An adjuvant free mouse model of oral allergenic sensitization to rice seeds protein

<p>Abstract</p> <p>Background</p> <p>Rice is commonly known as a staple crop consumed worldwide, though with several rice proteins being reported for allergic properties in clinical studies. Thus, there is a growing need for the development of an animal model to better understand the allergenicity of rice proteins and the immunological and pathophysiological mechanisms underlying the development of food allergy.</p> <p>Methods</p> <p>Groups of BALB/c mice were sensitized daily with freshly homogenized rice flour (30 mg or 80 mg) without adjuvant by intragastric gavage. In addition, the mice were challenged with extracted rice flour proteins at several time points intragastrically. Hypersensitivity symptoms in mice were evaluated according to a scoring system. Vascular leakage, ELISA of rice protein-specific IgE, histopathology of small intestine, and passive cutaneous anaphylaxis were conducted on challenged mice.</p> <p>Results</p> <p>An adjuvant free mouse model of rice allergy was established with sensitized mice showing increased scratching behaviors and increased vascular permeability. Rice protein-specific IgE was detected after eighteen days of sensitization and from the fifth challenge onwards. Inflammatory damage to the epithelium in the small intestine of mice was observed beyond one month of sensitization. Passive cutaneous anaphylaxis results confirmed the positive rice allergy in the mouse model.</p> <p>Conclusions</p> <p>We introduced a BALB/c mouse model of rice allergy with simple oral sensitization without the use of adjuvant. This model would serve as a useful tool for further analysis on the immunopathogenic mechanisms of the various rice allergens, for the evaluation of the hypersensitivity of rice or other cereal grains, and to serve as a platform for the development of immunotherapies against rice allergens.</p

The identification of novel marine bacteria, and the construction of single chain fragment variable antibodies for the control of a viral pathogen

Isolation, identification, and characterization of members of the yet uncultured bacterioplankton is fundamental in understanding the roles they play in the marine environment, and their relationships with other members of the marine environment, such as viruses, phytoplankton, and zooplankton etc. In the first part of the thesis, I have used modified culture media in the traditional cultivation approach and isolated three novel bacterial strains from the marine environment in Hong Kong and China. Using the powerful polyphasic taxonomic approach, I determined that the two isolates from Hong Kong belonged to two novel genera in the family Cryomorphaceae. Both strains were characterized and published as Owenweeksia hongkongensis gen. sp. nov. and Lishizhenia caseinilytica gen. sp. nov. in the 'International Journal of Systematic and Evolution Microbiology (IJSEM)', the leading journal in the field. The new strain isolated from a pearl-oyster culture pond, in Hainan Province, China, has been identified as a new species belonging to the genus Marinomonas, and was named and published as Marinomonas ostrestagni sp. nov. in IJSEM. With the increased demand of shrimp for food, shrimp aquaculture has expanded considerably in many countries. In the last decade, the production of shrimp from aquaculture was drastically reduced due to the infection of novel viruses like the White Spot Syndrome Virus (WSSV) and Yellow Head Virus. Traditional treatments such as the sterilization of culture ponds and/or usage of antibiotics did not stop the problem. The repeated infection incidences suggested that the native immune system of shrimp is not sufficient to defend against the viral infection. In the second part of the thesis, I have constructed murine single chain fragment variable antibodies (ScFvs) phage display libraries against two major structural proteins of WSSV, VP26 and VP28. The ScFvs consisted of variable light chains of immunoglobulin joined to variable heavy chains by a peptide linker of 7 amino acids (short linker) or 16 amino acids (long linker). The size of the short linker libraries and long linker libraries were 3 x 107 and 1.7 x 108 clones, respectively. BstO I fingerprinting patterns indicated that the independent clones in each phage library were diverse. Screening of these libraries led to the identification of three groups of ScFvs that bind to independent epitopes on r-VP28. Two VP28 binding ScFvs delayed the onset of mortality in an in vivo neutralization assay in the crayfish Procambarus clarkii, confirming the role of VP28 in WSSV infection. These ScFvs have potentials to be developed into prophylactic reagents against WSSV. The new Cryomorphaceae bacteria that I isolated and identified could also be evaluated for its potential probiotics due to their high carotenoids content that may relieve the oxidative stress caused by WSSV infection