Browsing by Author "Videira, P"
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- BD-2 and BD-3 Increase Skin Flap Survival in a Model of Ischemia and Pseudomonas Aeruginosa InfectionPublication . Casal, D; Iria, I; Ramalho, JS; Alves, S; Mota-Silva, E; Mascarenhas-Lemos, L; Pontinha, C; Guadalupe-Cabral, M; Ferreira-Silva, J; Ferraz-Oliveira, M; Vassilenko, V; Goyri-O'Neill, J; Pais, D; Videira, PThe main aim of this work was to study the usefulness of human β-defensins 2 (BD-2) and 3 (BD-3), which are part of the innate immune system, in the treatment of infected ischemic skin flaps. We investigated the effect of transducing rat ischemic skin flaps with lentiviral vectors encoding human BD-2, BD-3, or both BD-2 and BD-3, to increase flap survival in the context of a P. aeruginosa infection associated with a foreign body. The secondary endpoints assessed were: bacterial counts, and biofilm formation on the surface of the foreign body. A local ischemic environment was created by producing arterialized venous flaps in the left epigastric region of rats. Flaps were intentionally infected by placing underneath them two catheters with 105 CFU of P. aeruginosa before the surgical wounds were hermetically closed. Flap biopsies were performed 3 and 7 days post-operatively, and the specimens submitted to immunohistochemical analysis for BD-2 and BD-3, as well as to bacterial quantification. Subsequently, the catheter segments were analyzed with scanning electron microscopy (SEM). Flaps transduced with BD-2 and BD-3 showed expression of these defensins and presented increased flap survival. Rats transduced with BD-3 presented a net reduction in the number of P. aeruginosa on the surface of the foreign body and lesser biofilm formation.
- Blood Supply to the Integument of the Abdomen of the Rat: A Surgical PerspectivePublication . Casal, D; Pais, D; Iria, I; Videira, P; Mota-Silva, E; Alves, S; Mascarenhas-Lemos, L; Pen, C; Vassilenko, V; Goyri-O'Neill, JBACKGROUND: Many fundamental questions regarding the blood supply to the integument of the rat remain to be clarified, namely the degree of homology between rat and humans. The aim of this work was to characterize in detail the macro and microvascular blood supply to the integument covering the ventrolateral aspect of the abdominal wall of the rat. METHODS: Two hundred five Wistar male rats weighing 250-350 g were used. They were submitted to gross anatomical dissection after intravascular colored latex injection (n = 30); conversion in modified Spalteholz cleared specimens (n=10); intravascular injection of a Perspex solution, and then corroded, in order to produce vascular corrosion casts of the vessels in the region (n = 5); histological studies (n = 20); scanning electron microscopy of vascular corrosion casts (n = 10); surgical dissection of the superficial caudal epigastric vessels (n = 100); and to thermographic evaluation (n = 30). RESULTS: The ventrolateral abdominal wall presented a dominant superficial vascular system, which was composed mainly of branches from the superficial caudal epigastric artery and vein in the caudal half. The cranial half still received significant arterial contributions from the lateral thoracic artery in all cases and from large perforators coming from the intercostal arteries and from the deep cranial epigastric artery. CONCLUSIONS: These data show that rats and humans present a great deal of homology regarding the blood supply to the ventrolateral aspect of the abdominal integument. However, there are also significant differences that must be taken into consideration when performing and interpreting experimental procedures in rats.
- Development of Dl1.72, a Novel Anti-DLL1 Antibody with Anti-Tumor Efficacy against Estrogen Receptor-Positive Breast CancerPublication . Silva, G; Sales-Dias, J; Casal, D; Alves, S; Domenici, G; Barreto, C; Matos, C; Lemos, A; Matias, A; Kucheryava, K; Ferreira, A; Moita, MR; Braga, S; Brito, C; Cabral, MG; Casalou, C; Barral, D; Sousa, P; Videira, P; Bandeiras, T; Barbas, AThe Notch-signaling ligand DLL1 has emerged as an important player and promising therapeutic target in breast cancer (BC). DLL1-induced Notch activation promotes tumor cell proliferation, survival, migration, angiogenesis and BC stem cell maintenance. In BC, DLL1 overexpression is associated with poor prognosis, particularly in estrogen receptor-positive (ER+) subtypes. Directed therapy in early and advanced BC has dramatically changed the natural course of ER+ BC; however, relapse is a major clinical issue, and new therapeutic strategies are needed. Here, we report the development and characterization of a novel monoclonal antibody specific to DLL1. Using phage display technology, we selected an anti-DLL1 antibody fragment, which was converted into a full human IgG1 (Dl1.72). The Dl1.72 antibody exhibited DLL1 specificity and affinity in the low nanomolar range and significantly impaired DLL1-Notch signaling and expression of Notch target genes in ER+ BC cells. Functionally, in vitro treatment with Dl1.72 reduced MCF-7 cell proliferation, migration, mammosphere formation and endothelial tube formation. In vivo, Dl1.72 significantly inhibited tumor growth, reducing both tumor cell proliferation and liver metastases in a xenograft mouse model, without apparent toxicity. These findings suggest that anti-DLL1 Dl1.72 could be an attractive agent against ER+ BC, warranting further preclinical investigation.
- Inhibition of Fucosylation in Human Invasive Ductal Carcinoma Reduces E-Selectin Ligand Expression, Cell Proliferation, and ERK1/2 and p38 MAPK ActivationPublication . Carrascal, M; Silva, M; Ramalho, J; Pen, C; Martins, M; Pascoal, C; Amaral, C; Serrano, I; Oliveira, MJ; Sackstein, R; Videira, PBreast cancer tissue overexpresses fucosylated glycans, such as sialyl-Lewis X/A (sLeX/A ), and α-1,3/4-fucosyltransferases (FUTs) in relation to increased disease progression and metastasis. These glycans in tumor circulating cells mediate binding to vascular E-selectin, initiating tumor extravasation. However, their role in breast carcinogenesis is still unknown. Here, we aimed to define the contribution of the fucosylated structures, including sLeX/A , to cell adhesion, cell signaling, and cell proliferation in invasive ductal carcinomas (IDC), the most frequent type of breast cancer. We first analyzed expression of E-selectin ligands in IDC tissue and established primary cell cultures from the tissue. We observed strong reactivity with E-selectin and anti-sLeX/A antibodies in both IDC tissue and cell lines, and expression of α-1,3/4 FUTs FUT4, FUT5, FUT6, FUT10, and FUT11. To further assess the role of fucosylation in IDC biology, we immortalized a primary IDC cell line with human telomerase reverse transcriptase to create the 'CF1_T cell line'. Treatment with 2-fluorofucose (2-FF), a fucosylation inhibitor, completely abrogated its sLeX/A expression and dramatically reduced adherence of CF1_T cells to E-selectin under hemodynamic flow conditions. In addition, 2-FF-treated CF1_T cells showed a reduced migratory ability, as well as decreased cell proliferation rate. Notably, 2-FF treatment lowered the growth factor expression of CF1_T cells, prominently for FGF2, vascular endothelial growth factor, and transforming growth factor beta, and negatively affected activation of signal-regulating protein kinases 1 and 2 and p38 mitogen-activated protein kinase signaling pathways. These data indicate that fucosylation licenses several malignant features of IDC, such as cell adhesion, migration, proliferation, and growth factor expression, contributing to tumor progression.
- A Model of Free Tissue Transfer: The Rat Epigastric Free FlapPublication . Casal, D; Pais, D; Iria, I; Mota-Silva, E; Almeida, MA; Alves, S; Pen, C; Farinho, A; Mascarenhas-Lemos, L; Ferreira-Silva, J; Ferraz-Oliveira, M; Vassilenko, V; Videira, P; Gory O'Neill, JFree tissue transfer has been increasingly used in clinical practice since the 1970s, allowing reconstruction of complex and otherwise untreatable defects resulting from tumor extirpation, trauma, infections, malformations or burns. Free flaps are particularly useful for reconstructing highly complex anatomical regions, like those of the head and neck, the hand, the foot and the perineum. Moreover, basic and translational research in the area of free tissue transfer is of great clinical potential. Notwithstanding, surgical trainees and researchers are frequently deterred from using microsurgical models of tissue transfer, due to lack of information regarding the technical aspects involved in the operative procedures. The aim of this paper is to present the steps required to transfer a fasciocutaneous epigastric free flap to the neck in the rat. This flap is based on the superficial epigastric artery and vein, which originates from and drain into the femoral artery and vein, respectively. On average the caliber of the superficial epigastric vein is 0.6 to 0.8 mm, contrasting with the 0.3 to 0.5 mm of the superficial epigastric artery. Histologically, the flap is a composite block of tissues, containing skin (epidermis and dermis), a layer of fat tissue (panniculus adiposus), a layer of striated muscle (panniculus carnosus), and a layer of loose areolar tissue. Succinctly, the epigastric flap is raised on its pedicle vessels that are then anastomosed to the external jugular vein and to the carotid artery on the ventral surface of the rat's neck. According to our experience, this model guarantees the complete survival of approximately 70 to 80% of epigastric flaps transferred to the neck region. The flap can be evaluated whenever needed by visual inspection. Hence, the authors believe this is a good experimental model for microsurgical research and training.
- Optimization of an Arterialized Venous Fasciocutaneous Flap in the Abdomen of the RatPublication . Casal, D; Mota-Silva, E; Pais, D; Iria, I; Videira, P; Tanganho, D; Alves, S; Mascarenhas-Lemos, L; Martins Ferreira, J; Ferraz-Oliveira, M; Vassilenko, V; Goyri O'Neill, JBACKGROUND: Although numerous experimental models of arterialized venous flaps (AVFs) have been proposed, no single model has gained widespread acceptance. The main aim of this work was to evaluate the survival area of AVFs produced with different vascular constructs in the abdomen of the rat. METHODS: Fifty-three male rats were divided into 4 groups. In group I (n = 12), a 5-cm-long and 3-cm-wide conventional epigastric flap was raised on the left side of the abdomen. This flap was pedicled on the superficial caudal epigastric vessels caudally and on the lateral thoracic vein cranially. In groups II, III, and IV, a similar flap was raised, but the superficial epigastric artery was ligated. In these groups, AVFs were created using the following arterial venous anastomosis at the caudal end of the flap: group II (n = 13) a 1-mm-long side-to-side anastomosis was performed between the femoral artery and vein laterally to the ending of the superficial caudal epigastric vein. In group III (n = 14), in addition to the procedure described for group II, the femoral vein was ligated medially. Finally, in group IV (n = 14), the superficial caudal epigastric vein was cut from the femoral vein with a 1-mm-long ellipse of adjacent tissue, and an end-to-side arterial venous anastomosis was established between it and the femoral artery. RESULTS: Seven days postoperatively, the percentage of flap survival was 98.89 ± 1.69, 68.84 ± 7.36, 63.84 ± 10.38, 76.86 ± 13.67 in groups I-IV, respectively. CONCLUSION: An optimized AVF can be produced using the vascular architecture described for group IV.
- Reconstruction of a Long Defect of the Ulnar Artery and Nerve with an Arterialized Neurovenous Free Flap in a Teenager: A Case Report and Literature ReviewPublication . Casal, D; Pais, D; Mota-Silva, E; Pelliccia, G; Iria, I; Videira, P; Mendes, MM; Goyri-O'Neill, J; Mouzinho, MMThere is evidence that nerve flaps are superior to nerve grafts for bridging long nerve defects. Moreover, arterialized neurovenous flaps (ANVFs) have multiple potential advantages over traditional nerve flaps in this context. This paper describes a case of reconstruction of a long defect of the ulnar artery and nerve with an arterialized neurovenous free flap and presents a literature review on this subject. A 16-year-old boy sustained a stab wound injury to the medial aspect of the distal third of his right forearm. The patient was initially observed and treated at another institution where the patient was diagnosed with a flexor carpis ulnaris muscle and an ulnar artery section. The artery was ligated and the muscle was sutured. Four months later, the patient was referred to our institution with complaints of ulnar nerve damage, as well as hand pain and cold intolerance. Physical examination and ancillary tests supported the diagnosis of ulnar artery and nerve complete section. Surgery revealed an 8 cm hiatus of the ulnar artery and a 5 cm defect of the ulnar nerve. These gaps were bridged with a flow through ANVF containing the sural nerve and the lesser saphenous vein. The postoperative course was uneventful. Two years postoperatively, the patient had regained normal trophism and M5 strength in all previously paralyzed muscles according to the Medical Research Council Scale. Thermography revealed good perfusion in the right ulnar angiosome. The ANVF may be an expedite, safe and efficient option to reconstruct a long ulnar nerve and artery defect.
- Regulatory Cells, Cytokine Pattern and Clinical Risk Factors for Asthma in Infants and Young Children with Recurrent WheezePublication . Borrego, LM; Arroz, MJ; Videira, P; Martins, C; Guimarães, H; Nunes, G; Papoila, AL; Trindade, HSeveral risk factors for asthma have been identified in infants and young children with recurrent wheeze. However, published literature has reported contradictory findings regarding the underlying immunological mechanisms. OBJECTIVES: This study was designed to assess and compare the immunological status during the first 2 years in steroid-naive young children with >or= three episodes of physician-confirmed wheeze (n=50), with and without clinical risk factors for developing subsequent asthma (i.e. parental asthma or a personal history of eczema and/or two of the following: wheezing without colds, a personal history of allergic rhinitis and peripheral blood eosinophilia >4%), with age-matched healthy controls (n=30). METHODS: Peripheral blood CD4(+)CD25(+) and CD4(+)CD25(high) T cells and their cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), GITR and Foxp3 expression were analysed by flow cytometry. Cytokine (IFN-gamma, TGF-beta and IL-10), CTLA-4 and Foxp3 mRNA expression were evaluated (real-time PCR) after peripheral blood mononuclear cell stimulation with phorbol 12-myristate 13-acetate (PMA) (24 h) and house dust mite (HDM) extracts (7th day). RESULTS: Flow cytometry results showed a significant reduction in the absolute number of CD4(+)CD25(high) and the absolute and percentage numbers of CD4(+)CD25(+)CTLA-4(+) in wheezy children compared with healthy controls. Wheezy children at a high risk of developing asthma had a significantly lower absolute number of CD4(+)CD25(+) (P=0.01) and CD4(+)CD25(high) (P=0.04), compared with those at a low risk. After PMA stimulation, CTLA-4 (P=0.03) and Foxp3 (P=0.02) expression was diminished in wheezy children compared with the healthy children. After HDM stimulation, CTLA-4 (P=0.03) and IFN-gamma (P=0.04) expression was diminished in wheezy children compared with healthy children. High-risk children had lower expression of IFN-gamma (P=0.03) compared with low-risk and healthy children and lower expression of CTLA-4 (P=0.01) compared with healthy children. CONCLUSIONS: Although our findings suggest that some immunological parameters are impaired in children with recurrent wheeze, particularly with a high risk for asthma, further studies are needed in order to assess their potential as surrogate predictor factors for asthma in early life.
- Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune CellsPublication . Carrascal, M; Severino, P; Cabral, MG; Silva, M; Ferreira, JA; Calais da Silva, F; Quinto, H; Pen, C; Ligeiro, D; Lara Santos, L; Dall'Olio, F; Videira, PDespite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.
- Staining of E-Selectin Ligands on Paraffin-Embedded Sections of Tumor TissuePublication . Carrascal, M; Talina, C; Borralho, P; Gonçalo Mineiro, A; Henriques, AR; Pen, C; Martins, M; Braga, S; Sackstein, R; Videira, PBACKGROUND: The E-selectin ligands expressed by cancer cells mediate adhesion of circulating cancer cells to endothelial cells, as well as within tissue microenvironments important for tumor progression and metastasis. The identification of E-selectin ligands within cancer tissue could yield new biomarkers for patient stratification and aid in identifying novel therapeutic targets. The determinants of selectin ligands consist of sialylated tetrasaccharides, the sialyl Lewis X and A (sLeX and sLeA), displayed on protein or lipid scaffolds. Standardized procedures for immunohistochemistry make use of the antibodies against sLeX and/or sLeA. However, antibody binding does not define E-selectin binding activity. METHODS: In this study, we developed an immunohistochemical staining technique, using E-selectin-human Ig Fc chimera (E-Ig) to characterize the expression and localization of E-selectin binding sites on paraffin-embedded sections of different cancer tissue. RESULTS: E-Ig successfully stained cancer cells with high specificity. The E-Ig staining show high reactivity scores in colon and lung adenocarcinoma and moderate reactivity in triple negative breast cancer. Compared with reactivity of antibody against sLeX/A, the E-Ig staining presented higher specificity to cancer tissue with better defined borders and less background. CONCLUSIONS: The E-Ig staining technique allows the qualitative and semi-quantitative analysis of E-selectin binding activity on cancer cells. The development of accurate techniques for detection of selectin ligands may contribute to better diagnostic and better understanding of the molecular basis of tumor progression and metastasis.