Search

Understanding how asthma biomarkers relate to gene expression signatures could help identify drivers of pathogenesis.This post hoc exploratory analysis of the phase II tralokinumab trial MESOS (ClinicalTrials.gov identifier NCT02449473) aimed to profile baseline airway inflammation in patients with moderate-to-severe asthma.The T2 and T17 gene expression signatures, 3-gene mean and 5-gene mean, were calculated through transcriptomic analysis of baseline bronchial brushing samples. Clustering analysis using these signatures identified 3 distinct inflammatory subgroups: T2Fractional exhaled nitric oxide (Feno) levels were highest for T2Feno level may be useful to differentiate patients with T2 or T17 gene expression. Elevated Feno levels are associated with high CST1 expression.

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2 . On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Summary Background Tezepelumab is a human monoclonal antibody that blocks the activity of thymic stromal lymphopoietin (TSLP), an epithelial cell-derived cytokine. In phase 2b and 3 studies, tezepelumab significantly reduced exacerbations versus placebo in patients with severe uncontrolled asthma, irrespective of baseline levels of type 2 inflammatory biomarkers. We investigated the mechanism of action of tezepelumab by assessing its effects on airway inflammatory cells, airway remodelling, and airway hyperresponsiveness. Methods CASCADE was an exploratory, double-blind, randomised, placebo-controlled, parallel-group, phase 2 study done in 27 medical centres in Canada, Denmark, Germany, the UK, and the USA. Adults aged 18–75 years with uncontrolled, moderate-to-severe asthma were randomly assigned (1:1) to receive tezepelumab 210 mg or placebo administered subcutaneously every 4 weeks for a planned 28 weeks, extended to up to 52 weeks if COVID-19-related disruption delayed participants' end-of-treatment assessments. Randomisation was balanced and stratified by blood eosinophil count. The primary endpoint was the change from baseline to the end of treatment in the number of airway submucosal inflammatory cells in bronchoscopic biopsy samples. Eosinophils, neutrophils, CD3+ T cells, CD4+ T cells, tryptase+ mast cells, and chymase+ mast cells were evaluated separately. This endpoint was also assessed in subgroups according to baseline type 2 inflammatory biomarker levels, including blood eosinophil count. Airway remodelling was assessed via the secondary endpoints of change from baseline in reticular basement membrane thickness and epithelial integrity (proportions of denuded, damaged, and intact epithelium). Exploratory outcomes included airway hyperresponsiveness to mannitol. All participants who completed at least 20 weeks of study treatment, had an end-of-treatment visit up to 8 weeks after the last dose of study drug, and had evaluable baseline and end-of-treatment bronchoscopies were included in the primary efficacy analysis. All participants who received at least one dose of study drug were included in the safety analyses. This study is registered with ClinicalTrials.gov, NCT03688074. Findings Between Nov 2, 2018, and Nov 16, 2020, 250 patients were enrolled, 116 of whom were randomly assigned (59 to tezepelumab, 57 to placebo). 48 in the tezepelumab group and 51 in the placebo group completed the study and were assessed for the primary endpoint. Treatment with tezepelumab resulted in a nominally significantly greater reduction from baseline to the end of treatment in airway submucosal eosinophils versus placebo (ratio of geometric least-squares means 0·15 [95% CI 0·05–0·41]; nominal p 0·10). In assessment of secondary endpoints, there were no significant differences between treatment groups in reticular basement membrane thickness and epithelial integrity. In an exploratory analysis, the reduction in airway hyperresponsiveness to mannitol was significantly greater with tezepelumab versus placebo (least-squares mean change from baseline in interpolated or extrapolated provoking dose of mannitol required to induce ≥15% reduction in FEV1 from baseline: tezepelumab 197·4 mg [95% CI 107·9 to 286·9]; placebo 58·6 mg [−30·1 to 147·33]; difference 138·8 [14·2 to 263·3], nominal p=0·030). Adverse events were reported in 53 (90%) patients in the tezepelumab group and 51 (90%) patients in the placebo group, and there were no safety findings of concern. Interpretation The improvements in asthma clinical outcomes observed in previous studies with tezepelumab are probably driven, at least in part, by reductions in eosinophilic airway inflammation, as shown here by reduced airway eosinophil counts regardless of baseline blood eosinophil count. Tezepelumab also reduced airway hyperresponsiveness to mannitol, indicating that TSLP blockade might have additional benefits in asthma beyond reducing type 2 airway inflammation. Funding AstraZeneca and Amgen.

Background Eosinophilic inflammation has been implicated in the pathogenesis, severity, and treatment responsiveness of chronic rhinosinusitis with nasal polyps (CRSwNP). Objective We sought to assess the efficacy and safety of benralizumab-mediated eosinophil depletion for treating CRSwNP. Methods The phase 3 OSTRO study enrolled patients with severe CRSwNP who were symptomatic despite treatment with intranasal corticosteroids and who had a history of systemic corticosteroid (SCS) use and/or surgery for nasal polyps (NP). Patients were randomized 1:1 to treatment with benralizumab 30 mg or placebo every 4 weeks for the first 3 doses and every 8 weeks thereafter. Coprimary end points were change from baseline to week 40 in NP score (NPS) and patient-reported mean nasal blockage score reported once every 2 weeks. Results The study population comprised 413 randomized patients (207 in the benralizumab group and 206 in the placebo group). Benralizumab significantly improved NPS and nasal blockage score compared to placebo at week 40 (P ≤ .005). Improvements in Sinonasal Outcome Test 22 score at week 40, time to first NP surgery and/or SCS use for NP, and time to first NP surgery were not statistically significant between treatment groups. Nominal significance was obtained for improvement in difficulty in sense of smell score at week 40 (P = .003). Subgroup analyses suggested influences of comorbid asthma, number of NP surgeries, sex, body mass index, and baseline blood eosinophil count on treatment effects. Benralizumab was safe and well tolerated. Conclusion Benralizumab, when added to standard-of-care therapy, reduced NPS, decreased nasal blockage, and reduced difficulty with sense of smell compared to placebo in patients with CRSwNP. Trial registration: ClinicalTrials.gov NCT03401229

Fibrosis or cirrhosis is the major risk factor associated with morbidity and mortality in patients with non-alcoholic steatohepatitis (NASH)-driven chronic liver disease. While numerous efforts have been made to identify mediators of the initiation of liver fibrosis, the molecular underpinnings of fibrosis progression remain poorly understood and anti-fibrotic therapies to arrest liver fibrosis progression and promote resolution are elusive. Here, we identify a pathway involving WNT1 inducible signaling pathway protein 1 (WISP1) and myocardin related transcription factor (MRTF), as a central mechanism driving liver fibrosis progression through transcriptional reprogramming of myofibroblast cytoskeleton and motility. WISP1 expression is highly correlated with MRTF activity in human cirrhotic livers and can activate MRTF signaling in vitro. In mice, WISP1 deficiency protects against fibrosis progression but not fibrosis onset and leads to reduced myofibroblast accumulation in tissue interstitial areas and diminished expression of MRTF target cytoskeleton genes. WISP1 activates MRTF signaling via multiple integrins including αVβ1/3/5/8 and α11β1. Moreover, therapeutic administration of a novel antibody blocking WISP1-MRTF signaling axis halted the progression of existing liver fibrosis. These findings implicate the WISP1-MRTF axis as a crucial determinant of liver fibrosis progression and support targeting this pathway by antibody-based therapy for the treatment of NASH fibrosis and its associated complications.

Transforming growth factor-β (TGFβ) is a key driver of fibrogenesis. Three TGFβ isoforms (TGFβ1, TGFβ2, and TGFβ3) in mammals have distinct functions in embryonic development; however, the postnatal pathological roles and activation mechanisms of TGFβ2 and TGFβ3 have not been well characterized. Here, we show that the latent forms of TGFβ2 and TGFβ3 can be activated by integrin-independent mechanisms and have lower activation thresholds compared to TGFβ1. Unlike TGFB1, TGFB2 and TGFB3 expression is increased in human lung and liver fibrotic tissues compared to healthy control tissues. Thus, TGFβ2 and TGFβ3 may play a pathological role in fibrosis. Inducible conditional knockout mice and anti-TGFβ isoform-selective antibodies demonstrated that TGFβ2 and TGFβ3 are independently involved in mouse fibrosis models in vivo, and selective TGFβ2 and TGFβ3 inhibition does not lead to the increased inflammation observed with pan-TGFβ isoform inhibition. A cocrystal structure of a TGFβ2-anti-TGFβ2/3 antibody complex reveals an allosteric isoform-selective inhibitory mechanism. Therefore, inhibiting TGFβ2 and/or TGFβ3 while sparing TGFβ1 may alleviate fibrosis without toxicity concerns associated with pan-TGFβ blockade.

The dysregulation of multiple signaling pathways, including those through endosomal Toll-like receptors (TLRs), Fc gamma receptors (FcγR), and antigen receptors in B cells (BCR), promote an autoinflammatory loop in systemic lupus erythematosus (SLE). Here, we used selective small-molecule inhibitors to assess the regulatory roles of interleukin-1 receptor (IL-1R)-associated kinase 4 (IRAK4) and Bruton's tyrosine kinase (BTK) in these pathways. The inhibition of IRAK4 repressed SLE immune complex- and TLR7-mediated activation of human plasmacytoid dendritic cells (pDCs). Correspondingly, the expression of interferon (IFN)-responsive genes (IRGs) in cells and in mice was positively regulated by the kinase activity of IRAK4. Both IRAK4 and BTK inhibition reduced the TLR7-mediated differentiation of human memory B cells into plasmablasts. TLR7-dependent inflammatory responses were differentially regulated by IRAK4 and BTK by cell type: In pDCs, IRAK4 positively regulated NF-κB and MAPK signaling, whereas in B cells, NF-κB and MAPK pathways were regulated by both BTK and IRAK4. In the pristane-induced lupus mouse model, inhibition of IRAK4 reduced the expression of IRGs during disease onset. Mice engineered to express kinase-deficient IRAK4 were protected from both chemical (pristane-induced) and genetic (NZB/W_F1 hybrid) models of lupus development. Our findings suggest that kinase inhibitors of IRAK4 might be a therapeutic in patients with SLE.

Novel proteomics platforms, such as the aptamer-based SOMAscan platform, can quantify large numbers of proteins efficiently and cost-effectively and are rapidly growing in popularity. However, comparisons to conventional immunoassays remain underexplored, leaving investigators unsure when cross-assay comparisons are appropriate. We explored the correlation of results from immunoassays with relative protein quantification by SOMAscan. For 63 proteins assessed in two chronic obstructive pulmonary disease (COPD) cohorts, Subpopulations and Intermediate Outcome Measures in COPD Study (SPIROMICS) and COPDGene, using Myriad Rules Based Medicine (RBM) multiplex immunoassays and SOMAscan, Spearman correlation coefficients ranged from −0.13 to 0.97, with a median correlation coefficient of ~0.5 and consistent results across cohorts. A similar range was observed for immunoassays in the population based Multi-Ethnic Study of Atherosclerosis (MESA) and for other assays in COPDGene and SPIROMICS. Comparisons of relative quantification from the antibody-based Olink platform and SOMAscan in a small cohort of myocardial infarction patients also showed a wide correlation range. Finally, we integrated cis pQTL data, mass spectrometry aptamer confirmation, and other publicly available data to assess relationships with observed correlations. Correlation between proteomics assays shows a wide range and should be carefully considered when comparing and meta-analyzing proteomics data across assays and studies.

Periostin and dipeptidyl peptidase-4 (DPP-4) are proteins induced by type 2 cytokines interleukin (IL)-4 and IL-13 and show increased expression in asthma and diseases with type 2 inflammation, including atopic dermatitis and chronic rhinosinusitis. Both proteins can also be induced by other stimuli, such as profibrotic factors, which may confound their specificity as biomarkers of IL-13 pathway activation and type 2-driven disease. DPP-4 is important in glucose metabolism; therefore, serum concentrations may be confounded by the presence of concomitant metabolic disease. This review evaluates the potential of these biomarkers for anti-IL-13-directed therapy in asthma and diseases with type 2 inflammation.

Background & Aims The incidence of nonalcoholic steatohepatitis (NASH) is increasing. The pathophysiological mechanisms of NASH and the sequence of events leading to hepatic fibrosis are incompletely understood. The aim of this study was to gain insight into the dynamics of key molecular processes involved in NASH and to rank early markers for hepatic fibrosis. Methods A time-course study in low-density lipoprotein–receptor knockout. Leiden mice on a high-fat diet was performed to identify the temporal dynamics of key processes contributing to NASH and fibrosis. An integrative systems biology approach was used to elucidate candidate markers linked to the active fibrosis process by combining transcriptomics, dynamic proteomics, and histopathology. The translational value of these findings were confirmed using human NASH data sets. Results High-fat-diet feeding resulted in obesity, hyperlipidemia, insulin resistance, and NASH with fibrosis in a time-dependent manner. Temporal dynamics of key molecular processes involved in the development of NASH were identified, including lipid metabolism, inflammation, oxidative stress, and fibrosis. A data-integrative approach enabled identification of the active fibrotic process preceding histopathologic detection using a novel molecular fibrosis signature. Human studies were used to identify overlap of genes and processes and to perform a network biology-based prioritization to rank top candidate markers representing the early manifestation of fibrosis. Conclusions An early predictive molecular signature was identified that marked the active profibrotic process before histopathologic fibrosis becomes manifest. Early detection of the onset of NASH and fibrosis enables identification of novel blood-based biomarkers to stratify patients at risk, development of new therapeutics, and help shorten (pre)clinical experimental time frames., Graphical abstract

Background: In HIV infection, lymphoid tissue is disrupted by fibrosis. Angiotensin converting enzyme inhibitors have anti-fibrotic properties. We completed a pilot study to assess whether the addition of lisinopril to antiretroviral therapy (ART) reverses fibrosis of gut tissue, and whether this leads to reduction of HIV RNA and DNA levels. Methods: Thirty HIV-infected individuals on ART were randomized to lisinopril at 20mg daily or matching placebo for 24 weeks. All participants underwent rectal biopsies prior to starting the study drug and at 22 weeks, and there were regular blood draws. The primary end point was the change in HIV RNA and DNA levels in rectal tissue. Secondary outcomes included the change in 1) HIV levels in blood; 2) Gag-specific T-cell responses; 3) levels of T-cell activation; and 4) collagen deposition. Results: The addition of lisinopril did not have a significant effect on the levels of HIV RNA or DNA in gut tissue or blood, Gag-specific responses, or levels of T-cell activation. Lisinopril also did not have a significant impact on lymphoid fibrosis in the rectum, as assessed by quantitative histology or heavy water labeling. Conclusions: Treatment with lisinopril for 24 weeks in HIV-infected adults did not have an effect on lymphoid fibrosis, immune activation, or gut tissue viral reservoirs. Further study is needed to see if other anti-fibrotic agents may be useful in reversing lymphoid fibrosis and reducing HIV levels. Clinical Trials Registration: NCT01535235

Background: Chronic obstructive pulmonary disease (COPD) is a common, complex respiratory disease characterized by airway obstruction and accelerated lung function decline. Our understanding of systemic protein biomarkers associated with COPD remains incomplete. We tested whether aptamer-based protein profiling could identify markers and pathways related to impaired pulmonary function and lung function decline in multiple studies. Methods: We studied 3,827 non-Hispanic/European White participants across six cohort studies with both aptamer-based proteomic and spirometry data. In linear regression models we examined protein associations with baseline FEV1 and FEV1/FVC. In linear mixed effects models we investigated the associations of baseline protein levels with rate of FEV1 decline (mL/year) in 2,636 participants with up to 7 years of follow-up spirometry. Findings: We identified 198 proteins associated with FEV1, twelve of the proteins were also significantly associated with FEV1/FVC, including retinal binding protein 4 FEV1 : β=0·0307, Q= 2·18×10-4; FEV1/FVC: β=0·008, Q =4·0×10-3 ) and bactericidal permeability-increasing protein (FEV1 : β=-0·0280, Q =6·80×10-3; FEV1/FVC: β=0·0050, Q =0·04). We further identified 15 proteins associated with the rate of FEV1 decline ( Q

[Image: see text] IRAK4 kinase activity transduces signaling from multiple IL-1Rs and TLRs to regulate cytokines and chemokines implicated in inflammatory diseases. As such, there is high interest in identifying selective IRAK4 inhibitors for the treatment of these disorders. We previously reported the discovery of potent and selective dihydrobenzofuran inhibitors of IRAK4. Subsequent studies, however, showed inconsistent inhibition in disease-relevant pharmacodynamic models. Herein, we describe application of a human whole blood assay to the discovery of a series of benzolactam IRAK4 inhibitors. We identified potent molecule 19 that achieves robust in vivo inhibition of cytokines relevant to human disease.

A series of pyrazolopyrimidine inhibitors of IRAK4 were developed from a high-throughput screen (HTS). Modification of an HTS hit led to a series of bicyclic heterocycles with improved potency and kinase selectivity but lacking sufficient solubility to progress in vivo. Structure-based drug design, informed by cocrystal structures with the protein and small-molecule crystal structures, yielded a series of dihydrobenzofurans. This semisaturated bicycle provided superior druglike properties while maintaining excellent potency and selectivity. Improved physicochemical properties allowed for progression into in vivo experiments, where lead molecules exhibited low clearance and showed target-based inhibition of IRAK4 signaling in an inflammation-mediated PK/PD mouse model.

Chronic lymphocytic leukemia (CLL) is a progressive malignancy of mature B-cells that involves the peripheral blood (PB), lymph nodes (LNs) and bone marrow (BM). Although the majority of CLL cells are in a resting state, small populations of proliferating cells exist; however, the anatomical site of active cell proliferation remains to be definitively determined. Based on findings that CLL cells in LNs have increased expression of B-cell activation genes, we tested the hypothesis that the fraction of 'newly born' cells would be highest in the LNs. Using a deuterium oxide (2H) in vivo labeling method in which patients consumed deuterated (heavy) water (2H2O), we determined CLL cell kinetics in concurrently obtained samples from LN, PB and BM. The LN was identified as the anatomical site harboring the largest fraction of newly born cells, compared to PB and BM. In fact, the calculated birth rate in the LN reached as high a 3.3% of the clone per day. Subdivision of the bulk CLL population by flow cytometry identified the subpopulation with the CXCR4dimCD5bright phenotype as containing the highest proportion of newly born cells within each compartment, including the LN, identifying this subclonal population as an important target for novel treatment approaches.

Tumor progression locus 2 (TPL2; also known as MAP3K8) is a mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) that phosphorylates the MAPK kinases MEK1 and MEK2 (MEK1/2), which, in turn, activate the MAPKs extracellular signal-regulated kinase 1 (ERK1) and ERK2 (ERK1/2) in macrophages stimulated through the interleukin-1 receptor (IL-1R), Toll-like receptors (TLRs), or the tumor necrosis factor receptor (TNFR). We describe a conserved and critical role for TPL2 in mediating the effector functions of neutrophils through the activation of the p38 MAPK signaling pathway. Gene expression profiling and functional studies of neutrophils and monocytes revealed a MEK1/2-independent branch point downstream of TPL2 in neutrophils. Biochemical analyses identified the MAPK kinases MEK3 and MEK6 and the MAPKs p38α and p38δ as downstream effectors of TPL2 in these cells. Genetic ablation of the catalytic activity of TPL2 or therapeutic intervention with a TPL2-specific inhibitor reduced the production of inflammatory mediators by neutrophils in response to stimulation with the TLR4 agonist lipopolysaccharide (LPS) in vitro, as well as in rodent models of inflammatory disease. Together, these data suggest that TPL2 is a drug target that activates not only MEK1/2-dependent but also MEK3/6-dependent signaling to promote inflammatory responses.

Fibrotic disease is characterized by the pathological accumulation of extracellular matrix (ECM) proteins. Surprisingly, very little is known about the synthesis and degradation rates of the many proteins and proteoglycans that constitute healthy or pathological extracellular matrix. A comprehensive understanding of altered ECM protein synthesis and degradation during the onset and progression of fibrotic disease would be immensely valuable. We have developed a dynamic proteomics platform that quantifies the fractional synthesis rates of large numbers of proteins via stable isotope labeling and LC/MS-based mass isotopomer analysis. Here, we present the first broad analysis of ECM protein kinetics during the onset of experimental pulmonary fibrosis. Mice were labeled with heavy water for up to 21 days following the induction of lung fibrosis with bleomycin. Lung tissue was subjected to sequential protein extraction to fractionate cellular, guanidine-soluble ECM proteins and residual insoluble ECM proteins. Fractional synthesis rates were calculated for 34 ECM proteins or protein subunits, including collagens, proteoglycans, and microfibrillar proteins. Overall, fractional synthesis rates of guanidine-soluble ECM proteins were faster than those of insoluble ECM proteins, suggesting that the insoluble fraction reflected older, more mature matrix components. This was confirmed through the quantitation of pyridinoline cross-links in each protein fraction. In fibrotic lung tissue, there was a significant increase in the fractional synthesis of unique sets of matrix proteins during early (pre-1 week) and late (post-1 week) fibrotic response. Furthermore, we isolated fast turnover subpopulations of several ECM proteins (e.g. type I collagen) based on guanidine solubility, allowing for accelerated detection of increased synthesis of typically slow-turnover protein populations. This establishes the presence of multiple kinetic pools of pulmonary collagen in vivo with altered turnover rates during evolving fibrosis. These data demonstrate the utility of dynamic proteomics in analyzing changes in ECM protein turnover associated with the onset and progression of fibrotic disease.

BACKGROUND. Ibrutinib is an effective targeted therapy for patients with chronic lymphocytic leukemia (CLL) that inhibits Bruton's tyrosine kinase (BTK), a kinase involved in B cell receptor signaling. METHODS. We used stable isotopic labeling with deuterated water (2H2O) to measure directly the effects of ibrutinib on leukemia cell proliferation and death in 30 patients with CLL. RESULTS. The measured average CLL cell proliferation ("birth") rate before ibrutinib therapy was 0.39% of the clone per day (range 0.17%-1.04%); this decreased to 0.05% per day (range 0%-0.36%) with treatment. Death rates of blood CLL cells increased from 0.18% per day (average, range 0%-0.7%) prior to treatment to 1.5% per day (range 0%-3.0%) during ibrutinib therapy, and they were even higher in tissue compartments. CONCLUSIONS. This study provides the first direct in vivo measurements to our knowledge of ibrutinib's antileukemia actions, demonstrating profound and immediate inhibition of CLL cell proliferation and promotion of high rates of CLL cell death. TRIAL REGISTRATION. This trial was registered at clinicaltrials.gov (NCT01752426). FUNDING. This study was supported by a Cancer Center Support Grant (National Cancer Institute grant P30 CA016672), an NIH grant (CA081554) from the National Cancer Institute, MD Anderson's Moon Shots Program in CLL, and Pharmacyclics, an AbbVie company.

Excess collagen synthesis (fibrogenesis) in the liver plays a causal role in the progression of nonalcoholic fatty liver disease (NAFLD). Methods are needed to identify patients with more rapidly progressing disease and to demonstrate early response to treatment. We describe here a novel method to quantify hepatic fibrogenesis flux rates both directly in liver tissue and non-invasively in blood. Twenty-one patients with suspected NAFLD ingested heavy water (2H2O, 50 ml aliquots) 2-3 times daily for 3 to 5 weeks prior to a clinically indicated liver-biopsy. Liver collagen fractional synthesis rate (FSR) and plasma lumican FSR were measured based on 2H labeling using tandem mass spectrometry. Patients were classified by histology for fibrosis stage (F0-F4) and as having non-alcoholic fatty liver (NAFL) or non-alcoholic steatohepatitis (NASH). Magnetic resonance elastography measurements of liver stiffness were also performed. Hepatic collagen FSR in NAFLD increased with advancing disease stage (e.g. higher in NASH than NAFL, positive correlation with fibrosis score and liver stiffness) and correlated with hemoglobin A1C. In addition, plasma lumican FSR demonstrated a significant correlation with hepatic collagen FSR. Conclusion: Utilizing a well-characterized cohort of patients with biopsy-proven NAFLD, this study demonstrates that hepatic scar in NASH is actively remodeled even in advanced fibrosis, a disease that is generally regarded as static and slowly progressive. Moreover, hepatic collagen FSR correlates with established risks for fibrotic disease progression in NASH and plasma lumican FSR correlates with hepatic collagen FSR, suggesting applications as direct or surrogate markers, respectively, of hepatic fibrogenesis in humans. This article is protected by copyright. All rights reserved.

Pulmonary arterial remodeling characterized by increased vascular smooth muscle density is a common lesion seen in pulmonary arterial hypertension (PAH), a deadly condition. Clinical correlation studies have suggested an immune pathogenesis of pulmonary arterial remodeling, but experimental proof has been lacking. We show that immunization and prolonged intermittent challenge via the airways with either of two different soluble antigens induced severe muscularization in small- to medium-sized pulmonary arteries. Depletion of CD4+ T cells, antigen-specific T helper type 2 (Th2) response, or the pathogenic Th2 cytokine interleukin 13 significantly ameliorated pulmonary arterial muscularization. The severity of pulmonary arterial muscularization was associated with increased numbers of epithelial cells and macrophages that expressed a smooth muscle cell mitogen, resistin-like molecule α, but surprisingly, there was no correlation with pulmonary hypertension. Our data are the first to provide experimental proof that the adaptive immune response to a soluble antigen is sufficient to cause severe pulmonary arterial muscularization, and support the clinical observations in pediatric patients and in companion animals that muscularization represents one of several injurious events to the pulmonary artery that may collectively contribute to PAH.

Using a single vector targeting strategy, we have generated mice with a combined deficiency of interleukin (IL)-4 and IL-13 to clarify their roles in T helper type 2 (Th2) cell responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of the double-deficient mice with those generated by wild-type, IL-4–deficient, and IL-13–deficient mice. Using a pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that although eosinophil infiltration, immunoglobulin E, and IL-5 production are reduced in the IL-4–deficient mice and IL-13–deficient mice, they are abolished only in the combined absence of both cytokines. Furthermore, IL-4/13–deficient animals are severely impaired in their ability to expel the gastrointestinal nematode Nippostrongylus brasiliensis. Unexpectedly, N. brasiliensis–infected IL-4/13–deficient mice developed elevated IL-5 and eosinophilia, indicating that compensatory mechanisms exist for the expression of IL-5, although serum IgE remained undetectable. IL-4/13–deficient mice default to a Th1-like phenotype characterized by the expression of interferon γ and the production of IgG2a and IgG2b. We conclude that IL-4 and IL-13 cooperate to initiate rapid Th2 cell–driven responses, and that although their functions overlap, they perform additive roles.

We report that Th2 cell cultures generated using T cells or splenocytes from IL-13-deficient mice produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with wild-type. In contrast, IL-4 and IL-5 production by mast cells stimulated in vitro with PMA, ionomycin, or IgE cross-linking are unaffected. In vitro Th2 cell differentiation cannot be rescued by the addition of exogenous factors, but in vivo antigen challenge and administration of IL-13 can increase Th2-like cytokine responses as can infection with the parasitic nematode Nippostrongylus brasiliensis. IL-13-deficient mice also have lower basal levels of serum IgE and biased antigen-specific immunoglobulin responses. Thus, IL-13 is an important regulator of Th2 commitment and may therefore play a central role in atopy and infectious diseases.

Background Biomarkers facilitate early detection of disease and measurement of therapeutic efficacy, both at clinical and experimental levels. Recent advances in analytics and disease models allow comprehensive screening for biomarkers in complex diseases, such as asthma, that was previously not feasible. Objective Using murine and nonhuman primate (NHP) models of asthma, identify biomarkers associated with early and chronic stages of asthma and responses to steroid treatment. Methods The total protein content from thymic stromal lymphopoietin transgenic (TSLP Tg) mouse BAL fluid was ascertained by shotgun proteomics analysis. A subset of these potential markers was further analyzed in BAL fluid, BAL cell mRNA, and lung tissue mRNA during the stages of asthma and following corticosteroid treatment. Validation was conducted in murine and NHP models of allergic asthma. Results Over 40 proteins were increased in the BAL fluid of TSLP Tg mice that were also detected by qRT-PCR in lung tissue and BAL cells, as well as in OVA-sensitive mice and house dust mite-sensitive NHP. Previously undescribed as asthma biomarkers, KLK1, Reg3γ, ITLN2, and LTF were modulated in asthmatic mice, and Clca3, Chi3l4 (YM2), and Ear11 were the first lung biomarkers to increase during disease and the last biomarkers to decline in response to therapy. In contrast, GP-39, LCN2, sICAM-1, YM1, Epx, Mmp12, and Klk1 were good indicators of early therapeutic intervention. In NHP, AMCase, sICAM-1, CLCA1, and GP-39 were reduced upon treatment with corticosteroids. Conclusions and clinical relevance These results significantly advance our understanding of the biomarkers present in various tissue compartments in animal models of asthma, including those induced early during asthma and modulated with therapeutic intervention, and show that BAL cells (or their surrogate, induced sputum cells) are a viable choice for biomarker examination.

Background Interleukin (IL)-19 has been reported to enhance chronic inflammatory diseases such as asthma but the in vivo mechanism is incompletely understood. Because IL-19 is produced by and regulates cells of the monocyte lineage, our studies focused on in vivo responses of CD11c positive (CD11c+) alveolar macrophages and lung dendritic cells. Methodology/Principal Findings IL-19-deficient (IL-19-/-) mice were studied at baseline (naive) and following intranasal challenge with microbial products, or recombinant cytokines. Naive IL-19-/- mixed background mice had a decreased percentage of CD11c+ cells in the bronchoalveolar-lavage (BAL) due to the deficiency in IL-19 and a trait inherited from the 129-mouse strain. BAL CD11c+ cells from fully backcrossed IL-19-/- BALB/c or C57BL/6 mice expressed significantly less Major Histocompatibility Complex class II (MHCII) in response to intranasal administration of lipopolysaccharide, Aspergillus antigen, or IL-13, a pro-allergic cytokine. Neurogenic-locus-notch-homolog-protein-2 (Notch2) expression by lung monocytes, the precursors of BAL CD11c+ cells, was dysregulated: extracellular Notch2 was significantly decreased, transmembrane/intracellular Notch2 was significantly increased in IL-19-/- mice relative to wild type. Instillation of recombinant IL-19 increased extracellular Notch2 expression and dendritic cells cultured from bone marrow cells in the presence of IL-19 showed upregulated extracellular Notch2. The CD205 positive subset among the CD11c+ cells was 3-5-fold decreased in the airways and lungs of naive IL-19-/- mice relative to wild type. Airway inflammation and histological changes in the lungs were ameliorated in IL-19-/- mice challenged with Aspergillus antigen that induces T lymphocyte-dependent allergic inflammation but not in IL-19-/- mice challenged with lipopolysaccharide or IL-13. Conclusions/Significance Because MHCII is the molecular platform that displays peptides to T lymphocytes and Notch2 determines cell fate decisions, our studies suggest that endogenous IL-19 is a constituent of the regulome that controls both processes in vivo.

Milk fat globule epidermal growth factor 8 (Mfge8) is a soluble glycoprotein known to regulate inflammation and immunity by mediating apoptotic cell clearance. Since fibrosis can occur as a result of exaggerated apoptosis and inflammation, we set out to investigate the hypothesis that Mfge8 might negatively regulate tissue fibrosis. We report here that Mfge8 does decrease the severity of tissue fibrosis in a mouse model of pulmonary fibrosis; however, it does so not through effects on inflammation and apoptotic cell clearance, but by binding and targeting collagen for cellular uptake through its discoidin domains. Initial analysis revealed that Mfge8–/– mice exhibited enhanced pulmonary fibrosis after bleomycin-induced lung injury. However, they did not have increased inflammation or impaired apoptotic cell clearance after lung injury compared with Mfge8+/+ mice; rather, they had a defect in collagen turnover. Further experiments indicated that Mfge8 directly bound collagen and that Mfge8–/– macrophages exhibited defective collagen uptake that could be rescued by recombinant Mfge8 containing at least one discoidin domain. These data demonstrate a critical role for Mfge8 in decreasing the severity of murine tissue fibrosis by facilitating the removal of accumulated collagen.

Accumulation and degradation of scar tissue in fibrotic liver disease occur slowly, typically over many years. Direct measurement of fibrogenesis, the rate of scar tissue deposition, may provide valuable therapeutic and prognostic information. We describe here results from a pilot study utilizing in vivo metabolic labeling to measure the turnover rate of hepatic collagen and collagen-associated proteins in plasma for the first time in human subjects. Eight subjects with chronic liver disease were labeled with daily oral doses of 2H2O for up to 8 weeks prior to diagnostic liver biopsy and plasma collection. Tandem mass spectrometry was used to measure the abundance and fractional synthesis rate (FSR) of proteins in liver and blood. Relative protein abundance and FSR data in liver revealed marked differences among subjects. FSRs of hepatic type I and III collagen ranged from 0.2-0.6% per day (half-lives of 4 months to a year) and correlated significantly with worsening histologic fibrosis. Analysis of plasma protein turnover revealed two collagen-associated proteins, lumican and transforming growth factor beta-induced-protein (TGFBI), exhibiting FSRs that correlated significantly with FSRs of hepatic collagen. In summary, this is the first direct measurement of liver collagen turnover in vivo in humans and suggests a high rate of collagen remodeling in advanced fibrosis. In addition, the FSRs of collagen-associated proteins in plasma are measurable and may provide a novel strategy for monitoring hepatic fibrogenesis rates.

The gene encoding phosphoglycerate kinase (PGK) from the ArchaeonSulfolobus solfataricus, an organism growing optimally at 87°C, was inserted into a yeast expression vector under the control of the galactose-inducibleGAL1 yeast promoter. This vector was then transformed into apgk::TRP1 yeast mutant, a strain inhibited for growth on galactose or glucose due to its lack of PGK enzyme. Slow-growing transformants were obtained on galactose plates at 37°C, but not 28°C. These transformants contained low levels of transcripts of the heterologous gene and low amounts of thermostable PGK activity. Weak expression of the hyperthermophile gene in yeast a mesophile, therefore enabled complementation of the yeastpgk defect at 37°C but not at 28°C.

To the editor: In the July 29, 2010, issue of Blood , Pillay et al presented a novel study on the kinetics of neutrophils in humans.[1][1] Their study used deuterium in heavy water to label proliferating cells in the marrow. Using a first-order model, the authors found that the neutrophil

BACKGROUND: In patients with chronic lymphocytic leukemia (CLL), the Bruton tyrosine kinase(BTK) inhibitor ibrutinib induces a rapid reduction in lymphadenopathy, accompanied by a transient lymphocytosis. Continuation of therapy leads to a reduction in peripheral blood disease burden with achievement of objective responses in a majority of patients over time. Inhibition of B cell receptor (BCR) and chemokine receptor signaling are considered the mechanisms for redistribution lymphocytosis and CLL cell death. However, these mechanisms of action have not been formally documented directly in patients. To address this, before initiating therapy with ibrutinib, we labeled the DNA of proliferating CLL cells with deuterium (2H) by asking patients to drink deuterated water (2H2O) and then determined the effects of ibrutinib on leukemia cell kinetics (proliferation and death rates) and mobilization of cells from lymphoid tissues (trafficking) after ibrutinib administration. METHODS: 30 previously untreated CLL/SLL patients were enrolled between December 2012 and June 2013 at MD Anderson Cancer Center. Patients required treatment per iwCLL guidelines, had adequate hematopoietic (platelets > 50,000/μL, ANC > 750/μL) and organ functions. Patients drank 50 mL of 70% 2H2O 3 times a day for 5 days, followed by 60 mL daily for a total of 4 weeks (“labeling phase”). After a 6-12 week “washout phase”, patients started once-daily ibrutinib 420 mg continuously on 28 day cycles. Study objectives were to determine the impact on CLL cell proliferation and death rates, before and after ibrutinib, and on CLL cell trafficking after ibrutinib. PATIENT CHARACTERISTICS: Median age of the 30 patients was 64 years (range 48–78) with 19 males. 16 patients exhibited early stage (Rai stage 0-2), and 14 advanced stage (Rai stage 3-4) disease. 17 patients expressed unmutated IGHVs, 11 patients had mutated IGHVs, and 2 had inconclusive IGHV results. 8 were CD38+ and 15 ZAP-70 + by immunohistochemistry. FISH cytogenetics revealed 11 patients with del13q, 6 with trisomy 12, 3 with del17p or TP53 mutation, 4 with del11q, and 6 without abnormalities. Median β2 microglobulin level was 2.8 mg/L (1.6 - 7.4). RESPONSE TO THERAPY: At a median follow up of 13 months, 28 of 30 patients continued on therapy without disease progression. All patients were evaluable for response assessment per 2008 IWCLL guidelines: 28 (93%) achieved partial remission, 1 (3%) complete remission, and 1 (3%) had stable disease, yielding an ORR of 97%. Two patients came off study at 184 and 480 days, respectively. CLL CELL KINETICS AND TRAFFICKING: The average CLL cell birth rate determined before ibrutinib therapy was 0.42% per day (range 0.32 – 1.42%). After initiating ibrutinib, there was a rapid increase in absolute lymphocyte count in the blood in 26 of 30 patients; this contained previously labeled CLL cells rather than newly-divided unlabeled cells. Over time, the proportion of circulating CLL cells that were labeled did not decrease appreciably despite a concomitant fall in circulating lymphocytes, indicating that newly divided cells were not entering the circulation and strongly suggesting that ibrutinib had a major inhibitory effect on leukemia cell proliferation. In 3 of the 4 patients in whom there was a rapid influx of unlabeled cells with the onset of ibrutinib treatment, maintenance of circulating labeled cells concomitant with a significant fall in circulating lymphocytes suggested proliferation of CLL cells was inhibited with extended treatment. Prior to ibrutinib therapy, the measured proliferation rate of CLL cells was 0.42 % per day. Because after ibrutinib therapy both cell proliferation and exiting from the blood were markedly reduced, the elimination rate should equal the death rate. Notably, with treatment the elimination rate of CLL cells was much faster than before ibrutinib, revealing a “true” death rate of 1.48 % of the clone per day. CONCLUSIONS: Using 2H-labelling of dividing leukemic cells, we have shown for the first time directly in CLL patients that ibrutinib significantly inhibits leukemia cell proliferation, leads to an efflux of CLL cells from tissues into the blood, and promotes a remarkably high death rate of CLL cells in an apparently indirect manner, likely due to the interruption of survival signals from the BCR and other receptors that are engaged in lymphoid tissues. Disclosures Burger: Pharmacyclics, Inc.: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees. Li:KineMed, Inc.: Employment, Equity Ownership. O'Brien:Pharmacyclics, Inc.: Membership on an entity's Board of Directors or advisory committees, Research Funding. Hellerstein:KineMed Inc: Consultancy, Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Turner:KineMed, Inc.: Employment, Equity Ownership. Emson:KineMed, Inc.: Employment, Equity Ownership. Chiorazzi:Pharmacyclics, Inc.: Research Funding; KineMed: Equity Ownership.

Leishmania major infection is useful as an experimental model to define factors responsible for the development and maintenance of Th cell immune responses. Studies using inbred mouse strains have identified that the Th1 response characteristic of C57BL/6 mice results in healing, whereas BALB/c mice fail to control the infection due to the generation of an inappropriate Th2 response. We now demonstrate that IL-13 is a key factor in determining susceptibility to L. major infection. Overexpression of IL-13 in transgenic mice makes the normally resistant C57BL/6 mouse strain susceptible to L. major infection even in the absence of IL-4 expression. This susceptibility correlates with a suppression of IL-12 and IFN-γ expression. Furthermore, using BALB/c mice deficient in the expression of IL-4, IL-13, or both IL-13 and IL-4, we demonstrate that IL-13-deficient mice are resistant to infection and that there is an additive effect of deleting both IL-4 and IL-13.

Recent studies using interleukin (IL)-4–deficient animals have highlighted the existence of IL-4–independent immunoglobulin (Ig)E induction. We have established transgenic mice expressing IL-13 from a transgene comprising a genomic fragment containing the IL-13 gene and the human CD2 locus control region. The transgenes were expressed in lymphoid tissues and induced by T cell activators, suggesting regulation by elements of the IL-13 promoter. IL-13 transgenic lines expressed 10–100-fold higher levels of serum IgE than their littermate controls, but had normal levels of other serum Ig isotypes. Elevated IgE levels were also detected in sera from IL-4–deficient mice carrying IL-13 transgenes, indicating that IL-4 is not required for IL-13–induced IgE expression in the mouse. Expression of IL-13 also perturbed the development of thymocytes. Although thymocyte development was normal up to 4 wk of age, thymocyte number decreased dramatically thereafter, reaching 10% of normal by 10 wk, and despite normal size and appearance, histological examination demonstrated that transgenic thymi contained only small foci of thymocytes. The reduction in thymocyte number was due mainly to a depletion of CD4+CD8+ thymocytes, and did not affect significantly the composition of peripheral T cell populations. These data indicate that expression of IL-13 transgenes in vivo can regulate IgE production in the mouse, and that IL-13 may also influence thymocyte development.

Abstract 184 Chronic Lymphocytic Leukemia (CLL) is characterized by the accumulation of mature B cells in peripheral blood (PB), bone marrow (BM) and lymph node (LN). Recent studies identified the LN as an important site of tumor cell activation and proliferation (Herishanu et al 2011). Using in vivo labeling of “newly-born” cells with deuterated water (2H2O; heavy water), the proliferation rate of CLL cells was estimated to range between 0.1 to >1% of the clone per day (Messmer et al 2005). Furthermore, a CXCR4dimCD5bright population of CLL cells in the PB contained more deuterium (2H)-labeled DNA and hence “recently-born cells than the CXCR4brightCD5dim population (Calissano et al 2011). Possible explanations for this observation include that CXCR4dimCD5bright cells proliferate more rapidly in the PB or that these cells are recent emigrants from tissues where proliferation occurs. As prior studies were performed on PB cells, the growth rates and characterization of the proliferative fraction of CLL cells in the LN remain unknown. Here we used 2H-labelling to directly compare cellular growth rates in PB, BM, and LN. Patients drank 2H2O for 28 days; on day 13 an excisional LN biopsy and a BM aspirate were obtained. PB samples were obtained at baseline and on days 13 and 28. CLL cells were isolated using positive selection, or sorted based on reciprocal differences in CXCR4 and CD5 density for isolation of “proliferative” (CXCR4dimCD5bright), “intermediate” (CXCR4intCD5int), and “resting” (CXCR4brightCD5dim) fractions of CLL clones. 2H incorporation into the DNA of newly divided cells was measured by mass spectrometry. Raw values were normalized to the 2H2O content in total body water. Cellular growth rates were calculated by dividing the fraction of 2H-labeled cells by the number of days from the start of the labeling period. To date, samples from 6 treatment-naïve CLL patients have been analyzed. On day 13, up to 16%, 9%, and 24% of the CLL cells sampled from PB, BM, and LN, respectively were 2H-labeled. The resulting mean estimated growth rate in % of the clone per day was for PB 0.41 (0.09 – 1.13), for BM 0.41 (0.28 – 0.68), and for LN 0.83 (0.31 – 1.84). The difference in growth rates between PB and LN was statistically significant (P Taken together our data show that the proliferation rate of CLL cells is higher in the LN than in the BM and PB and suggest that some of the newborn cells exit the LN within days. A clonal subset of CXCR4dimCD5bright cells is present in both the LN and PB and might harbor the proliferative core of the disease. Disclosures: Gatmaitan: KineMed: Employment. Emson:KineMed: Employment. Chiorazzi:KineMed: Dr. Chiorazzi holds stock options in KineMed, Inc. Other.

Interleukin 19 (IL-19) is thought to have important roles in the immune control of pathogens, and in enhancing chronic inflammatory diseases of the skin (psoriasis), lung (asthma) and intestine (colitis). However, the biological function and the networked control of IL-19 are poorly understood. Naive mice deficient in IL-19 (IL-19-/-) on a 129xBL6 background had decreased alveolar macrophages and increased eosinophils in the broncho-alveolar lavage (BAL) and decreased B cells in the spleen and bone marrow. However, naïve IL-19-/- mice backcrossed to C57BL/6 or BALB/c, and mice housed in different locations exhibited an attenuated phenotype. Therefore, genetic backcross and challenge experiments with environmental stimuli (allergen, urban particulate matter) were conducted. The studies showed that abnormal composition of the cells in the BAL fluid, spleen and bone marrow in naïve IL-19-/- 129xBL6 mice was due to the deficiency in IL-19 combined with the activity of another gene inherited from the 129 strain. IL-19-/- mice on C57BL/6 or BALB/c backgrounds that were intranasally challenged with antigen or with antigen and urban particulate matter had decreased BAL eosinophils, alveolar macrophages and B cells when compared to wild type. Together our studies indicate that endogenous IL-19 is a critical component of immune homeostasis to the airborne environment and that mouse-strain specific genes control the function of IL-19.

Abstract 60 Chronic lymphocytic leukemia (CLL) was formerly considered an accumulative disease of immunologically incompetent lymphocytes that have a slow rate of cell turnover. However, recent studies suggest that CLL cells of some patients may have an unexpectedly high birth rate, which in some subjects, is counterbalanced by a relatively high death rate. In particular, studies on the leukemia cells of patients who ingested heavy water (2H2O) revealed that some patients had leukemia cells that incorporated deuterium into DNA at rates consistent with a birth rate of 1% of the leukemia population per day. This high birth rate, when counterbalanced with comparable rates of leukemia cell turnover, allows patients to maintain a steady state that was suggestive of indolent disease. Preliminary data also suggested a relationship between disease activity and CLL cell birth. To examine the association between measured rates of leukemia-cell turnover with established surrogate markers of aggressive disease, the CLL Research Consortium (CRC) recently completed enrolment of 119 patients with early stage disease. Patients were to be untreated, Rai Stage 0, 1, or 2 and diagnosed within the previous 3 years. The leukemia cells of each of these patients were evaluated by the central tissue core of the CRC for expression of ZAP-70, CD38, and immunoglobulin heavy chain variable region gene (IGHV) mutation status. We report here a subset analysis of baseline data for 59 patients (57% male, age 58.7 ± 10.1 y). Thirty-seven percent of patients (n=22) had leukemic cells that expressed unmutated IGHV, 39% (n=23) had cells that expressed ZAP-70 (> 20% positive by cytoplasmic flow cytometery), and 19% (n=11) had CD38 positive cells (> 30% positive by multiparameter flow cytometry). Associations between the traditional prognostic markers were exceedingly strong (ZAP-70 with mutation status, p < 0.001; ZAP-70 with CD38, p = 0.013; CD38 with mutation status, p =< 0.001). Birth rate was expressed as the peak percent new cells per day calculated from 2H enrichment in DNA using isotope ratio mass spectrometry (IRMS). Data from the previous study suggested more aggressive disease activity associated with a birth rate above 0.35% per day. Birth rates in the present study ranged from 0.07 - 1.31 % per day, spanning both sides of the previously published 0.35% per day cut-off. Using the Wilcoxon rank sum test, patients whose clones expressed unmutated IGHV, were ZAP-70 positive, or were CD38 positive had a significantly higher daily leukemia cell birth rates than subjects who were IGHV mutated (median of 0.39 v 0.24, p = 0.01), ZAP-70 negative (median of 0.36 v. 0.22, p = 0.002), or CD38 negative (0.62 V. 0.24, p = 0.001). Thus, we have shown that CLL cell birth rates can be determined in a multi-centered trial in a large number of patients, and that patients can be stratified into groups with varying rates of perceived cell turnover. The cell turnover data correlate directly in multivariate analysis with established surrogate markers of aggressive disease, a poor clinical course and decreased survival with higher birth rates associating with unmutated IGHV and expression of ZAP-70 and CD38. Clinical follow-up of these patients is underway to determine if these four parameters serve as complementary prognostic markers. Nevertheless, the strong association between established surrogate markers of aggressive disease with high leukemia-cell birth rates suggests that high-rates of leukemia-cell turnover define a biology that is associated with adverse outcome. As such, these studies strengthen the notion that leukemia cell populations that have high rates of cell turnover can more rapidly accumulate secondary and tertiary genetic mutations that potentially govern the progression of this disease. Disclosures: Murphy: KineMed, Inc.: Consultancy, Equity Ownership. Emson:KineMed, Inc.: Employment. Hayes:KineMed, Inc: Consultancy. Holochwost:KineMed, Inc: Employment. Hellerstein:KineMed, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding. Chiorazzi:KineMed, Inc: Membership on an entity's Board of Directors or advisory committees.