Praca oryginalna Original paper
Production of transgenic pigs expressing
human á1,2-fucosyltransferase to avoid
humoral xenograft rejection
DANIEL LIPIÑSKI*/**, JACEK JURA***, JOANNA ZEYLAND**, WOJCIECH JUZWA**, EWA MA£Y*, ROBERT KALAK**, MICHA£ BOCHENEK***, ANDRZEJ PLAWSKI*,
MARLENA SZALATA*/**, ZDZIS£AW SMOR¥G***, RYSZARD S£OMSKI*/**
*Institute of Human Genetics, Polish Academy of Sciences, Strzeszyñska 32, 60-479 Poznañ, Poland **Department of Biochemistry and Biotechnology, University of Life Sciences,
Wojska Polskiego 28, 60-637 Poznañ, Poland
***Department of Animal Reproduction, National Research Institute of Animal Production, 32-083 Balice, Kraków, Poland
Lipiñski D., Jura J., Zeyland J., Juzwa W., Ma³y E., Kalak R., Bochenek M., Plawski A., Szalata M., Smor¹g Z., S³omski R.
Production of transgenic pigs expressing human á1,2-fucosyltransferase
to avoid humoral xenograft rejection
Summary
The use of animals as a source of organs and tissues for xenotransplantation may overcome the growing shortage of human organ donors. However, the presence of xenoreactive antibodies in humans, directed against the swine Gal antigen present on the surface of xenograft donor cells, leads to the complement activation and an immediate xenograft rejection as a consequence of hyperacute reaction. In order to prevent a hyperacute rejection, it is possible to alter the swine genome with human genes modifying the set of the donors cell surface proteins. The aim of this study was to prepare a pCMVFut genetic construct and then introduce it into the swine genome in order to obtain transgenic pigs expressing human á1,2-fucosyltransferase and thereby avoid a humoral xenograft rejection. The pCMVFut gene construct containing the human gene encoding á1,2-fucosyltransferase enzyme under the human cytomegalovirus immediate early promoter was introduced by microinjection into a male pronucleus of a fertilized porcine oocyte. The screening procedure involved isolating genomic DNA from microsections of pigs ears, the amplification of two PCR fragments and the entire sequencing of positive samples. The mapping of the transgene was performed by fluorescence in situ hybridisation (FISH) and transgene expression, while its impact on the reduction of the Gal epitope level on the surface of pig cells was assessed by flow cytometry of primary cultured skin fibroblasts. The influence of the human complement was measured by testing the sensitivity of nontransgenic and transgenic cells to complement-mediated cytotoxicity upon exposure to human serum. As a result of this experiment, the founder male pig was obtained with the transgene mapping to chromosome 14q28. An RT-PCR analysis revealed the expression of the HT gene in different tissues of transgenic pigs. A flow cytometry analysis revealed a reduction in the level of epitop Gal on the cell surface of skin fibroblasts isolated from transgenic pigs. The complement-mediated cytotoxicity assay showed increased viability of transgenic cells in comparison with nontransgenic ones, which confirmed the protective influence of HT expression. In this study we demonstrated that the constitutive transgenic expression of human H-transferase (á1,2-fucosyltransferase) can decrease the amount of Galá1,3Gal (Gal epitope) on the surface of pig cells, which is consistent with the results of other researchers. The expression of á1,2-fucosyltransferase modified the cell surface carbohydrate phenotype of transgenic pig cells, resulting in the expression of the universally tolerated 0 blood group antigen (H antigen) and a subsequent reduction in the expression of Gal epitope, as evaluated by flow cytometry analysis. Apart from the principal data, the flow cytometry analysis revealed no significant differences between the Gal epitope level achieved by CMVFUT heterozygous boar founder TG 1154 and transgene homozygous pig 433 from the F2 generation. The flow cytometry results were confirmed by the cytotoxicity assay. We found no statistical difference in the survival rate between transgenic homozygous and heterozygous cells under the influence of 50% human serum with an active complement system. Both homozygous and heterozygous cells had the same level of lysis protection.
The transplantation of animal-derived organs could
save thousands of human beings who do not undergo
a transplant surgery of the heart, kidney or liver
because of the lack of a donor. All intensive and
increasingly numerous studies which could help to
solve this problem are focused on pigs. These animals
are easy and cheap in breeding, multiply fast and have
numerous progeny. The diversification between various
pig races makes it possible to select organs of
appro-priate size for recipients. Pigs and humans have
similar anatomical and physiological parameters, but
the significant genetic distance is among the main
causes of major immunological complications after
organ grafting. Organs are rejected almost immediately
after transplantation as a result of a hyperacute
rejec-tion (HAR). The main cause of HAR is the presence
of xenoreactive human antibodies against pig á-Gal
antigens present on glycolipids and glycoproteins. The
Gal antigen (Galá1,3Gal) develops from
á1,3-galacto-syltransferase which adds galactose to
N-acetyllacto-samine (N-lac) with á1,3-glycosyl bond (4). Both, the
enzyme and the sugar residue are absent in humans
and Old World monkeys (5). It is assumed that the gene
encoding á1,3-galactosyltransferase was inactivated in
the ancestors of higher primates (6). The introduction
of additional copies of a gene encoding human
á1,2--fucosyltransferase (HT, transferase H) into the pig
genome could prevent immunological response. It
cata-lyzes fucose addition, forming a neutral H structure.
The mechanism involved in the reduction of Gal
epi-tope as a result of á1,2-fucosyltransferase expression
is based on the competition between H transferase and
á1,3-galactosyltransferase for the same acceptor
substrate. The competition involves differences in the
location of both enzymes within compartments of the
Golgi apparatus: HT is specific to the medial region,
whereas á1,3-galactosyltransferase is found in the trans
region. Glycosylation occurs in a highly regulated
manner involving the movement of newly synthesized
molecules from the cis through medial to trans side of
Golgi stacks (16). Thus transferase H generates
fucosy-lated N-acetyllactosamine (H antigen) prior to N-lac
modification by the action of
á1,3-galactosyltrans-ferase. Furthermore á1,3-galactosyltransferase cannot
transfer the terminal galactose to fucosylated
N-ace-tyllactosamine. The process of replacing Gal epitope
with an alternative oligosaccharide H antigen has
been termed competitive glycosylation. The
introduc-tion of addiintroduc-tional copies of á1,2-fucosyltransferase
would result in a reduced number of Gal antigens on
the surface of donor cells and lead to a decrease in
immunogenicity (3, 15).
The aim of this study was to prepare a pCMVFut
genetic construct and then to introduce it into the
swine genome in order to obtain transgenic pigs
expres-sing human á1,2-fucosyltransferase so as to avoid
a humoral xenograft rejection.
Material and methods
The preparation of the gene construct. The pCMVFut construct contained human cytomegalovirus immediate early promoter (CMV-IE) (618 bp), human á1,2-fucosyltrans-ferase cDNA (1098 bp) and poly(A) signal from the hGH gene (104 bp) inserted in the pGT-N29 plasmid vector (New England BioLabs) (4662 bp) (fig. 1). It was prepared as follows: a) The CMV-IE promoter was amplified using pEGFP-C1 vector (Clontech) as the template and CMVF (5-GCGGTACCTAGTTATTAATAGTAATCAATTACGG--3) and CMVR (5-GCTCTAGACACCATGGTGGCGAC-CGG-3) as primers. 5 end was modified by the addition of KpnI restriction site, while 3 end of XbaI. PCR products were digested with KpnI and XbaI, purified on Wizard columns (Promega) and cloned into pGT-N29 plasmid vector within KpnI and XbaI restriction sites; b) The cDNA encoding human á1,2-fucosyltransferase (GenBank Acces-sion AB004863) was amplified by PCR, using first strand cDNA as the template and FutF (5-GATCTAGAATGTG-GCTCCGGAGCCATCGTCAG-3) and FutR (5-GAG-GATCCTCAAGGCTTAGCCAATGTCCAGAG-3) as primers. The 5 end was modified by the addition of an XbaI restriction site, while at 3 of BamHI. cDNA template was synthesized using total RNA isolated from human peri-pheral blood cells by MMLV reverse transcriptase with an oligo(dT)12-18 primer. PCR products were digested with re-striction enzymes, purified on Wizard columns and ligated to the pGT-N29 construct containing the CMV-IE promoter cleaved with XbaI and BamHI; c) The fragment containing the poly(A) signal of the hGH gene was amplified using human genomic DNA as the template and poly(A)F (5-AG-GATTCCTGCCCGGGTGGCATCCC-3) and poly(A)R (5-AAGGATTCCTGATGCAACTTAATTTTATTAGGA-CA-3) as primers. Both ends of the fragment were modi-fied by BamHI restriction site addition. PCR products were digested with BamHI and cloned into the plasmid containing the CMV-IE promoter and human á1,2-fucosyltransferase gene cleaved with the same enzyme. The proper orientation of the poly(A) signal of the hGH gene and the nucleotide sequence of the final pCMVFut construct were confirmed using a cycle sequencing kit and an ALFExpress sequencer (Pharmacia Biotech). The pCMVFut construct was purified with an UltraMobius Plasmid Kit (Novagen), digested to linear form with NotI and used for microinjection.
Microinjection, IVC, embryo transfer. Embryo donors were superovulated before artificial insemination (tab. 1). The zygotes for microinjection were obtained from indivi-duals representing Landrace, Duroc, Hampshire and pbz breeds. After collection, the zygotes were assayed
morpho-KpnI NotI XbaI BamHIBamHI CMV-IE promoter Human 1,2-á fucosyltransferase Poly(A)hGH CMV-6F CMV-7F FUT-3R 5’ 3’
Fig. 1. The scheme of the pCMVFut gene construct. An expres-sion plasmid (pCMVFut) was constructed by inserting human cytomegalovirus immediate early promoter, human á1,2-fuco-syltransferase gene and poly(A) signal from the hGH gene into the pGT-N29 vector. The location of primers for trans-gene integration analyses is shown
logically. Only zygotes with intact cytoplasm and two polar bodies were subjected. Prior to DNA insertion, the zygotes were centrifuged (19 000 × g for 5 min.) to reveal pronuclei. Microinjection was performed under an inverted microscope equipped with the Hoffman modulation contrast system, using micromanipulators. DNA was inserted into one of the pronuclei. After the microinjection of DNA, zygotes were subjected to repeated morphological assays. Transformed zygotes were surgically transferred to one of the oviducts of synchronized recipients under full anaesthesia (tab. 2). At least 20 transformed zygotes were inserted into the oviduct. Female recipients were tested for pregnancy about 50 days after the transplantation.
Screening for the presence of the transgene. The screen-ing procedure involved the isolation of genomic DNA with proteinase K from the pigs ear bioptats, amplification of two PCR fragments and entire sequencing of positive sam-ples. Forward primers were located in CMV promoter and reverse primer in the region coding á1,2-fucosyltransferase. PCR product of 144 bp was amplified with CMV-6F (5-GCAGAGCTGGTTTAGTGAA-3) and FUT-3R (5-ATATGGAGGAAGAAGATTACAGAG-3) primers, whereas the 343 bp fragment was obtained with CMV-7F ATGGTGATGCGGTTTTG-3) and FUT-3R (5--ATATGGAGGAAGAAGATTACAGAG-3) primers. One of each pair was labelled at 5 end with Cy5. PCR products were fractionated in 6% polyacrylamide gel (19 : 1, AA : BB) under denaturing conditions on an ALFExpress sequencer. Two microliter aliquots of PCR products were combined with the loading buffer and internal markers (113 bp and 388 bp). The PCR analysis was confirmed by sequencing, using a cycle sequencing kit and an ALFExpress sequencer (Phar-macia Biotech). For the result analysis, Fragment Manager and Sequence Manager software (Pharmacia Biotech) was applied.
The mapping of the transgene. The mapping of the trans-gene was performed by fluorescence in situ hybridisation (FISH), using porcine cell cultures of skin fibroblasts. Cells were cultured at 37°C in an atmosphere of 5% CO2, DMEM
medium with L-Glutamine, antibiotic and 20% foetal bovine serum. Metaphase plates were obtained by the addition of 0.05 µg/ml of colcemid. Cells were harvested with 0.25% tripsin-EDTA solution, and treated with hypotonic solution (0.075 M KCl) and then fixed with absolute methanol-glacial acetic acid (3 : 1). The analysis of karyotype was performed
using G-banding of methaphase chromosomes according to the routine procedure. The DNA probe specific for the trans-gene (CMVFut) was labelled with biotin-dUTP (Bio-dUTP) by random priming (Stratagene kit). Hybridisation with molecular probe was performed for 17 hours at 37°C. For signal detection, cell spreads were incubated with antibodies labelled with fluoresceine (FITC-avidin, goat anti-avidin antibodies, FITC-anti-goat antibody) at 37°C. The observa-tion of the transgene signal was performed using a fluo-rescence microscope after the standard staining of cells with DABCO/DAPI. The location of the transgene was set according to standard karyotype of domestic pig. Committee for the Standardized Karyotype of the Domestic Pig (7).
RNA isolation and RT-PCR analysis. Total RNA was prepared using TRIzol reagent as described by the manu-facturer (Invitrogen). The expression of the transgenes was evaluated by RT-PCR. First strand cDNA was prepared by reverse transcription, using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen). The analysis of human á1,2-fucosyltransferase expression was performed with the following primers: 5-ATG TCG GAG GAG CAC GCG G-3 and 5-CCA CGG TGT AGC CTC CTG TCC-3. The expected product was 426 bp. The â-actin primers 5--TGG AGA AGA TCT GGC ACC ACA-3 and 5-TCT TCA TGA GGT AGT CGG TCA-3 were used as a positive con-trol for each RNA sample and the 309 bp PCR product expec-ted from a correctly processed â-actin mRNA was obtained. The flow cytometry analysis of skin fibroblasts. Trans-gene expression and its impact on the reduction of the Gal epitope level on the surface of pig cells were assessed by flow cytometry of primary cultured skin fibroblasts. Direct fluorescence of cell-surface carbohydrate epitopes was per-formed with fluorescein isothiocyanate (FITC) conjugated BS-IB4 lectins detecting Gal epitope and UEA-1 lectins specific for H antigen (Sigma). Skin fibroblasts from the control, transgenic boar founder TG1154 and its litter from F2 generation CMVFUT homozygous pig 433 were cultured from ear tissue bioptats. The cells were harvested, washed with PBS containing 10% FBS, and then briefly centrifuged. Incubations with lectins were performed at 4°C for 30 minu-tes with 10 µg/ml of lectin in PBS + 1% fetal bovine serum. The cell surface expression of the Gal and H antigen was then measured by flow cytometry performed with a DAKO Galaxy cytometer.
Human serum cytotoxicity assay. Fully confluent cultu-red cells were trypsinized for 3 minutes at 37°C. The trypsin was inactivated with complete culture media containing 10% FBS. Cells were collected by centrifugation at 1000 rpm for 10 minutes at 20°C. Cell pellets were washed with 200 µl of Hanks Balanced Salt Solution. Cells were suspended in 200 µl of one of three possible treatment solutions: 50% DMEM and 15% FBS in PBS (A); 50% fresh human serum in PBS (B); 50% heat-inactivated human serum in PBS (C). Cells were incubated at 37°C for 30 minutes. Cytotoxicity assays were performed in twelve replications.
Calculating the percentage of viable cells. 20 µl of cell suspension was mixed with 20 µl of 0.2% trypan blue mix and incubated for 5 minutes at RT. The number of unstained cells and stained cells was counted in the Bürkers chamber. Dead cells took up trypan blue stain. The numbers were counted for 0.9 mm3. The percentage of viable cells was
determined by dividing the number of unstained cells by the total number of cells and multiplying by 100.
Tab. 2. Embryo recipients oestrus cycle synchronization draft Tab. 1. The superovulation draft
y a D Hour Treatment 1 800 Serogonadortopin,1500 .iu/.pig,inrtamusculalry 3 800 Biogonady,l1000 .iu/.pig,inrtamusculalry 4 800and2000 oesrtusdeteciton,selecitonofdonors,AI 5 1000 collecitonofembryos y a D Hour Treatment 1 800 Serogonadortopin,750 .iu/.pig,inrtamusculalry 3 800 Biogonady,l500 .iu/.pig,inrtamusculalry 4 800and2000 detecitonofoesrtus,selecitonofrecipients 5 about1200 rtansplantaitonofembryos
Results and discussion
The transgenic pig was generated by the microinjection of the linearized gene construct encompassing human cytomegalovirus immediate early promoter (CMV-IE), human á1,2-fucosyltransferase coding sequence and poly(A) signal from the hGH gene in the pGT-N29 vector into a zygote (fig. 1). The performed sequence analysis revealed that the cloned gene was 100% iden-tical with the sequence provided by a BLAST search.
The screening procedure involving 144 bp and 343 bp PCR products was applied to identify transgenic pigs. One individual male (number 1154) from the F0 gene-ration was found to have the transgene incorporated (0.45%). The screening results are presented in fig. 2. When male 1154 reached sexual maturity, his semen
was collected and used for artificial insemination of non-transgenic females. As a result, 247 piglets of the F1 were generated. The presence of the transgene was confirmed by PCR screening in 86 individuals (34.8%) (tab. 3). No change in the size of PCR screening frag-ments was observed in all transgenic animals of F1 (fig. 3). The performed sequence analysis confirmed that the transgene in F1 animals was 100% identical with the sequence of the gene construct used for the genera-tion of the founder animal. Further crossing of F1 hetero-zygotes generated male and female F2 homohetero-zygotes. The founder and its offspring showed no changes in phenotype and behaviour.
Transgenic animals were subjected to classical and molecular cytogenetic analysis after primary cell lines from skin fibroblasts of transgenic F1 and F2 pigs were
1150 piglet
Positive control (CMVFut) 1151 piglet
Negative control (no DNA)
Marker 50-400 bp Negative control (swine DNA)
1152 piglet 1153 piglet 1154 piglet 1155 piglet 1156 piglet 1157 piglet 1154 founder 162 piglet 163 piglet 164 piglet 165 piglet 166 piglet 167 piglet 168 piglet 169 piglet 170 piglet Negative control (no DNA) Negative control (swine DNA)
Positive control (CMVFut) Marker 50-400 bp
Fig. 2. Screening for the CMVFUT transgene in the popula-tion of F0 piglets. Screening for the transgene was performed by PCR and yielded 144 bp and 343 bp DNA fragments. Ar-rows indicate the presence of the transgene in piglet number 1154 of the same size as input DNA fragment (positive con-trol in line 11)
Fig. 3. The transmission of the CMVFUT transgene to the pigs of the F1 generation. Screening for the transgene was performed by PCR and yielded 144 bp and 343 bp DNA frag-ments. Arrows indicate the transgene in founder boar num-ber 1154 and his F1 offspring of the same size as input DNA fragment (positive control in line 13)
established. Based on the GTG-banding pattern, no chromosomal aberrations in pigs were observed. FISH with DAPI staining enabled the mapping of the trans-gene to the q28 region of chromosome 14 (SSC14q28). The performed FISH analysis of transgenic animals of F1 and F2 generations confirmed that the transgene was located in the same region as in the founder. In fig. 4, FISH mapping of the transgene in transgenic heterozy-gous and homozyheterozy-gous pigs is presented.
RT-PCR analysis was used to determine human HT expression in tissues derived from transgenic animals. The RT-PCR analysis revealed the expression of the HT gene driven by CMV promoter both in skin
fibro-blasts of the transgenic founder (no. 1154) and in all tested samples from transgenic animals of F1 and F2 generation. RT-PCR analysis in tissues derived from the transgenic female (no. 166) is presented in figure 5. No specific PCR products were observed in any of the non-transgenic samples. Endogenously expressed â-actin was used as a control to confirm the integrity of all samples tested by PCR (fig. 5).
Flow cytometry analysis was used to assess trans-gene expression at the protein level and determine whether the expression of functional enzyme á1,2-fuco-syltransferase, which was responsible for H antigen synthesis, was associated with a reduced level of Gal epitope on the surface of pig cells. Skin fibroblasts were established from the control and transgenic pigs, boar founder TG1154 and homozygous pig 433. Staining with the BS-IB4 lectin revealed significant reduction in mean fluorescence intensity of transgenic fibroblasts expressing á1,2-fucosyltransferase relative to the con-trol. Fibroblasts from transgenic boar TG1154 stained with UEA-1 lectin exhibited a marked increase in fluorescence intensity compared with the control. In both transgenic pigs, TG1154 and 433, the expression of the H epitope correlated inversely with that of Gal. It demonstrates that the reduction of the Gal epitope level on the surface of the transgenic boars cells was the result of á1,2-fucosyl-transferase expression, and it involved the competition between á1,3-galacto-syltransferase and H transferase for the same acceptor substrate molecule. What was particularly interesting was that transgene CMVFUT homozygous pig 433 demonstrated a lesser effect of reducing Gal epitope than CMVFUT heterozygous boar founder TG1154. This correlates with the level of H antigen expression which occurred to be slightly lower in pig 433 in relation to TG1154 (fig. 6).
The influence of the human com-plement was mea-sured by the sen-sitivity testing of nontransgenic and transgenic cells to complement-me-diated cytotoxicity upon exposure to human serum. The total number, the number of viable and the number of dead cells were counted using the trypan blue stain-ing method. The control
nontrans-Tab. 3. Number of transgenic pigs identified in studied gene-rations n o it a r e n e G Numbearniomfaalnsalyzed Numbearnoifmartalsnsgenic s g i p 0 F 186 11 s g i p 1 F 247 86 s g i p 2 F 115 11 l a t o T 448 98
Fig. 5. The RT-PCR analysis of human á1,2-fucosyltransferase expression in tissues of the transgenic female (number 166). (A) Whole RNAs were tested for genomic DNA contamination. (B) All mRNAs samples were tested for â-actin mRNA (positive control). (C) The RT-PCR analysis of CMVFUT trans-gene expression. Lanes 1-5, transgenic pig (no. 166); lanes 6-10, non transgenic pig. Lanes 1 and 6, heart; lanes 2 and 7, kidney; lanes 3 and 8, liver; lanes 4 and 9, muscle; lanes 5 and 10, ovary; lane 11, negative control (without template); lane 12, positive control (vector with transgene); lane 13, size marker (l DNA/HindIII, EcoRI). Human á1,2-fucosyltransferase gene expression, driven by CMV promoter is indicated by the arrow
A B C
1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13
A B
Fig. 4. The mapping of the CMVFUT transgene in transgenic heterozygous (A) (no. 431) and homozygous (B) (no. 437) pigs. The location of the transgene (arrow) on the q28 region of chromosome 14 was demonstrated by FISH
genic cells were effectively lysed by the human com-plement. In contrast, homozygotic transgenic cells, as well as heterozygotic cells were protected from human complement lysis. Incubation with the heat-inactivated human serum containing no or little complement system active elements hardly influenced the viability of all tested cells.
It was shown that the homozygous cells had statisti-cally about 26-fold higher odds ratio to stay alive than nontransgenic cells under the same conditions (50% HS with active complement system, two-sided test, p < 0.0001, OR = 26.44, 95% CI (17.46-40.06)) whereas heterozygous cells had about 22-fold higher odds ratio than nontransgenic cells (50% human serum with active complement system, two-sided test, p < 0.0001, OR = 22.31, 95% CI (15.08-33.01)). No statistically significant differences were found between homozygous and heterozygous cell viability under 50% HS with active complement system conditions (fig. 7). The presence in humans of xenoreactive antibodies directed against the swine Gal antigen found on the sur-face of xenograft donor cells leads to the complement activation and immediate xenograft rejection as a con-sequence of a hyperacute immunological reaction. The graft of a swine organ with knocked-out á1,3-galacto-sylotransferase, responsible for Gal antigen formation, would be tolerated with a simultaneous administration of medicines decreasing other less severe immuno-logical reactions. To prevent HAR it is also possible to modify the swine genome with human genes control-ling the enzymatic cascade of complement (CD59, CD55, CD46) or modifying the set of donor cell surface proteins (H transferase, á-galactosidase).
In this study it was demonstrated that the constitutive expression of human H transferase can decrease the amount of Gal epitope on the surface of transgenic pig cells, which is consistent with the results of other researchers (1, 3, 13, 15). The expression of á1,2-FUT modified the carbohydrate phenotype on the surface of transgenic pig cells, resulting in the expression of the universally tolerated 0 blood group antigen (H antigen) and a subsequent reduction in the expression of Gal epitope, as evaluated by flow cytometry analysis. Apart from the principal data, flow cytometry analysis reve-aled no significant differences between the Gal epitope level achieved by CMVFUT heterozygous boar foun-der TG1154 and transgene homozygous pig 433 from the F2 generation. The results of flow cytometry were confirmed by the cytotoxicity assay. It was found that there was no statistical difference in the survival rate between transgenic homozygous and heterozygous cells under the influence of 50% human serum with active complement system. Both homozygous and hetero-zygous cells had the same level of lysis protection.
The removal or a significant reduction in the number of Gal antigen molecules prevents complement activa-tion, resulting in the elimination of the hyperacute res-ponse of the recipients organ (4, 8). In this situation, the main cause of the rejection of the graft remains UEA-1
BS-IB4
Fig. 6. The flow cytometry analysis of fibroblasts isolated from the control, transgenic boar TG1154 and homozygous litter pig 433 after staining with UEA-1 lectin (detects H antigen) and BS-IB4 lectin (detects Gal epitope) as indicated. The x-axis shows the fluorescence intensity, and the y-axis shows the relative cell count for unstained cells (black thin dashed--dot line), the control (red thin solid line), boar TG1154 (blue thick solid line) and pig 433 (purple thick dotted line). The analysis demonstrates that an increased expression of the H antigen correlates with a reduced expression of Gal epitope on the cell surface of transgenic pigs
Fig. 7. The sensitivity of nontransgenic (blue), heterozygous (purple) and homozygous (green) cells to human serum as a source of complement system. The cell survival was determined by dye exclusion with trypan blue and then the percentage cytotoxicity was calculated. The x-axis shows the human serum concentration and the y-axis shows the cell survival percentage 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% 110% 0% 50% inactive
a malfunction of the system coagulation regulation inside the graft and cellular reactions involving macro-phages, neutrophiles, NK cells and T cells (10). With the increase of our knowledge on reactions taking place inside grafted tissue, it will be possible to introduce genes controlling the coagulation system into the swine genome (9).
Most of the strategies employed for successful pig--to-human xenotransplantation, including the one applied in our experiment, eliminate a hyperacute response, but do not prevent an acute vascular (delayed xenograft rejection) or acute cellular rejection. Those responses depend on an unknown number of different weaker antigens. At the present time, the only way to prevent such types of rejection is intense immunosuppression. However, it fails even in the case of autological, human transplantation with a high antigen concordance, there-fore its effectiveness when it comes to xenogenic, phylo-genetically distinct human-swine set must be signifi-cantly lower. The higher the level of immunosuppres-sion applied, the more complications and the greater the impairments of the immunological system are observed. The immunodeficiency increases the risk of the incidence of diseases and the necessity of taking medicines, additionally affecting transplanted organs, the kidney or liver, engaged in their metabolism. Extreme immunosuppression leaves the recipients organism defenseless to infections, making his life uncomfortable, and that is why we continue to search for alternatives. In addition, we should not forget about the proteins synthesized by those organs. The liver is responsible for the production of almost all plasma proteins; the kidney produces erythropoetin and the pancreas pro-duces insulin. Those proteins can also be responsible for immunological interactions.
The most reasonable solution to prevent the rejection of an organ is to induce tolerance to the graft antigens. The specific immunological tolerance is a state of the immunological system when there is no response to a specific antigen or group of antigens, but the ability to respond to other antigens is retained. Immunological tolerance can be induced by the triggering of T cells specific anergy, induction of regulating T cells, shifting Th1 cell response to Th2 and removal of specific T cells (12).
There are two different approaches to inducing xe-nograft specific tolerance. One of them is the induction of tolerance by molecular chimerism in the recipient of a graft. This can be done by the introduction of á1,3--galactosyltransferase encoding gene into bone marrow cells and their autological transplantation. The produc-tion of áGal antigen by genetically altered cells could result in tolerance to this antigen. The second approach concerns the chimerism of hematopoietic cells by the transplantation of xenogenic, progenitor, bone marrow cells parallely with thymectomy (2).
Animal studies confirmed the possibility of inducing immunological tolerance. Without additional immuno-suppression and radiation, an allogenic skin graft in
a mouse was able to survive for over 350 days. Tole-rance was induced by injections of the donors spleen cells into the portal vein of the graft recipient (11). Attempts were also made to induce immunological tolerance in a xenogenic system when researchers removed the thymus from the rat, eliminated NK cells, suppressed B cells, introduced hamster antigens and then grafted the heart and thymus of the hamster into the experimental rat. After the surgery no rejection occurred but autoimmunological reactions were observed (17).
It seems that the first part of research on the trans-plantation of animal organs into humans should lead to obtaining transgenic animals without Gal epitope molecules on the cell surface so that they would not bind with xenoreactive anti-Gal antibodies. There will be a great deal of problems to solve, such as physio-logical differences concerning the organ functioning, infections with animal diseases and, last but not least, ethical objections.
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Authors address: Dr Daniel Lipiñski, University of Life Sciences, Depart-ment of Biochemistry and Biotechnology, ul. Wo³yñska 35, 60-637, Poznañ, Poland; e-mail: lipin1@poczta.onet.pl
