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					Laboratory of
Immunobiochemistry

      Research update
Active research projects
   PI Slater
       Cockroach allergen standardization
            Determination of optimal surrogate test
            Depletion analysis of CR extracts
       Cockroach IgE/IgG combinatorial library
            Microarray analysis of allergen extracts
       Endotoxin in allergen vaccines
   PI Rabin
       MDR proteins in T cell activation
       RSV responses in human tonsil

                                                        2/20
  Publications
Trivedi B, Valerio C, Slater JE. Endotoxin content of
  standardized allergen vaccines. J Allergy Clin Immunol
  2003; 111:777-783.
Lockey RF, Slater JE, Esch R. Preparation and
  standardization of allergen vaccines. In Middleton’s
  Allergy: Principles and Practice, 6th ed. St. Louis: Mosby
  2003;573-584.
Rabin RL, Alston MA, Sircus JC, Knollmann-Ritschel B, Moratz
  C, Ngo D, Farber JM. CXCR3 is induced early on the
  pathway of CD4+ T cell differentiation and bridges central
  and peripheral functions. J Immunol. 2003;171:2812-24.
Zhang J, Alston MA, Huang H, Rabin RL. Multidrug Resistant
  Protein 1 (MRP1) is induced upon activation of human T
  cells, and its inhibition blocks T cell function, in
  preparation                                             3/20
 Abstracts
Valerio C, Murray P, Arlian LG, Slater JE. Endotoxin in dust mite allergen
    extracts. J Allergy Clin Immunol 2004; S136
Gam AA, Slater JE. Depletion of Specific Cockroach Allergens May Not
    Affect Overall Allergenicity Determinations. J Allergy Clin Immunol
    2004; S144
Finlay WJJ, Dobrovolskaia EN, Gam A, Slater JE. Generation of mono-
    specific recombinant antibody fragments from chicken to allergenic
    proteins Fel d 1, Amb a 1 and whole yellow jacket venom. J Allergy Clin
    Immunol 2004; S227
deVore N, Finlay W, Dobrovolskia E, Gam A, Slater J. Cloning and Analysis
    of Mono-specific scFv Fragments from Chicken to Allergenic Proteins of
    Periplaneta americana (American Cockroach). J Allergy Clin Immunol
    2004; S297
Alston MA, Zhang J, Huang H, Rabin RL. The Drug Pump Multi-Drug
    Resistance Protein 1 (MRP1) is Induced upon T cell Activation and its
    Inhibition Blocks T cell Function. J Allergy Clin Immunol 2004; S249
Chi B, Spann KM, Collins PL, Rabin RL. T Cell Response to Respiratory
    Syncytial Virus (RSV). J Allergy Clin Immunol 2004; S269
                                                                      4/20
Invited presentations - Rabin
   Smi Conference: Prevention and Treatment
    of Allergy, London, February 2004:
       The relationship between viral respiratory
        infections and asthma: chicken vs. egg
   AAAAI Annual Meeting, March 2004:
       FDA Food Drug and Cosmetics Act as it applies to
        research studies




                                                     5/20
Invited presentations - Slater
State of the Art Analytical Methods for the
  Characterization of Biological Products and
  Assessment of Comparability, June 2003:
   Characterization of allergen extracts
 AAAAI Annual Meeting, March 2004:
     IND process: what the FDA needs to know
     Standardization, and factors affecting stability
     Cockroach allergen standardization
     Diagnostic and therapeutic allergenic extracts

                                                         6/20
    Outside collaborations
   Larry G. Arlian, PhD                  Peter L. Collins, PhD
       Director, Microbiology and            Laboratory of Infectious
        Immunology, Department                 Diseases, National
        of Biological Sciences,                Institute of Allergy and
        Wright State University,               Infectious Diseases, NIH,
        Dayton, Ohio                           Bethesda, Maryland
   Patrick R. Murray, PhD                Mario Roederer, PhD
       Chief, Microbiology Service,          Vaccine Research Center,
        NIH Clinical Center,                   National Institute of
        Bethesda, Maryland                     Allergy and Infectious
                                               Diseases, NIH, Bethesda,
                                               Maryland

                                                                   7/20
Endotoxin content of allergen
vaccines – prior work
   Pollens < cat and mite
   Cat hair < cat pelt
   D. pteronyssinus << D. farinae
       Bioburden?
       Endogenous heat-stable ENP-binding LAL
        activator in D. farinae?
       Endogenous ENP in D. pteronyssinus?


                                            8/20
                                    Corrected endotoxin content (EU/mL)




                                0
                                       1
                                             10
                                                   100
                                                          1000
                                                                 10000
                                                                          100000




               G
                ra
                   ss
                      es

              R
               ag
                 w
                  ee
                        d

               C
                at
                     Pe
                        l   t

               C
                at
                     H
                      ai
                         r
                                                                                   p = 0.02




             D
                .f
                  ar
                     in
       D               ae
        .p
          te
            ro
               ny
                 ss
                    in
                       us
                                                                                   p = 0.0003




9/20
Plan
   Investigate differences between D.
    farinae and D. pteronyssinus
   Attempt to identify organisms




                                         10/20
Approach #1 - culture
   Fresh mites/eggs were washed free of
    media
       Sieve
       Sucrose gradients
   Material submitted for culture




                                      11/20
                                       D. pteronyssinus   D. farinae
Gram positive        Staph spp                11              10
                     Strep spp                 3               1
                     Bacillus spp              2               2
                     Micrococcus                               1
Gram negative        E. cloacae               1                2
                     R. picketii              1                2
                     S. maltophilia                            1
                     Acinetobacter            1                1
                     unspecified GNR          1                1
No growth                                     1                1
Number of cultures                           15               15


                                       D. pteronyssinus   D. farinae
                     Gram positive            16              14
                     Gram negative             4               7
                     No growth                 1               1

                                                                       12/20
Possible problems
   Culture data are non-quantitative –
    bioburdens may be very different
   Culture techniques have been optimized for
    human pathogens
   Organisms may be in “privileged sites”, and
    optimal conditions for extraction are
    uncertain
   Endosymbionts are notoriously difficult to
    culture

                                            13/20
Approach #2 - DNA
   Extract genomic DNA from fresh,
    washed mites
   Amplify with 16S rRNA sequences
   Identify clones with RFLP analysis
   Sequence
   Identify predominant organisms


                                         14/20
DNA from mites
      D. farinae D. pteronyssinus




          EcoR1 digests
          undigested DNA            15/20
16S rRNA primers
fD1 (most eubacteria):
ccgaattcgtcgacaacAGAGTTTGATCCTGGCTCAG

fD2 (enterics):
ccgaattcgtcgacaacAGAGTTTGATCATGGCTCAG

rP1 (most eubacteria):
cccgggatccaagcttACGGTTACCTTGTTACGACTT

      From Weisburg, J. Bacteriol. 1991; 172:697-703
                                             16/20
17/20
16S rRNA sequences recovered

   Bartonella/             Pseudomonas spp.
    Rochalimaea spp.            P. trivialis
       B. henselae             P. tolaasii
                                P. poae
       B. quintana
       B. vinsonii
                            Unidentified
       B. elizabethae
                             endosymbionts from
                                   Psoroptes ovis
                                   Brevipalpus lewisi

   Rhizobium spp.              Brevipalpus phoenicis
                                Encarsia pergandiella


                                                  18/20
Bartonella endotoxin
   High degree of LAL reactivity, but
   Minimal inflammatory responses in
    human cells and rats and
   Minimal activation of TLR’s 2 and 4


           Matera et al., Int Immunopharmacol 2003; 3:853-864
           Zahringer et al., J Biol Chem 2004 (Epub)

                                                           19/20
Next steps
   Need to verify the source of endotoxin
       high fidelity PCR
       additional mite sources
       “profiling” the LPS
   Use internal standards to quantify DNA
    in source materials
   Examine the effects of this endotoxin
    on immune responses

                                       20/20

				
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