Fibrous components of the extracellular matrix are light-microscopically classified into three types of fibers: collagen, reticular and elastic. The present study reviews the ultrastructure of these fibrous components as based on our previous studies by light, electron, and atomic force microscopy.
  Collagen fibers present a cord- or tape-shape 1-20 μm wide and run a wavy course in tissues. These fibers consist of closely packed thin collagen fibrils (30-100 nm thick in ordinary tissues of mammals), and exhibit splitting and joining in altering the number of the fibrils to form a three-dimensional network as a whole. Individual collagen fibrils (i.e., unit fibrils) in collagen fibers have a characteristic D-banding pattern whose length is ranges from 64 to 67 nm, depending on tissues and organs. During fibrogenesis, collagen fibrils are considered to be produced by fusing short and thin fibrils with tapered ends.
  Reticular fibers are usually observed as a delicate meshwork of fine fibrils stained black by the silver impregnation method. They usually underlie the epithelium and cover the surface of such cells of muscle cells, adipose cells and Schwann cells. Electron-microscopically, reticular fibers are observed as individual collagen fibrils or a small bundle of the fibrils, although the diameter of the fibrils is thin (about 30 nm) and uniform. Reticular fibers are continuous with collagen fibers through the exchange of these collagen fibrils. In silver-impregnated specimens, individual fibrils in reticular fibers are densely coated with coarse metal particles, probably due to the high content of glycoproteins around the fibrils.
  Elastic fibers and laminae are composed of microfibrils and elastin components. Observations of the extracted elastin have revealed that elastin components are comprised of elastin fibrils about 0.1-0.2μm thick. Elastic fibers and laminae are continuous with networks and/or bundles of microfibrils (or oxytalan fibers), and form an elastic network specific to individual tissues.
The fibrous components of the extracellular matrix are thereby morphologically categorized into two systems: the collagen fibrillar system as a supporting framework of tissues and cells, and the microfibril-elastin system for uniformly distributing stress to maintain the resilience adapted to local tissue requirements.

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This review proposes the possibility that malarial protection might not be achieved through the process of acquired immunity in which the constituents are conventional T and B (B-2) cells. On the other hand, malarial protection might be achieved by the process of innate immunity in which the constituents are extrathymic T cells and autoantibody-producing B-1 cells. Accordingly, mice infected with malaria exhibited severe thymic atrophy, and the expansion of IL-2Rβ⁺CD3^int cells and its subset of NK1.1^-CD3^int cells were simultaneously induced. In parallel with the expansion of extrathymic T cells in the liver, extramedullary erythropoiesis was found to begin in the liver of these mice. Interestingly, malarial protozoa were primarily seen in only these nucleated erythrocytes in the liver at the early stage of infection. These results suggest that malaria immunology falls into a new field of immunology, namely, innate immunity. The similarity of the immune states among malaria, aging, and autoimmune diseases also suggest that the immunosuppression of a conventional, acquired immune system is more likely the common mechanism underlying these diseases or physiological responses.

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We investigated the differential distribution of basement membrane type IV collagen α chains in the mouse brain by immunohistochemistry using α chain-specific monoclonal antibodies. Subendothelial basement membranes were found to contain α1 and α2 chains. Basement membranes surrounding smooth muscle cells on blood vascular walls were immunoreactive for α1 and α2 chains but not for α5 and α6 chains. Interestingly, the pia mater contained a thin basement membrane which was positive for α1, α2, α5, and α6 chains, suggesting that glia limitans superficialis coheres basement membranes containing [α1(IV)]₂α2(IV) and [α5(IV)]₂α6(IV) molecules. In contrast, capillaries always possessed thin basement membranes of [α1(IV)]₂α2(IV) molecules.
  Cerebrospinal fluid is produced through filtration of blood at the choroid plexus, where two distinct basement membranes were detected by anti-α1 and anti-α2 antibodies. The subendothelial basement membrane appeared to consist of
[α1(IV)]₂α2(IV) molecules, whereas the subependymal basement membrane in the choroid plexus was strongly positive for α3, α4, and α5 chains, indicating that the filtering unit was composed of α3(IV)α4(IV)α5(IV) molecules. That the specific localizations of these molecules are shared by renal glomeruli and the choroid plexus leads us to hypothesize that the supramolecular network containing α3(IV) α4(IV)α5(IV) molecules may function as a permeability selective barrier.

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Apoptosis commonly occurs in a variety of developmental processes in mammals. In this study, we investigated the relationship between apoptosis and the expression of both Bax and Bcl-2 during the early organogenesis period (9.5-11.5 days of gestation) of rat embryos. Apoptotic cells detected by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) method were extremely abundant in the foregut diverticulum at 9.5 days of gestation, while they largely disappeared at 10.5 and 11.5 days of gestation, although they were detected in newly formed mid- and hindgut diverticulum at these times. Real-time RT-PCR analysis of whole embryos revealed that the expression of bax mRNA was constant at days 9.5 to 11.5, while the expression of bcl-2 mRNA gradually increased.
Immunohistochemical studies of Bax and Bcl-2 expression revealed that these apoptotic cells were exactly positive to Bax in mirror sections, while their expression of Bcl-2 was generally too low to be detected. A disappearance of apoptotic cells was associated with strong Bcl-2 expression in the foregut diverticulum at 10.5 and 11.5 days of gestation. It was similarly observed that apoptotic cells detected in the cardiogenic area at 9.5 days of gestation disappeared with the formation of the primitive heart tube - accompanied by a strong expression of both Bcl-2 and Bax - in the developmental process of the primitive heart. Apoptotic cells were also observed in the primitive brain vesicle, optic vesicle, otic vesicle, and thyroid primordium at l0.5 and 11.5 days of gestation during the developmental process, with a strong expression of Bax. These results indicate that the Bax and Bcl-2 may be important in regulating the induction of embryonic cell apoptosis during early organogenesis.

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The olfactory bulb of the musk shrew, Suncus murinus, is characterized by the presence of various interneurons. Our previous report (KAKUTA et al., 2001) demonstrated that positive immunoreactions for calretinin were observed in periglomerular and perinidal cells in the glomerular layer, small ovoid neurons in the external plexiform layer, and granule cells in the granule cell layer of the olfactory bulb in the musk shrew aged 1 to 5 weeks, in addition to calretinin-immunoreactive bipolar cells distributed in the anterior subependymal layer and in each layer of the olfactory bulb. To examine the origin and migration of interneurons of the olfactory bulb, we labeled generated cells by injecting 28-day-old musk shrews with 5-bromo-2’-deoxyuridine (BrdU), and detected the labeled progeny cells that survived after several intervals. BrdU-labeled cells originated in the subependymal layer around the anterior horn of the lateral ventricle, and rostrally migrated in the subependymal layer from the anterior wall of the lateral ventricle into the center of the olfactory bulb, where they radially migrated into the granule cell layer, external plexiform layer, and glomerular layer. It took 2 days to migrate rostrally in the subependymal layer from the anterior lateral ventricle to the center of the olfactory bulb, and 2 to 6 days to migrate radially from the bulbar subependymal layer into the three layers mentioned. The rate of rostralward migration of the labeled cells was estimated to be 38 μm/h, while that of radial migration, 7 to 25 μm/h. The present BrdU-labeling study, together with our previous immunohistochemical study (KAKUTA et al., 2001), indicates that anterior subependymal cells differentiate into granule cells in the granule cell layer, into Van Gehuchten cells in the external plexiform layer, and into periglomerular and perinidal cells in the glomerular layer of the olfactory bulb in the musk shrew.

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We investigated the relationship between the motility of hepatic Ito cells and their myofibroblastic transformation in cultures. Ito cells were freshly isolated from rat liver and cultured in a 10% FBS-supplemented medium. On day 2 after beginning the culture, transmission electron microscopy and oil red O staining showed that Ito cells possessed numerous lipid droplets but not actin filaments in the cytoplasm. On day 8, actin filaments were abundantly found in the cytoplasm, whereas lipid droplets dramatically decreased in number. Western blot analysis also demonstrated that protein levels of α-smooth muscle actin in the cell markedly increased with time, but no obvious change was detected in those of desmin and tubulin β. In Boyden’s chamber assay, the migration of Ito cells, which was induced by platelet-derived growth factor-BB (PDGF-BB), was activated in a time-dependent manner. This migration of transformed Ito cells was independent of the degree of their adhesion to various substances of the extracellular matrix. Among these molecules, laminin showed the highest effect upon the migratory activity. The migration of transformed Ito cells on laminin was completely inhibited by cyto-chalasin D, colchicine, or taxol. Furthermore, their adhesion to the matrix was also decreased by cytochalasin D or colchicine, but not by taxol. These findings indicate that the motility of Ito cells is acquired in conjunction with their myofibroblastic transformation, which is accompanied by morphological changes with a new organization of actin filaments. The results also suggest that microtubules as well as the extracellular matrix are deeply associated with the motility of Ito cells.

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The question of whether odontoblast processes extend to the dentinal surface has been widely debated in previous studies. In this study odontoblast processes were investigated in the developing and aging dentin of rats and monkeys (Japanese macaques). For this purpose, F-actin of microfilaments and cellular membranes were stained with phalloidin and DiI, respectively. This dual staining demonstrated that positive signals for odontoblast processes were present in the dentinal surface in both the cusp and cervical regions of the dentin at 2 weeks of age. The tips of doubly positive processes were detectable in the dentinal surface in the cusp region even at 100 weeks of age, whereas in the cervical region they were retracted from the dentinal surface towards the pulp during the period of 3-6 weeks of age. During these stages, phalloidin-positive signals showing retracted odontoblast processes in the cervical region were closely associated with the interglobular dentin that was stained with sWGA-lectin. After 6 weeks of age, no association was observed between the processes and the interglobular dentin, since they were retracted approximately to the inner third portion of the dentinal tubules. This staining pattern can be detected until 100 weeks of age. Moreover, different distribution patterns of odontoblast processes between the two dentinal regions were also confirmed in dentin of monkey teeth. These results suggest that the existence of the regional differences in the extent of the odontoblast processes in the dentin, i.e., the persistence of the processes in the dentinal surface in the cusp region and their retraction from the dentinal surface in the cervical region.

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The synovial intima is composed of two types of synoviocytes: absorptive macrophages and secretory, fibroblast-like F cells. Many studies have tried to observe synoviocytes by scanning electron microscopy (SEM) but failed to reveal the entire shape of synoviocytes because they are deeply embedded in the interstitial matrix. The present study, primarily employing SEM observation of NaOH macerated samples, reveals the distribution and three-dimensional ultrastructure of the synoviocytes in the normal knee joint of rabbits, and the morphological changes of synoviocytes in an osteoarthritis model of this animal. F cells were broadly distributed throughout the synovial intima, while macrophages showed a restricted distribution on fatty tissues around the patella. F cells were classified into a flat type, which covered the surface of synovial membrane like an epithelium, and a dendritic type, which extended long processes to form a characteristic meshwork on the surface. The flat type predominated in regions adhering to the femur, while the dendritic type predominated in ambilateral parts of both the patella and tendon of the musculus quadriceps femoris, and on the peripatellar fatty tissue. Intermediate forms of flat and dendritic types appeared in middle regions between the patella and periphery of the joint capsule. In the synovial membrane of the osteoarthritis model, both types of synoviocytes increased in number and changed their morphology, indicating their elevated activities in absorption and secretion. It is suggested that the ultrastructural changes in synoviocytes reflect pathological conditions of the synovial membrane, and synoviocytes play important roles in the pathogenesis of osteoarthritis.

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The basal layer of the epithelium is usually thought to contain stem or progenitor cell populations. In order to analyze its biological characteristics, we attempted to produce novel monoclonal antibodies recognizing basal cells of the human esophageal epithelium. Hybridomas were generated from fusions between spleen cells of ddY mice immunized with esophageal epithelial cells (EECs) cultured at low calcium concentrations. A clone, NJ-E-H10, producing a monoclonal antibody (moAb) reacting with basal cells of human esophageal epithelia, was selected for its staining pattern on frozen sections of the epithelia. The immunoreactivities of NJ-E-H10 were detected in the cytoplasm of basal cells predominantly located in the interpapillary zones of the epithelia. In the primary culture of EECs, immunoreactive cells were present at the marginal area of the growing colonies, where EECs extend their cytoplasm and migrate out of the colonies. Immunoelectron microscopy demonstrated the immunoreactivities of the moAb NJ-E-H10 around the intermediate filaments of basal cells, but not on the filaments themselves. In the human skin, NJ-E-H10 positive cells were also observed in the basal layer of the epidermis as well as in keratinocytes in the outer layer of the outer root sheath in hair follicles and myoepitheial cells in the sweat glands. Since the distribution of NJ-E-H10 immunoreactivities differs from that reported by hithertoknown antibodies, the MoAb NJ-E-H10 is considered a new marker for clarifing the biological properties of the basal cell compartment in stratified epithelia.

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