US20010036642A1 - Antibodies specific for papillary fibroblasts as markers for skin quality - Google Patents

Antibodies specific for papillary fibroblasts as markers for skin quality Download PDF

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Publication number
US20010036642A1
US20010036642A1 US09/725,269 US72526900A US2001036642A1 US 20010036642 A1 US20010036642 A1 US 20010036642A1 US 72526900 A US72526900 A US 72526900A US 2001036642 A1 US2001036642 A1 US 2001036642A1
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skin
antibody
papillary
fibroblasts
dermis
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US09/725,269
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Daniel Asselineau
Arnold Caplan
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LOreal SA
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LOreal SA
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Assigned to SOCIETE L'OREAL S.A. reassignment SOCIETE L'OREAL S.A. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CAPLAN, ARNOLD, ASSELINEAU, DANIEL
Publication of US20010036642A1 publication Critical patent/US20010036642A1/en
Priority to US10/619,188 priority Critical patent/US7192719B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6881—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from skin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum
    • G01N2400/40—Glycosaminoglycans, i.e. GAG or mucopolysaccharides, e.g. chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparin, heparan sulfate, and related sulfated polysaccharides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/20—Dermatological disorders
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/971—Capture of complex after antigen-antibody reaction

Definitions

  • the present invention relates to the use of at least one antibody specific for papillary fibroblasts as a marker for the quality of skin, in particular of a skin equivalent.
  • human skin consists of two closely linked compartments or strata, namely, the epidermis and the dermis.
  • the epidermis is principally comprised of three cell types, keratinocytes, which themselves constitute the majority of the cells of the epidermis, melanocytes and Langerhans cells. These cells constitute a keratinized epithelium which is differentiated into superposed layers or strata surmounted by a layer of dead cells forming the stratum corneum.
  • the dermis provides the epidermis with a solid support. It is also the nourishing element of the epidermis. It principally comprises fibroblasts and an extracellular matrix which is itself principally collagen, elastin and a substance known as “ground substance”. This set of extracellular components is synthesized by the fibroblasts. Leukocytes, mastocytes and tissue macrophages also are present therein. Too, it also comprises blood vessels and nerve fibers. In normal skin, i.e., skin which is neither pathological nor cicatricial, the fibroblasts are in the quiescent state, i.e., non-proliferative, relatively inactive in metabolic terms and immobile.
  • the dermis is subdivided into two regions; firstly, a thin superficial dermis, termed papillary dermis, and secondly, the deep dermis, termed reticular dermis, which constitutes the great majority of the dermis.
  • the papillary dermis is the part of the dermis which is in contact with the epidermis, and it contains so-called papillary fibroblasts.
  • the reticular dermis is the region of the dermis which then extends down to the subcutaneous fatty layer, and it contains the reticular fibroblasts. In normal skin, these two regions reflect significant differences.
  • the papillary dermis is metabolically more active than the reticular dermis.
  • Papillary and reticular fibroblasts in culture exhibit differences in their growth potential. With immunolabelling, it is possible to demonstrate that decorin, small-sized dermatan sulfate proteoglycan (DSPG), is more abundant in the papillary dermis than in the reticular dermis. Papillary fibroblasts secrete up to approximately 6 times more decorin than reticular fibroblasts.
  • DSPG small-sized dermatan sulfate proteoglycan
  • the dermis comprises of at least two fibroblast populations, which can only have fundamental consequences on the skin itself.
  • papillary fibroblasts express a specific epitope which is not present, or present in only fractional amounts, in reticular fibroblasts.
  • the present invention features utilization of antibodies, in particular monoclonal antibodies, specific for this epitope, to label this particular population of dermal fibroblasts. Accordingly, using this antibody, it can be determined whether a skin equivalent has the two papillary and reticular fibroblast populations.
  • this invention features the use of at least one antibody specific for papillary fibroblasts as a marker for the quality of skin, particularly of a skin equivalent.
  • FIGURE of Drawing is a photomicrograph of a section of normal human skin immunolabelled with the PG4 mouse monoclonal antibody.
  • marker for the quality any marker which effectively indicates the presence in skin or in a skin equivalent of a biological element which is present in normal skin.
  • markers any element for which the presence, the absence, the modification of expression or the modification of distribution can be measured.
  • exemplary markers include epitopes, nucleic acids (ribonucleic or deoxyribonucleic acid), antibodies, proteins or a group of proteins which may or may not be linked, ions, cellular organelles, lipids or polysaccharides.
  • the marker is an antibody.
  • the antibody can be a polyclonal or monoclonal antibody. Preferably, the antibody is monoclonal.
  • the antibody can be an antibody originating from any origin, i.e., derived from any animal such as, for example, horses, goats, mice, rats or rabbits.
  • the antibody is a mouse antibody.
  • the antibody is a mouse monoclonal antibody.
  • a preferred antibody according to the invention is the antibody referred to under the designation PG4, described in the publication by Sorrell et al. ( The Histochemical Journal, 31, 549-558, 1999).
  • This mouse monoclonal antibody is described as recognizing in skin at least one epitope specific for glycosaminoglycans, and particularly described as an anti-chondroitin sulfate (CS) and anti-dermatan sulfate (DS) monoclonal antibody.
  • CS chondroitin sulfate
  • DS anti-dermatan sulfate
  • the present invention features the use of the PG4 monoclonal antibody as a marker for papillary fibroblasts, particularly for papillary fibroblasts of skin, very particularly papillary fibroblasts of the dermis.
  • This invention also features the use of the PG4 monoclonal antibody as a marker for the quality of skin, particularly of skin equivalents, in particular of dermis equivalents obtained in vitro.
  • Any immunological labelling technique which employs at least one known antibody of the prior art can be used to carry out the labelling with the antibodies of the invention.
  • representative is the method described by Asselineau et al. ( J.I.D., 86, 181-186, 1986), or, alternatively, by Sorrell et al. ( The Histochemical Journal, 31, 549-558, 1999).
  • the present invention thus also features a method for determining the quality of skin, particularly of a skin equivalent obtained in vitro, comprising carrying out immunological labelling on the skin and/or the skin equivalent employing at least one antibody specific for papillary fibroblasts, particularly the PG4 antibody.
  • FIGURE of Drawing illustrates the invention more clearly, without limiting the scope thereof.
  • the photograph is of a section of normal human skin after immunolabelling performed by indirect immunofluorescence using the PG4 monoclonal antibody, with propidium iodide counterstaining of the cell nuclei.
  • the presence of intense labelling (light grey zone) of the upper dermis at the level of the epidermis (recognizable by the many cell nuclei labelled with propidium iodide) is noted, demonstrating the presence in this zone of papillary fibroblasts which express the epitope recognized specifically by the PG4 monoclonal antibody.
  • mice conjugate 20 ml per section of an antibody directed towards mouse antibodies (mouse conjugate), obtained from the company Dako, were then placed on the sections and the sections were incubated according to the manufacturer's recommendation. After rinsing, the sections were contacted with a solution of PBS containing 0.5% propidium iodide, and then rinsed with PBS and mounted for observation under a fluorescence microscope, in order to stain the cell nuclei.

Abstract

Whether or not a sample of skin or of a skin equivalent contains such amount of a papillary fibroblast population as to be considered a normal skin is determined by labelling said skin or skin equivalent with at least one antibody specific for papillary fibroblasts and evaluating the extent of such labelling as a marker for skin or skin equivalent quality.

Description

    CROSS-REFERENCE TO PRIORITY APPLICATION
  • This application claims priority under 35 U.S.C. §119 of FR-99/15292, filed Dec. 3, 1999, hereby expressly incorporated by reference.[0001]
    • BACKGROUND OF THE INVENTION
  • 1. Technical Field of the Invention [0002]
  • The present invention relates to the use of at least one antibody specific for papillary fibroblasts as a marker for the quality of skin, in particular of a skin equivalent. [0003]
  • 2. Description of the Prior Art [0004]
  • It is of course well known that human skin consists of two closely linked compartments or strata, namely, the epidermis and the dermis. [0005]
  • The epidermis is principally comprised of three cell types, keratinocytes, which themselves constitute the majority of the cells of the epidermis, melanocytes and Langerhans cells. These cells constitute a keratinized epithelium which is differentiated into superposed layers or strata surmounted by a layer of dead cells forming the stratum corneum. [0006]
  • The dermis provides the epidermis with a solid support. It is also the nourishing element of the epidermis. It principally comprises fibroblasts and an extracellular matrix which is itself principally collagen, elastin and a substance known as “ground substance”. This set of extracellular components is synthesized by the fibroblasts. Leukocytes, mastocytes and tissue macrophages also are present therein. Too, it also comprises blood vessels and nerve fibers. In normal skin, i.e., skin which is neither pathological nor cicatricial, the fibroblasts are in the quiescent state, i.e., non-proliferative, relatively inactive in metabolic terms and immobile. [0007]
  • Indeed, the dermis is subdivided into two regions; firstly, a thin superficial dermis, termed papillary dermis, and secondly, the deep dermis, termed reticular dermis, which constitutes the great majority of the dermis. [0008]
  • The papillary dermis is the part of the dermis which is in contact with the epidermis, and it contains so-called papillary fibroblasts. [0009]
  • The reticular dermis is the region of the dermis which then extends down to the subcutaneous fatty layer, and it contains the reticular fibroblasts. In normal skin, these two regions reflect significant differences. The papillary dermis is metabolically more active than the reticular dermis. [0010]
  • Papillary and reticular fibroblasts in culture exhibit differences in their growth potential. With immunolabelling, it is possible to demonstrate that decorin, small-sized dermatan sulfate proteoglycan (DSPG), is more abundant in the papillary dermis than in the reticular dermis. Papillary fibroblasts secrete up to approximately 6 times more decorin than reticular fibroblasts. [0011]
  • Thus, in normal skin, the dermis comprises of at least two fibroblast populations, which can only have fundamental consequences on the skin itself. [0012]
  • In the domain of skin equivalents (or skin reconstructed in vitro), it is known to prepare dermis equivalents with each of the fibroblast populations isolated beforehand. It is also known to prepare dermis equivalents into which the two populations isolated beforehand are introduced. However, the problem remains of identifying the various fibroblast populations in dermis equivalents reconstructed from a random population of fibroblasts. After establishing in culture the dermis equivalent, does the latter have at least the two fibroblast populations, reticular and papillary, which are present in the dermis of normal skin? Now, it was previously recognized that normal skin has these two populations, and it is understood that skin reconstructed in vitro will be all the more similar to normal skin when it includes at least the two fibroblast populations. [0013]
  • To date, to applicants' knowledge, there exists no simple and effective means, particularly a means which does not impair the skin equivalent, which makes it possible to establish without ambiguity whether reconstructed skin either does or does not contain at least the two papillary and reticular fibroblast populations. [0014]
    • SUMMARY OF THE INVENTION
  • It has now surprisingly and unexpectedly been determined that papillary fibroblasts express a specific epitope which is not present, or present in only fractional amounts, in reticular fibroblasts. Thus, the present invention features utilization of antibodies, in particular monoclonal antibodies, specific for this epitope, to label this particular population of dermal fibroblasts. Accordingly, using this antibody, it can be determined whether a skin equivalent has the two papillary and reticular fibroblast populations. [0015]
  • Too, this invention features the use of at least one antibody specific for papillary fibroblasts as a marker for the quality of skin, particularly of a skin equivalent.[0016]
    • BRIEF DESCRIPTION OF THE DRAWING
  • The FIGURE of Drawing is a photomicrograph of a section of normal human skin immunolabelled with the PG4 mouse monoclonal antibody. [0017]
    • DETAILED DESCRIPTION OF BEST MODE AND SPECIFIC/PREFERRED EMBODIMENTS OF THE INVENTION
  • More particularly, according to the present invention, by the expression “marker for the quality” is intended any marker which effectively indicates the presence in skin or in a skin equivalent of a biological element which is present in normal skin. [0018]
  • Consistent herewith, by the term “marker” is intended any element for which the presence, the absence, the modification of expression or the modification of distribution can be measured. Exemplary markers include epitopes, nucleic acids (ribonucleic or deoxyribonucleic acid), antibodies, proteins or a group of proteins which may or may not be linked, ions, cellular organelles, lipids or polysaccharides. According to this invention, the marker is an antibody. [0019]
  • The antibody can be a polyclonal or monoclonal antibody. Preferably, the antibody is monoclonal. [0020]
  • The antibody can be an antibody originating from any origin, i.e., derived from any animal such as, for example, horses, goats, mice, rats or rabbits. Preferably, the antibody is a mouse antibody. Even more preferably, the antibody is a mouse monoclonal antibody. [0021]
  • A preferred antibody according to the invention is the antibody referred to under the designation PG4, described in the publication by Sorrell et al. ([0022] The Histochemical Journal, 31, 549-558, 1999). This mouse monoclonal antibody is described as recognizing in skin at least one epitope specific for glycosaminoglycans, and particularly described as an anti-chondroitin sulfate (CS) and anti-dermatan sulfate (DS) monoclonal antibody. To date, this antibody has not been described as being specific for a particular population of dermal fibroblasts, namely, papillary fibroblasts.
  • Thus, the present invention features the use of the PG4 monoclonal antibody as a marker for papillary fibroblasts, particularly for papillary fibroblasts of skin, very particularly papillary fibroblasts of the dermis. [0023]
  • This invention also features the use of the PG4 monoclonal antibody as a marker for the quality of skin, particularly of skin equivalents, in particular of dermis equivalents obtained in vitro. [0024]
  • Any immunological labelling technique which employs at least one known antibody of the prior art can be used to carry out the labelling with the antibodies of the invention. In this respect, representative is the method described by Asselineau et al. ([0025] J.I.D., 86, 181-186, 1986), or, alternatively, by Sorrell et al. (The Histochemical Journal, 31, 549-558, 1999).
  • The present invention thus also features a method for determining the quality of skin, particularly of a skin equivalent obtained in vitro, comprising carrying out immunological labelling on the skin and/or the skin equivalent employing at least one antibody specific for papillary fibroblasts, particularly the PG4 antibody. [0026]
  • The accompanying FIGURE of Drawing illustrates the invention more clearly, without limiting the scope thereof. In this FIGURE, the photograph is of a section of normal human skin after immunolabelling performed by indirect immunofluorescence using the PG4 monoclonal antibody, with propidium iodide counterstaining of the cell nuclei. The presence of intense labelling (light grey zone) of the upper dermis at the level of the epidermis (recognizable by the many cell nuclei labelled with propidium iodide) is noted, demonstrating the presence in this zone of papillary fibroblasts which express the epitope recognized specifically by the PG4 monoclonal antibody. [0027]
  • In order to further illustrate the present invention and the advantages thereof, the following specific example is given, it being understood that same is intended only as illustrative and in nowise limitative. [0028]
    • EXAMPLE Immunological Labelling of Papillary Fibroblasts in Normal Skin
  • Samples of normal skin derived from plastic surgery were embedded in Tissue-Tek, frozen in liquid nitrogen and stored in a freezer at −80° C. 4-micron-thick sections were prepared on a cryostat according to standard techniques. Labelling was performed using a conventional indirect immunofluorescence labelling technique (see Asselineau et al., [0029] J.I.D., 86, 181-186, 1986) with 20 ml per section of the PG4 monoclonal antibody in pure state (culture supernatant) (see Sorrell et al., The Histochemical Journal, 31, 549-558, 1999). 20 ml per section of an antibody directed towards mouse antibodies (mouse conjugate), obtained from the company Dako, were then placed on the sections and the sections were incubated according to the manufacturer's recommendation. After rinsing, the sections were contacted with a solution of PBS containing 0.5% propidium iodide, and then rinsed with PBS and mounted for observation under a fluorescence microscope, in order to stain the cell nuclei.
  • The presence of intense labelling of the dermis at the level of the epidermis was noted, demonstrating the presence in this zone of papillary fibroblasts expressing the epitope recognized specifically by the PG4 monoclonal antibody. [0030]
  • While the invention has been described in terms of various specific and preferred embodiments, the skilled artisan will appreciate that various modifications, substitutions, omissions, and changes may be made without departing from the spirit thereof. Accordingly, it is intended that the scope of the present invention be limited solely by the scope of the following claims, including equivalents thereof. [0031]

Claims (8)

What is claimed is:
1. A regime for determining whether or not a sample of skin or of a skin equivalent contains such amount of a papillary fibroblast population as to be considered a normal skin, comprising labelling said skin or skin equivalent with at least one antibody specific for papillary fibroblasts and evaluating the extent of such labelling as a marker for skin or skin equivalent quality.
2. The determinative regime as defined by
claim 1
, said at least one antibody comprising a monoclonal antibody.
3. The determinative regime as defined by
claim 1
, said at least one antibody comprising a horse, goat, mouse, rat or rabbit antibody.
4. The determinative regime as defined by
claim 3
, said at least one antibody comprising a mouse antibody.
5. The determinative regime as defined by
claim 1
, said at least one antibody comprising the PG4 antibody.
6. The determinative regime as defined by
claim 1
, comprising immunolabelling said skin or skin equivalent with at least one antibody specific for papillary fibroblasts.
7. A regime for determining the presence of papillary fibroblasts in a given sample, comprising labelling said sample with at least one antibody specific for papillary fibroblasts and evaluating the intensity of any such labelling as a marker for the presence of said papillary fibroblasts.
8. The determinative regime as defined by
claim 7
, said at least one antibody comprising the PG4 monoclonal antibody.
US09/725,269 1999-12-03 2000-11-29 Antibodies specific for papillary fibroblasts as markers for skin quality Abandoned US20010036642A1 (en)

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FR9915292A FR2801979B1 (en) 1999-12-03 1999-12-03 USE OF A SPECIFIC ANTIBODY OF PAPILLARY FIBROBLASTS AS A QUALITY MARKER FOR SKIN
FR99/15292 1999-12-03

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030059414A1 (en) * 2001-09-21 2003-03-27 Ho Tony W. Cell populations which co-express CD49c and CD90
US20070224177A1 (en) * 2002-09-20 2007-09-27 Ho Tony W Cell populations which co-express CD49c and CD90
US20090053183A1 (en) * 2007-06-15 2009-02-26 Neuronyx Inc. Treatment of Diseases and Disorders Using Self-Renewing Colony Forming Cells Cultured and Expanded In Vitro

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6042904B2 (en) * 2011-11-25 2016-12-14 シャネル パフュームズ ビューテ Novel markers of papillary and reticulated fibroblasts and uses thereof

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030059414A1 (en) * 2001-09-21 2003-03-27 Ho Tony W. Cell populations which co-express CD49c and CD90
US20050233452A1 (en) * 2001-09-21 2005-10-20 Neuronyx, Inc. Cell populations which co-express CD49c and CD90
US20070231309A1 (en) * 2001-09-21 2007-10-04 Neuronyx, Inc. Cell populations which co-express CD49c and CD90
US20070264232A1 (en) * 2001-09-21 2007-11-15 Neuronyx, Inc. Cell populations which co-express CD49c and CD90
US8486696B2 (en) 2001-09-21 2013-07-16 Garnet Biotherapeutics, Inc. Cell populations which co-express CD49c and CD90
US9969980B2 (en) 2001-09-21 2018-05-15 Garnet Biotherapeutics Cell populations which co-express CD49c and CD90
US10351826B2 (en) 2001-09-21 2019-07-16 Garnet Biotherapeutics, Inc. Cell populations which co-express CD49c and CD90
US20070224177A1 (en) * 2002-09-20 2007-09-27 Ho Tony W Cell populations which co-express CD49c and CD90
US9969977B2 (en) 2002-09-20 2018-05-15 Garnet Biotherapeutics Cell populations which co-express CD49c and CD90
US20090053183A1 (en) * 2007-06-15 2009-02-26 Neuronyx Inc. Treatment of Diseases and Disorders Using Self-Renewing Colony Forming Cells Cultured and Expanded In Vitro
US8354370B2 (en) 2007-06-15 2013-01-15 Garnet Biotherapeutics, Inc. Administering a biological composition or compositions isolated from self-renewing colony forming somatic cell growth medium to treat diseases and disorders

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EP1111389A1 (en) 2001-06-27
DE60026272T2 (en) 2006-11-23
ATE319090T1 (en) 2006-03-15
ES2259599T3 (en) 2006-10-16
CA2326713A1 (en) 2001-06-03
US20040082016A1 (en) 2004-04-29
US7192719B2 (en) 2007-03-20
FR2801979B1 (en) 2002-02-08
EP1111389B1 (en) 2006-03-01
JP3516263B2 (en) 2004-04-05
DE60026272D1 (en) 2006-04-27
JP2001215225A (en) 2001-08-10
CA2326713C (en) 2007-01-02

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