The goal of this interagency agreement is to provide support of National Toxicology Program (NTP) hazard identification (or hazard assessment) activities targeted toward the prevention of diseases or adverse effects caused by environmental exposure to chemical or physical agents.These cooperative studies continue to improve the risk assessment process by determining quantitatively what constitutes an adverse health effect on the immune system in humans. These studies evaluate unique cohorts of individuals from professions associated with immune-mediated occupational diseases including asthma, contact dermatitis, allergy to mold spores, chronic beryllium disease, allergic rhinitis, silicosis and latex allergy. These cohorts are being studied for a number of endpoints including, impact of genetic polymorphisms on inflammatory disease development and clinical outcomes, and identification of unique immunological biomarkers for disease. The NIOSH Laboratory for Occupational Genomics serves as a resource for obtaining samples from individuals with occupational and occupationally related diseases including chemical-induced contact dermatitis, musculoskeletal disease, chronic beryllium disease, pulmonary fibrosis, chronic obstructive pulmonary disease, HIV infection, occupationally related cancer, neurodegenerative disease, occupational asthma and rhinitis, and silicosis. A major emphasis has been placed on cytokine polymorphisms, especially within the MHC region, as a large number of occupational/environmental diseases are associated with chronic inflammatory responses and, thus, immune responses. Recruitment of subjects exposed to low- and high-molecular weight agents is still ongoing in occupational pulmonary clinics in Canada and the United States. This project has been expanded last year to study genetic variations in major histocompatibility complex (MHC) region. Genotyping has been completed using high density SNP microarray platform. Preliminary analyses comparing LMW-exposed specific inhalation challenge positive and negative groups showed that over 100 SNPs are statistically significantly associated with OA. Subject recruitment, data analysis and genotyping efforts are ongoing.. The Chronic Beryllium Disease (CBD) project investigates the contribution of genetic variations in the MHC region to the development of Be sensitization and CBD. Data has been collected using high density analysis of SN microarray (2,360 loci). An additional project examining the role of genetics in response to vaccination involved 150 healthy children (at age of 1) who visit a university hospital for routine examination. Data analysis has been completed for cytokine gene variations. The study will continue this year with the analysis of MHC region gene variations. We are also investigating the contribution of genetic variability in the immune-inflammatory-antioxidant responses to the development and/or severity of irritant contact dermatitis (ICD) in health care workers. Subject recruitment, sample collection and follow-ups are completed for approximately 350 individuals and recruitment of additional subjects is ongoing. We are currently processing collected samples. An additional aspect of our dermatitis studies will investigate underlying genetic profiles in nickel-sensitized patients, in patients sensitized to weak allergens, and in patients with reactions to more than 3 allergens of the 70 tested on the standard screening series (polysensitized) and in a non-sensitized group of controls. The association between allergic response and genetic variations in immune/inflammatory and antioxidant genes will be investigated. The study is in collaboration with Case Western Reserve University and Dartmouth?Hitchcock Medical Center. HSRB approvals have been obtained and subject recruitment has been started at both sites. Work continues on evaluating total antibody levels and atopy in a cohort of patients with systemic lupus erythematosus. Sera from an additional group of patients with pesticide exposures are also being evaluated for autoantibodies and markers of allergy. Diisocyanates are the leading cause of occupational asthma. To further current understanding of the pathobiology of respiratory disease induced by exposure to diisocyanates in the workplace we are using whole-genome techniques to identify major biological pathways that may serve as targets for biomarker development, disease diagnosis and/or treatment of disease. To fulfill this objective, we have initiated collaborations with extramural partners to investigate the mechanisms of occupational rhinitis in workers exposure to diisocyanates. Patient enrollment and assessment is currently underway. Nasal mucosal tissue will be obtained from workers that are confirmed as specific inhalation challenge positive to diisocyanate challenge. Nasal cytology will be performed on one sample to examine the cellular inflammation that characterizes this disease. Whole-genome microarrays will then be performed on a second sample to investigate the underlying biology and identify potential targets for biomarker development. Similar studies in diverse worker populations are required to develop biomarkers that may be useful for disease surveillance and diagnosis in geographically and ethnically distinct populations. We are conducting parallel studies in a murine model of diisocyanate-induced rhinitis. Using this animal model, we have identified an important role for Ccr3 chemokine receptor signaling in TDI rhinitis and have identified local antibody isotype class-switching within the nasal mucosa of mice with TDI rhinitis. In addition, we are investigating biomarkers for of toluene diisocyanate (TDI) exposure and asthma. At present, biomarkers for TDI exposure are chemical based, and instrument and labor intensive. They also do not distinguish between exposure to toluene diamine (starting material in the production of TDI) and TDI. BALB/c mice were sensitized with 2,4- and 2,6-TDI conjugated keyhole limpet hemocyanin (KLH), spleen cells isolated and hybridomas made. Resultant mAb produced by these hybridomas have been screened for their ability to bind TDI conjugated human serum albumin (HSA) by ELISA. MAbs were further characterized by ELISA and western blot against various monoisocyanate (mNCO) and dNCO and diisothiocyanate conjugated proteins. The 35 mAbs produced can differentiate between specific dNCOs in a carrier independent fashion and may be useful as hapten-specific reagents for immunoassay development and research into dNCO related diseases. Occupational rhinitis is a chronic inflammatory disease of the upper respiratory tract that is present in greater than 90% of workers with occupational asthma. A cohort of chronic rhinosinusitis patients is being evaluated for subjects with allergies to molds. One hundred sinusitis patients and 40 controls have been skin prick tested for a number of mold allergens and the sera have been analyzed for IgG and IgE to mold and other seasonal allergens. Preliminary analysis suggests that there is some increased SPT reactivity to Alternaria in the sinusitis group. As there is currently a lack of information on dry fungal aerosol exposures, we are utilizing aerosols from several organisms to investigate the consequences of dry fungal aerosol exposures in a rodent model. We have been using an insufflation technique to expose mice to dry preparations of spores and fragments from Aspergillus terreus. A new exposure chamber with an acoustical generator is currently being developed as a potential model of a more natural type of exposure. Critical evaluation of the insufflation exposure using Green Fluorescent Protein-labeled A fumagatus spores has shown that the bulk of the aerosol that is insufflated is being delivered to the trachea and not getting deep into the lungs, so modification of the procedure is currently being explored. A related project includes work to characterize the role of terrelysin as a marker of Aspergillus terreus exposure. Terrelysin has been successfully produced by recombinant technology using PCR and gene specific primers derived from the genomic DNA. We have identified the cDNA, cloned, and produced the molecule as a functionally active (hemolytic) recombinant protein. Monoclonal antibodies are being developed for use in environmental and personal exposure assessment methodologies. Finally, ongoing studies are identifying and quantifying the reaction products of gas-phase compounds present in the indoor environment, especially dicarbonyls, and investigating immunotoxic and hypersensitivity effects of these reaction products in both in vitro and in vivo models. The Vitrocell in vitro model is currently being used to expose lung cells to the gas-phase alpha-terpineol/ozone reaction products. Exposure to these aerosolized compounds (up to four hours), consistently increased gene expression of one or more inflammatory cytokines. One select chemical, 4-oxopentanal, is currently being tested in a murine model to investigate the role indoor air reaction products may play as potential asthmagens. In an attempt to confirm a Th2 mode of action, cytokine expression and airway hyper-reactivity after specific and non-specific challenge are being analyzed.
