Obtaining cells from colon of dog with leishmaniasis for flow cytometric analysis

Abstract

In dogs, a parasite burden is evident throughout the gastrointestinal tract but not is observed pathological alterations. Our aim was isolating lamina propria cells from dogs infected with leishmaniasis to further investigate if the parasite in the gut is correlated with the pathogenesis of the visceral infection. The biopsies were obtained of dogs and incubated with collagenase II. In the cells suspension was added antibodies against superficial and intracellular markers. Few studies of the markers used in this work have been done in the lamina propria cells in leishmaniasis. It is of great importance to determine markers in this tissue especially those who are described as being involved with the persistence of Leishmania infection within cells due to the fact that the infection in the gut does not induce lesions. Furthermore, work is ongoing in terms of investigating other segments of the GIT, a systematic study, from dogs with Leishmania

Expression of Regulatory T Cells in Jejunum, Colon, and Cervical and Mesenteric Lymph Nodes of Dogs Naturally Infected with Leishmania infantum

“…The results of the present study raise questions about whether these sites are solely involved in the induction and suppression of intestinal inflammation. In agreement with our previous findings (22), CD4 ϩ Foxp3 ϩ cells were recently reported in inflamed mucosae of colitic mice and in skin lesions in mice infected with L. major (37,38). There is compelling evidence that Tregs play a fundamental role in controlling immune responses (39), and our knowledge of established and potentially novel subsets of Tregs and their immunoregulatory mechanisms is expanding rapidly (40).…”

“…Six dogs of unknown age, obtained from the Control Zoonosis Center of the Municipality of Carandaí, Minas Gerais State, Brazil, and negative for Leishmania infection by all tests, were used as controls. Dogs were confirmed to be Leishmania-free by immunohistochemistry (21)(22)(23)(24)(25). This protocol received the approval of the CETEA-UFMG (protocol 139/2012).…”

“…Pellets were resuspended in 950 l complete RPMI medium. The final volume was adjusted to 1 ml, and the concentration was adjusted to 1 ϫ 10 7 cells/ml, as described by Figueiredo et al (21,22). A 20-l cell suspension was incubated with 20 l of a solution containing monoclonal antibodies (MAbs) in 96-well U-bottom plates (Limbro Biomedicals, Aurora, OH) at 4°C for 30 min in darkness.…”

“…A 20-l cell suspension was incubated with 20 l of a solution containing monoclonal antibodies (MAbs) in 96-well U-bottom plates (Limbro Biomedicals, Aurora, OH) at 4°C for 30 min in darkness. We diluted antibodies against the cell surface in PBS plus fetal bovine serum (0.1%), and antibodies against intracellular markers were diluted in wash buffer as described by Figueiredo et al (21,22). Nonconjugated, purified antibodies were conjugated using a Zenon tricolor kit (Molecular Probes) as described by the manufacturer.…”

“…After staining, samples were washed with 0.1% Na 3 N in PBS, fixed with 200 l 2% formaldehyde in PBS, and held at 4°C until analysis by flow cytometry (FACSVantage; Becton, Dickinson, San Jose, CA). Intracytoplasmic marking followed the protocol described by Figueiredo et al (21,22). An isotype control was used to define nonspecific markings.…”