<html><body><div style="color:#000; background-color:#fff; font-family:verdana, helvetica, sans-serif;font-size:10pt">Yes good idea<br>regards<br><br><div> </div><div><i><font color="#0000bf">---------------------------------------------------------------</font></i></div><i>sunil</i><div><br></div> <div style="font-family: verdana, helvetica, sans-serif; font-size: 10pt;"> <div style="font-family: times new roman, new york, times, serif; font-size: 12pt;"> <div dir="ltr"> <font face="Arial" size="2"> <hr size="1"> <b><span style="font-weight:bold;">From:</span></b> Peter Myler <peter.myler@seattlebiomed.org><br> <b><span style="font-weight: bold;">To:</span></b> Leish-L email post <Leish-l@lineu.icb.usp.br> <br><b><span style="font-weight: bold;">Cc:</span></b> Sunil Arora <skarora_in@yahoo.com>; Clos Joachim <clos@bni-hamburg.de>; Norma Windsor Andrews <andrewsn@umd.edu>; "'danz@bi.technion.ac.il'"
<danz@bi.technion.ac.il> <br> <b><span style="font-weight: bold;">Sent:</span></b> Friday, 5 April 2013 9:45 PM<br> <b><span style="font-weight: bold;">Subject:</span></b> RE: [Leish-l] Fwd: hot spots<br> </font> </div> <br>It seems that this topic has certainly generated some controversy and I suggest would be an excellent subject for discussion at WorldLeish5 (which is how this thread started).<br><br>Peter<br><br>Peter J. Myler, Ph.D.<br>Professor<br>Seattle BioMed<br>307 Westlake Ave N., Suite 500<br>Seattle, WA 98109-5219<br>USA <br>(206) 256-7332 (office)<br>(206) 256-7229 (FAX) <br><a ymailto="mailto:peter.myler@sbri.org" href="mailto:peter.myler@sbri.org">peter.myler@sbri.org</a><br>Affiliate Professor <br>Departments of Global Health &<br>Biomedical Informatics and Medical Education <br>University of Washington <br>Seattle, WA 98195 <br>Box 357238 <br><a ymailto="mailto:mylerpj@u.washington.edu"
href="mailto:mylerpj@u.washington.edu">mylerpj@u.washington.edu</a> <br><br>-----Original Message-----<br>From: <a ymailto="mailto:danz@bi.technion.ac.il" href="mailto:danz@bi.technion.ac.il">danz@bi.technion.ac.il</a> [mailto:<a ymailto="mailto:danz@bi.technion.ac.il" href="mailto:danz@bi.technion.ac.il">danz@bi.technion.ac.il</a>] <br>Sent: Friday, April 05, 2013 8:34 AM<br>To: Norma Windsor Andrews<br>Cc: Peter Myler; Sunil Arora; Clos Joachim; Leish-L email post; <a ymailto="mailto:danz@bi.technion.ac.il" href="mailto:danz@bi.technion.ac.il">danz@bi.technion.ac.il</a><br>Subject: Re: [Leish-l] Fwd: hot spots<br><br>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.<br><br>Dear Arora, dear all,<br>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.<br><br>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.<br><br>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.<br><br>My suggestion is that instead of being so negative about axenic amastigotes, we join efforts to make them a better tool to study leishmaniasis.<br><br>Dan<br><br>Professor Dan Zilberstein, Executive director Center for Pre Academic Education And The Faculty of Biology Technion-Israel Institute of Technology Haifa 32000, Israel<br><br>ב-5 באפר 2013, בשעה 11:10, "Norma Windsor Andrews" <<a ymailto="mailto:andrewsn@umd.edu" href="mailto:andrewsn@umd.edu">andrewsn@umd.edu</a><mailto:<a ymailto="mailto:andrewsn@umd.edu" href="mailto:andrewsn@umd.edu">andrewsn@umd.edu</a>>> כתב/ה:<br><br>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.<br><br>Norma<br><br><br>Norma Andrews<br>Professor and Chair<br>Cell Biology and Molecular Genetics<br>2134 Bioscience Research Building<br>University of Maryland<br>College Park, MD 20742-5815<br>Phone (301) 405-8418<br>Fax (301) 314-9489<br>Email: <a ymailto="mailto:andrewsn@umd.edu" href="mailto:andrewsn@umd.edu">andrewsn@umd.edu</a><<a href="https://exch.mail.umd.edu/owa/redir.aspx?C=i2B7cUGntEGPKDJ0HjEVDlgJZL3dm88IZ-48m2X5AOeXtJn1C0CwlzntujDpbzsSzzCTQNlv9Xw.&URL=mailto%3aandrewsn%40umd.edu" target="_blank">https://exch.mail.umd.edu/owa/redir.aspx?C=i2B7cUGntEGPKDJ0HjEVDlgJZL3dm88IZ-48m2X5AOeXtJn1C0CwlzntujDpbzsSzzCTQNlv9Xw.&URL=mailto%3aandrewsn%40umd.edu</a>><br><br>On Mar 29, 2013, at 4:09 PM, Peter Myler wrote:<br><br>Sunil:<br><br>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.<br><br>Peter<br><br>Peter J. Myler,
Ph.D.<br>Professor<br>Seattle BioMed<br>307 Westlake Ave N., Suite 500<br>Seattle, WA 98109-5219<br>USA<br>(206) 256-7332 (office)<br>(206) 256-7229 (FAX)<br><a ymailto="mailto:peter.myler@sbri.org" href="mailto:peter.myler@sbri.org">peter.myler@sbri.org</a><mailto:<a ymailto="mailto:peter.myler@sbri.org" href="mailto:peter.myler@sbri.org">peter.myler@sbri.org</a>><br>Affiliate Professor<br>Departments of Global Health &<br>Biomedical Informatics and Medical Education University of Washington Seattle, WA 98195 Box 357238 <a ymailto="mailto:mylerpj@u.washington.edu" href="mailto:mylerpj@u.washington.edu">mylerpj@u.washington.edu</a><mailto:<a ymailto="mailto:mylerpj@u.washington.edu" href="mailto:mylerpj@u.washington.edu">mylerpj@u.washington.edu</a>><br><br>From: <a ymailto="mailto:leish-l-bounces@lineu.icb.usp.br" href="mailto:leish-l-bounces@lineu.icb.usp.br">leish-l-bounces@lineu.icb.usp.br</a><mailto:<a
ymailto="mailto:leish-l-bounces@lineu.icb.usp.br" href="mailto:leish-l-bounces@lineu.icb.usp.br">leish-l-bounces@lineu.icb.usp.br</a>> [mailto:<a ymailto="mailto:leish-l-bounces@lineu.icb.usp.br" href="mailto:leish-l-bounces@lineu.icb.usp.br">leish-l-bounces@lineu.icb.usp.br</a>] On Behalf Of Sunil Arora<br>Sent: Monday, March 11, 2013 8:42 AM<br>To: Clos Joachim; Leish-L email post<br>Subject: Re: [Leish-l] Fwd: hot spots<br><br>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<br><br>best<br>sunil<br><br>---------------------------------------------------------------<br>Dr Sunil K.Arora<br>Professor<br>In Charge- HIV Diagnostic and Disease Monitoring Laboratory & NACO State Reference Laboratory,<br><br>Councillor- Federation of
Immunological Societies of Asia-oceania (FIMSA) Vice President- Indian Immunology Society (IIS) Secretary (Research)- The Cytometry Society of India (TCS)<br><br>Department of Immunopathology<br>PGIMER, Chandigarh-160 012<br>Ph.: 0091-172-2755192 (Off)<br>FAX: 0091-172-2744401, 2745078<br>Ph:+91-172-4666087 (Res); 9872866087 (cell)<br>email: <a ymailto="mailto:skarora_in@yahoo.com" href="mailto:skarora_in@yahoo.com">skarora_in@yahoo.com</a><mailto:<a ymailto="mailto:skarora_in@yahoo.com" href="mailto:skarora_in@yahoo.com">skarora_in@yahoo.com</a>> ; <a ymailto="mailto:skarorain@gmail.com" href="mailto:skarorain@gmail.com">skarorain@gmail.com</a><mailto:<a ymailto="mailto:skarorain@gmail.com" href="mailto:skarorain@gmail.com">skarorain@gmail.com</a>><br><br>________________________________<br>From: Clos Joachim <<a ymailto="mailto:clos@bni-hamburg.de" href="mailto:clos@bni-hamburg.de">clos@bni-hamburg.de</a><mailto:<a
ymailto="mailto:clos@bni-hamburg.de" href="mailto:clos@bni-hamburg.de">clos@bni-hamburg.de</a>>><br>To: Leish-L email post <<a ymailto="mailto:Leish-l@lineu.icb.usp.br" href="mailto:Leish-l@lineu.icb.usp.br">Leish-l@lineu.icb.usp.br</a><mailto:<a ymailto="mailto:Leish-l@lineu.icb.usp.br" href="mailto:Leish-l@lineu.icb.usp.br">Leish-l@lineu.icb.usp.br</a>>><br>Sent: Monday, 11 March 2013 8:16 PM<br>Subject: [Leish-l] Fwd: hot spots<br><br>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.<br>Cheers!<br><br><br><br><br>Am 07.02.2013 um 20:38 schrieb Carlos Costa:<br><br><br>What are the most polemic issues in leishmaniasis?<br><br>--<br>Carlos H. N. Costa, MD, DSc.<br>Sociedade Brasileira de Medicina Tropical (Brazilian Society of Tropical Medicine) President<br><br>Universidade Federal do Piauí<br>Instituto de Doenças Tropicais Natan Portella Rua Artur de Vasconcelos 151-Sul<br>64001-450 Teresina-PI<br>Brazil<br>Telephones: +55 86 3222-4377 (W),<br>+55 86 3221-3062 (W),<br>+55 86 3237-1075 (R).<br><br><br>Aviso: As informações contidas nesta mensagem são CONFIDENCIAIS, protegidas pelo sigilo legal, por direitos autorais e destinadas exclusivamente à pessoa ou organização para a qual a mensagem foi destinada.<br>Warning: This message is meant only for the intended recipient of the transmission. It is forbidden any unauthorized use, alteration, reproduction and distribution. If you are not the correct recipient,
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