Immunology & Inflammation

Characteristics of Humoral and Cellular Immunity

  • - 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.

Immunology- Analytical and Functional Assays

  • - 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.

Immunology-based 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.
  • - 3D colony formation and invasion assay (i.e. cancer cells and macrophages)

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
  • B lymphocytes IgE release assay
    • Stimulation of B lymphocyte cell line with IL4, IL5 and IL13
    • Measure IgE release by immunoassays

Immunology- Biomarker Analysis:

  • 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
  • Pathway activation biomarkers
    • - Measure phosphorylation of downstream signalling events
      • Multiplexing immunoassays
      • Western Blot
      • In-Cell ELISA
    • - Measure release of cytokines, chemokines, growth factors and inflammatory mediators (mRNA and protein)
      • Multiplexing immunoassays
      • ELISA
  • Disease Biomarkers

In vitro models for COX assays - Pain/Inflammation:

  • - 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
  • - 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.
    • 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


Inflammatory Bowel Disease (IBD):

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.
Example Study Design

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

  • chronic, complex allergic inflammatory skin disease that affects 10-20% of the population
  • initial immune response begins with the T helper (Th)2 response and is then shifted 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 the dependence of the keratinocyte-derived cytokine: Thymic Stromal Lymphoprotein (TSLP). TSLP targets dendritic cells (DCs) and drives a specific Th2 response.
  • AD physiopathology results in skin abnormalities, inflammation with IgE and histamine production, erythema, eosinophilia, alopecia and itching.

Example Data

In vitro models - Atopic Dermatitis (AD):

  • 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, IL1a, IL18, IFNa2, IFNß1, IL4R, IL8, MIP1a, RANTES, MIP3a, 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
  • AD inititation - 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, IL1a, IL18, IFNa2, IFN&beta1, IL4R, IL8, MIP1a, RANTES, MIP3a, 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
  • 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, TNFa, 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
  • 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
  • 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

Psoriasis:

  • an inflammatory skin disorder, triggered or exacerbated by a number of genetic, environmental, or immunological factors.
  • characterized by
    • - hyperproliferation of keratinocytes;
    • - abnormal epidermal differentiation; and
    • - infiltration of inflammatory cells

In Vitro assays - Psoriasis:

  • 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)
  • 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
  • Psoriasis physiopathology

    Keratinocytes & 3 D skin models (Cultures of keratinocytes grown on filter at the AirLiquid Interface), co-culture models (keratinocytes+dermal fibrobalsts) 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