Abstract: The state of operational tolerance has been detected sporadically in some renal transplanted patients that stopped immunosuppressive drugs, demonstrating that allograft tolerance might exist in humans. Several years ago, a study by Brouard et al. identified a molecular signature of several genes that were significantly differentially expressed in the blood of such patients compared with patients with other clinical situations. The aim of the present study is to analyze the role of one of these molecules over-expressed in the blood of operationally tolerant patients, SMILE or TMTC3, a protein whose function is still unknown.
Abstract: The endothelial protein C receptor (EPCR) negatively regulates the coagulopathy and inflammatory response in sepsis. Mechanisms controlling the expression of cell-bound and circulating soluble EPCR (sEPCR) are still unclear. Moreover, the clinical impact of EPCR shedding and its potential value to predict sepsis progression and outcome remain to be established.
Abstract: Targeting protective gene expression to porcine endothelium by genetic modification of the donor could improve xenograft survival by controlling cell activation and death. We previously found that, in endothelial cells (EC), the molecular adaptor Lnk (SH2B3) is a negative regulator of cytokine signaling. We also have shown that Lnk is upregulated in pig EC (PAEC) in response to tumor necrosis factor-α (TNF) and xenoreactive natural antibodies (XNA) binding. The present study investigated whether ectopic expression of human Lnk using gene transfer may be efficient to control signaling pathways associated with inflammation and apoptosis in porcine aortic endothelial cells (PAEC).
Abstract: Numerous reports have highlighted the central role of regulatory T cells in long-term allograft tolerance, but few studies have investigated the B-cell aspect. We analyzed the B-cell response in a rat model of long-term cardiac allograft tolerance induced by a short-term immunosuppression. We observed that tolerated allografts are infiltrated by numerous B cells organized in germinal centers that are strongly regulated in their IgG alloantibody response. Moreover, alloantibodies from tolerant recipients exhibit a deviation toward a Th2 isotype and do not activate in vitro donor-type endothelial cells in a pro-inflammatory way but maintained expression of cytoprotective molecules. Interestingly, this inhibition of the B-cell response is characterized by the progressive accumulation in the graft and in the blood of B cells blocked at the IgM to IgG switch recombination process and overexpressing BANK-1 and the inhibitory receptor Fcgr2b. Importantly, B cells from tolerant recipients are able to transfer allograft tolerance. Taken together, these results demonstrate a strong regulation of the alloantibody response in tolerant recipients and the accumulation of B cells exhibiting an inhibited and regulatory profile. These mechanisms of regulation of the B-cell response could be instrumental to develop new strategies to promote tolerance.
Abstract: Activated protein C (APC) is a natural anticoagulant protease that displays cytoprotective and antiinflammatory activities and has been demonstrated to reduce mortality of patients with severe sepsis. However, APC signaling is not fully understood. This study further investigated the antiinflammatory effects of APC in vascular endothelial cells (EC) and examined the cross talk between APC and TNF signaling. Analysis of the regulatory mechanisms mediated by APC on vascular human EC shows that APC impairs TNF signaling by triggering a preemptive activation of intracellular pathways. We found that APC signaling causes a moderate but significant induction of cell adhesion molecules (CAMs) including VCAM-1 at mRNA and protein levels. Activation of the noncanonical NF-κB and ERK1/2 are both pivotal to APC signaling leading to VCAM-1 expression. APC upregulates TNF receptor-associated factor 2 (TRAF2) and phosphorylates NF-κB p65 at Ser276 and Ser536 independently of IκB degradation. The ultimate protective antiinflammatory effect of APC in response to TNF is associated with a sustained activation of ERK1/2 and Akt while phosphorylation of NF-κB p65 is precluded. Inhibitors of ERK (PD98059 and U0126) abolish the antiinflammatory signal mediated by APC. Blocking antibodies and silencing assays also suggest that, in EC, protease-activated receptor 1 and endothelial protein C receptor (EPCR) both conduct ERK activation and VCAM-1 induction in response to APC. To conclude, APC protects EC by attenuating CAM expression during inflammation. APC engages a regulatory cross talk involving EPCR, ERK, and NF-κB that impairs TNF signaling.
Abstract: A better knowledge of the process by which inflammatory extracellular signals are relayed from the plasma membrane to specific intracellular sites is a key step to understand how inflammation develops and how it is regulated. This review focuses on Lnk (SH2B3) a member, with SH2B1 and SH2B2, of the SH2B family of adaptor proteins that influences a variety of signaling pathways mediated by Janus kinase and receptor tyrosine kinases. SH2B adaptor proteins contain conserved dimerization, pleckstrin homology, and SH2 domains. Initially described as a regulator of hematopoiesis and lymphocyte differentiation, Lnk now emerges as a key regulator in hematopoeitic and non hematopoeitic cells such as endothelial cells (EC) moderating growth factor and cytokine receptor-mediated signaling. In EC, Lnk is a negative regulator of TNF signaling that reduce proinflammatory phenotype and prevent EC from apoptosis. Lnk is a modulator in integrin signaling and actin cytoskeleton organization in both platelets and EC with an impact on cell adhesion, migration and thrombosis. In this review, we discuss some recent insights proposing Lnk as a key regulator of bone marrow-endothelial progenitor cell kinetics, including the ability to cell growth, endothelial commitment, mobilization, and recruitment for vascular regeneration. Finally, novel findings also provided evidences that mutations in Lnk gene are strongly linked to myeloproliferative disorders but also autoimmune and inflammatory syndromes where both immune and vascular cells display a role. Overall, these studies emphasize the importance of the Lnk adaptor molecule not only as prognostic marker but also as potential therapeutic target.
Abstract: The endothelial cell (EC) protein C receptor (EPCR) negatively regulates coagulation and inflammation. Factors and mechanisms regulating the expression of cell-bound EPCR and the release of soluble (s) EPCR are still unclear.
Abstract: Tumor-derived soluble factors, including soluble HLA molecules, can contribute to cancer immune escape and therefore impact on clinical course of malignant diseases. We previously reported that melanoma cells produce, in vitro, soluble forms of the non-classical MHC class I molecule HLA-E (sHLA-E). In order to investigate sHLA-E production by various tumors and to address its potential value as a tumor-associated marker, we developed a specific ELISA for the quantification of sHLA-E in biological fluids.
Abstract: To outline some recently described mechanisms that regulate endothelial cell function and vascular inflammation. A particular focus will be given to protective or adverse regulatory events, at molecular or cellular level, that could provide new therapeutic targets to control endothelial cell dysfunction in the xenotransplantation setting.
Abstract: It is likely that the polymorphic MICA (MHC class I related chain A) molecules on graft endothelial cells (ECs) may be a target for specific antibodies and T cells directed against solid organ grafts. Although there is evidence for a role of MICA in vascular and transplant biology, genotyping is not performed routinely, and thus there are few correlations between polymorphism and endothelial phenotype. The present study examined the frequency of the various alleles for the nonclassical MHCI MICA gene among a cohort of kidney transplant donors, particularly MICA genetic variants (MICA A5.1 and MICA-129) that may affect MICA expression and function on graft EC. Genotyping was performed on genomic DNA derived from primary cultures of EC established from transplant donors at the time of transplantation. Herein we have reported that among 106 alleles analyzed, 28/69 MICA alleles were distributed among 7 major variants (*00804, *00801, *004, *00201, *00901*, *011, *010), representing 70% of the donors. MICA*008 the most abundant allele (31.1%) was associated with the MICA A5.1 mutation. The majority of donors (52.8%) had at least one MICA A5.l allele, with 13.2% homozygous for this mutation. The MICA-129 val/val genotype, which encodes a low-affinity ligand, was predominant (49%), while the MICA-129 met/met, corresponding to a high-affinity ligand, was observed in 11.3% of the transplants. Our findings highlighted the MICA gene polymorphism that produces functional diversity in transplant recipients with variable interactions between MICA and its receptor NKG2D.
Abstract: C-type lectin receptors have recently been described as playing crucial roles in immunity and homeostasis since these proteins are able to recognize pathogens as well as self-Ags. We identified the C-type lectin-like receptor-1, CLEC-1, as being overexpressed in a model of rat allograft tolerance. We previously described in this model the expression of numerous cytoprotective molecules by graft endothelial cells and their interplay with regulatory CD4(+)CD25(+) T cells. In this study, we demonstrate that CLEC-1 is expressed by myeloid cells and specifically by endothelial cells in tolerated allografts and that CLEC-1 expression can be induced in endothelial cells by alloantigen-specific regulatory CD4(+)CD25(+) T cells. Analysis of CLEC-1 expression in naive rats demonstrates that CLEC-1 is highly expressed by myeloid cells and at a lower level by endothelial cells, and that its expression is down-regulated by inflammatory stimuli but increased by the immunoregulators IL-10 or TGFbeta. Interestingly, we demonstrate in vitro that inhibition of CLEC-1 expression in rat dendritic cells increases the subsequent differentiation of allogeneic Th17 T cells and decreases the regulatory Foxp3(+) T cell pool. Additionally, in chronically rejected allograft, the decreased expression of CLEC-1 is associated with a higher production of IL-17. Taken together, our data suggest that CLEC-1, expressed by myeloid cells and endothelial cells, is enhanced by regulatory mediators and moderates Th17 differentiation. Therefore, CLEC-1 may represent a new therapeutic agent to modulate the immune response in transplantation, autoimmunity, or cancer settings.
Abstract: Diagnosis of the specific cause of late allograft injury is necessary if more personalized and efficient immunosuppressive regimens are to be introduced. This study sought previously unrecognized biomarkers for specific histologic diagnoses of late graft scarring by comparison of gene sets from published microarray studies. Tribbles-1 (TRIB1), a human homolog of Drosophila tribbles, was identified to be a potentially informative biomarker. For testing this, mRNA expression in 76 graft biopsies, 71 blood samples, and 11 urine samples were profiled from independent cohorts of renal transplant patients with different histologic diagnoses recruited at two European centers. TRIB1 but not TRIB2 or TRIB3 was found to be a potential blood and tissue biomarker of chronic antibody-mediated rejection, an active immune-mediated form of chronic allograft failure associated with a poor prognosis. TRIB1 mRNA levels in peripheral blood mononuclear cells discriminated patients with chronic antibody-mediated rejection from those with other types of late allograft injury with high sensitivity and specificity. TRIB1 was also upregulated in a rodent model of chronic cardiac vasculopathy, suggesting that this biomarker may be useful in other solid-organ transplants and across species. It was determined that TRIB1 is expressed primarily by antigen-presenting cells and activated endothelial cells. Overall, these data support the potential use of TRIB1 as a biomarker of chronic antibody-mediated allograft failure.
Abstract: OBJECTIVE: Notch signaling pathway controls key functions in vascular and endothelial cells (EC). However, little is known about the role of Notch in allografted vessels during the development of transplant arteriosclerosis (TA). This study investigated regulation of the Notch pathway on cardiac allograft arteriosclerosis and further examined its implication in EC dysfunction. METHODS AND RESULTS: Here we show that, among Notch receptors, Notch2, -3, and -4 transcript levels were markedly downregulated in TA compared to tolerant and syngeneic allografts. TA correlates with high levels of tumor necrosis factor (TNF), transforming growth factor (TGF)beta, and IL10, which consistently decrease Notch4 expression in transplants and cultured ECs. We found that inhibition of Notch activity, reflected by both a reduced CBF1 activity and Hes1 expression, parallels the downregulation of Notch4 expression mediated by TNF in ECs. Notch4 and Hes1 knockdown enhances vascular cell adhesion molecule-1 expression and promotes EC apoptosis. Silencing Notch4 or Hes1 also drastically inhibits repair of endothelial injury. Overall, our results suggest that Notch4 and basal Notch activity are required to maintain EC quiescence and for optimal survival and repair in response to injury. CONCLUSIONS: Together, our findings indicate that impaired Notch4 activity in graft ECs is a key event associated with TA by triggering EC activation and apoptosis.
Abstract: Human leukocyte antigen (HLA)-E belongs, with HLA-G and HLA-F, to the non-classic major histocompatibility complex (MHC) class I (Ib) molecules, broadly defined by a limited polymorphism and a restricted pattern of cellular expression. In contrast to HLA-G, the expression and function of HLA-E and HLA-F in physiologic and pathologic processes remain poorly established. In the present study, we show that HLA-E protein expression in normal human nonlymphoid organs is mainly restricted to endothelial cells (ECs). HLA-E is also basally expressed by B and T lymphocytes, natural killer (NK) cells and by macrophages. We demonstrate that tumor necrosis factor alpha (TNFalpha), interleukin-1beta (IL-1beta), and interferon gamma (IFNgamma) up-regulate the cell-surface expression of HLA-E on ECs in vitro and induce the release of soluble HLA-E (sHLA-E). HLA-E up-regulation protects IFN-gamma-activated ECs from NK-mediated cell lysis, while sHLA-E protects bystander cells. Finally, sHLA-E is not detected in normal sera, and increased serum levels correlate with disease activity in patients with antineutrophil cytoplasmic antibody-associated systemic vasculitis. Thus, HLA-E expression and release of sHLA-E are features of EC activation and emphasize immunoregulatory functions of the endothelium. The present identification of soluble HLA-E molecules may have important implications in understanding the pathogenesis of immune-mediated vascular diseases and for the diagnosis and monitoring of patients.
Abstract: HLA-E are nonclassical MHC molecules with poorly characterized tissue distribution and functions. Because of their capacity to bind the inhibitory receptor, CD94/NKG2A, expressed by NK cells and CTL, HLA-E molecules might play an important role in immunomodulation. In particular, expression of HLA-E might favor tumor cell escape from CTL and NK immunosurveillance. To address the potential role of HLA-E in melanoma immunobiology, we assessed the expression of these molecules ex vivo in human melanoma biopsies and in melanoma and melanocyte cell lines. Melanoma cell lines expressed no or low surface, but significant intracellular levels of HLA-E. We also report for the first time that some of them produced a soluble form of this molecule. IFN-gamma significantly increased the surface expression of HLA-E and the shedding of soluble HLA-E by these cells, in a metalloproteinase-dependent fashion. In contrast, melanocyte cell lines constitutively expressed HLA-E molecules that were detectable both at the cell surface and in the soluble form, at levels that were poorly affected by IFN-gamma treatment. On tumor sections, a majority of tumor cells of primary, but a low proportion of metastatic melanomas (30-70 and 10-20%, respectively), expressed HLA-E. Finally, HLA-E expression at the cell surface of melanoma cells decreased their susceptibility to CTL lysis. These data demonstrate that HLA-E expression and shedding are normal features of melanocytes, which are conserved in melanoma cells of primary tumors, but become dependent on IFN-gamma induction after metastasis. The biological significance of these findings warrants further investigation.
Abstract: Lnk, with APS and SH2-B (Src homology 2-B), belongs to a family of SH2-containing proteins with potential adaptor functions. Lnk regulates growth factor and cytokine receptor-mediated pathways implicated in lymphoid, myeloid, and platelet homeostasis. We have previously shown that Lnk is expressed and up-regulated in vascular endothelial cells (ECs) in response to tumor necrosis factor-alpha (TNFalpha). In this study, we have shown that, in ECs, Lnk down-regulates the expression, at both mRNA and protein levels, of the proinflammatory molecules VCAM-1 and E-selectin induced by TNFalpha. Mechanistically, our data indicated that, in response to TNFalpha, NFkappaB/p65 phosphorylation and translocation as well as IkappaBalpha phosphorylation and degradation were unchanged, suggesting that Lnk does not modulate NFkappaB activity. However, Lnk activates phosphatidylinositol 3-kinase (PI3K) as reflected by Akt phosphorylation. Our results identify endothelial nitric-oxide synthase as a downstream target of Lnk-mediated activation of the PI3K/Akt pathway and HO-1 as a new substrate of Akt. We found that sustained Lnk-mediated activation of PI3K in TNFalpha-activated ECs correlated with the inhibition of ERK1/2 phosphorylation, whereas phosphorylation of p38 and c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinases (MAPKs) was unchanged. ERK1/2 inhibition decreases VCAM-1 expression in TNFalpha-treated ECs. Collectively, our results identify the adaptor Lnk as a negative regulator in the TNFalpha-signaling pathway mediating ERK inhibition and suggest a role for Lnk in the interplay between PI3K and ERK triggered by TNFalpha in ECs.
Abstract: Alloreactive T cells play a key role in mediating graft-vs-host disease and allograft rejection, and recent data suggest that most T cell alloreactivity resides within the CD4 T cell subset. Particularly, T cell responses to herpesvirus can shape the alloreactive repertoire and influence transplantation outcomes. In this study, we describe six distinct EBV-specific CD4(+) T cell clones that cross-reacted with EBV-transformed lymphoblastoid cell lines (LCLs), dendritic cells, and endothelial cells expressing MHC class II alleles commonly found in the population. Allorecognition showed exquisite MHC specificity. These CD4(+) T cell clones efficiently killed dendritic cells or LCLs expressing the cross-reactive allogeneic MHC class II molecules, whereas they did not kill autologous LCLs. Endothelial cells expressing the proper allogeneic MHC molecules were poorly killed, but they induced high-level TNF-alpha production by the EBV-specific CD4(+) T cell clones. As already proposed, the strong alloreactivity toward LCLs suggest that these cells could be used for selective depletion of alloreactive T cells.
Abstract: Lnk is an adaptator protein involved in B lymphocytes and platelet differentiation and in T lymphocyte activation. We previously reported on Lnk expression and regulation in endothelial cells (ECs) upon activation. In the present study, the involvement of Lnk in the tumor necrosis factor alpha (TNFalpha) pathway was investigated in vitro through Lnk overexpression in primary cultures of human endothelial cells. Using a recombinant adenovirus encoding human Lnk, we first demonstrated that Lnk overexpression does not induce vascular cell adhesion molecule-1 (VCAM-1) suggesting that Lnk does not promote ECs activation. However, Lnk overexpression significantly reduced TNFalpha-mediated expression of VCAM-1 (at mRNA and protein levels) in activated EC as compared with controls. Western blot analysis showed that Lnk overexpression in HUVEC was associated with phosphorylation of Akt kinase (at Ser 473) with no effect on IkappaBalpha, the specific inhibitor of NFkappaB, indicating that Lnk promotes activation of the phosphatidylinositol 3-kinase (PI3-kinase) pathway in ECs. Altogether, these results suggest that, in ECs, Lnk may participate to a regulatory pathway involving the PI3-kinase and modulating the inflammatory response.
Abstract: Originally designed to target elevated lipids, the "traditional" cause of atherosclerosis, statins might also confer vascular benefit by directly or indirectly modulating both the inflammatory and immune responses. Statins have been shown to downregulate MHC class II and CD40 expression on activated endothelial cells (EC). In this study, we investigate the potential effect of statins on MHC class I expression and regulation in response to IFNgamma. Primary cultures of human ECs have been treated with increasing doses of fluvastatin (0.01; 0.1 and 1 microM) with or without IFNgamma for 48 hours. Surface expression of MHC class I and class II has been analyzed by flow cytometry. Our data indicate that fluvastatin increases MHC class I expression on quiescent ECs by a dose-dependent effect. Furthermore, fluvastatin potentiates the MHC class I upregulation but prevents MHC class II induction triggered by IFNgamma. These effects are reversed by mevalonate. In conclusion, our results suggest that while decreasing MHC class II expression, fluvastatin (at 0.1 and 1 microM) upregulates MHC class I expression on ECs. Functional consequences of statin-mediated modulation of MHC on ECs have still to be elucidated in vitro and in vivo.
Abstract: HLA class I ligation on graft endothelial cells (EC) has been shown to promote graft arteriosclerosis and chronic allograft nephropathy. This study investigated transcriptional and functional changes mediated by anti-HLA antibodies (Ab), developed by transplant recipient, on vascular renal EC. For mimicking interactions that occur between alloantibodies and graft endothelium, HLA-typed primary cultures of human EC were incubated in vitro in the presence of monomorphic or polymorphic anti-HLA class I Ab. Gene expression analysis identified the upregulation of several molecules involved in cell signaling and proliferation, including the GTP-binding protein RhoA. It was demonstrated further that HLA class I ligation on EC induced a rapid translocation of RhoA to the cell membrane associated with F-actin stress fiber formation and cytoskeleton reorganization. Western blot analysis showed that anti-HLA class I Ab induced, in addition to RhoA, the activation of phosphatidylinositol 3-kinase, reflected by the phosphorylation of Akt (Ser473) and GSK3beta (Ser9), in EC. C3 exoenzyme, an inhibitor of RhoA, inhibited RhoA translocation in response to HLA class I ligation and reduced phosphatidylinositol 3-kinase activation. EC proliferation and cell cycle progression, examined by 5,6-carboxyfluorescein diacetate succinimidyl ester staining, demonstrated that anti-HLA-induced EC proliferation was efficiently prevented by the 3-hydroxy-3-methylglutaryl CoA reductase inhibitor simvastatin (0.1 micromol/L) through inhibition of RhoA geranylgeranylation. Taken together, these findings support the conclusion that RhoA is a key mediator of signaling pathways that lead to cytoskeletal reorganization and EC proliferation in response to alloantibodies that bind to HLA class I and demonstrate the specific and potent inhibitory effect of simvastatin on allostimulated EC growth.
Abstract: Control of molecular targets and signaling pathways which improve endothelial cell survival may be an attractive concept for interfering with dysregulated vascular injury and remodeling, a key mechanism for transplant arteriosclerosis and chronic allograft rejection. In addition to inhibiting matrix metalloproteinase activity, it has been suggested by recent studies that the tissue inhibitor of metalloproteinase (TIMP)-1 may inhibit apoptosis in various cell types. The present work examines the possibility that TIMP-1 belongs to a protective pathway via antiapoptotic properties and investigates the signaling pathway mediated by TIMP-1 in human ECs. We demonstrate that exogenous, recombinant, TIMP-1 efficiently prevents apoptosis induced by TNFalpha in cycloheximide-sensitized ECs. The antiapoptotic effect of TIMP-1 was dose-dependent and a maximal effect of TIMP-1 (30% protection) was reached using 250 ng/mL of recombinant TIMP-1. We present evidence that TIMP-1 induces activation of PI3-kinase but not NFkappaB pathway in ECs. Our findings further indicate that TIMP-1-induced EC survival is mediated through activation of PI3-kinase pathway and the downstream phosphorylation of Akt kinase. Blocking the PI3-kinase pathway with wortmannin or LY294002 restores TNFalpha-mediated EC death. In conclusion, our findings suggest that TIMP-1, generated upon inflammation, acts as an antiapoptotic molecule that can prevent EC apoptosis through activation of the PI3-kinase and phosphorylation of the Akt kinase.
Abstract: BACKGROUND: Human leukocyte antigen (HLA)-DR ligation mediates cell death of antigen-presenting cells (APC), including mature B cells, macrophages, and dendritic cells. This study investigates the apoptotic effects of HLA class II ligation mediated by anti-HLA antibodies on activated human vascular graft endothelial cells (ECs). METHODS: HLA class II expression was examined by flow cytometry using a panel of HLA-typed vascular ECs isolated from transplant donors and compared with that of B lymphocytes. The apoptotic effects of anti-HLA-DR monoclonal antibodies (mAbs) were investigated using viability assays, DNA content analysis, and annexin-V labeling. Intracellular signaling pathways mediated by HLA-DR ligation on ECs were examined by Western blotting. RESULTS: Even with optimal stimulation, the expression of HLA-DR on interferon (IFN)-gamma-treated ECs was quantitatively lower (3-5-fold) than that on B cells. Whereas anti-HLA-DR monomorphic mAbs induced apoptosis of B cells (approximately 22%), no significant apoptosis of IFN-gamma-activated (DR-positive) ECs ( < 5%), collected from the same donor, was observed under the same conditions. Similarly, specific polymorphic anti-HLA-DR11 or -DR16 antibodies were unable to induce EC apoptosis. Nevertheless, antibody-binding to HLA-DR on ECs is sufficient to induce intracellular signaling, as evident in the modulation of tyrosine phosphorylation and protein kinase (PK)C-alpha/beta and PKB/Akt activation. Our results suggest that HLA-DR ligation induces both common and divergent signaling events in ECs and B cells. CONCLUSION: Collectively, our data suggest that, in contrast with professional APC, graft ECs evade apoptosis mediated by HLA-DR ligation, not as a result of moderate HLA-DR expression but rather as a result of a specific signaling pathway.
Abstract: The vascular endothelium of transplanted organs represents an important target for allograft-directed immune responses. Although HLA antigens expressed on graft endothelial cells (EC) can become targets of the host immune response, the role of other, non-HLA-encoded EC antigens has been proposed but is still unclear. The aim of this study was to investigate the presence of and to characterize anti-EC antibodies (AECA) in 57 kidney transplant recipients according to their HLA-immunization status. Flow cytometry in pretransplant sera was used to detect AECA reactive with surface antigens on ABO and HLA-typed primary cultures of arterial ECs, stimulated or not with tumor necrosis factor-alpha (TNFalpha) or interferon-gamma (IFNgamma). FACS analysis revealed the presence of AECA in 47% of HLA-sensitized (PRA = 10%: mostly IgG) vs. 16.0% in nonsensitized patients (PRA < 10%) (p < 0.02). No significant correlation was found between the presence of AECA and acute rejection occurrence and graft outcome. Non-HLA reactive AECA are directed against TNFalpha- and IFNgamma-inducible membrane molecule(s), and react with two predominant antigens of approximately 35 kDa and approximately 50 kDa expressed on ECs but not on B cells. Binding of AECA decreases in vitro EC viability by 50-60% by promoting EC apoptosis, as demonstrated by DNA fragmentation assays.
Abstract: BACKGROUND: Fas ligand expression by cells of the vessel wall has been proposed to play a role in normal and pathologic conditions. Genetic engineering of vascularized organs for endothelial cell (EC) expression of FasL could protect the endothelium and underlying tissues from infiltrating Fas+ leukocytes. Nevertheless, the endogenous expression of FasL by ECs of different species and the potential deleterious effects of enforced FasL expression by ECs are largely unknown. In human ECs, levels of FLICE/caspase 8-inhibitory protein (FLIP) have been shown to control apoptosis mediated by Fas. MATERIALS AND METHODS: Cell surface expression of FasL in rat, mouse, human, and pig ECs was obtained using recombinant adenoviruses or transient plasmid transfection assays. FasL expression was evaluated by FACS analysis and cytotoxicity assays. Apoptosis was evaluated using annexin V, TUNEL, and cytotoxicity assays. FLIP levels were evaluated by Western blot analysis and overexpression was obtained by transient transfection. RESULTS: Analysis of ECs from different species showed that FasL was predominantly present in the cytoplasm, and depending on the species, little or no cell surface expression was detected. Enforced cell surface expression of FasL on rat or mouse ECs, either in culture or within the vessel wall resulted in massive apoptosis. In contrast, porcine or human ECs were completely resistant to apoptosis mediated by Fas-FasL interaction. Markedly reduced FLIP levels were observed in rat and mouse ECs compared to human and porcine ECs. Overexpression of FLIP in rat ECs conferred protection against cell surface expression of FasL. CONCLUSIONS: The consequences of FasL overexpression depend on the subcellular compartment and species in which FasL enforced expression is targeted and this is at least partially related to FLIP levels.
Abstract: BACKGROUND: RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations.Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha. MATERIALS AND METHODS: The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN. RESULTS: Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa. CONCLUSION: In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.
Abstract: BACKGROUND: A better understanding of inflammatory processes in endothelial cells (ECs) might reveal new ways of controlling inflammation and graft rejection. This study investigates EC genes regulated in response to human tumor necrosis factor (TNF)-alpha and xenogeneic natural antibodies (XNAs) that contribute to endothelial activation during transplantation. METHODS: Gene expression between resting and activated ECs was investigated by RNA differential display reverse-transcriptase polymerase chain reaction and confirmed by reverse-Northern blot. RESULTS: Forty-five cDNA fragments corresponding to genes up-regulated in activated ECs were identified. Our findings show that TNF-alpha-mediated EC activation was associated with increased levels of mRNA for the adaptor protein Lnk, the nuclear protein RED, and the initiation factor eIF4G. We further show that Lnk and eIF4G were also up-regulated in response to XNA binding to ECs. CONCLUSION: Our data suggest that TNF-alpha and XNAs could share common signaling pathways involving Lnk and eIF4G but may also drive specific transcriptional events.
Abstract: BACKGROUND: Effective improvement in xenograft survival is achieved using transplants from transgenic pigs expressing human complement (C) regulatory proteins, including decay-accelerating factor (DAF), CD59, and CD46 on endothelial cells (ECs). The aim of this study was to investigate whether human DAF expression in porcine ECs, as well as regulating C activation, can modify intercellular events through its interaction with its receptor, CD97, on human leukocytes. METHODS: Cellular interactions between human leukocytes and porcine ECs were investigated in vitro using ECs from either wild-type or DAF-transgenic pigs. Static leukocyte adhesion and T cell activation assays were performed using porcine ECs as target or effector cells, respectively. The role of the DAF-CD97 interaction was investigated using specific blocking monoclonal antibodies (mAbs) against human DAF and its receptor, CD97, in adhesion assays. RESULTS: Adhesion of U937 or Jurkat T cells, both expressing human DAF and CD97, was quantitatively similar for wild-type and transgenic-DAF-expressing pig ECs. Furthermore, blocking the CD97-DAF interaction did not inhibit xenogeneic leukocyte-endothelium adhesion, whereas blocking the very late antigen 4-vascular cell adhesion molecule-1 pathway reduced this adhesion by 50-80%. Furthermore, DAF and CD97 expression was not up-regulated during tumor necrosis factor-alpha- or lipopolysaccharide-mediated EC activation, unlike the adhesion molecules E-selectin, vascular cell adhesion molecule-1, and intracellular adhesion molecule (ICAM)-1. CONCLUSION: We found that high levels of human DAF expressed on ECs abrogates C-mediated cell damage but did not affect the in vitro adhesive properties or antigen-presenting cell function of genetically modified porcine ECs.
Abstract: The disaccharide galactose(alpha)1,3 galactose (the alphaGal epitope) is the major xenoantigen responsible for the hyperacute vascular rejection occurring in pig-to-primates organ transplantation. The synthesis of the alphaGal epitope is catalyzed by the enzyme alpha1,3-galactosyltransferase (alpha1,3GalT). To be able to control porcine alpha1,3GalT gene expression specifically, we have analyzed the upstream portion of the alpha1,3GalT gene, and identified the regulatory sequences. Porcine alpha1,3GalT transcripts were detected by 5' RACE analysis, and the corresponding genomic sequences were isolated from a phage library. The porcine alpha1,3GalT gene consists of at least 10 different exons, four of which contain 5' untranslated sequence. Four distinct promoters, termed A-D, drive alpha1,3GalT gene transcription in porcine cells. A combination of alternative promoter usage and alternative splicing produces a series of transcripts that differ in their 5' portion, but encode the same protein. Promoters A-C have been isolated, and functionally characterized using luciferase reporter gene assays in transfected porcine endothelial cells (PEC-A). Promoter preference in porcine endothelial cells was estimated on the basis of relative transcript levels as determined by real-time quantitative PCR. More than 90% of the alpha1,3GalT transcripts in PEC-A cells originate from promoter B, which has characteristics of a housekeeping gene promoter. While promoter preference remains unchanged, alpha1,3GalT mRNA levels increase by 50% in 12 h upon tumour necrosis factor alpha-activation of PEC-A cells. However, the magnitude of this change induced by inflammatory conditions could be insufficient to affect cell surface alpha1,3-galactosylation.
Abstract: In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.
Abstract: Endothelial cell (EC) interaction with antigraft antibodies (Abs) mediates EC injury and activation involved in vascular graft rejection. The aim of this study was to identify EC genes regulated in response to antigraft Ab binding that contribute to the endothelium alterations implicated in graft rejection or survival. By means of RNA differential display, 13 cDNA fragments corresponding to genes differentially expressed in ECs incubated with antigraft Abs were identified. Among these cDNAs were found the tissue inhibitor of metalloproteinase-1 (TIMP-1) and a desintegrin and metalloproteinase (ADAM-10). We demonstrated that TIMP-1 and ADAM-10 mRNA and protein expression was rapidly upregulated in ECs in response to antigraft Ab binding. Our data showed that TIMP-1 was upregulated in response to human IgG but not IgM and anti-galactosyl (Gal) alpha1-3Gal human xenogeneic Abs. In contrast, upregulation of ADAM-10 in ECs was shown to be mostly mediated by anti-Galalpha1-3Gal IgM Abs. Specific effects of human IgG and IgM xenogeneic Abs on endothelial transcripts indicate that different isotypes and specificities of Abs may mediate different EC changes. Our results suggest that interaction of ECs with antigraft Abs, according to their specificity, selectively induces synthesis and release of metalloproteinases and inhibitors, controlling proteolytic processes and immunological events that respectively contribute to graft rejection or survival.
Abstract: BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E-selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte-EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug-mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC.
Abstract: BACKGROUND: Cyclosporine (CsA) is currently given to recipients of vascularized xenografts as part of the immunosuppressive regimen required to prevent the hyperacute rejection phase. The effects of CsA on non-lymphoid immune cells, such as endothelial cells (ECs), have not been well characterized and sometimes seem contradictory, because both protective and adverse effects have been reported. In the present study, we investigated in vitro whether CsA could alter the antigenicity of activated porcine aortic endothelial cells (PAECs) by reducing class I and class II MHC antigen expression. METHODS: The effect of CsA on MHC antigen expression during tumor necrosis factor (TNF)-alpha- or lymphocyte-mediated PAEC activation was evaluated in vitro by flow cytometry and correlated to the ability of porcine ECs to promote human T lymphocyte proliferation. The effect of CsA on class II MHC antigen mRNA expression was also analyzed and related to class II transcriptional activator (CIITA) mRNA expression. RESULTS: Flow cytometry analysis showed that TNF-alpha-mediated induction of class II MHC antigen expression on PAECs was completely inhibited by CsA, whereas expression of class I MHC was reduced by 50%. The inhibition was dose dependent (at drug concentrations ranging from 2.5 microg/ml to 20.0 microg/ml) and was consistently observed at all time points (24-72 hr) during the activation period. Decreased MHC antigen expression dramatically reduced the ability of PAECs to further promote human T-cell proliferation. Similar levels of inhibition were achieved using an anti-porcine class II MHC blocking monoclonal antibody. Pretreatment of PAECs with CsA for 4 hr before coculture with human peripheral blood leukocytes efficiently blocked the induction on PAECs of E-selectin and class II MHC antigens and inhibited overexpression of class I antigens. Semiquantitative reverse transcriptase-polymerase chain reaction experiments showed that CsA markedly reduced the steady-state level of porcine class II (SLA-DRA and SLA-DQA) mRNA at 16 hr, compared with PAECs stimulated with TNF-alpha alone. The reduced level of class II MHC mRNA was associated with a lack of CIITA expression at this time point, suggesting that CsA could alter transcription or promote the rapid decay of CIITA mRNA. CONCLUSION: Our study indicates that CsA could play a role in preventing porcine MHC antigens being directly presented to human T lymphocytes by xenogeneic ECs.
Abstract: BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.
Abstract: Immunization of mice with TNF alpha-activated porcine endothelial cells led to the characterization of two monoclonal antibodies (MAbs), 5F3 and 8A7, specific for porcine VCAM-1. Upon flow cytometry, both antibodies increasingly labeled endothelial cells according to their degree of activation. They bound a band of MW 80 kDa on Western blots of endothelial cells, which is the apparent molecular weight of porcine VCAM-1. It was determined by surface plasmon resonance that the antibodies are directed to different antigenic sites. It was also found that 5F3 competes for binding the antigen with a MAb previously characterized as binding domain 1 of porcine VCAM-1. Subsequently, 5F3, but not 8A7, was found to inhibit the adhesion of human B lymphocyte Ramos cells to porcine endothelial cells in vitro. These antibodies, which do not cross-react with human VCAM-1, might be useful for diagnostic or therapeutic purposes in xenotransplantation.
Abstract: BACKGROUND: The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. METHODS: As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. RESULTS: After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. CONCLUSION: Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.
Abstract: Gal alpha 1,3Gal carbohydrate residues are present in the glycoproteins and glycolipids of lower mammals, and appear to be involved in the binding specificity of several membrane receptors. We report here that endothelial cells stimulated with lipopolysaccharide or inflammatory cytokines modulate their expression of UPD-Gal: beta-D-Gal alpha 1,3-galactosyltransferase (alpha 1,3GT), the Golgi enzyme that attaches a galactose in alpha 1,3 configuration to an N-acetyllactosamine acceptor. Upon activation, the steady state level of mRNA is transiently increased, the modifications being paralleled by a transcriptional regulation of the gene. Cell-associated enzyme activity, on the other hand, falls rapidly after activation, before being up- and downregulated with kinetics that parallel those of the mRNA, and after 3 days reaches a level representing 40-60% of the activity in cells before activation. Overall Gal alpha 1,3Gal expression at the cell surface follows enzyme activity, except that it is insensitive to the rapid and transient reduction of activity occurring shortly after activation. This reduced alpha 1,3GT activity in stimulated EC is correlated with lower stability of the protein, and with a switch in the expression of the isoform pattern, isoform 1 being predominant in resting cells whereas after activation it is isoform 2 that predominates. The two isoforms, however, appear to have similar intrinsic stability, so that the reduced stability of the enzyme in activated EC probably results from an induced proteolytic degradation pathway.
Abstract: Recombinant adenoviruses can be used for in vivo gene transfer with great efficiency. However, the duration of transgene expression and the possibility of readministering the virus are severely limited by the host anti-adenovirus immune response, which is controlled mainly by cytokine networks. Adenoviruses encoding IL-4 (AdIL-4) or IL-10 (AdIL-10) were administered to rats through the portal vein and the anti-adenovirus immune response was studied. As compared with administering adenoviruses without transgene (Addl324) or with the lacZ gene (AdlacZ), AdIL-4, but not AdIL-10, resulted in a significant increase in leukocytes in the liver, with a predominance of macrophages that peaked on days 7 and 14 after gene transfer and gradually returned to normal by day 28. AdIL-4 induced a significant increase in both neutralizing and ELISA-detected anti-adenovirus antibodies, whereas AdIL-10 caused an increase in ELISA-detected antibodies alone. Anti-adenovirus antibodies were predominantly of Th1-dependent immunoglobulin subclasses in rats receiving Addl324, AdlacZ, or AdIL-10, whereas animals receiving AdIL-4 showed a predominance of Th2-dependent immunoglobulin subclasses. Type 1 (IFN-gamma) and type 2 (IL-5) cytokines were increased only in livers from rats receiving AdIL-4. Rats receiving AdIL-4 showed increased anti-adenovirus cytotoxic T lymphocyte activity and CD8+ cell depletion prevented leukocyte infiltration in the liver. These results show that IL-4 increases local and systemic immune responses against recombinant adenoviruses.
Abstract: Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta 1 integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha 2 integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta 1 integrin and allowed the purification of the complex alpha 2 beta 1, also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta 1 integrin.
Abstract: Several immune responses are either limited to or concentrated in a given organ. Cytokines produced during ongoing immune responses have organ-localized effects that can be only partially mimicked upon their systemic delivery. Recombinant adenoviruses are efficient vectors to induce transient organ-localized cytokine expression. This allows in vivo analysis of the effects of cytokines produced spatially and temporally in a manner comparable to that observed during immune responses. The authors generated recombinant adenovirus for rat IL-4 (AdIL-4) and IL-10 (AdIL-10) to analyse the in vivo effects of these two important immunoregulatory molecules after gene transfer in the liver. It was first established that AdIL-4 and AdIL-10 were able to direct the production of biologically active cytokines by different rat cell types in vitro. Intraportal injection of doses of up to 10(10) pfu of AdIL-10 or control non-coding recombinant adenovirus were well tolerated, and hepatic histology showed only mild alterations. Conversely, animals receiving more than 2.5 x 10(9) pfu of AdIL-4 showed dose-dependent mortality, with clinical signs of hepatic dysfunction. Liver histology in animals receiving 2.5 x 10(9) pfu of AdIL-4 showed severe acute hepatitis with maximal lesions between day 7 and 14 and almost complete normalization by day 28 after gene transfer. The leukocyte infiltrate was composed primarily of mononuclear cells, but eosinophils and mast cells were significantly increased as compared to control animals. Hepatic function was also altered in animals that received AdIL-4, with kinetics similar to that of histological lesions. Our study describes a model for investigating cytokine function in vivo through liver-localized transgene expression mediated by adenoviral vectors and demonstrates that liver production of IL-4 but not IL-10 results in acute severe hepatitis.
Abstract: The investigation of human complement (C) inhibitors with a view to overcoming C-mediated tissue injury stands to benefit from the production of anatomically suitable transgenic animals. In this study, we used the CMV-IE1 enhancer/promoter to control the expression in vivo in transgenic rats of the human terminal C protein inhibitor CD59. Five transgenic rats were identified, of which four possessed at least one complete copy of the transgene. The presence of human CD59 transcripts and protein was demonstrated in two transgenic rat lines. A widespread tissue distribution of cells expressing human CD59, similar in the two lines, was observed-principally in pancreas, brain, heart, kidney, intestine and striated muscle. Whereas expression in pancreas and brain was uniform, mosaicism of CD59 expression was observed in some tissues such as heart and kidney, a proportion of cells within the tissue not expressing the transgene. Immunohistological analysis revealed surface expression of human CD59 in a variety of cells, including fibroblasts, epithelial cells and muscle cells, but not in endothelial cells. In conclusion, this paper analyses at the cellular level human CD59 expression directed by the CMV promoter in transgenic rats, amd discusses how they could be used to investigate in vivo the role of C in a variety of pathologies.
Abstract: The production of transgenic rats by DNA-microinjection into fertilized ova has now become an established procedure, although fewer than 20 lines have been described during the last 5 years. Overall, transgenic rats remain more difficult to produce than transgenic mice, but satisfactory yields have been obtained by several laboratories. A review of the methods used to generate transgenic rats shows considerable variation between different laboratories, particularly in choice of strain, superovulation protocols and the use of embryo culture before reimplantation. In some instances, the production of transgenic rats has provided data that are new and relevant, compared to data obtained in mice bearing the same transgene. Models have been developed for human diseases such as hypertension and autoimmunity, and applications have been found in the study of carcinogenesis and in pharmacological research. Transgenic rat technology also opens up interesting perspectives for transplantation research, in which microsurgery is an essential procedure. Intensive research is in progress in several laboratories to produce rat embryonic stem (ES) cell lines, but existing lines have not participated in germ line formation a prerequisite for their use in gene knock out experiments.
Abstract: We studied a rat-to-cynomolgous monkey model for xenotransplantation of vascularized organs and found that a rat heart was rejected in 5.5 +/- 1.4 min (n = 10). This hyperacute rejection (HAR) was consistent with kinetic experiments in vitro that showed damage to rat endothelial cells (ECs) after 3 min of incubation with primate serum. Histopathology and ultrastructural analysis of rejected hearts showed marked EC damage and early adherence of platelets and polymorphonuclear leukocytes to the endothelium. Immunohistochemical analysis revealed deposition along endothelial surfaces of IgG, IgM, and complement (C) components of the classical but not the alternative pathway, suggesting that, as in the pig-to-primate model, HAR is mediated by the binding of recipient xenogeneic natural antibodies and C activation. The effect of C depletion on xenograft survival was evaluated in two recipients that were treated with cobra venom factor (CVF). CVF caused complete C inactivation, demonstrated by lack of serum hemolytic activity and C-dependent EC cytotoxicity at engraftment and until the animals died. The rat cardiac transplants survived for at least 9 hr and 77 hr. Histology showed massive interstitial hemorrhage, edema, and cellular infiltration with scanty fibrin deposits. These results in CVF-treated recipient monkeys indicate that C activation mediates the development of HAR in this rat-to-primate model. We suggest that the model may be of interest as an alternative to the more expensive and time-consuming pig-to-primate model for testing the efficacy of transgenic modification of donor organs to prolong xenograft survival and for studying mechanisms of discordant xenograft rejection.
Abstract: The present study analyzed the ability of human decay-accelerating factor (DAF) and CD59 to protect rat endothelial cell (EC) clones from human and primate complement-mediated lysis. By flow cytometry and Scatchard analysis, we show that human DAF and/or CD59 cDNAs under the transcriptional control of elongation factor 1-alpha promoter were expressed at levels similar to or higher than that of a human EC line. Human DAF and CD59 were released from the surface of transfected rat cells by phosphatidylinositol phospholipase C, demonstrating that the two molecules were linked to the cell membrane by means of a glycolipid anchor. The functional activity of the two human C regulatory proteins expressed on rat EC lines was studied using an in vitro assay of C-dependent cytotoxic in which rat EC were incubated with human or nonhuman primate sera as sources of xenogeneic natural antibodies and C. We demonstrate that human and monkey xenogeneic natural antibodies bind to rat cells and induce lysis by a C-dependent mechanism involving mainly the C direct activation pathway. Our data indicate that human DAF and CD59, expressed either alone or in combination, abrogated all EC cytotoxicity, even in the presence of 50% human serum. This protective phenotype was correlated with decreased membrane attack complex (CD59 and/or DAF transfectants) and C3 deposition (DAF transfectants) on EC surface. Antibodies against the transfected molecules abolished the protection against C-mediated lysis.
Abstract: A xenotropic Moloney murine leukemia virus-derived recombinant retrovirus (MMuLVSVnlslacZ) has been utilized to study the mechanism of virus entry into endothelial and epithelial porcine cells. In the genome of this recombinant retrovirus, the nlslacZ reporter gene is under the transcriptional control of both LTR and SV40 early promoter. The entry of the retrovirus has been determined from the expression of this transduced reporter gene after its integration into the infected cells. This allows the detection of a very low level of viral infection and hence entry of the virus. Exposure of the virus-cell mixture to acidic pH (less than 6) during the early phase of interaction reduces the level of internalization. Cellular infection in presence of weak bases, ammonium chloride and amantadine and an ionophore monensin at concentrations sufficient to neutralize the endosomal pH does not modify the extent of viral entry into the cells. The results indicate that the entry of the recombinant retrovirus into porcine cells takes place by a pH-independent viral membrane-cell plasma membrane fusion mechanism.
Abstract: Swine testis (ST) cell lines producing a murine recombinant retrovirus (RRV) were established in order to transfer the bacterial lacZ gene fused to a nuclear location signal (nlslacZ) into animal cells. ST cells were infected with the supernatant of the cat G355.5LacZ2 cell line which produces amphotropic and xenotropic MMuLVSVnlslacZ-defective RRV and wild amphotropic and xenotropic MMuLV. Expression of the nlslacZ reporter gene was under the transcriptional control of both the SV40 early promoter and the retroviral LTR. ST cells expressing the reporter gene were sorted and cloned by limiting dilutions. Fourteen STLacZ-cell lines were isolated and subsequently tested for virus production. Depending on the host range of the retroviruses, two cell lines (STBF11 and STAA3) produced both a xenotropic recombinant pseudotype and wild retroviruses; another (STAB 10) produced both an amphotropic recombinant pseudotype and wild retroviruses. Southern blot analysis of the producer cell lines was carried out to verify proviral integration. The efficiency of the different pseudotypes in the transfer of the nlslacZ reporter gene to cultured animal cells, including porcine cells, was compared to the pseudotyped RRV produced by cat lines. Our results showed that the xenotropic RRV produced by the porcine STBF 11 cell line has a high titre for cells from different species and led to a higher number of porcine endothelial and lymphoblastoid cells expressing the reporter gene than did RRV produced by the cat packaging cell lines.
Abstract: The TV4 cell line is derived from sheep ovarian tissues trypsinized for 60 min and developed from a clone after serial dilutions. The TV4 cells had a doubling time of 24 h in B2 medium with 10% fetal calf serum and 10% BSA. TV4 cells synthesized progesterone (P4) in the presence of cholesterol. As the concentration of cholesterol increased (0, 92.5 and 125 mg/l), synthesis of P4 increased (P<0.01) from 1.05 +/- 0.20 to 30.6 +/- 3.03 ng/ml. Kinetics of P4 production were determined; a linear production response (y = 5.816 + 1.05 x, y = ng/ml, x = hour of incubation; R(2) = 0.97) was observed with up to 35 ng/ml of P4 obtained by 30 h of incubation. Follicle stimulating hormone (FSH), luteinizing hormone (LH), testosterone, or FSH and testosterone did not have any effect on estradiol-17beta (E2) or P4 production. Aromatase activity measured by RIA and HPLC following incubation with either nonradiolabeled or labeled testosterone was undetectable. In conclusion, this study established a cell line from the sheep ovary which has a high ability of divide and produce progesterone.
Abstract: Lymphocyte subpopulations derived from different pig organs (blood, spleen, mesenteric lymph nodes) were separated by a simple, rapid and cheap panning technique, using either normal or ozone treated Petri dishes with bovine serum albumin. Slg+ lymphocytes could thus be obtained with a purity of up to 95% by adhesion onto Corning plastic dishes. The purified cells retained their proliferative activity with regard to lectins. The subpopulation including PT4 and PT8 was then separated by another panning on ozone-treated plastic dishes with a purity of 80-90%.
