- Email: [email protected]
186 Functional analyses of the stratum corneum in experimental dermatophytosis
JSID Abstracts/Journal
of Dermatological Science IO (1995) 61-102
181
184
IMMUNOLCGICAL ANALYSIS OF CARBAhwZEFi NISRFACTIVB T CELL CLONES. N.Mochida*. A.Hashimoto. K.KatsuoLa.Depaltmem of IJmlatdogy , Kitasato university schclol of Medicine, Sagamiham 228. Japan.
ANTI-RO/SSA
Carbamazepiae (CBZ) hypersensitivity is thought to be mediaed by celhdm immunity. because lymphccyte proliferative response and patch test to CBZ are positive in mat of CBzaUergic petienls However pmcise meclmism of this hypersensitivity is still unknown. In this study, we esI&lished seven CBZ reaaive T cell clones from peripheral blood monom~clear cells of the patient The phenotypes of these clones were CD4+ and CDS-. These clones prod&d IFN-y, but not IL4, upon stimulation with CBZ. Mxeover we examined the cytokine expression in the skin lesions and positive sites of patch test, using RT-PCR nwhod. In the lesions, Tbl type cytokiw mRNAs such as IFN-y and TNF-p were relatively predominant These results suggest that CBZ hyPerse&tivity delayed-type hypenensitivity response
is a
ASSOCIATED
93
ANNULAR
ERYTHEMA:
AUTOIMMUNE RESPONSE TO RECOMBINANT 60- AND 52KD ROISSA PROTEINS. Q&gmva*. T. Fukumoto and T. Shirk Department of Dermatology,
Nara Medical University,
Nara, Japan
Recurrent annular erythema associated with anti-Ro/SSA antibody response has recently been recognized as a distinct cliicai entity. Sewn samples from 15 anti-Ro/SSA positive patients with recurrent anmdar erythema were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with fill-length recombinant human 60-kd and 52-kd Ro/SSA proteins. All of the 15 sera were positive for ami-60-kd Ro/SSA, and 11 sera (73.3%)
contained anti-52-kd
Ro/SSA. These
results suggest the importance of 60-kd polypeptide component of the Ro/SSA particle as a potential targel in anti-Ro/SSA associated annular erythema
182
185
INFLAMMATORY CYTOKINES ENHANCE C3 PRODUCTION BY HUMAN EPIDERMAL KERATINOCYTES. [email protected]~~~KJ&ij. ‘Department of Dermatology. 2Second Department of Pharmacology, Toboku University Schwl of Medicine. secdai. Jmm. it is known fhrU cultured human epidermal keratinocytes produce C3, but if remains to be elucidated how such pmducfion is controiied This study is designed to investigate what kind of cytokines a& able 10enhanceC3 production by cultured human keratinocyres and what kind of signal transductional pathways are related. Melhods: Human keratinocytes were cultured in serum free medium (KGM) in the absence or presence of a singlecymkine (IL% IFNs, TNFs, or TGFs) for 48 hs. Culture supematants were harvested and assayedfor C3 by a sandwich ELISA method.Changesof C3 mRNA were investigated with a Northern blot analysis fecbniqueusing C3 DNA probe made by PCR. To examine signal transductional pathways, keradnwytes were pretreated with either PKC or F’fK inhibitor Results: The resuifs showed [hat cultured human keratinocytes produce small anwunt of C3 consdwtiveiy and lhat the inflammamry cytokines enhanced remarkably with a prior increase in mRNA for C3, suggesting transcriptional control of C3 production by thesecytokines. Pretreatmentof keradnocyies with either PKC and PTK inhibitor caused abrogation of the enhancing effect of these cytukines. However. preincubation with an inhibitor of src-type FVK (herbimycin A) enhanced further C3 production. Coneiusion: Present study indicates that C3 omduction was enhanced bv the inflammatow cvrokines and that C3 orcduction was iegulated by PKC- and PTK&padent pathw&sj, which is alsoneg&ly regulatedby m-type PTK. We thin)r that inflammatory reactions prcduce a micro-environmar in the lesional skin where the complementacdvation tie place effectively via enhancementof C3 prcduction by the inflammarory cytokmes.
CLONING OF A PARTIAL cDNA FROM SPOROTHRlX SCHENCKII Y.Suaita*#. M. Murakami, I.Takahashi. H. Havakawa and H.Nakaiima Sagamino hospital#, Sagamihara and Department of Dermatology, Yokohama City University School of Medicine, Yokohama, Japan Sporothrix schenckii is a pathogenic fungus which causes sporotrichosisieve;;ave cloned a partial cDNA from Sschenckii. DNA fragments were generated by polymerase chain reaction (PCR) from S.schenckii using primers which were designed to amplify a 157 bp DNA fragment from Mycobacteriun leprae. The 231 bp DNA fragment was ligated into a plasmid vector and sequence analysis was performed. Nucleotide sequencing of the DNA fragment revealed a part of open reading frame encoding a polypeptide and sequence homology was observed with heat shock protein 70 in Mleprae. Molecular cloning of this cDNA fragment may provide not only genetical information of S.schenckii, but also potent tools for clinical use such as PCR primers to detect S.schenckii.
183
186
CULTURE SUPERNATANTS FROM KERATINOCYTES INDUCE *POPTOSIS OF LYMPHOCYTES. A m Dcpartmenr of Dermatology, Hokkaido Umvernty. Schoolof Medicine. Sappom.lapen Residentand mtiimrted Icukucyter bolh m theepidermisand dermware recogwed II, immunologic diseasesof the skin. Keraunocytes areknown LObe a rich source oi cytokim and growth factors to regulatemnmmdog~calevents. Many studies havelocused on kemtuwcyte-denvcd factam that regulateIhe survw.alandprdifemtiuu oi rhese ieukee)us; GM-CSF ior Langehans ceils and IL-7 da&tic epidamal T cells u1mice However. litdc is kmwo aboutkentinucyte-derived iactors lu induceamosis of lcukoeytes We hypothesized that kemtinocytes CM elaborateiactors to induce.apoptosisoi leukocytes in the skin. nsuiung io ceasing ~mmunoiogicaievenu tn Ihe healing prccess of uum~,~c skin dwases. The presentstudy was conductedtu determinewhether keratluocytes praluce iactors 10mducc apoptos~sof leukocytes. Cultwc supernalantsimm normal human keratmocyles or HaCaT keratmccyte ceil hoe equally mh~bitpmiiferatmu of a-2 T cells m a dose dependentiashiou either in rhc pesence or absenceoi IL-2. Supemaamsirom thesecells stimulatedwith 1FN-y alsodemoustmtedsinulpr iuhibtory eNects These mbtbitorv die& were due to iwxeasin~ tie number of awototic ceils. assessedby FACS an&is @mpxlium iodide staining7 Moreover, ind&thacm did not bic& theseinhibitory eflects audTNFlr did mt afiect IL-2.induced CIZLZ pmhfemtlou, suggestinglhat both TNF-a sod in Ihe supematantsmay no, be responsibleiactors to mduce apuplosisai a-2 T cells These results mdtca~ethal keratmocytes produce factors 10iuducc apuptosis uiT cells. which may parrxlpatc m the beaiiog process of mmunologic skin chseases
FUNCTIONAL ANALYSES OF THE STRATUM CORNEUM IN EXPERIMENTAL DERMATOPHYTOSIS. K.Kudoh. H.Tagami. Depatment of Dermatology. Toboku University School of Medicine, Send& Japan.
ior
Dermatophytosis is a unique infectious disease in that demmtophytes reside only in the stratum corneum. Ht?have postulated that skin
inflammation at the site of dennatophytosis plays an important role in the defence mechanism against de’matophytes by increasing turnover of the epidermis and stratum corneum, thereby fascilitaling the elimination of Ihe dermatophytes. In this study. using the infection model of self-healing
experimenlai dermatophytosis of guinea pigs, weevaluated the turnover of lesional stratum comeurn with the dansyl chloride method. immediately after T.mentagrophytes was inoculated on the back of guinea pigs with occlusive dressing method. 5% dansyl chloride ointment wis applied to the inoculation sites under occlusion for 24 hours. Intensity of fluorescence was evaluated wh a fluorometer over the subsequent 2 to 3
weeks. w also measured tmnsepidennai water loss (TEWL) and hydration state of the stratum comeurn with Evaporimeter and SKICON. respectevely. The intensity of fluorescence reduced LOthe half of the initial value in 7 days in the lesional skin. 10 lo 12 days in the control sites. The iesmnal skin showed extremely high TEWL with poorly hydrated suatum corneum during the course of dermatophytois.
of Dermatological Science IO (1995) 61-102
181
184
IMMUNOLCGICAL ANALYSIS OF CARBAhwZEFi NISRFACTIVB T CELL CLONES. N.Mochida*. A.Hashimoto. K.KatsuoLa.Depaltmem of IJmlatdogy , Kitasato university schclol of Medicine, Sagamiham 228. Japan.
ANTI-RO/SSA
Carbamazepiae (CBZ) hypersensitivity is thought to be mediaed by celhdm immunity. because lymphccyte proliferative response and patch test to CBZ are positive in mat of CBzaUergic petienls However pmcise meclmism of this hypersensitivity is still unknown. In this study, we esI&lished seven CBZ reaaive T cell clones from peripheral blood monom~clear cells of the patient The phenotypes of these clones were CD4+ and CDS-. These clones prod&d IFN-y, but not IL4, upon stimulation with CBZ. Mxeover we examined the cytokine expression in the skin lesions and positive sites of patch test, using RT-PCR nwhod. In the lesions, Tbl type cytokiw mRNAs such as IFN-y and TNF-p were relatively predominant These results suggest that CBZ hyPerse&tivity delayed-type hypenensitivity response
is a
ASSOCIATED
93
ANNULAR
ERYTHEMA:
AUTOIMMUNE RESPONSE TO RECOMBINANT 60- AND 52KD ROISSA PROTEINS. Q&gmva*. T. Fukumoto and T. Shirk Department of Dermatology,
Nara Medical University,
Nara, Japan
Recurrent annular erythema associated with anti-Ro/SSA antibody response has recently been recognized as a distinct cliicai entity. Sewn samples from 15 anti-Ro/SSA positive patients with recurrent anmdar erythema were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with fill-length recombinant human 60-kd and 52-kd Ro/SSA proteins. All of the 15 sera were positive for ami-60-kd Ro/SSA, and 11 sera (73.3%)
contained anti-52-kd
Ro/SSA. These
results suggest the importance of 60-kd polypeptide component of the Ro/SSA particle as a potential targel in anti-Ro/SSA associated annular erythema
182
185
INFLAMMATORY CYTOKINES ENHANCE C3 PRODUCTION BY HUMAN EPIDERMAL KERATINOCYTES. [email protected]~~~KJ&ij. ‘Department of Dermatology. 2Second Department of Pharmacology, Toboku University Schwl of Medicine. secdai. Jmm. it is known fhrU cultured human epidermal keratinocytes produce C3, but if remains to be elucidated how such pmducfion is controiied This study is designed to investigate what kind of cytokines a& able 10enhanceC3 production by cultured human keratinocyres and what kind of signal transductional pathways are related. Melhods: Human keratinocytes were cultured in serum free medium (KGM) in the absence or presence of a singlecymkine (IL% IFNs, TNFs, or TGFs) for 48 hs. Culture supematants were harvested and assayedfor C3 by a sandwich ELISA method.Changesof C3 mRNA were investigated with a Northern blot analysis fecbniqueusing C3 DNA probe made by PCR. To examine signal transductional pathways, keradnwytes were pretreated with either PKC or F’fK inhibitor Results: The resuifs showed [hat cultured human keratinocytes produce small anwunt of C3 consdwtiveiy and lhat the inflammamry cytokines enhanced remarkably with a prior increase in mRNA for C3, suggesting transcriptional control of C3 production by thesecytokines. Pretreatmentof keradnocyies with either PKC and PTK inhibitor caused abrogation of the enhancing effect of these cytukines. However. preincubation with an inhibitor of src-type FVK (herbimycin A) enhanced further C3 production. Coneiusion: Present study indicates that C3 omduction was enhanced bv the inflammatow cvrokines and that C3 orcduction was iegulated by PKC- and PTK&padent pathw&sj, which is alsoneg&ly regulatedby m-type PTK. We thin)r that inflammatory reactions prcduce a micro-environmar in the lesional skin where the complementacdvation tie place effectively via enhancementof C3 prcduction by the inflammarory cytokmes.
CLONING OF A PARTIAL cDNA FROM SPOROTHRlX SCHENCKII Y.Suaita*#. M. Murakami, I.Takahashi. H. Havakawa and H.Nakaiima Sagamino hospital#, Sagamihara and Department of Dermatology, Yokohama City University School of Medicine, Yokohama, Japan Sporothrix schenckii is a pathogenic fungus which causes sporotrichosisieve;;ave cloned a partial cDNA from Sschenckii. DNA fragments were generated by polymerase chain reaction (PCR) from S.schenckii using primers which were designed to amplify a 157 bp DNA fragment from Mycobacteriun leprae. The 231 bp DNA fragment was ligated into a plasmid vector and sequence analysis was performed. Nucleotide sequencing of the DNA fragment revealed a part of open reading frame encoding a polypeptide and sequence homology was observed with heat shock protein 70 in Mleprae. Molecular cloning of this cDNA fragment may provide not only genetical information of S.schenckii, but also potent tools for clinical use such as PCR primers to detect S.schenckii.
183
186
CULTURE SUPERNATANTS FROM KERATINOCYTES INDUCE *POPTOSIS OF LYMPHOCYTES. A m Dcpartmenr of Dermatology, Hokkaido Umvernty. Schoolof Medicine. Sappom.lapen Residentand mtiimrted Icukucyter bolh m theepidermisand dermware recogwed II, immunologic diseasesof the skin. Keraunocytes areknown LObe a rich source oi cytokim and growth factors to regulatemnmmdog~calevents. Many studies havelocused on kemtuwcyte-denvcd factam that regulateIhe survw.alandprdifemtiuu oi rhese ieukee)us; GM-CSF ior Langehans ceils and IL-7 da&tic epidamal T cells u1mice However. litdc is kmwo aboutkentinucyte-derived iactors lu induceamosis of lcukoeytes We hypothesized that kemtinocytes CM elaborateiactors to induce.apoptosisoi leukocytes in the skin. nsuiung io ceasing ~mmunoiogicaievenu tn Ihe healing prccess of uum~,~c skin dwases. The presentstudy was conductedtu determinewhether keratluocytes praluce iactors 10mducc apoptos~sof leukocytes. Cultwc supernalantsimm normal human keratmocyles or HaCaT keratmccyte ceil hoe equally mh~bitpmiiferatmu of a-2 T cells m a dose dependentiashiou either in rhc pesence or absenceoi IL-2. Supemaamsirom thesecells stimulatedwith 1FN-y alsodemoustmtedsinulpr iuhibtory eNects These mbtbitorv die& were due to iwxeasin~ tie number of awototic ceils. assessedby FACS an&is @mpxlium iodide staining7 Moreover, ind&thacm did not bic& theseinhibitory eflects audTNFlr did mt afiect IL-2.induced CIZLZ pmhfemtlou, suggestinglhat both TNF-a sod in Ihe supematantsmay no, be responsibleiactors to mduce apuplosisai a-2 T cells These results mdtca~ethal keratmocytes produce factors 10iuducc apuptosis uiT cells. which may parrxlpatc m the beaiiog process of mmunologic skin chseases
FUNCTIONAL ANALYSES OF THE STRATUM CORNEUM IN EXPERIMENTAL DERMATOPHYTOSIS. K.Kudoh. H.Tagami. Depatment of Dermatology. Toboku University School of Medicine, Send& Japan.
ior
Dermatophytosis is a unique infectious disease in that demmtophytes reside only in the stratum corneum. Ht?have postulated that skin
inflammation at the site of dennatophytosis plays an important role in the defence mechanism against de’matophytes by increasing turnover of the epidermis and stratum corneum, thereby fascilitaling the elimination of Ihe dermatophytes. In this study. using the infection model of self-healing
experimenlai dermatophytosis of guinea pigs, weevaluated the turnover of lesional stratum comeurn with the dansyl chloride method. immediately after T.mentagrophytes was inoculated on the back of guinea pigs with occlusive dressing method. 5% dansyl chloride ointment wis applied to the inoculation sites under occlusion for 24 hours. Intensity of fluorescence was evaluated wh a fluorometer over the subsequent 2 to 3
weeks. w also measured tmnsepidennai water loss (TEWL) and hydration state of the stratum comeurn with Evaporimeter and SKICON. respectevely. The intensity of fluorescence reduced LOthe half of the initial value in 7 days in the lesional skin. 10 lo 12 days in the control sites. The iesmnal skin showed extremely high TEWL with poorly hydrated suatum corneum during the course of dermatophytois.