Could anyone suggest how I can infect MACS purified B lymphocyte by promastigote of leishmania donovani? I am trying it with RPMI+10% FBS, keeping it for 24hrs incubation. Like to have your valuable suggestion.
Hi Koushik,
We exposed human tonsil B cells to Leishmania infantum pro and ama in RPMI-10%FBS overnight. For longer periods, depending on the protocol, we added 50ng/ml rhIL-10 or 50ng/ml rhIL-10+ 400U/ IL-4 to obtain a better viability. Li Pro and Ama will bind strongly to B cells and cause clumping. We have seen no evidence of infection of B cells. Be aware that Leishmania promastigotes continue to grow in RPMI-10% FBS. Some of the results are published in the attached publication. Cheers.
Article Leishmania infantum Amastigotes Trigger a Subpopulation of H...
I think the first thing to do is to activate B cells via the CD40 system, whether from the original spleen or tonsil, or blood.
The transient proliferation associated with activation of the CD40, may be enhanced by the addition of IL4 and IL10, but these cytokines may activate monocytes, which is not necessarily desirable.
Why do you want to infect B cells? To the best of my knowledge, they do not take up Leishmania but rather are activated by infected macrophages/monocytes. In situ, infected B cells definitely play no role for the Expression of the disease
I want to understand whether Leishmania has any affect on B cell or not, whether the parasite has any role on IL10 secretion by B cell, that's why I want to use purified B cell from BALB/c mice and exposing it with leishmania promastigote.
Dr. Solbach is right. Leishmanias do not infect nor are phagocytised by B cells. You may want to use peritoneal-derived macrophages for the infection (at 35 C) and co-culture with the B cells in different tissue culture time and conditions, to measure cytokines in the supernatant. Alternatively you may want to do ELISpot, the most adequate test for IL-4 and IL-10.
B cells can react to Leishmania directly even if they are not infected. You don't need macrophages or monocytes for a reaction to occur. B cells do play a role in Leishmania pathology as described in many papers.
For example, see http://www.ncbi.nlm.nih.gov/pubmed/10725726 and http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2944234/
If you want to see active replication of Leishmania, you need macrophages but if you just want to see the reaction of other immune cells to Leishmania, then you can use axenic pro or ama on purified B cells. You could also infect macrophages with Leishmania and coculture with b cells (or use the macrophage supernatant on b cells) but then you would see the reaction of B cells to Leish-infected macrophages (in response to macrophage cytokines) + the direct reaction of B cells to Leish (direct activation of B cells by the parasite). You don't answer to the same question.
Good luck,
B cells are not infected with leishmania. B cells from leishmania infected or ex infected patients can be stimulated but not from health non exported individuals
Yah, B cell interact with leishmania but not infected, you are right.
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