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PubMed1999

Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway.

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivation of bioactive peptides. Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lacks a signal peptide. To investigate the mechanisms responsible for this secretion, we examined by confocal microscopy the subcellular localization of these two enzymes in the neuroendocrine cell line AtT20. Both EP24.15 and EP24.16 were found by immunohistochemistry to be abundantly expressed in AtT20 cells. Western blotting experiments confirmed that the immunoreactivity detected in the soma of these cells corresponded to previously cloned isoforms of the enzymes. At the subcellular level, both enzymes colocalized extensively with the integral trans-Golgi network protein, syntaxin-6, in the juxtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH. These results demonstrate that both EP24.15 and EP24.16 are present within the classical secretory pathway. Their colocalization with ACTH further suggests that they may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.

Animals↗
PubMed1998

Loss of glucose transporter-2 precedes insulin loss in the nonobese diabetic and the low-dose streptozotocin mouse models: a comparative immunohistochemical study by light and confocal microscopy.

Glucose transporter-2 (glut2) is underexpressed in beta cells of several rodent models of non-insulin-dependent diabetes mellitus (NIDDM). This may also be true for rodent models of insulin-dependent diabetes mellitus (IDDM). The present study examines two murine models of autoimmune IDDM, the nonobese diabetic (NOD) and the low-dose streptozotocin (stz) murine models for changes in the expression of glut2 by double-label light and confocal microscopy during various stages of the disease. The spatial distribution of glut2 cells was also examined in relation to insulin immunoreactive cells and the islet inflammatory cells during these stages. In both the female NOD mouse and the female Swiss mouse without stz treatment, glut2 colocalized with insulin in virtually all the beta cells. In the NOD mouse, islets with moderate to advanced insulitis showed either an absence or considerably reduce expression of glut2 in insulin-containing beta cells. Cells with reduced glut2 expression were usually located adjacent to the region of insulitis. At onset of diabetes, glut2 immunolabeling was reduced despite the preservation of weak insulin immunoreactivity. In Swiss mice treated repeatedly with stz, glut2 labeling began to decline in select Beta cells after the fourth injection in approximately 50% of the islets, despite the lack of insulitis. At this stage expression of glut2 fell in a small number of islets with evidence of early macrophage infiltration. Loss of glut2 became more pronounced in nondiabetic Swiss mice after the fifth injection. At this stage glut2 labeling in the plasma membrane appeared diffuse and variable. At onset of stz-induced diabetes, glut2 expression significantly fell, despite weak immunoreactivity for insulin. This loss was associated with an enhanced influx of both macrophages and T lymphocytes within the islets of diabetes mice. In both the NOD and the low-dose stz mouse models, loss of glut2 thus occurs from an early stage and precedes hyperglycaemia. This loss may be mediated by immune and nonimmune mechanisms.

Animals↗
PubMed2003

The use of fluorescent nuclear dyes and laser scanning confocal microscopy to study the cellular aspects of arterial remodelling in human subjects with critical limb ischaemia.

Resistance arteries isolated from patients with critical limb ischaemia (CLI), a hypotensive/hypoperfusion state of the lower leg, have been shown to undergo morphological changes opposite to those observed in hypertension, that is, decreased wall thickness, reduced cross-sectional area and a decreased wall : lumen ratio. The aim of this present study was to use laser scanning confocal microscopy (LSCM) to study intact resistance arteries isolated from patients with CLI, specifically to identify the cellular aspects of the morphological changes identified in ischaemic subcutaneous and skeletal muscle resistance vessels. Using LSCM, a significant reduction in adventitial and medial thickness, cross-sectional area and wall : lumen ratio was confirmed in resistance arteries from both distal ischaemic subcutaneous and skeletal muscle vascular beds when compared with corresponding arteries from the proximal non-ischaemic sites. The cellular composition of the adventitial, medial and intimal layers of these distal ischaemic arteries was significantly different compared with proximal non-ischaemic arteries. These differences in the distal arteries were characterised by hypoplasia in the adventitial and medial layers of the arterial wall and hypertrophy in the intimal layer. The differences observed in both distal ischaemic vascular beds (subcutaneous and skeletal muscle) were similar.

Aged↗
PubMed2002

Confocal microscopy in vivo in corneas of long-term contact lens wearers.

PURPOSE: To compare keratocyte density, stromal backscatter, epithelial thickness, and corneal sensitivity between corneas of long-term contact lens wearers and those of non-contact lens wearers. METHODS: Twenty corneas of 20 daily contact lens wearers (>10 years' duration) and 20 corneas of 20 age-matched (+/-5 years) control subjects who had never worn contact lenses, were examined by confocal microscopy in vivo. The contact lens wearers removed their lenses 12 to 24 hours before the examination. Full-thickness images were recorded from the central and temporal cornea, and bright objects (keratocyte nuclei) in images were manually counted to calculate keratocyte density. Stromal intensity (backscatter) was measured by calculating the mean grayscale value (corrected for camera and light source variations) from the center of stromal images. Epithelial thickness was determined from the distance between images of the surface epithelium and subbasal nerve plexus. Central corneal sensitivity was measured by Cochet-Bonnet esthesiometry and correlated with the number of nerve fiber bundles in the subbasal nerve plexus. RESULTS: Full-thickness central and temporal keratocyte densities in contact lens wearers were 22,122 +/- 2,676 cells/mm(3) (mean +/- SD) and 20,731 +/- 2,627 cells/mm(3), respectively, and were not significantly different from central and temporal keratocyte densities in control subjects (P = 0.29). The minimum detectable difference in cell density was 11% (2346 cells/mm(3) and 2235 cells/mm(3) in central and temporal stroma, respectively). Temporal epithelial thickness was 46.3 +/- 4.7 microm in contact lens wearers and 50.9 +/- 4.7 microm in control subjects (P = 0.02). Central epithelial thickness and stromal backscatter did not differ between contact lens wearers and control subjects (P > 0.05). Corneal sensitivity was lower in contact lens wearers than it was in control subjects (P = 0.05) and did not correlate with the number of nerve fiber bundles in the subbasal nerve plexus. CONCLUSIONS: Long-term daily contact lens wear and its associated stromal hypoxia and acidosis have no demonstrable effect on keratocyte density. The temporal epithelium is thinner in corneas of long-term contact lens wearers than in control subjects. Decreased corneal sensitivity in contact lens wearers is not accompanied by decreased nerve fiber bundle density.

Adult↗
PubMed2003

Confocal microscopy of Spitzenkörper dynamics during growth and differentiation of rust fungi.

The membrane-selective fluorescent dye FM4-64, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridium dibromide, was used to stain the apical vesicle cluster within the specialized Spitzenkörper of the germ tube of the rust fungi Uromyces vignae and Puccinia graminis f. sp. tritici grown on glass surfaces. The Spitzenkörper stained within 15 min following addition of the dye. Optical sectioning by confocal microscopy of stained hyphal tips showed that the Spitzenkörper was asymmetrically positioned close to the cell-substratum interface during germ tube growth. The Spitzenkörper showed variations in shape and positioning over short (5 s) time intervals. The movement to a new location in the hyphal dome was followed by new growth in that region, consistent with the view that the Spitzenkörper supplies secretory vesicles for germ tube growth. A pronounced Spitzenkörper disappeared at the onset of appressorium differentiation during swelling of the germ tube. However, a stained structure, similar in appearance to a Spitzenkörper, was again observed during the formation of the highly polarized penetration peg.

Basidiomycota↗
PubMed1997

The epidermal nerve fibre network: characterization of nerve fibres in human skin by confocal microscopy and assessment of racial variations.

As a first line of defence, the skin is equipped with a complex and interactive nerve fibre system to detect irritants and maintain homeostasis. The dermal component of this fibre network has been well characterized and fibres are known to extend throughout the viable epidermis as free nerve endings. To date, this epidermal component remains poorly characterized. We have visualized human volar forearm epidermal nerve fibres by laser-scanning confocal microscopy using the pan-neuronal marker, protein gene-product 9.5 and specific antibodies to substance P. calcitonin gene-related peptide and nerve growth factor. In addition to the varicose free nerve endings, there is a 3-D fibre network in normal human epidermis, with frequent branching of fibres. Branching can be seen to converge on a central trunk apparently extending to the dermis. Thin unmyelinated fibres can be seen in all layers of the viable epidermis. Substance P staining is rarely observed and is much less intense than the protein gene-product 9.5 staining. Calcitonin gene-related peptide and nerve growth factor were not detected in volar forearm epidermis by this method. Pretreatment of the skin in vivo with the neuropharmacological agent, capsaicin, resulted in loss of epidermal fibre staining indicating that these are sensory fibres of the primary C-afferent type. Epidermal innervation in racial and ethnic skin types was also assessed. No apparent difference in innervation was observed between European caucasian and Japanese/Chinese skin at the architectural or biochemical level, i.e. the presence, properties and biochemical content of fibres was similar in all cases tested.

Asian People↗
PubMed1996

A quantitative study of the segmental distribution of somitic cells in the developing chick limb bud using laser-scanning confocal microscopy.

Our aim was to map the segmental distribution of somitic cells in the limb before and after the fusion of these cells into myotubes. Somitic cells of the brachial somites were labeled by injection of DiI and DiO into the somitoceles of embryonic Day 2 (E2) embryos. The quantitative distribution of dye-labeled cells from injected somites was examined in whole mounts of E4 wing buds and in sections of E5 wing buds using confocal microscopy. Dye derived from cells of anterior brachial somites 16 and 17 was highly concentrated in the anterior half of E4 wings, whereas dye from posterior brachial somites 20 and 21 was concentrated in the posterior half of the wing. Not more than 14% (on average) of the dye in the wing was found outside the preferred half. Dye was equally dispersed in the anterior and posterior halves of the wing when the middle two somites contributing to the wing musculature, numbers 18 and 19, were injected. The total amount of dye in the whole limb at E4 was not significantly different after injection of similar amounts of dye into either pair of brachial somites 16/17, 18/19, or 20/21. In E5 embryos the distribution of labeled cells in the newly formed muscle masses, identified with antibody 2H2 to myosin light chains, was examined in cryostat cross-sections of the wing. Dye was more widely distributed in the wing than at E4. As much as 32% (on average) of the dye was found in the muscle mass outside the preferred half after administration of dye into the anterior or posterior pair of somites. We conclude that the branchial somites contribute similar numbers of cells to the wing musculature and that the segmental origin of these cells is rigidly maintained during their lateral migration into the limb. Cells from adjacent segments then "mix" when they fuse to form myotubes at E5. In addition, we found a significantly greater amount of dye per unit of muscle mass in the proximal compared with distal parts of the limb. We argue that this finding is due to division of myogenic cells during the course of their migration in the wing.

Animals↗
PubMed2002

Spinning-disk confocal microscopy -- a cutting-edge tool for imaging of membrane traffic.

Confocal laser scanning microscopy is experiencing a revolution in speed from the world of seconds to that of milliseconds. The spinning Nipkow disk method with microlenses has made this remarkable innovation possible. In combination with the ultrahigh-sensitivity, high-speed and high-resolution camera system based on avalanche multiplication of photoconduction, we are now able to observe the extremely dynamic movement of small vesicles in living cells in real time.

Animals↗
PubMed2000

A novel method of virtual histopathology using laser-scanning confocal microscopy in-vitro with untreated fresh specimens from the gastrointestinal mucosa.

BACKGROUND AND STUDY AIMS: Histopathological examination for superficial gastrointestinal lesions has been mainly based upon the light microscopic examination of thin-slice specimens with hematoxylin and eosin (H&E) staining. However, it takes at least a couple of days to create a slide-glass for microscopic study. In order to obtain immediate microscopic images for untreated specimens, the authors used laser-scanning confocal microscopy (LCM) to study fresh samples of gastrointestinal mucosa. MATERIALS AND METHODS: Fresh untreated mucosal specimens from the esophagus, stomach, and colon, obtained by endoscopic pinch biopsy, polypectomy, or endoscopic mucosal resection (EMR), were fixed in normal saline and examined by LCM collecting the reflective light of a 488-nm wavelength argon laser beam. Findings from the LCM image were compared with those of conventional H&E staining in all specimens. For objective evaluation of the similarity of both pictures, the nucleus-to-cytoplasm ratio (N/C) of normal mucosa and that of cancer of the esophagus were calculated and statistically analyzed. The overall diagnostic accuracy for cancer was evaluated. RESULTS: The average scanning time to obtain the LCM image of a specimen was 1.6 seconds. The LCM images acquired corresponded well to the conventional H&E light microscopic images in the esophagus, stomach, and colon. Cell wall, nucleus, cytoplasm, and tissue structural elements were simultaneously visualized by LCM scanning. A difference in N/C ratios between normal mucosa and cancer in the esophagus was statistically apparent when Welch's test (P=0.05) was applied. The overall diagnostic accuracy of the LCM study for cancer was 89.7%. CONCLUSIONS: This novel method enables us to obtain an immediate serial virtual microscopic section through a fresh specimen, which has not actually been cut, although the resolution of the image obtained is still limited. These early results encourage us to develop imaging relevant to conventional histopathology alongside the development of LCM technology in the near future. We should aim at the in vivo application of LCM coupled to probes which can be introduced through the working channel of endoscopes.

Colon↗
PubMed2001

Statistical evaluation of confocal microscopy images.

BACKGROUND: The coefficient of variation (CV) is defined as the standard deviation (sigma) of the fluorescent intensity of a population of beads or pixels expressed as a proportion or percentage of the mean (mu) intensity (CV = sigma/mu). The field of flow cytometry has used the CV of a population of bead intensities to determine if the flow cytometer is aligned correctly and performing properly. In a similar manner, the analysis of CV has been applied to the confocal laser scanning microscope (CLSM) to determine machine performance and sensitivity. METHODS: Instead of measuring 10,000 beads using a flow cytometer and determining the CV of this distribution of intensities, thousands of pixels are measured from within one homogeneous Spherotech 10-microm bead. Similar to a typical flow cytometry population that consists of 10,000 beads, a CLSM scanned image consists of a distribution of pixel intensities representing a population of approximately 100,000 pixels. In order to perform this test properly, it is important to have a population of homogeneous particles. A biological particle usually has heterogeneous pixel intensities that correspond to the details in the biological image and thus shows more variability as a test particle. RESULTS: The bead CV consisting of a population of pixel intensities is dependent on a number of machine variables that include frame averaging, photomultiplier tube (PMT) voltage, PMT noise, and laser power. The relationship among these variables suggests that the machine should be operated with lower PMT values in order to generate superior image quality. If this cannot be achieved, frame averaging will be necessary to reduce the CV and improve image quality. There is more image noise at higher PMT settings, making it is necessary to average more frames to reduce the CV values and improve image quality. The sensitivity of a system is related to system noise, laser light efficiency, and proper system alignment. It is possible to compare different systems for system performance and sensitivity if the laser power is maintained at a constant value. Using this bead CV test, 1 mW of 488 nm laser light measured on the scan head yielded a CV value of 4% with a Leica TCS-SP1 (75-mW argon-krypton laser) and a CV value of 1.3% with a Zeiss 510 (25-mW argon laser). A biological particle shows the same relationship between laser power, averaging, PMT voltage, and CV as do the beads. However, because the biological particle has heterogeneous pixel intensities, there is more particle variability, which does not make as useful as a test particle. CONCLUSIONS: This CV analysis of a 10-microm Spherotech fluorescent bead can help determine the sensitivity in a confocal microscope and the system performance. The relationship among the factors that influence image quality is explained from a statistical endpoint. The data obtained from this test provides a systematic method of reducing noise and increasing image clarity. Many components of a CLSM, including laser power, laser stability, PMT functionality, and alignment, influence the CV and determine if the equipment is performing properly. Preliminary results have shown that the bead CV can be used to compare different confocal microscopy systems with regard to performance and sensitivity. The test appears to be analogous to CV tests made on the flow cytometer to assess instrument performance and sensitivity. Published 2001 Wiley-Liss, Inc.

Calibration↗
PubMed2000

A method for measuring colocalization of presynaptic markers with anatomically labeled axons using double label immunofluorescence and confocal microscopy.

Information concerning the location and distribution of presynaptic neurotransmitter release sites within anatomically labeled axons would be of value for a large number of studies in functional anatomy, development, and plasticity. Here we report a method for localizing presynaptic sites within identified arbors of interest using anterograde anatomical tracer injections to label axonal projections and synaptic vesicle protein (SVP) antibodies to label presumptive presynaptic terminals. The axons and presynaptic sites are independently visualized with double label immunofluorescence and confocal microscopy. Stacks of images representing adjacent focal planes are collected, and image processing techniques are applied to identify the location of each axonal branch segment and each cluster of SVP label in three-dimensional space. Segmentation of the SVP label into distinct pixel clusters in three-dimensional space, followed by colocalization of these clusters with the labeled axons (object-based analysis), yields much more reliable and sensitive measures of colocalization than a simple determination of the number (or summed intensities) of colocalized pixels in a single optical section (pixel-based analysis). The method has been extended to measure the colocalization of antigens that are not located at the presynaptic terminal with a labeled population of axons.

Animals↗
PubMed1991

Tandem scanning confocal microscopy (TSCM) of normal and ischemic living kidneys.

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20 x water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing rapidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush borders, and closure of the peritubular capillary spaces. Upon release of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.

Animals↗
PubMed2002

Confocal microscopy of a dense particle system.

We report experiments utilizing confocal microscopy to determine the position of cornflour starch granules in a paste, as a function of distance from a wall. The granules have an average diameter of 15 mum. If a solvent is chosen such as to approximately match the refractive index of the granules then images of them can be obtained to a depth of 120 mum, which is the limit imposed by the working distance of the x 40 oil immersion lens used in the experiment. An algorithm is presented which successfully identifies the particle centers in 3D. In addition to experiments on static systems we show how measurements of particle number density and velocity can be obtained from images of a system in pipe flow. We find that particles are depleted from the wall over a range of around 100 mum.

Journal Article↗
PubMed2002

Effect of myopic laser in situ keratomileusis on epithelial and stromal thickness: a confocal microscopy study.

PURPOSE: To determine changes in central epithelial and stromal thickness in human corneas in vivo after laser in situ keratomileusis (LASIK). DESIGN: Prospective, nonrandomized, comparative trial. PARTICIPANTS: Eighteen eyes of 12 patients received LASIK (performed using the VISX Star laser [VISX, Santa Ana, CA]) with a planned 180- micro m flap (created using an automated Hansatome microkeratome [Bausch & Lomb, Irvine, CA]) to correct refractive errors between -2.0 diopters (D) and -11.0 D. METHODS: Corneas were examined by using confocal microscopy in vivo before LASIK and at 1 week and 1, 3, 6, and 12 months after LASIK. Epithelial thickness was the distance between images of the surface epithelium and subbasal nerve plexus or, when nerves were not visible, the subbasal peak (if present in the light intensity profile). Total flap thickness was the distance between images of the surface epithelium and interface debris (or peak), and total stromal thickness was the distance between images of the most anterior keratocytes and endothelium. MAIN OUTCOME MEASURES: Corneal epithelial and stromal thickness. RESULTS: Epithelial thickness before LASIK was 46 +/- 5 micro m (mean +/- standard deviation) and increased 22% by 1 month after LASIK (56 +/- 5 micro m; P = 0.01). Thereafter, epithelial thickness did not change, but remained thicker at 12 months after LASIK (54 +/- 8 micro m) than before LASIK (P = 0.02). Total flap thickness at 1 month after LASIK was 160 +/-28 micro m and did not change thereafter. Changes in total stromal thickness between 1 and 12 months after LASIK were not significant. CONCLUSIONS: The central corneal epithelium was thicker in the first year after LASIK than before LASIK. There was no change in central stromal thickness between 1 month and 12 months after LASIK.

Adult↗
PubMed2001

Confocal microscopy of the normal human cornea.

The slit-scanning confocal microscope is a new clinical paradigm that allows the living human cornea to be viewed at a magnification of 680 x and a lateral resolution of 1 mum. As such, it allows corneal morphology to be inspected at a cellular level. The corneas of both eyes of 119 subjects who were evenly distributed in age from 10-80 years were examined using a Tomey ConfoScan P4 in-vivo slit-scanning real-time confocal microscope (Erlangen, Germany). Good quality representative images of the various corneal layers were selected for detailed qualitative analysis and are displayed here. A grid of corneal layer versus age was constructed from these images; this tool can be used as a normative confocal microscopy reference against which suspected corneal abnormalities can be assessed.

Journal Article↗
PubMed1994

Calcium waves and dynamics visualized by confocal microscopy in Xenopus oocytes expressing cloned TRH receptors.

Laser scanning confocal microscopy was used to analyse changes in free cytosolic calcium ([Ca2+]i) in Xenopus laevis oocytes expressing the cloned rat TRH receptor in response to TRH. In oocytes expressing TRH receptors, TRH invariably evoked a dose-dependent, biphasic calcium response. This response consisted of an initial transient planar wave of calcium propagating just below the surface of the membrane followed by a slower, secondary calcium phase. The TRH antagonist, chlordiazepoxide, markedly inhibited this calcium wave. The origins of calcium involved in this biphasic response were investigated using a variety of intra- and extra-cellular calcium antagonists. The intracellular calcium antagonists thapsigargin and TMB-8 reduced the initial and to a lesser extent the secondary phase of the planar calcium wave. In contrast, EGTA and the calcium channel blocker nifedipine produced a profound inhibition of the secondary phase while the initial phase was only slightly reduced. These results indicate that the release of intracellular calcium is predominantly responsible for the initial phase of the calcium wave while the influx of extracellular calcium is mainly involved in the secondary phase. Qualitative changes in the patterns of calcium release induced by TRH were observed following pretreatment with intracellular calcium antagonists. Following pretreatment with these compounds, TRH induced spiral or regenerative calcium waves. Addition of EGTA to the extracellular medium did not alter these responses confirming the importance of intracellular calcium in the generation of these spiral calcium waves. This study demonstrates the nature and multiplicity of regulating mechanisms of [Ca2+]i following activation of TRH receptors expressed in Xenopus oocytes.

Animals↗
PubMed1998

Confocal microscopy assessment of lymphoid tissues with follicular hyperplasia from patients infected with human immunodeficiency virus type 1.

OBJECTIVE: To characterize human immunodeficiency virus (HIV) infection of lymphoid tissues during follicular hyperplasia. METHODS: We examined 10 tonsil/adenoid, 3 parotid lymphoepithelial cyst, and 7 lymph node specimens that had been surgically removed from 13 patients infected with HIV-1. Characteristics of productive HIV-1 infection were assessed using immunocytochemistry for HIV-1 p24. Cellular colocalization was determined with the aid of a confocal microscope using double immunofluorescent staining for HIV-1 p24 and cell-specific markers. RESULTS: All specimens showed follicular hyperplasia. Using confocal microscopy with three-dimensional reconstruction, HIV-1 p24 was seen to be "intimately" colocalized with CD21 within the germinal centers. While lymphoid follicles were generally hyperplastic, only a subset of these follicles contained HIV-1 p24. Occasional HIV-1-expressing mononuclear cells identified outside follicles stained for CD68 or CD3. CONCLUSIONS: The differential involvement of hyperplastic follicles by HIV-1 within individual lymphoid tissues and the intimate colocalization of HIV-1 p24 and CD21 suggest that infected follicular dendritic cells may be an important reservoir of HIV-1 during follicular hyperplasia.

Adult↗
PubMed2004

Dynamics of endocytic traffic of Entamoeba histolytica revealed by confocal microscopy and flow cytometry.

Entamoeba histolytica, the protozoan parasite of humans, manifests constitutive endocytosis to obtain nutrients and, when induced to express invasive behavior, as a means of ingesting and processing host cells and tissue debris. E. histolytica trophozoites were grown in liquid axenic medium that contained fluorescently labeled fluid-phase markers, so that the kinetics of uptake, the transit of loaded endosomes through the cytoplasm, and the time of release of the markers could be monitored by flow cytometry. Confocal microscopy of live trophozoites revealed uptake of fluid by avid macropinocytosis and the occurrence of fusion between young and older endosomes, as well as between pinosomes and phagosomes containing bacteria. Endosomes were rapidly acidified, then gradually neutralized; finally, indigestible material was released. Transit of endosomes containing fluid-phase markers required about 2 h. Uptake and release of fluid-phase markers were impaired by drugs that inhibited actin dynamics and actin-myosin interaction; uptake was also impaired by inhibition of PI 3-kinase. A striking feature of the trophozoites was the great heterogeneity of their endocytic behavior.

Actin Cytoskeleton↗