Biallelic variants in PPP1R13L cause paediatric dilated cardiomyopathy.

Childhood dilated cardiomyopathy (DCM) is a leading cause of heart failure requiring cardiac transplantation and approximately 5% of cases result in sudden death. Knowledge of the underlying genetic cause can aid prognostication and clinical management and enables accurate recurrence risk counselling for the family. Here we used genomic sequencing to identify the causative genetic variant(s) in families with children affected by severe DCM. In an international collaborative effort facilitated by GeneMatcher, biallelic variants in PPP1R13L were identified in seven children with severe DCM from five unrelated families following exome or genome sequencing and inheritance-based variant filtering. PPP1R13L encodes inhibitor of apoptosis-stimulating protein of p53 protein (iASPP). In addition to roles in apoptosis, iASPP acts as a regulator of desmosomes and has been implicated in inflammatory pathways. DCM presented early (mean: 2 years 10 months; range: 3 months-9 years) and was progressive, resulting in death (n = 3) or transplant (n = 3), with one child currently awaiting transplant. Genomic sequencing technologies are valuable for the identification of novel and emerging candidate genes. Biallelic variants in PPP1R13L were previously reported in a single consanguineous family with paediatric DCM. The identification here of a further five families now provides sufficient evidence to support a robust gene-disease association between PPP1R13L and severe paediatric DCM. The PPP1R13L gene should be included in panel-based genetic testing for paediatric DCM.

Pathologic interpretation of transbronchial biopsy for acute rejection of lung allograft is highly variable.

Despite the standardization of pathologic grading of acute rejection in transbronchial lung biopsies following lung transplantation, the reproducibility of pathologic diagnosis has not been adequately evaluated. To determine the interobserver variability for pathologic grading of acute rejection, 1566 biopsies from 845 subjects in the Lung Allograft Rejection Gene Expression Observational study were regraded by a pathology panel blinded to the original diagnosis and compared to the grade of acute rejection assigned by individual center pathologists. The study panel confirmed 49.1% of center pathologists' A0 grades, but upgraded 5.7% to A1 and 2.7% to grade ≥ A2 rejection; 42.5% were regraded as AX. Of 268 grade A1 samples, 21.2% were confirmed by the pathology panel; 18.7% were upgraded to ≥ A2 and 35.8% were downgraded to A0 with 24.3% being regraded as AX. Lastly, 53.5% of ≥ A2 cases were confirmed, but 15.7% were downgraded to grade A0 and 18.4% cases to A1, while 12.4% were regraded as AX. The kappa value for interobserver agreement was 0.183 (95%CI 0.147-0.220, p < 0.001). The results for B grade interpretation were similar. Suboptimal sampling is common and a high degree of variability exists in the pathologic interpretation of acute rejection in transbronchial biopsies.

Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling.

Rejection diagnosis by endomyocardial biopsy (EMB) is invasive, expensive and variable. We investigated gene expression profiling of peripheral blood mononuclear cells (PBMC) to discriminate ISHLT grade 0 rejection (quiescence) from moderate/severe rejection (ISHLT > or = 3A). Patients were followed prospectively with blood sampling at post-transplant visits. Biopsies were graded by ISHLT criteria locally and by three independent pathologists blinded to clinical data. Known alloimmune pathways and leukocyte microarrays identified 252 candidate genes for which real-time PCR assays were developed. An 11 gene real-time PCR test was derived from a training set (n = 145 samples, 107 patients) using linear discriminant analysis (LDA), converted into a score (0-40), and validated prospectively in an independent set (n = 63 samples, 63 patients). The test distinguished biopsy-defined moderate/severe rejection from quiescence (p = 0.0018) in the validation set, and had agreement of 84% (95% CI 66% C94%) with grade ISHLT > or = 3A rejection. Patients >1 year post-transplant with scores below 30 (approximately 68% of the study population) are very unlikely to have grade > or = 3A rejection (NPV = 99.6%). Gene expression testing can detect absence of moderate/severe rejection, thus avoiding biopsy in certain clinical settings. Additional clinical experience is needed to establish the role of molecular testing for clinical event prediction and immunosuppression management.

Sirolimus (rapamycin) potentiates cyclosporine in prevention of acute lung rejection.

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.

Photopheresis for the prevention of rejection in cardiac transplantation. Photopheresis Transplantation Study Group.

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.

Myocardial osteopontin expression is associated with left ventricular hypertrophy.

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.

Adhesion molecules (E-selectin and ICAM-1) in pulmonary allograft rejection.

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.

Histological response to stent graft therapy.

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.

Talc pleurodesis: talc slurry versus thoracoscopic talc insufflation in a porcine model.

BACKGROUND: Pleurodesis using both talc slurry and thoracoscopic talc insufflation has been shown to be clinically effective. This study compares these two modalities of pleural talc instillation in an animal model. METHODS: Eleven immature pigs underwent general endotracheal anesthesia. On one side, a slurry of 5 g sterile United States Pharmacopeia talc in 50 mL of saline solution was instilled through a thoracostomy tube. On the other side, the lung was deflated and 5 g of dry talc was insufflated under thoracoscopic visualization. The animals were sacrificed 30 days later, and the quality of pleural adhesions was graded from 0 to 2 (0 = absent; 1 = light; 2 = dense) in each of six regions of each hemithorax. The distribution of adhesions on each side was graded from 0 to 6, according to the number of areas that contained adhesions. RESULTS: One animal died of anesthetic complications. Among the survivors, adhesions produced by both methods were dense and diffuse in 8 of 10 animals, and light and diffuse in 1 animal. One animal had light or absent adhesions on the talc slurry side, and dense and diffuse adhesions on the thoracoscopic talc insufflation side. There was no difference between the techniques for density of adhesion scores (talc slurry, 9.9 +/- 2.2; thoracoscopic talc insufflation, 10.0 +/- 2.5) or distribution of adhesion scores (talc slurry, 5.5 +/- 1.0; thoracoscopic talc insufflation, 5.8 +/- 0.4) (p > 0.1). CONCLUSIONS: Effective pleurodesis in a porcine model can be obtained with either talc slurry or thoracoscopic talc insufflation.

Bilateral lobar transplantation utilizing living related donors.

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.

Regulation of nitric oxide production in human coronary microvessels and the contribution of local kinin formation.

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.

Intravascular ultrasound imaging of human cerebral arteries.

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.

Short course single agent therapy with an LFA-3-IgG1 fusion protein prolongs primate cardiac allograft survival.

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.

Pathology of lung transplantation.

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.