[afro-nets] Advanced Laboratory Training Course on Molecular Immunology of Protozoan Infections

["Hamisi Malebo" <malebo@hotmail.com>]

Advanced Laboratory Training Course on Molecular Immunology of Protozoan Infections
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March 18-31, 2007
Buenos Aires, Argentina

Institute  for  Research on genetic Engineering and Molecular Biology,
CONICET

This  intensive  laboratory and lecture course, organized and directed by  HHMI  international  research  scholar  Mariano  Levin, is open to beginning scientists working in the field of protozoan parasitosis and will  provide students with an understanding of relevant immunological aspects  of  parasite infections. The course will be divided into five units:  Unit  1  will  consist  of an International Symposium covering different  aspects  of  basic  immunology  and  immunology of parasite infections.  Lectures  will emphasize recent progress in the fields of antigen   processing   and  presentation,  cytokine  action,  adhesion molecules,  and  co-receptor cell activation. Units 2 and 3 will teach students  how  to  construct  human recombinant antibody libraries and select  r-  hu  antibodies  using  different  panning strategies. Then students   will   learn  how  to  produce,  purify,  and  characterize recombinant  antibodies expressed in diverse cell types. Units 4 and 5 will   cover   cellular   immunological  topics  related  to  parasite infections   and   will  teach  students  how  to  analyze  lymphocyte differentiation and activation and characterize regulatory T cells.

Course  participants will develop contacts and strong scientific links among  themselves,  with  the  members  of  the international teaching staff,  and  with  the  invited  speakers.  These contacts should also encourage  sharing and exchanges between the labs of the participants, generating scientific collaborations and strong networks.

Detailed Information on Units 2-5
Unit 2: Construction of combinatorial antibodies libraries.
Library construction will begin with human VH and VL PCR amplification using  specific primers. VH and VL products, separately purified, will be  used  in  overlap  PCR  to create svFc fragments. Overlap products inserts  and  pComb3X  will  be prepared similarly by SfiI digestions, followed  by ethanol precipitations, resuspension, and purification in a  2%  agarose  gel.  Appropiate  DNA  bands will be electroeluted and ligated   overnight   at   room  temperature,  then  precipitated  and resuspended  for electroporation with electrocompetent cell. Bacterialcultures  will  be  incubated  with  VCS  M13  helper  phage and after infection, phage will be precipitated in PEG-8000/NaCl for first round panning.  Several  types  of  panning will be tested and/or discussed. After  a  round  of  selection,  fresh bacterias will be infected with eluted phages and the library will be reamplified.

Unit  3:  Production, purification and characterization of recombinant antibodies.
Expression  of  recombinant antibodies will include different cellular systems.  The  functional  effect  of  recombinant  antibodies on AMPc accumulation  will  be assessed. Recombinant antibodies will be tested by  indirect  immunofluorescence  assays  on  both fixed b1 adrenergic receptor stable transfected CHO cell lines and fixed Trypanosoma cruzi cells.   Passive  transfer  of  recombinant  antibodies.  Analysis  of individual  BALC/c  mouse  electrocardiogram (DI, DII, DIII, AVL, AVR, AVF);  heart  rates  will  be  measured  by counting QRS peaks in 10 s windows.

Units 4 and 5: Cellular immunology.
Polimorfonuclear  cells  (PBMC)  isolated  from  blood samples will be cultured  in  supplemented  RPMI 1640 medium with T. cruzi antigens at 25µg/ml  for 6 days at 37¼C in a 5% CO[2] humidified atmosphere. After incubation,  cells will be centrifugated and supernants will be stored for  cytokines  assays.  PMBC  will  be  washing with phosphate saline buffer/BSA  0.5% and stained with the manufactures recommended amounts of mAbs anti-CD4, anti-CD25, anti-HLA-DR, anti-CD8, anti-CD3, anti-28, anti-CD45RO,  anti-CD45RA  conjugated  with PE, FITC, PerCP or APC. To analyse  CD4^+CD25^+  Treg population, cells will also be stained with Ab anti-FoxP3. After incubation, the cell prepations will be fixed and analyze  by  flow  citometry  (FACS).  Citokine  productions  will  be assessed by ELISA with appropiate anti-cytokine monoclonal antibodies. It  will  be  considered  to  analyse  the production of interleukin 2 (IL-2), IL-4, IL-10, interferon g (IFN- g) and tumor necrosis factor a (TNF- a).

To Apply
Eligible participants include graduate students, postdoctoral fellows,  and  junior faculty. To apply, go to http://www.hhmi.org/argentina. The  deadline  for  applications  is  December 29, 2006. Notifications regarding acceptance will be made January 31, 2007.

Functional Genomics of Malaria Parasites
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National Center for Genetic Engineering and Biotechnology
Pathumthani, Thailand

Information on this course will be available soon.

Genetic  Engineering  in  the  Mouse  Genome  to Understand Human Gene Function and Disease
December 2007
Center for Scientific Studies
Valdivia, Chile

Information on this course will be available soon.

--
Hamisi Masanja Malebo,
National Institute for Medical Research,
P.O. Box 9653,
Dar es Salaam, Tanzania.
Mobile: +255 754 383143
Email: mailto:Malebo@hotmail.com or mailto:hmalebo@nimr.or.tz