Attachment Zone Research Articles

Distribution of MHC class I and of MHC class II molecules in macrophages infected with Leishmania amazonensis.

Macrophages, being apparently the only cells that in vivo allow the growth of the intracellular pathogen Leishmania, are likely candidates to present antigens to Leishmania-specific CD4+ and CD8+ T lymphocytes, known to be involved in the resolution or in the development of lesions induced by these parasites, and recognizing processed antigens bound to MHC class I and MHC class II molecules, respectively. In the present study, we analysed by confocal microscopy and by immunoelectron microscopy the subcellular distribution of both MHC class I and class II molecules in mouse (Balb/c and C57BL/6 strains) bone marrow-derived macrophages infected for 12 to 48 hours with Leishmania amazonensis amastigotes and activated with gamma interferon to determine the intracellular sites where Leishmania antigens and MHC molecules meet and can possibly interact. Double labelings with anti-MHC molecule antibodies and with either propidium iodide or an anti-amastigote antibody allowed localization of MHC molecules with regard to the endocytic compartments housing Leishmania amastigotes, organelles known as the parasitophorous vacuoles (PV) and which most likely contain the highest concentration of parasite antigens in the host cell. Both uninfected and infected macrophages from Balb/c mice expressed the MHC class I molecules H-2Kd and H-2Dd on their cell surface but no significant amount of these molecules could be detected in the PV, which indicates that, if infected macrophages play a role in the induction of Leishmania-specific CD8+ T lymphocytes, PV are probably not loading compartments for MHC class I molecules. In contrast, MHC class II molecules were found to be associated with the PV membranes as shown previously with microscopic techniques at lower resolution (Antoine et al. Infect. Immun. 59, 764-775, 1991). In addition, we show here that, 48 hours after infection of Balb/c macrophages, in about 90% of PV containing MHC class II molecules, the latter were mainly or solely localized at the attachment zone of amastigotes to PV membranes. This peculiar distribution, especially well demonstrated using confocal microscopy, was confirmed by subcellular fluorescence cytometry of infected macrophages stained for the MHC class II molecules. The following data agree with the idea that PV-associated MHC class II molecules establish specific interactions with plasma membrane components of amastigotes. First, the polarized localization of class II appeared specific to these molecules, since the distribution of the lysosomal glycoproteins Igp110 and Igp120, of the macrosialin (a macrophage-specific marker of endocytic compartments) and of the GTP-binding protein rab7p, shown here as being PV membrane components, was homogeneous.(ABSTRACT TRUNCATED AT 400 WORDS)

Injection‐attachment of Methylosinus trichosporium OB3b in a two‐dimensional miniature sand‐filled aquifer simulator

For some potentially useful and emerging in situ bioremediation technologies it is important to control bacterial attachment to subsurface materials during the injection of microbial cell suspensions. In this study the attachment patterns of Methylosinus trichosporium OB3b were measured after horizontal injections into a two‐dimensional miniature aquifer simulator containing a wet homogeneous sand. In preliminary sand column assays, bacterial attachment to the sand was increased nearly 2 orders of magnitude compared to attachment in the presence of distilled water by raising the concentration of a pH 7.0 sodium‐potassium phosphate buffer to 10 mM. The maximal concentration of attached cells was ∼4×108 cells/g dry sand with both sand minicolumns and the wet sand aquifer simulator. For the latter this occurred on streamlines directly between the horizontal injector and withdrawal ports, where injection‐withdrawal velocities were the highest. The effects of a simulated groundwater cross flow during suspension injection on bacterial attachment to the aquifer simulator sands were also studied, and a peristaltic pumping method to counteract these groundwater flow effects resulted in a more localized pattern, i.e., without extensive downstream skewing of the bacterial attachment zone. Phenol red was utilized as a nonbinding, red‐colored tracer compound. It proved to be very convenient for quantitatively measuring the earlier breakthroughs of cells versus an inert tracer during the aquifer simulator and subsequent capillary tube cell injection experiments and also for visualizing the anticipated boundaries of cell attachment in the aquifer simulator. The effect of injection velocity on the observed bacterial attachment patterns in these experiments appears to be accounted for by colloid filtration theory.

Developmental changes at the node and paranode in human sural nerves: morphometric and fine-structural evaluation

Developmental alterations of paranodal fiber segments have not been investigated systematically in human nerve fibers at the light- and electron-microscopic level. We have therefore analyzed developmental changes in the fine structure of the paranode in 43 human sural nerves during the axonal growth period up to 5 years of age, and during the subsequent myelin development up to 20 years and thereafter. The nodal, internodal, and paranodal axon diameters reach their adult values at 4-5 years of age. The ratio between internodal and paranodal axon diameters remains constant at 1.8-2.0. Despite a considerable increase in myelin sheath thickness, the length of the paranodal myelin sheath attachment zone at the axon does not increase correspondingly, because of attenuation, separation from the axolemma, and piling up of myelin loops in the paranode. Separation of variable numbers of terminal myelin loops from the underlying axolemma results in the formation of bracelets of Nageotte, whereas the transverse bands of these loops disappear. The adaptation of the paranodal myelin sheath to axonal expansion during development probably occurs by uneven gliding of the paranodal myelin loops simultaneously with internodal slippage of myelin lamellae. Since mechanically stabilizing structures (tight junctions and desmosomes between adjacent paranodal myelin processes; transverse bands between myelin loops and paranodal axolemma) are unevenly arranged, especially during rapid axonal growth, paranodal axonal growth with simultaneous adaptation of the myelin sheath is probably discontinuous with time.

The In-Vitro Cellular Response of Mouse and Human Fibroblasts to Collagen-Based Wound Dressings

Summary The in-vitro cellular response of 3T3, L929 mouse and RMF primary human fibroblasts to novel collagen laminated sponge dressings was evaluated in a direct contact cell culture model using the adhesive film test. Direct microscopical examination of test plates was followed by measurement of cell attachment areas, migration distances and zones of inhibition using a micrometer grid. Normal cell growth of each cell type was seen in the presence of a collagen sponge and a 4 layer film multilaminate. A cytotoxic response to a 6 film multilaminate was observed with RMF cells only; normal cell activity was observed with 3T3 and L929 fibroblasts.

Capsular tissues of the proximal interphalangeal joint: Normal composition and effects of Dupuytren's disease and rheumatoid arthritis

Three fibrocartilages associated with the proximal interphalangeal joint are described-at the attachment of the central slip to bone, within the slip where it passes over the joint, and the volar plate. Material was obtained at surgery following trauma, Dupuytren's disease and rheumatoid arthritis. The fibrocartilages were structurally distinct and immunolabelled differently with monoclonal antibodies to extracellular matrix components. All fibrocartilages from normal and Dupuytren's fingers contained chondroitin and keratan sulphate. Type II collagen was present in all attachment zones, although there was little in rheumatoid fingers. It was also present in the dorsal hood of some normal fingers, but not in pathological specimens or the volar plate. The results show that the fibrocartilages are dynamic tissues whose composition varies according to function and use, and changes in disease.

Ultrastructure of the life-cycle stages of Goussia janae (Apicomplexa, Eimeriidae), with X-ray microanalysis of accompanying precipitates

The ultrastructural features of the life-cycle stages of Goussia janae from the intestinal epithelium of the dace Leuciscus leuciscus and chub L. cephalus are described. All merogonial, gamogonial, and early sporogonial stages were localized in the microvillar region in an intracellular and extracytoplasmic position, covered by closely apposed enterocyte and parasitophorous vacuole membranes. Two types of location in the host cell were observed: (i) a more frequent "monopodial" type with a single zone of attachment to the host-cell cytoplasm, and (ii) a "spider-like" type with several isolated zones of attachment. Merozoites were formed by either ecto- or endo-merogony. Microgamonts produced elongated biflagellate microgametes at their periphery. The oocyst wall, produced exclusively by the parasite, was formed at the end of the intracellular phase of the life cycle. Exogenous sporulation resulted in the formation of elongated sporocysts with a thin sporocyst wall bearing a longitudinal suture accompanied by a narrow membranaceous veil. In the cytoplasm and cytoplasmic and parasitophorous vacuoles of the parasite, fine, dense precipitates were present. X-ray microanalysis of these precipitates from osmicated and non-osmicated samples revealed high levels of Ca and P, indicating the possible presence of hydroxyapatite.

Distribution of Filipin-Sterol Complexes in the Bloodstream Form of Trypanosoma brucei gambiense

The polyene antibiotic, filipin, was used as a probe for demonstrating sterols in the plasma membrane of Trypanosoma brucei gambiense. Three different regions of the continuous plasma membrane over the cell body proper, flagellar pocket, and flagellum, were compared as to density and distribution of the filipin-sterol complexes by freeze-fracture method. The density of the complexes was highest in the cell body membrane and lowest over the flagellar pocket. The filipin-sterol complexes in the cell body membrane were distributed homogeneously on both the protoplasmic and exoplasmic faces except in the zone of flagellar attachment. This junctional zone showed a linear, complex-poor region. In the flagellar membranes, a line of complex-poor regions was observed along the juncture of the flagellum to the cell body. At the neck of the flagellar pocket, the membranes of the flagellar pocket and flagellum were closely opposed, with few filipin-sterol complexes on either membrane. At the base of the flagellar shaft, the complexes were completely lacking on both faces. Based on these observations, the zones of juncture observed in T.b. gambiense seems to be similar in ultrastructure to mammalian cell junctions, such as tight, gap, septate junctions and desmosomes, which show a nearly complete absence of the filipin-sterol complexes.

Attachment of Blastocrithidia triatomae (trypanosomatidae) by flagellum and cell body in the midgut of the reduviid bug Triatoma infestans

The pathology and attachment of Blastocrithidia triatomae in two regions of the midgut of the reduviid bug Triatoma infestans (the stomach and the small intestine) were investigated by electron microscopy. In both regions the extracellular-membrane layers and the apical microvilli were often reduced, and some cells of the intestinal wall were vacuolated. In the stomach two types of attachment occurred: Flagella with intraflagellar swellings lay over and between the apices of the microvilli of the stomach cells, and in microvilli-free regions the cell body of B. triatomae seemed to be anchored to the host cell by corrugations of both cells in the attachment zone, a hitherto unknown mode of attachment of trypanosomatids in the intestinal tract. In the small intestine flagellar expansions held several microvilli, and rarely an unaltered flagellum seemed to be anchored between the microvilli. In both midgut regions, the flagellum of some epimastigotes was inserted into the apex of an intestinal cell.

Life Cycle of Goussia pannonica (Molnar, 1989) (Apicomplexa, Eimeriorina), an Extracytoplasmic Coccidium from the White Bream Blicca bjoerkna

ABSTRACT Life cycle stages of Goussia pannonica from naturally‐infected white bream Blicca bjoerkna were studied by light and electron microscopy. Fourteen of the sixteen fish examined were infected, with developmental stages found in all parts of the intestine. Merogonial, gamogonial, and sporogonial stages were localized intracellularly and extracytoplasmically in the microvillous region of enterocytes. They were separated from the gut lumen by closely apposed enterocyte and parasitophorous vacuole membranes. There were two types of extracytoplasmic attachment: 1) monopodial, with a single zone of attachment, and 2) spider‐like, with several isolated zones of attachment to the host cell. First‐generation merozoites were formed by ectomerogony. Second‐ or third‐generation merozoites were formed by endodyogeny and endopolygeny. Thirty to 50 biflagellated microgametes developed at the periphery of a microgamont. Macrogamonts contained lipid inclusions, amylopectin and dense granules; however, granules comparable to wall‐forming bodies type I and II were absent. At the beginning of sporogony, the sporont cytoplasm detached from two layers which subsequently became constituents of the oocyst wall. After the rupture of enterocyte and parasitophorous vacuole membranes, the sporont was released into the water where exogenous sporulation was completed within 48 h. The thin sporocyst wall contained a small longitudinal suture. Sporocyst and oocyts walls were of similar structure.

Development of functionally distinct fibrocartilages at two sites in the quadriceps tendon of the rat: the suprapatella and the attachment to the patella.

This paper describes the post-natal development of two fibrocartilages in the quadriceps tendon of the rat. The compression-resisting fibrocartilage of the suprapatella was derived from a cell population present in neonates and positioned on the deep surface of the tendon of vastus intermedius. The cells secreted a metachromatic, coarsely fibrous extracellular matrix that was rich in chondroitin sulphate but lacked keratan sulphate or type II collagen. The cells themselves accumulated large quantities of vimentin. The adult form of the suprapatella was attained 8 weeks after birth. The fibrocartilage of the attachment zone of the quadriceps tendon to the patella was formed in a different manner. In animals up to 4 weeks of age, the quadriceps tendon inserted directly into the cartilage model of the patella. When later this was resorbed, and replaced by bone, the cartilage at the attachment zone remained, along with that of the articular surface of the patella. Attachment-zone fibrocartilage was therefore rich in type II collagen, unlike that of the suprapatella. Thus two functionally different fibrocartilages have been shown to have different origins, even when separated by only a short distance within the same tendon. The compositional differences between attachment-zone and compressive region fibrocartilages are also due to their different origins.

Spreading of mouse fibroblasts on the substrate with multiple spikes

Mouse embryo fibroblasts were cultivated on special substrates with discontinuous surfaces. The substrates were silicon plates with multiple vertical (65-90 microns height) spike-like silicon microcrystals evenly distributed on the plate surfaces. It was shown that the cells were successfully spread and flattened on these substrates. The spread cells formed several discrete attachment zones at the tops and side surfaces of the spikes; these zones were separated from one another by distances considerably greater than the diameter of the unspread cell. At early stages of spreading the unspread cells attached to the tops of single spikes and extended long filopodia attached to the distant spikes. At later stages the lamellae were formed between the filopodia: probably these filopodia served as guidelines for extension of lamellae and progressive cell spreading. These experiments demonstrated that continuity of substrate surface is not a necessary condition for advanced cell spreading.

Cell and matrix biology of the suprapatella in the rat: A structural and immunocytochemical study of fibrocartilage in a tendon subject to compression

The structure, ultrastructure, histochemistry, and immunohistochemistry of the suprapatella have been described in the rat. The suprapatella is a fibrocartilaginous sesamoid within the tendon of quadriceps femoris that articulates with the femoral condyles during flexion of the knee joint and reduces the amount of bending required at the tendon-bone junction. The cells of the suprapatella were much larger and more numerous than those in the associated tendon and were packed with vimentin-containing, intermediate filaments. The tendon cells contained far fewer filaments. The cells of both regions contained actin and tubulin. Histochemical and immunohistochemical studies showed that the suprapatellar cells were embedded in a matrix that is rich in chondroitin sulphate, but does not contain keratan or heparan sulphate. The fibrocartilage of the adjacent attachment zone of the quadriceps tendon also contained chondroitin sulphate, but in addition was rich in type II collagen. The structure of the suprapatella was similar to that of the fibrocartilaginous regions of tendons that pass around bony pulleys. However, there were differences in matrix composition that could reflect functional differences between the fibrocartilages.

Purification and characterisation of cysteine proteinases from human osteoclastomas

The process of normal bone remodeling requires the continual resorption and replacement of bone mineral and bone matrix proteins. Excessive bone resorption is a feature of a number of pathological conditions. These include osteoporosis, rheumatoid arthritis and Paget's disease [l]. Bone resorption is accomplished by the osteoclast. Osteoclasts attached to bone are highly polarised cells [2]. The osteoclast attaches to bone through a peripheral attachment zone within which the surface (apical) membrane is thrown into complex ruffles. The space enclosed by the osteoclast apical surface and the bone surface is acidified, probably by the action of a proton pump, resulting in the dissolution of bone mineral. Lysosomal enzymes are released into the acidified subosteoclastic space where they are responsible for the degradation of bone matrix components. Bone matrix is largely composed of type I collagen with small amounts of types 111, V and VI collagens [31. In addition, bone contains a number of glycoproteins (e.g. osteopontin, osteonectin and bone sialoproteins) and proteoglycans [4]. In vitro studies using disaggregated osteoclasts have suggested that cysteine proteinases released by osteoclasts are responsible for the degradation of bone proteins 151. Investigations of the properties of osteoclast cysteine proteinases are hampered by the scarcity of osteoclasts in normal bone. As our starting material we have used osteoclastomas. These are primary bone tumours that contain large numbers of apparently normal osteoclasts. Using the synthetic peptide Z-PheArg-NHMec as substrate, we have separated six cysteine proteinase activities from osteoclastoma extracts by sequential chromatography on S-Sepharose, phenyl-Sepharose, heparin-Sepharose and Sephacryl S200HR. The proteinases have molecular weights ranging from 20,000 to 42,000 (determined by gel filtration). Z-Phe-Arg-NHMec was the preferred synthetic substrate. Four of the enzymes showed some activity towards Bz-Phe-Val-Arg-NHMec but only two demonstrated low activity against Z-Arg-Arg-NHMec. The kinetics of hydrolysis of these substrates gave values within the range expected for cathepsin B [61. All six activities were capable of degrading soluble and insoluble collagen. The pH optima for the hydrolysis of Z-Phe-Arg-NHMec and type I collagen by the six enzymes are shown in table 1. A variety of inhibitors of cysteine proteinases (eg. leupeptin, E-64, cystatin) were potent inhibitors of the osteoclastoma enzymes. The kinetics of inhibition by cystatin and the rate constants of inactivation by Z-PheTyr-(O-t-Bu)CHNz were similar to published values for cathepsin B [7,81. Immunolocalisation of cathepsin B in sections of osteoclastomas revealed that osteoclasts stained strongly with an antibody to cathepsin B whereas the neoplastic osteoblasts were only weakly stained. The Table 1 pH-Optima of osteoclastoma cysteiiie protciilases

Morphology of Pneumocystis carinii and activation of the plasmalemmal vesicular system in alveolar epithelial cells of the host

Lungs from rats with dexamethasone-induced Pneumocystis carinii pneumonia were examined. The ultrastructure of Pneumocystis carinii and their zone of attachment on type I alveolar epithelial cells are described. An activation of the plasmalemmal vesicular system of type I alveolar epithelial cells was observed and is described here for the first time. The significance of this observation is discussed.

Immunohistochemical Localization of the Matrix Glycoproteins-Tenascin and the ED-sequence-containing Form of Cellular Fibronectin—in Human Permanent Teeth and Periodontal Ligament

The expression of two matrix glycoproteins, tenascin and cellular fibronectin (cFN), has been studied in fully developed human permanent teeth, periodontal ligament, and alveolar bone, in both frozen and paraffin-processed material. Polyclonal antibodies to tenascin and a monoclonal antibody recognizing the ED sequence specific to at least some forms of cFN were used. Staining for both tenascin and cFN was positive in the dental pulp, odontoblastic layer, cementoblast-pre-cementum zone, and on the periosteal as well as endosteal surfaces of the alveolar bone. In the periodontal ligament, cFN was evenly distributed, whereas tenascin was accumulated in the attachment zones. Pre-dentin stained for tenascin but not for cFN. Mineralized dentin and cementum were tenascin- and cFN-negative. The relative staining intensity for tenascin was greater than that for cFN in the cementoblast-pre-cementum layer and in the attachment zones of the periodontal ligament, whereas cFN stained more intensely in the pulp. In frozen material, antigenicities were well-preserved. Paraffin processing facilitated precise recognition of tissue morphology, but the antigenicity of cFN was lost. The co-expression of tenascin and cFN in the dental pulp, cementogenic zone, and on the surfaces of the alveolar bone may reflect the ability of the cells to deposit mineralized tissue matrices. The pronounced expression of tenascin in the interfaces between mineralized and non-mineralized tissues suggests that it is functionally associated with mechanical stress and may thus have at least two distinct functions. The relative amounts of the two matrix glycoproteins may contribute to regulation of tissue structure.

Ultrastructural observations on the in vitro cytoadherence of Plasmodium falciparum infected red blood cells

In human malaria, the most serious clinical manifestation is cerebral malaria (CM) due to infection with Plasmodium falciparum. The pathology of CM is thought to relate to the fact that red blood cells containing mature forms of the parasite (PRBC) cytoadhere or sequester to post capillary venules of various tissues including the brain. This in vivo phenomenon has been studied in vitro by examining the cytoadherence of PRBCs to various cell types and purified proteins. To date, three Ijiost receptor molecules have been identified; CD36, ICAM-1 and thrombospondin. The specific changes in the PRBC membrane which mediate cytoadherence are less well understood, but they include the sub-membranous deposition of electron-dense material resulting in surface deformations called knobs. Knobs were thought to be essential for cytoadherence, lput recent work has shown that certain knob-negative (K-) lines can cytoadhere. In the present study, we have used electron microscopy to re-examine the interactions between K+ PRBCs and both C32 amelanotic melanoma cells and human umbilical vein endothelial cells (HUVEC).We confirm previous data demonstrating that C32 cells possess numerous microvilli which adhere to the PRBC, mainly via the knobs (Fig. 1). In contrast, the HUVEC were relatively smooth and the PRBCs appeared partially flattened onto the cell surface (Fig. 2). Furthermore, many of the PRBCs exhibited an invagination of the limiting membrane in the attachment zone, often containing a cytoplasmic process from the endothelial cell (Fig. 2).

Screening of in vitro cytotoxicity by the adhesive film test

A novel procedure, the adhesive film test, is proposed as a screening method to predict potential cytotoxicity of biomaterials. This in vitro test utilizes a sterile strip of acrylate-based medical adhesive as an anchorage substrate in cytotoxicity studies. The adhesive film allows direct fixation of test samples to the base of the petri dish, ensuring close contact between sample and cells. The test is based on the principle that toxic components present in the test material will readily leach out into the culture medium and adversely affect the local cell population. The main advantage of the adhesive film test is that a viable cell population can be added directly to the test plate and after an incubation period of 24 h, the cellular response can be recorded as either cytotoxic or cytocompatible. Microscopic examination can be followed by quantifying the results using a micrometer to measure cellular attachment areas, migration distances and zones of inhibition. In addition, the adhesive film used to attach the test samples is shown to support extensive fibroblast growth and attachment to its surface and hence can also function as a negative non-toxic control in cytotoxicity studies.

Scanning and transmission electron microscopic observations of bacteria adhering to ileal epithelial cells in growing broiler and white leghorn chickens

1. Bacteria adhering to the ileal epithelial cells in broiler and White Leghorn (WL) chickens aged 1 to 60 d were observed with scanning (SEM) and transmission (TEM) electron microscopes. 2. In SEM observations, bacteria were not found on day 1 after hatching in either breed. In 10-d-old broilers many bacteria were observed around the apical area of villi. The number decreased with age and disappeared by 50 d. In WL chicks, the bacteria were first observed at 20 d. Numbers were much fewer than in broilers and none were seen after 30 d. 3. TEM investigations showed that bacteria had a cytoplasmic membrane, cell wall and nucleus but no nuclear membrane and organella, were compartmentalised and resembled Streptobacillus moniliformis. At the attachment zone to the epithelium, many mitochondria were observed in the epithelial cells; the bacterial membrane did not fuse to the epithelial cell membrane except at the apex of the attachment end where the bacterial membrane seemed to undergo lysis, suggesting a possibility that some bacterial components were transferred to the epithelial cells. 4. It is possible that the bacterial aid in the functioning of ileal epithelial cells. Possible functions are discussed in relation to the morphological features.

The attachment of collagenous ligament to stereom in primary spines of the sea-urchin, Eucidaris tribuloides

The similar proximal and distal attachments to the stcrcom of primary spine ligament in the echinoid Eucidaris tribuloides are described, from thin sections and SEM studies on frozen and fractured spine articulations and ligaments from decalcified material. The orthogonal structure of the general stereom is modified on the attachment zones where bundles of collagen cylinders enter approximately hexagonally arranged channels. Straps of collagen extend in parallel scries between adjacent bundles via regularly placed ports and collagen loops rather than non-striated ‘tendons’ pass over skeletal trabeculac. The regular pattern of collagen straps is most evident on the proximal and distal attachment zones. Mechanical features of the nonadhesive mode of attachment are considered, together with similarities and differences between insertion of muscle cells and mutable eollagenous tissue (ligament) in echinoderms.

Metallurgy of open-bath plasma processes

Abstract