Roles of HAUSP-mediated p53 regulation in central nervous system development.

The deubiquitinase HAUSP (herpesvirus-associated ubiquitin-specific protease; also called USP7) has a critical role in regulating the p53-Mdm2 (murine double minute 2) pathway. By using the conventional knockout approach, we previously showed that hausp inactivation leads to early embryonic lethality. To fully understand the physiological functions of hausp, we have generated mice lacking hausp specifically in the brain and examined the impacts of this manipulation on brain development. We found that deletion of hausp in neural cells resulted in neonatal lethality. The brains from these mice displayed hypoplasia and deficiencies in development, which were mainly caused by p53-mediated apoptosis. Detailed analysis also showed an increase of both p53 levels and p53-dependent transcriptional activation in hausp knockout brains. Notably, neural cell survival and brain development of hausp-mutant mice can largely be restored in the p53-null background. Nevertheless, in contrast to the case of mdm2- and mdm4 (murine double minute 4)-mutant mice, inactivation of p53 failed to completely rescue the neonatal lethality of these hausp-mutant mice. These results indicate that HAUSP-mediated p53 regulation is crucial for brain development, and also suggest that both the p53-dependent and the p53-independent functions of HAUSP contribute to the neonatal lethality of hausp-mutant mice.

Blockade of RAGE suppresses alloimmune reactions in vitro and delays allograft rejection in murine heart transplantation.

The receptor for advanced glycation endproducts (RAGE), a multiligand member of the immunoglobulin superfamily, interacts with proinflammatory AGEs, the products of nonenzymatic glycation and oxidation of proteins; high-mobility group box 1 (HMGB1), also known as amphoterin and S100/calgranulins to amplify inflammation and tissue injury. Previous studies showed that blockade of RAGE suppressed recruitment of proinflammatory mechanisms in murine models. We tested the hypothesis that RAGE contributes to alloimmune responses and report that in vivo, acute rejection of fully allogeneic cardiac allografts in a murine model of heterotopic cardiac transplantation is significantly delayed by pharmacological antagonism of RAGE. In parallel, allogeneic T-cell proliferation in the mixed lymphocyte reaction is, at least in part, RAGE-dependent. These data provide the first insights into key roles for RAGE in allorecognition responses and suggest that antagonism of this receptor may exert beneficial effects in allogeneic organ transplantation.

Disturbed regulation of cholesterol synthesis in monocytes of obese patients with hypercholesterolemia.

The aim of the present study was to clarify the influence of obesity on the functions of low-density lipoprotein receptors (LDL-R) and 3-hydroxy-3-methylglutarate-coenyzme A (HMG-CoA) reductase both in healthy control subjects and in patients with hypercholesterolemia (HC). Experiments were performed on monocytes of 15 non-obese (C I) and 11 obese (C II) healthy control subjects and on 22 non-obese (HC I) and 26 obese (HC II) patients with HC. [(125)I]LDL was used to determine LDL-R activity by measuring binding and intracellular degradation. The rate of endogenous cholesterol synthesis was measured using [(14)C]acetate incorporation into the cholesterol fraction of monocytes. The binding ability of [(125)I]LDL was identical across all groups. The [(14)C]acetate incorporation in resting monocytes was increased only in obese HC group. The 50-microg/mL LDL protein-induced inhibition of [(14)C]acetate incorporation was significantly diminished (P <.001) in the same group. A strong positive correlation was detected between the [(14)C]acetate incorporation by resting cells and LDL-induced inhibition in all groups except the obese HC group, in which their correlation was negative (P <.001). Furthermore, in the obese HC group, a significant positive correlation was detected between body mass index (BMI) and the basal level of [(14)C]acetate incorporation, whereas a negative correlation was found between BMI and LDL-induced inhibition of [(14)C]acetate incorporation. The present data suggest that in patients with HC the concomitant obesity results in dysregulation of cholesterol homeostasis, which may contribute to the accelerated atherosclerosis.

Acute cardiac allograft rejection in nitric oxide synthase-2(-/-) and nitric oxide synthase-2(+/+) mice: effects of cellular chimeras on myocardial inflammation and cardiomyocyte damage and apoptosis.

BACKGROUND: The contribution of nitric oxide synthase (NOS)-2 to myocardial inflammation and cardiomyocyte necrosis and apoptosis during allograft rejection was investigated through heterotopic cardiac transplantation in mice. METHODS AND RESULTS: In the first experiments, hearts from C3H donor mice were transplanted into NOS-2(-/-) and NOS-2(+/+) C57BL/6J.129J recipients. A second series of experiments included NOS-2(-/-) donor hearts transplanted into NOS-2(-/-) recipients and wild-type NOS-2(+/+) donor hearts transplanted into wild-type NOS-2(+/+) recipients. (All donors were C57BL/6J and recipients were C57BL/6J.129J.) In the first series of experiments, no significant differences were observed in allograft survival, rejection score, total number of apoptotic nuclei (TUNEL), total number of apoptotic cardiomyocytes, or graft NOS-2 mRNA and protein. Positive NOS-2 immunostaining occurred in endothelial cells and cardiomyocytes in the allografts; the inflammatory infiltrate was NOS-2 positive only when recipients were NOS-2(+/+). In the second series of experiments, cardiac allograft survival was significantly increased in the NOS-2(-/-) mice (26+/-13 versus 17+/-8 days, P<0.05), along with significant reductions in inflammatory infiltrate, rejection score, and total number of apoptotic nuclei (23.5+/-9.5 versus 56.4+/-15.3, P<0.01) and of apoptotic cardiomyocytes (2.9+/-1.6 versus 6.9+/-2.7, P<0.05). No NOS-2 or nitrotyrosine, a marker of peroxynitrite exposure, was detected in NOS-2(-/-) allografts transplanted into NOS-2(-/-) recipients. CONCLUSIONS: The data suggest that NO derived from NOS-2 contributes to the inflammatory response and to cardiomyocyte damage and apoptosis during acute cardiac allograft rejection.

Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function.

Left ventricular remodeling is a major cause of progressive heart failure and death after myocardial infarction. Although neoangiogenesis within the infarcted tissue is an integral component of the remodeling process, the capillary network is unable to support the greater demands of the hypertrophied myocardium, resulting in progressive loss of viable tissue, infarct extension and fibrous replacement. Here we show that bone marrow from adult humans contains endothelial precursors with phenotypic and functional characteristics of embryonic hemangioblasts, and that these can be used to directly induce new blood vessel formation in the infarct-bed (vasculogenesis) and proliferation of preexisting vasculature (angiogenesis) after experimental myocardial infarction. The neoangiogenesis resulted in decreased apoptosis of hypertrophied myocytes in the peri-infarct region, long-term salvage and survival of viable myocardium, reduction in collagen deposition and sustained improvement in cardiac function. The use of cytokine-mobilized autologous human bone-marrow-derived angioblasts for revascularization of infarcted myocardium (alone or in conjunction with currently used therapies) has the potential to significantly reduce morbidity and mortality associated with left ventricular remodeling.

Analysis of CD154 and CD40 expression in native coronary atherosclerosis and transplant associated coronary artery disease.

T cells have roles in the pathogenesis of native coronary atherosclerosis (CA) and transplant-associated coronary artery disease (TCAD). The mechanisms by which T cells interact with other cells in these lesions are not fully known. CD154 is an activation-induced CD4+ T cell surface molecule that interacts with CD40+ target cells, including macrophages and endothelial cells, and induces the production of pro-inflammatory molecules, including CD54 (ICAM-1) and CD106 (VCAM-1). To investigate whether CD154-CD40 interactions might be involved in the pathogenesis of CA or TCAD we performed immunohistochemical studies of CD154 and CD40 expression on frozen sections of coronary arteries obtained from cardiac allograft recipients with CA (n=10) or TCAD (n=9). Utilizing four different anti-CD154 mAb we found that CD154 expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was markedly up-regulated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Dual immunolabeling demonstrated many CD40+ cells co-expressed CD54 and CD106. The extent of CD40, CD54 and CD106 expression showed statistical significant correlation with the severity of disease and the amount of intimal lymphocytes. Together these studies demonstrate the presence of activated CD154+ and CD40+ cells in both CA and TCAD lesions and suggest that CD154-mediated interactions with CD40+ macrophages, foam cells, smooth muscle cells and/or endothelial cells may contribute to the pathogenesis of these diseases.

Rapid in vivo monitoring of chemotherapeutic response using weighted sodium magnetic resonance imaging.

A novel pulse sequence strategy uses sodium magnetic resonance imaging to monitor the response to chemotherapy of mouse xenograft tumors propagated from human prostate cancer cell lines. An inversion pulse suppresses sodium with long longitudinal relaxation times, weighting the image toward intracellular sodium nuclei. Comparing these weighted sodium images before and 24 h after administration of antineoplastics, we measured a 36 +/- 4% (P < 0.001; n = 16) increase in signal intensity. Experiments with these same drugs and cells, treated in culture, detected a significant intracellular sodium elevation (10-20 mM) using a ratiometric fluorescent dye. Flow cytometry studies showed that this elevation preceded cell death by apoptosis, as determined by fluorescent end-labeling of apoptotic nuclei or Annexin V binding. Histopathology on formalin-fixed sections of explanted tumors confirmed that drug administration reduces proliferation (2.2 versus 8.6 mitotic figures per high power field; P < 0.0001), an effect that inversely correlates with the sodium magnetic resonance image response on a tumor-to-tumor basis (P < 0.02; n = 10). Morphological features, such as central zones of nonviable cells, rims of active apoptosis, and areas of viable tumor, could be distinguished by comparing weighted and unweighted images. Advantages of this sodium imaging technique include rapid determination of drug efficacy, improved diagnosis of lesions, ease of coregistration with high resolution proton magnetic resonance imaging, and absence of costly or toxic reagents.

Upregulation of COX-2 during cardiac allograft rejection.

BACKGROUND: The hypothesis that cyclooxygenase-2 (COX-2) is involved in the myocardial inflammatory response during cardiac allograft rejection was investigated using a rat heterotopic abdominal cardiac transplantation model. METHODS AND RESULTS: COX-2 mRNA and protein in the myocardium of rejecting cardiac allografts were significantly elevated 3 to 5 days after transplantation compared with syngeneic controls (n=3, P<0.05). COX-2 upregulation paralleled in time and extent the upregulation of iNOS mRNA, protein, and enzyme activity in this model. COX-2 immunostaining was prominent in macrophages infiltrating the rejecting allografts and in damaged cardiac myocytes. Prostaglandin (PG) levels in rejecting allografts were also higher than in native hearts. Because NO has been reported to modulate PG synthesis by COX-2, additional transplants were performed using animals treated with a selective COX-2 inhibitor (SC-58125) and a selective inhibitor of the inducible nitric oxide synthase (iNOS) N-aminomethyl-L-lysine. At posttransplant day 5, inhibitor administration resulted in a significant reduction of COX-2 mRNA expression (3764+/-337 versus 5110+/-141 arbitrary units, n=3, P<0.05) and iNOS enzymatic activity (1.7+/-0.4 versus 22.8+/-14. 4 nmol/mg protein, n=3, P<0.01) compared with vehicle-treated allogeneic transplants. Allograft survival in treated animals was increased modestly from 5.4 to 6.4 days (P<0.05). However, apoptosis of cardiac myocytes (TUNNEL method) was only marginally reduced relative to vehicle controls in treated graft recipients. The intensity of allograft rejection was also similar in the treated and untreated allografts. CONCLUSIONS: The data indicates that COX-2 expression is enhanced in parallel with iNOS in the myocardium during cardiac allograft rejection.

Coronary perfusate composition influences diastolic properties, myocardial water content, and histologic characteristics of the rat left ventricle.

BACKGROUND: Recent studies found that edema, histology, and left ventricular diastolic compliance exhibit quantitative relationships in rats. Edema due to low osmolarity coronary perfusates increases myocardial water content and histologic edema score and decreases left ventricular filling. The present study examined effects of perfusate osmolarity and chemical composition on rat hearts. METHODS: Arrested American Cancer Institute (ACI) rat hearts (4 degrees C) were perfused with different cardioplegia solutions, including Plegisol (289 mOsm/L), dilute Plegisol (172 mOsm/L), Stanford solution (409 mOsm/L), and University of Wisconsin solution (315 mOsm/L). Controls had blood perfusion (310 mOsm/L). Postmortem left ventricular pressure-volume curves and myocardial water content were measured. After glutaraldehyde or formalin fixation, dehydration, and paraffin embedding, edema was graded subjectively. RESULTS: Myocardial water content reflected perfusate osmolarity, being lowest in Stanford and University of Wisconsin solutions (p<0.05 versus other groups) and highest in dilute Plegisol (p<0.05). Left ventricular filling volumes were smallest in dilute Plegisol and Plegisol (p<0.05). Osmolarity was not a major determinant of myocardial edema grade, which was highest with University of Wisconsin solution and dilute Plegisol (p<0.05 versus other groups). CONCLUSIONS: Perfusate osmolarity determined myocardial water content and left ventricular filling volume. However, perfusate chemical composition influenced the histologic appearance of edema. Pathologic grading of edema can be influenced by factors other than osmolarity alone.

Disturbed LDL and scavenger receptor functions in monocytes from chronic haemodialysed patients.

BACKGROUND: The most frequent complication in patients with end-stage renal failure on chronic haemodialysis (HD) treatment is atherosclerosis, i.e. the different forms of heart and vascular diseases. The complete disorder of serum lipid and lipoprotein patterns is well demonstrated, whereas our knowledge about the low-density lipoprotein (LDL) and scavenger receptor expression and function are poorly understood. METHODS: In our current work, LDL and scavenger receptor expression and functions were simultaneously studied in monocytes obtained from 15 healthy male control subjects and from 11 chronic HD male patients applied with (125)I-labelled LDL, isolated from healthy volunteers. To study the scavenger LDL receptors, labelled acetylated LDL (acLDL) was used. RESULTS: LDL binding to the monocytes of the HD-group was found to be decreased in comparison to that of the controls. As a result, the 50 microg LDL protein-induced inhibition of endogenous cholesterol synthesis was also diminished. In contrast, acLDL binding was greatly increased, though it could trigger only a low apoE synthesis. Consequently the number of cholesterol inclusions in monocytes was increased. CONCLUSIONS: The disturbed expression and function of LDL and scavenger receptors both may play significant roles in pathogenesis of cardiovascular complications in chronic HD patients. Based on our present results, it can be assumed that dysfunction of scavenger receptors is at the centre of cardiovascular complications of HD patients with renal failure.

Nitric oxide triggers programmed cell death (apoptosis) of adult rat ventricular myocytes in culture.

Excessive nitric oxide (NO) production within the heart is implicated in the pathogenesis of myocyte death, but the mechanism whereby NO kills cardiac myocytes is not known. To determine whether NO may trigger programmed cell death (apoptosis) of adult rat ventricular myocytes in culture, the NO donor S-nitroso-N-acetylpenicillamine (SNAP) was shown to kill purified cardiac myocytes in a dose-dependent fashion. In situ analysis of ventricular myocytes plated on chamber slides using nick-end labeling of DNA demonstrated that SNAP induces cardiac myocyte apoptosis, which was confirmed by the identification of oligonucleosomal DNA fragmentation on agarose gel electrophoresis. Similarly, treatment of cardiac myocytes with cytokines that induce inducible NO synthase was shown to cause an NO-dependent induction of apoptosis. Addition of reduced hemoglobin to scavenge NO liberated by SNAP extinguished both the increase in percentage of apoptotic cells and the appearance of DNA ladders. Treatment with SNAP (but not with N-acetylpenicillamine or SNAP + hemoglobin) not only induced apoptosis but resulted in a marked increase in p53 expression in cardiac myocytes detected by Western blotting and immunohistochemistry. These data indicate that NO has the capacity to kill cardiac myocytes by triggering apoptosis and suggest the involvement of p53 in this process.

A comparison of frozen and paraffin sections in dermatofibrosarcoma protuberans.

BACKGROUND: Dermatofibrosarcoma protuberans (DFSP) is a tumor with a high local reoccurrence rate. Mohs micrographic surgery offers the highest cure rate. However, differentiating minimal residual tumor from normal skin can be difficult during Mohs surgery. OBJECTIVE: To clarify the problem of determining when a tumor-free plane had been achieved during Mohs surgery for a DFSP. METHODS: In two patients with DFSPs, we compared frozen and paraffin-embedded sections extending from tumor to normal skin, using both H&E and CD34 stains. RESULTS: On frozen, but not paraffin-embedded, sections scattered dermal spindle cells were seen in normal skin. CONCLUSIONS: Scattered dermal spindle cells in the dermis of normal skin make it difficult to differentiate minimal residual tumor from normal dermis during Mohs surgery. A biopsy of normal skin can be useful as a control in this setting.

Specific and scavenger low-density lipoprotein receptors involved in the disturbed lipid metabolism of patients with non-insulin-dependent diabetes mellitus are independent of obesity.

Comparative studies were performed on monocyte-derived macrophages (MDMs), prepared by a 72-hour incubation of blood monocytes obtained from patients with non-insulin-dependent diabetes mellitus (NIDDM) and age-matched obese and non-obese controls. The MDMs, after a 72-hour culturing, expressed both specific and scavenger low-density lipoprotein (LDL) receptors on their surfaces. To study the binding capacity of both receptor types, [125I]LDL and [125I] acetylated LDL (acLDL) were applied to cells and the labeled ligands were then monitored to estimate the rate of intracellular degradations. The LDL-induced inhibition of endogenous cholesterol synthesis and the acLDL-triggered apolipoprotein (apo) E secretion were also studied, as the biological marker of receptor activation. The results indicate that the binding capacities of both specific and scavenger LDL receptors were not reduced in MDMs of diabetic patients. However, the intracellular degradation after LDL incorporation was decreased. The LDL-induced inhibition of cholesterol synthesis and the acLDL-transmitted apo E secretion were also found to be decreased in the MDMs of patients with NIDDM as compared with the obese and non-obese control groups. The NIDDM-induced impaired signal transduction of both specific and scavenger LDL receptors suggests an unclarified functional alteration of both receptor structures.

Fast to slow transformation of denervated and electrically stimulated rat muscle.

1. Denervated fast extensor digitorum longus (EDL) muscles of adult rats were stimulated electrically for up to 4 months with a slow pattern resembling the activity in soleus (Sol) motor units and examined with antibodies against myosin heavy chains (MHCs). 2. The normal EDL contained, on average, 45% type IIB, 29% type IIX, 23% type IIA and 3% type I fibres. All type IIB and almost all type IIX fibres disappeared during the first 3 weeks of stimulation. They were replaced by type IIA and type I fibres, whose percentages increased to about 75 and 15, respectively. Type IIA fibres remained at 75% for nearly 2 months and were then gradually replaced by type I fibres during the next 2 months. The transformation occurred sequentially in the order IIB/IIX-->IIA-->I, the first step (IIB/IIX-->IIA) occurring after a short delay (2 weeks) and the last step (IIA-->I in originally IIB or IIX fibres) after a long delay (> 2 months). During the transformation coexpression of MHCs occurred. 3. It appears that the transformation to type I fibres occurred in pre-existing type II fibres since no signs of fibre damage or regeneration were observed. 4. Normal EDL was also stimulated through an intact nerve with the same pattern for up to 37 days. The effects on fibre type distributions were identical to those observed in the denervated EDL. The result indicated that the Sol-like pattern of evoked muscle activity, rather than nerve-derived trophic influences or denervation per se, was primarily responsible for the fast to slow transformation.

CNI-1493 prolongs survival and reduces myocyte loss, apoptosis, and inflammation during rat cardiac allograft rejection.

Cytokines and cytotoxic agents, including nitric oxide (NO) released by macrophages, play important roles during cardiac allograft rejection. In contrast to agents that suppress T-lymphocyte function, CNI-1493 is a multivalent guanylhydrazone compound that inhibits the synthesis and release of proinflammatory cytokines and NO from macrophages. This study investigated the effects of CNI-1493 on rejecting rat cardiac allografts by using Lewis to Wistar-Furth heterotopic cardiac transplants. CNI-1493 (2 mg/kg i.p., b.i.d.) or vehicle (water) was administered beginning the day before surgery. Rat cardiac allograft survival to cessation of heart beat, apoptosis of cardiac myocytes, degree of myocardial inflammation, and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), protein, and enzyme activity were studied at days 1, 3, 5, and 7 after transplantation. Allograft survival was increased significantly by 26% from 7.5 +/- 0.8 days in vehicle-treated rats (n = 6) to 9.5 +/- 1.2 days in CNI-1493-treated rats (n = 8, p < 0.05). Apoptotic cells per mm2 myocardium decreased from 2.25 +/- 1.25 to 0.84 +/- 0.49 at day 3 and 31.2 +/- 2.9 to 17.6 +/- 5.43 at day 5 after transplantation with CNI-1493 treatment (p < 0.05). The number of apoptotic myocytes and loss of cardiac muscle cells also decreased significantly at day 5 in the treated animals (p < 0.05). The reduction of myocyte loss at day 5 coincided with a significant decrease of the inflammatory response and reduced macrophage influx (p < 0.05). Myocardial iNOS mRNA, protein, and enzyme levels increased during the course of allograft rejection, and CNI-1493 did not significantly reduce iNOS expression in the rejecting rat allograft. CNI-1493 prolongs allograft survival and reduces myocyte loss, apoptosis, and inflammation during rat cardiac allograft rejection. These effects of CNI-1493 appear to be unrelated to altered NO synthesis but may be related to effects of the drug to inhibit macrophage synthesis of cytokines.

Inducible nitric oxide synthase expression in smooth muscle cells and macrophages of human transplant coronary artery disease.

BACKGROUND: The inducible isoform of the nitric oxide synthase (iNOS) produces large amounts of nitric oxide in response to cytokine stimulation. Previous investigations have demonstrated iNOS expression in the setting of acute and chronic rejection in experimental cardiac transplant models. The goal of this study was to investigate whether iNOS is upregulated in human transplant coronary artery disease (TCAD), a major cause of late mortality after cardiac transplantation. METHODS AND RESULTS: We studied 15 patients with TCAD and 10 with normal coronary arteries. In situ hybridization and immunohistochemistry were used in tissue sections to localize iNOS mRNA and protein, respectively. The presence of peroxynitrite was indirectly assessed by immunostaining with an anti-nitrotyrosine antibody. Normal coronary arteries had no evidence of iNOS expression. In contrast, 30 of 36 coronary artery segments with TCAD (83%) were immunostained by the iNOS antibody. The presence of iNOS mRNA was demonstrated in these vessels by in situ hybridization. Specific cell markers identified iNOS-positive cells as neointimal macrophages and smooth muscle cells. Nitrotyrosine immunoreactivity colocalized with iNOS expression in arteries with TCAD, distributed in macrophages and smooth muscle cells. CONCLUSIONS: iNOS mRNA and protein are expressed in human arteries with TCAD, where they are associated with extensive nitration of protein tyrosines. These findings indicate that the high-output nitric oxide pathway and possibly the oxidant peroxynitrite might be involved in the process leading to the development of TCAD.

Detection of CD5 antigen on B cell lymphomas in fixed, paraffin embedded tissues using signal amplification by catalyzed reporter deposition.

CD5 surface antigen is expressed on some categories of B cell lymphomas. The detection of CD5 coexpression on malignant B cell infiltrates, particularly in small biopsy specimens, is useful in distinguishing between small lymphocytic lymphoma, mantle cell lymphoma, low grade marginal zone B cell lymphoma, and follicular small cleaved cell lymphoma. However, conflicting results have been reported with regard to the detection of CD5 antigen expression on B cell non-Hodgkin's lymphomas (B-NHLs) in fixed, paraffin embedded tissues using routine immunohistochemical (IHC) staining techniques. We used catalyzed reporter deposition (CARD) as a strategy to amplify the IHC signal and consequently increase the sensitivity of antigen detection. CARD improved detection of CD5 antigen without sacrificing specificity of the test. In our study, virtually all malignant B-NHLs with CD5 antigen expression showed strong immunoreactivity for a commercially available anti-CD5 monoclonal antibody using CARD, whereas the majority of the same lymphomas did not label for CD5 using routine IHC without CARD amplification. The concordance between CD5 antigen detection by immunophenotyping of fresh or frozen tissues and immunostaining with CARD amplification on paraffin fixed tissue sections was 100%. It appears that this method can be applied in the diagnostic evaluation of B-NHLs or in other situations that a weak antigen signal is present.

Triple immunosuppression reduces mononuclear cell infiltration and prolongs graft life in pig-to-newborn baboon cardiac xenotransplantation.

OBJECTIVE: Pig hearts transplanted into unmedicated newborn baboons do not undergo hyperacute rejection by preformed xenoantibody and complement. These grafts are rejected at days 3 to 4 in association with the infiltration of macrophages and natural killer cells. We investigated whether an immunosuppressive regimen used widely in cardiac allotransplantation could reduce this cellular response and prolong xenograft life. METHODS: Ten newborn baboons underwent heterotopic pig cardiac xenotransplantation. Five baboons were immunosuppressed with mycophenolate mofetil (100 mg/kg), methylprednisolone acetate (0.8 mg/kg), and cyclosporine A (INN: ciclosporin; 10 mg/kg). Xenograft rejection was studied by light microscopy and immunofluorescence. The induced humoral response to porcine xenoantigens was documented by enzyme-linked immunosorbent assay using synthetic alpha-1,3-galactosyl epitopes coupled to bovine serum albumin. RESULTS: Graft life was extended from a mean of 3.6 +/- 0.5 days (n = 5) to a mean of 6.2 +/- 1.1 days (n = 5, p = 0.01). In comparison with controls, explanted grafts from medicated baboons demonstrated reduced infiltration with natural killer cells and macrophages, but increased evidence of complement-mediated rejection substantiated by increased deposition of immunoglobulin M, complement, and fibrin. In all baboons receiving transplants, levels of both immunoglobulin M and immunoglobulin G anti-galactose were significantly increased after transplantation, with immunoglobulin G levels remaining persistently elevated. CONCLUSIONS: These results indicate that cyclosporine-based triple immunosuppression marginally prolonged xenograft survival and appears to have reduced the natural killer cell and macrophage infiltrates. The immunosuppressive protocol, however, was not adequate to prevent the induced immunoglobulin M humoral response and prevent complement-mediated graft injury.