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Mapeamento de epítopos e produção de proteínas quiméricas como estratégia para o desenvolvimento de uma vacina multiepítopo contra Schistosoma mansoni

2026-01-27T13:04:56Z

Título: Mapeamento de epítopos e produção de proteínas quiméricas como estratégia para o desenvolvimento de uma vacina multiepítopo contra Schistosoma mansoni Autor(es): Khouri, Mariana Ivo Primeiro Orientador: Farias, Leonardo Paiva Abstract: Schistosomiasis is an endemic disease in 78 countries and presents the highest morbidity among helminth infections. Therefore, an effective vaccine against this disease would be a valuable addition to current control strategies and, ultimately, to its elimination. Most vaccines investigated to date have been based on single antigens, and none have reached the final stages of development. We believe that using multiple immunogenic epitopes associated with protective immune responses represents a more promising approach for developing an effective vaccine. It is possible to identify protection-associated epitopes in murine models immunized with attenuated cercariae and in self-curing rhesus macaques. Additionally, in endemic areas, some individuals develop resistance to reinfection after multiple treatments with praziquantel (PZQ). This thesis is divided into four chapters. In Chapter 1, a systematic review was conducted using articles indexed in PubMed, totaling 396 studies evaluated with respect to various aspects of vaccine development against schistosomiasis. It was observed that the emergence of new technologies has significantly impacted preliminary studies in animal models. The majority of published studies used S. mansoni, with over 80% employing mice as the animal model. Furthermore, most of the proteins evaluated originated from the parasite’s tegument. In Chapter 2, epitope mapping was performed in rhesus macaques experimentally infected with S. mansoni. The animals were classified as high or low responders according to the rate of parasite clearance. Subsequently, sera from these animals were used in peptide microarray assays targeting proteins at the parasite–host interface (tegument, esophageal gland, and gut lining). The targeted epitopes were similar between the groups; however, the intensity of the immune response was greater in high responders. Overall, animals exhibited a stronger response against gastrointestinal tract-associated antigens. Based on these findings, 18 candidate epitopes were selected for further analysis. In Chapter 3, a convenience study was conducted in the endemic region of Conde-BA, involving individuals classified as resistant to reinfection (RR) and susceptible to reinfection (SR) after PZQ treatment. Serological analysis revealed that RR individuals had significantly higher levels of IgG1 against tegumental membrane extract (SmTEG). Epitope mapping using microarrays identified three epitopes preferentially recognized by the RR group, located in the tegumental proteins Sm25 and ADP- ribosyl cyclase. When compared to animal models (mice immunized with attenuated cercariae and infected rhesus macaques), 4 shared epitopes were identified across all approaches, which warrant further investigation. In Chapter 4, three chimeric proteins were designed (P1 – Esophageal gland, P2 – Tegument, P3 – Gastrodermis) each containing epitopes previously mapped in the murine model immunized with attenuated cercariae. The proteins were expressed recombinantly and purified via nickel affinity chromatography. P1 yielded the highest production, while P2 exhibited the highest purity. Initially, these proteins were used to immunize BALB/c mice, aiming to generate polyclonal antibodies through hybridoma production. P1 induced the greatest number of positive clones, while P2 and P3 yielded fewer. Sera from immunized animals were able to recognize native parasite proteins, as demonstrated by immunohistochemistry assays. The next step will involve functional evaluation of these polyclonal antibodies against schistosomula in vitro. Finally, an immunization and challenge assay were conducted in C57BL/6 mice, which received three doses of the chimeric proteins (administered individually or in combination), formulated with Alum as an adjuvant. After 21 days, the animals were challenged with S. mansoni. A robust IgG1 response against the proteins was observed. The group immunized with P1 showed a significantly higher ratio of IgG1 response compared to IgG2c, especially when compared to the P3 and pool groups. There was a trend of reduced parasitic load across all groups. Group P1 showed a significant reduction in egg count and female fecundity, while Group P2 exhibited a significant reduction in the adult worm burden. Therefore, we intend to further investigate the protective potential of these proteins. Editora / Evento / Instituição: Universidade Federal da Bahia Tipo: Tese

Avaliação do potencial terapêutico de vesículas extracelulares derivadas de células-tronco mesenquimais de cordão umbilical humano em modelo experimental de dermatite atópica em camundongos

2026-01-23T18:41:48Z

Título: Avaliação do potencial terapêutico de vesículas extracelulares derivadas de células-tronco mesenquimais de cordão umbilical humano em modelo experimental de dermatite atópica em camundongos Autor(es): Daltro, Sérgio Ricardo Teixeira Primeiro Orientador: Soares, Milena Botelho Pereira Abstract: Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczematous lesions and intense pruritus, posing a significant public health challenge. Conventional therapies, primarily based on corticosteroids and immunosuppressants, have limited efficacy and are associated with significant adverse effects. The therapeutic use of mesenchymal stem cells (MSCs) or their secretome, including extracellular vesicles (VEs), has shown promising immunomodulatory effects in the treatment of immune-mediated diseases. In this study, the therapeutic potential of VEs derived from human umbilical cord MSCs was evaluated in BALB/c mice with atopic dermatitis induced by epicutaneous application of 2,4-dinitrochlorobenzene (DNCB). MSCs were isolated, characterized, and used to obtain VEs through ultracentrifugation. VEs were characterized by size, polydispersity index, zeta potential, and morphology using transmission electron microscopy. The results demonstrated that VEs significantly reduced inflammatory markers, such as the number of mast cells, epidermal thickness and pro-inflammatory cytokines IL-4 and TSLP, in addition to promoting the recovery of skin barrier integrity, evidenced by an increase in lipids in the stratum corneum. Histological analysis revealed superior epidermal organization in the treated groups, while clinical evaluation indicated significant improvement in lesions. In an in vitro model of keratinocyte activation, VEs modulated the gene expression of several genes associated with AD. The therapeutic effects observed were comparable to those of dexamethasone, establishing VEs as effective immunomodulatory agents. The findings underscore the potential of VEs derived from MSCs in the treatment of atopic dermatitis, offering an innovative and promising approach with a lower risk of adverse effects. Editora / Evento / Instituição: Universidade Federal da Bahia Tipo: Tese

Modulação da migração de macrófagos e células dendríticas por leishmania: alterações funcionais e moleculares em ambiente tridimensional e no contexto da infecção in vivo

2026-01-14T18:54:55Z

Título: Modulação da migração de macrófagos e células dendríticas por leishmania: alterações funcionais e moleculares em ambiente tridimensional e no contexto da infecção in vivo Autor(es): Luz, Yasmin da Silva Primeiro Orientador: Fullam, Juliana Perrone Bezerra de Menezes Abstract: Leishmaniases can present with different clinical manifestations, ranging from localized skin lesions to visceral disease. The dissemination of infected cells is essential for disease establishment, but the mechanisms driving cell migration after infection remain poorly understood. Studies with macrophages infected by L. amazonensis have shown reduced 2D migration following infection; however, the process of transendothelial migration of macrophages involves regulatory signals within a 3D environment. Additionally, infection with L. braziliensis induces inflammatory mediators that enhance host cell recruitment, but the in vivo effects on bone marrow cell activation and their influence on the migration and response of macrophages remain unclear. In this study, we investigated the migration of macrophages and dendritic cells (Dds) in a three-dimensional environment, as well as the alterations induced in the bone marrow of mice infected with L. braziliensis and their potential role in modulating the migration of bone marrow-derived macrophages (BMMΦ). BMMΦ from mice infected with L. braziliensis were isolated after 2, 5, and 10 weeks of infection. These macrophages, along with DCs, were subjected to 2D and 3D migration assays in transwell systems, and adhesion complex formation was evaluated by immunostaining for pFAK and pPaxillin. Actin cytoskeleton dynamics were analyzed using Phalloidin, Rac1, Cdc42, RhoA, and gelsolin, while podosome formation was assessed by talin and vinculin staining. Analyses were performed by confocal microscopy, and the expression of matrix metalloproteinases (MMPs) was measured by a Luminex-based assay. To identify differentially expressed proteins, samples were characterized by liquid chromatography coupled with mass spectrometry (LC-MS/MS). It was observed that human and murine macrophages exhibit reduced migratory capacity in 3D environments, associated with decreased adhesion complex formation and alterations in actin dynamics after infection with Leishmania spp. In contrast, DCs showed increased migration in 3D environments following L. infantum infection, accompanied by enhanced adhesion complex formation and increased actin dynamics. Furthermore, a significant reduction in both 2D and 3D migration of BMMΦ was observed after in vivo infection with L. braziliensis, together with decreased expression of pFAK and pPaxillin at all evaluated time points. Rac1 and Cdc42 expression increased at weeks 2 and 5 but decreased after 10 weeks, while RhoA expression was consistently reduced throughout the study period. Vinculin expression increased at week 2 and decreased at week 5. MMP expression was elevated at week 2 and reduced at week 5. Proteomic analysis identified 26 differentially expressed proteins after 2 weeks of infection, 11 proteins after 5 weeks, and 6 proteins after 10 weeks. Understanding how Leishmania infection affects parasite dissemination and the recruitment of bone marrow cells in vivo is essential for elucidating the pathogenesis of leishmaniasis. Editora / Evento / Instituição: Universidade Federal da Bahia Tipo: Tese

Avaliação do efeito das células-tronco estromais mesenquimais superexpressando fator inibitório de leucemia (LIF) em modelo de endotoxemia induzida por lipopolissacarídeo (LPS)

2026-01-14T16:34:02Z

Título: Avaliação do efeito das células-tronco estromais mesenquimais superexpressando fator inibitório de leucemia (LIF) em modelo de endotoxemia induzida por lipopolissacarídeo (LPS) Autor(es): Oliveira, Alâna Costa de Primeiro Orientador: Soares, Milena Botelho Pereira Abstract: INTRODUCTION: Dysregulation of the inflammatory response can trigger a state of systemic hyperinflammation, as observed in sepsis—a severe clinical condition with high lethality, characterized by an uncontrolled inflammatory response to infection, with challenging diagnosis and treatment. Given the limitations of available therapies, innovative approaches such as the use of genetically modified mesenchymal stromal cells (MSCs) aimed at enhancing their biological activities are being explored in the context of sepsis. Among the molecular targets is the leukemia inhibitory factor (LIF), a pleiotropic cytokine with anti-inflammatory and regenerative properties. Although its mechanisms are not yet fully elucidated, the overexpression of LIF in MSCs emerges as a promising strategy to amplify their immunomodulatory effects in systemic inflammation. OBJECTIVE: To evaluate the therapeutic effect of MSCs overexpressing leukemia inhibitory factor (MSC-LIF) in a model of endotoxemic shock induced by lipopolysaccharide (LPS). MATERIAL AND METHODS: Initially, in vitro assays were conducted using murine peritoneal macrophages stimulated with LPS and interferon-gamma (IFN-γ) and treated with unmodified MSCs (MSC-WT) or MSC-LIF. Immunomodulatory activity was assessed by quantifying nitric oxide (via the Griess method) and cytokines TNF-α, IL-6, IL-12, and IL-10 in the supernatant using ELISA. For in vivo experiments, male C57BL/6 mice were subjected to an endotoxemic shock model via intraperitoneal injection of LPS. Thirty minutes after the challenge, animals were treated with MSC-WT, MSC-LIF, dexamethasone, or glucose solution (control). Survival was monitored for four days. Hematological and biochemical analyses, as well as serum cytokine quantification (TNF-α, IL-1β, IL-6, IL-12, and IL-10), were performed to evaluate the systemic inflammatory response. RESULTS: In vitro assays, MSC-LIF inhibited nitric oxide production by 95% at a concentration of 2×10⁵ cells and modulated the inflammatory profile by reducing TNF-α and IL-12 and increasing IL-6 and IL-10 levels compared to the positive control. In the endotoxemic shock model, the dose of 1×10⁶ MSC-LIF ensured 100% survival, while MSC-WT resulted in 50% survival. The lower dose of MSC-LIF (2.5×10⁵) also maintained survival above 50%, suggesting a potential protective effect. In the evaluation of serum cytokines, the MSC-LIF treated group showed increased IL-10 levels. A reduction in the inflammatory cytokines IL-1β, IL-12, and TNF-α was also observed compared to controls. Regarding hematological parameters, thrombocytopenia was attenuated in animals treated with MSC-LIF and dexamethasone, with platelet counts approaching those of the naive group (p < 0.05). Additionally, biochemical parameters such as creatinine and urea were normalized in the treated groups, suggesting preservation of renal function in the face of systemic inflammatory insult. CONCLUSION: LIF overexpression enhanced the immunomodulatory action of MSCs, contributing to improved survival and inflammation modulation in an endotoxemia model. Editora / Evento / Instituição: Universidade Federal da Bahia Tipo: Dissertação