2.1 Giardia antigen preparation
2.1.1 Origin of Giardia antigens
In October 2013, Giardia antigens were sent to the Department of Clinical Science, University of Bergen, Bergen, Norway from the Department of cell and molecular biology, Uppsala University, Uppsala, Sweden. The laboratory work regarding growing trophozoites, harvesting and acquiring of the proteins in the sonicated lysates was done in Uppsala, Sweden.
2.1.2 Harvesting, lysation and sonication of Giardia trophozoites
Giardia assemblage A (WB-C6, ATTC 50803) and B (GS/M, ATTC 50581) trophozoites were grown in separate Diamond- and Keister medium (TYDK medium) supplemented with bile, supporting the methods of Keister [68], at a temperature of 37 °C.
The trophozoites were collected from a 50 mL falcon tube with an 80 % confluence (approximately 5x106 cells) and washed 3 times in cold sterile PBS.
The cells were harvested at 4 °C using centrifugation at 2500 rounds per minute (RPM) for 5 minutes and re-suspended in 5 mL sterile PBS. The re-suspended cells were snap-freezed-thawed in liquid nitrogen twice and sonicated (3 times for 30 seconds at 50 Watts). Membrane and cell debris were removed by centrifugation at 4 °C at 13000 RPM for 15 minutes. The supernatants containing Giardia soluble protein fractions were sent on dry ice to the Department of Clinical Science, University of Bergen, Bergen, Norway and stored in at – 70
°C until further investigation.
2.1.3 Concentrations of the Giardia soluble proteins
The protein concentrations were measured in the received Giardia soluble proteins solutions.
Measurement was done using the DIRECT DETECT™ system (EMD Millipore corporation, Billerica, MA, USA). The Giardia protein solutions were then diluted to 50 µg/mL in X-vivo medium and stored at -20°C1.
These Giardia soluble proteins, named SSA for Giardia assemblage A and SSB for Giardia assemblage B, were later used to stimulate peripheral blood mononuclear cells (PBMC) in order to elicit Giardia-specific T cell responses. The concentrations used for PBMC
1 This work was done by lab technician Steinar Sørnes
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Table 2.1: Concentrations of the two Giardia isolates used in the project and final concentrations used in stimulation culture. The measured concentrations of the received solutions of Giardia proteins, the concentration of the stock solutions and the final concentration used to stimulate PBMCs. The sonicated supernatant proteins were used to stimulate cells on day one and day six.
Giardia
2.2 Reagents for positive and negative controls
Reagents used as positive controls were chosen depending on their stimulation capacity.
Lipopolysaccharide (LPS) is a powerful macrophage activator, and macrophages can activate T cells [1]. Staphylococcal enterotoxin B (SEB) is classified as a superantigen and can stimulate more T cells than conventional antigens. SEB has a capacity to stimulate naïve CD4+ cells into proliferation [1]. Purified protein derivative (PPD) was used, as this antigen can stimulate T cells of previously vaccinated individuals [1]. Phorbol 12-myristate 13-acetate (PMA) and Ionomycin calcium salt (IC) were used in combination. PMA can diffuse directly through T cell membranes and activate cells without MHC presentation of antigens.
IC is a reagent triggering calcium release and works synergistically with PMA [69].
2 Mean of two protein concentration measurements
3 Kindly provided by Staffan Svärd and his group in Uppsala.
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2.2.1 Reagents used for positive controls
Positive controls were used in every experiment to ascertain cell responses. Table 2.2 shows the reagents used for positive controls in the project.
Table 2.2: Concentrations of reagents for the PBMCs stimulation and which assay they were used.
Name of antigen reagents Stock concentration
4 Sigma Aldrich, product number: L9516, 5mg dissolved in 10 mL sterile NaCl and stored at -20°C in aliquots.
5 Sigma-Aldrich, product number: P8139, 1 mg, diluted in 1 mL DMSO and stored at -20°C in aliquots.
6 Sigma-Aldrich. Product number I0634, 1 mg, diluted in 1 mL DMSO and stored at -20°C in aliquots.
7 Statens Serum Institut, Copenhagen, Merida number: 3704627. 1mg/mL 1 mL test tubes stored at 4-8°C.
8 Kindly provided by Ida Wergeland, originally from Sigma-Aldrich, concentration 500 µg/mL stored at 4-8°C
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2.2.2 Negative control
Table 2.3: Cells in medium without stimulating agents was used as a negative control in this project
Name of medium Application day Supplier
X-vivo 15 with Gentamicin and Phenol red (MED)
Day 1 and day 6 Lonza via BioNordika
2.3 Solutions made or diluted in the laboratory
Table 2.4: Solutions made or diluted for this project
Solution Ingredients and storage L with Milli-Q water. Stored at 2-8°C
Paraformaldehyde 2 % (w/v) in PBS. (PFA).
Filtered before usage.
2 g paraformaldehyde (Sigma-Aldrich P-6148) was added to every 100 mL PBS and heated to 65 °C until dissolved. Fresh solution was made every 2 weeks. Stored at 2-8 °C
Perm/Wash 1:10 dilution (PW).
Filtered before usage.
1 mL 10x Perm/Wash (BD Biosciences Franklin Lakes, New Jersey, USA) was added to every 9 mL milli-Q water making a solution of 10 % Perm/Wash. Stored at 4 °C. Throughout the project, a fresh made solution was made for every new experiment.
Brefeldin 5 mg/mL
5 mg Brefeldin (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 1 mL Dimethyl sulfoxide (DMSO). Stored in aliquots at
-20 °C.
Serum 10 % in PBS
550 µL of Pooled human serum, drawn the 17.04.2008 (Infectious laboratory, Haukeland University hospital, Bergen, Norway), was diluted in 4950 µL PBS and stored at 4-8 °C.
Fresh solution was made for every new experiment.
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2.4 Kits used in the project
Table 2.5: Purchased kits used in this project
Kit name Supplier Catalog nr
LIVE/DEAD Fixable Near-IR Dead cell Stain Kit
(Dye coupled to APC-H7) Life Technologies L10119
Anti-Mouse Ig, κ/Negative Control (FBS)
Compensation Particles Set BD Biosciences 552843
Anti-Rat and Anti-Hamster Ig κ/Negative Control
Compensation Particles Set BD Biosciences 552845
BD Cytofix/Cytoperm™
Fixation/Permeabilization Kit BD Biosciences 554714
CellTrace™ Violet Cell Proliferation Kit (Dye
coupled to the fluorochrome Pacific-Blue) Life Technologies C34557
2.5 Equipment for cell harvesting and culturing
Table 2.6 Tubes and plates used for cell harvesting and stimulation
Equipment Supplier
BD Vacutainer CPT Na-Heparin 8 mL BD Biosciences
Centrifuge tube 15 mL Polypropylene Sarstedt
Centrifuge tube 50 mL Polypropylene Sarstedt
Tissue Culture 96-well Vee bottom (96 V-well plate) Sarstedt
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2.6 Fluorochrome-conjugated antibodies (FABs)
Table 2.7: Overview of the FABs used in this project.
All the antibodies and dyes used in this project were directly coupled to a fluorochrome.
Antibody Clone Fluorochrome Isotype Concentration Supplier Catalog nr.
Two groups of people were recruited in this experiment in order to evaluate differences between a Giardia exposed group and a control group. All the individuals in the study had previously received BCG vaccine against tuberculosis.
2.7.1 Giardia exposed group
Fifteen consecutively identified adults with recent (last 26 months) symptomatic chronic or acute giardiasis were eligible for inclusion. The majority of these individuals were returning travelers. The infection was laboratory confirmed by routine light microscopy. Participants in the Giardia exposed group were given study IDs starting with Ag.
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2.7.2 Low risk healthy controls
Eleven age and sex matched controls with a low risk of ever having had giardiasis were eligible for inclusion. A low risk healthy control was defined as never having travelled to highly endemic areas (low and middle income countries), not drinking contaminated water in Bergen the Autumn 2004, or known previous giardiasis and having no relatives with known giardiasis in the past.
Participants in the low risk healthy control group were given study IDs starting with LR.
2.7.3 Exclusion criteria for both groups
Exclusion criteria for all groups were age below 18 or above 70, known immunosuppression or ongoing treatment with immunosuppressive medication and autoimmune diseases.
2.8 Instruments and incubator
2.8.1 Flow cytometer
BD LSR Fortessa™ Cell Analyzer (BD BioSciences, Franklin lakes, New Jersey, USA) was used to gather fluorescence properties of cells.
2.8.2 Cell counting
MUSE™ Cell Analyzer (Millipore Corporation, Billerica, USA) was used to count cell concentrations and to assess viability of freshly acquired PBMCs before culture stimulation.
2.8.3 Centrifuge
The centrifuge used for both tubes and plates was a Centrifuge 5810 R (Eppendorf, Hamburg, Germany).
2.8.4 Eppendorf centrifuge
The centrifuge used for spinning down aggregates in FAB mixes was a Centrifuge 5417 C (Eppendorf, Hamburg, Germany).
2.8.5 CO2 incubator
For the stimulation of PBMCs with antigens, a CO2 incubator model MCO-15AC (Sanyo Electric Co., Ltd, Moriguchi, Osaka, Japan) was used.
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2.9 Computer software
2.9.1 Word
The project was written and edited using Word 2013 (Microsoft corporation, Redmond, Washington, USA).
2.9.2 Excel
Graphs and histograms were made using Excel 2013 (Microsoft corporation, Redmond, Washington, USA).
2.9.3 Flow cytometer software
For acquiring data from cell samples, BD Facsdiva version 8 (BD Biosciences, Franklin Lakes, New Jersey, USA) was used. The data was collected as FCS-files, and was transferred to other computers for further investigations.
2.9.4 Flow cytometric analysis program
Analysis of FCS-file data from BD Facsdiva was done in FlowJo version X10 (Tree star Inc., Ashland, Oregon,USA).
2.9.5 Statistical analysis program
To assess statistical significance of comparisons between the groups and responses, Mann-Whitney U non-parametric test, linear regression using Pearson’s correlation coefficient test, Fisher’s exact test and non-parametric Kruskal Wallis test were applied.
The Statistics software used was IBM SPSS 21 (IBM corp, Armonk, New York, USA).
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