Miya Kobayashi

Expression of basigin, a member of the immunoglobulin superfamily, in the mouse central nervous system

Basigin (Bsg) is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. Chicken Bsg (HT7/neurothelin/ 5A11) is expressed in neuroblasts, but disappears from neurons after a specific stage of cytodifferentiation, and becomes restrictedly expressed in the capillary endothelium in the adult brain. We show herein by means of in situ hybridization that Bsg mRNA was expressed in neuroblasts in 13.5 day old mouse embryos. In the adult mouse, Bsg was differentially expressed in subregions of the brain. Strong Bsg expression was detected in the limbic system, including the olfactory system, hippocampal formation, septal area, amygdala, thalamic anterior nuclei, hypothalamus, mesencephalic tegmentum, entorhinal cortex, and cingulate gyrus. Bsg was also intensely expressed in the retinal neuronal layers, the Vth layer of the cerebral neocortex, Purkinje cells of the cerebellum, several nuclei of the brain stem, and the gray matter of the spinal cord. Although in situ hybridization showed a weak signal in the brain capillary endothelium, protein expression of Bsg was strong enough to be detected by immunohistochemistry. Northern blot analysis confirmed the strong expression of Bsg in the central nervous system. Taking into account that Bsg knockout mice exhibit abnormalities in behavior, but a normal blood-brain barrier function, the present findings suggest that Bsg functions actively in neuronal interactions in the central nervous system.

SEPARATION OF CORNEAL STROMA AND DESCEMET’S MEMBRANE DURING DEEP LAMELLAR KERATOPLASTY

Purpose. To study the interface of the corneal stroma and Descemets membrane and to report the histologic findings of the deep corneal stromal tissue removed during deep lamellar keratoplasty (DLKP). Methods. The deep stromal tissues of four corneas were removed during DLKP and were examined histologically by light microscopy (LM) and transmission electron microscopy (TEM). Results. Descemets membrane appeared as a smooth surface under the surgical microscope during the removal of deep corneal stroma. Two of the four excised tissues showed a thin layer attachment to the deep stromal tissue by LM and TEM. The layer measured approximately 3.0 to 4.0 &mgr;m and was observed as a striated structure attached to the stromal tissue of both specimens. The other two specimens contained only stromal tissue. Conclusions. Separation of the deep stromal tissue from Descemets membrane may occur within the Descemets membrane, and the separation is probably between the anterior banded and the posterior nonbanded layer of Descemets membrane in some cases during DLKP. The lamellar structure of the delaminated region suggests a mechanically weak segment of Descemets membrane. The smooth surface of Descemets membrane observed under a surgical microscope is not the actual interface of corneal stroma and Descemets membrane.

Development of ATPase-positive, immature Langerhans cells in the fetal mouse epidermis and their maturation during the early postnatal period

SummaryThe development and maturation of Langerhans cells during the differentiation of skin was studied in mice from fetal day 13 to adult using 3 indices: (1) ATPase activity; (2) ultrastructure; and (3) quantitative evaluation of the cell population.ATPase-positive Langerhans cells appeared in the epidermis at first at fetal day 16, and they increased in number in the differentiating epidermis during the late fetal period. The earliest appearance of Birbeck granules was at postnatal day 4. Cored tubules were also formed in the Langerhans cells in the dermis at around the same age. The cells containing Birbeck granules or cored tubules are considered to be mature Langerhans cells. In the Langerhans-cell lineage, those cells in the epidermis at stages earlier than postnatal day 4 and not yet containing specific organelles are considered to be immature Langerhans cells. These immature Langerhans cells can be identified ultrastructurally in the epidermis at fetal day 16, coinciding with the appearance of ATPase-positive cells. The increase in the number of immature Langerhans cells during the perinatal period was shown by quantitative analysis of nuclear density and relative Langerhans-cell area on the electron micrographs.It is concluded that ATPase is a marker of the Langerhans-cell lineage from the early development stages, while Birbeck granules and cored tubules are markers that identify mature Langerhans cells in electron micrographs.

Elongated dermatan sulphate in post-inflammatory healing skin distributes among collagen fibrils separated by enlarged interfibrillar gaps.

It has been reported that the disaccharide composition of dermatan sulphate shows transient changes after epicutaneous application of the hapten 2,4-dinitrofluorobenzene to mouse skin, and that these changes are most conspicuous in healing skin on day 15 after chemical insult [Kuwaba, Nomura, Irie and Koyama (1999) J. Dermatol. Sci. 19, 23-30]. In the present study it was found that the molecular size of dermatan sulphate was increased on day 15 after hapten application. The molecular size of decorin increased in healing skin, whereas the size of dermatan-sulphate-depleted core protein did not increase. The length and localization of decorin dermatan sulphate were investigated by electron microscopy. Dermatan sulphate filaments oriented orthogonally to collagen fibrils were longer in healing skin than in control skin. In control skin, dermatan sulphate filaments were found among tightly packed collagen fibrils. In contrast, the interfibrillar gaps between each collagen fibril were enlarged in healing skin; elongated dermatan sulphate filaments extended from the surface of collagen fibrils across the enlarged gap. These results suggest that the increase in molecular size of decorin dermatan sulphate is important in organizing collagen fibrils separated by enlarged interfibrillar gaps in healing skin.

Histopathologic Findings of Cornea Verticillata in a Woman Heterozygous for Fabry's Disease

Purpose. To report the histopathologic findings of the cornea verticillata observed in a woman who was heterozygous for Fabrys disease. Method. A 67-year-old woman was found to have a whorl-like corneal opacity on her visit to the Department of Ophthalmology, Aichi Saiseikai Hospital. Her visit was because of a sudden loss of vision in her right eye owing to a central retinal artery occlusion in association with an ophthalmic artery occlusion. The patient died suddenly of an acute heart failure; with family consent, an autopsy was performed and the right eye was removed for histopathologic examination by light and electron microscopy. Results. Low levels of &agr;-galactosidase in the leukocytes together with the corneal finding led to the diagnosis of heterozygous Fabrys disease. Light microscopy revealed a 0.3-to 0.5-&mgr;m thick layer between the epithelial and Bowmans layers. Oil red O positive deposits were accumulated in the subepithelial layer, and the density varied in different regions. Electron microscopy showed that subepithelial layer differed in thickness, and the basal lamina reduplicated regionally. We were not able to determine the structure that correlated with the “ridge” in the central part of the cornea. Conclusion. The oil red O positive deposits and their variation in density in the subepithelial area of the cornea may have caused the characteristic whorl-like corneal opacity in this woman who was heterozygous for Fabrys disease.

Isolation and Culture of Panning Method‐enriched Langerhans Cells from Dispase‐dissociated Epidermal Cells of the Mouse

Langerhans cells (LCs) are bone marrow‐derived, Ia‐positive antigen‐presenting cells in the epidermis which constitute 2–4% of the total epidermal cells. We examined the usefulness of a combination of dispase treatment and the panning method for enriching and culturing mouse LCs. Trunk skin was treated with partially purified dispase (Godo Shusei, type II) to separate epidermal sheets and to dissociate epidermal cells. Suspended cells were treated with ascites or culture supernatant containing anti‐Ia monoclonal antibody, and LCs were enriched by the Ia‐mediated panning method. Per mouse, 3–4 × 105 LCs were recovered with >95% purity and >90% viability. Enriched LCs potently stimulated the allogeneic mixed‐leukocyte reaction. Ultrastructural observations revealed that enriched LCs contained many vesicles but almost no Birbeck granules. A laminal structure, which was apparently adhesive to the surface of LCs, was observed when ascites were employed as the anti‐Ia antibody. These results indicate that a combination of dispase treatment and the Ia‐mediated panning method is very useful for isolating high yields of functionally mature murine Langerhans cells with high purity and viability.

Size control of decorin dermatan sulfate during remodeling of collagen fibrils in healing skin

Recently it has been reported that the molecular size of decorin dermatan sulfate (DS) was increased in healing skin after hapten application and that the elongated DS was distributed in enlarged interfibrillar space among thin collagen fibrils in situ. Here we show that such modulation of the length of decorin DS is temporary. Although the size of decorin DS was evidently increased on day 15, it decreased to almost normal size on day 35 when the altered disaccharide composition of DS was also recovered. Electron microscopic observation revealed that elongated decorin DS was localized among thin collagen fibrils packed loosely in hapten-treated skin on day 15. In contrast, decorin DS of normal size was distributed among thick collagen fibrils packed tightly on day 35. These results suggest that size control of decorin DS plays important roles in organization of collagen fibrils into bundles by regulating interfibrillar space in healing skin, particularly in maturation of collagen fibrils through shortening of decorin DS in later stages of healing.

Surface Ultrastructure of Collagen Fibrils and Their Association With Proteoglycans in Human Cornea and Sclera by Atomic Force Microscopy and Energy-filtering Transmission Electron Microscopy

Purpose. We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using energy-filtering transmission electron microscopy (EF-TEM) and atomic force microscopy (AFM). Methods. Human cornea and sclera were digested with keratanase to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observation, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. Results. With CB staining, numerous CB-positive short filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic collagen fibrils in keratanase-treated corneal collagens were deeper and more regular than in untreated ones. Moreover, the depth and periodicity of keratanase-untreated corneal collagens were shallow and irregular in comparison with keratanase-untreated scleral collagens. On the other hand, there was no difference in depth or regularity between keratanase-treated and -untreated scleral collagen fibrils. Using AFM imaging, additional thin grooves sub-bands were detected on the surface of keratanase-treated corneal collagen fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestion. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. Conclusion. Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with proteoglycans, we conclude that KSPG is found in ample amounts in the human cornea in comparison with sclera. Moreover, we topologically detected KSPG attached to a and c bands of collagen fibrils.

Relationship between morphological changes and endotoxin release induced by carbapenems in Pseudomonas aeruginosa

The relationship between morphological changes and endotoxin release induced in vitro by carbapenems in a clinical isolate of Pseudomonas aeruginosa was examined. The time-course and magnitude of endotoxin release induced varied among imipenem, panipenem, meropenem and biapenem and related to the morphological changes caused by these agents which variously affected cell shape, cell-wall disintegration and cell lysis. The amount of endotoxin released by carbapenem-treated cells correlated with both the cell-wall morphology and bacterial shape immediately before lysis. Meropenem and biapenem caused markedly increased endotoxin release during cell lysis and cell-wall disintegration, whereas imipenem and panipenem caused much less release of endotoxin.

Ultrastructural study on adhesions in internal derangement of the temporomandibular joint

PURPOSE This study examined the ultrastructural characteristics of adhesions in the upper joint compartment of temporomandibular joint (TMJ). MATERIALS AND METHODS Tissue biopsy specimens of adhesions were obtained during arthroscopic operation on 36 joints in 22 patients with internal derangement (ID). The biopsy specimens were examined by light and transmission electron microscopy. RESULTS Adhesions were grossly divided into two types based on arthroscopic observation: 1) a band-like type, which connected the articular fossa and TMJ disc, and 2) a pseudowall-like type, which faced the synovial fluid and was lined by articular tissue. Two types of collagen arrangement were observed at the electron microscopic level: orderly arranged collagen bundles and randomly arranged collagen bundles. Orderly arranged collagen bundles were prominent in the band-like adhesions. In pseudowall-like adhesions, mainly the randomly arranged collagen bundles were seen. However, in some dense fiber parts, orderly arranged collagen bundles also were observed. In other pseudowall-like adhesions, only orderly arranged collagen bundles were seen. Elastic fibers were abundant in some pseudowall-like adhesions with randomly arranged collagen bundles. There were no elastic fibers in the band-like adhesions, some dense fiber parts of the pseudowall-like adhesion, pseudowall-like adhesions consisting of only orderly arranged collagen bundles, and in the synovial membrane. CONCLUSION The different arrangement of collagen fibers and presence or absence of elastic fibers were observed in the two types of adhesions. These findings served to show that extracellular components correspond to a dysfunction involving an ID of TMJ.