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.

Aging and Alzheimer's disease: Searching for novel molecular cues

Tese de Doutoramento em Ciências da Saúde

Assessment of a protease inhibitor peptide for anti-ageing

Ageing and skin exposure to UV radiation induces production and activation of matrix metalloproteinases (MMPs) and human neutrophil elastase (HNE). These enzymes are known to break down the extracellular matrix (ECM) which leads to wrinkle formation. Here, we demonstrated the potential of a solid-in-oil nanodispersion containing a competitive inhibitor peptide of HNE mixed with hyaluronic acid (HA), displaying 158 nm of mean diameter, to protect the skin against the ageing effects. Western blot analysis demonstrated that activation of MMP-1 in fibroblasts by HNE treatment is inhibited by the solid-in-oil nanodispersion containing the peptide and HA. The results clearly demonstrate that solid-in-oil nanodispersion containing the HNE inhibitor peptide is a promising strategy for anti-ageing effects. This effect can be seen particularly by ECM regulation by affecting fibroblasts. The formulation also enhances the formation of thicker bundles of actin filaments.

Effect of short chain fructooligosaccharides (scFOS) on immunological status and gut microbiota of gilthead sea bream (Sparus aurata) reared at two temperatures

The effects of dietary short chain fructooligosaccharides (scFOS) incorporation on hematology, fish immune status, gut microbiota composition, digestive enzymes activities, and gut morphology, was evaluated in gilthead sea bream (Sparus aurata) juveniles reared at 18 °C and 25 °C. For that purpose, fish with 32 g were fed diets including 0, 0.1, 0.25 and 0.5% scFOS during 8 weeks. Overall, scFOS had only minor effects on gilthead sea bream immune status. Lymphocytes decreased in fish fed the 0.1% scFOS diet. Fish fed the 0.5% scFOS diet presented increased nitric oxide (NO) production, while total immunoglobulins (Ig) dropped in those fish, but only in the ones reared at 25 °C. Red blood cells, hemoglobin, bactericidal activity and NO were higher at 25 °C, whereas total white blood cells, circulating thrombocytes, monocytes and neutrophils were higher at 18 °C. In fish fed scFOS, lymphocytes were higher at 18 °C. Total Ig were also higher at 18 °C but only in fish fed 0.1% and 0.5% scFOS diets. No differences in gut bacterial profiles were detected by PCR-DGGE (polymerase chain reaction denaturing gradient gel electrophoresis) between dietary treatments. However, group's similarity was higher at 25 °C. Digestive enzymes activities were higher at 25 °C but were unaffected by prebiotics incorporation. Gut morphology was also unaffected by dietary prebiotic incorporation. Overall, gut microbiota composition, digestive enzymes activities and immunity parameters were affected by rearing temperature whereas dietary scFOS incorporation had only minor effects on these parameters. In conclusion, at the tested levels scFOS does not seem worthy of including it in gilthead sea bream juveniles diets.

A breakthrough on Amanita phalloides poisoning: an effective antidotal effect by polymyxin B

Amanita phalloides is responsible for more than 90 % of mushroom-related fatalities, and no effective antidote is available. a-Amanitin, the main toxin of A. phalloides, inhibits RNA polymerase II (RNAP II), causing hepatic and kidney failure. In silico studies included docking and molecular dynamics simulation coupled to molecular mechanics with generalized Born and surface area method energy decomposition on RNAP II. They were performed with a clinical drug that shares chemical similarities to a-amanitin, polymyxin B. The results show that polymyxin B potentially binds to RNAP II in the same interface of a-amanitin, preventing the toxin from binding to RNAP II. In vivo, the inhibition of the mRNA transcripts elicited by a-amanitin was efficiently reverted by polymyxin B in the kidneys. Moreover, polymyxin B significantly decreased the hepatic and renal a-amanitin-induced injury as seen by the histology and hepatic aminotransferases plasma data. In the survival assay, all animals exposed to a-amanitin died within 5 days, whereas 50 % survived up to 30 days when polymyxin B was administered 4, 8, and 12 h post-a-amanitin. Moreover, a single dose of polymyxin B administered concomitantly with a-amanitin was able to guarantee 100 % survival. Polymyxin B protects RNAP II from inactivation leading to an effective prevention of organ damage and increasing survival in a-amanitin-treated animals. The present use of clinically relevant concentrations of an already human-use-approved drug prompts the use of polymyxin B as an antidote for A. phalloides poisoning in humans.

Autoimmune diseases and pregnancy: analysis of a series of cases

BACKGROUND: An autoimmune disease is characterized by tissue damage, caused by self-reactivity of different effector mechanisms of the immune system, namely antibodies and T cells. All autoimmune diseases, to some extent, have implications for fertility and obstetrics. Currently, due to available treatments and specialised care for pregnant women with autoimmune disease, the prognosis for both mother and child has improved significantly. However these pregnancies are always high risk. The purpose of this study is to analyse the fertility/pregnancy process of women with systemic and organ-specific autoimmune diseases and assess pathological and treatment implications. METHODS: The authors performed an analysis of the clinical records and relevant obstetric history of five patients representing five distinct autoimmune pathological scenarios, selected from Autoimmune Disease Consultation at the Hospital of Braga, and reviewed the literature. RESULTS: The five clinical cases are the following: Case 1-28 years old with systemic lupus erythematosus, and clinical remission of the disease, under medication with hydroxychloroquine, prednisolone and acetylsalicylic acid, with incomplete miscarriage at 7 weeks of gestation without signs of thrombosis. Case 2-44 years old with history of two late miscarriages, a single preterm delivery (33 weeks) and multiple thrombotic events over the years, was diagnosed with antiphospholipid syndrome after acute myocardial infarction. Case 3-31 years old with polymyositis, treated with azathioprine for 3 years with complete remission of the disease, took the informed decision to get pregnant after medical consultation and full weaning from azathioprine, and gave birth to a healthy term new-born. Case 4-38 years old pregnant woman developed Behcet's syndrome during the final 15 weeks of gestation and with disease exacerbation after delivery. Case 5-36 years old with autoimmune thyroiditis diagnosed during her first pregnancy, with difficult control over the thyroid function over the years and first trimester miscarriage, suffered a second miscarriage despite clinical stability and antibody regression. CONCLUSIONS: As described in literature, the authors found a strong association between autoimmune disease and obstetric complications, especially with systemic lupus erythematosus, antiphospholipid syndrome and autoimmune thyroiditis.

Facilitative effects of bi-hemispheric tDCS in cognitive deficits of Parkinson Disease patients

Parkinson’s disease (PD) is a progressive neurodegenerative disorder, primarily characterized by motor symptoms such as tremor, rigidity, bradykinesia, stiffness, slowness and impaired equilibrium. Although the motor symptoms have been the focus in PD, slight cognitive deficits are commonly found in non-demented and non-depressed PD patients, even in early stages of the disease, which have been linked to the subsequent development of pathological dementia. Thus, strongly reducing the quality of life (QoL). Both levodopa therapy and deep brain stimulation (DBS) have yield controversial results concerning the cognitive symptoms amelioration in PD patients. That does not seems to be the case with transcranial direct current stimulation (tDCS), although better stimulation parameters are needed. Therefore we hypothesize that simultaneously delivering cathodal tDCS (or ctDCS), over the right prefrontal cortex delivered with anodal tDCS (or atDCS) to left prefrontal cortex could be potentially beneficial for PD patients, either by mechanisms of homeostatic plasticity and by increases in the extracellular dopamine levels over the striatum.

MicroRNA-375 plays a dual role in prostate carcinogenesis

Background: Prostate cancer (PCa), a highly incident and heterogeneous malignancy, mostly affects men from developed countries. Increased knowledge of the biological mechanisms underlying PCa onset and progression are critical for improved clinical management. MicroRNAs (miRNAs) deregulation is common in human cancers, and understanding how it impacts in PCa is of major importance. MiRNAs are mostly downregulated in cancer, although some are overexpressed, playing a critical role in tumor initiation and progression. We aimed to identify miRNAs overexpressed in PCa and subsequently determine its impact in tumorigenesis. Results: MicroRNA expression profiling in primary PCa and morphological normal prostate (MNPT) tissues identified 17 miRNAs significantly overexpressed in PCa. Expression of three miRNAs, not previously associated with PCa, was subsequently assessed in large independent sets of primary tumors, in which miR-182 and miR-375 were validated, but not miR-32. Significantly higher expression levels of miR-375 were depicted in patients with higher Gleason score and more advanced pathological stage, aswellaswithregionallymph nodesmetastases. Forced expression of miR-375 in PC-3 cells, which display the lowest miR-375 levels among PCa cell lines, increased apoptosis and reduced invasion ability and cell viability. Intriguingly, in 22Rv1 cells, which displayed the highest miR-375 expression, knockdown experiments also attenuated the malignant phenotype. Gene ontology analysis implicated miR-375 in several key pathways deregulated in PCa, including cell cycle and cell differentiation. Moreover, CCND2 was identified as putative miR-375 target in PCa, confirmed by luciferase assay. Conclusions: A dual role for miR-375 in prostate cancer progression is suggested, highlighting the importance of cellular context on microRNA targeting.

SMYD3 contributes to a more aggressive phenotype of prostate cancer and targets Cyclin D2 through H4K20me3

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa.

Stress-triggered synaptic malfunction: a gate along the path from depressionto dementia

Tese de Doutoramento em Ciências da Saúde.

Reprogramming energy metabolism and inducing angiogenesis : co-expression of monocarboxylate transporters with VEGF family members in cervical adenocarcinomas

Reprogramming energy metabolism and inducing angiogenesis: co-expression of monocarboxylate transporters with VEGF family members in cervical adenocarcinomas.

The role of the mammalian DNA end-processing enzyme polynucleotide kinase 3'-phosphatase in spinocerebellar ataxia Type 3 pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.

Stage at presentation of breast cancer in Luanda, Angola - a retrospective study

Background: It is expected that, by 2020, 15 million new cases of cancer will occur every year in the world, one million of them in Africa. Knowledge of cancer trends in African countries is far from adequate, and improvements in cancer prevention efforts are urgently needed. The aim of this study was to characterize breast cancer clinically and pathologically at presentation in Luanda, Angola; we additionally provide quality information that will be useful for breast cancer care planning in the country. Methods: Data on breast cancer cases were retrieved from the Angolan Institute of Cancer Control, from 2006 to 2014. For women diagnosed in 2009 (5-years of follow-up), demographic, clinical and pathological information, at presentation, was collected, namely age at diagnosis, parity, methods used for pathological diagnoses, tumor pathological characteristics, stage of disease and treatment. Descriptive statistics were performed. Results: The median age of women diagnosed with breast cancer in 2009 was 47 years old (range 25–89). The most frequent clinical presentation was breast swelling with axillary lymph nodes metastasis (44.9 %), followed by a mass larger than 5 cm (14.2 %) and lump (12.9 %). Invasive ductal carcinoma was the main histologic type (81.8 %). Only 10.1 % of cancer cases had a well differentiated histological grade. Cancers were diagnosed mostly at advanced stages (66.7 % in stage III and 11.1 % in stage IV). Discussion: In this study, breast cancer was diagnosed at a very advanced stage. Although it reports data from a single cancer center in Luanda, Angola it reinforces the need for early diagnosis and increasing awareness. According to the main challenges related to breast cancer diagnosis and treatment herein presented, we propose a realistic framework that would allow for the implementation of a breast cancer care program, built under a strong network based on cooperation, teaching, audit, good practices and the organization of health services. Conclusion: Angola needs urgently a program for early diagnosis of breast cancer.

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

Tese de Doutoramento em Ciências da Saúde

Macroscopic and histopathological study of the placenta: an essential resource in litigation processes

The pathological study of the placenta is of upmost importance in cases of unexplained fetal/perinatal loss and often these carry litigation implications. Integrating pathological findings and the underlying pathophysiological processes, leading to placental lesions, is fundamental for the evaluation of poor fetal and perinatal outcomes and to distinguish from cases of true negligence.