculatory dysfunctions and BT, attenuating local and remote damage in a model of strangulated small bowel obstruction.

Bacterial translocation has been shown to occur in critically ill patients after extensive trauma, shock, sepsis, or thermal injury. The present study investigates mesenteric microcirculatory dysfunctions, the bacterial translocation phenomenon, and hemodynamic/metabolic disturbances in a rat model of intestinal obstruction and ischemia.Anesthetized (pentobarbital 50 mg/kg, i.p.) male Wistar rats (250-350 g) were submitted to intestinal obstruction or laparotomy without intestinal obstruction (Sham) and were evaluated 24 hours later. Bacterial translocation was assessed by bacterial culture of the mesenteric lymph nodes (MLN), liver, spleen, and blood. Leukocyte-endothelial interactions in the mesenteric microcirculation were assessed by intravital microscopy, and P-selectin and intercellular adhesion molecule (ICAM)-1 expressions were quantified by immunohistochemistry. Hematocrit, blood gases, lactate, glucose, white blood cells, serum urea, creatinine, bilirubin, and hepatic enzymes were measured.About 86% of intestinal obstruction rats presented positive cultures for E. coli in samples of the mesenteric lymph nodes, liver, and spleen, and 57% had positive hemocultures. In comparison to the Sham rats, intestinal obstruction induced neutrophilia and increased the number of rolling (approximately 2-fold), adherent (approximately 5-fold), and migrated leukocytes (approximately 11-fold); this increase was accompanied by an increased expression of P-selectin (approximately 2-fold) and intercellular adhesion molecule-1 (approximately 2-fold) in the mesenteric microcirculation. Intestinal obstruction rats exhibited decreased PaCO2, alkalosis, hyperlactatemia, and hyperglycemia, and increased blood potassium, hepatic enzyme activity, serum urea, creatinine, and bilirubin. A high mortality rate was observed after intestinal obstruction (83% at 72 h vs. 0% in Sham rats).Intestinal obstruction and ischemia in rats is a relevant model for the in vivo study of mesenteric microcirculatory dysfunction and the occurrence of bacterial translocation. This model parallels the events implicated in multiple organ dysfunction (MOD) and death.