[Leish-l] Fwd: hot spots

Sat Mar 30 01:39:26 BRT 2013

Ya Peter, I know what you are saying about studying the expression profile of the transcriptome, one has to be careful regarding contaminating RNA from macrophages, in that case yes, one has to depend on axenic amastigotes which are much better purity wise as well as number wise, Thanks

---------------------------------------------------------------
sunil

From: Peter Myler <peter.myler at seattlebiomed.org>
To: 'Sunil Arora' <skarora_in at yahoo.com>; Clos Joachim <clos at bni-hamburg.de>; Leish-L email post <Leish-l at lineu.icb.usp.br> 
Cc: "danz at bi.technion.ac.il" <danz at bi.technion.ac.il> 
Sent: Saturday, 30 March 2013 1:39 AM
Subject: RE: [Leish-l] Fwd: hot spots

Sunil:

Axenic amastigotes (Ax) are much more than “stressed-out promastigotes”.  We (and others) have shown they are very similar (although not identical) to macrophage-derived amastigotes (Am) in terms of global mRNA and protein expression levels.  While it would obviously be better to work with Am, it is often technically difficult due to low numbers and contamination with macrophage RNA and protein.  In addition, it is my contention that the time taken to isolate Am from the macrophages makes them “stressed-out amastigotes”, which are less physiological relevant than Ax.  We need to approach this discussion from a position of knowledge and data (i.e. understanding exactly which genes are expressed differently in Am and Ax) than belief.  Hopefully, we will be able to publish the results of our RNA-seq studies comparing promastigotes, axenic amastigotes and macrophage-derived amastigotes (without purification) in the not too distant future.

Peter  

Peter J. Myler, Ph.D.
Professor
Seattle BioMed
307 Westlake Ave N., Suite 500
Seattle, WA  98109-5219
USA
(206) 256-7332 (office)
(206) 256-7229 (FAX)
peter.myler at sbri.org
Affiliate Professor 
Departments of Global Health &
Biomedical Informatics and Medical Education 
University of Washington 
Seattle, WA  98195 
Box 357238 
mylerpj at u.washington.edu

From:leish-l-bounces at lineu.icb.usp.br [mailto:leish-l-bounces at lineu.icb.usp.br] On Behalf Of Sunil Arora
Sent: Monday, March 11, 2013 8:42 AM
To: Clos Joachim; Leish-L email post
Subject: Re: [Leish-l] Fwd: hot spots

I agree...axenic amastigotes are more like stressed out promastigotes... I always prefer working with something inside the macrophages as a model for testing drugs or immunomodulators which I believe are closer to actual intracellular amastigotes in the infected host

best
sunil

---------------------------------------------------------------
Dr Sunil K.Arora
Professor 
In Charge- HIV Diagnostic and Disease Monitoring Laboratory &
NACO State Reference Laboratory,

Councillor- Federation of Immunological Societies of Asia-oceania (FIMSA)
Vice President- Indian Immunology Society (IIS)
Secretary (Research)- The Cytometry Society of India (TCS)

Department of Immunopathology
PGIMER, Chandigarh-160 012
Ph.: 0091-172-2755192 (Off)
FAX: 0091-172-2744401, 2745078
Ph:+91-172-4666087 (Res); 9872866087 (cell)
email: skarora_in at yahoo.com ; skarorain at gmail.com 

From:Clos Joachim <clos at bni-hamburg.de>
To: Leish-L email post <Leish-l at lineu.icb.usp.br> 
Sent: Monday, 11 March 2013 8:16 PM
Subject: [Leish-l] Fwd: hot spots

Are axenic amastigotes a valid model (and for what?), the first developmental step towards real amastigotes, or just stressed-out promastigotes? Many of us use axenics and swear by them, others are skeptical, others will accept what's inside a macrophage, still others only want amastigotes isolated from something with a fur. I believe we should come to some sort of common language and interpretation. I am somewhere in the middle myself, preferring to do things in macrophages whenever possible.
>Cheers!

Am 07.02.2013 um 20:38 schrieb Carlos Costa:

What are the most polemic issues in leishmaniasis?

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