↓37 |
Autoclave SL-216/1 |
WEBECO, Bad Schwartau |
Balance |
Sartorius, Berlin |
Electrophoresis Power supply EPS 3501 XL |
Amersham Biosciences, Freiburg |
Freezers |
New Brunswick Scientific GmbH, Nürtingen |
Glas material |
Schott, Mainz |
ZIEGRA Chip-ice automat |
Ziegra Eismachinen GmbH, Isernhagen |
Pasteur pipettes |
Sarstedt, Nümbrecht |
pH-Meter PH 211 |
Hanna Instruments, Padova, Italy |
Refrigerators |
Liebherr, Ochsenhausen |
↓38 |
15 ml Tubes BD FalconTM |
BD Labware, Franklin Lakes, USA |
50 ml Tubes BD Falcon TM |
BD Labware, Franklin Lakes, USA |
Cell culture flasks BD FalconTM |
BD Labware, Franklin Lakes, USA |
Cell culture plates BD FalconTM |
BD Labware, Franklin Lakes, USA |
Cell strainers (100 µm pores) BD FalconTM |
BD Biosciences Discovery Labware, |
Centrifuge Sigma 3K15 |
Sigma Laborzentrifugen GmbH, |
Cryo TubeTM Vials |
Nunc, Roskilde, Denmark |
Descosept |
Dr. Schumacher GmbH, Melsungen |
Filters 0,2 µm |
Schleicher & Schuelll, Dassel |
Filters 0,45 µm |
Schleicher & Schuelll, Dassel |
Humified incubator |
Heraeus, Berlin |
Laminair HBB2448 |
Heraeus, Berlin |
Light microscope Olympus CK2 |
Olympus, Hamburg |
Neubauer counting camera |
Fein-Optik, Blankenburg |
Nitrogen tank MVE Cryosystem 2000 |
CHART/MVE, Burnsville, USA |
S1 Laminar Flow Nuaire |
Sarstedt, Nümbrecht |
Sterile Pipetes (5,10 and 15 ml) BD FalconTM |
BD Labware, Franklin Lakes, USA |
Vacuum Filtration Unit |
Schleicher & Schuelll, Dassel |
Vivaspin 20 |
Vivascience, Hannover |
Biometra Fastblot |
Biometra, Göttingen |
Biometra Minigel |
Biometra, Göttingen |
Chromatography paper Whatman® |
Whatman International Ltd, Maidstone, |
ECLTM detection agents |
Amersham Biosciences, Buckinghamshires, England |
HypercassetteTM |
Amersham Biosciences, Buckinghamshires, England |
Kodak BioMax MR film |
Kodak, Cedex, France |
Mini Trans-Blot® cell |
Bio-Rad Laboratories, Hercules, USA |
Mini-PROTEAN® 3 electrophoresis cell |
Bio-Rad Laboratories, Hercules, USA |
Power supply |
Amersham Biosciences, Freiburg |
Protec 45 Compact |
PMA Bode GmbH, Hamburg |
Trans-Blot® Transfer Medium |
Bio-Rad Laboratories, Hercules, USA |
5 ml Polystyrene round-bottom tubes |
BD Falcon, Erembodegem, Belgium |
Cell strainer, 100 μm Nylon |
BD Falcon, Erembodegem, Belgium |
BD Plastipak, 1 ml Syringe |
BD Falcon, Erembodegem, Belgium |
FACS Calibur |
Becton Dickinson, Heidelberg, Germany |
↓39 |
ABI PRISM 7700 sequence detection system |
Applied Biosystems, Darmstadt |
Agilent 2100 bioanalyzer |
Agilent Technologies, Waldbronn |
Filter tips RNase/DNase free 100 (100 μl) |
Greinder BIO-ONE GmbH, Frickenhausen |
Filter tips RNase/DNase free 1000 (G) (1000 μl) |
Greinder BIO-ONE GmbH, Frickenhausen |
Filter Tips RNase/DNase free 10E (10 μl) |
Greinder BIO-ONE GmbH, Frickenhausen |
HE 99X Max submarine unit |
Amersham Biosciences, San Francisco, USA |
HP 845x UV-Visible system software |
Hewlett Packard, Palo Alto, USA |
HP spectrophotometer 8453 |
Hewlett Packard, Palo Alto, USA |
iCycler iQ Real-Time PCR Detection System |
Bio-Rad, Munich |
Mastercycler personal |
Eppendorf, Hamburg |
Precision cells made of Quartz SUPRASIL® |
Hellma, Müllheim |
Safe lock tubes (0.5 ml, 1.5 ml, 2.0 ml) |
Eppendorf AG, Hamburg |
Thermo-Fast® 96, non skirted PCR plates |
ABgene, Epsom, UK |
Ultra Clear cap strips |
ABgene, Epsom, UK |
Ultra-Turrax T25 homogenisator |
Janke & Kunkel, IKA Labortechnik, Staufen |
Work station |
ABgene, Epsom, UK |
ABComplex/HRP |
DakoCytomation, Glostrup, Denmark |
Accustain® Eosin Y solution aqueous |
Sigma-Aldrich Chemie GmbH, Steinheim |
AEC+ substrate |
DakoCytomation, Glostrup, Denmark |
Aquatex® |
Merck, Darmstadt |
Cover glasses |
Menzel-Glaser, Braunschweig |
Cryostat HM 500 OM |
Microm, Walldorf |
Jung tissue freezing medium® |
Leica Microsystems Nussloch GmbH, |
Light microscope Olympus BX40 |
Olympus, Hamburg |
Microscope slides |
R. Langenbrinck, Teningen |
Microtome HM 400 |
Microm, Walldorf |
Roti®-Histokitt |
Roth, Karlsruhe |
VECTASHIELD® Mounting medium for fluorescence with DAPI |
Vector Laboratories, Burlingame, USA |
Leica MZ75 high performance |
Leica Microsystems GmbH, Wetzlar |
Sterile Petri dishes (35 mm) |
BD Biosciences, Edembodegem, Belgium |
Sterile Petri dishes (60 mm) |
BD Biosciences, Edembodegem, Belgium |
↓40 |
1,4-Dithio-DL-threitol (DTT) |
Fluka Chemie GmbH, Buchs |
30% Acrylamid/Bis solution |
Bio-Rad, Munich |
3-aminopropyltriethoxysilane |
Sigma-Aldrich Chemie, Steinheim |
[3-[3-Cholamidopropyl)dimethyl | |
ammonio]-1-propansulfonate] (CHAPS) |
Fluka Chemie GmBH, Buchs |
Agarose 1000 |
Invitrogen Ltd., Paisley, UK |
Ammonium persulfate |
Bio-Rad Laboratories, Hercules, USA |
Ampicilin |
Merck, Darmstadt |
AmpliTaq DNA-Polymerase, Buffer + MgCl2 |
Applied Biosystems, PE, Rodgau-Jügesheim |
Aqua ad injectabilia |
Delta Pharma, Pfüllingen |
Boric Acid |
Sigma-Aldrich Chemie GmbH, Steinheim |
Bovine serum albumin, Fraktion V (BSA) |
Fluka, Buchs, Switzerland |
Bromophenol blue |
Sigma-Aldrich Chemie, Steinheim |
Calcium Chloride |
Merck, Darmstadt |
Carboxyfluorescein diacetate succinimidyl ester |
Molecular Probes, Leiden, |
(CFDA-SE) |
The Netherlands |
Chloroform |
Sigma-Aldrich Chemie GmbH, Steinheim |
Citric acid monohydrate |
Merck, Darmstadt |
Cobalt protoporphyrin chloride (CoPPIX) |
Sigma, Steinheim |
Dimethyl sulfoxide (DMSO) |
Sigma-Aldrich Chemie GmbH, Steinheim |
Di-sodium hydrogen phosphate heptahydrate |
Merck, Darmstadt |
DMEM |
Lifetechnologies, Karlsruhe |
DNase I RNase free |
Stratagene, Amsterdam, The Netherlands |
DNase RNase free |
Ambion, Huntingdon, UK |
dNTPs |
Amersham Pharmacia, Uppsala, Sweden |
Dulbecco´s Phosphate Buffer Solution (PBS) |
PAA Laboratories, Pasching |
Ethanol absolut puriss. p.a. |
Riedel-de Haёn, Seelze |
Ethanol |
Herbeta Arzneimittel, Berlin |
Ethidiumbromide |
Carl Roth GmbH, Karlsruhe |
Ethyldiamintetracetic acid (EDTA) |
Calbiochem, Darmstadt |
Fetal bovine serum (FBS) |
Biochrom, Berlin |
Fetal bovine serum (FBS) |
Cambrex Bio Science Verviers, Verviers, |
Fetal calf serum (FCS) |
Seromed, Berlin |
FicoLite-M |
Linaris, Bettingen am Main |
Geneticin (G418 Sulfate) |
Gibco Invitrogen, Paisley, UK |
Glycerol |
Serva, Heidelberg |
Glycine |
Serva, Heidelberg |
Guanidine thiocyanate |
Fluka Chemie GmbH, Buchs |
Hematoxylin solution according to Mayer |
Fluka Chemie GmbH, Buchs |
Horse serum |
Gibco Invitrogen, Paisley, UK |
Hydrocloric acid fuming 37% |
Merck, Darmstadt |
Ionomycin |
Sigma-Aldrich Chemie GmbH, Steinheim |
L-Glutamine |
Sigma-Aldrich Chemie GmbH, Steinheim |
Methanol |
JT Baker |
Mineral oil (mouse embryo tested) |
Sigma-Aldrich Chemie GmbH, Steinheim |
N,N,N´,N´-Tetramethyl-ethylene diamine (TEMED) |
Bio-Rad, Hercules, USA |
Oligo DT |
Amersham Pharmacia, Freiburg |
Paraformaldehyd |
Merck, Darmstadt |
PCR-Mastermix |
Eurogentec, Seraim, Belgium |
Penicillin, Streptomycin |
Gibco Invitrogen, Paisley, UK |
Peracetic acid |
Herbeta Arzneimittel, Berlin |
Piruvic acid- sodium salt |
Sigma-Aldrich Chemie GmbH, Steinheim |
Phorbol 12-mystirate 13-acetat (PMA) |
Sigma-Aldrich Chemie GmbH, Steinheim |
Polybrene |
Sigma-Aldrich Chemie GmbH, Steinheim |
Poly-L-lisine |
Gibco Invitrogen, Paisley, UK |
Potasssium dihydrogen phosphate |
Merck, Darmstadt |
Potassium chloride |
Merck, Darmstadt |
Potassium hydrogen carbonate |
Merck, Darmstadt |
Protease inhibitor |
Sigma-Aldrich Chemie GmbH, Steinheim |
Reaction buffer |
Promega, Mannheim |
Reverse transcriptase |
Promega, Mannheim |
RNA-marker |
Ambion, Huntingdon, UK |
RNase inhibitor |
Promega, Mannheim |
Saponin |
Sigma-Aldrich Chemie GmbH, Steinheim |
Skim milk |
Fluka Chemie GmbH, Buchs |
Sodium azide |
Merck, Darmstadt |
Sodium carbonate |
Sigma-Aldrich Chemie GmbH, Steinheim |
Sodium chloride |
Merck,Darmstadt |
Sodium hydrogen carbonate |
Merck, Darmstadt |
Sodiumdodecylsulphate (SDS) |
Sigma-Aldrich Chemie GmbH, Steinheim |
Trizma®-base |
Sigma-Aldrich Chemie GmbH, Steinheim |
TRIzol® reagent |
Invitrogen, Paisley, UK |
Trypan blue |
Biochrom AG, Berlin |
Trypsin-EDTA |
Gibco Invitrogen, Paisley, UK |
Zinc (II) protophorphyrin IX (ZnPPIX) |
Frontier-Scientific, Lancashire, UK |
β-mercaptoethanol |
Sigma-Aldrich Chemie GmbH, Steinheim |
Annexin-PE apoptosis detection kit I |
BD Pharmingen, San Diego, USA |
Avidin/Biotin blocking kit |
Vector Laboratories, Burlingame, USA |
Biorad protein assay |
Bio-Rad Laboratories, Munich |
CD4+ T cell isolation kit |
Miltenyi Biotec, Bergisch Galdbach |
CD4+CD25+ regulatory T cell isolation kit |
Miltenyi Biotec, Bergisch Galdbach |
Caspase-3 assay kit, colorimetric |
Sigma-Aldrich Chemie GmbH, Steinheim |
ChariotTM |
Active Motif, Rixensart, Belgium |
In Situ Cell Death Detection kit, POD |
Roche Diagnostics GmbH, Penzberg |
OptEIATM Mouse TNF-α ELISA set |
BD Pharmingen, San Diego, USA |
OptEIATM Mouse IFN-γ ELISA set |
BD Pharmingen, San Diego, USA |
OptEIATM Mouse IL-4 ELISA set |
BD Pharmingen, San Diego, USA |
OptEIATM Mouse IL-10 ELISA set |
BD Pharmingen, San Diego, USA |
peqGOLD Tissue DNA Mini Kit |
Peqlab Biotechnologie GmbH, Erlangen |
RNase-free DNase Set |
Qiagen, Hilden |
RNeasy Mini Kit |
Qiagen, Hilden |
Strata Prep Total RNA Miniprep kit |
Stratagene, Amsterdam, The Netherlands |
Anti-Bcl-2 (Clone N-19) |
Santa Cruz Biotechnology, Santa Cruz, USA |
Anti-goat Ig, biotinylated |
Vector Laboratories, Burlingame, UK |
Anti-Heme Oxygenase-2 |
Stressgen, Victoria, Canada |
Anti-rabbit Ig, horseradish peroxidase |
Santa Cruz Biotechnology, Santa Cruz, USA |
Anti-VEGF (Clone P-20), goat plyclonal antibody |
Santa Cruz Biotechnology, Santa Cruz, USA |
CD3-e (Clone 48-2b) armenian hamster monoclonal IgG |
Santa Cruz Biotechnology, Santa Cruz, USA |
Goat anti-armenian hamster IgG, biotinylated |
Santa Cruz Biotechnology, Santa Cruz, USA |
Goat anti-rabbit Ig, biotinylated |
Dako, Hamburg |
pAb to HO-1 (made in rabbit) |
Alexis, San Diego, USA |
↓41 |
Alexa Fluor® 647-Conjugated anti-mouseCD4 |
BD Pharmingen, San Diego, CA, USA |
(L3T4) monoclonal antibody (Clone RM4-5) | |
FITC anti-mouse CD4 (Clone GK1.5) |
BD Pharmingen, San Diego, CA, USA |
PE-Cy5 anti-mouse CD8a (Ly-2) (Clone 53-6.7) |
BD Pharmingen, San Diego, CA, USA |
PE anti-mouse CD25(Clone PC61) |
BD Pharmingen, San Diego, CA, USA |
PE anti-mouse IFN-γ |
BD Pharmingen, San Diego, CA, USA |
PE anti-mouse IL-10 |
BD Pharmingen, San Diego, CA, USA |
PE anti-mouse IL-4 |
BD Pharmingen, San Diego, CA, USA |
PE anti-mouse TNF-α |
BD Pharmingen, San Diego, CA, USA |
PE-Cy7-conjugated hamster anti-mouse CD69 |
BD Pharmingen, San Diego, CA, USA |
Monoclonal antibody (Clone H1.2F3) | |
PE-Cy7-conjugated hamster anti-mouse CD95 |
BD Pharmingen, San Diego, CA, USA |
(Fas) monoclonal antibody (Clone Jo2) | |
PerCP anti-mouse CD3e (145-2C11) |
BD Pharmingen, San Diego, CA, USA |
Anti-mouse CD3ε NA/LE (Clone 145-2C11) |
BD Pharmingen, San Diego, CA, USA |
Anti-mouse CD28 NA/LE (Clone 37.51) |
BD Pharmingen, San Diego, CA, USA |
Anti-mouse IFN-γ NA/LE (Clone XMG1.2) |
BD Pharmingen, San Diego, CA, USA |
Anti-mouse IL-12 (p40/p70) (Clone C17.8) |
BD Pharmingen, San Diego, CA, USA |
Anti-β-actin |
Delta Biolabs, Gilroy, USA |
Anti-Heme Oxygenase-1 |
Stressgen, Victoria, Canada |
Anti-GAPDH |
Santa Cruz Biotechnology, Santa Cruz, USA |
Anti-goat IgG, biotinylated |
Vector Laboratories, Burlingame, UK |
Anti-rabbit IgG, biotinylated |
Vector Laboratories, Burlingame, UK |
Anti-rabbit IgG, HRP conjugated |
Santa Cruz Biotechnology, Santa Cruz, USA |
Rabbit anti-prolactin-like protein A |
Chemicon International, Temecula, USA |
↓42 |
5-(and-6)-CFDA-SE |
Molecular Probes, Leiden, Holland |
293 cells |
American Type Culture Collection (ATCC) |
GP+E 86 packaging cell line |
Dr. A. Flügel, MPI für Neurobiologie, |
NIH 3T3, mouse fibroblasts |
DSMZ, Braunschweig |
Rcho-1 trophoblast cell line |
Dr. Michael Soares, Department of |
PLXSN Amp R , Neo R , pBR322ori, Ψ + ,PSV40, 5/3-LTR-Promoter |
Clontech |
↓43 |
Human chorionic gonadotropin (hCG) |
Sigma-Aldrich Chemie GmbH, Steinheim |
Pregnant mare serum gonadotropin (PMSG) |
Sigma-Aldrich Chemie GmbH, Steinheim |
BALB/c males |
Harlan Winkelmann, Borchen or |
CBA/J females |
Charles River, Les Oncins, France |
DBA/2J males |
Charles River, Boston, USA |
Hmox-1 +/+ , Hmox-1 +/- and Hmox-1 -/- females and males were originally generated by Dr. Yet, Harvard Medical School, Boston, USA. The colony of mice used in this work were maintained at the Instituto Gulbenkian de Ciencia, Oeiras, Portugal, at the group of Prof. Miguel Soares. After a MTA agreement with Dr. Yet, animals were kindly provided by Prof. Soares and the colony is being currently maintained in our group.
↓44 |
DMEM |
Invitrogen, Gibco, Karlsruhe |
HBSS |
Invitrogen, Gibco, Karlsruhe |
RPMI with L-Glutamin |
Invitrogen, Gibco, Karlsruhe |
Embryomax Human® Tubal Fluid (HTF) |
Millipore Corporation, Millerica, MA, USA |
M2 Media |
Sigma-Aldrich Chemie GmbH, Steinheim |
Packaging cell line media:
DMEM with |
1 mM |
Natriumpiruvat |
|
2 mM |
L-Glutamin |
||
100 U/ml |
Penicillin |
||
100 µg/ml |
Streptomycin |
||
10% |
Fetal Calf serum |
||
+/- |
1 mg/ml |
G-418 |
|
2 x HBSP Buffer: |
50 mM |
Hepes |
|
10 mM |
KCl |
||
12 mM |
Dextrose |
||
280 mM |
NaCl |
||
1,5 mM |
Na2PO4 x 2 H2O |
↓45 |
T-cell media:
RPMI with |
2,05 mM |
L-Glutamin |
50 μM |
β-mercaptoethanol |
|
1 mM |
Na-Piruvate |
|
100 μg/ml |
Penicillin |
|
100 U/ml |
Streptomycin |
|
10% (v/v) |
Fetal Bovine Serum (Cambrex) |
Rcho-1 proliferative cell media:
↓46 |
RPMI with |
2,05 mM |
L-Glutamin |
50 μM |
β-mercaptoethanol |
|
1 mM |
Na-Piruvate |
|
100 μg/ml |
Penicillin |
|
100 U/ml |
Streptomycin |
|
20% (v/v) |
Fetal Bovine Serum (Cambrex) |
Rcho-1 differentiation cell media:
RPMI with |
2,05 mM |
L-Glutamin |
50 μM |
β-mercaptoethanol |
|
1 mM |
Na-Piruvate |
|
100 μg/ml |
Penicillin |
|
100 U/ml |
Streptomycin |
|
10% (v/v) |
Horse serum |
↓47 |
Solutions for flow cytometry
FACS Buffer: |
1% |
BSA |
0.1% |
NaN3 |
|
in PBS | ||
Saponin solution: |
0.1% Saponin | |
in PBS | ||
Lysis buffer |
1.5 M |
NH4Cl |
10 mM |
KHCO3 |
|
100 mM |
EDTA |
|
in distilled water |
Solutions for magnetic cell isolation
↓48 |
MACS Buffer |
0.5% BSA |
2mM EDTA |
|
in PBS, pH 7,4 |
Solutions for molecular biology
Solution D: |
3.676 g |
(tri)-Nacitrat-Dihydrat |
236.32 g |
Guanidine-Isothiocyanate |
|
2.5 g |
N-Lauroylsarcosin |
|
ad 500 ml |
DEPC-H2O, pH 7,0 |
|
Loading buffer |
34 mg |
Bromophenol blue |
3.4 ml |
Ficolite-M (δ=1,091) |
|
10.2 ml |
milli Q water |
|
5x TBE |
27 g |
Trizma®-base |
13.75 g |
Boric acid |
|
10 ml |
EDTA 0,5 M pH 8 |
↓49 |
Solutions for immunohistochemistry
Citrate buffer |
1.8 mM |
Citric acid |
8.2 mM |
Sodium citrate |
Solutions for SDS-PAGE and Western Blot
↓50 |
Transfer buffer pH 8,3 |
25 mM |
Tris-Base |
(for semi-dry blot) |
150 mM |
Glicine |
10% |
Methanol |
|
in distilled water | ||
Transfer buffer pH 8,3 |
25 mM |
Tris-Base |
(for wet blot) |
150 mM |
Glicine |
20% |
Methanol |
|
in distilled water | ||
Running buffer |
25 mM |
Tris-Base |
192 mM |
Glycine |
|
0,1% |
SDS |
|
in distilled water | ||
4x sample buffer |
1 ml |
0,5 Tris/HCl pH 6,8 |
1,6 ml |
10% SDS |
|
3,7 ml |
Ficolite-M (=1,091) |
|
0,4 ml |
-mercapthoethanol |
|
0,2 ml |
bromophenol blue (1% in ethanol) |
|
1,2 ml |
dist. H2O |
|
10x TBS |
200 mM |
Tris-base |
80 g/L |
NaCl |
|
10% |
SDS |
|
pH 7,6 in distilled water | ||
Stacking gel (5%) |
1.3 ml |
29% acrylamide, 1% bisacrylamide |
2.5 ml |
0,5 M Tris/HCl pH 6,8 |
|
0.1 ml |
10 % SDS |
|
6.1 ml |
dist. H2O |
|
0.01 ml |
TEMED |
|
0.05 ml |
10% ammonium persulphate sol. |
|
Running gel (10%) |
3.3 ml |
29% acrylamide, 1% bisacrylamide |
2.5 ml |
1.5 M Tris/HCl pH 8,8 |
|
0.1 ml |
10 % SDS |
|
4.1 ml |
dist. H2O |
|
0.005 ml |
TEMED |
|
0.05 ml |
10% ammonium persulphate sol. |
An adenovirus coding for HO-1 and GFP (AdHO-1/GFP) was constructed using the pAdEasy and pAdTrack-CMV system in 293 cells. AdHO-1/GFP contains two expression cassettes, one for the HO-1 and the other for the GFP, both with the human cytomegalovirus (CMV) promoter, with the human HO-1 cDNA fused to a Flag sequence in its 3´end and a polyA sequence. This adenovirus was designed and produced in INSERM (France) and was a kind gift of Dr. Ignacio Anegon. The adenovirus containing EGFP, which was used as a control, was a kind gift of Dr. Michael Willem (Institute of Neurobiology, Max Planck, Martinsried, Germany). The propagation and purification of both recombinant adenoviruses was performed in 911 cells as previously described (Fallaux et al., 1996; Ritter et al., 1999). In brief, 911 cells were infected at a multiplicity of 5-10 and harvested after 36-48 hrs. The virus was released by five freeze-thaw cycles and purified by two CsCl-gradients (Graham and Prevec, 1991). The banded virus was recovered, desalted over sephadex columns (Pharmacia, Erlangen, Germany) and kept in virus storage buffer after addition of 10% Glycerol at –80°C. Titration of the virus concentration after elution from the column was performed by plaque assay on 911 cells. The propagation of the adenoviruses as well as the determination of their viral titer was routinely performed by Heinz Tanzmann, member of the Institute of Medical Immunology.
All animals were maintained in a barrier animal facility with a 12 h light/dark cycle. Animal care and experimental procedures were followed according to institutional guidelines and conformed to the requirement of the state authority for animal research conduct (LaGeSo Nr. 0062/03 and 0070/03, Berlin). The previously described immunological murine model of abortion was used, in which the mating combination CBA/J x DBA/2J represents the abortionprone group, being the combination CBA/J x BALB/c the normal pregnancy control. Two months old CBA/J females were mated with 2-4 months old BALB/c or DBA/2J males, checked twice a day for vaginal plugs and separated from the males if pregnant. The day of vaginal plug detection was considered as day 0 of pregnancy. Pregnant females mated with BALB/c were considered as group (1) and received PBS intraperitoneally (i.p.) (n=15). DBA/2J-mated females were then randomized and divided in the following groups:
↓51 |
2) Abortion-prone group + PBS i.p. (n=11)
3) Abortion-prone group + 1.105 PFU AdHO-1/GFP in PBS i.p. (n=7)
4) Abortion-prone group + 1.105 PFU AdEGFP in PBS i.p. (n=7).
↓52 |
5) Abortion-prone group + 1.108 PFU AdHO-1/GFP in PBS i.p. n=8 )
6) Abortion-prone group + 1.108 PFU AdEGFP (n=7 )
Fig. 15 : Experimental setting. | ||
CBA/J females were mated either with BALB/c males (normal pregnant combination) or with DBA/2J males (abortion-prone combination). The day of the plug detection was considered as day 0 of pregnancy. Injections were made intraperitoneally (i.p.) on day 5 of pregnancy. |
↓53 |
All injections were made i.p. on day 5 of pregnancy, which is known to be in the implantation window (Psychoyos, 1986). A schematic representation of the experimental setting is shown in Fig. 15.
The groups 5 and 6 are not included in the schematic representation, since its data will be shown as a table in the appendix. On day 14 of pregnancy, the females were sacrificed, the uteri removed and the implantation sites were documented. The abortion sites were identified by their small size accompanied by a necrotic, haemorrhagic appearance, compared with normal embryos and placentas.
The percentage of abortion was calculated as the ratio of abortion sites and total implantation sites using the following formula:
↓54 |
From each animal, different organs were taken for further analysis. Spleens were kept in cold RPMI for flow cytometry until further use. Two small portions of decidua were snapfrozen and kept at -80°C for protein and RNA isolation. The rest of the decidua was used for flow cytometry and for that, it was cut in small pieces and kept in HBSS at 4°C until further use. Placentas were snapfrozen and kept at -80° until use (for protein and RNA isolation).
Flow cytometry analysis was performed using decidual and spleen lymphocytes. The isolation of mononuclear cells (MC) from decidua was performed as previously described (Zenclussen et al., 2003; Zenclussen et al., 2004). Briefly, decidual samples were washed with PBS, cut into small pieces, collected in HBSS medium containing 1mM DTT and incubated with agitation for 20 min at 37°C. Thereafter, cells were filtered through a 100 μm net and collected in a fresh tube where they were washed with RPMI medium containing 10% FCS. The procedure was repeated twice, with HBSS without DTT, all supernatants were collected and washed with RPMI medium containing 10% FCS. After that, a Ficolite-M gradient was performed following the instructions of the manufacturer (Linaris, Bettingen am Main, Germany).
↓55 |
Spleen tissues were crushed against a 100 μm net, collected in medium containing 10% FCS and the erythrocytes were lysed with lysis buffer containing NH4Cl, KHCO3 and EDTA for 10 min at RT. After erythrocyte lysis, cells were washed once with RPMI containing 10% FBS.
Flow cytometry is a technique that allows the analysis of different cells in a cell mixed population, as well as to phenotypically characterize these cells based on their size, granularity and cell surface or intracellular expression of different molecules.
This technique is commonly performed by staining the cells with fluorochrome-labeled antibodies that are specific for molecules expressed in their surface or being produced by them. The flow cytometer is an instrument capable of detecting fluorescence on individual cells in a suspension and thereby determining the number of cells expressing the molecule labeled with the specific fluorochrome-labeled antibody. A representative scheme of the principle of flow cytometry is depicted in Fig. 16 (from Abbas and Lichtmann in: Cellular and Molecular Immunology).
↓56 |
Spleen and decidual cells were analyzed in order to determine the systemic (in spleen cells) and local (in decidua cells) immune response after the different treatments.
Extra- and intracellular staining for tissue samples
Isolated spleen or decidual lymphocytes were incubated for 1 hour with 50 ng/ml PMA and 1 μg/ml ionomycin at 37°C with 5% CO2 for stimulation of cytokine secretion. The adition of PMA and the ionophore ionomycin is a powerful activating stimulus acting on protein kinase C and calcium ion influx, and is used both to induce cytokine expression of cells previously activated by physiological stimuli (Pala et al., 2000). After this, 2 μM of monensin, which acts by blocking the transport between the endoplasmic reticulum and the Golgi complex (Luttmann et al. in: The Experimentator Immunologie), was aggregated and incubated for further 3 hours to allow intracellular accumulation of secreted proteins. Cells were further washed and antibodies against surface markers were incubated for 10 min at 4°C in darkness.
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For fixation, paraformaldehyde solution (PFA) at a concentration of 1% (p/v) was used, and cells were incubated overnight (O.N.) at 4°C. After washing the cells, antibodies for the detection of intracellular proteins were incubated 20 min at 4°C in darkness, diluted in saponin 0.1% (p/v), a detergent solution for permeabilization of the cells. After this incubation, cells were washed with saponin solution, in order to remove excess of intracellular antibodies. The labelled cells were finally resuspended in FACS buffer and analyzed in a FACS Calibur (Becton Dickinson) cytometer. The lymphocyte population was gated based on size and granularity and used for further analysis. When only analysing extracellular markers, incubation with PMA, ionomycin and monensin was avoided.
Fig. 16 : Principle of flow cytometry. | ||
Figure modified from Abbas and Lichtmann, 2003. Briefly, cells pass one at a time through a fluorimeter with a laser-generated incident beam and the light that emerges from the sample is analyzed for forward and side scatter (size and granularity, respectively) as well as fluorescent light of two or more wavelengths that depend on the fluorochrome labels attached to the antibodies. The example represented here is based on a staining of two antigenic markers. |
All washing steps were performed using FACS washing buffer (1% bovine albumin serum in PBS). Samples were analyzed in a FACS Calibur (Becton Dickinson, Heidelberg, Germany). Negative controls were performed by using the respective isotype controls, and cells without antibodies were also included as negative controls.
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The extra- and intra-cellular markers analyzed included:
Extra-cellular markers: |
CD3: PerCP anti-mouse CD3e (CD3ε chain), diluted 1:100 |
CD4: detected with FITC anti-mouse CD4 (L3T4), diluted 1:100 |
CD8: detected with PE-Cy5 anti-mouse Cd8a (Ly-2), diluted 1:100 |
CD69: detected with PE-Cy7 labeled anti-mouse CD69 (VEA Antigen) (H1.2F3) |
CD95: detected with PE-Cy7 labeled anti-mouse CD95 (Jo2) |
Intra-cellular markers: |
IL-10: detected with PE-rat anti-mouse IL-10, diluted 1:200 |
IL-4: detected with PE-rat anti-mouse IL-4, diluted 1:200 |
TNF-α: detected with PE labelled anti-mouse TNF-α, diluted 1:200 |
IFN-γ: detected with PE anti-mouse IFN-γ, diluted 1:200 |
Placenta, liver and fetuses from animals receiving adenoviruses were snapfrozen in liquid nitrogen and kept at –80°C until use. DNA extraction from tissue homogenates was performed using the peqGOLD Tissue DNA Mini Kit. For that, livers, fetuses and placentas (30 mg of tissue or less) were grinded in a mortar using a pistil under the influence of liquid nitrogen. Samples were taken up in TL Buffer, and after adding OBTM protease, samples were incubated at 55°C in a shaking water bath for at least three hours. After that, isolation was performed following manufacturer´s instructions, using HiBind® DNA columns for the specific binding of DNA. After two washing steps, samples were eluted with elution buffer and kept at -20°C until further use.
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SYBR Green chemistry is an alternate method to the method using fluorescent probes used to perform real-time PCR analysis. SYBR Green is a dye that binds the Minor Groove of double stranded DNA (Applied Biosystems Manual). When SYBR Green dye binds to double stranded DNA, the intensity of the fluorescent emission increases. As more double stranded amplicons are produced, SYBR Green dye signal will increase. The SYBR Green dye will bind to any double stranded DNA molecule, while the 5′ Nuclease assay (explained later) is specific to a pre-determined target. For this reason, primers designed for this methodology have to be very specific and should not form any dimmers. To analyze whether this occurs, it is necessary to perform a melting curve, which plots the fluorescence as a function of the temperature as the thermal cycler heats through the dissociation temperature of the product. The shape and position of this melting curve are functions of the GC/AT ratio, length and sequence and can be used to differentiate desired and undesired amplification products (Ririe et al., 1997).
In the real time PCR methodology, the reactions are characterized by the time point during cycling when amplification of a PCR product is first detected. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in the fluorescence is observed. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the reporter fluorescence generated by the cleavage of the probe passes a fixed threshold above base line.
Amplifications reactions (13 μl) were performed by mixing 1 μl DNA, 6,25μl Master mix (Stratagene, Heidelberg, Germany) containing PCR Buffer, dNTPs, MgCl2, Ampli-Taq DNA Polymerase and SYBR-Green, 3 μl of the primer mix and 2,75 μl water. PCR reaction was performed as follows: 2 min at 50° C followed by an initial denaturation step of 10 min at 95°C, followed by 15s at 95°C and 1 min at the appropriate annealing temperature (60°C) for 40 cycles. The measurement of adenoviral particles was performed by detecting GFP or viral constructs. All reactions were performed on the ABI Prism 7700 Sequence Detection System (Perkin Elmer Applied Biosystems). As intra-laboratory controls, non-related DNA samples were checked for their viral expression showing negative values. The sequences of the primers used are shown in Table 1.
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Table 1 : Sequences of the primers used for GFP detection by real time PCR
Primer sequence (5´ 3´) |
|
Apob |
fw: CGTGGGCTCCAGCATTCTA |
rev: TCACCAGTCATTTCTGCCTTTG |
|
GFP |
fw: TGCAGTGCTTCAGCCGCTA |
rev: AAGATGGTGCGCTCCTGGA |
The primers for GFP were used at a concentration of 900 nM for forward and 900 nM for reverse primers. The primers for Apob were used at a concentration of 300 nM for forward and 300 nM for reverse primers. Optimal primer concentrations were determined by titration of the primers with DNA samples from a GFP+ animal (titration done by Nadja Ahmad during her Diploma Thesis in our laboratory), where the expression of GFP was linked to Apo-b expression.
Solutions of APES are normally used to treat glass slides. The silane reacts with the –OH group of glass, covalently linking aminoalkyl groups to the glass. The resultant surface acts “sticky”, promoting the binding of the tissue to the glass. For the treatment of the glass slides, they were soaked in a solution of 2% APES in methanol and washed twice with distilled water. Slides were dried completely in the oven and were then ready to use for paraffin as well for cryosections.
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In order to detect the expression of the green fluorescent protein by fluorescence microscopy, placental tissues as well as placental tissue bound to fetuses were cut in cryostat in 8 μm thick samples. After fixing the samples for 10 min with PFA 4%, samples were dried and kept at 20°C until use. For analysis of GFP expression, samples were mounted with DAPI and observed under fluorescence microscope.
Paraffin embedding of the tissues was performed following a well-established protocol (SaintMarie, 1963). For doing that, tissues were fixed in 96° ethanol at 4°C for 1-7 days, following the proportion of 50 ml alcohol/g tissue. After that, following incubations were performed:
Dehydratation: |
-Ethanol 100° at 4°C, 1-2 h, with 4x changes |
-Xylol at 4°C, 1-2 h, with 2 changes |
|
-Xylol at RT, 1-2 h, with 1x change |
|
Inclusion: |
-Paraffin at 56°C, 1-2 h |
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After inclusion in paraffin, tissues were kept at 4°C.
For cutting the tissues, a microtome was used, and all tissues were cut in 5 μm thick specimens and placed in slides previously covered with 3-aminopropyltriethoxysilane. Samples were allowed to dry at 37°C O.N. and were finally kept at R.T.
In order to perform an immunostaining, it is necessary to first remove the paraffin in the cuts. For doing that, the following incubations were performed:
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Dewaxation: |
Xylol, 2 x 20 min |
Fixation/Hydration: |
Ethanol 100°, 10 min |
Ethanol 95°, 10 min |
|
Ethanol 75°, 10 min |
|
Distilled water, 5 min |
In order to analyze the morphology of the tissues (placenta, decidua and resorptions), a staining with Hematoxylin and Eosin, which stain nucleus and cytoplasm of the cells respectively, was performed. For doing this, following incubation steps were performed:
- Distilled water, 5 min |
- Hematoxylin, 2 min |
- Tap water, 5 min |
- Eosin, 10 sec. |
- Ethanol 75°, 10 sec, with agitation |
- Ethanol 95°, 10 sec, with agitation |
- Ethanol 100°, 2 x 10 sec, with agitation |
- Xylol, 2 x 5 min |
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After that, slides were mounted with Roti-Histokitt and analyzed under light microscope.
Immunohistochemistry (IHC) consists in the detection of specific antigens in histologic tissue sections by the use of a system consisting in a specific antibody for the antigen of interest. This enzyme coupled system allows the conversion of a colorless substrate into a colored insoluble substance that precipitates at the site where the antibody an thus the antigen are localized (Abbas and Lichtmann in: Cellular and Molecular Immunology). The specific antibody can be directly coupled to the enzyme, or a secondary antibody recognizing the first specific antibody can be the one coupled to the enzyme. Another possibility, normally used to amplify the signal and thus detect low expressed antigens, is the use of a specific primary antibody, which is recognized by a secondary antibody coupled to biotin. This biotin is then recognized by avidin coupled to an enzyme, normally horse radish peroxidase (HRP). This methodology is normally known as ABC system (Avidin-Biotin-Complex), and was routinely used in the experiments done in the course of this doctoral thesis.
IHC was performed for each molecule of interest on tissue pieces containing placenta and decidua. After dewaxation, the sections were washed with Tris buffered saline solution (TBS, pH = 7.40) for 10 min and treated with 3% hydrogen peroxide in methanol for 30 min at RT to block the endogenous peroxidase activity, which is very high in placental tissue. The tissues were then washed and exposed to 5% BSA in TBS for 20 min at RT for protein blocking, stained with the primary antibody (Ab) diluted in 5% BSA in TBS, and incubated O.N. at 4°C. The tissues were then washed and further stained with the secondary Ab diluted in 5% BSA for 1 h at RT. After washing, the samples were incubated for 30 min with an ABComplex/HRP solution. Finally, the sections were developed with AEC+ Substrate Chromogen, counterstained with Hematoxylin and mounted. Negative controls were obtained by replacing the first antibody with 5% BSA or 10% rabbit serum.
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The dilutions of the primary and secondary antibody used in each case are indicated in Table 2.
Table 2 : Dilutions of the antibodies used for immunohistochemical staining
Molecule of interest |
First ant i body |
Secondary antib o dy |
HO-1 |
1:100 |
1:100 |
HO-2 |
1:500 |
1:100 |
CD3 |
1:20 |
1:100 |
VEGF |
1:100 |
1:200 |
For the analysis of the results, samples were analyzed under a light microscope, by counting the number of positive cells (for VEGF and CD3 staining). In these cases, where only some cells were positive for the markers, the number was determined counting the number of cells per field. Each field consisted in a 0.25 mm2 square coupled to the ocular of the microscope. Using a magnification of 200X (20X objective and 10X ocular magnification), at least 20 representative fields per sample were counted, and the results were expressed for each sample as the mean of cells per mm2. The analysis was performed without knowing the nature of the samples and was confirmed by an independent observer.
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In the case of HO-1 and HO-2 staining, where almost all cells were stained for HO-1 and HO2 expression, the analysis was performed for each cell type independently. The expression levels were set by using scores from 0 (no staining) to 6 (very intense staining) for every cell type without knowing the nature of the samples, and the results were again confirmed by an independent observer.
For protein extraction from tissues, frozen specimens were re-suspended in 500 µl Lysis-buffer containing CHAPS, HEPES and DTT. The buffer used was the one recommended for caspase activity measurement (explained later). The samples were homogenized using a glass homogenizer. After isolation, homogenates were centrifuged for 10 min at 10.000 g, and the supernatant containing the proteins was transferred to a fresh tube. Protein concentration was assessed using the BioRad Protein Assay as indicated by the manufacturer. Protein samples were kept at 80°C and when working with them, they were always kept on ice.
For isolation of proteins from cells, 5 x 105 cells were washed with PBS and resuspended in 50 μl of Lysis Buffer containing Protease Inhibitor and used immediately or kept frozen at 70°C until use. For the preparation of the samples, the cell suspension was centrifuged 5 min at 15000 g at 4°C and the supernatant containing the proteins was further transferred to a fresh tube.
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The caspase-3 colorimetric assay is based on the hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) by caspase-3, resulting in the release of the p-nitroaniline (pNA) moiety by the following reaction:
For doing this assay, placentas were snapfrozen in liquid nitrogen and kept in –80°C until use. For extracting proteins, a lysis buffer containing 5 mM CHAPS, 50 mM HEPES and 5 mM DTT was used. It is very important that the proteins are lysed in a buffer without protease inhibitors, since they can interfere in the reaction, especially cysteine protease inhibitors. The determination of total protein concentration was performed using the Bio-Rad Protein Assay Dye Reagent with a calibration curve performed with bovine serum albumin standards. The determination of the caspase-3 activity was performed in 96-well plates by means of the colorimetric Caspase-3 Assay Kit (Sigma) using 50 μg of total protein/sample and following manufacturer’s instructions. Three controls were included: an inhibitor-treated lysate control for measuring the non-specific hydrolysis of the substrate, a caspase-3 positive control provided with the kit, and a reagent blank. Briefly, the following schema was followed (Table 3):
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Table 3 : Scheme of the procedure for the determination of caspase3 activity.
Cell lysate |
Caspase 3 2 μ g/ml |
1x Assay Buffer |
Ca s pase-3 Inhibitor |
Ca s pase-3 Su b strate |
|
Reagent Blank |
- |
- |
90 μl |
- |
10 μl |
Sample |
5 μl |
- |
85 μl |
- |
10 μl |
Sample + I n hib i tor |
5 μl |
- |
75 μl |
10 μl |
10 μl |
Caspase-3 positive co n trol |
- |
5 μl |
85 μl |
- |
10 μl |
Caspase-3 positive co n trol + Inhib i tor |
- |
5 μl |
75 μl |
10 μl |
10 μl |
The test was performed in 96-well plates. Each setting was made in duplicate for each sample. Plates were incubated O.N. at 37°C, and the optical density (O.D.) was finally measured at 405 nm.
The calibration curve was made using a p-nitroaniline standard provided by the manufacturer. The concentration of the stock solution was determined by measuring the O.D. at 405 nm and calculating it taking into account the εmM of 10.5. In each plate, the calibration curve was done in duplicate in serial 1:2 dilutions ranging from 200 μM until 12.5 μm.
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For calculating the Caspase-3 Activity, the following formula was used:
t= incubation time (min); d= dilution; V= volume of sample in ml μmol pNA was calculated for each well using the pNA calibration curve. |
where:
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The cleavage of genomic DNA during apoptosis may yield double-stranded, low molecular weight DNA fragments as well as single strand breaks, or nicks, in high molecular weight DNA. Those DNA strand breaks can be identified by labelling free 3’-OH termini with modified nucleotides in an enzymatic reaction. The In Situ Cell Death Detection Kit labels DNA strand breaks by Terminal deoxynucleotidyl transferase (TdT), which catalyses the polimerization of labeled nucleotides to free 3’-OH DNA ends in a template-independent manner (TUNEL reaction). The incorporated fluorescein is detected by anti-fluorescein antibody Fab fragments, conjugated with horse-radish peroxidase (POD). After substrate reaction, stained cells can be analyzed under light microscope.
The detection of apoptotic cell death at single cell level was performed in tissues containing placenta and decidua following manufacturer’s instructions. However, a modification of the temperature of incubation was performed. In the current protocol, the TUNEL reaction mixture and the Converter-POD were incubated at RT instead of 37°C. The modification was performed based on the observation of unspecific staining of the cytoplasm of the cells when using the 37°C incubation while establishing the staining. Briefly, paraffin sections were dewaxed and hydrated as usual, followed by a permeabilization of the samples. For that, slides were treated with citrate buffer pH 6 for 5 min at 600 W in a microwave. After cooling of the slides, they were washed twice with PBS. The endogenous peroxidase activity of the samples was blocked with 3% H2O2 in methanol for 30 min at RT. After washing the slides twice with PBS, a protein blocking was performed during 20 min at RT using a buffer containing 3% BSA and 20% SFB in Tris-HCl pH 7.5. The TUNEL reaction mixture was added to the samples, and incubated in a humified chamber for 60 min at RT. For negative controls, label solution instead of TUNEL reaction mixture was added. After the incubation, the samples were washed twice with PBS, and a Converter-POD solution was added. After 30 min of incubation at RT, tissues were washed twice with PBS and developed using the AEC+ Substrate Chromogen, counterstained with Hematoxylin and mounted with Aquatex.
For the quantification of the number of apoptotic cells, stained nuclei were identified and counted per field. Each field consisted in a 0.25 mm2 square coupled to the ocular of the microscope. Using a magnification of 200X (20X objective and 10X ocular magnification), at least 20 representative fields per sample were counted, and the results were expressed for each sample as the mean of cells per mm2. The analysis was performed without knowing the nature of the samples and was confirmed by an independent observer.
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When an electric field is applied to a solution containing protein molecules, these molecules migrate in a direction and at a speed that reflects their size and net charge. This is the basis of the technique called electrophoresis (Alberts et al. in: Essential Cell Biology, 2004). When proteins are mixed with negatively charged molecules of sodium dodecyl sulfate (SDS), the negatively charged SDS-protein complex will migrate through a porous polyacrilamide gel to the anode. When protein samples are treated like this, they will migrate at a rate that reflects only their molecular weight, since they are all be negatively charged by an excess of SDS. A reducing agent as like mercaptoethanol is usually added to break any S-S linkage in or between proteins.
Proteins isolated from tissues or from cells were mixed with a sample buffer containing SDS, βmercaptoethanol and bromophenol blue and heated for 5 min at 95°C in order to allow the complete denaturalisation of the proteins. The same procedure was carried out with the protein marker. Samples were transferred to an acrylamid/polyacrylamid running gel with a 5% stacking gel. Electrophoresis was performed at 150 V until its end.
Blotting is the technique of transferring electrophoretic products onto other materials prior to visualisation. Western Blot, specifically, is the transfer (or blotting) of proteins to a membrane (Acquaah in: Understanding Biotechnology, 2004). The membrane is then sequentially exposed to solutions containing a primary antibody, followed by a secondary antibody to which an enzyme or biotin is coupled. Here, after finishing of the SDSPAGE, proteins were transferred into a nitrocellulose membrane. For doing this, the following schema was followed:
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When using the semi-dry blot method, the protein transfer took place during 30 min at 250 mA. When using the wet blot method, protein transfer took place during 2 h at 180 mA. For verifying the full protein transfer, the nitrocellulose membranes were stained with Ponceau Red for 3 min, and further decoloured using distilled water (dH2O).
The expression of HO-1 was analyzed (in the membrane) by immunoblot as follows:
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The membrane was blocked with 5% milk powder in TBS for 1h at R.T., and further washed with TBST, 3 x 5 min each time. The first antibody (rabbit anti rat-HO-1 polyclonal antibody, diluted 1:2000 in 5% milk in TBS) was applied for 2 h at R.T. After washing with TBST, the samples were incubated with the secondary antibody (anti-rabbit horseradish peroxidase, diluted 1:5000 in 5% milk in TBS) for 1 h at R.T. The membrane was washed again, and the reaction was finally developed by using chemoluminiscence (ECL Assay of Amersham) and exposed onto Kodak Miomax MR Imaging film. The intensity of the bands was quantified by using the Quantity One® Software, Version 4.5.2 from Bio-rad.
One placenta and decidua of each animal were snapfrozen in liquid nitrogen and kept in 80°C until preparation. RNA extraction was performed using Trizol Reagent, which is a monophasic solution of phenol and guanidine isothiocyanate, and represents an improvement to the single step RNA isolation method developed by Chomczynski and Sacchi (1987). Briefly, tissues were homogenized in Trizol using an Ultra-Turrax T25 homogenizer. After adding chloroform and vortexing for 2 min at RT, samples were centrifuged at 10.000 g for 10 min at 4°C. The upper phase obtained after the centrifugation was then transferred to a new tube, and ice-cold ethanol was added. After an incubation of 10 min at 20°C, samples were centrifuged for 10 min at 10.000 g at 4°C. The pellet obtained after this centrifugation was then washed three times with ethanol 80°, in order to remove the rest of Trizol reagent. Between each wash step, cells were centrifuged for 10 min at 10.000 g and 4°C. After the last wash, the pellet was allowed to dry and it then was resuspended with RNase free water. In case that the pellet was not immediately resuspended, samples were kept at 4°C to allow pellet resuspension. RNA concentration was determined by measuring the O.D. at 260 nm and at 280 nm. To calculate the RNA concentration, the following formula was used:
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Samples containing 2 μg of total RNA were placed for 2 min on ice and added with dNTPs (2,5 mM), Amersham Pharmacia), DNase I (2U/μl, Stratagene) and RNase-Inhibitor (40 U/μl) mixed in reaction buffer. The mix was incubated for 30 min at 37°C and further heated to 75°C for 5 min. The addition of the reverse transcriptase (200 U/μl) and RNase-inhibitor in distilled water started the reverse transcription. This reaction mixture was incubated at 42°C for 60 min followed by incubation at 94° C for 5 min.
As negative controls, we performed a so called RT- control, which consists in the same mixture, but avoiding the aggregate of Reverse Transcriptase. This control was done in order to check the samples for contamination with genomic DNA.
Once the cDNA synthesis was completed, the samples were immediately used or kept at 20°C.
↓75 |
The Real-Time TaqManTM PCR is a sensitive, reproducible and specific method for the quantification of mRNA. Together with the specific primers for the gene of interest, an oligonucleotid fluorescence probe is used. The probe is marked at the 5´end with a fluorescent reporter-dye (6-Carboxyfluorescein, FAM) and at the 3´end with a quencher-dye (6-carboxy-tetramethyl-rodhamin, TAMRA). While the probe is intact, the proximity of the quencher reduces the fluorescence emitted by the fluorescent dye. During the PCR, if the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5´-exonuclease activity of Taq polymerase as the primer is extended. Then, the cleavage of the probe separates the reporter dye from the quencher, increasing the reporter dye signal. Additional reporter dye molecules are cleaved with each cycle, which produces an exponential increase in the fluorescence intensity that is proportional to the amount of the amplicon produced (Fig. 17).
In the real time PCR methodology, the reactions are characterized by the time point during cycling when amplification of a PCR product is first detected. The higher the starting copy number of the nucleic acid target, the sooner a significant increase in the fluorescence is observed. The parameter CT (threshold cycle) is defined as the fractional cycle number at which the reporter fluorescence generated by the cleavage of the probe passes a fixed threshold above base line.
Fig. 17 : Principle of the TaqManTM PCR Reaction (Figure taken from the Applied Biosystems´s user manual) | ||
A) The 5´nuclease assay B) Polymerase collides with TaqMan® probe C) Cleavage of the TaqMan® probe |
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The PCR reaction was developed using the ABI PRISMTM 7700 Sequence Detection Systems, which detects the fluorescence intensity.
The primers and probes were designed using the Primer Express Software. The establishment of the primers, their titration and their efficiency determination through standard curves were performed at the Institute of Medical Immunology (Charité, Berlin) by Katrin Vogt, a member of the Institute. The sequences for primers and probes are detailed in Table 4.
For each sample, 1 μl of template was used together with 6 μl primer mix and 1 μl probe. The final volume of 25 μl was reached aggregating 12.5 μl Mastermix and 4.5 μl distilled water.
↓77 |
For the PCR, the following cycle conditions were applied:
50°C |
2:00 min |
95°C |
10:00 min |
95°C |
0:15 min |
60°C |
1:00 min |
40 cycles for each determination were performed.
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Table 4 : Sequences of primers and probes
Primer (5´ 3´) |
Probe (5´ 3´) |
|
β-actin |
Fw: GCTTCTTTGCAGCTCCTTCGTT |
CAGCCTTCCTTCTTGGGTATGGAATCCT |
rev: GTTGTCGACGACCAGCGC |
||
Bag-1 |
fw:GCTAACCACCTGCAAGAATTGAAT |
TTCTGACATCCAGCAGGGTTTTCTGGC |
rev: GTTTGCAGAGAGCCTCCGC |
||
Bcl-2 |
fw: TGAACCGGCATCTGCACA |
AACGGAGGCTGGGATGCCTTTGTG |
rev: CAGAGGTCGCATGCTGGG |
||
Bcl-xl |
Fw: GGTGAGTCGGATTGCAAGTTG |
CCTGAATGACCACCTAGAGCCTTGGATCC |
r:GTAGAGATCCACAAAAGTGTCCCAG |
||
HO-1 |
fw: GGCTACCATGCCAACTTCTGTCT |
CACACAGTACAGCAAGGTCCTTGCCCT |
Rev: CCGGGTTGTGTTGGTTGTAGA |
||
CD3 |
fw:ATTGCGGGACAGGATGGAG |
TCGCCAGTCAAGAGCTTCAGACAACGA |
rev:CTTGGAGATGGCTGTACTGGTCA |
||
FoxP3 |
Fw: CCCAGGAAAGACAGCAACCTT |
ATCCTACCCACTGCTGGCAAATGGAGTC |
Rev: TTCTCACAACCAGGCCACTTG |
||
TGF-β |
fw: GGCTACCATGCCAACTTCTGTCT |
CACACAGTACAGCAAGGTCCTTGCCCT |
Rev: CCGGGTTGTGTTGGTTGTAGA |
A relative quantification of the mRNA expression is possible because of the use of a “house keeping gene”, which is constitutively expressed in all cell types. In all cases, β-actin was used as house keeping gene after confirming its constitutive expression in the analyzed tissues (decidua and placenta).
The determination of the ΔCT value is made using the formula:
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ΔCT= CT (gene of interest) – CT (“house keeping gene”)
Due to the inverse proportional relationship between the Ct and the original gene expression level, and the doubling of the amount of the product with every cycle, the original expression level for each gene of interest is expressed as following:
↓80 |
Spleen and lymph nodes were isolated from CBA/J females. Cells from spleen and lymph nodes were separately isolated. For spleen cells isolation, spleen tissues were crushed against a 100 μm net, collected in medium containing 10% FCS and the erythrocytes were lysed with lysis buffer containing NH4Cl, KHCO3 and EDTA for 10 min at R.T. After centrifugation, cells were washed once with complete medium and finally resuspended in RPMI containing 10% FBS, Na-Piruvate, Penicillin, Streptomycin and β-mercaptoethanol and kept on ice until use. For lymph node cell isolation, lymph nodes were crushed against a 100 μm net, washed with complete medium, and after centrifugation resuspended in RPMI containing 10% FBS, Penicillin, Streptomycin, Na-Piruvate and β-mercaptoethanol.
Different culture conditions were tested: a) stimulation of the cells with irradiated APCs from the male (DBA/2J) or b) polyclonal stimulation with anti-CD3 and anti-CD28. For stimulation with male antigens, spleen and lymph node cells from DBA/2J were irradiated at 30 Gy, and immediately used. The cell amount was always the same as the one from the CBA/J female cells. For polyclonal stimulation, 1μg/ml anti-CD3 and 0.1 μg/ml anti-CD28 were used. In both cases, 10 ng/ml of recombinant murine IL-2 (rmIL-2) were added to the culture (final concentration). Cells were routinely maintained at 37°C in a humified incubator with 5% CO2. When necessary, medium was changed for fresh medium containing rmIL-2 (normally at a 2-3 days interval). After one week in culture, cells were restimulated with either APC or anti-CD3/anti-CD28. The optimal cell amount was tested and 1.5 x 106 cells/ml were finally used.
The effect of antibodies against Th1 phenotype was also tested in our culture at a concentration of 5 μg/ml anti-IFN-γ and 5 μg/ml anti-TNF-α. In another setting, IL-4 was added to the cultures at a 10% of the final volume. IL-4 was obtained as a supernatant of the 3T3 BMG IL4 cell line (kind gift of Prof. Werner Müller, Braunschweig). This was done in order to obtain majority of Th2 cells.
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Two different retroviral vectors expressing HO-1 were generated. Both retroviral vectors encode for the rat HO-1 gene, while the second construct express in addition the EGFP gene for tracing the transduced cells. The generation of these constructs was done according to standard procedures. Briefly, the rat HO-1 cDNA was used as template for PCR-amplification with specific primers introducing Xho-1 restriction sites on both ends of the PCR product. The resulting PCR-fragment was subsequently cloned into the pdrive cloning vector (Qiagen) to allow DNA-sequencing. Then, the HO-1 fragment was excised from pdrive and cloned into the pLXSN retroviral vector (Clontech). The correct orientation of the HO-1 transgene was confirmed both by restriction analysis and DNA-sequencing. For the generation of the bicistronic retrovirus expressing both HO-1 and EGFP, a pLXSN-based construct containing an internal ribozyme binding site (IRES) followed by the EGFP-gene (kind gift of Dr. A. Flügel, Max-Planck-Institute of Neurobiology, Martinsried) was employed. A similar strategy as described for the generation of pLXSNHO-1 was performed to obtain the bi-cistronic construct. The generation of both vectors was performed previously to the beginning of this thesis by Katrin Vogt, member of the Institute of Medical Immunology.
The retroviral gene transfer system needs basically two components: the packaging cell line and the retroviral vector. The retroviral vector is modified, being the genes gag, pol and env deleted. It contains the packaging signal Ψ+, and the 5 - and 3 –LTRs (Long Terminal Repeats), which flank the transgen and a selection marker. The packaging cell line contains the genes gag, env and pol. Once the packaging cell line is transfected with the retroviral vector, it is able to produce retroviral particles which are able to infect other cells, but are unable to replicate by themselves.
The packaging cell line used was one derived from the NIH/3T3, called GP+E 86 (Dr. Flügel, Neurobiology, Munich). It was cultured under normal conditions, in T75 cm2 flasks, using DMEM with 10% FCS, penicillin, streptomycin and glutamine, in an incubator with 5% CO2 at 37°C. Routine passaging of the cells was performed using brief exposure to a trypsin-EDTA solution followed by vigorous agitation of the culture flask. The cells were frozen employing a solution containing 90% FCS and 10% DMSO.
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24 h before transfection, 2.2x106 GP+E 86 cells were seeded in 60 mm (21,5 cm2) dishes in DMEM with 10% FCS, in order to allow about 60-70% confluence on the next day. From two to four hours before the transfection, culture medium was replaced by 5 ml of fresh medium. The preparation of the phosphate-DNA suspension was made using 15 μg Plasmid DNA, 31 μl 2M CaCl2, ad 250 μl distillate water and 250 μl 2x HBSP-Buffer. After 15 min at R.T., the suspension was mixed and dispensed drop by drop onto the cells. 12-24 h after the transfection, medium was replaced for one containing 1 mg/ml G-418 in order to select the G418 resistant cells.
Two different constructs, the pLXSN-HO-1, and the pLXSN-HO-1iresEGFP, were used. The efficient transfection and the over-expression of HO-1 in the both cell lines were verified by Western Blot analysis. Cells transfected with the HO-1iresEGFP vector were routinely checked for their EGFP expression under fluorescence microscope.
After verifying the HO-1 transgene expression through Western Blot, a limiting dilution method was used to obtain monoclonal cell lines from the polyclonal packaging cell line. This approach offers the possibility of select those clones with an optimal viral titer and transgene expression. To perform this, the following schemas followed:
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1 x 96 plates with 5 cells/well |
2 x 96 plates with 1 cells/well |
1 x 96 plates with 0.3 cells/well |
After 4 weeks under selection, the positive clones were tested again through Western Blot for the transgene expression, and those ones with the higher HO-1 expression were selected and further analyzed for their viral titer.
The viral titer determination was analyzed following the protocol proposed by the manufacturer (Clontech). In brief, 5 x 105 GP+E 86 cells were harvested two days before of the beginning of the viral titer determination in a 25 cm2 flask. One day before transfection, NIH 3T3 cells were plated at a density of 1.105 cells/well with 2 ml medium/well in 6-well plates.
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Lastly, for viral titer determination, the supernatants of the packaging cell lines were filtered through 0.45 μm cellulose acetate filters. Serial dilutions of the supernatant were made, from 1.10-1 until 1.10-8, and the NIH 3T3 cells were infected by adding 1 ml of the diluted virus medium (day 0). The dilution medium contained polybrene (final concentration = 8 μg/ml). The selection with G-418 began 24 h after the infection, followed by a medium change, on day 6. On day 12 after infection, the colonies were counted under the microscope, and the viral titer corresponded to the number of colonies present at the highest dilution which contains colonies, multiplied by the dilution factor.
Since the titer of retroviral particles was not always sufficient to achieve good percentages of transduction, a concentration of the viral particles was aimed by means of centrifugation of the supernatant containing the viral particles. For that, supernatants from the packaging cell lines were collected and filtered through 0.45 μm filters to remove possible dead cells o cell debris present in the supernatant. Filtered supernatants (15 ml) containing the retroviruses were transferred into Vivaspin 20 tubes with a 100000 MWCO PES membrane and centrifuged for 30 min at 3300 r.p.m. at 4°C. After this first centrifugation, 15 ml of filtered supernatant were further added and centrifuged for 45 min at 3300 r.p.m. Concentrated supernatants were immediately used.
T cells were isolated from spleen and lymph nodes from CBA/J females, and treated for 10 min at RT with lysis buffer containing ammonium chloride, potassium hydrogen carbonate and EDTA, and further washed twice with medium containing 10% FBS. These cells were then cultured under the presence of antigen presenting cells (APCs) from a DBA/2J male isolated similarly from spleen and lymph nodes and treated with lysis buffer. These APCs were irradiated at 30 Gray (Gy) and co-cultured with the CBA/J cells, putting the same cell number of CBA/J and irradiated DBA/2J cells.
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After observing that these cells were poorly stimulated with DBA/2J cells, other stimulation condition was tested, namely stimulation with anti-CD3 and anti-CD28 antibodies. For that, cells were cultured in the presence of 1 μg/ml anti-CD3 and 10 ng/ml anti-CD28 and in the presence of 10 ng/ml rmIL-2. For maintenance of the culture, medium was changed every two days, and rmIL2 concentration was maintained at the same concentration, but no further anti-CD3 or antiCD28 was added to the culture. Although a cell suspension containing different type of cells was isolated, the stimulation protocols tested were aimed to stimulate only T cells to proliferate.
CD4+ T cells were isolated from lymph nodes of CBA/J females using the MACS CD4+ T cell negative selection kit following the manufacturer´s instructions. Briefly, a cell suspension from lymph nodes was stained with a cocktail of streptavidin conjugated antibodies against all cells except CD4+ T cells. Subsequently, cells were incubated with biotin conjugated ferromagnetic microbeads. The suspension was passed through a magnetic column, allowing the CD4+ T cells to be collected as a negative fraction. After centrifugation of the cells, the cell pellet was resuspended in T-cell media and cells were counted. A representative fraction of the sample was taken in order to analyze the purity of the isolated cells by flow cytometry.
CD4+ T cells were then cultured at a concentration of 1.5 x 106 cells/ml in 12 well plates with 1.5 ml/well. Polyclonal stimulation was performed as previously described and cells were routinely maintained in medium containing 10 ng/ml rmIL2.
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CD4+CD25+ T cells (known as Treg) and CD4+CD25- T cells (reportedly T-effector cells) were isolated from thymus using the MACS CD4+CD25+ regulatory T cell isolation kit following manufacturer´s instructions. Briefly, cells were resuspended in MACS Buffer, followed by addition of Biotin-Antibody Cocktail. After 10 min of incubation at 4°C, MACS buffer, biotin microbeads and CD25-PE antibody were added. Cells were additionally incubated for 15 min at 4°C and the reaction was stopped by adding MACS buffer. Cells were centrifuged at 300 g for 10 min, and the cell pellet was resuspended in MACS Buffer. Non-CD4+ T cells were depleted by using a LD Column, which allows the CD4+ T cells to pass through as a negative fraction. After centrifugation of the isolated CD4+ T cell fraction, the magnetic labelling of CD25+ cells was performed. For doing this, cells were resuspended in MACS buffer, and antiPE microbeads were added. Cells were incubated in darkness for 15 min at 4°C, and the reaction was stopped by adding MACS buffer and centrifuging at 300 g for 10 min. Cells were resuspended in MACS buffer and the magnetic separation was performed in a MS column. CD4+CD25- cells were allowed to pass through the magnetic column and collected in a tube, and after removing the column from the magnetic field, CD4+CD25+ cells were flushed out by adding MACS buffer. Both fractions were centrifuged at 300 g for 10 min, resuspended in T cell media and counted. Representative samples of each fraction were taken in order to analyze the purity of the isolated cells by flow cytometry.
The aim of this part of the study was to obtain T lymphocytes over-expressing HO-1. Since retroviral vectors are able to permanently transduce cells, different conditions were tested in order to obtain enough number of T cells over-expressing HO-1.
The following conditions were tested: co-culture of packaging cell line and T-lymphocytes, culture of T-lymphocytes with supernatant containing the retroviruses, and culture of the cells with a concentrated supernatant. Every condition is described below:
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This co-culture was performed in order to have the packaging cell line constantly producing retroviral particles during the proliferation process of the T cells under stimulation with antigen presenting cells (APCs) or under the stimulation with anti-CD3 and anti-CD28 antibodies in the presence of rmIL-2. For that, cells from the packaging cell line were cultured O.N. in 96-well plated with U-bottom in a concentration from 1x103 to 1x105 (different conditions tested). Once adhered to the plate, the supernatant was taken and CBA/J cells (cultured under the conditions explained in 3.2.2.8) were added, at a concentration from 1.103 to 1x105 (different conditions tested). After 48 h of co-culture in the presence of polybren (tested at 4, 6 and 8 μg/ml), supernatant containing cells in suspension was taken, aiming to let the packaging cells adhered to the plate. Cells previously obtained from CBA/J females were further cultured and analyzed for transduction efficiency.
In order to avoid contamination of the T-lymphocyte culture with cells from the packaging cell line, transduction was attempted by means of the supernatant containing the retroviral particles, following a protocol described by Hori et al. (Hori et al., 2003). For that, 2.5 x 105 T cells from a CBA/J female were cultured in the presence of 5 x 106 APCs from the male (DBA/2J), previously irradiated at 30 Gy in a 24 well plate, and after 24 h of culture half of the volume was replaced by virus supernatant, previously filtered through 0.45 μm filters and containing polybren (at a final concentration of 4, 6 or 8 μg/ml). This procedure was also tested stimulating cells with anti-CD3 and anti-CD28 as previously described (3.2.2.8), as well as in 12 and 96 well plates.
Since the transduction of total cells from CBA/J using the previous conditions was not successful, a new protocol was tested using CD4+ T cells previously isolated using magnetic cell separation (as explained in 3.2.2.8.1). In brief, cells from CBA/J females were cultured for 24 h using anti-CD3/anti-CD28 stimulation in the presence of rmIL-2 in 12-well plates with a total volume of 1.5 ml/well with 1.5 x 106 cells/ml. 1 ml of cell supernatant was carefully removed, and 500 μl of concentrated retroviral supernatant as well as 500 μl of media containing rmIL-2, antiCD3 and polybren were added. The final concentration of polybren in the culture was 4 μg/ml. The transduction took place for 24 h, and 1 ml of media of the cells was replaced for fresh medium containing anti-CD3 and rmIL-2. Cells were further incubated at 37°C and were eventually measured for HO-1iresEGFP expression by flow cytometry.
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The protein transfection was performed by using ChariotTM, which forms a non-covalent compound with the protein of interest, in our case HO-1. This complex bypasses the transcription-translation process associated with gene expression. The complex formed stabilizes the protein, and helps to protect it from degradation during the transfection process. Upon internalisation, the complex dissociates and the macromolecule is free to proceed to its target organelle.
No protocol was available for the transfection of primary lymphocytes, therefore various conditions were tested in this work and the protocol was adapted for CD4+ lymphocytes in culture. Briefly, for each well to be transfected, 3.5 μl of Chariot were dissolved in 100 μl of sterile water, and 2 μg of the protein of interest (HO-1, Stressgen) was dissolved in 100 μl of PBS. These two solutions were mixed up carefully and incubated at RT for 30 min. During this incubation time, a specific number of cells (2 x 106 per well) were washed twice with PBS and further centrifuged. After incubation, the pellet of washed cells was resuspended in 200 μl of the Chariot-protein complex, and placed in a 12-well plate. After adding 150 μl of RPMI without serum, cells were incubated for 30 min at 37°C in a CO2 incubator, and 1 ml of RPMI with serum was added to stop the protein transfection and to avoid the damage of the cells. Cells were further incubated for 30 min-1h and used.
Protein transfection using β-galactosidase (provided by the manufacturer of the transfection kit) was used as a positive control to determine the percentage of transfection. Although it has to be considered that both proteins differ in structure and the transfection may not be the same, it was used as an internal control to verify whether the transfection has worked, since it was no colorimetric method available to analyze whether HO-1 has entered the cells. In addition, for HO1, the transfection efficiency was checked by analysing the HO-1 protein content by Western Blot.
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The principle of the measurement of the β-galactosidase activity is based in the stepwise hydrolysis of fluorescein-di-β-galactosidase (FDG) by β-galactosidase (Huang, 1991), which is measured by flow cytometry. The measurement of the activity of the β-galactosidase was performed following a protocol described by Nolan et al., 1988. The reaction was performed at 37°C. Briefly, up to 1 x 106 cells were resuspended in 100 μl pre-warmed media and placed in pre-warmed tubes. After that, 100 μl of a pre-warmed 2mM solution of FDG was added, and the reaction took place for 1 min 30 sec at 37°C. The reaction was stopped by adding 1.8 ml of ice cold medium and vortexing. After incubation of the cells for 1 h on ice protected from light, cells were placed in 5 ml Falcon tubes and washed with FACS Buffer. After centrifugation of the cells, they were fixed with 1% PFA O.N. After washing the cells, they were measured on a flow cytometer, being FDG detectable on FL-1H.
The mixed leukocyte culture (MLC) test (Bain et al., 1964, Bach and Hirschhorn, 1964) is widely used in the transplantation field in order to study the histocompatibility and cellular mechanisms of graft rejection. As pregnancy involves antigens from two genetically different organisms, MLC reactions are a useful tool to study the histocompatibility and the cellular mechanisms involved when cells from both organisms are put in contact with each other.
For this study, cells obtained from pregnant DBA/2J-mated CBA/J females (day 5 of pregnancy) were put in contact with mitomycin-treated DBA/2J cells. In this assay, CD4+ T cells over-expressing HO-1 were also added in order to analyze the effect of HO-1 over-expressing lymphocytes on this in vitro setting. CD4+ T cells receiving the same transfection procedure but without HO-1 were used as control. The study involved the following steps:
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Treatment of mitomycin C is known to arrest the cell cycle in the stimulating cells, so that they remain viable but are unable to proliferate or divide (Malinowski et al., 1992). Mitomycin C crosslinks with DNA and selectively inhibits DNA synthesis; RNA and protein synthesis are less affected, with a slowing in progress of the cells through the S and into the G2 phase (Anderson and Williams, 1977).
DBA/2J male cells were used as stimulator cells and were therefore treated with mitomycin C. Spleen and lymph node from DBA/2J males were crushed against at 100 μm net and collected in RPMI. After lysis of erythrocytes with a lysis buffer containing NH4Cl, KHCO3 and EDTA, the cells were finally re-suspended in RPMI containing 10% FBS. For treatment of the cells with mitomycin C, they were washed twice with RPMI without FBS, and finally resuspended in RPMI without FBS containing 50 μg/ml of mitomycin C, at a ratio of 1.107 cells/ml. Cells were incubated for 30 min at 37°C with 5 % CO2. The reaction was stopped by washing the cells three times with medium containing 10% FBS.
In order to measure the proliferation of responder cells by flow cytometry, cells were first stained with CFDA-SE, which is a dye known to intercalate in the membrane of the cells.
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Spleen and lymph node cells were isolated as previously described from DBA/2J-mated CBA/J females (day 5 of pregnancy). CD4+CD25- and CD4+CD25+ cells were isolated from thymus of these animals as previously described (3.2.2.8.2). All these cells were stained as followed: after washing the cells twice with PBS to remove rest of FBS from the medium, cells were resuspended in medium containing 1 μM of CFDA-SE in PBS, at a ratio of 1.107 cells/ml. Cells were incubated for 1 min 30 sec and the reaction was stopped by adding RPMI containing 10% FBS. After two washes with RPMI with FBS the, cells were re-suspended in the same medium. The number of viable cells was assessed by means of trypan blue as it is known that many cells die during the procedure.
The protein transfection was performed as mentioned above. CD4+ T cells were transfected with HO-1 protein and these cells will be mentioned as HO-1 over-expressing CD4+ T cells in the following sections. CD4+ T cells that were treated with the protein transfection reagent but with PBS instead of HO-1 will be referred from now on as CD4+ control T cells. For this study, purified CD4+ T cells that were already at least one week in culture were used.
3.105 CFSE-responder CBA/J cells (HO-1+ or control cells) were put in contact with 3.105 mitomycin-treated stimulator DBA/2J cells in RPMI containing 10% FBS, in a total volume of 100 μl. CD4+ T cells over-expressing HO-1 or CD4+ control T cells were added at a concentration of 1.105 cells per well. The schema indicated in Table 5 was followed.
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Table 5 : Scheme of reaction in 96 well plates
CFSE + CBA/J ♀ cells |
DBA/2J ♂cells |
HO-1 + CD4 + Tcells |
CD4 + control Tcells |
|
Spleen cells |
1.105 |
1.105 |
- |
- |
1.105 |
1.105 |
1.105 |
- |
|
1.105 |
1.105 |
- |
1.105 |
|
Lymph node cells |
1.105 |
1.105 |
- |
- |
1.105 |
1.105 |
1.105 |
- |
|
1.105 |
1.105 |
- |
1.105 |
|
CD4 + CD25 + cells |
1.105 |
1.105 |
- |
- |
1.105 |
1.105 |
1.105 |
- |
|
1.105 |
1.105 |
- |
1.105 |
|
CD4 + CD25 - cells |
1.105 |
1.105 |
- |
- |
1.105 |
1.105 |
1.105 |
- |
|
1.105 |
1.105 |
- |
1.105 |
The cells were cultured for 48 h in 96-well plates, at 37°C in humified incubator with 5% CO2 atmosphere. Each experiment setting was performed in triplicate, and the proliferation of CFSE+ cells was analyzed by flow cytometry at two time points, namely 0 h and 48 h.
Since the CFSE membrane fluorescence dye is transferred to daughter cells in the same proportion, a decrease in fluorescence intensity can be followed (as cells multiply). CFSE is therefore widely used as an indirect proliferation marker. Unstained cells were used as controls.
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Rcho-1 trophoblast cells represent a stem cell population capable of differentiation along the trophoblast giant cell lineage (Faria and Soares, 1991). The cells can be manipulated to proliferate or differentiate depending upon culture conditions (Peters et al. in: Methods in Molecular Biology, 2000). This cell line was a kind gift of Dr. Michael Soares, and was first established in his laboratory from a rat choriocarcinoma.
Cells were cultured in proliferation medium consisting of RPMI containing L-glutamine supplemented with 20% FBS (Cambrex), 1 mM sodium piruvate, 100 μg/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. Cells were grown at a density of 12.106 cells/T 75 cm2 flask. Cells were routinely maintained at subconfluent conditions and fed at a 2-day interval. It is very important to maintain the cells at subconfluent conditions, since high cell density facilitate trophoblast giant cell formation.
For differentiation of the Rcho-1 trophoblast stem cells into trophoblast giant cells, FBS was replaced by horse serum. Since a 1 to 10% of horse serum was mentioned in the literature, different concentrations of horse serum were tested. Finally, cells were differentiated using 10% of horse serum since it showed the best differentiation. The differentiation process takes normally place during 7 days, period after which the cells are fully differentiated.
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In order to determine the best concentration of protoporphyrin necessary to cause a diminution or augmentation of HO-1 protein expression in Rcho-1 cells, different concentrations of CoPPIX and ZnPPIX were tested, namely 25, 50, 100 and 200 μM. It was found that 50 and 100 μM of ZnPPIX were able to down-regulate the expression of HO-1, as measured by Western Blot. CoPPIX did not induce a down- or up-regulation of HO-1 in these cells. Nevertheless, CoPPIX was used as a control for the use of ZnPPIX, at the same concentrations in order to assess whether the effects of ZnPPIX observed in the cells is due to the down-regulation of HO-1 or to the toxic effect of the porphyrin.
Two approaches using CoPPIX and ZnPPIX were performed. First, the porphyrins were employed to test the effect of a down-regulation of HO-1 in undifferentiated stem cells. In this case, cell viability was assessed by trypan blue staining.
In a second approach, CoPPIX or ZnPPIX were used during the whole differentiation process of the cells, changing the media when necessary (normally every two days) in order to investigate the influence of HO-1 down- or up-regulation in the differentiation from stem cells into giant cells. A control of differentiation using differentiation media without CoPPIX or ZnPPIX was always included.
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For protein isolation, cells were trypsinized as usual and then washed with PBS. For protein isolation, cells were lysed in a protein isolation buffer containing HEPES, CHAPS and DTT. After isolation, homogenates were centrifuged for 10 min at 10.000 g, and the supernatant containing the proteins was transferred to a fresh tube. Protein concentration was assessed using the BioRad Protein Assay as indicated by the manufacturer. Protein samples were kept at 80°C and when working with them, they were always kept on ice.
For Western Blot analysis, 15 μg of protein were transferred into a 10% polyacrilamide gel and a SDS-PAGE in denaturizing conditions was performed at 120V using a Mini-Protean 3 electrophoresis gel system. Then, proteins were transferred into a nitrocellulose membrane using a Mini-trans-Blot system for 1h at 180 mA. After the transfer, efficient protein transfer was assessed by staining of the membrane of Ponceau Red. The expression of HO-1 was analyzed (in the membrane) by immunoblot as follows: the membrane was blocked with 5% milk powder in TBS O.N. at 4°C, and further washed with TBST, 3 x 5 min each time. The first antibody (rabbit anti-HO-1 polyclonal antibody, diluted 1:500 in 5% milk in TBS) was applied for 2 h at R.T. After washing with TBST, the samples were incubated with the secondary antibody (anti-rabbit horseradish peroxidase, diluted 1:1000 in 5% milk in TBS) for 1 h at RT. The membrane was washed again, and the reaction was finally developed by using chemoluminiscence (ECL Assay of Amersham) and exposed onto Kodak Miomax MR Imaging film. The intensity of the bands was quantified by using the Quantity One® Software, Version 4.5.2 from Bio-Rad.
In order to be able to analyze the phenotype of Rcho-1 cells by immunohistology, the cells were grown on glass slides previously treated with poly-L-lysine (Sigma). Poly-Lysine enhances electrostatic interaction between negatively-charged ions of the cell membrane and positively-charged surface ions of attachment factors on the culture surface. When adsorbed to the culture surface, it increases the number of positively charged sites available for cell binding. Briefly, slides were incubated in sterility for 5 min with a 0.01% solution of polyLlysine at RT, then washed with sterile distilled water and allowed to dry for at least 2 hours. Covered slides were kept under sterility until use, and the cells have shown normal growth patterns on these slides.
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The fact that a homozygous mating of Hmox1 deficient mice does not yield progeny (Poss and Tonegawa, 1997; Yet et al., 1999), and that the mating of heterozygous mice does not yield the expected Mendelian rate suggests that HO-1 plays a very important role in pregnancy. For this part of the work, animals partially or totally deficient in Hmox1 in a BALB/c genetic background- first established in the lab of Dr. Lee by Shaw-Fang Yet (Yet et al., 1999) were used as a part of a cooperation project with Prof. Miguel Soares, from the Instituto Gulbenkian de Ciencia in Oeiras, Portugal, who provided us with the mice, after MTA agreement with Dr. Yet from Harvard. This part of the work was divided in different parts, which are explained below.
For this, different mating combinations of females and males partially or totally deficient in Hmox1, as well as of wild type mice were performed. A schematic representation of these mating combinations is shown in Fig. 18.
This was performed in a syngeneic (BALB/c x BALB/c) and allogeneic (C57/BL6 x BALB/c) background. A schematic representation of the groups analyzed is shown in Fig. 18.
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Part of the mating combinations as well as the analysis of the abortion rates and collection of the samples of some of the animals was performed by Ivonne Wollenberg and Prof. Dr. Ana Claudia Zenclussen from our group at the Instituto Gulbenkian de Ciencia. This work was continued in our laboratories by me and the Hmox1 -/- colony is currently being maintained at the animal facility of Virchow Klinikum, Charité.
The isolation of DNA from the fetuses and resorptions was performed using the peqGOLD Tissue DNA Mini Kit. For that, fetuses and resorptions were previously washed with PBS in order to remove the rests of blood from the mother, and the samples were grinded in a mortar using a pistil under the influence of liquid nitrogen. The samples were taken up in TL Buffer, and after adding OBTM protease and were further incubated at 55°C in a shaking water bath for at least three hours. After that, isolation was performed following manufacturer´s instructions, using HiBind® DNA columns for the specific bindng of DNA. After two washing steps, samples were eluted with elution buffer and kept at -20°C until further use.
Fig. 18 : Experimental setting. | ||
Heterozygous females for Hmox1 as well as wild type females were mated with knockout, heterozygous or wild type BALB/c males. For the syngeneic combination, BALB/c females were mated to BALB/c males, whereas for the allogeneic combination C57BL/6 wild type females were mated with BALB/c males knockout or wild type for Hmox1. |
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The fundament for the PCR reaction used for genotyping of the fetuses and resorptions is based in the way that the knockouts were generated, which is schematized in Fig. 19. The mutated gene lacks the E3 region of the Hmox1 gene, and possesses a Neo resistance gene instead. As it can be deduced from this Fig. 19, knockout fetuses show amplification only with the HO/E4-Neo1 primers which amplifies a 400-bp fragment of the mutated allele, whereas wild type fetuses will show amplification only with the HO/E3HO/I3R set of primers, which amplifies a 456-bp fragment of the wild type allele. Accordingly, heterozygous fetuses show amplification with both set of primers. Table 6 shows the sequences of the primers used for the PCR reaction.
Fig. 19 : Deletion in the Hmox1 gene for the generation of the knockout colony (by Yet et al., 1999). | ||
The mutated gene lacks the E3 region and possesses a gene of resistance to Neomycin (Neo). |
Fetuses and resorptions were genotyped using the standard procedure used for genotype the colony. This was done by means of traditional PCR, which was optimized for the GoTaq® DNA Polymerase (Promega) using 2.5 units of polymerase with Green GoTaq® Buffer containing 1.5 mM MgCl2 (final concentration). This buffer allows the direct transfer of the sample into agarose gel for visualization with ethidium bromide. The reaction mixture was completed with 25 pmol of each primer (forward and reverse) and completed with water to a final volume of 50 μl.
↓99 |
The PCRs for the HO/E3HO/I3R and for the Neo1-HO/E4 set of primers were performed in different tubes, making duplicates for each sample. Every time that PCR was performed, wild type, knockout and heterozygous controls were included. The PCR was performed by 3 cycles of 5 min at 94°C followed by 30 s at 58°C and 30 s at 72°C. This was followed by 25 cycles at 94°C, 58°C and 72°C, for 30 s each, followed by a final elongation step of 7 min at 72°C. Samples were transferred into an agarose gel and visualized using ethidium bromide.
Table 6 : Sequences of the primers used for genotyping of fetuses and resorptions
Primer |
Sequence |
Neo1 |
5´-TCT TGA CGA GTT CTT CTG AG-3´ |
HO/E4 |
5´-ACG AAG TGA CGC CAT CTG T-3´ |
HO/E3 |
5´-GGT GAC AGA AGA GGC TAA G-3´ |
HO/I3R |
5´-CTG TAA CTC CAC CTC CAA C-3´ |
In vitro fertilization (IVF) was carried out during my research period at the Instituto Gulbenkian de Ciencia, Oeiras, Portugal, according to the host laboratory´s procedure and with the assistance of Sofia Rebelo, an expert in the field. It included the following steps: induction of superovulation by hormonal treatment, preparation of the dishes for IVF, sperm collection, collection of the oviducts, fertilization, washing of the oocytes, and assessment of the fertilization rates, which will be briefly described below:
↓100 |
For superovulation, 2-3 months old females were intraperitoneally injected with 5.0 IU of PMSG (Pregnant Mare Serum Gonadotropin), followed by an injection of 5.0 IU of hCG (human chorionic gonadotropin) 48 h later.
A small Petri dish with one drop (500 μl) of HTF medium in the middle of the dish was prepared (“sperm dish”). This drop was covered with oil and placed O.N. at 37°C. The “fertilization dish” (1 per three females) consisted in a 60 mm Petri dish with 500 μl of HTF medium covered with oil. This dish was also incubated O.N. at 37°C. One “wash dish” per each fertilization dish was prepared using 4 drops x 125 μl of HTF in a small Petri dish, covered with oil, which was also incubated O.N. at 37°C.
For sperm collection, males were sacrificed by cervical dislocation 12 h after hCG was injected to the females. The cauda epididymides and vasa deferentia were dissected out and placed in the sperm dish, inside the medium drop. The cauda epididymis was dissected out making 5 slashes with a 30-gauge needle on a syringe and the sperm was gently squeezed out from the vasa deferentia by using forceps. Sperm was collected 15 to 45 min before the females reach 13 h post hCG injection. After 10 min-1 h incubation of the sperm in a 37° C incubator in a dish containing HTF medium, 10 μl of sperm was transferred into each fertilization dish.
↓101 |
For collection of the oocytes, females were sacrificed 13 to 14 h after hCG injection by cervical dislocation and oviducts were taken as shown in Fig. 20. The oocytes were collected from the swollen ampulla (Fig. 21) and were found in a cloud together with cumulus cells. This “cloud” was taken to the drop and put in contact with the sperm. This procedure had to be very quick and not to take more than 5 minutes per plate.
Fig. 20 : Dissection of the oviduct | ||
Figure taken from Nagy et al. in: Manipulating the mouse embryo, 2003. A) The ovary, oviduct and the end of the uterus are separated from the mesometrium. B) A cut is made between the oviduct and the ovary. A second cut separated the oviduct from the uterus. |
Fig. 21 : Isolation of oocytes from dissected oviduct | ||
When the oviduct is removed soon after hormonal treatment, the oocytes surrounded by cumulus cells in the upper part of the oviduct (ampulla), and can be released by tearing the ampulla with fine forceps. |
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The manipulation of the oocytes was always carried out with a mouth pipette assembly, consisting of an aspirator mouthpiece, tubing, and a glass Pasteur pipette previously pulled on a flame (to create a narrow opening). Oocytes were placed in the fertilization drop, which already contained the sperm. After 4 to 6 h of incubation, oocytes were washed in order to remove the excess of sperm, and further incubated O.N. at 37°C. In this step, the number of total oocytes was counted and will be referred as one-cell stage.
Two-cell stage was evaluated in order to address fertilization efficiency. The number of cells in two cell stage was determined under the microscope, and the fertilization rate was calculated according to the formula:
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Two-cell stage embryos were washed in M2 Medium, in order to remove the excess of HTF Medium.
For the vasectomisation procedure, males were anesthetized and both testes were pushed down into the scrotal sac by applying pressure to the abdomen. A small incision was made through the skin along the midline of the scrotal sac. Testes were taken out one at a time, and the membrane of the testis suffered a small incision close to the vas deferens. The vas deferens was identified as a bright white tubule with a single blood vessel. Using forceps, both the vas deferens and the blood vessels were hold and cauterized in two positions with red hot tips, taking out a small portion of the vas deferens. Testes were put back in place, and the skin was clipped together. Males were kept on a heating pad until they recovered from the injected anesthetic, and were mated with the females after at least one week.
Vasectomized males (BALB/c background) were bred with females to produce pseudopregnant recipients for oviduct and uterine transfer.
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The surgical procedure of embryo transfer was performed by Sofia Rebelo, member of the Instituto Gulbenkian de Ciencia, an expert on the field. Two-cell stage embryos were transferred into female recipient mice that have been previously mated with vasectomized males and which exhibited plug in the morning of the transfer. In order to assure that females will show plug, recipient females were injected with hormones and mated with the males immediately after HCG injection. After mating with the vasectomised (sterile) male, the female´s reproductive tract is supposed to become receptive for transferred embryos, even though their own unfertilized oocytes degenerate. The day of the embryo transfer was considered as day 0 of pregnancy.
The transfer was performed anesthetizing the female and making a small incision in the skin along the dorsal midline, at the level below the last rib. As shown in Fig.22, the ovarian fat pad, attached to the ovary, oviduct and uterus was pulled out in order to make the transfer into the oviduct (infundibulum). Embryos in two-cell stage in M2 Medium were transferred into the oviduct using a transfer pipette (mouth-pipetting device). Once transferred in both oviducts, the uterus, oviducts and ovaries were put back inside the body cavity and the skin was closed with wound clips. Each side of the uterus received 10 embryos in two-cell stage.
Fig. 22 : Embryo transfer into the oviduct | ||
Fig. taken from Nagy et al. in: Manipulating the mouse embryo, 2003. |
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On day 14 of pregnancy, females were sacrificed, the uteri removed, and the implantation sites were documented. Resorptions were kept for further analysis, as well as spleen, liver, thymus, ovaries and decidua. Abortion rates were calculated as the ratio of abortion sites to total implantation sites as previously described.
Ovaries from Hmox-1 +/+ and Hmox-1 -/- females used as donors were embedded in paraffin as previously described in 4.2.1.9. Samples were cut in 5 μm slides and stained with Hematoxilin and Eosin as previously described in 4.2.1.11. The analysis of the follicles was carried out under light microscope, counting the follicles in each stage of maturation (primordial, primary, secondary and mature) using a 20X magnification. Both ovaries of each animal were analyzed, counting at least three slides per ovary.
Most of the data presented in this thesis is represented by boxplots. This type of graphic was chosen since it is the best way to present data that is analyzed by non-parametric tests.
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An example of a boxplot representation is shown in Fig. 23. The boxes show the lower quartile (25th percentile), the median and the upper quartile (75th percentile). The difference between the upper and lower quartiles is the inter-quartile range (IQR) and it contains 50% of the sample. The smallest and largest values are indicated by the small horizontal bars at the end of the whiskers. Outliers (defined as 1.5-fold the IQR, Henderson, 2006) are normally shown as circles and/or asterisks below or above boxplots.
Fig. 23 : Representation of the data with boxplots | ||
Boxplots were used as a representation for non-parametric data, showing between the maximal and minimal value the 100% of the data obtained. |
Data from the study using the murine model of abortion are presented as medians or medians ± 75% quartiles. Analysis and graphics were made with the SPSS 11.5 software. When appropriate, statistical analysis of the data was performed using the non-parametric KruskalWallis test followed by Mann-Whitney U-test. In all cases, p<0.05 was considered a statistically significant difference.
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Data obtained using the Hmox1 deficient mice are presented as mean ± SD when analyzing the in vitro fertilization results. The statistical analysis of the in vitro fertilization was performed with the total number of oocytes, fertilized and unfertilized, using the Fisher´s exact test. The graphics and statistics of this part of the work were obtained with the GraphPad Prism 4 Software. The data regarding follicle development is shown as median ± interquartile range and were analyzed by the Mann-Whitney U-test.
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