SOLICITATION NOTICE
B -- CELLULAR RECEPTORS AND NEURAL REGULATION IN AN ANIMAL MODEL OF POPCORN WORKER'S LUNGS
- Notice Date
- 1/13/2010
- Notice Type
- Presolicitation
- NAICS
- 611310
— Colleges, Universities, and Professional Schools
- Contracting Office
- Department of Health and Human Services, Centers for Disease Control and Prevention, Acquisition and Assistance Field Branch (Morgantown), 1095 Willowdale Road, Morgantown, West Virginia, 26505
- ZIP Code
- 26505
- Solicitation Number
- 000HCCC-2010-79644
- Archive Date
- 2/28/2010
- Point of Contact
- Rebecca S Mullenax, Phone: 304-285-5880, Kimberly P Groves, Phone: 304-285-5885
- E-Mail Address
-
rmullenax@cdc.gov, kgroves@cdc.gov
(rmullenax@cdc.gov, kgroves@cdc.gov)
- Small Business Set-Aside
- N/A
- Description
- NOTICE OF INTENT TO ISSUE A PURCHASE ORDER. The Centers for Disease Control and Prevention (CDC), National Institute for Occupational Safety and Health (NIOSH), Pathology and Physiology Research Branch (PPRB), would like to add a modification to a purchase order to West Virginia University Research Corporation for the following: This announces the Governments intent to issue a modification. STATEMENT OF WORK Title: Cellular Receptors and Neural Regulation in an Animal Model of Popcorn Workers' Lung I. Background and Purpose The purpose of this contract is to provide data concerning the role of neural regulation in the airway toxicity of butter flavoring vapors. The project investigates the 2 hypotheses: 1. Substance P (SP) production in sensory neurons projecting to the airways is enhanced after exposure to butter flavoring vapor components, 2. Mice with an absence of the diacetyl receptor will display early death from epithelial and endothetial cancer but decreased sensitivity to diacetyl-induced epithelial and endothelial injury. II. Scope of Work The contractor will be responsible for determining the SP content of sensory neurons of the airways and SP nerve fiber density in airways following inhalation of acetoin, diacetyl, and acetic acid. Diacetyl, acetoin and acetic acid exposures will be the responsibility of NIOSH, Morgantown, WV. III. Detailed Technical Requirements Fluorescent labeling of sensory neurons: Procedures for labeling the sensory neurons of the tracheal and nasal airways have previously been described in peer-reviewed publications from the laboratory of Richard Dey, PhD. (Dey et at., 1996; Dey et at., 1999; Hunter et a!., 2000; Wilfong and Dey, 2004). In brief, 10 to 15 days prior to exposure to flavoring vapors, rats will be anesthetized with methohexital sodium (35-50 mg/kg, i.p.). Once anesthetized, rhodamine-labeled latex microspheres (Lumafluor) will be instilled into the trachea through a feeding needle. Prior to the labeling of the SP nerve fibers, the contractor may label another group of rats to assure the procedure is working correctly. By mutual agreement between the P1 and the contractor, the contractor may also label nasal sensory neurons in addition to tracheal sensory neurons and determined their substance P content and nerve fiber density as previously described (Hunter and Dey, 1998; Hunter et at., 2000; Wilfong and Dey, 2004). Instillation of rhodam inc-labeled beads in animals used for inhalation exposures will be performed in the Animal Quarters at NIOSH. Diacetyl, acetoin and acetic acid exposures. Diacetyl, acetoin and acetic acid exposures will be conducted in the Inhalation Facility at NIOSH 10- 15 days after rhodamine tracer instillation. The animals will then be exposed by inhalation of filtered air, diacetyl, acetoin or acetic acid as detailed in the table below for 6 hours. With written permission of the NIOSH project officer, the target concentrations may be changed based upon findings from ongoing research. In addition, by mutual agreement between the P1 and contractor, the number of animals exposed may be increased and reagents for use in those animals may be provided by NIOSH but additional animals will not affect the cost of the unmodified contract. Immunocytochemistry and morphometric measurements in sensory nerves. The airways and nodose and jugular ganglia will be removed, fixed in picric acid- formaldehyde fixative for 3 hours and rinsed 3 times with a 0.1 M phosphate- buffered saline containing 0.3% Triton-X-1 00 (PBS-Tx). The airways will be dissected to identify: nasal mucosa, upper, middle, and lower trachea, extra- pulmonary bronchi, proximal (generations 1-3) and distal bronchi (generation 4 and beyond) and bronchioles. The airways and ganglia will be frozen in isopentane, cooled with liquid nitrogen, and stored at -80°C. Cryostat sections (12 j.m) on coated cover slips will be treated with rabbit lgGs against SP, glutamate, VIP, NOS and then treated with fluorescein isothiocyanate-labeled goat anti-rabbit antibody. The control for specificity of primary antiserum consists of absorption with I mg/mi of the specific antigen. Nonspecific background labeling is determined by omission of primary antiserum. Images of airway sections and neurons will be digitally recorded. Images of fluorescent nerve fibers and cell bodies will be obtained with a digital camera at a resolution of 300 dpi and a size of at least 2000 x 2000 pixels. For measuring nerve fiber density, immunoreactive nerve fibers in the defined airway regions will be digitally captured and processed. The number of irnmunoreactive nerve fibers and the total area of the smooth muscle or airway epithelium in the entire section will be determined and used to calculate the value: % nerve fiber density (% of area occupied by nerve fibers). At least 10 measurements will be made for each section. The means of three sections will be used to calculate a mean for each region. The number of separate experiments will be 9 for each exposure and control group. For measuring fluorescence intensity in airway neurons, SP in the cell bodies in the nodose and jugular ganglia containing rbodamine-labeied latex microspheres will be evaluated. The intensity of immunofluorescence will be measured using morphometry. Sensory and intrinsic airway neurons will be identified by drawing the perimeter of the cell. The immunofluorescence intensity will be computed as a gray level for each neuron. Neurons with a gray Level less than 50 will be considered negative because they are at or below the general background. Fluorescence intensity of 50 or greater will be considered to signify immunoreactivity and such cells will be counted as a labeled neuron, The mean percentage of labeled neurons will be calculated for each experimental group and control group at each recovery time period. Phenotyping of Dicarbonyl/L-xylulose reductase knockout mice The receptor for diacetyl is dicarbonyl/L-xylulose reductase (DCXR). DCXR is located at intercellular junctions of epithelial and endothellal cells and colocalizes with the adhesion molecules e-cadherin and f3-catenin (Cho-Vega et a!., 2007a; Cho-Vega eta!., 200Th). To investigate the role of DCXR in diacetyl toxicity, we have generated DCXR knockout obimeras and are in the process of breeding DCXR knockout mice. The second job in this contract will be to phenotype the DCXR knockout mouse with emphasis on establishing the viability of the homozygous knockout mice, developing a rapid test for DCXR knockout mice, evaluate morbidity and mortality in the homozygous mice, and evaluate the toxicity of aspirated diacetyl in knockout mice. We believe the homozygous knockout mice will be viable based upon the human condition, pentosuria, that results from deficiencies in DCXR (http://www.ncbi.nlm.nih.aov/entrez/disomim.cgi?id-260800). There is excretion of pentose in the urine of people who are missing both copies of the DCXR gene (Lane, 1985; Lane and Jenkins, 1985). Heterozygotes can be detected by decreases in erythrocyte L-xylulose reductase activity (Lane and Jenkins, 1985). In humans, both homozygotes and heterozygotes are healthy (Flynn, 1955). However, homozygotes excrete pentose in the urine and show up positive for reducing agents in the urine, a test once used to detect diabetes using Benedict's reagent (Flynn, 1955). NIOSH will be responsible for determining the genotype of DCXR knockout mice. The contractor will then collect urine from at least 5 DCXR knockout mice and 5 control mice and attempt a rapid test for urine pentose to correlate with genotype, In this project, the contractor will also perform necropsies on up to 25 untreated DCXR knockout mice. These will include at least 5 healthy mice and up to 25 DCXR mice with signs of clinical signs. Sections for histopathology will be taken by the contractor from lung, kidney, any gross lesions, and 4 sections of nose. In addition, immunostaining for e-cadherin, f3-catenin, and DCXR will be quantified using morphometry in frozen sections of lung from at least 5 mice. Once the DCXR knockout mice are established and 15 DCXR knockout mice are available for exposure. The contractor will expose DCXR and wildtype mice to 0, 200, or 400 mg diacetyl/kg (n = 5/group) body weight in sterile water by aspiration as previously described (Morgan et a!., 2008). Lung will be collected and perfbsion fixed for histopathology and brain will be harvested for PCR. The contractor will then stain lung sections from the 400 mg diacetyl/kg group of DCXR knockout and control mice for e-cadherin, 3-catenin, DCXR and quantif' changes using morphometry. Safety The contractor and any employees and/or students who conduct these studies must have appropriate training for the safe conduct of the work including in the safe handling of diacetyl for the aspiration exposures. The exposures will be conducted at NIOSH and safely and animal exposure procedures will be reviewed by the NIOSH Safety Office and the Animal Care and Use Commitee. Investigative staff The work performed in this contract may be conducted by employment and/or student fellowship under the direction of contractor. Publication and dissemination of results The results of the experiments may be published in the peer-reviewed literature and/or presented in oral and/or poster form at scientific meetings, conferences or other forums only after internal review and approval by NIOSH. Authorship of such publications will be decided by consensus of the Contractor and the Project Officer. No other dissemination of results is authorized. Progress reports Progress of the research will be reported regularly, at least quarterly, to the Project Officer. The format may involve written reports or informal meetings and discussions. lance (25%) will be authorized when a final report is supplied to the NIOSH Project Officer. Responsible sources that believe they possess the expertise and capabilities identified above are encouraged to submit to the Contracting Officer within 15 days from the posting date of this notice, their written capabilities statement and pricing information in the format they choose, not to exceed 10 pages. Please forward the capability statement and pricing information to Rebecca Mullenax, Purchasing Agent, Reference 000HCCC-2010-79644, Centers for Disease Control, NIOSH, ATTN: Rebecca Mullenax GS: GD, 1095 Willowdale Road, Morgantown, WV 26505. All vendors must be registered in the Central Contractor Registry (CCR) prior to an award of a federal contract. The website is: www.ccr.gov. The Government will review any/all capabilities statements and pricing information submitted and determine if other qualified sources do exist that could provide this requirement. Information received in response to this announcement will be used solely for the purpose of determining whether to conduct a competitive procurement. If no affirmative responses are received within 15 days, in accordance with FAR 13.106-1(b)(1), negotiations will be conducted with West Virginia University Research Corporation, 886 Chestnut Ridge Road, Morgantown, WV 26505-2742 as the only source and a modification to the purchase order will be issued without any additional notices being posted.
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