Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-08-31
2003-02-04
Wortman, Donna C. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S347000, C435S371000, C435S373000, C435S402000, C435S408000
Reexamination Certificate
active
06514711
ABSTRACT:
BACKGROUND OF THE INVENTION
Skin is a continually renewing tissue that acts as one of the body's primary defense systems against environmental insult. Skin is composed of two layers, the dermis and the epidermis. The dermis, whose primary cellular components are fibroblasts, forms the foundation upon which the epidermis lies. Dermal fibroblasts, the primary cellular component of the dermis, exist in a sea of extracellular matrix. These matrix components not only physically support the overlying layers, but are also involved in biochemical signaling pathways. Clark, E. A. and J. S. Brugge,
Science
268: 233-9 (1995). In addition to secreting matrix components, fibroblasts also secrete a spectrum of growth factors, which act on the overlying epidermis. Mass-Szabowski and N. E. Fusenig,
J. Invest. Dermatol.
107: 849-55 (1996); Smola, H. et al.,
J. Cell Biol.
122: 417-29 (1993). The epidermis is a stratified squamous epithelium, composed primarily of keratinocytes. Keratinocytes within the epidermis are organized into four layers based on morphological and biochemical properties; these are the basal, spinous, granular, and cornified layers. Eckert, R. L. et al.,
Physiol Rev
77: 397-424 (1997). The basal layer rests atop the dermis and is in direct contact with the specialized extracellular matrix proteins of the basement membrane. Basal keratinocytes have the ability to replicate and are the source of all suprabasal keratinocytes within the epidermis. As basal cells divide, certain daughter cells lose contact with the basement membrane. These cells assume a suprabasal position and continue movement upward through the layers of the epidermis until they enucleate and are sloughed from the surface of the skin, a process known as terminal differentiation. The differentiation process in keratinocytes is accompanied by a pattern of characteristic changes in gene expression; a number of proteins serve as markers of terminal differentiation. Undifferentiated basal keratinocytes express the keratins K5 and K14 while differentiating cells express K1 and K10. Other proteins that are expressed in a specific pattern during keratinocyte terminal differentiation include precursors of the cornified envelope such as involucrin, filaggrin, pancornulin, cornifin, loricrin and elafin, as well as the cross-linking enzyme, epidermal transglutaminase. Morley, S. M. and E. B. Lane,
The Keratinocyte Handbook,
293-321 (1994); Simon, M.,
The Keratinocyte Handbook,
275-292 (1994); Steinert, P. M. and L. N. Marekov,
The Journal of Biological Chemistry
270: 17702-17711 (1995).
As one of the body's primary defense systems against environmental insult, skin often comes into contact with a wide spectrum of toxins, which may damage the skin. For example, human exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), one congener of a family of ubiquitous environmental toxins called dioxins, produces an array of clinical manifestations. Chloracne, a hyperkeratotic skin disorder, is the most consistently observed pathology in exposed humans. Historically, the effect of a toxin on human skin has been studied in vitro using monolayer culture systems. Human keratinocytes isolated from stratified squamous epitheliums can be readily cultivated in vitro. Leigh, I. M. and F. M. W.,
The Keratinocyte Handbook,
43-52 (1994). Cultivated keratinocytes replicate readily during early passage and can generate large numbers of cells which exhibit certain features of squamous differentiation in vivo. However, monolayer cultures of human keratinocytes do not form the epidermal tissue architecture that mimics a stratified squamous epithelium in human epidermis. Thus, studying a toxin's effects on human skin in monolayer keratinocyte cultures is flawed in that monolayer cultures do not reflect the normal in vivo tissue context. For example, although several laboratories have previously reported on the cellular responses of normal human or murine keratinocytes to TCDD in vitro, little evidence has been available to link these observations to the specific pathology observed in human skin.
When cultured normal human keratinocytes are transplanted onto mice, epidermal tissue architecture is regenerated over time in an orderly fashion. Breitkruetz, D. et al.,
Differentiation
61(3): 195-209 (1997). However, studying the effect of a toxin on human skin using transplanted animals can be time consuming and expensive. In addition, when primary human keratinocytes are used in monolayer cultures and transplanted animals, different batches of cells may vary one way or another, which further complicates the toxin testing process.
BRIEF SUMMARY OF THE INVENTION
In one embodiment, the present invention is a method for testing the effects of various factors on human skin equivalents. The method involves providing a human stratified squamous epithelial cell culture of an immortalized human keratinocyte cell line that forms a reconstructed epidermis, exposing the reconstructed epidermis to a test factor, and evaluating the effect of the factor on the reconstructed epidermis. Preferably, the culture further comprises a base layer of collagen and fibroblasts.
In a particularly advantageous embodiment of the invention, the spontaneously immortalized human keratinocytes are ATCC CRL 12191 cells. In another embodiment, the spontaneously immortalized human keratinocytes carry an exogenous gene, preferably green fluorescence protein gene.
In another preferred embodiment of the present invention, the factor is a material that may contact human skin or is selected from the group consisting of UV light, radiation, air pressure, environmental temperature, and friction.
In an preferred embodiment of the present invention, the effect of the factor on the reconstructed epidermis is selected from the group consisting of cell differentiation, cell proliferation, cell survival, cell damage, and cell death. In another embodiment, the effect may be selected from the group consisting of tissue culture morphology change, barrier function, and tissue strength.
Another embodiment of the present invention is a method for selecting preventive or therapeutic agents for skin damages caused by a test factor. The method involves providing a human stratified squamous epithelial cell culture of an immortalized human keratinocyte cell line that forms a reconstructed epidermis, exposing the reconstructed epidermis to a factor that can cause damage in the reconstructed epidermis, exposing the reconstructed epidermis to an agent being screened for preventive or therapeutic use, and evaluating the effect of the agent on the reconstructed epidermis.
In a preferred embodiment of the present invention, the exposure of the reconstructed epidermis to the factor may occur prior to, after, or at the same time as the exposure of the reconstructed epidermis to the agent being screened for production of a therapeutic use.
In separate embodiments of the invention, the test factors described above in various embodiments may be in liquid, solid, emulsion, gaseous, or solvent form. The agent to be screened may also be in solvent, liquid, solid, gaseous, or emulsion form.
It is an advantage of the present invention that the effects of a factor on a tissue culture system mimics the effects of the factor on human skin in vivo.
Further objects, features and advantages of the invention will be apparent from the following detailed description when taken in conjunction with the accompanying claims and drawings.
REFERENCES:
patent: 5491084 (1996-02-01), Chalfie et al.
patent: 6214567 (2001-04-01), Allen-Hoffman et al.
L. Allen-Hoffman, et al., Proc. Nat'l. Acad. Sci. USA 81:7802-7806, 1984.
B.L. Allen-Hoffman, et al., “Use of RHEK-1 Immortalized Human Keratinocytes for Detection of Induced Mutation at the Hypoxanthine-guanine Phosphoriboxyltransferase Locus,” Inter. J. Oncol. 3:619-625, 1993.
H.P. Baden, et al., “Isolation and Characterization of a Spontaneously Arising Long-lived Line of Human Keratinocytes (NM1),” In Vitro Cell. Dev. Biol. 23(3):205-213, 1987.
P. Boukamp
Quarles & Brady LLP
Wisconsin Alumni Research Foundation
Wortman Donna C.
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