Respiratory

Respiratory diseases have been identified to be an immense health burden worldwide with approximately 1 billion persons suffering from chronic respiratory conditions.

Estimates suggest that:

  • 235 million people suffer from asthma
  • More than 200 million people have chronic obstructive pulmonary disease (COPD)
  • 65 million endure moderate-to-severe COPD
  • 8.7 million people develop tuberculosis (TB) annually
  • Millions live with pulmonary hypertension and more than 50 million people struggle with occupational lung diseases
  • Each year, 4 million people die prematurely from chronic respiratory disease
  • Asthma is the most common chronic disease, affecting about 14% of children globally and rising
  • COPD is the fourth leading cause of death worldwide

 

Cellomatics Biosciences Ltd. is a respiratory CRO that offers well validated preclinical airway models to investigate the effects of compounds in a variety of pathological settings including inflammation, gel contraction, oxidative stress, mast cell degranulation, extracellular matrix deposition (ECM) and epithelial to mesenchymal transition. These models are suitable for low to high throughput studies.

Asthma and Airway Inflammation

Asthma is the most common chronic disease, affecting about 14% of children globally and rising. Asthma is a chronic inflammatory disorder arising from not fully understood heterogenic gene-environment interactions. It features variable airway obstruction and bronchial hyperresponsiveness. This leads to breathlessness associated with wheezing and cough. It is frequently known to coexist with atopic diseases, particularly allergic rhinitis.

Human Airway Smooth Muscle cells stimulated with Poly I:C

The effect of PDE4 inhibitor on IL6 and IL8 release from Poly I:C stimulated Human Airway Smooth muscle cells (HASMs) was assessed. HASMs were pretreated with the PDE4 inhibitor (1 nM) for 1 hour prior to stimulation with Poly I:C (10 µg/ml) for 24 hours. Cell culture supernatants were then collected and analysed for IL6 and IL8 release by ELISA (n=3±SEM; **p < 0.01).

Human Lung Fibroblasts stimulated with Poly I:C

Primary human lung fibroblasts were treated with TL3 in conjunction with Poly I:C stimulation for 48 hours. The levels of IL-6, IL-8 and MCP-1 released in the supernatants were determined using Luminex Multiplex assay. One-way ANOVA was performed to determine statistical significance (*p<0.05; **p<0.01; ***p <0.001).

Human Lung Fibroblasts stimulated with TNFα

Primary human lung fibroblasts were treated with reference compound – Hydrocortisone in conjunction with TNF-α-mediated stimulation for 48 hours. Expression levels of IL1b, IL6, IL8, IFNγ, MMP1 and HMGB1 were determined using an ELISA-based assay. One-way ANOVA was performed to determine statistical significance (*p<0.05; **p<0.01; ***p <0.001).

Human Lung Fibroblasts stimulated with TNFα

Primary human lung fibroblasts were treated with reference compound – Hydrocortisone along with TNF-α for 48 hours. Expression levels of PAI-1 and a-SMA were determined using an ELISA-based assay. One-way ANOVA was performed to determine whether TNF-α increased the expression of PAI-1 and if hydrocortisone subsequently resulted in a reduction of levels (***p <0.001; n=3±SEM).

Percentage change in gel contraction upon pre-treatment with 2,3 BDM followed by incubation for 4 hours with Carbachol. For each treatment the percentage change in the gel contraction was calculated based on the values obtained at time 0. Each condition is representative of 3 biological replicates (n=3); mean+SEM. Data analysis was performed using the 2-WAY ANOVA analysis, *p<0.05; **p<0.01, ***p<0.0001; Carbachol vs. test ingredients groups. 

Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is the commonest interstitial lung disease (ILD) and is characterised by progressive scarring of the lungs. Impaired pulmonary would healing response following lung injury coupled with excessive production of collagens leads to a pathogenic lung fibrosis.

Human Lung Fibroblasts stimulated with LPS

Primary Human Lung Fibroblasts were treated with 10 µg/ml LPS for 24 hours in the presence or absence of ± hydrocortisone. Cell culture supernatants were then collected and levels of IL6 and IL8 measured by ELISA (n=5±SEM; *p<0.05; **p<0.01).

Collagen biomarkers in Human Lung Fibroblasts

Primary Human Lung Fibroblasts were pre-treated with Pirfenidone for 1 hour followed by 24 hours stimulation with H2O2. Cell lysates were analysed using QuantiGene multiplex for Collagen gene expressions. The fold changes were normalised to house keeping genes (HKG) such as GAPDH and HPRT1. All treatment conditions were compared to the H2O2 treated samples (***p<0.001; **p<0.01; *p<0.05; n=3±SEM).

Inflammatory mediators

Primary Human Lung Fibroblasts were pre-treated with Pirfenidone for 1 hour followed by 24 hours stimulation with TGFβ. Supernatants were analysed using Luminex multiplexed assays for inflammatory and extracellular matrix component markers. All treatment conditions were compared to the TGFβ treated samples (***p<0.001; **p<0.01; *p<0.05; n=3±SEM).

Extracellular Matrix Deposition

Primary Human Lung Fibroblasts were pre-treated with Pirfenidone for 1 hour followed by 24 hours stimulation with TGFβ. Cell lysates were analysed using QuantiGene multiplexed assays for gene expression. The fold changes were normalised to house keeping genes (HKG) such as GAPDH and HPRT1. All treatment conditions were compared to the TGFβ treated samples (***p<0.001; **p<0.01; *p<0.05; n=3±SEM).

Oxidative Stress - Reactive Oxygen Species (ROS)

Human Lung Fibroblasts were pre-treated with Pirfenidone as an inhibitor for 1 hour  followed by treatment with 200 µM H2O2 for 48 hours. Lysates were analysed for intracellular ROS using fluorescence detection kit. A significant increase in intracellular ROS (indicated by increased fluorescence signal) was observed in 200 µM H2O2 when compared to untreated controls (n=3±SEM; **p<0.01).

COPD

The GOLD initiative defines COPD as “a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases.” The American Thoracic Society defines COPD in terms of chronic bronchitis and emphysema. Chronic bronchitis is characterized by the clinical symptoms of excessive cough and sputum production; emphysema refers to chronic dyspnea, resulting from enlarged air spaces and destruction of lung tissue. COPD is the fourth leading cause of death worldwide.

Goblet cell hyperplasia

Human airway epithelial cells were differentiated into goblet cells and then treated with pulsed 5% cigarette smoke extract (CSE) and 1 µM Acrolein (continuous) for 28 days. Immunofluorescence staining for MUC5AC was performed at Day 14 and Day 28 and imaged using the Molecular Devices Xpress Pico machine

Goblet cell hyperplasia

MUC5AC and MUC5AB mRNA expression was assessed in NHBE-Bronchial Epithelial Cells following 28 days of differentiation and treatment with cigarette smoke extract (CSE) or Acrolein. Gene expression was analysed using a Quantigene Luminex Assay and fold change normalised to Day 0 differentiation (Control-untreated) (n=5).

Mast Cell Degranulation

Mast cells are immune cells of the myeloid lineage and are present in connective tissues throughout the body. The activation and degranulation of mast cells significantly modulates many aspects of physiological and pathological conditions in various settings. With respect to normal physiological functions, mast cells are known to regulate vasodilation, vascular homeostasis, innate and adaptive immune responses, angiogenesis, and venom detoxification. On the other hand, mast cells have also been implicated in the pathophysiology of many diseases, including allergy, asthma, anaphylaxis, gastrointestinal disorders, many types of malignancies, and cardiovascular diseases (Krystel-Whittemore et al., 2016).

HMC1.2 cells were treated with Calcium Ionophore (CaI) and Compound 48/80 (48/80) ±Protamine or Dexamethasone. The vehicle control was 0.1% DMSO in culture media. The beta-hexosaminidase alpha (HEXA) was analysed in the cell lysates by ELISA. An increase in HEXA was observed in cells treated with CaI and 48/80 when compared to vehicle control/untreated cells. These levels of HEXA reduced when treated with inflammatory inhibitors such as Protamine and Dexamethasone.

Allergic Rhinitis

Allergic rhinitis is an inflammatory disorder of the nasal mucosa induced by allergen exposure triggering IgE-mediated inflammation. It can also be associated with co-morbid conditions as Asthma, Atopic Dermatitis & Nasal polyps (Varshney et al., 2015).

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