Identification and characterization of two genes (MIP-1beta, VE-CADHERIN) implicated in acute rejection in human heart transplantation: use of murine models in tandem with cDNA arrays.

BACKGROUND: Genes and mechanisms of action involved in human acute rejection after allogeneic heart transplantation remain to be elucidated. The use of a murine allograft model in tandem with cDNA arrays and quantitative real-time polymerase chain reaction (Q-PCR) can greatly help in identifying key genes implicated in human heart acute rejection. METHODS AND RESULTS: Hearts from Balb/c mice were either not transplanted or transplanted heterotopically in the abdomen of Balb/c (isografts) and C57BL/6 (allografts) mice. Histological analysis showed acute rejection only in allografts. Total RNA was extracted from isografts (n=3), allografts (n=4), and not transplanted hearts (n=4); reverse transcribed; and labeled with P32. Each probe was hybridized to cDNA macroarrays. Eight genes were overexpressed and 7 genes were underexpressed in allografts compared with isografts. Macrophage inflammatory protein-1beta (MIP-1beta), an overexpressed gene, and VE-cadherin, an underexpressed gene, were validated by immunohistochemistry and Q-PCR in the murine models. Genes of interest, validated in the 3 murine groups, were then investigated in human heart tissues. Immunohistochemistry and Q-PCR performed on endomyocardial biopsies after heart transplantation showing no rejection (n=10) or grade IB (n=10) or IIIA (n=10) rejection, according to International Society of Heart and Lung Transplantation criteria, confirmed the results obtained from the murine model. CONCLUSIONS: We have demonstrated that the upregulation of MIP-1beta and downregulation of VE-cadherin may strongly participate in human acute heart rejection.

Spectral analysis of blood pressure variability in heart transplant patients.

The cardiac transplant patient provides a unique model for the study of blood pressure variability in the absence of heart rate variability. We examined the harmonic and fractal components of blood pressure variability in 14 heart transplant patients (12 men, 2 women; 21 to 62 years of age) and in age-and sex-matched control subjects during seated rest, supine rest, and supine rest with fixed-pace breathing (12 respirations per minute). Heart rate was faster in transplant patients than in control subjects, with much less heart rate variability (P < .0001). Spectral analysis of blood pressure variability revealed no difference in total power for either systolic or diastolic pressure, but transplant patients had less low-frequency (0 to 0.15 Hz) harmonic spectral power in both systolic (P < .01) and diastolic (P < .03) pressure and more high-frequency power (0.15 to 0.5 Hz) in diastolic pressure than control subjects. The ratio of high-frequency power in diastolic relative to systolic pressure was consistently higher (P < .0001) in the transplant patients (0.29 to 0.51) than in control subjects (0.11 to 0.13). The slope of the fractal component of systolic pressure was approximately 1.8 in both transplant patients and control subjects. This was greater than the slope for heart rate variability (approximately 1.1 in control subjects). These data provide clear evidence of independence of the fractal component of heart rate and blood pressure variabilities in both transplant patients and control subjects. The heart rate component of the arterial baroreflex minimized high-frequency diastolic pressure changes while contributing to low-frequency variations in both systolic and diastolic pressures.

High prevalence of thromoembolic complications in heart transplant recipients. Which preventive strategy.?

Consecutive patients transplanted between January 1984 and December 1988 were followed until August 1992 to detect fatal and nonfatal thromboembolic complications, including sudden death, acute and chronic myocardial infarction, pulmonary and peripheral embolisms, stroke, and thrombophlebitis. The probability of developing such complications was 9.86 per 100 patients per year. The probability of fatal complications was 3.97% per year; the mean interval between transplant and death was 1247 days versus 29.5 days for nonthromboembolic deaths. Thromboembolic deaths represented 5.1% of total mortality at the first year posttransplant but 57, 30, 67 and 73% at the second, third, fourth, and fifth years, respectively. Among the prognosis factors that were analyzed, none was significant predictor of thromboembolic complication. This high prevalence of thromboembolic complications suggests that effective antithrombotic strategy should be defined in heart transplant recipients.

The beneficial effect of dietary antioxidant supplementation on platelet aggregation and cyclosporine treatment in heart transplant recipients.

To determine whether dietary antioxidant supplementation can reduce platelet reactivity in heart transplant recipients, 20 patients were prospectively randomized to receive either 500 IU vitamin E orally per day in the form of acetate for 2 months or no vitamin E. Blood creatinine (P = 0.01) and lymphocyte count (P = 0.009) significantly decreased only in supplemented patients, whereas the cyclosporine blood level was not modified. Platelet aggregation was stable in control patients but significantly decreased in supplemented patients in response to either thrombin (from 8.3 +/- 0.9% of maximum aggregation to 3.7 +/- 0.7, P = 0.001) or ADP (secondary wave: from 44.7 +/- 5.9% to 33.2 +/- 7.0, P = 0.02). Thus antioxidant supplementation tended to improve immunosuppression (by reducing lymphocyte count), to reduce cyclosporine nephrotoxicity, and to decrease the high thrombotic risk associated with heart transplantation.

Thiopurine S-methyltransferase gene polymorphism is predictive of azathioprine-induced myelosuppression in heart transplant recipients.

Azathioprine (AZA) is metabolized via the cytosolic enzyme thiopurine S-methyltransferase (TPMT). TPMT activity exhibits genetic polymorphism with four prevalent (75%) mutant alleles TPMT*2 (G238C) and TPMT*3 (A719G and/or G460A) and a wild-type allele TPMT*1. To test the hypothesis that presence of these mutations is associated with greater toxicity of AZA in heart transplant recipients, 30 consecutive patients treated with AZA were followed up for the first month after heart transplant. Mutation of TPMT gene (mutation-specific polymerase chain reaction-based methods) was observed in four patients (A719G: n = 2; A719G plus G460: n = 2). Agranulocytosis did not occur in patients with the wild genotype. It occurred in the two patients with mutation A719G and there was a 40% drop in neutrophils in the two other patients. Discontinuation of AZA in the four mutant patients corrected for the drop. Presence of TPMT mutations is associated with a greater likelihood of agranulocytosis. Determination of these mutations could reduce the risk for hematological side-effects.

A randomized active-controlled trial of mycophenolate mofetil in heart transplant recipients. Mycophenolate Mofetil Investigators.

BACKGROUND: After heart transplantation, 1-year and 5-year survival rates are 79% and 63%, respectively, with rejection, infection, and allograft coronary artery disease accounting for the majority of deaths. Mycophenolate mofetil (MMF), an inhibitor of the de novo pathway for purine biosynthesis, decreases rejection in animals and in human renal transplantation. METHODS: In a double-blind, active-controlled trial, 28 centers randomized 650 patients undergoing their first heart transplant to receive MMF (3000 mg/day) or azathioprine (1.5-3 mg/kg/day), in addition to cyclosporine and corticosteroids. Rejection and survival data were obtained for 6 and 12 months, respectively. Because 11% of the patients withdrew before receiving study drug, data were analyzed on all randomized patients (enrolled patients) and on patients who received study medications (treated patients). RESULTS: Survival and rejection were similar in enrolled patients (MMF, n=327; azathioprine, n=323). In treated patients (MMF, n=289; azathioprine, n=289), the MMF group compared with the azathioprine group was associated with significant reduction in mortality at 1 year (18 [6.2%] versus 33 deaths [11.4%]; P=0.031) and a significant reduction in the requirement for rejection treatment (65.7% versus 73.7%; P=0.026). There was a trend for fewer MMF patients to have > or = grade 3A rejection (45.0% versus 52.9%; P=0.055) or require the murine monoclonal anti-CD3 antibody or antithymocyte globulin (15.2% versus 21.1%; P=0.061). Opportunistic infections, mostly herpes simplex, were more common in the MMF group (53.3% versus 43.6%; P=0.025). CONCLUSIONS: Substitution of MMF for azathioprine may reduce mortality and rejection in the first year after cardiac transplantation.

Safety, tolerability and efficacy of cyclosporine microemulsion in heart transplant recipients: a randomized, multicenter, double-blind comparison with the oil based formulation of cyclosporine--results at six months after transplantation.

BACKGROUND: The introduction of cyclosporine has resulted in significant improvement in the survival of cardiac allograft recipients due to decreased mortality from infection and rejection. The original oil-based cyclosporine formulation exhibits variable and unpredictable bioavailability that correlates with an increased incidence of acute and chronic rejection in those patients in whom this is most pronounced. The primary objectives of this prospective, multicenter, randomized, double-blind study in cardiac transplant patients were: to compare the efficacy of cyclosporine microemulsion (CsA-NL) with oil-based cyclosporine (CsA-SM) as measured by cardiac allograft and recipient survival and the incidence and severity of acute rejection episodes; and to assess the safety and tolerability of CsA-NL compared with CsA-SM in this population. This report represents the analysis of results 6 months after transplantation. METHODS: A total of 380 patients undergoing their first cardiac transplant at 24 centers in the United States, Canada, and Europe were enrolled in this double-blind, randomized trial examining the safety and efficacy of CsA-NL versus CsA-SM. Rejection was diagnosed using endomyocardial biopsy and were graded according to standardized criteria of the International Society of Heart and Lung Transplantation (ISHLT). Clinical parameters were monitored during the study. Survival and freedom from were used for analysis as was Fisher's exact test for comparisons between groups. RESULTS: At 6 months after transplantation, allograft and patient survival were the same for both groups. The frequency of ISHLT grade 3A or greater episodes in the two groups was identical. Fewer CsA-NL patients (5.9%) required antilymphocyte antibody (ATG or OKT-3) therapy for rejection compared with the CsA-SM-treated patients (14.1%, P=0.01). Females with ISHLT rejection grade > or = 3A treated with CsA-NL had a 46% lower incidence of rejection compared with the CsA-SM-treated group (31.3% vs. 57.6%, P=0.032). Fewer infections were seen in the CsA-NL. With the exception of baseline and 1 week posttransplant creatinines which were higher in the CsA-NL group, the overall creatinine was not significantly different between the two groups. CONCLUSIONS: This multicenter, randomized study of cardiac transplant recipients documented less severe rejection (in particular those requiring antibody therapy) and a lower incidence of infection in CsA-NL-treated patients. Results from the female subgroup analysis suggest that the improved bioavailability of CsA-NL might reduce the frequency of rejection episodes in female patients. The use of CsA-NL was not associated with an increased risk of adverse events.

Safety, tolerability, and efficacy of cyclosporine microemulsion in heart transplant recipients: a randomized, multicenter, double-blind comparison with the oil-based formulation of cyclosporine--results at 24 months after transplantation.

BACKGROUND: The widespread use of cyclosporine has improved the survival of cardiac transplant patients as a result of reduced morbidity and mortality from rejection and infection. The original oil-based form of cyclosporine demonstrated unpredictable absorption resulting in an increased frequency of acute and chronic rejection in patients with poor bioavailability. The primary end. points of the present, prospective, randomized multicenter, double-blind trial were to compare the efficacy of the micro-emulsion form of cycolsporine (CsA-NL) with the oil-based formulation as determined by cardiac allograft and recipient survival and the incidence and severity of the acute rejection episodes and to determine the safety and tolerability of CsA-NL compared with Sandimmune CsA-(SM) in the study population. The 6-month analysis of the study showed reduced number of CsA-NL patients requiring antilymphocyte antibody therapy for rejection, fewer International Society of Heart and Lung Transplantation grade > or =3A rejections in female patients and fewer infections. Our report represents the final analysis of the results 24 months after transplantation. METHODS: A total of 380 patients undergoing de novo cardiac transplants at 24 centers in the United States, Canada, and Europe were enrolled in this double-blind, randomized trial evaluating the efficacy and safety of CsA-NL versus CsA-SM. Acute allograft rejection was diagnosed by endomyocardial biopsy and graded according to the International Society of Heart and Lung Transplantation nomenclature. Kaplan-Meier analysis and Fisher's exact test were used for comparisons between groups. RESULTS: After 24 months, allograft and recipient survival were identical in both groups. There were fewer CsA-NL patients (6.9%) requiring antilymphocyte antibody therapy for rejection than in the CsA-SM-treated patient group (17.7%, P=0.002). There were fewer discontinuations of study drug for treatment failures in the CsA-NL groups (7; 3.7%) compared with the CsA-SM group (18; 9.4%, P=0.037). The average corticosteroid dose was lower in the CsA-NL group (0.37 mg/kg/day) compared with the CsA-SM group (0.48 mg/kg/day, P=0.034) over the 24-month study period. Overall, there was no difference in blood pressure or creatinine between the two study groups. CONCLUSIONS: The final results of this multi-center, randomized study of two forms of cyclosporine confirmed that there were fewer episodes of rejection requiring antilymphocyte antibodies and fewer study discontinuations for treatment failures in CsA-NL-treated patients compared to those treated with CsA-SM. The use of CsA-NL did not predispose these patients to a higher risk of adverse events.

In vivo release of a heparin-like factor in dogs during profound hypothermia.

Experimentally induced hypothermia (20 degrees C) for 60 minutes in dogs provokes a significant decrease in the platelet count, which reverses during subsequent rewarming, and the constant release of a heparin-like factor, which reacts as a specific inhibitor of factor Xa. This phenomenon is also rapidly reversible, and heparin values are not significantly different from control levels after 90 minutes of rewarming. The mean maximal concentration of heparin-like material is 0.54 U/ml, or about double control levels. Its half-life is approximately 90 minutes. The level of circulating antithrombin III was not modified during hypothermia and rewarming.

Haemocompatibility and biological course of carbonaceous composites for cardiovascular devices.

A new class of carbonaceous composites has been developed for cardiovascular devices. The aim of the present study, performed in dogs, was to test the immediate blood compatibility of these materials when inserted within the vascular bed. Biocompatibility studies were performed on vascular cylinders (6 mm i.d.) and intra-atrial implants. The specimens were examined sequentially by SEM at 10, 20, 30, 180 s and 10 min after re-establishment of the blood flow. Patency of the vascular cylinders was tested during the second and third postoperative month by Doppler ultrasound investigations; specimens were examined by light and electron microscopy (scanning and transmission) at 15, 60 and 110 d following implantation. As early as 10 s after re-establishment of the blood flow platelet adhesion and a limited fibrin mesh with few erythrocytes developed on the material. Platelet aggregates were only observed on intravenous implants. Except in the case of the intravenous insert, no thrombosis developed at the contact of intra-arterial or intracardiac implants. After 15 d it was completely covered by a fibrocellular layer (3-5 cells thick) consisting of large myofibroblasts with microfilaments, newly synthesized collagen and elastin. Endothelial-like cells developed and were completed 2 mnth after implantation. However, deposits present inside and outside the fibrocytic cells of the newly developed tissue were observed corresponding to carbon peaks as indicated by wavelength dispersive X-ray microanalysis.

Lack of successful reanimation of pig hearts harvested more than 10 minutes after death.

BACKGROUND: The aim of this study was to evaluate the viability of arrested pig hearts harvested after animal death. METHODS: Hearts (n = 25) were preserved for 2 hours by cold storage (4 degrees C) with St. Thomas' cardioplegic solution no warm ischemia (0 minutes; control) or 10, 20, 30, or 60 minutes of in situ warm ischemia (animal exsanguination). Hearts were then reperfused for 1 hour with whole blood with an in vitro functional testing system. Left ventricular developed pressure and coronary flow were measured during reperfusion. Energetic compound measurements and histologic analysis were performed on tissue biopsy specimens. RESULTS: After 10- and 20-minute warm ischemia, hearts showed a significant decrease in energetic compounds, a 51% and 73% decreases of left ventricular developed pressure, and 38% and 65% decreases in coronary flow, respectively. After 30 minutes hearts showed irreversible ischemic injury with ultrastructural tissue damage, a large decrease in energetic adenine nucleotide compounds, and an inability to beat more than 15 minutes after reperfusion. CONCLUSION: We conclude that in contrast with other species, pig hearts harvested 10 minutes or more after animal exsanguination fail to be successfully reanimated.

Comparison of continuous microperfusion and cold storage for pig heart preservation.

The aim of this study was to compare two methods of hypothermic heart preservation. Isolated hearts of pigs were preserved in cold cardioplegic solution (St. Thomas Hospital solution) either by simple storage or continuous microperfusion (with a new perfusion device) for 6 hours (group I, n = 12), 12 hours (group II, n = 12) and 24 hours (group III, n = 12). After storage, the myocardial function was studied for 60 minutes under nonworking conditions with an ex vivo functional testing system. Hearts preserved 24 hours by cold storage (group III) showed ventricular compliance and mean spontaneous left ventricular developed pressure significantly lower than hearts preserved by microperfusion (respectively, 63 +/- 47 versus 14 +/- 18 mm Hg and 16.8 +/- 22.0 versus 83 +/- 33 mm Hg). After 12 hours (group II) of preservation, mean left ventricular developed pressure was higher in microperfused hearts compared to immersed hearts (respectively, 133.3 +/- 39.0 versus 83.1 +/- 27.0 mm Hg, p < 0.05), whereas after 6 hours of preservation, no functional difference was observed between the microperfused and the immersed hearts. Hearts were also studied using myocardial biopsy specimens taken at the end of the preservation. The biopsy specimens were analyzed for high-energy phosphates. After 6 hours of preservation, the microperfusion group showed higher levels of adenosine triphosphate and total adenine nucleotides (adenosine triphosphate + adenosine diphosphate + adenosine monophosphate) (respectively, 4.60 +/- 0.5 mumol/gm and 5.98 +/- 0.5 mumol/gm fresh tissue) versus the cold storage group (respectively, 3.10 +/- 0.4 mumol/gm and 3.75 +/- 0.4 mumol/gm).(ABSTRACT TRUNCATED AT 250 WORDS)

Long conservation organs in heart transplantation: postoperative results and long-term follow-up in fourteen patients.

Between 1988 and 1995, 14 heart transplantations were performed after a long preservation period (10 to 13 hours). The transplantation procedure (Shumway) was standard, and our results were achieved through the implementation of a very strict reperfusion technique that included low pressure and low cardiopulmonary bypass output for the first 10 minutes. Three patients died during the postoperative period, and the survival rate was 75% at 1 year and 71% at 5 years. The results obtained with hearts stored for such long periods are comparable to the results obtained with hearts stored for less than 4 hours.

Increased platelet aggregation after heart transplantation: influence of aspirin.

Accelerated graft coronary artery disease remains the most dramatic complication in long-term survivors of heart transplantation. The main purpose of this study was to evaluate ex vivo platelet function of heart transplant recipients as compared with that of healthy subjects and nontransplant coronary patients. The influence of aspirin, the chief antiplatelet agent, was also evaluated. The heart transplant recipients exhibited a marked platelet hyperaggregation to adenosine diphosphate as compared with the two control groups. In addition, platelets of the heart transplant recipients appeared to be resistant to the inhibitory effect of aspirin. These results could, at least partly, explain the failure of antiplatelet agents to prevent myocardial infarction in these patients.

Fluid-regulating and sympathoadrenal hormonal responses to peak exercise following cardiac transplantation.

Orthotopic heart transplantation results in cardiac denervation that can disrupt the normal regulation of hydromineral balance. This study compared the exercise-induced variations in plasma osmolality; atrial natriuretic peptide (ANP), arginine vasopressin (AVP), norepinephrine (NE), epinephrine (E), and dopamine (DA) concentrations; and plasma renin activity (PRA) of six cardiac transplant recipients (HTX) and six healthy age-matched controls (C) submitted to graded upright maximal cycling. Venous blood samples were obtained at rest, at submaximal (70% O2 uptake) and peak exercise, and after 10 and 30 min of sitting recovery. Peak O2 uptake was not different between groups despite lower maximal heart rate in HTX (136 +/- 6 vs. 183 +/- 9 beats/min). Baseline plasma ANP and PRA were higher in HTX (203 +/- 55 pg/ml and 29.9 +/- 7.4 ng.ml-1 x h-1) than in C (71 +/- 17 pg/ml and 5.4 +/- 0.96 ng.ml-1 x h-1); AVP was lower in HTX than in C (1.1 +/- 0.3 vs. 3.2 +/- 0.8 pg/ml; P < 0.05); and circulating E, NE, and DA were not different between groups. Exercise resulted in more marked increases in HTX than in C for ANP (300 vs. 100%), AVP (2,000 vs. 300%), NE (860 vs. 500%), and DA (611 vs. 187%) but not for PRA and a higher E response in C than in HTX (455 vs. 1,258%). These observations confirm that the potential for ANP release to central volume loading is independent of intact cardiac innervation. The exaggerated AVP response in HTX could, however, reflect the absence of inhibitory influences consecutive to denervation.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of different techniques of hypothermic pig heart preservation.

The aim of this study was to compare several methods of hypothermic heart preservation. Isolated pig hearts were preserved for 24 hours in cold cardioplegic solution (St. Thomas' Hospital modified solution) by continuous perfusion (group I), microperfusion (group II), or simple storage (group III). The findings were then compared with those from hearts harvested and immediately reperfused (the control group). Group III hearts showed lower adenosine triphosphate preservation (0.47 +/- 0.18 mumol/g) than did group I and II hearts and the control hearts (1.86 +/- 0.40, 1.98 +/- 0.27, and 1.84 +/- 0.55 mumol/kg, respectively). Electronic microscopy studies also revealed that the myocardial cells in the group III hearts appeared to be damaged. After the hearts had undergone preservation, myocardial function was studied for 60 minutes under nonworking conditions using an ex vivo functional testing system. For group III, the mean left ventricular developed pressure and ventricular compliance (16 +/- 22 and 63 +/- 48 mm Hg, respectively) differed significantly from those for group I (83 +/- 26 and 0 +/- 0 mm Hg, respectively), group II (83 +/- 33 and 14 +/- 18 mm Hg, respectively), and the control group (115 +/- 13 and 0 +/- 0 mm Hg, respectively). We concluded from our findings that perfusion methods are superior to cold storage but inadequate to maintain heart viability for the long term during hypothermia. These techniques must be improved before they can be adopted for clinical use.

Human papillomavirus type 1-associated squamous cell carcinoma in a heart transplant recipient.

The occurrence of squamous cell carcinomas in organ transplant recipients with warts represents a good model to study viral carcinogenesis. Most of the cases were reported in renal transplant recipients. We present the case of a heart transplant recipient in whom multiple common warts, preepitheliomatous keratoses, and squamous cell carcinomas developed. The warts began 4 years after the transplantation and the first carcinoma occurred 2 years after the warts, all the lesions being on sun-exposed areas. Histologic signs of human papillomavirus infection were seen in all premalignant and malignant lesions. Furthermore, human papillomavirus type 1 DNA was detected by in situ molecular hybridization within one of the carcinomas. Human papillomaviruses, along with other carcinogenic factors, play an important role in the development of carcinomas, and benign types could be implicated. Further studies are required to evaluate the frequency of cutaneous malignant neoplasms in heart transplant recipients as compared with renal transplant recipients.

Course of the initial epithelial lesions associated with autologous bile duct replacement. Experimental study in rats.

Mucosal lesions and healing of the common bile duct were analyzed after various chemical and surgical insults in the rat by means of histologic and scanning electron microscopic investigations. Autogenous bile duct replacement is rapidly associated within the first 72 postoperative hours with complete epithelial cell desquamation, and its precocious consequence is biliary sludge. These lesions also occurred when the bile flow was diverted; the extrinsic devascularization and denervation did not produce extensive epithelial cell loss. Heparin perfusion within the biliary tree or the induction by glycerol of epithelial cell loss before bile duct transplantation is suggested in order to avoid extensive biliary sludge after bile duct transplantation.

Increased production of reactive oxygen species in pharmacologically-immunosuppressed patients.

HIV-infected patients and transplanted patients share similar immunosuppressed status. Recent insights gained through the field of heart transplantation may help to clarify the role of reactive oxygen species in HIV-infected patients.

Tolerance and resistance of myocardium to anoxia: experimental studies.

We shall emphasize the notion of wide variability in the tolerance and resistance to anoxia of the myocardium, although the most important parameters are temperature and time. They can be modified widely by accompanying physical or biochemical conditions: --Cardiac distension and drugs inducing cardiac arrest; --Resuscitative perfusion; --The effect of some biochemical components is more likely explained by their possible action on the mitochondriae (glyoxilic acid-Isoproterenol). The study of the mitochondriae alone can explain the different behaviours of hearts subjected to anoxia in various situations.