Mark D. Crew

Transgenic expression of proximal tubule peroxisome proliferator–activated receptor-α in mice confers protection during acute kidney injury

Our previous studies suggest that peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a critical role in regulating fatty acid beta-oxidation in kidney tissue and this directly correlated with preservation of kidney morphology and function during acute kidney injury. To further study this, we generated transgenic mice expressing PPARalpha in the proximal tubule under the control of the promoter of KAP2 (kidney androgen-regulated protein 2). Segment-specific upregulation of PPARalpha expression by testosterone treatment of female transgenic mice improved kidney function during cisplatin or ischemia-reperfusion-induced acute kidney injury. Ischemia-reperfusion injury or treatment with cisplatin in wild-type mice caused inhibition of fatty-acid oxidation, reduction of mitochondrial genes of oxidative phosphorylation, mitochondrial DNA, fatty-acid metabolism, and the tricarboxylic acid cycle. Similar injury in testosterone-treated transgenic mice resulted in amelioration of these effects. Similarly, there were increases in the levels of 4-hydroxy-2-hexenal-derived lipid peroxidation products in wild-type mice, which were also reduced in the transgenic mice. Similarly, necrosis of the S3 segment was reduced in the two injury models in transgenic mice compared to wild type. Our results suggest proximal tubule PPARalpha activity serves as a metabolic sensor. Its increased expression without the use of an exogenous PPARalpha ligand in the transgenic mice is sufficient to protect kidney function and morphology, and to prevent abnormalities in lipid metabolism associated with acute kidney injury.

Molecular cloning and functional characterization of a vasotocin receptor subtype expressed in the pituitary gland of the domestic chicken (Gallus domesticus): avian homolog of the mammalian V1b-vasopressin receptor.

The neurohypophysial hormone arginine vasotocin (AVT) stimulates adrenocorticotropin hormone (ACTH) secretion from the avian anterior pituitary gland resulting in increased adrenal secretion of corticosterone in response to stress. Here, we report molecular cloning and functional characterization of a gene encoding an AVT receptor subtype, designated the VT2 receptor, that may mediate the stimulatory effect of AVT on ACTH secretion in birds. The open reading frame predicts a 425 amino acid polypeptide that includes seven segments of 19 to 24 hydrophobic amino acids, typical of guanine nucleotide-protein coupled receptors. Phylogenetic analysis revealed that the VT2 receptor shares highest identity with the mammalian V1b-vasopressin receptor subtype. Expressed VT2 receptors in COS7 cells mediate AVT-induced phosphatidylinositol turnover and Ca(2+) mobilization. In the domestic chicken, expression of VT2 receptor gene transcripts is limited to the pituitary gland. Based on similarities in sequence, site of expression and coupled signal transduction pathways, we conclude that the VT2 receptor is the avian homolog of the mammalian V1b-vasopressin receptor, and therefore may play an important role in the avian stress response.

Transgenic expression of HLA-E single chain trimer protects porcine endothelial cells against human natural killer cell-mediated cytotoxicity

Abstract:  Background:  The susceptibility of porcine endothelial cells (pEC) to human natural killer (NK) cells is related to the failure of human major histocompatibility complex (MHC)‐specific killer inhibitory receptors to recognize porcine MHC class I molecules. The aims of this study were (i) to assess the protection of pEC against xenogeneic NK‐mediated cytotoxicity afforded by the stable expression of HLA‐E single chain trimers (SCT) composed of a canonical HLA‐E binding peptide antigen, VMAPRTLIL, the mature human β2‐microglobulin, and the mature HLA‐E heavy chain, and (ii) to test whether HLA‐E expression on pEC and porcine lymphoblastoid cells affects the adhesion of human NK cells.

Porcine UL16-Binding Protein 1 Expressed on the Surface of Endothelial Cells Triggers Human NK Cytotoxicity through NKG2D

Cellular rejection mechanisms, including NK cells, remain a hurdle for successful pig-to-human xenotransplantation. Human anti-pig NK cytotoxicity depends on the activating receptor NKG2D. Porcine UL16-binding protein 1 (pULBP1) and porcine MHC class I chain-related protein 2 (pMIC2) are homologues of the human NKG2D ligands ULBP 1–4 and MICA and B, respectively. Although transcribed in porcine endothelial cells (pEC), it is not known whether pULBP1 and pMIC2 act as functional ligands for human NKG2D. In this study, surface protein expression of pULBP1 was demonstrated by flow cytometry using a novel pULBP1-specific polyclonal Ab and by cellular ELISA using NKG2D-Fc fusion protein. Reciprocally, pULBP1-Fc bound to primary human NK cells, whereas pMIC2-Fc did not. Transient and stable down-regulation of pULBP1 mRNA in pEC using short-interfering RNA oligonucleotide duplexes and short hairpin RNA, respectively, resulted in a partial inhibition of xenogeneic NK cytotoxicity through NKG2D in 51Cr release assays. In contrast, down-regulation of pMIC2 mRNA did not inhibit NK cytotoxicity. Human NK cytotoxicity against pEC mediated by freshly isolated or IL-2-activated NK cells through NKG2D was completely blocked using anti-pULBP1 polyclonal Ab. In conclusion, this study suggests that pULBP1 is the predominant, if not only, functional porcine ligand for human NKG2D. Thus, the elimination of pULBP1 on porcine tissues represents an attractive target to protect porcine xenografts from human NK cytotoxicity.

Influence of mast cells on outcome after heterotopic cardiac transplantation in rats

Correlative data suggest that mast cells adversely affect cardiac transplantation. This study uses a mast cell‐deficient rat model to directly address the role of mast cells in cardiac allotransplantation. Standardized cardiac heterotopic transplantation with cyclosporine immunosuppression was performed in mast cell‐deficient and mast cell‐competent rats. Rejection, ischemia, fibrosis, fibrin deposition, numbers of T‐cell receptor α/β positive cells, expression of transforming growth factor‐β (TGF‐β), and of endothelin‐1 (ET‐1) and its receptors ETA and ETB were assessed. Differences in baseline cardiac gene expression were quantified by real‐time PCR in a separate group of untransplanted animals. Baseline cardiac gene expression levels of all investigated growth factors, cytokines, ET‐1, ETA, and ETB were similar in mast cell‐deficient and mast cell‐competent rats. Surprisingly, upon heterotopic transplantation, donor heart survival was significantly reduced in mast cell‐deficient rats. Moreover, in mast cell‐deficient donor hearts rejection was more severe, although nonsignificant, and extracellular matrix associated TGF‐β immunoreactivity was significantly lower than in mast cell‐competent donor hearts. Fibrin immunoreactive area, on the other hand, was only increased in mast cell‐deficient donor hearts, but not in mast cell‐competent donor hearts. Histopathological changes in all donor hearts were accompanied by increased immunoreactivity for ET‐1. In conclusion, this study shows that mast cells play a protective role after cardiac transplantation.

Play it in E or G: utilization of HLA-E and -G in xenotransplantation.

Abstract Human NK cell‐mediated graft rejection is likely to be one of several biological obstacles to routine pig‐to‐human xenotransplantation. Abrogating NK cell activation by either elimination of activating ligands on porcine cells or expression of molecules serving as ligands for NK cell inhibitory receptors, or both, could overcome this hurdle. HLA‐E and ‐G exhibit very limited polymorphism and are ligands for NK cell inhibitory receptors. This review summarizes successes and limitations of their use in xenotransplantation as inferred from ex vivo analyses of NK cell activity, highlights potential effects they may have on T‐cell responses, and considers prospects of preclinical trials and potential outcomes.

Expressed Peromyscus maniculatus (Pema) MHC class I genes: evolutionary implications and the identification of a gene encoding a Qa1-like antigen

Abstract To gain insight into the evolution of rodent major histocompatibility complex (MHC) class I genes and identify important (conserved) nonclassical class I (class I b) gene products and residues in these proteins, six Peromyscus maniculatus MHC (Pema) class I cDNA clones were isolated and sequenced. Five Pema class I cDNAs appeared most similar to mouse and rat classical class I (class I a) genes. One exhibited highest similarity to an H2 class I b gene, H2-T23 (encoding the Qa1 antigen). Phylogenetic trees constructed with Pema, RT1, and H2 class I sequences suggested that the lineages of some rodent class I b genes (e. g., T23 and T24) originated prior to Mus and Peromyscus speciation [>50 million years (My) ago]. Sequences of four Qa1-like proteins from three species permitted the identification of ten Qa1-specific amino acids. On the basis of molecular modeling, three residues showed the potential to interact with T-cell receptors and three residues (all corresponding to polymorphic positions among H2 class I a proteins) were predicted to influence antigen binding. The recognition of mouse Qa1 proteins by a subset of T-cells in influenced by a locus, Qdm, which encodes the H2-D leader peptide. One of the Pema class I cDNA clones classified as H2-K, D/L-like (class I a) is predicted to encode an identical peptide, implying that an antigen binding protein (Qa1) and the antigen to which it binds (the product of Qdm) has been conserved for over 50 My.

Sequence and mRNA expression of nonclassical SLA class I genes SLA-7 and SLA-8

Given the prominent position of pig endothelial cells in pig-to-human xenotransplantation and the role of classical and nonclassical MHC class I proteins in T and NK cell recognition, the expression of pig MHC (SLA) class I genes in a pig aortic endothelial cell line (AOC cells) was examined. Using a primer corresponding to a highly conserved region of exon 4, RT-PCR analysis of SLA class I expression in AOC cells revealed not only expression of the classical SLA class I (SLA-1, -2, and -3) genes, but also SLA class I transcripts corresponding to SLA nonclassical class I (class Ib) genes SLA-6 and SLA-8. Further analysis of SLA class Ib expression in porcine aortic endothelial cells using SLA class I gene-specific primers confirmed SLA-6 and SLA-8 expression and also demonstrated expression of SLA-7. While SLA-6 has been relatively well characterized, no data regarding bona fide SLA-7 and SLA-8 transcripts have been reported. Therefore, cDNAs containing the complete open reading frames of SLA-6, -7, and -8 were obtained. Compared to an SLA-1 protein sequence, the predicted SLA-7 and -8 protein sequences exhibited most sequence divergence in α1, α2, and cytoplasmic domains. Expression of SLA-6, -7, and -8 was examined by RT-PCR using RNA prepared from a variety of tissues. SLA-6 transcripts were detected in every tissue examined. Except for brain, SLA-8 transcripts were similarly widespread. SLA-7 exhibited more limited tissue distribution.

Novel target antigens for dendritic cell-based immunotherapy against ovarian cancer.

Identification of tumor-specific target antigens has been a major hurdlefor the treatment of malignant disease by vaccination or immunotherapy. A second challenge has been the induction of therapeutically effective immune responses to these ‘self’ antigens. The recent recognition of dendritic cells as powerful antigen-presenting cells capable of inducing primary T-cell responses in vitro and in vivo – in combination with identification of tumor-specific antigens – has generated widespread interest in dendritic cell-based immunotherapy against a wide variety of tumors. In this review, a series of recently identified novel ovarian tumor antigens is discussed, and the potential for therapeutic dendritic cell vaccination targeted against these antigens is assessed.

Definition of an immunogenic region within the ovarian tumor antigen stratum corneum chymotryptic enzyme

Purpose: The serine protease stratum corneum chymotryptic enzyme (SCCE) is overexpressed by ovarian tumor cells, but is not expressed by normal tissues, suggesting that SCCE may be an attractive target for immunotherapy. In this study, we tested the hypothesis that dendritic cells loaded with SCCE peptides will induce ovarian tumor antigen–specific CD8+ CTL responses and antigen-specific CD4+ helper T cell responses. Experimental Design: Computer algorithms were used to identify candidate HLA-A2.1-restricted CD8+ CTL epitopes and HLA-DR-binding CD4+ helper T cell epitopes within SCCE. CD8+ CTL stimulated with peptide-loaded dendritic cells were tested against targets expressing endogenous SCCE, including HLA-A2.1-matched ovarian tumor cells. Dendritic cells were also loaded with an extended SCCE peptide, SCCE 110-139, which encompassed a defined CD8+ CTL epitope and multiple candidate CD4+ T helper cell epitopes. Results: CD8+ CTL specific for SCCE 123-131 lysed autologous macrophages infected with an SCCE-expressing recombinant adenovirus, and also lysed HLA-A2.1-matched, SCCE-expressing ovarian tumor cells. Dendritic cells loaded with SCCE 5-13 peptide stimulated an HLA-A2.1-restricted CD8+ CTL response, but with a reduced level of lysis against ovarian tumor cells. Dendritic cells loaded with SCCE 110-139 induced antigen-specific CD4+ T cell and CD8+ T cell responses. Although SCCE 110-139-loaded dendritic cells processed and presented the 123-131 epitope, the dominant CD8+ CTL response was directed against alternative epitopes within SCCE 110-139. Conclusions: The 110-139 region of SCCE incorporates multiple CD8+ CTL and CD4+ helper T cell epitopes, and represents an attractive target antigen for immunotherapy of ovarian cancer.