Method for determining the homeostasis of hairy skin

• US 20060088852A1
• Filed: 06/20/2005
• Published: 04/27/2006
• Est. Priority Date: 12/20/2002
• Status: Abandoned Application

• Current Assignee: Henkel KGaA (Henkel AG & Co KGaA)
• Original Assignee: Henkel KGaA (Henkel AG & Co KGaA)
• Inventors: Schlotmann, Kordula, Gassenmeier, Thomas, Hofmann, Kay, Petersohn, Dirk, Conradt, Marcus, Holtkoetter, Olaf
• Application Number:
  US11/158,209
• Publication Number:
  US 20060088852A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/148 Screening for cosmetic compounds
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The invention relates to a method for determining the homeostasis of hairy skin in vitro, to test kits and biochips for determining markers of hairy skin, in addition to the use of proteins, mRNA molecules or fragments thereof as markers of hairy skin. The invention also relates to a test method for detecting the effectiveness of cosmetic or pharmaceutical active substances for treating hairy skin in addition to a screening method for identifying cosmetic or pharmaceutical active substances for treating hairy skin and to a method for producing a cosmetic or pharmaceutical preparation for treating hairy skin.

Claims

1. An in vitro method for identifying genetically encoded molecules which are differentially expressed in hairy skin when compared to hairless skin, comprising:

a) providing a first sample of human hairy skin comprising genetically encoded molecules which are transcribed and optionally translated in said hairy skin;

b) providing a second sample of human hairless skin comprising genetically encoded molecules which are transcribed and, optionally translated in said hairless skin; and

c) subjecting said first and second samples to a serial analysis of gene expression (SAGE), thereby identifying genetically encoded molecules which are differentially expressed in hairy as compared to hairless skin.

2. The method as claimed in claim 1, wherein said first sample is obtained from hairy scalp and said second sample is obtained from hairless facial skin.

3. The method of claim 1, wherein said genetically encoded molecules are selected from the group consisting of at least one mRNA molecule, at least one protein or polypeptide, or fragments thereof,

4. The method of claim 3, wherein said encoded molecule is translated and is selected from the group consisting of a protein, a polypeptide or a fragment of either, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. one- or two-dimensional gel electrophoresis

ii. affinity chromatography

iii. protein-protein complexation in solution

iv. mass spectrometry, especially matrix-assisted laser desorption ionization (MALDI) and,

v. use of protein chips.

5. The method of claim 3, wherein said encoded molecule is an mRNA molecule or fragment thereof, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. Northern blots

ii. reverse transcriptase polymerase chain reaction (RT-PCR),

iii. RNase protection experiments,

iv. dot blots,

v. cDNA sequencing,

vi. clone hybridization,

vii. differential display,

viii. subtractive hybridization,

ix. cDNA fragment fingerprinting,

x. total gene expression analysis (TOGA),

xi. serial analysis of gene expression (SAGE), and

xii. massively parallel signature sequencing (MPSS®

) and,

xiii. use of nucleic acid chips.

6. An in vitro method for determining the homeostasis of hairy skin in humans, said method comprising:

a) performing the method of claim 1, thereby identifying a population of hairy skin associated genetically encoded molecules

b) obtaining a hairy skin sample from a patient and determining the presence and optionally the amount of hairy skin associated genetically encoded molecules present in the sample and

c) designating the sample of b) as

i) healthy or homeostasis-undergoing hairy skin if it comprises molecules which are expressed at higher levels in hairy skin relative to hairless skin, or

ii) diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at higher levels in hairless skin relative to hairy skin.

7. The method of claim 6, wherein said genetically encoded molecules are selected from the group consisting of at least one mRNA molecule, at least one protein or polypeptide, or fragments thereof

8. The method as claimed in claim 6, comprising:

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Tables 11 and 12;

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Tables 11 and 12, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 1.9-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 1.9-fold higher in hairless skin when compared to hairy skin.

9. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Tables 9 and 10,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Tables 9 and 10, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 3-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 3-fold higher in hairless skin when compared to hairy skin.

10. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Tables 7 and 8,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Tables 7 and 8, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 5-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 5-fold higher in hairless skin when compared to hairy skin.

11. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Table 6,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Table 6, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 1.9-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 1.9-fold higher in hairless skin when compared to hairy skin.

12. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Table 5,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Table 5, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 3-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 3-fold higher in hairless skin when compared to hairy skin.

13. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Table 4,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Table 4, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 5-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 5-fold higher in hairless skin when compared to hairy skin.

14. The method as claimed in claim 6, comprising

a) determining the presence and optionally the amount of at least one of the genetically encoded molecules or fragments thereof in said patient sample, said molecule having a UniGene accession number set forth in column 7 of Table 3,

b) comparing the results from a) with the expression quotients set forth in column 3 and column 5 in Table 3, and

c) designating the patient sample as healthy hairy skin if it comprises molecules which are expressed at levels at least 10-fold higher in healthy hairy skin when compared to hairless skin, or designating the patient sample as diseased or homeostasis-impaired hairy skin if it comprises molecules which are expressed at levels at least 10-fold higher in hairless skin when compared to hairy skin.

15. The method of claim 6, wherein said encoded molecule is translated and is selected from the group consisting of a protein, a polypeptide or a fragment of either, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. one- or two-dimensional gel electrophoresis

ii. affinity chromatography

iii. protein-protein complexation in solution

iv. mass spectrometry, especially matrix-assisted laser desorption ionization (MALDI) and,

v. use of protein chips.

16. The method of claim 6, wherein said encoded molecule is an mRNA molecule or fragment thereof, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. Northern blots

ii. reverse transcriptase polymerase chain reaction (RT-PCR),

iii. RNase protection experiments,

iv. dot blots,

v. cDNA sequencing,

vi. clone hybridization,

vii. differential display,

viii. subtractive hybridization,

ix. cDNA fragment fingerprinting,

x. total gene expression analysis (TOGA),

xi. serial analysis of gene expression (SAGE), and

xii. massively parallel signature sequencing (MPSS®

) and,

xiii. use of nucleic acid chip.

17. An in vitro method for determining the homeostasis of hairy skin in humans, comprising:

a) obtaining a sample of genetically encoded molecules or fragments thereof from hairy human skin,

b) quantifying at least two of the genetically encoded hairy skin associated molecules or fragments thereof present in the sample, said hairy skin associated molecules or fragments thereof being identified by the method of claim 1,

c) determining the expression ratios of the at least two genetically encoded hairy skin associated molecules, thereby generating an expression quotient,

d) comparing the expression quotients from c) with the expression quotients provided in at least one table selected from the group consisting of Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11 and Table 12 and

e) designating the patient sample as healthy hairy skin if the expression ratios of the sample correspond to the expression ratios in present in hairy skin, or designating the sample as diseased skin if the expression ratios of the patient sample correspond to the expression ratios of hairless skin.

18. The method of claim 17, wherein said encoded molecule is translated and is selected from the group consisting of a protein, a polypeptide or a fragment of either, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. one- or two-dimensional gel electrophoresis

ii. affinity chromatography

iii. protein-protein complexation in solution

iv. mass spectrometry, especially matrix-assisted laser desorption ionization (MALDI) and,

v. use of protein chips.

19. The method of claim 17, wherein said encoded molecule is an mRNA molecule or fragment thereof, said molecule being characterized and optionally quantitated by at least one method selected from the group consisting of

i. Northern blots

ii. reverse transcriptase polymerase chain reaction (RT-PCR),

iii. RNase protection experiments,

iv. dot blots,

v. cDNA sequencing,

vi. clone hybridization,

vii. differential display,

viii. subtractive hybridization,

ix. cDNA fragment fingerprinting,

x. total gene expression analysis (TOGA),

xi. serial analysis of gene expression (SAGE), and

xii. massively parallel signature sequencing (MPSS®

) and,

xiii. use of nucleic acid chips.

20. The method as claimed in claim 1, comprising detecting and optionally quantitating from about 1 to about 5000 genetically encoded molecules which are defined by their UniGene accession number in column 7 in a Table selected from the group consisting of Tables 3 to 12.

21. A test kit for determining the homeostasis of hairy skin in humans in vitro, said test kit comprising reagents suitable for carrying out the method of claim 1.

22. The test kit of claim 21 further comprising reagents suitable for performing at least one of the methods of claim 4.

23. The test kit of claim 21, further comprising reagents suitable for performing at least one of the methods of claim 5.

24. A biochip for determining the homeostasis of hairy skin in humans in vitro, including

i. a solid, i.e. rigid or flexible support and

ii. probes immobilized thereon which are capable of specific binding to at least one of the proteins, mRNA molecules or fragments thereof defined by their UniGene accession number in column 7 in a Table selected from the group of Tables 3 to 12.

25. The biochip of claim 24, comprising probes having at least one sequence selected from the group consisting of 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, and 74.

26. The biochip as claimed in claim 24, including from about 1 to about 5000, different probes.

27. The biochip as claimed in claim 24, wherein said probes are selected from the group consisting of DNA probes, RNA probes, and PNA probes.

28. The biochip as claimed in claim 24, including probes with a length of about 10 to about 1000 nucleotides.

29. The biochip as claimed in claim 24, wherein said probes are selected from the group consisting of peptide, protein and antibody probes.

30. A test method for determining the efficacy of cosmetic or pharmaceutical active ingredients for disorders or impairments of homeostasis of hairy skin in vitro, said method comprising

a) obtaining a hairy skin sample and determining the status of said skin;

b) contacting said skin sample with an active ingredient for disorders or impairments of homeostasis of hairy skin at least once,

c) determining the skin status of hairy skin following step b) and

d) comparing the skin status results from a) and c), agents which improve said skin status being efficacious for disorders or impairments of homeostasis of hairy skin.

31. The method of claim 30, further comprising formulating said efficacious agent into a cosmetic or pharmaceutically acceptable carrier.

32. A test kit for determining the efficacy of cosmetic or pharmaceutical active ingredients for disorders or impairments of homeostasis of hairy skin, including reagents suitable for performing the method of claim 30.

33. The test kit of claim 32, comprising a biochip.