Viral infection modulates Qa-1b in infected and bystander cells to properly direct NK cell killing.

Natural killer (NK) cell activation depends on the signaling balance of activating and inhibitory receptors. CD94 forms inhibitory receptors with NKG2A and activating receptors with NKG2E or NKG2C. We previously demonstrated that CD94-NKG2 on NK cells and its ligand Qa-1b are important for the resistance of C57BL/6 mice to lethal ectromelia virus (ECTV) infection. We now show that NKG2C or NKG2E deficiency does not increase susceptibility to lethal ECTV infection, but overexpression of Qa-1b in infected cells does. We also demonstrate that Qa-1b is down-regulated in infected and up-regulated in bystander inflammatory monocytes and B cells. Moreover, NK cells activated by ECTV infection kill Qa-1b-deficient cells in vitro and in vivo. Thus, during viral infection, recognition of Qa-1b by activating CD94/NKG2 receptors is not critical. Instead, the levels of Qa-1b expression are down-regulated in infected cells but increased in some bystander immune cells to respectively promote or inhibit their killing by activated NK cells.

Regulatory functions of NK cells during infections and cancer.

After recognition, NK cells can kill susceptible target cells through perforin-dependent mechanisms or by inducing death receptor-mediated apoptosis, and they can also secrete cytokines that are pivotal for immunomodulation. Despite the critical role as effector cells against tumors and virus-infected cells, NK cells have been implicated in the regulation of T cell-mediated responses in different models of autoimmunity, transplantation, and viral infections. Here, we review the mechanisms described for NK cell-mediated inhibition of adaptive immune responses, with spotlight on the emerging evidence of their regulatory role that shapes antitumor immune responses.

Transposon-mediated generation of CAR-T cells shows efficient anti B-cell leukemia response after ex vivo expansion.

CAR-T-cell therapy has shown considerable advance in recent years, being approved by regulatory agencies in US, Europe, and Japan for the treatment of refractory patients with CD19+ B-cell leukemia or diffuse large B-cell lymphoma. Current methods for CAR-T-cell production use viral vectors for T-cell genetic modification and can take up to 15 days to generate the infusion product. The development of simple and less costly manufacturing protocols is needed in order to meet the increasing demand for this therapy. In this present work, we generated 19BBz CAR-T cells in 8 days using a protocol based on the non-viral transposon-based vector Sleeping Beauty. The expanded cells display mostly a central memory phenotype, expressing higher levels of inhibitory receptors when compared with mock cells. In addition, CAR-T cells were cytotoxic against CD19+ leukemia cells in vitro and improved overall survival rates of mice xenografted with human RS4;11 or Nalm-6 B-cell leukemias. Infused CAR-T cells persisted for up to 28 days, showing that they are capable of long-term persistence and antitumor response. Altogether, these results demonstrate the effectiveness of our protocol and pave the way for a broader application of CAR-T-cell therapy.

Lipopolysaccharides' Cytotoxicity on Human Umbilical Cord Mesenchymal Stem Cells

Abstract Objective: To show the cytotoxicity of Porphyromonas gingivalis lipopolysaccharide (LPS) on human umbilical cord mesenchymal stem cells (HUCMSCs) to better understand the characteristics for its application in regenerative procedures under periodontopathogen LPS influence. Material and Methods: Ultrapure Porphyromonas gingivalis LPS was used in this study. This research used a frozen stock HUCMSCs, previously confirmed by flow cytometry. The biological characteristics, such as cell morphology, proliferation, and protein expression, were screened. To check the cytotoxicity, HUCMSCs were cultured and divided into two groups, the control group and LPS group with various concentrations from 25 to 0.39 µg/mL. MTT assay was done and the cells were observed and counted. The significance level was set at 5%. Results: The percentage of living HUCMSCs on LPS group were not significantly different among concentrations (p>0.05) from 25 to 0.39 µg/mL, even though there were slight mean decrease between groups, but they were not significant. The duration of 24 hours of exposure of LPS does not significantly lower HUCMSCs viability. Conclusion: LPS does not affect the viability of HUCMSCs. The lower the concentration of LPS, the higher the viability of HUCMSCs.

Robo1-specific CAR-NK Immunotherapy Enhances Efficacy of 125I Seed Brachytherapy in an Orthotopic Mouse Model of Human Pancreatic Carcinoma.

BACKGROUND/AIM: The aim of the current study was to investigate the synergistic efficacy of Robo1 bichimeric antigen receptor-natural killer cell (BiCAR-NK) immunotherapy and 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model. MATERIALS AND METHODS: The orthotopic pancreatic tumor model was established with human pancreatic cancer BxPC-3 cells expressing red fluorescent protein. The mice were treated with 125I seed implantation alone or the combination of 125I seeds with Robo1-specific CAR-NK cells. To assess tumor inhibition, in vivo fluorescence imaging was conducted. 7 Tesla magnetic resonance (7T-MR) scanning was applied to measure the changes in the metabolic profiles of tumor tissues. RESULTS: Tumor size was significantly reduced in the 125I and 125I +CAR-NK treated group compared to the untreated group (p<0.05). The 125I seed +CAR-NK treated group showed significantly higher tumor reduction than 125I seed treatment alone (p<0.05). T1 diffusion weighted imaging (T1DWI) sequence showed that the tumors of the 125I +BiCAR-NK treated group had a significantly higher grey scale value than the tumors from the untreated control and the group treated with 125I seed alone (p<0.05). CONCLUSION: Robo1 specific CAR-NK immunotherapy enhances efficacy of 125I seed brachytherapy in an orthotopic pancreatic cancer mouse model.

Human CD8 T-cell activation in acute and chronic chikungunya infection.

There is a need for more detailed elucidation of T-cell immunity in chikungunya infection. CD8 T cells are one of main actors against viruses. Here, we analysed CD8+ T lymphocytes from patients in the acute and chronic phases of chikungunya disease (CHIKD). Our results demonstrate that CD8+ T cells expressed higher ex vivo granzyme B, perforin and CD107A expression in patients in the acute phase of CHIKD compared with healthy individuals and higher ex vivo expression of CD69, interleukin-17A, interleukin-10 and CD95 ligand, and co-expression of CD95/CD95 ligand. These results elucidate the importance of these lymphocytes, demonstrating immune mechanisms mediated in human chikungunya infection.

Evaluation of the cytotoxic response mediated by perforin and granzyme B in patients with non-Hodgkin lymphoma.

This study quantified the perforin and granzyme B in patients with non-Hodgkin lymphoma (NHL) at the time of diagnosis. Protein quantification was performed by flow cytometry. NHL patients had a higher number of cytotoxic T lymphocytes (CTLs) expressing perforin as well as a greater number of activated CTLs than the control group. However, intracellular perforin levels in natural killer cells were lower in the NHL patients compared to the control group. Quantitative real time PCR showed that patients had more expression of perforin and granzyme B transcripts compared to the control group. In addition, patients who had expression of both genes below the median found for the NHL group had lower survival rates. Considering this, we believe that perforin and granzyme B are potential prognostic markers in NHL and thus it is fundamental to pay attention to their expressions in these patients.

Avaliação da capacidade de limpeza e biocompatibilidade de diferentes soluções irrigadoras utilizadas em Endodontia / Evaluation of the cleaning capacity and biocompatibility of different irrigation solutions used in Endodontics

Os objetivos deste estudo foram: Artigo 1 ­ Analisar a extrusão apical de hipoclorito de sódio (NaOCl), debris e sua citotoxicidade após o preparo dos canais radiculares utilizando NaOCl líquido ou gel. Artigo 2- avaliar a dissolução de matéria orgânica do NaOCl líquido e gel, e a limpeza das paredes dentinárias após a instrumentação; Métodos: Artigo 1 - A avaliação da extrusão apical de NaOCl e debris foi feita pela espectrofotometria do conteúdo extruído após o preparo biomecânico. A citotoxicidade foi avaliada pela resposta de culturas celulares de fibroblastos de ligamento periodontal (PDFL) frente as soluções irrigadoras extruídas, pelo teste XTT para análise da viabilidade celular. Para isso, oitenta dentes foram instrumentados com limas Reciproc #25 e #40 (VDW Munique, Alemanha) e utilizado NaOCl gel e líquido ativados por ultrassom. Os dados foram analisados estatisticamente pelo teste de ANOVA e as diferenças estatísticas pelo teste de Tukey e Dunn (p<0,05). Artigo 2 - A dissolução de matéria orgânica foi realizada usando cubos de carne com tamanho e peso determinado, os quais foram deixados em contato com 1 mL das amostras dos seguintes grupos: NaOCl gel 3% (ChlorCid V); NaOCl gel 3%(VIM); NaOCl líquido 2,5%; NaOCl líquido 5,25%; Solução fisiológica estéril (SF) (controle) por um período de 3 min, os fragmentos foram removidos e pesados novamente para quantificar a matéria orgânica não dissolvida. A comprovação da limpeza das paredes dos canais foi avaliada através do MEV e da estereomicroscopia para isso, oitenta dentes foram instrumentados com limas Reciproc #25 e #40 (VDW Munique, Alemanha) e utilizado NaOCl líquido e gel ativados por ultrassom. Os dados foram analisados estatisticamente pelo teste de ANOVA e as diferenças estatísticas pelo teste de Tukey (p<0,05); Resultados: Artigo 1 ­ A extrusão apical de hipoclorito ocorreu em todos os grupos, sento estatisticamente significante ao controle de SF. O grupo NaOCl 5,25 % foi o que teve a maior quantidade de hipoclorito nas amostras (4,76 µL) e o NaOCl gel 3% VIM a menor extrusão (2,32 µL) sendo estatisticamente significante. Quando comparado os grupos de NaOCl gel 3%, o VIM teve menor quantidade de hipoclorito nas amostras, comparado ao CV, com relevância estatística. A extrusão apical de debris esteve presente em todos os grupos, sendo maior no grupo da SF, porém não houve diferença estatística entre os grupos. Quanto a citotoxicidade, todos os grupos foram citotóxicos estatisticamente quando comparado ao controle (SF); O NaOCl 5,25% foi o mais citotóxico perante todos os grupos. O NaOCl 2,5% foi o menos citotóxico comparado ao NaOCl 5,25% e o NaOCl gel 3%VIM, sendo estatisticamente significante; Artigo 2- O NaOCl Liq 5,25 % foi o que mais dissolveu matéria orgânica comparado aos demais grupos, sendo estatisticamente significante. Todos os grupos contendo NaOCl foram estatisticamente eficientes em dissolução tecidual comparado ao grupo controle (SF). A avaliação da limpeza das paredes radiculares feita por MEV mostrou melhor eficiência no grupo do NaOCl 5,25% e pior resultado no grupo do NaOCl gel 3% VIM, com menor porcentagem de túbulos dentinários abertos. Conclusão: Artigo 1- Houve a extrusão apical de debris e hipoclorito em todos os grupos experimentais após a instrumentação. Sendo o NaOCl 5,25% o grupo com a maior extrusão de hipoclorito e o grupo do NaOCl gel 3% VIM o com menor extrusão apical comparado aos demais grupos. O NaOCl 5,25% foi o mais citotóxico para os fibroblastos, e o NaOCl 2,5% foi o menos citototóxico, sendo recomendado o seu uso no tratamento endodôntico; Artigo 2 ­ O NaOCl 5,25% foi o grupo com maior capacidade de dissolução tecidual e limpeza das paredes dentinarias, seguido pelo grupo NaOCl gel 3% CV.(AU)

The objectives of this study were: Article 1 - Analyze the apical sodium hypochlorite (NaOCl) extrusion, debris and it´s cytotoxicity after the root canals preparation using liquid or gel NaOCl. Article 2 - Evaluate the tissue dissolution of NaOCl liquid and gel, and it´s cleaning efficiency of dentin walls after instrumentation; Methods: Article 1 - The evaluation of the apical extrusion of NaOCl and debris was done by the spectrophotometry of the extruded contents after the biomechanical preparation. Cytotoxicity was evaluated by the cell cultures response of periodontal ligament fibroblasts (PDFL) against extruded irrigation solutions by the XTT test for cell viability analysis. For this, eight teeth were instrumented with Reciproc # 25 and # 40 files (VDW Munich, Germany) and used NaOCl gel and liquid activated by ultrasound. Data were analyzed statistically by the ANOVA test and the statistical differences by the Tukey and Dunn test (p <0.05). Article 2 - The dissolution of organic matter was carried out using meat cubes of determined size and weight, which were left in contact with 1 mL of samples from the following groups: NaOCl gel 3% (ChlorCid V); NaOCL gel 3% (VIM); NaOCl liquid 2.5%; NaOCl liquid 5.25%; Sterile physiological solution (SP) (control) for a period of 3 min, the fragments were removed and weighed again to quantify the undissolved organic matter. The verification of the cleansing of the canal walls was evaluated through the SEM and the stereomicroscopy for this, eight teeths were instrumented with Reciproc # 25 and # 40 files (VDW Munich, Germany) and used NaOCl gel and liquid activated by ultrasound. Data were analyzed statistically by the ANOVA test and statistical differences by the Tukey test (p <0.05); Results: Article 1 ­ Hypochlorite apical extrusion occurred in all groups, and was statistically significant at SP control. The NaOCl 5.25% group had the highest amount of hypochlorite in the samples (4.76 µL) and the NaOCl gel 3% VIM the lowest extrusion (2.32 µL) was statistically significant. When compared to the 3% NaOCl gel groups, the VIM had lower amount of hypochlorite in the samples, compared to the CV, with statistical relevance. Apical extrusion of debris was present in all groups, being higher in the SP group, but there was no statistical difference between the groups. As for cytotoxicity, all groups were statistically cytotoxic when compared to control (SP); NaOCl 5.25% was the most cytotoxic in all groups. 2.5% NaOCl was the least cytotoxic compared to NaOCl 5.25% and NaOCl 3% VIM gel, being statistically significant; Article 2 -The NaOCl Liq 5.25% was the one that dissolved organic matter more compared to the other groups, being statistically significant. All groups containing NaOCl were statistically efficient in tissue dissolution compared to the control group (SP). The SEM evaluation showed a better efficiency in the NaOCl 5.25% group and a worse result in the NaOCl gel group 3% VIM, with a lower percentage of open dentinal tubules. Conclusion: Article 1 - There was the apical extrusion of debris and hypochlorite in all experimental groups after instrumentation. The NaOCl 5.25% group had the highest hypochlorite extrusion and the NaOCl 3% VIM group had the lowest apical extrusion compared to the other groups. NaOCl 5.25% was the most cytotoxic for fibroblasts, and 2.5% NaOCl was the least cytotoxic, being recommended for endodontic treatment; Article 2 - NaOCl 5.25% was the group with the greatest capacity for tissue dissolution and cleaning of the dentin walls, followed by the NaOCl group 2.5% (AU)

Ação antimicrobiana e citotoxicidade de extratos aquoso e glicólico de própolis sobre bactérias anaeróbias de importância odontológica / Antimicrobial action and cytotoxicity of aqueous and glycolic extracts of propolis on anaerobic bacteria of dental importance

As plantas medicinais e os fitoterápicos têm sido utilizados como coadjuvantes e alternativos no combate a diversas doenças com crescente frequência, no entanto, na Odontologia seu uso ainda é bastante limitado. Com isso, o objetivo deste estudo é avaliar a ação antimicrobiana dos extratos aquoso e glicólico de própolis verde sobre micro-organismos anaeróbios de interesse odontológico, bem como sua citotoxicidade, a fim de introduzir e incentivar o uso efetivo e sistemático desse fitoterápico em produtos como dentifrícios e enxaguatórios bucais no combate a cáries e doenças periodontais. Os extratos comerciais aquoso e glicólico de própolis foram obtidos das empresas Apis Flora e Mapric, respectivamente. Para avaliação da atividade antimicrobiana foram utilizadas cepas-padrão (ATCC) de Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis Porphyromonas gingivalis e Prevotella intermedia em cultura planctônica, verificando a concentração inibitória mínima e concentração microbicida mínima (CIM e CMM), segundo Clinical and Laboratory Standards Institute. Para biofilmes monotípicos, suspensões padronizadas (107 céls/mL) foram adicionadas em poços de microplacas e após 48 h em anaerobiose foram tratados com 3 concentrações do extrato de própolis (n=12) por 5 min. Foi incluído um controle positivo (solução fisiológica) e um controle negativo (clorexidina). O biofilme foi mensurado pelos testes MTT e Cristal Violeta. Para análise de citotoxicidade, queratinócitos humanos (HaCaT) foram cultivados e colocados em contato com os extratos por 5 min e 24h. Os dados foram analisados estatisticamente pelos testes ANOVA e Tukey Test (5%). Os resultados mostraram que os extratos tiveram ação antimicrobiana contra as suspensões planctônicas e os biofilmes monotípicos dos patógenos, sendo tão ou mais eficazes que a clorexidina. Quanto à citotoxicidade, observou-se diminuição da viabilidade celular dos queratinócitos humanos após a aplicação dos extratos, do mesmo modo que a clorexidina, sendo o extrato glicólico menos citotóxico que o aquoso. Com isto conclui-se que os extratos comerciais aquoso e glicólico de própolis verde tem ação antimicrobiana contra os micro-organismos anaeróbios orais estudados e apresentam potencial para serem utilizados nos tratamentos contra os referidos patógenos, já que no que se refere à citotoxicidade, se comportaram de forma semelhante à Clorexidina, cujo uso é conhecidamente seguro e eficaz(AU)

Medicinal plants and herbal medicines have been used as adjuvants and alternatives in fighting various diseases with increasing frequency. However, in dentistry its use is still quite limited. Therefore, the objective of this study is to evaluate the antimicrobial action of the aqueous and glycolic extracts of green propolis on anaerobic microorganisms of dental interest, in order to introduce and encourage the effective and systematic use of this herbal medicine in products such as dentifrices and mouthwashes in the fight against tooth decay and periodontal diseases. Aqueous and glycolic commercial extracts of propolis were obtained from Apis Flora and Mapric companies, respectively.To evaluate the antimicrobial activity, standard strains (ATCC) of Fusobacterium nucleatum, Parvimonas micra, Porphyromonas endodontalis, Porphyromonas gingivalis, and Prevotella intermedia in planktonic culture was used, verifying the minimum inhibitory concentration and minimum microbicide concentration (MIC and CMM), according to Clinical and Laboratory Standards Institute. For monotypic biofilms, standardized suspensions (107 cells / mL) was added to microplate wells and after 48 h in anaerobiosis was treated with 3 concentrations of propolis extracts (n = 12) for 5 min. A positive control (saline solution) and a negative control (chlorhexidine) was included. The biofilm was measured by the MTT and Violet Crystal tests. For cytotoxicity analysis, human keratinocytes (HaCaT) were cultured and placed in contact with the extracts for 5 min and 24 h. Data were analyzed statistically by ANOVA and Tukey Test (5%). The results showed that the extracts had antimicrobial action against planktonic suspensions and monotypic pathogen biofilms, being as or more effective than chlorhexidine. As for cytotoxicity, the cellular viability of human keratinocytes was observed after application of the extracts, in the same way as chlorhexidine, the glycolic extract being less cytotoxic than aqueous. It is concluded that the aqueous and glycolic extracts of green propolis have an antimicrobial action against the studied oral anaerobic microorganisms and and have the potential to be used in the treatments against these pathogens, since in cytotoxicity they behaved in a way similar to chlorhexidine, the use of which is known to be safe and effective(AU)

Cytotoxic activity in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.

Cytotoxic activity in cutaneous leishmaniasis

Cutaneous leishmaniasis (CL) is a chronic disease caused by species of the protozoan Leishmania and characterised by the presence of ulcerated skin lesions. Both parasite and host factors affect the clinical presentation of the disease. The development of skin ulcers in CL is associated with an inflammatory response mediated by cells that control parasite growth but also contribute to pathogenesis. CD8+ T cells contribute to deleterious inflammatory responses in patients with CL through cytotoxic mechanisms. In addition, natural killer cells also limit Leishmania infections by production of interferon-γ and cytotoxicity. In this review, we focus on studies of cytotoxicity in CL and its contribution to the pathogenesis of this disease.

Avaliação da biocompatibilidade e atividade antimicrobiana in vitro da matriz de Hidrogel associada ao extrato glicólico do Punica granatum L. (romã) / Evaluation of biocompatibility and antimicrobial action in vitro of a hydrogel scaffolds Hydromatrix associated to glycolic extract of Punica granatum (pomegranate)

A matriz de hidrogel é um biomaterial de nanofibra peptídica tridimensional que induz o crescimento, migração e proliferação celular, assim favorecendo a regeneração tecidual. O objetivo deste trabalho foi avaliar a biocompatibilidade e a atividade antimicrobiana da matriz de hidrogel associado ao extrato romã e ao antimicrobiano ciprofloxacino. Para isso, foram realizadas análises microbiológicas sobre Enterococcus faecalis (ATCC 4083) em cultura planctônica, por meio d ensaio de microdiluição em caldo e em biofilme pelo teste de MTT. Para os ensaios da biocompatibilidade, foi utilizada cultura de macrófagos (RAW 264.7). A citotoxicidade foi avaliada pelo ensaio de MTT e a genotoxicidade foi realizado pelo teste de micronúcleo. A análise estatística foi realizada pelos testes ANOVA e Tukey, adotando o nível de significância 5%. Os resultados demonstraram que a matriz de hidrogel associado ao extrato de romã não apresentou atividade antimicrobiana para a cultura planctônica como para o biofilme de E. faecalis. Porém, não foi citotóxico e genotóxico para RAW 264.7. Por outro lado, o antimicrobiano ciprofloxacino apresentou atividade antimicrobiana sobre E. faecalis,além de ter apresentado efeitos citotóxico e genotóxico para os macrófagos. Com isso, foi possível concluir que o extrato de romã associado ou não a matriz de hidrogel apresenta ausência de citotóxico, genotóxico e efeito antimicrobiano

The hydrogel matrix is a three-dimensional peptide nanofiber biomaterial that induces cell growth, migration and proliferation, thus favoring tissue regeneration. The objective of this work was to evaluate the biocompatibility and antimicrobial activity of the hydrogel matrix associated with the pomegranate extract and the antimicrobial ciprofloxacin. For this, microbiological analyzes on Enterococcus faecalis (ATCC 4083) were carried out in planktonic culture, by means of the microdilution test in broth and in biofilm by the MTT test. For the biocompatibility assays, macrophage culture (RAW 264.7) was used. Cytotoxicity was assessed by the MTT assay and genotoxicity was performed by the micronucleus test. Statistical analysis was performed by the ANOVA and Tukey tests, adopting the significance level 5%. The results showed that the hydrogel matrix associated with the pomegranate extract did not show antimicrobial activity for the planktonic culture as for the E. faecalis biofilm. However, it was not cytotoxic and genotoxic for RAW 264.7. On the other hand, the antimicrobial ciprofloxacin showed antimicrobial activity on E. faecalis, besides having cytotoxic and genotoxic effects for the macrophages. With this, it was possible to conclude that the pomegranate extract associated or not with the hydrogel matrix shows absence of cytotoxic, genotoxic and antimicrobial effect

Individual Human Cytotoxic T Lymphocytes Exhibit Intraclonal Heterogeneity during Sustained Killing.

The killing of antigen-bearing cells by clonal populations of cytotoxic T lymphocytes (CTLs) is thought to be a rapid phenomenon executed uniformly by individual CTLs. We combined bulk and single-CTL killing assays over a prolonged time period to provide the killing statistics of clonal human CTLs against an excess of target cells. Our data reveal efficiency in sustained killing at the population level, which relied on a highly heterogeneous multiple killing performance at the individual level. Although intraclonal functional heterogeneity was a stable trait in clonal populations, it was reset in the progeny of individual CTLs. In-depth mathematical analysis of individual CTL killing data revealed a substantial proportion of high-rate killer CTLs with burst killing activity. Importantly, such activity was delayed and required activation with strong antigenic stimulation. Our study implies that functional heterogeneity allows CTL populations to calibrate prolonged cytotoxic activity to the size of target cell populations.

Interleukin-21 expanded NKDC in vitro reduces the B16F10 tumor growth in vivo.

Innate immunity to tumors is mediated mainly by natural killer cells (NKs) and dendritic cells (DCs). The function of these cells is coordinated by cytokines produced during the inflammatory process. NK cells are highly active against tumors, being an important source of IFN-γ. Natural killer dendritic cells (NKDCs) were recently identified as a group of hybrid cells; some studies claim that they have lytic activity, produce IFN-γ and can also stimulate antigen-specific T cells. Interleukin 21 (IL-21) regulates the proliferation capacity and cytotoxicity of NK and T cells. The main objective of this study was to investigate if IL-21 influences the frequency of NKDCs in vitro as well as IFN-γ production and also to verify if these cells could enhance the antitumor activity against B16F10 tumor model in vivo. Splenocytes from C57BL/6 mice were isolated and the DC were enriched by immunomagnetic beads and cultured for four days with recombinant IL-21 (10, 20, 40 or 100 ng/ml). NKDC population was characterized as CD11clow/medB220+NK1.1+. Expanded cells were used to treat B16F10 tumor bearing mice and tumor growth was compared between the doses of IL-21 10 ng/ml and 20 ng/ml. The results indicate that IL-21 increases the expansion of splenic NKDCs in vitro in doses of 10 ng/ml and 20 ng/ml and these cells produce IFN-γ. In vivo, cells expanded with IL-21 and injected directly into the growing tumor efficiently reduced the tumor size. Together, these results showed for the first time that IL-21 influences the biology and the effector activity of NKDCs.

A polymeric protein induces specific cytotoxicity in a TLR4 dependent manner in the absence of adjuvants.

Lumazine synthase from Brucella spp. (BLS) is a highly immunogenic decameric protein. It is possible to insert foreign peptides or proteins at its ten-amino acid termini. These chimeras elicit systemic and oral immunity without adjuvants, which are commonly needed in the formulation of subunit-based vaccines. Here, we show that BLS induces the cross presentation of a covalently attached peptide OVA(257-264) and a specific cytotoxic response to this peptide in the absence of adjuvants. Unlike other subunit-based vaccines, this chimera induces rapid activation of CTLs and a specific cytotoxic response, making this polymeric protein an ideal antigen carrier for vaccine development. Adoptive transfer of transgenic OT-I T cells revealed efficient cross presentation of BLS-OVA(257-264)in vivo. BLS-OVA(257-264) immunization induced the proliferation of OVA(257-264)-specific CD8+ lymphocytes and also increased the percentage of OVA(257-264)-specific CD8+ cells expressing the early activation marker CD69; after 5 days, the percentage of OVA(257-264)-specific CD8+ cells expressing high levels of CD44 increased. This cell subpopulation showed decreased expression of IL-7Rα, indicating that BLS-OVA(257-264) induced the generation of CD8+ effector cells. BLS-OVA(257-264) was cross presented in vitro independently of the presence of a functional TLR4 in the DCs. Finally, we show that immunization of wild type mice with the chimera BLS-OVA(257-264) without adjuvants induced a strong OVA(257-264)-specific effector cytotoxic response. This cytotoxicity is dependent on TLR4 as is not induced in mice lacking a functional receptor. These data show that TLR4 signaling is necessary for the induction of a cytotoxic response but not for antigen cross presentation.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

Characterization of CD4⁺ cytotoxic lymphocytes and apoptosis markers induced by Trypanossoma cruzi infection.

Although the pathophysiology of Chagas disease is not completely understood, it is widely accepted that involvement of the immune response is critical in determining the outcome of the disease. In this context, CD4⁺ T cells may play an important role in generating different mechanisms of protection. In addition to effector and regulatory functions, CD4⁺ T cells may be also involved with lytic activities against the parasite and may have a relevant role on control of the infection. In this study, we have evaluated CD4⁺ T cells expressing cytotoxic and apoptosis markers in response to Trypanossoma cruzi infection in indeterminate (IND) and cardiac (CARD) patients with Chagas disease and non-infected individuals (NI). Our data demonstrated that: (1) CD4⁺ T cells presented higher ex vivo granzyme B expression in patients with Chagas disease compared with healthy individuals and that antigen induced a greater granzyme B expression in IND patients; (2) CD95L expression in CD4⁺ CD95⁺ T cells from IND patients is higher than in CARD and NI; (3) IND and CARD patients had an increased frequency of caspase-3 after in vitro stimulation and also expressed a high frequency of annexinV⁺ 7ADD⁺ within CD4⁺ T cells; (4) Lastly, a positive correlation was seen between cytotoxic molecules and CD45RO memory marker in CD4⁺ T cells and between caspase-3 and CD95L within CD4⁺ CD95⁺ T cells. These results suggest new insights into the functional competence of CD4⁺ T cells among the different clinical forms of Chagas disease, which will lead to a better understanding of their influence during immune responses against T. cruzi.

Phenotypic and functional characterization of NK cells in human immune response against the dimorphic fungus Paracoccidioides brasiliensis.

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56(+)granulysin(+) cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6 percent, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4 percent) and CD86 (80.13 ± 2.81 percent)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells.

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMax™ Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD(450) = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.