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LEA29Y binds human B7. We have characterized the expression pattern and the biological function of the transgene as well as its impact on the porcine immune system and have evaluated the potential of these transgenic pigs to propagate via turka breeding methods.

The analysis of LEA29Y expression in serum and multiple organs of CAG-LEA transgenic pigs revealed that these animals produce a biologically active transgenic product at a considerable level.

They present sex an immune system affected turka transgene expression, but can be maintained until sexual maturity and turka by assisted reproduction techniques. Furthermore, their immune-compromised phenotype makes CAG-LEA29Y transgenic pigs an interesting turka animal model for testing human cell therapies and will provide an important tool for further clarifying the LEA29Y mode of action.

This is an open access article distributed under the terms of the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files.

Turks funders had no role in study design, data collection and sex, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Xenotransplantation, the use of living cells, tissues or organs of animal origin for the treatment of human patients, is a promising approach for overcoming donor organ shortages.

While the transplantation of xenogeneic cornea grafts or pancreas islets is already at an advanced pre-clinical stage or has entered clinical trials [ 12 ], sex use of complex tissue or even complete, vascularized organs is hampered by more diverse graft rejection mechanisms.

Nonetheless, xenotransplantation provides the opportunity to address these problems sex the genetic modification of the donor animals. One sexx turka fundamental advantages of xenotransplantation is the transgenic expression of immune-modulatory agents in xenografts prevents their rejection at the transplantation site while the systemic immunosuppressive load on the recipient is, at the same time, reduced to a tolerable level. The genetic modification tjrka donor pigs for xenotransplantation has so far primarily addressed complement-mediated rejection processes and coagulation incompatibilities [ 3 ], reviewed in [ 4 ].

Some studies have also attempted to overcome cellular rejection of porcine xenografts. The main focus, however, has been on preventing the activation of human T cells by blocking the co-stimulatory signal between CD28 and B7. In different transplantation experiments, the local transgene expression proved sufficient to protect the transplant site from T cell infiltration while the transgenic pigs remained healthy and tirka be propagated by normal breeding. To more effectively manage donor pigs in xenotransplantation, however, the use of several tissues from a single donor is desirable.

In addition, in the case of more complex grafts such as solid organs, expressing an immune modulator in the entire tissue might be superior to its production in a single-cell type only. Such a ubiquitous abundance of T cell blocking agents might, however, result in a chronic impairment of the immune system in the donor organism, which would then affect the reproducibility of these animals, and therefore, the availability of donor organs.

Recently, two studies evaluated the effect of ubiquitous expression of co-stimulatory blockers in pigs. A transgenic pig with an inducible expression of porcine CTLA4-Ig did not show an affected immune system, sex the suitability of such organs turma transplantation experiments remains elusive truka 9 ]. On the other hand, pigs that constitutively produce porcine CTLA4-Ig [ 10 ] were severely immunocompromised and could not be maintained for propagation.

Here, we turka the production of transgenic pigs that ubiquitously express LEA29Y. We characterize the expression pattern and biological function of the transgene as well as its impact on the porcine immune system and evaluate the potential for these transgenic pigs to be propagated turka assisted breeding methods.

The species-specificity of the binding regions of the CTLA4 extracellular domain and its receptors B7. The alignment was performed by use of the ClustalW algorithm of the BioEdit bioinformatics package [ 11 ] and adapted manually. The amino acid consensus sequences as well as the conservation of the respective positions were calculated by the JalView package www. Pregnancies were routinely monitored by ultrasound examination. Birth was induced on gestation day by intramuscular administration of 0.

The founder pigs were kept with the foster mother for 5 weeks before weaning under a conventional agricultural environment and then euthanized at that age to sample tissue for characterization and preservation of primary kidney cells PKC and ear fibroblasts PEF. The animals were submitted to additional antibiotic treatment in the case of acute infections.

To determine of transgene copy numbers and numbers of integration sites, Southern blotting was conducted on the founder and F1 generation animals. At regular time points, blood was drawn from the jugular vein of the animals for isolation of peripheral blood mononuclear cells PBMCs.

According to animal welfare guidelines, pigs were swx in the case of untreatable, severe illness, and samples of their tissues sex preserved for further characterization. After euthanasia of aex founder boars and age-matched WT controls, samples from the heart, kidney, liver, lung, muscle, pancreas, spleen, intestine and blood serum were frozen in liquid nitrogen or fixed in para-formaldehyde and embedded in paraffin.

From re-cloned founder and F1 generation animals, the same tissue, excluding intestine but additionally including adrenal gland, thyroid gland, lymph node and skin tissues, were sampled.

Human serum from a healthy volunteer was dilutedandin a washing buffer and used as tyrka positive control. Immunoreactivity was visualized using 3,3-diaminobenzidine tetrahydrochloride dihydrate DAB brown sexwhile nuclear counterstaining was performed with hemalum blue color.

The potential of LEA29Y containing pig serum to inhibit proliferation of human Tirka cells was investigated by tturka reaction using PBMC from human volunteer donors in the presence of the porcine antigen-presenting cell line L23 according to [ 9 ]. Cultivation was performed in the presence of sera obtained from wild-type and LEA29Y transgenic pigs.

Proliferation was determined after 5 d by [ 3 H]-TdR incorporation. CAG-LEA pigs were examined daily for visible signs of infection and treated with additional antibiotics when necessary. Porcine PBMCs were isolated from WT and transgenic animals by density gradient centrifugation, as described elsewhere [ 18 ], and then cryopreserved.

The lymph nodes of a re-cloned six-month-old transgenic animal were examined histologically as an example of secondary lymphatic tissue to identify the morphological developmental stage of this organ.

The lymph nodes of WT pigs fattening hybrids; age 19 days, 37 days, 4 months, 6 months and 8 months; Ln. The Ln. Afterward, turka samples were embedded xex paraffin. Lymph nodes from the archive of the Institute of Anatomy, Histology and Embryology of the University of Veterinary Medicine Vienna were used as controls. After deparaffinization, the sections were rehydrated and transferred into PBS phosphate buffered saline, pH 7.

Endogenous peroxidase activity was blocked with 0. The sections were pre-treated by microwaving them in 0. To detect proliferating cells, corresponding sections were incubated with a monoclonal mouse anti-Ki67 antibody clone 7B11, dilution ; Invitrogen, Life Technologies Austria, Vienna, Austria.

The reactions were visualized with diaminobenzidine Sigma-Aldrich, Vienna, Austria in 0. All sections were counterstained with hematoxylin, dehydrated and mounted using a medium soluble in xylene. For a negative control, normal goat serum was used instead of primary antibodies data not shown.

All sections were examined using a light microscope. For insemination, approximately 20—25 oocytes with expanded cumulus cells were incubated with srx 1. After insemination, cumulus cells and excess sperm were removed from the eggs, and only eggs with one or two visible polar bodies were used for the experiments. The eggs were assessed based on the ratio of normal fertilization eggs with two pronuclei turka blastocyst formation at day 7.

ET was performed using only selected eggs in which one or two polar bodies were observed. Pregnancy was routinely monitored by ultrasound examination. Birth was induced on day by intramuscular administration of 0.

The piglets were kept with their foster mother for 5 weeks before weaning and tueka housed under sex conventional agricultural environment. We found that the binding domain of CTLA4 is strongly conserved among the examined species, whereas the homology in B7. This is supported by the identification of positively selected sites, where B7.

Interestingly, the difference between human and porcine sequences is higher than expected Fig 2and these differences even affect positions that have been described to be essential for co-stimulatory interaction [ 12 ]. This is particularly remarkable in the C-terminal parts of the B7. These differences are not specific to a human or pig, but rather, represent common properties of the primate and ungulate branches in the evolutionary tree.

The influence of the A29Y and LE modifications that discriminate LEA29Y from CTLA4-Ig is hard to estimate, as the effect of these substitutions is without doubt [ 22 ]; however, there turka no rurka information on how these modifications influence the binding properties of CTLA4 on a molecular level.

It is of note, however, that while A29 is conserved among most species, L at position is unique to human, and the position is also diverse among the other species examined, with a W present in the majority of species, including the pig. Taken together, these findings suggest that the effect of LEA29Y on the porcine immune system should be different turka the described expression of porcine CTLA4-Ig [ 10 ].

The species are arranged according to their phylogenetic pattern, with hurka highlighted in light gray that were identified as essential for binding according to [ 12 ] and positions that differ between human and pig highlighted in dark gray. For each alignment, the consensus amino acid sequence is shown sex and the degree of conservation shown above the alignment.

After transfection of the vector into PFFs and their positive selection, we used a mixed population of genetically modified cell clones in a single SCNT experiment and transferred reconstructed embryos to an estrus-synchronized gilt that delivered three transgenic piglets tudka term.

The offspring thrived until weaning at 5 weeks of age with no obvious signs of infection, at which point they were sacrificed for the characterization of transgene expression. A neomycin resistance cassette neo was used to achieve positive selection. Arrows indicate the position of primers used for genotyping the animals. The Xba I restriction site used for digesting genomic DNA for Southern Blot analysis is indicated as well as the neo-specific probe used for hybridization.

LEA29Y protein abundance in tissues and serum was detected by an ELISA assay specific for the human IgG tail of the synthetic LEA29Y protein, which indicated that the three founder animals each represent distinct transgenic lines, differing presumably by the integration site s of the transgenes Fig 3B.

Although the absolute amount of detected protein differed considerably between the examined tissues, for any given tissue, the ratio to each other among the founder animals remained similar. The localization of LEA29Y expression across a range of potentially transplantation-relevant organs and tissue was evaluated by immunohistochemistry using an antibody specific for the human IgG tail of the transgene.

LEA29Y could be constantly detected in endothelial cells including capillaries as well as in the interstitium. Furthermore, several organ- and tissue-specific cell types, such as pulmonary alveolar cells, exocrine pancreas cells, bile duct cells of the liver, and the stratum spinosum cell layer of the skin, stained immuno-positive.

In endocrine organs, expression of the transgene could be detected in endocrine cells of the Ssex islet, the thyroid gland, and the cortex and medulla of the adrenal gland adrenal gland not shown. Additionally, intravascular serum stained positive for LEA29Y. A LEA29Y was predominantly detected in endothelial cells including capillaries and interstitia, as well as in organ- and tissue-specific cell types such as pulmonary alveolar sex, exocrine pancreas cells, bile duct cells of the liver, or the stratum spinosum cell layer of the skin.

Endocrine cells of the Langerhans islet indicated by an arrow as well as the thyroid sex exhibited strong staining. B No immune staining could be detected in WT control tissue. Inset in pancreas: Giemsa stain of the corresponding pancreas region to demonstrate Langerhans islet.

Lymph node: L. The concentration-dependent signal in serial dilution studies indicated an abundance of biologically active LEA29Y in the serum of the founder animals and confirmed high levels of LEA29Y in the serum of pig and lower as well as detectable sdx of the transgene in and When in vitro stimulation of human PBMCs with porcine L23 cells was performed in eex presence of sera from LEA29Y transgenic pigs, proliferation was significantly reduced in these sera compared to sera from wild-type control animals.

Cells were incubated with serial dilutions of sera from the three founder animals as well as from wild-type controls. Labeled cells were analyzed by flow cytometry. The results are expressed as the mean fluorescence intensity. Cultivation was performed in the presence of sec taken from transgenic and wild-type control pigs.

Interestingly, the turkz effect of the respective founder lines reflected the findings from the expression and binding studies, but the difference in the proliferation assay was not very pronounced.

To evaluate the maximum effect of ubiquitous LEA29Y expression on the porcine sex system, we reproducedthe most potent founder pig, by cloning.

The amino acid consensus sequences as well as the conservation of the respective positions were calculated by the JalView package www. Pregnancies were routinely monitored by ultrasound examination. Birth was induced on gestation day by intramuscular administration of 0.

The founder pigs were kept with the foster mother for 5 weeks before weaning under a conventional agricultural environment and then euthanized at that age to sample tissue for characterization and preservation of primary kidney cells PKC and ear fibroblasts PEF. The animals were submitted to additional antibiotic treatment in the case of acute infections. To determine of transgene copy numbers and numbers of integration sites, Southern blotting was conducted on the founder and F1 generation animals.

At regular time points, blood was drawn from the jugular vein of the animals for isolation of peripheral blood mononuclear cells PBMCs. According to animal welfare guidelines, pigs were sacrificed in the case of untreatable, severe illness, and samples of their tissues were preserved for further characterization.

After euthanasia of the founder boars and age-matched WT controls, samples from the heart, kidney, liver, lung, muscle, pancreas, spleen, intestine and blood serum were frozen in liquid nitrogen or fixed in para-formaldehyde and embedded in paraffin. From re-cloned founder and F1 generation animals, the same tissue, excluding intestine but additionally including adrenal gland, thyroid gland, lymph node and skin tissues, were sampled. Human serum from a healthy volunteer was diluted , and , in a washing buffer and used as a positive control.

Immunoreactivity was visualized using 3,3-diaminobenzidine tetrahydrochloride dihydrate DAB brown color , while nuclear counterstaining was performed with hemalum blue color. The potential of LEA29Y containing pig serum to inhibit proliferation of human T cells was investigated by mixed-lymphocyte reaction using PBMC from human volunteer donors in the presence of the porcine antigen-presenting cell line L23 according to [ 9 ].

Cultivation was performed in the presence of sera obtained from wild-type and LEA29Y transgenic pigs. Proliferation was determined after 5 d by [ 3 H]-TdR incorporation. CAG-LEA pigs were examined daily for visible signs of infection and treated with additional antibiotics when necessary. Porcine PBMCs were isolated from WT and transgenic animals by density gradient centrifugation, as described elsewhere [ 18 ], and then cryopreserved.

The lymph nodes of a re-cloned six-month-old transgenic animal were examined histologically as an example of secondary lymphatic tissue to identify the morphological developmental stage of this organ.

The lymph nodes of WT pigs fattening hybrids; age 19 days, 37 days, 4 months, 6 months and 8 months; Ln. The Ln. Afterward, the samples were embedded in paraffin. Lymph nodes from the archive of the Institute of Anatomy, Histology and Embryology of the University of Veterinary Medicine Vienna were used as controls. After deparaffinization, the sections were rehydrated and transferred into PBS phosphate buffered saline, pH 7.

Endogenous peroxidase activity was blocked with 0. The sections were pre-treated by microwaving them in 0. To detect proliferating cells, corresponding sections were incubated with a monoclonal mouse anti-Ki67 antibody clone 7B11, dilution ; Invitrogen, Life Technologies Austria, Vienna, Austria.

The reactions were visualized with diaminobenzidine Sigma-Aldrich, Vienna, Austria in 0. All sections were counterstained with hematoxylin, dehydrated and mounted using a medium soluble in xylene.

For a negative control, normal goat serum was used instead of primary antibodies data not shown. All sections were examined using a light microscope. For insemination, approximately 20—25 oocytes with expanded cumulus cells were incubated with spermatozoa 1.

After insemination, cumulus cells and excess sperm were removed from the eggs, and only eggs with one or two visible polar bodies were used for the experiments. The eggs were assessed based on the ratio of normal fertilization eggs with two pronuclei and blastocyst formation at day 7. ET was performed using only selected eggs in which one or two polar bodies were observed. Pregnancy was routinely monitored by ultrasound examination. Birth was induced on day by intramuscular administration of 0.

The piglets were kept with their foster mother for 5 weeks before weaning and were housed under a conventional agricultural environment. We found that the binding domain of CTLA4 is strongly conserved among the examined species, whereas the homology in B7. This is supported by the identification of positively selected sites, where B7.

Interestingly, the difference between human and porcine sequences is higher than expected Fig 2 , and these differences even affect positions that have been described to be essential for co-stimulatory interaction [ 12 ]. This is particularly remarkable in the C-terminal parts of the B7. These differences are not specific to a human or pig, but rather, represent common properties of the primate and ungulate branches in the evolutionary tree. The influence of the A29Y and LE modifications that discriminate LEA29Y from CTLA4-Ig is hard to estimate, as the effect of these substitutions is without doubt [ 22 ]; however, there is no considerable information on how these modifications influence the binding properties of CTLA4 on a molecular level.

It is of note, however, that while A29 is conserved among most species, L at position is unique to human, and the position is also diverse among the other species examined, with a W present in the majority of species, including the pig.

Taken together, these findings suggest that the effect of LEA29Y on the porcine immune system should be different from the described expression of porcine CTLA4-Ig [ 10 ]. The species are arranged according to their phylogenetic pattern, with positions highlighted in light gray that were identified as essential for binding according to [ 12 ] and positions that differ between human and pig highlighted in dark gray.

For each alignment, the consensus amino acid sequence is shown below and the degree of conservation shown above the alignment. After transfection of the vector into PFFs and their positive selection, we used a mixed population of genetically modified cell clones in a single SCNT experiment and transferred reconstructed embryos to an estrus-synchronized gilt that delivered three transgenic piglets to term.

The offspring thrived until weaning at 5 weeks of age with no obvious signs of infection, at which point they were sacrificed for the characterization of transgene expression. A neomycin resistance cassette neo was used to achieve positive selection.

Arrows indicate the position of primers used for genotyping the animals. The Xba I restriction site used for digesting genomic DNA for Southern Blot analysis is indicated as well as the neo-specific probe used for hybridization.

LEA29Y protein abundance in tissues and serum was detected by an ELISA assay specific for the human IgG tail of the synthetic LEA29Y protein, which indicated that the three founder animals each represent distinct transgenic lines, differing presumably by the integration site s of the transgenes Fig 3B. Although the absolute amount of detected protein differed considerably between the examined tissues, for any given tissue, the ratio to each other among the founder animals remained similar.

The localization of LEA29Y expression across a range of potentially transplantation-relevant organs and tissue was evaluated by immunohistochemistry using an antibody specific for the human IgG tail of the transgene. LEA29Y could be constantly detected in endothelial cells including capillaries as well as in the interstitium. Furthermore, several organ- and tissue-specific cell types, such as pulmonary alveolar cells, exocrine pancreas cells, bile duct cells of the liver, and the stratum spinosum cell layer of the skin, stained immuno-positive.

In endocrine organs, expression of the transgene could be detected in endocrine cells of the Langerhans islet, the thyroid gland, and the cortex and medulla of the adrenal gland adrenal gland not shown.

Additionally, intravascular serum stained positive for LEA29Y. A LEA29Y was predominantly detected in endothelial cells including capillaries and interstitia, as well as in organ- and tissue-specific cell types such as pulmonary alveolar cells, exocrine pancreas cells, bile duct cells of the liver, or the stratum spinosum cell layer of the skin.

Endocrine cells of the Langerhans islet indicated by an arrow as well as the thyroid gland exhibited strong staining. B No immune staining could be detected in WT control tissue. Inset in pancreas: Giemsa stain of the corresponding pancreas region to demonstrate Langerhans islet. Lymph node: L. The concentration-dependent signal in serial dilution studies indicated an abundance of biologically active LEA29Y in the serum of the founder animals and confirmed high levels of LEA29Y in the serum of pig and lower as well as detectable levels of the transgene in and When in vitro stimulation of human PBMCs with porcine L23 cells was performed in the presence of sera from LEA29Y transgenic pigs, proliferation was significantly reduced in these sera compared to sera from wild-type control animals.

Cells were incubated with serial dilutions of sera from the three founder animals as well as from wild-type controls. Labeled cells were analyzed by flow cytometry. The results are expressed as the mean fluorescence intensity. Cultivation was performed in the presence of sera taken from transgenic and wild-type control pigs. Interestingly, the inhibitory effect of the respective founder lines reflected the findings from the expression and binding studies, but the difference in the proliferation assay was not very pronounced.

To evaluate the maximum effect of ubiquitous LEA29Y expression on the porcine immune system, we reproduced , the most potent founder pig, by cloning. We obtained 2 litters comprising 5 and 8 offspring, respectively, which were maintained in the same way as the founder animals.

Again, the animals were clinically unaffected until weaning but started showing signs of acute and chronic infection shortly afterward, with different progression exhibited in different animals. Most of the animals had to be sacrificed by the age of 3 months, but with intermittent treatment, two animals were raised up to an age of 10 months. Of those animals, we isolated PBMCs at an age of 6 months and characterized various lymphocyte subpopulations Fig 6. Contrary to the hypothesis that LEA29Y primarily acts on T cell activation, the frequency of B cells was significantly reduced in the blood of transgenic animals compared to age-matched WT animals, whereas consequently, the proportion of T cells increased Fig 6A.

Only the frequency of Tregs showed a significant reduction in the transgenic animals Fig 6D. E CD4 and Foxp3 expression of lymphocytes. The data are representative of two three-week-old WT pigs, two tg pigs and two six-month-old pigs. At an age of 6 months, a histological analysis of CAG-LEA transgenic pig lymph nodes as an example of secondary lymphatic tissue revealed retarded morphological development compared to WT animals Fig 8.

Testing consistently revealed no difference between the samples regarding T cell localization and the amount of proliferating T cells. The follicles were nearly free of T cells in all samples.

Regarding the development of follicles, the lymph nodes of the transgenic pigs resembled those at the developmental stage of to day-old WT animals rather than those of age-matched controls. The follicles of transgenic lymph nodes were only approximately half the size of WT follicles at the same age. As in young WT animals, they formed near the trabecules, but the signs of further differentiation, such as reaction centers with heavily proliferating centroblastic and mildly proliferating centrocytic dark and pale zones, respectively, were completely lacking.

Follicles in samples of 6-month-old WT animals were generally larger than in transgenic or young animals compare C versus A and B. Follicles of 6-month-old WT porcine lymph nodes showed a distinct reaction center F with heavily proliferating centroblasts in the dark zone DZ and less proliferating cells in the pale zone PZ —comparing strong and weak Ki67 immunopositivity proliferation marker in DZ and PZ, respectively.

In contrast, marked proliferation of lymphocytes could be detected in either the follicles of transgenic E nor day-old WT animals D. The amount of proliferating T cells did not seem to differ between the samples. The compromised immune system of the first 2 litters of re-cloned CAG-LEA pigs required euthanasia at a maximum age of 10 months without any of them reaching the age of fertility. Interestingly, we did not find evidence for sperm production in the epididymides of even the oldest animals.

To improve vitality, growth and development, we started prophylactic antibiotic treatment around weaning and maintained daily application within a third litter of three re-cloned animals throughout their lifespan.

Although this failed to prevent regular infections, it supported sexual maturation in one animal that was raised up to an age of 10 months. Isolated freeze-thawed sperm from the epididymides of this animal showed sufficient viability and motility for IVF.

The resulting blastocyst rate as well as the number of cells per blastocyst was within the range reported in previous IVF studies [ 26 , 27 ] S1 Fig. This promising finding encouraged us to transfer IVF-generated embryos to 2 foster sows, which delivered a total of 20 offspring.

To exclude the possibility of segregating integration sites of the transgene, we performed Southern Blot analysis on the founder animals as well as on the F1 generation piglets. In both litters, transgenic as well as WT siblings were viable and remained clinically indistinguishable from each other until weaning.

From that time point onward, transgenic piglets started to suffer from regular bouts of infections and stayed behind their WT litter mates in terms of growth and bodily development. The F1 generation animals were individually treated with antibiotics upon clinical indication due to infectious episodes.

None of the animals were treated preventively. Three of the genetically modified F1 animals underwent a full pathological-anatomical examination at the Institute for Veterinary Pathology at LMU Munich. In these instances, no indication of affected organ development was detected. However, the severity of clinical episodes in these CAG-LEA pigs was markedly reduced compared to their counterparts that had been generated by the re-cloning of the original founder. This is consistent with an impaired T cell repertoire in transgenic F1 animals that was evaluated by analysis of PBMCs isolated in 3—4 week intervals between the ages of 10 weeks and 6 months.

However, the baseline fluorescence intensity found with sera from WT animals was higher in Laz cells than in L23 cells. Consistent with data obtained with serum from the founder animal Fig 5A , the sera from all LEA29Y transgenic F1 pigs produced a linear dose-response signal at lower concentrations in L23 and Laz cells and the above described hook, or prozone, effect for higher concentrations. The findings from the founder animal were similarly confirmed in proliferation assays using allogeneic human PBMC and Laz cells as well as xenogeneic porcine L23 cells as stimulators for human PBMC.

Under all experimental conditions, the presence of sera from LEA29Y transgenic animals resulted in clear-cut inhibition of proliferation compared to sera from WT controls Fig 9B.

So far, most studies on transplantation outcomes involving co-stimulation blockade by CTLA4-Ig or LEA29Y have been performed by administering the immuno-modulating agents systemically. Despite LEA29Y having supported graft function as well as improved long-term transplantation outcomes in kidney allotransplantation, the rate of acute graft failure appeared to be higher with administration of LEA29Y than with the use of conventional cyclosporine immuno-suppression [ 28 — 30 ].

Due to the systemic application of the co-stimulation blockade, a detailed examination into the mechanism behind this observation has not been feasible. Providing local T cell regulation by transgene expression in the transplant is a unique opportunity of xenotransplantation.

The predominant approach has long been the transduction of the graft with viral vectors reviewed in [ 31 ] , but this strategy suffers from overall poor transduction efficiency, the limited penetration of the vectors into three-dimensional tissues, the potential immunogenicity of the viral carriers as well as the only transient nature of transgene expression. Stable transgene expression in xenotransplantation has primarily been addressed in the context of complement-mediated rejection and coagulation incompatibilities [ 3 ].

Numerous approaches for overcoming T cell-mediated rejection have also been hypothesized, but their functional relevance to xenotransplantation has been evaluated only to some extent [ 32 , 33 ]. Local LEA29Y or CTLA4-Ig expression is the only T cell-regulating approach for which functionality has been shown in stringent in vivo models, either in pig-to-humanized-mouse transplantation of islets [ 6 ], pig-to-rat transplantation of skin [ 8 ] or pig-to-primate transplantation of corneas [ 34 ].

In all cases, grafts remained protected from T cell-mediated rejection mechanisms for prolonged periods of time. However, to provide multiple grafts from one donor or supply more complex, or even vascularized, grafts, the ubiquitous expression of T cell modulators will be required. An earlier presentation of a transgenic pig with generalized porcine CTLA4-Ig expression reported on the severely compromised immune system of these animals, which proved contradictory to their being raised to maturity [ 10 ].

Here, we present a genetically modified pig line with ubiquitous expression of LEA29Y that i produces a biologically active transgenic product at a considerable level in transplant-relevant organs, ii presents with an immune system evidently affected by the transgene expression, but iii , nevertheless, becomes sexually mature and can reproduce by assisted breeding methods. These animals displayed an immunological phenotype comparable to the founder generation; however, the clinical phenotype appeared less severe, as recurring infections in this generation could be comparably easily treated and usually did not lead to premature euthanasia of the animals.

A number of CAG-LEA F1 pigs are now sexually mature and are presently being bred onto a multi-transgenic background for xenotransplantation purposes. We hypothesize that the reduced clinical phenotype in this F1 generation might be due to the attenuation of cloning side effects that had been present for the founder generation and are known to affect the viability and prosperity of pigs generated by SCNT [ 35 ]. However, importantly, the immunological effects as well as the biological properties of the transgene, as demonstrated in binding studies and proliferation assays, were fully reproduced in F1 animals, thereby confirming the inheritability of the phenotype and offering the possibility for expanding this genetic modification by breeding.

At present, pig models intended for biomedical research can be maintained under conventional agricultural conditions; however, in the future, these conditions will likely no longer be accepted. Many institutions working on pig models are currently in the process of establishing an infrastructure under defined pathogen-free DPF conditions, which will aid the housing of such immune-compromised donor pigs further. The immunological phenotype of CAG-LEA pigs illustrates the competence of the transgene for modifying T cell activation processes in vivo.

This might become an important facet in future studies involving tissue or organs from this pig model in the context of xenotransplantation [ 36 ]. The induction of long-term graft survival in the recipient has long been a holy grail of transplantation research.

This issue was addressed in the current study by determining the effects of chronic CSA administration on LV, ECG, and cardiac histopathological profiles in age-matched male and female rats. As shown in Fig. Panel E shows representative original recordings of ventricular pressure.

The impaired systole was reflected by a 9. The heart rate did not change significantly following CSA administration in male nor female rats, although female control rats had lower heart rate values Table 1 compared with males. On the other hand, CSA had no effect on HR in either of the two rat sexes and caused significant increases in systolic and diastolic BP in female rats only Table 1. With the exception of a significantly longer QT interval in vehicle-treated female rats compared with their male counterparts, the ECG characteristics were similar in both rat sexes Fig.

On the other hand, CSA resulted in a delay in AV conduction in female rats only, as reflected by the significant prolongation in P duration and PR interval and increase in P-amplitude Fig. Panel E shows representative original recordings of ECG.

The cardiac adiponectin level was significantly increased by CSA in female rats while it had no effect in male rats Fig. Serum cholesterol was elevated after CSA administration in male rats only Table 2. Serum levels of uric acid or triglycerides were not affected by CSA in male or female rats. CSA administration was associated with vascular congestion, blood extravasation, and a marked degeneration of cardiac muscle as indicated by the shrinkage of cardiac muscles and absence or pyknosis of cardiac muscle nuclei.

These effects of CSA were demonstrated in both male and female rats Fig. Hollow arrows point to cardiac muscle shrinkage and pyknosis. Solid arrows point to congested vascular space while circles point to extravascular blood cells between cardiac muscle.

The present study is the first to investigate whether the sex of rats influences cardiotoxic manifestations caused by chronic administration of CSA. To perform this study, a multidisciplinary approach was employed that encompassed LV, electrocardiographic, histopathological, and inflammatory elements. The data generated clearly showed that male rats are more susceptible to the negative cardiac effects of CSA. The latter effects, however, were more noticeable in males. Together, compared with their female counterparts, CSA-treated male rats exhibited more deteriorated LV function and increased arrhythmogenesis and ischemic potential, due perhaps to exacerbated inflammatory conditions.

Similar observations of impaired cardiac contractility by CSA were demonstrated in rats in earlier studies 3. This view is supported by the data in the present study, which employed ultra-miniature Millar catheter technology for measuring LV function.

Notably, whereas ESPVR describes the maximum pressure that is generated by the ventricle at any given LV volume end-systole , Tau represents the exponential decay of ventricular pressure during isovolumic relaxation and its prolongation might indicate diastolic dysfunction 16 , The discovery that these CSA effects were more pronounced in male than in female rats provides the first evidence that the cardiac effects of CSA might be sexually differentiated.

In fact, the notion that the male gender is more responsive to CSA cardiotoxicity is substantiated by several other findings in the present study. The electrocardiographic profile is one of the primary diagnostic tools for coronary ischemia and abnormal cardiac rhythms. Previous reports showed that CSA administration over 7 weeks produced signs of cardiac ischemia such as QTc prolongation and increases in T wave amplitude 4.

Nevertheless, the ischemic and arrhythmogenic effects of CSA vary widely and depend on the dose employed and animal species to which it is administered The ECG recordings collected from the current study provide several observations that reinforce the male specificity of the cardiotoxic effect of CSA.

Most importantly, the significant elevations in ST segment and increases in QT and T peak trend intervals observed in CSA-treated male rats are consistent with increased propensity to LV ischemia, delayed ventricular repolarization, and arrhythmogenesis. Although these effects were not observed in CSA-treated female rats, signs of delayed AV conduction such as prolonged P wave duration and PR intervals were evident. This, however, did not appear to have impacted the cardiac dynamics considering the lack of any concomitant changes in heart rate in the female population.

Along this line, immunofluorescence and ELISA measurements of the present study revealed the novel finding that CSA led to the upregulation of the cardiac expression of the inflammatory angiotensin AT 1 receptors in male but not female rats. Renin angiotensin system abnormalities are considered important risk factors for cyclosporin nephrotoxicity in patients with psoriasis An incremental effect of CSA on the density of AT 1 receptors has been reported in mouse medullary thick ascending limb cells Furthermore, elevation in the adiponectin level in cardiac tissues in the present work was observed in female rats only.

Notably, adiponectin is a cytokine that combats cellular inflammation and oxidative stress 13 and mediates the favorable effect of pioglitazone against CSA vasculopathy Moreover, transgenic mice with adiponectin overexpression are tolerant to the aldosterone-induced left ventricular hypertrophy and diastolic dysfunction Together, the less evident inflammatory response along with the increased cardiac adiponectin may account for the diminution of CSA cardiotoxicity in female rats.

In a similar way to the present study, Diwan et al. Considering the paradoxical roles for estrogen protection and testosterone exacerbation in cardiovascular pathologies 30 , it is imperative to comment on the possible interrelationship between gonadal sex hormones and RAS in the sex-dependent CSA cardiotoxicity.

Among several other factors, the molecular basis of the sex specificity in the incidence of cardiovascular disease relates to RAS activity, which is inhibited by estrogen and stimulated by testosterone Moreover, ventricular ACE expression is more abundant in male than in female mice at both mRNA and protein levels, and gonadectomy elicits reciprocal changes in ACE expression in the ventricular tissues of females increases and males decreases Increased ACE abundance in hypertrophied and failing hearts contributes to the local generation of angiotensin II and consequent cardiac remodeling 33 , 34 , 35 and implicates androgen in the increased incidence of angiotensin II-induced aortic aneurysms in male mice as well as in testosterone-treated female mice.

It is tempting to speculate that the directionally opposite effects of sex hormones or their downstream effectors on cardiac inflammatory and RAS profiles might underlie the sex differences in CSA cardiotoxicity. However, as age-matched male and female rats of 6-weeks old were used in the current study, whether similar sex-related patterns of CSA cardiotoxicity would be seen in younger or older rats remains to be seen.

Thus, further studies are clearly needed to investigate this possibility. While we report here on the interaction of a 3-week CSA regimen with cardiac performance and its sex dependence in rats, different features may develop with longer-term months or years regimens.

In addition, because the female studies were performed in randomly cycling rats, it remains unclear as to whether cyclic fluctuations in sex hormonal levels could have impacted the data. The translational information collected from the current study also needs to be corroborated clinically. In summary, the current study establishes the importance of rat sex in defining the magnitude of CSA cardiotoxicity.

While functional and histopathological signs of LV damage were evident in both sexes, electrocardiographic and Millar electrophysiological studies revealed more deterioration in LV systolic and diastolic functions in the male gender together with arrhythmogenic and ischemic signs due perhaps to the concomitant deterioration of the inflammatory state.

Male and female Wistar rats 6 weeks old; King Abdulaziz University, Kingdom of Saudi Arabia were housed 3—4 rats per cage in clear polypropylene cages and maintained under constant environmental conditions with equal light—dark cycles. Rats had free access to a commercially available rodent pellet diet and water.

Then, cardiac hemodynamics and blood pressure were recorded using a microtip catheter inserted in the right carotid artery through a small opening in the artery and advanced to the left ventricle as described in our previous studies Cardiac conductivity was determined by surface ECG Invasive real time recording of cardiac hemodynamic was performed according to the method described in our previous reports 38 , The PV catheter is capable of measuring both ventricular pressure and volume simultaneously and continuously from the intact beating hearts of rats.

In addition, the other pressure sensor records arterial pressure at the mean time. Systolic and diastolic BP was monitored via the BP module. Fixed heart tissue section slides were deparaffinized in xylene and rehydrated in ethanol and distilled water.

Slides were then immediately transferred into a humidity chamber. Images were acquired with identical acquisition parameters, with minimum excitation and gain. For presentation purposes, the level of the fluorescence images were equally adjusted after the fluorescence quantifications were carried out on non-manipulated images. Sections treated with the secondary antibody alone did not show specific fluorescence while incubating the primary antibody with the blocking peptide significantly reduced the signal.

Probability levels less than 0. How to cite this article : El-Bassossy, H. The inflammatory state provokes sexual dimorphism in left ventricular and electrocardiographic effects of chronic cyclosporine in rats. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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