A Role for the Glycolipid Exoantigen (GLXA) in Chlamydial Infectivity

The chlamydial glycolipid exoantigen, GLXA, is associated with the bacterial membrane, intracellular inclusion, and can also be found secreted into the microenvironment of Chlamydia trachomatis-infected cells. The aim of this study was to investigate the function of GLXA in chlamydial pathogenesis. Pretreatment of HeLa 229 cells with affinity-purified GLXA resulted in a significant enhancement of chlamydial infectivity as determined by inclusion body enumeration. The GLXA-mediated enhancement was shown to be time- and dose-dependent and, more importantly, GLXA-specific, as the effect was abrogated by anti-GLXA antibody. In vitro neutralization assays on HEp-2 cells revealed that an anti-anti-idiotypic antibody to GLXA effectively reduced the infectivity of C. trachomatis, C. pneumoniae, and C. psittaci. In vivo, the co-inoculation of GLXA at the time of C. trachomatis serovar K intravaginal challenge of C3H/HeJ mice resulted in a significant increase in the numbers of shed organisms on days 4, 7, 14, 21, and 28. Taken together, these observations suggest that GLXA, both organism bound and secreted, is important in facilitating the initiation of infection. Received: 12 April 2002 / Accepted: 8 June 2002     

Cell Surface Display of the Chlamydial Glycolipid Exoantigen (GLXA) Demonstrated by Antibody-Dependent Complement-Mediated Cytotoxicity

The chlamydial species are Gram-negative bacterial pathogens critical to human health. Their developmental cycle is associated with the formation and release of the broadly conserved glycolipid exoantigen (GLXA), which has been implicated in the chlamydial elementary body–host cell interaction. This study examines the potential surface display of this glycolipid by chlamydiae-infected cells and the ability of the GLXA they secrete to associate with the plasma membranes of uninfected cells, a prerequisite for exerting influence on them. The sequential incubation of anti-GLXA antibody and complement with Chlamydia trachomatis serovar K or C. pneumoniae AR-39-infected HeLa 229 or macrophage cells resulted in significant cellular cytotoxicity, which preceded the formation of mature elementary bodies. For uninfected cells, co-incubation of GLXA, purified from supernatants of either C. trachomatis or C. pneumoniae-infected HeLa 229 cells, followed by the successive addition of mouse anti-GLXA antibody and complement, yielded similar levels of cellular cytotoxicity. Thus, GLXA indeed is displayed on the surface of infected cells and, therefore, if antibody of appropriate specificity were present, this GLXA could serve to target these infected cells for elimination. Furthermore, released GLXA can associate with uninfected cells and therefore would be positioned to influence their behavior, especially in the context of infection.     

Establishment and characterization of an immortalized but non-transformed human prostate epithelial cell line: BPH-1

Summary This report describes the development and characterization of an epithelial cell line (BPH-1) from human prostate tissue obtained by transurethral resection. Primary epithelial cell cultures were immortalized with SV40 large T antigen. One of the isolated clones was designated BPH-1. These cells have a cobblestone appearance in monolayer culture and are non-tumorigenic in nude mice following subcutaneous injection or subrenal capsule grafting. They express the SV40 large T antigen and exhibit increased levels of p53, as determined by immunocytochemistry. Cytogenetic analysis by G-banding demonstrated an aneuploid karyotype with a modal chromosome number of 76 (range 71 to 79,n=28) and 6 to 8 marker chromosomes. Some structurally rearranged chromosomes were observed, but the Y chromosome was normal. The expressed cytokeratin profile was consistent with a prostatic luminal epithelial cell. This profile was the same as that of primary prostatic epithelial cultures from which the BPH-1 cells were derived. In serum-free culture in plastic dishes epidermal growth factor (EGF), transforming growth factor (TGF)-α, fibroblast growth factor (FGF) 1 (aFGF), and FGF 7 (KGF) induced increased proliferation in these cells whereas FGF 2 (bFGF), TGF-β1, and TGF-β2 inhibited proliferative activity. Testosterone had no direct effect on the proliferative rate of BPH-1 cells. 5α-Reductase, 3α-hydroxysteroid oxidoreductase, and 17β-hydroxy-steroid oxidoreductase activities were detected in BPH-1 cells. Expression of androgen receptors and the secretory markers, prostate specific antigen and prostatic acid phosphatase, were not detectable by immunocytochemistry, biochemical assay, or RT-PCR analysis.     

Characterization of a monoclonal antibody reactive with a glycolipid antigen expressed by tumorigenic and certain immortalized,non-tumorigenic rat esophageal epithelial cell lines

A monoclonal antibody (mAb 5G) was produced against a tumorigenic rat esophageal epithelial cell line, designated B2T. Using an enzyme-linked immunosorbent assay, immunofluorescence assay (IFA), thin-layer chromatography (TLC) and immunoperoxidase staining, it was found that mAb 5G reacted specifically with a glycolipid antigen expressed by three tumorigenic rat esophageal epithelial cell lines, and two out of the three nontumorigenic, immortalized rat esophageal epithelial cell lines tested; but did not react with primary cultures of normal rat esophageal epithelial cells or fibroblasts. mAb 5G did not bind to rat respiratory tract carcinoma cell lines, to immortalized rat tracheal epithelial cell lines, or to primary cultures of normal rat tracheal epithelial cells. In addition, mAb 5G did not react with any of the human or mouse cell lines tested. In IFA experiments, mAb 5G stained imprints prepared from in vivo propagated B2T tumor tissues, but did not react with normal rat esophageal, tracheal, lung, liver, and kidney tissues. The antigen was identified by TLC as a neutral glycolipid, consisting of two bands, withR _F = 0.45 and 0.41, which migrated in proximity to the ceramide trihexoside standard on TLC plates. Densitometric scanning of the antigen bands indicated that the tumorigenic rat esophageal cell lines possessed 50%–90% more mAb-5G-reactive antigen than the nontumorigenic esophageal cell lines. The results show that mAb 5G reacts specifically with a glycolipid antigen expressed by tumorigenic and certain non-tumorigenic, immortalized rat esophageal epithelial cell lines that might be at the late stages of transformation and early malignancy.     

Accumulation of betulinic,oleanolic, and ursolic acids in <Emphasis Type="Italic">In vitro</Emphasis> cell cultures of <Emphasis Type="Italic">Lantana camara</Emphasis> L. and their significant cytotoxic effects on HeLa cell lines

This study reports the accumulation of three pentacyclic triterpenoids, betulinic acid (BA), oleanolic acid (OA), and ursolic acid (UA), in cell cultures of Lantana camara using leaf disc explants. Thin layer chromatography (TLC), high performance liquid chromatography (HPLC), and mass spectroscopy (MS) of organic extract followed by comparison with the standards revealed the presence of the above three triterpenoids. By following the extraction procedure mentioned here the recovery of all these three compounds were > 95%. The bioactivity of the cell-derived extract was demonstrated using cancerous HeLa cell lines. Specifically, the effect of this extract on HeLa cells was noticed from 36 h (at 100 μg/mL) to 72 h (at 25 μg/mL) by employing 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) cytotoxicity assay. The results show that an increase in the concentration or duration of the extract treatment was effective in killing cancerous cells. DNA laddering assay confirmed that cell death was due to apoptosis. Further, BA was identified for the first time from an in vitro source. Moreover, this was the first report describing this kind of studies conducted using Lantana plants.     

Immunofluorescent Cell Assay of Infectious Pancreatic Necrosis Virus

An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.     

Helicobacter pylori induces urease subunit B-specific CD8+ T cell responses in infected individuals via cytosolic pathway of cross-presentation

Background

Urease subunit B (UreB), a conserved and key virulence factor of Helicobacter pylori (H. pylori), can induce the host CD4⁺ T cell immune responses to provide protection, but less is known regarding CD8⁺ T cell responses. The characteristics of H. pylori-specific CD8⁺ T cell responses and the mechanism underlying antigen processing and presentation pathways remain unclear. This study was focus on protective antigen recombinant UreB (rUreb) to detect specific CD8⁺ T cell responses in vitro and elucidate the mechanism of UreB antigen processing and presentation.

Methods

The peripheral blood mononuclear cells (PBMCs) collected from H. pylori-infected individuals were stimulated with rUreB in vitro to detect specific CD8⁺ T cell responses after co-culture with rUreB-pulsed autologous hMDCs. Through blocking assay, we investigated the potential pathway of UreB antigen processing and presentation via the cytosolic pathway or vacuolar pathway. The cytokines production of UreB specific CD8⁺ T cell were evaluated as well.

Results

We demonstrated UreB can induce specific CD8⁺ T cell immune responses in H. pylori infected individuals. Importantly, we characterized that UreB were mainly processed by proteasome instead of lysosomal proteases and presented through cytosolic pathway of cross-presentation, which requires endoplasmic reticulum–Golgi transport and newly synthesized MHC-I molecules, to induce functional-specific CD8⁺ T cell (IFN-γ + TNF-α + Grz A+ Grz B+) responses.

Conclusions

These results suggest that H. pylori UreB induces specific CD8⁺ T cell responses through cytosolic pathway of cross-presentation in infected individuals.     

An ex vivo readout for evaluation of dendritic cell-induced autologous cytotoxic T lymphocyte responses against esophageal cancer

Esophageal cancer is a highly malignant disease that despite surgery and adjuvant therapies has an extremely poor outcome. Dendritic cell (DC) immunotherapy as a novel promising strategy could be an alternative for treating this malignancy. Effective DC-mediated immune responses can be achieved by raising cytotoxic T lymphocyte (CTL) response against multiple antigens through loading DCs with total tumor RNA. However, the efficacy of this strategy first needs to be evaluated in a pre-clinical setting. The aim of the study was to set up an ex vivo autologous human readout assay for assessing the effects of DC-mediated cytotoxic responses, using total tumor RNA as an antigen load. Biopsy specimens of seven esophageal cancer patients were used to establish primary cultures of normal and cancer cells and to obtain autologous RNA for loading DCs. Mature DCs loaded with either normal or tumor RNA were obtained and subsequently used to raise various lymphocytes populations. Apoptosis levels of the autologous cultures were measured before and after incubating the cultures with the different lymphocytes populations. The mean apoptosis levels in the tumor cell cultures, induced by lymphocytes instructed by DCs loaded with tumor RNA, significantly increased with 15.6% ±2.9 SEM (range 3.4–24.5%, t-test, P < 0.05). Incubation of the normal cultures with the lymphocytes populations showed a mean non-significant increase in apoptosis of 0.4% ±3.4 SEM (range −13.9 to 9.8%, t-test, P = 0.7). Here, we introduce a practical, patient-specific autologous readout assay for pre-clinical testing of DC-mediated cytotoxic responses. Additionally, we demonstrated that the use of autologous tumor RNA as a strategy for raising cytotoxic responses against multiple tumor antigens could be effective for treating esophageal cancer. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular analyses of a Lesch-Nyhan syndrome mutation (hprt Montreal) by use of T-lymphocyte cultures

Summary The frequency of hprt mutants in peripheral blood T-lymphocytes of two putative Lesch-Nyhan individuals and their parents was determined by a cell cloning assay to quantify the frequency of thioguanine-resistant mutants. The results confirmed the Lesch-Nyhan diagnosis and demonstrated that the mother has an elevated mutant frequency consistent with being heterozygous for an hprt mutation. Mass cultures of T-lymphocytes from both the children and their mother, as well as cultures of hprt mutant clones from the mother, were employed as sources of mRNA for cDNA sequence analysis. These hprt mutants show a single base substitution (TC transition) at position 170 (exon 3). The predicted amino acid change is the substitution of threonine for methionine₅₆. We have designated this new Lesch-Nyhan mutation hprt _Montreal. The use of T-lymphocyte cultures allows rapid sequence analyses of hprt mutations, as well as family studies to define the origin of a particular mutation.     

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.     

Application of Colorimetric Assays to Assess Viability, Growth and Metabolism of Hydrogel-Encapsulated Cells

The applicability of the colorimetric 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays to measure cell growth and viability in hydrogel encapsulation systems was investigated using HepG2 liver cells encapsulated in alginate matrices. The MTT assay was effective in measuring viable cell density in alginate-encapsulated cell systems, demonstrating less variance and higher throughput capability than hemocytometry. The LDH assay was effective in measuring dead cell density in monolayer cultures and in alginate-encapsulated cells simply by measuring the LDH concentration secreted into the medium. Further validation of these assays was shown in two additional cell lines (rat muscle and mouse embryonic fibroblasts). The MTT and LDH assays are particularly significant in the rapid evaluation of in vitro cell encapsulation device design.     

Quiescence, cell viability, apoptosis and necrosis of smooth muscle cells using different growth inhibitors

Smooth muscle cells and endothelial cells play an important role in cardiovascular diseases and may therefore be a potential target for gene therapy. Most in vitro experiments are performed using proliferating cell cultures. Nevertheless, non-dividing cells would represent more realistic in vivo conditions for gene therapy. Therefore, a simple method to achieve physiologically quiescence in cell cultures is needed for experiments. Growth to confluence is sufficient for endothelial cells to reach quiescence, in contrast to smooth muscle cells. Alternative techniques were investigated to achieve quiescence for smooth muscle cells. N-acetyl-cysteine, heparin, aphidicolin and serum-free medium are known inhibitors of smooth muscle cell proliferation and were tested for cell viability, necrosis and apoptosis. The inhibition status was evaluated counting cells in a cell counter. Toxicity, necrosis and apoptosis were determined using FACS analysis. Then, smooth muscle cells and endothelial cells were transfected with plasmid containing the beta-galactosidase gene using liposomes. Analysis of gene expression in transfected cells included a quantitative beta-galactosidase assay and X-gal staining. Growth inhibition was achieved with all agents tested. Using N-acetyl-cysteine, only slightly reduced growth rates were observed. Aphidicolin stopped cell growth almost immediately, but demonstrated enhanced toxicity. The amount of apoptotic and necrotic cells was lowest using heparin in the presence of foetal calf serum. Transfection experiments using stationary cultures of smooth muscle cells using heparin or aphidicolin demonstrated 5-10-fold lower transfection rates compared to transfected proliferating cell cultures serving as controls. Transfection experiments using stationary cultures of endothelial cells using growth inhibition through confluence demonstrated 40-fold lower transfection rates than transfected proliferating cell cultures. Transfer efficiency was much lower in endothelial cells compared to smooth muscle cells. In conclusion, quiescent cells simulate more realistically the in vivo situation and may therefore represent a better model for future in vivo experiments based on in vitro findings.     

Establishment and characterization of the NEYS cell line derived from carcinosarcoma of human ovary with special reference to the susceptibility test of anticancer drugs

A cell line designated as NEYS was established from ovarian carcinosarcoma (stage IIIc) of a 56-year-old Japanese woman. The extirpated original tumor was carried in growth medium at 0 °C to the culture room. The primary culture was done on 20 August 2003. The cell line was composed of angular adhesive cells and showed neoplastic and pleomorphic features, such as bizarre aggregation of chromatin granules, an irregular thickening nuclear membrane and multiple large nucleoli. They grew as multi-layered cultures without contact inhibition. The cells proliferated moderately, and population doubling time was about 56 h. The chromosome number showed an underdiploidy of aneuploidy. The modal chromosome numbers were 37 (36%) and 38 (26%). The cultures produced carcinoembryonic antigen (27.4 ng/mL), carbohydrate antigen 19-9 (210 U/mL), and carbohydrate antigen 125 (526 U/mL). The NEYS cells did not give rise to transplant tumors in nude mice, and showed no susceptibility against cisplatin (CDDP), CPT-11, carboplatin, Paclitaxel, Taxotere and 5-FU. This cell line is useful for studies on the histogenesis of carcinosarcoma and susceptibility of cancer drugs in human ovarian carcinosarcoma. The immunohistochemical and ultrastructual analysis demonstrated that NEYS cells showed epithelial and mesenchymal differentiation, and supported the metaplasis theory as the cause of carcinosarcoma.     

Continous replication of potato spindle tuber viroid (PSTV) in permanent cell cultures of potato and tomato

The continuous replication of potato spindle tuber viroid (PSTV) in callus cultures from PSTV-infected wild-type potato (Solanum dem/ssum L.) and tomato (Lycopersicon peruvianum L. Mill) plants and in cell suspensions derived from potato protoplasts (Solanum tuberosum L.) inoculatedin vitro is described. The persistence of PSTV replication in these cell lines through at least 14 subculture passages, which corresponds to a continous replication over a period of more than one year, was demonstrated by infectivity assay and by polyacrylamide-gel electrophoresis of isolated nucleic acids. This continuous synthesis denovo of PSTV was substantiated by the incorporation of [³H]uridine and of [³²P]orthophosphate into viroid RNA.     

Cell growth and hemoglobin synthesis in murine erythroleukemic cells propagated in high density microcapsule culture

Summary A new microencapsulation technology, developed for the encapsulation of living cells, has been demonstrated to be useful for the study of growth and differential gene expression using Friend erythroleukemic cells cultured at high cell densities. Using this technology, cultures of FL Clone 745 cells were encapsulated within semipermeable membranes composed of cross-linked alginic acid and poly-l-lysine. Cell growth studies measuring total cell number demonstrated an average generation time of 8.5 h in 5% (vol/vol) microcapsule cultures vs. 8.0 h in suspension cultures. Similar microcapsule cultures were serially propagated for more than 90 cell generations (13 sequential passages) with no significant change in this growth rate. In addition, final culture densities of greater than 1.0×10⁸ cells/ml of intracapsular volume were attained using a 3% (vol/vol) microcapsule culture in conjunction with a standard refeeding schedule. Comparison of the level of dimethyl sulfoxide-induced hemoglobin production in suspension and microcapsule cultures demonstrated that the total amount of hemoglobin produced on a per cell basis was comparable in both systems. Due to the retention characteristics of the semipermeable membrane, the concentration of detergent-released hemoglobin, relative to other released protein, was approximately twofold higher in microcapsule cultures than in control suspension cultures.     

CANDLES,an assay for monitoring GPCR induced cAMP generation in cell cultures

Background

G protein-coupled receptors (GPCRs) represent a physiologically and pharmacologically important family of receptors that upon coupling to Gα_S stimulate cAMP production catalyzed by adenylyl cyclase. Thus, developing assays to monitor cAMP production is crucial to screen for ligands in studies of GPCR signaling. Primary cell cultures represent a more robust model than cell lines to study GPCR signaling since they physiologically resemble the parent tissue. Current cAMP assays have two fundamental limitations: 1) absence of cAMP kinetics as competition-based assays require cell lysis and measure only a single time-point, and 2) high variation with separate samples needed to measure consecutive time points. The utility of real-time cAMP biosensors is also limited in primary cell cultures due to their poor transfection efficiency, variable expression levels and inability to select stable clones. We therefore, decided to develop an assay that can measure cAMP not only at a single time-point but the entire cAMP kinetics after GPCR activation in untransfected primary cells.

Results

CANDLES (C yclic A MP iN direct D etection by L ight E mission from S ensor cells) assay for monitoring cAMP kinetics in cell cultures, particularly in primary cultures was developed. The assay requires co-culturing of primary cells with sensor cells that stably express a luminescent cAMP sensor. Upon GPCR activation in primary cells, cAMP is transferred to sensor cells via gap junction channels, thereby evoking a luminescent read-out. GPCR activation using primary cultures of rat cortical neurons and mouse granulosa cells was measured. Kinetic responses of different agonists to adrenergic receptors were also compared using rat cortical neurons. The assay optimization was done by varying sensor-test cell ratio, using phosphodiesterase inhibitors and testing cell-cell contact requirement.

Conclusions

Here we present CANDLES assay based on co-culturing test cells with cAMP-detecting sensor cells. This co-culture setup allows kinetic measurements, eliminates primary cell transfections and reduces variability. A variety of cell types (rat cortical neurons, mouse granulosa cells and established cell lines) and receptors (adrenergic, follicle stimulating hormone and luteinizing hormone/chorionic gonadotropin receptors) were tested for use with CANDLES. The assay is best applied while comparing cAMP generation curves upon different drug treatments to untransfected primary cells.

     

Investigation of the effectiveness of disinfectants against planktonic and biofilm forms of P. aeruginosa and E. faecalis cells using a compilation of cultivation,microscopic and flow cytometric techniques

This study evaluated the effectiveness of selected disinfectants against bacterial cells within a biofilm using flow cytometry, the conventional total viable count test and scanning electron microscopy (SEM). A flow cytometric procedure based on measurement of the cellular redox potential (CRP) was demonstrated to have potential for the rapid evaluation of activity against biofilm and planktonic forms of microbes. Quaternary ammonium compound-based disinfectant (QACB) demonstrated a higher level of anti-microbial activity than a performic acid preparation (PAP), with mean CRP values against P. aeruginosa cells of 2 and 1.33 relative fluorescence units (RFU) vs 63.33 and 61.33 RFU for 8 and 24 h cultures respectively. Flow cytometric evaluation of the anti-biofilm activity demonstrated a higher efficacy of QACB compared to PAP for P. aeruginosa cells of 1 and 0.66 RFU vs 18.33 and 22.66 RFU for 8 and 24 h cultures respectively. SEM images of treated P. aeruginosa cells demonstrated disinfectant-specific effects on cell morphology.     

Involvement of arabinogalactan proteins in the control of cell proliferation of <Emphasis Type="Italic">Cucurbita pepo</Emphasis> suspension cultures

Arabinogalactan proteins (AGPs) secreted by zucchini squash (Cucurbita pepo L.) cell cultures into the medium are implicated in cell proliferation. Conditioned medium derived from cell suspensions of squash cultivar Dundoo could enhance multiplication rate of slow-growing cell line Cx3005. To examine the role of AGPs, a precipitation assay was performed using Yariv reagent which binds selectively to AGPs. This AGP precipitation as well as proteinase application arrested cell division. However, chitinase treatment successfully increased embryogenic callus mass. A growth promotion was also obtained by arabinogalactan addition to the culture medium. Immunoblotting analysis using the MAC 207 anti-AGP monoclonal antibody showed high AGP expression in Dundoo cell cultures.