Esse trabalho objetivou avaliar o perfil genômico de pacientes HIV+ submetidos à imunoterapia com células dendríticas (DC). Os resultados obtidos mostraram que as DC utilizadas na imunoterapia podem ser diferentes em termos de perfil de expressão gênica entre os pacientes selecionados para o ensaio clínico e essas diferenças podem afetar a qualidade do produto administrado ao paciente. Nossos dados não correlacionaram diferenças na ativação das DC com esse perfil de expressão gênica distinto, possivelmente pelo número limitado de amostras disponíveis ou pela escassez de marcadores de ativação adequados. O único paciente que respondeu à imunoterapia mostrou, em leucócitos circulantes, um perfil de expressão específico e relacionado com um melhor controle da carga viral plasmática e com uma resposta imune de tipo Th1 quando comparado com os indivíduos que não responderam ao tratamento. A genotipagem revelou que esse paciente também carrega polimorfismos relacionados a menor progressão à AIDS o que também pode estar relacionado com a boa resposta à imunoterapia. / Aim of this project was the evaluation of genomic profile of HIV+ patients submitted to dendritic cell (DC)-based immunotherapy. Results showed that expression profile of DC used in immunotherapy could be different among patients selected for immunotherapy, and that those differences could affect the quality of final vaccine product, even if our data did not evidence significant difference in DC activation state with regard to expression profile, maybe due to limited size of available samples or to the lack of appropriate DC activation markers. The expression profile of peripheral blood leukocytes from the only good responder of immunotherapy trial was associated with better control of plasma viral load and a Th1 immune response compared to weak or transient responders. Genotyping analysis revealed that the good responder carries polymorphisms associated with a less progression toward AIDS, suggesting that also the genetic background could affect the response to immunotherapy.

OBJETIVO: Avaliar a eficácia e segurança da imunoterapia tópica com imiquimode creme 5% no tratamento do carcinoma basocelular nodular periocular. MÉTODOS: Pacientes com carcinoma basocelular confirmado por biopsia e com contraindicação clínica para a cirurgia reconstrutiva devido ao alto risco ou que recusaram a cirurgia por razões estéticas ou fobia foram incluídos no estudo. O tratamento foi iniciado após treinamento do paciente e de acompanhante. A posologia foi de 5 vezes por semana por 8 a 16 semanas. Acompanhamento quinzenal foi realizado durante a vigência do tratamento com questionário, exame biomicroscópico, medida da acuidade visual e documentação fotográfica. As características clínicas das lesões foram mensuradas através do software ImageJ. Após 12 semanas do fim da terapia, uma nova biópsia na região da lesão foi guiada por fotografia. O seguimento dos pacientes foi semestral, após fim do tratamento, com biópsias anuais da região até o presente momento. RESULTADOS: 19 foram tratadas. A taxa de cura histológica foi de 89,5% após três meses do final do tratamento, e de 84,2% nos três anos de seguimento (39,5 meses). A taxa de cura histológica em três anos foi de 100% para lesões menores que 10 mm, e de 81,8% para lesões maiores que 10 mm. De uma forma geral, os efeitos colaterais da medicação foram mais frequentes durante as oito primeiras semanas de tratamento. Quanto menor foi a distância da lesão à margem palpebral, maior foi chance de o paciente desenvolver ectrópio no tratamento (p = 0,045). Assim, como quanto maior foi a inflamação, maior foi a chance de desenvolver ectrópio, dor e edema durante o tratamento (p = 0,017, p = 0,016 e p = 0,044, respectivamente). CONCLUSÕES: Imiquimode creme 5% mostrou-se eficaz para o tratamento alternativo do carcinoma basocelular periocular, principalmente em lesões menores que 10 mm. Em adição, demonstrou um interessante efeito neoadjuvante sobre as lesões maiores que 10 mm que não foram curadas. Mostrou-se um tratamento seguro; entretanto, um cuidado maior deve ser dado às lesões próximas à margem palpebral devido ao maior risco de complicações e desenvolvimento de ectrópio / Objective: to evaluate the efficacy and safety of topical administration of 5% imiquimod cream in the treatment of periocular nodular basal cell carcinoma (BCC). Methods: Patients with periocular nodular basal cell carcinoma confirmed by biopsy and clinical contraindication to reconstructive surgery due to high risk or who decline surgery for aesthetic reasons or phobia were included in the study. The medication was applied once a day, five days a week for 8-16 weeks. Treatment was initiated after provision of patient and caretaker training. During treatment, patients were followed up twice a month with questionnaires, biomicroscopic examinations, measurement of visual acuity and photographic documentation. The clinical characteristics of the tumors were registered with the software ImageJ. Twelve weeks after the end of treatment, an image-guided biopsy of the tumor site was performed. Patients have since been attending follow-up visits every six months, and biopsies of the region are performed annually. Results: 19 tumors were treated with imiquimod. The average histological cure rate was 89.5% after 3 months at the end of the treatment and 84.2% after 3 years of follow-up (39.5 months). The 3-year histological cure rate was 100% for smaller tumors and 81.8% for larger tumors (>10 mm). In general, drug-related side effects were more frequent during the first 8 weeks of treatment. The smaller the distance between tumor and lid margin, the greater the probability of developing ectropion during treatment (p=0.045). Likewise, the more severe the inflammation, the greater the probability of developing ectropion, pain and edema during treatment (p=0.017, p=0.016 and p=0.044 respectively). Conclusion: Topical administration of 5% imiquimod cream was found to be an efficacious and relatively safe alternative treatment for periocular BCC, especially for tumors smaller than 10 mm, with interesting neoadjuvant effects on uncured tumors larger than 10 mm. However, special care is required when treating tumors near the eyelid margin due to the risk of complications and development of ectropion

The functions of the immune system include immunosurveillance of transformed cells, i.e., the ability to eliminate these cells before they become harmful to the organism. If the transformed cells succeed to escape the immune system surveillance, an oncological disease develops. The tumour immunotherapy aims to stimulate the immune system mechanisms to fight against the tumour. Lately, there's an interest in using NK cells in the immunotherapy of tumours. These cells appertain to the innate immune system and participate in immunosurveillance. When an NK cell encounters a target cell, its activation depends on the integration of signals from the surface activating and inhibiting receptors which bind ligands on the surface of the target cell. Upon activation, NK cell exhibits a cytotoxic response against the target cell. The use of NK cells in immunotherapy includes, among others, the testing of bispecific fusion proteins which can bind a tumour surface antigen by one part and NK cell activating receptor by the other part. Thus, these fusion proteins mediate a contact between both cells and trigger the cytotoxic response. This work presents a preparation of bispecific fusion proteins which consist of an activating ligand MICA (for the receptor NKG2D) or B7H6 (for the receptor NKp30), and a nanobody...

Background: Exposure of skin to UVB radiation (290-320 nm) modulates the immune system, with most studies showing a suppression of Th1-driven immune responses. Investigations into the effects of UVB exposure on allergic respiratory responses have been limited. This study investigated the systemic effects of UVB on Th2-associated immune responses using two different murine models of allergic respiratory inflammation. The mechanism of immune regulation was also examined. Methods and Results: Two murine models of asthma were used: the papain model and the ovalbumin (OVA) model using papain and OVA, respectively, as the allergens. In the papain model, C57BL/6, histamine receptor-1 knockout (H1RKO) and histamine receptor-2 knockout (H2RKO) mice were exposed to a single 4 kJ/m2 dose of UVB (twice a minimal oedemal dose) on shaved dorsal skin three days prior to intranasal sensitisation with papain, a cysteine protease homologue of the house dust mite (Dermatophagoides pteronyssinus) allergen Der p 1. Sensitisation and boost each consisted of five daily intranasal doses of 1 µg papain whilst the challenge consisted of three daily intranasal doses of 100 µg papain. Asthmatic symptoms were assessed 24 h after the final challenge dose. H1RKO mice demonstrated enhanced papain-specific inflammatory responses in the lung-draining lymph nodes (LDLNs) whilst the responses of H2RKO mice closely mimicked those of C57BL/6 mice. UVB irradiation three days before sensitisation reduced in vitro papain-specific proliferation of LDLN cells from C57BL/6 and H1RKO mice but not H2RKO mice 24 h after challenge. The regulatory effect of UVB was transferred by adoptive transfer of 5 x 106 unfractionated LDLN cells from UVB-irradiated, papain-sensitised and -challenged C57BL/6 and H1RKO donor mice into naïve recipients of the corresponding strain that were ii subsequently sensitised and challenged with papain. Additionally, UVB exposure suppressed papain-induced IL-5 and IL-10 production in vitro by LDLN cells from H1RKO mice but not from C57BL/6 mice or H2RKO mice. The results of this study demonstrate systemic immunomodulation of responses to intranasally delivered antigen by UVB irradiation and the induction of regulatory cells in the LDLN following UVB exposure. Furthermore, these results implicate a role for the H2R in UVB-induced suppression of antigen-specific responses in the draining lymph nodes.

<p>Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. </p><p>Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.</p>

<p>Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses.</p><p>We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy.</p><p>In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.</p>

<p>This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy.</p><p>The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated <i>ex vivo</i>.</p><p>Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3⁺ to CD3⁺ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications.</p><p>Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. <i>In vitro</i> expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4⁺ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048).</p><p>In conclusion, human solid tumours are recognised in sentinel nodes and <i>in vitro</i> expanded sentinel node-acquired CD4⁺ T lymphocytes seem useful in the treatment of patients with disseminated cancer.</p>

Immunoglobulin Y (IgY) is the major antibody found in eggs from chicken (Gallus domesticus). IgY can be used as an alternative to mammalian antibodies normally used in research, and its use in immunotherapy has recently been proposed. Compared to mammalian antibodies, IgY possesses several biochemical advantages and its simple purification from egg yolk prevents a stressful moment in animal handling, as no bleeding is necessary. Small amount of antigen (1 mg) can be used to elicit an immune response in chickens and there are low intra-individual differences regarding antibody concentration found in yolk. By studying two chicken breeds and their cross, a genetic correlation was shown regarding the IgY concentration, which implies a possibility by breeding to increase IgY concentrations. By using IgY instead of goat antibody as capture antibody in ELISA, it is possible reduce interferences by complement activation. After oral administration of IgY to healthy volunteers, IgY activity was present in saliva 8 hours later, indicating a protective effect. This effect has been studied in an open clinical trial with cystic fibrosis patients. Specific IgY against Pseudomonas aeruginosa given orally prolongs the time of intermittent colonization by six months, decrease the number of positive colonizations and might be a useful complement to antibiotic treatment. Immunoglobulin therapy may diminish the development of antibiotic resistant microorganisms. The use of immunoglobulin therapy broadens the arsenal available to combat pathogens in medicine and IgY is a promising candidate, both as an alternative to antibiotics and as a useful tool in research and diagnostics.

Cancer immunotherapy aims at reversing the immunosuppressive tumor environment and enhancing anti-tumor immunity. This thesis comprises studies on murine models for prostate (TRAMP-C2) and bladder (MB49) cancer with the aim to explore if the introduction of an adenoviral vector expressing CD40 ligand (AdCD40L) can induce anti-tumor immune responses. We show in subcutaneous mouse models that AdCD40L treatment suppresses tumor growth. Bladder cancer is known to secrete immunosuppressive IL-10 which may inhibit T cell function. We show that introducing AdCD40L into mouse bladder tumors inhibits IL-10 production and reverses immunosuppression. AdCD40L-transduced mouse prostate cancer cells showed caspase activation and reduced cell viability. Vaccination with CD40L-modified prostate cancer cells induces anti-tumor responses and protects mice against rechallenge with native TRAMP-C2 cells. In order to enhance AdCD40L therapy, we explored the possibility of combining it with the histone deacetylase inhibitor FK228, also known as depsipeptide. We show that FK228 upregulates coxsackie and adenovirus receptor expression and thereby enhances adenoviral-mediated CD40L expression in both murine and human prostate cancer cells. Increasing amounts of FK228 or AdCD40L reduces prostate cancer cell viability, while the combined treatment gives at least an additive therapeutic effect. Moreover, we show that AdCD40L transduction of prostate cancer cells induces endogenous CD40 expression and sensitize them for CD40L-mediated therapy. In order to conduct prostate-specific gene therapy, prostate-specific promoters can be used to drive transgene expression. However, there are no reports on prostate-specific promoters that are transcriptionally active in mouse cells. Here we show that by using the two-step transcription activation system (TSTA), we can enhance the activity of a recombinant human promoter sequence and obtain activity in mouse prostate cancer cells as well. This finding paves the way for future studies of prostate-specific gene therapy in immunocompetent mouse models.

This thesis deals with recognition and elimination of tumours by T lymphocytes and their use in adoptive immunotherapy. The first tumour-draining lymph node; the sentinel node, is identified by peritumoural injection of a tracer. This is the hypothesised location for the activation of tumour-reactive lymphocytes. Accordingly, proliferation and IFN-γ production in response to autologous tumour extract was detected in sentinel nodes from patients with colon and urinary bladder cancer. Reactivity in metastatic nodes was generally lower or absent, but the non-responsiveness could be subdued in long-term cultures by addition of tumour antigen and IL-2. A novel padlock-probe based method was developed for measuring the T cell receptor Vβ repertoire. Common Vβ gene expansions were detected in tumour-infiltrating lymphocytes and sentinel nodes. Thus, tumour antigens are recognised in sentinel nodes by Th1 lymphocytes, resulting in a clonally expanded cell population that can be further propagated ex vivo. Regulatory T cells (Tregs) may contribute to tumour-induced immunosuppression. Immunohistochemical stainings against the pan-T cell marker CD3 and Treg marker FOXP3 was performed on tumour tissue from 20 historical urinary bladder cancer patients. The ratio of FOXP3+ to CD3+ cells was lower in patients alive 7 years post-cystectomy, suggesting that Tregs in bladder cancer have prognostic implications. Lymphocytes were isolated from sentinel nodes from sixteen patients with advanced or high-risk colon cancer. In vitro expansion with addition of autologous tumour extract and IL-2 mainly promoted the outgrowth of CD4+ Th1 lymphocytes, which were safely re-transfused to the patients. Four patients responded with complete tumour regression. Survival time in the Dukes’ D patients was significantly increased compared with conventionally treated controls (2.6 versus 0.8 years; p=0.048). In conclusion, human solid tumours are recognised in sentinel nodes and in vitro expanded sentinel node-acquired CD4+ T lymphocytes seem useful in the treatment of patients with disseminated cancer.