Recently Identified Forms of Epidermolysis Bullosa

Epidermolysis bullosa (EB) comprises a collection of clinically diverse inherited blistering diseases that affect the skin and, in some subtypes, mucous membranes and other organs. Currently classified into four main subtypes (EB simplex, junctional EB, dystrophic EB, and Kindler syndrome, mainly based on the level of skin cleavage), the spectrum of EB extends to more than 30 clinical subtypes with pathogenic mutations in at least 18 distinct genes. This review focuses on three recent additions to variants of EB: all are autosomal recessive, and result from mutations in either DST-e (coding for epidermal dystonin, also known as the 230 kDa bullous pemphigoid antigen, BP230), EXPH5 (coding for exophilin-5, also known as Slac2-b), or ITGA3 (coding for the integrin alpha-3 subunit). Each of these new forms of EB is reviewed with respect to the initial gene discovery, clinical features, the current mutation database, and skin pathology. Awareness of these recently described forms of EB is helpful in the clinical evaluation of patients with EB and in defining genotype-phenotype correlation for inherited blistering skin diseases.

Effect of Amniotic Membrane on Epithelial Thickness and Formation of Hemidesmosomes after Corneal Stromal Wound

PURPOSE: To investigate the effects of an amniotic membrane patch on corneal epithelial thickness and formation of hemidesmosomes during corneal stromal wound healing. METHODS: A stromal wound 9 mm in diameter and 130 microm in depth was created on rabbit cornea using a microkeratome. The changes in corneal epithelial thickness and hemidesmosome formations were compared between the amniotic membrane, contact lens, and control groups. Changes in the corneal epithelium were examined using H&E staining and hemidesmosome formation was examined using an electron microscope at 2 and 4 weeks after flap removal. RESULTS: Two weeks after treatment, the corneal epithelial thickness was 95.3 +/- 6.3 microm in the amniotic membrane group being significantly thicker than 76.4 +/- 5.1 microm in the contact lens group and 68.3 +/- 6.1 microm in the control group. Furthermore, more hemidesmosome formations were observed in the amniotic membrane group compared to the other 2 groups. However, there were no significant differences in corneal epithelial thickness or hemidesmosome formation among the 3 groups at week 4. CONCLUSIONS: The amniotic membrane group showed a thicker corneal epithelium and more hemidesmosome formation than the other 2 groups 2 weeks after flap removal. Thus, the use of an amniotic membrane patch appears to be effective in the early stages of corneal stromal wound healing.

Matrix Metalloproteinases and Autoimmune Bulbous Diseases: Expression of MMP-2, -3, and -9 in Bullous Pemphigoid, Pemphigus Vulgaris, and Pemphigus Foliaceus / 대한피부과학회지

BACKGROUND: Matrix metalloproteinases (MMPs) participate in extracellular matrix degradation and may play an important role in basal membrane damage in many dermatologic diseases. Recent studies implicated the importance of MMP-9 in the pathogenesis of bulla formation of bullous pemphigoid (BP). Various autoimmune bullous diseases are strongly associated with desmosome or hemidesmosome pathologies, and show an increased level of lesional MMP and exposed autoantigens from these structures. OBJECTIVE: This study evaluated the level of MMP-2, -3, and -9 in three types of autoimmune bullous disease [BP, pemphigus vulgaris (PV), pemphigus foliaceus (PF)] with the aim of investigating the role of MMPs in the pathogenesis of autoimmune bullous diseases. METHODS: Sample specimens were obtained from skin lesions of patients with BP (n=12), PV (n=10), and PF (n=12), and from normal controls (n=8). The immunohistochemical expression of MMP-2, -3, and -9 was analyzed and serum levels of MMP-2, -3, and -9 were measured by enzyme-linked immunosorbant assay (ELISA). The results were analyzed with reference to graded levels of clinical severity. RESULTS: Expression of dermal MMP-2, -3, and -9 were increased in BP, PV, and PF (p=0.036, 0.022, and 0.015, respectively). However, decreased expression of the three MMPs in the epidermis of skin lesions may have resulted from epidermal destruction. ELISA-determined serum levels of MMP-2, -3, and -9 increased in BP, PV and PF. Interestingly, MMP-2 was significantly increased in the sera of BP patients (p=0.015), consistent with the previous studies concerning the role of gelatinase (MMP-2 and -9) in the pathogenesis of BP. In BP patients, clinical severity was proportional to increased levels of MMP-2 in both skin lesions and and sera. CONCLUSION: The increased expression of MMP-2, -3, and -9 in skin lesions and sera may reflect the involvement of these enzymes in the mechanism of bulla formation in autoimmune bullous diseases including BP. In addition, expression of MMP and clinical severity may be closely connected.

Cell-matrix adhesions of soft tissue cells around dental implants / 대한치과보철학회지

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.

Cell-matrix adhesions of soft tissue cells around dental implants / 대한치과보철학회지

The importance of soft tissue response to implant abutments has become one of the major issues in current implant dentistry. To date, numerous studies have emphasized on maintaining connective tissue barriers in quantity, as well as in quality for the long term success of dental implants. The cells mainly consisting the soft tissue around dental implants are fibroblasts and epithelial cells. The mechanism of the fibroblasts'adhesions to certain substrata can be explained by the 'focal adhesion'theory. On the other hand, epithelial cells adhere to the substratum via hemidesmosomes. The typical integrin-mediated adhesions of cells to certain matrix are called 'cell-matrix adhsions'. The focal adhesion complex of fibroblasts, in relation to the cell-matrix adhsions, consists of the extracellular matrix(ECM) such as fibronectin, the transmembrane proteins such as integrins, the intracellular cytoplasmic proteins such as vinculin, talin, and more, and the cytoskeletal structures such as filamentous actin and microtubules. The mechanosensory function of integrins and focal adhesion complexes are considered to play a major role in the cells'adhesion, migration, proliferation, differentiation, division, and even apoptosis. The '3-D matrix adhesions'defined by Cukierman et al. makes a promising future for the verification of the actual process of the cell-matrix adhesions in vivo and can be applied to the field of implant dentistry in relation to obtaining strong soft tissue attachment to the implant abutments.

Formation of basement membrane and stratification of Rabbit oral keratinocytes cultured on human acellular dermal matrix

Ultrastructural Changes in Corneas of Diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) Rats

PURPOSE: To evaluate the changes in corneal fine structure of diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats, compared with age-matched nondiabetic rats. METHODS: At the ages of 30 and 50 weeks, we measured the body weight and blood glucose level of OELTF and control rats (n=5 in each age-matched group). Using the transmission electron micrography (TEM), we examined the ultrastructural changes between the corneas of 30-and 50-week-old OLETF and control rats. RESULTS: At the age of 30 and 50 weeks, the mean body weight and blood glucose levels of OLETF rats were significantly higher than those of control rats (p0.05). CONCLUSIONS: The author could observe the changes of the corneal adherent complex and basement membrane in OLETF rats, a new strain of spontaneous non-insulin dependent diabetes mellitus (NIDDM) model. OLETF rats may be useful as an animal model of NIDDM to examine the diabetic corneal changes.

Adhesion Complex in Cultivated Limbal Epithelium on Amniotic Membrane after Transplantation into Chemical Burn Model

PURPOSE: To investigate adhesion complex formation in cultivated human limbal epithelium after transplantation into the chemical burn model. METHODS: human limbal epithelial cells were cultured on amniotic membrane that had not undergone dispase treatment. Laminin V was evaluated using immunohistochemistry. The adhesion complex was examined by electron microscopy. Cultured epithelium was transplanted into limbal deficient rabbits induced by chemical burn and mechanical limbal removal. The transplanted rabbits and the controls with mechanical wounding were sacrificed at 1, 2, 3, and 4 weeks. The adhesion complex was examined by electron microscopy. RESULTS: Linear staining was observed against laminin V at 4-week culture but matured adhesion complex was not found. Graft failure developed in 3 rabbits (25%) after transplantation. Morphologically identifiable hemidesmosomes appeared at 1 week and matured adhesion complex with continuous basement membrane was found at 3 weeks. The mean numbers of hemidesmosomes/2.25 micro meter were 2.3 +/- 0.9, 2.5 +/- 0.5, 5.2 +/- 1.0, and 4.0 +/- 0.9 at 1, 2, 3, and 4 weeks, respectively. The adhesion assembly nearly recovered to the level of that in the human cornea (3.7 +/- 60.11) at 3 weeks. CONCLUSIONS: Adhesion complex of cultivated limbal epithelium did not developed in vitro, but the assembly was almost completed at 3 weeks after transplantation in vivo.

Corneal Epithelial Cell Survival and Electron Microscopic Changes after Ethanol Treatment

PURPOSE: In LASEK models, the viability of corneal epithelial cells after ethanol treatment was investigated in addition to morphological analysis. METHODS: Cell viability was assayed using a confocal laser scanning microscopy(CLSM) and MTT assay for in vivo and cultured rabbit corneal epithelial cells after treatment with various concentrations of ethanol. Ethanol treated human corneas were compared with control in terms of structural changes. RESULTS: CLSM assay showed that cells were viable at the ratio of 78.5, 75.0, 57.3, 43.3, 23.9, 4.3% for 0, 10, 20, 30 40, 60 % ethanol groups, respectively. The relative survival rates in comparison to 0% ethanol-treated (control) corneas were 95.5(10%), 72.9(20%), 55.2(30%), 30.4(40%), 5.5(60%). On MTT assay after 10, 20, 25, 30, 40, 60% ethanol treatment, cultured epithelial cells were still alive at the percentage of 92.7, 92.2, 36.6, 30.7, 5.4, 5.1% (20 sec), 90.8, 57.1, 29.0. 28.6, 5.0, 4.7% (40 sec) at 0 hr with decreasing cell survival over time in 20% ethanol group. TEM showed multiple vacuole-like loosenings along the intercellular junction of superficial squamous and wing cells. The loss and break-up of hemidesmosome and basement membrane were also demonstrated in conjunction with the loosening of sub-basal interface. CONCLUSIONS: After 20% ethanol exposure for 40 seconds, 72.9% and 57.1% of in vivo and cultured rabbit corneal epithelial cells were in viable state with decreasing cell survival over increasing ethanol concentration and time. The loss and break-up of hemidesmosome and basement membrane, resulting in loosening of sub-basal interface, might be the mechanism for the detachment of LASEK flap en bloc.

The Change of Cornea after 20% Ethanol Treatment in LASEK

PURPOSE: Recently, 20% ethanol has been used to make a corneal epithelial flap effectively. We investigated the toxicity of ethanol using the corneal epithelial cell line and rat cornea. METHODS: Cultured corneal epithelial cells were exposed to different concentrations (5~60%) of ethanol for different exposure time (20~120 seconds). And then cell viability was measured with MTT assay. In vivo, rat central corneas (3mm in diameter) were exposed to 20% ethanol for 30 seconds. The eyes were enucleated, fixed and processed for light microscopy (LM) and transmission electron microscopy (TEM) at indicated interval (0, 4, 8 and 12hours, 1, 3 and 7 days). TUNEL assay was used to detect apoptotic cells. RESULTS: Cultured epithelial cells showed survival rate of over 50% in ethanol concentration of below 20% (76.2 +/- 7.4%) and exposure time of below 1 minute (54.8 +/- 6.5%). In vivo experiment, after 20% ethanol exposure for 30 seconds, vacuoles were detected in the nuclei of epithelial cells and gradually disappeared. Hemidesmosomes were detected in the basement membrane at all times. TUNEL positive cells were detected in the epithelial layer and anterior stroma at the edge until 8hrs. CONCLUSIONS: Our results showed 20% ethanol exposure for 30 seconds did not seems to induce significant damage to the cornea. Corneal epithelial damage could occur by physical force during epithelial flap making.

Effect of Corneal Epithelial Flap on the corneal wound healing of canine eyes

PURPOSE: LASEK is a newly developed refractive surgery technique that can make up for the complications from PRK and LASIK. The most unique procedures in LASEK is covering of the cornea with epithelial flap after keratectomy. We examined the effect of corneal epithelial flap on the wound healing of canine cornea. METHODS: Operation was performed in eyes from 12 dogs, and the 12 eyes were recovered with epithelial flap and the remaining 12 eyes were recovered without epithelial flap. Wound healing process was compared using fluorescein staining, light and transmission electron microscopic examination. RESULTS: Fluorescein stained area of the cornea was reduced with time in both groups, and from 9 hours after the operation, it was significantly reduced in the group with epithelial flap compared with those of the group without epithelial flap (p< 0.05). On light microscopic examination of the group with epithelial flap, and normal epithelial structure was found at 24 and 48 hours, respectively. However, in the group without epithelial flap, no complete reepithelialization had occurred on center at 48 hours after the operation. On transmission electron microscopic examination, eyes of the group with epithelial flap showed hemidesmosomes in the area where epithelial flap was closely contacted with the stroma at 24 hours, and they were completely developed at 48 hours. On the other hand, in the group without epithelial flap, hemidesmosomes developed only in the proximal portion but not at the leading edge even at 48 hours. CONCLUSIONS: These results suggest that corneal epithelial flap accelerate the wound healing process of the cornea and the wound healing process depend on the vitality of the epithelial flap.

A Case of Bullous Pemphigoid with Severe Pruritus and Elevated Serum IgE Level / 대한피부과학회지

Bullous Pemphigoid(BP) is a chronic non-hereditary autoimmune disease of the elderly characterized by subepidermal blisters due to autoantibodies targeted to hemidesmosome. An elevated serum IgE level and perepheral blood eosinophilia are observed in one-half of cases though their clinical relevance on disease activity is unclear. A 48-year-old woman presented with severe pruritus and recently developed multiple bullae. IgG antibody was deposited on the epidermal roof side on direct immunofluorescence study and 180 & 230 kD BP antigens were identified with immunoblotting analysis. Pruritus was so severe as not to be controlled by conventional antihistamine therapy, and serum IgE level was highly elevated during the intensely itching period. However, it dropped gradually with improvement of skin lesions and pruritus in response to systemic corticosteroid and azathioprine. This finding suggests a long-term follow-up of IgE levels is necessary to determine the disease activity in a peculiar BP.

Ultrastructural Changes of Corneal Epithelium and Basement Membrane in Neurotrophic Corneal Ulcer

PURPOSE: Neurotrophic keratitis is corneal epithelial defects and stromal thinning associated with loss of sensory function in the ophthalmic branch of the trigeminal nerve. The purpose of this study was to determine the ultrastructural changes of corneal epithelium and basement membrane in neurotrophic keratitis. METHODS: The corneal tissues were obtained from the elevated margin of corneal ulcer in 5 patients with neurotrophic keratitis. Electron microscopic studies were performed. RESULTS: Degenerated epithelial cells, widened intercellular spaces and infiltrated inflammatory cells were observed. In addition, discontinuous basement membrane and loss of adhesion complex including hemidesmosome, anchoring fibril and anchoring plaque were found. CONCLUSIONS: These results suggest that ultrastructural pathology of persistent epithelial defect and inadequate healing of neurotrophic keratitis are loss of adheson complex, discontinuous basement membrane, degenerated epithelial cells, and infiltrating inflammatory cells.

Electron microscopic studies of epithelial adhesion complex of keratoconus

PURPOSE: Keratoconus is a bilateral noninflammatory ecstatic disease of cornea. Clinical manifestations and treatments are well-described , but the exact pathophysiology has many debates. There are many reports on pathologic abnormalities of keratoconus, but few reports on epithelial adhesion complex. The authors investigated the abnormalities in epithelial adhesion complex of keratoconus. METHODS: Using 4 corneas from 4 recipients of penetrating keratoplasty, examination was done with transmission electron microscope (Hitachi-600, Japan) after proper fixation and staining. Central and peripheral portion of each corneal tissues were examined. RESULTS: In two tissues, severe degeneration of basement membrane and Bowman's layer were found. Some degree of abnormalities was found in other tissues, which had minimal change. Some of hemidesmosomes, the most distinct part of adhesion complex, were found only in well-maintained tissue but the distribution was abnormal. CONCLUSIONS: The fact that basal plasma membrane had selectively more degenerations and changes than intercellular plasma membrane implies pathophysiology of keratoconus on adhesion complex, basal plasma membrane, basement membrane and Bowman's layer. Further study on this issue will reveal more information as to its pathophysiology.

The Investigation of the Melanocytes in a Cultured Skin Equivalent Model / 대한해부학회지

Melanocytes grown in the pure monolayer culture lack the three-dimentional organization and the cellular interactions that exist in vivo. These can be partially overcome by growing melanocytes together with other epidermal cells in cultured skin equivalent models. In this study, skin equivalents were prepared by seeding mixtures of cultured human keratinocytes and melanocytes in ratio 10 : 1 onto artificially constructed dermis. They were cultured in DMEM/F12 (1 : 1) for 4 days and then lifted to the air-liquid interface and maintained in DMEM/F12 (3 : 1) for 10 days. Histological and electronmicroscopic examinations of the cultured skin equivalants revealed a structure that closely resembled human interfollicular epidermis; 1. Melanocytes, were identified by positive staining with melanoma-specific antibodies (NKI/C3 and S-100 protein) and prominent cytoplasm with rich cell organelles, were located in the basal layer. 2. Melanocytes contained predominently early stage melanosomes and prominent Golgi apparatus. Mature melanins, were usually abundant in normal skin, were hardly seen both in melanocytes and in neighbouring keratinocytes. 3. Melanocytes were surrounded by keratinocytes but did not form intercellular junctions with them. 4. Keratinocytes were charaterized by microfilament bundles and intercellular junctions such as desmosomes and hemidesmosomes with neighbouring keratinocytes and artificial dermis. The melanocyte in the above skin equivalents had a strong resemblance to the one of normal human skin and therefore this model can be used as artificial skin for the transplantation and in the investigation of melanocytes' role to the UV stimuli.

Effects of Interleukin-6 on Corneal Epithelial Wound Closure after Excimer Laser Surface Ablation in Rabbits

To evaluate the effects of interleukin-6(IL-6) on epithelial migration in corneal epithelial wound healing process after excimer laser surface ablation, the morphologic changes as well as the healing rate of the defect area were observed on 30 rabbits(60 eyes). The animals were divided into three groups of twenty each: the control group and the two IL-6 treated groups(eye dropping with 0.1mg/L and 1.0mg/L of IL-6). On light microscopic examination, after 9 hours, centripetal migration of epithelial cells and covering of bare stroma with monolayered epithelial cells were more remarkable in the 1.0mg/L group than in the control group, reaching the peak at 18 hours. On transmission electron microscopic examination after 18 hour, the 1.0mg/L group showed the loss of hemidesmosome beneath the leading edge of sliding epithelial cell, other intracellular structures were well preserved. On scanning electron microscopic examination, epithelial migration started at 9 hour in all three groups and the epithelial migration was more rapid in the 1.0mg/L group than in the control group. Fifteen hours and afterwards, the healing rate was significantly accelerated in the 1.0mg/L group than in the control group. In view of the above findings, it was speculated that IL-6 could be used effectively in the early treatment of excimer laser keratectomy and other corneal epithelial injury.

Effect of Hyaluronic Acid on Corneal Re-epithelization in Rats following Excimer Laser Keratectomy

The effect of hyaluronic acid(HA) on corneal reepithelization following excimer laser keraectomy was evaluated in rats. An argon fluoride excimer laser(193nm) was used to perform laser keratectomy in each eye of 44 rats (4mm in diameter and 38micrometer in depth). The animals were divided into two guoups: the control group(dropping with PBS) and the HA treated group (dropping with 10mg/ml HA). The healing rate of the epithelial defectarea as well as morphologic changes were observed and the healing response of the cornea was analyzed immunohistochemically to determine the dirsribution of fibronectin. The results were as follows: 1. The reduction of epithelial defect area after 15 hours following laser keratectomy was significantly accelerated in HA treated group compared with the control group(P<0.005). The mean epithelial healing rate was 0.38+/-0.05mm2/hr in the HA treated group and 0.32+/-0.05mm2/hr in the control group. 2. Histologically, remarkable epithelial thickening and less polymorphonuclear leukocyte infiltration under leading epithelium in the HA treated group were observed, which were limited in anterior stroma. 3. Under the transmission electrom microscope, after 72 hours, the intercellular spaces of wing cell layer were bridged by desmosoes more tightly, and remarkable hemidesmosomes were shown in Ha treated group compared with the control group. 4. Fibronectin immunoreactivity observed was under the leading epithelium by using immunohistochemistry at 15 hours following keratectomy in both groups. The results indicate that hyaluronic acid stimulates epithelial wound healing following excimer laser keratectomy by enhancing cell migration and promoting the tight ntercellular junction. The expression of fibronectin was not influenced by HA.

Effect of Topical Na-Hyaluronan on Epithelial Cell Morphogenesis and Superficial Stromal Healing in Corneal Wound

The effect of 1% Na-Hyaluronan(Na-HA) on the morphogenesis of microvilli in superficial epithelial cells and of hemidesmosomes in basal epithelial cells together with the organization of superficial stromal collagen were evaluated in n-heptanol induced corneal epithelial wounds. Epithelial wounds were produced by applying a 5.5mm round filter paper, soaked in n-heptanol, on the central cornea for 60 seconds. 1% Na-HA in phosphate buffered saline(PBS) or PBS alone were instilled 4 times a day for 3 days. Epithelial healing rates determined during the first two days were not altered by Na-HA. The number of microvilli in superficial epithelial cells and of collagens fibers in superficial stroma were approximately the same between two groups. However, the number of hemidesmosome in the central cornea, which was counted in 2micrometer length of the basement membrane, significantly increased by the treatment with 1% Na-HA, being 10.0+/-1.1 in the 1% Na-HA treated group and 6.5+/-2.5 in the control group. The results suggest that topically applied 1% Na-HA may enhance the formation of hemidesmosome in n-heptanol wounded cornea.

Interaction of Attachment Complex and Anchoring Fibrils following Corneal Culture

This study was performed to elucidate the structural changes of the attachment complex in the cultured cornea. The three normal corneal tissues were used in this study. The ultrastructural changes of the attachment complex were observed by electron microscopy in one corneal tissue which was not cultured and cultured for 6 days and two corneal tissues which were cultured 10 days and 20 days, respectively. The cornea cultured for 6 days showed changes in the electron density of the hemidesmosome. The basal lamina was focally detached from the cytoplasmic wall of the basal epithelial cells in the cornea cultured for 10 days. The anchoring fibrils within the nuded corneal stroma, which was cultured for 20 days, were markedly decreased in number. These findings suggest that the normal basal epithelial cell, hemidesmosomes and basal lamina might be the essential factors to maintain the networks of normal anchoring fibrils in corneal stroma.

The Changes of Marginal Epithelial Cells in Corneal Wound Healing

The changes of marginal epithelial cells in corneal wound healing were observed in a rabbit. The randomly assigned three eyes in the rabbits were extracted at first, third, and eighteenth day after full thickness epithelial removal, then observed under the electronmicroscope. At the 1st and 3rd day ,the thickness of the epithelium at the wound margin was reduced at the leading edge. These flattened epithelial cells showed ruffling and folding of the plasma membrane near free edge to form filopodia or lamellipodia processes, extending onto wound surface. Cytoskeletons reorganized and rearranged in leading edge. Basement membrane of the wound was relatively intact, but on which cellular debris were observed, and cell migration undergone and hemidesmosomes developed incompletely. In eighteenth day, basal cell recovered original cylindric shape, cytoskeletons was originally redistributed in cytoplasm after migratory phase, and hemidesmosome developed completely.