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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.
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<div>Norma</div>
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<div style="font-size: 12px; ">Norma Andrews</div>
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<div style="font-size: 12px; ">Email: <a href="https://exch.mail.umd.edu/owa/redir.aspx?C=i2B7cUGntEGPKDJ0HjEVDlgJZL3dm88IZ-48m2X5AOeXtJn1C0CwlzntujDpbzsSzzCTQNlv9Xw.&URL=mailto%3aandrewsn%40umd.edu">andrewsn@umd.edu</a></div>
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<div>On Mar 29, 2013, at 4:09 PM, Peter Myler wrote:</div>
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<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125); ">Sunil:<o:p></o:p></span></div>
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<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125); ">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.<o:p></o:p></span></div>
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<span style="font-size: 11pt; font-family: Calibri, sans-serif; color: rgb(31, 73, 125); ">Peter <o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Arial, sans-serif; color: navy; ">Peter J. Myler, Ph.D.</span><span style="color: navy; "><o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Arial, sans-serif; color: navy; ">Professor</span><span style="color: navy; "><br>
</span><span style="font-size: 10pt; font-family: Arial, sans-serif; color: navy; ">Seattle BioMed<br>
307 Westlake Ave N., Suite 500<br>
Seattle, WA 98109-5219<br>
USA</span><span style="color: navy; "><span class="Apple-converted-space"> </span><br>
</span><span style="font-size: 10pt; font-family: Arial, sans-serif; color: navy; ">(206) 256-7332 (office)</span><span style="color: navy; "><br>
</span><span style="font-size: 10pt; font-family: Arial, sans-serif; color: navy; ">(206) 256-7229 (FAX)</span><span style="color: navy; "><span class="Apple-converted-space"> </span><br>
</span><span style="color: rgb(31, 73, 125); "><a href="mailto:peter.myler@sbri.org" style="color: blue; text-decoration: underline; ">peter.myler@sbri.org</a><o:p></o:p></span></div>
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<span style="color: navy; ">Affiliate Professor<span class="Apple-converted-space"> </span><br>
Departments of Global Health &<br>
Biomedical Informatics and Medical Education<span class="Apple-converted-space"> </span><br>
University of Washington<span class="Apple-converted-space"> </span><br>
Seattle, WA 98195<span class="Apple-converted-space"> </span><br>
Box 357238<span class="Apple-converted-space"> </span><br>
</span><span style="color: rgb(31, 73, 125); "><a href="mailto:mylerpj@u.washington.edu" style="color: blue; text-decoration: underline; ">mylerpj@u.washington.edu</a></span><span style="color: navy; "><o:p></o:p></span></div>
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<b><span style="font-size: 10pt; font-family: Tahoma, sans-serif; ">From:</span></b><span style="font-size: 10pt; font-family: Tahoma, sans-serif; "><span class="Apple-converted-space"> </span><a href="mailto:leish-l-bounces@lineu.icb.usp.br" style="color: blue; text-decoration: underline; ">leish-l-bounces@lineu.icb.usp.br</a><span class="Apple-converted-space"> </span>[mailto:leish-l-bounces@lineu.icb.usp.br]<span class="Apple-converted-space"> </span><b>On
Behalf Of<span class="Apple-converted-space"> </span></b>Sunil Arora<br>
<b>Sent:</b><span class="Apple-converted-space"> </span>Monday, March 11, 2013 8:42 AM<br>
<b>To:</b><span class="Apple-converted-space"> </span>Clos Joachim; Leish-L email post<br>
<b>Subject:</b><span class="Apple-converted-space"> </span>Re: [Leish-l] Fwd: hot spots<o:p></o:p></span></div>
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<o:p> </o:p></div>
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<span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; ">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<o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; ">best<o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; ">sunil<o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; "> <o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: rgb(0, 0, 191); ">---------------------------------------------------------------</span></i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; "><o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: rgb(0, 0, 191); ">Dr Sunil K.Arora<br>
Professor<span class="Apple-converted-space"> </span><br>
In Charge- HIV Diagnostic and Disease Monitoring Laboratory &<br>
NACO State Reference Laboratory,</span></i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; "><o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: rgb(0, 0, 191); ">Councillor- Federation of Immunological Societies of Asia-oceania (FIMSA)</span></i><span style="font-size: 10pt; font-family: Verdana, sans-serif; color: black; "><o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); ">Vice President- Indian Immunology Society (IIS)</span></i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); "><o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); ">Secretary (Research)- The Cytometry Society of India (TCS)</span></i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); "><o:p></o:p></span></div>
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<i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); ">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:<span class="Apple-converted-space"> </span><a href="mailto:skarora_in@yahoo.com" target="_blank" style="color: blue; text-decoration: underline; ">skarora_in@yahoo.com</a><span class="Apple-converted-space"> </span>;<span class="Apple-converted-space"> </span><a href="mailto:skarorain@gmail.com" target="_blank" style="color: blue; text-decoration: underline; ">skarorain@gmail.com</a></span></i><span style="font-size: 10pt; font-family: Arial, sans-serif; color: rgb(0, 0, 191); "><o:p></o:p></span></div>
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<b><span style="font-size: 10pt; font-family: Arial, sans-serif; color: black; ">From:</span></b><span style="font-size: 10pt; font-family: Arial, sans-serif; color: black; "><span class="Apple-converted-space"> </span>Clos Joachim <<a href="mailto:clos@bni-hamburg.de" style="color: blue; text-decoration: underline; ">clos@bni-hamburg.de</a>><br>
<b>To:</b><span class="Apple-converted-space"> </span>Leish-L email post <<a href="mailto:Leish-l@lineu.icb.usp.br" style="color: blue; text-decoration: underline; ">Leish-l@lineu.icb.usp.br</a>><span class="Apple-converted-space"> </span><br>
<b>Sent:</b><span class="Apple-converted-space"> </span>Monday, 11 March 2013 8:16 PM<br>
<b>Subject:</b><span class="Apple-converted-space"> </span>[Leish-l] Fwd: hot spots</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="color: black; "><o:p> </o:p></span></div>
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<span style="font-size: 10pt; font-family: Baghdad, serif; color: rgb(1, 1, 1); ">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.</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-size: 10pt; font-family: Baghdad, serif; color: rgb(1, 1, 1); ">Cheers!</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="color: black; ">Am 07.02.2013 um 20:38 schrieb Carlos Costa:<o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">What are the most polemic issues in leishmaniasis?<br clear="all">
</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="color: black; "><o:p> </o:p></span></div>
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<span style="color: black; ">--<o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Carlos H. N. Costa, MD, DSc.</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Sociedade Brasileira de Medicina Tropical </span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">(Brazilian Society of Tropical Medicine)</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">President</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="color: black; "><o:p> </o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Universidade Federal do Piauí</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Instituto de Doenças Tropicais Natan Portella</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Rua Artur de Vasconcelos 151-Sul</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">64001-450 Teresina-PI</span><span style="color: black; "><o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Brazil<o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">Telephones: +55 86 3222-4377 (W), <o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">+55 86 3221-3062 (W), <o:p></o:p></span></div>
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<span style="font-family: Verdana, sans-serif; color: black; ">+55 86 3237-1075 (R). <o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Joachim Clos, Dr. rer. nat.</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Senior Staff Scientist</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Group Leader</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Bernhard Nocht Institute for Tropical Medicine</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Bernhard Nocht St. 74, D-20359 Hamburg, FRG</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Phone +49 40 42818-481; Fax -512</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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<span class="yiv1658093374apple-style-span"><span style="font-size: 9pt; font-family: Helvetica, sans-serif; color: black; ">Skype: joachim.clos</span></span><span style="font-size: 9pt; font-family: Arial, sans-serif; color: black; "><o:p></o:p></span></div>
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