Patent Application
20060088852
The present invention relates to a method for determining the homeostasis of hairy skin in vitro, to test kits and biochips for determining genetic and protein markers of hairy skin, and to the use of proteins, mRNA molecules or fragments thereof as molecular markers of hairy skin. Also provided is a test method for determining the efficacy of cosmetic or pharmaceutically active ingredients for the treatment of hairy skin, and a method for producing a cosmetic or pharmaceutical preparation so identified.
Every living cell responds to signals from its surroundings. The responses of cells are implemented by an ordered regulation of gene expression such that cellular metabolism is a dynamic, rather than static state. The human genome comprises, according to the most recent estimates, between 30,000 and 140,000 genes. However, each cell uses only a small portion of this genetic information which is specific for that particular cell. Thus, gene expression patterns differ from cell type to cell type. Exogenous signals often trigger complex signal transduction cascades which effect changes in gene expression patterns. In this way, signals from cellular surroundings give rise to alterations in cellular metabolism.
Besides these relatively short-lived changes in gene expression patterns, each living cell is subject to an aging phenomenon, a process which is associated with a slow change in gene expression.
The human skin is complex in structure and is the largest organ of the human body. The skin forms the body's interface with its surroundings and is comprised of many different cell types. Most cells of the skin are found in the epidermis and dermis.
Analysis of gene expression is crucially important for understanding the general biological responses of the skin and in particular the modulation of cutaneous structure formation in response to exogenous stimuli. Cutaneous macromolecular structures or appendages, including, without limitation, hair follicles, sebaceous glands, sweat glands etc. are formed by a relatively small proportion of specialized cells (e.g., fewer than 5% of skin cells are involved in hair follicle structure). Accordingly, analyzing the cells which contribute to these structures (e.g., by gene expression profiling) is performed only with difficulty.
Isolation of cutaneous structures is technically difficult and very time-consuming. Removal of cells from their natural biological state tends to inhibit realistic visualization of biochemical processes in the skin or its appendages. Every manipulation of tissue (e.g., to isolate or concentrate particular structures) perturbs the cells, resulting in an alteration of gene expression patterns. This state no longer reflects the natural cellular state and thus cannot be regarded as representative.
Hairs are present on most regions of human skin, with the exception of the lips, the palms of the hands and the soles of the feet. Inapparent vellus hairs differ macroscopically from the cosmetically relevant cephalic hairs on the head. Microscopic differentiation is possible for both types of hair follicles and for the hairs themselves. However, the biochemical and molecular biological mechanisms underlying these differences are substantially unknown.
A relevant feature of hairs and their follicles is that with increasing age, the cells lose their ability to maintain the homeostasis of the organ. Thus, for example, over time the number of hair follicles per unit area decreases. There is likewise a change in the structure of hairs in that, for example, the hair diameter becomes smaller. Frequently pigment producing cells in the hair follicles lose this ability with increasing age resulting the graying of the hair. The molecular mechanisms underlying this development are as yet substantially unclear.
Effective cosmetic or pharmaceutical hair products should provide beneficial cosmetic and/or therapeutic effects on a variety of molecular processes in the hair follicle. However, to date, only a few molecular reaction mechanisms in the hair follicle have been described, thereby limiting the number of suitable targets for new cosmetic hair products.
Every cell type in the skin and its appendages expresses about 15,000 different genes and synthesizes therefrom a corresponding number of proteins. However, it is as yet substantially unclear which particular genes are important in hair follicles.
The skin consists of a plurality of different cell types (e.g. fibroblasts, keratinocytes in various states of differentiation, melanocytes, Merkel cells, Langerhans cells, a large number of different cells of the hair follicle or other cutaneous appendages) so that the complexity of genes expressed in the skin is very great. To date, it has not been possible to identify the particular genes or markers associated with the hair follicle from this complex mixture. An additional difficulty is that mRNA molecules occur in the cell in widely ranging concentrations (e.g., between a few and several hundred copies). Thus, weakly expressed genes are difficult to assess with currently available analytical techniques but may very well be of crucial importance in the hair follicle.
The transcriptome of the human hair follicle, that is the totality of all transcribed genes therein, has not been described to date.
Transcriptome analyses of the skin by various methods, including SAGE™ analysis reflects the current state of the art. However, previously described approaches employed isolated keratinocytes (in vitro) or epidermis explants, but as explained above, these artificial models are not representative of the naturally occurring complex events in the skin.
DE-A-101 00 127.4-41 to the present inventors discloses subjecting skin cells to a SAGE™ analysis in order to characterize the complete transcriptome of the skin. DE-A-101 00 121.5-41 of the applicant discloses the identification of markers of stressed or aged skin on the basis of a comparative SAGE™ analysis between stressed and aged skin and unstressed and young skin, respectively. However, information about specific markers of hairy skin is not to be found in these publications.
J Invest Dermatol 2002 July; 119(1):3-13; “A serial analysis of gene expression in sun-damaged human skin”; Urschitz J. et al.; discloses the determination of markers of sun-damaged skin by means of a comparative SAGE™ analysis of full-thickness skin explants taken from in front of the oracle (sun-damage) and behind the oracle (protected from the sun's rays). It is likewise impossible to obtain any information about specific markers of hairy skin from this publication.
There is thus a need to identify as many as possible, and preferably all, of the genes which contribute to the hairiness of the skin.
It is an object of the present invention to identify a large portion, if not all, of the genes important for the hairiness of skin. Also encompassed by the invention is the use of this genetic information in methods for determining the homeostasis of hairy skin.
According to a first object of the invention, a method is provided for identifying differentially expressed genes which modulate the hairiness of skin in humans in vitro. An exemplary method comprises:
For the purposes of the present invention “hairless” also means hairiness comprising the fine and scarcely visible vellus hairs.
The method of the invention facilitates an understanding of the complex processes involved in the development of hair and the causal relationships of the changes in hair. Only with this knowledge, is it possible to develop novel targets for cosmetic hair products which exert their effect on the variety of genes expressed in the hair follicle. The present inventors, employing SAGE™ analysis have identified for the first time the differential gene expression patterns present in hairy (with cephalic hair) scalp skin and essentially hairless skin.
According to a second object of the present invention, a method is provided for determining the homeostasis of hairy skin in humans, especially in women, in vitro. An exemplary method entails
A third object of the invention relates to yet another embodiment for determining the homeostasis of hairy skin in humans. An exemplary method entails performing SAGE analysis as described above to identify molecules characteristic of healthy hairy human skin. At least two molecules (e.g., mRNA or proteins) identified as important in hairy skin are quantified and the expression ratios relative one to another determined, thereby providing an expression quotient. A hairy skin sample is obtained from a patient and the sample is assessed for the expression ratios of the at least two molecules, the expression quotients in the patient sample are then compared with those provided in Tables 3-12, columns 3 and 5, the patient sample is then designated as healthy hairy skin (homeostasis-undergoing) if the expression ratios of the sample correspond to the expression ratios in hairy skin, or designated as diseased (homeostasis-impaired) skin if the expression ratios of the patient skin sample correspond to the expression ratios of hairless skin.
Examples of disorders or impairments of homeostasis of hairy skin are: pili torti (corkscrew hairs, twisted hairs), monilethrix (beaded hair), wooly hairs (crinkled hair), hair shaft changes with breaks [trichorrhexis nodosa, trichorrhexis invaginata, trichoschisis, trichoptilosis (hair splitting)], hair shaft changes associated with metabolic disorders, pili recurvati, ingrowing hairs, changes in hair color [heterochromia, albinism, poliosis (acquired focal lack of hair pigment), canitis (physiological graying)], hypertrichoses, hirsutism, alopecias (irreversible alopecia: e.g. androgenic alopecia of men and women); reversible alopecia: e.g. symptomatic diffuse alopecias due to infections, noxious chemicals and drugs, hormonal disorders, diseases, etc.) and alopecia greata.
The present invention also provides a kits for performing the methods described above. Also provided in the invention is a biochip which facilitates the practice of the present invention. An exemplary biochip comprises
A biochip which is particularly preferred according to the invention includes probes which are selected from those capable of specific binding to at least one of the proteins, mRNA molecules or fragments thereof which are defined in column 1 in Tables 3 to 6 by the following consecutive serial number: 2, 4, 9, 12, 14, 16, 22, 25, 29, 31, 35, 36, 38, 39, 40, 42, 43, 44, 46, 59, 62, 63, 65, 67, 68, 69, 74.
The present invention also relates to a method for screening pharmaceutically and/or cosmetically active ingredients for efficacy in the treatment of disorders or impairment of homeostasis of hairy skin in vitro. The method entails incubating the hairy skin sample in the presence and absence of the ingredient and determining whether the homeostasis of hairy skin using the methods set forth above is improved. Ingredients which improve the state of the hairy skin can then be isolated and formulated with cosmetically and/or pharmaceutically suitable and acceptable carriers.
Cosmetic hair products available on the market usually exert their effects on the hair shaft (e.g. hair colorations). In accordance with the present invention, it is now possible for the first time to gain an understanding of the complex genetic and biological processes in the hair follicle. Identification of suitable markers present in the hair follicle thus permits a targeted search for substances or combinations of substances having a broad range of effects on gene expression in the hair follicle. It has not been possible until the present time to develop products of this type, because a large number of the hair follicle markers were as yet, unknown.
The technique employed to establish the transcriptome of hairy skin was “serial analysis of gene expression” (SAGE™). This technique permits simultaneous identification and quantification of all genes expressed in hairy skin. It is true that gene expression can also be analyzed by quantifying specific mRNA molecules (e.g. Northern blotting, RNase protection experiments). However, often these techniques measure only a relatively limited number of genes. It would theoretically be possible for the techniques of MPSS (massive parallel signature sequencing) or techniques based on differential display to replace the SAGE™ analysis. However, in practice, the SAGE™ technique is faster and more reliable than alternative methods and is thus preferred.
Comparison of the transcriptome of hairy skin with the transcriptome of hairless skin permits differentiation between genes relevant and not relevant for the hairiness of the skin.
Human skin from healthy female donors was used for the SAGE™ analysis. The SAGE™ analysis was carried out as described in EP-A-0 761 822 and by Velculescu, V. E. et al., 1995 Science 270, 484-487. This technique permits simultaneous identification and quantification of the genes expressed in the hairy scalp. Comparison of the transcriptome of hairy scalp with the transcriptome of facial skin permits the identification of relevant genes for hairy scalp. These may be genes which are highly expressed in hairy scalp or alternatively, genes which are characterized in that their expression is diminished when compared with hairless facial skin. SAGE™ analysis is a technique with particular sensitivity and even surprisingly reveals interindividual differences in gene expression profiles. For description of the transcriptome of hairy skin, therefore, comparison with the transcriptome of hairless skin is especially effective when the analyzed tissues are derived from one individual, that is one donor. Thus, interindividual differences in gene expression do not apply. Hairy tissues from the region above the ear and the region behind the ear are obtained from a patient during plastic surgery operations such as, for example, “lower facelifts”. At the same time, cutaneous tissue that—at least in female donors—bears only vellus hair is removed in front of the ear. Analysis of such tissue samples therefore permits description of the transcriptomes of hairy scalp and hairless (or only having vellus hair) facial skin while avoiding interindividual differences in gene expression.
Human skin from a healthy female donor (65 years old) was used for the SAGE™ analysis. The SAGE™ analysis was carried out as described in EP-A-0 761 822 and by Velculescu, V. E. et al., 1995 Science 270, 484-487. Two SAGE™ libraries from a patient's skin samples from various locations were analyzed. The first sample was derived from a removal point above the ear and provided skin with hair of the head. The second sample was derived from the same operation, was taken in front of the ear and provided skin with only vellus hair. For further analysis, the two SAGE™ libraries were normalized to the average number of tags. The two libraries were compared with one another in order to identify genes with hair-specific regulation. As expected for two libraries of the same tissue type, the tag repertoire of the two skin libraries is substantially similar. Despite the similarity of the tissues and the relatively small number of tags, 74 tags show a significantly differential expression at a significance level of p>0.05.
For hairy skin, it is possible to show an increased number of tags for genes which are expressed in the hair follicle or in hair appendages. Besides an increased level of hair- and hair follicle-typical keratins, hairy skin also shows, for example, elevated FGF7 levels, a transcription factor involved in hair development. Dermcidin and cystatin, both involved in the defense against bacterial and viral invasions in the hair shaft, likewise show significantly increased expression levels in hairy skin. Expression of differentiation-dependent genes of the interfollicular epidermis is increased in hairless skin as reflected in the results of the investigation.
The differentially expressed genes are led by keratin 10, a typical marker for the differentiation of stratified epithelia. Keratin 1, the partner of keratin 10, does not show these differences in expression. Analysis of further differentiation-dependently expressed genes likewise confirms increased expression thereof in hairless skin (See Table 2). Another keratin, keratin 2e, is according to the literature, expressed mainly in hairless skin, and this is likewise observed in the analysis carried out. Expression of keratins 5, 14 and 15, which are expressed in the basal layer of the epidermis and in the hair follicles, is, as expected, virtually identical in the two samples (Table. 2).
Table 1 lists markers which are indicated in the literature to be differentially expressed in the hair follicle compared with the remainder of the skin (interfollicular skin). They serve as positive controls for the experiment.
Table 2 shows differentiation-dependent genes of the interfollicular epidermis.
Further SAGE™ analyses of human skin were taken into account for describing and verifying subsignificant differences in gene expression (Tables 7 to 12). Thus, low expression of a hair-specific gene is to be expected not only in hairless facial skin but also in other hairless body skin. The subsignificant differences from the comparison of the transcriptome of hairy scalp with the transcriptome of hairless facial skin were therefore compared with the expression profile of hairless breast skin. Breast skin from a healthy female donor (69 years old) was used for the SAGE™ analysis of breast skin. It was possible thereby to confirm some significant differences in gene expression.
Tables 3 to 12 contain a detailed listing of genes differentially expressed in hairy and hairless skin identified with the aid of the method of the invention, and include:
The quotient in column 3 indicates the strength of differential expression, i.e. the factor by which the particular gene is more highly or strongly expressed in hairy scalp (scalp) than in hairless facial skin (face), or vice versa.
The quotient in column 5 indicates the strength of differential expression, i.e. the factor by which the particular gene is more strongly expressed in hairy scalp (scalp) than in hairless body skin (breast), or vice versa.
The respective genes and gene products are disclosed under their UniGene accession number in the database of the National Center for Biotechnology Information (NCBI). This database can be accessed in the internet under the following address:
The genes and gene products can additionally be accessed directly under the internet addresses
Table 3 lists all the genes which exhibit at least a 10-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p>0.05 (signif>1.3).
Table 4 lists all the genes which exhibit at least a 5-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p>0.05 (signif>1.3)
Table 5 lists all the genes which exhibit at least a 3-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p>0.05 (signif>1.3)
Table 6 lists all the genes which exhibit at least a 1.9-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p>0.05 (signif>1.3)
Table 7 lists all the genes which exhibit at least a 5-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and which exhibit at least a 5-fold differential in expression levels in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p>0.05 (signif>1.3). Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
Table 8 lists all the genes which exhibit at least a 5-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and which exhibit at least a 5-fold differential in expression levels in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p<0.05 (signif<1.3) whose expression differs by less than one power of 10, i.e. the quotient (scalp/face)/(scalp/breast) is less than 10 or greater than 0.1. Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
Table 9 lists all the genes which exhibit at least a 3-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and which exhibit at least a 3-fold differential in expression level in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p>0.05 (signif>1.3). Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
Table 10 lists all genes which exhibit at least a 3-fold differential in expression level when expressed in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and exhibit at least a 3-fold differential in expression level in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p<0.05 (signif<1.3) whose expression differs by less than one power of 10, i.e. the quotient (scalp/face)/(scalp/breast) is less than 10 or greater than 0.1. Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
Table 11 lists all genes which exhibit at least a 1.9-fold differential in expression levels in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and which exhibit at least a 1.9-fold differential in expression level in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p>0.05 (signif>1.3). Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
Table 12 lists all genes which exhibit at least a 1.9-fold differential in expression level in hairy scalp (scalp) when compared with hairless facial skin (face) with a p value of p<0.05 (signif<1.3) and which exhibit at least a 1.9-fold differential in expression level in hairy scalp (scalp) when compared with hairless body skin (breast) with a p value of p<0.05 (signif<1.3) whose expression differs by less than one power of 10, i.e. the quotient (scalp/face)/(scalp/breast) is less than 10 or greater than 0.1. Comparison of the subsignificant scalp/face data with independent SAGE™ experiments (scalp/breast) confirms the differential gene expression and validates the markers of hairy skin.
According to a second object of the present invention, a method is provided for determining the homeostasis of hairy skin in humans, especially in women, in vitro. An exemplary method entails
Examples of disorders or impairments of homeostasis of hairy skin are: pili torti (corkscrew hairs, twisted hairs), monilethrix (beaded hair), wooly hairs (crinkled hair), hair shaft changes with breaks [trichorrhexis nodosa, trichorrhexis invaginata, trichoschisis, trichoptilosis (hair splitting)], hair shaft changes associated with metabolic disorders, pili recurvati, ingrowing hairs, changes in hair color [heterochromia, albinism, poliosis (acquired focal lack of hair pigment), canitis (physiological graying)], hypertrichoses, hirsutism, alopecias (irreversible alopecia: e.g. androgenic alopecia of men and women); reversible alopecia: e.g. symptomatic diffuse alopecias due to infections, chem. noxae and drugs, hormonal disorders, diseases, etc.) and alopecia greata.
The sample of step b) of the method of the invention for determining the homeostasis of hairy skin in a patient may be obtained from full-thickness skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hairy skin.
It may be sufficient in step c) of the method to investigate the obtained sample for the presence of at least one of the proteins, mRNA molecules or fragments thereof which are identified by serial analysis of gene expression (SAGE) as being exclusively expressed in hairy or exclusively in hairless skin. In all other cases (e.g., genes which are differentially expressed in both tissue types), the amount of the differentially expressed molecules must also be investigated in step b), i.e. the expression must be quantified.
In step c) of the method for determining the homeostasis of hairy skin, the sample investigated in b) is designated as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments of proteins or mRNA molecules which are expressed at higher levels in hairy skin when compared to hairless skin, i.e. that the sample either comprises a greater number of molecules (e.g., proteins or mRNA) typically expressed in hairy skin as compared to those typically expressed in hairless skin (qualitative differentiation), or comprises more copies of molecules typically expressed in hairy skin than those typically present in hairless skin (quantitative differentiation). A complementary procedure is used to assign to diseased or homeostasis-impaired hairy skin.
A preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises determining whether at least one of the proteins, mRNA molecules or fragments of proteins or mRNA molecules which are defined by their UniGene accession number in column 7 in Tables 11 and 12 are present in the sample, and further comparing the expression quotients indicated in column 3 and column 5 in Tables 11 and 12. In step c) the sample is assigned to healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at approximately 1.9-fold higher levels in healthy hairy skin as compared to hairless skin. Alternatively the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or thereof which are expressed at approximately 1.9-fold higher levels in hairless skin as compared to hairy skin.
A further preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises determining the presence and, where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Tables 9 and 10 in the sample, and comparing the results with the expression quotients indicated in column 3 and column 5 in Tables 9 and 10. The sample is designated as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 3-fold higher in healthy hairy skin as compared to hairless skin. Alternatively, the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 3-fold higher in hairless skin as compared to hairy skin.
A further preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises assessing the sample for the presence and where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Tables 7 and 8, comparing the expression quotients indicated in column 3 and column 5 in Tables 7 and 8, and designating the sample as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 5-fold higher in healthy hairy skin when compared to hairless skin. Alternatively, the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 5-fold higher in hairless skin when compared to hairy skin.
A particularly preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises assessing the patient sample for the presence and where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Table 6, comparing the results with the expression quotients indicated in column 3 and column 5 in Table 6, and designating the sample as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at a level at least 1.9-fold higher in healthy hairy skin when compared to hairless skin. Alternatively, the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof proteins which are expressed at a level at least 1.9-fold higher in hairless skin when compared to hairy skin.
A further particularly preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises assessing the sample for the presence and where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Table 5, comparing the results with the expression quotients indicated in column 3 and column 5 in Table 5, and designating the sample as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at levels at least 3-fold higher in healthy hairy skin when compared to hairless skin. Alternatively, the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof proteins which are expressed at levels at least 3-fold higher in hairless skin when compared to hairy skin.
A further particularly preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises assessing the sample for the presence and where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Table 4, comparing the results with the expression quotients indicated in column 3 and column 5 in Table 4, and designating the sample as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 5-fold higher in healthy hairy skin when compared to hairless skin. Alternatively, the sample is designated as diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 5-fold higher in hairless skin when compared to hairy skin.
A very particularly preferred embodiment of the method of the invention for determining the homeostasis of hairy skin comprises assessing the sample for the presence and where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof as defined by their UniGene accession number in column 7 in Table 3, comparing the results with the expression quotients indicated in column 3 and column 5 in Table 3, and designating the sample as healthy hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 10-fold higher in healthy hairy skin when compared to hairless skin. Alternatively, the sample is designated diseased or homeostasis-impaired hairy skin if it comprises predominantly proteins, mRNA molecules or fragments thereof which are expressed at least 10-fold higher in hairless skin when compared to hairy skin.
The state of the skin can also be described by quantifying a plurality of expression products of genes important for hairy skin (e.g., markers) which must then be active among themselves in a characteristic ratio in order to represent healthy (homeostasis-undergoing) hairy skin, or must be active in the characteristic ratio differing therefrom in order to represent diseased (homeostasis-impaired) skin.
The third object of the present invention therefore further relates to yet another embodiment of a method for determining the homeostasis of hairy skin in humans, especially in women, in vitro.
An exemplary method entails
The mixture in step a) of the method of the invention for determining the homeostasis of hairy skin is preferably obtained from a skin sample, in particular from a full-thickness skin sample or from an epidermis sample. In this connection, the full-thickness skin sample facilitates a more comprehensive comparison if the SAGE libraries which are likewise obtained from full-thickness skin. The epidermis sample is, on the other hand, easier to obtain, for example by applying an adhesive tape to the skin and peeling it off, as described in WO 00/10579, which is incorporated herein by reference.
In a further embodiment of the method of the invention for determining the homeostasis of hairy skin, the mixture is obtained in step a) by microdialysis. The technique of microdialysis is described for example in “Microdialysis: A method for measurement of local tissue metabolism”, Nielsen P S, Winge K, Petersen L M; Ugeskr Laeger 1999 Mar. 22 161:12 1735-8; and in “Cutaneous microdialysis for human in vivo dermal absorption studies”, Anderson, C. et al.; Drugs Pharm. Sci., 1998, 91, 231-244; and also in the internet under www.microdialysis.se/techniqu which are incorporated herein by reference.
When microdialysis is used, typically a probe is introduced into the skin and a suitable carrier solution is used to rinse the probe slowly. After the acute reactions following the puncture have subsided, the microdialysis provides proteins, mRNA molecules or fragments of proteins or mRNA molecules which occur in the extracellular space and which can then be isolated, for example by fractionation of the carrier liquid, and analyzed in vitro. Microdialysis is less invasive than removal of a full-thickness skin sample; however, a disadvantage is that it is confined to obtaining molecules occurring in the extracellular space.
A further preferred embodiment of the methods of the second and third objects of the invention for determining the homeostasis of hairy skin comprises assessing the sample(s) for the presence and, where appropriate, the amount of at least one of the proteins or fragments thereof. Such determinations can be carried out by a method selected from
These methods which can be employed according to the invention are described in the review article by Akhilesh Pandey and Matthias Mann: “Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and the references indicated therein, which are incorporated herein by reference.
2D gel electrophoresis is described for example in L. D. Adams, Two-dimensional Gel Electrophoresis using the Isodalt System or in L. D. Adams & S. R. Gallagher, Two-dimensional Gel Electrophoresis using the O'Farrell System; both in Current Protocols in Molecular Biology (1997, Eds. F. M. Ausubel et al.), Unit 10.3.1-10.4.13; or in 2-D Electrophoresis Manual; T. Berkelman, T. Senstedt; Amersham Pharmacia Biotech, 1998 (Order No. 80-6429-60).
Characterization of the proteins or protein fragments by mass spectrometry takes place in a manner known to those skilled in the art, for example as described in the following references: Methods in Molecular Biology, 1999; Vol. 112; 2-D Proteome Analysis Protocols; Editor: A. J. Link; Humana Press; Totowa; N.J. Especially: Courchesne, P. L. and Patterson, S. D.; pp. 487-512 therein and Carr, S. A. and Annan, R. S.; 1997; in: Current Protocols in Molecular Biology; Editor: Ausubel, F. M. et al.; John Wiley and Sons, Inc. 10.2.1-10.21.27.
A further preferred embodiment of the methods of the second and third objects of the invention for determining the homeostasis of hairy skin comprises assessing the sample(s) for the presence and, where appropriate, the amount of at least one of mRNA molecule and/or fragments thereof. Such determinations can be carried out by a method selected from
These methods which can be employed according to the invention are described in the review articles by Akhilesh Pandey and Matthias Mann: “Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and “Genomics, gene expression and DNA arrays”, Nature, Volume 405, Number 6788, 827-836 (2000), and the references indicated therein, which are incorporated herein by reference.
The TOGA method is described in “J. Gregor Sutcliffe et al, TOGA: An automated parsing technology for analyzing expression of nearly all genes, Proceedings of the National Academy of Sciences of the United States of America (PNAS), Vol. 97, No. 5, pp. 1976-1981 (2000)”, which is incorporated herein by reference.
The MPSS® method is described in U.S. Pat. No. 6,013,445, which is incorporated herein by reference.
However, it is also possible according to the invention to employ other methods known to the skilled worker for investigating for the presence and, where appropriate, the amount of at least one of the proteins, mRNA molecules or fragments thereof in a sample.
A further preferred embodiment of the method of the invention for determining the homeostasis of hairy skin entails assessing the sample of step b) for the presence and, optionally, the amount of from 1 to about 5000, preferably from 1 to about 1000, in particular from about 10 to about 500, preferably from about 10 to about 250, particularly preferably from about 10 to about 100 and very particularly preferably from about 10 to about 50 of the proteins, mRNA molecules or fragments thereof which are defined by their UniGene accession number in column 7 in Tables 3 to 12.
The present invention further relates to a test kit for determining the homeostasis of hairy skin in humans in vitro, including means for carrying out the methods of the invention for determining the homeostasis of hairy skin.
The present invention also relates to a biochip for determining the homeostasis of hairy skin in humans in vitro, including
A biochip which is particularly preferred according to the invention includes probes which are selected from those capable of specific binding to at least one of the proteins, mRNA molecules or fragments thereof which are defined in column 1 in Tables 3 to 6 by the following SEQ ID NOS: 2, 4, 9, 12, 14, 16, 22, 25, 29, 31, 35, 36, 38, 39, 40, 42, 43, 44, 46, 59, 62, 63, 65, 67, 68, 69, 74.
A biochip is a miniaturized functional element having molecules, in particular biomolecules, which can act as specific interaction partners and are immobilized on a surface. The structure of these functional elements frequently comprises rows and columns; the term used is then chip arrays. Since thousands of biological or biochemical functional elements can be arranged on one chip, they must generally be fabricated by microengineering methods. Particularly suitable biological and biochemical functional elements are: DNA, RNA, PNA (nucleic acids and their chemical derivatives may be for example in the form of single strands, triplex structures or combinations thereof), saccharides, peptides, proteins (e.g. antibodies, antigens, receptors) and derivatives of combinatorial chemistry (e.g. organic molecules).
Biochips generally have a 2D basic area for coating with biologically or biochemically functional materials. The basic areas may for example also be formed by walls of one or more capillaries or by channels. Reference may be made to the following publications for example for the prior art: Nature Genetics, Vol. 21, supplement (complete), January 1999 (BioChips); Nature Biotechnology, Vol. 16, pp. 981-983, October 1998 (BioChips); Trends in Biotechnology, Vol. 16, pp. 301-306, July 1998 (BioChips) and the previously mentioned review articles by Akhilesh Pandey and Matthias Mann: “Proteomics to study genes and genomes”, Nature, Volume 405, Number 6788, 837-846 (2000), and “Genomics, gene expression and DNA arrays”, Nature, Volume 405, Number 6788, 827-836 (2000), and the references cited therein, which are incorporated herein by reference.
A clear description of the practical methods for using DNA chip technology is provided by the books “DNA Microarrays: A Practical Approach” (editor: Mark Schena, 1999, Oxford University Press) and “Microarray Biochip Technology” (editor: Mark Schena, 2000, Eaton Publishing), which are incorporated herein by reference.
The DNA chip technology which is particularly preferred for the purposes of the present invention is based on the ability of nucleic acids to enter into complementary base pairings. This technical principle, which is referred to as hybridization, has been employed for some years in Southern blotting and Northern blotting analyses. Compared with these conventional methods, in which only a few genes are analyzed, DNA chip technology permits parallel investigation of some hundreds and up to several ten thousands of genes. A DNA chip consists essentially of a support material (e.g. glass or plastic) on which single-stranded, gene-specific probes are immobilized in high density at a defined site (spot). Problems associated therewith are assessed as being the technique of probe application and the chemistry of probe immobilization.
In the current state of the art, several ways for immobilizing probes are implemented:
E. M. Southern (E. M. Southern et al (1992), Nucleic Acid Research 20, 1679-1684 and E. M. Southern et al. (1997), Nucleic Acid Research 25, 1155-1161) describes the production of oligonucleotide arrays by direct synthesis on a glass surface which has been derivatized with 3-glycidoxypropyltrimethoxysilane and then with a glycol.
A similar method achieves the in situ synthesis of oligonucleotides by means of a photosensitive, combinatorial chemistry which can be compared with photolithographic techniques (Pease, A. C. et al. (1994), Proc. Natl. Acad Sci USA 91, 5022-5026).
Besides these techniques which are based on the in situ synthesis of oligonucleotides, it is likewise possible for already present DNA molecules to be bound to surfaces of support material.
P. O. Brown (DeRisi et al. (1997), Science 278, 680-686) describes the immobilization of DNA on polylysine-coated glass surfaces.
The publication by L. M. Smith (Guo, Z. et al. (1994), Nucleic Acid Research 22, 5456-5465) discloses a similar method: oligonucleotides having a 5′ terminal amino group can be bound to a glass surface which has been treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyl diisothiocyanate.
The DNA probes can be applied to a support with a so-called pin spotter. For this purpose, thin metal needles with, for example, a diameter of 250 μm are dipped into probe solutions and then transfer the pendant sample material with defined volumes to the support material of the DNA chip.
The probes are preferably applied by means of a piezo-controlled nanodispenser which, similar to an inkjet printer, applies probe solutions with a volume of 100 picoliters without contact to the surface of the support material.
The probes are immobilized for example as described in EP-A-0 965 647. In this case, DNA probes are generated by PCR using a sequence-specific primer pair, one primer being modified at the 5′ end and having a linker with a free amino group. This ensures that a defined strand of the PCR products can be bound to a glass surface which has been treated with 3-aminopropyltrimethoxysilane and then with 1,4-phenyl diisothiocyanate. The gene-specific PCR products should ideally have a defined nucleic acid sequence in a length of 200-400 bp and not include redundant sequences. After the immobilization of the PCR products via the derivatized primer, the complementary strand of the PCR product is removed by incubation at 96° C. for 10 min.
In a typical application for DNA chips, mRNA is isolated from the two cell populations to be compared. The isolated mRNAs are converted into cDNA by reverse transcription using, for example, fluorescence-labeled nucleotides. In this case, the samples to be compared are labeled with, for example, red or green fluorescent nucleotides. The cDNAs are then hybridized with gene probes immobilized on the DNA chip and subsequently the bound fluorescences are quantified.
The analysis chips mentioned in DE-A-100 28 257.1-52 and in DE-A-101 02 063.5-52 are very particularly preferred for producing small biochips (including up to about 500 probes). These analysis chips have an electrically addressable structure which allows electrofocusing of the samples. This advantageously makes it possible for samples to be focused and immobilized irrespective of their viscosity with the aid of electrodes at defined points in a grid of points (array). The focusability simultaneously results in an increase in the local concentration of the samples and thus a higher specificity. During the analysis itself it is possible to address the test material on the individual positions of the array. It is thus potentially possible for all the information investigated to be traced with the highest possible sensitivity. Cross-contamination by adjacent spots is virtually precluded.
The biochip of the invention preferably includes from 1 to about 5000, more preferably from 1 to about 1000, in particular from about 10 to about 500, preferably from about 10 to about 250, particularly preferably from about 10 to about 100 and very particularly preferably from about 10 to about 50 mutually different probes. The mutually different probes may in each case be present in more than one copy on the chip.
The biochip of the invention preferably includes nucleic acid probes, especially RNA or PNA probes, particularly preferably DNA probes. The nucleic acid probes preferably have a length from about 10 to about 1000, in particular from about 10 to about 800, preferably from about 100 to about 600, particularly preferably from about 200 to about 400 nucleotides.
In a further preferred form, the biochip of the invention includes peptide or protein probes, in particular antibodies.
The present invention further relates to the use of the proteins, mRNA molecules or fragments thereof defined by their UniGene accession number in column 7 in Tables 3 to 12 as markers of hairy skin in humans.
In this connection, some of the genes and gene products listed in Tables 3 to 12 are of particular interest, for example the protein CDT6, whose role as a hair cycle marker is particularly surprising. It was anticipated that VEGF or angiopoietin would be more appropriate markers in this regard. The same applies to proteins of the 14-3-3 family. Although these are expressed ubiquitously, it was not easy to predict which isoforms are involved in the hair cycle, nor which expression pattern these isoforms will possess.
In the overrepresented groups, especially the DPPIV family and the DNA helicases are noteworthy. The skilled worker would have been unable to predict the involvement of either of these gene families in the hair cycle.
Mention should also be made of the genes of melanin biosynthesis. It is unexpected and surprising that the genes of melanin biosynthesis are more strongly expressed in the catagenic than in the anagenic hair follicle.
The present invention further relates to a test method for detecting the efficacy of cosmetic or pharmaceutical active ingredients for disorders or impairments of homeostasis of hairy skin in vitro. An exemplary method entails
The test method of the invention can be carried out with full-thickness skin samples, hairy skin equivalents, isolated hair follicles, hair follicle equivalents or cells of hairy skin.
The present invention further relates to a test kit for detecting the efficacy of cosmetic or pharmaceutically active ingredients for disorders or impairments of homeostasis of hairy skin, including means for carrying out the test method of the invention.
The present invention further relates to the use of the proteins, mRNA molecules or fragments thereof which are defined by their UniGene accession number in column 7 in Tables 3 to 12 for detecting the efficacy of cosmetic or pharmaceutical active ingredients for disorders or impairments of homeostasis of hairy skin.
The present invention further relates to a screening method for identifying cosmetic or pharmaceutically active ingredients for disorders or impairments of homeostasis of hairy skin in vitro. An exemplary method entails
The present invention further relates to the use of the proteins, mRNA molecules or fragments thereof which are defined by their UniGene accession number in column 7 in Tables 3 to 12 for identifying cosmetic or pharmaceutical active ingredients for disorders or impairments of homeostasis of hairy skin.
The present invention further relates to a method for producing a cosmetic or pharmaceutical preparation for disorders or impairments of homeostasis of hairy skin. An exemplary method entails
Tables:
H. sapiens mRNA; cDNA
H. sapiens mRNA; cDNA
H. sapiens mRNA; cDNA
Homo sapiens cDNA:
Homo sapiens mRNA;
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
C. elega
Homo sapiens cDNA:
Homo sapiens cDNA:
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens,
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens cDNA
Homo sapiens mRNA;
Homo sapiens cDNA:
Homo sapiens, clone
Homo sapiens cDNA:
Homo sapiens mRNA;
Homo sapiens cDNA:
Homo sapiens,
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Home sapiens
Home sapiens
Homo sapiens
Home sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens,
Homo sapiens
Homo sapiens
Homo sapiens,
| Number | Date | Country | Kind |
|---|---|---|---|
| 102 60 931.4 | Dec 2002 | DE | national |
This application is a §365 (c) continuation application of PCT/EP2003/014070 filed 11 Dec. 2003, which in turn claims priority to DE Application 102 60 931.4 filed 20 Dec. 2002, each of the foregoing applications is incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP03/14070 | Dec 2003 | US |
| Child | 11158209 | Jun 2005 | US |
