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ABSTRACT: Some biomedical research procedures, such as organ xenotransplantation, usually require intensive hemotherapy. Knowledge of the whole phenotype of blood donor and graft could be useful in the field of xenotransplantation. Human and simian-type categories of blood groups have been established and they can be tested by standard methods used for human blood grouping. The aim of this work was to study the incidence of non-ABO blood group systems in different species of non-human primates, which are employed in biomedical research. The phenotype of Rh, Lewis, Kidd, Kell, MNSs, Lutheran, P and Duffy antigens was investigated in olive baboon (n = 48), chacma baboon (n = 9), Guinea baboon (n = 14), Rhesus macaque (n = 38) and squirrel monkey (n = 30) by using commercial microtyping cards. Kell, Lutheran, Kidd and Duffy antigens have been detected in all species, Rh in squirrel monkey, MNSs in rhesus macaque and squirrel monkey, and Lewis in baboon and rhesus macaque. There were differences in frequency and haemagglutination scores between species regardless of their gender and age. The main differences were found in squirrel monkey when compared with baboons and macaques. This typing system provides a tool to assess the presence of antigens in animals used for experimental procedures, such as xenotransplantation and xenotransfusion.
Laboratory Animals 04/2013; 47(2):100-5. · 1.21 Impact Factor
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L Mendonça,
L Martínez-Alarcón,
A Ríos,
G Ramis, J J Quereda,
J M Abellaneda,
A Saez-Acosta,
J M Herrero,
A Muñoz,
P Ramírez,
P Parrilla
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ABSTRACT: The shortage of organs has made it necessary to search for new alternatives such as xenotransplantation. However, the use of animal organs could be opposed by society and the personnel involved in its implementation. This study aimed to analyze the attitude of veterinary degree students in a Brazilian university towards xenotransplantation, to determine factors that affect its acceptance, and to compare the attitudes among a control group of veterinary degree students in a Spanish university.
Of the 422 students registered for a veterinary course from 2010 to 2011, 374 were surveyed with a questionnaire completion rate of 89%. Attitudes were evaluated using a validated questionnaire that was self-administered administered anonymously. The process was coordinated by an independent health care worker. We applied the student t and the chi-squared-tests for statistical analysis.
If xenotransplantation was confirmed as a clinical reality, 90% (n = 338) of Brazilian students would accept the use of a xenotransplanted organ; 94% (n = 350), tissue; and 97% (n = 360), cell xenotransplantation. Attitudes toward xenotransplantation were not determined by the academic year, any psychosocial variable, or attitudes toward deceased human organ donation (P = .167). However, the attitudes would be affected by a belief that the transplanted animal organ would not change anything (P = .001). Interaction with other people was also related to more favorable attitudes (P = .015). Subjects who expressed a more favorable attitude tended to more readily accept cell (P = .000) or tissue xenotransplantation (P = .000). In Spain (control group), the results were similar: 91% (n = 436) would accept a xenotransplantation; 95% (n = 457) tissue; and 97% (n = 467), cell xenotransplantation. Also, this attitude was not affected by the academic year, any psychosocial variable, or attitude toward organ donation (P = .779).
Both Brazilian and Spanish veterinary students had favorable attitudes toward xenotransplantation.
Transplantation Proceedings 04/2013; 45(3):1046-9. · 1.00 Impact Factor
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J M Abellaneda,
G Ramis,
L Martínez-Alarcón,
M J Majado, J J Quereda,
J M Herrero-Medrano,
L Mendonça,
O García-Nicolás,
M Reus,
C Insausti,
A Ríos,
A López-Navas,
M R González,
F J Pallarés,
A Munoz,
P Ramírez,
P Parrilla
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ABSTRACT: Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported.
The two types of human cells included umbilical cord blood mononuclear elements and mesenchymal stem cells cultured from bone marrow. Four sows at gestational day 50 were anesthetized. A needle was inserted through the skin and uterine wall to reach the peritoneal cavity of the fetuses under continuous ultrasound guidance. Fourteen piglets were injected with various cell concentrations.
All sows carried pregnancies to term yielding 69 piglets, among which 67 were alive and two mummified. Two piglets died during the first 48 hours of life. Chimerism was detected using flow cytometry and by quantitative polymerase chain reaction (q-PCR) to detect Alu gene in blood or tissues samples. The analysis detected blood chimerism in 13 piglets (21%) by flow cytometry and the presence of the human Alu gene in 33 (51%) by q-PCR. The results suggest cell trafficking between littermates after in utero injection.
Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death.
Transplantation Proceedings 07/2012; 44(6):1574-8. · 1.00 Impact Factor
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G Ramis,
L Martínez-Alarcón,
M J Majado, J J Quereda,
L Mendonça,
J M Herrero-Medrano,
J M Abellaneda,
A Ríos,
A López-Navas,
P Ramírez,
A Muñoz
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ABSTRACT: To assess the effect of sodium heparin concentrations on antibody- and complement-mediated cytolysis by means of a real-time cell analyzer system (RTCA) investigating the complement regulation ability of heparin to reduce or prevent hyperacute in an in vitro model of pig-to-baboon xenotransplantation.
Fibroblasts isolated from the skin of two transgenic pigs were cultured in microelectronic 96-well plates for 9 hours. Then, we added 20 μL of normal sera from two healthy adult olive baboons (Papio anubis) or two volunteer healthy humans. Simultaneous cultures had added heparin at 3.5, 5, 7.5, 15, and 30 IU. Moreover, rabbit complement was added for the exogenous complement group (ExC) versus the other group only with the complement present in the sera as an endogenous complement group (EnC). Cellular cultures were monitored over 150 hours after challenge. With cellular index (CI) data recorded by the xCELLigence software system, we calculate area under the curve versus concentration (AUC) and minimum CI (CImin) versus concentration.
All cultures showed decreased CI after challenge with human or baboon sera. There was a high correlation for AUC (r(2) > 0.90) and CImin versus concentration (r(2) > 0.970) during the first 40 hours postchallenge among the EnC group, regardless of human or baboon sera. However, there was no correlation for AUC and CImin for the ExC group. There was a reduction of CImin related to increased heparin concentrations.
The addition of heparin did not reduce antibody- and complement-mediated cytolysis assessed in vitro by RTCA in pig-to-baboon compatibility assays.
Transplantation Proceedings 07/2012; 44(6):1584-8. · 1.00 Impact Factor
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ABSTRACT: The risk of zoonoses is a major obstacle to xenotransplantation. Porcine endogenous retrovirus (PERV) poses a potential risk of zoonotic infection, and its control is a prerequisite for the development of clinical xenotransplantation. The copy number of PERV varies among different breeds, and it has been suggested that the PERV integrations number is increased by inbreeding. The purpose of this study was (i) to examine the copy number of PERV in different Spanish pig breeds, Spanish wild boar and commercial cross-bred pigs from five different farms and genetic background (CCP1-CCP5) and (ii) to investigate the correlation between PERV copy number and the genetic background of the pigs in order to improve the selection of pigs for xenotransplantation. PERV copy number was determined by quantitative, real-time polymerase chain reactions. Thirty-four microsatellite markers were genotyped to describe the genetic diversity within populations (observed and expected heterozygosities, Ho and He, respectively) and the inbreeding coefficient (F). Pearson's correlation coefficient was used to determine the relationship between PERV copy number and Ho, He and F. The copy number of PERV among different pig breeds was estimated to range between three (CCP1) and 43 copies (Iberian Pig). Statistical differences were found among the studied populations concerning PERV copy number. No correlation was found between the PERV copy number and the heterozygosity (calculated at an individual level or at a population level) or the inbreeding coefficient of each population. Our data suggest that pigs inbreeding does not increase PERV copy number and support the idea that careful selection of pigs for organ donation with reduced PERV copy number will minimize the risk of retrovirus transmission to the human receptor.
Zoonoses and Public Health 02/2012; 59(6):401-7. · 1.89 Impact Factor
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ABSTRACT: The concentrations of haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty-three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty-one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).
The Veterinary record. 11/2011; 170(1):21.
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ABSTRACT: To design a real-time quantitative polymerase chain reaction (q-PCR) to assess gene expression for hCD55, hCD59, and hCD46 in polytransgenic (PT) pigs used as xenograft donors for orthotopic liver xenotransplantation using a pig-to-baboon model.
Three pairs of primers were designed using PrimerBlast and mRNA of hCD55, hCD59, and hCD46 sequences. Blood samples from five PT pigs (two males and three females) were used to isolated peripheral blood mononuclear cells (PBMCs) by means of Ficoll gradients. After DNAase digestion of isolated mRNA, we synthesized cDNA. Using SYBR-Green chemistry of q-PCR, we constructed a standard curve. Two wild-type (WT) pigs were used as negative controls, and PBMCs from two healthy human volunteers as positive controls. The amplicon length was assessed by means of agarose gel electrophoresis and PCR products, sequenced.
We observed amplification for hCD55, hCD59, and hCD46 in all samples from the five PT pigs except for hCD55 and hCD46 in one male PT pig. Neither the human samples nor the negative controls showed amplification. The expected amplicon length was confirmed; sequencing showed high homology with human mRNA for the three proteins and no match with any known pig sequence.
The q-PCR allowed detection of animals with the highest gene expression for hCD55, hCD59, and hCD46 for xenograft donors in transplantation experiments.
Transplantation Proceedings 10/2010; 42(8):3235-8. · 1.00 Impact Factor
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ABSTRACT: To validate the use of a microelectronic real-time cell analyzer system (RTCA) we developed a complement-mediated antibody cytotoxicity assay to investigate the compatibility of a graft and a recipient in pig-to-baboon xenotransplantation.
Fibroblasts isolated from the skin of five hCD55, hCD59, and hCD46 transgenic pigs (TP) were cultured in 96 microelectronic well plates for 17 hours. Then, we added to each microwell 20 μL of normal sera from nine healthy adult olive baboons (Papio anubis)-three males and six females. The evolution of the cell culture was assessed every 3 minutes during the pretreatment period, at 11 hours postaddition, and every 30 minutes from 12 to 96 hours. Simultaneously, we performed a 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Fibroblasts from wild-type (WT) pigs were used as positive controls and microwells without serum addition from each TP as negative controls. The RTCA results were expressed as a normalized cellular index (NCI).
Differences were observed between the five TP fibroblasts and the WT fibroblasts, with greater cytotoxicity on WT cells. Among TP, a higher cytolytic level was observed in males than females. The MTT results correlated with NCI at different times, with the minimum NCI and with the time to for NCI recovery before serum addition. The correlation was lower than that previously reported in environmental toxicity assays.
RTCA allows a long-term assessment of the immunocytotoxic effect of baboon sera on pig cells, providing a suitable tool to perform compatibility tests for xenotransplantation.
Transplantation Proceedings 10/2010; 42(8):3239-43. · 1.00 Impact Factor
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ABSTRACT: Transplantation or transfusion with ABO disparity is a cause for rejection or for severe hemodynamic alterations. ABO groups in pigs are commonly an unknown variable, which has been previously assessed by means of hemagglutination tests or immunohistochemical procedures on tissues. Herein, we have reported a simple method using commercial microcards for human ABO typing. However, the reagents directly derived from human sera included in these cards can result in false determinations due to alpha-gal interference. The ABO groups of 19 wild-type pigs (Landrace x Large White) were assessed using 2 commercial cards: Human sera-based and monoclonal antibody-based cards. The human sera cards determined that 8 pigs belonged to the AB group and 11 to the B group. The monoclonal antibody cards determined that 8 pigs belonged to the A group and 11 to the O group. None of the pigs showed reactions to Rh1 antibodies. Because the B group has not been described in pigs, the reaction in human sera cards represented an interference with alpha-gal antigen, a molecule structurally similar to the B blood antigen. Thus, microtyping cards based on monoclonal antibodies provided simple, quick way to assess ABO groups in pigs used for xenotransplantation. ABO concordance should always be investigated for these types of procedures.
Transplantation Proceedings 42(6):2146-8. · 1.00 Impact Factor
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G Ramis,
L Martínez-Alarcón,
M J Majado, J J Quereda,
L Mendonça,
J M Herrero-Medrano,
J M Abellaneda,
K Gomes-Coelho,
A López-Navas,
A Ríos,
P Ramírez,
A Muñoz
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ABSTRACT: Various strategies have been designed to assess in vitro donor-graft compatibility in pig-to-primate xenotransplantation models. Most of them are based on a cytolysis assessment by exposing donor tissue to host serum with investigations by flow cytometry, and photocolorimetric levels. The aim of this study was to analyze the difference in cytolysis produced by sera and plasma obtained using various anticoagulants, or containing high versus low levels of platelets.
The cytolysis trials were performed using an xCELLigence real-time cell analyzer (RTCA) in a cell model involving transgenic pig fibroblasts exposed to sera (S) or plasma obtained using EDTA, Li-heparin, or Na-heparin in combination with plasma containing high versus low content of platelets. Samples were obtained from two baboons and five volunteer human donors. Evolution of fibroblast cell growth was assessed by RTCA as the cell index (CI). After 9 hours of growth, cells were exposed to 20 μL of each sample. The minimum CI (CImin), time to CImin (TCImin), and time to reach the CI observed before compound addition (Trec) were recorded for each microwell.
The lowest CImin, highest TCImin, and Trec observed for EDTA plasma showed significant differences from other samples (P < .001).
On the basis of this study, using the RTCA assay, heparinized plasma produced complement inhibition and with undervaluation of the cytolysis reaction. EDTA plasma produced total death of most of cultures. The most accurate sample matrix seems to be serum.
Transplantation Proceedings 43(1):249-53. · 1.00 Impact Factor
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ABSTRACT: To assess the presence of irregular xenoantibodies against human red blood cells (RBCs) in 6 primate species used in xenotransplantation and other experimental procedures.
Serum samples from 109 baboons of 4 different species (olive, chacma, sacred, and Guinea), 38 rhesus macaques, and 30 squirrel monkeys were tested for irregular xenoantibodies using an agglutination test using human RBCs of known phenotype for Rh, Kell, Kidd, Lewis, Lutheran, P1, and Duffy antigens, commercially available as RBC I, II, and III.
We found hemagglutination for RBC I in 49%, 22%, 100%, 57%, 32%, and 33% of olive, chacma, sacred, and Guinea baboons, rhesus macaques, and squirrel monkey, respectively. The frequency for RBC II was 49%, 50%, 100%, 57%, 37%, and 33%, respectively, and for RBC III was 56%, 37%, 100%, 79%, 34%, and 33%, respectively. There were differences in frequency depending on the sex of the rhesus macaques; all 3 RBCs tested were higher in the females: 44% vs 0%, P = .008; 48% vs 1%, P = .02, and 44% vs 9.1%, P = .04 for RBC I, II, and III, respectively. There were differences due to age in only olive baboons, and a higher frequency in younger animals compared with juvenile, subadult, and adult animals for all 3 human RBCs.
Assessment of irregular antibodies in the presence of primate serum should be taken into account during any experimental xenotransplantation protocol.
Transplantation Proceedings 42(1):387-9. · 1.00 Impact Factor
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ABSTRACT: The shortage of available transplant organs has made it necessary to search for new alternatives, one of which is xenotransplantation. However, the use of animal organs and the personnel involved in its implementation could face opposition. Our objective was to analyze the attitudes of veterinary degree students in a Spanish university toward xenotransplantation and to determine the factors that affect its acceptance.
Of the 515 students registered in a veterinary degree course (2007-2008), we surveyed 482 94% of whom completed the questionnaire. Attitudes toward organ xenotransplantation were evaluated using a validated, self-administered questionnaire concerning organ donation, which was completed anonymously. We applied Student's t-tests and chi(2) tests.
If xenotransplantation was confirmed as a clinical reality, 91% (n = 436) of those students surveyed would accept a xenotransplanted organ, whereas 9% (n = 46) would not. Furthermore, 95% (n = 457) would accept tissue xenotransplantation and 97% (n = 467) cell xenotransplantation. Attitudes toward xenotransplantation were not affected by the academic year in which a student was studying, even when this was the year in which it was taught as a subject. Attitudes were not associated with any pyschosocial variable or attitude toward deceased organ donation; (P = .779). The students who believed that the demand for organs is not covered had a more favorable attitude toward xenotransplantation than those who think that there is no shortage (91% vs 70%; P = .027).
Veterinary students had favorable attitudes toward xenotransplantation, assuming that the animal organs functioned as well as human organs. Therefore, these students could play important roles in the future promotion of this technique.
Transplantation Proceedings 42(6):2130-3. · 1.00 Impact Factor
