Optimization of cytotoxicity assay by real-time, impedance-based cell analysis.

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.

Farm-by-farm analysis of microsatellite, mtDNA and SNP genotype data reveals inbreeding and crossbreeding as threats to the survival of a native Spanish pig breed.

The Chato Murciano (CM), a pig breed from the Murcia region in the southeastern region of Spain, is a good model for endangered livestock populations. The remaining populations are bred on approximately 15 small farms, and no herdbook exists. To assess the genetic threats to the integrity and survival of the CM breed, and to aid in designing a conservation program, three genetic marker systems - microsatellites, SNPs and mtDNA - were applied across the majority of the total breeding stock. In addition, mtDNA and SNPs were genotyped in breeds that likely contributed genetically to the current CM gene pool. The analyses revealed the levels of genetic diversity within the range of other European local breeds (H(e) = 0.53). However, when the eight farms that rear at least 10 CM pigs were independently analyzed, high levels of inbreeding were found in some. Despite the evidence for recent crossbreeding with commercial breeds on a few farms, the entire breeding stock remains readily identifiable as CM, facilitating the design of traceability assays. The genetic management of the breed is consistent with farm size, farm owner and presence of other pig breeds on the farm, demonstrating the highly ad hoc nature of current CM breeding. The results of genetic diversity and substructure of the entire breed, as well as admixture and crossbreeding obtained in the present study, provide a benchmark to develop future conservation strategies. Furthermore, this study demonstrates that identifying farm-based practices and farm-based breeding stocks can aid in the design of a sustainable breeding program for minority breeds.

Presence of Pig IgG and IgM in Sera Samples From Baboons After an Orthotopic Liver Xenotransplantation.

INTRODUCTION: The immunorejection in xenotransplantation has mostly been studied from the host's immune system activation point of view and there is very little information about the graft-vs-host reaction. OBJECTIVES: To validate an enzyme-linked immunosorbent assay (ELISA) test for porcine IgM and IgG quantitation, the assessment of porcine IgG and IgM in sera samples from baboons after liver orthotopic xenotransplantation or in human plasma after xenotransfusion through pig organs, and to assess the presence of porcine immunoglobulin in a baboon after plasmapheresis to a complete change of plasma after 4 passages through pig liver. MATERIALS AND METHODS: Two commercial ELISA kits for pig IgG and IgM quantitation were evaluated for cross reactivity with samples from baboons, Rhesus monkeys, squirrel monkeys, and humans. Then, samples from 18 baboons after orthotopic liver xenotransplantation were studied for porcine IgG and IgM. To understand the phenomenon, human plasma samples after xenotransfusion 1, 2, 3, or 4 times through liver or kidney were assessed for porcine IgG presence and finally, the porcine IgG were quantified in sera samples obtained during more than 4 years from a baboon after plasmapheresis with baboon plasma after xenotransfusion 4 times through a pig liver. RESULTS: Porcine IgG and IgM were found in samples from xenotransplanted baboon during all survival. The quantity of porcine IgG in plasma after xenotransfusion correlated with the number of passages through the pig liver, and the IgG were completely cleared from the baboon 16 days after plasmapheresis and complete substitution of plasma after 4 xenotransfusions through a pig liver.

Evidence that periweaning failure-to-thrive syndrome (PFTS) has a genetic predisposition.

Genetic susceptibility or resistance to diseases is currently drawing increasing attention. This work describes two different breeding herds showing signs of periweaning failure-to-thrive syndrome (PFTS), an emergent swine disease. The disease was diagnosed based on clinical picture and confirmed by histopathology. The possibility of main infectious pathogens was ruled out by immunohistochemistry and PCR. In a simple approach, sires of the affected piglets have been determined using microsatellite paternity analysis, including a healthy group in each case. In each of the two farms, a single boar was found to have sired 45-50 per cent sick animals. Removal of this sire from two farms resulted in a significant decrease in the prevalence of the disease among the offspring, in accordance with other two cases diagnosed, although without including a control group. Since the analysed animals belonged to three different genetic lines, these findings point to the existence of individual genetic susceptibility to this syndrome.

Validation of a quantitative polymerase chain reaction method for human Alu gene detection in microchimeric pigs used as donors for xenotransplantation.

This work was undertaken to evaluate whether a real-time quantitative polymerase chain reaction (qPCR) is as an adequate method for detection and quantification of human-specific DNA elements (Alu gene) in tissues and blood samples of pigs in which human stem cells were engrafted. Real-time qPCR quantification was performed with the use of previously described primers. The human DNA was mixed with different quantities of porcine DNA. The primer concentration and specificity, the qPCR efficiency, the quantification variations due to different porcine DNA concentrations, and the dissociation curve produced by the assay were evaluated. The qPCR proved to be specific, robust, with a reproducible and specific bimodal melting curve. High porcine DNA concentration produced subquantification, especially with low human DNA quantity. However, the assay proved to be useful for the detection of chimeric piglets produced by human cells injected in utero, because the effect caused by the porcine DNA interference was corrected in quantification of human DNA from piglets.

Are veterinary students in favour of xenotransplantation? Comparative opinion study in a Brazilian and a Spanish university.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: Both Brazilian and Spanish veterinary students had favorable attitudes toward xenotransplantation.

Porcine endogenous retrovirus copy number in different pig breeds is not related to genetic diversity.

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.

Assessment of in vitro heparin complement regulation capacity during real-time cell analyzer antibody-mediated cytolysis assay: compatibility studies for pig-to-baboon xenotransplantation.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: The addition of heparin did not reduce antibody- and complement-mediated cytolysis assessed in vitro by RTCA in pig-to-baboon compatibility assays.

Generation of human-to-pig chimerism to induce tolerance through transcutaneous in utero injection of cord blood-derived mononuclear cells or human bone marrow mesenchymals cells in a preclinical program of liver xenotransplantation: preliminary results.

OBJECTIVE: Using a percutaneous ecoguided injection system to obtain chimeric piglets through a less invasive and traumatic technique than previously reported. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: Transcutaneous echo-guided injection succeeded to produce chimeric piglets without disadvantages to the sow or the fetuses and avoiding abortions or fetal death.

Donor-graft compatibility tests in pig-to-primate xenotransplantation model: serum versus plasma in real-time cell analyzer trials.

INTRODUCTION: 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. METHODS: 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. RESULTS: The lowest CImin, highest TCImin, and Trec observed for EDTA plasma showed significant differences from other samples (P < .001). DISCUSSION: 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.