ABSTRACT
INTRODUCTION. Elastofibromatous alteration of the gastrointestinal tract is generally characterized as a polyp with anomalous accumulation of metallic fibers mainly in the submucosa. It is little studied and described as isolated cases or small series. The largest series describes 13 cases. There are no reports of cases in Brazil. With the systematic use of colonoscopy in the prevention and early detection of colorectal cancer, polypoid lesions with characteristics of this change have been seen, but must be underdiagnosed. Differential diagnoses are important in the practice of gastroenterologists and pathologists. Its pathogenesis has not yet been clarified. OBJECTIVE. To the clinical and histopathological characteristics of a series of cases of colorectal elastofibromatous changes visible in a reference laboratory in gastrointestinal pathology. Check its frequency in relation to colonoscopy exams with biopsies submitted for histopathological evaluation. Based on specular morphological observations about the possible pathogenesis of this change. MATERIAL AND METHODS. Descriptive study of a series of cases presented in a laboratory in Salvador-Bahia, a reference in gastrointestinal pathology from January 2016 to October 2023. The reports were reviewed and information was recorded on age, sex, clinical manifestations, indication for the exam, topography and endoscopic description of the lesions. The histological slides, cut by hematoxylin and eosin, in addition to special stains to identify metallic fibers and connective matrix (Orcein, Verhoeff-Van Gieson, Weigert and Masson's trichrome) and amyloid deposits (Congo red), were reviewed. In eight cases, immunohistochemical evaluation was performed with CD34 and anti-smooth muscle antibodies to characterize blood vessels in the lesions. RESULTS. In 26 reports, 27 lesions were identified as elastosis and/or elastofibromatous changes in the topography of the large intestine. One patient had two synchronous lesions. Twenty-six lesions were identified by Hematoxylin-Eosin skin staining in conjunction with metallurgical fiber stains. One lesion required immunohistochemical evaluation for differential diagnosis with mesenchymal neoplasms. Eleven patients were male and 16 female. The average age was 60.31 years (ranging from 49 to 73 years). All lesions were described as polyps on endoscopy. For the eventual differential diagnosis with amyloidosis, Congo red staining under polarized light was used. Immunohistochemical evaluation with blood vessel markers showed an association of the lesion with anomalous blood vessels, consistent with vascular malformation. In none of the trained cases was there reference to extra-intestinal elastosis. CONCLUSION: Despite some frequencies, polyps with elastofibromatous changes are reported in services that receive a significant volume of colon and rectal polyps for histopathological evaluation. In this series of cases, these lesions were identified in 0.03% of colonoscopy exams performed from January 2016 to October 2023. Their morphological characteristics are distinct from polyps of a neoplastic or non-neoplastic epithelial nature, as well as other polyps. mesenchymal in nature and recognized by pathologists. The diagnosis is suspected on sections cut by hematoxylin-eosin and confirmed by using a histochemical method for metallurgical fibers. The histopathological characteristics
are peculiarities of amyloid accumulations and, when necessary, Congo Red staining excludes this possibility. In our series, all cases were submucosal in location and several of them showed characteristics similar to mucous-submucosal polyps with vascular anomalies, with vessels with muscular walls larger than normal in the submucosa. Histopathological analysis plus immunohistochemical evaluation suggests that the elastotic change is related to the wall of anomalous vessels with increased and disorganized deposition of metallurgical fibers and other elements of the connective matrix. Associated with this, there is a reduction in the lumen of the vessels, not only due to the hyperplasia of the metalworker, but also due to the deposition of conjunctiva matrix, indicating that these anomalous vessels, in their most distal portions, suffered thrombosis and a process of anomalous organization with reduction or disappearance of the lumen. vascular. Our data suggest an association between these elastofibromatous polyps with focal vascular anomalies in the submucosa of the large intestine.

ABSTRACT
Leishmania spp. infect several vertebrate hosts, including humans. The diseases caused by this protozoan, leishmaniasis, can present different clinical manifestations, from localized skin lesions that heal spontaneously, to disseminated skin lesions, mucocutaneous form of the disease, and visceral leishmaniasis, the most severe form that can lead to death if left untreated. The dissemination and homing of infected cells containing Leishmania antigens are crucial for the parasite's survival in the host and the establishment of the lesions. However, the understanding of the mechanisms underlying host cell adhesion and migration during infection by this protozoan remains limited. Therefore, this study aimed to evaluate the migration of human monocytes, macrophages, and dendritic cells in Leishmania infection, and the mechanisms involved in this process. Human host cells from healthy donors were obtained, infected with different isolates of Leishmania, and subjected to directional and random migration using a transwell system and real-time tracking, respectively. We also assessed the formation of adhesion complexes by immunostaining p-FAK and p-paxillin. Additionally, we investigated actin dynamics by analyzing the expression of Rac1, Rho-A, Cdc42, and phalloidin staining in these cells, as well as the formation of podosomes by immunostaining vinculin. Finally, we investigated the role of the AKT/PI3K signaling pathway in the migration of host cells infected by these parasites, through the analysis of protein expression by western blot, and the effect of inhibition of this pathway on the migration of infected host cells. Our results showed a reduction in the two-dimensional migration of human monocytes and macrophages infected with L. amazonensis, L. braziliensis, or L. infantum, associated with a decrease in the formation of adhesion complexes, podosomes, and actin dynamics in these cells. However, we observed an increase in the migration of dendritic cells infected by L. infantum or Leishmania isolates from patients with the disseminated or diffuse form of the disease. Additionally, we observed that the increase in migration in these cells is associated with an increase in the formation of adhesion complexes, podosomes, actin dynamics, and CCR7 expression. Finally, we observed that Leishmania infection induces activation of the AKT/PI3K pathway. Taken together, our data point to a role of dendritic cells, but not macrophages and monocytes, in the dissemination of Leishmania parasites in the vertebrate host.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is a health concern worldwide. Cellular interactions contribute to tumor aggressiveness through mechanisms such as the formation of cell-in-cell structures (CIC). However, only with the development of in vitro 3D models, it has become possible to study the biology of these events. OBJECTIVE: The objective of this study is to evaluate the model of cellular magnetization (bioprinting) for spheroid formation as a tool for the evaluation of cell-in-cell structures in oral squamous cell carcinoma. MATERIALS AND METHODS: OSCC metastatic cell line (HSC3) and cancer-associated fibroblast (CAF) line (CAF1) were selected to obtain tumor spheroids using the bioprinting system with NanoshuttlesTM (Greiner Bio-One). In 24-well repellent plates (Greiner Bio-One), cells were plated in groups of homotypic and heterotypic spheroids. The plating protocol was modified to eliminate the levitation step and reduce the number of magnetic beads. The spheroids were evaluated at 6h, 12h, 24h, 48h and 72h (Evos™ XL) and their unidimensional measurements were recorded (Image J version 1.8). Spheroids were sectioned to obtain slides, processed, and stained with hematoxylin and eosin (H/E). Slides were scanned (Axio Imager Z2/VSLIDE) and analyzes were performed using the OlyVIA viewer. Histomorphological evaluation was performed considering cellular morphology and formation of tumor zones (hypoxia and proliferative zone). CIC structures were counted considering the percentage of CIC events in the total number of cells in areas chosen from the center and periphery of the spheroids. To identify the CIC structures, the morphological pattern of “signet ring” or “bird's eye” was considered. RESULTS: With only 6h of bioprinting, tumor spheroids were already formed. The modified protocol allowed the formation of homotypic and heterotypic spheroids of reproducible size and integrity. In the analysis of H/E stains, there was a reproduction of spatiality and morphology aspects compatible with the tumor microenvironment, with formation of hypoxia (center) and proliferative zones (periphery). Cellular loosening and hyperchromatism was observed at the periphery of the spheroids. CIC structures were found in all groups, predominantly in the proliferative zone of mixed spheroids with CAFs. Events of “complex cannibalism” have been recorded, in which an encompassed cell contains another within it. CONCLUSION: The bioprinting model is useful to obtain homotypic and heterotypic tumor spheroids that reproduce histomorphological characteristics of oral squamous cell carcinoma. The occurrence of CIC structures was higher in the presence of FACs and in the proliferative zone of spheroids. This model can be used in further studies of CIC events, their formation mechanisms, and their relationship with tumor prognosis markers.

ABSTRACT

INTRODUCTION: Leishmaniasis are diseases of public health importance, endemic in 98 countries, affecting 12 million people worldwide. In Brazil, the prevalent clinical form is LCL, and the main etiologic agent is Leishmania braziliensis. Control and pathology in LCL are determined by a complex interaction between a variety of cell types and soluble factors, associated with Th1/Th2/Th17 cellular responses, which is poorly understood. In this sense, analysis and comparison of the immunological profiles of different biological compartments can be useful to understand the interaction and activation of the immune system, and the mechanisms associated with the systemic and in situ response. OBJECTIVE: To characterize and identify the predominant immune response in Localized Human Cutaneous Leishmaniasis caused by L. braziliensis. METHOD: We evaluated the systemic and in situ immune response of patients with LCL recruited from a reference clinic in Jiquiriçá, an endemic area of Bahia. Plasma and lesion biopsy samples were used to perform immunoassays using Luminex™ to identify levels of cytokines, chemokines, growth factors and soluble receptors. RESULTS: The results showed a predominance of the Th1 profile in patients with LCL, with high levels of pro-inflammatory and regulatory molecules. Plasma from patients with LCL showed a significant increase in IL-8, IL-1β, TNF-α, IL-6, MIP-1α, MIP-1β, GM-CSF, VEGF and TGFβ-1. In the lesion we found a significant increase in IL-8, IL-6, TNF-α, IL-1β, IFN-γ, IL- 12(P40), IL-17A, IL-2, IL-7, MCP-1, MIP-1α, MIP-1β, IP-10, Eotaxin, G-CSF, GM-CSF and VEGF and IL-10. Only sIL-2Rα and sTNFRII receptors showed significantly increased levels in the lesion biopsy. CONCLUSION: It was possible to demonstrate that the LCL systemic and local immune response profiles converge, however the local immune response is more expressive. The predominant immune response, in the late phase, is of the Th1 type, mediated by inflammatory molecules, but regulatory elements and soluble receptors can modulate the possible damage caused by inflammation.

ABSTRACT
INTRODUCTION: Leishmaniasis is a tropical disease caused by an obligatory intracellular protozoan of the genus Leishmania. Different species of Leishmania have developed distinct mechanisms of immune evasion, such as the interruption of cell signaling and metabolic processes, in order to obtain an effective infection in the host’s mononuclear cells. However, how different Leishmania species impact metabolism remains poorly understood. AIM: Therefore, the aim of this study is to dissect the metabolic signature of the two species of Leishmania: L. amazonensis and L. braziliensis, in the infection of human monocytes. MATERIALS AND METHODS: Human monocytes were collected from the peripheral blood of healthy donors and infected with Leishmania spp. for 18 hours, and the rates of extracellular acidification (ECAR) and oxygen consumption (OCR) were measured using Seahorse. Additionally, lactate and nitric oxide production were assessed using spectrophotometry. Monocytes were pre-incubated with different inhibitors of the main metabolic pathways to verify the minimum inhibitory concentrations that do not provoke cell death. Furthermore, the percentage of infected cells and the number of parasites per cell were evaluated following treatment with metabolic inhibitors. In addition, we evaluated mitochondrial mass and membrane potential using “MitoTrackers” probes by flow cytometry. The production of mitochondrial and cellular reactive oxygen species (ROS) was measured using “MitoSox” and “Cellrox” probes, respectively, through fluorescence microscopy. Cytokine levels were determined using Luminex. The obtained data were then subjected to statistical analysis, and values were considered significant when p ≤ 0.05. RESULTS: Our data demonstrate that monocytes infected with L. amazonensis and L. braziliensis exhibit a similar metabolic profile, leading to reduced extracellular acidification and oxygen consumption. When monocytes are treated with different inhibitors of metabolic pathways prior to infection, there is an increased lactate production, however, when monocytes are treated after infection, there is a decrease in lactate production. Infected monocytes treated with inhibitors targeting glycolytic, mitochondrial, β-oxidation, and
fatty acid synthesis pathways showed a decrease in parasite load, highlighting the importance of these pathways for parasite survival. Furthermore, we observed that infection with both Leishmania species increased mitochondrial mass and membrane potential, as well as interferences in monocytes metabolism are related to increased oxidative stress, leading to the production of cellular reactive oxygen species (ROS) and mitochondrial ROS (mtROS), which aid in parasite elimination. CONCLUSION: This study demonstrates that infection with L. amazonensis and L. braziliensis exhibits a similar profile in altering cellular metabolism. Inhibiting host metabolism is relevant for parasite elimination, underscoring the importance of interfering with cellular metabolism to eradicate the infection. This work highlights the potential of monocyte cellular metabolism as a future therapeutic target for modulating Leishmania spp. infection.

ABSTRACT Introduction: Chikungunya disease, caused by the Chikungunya virus (CHIKV), is characterized by an acute febrile syndrome with frequent arthralgia and a progression to chronic musculoskeletal manifestations in 15-86% of cases. Little is known about the infection in children and adolescents. Objective(s): We aim to understand the characteristics and dynamics of CHIKV infection in a pediatric population in epidemic and non-epidemic settings. Materials and Methods: This is a nested cohort study within the phase III clinical trial of the dengue vaccine at the Butantan Institute - DEN-03-IB. Children and adolescents from the municipality of Simões Filho, Bahia, Brazil, were included between 2018 and 2023. Arbovirus surveillance was conducted during medical visits for: 1. routine check-ups, scheduled periodically with blood collection; 2. adverse events, in case of any symptoms or illness; and 3. fever, with blood collection for Chikungunya, Dengue, and Zika RT-PCR testing. During the latter two types of visits, a structured questionnaire was administered to assess arbovirus symptoms (headache, arthralgia, arthritis, myalgia, retroocular pain, diarrhea, abdominal pain, and vomiting). For the sub-study, clinical and laboratory data from visits with up to 4 years of follow-up were evaluated, including arbovirus symptom persistence. Additionally, ELISA for Anti-CHIKV IgG was performed on admission (T1) and at the end of follow-up (T2). Chronic Chikungunya was defined by the persistence of arthralgia for more than three months after infection. Results: A total of 348 volunteers were included, with 52% females, and a median age of six years (interquartile range - IQR - 3-10). At T1, 23 out of 348 (7%) tested positive for Anti-Chikungunya IgG. Among the 325 seronegative volunteers at T1, 311 completed the follow-up, with an average duration of 41 months (IQR 38-43). Of the 311, 53 (17%) were infected with CHIKV, with 25 cases detected through RT-PCR and 28 through serology only. Among these 28 cases, 2 (7%) were asymptomatic. The 25 RT-PCR positive cases were followed for 29 months (IQR=25-34) after acute infection, with 3 (12%) developing Chronic Chikungunya. One of them had clinical suspicion of juvenile rheumatoid arthritis after CHIKV infection. Regarding the distribution of cases in the municipality, 17 out of 37 analyzed neighborhoods (46%) had positive cases. The regions with the highest number of cases were Region 2 (39) and Region 1 (10). Conclusion: Approximately one-fifth of children and adolescents were exposed to CHIKV before or during the follow-up period. Among those followed, infection was symptomatic for at least one-fifth of them. Although with low frequency, chronic arthralgia was observed in the pediatric population.

ABSTRACT
INTRODUCTION: Oral squamous cell carcinoma (OSCC) is an aggressive neoplasm with high morbidity and mortality, and despite scientific advances related to its biology, these have not yet had a positive impact on the therapy and prognosis of this tumor. The Hedgehog (Hh) signaling pathway's reactivation stands out in the CEO's pathogenesis and its inhibition represents a promising therapeutic target. In this context, the Hh pathway interacts with autophagy and this relationship represents an emerging opportunity to optimize this strategy, and among the autophagy inhibitors only chloroquine and hydroxychloroquine have approval for therapeutic use in humans. OBJECTIVE: To study the biological effect of GANT61 on autophagy in metastatic CEO cells. METHODOLOGY: The cytotoxicity of GANT61 (18 and 36 μM) and chloroquine (10 μM) in HSC3 cells was initially evaluated by Alamar Blue method for 72 hours. Cell viability assays were performed using CellTiter-Glo®️ for 12, 24 and 48 hours of treatment of GANT61 (18 and 36 μM), chloroquine (10 μM) and the association of these drugs. Cell morphology was evaluated in the high-content system by marking the cytoskeleton with phalloidin 568 after treatment of the conditions for 24 hours. The expression of autophagic flux proteins (LC3 and P62) was evaluated after treatments by Western Blot and immunofluorescence, and subsequently analyzed using the Operetta HTS imaging system. Ultrastructural evaluation of HSC3 cells after 24 hours of treatment was performed by transmission electron microscopy (MET). RESULTS: Analysis of the cytotoxicity of GANT61 and chloroquine in HSC3 cells demonstrated CI50 of 36 μM and 11.6 μM, respectively. The therapeutic association of GANT61 with chloroquine demonstrated reduced cell viability and increased changes in HSC3 cell morphology. The accumulation of LC3 and P62 expression was observed more markedly in combinations of GANT61 (18 and 36 μM) with chloroquine (10 μM) within 24 hours of treatment, as well as the presence of autophagosomes in HSC3 cells treated with GANT61 alone and in combination. CONCLUSION: The association of the compound GANT61 with the autophagic inhibitor, chloroquine, promotes an accentuation of the cytotoxic effect of this compound in HSC3 cells, which may indicate the cytoprotective role of autophagy in oral squamous cell carcinoma cells.

ABSTRACT
Introduction: Pandemics are recurrent events in human history, and COVID-19 stands out
with 767 million cases and 6.9 million registered deaths. In Brazil, the health crisis was
exacerbated by socioeconomic inequality and the country's magnitude, resulting in a rapid
increase in cases that overwhelmed healthcare services and put great pressure on frontline
professionals. With the aim of optimizing resource allocation, understanding disease-related
factors, and supporting healthcare professionals, we sought to identify clinical and
epidemiological determinants associated with patient diagnosis and prognosis as well as
professional burnout. Methodology: This thesis comprises four longitudinal and analytical
manuscripts addressing different aspects related to COVID-19. Results: Initially, differences
were observed in the epidemiological and symptomatic profiles of viral agents causing severe
lower respiratory tract infections in children during the pandemic. The atypical and exacerbated
prevalence of Respiratory Syncytial Virus (RSV) throughout the year stands out. Furthermore,
after adjustments for comorbidities, age, seasonality, and country region, an inverse association
was found between SARS-CoV-2 and respiratory discomfort symptoms, which were directly
related to RSV. On the other hand, Influenza was associated with fever and sore throat. The
second manuscript describes the development and internal validation of the COPS score,
demonstrating an accuracy of 88% in the prognosis of patients attended in the emergency
department. Additionally, we calibrated the pShock score, previously developed by our team,
to assess the prognosis of COVID-19 patients in the intensive care unit. The pShock score
showed an accuracy of 80% and outperformed the QSOFA and CURB-65 scores. Lastly, it was
identified that alcohol consumption was associated with the development of burnout syndrome
among healthcare professionals working in the emergency department or ICU. On the other
hand, working in the intensive care unit and engaging in daily prayers were inversely associated
with this phenomenon. Conclusion: The manuscripts comprising this thesis provide relevant
information about viral manifestation, prognosis, and professional burnout in COVID-19,
serving as a basis for fair resource allocation and addressing future challenges.

INTRODUCTION: LPG is a virulence factor that, along with other molecules such as PPGs,
PGs, and sAP, participates in a variety of processes during the establishment of infection in the
mammalian host. The GDP-mannose transferase encoded by the lpg2 gene is one of the essential enzymes for the synthesis of these molecules containing phosphoglycans (PGs) in their structures. The CRISPR/Cas9 system has been effective in obtaining Leishmania parasites deficient in various key molecules in the parasite-host cell interaction. OBJECTIVES: This
study aims to use the CRISPR/Cas9 system to target the lpg2 gene in L. infantum and L.
amazonensis (LCL and DCL), etiological agents of visceral and cutaneous forms of leishmaniasis, respectively. METHODS: In L. infantum, genome editing was performed by inserting stop codons with subsequent selection using agglutination assays. In L. amazonensis,
we inserted a resistance marker gene at the Cas9 cleavage site. In in vitro assays, human
neutrophils or BMDM were infected with L. infantum or L. amazonensis WT or Δlpg2. The in
vivo evaluation involved the inoculation of L. amazonensis WT or Δlpg2 promastigotes into the
ear of BALB/c mice. RESULTS: During the process of obtaining L. infantum Δlpg2, we detected and characterized that this gene was duplicated in this species. The sequencing results showed the expected genome editing occurrence in all clones of the two species. Furthermore, the Western blot result showed the complete loss of LPG and PG expression. We found that the deletion of the lpg2 gene in L. infantum decreased the infection rate (83%) in neutrophils, while for L. amazonensis, in vitro assays did not reveal differences in infection and replication rates between WT and Δlpg2 parasites. In vivo, the DCL Δlpg2 isolate showed minimal differences in lesion development compared to the WT, while the LCL Δlpg2 isolate failed to induce any lesion development. CONCLUSION: The results reinforce the importance of LPG and other PGs as virulence factors for L. infantum. For L. amazonensis, the absence of PGs does not seem to be essential for in vitro virulence assays. In contrast, PGs appear to play an essential role in lesion development induced by the LCL isolate. This work expands the knowledge of the function of glycoconjugates in New World Leishmania species.

ABSTRACT
INTRODUCTION: Tuberculosis (TB) is an infection caused in humans mainly by Mycobacterium tuberculosis (Mtb) and constitutes a serious global public health problem. Mtb is an intracellular pathogen that has a tropism for humans and mainly causes pulmonary tuberculosis. Tuberculosis co-infection in people living with HIV (PLHIV) is of great concern at a global level, since TB is the infection that affects them most and the main cause of death due to intracellular pathogens in PLWH. Autophagy, in turn, is a physiological process responsible for the removal of non-functional cellular components and under conditions of nutrient deprivation, functional cellular components can be digested to generate energy for the cell. This mechanism involves the formation of a double-membrane vesicle called an autophagosome, which encompasses components of the cytoplasm, including macromolecules and organelles and, after fusion with the lysosome, digests the contents through the action of lysosomal enzymes. Autophagy has been associated with the intracellular elimination of pathogens such as Mtb and HIV. METHODS: The present study evaluated the expression of genes related to autophagy in volunteers infected with Mtb or HIV and co-infected by Mtb and HIV and the expression of LC3 in macrophages derived from THP-1 lineage monocytes infected with different mycobacteria to understand how autophagy can modulate and influence tuberculosis infection and HIV co-infection. RESULTS: From the evaluation of the gene expression profile related to autophagy, it was possible to observe that most of the autophagy pathway genes had a higher expression in the TB and TB-HIV groups. The ATG4B, ATG4C, ATG7 and LGALS3 genes separated the TB and TB-HIV groups, while the ATG9A, IFNA10 and THBD genes separated the LTBI and HIV groups from the others. In experimental conditions, the expression of LC3 showed different levels of LC3 expression in different conditions, while LC3 expression was higher in the condition without stimulation and with Mtb H37Rv infection after 24 hours, this expression was higher after 48 hours. In the condition of cells infected and incubated with rapamycin, it was possible to observe a higher expression of LC3 after 48 hours in the conditions, without infection, infection with M. bovis (BCG) and in the condition of infection with Mtb from the highly prevalent clinical isolate 76937 CONCLUSIONS: The ATG4B, ATG4C, ATG7 and LGALS3 genes were able to separate the active TB and TB-HIV groups from the others, suggesting a relationship between these and active TB, while the ATG9A, IFNA10 and THBD genes were able to separate the groups LTBI and HIV. Mtb virulence factors may influence the development of autophagy, since the LC3 gene has been suggested to have different levels of expression in different Mtb clinical isolates; The time of autophagy induction through rapamycin may influence the different levels of LC3 expression observed in macrophages infected by different mycobacteria.

ABSTRACT Oral squamous cell carcinoma (OSCC) is a serious public health problem with a high prevalence in populations with low socioeconomic status, and is often diagnosed at a late stage, when the prognosis becomes poor. There are still few drug options for the treatment of this tumor in patients. Previously, we demonstrated that the embryonic Hedgehog (HH) pathway is reactivated in this tumor, leading to a more aggressive profile. Thus, the search for drugs that inhibit the HH pathway, either by inhibiting it’s canonical or non-canonical activation, becomes of great relevance for the treatment of OSCC. Among the non-canonical activation mechanisms of the HH pathway, the interaction with PI3K/AKT, an important cell survival pathway, stands out. The aim of this work was to evaluate in vitro antitumor activity and the pharmacological inhibition of components of the HH pathway by a pharmacological PI3K inhibitor, Pictilisib, in a metastatic line of OSCC. Thus, treatment of HSC3 cells with Pictilisib at different concentrations was performed to evaluate cytotoxicity, cell viability, apoptosis analysis by annexin-PI assay in flow cytometry, cell migration analysis by scratch assay, in addition to the evaluation of expression of the components of the HH pathway by Western blot, Immunofluorescence and RT-qPCR. Pictilisib presented a cytotoxicity profile in the HSC3 line, with an IC50 value of 0,05 µM. At concentrations of 0.97 μM and 1.94 μM, a significant reduction in cell viability was demonstrated, in addition to an increase in the percentage of cells in the sub-G1 phase after 48 and 72 h of incubation with Pictilisib at a concentration of 0.97 μM. The Annexin-PI assay demonstrated induction of apoptosis 48 and 72 h after incubation with Pictilisib at concentrations of 0.97 µM and 1.94 µM. The cell migration assay demonstrated that treatment with Pictilisib reduced cell migration at 6 and 24 h. Through the Western blot technique, PTCH1, SHH and GLI1 proteins were analyzed after treatment with Pictilisib in a period of 24h at concentrations of 0.97 and 1.94 µM, but no difference was observed in the expression of these proteins with the treatment . Using the immunofluorescence technique, the proteins GLI1, GLI2 and SMO were analyzed in relation to their presence and location. The results demonstrate strong immunoexpression of the mentioned components, with nuclear labeling of proteins GLI1, GLI2 and SMO at both concentrations with Pictilisib. The gene expression of the components of the Hedgehog pathway (GLI1, GLI2 and GLI3) was evaluated by RT-qPCR in the HSC3 line after 24 h of incubation with 1.94 µM Pictilisib. No inhibition of GLI1 and GLI2 mRNA expression was observed when treated cells were compared with untreated control. However, an increase in GLI3 mRNA was observed after incubation with Pictilisib at 1.94 µM. In conclusion, the data from this study support a potential role of Pictilisib as a promising drug for therapeutic use in OSCC. The role of this drug in the interaction of PI3K/AKT with the HH pathway deserves further investigation in future studies.

ABSTRACT
Introduction: Visceral leishmaniasis (VL) is an infectious disease caused by Leishmania infantum, which internal organs are compromised. In the spleen the infection is chronic and progressive. In addition, in severe VL, there is disruption of splenic compartments, a decrease of B cells number in the white pulp (WP) and plasmacytosis in the red pulp (RP). Plasma cells have extended survival and accumulated in many chronic diseases. However, poorly is known about the functions and mechanisms of generation and maintenance of cell survival. Objective: In this work, we investigated the potential mechanisms associated with the abnormal development of B cells that lead to plasmacytosis during VL. Methods: 56 Golden Syrian hamsters with 6 to 8 weeks were used in this study: 28 were injected intraperitoneally with 1x107 L. infantum promastigotes and 28 received the diluent solution by the same route. Seven animals from each group were euthanized 30-, 60-, 120- and 150-days post-injection (dpi). The animals were necropsied and organ samples were fixed in paraffin for histological study and evaluation of B cell and plasma cell distribution by immunohistochemistry. For three animals from each group and at each point, a spleen fragment was collected for transcriptomic evaluation of gene expression and association between molecules related to B cell development, including a molecule called DLK1. Results: There was an increase in the WP of the infected animals at 120 dpi, while at the 150 dpi there was atrophy affecting the follicle, which might be related to a decrease in B cells. We also observed a reduction in B cell chemotaxis, due to the decrease in CXCL13 and CXCL12 that occurred at 120 and 150 dpi. We noticed a toward to decrease molecular factors related to the germinal center (GC) response, observed by the gene expression of infected hamsters. There were no changes in the amount of B cells in the RP of the infected animals, but there was progressive plasmacytosis in the RP of the animals at the later stages of the disease, 120 and 150 dpi and the accumulation of these cells in the T cell zone at 120 dpi, DLK1 was associated with the plasmacytosis in RP. The gene expression of infected animals confirmed the differentiation of plasma cells at 150 dpi and associated signaling by IL-21 and IFN-γ such the main molecules involved. In addition, there was an increase in IL-6 150 dpi. Conclusions: In this study we correlated structural and cellular changes with molecular expression to investigate the determinants of B cell anomaly in severe forms of VL. There were increased of IL-21 and IFN-γ related with the B cell differentiation and plasma cell generation to latter disease stages. In addition, we demonstrated a decrease in CXCL13, CXCL12 and increase in IL-6. Moreover, there was an increase in the proportion of B cells at 120 dpi but decrease at 150 dpi in WP and plasmacytosis in RP.

ABSTRACT
INTRODUCTION: The Dengue virus (DENV) has been circulating in Brazil for approximately 35 years, in the year 2014, the Chikungunya virus (CHIKV) was first identified in the country, succeeded by the introduction of the Zika virus (ZIKV) the following year. The circulation of these 3 arboviruses through the Brazilian territory resulted in major epidemics that hit with greater force the Southeast and Northeast regions of the country, in addition to acute cases, severe sequelae of these infections such as chronic arthralgia and microcephaly, contributed to the understanding that in this epidemic scenario of high complexity the development of research and strengthening in the surveillance was not only necessary but also urgent. Despite the efforts of the scientific community, surveys aimed at evaluating the impact and susceptibility of populations to these arboviruses are still scarce in the literature, especially in rural regions of the country far from large urban centers. OBJECTIVE: To estimate the prevalence of positivity for specific antibodies markers of previous infections by Zika virus, Dengue virus, and Chikungunya virus, in residents of urban and rural areas of a municipality in the state of Bahia, far from the capital Salvador and its metropolitan region. METHODOLOGY: This is a cross-sectional study conducted in the municipality of Conde-BA, in three rural villages and urban areas near the city center, in which individuals aged 4 years and older were included from whom blood serum samples were collected. These samples were used to perform ELISA to detect anti-DENV, anti-ZIKV, and anti-CHIKV antibodies. All samples that tested positive on the anti-ZIKV ELISA were submitted to a PRNT, with an endpoint of 90% (PRNT90). RESULTS: A total of 328 samples were evaluated. Of the total 144 (43.9%) seropositive for DENV, 57 (17.4%) for Zika, and 18 (5.5%) seropositive for CHIKV. Among the seropositive samples, 118 (77.6%) were from urban areas of the municipality and 109 (51.7%) samples were from female participants. In the age group between 30 and 59 years, the highest frequency of positivity was observed, in this same age group, an association with seropositivity for arbovirus was observed mainly among residents of rural areas (PR: 6.86; 95%CI: 2.16-21.78), a positive association is also observed among participants with income over 1 minimum wage (PR: 1.44; 95%CI: 1.12-1.84). After adjustment of the regression model, an association with seropositivity is observed only among individuals with monthly family income greater than 1 minimum wage (PR: 1.30 95%CI: 1.02-1.65) and non-black participants (PR: 1.31; 95%CI: 1.00-1.69). CONCLUSION: The evaluated data indicate that previous exposure to DENV and CHIKV in the population living in urban sectors of the city is similar to that found in other studies developed even in large urban centers. It is also possible to observe that there has probably not yet been expressive circulation of ZIKV and CHIKV in the rural areas studied, which indicates these individuals as susceptible to future epidemics. The identification of susceptibility is an important factor for the implementation of prevention and control measures and encourages the development of studies in this and other regions with similar characteristics.

ABSTRACT
INTRODUCTION: Macrophages are important cells in inflammatory processes, and their
deregulation can affect several diseases, such as autoimmune diseases and parasitic diseases.
Leishmaniasis, a group of neglected tropical diseases, is included in this category of diseases.
Cutaneous leishmaniasis is characterized by ulcers in the tegument area and its severity is
related to immunopathogenesis. The available treatment is cytotoxic, associated with
therapeutic failure, parasite resistance, high cost and prolonged administration time. Physalin
F has promising pharmacological properties with potent antileishmanial and
immunomodulatory activity in mice. OBJECTIVE: This study aimed to evaluate the
immunomodulatory role in human macrophages and the anti-Leishmania braziliensis activity
of physalin F in vitro. MATERIAL AND METHODS: Human macrophages were obtained
from PBMC from healthy donors. The dosage of inflammatory cytokines was performed by
the ELISA method in the supernatant of human macrophages stimulated with LPS and with or
without the addition of physalin F and dexamethasone. The cytotoxicity of physalin F on
human macrophages and the viability of L. braziliensis promastigotes were measured using
Alamar Blue®. The action of physalin F on human macrophages was determined after 24
hours of infection with L. braziliensis. Flow cytometry and electron microscopy assays were
performed to evaluate the possible mechanisms of action of physalin F in L. braziliensis
promastigotes. RESULTS: Physalin F reduced the production of the pro-inflammatory
cytokines IL-6, IL-1β and TNF-α by activated macrophages. In the cytotoxicity assays, it
presented a CC50 value of 6.07 ± 1.17 μM. This compound also inhibited the proliferation of
promastigote forms of L. braziliensis, with an IC50 value of 10.85 ± 0.99 μM, and reduced the
number of infected human macrophages and the number of amastigotes per macrophage,
when compared to controls. In promastigotes, the appearance of lipid inclusions, rounding of
the cell body, destruction and loosening of the cell membrane were observed in the
ultrastructural analysis by MEV and MET after treatment with physalin F. Flow cytometry
analyzes indicate that physalin F induces apoptosis in L. braziliensis. CONCLUSIONS:
Physalin F inhibited the activation of human macrophages and promoted an effective anti-L.
braziliensis activity, in both promastigotes and amastigotes.

INTRODUCTION: MSCs have become targets of clinical and preclinical studies due to their anti-inflammatory and immunoregulatory potential. It is known that part of the effects of MSCs are related to their secretome, which includes soluble factors such as cytokines, proteins and the release of extracellular vesicles (EVs). EVs are small cellular components, which have bioactive molecules (small RNAs, proteins, among others) that were inherited from the cells of origin and develop immunomodulatory and trophic actions. The analysis of parameters involving the immunomodulatory profile of MSC-derived EVs (MSC-EV) is essential for important assays for clinical development. OBJECTIVE: To characterize the in vitro immunomodulatory activity of hucMSC-EVs in a lymphoproliferation assay. MATERIAL AND METHODS: Human Wharton's jelly-derived MSCs (hucMSCs) were used for the isolation and purification of hucMSC-EVs. The hucMSCs were characterized according to ISCT by analyzing morphology, immunophenotyping and differentiation assays. By RT-PCR we evaluated the expression of genes related to EVs biogenesis (CD81, CD63) and immunomodulatory potential (IDO1 and TNFAIP6). HucMSC-EVs were isolated in passage 5 and characterized following ISEV recommendations,by transmission electron microscopy (TEM), nanoflow analysis and nanoparticle tracking analysis (NTA). Blood mononuclear cells (PBMC) from healthy donors were stimulated with anti-CD3/ anti-CD28 beads and incubated with different concentrations of hucMSC-EVs (10,25 and 50 μg/ml) for 72 hours at 37 °C with 5% CO2, lymphoproliferation analysis was performed by luminescence ATP assay. Regulatory T cells (Treg-CD4+CD25+FoxP3+) were evaluated by flow cytometry. Cytokines in the supernatant were quantified by ELISA. RESULTS: Cultured hucMSCs showed fibroblastoid morphology, expression > 95% for CD90, CD105, CD44, and CD73, with <0.05% of hematopoietic markers, and the ability to differentiate into adipocytes, osteocytes, and chondroblasts. Deprivation of human platelet lysate was associated with a peak in gene expression of CD81, CD63 and TNFAIP6 after 24h, 48h and 72h. IDO1 expression was not detected. We observed that CM stability remains when stored at -20°C for 7 days. The hucMSC-EVs showed an average diameter <200 nm, showed typical morphology in TEM and phenotypic analysis demonstrated positive markers for EVs (Annexin, CD81, CD63) and for hucMSC origin (CD90). Treatment with hucMSC-EVs inhibited lymphoproliferation in vitro at all concentrations tested. Stimulation of Tregs was slightly increased after treatment with 50 μg/ml MSC-EVs. The production of TNF-a, IFN-y, and IL-10 was inhibited by incubation with MSC-EVs, whereas IL-6 showed no significant changes. CONCLUSION: HucMSC-EVs showed immunoregulatory effects through in-vitro lymphocyte inhibition and moderate Treg cell induction, but additional tests need to be performed to elucidate further effects on large-scale application of hucMSC-EVs.

ABSTRACT
Sickle cell disease (SCD) is one of the types of sickle cell disease (SCD), which is
characterized by hematological events such as hemolysis and endothelial damage that
culminate in vaso-occlusion, leading to the appearance of several clinical manifestations.
SCD is the result of erythrocyte alteration conditioned by a mutation in the HBB gene that
leads to the formation of hemoglobin S (HbS). Fetal hemoglobin (HbF) is known to
positively affect the clinical condition of individuals with SCA, by interfering with the role of
HbS in erythrocyte alteration. Genetic polymorphisms are known to positively modulate
clinical phenotypes of SCD, with the BCL11A gene being an important marker in this regard.
However, polymorphisms in the BCL11A gene have been described as negatively change the
clinical status of patients with SCD. The aim of this present study was associate laboratory
biomarkers in the presence of polymorphisms rs766432 (C>A) and rs6732518 (C>T) in the
BCL11A gene. Hematological and biochemical markers were investigated by automated
methods and genetic markers were identified by polymerase chain reaction and Restriction
Fragment Length Polymorphism (PCR-RFLP) techniques. Statistical analyses were
performed using the Graphpad prism software, version 9.0. HbF showed a statistically
significant association in the presence of the rs766432 polymorphism (p=0.0306) as well as
simultaneously with the rs6732518 polymorphism (p = 0.0302), and HbS concentration
(p=0.0464). The concentration of hemoglobin (Hb) (p=0.0008), hematocrit (Ht) (p=0.0243)
(p=0.0329) and red blood cell count (Hm) (p=0.0069) (p= 0.00466) were high in the presence
of polymorphisms. Total (p=0.0264) and direct (p=0.0039) bilirubin concentrations showed
lower values in the presence of the rs766432 polymorphism, as well as in the co-inheritance
of both polymorphisms (p=0.0073) (p=0, 0154). High concentrations of HDL cholesterol
were associated with the presence of the variant allele of the polymorphism rs766432
(p=0.0402) and high levels of alpha1-antitrypsin were associated with the presence of the
variant allele of the polymorphism rs6732518 (p=0.0320). There was no correlation between
HbF and HDL (r=0.03705). It is concluded that polymorphisms in the BCL11A locus are
important for the variation in HbF levels, and that they may have a pleiotropic effect by
determining laboratory parameters not related to HbF levels.

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis is still a worldwide public health problem. The transmission of TB occurs through the air, when the infected person releases the bacilli of Koch through the expulsion of droplets of Flügge, through coughing, sneezing or speaking, being suspended in the air and being inhaled by other people. Thus, the exposed person can become sick or with latent infection (ILTB). Over time, several research and initiatives in the world have been carried out to control TB, among them The Strategy for the End of TB of the World Health Organization, whose objective is to eliminate TB by the year 2035. Despite the implementation of the strategy, the scenario for the American region has not improved much due to the increase in the incidence of TB cases, with Brazil and Peru being the countries with the highest burden of the disease. Current knowledge of TB is needed to implement new approaches in health systems. This thesis work brings together eleven manuscripts that identify potential clinical and epidemiological determinants of susceptibility to M. tuberculosis infection and therapeutic response in TB patients. The first study uses innovative statistical tools to characterize the RePORT-Brasil cohort sample and compares it with the Brazilian national TB population, in addition to identifying factors associated with unfavorable outcomes in anti-TB treatment, such as the use of illicit drugs and co-infection with HIV and diabetes (DM) only in the RePORT-Brasil cohort. Screening contacts of TB cases and prophylactic treatment of those with ILTB are important for controlling disease transmission, we found that not completing the ILTB follow-up cascade was independently associated with increasing age, low socioeconomic status, and HIV co-infection. Furthermore, following the results of the first study, we demonstrated once again that DM is associated with an increased risk of unfavorable treatment outcomes, as well as mortality in patients with pulmonary TB, and we contrasted these results with those of SINAN. In the Peruvian court we find that persistent dysglycemia is also associated with unfavorable outcomes without treatment. Further, we also found that the presence of HIV did not substantially affect the clinical presentation in people with TBDM. Additionally, contacts of patients with TB and pre-DM were at risk of positive QuantiFERON at baseline and contacts of patients who had TBDM were at increased risk of having a conversion (QuantiFERON negative at baseline to positive at month 6). In screening for DM in TB cases, we identified differences regarding HbA1c and FPG values for the diagnosis of DM in the cohorts of Peru (where we found a high prevalence of DM and pre-DM in TB cases) and Brazil. Finally, in the Peru cohort, dysglycemia (DM and preDM) affected the presentation of lung lesions in TB cases and dietary patterns were identified in the food intake profile of the study participants. Thus, the manuscripts that make up the thesis add relevant information in identifying the clinical and epidemiological determinants of TB infection, transmission, and treatment, which aims to support strategies implemented in the health system.

ABSTRACT Mycobacterium tuberculosis (Mtb) infection affects approximately a quarter of the global population. Host genetic polymorphisms may be important in determining susceptibility to Mtb infection, but their role is not fully understood. In a first study, different SNPs were tested as risk factors for tuberculin skin test (TST)conversion and development of Tuberculosis (TB): TLR2 (rs5743708), TLR4 (rs4986791), TNFA (rs361525), IFNG (rs2430561), IL1B (rs1143627). In a second study, seven additional SNPs were tested for association with TT positivity: candidate genes IFI16-PYHIN1-AIM2 (rs1101998, rs1633256, rs866484), IFIT5 (rs59633641, rs10887959), IFIT1 (rs304478, rs730449.8), and IRF7 (rs11246213). Both studies were conducted on contacts of microbiologically confirmed pulmonary TB cases in reference laboratories. Finally, we performed a systematic review to assess the association between CD14 and NOD2 reported polymorphisms and Mtb diseases, and how this association might differ in distinct ethnic populations. In the prospective study, among the 526 participants, 60 had a conversion to TT, and 44 developed active TB during follow-up. Multivariate regression analysis demonstrated that SNPs in TLR4 genes (odds ratio [OR]: 62, 8, 95% confidence interval [95% CI: 7.5–525.3) and TNFA (OR: 4.2, 95% CI: 1.9– 9.5) were independently associated with TT conversion. In the retrospective studio outside 482 contacts were examined, of which 296 contacts had positive TT. In a multivariate model, we observed in the recessive model that PYHIN1-IFI16-AIM2 rs1101998 (adjusted OR [aOR] = 2.90; 95% CI = 1.24-6.78; p = 0.014) and rs1633256 (aOR = 10, 1; 95% CI = 2.20-46.28; p = 0.003) were associated with an increased risk of TT positivity. In the systematic review, thirteen studies met the selection criteria. Of these, nine investigated CD14 SNPs and six reported a significant association between the T allele and the TT genotypes of SNP rs2569190 and increased risk of Mtb disease. In addition, four studies reported data finding the relationship between NOD2 SNPs and Mtb disease risk, with two reporting significant associations of rs1861759 and rs7194886 and increased risk of Mtb disease in a Han Chinese population. The results suggest associations between immunityrelated genes polymorphisms and the probabilities of Mtb infection. This study contributes to the understanding of associations between immunity-related genes polymorphisms and the probabilities of Mtb infection

ABSTRACT
INTRODUCTION: The diagnosis of tuberculosis (TB) is performed by means of the detection of M. tuberculosis (MTB) in microbiological tests, such as smear and culture and molecular biology assays. The interpretation of these tests is limited, as they do not provide information on the systemic, cellular, and inflammatory repercussions and they have low sensitivity in paucibacillary individuals. OBJECTIVE: To evaluate cell activation and phenotypic changes in CD4+ T lymphocytes in individuals with tuberculosis, with or without HIV. METHODS: Initially, in a cohort from Brazil, the use of CD38, HLADR and/or Ki67 cell activation markers on MTB-specific CD4+ T cells to discriminate pulmonary tuberculosis (PTB) from extrapulmonary tuberculosis (EPTB) from latent tuberculosis infection (LTBI), as well as EPTB from PTB, was validated. We also tested the effect of HIV coinfection on diagnostic performance. Added to this thesis, another manuscript covering South Indian patients with TB-HIV before and after initiation of ART, determining the phenotypic changes in CD4+ T cells during Tuberculosis-immune reconstitution inflammatory (SIRI) and their relationship with systemic inflammation. In this study, we analyzed subpopulations of T lymphocytes and biomarkers in peripheral blood. RESULTS: EPTB and PTB patients had a higher frequency of T CD4+ IFN-γ+ cells expressing CD38, HLADR compared to LTBI. Furthermore, the frequencies of HLADR+ or Ki67+ cells accurately distinguished EPTB from PTB. HIV infection did not affect the ability of these markers to distinguish active tuberculosis from LTBI or EPTB from PTB. In the second study, the frequency of naive CD4 T cells (CD27+CD45RO−), as well as of effector memory (CD27−CD45RO+) distinguished individuals who developed the inflammatory reconstitution syndrome from those who did not, in the 2nd-6th week after starting ART. Further analysis revealed that ART reconstituted different amounts of subsets of CD4+ T lymphocytes with preferential expansion of CXCR3+ CCR6− cells in TB-SIRI patients. In addition, there was an expansion and functional restoration of CXCR3−CCR6 CD4+ lymphocytes expressing central memory markers (CD27+ CD45RO+) and corresponding cytokines, with a reduction in CXCR3−CCR6+ cells after ART only in those who developed TB-SIRI. CONCLUSION: Cell activation markers on CD4+ T cells specific for MTB distinguished active TB from LTBI and EPTB from PTB, regardless of HIV infection status. CD4+ T cell subsets are strongly associated with SIRI.

Trabalho com sigilo

ABSTRACT
INTRODUCTION: With millions of confirmed cases and deaths worldwide, the coronavirus disease 2019 (COVID-19) pandemic caused by the infection of the novel coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a worldwide concern. It is known that high levels of glucose in the bloodstream is one of the main risk factors for the worsening of the disease. However, the real mechanisms involved in the aggravation of COVID-19 in individuals with prediabetes and diabetes are not yet elucidated. OBJECTIVE: Thus, the aim of the present study was to evaluate the influence of diabetes and pre-diabetes on the worsening of COVID-19. MATERIAL AND METHODS: Initially, possible genes related to the severity of COVID-19 in individuals with diabetes were identified from public gene expression data in peripheral blood mononuclear cells (PBMCs). Then, we validated important targets for SARS-CoV-2 infection in PBMCs of patients with and without diabetes. Systemic inflammatory mediators and correlation analyzes were performed to identify factors associated with COVID-19 severity in patients with altered blood glucose. RESULTS: Public data analyzes revealed the leukotriene metabolism as a pathway potentially associated with respiratory conditions in patients with diabetes. Thus, we identified, during the acute phase of COVID-19, an increase in the expression of ALOX5 and ACE2/TMPRSS2 in PBMCs of individuals with diabetes. In these patients, we also found an increase in serum levels of LTB4 and IL-6 compared to patients without diabetes. The increase in IL-6 observed in individuals with diabetes was associated with longer intensive care unit admission. Taken together, the data show that diabetes, with the participation of the LTB4 pathway, induces a more severe disease, with more intense lung damage (gas exchange rates) and longer disease duration. Interestingly, patients with prediabetes, under the participation of IL-6 production, also develop to severe COVID-19 more frequently, considering the reduction in gas exchange rates and longer hospitalization time. However, prediabetes did not induce sequelae of COVID-19 distinct from those of subjects without diabetes. In addition, we also show that routine laboratory tests can be used to identify different producers of IL-6, which is related to the severity of COVID-19. CONCLUSION: The increased production of LTB4 and IL-6 seen in individuals with diabetes and prediabetes, respectively, may worsen the outcome of COVID-19.

Tuberculosis (TB) is a chronic infectious disease difficult to manage and the
mechanisms involved in disease progression are not fully established. The gene
expression profile has been studied with the objective of validating potential
biomarkers that help in the understanding of the processes involved in TB and in the
development of strategies against disease. Vaccination with BCG has been pointed
out with the possibility of modulating the host response and its efficacy against TB
has been widely discussed, but it is still not fully known about what these effects
would be and their duration in the organism. Through a meta-analysis of
transcriptome data using GEO repository, we were able to demonstrate that
vaccination with BCG is capable of generating lasting effects in the host affecting the
modulation of specific genes. When comparing the groups of uninfected, infected and
sick that were unvaccinated, 100 genes were observed (92 down-regulated and 8
up-regulated), mainly associated with pathways and functions linked to metabolism.
Among vaccinated individuals, comparing the same groups, 53 genes were
observed, being 18 overexpressed and 35 underexpressed, mainly linked to
immunity processes. In a secondary meta-analysis, we also found distinct
vaccine-linked signatures, with 5 underexpressed genes identified for the vaccinated
and 2 genes (1 over and 1 underexpressed) for the unvaccinated. Our results also
showed that a gene of the Rab11 subfamily (composed of two other members of high
homology, RAB11A and RAB11B), RAB25, was overexpressed in sick individuals
unvaccinated with BCG, when also compared sick individuals, but vaccinated. During
our evaluations, we identified RAB11A as the target of our study. In whole blood
samples from a cohort of patients with active tuberculosis from our population, we
observed higher mean expression values for the RAB11A gene in sick and
unvaccinated individuals compared to vaccinated, corroborating the meta-analysis
data. We also saw that the expression values of this gene are lower in THP-1 cells
infected with BCG strain compared to uninfected. For future investigation of the role
of this gene, we successfully performed gene silencing in THP-1 cells using RNA
interference methodology. With this work, we were able to identify the transcriptional
signatures associated with BCG vaccination and point out a target that deserves to
be better studied in tuberculosis.

ABSTRACT
INTRODUCTION: People living with HIV (PLHIV) often have anemia as a complication. Anemia in HIV patients may be associated with an exacerbation of the inflammatory profile, with an increase in blood cytokines and more accelerated disease progression. However, little has been reported on the impact of anemia severity on unfavorable clinical outcomes during treatment of these patients with antiretroviral drugs, such as incident tuberculosis (TB), immunoreconstitution inflammatory syndrome (IRIS) and death. OBJECTIVE: To characterize the inflammatory profile of anemic patients with HIV, as well as to identify the associations between the presence of anemia and its severity with the unfavorable treatment outcomes of these patients. METHODS: This thesis brings together a set of six manuscripts that aim to characterize and associate anemia with the clinical outcomes of PLHIV. Altogether, data from 1617 PLHIV were evaluated. Anemia and its severity were defined according to World Health Organization criteria. For each manuscript, a different PLHIV cohort was used, and in each of them the plasma levels of inflammatory markers were analyzed, as well as clinical, socioeconomic and laboratory data. Descriptive statistical methods and a set of multidimensional techniques were applied, including network analyses, integrative analyses, logistic regressions and calculation of the degree of inflammatory perturbation (DIP). RESULTS: In the first study, we concluded that anemic PLHIV are more likely to develop incident TB after starting antiretroviral treatment (ART) compared to non-anemic individuals. In the second, it was found that severe anemia increases the risk of IRIS, mainly from TB, during ART. In the third study, it was observed that anemia is associated with a greater dissemination of TB in PLHIV. In the three aforementioned studies, moderate/severe anemia are risk factors for death. The fourth study demonstrated that low hemoglobin levels were associated with higher DIP in HIV-TB patients. Next, we observed that HIV-TB patients with persistent anemia have a higher risk of dying than non-anemic patients during anti-TB treatment (ATT). This last result was reinforced by the sixth study, where moderate/severe anemia pre-ATT was a risk factor for death. CONCLUSIONS: Together, the manuscripts of this thesis add important information regarding the importance of anemia in the context of HIV-TB co-infection. By identifying moderate and severe anemia as a risk factor for incident TB, IRIS, disseminated TB, inflammatory disturbance, and death, we demonstrate that anemic PLHIV should be carefully monitored and, if possible, anemia should be carefully evaluated as a hallmarker of possible opportunistic infection and worse prognosis. In this context, this information is useful in directing new approaches to optimize clinical management and improve the quality and life expectancy of PLHIV.

Abstract

INTRODUCTION: Tuberculosis-associated immune reconstitution inflammatory syndrome (SIRI) is a clinical picture of tuberculosis symptoms observed in a disease of patients co-infected with HIV shortly after initiation of antiretroviral therapy (ART). It is well known that TB-IRIS occurs as a function of exacerbated inflammation and tissue damage in response to the overproduction of IFN-γ derived from CD4+ T cells. Concomitant to several lymphocyte reconstitution, changes and different layers of biological organization as alterations and support to the pathological phenomena associated with SIRI. Several immunological studies stand out, but that contribute to the great evolution of the role of genetics in the development of T lymphocytes, but that contribute to the great evolution of the development of T lymphocytes, remains highlighted. OBJECTIVE: To evaluate the lymphocyte profile of patients starting antiretroviral treatment. This thesis was joined by another manuscript that investigated the application of multi-omics techniques to bioprospecting for indicators associated with TB-SIRI. METHODS: This was a retrospective study of TB-HIV patients from South India before and weeks after initiation of ART, in which there was a phenotypic characterization of lymphocytes and determination of the degree of systemic inflammation in these patients. RESULTS: We observed that SIRI is related to alterations in amino acid and lipid metabolism. Such metabolic pathways have been correlated with elevated pro-inflammatory mediators during SIRI. The second manuscript of this thesis revealed that patients who developed IRIS had marked CD4+ T-cell lymphopenia before starting ART and had high frequencies of CD8+ T-cells. In addition, IRIS episodes were associated with dysfunctions of lymphocyte activation dynamics, with elevation of CD4+ HLA-DR+ T lymphocytes and CD4+ and CD8+ T cells expressing granzyme B. We developed models based on machine learning algorithms that were able to predict and diagnose IRIS taking into account the frequencies of lymphocytes expressing molecules associated with cell activation. In the third manuscript, we observed that the reconstitution of the memory CD8+ T cell compartment is predominated by effector memory cells in patients who develop IRIS. In turn, the magnitude of the frequency variation of these cells is positively correlated with the abundance of pro-inflammatory cytokines. In patients who manifested TB-associated SIRI, there is retention of CD8+ T cells expressing CXCR3 in persistently inflamed sites and the frequency of these cells was able to distinguish patients with great accuracy. CONCLUSION: The data presented in this thesis confirm the role of lymphocyte activation in the immunopathogenesis of IRIS, and the subsets of these cells and the profile of metabolic alterations are capable of diagnosing or predicting the manifestation of this syndrome.

INTRODUCTION: Leishmaniasis present different clinical manifestations. Some Leishmania spp. cause chronic, slow-to-heal diseases that are known as cutaneous or mucocutaneous leishmaniasis. Other Leishmania spp. disseminate to internal organs such as the liver, spleen, and bone marrow to cause visceral leishmaniasis, leading to death if left untreated. The dissemination and homing of infected cells containing Leishmania antigens are essential for the survival of this parasite in the host. However, the mechanisms involved in the adhesion and migration of host cells during Leishmania infection is still poorly understood. A previous study showed that Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane– extracellular matrix interactions. However, in the tissues, cells migrate through the extracellular matrix, a three-dimensional fiber mesh. AIM: Thus, the present study aims to evaluate the migration of macrophages infected by Leishmania and the mechanisms involved in this process, using a three-dimensional environment. METHODOLOGY: For this, bone marrow-derived macrophages were infected by L. amazonensis, L. braziliensis, or L. infantum and placed in a collagen I matrix to assess migration using Operetta. Additionally, we evaluated adhesion complexes formation by the immunostaining of p-FAK and p-paxillin and actin polymerization by phalloidin, Cdc42, RhoA, Rac1, and gelsolin, using confocal microscopy or Operetta. Also, using matrigel, we evaluated the secretion of proteases by these cells. RESULTS: Our results show a reduced migration of macrophages infected by L. amazonensis, L. braziliensis, and L. infantum in a three-dimensional environment. In addition, we show that infected macrophages have lower levels of phosphorylated paxillin and FAK when compared to noninfected macrophages. Analysis of the dynamics of actin polymerization show an increase Rac1 expression in L. braziliensis- and L. infantum infected cells, in contrast with a reduction in Cdc42 in these cells. In addition, we show a reduced expression of RhoA and gelsolin in L. amazonensis- and L. infantuminfected cells. We also showed that the infection by L. amazonensis, L. braziliensis, and L. infantum does not alter the secretion of proteases by macrophages. CONCLUSIONS: Taken together, our results show that Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions in a threedimensional environment

Introduction: Leishmaniasis is a zoonosis caused by the protozoan parasite Leishmania ssp., which presents different clinical manifestations such as cutaneous leishmaniasis, mucocutaneous leishmania, and visceral leishmaniasis. The dissemination and homing of infected cells containing Leishmania antigens are crucial for the survival of the parasite inside the host and the establishment of infection. In addition to host factors, parasite factors are also important components for disease development. Recent studies have already demonstrated the relevance of LPG from Leishmania in host-parasite interaction, parasite survival inside the host, and the establishment of the infection. However, the role LPG plays in the migration and adhesion of human host cells is unknown. Aim: Thus, the objective of this work is to evaluate the role of lpg2 in the adhesion and migration of human host cells infected by L. infantum. Material and methods: For this, human dendritic cells and macrophages obtained from healthy donors were cultivated and infected by wildtype L. infantum or parasites knockout for the lpg2 gene and submitted to migration using a specific chemoattractant for these cells in a transwell system. Additionally, we evaluated the formation of adhesion complexes through p-FAK and p-paxillin evaluation, the polymerization of the actin cytoskeleton by phalloidin, and essential proteins in this process such as Cdc42, Rho and Rac-1, using confocal microscopy. Results: Our results show an increase in the migration of dendritic cells after infection by wildtype L. infantum. However, there was a reduction in the migration of dendritic cells infected by L. infantum lpg2 knockout when compared to those infected with the wildtype parasites. We also showed an increase in the adhesion complexes formation and polymerization of the actin cytoskeleton in dendritic cells infected by wildtype L. infantum when compared to those infected with the lpg2 knockout parasites. On the other hand, we found a reduction in the migration of macrophages infected by wildtype L. infantum. However, there was an increase in the migration of macrophages infected by L. infantum lpg2 knockout when compared to those infected with the wildtype parasites. Also, we found an increase in the adhesion complexes formation in macrophages infected by wildtype L. infantum and parasites lpg2 knockout. We also show a reduction in phalloidin and Rac1 expression in macrophages infected by wildtype L. infantum and lpg2 knockout parasites compared to noninfected cells. Also, we found an increase in RhoA expression in macrophages infected by wildtype L. infantum when compared to uninfected cells. However, there was a reduction in RhoA expression in macrophages infected by lpg2 knockout parasites when compared to those infected by the wildtype parasites. Conclusions: In conclusion, our results suggest that LPG from L. infantum is important for the modulation induced by these parasites in human host cells. Further experiments should be performed to confirm and better understand LPG's role in host cell migration during L. infantum infection.

ABSTRACT

INTRODUCTION: Sickle Cell Anemia (SCA) is an autosomal recessive hemoglobinopathy characterized by the mutation in the sixth position of the β-globin gene (GAG àGTG) that leads to the replacement of glutamic acid by valine, generating hemoglobin S (HbS). Its pathogenesis involves HbS polymerization, vaso-occlusion and sterile inflammation with subsequent complications and systemic changes. Sickle cell ulcers are complications that affect 8% to 10% of SCA patients. While it is well-established that sickle cell ulcer both results from genetic predisposition and induces inflammatory processes, the mechanisms involved, in particular, the participation of lipid mediators, remain to be better investigated.  OBJECTIVE: To characterize the hematological, biochemical, and inflammatory profile of transgenic sickle cell anemia mice and to evaluate the local and systemic alterations induced by cutaneous ulcer in these animals. MATERIALS AND METHODS:  After anesthesia, samples of whole blood, serum, organs and skin fragments of Townes B6:129 mice were collected in order to determine the baseline differences in biochemical, hematological and inflammatory parameters of healthy (AA), sickle cell trait (AS) and sickle cell anemia (SS) mice. In the murine model of sickle cell ulcer, four 4 mm skin lesions were induced on the back of the animals, and samples of these lesions were collected at the time-points 0, 24 and 72 h after ulcer induction. In these same evaluation periods, whole blood samples were separated for blood counts and leukogram analysis by semi-automated analysis, using URIT 5160 Vet equipment and blood smears with the Romanowsky method. Biochemical analyses of total cholesterol (TC), triglycerides, AST, ALT, urea and creatinine were performed in the serum by spectrophotometry by automated method in a smart vet 200+ equipment. The concentrations of PGE₂ and LTB₄ in the serum were evaluated by ELISA. The expression of genes associated with the production of eicosanoids such as COX-1, COX-2, 5-LO, PLA₂ and PLIN₂ in skin lesions was determined by RT-qPCR.  Histological analyses of inflammatory infiltrates in the skin were performed by optical microscopy using H&E-stained slides and quantifications were performed using the Imaje J program version 1.53. The differences between the groups and their respective time-points were evaluated by One-way ANOVA, Mann-Whitney and Kruskal-Wallis tests, using the Graphpad Prism program version 8.0. RESULTS: The characterization of baseline parameters showed that SS animals presented severe hemolytic anemia and leukocytosis, while the AS group showed no significant differences compared to the control group. High levels of AST were observed in AS and SS animals, compared to the AA group, while ALT was found to be increased in the SS group compared to both AA and AS animals, and creatinine levels were increased in the SS group compared to the AA group. In the SS group, the levels of PGE₂ in the serum were increased while LTB₄ levels were decreasedin comparison with AA and AS mice. Negative correlations between hemoglobin, red blood cells, hematocrit and PGE₂ were observed. The changes induced by the ulcer protocol in the blood counts of the SS group were not significant. However, we observed a reduction in total leukocytes within 24 h and an increase in 72 h in this group. On the other hand, the tissue leukocyte infiltrate increased in 24 h and a slight reduction in 72 h. In the SS group in 24 h, there was a reduction in total cholesterol (TC) and AST. In 72 h, we observed an increase in TC and a reduction of ALT and creatinine in the same group. Also in the SS group, tissue COX-2 expression was increased in 72 h, while systemic PGE₂ and LTB₄ did not present significant alterations. CONCLUSION: Transgenic sickle cell anemia mice present biochemical and hematological alterations that are correlated with an imbalance in lipid metabolism and eicosanoid production, contributing to a greater inflammatory state at the basal level. The induction of skin ulcers in this model leads to greater leukocyte recruitment in sick animals compared to healthy and sickle cell trait animals, which seems to be related to the biochemical, hematological, and inflammatory changes presented by these animals.

ABSTRACT INTRODUCTION: Schistosomiasis is a neglected disease caused by helminths of the genus Schistosoma, and recognized as an important public health problem that especially affects the poorest communities around the world. Schistosomiasis has a wide distribution around the world, however, it is restricted only to the tropical and subtropical zones, where the parasites and their intermediate hosts live, a factor that influences directly into the global distribution of the disease. According to the World Health Organization (WHO), the people most susceptible to contracting schistosomiasis are those who live in poor areas, whose main source of income is fishing or agriculture in rivers and lakes with infected snails. Diagnosis is performed by parasitological examination of feces, using the Kato-Katz method, which represents a limitation of studies and parasitological surveys, considering the current epidemiological scenario in Brazil. Some diagnostic tests are being proposed and developed all over the world, among them, the POC-CCA rapid urine test stands out. OBJECTIVE: To determine the frequency of positivity of the rapid urine test for the diagnosis of schistosomiasis, POC-CCA, in a population of high prevalence in the state of Bahia, before and after treatment for schistosomiasis. METHODOLOGY: This is a cohort carried out in the city of Conde-BA, located about 170 km from Salvador-BA. Individuals of both sexes aged between 4 and 70 years who delivered stool and urine samples were included. Fecal samples were used to make two Kato-Katz slides, and urine samples were used to perform the POC-CCA and urine reagent strip. For post-treatment analyses, all subjects who received treatment for schistosomiasis and submitted stool and urine samples were included. Follow-up was performed at five times: pre-treatment evaluation (D0), treatment (D30), cure control (D30), follow-up 180 (D180) and 360 (D360) days after treatment. RESULTS: The presence of S. mansoni eggs was identified in 189 individuals (55.59%) with a median parasite load of 36 (12–108) eggs per gram of feces (OPG). For the evaluation of POC-CCA among 273 samples on D0, 58.24% (n=159) were positive for the Circulating Cathodic Antigen (CCA), of these, 18.32% (n=50) were considered false-negative; 19.05% (n=52) false positives; 23.44% (n=64) true-negative and 39.19% (n=107) true-positive. We further demonstrate that urine density is a factor that interferes with the performance of the POC-CCA, where higher density urines increase the chance of false-negative results in the POC-CCA CONCLUSION: The POC-CCA, despite presenting a frequency of positive results greater than that brought about by two Kato-Katz slides, presents a considered number of false-negatives, especially among individuals with low parasite load. The POC-CCA showed the same flaws as the Kato-Katz, with regard to sensitivity in samples with low parasite load, in addition, the method showed to be influenced by urinary density. For this reason, the largescale use of the POC-CCA cannot be recommended in its current format. It is possible that the adequacy of the test or the patient's condition, such as the recommendation to hydrate the patient before collecting the material, or using a buffer solution during sample application, may help to improve the performance of this test in highly endemic regions from Brazil.

ABSTRACT INTRODUCTION: Leishmaniasis are a group of neglected diseases, according to the World Health Organization, that affects one billion of people that are at risk of infection. Clinical status vary according to the parasite species and the host immune response and genetic background. The most used drugs to the treatment of leishmaniasis are pentavalent antimonials. However, these drugs present high toxicity and uncomfortable collateral effects. In this context, it is necessary the search for new medicines with a high leishmanicidal action and reduced toxicity. An interesting alternative has been the use of drugs that act in the mechanisms of immune response as it has shown promising results. In regard of these drugs, there is the diethyldithiocarbamate,which is a copper-quelating compound that inhibits the function of the superoxide dismutase and it has been demonstrated that this compound increases the production of superoxide and decrease the parasitic load in the experimental cutaneous leishmaniosis. Another alternative drug is the hydroxylamine that reacts with the superoxide and during the methabolism promoves the increase of nitrite peroxide, a molecule capable of destroying intracelular parasites. OBJECTIVE: That being said, this study objetive was to test the in vitro action of the association of Hydroxylamine and DETC against Leishmania braziliensis, amazonensis e infantum. MATERIALS E METHODS: It has been performed toxicity tests in Leishmania braziliensis, amazonensis e infantum and also in murine macrophages derivatived from BALB/C bone marrow. In addition, BALB/C bone marrow-derived macrophages were infected by L. amazonensis, L. braziliensis and L. infantum and treated for 48 hours to assess the percentage of infection and number of amastigotes by counting under a fluorescence microscope. RESULTS: Our results showed a reduction in the growth and viability of the parasites when treated from 2 µM onwards of DETC and at the highest concentration of hydroxylamine (5 mM). Inhibition index of 50% was 33.03 µM to hydroxylamine, 0.247 µM to DETC and 1.676 µM to the association between them agaisnt the Leishmania braziliensis. Regarding the murine macrophages the inhibition index of 50% was 237.0 µM to hydroxylamine, 11.47 µM to DETC and 67.16 µM to the association between the two drugs (DETC/Hydroxylamine=1:2,5). An antagonistic action was observed against the murine macrophages when using the combined concentration of 20 uM from DETC and 500 uM from hydroxylamine. On the other hand, this combined concentrations was highly synergistic to the L. braziliensis. The association between these concentrations was also capable of reducing significantly the infecction percentage and the number of amastigotes per 100 cells. CONCLUSION: Our results demonstrated that the association of hydroxylamine and DETC has powerful antiproliferative effects in promastigotes and amastigotes from L. braziliensis, amazonensis e infantum and presents low toxicity to the murine macrophages.

Lung cancer is the most common cause of cancer-related mortality worldwide, accounting for 18% of all cancer deaths. The new treatment strategies require an integrated multidisciplinary approach between pathologists, clinicians, molecular biologists, radiologists and surgeons. Investigation through imaging and metabolic techniques, such as PET-CT, in the investigation of metastasis to mediastinal lymph nodes has been recommended; however, the analysis of results is hampered primarily by false positives. The present study investigated neoplastic and, especially, non-neoplastic histopathological changes, as well as the immunoexpression of GLUT1 and Ki67 for the evaluation of cell metabolism and cell proliferation, respectively, correlating them to the results of the 18-FDG PET-CT in a sample of patients with NSCLC. There was no association between lymph node histological pattern and PET uptake; there was a statistically significant association between histological patterns of histiocytosis with paracortical extension with anthracosis (p = 0.0001) and follicular hyperplasia with number of cigarettes/day (p = 0.001), respectively. The granulomatous reaction was uncommon, affecting 4/54 patients with a corresponding false positive result in only 1/54 (1.8%). There was a statistically significant correlation between the cell proliferation index (Ki67) and cell metabolism (GLUT1); as well as between the cell proliferation index (Ki67) and SUV 1h (p = 0.013) and SUV 2h (p = 0.020); however, the same does not occur for the evaluation of cellular metabolism (GLUT1 - EIR), possibly due to glucose uptake by another transporter in reactive lymph nodes. The association of imaging and metabolism techniques, combining computed tomography and PET, increases the specificity of the exam from 54% and 57%, respectively, to 71% and with a negative predictive value of 71%. In addition, the finding of a distant lesion through PET-CT, indicates the biopsy of a potentially more advanced lesion, avoiding unnecessary surgery in patients with distant metastases.

ABSTRACT Introduction: Arthrogryposis multiplex congenita (AMC) are joint contractures in more than two joints in different areas of the body, seen since birth. It is a clinical finding, observed in about 400 different diseases, most of which are of genetic origin. All of them result in fetal akinesia due to reduced intrauterine fetal movements, with joint limitations, which may be associated with other findings such as dysmorphia, muscle weakness, cognitive impairment and involvement of other organs. Neuromuscular diseases are a frequent cause of AMC. The etiological definition is fundamental for therapeutic definition, genetic counseling and prognosis. The frequency of AMC is unknown in Brazil and there are no demographic studies for this population in our country. Aim: The present study aimed to identify the etiology of AMC in individuals assisted in a public service specialized in neuromuscular and genetic diseases at Professor Edgard Santos University Hospital, in addition to collecting demographic and clinical data of these patients. Material and Methods: A prospective study, formed by a convenience sample of patients referred for evaluation, in which a rational investigation protocol was developed based on clinical aspects and complementary exams. After filling out the standard form, patients underwent investigation with other exams, such as creatinine phosphokinase measurement, electroneuromyography, cranial magnetic resonance, karyotype, muscle biopsy and molecular studies. Results: During the 42-month period, 41 patients were evaluated. 53.7% were female and the average age at the first assessment was 8.8 years. About 30% of patients reported a positive family history. The average number of joints involved was 6, with clubfoot being the most observed alteration (85.4%). The etiological diagnosis was established in 75.6% of the cases, based on the phenotype and on complementary exams. In four patients from three different families, the diagnosis was made by exome. Of the total diagnosed cases, 39% were caused by neuromuscular and connective tissue diseases, 24% classified as amyoplasia, in 12% the cause was involvement of the central nervous system and in 24.4% an etiological diagnosis was not established. Rare forms of AMC and pathogenic variants previously not described in genes associated with myopathies were found in this series. Conclusion: The present study demonstrated the main clinical and morphological characteristics of different forms of AMC in the studied population. It was able to establish an etiological diagnosis in most cases with AMC, based on data from clinical examination, muscle biopsy, electromyography and genetic study.

INTRODUCTION: Sickle cell disease (SCD) is a group of hemoglobinopathies and
sickle cell anemia (SCA), characterized by HbSS homozygous, is the most severe form,
while SC hemoglobinopathy is milder. The main clinical manifestations in SCD results
from continuous hemolysis and the chronic inflammatory process, leading to anemia
and vaso-occlusion process. The vaso-occlusive crises have been associated with the
presence of sickle cell, reticulocytes, leukocytes and activated platelets, which alters the
vascular endothelium and enhancer blood hypercoagulability. Several genetic factors
have been associated with the clinical profile variability in SCD. OBJECTIVE:
identify genetic biomarkers of endothelial dysfunction and associations with laboratory
parameters and clinical manifestations in individuals with SCA and SC disease
METHODS: a cross-sectional study it was carried, whose clinical data were collected
using a standardized questionnaire (self-reported or reported by the parents);
hematological and biochemical analyzes were performed by automated methods;
molecular analyzes were performed to determine the βS haplotypes, α-thalassemia and
polymorphisms in SELP (rs6127, rs6131 and rs6136); SELPLG (rs2228315); CD40
(rs1883832); and CD40L (rs3092952); patients are followed-up at the Bahia
Hemotherapy and Hematology Foundation (HEMOBA). RESULTS: the first
manuscript involved 80 individuals with SCA and 51 individuals with HbSC, whose
laboratory parameters and clinical manifestations were compared. It was observed that
SCA patients exhibited a more prominent anemia, hemolysis, leukocytosis and
inflammation, and that HbSC patients had altered lipid markers. The main cause of
hospitalization in both groups was pain crises, which were associated with high red
blood cell counts in patients with SCA. The occurrence of dactylitis was significantly
higher in SCA patients and was associated with increased RDW, monocytes and CRP,
as well as with hydroxyurea therapy. Pneumonia and ACS were associated with high
CRP and LDL-C levels, respectively. In individuals with HbSC disease, a previous
history of splenomegaly was associated with reduction on platelet counts. The second
manuscript involved 80 patients with SCA and investigated the association of clinical
manifestations and laboratory parameters with the rs6127, rs6131 and rs6136
polymorphisms in the SELP gene; the rs2228315 polymorphism in the SELPLG gene;
rs1883832 in CD40 gene; and rs3092952 in CD40L gene. It was possible to identify an
association of the variant (rs3092952) CD40L gene with a previous history of painful
crises and dactylitis in SCA patients. Furthermore, our results suggest an independent
association of the variant (rs6127) SELP with increased ET-1 levels and of the variant
(rs6131) SELP with elevated glucose levels. It was also observed that SCA patients
under hydroxyurea treatment presented a reduction in monocyte count and LDH levels.
The third manuscript demonstrated that SCA individuals carriers of TTC haplotype in
the SELP gene presented lower levels of glucose than individuals with non-TTC
haplotypes. SAC individuals with the TTT haplotype presented reduced total cholesterol
and LDL-C levels when compared to those with non-TTT haplotype. Furthermore, it
was observed that SCA individuals carriers of βS CAR/BEN or CAR/CAR haplotypes
had high levels of total cholesterol.CONLUSIONS: Our results corroborated the
differences of the clinical and laboratory profiles between individuals with SCA and SC
hemoglobinopathy. Our study also confirmed that hydroxyurea treatment is associated
with improved inflammatory and hemolytic profiles; and that SCA individuals carriers
of the βs CAR/BEN or CAR/CAR haplotype may present a most severe clinical profile.
Furthermore, our data suggest that variants in the SELP and CD40L genes are
associated with clinical complications and laboratory markers related with vasoocclusion; and that haplotypes in the SELP gene may regulate classic biomarkers
involved in endothelial dysfunction in SCA patients.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is the tumor subtype that most affects oral tissues. The Hedgehog (HH) pathway is an important cascade in embryonic development, which is also associated with tumor development, including the OSCC. The HH pathway occurs canonically in the Primary Cilia (PC), a structure that functions as a cellular sensor and is responsible for housing several signaling pathways. An alteration in the PC function can lead to several problems, including neoplasms. HYPOTHESIS: There is loss of CP in the CECO, which favors the maintenance of cell proliferation and dysregulation of the HH pathway. AIM: To study the ciliogenesis process and the presence of primary cilium in OSCC tissues and cell lines, as well as the relationship between PC, HH molecules and proliferation of cells of this type of tumor. MATERIAL AND METHODS: Tumor cell lines (TC), non-metastatic (CAL27) and metastatic (HSC3), non-tumor cell line (HGK, control) and 22 samples of human tumors (HT) were used. Tumor lines were cultivated in complete DMEM medium supplemented with 10% Fetal Bovine Serum, 1% antibiotics and 8% hydrocortisone; the HGK strain was cultivated in Keratinocyte SFM medium. Cells were analyzed at times 0h and, for TC, also at times 6, 12, 24 and 48h in serum deprivation (synchronization in G0). qPCR was performed at time 0h (TC and HGK) and at times 6, 12 and 24h (TC), as well as for HT samples for gene expression analysis of the HH pathway (PTCH1, SMO, GLI1 and SHH), PC (KIF3A, IFT88, AURKA and BORA) and proliferation (Cyclin D1). For characterization of PC proteins (α-acetylated tubulin), via HH (SMO and GLI1) and proliferation (Ki-67 and profilin), Western Blot (WB) and Immunofluorescence (IF) assays were performed on TC (times 0, 24 and 48h) and HGK (time 0h), as well as Immunohistochemistry in HT. Statistical analysis was performed using the ANOVA test (p<0.05). RESULTS: Assembly and disassembly genes showed lower or similar expression levels in TC when compared to HGK; There was no statistically significant difference between PC assembly and disassembly components, cell proliferation and HH molecules. SHH was not found in the strains studied. The presence of CP in CT was verified, as well as activation of the HH pathway (nuclear GLI1), nuclear and cytoplasmic SMO, as well as cell proliferation (Ki-67 nuclear) greater in TC than in HGK. CONCLUSIONS: CP is present in metastatic and non-metastatic OSCC cells, as well as in human tumors. In these cells and tumors, GLI1 and nuclear SMO were described regardless of the presence of CP, but also in cells that exhibit this organelle. Together, these results indicate activation of the HH pathway, in a canonical and non-canonical manner, in OSCC. Furthermore, cell proliferation occurred independently of CP in cell lines.

Material Sigiloso

ABSTRACT
INTRODUCTION: Considered a rare condition of Cutaneous Leishmaniasis, Cutaneous Recurrent Leishmaniasis (LCR) is characterized by nodular lesions around or within the scar of an ulcer previously produced by Leishmania sp., with late onset and long lasting. OBJECTIVE: To characterize cytokines, chemokines and immune memory T cells in patients with LRC and LCL. MATERIALS AND METHODS: The sample group is represented by 36 patients with LRC and 36 with LCL, diagnosed as positive by the parasitological test and the Montenegro test. Cytokines from the “inflammatory” (IL-1β, IL-8, IL-10, and IL-12p40), “Th1/ Th2/ IL 17” (IL2, IL-4, IL-6, TNF, IFN and IL17) and "chemokines" (Rantes, MIG, MCP-1 and IP-10) using plasma and in vitro Cytometric Bead Array (CBA) kits. For extracellular labeling of memory cells specific fluorescence-conjugated monoclonal antibodies were used for cell characterization molecules (CD4+ or CD8+). For the panel was inserted the marking for memory cells CD45RO+ and CCR7+ (naive, effector memory (TME), central memory (TMC) and effector cells); cell activation molecules (CD25, CD69 and HLA-DR) and adhesion molecules (CD62Llow and CD62high). Samples were purchased from FACS Fortessa (BD) and analyzed on GraphPad. RESULTS: The plasma cytokines dosed in the “inflammatory” and “Th1/ Th2/IL-17” kits in LRC and LCL were higher among active or healed individuals than healthy controls. In LRC IL-8 and IL-12p40 presented significance only of the cured in relation to healthy controls. In vitro, in the “Th1/Th2/ IL-17” kit, no differences were evidenced when analyzing the clinical forms alone (active versus cured) and neither (LRC versus LCL). IL-8 levels in LRC and LCL were higher among active and cured after stimulation with Leishmania braziliensis. Comparing LRC versus LCL MIG (CXCL9) and IP-10 (CXCL10), responsible for cellular recruitment to the site of infection, were decreased in the plasma of individuals with active lesion. No difference was detected between clinical forms in CD45RO+ expression in immune memory T lymphocytes (CD4+ and CD8+). The markers of CD25 activation (intermediate activation) and HLA-DR (late activation) showed lower expression in patients with LRC compared to those active by LCL, and CD4+ and CD8+ T cells of patients with LRC compared to patients. LCL showed lower CD62Llow expression, higher amount of CD62Lrigh and lower number of TME cells in peripheral blood. CONCLUSÕES: Our data suggest that in LRC, the decrease in IP-10 could be characterized as a biomarker for early identification of this clinical form and may be related to other cellular response events (activation, migration and effector memory), associated with difficult therapeutic response. and resistance to treatment.

Patients with sickle cell anemia (SCA) have high phenotypic variability as well as several
clinical manifestations, including stroke. In the context of SCA, impairment of the cerebral
arteries is primarily associated with chronic inflammation, hemolysis and anemia. Thus, the aim
of the present study was to identify the presence of polymorphisms in inflammatory cytokine
genes, in order to associate the frequency of such genetic alteration with the development of
stroke in children with SCA. For this, 66 individuals were investigated, 24 individuals who had a
stroke, considered the case group, and 42 individuals who never had a stroke, designated as the
control group. Hematological, biochemical and inflammatory profile parameters were analyzed,
as well as the presence of polymorphisms in inflammatory cytokine genes, as TNF -308 G> A
(rs1800629), IL1B +3954 C> T (rs1143634), IL4 - 590 C> T (rs2243250) and IFNG +874 T> A
(rs2430561). Hematological, biochemical and immunological markers were investigated using
automated methods, and genetic markers were identified by polymerase chain reaction and
Restriction Fragment Length Polymorphism techniques. Analyzing the laboratory parameters, a
high reticulocyte count was found in individuals belonging to the case group (p = 0.0101).
Evaluating the laboratory data, we found an association between the dosage of albumin (p =
0.0006), total proteins (p = 0.0087) and uric acid (p = 0.0395). An association of the presence of
the TNF -308 G>A polymorphism, where the A allele was associated with the occurrence of
stroke (p = 0.430). A multivariate analysis showed an association between the presence of the
TNF -308 G> A polymorphism and the occurrence of stroke (p = 0.016). A high frequency of
mutants was found for the IFNG gene polymorphism. A significant association was observed
between the presence of the IL4 -590 C> T polymorphism and some laboratory parameters, such
as lymphocyte count (p = 0.0152), platelets (p = 0.0203), total proteins (0.0227) and direct
bilirubin (p = 0.0054). Analyzing the laboratory parameters in individuals with SCA, a
significant association was identified between the presence of the T allele in the IL4 -509 C> T
polymorphism and some laboratory parameters, such as MCV (p = 0.0105), MCH (0.0421),
RDW (p = 0.0105), LDL (p = 0.0351), AST (p = 0.0292) and total bilirubin (p = 0.0468). In
addition, an association was found between pro-inflammatory cytokines, as IL-12 (p = 0.0095),
IL1-β (p = 0.000) and IL-8 (p = 0.0167) levels in individuals with the mutant allele of the IL4 -
509 C> T polymorphism. No statistical association was found between the investigated
parameters and the presence of polymorphisms in the IL1B and IFNG genes. Therefore, patients
with SCA with a previous history of stroke present alterations in laboratory parameters and have
a higher frequency of the allele A of the TNF -308 G> A polymorphism. In addition, it can be 

seen that the IL4 -590 C> T polymorphism was associated with changes in laboratory markers,
altering the level of expression of inflammatory cytokines. Our results demonstrate the need to
investigate possible genetic interferences, in order to establish the effects of such variables on the
stroke outcome in patients with SCA.

INTRODUCTION: Schistosomiasis is a parasitic disease considered a prevalent health problem in tropical developing countries, and the Praziquantel (PZQ) is the drug of choice for treatment. The adult worm of S. mansoni resides in the vascular lumen, although little is known about the interaction of endothelial cells with the adult worm and the participation of these cells during progression or protection from infection. In addition, the expression of antioxidant genes by these cells has not yet been evaluated during schistosomiasis and after treatment. AIM: To evaluate the expression of antioxidant genes in HUVEC cells grown with adult S. mansoni worms and exposed to treatment, as well as the survival capacity of worms with HUVEC and exposed to oxidizing agents. METHODOLOGY: The names of the genes, enzymes and substrates used in this work were presented in a coded form to keep confidentiality because it is sensitive information for innovation. HUVEC cells were cultured and challenged with adult S. mansoni worms and subjected to PZQ treatment at 1, 3 and 6 hours. After the challenge, RNA extraction was performed using trizol. Then, the qPCR reaction of antioxidant genes was performed. To evaluate the adult worms ability to survive oxidative stress, adult worms were cultivated with the oxidizing agent OX and adult worms with OX in the presence of HUVEC. The worms were evaluated by optical microscopy. RESULTS: HUVEC cells showed increased expression of the antioxidant genes αOX-A and αOX-B when challenged only with PZQ. In addition, it was interesting to note that adult worms were not able to induce or suppress the antioxidant genes evaluated, with the exception of αOX-E, which had its expression significantly increased in HUVEC cells after 6 hours of exposure to an adult worm couple . When the survival capacity was evaluated, adult worms grown with HUVEC cells and challenged with OX showed a higher percentage of survival and resisted higher concentrations of OX when compared to adult worms challenged with OX without the presence of HUVEC. The antioxidant protein αOX-E was added to the culture of adult worms together with the oxidizing agent OX, demonstrating an increased survival of worms in this condition even in high concentrations of OX. Thus, adult worms grown with HUVEC cells behave similarly to the culture of the worms in which αOX-E was added. CONCLUSION: The adult S. mansoni worm seems to induce HUVEC cells to express more αOX-E, a molecule that is involved in the control of oxidative stress. Thus making it an environment conducive to their survival. This information can help new approaches to adjuvant treatments to PZQ.

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INTRODUCTION: In the New World, dogs are considered the main reservoir of visceral leishmaniasis (VL). Due to inefficiencies in existing treatments and the lack of an effective vaccine, euthanasia in dogs is one of the main strategies for controlling the disease, making the development of new therapeutic interventions mandatory. Previously, our group has shown that 17-AAG, an HSP90 inhibitor, has demonstrated potential for use in the treatment of leishmaniasis. OBJECTIVE: The present study aimed to test the safety of 17-AAG in dogs, evaluating the plasma pharmacokinetics, dose proportionality and tolerability of 17-AAG in response to one or more intravenous (IV) doses in healthy dogs. MATERIAL AND METHODS: Two research protocols were used. Protocol 1 (P1): healthy dogs received variable doses (50, 100, 150, 200 or 250 mg / m²) of 17-AAG or placebo (n = 4) intravenously, using a cross-over design with a wash-out period seven days between treatments. Protocol 2 (P2) involved nine healthy dogs that received three doses of 150 mg / m² 17-AAG administered IV at 48 hour intervals. RESULTS: All dogs successfully completed both protocols. At P1, 17-AAG was well tolerated, however, increased levels of liver enzymes and diarrhea were observed in all four dogs that received a dosage of 250 mg / m². The parameters maximum plasma concentrations (Cmax) and area under the curve (AUC) were proportional to the dose administered between the doses of 50 and 200 mg / m². Regarding P2, 17-AAG was considered to be well tolerated at multiple doses of 150 mg / m². Increased levels of liver enzymes and diarrhea were observed in 3/9 and 1/9 of these dogs, respectively. CONCLUSION: Our results demonstrated safety for conducting the 17-AAG efficacy test in dogs with VL when administered in concentrations equal to or less than 150 mg / m² at intervals of 48 hours. However, the hepatotoxicity observed with the interval dosing scheme, despite being reversible, points to efforts to reduce toxicity such as the use of nanoformulations or liposomes.

ABSTRACT

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is an invasive neoplasm that still has a high mortality rate. It is known that activation of the Hedgehog signaling pathway (HH) in adult tissues may result in tumor development. Cancer-associated fibroblasts (CAFs) represent the most abundant cell population in the stroma and favor tumor progression, and the participation of the HH pathway has already been described in the activation of these cells. HYPOTHESIS: FACs contribute to cell proliferation and tumor angiogenesis through the HH signaling pathway. AIM: To evaluate the participation of CAFs in HH pathway activation, cell proliferation and angiogenesis in OSCC. MATERIAL AND METHODS: OSCC lines (CAL27, SCC4, SCC9 and HSC3) and primary cultures of normal oral cavity fibroblasts (NOF) and cancer-associated fibroblasts (CAF) were used. In human OSCC, immunostaining of S100A4, α-SMA, CD105 and MCM3 was performed. Gene expression of HH pathway components was performed in OSCC lines, NOF and CAF. HSC3 and CAL27 were stimulated with recombinant SHH and expression of PCTH1, GLI1 and GLI2 genes was analyzed. The alamar blue assay was performed to evaluate the cytotoxicity of GANT61, GLI1 inhibitor, in CAL27, NOF and CAF. NOF conditioned medium (CM) and CAF CM (before and after GANT61 treatment) was incubated with CAL27 and then evaluated for expression. of genes related to the HH pathway, cell proliferation and angiogenesis. CAL27 cells were also treated with GANT61 and the above genes were also evaluated. The CAL27 cell cycle assay was performed after different stimuli as well as angiogenesis assay using the endothelial cell line EA.hy926. RESULTS: In human OSCC, a positive association was found between α-SMA and S100A4 proteins and cell proliferation marker MCM3 as well as between α-SMA and S100A4 with CD105 protein, positive in neoformed vessels. Gene expression of HH pathway components was observed in OSCC line as well as NOF and CAF. In tumor cells (HSC3 and CAL27), as well as NOF and CAF, protein labeling of the HH pathway was observed, which was lower in NOF. After stimulation with recombinant SHH, HSC3 and CAL27 cells showed a higher expression of HH pathway genes (PTCH1 and GLI1). CAF CM, when incubated with CAL27, increased levels of HH pathway gene transcripts (PTCH1, GLI1, GLI2) as well as target genes of this cascade that are involved in proliferation (MYC, Cyclin D1) and angiogenesis (HIF-1, VEGF-A). On the other hand, CAF CM post GANT61 prevented the increased expression of the above genes at different times. Furthermore, this compound was also able to prevent higher levels of expression of the above genes after incubation with CAL27. In the cell cycle, we observed a larger cell population in the G0-G1 phase in the CAF CM treated group, as well as a smaller apoptotic cell population. Finally, we observed that despite the fibroblast type (NOF or CAF), the factors secreted by these cells exert an effect on induction of angiogenesis. CONCLUSIONS: CAFs secreted molecules can mediate paracrine activation of the HH pathway and contribute to proliferation and angiogenesis in OSCC.

INTRODUCTION: Oral squamous cell carcinoma (OSCC) is the most common tumor of the head and neck region, constituting an important public health problem in Brazil. Despite advances in the molecular studies of this disease, especially in relation to data available in the TCGA, therapy of this tumor type is still a challenge, especially when the disease is in stages 3 and 4. The PI3K-AKT pathway is one of the most important for tumorigenesis and activation of this cascade may also be related to the Hedgehog (HH) pathway. Studies point to the relationship between the expression of insulin-like growth factor-1 (IGF-1) in stromal cells, non-canonical activation of the HH pathway and maintenance of AKT activity, thus contributing to the more normal behavior. aggressive tumors. OBJECTIVE: To evaluate the effects of IGF-1 on the expression of HH and PI3K-AKT pathway molecules, and its participation in pro-tumorigenic events such as proliferation, angiogenesis, migration, invasion and tumor stem cell population in vitro. MATERIAL AND METHODS: Skin fibroblasts (MF1) were genetically engineered for IGF-1 overexpression by lentiviral transduction (MF1-IGF-1). SCC-4 cells were incubated with fibroblast conditioned medium (CM) or rIGF-1 for proliferation, migration, pluripotency gene expression, tumor bead formation, and protein identification of the AKT and HH pathways. CM was also used for the angiogenesis assay, performed in endothelial strain EA.hy926. Co-culture conditions (fibroblasts and SCC-4) were adopted for immunostaining of pluripotency factors and the HH pathway. Fibroblasts and SCC-4 were seeded in different transwell compartments for invasion evaluation. RESULTS: Analysis of gene expression confirmed IGF-1 overexpression in MF1-IGF-1 and absence of IGF-1 mRNA in SCC-4, however, these cells showed high IGF1R expression. Ihh ligand was observed in MF1 and MF1-IGF-1, and increased GLI1 mRNA in CM MF1-stimulated SCC-4. HH (GLI1, Ihh and SMO) and AKT (total and phosphorylated AKT) pathway proteins were identified in SCC-4 cultured in the presence of CM MF-1-IGF-1. rIGF-1 promoted: proliferation, migration, invasion and formation of tumor spheres. MC MF1 was the most important stimulus for angiogenesis. CONCLUSIONS: rIGF-1 exerted pro-tumorigenic effects on SCC-4 (proliferation, migration, invasion, and tumor spheres). However, CM MF1 had greater influence on angiogenesis and gene expression of GLI1. Proteins of the AKT and HH pathways were identified in SCC-4 against different stimuli, suggesting that IGF-1 regulation of these pathways may be related to the observed events. However, further studies are needed to prove this relationship.

Introduction: hydroxyurea (HU) is the treatment of choice for individuals with sickle cell anemia (SCA) who have a severe clinical profile. Approved in 1998 by the FDA, HU is known to increase fetal hemoglobin (HbF) concentrations and reduce polymerization of variant hemoglobin S (HbS), with improvement in the clinical profile, as well as some laboratory parameters. However, despite these benefits, there is variation in response to treatment, from non-improvement to toxicity. Objective: the present study aimed to validate a model that can explain variability in HU response and, investigate the effects of HU, in association with some classical genetic biomarkers of the disease, in children with SCA. Method: first, we performed a literature review for identifying the candidate genetic factors, which may influence HU response, focusing on polymorphisms in genes encoding enzymes metabolizing drugs (EMD) and solute carriers. In addition, we performed 02 types of study: longitudinal and crosssectional. The longitudinal study involved 22 children with SCA, whose blood samples were collected before and during HU use. The cross-sectional study involved 90 children with SCA with or without HU use. Blood samples collected were used for laboratory determinations and investigation of polymorphisms. Results: the literature review resulted in the identification of several polymorphisms in genes related to HbF expression (HBG2, BCL11A, HMIP, OR51B5/6, MAP3K5, FLT1, KLF10), as well as drug metabolism and solute carriers (CYP450, CAT, SLC), which are associated with variations in HU response. Furthermore, we proposed the differential HU metabolism model, which together with the two models (differential baseline HbF model and differential susceptibility model), previously described in the literature, could explain the inter-individual variability in HU response. The laboratory parameters analysis, before and during HU treatment, demonstrated the association of HU with increase in HbF in a fraction of the children as well as increases in glucose, HDL-C, protein and albumin and, reduction in AST. In addition, variants alleles 1934A, -21T, -262T and A of polymorphisms CYP2D6 1934G>A, CAT -21A>T, CAT -262C>T and SLC14A1 G>A rs2298720, respectively, were associated with increase in HU effects. Conclusion: the present study demonstrates the importance of evaluate metabolic profile during the HU therapy, in addition to classical parameters investigated, since the drug seems possess large spectra of action. Moreover, the results of the association between the polymorphisms in genes encoding DME and solute carriers corroborate our model (differential HU metabolism model) and demonstrate the necessity to identify genes candidates involved in HU metabolism.

Transforming growth factor beta (TGF-β) is a cytokine that plays an important role in biological process, such as endothelial and vascular dysfunction, inflammation and hematopoietic homeostasis. This study aimed to investigate associations of TGF-β1 levels and transforming growth factor beta receptor 3 (TGFBR3) gene polymorphisms with genetic, hematological, biochemical and immunological biomarkers in individuals with sickle cell disease (SCD), as well as clinical complications. A cross-sectional study was conducted, which were investigated 175 individuals with SCD (120 HbSS and 55 HbSC genotype). TGF-β, tissue inhibitor of metalloproteases-1 (TIMP-1) and matrix metalloproteinase 9 (MMP-9) plasma measurements were performed by ELISA technique. Hematological, biochemical and immunological markers were carried out by automated methods and TGFBR3 polymorphisms genotyping was performed using TaqMan SNP Genotyping Assays. TGF-β1 plasma levels were higher in HbSS individuals than in HbSC and health controls. In individuals with HbSS, TGF-β1 levels were positively correlated with red blood cells, hemoglobin, hematocrit, platelets and TIMP-1. In addition, HbSS individuals with TGF-β1 levels above the median (≥72.29 ng/mL) also presented increased monocyte counts and decreased albumin levels. In patients with HbSC, TGF-β1 levels were positively correlated with leukocytes, eosinophils, lymphocytes, monocytes, platelets, TIMP-1, VLDL-C, triglycerides, heme and AST. Additionally, HbSC individuals with TGF-β1 levels above the median (≥47.80 ng/mL) presented increased leukocyte and platelet counts, as well as increased levels of triglycerides, VLDL-C, MMP-9 and TIMP-1, and decreased HDL-C. In HbSS individuals, the minor allele (A) of the TGFBR3 rs1805110 polymorphism was associated with increased hemoglobin, hematocrit, reticulocyte counts, low density lipoprotein, uric acid and endothelin levels, as well as decreased platelet distribution width (PDW) and the occurrence of bone alterations. The minor allele (T) of TGFBR3 rs7526590 was associated with increased red cell distribution width, PDW, alkaline phosphatase, aspartate aminotransferase, indirect bilirubin and lactate dehydrogenase levels, as well as lower ferritin levels and the occurrence of leg ulcers. Our data suggest that the minor allele (A) of TGFBR3 rs1805110 is associated with inflammation and bone alterations, while the minor allele (T) of TGFBR3 rs7526590 is related to hemolysis and the occurrence of leg ulcers. SCD individuals carries of GG haplotype presented higher levels of total cholesterol (T-CHOL), low density lipoprotein cholesterol (LDLC), triglycerides, non-HDL cholesterol, total proteins and globulin than individuals with non-GG haplotypes. SCD individuals with the CGG haplotype presented increased plateletcrit, T-CHOL, LDL-C levels and non-HDL cholesterol. Both haplotypes were associated with a previous history of pneumonia. In addition, the GG haplotype was associated with a previous history of pneumonia. Our findings suggest the importance of TGF-β1 in vascular remodeling, vasculopathy, angiogenesis and inflammation in pediatric patients with SCD. TGFBR3 polymorphisms were associated to inflammatory status, hemolysis as well as clinical complications, bone alterations and leg ulcers occurrence. In addition, individuals with the GG and CGG haplotypes of TGFBR3 present significant lipid profile alterations and could be associated with the occurrence of pneumonia.

INTRODUCTION: Pregnancy is a high risk condition for women with sickle cell disease and is associated with an increased risk of maternal and fetal morbidity and mortality. Increased frequency and severity of pain crises occur during pregnancy in women with sickle cell disease. OBJECTIVE: The objective of this study was to analyze the clinical and laboratory characteristics of pregnant women with sickle cell disease in the presence of pain crisis. MATERIALS AND METHODS: A prospective, descriptive, exploratory, quantitative-qualitative, observational longitudinal study. Twenty-seven women with sickle cell disease were included in the study, 13 nulliparous and 14 pregnant women, accompanied by the Ambulatório Municipal Especializado em Doença Falciforme and the Ambulatório da Maternidade de Referência Professor José Maria de Magalhães Netto, respectively. We performed structured interview and follow-up of laboratory tests. Statistical analyzes were performed using the software Graph Pad Prism version 6, SPSS version 22, and program Excel version 2010. RESULTS: Most nulliparous and pregnant women classified the pain crisis as frequent. Regarding the duration of pain, most of the nulliparous women took more days to resolve their pain when compared to the pregnant women. Regarding the intensity of pain, there was variation between mild to moderate pain in the two groups, with emphasis on moderate pain in the nulliparous group; and moderate and intense pain in the pregnant group. The most affected anatomic sites were the upper and lower limbs in nulliparous women; and lower limbs and lumbar spine in the group of pregnant women. It is noteworthy that the sensorial and affective factors presented a greater influence on the perception of pain by the participants of this study. The resuls of the laboratory tests analyzed in this study, showed that pregnant women in pain crisis presented significantly lower levels of red blood cells, hematocrit and hemoglobin; significantly higher CHGM value; leukocyte count, percentage of neutrophils, bands and segmented cells were significantly higher; percentage of eosinophils and lymphocytes were significantly lower when compared to nulliparous women with stable disease. CONCLUSIONS: The pain episodes presented by pregnant women with sickle cell disese are associated with altered hematological and hemolytic parameters. Thus, future preventive interventions can be directed to the control of the pain crisis and the blood levels of these parameters in these patients. The results of this work consolidate the idea that pregnancy is a phenomenon associated with pain crisis in sickle cell disease and is able to induce alterations in some laboratory parameters, which should be evaluated to ensure adequate health care, with the purpose of avoiding complications and providing the well- being of pregnant women with sickle cell disease.

Leishmaniasis is an infectious-parasitic disease caused by the protozoan of the genus Leishmania and it is considered a public health problem in Brazil and in the world. It presents a wide spectrum of clinical forms, with Visceral Leishmaniasis (LV) being the most severe. In Brazil, the agent of VL is the species Leishmania infantum, and it is characterized by high fever, weight loss, hepatosplenomegaly, anemia, leukopenia and haemorrhage in the most severe cases. Neutrophils have been shown to play an important role in the immunopathogenesis of Leishmaniasis, especially in LV. It is known that different cell death pathways can be activated in infected neutrophils, influencing the course of infection by different pathogens. Necroptosis, a programmed pathway of death, is canonically activated in the absence of caspase 8 and has proinflammatory characteristics. Necroptosis has been described in the context of different diseases. The general objective of this work was to evaluate the effect of the blockade of caspases in the path of death by necroptosis in human and murine neutrophils infected by L. infantum and its effect on the viability of the parasite. Patients with LV showed elevated serum levels of Lactate Dehydrogenase (LDH), characteristic of cellular / tissue damage. Using the in vitro system of infection of human and murine neutrophils with caspase inhibitors, we demonstrated the activation of the necroptosis death pathway with the involvement of RIPK1, RIPK3 and MLKL. Next, the role of necroptosis in the parasite load of human neutrophils was tested in vitro. Pre-treatment with caspase inhibitors, followed by infection, increased LDH levels, RIPK3 and MLKL expression in human neutrophils, while decreasing the viability of the parasites. This effect was reversed by Necrosulfonamide (NSA), a pharmacological inhibitor of MLKL. In murine neutrophils infected by L. infantum, we also observed the involvement of necrotic pathway molecules, RIPK1 and RIPK3 in the control of parasite viability. In these groups, neutrophils pretreated with caspase inhibitors showed high levels of Reactive Oxygen Species (ROS) and LDH, suggesting the participation of these proinflammatory molecules in the control of infection. Pharmacological inhibition of RIPK1 by Nec-1 and RIPK3 by GSK'872 reversed the death of parasites. Finally, transmission electron microscopy (MET) revealed morphological features associated with necroptosis in neutrophils infected with L. infantum when these were pre-treated with caspase inhibitors, whereas cells infected in the presence of necroptosis pathway inhibitors showed no structural changes. These data suggest that inhibition of caspase 8 on neutrophils may contribute to control of L. infantum infection, possibly by stimulating an inflammatory response associated with necroptotic death.

Cardiovascular diseases are the leading cause of death in the world. Among the pathologies of the group, acute myocardial infarction, further by the extreme loss of cardiomyocytes and consequent negative remodeling, is a more prevalent clinical manifestation. Conventional therapeutic interventions are able to ameliorate the symptoms, but do not result in tissue regeneration and, consequently, in the reestablishment of cardiac function. For some patients the only alternative is transplantation, however there are limitations on the number of donors and compatibility. In this sense, new strategies have been investigated to regenerate the injured myocardium, including gene therapy, cell therapy and the use of biomaterials. This latter approach plays a crucial role in the administration of stem cells in view of the need for a physical platform and a microenvironment that allows cell viability and differentiation in cardiac tissue. Thus the development of 3D polymer matrices (scaffolds) that mimic the extracellular matrix and allow the anchoring of cells represents an important approach in the context of the translational potential of cellular therapy in acute myocardial infarction. The present work is based on the development of a matrix from silk fibroin as a platform for administration of stem cells in the infarcted myocardium. Matrices in the form of an injectable hydrogel were prepared by the chemical crosslink method through an organic solvent (methanol). Hydrogels were submitted to physical-chemical characterization, morphology, in vitro cytocompatibility and cell retention in a pharmacological model of acute infarction in mice. After synthesized and dried the hydrogels had the capacity to absorb water. Fourier-transform infrared spectroscopy (FTIR) indicated the protein conformational change, proving the gelation of the material. There was no significant variation in the pH of the hydrogel at the storage temperatures evaluated (4 ° C and 37 ° C). The morphological analysis by scanning electron microscopy (SEM) indicated a three - dimensional arrangement with pores, which demonstrates the porous architecture in the form of scaffold. This spatial pattern allowed the adhesion of L929 fibroblasts, H9c2 cardiomyocytes, mesenchymal stem cells (CTMs) and induced cardiac progenitor cells (CPCi) in the fibroin matrix. These cells were also found to be viable and proliferative when cultured on a plastic substrate coated with the hydrogel. This scaffold was evaluated for its ability to provide cellular retention in a murine model of acute myocardial infarction. It was possible to observe iCPCs in cardiac tissue 72 hours after its administration via intramyocardial guided ultrasound. In the context of research into new biomaterials for cell therapy, this work demonstrates the potential of the fibroin hydrogel as an administration system capable of optimizing the retention of stem cells in cardiac tissue.

INTRODUCTION: Chronic chagasic cardiomyopathy is a debilitating disease of fatal course, with cardiac arrhythmias being the main cause of sudden death in individuals with this disease. Due to the lack of adequate therapeutic options, the understanding of the mechanisms that lead to the development of severe arrhythmias is of great relevance. Connexin 43 is an important protein component of the communicating junctions in cardiomyocytes, and the altered expression of this protein is related to conduction disorders in other cardiomyopathies. OBJECTIVE: To characterize the changes in expression and distribution of Cx43 in chronic chagasic hearts. MATERIAL AND METHODS: C57BL/6 mice with 6 and 12 months of infection by the Colombian Trypanosoma cruzi strain and uninfected controls were submitted to functional evaluation (ergometric and ECG). After euthanasia, the hearts were collected and destined for histopathological analysis, to evaluate the inflammatory infiltrate and fibrosis; analysis by immunofluorescence to evaluate the distribution pattern of total and phosphorylated Cx43 (S368); analysis by the Immunogold technique, to evaluate the presence of Cx43 in the intercalated discs; gene expression analysis by RT-qPCR, to evaluate the gene expression of total Cx43, TNF-α and IL-1β. Samples from explanted hearts of patients with chronic chagasic cardiomyopathy submitted to cardiac transplantation were used for immunofluorescence analysis of Cx43 expression. RESULTS: Animals with 6 and 12 months of infection lost the ability to exercise on the treadmill and presented conduction disorders in the heart, observed on the ECG. Histopathological analyzes confirmed intense inflammatory and fibrotic infiltration in chagasic hearts (6 and 12 months), different from that observed in control animals. In the immunofluorescence analyses, we observed a change in the total Cx43 distribution and with lateral membrane or cardiomyocyte markings in the infected animals (6 and 12 months), while the hearts of uninfected animals presented Cx43 located in the intercalated discs. The staining for Cx43 (S368) was more intense in infected (6 and 12 months) than in uninfected animals. In the analysis by Immunogold, uninfected animals presented more intense localization of total Cx43 in the intercalated discs, whereas in the infected ones the markings were present in structures similar to autophagic vacuoles. The RT-qPCR analyses showed an increase in the gene expression of TNF-α and IL-1β in the infected (6 and 12 months) compared to uninfected controls. A reduction of Cx43 gene expression was reduced in the animals with 6 months, but not witl 12 months of infection, compared to uninfected controls. Finally, the labeling of total Cx43 and Cx43 (S368) in the human material presented a pattern similar to that observed in the experimental model. CONCLUSION: Chronic chagasic hearts present alterations in Cx43 expression, as well as in the distribution pattern of total and phosphorylated Cx43, which may be associated with the high production of proinflammatory cytokines TNF-α and IL-1β, and contribute to the establishment of conduction disorders in Chagas disease.

Propolis is a natural product with several biological activities tested and its extracts obtained by various techniques have been used in the food, pharmaceutical and cosmetic industries. Variations in the chemical composition are directly associated to the type and geographic origin of the propolis, consequently, interferes in the biological activity of the propolis. The aimed of this work was to characterize the chemical and biological activity of samples of propolis extract: red, green and brown, collected in different regions of the State of Bahia, Brazil, obtained by the conventional method (ethanolic extraction) or the supercritical extraction technique. First, we identified the physical-chemical profile of the raw samples, as for humidity content, water activity, total ash content, protein, lipids and fibers. The extracts of propolis obtained by the ethanolic and supercritical extraction were evaluated for the content of phenolic compounds, flavonoids, antioxidant activity (DPPH), catechin, ferulic acid and luteolin. The results demonstrated that ethanolic extracts, especially those obtained from red propolis, have a higher selectivity for antioxidant compounds, as well as a better antimicrobial activity against two strains of bacteria (Staphylococcus aureus and Escherichia coli), when compared to the extracts obtained by supercritical extraction, indicating that there was no significant efficiency of the parameters evaluated in this condition. Finally, we compared the leishmanicidal action of ethanolic extracts obtained from green and red propolis on the in vitro infection of macrophages by Leishmania braziliensis, the main etiological agent of the cutaneous clinical form of the disease. In this context, red propolis had a greater potential for leishmanicidal effect. The results confirm the influence of the raw material type as well as the extraction methods regarding the composition and biological activity of the different extracts of propolis, including their use as antimicrobial and leishmanicidal agents. The present study contributes with new perspectives in order to enable technological applications of propolis and its derivatives in the natural products industry.

Introduction: sickle cell disease (SCD) has a high global prevalence and autosomal recessive inheritance, being considered a public and social health problem. Hydroxyurea (HU) has been used as a pharmacological therapy for sickle cell anemia (SCA), and in the last thirty years it has become the primary modality of treatment modifying the disease, with a reduction in the incidence of acute pain episodes, hospitalizations, chest syndrome transfusion and mortality. HU has been described as well tolerated and with low toxicity. The use of HU in Brazil was standardized in 2010, and there is a clinical protocol and therapeutic guidelines for its use. However, the investigation of the clinical evolution of patients with SCD using HU is necessary to have data regarding the clinical follow-up of this group of individuals on a prolonged basis. Objective: The objective of this study was to evaluate the clinical and laboratory evolution of patients with SCA using HU in a reference outpatient clinic, before and after the use of the therapy. Methods: the population was composed of SCA patients followed at the pediatric hematology outpatient clinic of the University Hospital Complex Professor Edgard Santos of the Federal University of Bahia (HUPES-UFBA), using HU for at least six months. Demographic, clinical, and laboratory data on the use of HU were evaluated. Results: the sample consisted of 58 patients with SCA ranging from 5 to 19 years of age, with a mean age of 12.81 years (SD ± 3.82), 55.2% female, with an 8.8 years of age and mean time of use of HU around 50.5 months. The treatment improved the clinical variables (number of hospitalizations, transfusions, acute pain episodes, acute chest syndrome and infections), general reduction of transcranial Doppler velocity and laboratory improvement with a significant increase in the haematological parameters of fetal hemoglobin, hemoglobin and volume significant reduction in platelet, leukocyte, neutrophil, monocyte and lymphocyte counts. Conclusion: the study showed clinical and laboratory improvement in patients with SCA after the use of HU, without serious adverse effects, with a decrease in hospitalizations and transfusions; however, use requires regular follow-up with specialist and frequent monitoring, due to possible causes of drug toxicity. The use of HU reduced the speed of the transcranial Doppler in both children with conditional speed and in children with normal speed. According to the results obtained, it is believed that HU could be prescribed more precociously and frequently in FA, based on the benefits achieved.

Cutaneous Leishmaniasis, caused by Leishmania parasites, affects millions of individuals worldwide. Despite its side-effect and cytotoxicity, antimonial compounds remain the first choice treatment to leishmaniasis. In order to discover novel leishmanicidal compounds in silico approaches (pharmacophore models) were employed to identify putative inhibitors of arginase, a key enzyme in polyamines biosynthesis that is essential for parasite proliferation. Herein, we report the in vitro assays for three of the compounds selected (A4021, M5171 e S783579) by this approach, against recombinant arginase, promastigote stages of Leishmania amazonensis and in infected macrophages. Drugs A4021, M5171 and S783579 showed no inhibitory effect of purified recombinant arginase at any of the concentrations used (500; 125; 31,25; 7,81; 1,95 and 1,95μM). Regarding the L.amazonensis axenic culture, we detected a reduction in parasite proliferation at the highest concentrations (1000,500 and 100 μM) and observed a reduction in arginase activity in the M5171 and S783579 treated ones. In addition, through flowering microscopy nuclear, dilation and propidium iodide staining were observed by altering the membrane permeability, suggesting that the drugs cause secondary necrosis or late apoptosis. Concerning intracellular morphology, evaluated by transmission electron microscopy, structures indicative of cell death process in those treated with M5171 and S783579. Regarding the effect of M5171 (77.88μM) and S783579 (379.5μM) on parasite-host interaction, we observed a reduction in parasitic load, being M5171 the most effective, although no reduction in arginase activity has been detected. The study with A4021 was discontinued due to dilution difficulty and low efficacy. Although the compounds presented toxicity to leishmania, the viability in human macrophages was not affected at any concentrations evaluated by the Alamar Blue assay (1000,100,10,1 and 0,1μM).These results demonstrate the effectiveness of the in silico approach to select compounds as potential candidates for controlling L. amazonensis replication. In addition, the lack of cellular toxicity and chemical complexity of A4021, M5171 and S783579 make them good candidates for optimization and design of new drugs with the prospect of developing more effective treatments for cutaneous leishmaniasis caused by L. amazonensis.

INTRODUCTION: Diabetes is classified as a risk factor for infectious and endemic diseases, in
tropical countries. The increased incidence in the severity of clinical manifestations in patients with
metabolic diseases such as T2DM is due to chronic inflammation induced by increased plasma
glucose and exogenous lipid levels. Therefore, cutaneous infections caused by pathogens in Brazil,
such as L. braziliensis (Lb), which causes cutaneous leishmaniasis (LC), represent a proposed
model to evaluate the effect of the association between DM, metformin use. Metformin is a
hypoglycemic drug of first choice for hypoglycemic treatment of DM and is widely used in the
Unified Health System (SUS), despite its interference in the host's in situ and systemic immune
response against the protozoan. Given the additional effects of metformin (MET), it is capable of
modulating the innate and adaptive host immune response. OBJECTIVE: To evaluate the
immunomodulatory effect of metformin in an experimental model of Lb infection and diabetic
patients. MATERIAL AND METHODS: This is a case-control study, convenience sample,
investigative and descriptive study in patients with LC with and without DM. Additionally, an
experimental study of Lb infection and MET treatment in vivo in BALB / c mice and in vitro in
Raw 264.7 macrophages. RESULTS: Patients with DM develop atypical LC, with less frequency
of necrosis and CD8 + T cells in lesions. MET in the in vivo experimental model induced a decrease
in serum TNF-α and IL-12p70, interfered with lesion kinetics and was associated with increased
parasite load at the inoculation site and ipsilateral lymph nodes. In MET-treated macrophages, cell
proliferation reduction was observed and there was no interference on cell viability. Small MET
concentration in L.b culture allows the maintenance of the stationary phase of growth. In vitro
infection with Lb and MET-treated cells increased the frequency of infected macrophages and
parasite load. Although MET and Lb infection alone are able to promote the production of
intracellular ROS, ROS levels are reduced in MET-treated macrophages undergoing L.b infection.
CONCLUSION: DM and MET can interfere with the phenotypic of skin lesions by modulating
cell infiltrate at the inflammation site, as well as the extent and frequency of LC necrosis in diabetic
patients. Systemically, the experimental treatment with MET was able to reduce the levels of
cytokines that induce a Th1 response, described as protective for parasitic control, as well as to
interfere with cutaneous ulceration kinetics and causing increased parasite load. Experimental
evidence was confirmed in cell culture assays in which MET increased parasitic load, altered ROS
production, and modulated cell proliferation. Based on this evidence, it can be concluded that the
use of MET worsens the evolution of cutaneous leishmaniasis and the use in diabetics in endemic
regions for LC should be evaluated.

INTRODUCTION: Visceral leishmaniasis (VL) is a disease caused by parasites of the genus Leishmania, affecting humans and other mammals worldwide. Spleen is involved in VL immunopathogenesis and presents alterations in white pulp microenvironments that are associated with increased susceptibility to coinfections and patient death. Plasmacytosis in splenic red pulp (RP) is one of the main alterations observed, but the specificity of secreted antibodies and distribution of these cells had not yet been evaluated. AIM: The aim of this study was to standardize the technique for detection of anti-L. infantum antibody producing cells and to determine the proportion of these cells in the spleen of dogs with VL. MATERIAL AND METHODS: L. infantum soluble biotinylated antigens were used as probes in a modified immunohistochemistry reaction to detect the presence of anti-L. infantum antibody secreting cells. Were used spleens from eight dogs from the endemic area for VL and as a control, three dogs from Instituto Gonçalo Moniz kennel. The samples were cryopreserved and sectioned on slides for immunohistochemistry. The amount of labeled and unlabeled cells was quantified, and the ratio in spleen RP and Periarteriolar lymphatic sheath (PALS) was calculated. RESULTS: Dogs with VL present hyperglobulinemia and more plasma cells in RP than dogs without VL. Among these dogs, 75% (6/8) had less than 42% of anti-L. infantum antibody-secreting cells in RP. The proportion of labeled plasma cells is higher in PALS (59.3%) than in RP (29.8%) and as higher the proportion in PALS, smaller is the proportion of positive cells in RP. CONCLUSIONS: The low frequency of anti-L.infantum Ig-producing plasma cells may be due to polyclonal activation of B lymphocytes, resulting in production of non-specific immunoglobulin and hyperglobulinemia

Visceral Leishmaniasis (VL) is among the most relevant zoonoses transmitted by vectors. Severe patients with VL present blood disorders, such as hemolysis and heme free, which may alter the behavior of neutrophils. In the present study, we noticed that in patients with VL there is a direct association between heme levels and neutropenia. From in vitro tests, we have seen that heme reduces the survival of human neutrophils infected with Leishmania infantum through the production of reactive oxygen species and release of neutrophilic enzymes, while favoring the viability of the parasite. In addition, neutrophils infected with heme showed an increase in the enzymatic activity of superoxide dismutase-1 (SOD-1) and with interference in the oxidative pathways there was a reduction in the proliferation rate of L. infantum in these cells. Thus, the activation of neutrophils in the presence of heme, causes the death of the cell and the survival of the parasites. We then investigated the lipofosfoglican (LPG) of L. infantum as well as the activation trigger of neutrophils at the initial moments of infection. In order to evaluate how the L. infantum surface LPG interferes with the activation of neutrophils, we infected human neutrophils with LPG deficient parasites (∆lpg1) and compared them with wild promastigotes (WT) or parasites that had their LPG reconstituted (∆lpg1+LPG1). The absence of LPG in ∆lpg1 forms increased the infection rate, but favored the mortality of these forms in neutrophils compared to WT or ∆lpg1+LPG1. Regarding the activation of neutrophils, there was an expressive production of reactive oxygen species (ROS) in cultures infected with L. infantum ∆lpg1, able to modulate the survival of these promastigotes. These findings stimulate new interpretations in the context of VL and evidence the complexity of neutrophil-parasite interactions depending on the stimulus.

Neutrophils are able to modulate the immune response by cytokines and chemokines
production, degranulation, extracellular traps or direct interaction with other cells present in
the site of infection. Dendritic cells (DCs) are antigen-presenting cells, which can interact
with different cells of the immune system. This interaction between cells of the innate
immune system is essential for targeting the adaptive immune response, which is responsible
for eliminating microorganisms and the maintenance of immunological memory. Extracellular
ATP produced after cells’ injury exhibits proinflammatory properties, while adenosine, ATP
catabolism product, exhibits antiinflamatory properties. In this study, we evaluated the effect
of the interaction between human DCs and neutrophils infected by L. braziliensis or L.
amazonensis, and the participation of adenosine receptors. Methodology: Neutrophils
purified from peripheral blood of healthy donors were infected or not with L. braziliensis or
L. amazonensis and co-cultured with DCs generated in vitro with IL-4/GM-CSF. Analyzing
DCs in these co-cultures we assessed their infection rate, expression of surface molecules and
cytokine production. To investigate the involvement of adenosine, the parasite load on these
cells was assessed following inhibition of CD39, CD73, or A2A and A2B adenosine receptors.
Results: DCs co-cultured with L. amazonensis-infected neutrophils showed a significant
increase in parasite load, reduction on the expression of surface molecules and production of
pro-inflammatory cytokines, as well as increase in anti-inflammatory cytokines production.
Opposite results were obtained in the co-cultures of DCs and L. braziliensis-infected
neutrophils. Blocking CD39 but not CD73 on neutrophils or DCs induces a decrease in
parasite load in DCs co-cultured with L. amazonensis-infected neutrophils. Additionally,
inhibition of A2B or A2A adenosine receptor leads to a decrease of the parasite load after the
co-culture of DCs with L. amazonensis-infected neutrophils or when these cells were
cultivated with adenosine. This same treatment only altered the parasite load in DCs cocultivated
with neutrophils infected by L. braziliensis in presence of adenosine. Neutrophils
could be an important cell in modulating the activation or inhibition of DCs and different
species of Leishmania present differential behaviors in the interactions between neutrophils
and DCs.

Sickle cell disease (SCD) is a group of hemoglobinopathies and sickle cell anemia (SCA), characterized by HbSS homozygous, is the most severe form, while SC hemoglobinopathy (HbSC) is milder. Clinical manifestations in SCD are heterogeneous, thus, laboratory and inflammatory biomarkers are very useful in the clinical practice. Several pathophysiological mechanisms were suggested as significant associations were found with clinical manifestations and systemic inflammatory involvement of the disease. Therapy for SCD is based on hydroxyurea (HU), capable to improve laboratory biomarkers as well as to reduce clinical manifestations. Therefore, the aim of the present study was to investigate laboratory and inflammatory biomarkers associated to clinical manifestations in SCD as well as HU therapy. This study was approved by the Institutional Research Board (CAAE: 52280015.1.0000.0048), laboratory analyses were carried by automated methods and patients were followed-up at the Bahia Hemotherapy and Hematology Foundation (HEMOBA). In the first manuscript we included 181 SCD patients (126 HbSS and 55 HbSC) and we compared the groups based on each genotype. Our results suggest that SCA individuals more prominent exhibit anemia, hemolysis, leukocytosis and inflammation as well as increased frequency of clinical manifestations, while HbSC individuals exhibit less hemolysis and clinical complications. In the second manuscript we investigated among the 126 SCA individuals the association between clinical manifestations and lipid profile. History of pneumonia was found to be associated with higher total cholesterol levels, leg ulcers were associated with decreased low density lipoprotein (LDL) and pain crises were associated with increased high density lipoprotein (HDL). We also found associations between total cholesterol, LDL and HDL levels with hemolysis biomarkers as well as HbF levels, which was confirmed by the correlation analyses between individuals with previous history of pneumonia and pain crises. In the third manuscript 43 SCA patients were included and we evaluated interleukin-8 (IL-8) plasma levels as well as the rs4073 polymorphism. We verified that individuals carriers of the A allele have increased IL-8 levels, which was also associated with biomarkers of inflammation and hemolysis. In addition, lower IL-8 levels were associated with previous history of splenomegaly. In the last manuscript we investigated the effect of HU treatment on monocytes of SCA patients. Thirty seven patients were included, 17 were undergoing HU therapy while 20 were not receiving the medication. Our results suggest that HU was capable to decrease monocyte counts in peripheral blood, decrease classical monocytes (CD14++CD16-) frequency and increase non-classical monocytes (CD14dimCD16+). HU also decreased TNF-α, IL-1β, IL-6 and tissue factor (TF) production by the monocytes, as well as their polyfunctionality. Monocytes producers of TF were found to be associated with vaso-occlusion, in addition, classical monocytes are responsible for multiple cytokine production. Our data corroborate with previous studies regarding inflammatory and clinical aspects of SCA, in addition new associations between physiopathological mechanisms, laboratory biomarkers and pro-inflammatory molecules were found.

INTRODUCTION: Leishmaniasis is a zoonotic disease in more than 90 countries, which already affects 15 million people worldwide and grows at a rate of 1.6 million new cases per year. The most common clinical form of the disease is localized cutaneous leishmaniasis (LCL). Usually, patients with LCL present a single lesion that begins at the entry site of the parasite (Leishmania braziliensis), as small papules that develop into nodules and ulcerate in the center. Some studies have sought to clarify the mechanisms that control the development of these lesions and the immune response involved. In this sense, medium/high-throughput techniques have revealed important genes and immunological mechanisms in the progression of the disease. AIM: To identify the main molecules and pathways related to the pathogenesis of human cutaneous leishmaniasis ex vivo and to define, through in silico analysis, mechanisms of protection/susceptibility. METHOD: We explored skin lesion biopsies using medium-throughput techniques to quantify the expression of nCounter (NanoString) genes in patients recruited during medical visits to endemic areas of the Jiquiriçá Valley, Bahia. We associate in silico flow cytometry techniques and Ingenuity Pathway Analysis (IPA) to analyze the gene expression and the biological mechanisms involved in human LCL. RESULTS: Transcriptomic analysis of 601 genes of immune response, 4 genes of Leishmania braziliensis and 1 gene of Staphylococcus aureus was able to differentiate two groups of lesions with distinct immune response profiles associated with the presence or absence of Leishmania braziliensis. Epidemiological clinical data indicates that lesions with presence of Leishmania braziliensis transcripts are more recent than lesions that were not detected transcribed. The in silico flow cytometry evaluation revealed a predominance of cells belonging to the innate immunity in lesions with presence of Leishmania braziliensis transcripts (initial) and the predominance of adaptive immunity cells in lesions that were not detected Leishmania braziliensis (late) transcripts. The analyzes of integrated gene expression by IPA revealed mainly a continuous mechanism of extracellular matrix destruction. CONCLUSION: Our data suggest a relation between the transcriptomic signature of initial lesions with the presence of the parasite and the cells of the innate immune response. In addition, we confirmed the involvement of pathways associated with inflammation in the progression of the disease. Finally, we revealed the presence of a durable extracellular matrix degradation pathway in lesions of patients with LCL.

ABSTRACT
INTRODUCTION: Leishmaniasis remains one of the most neglected tropical diseases in the world. The immune response of the host against Leishmania plays a critical role in the elimination of parasites, but is also responsible for the inflammation and severity of the disease. The localized cutaneous form (LCL) is characterized by the presence of a single ulcerated lesion on the skin and uncontrolled inflammation. The increased susceptibility of skin infections is one of the complications of diabetes. When compared to the euglycemic state, hyperglycemia leads to alterations in the production of proinflammatory cytokines and chemokines, mainly due to increased production of leukotriene B4 (LTB4), an inflammatory lipid mediator. OBJECTIVE: In this context, the present study aimed to evaluate the influence of diabetes on the outcome of human LCL, considering the role of LTB4. MATERIAL AND METHODS: Serum levels of inflammatory mediators such as LTB4, PGE2, IL-1β, IL-6, TNF-α and IL-10 were measured by immunoenzymatic assay (ELISA) in euglycemic or hyperglycemic patients with LCL. A correlation was then made with clinical data, such as glucose, time, size, number and area of the lesion. To determine the susceptibility to infection by Leishmania braziliensis, macrophages were infected from diabetic or non-diabetic individuals, cultured under conditions with low and high glucose concentration. The culture supernatant was used to measure LTB4 and the cells for analysis of the Toll-like receptor (TLR) and BLT1 gene expression. RESULTS: We detected increased LTB4, IL-6 and TNF-α in the plasma of diabetic patients with LCL compared to non-diabetic patients. Interestingly, only LTB4 showed a positive correlation with healing time. In vitro assays indicate that glucose increases the rate of infection and parasite viability in a dose- dependent manner in L. braziliensis-infected human macrophages. In addition, high glucose concentration negatively modulates BLT1, TLR2 and TLR4. CONCLUSION: Together, these results suggest that diabetic patients produce high levels of LTB4 and that this is related to the healing time. In addition, high glucose concentrations increase the susceptibility of macrophages to L. braziliensis infection through the negative modulation of BLT1, TLR2 and TLR4 thus decreasing the action of the exacerbated production of LTB4.

ABSTRACT
INTRODUCTION: Visceral leishmaniasis (VL) is a major public health problem in Brazil, where it persists with elevated fatality rates despite of controlling measures. AIM: Identify clinical and laboratorial admission aspects associated to death in VL. MATERIAL AND METHODS: It’s a cross-sectional retrospective study including patients admitted to the Hospital Couto Maia (Salvador/Ba) with a diagnosis of VL between January 2010 and December 2014. Epidemiological, clinical and laboratory admission variables were collected in a standardized instrument and analyzed in the software Stata ® version 13.0 in the comparison between groups of clinical outcome (CO), discharge (n=106) or death (n=12). RESULTS: predominated male patients (62.7%), pediatric age (53.4%), origin of the urban area of municipalities in the interior of Bahia with low Human Development Index and classical clinical presentation with hepatosplenomegaly feverish and cytopenias. The average fatality rate was 10.2%, higher than 40 years of age. The main declared causes of death were renal and liver insufficiencies/hepatitis, sepsis/septic shock, unspecified shock and disseminated intravascular coagulation. Comorbidities were referred in 19.5% of patients and was associated with the CO (p=0.043), as well as the presence of abdominal pain (p=0.022), mucosal bleeding (p=0.034), edema (p=0.029), jaundice (p=0.000), sensory reduction (p = 0.001), lung crepitation (p=0.014), arrhythmic cardiac sounds (p=0.027), bacterial coinfection (p=0.019) and electrocardiographic abnormalities (p=0.001). Presented death association platelet levels < 81,000/mm ³ (p=0.004), serum albumin ≤ 2, 2 g/dL (p=0.032), urea > 37 mg/dL (p=0.009), total bilirubin > 0.9 mg/dL (p=0.001), direct bilirubin > 0, 4 mg/dL (p=0.002), prothrombin activity < 45% (p=0.008) and serum creatinine elevated to the age (p=0.003). The scores obtained in clinical (CP) and clinicolaboratorial (CLP) predictor models adopted by the Ministry of Health of Brazil showed agreement with the CO; CP ≥ 4 and CLP ≥ 6 were significantly associated with death in this sample, with greater strength of association for the last (p=0.000). CONCLUSIONS: the clinical profile of the sample was concordant with the literature, though some variables classically associated with death in VL showed no significant, or if made in different cut-offs, which may be due to population variations or study design, requiring more robust research for verification. The results for the CP and CLP scores in this sample reflect the importance of their achievement as a risk stratification tool of patients diagnosed with VL. It is hoped that this study will help in situational diagnosis of the disease in local population and in the identification of key issues to be deepened through different methodologies.

INTRODUCTION: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The disease is among the top neglected tropical diseases. In Brazil, schistosomiasis is endemic, being distributed, mainly, in Northeastern states. The gold-standard method for diagnosing the disease, according to the World Health Organization, is Kato-Katz. However, the technique shows a decrease in sensitivity in areas of low endemicity, besides the difficulty of acquiring faecal samples on different and alternating days and the need for professional expertise to analyze the blades. Within this context, other methodologies have been presented as good alternatives for the diagnosis of schistosomiasis in areas of low endemicity, such as the POC-CCA immunological test and the molecular rt-PCR test. AIM: The present study aimed to evaluate the performance of the POC-CCA immunological method and the real-time PCR molecular method for the diagnosis of schistosomiasis mansoni in areas of low endemicity, in relation to the gold standard, the Kato-Katz parasitological method. MATERIAL AND METHODS: 121 individuals from the rural community of Jenipapo, Ubaíra, Bahia, Brazil, aged between 2 and 80 years, were evaluated. Each individual provided a urine sample and a stool sample. For the Kato-Katz and real-time PCR, fecal samples were used. For the implementation of POC-CCA, the urine samples were used. Infection intensity and prevalence rates were determined and diagnostic performance was assessed through the parameters of sensitivity, specificity and accuracy. The Kappa index assessed the agreement of results among observers of the POC-CCA rapid test. The gold standard method of study is the Kato-Katz. RESULTS: The highest number of positive individuals was found by Kato-Katz (32), followed by POC-CCA (25) and rt-PCR (18). The POC-CCA presented a more inconsistent performance, with sensitivity of 43.8%, specificity of 87.6% and accuracy of 76%. The color intensity of the reaction was stronger in individuals with high and medium parasitic loads. The rt-PCR showed sensitivity of 56.2%, specificity of 100% and accuracy of 88.4%. The Kappa index showed that there was a strong agreement among the observers of the POC-CCA test (k = 0.94). According to the accuracy indexes obtained, the best test was rt-PCR (88.4%). CONCLUSIONS: The results demonstrate that the Kato-Katz method is a good technique to use for screening and morbidity control. The POC-CCA presented inconsistent results, not being a good alternative for screening or diagnosis. There was a correlation between the parasite load intensity and the reaction staining intensity in the POC-CCA test. The rt-PCR proved to be a viable alternative, despite the high cost. The association between the Kato-Katz parasitological method and the molecular method of rt-PCR is a good alternative for the control of transmission and diagnosis, since it presents an increase in sensitivity, which may improve the diagnosis in areas of low endemicity.

ABSTRACT
INTRODUCTION: CBA mouse macrophages (MΦ) control Leishmania major
infection yet are permissive to Leishmania amazonensis. The role played by
autophagy in Leishmania infection needs further investigation. OBJECTIVE:
Thus, we assessed whether activation of autophagic pathway may account for
differences in the response of infected MΦ to these two parasite strains.
MATERIAL and METHODS and RESULTS: First, we demonstrated by qPCR
and by analysis of publicly available microarray data that a greater number of
autophagy-related genes (Atg) are positively modulated in cells infected by L.
amazonensis compared to those infected by L. major. Ingenuity Pathway
Analyses (IPA) demonstrated opposite modulation in genes in L. amazonensisand
L. major-infected MΦ. After 24 h of infection, the autophagic flux measured
by LC3-II/Act ratio was similarly increased in either L. amazonensis- or L. majorinfected
MΦ compared to uninfected cells. Although L. major-induced
parasitophorous vacuoles exhibited greater positivity for the degradative marker,
DQ-BSA, LC3 recruitment was increased in L. amazonensis-induced
parasitophorous vacuoles. Interestingly, autophagy induction enhanced
intracellular L. amazonensis and L. major viability, although autophagy inhibition
caused no effect on infection profile. We also demonstrated that autophagy
induction reduced NO production by Leishmania-infected MΦ, yet did not alter
arginase activity. Moreover, principal component analysis completely
discriminated L. major-infected MΦ from L. amazonensis-infected cells regarding
infection intensity and autophagic features of parasite-induced PV.
CONCLUSION: In conclusion, infection by L. amazonensis or L. major, although
similarly activates the autophagic flux in infected MΦ and the parasites have their
viability favored by autophagy induction, these Leishmania species cause
differentiated expression of Atg and distinct interaction of their parasitophorous
vacuoles with autophagic vacuoles. These differences are capable to
discriminate MΦ infected by L. amazonensis from those infected by L. major.

ABSTRACT
INTRODUCTION: the pathophysiology of sickle cell anemia (SCA) is marked by
intermittent hemolytic and vasoconstrictive seizures with increased redox status in
the vascular microenvironment that favors inflammatory chronicity. OBJECTIVES: to
evaluate the association of heme with clinical-laboratory markers in patients with
SCA; the role of intact and lysed erythrocytes in the gene expression of NLRP3
inflammation, and whether the treatment of patients with hydroxyurea (HU) interferes
with this expression and the release of IL-1β and leukotriene B4 (LTB4); and
investigate the antioxidant properties of HU. METHODS: plasma heme of patients
with SCA (n = 80) was dosed by ELISA, laboratory markers were determined by
automated methods, and genetic analyzes by DNA fragment length polymorphism
(RFLP). The clinical history of the patients was obtained through medical records.
The gene expression assays of NLRP3 (NRLP3, IL1B, CASP1 and IL18)
components of RT-qPCR were performed on human peripheral blood mononuclear
cells (PBMCs) of healthy volunteers challenged with whole and lysed sickle cells (n =
8), and normal erythrocytes (n = 10); and total leukocytes from treated (n = 13) or not
treated with HU (n = 15) and and healthy volunteers (n = 20). The determinations of
IL-1β and LTB4 were performed by ELISA. The antioxidant activity was assessed by
sweep tests using the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH). Expression of
superoxide dismutase 1 (SOD1), glutathione peroxidase (GPx), glutathione Sreductase
(GSR) and heme oxygenase-1 (HMOX1) was performed by RT-qPCR in
human umbilical vein endothelial cells (HUVEC) and PBMC. Microarray analyzes
were performed on HUVEC treated with HU. RESULTS: increased free heme was
associated with increased hemoglobin S (HbS), monocyte count, hepatic markers,
triglycerides and very low density lipoprotein (VLDL-C); and decreased levels of fetal
hemoglobin (HbF) and high-density lipoprotein (HDL-C). The BEN/BEN genotype
was associated with higher levels of HbF than the CAR/CAR. The release of free
heme was not associated with haplotypes, but was associated with the clinical history
of stroke. Intact and lysed red blood cells (SS-RBC) induced the expression of
NLRP3 inflammation components and secretion of IL-1β and LTB4. SS-RBC induced
IL1B expression with secretion of IL-1β and LTB4, compared to lysed SS-RBC or
erythrocytes of healthy volunteers (AA-RBC). Significant decrease in NLRP3
expression and LTB4 secretion were observed in patients treated with HU. HU
presented antioxidant activity at concentrations equivalent to that found in the plasma
of patients treated with the drug (~200 μM). Treatments with HU or HU+hemin did not
induce toxicity in PBMC and HUVEC. The HU+hemin treatment stimulated
nitrate/nitrite production in PBMC and HUVEC. HU increased expression of SOD1
and GPx in PBMC and HUVEC. The increase in GSR expression was observed in
PBMC and HUVEC treated with HU+hemin. Treatment with HU (or in combination
with hemin) did not interfere in the expression of HMOX1 in PBMC as in HUVEC. HU
induced expression of SOD2, GSR, GST1 (glutathione S-transferase-1), GSTM2
(glutathione S-transferase mu 2), MGST1 (glutathione S-transferase 1), CBR1
(carbonyl reductase 1), protein kinases phosphatidylinositol 3 -phosphate C (PRKCB,
PRKCZ, PIK3C2B) and sequestosome-1 (p62/SQSTM1). In contrast, the decrease in
transcriptional factor BACH1 expression was observed. Upstream analyzes
demonstrated prediction of activation of Jun, miR-155 and mir-141-3p.
CONCLUSIONS: excessive heme release from recurrent hemolytic events may
contribute substantially to the clinical severity of SCA. Whole and lysed blood cells
can act as DAMPs by inducing the expression of inflammatory components and the
production of IL-1β and LTB4, contributing to the establishment of inflammation.
Treatment with HU appears to decrease inflammation by NLRP3 and LTB4
dependent pathways. HU has direct antioxidant properties by eliminating free
radicals and inducing the enzymatic system of antioxidant response via Nrf2 under
sequestosome-1 regulation. These findings may aid in the development of new
therapeutic strategies that can be used in conjunction with the induction of HbF in
order to minimize the inflammatory chronicity of SCA.

INTRODUCTION: Chagas' disease is a parasitic disease caused by Trypanosoma cruzi, which is one of the main causes of cardiovascular morbidity and mortality in Latin America. Existing therapeutic interventions are not fully effective, and heart transplantation is the only alternative for patients with severe chronic Chagas' heart disease. In this sense, the absence of therapies capable of acting directly on the pathophysiological determinants of the disease demonstrates the necessity of identifying new therapeutic approaches. Studies previously conducted by our group demonstrated that the use of stem cells obtained from bone marrow and other sources had beneficial effects in the treatment of experimental chagas disease. Additionally, the possibility of enhancing stem cell paracrine effects through genetic modification has been the subject of scientific investigations. OBJECTIVE: To evaluate the effects of genetically modified mesenchymal stem cell therapy to overexpress granulocyte colony stimulating factor (hG-CSF) or insulin-like growth factor 1 (hIGF-1) in an experimental model of Chagas disease. MATERIAL AND METHODS: C57BL/6 mice were infected with 1000 trypomastigotes from the Colombian strain of T. cruzi and, after six months of infection, were treated with CTM, CTM-G-CSF, or CTM-IGF-1. Groups of uninfected or infected animals treated with saline (vehicle) were used as controls. All animals were euthanized under anesthesia after two months of treatment for histopathological and morphometric analysis of the heart or skeletal muscle, as well as for evaluation of inflammatory cytokine expression. RESULTS: Mouse heart sections from groups treated with CTM, CTM-GCSF or CTM-IGF-1 showed a significant reduction in the number of inflammatory cells and the percentage of fibrosis when compared to chagasic animals treated with saline.This difference was more evident in the group that was treated with stem cells overexpressing G-CSF. In addition, CTM-G-CSF therapy induced mobilization of immunomodulatory cells to the heart, including myeloid suppressor cells (MDSC) and Foxp3 + regulatory T cells expressing IL-10. Expression of inflammatory cytokine genes in cardiac tissue revealed an increase in inflammatory cytokines in chronic chagasic animals when compared to uninfected controls, where most cytokines were significantly modulated in groups treated with CTM or CTM-G-CSF. Although CTM-IGF-1 therapy demonstrated no additional benefit to cardiac tissue when compared to the group treated with unmodified CTM, a regenerative effect of this therapy was observed in chagasic mice skeletal muscle, resulting in an increase in skeletal muscle fibers 60 days after treatment. CONCLUSION: Our results demonstrate that bone marrow derived CTM treatment overexpressing hG-CSF or hIGF-1 enhanced the therapeutic effects of MSCs through immunomodulation and pro-regenerative actions in the heart and skeletal muscle of mice chronically infected wthT. cruzi. Thus, the genetic modification of CTMs for overexpression of factors with therapeutic potential represents a promising strategy for the development of new therapies for chronic chagasic cardiomyopathy.

ABSTRACT
Forensic entomology is the science that studies the insects biology and their relationship to any criminal acts. In investigations, particularly in forensic medicine, it science can be an important tool in estimate postmortem interval (PMI). Calliphoridae (Diptera) is the most important forensic interest familie. They are the first to locate the substrate, develop therein, and are found throughout all the decomposition process. This study aimed to verify the occurrence of the species of this family in swine carcasses exposed during the seasons in an atlantic forest fragment of. Daily, adult insects were collected in specific traps, and immature stages were collected daily too, on the carcass and were transfer to the Forensic Entomology Laboratory of the Department of Technical Police of Bahia, to compainnin its development, until emmergence. After they were identified. Furthermore, we found the correlation between the species that visit the carcass with those that develop in it, relating it to the decomposition stages. Twenty-nine thousand and one hundred and thrirty six (29,136) adults were collected and total larvae collected in, only 1278 emerged in the laboratory. Adults captured belonged to the following species: Cochliomyia macellaria (Sum: 1,5%; Aut: 0,1%; Win:0,5%; Spr:1,3%), Chrysomya megacephala (Sum:11,3%; Aut17,6%; Win:2,7%; Spr:7,6%), Chrysomya albiceps (Sum:83,5% Aut:67,5%; Win:89,2%; Spr:47%), Chrysomya putoria (Sum:2,1% Aut:7,8%; Win:1,8%; Spr:0,8%), Lucilia eximia (Sum: 0,9% Aut:2,1%; Win:2,9%; Spr:0,5%), Mesembrinella bellardiana (Sum:0,6% Aut:3,7%; Win:0,8%; Spr:3,4%), Hemilucilia segmentaria (Sum:0,2% Aut:1,1%; Win:1,6%; Spr:6,2%) e Chloroprocta idioidea (Win: 0,4%; Spr:33,1%). The immature stages collected and followed untill adult stage were identified: Chrysomya albiceps (Sum: 97,2%; Aut:79,1%; Win:82,2% Spr:53,5), Chrysomya putoria (Aut: 5,9%; Win: 0,3%; Spr: 1,3%), Hemilucilia segmentaria (Aut: 12,8%; Win: 15,2%; Spr:40,3%), Lucilia eximia (Sum: 2,4%; Aut:2,2%; Win:2,3%; Spr: 5,0%). We conclude that there are differences in abundance between species during the experiment and the species Chrysomya albiceps was the most prevalent. With our observations we can´t establish patterns of succession among the species of this family. It was found that in the first moments after colonization, to about 24 hours, there is a more chance to collect Calliphoridae other species, however, after this period, C albiceps occupies almost all of the niche, each more rare you see the meeting of larvae of other species, probably due to predation. This work contributed essential information to implement this type of expertise in the routine of the Forensic Entomology Laboratory of Bahia´s Technical Police Department.