Lawrence Chan, B.A. - US grants

The candidate, Lawrence S. Chan, is an Assistant Professor of Dermatology at the Northwestern University Medical School. After graduating form the University of Pennsylvania School of Medicine in 1985, Dr. Chen obtained his Dermatology residency training at the University of Michigan Medical School under the leadership of Dr. John J. Voorhees. He was also trained as an Immunodermatology fellow at the same institution under the mentorship of Dr. Kevin D. Coper, a cellular immunologist, and Dr. James T Eler, a molecular biologist. The candidate's career interest is in the area of human autoimmunity against skin basement membrane zone (BMZ) components. During his residency, Dr. Chan discovered a new immunemediated blistering skin disease characterized by autoantibodies against a 105-kDa BMZ component at the lower lamina lucida. The candidate's short-term goals are to purify this new BMZ component and clone the human gene encoding for this new component. The candidate's long-term goals are to understand the functions of this new BMZ component: its relationship to other BMZ components, its influence on epidermal cell biology, its antigenic domains, and its pathophysiologic role in inducing autoimmune reaction. The research specific aims for the next five years, are to purify this 105- kD component, to isolate the human cDNA that encodes for this component, and to determine its functional domains. To achieve these specific aims, fibroblast protein will be subjected to multiple chromatographic columns, including Mono Q anion-exchange and reverse phase columns, for purification. The purified fractions will be analyzed by 2-dimensional gel electrophoresis and immunoblotting. The internal amino acid sequences will be obtained after proteolytic digestion of th purified protein. Monoclonal antibodies raised against the 105-kDa component and polyclonal antibodies raised against the amino-terminal peptide will be utilized to screen a human fibroblast cDNA expression library. Positive plaques will be subjected to cloning and DNA sequencing. Once the entire cDNA is delineated, multiple fusion proteins will be generated to examine the antigenic domains, the cellular and extracellular attachment domains, and the domains that influence keratinocyte biology. Understanding the structure and function of this new lamina lucida component will shed light on our understanding of the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering diseases, gestational development, and wound healing. The research environment provided for Dr. Chan at the northwestern University include; a Biotechnology Core Facility for protein microsequencing, peptide and oligonucleotide synthesis, monoclonal antibody production, two-dimensional gel electrophoresis, flow cytometry, molecular biology supplies, and an Animal Care Facility, all in the same research building as Dr. Chan's laboratory. The Department of Dermatology has several faculty members who can assist Dr. Nageswararao, another collaborator, is a protein biochemist, Dr. Woodley, the candidate's primary sponsor, is a protein biochemist and is well acquainted with epithelial cell biology. The Department of Dermatology has a departmental library which contains many major scientific journals and a state-of-the-art Fast Protein Liquid Chromatography system that can facilitate Dr. Chan's protein purification. Dr. Chan has been provided a fully equipped 660 square-feet laboratory space, a 200 square-feet office space adjacent to his laboratory, a culture room, and an electron microscope. In addition, the candidate has 80% projective time devoted to research. Together, this environment provides sufficient support for the candidate to succeed in his research effort.

DESCRIPTION: (Verbatim) Skin basement membrane zone (BML) serves as an interface barrier and as an adherent connection between the outer layer of skin, the epidermis, and inner 'layer of skin, the dermis. Some of the BMZ components, such as 8P230, BP180 (type XVII collagen) were discovered as a result of being targeted by an autoimmune reaction. Using sera from patients with autoimmune blistering diseases and autoantibodies to BMZ components, the cDNAs encoding BMZ components, such as BP18O and type VII collagen, were determined and became very valuable in biomedical research. The autoantibody-delineated nucleotide sequences allow synthesis of large quantities of recombinant proteins, which in turn significantly facilitates the understanding of pathogenesis of blistering process and the normal connecting function of BMZ components. We now have the opportunity to discover and understand another previously unknown BMZ component, termed p105, also identified as a result of an autoimmune reaction. This novel autoimmune blistering skin disease is characterized by extensive blisters and erosions clinically, subepidermal separation with neutrophilic infiltration histologically, in vivo lgG deposition at BMZ and circulating lgG binding to the dermal side of salt-split skin substrate immunopathologically, in vivo lgG deposition and circulating lgG binding to the lower lamina lucida ultrastructural!y, and lgG autoantibodies recognizing a 1O5-kDa epidermal protein immunochemically. This p105 has been further characterized for its distinction from the HO5-kDa laminin-5 -y2 chain by immunochemical methods, its isoelectric point by two-dimensional gel electrophoresis and immunoblotting, its ionic strength by Mono 0 anion-exchange column chromatography, and its N-terminal amino acid sequence by protein sequencing. Furthermore, a monoclonal antibody has been generated against this p105 protein. Having cloned two cDNAs encoding BMZ components, the principal investigator has gained experience in molecular cloning and is now ready to propose the following works to study the structure and function of this novel BMZ component p105. In this proposal, we aim to molecularly clone the human p105 cDNA sequence, to express the human p105 recombinant protein, to study the in vitro functions of human p105 protein, to molecular clone the mouse p105 cDNA, and to passive transfer of anti-mouse p105 antibodies to new born mice. Understanding of the structure and function of this new BMZ component p105 will shed light on the complex structure of the skin BMZ, the relationship between different components of skin BMZ, and their roles in epidermal-dermal adhesion, human blistering skin diseases, gestational development, epidermal cancer metastasis, and cutaneous wound healing.

DESCRIPTION (provided by applicant): Alopecia areata is a chronic autoimmune hair loss disorder that affects 0.2-2 % of population. Although alopecia areata is a non-fatal disease, the disease itself and the treatment complication can cause significant morbidity. Clinical and laboratory data from studying of human patients suggests that alopecia areata may be caused by autoreactive lymphocytes, particularly CD8+ T lymphocytes. Although passive transfer experiments delineated the hair loss process once the autoreactive T lymphocytes are formed, the step-by-step immunological sequence of events accounting for the initiation, progression, and maintenance of the disease remain unclear. Moreover, the antigen targeted by the autoreactive lymphocytes remains unknown. Furthermore, currently there is no active experimental animal model of alopecia areata for dissecting these step by-step events. The PI, Lawrence S. Chan, M.D., was trained as a fellow in Immuno-dermatology under Dr. Kevin D. Cooper, a cellular immunologist at the Univ. of Michigan. For the current proposal, the PI aims at characterizing a newly generated mouse model of hair loss disorder, induced by subcutaneous immunization of a mouse hair follicle basement membrane component. This newly generated active mouse model of hair loss disorder has clinical and histologic findings resembling the human disease alopecia areata. Some affected mice developed autoantibodies to the immunogens. The same strain of mice developed autoreactive T cell response to the self proteins. The PI proposes to authenticate this model as a mouse model for human alopecia. areata with the following specific aims: 1). Authentication of the autoimmune nature of this hair loss disorder model 2). Characterization of the effector immune cell types. If established as a model of human disease, it may shed light to the pathogenesis of alopecia areata in human patients and thereby lead to eventual target-specific immunological treatments for human patients.

DESCRIPTION (Taken from the application): Atopic dermatitis is a chronic, inflammatory skin disease that affects about 20% children between ages 3 and 11. The prevalence of atopic, dermatitis is increasing, particularly in the industrialized nations. Atopic dermatitis is characterized by pruritic skin rash clinically, T lymphocyte and mast cell infiltration histopathologically, and elevation of total serum IgE serologically. Although usually non-fatal, atopic dermatitis can cause significant morbidity. Clinical and laboratory data from studying of human patients suggests that atopic dermatitis may be caused by an imbalance of excessive activation of Th2-type lymphocytes over Thl-type lymphocytes, resulting in a Th2-biased immune response. However, the step-by-step immunological sequence of events accounting for the initiation, progression, and maintenance of the disease remain unclear. Furthermore, currently there is no available experimental animal model of atopic derrnatitis for dissecting these step-by-step events. The PI, Lawrence S. Chan, M.D., was trained as a fellow in Immuno-dermatology under Dr. Kevin D. Cooper, a cellular immunologist at the Univ. of Michigan. For the current proposal, the PI aims at characterizing a transgenic (Tg) mouse model that the PI has recently created. This experimental mouse model was generated by transgenically introduced critical Th2 cytokine IL-4 to the basal epidermis of Tg mice and the affected Tg mice has identical clinical, histopathological, microbiological, and serological characteristics as human atopic dermatitis. With the availability of this newly created mouse disease model, the PI can now move forward to further characterize this experimental model of atopic dermatitis with the following specific aims: 1). Determining the correlation of epidermal IL-4 in vivo protein expression and total serum IgE levels with clinical phenotype. 2). Determining the inflammatory cell types of skin lesions. 3). Characterizing the cytokine profiles of skin lesions. By parallel studying the natural history of atopic dermatitis and the immunological parameters, including lesional inflammatory cell and T cell subsets, lesional T cell cytokines, adhesion molecules, and total serum IgE, the PI aims at delineating the step-by-step immune events accounting for the initiation, progression, and maintenance of the disease. The likelihood of achieving these aims is supported by the PI's past experience in these areas of investigation and the assistance of Dr. Stephen D. Miller, an experienced cellular immunologist and the coinvestigator of the project. Delineating the characteristics of atopic dermatitis in this mouse disease model may shed light to the pathogenesis of atopic dermatitis in human patients, thereby lead to eventual target-specific immunological treatments for human patients suffering from atopic dermatitis.

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