Papulonecrotic tuberculid caused by Bacille Calmette-Guerin vaccination requiring no antitubercular agents.

This paper describes a case of papulonecrotic tuberculid after Bacille Calmette-Guerin (BCG) vaccination. A 6-month-old boy was seen in our emergency room with a fever and erythematous papular. He received BCG vaccination at the age of 5 months and there were no systemic problems after the vaccination. There were no abnormal physical findings except for skin lesions and fever. Histopathologically, the lesion was associated with pyogenic granulomatous inflammation and fibrosis, with multiple instances of vascularization and infiltration of various inflammatory cells. Acid-fast stain, culture,and polymerase chain reaction for tuberculous bacillus DNA of the papulonecrotic skin lesions were negative. He was diagnosed papulonecrotic tuberculid caused by BCG vaccination. The fever lasted 10 days, and all lesions healed spontaneously in 2 months. Papulonecrotic tuberculid is a vasculitis caused by an allergic reaction to an internal focus of Mycobacterium tuberculosis or its metabolites. The appearance of an unexplained skin rash may cause parental anxiety, nevertheless the disease may require no treatment unless the patient is immunocompromised. It is necessary to improve awareness regarding the disease as a side effect of BCG vaccination.

SIRPα signaling regulates podocyte structure and function.

Signal-regulatory protein-α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylation-dependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knockin mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα-mutant mice). Light microscopic examination revealed no apparent morphological abnormalities in the kidneys of the SIRPα-mutant mice. On the other hand, electron microscopic examination revealed abnormal podocytes with irregular major processes and wider and flattened foot processes in the SIRPα-mutant mice compared with their wild-type counterparts. Significantly impaired renal functions and slight albuminuria were demonstrated in the SIRPα-mutant mice. In addition, adriamycin injection induced massive albuminuria together with focal glomerulosclerosis in the SIRPα-mutant mice, while their wild-type counterparts were resistant to adriamycin-induced nephropathy. These data demonstrate that SIRPα is involved in the regulation of podocyte structure and function as a filtration barrier under both physiological and pathological conditions.

Nephrotic syndrome caused by immune-mediated acquired LCAT deficiency.

Lecithin-cholesterol acyltransferase (LCAT) is an enzyme involved in maintaining cholesterol homeostasis. In familial LCAT deficiency (FLD), abnormal lipid deposition causes renal injury and nephrotic syndrome, frequently progressing to ESRD. Here, we describe a 63-year-old Japanese woman with no family history of renal disease who presented with nephrotic syndrome. The laboratory data revealed an extremely low level of serum HDL and undetectable serum LCAT activity. Renal biopsy showed glomerular lipid deposition with prominent accumulation of foam cells, similar to the histologic findings of FLD. In addition, she had subepithelial electron-dense deposits compatible with membranous nephropathy, which are not typical of FLD. A mixing test and coimmunoprecipitation study demonstrated the presence of an inhibitory anti-LCAT antibody in the patient's serum. Immunohistochemistry and immunofluorescence detected LCAT along parts of the glomerular capillary walls, suggesting that LCAT was an antigen responsible for the membranous nephropathy. Treatment with steroids resulted in complete remission of the nephrotic syndrome, normalization of serum LCAT activity and HDL level, and disappearance of foam cell accumulation in renal tissue. In summary, inhibitory anti-LCAT antibody can lead to glomerular lesions similar to those observed in FLD.

Effects of repeated administration of pilocarpine and isoproterenol on aquaporin-5 expression in rat salivary glands.

Aquaporins are water channel proteins which enable rapid water movement across the plasma membrane. Aquaporin-5 (AQP5) is the major aquaporin and is expressed on the apical membrane of salivary gland acinar cells. We examined the effects of repeated administration of pilocarpine, a clinically useful stimulant for salivary fluid secretion, and isoproterenol (IPR), a stimulant for salivary protein secretion, on the abundance of AQP5 protein in rat salivary glands by immunofluorescence microscopy and semi-quantitative immunoblotting. Unexpectedly AQP5 was decreased in pilocarpine-administered salivary glands, in which fluid secretion must be highly stimulated, implying that AQP5 might not be required for fluid secretion at least in pilocarpine-administered state. The abundance of AQP5, on the other hand, was found to be significantly increased in IPR-administered submandibular and parotid glands. To address the possible mechanism of the elevation of AQP5 abundance in IPR-administered animals, changes of AQP5 level in fasting animals, in which the exocytotic events are reduced, were examined. AQP5 was found to be decreased in fasting animals as expected. These results suggested that the elevation of cAMP and/or frequent exocytotic events could increase AQP5 protein. AQP5 expression seems to be easily changed by salivary stimulants, although these changes do not always reflect the ability in salivary fluid secretion.

Function of the membrane water channel aquaporin-5 in the salivary gland.

The process of saliva production in the salivary glands requires transepithelial water transfer from the interstitium to the acinar lumen. There are two transepithelial pathways: the transcellular and paracellular. In the transcellular pathway, the aquaporin water channels induce passive water diffusion across the membrane lipid bilayer. It is well known that aquaporin-5 (AQP5) is expressed in the salivary glands, in which it is mainly localized at the apical membrane of the acinar cells. This suggests the physiological importance of AQP5 in transcellular water transfer. Reduced saliva secretion under pilocarpine stimulation in AQP5-null mice compared with normal mice further indicates the importance of AQP5 in this process, at least in stimulated saliva secretion. Questions remain therefore regarding the role and importance of AQP5 in basal saliva secretion. It has been speculated that there would be some short-term regulation of AQP5 such as a trafficking mechanism to regulate saliva secretion. However, no histochemical evidence of AQP5-trafficking has been found, although some of biochemical analyses suggested that it may occur. There are no reports of human disease caused by AQP5 mutations, but some studies have revealed an abnormal subcellular distribution of AQP5 in patients or animals with xerostomia caused by Sjögren's syndrome and X-irradiation. These findings suggest the possible pathophysiological importance of AQP5 in the salivary glands.

Close association of aquaporin-2 internalization with caveolin-1.

Aquaporin 2 (AQP2) is a membrane water channel protein that traffics between the intracellular membrane compartment and the plasma membrane in a vasopressin-dependent manner in the renal collecting duct cell to control the amount of water reabsorption. We examined the relation between AQP2 internalization from the plasma membrane and caveolin-1, which is a major protein in membrane microdomain caveolae, in Mardin-Darby canine kidney cells expressing human AQP2 (MDCK-hAQP2 cells). Double-immunofluorescence microscopy showed that AQP2 is colocalized with caveolin-1 in the apical plasma membrane by stimulating the intracellular signaling cascade of vasopressin with forskolin. After washing forskolin, both AQP2 and caveolin-1 were internalized to early endosomes and then separately went back to their individual compartments, which are subapical compartments and the apical membrane, respectively.Double-immunogold electron microscopy in ultrathin cryosections confirmed the colocalization of AQP2 with caveolin-1 at caveolar structures on the apical plasma membrane of forskolin-treated cells and the colocalization within the same intracellular vesicles after washing forskolin. A co-immunoprecipitation experiment showed the close interaction between AQP2 and caveolin-1 in forskolin-treated cells and in cells after washing forskolin. These results suggest that a caveolin-1-dependent and possibly caveolar-dependent pathway is a candidate for AQP2 internalization in MDCK cells.

Pre-embedding immunoelectron microscopy of chemically fixed mammalian tissue culture cells.

Immunoelectron microscopy is one of the best methods for detecting and localizing protein molecules in cells and tissues. Gold particles of 1.4 nm in diameter (Nanogold) conjugated with Fab' fragments easily penetrate into the cell interior and are used for pre-embedding immunoelectron microscopy. To obtain a contrast for the gold label, silver enhancement of the gold particles is essential. By changing the intensity of the silver enhancement, the size of the granules can be controlled. In this chapter, we described the use of Nanogold for pre-embedding immunoelectron microscopy of paraformaldehyde-fixed cultured cells.

Double-label immunoelectron microscopy for studying the colocalization of proteins in cultured cells.

Multiple label immunoelectron microscopy localizes and detects multiple antigens in cells and tissues. In double labeling, two kinds of primary antibodies from different animal species are used after being mixed in a single solution. To distinguish the different antigens, secondary antibodies should be labeled with colloidal gold particles of different diameter. Generally, the secondary antibody that is used for detecting the antigen with lower distribution density is labeled with smaller-sized gold particles. In this chapter, double-label immunoelectron microscopy of gelatin-embedded cultured cells using the cryosectioning technique is described.

Effect of passenger position on fear of danger experienced during sudden bus stops.

The purpose of this study was to identify the effect of bus passengers' positions on their fear of danger when a bus stopped suddenly. A temporary bus running course with one bus stop was set up on the campus of the Tokyo University of Agriculture and Technology (TUAT). The bus ran the course 14 times with the bus stopping twice during the course, once at the bus stop and again just after re-starting from the bus stop. The driver was asked to brake more strongly than usual when stopping. Sixteen students (15 males and 1 female) between the ages of 18 and 21 years participated. In turn, all participants were asked to take 14 different postures in the bus. Participants were also asked to report their level of fear on a rating scale each time the bus stopped. The study showed that (1) passengers' fear of danger at the first sudden stop was typically higher than that at the second stop, (2) standing passengers who held hand straps experienced more fear than those who held fixed safety devices, (3) bus passengers sitting on the centre of the rear seat had a great risk of injury if the bus stopped suddenly, and (4) when passengers faced the window and stood transversely with respect to from the moving direction of the bus and the bus stopped suddenly, passengers' fear of danger was affected by the side of the bus on which they stood as well as which hand they used to grasp a safety device.

The primary cilia of secretory cells in the human oviduct mucosa.

The human oviduct is lined with a simple columnar epithelium composed of ciliated cells and secretory cells. Primary cilia or solitary cilia usually extend from the apical surface of the secretory cells. The axoneme of the primary cilia is composed of nine peripheral microtubule doublets (9 + 0 pattern) that lack dynein arms and nexin links. Displacement of peripheral doublets to the central region, which is suggested to be attributable to the lack of nexin links, is one of the distinctive features of oviductal primary cilia. The basal body that extends the primary cilium connects to its paired centriole by the striated connector. The basal body is associated with the accessory structures, such as alar sheets, basal feet, and striated rootlets. Several basal feet project laterally from the basal body. The cap of the basal foot serves as the microtubule organizing center. Several striated rootlets radiate from the basal body toward the nucleus. The basal body, the paired centriole, and the basal body-associated structures are considered to play important roles in the stabilization and fixing of the cilium in the proper position on the apical cell surface.

Immunohistochemical and electron microscopic observations of stromal cells in the human oviduct mucosa.

Stromal cells in the lamina propria of the human oviduct mucosa are unique cells that can differentiate into decidual cells during ectopic pregnancy in the oviduct. The nature of stromal cells is still unknown. In the present study, we investigated human oviductal stromal cells with transmission electron microscopy and immunohistochemistry and revealed that they had ultrastructural features similar to myofibroblasts and expressed alpha-smooth muscle actin, a marker used to identify myofibroblasts. Primary cilia were also one of the characteristic profiles of the stromal cells. These findings showed that the connective tissue-stromal cells in the human oviduct mucosa are myofibroblasts. They are considered to play an important role in the transport of oocytes by bringing about contraction of the mucosal folds.

Immunolocalization of water channel aquaporins in the vomeronasal organ of the rat: expression of AQP4 in neuronal sensory cells.

The vomeronasal organ comprises a pair of narrow tubes in the mammalian nasal septum, serving as a chemosensory system for pheromones. We examined the expression and localization of water channel aquaporins (AQPs) in the rat vomeronasal organ. AQP1 was localized in blood vessels, being particularly abundant in cavernous tissues of the nonsensory mucosa. AQP5 was found in the apical membrane of the gland acinar cells in the vomeronasal organ. AQP3 was detected in the basal cells of the nonsensory epithelium, whereas it was absent in the sensory epithelium. AQP4 was found in both the sensory and the nonsensory epithelia. Interestingly, AQP4 was highly concentrated in the sensory cells of the sensory epithelium. Immunoelectron microscopic examination clearly showed that AQP4 was localized at the plasma membrane in the cell body and lateral membrane of the dendrite, except for the microvillous apical membrane. Nerve fiber bundles emanating from neuronal sensory cells were positive for AQP4, whereby the plasma membrane of each axon was positive for AQP4. These observations clearly show that neuronal sensory cells in the vomeronasal organ are unique in that they express abundant AQP4 at their plasma membrane. This is in marked contrast to the olfactory and central nervous systems, where AQPs are not detectable in neurons, and instead, AQP4 is abundant in the supporting cells and astrocytes surrounding them. The present findings suggest a unique water-handling feature in neuronal sensory cells in the vomeronasal organ.

Experimental model of lacunar infarction in the gyrencephalic brain of the miniature pig: neurological assessment and histological, immunohistochemical, and physiological evaluation of dynamic corticospinal tract deformation.

BACKGROUND AND PURPOSE: Lacunar infarction accounts for 25% of ischemic strokes, but the pathological characteristics have not been investigated systematically. A new experimental model of lacunar infarction in the miniature pig was developed to investigate the pathophysiological changes in the corticospinal tract from the acute to chronic phases. METHODS: Thirty-five miniature pigs underwent transcranial surgery for permanent anterior choroidal artery occlusion. Animals recovered for 24 hours (n=7), 2 (n=5), 3 (n=2), 4 (n=2), 6 (n=1), 7 (n=7), 8 (n=2), and 9 days (n=1), 2 weeks (n=2), 4 weeks (n=3), and more than 4 weeks (n=3). Neurology, electrophysiology, histology, and MRI were performed. Seven additional miniature pigs underwent transient anterior choroidal artery occlusion to study muscle motor-evoked potentials and evaluate corticospinal tract function during transient anterior choroidal artery occlusion. RESULTS: The protocol had a 91.4% success rate in induction of internal capsule infarction 286+/-153 mm(3) (mean+/-SD). Motor-evoked potentials revealed the presence of penumbral tissue in the internal capsule after 6 to 15 minutes anterior choroidal artery occlusion. Total neurological deficit scores of 15.0 (95% CI, 13.5 to 16.4) and 3.4 (0.3 to 6.4) were recorded for permanent anterior choroidal artery occlusion and sham groups, respectively (P<0.001, maximum score 25) with motor deficit scores of 3.4 (95% CI, 2.9 to 4.0) and 0.0 (CI, 0.0 to 0.0), respectively (P<0.001, maximum score 9). Histology revealed that the internal capsule lesion expands gradually from acute to chronic phases. CONCLUSIONS: This new model of lacunar infarction induces a reproducible infarct in subcortical white matter with a measurable functional deficit and evidence of penumbral tissue acutely.

Internalization of caveolae and their relationship with endosomes in cultured human and mouse endothelial cells.

Treatment of cells with pervanadate or vanadate induces the phosphorylation of caveolin-1 and its internalization from the cell surface, but the intracellular fate of caveolae has not been fully elucidated. In the present study, we examined the fate of endocytosed caveolae in human umbilical vein endothelial cells and mouse endothelial KOP2.16 cells. The localization of internalized caveolae and their relationship with the endosomes were examined by immunofluorescence microscopy as well as by immunoprecipitation and chasing of biotinylated transferrin. In untreated cells, caveolin-1 was mostly confined to the cell surface. When cells were treated with either pervanadate for 30 min or vanadate for 3 h, many caveolin-1-labeled vesicles were formed inside the cells, some of which were colocalized with Rab5 or Rab4. The internalized caveolin-1 was colocalized with the endocytosed transferrin in the Rab5-, Rab4- or early endosome antigen-1-labeled compartment where caveolin-1 was phosphorylated. It then moved to the Rabl 1-associated compartment. Immunogold electron microscopy revealed that internalized caveolin-1 colocalized with Rab5 or Rab4 in vesicles larger than caveolae. These results suggest that the internalized caveolae interact with early endosomes.

Differential localization of aquaporin-2 and glucose transporter 4 in polarized MDCK cells.

Membrane water channel aquaporin-2 (AQP2) and glucose transporter 4 (GLUT4) exhibit a common feature in that they are stored in intracellular storage compartments and undergo translocation to the plasma membrane upon hormonal stimulation. We compared the intracellular localization and trafficking of AQP2 and GLUT4 in polarized Madin-Darby canine kidney cells stably transfected with human AQP2 (MDCK-hAQP2) by immunofluorescence microscopy. When expressed in MDCK-hAQP2 cells, GLUT4 and GLUT4-EGFP were predominantly localized in the perinuclear region close to and within the Golgi apparatus, similar to endogenous GLUT4 in adipocytes and myocytes. In addition, GLUT4 was occasionally seen in EEA1-positive early endosomes. AQP2, on the other hand, was sequestered in subapical Rab11-positive vesicles. In the basal state, the intracellular storage site of GLUT4 was distinct from that of AQP2. Forskolin induced translocation of AQP2 from the subapical storage vesicles to the apical plasma membrane, which did not affect GLUT4 localization. When forskolin was washed out, AQP2 was first retrieved to early endosomes from the apical plasma membrane, where it was partly colocalized with GLUT4. AQP2 was then transferred to Rab11-positive storage vesicles. These results show that AQP2 and GLUT4 share a common compartment after retrieval from the plasma membrane, but their storage compartments are distinct from each other in polarized MDCK-hAQP2 cells.

Recent advances in fluorescent labeling techniques for fluorescence microscopy.

Tremendous progress in recent computer-controlled systems for fluorescence and laser-confocal microscopy has provided us with powerful tools to visualize and analyze molecular events in the cells. Various fluorescent staining and labeling techniques have also been developed to be used with these powerful instruments. Fluorescent proteins such as green fluorescent protein (GFP) allow us to directly label particular proteins of interest in living cells. This technique has been extended over a large area of cell biology, and a variety of fluorescent protein-derived techniques have been developed to visualize the functions and conditions of the molecules within living cells. In this review, we summarize the techniques for fluorescent staining and labeling for recent fluorescence microscopy.

Changes of aquaporin 5-distribution during release and reaccumulation of secretory granules in isoproterenol-treated mouse parotid gland.

Water channel aquaporin 5 (AQP5) is present in the apical membrane of the salivary gland acinar cells. We examined changes of AQP5-distribution during the fusion process of secretory granule membranes into the apical membrane and subsequent recovery process in the mouse parotid gland by administering isoproterenol (IPR) in vivo. We performed immunoperoxidase, immunofluorescence and immunoelectron microscopy. In the basal state, AQP5 was localized mainly in the apical membrane of the acinar cell. It was also present in the basolateral membrane to a lesser extent. When IPR was administered to mice, dot-like, vesicle-like and vacuole-like labeling for AQP5 was seen in the subapical regions by light microscopy. By immunoelectron microscopy, AQP5 was localized at both the apical and basolateral plasma membranes in the basal state. At 5 and 30 min after the IPR-administration, acinar lumen became enlarged and small invaginations formed by fusion of secretory granules were seen. AQP5 was positive along the apical plasma membrane and its small invaginations. At 60 min, large invaginations of the lumen were formed. AQP5 remained positive in the membrane of these large invaginations. At 6 h, large invaginations disappeared and AQP5 was localized in the apical plasma membrane. AQP5 was restricted to plasma membranes and continuous invaginations formed by the exocytosis of secretory granules. AQP5 was not detected in the cytoplasm. These observations show that AQP5 does not seem to be endocytosed during the membrane recycling process following the exocytosis.

Immunolocalization of water channel aquaporins in the nasal olfactory mucosa.

Aquaporins (AQPs), membrane water channel proteins expressed in various tissues and organs, serve in the transfer of water and small solutes across the membrane. We raised antibodies to AQPs using isoform-specific synthetic peptides and surveyed their expression in the rat nasal olfactory and respiratory mucosae. AQP1, AQP3, AQP4, and AQP5 were detected by immunohistochemical and immunoblotting analyses. AQP1 was expressed in the endothelial cells of blood vessels and the surrounding connective tissue cells in the olfactory and respiratory mucosae. AQP1 may be involved in water transfer across the blood vessel wall. In the olfactory epithelium, no AQP was detected in the olfactory sensory cells. Instead, AQP3 was abundant in the olfactory epithelium, where it was localized in the supporting cells and basal cells. Expression of AQP3 was mostly restricted to the basal cells in the respiratory epithelium. In marked contrast, AQP4 was abundant in the respiratory epithelium, but its abundance was limited to the basal cells in the olfactory epithelium. In the Bowman's gland, AQP5 was localized in the apical membrane in the secretory acinar cells, whereas AQP3 and AQP4 were found in the basolateral membrane. Similar localization was seen in its duct cells. These results showed a distinct localization pattern for AQPs in the olfactory epithelium. AQP3 and AQP4 in the supporting cells and basal cells may play an important role in generating and maintaining the specific microenvironment around the olfactory sensory cells. AQP3, AQP4, and AQP5 in the Bowman's gland may serve in the secretion to generate the microenvironment at the apical surface of the olfactory dendrites for odorant reception.

Localization of Golgi 58K protein (formiminotransferase cyclodeaminase) to the centrosome.

In vertebrate cells, the centrosome consists of a pair of centrioles and surrounding pericentriolar material. Using anti-Golgi 58K protein antibodies that recognize formiminotransferase cyclodeaminase (FTCD), we investigated its localization to the centrosome in various cultured cells and human oviductal secretory cells by immunohistochemistry. In addition to the Golgi apparatus, FTCD was localized to the centrosome, more abundantly around the mother centriole. The centrosome localization of FTCD continued throughout the cell cycle and was not disrupted after Golgi fragmentation, which was induced by colcemid and brefeldin A. Centriole microtubules are polyglutamylated and stable against tubulin depolymerizing drugs. FTCD in the centrosome may be associated with polyglutamylated residues of centriole microtubules and may play a role in providing centrioles with glutamate produced by cyclodeaminase domains of FTCD.

Apical localization of sodium-dependent glucose transporter SGLT1 is maintained by cholesterol and microtubules.

A GFP-labeled sodium-dependent glucose transporter SGLT1 (SGLT-GFP) was transfected into MDCK cells. SGLT-GFP was localized at the apical membrane in confluent cells. When cellular cholesterol was depleted by methyl-beta-cyclodextrin (MbetaCD) treatment, the localization of SGLT-GFP gradually switched from apical to whole plasma membrane. Time-lapse microscopy revealed that the effect of MbetaCD appeared within 30 min, and that the transition of SGLT-GFP to the whole plasma membrane was completed within 2 hr after the administration. Immunofluorescence microscopy revealed that the tight junction framework remained steady during this process. The effect of MbetaCD on SGLT-GFP localization was counter-balanced by the addition of cholesterol into the culture medium. Disruption of microtubules by colcemid also perturbed SGLT-GFP localization. SGLT-GFP localized to the whole plasma membrane by colcemid treatment, and apical localization was restored within 1 hr after -removal of colcemid. Inhibition of protein synthesis by cycloheximide had no effect on the transition of SGLT-GFP induced by the MbetaCD or colcemid. These results indicated that the apical localization of SGLT-GFP is maintained by cellular cholesterol and microtubules, possibly with an apical recycling machinery.