Human Membrane Cofactor Protein Research Articles

Computational Analysis of CD46 Protein Interaction with SARS-CoV-2 Structural Proteins: Elucidating a Putative Viral Entry Mechanism into Human Cells.

This study examines an unexplored aspect of SARS-CoV-2 entry into host cells, which is widely understood to occur via the viral spike (S) protein's interaction with human ACE2-associated proteins. While vaccines and inhibitors targeting this mechanism are in use, they may not offer complete protection against reinfection. Hence, we investigate putative receptors and their cofactors. Specifically, we propose CD46, a human membrane cofactor protein, as a potential putative receptor and explore its role in cellular invasion, acting possibly as a cofactor with other viral structural proteins. Employing computational techniques, we created full-size 3D models of human CD46 and four key SARS-CoV-2 structural proteins-EP, MP, NP, and SP. We further developed 3D models of CD46 complexes interacting with these proteins. The primary aim is to pinpoint the likely interaction domains between CD46 and these structural proteins to facilitate the identification of molecules that can block these interactions, thus offering a foundation for novel pharmacological treatments for SARS-CoV-2 infection.

HEPCR.hTBM.hCD47.hHO-1 with donor clodronate and DDAVP treatment improves perfusion and function of GalTKO.hCD46 porcine livers perfused with human blood.

Platelet sequestration, inflammation, and inappropriate coagulation cascade activation are prominent in liver xenotransplant models and are associated with poor outcomes. Here, we evaluate a cassette of six additional genetic modifications to reduce anti-pig antibody binding (α-1,3-galactosyl transferase knockout [GalTKO]) and target coagulation dysregulation (human endothelial protein C receptor [hEPRC] and thrombomodulin [hTBM]), complement pathway regulation (human membrane cofactor protein, hCD46), inflammation heme oxygenase 1 [hHO-1]), and a self-recognition receptor (integrin-associated protein [hCD47]), as well as donor pharmacologic treatments designed to blunt these phenomena. Livers from GaltKO.hCD46 pigs ("2-gene," n=3) and GalTKO.hCD46 pigs also transgenic for hEPRC, hTBM, hCD47, and hHO-1 ("6-gene," n=4) were perfused ex vivo with whole human blood. Six-gene pigs were additionally pretreated with desmopressin (DDAVP) and clodronate liposomes to deplete vWF and kupffer cells, respectively. The average perfusion times increased from 304 (±148) min in the 2-gene group to 856(±61) min in the 6-gene group (p=.010). The average heparin administration was decreased from 8837 U/h in the 2-gene to 1354 U/h in the 6-gene group (p=.047). Platelet sequestration tended to be delayed in the 6-gene group (p=.070), while thromboxane B2 (TXB2, a platelet activation marker) levels were lower over the first hour (p=.044) (401±124vs. 2048±712 at 60min). Thrombin production as measured by F1+2 levels tended to be lower in the 6-gene group (p=.058). The combination of the hEPCR.hTBM.hCD47.hHO-1 cassette along with donor pig DDAVP and clodronate liposome pretreatment was associated with prolonged function of xenoperfused livers, reduced coagulation pathway perturbations, and decreased TXB2 elaboration, and reflects significant progress to modulate liver xenograft injury in a pig to human model.

Stable Regulation of Senescence-Related Genes in Galactose-alpha1,3-galactose Epitope Knockout and Human Membrane Cofactor Protein hCD46 Pig

BackgroundPigs are considered suitable animal donor models for xenotransplantation. For successful organ transplantation, immune rejection must be overcome. Xenotransplantation has recently been successfully performed using galactose-alpha1,3-galactose epitopes knockout (GalTKO) and a human membrane cofactor protein (hCD46) in a pig model. However, the growth and lifespan of the grafted organ have not been evaluated. Therefore, in the present study we evaluated aging and 84 senescence-related genes using the RT2 Profiler PCR array and whole blood samples from GalTKO/hCD46 Massachusetts General Hospital (MGH) pigs. MethodsExperimental groups were double GalTKO/hCD46 (5-month-old), single GalTKO/hCD46 (2-year-old), and non-genetically modified (>3.5-year-old; control group within the same strain). Age-matched white hairless Yucatan (WHY) miniature pig groups were used as controls. ResultsAmong the 19 senescence-related genes selected from the 84 genes for further evaluation, 13 were upregulated in the double GalTKO/hCD46 MGH pigs compared to control MGH pigs; however, in WHY pigs, only 4 genes were up- or down-regulated among the 19 genes. Moreover, in double GalTKO/hCD46 MGH and WHY pigs, the expression of the 19 genes changed only 1- to 2-fold, suggesting that there were no significant differences in senescence signals between the 2 pig lines. ConclusionsThe present results indicate that the double GalTKO/hCD46 MGH pig might be a suitable model for human xenotransplantation studies. However, we used a limited number of experimental individuals, so further studies using larger experimental groups should be conducted to verify the present results.

Consistent success in life-supporting porcine cardiac xenotransplantation.

Heart transplantation is the only cure for patients with terminal cardiac failure, but the supply of allogeneic donor organs falls far short of the clinical need1-3. Xenotransplantation of genetically modified pig hearts has been discussed as a potential alternative4. Genetically multi-modified pig hearts that lack galactose-α1,3-galactose epitopes (α1,3-galactosyltransferase knockout) and express a human membrane cofactor protein (CD46) and human thrombomodulin have survived for up to 945days after heterotopic abdominal transplantation in baboons5. This model demonstrated long-term acceptance of discordant xenografts with safe immunosuppression but did not predict their life-supporting function. Despite 25years of extensive research, the maximum survival of a baboon after heart replacement with a porcine xenograft was only 57days and this was achieved, to our knowledge, only once6. Here we show that α1,3-galactosyltransferase-knockout pig hearts that express human CD46 and thrombomodulin require non-ischaemic preservation with continuous perfusion and control of post-transplantation growth to ensure long-term orthotopic function of the xenograft in baboons, the most stringent preclinical xenotransplantation model. Consistent life-supporting function of xenografted hearts for up to 195days is a milestone on the way to clinical cardiac xenotransplantation7.

Development of α 1,3-galactosyltransferase Inactivated and Human Membrane Cofactor Protein Expressing Homozygous Transgenic Pigs for Xenotransplantation

Development of α 1,3-galactosyltransferase Inactivated and Human Membrane Cofactor Protein Expressing Homozygous Transgenic Pigs for Xenotransplantation

Reduction of myocardial ischemia reperfusion injury in pigs by (over) expression of human membrane co-factor protein

Edible films and barriers derived from polysaccharides, proteins and lipids provide several functions which prevent mass transfer, for example, water vapor, gas, and volatile compounds, between foods and environment. The preparation of materials can be either by solution casting commonly used in the laboratory or a more commercially extrusion process. Plasticizers are used to increase deformability reducing the glass transition temperature (Tg) of the materials which can be determined using differential scanning calorimetry (DSC) and dynamic mechanical thermal analysis (DMTA). This chapter also demonstrates the effects of plasticizers, other components, and treatments on the Tg values of various edible films. Moreover, the non-equilibrium state of the edible film matrices causes several time-dependent changes during storage or “aging,” for example, plasticizer loss, lipid destabilization, and crystallization which subsequently impacts functional and mechanical properties of the films.

Transgenic expression of human leukocyte antigen‐E attenuates GalKO.hCD46 porcine lung xenograft injury

Lung xenografts remain susceptible to loss of vascular barrier function within hours in spite of significant incremental advances based on genetic engineering to remove the Gal 1,3-αGal antigen (GalTKO) and express human membrane cofactor protein (hCD46). Natural killer cells rapidly disappear from the blood during perfusion of GalTKO.hCD46 porcine lungs with human blood and presumably are sequestered within the lung vasculature. Here we asked whether porcine expression of the human NK cell inhibitory ligand HLA-E and β2 microglobulin inhibits GalTKO.hCD46 pig cell injury or prolongs lung function in two preclinical perfusion models. Lungs from pigs modified to express GalTKO.hCD46 (n=37) and GalTKO.hCD46.HLA-E (n=5) were harvested and perfused with human blood until failure or elective termination at 4hours. Airway pressures and pulmonary artery hemodynamics were recorded in real time. Blood samples were also collected throughout the experiment for analysis. Porcine aortic endothelial cells (PAECs) from each genotype were cultured in monolayers in microfluidic channels and used in fluorescent cytotoxicity assays using human NK cells. HLA-E expression on GalTKO.hCD46 PAECs was associated with significantly decreased antibody-dependent and antibody-independent NK-mediated cytotoxicity under in vitro conditions simulating physiologic shear stress. Relative to GalTKO.hCD46 pig lungs perfused with human blood on an ex vivo platform, additional expression of HLA-E increased median lung survival (>4hours, vs 162minutes, P=.012), and was associated with attenuated rise in pulmonary vascular resistance, and decreased platelet activation and histamine elaboration. As expected, HLA-E expression was not associated with a significant difference in NK cell adhesion to endothelial cells in vitro, or NK cell and neutrophil sequestration during organ perfusion. We conclude human NK cell activation contributes significantly to GalTKO.hCD46 pig endothelial injury and lung inflammation and show that expression of HLA-E is associated with physiologically meaningful protection of GalTKO.hCD46 cells and organs exposed to human blood.

A highly susceptible CD46 transgenic mouse model of subcutaneous infection with Streptococcus dysgalactiae subspecies equisimilis.

The Streptococcus dysgalactiae subspecies equisimilis (SDSE) possesses clinical similarities to group A streptococcus (GAS) and has recently been recognized as a causative pathogen of life-threatening streptococcal infections. Human membrane cofactor protein (CD46), a complement regulatory protein ubiquitously expressed on every cell type except for erythrocytes, has been implicated as a receptor for human-specific pathogens including GAS. In the present report, SDSE strain GGS_124 was isolated from a patient suffering from streptococcal toxic shock syndrome. When CD46-expressing transgenic (Tg) and non-Tg mice were infected subcutaneously into a hind footpad with 1×10(7)colony-forming units of GGS_124, both CD46 Tg and non-Tg mice showed similar levels of colonization in the popliteal lymph nodes at day 3 after infection. However, the following differences were found between CD46 Tg and non-Tg mice after infection. First, there was a statistically significant difference in mortality rates between CD46 Tg (33%) and non-Tg (0%) mice within 35 days after infection. Second, all surviving CD46 Tg mice developed ankle arthritis at day 35 after infection, whereas non-Tg mice did not develop ankle arthritis on the infected hind paws. Finally, CD46 Tg mice developed a pus-filled abscess accompanied by renal failure at day 6 or later after infection. These observations suggest that CD46, the host cell-surface pathogen receptor, functioned to attract GGS_124 into deep tissues, so that the subcutaneous infection with GGS_124 induced invasive streptococcal diseases in CD46 Tg mice.

Tissue-specific deletion of Crry from mouse proximal tubular epithelial cells increases susceptibility to renal ischemia–reperfusion injury

The murine cell surface protein Crry is a key complement regulator with similar activities to human membrane cofactor protein (MCP) and decay-accelerating factor. MCP plays a critical role in preventing complement-mediated tissue injury and its mutation has been implicated in several human kidney diseases. Study of Crry in mice has relevance to understanding MCP activity in human diseases but such efforts have been hampered by the embryonic lethality phenotype of Crry gene knockout. Here we used a conditional gene targeting approach and deleted Crry from the mouse proximal tubular epithelial cells where Crry is prominently expressed. Absence of Crry from proximal tubular epithelial cells resulted in spontaneous C3 deposition on the basolateral surface but no apparent renal disease in unchallenged mice. However, mice deficient in Crry on proximal tubular epithelial cells developed exacerbated renal injury when subjected to renal ischemia reperfusion showing increased blood urea nitrogen levels, higher tubular injury scores, more tubular epithelial cell apoptosis, and inflammatory infiltrates. Renal ischemia reperfusion injury in the Crry conditional knockout mice was prevented by blocking C3 and C5 activation using an anti-properdin or anti-C5 mAb, respectively. Thus, Crry has a critical role in protecting proximal tubular epithelial cells during ischemia reperfusion challenge. Our results highlight the latent risk for inflammatory kidney injury associated with defects in membrane complement regulators.

Complement Regulation in Renal Disease Models

Activation of the complement system is tightly regulated by plasma and cell-associated complement regulatory proteins (CRPs), such as factor H (fH), decay-accelerating factor, and membrane cofactor protein. Animal models of disease have provided considerable insights into the important roles for CRPs in the kidney. Mice deficient in fH have excessive fluid phase C3 activation and inactivation, leading to deposition of inactivated C3b in glomerular capillary walls (GCW), comparable with dense deposit disease. In contrast, when fH lacks C-terminal surface targeting regions, local activation on the GCW leads to a disease reminiscent of thrombotic microangiopathy. The uniquely rodent protein, CR1-related y (Crry), has features analogous to human membrane cofactor protein. Defective Crry leads to unrestricted alternative pathway activation in the tubulointerstitium, resulting in pathologic features ranging from thrombotic microangiopathy (TMA), acute kidney injury, and tubulointerstitium nephritis. In the presence of initiators of the classic or lectin pathways, commonly in the form of immune complexes in human glomerular diseases, complement regulation is stressed, with the potential for recruitment of the spontaneously active alternative pathway. The threshold for this activation is set by CRPs; pathology is more likely when complement regulation is defective. Within the endocapillary region of the GCW, fH is key, while decay-accelerating factor and Crry are protective on mesangial cells and podocytes. Arguably, acquired alterations in these CRPs is a more common event, extending from pathologic states of cellular injury or production of inhibitory antibodies, to physiological fine tuning of the adaptive immune response.

Domain Swapping Reveals Complement Control Protein Modules Critical for Imparting Cofactor and Decay-Accelerating Activities in Vaccinia Virus Complement Control Protein

Vaccinia virus encodes a structural and functional homolog of human complement regulators named vaccinia virus complement control protein (VCP). This four-complement control protein domain containing secretory protein is known to inhibit complement activation by supporting the factor I-mediated inactivation of complement proteins, proteolytically cleaved form of C3 (C3b) and proteolytically cleaved form of C4 (C4b) (termed cofactor activity), and by accelerating the irreversible decay of the classical and to a limited extent of the alternative pathway C3 convertases (termed decay-accelerating activity [DAA]). In this study, we have mapped the VCP domains important for its cofactor activity and DAA by swapping its individual domains with those of human decay-accelerating factor (CD55) and membrane cofactor protein (MCP; CD46). Our data indicate the following: 1) swapping of VCP domain 2 or 3, but not 1, with homologous domains of decay-accelerating factor results in loss in its C3b and C4b cofactor activities; 2) swapping of VCP domain 1, but not 2, 3, or 4 with corresponding domains of MCP results in abrogation in its classical pathway DAA; and 3) swapping of VCP domain 1, 2, or 3, but not 4, with homologous MCP domains have marked effect on its alternative pathway DAA. These functional data together with binding studies with C3b and C4b suggest that in VCP, domains 2 and 3 provide binding surface for factor I interaction, whereas domain 1 mediates dissociation of C2a and Bb from the classical and alternative pathway C3 convertases, respectively.

Structure of the Extracellular Portion of CD46 Provides Insights into Its Interactions with Complement Proteins and Pathogens

The human membrane cofactor protein (MCP, CD46) is a central component of the innate immune system. CD46 protects autologous cells from complement attack by binding to complement proteins C3b and C4b and serving as a cofactor for their cleavage. Recent data show that CD46 also plays a role in mediating acquired immune responses, and in triggering autophagy. In addition to these physiologic functions, a significant number of pathogens, including select adenoviruses, measles virus, human herpes virus 6 (HHV-6), Streptococci, and Neisseria, use CD46 as a cell attachment receptor. We have determined the crystal structure of the extracellular region of CD46 in complex with the human adenovirus type 11 fiber knob. Extracellular CD46 comprises four short consensus repeats (SCR1-SCR4) that form an elongated structure resembling a hockey stick, with a long shaft and a short blade. Domains SCR1, SCR2 and SCR3 are arranged in a nearly linear fashion. Unexpectedly, however, the structure reveals a profound bend between domains SCR3 and SCR4, which has implications for the interactions with ligands as well as the orientation of the protein at the cell surface. This bend can be attributed to an insertion of five hydrophobic residues in a SCR3 surface loop. Residues in this loop have been implicated in interactions with complement, indicating that the bend participates in binding to C3b and C4b. The structure provides an accurate framework for mapping all known ligand binding sites onto the surface of CD46, thereby advancing an understanding of how CD46 acts as a receptor for pathogens and physiologic ligands of the immune system.

Co-expression and Synergic Effect of Human Complement Regulatory Proteins DAF and MCP

Recombinant expression vector pcDNA3-DAFMCP-DP containing human membrane complement regulatory proteins (hCRPs) decay accelerating factor (DAF) and membrane cofactor protein (MCP) cDNA was constructed by using two independent promoters. After transfected into NIH3T3 cells by calcium phosphate-DNA precipitate method, stable NIH3T3 pcDNA3 -DAFMCP-DP transfectants were obtained by G418 selection. Extraneous genes integration was identified by PCR. The co-expression of human DAF and MCP at both mRNA and protein levels was confirmed by using RT-PCR and Western blot analysis. Human DAF and MCP cDNA were integrated into NIH3T3 pcDNA3-DAFMCP-DP genomic DNA after continuous 30 times passages, indicating that NIH3T3 pcDNA3-DAFMCP-DP were stable cell lines. Human C-mediated cytolysis assays showed that NIH3T3 cells, transfected stably with pcDNA3-DAF, pcDNA3-MCP, and pcDNA3-DAFMCP-DP, were protected from C-mediated damage, and co-expressed human DAF and MCP provided more excellent protection against C-mediated attack than either DAF or MCP alone. These results suggest that DAF and MCP synergize the action of each other and the dicistronic vector could improve the efficiency of multi-gene delivery.

Life-supporting function of genetically modified swine lungs in baboons

During ex vivo perfusion with human blood, homozygous galactosyl transferase knockout swine lungs exhibit prolonged survival (approximately 2 hours) relative to wild-type (<15 minutes) and swine lungs expressing human decay accelerating factor (<1 hour). In this study, the in vivo behavior of galactosyl transferase knockout lungs was evaluated. Three galactosyl transferase knockout swine left lungs were transplanted into baboons in a life-supporting model. One baboon lung allograft and two swine lung xenografts transgenic for human membrane cofactor protein (CD46) served as controls. Whereas two membrane cofactor protein lungs exhibited high pulmonary vascular resistance (>500 mm Hg x min/L) and failed to support life within 21 minutes, two of three galactosyl transferase knockout lungs supported life, for 90 and 215 minutes, and displayed low peripheral vascular resistance (48 +/- 12 mm Hg x min/L at 60 minutes), similar to the allogeneic control. Complement activation (delta C3a < 250 ng/mL through 60 minutes) and C5b-9 deposition were minimal in both galactosyl transferase knockout and membrane cofactor protein lungs. Neutrophils, monocytes, and platelets were rapidly sequestered in galactosyl transferase knockout and human membrane cofactor protein lung recipients, unlike the allogeneic control (<20%); and thrombin formation (delta plasma fraction 1+2 > 0.5 nmol/L) was seen in the galactosyl transferase knockout recipients. Platelet activation (beta-thromboglobulin rise > 200) and appearance of capillary congestion and vessel thrombosis confirmed coagulation activation associated with galactosyl transferase knockout lung failure. Galactosyl transferase knockout swine lungs are significantly protected in vivo from the physiologic consequences (increased pulmonary vascular resistance, capillary leak) associated with hyperacute lung rejection. As during ex vivo perfusion, dysregulated coagulation-thrombin elaboration, platelet activation, and intravascular thrombosis-mediates galactosyl transferase knockout lung xenograft injury.

Positive expression of the immunoglobulin superfamily protein IZUMO on human sperm of severely infertile male patients

Recently, the immunoglobulin superfamily protein IZUMO on the sperm surface was identified as being essential for sperm-egg fusion. Although we examined the expression of IZUMO on sperm of 13 male infertile patients with severe oligozoospermia and/or athenozoospermia and 12 infertile patients with fertilization failure in previous conventional IVF to determine whether it is varied in infertility patients, we could not identify any patients with IZUMO negative by immunocytochemistry.

Co‐stimulation blockade targeting CD154 and CD28/B7 modulates the induced antibody response after a pig‐to‐baboon cardiac xenograft

The induced antibodies against Galalpha1,3Gal (Gal) and non-Gal epitopes may contribute to delayed xenograft rejection (DXR). We asked whether blockade of the CD40/CD154 and CD28/B7 co-stimulatory pathways modulates the baboon elicited antibody response to pig Gal and non-Gal antigens. Eighteen baboons received heterotopic heart transplants from pigs transgenic for human decay-accelerating factor (n = 13) or membrane cofactor protein (n = 5). Ten reference ''conventional therapy'' animals received cyclosporin A, cyclophosphamide and mycophenolate mofetil, with (n = 4) or without (n = 6) anti-CD20. Eight ''co-stimulation blockade'' animals received anti-CD154 mAb (IDEC-131) and anti-thymocyte globulin, with (n = 4) or without (n = 4) anti-CD20; two of these animals also received CTLA4-Fc. Anti-alphaGal IgG and IgM, anti-non-Gal antibodies and graft histology were assessed serially. Excluding two early graft failures, median graft survival with conventional therapy was 15 days (range 6 to 36 days, n = 8). Anti-Gal IgG antibody remained low through day 6 to 10, only one graft failure was accompanied by significant rise in anti-Gal IgG, and the anti-non-Gal response was weak (n = 2) or absent (n = 7). However many recipients succumbed with infection (n = 4) or coagulopathy (n = 2); DXR and ICOS+ T cells were prevalent in long-surviving grafts. With co-stimulation blockade, excluding three early graft failures, median graft survival was 7 days (range 6 to 11 days, n = 5). This regimen was very well tolerated, but increased anti-Gal antibody titer within 14 days was associated with graft failure in four of six animals. Although an anti-non-Gal response was present in three of six animals during IDEC-131 monotherapy (one strong, two weak), it was absent in both cases with additional CTLA4-Fc treatment. As used here, CD154 blockade alone does not completely prevent induction of Gal and non-Gal anti-pig antibodies. Our preliminary data suggest that other co-stimulation pathways, including CD28/B7 and ICOS, are sufficient to mediate high-titer anti-non-Gal antibody to porcine antigens in baboons, and contribute significantly to the pathogenesis of DXR.

Transgenic pigs expressing human CD59, in combination with human membrane cofactor protein and human decay‐accelerating factor

The expression of human complement regulators has been proved as an effective strategy to overcome hyperacute rejection in discordant xenogeneic organ transplantation. In this study, we tested the hypotheses that expression of triple transgenes for human complement regulators and provide more effective protection to the transplanted pig tissues. Pigs transgenic for human complement regulatory proteins, human CD59 (hCD59) and human membrane cofactor protein (hMCP), have been generated using large genomic constructs. Heterozygous human decay-accelerating factor (hDAF) transgenic pigs, from a previously established line, were bred with hCD59 or hCD59 plus hMCP pigs to produce animals that expressed both hCD59 and hDAF, or expressed triple transgenes hCD59, hDAF and hMCP. All three transgenes were widely expressed in most of the tissues analyzed, but the expression of hMCP was at low levels. In cytotoxicity assays on porcine peripheral blood mononuclear cells, the expression of a single transgenic protein, hCD59, or hCD59 in combination with hMCP provided similar protection against human complement-mediated damage as the single expression of hDAF. However, the expression of triple transgenic proteins or double hCD59 and hDAF transgenic proteins provided greater protection than either hCD59 or hDAF alone. Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.

215 COMPARISON OF STRONG ENDOTHELIAL CELL-SPECIFIC PROMOTERS FOR EXPRESSION OF HUMAN COMPLEMENT REGULATORY PROTEINS IN PORCINE XENOGRAFT ENDOTHELIAL CELLS

The complement system is composed of a complex group of soluble proteins that have important roles in the immune response against foreign cells such as xenografted tissue cells. Activation of complement pathways results in the formation of the membrane attack complex (MAC). Several cell surface regulators known as membrane complement regulatory proteins (mCRPs) prevent the formation of MAC. For blocking the formation of MAC in the xenotransplanted cells, we tried to overexpress mCRPs in the endothelial cells to prevent the hyperacute rejection (HAR), because one of the major obstacles to xenotransplantation of vascularized organs was reported to be HAR. For this purpose, we isolated human MCP (membrane cofactor protein, CD46) (1.5 kb) and DAF (decay-accelerating factor, CD55) (1.1 kb) promoter regions from human genomic DNA isolated from the human embryonic kidney cell line HEK293, and cloned into pGL3 plasmid for luciferase reporter assay. We also inserted other known endothelial cell-specific promoters, such as Flk-1 (fetal liver kinase-1) and ICAM-2 (intercellular adhesion molecule-2), thrombomodulin promoters, into pGL3 vector to compare their promoter activities. We transfected the plasmids into several endothelial cell lines such as bovine aortic endothelial cells (BAEC), human umbilical vein endothelial cells (HUVEC), and mouse pancreatic microvascular endothelial cells (MS1). We also used two epithelial cell lines, human embryonic kidney epithelial cells (HEK293) and human epidermal keratinocytes (HaCaT). The endothelial specific expression of the promoters were compared with those of SV40, CMV, and EF-2a (elongation factor) promoters which are generally used in mammalian gene expression. Luciferase assays showed that among the endothelial cell-specific promoters, the 1.1 kb DAF promoter was the strongest, followed by the Flk-1 promoter. The promoter activity of 1.1 kb DAF was about 2-fold that of the Flk-1 promoter. Moreover, the 1.1 kb DAF and Flk-1 promoters were chosen as the best endothelial specific promoters, as both showed about 5 times more luciferase activity in the endothelial cells compared to that in the epithelial cell lines. We also found that the 5′-flanking region between −1126 and −968 of the 1.1 kb DAF promoter was very important for the endothelial-specific strong gene expression. On the 0.2 kb region of DAF promoter, we could detect several conserved nucleotide sequences interacting with the specific transcription factors, such as GATA-1, USF, CdxA, SRY, Sox-5, and Sp1. Interestingly, deletion of the GATA-1 motif between −1113 and −1104 reduced the promoter activity of the DAF promoter by about 25%. We conclude that the 1.1 kb DAF promoter is a suitable candidate promoter for strong endothelial cell-specific expression of mCRP genes.

Transgenic expression in pig hearts of both human decay-accelerating factor and human membrane cofactor protein does not provide an additional benefit to that of human decay-accelerating factor alone in pig-to-baboon xenotransplantation.

This study investigated whether the coexpression of human decay-accelerating factor (hDAF) and human membrane cofactor protein (hMCP) on porcine organs provides an additional benefit to that of hDAF alone to prevent rejection. Heterotopic heart xenotransplantation was performed in baboons with either hDAF (n=5) or hDAF/hMCP (n=5) transgenic pig organs. The only immunosuppression given was GAS914 (a soluble Gal [alpha1-3] Gal polymer) and cyclosporine A. With the exception of one hDAF organ that failed from a left atrium thrombosis, all xenografts developed acute humoral xenograft rejection. Acute humor xenograft rejection occurred at a median time of 152 hr in hDAF hearts and 162 hr in hDAF/hMCP organs. Recipients of hDAF or hDAF/hMCP hearts did not differ in their patterns of serum antiporcine antibodies or in plasma levels of the soluble terminal complement complex sC5b-9. It is concluded that in this pig-to-baboon heterotopic heart transplant, model expression of hDAF/hMCP does not provide an additional benefit in prevention of rejection to that of hDAF alone.

Characterization of a CD46 transgenic pig and protection of transgenic kidneys against hyperacute rejection in non‐immunosuppressed baboons

Abstract: Human membrane cofactor protein (CD46) controls complement activation and when expressed sufficiently as a transgene protects xenografts against complement‐mediated rejection, as shown here using non‐immunosuppressed baboons and heterotopic CD46 transgenic pig kidney xenografts. This report is of a carefully engineered transgene that enables high‐level CD46 expression. A novel CD46 minigene was validated by transfection and production of a transgenic pig line. Pig lymphocytes were tested for resistance to antibody and complement‐mediated lysis, transgenic tissues were characterized for CD46 expression, and kidneys were transplanted to baboons without immunosuppression. Absorption of anti‐Galα(1,3)Gal epitope (anti‐GAL) serum antibodies was measured. Transgenic pigs expressed high levels of CD46 in all tissues, especially vascular endothelium, with stable expression through three generations that was readily monitored by flow cytometry of transgenic peripheral blood mononuclear cells (PBMC). Transgenic PBMC pre‐sensitized with antibody were highly resistant to human complement‐mediated lysis which readily lysed normal pig PBMC. Normal pig kidneys transplanted without cold ischemia into non‐immunosuppressed adult baboons survived a median of 3.5 h (n = 7) whereas transgenic grafts (n = 9), harvested at ∼24‐h intervals, were either macroscopically normal (at 29, 48 and 68 h) or showed limited macroscopic damage (median &gt; 50 h). Microscopic assessment of transplanted transgenic kidneys showed only focal tubular infarcts with viable renal tissue elsewhere, no endothelial swelling or polymorph adherence and infiltration by lymphocytes beginning at 3 days. Coagulopathy was not a feature of the histology in four kidneys not rejected and assessed at 48 h or later after transplantation. Baboon anti‐GAL serum antibody titers were high before transplantation and, in one extensively analyzed recipient, reduced ∼8‐fold within 5.5 h. The data demonstrate that a single CD46 transgene controls hyperacute kidney graft rejection in untreated baboons despite the presence of antibody and complement deposition. The expression levels, tissue distribution and in vitro functional tests indicate highly efficient CD46 function, controlling both classical and alternative pathway complement activation, which suggests it might be the complement regulator of choice to protect xenografts.