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PubMed1980

Absence of immunoglobulins in rheumatoid cartilage-pannus junctions.

Immune complexes (IC) present in the superficial region of rheumatoid articular cartilage may be an important factor in the invasion of the cartilage by pannus. The presence of such IC at the cartilage-pannus junction in rheumatoid articular cartilage invaded by pannus was investigated by using immunoelectron microscopy with horseradish peroxidase anti-human immunoglobulin (Ig). It was observed that, although the pannus-free surface of the cartilage contained easily detectable deposits of Ig, the areas covered by pannus failed to show any Ig in the 5 5 patients studied. This was true even in areas immediately adjacent to the advancing edge of the pannus which presumably had only recently been invaded by the granulation tissue. These results were confirmed by fluorescent antibody ultraviolet microscopy. It was concluded that rheumatoid pannus invasion of the free cartilage surface containing trapped IC results in the disappearance of such complexes.

Antibodies↗

PubMed1980

Influence of S-adenosylmethionine on DAPI-induced fluorescence of polyphosphate in the yeast vacuole.

Use of the fluorochrome 4',6-diamidino-2-phenylindole.2 HCl (DAPI) in ultraviolet microscopy revealed fluorescent objects in Brownian motion within the vacuoles of seven species of yeast. The abundance of these bodies increased when cells of Saccharomyces cerevisiae were transferred from growth medium to a glucose-phosphate solution, indicating that they contain polyphosphate. In addition, the effect on vacuolar fluorescence of supplementing a defined growth medium with amino acids provided evidence that they also contained S-adenosylmethionine. These deductions were supported by in vitro studies of the interaction and fluorescence of polyphosphate, S-adenosylmethionine, and DAPI. Vacuolar fluorescence of cells in suspension in flucose-phosphate solution was less after addition of exogenous arginine, lysine, or glutamine but not after addition of alanine, aspartic acid, or methionine. Mithramycin was superior to DAPI as a fluorochrome for ultraviolet demonstration of yeast nuclei since it stained the nuclei much more intensely and did not fluoresce with other material in the cells.

Amidines↗

PubMed1991

Triple immunofluorescence confocal laser scanning microscopy: spatial correlation of novel cellular differentiation markers in human muscle biopsies.

Two different methods for triple immunofluorescence imaging with a confocal laser scanning microscope (CLSM) are described. The methods enable spatial "mapping" of 3 different epitope distribution patterns simultaneously in one tissue section. The key to triple imaging includes: (a) specific immunolabeling with 3 different mouse monoclonal antibodies (mAbs), (b) localization of the antibody binding sites by 3 different dyes, (c) spectral isolation of each dye by using selective band pass or long pass emission filters, (d) computerized imaging of the fluorescences as colored overlays or as selective signals in each optical section through the tissue in the z-direction. Method 1 consists of the combination of fluorescein isothiocyanate (FITC), phycoerythrin R (PE), and Texas Red (TR) as fluorescent markers. These dyes can be imaged by using 488 nm and 543 nm excitation laser lines. In method 2 aminomethyl coumarin acetic acid (AMCA) was combined with FITC and PE. For this application the CLSM was adapted to ultraviolet microscopy that enabled the use of 3 laser lines (364 nm, 488 nm, 543 nm) for excitations. Cryostat sections of diagnostic human muscle biopsies (n = 9) were studied which were normal by ordinary light microscopic examination. Sections were incubated with mAbs specific for: (1) the fast myosin heavy chain (My32); (2) the major histocompatibility class II antigen HLA-DR; (3) the lymph node homing receptor Leu8, and (4) the cell adhesion receptor OKM5 (CD36). By combining these mAbs in triple staining procedures, 3 capillary types and 4 different phenotypes expressed by muscle fibers were identified simultaneously. The mAbs Leu8 and OKM5, widely used as leukocyte typing antibodies in the blood, exhibit hitherto unrecognized specificities for antigens displayed by muscle fibers. At the level of these markers, specific spatial correlations between OKM5 reactive capillaries and both OKM5 reactive and nonreactive muscle fiber types become visible. The presented results provide direct evidence for cellular complexity and novel insight into the immunoanatomical architecture of skeletal muscle. The methods may be of general significance for the construction and quantification of three-dimensional multiparameter "maps" of cells and tissues.

Adult↗

PubMed2002

Stromal cell-derived factor 1 (CXCL12) induces monocyte migration into human synovium transplanted onto SCID Mice.

OBJECTIVE: The mechanisms by which monocyte/macrophage cells migrate to the joint involve a series of integrated adhesion and signaling events in which chemokines and their receptors are strongly implicated. This study was undertaken to investigate the hypothesis that stromal cell-derived factor 1 (SDF-1), a CXC chemokine (CXCL12), plays a critical role in monocyte/macrophage localization to synovium. METHODS: SDF-1 and CXC receptor 4 (CXCR4) expression in rheumatoid arthritis (RA) and osteoarthritis synovium and graft SDF-1, tumor necrosis factor alpha (TNF alpha), and human and murine vascular markers were examined by immunohistochemistry and double-immunofluorescence. The functional capacity of SDF-1 to modulate monocyte migration into joints was investigated by examining the localization of pro-myelomonocytic U937 cells into synovial tissue transplanted into SCID mice. SDF-1, TNF alpha, or saline was injected into graft sites and response determined by the number of fluorescently labeled U937 cells (injected intravenously) detected in grafts by ultraviolet microscopy. RESULTS: SDF-1 and CXCR4 were highly expressed in CD68+ cells in the RA synovium. SDF-1 induced U937 cell migration in vitro and in vivo in a dose-dependent manner and, in vivo, SDF-1 was more effective than TNF alpha. In contrast to TNF alpha, SDF-1 did not induce intracellular adhesion molecule 1 in transplant microvasculature. Furthermore, intragraft injection of SDF-1 did not up-regulate TNF alpha, or vice versa. CONCLUSION: This study demonstrates, for the first time, that SDF-1 is functional in vivo when injected into synovial grafts. In addition, SDF-1 is more potent than TNF alpha, and its mechanisms of action appear to be autonomous. Therefore, SDF-1 may be an important TNF-independent molecule involved in the migration to and retention of inflammatory effector cells in the joint.

Aged↗

PubMed2000

New tools in diagnosing catheter-related infections.

Clinical criteria alone are insufficient to allow a diagnosis of intravascular catheter-related sepsis (CRS). A definite diagnosis of CRS usually requires removal of the catheter for quantitative catheter tip culture. However, only about 15-25% of central venous catheters (CVC) removed because infection is suspected actually prove to be infected, and the diagnosis is always retrospective. Other diagnostic tests, such as differential quantitative blood cultures from samples taken simultaneously from the catheter and a peripheral vein, have been proposed to avoid unjustified removal of the catheter and the potential risks associated with the placement of a new catheter at a new site: a central-to-peripheral blood culture colony count ratio of 5:1 to 10:1 is considered indicative of CRS. Despite its high specificity, the latter diagnostic technique is not routinely used in clinical practice because of its complexity and cost. The measurement of the differential time to positivity between hub blood (taken from the catheter port) and peripheral blood cultures might be a reliable tool facilitating the diagnosis of CRS in situ. In an in vitro study, we found a strong relationship between the inoculum size of various microorganisms and the time to positivity of cultures. When the times to positivity of cultures of blood taken simultaneously from central and peripheral veins in patients with and without CRS were examined, we found that earlier positivity of central vs peripheral vein blood cultures was highly correlated with CRS. Using a cut-off value of +120 min, the "differential time to positivity" of the paired blood samples, defined as time to positivity of the peripheral blood minus that of the hub blood culture, had 91% specificity and 94% sensitivity for the diagnosis of CRS. This method may be coupled with other techniques that have high negative predictive value, such as skin cultures at the catheter exit site. This diagnostic test can be proposed for routine clinical practice in most hospitals using automatic devices for blood cultures positivity detection. Endoluminal brushing of the catheter is considered sensitive and specific for the diagnosis of CRS, but the risk of embolisation or subsequent bacteraemia should be considered. Gram staining and the acridine-orange leucocyte cytospin test on through-catheter blood culture have been proposed for rapid diagnosis of CRS without catheter removal. The technique, which requires 100 microl catheter blood and the use of light and ultraviolet microscopy, is considered simple, rapid (30 min) and inexpensive. In conclusion, diagnostic tools such as paired blood cultures or Gram staining and the acridine-orange leucocyte cytospin test should allow a diagnosis of CRS without catheter removal in cancer patients.

Algorithms↗

PubMed1992

A new method for measuring clustering in suspension between accessory cells and T lymphocytes.

Specifying the molecular basis and clinical significance of cluster formation between antigen-presenting cells and T lymphocytes will be important in many areas of immunology. In this paper we describe a novel and reproducible technique for measuring cluster formation in suspension between purified human blood monocytes and purified autologous T lymphocytes, and its application to determining the effects of recall antigens and mitogen. Blood monocytes and T lymphocytes from eight normal subjects were separately prelabelled with two different carbocyanine dyes prior to co-culture in suspension with or without antigen (PPD, SKSD) or mitogen (PHA). At 24 h the co-cultures were examined for cluster formation by ultraviolet microscopy and flow cytometry. Control experiments showed that the carbocyanine dyes were non-toxic in vitro, that cell labelling was stable for culture periods up to 120 h, and that the two dyes did not leak from cell to cell. By this technique we measured the proportion of monocytes clustering one or more T lymphocytes in the presence and absence of recall antigen or PHA. There was a close correlation between visual and flow cytometric measurement of monocyte: T lymphocyte clustering (p < 0.001) as well as a close relationship between the ability of the two recall antigens to increase the extent of clustering above baseline (p < 0.001). Antigen-increased cluster formation did not correlate with baseline clustering, unlike PHA-increased clustering, which was related to baseline levels (p = 0.02), suggesting the operation of distinct mechanisms. The method is applicable to measuring cell-cell associations in suspension during extended periods of culture, as well as for the study of agents which might modify intercellular adhesion processes.

Antigen-Presenting Cells↗

PubMed1987

Interaction of soybean agglutinin with human leukemia-lymphoma lines at various stages of differentiation.

Human leukemia-lymphoma cell lines reflecting hematopoietic clones at various stages of differentiation were examined for reactivity with soybean agglutinin (SBA). The binding and redistribution pattern of soybean surface receptors was determined with fluorescein-isothiocyanate conjugated SBA (F-SBA) by ultraviolet microscopy, and with a fluorescent activated cell sorter (FACS). The results indicate that there is a correlation between SBA labelling--distribution and the stage of lymphoid cell differentiation. The SBA labelling on the membrane of null lines was undetectable by U.V. microscopy and flow cytometry. A gradual increase in SBA labelling correlating with the stage of differentiation was observed on cell lines of both B and T origin. However the maximal fluorescence intensity of the T lines was lower than the B lines. The redistribution pattern of SBA on the membrane of T lines was rings and mild patches, whereas that on the B lines was moderate to large patches. The reactivity of the lymphoid lines with SBA was not affected by growth conditions. The binding of SBA to normal lymphoblastoid lines was generally low and the fluorescence intensity weak. The reactivity of these lines with SBA was not associated with their origin or "age". It is suggested that the differences in the reactivity of SBA with human hematopoietic lines at various stages of maturation may be of value in future understanding the differences in structure and function of the surface membrane between normal and malignant cells, and the relation to normal and abnormal cell differentiation.

Cell Differentiation↗

PubMed2004

Human ATP-binding cassette transporter-2 (ABCA2) positively regulates low-density lipoprotein receptor expression and negatively regulates cholesterol esterification in Chinese hamster ovary cells.

We present evidence that the ATP binding-cassette transporter-2 (ABCA2) is a sterol-responsive gene that has a role in the trafficking of low-density lipoprotein-derived free cholesterol (LDL-FC). In HepG2 cells ABCA2 was coordinately expressed with other sterol-responsive genes. Stable constitutive expression of ABCA2 in Chinese hamster ovary cells (CHOA2) was accompanied by an increase the expression of the low-density lipoprotein receptor (LDLR) and other genes involved in the regulation of cholesterol homeostasis. LDLR mRNA was elevated greater than ninefold and 3-hydroxy-3-methylglutaryl CoA synthase (HMGCoA S) expression was elevated sevenfold in CHOA2 cells. The increase in LDLR expression was regulated at the level of transcription; however, culture of CHO and CHOA2 cells in medium containing lipoprotein-deficient serum (LPDS) results in similar levels of LDLR promoter expression. No differences were measured in the dose-dependent uptake of fluorescently labeled 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchorate-LDL (DiI-LDL) between CHO and CHOA2 cells cultured in medium containing LPDS. Ultraviolet microscopy revealed a similar distribution of the DiI-LDL label in cytoplasmic vesicles. We measured an LDL dose-dependent reduction in esterification of LDL-FC in intact CHOA2 cells cultured in medium containing LPDS, however, no significant difference was measured in acylcoenzyme A:cholesterol acyltransferase (ACAT) activity in cell-free extracts of CHO and CHOA2 cells. CHO cells or CHOA2 cells treated with the hydrophobic amine, U18666A, showed similar filipin staining of unesterified cholesterol in cytoplasmic vesicles. Addition of progesterone or U18666A to CHO cells elevated ABCA2 expression. Finally, we found that ABCA2 expression was elevated in Niemann-Pick type C1 (NPC1) fibroblasts and in Familial Hypercholesterolemia (FHC) fibroblasts.

ATP-Binding Cassette Transporters↗

PubMed1985

Schistosoma mansoni: lectin-dependent cytotoxicity of hemocytes from susceptible host snails, Biomphalaria glabrata.

Normally benign hemocytes from a strain (M-line) of the snail, Biomphalaria glabrata, susceptible to Schistosoma mansoni, became cytotoxic toward the sporocyst stage if the parasite was first treated with the lectin, concanavalin A. Concanavalin A binding was inhibitable with alpha-methyl mannoside and killing was dose-dependent. Maximal levels of concanavalin A-induced cytotoxicity were comparable with levels observed when hemocytes from a resistant snail strain (13-16-R1) encountered untreated sporocysts. Induction of the cytotoxic response did not occur if hemocytes alone were pretreated with the lectin. A unique method incorporating ultraviolet microscopy and the vital fluorescent dye, eosin Y, was used for discriminating between live and dead sporocysts. This model may prove useful in understanding mechanisms used by invertebrate effector cells in recognition and killing of invading organisms.

Animals↗

PubMed1999

Rapid diagnosis of central-venous-catheter-related bloodstream infection without catheter removal.

BACKGROUND: Current methods for the diagnosis of bloodstream infection related to central venous catheters (CVC) are slow and in many cases require catheter removal. Since most CVC that are removed on suspicion of causing infection prove not to be infected, removal of catheters unnecessarily exposes patients to the risks associated with reinsertion. METHODS: The gram stain and acridine-orange leucocyte cytospin test (AOLC) is rapid (30 min), inexpensive, and requires only 100 microL catheter blood (treated with edetic acid) and the use of light and ultraviolet microscopy. We assessed the gram stain and AOLC test in suspected cases of catheter-related bloodstream infection, in comparison with two methods requiring catheter removal (tip roll and tip flush), and a third technique, done in situ (endoluminal brush) in conjunction with quantitative peripheral-blood cultures. FINDINGS: 128 cases of suspected catheter-related bloodstream infection were assessed in 124 adult surgical patients (median duration of CVC placement was 16 days). In 112 (88%) cases CVC blood was obtainable. Catheter-related bloodstream infection was diagnosed in 50 cases (culture of the same organism from the catheter, in significant numbers, and from peripheral-blood culture). The sensitivity of the gram stain and AOLC test was 96% and the specificity was 92%, with a positive predictive value of 91% and a negative predictive value of 97%. By comparison, the tip-roll, tip-flush, and endoluminal-brush methods had sensitivities of 90%, 95%, and 92%, and specificities of 55%, 76%, and 98%, respectively. INTERPRETATION: The gram stain and AOLC test is a simple, and rapid method for the diagnosis of catheter-related bloodstream infection. This diagnostic method compares favourably with other diagnostic methods, particularly those that require the removal of the catheter, and can permit early targeted antimicrobial therapy.

Acridine Orange↗

PubMed2000

Co-expression of interleukin-2 and green fluorescent protein reporter in mycobacteria: in vivo application for monitoring antimycobacterial immunity.

Recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing green fluorescent protein (rBCG-GFP), driven by the mycobacterial heat shock protein 70 (HSP 70) promoter from an autonomously replicating plasmid, was genetically engineered to co-express mouse interleukin-2 (IL-2) by introduction of an independent HSP 60 promoter. To monitor host antimycobacterial immunity, C57BL/6 mice were intravenously infected with IL-2 expressing and non-expressing GFP rBCGs. Both rBCGs were clearly imaged and easily quantified with ultraviolet microscopy of tissue sections and whole organ suspensions. Enhanced mycobacterial clearance from the spleens of mice infected with the rBCG-IL-2/GFP strain was apparent by both diminished bacterial counts and spleen weights during the first 6 weeks post-infection relative to rBCG-GFP. T helper type 1 (TH1) cytokine production and proliferative response to BCG restimulation was also elevated from in vitro splenocyte cultures taken from the rBCG-IL-2/GFP-infected group. Taken together, these results suggest that IL-2 expression from rBCG augmented host protective immunity to mycobacterial infection via an enhanced TH1 immune response. Mycobacterial expression vectors that allow simultaneous but independent production of reporter proteins and bioactive substances provide an ideal means for monitoring the in vivo fate of recombinant mycobacteria.

Animals↗

PubMed2003

Synovial sheath cell migratory response to flexor tendon injury: an experimental study in rats.

PURPOSE: We aimed to investigate the degree of participation of synovial sheath cells in the process of tendon healing by selective cell labeling and direct observation of migrational pathways. METHODS: We designed a novel rat animal model that employed vital dye staining of synovial sheath cells. The flexor digitorum profundus (FDP) tendon was removed from its sheath and vital dye was applied directly to the synovial sheath cells. A window was cut in the removed tendon before being returned to the sheath, thus placing a tendon injury adjacent to the labeled synovial sheath cells. The synovium remained intact at all times, and labeling was confirmed to be localized to the synovium. The migrational response of the synovial sheath cells to the tendon injury was observed by harvesting the tendons at 1, 3, 5, and 7 days (n = 6 for each time period) after injury and assessing tendon response with frozen sections under ultraviolet microscopy. RESULTS: Labeled synovial sheath cells were observed within the substance of the healing tendon 24 hours after injury, with numbers increasing with time for up to 5 days, but decreasing by day 7. CONCLUSIONS: This study confirms that in the rat model synovial sheath cells move into the healing tendon area and then migrate into the tendon core.

Animals↗

PubMed1959

Observations on the microanatomy of the spermatozoid of the bracken fern (Pteridium aquilinum).

Salient features of structure of the spermatozoid of a fern (Pteridium aquilinum) have been determined by a combination of visual and ultraviolet microscopy, with electron microscopy of shadow-cast whole mounts and thin sections, using magnifications up to but not exceeding 50,000 diameters. Attention has been concentrated on the arrangement rather than on the internal details of the various parts. The most important component, apart from the spirally wound nucleus, numerous (about 40) cilia, and mitochondria, is a sheet of parallel fibres spirally wound near the surface of a cone of cytoplasm to which all the other major components are in various ways attached. The diameter of the individual fibres is of the order of 200 A. A few details are given of other minor cell constituents including additional mechanical materials, starch-containing leucoplasts, and the smaller cytoplasmic inclusions.

Cell Nucleus↗

PubMed1991

The effects of early postoperative radiation on vascularized bone grafts.

The effects of early postoperative radiation were assessed in free nonvascularized and free vascularized rib grafts in the canine model. The mandibles of one-half of the dogs were exposed to a cobalt 60 radiation dose of 4080 cGy over a 4-week period, starting 2 weeks postoperatively. The patency of vascularized grafts was confirmed with bone scintigraphy. Histological studies, including ultraviolet microscopy with trifluorochrome labeling, and histomorphometric analyses were performed. Osteocytes persist within the cortex of the vascularized nonradiated grafts to a much greater extent than in nonvascularized, nonradiated grafts. Cortical osteocytes do not persist in either vascularized or nonvascularized grafts subjected to radiation. New bone formation is significantly retarded in radiated grafts compared with nonradiated grafts. Periosteum and endosteum remained viable in the radiated vascularized grafts, producing both bone union and increased bone turnover, neither of which were evident to any significant extent in nonvascularized grafts. Bone union was achieved in vascularized and non-vascularized nonradiated bone. In the radiated group of dogs, union was only seen in the vascularized bone grafts.

Animals↗

PubMed1961

Purity control of the production of baker's yeast employing a fluorescent antiserum.

A simple staining procedure for the rapid detection of wild yeasts contaminating baker's yeast during the course of industrial production is described. Fluorescein-labeled, specific antiserum against Saccharomyces cerevisiae is applied to smears of baker's yeast which are then examined by ultraviolet microscopy. Optimal results are obtained with the combined phase contrast and fluorescence which makes the S. cerevisiae appear green, whereas cells of wild yeasts are visible in bright red counterstain. With this method, wild yeasts could be identified in baker's yeast at a dilution of 1:10,000.

Immune Sera↗

PubMed1992

Remodelling of subchondral bone in osteoarthritis: a histomorphometric study.

AIMS: To determine whether remodelling of subchondral bone in osteoarthritis is related to anatomical and mechanical factors operating on the joint surface. METHODS: Ten femoral heads were examined. Patients due to have the femoral head removed because of osteoarthritis were given a double tetracycline label before surgery. The specimens were divided in three roughly equal parts, corresponding to the medial, central (weight bearing), and lateral aspects of the femoral heads. Undecalcified methylmethacrylate embedded sections were prepared. The subchondral bone was assessed for appositional bone formation by ultraviolet light microscopy, for resorptive activity by osteoclast count, and for trabecular bone volume by semiautomatic computerised image analysis. Appositional bone rate was also correlated with the presence or absence of overlying cartilage. RESULTS: Bone density was reduced in the medial aspect of the femoral heads but there was no significant difference between the appositional bone rate or the resorptive activity in the three areas. Nor was there any correlation between appositional bone rate in the subchondral bone and the presence or absence of overlying cartilage. CONCLUSION: Remodelling of subchondral bone in osteoarthritis, at least in the short term, is fairly constant and is not related to weight bearing.

Aged↗

PubMed1975

Adhesion of Phytophthora palmivora zoospores: detection and ultrastructural visualization of concanavalin A-receptor sites appearing during encystment.

The binding of concanavalin A (Con A) to the cell surface of zoospores and cysts of Phytophthora palmivora was studied by radiometry (125I-Con A), ultraviolet microscopy (fluorescein-Con A) and electron microscopy peroxidase-diaminobenzidine technique). Zoospores were found to secrete during the early stages of encystment a Con A-binding material susceptible to trypsin digestion. This glycoprotein is contained in the so-called peripheral vesicles and is probably responsible for the adhesion of the encysting zoospores to solid surfaces.

Binding Sites↗

PubMed1985

Eimeria meleagrimitis sporozoites: effect of lectins on invasion of cultured cells.

Exposure of primary turkey kidney cell cultures to 100 micrograms/ml wheat germ agglutinin (WGA), a lectin that binds N-acetylglucosamine (NAcGl), significantly inhibited invasion of the cells by Eimeria meleagrimitis sporozoites. However, neither succinyl-WGA, a lectin that retains an affinity for NAcGl but does not bind sialic acid, nor pokeweed mitogen, another lectin that binds NAcGl, similarly inhibited invasion. Collectively, the data suggested that the inhibition of invasion may be caused primarily by binding of WGA to anionic moieties on the host cell surface and not to specific binding to NAcGl. Exposure of cells to concanavalin A, phytohemagglutinin, or Ricinus communis lectin, as well as with pokeweed mitogen, failed to inhibit invasion by the sporozoites. Ultraviolet microscopy, using fluorescein-conjugated lectins, showed that the lectins had bound to the surface of the cultured cells. No binding of NAcGl or lectins to the surfaces of the sporozoites was demonstrated by either the clumping of the parasites in different lectin concentrations or the use of fluorescein-conjugated lectins. However, exposure of E. meleagrimitis sporozoites to NAcGl increased invasion of untreated cells by 50%.

Animals↗