Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

The jigsaw of PRRSV virulence.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of the, probably, most economically important disease for the pig industry worldwide. This disease, characterised by producing reproductive failure in sows and respiratory problems in growing pigs, appeared in the late 1980s in the United States and Canada. Since its appearance, strains capable of producing higher mortality rates as well as greater severity in clinical signs and lesions than classical strains have been identified. However, since the first reports of these "virulent" PRRSV outbreaks, no homogeneity and consensus in their description have been established. Moreover, to the authors' knowledge, there is no published information related to the criteria that a PRRSV strain should fulfil to be considered as a "virulent" strain. In this review, we revise the terminology used and gather the information related to the main characteristics and differences in clinical signs, lesions, viral replication and tropism as well as immunological parameters between virulent and classical PRRSV strains and propose a first approximation to the criteria to define a virulent PRRSV strain.

Prevalence of mycoplasma-like lung lesions in pigs from commercial farms from Spain and Portugal.

BACKGROUND: Mycoplasma hyopneumoniae causes a chronic respiratory disease that produces important economic losses due to poor productive performance, increased mortality and costs for several control strategies. The prevalence of mycoplasma-like lesions (MLL) at abattoir has been widely studied in different countries, making use of different scoring systems. However, most of them are difficult to apply in abattoirs with high number of pigs sacrificed per hour. For that reason, it is necessary to adapt the scoring system to the reality of the modern abattoir, even if there is a loss of accuracy. Our purpose was to investigate the prevalence and severity of MLL at abattoirs in Spain and Portugal using a 0 to 5 scoring system adapted to abattoirs with high number of sacrificed pigs per hour and to highlight the histopathological diagnosis as confirmatory method to identify patterns of pneumonia correlated to gross lesions. RESULTS: Cranioventral pulmonary consolidation, a typical MLL, was the most frequent lung lesion (30.97 %) detected at the abattoir, followed by dorsocaudal infarcts with pleurisy (12.51 %) and pleurisy alone (6.26 %). The average score for all examined lungs at abattoir was 1.99 out of 5 points. The histopathological study revealed that the 78.17 % of the randomly selected lungs with MLL presented microscopic lesions compatible with M. hyopneumoniae infection. Most bronchointerstitial and interstitial pneumonia lesions had a chronic course while most suppurative and fibrinous bronchopneumonia lesions had an acute course and a higher degree of severity. The combination of microscopic lesions more frequently observed was bronchointerstitial pneumonia + interstitial pneumonia + suppurative bronchopneumonia. CONCLUSIONS: The prevalence of MLL at abattoir was 30.97 %, however, after microscopic examination the real prevalence of lungs with lesions compatible with M. hyopneumoniae infection was reduced up to 24.21 %. The six more prevalent combinations of lesions in the microscopic study involved the 66.13 % of examined lungs, and in all of them, microscopic lesions characteristic of M. hyopneumoniae infection were found, what supports the importance of M. hyopneumoniae as a primary pathogen in cases of PRDC.

Activation of pro- and anti-inflammatory responses in lung tissue injury during the acute phase of PRRSV-1 infection with the virulent strain Lena.

Porcine reproductive and respiratory syndrome virus (PRRSV) plays a key role in porcine respiratory disease complex modulating the host immune response and favouring secondary bacterial infections. Pulmonary alveolar macrophages (PAMs) are the main cells supporting PRRSV replication, with CD163 as the essential receptor for viral infection. Although interstitial pneumonia is by far the representative lung lesion, suppurative bronchopneumonia is described for PRRSV virulent strains. This research explores the role of several immune markers potentially involved in the regulation of the inflammatory response and sensitisation of lung to secondary bacterial infections by PRRSV-1 strains of different virulence. Conventional pigs were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6 and 8 dpi. Lena-infected pigs exhibited more severe clinical signs, macroscopic lung score and viraemia associated with an increase of IL-6 and IFN-γ in sera compared to 3249-infected pigs. Extensive areas of lung consolidation corresponding with suppurative bronchopneumonia were observed in Lena-infected pigs. Lung viral load and PRRSV-N-protein+ cells were always higher in Lena-infected animals. PRRSV-N-protein+ cells were linked to a marked drop of CD163+ macrophages. The number of CD14+ and iNOS+ cells gradually increased along PRRSV-1 infection, being more evident in Lena-infected pigs. The frequency of CD200R1+ and FoxP3+ cells peaked late in both PRRSV-1 strains, with a strong correlation between CD200R1+ cells and lung injury in Lena-infected pigs. These results highlight the role of molecules involved in the earlier and higher extent of lung lesions in piglets infected with the virulent Lena strain, pointing out the activation of routes potentially involved in the restraint of the local inflammatory response.

Relationship between ultrasonographic and histopathological measurements of small intestinal wall layers in fresh cat cadavers.

The relationship between histological and ultrasonographic thickness of the intestinal wall and its layers in cats is unknown so far. The aims of this study were to establish the relationship between ultrasonographic measurements in the transverse and longitudinal planes of the small intestine and to establish the agreement between ultrasonographic and histologic thickness of the overall intestinal wall and layers in cat cadavers. Seventeen adult cats were euthanased for reasons unrelated to gastrointestinal tract disease and ultrasonography was performed immediately after death using a high-frequency linear transducer. Ultrasound images of the duodenum, jejunum, ileum, and distal ileum were acquired in both the longitudinal and transverse planes. Small intestinal samples were collected close to where ultrasonographic images were obtained, fixed in formalin, and histological sections were obtained. Measurements of the intestinal layers and the overall wall thickness were performed on the ultrasonographic images and histological sections. No statistical differences were found between the ultrasonographic measurements of thickness obtained in the transverse and longitudinal planes except for the distal ileum (P<0.05). There was good agreement between the ultrasonographic and histologic measurements of the overall wall thickness and the layers of the different intestinal segments except at the submucosa and muscularis of the duodenum. Immediate postmortem ultrasonographic and histological thickness measurements of the different layers of the small intestine obtained in this study could serve as a reference for ultrasonographic scans and histological samples in cats.

Pathology and Virus Distribution in the Lung and Lymphoid Tissues of Pigs Experimentally Inoculated with Three Distinct Type 1 PRRS Virus Isolates of Varying Pathogenicity.

Porcine reproductive and respiratory syndrome (PRRS) continues to be the most economically important disease of swine worldwide. The appearance of highly pathogenic PRRS virus (PRRSV) strains in Europe and Asia has raised concerns about this disease and initiated increased efforts to understand the pathogenesis. In this study, we have compared the pathology and the virus distribution in tissues of pigs experimentally inoculated with three different genotype 1 PRRSV isolates. Sixty 5-week-old pigs were inoculated intranasally with a) the Lelystad virus (LV), b) a field strain from the UK causing respiratory clinical signs (UK) or c) a highly pathogenic strain from Belarus (BE). Sixteen animals were mock-infected and used as controls. The animals were euthanized at 3, 7 and 35 days post-infection (dpi), and lung and lymphoid tissues collected for histopathological examination and PRRSV detection by immunohistochemistry (IHC). Histopathological lesions consisted of interstitial pneumonia with mononuclear cell infiltrates in the lungs, lymphoid depletion, apoptosis and follicular hyperplasia in the spleen, lymph nodes and tonsil and lymphoid depletion in the thymus. Porcine reproductive and respiratory syndrome virus was detected mainly in monocytes-macrophages. BE-infected animals showed the highest pathological scores and the highest presence of virus at 3 and 7 dpi, followed by the UK field strain and then LV. Moderate lesions were observed at 35 dpi with lesser detection of PRRSV by IHC in each infected group. The highly pathogenic BE strain induced more severe pathology in both lungs and lymphoid organs of pigs compared with the classic field isolate and the prototype LV. The increased severity of pathology was in correlation with the presence of a higher number of PRRSV-infected cells in the tissues.

Use of tylvalosin in the control of porcine enzootic pneumonia.

OBJECTIVES: The purpose of this study was to investigate the efficacy of tylvalosin (Aivlosin Water Soluble Granules, ECO Animal Health) in drinking water for control of Mycoplasma hyopneumoniae (M hyo) on a farm with chronic enzootic pneumonia (EP) problems and high prevalence of mycoplasma-like lesions at slaughter. DESIGN: On a 4000-sow farm in the southeast of Spain, 1500 animals of same age were randomly divided into two groups: 900 pigs in the treated group (TG) and 600 pigs in the non-treated control group (CG). TG was medicated for seven days with tylvalosin in drinking water (2.5 mg tylvalosin/kg bodyweight (BW)) at weaning (from 21st to 28th day of life) and a second treatment when moved to finisher barn (from 63rd to 70th day of life). RESULTS: In the TG, there was a significant reduction in the severity (P<0.001) and number of animals with lung lesions (P<0.001) compared with CG. TG had an increased average daily gain and decreased average number of days in finishing. TG had a lower average carcase weight, but improved homogeneity. M hyo was not detected by q-PCR in samples, taken from lungs with characteristic EP lesions in the TG (0/9), in contrast to the CG (8/9 positive). CONCLUSIONS: A strategic medication with Aivlosin at 2.5 mg tylvalosin/kg BW in drinking water for seven days at weaning and when moved to finisher barn significantly reduces mycoplasma-like lung lesions and improves productivity parameters.

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.

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.

Downregulation of antigen-presenting cells in tonsil and lymph nodes of porcine reproductive and respiratory syndrome virus-infected pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) can persist in different organs of infected pigs, which suggests a failure in the immune response. Antigen-presenting cells (APCs) play a pivotal role in the induction of effective T- and B-cell responses. In this study, we investigated the changes in the different APC subpopulations and T- and B-cell counts in the tonsil, retropharyngeal and mediastinal lymph nodes of pigs experimentally infected with a European PRRSV field isolate. Our results demonstrated that the expression of S100, SWC3, HLA-DR molecule and CD3 was diminished in the studied organs throughout the study, observing a significant negative correlation between viral antigen and HLA-DR expression in both retropharyngeal and mediastinal lymph nodes. In contrast, λ-light chains showed an increase during the study. Taking all into account, after PRRSV infection, no enhancement in the number of APCs and T cells was observed, suggesting an impairment of the immune function which may allow the persistence of PRRSV into the organism.

Immunohistochemical expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs.

Despite the numerous studies carried out, the mechanisms used by porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) to impair the host immune response are not yet clear. The aim of this study was to determine the expression of IL-12, IL-10, IFN-α and IFN-γ in lymphoid organs of PRRSV experimentally-infected pigs. Twenty eight piglets were inoculated with PRRSV field isolate 2982 and killed in batches of four at 3, 7, 10, 14, 17, 21 and 24 days post-inoculation (dpi). Control animals were mock-inoculated and killed at the end of the study. Samples from mediastinal and retropharyngeal lymph nodes and tonsil were collected and fixed for histopathological and immunohistochemical analyses. PRRSV antigen was mainly detected in the cytoplasm of macrophages, displaying a bimodal expression with a first peak at 3-7 dpi and a second peak at 14 dpi. The expression of IFN-α showed an early enhancement at 3 dpi, and both IL-12 and IFN-γ displayed a similar trend in all the lymphoid organs analysed, showing an increase at 3-7 dpi and at 14-17 dpi. On the other hand, the expression of IL-10 was lower than the one observed for the other cytokines. The expression of IL-10 compared with the higher expression of IL-12, IFN-α and IFN-γ detected in this study, indicates that other mechanisms besides the expression of IL-10 play a role in the inducement of an erratic host immune response. Taking into account the enhanced expression of IFNs together with the detection of PRRSV antigen until the end of the study in the examined lymphoid organs, further studies are being conducted to rule out a down-regulation in IFN signalling pathway.

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.

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.

Acute phase proteins as a tool for differential diagnosis of wasting diseases in growing pigs.

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).

Local identification of porcine haptoglobin in salivary gland and diaphragmatic muscle tissues.

In order to clarify the origin of the haptoglobin (Hp) quantified in saliva and meat juice samples, the extrahepatic localization of Hp in salivary gland and in diaphragmatic muscle, as part of the systemic acute phase response in pigs, was studied by immunohistochemistry. For this purpose a specific monoclonal antibody (mAb) produced by immunising mice with purified porcine Hp was used. Reactivity of the mAb was assessed by direct ELISA and by western blot, which showed the ability and specificity of the mAb to identify porcine haptoglobin as a purified antigen or in porcine serum in a native or denatured but non-reduced state. Five healthy and five diseased pigs were sampled at slaughter for serum and tissue procurement. Hepatic immunohistochemical analysis was used as control of the acute phase reaction status. In the liver, cell immunostaining revealed a perinuclear, cytoplasmic localization of Hp within hepatocytes, following mainly a periacinar pattern. Extrahepatic immunohistochemical analysis revealed positive cells in the glandular acini and duct epithelial cells of the salivary gland and intrasarcoplasmic immunolabelling of random diaphragmatic myofibers. A possible role of both salivary gland and diaphragmatic muscle on local Hp production could be postulated based on the present immunohistochemical study, which supports the concept that other cells besides hepatocytes may have the potential to produce Hp in the pig.

Differential expression of proinflammatory cytokines in the lymphoid organs of porcine reproductive and respiratory syndrome virus-infected pigs.

Porcine reproductive and respiratory syndrome (PRRS) is considered one of the most important diseases in the swine industry. Although several studies have been carried out to elucidate the host immune response evoked against PRRS virus (PRRSV), there are several aspects that still remain unclear. The aim of this study was to determine the expression of IL-1α, IL-6 and TNF-α in the lymphoid organs (mediastinal and retropharyngeal lymph nodes and tonsil) of PRRSV-infected pigs and to determine their correlation with the expression of PRRSV antigen. Proinflammatory cytokine expression was different depending on the body compartment examined. Thus, whereas IL-1α and TNF-α were the main cytokines expressed in the mediastinal lymph node, IL-6 was the most highly expressed cytokine in the retropharyngeal lymph node, and no expression of proinflammatory cytokines was observed in the tonsil. These findings may be related to the impairment of the host immune response evoked after PRRSV infection. Therefore, lymphoid organs and proinflammatory cytokines represent an important target of study for clarifying the immunopathogenesis of PRRS.

Design of a real-time quantitative polymerase chain reaction to assess human complement regulatory protein gene expression in polytransgenic xenograft pigs.

OBJECTIVE: 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. MATERIALS AND METHODS: 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. RESULTS: 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. CONCLUSIONS: The q-PCR allowed detection of animals with the highest gene expression for hCD55, hCD59, and hCD46 for xenograft donors in transplantation experiments.

Validation of xCELLigence real-time cell analyzer to assess compatibility in xenotransplantation with pig-to-baboon model.

OBJECTIVE: 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. MATERIALS AND METHODS: 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). RESULTS: 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. CONCLUSIONS: 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.

Cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome (PRRS) is caused by a virus that predominantly replicates in alveolar macrophages. The aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the PRRS virus (PRRSV). Expression of interleukin (IL) 1alpha, IL-6 and tumour necrosis factor (TNF)-alpha correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. Significant correlations were observed between PRRSV infection and the expression of IL-10, between the expression of IL-12p40 and interferon (IFN)-gamma, and between the expression of TNF-alpha and IFN-gamma. These findings suggest that PRRSV modulates the immune response by the up-regulation of IL-10, which may in turn reduce expression of cytokines involved in viral clearance (e.g. IFN-alpha, IFN-gamma, IL-12p40 and TNF-alpha). The results also suggest that expression of IFN-gamma is stimulated by IL-12p40 and TNF-alpha, but not by IFN-alpha. All of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. There appears to be differential activation of septal and alveolar macrophages in PRRSV infection, with septal macrophages being the major source of cytokines.