A J d'Apice

St. Vincent Hospital, Green Bay, Wisconsin, United States

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Publications (105)335.02 Total impact

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    ABSTRACT: The search for alternative sources for transplant organs leads us to the search for animals as an inexhaustible source of organs. The objective of this study was to analyze whether livers from polytransgenic pigs expressing the human complement regulatory proteins CD55 (hDAF), CD59, and alfa alpha1,2-fucosyltransferase (H-transferase), protected against hyperacute rejection after orthotopic liver xenotransplantation to a baboon and also to study pig liver function in a nonhuman primate. Nine liver transplants from pig to baboon were divided into two groups: a control group (n = 4) of genetically unmodified pigs and an experimental group (n = 5) of pigs transgenic for CD55, CD59, and H-transferase as donors. All the donating piglets obtained through hysterectomy were maintained in specific pathogen-free conditions. The selection of transgenic pig donors followed demonstration of transgene expression using monoclonal antibodies (antiCD55, antiCD59) and immunohistological studies on liver biopsies. All animals in the control group developed hyperacute rejection with survival rates less than 16 hours without function of transplanted livers. In the experimental group none of the animals suffered hyperacute rejection. Survival in this group was between 13 and 24 hours. The livers were functional, producing bile and maintaining above 35% prothrombin activity. Only in one case was there primary dysfunction of the xenograft. Polytransgenic livers for complement regulatory proteins prevent hyperacute rejection when xenotransplanted into a baboon.
    Transplantation Proceedings 12/2005; 37(9):4103-6. DOI:10.1016/j.transproceed.2005.09.186 · 0.95 Impact Factor
  • Nephrology 02/2002; 7(1). DOI:10.1046/j.1440-1797.2002.00007-1-59.x · 1.86 Impact Factor
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    ABSTRACT: 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.
    Transgenic Research 01/2002; 10(6):523-31. DOI:10.1023/A:1013007329936 · 2.28 Impact Factor
  • A J d'Apice, M B Nottle, P J Cowan
    Transplantation Proceedings 11/2001; 33(7-8):3053-4. DOI:10.1016/S0041-1345(01)02305-3 · 0.95 Impact Factor
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    ABSTRACT: 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.
    Gene Therapy 07/2001; 8(12):897-904. DOI:10.1038/sj.gt.3301468 · 4.20 Impact Factor
  • A J D'Apice, P J Cowan
    Transplantation 12/2000; 70(9):1273-4. DOI:10.1097/00007890-200011150-00003 · 3.78 Impact Factor
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    Transplantation Proceedings 12/2000; 32(7):2075. DOI:10.1016/S0041-1345(00)01574-8 · 0.95 Impact Factor
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    ABSTRACT: 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.
    Xenotransplantation 12/2000; 7(4):237-46. · 1.78 Impact Factor
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    ABSTRACT: It has been proposed that hyperacute rejection (HAR) of pig-to-primate vascularized xenografts is due in large part to ineffective regulation of recipient complement by pig complement regulatory proteins (CRPs), and indeed transgenic expression of human CRPs in pigs can prevent hyperacute rejection. However, at least one pig CRP (CD59) efficiently regulates human complement in vitro, suggesting that it is the level of expression of a particular CRP(s) rather than cross-species incompatibility that explains the HAR of porcine xenografts. We investigated the relative effectiveness of transgenically expressed pig and human CD59 in providing protection of mouse hearts from human complement in an ex vivo setting. Transgenic mice expressing pig CD59 or human CD59 under the control of the human ICAM-2 promoter, which restricts expression in tissues to vascular endothelium, were used. Hearts from mice expressing similar levels of pig CD59 or human CD59 were perfused ex vivo with 10% human plasma and heart function was monitored for 60 min. Sections of perfused hearts were examined for deposition of the membrane attack complex (MAC). Control nontransgenic hearts (n=5) were rapidly affected by the addition of human plasma, with mean function falling to less than 10% of the initial level within 15 min. In contrast, hearts expressing either pig CD59 (n=6) or human CD59 (n=8) were protected from plasma-induced injury, maintaining 31 and 35% function, respectively, after 60 min of perfusion. MAC deposition was markedly reduced in both pig CD59 and human CD59 transgenic hearts compared to nontransgenic control hearts. When highly expressed on endothelium in transgenic mice, pig CD59 provided equivalent protection to human CD59 in a model of human complement-mediated xenograft rejection. Thus supranormal expression of endogenous porcine CRPs may be a feasible alternative to the expression of human CRPs in preventing HAR of pig-to-primate xenografts.
    Transplantation 10/2000; 70(6):963-8. DOI:10.1097/00007890-200009270-00014 · 3.78 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Transplantation 10/2000; 70(5):828-36. DOI:10.1097/00007890-200009150-00021 · 3.78 Impact Factor
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    ABSTRACT: The genetic modification of pigs is a powerful strategy that may ultimately enable successful xenotransplantation of porcine organs into humans. 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. 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. 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.
    Transplantation 07/2000; 69(12):2504-15. DOI:10.1097/00007890-200006270-00008 · 3.78 Impact Factor
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    ABSTRACT: 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.
    Xenotransplantation 03/2000; 7(1):42-7. · 1.78 Impact Factor
  • D J Goodman, A J d'Apice
    Transplantation 12/1999; 68(11):1630-1. DOI:10.1097/00007890-199912150-00004 · 3.78 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Molecular Medicine 09/1999; 5(9):617-30. · 4.82 Impact Factor
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    ABSTRACT: 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.
    Sub-cellular biochemistry 02/1999; 32:281-310.
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    ABSTRACT: 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. 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. 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. 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.
    Transplantation 01/1999; 66(11):1401-6. DOI:10.1097/00007890-199812150-00001 · 3.78 Impact Factor
  • A J d'Apice, D J Goodman, M J Pearse
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    ABSTRACT: Over the past 20 years, the mortality and morbidity associated with cardiac allotransplantation has fallen significantly, providing a viable treatment for patients with terminal cardiac failure. Unfortunately, the increase in the number of patients who could benefit from cardiac transplantation has not been matched with an increase in the number of organs available for transplantation. Thus, many patients with cardiac failure die waiting for a suitable organ, unlike patients with renal failure, who can be maintained on dialysis while waiting for a kidney. Although the development of artificial hearts may provide a life-sustaining bridging therapy until a donor organ becomes available, the quality of life associated with cardiac prostheses is currently less than that following successful cardiac allotransplantation.
    Trends in Cardiovascular Medicine 11/1998; 8(7):319-25. DOI:10.1016/S1050-1738(98)00021-8 · 2.07 Impact Factor
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    ABSTRACT: 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. 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. 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"). 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.
    Transplantation 09/1998; 66(6):748-54. · 3.78 Impact Factor
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    ABSTRACT: 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. 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. 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. 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.
    Transplantation 07/1998; 65(12):1599-604. · 3.78 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 06/1998; 273(19):11737-44. DOI:10.1074/jbc.273.19.11737 · 4.60 Impact Factor

Publication Stats

2k Citations
335.02 Total Impact Points


  • 1991–2002
    • St. Vincent Hospital
      Green Bay, Wisconsin, United States
  • 1989–2000
    • St. Vincent's Hospital Melbourne
      • Immunology Research Centre
      Melbourne, Victoria, Australia
    • Oregon Health and Science University
      • Department of Medicine
      Portland, OR, United States
    • The Royal Children's Hospital
      Melbourne, Victoria, Australia
  • 1993
    • Royal Victorian Eye and Ear Hospital
      Melbourne, Victoria, Australia
  • 1980–1991
    • Royal Melbourne Hospital
      • Department of Nephrology
      Melbourne, Victoria, Australia
  • 1987
    • University of Melbourne
      • Department of Surgery
      Melbourne, Victoria, Australia