Laboratory of Immunobiochemistry

1. Laboratory of Immunobiochemistry Research update

2. 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

3. 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

4. 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

5. 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

6. 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

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

8. 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?

10. Plan • Investigate differences between D. farinae and D. pteronyssinus • Attempt to identify organisms

11. Approach #1 - culture • Fresh mites/eggs were washed free of media • Sieve • Sucrose gradients • Material submitted for culture

13. 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

14. Approach #2 - DNA • Extract genomic DNA from fresh, washed mites • Amplify with 16S rRNA sequences • Identify clones with RFLP analysis • Sequence • Identify predominant organisms

15. D. farinae D. pteronyssinus EcoR1 digests undigested DNA DNA from mites

16. 16S rRNA primers fD1 (most eubacteria): ccgaattcgtcgacaacAGAGTTTGATCCTGGCTCAG fD2 (enterics): ccgaattcgtcgacaacAGAGTTTGATCATGGCTCAG rP1 (most eubacteria): cccgggatccaagcttACGGTTACCTTGTTACGACTT From Weisburg, J. Bacteriol. 1991; 172:697-703

18. Bartonella/ Rochalimaea spp. B. henselae B. quintana B. vinsonii B. elizabethae Rhizobium spp. Pseudomonas spp. P. trivialis P. tolaasii P. poae Unidentified endosymbionts from Psoroptes ovis Brevipalpus lewisi Brevipalpus phoenicis Encarsia pergandiella 16S rRNA sequences recovered

19. 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)

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