[Leish-l] Fwd: hot spots

danz at bi.technion.ac.il danz at bi.technion.ac.il
Fri Apr 5 12:34:11 BRT 2013


A few days ago I sent my respond to Arora's criticism on axenic amastigotes being stressed cells. For some reason leish-l did not publish the message. So I decided to send the message again.

Dear Arora, dear all,
We have been studying axenic amastigotes in great detail and have published over dozen papers that characterize several aspects of promastigote to amastigote differentiation in host-free culture.

I totally disagree with Sunil’s statement that culture amastigotes are stressed parasites. Evidently, only combined high temperature and acidic pH induce morphogenesis pf axenic promastigotes into amastigotes. Time course analysis of differentiation clearly indicate a highly regulated and consistent process. Axenic amastigotes easily cycle back to promastigotes (paper submitted). If you read our publications you will agree with me that axenic parasites, i.e. both promastigotes (yes, culture promastigotes are axenic!) and amastigotes are the best simulators of Leishmania life cycle.

I agree that some of our colleagues use none-controlled protocols and their “axenic amastigotes” are actually stressed cells. But if one uses quality control assays like those we have developed, will work with a reliable system that simulates in vivo promastigote to amastigote differentiation.

My suggestion is that instead of being so negative about axenic amastigotes, we join efforts to make them a better tool to study leishmaniasis.

Dan

Professor Dan Zilberstein, Executive director
Center for Pre Academic Education
And The Faculty of Biology
Technion-Israel Institute of Technology
Haifa 32000, Israel

ב-5 באפר 2013, בשעה 11:10, "Norma Windsor Andrews" <andrewsn at umd.edu<mailto:andrewsn at umd.edu>> כתב/ה:

I fully agree with Peter that purification of intracellular amastigotes causes serious stress problems. Danilo Miguel in my lab just quantified the intracellular growth of L. amazonensis in bone marrow-derived mouse macrophages, comparing axenic amastigotes and amastigotes isolated from infected macrophages. The axenic amastigotes were much more infective, and grew much more robustly that the ones isolated from disrupted macrophages.

Norma


Norma Andrews
Professor and Chair
Cell Biology and Molecular Genetics
2134 Bioscience Research Building
University of Maryland
College Park, MD   20742-5815
Phone (301) 405-8418
Fax (301) 314-9489
Email: andrewsn at umd.edu<https://exch.mail.umd.edu/owa/redir.aspx?C=i2B7cUGntEGPKDJ0HjEVDlgJZL3dm88IZ-48m2X5AOeXtJn1C0CwlzntujDpbzsSzzCTQNlv9Xw.&URL=mailto%3aandrewsn%40umd.edu>

On Mar 29, 2013, at 4:09 PM, Peter Myler wrote:

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<mailto: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<mailto:mylerpj at u.washington.edu>

From: leish-l-bounces at lineu.icb.usp.br<mailto: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<mailto:skarora_in at yahoo.com> ; skarorain at gmail.com<mailto:skarorain at gmail.com>

________________________________
From: Clos Joachim <clos at bni-hamburg.de<mailto:clos at bni-hamburg.de>>
To: Leish-L email post <Leish-l at lineu.icb.usp.br<mailto: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?

--
Carlos H. N. Costa, MD, DSc.
Sociedade Brasileira de Medicina Tropical
(Brazilian Society of Tropical Medicine)
President

Universidade Federal do Piauí
Instituto de Doenças Tropicais Natan Portella
Rua Artur de Vasconcelos 151-Sul
64001-450 Teresina-PI
Brazil
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