Isolation and Culture of Primary Aortic Endothelial Cells from Miniature Pigs.

Xenotransplantation is a promising way to resolve the shortage of human organs for patients with end-stage organ failure, and the pig is considered as a suitable organ source. Immune rejection and coagulation are two major hurdles for the success of xenotransplantation. Vascular endothelial cell (EC) injury and dysfunction are important for the development of the inflammation and coagulation responses in xenotransplantation. Thus, isolation of porcine aortic endothelial cells (pAECs) is necessary for investigating the immune rejection and coagulation responses. Here, we have developed a simple enzymatic approach for the isolation, characterization, and expansion of highly purified pAECs from miniature pigs. First, the miniature pig was anaesthetized with ketamine, and a length of aorta was excised. Second, the endothelial surface of aorta was exposed to 0.005% collagenase IV digestive solution for 15 min. Third, the endothelial surface of the aorta was scraped in only one direction with a cell scraper (<10 times), and was not compressed during the process of scraping. Finally, the isolated pAECs of Day 3, and after passage 1 and passage 2, were identified by flow cytometry with an anti-CD31 antibody. The percentages of CD31-positive cells were 97.4% ± 1.2%, 94.4% ± 1.1%, and 92.4% ± 1.7% (mean ± SD), respectively. The concentration of Collagenase IV, the digestive time, the direction, and frequency and intensity of scraping are critical for decreasing fibroblast contamination and obtaining high-purity and a large number of ECs. In conclusion, our enzymatic method is a highly-effctive method for isolating ECs from the miniature pig aorta, and the cells can be expanded in vitro to investigate the immune and coagulation responses in xenotransplantation.

Circulating pig-specific DNA as a novel biomarker for monitoring xenograft rejection.

Monitoring for immune rejection is crucial for long-term survival of pig xenografts. Circulating DNA is a promising non-invasive biomarker for either organ injury or response to therapy. In this study, circulating pig-specific DNA (cpsDNA) was monitored during xenograft rejection. Potential targets of cpsDNA were selected by in silico analysis, and species specificity of selected primers was confirmed by PCR. Subsequently, cpsDNA as a biomarker was evaluated using a complement-dependent cytotoxicity (CDC) assay in vitro. Then, early diagnosis and response to rapamycin were assessed by an in vivo imaging model of pig-to-mouse cell transplantation. Finally, cpsDNA was monitored in a pig-to-monkey artery patch transplantation model. The results showed that (a) a method of cpsDNA quantitation was established for application in mouse and nonhuman primate models; (b) cpsDNA reflected CDC in vitro; (c) cpsDNA in vivo mirrored xenograft rejection, and correlated with xenograft loss in pig-to-mouse cell transplantation; (d) cpsDNA was significantly reduced when rapamycin was administered; and (e) dynamic cpsDNA was detectable in pig-to-monkey artery patch transplantation. In conclusion, measurement of cpsDNA could prove to be a less invasive, but more specific and sensitive low-cost biomarker enabling monitoring of xenograft rejection and the response to immunosuppressive therapy.

Oleate inhibits hepatic autophagy through p38 mitogen-activated protein kinase (MAPK).

Hepatic autophagy plays an important role in lipid metabolism, especially in nonalcoholic fatty liver disease. The relationship between Oleate acid and autophagy is not yet clear. In this work, using mouse epithelial cell hepa1c1c7, we investigated the role of Oleate acid on autophagy and explored its potential mechanisms. The exposure of hepatic cells to Oleate acid resulted in a significant reduction of LC3 accumulation together with enhancement of p62 protein expression and the mRNA levels of ATG7 and BECN1 were reduced as well. Mechanistically, the inhibitory effects of Oleate acid on rapamycin-induced autophagy were completely blocked by treatment with dominant negative p38α and p38 inhibitor SB203580. Furthermore, ATF-2, downstream of p38, was activated by Oleate treatment. Oleate treatment also inhibited the ULK1 promoter and decreased the ULK1 mRNA level. Our data therefore suggest that Oleate activated the ATF-2 via p38 kinase which inhibited the ULK1 via binding to ULK1 promoter, and eventually the rapamycin-induced autophagy was suppressed.

Rh-Matched Transfusion through Molecular Typing for ß-Thalassemia Patients Is Required and Feasible in Chinese.

BACKGROUND: Molecular typing for RHCE blood group alleles has been established in many countries for patients and blood donors. In the Chinese literature nearly 80% of transfused patients with alloimmunization have antibodies specific for antigens of the Rh blood group system. We investigated if it is feasible to match packed red blood cells (RBCs) for Chinese ß-thalassemia patients by RHCE genotyping. METHODS: In this study, 481 patients with ß-thalassemia were enrolled. They were genotyped for RHCE alleles by a simple PCR method with sequence-specific primers (PCR-SSP). Among these patients, 203 continuously received RBCs of the identical Rh subgroups according to the genotyping results for at least 3 months. Subsequently, their phenotypes were tested through a micro-column gel card method. For validation purposes, 400 donors were serologically typed with the same technology, of which 164 were genotyped too. Finally, the C, c, E, and e frequencies and the feasibility of the simple genotyping method were analyzed. RESULTS: All patients showed mixed-field agglutination in the Rh subgroup gel cards before the same Rh subgroups in blood donors were selected for blood transfusion. The results, however, lacked mixed-field agglutination in all 203 cases after transfusion with RBC concentrates selected for the patient's C, c, E, and e antigens for at least 3 months. The genotyping results of 164 donors were all consistent with the serological results. Whole coding regions of RHCE were sequenced in 7 individuals with weak c, E, or e antigens. In only one sample we observed a 1059G>A nucleotide mutation coding for a truncated RhCE polypeptide (GenBank KT957625), in the other 6 samples no sequence variant was found. Both patients and donors were predominantly CcEe and CCee, with a prevalence of 55.3% and 24.9% for patients or 49.3% and 31.3% for donors, respectively. It revealed that about 80% of Chinese could receive Rh-matched RBCs easily. CONCLUSION: A simple RHCE genotyping technique is safe enough for Rh-matched transfusion of ß-thalassemia patients in Chinese Han.

Drug repurposing: Ibrutinib exhibits immunosuppressive potential in organ transplantation.

Long-term administration of classic immunosuppressants can induce severe adverse effects. The development of novel immunosuppressants confronts great challenges and opportunities. Ibrutinib, an approved drug for B-cell lineages and chronic graft versus host disease (cGVHD), exhibits immunosuppressive efficacy in autoimmune diseases. Ibrutinib's potential as an immunosuppressant in organ transplantation has not been investigated to date. In a xeno-artery patch model ex vivo, ibrutinib inhibited the proliferation of PBMCs (POD 14-42), mainly CD3+CD4+ and CD3+CD8+ T cells ex vivo. The secretion of cytokines (IL-6, IL-2 and IFN-γ) was suppressed in response to ibrutinib. In allo-skin transplantation models, ibrutinib delayed the rejection of grafted skins. Ibrutinib decreased the amount of T/B cells and lymphocyte infiltration. Altogether, ibrutinib exhibited immunosuppressive potential through cytokine regulation and T cell inhibition ex vivo and in vitro. Repositioning of ibrutinib as an immunosuppressant will greatly facilitate novel immunosuppressant development.

The Sda and Cad glycan antigens and their glycosyltransferase, ß1,4GalNAcT-II, in xenotransplantation.

Antibody-mediated rejection is a barrier to the clinical application of xenotransplantation, and xenoantigens play an important role in this process. Early research suggested that N-acetyl-D-galactosamine (GalNAc) could serve as a potential xenoantigen. GalNAc is the immunodominant glycan of the Sda antigen. Recently, knockout of ß1,4-N-acetylgalactosaminyltransferase 2 (ß1,4GalNAcT-II) from the pig results in a decrease in binding of human serum antibodies to pig cells. It is believed that this is the result of the elimination of the GalNAc on the Sda antigen, which is catalyzed by the enzyme, ß1,4GalNAcT-II. However, research into human blood group antigens suggests that only a small percentage (1%-2%) of people express anti-Sda antibodies directed to Sda antigen, and yet a majority appear to have antibodies directed to the products of pig B4GALNT2. Questions can therefore be asked as to (i) whether the comprehensive structure of the Sda antigen in humans, that is, the underlying sugar structure, is identical to the Sda antigen in pigs, (ii) whether the human anti-Sda antibody binds ubiquitously to pig cells, but not to human cells, and (iii) what role the Sda++ (also called Cad) antigen is playing in this discrepancy. We review what is known about these antigens and discuss the discrepancies that have been noted above.

The impact of serum incubation time on IgM/IgG binding to porcine aortic endothelial cells.

The results of the assay for measuring anti-non-Gal antibodies (which affect pig xenograft survival) in recipients are important. Serum incubation time and concentration may be important factors in the extent of antibody binding to the graft. The aim of this in vitro study was to determine the optimal incubation time and serum concentration for measuring anti-non-Gal antibody binding to porcine aortic endothelial cells (pAECs). Pooled human, naive, and sensitized baboon sera were incubated with wild-type, α1,3-galactosyltransferase gene-knockout (GTKO), and GTKO/human CD55 pAECs. IgM/IgG binding to pAECs after varying serum incubation times (0.5, 1, 2, and 3 hour) and concentrations (5, 10, 20, and 40 µL) was determined by flow cytometry. An increase in incubation time from 30 minutes to 2 hour was associated with increases in anti-non-Gal IgM/IgG binding to GTKO and GTKO/hCD55 pAECs of pooled human, naive and sensitized baboon sera (P<.05). Pooled human serum showed a significant increase in anti-non-Gal IgM (1.5 times) and a minimal increase in anti-non-Gal IgG antibody binding. IgM/IgG binding of sensitized baboon serum to GTKO pAECs after 2-hour incubation was 1.5 times and 2 times greater than after 30-minutes incubation, respectively, whereas naïve baboon sera showed minimal (non-significant) increase in anti-non-Gal IgM/IgG antibody binding. With 2-hour incubation, increasing the serum concentration from 5 µL to 20 µL significantly increased antibody binding to non-Gal antigens in pooled human and sensitized baboon serum. With naïve baboon serum, only IgG was significantly increased. Increasing the serum incubation time contributed to improve the sensitivity of detecting anti-non-Gal antibodies, without affecting cell viability in vitro.

Production of α1,3-galactosyltransferase and cytidine monophosphate-N-acetylneuraminic acid hydroxylase gene double-deficient pigs by CRISPR/Cas9 and handmade cloning.

Gene-knockout pigs hold great promise as a solution to the shortage of organs from donor animals for xenotransplantation. Several groups have generated gene-knockout pigs via clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) and somatic cell nuclear transfer (SCNT). Herein, we adopted a simple and micromanipulator-free method, handmade cloning (HMC) instead of SCNT, to generate double gene-knockout pigs. First, we applied the CRISPR/Cas9 system to target α1,3-galactosyltransferase (GGTA1) and cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes simultaneously in porcine fetal fibroblast cells (PFFs), which were derived from wild-type Chinese domestic miniature Wuzhishan pigs. Cell colonies were obtained by screening and were identified by Surveyor assay and sequencing. Next, we chose the GGTA1/CMAH double-knockout (DKO) cells for HMC to produce piglets. As a result, we obtained 11 live bi-allelic GGTA1/CMAH DKO piglets with the identical phenotype. Compared to cells from GGTA1-knockout pigs, human antibody binding and antibody-mediated complement-dependent cytotoxicity were significantly reduced in cells from GGTA1/CMAH DKO pigs, which demonstrated that our pigs would exhibit reduced humoral rejection in xenotransplantation. These data suggested that the combination of CRISPR/Cas9 and HMC technology provided an efficient and new strategy for producing pigs with multiple genetic modifications.

Identification of a Vav2-dependent mechanism for GDNF/Ret control of mesolimbic DAT trafficking.

Dopamine (DA) homeostasis is essential for a variety of brain activities. Dopamine transporter (DAT)-mediated DA reuptake is one of the most critical mechanisms for normal DA homeostasis. However, the molecular mechanisms underlying the regulation of DAT activity in the brain remain poorly understood. Here we show that the Rho-family guanine nucleotide exchange factor protein Vav2 is required for DAT cell surface expression and transporter activity modulated by glial cell line-derived neurotrophic factor (GDNF) and its cognate receptor Ret. Mice deficient in either Vav2 or Ret displayed elevated DAT activity, which was accompanied by an increase in intracellular DA selectively in the nucleus accumbens. Vav2(-/-) mice exposed to cocaine showed reduced DAT activity and diminished behavioral cocaine response. Our data demonstrate that Vav2 is a determinant of DAT trafficking in vivo and contributes to the maintenance of DA homeostasis in limbic DA neuron terminals.

[Effects of curcumin on bleomycin-induced damages in pulmonary functions in rats].

OBJECTIVE: To determine the effects of curcumin on bleomycin (BLM)-induced pulmonary fibrosis in rats. METHOD: One hundred and forty-four male Sprague-Dawley rats were randomized into 6 groups (24 rats in each group, model group, sham group, prednisone group (0.56 mg x kg(-1) x d(-1)), curcumin with low dose 5 mg group, curcumin with middle dose group 10 mg and curcumin with high dose group 20 mg per 100 g of body weight). Rats in all groups except in sham group were injected with BLM intratracheally. Curcumin with different doses were given by gavage one time everyday for 7, 14 and 28 days. Prednisone were given to rats in prednisone group, po, serving as the positive treatment group. On the 7th, 14th, 28th day, the lung functions (inspiratory resistance, maximal volutary ventilation, forced vital capacity, Fev 0.2/FVC, peak expiratory flow) were determinated in experimental rats, respectively, and the concentrations of hydroxyproline in lung homogenates of each rat were assayed. RESULT: Administration of curcumin in different doses improved lung functions of BLM-induced fibrotic rats in the all experimental days; and it decreased the concentration of hydroxyproline in lung homogenates compared with those levels in model control group; and it also lessened the hyperplasia of BLM-induced pulmonary fibrosis in rats. CONCLUSION: Administration of curcumin can suppress BLM induced pulmonary fibrosis indicated by improved respiratory function, as well as companied with low content of hydroxyproline in lung tissue of rats.