Lung allograft standardized histological analysis (LASHA) template: A research consensus proposal.

BACKGROUND: Routine monitoring of lung-transplanted patients is crucial for the identification of immunological and non-immunological complications. Determining the etiology of acute allograft dysfunction, particularly in alloimmune-mediated disorders, relies heavily on the lung biopsy with histopathologic analysis. Standardization of the pathologic diagnosis of rejection (e.g., cellular and antibody-mediated) is based on consensus statements and guidelines, indicating the importance of a multidisciplinary approach to achieve a definitive etiological diagnosis. In addition to these statements and guidelines, refinements and standardizations are feasible through systematic analysis morphological, immunophenotypic and molecular alterations observed in transbronchial biopsies. This study is to identify key morphologic features to be assessed, select consistent and reproducible terminology for each histological feature, and provide standardized definitions for pathological assessment and grading. METHODS: A template was created by experts in lung transplantation including pathologists, pulmonologists, immunologists. An initial draft was circulated, followed by discussions and multiple revisions by email and conference calls. RESULTS: The "lung allograft standardized histological analysis - LASHA" template was created and structured as multiple-choice questions with number of fields to be filled in to allow for standardization of results and easy transfer into a future electronic spreadsheet. CONCLUSION: This template will help facilitate multicenter studies through a uniform protocol and correlations with new diagnostic modalities. After validation in large-scale studies, an optimized template could be included in routine clinical practice to enhance graft assessment and medical decision-making.

[A clinical radiological score for femoral head grafts : Establishment of the Tabea FK score to ensure the quality of human femoral head grafts]. / Ein klinisch-radiologischer Score für Femurkopftransplantate : Etablierung des Tabea-FK-Scores zur Sicherung der Qualität humaner Femurkopftransplantate.

INTRODUCTION: Transplantation of cancellous tissue from human femoral heads (FK) is an established method in the reconstruction of bony defects in orthopedic and trauma surgery. Standardized rating systems with respect to the morphological quality of this tissue are not available. MATERIALS AND METHODS: In 91/105 patients who had been a regular, clinically-indicated surgery (arthroplasty of the hip joint) the respective femoral head (FK) was taken under standardized conditions. Using a checklist defined clinical and radiological criteria of FK are judged in terms of their quality (cysts, necrosis, calcification, deformities, osteoporosis) and divided by the Tabea FK score into three classes (best/middle/poor quality). This was followed by a blinded repeated scoring, now as macroscopic assessment of three sawed layers from the same femoral head. The femoral heads are examined by peripheral quantitative computed tomography (pQCT) and a standardized histological examination of the bony tissue. We evaluated the accordance of the Tabea FK score with complementary assessments by calculation of sensitivity and specificity. RESULTS: Femoral heads from 91/105 patients (ages: 68.4 ± 9.9 , n = 60 women, n = 31 men) were explanted and included in the study. The correlation between the primary radiologic clinical score (Tabea FK score) and the macroscopic second review of the sawn FK with respect to middle/best and poor/middle quality was classified as good (sensitivity 77% and 81%, respectively; specificity 76% and 84%, respectively). The correlation of histology and macroscopic second review was worse and in relation to discrimination of middle/best and poor/middle quality had a sensitivity of 85% and 54%, respectively, and a specificity of 66% and 97%, respectively. The pQCT showed a sensitivity of 82% only in discrimination of middle/best, while sensitivity in discrimination of poor/middle and poor/middle + best, respectively, was <10%. DISCUSSION: The corresponding correlation between the primary and the second clinical score was evaluated as good. This emphasizes the long-standing skills of operationally active orthopedic surgeons to classify the quality of cancellous bone correctly already on the basis of X­ray images and intraoperative findings. In this respect, the introduction of the Tabea FK score as a quality assurance tool in the routines of bone banks can be recommended.

Cardiac Myxomas Show Elevated Native T1, T2 Relaxation Time and ECV on Parametric CMR.

Introduction: While cardiac tumors are rare, their identification and differentiation has wide clinical implications. Recent cardiac magnetic resonance (CMR) parametric mapping techniques allow for quantitative tissue characterization. Our aim was to examine the range of values encountered in cardiac myxomas in correlation to histological measurements. Methods and Results: Nine patients with histologically proven cardiac myxomas were included. CMR (1.5 Tesla, Philips) including parametric mapping was performed in all patients pre-operatively. All data are reported as mean ± standard deviation. Compared to myocardium, cardiac myxomas demonstrated higher native T1 relaxation times (1,554 ± 192 ms vs. 1,017 ± 58 ms, p < 0.001), ECV (46.9 ± 13.0% vs. 27.1 ± 2.6%, p = 0.001), and T2 relaxation times (209 ± 120 ms vs. 52 ± 3 ms, p = 0.008). Areas with LGE showed higher ECV than areas without (54.3 ± 17.8% vs. 32.7 ± 18.6%, p = 0.042), with differences in native T1 relaxation times (1,644 ± 217 ms vs. 1,482 ± 351 ms, p = 0.291) and T2 relaxation times (356 ± 236 ms vs. 129 ± 68 ms, p = 0.155) not reaching statistical significance. Conclusions: Parametric CMR showed elevated native T1 and T2 relaxation times and ECV values in cardiac myxomas compared to normal myocardium, reflecting an increased interstitial space and fluid content. This might help in the differentiation of cardiac myxomas from other tumor entities.

Rare non-coding Desmoglein-2 variant contributes to Arrhythmogenic right ventricular cardiomyopathy.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to variants in the coding sequence of desmosomal genes. The potential contribution of non-coding desmoglein-2 (DSG2) variants for development of ARVC is undescribed. We sequenced 1450 base pairs upstream of ATG in the DSG2 gene in 65 unrelated patients diagnosed with ARVC (10 borderline cases). Identified variants was evaluated by cosegregation and allele population frequency analysis, in silico tools, immunohistological investigations of myocardial biopsies, gene reporter assays, electrophoretic mobility shift assays (EMSA), and chromatin immunoprecipitation. The genetic analysis identified one novel, rare heterozygous DSG2 upstream variant (-317G > A) in a genetically unexplained ARVC patient. The variant segregated with signs of disease, was absent in publicly available databases, and affected a predicted binding site for activating protein-1 (AP-1). Immunohistochemical analysis of a myocardial biopsy from the -317G > A patient showed a marked reduction in DSG2 protein levels compared to healthy controls. Luciferase reporter gene assays showed promoter activity of the identified DSG2 upstream region and a general reduction in transcriptional activity in the presence of the minor DSG2_A allele (p < .01). Moreover, the DSG2_A allele reduced DSG2 activation by TGF-beta1 and a protein kinase C pathway activator (PMA; all p < .001 vs. DSG2_G). EMSAs showed altered transcription factor binding in presence of the DSG2_A allele. Chromatin immunoprecipitation assays in wild type epithelial cells identified AP-1 components c-FOS and c-JUN at the -317 locus. In conclusion, the non-coding DSG2 promoter variant -317G > A reduces DSG2 transcription in vitro and reduced myocardial DSG2 protein levels were observed in vivo. Our data support a contribution of non-coding DSG2 variants to the pathogenesis of ARVC.

Long-Term Lung Function and Exercise Capacity in Postinfectious chILD.

Background: Severe postinfectious diffuse pulmonary disease may clinically mimic other entities of children's interstitial lung disease and is clinically challenging comprising various disease severities despite treatment. Long-term lung function trend and physical capacity in children with postinfectious diffuse pulmonary disease are rarely reported. We investigated trends in pulmonary function by long-term follow-up and assessed physical capacity in such patients. Methods: We performed a descriptive, single-center follow-up study in children with biopsy-verified postinfectious diffuse pulmonary disease. Patients with completed primary treatment course were eligible for follow-up, including pulmonary function and exercise (VO2peak) testing. Results: Thirty patients with postinfectious diffuse pulmonary disease were identified and included. Median (range) age at diagnose was 27.5 (2-172) months after a mean lag time of 23 months. H. influenzae and rhinovirus were the most frequent pathogens. Fifteen patients were available for follow-up after mean (range) 7.6 (2-15) years of treatment completion. Lung clearance index (LCI2.5), forced expiratory volume in 1 second (FEV1), and bronchodilator responsiveness were abnormal in 80%, 53%, and 44%, respectively. Diffusion capacity for monoxide was abnormal in 7% and total lung capacity in 33%. Only 8% demonstrated low VO2peak, while 40% reported difficulties during physical exertion. Longitudinal data on spirometry (n = 14) remained unchanged from end of treatment throughout follow-up. A significant association was found between zLCI2.5 and zFEV1 (multiple linear regression; r 2 = 0.61; P = 0.0003). Conclusion: Postinfectious diffuse pulmonary disease in children carries a varying degree of chronic pulmonary impairment with onset of symptoms in the first months of life and a typical considerable lag time before diagnosis. Follow-up several years after the initial injury demonstrated moderate-to-severe peripheral airway impairment although no further lung function decline was found years after completion of treatment. Despite acceptable VO2peak, a considerable proportion struggled during heavy exercise.

Immune regulation by fibroblasts in tissue injury depends on uPARAP-mediated uptake of collectins.

Collectins such as mannose-binding lectin (MBL) and surfactant protein D (SP-D) become temporarily deposited in extravascular compartments after tissue injury and perform immune-stimulatory or inflammation-limiting functions. However, their turnover mechanisms, necessary to prevent excessive tissue damage, are virtually unknown. In this study, we show that fibroblasts in injured tissues undertake the clearance of collectins by using the endocytic collagen receptor uPARAP. In cellular assays, several types of collectins were endocytosed in a highly specific uPARAP-dependent process, not shared by the closely related receptor MR/CD206. When introduced into dermis or bleomycin-injured lungs of mice, collectins MBL and SP-D were endocytosed and routed for lysosomal degradation by uPARAP-positive fibroblasts. Fibroblast-specific expression of uPARAP governed endogenous SP-D levels and overall survival after lung injury. In lung tissue from idiopathic pulmonary fibrosis patients, a strong up-regulation of uPARAP was observed in fibroblasts adjacent to regions with SP-D secretion. This study demonstrates a novel immune-regulatory function of fibroblasts and identifies uPARAP as an endocytic receptor in immunity.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) in clinical practice.

Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited myocardial disease characterized by fibro-fatty replacement of the right ventricular myocardium, and associated with paroxysmal ventricular arrhythmias and sudden cardiac death (SCD). It is currently the second most common cause of SCD after hypertrophic cardiomyopathy in young people <35 years of age, causing up to 20% of deaths in this patient population. This condition has a male preponderance and is more commonly found in individuals of Italian and Greek descent. To date, there is no single diagnostic test for ARVC/D and the diagnosis is made based on clinical, electrocardiographic, and radiological findings according to the Revised 2010 Task Force Criteria. In this review, we will discuss the mainstay treatment which includes pharmacotherapy, implantable cardioverter-defibrillator insertion for abortion of sudden cardiac death, and in the advanced stages of the disease cardiac transplantation.

Identification of myocardial diffuse fibrosis by 11 heartbeat MOLLI T 1 mapping: averaging to improve precision and correlation with collagen volume fraction.

OBJECTIVES: Our objectives involved identifying whether repeated averaging in basal and mid left ventricular myocardial levels improves precision and correlation with collagen volume fraction for 11 heartbeat MOLLI T 1 mapping versus assessment at a single ventricular level. MATERIALS AND METHODS: For assessment of T 1 mapping precision, a cohort of 15 healthy volunteers underwent two CMR scans on separate days using an 11 heartbeat MOLLI with a 5(3)3 beat scheme to measure native T 1 and a 4(1)3(1)2 beat post-contrast scheme to measure post-contrast T 1, allowing calculation of partition coefficient and ECV. To assess correlation of T 1 mapping with collagen volume fraction, a separate cohort of ten aortic stenosis patients scheduled to undergo surgery underwent one CMR scan with this 11 heartbeat MOLLI scheme, followed by intraoperative tru-cut myocardial biopsy. Six models of myocardial diffuse fibrosis assessment were established with incremental inclusion of imaging by averaging of the basal and mid-myocardial left ventricular levels, and each model was assessed for precision and correlation with collagen volume fraction. RESULTS: A model using 11 heart beat MOLLI imaging of two basal and two mid ventricular level averaged T 1 maps provided improved precision (Intraclass correlation 0.93 vs 0.84) and correlation with histology (R 2 = 0.83 vs 0.36) for diffuse fibrosis compared to a single mid-ventricular level alone. ECV was more precise and correlated better than native T 1 mapping. CONCLUSION: T 1 mapping sequences with repeated averaging could be considered for applications of 11 heartbeat MOLLI, especially when small changes in native T 1/ECV might affect clinical management.

Myocardial T1 maps reflect histological findings in acute and chronic stages of myocarditis in a rat model.

BACKGROUND: Cardiovascular magnetic resonance offers both diagnostic and prognostic information in myocarditis. Using an established animal model of myocarditis, the aim of this study was to measure myocardial T1 before the onset, in the acute and in the chronic phases of the disease and to compare its course with histological and immunohistochemistry findings. METHODS: Male young Lewis rats were immunized with 0.25 mg porcine myocardial myosin into the rear footpads on day 0. Native and contrast-enhanced ECG-triggered cardiac MRI examinations were performed before immunization on day 0 and on days 14, 21 and 35. Left ventricular function, pre- and post- contrast T1 parameters and LGE images were assessed using Small animal look-locker inversion recovery (SALLI). For each of the indicated time points a minimum of 4 rats were randomly sacrificed for pathological investigations including conventional histology (HE and Sirius-Red staining) and immunohistochemistry (CD 68) investigations. RESULTS: All immunized rats developed myocarditis (morbidity 100%). Histologically we observed increased wall thickness with biventricular macrophage-rich mixed inflammatory infiltrates. All rats with a histologically severe myocarditis showed increased native T1 and decreased post-contrast T1 of the myocardium. CONCLUSIONS: The assessment of native T1 and post-contrast T1 allows accurate differentiation between healthy myocardium and myocardium with inflammation and also between the acute and chronic phases of the disease.

Ultrastructural morphometric findings of cardiomyocytes in patients with impaired ventricular function--a comparative clinicopathological study.

AIM: The present study aims to analyze the differences in ultrastructural changes between right ventricular myocardium in clinically determined grades of heart failure (HF) [New York Heart Association (NYHA) classes I-IV] and their value in the routine diagnostic setting. METHODS: We investigated consecutive right ventricular endomyocardial biopsies of 12 patients presenting with HF (49±11.2years; male=10) by light microscopy and ultrastructural morphometric analysis. The patients were divided into four groups according to their NYHA classes (NYHA I: n=1, II: n=2, III: n=8, IV: n=1). We used a stereological point counting method on electron micrographs to determine the volume, surface, and numerical density of cardiomyocyte myofibrils; z-lines; mitochondria; and cristae as required. Further, secondary parameters were calculated. RESULTS: Myofibrillar parameters increased between NYHA class I and II (P<.01), which matched with more pronounced cardiomyocyte hypertrophy on the light microscopic level. In NYHA classes III and IV, the myofibrillar parameters dropped, while parameters concerning the mitochondria and their cristae rose (P<.01). This resulted in an elevated mitochondria to myofibril ratio (P<.05) and correlated with histologically evident atrophic cardiomyocytes, perinuclear loss of myofibrils and dot-like perinuclear staining positive on peroxide acid shift. CONCLUSION: In this present study, right ventricular myocardial ultrastructure differed between patients diagnosed with HF of different degrees in distinct subcellular changes. These findings suggest that ultrastructural analysis, while correlated with histopathological features, adds to the diagnosis in the routine diagnostic setting, specifically in lower NYHA grades, in which only minor changes are observed histologically.

Human cardiac extracellular matrix supports myocardial lineage commitment of pluripotent stem cells.

OBJECTIVES: Cross-talk between organ-specific extracellular matrix (ECM) and stem cells is often assumed but has not been directly demonstrated. We developed a protocol for the preparation of human cardiac ECM (cECM) and studied whether cECM has effects on pluripotent stem cell differentiation that may be useful for future cardiac regeneration strategies in patients with end-stage heart failure. METHODS: Of note, 0.3 mm-thick cECM slices were prepared from samples of myocardium from patients with end-stage non-ischaemic dilated cardiomyopathy, using a three-step protocol involving hypotonic lysis buffer, sodium dodecyl sulphate (SDS) and foetal bovine serum (FBS). Murine embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and mesenchymal stromal cells (MSCs) were seeded and grown in standard culture, on cECM or on non-specific ECM preparations (Matrigel® or Geltrex®). Cell attachment, apoptosis induction (Caspase 3/7 activity) and metabolic activity (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium conversion) were followed. Transcriptional activation of genes involved in pluripotency; early and late myocardial development; and endothelial, ectodermal or endodermal commitment were monitored by quantitative real-time polymerase chain reaction (rtPCR). Protein expression of selected markers was confirmed by immunohistology. RESULTS: cECM supported the proliferation of ESCs and iPSCs, and Caspase 3/7 activity was significantly lower compared with standard culture. Cardiac lineage commitment was favoured when ESCs or iPSCs were grown on cECM, as evidenced by the significantly increased mRNA expression of cardiac alpha myosin heavy polypeptide 6 (Myh6), cardiac troponin T2 (Tnnt2) and NK2 homeobox 5 (Nkx2.5) as well as positive immunohistology for cardiac troponin T and heavy-chain cardiac myosin protein. In contrast, Matrigel or Geltrex did not induce cardiac-specific markers. MSCs showed no evidence of cardiomyocyte differentiation. CONCLUSIONS: Human cardiac ECM seems to direct differentiation of pluripotent stem cells towards a cardiomyocyte phenotype. This phenomenon supports the use of cardiac ECM preparations for guided stem cell differentiation and myocardial repair, and may ultimately increase the therapeutic efficacy of cell therapy in heart failure patients.

Peri-operative chemotherapy in the management of resectable colorectal cancer pulmonary metastases.

BACKGROUND: Surgery is often advocated in patients with resectable pulmonary metastases from colorectal cancer (CRC). Our study aims to evaluate peri-operative chemotherapy in patients with metastastic CRC undergoing pulmonary metastasectomy. METHODS: Patients treated for CRC who underwent pulmonary metastasectomy by a single surgeon were identified. Outcome measures included survival, peri-operative complications, radiological and histological evidence of chemotherapy-induced lung toxicities. RESULTS: Between 1997 and 2009, 51 eligible patients were identified undergoing a total of 72 pulmonary resections. Thirty-eight patients received peri-operative chemotherapy, of whom 9 received an additional biological agent. Five-year overall survival rate was 72% in the whole cohort - 74% and 68% in those who received peri-operative chemotherapy (CS) and those who underwent surgery alone (S) respectively. Five-year relapse free survival rate was 31% in the whole cohort - 38% and ≤18% in CS and S groups respectively. Only 8% had disease progression during neoadjuvant chemotherapy. There were no post-operative deaths. Surgical complications occurred in only 4% of patients who received pre-operative chemotherapy. There was neither radiological nor histological evidence of lung toxicity in resected surgical specimens. CONCLUSIONS: Peri-operative chemotherapy can be safely delivered to CRC patients undergoing pulmonary metastasectomy. Survival in this selected group of patients was favourable.