Calpain I is expressed in early normal skin development but is reduced in neonatal harlequin ichthyosis

S61

ESDR I JSID / SID Abstracts

0364

0361 DECREASED

LEVELS

ARE CAUSED

OF EPIDERMAL

SKALP

BY POST-TRANSLATIONAL

and

fN PUSTULAR

MECHANISMS.

FORMS

OF PSORJASIS

Artrid

Depertment of Dermatology, University Hospital Nijmegen, Ndmegen,

*Depanment of Pubnonology, Leiden Univewty Hospital, Leiden, the Netherlands. The proteinase inhibitor SKALP

(also known es &fin)

is absent in normal skin bur highly

expressed in plaque-type psoriatic epidermis. Because SKALP derived elastase and proteinaxPMN

is a major inhibitor of PMN-

it is speculatively involved in regulation of tissue damage and

migration. We have recently shown that SKALP

protein lwels are strongly decreased I”

lesional epidermis of pushller psoriasis compared to plaque type psoriasis. Here we have investigated whether there is an intrinsic defect in SKALP gene expression of keratinocyter from put&r

psoriesis patients, and we have addressed the possibility

tmnslarionally inactivated by PMN-derived

that SKALP

1s post-

mediators which are abundantly present in pustolar

epidermis. In addition we have studied to what extent the reduction of SKALP levels is a specific phenomenon, by comparison with another epidermal proteinare inhibitor Secretory Leukocyte Proteinase Inhibitor (SLPI).

Using Northern blot analysis and ELlSA

we found no significant

differences in trewcript size or expression levels of SKALP between cultured kentinocytes from patients with peat&or

plaque t&e psoriasis. Recombinerd SKALP

resistant to degadatian

or inactivation by PMN-derived

was found to be exrremely

mediators such as elastase, superoxide

anion and hydrogen peroxyde. Hypochlomos acid was found to cause oxidation of SKALP end, et high concentrations, caused a decrease in functional activity Remarkably, we found that the levels

of SLPI, a homologous epidermal proteinare inhibitor, were not significantly different in rhe epidermis of pustukx and plaque-type psoriasis. We conolude that low levels of SKALP in lesional skin of pustular psoriasis patients appear to be specific for this inhibitor, cannot be explained by decreased synthesis or secretion, end are due lo pattranslational

mechanisms

TRANSGLUTAMINASE NORMAL

1 IS INDUCED

HUh4AN

Steinert,

DERMAL

zSoo-Youl

University,

Kim.

Seoul, Korea,

BY

IRRITANT ‘Jun-MO

FIBROBLASTS.

‘Department

of

and *Laboratory

TREATMENT

Dermatology,

of Skin Biology,

Yang, Sung

IN

*Pet,

M.

Kyun

NIAMS,

Kwan

NIH,

Bethesda,

to certain

chemicals.

MD, USA. Irritant

contact

dermatitis

tindings

the dermal

layer. We have proposed

normal human PCR, TGase

fibroblasts

By

were reduced

microscopic

studies

revealed

that MEKKT-1

contained

a few characteristic

to control

in the membrane amount ofhigh precipitated included

which

moleculzu

1 antibody proteins

to the toxicity

proteins.

antibodies,

and activity

with a two-fold

fractions.

weight insoluble

by the TGase

There

levels

(SLS) as by RT-

at 1, 3 and 5

of the TGase

of necrosis of TGase

2

or induced

and actin.

in the

Antigens

c+

by amino acid sequencing,

and

These

of TGase

that a similar process

1 activity

increase

due to crosslinking

lucre identified

vim&n

increase

was also a significant

proteins

of SLS by induction

We suggest

sulfate

expression

level of the TGase 1 enzyme WBS increased 200% in

correlated

and cytosolic

the cytoskeletal

cells respond of cellular

the mRNA

deep into

might be related

lamyl

the level of TGase with specitic

the mP.NA

ATMOSPHERIC OXYGEN ACCELERATES DIFFER&T L4TlON OF BUMAN DEBMAL. FIBROBLASTS TOWARDS A POST-hUTOl’IC PHBNOTYF’B. &Al&f. H.Mvir-Howie. ~ Uailever Rssurch, Cotwortb Lboretmy. Sbanibmok, Bedford. UK. Humea demul tibmb&ls have previously been shown to differeat& along a tennimlo~1 Iii, pass&g tbrwgb 7 diet& phcllotypic sagea tb# are chrnctarised by altered morphology, inereasieg size and dsweiag proliferative capacity. The sire of tbc current btudy we to inveeti@e the e&t of

To test this idea, we incubated

by SO%, which might suggest the absence

However,

comparison

5 days,

reaction

(TGases)

with IO-6M sodium

and immunoprecipitation

at&r treatment

apoptosis.

in the dermis.

(NHDF)

for up 10 5 days. We analyzed

activity,

skin response to an eczcmatoos

that transglutaminases

of ICD symptoms

dermal

an acute irritant,

enzyme

(ICD) is a well known

of ICD vary fmm mild erythema

to the progression

days

Electron

membrane bound secretory graoules in the cytoplasm. ‘Ilxse observations strongly indicate that MEKKT-1 is a new Mcrkel cell tomor cell line to be useful for studiing Merkcl cell biology.

0365

0362

Clinical

ESTABLISHMENT AND CHARACfERIZATION OF A NEW MERKEL CELL TUMOR CELL LINE, MEKKT-I. Ken-Ichi Toda, Norihisa Mslsuvoshi. Satoshi Kerceda, Kaeemasa Kuribavashi. Sadao Imamura, Department of Dermatology, Postgraduate School of Medicine, Kyoto University, Kyolo, Japan A new tumorigenic muine cell line, deign&d MEKKT-I, was established through cultivating cutaneous tumors generated by repetitive UVF&irradiations on the denuded C57BIJ6 mice skin. The tumors were adaplcd to cultures from the first explant generation, and several clones including MEKICT-1 were obtained by limiting dilution techniques and maintained in lO%FCSsupp1emented DMEM. Xx morphological observations of MEKKT-1 showed that the cell was long-spindle shaped and the cells grew to form dendridc patterns under sparse culture conditions, followed by a flat sheet with some holes on the flasks under dense conditions. The doubling time was 78hn and the chromosomes number of mosf cells revealed diploid pattern. The nude mice injected with MEKKT-1 cells rapidly developed a subcutanmus red-colored tumor. The histological examinations showed that tumor cells were arranged in an orderly manner lo be resulted in forming Ihe trabecolar pattern. ‘Ihc tumor vasculatures was more prominent, in comparison with fibmsarcoma cell lines or squamous cell carcinoma cell lines induced lumors. The expressions of cytokeratin 20 and neuron-specific eaplase in both MEKKT-1 cells in vitro and the eeneratcd tumors were msitive in the cvto~lasm. The cells were also positively labeled >th qoinacrlne under & vitro culture &&ions.

data show that NHDF

I activity

and crosslinking

may occur in ICD.

awleus. Compwisoe d these cell populations h rtmmpheric (MB) 4 physiological (4%) 4 revealed no difference in elotig eficiary (68.1*22.1% ve 75.6+14.52), es d&ermb& by the proportion of wells containing a1 leasi one cell * weeks e&r seeding. However, ei@?untly feww Type- 1 aed more of the differwdeted Type 2 ad Type 3 cells WMO obsavrd uada atmos+nc (20%) O2 conditions tbln under pbysiologicsl (4%) 0, eonditims: m X5.6+14.5% (zO% 4) ys 73.4*10.6.% (4% 4, p=“.@X. ,114); && 3X3+9.9% (20% 0,) y8 1X4*6.3% (4% 0,. p=O.oZ, n=4): 32.tf8.6% (20% 4) “s 13.2*4.9% (4% 4. p-0.037, n=4). lhcsa results eugg& &.I ieoletcd dcrmel fibr&tas& cultwed in etmxpbetic (20%) oxygen uperiencc en axidative strea lbet eecellcratcsdiffereatmtian from a tit&c to e poet-mitotic pbeaotype. Xx date tberefom support the hypothesis Whetoridnhve stress is a urotributory faecor in premature eelbder md dermel e&&g.

0363

0366

!LXW&SDlNEARLYNcRMAL SmJ-mIs-IN cAi.R4NIl8 mATAL HARLEQUIN ICHIHYrXS M.Michel. P.Fle&man. B.A.Dale. Depts. of Oral Biology and Medicine/Dmnalology , U.of Washington, Seattle, WA. Human barleqoin ichthyosis @II) is a rare scaling skin disorder with consistent hyperkeradnization and abnormality in lipid-rich lamellar bodies. Initial studies showed alteration in keratin and filaggrin expression. Working fmm the map generated from a spontaneous moose mutation lie stody and the homologous region of the human genome, and considering events of normal epidermal differentiation, Calpain I was identified as a candidate gene. We utilized Calpain I antibody for staining of human samples. In early skin development (SSd), Calpain I is expressed in basal and first suprabwal cell layers. A&r 84d, the antibody stro”~ly reacts with the basal layer and pcriderm. As soon as appendages start forming. Calpein I is present in the outer root sheath of hair follicles and in sweat ducts and glands. The antibody strongly reacts with basal and granular layers in neonatal foreskin, but moderately in adult epidermis. However, 5 Hl cases showed significantly decreased levels of staining. Western blot analysis allows demonstration of the presence of the active form which is generated via limited proteolysis oftbe 8OkD to yield a 78 and/or 76kD catalytic subunit as previously demonstrated in cultured rat keradnocytes. Immuooblots of human epidcrmal extracts show the occurrence of mainly 78 and 76kD forms; however, all forms are weaker in HI samples. Limited protcolysis also occurs in normal and HI keratinocytes but the signal was weaker in HI cells consistent with findings in epidennal extracts and tissue sectioos. Thus, Calpain I is abnormally expressed in HI and further characterization of this enzyme is warrenfed, regardless of whether e mutation in tbis gene is responsible for the disorder.

TELOMERASE ACTMTY OF SKIN-HOMING T LYMPHOCYTES INDICATES T CELL IMMATURITY. Kaida Wu. Marianne Lund. Karen Bana. Kristian Thestruo-Pedersen. Department of Dermatology, Marselisborg Hospital, University of Aarhus, 8000 Aarhus C, Denmark. Telomeres shorten with successive cell divisions in normal somatic cells. Telomerase is an enzyme associated with cellular immortality and plays an important role in maintaining the stability of DNA and length of DNA telomere lengths. Telomerase activity is expressed in human germline cells and malignant cells. It has recently been demonstrated that telomerase activity is also detected in different kinds of diseases and lymphocytes at specific stages of development and activation. In this study ws measured the levels of telomerase activity in IO cell lines established from skin of atopic dermatitis, 5 from mycosis fimgoides, 4 from thymus and 15 samples of peripheral blood mononuclear cells using the telomarase PCR Kit (Boehringer Mannheim). We found that peripheral blood lymphocytes from normal donors expressed low levels of telomerase activity. In T lymphocytes from patients with MF, the activity was much higher (3-5 fold) and at the same level as thymocyte in some cell lines. Relative to HF and thymus, the T hphocytes from patients with AD ewressed an intermediate level of activity. Finding higher levels of telomerase activity in thymocytes from the thymus. a main organ of T cell development and in skin-homing T lymphocytes from patients with MF and AD, indicate that there is a presence of T cells with a much stronger T cell proliferation capacity than normal and matured T lymphocyte in peripheral blood.