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Tissue-resident memory T cells (T RM ) maintain immunity in diverse sites as determined in mouse models, whereas their establishment and role in human tissues have been difficult to assess. Here, we investigated human lung T RM generation, maintenance, and function in airway samples obtained longitudinally from human leukocyte antigen (HLA)-disparate lung transplant recipients, where donor and recipient T cells could be localized and tracked over time. Donor T cells persist specifically in the lungs (and not blood) of transplant recipients and express high levels of T RM signature markers including CD69, CD103, and CD49a, whereas lung-infiltrating recipient T cells gradually acquire T RM phenotypes over months in vivo. Single-cell transcriptome profiling of airway T cells reveals that donor T cells comprise two T RM -like subsets with varying levels of expression of T RM -associated genes, whereas recipient T cells comprised non-T RM and similar T RM -like subpopulations, suggesting de novo T RM generation. Transplant recipients exhibiting higher frequencies of persisting donor T RM experienced fewer adverse clinical events such as primary graft dysfunction and acute cellular rejection compared with recipients with low donor T RM persistence, suggesting that monitoring T RM dynamics could be clinically informative. Together, our results provide spatial and temporal insights into how human T RM develop, function, persist, and affect tissue integrity within the complexities of lung transplantation., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Allograft failure is common in lung-transplant recipients and leads to poor outcomes including early death. No reliable clinical tools exist to identify patients at high risk for allograft failure. This study tested the use of donor-derived cell-free DNA (%ddcfDNA) as a sensitive marker of early graft injury to predict impending allograft failure., Methods: This multicenter, prospective cohort study enrolled 106 subjects who underwent lung transplantation and monitored them after transplantation for the development of allograft failure (defined as severe chronic lung allograft dysfunction [CLAD], retransplantation, and/or death from respiratory failure). Plasma samples were collected serially in the first three months following transplantation and assayed for %ddcfDNA by shotgun sequencing. We computed the average levels of ddcfDNA over three months for each patient (avddDNA) and determined its relationship to allograft failure using Cox-regression analysis., Findings: avddDNA was highly variable among subjects: median values were 3·6%, 1·6% and 0·7% for the upper, middle, and low tertiles, respectively (range 0·1%-9·9%). Compared to subjects in the low and middle tertiles, those with avddDNA in the upper tertile had a 6·6-fold higher risk of developing allograft failure (95% confidence interval 1·6-19·9, p = 0·007), lower peak FEV1 values, and more frequent %ddcfDNA elevations that were not clinically detectable., Interpretation: Lung transplant patients with early unresolving allograft injury measured via %ddcfDNA are at risk of subsequent allograft injury, which is often clinically silent, and progresses to allograft failure. FUND: National Institutes of Health., (Published by Elsevier B.V.)

Background: Antibody-mediated rejection (AMR) often progresses to poor health outcomes in lung transplant recipients (LTRs). This, combined with the relatively insensitive clinical tools used for its diagnosis (spirometry, histopathology) led us to determine whether clinical AMR is diagnosed significantly later than its pathologic onset. In this study, we leveraged the high sensitivity of donor-derived cell-free DNA (ddcfDNA), a novel genomic tool, to detect early graft injury after lung transplantation., Methods: We adjudicated AMR and acute cellular rejection (ACR) in 157 LTRs using the consensus criteria of the International Society for Heart and Lung Transplantation (ISHLT). We assessed the kinetics of allograft injury in relation to ACR or AMR using both clinical criteria (decline in spirometry from baseline) and molecular criteria (ddcfDNA); percent ddcfDNA was quantitated via shotgun sequencing. We used a mixed-linear model to assess the relationship between and ddcfDNA levels and donor-specific antibodies (DSA) in AMR + LTRs., Results: Compared with ACR, AMR episodes (n = 42) were associated with significantly greater allograft injury when assessed by both spirometric (0.1 liter vs -0.6 liter, p < 0.01) and molecular (ddcfDNA) analysis (1.1% vs 5.4%, p < 0.001). Allograft injury detected by ddcfDNA preceded clinical AMR diagnosis by a median of 2.8 months. Within the same interval, spirometry or histopathology did not reveal findings of allograft injury or dysfunction. Elevated levels of ddcfDNA before clinical diagnosis of AMR were associated with a concurrent rise in DSA levels., Conclusion: Diagnosis of clinical AMR in LTRs lags behind DSA-associated molecular allograft injury as assessed by ddcfDNA., (Copyright © 2018. Published by Elsevier Inc.)

Background:  This study examines the effects of the empty-and-refill patency test on rat femoral arteries in the longer postoperative time period., Methods:  A simple arterial anastomosis was performed bilaterally on 20 rats. The empty-and-refill test was performed unilaterally in all rats, leaving the contralateral artery as an internal control. Rats were divided into two cohorts of 10 rats and survived for 48 hours and 2 weeks. Vessel patency was assessed prior to closing and immediately prior to sacrifice. The femoral arteries were harvested bilaterally and hematoxylin and eosin stains were performed. The femoral artery distal to the anastomosis in the region of the empty-and-refill test was histologically evaluated., Results:  All vessels were patent at the time of sacrifice. There was no statistical difference in the numeric scoring between the experimental and control vessels in the 48-hour cohort. Almost all vessels harvested at 48 hours showed endothelial cell loss distal to the anastomosis regardless of whether they underwent the empty-and-refill test. The only statistically significant difference in the 2-week cohort was an increase in adventitial smooth muscle proliferation in the experimental group. There were no other statistically significant results between the experimental and control groups at 2 weeks. An overall comparison of both cohorts revealed a statistically significant increase in endothelial cell number and intimal proliferation by 2 weeks postsurgery., Conclusion:  The empty-and-refill test does not compromise rat femoral artery anastomotic patency, nor does it produce histological damage either 48 hours or 2 weeks postsurgery., Competing Interests: Authors A.N., R.N., J.G.L., Y.A., and C.M. report grants from Orthopedic Scientific Research Foundation, during the conduct of the study. Author R.S. reports no conflict of interest., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Background: Cardiac allograft vasculopathy (CAV) has been associated with graft-infiltrating B cells, although their characteristics are still unclear. In this study we examined the frequency, localization and reactivity profile of graft-infiltrating B cells to determine their contribution to the pathophysiology of CAV., Methods: B cells, plasma cells and macrophages were examined by immunohistochemistry in 56 allografts with CAV, 49 native failed hearts and 25 autopsy specimens. A total of 102 B-cell clones were immortalized directly from the infiltrates of 3 fresh cardiac samples with CAV. Their secreted antibodies were assessed using enzyme-linked immunoassay and flow cytometry., Results: B-cell infiltration was observed around coronary arteries in 93% of allograft explants with CAV. Comparatively, intragraft B cells were less frequent and less dense in the intraventricular myocardium from where routine biopsies are obtained. Plasma cells and macrophages were also detected in 85% and 95% of explants, respectively. Remarkably, B-cell infiltrates were not associated with circulating donor-specific antibodies (DSA) or prior episodes of antibody-mediated rejection (AMR). Among all B-cell clones generated from 3 explants with CAV, a majority secreted natural antibodies reactive to multiple autoantigens and apoptotic cells, a characteristic of innate B cells., Conclusions: Our study reveals a high frequency of infiltrating B cells around the coronary arteries of allografts with CAV, independent of DSA or AMR. These cells are enriched for innate B cells with a polyreactive profile. The findings shift the focus from conventional DSA-producing B cells to the potentially pathogenic polyreactive B cells in the development of clinical CAV., (Copyright © 2018 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Use of new genomic techniques in clinical settings requires that such methods are rigorous and reproducible. Previous studies have shown that quantitation of donor-derived cell-free DNA (%ddcfDNA) by unbiased shotgun sequencing is a sensitive, non-invasive marker of acute rejection after heart transplantation. The primary goal of this study was to assess the reproducibility of %ddcfDNA measurements across technical replicates, manual vs automated platforms, and rejection phenotypes in distinct patient cohorts., Methods: After developing and validating the %ddcfDNA assay, we subjected the method to a rigorous test of its reproducibility. We measured %ddcfDNA in technical replicates performed by 2 independent laboratories and verified the reproducibility of %ddcfDNA patterns of 2 rejection phenotypes: acute cellular rejection and antibody-mediated rejection in distinct patient cohorts., Results: We observed strong concordance of technical-replicate %ddcfDNA measurements across 2 independent laboratories (slope = 1.02, R 2 > 0.99, p < 10 -6 ), as well as across manual and automated platforms (slope = 0.80, R 2 = 0.92, p < 0.001). The %ddcfDNA measurements in distinct heart transplant cohorts had similar baselines and error rates. The %ddcfDNA temporal patterns associated with rejection phenotypes were similar in both patient cohorts; however, the quantity of ddcfDNA was significantly higher in samples with severe vs mild histologic rejection grade (2.73% vs 0.14%, respectively; p < 0.001)., Conclusions: The %ddcfDNA assay is precise and reproducible across laboratories and in samples from 2 distinct types of heart transplant rejection. These findings pave the way for larger studies to assess the clinical utility of %ddcfDNA as a marker of acute rejection after heart transplantation., (Copyright © 2017. Published by Elsevier Inc.)

Eosinophilic infiltration of the myocardium is occasionally observed as an incidental histological finding in endomyocardial biopsy specimens before heart transplantation (HTx) as well as in explanted heart obtained at the time of HTx. However, the indications for HTx in these patients have not yet been fully established. We investigated the pre-HTx characteristics of the recipients with myocardial eosinophilic infiltration in the explanted heart and diagnosed as hypersensitivity myocarditis (HSM) (21 among 761 recipients, 2.8%). Dobutamine, a common cause of HSM, was administered to 12 patients (57%). Ten patients (47.6%) were on milrinone and 4 (19.0%) were on ventricular assist devices. Post-transplant survival of HSM patients was comparable to that of patients transplanted for active myocarditis or other cause of heart failure. In conclusion, myocardial eosinophilic infiltration is associated with multiple medications in patients with advanced heart failure; however, it does not affect the post-transplant prognosis.

Cardiac transplantation is a treatment option for patients with hypertrophic cardiomyopathy (HC) who developed refractory heart failure and/or intractable arrhythmia. However, the pretransplant characteristics and post-transplant prognosis for patients with nondilated idiopathic HC has not yet fully elucidated. Therefore, we retrospectively reviewed 813 consecutive transplant recipients undergoing cardiac transplantation at Columbia University Medical Center from 1999 to 2010 and compared the clinical course of 41 patients with idiopathic HC with that of 373 patients with ischemic heart disease and 398 patients with other heart disease. The patients with HC were younger than those with ischemic heart disease (47.8 ± 14.0 vs 57.1 ± 9.4 years; p <0.0001). The proportion of patients undergoing ventricular assist devise surgery for bridge-to-transplant was lower in patients with HC than in those with ischemic heart disease or other heart disease (14.6% vs 31.1% vs 35.7%, all p <0.01). The post-transplant survival of those with HC was better than that for those with ischemic heart disease (90.1% vs 85.8% and 83.9% vs 67.1% at 1 and 5 years, respectively; p = 0.0359), although it was not significantly different from those with other heart disease. Proportional hazards analysis revealed that the subjects with HC had reduced post-transplant mortality (hazard ratio 0.4760, 95% confidential interval 0.1889 to 0.9762; p = 0.042) on univariate, but not multivariate, analysis. Most patients with HC had nondilated left ventricles (left ventricular end-diastolic dimension ≤ 55 mm; n = 27), and post-transplant survival did not differ from that for those with dilated left ventricles (left ventricular end-diastolic dimension >55 mm; n = 14). In conclusion, the post-transplant survival of those with HC did not differ from those of the subjects who underwent transplant for other non-HC indications., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We sought to delineate further the immunological significance of T lymphocytes infiltrating the valve leaflets in calcific aortic stenosis (CAS) and determine whether there were associated alterations in circulating T cells. Using clonotypic TCR β-chain length and sequence analysis we confirmed that the repertoire of tricuspid CAS valves contains numerous expanded T cell clones with varying degrees of additional polyclonality, which was greatest in cases with severe calcification. We now report a similar proportion of clonal expansions in the much younger bicuspid valve CAS cases. Peripheral blood flow cytometry revealed elevations in HLA-DR(+) activated CD8 cells and in the CD8(+)CD28(null)CD57(+) memory-effector subset that were significantly greater in both bicuspid and tricuspid CAS cases with more severe valve calcification. Lesser increases of CD4(+)CD28(null) T cells were identified, principally in cases with concurrent atherosclerotic disease. Upon immunostaining the CD8 T cells in all valves were mainly CD28(null), and CD8 T cell percentages were greatest in valves with oligoclonal repertoires. T cell clones identified by their clonotypic sequence as expanded in the valve were also found expanded in the circulating blood CD28(null)CD8(+) T cells and to a lesser degree in the CD8(+)CD28(+) subset, directly supporting the relationship between immunologic events in the blood and the valve. The results suggest that an ongoing systemic adaptive immune response is occurring in cases with bicuspid and tricuspid CAS, involving circulating CD8 T cell activation, clonal expansion, and differentiation to a memory-effector phenotype, with trafficking of T cells in expanded clones between blood and the valve.

The involvement of humoral response in allograft rejection has been suggested by both immunologic and histochemistry studies. In the present study, we explored the role of alloantibodies in a large cohort of heart allograft recipients followed for 15 years. Sequential samples of sera were obtained from 950 recipients of heart allografts before and after transplantation at the time when protocol endomyocardial biopsies were performed. The presence of anti-human leukocyte antigen (HLA) antibodies was investigated using complement mediated cytotoxicity and solid phase assay (SPA). Our data reveal an inverse correlation between the development of alloantibodies after transplantation and heart allograft survival. The 15-year graft survival was highest in patients who never developed alloantibodies (70%) or who displayed them only before transplantation (71%); graft survival in recipients who showed antibodies both before and after transplantation (56%), or only after transplantation (47%), was lower. The deleterious effect of antibodies on graft survival started 8 years after transplantation, suggesting that the production of de novo antibodies may have been triggered by some later event. We found that patients with de novo antibodies appearing more than 1 year after transplantation had the poorest survival. Furthermore, the development of de novo antibodies was preceded in 76% of these patients by cellular rejection grade 3 or higher, according to the International Society for Heart Transplantation (ISHT) grading criteria. Development of antibody-mediated rejection (AMR) had a significant negative impact on graft survival (16% in AMR(+) vs 63% in AMR(-) patients, p = 0.0008). Of the 23 patients with AMR, 21 displayed cytotoxic donor-specific antibodies (DSA) at the time of diagnosis, and in 18 of these cases SPA showed that they were directed against the donors' HLA. The data demonstrate that the detection of alloantibodies permits a better definition of AMR in heart allograft recipients. Identification of patients at risk for developing AMR is of great importance for early treatment of rejection episodes., (Copyright © 2011 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Transplant allograft vasculopathy (TAV) was a leading cause of death in cardiac transplant recipients after the first year of transplantation. Whether drug-eluting stents (DESs) performed better than bare-metal stents (BMSs) for the treatment of patients with discrete epicardial stenosis was unknown. The aim was to determine the safety and efficacy of DESs compared with BMSs in the treatment of patients with TAV. Outcomes of 32 patients sequentially treated using DESs for TAV were retrospectively reviewed and compared with a historic cohort of 35 patients treated sequentially with BMSs for TAV. Patients treated with DESs were also compared with age- and gender-matched cardiac transplant controls to determine differences in survival. After adjustment for baseline risk factors, there was no difference in 1-year survival between patients treated with DESs or BMSs for TAV. Restenosis rates at 1 year were 49% in lesions treated using BMSs and 19% in those treated using DESs. Compared with an age- and gender-matched control group of cardiac transplant patients who did not have discrete obstructive epicardial TAV, patients who required treatment with DESs for epicardial obstructive disease had significantly worse survival. In conclusion, treatment of patients with TAV with DESs did not seem to alter the natural deleterious history of this disease process.

Mitral regurgitation, when it arises from functional restriction of mitral leaflet closure, can be relieved by surgical cutting of the mitral tendineae chordae. We hypothesized that high intensity focused ultrasound (HIFU) might be useful as a noninvasive extracorporeal technique for cutting mitral chordae. As a pilot study to test this hypothesis, we examined the in vitro feasibility of using HIFU to cut calf mitral chordae with diameters from 0.2 to 1.6 mm. Sixty-seven percent of chordae were completely cut with HIFU, operated at 4.67 MHz and 45 W acoustic power, with up to 120 pulses of 0.3-s duration at 2-s intervals. Forty-five percent were completely cut when the pulse duration was reduced to 0.2 s. The average diameter of those chordae, which were completely cut, was significantly smaller than that of incompletely cut chordae (0.59 +/- 0.30 versus 1.14 +/- 0.30 mm with a pulse duration of 0.2 s, p < 0.0001; 0.68 +/- 0.29 versus 1.32 +/- 0.20 mm with a pulse duration of 0.3 s, p < 0.0001). For each pulse duration, the number of pulses required for complete cutting exhibited a strong positive correlation with the chordae diameter. In conclusion, in vitro feasibility of mitral chordal cutting by HIFU depended on the diameter of chordae but was controllable by HIFU settings. (E-mail: abeyukio@aol.com).

Background: High-intensity focused ultrasound (HIFU) produces immediate focal lesions without direct tissue contact. Previously, we reported the HIFU potential for cardiac ablation. The purpose of this study was to evaluate the possibility of myocardial ablation in the left ventricle of beating dog hearts with monitoring by 2-dimensional echocardiography., Methods: The operating frequency and the acoustic intensity were 5.25 MHz and 23 kW/cm(2), and the focal length and diameter were 3.3 mm axial and 0.37 mm wide at a distance of 35 mm from the transducer. Three dogs underwent a left-sided thoracotomy. The right ventricular surface was coupled with the transducer. The timing of the HIFU exposure was set during the early systolic phase using an electrocardiographic triggering system. The focal point was set in the left ventricular septum using 2-dimensional echocardiography mounted in the HIFU transducer. Ultrasound energy was delivered for 0.2 seconds. For each dog, we created 18 lesions. Exposures were performed 20, 30, or 40 times. Lesion size was assessed by manually measuring its length and width., Results: All lesions except one were clearly visible. The histologic lesion area was 18.7 +/- 8.3, 26.3 +/- 8.7, and 35.5 +/- 15.7 mm(2) (20, 30, and 40 times, respectively). The intraclass correlation coefficients were found to be 0.72, 0.63, 0.75, and 0.73 for lesion length, width, area, and depth, respectively., Conclusion: HIFU can be used to create targeted, well-demarcated thermal lesions in the ventricular septum myocardium during cardiac contraction.

Background: Most humoral rejection (HR) episodes occur early after cardiac transplantation and are associated with hemodynamic compromise and poor prognosis. Late cases of HR (>6 months after transplant) have been reported. We examined the differences in clinical characteristics and outcomes in patients presenting with HR in the early (<6 months) and late transplant periods., Methods: A retrospective chart review was performed of all cases of HR at a single large transplant center from January 1, 1995 to March 1, 2006., Results: A total of 37 adult transplants had biopsy-proven HR; 13 patients had early HR and 24 patients had HR a mean of 5 yr after transplantation (range, 7 months to 17 yrs). Treatment for HR included plasmapheresis, cyclophosphamide, and rituximab. The age of the early and late humoral rejecters was similar (58+/-14 vs. 50+/-14 yrs; P=0.12). There was a trend toward more women in the early HR group (54% vs. 33%). Use of left ventricular assist devices was similar (38% vs. 33%). Early rejecters were more likely to have positive cross-matches (46% vs. 8%; P<0.01). Patients with late HR had a coexistent diagnosis of malignancy, or significant recent infection in 50% vs. 8% for early HR, suggesting an activation of a nonhuman leukocyte antigen antibody-mediated immune response to an acute illness. One-year survival after the diagnosis of HR was 78% for the both groups (P=NS)., Conclusions: Humoral rejection occurs now more frequently in patients with remote transplants and is commonly associated with the presence of malignancy or infection.

Background: Systemic amyloidosis complicated by heart failure is associated with high cardiovascular morbidity and mortality. Heart transplantation for patients with systemic amyloidosis is controversial due to recurrence of disease in the transplanted organ or progression of disease in other organs., Methods: All patients with systemic amyloidosis and heart failure referred for heart transplant evaluation from 1997 to 2004 were included in this retrospective cohort analysis. An interdisciplinary protocol for cardiac transplantation using extended-donor criteria organs, followed in 6 months by either high-dose chemotherapy and stem cell transplantation for patients with primary (AL) or by orthotopic liver transplantation for familial (ATTR) amyloidosis, was developed. Survival of the transplanted amyloid cohort was compared to survival of those amyloid patients not transplanted and to patients transplanted for other indications., Results: A total of 25 patients with systemic amyloidosis and heart failure were included in the study; 12 patients received heart transplants. Amyloid heart transplant recipients were more likely female (58% vs. 8%, P=0.02) and had lower serum creatinine (1.3+/-0.5 vs. 2.0+/-0.7 mg/dL, P=0.01) than nontransplanted amyloid patients. Survival at 1-year after heart transplant evaluation was higher among transplanted patients (75% vs. 23%) compared to patients not transplanted (P=0.001). Short-term survival posttransplant did not differ between transplanted amyloid patients and contemporaneous standard and extended-donor criteria heart transplant patients (P=0.65)., Conclusions: Cardiac transplantation for amyloid patients with extended-donor criteria organs followed by either stem cell or liver transplantation is associated with improved survival compared to patients not transplanted. Short- to intermediate-term survival is similar to patients receiving heart transplantation for other indications. This clinical management strategy provides cardiac amyloid patients a novel therapeutic option.

The purpose of this study was to evaluate the possibility of using high-intensity focused ultrasound (US), or HIFU, to create lesions in cardiac valves in vitro. Calf mitral valves and aortic valves were examined. Focused US energy was applied with an operating frequency of 4.67 MHz at a nominal acoustic power of 58 W for 0.2, 0.3 and 0.4 s at 4-s intervals. Mitral valve perforation was achieved with 20.8 +/- 3.7 exposures of 0.2 s, 15.4 +/- 2.1 exposures of 0.3 s or 11.2 +/- 2.3 exposures of 0.4 s. Aortic valve perforation was achieved with 13.3 +/- 2.4 exposures of 0.2 s, 10.3 +/- 2.2 exposures of 0.3 s or 8.4 +/- 1.8 exposures of 0.4 s. The mean diameter of the perforated area was 1.09 +/- 0.11 mm. The lesions were slightly discolored and coagulation of tissue around the perforation was observed. HIFU was successful in perforating cardiac valves. With further refinement, HIFU may prove useful for valvulotomy or valvuloplasty.

Endothelial cells have been shown to activate T cell responses to alloantigens, triggering transplant rejection. However, they may also play a role in tolerance induction. Using RT-PCR we show here that alloantigen specific CD8(+)CD28(-) T suppressor cells generated in vitro are FOXP3 positive and interact with human endothelial cells. This interaction results in the induction of inhibitory receptors and down-regulation of costimulatory and adhesion molecules, thus rendering endothelial cells tolerogenic. In turn, tolerized endothelial cells elicit the differentiation of CD8(+)CD28(-) FOXP3(+) T suppressor cells. Taken together our data demonstrate a functional and phenotypic overlap between tolerogenic dendritic cells and endothelial cells. Furthermore, alloantigen specific CD8(+)CD28(-) FOXP3(+) T cells, which trigger the upregulation of inhibitory receptors in endothelial cells, are present in the circulation of heart allograft recipients in quiescence as demonstrated by flow cytometry, RT-PCR and luciferase transcription assays. Their detection facilitates the identification of patients who may benefit from partial or complete cessation of immunosuppressive therapy, a goal of obvious importance given the morbidity and mortality associated with chronic immunosuppression. Modulation of endothelial cells in favor of promoting tolerance may be important for long-term survival of organ allografts.

We have analyzed the relationship between the development of transplant-related coronary artery disease (TRCAD) and the following potential risk factors: (a). number of HLA mismatches between recipient and donor; (b). production of anti-HLA antibodies; (c). growth of lymphocytes infiltrating the graft; and (d). frequency of biopsy proven episodes of acute rejection. The study population consisted of 285 adult heart allograft recipients who were monitored over a period of two years or more. The results demonstrate a significant correlation between TRCAD, generation of anti-HLA class II antibodies and potential of lymphocytes infiltrating the graft to proliferate ex-vivo in medium containing IL-2. Humoral and cellular immune responses to HLA-DR antigens expressed by the graft seem to underlie the development of TRCAD.

Background: Cyclosporine-based immunosuppressive regimens (INN: ciclosporin) in human lung transplantation continue to result in a high incidence of acute cellular rejection. We investigated the use of sirolimus, a macrolide with structural similarity to tacrolimus, as monotherapy and in combination with cyclosporine in a rodent lung transplant model., Methods: Orthotopic left lung transplantation was performed in Lewis recipients from Brown-Norway donor rats with syngeneic Lewis-to-Lewis controls. Open biopsies were performed on postoperative day 7, and the severity of acute lung rejection was graded by a pathologist blinded to the protocol., Results: All recipients survived despite the amount of acute rejection seen on examination of the biopsy tissue. Lewis-to-Lewis isografts demonstrated near normal pulmonary architecture. Allogeneic recipients receiving high-dose cyclosporine (25 mg/kg) monotherapy showed mild to moderate acute rejection with some perivascular focal interstitial infiltrates. Recipients receiving low-dose cyclosporine (5 mg/kg) monotherapy or low- or high-dose sirolimus (0.5 or 2.0 mg/kg, respectively) monotherapy demonstrated massive cellular infiltration leading to necrosis and infarction and could not be graded. However, the addition of low-dose sirolimus (0.5 mg/kg) to low-dose cyclosporine (5 mg/kg) demonstrated a significant potentiating immunosuppressive effect, and the addition of high-dose sirolimus (2.0 mg/kg) to low-dose cyclosporine (5.0 mg/kg) demonstrated an even greater effect, with rejection scores better than those obtained with high-dose cyclosporine monotherapy and similar to those obtained with isografts., Conclusions: This study demonstrates that low-dose sirolimus has a cyclosporine-sparing effect and that a higher dose of sirolimus in combination with cyclosporine strongly protects lung allografts from acute cellular rejection. These results suggest that sirolimus may be indicated as an adjunct to current cyclosporine-based immunosuppressive regimens in clinical lung transplantation.

Background: Photopheresis is an immunoregulatory technique in which lymphocytes are reinfused after exposure to a photoactive compound (methoxsalen) and ultraviolet A light. We performed a preliminary study to assess the safety and efficacy of photopheresis in the prevention of acute rejection of cardiac allografts., Methods: A total of 60 consecutive eligible recipients of primary cardiac transplants were randomly assigned to standard triple-drug immunosuppressive therapy (cyclosporine, azathioprine, and prednisone) alone or in conjunction with photopheresis. The photopheresis group received a total of 24 photopheresis treatments, each pair of treatments given on two consecutive days, during the first six months after transplantation. The regimen for maintenance immunosuppression, the definition and treatment of rejection episodes, the use of prophylactic antibiotics, and the schedule for cardiac biopsies were standardized among all 12 study centers. All the cardiac-biopsy samples were graded in a blinded manner at a central pathology laboratory. Plasma from the subgroup of 34 patients (57 percent) who were enrolled at the nine U.S. centers was analyzed by polymerase-chain-reaction amplification for cytomegalovirus DNA., Results: After six months of follow-up, the mean (+/-SD) number of episodes of acute rejection per patient was 1.44+/-1.0 in the standard-therapy group, as compared with 0.91+/-1.0 in the photopheresis group (P=0.04). Significantly more patients in the photopheresis group had one rejection episode or none (27 of 33) than in the standard-therapy group (14 of 27), and significantly fewer patients in the photopheresis group had two or more rejection episodes (6 of 33) than in the standard-therapy group (13 of 27, P=0.02). There was no significant difference in the time to a first episode of rejection, the incidence of rejection associated with hemodynamic compromise, or survival at 6 and 12 months. Although there were no significant differences in the rates or types of infection, cytomegalovirus DNA was detected significantly less frequently in the photopheresis group than in the standard-therapy group (P=0.04)., Conclusions: In this pilot study, the addition of photopheresis to triple-drug immunosuppressive therapy significantly decreased the risk of cardiac rejection without increasing the incidence of infection.

Background: Osteopontin (OP) has been identified in cultured rat cardiac fibroblasts, where it contributes to angiotensin II (AII)-induced remodeling processes; in cultured cardiomyocytes; and in macrophages in cardiac tissues with inflammation. However, the presence of OP has not been reported in histological sections of myocardial tissue. In the present study, we investigated (1) the regulation of OP mRNA expression in cultured rat cardiomyocytes; (2) the localization of OP mRNA in neonatal and adult normal and hypertrophied rat hearts; and (3) the histology of OP expression in myocardial specimens from humans either with myocyte hypertrophy or with no pathological changes., Methods and Results: Cultured neonatal cardiomyocytes expressed OP mRNA and were immunoreactive for OP. Endothelin-1 (ET-1) and norepinephrine (NE) increased both OP and atrial natriuretic peptide (ANP) mRNA levels twofold to threefold (P<.01). OP mRNA was prominent in ventricular tissue from neonatal and adult rats with renovascular hypertension and aortic banding, whereas barely detectable levels were observed in normal adult cardiac tissue. ANP and OP mRNA levels in normal and hypertrophied ventricles correlated (r2=.87, P<.001). OP immunoreactivity and mRNA transcripts were predominantly found in cardiomyocytes not associated with inflammatory cells in sections from neonatal and adult hypertrophied hearts. No staining was detectable in normal adult hearts. Human myocardium with extensive fibrosis and cardiomyocyte hypertrophy obtained from explanted hearts with either idiopathic (n=5) or ischemic cardiomyopathy (n=7) demonstrated substantial myocyte immunoreactivity for both OP and ANP in right and left ventricles that was not associated with leukocyte infiltration. In situ hybridization identified cardiomyocytes as the major source of OP mRNA transcripts in these hearts. In contrast, OP immunoreactivity was not detectable in four of five endomyocardial biopsies with normal histology., Conclusions: The present study provides the first evidence that cardiomyocytes are a prominent source of OP in vivo and suggests that induction of OP expression is strongly associated with ventricular hypertrophy.

Vascular endothelial cells act as antigen-presenting cells in the lung allograft and stimulate alloreactive host lymphocytes. Activated lymphocytes and cytokines can induce expression of leukocyte-endothelial adhesion molecules that facilitate invasion of the allograft by circulating leukocytes. To define the role of endothelial HLA class II antigen and adhesion molecule expression in lung allograft rejection, we prospectively analyzed endothelial expression of HLA class II, E-selectin, and intercellular adhesion molecule-1 (ICAM-1) antigens in 52 transbronchial biopsy specimens from 24 lung allograft recipients as compared to normal control subjects. Thirty-one of 52 specimens showed histologic rejection and 8 of 24 patients developed histologic obliterative bronchiolitis (OB) by the end of the study period. Increased expression of HLA class II antigen was seen in 32 of 52 (62%) lung allograft specimens, but increased expression did not correlate with acute rejection or OB. In contrast, E-selectin expression was seen in 30 of 52 (58%) biopsy specimens and was associated with acute rejection (p < 0.005) and with the development of OB (p < 0.05). Increased expression of ICAM-1 was seen in only 18 of 52 (35%) biopsy specimens and did not correlate with acute rejection or OB. These data suggest that E-selectin expression may be a tissue marker of acute and chronic lung rejection possibly by promoting leukocyte adhesion to the allograft endothelium. The high levels of endothelial HLA class II expression may reflect long-term antigenic stimulation of the allograft even in the absence of rejection.

Background: The purpose of the present study was to examine the histological consequences of the endoluminal exclusion of blood flow effected with stent graft technology., Methods and Results: In 25-kg mongrel dogs, patulous vein patch infrarenal aortoplasty with iliac vein produces a fusiform abdominal aortic dilation (AAD). All aortic tributaries were preserved. Endoluminal exclusion via transfemoral placement of a thin-wall Dacron graft occurred 4 +/- 2 months later (n = 23). Balloon-expandable stents anchored the ends of the graft to the aorta. Hematoxylin and eosin, elastin van Gieson's, and Masson's trichrome staining was performed 6 and 12 months later at death. In control nongrafted AADs, the arterial portion of the AAD was lined by elastin -and collagen-rich intimal hyperplasia, and the venous portion developed medial hyperplasia containing collagen but little elastin. After stent graft placement, the stent struts and the graft were completely incorporated into an elastin-poor, collagen-rich neointima. Fibrosis of the vein patch was observed at 1 year. laminated thrombus did not form in the AAD until immediately after stent graft placement; flow arrest occurred in the space between the graft and the AAD intima despite the patent tributaries. At 6 and 12 months, microscopic recanalization was seen in this thrombus, although macroscopic flow was not discernible by duplex imaging or angiography. No AAD growth was measured., Conclusions: Aortic dilation was not observed at 1 year after stent graft placement within AADs with patent side branches despite microscopic evidence of thrombus recanalization. A collagen-rich and elastin-poor neointima incorporated the entire stent graft.

As the recipient list for patients requiring lung transplantation continues to increase, cadaveric donor lung availability has remained static. Our experience with utilizing lobes from living related donors for bilateral pulmonary transplantation in 20 patients has yielded a 75% survival at 1 year follow-up. Morbidity and mortality have been predominately due to infection. Rejection episodes have been mild and unilateral and have responded to augmented corticosteroids. Pulmonary function tests in the recipients tend to improve steadily during the first year postoperatively, and the patients have excellent functional capacity. There have been no significant complications in the donors. On the basis of our clinical experience, we have found that bilateral lobar transplantation utilizing living related donors has resulted in organ availability that can be lifesaving in critically ill patients and can provide a good alternative in certain noncritical, deteriorating patients.

Background: The goal of this study was to define the regulation of nitric oxide release by coronary microvessels from the failing and nonfailing human heart and to determine the role of local kinin production in the elaboration of nitric oxide by human coronary microvascular endothelium., Methods and Results: Ten hearts from humans with end-stage heart failure and two hearts from patients without heart failure were harvested at the time of orthotopic cardiac transplantation. Microvessels were sieved and the production of nitrite was determined by the Griess reaction. Microvessels were incubated in the presence of agonists for nitric oxide production (acetylcholine and bradykinin), which caused dose-dependent increases in nitrite, a response that was blocked by NG-nitro-L-arginine methyl ester and receptor-specific antagonists (atropine and HOE 140, respectively). In addition, the production of nitrite by microvessels from the failing heart appeared to be less than that produced by microvessels from the nonfailing heart. Incubation with norepinephrine or the alpha2-adrenergic agonist BHT 920 also caused dose-dependent increases in nitrite production, which were blocked by the B2-receptor antagonist HOE 140. This implicated local kinin synthesis as an intermediate step in the production of nitric oxide in response to alpha2-adrenoceptor stimulation. The production of nitric oxide was also prevented by the addition of serine protease inhibitors, which blocked the action of local kallikrein, again suggesting a role for local kinin synthesis., Conclusions: Our results indicate that nitric oxide is produced by human coronary microvessels, that nitric oxide production may be reduced but certainly not increased in microvessels from the failing human heart, and that there is active local kinin generation in these blood vessels.

The aim of this study was to assess the feasibility of imaging cerebral arteries in vitro with intravascular ultrasound and to establish a correlation between echographic images and corresponding histological architecture. Intravascular ultrasound imaging was performed using a 30-MHz, 4.3F ultrasound probe. Twenty-two arterial segments were obtained at autopsy from 6 patients and were imaged fresh. Arteries were then processed for histological examination and comparisons were made between echographic and histological findings. The correlation between luminal area measurements as determined histologically and by intravascular ultrasound was tested by linear regression analysis. Intravascular ultrasound demonstrated a three-layered appearance in normal cerebral arteries but not in those affected by severe atherosclerosis. Overall, ultrasound correctly identified the presence of a plaque in 83% of patients. Intravascular ultrasound sensitivity and specificity, respectively, were 100 and 80% for calcium deposits and 83 and 75% for fibrous tissue. Intravascular ultrasound and histological measurements correlated well for the determination of luminal area (r = 0.89). Intravascular ultrasound provides accurate characterization of the arterial lumen and geometry, as well as the presence and histological features of atherosclerotic plaque. Thus, it appears to have a great potential for an earlier and more accurate diagnosis of atherosclerosis and may serve to guide new interventional techniques being utilized in the treatment of cerebrovascular diseases.

The interaction of T cell costimulatory molecules with their ligands is required for optimal T cell activation. Interference with such interactions can induce antigen unresponsiveness and delay xeno- and allograft rejection. We have previously shown that LFA3TIP, a soluble human lymphocyte function-associated antigen (LFA)-3 construct, binds CD2 and inhibits responses of human T cells in vitro. This study reports the first use of a human fusion protein, LFA3TIP, to significantly prolong primate cardiac allograft survival. Based on our observations that LFA3TIP inhibits baboon allogeneic mixed lymphocyte reactions, we gave baboon recipients of heterotopic cardiac allografts injections of LFA3TIP, 3 mg/kg i.v., for 12 consecutive days, starting 2 days before transplantation. This regimen delayed graft rejection from an average of 10.6 +/- 2.3 days for human IgG-treated controls (n = 5) to an average of 18.0 +/- 5.3 days for LFA3TIP-injected animals (n = 7; P < or = 0.01). Grafts from LFA3TIP-treated animals showed markedly diminished coronary endothelialitis as compared with control animals. LFA3TIP reached peak serum levels of approximately 100 micrograms/ml after 7-9 injections and persisted in the 10-micrograms/ml range for 1 to 2 weeks after the final injection. Despite these blood levels, circulating antibodies to LFA3TIP were not detected in the serum. No renal or hepatic toxicity was noted. The possible mechanism by which LFA3TIP acts to inhibit graft rejection is discussed; success in prolonging graft survival when LFA3TIP is used as a single-agent therapy suggests great potential for this novel therapeutic agent.

As lung transplant patients are being returned more quickly to their own communities and physicians, pathologists will be challenged more frequently to provide immediate diagnoses to distinguish infection from rejection. This chapter provides a guide for meeting this challenge and discusses in detail rejection of the graft, infection in the graft, other pathology in the graft, and the procedures for evaluating and distinguishing among these conditions.

Objectives: This study examined the long-term efficacy of intracoronary irradiation for limiting neointimal proliferation after overstretch balloon angioplasty in a porcine model of restenosis. In addition, this study sought to identify any adverse late sequelae of this novel therapy for restenosis., Background: Restenosis after coronary angioplasty represents in part a proliferative response of vascular smooth muscle at the site of injury. We have shown previously that high dose intracoronary radiation induces focal medial fibrosis and markedly reduces neointimal proliferation early after balloon angioplasty in swine., Methods: Twenty-two juvenile swine underwent intervention at a target segment of the left coronary artery. In 11 swine, a 2-cm ribbon of iridium-192 was positioned at the target segment and 2,000 cGy was delivered to the vessel wall. Subsequently, overdilation balloon angioplasty was performed at the irradiated segment. In 11 control swine, overdilation balloon angioplasty was performed without previous irradiation. Twenty animals survived and underwent histopathologic analysis at 180 +/- 8 days., Results: Mean (+/- SD) neointimal area was 1.59 +/- 0.78 and 0.46 +/- 0.35 mm2 (p < 0.001) in control and irradiated animals, respectively. Mean percent area stenosis was 37.9 +/- 12.4% and 14.2 +/- 9.0% (p < 0.001) in the control and irradiated animals, respectively. Thus, by 6-month follow-up, intracoronary irradiation before balloon angioplasty had reduced the bulk of the neointimal lesion by 71.1% and reduced percent area stenosis by 62.5% compared with that in control animals. There was no evidence of radiation vasculopathy or myocardial damage at 6 months., Conclusions: Intracoronary irradiation (2,000 cGy) produces persistent impairment of neointimal proliferation 6 months after balloon injury, with no evidence of late radiation sequelae.

Objective: The authors review their experience and that of others who have reported cases in the literature on the surgical management of cardiac pheochromocytomas., Summary Background Data: Cardiac pheochromocytomas are rare cathecolamine-producing tumors that can be densely adherent to myocardium. Because resection can be associated with significant morbidity, we sought to determine the best mode of treatment for these difficult tumors., Method: The authors reviewed the experience for management of cardiac pheochromocytomas in their two institutions and those reported in the literature. Follow-up was available for 21 of 26 patients up to 9 years after resection., Results: Twenty-five patients had reconstruction of the native heart; five (20%) died intraoperatively from hemorrhage, one (4%) died postoperatively from sepsis, three (12%) sustained myocardial infarction, one (4%) required a mitral valve replacement, and three (12%) had incomplete resections, two of whom subsequently developed metastatic disease and died. One patient, thought to be a high risk for resection, received an orthotopic heart transplantation., Conclusions: Surgical resection of cardiac pheochromocytomas can be performed successfully. However, resection of lesions that aggressively invade adjacent myocardium is associated with significant mortality and inadequate control of the neoplasm. Cardiac transplantation should be available as an option before embarking on resection, and it should be performed if mandated by intraoperative findings.

15-Lipoxygenase (15-LO) catalyzes the oxygenation of arachidonic and linoleic acids and has been implicated in the oxidative modification of low-density lipoproteins (LDL). 15-LO mRNA and protein have previously been demonstrated in macrophages of rabbit and human atherosclerotic lesions. The purpose of this study was to investigate whether 15-LO is also present in the accelerated form of coronary artery disease that can complicate cardiac transplantation (TCAD). Immunohistochemical analysis of coronary arteries with TCAD was carried out by using a rabbit polyclonal antibody raised against human recombinant 15-LO and an avidin-biotin-immunoperoxidase system. Normal coronary and pulmonary arteries showed no immunostaining for 15-LO. Two different types of TCAD were observed. One type consisted of concentric intimal proliferation of smooth muscle cells, without lipid or calcium deposits. No immunoreactivity for 15-LO was present in these lesions. The second type of graft arteriosclerosis consisted of complex atheromatous lesions, containing myointimal cells, lipid-laden foam cells, fragmented internal elastic laminae, and calcifications. 15-LO immunostaining of myointimal cells, lipid-laden foam cells, and endothelial cells was consistently present in these atheromatous lesions. The majority of the myointimal and foam cells positive for 15-LO were recognized by antisera to alpha-smooth muscle actin; the others were identified as macrophages. The results indicate that 15-LO expression is present in endothelial, myointimal, and foam cells in complex atheromatous lesions of TCAD, and suggest that 15-LO may play a role in the pathogenesis of this form of the disease.

Cardiac papillary fibroelastomas are unusual, frond-like growths typically found on cardiac valves, diagnosed incidentally on autopsy or cardiac surgery, but rarely during life. We report a rare case of an aortic valve papillary fibroelastoma detected by transthoracic echocardiography and confirmed by histologic study.

A 66-year-old man without symptoms, referred for the treatment of moderate hypertension, was found to have a right atrial mass by two-dimensional echocardiography. On transesophageal echocardiography two masses were detected on the tricuspid valve, the first one on the posterior leaflet and the second smaller one on the anterior leaflet. These findings were confirmed at surgery. Histologic analysis revealed that the masses represented papillary fibroelastomas.

Objectives: This study examined the effects of intracoronary irradiation on neointimal proliferation after overstretch balloon angioplasty in a normolipemic swine model of restenosis., Background: Restenosis after percutaneous transluminal coronary angioplasty represents, in part, a proliferative response of vascular smooth muscle at the site of injury. We have previously shown that ionizing radiation, delivered by means of an intracoronary source, causes focal medial fibrosis. We therefore hypothesized that intracoronary irradiation delivered at the time of balloon angioplasty might impair the restenosis process., Methods: Nineteen juvenile swine underwent coronary angiography; a segment of the coronary artery was chosen as a target for balloon injury. In 10 swine, a ribbon of iridium-192 was positioned at the target segment, and 2,000 cGy was delivered at the vessel wall. Subsequently, overdilation balloon angioplasty was performed at the irradiated segment. In nine control swine, overdilation balloon angioplasty was performed without previous irradiation. Eighteen animals survived and were killed at 30 days. Histopathologic analysis was performed by a pathologist in blinded manner. The area of maximal lumen compromise within the target segment was analyzed by computer-assisted planimetry., Results: In the control group, mean (+/- SD) neointimal area was 0.84 +/- 0.60 mm2 compared with that in the irradiated group, 0.24 +/- 0.13 mm2 (p = 0.01). In the control group, mean percent area stenosis was 47.6 +/- 20.7%, whereas that in the irradiated group was 17.6 +/- 10.5% (p = 0.001). This represents a 71.4% reduction in neointimal area and a 63.0% reduction in percent area stenosis in the irradiated group. Adjacent coronary segments and surrounding myocardium were unaffected., Conclusions: Intracoronary irradiation (2,000 cGy) delivered to a target porcine coronary segment before balloon overdilation markedly reduces neointima formation at 30 days and thus significantly impairs the restenosis process.

Background: Cardiac preservation for transplantation disrupts normal vascular homeostatic mechanisms. Hypoxia and reoxygenation increase endothelial cell permeability, induce procoagulant activity, and alter endothelial cell/leukocyte interactions, with a parallel reduction in endothelial cAMP and nitric oxide levels. Because hypoxia/reoxygenation simulates a significant component of the global ischemia/reperfusion of cardiac transplantation, we hypothesized that preservation failure may be related to these perturbations. This work focuses on repleting the intercellular/intracellular second messengers nitric oxide/cGMP and cAMP in the donor heart to enhance cardiac preservation for transplantation., Methods and Results: A heterotopic rat heart transplant model was used to compare lactated Ringer's (LR) and University of Wisconsin (UW) solutions to a novel storage solution (Columbia University solution, CU), which contains a cAMP analogue (dibutyryl cAMP) and nitroglycerin (to enhance nitric oxide-related mechanisms). By 24 to 28 hours of preservation, no LR hearts survived (n = 9), 35% of UW hearts survived (n = 20), and 88% of CU-preserved hearts survived (n = 8; P < .05) versus LR or UW). The viability of preserved hearts was explored by determining whether CU preservation enabled myocytes to maintain resting membrane potentials and preserve their ability to generate an action potential in response to a field stimulus. Of 24 sites explored with a microelectrode in UW-preserved hearts, only 4% were able to generate an action potential, compared with 75% of 36 sites in CU-preserved hearts (P < .001), with corresponding preservation of resting membrane potential in the CU-preserved hearts (-13 mV for UW versus -54 mV for CU, P < .001). Orthotopic baboon cardiac transplantation performed after 24-hour simple hypothermic preservation demonstrated that no UW-preserved heart (n = 4) survived the perioperative period; in contrast, four of five hearts stored for 24 hours in CU solution sustained the recipient with minimal inotropic support, and two animals survived long-term., Conclusions: Sustaining higher levels of intercellular/intracellular second messengers cAMP and nitric oxide/cGMP provides a new approach to enhancing cardiac preservation.