1CONFIDENTIAL

Immunology/Inflammation

Capabilities

■ Cellomatics Biosciences Ltd is a Specialised laboratory-based

Contract Research Organisation (CRO)

■ We support our customers in every aspect of their Preclinical in vitro

R&D studies

■ With an experienced team of scientists and clinical professionals, we

understand your requirements and can deliver innovative and

practical solutions

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Who are we?

Our team

3CONFIDENTIAL

Dr. Anushuya Tamang

Bioassay Study ScientistAnushuya holds a MSc in

Biotechnology , MRes in Cancer

Biology and PhD in Developmental

Biology. She has more than 6 years

experience in Molecular and Cellular

biology as well as Histology.

Dr. Federica Riu

Bioassay Study ManagerFederica holds a MSc in Cellular

Biology and a PhD in Clinical and

Experimental Microbiology. She has

over 10 years of experience in

Molecular, Cellular and Vascular

Biology, Microbiology and Stem Cells.

Dr. Shailendra Singh

Founder and CEOShailendra’s scientific background

lies in target identification and

validation for the management of

respiratory diseases. Overall, he has

over 14 years of experience in

research and development within the

academia and industry together with

clinical diagnostics.

Camille Hetez, MSc

Head business development Camille holds a MSc in Biotechnology

Engineering from Paris and an MBA

Entrepreneurship. She has experience

in various Business Development

positions and also 3 years experience

as a Co-founder and CEO in a

innovative biotechnology company.

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Area of expertise

RespiratoryImmunology CardiovascularOncology

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Overview of our services

Toxicity assays

Target validation & qualification

Biomarker assays

Multiplex nucleic acid & proteins test

In vitro Cell-based assays

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Receive your request

Understand your need

Personalized quotation

Sign contract

Weekly project

updates

Deliver High quality report

We are committed to

1. Understanding your needs and tailoring

our services to meet your requests

2. Meeting your timelines

3. Providing interactive and efficient

customer support

4. Delivering high quality services at a very

cost effective price

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Our approach

■ Immunology is the study of the immune response of a body towards

antigens.

■ There are two types of immune-responses:

– Innate (in-born)

– Adaptive (acquired)

■ They can be further divided into: Humoral and Cellular

■ Humoral immunity is carried out by the production of antibodies by B

lymphocytes (B-cells).

■ Cellular immunity is mediated by thymus derived lymphocytes (T-cells).

■ Both mechanisms recruit other cells of the immune system, such as

macrophages, dendritic cells, to further eliminate the antigens.

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Immunology

TYPE DESCRIPTION METHOD END-POINT DATA

HUMORAL

IMMUNITY

Antibody Production

ELISA Colorimetric Quantitative

Luminex Bead-based Quantitative

cell-ELISA Colorimetric Quantitative

Macrophage

Endocytosis Fluorometric Quantitative

Phagocytosis Colorimetric Quantitative

PeroxidaseColorimetric/

FluorometricQuantitative

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Analytical and Functional Assays

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Analytical and Functional Assays

CELLULAR

IMMUNITY

CytokinesELISA Colorimetric Quantitative

Luminex Bead-based Quantitative

Cell Surface antigens

ELISA Colorimetric Quantitative

Immunofluorescent

stainingImaging Qualitative

cell-ELISA Colorimetric Quantitative

Proliferation BrdU incorporation Imaging Qualitative

Toxicity assays:

• Antibody dependent cellular cytotoxicity (ADCC)

• Antibody-dependent cellular phagocytosis (ADCP)

• Complement-dependent cytotoxicity (CDC)

Cytotoxicity Assay Chemiluminescence Quantitative

Cell viability, apoptosis / necrosis Colorimetric/Fluorometric Quantitative

Chemotaxis Boyden chamber Colorimetric/Fluorometric Quantitative

Macrophage

Endocytosis Fluorometric Quantitative

Phagocytosis Colorimetric Quantitative

Peroxidase Colorimetric/Fluorometric Quantitative

TYPE DESCRIPTION METHOD END-POINT DATA

■ Assays can be performed with combination of cell co-cultures:

– Such as leukocytes, fibroblasts, macrophages etc.

– Primary or immortalised cell lines.

■ We also assist in isolating the primary cells in-house, such as PBMCs,

eosinophils, neutrophils, lymphocytes etc.

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Functional Assays

■ Proliferation/Apoptosis: BrdU incorporation, Alamar Blue, Ki-67

Caspase 3/7, TUNEL assay

■ Cytotoxicity: MTT assay, mitochondrial damage, genotoxicity, LDH

■ Migration: Boyden chamber, scratch assay, Matrigel degradation,

haptotaxis assay

■ Cell adhesion assay: to test the ability of immune cells to adhere to

specific extracellular matrix proteins.

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Proliferation/apoptosis, cytotoxicity, migration

■ Macrophage Activation

– Blood samples are collected from healthy donors and cells are isolated.

– Isolated cells will then be exposed either to the CLIENT’s proprietary molecules

for defined timings or to control Antibody, along with a positive (i.e. LPS) or

negative (to be defined by CLIENT) control.

– Stimulated cells will be then processed for the following activation parameters:

■ Molecular Biology

– Quantitative gene expression of target inflammatory genes

■ Biochemical Characterization

– Inflammatory cytokine production (i.e. IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, VEGF)

– Total ROS production

– Phagocytic activity

– Mitochondrial damage

– Cell viability and toxicity

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Immune Cell Activation Assays

■ Mast Cells Activation

– HBEC/BEAS 2B and HMC-1 Co-culture model

■ Sensitisation using either 2.5 µg/ml human IgE or Calcium Ionophore to induce

mast cell degranulation

■ Mast cell activation will be monitored by quantitative analysis of allergen-induced

histamine release. In particular, the following parameters will be measured:

– Release of β−hexosaminidase

– Mast cell degranulation assays

■ Measure the mast cell tryptase activity using a spectrophotometric method as an

indicator of mast cell degranulation

■ Measure the release of other mediators of allergy and inflammation including

histamine, lipoxin A4 by immunoassays

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Immune Cell Activation Assays

■ B lymphocytes IgE release assay

– Stimulation of B lymphocyte cell line with IL4, IL5 and IL13

– Measure IgE release by immunoassays

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Immune Cell Activation Assays

■ Intestinal epithelial cell lines

– Caco-2, HT-29, T-84

■ Treatment with pro-inflammatory compounds

– LPS, IL-1β

■ End points

– Release of pro-inflammatory markers

■ e.g. IL8, TNF-alpha: ELISA, Multiplex immunoassay

– Rearrangement of tight junction proteins

■ e.g. ZO-1, occludin & claudin-1: Immunofluorescence (IF)

– Increased mucus production

■ Mucin staining: Periodic acid/ Schiff reagent; Mucus staining: Alcian blue

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Monolayer: Single-culture

Monolayer: Single culture

■ Intestinal epithelial cell lines

– Caco-2, HT-29, T-84

■ Treatment with pro-inflammatory compounds

– LPS, IL-1β

■ End points

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Monolayer: Trans-well Single-culture

- Release of pro-inflammatory markers:

e.g. IL8, TNF-alpha: ELISA, Multiplex

immunoassay

- Increased mucus production

Mucin staining: Periodic acid/ Schiff

reagent; Mucus staining: Alcian blue

- Rearrangement of tight junction

proteins

e.g. ZO-1, occludin & claudin-1:

Immunofluorescence (IF)

- Reduction of barrier function

TEER

Fluorescein transport

Monolayer: Trans-well Single culture

■ Intestinal epithelial cell lines

– Caco-2, HT-29, T-84

■ Co-culture

– Human blood-derived macrophages and dendritic cells, PBMC

■ Treatment with pro-inflammatory compounds

– LPS, IL-1β

■ End points

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3-D: Trans-well Co-culture

- Release of pro-inflammatory markers:

e.g. IL8, TNF-alpha: ELISA, Multiplex

immunoassay

- Increased mucus production

Mucin staining: Periodic acid/ Schiff

reagent; Mucus staining: Alcian blue

- Rearrangement of tight junction

proteins

e.g. ZO-1, occludin & claudin-1:

Immunofluorescence (IF)

- Reduction of barrier function

TEER

Fluorescein transport

3-D: Trans-well Co-culture

BIOMARKERS

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■ The regulation and initiation of immune

response involves multiple changes in gene and

protein expressions.

■ These variations can be monitored to

investigate the progression of diseases and

validate drug treatment.

■ Some of the Biomarker panels available for

Luminex Platform:

– Immunology

– Inflammation

– Cytokines/Chemokines

– Sepsis

– Skin

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Biomarker Analysis

ASSAY

CATEGORYASSAY

Protein

expression

ELISA

Multiplex Immunoassays

(Luminex Platform)

Gene expression

PCR

RT-PCR

Multiplex Nucleic Acid Testing

(Luminex Platform)

LocalisationImmunohistochemistry (IHC)

Immunocytochemistry (ICC)

Different types of biomarker assays

■ Pathway activation biomarkers

– Measure phosphorylation of downstream signalling events

■ Multiplexing immunoassays

■ Western Blot

■ In-Cell ELISA

■ Disease Biomarkers

– Measure release of cytokines, chemokines, growth factors and inflammatory

mediators (mRNA and protein)

■ Multiplexing immunoassays

■ ELISA

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Immunology/Inflammation Assays

■ Advantages

– Multiplex Technology allows to detect up to 50 targets per well

– No need to process the samples

– No RNA isolation: no risk to loose material, RNA degradation, DNA

contamination

– No cDNA: some mRNA are low represented and difficult to retrotranscribe

– No PCR: low number transcript might be masked by high copies transcripts; no

requirement for specific primers

– High specificity of probes and antibodies.

– Large panel of targets and species

– Plated and panels customizable

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Multiplex gene assays

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Inflammatory Pathologies

■ Whole blood assay (WBA) (Laufer et al) - Among several in vitro testing

systems the whole blood assay (WBA) is a well-known method to

examine non-steroidal anti-inflammatory drugs (NSAIDs) in view of

their potency to inhibit COX activity.

– Measurement of COX-1 activity in whole blood:

■ Using heparinised blood

■ Using coagulated blood

– Measurement of COX-2 activity in whole blood

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In vitro models for COX assays - Pain/Inflammation

■ Cell-based models for COX assays

– Specific Cell lines

■ A549, a human epithelial carcinoma cell line (ECACC Ref. No. 86012804),

expresses COX-2 when exposed to IL-1β (Mitchell et al., 1994). Production of PGE2

by this cell line can therefore be used as an index of COX-2 activity.

– Other cell lines where COX inhibition can be investigated:

■ COS-1 cells

■ CHO cells

■ RBL-1 cells

■ melanoma (MeLiM)

■ histiocytic lymphoma cell line U937

■ prostate carcinoma line PC3

■ P493-6, a B cell line carrying a conditional, tetracyclin-regulated MYC gene bovine

aortic endothelial cells.

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In vitro models for COX assays - Pain/Inflammation

■ Cell-based models for COX assays

– Intact cells: Detection of PGE2 or TXB2 levels in the cell culture medium.

■ COX-1 activity

■ COX-2 activity

– Cell Lysate preparation: Detection of COX activity in the cell lysates

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In vitro models for COX assays - Pain/Inflammation

■ A chronic or recurrent inflammatory conditions of the colon and small

intestine (Crohn's Disease and Ulcerative Colitis).

Causes: Defects in the intestinal barrier

■ Impaired immune function

■ Genetic predispositions (e.g. NOD2 mutation)

■ Environmental factors

Characteristic:

Increased proinflammatory immune response to the commensal intestinal microbial flora.

■ Leads to increased permeability of the intestinal epithelial barrier.

■ Allows toxins and microbes to reach the underlying tissues.

■ Alterations in the mucosal architecture, e.g. transcellular bridge formation in epithelial cells

and goblet cell hyperplasia or hypertrophy or both.

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Inflammatory Bowel Disease (IBD)

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IBD cell models

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IBD – STUDY DESIGN EXAMPLE

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IBD – STUDY DESIGN EXAMPLE

IMMUNE PATHOLOGIES

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■ An inflammatory skin disorder,

triggered or exacerbated by a

number of genetic,

environmental, or immunological

factors.

■ Characterized by

– Hyperproliferation of keratinocytes

– Abnormal epidermal differentiation

– Infiltration of inflammatory cells

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Psoriasis

Mahajan R, Handa S. Pathophysiology of psoriasis. Indian J Dermatol Venereol Leprol 2013;79,

Suppl S1:1-9

■ Psoriasis initiation

– Keratinocyte & blood cell models – 3D, co-culture (keratinocytes + dermal

fibrobalsts)

– Proliferation (BrdU, MTT, Alamar Blue, Ki-67, Caspase3/7, TUNEL assay)

– Epidermal differentiation - Cultures of keratinocytes grown on filter at the

AirLiquid Interface

■ measure epidermal differentiation markers (involucrin, keratin10, keratin 14,

filaggrin)– mRNA and protein

– Membrane integrity assay

– LL-37 production by keratinocytes, macrophages & neutrophils (mRNA &

protein)

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In vitro assays - Psoriasis

■ Psoriasis progression

– T cell models (including T cells from psoriatic lesions)

■ Th17 cytokine production/release mRNA and protein)

– Granulocyte & monocyte/macrophage models

■ Pro-inflammatory cytokine production/release

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In vitro assays - Psoriasis

■ Psoriasis physiopathology

Keratinocytes & 3 D skin models (Cultures of keratinocytes grown on

filter at the AirLiquid Interface), co-culture models (keratinocytes +

dermal fibroblasts) induction by validated cytokine cocktails

– Modifications of epidermal differentiation and disease markers (involucrin,

keratin10, keratin 14, filaggrin; mRNA & protein)

– Antimicrobial peptide production/release (mRNA & protein)

– Membrane integrity assay

– Chemokine production/release (mRNA & protein)

– Cytokine signaling; STAT-3/STAT-1, MAPK, NFkB & other involved pathways

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In vitro assays - Psoriasis

■ Atopic Dermatitis - Also known as atopic eczema, is a chronic inflammatoryfamilial disease of the skin. It is characterized by an increased ability to formimmunoglobulin E and a tendency to develop other allergic conditions suchas allergic rhinitis and asthma.

■ Psoriasis - Psoriasis is an autoimmune disease in which the skin becomesinflamed, producing red, flaky areas. It affects 1-2% of the population and isthought to be caused by misguided T cell attacks on the skin.

■ Arthritis - is an umbrella term that describes a group of disorderscharacterized by joint inflammation involving the immune system andsudden, painful onset.

■ Asthma - Asthma is a form of bronchial disorder caused by inflammation ofthe bronchi. It is characterized by spasmodic contraction of airway smoothmuscle, difficulty breathing, wheezing and coughing.

■ Inflammatory Bowel Disease (IBD): Chronic or recurrent inflammatoryconditions of the colon and small intestine (Crohn’s Disease and UlcerativeColitis).

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Pathology involving immune system

■ Chronic, complex allergic inflammatory skin disease that affects 10-20% ofthe population

■ Initial immune response begins with the T helper (Th)2 response and is thenshifted to Th1 in the chronic phase.

■ Genetic background (e.g. mutations in filaggrin) and defective skin barrier -an additional risk factor for developing AD.

■ Barrier dysfunction exposes immune system cells to external allergens,eliciting an immune response.

■ Initiation and progression of this "Th2"-type dermatosis is under thedependence of the keratinocyte-derived cytokine: Thymic StromalLymphoprotein (TSLP). TSLP targets dendritic cells (DCs) and drives aspecific Th2 response.

■ AD physiopathology results in skin abnormalities, inflammation with IgE andhistamine production, erythema, eosinophilia, alopecia and itching.

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Atopic dermatitis (AD)

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Atopic dermatitis

▪ Carr WW. Topical calcineurin inhibitors for atopic dermatitis: review and treatment recommendations. Paediatr Drugs 2013;

15:303–10.

▪ Elias PM, Hatano Y, Williams ML. Basis for the barrier abnormality in atopic dermatitis: outside-inside-outside pathogenic

mechanisms. J Allergy Clin Immunol 2008; 121:1337–43.

■ AD initiation - Keratinocyte model

– Involves stimulation of cultured keratinocytes (NHEK) using a cocktail of

proinflammatory agents, including Poly(I:C) and (TNF)-a (Th1cytokine) Or

Poly(I:C), IL-4 and IL-13 (Th2 cytokines)

– Measure expression of TSLP, IL1α, IL18, IFNα2, IFNβ1, IL4R, IL8, MIP1α,

RANTES, MIP3α, MDC, CCL27, involucrin and filaggrin in stim vs unstim human

epidermal keratinocytes (NHEKs): Multiplex immunoassay/gene expression

– Measure anti-microbial peptides including cathelicidin, RNA SE7, S100A11,

psoriasin – immunoassays

– TLR3/RLRs signaling: NFkB, IRF3 translocation/ phosphorylation.

– JAK/STAT, PI3K signalling

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In vitro models

■ AD initiation - Atopic dermatitis reconstructed human epidermis model

(3D)

– Consists of normal, human-derived epidermal keratinocytes (NHEK) cultured on

specially prepared tissue culture inserts.

– Biology of whole-tissue models closely aligned with in vivo modelling than cell

culture alone

– Stimulated with Th2 cytokines IL-4 and IL-13, and the TLR ligands Poly (I:C) and

Pam3CKS4

– Measure expression of TSLP, IL1α, IL18, IFNα2, IFNβ1, IL4R, IL8, MIP1α,

RANTES, MIP3α, MDC, CCL27, involucrin and filaggrin in stim vs unstim human

epidermal keratinocytes (NHEKs): Multiplex immunoassay/gene expression

– Measure anti-microbial peptides including cathelicidin, RNA SE7, S100A11,

psoriasin – immunoassays

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In vitro models

■ AD progression - Adaptive immunity model (T cell models )

– Purifying human CD4+ lymphocytes from circulating peripheral blood

mononuclear cells isolated from healthy donors stimulated with staphylococcal

enterotoxin B (SEB)

– Measure expression of IL2, IL12, IL1β, TNFα, IL4, IL5, IL13, IL6, IL18, IL10,

IL17, IL31 (mRNA and protein by multiplexing)

■ AD progression - Innate immunity model (antimicrobial peptides)

– Stimulation of NHEK cells with IL1β

– Measure expression of cathelicidin antimicrobial peptide LL-37 (CAMP) and

human b-defensin (hBD)2

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In vitro models

■ AD progression - Monocyte-derived dendritic cell (MoDC) models

– Isolation and stimulation of dendritic cells with TSLP

– Measure TSLP-induced expression of activation markers: OX40L, MHCII, CD80,

CD86, CCL17, CCL22 (mRNA & protein by multiplex)

– TSLP signaling: STAT-5 phosphorylation (JAK-independent)

■ AD physiopathology - Mast cell model

– Stimulation of human mast cell (LUVA) by IgE

– Measure release of histamine and NGF

– Measure release of mast cell proteinases enzyme tryptase and chymase

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In vitro models

■ AD physiopathology - B cell model

– Stimulation of human B lymphocyte with IL4/IL13

– Measure release of IgE

■ AD physiopathology

– Cytokine signaling: STAT-6, MAPK & other involved pathways – Western Blotting

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In vitro models

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Example of AD initiation – Keratinocyte model

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Example of AD initiation – Keratinocyte model

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Example of AD pathophysiology – Mast cell model

“At Cellomatics Biosciences, we believe

in growing together. We welcome the

opportunity to partner and achieve

excellence together”

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