Indications for dental-CT. Case reports

In an earlier study we demonstrated that Concanavalin-A stimulated bovine T cell supernatants inhibited the growth of Cowdria ruminantium in bovine endothelial cells in vitro. An investigation was conducted to identify the cytokines which were responsible for this growth inhibition. Addition of antiserum against bovine interferon gamma (IFN gamma) reproducibly neutralized the inhibitory effect of the T cell supernatants, whereas addition of antisera against bovine tumor necrosis factor alpha (TNF alpha) had no effect. The inhibitory effect of IFN gamma on C. ruminantium growth was not mediated by the production of nitric oxide as there was no detectable difference in nitric oxide levels in cultures that were supplemented with T cell supernatants compared with those that were not. The IFN gamma mediated anti-C. ruminantium effect highlights the importance of cell mediated immune responses in control of these infections and in particular incriminates the protective role of T cells, or cells that secrete IFN gamma.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.     

Immunoaffinity localization of the enzyme xanthine oxidase on the outside surface of the endothelial cell plasma membrane

BACKGROUND: Reactive oxygen metabolites generated from endothelial xanthine oxidase (XO) trigger reperfusion injury in many organs. We evaluated the possibility that endothelial XO was localized on the endothelial cell surface, as well as within the cytoplasm. METHODS: Primary cultures of bovine (BAECs) and porcine (PAECs) aortic endothelial cells were grown in media documented to be free of XO. Polyclonal and monoclonal antibodies were developed against XO. These antibodies were used to evaluate BAEC and PAEC for cell surface XO through immunofluorescence staining, hybridoma cell surface labeling, and endothelial cell surface binding. RESULTS: These antibodies bound specifically to the surface of these cells when the membrane was shown to be intact and impermeable (and the cytoplasm inaccessible) to immunoglobulins Moreover, hybridoma cells expressing monoclonal antibody to XO bound specifically to the endothelial cell surface. Finally, intact endothelial cells bound specifically to the anti-XO polyclonal antibodies immobilized to the surface of a Petri dish. The integrity of these endothelial cell plasma membranes was demonstrated by the subsequent growth and replication of these cells in culture. CONCLUSIONS: These findings indicate that XO is present on the outside surface of the endothelial cell plasma membrane. This would not only explain the known in vivo efficacy of intravascularly administered large molecular weight antioxidants (such as superoxide dismutase) but could have important implications for inflammatory signaling.     

Production of calves by transfer of nuclei from cultured inner cell mass cells

We report here the isolation and in vitro culture of bovine inner cell mass (ICM) cells and the use of ICM cells in nuclear transfer to produce totipotent blastocysts that resulted in calves born. Of 15 cell lines represented in this study, 13 were derived from immunosurgically isolated ICM of 3 in vitro produced day 9-10 bovine blastocysts, while 2 lines were derived from single blastocysts. Approximately 70% of attempted cell lines became established cell lines when started from 3 ICMs. The ability to establish cell lines was dependent on the number of ICMs starting the line. Sire differences were noted in the ability of ICMs to establish cell lines and to form blastocysts. The cell lines were cultured as a low cell density suspension in the medium CR1aa plus selenium, insulin, and transferrin (SIT) and 5% fetal calf serum (FCS) for 6-101 days before use in nuclear transfer, at which time some had multiplied to more than 2000 cells. If allowed to aggregate, cells of established cell lines formed embryoid bodies. A total of 659 nuclear transfer clones were made by fusing the ES cells into enucleated oocytes with polyethylene glycol; 460 of these fused, based on cleavage (70%). After culture of the clones for 7 days in vitro in CR1aa/SIT/5% FCS, 109 (24%) of those fused became blastocysts. Thirty-four blastocysts were transferred into uteri of 27 cows, and 13 cows (49%) became pregnant. Four of the 13 cows gave birth to 4 normal calves. DNA typing showed the calves to be derived from the respective sires of the cell lines. The calves were derived from cultures of less than 28 days.     

Detection of HeLa cell contamination--presence of human papillomavirus 18 DNA as HeLa marker in JTC-3, OG and OE cell lines

There are warnings of the contamination of cell cultures with HeLa cells in many laboratories in the world. The cell lines JTC-3, OG and OE that were established in Okayama in 1959, 1969 and 1971, respectively, were examined for human papillomavirus (HPV) 18 DNA by Southern blot hybridization. The HPV 18 DNA detected in these three cell lines showed hybridization patterns characteristic of the HPV 18 DNA in the HeLa cell line established in 1951. Southern hybridization patterns of HPV 18 DNA in the cellular DNA of the C4-II cervical cancer cell line that was established in the USA in 1962 was different from that of HeLa cells. These results suggest that the JTC-3, OG and OE cell lines have been contaminated by HeLa cells.     

Establishment and characterization of high- and low-lung-metastatic cell lines derived from murine colon adenocarcinoma 26 tumor line

We established and characterized high- (LuM1) and low-lung-metastatic (NM11) cell lines derived from murine colon adenocarcinoma 26 tumor line. LuM1 cell line was established as a clonal cell line from a cultured cell mixture derived from a lung-metastatic nodule after 7 sequential subcutaneous transplantations of lung-metastatic tumors in the abdominal wall of BALB/c mice. NM11 cell line was established from a cultured cell mixture derived from a subcutaneous transplant of murine colon adenocarcinoma 26 tumor cells. LuM1 cells showed marked spontaneous lung metastases, but NM11 cells rarely did. High invasive potential of LuM1 cells was revealed by in vitro invasion assay using Matrigel reconstituted membranes. Rapid retraction was observed in monolayers of human umbilical vein endothelial cells and bovine aortic endothelial cells when LuM1 cells were added on the monolayers. Gelatin zymography and immunochemical examinations with monoclonal antibodies against gelatinase B (Mr 95,000 type IV collagenase) showed secretion of large amounts of the gelatinase by LuM1 cells.     

Vascular endothelial growth factor (VEGF)-C synergizes with basic fibroblast growth factor and VEGF in the induction of angiogenesis in vitro and alters endothelial cell extracellular proteolytic activity

Vascular endothelial growth factor-C (VEGF-C) is a recently characterized member of the VEGF family of angiogenic polypeptides. We demonstrate here that VEGF-C is angiogenic in vitro when added to bovine aortic or lymphatic endothelial (BAE and BLE) cells but has little or no effect on bovine microvascular endothelial (BME) cells. As reported previously for VEGF, VEGF-C and basic fibroblast growth factor (bFGF) induced a synergistic in vitro angiogenic response in all three cells lines. Unexpectedly, VEGF and VEGF-C also synergized in the in vitro angiogenic response when assessed on BAE cells. Characterization of VEGF receptor (VEGFR) expression revealed that BME, BAE, and BLE cell lines express VEGFR-1 and -2, whereas of the three cell lines assessed, only BAE cells express VEGFR-3. We also demonstrate that VEGF-C increases plasminogen activator (PA) activity in the three bovine endothelial cell lines and that this is accompanied by a concomitant increase in PA inhibitor-1. Addition of alpha2-antiplasmin to BME cells co-treated with bFGF and VEGF-C partially inhibited collagen gel invasion. These results demonstrate, first, that by acting in concert with bFGF or VEGF, VEGF-C has a potent synergistic effect on the induction of angiogenesis in vitro and, second, that like VEGF and bFGF, VEGF-C is capable of altering endothelial cell extracellular proteolytic activity. These observations also highlight the notion of context, i.e., that the activity of an angiogenesis-regulating cytokine depends on the presence and concentration of other cytokines in the pericellular environment of the responding endothelial cell.     

Species-specific patterns of urine investigation and flehmen in Grant's gazelle (Gazella granti), Thomson's gazelle (G. thomsoni), impala (Aepyceros melampus), and eland (Taurotragus oryx).

During sexual encounters in most antelope species, males routinely investigate recently voided urine or the genitalia of females and often perform flehmen following such investigations. Quantitative data from field studies of gazelles and eland confirmed previous observations that the most common result of the female's urinating when being sexually pursued is to terminate the male's sexual pursuit, presumably by communicating the state of anestrus. Impala females do not urinate when being sexually pursued. This difference between impala and the other antelope was attributed to the ease with which impala females can escape sexual pursuit of males by dashing into the tightly clustered herd of females or losing the male in light woodland brush. Eland males direct 93% of all flehmen responses to genital contact or the urine stream of females they are pursuing, in contrast to gazelles, which devote less than 10% of flehmen responses to these stimuli. These species differences were related to differences between the multimale groups characterizing eland herds and the territorial, single-male social system characterizing gazelle. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Production of heparin-binding endothelial mitogens by bovine uterine fibroblastic and epithelial cells

This study was conducted to determine whether early-passage cultures of bovine endometrial fibroblastic (Fb, n = 7 uteri) and epithelial (Ep, n = 3 uteri) cells produce endothelial mitogens in vitro and to begin characterization of these mitogens. Confluent cultures of Fb and Ep were incubated for 72 h in serum-free media, and the resulting conditioned media (CM) were evaluated for effects on proliferation of bovine aortic endothelial cells. CM from these Fb cultures (n = 8) and Ep cultures (n = 4) stimulated (147 +/- 10% (mean +/- SEM), p < 0.01, and 124 +/- 8%, p < 0.10, respectively) proliferation of endothelial cells compared with control (unconditioned) media. Most of the mitogenic activity of a sample of Fb CM and a sample of Ep CM from one individual uterus bound to heparin-agarose, and each exhibited two major peaks of activity that eluted at 0.9-1.0 and 1.7-1.8 M NaCl; the Fb CM also exhibited an additional heparin-binding peak eluting at 0-0.1 M NaCl. Pooled Fb CM (n = 8 cultures from 7 animals) also contained mitogenic activity for endothelial cells that bound to heparin-agarose, but exhibited three major peaks, eluting at 0.6, 1.1, and 1.8 M NaCl. Pooled Ep CM (n = 4 cultures from 3 animals) showed only one peak of mitogenic activity, which eluted at 0.9 M NaCl. Further characterization indicated that heat treatment reduced the activity of all heparin-binding Fb CM and Ep CM peaks, except the Fb CM peak eluting at 1.7 M NaCl. Trypsin reduced the activity of all peaks except one. Protein-A-purified antibody against fibroblast growth factor 1 (FGF-1) had no or only a slight effect on the mitogenic activity of the peaks. Mitogenic activity of the Fb CM peak eluting at 0.6 M NaCl was reduced by antibody against FGF-2. Activity of the Fb CM and Ep CM peaks that eluted at 1.7-1.8 M NaCl also was immunoneutralized by antibody to FGF-2. These data demonstrate that early passage cultures of endometrial Fb and Ep cells produce heparin-binding endothelial mitogens that appear to be immunologically related to FGF-2. These heparin-binding endothelial mitogens may influence endometrial vascular growth.     

Synthetic analogues of TNP-470 and ovalicin reveal a common molecular basis for inhibition of angiogenesis and immunosuppression

TNP-470 (1), a synthetic derivative of the natural product fumagillin (2), potently inhibits angiogenesis in vivo and the growth of endothelial cell cultures in vitro. The structurally related natural product ovalicin (3) also inhibits angiogenesis but possesses potent immunosuppressive activity. The recent finding that all three drugs bind and inhibit the same target, methionine aminopeptidase 2 (MetAP2), raised the question of whether TNP-470 is also immunosuppressive and whether inhibition of MetAP2 underlies both activities of ovalicin. To address these questions, we synthesized a series of analogues of TNP-470 and ovalicin and tested them for their abilities to inhibit the proliferation of either endothelial cell or mixed lymphocyte cultures. TNP-470 and its analogues were found to possess both immunosuppressive and anti-angiogenic activities. A strong correlation was observed between the ability of compounds to inhibit bovine and human endothelial cell growth and their ability to inhibit the mouse mixed lymphocyte reaction (MLR), implying that the two activities share a common molecular basis, i.e., inhibition of MetAP2. Interestingly, ovalicin and several other compounds behaved differently in the human MLR than in either the mouse MLR or human endothelial cell proliferation assays, pointing to possible species-specific and cell type-specific differences in the metabolism or uptake of these compounds.     

Cell, tissue and organ culture as in vitro models to study the biology of squamous cell carcinomas of the head and neck

In vitro models are currently being used to study head and neck squamous cell carcinoma (HNSCC). Several hundred HNSCC cell lines have been established by various investigators and used to study a broad spectrum of questions related to head and neck cancer. The head and neck model with respect to multistage carcinogenesis is now complete. Several techniques exist for the culture of normal epithelial cells from the upper aerodigestive tract (UADT). The biology of these UADT cells (oral cavity, oropharynx, hypopharynx and larynx) is being studied. Successful culture of premalignant lesions (dysplastic mucosa, leukoplakia, erythroplakia) has resulted in establishment of a limited number of premalignant cell lines and cell cultures. HPV infection of normal oral epithelial cells for immortalization (approximately premalignant cells) coupled with transformation with carcinogens (malignant cells) has established an experimental model for progression. Two in vivo models for oral carcinogenesis, the 7,12 dimethylbenz(a)anthracene-induced hamster cheek pouch model and the 4-nitroquinoline-N-oxide rat oral model, have been established in culture. Thus, multistage carcinogenesis models have been established from both human tissues and animal models and include cultures of normal, premalignant and malignant cells. Culture techniques for growing dissociated primary tumor cells for short term experimental analysis are being used. The culture of normal or tumor tissue as organ/explant cultures allows for the maintenance of normal cell-cell and cell-matrix interaction, but limits experimentation since these cultures cannot be propagated. Several three dimensional model systems are being used to obtain this histological complexity but allow for experimentation. The ability to culture normal, premalignant and malignant cells coupled with the use of a variety of culture techniques, should allow for the continued growth and experimentation in head and neck cancer research.     

Thermal unfolding of small proteins with SH3 domain folding pattern

We have generated two conditionally immortalized neuronal cell lines from primary cultures of embryonic day 13 (E13) and postmitotic (postnatal day 0; P0) cortical neurons transformed with the temperature-sensitive SV-40 large-T antigen. Two clonal cell lines (CN1.4 from E13 cultures and SJ3.6 from P0 cultures) were isolated and stable maintained in vitro. Both cell lines expressed a number of neuronal markers such as the neurofilaments, glutamic acid decarboxylase 67, neuron-specific enolase, and the BG21 isoform of the myelin basic protein gene. At 34 degrees C, the CN1.4 cell line had elaborated short processes, whereas the SJ3.6 cell line produced long processes that formed a delicate network. When these cell lines were cultured at 39 degrees C, some of the cellular processes grew longer, adopting a more mature neuronal morphology. Interestingly, at 39 degrees C, the in vitro survival of these cell lines differed significantly. Whereas the survival of CN1.4 cell line was greatly unaffected, SJ3.6 cells died soon after they were cultured at 39 degrees C. The cell death of SJ3.6 cells was accompanied by fragmentation and condensation of DNA in their nuclei, indicative of an apoptotic event. Under these conditions, SJ3.6 showed an upregulation of the p75 receptor. When this cell line was cocultured with oligodendrocytes, astrocytes, or glial conditioned media (GCM), there was a marked increase in survival. In contrast, little effect of glial cells or GCM was observed on the CN1.4 cell line. These lines appear to be useful models to study neuronal-glial interactions in addition to neuronal cell death and the effects of glial factors that promote the survival of neurons.     

Differential effects of SPARC and cationic SPARC peptides on DNA synthesis by endothelial cells and fibroblasts

SPARC (secreted protein, acidic and rich in cysteine), also known as osteonectin, is an extracellular Ca+2-binding glycoprotein that inhibits the incorporation of [3H]-thymidine and delays the onset of S-phase in synchronized cultures of bovine aortic endothelial (BAE) cells. This effect appears not to be dependent on the functional properties of SPARC associated with changes in cell shape or inhibition of cell spreading. In this study we investigate the conditions under which cell cycle modulation occurs in different types of cells. Human umbilical vein endothelial cells, a transformed fetal BAE cell line, and bovine capillary endothelial cells exhibited a sensitivity to SPARC and a cationic peptide from a non-Ca+2-binding region of SPARC (peptide 2.1, 0.2-0.8 mM) similar to that observed in BAE cells. In contrast, human foreskin fibroblasts and fetal bovine ligament fibroblasts exhibited an increase in the incorporation of [3H]-thymidine in the presence of 25 microM-0.2 mM peptide 2.1; inhibition was observed at concentrations in excess of 0.4 mM. This biphasic modulation could be further localized to a sequence of 10 amino acids comprising the N-terminal half of peptide 2.1. A synthetic peptide from another cationic region of SPARC (peptide 2.3) increased [3H]-thymidine incorporation by BAE cells and fibroblasts in a dose-dependent manner. In endothelial cells, a stimulation of 50% was observed at a concentration of 0.01 mM; fibroblasts required approximately 100-fold more peptide 2.3 for levels of stimulation comparable to those obtained in endothelial cells. The observation that SPARC and unique SPARC peptides can differentially influence the growth of fibroblasts and endothelial cells in a concentration-dependent manner suggests that SPARC might regulate proliferation of specific cells during wound repair and remodeling.     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

Human bone marrow-derived mitogenic stimulation selective for breast carcinoma and neuroblastoma cells

In patients with neuroblastoma (NB) or breast carcinoma (BC), metastatic disease in the bone marrow (BM) is observed more frequently than at any other site, and a high incidence of BM metastases in these patients is associated with advanced disease and poor prognosis. These observations suggest the presence of BM micro-environmental elements that are favorable for NB and BC tumor cell growth. The influence of normal human BM cell-derived conditioned medium (CM) on clonogenic growth of BC and NB cell lines was investigated in vitro. The effects obtained were compared with those on tumor cells with a lower potential for BM metastasis. CM from unstimulated cultures of normal, healthy, low-density BM cells reproducibly and markedly augmented clonogenic growth of 3 BC and 3 NB cell lines. In contrast, growth of cell lines established from human tumors with differing metastatic propensity was unaffected by BM CM. Initial characterization, using crude BM CM, indicated that mitogenic activity (i) is mediated by peptides released by the non-adherent fraction of low-density BM cells and (ii) is not abolished by neutralizing antibodies against various cytokines known to be produced by BM cells and to regulate hematopoietic cell growth. Our observations suggest that certain specific peptides in the BM micro-environment may be responsible for the preferential growth of NB and BC metastases in BM.     

Na+ channel modulation and force-frequency relationship in human myocardium

Insect cell lines in culture are used for a variety of studies. In this laboratory imaginal disc cell lines have been established from primary cultures from third instar larvae, and used for a number of experiments. The effect of ageing on the morphology and physiology of Drosophila cell lines has received very little attention, although problems of genotypic or phenotypic changes in cell lines with age are recognized in other areas of animal cell culture. We tested our cell line Cl8+ for any difference in growth, morphology and response to 20-hydroxyecdysone (20HE) at different ages (passage numbers). The cells were found to multiply faster, adhere less firmly to the substrate and to lose the tendency to aggregate at higher passages. The response to 20HE in terms of cell numbers and induction of beta-galactosidase was similar at all passage numbers but morphological changes in hormone-treated cells were less obvious in the higher passages. Cell lines are likely to vary in the extent of ageing effects but workers are advised to be aware of the possibilities. We suggest the effects of age on cell lines should be established, and passage numbers noted in experimental reports.