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Scleroderma fibroblasts express c-kit ligand in vitro
ESDR I JSID I SID Abstracts
s141
0841
0844
IDENTIFICATION KERATINOCYTE
OF VITAMIN D RESPONDING GENES IN CULTURES BY DIFFERENTIAL DISPLAY. L&! MW &&ni,l~. JJ Hamll). PH Andrw G eaersen(3). I Bolui3sl(41. I Binderuo(lband K Kre 1: D:oartment of Biochemistrv. LEO Pharmaceutical Products. Balleruo. 2: Department of Dermatology, Marselisborg Hospital, Aarhus3: Mole&rlar Medical Research Unit, Skejby Hospital, Aarhus, 4: Institute of Human Genetics, University of Aarhus, Aarhus, Denmark. The active form of vitamin D, 1,25dihydroxyvitamin D, (VD) and VD analogues have been shown to improve psoriasis. However the mode of action of D-vitamins in psoriasis is unknown. Thus, it is not known which genes are regulated when the skin is treated with VD or VD analogues. Only few VD responding genes have been identified in the skin or in cultures of human keratinocytes. In order to identify new targets for the actions of D-vitamins in human skin we have used the differential display (DD) technique for the identification of VD responsive genes in cultures of normal human epidermal keratinocytes. Using the DD-procedure with 24 different primer combinations we identified 5 cDNA fragments, all upregulated in VD treated cultures compared to vehicle treated cultures. Clonina and seauencina of these fraaments followed bv searchina aene banks‘ior sequence similarity, it wai found that 3 fragments h&-high similarity with the 3’ ends of known genes. Currently work is in progress to verlfy these findings and elucidate the biological significance
A MODEL TO STUDY TOPICAL SELECTION OF KERATlNGCYTES CONTAINING THE MULTI-DRUG RESISTANCE GENE. W Pfutzner. U Heggg&_l e. RA Fo . IC VoeeL Dertnatology Branch, NCI, Bethesda, MD: #Dermatology, University of EiiEi In gene therapy, persistent expression of rut inserted gene has been problematic. Introduction of a selectable marker gene, such as the multi-dmg resistance (MDR) gene, into keratinocytes (KC) might allow topical selection of transduced KC, and ensure sustained expression of the marker gene. Topical applicatton of a selecting agent, such as the anti-mitotic agent colchicine, would inhibit cell division of KCs lacking a MDR gene and select for sustained expression and pmliferstion of KCs expressing MDR. To test the feasibility in both in vitro and in vivo models, pig and hums” KC were transduced by the MDR-expressing retrovirtd vector pHsMDRl/A. A transduction efficiency of 7085% (demonstrated by flow cytometry) was obtained by optimizing media conditions, incubation time, temperature,and centrifogation. Ski” equivalents were created by seeding transduced KC onto acellular pig dermis and MDR expression demonstrated immunohistochemicslly at various tie points. Under colchicine selective pressure, MDRtransduced KCs were able to form a differentiated epidermis in vitro while non-transduced KC were not. Skin equivalents with MDR-transduced pig KCs were grafted back successfolly onto the autologous pig following placement onto muscle fascia in correct anatomical location along with skin flap coverage for4-6 days. Histologic exsmination of skin biopsies taken at weekly intervals showed that MDR-transduced KCs were able to fort” a diffenntiated matote epidetmis in viva, however MDRexptession was lost sfter 46 weeks in the absence of selection. Techniques applying colchicine topically see ctmently being developed to tncresse the duration of MDR-expression in g&ted KC. This topical selection approach holds promise for prolonged expression of not only the MDR marker gene, but also a therapeutic gene that is linked to MDR.
0842
0845
SCLERODERMA FIBROBLASTS EXPRESS C-KIT LIGAND IN VITRO Janina Wv&Ikotis’. Ansstwx Omulecki# Aotelis Wskz&Dtzewiecka’. Arms SvssJedrseiowska#. Bo~omiln Kolsno’. Botens Dziwkowsks-Bar!kowdsk#isk#. Jsto&w I&tvch*. Arms Z”lew&&. Depanment of Biogenic Aniines Polish Academy of Sciences’ and Depsrhnent of Dumntology, Medical Univetsity of L&i&+,L6di, Poland. Scleeodemts is a chrontc connective tissue disease of unknown etiology chsrscterined by excessive production of collsgen in the &lo and iotemsl organs. Ihe changes in mast cell “““lber have been sssoctsted with tbe progress of this disease so we h&e decided to explore whether sclerodenns tlbxoblssis preserve expression of ckit linsnd called also stem cell tbctor (SCFl. whtch is the msi” cvtokiie able to in&t&e humsn w cell growth and di0b”e”tt”tio”. Ftboblnsts obiaioed ikont the sktn punch biopstes (3 mm) ikom patients with systemic sclerosis of two groups disease domtio” less than three years and mote than three yeats, were colhued in the modifiedEagle’s mediunt. ARet foot to six weeks m colhtte the sclerodetms fibroblnsts of the lirst passage have bee” used in the RT-PCR expwbmmt. We used specdlc printers for SCF and 8-&n (control) and observed thst sclerodetms 8btobkw.s cultured in vitro express SCF ntRNA. This prehminssy observation soggests that iiksoblasts present in sclerodetms lesions “tight express c-kit hgsnd and thus be able to sttpport mast cell survival
PRENATAL ASSESSMENT OF CLINICAL PHENOTYPE BY MUTATIONAL SCREENING IN A FAMILY WITH BHLBRS DANLOS SYNDROME TYPE VI. HN Yeowell sod LC Wnlker. Duke University Medical Center, Dorhsni, NC, USA. We have performed the first prenatal assessment of clinical phenotype in a family affected by the autosomsl recessive collsge” disorder Bhlet’sD&OS Syndrome Vl (BDS VI) by screening the fetal DNA for mutstions in the lysyl hydmxylsse (LH) gene. We have recently characterized mot&ions that are responsible for the deficiency of LH activity (GO% of normal) in ski” fihtoblnsts ikom the sifected child in this family. The proband, who is compownl hetemzygotts for these mutations, has the chsmcteristic clinical EDS VI phenotype of extensible ski” and joints, and kyphoscoliosis. One allele has a patemslly inherited C,,,, to G change coding for a premature stop codon (Y’S1IX) and introduces so Nhe I restriction site in exon 14 of the LH gene. The mutation in the other allele is a deletion of exon 5. Sequencing of gettomic DNAs spsnning exe” 5 shows a ntstemslly-inherited mutation in the consensus donor splice site at the beginning of intmn 5 @‘at). To perform the prenatal evsloation on the fetus, we cultured chorionic vilhts cells at 10 weeks gestation, and analyzed genomic DNA for the previously-chsed mutations in the gene. Sequencing of a PCR-amplified 199bp fmgnlent covering exon 5 showed thst one allele h&l the mstemslly-inherited g&&at splice-site motstion. However, Nhe I reshiction and sequencing of a 430hp t&went amplified ovet exe” 14 showed the absence of the abnonnsl pstemsl allele. As EDS VI is a recessive disorder, we predict that although the fetus is a carrier due to inheritance of the ahnonnsl matemsl allele, the baby should be o”stTected due to the presence of a normal sllele. This diagnosis is supported by the notntal LH activity in the cells, and provides the first example of a mutation-bssedprensts.1 sssessntent of EDS VI.
0846
0843 ELEVATED EXPRESSION OF ORNrIIIINB SCIERODERMA EPIDERMIS.
Tsutomu
DBCARBOXYL4SE “RNA
‘*
IN
.. pmaTdkiDepsrtment
of Demtstology, Physiology* and Biochemistty*‘, Dokkyo University Sdtool of Medicine, Mibu, Tochigi, Japan. Only a little attention has bee” paid to the biochemical changes of the epidermis in systemic sclerosis (SSc). We examined the expression of ornithine dexxrboxylase (ODC) mRNA in the ski” sections of 5 patients with SSc and those of normal controls, using Northern blot analysis and in siti hybridizstion techniques. Northern blot analysis showed that the expression of ODC mRNA in SSc patients was mote intense than those of “onnal controls. Sections nested with ss antisense probe showed concentrated grains exclusively in the epidermis of the patients with SSc, hut not in that of “orntnl mntiols. The grain densities in SSc epidermis were significantly higher than those of normal controls (PcO.02). Because out subcloned antisense probe specifknlly hybridize with ODC mRNA, these fmdings indicate that the expression of ODC ntRNAis elevated in the epidermis of SSc. These results also raise the possibility that polysmines play an important role in the process of skin changes in SSc.
MUTATIONS IN THE LYSYL HYDROXYLASE (LH) GENE ARE RESPONSIBLE FOR LH DEFIClENCY lN EIGHT PATIENTS WITH EHLERS DANLOS TYPE VI. p Duke University, Durham, NC 27710, USA. Screening of frill-length (29kb) cDNAs for lysyl hydmxylsse (LH) amplified from dermsl fibtoblssts Rooma gmttp of 8 unrelated patients with the sutosomsl recessive disorder Ehlers Dsnlos Syndtmne VI (BDS VI) has shown them to be compound heterozygotts for mutations in the LH gene. These mntstions were verified in genomic DNA and by sllelic inheritance, and result in s deficiency of LH (QS% of normal) in the probsnds who sre clinically characterized by kyphoscoliosis and extensibility of skin and joints, Interestingly, we hsve found 3 different types of mutations thst octet in more than one patient in this group. Of the five mutations defined prior to this study, one mutation, s large duplication of 7 exons that has bee” show” to be a common mutation, was identified in one patient JH790. I” the other allele this patient had a point mutation in exe” 14 that cc&d for apremstote termination codon (PTC) Y512X. We have identified this point mutation, thst in some clones results in a psrtial or complete deletion of exon 14, in 2 othet patients, SF996 and 081122. A third mutation has been observed in 2 other patients, JH716 and CC959, of a 15 bp deletion in exe” 11 that codes for amino acids 367-371 (DLCRQ). Deletion of the cysteine residue has been shown by beculovims expression to be essential for LH activity. InJH716, the mutation m the second allele is an early PTC at Q49X. Two other point mutations resulting in PTCs occor in patient xK1072 (Y427X in exon 12). and in patient MCI200 (R67OXin exe” 18). Patient AF1199 is affected by a large deletion ofexons 3,4, and 5. Analysis of these novel mutations has enabled us to begin stmchne/6mction studies to define the active siteof LH, an essential enzyme for the stmctttrsl integrity of extracelhdst matrix.
s141
0841
0844
IDENTIFICATION KERATINOCYTE
OF VITAMIN D RESPONDING GENES IN CULTURES BY DIFFERENTIAL DISPLAY. L&! MW &&ni,l~. JJ Hamll). PH Andrw G eaersen(3). I Bolui3sl(41. I Binderuo(lband K Kre 1: D:oartment of Biochemistrv. LEO Pharmaceutical Products. Balleruo. 2: Department of Dermatology, Marselisborg Hospital, Aarhus3: Mole&rlar Medical Research Unit, Skejby Hospital, Aarhus, 4: Institute of Human Genetics, University of Aarhus, Aarhus, Denmark. The active form of vitamin D, 1,25dihydroxyvitamin D, (VD) and VD analogues have been shown to improve psoriasis. However the mode of action of D-vitamins in psoriasis is unknown. Thus, it is not known which genes are regulated when the skin is treated with VD or VD analogues. Only few VD responding genes have been identified in the skin or in cultures of human keratinocytes. In order to identify new targets for the actions of D-vitamins in human skin we have used the differential display (DD) technique for the identification of VD responsive genes in cultures of normal human epidermal keratinocytes. Using the DD-procedure with 24 different primer combinations we identified 5 cDNA fragments, all upregulated in VD treated cultures compared to vehicle treated cultures. Clonina and seauencina of these fraaments followed bv searchina aene banks‘ior sequence similarity, it wai found that 3 fragments h&-high similarity with the 3’ ends of known genes. Currently work is in progress to verlfy these findings and elucidate the biological significance
A MODEL TO STUDY TOPICAL SELECTION OF KERATlNGCYTES CONTAINING THE MULTI-DRUG RESISTANCE GENE. W Pfutzner. U Heggg&_l e. RA Fo . IC VoeeL Dertnatology Branch, NCI, Bethesda, MD: #Dermatology, University of EiiEi In gene therapy, persistent expression of rut inserted gene has been problematic. Introduction of a selectable marker gene, such as the multi-dmg resistance (MDR) gene, into keratinocytes (KC) might allow topical selection of transduced KC, and ensure sustained expression of the marker gene. Topical applicatton of a selecting agent, such as the anti-mitotic agent colchicine, would inhibit cell division of KCs lacking a MDR gene and select for sustained expression and pmliferstion of KCs expressing MDR. To test the feasibility in both in vitro and in vivo models, pig and hums” KC were transduced by the MDR-expressing retrovirtd vector pHsMDRl/A. A transduction efficiency of 7085% (demonstrated by flow cytometry) was obtained by optimizing media conditions, incubation time, temperature,and centrifogation. Ski” equivalents were created by seeding transduced KC onto acellular pig dermis and MDR expression demonstrated immunohistochemicslly at various tie points. Under colchicine selective pressure, MDRtransduced KCs were able to form a differentiated epidermis in vitro while non-transduced KC were not. Skin equivalents with MDR-transduced pig KCs were grafted back successfolly onto the autologous pig following placement onto muscle fascia in correct anatomical location along with skin flap coverage for4-6 days. Histologic exsmination of skin biopsies taken at weekly intervals showed that MDR-transduced KCs were able to fort” a diffenntiated matote epidetmis in viva, however MDRexptession was lost sfter 46 weeks in the absence of selection. Techniques applying colchicine topically see ctmently being developed to tncresse the duration of MDR-expression in g&ted KC. This topical selection approach holds promise for prolonged expression of not only the MDR marker gene, but also a therapeutic gene that is linked to MDR.
0842
0845
SCLERODERMA FIBROBLASTS EXPRESS C-KIT LIGAND IN VITRO Janina Wv&Ikotis’. Ansstwx Omulecki# Aotelis Wskz&Dtzewiecka’. Arms SvssJedrseiowska#. Bo~omiln Kolsno’. Botens Dziwkowsks-Bar!kowdsk#isk#. Jsto&w I&tvch*. Arms Z”lew&&. Depanment of Biogenic Aniines Polish Academy of Sciences’ and Depsrhnent of Dumntology, Medical Univetsity of L&i&+,L6di, Poland. Scleeodemts is a chrontc connective tissue disease of unknown etiology chsrscterined by excessive production of collsgen in the &lo and iotemsl organs. Ihe changes in mast cell “““lber have been sssoctsted with tbe progress of this disease so we h&e decided to explore whether sclerodenns tlbxoblssis preserve expression of ckit linsnd called also stem cell tbctor (SCFl. whtch is the msi” cvtokiie able to in&t&e humsn w cell growth and di0b”e”tt”tio”. Ftboblnsts obiaioed ikont the sktn punch biopstes (3 mm) ikom patients with systemic sclerosis of two groups disease domtio” less than three years and mote than three yeats, were colhued in the modifiedEagle’s mediunt. ARet foot to six weeks m colhtte the sclerodetms fibroblnsts of the lirst passage have bee” used in the RT-PCR expwbmmt. We used specdlc printers for SCF and 8-&n (control) and observed thst sclerodetms 8btobkw.s cultured in vitro express SCF ntRNA. This prehminssy observation soggests that iiksoblasts present in sclerodetms lesions “tight express c-kit hgsnd and thus be able to sttpport mast cell survival
PRENATAL ASSESSMENT OF CLINICAL PHENOTYPE BY MUTATIONAL SCREENING IN A FAMILY WITH BHLBRS DANLOS SYNDROME TYPE VI. HN Yeowell sod LC Wnlker. Duke University Medical Center, Dorhsni, NC, USA. We have performed the first prenatal assessment of clinical phenotype in a family affected by the autosomsl recessive collsge” disorder Bhlet’sD&OS Syndrome Vl (BDS VI) by screening the fetal DNA for mutstions in the lysyl hydmxylsse (LH) gene. We have recently characterized mot&ions that are responsible for the deficiency of LH activity (GO% of normal) in ski” fihtoblnsts ikom the sifected child in this family. The proband, who is compownl hetemzygotts for these mutations, has the chsmcteristic clinical EDS VI phenotype of extensible ski” and joints, and kyphoscoliosis. One allele has a patemslly inherited C,,,, to G change coding for a premature stop codon (Y’S1IX) and introduces so Nhe I restriction site in exon 14 of the LH gene. The mutation in the other allele is a deletion of exon 5. Sequencing of gettomic DNAs spsnning exe” 5 shows a ntstemslly-inherited mutation in the consensus donor splice site at the beginning of intmn 5 @‘at). To perform the prenatal evsloation on the fetus, we cultured chorionic vilhts cells at 10 weeks gestation, and analyzed genomic DNA for the previously-chsed mutations in the gene. Sequencing of a PCR-amplified 199bp fmgnlent covering exon 5 showed thst one allele h&l the mstemslly-inherited g&&at splice-site motstion. However, Nhe I reshiction and sequencing of a 430hp t&went amplified ovet exe” 14 showed the absence of the abnonnsl pstemsl allele. As EDS VI is a recessive disorder, we predict that although the fetus is a carrier due to inheritance of the ahnonnsl matemsl allele, the baby should be o”stTected due to the presence of a normal sllele. This diagnosis is supported by the notntal LH activity in the cells, and provides the first example of a mutation-bssedprensts.1 sssessntent of EDS VI.
0846
0843 ELEVATED EXPRESSION OF ORNrIIIINB SCIERODERMA EPIDERMIS.
Tsutomu
DBCARBOXYL4SE “RNA
‘*
IN
.. pmaTdkiDepsrtment
of Demtstology, Physiology* and Biochemistty*‘, Dokkyo University Sdtool of Medicine, Mibu, Tochigi, Japan. Only a little attention has bee” paid to the biochemical changes of the epidermis in systemic sclerosis (SSc). We examined the expression of ornithine dexxrboxylase (ODC) mRNA in the ski” sections of 5 patients with SSc and those of normal controls, using Northern blot analysis and in siti hybridizstion techniques. Northern blot analysis showed that the expression of ODC mRNA in SSc patients was mote intense than those of “onnal controls. Sections nested with ss antisense probe showed concentrated grains exclusively in the epidermis of the patients with SSc, hut not in that of “orntnl mntiols. The grain densities in SSc epidermis were significantly higher than those of normal controls (PcO.02). Because out subcloned antisense probe specifknlly hybridize with ODC mRNA, these fmdings indicate that the expression of ODC ntRNAis elevated in the epidermis of SSc. These results also raise the possibility that polysmines play an important role in the process of skin changes in SSc.
MUTATIONS IN THE LYSYL HYDROXYLASE (LH) GENE ARE RESPONSIBLE FOR LH DEFIClENCY lN EIGHT PATIENTS WITH EHLERS DANLOS TYPE VI. p Duke University, Durham, NC 27710, USA. Screening of frill-length (29kb) cDNAs for lysyl hydmxylsse (LH) amplified from dermsl fibtoblssts Rooma gmttp of 8 unrelated patients with the sutosomsl recessive disorder Ehlers Dsnlos Syndtmne VI (BDS VI) has shown them to be compound heterozygotts for mutations in the LH gene. These mntstions were verified in genomic DNA and by sllelic inheritance, and result in s deficiency of LH (QS% of normal) in the probsnds who sre clinically characterized by kyphoscoliosis and extensibility of skin and joints, Interestingly, we hsve found 3 different types of mutations thst octet in more than one patient in this group. Of the five mutations defined prior to this study, one mutation, s large duplication of 7 exons that has bee” show” to be a common mutation, was identified in one patient JH790. I” the other allele this patient had a point mutation in exe” 14 that cc&d for apremstote termination codon (PTC) Y512X. We have identified this point mutation, thst in some clones results in a psrtial or complete deletion of exon 14, in 2 othet patients, SF996 and 081122. A third mutation has been observed in 2 other patients, JH716 and CC959, of a 15 bp deletion in exe” 11 that codes for amino acids 367-371 (DLCRQ). Deletion of the cysteine residue has been shown by beculovims expression to be essential for LH activity. InJH716, the mutation m the second allele is an early PTC at Q49X. Two other point mutations resulting in PTCs occor in patient xK1072 (Y427X in exon 12). and in patient MCI200 (R67OXin exe” 18). Patient AF1199 is affected by a large deletion ofexons 3,4, and 5. Analysis of these novel mutations has enabled us to begin stmchne/6mction studies to define the active siteof LH, an essential enzyme for the stmctttrsl integrity of extracelhdst matrix.