Student Awarded

Muluneh Ademe Worku

Muluneh Ademe Worku

Country: Ethiopia

Project Title: Mechanisms of Immune Suppression in Human Visceral Leishmaniasis and HIV co-infection

Institution: Addis Ababa University

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is a systemic disease in which the mortality rate can be as high at 100%. The majority of VL cases occur in six countries, namely Bangladesh, Brazil, Ethiopia, India, South Sudan, and Sudan. There are many knowledge gaps in the understanding of the pathogenesis of leishmaniasis. The immunopathogenic mechanisms of leishmaniasis in humans are not fully understood. Therefore in this study, we will characterize the phenotype of myeloid cells in whole blood and spleen of patients with VL and investigate the mechanisms that regulate the expression of immunosuppressive genes. We will particularly focus on expression of the enzymes arginase 1, indoleamine dioxygenase 1, and cysteine dioxygenase 1, all of which can cause immunosuppression through amino acid depletion. Identification of the cell source of these enzymes at the site of infection (spleen) will be an important step in understanding the pathogenesis of active disease. Blood will be collected by venipuncture, and stool will be collected with leak proof sterile containers. Buffy coat will be isolated and plasma will be separated. The buffy coat will be used for multiparameter flow cytometry, immunofluorescent staining and isolation of RNA. Smears from splenic and bone marrow aspirate will be used for confocal immunofluorescent analysis. DNA extracted from stools will be used to measure dysbiosis and how this influences the pathogenesis and identify factors responsible for immunosuppression observed in VL patients.

Report Back – Skills Gained

Visit to Dr Peter C Melby’s Laboratory at the University of Texas Medical Branch (UTMB), USA.

The aim of my trip to UTMB is two-fold. At one hand, I took hands-on training on different techniques including immunofluorescence staining and microscopy, multi-parameter flow cytometer staining, acquiring and analysis of results. On the other hand, I have done optimization of protocols. For instance, in my PhD study, leftover splenic aspirate sample will be used and processed for immunofluorescence microscopy. However, due to the low volume and cell number of the splenic sample, I have tried to check if cell block works and allows us to do immunofluorescence microscopy from sections as many times as necessary. Moreover, immunohistochemistry (IHC) protocols require microwave oven for antigen retrieval. However, there is no microwave in the field where I collect and process samples. In this regard, testing whether boiling in a water bath replaces the microwave antigen retrieval was necessary.

African Supervisor

Tamrat Abebe

Tamrat Abebe

African Host Country: Ethiopia

Institution: Department of Microbiology, Immunology and Parasitology (DMIP), College of Health Sciences, Addis Ababa University

Laboratory: Molecular and Cell & Tissue culture laboratory

Department of Microbiology, Immunology and Parasitology (DMIP) owns one undergraduate and four post graduate laboratories. The Undergraduate laboratory mainly supports undergraduate student’s practical demonstration. There are four post graduate laboratories, namely, Molecular lab, Cell & Tissue culture lab, Microbiology lab and Parasitology lab. Moreover, there is a core laboratory commonly used by all departments in the the college. The core lab is where the real-time PCR is placed.

Some of the major equipment’s in these laboratories include Class I &II Bio safety cabinets, Fume hoods, MX3000P real-time PCR, 3500XL genetic analyzer, Shaking incubator 3000, Polaroid MP-4 land camera, Electrolyte analyzer, Universal 320R centrifuge, Deep Freezer (-80), 2470 Automatic gamma counter, Hot plate stirrer, Vortex mixer, Laboratory carts stainless steel, Low temperature incubator, Binocular phase contrast microscope, Ultrasonic homogenizer with temperature control, Liquid nitrogen Dewar, LOCATOR8 with Trolley, Eppendrof variable speed micro centrifuge (5417c), Pneumatic-lift clean chairs and Centrifuge bench, Micro Tissue Grinder W/Accessories, Harvard trip balance, High Current DC-power Supply, Ultra violet-visible scanning spectrophotometers, Dry ice machine, hybridization incubator(Grant), pH meter, Membrane-vacuum pump, Analytical Balance, Top Loading Balance, Ice maker , Vertical Cell, Ultraviolet fluorescent table and Pneumatic-lift clean chairs, MFMBRAN-VAKUUM PUMPE, Nap CO2 incubator, ELISA reader, Inverted microscope, Microplate washers and Water bath with accessories.

Therefore, plasma biomarker evaluation, RNA isolation from the peripheral blood leukocyte buffy coat and DNA isolation from fecal samples will be done in these laboratories.

International Supervisor

Peter C Melby

Peter C Melby

International Host Country: United States of America

Institution: University of Texas Medical Branch (UTMB), Galveston, Texas, USA

Laboratory: Department of Internal Medicine, Division of Infectious Disease

Website: www.utmb.edu

Dr. Melby’s lab has extensive experience in working with reservoirs, animal models and patients with cutaneous and visceral leishmaniasis. The major facilities of laboratories in UTMB center for Tropical disease include (1) Mass spectrometry core: provides MALDI-TOF/TOF MS/MS analysis, including liquid chromatography (LC/MS/MS), Protein ID analysis, post-translational modification identifications and stable isotope methods. (2) Proteomics Bioinformatics Core: provides nonlinear 2D gel analysis, genologics LIMS for data/sample management and access and database searching. (3) Separations Technology Core: provides HPLC analysis, 1D and 2D gel electrophoresis, fluorescence gel staining and imaging and robotic gel spot picking and processing for MS analysis. (4) Flow cytometry facility: equipped with one high-speed cell sorter as well as three analyzers suitable for various tasks and levels of user friendliness. The BD LSRFortessa and FacsCanto are advanced analyzers that can detect 18 and 6 colors, respectively. The Fortessa offers automatic sample loading with similar convenience features. (5) Tissue Culture Core Facility: provides products for tissue culture, molecular biology and immunology research. (6) Molecular Genomics Core: provides services in the area of genomic analysis. It also provides gene expression analysis through the use of Affymetrix gene chips and real time arrays, SNP analysis, RTPCR, miRNA analysis, DNA sequence analysis and high resolution melt analysis. (7) Recombinant DNA lab: supports recombinant DNA techniques including the production of plasmid DNA, transformation and screening of recombinant plasmids, bacterial expression, site directed mutagenesis, construction of transgenic and null gene plasmids and various DNA based technologies.