Margarita Romanella

The role of leukemia inhibitory factor in skeletal muscle regeneration.

Although a number of cytokines have been implicated in tissue regeneration, it is unknown which ones actually function in vivo. Here, we use mice with a targeted mutation in the leukemia inhibitory factor (LIF) gene to examine the role of LIF in muscle regeneration. Using a muscle crush model, we show that muscle regeneration in LIF knockout mice is significantly reduced compared to control littermates. Further, targeted infusion of LIF in both normal and LIF knockout animals stimulated muscle regeneration, but the stimulation observed was much greater in the mutant animals than in controls. In contrast, interleukin‐6 and transforming growth factor‐α, which also stimulate myoblast proliferation in vitro, had no effect on regeneration. These findings demonstrate directly that LIF is involved in regeneration of injured muscle and points to the use of LIF as a therapeutic agent in the treatment of neuromuscular disease.

Anti-gal Antibody-mediated Allograft Rejection In α1,3-galactosyltransferase Gene Knockout Mice: A Model of Delayed Xenograft Rejection

Background. The key role of anti-galactosea1,3-galactose (anti-aGal) 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-aGal 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 a1,3-galactosyltransferase knockout (Gal KO) mice, which like humans develop antibodies to the disaccharide galactosea1,3-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-aGal 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-aGal antibody in graft rejection can be studied in isolation from other rejection mechanisms. The titer of antiaGal 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.

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.

Leukaemia inhibitory factor treatment stimulates muscle regeneration in the mdx mouse

A number of growth factors are involved in coordinating muscle cell proliferation and differentiation, particularly after injury and in disease. Leukaemia inhibitory factor (LIF) strongly stimulates the proliferation of myoblasts in vitro and in vivo and its expression in muscle after injury suggests that LIF may have a role as a trauma factor. The mdx mouse was used to study the effects of LIF on in vivo muscle regeneration during disease. The rationale for using trophic factors such as LIF to treat neuromuscular disease includes the understanding that these molecules show some degree of selectivity for the population of cells in which they are effective. LIF was administered to muscle of the mdx mouse using osmotic pumps implanted subcutaneously in unrestrained mice. The growth factor was continuously delivered into the vastus lateralis muscle at 7 U/mu 1 for 7 days via a catheter. The results show that LIF increased the rate of muscle regeneration in mdx mice by stimulating the formation of larger myotubes. LIF treatment also increased the number of regenerating myotubes in the perfused area. This myotrophic action indicates that LIF contributes to muscle regeneration. Together with its known neurotrophic action, LIF is a potential therapeutic agent for the treatment of neuromuscular disease.

Transgenic expression of human α1,2-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.

High-level co-expression of complement regulators on vascular endothelium in transgenic mice: CD55 and CD59 provide greater protection from human complement-mediated injury than CD59 alone

Abstract: High‐level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement‐mediated injury in an ex vivo perfusion model. In this study we examine whether co‐expression of CD55 provides additional protection. CD55/CD59 double‐transgenic mice were generated by co‐injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM‐2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild‐type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high‐level endothelial co‐expression of CD55 and CD59 provides greater protection from human complement‐mediated injury in this model than expression of CD59 alone.

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.

Involvement Of Both The Classical And Alternate Pathways Of Complement In An Ex Vivo Model Of Xenograft Rejection

BACKGROUND It is now generally accepted that complement activation is critical for the hyperacute rejection of xenografts. Activation of the classical pathway as the result of the interaction of xenoreactive IgM xenoantibodies with the vascular endothelium has been observed in all species combinations examined to date. A number of studies using a variety of species combinations have also implicated alternate pathway involvement; however, these studies do not enable a conclusion to be drawn as to whether the alternate pathway can be activated in the complete absence of classical pathway activation. METHODS In this study, human plasma was depleted of both Clq and factor D and then reconstituted with purified Clq or factor D to restore the classical and alternate complement pathways, respectively. The ability of these modified plasmas to prosecute hyperacute rejection was then examined using an ex vivo isolated mouse heart perfusion model based on the Langendorff system. RESULTS AND CONCLUSIONS In the mouse to human species combination, both the classical and alternate pathways of complement are independently capable of initiating complement activation and mediating xenograft rejection.

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.

Demonstration of the functional importance of the Gal epitope in an ex vivo model of xenotransplantation

Abstract: The galactose a 1‐3 galactose terminal disaccharide (Gal epitope) has been identified as the major porcine xenoantigen recognised by xenoantibody in human plasma. Elimination or suppression of the epitope or antibody will be a major factor in overcoming hyperacute rejection. Inhibition of the antibody by depletion or elimination of the epitope by gene knockout may reveal the importance of other xenoantibodies, and in addition elimination of the epitope may unmask or produce other xenoantibody combinations. This study aims to determine the relative importance of anti‐Gal antibody and Gal epitope elimination in a functional model of xenotransplantation, ex vivo perfusion of mouse hearts with human plasma on a Langendorff apparatus. Perfusion of mouse hearts with human plasma depleted of anti‐Gal antibody demonstrates a protective effect compared to hearts perfused with undepleted plasma with prolongation of survival time from 24.1 to 44.5 min. Similarly, elimination of the epitope is also protective. Hearts from Gal knockout mice, which were generated by gene targeting of the al,3 galactosyltransferase gene, and hearts from appropriate control mice were perfused with human plasma. Gal knockout mice hearts demonstrated an increase in survival time from 10.2 to 33.8 min compared to control hearts. This was accompanied by a decrease in C3c and IgM, but little change in IgG deposition. The protective effect is incomplete, probably due to the effect of antibodies against non‐Gal xenoantigens. There was no functional evidence for generation of neo‐antigens in the Gal KO mice that were I recognised by naturally occurring human xenoantibodies.