Studying T Cells N-Glycosylation by Imaging Flow Cytometry

Imaging flow cytometry is an emerging imaging technology that combines features of both conventional flow cytometry and fluorescence microscopy allowing quantification of the imaging parameters. The analysis of protein posttranslational modifications by glycosylation using imaging flow cytometry constitutes an important bioimaging tool in the glycobiology field. This technique allows quantification of the glycan fluorescence intensity, co-localization with proteins, and evaluation of the membrane/cytoplasmic expression. In this chapter we provide the guidelines to analyze glycan expression, particularly the ß1,6 GlcNAc branched N-glycans, on the membrane of intestinal T cells from inflammatory bowel disease patients.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/DTPPIC/0560/2014; PTDC/BBB-EBI/0786/2012; EXPL/BIMMEC/0149/2012), “financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER,” e do Quadro de Referência Estratégia Nacional QREN. This work was further supported by a Portuguese grant from “Grupo de Estudo da Doença Infl amatória Intestinal” (GEDII). This work had also the fi nantial support of FCT/MEC through National Funds and, when applicable, co-fi nanced by the FEDER via the PT2020 Partnership Agreement under the 4293 Unit I&D. S.S.P. (SFRH/BPD/63094/2009) also acknowledges FCT. A.M.D. PD/BD/105982/2014 also acknowledges FCT and BiotechHealth Doctoral Programme. The Institute of Molecular Pathology and Immunology of the University of Porto (IPATIMUP) integrates the Institute for Research and Innovation in Health (I3S), which is partially supported by the Portuguese Foundation for Science and Technology (FCT). Data was acquired at the Bioimaging Center for Biomaterials and Regenerative Therapies (b.IMAGE, INEB, Porto, Portugal)

Molecular Evolutionary Characterization of a V1R Subfamily Unique to Strepsirrhine Primates

Vomeronasal receptor genes have frequently been invoked as integral to the establishment and maintenance of species boundaries among mammals due to the elaborate one-to-one correspondence between semiochemical signals and neuronal sensory inputs. Here, we report the most extensive sample of vomeronasal receptor class 1 (V1R) sequences ever generated for a diverse yet phylogenetically coherent group of mammals, the tooth-combed primates (suborder Strepsirrhini). Phylogenetic analysis confirms our intensive sampling from a single V1R subfamily, apparently unique to the strepsirrhine primates. We designate this subfamily as V1Rstrep. The subfamily retains extensive repertoires of gene copies that descend from an ancestral gene duplication that appears to have occurred prior to the diversification of all lemuriform primates excluding the basal genusDaubentonia (the aye-aye). We refer to the descendent clades as V1Rstrep-a and V1Rstrep-b. Comparison of the two clades reveals different amino acid compositions corresponding to the predicted ligand-binding site and thus potentially to altered functional profiles between the two. In agreement with previous studies of the mouse lemur (genus, Microcebus), the majority of V1Rstrep gene copies appear to be intact and under strong positive selection, particularly within transmembrane regions. Finally, despite the surprisingly high number of gene copies identified in this study, it is nonetheless probable that V1R diversity remains underestimated in these nonmodel primates and that complete characterization will be limited until high-coverage assembled genomes are available

Geogenetic patterns in mouse lemurs (genus Microcebus) reveal the ghosts of Madagascar's forests past.

Phylogeographic analysis can be described as the study of the geological and climatological processes that have produced contemporary geographic distributions of populations and species. Here, we attempt to understand how the dynamic process of landscape change on Madagascar has shaped the distribution of a targeted clade of mouse lemurs (genus Microcebus) and, conversely, how phylogenetic and population genetic patterns in these small primates can reciprocally advance our understanding of Madagascar's prehuman environment. The degree to which human activity has impacted the natural plant communities of Madagascar is of critical and enduring interest. Today, the eastern rainforests are separated from the dry deciduous forests of the west by a large expanse of presumed anthropogenic grassland savanna, dominated by the Family Poaceae, that blankets most of the Central Highlands. Although there is firm consensus that anthropogenic activities have transformed the original vegetation through agricultural and pastoral practices, the degree to which closed-canopy forest extended from the east to the west remains debated. Phylogenetic and population genetic patterns in a five-species clade of mouse lemurs suggest that longitudinal dispersal across the island was readily achieved throughout the Pleistocene, apparently ending at ∼55 ka. By examining patterns of both inter- and intraspecific genetic diversity in mouse lemur species found in the eastern, western, and Central Highland zones, we conclude that the natural environment of the Central Highlands would have been mosaic, consisting of a matrix of wooded savanna that formed a transitional zone between the extremes of humid eastern and dry western forest types

Dysregulation of T cell receptor N-glycosylation: A molecular mechanism involved in ulcerative colitis

The incidence of inflammatory bowel disease is increasing worldwide and the underlying molecular mechanisms are far from being fully elucidated. Herein, we evaluated the role of N-glycosylation dysregulation in T cells as a key mechanism in the ulcerative colitis (UC) pathogenesis. The evaluation of the branched N-glycosylation levelsandprofile of intestinalTcell receptor (TCR)wereassessedin colonic biopsies fromUCpatientsand healthy controls. Expression alterations of the glycosyltransferase gene MGAT5 were also evaluated. We demonstrated thatUCpatients exhibit a dysregulation ofTCRbranchedN-glycosylationonlamina propriaTlymphocytes. Patients with severe UC showed the most pronounced defect on N-glycan branching in T cells. Moreover, UC patients showed a significant reduction of MGAT5 gene transcription in T lymphocytes. In this study, we disclose for the first time that a deficiency in branched N-glycosylation on TCR due to a reduced MGAT5 gene expression is a new molecular mechanism underlying UC pathogenesis, being a potential novel biomarker with promising clinical and therapeutic applications.This work was supported by grants from the Portuguese Foundation for Science and Technology (FCT), project grants (PTDC/ CVT/111358/2009; PTDC/BBB-EBI/0786/2012; EXPL/ BIM-MEC/0149/2012), ‘financiados no âmbito do Programa Operacional Temático Factores de Competitividade (COMPETE) e comparticipado pelo fundo Comunitário Europeu FEDER’, e do Quadro de Referência Estratégia Nacio-nal QREN. This work was further supported by a portuguese grant from ‘Grupo de Estudo da Doenc¸a Inflamatória Intestinal’ (GEDII). S.S.P. (SFRH/BPD/63094/2009); S.C. (SFRH/BD/ 77386/2011) also acknowledge FCT. IPATIMUP is an Associate Laboratory of the Portuguese Ministry of Science, Technology and Higher Education, and is partially supported by FCT

Delivery of sTRAIL variants by MSCs in combination with cytotoxic drug treatment leads to p53-independent enhanced antitumor effects

Mesenchymal stem cells (MSCs) are able to infiltrate tumor tissues and thereby effectively deliver gene therapeutic payloads. Here, we engineered murine MSCs (mMSCs) to express a secreted form of the TNF-related apoptosis-inducing ligand (TRAIL), which is a potent inducer of apoptosis in tumor cells, and tested these MSCs, termed MSC.sTRAIL, in combination with conventional chemotherapeutic drug treatment in colon cancer models. When we pretreated human colorectal cancer HCT116 cells with low doses of 5-fluorouracil (5-FU) and added MSC.sTRAIL, we found significantly increased apoptosis as compared with single-agent treatment. Moreover, HCT116 xenografts, which were cotreated with 5-FU and systemically delivered MSC.sTRAIL, went into remission. Noteworthy, this effect was protein 53 (p53) independent and was mediated by TRAIL-receptor 2 (TRAIL-R2) upregulation, demonstrating the applicability of this approach in p53-defective tumors. Consequently, when we generated MSCs that secreted TRAIL-R2-specific variants of soluble TRAIL (sTRAIL), we found that such engineered MSCs, labeled MSC.sTRAIL DR5, had enhanced antitumor activity in combination with 5-FU when compared with MSC.sTRAIL. In contrast, TRAIL-resistant pancreatic carcinoma PancTu1 cells responded better to MSC.sTRAIL DR4 when the antiapoptotic protein XIAP (X-linked inhibitor of apoptosis protein) was silenced concomitantly. Taken together, our results demonstrate that TRAIL-receptor selective variants can potentially enhance the therapeutic efficacy of MSC-delivered TRAIL as part of individualized and tumor-specific combination treatments. © 2013 Macmillan Publishers Limited All rights reserved

Assessment of the impact of the scanner-related factors on brain morphometry analysis with Brainvisa.

BACKGROUND: Brain morphometry is extensively used in cross-sectional studies. However, the difference in the estimated values of the morphometric measures between patients and healthy subjects may be small and hence overshadowed by the scanner-related variability, especially with multicentre and longitudinal studies. It is important therefore to investigate the variability and reliability of morphometric measurements between different scanners and different sessions of the same scanner. METHODS: We assessed the variability and reliability for the grey matter, white matter, cerebrospinal fluid and cerebral hemisphere volumes as well as the global sulcal index, sulcal surface and mean geodesic depth using Brainvisa. We used datasets obtained across multiple MR scanners at 1.5 T and 3 T from the same groups of 13 and 11 healthy volunteers, respectively. For each morphometric measure, we conducted ANOVA analysis and verified whether the estimated values were significantly different across different scanners or different sessions of the same scanner. The between-centre and between-visit reliabilities were estimated from their contribution to the total variance, using a random-effects ANOVA model. To estimate the main processes responsible for low reliability, the results of brain segmentation were compared to those obtained using FAST within FSL. RESULTS: In a considerable number of cases, the main effects of both centre and visit factors were found to be significant. Moreover, both between-centre and between-visit reliabilities ranged from poor to excellent for most morphometric measures. A comparison between segmentation using Brainvisa and FAST revealed that FAST improved the reliabilities for most cases, suggesting that morphometry could benefit from improving the bias correction. However, the results were still significantly different across different scanners or different visits. CONCLUSIONS: Our results confirm that for morphometry analysis with the current version of Brainvisa using data from multicentre or longitudinal studies, the scanner-related variability must be taken into account and where possible should be corrected for. We also suggest providing some flexibility to Brainvisa for a step-by-step analysis of the robustness of this package in terms of reproducibility of the results by allowing the bias corrected images to be imported from other packages and bias correction step be skipped, for example.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

TRAIL-receptor preferences in pancreatic cancer cells revisited: Both TRAIL-R1 and TRAIL-R2 have a licence to kill

Background TRAIL is a potent and specific inducer of apoptosis in tumour cells and therefore is a possible new cancer treatment. It triggers apoptosis by binding to its cognate, death-inducing receptors, TRAIL-R1 and TRAIL-R2. In order to increase its activity, receptor-specific ligands and agonistic antibodies have been developed and some cancer types, including pancreatic cancer, have been reported to respond preferentially to TRAIL-R1 triggering. The aim of the present study was to examine an array of TRAIL-receptor specific variants on a number of pancreatic cancer cells and test the generality of the concept of TRAIL-R1 preference in these cells. Methods TRAIL-R1 and TRAIL-R2 specific sTRAIL variants were designed and tested on a number of pancreatic cancer cells for their TRAIL-receptor preference. These sTRAIL variants were produced in HEK293 cells and were secreted into the medium. After having measured and normalised the different sTRAIL variant concentrations, they were applied to pancreatic and control cancer cells. Twenty-four hours later apoptosis was measured by DNA hypodiploidy assays. Furthermore, the specificities of the sTRAIL variants were validated in HCT116 cells that were silenced either for TRAIL-R1 or TRAIL-R2. Results Our results show that some pancreatic cancer cells use TRAIL-R1 to induce cell death, whereas other pancreatic carcinoma cells such as AsPC-1 and BxPC-3 cells trigger apoptosis via TRAIL-R2. This observation extended to cells that were naturally TRAIL-resistant and had to be sensitised by silencing of XIAP (Panc1 cells). The measurement of TRAIL-receptor expression by FACS revealed no correlation between receptor preferences and the relative levels of TRAIL-R1 and TRAIL-R2 on the cellular surface. Conclusions These results demonstrate that TRAIL-receptor preferences in pancreatic cancer cells are variable and that predictions according to cancer type are difficult and that determining factors to inform the optimal TRAIL-based treatments still have to be identified

The Liver Plays a Major Role in Clearance and Destruction of Blood Trypomastigotes in Trypanosoma cruzi Chronically Infected Mice

Intravenous challenge with Trypanosoma cruzi can be used to investigate the process and consequences of blood parasite clearance in experimental Chagas disease. One hour after intravenous challenge of chronically infected mice with 5×106 trypomastigotes, the liver constituted a major site of parasite accumulation, as revealed by PCR. Intact parasites and/or parasite remnants were visualized at this time point scattered in the liver parenchyma. Moreover, at this time, many of liver-cleared parasites were viable, as estimated by the frequency of positive cultures, which considerably diminished after 48 h. Following clearance, the number of infiltrating cells in the hepatic tissue notably increased: initially (at 24 h) as diffuse infiltrates affecting the whole parenchyma, and at 48 h, in the form of large focal infiltrates in both the parenchyma and perivascular spaces. Phenotypic characterization of liver-infiltrating cells 24 h after challenge revealed an increase in Mac1+, CD8+ and CD4+ cells, followed by natural killer (NK) cells. As evidence that liver-infiltrating CD4+ and CD8+ cells were activated, increased frequencies of CD69+CD8+, CD69+CD4+ and CD25+CD122+CD4+ cells were observed at 24 and 48 h after challenge, and of CD25−CD122+CD4+ cells at 48 h. The major role of CD4+ cells in liver protection was suggested by data showing a very high frequency of interferon (IFN)-γ-producing CD4+ cells 24 h after challenge. In contrast, liver CD8+ cells produced little IFN-γ, even though they showed an enhanced potential for secreting this cytokine, as revealed by in vitro T cell receptor (TCR) stimulation. Confirming the effectiveness of the liver immune response in blood parasite control during the chronic phase of infection, no live parasites were detected in this organ 7 days after challenge

Predictors of linkage to care following community-based HIV counseling and testing in rural Kenya

Despite innovations in HIV counseling and testing (HCT), important gaps remain in understanding linkage to care. We followed a cohort diagnosed with HIV through a community-based HCT campaign that trained persons living with HIV/AIDS (PLHA) as navigators. Individual, interpersonal, and institutional predictors of linkage were assessed using survival analysis of self-reported time to enrollment. Of 483 persons consenting to follow-up, 305 (63.2%) enrolled in HIV care within 3 months. Proportions linking to care were similar across sexes, barring a sub-sample of men aged 18–25 years who were highly unlikely to enroll. Men were more likely to enroll if they had disclosed to their spouse, and women if they had disclosed to family. Women who anticipated violence or relationship breakup were less likely to link to care. Enrolment rates were significantly higher among participants receiving a PLHA visit, suggesting that a navigator approach may improve linkage from community-based HCT campaigns.Vestergaard Frandse