Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Stimulating immune responses to fight cancer: Basic biology and mechanisms

Chronic inflammation is now recognized as a major cause of malignant disease. In concert with various mechanisms (including DNA instability), hypoxia and activation of inflammatory bioactive lipid pathways and pro-inflammatory cytokines open the doorway to malignant transformation and proliferation, angiogenesis, and metastasis in many cancers. A balance between stimulatory and inhibitory signals regulates the immune response to cancer. These include inhibitory checkpoints that modulate the extent and duration of the immune response and may be activated by tumor cells. This contributes to immune resistance, especially against tumor antigen-specific T-cells. Targeting these checkpoints is an evolving approach to cancer immunotherapy, designed to foster an immune response. The current focus of these trials is on the programmed cell death protein 1 (PD-1) receptor and its ligands (PD-L1, PD-L2) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Researchers have developed anti-PD-1 and anti-PDL-1 antibodies that interfere with the ligands and receptor and allow the tumor cell to be recognized and attacked by tumor-infiltrating T-cells. These are currently being studied in lung cancer. Likewise, CTLA-4 inhibitors, which have had success treating advanced melanoma, are being studied in lung cancer with encouraging results.

Rapid complete response of metastatic melanoma in a patient undergoing ipilimumab immunotherapy in the setting of active ulcerative colitis.

While blockade of the cytotoxic T-lymphocyte antigen-4 (CTLA-4) T cell regulatory receptor has become a commonly utilized strategy in the management of advanced melanoma, many questions remain regarding the use of this agent in patient populations with autoimmune disease. We present a case involving the treatment of a patient with stage IV melanoma and ulcerative colitis (UC) with anti-CTLA-4 antibody immunotherapy. Upon initial treatment, the patient developed grade III colitis requiring tumor necrosis factor-alpha (TNF-α) blocking antibody therapy, however re-treatment with anti-CTLA-4 antibody following a total colectomy resulted in a rapid complete response accompanied by the development of a tracheobronchitis, a previously described extra-intestinal manifestation of UC. This case contributes to the evolving literature on the use of checkpoint inhibitors in patients also suffering from autoimmune disease, supports future clinical trials investigating the use of these agents in patients with autoimmune diseases, and suggests that an understanding of the specific molecular pathways involved in a patient's autoimmune pathology may provide insight into the development of more effective novel combinatorial immunotherapeutic strategies.

In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

The CTLA4 + 49A/G and CT60 polymorphisms and chronic inflammatory arthropathies in Northern Ireland

Rheumatoid and juvenile idiopathic arthritis (RA, JIA) are chronic inflammatory arthropathies with an autoimmune background. The cytotoxic T-lymphocyte antigen-4 (CTLA-4) protein plays a key role in the down-regulation of T cell activation. We analyzed the CTLA4 +49A/G and CT60 polymorphisms in cohorts of Northern Irish RA and JIA patients and healthy control subjects using restriction fragment length polymorphism methods. The +49 A allele was increased in RA (61.2%; P=0.02; OR=1.28; 95% C.I.=1.04-1.58) and JIA (61.8%; P=0.14) patients compared to the control population (55.3%). No significant association was observed for the CT60 polymorphism. Haplotype analysis revealed a significantly different distribution of +49 A/G-CT60 haplotypes in RA and JIA patients compared to controls (P value

BTLA mediates inhibition of human tumor-specific CD8+ T cells that can be partially reversed by vaccination.

The function of antigen-specific CD8+ T cells, which may protect against both infectious and malignant diseases, can be impaired by ligation of their inhibitory receptors, which include CTL-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1). Recently, B and T lymphocyte attenuator (BTLA) was identified as a novel inhibitory receptor with structural and functional similarities to CTLA-4 and PD-1. BTLA triggering leads to decreased antimicrobial and autoimmune T cell responses in mice, but its functions in humans are largely unknown. Here we have demonstrated that as human viral antigen-specific CD8+ T cells differentiated from naive to effector cells, their surface expression of BTLA was gradually downregulated. In marked contrast, human melanoma tumor antigen-specific effector CD8+ T cells persistently expressed high levels of BTLA in vivo and remained susceptible to functional inhibition by its ligand herpes virus entry mediator (HVEM). Such persistence of BTLA expression was also found in tumor antigen-specific CD8+ T cells from melanoma patients with spontaneous antitumor immune responses and after conventional peptide vaccination. Remarkably, addition of CpG oligodeoxynucleotides to the vaccine formulation led to progressive downregulation of BTLA in vivo and consequent resistance to BTLA-HVEM-mediated inhibition. Thus, BTLA activation inhibits the function of human CD8+ cancer-specific T cells, and appropriate immunotherapy may partially overcome this inhibition.

Étude de protection conférée par vaccination ADN dans un modèle murin d’hépatite auto-immune

La vaccination ADN à l’aide de plasmides codant pour des autoantigènes s’est avérée efficace dans la protection contre plusieurs maladies auto-immunes. Le but de ce mémoire était dans un premier temps d’établir si un protocole de vaccination ADN composé de 3 injections de pCMV-CTLA-4-NP et de pVR-IL-12 à deux semaines d’intervalle avait un effet protecteur contre le développement d’une hépatite auto-immune chez la souris TTR-NP, un modèle murin transgénique de la maladie et précédemment développé au laboratoire. Dans un deuxième temps, le but était d’élucider, le cas échéant, les mécanismes sous-tendant la protection conférée par la vaccination ADN. Les hypothèses initiales étaient qu’une protection allait effectivement être conférée par la vaccination ADN et que celle-ci pouvait être attribuable à une déviation de la réponse typiquement Th1 de la maladie vers une réponse Th2, à un épuisement des cellules immunitaires et/ou à l’activation et à l’induction de prolifération de cellules régulatrices. Les résultats montrent que la vaccination ADN induit une protection transitoire contre le développement d’infiltrations lymphocytaires au foie. Cette protection se ferait via un épuisement des cellules CD4+, CD8+ et CD19+ se retrouvant à la rate et exprimant PD 1 dans une plus forte proportion à 3 mois, et ne serait médiée ni par les lymphocytes T régulateurs CD4+CD25+FoxP3+, ni par les cellules CD8+FoxP3+. Une déviation de la réponse Th1 vers une réponse Th2 demeure une explication supplémentaire plausible à la protection conférée mais nécessiterait une caractérisation en situation plus physiologique avant de pouvoir inférer sur son implication réelle. La vaccination ADN n’influe ni sur la présence d’autoanticorps, ni sur les niveaux d’alanine aminotransférase, deux marqueurs de la maladie.

Enfermedad tiroidea autoinmune en pacientes colombianos con lupus eritematoso sistémico

Objetivos: Determinar la prevalencia y los factores asociados con el desarrollo de hipotiroidismo autoinmune (HA) en una cohorte de pacientes con lupus eritematoso sistémico (LES), y analizar la información actual en cuanto a la prevalencia e impacto de la enfermedad tiroidea autoinmune y la autoinmunidad tiroidea en pacientes con LES. Métodos: Este fue un estudio realizado en dos pasos. Primero, un total de 376 pacientes con LES fueron evaluados sistemáticamente por la presencia de: 1) HA confirmado, 2) positividad para anticuerpos tiroperoxidasa/tiroglobulina (TPOAb/TgAb) sin hipotiroidismo, 3) hipotiroidismo no autoinmune, y 4) pacientes con LES sin hipotiroidismo ni positividad para TPOAb/TgAb. Se construyeron modelos multivariados y árboles de regresión y clasificación para analizar los datos. Segundo, la información actual fue evaluada a través de una revisión sistemática de la literatura (RLS). Se siguieron las guías PRISMA para la búsqueda en las bases de datos PubMed, Scopus, SciELO y Librería Virtual en Salud. Resultados: En nuestra cohorte, la prevalencia de HA confirmado fue de 12% (Grupo 1). Sin embargo, la frecuencia de positividad para TPOAb y TgAb fue de 21% y 10%, respectivamente (Grupo 2). Los pacientes con LES sin HA, hipotiroidismo no autoinmune ni positividad para TPOAb/TgAb constituyeron el 40% de la corhorte. Los pacientes con HA confirmada fueron estadísticamente significativo de mayor edad y tuvieron un inicio tardío de la enfermedad. El tabaquismo (ORA 6.93, IC 95% 1.98-28.54, p= 0.004), la presencia de Síndrome de Sjögren (SS) (ORA 23.2, IC 95% 1.89-359.53, p= 0.015) y la positividad para anticuerpos anti-péptido cíclico citrulinado (anti-CCP) (ORA 10.35, IC 95% 1.04-121.26, p= 0.047) se asociaron con la coexistencia de LES-HA, ajustado por género y duración de la enfermedad. El tabaquismo y el SS fueron confirmados como factores predictivos para LES-HA (AUC del modelo CART = 0.72). En la RSL, la prevalencia de ETA en LES varío entre 1% al 60%. Los factores asociados con esta poliautoinmunidad fueron el género femenino, edad avanzada, tabaquismo, positividad para algunos anticuerpos, SS y el compromiso articular y cutáneo. Conclusiones: La ETA es frecuente en pacientes con LES, y no afecta la severidad del LES. Los factores de riesgo identificados ayudarán a los clínicos en la búsqueda de ETA. Nuestros resultados deben estimular políticas para la suspensión del tabaquismo en pacientes con LES.

Inmunoterapia en pacientes con cáncer de pulmón de células no pequeñas versus terapia convencional. Revisión sistemática

RESUMEN Antecedentes y Justificación: El cáncer de pulmón es la principal causa de muerte relacionada con Cáncer en el mundo. El cáncer pulmonar de células no pequeñas (Non-Small-cell lung cancer NSCLC) representa el 85% de todos los cánceres de pulmón y en un 40% es diagnosticado tardíamente y con los tratamientos disponibles actualmente (cirugía, radioterapia y quimioterapia) presenta una supervivencia a 5 años entre el 10 y el 15%. En los últimos años han surgido nuevos tratamientos basados en la inmunoterapia que prometen mejorar la supervivencia de estos pacientes. Objetivo: Determinar la eficacia de la inmunoterapia en el tratamiento del cáncer de pulmón de células no pequeñas (NSCLC) con el fin de integrar la información disponible para su posterior uso en la clínica. Metodología: Se realizó búsqueda exhaustiva de la literatura disponible del 1 de Enero de 2003 al 31 de Diciembre de 2013. Se examinaron las siguientes bases de datos: Pubmed, Scielo, Medline, Lilacs, EMBASE, Bandolier, peDRO y Cochrane. Se utilizaron los términos MeSH de búsqueda: immunotherapy, NSCLC, clinical trials. Resultados: de 163 referencias identificadas en las bases de datos, 12 fueron seleccionadas para la revisión. Se identificaron 11 estrategias inmunoterapéuticas que fueron complementarias al uso de quimioterapia, radioterapia o ambas. No se encontró diferencia significativa entre la supervivencia global de los grupos de intervención y controles con excepción de 1 artículo. La mayoría de efectos secundarios fueron de leves a moderados y no hubo diferencias significativas entre los grupos. Discusión: no se evidenció un aumento significativo de la supervivencia global con la utilización de inmunoterapias, a excepción de la que emplea células asesinas inducidas por citocinas junto a células dendríticas. Sin embargo es necesario esperar resultados de estudios fase III en curso.

Avaliação do efeito do benzonidazol sobre linfócitos, de portadores crônicos da doença de Chagas, submetidos experimentalmente à exposição ao Trypanosoma cruzi

O único medicamento disponível para o tratamento da doença de Chagas (DC) no Brasil é o benzonidazol (Bz). O beneficio da medicação aos portadores crônicos da doença ainda demonstra controvérsias, mas seu uso nessa fase pode ter a finalidade de prevenir/retardar a evolução da DC para formas mais graves. Os fenômenos imunológicos que ocorrem após a terapia com o Bz ainda não estão elucidados. Assim, este estudo propôs avaliar o efeito do Bz sobre o perfil imunológico de linfócitos T e a produção de citocinas por células da resposta imune expostas in vitro ao T. cruzi. Amostras de sangue de portadores crônicos da DC foram coletadas para obtenção de células mononucleares de sangue periférico com posterior infecção com tripomastigotas de T. cruzi, seguida de adição do Bz às culturas. Após o tempo de cultivo, os sobrenadantes foram estocados para posterior análise das citocinas IFN-gama, TNF, IL-10, IL-6, IL-4 e IL-2 por CBA e as células foram avaliadas por citometria de fluxo, quanto à expressão das moléculas CD28+ e CTLA-4+ e a produção de citocinas (IL-10 e IFN-gama) em linfócitos T CD4+ e CD8+. Nossos resultados mostraram que o tratamento com o Bz aumentou a expressão da molécula CTLA-4+ em linfócitos T CD8+, indicando que o Bz pode induzir uma modulação da resposta imune e, consequentemente, diminuir a ativação exacerbada dessas células. Além disso, verificamos uma diminuição da produção da citocina IL-10 por linfócitos T CD4+ na presença do Bz. Com relação à produção global de citocinas por células aderentes e não aderentes, observamos que o Bz causou uma diminuição da citocina pró-inflamatória TNF e da citocina anti-inflamatória IL-10, enquanto que as outras citocinas (IFN-gama, IL-6, IL4 e IL-2) permaneceram com níveis elevados de produção na presença desse fármaco. Nossos resultados sugerem que o Bz induz uma regulação da ativação de linfócitos T CD8+ e a produção de citocinas do perfil Th1 modulado por citocinas do perfil Th2, em células de portadores crônicos da DC. Assim, acreditamos que a administração do Bz aos portadores de formas clínicas brandas da DC pode ser benéfica a esses pacientes, visto que o Bz não promoveu um perfil inflamatório exacerbado

CD4(+) CD25(+) Foxp3(+) Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4(+) CD25(+) Foxp3(+) Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123(+)), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-alpha) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and longlasting protective immunity to malaria.

Gene Expression Profiles Stratified according to Type 1 Diabetes Mellitus Susceptibility Regions

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Differential expression of the costimulatory molecules CD86, CD28, CD152 and PD-1 correlates with the host-parasite outcome in leprosy

Leprosy is a spectral disease exhibiting two polar sides, namely, lepromatous leprosy (LL) characterised by impaired T-cell responses and tuberculoid leprosy in which T-cell responses are strong. Proper T-cell activation requires signalling through costimulatory molecules expressed by antigen presenting cells and their ligands on T-cells. We studied the influence of costimulatory molecules on the immune responses of subjects along the leprosy spectrum. The expression of the costimulatory molecules was evaluated in in vitro-stimulated peripheral blood mononuclear cells of lepromatous and tuberculoid patients and healthy exposed individuals (contacts). We show that LL patients have defective monocyte CD86 expression, which likely contributes to the impairment of the antigen presentation process and to patients anergy. Accordingly, CD86 but not CD80 blockade inhibited the lymphoproliferative response to Mycobacterium leprae. Consistent with the LL anergy, there was reduced expression of the positive signalling costimulatory molecules CD28 and CD86 on the T-cells in these patients. In contrast, tuberculoid leprosy patients displayed increased expression of the negative signalling molecules CD152 and programmed death-1 (PD-1), which represents a probable means of modulating an exacerbated immune response and avoiding immunopathology. Notably, the contacts exhibited proper CD86 and CD28 expression but not exacerbated CD152 or PD-1 expression, suggesting that they tend to develop a balanced immunity without requiring immunosuppressive costimulatory signalling.

Increased Expression of Regulatory T Cells and Down-Regulatory Molecules in Lepromatous Leprosy

T regulatory cells (Tregs) play an important role in the mechanism of host's failure to control pathogen dissemination in severe forms of different chronic granulomatous diseases, but their role in leprosy has not yet been elucidated; 28 newly diagnosed patients (16 patients with lepromatous leprosy and 12 patients with tuberculoid leprosy) and 6 healthy Mycobacterium leprae-exposed individuals (contacts) were studied. Tregs were quantified by flow cytometry (CD4+ CD25+ Foxp3+) in peripheral blood mononuclear cells stimulated in vitro with a M. leprae antigenic preparation and phytohemagglutinin as well as in skin lesions by immunohistochemistry. The lymphoproliferative (LPR), interleukin-10 (IL-10), and interferon-gamma (IFN-gamma) responses of the in vitro-stimulated peripheral blood mononuclear cells and the in situ expression of IL-10, transforming growth factor-beta (TGF-beta), and cytotoxic T-lymphocyte antigen 4 (CTLA-4) were also determined. We show that M. leprae antigens induced significantly lower LPR but significantly higher Treg numbers in lepromatous than tuberculoid patients and contacts. Mitogen-induced LPR and Treg frequencies were not significantly different among the three groups. Tregs were also more frequent in situ in lepromatous patients, and this finding was paralleled by increased expression of the antiinflammatory molecules IL-10 and CTLA-4 but not TGF-beta. In lepromatous patients, Tregs were intermingled with vacuolized hystiocyte infiltrates all over the lesion, whereas in tuberculoid patients, Tregs were rare. Our results suggest that Tregs are present in increased numbers, and they may have a pathogenic role in leprosy patients harboring uncontrolled bacillary multiplication but not in those individuals capable of limiting M. leprae growth.

Early skin immunological disturbance after Plasmodium-infected mosquito bites

Although the role of regulatory T cells (Tregs) during malaria infection has been studied extensively, such studies have focused exclusively on the role of Treg during the blood stage of infection; little is known about the detailed mechanisms of Tregs and sporozoite deposition in the dermis by mosquito bites. In this paper we show that sporozoites introduced into the skin by mosquito bites increase the mobility of skin Tregs and dendritic cells (DCs). We also show differences in MHC class II and/or C086 expression on skin-resident dendritic cell subtypes and macrophages. From the observed decrease of the number of APCs into draining lymph nodes, suppression of CD28 expression in conventional CD4 T cells, and a low homeostatic proliferation of skin-migrated CD4 T found in nude mice indicate that Tregs may play a fundamental role during the initial phase of malaria parasite inoculation into the mammalian host. (C) 2012 Elsevier Inc. All rights reserved.