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Results for “Laser Scanning Cytometry”

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PubMed1994

[Damage to tumor cells by photodynamic therapy].

Photodynamic therapy (PDT) is a new, promising method in the treatment of cancer. To gain insights into PDT-mediated tumour destruction we studied the influence of treatment with Photofrin and laser light on changes in cell volume and cell viability. A-Mel-3 tumour cells were subjected to Photofrin or illumination with laser light, or a combination of both (PDT). Cell volume was measured by flow cytometry and cell viability by the trypan blue exclusion test for up to 60 min after PDT and the respective controls. In addition, scanning and transmission electron microscopy were performed. Tumour cells incubated in concentrations of 0.75, 1.5 and 3.0 micrograms Photofrin/ml revealed a rapid increase in cell volume to 117%, 207% and 235% 30 min after PDT and to 147%, 210% and 199% 60 min after PDT. Cell viability with 1.5 and 3.0 micrograms Photofrin/ml and laser light was reduced to 83% and 44% at 30 min after PDT and to 38% and 17% 60 min after PDT. At Photofrin concentrations of 1.5 micrograms/ml and exposure to laser light scanning electron microscopy revealed extreme loss of microvilli and formation of blebs on the cellular surface. Transmission electron microscopy showed swollen mitochondria and ruptures of the cell membrane. This study demonstrates that PDT induces a significant time-dependent and dose-related increase in tumour cell volume. We suggest that the PDT-induced swelling of tumour cells contributes to the increase of interstitial fluid pressure and to impairment of microvascular perfusion of tumours.

Cell Division↗

PubMed1997

Role of epithelial interleukin-8 (IL-8) and neutrophil IL-8 receptor A in Escherichia coli-induced transuroepithelial neutrophil migration.

Escherichia coli stimulates neutrophil migration across human uroepithelial cell layers. This study investigated the role of the neutrophil chemokine interleukin-8 (IL-8) in this process. E. coli and IL-1alpha stimulated urinary tract epithelial layers to secrete IL-8 and induced transepithelial neutrophil migration. Anti-IL-8 antibody reduced neutrophil migration across epithelial cell layers, indicating a central role for this chemokine in the migration process. Furthermore, addition of recombinant IL-8 to unstimulated cell layers was sufficient to induce migration. The IL-8 dependence of neutrophil migration was maintained after removal of soluble IL-8 by washing of the cell layers. Flow cytometry analysis with fluorescein isothiocyanate-labelled IL-8 confirmed IL-8's ability to bind to the epithelial cell surface. Indirect immunofluorescence with confocal laser scanning microscopy showed that IL-8 associated with the epithelial cell layers. Prior incubation of neutrophils with antibodies to IL-8 receptor A (IL-8RA) reduced neutrophil migration. Anti-IL-8 RB antibody had no effect on neutrophil migration. These results demonstrate that IL-8 plays a key role in E. coli- or IL-1alpha-induced transuroepithelial migration and suggest that epithelial cell-produced IL-8 interacts with IL-8RA on the neutrophil surface.

Antigens, CD↗

PubMed2001

[The effects of sodium butyrate on proliferation, differentiation and apoptosis in immortalized esophageal epithelial cells].

OBJECTIVE: To study the effects of sodium butyrate on proliferation, differentiation and apoptosis of immortalized esophagus epithelial cells. METHODS: SHEE, an immortalized human fetal esophageal epithelial cell line induced by HPV18 E6E7, was cultivated in culture flasks and 24-well plates. Two experiment groups of cultured cells were treated with 1 and 5 mmol/L of sodium butyrate respectively for 4 days, and one group of untreated cells set aside as control. The numbers of cloned cells were calculated. The ultra-structure of SHEE cells was examined by transmission electron microscopy (TEM). The cell cycle and number of apoptotic cells were measured by flow cytometry, Ki-67 and cytokeratin of cells were detected by immunohistochemistry method and F-actin of cells labeled with phalloidin was examined by laser confocal scanning microscopy. RESULTS: Colony formations showed a significant decrease in the 2 experiment groups after 4 days of culture (P < 0.01). In the 1 mmol/L group, the cells at S phase were diminished and arrested at G(0)/G(1) phase. Compared with control group, Ki-67 positive cells were found decreased, while F-actin and cytokeratin were increased. Apoptotic cells in 5 mmol/L group were increased markedly. CONCLUSIONS: Sodium butyrate may induce SHEE cells growth arrest, differentiation and apoptosis. The effects depend on sodium butyrate concentration and time of exposure. Whether it can be used in combination with other anticancer drugs should be further studied.

Actins↗

PubMed2004

Transport of dendrimer nanocarriers through epithelial cells via the transcellular route.

The mechanism of transport of G3 PAMAM and surface-modified (with lauroyl chains) G3 PAMAM dendrimer nanocarriers across Caco-2 cell monolayers has been investigated. Flow-cytometry studies following quenching of extracellular fluorescence demonstrated the cellular internalisation of dendrimers. Optical sectioning of cells incubated with fluorescein isothiocyanate (FITC)-conjugated dendrimer and lauroyl-dendrimer using confocal laser scanning microscopy revealed colocalisation of a marker for cell nuclei (4',6-diamidino-2-phenylindole, DAPI) and FITC fluorescence, also suggesting cellular internalisation of dendrimers. Transmission electron microscopic analyses of cells incubated with gold-labelled G3 PAMAM dendrimers confirmed endocytosis-mediated cellular internalisation when dendrimers were applied to the apical domain of Caco-2 cells. These findings are in agreement with our previous studies using Caco-2 cell monolayers that showed a significant decrease of dendrimer uptake in the presence of colchicine (endocytosis inhibitor) and when temperature was reduced from 37 to 4 degrees C.

Biological Transport↗

PubMed1998

Identification of cytokeratin 1 as a binding protein and presentation receptor for kininogens on endothelial cells.

A kininogen binding protein(s), a putative receptor, was identified on endothelial cells. A 54-kDa protein was isolated by a biotin-high molecular mass kininogen (HK) affinity column that, on aminoterminal sequencing of tryptic digests, was identified as cytokeratin 1. Multiple antibodies directed to cytokeratin 1 reacted with a 54-kDa band on immunoblot of lysates of endothelial cells. On laser scanning confocal microscopy, cytokeratin 1 antigen was found on the surface of endothelial cells. Cytokeratin 1 antigen also was detected on endothelial cell membranes by flow cytometry. Moreover, an antipeptide antibody to a sequence unique to cytokeratin 1 also specifically bound to nonpermeabilized endothelial cells. Biotin-HK specifically bound to cytokeratin only in the presence of Zn2+, and cytokeratin blocked biotin-HK binding to endothelial cells. Further, HK and low molecular mass kininogen, but not factor XII, blocked biotin-HK binding to cytokeratin, and peptides of each cell binding region of HK on domains 3,4, and 5 blocked biotin-HK binding to cytokeratin. gC1qR and soluble urokinase-like plasminogen activator receptor also inhibited biotin-HK binding to cytokeratin. These investigations identify a new function for cytokeratin 1 as a kininogen binding protein. Cytokeratins, members of the family of intermediate filament proteins, may represent a new class of receptors.

Endothelium, Vascular↗

PubMed2006

40 nm, but not 750 or 1,500 nm, nanoparticles enter epidermal CD1a+ cells after transcutaneous application on human skin.

Although conventional vaccines have generated major successes in the control of infectious diseases, several obstacles remain in their development against chronic diseases (HIV, tuberculosis), against which no current candidate vaccines yet ensure protection. The transcutaneous route of vaccine administration appears to be a promising approach of targeting vaccines toward antigen-presenting cells (APCs) and thus improving immune responses. We investigated the suitability of nanoparticles in this approach. We found a high density of Langerhans cells (LCs) around hair follicles that, when sorted, readily internalized all size particles. However, flow cytometry after transcutaneous application of 40, 750, or 1,500 nm nanoparticles on human skin samples revealed that only 40 nm particles entered epidermal LC. Fluorescence and laser scan microscopies, which were carried out to identify the penetration pathway of transcutaneously applied nanoparticles, revealed that only 40 nm particles deeply penetrate into vellus hair openings and through the follicular epithelium. We conclude that 40 nm nanoparticles, but not 750 or 1,500 nm nanoparticles, may be efficiently used to transcutaneously deliver vaccine compounds via the hair follicle into cutaneous APCs.

Administration, Cutaneous↗

PubMed1995

Shape, F-actin, and surface morphology changes during chemotactic peptide-induced polarity in human neutrophils.

BACKGROUND: The exposure of human neutrophils to uniform concentrations of chemoattractants, such as N-formyl peptides, induces morphological cell polarization. In this study we report the temporal sequence of changes in cell shape, F-actin, and cell surface morphology during cellular polarization induced by N-formylmethionyl-leucyl-phenylalanine (fMLP) in human neutrophils in suspension. METHODS: Neutrophil shape changes induced by 10(-8) M fMLP were observed with DIC microscopy. Size and cellular granularity were analyzed by flow cytometry measuring their forward and side scattered light. To visualize F-actin distribution, neutrophils were labeled with the fluorescence probe FITC-phalloidin, and were examined with fluorescence and confocal laser scanning microscopy. Cell surface morphology was assessed with scanning electron microscopy (SEM). RESULTS: The stimulation of round-smooth neutrophils with nanomolar concentrations (10(-8) M) of fMLP in suspension induced a temporal sequence of morphological changes during cell polarization, characterized by 1) increase in size as determined by forward angle scattered light, 2) rapid redistribution of F-actin from a diffuse cytoplasmic localization to the cell periphery, and 3) rapid reorganization of cell surface morphological features, with accumulation of plasma membrane in the front of polar cells. Four cell shapes were identified with SEM after stimulation of round-smooth neutrophils: round-ridged, round-ruffled, nonpolar ruffled, and polar cells. These cell shapes were correlated with a cortical localization, focal aggregates, and multipolar distribution of F-actin. In polar neutrophils, F-actin became concentrated in the front of the cell. CONCLUSIONS: These findings show the relation between reorganization of the microfilamentous cytoskeleton and modifications in cell shape and surface features during cell polarization induced after fMLP activation in neutrophils. This approach offers a powerful tool for further analysis of receptor distribution in polarized, motile neutrophils.

Actins↗

PubMed2002

Analysis of cytotoxic T lymphocytes and Fas/FasL in Japanese patients with non-small cell lung cancer associated with HLA-A2.

PURPOSE: In recent years, the use of immunotherapy for malignant tumors has been proposed. To explore the significance of immunotherapy for lung cancer, we examined two systems : the HLA class I and cancer-reactive CTL system, and the Fas-FasL system. METHODS: HLA class I (HLA-A, -B, and -C) antigens were determined in 61 patients with lung cancer and in 30 healthy controls. The HLA class I phenotype was investigated by serological techniques. HLA-A2 alleles were investigated by polymerase chain reaction sequence-specific primer analysis. We analyzed lymphocytes isolated from 61 patients with two-color surface labeling and flow cytometry. Furthermore, we analyzed sFas and sFasL expression by enzyme-linked immunosorbent assay (ELISA). We also examined lung cancer cell line-induced apoptosis of CD8(+) lymphocytes using confocal laser scanning microscopy. RESULTS: The HLA-A2 allele was observed in 27 of 61 patients with lung cancer. There were no differences in HLA-A2 allele classifications between lung cancer patients and controls. Thirty-six of the 61 lung cancer patients (57%) had elevated levels of sFas, and 16 of the 61 patients (26.2%) had elevated levels of sFasL. The sFas level of lung cancers with HLA-A2 was significantly higher than that of cancers without HLA-A2 ( P<0.01). This tendency was observed in every lung cancer tissue type, and the sFas levels of lung cancers with HLA-A2 associated significantly with the CTL levels of lung cancers with HLA-A2. Furthermore, compared to lung cancers without HLA-A2, CD8(+) T-cell levels were elevated in lung cancers with HLA-A2. In contrast, sFas levels of non-small cell lung cancers without HLA-A2 were higher than those in lung cancers with HLA-A2. In an in vitro experiment using lung cancer cell lines, we observed apoptosis of CD8(+) lymphocytes induced by lung cancer cells. Lung cancer-reactive CTLs are mobilized easily by restriction of HLA-A2, but this restriction is not always specific. In addition, FasL derived from lung cancer cells can induce apoptosis of CD8(+) T-cells, and the frequency of this phenomenon is increased in small cell lung cancers without HLA-A2. CONCLUSION: Our findings suggest that the effect of immunotherapy may be insufficient for non-small cell lung cancer without HLA-A2.

Adult↗

PubMed2003

Visualization of abscisic acid-perception sites on the plasma membrane of stomatal guard cells.

Abscisic acid (ABA) is a phytohormone that plays a key role as a stress signal, regulating water relations during drought conditions, by inducing stomatal closure. However, to date, no putative ABA receptor(s) has been reported at the protein sequence, gene family, or cellular localization levels. We used biotinylated ABA (bioABA) to characterize the ABA-perception sites in the stomatal guard cells of Vicia faba. Treatment with bioABA induced stomatal closure and shrinkage of guard cell protoplasts (GCPs). The ABA-perception sites were visualized by fluorescence microscopy and confocal laser scanning microscopy (CLSM), using bioABA and fluorescence-labeled avidin. Fluorescent particles were observed in patches on the surface of the GCPs. Fluorescence intensity was quantified by flow cytometry (FCM) as well as by CLSM. Binding of bioABA was inhibited by ABA in a dose-dependent manner. Pre-treatment of GCPs with proteinase K also blocked the binding of bioABA. Binding of bioABA was inhibited by RCA-7a, an ABA analog that induces stomatal closure, but not by RCA-16, which has no effect on stomatal aperture. Another ABA analog, PBI-51, inhibited ABA-induced stomatal closure. This ABA antagonist also inhibited binding of bioABA to the GCPs. These results suggest that ABA is perceived on the plasma membrane of stomatal guard cells, and that the present experimental methods constitute valuable tools for characterizing the nature of the ABA receptor(s) that perceives physiological ABA signals. These imaging studies allow us to demonstrate the spatial distribution of the ABA-perception sites. Visualization of the ABA-perception sites provides new insights into the nature of membrane-associated ABA receptor(s).

Abscisic Acid↗

PubMed2005

Allyl alcohol and garlic (Allium sativum) extract produce oxidative stress in Candida albicans.

Both the growth and respiration of Candida albicans are sensitive to extracts of Allium sativum and investigations into the anticandidal activities are now focussing on the purified constituents to determine the targets of inhibition. Of particular interest is allyl alcohol (AA), a metabolic product that accumulates after trituration of garlic cloves. Putative targets for AA were investigated by monitoring changes in intracellular responses after exposure of C. albicans cells to AA or a commercially available garlic extract. Two-photon laser scanning microscopy and other techniques were used. Changes typical of oxidative stress--NADH oxidation and glutathione depletion, and increased reactive oxygen species--were observed microscopically and by flow cytometry. Known targets for AA are alcohol dehydrogenases Adh1 and 2 (in the cytosol) and Adh3 (mitochondrial), although the significant decrease in NAD(P)H after addition of AA is indicative of another mechanism of action.

Candida albicans↗

PubMed2005

Phenotype-specific cells with proliferative potential are produced by polyethylene glycol-induced fusion of mouse embryonic stem cells with fetal cardiomyocytes.

Because cardiomyocytes lose the ability to divide upon differentiation, myocardial failure is assumed to be generally irreversible. For terminal cardiac insufficiency, the potential for regenerative treatment by stem cells, especially embryonic stem (ES) cells, offers hope for the future. Recent studies showed that stem cells fuse spontaneously with cells remaining in damaged tissues, and restore tissue function. To imitate spontaneous fusion in vivo, we used polyethylene glycol (PEG) in vitro to fuse mouse ES cells and fetal cardiomyocytes and analyzed the cytochemical properties of the fused cells. Confocal laser scanning microscopy coupled with lipophilic dye labeling of the living cell membranes showed that there were fused cells of ES cells and cardiomyocytes after PEG treatment. By flow cytometry, the fusion efficiency between ES cells and cardiomyocytes was estimated to be about 45% of the total resulting cells. When green fluorescent protein (GFP)-expressing ES cells were fused with cardiomyocytes, the fused cells had immunoreactivity for GFP in their cytoplasm and cardiac troponin I in their myofibrils. Some of these cells also expressed proliferating cell nuclear antigen up to 11 days after fusion, the last time point examined. This study shows that PEG-induced fusions of mouse ES cells and cardiomyocytes have the cardiomyocyte phenotype and proliferation potential.

Animals↗

PubMed2005

[Effects of transforming growth factor-beta/Smad signaling on the growth and apoptosis of human rhabdomyosarcoma cell line RD].

OBJECTIVE: To study the effects of TGF-beta/Smad signaling on the growth and apoptosis of human rhabdomyosarcoma cell line RD. METHODS: Biosynthesized short hairpin RNA (shRNA) was transfected into RD cells by cation liposome vector to suppress Smad4 expression. The mRNA and protein expression of Smad4 in RD after shRNA-transfection were examined by RT-PCR and Western blot respectively. Immunofluorescent staining was used to detect the location of Smad2/3 in RD by laser scanning confocal microscopy. The viability of RD cells was examined by MTT method and (3)H-thymidine incorporation assay. The apoptosis of RD was examined by flow cytometry analysis and fluorescent staining. RESULTS: The expression of mRNA and protein of Smad4 in RD were effectively suppressed by shRNA interference. Such suppression effectively interrupted the endogenous TGF-beta/Smad signaling and consequently blocked the translocation of Smad2/3. The interruption of endogenous TGF-beta/Smad signaling not only inhibited the growth of RD but also induced apoptosis of RD. Exogenous TGF-beta1 inhibited the growth of RD but did not influence the apoptosis of RD. CONCLUSION: shRNA interference can effectively interrupt the TGF-beta/Smad signaling by suppressing the expression of Smad4. TGF-beta/Smad signaling subtly regulates the growth and apoptosis of RD.

Apoptosis↗

PubMed2002

Presence of anaerobic bacteroides in aerobically grown microbial granules.

Microbial granules were grown in a column-type sequential aerobic sludge blanket reactor inoculated with activated sludge flocs taken from a wastewater treatment plant and containing a medium with glucose as the main carbon source. The reactor selected for granules that could settle rapidly by employing a short settling time of 2 min. Matured granules with diameters between 2 and 3 micro m were examined for anaerobic bacteria as their presence can signal the onset of diffusion limitation problems that can potentially diminish granule stability due to the bacterial production of fermentation gases and organic acids under anaerobic conditions. To detect the anaerobes in the granules, clones were constructed from 16S rRNA PCR amplicons. Two sequence types associated with a strict anaerobe Bacteroides spp. were identified from these clones. Fluorescence in situ hybridization (FISH) followed by confocal laser scanning microscopy (CLSM) demonstrated that cells of Bacteroides spp. were concentrated at a depth of approximately 800 micro m below the surface of the granule. Cell enumeration using flow cytometry showed that the percentage of labeled cells of Bacteroides spp. compared to total bacterial cells in the granules was 0.56%. This is the first study to use a suite of culture-independent techniques to report the presence of a defined species of anaerobic bacteria in aerobically grown microbial granules.

Aerobiosis↗

PubMed2004

Anti-neoplastic efficacy of Haimiding on gastric carcinoma and its mechanisms.

AIM: To study the anti-neoplastic effect of Haimiding and its mechanisms of action. METHODS: Experiments using MTT and colony formation were carried out to study the in vitro anti-neoplastic action of Haimiding, its in vivo anti-neoplastic action was studied by observing its effect on the weight of tumors in FC mice and S(180), H(22) tumor bearing mice, as well as their life spans. The effect of Haimiding on cell apoptosis and different stages of cell cycles in human gastric carcinoma cells were studied by flow cytometry. Its effect on [Ca(2+)](i) of human gastric carcinoma cells and the source of Ca(2+) during the change of [Ca(2+)](i) were observed by confocal laser scanning technique. RESULTS: Haimiding showed a definite cytotoxicity to 8 human tumor cell lines, which was most prominent against BGC-823, E(ca-109) and HCT-8 tumor cells. It also exhibited an obvious inhibition on colony formation of the above tumor cell lines, which was most prominent in E(ca-109) tumor cells. It showed obvious inhibition on the growth of tumor in FC mice and S(180) bearing mice as well as prolonged the life span of H(22) bearing mice. It was able to induce apoptosis and elevate intracellular [Ca(2+)](i) concentration of tumor cells. The source of Ca(2+) came from both extracellular Ca(2+) influx and intracellular Ca(2+) release. CONCLUSION: Haimiding is composed of a TCM preparation and 5-flurouracil. Its anti-neoplastic potency is highly enhanced by synergism as compared with either one of its components. Its mechanisms of anti-neoplastic action can be attributed to its action to initiate apoptosis of tumor cells by opening the membrane calcium channel and inducing intracellular Ca(2+) release to elevate [Ca(2+)](i) of the tumor cells.

Adenocarcinoma↗

PubMed1987

Design for a fast fluorescence laser scanning microscope.

The design of a fast fluorescence laser scanning microscope is described and illustrated, with discussion of the design consideration of the principal components, including the optical elements. The system, now under construction at the Optical Sciences Center of the University of Arizona, is expected to provide very-high-speed scanning, at a high spatial sampling density, of large object areas while retaining a flexibility of applications. The projected scanning rate approximates the rate achieved by flow cytometry; the projected rates of information generation should be orders of magnitude higher.

Equipment Design↗

PubMed2000

Promotion of adhesion and migration of RPE cells to provisional extracellular matrices by TNF-alpha.

PURPOSE: Adhesion and migration of retinal pigment epithelial (RPE) cells to provisional extracellular matrices (ECM) is important in the development of epiretinal membranes found in proliferative vitreoretinopathy (PVR). Tumor necrosis factor alpha (TNF-alpha) is found in PVR membranes and regulates many functions of RPE cells. In this study, the effects of TNF-alpha on adhesion and migration of RPE cells to various components of ECM were examined and elucidation of the mechanism of the response was attempted. METHODS: Mitogen activated protein kinase (ERK1/2; MAPK) activation was measured by immunoblot. RPE cells pretreated with TNF-alpha (10 ng/ml) or TNF-alpha + PD98059 (a specific inhibitor of MAPK, 30 microM) for 24 hours were compared with control RPE. Attachment was measured by modified MTT assay on fibronectin and collagen types I and IV. Spreading was measured by staining with fluo3-AM and confocal laser scanning microscopy. Migration of RPE cells on substrates was determined by Boyden chamber assay using PDGF-BB (20 ng/ml) as a chemotactic factor. Integrin expression was determined by flow cytometry and RT-PCR. RESULTS: TNF-alpha rapidly activated MAPK and increased the extent of attachment, spreading and migration on fibronectin and collagen type I (P < 0.01) but not on collagen type IV. TNF-stimulated RPE cells showed increased mRNA and surface protein expression for alpha1 and alpha5 integrin (P < 0.01) but not alpha3 integrin subunit. Neutralizing the anti-alpha1 antibody inhibited migration on collagen type I, whereas alpha5 antibody inhibited fibronectin-induced migration. Treatment with both TNF and PD98095 reduced attachment and migration on provisional ECM and reduced the upregulated integrin expression to control levels. CONCLUSIONS: After treatment with TNF-alpha, there is increased expression of specific integrins associated with increased adhesion and migration on provisional ECM (fibronectin and collagen type I). This effect is mediated, at least in part, by activation of MAPK signaling pathway.

Calcium-Calmodulin-Dependent Protein Kinases↗

PubMed1999

Detection of Cryptosporidium oocysts in water: techniques for generating precise recovery data.

When determining the recovery efficiency of a procedure for the detection of Cryptosporidium or the removal efficiency of a treatment process, it is necessary to accurately enumerate a 'seed dose'. Conventional techniques for this are highly variable and consequently, can result in misleading data. In this study, a flow cytometric method was developed for the production of suspensions of Cryptosporidium oocysts in which the number of organisms could be precisely determined. A Becton Dickinson FACScalibur flow cytometer was employed to produce oocyst suspensions containing 100 oocysts. Analysis of these suspensions resulted in a mean dose of 99.5 oocysts (S.D. = 1.1, %cv = 1.1). These results indicate that the use of such suspensions to seed test systems generates far more accurate data than is presently possible using conventional techniques. In addition, the use of immunomagnetic separation (IMS) for the isolation of oocysts from three different water matrices, after seeding with oocysts counted using flow cytometry, was investigated. The recovery efficiency of the IMS procedure was found to be high, with the percentage recovery of oocysts ranging from 82.3 to 86.3%, and the use of precise numbers of oocysts allowed accurate recovery efficiency data to be generated. A laser scanning instrument (ChemScan RDI) was employed for the rapid detection and enumeration of oocysts after capture using membrane filtration. This technique was found to be faster and easier to perform than conventional epifluorescence microscopy. These findings demonstrate that the ChemScan RDI system may be used as alternative procedure for the routine examination of IMS supernatant fluids for the presence of Cryptosporidium.

Animals↗

PubMed1998

Internalin B is essential for adhesion and mediates the invasion of Listeria monocytogenes into human endothelial cells.

Listeria monocytogenes causes rhombencephalitis in humans and animals and also affects the fetus in utero, causing disseminated sepsis. In both instances, the infection occurs by the crossing of endothelial cells lining a physiological barrier, the blood-brain barrier or the transplacental barrier. In this study, the ability of L. monocytogenes wild-type EGD to invade human umbilical vein endothelial cells (HUVECs) was evaluated using wild-type bacteria and isogenic Listeria mutants. Here, we show that invasion of HUVECs by L. monocytogenes is dependent on the expression of the internalin B gene product. This was demonstrated in several ways. First, L. monocytogenes strains lacking the inlB gene did not invade HUVECs. Secondly, avid invasion was obtained when a strain deleted for inlAB was complemented with a plasmid harbouring inlB only, whereas strains expressing inlA did not enter HUVECs. Thirdly, entry of wild-type EGD could be blocked effectively with antibodies to InlB. Fourthly, cell binding assays and flow cytometry with HUVECs showed binding of purified InlB, but not InlA, suggesting a tropism of InlB for this cell type. Finally, physical association of purified native InlB with the surface of non-invasive mutants dramatically increased their ability to invade HUVECs. In laser-scanning confocal microscopy, binding of InlB was observed as focal and localized patches on the cell surface of HUVECs. Qualitative examination of the entry process by scanning electron microscopy revealed that both wild-type EGD and a recombinant strain overexpressing only InlB enter HUVECs in a similar fashion. The entry process was polarized, involved single bacteria and occurred over the entire surface of endothelial cells.

Antibodies, Bacterial↗