Identification of mechanisms controlling the transcriptional activity of nuclear factor kappa B (NF-κB) p65/RelA

Dissertação para a obtenção do grau de doutor em Biologia pelo Instituto de Tecnologia Química e Biológica. Universidade Nova de Lisboa.

The hamster (mesocricetus auratus) as experimental model in chagas' disease: parasitological and histopathological studies in acute and chronic phases of Trypanosoma cruzi infection

This research characterizes the acute and chronic phases of Chagas ' disease in hamster through parasitological and histopathological studies. The acute phase was achieved with 44 young hamsters injected intraperitoneally with 100.000 blood trypomastigotes of Benedito and Y strains of T. cruzi. The chronic phase was induced in 46 hamsters injected intraperitoneally with 35.000 trypomastigotes ofVicentina, Benedito and Y strains. Animals were sacrificed at regular intervals of 24 hours of acute phase and from the 3rd to the 10th month of infection ofchronic phase. In the acute phase, parasites were easily recoveredfrom all animals and there was an inflammatory reaction characterized by mononuclear and polymorphous leukocyte infiltration of variable degree in the majority of tissues and organs, specially in the connective loose and fatty tissues, smooth muscle myocardium and skeletal muscle. In the chronic phase the lesions occurred in the same tissues and organs, but the inflammatory response was less severe and characterized by mononuclear infiltration mainly with focal or zonalfibrosis in the myocardiun. In 50% of infected animals parasites were found inmyocardiun and recoveredfrom pericardic, peritoneal and ascitic fluids in some animals. Signs of heart failure, sudden death and enlargement of bowel were observed regularly. We concluded that the hamster is an useful model for Chagas' disease studies.

Dissecting cross-talk between microglia and motoneurons in ALS: signaling events and soluble factors

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Glia-Motoneuron dialogue in ALS onset and progression in SOD1G93A-mice model

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the pro-gressive loss of motoneurons (MN). Increasing evidence points glial cells as key players for ALS onset and progression. Indeed, MN-glia signalling pathways involving either neuroprotection or inflammation are likely to be altered in ALS. We aimed to study the molecules related with glial function and/or reactivity by evaluating glial markers and hemichannels, mainly present in astrocytes. We also studied molecules involved in mi-croglia-MN dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8), as well as proliferation (Ki-67) and inflammatory-related molecules (TLR2/4, NLRP3; IL-18) and alarming/calming signals (HMGB1/autotaxin). We used lumbar spinal cord (SC) homogenates from mice expressing a mutant human-SOD1 protein (mSOD1) at presymptomatic and late-symptomatic ALS stages. SJL (WT) mice at same ages were used as controls. We observed decreased expression of genes associated with astrocytic (GFAP and S100B) and microglial (CD11b) markers in mSOD1 at the presymptomatic phase, as well as diminished levels of gap junction components pannexin1 and connexin43 and expression of Ki-67 and decreased autotax-in. In addition, microglial-MN communication was negatively affected in mSOD1 mice as well as in-flammatory response. Interestingly, we observed astrocytic (S100B) and microglial (CD11b) reactivity, increased proliferation (Ki-67) and increased autotaxin expression in symptomatic mSOD1 mice. In-creased MN-microglial dialogue (CXCR3/CCL21; CX3CR1/CX3CL1; MFG-E8) and hemichannel activ-ity, namely connexin43 and pannexin1, were also observed in mSOD1 at the symptomatic phase, along with an elevated inflammatory response as indicated by increased levels of HMGB1 and NLRP3. Our results suggest that decreased autotaxin expression is a feature of the presymptomatic stage, and precede the network of pro-inflammatory-related symptomatic determinants, including HMGB1, CCL21, CX3CL1, and NLRP3. The identification of the molecules and signaling pathways that are dif-ferentially activated along ALS progression will contribute for a better design of therapeutic strategies for disease onset and progression.

Severe visceral leishmaniasis in children: the relationship between cytokine patterns and clinical features

Introduction The relationship between severe clinical manifestations of visceral leishmaniasis (VL) and immune response profiles has not yet been clarified, despite numerous studies on the subject. This study aimed to investigate the relationship between cytokine profiles and the presence of immunological markers associated with clinical manifestations and, particularly, signs of severity, as defined in a protocol drafted by the Ministry of Health (Brazil). Methods We conducted a prospective, descriptive study between May 2008 and December 2009. This study was based on an assessment of all pediatric patients with VL who were observed in a reference hospital in Maranhão. Results Among 27 children, 55.5% presented with more than one sign of severity or warning sign. Patients without signs of severity or warning signs and patients with only one warning sign had the highest interferon-gamma (IFN-γ) levels, although their interleukin 10 (IL-10) levels were also elevated. In contrast, patients with the features of severe disease had the lowest IFN-γ levels. Three patients who presented with more than two signs of severe disease died; these patients had undetectable interleukin 2 (IL-2) and IFN-γ levels and low IL-10 levels, which varied between 0 and 36.8pg/mL. Conclusions Our results showed that disease severity was associated with low IFN-γ levels and elevated IL-10 levels. However, further studies with larger samples are needed to better characterize the relationship between disease severity and cytokine levels, with the aim of identifying immunological markers of active-disease severity.

Improving silicon probe performance through layer-by-layer coating

Fully comprehending brain function, as the scale of neural networks, will only be possi-ble with the development of tools by micro and nanofabrication. Regarding specifically silicon microelectrodes arrays, a significant improvement in long-term performance of these implants is essential. This project aims to create a silicon microelectrode coating that provides high-quality electrical recordings, while limiting the inflammatory response of chronic implants. To this purpose, a combined chitosan and gold nanoparticles coating was produced allied with electrodes modification by electrodeposition with PEDOT/PSS in order to reduce the im-pedance at 1kHz. Using a dip-coating mechanism, the silicon probe was coated and then charac-terized both morphologically and electrochemically, with focus on the stability of post-surgery performance in anesthetized rodents. Since not only the inflammatory response analysis is vital, the electrodes recording degradation over time was also studied. The produced film presented a thickness of approximately 50 μm that led to an increase of impedance of less than 20 kΩ in average. On a 3 week chronic implant, the impedance in-crease on the coated probe was of 641 kΩ, compared with 2.4 MΩ obtained for the uncoated probe. The inflammatory response was also significantly reduced due to the biocompatible film as proved by histological tests.

Bronchiolitis, respiratory syncytial virus, and recurrent wheezing: what is the relationship?

Various follow-up studies of children hospitalized with bronchiolitis caused by respiratory syncytial virus have demonstrated that a significant proportion of infants (50%) have recurrent wheezing during childhood. Nevertheless, the relationship between these two entities, if any, has not been established. In order to explain this observation, several hypotheses have been proposed. The first suggests that some children could have an individual predisposition to bronchiolitis caused by respiratory syncytial virus and recurrent wheezing. The virus could be a marker of this condition, and the individual predisposition could in turn be related to an individual hypersensitivity to common allergens (atopy), airway hyperreactivity, or to some disorder related to pulmonary anatomy or physiology that was present before the acute episode of bronchiolitis. Another hypothesis proposes that respiratory syncytial virus could be directly responsible for recurrent wheezing. During an episode of bronchiolitis, the damage in the airway mucosa caused by the vital inflammatory response to infection contributes to sensitivity to other allergens or exposes irritant receptors, resulting in recurrent wheezing. For this review, we analyzed the studies that discuss these hypotheses with the purpose of clarifying the mechanisms for the important issue of recurrent wheezing in childhood.

Effect of clarithromycin on the cell profile of bronchoalveolar lavage fluid in mice with neutrophil-predominant lung disease

OBJECTIVE: Macrolide antibiotics have anti-inflammatory properties in lung diseases. The aim of this study was to investigate the effect of clarithromycin in pulmonary cellular inflammatory response in mice. METHOD: Eight adult Swiss mice were studied. All animals received an intranasal challenge (80 µL) with dead Pseudomonas aeruginosa (1.0 x 10(12) CFU/mL). Bronchoalveolar lavage was performed 2 days later, with total cell count and differential cell analysis. The study group (n = 4) received clarithromycin treatment (50 mg/kg/day, intraperitoneal) for 5 days. Treatment was initiated 2 days before intranasal challenge. RESULTS: There was no significant difference in total cell count between the groups (mean: 2.0 x 10(6) and 1.3 x 10(6), respectively). In both groups, there was a predominance of neutrophils. However, the study group had a higher percentage of lymphocytes in the bronchoalveolar lavage than the control group (median of 19% vs 2.5%, P = .029). CONCLUSION: Clarithromycin alters the cytological pattern of bronchoalveolar lavage of Swiss mice with neutrophil pulmonary inflammation, significantly increasing the percentage of lymphocytes.

Involvement of C4 allotypes in the pathogenesis of human diseases

The complement system is an important humoral defense mechanism that plays a relevant role against microbial agents, inflammatory response control, and immunocomplex clearance. Classical complement pathway activation is antibody-dependent. The C4 component participates in the initial step of activation, and C4 expression is determined by 2 pairs of allotypes: C4A and C4B. Deficiencies in C4 allotypes have been associated with several diseases. The aim of the present review is evaluate the reported data in the literature regarding specific C4A and C4B deficiencies and characterize their clinical relevance. We searched the MEDLINE and LILACS databases. Papers referring to total C4 deficiency without allotype evaluation and case reports of primary C4 deficiency were not included. Deficiencies in C4 allotypes have been associated with Mycobacterium leprae infection, erythema nodosum, systemic sclerosis with anti-topoisomerase I antibodies, intermediate congenital adrenal hyperplasia with DR5 genotype, diabetes mellitus type 1 with DR3,4 genotype, and diabetes mellitus with antibodies against islet cells. C4 allotype deficiency is also related to C4B deficiency and autoimmune-associated diseases, such as systemic lupus erythematosus, or diseases with an autoimmune component, such as autism. Some reports associate C4A with thyroiditis after delivery as well as limited and systemic sclerosis without anti-topoisomerase I antibodies. However, the studies with C4A and C4B have been concentrated in isolated populations, and some of the studies could not be reproduced by other authors.

Hypophosphatemia in critically ill children

The purpose of this paper is to review clinical studies on hypophosphatemia in pediatric intensive care unit patients with a view to verifying prevalence and risk factors associated with this disorder. We searched the computerized bibliographic databases Medline, Embase, Cochrane Library, and LILACS to identify eligible studies. Search terms included critically ill, pediatric intensive care, trauma, sepsis, infectious diseases, malnutrition, inflammatory response, surgery, starvation, respiratory failure, diuretic, steroid, antiacid therapy, mechanical ventilation. The search period covered those clinical trials published from January 1990 to January 2004. Studies concerning endocrinological disorders, genetic syndromes, rickets, renal diseases, anorexia nervosa, alcohol abuse, and prematurity were not included in this review. Out of 27 studies retrieved, only 8 involved pediatric patients, and most of these were case reports. One clinical trial and one retrospective study were identified. The prevalence of hypophosphatemia exceeded 50%. The commonly associated factors in most patients with hypophosphatemia were refeeding syndrome, malnutrition, sepsis, trauma, and diuretic and steroid therapy. Given the high prevalence, clinical manifestations, and multiple risk factors, the early identification of this disorder in critically ill children is crucial for adequate replacement therapy and also to avoid complications.

Acellular Gellan-gum based bilayered structures for the regeneration of osteochondral defects: a preclinical study

 In orthopaedics, the management and treatment of osteochondral (OC) defects remains an ongoing clinical challenge. Autologous osteochondral mosaicplasty has been used as a valid option for OC treatments although donor site morbidity remains a source of concern [1]. Engineering a whole structure capable of mimicking different tissues (cartilage and subchondral bone) in an integrated manner could be a possible approach to regenerate OC defects. In our group we have been proposing the use of bilayered structures to regenerate osteochondral defects [2,3]. The present study aims to investigate the pre-clinical performance of bilayered hydrogels and spongy-like hydrogels in in vivo  models (mice and rabbit, respectively), in both subcutaneous and orthotopic models. The bilayered structures were produced from Low Acyl Gellan Gum (LAGG) from Sigma-Aldrich, USA. Cartilage-like layers were obtained from a 2wt% LAGG solution. The bone-like layers were made of 2wt% LAGG with incorporation of hydroxyapatite at 20% and 30% (w/v). Hydrogels and spongy-like were subcutaneouly implanted in mice to evaluate the inflammatory response. Then, OC defects were induced in rabbit knee to create a critical size defect (4 mm diameter and 5 mm depth), and then hydrogels and sponges implanted. Both structures followed different processing methods. The hydrogels were injected allowing in situ  crosslinking. Unlike, the spongy-like were pre-formed by freeze-drying. The studies concerning subcutaneous implantation and critical size OC defect were performed for 2 and 4 weeks time, respectively. Cellular behavior and inflammatory responses were assessed by means of histology staining and biochemical function and matrix deposition by immunohistochemistry. Additionally, both OC structures stability and new cartilage and bone formation were evaluated by using vivo- computed tomography (Scanco 80). The results showed no acute inflammatory response for both approaches. New tissue formation and integration in the adjacent tissues were also observed, which present different characteristic behaviors when comparing hydrogels and sponges response. As future insights, a novel strategy for regeneration of OC defects can be designed encompassing both, hydrogels and spongy-like structures and cellular approaches. References: 1. Espregueira-Mendes J. et al. Osteochondral transplantation using autografts from the upper tibio-fibular joint for the treatment of knee cartilage lesions. Knee Surgery, Sports Traumatology, Arthroscopy 20,1136, 2012. 2. Oliveira JM. et al, Novel hydroxyapatite/chitosan bilayered scaffold for osteochondral tissue-engineering applications: Scaffold design and its performance when seeded with goat bone marrow stromal cells. Biomaterials 27, 6123, 2006. 3. Pereira D R. et al. Gellan Gum-Based Hydrogel Bilayered Scaffolds for Osteochondral Tissue Engineering. Key Engineering Materials 587, 255, 2013.

Release of insulin from PLGA-alginate dressing stimulates regenerative healing of burn wounds in rats

Burn wound healing involves a complex set of overlapping processes in an environment conducive to ischemia, inflammation, and infection costing $7.5 billion/year in the US alone, in addition to the morbidity and mortality that occur when the burns are extensive. We previously showed that insulin, when topically applied to skin excision wounds, accelerates re-epithelialization, and stimulates angiogenesis. More recently, we developed an alginate sponge dressing (ASD) containing insulin encapsulated in PLGA microparticles that provides a sustained release of bioactive insulin for >20days in a moist and protective environment. We hypothesized that insulin-containing ASD accelerates burn healing and stimulates a more regenerative, less scarring, healing. Using a heat-induced burn injury in rats, we show that burns treated with dressings containing 0.04mg insulin/cm2, every three days for 9 days, have faster closure, faster rate of disintegration of dead tissue, and decreased oxidative stress.In addition, in insulin-treated wounds the pattern of neutrophil inflammatory response suggests faster clearing of the burn dead tissue. We also observe faster resolution of the pro-inflammatory macrophages. We also found that insulin stimulates collagen deposition and maturation with the fibers organized more like a basket weave (normal skin) than aligned and crosslinked (scar tissue). In summary , application of ASD-containing insulin-loaded PLGA particles on burns every three days stimulates faster and more regenerative healing. These results suggest insulin as a potential therapeutic agent in burn healing and, because of its long history of safe use in humans, insulin could become one of the treatments of choice when repair and regeneration are critical for proper tissue function.

The role of IFNγ in higher brain function: in health and under chronic stress

Tese de Doutoramento em Ciências da Saúde

Invasive hemodynamic monitoring in the postoperative period of cardiac surgery

OBJETIVE: To assess the hemodynamic profile of cardiac surgery patients with circulatory instability in the early postoperative period (POP). METHODS: Over a two-year period, 306 patients underwent cardiac surgery. Thirty had hemodynamic instability in the early POP and were monitored with the Swan-Ganz catheter. The following parameters were evaluated: cardiac index (CI), systemic and pulmonary vascular resistance, pulmonary shunt, central venous pressure (CVP), pulmonary capillary wedge pressure (PCWP), oxygen delivery and consumption, use of vasoactive drugs and of circulatory support. RESULTS: Twenty patients had low cardiac index (CI), and 10 had normal or high CI. Systemic vascular resistance was decreased in 11 patients. There was no correlation between oxygen delivery (DO2) and consumption (VO2), p=0.42, and no correlation between CVP and PCWP, p=0.065. Pulmonary vascular resistance was decreased in 15 patients and the pulmonary shunt was increased in 19. Two patients with CI < 2L/min/m² received circulatory support. CONCLUSION: Patients in the POP of cardiac surgery frequently have a mixed shock due to the systemic inflammatory response syndrome (SIRS). Therefore, invasive hemodynamic monitoring is useful in handling blood volume, choice of vasoactive drugs, and indication for circulatory support.

Control of bovine mastitis: old and recent therapeutic approaches

Mastitis is defined as the inflammatory response resulting of the infection of the udder tissue and it is reported in numerous species, namely in domestic dairy animals. This pathology is the most frequent disease of dairy cattle and can be potentially fatal. Mastitis is an economically important pathology associated with reduced milk production, changes in milk composition and quality, being considered one of the most costly to dairy industry. Therefore, the majority of research in the field has focused on control of bovine mastitis and many efforts are being made for the development of new and effective anti-mastitis drugs. Antibiotic treatment is an established component of mastitis control programs; however, the continuous search for new therapeutic alternatives, effective in the control and treatment of bovine mastitis, is urgent. This review will provide an overview of some conventional and emerging approaches in the management of bovine mastitis infections.