Time-course transcriptome analysis of a double challenge bleomycin-induced lung fibrosis rat model uncovers ECM homoeostasis-related translationally relevant genes.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is an irreversible disorder with a poor prognosis. The incomplete understanding of IPF pathogenesis and the lack of accurate animal models is limiting the development of effective treatments. Thus, the selection of clinically relevant animal models endowed with similarities with the human disease in terms of lung anatomy, cell biology, pathways involved and genetics is essential. The bleomycin (BLM) intratracheal murine model is the most commonly used preclinical assay to evaluate new potential therapies for IPF. Here, we present the findings derived from an integrated histomorphometric and transcriptomic analysis to investigate the development of lung fibrosis in a time-course study in a BLM rat model and to evaluate its translational value in relation to IPF. METHODS: Rats were intratracheally injected with a double dose of BLM (days 0-4) and sacrificed at days 7, 14, 21, 28 and 56. Histomorphometric analysis of lung fibrosis was performed on left lung sections. Transcriptome profiling by RNAseq was performed on the right lung lobes and results were compared with nine independent human gene-expression IPF studies. RESULTS: The histomorphometric and transcriptomic analyses provided a detailed overview in terms of temporal gene-expression regulation during the establishment and repair of the fibrotic lesions. Moreover, the transcriptomic analysis identified three clusters of differentially coregulated genes whose expression was modulated in a time-dependent manner in response to BLM. One of these clusters, centred on extracellular matrix (ECM)-related process, was significantly correlated with histological parameters and gene sets derived from human IPF studies. CONCLUSIONS: The model of lung fibrosis presented in this study lends itself as a valuable tool for preclinical efficacy evaluation of new potential drug candidates. The main finding was the identification of a group of persistently dysregulated genes, mostly related to ECM homoeostasis, which are shared with human IPF.

Rostro-caudal Connectional Heterogeneity of the Dorsal Part of the Macaque Prefrontal Area 46.

Based on neural tracer injections we found evidence for 3 connectionally distinct sectors of the dorsal part of the macaque prefrontal area 46 (46d), located at different rostro-caudal levels. Specifically, a rostral sector displayed an almost exclusive and extensive intraprefrontal connectivity and extraprefrontal connections limited to superior temporal areas and the caudal cingulate area 31. Conversely, both a middle and a caudal sector were characterized by robust, topographically organized connections with parietal and frontal sensorimotor areas. Both these sectors shared connections with caudal and medial superior parietal areas (V6A and PGm) where visuospatial information is combined with gaze- and arm-related signals for visuomotor control of arm reaching and/or eye movements. However, the caudal sector was preferentially connected to parietal and frontal oculomotor areas, whereas the middle one was preferentially connected to skeletomotor, mostly arm-related, parietal and premotor areas. The present study provides evidence for a rostro-caudal organization of area 46d similar to that described for the ventrolateral prefrontal cortex, in which more caudal areas are relatively more directly involved in controlling different aspects of motor behavior and more rostral areas are most likely involved in higher order, possibly more abstract, cognitive functions.

Blood and lymphatic vessels contribute to the impact of the immune microenvironment on clinical outcome in non-small-cell lung cancer.

OBJECTIVES: Lymphangiogenesis plays a critical role in the immune response, tumour progression and therapy effectiveness. The aim of this study was to determine whether the interplay between the lymphatic and the blood microvasculature, tumour-infiltrating lymphocytes and the programmed death 1 (PD-1)/programmed death ligand 1 (PD-L1) immune checkpoint constitutes an immune microenvironment affecting the clinical outcome of patients with non-small-cell lung cancer. METHODS: Samples from 50 squamous cell carcinomas and 42 adenocarcinomas were subjected to immunofluorescence to detect blood and lymphatic vessels. CD3pos, CD8pos and PD-1pos tumour-infiltrating lymphocytes and tumour PD-L1 expression were assessed by immunohistochemical analysis. RESULTS: Quantification of vascular structures documented a peak of lymphatics at the invasive margin together with a decreasing gradient of blood and lymphatic vessels from the peritumour area throughout the neoplastic core. Nodal involvement and pathological stage were strongly associated with vascularization, and an increased density of vessels was detected in samples with a higher incidence of tumour-infiltrating lymphocytes and a lower expression of PD-L1. Patients with a high PD-L1 to PD-1 ratio and vascular rarefaction had a gain of 10 months in overall survival compared to those with a low ratio and prominent vascularity. CONCLUSIONS: Microvessels are an essential component of the cancer immune microenvironment. The clinical impact of the PD-1/PD-L1-based immune contexture may be implemented by the assessment of microvascular density to potentially identify patients with non-small-cell lung cancer who could benefit from immunotherapy and antiangiogenic treatment.

Low PD-1 Expression in Cytotoxic CD8+ Tumor-Infiltrating Lymphocytes Confers an Immune-Privileged Tissue Microenvironment in NSCLC with a Prognostic and Predictive Value.

Purpose: The success of immune checkpoint inhibitors strengthens the notion that tumor growth and regression are immune regulated. To determine whether distinct tissue immune microenvironments differentially affect clinical outcome in non-small cell lung cancer (NSCLC), an extended analysis of PD-L1 and tumor-infiltrating lymphocytes (TIL) was performed.Experimental Design: Samples from resected adenocarcinoma (ADC 42), squamous cell carcinoma (SCC 58), and 26 advanced diseases (13 ADC and 13 SCC) treated with nivolumab were analyzed. PD-L1 expression and the incidence of CD3, CD8, CD4, PD-1, CD57, FOXP3, CD25, and Granzyme B TILs were immunohistochemically assessed.Results: PD-L1 levels inversely correlated with N involvement, although they did not show a statistically significant prognostic value in resected patients. The incidence and phenotype of TILs differed in SCC versus ADC, in which EGFR and KRAS mutations conditioned a different frequency and tissue localization of lymphocytes. NSCLC resected patients with high CD8pos lymphocytes lacking PD-1 inhibitory receptor had a longer overall survival (OS: HR = 2.268; 95% CI, 1.056-4.871, P = 0.03). PD-1-to-CD8 ratio resulted in a prognostic factor both on univariate (HR = 1.952; 95% CI, 1.34-3.12, P = 0.001) and multivariate (HR = 1.943; 95% CI, 1.38-2.86, P = 0.009) analysis. Moreover, low PD-1 incidence among CD8pos cells was a distinctive feature of nivolumab-treated patients, showing clinical benefit with a prolonged progression-free survival (PFS: HR = 4.51; 95% CI, 1.45-13.94, P = 0.004).Conclusions: In the presence of intrinsic variability in PD-L1 expression, the reservoir of PD-1-negative effector T lymphocytes provides an immune-privileged microenvironment with a positive impact on survival of patients with resected disease and response to immunotherapy in advanced NSCLC. Clin Cancer Res; 24(2); 407-19. ©2017 AACR.

Corticostriate Projections from Areas of the "Lateral Grasping Network": Evidence for Multiple Hand-Related Input Channels.

Corticostriatal projections from the primate cortical motor areas partially overlap in different zones of a large postcommissural putaminal sector designated as "motor" putamen. These zones are at the origin of parallel basal ganglia-thalamocortical subloops involved in modulating the cortical motor output. However, it is still largely unknown how parietal and prefrontal areas, connected to premotor areas, and involved in controlling higher order aspects of motor control, project to the basal ganglia. Based on tracer injections at the cortical level, we analyzed the corticostriatal projections of the macaque hand-related ventrolateral prefrontal, ventral premotor, and inferior parietal areas forming a network for controlling purposeful hand actions (lateral grasping network). The results provided evidence for partial overlap or interweaving of these projections in correspondence of 2 putaminal zones, distinct from the motor putamen, one located just rostral to the anterior commissure, the other in the caudal and ventral part. Thus, the present data provide evidence for partial overlap or interweaving in specific striatal zones (input channels) of projections from multiple, even remote, areas taking part in a large-scale functionally specialized cortical network. Furthermore, they suggest the presence of multiple hand-related input channels, possibly differentially involved in controlling goal-directed hand actions.

Late Na(+) current and protracted electrical recovery are critical determinants of the aging myopathy.

The aging myopathy manifests itself with diastolic dysfunction and preserved ejection fraction. We raised the possibility that, in a mouse model of physiological aging, defects in electromechanical properties of cardiomyocytes are important determinants of the diastolic characteristics of the myocardium, independently from changes in structural composition of the muscle and collagen framework. Here we show that an increase in the late Na(+) current (INaL) in aging cardiomyocytes prolongs the action potential (AP) and influences temporal kinetics of Ca(2+) cycling and contractility. These alterations increase force development and passive tension. Inhibition of INaL shortens the AP and corrects dynamics of Ca(2+) transient, cell contraction and relaxation. Similarly, repolarization and diastolic tension of the senescent myocardium are partly restored. Thus, INaL offers inotropic support, but negatively interferes with cellular and ventricular compliance, providing a new perspective of the biology of myocardial aging and the aetiology of the defective cardiac performance in the elderly.

c-Kit-positive cardiac stem cells nested in hypoxic niches are activated by stem cell factor reversing the aging myopathy.

RATIONALE: Hypoxia favors stem cell quiescence, whereas normoxia is required for stem cell activation, but whether cardiac stem cell (CSC) function is regulated by the hypoxic/normoxic state of the cell is currently unknown. OBJECTIVE: A balance between hypoxic and normoxic CSCs may be present in the young heart, although this homeostatic control may be disrupted with aging. Defects in tissue oxygenation occur in the old myocardium, and this phenomenon may expand the pool of hypoxic CSCs, which are no longer involved in myocyte renewal. METHODS AND RESULTS: Here, we show that the senescent heart is characterized by an increased number of quiescent CSCs with intact telomeres that cannot re-enter the cell cycle and form a differentiated progeny. Conversely, myocyte replacement is controlled only by frequently dividing CSCs with shortened telomeres; these CSCs generate a myocyte population that is chronologically young but phenotypically old. Telomere dysfunction dictates their actual age and mechanical behavior. However, the residual subset of quiescent young CSCs can be stimulated in situ by stem cell factor reversing the aging myopathy. CONCLUSIONS: Our findings support the notion that strategies targeting CSC activation and growth interfere with the manifestations of myocardial aging in an animal model. Although caution has to be exercised in the translation of animal studies to human beings, our data strongly suggest that a pool of functionally competent CSCs persists in the senescent heart and that this stem cell compartment can promote myocyte regeneration effectively, partly correcting the aging myopathy.