Pulmonary Fibrosis (PF), a devastating lung disease with rising prevalence, is defined by a physiologic defect in gas exchange owing to two cardinal pathological features: intrusion of myofibroblasts into the distal lung parenchyma and an alteration of normal alveolar epithelial composition most clearly defined by a loss of the squamous alveolar epithelial type 1 cell (AEC1) and the presence of a recently identified aberrant alveolar epithelial "transitional state" enriched in profibrotic mediators. While this transitional cell state has been identified transiently in the epithelium of murine lung injury models, the mechanism by which it arises in human fibrotic lung disease is unknown. We previously reported the in vivo modeling of PF utilizing inducible, knock-in expression of a clinical Surfactant Protein C (SP-C) mutation in alveolar type 2 cells (AEC2). Expression of the SP-C mutation in the adult mouse AEC2s led to activation of Unfolded Protein Response (UPR) signaling pathways, endoplasmic reticulum (ER) stress and spontaneous fibrosis providing proof of concept for disruption to proteostasis as a proximal driver of PF. We hypothesized that disruption of AEC2 protein quality control and specifically UPR signaling causes cell autonomous AEC2 reprogramming to the transitional state in the absence of an exogenous lung injury. Using two clinical SP-C mutation models we discovered that AEC2s experiencing significant ER stress lose quintessential AEC2 features and develop the recently identified transitional cell state. Using single cell RNA sequencing of the epithelium in our murine models we identify that UPR activated AEC2s develop the transitional cell phenotype in the absence of an exogenous lung injury. Through organoid based modeling we validated that this state arises de novo from intrinsic AEC2 dysfunction. The cell autonomous AEC2 reprogramming is mediated through IRE1 signaling as use of a novel IRE1 inhibitor attenuated the development of the transitional cell state and diminished AEC2 driven recruitment of granulocytes, alveolitis, and lung injury arising from the loss of proteostasis. We further show that this transitional state persists into the fibrotic phase of our murine models and is enriched in pro-fibrotic mediators. These findings identify AEC2 proteostasis, and specifically IRE1 signaling, as a driver of a key AEC2 phenotypic change that has been identified in lung fibrosis.

The oxycyclohexyl acid BMS-986278 (33) is a potent lysophosphatidic acid receptor 1 (LPA1) antagonist, with a human LPA1 Kb of 6.9 nM. The structure-activity relationship (SAR) studies starting from the LPA1 antagonist clinical compound BMS-986020 (1), which culminated in the discovery of 33, are discussed. The detailed in vitro and in vivo preclinical pharmacology profiles of 33, as well as its pharmacokinetics/metabolism profile, are described. On the basis of its in vivo efficacy in rodent chronic lung fibrosis models and excellent overall ADME (absorption, distribution, metabolism, excretion) properties in multiple preclinical species, 33 was advanced into clinical trials, including an ongoing Phase 2 clinical trial in patients with lung fibrosis (NCT04308681).

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) causes irreversible loss of lung function. The lysophosphatidic acid receptor 1 (LPA(1)) pathway is implicated in IPF etiology. Safety and efficacy of BMS-986020, a high-affinity LPA(1) antagonist, was assessed vs placebo in a phase 2 study in patients with IPF. METHODS: IM136003 was a phase 2, parallel-arm, multicenter, randomized, double-blind, placebo- controlled trial. Adults with IPF (FVC, 45%-90%; diffusing capacity for carbon monoxide, 30%-80%) were randomized to receive placebo or 600 mg BMS-986020 (once daily [qd] or bid) for 26 weeks. The primary end point was rate of change in FVC from baseline to week 26. RESULTS: Of 143 randomized patients, 108 completed the 26-week dosing phase. Thirty-five patients discontinued prematurely. Patient baseline characteristics were similar between treatment groups (placebo: n = 47; 600 mg qd: n = 48; 600 mg bid: n = 48). Patients treated with BMS-986020 bid experienced a significantly slower rate of decline in FVC vs placebo (-0.042 L; 95% CI, -0.106 to -0.022 vs -0.134 L; 95% CI, -0.201 to -0.068, respectively; P =.049). Dose-related elevations in hepatic enzymes were observed in both BMS-986020 treatment groups. The study was terminated early because of three cases of cholecystitis that were determined to be related to BMS-986020 after unblinding. CONCLUSIONS: BMS-986020 600 mg bid treatment for 26 weeks vs placebo significantly slowed the rate of FVC decline. Both regimens of BMS-986020 were associated with elevations in hepatic enzymes.

Fibrosis, resulted from the imbalance of fibrogenesis and fibrolysis, is a key readout of disease progression in nonalcoholic steatohepatitis (NASH) and reflects mortality risk. Non-invasive biomarkers capable of diagnosing fibrosis stages and monitoring fibrosis changes in NASH patients are urgently needed. This study is to evaluate collagen formation and degradation biomarkers, reflective of fibrogenesis or fibrolysis, in patients with biopsy proven NASH. Collagen formation biomarker PRO-C3 and PRO-C6 levels were significantly higher in patients with advanced fibrosis stage 3-4 than those with fibrosis stage 0-2. Elevated PRO-C3 levels were also associated with severe lobular inflammation and ballooning, but not with steatosis. Multivariate logistic regression analysis identified PRO-C3 and PRO-C6 to be independently related to fibrosis stage. PRO-C3 showed similar performance to identify patients with advanced fibrosis in discovery and validation cohorts. Furthermore, in a longitudinal study cohort with paired biopsies, mean PRO-C3 increased with worsening of fibrosis and decreased with fibrosis improvement. The results suggest that PRO-C3 may be a potentially useful biomarker in identifying patients with advanced fibrosis and active fibrogenesis, as well as in assessing changes in fibrosis over time. It is worthy of further evaluation to confirm its diagnostic value and clinical utility.

Recently, the US Food and Drug Administration (FDA) approved the first two drugs (pirfenidone and nintedanib) indicated for the treatment of idiopathic pulmonary fibrosis (IPF). The purpose of this analysis was to leverage publicly available data to quantify comparative efficacy of compounds that are approved or in development. An analysis‐ready database was developed, and the analysis dataset is composed of summary‐level data from 43 arms in 20 trials, with treatment durations ranging from 8–104 weeks. A hierarchical multivariable regression model with nonparametric placebo estimation was used to fit the longitudinal profile of change from baseline of percent predicted forced vital capacity (%predicted FVC) data. Pirfenidone and nintedanib were the only drugs identified to have significant estimated positive treatment effects. Model simulations were performed to further evaluate the covariate and time course of treatment effects on longitudinal change from baseline %predicted FVC to inform future trial designs and support decision making.

Idiopathic pulmonary fibrosis (IPF) is a chronic fibrosing lung disease with limited effective treatment options. The LPA1 pathway has been implicated in the etiology and pathogenesis of IPF and is a promising therapeutic target for fibrotic diseases. LPA1 antagonists, including BMS‑986020 and BMS-986234, are being investigated for IPF. Differences in structure and pharmacology of LPA1 antagonists could impact their efficacy and safety profile. In a Phase 2 trial, BMS-986020 compared with placebo significantly slowed lung function decline but, in some patients, showed hepatobiliary effects; the mechanisms underlying these effects were investigated in vitro and in vivo. In vitro, BMS-986020 inhibits bile acid and phospholipid transporters, BSEP (IC50=4.8 µM), MRP4 (6.2 µM), and MDR3 (7.5 µM), which may reduce bile acid and phospholipid efflux, and alter bile composition and flow. BMS-986020 altered bile homeostasis in vivo, yielding elevated systemic bile acids in rats and humans. In contrast, a structurally distinct LPA1 antagonist BMS-986234, at projected clinically relevant concentrations, did not inhibit BSEP (IC50=19.6 µM), MRP4 (>50 µM), or MDR3 (>50 µM) in vitro, or inhibit bile acid efflux in human hepatocytes (≤50 µM). Additionally, BMS-986234 did not increase bile acids in rats or monkeys. In conclusion, the hepatobiliary effects observed with BMS‑986020 are likely off-target effects specific to this molecule and not mediated via antagonism of LPA1. These results suggest that structural variations in LPA1 antagonists may result in different safety profiles in patients with IPF and other fibrotic diseases.

BACKGROUND: Despite Food and Drug Administration approval of 2 new drugs for idiopathic pulmonary fibrosis (IPF), curative therapies remain elusive and mortality remains high. Preclinical and clinical data support the safety of human mesenchymal stem cells as a potential novel therapy for this fatal condition. The Allogeneic Human Cells (hMSC) in patients with Idiopathic Pulmonary Fibrosis via Intravenous Delivery (AETHER) trial was the first study designed to evaluate the safety of a single infusion of bone marrow-derived mesenchymal stem cells in patients with idiopathic pulmonary fibrosis.METHODS: Nine patients with mild to moderate IPF were sequentially assigned to 1 of 3 cohorts and dosed with a single IV infusion of 20, 100, or 200 x 10(6) human bone marrowderived mesenchymal stem cells per infusion from young, unrelated, men. All baseline patient data were reviewed by a multidisciplinary study team to ensure accurate diagnosis. The primary end point was the incidence (at week 4 postinfusion) of treatment-emergent serious adverse events, defined as the composite of death, nonfatal pulmonary embolism, stroke, hospitalization for worsening dyspnea, and clinically significant laboratory test abnormalities. Safety was assessed until week 60 and additionally 28 days thereafter. Secondary efficacy end points were exploratory and measured disease progression.RESULTS: No treatment-emergent serious adverse events were reported. Two nontreatmentrelated deaths occurred because of progression of IPF (disease worsening and/or acute exacerbation). By 60 weeks postinfusion, there was a 3.0% mean decline in % predicted FVC and 5.4% mean decline in % predicted diffusing capacity of the lungs for carbon monoxide.CONCLUSIONS: Data from this trial support the safety of a single infusion of human mesenchymal stem cells in patients with mild-moderate IPF.

Idiopathic pulmonary fibrosis (IPF) is a lethal, chronic, progressive disease characterized by formation of scar tissue within the lungs. Because it is a disease of unknown etiology, it is difficult to diagnose, to predict disease course and to devise treatment strategies. Recent evidence suggests that activated macrophages play key roles in the pathology of IPF. Therefore, imaging probes that specifically recognize these pools of activated immune cells could provide valuable information about how these cells contribute to the pathobiology of the disease. Here we demonstrate that cysteine cathepsin-targeted imaging probes can be used to monitor the contribution of macrophages to fibrotic disease progression in the bleomycin-induced murine model of pulmonary fibrosis. Furthermore, we show that the probes highlight regions of macrophage involvement in fibrosis in human biopsy tissues from IPF patients. Finally, we present first-in-human results demonstrating non-invasive imaging of active cathepsins in fibrotic lesions of patients with IPF. Together, our findings validate small molecule cysteine cathepsin probes for clinical PET imaging and suggest that they have the potential to be used to generate mechanistically-informative molecular information regarding cellular drivers of IPF disease severity and progression.

Malformations of cortical development (MCD) have been observed in human reading and language impaired populations. Injury-induced MCD in rodent models of reading disability show morphological changes in the auditory thalamic nucleus (medial geniculate nucleus; MGN) and auditory processing impairments, thus suggesting a link between MCD, MGN, and auditory processing behavior. Previous neuroanatomical examination of a BXD29 recombinant inbred strain (BXD29-Tlr4(lps-2J)/J) revealed MCD consisting of bilateral subcortical nodular heterotopia with partial callosal agenesis. Subsequent behavioral characterization showed a severe impairment in auditory processing-a deficient behavioral phenotype seen across both male and female BXD29-Tlr4(lps-2J)/J mice. In the present study we expanded upon the neuroanatomical findings in the BXD29-Tlr4(lps-2J)/J mutant mouse by investigating whether subcortical changes in cellular morphology are present in neural structures critical to central auditory processing (MGN, and the ventral and dorsal subdivisions of the cochlear nucleus; VCN and DCN, respectively). Stereological assessment of brain tissue of male and female BXD29-Tlr4(lps-2J)/J mice previously tested on an auditory processing battery revealed overall smaller neurons in the MGN of BXD29-Tlr4(lps-2J)/J mutant mice in comparison to BXD29/Ty coisogenic controls, regardless of sex. Interestingly, examination of the VCN and DCN revealed sexually dimorphic changes in neuronal size, with a distribution shift toward larger neurons in female BXD29-Tlr4(lps-2J)/J brains. These effects were not seen in males. Together, the combined data set supports and further expands the observed co-occurrence of MCD, auditory processing impairments, and changes in subcortical anatomy of the central auditory pathway. The current stereological findings also highlight sex differences in neuroanatomical presentation in the presence of a common auditory behavioral phenotype. (C) 2014 Elsevier B.V. All rights reserved.

Genome-wide association studies are used to identify common genetic variants that affect the structure of selected subcortical regions of the human brain; their identification provides insight into the causes of variability in brain development and may help to determine mechanisms of neuropsychiatric dysfunction.

BACKGROUND:The beneficial outcome associated with the use of proton pump inhibitors (PPIs) in idiopathic pulmonary fibrosis (IPF) has been reported in retrospective studies. To date, no prospective study has been conducted to confirm these outcomes. In addition, the potential mechanism by which PPIs improve measures of lung function and/or transplant-free survival in IPF has not been elucidated.METHODS:Here, we used biochemical, cell biological and preclinical studies to evaluate regulation of markers associated with inflammation and fibrosis. In our in vitro studies, we exposed primary lung fibroblasts, epithelial and endothelial cells to ionizing radiation or bleomycin; stimuli typically used to induce inflammation and fibrosis. In addition, we cultured lung fibroblasts from IPF patients and studied the effect of esomeprazole on collagen release. Our preclinical study tested efficacy of esomeprazole in a rat model of bleomycin-induced lung injury. Furthermore, we performed retrospective analysis of interstitial lung disease (ILD) databases to examine the effect of PPIs on transplant-free survival.RESULTS:The cell culture studies revealed that esomeprazole controls inflammation by suppressing the expression of pro-inflammatory molecules including vascular cell adhesion molecule-1, inducible nitric oxide synthase, tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1β and IL-6). The antioxidant effect is associated with strong induction of the stress-inducible cytoprotective protein heme oxygenase-1 (HO1) and the antifibrotic effect is associated with potent inhibition of fibroblast proliferation as well as downregulation of profibrotic proteins including receptors for transforming growth factor β (TGFβ), fibronectin and matrix metalloproteinases (MMPs). Furthermore, esomeprazole showed robust effect in mitigating the inflammatory and fibrotic responses in a murine model of acute lung injury. Finally, retrospective analysis of two ILD databases was performed to assess the effect of PPIs on transplant-free survival in IPF patients. Intriguingly, this data demonstrated that IPF patients on PPIs had prolonged survival over controls (median survival of 3.4 vs 2 years).CONCLUSIONS:Overall, these data indicate the possibility that PPIs may have protective function in IPF by directly modulating the disease process and suggest that they may have other clinical utility in the treatment of extra-intestinal diseases characterized by inflammatory and/or fibrotic phases.

Introduction: Disease inclusion in the newborn screening (NBS) panel should consider the opinions of those most affected by the outcome of screening. We assessed the level and factors that affect parent attitudes regarding NBS panel inclusion of Duchenne muscular dystrophy (DMD), Becker muscular dystrophy (BMD), and spinal muscular atrophy (SMA). Methods: The attitudes toward NBS for DMD, BMD, and SMA were surveyed and compared for 2 categories of parents, those with children affected with DMD, BMD, or SMA and expectant parents unselected for known family medical history. Results: The level of support for NBS for DMD, BMD, and SMA was 95.9% among parents of children with DMD, BMD, or SMA and 92.6% among expectant parents. Conclusions: There was strong support for NBS for DMD, BMD, and SMA in both groups of parents. Given advances in diagnostics and promising therapeutic approaches, discussion of inclusion in NBS should continue. Muscle Nerve 49: 822-828, 2014

for all combinations of (n1, n2, n3) with n1 + n2 + n3 = N and n1,2,3 2 N. Eq. (1) yields N+2 2 templates R(n1,n2,n3) and forms multiscale steerable filterbanks when coupled with a multi–resolution framework based on isotropic band–limited wavelets (e.g., Simoncelli) [11]. The Riesz transform allows for a complete coverage of image scales and directions. The angular selectivity of the filters can be tuned with the ord r N of the transform. The second–order Riesz filterbank is depicted in Fig. 2.

Objectives: There is a need for better, noninvasive quantitative biomarkers for assessing the rate of progression and possible response to therapy in spinal muscular atrophy (SMA). In this study, we compared three electrophysiological measures: compound muscle action potential (CMAP) amplitude, motor unit number estimate (MUNE), and electrical impedance myography (EIM) 50 kHz phase values in a mild mouse model of spinal muscular atrophy, the Smn1(c/c) mouse.Methods: Smn1(c/c) mice (N = 11) and wild type (WT) animals ((-/-), N = 13) were measured on average triweekly until approximately 1 year of age. Measurements included CMAP, EIM, and MUNE of the gastrocnemius muscle as well as weight and front paw grip strength. At the time of sacrifice at one year, additional analyses were performed on the animals including serum survival motor neuron (SMN) protein levels and muscle fiber size.Results: Both EIM 50 kHz phase and CMAP showed strong differences between WT and SMA animals (repeated measures 2-way ANOVA, P < 0.0001 for both) whereas MUNE did not. Both body weight and EIM showed differences in the trajectory over time (p < 0.001 and p = 0.005, respectively). At the time of sacrifice at one year, EIM values correlated to motor neuron counts in the spinal cord and SMN levels across both groups of animals (r = 0.41, p = 0.047 and r = 0.57, p = 0.003, respectively), while CMAP did not. Motor neuron number in Smn1(c/c) mice was not significantly reduced compared to WT animals.Conclusions: EIM appears sensitive to muscle status in this mild animal model of SMA. The lack of a reduction in MUNE or motor neuron number but reduced EIM and CMAP values support that much of the pathology in these animals is distal to the cell body, likely at the neuromuscular junction or the muscle itself.

A characteristic of dysregulated wound healing in IPF is fibroblastic-mediated damage to lung epithelial cells within fibroblastic foci. In these foci, TGF-β and other growth factors activate fibroblasts that secrete growth factors and matrix regulatory proteins, which activate a fibrotic cascade. Our studies and those of others have revealed that Akt is activated in IPF fibroblasts and it mediates the activation by TGF-β of pro-fibrotic pathways. Recent studies show that mTORC2, a component of the mTOR pathway, mediates the activation of Akt. In this study we set out to determine if blocking mTORC2 with MLN0128, an active site dual mTOR inhibitor, which blocks both mTORC1 and mTORC2, inhibits lung fibrosis. We examined the effect of MLN0128 on TGF-β-mediated induction of stromal proteins in IPF lung fibroblasts; also, we looked at its effect on TGF-β-mediated epithelial injury using a Transwell co-culture system. Additionally, we assessed MLN0128 in the murine bleomycin lung model. We found that TGF-β induces the Rictor component of mTORC2 in IPF lung fibroblasts, which led to Akt activation, and that MLN0128 exhibited potent anti-fibrotic activity in vitro and in vivo. Also, we observed that Rictor induction is Akt-mediated. MLN0128 displays multiple anti-fibrotic and lung epithelial-protective activities; it (1) inhibited the expression of pro-fibrotic matrix-regulatory proteins in TGF-β-stimulated IPF fibroblasts; (2) inhibited fibrosis in a murine bleomycin lung model; and (3) protected lung epithelial cells from injury caused by TGF-β-stimulated IPF fibroblasts. Our findings support a role for mTORC2 in the pathogenesis of lung fibrosis and for the potential of active site mTOR inhibitors in the treatment of IPF and other fibrotic lung diseases.

Rationale: The cardiovascular enzyme dimethylarginine dimethylaminohydrolase (DDAH) has recently been implicated in the progression of idiopathic pulmonary fibrosis (IPF). By degrading asymmetric dimethylarginine (ADMA), an inhibitor of inducible nitric oxide (NO) synthase (iNOS), DDAH releases the brake on a pathway implicated in lung injury and fibrosis. In addition, DDAH promotes the pro-fibrotic activity of the TGFβ pathway. Objectives: We embarked on studies to discover DDAH inhibitors, and to assess the effect of such inhibitors on processes involved in IPF. Methods: We performed high throughput screening (HTS) of over 130,000 small molecules and discovered that the proton pump inhibitors (PPIs) modestly inhibit DDAH. We used cellular and molecular assays to study the effect of the PPIs on DDAH activity by measuring the concentration of ADMA. In addition, we assessed the effect of PPIs on collagen synthesis by IPF lung fibroblasts, and on the survival and proliferation of lung alveolar epithelial cells. Finally, a retrospective analysis of the Stanford University interstitial lung disease (ILD) database was performed to assess the effect of PPIs on transplant-free survival. Results: We found that the PPIs inhibit DDAH activity in biochemical assays, cell culture and in vivo. The resulting increase in ADMA level was mirrored by a reduction in NO production. In addition, the PPIs reduced the production of soluble collagen by primary IPF lung fibroblasts in response to TGFβ and regulated both proliferation and survival of lung alveolar epithelial cells. Retrospective review of the ILD database of 131 IPF patients (of who 85 were on PPIs for > 1 year) revealed a survival advantage (i.e. time to transplant or death when calculated from the time of diagnosis) of the patients on PPIs, including those without symptoms of Gastroesophageal disease (GERD); an indication for which the PPIs are primarily prescribed for, suggesting the involvement of a mechanism other than acid-suppression. Conclusions: Our data reveal a plausible biological explanation for the clinical benefit of the PPIs in IPF. The reported reduction in radiologic fibrosis score and significant survival advantage of IPF patients on PPIs may be due to modulation of the DDAH/NOS pathway.

Developmental dyslexia is a language learning disorder that affects approximately 4-10% of the population. A number of candidate dyslexia susceptibility genes have been identified, including DCDC2 and KIAA0319 on Chromosome (Chr) 6p22.2 and DYX1C1 on Chr 15q21. Embryonic knockdown of the function of homologs of these genes in rat neocortical projection cell progenitors by in utero electroporation of plasmids encoding small hairpin RNA (shRNA) revealed that all three genes disrupted neuronal migration to the neocortex. Specifically, this disruption would result in heterotopia formation (Dyx1c1 and Kiaa0319) and/or overmigration past their expected laminar location (Dyx1c1 and Dcdc2). In these experiments, neurons normally destined for the upper neocortical laminæ were transfected on embryonic day (E) 15.5, and we designed experiments to test whether these migration phenotypes were the result of targeting a specific type of projection neuron. We transfected litters with Dcdc2 shRNA, Dyx1c1 shRNA, Kiaa0319 shRNA, or fluorescent protein (as a control) at each of three gestational ages (E14.5, E15.5, or E16.5). Pups were allowed to come to term, and their brains were examined at 3 weeks of age for the position of transfected cells. We found that age of transfection did not affect the percentage of unmigrated neurons--transfection with Kiaa0319 shRNA resulted in heterotopia formation at all three ages. Overmigration of neurons transfected with Dcdc2 shRNA, while present following transfections at the later ages, did not occur following E14.5 transfections. These results are considered in light of the known functions of each of these candidate dyslexia susceptibility genes.

Disruption of neuronal migration in humans is associated with a wide range of behavioral and cognitive outcomes including severe intellectual disability, language impairment, and social dysfunction. Furthermore, malformations of cortical development have been observed in a number of neurodevelopmental disorders (e.g. autism and dyslexia), where boys are much more commonly diagnosed than girls (estimates around 4 to 1). The use of rodent models provides an excellent means to examine how sex may modulate behavioral outcomes in the presence of comparable abnormal neuroanatomical presentations. Initially characterized by Rosen et al. 2012, the BXD29- Tlr4(lps-2J) /J mouse mutant exhibits a highly penetrant neuroanatomical phenotype that consists of bilateral midline subcortical nodular heterotopia with partial callosal agenesis. In the current study, we confirm our initial findings of a severe impairment in rapid auditory processing in affected male mice. We also report that BXD29- Tlr4(lps-2J) /J (mutant) female mice show no sparing of rapid auditory processing, and in fact show deficits similar to mutant males. Interestingly, female BXD29- Tlr4(lps-2J) /J mice do display superiority in Morris water maze performance as compared to wild type females, an affect not seen in mutant males. Finally, we report new evidence that BXD29- Tlr4(lps-2J) /J mice, in general, show evidence of hyper-social behaviors. In closing, the use of the BXD29- Tlr4(lps-2J) /J strain of mice - with its strong behavioral and neuroanatomical phenotype - may be highly useful in characterizing sex independent versus dependent mechanisms that interact with neural reorganization, as well as clinically relevant abnormal behavior resulting from aberrant neuronal migration.