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Batista S.A.,Federal University of Minas Gerais | Rocha G.A.,Federal University of Minas Gerais | Rocha A.M.C.,Federal University of Minas Gerais | Saraiva I.E.B.,Federal University of Minas Gerais | And 3 more authors.
BMC Microbiology | Year: 2011

Background: Helicobacter pylori infection is one of the most common infections worldwide and is associated with gastric cancer and peptic ulcer. Bacterial virulence factors such as CagA have been shown to increase the risk of both diseases. Studies have suggested a causal role for CagA EPIYA polymorphisms in gastric carcinogenesis, and it has been shown to be geographically diverse. We studied associations between H. pylori CagA EPIYA patterns and gastric cancer and duodenal ulcer, in an ethnically admixed Western population from Brazil. CagA EPIYA was determined by PCR and confirmed by sequencing. A total of 436 patients were included, being 188 with gastric cancer, 112 with duodenal ulcer and 136 with gastritis. Results: The number of EPIYA C segments was significantly associated with the increased risk of gastric carcinoma (OR = 3.08, 95% CI = 1.74 to 5.45, p < 10-3) even after adjustment for age and gender. Higher number of EPIYA C segments was also associated with gastric atrophy (p = 0.04) and intestinal metaplasia (p = 0.007). Furthermore, patients infected by cagA strains possessing more than one EPIYA C segment showed decreased serum levels of pepsinogen I in comparison with those infected by strains containing one or less EPIYA C repeat. Otherwise, the number of EPIYA C segments did not associate with duodenal ulcer. Conclusions: Our results demonstrate that infection with H. pylori strains harbouring more than one CagA EPIYA C motif was clearly associated with gastric cancer, but not with duodenal ulcer. Higher number of EPIYA C segments was also associated with gastric precancerous lesions as demonstrated by histological gastric atrophic and metaplastic changes and decreased serum levels of pepsinogen I. © 2011 Batista et al; licensee BioMed Central Ltd.

Duc T.N.,VIB | Duc T.N.,Laboratory of Cellular and Molecular Immunology | Hassanzadeh-Ghassabeh G.,VIB | Hassanzadeh-Ghassabeh G.,Laboratory of Cellular and Molecular Immunology | And 6 more authors.
Methods in Molecular Biology | Year: 2012

Chromatin immunoprecipitation (ChIP), followed by microarray hybridization (ChIP-chip) or high-throughput sequencing (ChIP-seq), is becoming a widely used powerful method for the analysis of the in vivo DNA-protein interactions at genomic scale. The success of ChIP largely depends on the quality of antibodies. Although polyclonal antibodies have been successfully used for ChIP, their production requires regular immunization and they exhibit high aspecificity and batch to batch variability. These problems can be circumvented by generating monoclonal antibodies (mAbs) via hybridoma technology. However, such mAbs do not often capture DNA-protein complexes and are not amenable to engineering. Nanobodies are recombinant single domain antibody fragments derived from camelid Heavy-Chain antibodies. Nanobodies exhibit high affinity and specificity towards their cognate antigens and often capture their target antigens in solution. Moreover, the Nanobody genes can be easily tailored to streamline ChIP. Here, we describe a Nanobody-based ChIP protocol which we have successfully used for genome-wide identification of the binding sites of the low-abundant transcription factor Ss-LrpB from the hyperthermoacidophilic archaeon Sulfolobus solfataricus. © 2012 Springer Science+Business Media, LLC.

Buelens K.,Catholic University of Leuven | Hassanzadeh-Ghassabeh G.,Laboratory of Cellular and Molecular Immunology | Hassanzadeh-Ghassabeh G.,Vrije Universiteit Brussel | Muyldermans S.,Laboratory of Cellular and Molecular Immunology | And 3 more authors.
Journal of Thrombosis and Haemostasis | Year: 2010

Background and objective: As activated thrombin-activatable fibrinolysis inhibitor (TAFIa) is a potent antifibrinolytic enzyme, the development of TAFI inhibitors is a new promising approach in the development of profibrinolytic drugs. We, therefore, aimed to generate nanobodies, camelid-derived single-domain antibodies towards TAFI. Methods and results: This study reports the generation and characterization of a panel of 22 inhibitory nanobodies. This panel represents a wide diversity in mechanisms for interference with the functional properties of TAFI as the nanobodies interfere with various modes of TAFI activation, TAFIa activity and/or TAFI zymogen activity. Nanobodies inhibiting TAFIa activity and thrombin/thrombomodulin-mediated TAFI activation revealed profibrinolytic properties in a clot lysis experiment with exogenously added thrombomodulin (TM), whereas nanobodies inhibiting plasmin-mediated TAFI activation only revealed profibrinolytic properties in a clot lysis experiment without TM. The results of in vitro clot lysis experiments provided evidence that inhibitory nanobodies penetrate the clot better compared with inhibitory monoclonal antibodies. Conclusions: These data suggest that the generated nanobodies are potent TAFI inhibitors and are a step forward in the development of a profibrinolytic drug. They might also be an excellent tool to unravel the role of the physiological activators of TAFI in various pathophysiological processes. © 2010 International Society on Thrombosis and Haemostasis.

Luyckx A.,Catholic University of Leuven | Schouppe E.,Laboratory of Cellular and Molecular Immunology | Schouppe E.,Vrije Universiteit Brussel | Rutgeerts O.,Catholic University of Leuven | And 9 more authors.
Bone Marrow Transplantation | Year: 2012

To date, myeloid-derived suppressor cells (MDSC) have been best studied in cancer, where they represent an escape mechanism for immune surveillance. MDSC are now also gaining interest in the context of transplantation. Suppressive CD11b myeloid progenitor cells have been reported to expand endogenously during BM chimerism induction in mice; in particular, in irradiated MHC-matched BM chimeras and in parent-in-F1 BM chimeras. Myeloid cell expansion coincided with a time frame where donor lymphocyte infusion (DLI) therapy-mediated GVL effects without GVHD. Hypothesizing that regulatory myeloid cells may have a role in regulating post-transplant T-cell alloreactivity, we performed a detailed phenotypic and functional characterization of these cells in the parent-in-F1 C57BL/6 C57BL/6xDBA2 model. We found that transiently expanding CD11b myeloid progenitor cells comprise the two phenotypically and functionally distinct mononuclear and polymorphonuclear MDSC subsets that were recently described in tumor-bearing mice. Both MDSC subsets suppressed in vitro and in vivo alloreactive T-cell proliferation. Also, both the subsets mediated enhanced in vitro suppression when harvested from chimeras, given a prior in vivo challenge with non-tolerant donor T cells, indicating that allo-activated T cells can activate MDSC in vivo. This study provides the basis to investigate thepotentially beneficialrole of expanding MDSC in influencing the risk of GVHD during chimerism induction. © 2012 Macmillan Publishers Limited.

Muller G.C.,Laboratory of Cellular and Molecular Immunology | Pitrez P.M.,Grande Rio University | Teixeira A.L.,Federal University of Minas Gerais | Pires P.S.,Grande Rio University | And 3 more authors.
Clinical and Experimental Allergy | Year: 2010

Background Asthma is characterized by chronic inflammation of the airways with significant changes in leucocyte trafficking, cellular activation and tissue remodelling. Brain-derived neurotrophic factor (BDNF) has been involved with asthma and allergic diseases but its role as a severity marker in paediatric asthma has not been clinically assessed.Objectives To evaluate plasma BDNF and inflammatory markers in order to address their relationships with disease severity in children (6-15 years) with controlled persistent asthma.Methods Children with persistent asthma were selected and lung function and skin prick tests were performed in all patients. Plasma BDNF levels and various inflammatory markers (CCL3, CCL11, CCL22, CCL24, CXCL8, CXCL9, CXCL10, soluble TNF receptors) were assessed by ELISAs.Results Subjects with moderate and severe asthma had higher BDNF levels than mild asthma and controls (P<0.001). The chemokines studied and soluble TNF receptors did not differ between the studied groups.Conclusion and Clinical Relevance Our results indicate BDNF as a potential biomarker for clinical severity in children with asthma. © 2010 Blackwell Publishing Ltd.

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