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.

Anti-CD25 treatment and FOXP3-positive regulatory T cells in heart transplantation.

The interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part in shaping the dynamics of T cell populations following immune activation, due to its role in T cell proliferation and survival. Strategies to blunt the effector responses in transplantation have been developed by devising pharmaceutical agents to block the IL-2 pathways. However, such strategies could adversely affect the CD25(+)FOXP3(+)T regulatory (T reg) populations which also rely on intereukin-2 signaling for survival. The present study shows that a cohort of heart allograft recipients treated with Daclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patterns consistent with functional T regulatory cell populations. High levels of FOXP3 were observed to correlate with lower incidence of and recovery from acute rejection, as well as lower levels of anti-donor HLA antibody production. Therefore, T reg populations appear fully functional in patients treated with Daclizumab, even when 5 doses were administered. By comparison, patients treated with fewer doses or no Daclizumab had a higher incidence of acute rejection, antibody production and graft failure. Therefore, our data indicates that Daclizumab treatment does not interfere with the generation of regulatory T cells and has a beneficial effect on heart allograft survival.

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.

Porous balloon delivery of S-dC28 does not prevent restenosis in the porcine coronary artery model of balloon injury.

Phosphorothioate (PS) oligodeoxynucleotides (ODN) inhibit vascular smooth muscle cell proliferation through antisense and G-quartet aptameric mechanisms. PS-ODN such as the cytidine homopolymers, have been demonstrated to have non-G-quartet, nonsequence-specific inhibitory effects in a rat carotid balloon injury model of neointimal proliferation. We sought to test the efficacy of S-dC28, a cytidine homopolymer lacking G-quartets, on neointimal proliferation in the porcine coronary artery model of balloon injury. A total of 23 animals (11 controls, 12 treated) were subjected to balloon injury in a coronary artery, followed by infusion of control solution or S-dC28 via porous balloon, the Scimed Dispatch Coronary Infusion Catheter. After a mean interval of 49 days, the animals were killed, and the target coronary segments were examined histologically. S-dC28 did not significantly inhibit neointimal formation. Fluorescein isothiocyanate (FITC)-labeled S-dC28 was present in the intima and media immediately after administration but was present mainly within the adventitia 3 hours after administration. S-dC28, when delivered by a Scimed Dispatch Coronary Infusion Catheter (Maple Grove, MN), did not significantly affect neointimal proliferation after balloon injury in a porcine coronary artery model.

Radioactive beta-emitting solution-filled balloon treatment prevents porcine coronary restenosis.

PURPOSE: Intracoronary gamma or beta radiation from centrally located sources at the time of overstretch balloon injury inhibits neointimal proliferation. In an effort to deliver homogeneous, centered radiation fields in a technically straightforward fashion, we studied the effects of a beta-emitting solution used as a balloon inflation fluid to deliver radiation at the time of coronary injury. METHODS: Twenty-one coronary arteries in 13 juvenile swine underwent irradiation (control and 11 or 25 Gy media dose). Radiation was delivered using a perfusion balloon inflated with an Re-188 solution. Subsequently, overdilatation percutaneous transluminal coronary angioplasty was performed at the pretreated segment. Histopathologic and histomorphometric analysis was performed at 30 days after injury on the entire irradiated artery. RESULTS: Balloon overdilation was associated with significant vascular injury and marked neointimal proliferation in control and low-dose (11 Gy)-treated arteries. High-dose radiation (25 Gy) significantly inhibited neointima formation compared with controls (neointimal area: 0.49 +/- 0.29 mm2 vs. 1.51 +/- 0.22 mm2, respectively; p = 0.02) and low-dose radiation (neointimal area 1.75 +/- 0.54 mm2, p > 0.1 compared with controls). CONCLUSIONS: Liquid Re-188 is an effective beta-emitting vehicle to deliver intracoronary radiation and prevent restenosis in this model. Intracoronary radiation treatment using aqueous radioisotope sources is technically straightforward and provides the optimally achievable radiation dose distribution.

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.

Transesophageal echocardiography-guided transvenous biopsy of a cardiac sarcoma.

Transvenous endomyocardial biopsy is a well established procedure to diagnose rejection after heart transplantation as well as to assess for other cardiomyopathic processes. However, it is rarely used to obtain samples of unidentified cardiac masses. We report a case of a primary cardiac sarcoma in which the histologic diagnosis was provided by transesophageal echocardiography-guided transvenous biopsy. This procedure is accurate and can avoid the potential risk of a diagnostic thoracotomy.

Intracoronary irradiation: dose response for the prevention of restenosis in swine.

PURPOSE: 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 high-dose radiation (20 Gy), delivered via an intracoronary 192Ir source, causes focal medial fibrosis and markedly impairs the restenosis process after balloon angioplasty in swine. This study sought to delineate the dose-response characteristics of this effect. METHODS AND MATERIALS: Forty juvenile swine underwent coronary angiography; a segment of the left coronary artery was chosen as a target for balloon injury. In 30 swine, a 2 cm ribbon of 192Ir was positioned at the target segment and 20, 15, or 10 Gy were delivered to the vessel wall (10 animals/dose). Subsequently, overdilatation balloon angioplasty was performed at the irradiated segment. In 10 control swine, overdilatation balloon angioplasty was performed without previous irradiation. Thirty-eight animals survived until sacrifice at 30 +/- 3 days. Histopathological analysis was performed by a pathologist in a blinded manner. The area of maximal luminal compromise within the target segment was analyzed via computer-assisted planimetry. RESULTS: Neointimal area was decreased by 71.4% at 20 Gy and by 58.3% at 15 Gy compared with control animals (p < 0.05 for both). A stimulatory effect on smooth muscle cell proliferation was noted at 10 Gy, with a 123% increase in neointimal area compared with controls (p < 0.05). Mean percent area stenosis was also reduced by 63% at 20 Gy and by 74.8% at 15 Gy compared with controls (p < 0.05 for both). CONCLUSIONS: Intracoronary irradiation prior to overstretch balloon angioplasty markedly reduces neointima formation; this effect is dose dependent, with evidence of a significant stimulatory effect at 10 Gy. The effective therapeutic dose range for the prevention of restenosis in this model begins at approximately 15 Gy delivered to the vessel wall.

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.

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.

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.