Slide 1

Update on the Pathogenesis of Canine Atopic Dermatitis: a comparative review Rosanna Marsella University of Florida Slide 2 R Marsella ACVD Resident Review 2003 Important points Pathogenesis Pathogenesis The old theory: Type I hypersensitivity The old theory: Type I hypersensitivity IgE, mast cells, histamine, LT Increased PDE The new theories: T cell imbalances The new theories: T cell imbalances Biphasic responses of T helper cells Cytokines and chemokines Implications for therapy Implications for therapy Slide 3 R Marsella ACVD Resident Review 2003 Canine Atopic Dermatitis (cAD) Definition: Relapsing dermatitis Relapsing dermatitis with characteristic clinical features with characteristic clinical features familiar predilection familiar predilection mostly associated with increased IgE antibodies against environmental allergens (Task Force, 2001) mostly associated with increased IgE antibodies against environmental allergens (Task Force, 2001) Slide 4 R Marsella ACVD Resident Review 2003 Canine Human Slide 5 R Marsella ACVD Resident Review 2003 Canine Human Slide 6 R Marsella ACVD Resident Review 2003 Experimental canine AD Slide 7 R Marsella ACVD Resident Review 2003 Experimental canine AD Slide 8 R Marsella ACVD Resident Review 2003 Canine Human Slide 9 R Marsella ACVD Resident Review 2003 Canine Human Slide 10 R Marsella ACVD Resident Review 2003 CanineHuman Slide 11 R Marsella ACVD Resident Review 2003 Human Canine Slide 12 R Marsella ACVD Resident Review 2003 Spongiotic superficial perivascular mononuclear dermatitis Slide 13 R Marsella ACVD Resident Review 2003 HumanCanine Slide 14 R Marsella ACVD Resident Review 2003 Prevalence and risk factors Increased prevalence in humans Increased prevalence in humans Risk factors Risk factors Genetics Environmental exposure Nature of the allergen, dose, timing Cytokine profile Foods Slide 15 R Marsella ACVD Resident Review 2003 Co-factors in canine AD Bacteria Bacteria IgE against Staphylococcus Modulation of T cells (e.g.Protein A) Yeast Yeast IgE against Malassezia Zymogen and direct complement activation Slide 16 R Marsella ACVD Resident Review 2003 Epidemiology of AD Human AD Children 10-20% Children 10-20% Adults 1-3% Adults 1-3% Increase in prevalence in developed countries Increase in prevalence in developed countries Hygiene hypothesis? Canine AD 10% of canine population 10% of canine population Increased prevalence Increased prevalence More cases diagnosed? Genetic selection? Slide 17 R Marsella ACVD Resident Review 2003 Genetics and AD Numerous genetic abnormalities reported in humans Numerous genetic abnormalities reported in humans Little is known in dogs Little is known in dogs Severity of disease in individual patient may depend on the cumulative effect Severity of disease in individual patient may depend on the cumulative effect Genotype Phenotype Severity of AD Slide 18 R Marsella ACVD Resident Review 2003 Human AD Familial history Familial history Gene candidates Gene candidates IL-3 IL-4 IL-5 IL-13 IL-4 R CD 80 and CD 86 Canine AD Familial history Strong breed predilection Increased frequency of haplotype DL-A3 and R15 Only evaluated genetic inheritance of IgE production (Dominant) Genetics Slide 19 R Marsella ACVD Resident Review 2003 Genetics and AD Response to treatment varies in individual patients depending on the specific abnormality Response to treatment varies in individual patients depending on the specific abnormality Identification of genotype may be useful in predict clinical response to therapies Identification of genotype may be useful in predict clinical response to therapies Slide 20 R Marsella ACVD Resident Review 2003 The old theory: Role of IgE, mast cells and type I hypersensitivity in AD Slide 21 R Marsella ACVD Resident Review 2003 IgE Th1 Th2 MC APC Eos IL-4 TNF-a Lets not put all our atopic eggs in one basket. Slide 22 R Marsella ACVD Resident Review 2003 IgE Destroyed by heating to 56 o C for 4 hrs Destroyed by heating to 56 o C for 4 hrs Transferable by ID injection Transferable by ID injection Persistence at the site of injection for > 48 hrs Persistence at the site of injection for > 48 hrs Isotype switching regulated by: Isotype switching regulated by: IL-4, IL-13 -IFN and TGF- Slide 23 R Marsella ACVD Resident Review 2003 Canine IgE Constant region gene isolated and sequenced Constant region gene isolated and sequenced Heavy chain 75kDa Heavy chain 75kDa Heterogeneity in canine IgE Heterogeneity in canine IgE Different subisotypes Different chromatic properties Different ability to bind antibodies Slide 24 R Marsella ACVD Resident Review 2003 IgE receptors High affinity (Fc RI) High affinity (Fc RI) Tetrameric ( 2) expressed constitutively on mast cells and basophils Trimeric ( 2) only in patients with extrinsic AD (skin and monocytes) Low affinity (Fc RII, CD 23) Low affinity (Fc RII, CD 23) IgE binding lectin galectin-3 IgE binding lectin galectin-3 Slide 25 R Marsella ACVD Resident Review 2003 Fc RI on APC in AD Antigen uptake Antigen uptake APC maturation APC maturation Release of IL-10 Slide 26 R Marsella ACVD Resident Review 2003 Route of allergen exposure Inhalatory Inhalatory Percutaneous Percutaneous Slide 27 R Marsella ACVD Resident Review 2003 Y Y Y Y Y Y Y Y Y Pathogenesis: Route of allergen exposure - Inhalation Y IgE Allergen Allergen is inhaled and systemically absorbed IgE are produced systemically IgE migrate to tissue and bind to mast cells Slide 28 R Marsella ACVD Resident Review 2003 Pathogenesis: Route of allergen exposure Percutaneous absorption 1) Allergen is captured by LC in the skin Y Y Y 2) Local production of IgE Y IgE Allergen Slide 29 R Marsella ACVD Resident Review 2003 Canine AD Increased expression of surface bound IgE on LC in lesional skin (Olivry et al, 1996) Increased expression of surface bound IgE on LC in lesional skin (Olivry et al, 1996) Human AD LC capture allergen via their high affinity IgE receptor (Fc RI) Important for LC maturation Role of IgE- Facts in support 1) Role in allergen capture Slide 30 R Marsella ACVD Resident Review 2003 Role in the effector pathway Role of IgE - Facts in support 2) Role in the effector pathway Canine AD Most dogs with AD have detectable allergen-specific IgE Most dogs with AD have detectable allergen-specific IgE Human AD Most patients with AD exhibit both elevated total and allergen specific IgE (extrinsic AD, 80% of patients) Slide 31 R Marsella ACVD Resident Review 2003 Canine AD IgE do not correlate with severity of disease IgE do not correlate with severity of disease No difference in serum total IgE between normal and atopic dogs No difference in serum total IgE between normal and atopic dogs Negative IDST and serology testing in some dogs with clinical AD Negative IDST and serology testing in some dogs with clinical AD Human AD People with genetic inability to produce IgE may develop AD Patients treated with -IFN improve despite raising IgE Intrinsic AD or non- allergic AD (20%) Role of IgE - Controversies Slide 32 R Marsella ACVD Resident Review 2003 Role of IgE Controversies Canine AD Negative IDST and serology testing in some dogs with clinical AD Negative IDST and serology testing in some dogs with clinical AD Anergy? Anergy? Allergens not included in the test? Allergens not included in the test? Wrong season? Wrong season? Heterogeneity of IgE Heterogeneity of IgE Slide 33 R Marsella ACVD Resident Review 2003 Positive IDST and serology testing in normal dogs with no signs of AD Positive IDST and serology testing in normal dogs with no signs of AD IgE heterogeneity IgE heterogeneity Positive IDST and serology testing: a secondary criteria for diagnosis Positive IDST and serology testing: a secondary criteria for diagnosis Role of IgE- Controversies Canine AD Slide 34 R Marsella ACVD Resident Review 2003 Past attempts to create a model for cAD in high IgE producing dogs have failed Past attempts to create a model for cAD in high IgE producing dogs have failed No correlation existed between serum levels of IgE and development of disease No correlation existed between serum levels of IgE and development of disease Role of IgE- Controversies Canine AD Slide 35 R Marsella ACVD Resident Review 2003 Evaluation of total and cell bound IgE in normal and atopic dogs (Jackson, 2002) Evaluation of total and cell bound IgE in normal and atopic dogs (Jackson, 2002) No significant difference in levels of total IgE ** No significant difference in % of B cells expressing IgE Total IgE did not correlate with cell bound IgE in any of the leukocyte populations studied ** **difference between canine and human AD IgE in canine AD Slide 36 R Marsella ACVD Resident Review 2003 IgE - Controversies Additional factors besides IgE are necessary to cause disease Additional factors besides IgE are necessary to cause disease T cells abnormalities Abnormality in lipid composition in the skin Altered reactivity in the skin adrenergic hyporesponsiveness Increased PDE, decreased cAMP Slide 37 R Marsella ACVD Resident Review 2003 Intrinsic AD or non allergic atopiform dermatitis 10-30% of humans with AD do not have increased IgE levels 10-30% of humans with AD do not have increased IgE levels Negative IDST Negative IDST Negative APT Negative APT Negative serology Negative serology Slide 38 R Marsella ACVD Resident Review 2003 Human AD Extrinsic AD IgE and Fc RI IgE and Fc RI + APT + APT + serology + serology + IDST + IDST IL-13 IL-13 IL-5 IL-5 IL-4 IL-4 Intrinsic AD Normal IgE, low Fc RI - APT - serology - IDST IL-13 IL-5 IL-4 but IL-4R (receptor for both IL-4 and IL-13) Slide 39 R Marsella ACVD Resident Review 2003 IL-13 and IL-4 important for IgE production, but non sufficient IL-13 and IL-4 important for IgE production, but non sufficient Additional co-stimulatory factors missing in patients with intrinsic AD? IL-13 may cause disease directly bypassing production of IgE IL-13 may cause disease directly bypassing production of IgE Intrinsic AD Slide 40 R Marsella ACVD Resident Review 2003 IgE as epiphenomena in human AD? Increased LC in lesions and increased expression of Fc RI may derive from excessive transcutaneous allergen exposure in traumatized skin Increased LC in lesions and increased expression of Fc RI may derive from excessive transcutaneous allergen exposure in traumatized skin Slide 41 R Marsella ACVD Resident Review 2003 ROLE OF MAST CELLS AND HISTAMINE IN AD Slide 42 R Marsella ACVD Resident Review 2003 Role of histamine in AD Circulating levels Human AD Elevated during exacerbation of disease (plasma) and return to normal during clinical remission Elevated during exacerbation of disease (plasma) and return to normal during clinical remission Correlation between histamine release and IgE levels is controversial Correlation between histamine release and IgE levels is controversial Canine AD Serum concentrations in dogs with AD are same or lower than in controls Serum concentrations in dogs with AD are same or lower than in controls No correlation with IgE No correlation with IgE Slide 43 R Marsella ACVD Resident Review 2003 Human AD Increased concentrations in AD patients Increased concentrations in AD patients Good correlation between cutaneous and circulating levels of histamine Good correlation between cutaneous and circulating levels of histamine Canine AD Levels are greater in dogs with AD than normal dogs No correlation between cutaneous and plasma histamine concentrations Role of histamine in AD Skin Slide 44 R Marsella ACVD Resident Review 2003 IDST reactivity to histamine in AD Human AD Decreased response in people with AD Decreased response in people with AD Down regulation of target structures? Down regulation of target structures? Canine AD Decreased response in dogs with AD Down regulation of target structures? Slide 45 R Marsella ACVD Resident Review 2003 Role of histamine in AD Peripheral leukocytes Human AD Increased histamine releasability after stimulation Increased histamine releasability after stimulation High and low histamine responders High and low histamine responders Pre-medication with PDE inhibitors normalized histaminereleasability Canine AD Greater tendency to release histamine than normal dogs Slide 46 R Marsella ACVD Resident Review 2003 Role of Mast cells in AD Traditionally seen as the effector cell for Type I hypersensitivity Traditionally seen as the effector cell for Type I hypersensitivity Insufficient evidence to support an important role in canine AD Insufficient evidence to support an important role in canine AD Slide 47 R Marsella ACVD Resident Review 2003 Fc RI Y Y Allergen IgE MAST CELL Y Y Y Anti-IgE Y Anti-Fc RI Substance P Stem cell factor C5a Codein Slide 48 R Marsella ACVD Resident Review 2003 MAST CELL DEGRANULATION Cytokines (e.g. IL-1, 2, 3, 4, 5, 6, 8, 9, 13, GM-CSF, TNF- , IFN- , MIP-1a and MIP-1b) Pre-formed mediators Proteases (e.g. tryptase) Heparin Histamine Newly-formed mediators PGD2 LTC4 PAF Slide 49 R Marsella ACVD Resident Review 2003 TYPE I HYPERSENSIVITY Blood vessel Histamine Fc RI Y Y Allergen IgE Y Y Y Y LT IL-1, 2, 3, 4, 5, 13 IL-6, TNF- Slide 50 R Marsella ACVD Resident Review 2003 Canine Mast cells Heterogeneity, not tissue specific Heterogeneity, not tissue specific Highest number on pinnae and feet Highest number on pinnae and feet Slide 51 R Marsella ACVD Resident Review 2003 Total histamine content, per skin mast cell, is higher in dogs with AD than controls (Nimmo Wilkie, 1990) Total histamine content, per skin mast cell, is higher in dogs with AD than controls (Nimmo Wilkie, 1990) Increased histamine/cell? Increased reactivity to non-immunological and immunological stimuli (Demora, 1996) Increased reactivity to non-immunological and immunological stimuli (Demora, 1996) Mast cells in canine AD Slide 52 R Marsella ACVD Resident Review 2003 Number of mast cells in atopic skin is controversial Number of mast cells in atopic skin is controversial Increased, decreased, normal (Scott et al, 1981) Same as normal dogs (Olivry, 1997 and Welle 1999) Increased but not in all sites (Nimmo Wilkie, 1990) Mast cells in canine AD Slide 53 R Marsella ACVD Resident Review 2003 Leukotrienes in AD Peripheral leukocytes Human AD Enhanced release of LTB 4 and LTC 4 in patients with AD when compared to controls Enhanced release of LTB 4 and LTC 4 in patients with AD when compared to controls Increased LTA 4 hydrolase Increased LTA 4 hydrolase Canine AD No differences in s- LT synthesis between normal and dogs with AD after challenge No differences in s- LT synthesis between normal and dogs with AD after challenge Slide 54 R Marsella ACVD Resident Review 2003 Human AD Increased LTB4 in lesional skin of atopic patients Increased LTB4 in lesional skin of atopic patients Increased production of LT after allergen challenge Increased production of LT after allergen challenge Canine AD No differences in s-LT between controls and non-lesional skin of AD dogs Within AD,no differences in s-LT between lesional and non-lesional skin Leukotrienes in AD Skin Slide 55 R Marsella ACVD Resident Review 2003 The new theories: Role of T cells, cytokines, chemokines and type IV hypersensitivity in AD Slide 56 R Marsella ACVD Resident Review 2003 Additional players T cells T cells Dendritic cells Dendritic cells Cytokines Cytokines Function Kinetics APC APC Chemokines Chemokines Neuropeptides Neuropeptides Slide 57 R Marsella ACVD Resident Review 2003 T cell sub-populations - Human AD Cutaneous lesions Cutaneous lesions CD4+, CLA+ Imbalance in the T helper cells (BIPHASIC response) Imbalance in the T helper cells (BIPHASIC response) Acute phase Th2 cytokines (IL-3, 4, 5, and 13) Chronic phase Th1 cytokines (IL-2, 12 and -IFN) Slide 58 R Marsella ACVD Resident Review 2003 Atopic Dermatitis (acute lesions) T helper 2 T helper 1 IL-4 IL-5 IL-2 -IFN Slide 59 R Marsella ACVD Resident Review 2003 Atopic Dermatitis (chronic lesions) T helper 2 T helper 1 IL-4 IL-5 IL-2 -IFN Slide 60 R Marsella ACVD Resident Review 2003 Epidermal dendritic cells in AD Langerhans cells (LC) Langerhans cells (LC) Resident cells Polarization toward Th2 response (IL-4, IL-13) Stimulation by TLR (early phase) Stimulation of Fc RI (later phase) Inflammatory dendritic epidermal cells (IDEC) Inflammatory dendritic epidermal cells (IDEC) Recruited ex novo from dermis Upon stimulation of Fc RI they release IL-12 and IL18 switch of Th response into Th1 Slide 61 R Marsella ACVD Resident Review 2003 Dendritic cells in AD Receptors Receptors Fc RI, Fc RII, TLR2 and 4 Important for antigen presentation Important for antigen presentation Important for cytokine release and modulation of T cell population Important for cytokine release and modulation of T cell population Increased number in chronic lesions of AD Increased number in chronic lesions of AD Slide 62 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines IL-1, IL-6 and TNF- Th2 cytokines Th2 cytokines IL3, 4, 5, 13 Th1 cytokines Th1 cytokines IL-2, -IFN, IL-12 IL-18 IL-18 Suppressive cytokines Suppressive cytokines TGF- and IL-10 Slide 63 R Marsella ACVD Resident Review 2003 TNF- Produced by: Produced by: Mononuclear cells Neutrophils Activated T cells NK cells Mast cells Keratinocytes Slide 64 R Marsella ACVD Resident Review 2003 Inducers Inducers Trauma LPS Toll-like receptors (TLR2 and TLR4) TNF- Slide 65 R Marsella ACVD Resident Review 2003 Role of TNF- in AD Induces adhesion molecules expression Induces adhesion molecules expression ICAM-1, ELAM-1, VCAM-1 No direct effect on lymphocyte proliferation No direct effect on lymphocyte proliferation TNF- increases: TNF- increases: IL-1 (activation and recruiting of inflammatory cells, expression of adhesion molecules) IL-1 (activation and recruiting of inflammatory cells, expression of adhesion molecules) IL-3 (mast cell growth factor) IL-3 (mast cell growth factor) IL-4 (IgE synthesis) IL-4 (IgE synthesis) Slide 66 R Marsella ACVD Resident Review 2003 Increases: Increases: IL-5 (maturation and activation of eosinophils) IL-5 (maturation and activation of eosinophils) IL-6 (important for IL-4 induced IgE synthesis) IL-6 (important for IL-4 induced IgE synthesis) IL-8 (chemotactic for neutrophils) IL-8 (chemotactic for neutrophils) GM-CSF (activation and prolonged survival of eosinophils) GM-CSF (activation and prolonged survival of eosinophils) Role of TNF- in AD Slide 67 R Marsella ACVD Resident Review 2003 TNF- and AD Expression of TNF- up-regulated 2-4 hours after allergen challenge in atopic people (Gosset et al, 1992) Expression of TNF- up-regulated 2-4 hours after allergen challenge in atopic people (Gosset et al, 1992) Expression of TNF- correlates with severity of late phase cutaneous reactions after allergen challenge in atopic people (Gosset et al, 1992) Expression of TNF- correlates with severity of late phase cutaneous reactions after allergen challenge in atopic people (Gosset et al, 1992) Slide 68 R Marsella ACVD Resident Review 2003 TNF- and AD TNF- produced by mast cells after IgE mediated activation (Gordon et al, 1990) TNF- produced by mast cells after IgE mediated activation (Gordon et al, 1990) Anti-TNF- antibodies abrogate LPR in mice (Wershill et al, 1991) Anti-TNF- antibodies abrogate LPR in mice (Wershill et al, 1991) Slide 69 R Marsella ACVD Resident Review 2003 IL-1 Family of peptides Family of peptides IL-1 , IL-1 IL-1ra, IL-18 Two receptors Two receptors Type I - Active Type II Inactive, anti-inflammatory functions Slide 70 R Marsella ACVD Resident Review 2003 IL-1 Produced by: Produced by: Mononuclear cells Endothelial cells Keratinocytes Slide 71 R Marsella ACVD Resident Review 2003 IL-1 Activation of T cells Activation of T cells Increased IL-2 and IL-2 receptors Expression of adhesion molecules Expression of adhesion molecules ICAM-1, VCAM-1, E-selectin Slide 72 R Marsella ACVD Resident Review 2003 IL-1ra (receptor antagonist) Important for down-regulation of inflammatory process Important for down-regulation of inflammatory process Up-regulated by: Up-regulated by: IL-4, IL-13, IL-6 Slide 73 R Marsella ACVD Resident Review 2003 IL-6 Produced by: Produced by: Mononuclear cells T cells B cells Fibroblasts Endothelial cells Keratinocytes Slide 74 R Marsella ACVD Resident Review 2003 IL-6 Pro-inflammatory properties Pro-inflammatory properties T cell activation and differentiation B cell differentiation and Ig synthesis Anti-inflammatory properties Anti-inflammatory properties Inhibits IL-1 and TNF- Increases IL-1ra Slide 75 R Marsella ACVD Resident Review 2003 IL-6 IL-6 Role in IL-4 induced IgE synthesis Released after allergen challenge in humans with AD Correlates with the size of cutaneous LPR Cytokines in human AD Slide 76 R Marsella ACVD Resident Review 2003 IL-6 and AD IL-6 released after allergen challenge in atopic people (Lee et al, 1992) IL-6 released after allergen challenge in atopic people (Lee et al, 1992) IL-6 production correlates with severity of LPR (Yamada et al, 1995) IL-6 production correlates with severity of LPR (Yamada et al, 1995) Obligatory role of IL-6 in IL-4 induced IgE synthesis in humans (Vercelli et al, 1989) Obligatory role of IL-6 in IL-4 induced IgE synthesis in humans (Vercelli et al, 1989) IL-6 produced by mast cells after IgE mediated activation (Gordon et al, 1990) IL-6 produced by mast cells after IgE mediated activation (Gordon et al, 1990) Slide 77 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines IL-1, IL-6 and TNF- Th2 cytokines Th2 cytokines IL 4, 5, 13 Th1 cytokines Th1 cytokines IL-2, -IFN, IL-12 IL-18 IL-18 Suppressive cytokines Suppressive cytokines TGF- and IL-10 Slide 78 R Marsella ACVD Resident Review 2003 IL-4 Produced by: Produced by: T cells Eosinophils Mast cells Basophils Slide 79 R Marsella ACVD Resident Review 2003 IL-4 IL-4 and 13 share IL-4 and 13 share Common receptor subunit (IL-4R ) 8 allelic variants reported, some associated with AD 25% homology Overlap in functions Stimulates antigen presentation Stimulates antigen presentation MHCII, B7, CD40 Increases expression of CD23 (Fc RII) Increases expression of CD23 (Fc RII) On B cells, Macrophages, APC Isotype switching (IgE) Isotype switching (IgE) Increases LTC4 synthetase Increases LTC4 synthetase Slide 80 R Marsella ACVD Resident Review 2003 Increases expression of adhesion molecules Increases expression of adhesion molecules Differentiation of Th0 into Th2 (function not shared with IL-13) Differentiation of Th0 into Th2 (function not shared with IL-13) Prevents apoptosis of T cells Prevents apoptosis of T cells Increases resistance to glucocorticoids Increases resistance to glucocorticoids IL-4 and AD Slide 81 R Marsella ACVD Resident Review 2003 Anti-inflammatory properties Anti-inflammatory properties Decreases IL-1, IL-6, TNF- Increases IL-1ra Actions of IL-4 Slide 82 R Marsella ACVD Resident Review 2003 IL-13 and AD Receptor Receptor IL-4R chain of the receptor IL13R chain (not expressed on mast cells and T cells) No ability to induce Th2 differentiation or mast cells activation No ability to induce Th2 differentiation or mast cells activation Shares many actions with IL-4 Shares many actions with IL-4 IgE isotype switching Expression of VCAM-1 Slide 83 R Marsella ACVD Resident Review 2003 IL-5 Produced by: Produced by: Th2 cells Mast cells Eosinophils? Actions on eosinophils Actions on eosinophils Stimulation of eosinophil production Activation of mature eosinophils Increased survival (blockade of apoptosis) Up-regulation of responses to chemokines (increases expression of CCR3) Slide 84 R Marsella ACVD Resident Review 2003 IL-5 and AD Positive correlation between IL-5 release and size of LPR after allergen challenge (Okada et al, 2002) Positive correlation between IL-5 release and size of LPR after allergen challenge (Okada et al, 2002) Prevention of blockade of eosinophil apoptosis by oxatomide as a potential treatment for atopic disease (Domae et al, 2003) Prevention of blockade of eosinophil apoptosis by oxatomide as a potential treatment for atopic disease (Domae et al, 2003) Slide 85 R Marsella ACVD Resident Review 2003 Eosinophils and s-LT Number of Eosinophils RecruitmentSurvival S-LT ++ IL-4, IL-5, IL-13 + + Slide 86 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines IL-1, IL-6 and TNF- Th2 cytokines Th2 cytokines IL3, 4, 5, 13 Th1 cytokines Th1 cytokines IL-2, -IFN, IL-12 IL-18 IL-18 Suppressive cytokines Suppressive cytokines TGF- and IL-10 Slide 87 R Marsella ACVD Resident Review 2003 IL-2 Produced by T cells (Th1) after antigen stimulation Produced by T cells (Th1) after antigen stimulation Clonal T cell proliferation and differentiation Activation of NK cells Activation of cytotoxic T cells Activation of macrophages Slide 88 R Marsella ACVD Resident Review 2003 IL-2 and AD IL-2 levels have significant inverse correlation with serum IgE levels (Yoshizawa et al, 2002) IL-2 levels have significant inverse correlation with serum IgE levels (Yoshizawa et al, 2002) IL-2 may inhibit IgE synthesis in patients with AD Prevents apoptosis of T cells in skin of patients with AD Prevents apoptosis of T cells in skin of patients with AD Slide 89 R Marsella ACVD Resident Review 2003 -IFN Produced by: Produced by: Th1 cells Cytotoxic T cells NK cells Increases expression of MHCI and II Increases expression of MHCI and II Stimulation of cytokines release by monocytes Stimulation of cytokines release by monocytes IL-12, IL-18 and IL-23 Activation of macrophages Activation of macrophages Improves killing by NK cells Improves killing by NK cells Inhibits IL-4 and regulates IgE synthesis Inhibits IL-4 and regulates IgE synthesis Slide 90 R Marsella ACVD Resident Review 2003 Reduced production by peripheral mononuclear cells in AD Reduced production by peripheral mononuclear cells in AD Important in chronic lesions of AD Important in chronic lesions of AD Recruitment of mononuclear cells Up-regulation of Fas on keratinocytes which increases susceptibility to apoptosis and leads to spongiosis -IFN and AD Slide 91 R Marsella ACVD Resident Review 2003 IL-12 Produced by: Produced by: Monocytes Macrophages B cells Dendritic cells (IDEC) Induces Th1 response Induces Th1 response Proliferation of cytotoxic cells Proliferation of cytotoxic cells Slide 92 R Marsella ACVD Resident Review 2003 IL-12 and AD IL-12 p40 polymorphism is associated with development of AD (Tsunemi et al, 2002) IL-12 p40 polymorphism is associated with development of AD (Tsunemi et al, 2002) IL-12 serum levels correlate with IL-10 (Yoshizawa et al, 2002) IL-12 serum levels correlate with IL-10 (Yoshizawa et al, 2002) Slide 93 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines IL-1, IL-6 and TNF- Th2 cytokines Th2 cytokines IL3, 4, 5, 13 Th1 cytokines Th1 cytokines IL-2, -IFN, IL-12 IL-18 IL-18 Suppressive cytokines Suppressive cytokines TGF- and IL-10 Slide 94 R Marsella ACVD Resident Review 2003 IL-18 Produced by: Produced by: Macrophages IDEC Keratinocytes Pleiotropic properties Pleiotropic properties Inducer of Th-1 response Inducer of Th-1 response Stimulates production of -IFN Co-stimulant for IL-12 Increases expression of ICAM-1 Slide 95 R Marsella ACVD Resident Review 2003 IL-18 Inducer of Th-2 response Inducer of Th-2 response In presence of IL-3, stimulates basophil degranulation (in absence of IgE) Stimulates IL-4 and IL-13 synthesis IgE Slide 96 R Marsella ACVD Resident Review 2003 IL-18 in AD Elevated serum levels in human patients with AD (Shida et al 2002, El- Mezzein et al, 2001) Elevated serum levels in human patients with AD (Shida et al 2002, El- Mezzein et al, 2001) Marked expression in skin biopsies from lesional skin in AD patients (Higashi et al, 2002) Marked expression in skin biopsies from lesional skin in AD patients (Higashi et al, 2002) Slide 97 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines IL-1, IL-6 and TNF- Th2 cytokines Th2 cytokines IL3, 4, 5, 13 Th1 cytokines Th1 cytokines IL-2, -IFN, IL-12 IL-18 IL-18 Suppressive cytokines Suppressive cytokines TGF- and IL-10 Slide 98 R Marsella ACVD Resident Review 2003 TGF- Produced by Th3 or T repressors Produced by Th3 or T repressors Inhibits B cells and T helper and cytotoxic Inhibits B cells and T helper and cytotoxic Inhibits IgE and mast cell proliferation Inhibits IgE and mast cell proliferation Slide 99 R Marsella ACVD Resident Review 2003 Patients with AD have decreased ability to produce TGF- Patients with AD have decreased ability to produce TGF- TGF- suppresses AD lesions in NC/Nga mice through down-regulation of IFN- TGF- suppresses AD lesions in NC/Nga mice through down-regulation of IFN- TGF- and AD Slide 100 R Marsella ACVD Resident Review 2003 IL-10 Pleiotropic properties according to circumstances Pleiotropic properties according to circumstances Generally considered as a Th-2 cytokine Generally considered as a Th-2 cytokine Suppression of Th-1 response Enhancement of Th-2 response Suppression of pro-inflammatory cytokines Suppression of pro-inflammatory cytokines Used as a marker of response to IT Used as a marker of response to IT Slide 101 R Marsella ACVD Resident Review 2003 Redundancy and divergence in the cytokine system Redundancy Redundancy IL 4 and IL13 IL-1 and TNF- Cross-reaction of pathways Cross-reaction of pathways IL-4 Th2 IL-12 Th1 Divergence Divergence IL-18 IL-10 Slide 102 R Marsella ACVD Resident Review 2003 Chemokines (Chemotactic cytokines) Group of small molecules (8-14kD) Group of small molecules (8-14kD) Released at site of infection or trauma Released at site of infection or trauma 4 families, classified based on the position of cysteine residues 4 families, classified based on the position of cysteine residues CXC target neutrophils and lymphocytes CC - target eosinophils, basophils, dendritic cells, T cells, and monocytes C CX3C Slide 103 R Marsella ACVD Resident Review 2003 Chemokines Link with innate immune system Link with innate immune system Triggered by: Triggered by: Bacterial products Pro-inflammatory cytokines Released in a short time (within 1-2 hours) Released in a short time (within 1-2 hours) Binding to seven specific trans- membrane G protein coupled surface receptors (GPCR) Binding to seven specific trans- membrane G protein coupled surface receptors (GPCR) Slide 104 R Marsella ACVD Resident Review 2003 Chemokines Induce chemotaxis Induce chemotaxis By up-regulating selectins Affect T cell differentiation Affect T cell differentiation Altering cytokine secretion Altering APC trafficking Slide 105 R Marsella ACVD Resident Review 2003 Chemokines and Th cytokines -IFN induced chemokines -IFN induced chemokines 10kd IFN Inducible Protein (IP-10) Monokine Induced by -IFN (MIG) IFN-Inducible T cell chemoattractant (I- TAC) IL-4 and IL-13 induced chemokines IL-4 and IL-13 induced chemokines Macrophage Chemoattractant Protein (MCP) Eotaxin Slide 106 R Marsella ACVD Resident Review 2003 Chemokine receptors and Th cells Th1 Th1 CXCR3 - binds MIG and IP-10 CCR5 - binds RANTES Th2 Th2 CCR3 - binds MCP 4 CCR4 - binds TARC, MDC CCR8 Slide 107 R Marsella ACVD Resident Review 2003 Chemokines and eosinophils CCR3 (high expression) CCR3 (high expression) CCR1 (low expression) CCR1 (low expression) Chemotactic stimuli Chemotactic stimuli Macrophage Inflammatory Protein (MIP) 1 RANTES Eotaxin-1, 2, 3 MCP-2, 3, 4 Slide 108 R Marsella ACVD Resident Review 2003 Antagonism between Chemokines MIG inhibits CCR3 mediated responses MIG inhibits CCR3 mediated responses IV administration before allergen challenge inhibits recruitment of eosinophils Slide 109 R Marsella ACVD Resident Review 2003 Chemokines and AD Macrophage Chemoattractant Proteins (MCP) - MCP 2, 3, 4 Macrophage Chemoattractant Proteins (MCP) - MCP 2, 3, 4 Bind to CCR3 - Expressed on eosinophils and Th2 Bind to CCR2- Expressed on monocytes (constitutively) and memory T cells (after IL-2 stimulation) Slide 110 R Marsella ACVD Resident Review 2003 Chemokines and AD Macrophage Derived Chemokine (MDC, CCL22) Macrophage Derived Chemokine (MDC, CCL22) Produced by DC and macrophages Binds to CCR4 - Expressed on Th2 cells Increased expression in patients with AD (both serum and lesional skin) Slide 111 R Marsella ACVD Resident Review 2003 Eotaxin (CCL11) Eotaxin (CCL11) Attracts eosinophils Binds to CCR3 - Expressed on eosinophils and Th2 cells RANTES (Regulated on Activation Normal T-cell-Expressed and Secreted or CCL5) RANTES (Regulated on Activation Normal T-cell-Expressed and Secreted or CCL5) Attracts eosinophils Binds to CCR1, CCR3 and CCR5 Chemokines and AD Slide 112 R Marsella ACVD Resident Review 2003 CC Chemokines and AD Spontaneous production of RANTES, MCP-1 and eotaxin by peripheral mononuclear cells in patients with AD Spontaneous production of RANTES, MCP-1 and eotaxin by peripheral mononuclear cells in patients with AD Slide 113 R Marsella ACVD Resident Review 2003 TARC and AD Thymus and activation-regulated chemokine (TARC, CCL17) Thymus and activation-regulated chemokine (TARC, CCL17) Produced by keratinocytes, peripheral mononuclear cells, dendritic cells, macrophages, endothelial cells Produced by keratinocytes, peripheral mononuclear cells, dendritic cells, macrophages, endothelial cells Production is stimulated by: Production is stimulated by: IL-4 and IL-13 Pro-inflammatory cytokines such as -IFN and TNF- LPS Slide 114 R Marsella ACVD Resident Review 2003 Binds to CCR4 - Expressed on Th2 cells Binds to CCR4 - Expressed on Th2 cells Recruits CLA+, CCR4+ T cells (Th2) into lesional skin Recruits CLA+, CCR4+ T cells (Th2) into lesional skin Increases integrin-dependent adhesion of T cells to endothelial ICAM-1 Increased expression in patients with AD Increased expression in patients with AD TARC and AD Slide 115 R Marsella ACVD Resident Review 2003 TARC and AD Plasma TARC levels Plasma TARC levels Significantly increased in patients with AD and correlate with severity of AD TARC expression in the skin TARC expression in the skin Not expressed in normal skin or psoriatic skin Highly expressed in lesional atopic skin Higher expression of TARC in acute lesions than chronic Keratinocytes in the basal layer showed the strongest staining for TARC Slide 116 R Marsella ACVD Resident Review 2003 CCR4 and AD Circulating CCR4+ cells (CD4+ T cells) Circulating CCR4+ cells (CD4+ T cells) Significantly higher in AD patients than that in healthy controls CCR4+ cells in the skin CCR4+ cells in the skin Present only in the lesional skin of AD patients, but not in the non-lesional skin Slide 117 R Marsella ACVD Resident Review 2003 Pre-administration of CCR4 neutralizing antibody before challenge prevents recruitment of eosinophils and T cells after allergen challenge (Kawasaki et al, 2003) Pre-administration of CCR4 neutralizing antibody before challenge prevents recruitment of eosinophils and T cells after allergen challenge (Kawasaki et al, 2003) CCR4 and AD Slide 118 R Marsella ACVD Resident Review 2003 Cytokines in Canine AD (Nuttal et al, 2002) RNA isolated from atopic and normal skin IL-4 in atopic skin TGF- IFN- TNF- and IL-2 in lesional vs. non lesional (infections?) Slide 119 R Marsella ACVD Resident Review 2003 mRNA isolated from atopic and normal skin TARC mRNA selectively expressed in lesional skin of dogs with AD IL-1 , -IFN and TNF- in lesional skin compared to non-lesional skin of dogs with AD CCR4 mRNA preferentially expressed in lesional skin of dogs with AD Cytokines in Canine AD (Maeda et al, 2002) Slide 120 R Marsella ACVD Resident Review 2003 T cell sub-populations in canine AD (Hayahiya et al, 2002) Expression of cytokine mRNA from peripheral blood mononuclear cells Expression of cytokine mRNA from peripheral blood mononuclear cells -IFN and IL-5 in dogs with AD No difference in IL-4 and IL-10 between normal and atopic dogs Slide 121 R Marsella ACVD Resident Review 2003 T cell populations in canine AD (Olivry et al, 1999) Increase in CD4+ and CD8+ T-cells in lesional skin of dogs with AD Increase in CD4+ and CD8+ T-cells in lesional skin of dogs with AD Predominance of CD4+ T-cells in the epidermis Predominance of CD4+ T-cells in the epidermis IL-4 and IL-5 cytokine-gene transcripts are detected more commonly in atopic skin biopsies IL-4 and IL-5 cytokine-gene transcripts are detected more commonly in atopic skin biopsies of atopic samples has type-2 cytokine profiles of atopic samples has type-2 cytokine profiles Slide 122 R Marsella ACVD Resident Review 2003 The Hygiene Hypothesis A protective role for endotoxin? Slide 123 R Marsella ACVD Resident Review 2003 Bacterial infections and AD Protective effect (hygiene hypothesis) Protective effect (hygiene hypothesis) Toll-like receptors (TLR) and DC Contributing factor in clinical relapses Contributing factor in clinical relapses Serving as antigen Stimulating TLR on mast cells and LC Activating keratinocytes Recruiting macrophages Slide 124 R Marsella ACVD Resident Review 2003 Protective effects of innate immunity Toll-like receptors Toll-like receptors Expressed on variety of cells (DC, Mast cells, B and T cells) Recognize invariant pathogen associated molecular patterns 10 different types recognized in humans and 11 in mice Type of antigen, dose and receptor determine the outcome Slide 125 R Marsella ACVD Resident Review 2003 Toll-like receptors Stimulation of receptor TLR4 on APC by high doses of LPS Stimulation of receptor TLR4 on APC by high doses of LPS Release of antibacterial peptides Release of cytokines important for Th-1 (IL-12, IL-18) Maturation of APC (expression of co- stimulatory molecules, CD 80) Slide 126 R Marsella ACVD Resident Review 2003 Endotoxin (high doses) INNATE IMMUNITY Dendritic cell Macrophage Monocyte TLR4 -IFN ACQUIRED IMMUNITY IL-12 IL-10 IL-18 Th1 cell NK cell -IFN Memory Th2 cell IL-4, 5, 13 ATOPY X Slide 127 R Marsella ACVD Resident Review 2003 Endotoxin (low doses) INNATE IMMUNITY Dendritic cell Macrophage Monocyte TLR4 ACQUIRED IMMUNITY IL-4 Th2 cell IL-4 IL-5 IL- 13 Memory Th2 cell ATOPY Slide 128 R Marsella ACVD Resident Review 2003 Deleterious effects of bacteria TLR on mast cells (TLR 2 and TLR 4) TLR on mast cells (TLR 2 and TLR 4) Mast cell degranulation in absence of IgE cross-linking Release of s-LT Release of IL-1, TNF- , IL-4, IL-5, IL-13 Slide 129 R Marsella ACVD Resident Review 2003 Different actions of bacteria in AD patients IL-1, IL-6, TNF- TARC Staphilococcus LC MC Y Y Y IL-4 Th2 IL-4, 5, 13 IL-1, IL-6 TNF- , LT, Histamine Th2 IL-4, 5, 13 Slide 130 R Marsella ACVD Resident Review 2003 Trauma of the skin in AD Trauma leads to release of Trauma leads to release of IL-10 (stimulation of Th2) C3 TARC (chemotactic for Th2) Eotaxin (chemotactic for eosinophils) IL-1, TNF- (increased expression of adhesion molecules leading to leukocytes extravasation) Slide 131 R Marsella ACVD Resident Review 2003 Immunologic phases of AD IL-1, IL-6, TNF- TARC, Eotaxin Staph MC Y Y Y IL-1, IL-6 TNF- , LT, Histamine Allergens LC Th2 IL-4, 5, 13 Y Y Y Th2 CLA+ Trauma Circulation Eos IDEC YY IL-12, 18 Th0 IL-2, -IFN Th1 Slide 132 R Marsella ACVD Resident Review 2003 Additional theories on AD Abnormalities in cyclic nucleotide regulation Abnormalities in cyclic nucleotide regulation Excessive activity of PDE Decreased cAMP Hyperreactivity and excessive releasability of mediators Role of neuropeptides Role of neuropeptides Slide 133 R Marsella ACVD Resident Review 2003 PDE and AD Increased PDE Increased PDE Dogs with AD (Chan et al, 1985) Dogs with AD (Chan et al, 1985) People with AD (Hanifin et al, 1992) People with AD (Hanifin et al, 1992) Atopic PDE in man has higher sensitivity to a variety of enzyme inhibitors than healthy controls (Crocker et al, 1998) Atopic PDE in man has higher sensitivity to a variety of enzyme inhibitors than healthy controls (Crocker et al, 1998) Deficient cAMP response Deficient cAMP response Dogs with AD (Emala et al, 1995) Dogs with AD (Emala et al, 1995) People with AD (Butler et al, 1983) People with AD (Butler et al, 1983) Slide 134 R Marsella ACVD Resident Review 2003 PDE Various PDE isoenzymes exist Various PDE isoenzymes exist PDE-4 important for mediator release PDE-4 important for mediator release Mast cells Macrophages T-lymphocytes Eosinophils Keratinocytes Slide 135 R Marsella ACVD Resident Review 2003 Substance P in human AD Substance P has numerous modulatory effects on inflammation Substance P has numerous modulatory effects on inflammation Chemotactic for neutrophils, monocytes, T cells, and eosinophils Induces the expression of cell adhesion molecules Induces proliferation of T helper lymphocytes Slide 136 R Marsella ACVD Resident Review 2003 Substance P in human AD SP receptors identified on mast cells SP receptors identified on mast cells Stimulation of SP receptors triggers degranulation and histamine release Stimulation of SP receptors triggers degranulation and histamine release Slide 137 R Marsella ACVD Resident Review 2003 Substance P and AD Human AD Decreased reactivity of ID injections of SP Decreased reactivity of ID injections of SP SP release is triggered by allergen challenge and by histamine SP release is triggered by allergen challenge and by histamine Canine AD Decreased reactivity to ID injections of SP in dogs with AD Altered sensitivity? Altered sensitivity to histamine (triggered by SP)? Slide 138 R Marsella ACVD Resident Review 2003 Human AD Patients with AD have increased SP immunoreactivity compared to controls Patients with AD have increased SP immunoreactivity compared to controls Canine AD No difference in SP concentration between normal and AD dogs Substance P and AD Slide 139 R Marsella ACVD Resident Review 2003 Treatments for AD Targeted treatments Targeted treatments Antihistamines PDE inhibitors (e.g. pentoxifylline) Anti-IgE, Anti-LT, Anti-TNF- Non-specific treatments Non-specific treatments Glucocorticoids Calcineurin inhibitors (e.g. Cyclosporine, FK-506) PG analogues Slide 140 R Marsella ACVD Resident Review 2003 Considerations Advantage of targeted treatments Advantage of targeted treatments Mediator targeted is produced in large quantities Receptors are expressed Other pathways are not affected Disadvantages Disadvantages Alternative circuits may compensate for blockade of mediators Insignificant clinical improvement Slide 141 R Marsella ACVD Resident Review 2003 Considerations Advantages of non-specific treatments Advantages of non-specific treatments Broad spectrum effects Disadvantages Disadvantages Unwanted effects Steroids Decrease IL-10Decrease IL-10 Increase LTB4 receptor expressionIncrease LTB4 receptor expression Poor inhibitors of LTPoor inhibitors of LT Slide 142 R Marsella ACVD Resident Review 2003 Alternative treatments for AD The present Misoprostol Misoprostol PDE inhibitors PDE inhibitors Pentoxifylline Pentoxifylline Leukotriene inhibitors Leukotriene inhibitors Zileuton Zileuton Calcineurin inhibitors Calcineurin inhibitors Cyclosporine Cyclosporine Tacrolimus Tacrolimus Neuropeptide modulators Neuropeptide modulators Slide 143 R Marsella ACVD Resident Review 2003 Misoprostol PGE1 analog Rationale for use in cAD: PGE1 increases cAMP stabilizes cells and blocks secretion of pro- inflammatory cytokines PGE1 increases cAMP stabilizes cells and blocks secretion of pro- inflammatory cytokines Strong anti-allergic properties in people Strong anti-allergic properties in people Slide 144 R Marsella ACVD Resident Review 2003 Open, uncontrolled study (Olivry) 20 dogs with AD received 3-6mcg/kg of misoprostol PO 3x/day for 30 days 20 dogs with AD received 3-6mcg/kg of misoprostol PO 3x/day for 30 days Pruritus decreased by 50% in 56% of dogs Pruritus decreased by 50% in 56% of dogs Skin lesions decreased by 50% in 61% of dogs Skin lesions decreased by 50% in 61% of dogs Misoprostol (Cytotec ) Slide 145 R Marsella ACVD Resident Review 2003 Blinded, placebo controlled, study (Olivry) 20 dogs were either given 5mcg/kg of misoprostol TID for 3 weeks or placebo 20 dogs were either given 5mcg/kg of misoprostol TID for 3 weeks or placebo At the end of the trial, in the misoprostol group At the end of the trial, in the misoprostol group P ruritus decreased by 30% Skin lesions decreased by 30% No change was noted in placebo group No change was noted in placebo group Misoprostol (Cytotec ) Slide 146 R Marsella ACVD Resident Review 2003 PDE inhibitors Individuals with AD have high levels of PDE and low cAMP Individuals with AD have high levels of PDE and low cAMP PDE inhibitors PDE inhibitors Decrease production of IL-10 Decrease production of IL-10 Decrease production of PGE2 by monocytes and of IL-4 by T cells Decrease production of PGE2 by monocytes and of IL-4 by T cells PDE inhibitors might reduce inflammation in AD Slide 147 R Marsella ACVD Resident Review 2003 Pentoxifylline Methyl-xanthine derivative Methyl-xanthine derivative Strong PDE inhibitor Strong PDE inhibitor Rationale for use in cAD: It inhibits PDE increases cAMP stabilizes cells It inhibits PDE increases cAMP stabilizes cells It suppresses synthesis of pro- inflammatory cytokines (IL-1, IL-6 and TNF-) It suppresses synthesis of pro- inflammatory cytokines (IL-1, IL-6 and TNF-) Slide 148 R Marsella ACVD Resident Review 2003 Double blinded, placebo controlled, cross over clinical trial (Marsella et al, 2000) 10 dogs with AD were randomly divided into 2 groups (A and B) Group A received PTX at 10mg/kg BID for 4 weeks Group A received PTX at 10mg/kg BID for 4 weeks Groups B received placebo for 4 weeks Groups B received placebo for 4 weeks Pentoxifylline (Trental ) Slide 149 R Marsella ACVD Resident Review 2003 After 4 weeks, a 2 week wash-out period was observed and then the treatments were switched After 4 weeks, a 2 week wash-out period was observed and then the treatments were switched Pentoxifylline (Trental ) Slide 150 R Marsella ACVD Resident Review 2003 At the end of the trial Significant decrease in pruritus and erythema was observed in PTX treated dogs when compared to placebo Significant decrease in pruritus and erythema was observed in PTX treated dogs when compared to placebo Residual pruritus was observed in all dogs Residual pruritus was observed in all dogs Pentoxifylline (Trental ) Slide 151 R Marsella ACVD Resident Review 2003 Conclusions PTX is a moderately effective adjunctive treatment for canine AD PTX is a moderately effective adjunctive treatment for canine AD Well tolerated in dogs Well tolerated in dogs Pentoxifylline (Trental ) Slide 152 R Marsella ACVD Resident Review 2003 Efficacy seems to be dose dependent Efficacy seems to be dose dependent Half life is short thus 3x/day is better than 2x/day Half life is short thus 3x/day is better than 2x/day Dogs with grass allergy respond better than dogs allergic to HDM and molds Dogs with grass allergy respond better than dogs allergic to HDM and molds Older dogs respond better than younger dogs Older dogs respond better than younger dogs Pentoxifylline (Trental ) Slide 153 R Marsella ACVD Resident Review 2003 Arofylline (Almirall ) Oral PDE-4 inhibitor Oral PDE-4 inhibitor Controlled study in dogs with AD (Ferrer et al, 1999) Controlled study in dogs with AD (Ferrer et al, 1999) Arophylline (1 mg/kg BID) for 4 weeks Prednisone (0.5 mg/kg BID for the first week, SID for the second week and EOD for two additional weeks) Pruritus and skin lesions were evaluated and graded (scale from 0 to 3) weekly Pruritus and skin lesions were evaluated and graded (scale from 0 to 3) weekly Slide 154 R Marsella ACVD Resident Review 2003 Arofylline (Almirall ) No significant difference was noted among treatments No significant difference was noted among treatments Frequent adverse effects in the arofylline group Frequent adverse effects in the arofylline group Vomiting, diarrhea and anorexia Slide 155 R Marsella ACVD Resident Review 2003 Other PDE inhibitors In human medicine In human medicine Topical PDE inhibitors (e.g. Ro 20-1724) Slide 156 R Marsella ACVD Resident Review 2003 Capsaicin is a derivative of chili pepper Capsaicin is a derivative of chili pepper Decreases pain and itch Decreases pain and itch Mechanism of action unknown Mechanism of action unknown Depletion of Substance P (SP) from sensory nerve endings? Desensitization of C fibers? Capsaicin Slide 157 R Marsella ACVD Resident Review 2003 Rationale for use in AD In humans, there is evidence that SP plays a role in AD In humans, there is evidence that SP plays a role in AD SP release is triggered by allergen challenge and by histamine SP release is triggered by allergen challenge and by histamine SP receptors have been identified on mast cells SP receptors have been identified on mast cells Capsaicin Slide 158 R Marsella ACVD Resident Review 2003 Double blinded, placebo-controlled clinical trial (Marsella et al, 2002) 12 dogs with AD were selected and randomly divided into 2 groups 12 dogs with AD were selected and randomly divided into 2 groups Group A received 0.025% capsaicin lotion 2x/day for 6 weeks Group A received 0.025% capsaicin lotion 2x/day for 6 weeks Group B received placebo lotion Group B received placebo lotion Capsaicin clinical trial Slide 159 R Marsella ACVD Resident Review 2003 After 6 weeks, there was a 4 week washout period and groups were crossed over After 6 weeks, there was a 4 week washout period and groups were crossed over Investigator scored pruritus before and after each treatment Investigator scored pruritus before and after each treatment Owner scored pruritus on a weekly basis Owner scored pruritus on a weekly basis On week 0 and 6 skin biopsies were taken (to extract and measure SP (via ELISA) On week 0 and 6 skin biopsies were taken (to extract and measure SP (via ELISA) Capsaicin clinical trial Slide 160 R Marsella ACVD Resident Review 2003 Results Owner scores of pruritus A week 6 in the capsaicin group A week 6 in the capsaicin group Scores were significantly lower scores than the placebo group (p=0.005) Scores were significantly lower than the beginning of the study (p=0.0006) Scores worsened after first week of treatment (not significant) Scores worsened after first week of treatment (not significant) Slide 161 R Marsella ACVD Resident Review 2003 Correlation between pruritus and SP No correlation was found between pruritus and SP concentrations in the skin No correlation was found between pruritus and SP concentrations in the skin Slide 162 R Marsella ACVD Resident Review 2003 Capsaicin Conclusions Capsaicin at 0.025% was well tolerated Capsaicin at 0.025% was well tolerated Alternative topical treatment for cAD Alternative topical treatment for cAD Initial worsening may be observed Initial worsening may be observed Slide 163 R Marsella ACVD Resident Review 2003 Leukotrienes inhibitors in hAD Leukotriene receptor antagonists and inhibitors have been used successfully in the management of atopic disease in man Leukotriene receptor antagonists and inhibitors have been used successfully in the management of atopic disease in man Slide 164 R Marsella ACVD Resident Review 2003 Zileuton in man Zileuton inhibits LT production (5-LO inhibitor) Zileuton inhibits LT production (5-LO inhibitor) Used successfully in the management of atopic disease in man Used successfully in the management of atopic disease in man Slide 165 R Marsella ACVD Resident Review 2003 Zileuton in dogs Zileuton is rapidly absorbed in the dog after oral administration Zileuton is rapidly absorbed in the dog after oral administration Peak levels are achieved within 1 hour Peak levels are achieved within 1 hour Half-life (t 1/2 ) is approximately 7.5 hours Half-life (t 1/2 ) is approximately 7.5 hours Slide 166 R Marsella ACVD Resident Review 2003 Clinical trial with Zileuton Controlled study (Crow and Marsella, 2001) Controlled study (Crow and Marsella, 2001) 9 dogs with AD were selected and randomly divided into two groups 9 dogs with AD were selected and randomly divided into two groups Group A received zileuton (Zyflo ) orally at 2 mg/kg TID for 30 days Group B received placebo carrier suspension TID for 30 days Slide 167 R Marsella ACVD Resident Review 2003 Zileuton for canine AD Double-blinded, placebo controlled, cross-over pilot study Double-blinded, placebo controlled, cross-over pilot study Dogs were assigned to either Zileuton or placebo for 30 days Dogs were assigned to either Zileuton or placebo for 30 days After a wash out period of 5 days, the treatments were switched After a wash out period of 5 days, the treatments were switched Slide 168 R Marsella ACVD Resident Review 2003 Zileuton for canine AD Clinical signs (erythema and pruritus) were evaluated and scored by the investigator on day 0 and 30 of each treatment Clinical signs (erythema and pruritus) were evaluated and scored by the investigator on day 0 and 30 of each treatment Slide 169 R Marsella ACVD Resident Review 2003 Results Investigator scores of erythema On Day 30 in the zileuton group On Day 30 in the zileuton group Erythema scores were lower than the placebo treated dogs (p=0.02) Erythema was significantly decreased (p=0.02) compared to day 0 On Day 30 in the placebo group On Day 30 in the placebo group No significant change compared to day 0 Slide 170 R Marsella ACVD Resident Review 2003 Results Investigator scores of pruritus On day 0 and 30 On day 0 and 30 There were no differences between zileuton or placebo treated dogs Scores did not change over time between treatment groups or within each group Scores did not change over time between treatment groups or within each group Slide 171 R Marsella ACVD Resident Review 2003 Results Owners scores of Pruritus No significant changes over time in either group No significant changes over time in either group Slide 172 R Marsella ACVD Resident Review 2003 Conclusions Pilot study using Zileuton Zileuton was well tolerated Zileuton was well tolerated 4 weeks of treatment significantly decreased erythema, but had no effect on pruritus 4 weeks of treatment significantly decreased erythema, but had no effect on pruritus Slide 173 R Marsella ACVD Resident Review 2003 Cyclosporine A (Sandimmune , Neoral ) Fungal metabolite with Fungal metabolite with immuno-modulatory properties Rationale for use in cAD: It suppresses T cell proliferation and cytokine production (IL-1, IL-2, IL4, IL-6, and TNF-) It suppresses T cell proliferation and cytokine production (IL-1, IL-2, IL4, IL-6, and TNF-) It suppresses histamine release from mast cells It suppresses histamine release from mast cells Slide 174 R Marsella ACVD Resident Review 2003 Cyclosporine: Mode of Action calcineurin cyclophilin CycloP NFAT AP1 + + CaN P- NFAT TCR CM calmodulin Ca 2+ Slide 175 R Marsella ACVD Resident Review 2003 MC Y Y Y IL-1, IL-6 TNF-a, LT, Histamine LC Th2 IL -4 Y Y Y Circulation IL-2 Th1 Cyclosporine in Atopic Dermatitis X Th1 ACUTE CHRONIC Slide 176 R Marsella ACVD Resident Review 2003 Cyclosporine A Pharmacokinetics Pharmacokinetics Absorption Metabolism Drug interactions Different formulations Different formulations Adverse effects Adverse effects Slide 177 R Marsella ACVD Resident Review 2003 Cyclosporine A Very effective in human AD after oral administration Very effective in human AD after oral administration Not effective after topical application Not effective after topical application Slide 178 R Marsella ACVD Resident Review 2003 Open study (Fontaine et al, 2001) 14 dogs with AD received 5mg/kg once daily for 2 weeks 14 dogs with AD received 5mg/kg once daily for 2 weeks At the end of the trial: At the end of the trial: Pruritus decreased by 100% Skin lesions decreased by 60% Cyclosporine A (Neoral ) Slide 179 R Marsella ACVD Resident Review 2003 Controlled study (Olivry et al, 2002) 30 dogs with AD were randomly allocated to receive either: Oral solution of Neoral once daily (5 mg/kg) or Oral solution of Neoral once daily (5 mg/kg) or Prednisolone (0.5 mg/kg) for 6 weeks Prednisolone (0.5 mg/kg) for 6 weeks Cyclosporine A (Neoral ) Slide 180 R Marsella ACVD Resident Review 2003 Controlled study (Olivry et al, 2002) % reduction of skin lesions at 6 weeks: % reduction of skin lesions at 6 weeks: 68-70% in the prednisolone group 58% in the cyclosporine group % reduction in pruritus at 6 weeks: % reduction in pruritus at 6 weeks: 81% in the prednisolone group 78% in the cyclosporine group Cyclosporine A (Neoral ) Slide 181 R Marsella ACVD Resident Review 2003 Cyclosporine A (Neoral ) Controlled study (Steffan et al, 2003) Multicentre, parallel, blinded, randomized, controlled, long-term study Multicentre, parallel, blinded, randomized, controlled, long-term study Comparison between cyclosporine (117 dogs) and methylprednisolone (59 dogs) for cAD for 4 months Comparison between cyclosporine (117 dogs) and methylprednisolone (59 dogs) for cAD for 4 months Cs (induction): 5mg/kg Cs (induction): 5mg/kg Tapered according to clinical response Slide 182 R Marsella ACVD Resident Review 2003 Controlled study (Steffan et al, 2003) % reduction of lesion scores % reduction of lesion scores 52% in Cs 45% in MP % reduction in pruritus score % reduction in pruritus score 36% in Cs 33% in MP No significant changes in CBC and chemistry No significant changes in CBC and chemistry Cs dogs had more GI problems Cs dogs had more GI problems MP dogs tended to have more skin infections MP dogs tended to have more skin infections Cyclosporine A (Neoral ) Slide 183 R Marsella ACVD Resident Review 2003 Cyclosporine A Conclusions Effective treatment for refractory cases of cAD Effective treatment for refractory cases of cAD Same efficacy of prednisone Same efficacy of prednisone It may work in cases where steroids are not effective or stop working It may work in cases where steroids are not effective or stop working No increased incidence of skin infections and demodicosis was noted No increased incidence of skin infections and demodicosis was noted Slide 184 R Marsella ACVD Resident Review 2003 Conclusions Efficacy may be noted within the first week of therapy Efficacy may be noted within the first week of therapy Side effects: Side effects: Vomiting, diarrhea, bone marrow suppression, nephropathy, papillomatous dermatitis, gingival hyperplasia Cyclosporine A Slide 185 R Marsella ACVD Resident Review 2003 Tacrolimus Tacrolimus (FK-506) is a macrolide lactone produced by the fungus Streptomyces tsukubaensis Tacrolimus (FK-506) is a macrolide lactone produced by the fungus Streptomyces tsukubaensis Mechanism of action similar to Cyclosporine A Mechanism of action similar to Cyclosporine A Slide 186 R Marsella ACVD Resident Review 2003 Tacrolimus and DC IDEC are reduced after only one week IDEC are reduced after only one week Decreased inflammation LC are not affected LC are not affected No effect on risk for secondary infections Slide 187 R Marsella ACVD Resident Review 2003 Tacrolimus and cytokines Suppression of synthesis of: Suppression of synthesis of: IL-2 IL-4 IL-5 -IFN Slide 188 R Marsella ACVD Resident Review 2003 Tacrolimus Advantage of being effective after topical application Advantage of being effective after topical application Successfully used to decrease signs of AD in people Successfully used to decrease signs of AD in people Clinical improvement is marked and rapid Clinical improvement is marked and rapid Slide 189 R Marsella ACVD Resident Review 2003 Tacrolimus in human AD 0.1% ointment (controlled studies) 0.1% ointment (controlled studies) 83% improvement Significant difference with placebo was evident as early as day 8 0.3% ointment (controlled studies) 0.3% ointment (controlled studies) On day 3, significant improvement noted in 81% of adults and 88% of children On day 8, improvement was evident in 94% of adults and 100% of children Slide 190 R Marsella ACVD Resident Review 2003 Minimally absorbed and well tolerated Minimally absorbed and well tolerated 0.3% ointment applied to up to 30% of total body surface 0.3% ointment applied to up to 30% of total body surface Highest blood level of tacrolimus was 1.6ng/ml (Toxic levels are > 20ng/ml) No accumulation was found with repetitive applications Tacrolimus in human AD Slide 191 R Marsella ACVD Resident Review 2003 Clinical trial in dogs with AD Marsella et al, 2002 Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.3% tacrolimus lotion in dogs with AD Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.3% tacrolimus lotion in dogs with AD Marsella et al, 2003 (unpublished) Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.1% tacrolimus ointment in dogs with AD Randomized, double blind, placebo controlled, cross-over study to evaluate the efficacy and safety of 0.1% tacrolimus ointment in dogs with AD Slide 192 R Marsella ACVD Resident Review 2003 TAC (0.3% lotion) clinical trial Six dogs with AD were selected Six dogs with AD were selected Selected dogs were randomly assigned to either group A (0.3% tacrolimus lotion at 0.1ml/kg/day) or group B (vehicle) for 4 weeks Selected dogs were randomly assigned to either group A (0.3% tacrolimus lotion at 0.1ml/kg/day) or group B (vehicle) for 4 weeks After 2-week wash out, treatments were switched After 2-week wash out, treatments were switched Slide 193 R Marsella ACVD Resident Review 2003 Evaluation of efficacy and safety Owners evaluated pruritus weekly Owners evaluated pruritus weekly Investigator evaluated pruritus and erythema at at week 0 and 4 (beginning and end of each treatment) Investigator evaluated pruritus and erythema at at week 0 and 4 (beginning and end of each treatment) Blood was collected for CBC and chemistry panel and to monitor drug absorption (ELISA for TAC) on week 0 and 4 of each treatment Blood was collected for CBC and chemistry panel and to monitor drug absorption (ELISA for TAC) on week 0 and 4 of each treatment Slide 194 R Marsella ACVD Resident Review 2003 Results CBC and Chemistry No significant changes were detected No significant changes were detected Tacrolimus values On day 28 tacrolimus levels were significantly higher at 2 and 4 hours compared to day 0 On day 28 tacrolimus levels were significantly higher at 2 and 4 hours compared to day 0 Peak concentrations noted at 2 hours Peak concentrations noted at 2 hours All mean values were below toxicity levels All mean values were below toxicity levels Slide 195 R Marsella ACVD Resident Review 2003 Results Owner score of pruritus No difference was found for both groups overtime Slide 196 R Marsella ACVD Resident Review 2003 Results Investigator score of pruritus Pruritus decreased significantly within the TAC group (p=0.03) Slide 197 R Marsella ACVD Resident Review 2003 Results Investigator score of erythema Erythema decreased significantly in the TAC group (p=0.015) Slide 198 R Marsella ACVD Resident Review 2003 Conclusions Pilot study using 0.3% tacrolimus lotion Tacrolimus was well tolerated Tacrolimus was well tolerated No adverse effects were noted No adverse effects were noted Tacrolimus was effective in decreasing scores according to the investigator but not the perception of pruritus by owners Tacrolimus was effective in decreasing scores according to the investigator but not the perception of pruritus by owners Slide 199 R Marsella ACVD Resident Review 2003 New trial using TAC 0.1% ointment (Protopic ) Stability of lotion could not be guaranteed Stability of lotion could not be guaranteed Need to repeat a study using the ointment, once commercially available Need to repeat a study using the ointment, once commercially available Efficacy Safety 2 formulations available in the US 2 formulations available in the US 0.1% 0.03% Slide 200 R Marsella ACVD Resident Review 2003 Experimental Design Randomized, double-blinded, placebo- controlled cross-over study Randomized, double-blinded, placebo- controlled cross-over study Selected dogs were allocated to either tacrolimus or placebo for 4 weeks Selected dogs were allocated to either tacrolimus or placebo for 4 weeks Wash out period of 2 weeks Wash out period of 2 weeks Treatments were switched (4 additional weeks) Treatments were switched (4 additional weeks) Slide 201 R Marsella ACVD Resident Review 2003 Materials and Methods All dogs were evaluated on week 0 and 4 of each treatment All dogs were evaluated on week 0 and 4 of each treatment PE Blood samples CBC Chemistry panels Tacrolimus levels Cytology Scoring of clinical signs (CADESI) Slide 202 R Marsella ACVD Resident Review 2003 Modified CADESI used by Investigator Body divided into 40 sites Body divided into 40 sites Evaluated for: Evaluated for: Erythema Lichenification Excoriations Papules Pruritus On a scale of 0 - 3 (3 = severe) On a scale of 0 - 3 (3 = severe) Slide 203 R Marsella ACVD Resident Review 2003 Materials and Methods Owners evaluation of pruritus Owners evaluation of pruritus Required to fill out a scoring sheet once a week Body divided into 22 sites Evaluated for pruritus once weekly On a scale of 0 - 3 Slide 204 R Marsella ACVD Resident Review 2003 Results Owners evaluation of pruritus Within the TAC group Within the TAC group Significant decrease of scores at week 3 and 4, compared to week 0 (p< 0.01 and p=0.055, respectively) Within the placebo group Within the placebo group No differences Slide 205 R Marsella ACVD Resident Review 2003 Results Owners evaluation of pruritus Slide 206 R Marsella ACVD Resident Review 2003 Results Investigator scores Within the TAC group Within the TAC group Scores at week 4 were significantly lower than week 0 (p=0.0019) Within the placebo group Within the placebo group No differences Slide 207 R Marsella ACVD Resident Review 2003 Results Investigator scores Slide 208 R Marsella ACVD Resident Review 2003 Results TAC blood concentrations Week 0Week 4 Slide 209 R Marsella ACVD Resident Review 2003 Results CBC and Chemistry No changes in CBC and Chemistry parameters were detected between groups or within groups No changes in CBC and Chemistry parameters were detected between groups or within groups No adverse effects was noted during the trial in either group No adverse effects was noted during the trial in either group Slide 210 R Marsella ACVD Resident Review 2003 Results Dogs with localized disease responded better on TAC than dogs with generalized Dogs with localized disease responded better on TAC than dogs with generalized Investigator scores Generalized disease: 24% decrease Localized disease: 61% decrease Owners scores Generalized disease: 19% decrease Localized disease: 58% decrease Slide 211 R Marsella ACVD Resident Review 2003 Conclusions Clinical trial using 0.1% ointment TAC is well tolerated and minimally absorbed in dogs with AD TAC is well tolerated and minimally absorbed in dogs with AD Protopic is a useful topical treatment for canine AD Protopic is a useful topical treatment for canine AD Protopic is best used in dogs with localized disease due to the difficulty in application on extensive parts of the body Protopic is best used in dogs with localized disease due to the difficulty in application on extensive parts of the body Slide 212 R Marsella ACVD Resident Review 2003 Additional calcineurin inhibitors SDZ ASM 981 or Pimecrolimus (Elidel , 1%) SDZ ASM 981 or Pimecrolimus (Elidel , 1%) Sirolimus (Rapamycin) Sirolimus (Rapamycin) Efomycin Efomycin Everolimus (SDZ RAP) Everolimus (SDZ RAP) Slide 213 R Marsella ACVD Resident Review 2003 Conclusions Cause of AD is a complex interaction Cause of AD is a complex interaction Genetic susceptibility Environmental factors Abnormal immune responses Likely that not one single mediators is responsible for clinical signs Likely that not one single mediators is responsible for clinical signs So, what do we know about AD? Slide 214 R Marsella ACVD Resident Review 2003 Rejection of old dogma AD may represent a type IV hypersensitivity more than originally thought AD may represent a type IV hypersensitivity more than originally thought AD encompasses a spectrum of abnormalities, different for each patient AD encompasses a spectrum of abnormalities, different for each patient AD is a dynamic process AD is a dynamic process Slide 215 R Marsella ACVD Resident Review 2003 Directions for the future Identification of mediators of disease in order to develop targeted treatments Identification of mediators of disease in order to develop targeted treatments Anti-IL-5, anti-TNF-? anti- CCR4? Anti-IgE? Evaluation of genotypes to better predict clinical response of individual patients Evaluation of genotypes to better predict clinical response of individual patients Slide 216 R Marsella ACVD Resident Review 2003 Questions?