Estudo da actividade angiogénica na nurse cell de trichinella spiralis no decurso da triquinelose em modelo roedor

A triquinelose é uma zoonose parasitária que é transmitida aos humanos e animais, através da ingestão de carne crua ou insuficientemente cozinhada, que contenha larvas infectantes de Trichinella spp., sendo actualmente considerada uma doença emergente e/ou re-emergente. O sucesso do parasitismo do nemátode Trichinella spiralis está intimamente ligado com o processo de angiogénese, ou seja, a formação de novos vasos a partir de vasos pré-existentes. Com o objectivo de estudar a actividade angiogénica na nurse cell de T. spiralis, realizaram-se técnicas imunohistoquímicas e imunofluorescentes para o factor de crescimento endotelial vascular (VEGF), molécula-1 de adesão celular endotelial a plaquetas (PECAM-1) e actina músculo liso (AML), em tecido muscular de Rattus rattus infectado com T. spiralis. Através destas técnicas observou-se marcação intensa no infiltrado inflamatório adjacente à nurse cell e também na larva. Já o citoplasma da nurse cell apresentou uma marcação moderada. Este padrão de marcação manteve-se desde os 45 até aos 120 dias após a infecção. A avaliação da densidade vascular (PECAM-1) e da densidade da expressão de células positivas para AML permitiu estabelecer uma correlação positiva entre o aumento da densidade vascular e o número de dias de infecção. Adicionalmente estabeleceu-se uma correlação negativa entre o aumento da densidade de células que expressam AML e o número de dias de infecção. Os resultados indicam uma produção constante de VEGF pela larva, pelo citoplasma da nurse cell e pelo hospedeiro (infiltrado inflamatório), durante todo período de infecção, levando à formação de uma rede vascular crescente (com um aumento médio de 79% face ao controlo), acompanhada de células murais que promovem a sua estabilização (com um aumento médio de 50% face ao controlo) com particular incidência no primeiro período estudado (45 a 55 dias).Trichinellosis is a parasitic zoonosis, transmitted to humans and animals through the ingestion of raw or undercooked meat, containing infective larvae of Trichinella spp. This disease is currently considered as emerging and / or re-emerging. Nematode Trichinella spiralis success as a parasite is strongly related to the angiogenesis process, that is, the formation of new vessels from pre-existing ones. In order to study the angiogenic activity in T. spiralis nurse cell, immunohistochemistry and immunofluorescence for vascular endothelial grow factor (VEGF), platelet/endothelial cell adhesion molecule-1 (PECAM-1), and α- smooth muscle actin (SMAα) was performed on Rattus rattus muscular tissue infected with T. spiralis. Intense immunostaining of VEGF was observed in the surrounding inflammatory cell infiltrate and larva. Moderate immunostaining was noted in the developing nurse cell cytoplasm. This pattern persisted from day 45 to day 120 after initial infection. Vessel density (PECAM-1) and positive cells for SMAα density were evaluated. A positive correlation was found between vascular density and the number of days after the initial infection. A negative correlation was established between the density of cells expressing AML and the number of days. These results show that there is a constant production of VEGF by the larva, nurse cell cytoplasm and the host (inflammatory infiltrate) during the full period of infection, which results in the formation and enlargement of the vascular network (with an average increase of 79% comparing with the control), accompanied by the maturation and stabilization of the vessels (with an average increase of 50% comparing with the control), mainly in the first observed period (45-55 days). Vessel density (PECAM-1) and positive cells for SMAα density were evaluated. A positive correlation was found between vascular density and the number of days after the initial infection. A negative correlation was established between the density of cells expressing AML and the number of days. These results show that there is a constant production of VEGF by the larva, nurse cell cytoplasm and the host (inflammatory infiltrate) during the full period of infection, which results in the formation and enlargement of the vascular network (with an average increase of 79% comparing with the control), accompanied by the maturation and stabilization of the vessels (with an average increase of 50% comparing with the control), mainly in the first observed period (45-55 days). Vessel density (PECAM-1) and positive cells for SMAα density were evaluated. A positive correlation was found between vascular density and the number of days after the initial infection. A negative correlation was established between the density of cells expressing AML and the number of days. These results show that there is a constant production of VEGF by the larva, nurse cell cytoplasm and the host (inflammatory infiltrate) during the full period of infection, which results in the formation and enlargement of the vascular network (with an average increase of 79% comparing with the control), accompanied by the maturation and stabilization of the vessels (with an average increase of 50% comparing with the control), mainly in the first observed period (45-55 days). Vessel density (PECAM-1) and positive cells for SMAα density were evaluated. A positive correlation was found between vascular density and the number of days after the initial infection. A negative correlation was established between the density of cells expressing AML and the number of days. These results show that there is a constant production of VEGF by the larva, nurse cell cytoplasm and the host (inflammatory infiltrate) during the full period of infection, which results in the formation and enlargement of the vascular network (with an average increase of 79% comparing with the control), accompanied by the maturation and stabilization of the vessels (with an average increase of 50% comparing with the control), mainly in the first observed period (45-55 days)

Inhibition of fucosylation in human invasive ductal carcinoma reduces E‐selectin ligand expression, cell proliferation, and ERK1/2 and p38 MAPK activation

Breast 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

Sialyl Tn-expressing bladder cancer cells induce a tolerogenic phenotype in innate and adaptive immune cells

This work was supported by the Portuguese Foundation for Science and Technology (FCT) - PTDC/SAU-MII/67561/2006 and Premio Santander Totta - UNL (Paula A. Videira), LPCC/Pfizer2011 (Mylene A. Carrascal), SFRH/BPD/21619/2005 (M. Guadalupe Cabral), SFRH/BD/81860/2011 (Mariana Silva), SFRH/BD/45120/2008 (Paulo F. Severino) and SFRH/BPD/ 66288/2009 (Jose Alexandre Ferreira). FCT is co-financed by European Social Fund (ESF) under Human Potential Operation Programme (POPH) from National Strategic Reference Framework (NSRF).) and European Union, QREN, FEDER, COMPETE, for funding the Organic Chemistry Research Unit (QOPNA) (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296).Despite 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. Invitro, 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.publishersversionpublishe

Reconstruction of a 10-mm-long median nerve gap in an ischemic environment using autologous conduits with different patterns of blood supply: A comparative study in the rat

The aim of this study was to evaluate in the Wistar rat the efficacy of various autologous nerve conduits with various forms of blood supply in reconstructing a 10-mm-long gap in the median nerve (MN) under conditions of local ischemia. A 10-mm-long median nerve defect was created in the right arm. A loose silicone tube was placed around the nerve gap zone, in order to simulate a local ischemic environment. Rats were divided in the following experimental groups (each with 20 rats): the nerve Graft (NG) group, in which the excised MN segment was reattached; the conventional nerve flap (CNF) and the arterialized neurovenous flap (ANVF) groups in which the gap was bridged with homonymous median nerve flaps; the prefabricated nerve flap (PNF) group in which the gap was reconstructed with a fabricated flap created by leaving an arteriovenous fistula in contact with the sciatic nerve for 5 weeks; and the two control groups, Sham and Excision groups. In the latter group, the proximal stump of the MN nerve was ligated and no repair was performed. The rats were followed for 100 days. During this time, they did physiotherapy. Functional, electroneuromyographic and histological studies were performed. The CNF and ANVF groups presented better results than the NG group in the following assessments: grasping test, nociception, motor stimulation threshold, muscle weight, and histomorphometric evaluation. Radial deviation of the operated forepaw was more common in rats that presented worse results in the other outcome variables. Overall, CNFs and ANVFs produced a faster and more complete recovery than NGs in the reconstruction of a 10-mm-long median nerve gap in an ischemic environment in the Wistar rat. Although, results obtained with CNFs were in most cases were better than ANVFs, these differences were not statistically significant for most of the outcome variables.publishersversionpublishe

Additional file 1: of Staining of E-selectin ligands on paraffin-embedded sections of tumor tissue

Figure S1. Example of application of staining of E-selectin ligands by Immunofluorescence. Staining of E-selectin ligands in CF1_T cells. The breast cancer cell line CF1_T has a high content of E-selectin ligands and it was obtained and cultured as described by Carrascal et al. (2017). Cells were cultured on glass coverslips overnight and then fixed with 3.7% paraformaldehyde. After blocking with 1% bovine serum albumin, cells were stained with E-Ig chimera in the presence of 2 mM CaCl2. The final step included anti-human Ig antibody conjugated with fluorescein (FITC, green), in the presence of PBS containing 2 mM CaCl2 (A). Control experiments were processed in the absence of CaCl2 (B). After permeabilization with 0.1% TritonX-100, F-actin was stained with Alexa Fluor 568 phalloidin (Molecular Probes, Leiden, Netherlands). Images were acquired with a Leica TCS SP2 AOBS confocal microscope. A representative cross-section confocal images were selected after Z-stacking. (PPTX 230 kb

Blood Supply to the Integument of the Abdomen of the Rat: A Surgical Perspective

Background:. 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

Representative histological features of the different experimental groups.

<p>HE, hematoxylin-eosin staining; MT, Masson’s trichrome staining; NF, neurofilament immunohistochemical staining; Per, peripherin immunohistochemical staining; ACHE, acetylcholinesterase immunohistochemical staining. Calibration bar (A to E) = 10 μm Calibration bar (F to I’) = 100 μm.</p

Average velocity in the ladder running test in the different experimental groups during the experiment.

<p>Vertical bars represent 95% confidence intervals. Horizontal lines in the upper portion of the figure indicate statistically significant differences between groups on the 90^th day postoperatively (p<0.05). *, p<0.05; **, p<0.01; ***, p<0.001.</p

Walking tracks measurements using forepaw impressions.

<p><b>(A)</b> Photograph of a typical print of the left forepaw (uninjured). <b>(B)</b> Contrast-enhanced image of the photograph in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195692#pone.0195692.g003" target="_blank">Fig 3A</a>, using the software Fiji®. Similar images were used for measurement purposes, namely of determination of the stance factor (paw impression area on the paper sheet). <b>(C)</b> Typical forepaw prints of a rat in the nerve graft group. 1, Intermediate finger spread factor: widest width between the second and third fingers; 2, Finger spread factor: widest width of the paw impression; 3, Print length factor: longest length of the paw impression; 4, Stride length: distance between homologous points of successive paw impressions on a given side; 5, Base of support: perpendicular distance between the central portion of the paw impression and the direction of movement.</p