Induction of myocardial nitric oxide synthase by cardiac allograft rejection.

Cardiac transplantation, effective therapy for end-stage heart failure, is frequently complicated by allograft rejection, the mechanisms of which remain incompletely understood. Nitric oxide (NO), a vasodilator which is cytotoxic and negatively inotropic, can be produced in large amounts by an inducible NO synthase (iNOS) in response to cytokines. To investigate whether iNOS is induced during cardiac allograft rejection, hearts from Lewis or Wistar-Furth rats were transplanted into Lewis recipients. At day 5, allogeneic grafts manifested reduced contractility and histologic evidence of rejection (inflammatory infiltrate, edema, necrosis of myocytes). The mRNA for iNOS and iNOS protein were detected in ventricular homogenates and in isolated cardiac myocytes from rejecting allogeneic grafts but not in tissue and myocytes from syngeneic control grafts. Immunocytochemistry showed increased iNOS staining in infiltrating macrophages and in microvascular endothelial cells and cardiac muscle fibers and also in isolated purified cardiac myocytes from the rejecting allografts. Using a myocardial cytosolic iNOS preparation, nitrite formation from L-arginine and [3H] citrulline formation from [3H]L-arginine were increased significantly in the rejecting allogeneic grafts (P < 0.01). Myocardial cyclic GMP was also increased significantly (P < 0.05). The data indicate myocardial iNOS mRNA, protein and enzyme activity are induced in infiltrating macrophages and cardiac myocytes of the rejecting allogeneic grafts. Synthesis of NO by iNOS may contribute to myocyte necrosis and ventricular failure during cardiac allograft rejection.

Indirect recognition of donor HLA-DR peptides in organ allograft rejection.

To determine whether indirect allorecognition is involved in heart allograft rejection T cells obtained from peripheral blood and graft biopsy tissues were expanded in the presence of IL-2 and tested in limiting dilution analysis (LDA) for reactivity to synthetic peptides corresponding to the hypervariable regions of the mismatched HLA-DR antigen(s) of the donor. Serial studies of 32 patients showed that T cell reactivity to donor allopeptides was strongly associated with episodes of acute rejection. The frequency of allopeptide reactive T cells was 10-50-fold higher in the graft than in the periphery indicating that T cells activated via the indirect allorecognition pathway participate actively in acute allograft rejection. In recipients carrying a graft differing by two HLA-DR alleles the response appeared to target only one of the mismatched antigens of the donor. Indirect allorecognition was restricted by a single HLA-DR antigen of the host and directed against one immunodominant peptide of donor HLA-DR protein. However, intermolecular spreading was demonstrated in patients with multiple rejection episodes by showing that they develop allopeptide reactivity against the second HLA-DR antigen. These data imply that early treatment to suppress T cell responses through the indirect pathway of allorecognition, such as tolerance induction to the dominant donor determinant, may be required to prevent amplification and perpetuation of the rejection process.

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.

The evolving management of acute right-sided heart failure in cardiac transplant recipients.

Avoidance of the clinical syndrome of acute right-sided heart failure after heart transplantation is, unfortunately, not possible. Clinical experience and the literature certainly suggest that a significant factor in the successful management of right ventricular (RV) failure is recipient selection. Moreover, threshold hemodynamic values beyond which RV failure is certain to occur and heart transplantation is contraindicated do not exist. Nor are there values below which RV failure is always avoidable. Acute RV failure will remain a difficult and ever-present clinical syndrome in the transplant recipient. Goals in the treatment of this clinical problem include: 1. Preserving coronary perfusion through maintenance of systemic blood pressure. 2. Optimizing RV preload. 3. Reducing RV afterload by decreasing pulmonary vascular resistance (PVR). 4. Limiting pulmonary vasoconstriction through ventilation with high inspired oxygen concentrations (100% FiO(2)), increased tidal volume and optimal positive end expiratory pressure ventilation. Inhaled nitric oxide is recommended before leaving the operating room in cases where the initial therapies have had little impact. Intra-aortic balloon counterpulsation is employed in patients with impaired left ventricular (LV) function and may be of benefit in patients with RV dysfunction resulting from ischemia, preservation injury or reperfusion injury. Optimal LV function reduces RV afterload and PVR. A proactive decision regarding RV assist device implantation is made before leaving the operating room and is highly dependent upon overall hemodynamics, size and function of the ventricles as seen on transesophageal echocardiography, renal function and surgical bleeding. Only through careful preoperative planning can this life-threatening condition be managed in the postoperative period.

Heart transplantation in children with congenital heart disease.

OBJECTIVES: The aim of this study was to describe heart transplantation in children with congenital heart disease and to compare the results with those in children undergoing transplantation for other cardiac diseases. BACKGROUND: Reports describe decreased survival after heart transplantation in children with congenital heart disease compared with those with cardiomyopathy. However, transplantation is increasingly being considered in the surgical management of children with complex congenital heart disease. Present-day results from this group require reassessment. METHODS: The diagnoses, previous operations and indications for transplantation were characterized in children with congenital heart disease. Pretransplant course, graft ischemia time, post-transplant survival and outcome (rejection frequency, infection rate, length of hospital stay) were compared with those in children undergoing transplantation for other reasons (n = 47). RESULTS: Thirty-seven children (mean [+/- SD] age 9 +/- 6 years) with congenital heart disease underwent transplantation; 86% had undergone one or more previous operations. Repair of extracardiac defects at transplantation was necessary in 23 patients. Causes of death after transplantation were donor failure in two patients, surgical bleeding in two, pulmonary hemorrhage in one, infection in four, rejection in three and graft atherosclerosis in one. No difference in 1- and 5-year survival rates (70% vs. 77% and 64% vs. 65%, respectively), rejection frequency or length of hospital stay was seen between children with and without congenital heart disease. Cardiopulmonary bypass and donor ischemia time were significantly longer in patients with congenital heart disease. Serious infections were more common in children with than without congenital heart disease (13 of 37 vs. 6 of 47, respectively, p = 0.01). CONCLUSIONS: Despite the more complex cardiac surgery required at implantation and longer donor ischemic time, heart transplantation can be performed in children with complex congenital heart disease with success similar to that in patients with other cardiac diseases.

Incidence of pulmonary vein complications after lung transplantation: a prospective transesophageal echocardiographic study.

OBJECTIVES: This study attempted to document the incidence of pulmonary vein complications and their potential relation to clinical outcome in patients after lung transplantation. BACKGROUND: Several case reports have documented the presence of pulmonary venous thrombosis causing graft failure in patients after lung transplantation. Because the presentation of these complications mimics that of other postoperative problems, the true incidence of pulmonary vein abnormalities remains unclear. Transesophageal echocardiography is ideally suited to examine the pulmonary veins in the postoperative setting. METHODS: Twenty-one consecutive patients undergoing lung transplantation at our institution underwent transesophageal echocardiography within 32 days of transplantation (mean [+/- SD] 6.5 +/- 7.8 days). Special attention was placed on visualizing the pulmonary veins. RESULTS: Six (29%) of the 21 patients were noted to have abnormalities of the pulmonary veins in the vicinity of the anastomotic site. After follow-up of 30 days, 4 of these patients (67%) had significant cardiovascular morbidity, and 2 died, compared with 1 (7%) of 15 patients with normal pulmonary veins (p = 0.03). The degree of obstruction of the pulmonary vein appeared to correlate with short-term outcome. CONCLUSIONS: Abnormalities of the pulmonary veins are common after lung transplantation and are easily identified by transesophageal echocardiography. Occlusive thrombi appear to be detrimental to short-term outcome.

Imaging of cardiac allograft rejection in dogs using indium-111 monoclonal antimyosin Fab.

The acute rejection of cardiac allografts is currently diagnosed by the presence of myocyte necrosis on endomyocardial biopsy. We evaluated the efficacy of noninvasive scintigraphic imaging with indium-111-labeled anticardiac myosin Fab fragments (indium-111 antimyosin) to detect and quantify cardiac allograft rejection. Six dogs that had intrathoracic heterotopic cardiac allograft transplantation were injected with indium-111 antimyosin and planar and single photon emission computed tomographic (SPECT) images were obtained in various stages of acute and subacute rejection. Four dogs had an allograft older than 8 months and had been on long-term immunosuppressive therapy; two dogs had an allograft less than 2 weeks old and were not on immunosuppressive therapy. Count ratios comparing heterotopic with native hearts were calculated from both SPECT images and in vitro scans of excised and sectioned hearts and were compared with the degree of rejection scored by an independent histopathologic review. Indium-111 antimyosin uptake was not visible in planar or SPECT images of native hearts. Faint diffuse uptake was apparent in cardiac allografts during long-term immunosuppression and intense radioactivity was present in hearts with electrocardiographic evidence of rejection. The heterotopic to native heart count ratios in SPECT images correlated significantly with the count ratios in the excised hearts (r = 0.93) and with the histopathologic rejection score (r = 0.97). The distribution of indium-111 antimyosin activity in right and left ventricles corresponded to areas of histopathologic abnormalities. Immunoperoxidase studies showed deposition of indium-111 antimyosin only in areas of myocyte necrosis. The results demonstrate that indium-111 antimyosin imaging can noninvasively detect the presence, location and severity of canine cardiac allograft rejection.

Transmembrane action potentials and intracellular potassium activity of baboon cardiac tissues.

Preparations of baboon cardiac tissue were studied using standard microelectrode and double barrelled potassium ion selective microelectrode techniques. Transmembrane action potentials were recorded from Purkinje fibres and from atrial and ventricular muscle cells. Purkinje fibres were impaled frequently on the ventricular septum but were sparse on the endocardium of the free wall. Purkinje fibres had longer action potential durations than ventricular muscle cells, and endocardial muscle cells had longer action potential durations than epicardial muscle cells. Tissue bundles resembling the false tendons of the canine heart were found in the ventricles. Most of these false tendons consisted of ventricular muscle as indicated by transmembrane action potentials. On rare occasions, false tendon preparations were studied in which the cells had action potentials similar to those of canine Purkinje fibres. In these preparations, normal automaticity occurred from maximum diastolic potentials greater than or equal to -85 mV. Intracellular potassium activity in the baboon ventricular muscle cells was 96(3) mmol.litre-1 (mean(SEM), n = 32), which is lower than that reported for canine tissues but higher than that reported for feline or rabbit tissues. It is concluded that, although the cellular electrophysiology of primate and canine cardiac tissues is superficially similar, consistent and perhaps important differences do occur.

Methylation of the estrogen receptor-alpha gene promoter is selectively increased in proliferating human aortic smooth muscle cells.

OBJECTIVE: Atherosclerosis is a multigenic process leading to the progressive occlusion of arteries of mid to large caliber. A key step of the atherogenic process is the proliferation and migration of vascular smooth muscle cells into the intimal layer of the arterial conduit. The phenotype of smooth muscle cells, once within the intima, is known to switch from contractile to de-differentiated, yet the regulation of this switch at the genomic level is unknown. Estrogen has been shown to regulate cell proliferation both for cancer cells and for vascular cells. However, methylation of the estrogen receptor-alpha gene (ERalpha) promoter blocks the expression of ERalpha, and thereby can antagonize the regulatory effect of estrogen on cell proliferation. We sought to determine whether methylation of the ERalpha is differentially and selectively regulated in contractile versus de-differentiated arterial smooth muscle cells. METHODS: We used Southern blot assay, combined bisulfite restriction analysis (Cobra) and restriction landmark genome scanning (RLGS-M) to determine the methylation status of ERalpha in human aortic smooth muscle cells, either in situ (normal aortic tissue, contractile phenotype), or the same cells explanted from the aorta and cultured in vitro (de-differentiated phenotype). RESULTS: We provide evidence that methylation of the ERalpha in smooth muscle cells that display a proliferative phenotype is altered relative to the same cells studied within the media of non-atherosclerotic aortas. Thus, the ERalpha promoter does not appear to be methylated in situ (normal aorta), but becomes methylated in proliferating aortic smooth muscle cells. Using a screening technique, RLGS-M, we show that alteration in methylation associated with the smooth muscle cell phenotypic switch does not seem to require heightened activity of the methyltransferase enzyme, and appears to be selective for the ERalpha and a limited pool of genes whose CpG island becomes either demethylated or de novo methylated. CONCLUSIONS: Our data support the concept that the genome of aortic smooth muscle cells is responsive to environmental conditions, and that DNA methylation, in particular methylation of the ERalpha, could contribute to the switch in phenotype observed in these cells.

Concomitant MAZE procedure during cardiac surgical procedures: is there any survival advantage in conversion to sinus rhythm?

AIM: The MAZE procedure, or concomitant intraoperative ablation, is an effective technique to restore long-term sinus rhythm (SR). The survival benefit of conversion to SR has been questioned recently. METHODS: We retrospectively evaluated the conversion rate to SR and its correlation with long-term survival in 209 patients with chronic AF, who had a MAZE procedure during cardiac surgical procedures between the years 2006 and 2011 at our institution. The mean age was 67.2 ± 12.0 years and 52.2% were female (N. = 109). Perioperative mortality was 5.74% (N. = 12). RESULTS: In univariate analysis, significant risk factors for perioperative mortality were age (P = 0.0033), duration of perfusion time (P = 0.0093), elevated creatinine (≥ 1.6 mg/dL, P = .02), and cross clamp time (P = 0.016). In multivariate analysis age (HR 2.97) and duration of perfusion time (HR 1.48) were the only independent predictors of perioperative mortality. The overall one and five-year survival rates were 88% ± 2.2%, and 76% ± 3.3%, respectively. The one and five-year survival rates for patients who converted and were in sinus rhythm (SR) upon discharge (N. = 154) were 88% ± 2.6% and 80% ± 3.5%, respectively. While the one and five-year survival rates for patients who were still in AF upon discharge (N. = 55) were 94% ± 3% and 82% ± 6.6%, respectively, this survival difference was not statistically significant (P = 0.24). Significant risk factors for long-term mortality included DM (P = 0.023), preoperative MI (P = 0.043), preoperative renal insufficiency (creatinine, ≥ 1.6 mg/dL, P = 0.02) and asthma/COPD (P = 0.040). In multivariate analysis, age (HR 1.048) and preoperative MI (HR 1.948) were the only independent predictors of long-term mortality. CONCLUSION: The surgical MAZE procedure has a high conversion rate, however, our data did not show improved survival in patients who converted to SR prior to discharge.

Fracture after cardiac transplantation: a prospective longitudinal study.

Cardiac transplantation is associated with increased prevalence of vertebral fractures, but the natural history of and risk factors for fracture after this life-saving procedure are unclear. We evaluated 47 patients (34 men and 13 postmenopausal women) before transplantation with spinal radiographs, determination of bone density by dual energy x-ray absorptiometry, and measurement of biochemical indexes of mineral metabolism. During the first year after transplantation, incident fractures were documented radiographically. Associations among demographic characteristics, bone density, biochemistries, and fracture risk were evaluated with logistic regression analysis. Despite supplementation with elemental calcium (1000 mg/day) and vitamin D (400 IU/day), 17 patients (7 women and 10 men) sustained a total of 34 fractures. Most fractures involved the spine, and 85% of the patients who experienced fracture did so within 6 months of transplantation. Fifty-four percent of the women and 29% of the men experienced fracture. Femoral neck bone mineral density was significantly lower in women who experienced fracture than in those who did not (0.604 +/- 0.11 vs. 0.760 +/- 0.12 g/cm2; P < 0.04), but did not differ in men according to fracture outcome. The amount of bone loss at the femoral neck by 6 months after transplantation was significantly greater in men with fracture than in men without fracture (12.0 +/- 6.4% vs. 6.8 +/- 5.3%; P < 0.04), but did not differ in women according to fracture outcome. Pretransplant 1,25-dihydroxyvitamin D levels were significantly lower (25 +/- 9 vs. 39 +/- 17 pg/mL; P < 0.007) and intact PTH levels tended to be higher in men who did not experience fracture (37 +/- 15 vs. 69 +/- 46 pg/mL; P < 0.06). Individual pretransplant bone density measurements demonstrated substantial overlap between patients who did and did not experience fracture, and normal bone density did not necessarily protect against fracture after transplantation. We conclude that fractures are a common and early complication of cardiac transplantation. No pretransplant measurement has yet been identified that reliably predicts fracture after transplantation in the individual patient.

Bone loss and turnover after cardiac transplantation.

Cardiac transplantation is associated with increased prevalence and incidence of fracture, and rapid bone loss has been reported during the first posttransplant year. To define further the pattern and etiology of bone loss after cardiac transplantation, we enrolled 70 patients (52 men and 18 women) in a prospective 3-yr study. Bone densitometry (BMD) and biochemical indexes of mineral metabolism were performed before and at defined times after transplantation. Despite supplementation with elemental calcium (1000 mg/day) and vitamin D (400 IU/day), the mean rate of bone loss during the first year was 7.3 +/- 0.9% (+/- SEM) at the lumbar spine and 10.5 +/- 1.1% at the femoral neck. The rate of bone loss slowed (P < 0.001 compared to year 1) at both sites (0.9 +/- 0.9% and 0.1 +/- 1.0%, respectively) during the second year. During the third year, lumbar spine BMD increased at a rate of 2.4 +/- 0.8%/yr (P < 0.02 compared to year 2), but femoral neck BMD did not change. At the radius, the rate of decline in BMD was negligible during the first year (0.9 +/- 0.5%), but was significant during the second (2.1 +/- 0.6%; P < 0.01) and third (2.9 +/- 0.8%; P < 0.03) years. Evaluation of the pattern of bone loss during the first year demonstrated that mean lumbar spine BMD decreased rapidly during the first 6 months, after which there was no further decline. In contrast, femoral neck BMD continued to fall at an annualized rate of 8.2 +/- 1.3% during the second half of the year. The pattern and rates of bone loss were similar in men and women. Biochemistries revealed decreases in serum testosterone and osteocalcin and increases in all bone resorption markers 1 and 3 months after transplantation, with a return to baseline by 6 months. Higher rates of bone loss were associated with greater exposure to prednisone, lower serum concentrations of vitamin D metabolites, greater suppression of osteocalcin, higher levels of bone resorption markers, and, in men, lower serum testosterone concentrations. We conclude that rapid bone loss is primarily confined to the initial year after transplantation. During the first 6 months, bone loss is accompanied by alterations in markers of bone turnover consistent with biochemical uncoupling of bone formation and resorption. Greater exposure to glucocorticoids, lower serum concentrations of vitamin D metabolites and testosterone, and higher bone turnover were associated with more rapid bone loss.

Treatment of cardiac tumors by orthotopic cardiac transplantation.

Primary soft tissue tumors of the heart usually cannot be excised with adequate margins. Orthotopic heart transplantation (OHT) could allow complete resection of cardiac tumors and has been performed in selected patients. However, most are not transplanted because of the high risk of tumor recurrence or metastasis and the possible enhancement of tumor growth by immunosuppressive drugs. Six patients with soft tissue cardiac tumors have been transplanted at the Columbia Presbyterian Medical Center: one paraganglionoma and one fibroma (both benign), and four malignant primary sarcomas. In all cases, there was no preoperative evidence of metastasis. In all but one case, the tumor was completely resected with adequate margins at the time of transplantation. One sarcoma patient who had not received preoperative or postoperative chemotherapy died suddenly 2.6 months after operation with no evidence of tumor. One patient is alive at 38 months with diffuse metastatic disease; two patients were treated preoperatively with intensive doxorubicin-based chemotherapy (one of whom also received postoperative external-beam radiotherapy to a positive surgical margin) and are tumor free 16 and 6 months after transplantation. Our experience compares favorably with the worldwide results of OHT for cardiac tumors (an additional 13 patients).

Apoptosis and increased expression of inducible nitric oxide synthase in human allograft rejection.

BACKGROUND: The mechanisms of myocyte death during cardiac allograft rejection are incompletely understood. In a previous study using a rat heterotopic cardiac allograft model, we showed that cardiac myocyte apoptosis, inducible nitric oxide synthase (iNOS) mRNA, protein and enzyme activity, and nitrotyrosine increased simultaneously during cardiac allograft rejection. This study was designed to investigate whether apoptosis and expression of iNOS occur in human cardiac allograft rejection. METHODS: Right ventricular endomyocardial biopsies from 30 cases of allograft rejection (International Society of Heart and Lung Transplantation grade 3A/B) were compared with 12 biopsies with no rejection (International Society of Heart and Lung Transplantation grade 0). Samples were co-labeled for apoptosis and muscle actin. Serial sections were stained for iNOS, nitrotyrosine, and the leukocyte markers CD3, CD4, CD8, and CD68 to identify T-cell subpopulations and macrophages. RESULTS: Biopsies with cardiac allograft rejection showed a 30-fold increase of apoptotic cells when compared with controls. Most apoptotic cardiac myocytes were found in proximity to macrophage (CD68+)-rich inflammatory infiltrates. iNOS immunoreactivity was strongest in macrophages and adjacent myocytes, which also showed high levels of nitrotyrosine, representing damage by peroxynitrite. CONCLUSIONS: Apoptosis is a major form of myocyte death during human cardiac allograft rejection. Cardiac myocyte apoptosis is closely associated with expression of iNOS in macrophages and myocytes and with nitration of myocyte proteins by peroxynitrite.

Newborn baboon serum lacks natural anti-pig xenoantibody.

Discordant xenotransplantation represents an attractive alternative to allotransplantation in light of the shortage of donor organs currently available for cardiac allotransplantation. Unfortunately, discordant xenotransplantation is still limited by hyper-acute rejection, a process thought to be mediated by natural anti-xenodonor antibody. Based on data that cytotoxic natural xenoantibodies are IgM in nature, we postulated that natural xenoantibodies may be absent from newborn serum. Baboon sera were collected from infant baboons. Pooled adult baboon sera were used as controls. A whole cell ELISA was performed to determine the binding of xenoantibodies to pig aortic endothelial cells and pig lymphocytes. The cytotoxicity of both adult and newborn baboon sera to pig aortic endothelial cells was measured by a MTT (3-(4,5-dimethyl-thiazoyl-2-y) 2,5 diphenyl-tetrazolium bromide) assay. Newborn baboon sera demonstrated very low levels of binding of natural IgM xenoantibodies to pig endothelial cells and lymphocytes, whereas natural IgM xenoantibodies from adult baboon sera bound significantly to both pig aortic endothelial cells and lymphocytes. IgG natural antibodies in both adult and newborn sera bound to pig endothelial cells and pig lymphocytes. The MTT assay demonstrated high levels of cytotoxicity to pig endothelial cells from adult baboon sera and very low levels of cytotoxicity from newborn baboon sera. In this study, newborn baboon sera were demonstrated to be free of natural IgM xenoantibodies to pig endothelial cells and lymphocytes. Although natural anti-pig IgG antibodies were present in newborn sera, newborn baboon sera lack cytotoxicity to pig target cells. These findings suggest that IgM is the more important xenoantibody and that hyperacute rejection of discordant cardiac xenografts may be avoidable in the newborn.

Abrogation of baboon natural xenoantibody to pig splenocytes by DL-penicillamine.

Natural xenoantibodies are believed to be IgM in nature and are known to play a critical role in the hyperacute rejection of distantly related xenografts. The purpose of this study was to determine whether the reducing agent DL-penicillamine could inactivate baboon natural xenoantibodies to pig splenocytes. Pooled baboon serum was treated with varying concentrations of DL-penicillamine over different lengths of time and a complement-mediated cytotoxicity assay was used to determine the reactivity of baboon natural xenoantibodies to pig splenocytes. A whole-cell ELISA assay was used to assess the binding of both IgG and IgM xenoantibodies to pig splenocytes. In addition, DL-penicillamine-treated serum was dialyzed to assess its potential clinical application. These in vitro experiments indicate that both IgM and IgG baboon natural xenoantibodies bind to pig splenocytes, but only IgM xenoantibody is cytotoxic. The binding of baboon natural IgM xenoantibody can be eliminated, and the cytotoxicity of IgM xenoantibody markedly reduced by DL-penicillamine treatment despite continued binding of IgG xenoantibody to pig splenocytes. In addition, DL-penicillamine can be dialyzed, suggesting that it may be an efficacious clinical treatment, the toxicity of which can be regulated with hemodialysis.

Prolongation of primate cardiac xenograft survival with cyclosporine.

Cardiac xenotransplantation in nonprimates using traditional immunosuppression (azathioprine and prednisone) or cyclosporine has been unsuccessful or has required doses of immunosuppressants not tolerated by man. This study sought to determine if primate hearts could be transplanted successfully across genus boundaries using a dose of cyclosporine applicable to human transplantation. The hearts of outbred cynomolgus monkeys (Macaca fascicularis) were heterotopically transplanted into the necks of outbred baboons (Papio anubis). Hyperacute rejection did not occur and there were no cyclosporine-induced malignancies or nephrotoxicity. A 12-fold prolongation of mean cardiac xenograft survival to 77 days was accomplished using parenteral cyclosporine and steroids. The histology of rejection was notable for the appearance of reversible rejection on the 30-day biopsies. The histopathologic and immunologic data support the role of both cell-mediated and humoral mechanisms in primate cardiac xenograft rejection. Neither mixed lymphocyte cultures or cytotoxic antibody assays were predictive of graft loss, but there was a significant increase in their respective levels at the time of cessation of graft function. Thus, significant prolongation of primate cardiac graft survival across a genus boundary was accomplished using a dose of cyclosporine similar to that used in human transplantation.

Carcinoma of the lung after heart transplantation.

We have recently noted an unexpected high incidence of lung cancer in our population of cardiac allograft recipients. We conducted a retrospective review of cardiac transplantation at our institution to investigate the incidence, clinical course, and outcome of patients who developed lung cancer following heart transplantation. Nine patients--each with a history of smoking at 30 pack-years--developed lung cancer following heart transplantation, for an incidence of 1.56% of patients at risk. Eight of the patients were male > or = 50 years of age, representing 3.3% of the male transplant recipients in this age group. The interval from transplantation to diagnosis clustered around 3-5 years after transplantation, but in two instances (22%), a neoplasm was discovered within 6 months of transplantation. Almost half of the cancers were discovered incidentally, despite routine radiographic surveillance. Seven of 9 (78%) patients had stage IV disease at presentation. Median survival after diagnosis was 3 months, and five of the seven patients who died survived less than 4 months after diagnosis. We conclude that cardiac transplant recipients are at increased risk for development of lung cancer. Patients with a moderate to heavy smoking history might well be advised to undergo chest CT scanning in an aggressive search for occult lung cancer before cardiac transplantation is considered further. Finally, despite frequent radiologic examinations, these lung cancers are often diagnosed incidentally, are far advanced at the time of diagnosis, are not surgically resectable, and are poorly responsive to adjuvant therapy.

Circulating human mononuclear cells exhibit augmented lysis of pig endothelium after activation with interleukin 2.

In immunohistochemical studies investigating the cellular infiltrates in pig xenografts undergoing delayed rejection by newborn and adult primate recipients, we observed extensive infiltration with primate macrophages and natural killer (NK) cells. To extend these studies in vitro, we investigated the functional properties of human NK cell precursors with respect to their potential interactions with pig aortic endothelial cells (PAEC). Using a short-term 51Cr release assay, human peripheral blood mononuclear cells (PBMC) demonstrated spontaneous and interleukin (IL) 2 augmented lytic activity against PAEC which increased with increasing effector to target cell ratio. Treatment of human PBMC with anti-CD2 significantly reduced this NK lytic activity by IL-2-activated PBMC. Finally, we investigated the effects of PAEC treatment with certain macrophage-derived human cytokines on adhesion of IL-2-activated human PBMC. Treatment of PAEC with IL-1 and tumor necrosis factor-alpha, in a dose-dependent manner, increased adherence of IL-2-activated human PBMC. These results demonstrate that humans contain circulating NK cells capable of lysing PAEC after activation with IL-2, that the mechanism involves interactions between CD2 and its ligand on porcine endothelium, and that these interactions may be influenced by macrophage-derived cytokines produced at the site of xenograft rejection.

Anti-GaL IgG antibodies in sera of newborn humans and baboons and its significance in pig xenotransplantation.

We have previously demonstrated that hyperacute rejection does not occur in a pig-to-newborn baboon heart transplant model, presumably because of low levels of cytotoxic antipig antibodies present in the serum of newborn baboons. Cytotoxic antipig antibodies are primarily directed to alpha-1,3-galactosyl (alpha Gal) residues on endothelial cell surface structures Twenty-one full-term humans and 5 full-term baboons were tested for complement mediated lysis (CML) of pig kidney (PK-15) cells and anti-alpha Gal activity with an ELISA using BSA-conjugated alpha Gal residues as target. To evaluate the significance of the anti-alpha Gal titers in vivo 5 newborn baboons underwent heterotopic pig cardiac xenotransplantation. Six of 21 human samples and 1 of 5 baboon samples demonstrated significant cytotoxicity to PK-15 cells. Twelve of 21 newborn humans had anti-alpha Gal IgG antibodies at titers of 1:80 or greater. None of the samples had anti-alpha Gal IgM. In newborn baboons, 1 of 5 sera had anti-alpha Gal IgG antibodies at titers greater than 1:80 and none of these samples had anti-alpha Gal IgM. Xenografts survived for an average of 3.6 days, even in the baboon with high anti-alpha Gal IgG titers. Analysis of the explanted grafts showed minimal evidence of complement-mediated hyperacute rejection (HAR), but prominent mononuclear cell infiltrates. In serum tested posttransplant there was an induced anti-alpha Gal response with cytotoxicity against PK-15 cells. These results show that anti-alpha Gal IgM is absent in newborn human and baboon sera, allowing pig grafts to avoid HAR. However, the presence of anti-alpha Gal IgG may be associated with mononuclear cell infiltration of the xenograft and its subsequent rejection.