Crosssensitization to milk proteins: Reactivity to a kosher epitope

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Cross-sensitization to milk proteins Reactivity
to a kosher epitope?

• Michael Goldberg, MD, PhD
• Institute of Allergy and Immunology
• Assaf Harofeh Medical Center

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Epidemiology Who is really allergic?

• The literature reports between 25 during the
  first three years of life.
• The prevalence of self-diagnosed CMA in the
  community is 10-fold higher than the clinically
  proven incidence, suggesting a sizable population
  is unnecessarily eschewing dairy products (J Am
  Coll Nutr. 2005 Dec24(6 Suppl)582S-91S ).
• True incidence in Israel is less than 0.5 of the
  population (Katz, et al. JACI abstract 2009).
• Near-fatal or fatal outcomes occurs primarily in
  the older children and adolescents.

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Kosher
Nonkosher
Crossreacting
Not Crossreacting
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Hypothesis

• Milk derived from kosher animals and not from
  nonkosher animals are reactive in IgE-mediated
  cows milk protein allergy (Katz, et al. Isr Med
  Assoc J. 101-4.)

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SKIN PRICK TEST (SPT)
Cows milk allergy or drug allergy ????
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SPT results in cows milk protein allergic
patients
Wheal/flare
(Katz, et al. Isr Med Assoc J. 101-4.)
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SPT results in Control Group
(Katz, et al. Isr Med Assoc J. 101-4.)
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Cross reactivity of CMP with proteins from milk
of other animals response to Non- Kosher age
dependent
Kosher Cross Allergenicity
Non Kosher No Cross Allergenicity

• Camel 2/88/47
• Pig 5/24 12/47
• Rabbit 4/47
• Dog 16/47
• Horse 3/42
• Alpaca 10/47

• Cow 24/24 47/47
• Buffalo 24/24
• Goat 46/47
• Ibex 24/2446/47
• Deer 24/24
• Giraffe 46/47

• p 0.02 (Fisher test)
• p gt 0.35

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Cross reactivity of CMP with proteins from milk
of other animals response to Non- Kosher age
dependent
Below 30 m/ over 30 m
Kosher Cross Allergenicity
Non Kosher No Cross Allergenicity

• Camel 2/88/47 1/8
• Pig 5/24 12/47 1/11
• Rabbit 4/47 0/4
• Dog 16/47 4/12
• Horse 3/42 0/3
• Alpaca 10/47 1/9

• Cow 24/24 47/47
• Buffalo 24/24
• Goat 46/47
• Ibex 24/2446/47
• Deer 24/24
• Giraffe 46/47

• p 0.02 (Fisher test)
• p gt 0.35

Logistic regression for moth of life OR 1.008,
OR1.026
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Is there a common epitope found only in kosher
animals?
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Main characteristics of the bovine milk proteins
Wal, JM. Ann Allergy Asthma Immunol. 2004
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Beta-lactoglobulin
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Beta-Casein
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Alpha-lactalbumin
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Cow Buffalo Deer Ibex Sheep
Camel Horse Pig Human
Camel Horse Pig Human
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This eight amino acid sequence creates a
completely new structure
CC
C
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Schematic model of kappa-casein multimers in
bovine and human milk
Rasmusssen, International Dairy Journal, 1999
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VPAKSCQA/D-Sequence responsible for
allergenicity? Hypothesis

• Aggregation of casein monomers reveals new
  determinants that seem to be absent in the
  monomeric forms, since some sera show IgE
  reactivity only against aggregates, but not
  against individual components (Allergy. 1996
  Jun51(6)412-6)
• When bovine kappa-casein was expressed in the
  mouse a a different micellar structure was noted
  (J Dairy Sci 79791-799, 1996)
• This region may therefore lead to the generation
  of multiple allergenic epitopes in susceptible
  individuals.

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Approach

• Structural analysis of the native and mutated
  kappa casein
• Modeling
• Limited proteolytic digestion maintaining the
  disulfide bond intact
• Generation of wildtype and mutated kappa-casein
• In vitro-mammalian expression systems
• In vivo- transgenic mouse lines
• Analyze mutated kappa-casein both by SPT on
  milk-allergic patients and their sera for IgE
  binding antibodies

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Ser88
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Trypsin digests reveals Arg at 97 and Lys at 111
112 are sensitive sites
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Chymosin digestion of kappa-casein
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• Difference in micellar structure between wildtype
  mice and mice carrying bovine kappa-casein gene
• Milk derived from transgenic mice with the bovine
  kappa-casein gene form a stronger curd.

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Acknowledgements

• Yitzhak Katz, Director, Institute of Allergy and
  Immunology, Assaf Harofeh Medical Center
• Richard Friedman, Columbia University, NY
• Menachem Shoham, Case Western University
• Alberto Huberman, University of Mexico, UNAM
• James D. Murray, UC Davis
• Elizabeth Maga, UC Davis

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