[show abstract][hide abstract] ABSTRACT: This paper presents an optimized procedure for assessing an immune-mediated cytotoxicity, produced after the addition of human and baboon serum to transgenic porcine fibroblasts. This procedure is performed with the xCELLigence Real-Time Cell Analyzer (RTCA). The xCELLigence system measures the impedance variations in the culture media of a 96-well microelectronic plate, and shows the changes in cell number and morphology in a real-time plot. However, different factors need to be optimized before developing an RTCA assay. Thus, we studied the influence of several variables, such as the number of cells seeded, the time the cells were allowed to grow before the tests, the serum concentration and the addition of rabbit complement. The findings were confirmed by the WST-1 classical cytotoxicity test. The results showed that 7.5 × 10(3) cells seeded per well produced the adequate CI in 10 h. The area under the curve and the CImin versus concentration values showed a very high correlation index (r(2) = 0.966 and r(2) = 0.92 for the first 50 h after challenge, respectively), proving that CI variations are directly proportional to the quantity of serum added. The addition of complement resulted in lower CImin values. Therefore, both the cytolysis level with and without exogenous complement addition had to be assessed. There was a high correlation between the relative cytotoxicity assessed by WST-1 and the CI obtained by RTCA when exogenous complement was not added (r(2) = 0.827; p < 0.001). The correlation was average when rabbit complement was added (r(2) = 0.523; p = 0.046). In conclusion, culture conditions have an important influence on RTCA cytotoxicity assays.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] 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.
[show abstract][hide abstract] ABSTRACT: Our goal was to determine the hemodynamic changes that are witnessed during the initial minutes of reperfusion of the graft in liver xenotransplantation from pig to baboon.
We studied a group of 12 baboons undergoing transplantation of a pig liver via the classic technique with arterial anastomosis to the aorta. The anesthesia technique was similar to that used in humans. Hemodynamic monitoring, due to the size of the recipient, consisted of heart rate (HR), mean arterial pressure (MAP), and central venous pressure (CVP) recorded at the beginning and end of each of the three phases: preanhepatic (A1, A2), anhepatic (B1, B2), and neohepatic (C1 and C2). We aimed to maintain the following values by means of crystalloids, colloids, and blood derivates: HR >50 beats/minute; MAP >60 mm Hg; and CVP >10 mm Hg.
Both HR and CVP remained unchanged throughout the procedure. MAP droped briefly after vascular clamping (B1) but not on reperfusion (C1).
In cirrhotic patients there is an autonomic dysfunction, demonstrated as cardiovascular instability at times like the clamping of major vessels and reperfusion of the graft. On the other hand, the intact baboon has an intact nervous system. After vascular clamping, the sharp decrease in venous return lead to an adequate vasopressor response. Likewise, the extreme vasodilatation involved with reperfusion managed to maintain MAP above 70 mm Hg.
[show abstract][hide abstract] ABSTRACT: The search for alternative sources for transplant organs leads us to the search for animals as an inexhaustible source of organs. The objective of this study was to analyze whether livers from polytransgenic pigs expressing the human complement regulatory proteins CD55 (hDAF), CD59, and alfa alpha1,2-fucosyltransferase (H-transferase), protected against hyperacute rejection after orthotopic liver xenotransplantation to a baboon and also to study pig liver function in a nonhuman primate.
Nine liver transplants from pig to baboon were divided into two groups: a control group (n = 4) of genetically unmodified pigs and an experimental group (n = 5) of pigs transgenic for CD55, CD59, and H-transferase as donors. All the donating piglets obtained through hysterectomy were maintained in specific pathogen-free conditions. The selection of transgenic pig donors followed demonstration of transgene expression using monoclonal antibodies (antiCD55, antiCD59) and immunohistological studies on liver biopsies.
All animals in the control group developed hyperacute rejection with survival rates less than 16 hours without function of transplanted livers. In the experimental group none of the animals suffered hyperacute rejection. Survival in this group was between 13 and 24 hours. The livers were functional, producing bile and maintaining above 35% prothrombin activity. Only in one case was there primary dysfunction of the xenograft.
Polytransgenic livers for complement regulatory proteins prevent hyperacute rejection when xenotransplanted into a baboon.
[show abstract][hide abstract] ABSTRACT: The deficit in transplant organs is encouraging research into stem cells and xenotransplantation. However, many studies have shown that using animals for human transplantation could be rejected by society. The objective here was to analyze the attitude of patients on the waiting list toward a possible transplant of an organ of animal origin.
Patients on the waiting list for kidney and liver transplants including last year (n = 96) underwent a direct interview by an independent health professional from the transplant unit. Using a psychosocial survey, an evaluation was made of attitudes toward donation of organs of animal origin and its various options. Student t test and the chi-square test were used for analysis.
If results from xenotransplantation could be superimposed onto those of human transplantation, 71% would accept such an organ. In the case of the kidney, 83% would accept, 4% would not, and 13% have doubts; as opposed to 60%, 12%, and 28%, respectively, of liver cases (P < .05). Supposing that the results were worse than in human organs, only 26% would accept an animal organ. Thus, for kidney, 33% would accept it, 48% would not, and 20% would have doubts; and for liver, it would be 20%, 50%, and 30%, respectively. In a life-threatening situation 98% would accept an animal organ as a bridge of hope in the wait for a human organ. In addition, if the organ functioned correctly, 98% would keep the animal organ, thus avoiding an intervention to substitute a human organ.
If xenotransplantation became a clinical reality, acceptance of an animal organ by patients on the waiting list would be low, especially if the results could not be superimposed onto human ones. Only its use as a bridge until the arrival of a human organ would increase its acceptance.
[show abstract][hide abstract] ABSTRACT: The aim of this study was to analyse the efficacy of 2 second mobilization (MB) protocols in 2 groups of patients who failed to obtain enough peripheral blood progenitor cells (PBPC) in the first MB. In 1 group (8 patients), 10 microg/kg of G-CSF was administered, and in the other group (8 patients), a double dosage (10 microg/kg twice a day) was administered. Both groups of patients received Cyclophosphamide (1.5 g/kg) 10 days before the apheresis. No difference was found among both groups of patients in diagnosis, previous chemotherapy, and time elapsed after the first MB. Administration of higher doses of G-CSF decreased the number of apheresis needed in the second MB to complete 2 x 10(6)/kg of CD34+ cells. It also increased the number of patients who achieved sufficient CD34+, namely, 75% versus 50%.
[show abstract][hide abstract] ABSTRACT: hDAF transgenic pigs do not display the inherent hyperacute rejection reactions of pig-to-primate xenotransplants. The purpose of this study was to determine the immunopathologic phenomena following an hDAF transgenic pig hepatic orthotopic xenotransplant into a baboon.
Donor animals were unmodified pigs (n=4) and hDAF transgenic pigs (n=2). Recipient animals were baboons (Papio anubis). Liver biopsies were immunostained using monoclonal antibodies to C3, C5b-9, IgG, IgM, CD2, CD4, CD8, CD68, CD20, Bric 216, CD31, and fibrin, and polyclonal antibody to C4.
hDAF transgenic grafts showed IgG, IgM, and C4 endothelial deposits. However, no fibrin, C3, or C5b9 deposits were observed after reperfusion. hDAF xenografts displayed CD31 staining in the portal spaces, perilobular areas, and at hepatic sinuisoidal levels. The baboon that lived for 4 days displayed either CD4 or CD8 T-cells periportal infiltrate.
Future studies will seek to determine the physiologic role of CD31 hepatic sinusoidal expression in transgenic xenotransplants, and will also study the role of T-cell infiltrates in xenograft rejection.