# Presentation: 236301

## 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

## 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*

## 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

## 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

## 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

## Outside collaborations

**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

## 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?

## Slide 9

## Plan

- Investigate differences between D. farinae and D. pteronyssinus
- Attempt to identify organisms

## Approach #1 - culture

- Fresh mites/eggs were washed free of media
- Sieve
- Sucrose gradients
- Material submitted for culture

## Slide 12

## 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

## Approach #2 - DNA

- Extract genomic DNA from fresh, washed mites
- Amplify with 16S rRNA sequences
- Identify clones with RFLP analysis
- Sequence
- Identify predominant organisms

## DNA from mites

## 16S rRNA primers

- fD1 (most eubacteria):
- ccgaattcgtcgacaacAGAGTTTGATCCTGGCTCAG
- fD2 (enterics):
- ccgaattcgtcgacaacAGAGTTTGATCATGGCTCAG
- rP1 (most eubacteria):
- cccgggatccaagcttACGGTTACCTTGTTACGACTT

- From Weisburg, *J. Bacteriol. *1991; 172:697-703

## 16S rRNA sequences recovered

- 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

**Notes:**
Bartonella-associated diseases: cat-scratch disease, bacillary angiomatosis, bacillary peliosis, bacteremia, endocarditis, and trench fever

## 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)

## 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