Billie Fyfe

Annexin-V imaging for noninvasive detection of cardiac allograft rejection

Heart transplant rejection is characterized pathologically by myocyte necrosis and apoptosis associated with interstitial mononuclear cell infiltration. Any one of these components can be targeted for noninvasive detection of transplant rejection. During apoptotic cell death, phosphatidylserine, a phospholipid that is normally confined to the inner leaflet of cell membrane bilayer, gets exteriorized. Technetium-99m-labeled annexin-V, an endogenous protein that has high affinity for binding to phosphatidylserine, has been administered intravenously for noninvasive identification of apoptotic cell death. In the present study of 18 cardiac allograft recipients, 13 patients had negative and five had positive myocardial uptake of annexin. These latter five demonstrated at least moderate transplant rejection and caspase-3 staining, suggesting apoptosis in their biopsy specimens. This study reveals the clinical feasibility and safety of annexin-V imaging for noninvasive detection of transplant rejection by targeting cell membrane phospholipid alterations that are commonly associated with the process of apoptosis.

Identification of Active Tissue Factor in Human Coronary Atheroma

BACKGROUND Recent observations suggest that thrombosis in vivo is initiated via the tissue factor (TF) pathway. The TF activity of human coronary atheroma has not been reported. METHODS AND RESULTS Directional coronary atherectomy (DCA) specimens from 63 lesions were analyzed with the use of a quantitative TF-specific activity assay. The median content of TF was 10 ng/g plaque (95% CI, 6 to 13 ng/g; range, 0 to 47 ng/g). After homogenization of the specimens, TF activity was detected in 28 of 31 lesions (90%). With a polyclonal anti-human TF antibody, the use of immunohistochemistry detected TF antigen in 43 of 50 lesions (86%); TF antigen was expressed in cellular and acellular areas of the plaque. Histologically defined thrombus was present in 19 of the 43 lesions with detectable TF antigen and in none of the 7 lesions without detectable TF antigen (19 of 43 versus 0 of 7; P < .02). TF antigen was undetectable with immunohistochemistry in 4 of 13 restenotic lesions (31%) and in 3 of 37 de novo lesions (8%) (P < .05). CONCLUSIONS TF contributes to the procoagulant activity of most atherosclerotic lesions treated with DCA. The association of immunohistochemically detectable TF with plaque thrombus suggests that TF plays a role in coronary thrombosis. Diminished TF expression in restenotic lesions may in part account for the lower complication rate that has been associated with DCA of restenotic versus de novo lesions. Inhibition of TF may represent a therapeutic goal for the prevention of thrombotic complications associated with percutaneous coronary interventions.

Differential regulation of two types of intracellular calcium release channels during end-stage heart failure.

The molecular basis of human heart failure is unknown. Alterations in calcium homeostasis have been observed in failing human heart muscles. Intracellular calcium-release channels regulate the calcium flux required for muscle contraction. Two forms of intracellular calcium-release channels are expressed in the heart: the ryanodine receptor (RyR) and the inositol 1,4,5-trisphosphate receptor (IP3R). In the present study we showed that these two cardiac intracellular calcium release channels were regulated in opposite directions in failing human hearts. In the left ventricle, RyR mRNA levels were decreased by 31% (P < 0.025) whereas IP3R mRNA levels were increased by 123% (P < 0.005). In situ hybridization localized both RyR and IP3R mRNAs to human cardiac myocytes. The relative amounts of IP3 binding sites increased approximately 40% compared with ryanodine binding sites in the failing heart. RyR down-regulation could contribute to impaired contractility; IP3R up regulation may be a compensatory response providing an alternative pathway for mobilizing intracellular calcium release, possibly contributing to the increased diastolic tone associated with heart failure and the hypertrophic response of failing myocardium.

Tissue factor is rapidly induced in arterial smooth muscle after balloon injury.

Tissue factor (TF) is a major activator of the coagulation cascade and may play a role in initiating thrombosis after intravascular injury. To investigate whether medial vascular smooth muscle provides a source of TF following arterial injury, the induction of TF mRNA and protein was studied in balloon-injured rat aorta. After full length aortic injury, aortas were harvested at various times and the media and adventitia separated using collagenase digestion and microscopic dissection. In uninjured aortic media, TF mRNA was undetectable by RNA blot hybridization. 2 h after balloon injury TF mRNA levels increased markedly. Return to near baseline levels occurred at 24 h. In situ hybridization with a 35S-labeled antisense rat TF cRNA probe detected TF mRNA in the adventitia but not in the media or endothelium of uninjured aorta. 2 h after balloon dilatation, a marked induction of TF mRNA was observed in the adventitia and media. Using a functional clotting assay, TF procoagulant activity was detected at low levels in uninjured rat aortic media and rose by approximately 10-fold 2 h after balloon dilatation. Return to baseline occurred within 4 d. These data demonstrate that vascular injury rapidly induces active TF in arterial smooth muscle, providing a procoagulant that may result in thrombus initiation or propagation.

Tissue factor expression in human arterial smooth muscle cells. TF is present in three cellular pools after growth factor stimulation.

Tissue factor (TF) is a transmembrane glycoprotein that initiates the coagulation cascade. Because of the potential role of TF in mediating arterial thrombosis, we have examined its expression in human aortic and coronary artery smooth muscle cells (SMC). TF mRNA and protein were induced in SMC by a variety of growth agonists. Exposure to PDGF AA or BB for 30 min provided all of the necessary signals for induction of TF mRNA and protein. This result was consistent with nuclear runoff analyses, demonstrating that PDGF-induced TF transcription occurred within 30 min. A newly developed assay involving binding of digoxigenin-labeled FVIIa (DigVIIa) and digoxigenin-labeled Factor X (DigX) was used to localize cellular TF. By light and confocal microscopy, prominent TF staining was seen in the perinuclear cytoplasm beginning 2 h after agonist treatment and persisting for 10-12 h. Surface TF activity, measured on SMC monolayers under flow conditions, increased transiently, peaking 4-6 h after agonist stimulation and returning to baseline within 16 h. Peak surface TF activity was only approximately 20% of total TF activity measured in cell lysates. Surface TF-blocking experiments demonstrated that the remaining TF was found as encrypted surface TF, and also in an intracellular pool. The relatively short-lived surface expression of TF may be critical for limiting the thrombotic potential of intact SMC exposed to growth factor stimulation. In contrast, the encrypted surface and intracellular pools may provide a rich source of TF under conditions associated with SMC damage, such as during atherosclerotic plaque rupture or balloon arterial injury.

Safety and efficacy of steroid withdrawal two days after kidney transplantation: Analysis of results at three years

Background. Chronic steroid therapy in spite of myriad side effects is widely used in kidney transplantation. This prospective controlled study evaluated safety and efficacy of steroid withdrawal at 2 days in kidney recipients monitored by surveillance biopsy. Methods. In all, 300 kidney recipients were studied; 150 in second-day steroid withdrawal group and 150 in steroid treated group (control group). Immunosuppression was basiliximab induction and maintenance was a calcineurin inhibitor and mycophenolate mofetil or sirolimus. Biopsy-proven acute rejection (BPAR) was treated by methylpredisolone. Surveillance biopsies were completed to evaluate subclinical acute rejection (SCAR) and chronic allograft nephropathy (CAN). Primary end point was acute rejection. Three-year patient and graft survival, new onset diabetes mellitus (NODM), serum creatinine and creatinine clearance were evaluated. Results. Acute rejection was diagnosed in 14% in control group and 16% in steroid withdrawal group. Three-year patient and graft survival was 89% and 79% in control and 91% and 78% in steroid withdrawal group. Serum creatinine and creatinine clearance was 1.9±0.8 and 59±11 in control group and 1.8±0.9 mg/dl and 61±10 mls/minute in steroid withdrawal group. Incidence of SCAR and progression of CAN were comparable in the 2 groups. At 3-years NODM was diagnosed in 21% in control group and 4% in steroid withdrawal group (P<0.01). Conclusions. Two-day steroid withdrawal in kidney transplant recipients did not affect BPAR, SCAR, CAN, graft function and patient and graft survival compared to control group up to 3 years. NODM was significantly less in steroid withdrawal group. Two-day steroid withdrawal is safe and beneficial in kidney transplant recipients.

Comparison of steroid avoidance in tacrolimus/mycophenolate mofetil and tacrolimus/ sirolimus combination in kidney transplantation monitored by surveillance biopsy

Background. Chronic steroid therapy in kidney transplantation has myriad side effects and steroid avoidance has become feasible. This prospective study compared the safety and efficacy of steroid avoidance in tacrolimus (TAC)/mycophenolate mofetil (MMF) and TAC/sirolimus (SRL) combinations in kidney transplantation. Methods. In all, 150 kidney recipients were analyzed: 75 each in TAC/MMF and TAC/SRL groups. The primary endpoint was acute rejection. Surveillance biopsies were completed to analyze subclinical acute rejection (SCAR) and chronic allograft nephropathy (CAN). Acute rejection and SCAR were treated by methylprednisolone. Two-year patient and graft survival, renal function, and adverse effects were monitored. Results. Acute rejection was seen in 12% of TAC/MMF and 8% of TAC/SRL patients. Two-year actuarial patient survival was 95% and 97%, and graft survival 90% and 90% in TAC/MMF and TAC/SRL groups, respectively. Surveillance biopsy showed cumulative incidence of SCAR was 27 % in TAC/MMF and 16 % in TAC/SRL groups at 2 years (P=0.04). Overall, 33% of recipients in TAC/MMF and 20% in TAC/SRL received methylprednisolone for acute rejection/SCAR. Moderate/severe CAN was 10% in TAC/SRL group and 22% in TAC/MMF group(P=0.06). New-onset diabetes mellitus (NODM) was 4% each in both groups. All recipients remain free of maintenance steroid therapy. Conclusions. Steroid avoidance in tacrolimus-based immunosuppression with MMF or SRL provides equivalent 2-year patient and graft survival with a low incidence of acute rejection and NODM. SCAR and CAN are lower in TAC/SRL compared to TAC/MMF group. The impact of decreased SCAR and CAN in TAC/SRL group on longer-term graft survival and function is to be evaluated.

Successful Transplantation of Kidneys from Deceased Donors with Acute Renal Failure: Three-year Results

Background. Kidneys from deceased donors with acute renal failure (ARF) are generally not accepted for transplantation because of the expected poor outcome. This prospective study examined the utilization of kidneys from donors with ARF for transplantation and the outcomes. Methods. Fifty-five kidneys from donors with ARF were transplanted. The outcome was compared with concurrent and matched 55 recipients of standard criteria donor (SCD) kidneys and 55 expanded criteria donor (ECD) kidneys. ARF kidneys were accepted from donors aged <50 years, a negative history for kidney disease, and a negative pretransplant biopsy for chronic structural changes. The immunosuppression was similar in all three groups. The outcome measurements included three-year patient and graft survival, biopsy-proven acute rejection (BPAR), subclinical acute rejection (SCAR), and chronic allograft nephropathy (CAN), serum creatinine, and creatinine clearance. Results. Three-year patient and graft survival was 90% and 90% in ARF group, 100% and 89% in SCD group and 83% and 66% in ECD group. BPAR and SCAR were comparable in the groups but CAN was significantly higher in ECD group. Mean serum creatinine levels were 1.9±1.1, 1.9±0.9, and 2.2±1.3 mg/dl and mean creatinine clearances were 66±15, 68±14, and 58±10 mls/minute in ARF, SCD, and ECD groups, respectively (SCD and ARF vs. ECD P=0.04). Conclusions. Transplantation of kidneys from selected deceased donors with ARF provides comparable survival and function compared to kidneys from non-ARF donors and may be considered for transplantation to expand the donor pool to overcome the current acute shortage of kidneys.

Steroid avoidance in renal transplantation using basiliximab induction, cyclosporine-based immunosuppression and protocol biopsies

Abstract:  Background:  Reducing chronic steroid exposure is important to minimize steroid‐related morbidity, particularly for susceptible renal transplant recipients. Steroid‐free and steroid‐sparing protocols have shown benefits, but safety has not been established for all populations. We investigated the safety of steroid avoidance (SA) in a population including African‐Americans, using modern immunosuppression with protocol biopsy monitoring.

Heart Transplantation–Associated Perioperative Ischemic Myocardial Injury Morphological Features and Clinical Significance

BACKGROUND The frequency and clinical significance of perioperative ischemic myocardial injury (PIMI) after heart transplantation and the diagnostic features distinguishing PIMI from rejection are not well defined. METHODS AND RESULTS We evaluated PIMI in the first four weekly endomyocardial biopsies and/or autopsy myocardium from 140 consecutive orthotopic heart transplantation recipients (1984 to 1991) by grading the severity of coagulative myocyte necrosis (CMN) as absent, 0; mild-focal, 1; moderate-multifocal, 2; or severe-confluent, 3, and determining the evolution of morphological features of its healing. CMN (often with contraction bands) was noted in 124 patients (89%); 24 patients (17%) had grade 3 CMN, of which 4 died within 30 days of transplantation. Nevertheless, at 1 year after surgery, survival was similar in patients with and without severe injury. Increased cold ischemic time but neither donor age nor intensity of inotropic support correlated with more severe early ischemic injury. PIMI inflammation was characterized by a predominantly polymorphonuclear/histiocytic infiltrate that contained lymphocytes and plasma cells, expanding the interstitium but not encroaching upon and separable from adjacent viable myocytes. Histological features of PIMI developed and resolved more slowly than those of typical myocardial infarct necrosis in nonimmunosuppressed patients; at 4 weeks, CMN persisted in 20% of patients and residual healing in nearly half. Diagnostic rejection was observed concurrently with PIMI in 54 of 533 biopsies (10%). CONCLUSIONS Diagnosed by conventional histological criteria, PIMI is prevalent early after heart transplantation and has a protracted healing phase that can mimic or coexist with rejection. Extensive PIMI has deleterious impact on short-term survival, but the long-term impact of PIMI remains to be established.