The discovery of epidermal tight junctions.

It was previously thought that the skin barrier is composed singly by the stratum corneum. However, this concept was overturned by the report of Tsukita's group in 2002. They convinced us that tight junctions exist in the stratum granulosum of the epidermis, with the constituent proteins being occludin, claudin-1 and claudin-4. However, more than 30 years before this, Hashimoto et al. described the possible existence of tight junctions in the epidermis in 'Intercellular spaces of the human epidermis as demonstrated with lanthanum' in 1971. Dr. Hashimoto observed lanthanum nitrate-injected human skin by electron microscopy. He discovered that the injected lanthanum penetrated the intercellular spaces of the basal and spinous layers of the epidermis and then moved towards the skin surface until penetration was halted in the granular cell layer near the stratum corneum. He described the cell-to-cell adhesion structures that blocked the movement of lanthanum as 'truly tight junctions'. Thus, this was the first description of the existence of tight junctions in the epidermis. However, the presence of these structures was denied by others and was forgotten. Thanks to the discovery of claudin, the existence of tight junctions between epidermal keratinocytes was finally confirmed. It is interesting that Hashimoto's finding was eventually proved to be correct three decades later as a result of progress in molecular biology. This article encourages us to recognize the importance of careful observation in the molecular biology era.

Detection of HLA-DR on microglia in the human brain is a function of both clinical and technical factors.

Detection of HLA-DR, a class II major histocompatibility antigen, on glial cells is dependent not only on duration and type of tissue fixation and processing, but also on clinical factors. Glial cells labeled by anti-HLA-DR were consistent with microglia by light microscopic and ultrastructural criteria, and were colabeled with other microglial markers, including LN-1, Leu-M5, and leukocyte common antigen (LCA). In young and elderly subjects who died suddenly, anti-HLA-DR labeled microglia in the white matter, but far fewer cells in the gray matter. In subjects who died of chronic debilitating illness, such as Alzheimer's disease and carcinomatosis, anti-HLA-DR labeled numerous microglia throughout both the gray and white matter. In Alzheimer's disease, microglia were aggregated in compact senile plaques, but loosely associated with diffuse amyloid deposits. These results suggest that HLA-DR may be constitutively expressed in white matter, but induced in gray matter microglia in chronic disease states or in association with amyloid deposits.

Decreased cytosolic calcium and prostaglandin synthesis in prehypertensive rats.

The capacity of cultured renal medullary interstitial cells derived from Dahl salt-sensitive and salt-resistant rats to synthesize prostaglandin E2 (PGE2) was compared. Basal and arginine vasopressin (AVP)-induced PGE2 production by interstitial cells from salt-resistant rats was fourfold to fivefold higher than corresponding values of those from the salt-sensitive rats. Similarly, basal and AVP-responsive release of [3H]arachidonate were twofold higher by interstitial cells from salt-resistant compared with salt-sensitive rats. Differences in PGE2 production were abolished by the calcium inophore A23187 or the addition of exogenous arachidonate. The latter findings suggested a role for altered availability of endogenous arachidonate, possibly mediated by reduced calcium-responsive lipase activity. Basal and AVP-induced increases in cytosolic free calcium concentration, assessed by the aequorin method, were significantly lower in interstitial cells from salt-sensitive versus salt-resistant rats, further supporting a possible role for altered cellular calcium homeostasis. Studies of the potential contribution of various phospholipases and of triglyceride lipase to the release of arachidonate for PGE2 synthesis in interstitial cells implicated phospholipase A2 activity as a major pathway. When assessed in vitro in cell cytosolic fractions at identical calcium concentration, phospholipase A2 activity was lower in interstitial cells from salt-sensitive versus salt-resistant rats. Thus, both reduced cytosolic free calcium and phospholipase A2 activity of interstitial cells from salt-sensitive rats may contribute to the diminished capacity of these cells to liberate endogenous arachidonate for PGE2 synthesis.

Pressure induced deformation of the interstitial route across synovium and its relation to hydraulic conductance.

The ease with which fluid passes across the synovial lining (i.e., the lining's hydraulic conductance) is enhanced when intraarticular fluid pressure (IAP) is raised acutely to pathological levels in rabbit knees. A structural basis for this pathophysiological change was sought by morphometry of synovial sections from rabbit knees fixed in situ at less than or equal to 5 cm H2O and 25 cm H2O IAP. Light and electron microscopy showed that the main structural changes induced in areolar synovium by raising IAP to 25 cm H2O were (1) a reduction in synovial thickness to 56% control value; (2) an increase in the area of interstitium exposed at the synovial surface (3) an increased proximity of the synovial capillaries to the joint lumen, the mean distance of capillaries from the surface falling from 8.9 +/- 0.5 microns (n = 391) to 3.3 +/- 0.3 microns (n = 92: p less than 0.001). The capillary profiles showed slight compression under 25 cm H2O IAP, but no collapse. The ratio of interstitial area to thickness is the geometric factor governing hydraulic conductance. The maximum change in interstitial area/thickness was 6.8 times for the blood-joint barrier. A change of this magnitude accounts partly (but not fully) for the experimentally observed conductance changes; and it highlights the importance of capillary depth as a factor governing exchange in joints.

Systemic glutathione deficiency in symptom-free HIV-seropositive individuals.

To find out whether systemic glutathione deficiency is associated with human immunodeficiency virus (HIV) infection, thus contributing to the immunodeficiency state, glutathione concentrations in venous plasma and lung epithelial lining fluid (ELF) of symptom-free HIV-seropositive and normal individuals were measured. Total and reduced glutathione concentrations in the plasma of the HIV-infected subjects were about 30% of those in the normal individuals. Concentrations of these substances in the ELF of HIV-infected subjects were about 60% of those in the controls. There was no correlation between ELF and plasma concentrations of total or reduced glutathione. Since glutathione enhances immune function, glutathione deficiency may contribute to the progressive immune dysfunction of HIV infection.

Proliferation of cardiomyocytes and interstitial cells in the cardiac muscle of the mouse during pre- and postnatal development.

Proliferation of cardiomyocytes and interstitial cells in the cardiac ventricle of the mouse during pre- and postnatal development was studied. Furthermore, the number of cardiomyocyte and interstitial cell nuclei per unit area was determined on histological sections. The labelling index of cardiomyocytes decreases from 23% on day 14 of gestation to about zero at 3 weeks after birth. The number of cardiomyocyte nuclei per unit area increases up to day 16 of gestation and then continuously declines. This coincides with the concept that the increase in size of the heart during early fetal life is mainly due to hyperplasia, while during late fetal life and after birth it is mainly, and during adult life exclusively, due to hypertrophy of cardiomyocytes. Proliferation of interstitial cells continues up to 5 days after birth and then decreases. The ratio of cardiomyocytes to interstitial cells decreases by a factor of about 10 between day 14 of gestation and 3 weeks after birth.

Ethylene dimethanesulfonate destroys Leydig cells in the rat testis.

Ultrastructural changes in the interstitial cells of the adult rat testis were studied up to 45 days after administration of a single dose (100 mg/kg) of the antifertility compound ethylene dimethanesulfonate (EDS). Most Leydig cells showed degenerative changes 12 h after treatment. Twenty-four and 48 h after injection, all Leydig cells observed showed gross degenerative changes. At 4 and 14 days, intact Leydig cells could not be identified in the interstitial spaces. Twenty-one days after treatment with EDS, small Leydig cells were visible, and at 45 days, Leydig cells appeared normal. The seminiferous epithelium appeared morphologically normal until 4 days after injection of EDS, when slight abnormalities were observed. At 14 and 21 days, the seminiferous epithelium was grossly abnormal, but at 48 days, spermatogenesis appeared normal. Twelve, 24, and 48 h after treatment, large quantities of material, presumably from dead Leydig cells, were observed within the macrophage cytoplasm. The predominant cell in the interstitial space 4 and 14 days after EDS was the macrophage. Inclusions from the dead Leydig cells within the cytoplasm of the macrophages had almost disappeared. LH receptors (hCG binding) in testicular homogenates were consistent with the cytological changes in Leydig cells. Receptor concentration was low at 24 h and was almost zero at 4 days. This change was accompanied by a decrease in serum testosterone to castrate levels by 2 days. The responses of the endocrine system to destruction of the Leydig cell by EDS, as monitored by serum FSH, LH, and testosterone, were slower than those after castration, indicating that the response to EDS reflects the time required to kill the Leydig cell rather than direct impairment of the steroidogenic pathway. These experiments demonstrate that Leydig cells can be specifically destroyed by a cytotoxic drug. The availability of a specific cytotoxic agent for Leydig cells offers further opportunities to study the interrelationships between the Leydig cell and the seminiferous tubule.

Pulmonary macrophages: alveolar and interstitial populations.

The sizes of the alveolar macrophage (AM) and interstitial macrophage (IM) populations in the lungs of adult Fischer-344 rats were determined during steady state. AM labeled with opsonized erythrocytes during an in situ phagocytic assay were lavaged from excised lungs. The lungs were then dispersed into single-cell suspensions with collagenase and mechanical agitation, and the remaining mononuclear phagocytes were identified following a second labeling step. The size of the AM population was 1.3 X 10(7) cells, or approximately equal to 3% of the total lung cell population. The AM were negative for cytoplasmic myeloperoxidase granules. The size of the IM population was 8 X 10(6) cells, or approximately equal to 2% of the total lung cell population. IM were also negative for myeloperoxidase and, like AM, demonstrated marked Fc gamma R-mediated phagocytic activity. The high cell yields (approximately equal to 4 X 10(8) cells/lung; viability, greater than 85%) and the percentages of type II cells (11%) and ciliated epithelial cells (less than 0.5%) indicated the enzymatic dispersion method resulted in a highly efficient and representative sampling of the lung parenchyma. The collagenase method used in this study to disperse the lung cells into single-cell suspensions, in conjunction with additional cell separation techniques, may be of potential use for isolating enriched populations of IM, as well as other lung cell types, for in vitro study.

[Development of the nasal septum in rabbits. III. Quantitative observations on cells and intercellular substance]. / Die Entwicklung des Septum Nasi beim Kaninchen. III. Quantitative Beobachtungen an Zellen und Interzellularsubstanz.

With the aim to further characterize the various regions of the nasal septum, the following measurements were made: In the thin anterior region and in the thick regions in the middle and posterior parts of the septum the quantitative relationships between cells and intercellular substance as well as the size of the cells were determined. The material used, included transverse and horizontal sections through the nasal septum of rabbits of ages of 1 day and 2, 4, 6, 8, 12 and 24 weeks. The results showed that in the thin anterior region the relationship between cells and intercellular substance remains constant, while in the two thick regions the amount of intercellular substance proportionately increases. In the thick regions, the cells are always larger than in the thin region. The rapid growth of the cells between the 4th and the 6th week coincides with a growth-spurt of the whole head.

Changes in extracellular space in optic nerves of regenerative and nonregenerative animals after hypertonic fixation: implications for axon regeneration.

The optic nerves of some regenerative and nonregenerative animals were compared using electron microscopy, after hypertonic perfusion. Optic axons and glial cell processes separated more readily in regenerative animals, creating large extracellular spaces. In mammals, cell processes remained in close proximity. These findings may indicate lesser adhesion between cell processes in optic nerves of regenerative animals, a characteristic that could allow "potential" space for axon regrowth after nerve injury.

Meningeal relations of the rat hypothalamo-hypophyseal system. Extravascular fluid spaces in and around the median eminence.

Meningeal relations of the rat pituitary and basal hypothalamus including the pituitary stalk and median eminence, were studied by both light and electron microscopy, and by ink perfusion techniques. The dura mater encapsulates the pituitary gland. The arachnoid covers the basal surface of the hypothalamus except the caudal two-thirds of the median eminence and the pituitary stalk. No arachnoidea, i.e. no subarachnoideal space, was found in the sella. Pia mater covers the entire basal surface of the hypothalamus, including the median eminence and the pituitary stalk, but encapsulates only the posterior lobe of the pituitary. The superficial portal vessels (primary plexus and portal veins) are located in the subdural space, covered by the pia mater. The deep vessels in the median eminence are surrounded by the perivascular space, which is bordered by the vascular and neuropil basement membranes, and separated from the external fluid space around the median eminence only by the pia mater. This special topography suggests that molecules present in the portal vessels or in the cerebrospinal fluid, or in and around the pituitary gland, readily move from one liquid space to another.

Stimulating effect of the low dose laser - a new hypothesis.

Increase in the amount of secretary material (interreceptor matrix) surrounding the visual cells was observed after laser treatment. At the same time, an increase in labelled uridine uptake by the pigment epithelium was detected. The increase uptake was highest 4 h after laser treatment.

Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen.

The lungs of rats exposed to 100% O2 until death and to 85% O2 for as long as 14 days were studied using morphometric and biochemical techniques. The primary injury leading to death in rats exposed to 100% O2 was injury to the pulmonary capillary endothelium, where 44% of the endothelial cells were destroyed; there was a corresponding decrease in capillary surface area and in capillary lumen volume. Animals exposed to 85% O2 had proliferation and hypertrophy of alveolar Type II epithelial cells. In addition, 41% of the capillary endothelial cells were destroyed, but the endothelial cells that survived 7 days in 85% O2 were hypertrophied, and after this point no further destruction of the pulmonary capillary bed took place. Exposures to 85% O2 led to enhanced activity of the copper-zinc and manganese superoxide dismutases, which might be related to the apparently adaptive structural changes that occurred in the alveolar Type II epithelium and in the capillary endothelial cells.

Role of monocytes and interstitial cells in the generation of alveolar macrophages II. Kinetic studies after carbon loading.

Increased production of alveolar macrophages after carbon administration to the lung is biphasic; initially the increase is unrelated to cell division in the lung, whereas later, mitotic activity is observed in the interstitium. The role of monocytes and interstitial cells in this dual response is now investigated by injecting 3H-thymidine 1 day before administering 4 mg. of carbon to mice, and following the sequential labeling and grain counts of monocytes, interstitial cells, and free alveolar macrophages. The mice also received colchicine 4 hours before sacrifice. The results suggest that the half life of circulating monocytes is reduced after carbon, indicating that more rapid monocyte production in the marrow is balanced by faster migration from the blood. The kinetic data also suggest that increased cellularity of the interstitium in response to carbon is related initially to monocytic passage from blood to alveoli, and later is associated with division of interstitial cells. The slight increase in mitotic activity observed in alveolar macrophages is not sufficient to account for the large increase in free cells. It is concluded that the adaptive outpouring of macrophages following carbon is an acceleration of the normal dual mechanism whereby most cells are derived from monocytes crossing the interstitium without division and a smaller proportion arising by division of interstitial cells with migration to the alveoli.

Origin of cushion tissue in the developing chick heart: cinematographic recordings of in situ formation.

Differential interference microscopy and time-lapse cinematography were used to determine unequivocally the origin of cushion tissue cells migrating in situ in the atrioventricular region of the embryonic chick heart. These studies have verified the presumed endocardial origin of cushion tissue mesenchyme.

Dorsal root potentials and changes in extracellular potassium in the spinal cord of the frog.

1. In the present study changes in extracellular potassium, ([K]e), were recorded in the isolated spinal cord of the frog with glial cell recordings and K-selective micro-electrodes to test the hypothesis that elevations in [K]e during neuronal activity produce the dorsal root potential (d.r.p.). 2. Sucrose gap recording from the dorsal root (d.r.) was used to record responses to root stimulation and to exogenously applied K+. 3. Stimulation of the ventral root, which elicits a d.r.p. in the frog spinal cord, was not associated with any change in [K]e, suggesting that d.r.p.s produced by ventral root stimulation are not due to changes in [K]e. 4. The largest change in [K]e observed following single stimuli to the dorsal root was 0.4 mM. Such a change in [K]e, if evenly distributed, would depolarize the dorsal root by about 1 mV and yet the simultaneously recorded d.r.p. evoked by stimulating an adjacent dorsal root (d.r.-d.r.p.) was over 10 mV. 5. The time-to-peak of the glidal cell responses was 10 times that of the d.r.-d.r.p. Low frequency (1-10 Hz) d.r. stimulation caused a decremental summation of glial cell responses, while there was no summation in the d.r.-d.r.p. These results suggest that the d.r.p. produced by single d.r. stimulation is generated in large part by a mechanism other than a change in [K]e. 6. During high frequency d.r. stimulation, which evoked 6-8 mM increases in [K]e, the adjacent d.r. was depolarized to a greater extent than that produced by single stimuli. The magnitude of this depolarization was similar to that produced by applying a [K]e equivalent to that observed in the spinal cord during high frequency stimulation. Thus, a substantial component of the sustained d.r. depolarization during high frequency d.r. stimulation may result from changes in [K]e. 7. In the presence of magnesium, high frequency d.r. stimulation evoked a picrotoxin resistant depolarization of an adjacent d.r. whose magnitude correlated well with the changes in [K]e recorded in the spinal cord. 8. In the presence of picrotoxin a slow, long duration depolarization of the d.r. occurred following single stimuli to the adjacent d.r. and the appearance and time course of this response correlated well with the time course of changes in [K]e. 9. Addition of K+ to the Ringer solution in concentrations up to 12 mM had a facilitatory action on reflex activity in the frog spinal cord. 10 The present results suggest that although changes in [K]e play a relatively minor role in generating d.r.p.s. elicited by single d.r. stimulation, the sustained dorsal root depolarization evoked either by high frequency stimulation or by single stimuli in the presence of picrotoxin may be due to a considerable extent to [K]e.