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Update on the Pathogenesis of Canine Atopic Dermatitis: a comparative review Rosanna Marsella University of Florida.

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Presentation on theme: "Update on the Pathogenesis of Canine Atopic Dermatitis: a comparative review Rosanna Marsella University of Florida."— Presentation transcript:

1 Update on the Pathogenesis of Canine Atopic Dermatitis: a comparative review Rosanna Marsella University of Florida

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

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)

4 R Marsella ACVD Resident Review 2003 Canine Human

5 R Marsella ACVD Resident Review 2003 Canine Human

6 R Marsella ACVD Resident Review 2003 Experimental canine AD

7 R Marsella ACVD Resident Review 2003 Experimental canine AD

8 R Marsella ACVD Resident Review 2003 Canine Human

9 R Marsella ACVD Resident Review 2003 Canine Human

10 R Marsella ACVD Resident Review 2003 CanineHuman

11 R Marsella ACVD Resident Review 2003 Human Canine

12 R Marsella ACVD Resident Review 2003 Spongiotic superficial perivascular mononuclear dermatitis

13 R Marsella ACVD Resident Review 2003 HumanCanine

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

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

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?

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

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

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

20 R Marsella ACVD Resident Review 2003 The old theory: Role of IgE, mast cells and type I hypersensitivity in AD

21 R Marsella ACVD Resident Review 2003 IgE Th1 Th2 MC APC Eos IL-4 TNF-a Let’s not put all our “atopic” eggs in one basket….

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- 

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

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

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

26 R Marsella ACVD Resident Review 2003 Route of allergen exposure Inhalatory Inhalatory Percutaneous Percutaneous

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

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

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

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)

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

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

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

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

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

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  β adrenergic hyporesponsiveness  Increased PDE, decreased cAMP

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

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)

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

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

41 R Marsella ACVD Resident Review 2003 ROLE OF MAST CELLS AND HISTAMINE IN AD

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

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

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?

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 histamine”releasability” Canine AD Greater tendency to release histamine than normal dogs

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

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

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

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- 

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

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

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

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

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

55 R Marsella ACVD Resident Review 2003 The new theories: Role of T cells, cytokines, chemokines and type IV hypersensitivity in AD

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

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)

58 R Marsella ACVD Resident Review 2003 Atopic Dermatitis (acute lesions) T helper 2 T helper 1 IL-4 IL-5 IL-2  -IFN

59 R Marsella ACVD Resident Review 2003 Atopic Dermatitis (chronic lesions) T helper 2 T helper 1 IL-4 IL-5 IL-2  -IFN

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

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

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

63 R Marsella ACVD Resident Review 2003 TNF-  Produced by: Produced by:  Mononuclear cells  Neutrophils  Activated T cells  NK cells  Mast cells  Keratinocytes

64 R Marsella ACVD Resident Review 2003 Inducers Inducers  Trauma  LPS  Toll-like receptors (TLR2 and TLR4) TNF- 

65 R Marsella ACVD Resident Review 2003 Role of TNF-α 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)

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

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)

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)

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

70 R Marsella ACVD Resident Review 2003 IL-1 Produced by: Produced by:  Mononuclear cells  Endothelial cells  Keratinocytes

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

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

73 R Marsella ACVD Resident Review 2003 IL-6 Produced by: Produced by:  Mononuclear cells  T cells  B cells  Fibroblasts  Endothelial cells  Keratinocytes

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

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

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)

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

78 R Marsella ACVD Resident Review 2003 IL-4 Produced by: Produced by:  T cells  Eosinophils  Mast cells  Basophils

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

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

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

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

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)

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)

85 R Marsella ACVD Resident Review 2003 Eosinophils and s-LT Number of Eosinophils RecruitmentSurvival S-LT ++ IL-4, IL-5, IL-13 + +

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

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

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

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

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

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

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)

93 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines  IL-1, IL-6 and TNF-α 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-β and IL-10

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

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

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)

97 R Marsella ACVD Resident Review 2003 Cytokines in AD Pro-inflammatory cytokines Pro-inflammatory cytokines  IL-1, IL-6 and TNF-α 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-β and IL-10

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

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

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

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

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

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)

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

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

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

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

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

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)

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)

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

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

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-α  LPS

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

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

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

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

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?)

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)

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

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 ¼ of atopic samples has type-2 cytokine profiles ¼ of atopic samples has type-2 cytokine profiles

122 R Marsella ACVD Resident Review 2003 The Hygiene Hypothesis A protective role for endotoxin?

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

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

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)

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

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

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

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

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-α (increased expression of adhesion molecules leading to leukocytes extravasation)

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

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

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)

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

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

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

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)?

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

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

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

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

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

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

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 ® )

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 ® )

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

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-α) It suppresses synthesis of pro- inflammatory cytokines (IL-1, IL-6 and TNF-α)

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 ® )

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 ® )

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 ® )

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 ® )

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 ® )

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

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

155 R Marsella ACVD Resident Review 2003 Other PDE inhibitors In human medicine In human medicine  Topical PDE inhibitors (e.g. Ro 20-1724)

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

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

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

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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-α) It suppresses T cell proliferation and cytokine production (IL-1, IL-2, IL4, IL-6, and TNF-α) It suppresses histamine release from mast cells It suppresses histamine release from mast cells

174 R Marsella ACVD Resident Review 2003 Cyclosporine: Mode of Action calcineurin cyclophilin CycloP NFAT AP1 + + CaN P- NFAT TCR CM calmodulin Ca 2+

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

176 R Marsella ACVD Resident Review 2003 Cyclosporine A Pharmacokinetics Pharmacokinetics  Absorption  Metabolism  Drug interactions Different formulations Different formulations Adverse effects Adverse effects

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

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 ® )

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 ® )

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 ® )

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

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 ® )

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

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

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

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

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

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

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

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

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

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

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

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

195 R Marsella ACVD Resident Review 2003 Results – Owner score of pruritus No difference was found for both groups overtime

196 R Marsella ACVD Resident Review 2003 Results – Investigator score of pruritus Pruritus decreased significantly within the TAC group (p=0.03)

197 R Marsella ACVD Resident Review 2003 Results – Investigator score of erythema Erythema decreased significantly in the TAC group (p=0.015)

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

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%

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)

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)

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)

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

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

205 R Marsella ACVD Resident Review 2003 Results – Owners evaluation of pruritus

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

207 R Marsella ACVD Resident Review 2003 Results – Investigator scores

208 R Marsella ACVD Resident Review 2003 Results – TAC blood concentrations Week 0Week 4

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

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

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

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)

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?

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

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-α? 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

216 R Marsella ACVD Resident Review 2003 Questions?


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