PubMed Health⌕ Search

Biomedical subjects

A J d'Apice

Publications and source records attributed to A J d'Apice.

At least 19 recordsLinked to original sources
PubMed2001

Blockade of the CD28 and CD40 pathways result in the acceptance of pig and rat islet xenografts but not rat cardiac grafts in mice.

Previously, we demonstrated that combination CTLA4-Fc and anti-CD40L mAb treatment results in tolerance to concordant, cellular islet xenografts. The aim of this study was to determine its effectiveness in a model of fetal pig pancreas (FPP) xenotransplantation. Survival of FPP fragment grafts were compared to the survival of rat islet or cardiac xenografts following short term CTLA4-Fc and anti-CD40L mAb treatment. Rat islet and FPP fragment grafts survived long-term. However, rat cardiac grafts were rejected by 52-91 days. Both rat islet and FPP grafts showed similar histology with intact islet structures and adjacent 'nests' of lymphocytes. Concordant vascularised rat hearts showed extensive polymorphonuclear infiltrate, concentric vasculitis and a perivascular infiltrate predominantly of CD8+ T cells. This suggests that this therapy is effective for prolonging islet xenografts and demonstrates that the cellular mechanism of rejection for vascularised and non-vascularised xenografts are different.

Abatacept↗
PubMed2001

Effect of DNA concentration on transgenesis rates in mice and pigs.

A retrospective analysis of transgenesis rates obtained in seven pronuclear microinjection programs was undertaken to determine if a relationship existed between the amount of DNA injected and transgenesis rates in the pig. Logistic regression analysis showed that as the concentration of DNA injected increased from 1 to 10 ng/microl, the number of transgenics when expressed as a proportion of the number liveborn (integration rate) increased from 4% to an average of 26%. A similar relationship was found when the number of molecules of DNA injected per picolitre was analysed. No evidence was obtained to suggest either parameter influenced integration rate in mice when the same constructs were injected. The number of transgenics liveborn when expressed as a proportion of ova injected (efficiency rate), increased as DNA concentration increased up to 7.5 ng/microl and then decreased at 10 ng/microl for both species suggesting that at this concentration DNA (or possible contaminants) may have influenced embryo survival. The relationship between efficiency and the number of molecules injected per picolitre was complex suggesting that the concentration at which DNA was injected was a better determinant of integration and efficiency rates. In conclusion, the present study suggests that transgenes need to be injected at concentrations of between 5 and 10 ng/microl to maximise integration and efficiency rates in pigs.

Animals↗
PubMed2001

Vascular gene transfer driven by endoglin and ICAM-2 endothelial-specific promoters.

The involvement of the vascular endothelium in a large number of diseases supports the importance of vascular-specific gene delivery for their treatment. The hereditary hemorrhagic telangiectasia type 1 is an example of a vascular inherited disease (OMIM 187300). This is an autosomal dominant vascular disorder originated by mutations in the endoglin gene and associated with frequent epistaxis, telangiectases, gastrointestinal bleedings, and arteriovenous malformations in brain, lung and liver. Here, we address for the first time the possibility of using in vivo gene transfer to target endoglin expression to the vasculature. The promoter of the endothelial gene, ICAM-2, was used to generate transgenic animals which demonstrated endothelial expression of endoglin. Next, the promoters of the human endothelial genes, endoglin and ICAM-2, were inserted upstream of the human endoglin cDNA, and the resulting constructs were systemically or locally delivered, demonstrating endoglin expression in the vessel walls of liver, lung and skin. These gene transfer experiments represent an initial step in the treatment of the hereditary hemorrhagic telangiectasia type 1 by gene therapy, and suggest that endoglin and ICAM-2 promoters can be used to deliver other genes to the endothelium specifically.

Animals↗
PubMed2000

Synthetic peptides which inhibit the interaction between C1q and immunoglobulin and prolong xenograft survival.

BACKGROUND: Acute vascular xenograft rejection (AVXR), also termed delayed xenograft rejection (DXR), occurs when hyperacute rejection (HAR) is prevented by strategies directed at xenoreactive natural antibodies and/or complement activation. We have hypothesized that AVXR/DXR is initiated in part by early components of the complement cascade, notably C1q. We have developed synthetic peptides (termed CBP2 and WY) that interfere with the interaction between C1q and antibody. METHODS: CBP2 and the WY-conjugates were used as inhibitors of immunoglobulin aggregate binding to solid phase C1q. Inhibition of complement activation by the peptides of the classical system was determined using lysis assays with sensitized sheep red blood cells or porcine aortic endothelial cells as targets and of the alternate complement pathway using guinea pig red blood cells as targets. Two transplant models were used to study the effects of administering peptides to recipients: rat heart transplant to presensitized mouse, and guinea heart transplant to PVG C6-deficient rats. RESULTS: CBP2 and WY-conjugates inhibited immunoglobulin aggregate binding to C1q. The peptides also inhibited human complement-mediated lysis of sensitized sheep red blood cells and porcine aortic endothelial cells in a dose-dependent manner and the WY-conjugates prevented activation of the alternate complement pathway as shown by inhibition of guinea pig red blood cells lysis with human serum. In addition, the use of the peptides and conjugates resulted in significant prolongation of xenograft survival. CONCLUSIONS: The CBP2 and WY peptides exhibit the functional activity of inhibition of complement activation. These peptides also prolong xenograft survival and thus provide reagents for the study of the importance of C1q and other complement components in transplant rejection mechanisms.

Animals↗
PubMed2000

Renal xenografts from triple-transgenic pigs are not hyperacutely rejected but cause coagulopathy in non-immunosuppressed baboons.

BACKGROUND: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. METHODS: Transgenic pigs were produced by microinjection of gene constructs for human complement regulatory proteins CD55 and CD59 and the enzyme alpha1,2-fucosyltransferase (H-transferase, HT), which reduces expression of the major xenoepitope galactose-alpha1,3-galactose (alphaGal). Kidneys from CD55/HT and CD55/CD59/HT transgenic pigs were transplanted into nephrectomised, nonimmunosuppressed adult baboons. RESULTS: In several lines of transgenic pigs, CD55 and CD59 were expressed strongly in all tissues examined, whereas HT expression was relatively weak and did not significantly reduce alphaGal. Control nontransgenic kidneys (n=4) grafted into baboons were hyperacutely rejected within 1 hr. In contrast, kidneys from CD55/HT pigs (n=2) were rejected after 30 hr, although kidneys from CD55/CD59/HT pigs (n=6) maintained function for up to 5 days. In the latter grafts, infiltration by macrophages, T cells, and B cells was observed at days 3 and 5 posttransplantation. The recipients developed thrombocytopenia and abnormalities in coagulation, manifested in increased clotting times and an elevation in the plasma level of the fibrin degradation product D-dimer, within 2 days of transplantation. Treatment with low molecular weight heparin prevented profound thrombocytopenia but not the other aspects of coagulopathy. CONCLUSIONS: Strong expression of CD55 and CD59 completely protected porcine kidneys from hyperacute rejection and allowed a detailed analysis of xenograft rejection in the absence of immunosuppression. Coagulopathy appears to be a common feature of pig-to-baboon renal transplantation and represents yet another major barrier to its clinical application.

Animals↗
PubMed2000

Naturally acquired anti-alpha Gal antibodies in a murine allograft model similar to delayed xenograft rejection.

Antibodies directed against galactose-alpha1,3-galactose (alphaGal) are believed to play an important role in the pathogenesis of delayed xenograft rejection (DXR). This study was designed to determine whether alpha1,3-galactosyltransferase-deficient (Gal KO) mice can naturally acquire a sufficient anti-alphaGal titre to cause the delayed type rejection of alphaGal-expressing hearts. Gal KO mice of various ages were assessed for anti-alphaGal antibody levels. alphaGal-expressing hearts were transplanted heterotopically into these mice and monitored daily. Rejecting and surviving hearts were evaluated histologically. In Gal KO mice greater than 6-month-old, 64% had an anti-alphaGal antibody titre above the background level. When wild-type alphaGal-expressing hearts were transplanted into this group, 45% of grafts rejected within 5 to 13 days. Histological examination of the rejected hearts displayed marked tissue damage and an inflammatory infiltrate of predominantly macrophage/monocytes. Surviving grafts showed preserved morphology. Like humans, Gal KO mice naturally develop anti-alphaGal antibodies with age. The titre in these mice was sufficient to cause a "delayed-type" rejection of a significant proportion of alphaGal-expressing cardiac grafts. This model thus provides an opportunity to investigate the role of naturally acquired anti-alphaGal antibodies in the pathogenesis of DXR.

Animals↗
PubMed2000

Disordered regulation of coagulation and platelet activation in xenotransplantation.

Rejection of xenografts is associated with vascular-based inflammation, thrombocytopenia and the consumption of coagulation factors that may evolve into disseminated intravascular coagulation (DIC). Similarly, bone marrow-derived cellular xenotransplantation procedures are associated with endothelial cell activation and thrombotic microangiopathic injury. These complications generally develop despite the best available measures for depletion of xenoreactive natural antibody, inhibition of complement activation and suppression of T- and B-cell mediated immune responses. The mechanisms underlying the DIC and thrombotic microangiopathy associated with xenotransplantation are unclear. A proposed primary biological dysfunction of xenografts with respect to regulation of clotting could amplify vascular injury, promote immunological responses and independently contribute to graft failure. Disordered thromboregulation could have deleterious effects, comparable to unregulated complement activation, in the pathogenesis of xenograft rejection and may therefore represent a substantive barrier to xenotransplantation.

Animals↗
PubMed2000

Hyperacute rejection of vascularized heart transplants in BALB/c Gal knockout mice.

Pig-to-primate vascularized xenografts undergo hyperacute rejection (HAR). This results from pre-formed xenoreactive antibodies directed against galactose-alpha1,3-galactose (alphaGal) in the donor organ and activation of the complement cascade. We describe an in vivo murine model of HAR using a BALB/c mice system devoid of histocompatibility or complement differences between donor and recipient to investigate in isolation, the effects of alphaGal epitope and anti-alphaGal antibody interactions in causing rejection of vascularized heart transplants. Gal KO mice were immunized with rabbit red blood cell membranes to induce high anti-alphaGal antibody titers that were predominantly IgM by ELISA (enzyme-linked immunosorbent assay). When alphaGal-expressing mice hearts were transplanted heterotopically into these recipients (n= 12), 67% of grafts rejected within 24 h, the majority within 16 h with histological features of HAR. In contrast, none of the grafts in the non-immunized Gal KO recipient control group (n=11) underwent HAR. Interestingly, approximately 50% of the remaining grafts in both the immunized and non-immunized Gal KO recipient group were rejected between 7 and 27 days by a rejection process characterized by a dense infiltrate of macrophage/monocytes, perivascular cuffing and tissue destruction similar to recent descriptions of delayed xenograft rejection (DXR). In addition, some grafts (21.5%) continued to survive in the immunized Gal KO recipients despite the presence of anti-alphaGal antibody and normal complement activity and these showed well-preserved myocardium when harvested whilst still functioning well at days 30 or 90. No rejection was seen when Gal KO donors were used in this system (n=4), nor when alphaGal-expressing BALB/c hearts were transplanted into alphaGal-expressing BALB/c recipients (n=5). This in vivo small animal model offers the opportunity to test a variety of strategies to overcome HAR prior to more resource intensive pig-to-primate studies, and may provide insights into the processes similar to DXR and accommodation.

Acute Disease↗
PubMed1999

Anti-xenograft immune responses in alpha 1,3-galactosyltransferase knock-out mice.

Although originally generated to test the effect of eliminating the alpha-Gal epitope on HAR, it is becoming increasingly clear that GalT KO mice offer a convenient and inexpensive model to investigate many aspects of the anti-xenorgraft immune response. Clearly, not all aspects of anti-xenograft rejection responses are identical in mice and primates, which should be kept in mind when interpreting results of GalT KO mouse studies. However, with this and other mouse models it is possible to test a large number of variables, which is impractical for both logistical and financial reasons with primates. Furthermore the short gestation time and large litter size of mice means that genetic strategies targeting different aspects of the anti-xenograft immune response can be combined and subsequently tested to identify the optimal combination of genetic and therapeutic approaches to achieve long term xenograft survival. In this regard the GalT KO mouse has been and will continue to be a valuable small animal model for the study of all facets of xenograft rejection involving anti-Gal antibodies.

Animals↗
PubMed1998

Expression and functional analysis of glycosyl-phosphatidyl inositol-linked CD46 in transgenic mice.

BACKGROUND: Complement activation plays a pivotal role in hyperacute xenograft rejection. In humans, activation of complement is regulated by a number of cell surface regulatory proteins. Membrane cofactor protein (CD46) is one such regulator that protects cells by acting as a cofactor for the factor I-mediated cleavage of C3b and C4b. Transgenic animals expressing human CD46 may provide organs that are resistant to complement attack. However, attempts to generate mice expressing human CD46 using cDNA-based constructs have been largely unsuccessful. METHODS: Transgenic mice expressing a glycosylphosphatidyl inositol (GPI)-linked form of CD46 were generated by microinjection of a hybrid CD46/CD55 cDNA under the control of the human intercellular adhesion molecule-2 promoter. Expression of CD46-GPI on the vascular endothelium was determined by immunohistochemistry. The ability of CD46-GPI to protect mouse tissues from human complement attack was determined using an ex vivo isolated perfused heart model. RESULTS: Three founder animals expressing CD46-GPI were identified. Histological analysis showed strong and uniform expression of CD46-GPI on the vascular endothelium of all organs examined. Ex vivo perfusion of transgenic mouse hearts with human plasma showed a reduction in C3c deposition and a slightly prolonged function compared with controls. CONCLUSIONS: High-level expression of CD46-GPI was achieved in transgenic mice by using a modified cDNA-based construct. The CD46-GPI was functional, providing some protection from complement-mediated damage in the ex vivo model, and may be useful in xenotransplantation if expressed in combination with CD55 and CD59.

Animals↗
PubMed1998

Anti-Gal antibody-mediated allograft rejection in alpha1,3-galactosyltransferase gene knockout mice: a model of delayed xenograft rejection.

BACKGROUND: The key role of anti-galactose alpha1,3-galactose (anti-alphaGal) xenoantibodies in initiating hyperacute xenograft rejection has been clearly demonstrated using a variety of in vitro and in vivo approaches. However, the role of anti-alphaGal antibodies in mediating post-hyperacute rejection mechanisms, such as antibody-dependent cellular cytoxicity, remains to be determined, primarily because of the lack of a small animal model with which to study this phenomena. METHODS: Hearts from wild-type mice were transplanted heterotopically into alpha1,3-galactosyltransferase knockout (Gal KO) mice, which like humans develop antibodies to the disaccharide galactose alpha1,3-galactose (Gal). At the time of rejection, hearts were examined histologically to determine the mechanism of rejection. RESULTS: Hearts from wild-type mice transplanted into high-titer anti-alphaGal recipients were rejected in 8-13 days. Histological examination demonstrated a cellular infiltrate consisting of macrophages (80-90%), natural killer cells (5-10%), and T cells (1-5%). In contrast, wild-type hearts transplanted into low anti-Gal titer recipients demonstrated prolonged (>90 day) survival. However, a significant proportion (30-40%) of these underwent a minor rejection episode between 10 and 13 days, but then recovered ("accommodated"). CONCLUSIONS: The results of this study suggest that the Gal KO mouse is a useful small animal vascularized allograft model, in which the role of anti-alphaGal antibody in graft rejection can be studied in isolation from other rejection mechanisms. The titer of anti-alphaGal antibody was found to be the critical determinant of rejection. The histopathological features of rejection in this model are very similar to other models of delayed xenograft rejection, in both the timing and composition of the cellular infiltrate. The Gal KO mouse therefore provides a new rodent model, which will aid in the identification of the distinct components involved in the pathogenesis of delayed xenograft rejection.

Animals↗
PubMed1998

Knock out of alpha1,3-galactosyltransferase or expression of alpha1,2-fucosyltransferase further protects CD55- and CD59-expressing mouse hearts in an ex vivo model of xenograft rejection.

BACKGROUND: Organs from transgenic animals with high-level endothelial expression of the human complement regulatory factors CD55 and CD59 are significantly protected from human complement-mediated injury. Elimination or reduction of the major xenoepitope alphaGal, achieved by knocking out the alpha1,3-galactosyltransferase gene (Gal KO) or expressing human alpha1,2-fucosyltransferase (H transferase or HTF), also affords protection, although to a lesser degree. In this study, we examined whether the protection provided by strong CD55 and CD59 expression can be augmented by the Gal KO or HTF modifications. METHODS: Hearts from four groups of mice (wild type, CD55/CD59, CD55/CD59/HTF, and CD55/CD59/Gal KO) were perfused ex vivo with 40% human plasma. Mean heart work for each group was compared over a 60-min period. RESULTS: Wild-type hearts ceased to function effectively within 15 min of plasma addition. CD55/CD59 hearts displayed prolonged survival and maintained approximately 10% maximum work at the end of perfusion. Introduction of Gal KO or HTF onto the CD55/CD59 background resulted in a further prolongation, with work maintained at 20-30% of the maximum level. CONCLUSIONS: We used an ex vivo model to demonstrate that eliminating alphaGal expression further prolongs the function of mouse hearts expressing high levels of CD55 and CD59. In addition, we showed that reducing alphaGal by expressing HTF is equally as effective in prolonging CD55/CD59 heart function as knocking out Gal transferase, thus providing a feasible strategy for translating these advances to the pig.

Animals↗
PubMed1998

The human ICAM-2 promoter is endothelial cell-specific in vitro and in vivo and contains critical Sp1 and GATA binding sites.

The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes. We have previously shown that the endothelial-specific in vivo activity of the human ICAM-2 promoter is contained within a small (0.33-kilobase (kbp)) 5'-flanking region of the gene. Here we describe the in vitro characterization of this region. The ICAM-2 promoter is TATA-less, and transcription in endothelial cells initiates at four sites. Reporter gene expression directed by the promoter was 125-fold greater than vector alone in bovine aortic endothelial cells but less than 2-fold vector alone in non-endothelial (COS) cells, confirming that specificity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5'-flanking region to the 0.33-kbp fragment had no effect on promoter activity or specificity. The mutation of an Sp1 motif centered at base pair -194 or an eight-base pair palindrome at -268 each reduced promoter activity by 70%. Mutation of GATA motifs at -145 and -53 reduced promoter activity by 78 and 61%, respectively. Specific binding of bovine aortic endothelial cells nuclear proteins to the Sp1 and GATA sites was demonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-2. The results presented here suggest that transcription from the ICAM-2 promoter in endothelial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-specific gene expression.

Animals↗
PubMed1998

High-level endothelial expression of human CD59 prolongs heart function in an ex vivo model of xenograft rejection.

BACKGROUND: Hyperacute rejection of discordant xenografts is dependent on activation of the complement system of the recipient. Transgenic expression of recipient complement regulatory factors in donor tissue has proved to be a promising approach to dealing with hyperacute rejection, although the relationship between the level of complement regulatory factor expression and the degree of protection is not well established. Here, we examine this relationship using CD59 transgenic mouse hearts in an ex vivo model of xenograft rejection. METHODS: The level of expression of CD59 in two lines of transgenic mice, in which CD59 is expressed under the control of either the murine H2Kb (MHC class I) promoter (line CA-17) or the endothelium-specific human intercellular adhesion molecule-2 promoter (line 237-7), was compared by immunohistochemistry and flow cytometry. Hearts from both groups and wild-type controls were perfused ex vivo with human plasma, and mean heart work for each group was compared over a 60-min period. RESULTS: CD59 expression on cardiac endothelial cells isolated from homozygous CA-17 mice was 25- to 30-fold lower than that on cardiac endothelial cells from heterozygous 237-7 mice. CA-17 hearts perfused with 6% human plasma exhibited a reduction in deposition of the membrane attack complex, but not a prolongation of function, compared with nontransgenic mouse hearts. In contrast, 237-7 hearts showed significantly prolonged function during perfusion with 20% plasma. CONCLUSIONS: High-level endothelial-specific expression of CD59 was effective in prolonging the function of mouse hearts perfused with 20% human plasma, whereas low-level, broader expression did not provide protection from 6% plasma.

Animals↗
PubMed1998

Transgenic expression of human alpha1,2-fucosyltransferase (H-transferase) prolongs mouse heart survival in an ex vivo model of xenograft rejection.

BACKGROUND: The expression of human alpha1,2-fucosyltransferase (H-transferase, HT) has been proposed as an alternative strategy to alpha1,3-galactosyltransferase (GT) gene knockout, which is not currently feasible in pigs, to reduce the galactose-alpha1,3-galactose (Gal) epitope expression. HT expression has recently been shown in transgenic mice and pigs to significantly reduce Gal expression on a variety of cells; however, its ability to do so on endothelial cells and its effectiveness at prolonging xenograft survival are yet to be determined. METHODS: HT-transgenic, Gal knockout (Gal KO) mice, and mice containing both genetic modifications (HT-transgenic/Gal KO) were tested for H-substance and Gal expression on splenocytes and endothelial cells by flow cytometric analysis. In addition, the hearts of these mice were perfused ex vivo with 6% human plasma, and the effect on cardiac function was determined. RESULTS AND CONCLUSION: H-substance expression was detected on both splenocytes and endothelial cells of HT-transgenic mice. The level of H-substance expression was not affected by the presence or absence of GT in the cells, consistent with HT being dominant over GT. The ability of HT expression to reduce Gal expression was highly variable depending on the cell type. Gal expression on splenocytes was almost completely eliminated, whereas on endothelial cells, substantial Gal remained despite a 70% reduction. When perfused ex vivo with human plasma, hearts from HT-transgenic, Gal KO, and HT-transgenic/Gal KO mice demonstrated a similar prolongation in survival, compared with wild-type controls. Therefore, as far as hyperacute rejection is concerned, HT expression may be as effective as Gal KO in protecting against xenoantibody and complement mediated injury. However, the effect of residual Gal on non-hyperacute rejection responses remains to be determined.

Animals↗