To see the other types of publications on this topic, follow the link: In toto immunofluorescence.
Journal articles on the topic 'In toto immunofluorescence'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 33 journal articles for your research on the topic 'In toto immunofluorescence.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
1
Pardanaud, L., C. Altmann, P. Kitos, F. Dieterlen-Lievre, and C. A. Buck. "Vasculogenesis in the early quail blastodisc as studied with a monoclonal antibody recognizing endothelial cells." Development 100, no. 2 (June 1, 1987): 339–49. http://dx.doi.org/10.1242/dev.100.2.339.
Full textAbstract:
QH1, a monoclonal antibody that recognizes quail endothelial and haemopoietic cells, was applied to quail blastodiscs in toto, in order to analyse by immunofluorescence the emergence of the vascular tree. The first endothelial cells were detected in the area opaca at the headfold stage and in the area pellucida at the 1-somite stage. Single cells then interconnected progressively, especially in the anterior intestinal portal and along the somites building up the linings of the heart and dorsal aortas. This study demonstrates that endothelial cells differentiate as single entities 4 h earlier in development than hitherto detected and that the vascular network forms secondarily. The horseshoe shape of the extraembryonic area vasculosa is also a secondary acquisition. A nonvascularized area persists until later (at least the 14-somite stage) in the region of the regressing primitive streak.
2
Ribet, David, Sophie Louvet-Vallée, Francis Harper, Nathalie de Parseval, Marie Dewannieux, Odile Heidmann, Gérard Pierron, Bernard Maro, and Thierry Heidmann. "Murine Endogenous Retrovirus MuERV-L Is the Progenitor of the “Orphan” Epsilon Viruslike Particles of the Early Mouse Embryo." Journal of Virology 82, no. 3 (November 28, 2007): 1622–25. http://dx.doi.org/10.1128/jvi.02097-07.
Full textAbstract:
ABSTRACT Viruslike particles which displayed a peculiar wheellike appearance that distinguished them from A-, B- or C-type particles had previously been described in the early mouse embryo. The maximum expression of these so-called epsilon particles was observed in two-cell-stage embryos, followed by their rapid decline at later stages of development and no particles detected at the zygote one-cell stage. Here, we show that these particles are in fact produced by a newly discovered murine endogenous retrovirus (ERV) belonging to the widespread family of mammalian ERV-L elements and named MuERV-L. Using antibodies that we raised against the Gag protein of these elements, Western blot analysis and in toto immunofluorescence studies of the embryos at various stages disclosed the same developmental expression profile as that observed for epsilon particles. Using expression vectors for cloned, full-length, entirely coding MuERV-L copies and cell transfection, direct identification of the epsilon particles was finally achieved by high-resolution electron microscopy.
3
Weber, Michael H. W., Arsen V. Volkov, Ingo Fricke, Mohamed A. Marahiel, and Peter L. Graumann. "Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in Bacillus subtilis Depends on Active Transcription." Journal of Bacteriology 183, no. 21 (November 1, 2001): 6435–43. http://dx.doi.org/10.1128/jb.183.21.6435-6443.2001.
Full textAbstract:
ABSTRACT Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal incspB cspC and cspB cspDdouble-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.
4
Turner, Joel G., Thomas C. Rowe, David A. Ostrov, Elizabeth J. Ciaravino, Jana L. Gump, and Daniel M. Sullivan. "Blocking a Topoisomerase IIα Nuclear Export Signal Sensitizes Human Multiple Myeloma Cells to Topoisomerase II Inhibitors." Blood 114, no. 22 (November 20, 2009): 2850. http://dx.doi.org/10.1182/blood.v114.22.2850.2850.
Full textAbstract:
Abstract Abstract 2850 Poster Board II-826 Background We have previously demonstrated that topoisomerase IIa (topo IIa) is exported from the nucleus of high density human multiple myeloma (MM) cells by a CRM1-dependent mechanism (Engel et al, Exp. Cell Res. 295(2):421-31, 2004). We have also identified the nuclear export signals (NES) for topo IIa at amino acids 1017-28 (site 1) and 1054-66 (site 2) using mutated full-length FLAG-tagged topo IIa protein and immunofluorescence microscopy (Turner et al, J. Cell Sci. 117:3061-71, 2004). Drug resistance to topo II poisons occurs when topo II is trafficked to the cytoplasm where it is not in contact with the DNA and thus unable to induce cell death in response to topo II inhibitors. In addition, we have recently shown that blocking nuclear export of topo IIα with a CRM1 inhibitor or by siRNA sensitizes MM cells to topo II poisons (Turner et al, Cancer Res. In press). Materials and Methods The structure of S. cerevisiae topo II was used to create a model of human topo IIα using the program Protein Homology/analogy Recognition Engine (Phyre), available on the Web (http://www.imperial.ac.uk/phyre). The procedure for molecular docking involved selection of structural pockets of the NES in topo IIa that were suitable for interactions with drug-like small molecule inhibitors (SMI). Molecular docking simulations screened 140,000 small molecules (mw < 500) from the NCI database. The top scoring SMI for each of the two NES (20 total) were obtained from NCI and tested for induction of apoptosis (caspase 3) and anti-proliferative activity using CellTiter-Blue (Promega). Cell types used included human MM RPMI 8226 and NCI-H929 cells. SMI were tested both as single agents and in combination experiments with the topo II inhibitor doxorubicin. In addition, immunofluorescence microscopy for the intracellular location of topo IIα in SMI-treated MM cells was also performed. Results Low density myeloma cells: Robotic cell viability assays determined that several of the SMI had anti-proliferative activity. However, only SMI that docked to NES site 1 showed any inhibition of viability. The IC50 values obtained from single-drug cell viability assays in low density cells revealed two SMI compounds with IC50 values of 4.7 and 11.1 μM. None of the SMI affected the viability of high density cells (IC50 >100 μM). High density drug-resistant myeloma cells: Data from apoptosis assays indicated that four of the SMI that docked to NES site 1 significantly (p<0.05) sensitized high density MM cells to doxorubicin. Immunofluorescence microscopy revealed an increase in topo IIα in the cell nucleus of cells treated for 20 hours with the four lead SMI. Conclusions Using computer-generated molecular modeling, we have identified four lead compounds from the NCI database that bind to the site 1 NES of topo IIα. These lead compounds are SMI that synergize with the topo II inhibitor doxorubicin. In vitro apoptosis assays indicate that these drugs may be effective as single agents or in combination with currently used cancer drugs that target topo II. These data may have potential clinical implications in the treatment of multiple myeloma. Disclosures: Sullivan: Merck: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Merrion: Membership on an entity's Board of Directors or advisory committees.
5
Moulder, S. L., N. Valkov, S. Minton, P. Munster, J. Gump, M. Lacevic, C. Rocha-Lima, J. Horton, R. Lush, and D. Sullivan. "A single arm phase II trial of gemcitabine (G) and irinotecan (I) in metastatic breast cancer: Can localization of topoisomerase I (topo I) predict response to topo I inhibitors?" Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 661. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.661.
Full textAbstract:
661 Background: The incorporation of G into DNA enhances cleavage complexes in vitro when combined with a topo I inhibitor. Topo I poisons require enzyme interaction with DNA to exert activity. Methods: Two stage accrual design, primary endpoint: response (RR) using RECIST criteria. Inclusion criteria: male and female patients (pts) with MBC, prior anthracycline therapy, measurable disease, ECOG PS of ≤ 2, adequate organ function, and ≤ 3 prior chemotherapy regimens for MBC. 51 eligible pts received therapy with G at 1000mg/m2 and I at 100mg/m2 on days 1 and 8 of a 21-day cycle. Optional tumor biopsies were obtained in 9 pts (18%) prior to therapy to determine localization of topo I using immunofluorescence. PK: Irinotecan: A validated limited sampling strategy was used. Gemcitabine: Serial blood samples were collected over 24 hrs following the first dose. Intracellular nucleotides were quantitated in PBMCs. Results: 45 pts have been evaluated with a RR of 27% (CR=0, PR=12; 95% CI 13–37%). 4 pts had SD for ≥6 months for a clinical benefit rate (PR+SD) of 36%. 3 pts received < 1 cycle of therapy before protocol withdrawal and were not evaluable for RR. RR for the final 3 patients will be available at the time of presentation. 7/9 tissue biopsies were assessable for topo I with results listed below. PK and toxicity data will be available at presentation.Conclusion: GI is active in MBC. Topo I localization can be measured in MBC. In this limited data set, the two lowest nuclear to cytoplasmic (N/C) ratios were associated with lack of response to irinotecan. Further validation is needed. [Table: see text] [Table: see text]
6
Whalen, A. M., M. McConnell, and P. A. Fisher. "Developmental regulation of Drosophila DNA topoisomerase II." Journal of Cell Biology 112, no. 2 (January 15, 1991): 203–13. http://dx.doi.org/10.1083/jcb.112.2.203.
Full textAbstract:
Affinity-purified polyclonal antibodies were used to quantitate steady-state levels of DNA topoisomerase II (topo II) throughout Drosophila development. Although wide fluctuations were recorded at different stages, these fluctuations were paralleled by changes in levels of the nuclear lamin, a nuclear structural protein used as an internal standard. The exception to this was adult males where lamin levels were significantly elevated relative to topo II. Northern blot analyses of topo II and lamin mRNA, performed in conjunction with immunoblot analyses of protein revealed fluctuations in levels of the two different messages that paralleled changes in each other and in their respective translation products. Biochemical and immunochemical analyses were complemented by indirect immunofluorescence and immunoperoxidase experiments performed in situ. topo II was found distributed throughout nuclei in most but not all cell types examined. These results for Drosophila topo II are apparently at odds with those obtained by others working in vertebrate systems (see, for example, Heck, M. M. S. and W. C. Earnshaw. 1986. J. Cell Biol. 103:2569-2581; Heck, M. M. S., W. N. Hittelman, and W. C. Earnshaw. 1988. Proc. Natl. Acad. Sci. USA. 85:1086-1090) and suggest that in Drosophila, topo II may not be a useful marker for the proliferative state.
7
Hirano, T., and T. J. Mitchison. "Topoisomerase II does not play a scaffolding role in the organization of mitotic chromosomes assembled in Xenopus egg extracts." Journal of Cell Biology 120, no. 3 (February 1, 1993): 601–12. http://dx.doi.org/10.1083/jcb.120.3.601.
Full textAbstract:
We have investigated the role of topoisomerase II (topo II) in mitotic chromosome assembly and organization in vitro using Xenopus egg extracts. When sperm chromatin was incubated with mitotic extracts, the highly compact chromatin rapidly swelled and concomitantly underwent local condensation. Further incubation induced the formation of entangled thin chromatin fibers that eventually resolved into highly condensed individual chromosomes. This in vitro system made it possible to manipulate mitotic chromosomes in their assembly condition without any isolation or stabilization steps. Two complementary approaches, immunodepletion and antibody blocking, demonstrated that topo II activity is required for chromosome assembly and condensation. Once condensation was completed, however, blocking of topo II activity had little effect on the chromosome morphology. Immunofluorescent studies showed that topo II was uniformly distributed throughout the condensed chromosomes and was not restricted to the chromosomal axis. Surprisingly, all detectable topo II molecules were easily extracted from the chromosomes under mild conditions where the shape of chromosomes was well preserved. Our results show that topo II is essential for mitotic chromosome assembly, but does not play a scaffolding role in the structural maintenance of chromosomes assembled in vitro. We also present evidence that changes of DNA topology affect the distribution of topo II in mitotic chromosomes in our system.
8
Morse-Gaudio, M., and M. S. Risley. "Topoisomerase II expression and VM-26 induction of DNA breaks during spermatogenesis in Xenopus laevis." Journal of Cell Science 107, no. 10 (October 1, 1994): 2887–98. http://dx.doi.org/10.1242/jcs.107.10.2887.
Full textAbstract:
The relative content of topoisomerase II (topo II) and the induction of topo-II-mediated DNA damage and cellular abnormalities have been characterized in developing spermatogenic cells of Xenopus laevis to gain an insight into the role of topo II during spermatogenesis. Decatenation assays identified topo II activity in nuclear extracts from spermatocytes and pre-elongate spermatids, but not in extracts from elongate spermatids or sperm. Extracts from early-mid spermatids contained 14% (per cell) of the decatenation activity found in spermatocyte extracts. Immunoblots of SDS extracts from whole cells and nuclei from both spermatocytes and pre-elongate spermatids, but not elongate spermatids or sperm, resolved a 180 kDa polypeptide that reacts with polyclonal antisera to Xenopus oocyte topo II, an antipeptide antibody (FHD29) to human topo II alpha and beta, and an antipeptide antibody to human topo II alpha, suggesting homology between Xenopus spermatogenic cell topo II and mammalian topo II alpha. Immunofluorescence microscopy of topo II in testis cryosections revealed the presence of topo II in nuclei of all spermatogenic stages, but not in sperm. The relative levels of topo II estimated from fluorescence intensity were highest in spermatogonia and spermatocytes, then early-mid spermatids, followed by elongate spermatids and somatic cells. Incubation of isolated spermatogenic cells with teniposide (VM-26), a topo II-targetted drug, resulted in a dose-dependent induction of DNA breaks in all spermatocytes and spermatid stages to nuclear elongation stages, as analyzed by alkaline single cell gel electrophoresis. Addition of 0.5-50 microM VM-26 to spermatogenic cell cultures for 27 hours resulted in stage-dependent abnormalities. Mid-late spermatid stages were relatively resistant to VM-26-induced damage. In contrast, meiotic division stages were arrested and spermatogonia B were killed by VM-26, and VM-26 induced abnormal chromosome condensation in pachytene spermatocytes. The results of these studies show that cellular levels of topo II are stage-dependent during spermatogenesis, that most spermatogenic stages are sensitive to topo II-mediated DNA damage, and that spermatogonia B, meiotic divisions and pachytene spermatocytes are particularly sensitive to induction of morphological abnormalities and cell death during acute exposure to topo II-targetted drugs.
9
Andrade, Luis E. C., Werner Klotz, Manfred Herold, Karsten Conrad, Johan Rönnelid, Marvin J. Fritzler, Carlos A. von Mühlen, et al. "International consensus on antinuclear antibody patterns: definition of the AC-29 pattern associated with antibodies to DNA topoisomerase I." Clinical Chemistry and Laboratory Medicine (CCLM) 56, no. 10 (September 25, 2018): 1783–88. http://dx.doi.org/10.1515/cclm-2018-0188.
Full textAbstract:
Abstract The indirect immunofluorescence assay (IFA) on HEp-2 cells is the reference method for autoantibody screening. The HEp-2 IFA pattern provides useful information on the possible autoantibodies in the sample. The International Consensus on Antinuclear Antibody Patterns (ICAP) initiative seeks to define and harmonize the nomenclature of HEp-2 IFA patterns. The most relevant and usual patterns have been assigned an alphanumeric code from anti-cell (AC)-1 to AC-28 and were organized into a classification algorithm (www.ANApatterns.org). The systemic sclerosis-associated autoantibodies to DNA topoisomerase I (Topo I) produce a peculiar composite 5-element HEp-2 IFA pattern (Topo I-like pattern) comprising the staining of the nucleus, metaphase chromatin plate, nucleolar organizing region, cytoplasm and nucleolus. In a recent assessment of the European Consensus Finding Study Group on autoantibodies, a well-defined anti-Topo I sample was blindly analyzed and classified according to ICAP AC patterns by 43 participant laboratories across Europe. There were wide variations among these laboratories in reporting nuclear, nucleolar and cytoplasmic patterns, indicating the inadequacy of the existing AC patterns to report the Topo I-like pattern. Several ICAP member laboratories independently demonstrated the overall consistency of the HEp-2 IFA Topo I-like pattern using HEp-2 slides from different manufacturers. The ICAP committee reviewed 24 candidate images and selected the four most representative images to be available on the ICAP website. The proper recognition of the AC-29 pattern should trigger suspicion of the presence of anti-Topo I antibodies, which may engender appropriate analyte-specific reflex tests to confirm the autoantibody specificity.
10
CODULLO, VERONICA, ILARIA CAVAZZANA, CLAUDIA BONINO, CLAUDIA ALPINI, LORENZO CAVAGNA, FRANCO COZZI, NICOLETTA DEL PAPA, et al. "Serologic Profile and Mortality Rates of Scleroderma Renal Crisis in Italy." Journal of Rheumatology 36, no. 7 (June 1, 2009): 1464–69. http://dx.doi.org/10.3899/jrheum.080806.
Full textAbstract:
Objective.To analyze clinical and serological characteristics of subjects with scleroderma renal crisis (SRC) in Italian patients with systemic sclerosis (SSc).Methods.A retrospective analysis of medical records from 9 Italian rheumatologic referral centers was carried out. All patients with SRC and an available serum sample at the time of crisis were included. Antinuclear antibodies (ANA) by indirect immunofluorescence, anti-topoisomerase (topo) I by enzyme-linked assay (ELISA), anti-RNA polymerases (RNAP) by ELISA for the subunit III, and immunoprecipitation (IP) were performed.Results.Forty-six cases (38 female; 40 diffuse cutaneous SSc) were identified. Mean age at SSc and SRC onset was 52.8 years ± 13.2 and 55.4 years ± 11.8, respectively. ANA were present in 44 patients (96%). Anti-topo I antibodies were detected in 30 (65%), anti-RNAP I–III in 7 (15%). No differences emerged between these 2 groups for their main clinical characteristics. The proportion of patients in the anti-RNAP I–III group developing SRC early (< 18 mo) in the course of SSc was significantly higher (p = 0.03). Cumulative survival rates were 64%, 53%, and 35% at 1, 2, and 10 years of followup, respectively. Survival rates of SSc patients significantly differed according to their autoantibody profile, being lower in the anti-topo I than in the anti-RNAP I–III group (p = 0.034).Conclusion.SRC is a rare manifestation of SSc in Italy but it is still associated with severe prognosis. Anti-topo I reactivity was more frequent than anti-RNAP I–III in our patients with SRC and was associated with delayed onset and high mortality rates.
11
Turner, Joel G., Jana L. Dawson, and Daniel M. Sullivan. "Casein Kinase II Modulates Topoisomerase II Alpha Nuclear Export and Drug Sensitivity in Multiple Myeloma." Blood 120, no. 21 (November 16, 2012): 4032. http://dx.doi.org/10.1182/blood.v120.21.4032.4032.
Full textAbstract:
Abstract Abstract 4032 Nuclear export of topoisomerase II alpha (topo IIα) to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. We have previously demonstrated that topo IIα is exported from the nucleus of human multiple myeloma cells by a CRM1-dependent mechanism at densities similar to those in myeloma patient bone marrow (Engel et al, Exp. Cell Res. 295:421-31, 2004). We have also identified the nuclear export signals for topo IIα at amino acids 1017–1028 and 1054–1066 using mutated full-length FLAG-topo IIα protein and immunofluorescence microscopy, (Turner et al, J. Cell Sci. 117:3061-71, 2004). In addition, blocking nuclear export with a CRM1 inhibitor or by using CRM1-siRNA sensitizes myeloma cells to topo II poisons (Turner et al. Cancer Res. 69:6899-905, 2009). Therefore, preventing nuclear export may sensitize myeloma cells to topo II inhibitors. To determine what signals CRM1-mediated nuclear export of topo IIα we investigated the phosphorylation status of topo IIα, both in the nucleus and in the cytoplasm, using proteomic technologies. Topo IIα was isolated from the nuclei and cytoplasm of human myeloma cells by immunoprecipitation. Purified topo IIα fractions were analyzed by Orbitrap mass spectroscopy. We found that serine 1524 was highly phosphorylated in the cytoplasmic fraction. Using site-directed mutagenesis we converted serine 1524 to an alanine to determine if mutated FLAG-tagged topo IIα export was reduced as compared to wild-type FLAG-tagged topo IIα. Serine 1524 is a CK2 phosphorylation site, therefore, we tested a recently published and highly specific inhibitor of CK2; CX-4945 (Ferguson et al, FEBS Letters 585:104–110, 2011). This drug was used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry both in vitro using myeloma cell lines and ex vivo using patient bone marrow mononuclear cells. Using mass spectroscopy we found that cytoplasmic but not nuclear topo IIα was phosphorylated at serine 1524, a published CK2 motif. Using site-directed mutagenesis we converted serine 1524 to an alanine and found that mutated FLAG-topo IIα export was reduced, as compared to wild-type FLAG- topo IIα. The CK2 inhibitor CX-4945 induced apoptosis in both myeloma cell lines and in myeloma patient bone marrow aspirates. In addition, we found that CX-4945 prevented nuclear export of topo IIα in high-density cells. These data were duplicated using a CK2 specific siRNA to knockdown CK2 expression (90% knockdown). Blocking nuclear export of topo IIα with the CK2 inhibitor CX-4945 or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poison doxorubicin. These data may have potential clinical implications in the treatment of multiple myeloma. Disclosures: No relevant conflicts of interest to declare.
12
Gallego-Paez, Lina Marcela, Hiroshi Tanaka, Masashige Bando, Motoko Takahashi, Naohito Nozaki, Ryuichiro Nakato, Katsuhiko Shirahige, and Toru Hirota. "Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells." Molecular Biology of the Cell 25, no. 2 (January 15, 2014): 302–17. http://dx.doi.org/10.1091/mbc.e13-01-0020.
Full textAbstract:
The structural maintenance of chromosomes (SMC) proteins constitute the core of critical complexes involved in structural organization of chromosomes. In yeast, the Smc5/6 complex is known to mediate repair of DNA breaks and replication of repetitive genomic regions, including ribosomal DNA loci and telomeres. In mammalian cells, which have diverse genome structure and scale from yeast, the Smc5/6 complex has also been implicated in DNA damage response, but its further function in unchallenged conditions remains elusive. In this study, we addressed the behavior and function of Smc5/6 during the cell cycle. Chromatin fractionation, immunofluorescence, and live-cell imaging analyses indicated that Smc5/6 associates with chromatin during interphase but largely dissociates from chromosomes when they condense in mitosis. Depletion of Smc5 and Smc6 resulted in aberrant mitotic chromosome phenotypes that were accompanied by the abnormal distribution of topoisomerase IIα (topo IIα) and condensins and by chromosome segregation errors. Importantly, interphase chromatin structure indicated by the premature chromosome condensation assay suggested that Smc5/6 is required for the on-time progression of DNA replication and subsequent binding of topo IIα on replicated chromatids. These results indicate an essential role of the Smc5/6 complex in processing DNA replication, which becomes indispensable for proper sister chromatid assembly in mitosis.
13
Turner, Joel G., Thomas C. Rowe, David Ostrov, Jana L. Dawson, Danielle Pernazza, Nicholas J. Lawrence, and Daniel M. Sullivan. "Targeting The Nuclear Export Signal In Multiple Myeloma." Blood 122, no. 21 (November 15, 2013): 1925. http://dx.doi.org/10.1182/blood.v122.21.1925.1925.
Full textAbstract:
Abstract Introduction In multiple myeloma (MM), de novo drug resistance to topoisomerase (topo) II poisons occurs at high cell densities due to trafficking of topo IIα from the nucleus to the cytoplasm where it is no longer in contact with the DNA and thus unable to induce cell death (Turner et al. 2009, Cancer Res, 69, 6899-905, Engel et al. 2004, Exp Cell Res, 295, 421-31). We have previously demonstrated that topo IIα is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism ( Valkov et al 2000, Br J Haematol, 108, 331-45, Engel et al. 2004, Exp Cell Res, 295, 421-31) and we have also identified the nuclear export signals (NES) for topo IIα at amino acids 1017-28 (site A) and 1054-66 (site B) ( Turner et al. 2004, J Cell Sci, 117, 3061-71). Blocking nuclear export with a CRM1 inhibitor or by siRNA has been shown to sensitize drug-resistant MM cells to topo II poisons ((Turner et al. 2009, Cancer Res, 69, 6899-905, Turner et al 2013, J. of Cancer, In press). Even though the active NES site (site A) of topo IIα conforms to the hydrophobic amino acid motif for an NES, the amino acid sequence does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo IIα protein. This specificity of the NES could lead to the development of drugs that may exclusively block the NES of topo IIα and not affect the CRM 1-dependent export of other nuclear proteins. Methods In this study, our aims were to; 1) generate an atomic homology model of human topo IIα to identify lead compounds targeting the NES of topo IIα (nuclear export signal inhibitors, NESi), 2) determine if the compounds bind specifically to topo IIα and inhibit nuclear export by immunofluorescence microscopy, nuclear-cytoplasmic fractionation, immunoprecipitation, proximity ligation assay, and Biacore kinetic/affinity assay, and 3) test the anticancer activity of these compounds in multiple myeloma and normal cells with and without a topo IIα inhibitor. Results and Conclusions We generated an atomic homology model of human topo IIα to provide the basis for in silico molecular docking screenings of 139,735 small molecules (MW < 500) to identify lead compounds or NESi targeting the NES of topo IIα. The highest docking score NESi compounds (46 total) were assayed for their ability to induce apoptosis in drug-resistant human H929 myeloma cells when used in combination with the topo II inhibitor doxorubicin. We isolated four lead compounds that induced apoptosis (caspase 3 cleavage) when used with doxorubicin and that inhibited nuclear export of topo IIα, as shown by immuno-fluorescence microscopy and nuclear-cytoplasmic fractionation. Immunoprecipitation, proximity ligation assay and Biacore kinetic/affinity assay showed that the most potent lead compound, NCI-9138, inhibited binding of topo IIα to the export receptor CRM1. Inhibition was specific to topo IIα because p53 trafficking was unaffected and the inhibitor did not affect topo IIα protein expression or topo IIα function (decatenation). The NESi were found to sensitize drug-resistant human myeloma cells to doxorubicin but did not affect normal fibroblast cell lines (Flow2000 or WI-38) or human PBMCs. These topo IIα-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance in multiple myeloma. Disclosures: No relevant conflicts of interest to declare.
14
CERIBELLI, ANGELA, ILARIA CAVAZZANA, FRANCO FRANCESCHINI, PAOLO AIRÒ, ANGELA TINCANI, ROBERTO CATTANEO, BRAD A. PAULEY, EDWARD K. L. CHAN, and MINORU SATOH. "Anti-Th/To Are Common Antinucleolar Autoantibodies in Italian Patients with Scleroderma." Journal of Rheumatology 37, no. 10 (August 3, 2010): 2071–75. http://dx.doi.org/10.3899/jrheum.100316.
Full textAbstract:
Objective.Patients with scleroderma (systemic sclerosis; SSc) can be classified into subsets based on autoantibody profile and clinical features. Specificities such as anti-Th/To and anti-fibrillarin (U3RNP) are detectable mainly by immunoprecipitation (IP), which is not widely used in clinical laboratories. We examined the autoantibody profiles and clinical manifestations in a cohort of Italian patients with SSc, focusing on anti-Th/To and anticentromere (ACA) antibodies, associated with limited cutaneous SSc (lcSSc).Methods.Sera from 216 consecutive patients with SSc were tested for ACA (by indirect immunofluorescence), antitopoisomerase I (topo I; by counterimmunoelectrophoresis), and anti-RNA polymerase III (RNAPIII; by ELISA). Forty-one sera negative for these specificities were tested by IP analysis of proteins (35S-methionine labeled K562 cell extract) and RNA (silver staining).Results.Among 216 SSc patients analyzed, anti-topo I, ACA, and anti-RNAPIII were detected in 38% (81/216), 31% (67/216) and 7% (15/216), respectively. Among 41 sera negative for ACA, anti-topo I, and anti-RNAPIII and which were tested by IP, 14 were nucleolar stain-positive. Eight out of 14 (57%) showed anti-Th/To reactivity, but no anti-U3RNP was found. In comparison with ACA-positive patients, anti-Th/To-positive patients were younger (p = 0.0046) and more commonly were male (p = 0.0006). All 8 anti-Th/To-positive and all but one ACA-positive patients had lcSSc. Interstitial lung disease (ILD) and pericarditis were more frequent in anti-Th/To-positive patients.Conclusion.Anti-Th/To are common in antinucleolar antibody-positive Italian patients with SSc. Anti-Th/To and ACA patients had lcSSc, with excellent prognosis. The anti-Th/To group had frequent pericarditis and ILD, although impairment of pulmonary function appeared mild.
15
Hirano, T., and T. J. Mitchison. "Cell cycle control of higher-order chromatin assembly around naked DNA in vitro." Journal of Cell Biology 115, no. 6 (December 15, 1991): 1479–89. http://dx.doi.org/10.1083/jcb.115.6.1479.
Full textAbstract:
We have developed an in vitro system in which higher-order chromatin structures are assembled around naked DNAs in a cell cycle-dependent manner. Membrane-free soluble extracts specific to interphase and mitotic states were prepared from Xenopus eggs. When high molecular weight DNA is incubated with interphase extracts, fluffy chromatin-like structures are assembled. In contrast, mitotic extracts produce highly condensed chromosome-like structures. Immunofluorescence studies show that a monoclonal antibody MPM-2, which recognizes a class of mitosis-specific phosphoproteins, stains the "core" or "axis" of condensed mitotic chromatin but not interphase chromatin. By adding mitotic extracts, interphase chromatin structures are synchronously converted into the condensed state. The increasingly condensed state of chromatin correlates with the appearance and structural rearrangements of the MPM-2-stained structures. These results suggest that mitosis-specific phosphoproteins recognized by MPM-2 may be directly involved in the assembly of the chromosome scaffold-like structures and chromatin condensation. Although both extracts promote nucleosome assembly at the same rate, topoisomerase II (topo II) activity is four to five times higher in mitotic extracts compared with interphase extracts. The addition of a topo II inhibitor VM-26 into mitotic assembly mixtures disturbs the organization of the MPM-2-stained structures and affects the final stage of chromatin condensation. This in vitro system should be useful for identifying cis- and trans-acting elements responsible for higher-order chromatin assembly and its structural changes in the cell cycle.
16
Turner, Joel G., Thomas C. Rowe, David Ostrov, Jana L. Dawson, Elizabeth Ciaravino, and Daniel Sullivan. "Novel Drug Targets to Overcome De Novo Drug-Resistance In Multiple Myeloma." Blood 116, no. 21 (November 19, 2010): 3006. http://dx.doi.org/10.1182/blood.v116.21.3006.3006.
Full textAbstract:
Abstract Abstract 3006 Abstract Human multiple myeloma (MM) still remains an incurable disease despite improved treatment regimens that include bortezomib, lenalidomide and thalidomide. New therapeutic targets are needed to further improve treatment outcomes. We have shown that targeting intracellular trafficking of proteins may sensitize cells to antitumor agents (Turner et al. 2009, Cancer Res, 69, 6899-905). We have previously demonstrated that topoisomerase II alpha (topo IIα) trafficking from the nucleus to the cytoplasm in myeloma cells occurs by a CRM1-dependent mechanism and resulting in drug resistance to topo II inhibitors (Engel et al. 2004, Exp Cell Res, 295, 421-31). We have also identified the nuclear export signals (NES) for topo IIα at amino acids 1017-28 (site 1) and 1054-66 (site 2) (Turner et al. 2004, J Cell Sci, 117, 3061-71). Blocking nuclear export of topo IIα with a CRM1 inhibitor or by siRNA has been shown to sensitize MM cells to topo II poisons (Turner et al. 2009, Cancer Res, 69, 6899-905). The NES amino acid sequence of topo IIα at 1017–1028 is a unique site. Even though this site conforms to the hydrophobic amino acid motif for an NES, the amino acid sequence does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo IIα protein. These factors allow the potential for the development of drugs that will exclusively block the NES of topo IIα and not affect the export of other nuclear proteins, as occurs with other known CRM1 inhibitors. Drug resistance to topo II inhibitors occurs when topo IIα is trafficked from the nucleus to the cytoplasm where it is no longer in contact with the DNA, and thus unable to induce cell death (Valkov et al 2000, Br J Haematol, 108, 331-45, Engel et al. 2004, Exp Cell Res, 295, 421-31). We therefore hypothesize that targeting a specific NES in topo IIα is an innovative treatment approach in MM and may allow a very focused and potent combination with topo II inhibitors, possibly overcoming de novo drug resistance in this malignancy. To date, we know of no agents that target the NES of a specific protein that are being developed to treat cancer. A computer generated hybrid molecule using the known three dimensional structure of yeast topo II and the human NES sequences of topo IIα was produced. Molecules were docked in silico using the NCI small molecule database (140,000 compounds). The molecules with the highest docking scores were obtained from NCI and assayed for IC50 values and synergy with the topo II inhibitor doxorubicin. All NES site 1 molecules tested showed activity, however, none of the NES site 2 molecules exhibited any anti-neoplastic activity with or without a topo II inhibitor. CT blue (Promega) robotic cell viability assays determined that several of the site 1 inhibitors had anti-proliferative activity. The IC50 values obtained from single drug cell viability assays in low density cells revealed two site 1 inhibitors compounds with IC50 values of 4.7 (NCI-36400) and 11.1 μM (NCI-35847). None of the site 1 inhibitors affected the viability of high-density cells (IC50>100 μM). Data from apoptosis assays indicate that three of the site 1 inhibitors (NCI-36400, NCI-35847, NCI-35024) that dock to NES site 1 do significantly (p<0.05) sensitize high density MM cells to doxorubicin. Immunofluorescence microscopy revealed an increase in topo IIα in the cell nucleus of cells treated for 20 hours with the three lead site 1 inhibitors. Nuclear-cytoplasmic fractionation revealed that the NES site 1 docking molecules prevent nuclear export of topo IIα. These compounds may lead to new chemotherapeutic treatments of myeloma. Disclosures: No relevant conflicts of interest to declare.
17
Chung, Hye Won, Roberto A. Salas Fragomeni, Sharon Shacham, Michael Kauffman, and James C. Cusack. "Use of selective inhibition of nuclear export (SINE) using a CRM1/XPO1 antagonist to overcome resistance to CPT-11 in colon cancer in preclinical models." Journal of Clinical Oncology 31, no. 4_suppl (February 1, 2013): 396. http://dx.doi.org/10.1200/jco.2013.31.4_suppl.396.
Full textAbstract:
396 Background: Overcoming the resistance of topoisomerase I (TOP1) inhibitors has great clinical potential for treatment of advanced colon cancer. Previously, we showed a kind of novel CRM1-dependent SINEs leads to increase the anti-cancer effects of topoisomerase-1 (topo-1) inhibitor, CPT-11 (SN38), in colon cancer in vitro. However, its underlying mechanisms have not been fully understood. Here, we showed CRM1-dependent SINEs induced the sequestration of TOP1 into nucleus as well as other tumor suppressors or growth regulatory proteins. Methods: The synergism of KPT-251, a novel CRM1-dependent SINE, with SN38 were evaluated using Chou-Talalay method in both CPT-11/SN38-sensitive (SW480) and CPT-11/SN38-resistant colon cancers cells (WiDr) in vitro and animal experiments bearing CPT-sensitive and CPT-resistant cells in vivo. The expression change of TOP1, p-FOXO3a, p27, p53, p21, and IkBa in nucleus were evaluated by western blot. Nuclear trafficking of TOP1 was confirmed by immunofluorescence microscopy and measuring nuclear TOP1 catalytic activity. Increased apoptosis through TOP1 nuclear sequestration were evaluated by comet assay. Results: We found the dramatic synergism of KPT-251 with CPT-11 in both CPT-11/SN38-sensitive and CPT-11/SN38-resistant colon cancers cells in vitro and in vivo. We also found TOP1 was sequestrated into nucleus by KPT-251 through CRM1 inhibition, and this induced overcoming the resistance of CPT-11 in colon cancers, which were demonstrated by western blot, immunofluorescence microscopy, TOP1 catalytic activity, and comet assays. Conclusions: Our results suggest blocking TOP1 nuclear export by a novel CRM1-dependent SINE sensitizes colon cancer to TOP1 inhibitors. Our study supports the development of a novel treatment strategy to overcome the resistance of CPT-11 and other TOP1 inhibitors in colon and potentially other cancers.
18
Vire, Berengere, Alexandre David, and Adrian Wiestner. "FcμR (TOSO/FAIM3) Is An O-Glycosylated Endocytic Receptor That Shuttles IgM From the Cell Surface to the Lysosome and Whose Expression Is Regulated by TLR Signaling." Blood 118, no. 21 (November 18, 2011): 2828. http://dx.doi.org/10.1182/blood.v118.21.2828.2828.
Full textAbstract:
Abstract Abstract 2828 TOSO/FAIM3 has recently been identified as the long sought after Fc receptor for IgM (FcμR). Previous studies have shown that FcμR is selectively overexpressed in chronic lymphocytic leukemia (CLL) cells as compared to normal B-cells or other B cell malignancies. In this study, we extend the characterization of FcμR and identify the mechanisms regulating its trafficking and expression. In CLL cells, analysis of FcμR protein expression by immunoblotting revealed one major band around 60 kDa, a series of fainter migrating species, and a band at 41 kDa corresponding to its predicted molecular weight. By contrast, normal B cells express predominantly the 60 kDa form. Inspection of FcμR sequence revealed numerous potential O-glycosylation sites in the extracellular domain and treatment with a general inhibitor of O-glycosylation, benzyl-N-acetyl- α-galactosaminide, confirmed that FcμR is indeed heavily O-glycosylated. By immunofluorescence confocal microscopy, we determined that O-glycosylation is critical for trafficking of FcμR to the cell surface as mutations in the predicted O-glycosylation sites led to intracellular retention of FcμR in HeLa cells transfected with the appropriate expression vectors. In addition, pharmacologic inhibition of O-glycosylation decreased FcμR expression on CLL cells. Next, we used biotinylated IgM and streptavidin DyLight 488 to determine the fate of IgM bound to FcμR. Addition of IgM to CLL cells prompted rapid internalization of both IgM and FcμR, reaching half maximal internalization of cell bound IgM within one minute and virtually complete internalization within 5 minutes. By contrast, a monoclonal antibody specific for FcμR was not internalized. Using immunofluorescence confocal microscopy and co-staining with transferrin and anti-LAMP-1 antiserum, we followed IgM trafficking. FcμR transported IgM through the endocytic pathway to the lysosome, where it was degraded. This last step was chloroquine but not bortezomib sensitive, confirming the role of the lysosome in FcμR degradation. Using a series of FcμR deletion mutants, we identified a proline-rich domain that is essential for cell surface expression of FcμR and a second domain, containing a YXX Φ motif, that controls internalization. Of particular interest, but so far elusive, is a possible functional role of FcμR in CLL pathogenesis. Our data suggest that a major physiologic role of FcμR may be internalization of IgM bound cargo into cells. Given the broad reactivity of IgM such cargo likely includes a variety of infectious agents as well as cellular debris that will be transported into the lysosome and thereby brought into contact with intracellular Toll-like Receptors (TLRs). To address a possible interaction between FcμR and TLRs, we investigated the effect of TLR activation on FcμR expression. There was a striking downregulation of FcμR on CLL cells in response to activation of TLR7 (using imiquimod) or TLR9 (using CpG-ODN) that involved both inhibition of transcription as well as degradation of FcμR through the lysosome. Following CpG stimulation, FcμR mRNA decreased with a half life of approximately 3 hours; that is at the same rate as after actinomycin D treatment. At 24 hours, FcμR protein was greatly reduced in response to either CpG or imiquimod, but interestingly there was less response to CpG in IGHV unmutated than in IGHV mutated CLL samples (p<0.001). In summary, FcμR is an O-glycosylated endocytic receptor that shuttles IgM from the cell surface to the lysosome. In vivo, FcμR could play a role in presenting IgM opsonized immune complexes to intracellular TLRs resulting in B-cell activation. Further studies are needed to clarify a potential contribution of FcμR to the activation of CLL cells in vivo. In addition, owing to the rapid internalization of IgM, we are pursuing FcμR as a potential pathway for the delivery of therapeutic antibody-drug conjugates into CLL cells (to be presented in a separate abstract). This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute. Disclosures: No relevant conflicts of interest to declare.
19
Miller, R. K., K. Vikstrom, and R. D. Goldman. "Keratin incorporation into intermediate filament networks is a rapid process." Journal of Cell Biology 113, no. 4 (May 15, 1991): 843–55. http://dx.doi.org/10.1083/jcb.113.4.843.
Full textAbstract:
The properties of keratin-containing intermediate filament (IF) networks in vivo were studied following the microinjection of biotinylated keratin. Keratin-IFs were biotinylated, disassembled, and separated into type I and type II proteins by ion exchange chromatography. Recombination of these derivatized type I and type II keratins resulted in the formation of 10-nm diameter IF. The type I keratins were microinjected into epithelial cells and observed by immunofluorescence microscopy. Biotin-rich spots were found throughout the cytoplasm at 15-20 min after injection. Short biotinylated fibrous structures were seen at 30-45 min after injection, most of which colocalized with the endogenous bundles of IF (tono-filaments). By 1 1/2 to 2 h after microinjection, extensive biotinylated keratin IF-like networks were evident. These were highly coincident with the endogenous tonofilaments throughout the cell, including those at desmosomal junctions. These results suggest the existence of a relatively rapid subunit incorporation mechanism using numerous sites along the length of the endogenous tonofilament bundles. These observations support the idea that keratin-IFs are dynamic cytoskeletal elements.
20
Vire, Berengere, Alexandre David, Joshua D. Thomas, Terrence R. Burke, Christoph Rader, and Adrian Wiestner. "Fcμ-Receptor (FcμR; TOSO/FAIM3) Mediated Internalization of Antibody-Drug Conjugates: A Novel Approach to Selectively Target Chronic Lymphocytic Leukemia Cells." Blood 118, no. 21 (November 18, 2011): 734. http://dx.doi.org/10.1182/blood.v118.21.734.734.
Full textAbstract:
Abstract Abstract 734 The therapeutic principle of antibody-drug conjugates (ADCs) in cancer relies on an antibody to deliver a cytotoxic agent into malignant cells that express a tumor-associated antigen. Recent progress in the field has shown that the use of such conjugates can increase drug activity and reduce cytotoxicity through selective targeting. Recently, TOSO/FAIM3 was identified as the long sought FcR for IgM (FcμR). FcμR is a transmembrane protein expressed on CD19+ B cells, on some CD4+/CD8+ T cells, and weakly on CD56+/CD3− NK cells. We and others have shown that FcμR is consistently overexpressed on chronic lymphocytic leukemia (CLL) cells compared to normal B-cells. In addition, we detected FcμR expression in the mantle cell lymphoma cell line Mino. Using immunofluorescence staining, we found that FcμR can rapidly internalize IgM and transport it through the endocytic pathway to the lysosome. Interestingly, aggregation of FcμR with IgM lead to rapid internalization (>80% internalized within 5 minutes, n=5) whereas mAb bound FcμR was not internalized (n=3). Because of its restricted expression on CLL cells, its cell surface localization and its rapid internalization, FcμR represents an excellent target to deliver a cytotoxic drug into CLL cells using an ADC. To make use of the rapid internalization of FcμR when bound by the Fc-portion of IgM, we engineered a protein scaffold derived from the CH3-CH4 IgM constant region, and inserted a C-terminal selenocysteine, that allows site specific and covalent conjugation of drugs to the protein scaffold. Using gel filtration, we found that purified CH3-CH4 exists mostly as a multimer including pentameric and hexameric forms, similar to native IgM. First, we verified that the CH3-CH4 protein scaffold also binds FcμR, is internalized and addressed to the lysosomes. Next, we conjugated the CH3-CH4 protein scaffold to cemadotin, a synthetic anti-mitotic agent that inhibits tubulin polymerization. This CH3-CH4-cemadotin conjugate was designed to specifically release the drug inside the cell by inserting a peptide linker that can by cleaved by cathepsin B in the lysosomal compartment. In vitro cell killing showed that this CH3-CH4-cemadotin conjugate potently and selectively killed FcμR expressing cells (IC(50) <0.16 μM) while it was about 200-fold less efficient for cells with undetectable FcμR (IC(50) >31 μM). Control experiments showed equal, i.e. non-selective cytotoxicity of free cemadotin against FcμR expressing and non-expressing cells and absence of cell killing with the unconjugated CH3-CH4 protein scaffold. Taken together, these data identify FcμR as a promising therapeutic target in CLL and possibly select other malignanices. IgM-derived scaffolds constitute ideal carriers for drugs or toxins as they are rapidly internalized. In addition, trafficking of IgM scaffolds to the lysosome provides an additional layer of selectivity as a precursor inactive drug can be activated through lysosomal peptidases. Ongoing efforts aim to test modifications of the scaffold and evaluate additional cytotoxic drugs. Furthermore, we are scaling up production of the lead scaffold drug conjugate in order to test its efficacy in xenograft mouse models. This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute. Disclosures: No relevant conflicts of interest to declare.
21
Liu, H., M. I. Boulton, C. L. Thomas, D. A. M. Prior, K. J. Oparka, and J. W. Davies. "Maize Streak Virus Coat Protein Is Karyophyllic and Facilitates Nuclear Transport of Viral DNA." Molecular Plant-Microbe Interactions® 12, no. 10 (October 1999): 894–900. http://dx.doi.org/10.1094/mpmi.1999.12.10.894.
Full textAbstract:
Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.
22
Halicka, Dorota H., Fevzi M. Ozkaynak, Karen Seiter, Malgorzata A. Kajstura, Delong Liu, Toshiki Tanaka, Frank Traganos, and Zbigniew Darzynkiewicz. "Assessing DNA Damage Ex Vivo Induced by Chemotherapy Targeting Topoisomerase Inhibitors in Acute Leukemias." Blood 108, no. 11 (November 16, 2006): 4372. http://dx.doi.org/10.1182/blood.v108.11.4372.4372.
Full textAbstract:
Abstract Our previous experiments on leukemic cell lines demonstrated that the intensity of immunofluorescence (IF) that is proportional to the level of the phosphorylated histone H2AX (γH2AX) reveals the frequency of DNA double-strand breaks (DSBs) in chromatin induced by DNA topoisomerase I and II (topo I and II) inhibitors. The aim of the present study was to explore whether ex vivo assessment of the extent of DNA damage in leukemic blasts induced during chemotherapy is feasible. The long term goal is to develop a rapid assay to assess the effectiveness of chemotherapy early in the course of treatment of leukemia. Mononuclear cells were isolated from blood samples of 1 AML and 10 ALL patients with peripheral blasts, 4 of whom were, at diagnosis, high-risk ALL while 4 were in relapse. The median age of the patients was 17 yrs (2–25 yrs) and all were entered into an anthracycline-containing drug regimen. Samples were collected: prior to drug administration; and, one hour after completion of the drug infusion. The extent of DNA damage was assessed by analysis of histone H2AX phosphorylation occurring at sites of DSBs, which could be detected immunocytochemically and measured by multiparameter flow cytometry in conjunction with DNA content. In the only patient not responding to treatment, one of 4 relapsed ALL patients, there was no induction of DSBs and no activation of ATM. In all patients phosphorylation of ATM appeared to be strongly linked to induction of DSBs after treatment targeting topo inhibitors. In a pilot study of 3 adult patients with acute leukemia (two AML and one ALL in relapse) treated with the topo II inhibitor Mitoxantrone (MTX), ATM activation was strongly linked to induction of DSBs 1 h post drug infusion. ATM activation and induction of DSBs was several times higher in two adult responders in comparison to one non responder to therapy. A new approach to detect DNA damage in leukemic cell by flow cytometry using gating strategy which combines CD45 expression on the cell surface with side scatter (SS) is being developed and was applied in 5 cases. This approach will be useful when the proportion of blasts in peripheral blood is low. Although the number of patients is very small in this study, the results suggest that a cytometric assay that reveals the extent of DNA damage based on detection of H2AX phosphorylation during induction therapy targeting topo I and II inhibitors may provide an early prognostic marker.
23
Nandiwada, Sarada L., Lisa K. Peterson, Maureen D. Mayes, Troy D. Jaskowski, Elisabeth Malmberg, Shervin Assassi, Minoru Satoh, and Anne E. Tebo. "Ethnic Differences in Autoantibody Diversity and Hierarchy: More Clues from a US Cohort of Patients with Systemic Sclerosis." Journal of Rheumatology 43, no. 10 (August 1, 2016): 1816–24. http://dx.doi.org/10.3899/jrheum.160106.
Full textAbstract:
Objective.To determine the autoantibody repertoire and clinical associations in a multiethnic cohort of American patients with systemic sclerosis (SSc).Methods.There were 1000 patients with SSc (196 Hispanic, 228 African American, 555 white, and 21 other) who were screened for antinuclear antibodies (ANA), including anticentromere antibodies (ACA) by indirect immunofluorescence assay, antitopoisomerase-1 (topo-1/Scl-70) by immunodiffusion, and anti-RNA polymerase III (RNAP III) by ELISA. Sera from 160 patients with mainly nucleolar and/or speckled ANA pattern, but negative for ACA, Scl-70, and RNAP III, were further characterized by immunoprecipitation for SSc-specific antibodies.Results.The prevalence of antibodies against RNAP III, Th/To, and PM/Scl did not differ significantly among the ethnic groups. The frequency of anti-Scl-70 was lowest in whites (18.0%) compared with 24.0% and 26.8% in Hispanics and African Americans (p = 0.01), respectively. Compared with African American patients, Hispanic and white subjects had a higher frequency of ACA (p < 0.0001) and lower frequency of U3-RNP (p < 0.0001). U3-RNP antibodies were uniquely higher in African American patients, independent of clinical subset, while Th/To autoantibodies were associated with limited cutaneous SSc in white subjects. Overall, Hispanic and African American patients had an earlier age of onset and a predominance of diffuse cutaneous SSc compared with their white counterparts.Conclusion.SSc-specific antibodies may predict disease subset; however, the hierarchy of their prevalence differs across ethnic groups. This study provides the most extensive analysis to date on the relevance of autoantibodies in the diagnosis and clinical manifestations of SSc in Hispanic American patients.
24
Kalter, D. C., J. J. Greenhouse, J. M. Orenstein, S. M. Schnittman, H. E. Gendelman, and M. S. Meltzer. "Epidermal Langerhans cells are not principal reservoirs of virus in HIV disease." Journal of Immunology 146, no. 10 (May 15, 1991): 3396–404. http://dx.doi.org/10.4049/jimmunol.146.10.3396.
Full textAbstract:
Abstract Several reports implicate Langerhans cells of skin as susceptible targets, reservoirs, and vectors for transmission of HIV: 1) numbers of Langerhans cells in skin of HIV-infected patients were decreased about 50% of that in control skin; 2) as many as 30% of Langerhans cells in the skin of HIV-infected patients were morphologically abnormal; 3) viral particles typical for HIV were identified in or around 2 to 5% of these cells; and 4) infectious HIV was isolated from skin biopsies of infected patients. These results were consistent with similar observations of HIV-infected macrophages in such tissues as brain, lung, and lymph node. Despite these findings, other investigators find no evidence for virus infection in the epidermis of HIV-infected patients by any of several immunohistochemical or ultrastructural criteria. To address this controversy, we obtained skin from 28 HIV-seropositive subjects at various clinical stages by full thickness biopsy or suction blister. Samples were analyzed by transmission electron microscopy for presence of HIV virions, by immunofluorescent staining for viral proteins, by in situ hybridization for HIV-specific mRNA, by polymerase chain reaction amplification of virus-specific DNA, and by direct virus isolation by coculture of epidermis onto monocyte target cells. By any of these techniques, demonstration of HIV in the epidermis of infected patients was equivocal and even then, infrequent. In contrast, viral DNA was detected from the dermis of the same skin samples (26 of 28 samples). Moreover, the number and morphology of Langerhans cells in skin of infected patients were within normal limits, regardless of stage of disease. These studies in toto suggest that a role for Langerhans cells as a principal viral reservoir or vector of transmission is highly unlikely.
25
ZAHARI, NORMAWATI MOHAMAD, and TEOH TEOW CHONG. "THE CHARACTERIZATION, EXPRESSION AND IN SILICO STUDIES ON THE SLC39A13 GENE; IT'S INVOLVEMENT IN BREAST CANCER." International Journal of Modern Physics: Conference Series 09 (January 2012): 163–73. http://dx.doi.org/10.1142/s2010194512005223.
Full textAbstract:
The zinc transporters superfamily is divided into four subfamilies, and SLC39A is one of the subfamilies. The SLC39A subfamily has 9 members. Based on our computer searchers, all 9 sequences each contain 8 transmembranes domains. Since it is related to the zinc transporters superfamily, the SLC39A subfamily may have the same function that is to transport zinc ion. This paper focus on SLC39A13 studies and using the recombinant technology with CHO cells, it is shown that the recombinant protein, pcDNA5/FRT/V5-His-TOPO®-SLC39A13, has 43kD molecular weight. A second study using immunofluorescence technique with MCF-7 cells, it is shown that the recombinant protein expresses intracellularly. Both studies demonstrate that SLC39A13 expresses in breast cancer cells line, therefore the gene has involvement in the development of breast cancer disease. In our computational studies which is divided into two; the homologous study and sequence analysis, both results are supporting our laboratory results. The homologous study using EMBL-EBI and UniProt tools concluded that SLC39A13 is a member to SLC39A subfamily and it is closely related to SLC39A7 member. Although the sequence analysis shows that the molecular weight of SLC39A13 is 38.35kD it is still comparable to our laboratory result. Separately, using Swiss-EMBnet tools, TMpred, has shown that SLC39A13 has 8 transmembranes domains similar to other family members of SLC39A subfamily. Another analysis using EMBL-EBI tools, PPsearch, shows that SLC39A13 has various protein motif such as the protein kinase C phospho, casein kinase II phospho, leucine zipper and ASN-glycosylation sites. These are the useful information that we need when we study its tertiary structure and simulation in the future.
26
Francoeur, A. M., C. L. Peebles, P. T. Gompper, and E. M. Tan. "Identification of Ki (Ku, p70/p80) autoantigens and analysis of anti-Ki autoantibody reactivity." Journal of Immunology 136, no. 5 (March 1, 1986): 1648–53. http://dx.doi.org/10.4049/jimmunol.136.5.1648.
Full textAbstract:
Abstract Anti-Ki (Ku, p70/p80) autoantibodies, named after the prototype patient Kikuta by Tojo et al., occur in approximately 10% of patients with SLE, often in association with anti-Sm autoantibodies. The immunofluorescent staining pattern characteristic of anti-Ki antibodies is diffuse speckled nuclear, although some substrates show nucleolar staining as well. Anti-Ki sera specifically immunoprecipitated two protein antigens, Ki86 (Mr 86,000) and Ki66 (Mr 66,000), from radiolabeled cell extracts. The Ki system was found to be immunologically identical to the Ku system described by Mimori et al. and the p70/p80 system described by Reeves. The Ki primary in vitro translation products were identified and proved similar in size to the cellular antigens. The Ki antigens were purified from human spleen by immunoaffinity chromatography followed by SDS-PAGE. The purified Ki antigens proved to be closely related by amino acid composition, and did not appear to be phosphorylated, glycosylated, or associated with RNA. The Ki antigens were found to bind to DNA, in agreement with the observations on the Ku and p70/p80 antigens. They were found to be widely conserved in mammals and were coordinately expressed in all tissues tested. Anti-Ki autoantibodies were purified by antigen-affinity chromatography and were tested by immunoblotting. The antibodies were classified as class I, II, or III, depending on their reactivity with the Ki antigens in immunoblots. Class I antibodies cross-reacted with both Ki antigens, class II antibodies reacted solely with Ki66, and class III antibodies reacted solely with Ki86. These results suggest that at least three different epitopes are present on the Ki autoantigens and that patients differ in their autoantibody response to each epitope.
27
Temiz Karadağ, D., A. Yazici, A. Komac, Y. Erez, M. Birlik, A. Akdoğan, A. Sari, et al. "AB0618 COMPREHENSIVE ANALYSIS OF AUTOANTIBODY PROFILE IN A TURKISH SYSTEMIC SCLEROSIS COHORT." Annals of the Rheumatic Diseases 79, Suppl 1 (June 2020): 1604.2–1605. http://dx.doi.org/10.1136/annrheumdis-2020-eular.5030.
Full textAbstract:
Background:Serum autoantibodies closely reflect patterns of organ involvement and disease progression in systemic sclerosis (SSc). The entire autoantibody profile is less well defined in many cohorts and the data regarding their clinical associations and frequencies is limited.Objectives:To determine the autoantibody profile of patients with SSc, as well as their clinical associations, in well-characterized inception- cohort with disease duration less than 3 years.Methods:Serum samples of 100 patients out of 105 enrolled in the study were analyzed for ANA patterns with indirect immunofluorescence (IIF) assay using HEp-20-10/primate liver mosaic IIFT kit. Sera of 96 patients were subjected to commercial line immunoassay to quantify autoantibodies against 13 different autoantigens.Results:92 (92%) out of 100 patients were positive for ANA by IIF (Table 1). The speckled staining was the most pattern followed by nucleolar in 10 patients, centromere in 4, reticular in 1, nuclear in 2 and homogenous in 1. All patients (n= 96) patients were positive for at least 1 autoantibody by immunoblotting (Table 2). Twenty-two (49%) of patients with antiTopo I, 12 (44%) of the patients with antiCENP and 4 (22%) of the patients with antiRNAPIII were single positive. There was no difference in terms of the clinical findings when the patients with single and coexpression of these antibodies were compared. The distributions of the most frequent autoantibodies are shown in Figure 1. Interstitial lung disease was more frequent in the patients positive for anti-Topo I (78.8%) and anti-RNAPIII (27.3%). One of the two patients with breast cancer was anti-RNAPIII positive and none of the patients have diagnosed scleroderma renal crisis. Anti-Topo I was more common in patients with dcSSc (75%) and anti-CENP in lcSSc (46.4%).Table 1.Demographic, clinical and laboratory characteristics of the SSc patients.Sex Female N, %91 (86.7%) Male N, %14 (13.3) Female-to-male ratioAge, mean±SD years48.6±12.7Disease duration, mean±SD years2±1.4Disease classification N, % Diffuse39 (36.5%) Limited65 (62.5%) Sine scleroderma1 (1%)Interstitial lung disease37 (34.3%)Pulmonary arterial hypertension3 (2.8%)Scleroderma renal crisis0Digital ulcer14 (13.3%)Raynaud phenomenon105(100%)Telengiectasia31 (28.8%)Calcinosis1 (1%)Malignancy3 (2.9%)Antinuclear antibody profile N*, % Positive92 (92%)Staining pattern Speckled65 (65%) Nucleolar13 (13%) Centromere29 (29%) Homogeneous3 (3%) Reticular3 (3%)Table 2.Numbers and combinations of autoantibodies identified in the 96 SSc patients.Topo-ICENPRNAP IIIFibrillarinNOR90Th/ToPm/SclKuPDGFRRo52Topo-I22170159507CENP12202222011RNAPIII40121307Fibrillarin0000000NOR90011002Th/To04202Pm/Scl4306Ku102PDGFR00Ro522Single positive221240004102Total452718058179024Figure 1.Diagram of disease-related antibodies against the four main autoantibodies [anti-centromere (antiCENP) anti-Topoisomerase I (antiTopo I), anti-RNA polymerase III (antiRNAP III) and anti-Ro52).Conclusion:We presented the clinical and serologic features of the Turkish SSc patients from a new inception cohort. Clinical features of the SSc patients with single or multiple antibody positivity were not different.References:NoneDisclosure of Interests:None declared
28
Kimura, Yuki, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, and Joji Kitayama. "Abstract 6284: Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6284. http://dx.doi.org/10.1158/1538-7445.am2022-6284.
Full textAbstract:
Abstract Background: Mesothelial cells play tumor-promoting roles in the development of peritoneal metastasis (PM) through mesothelial-mesenchymal transition (MMT). We reported miR-29b suppressed MMT of human peritoneal mesothelial cell (HPMC). Methods: MiR-29b mimics and negative control miRNA (NC) were transfected to human bone marrow derived MSC by lipofection method. Exosomes were purified from the culture supernatants of the MSC with ultracentrifugation, and added to HPMC. Proliferation and migration of HPMC were examined with MTT assay and transwell assay, respectively. MMT of HPMC induced by 10 ng/ml TGF-β, and evaluated with immunofluorescence staining. Results: MSC transfected with miR-29b mimics produced significantly increased amounts of miR-29b-containing exosomes (p<0.0001, n=3) as compared with negative control. After 24h-culture with PKH-26 stained exosomes, HPMC actively took the exosomes. After 48 hr-culture with TGF-β, HPMC apparently changed the morphology from round to spindle shape. The expression level of E-cadherin and Calretinin in HPMC was decreased while vimentin and Fibronectin tended to be upregulated by TGF-β. However, exosomes from miR-29b transfected MSC significantly suppressed the morphological changes and reduced the expression levels of vimentin and Fibronectin with restored expression of E-cadherin and Calretinin, suggesting the inhibition of MMT in HPMC. The exosome inhibited the proliferation and migration of HPMC by 31±14% (p<0.01, n=5) and 67±9% (p<0.01, n=3), and inhibited the adhesion of NUGC by 90±3% (p<0.001, n=5) as compared with NC exosome. Conclusion: Exosomes from MSC transfected with miR-29b mimic efficiently suppresses MMT. Replacement of the miR-29b in peritoneal cavity might be used for the treatment of PM partially via the effects on mesothelial cell. Citation Format: Yuki Kimura, Hideyuki Ohzawa, Yuki Kaneko, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Joji Kitayama. Exosomal miR-29b derived from mesenchymal stem cell (MSC) may suppress peritoneal metastasis via the effects on peritoneal mesothelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6284.
29
Awsare, N. S., T. A. Martin, H. G. Kynaston, R. E. Mansel, P. N. Matthews, and W. G. Jiang. "Overexpression of claudin-11 decreases the invasive potential of bladder cancer cells in vitro, but has no effect on their electrical resistance." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 21038. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.21038.
Full textAbstract:
21038 Background: An increasing body of evidence suggests that tight junctions (TJ) play an important role in invasion and metastasis. Claudins (Cl), a group of integral membrane proteins play an important role in maintaining TJ structure and function. Hepatocyte growth factor (HGF) has been shown to disrupt TJ structure. We investigated the effect of forced overexpression of Cl-11 on TJ function and invasive potential of T24 bladder cancer cells, in response to HGF. Methods: Expression of Cl-11 in bladder cancer cells was determined by RT-PCR. Cl-11 expression construct was made from full length human Cl-11 obtained from normal human cDNA bank, using pcDNA 3.1/ NT-GFP-TOPO expression vector. T24 cells were transfected with the resultant expression cassette (T24Cl-11) or an empty plasmid control (T24GFP). Change in TJ function of stably transfected cells was assessed by trans-urothelial resistance (TUR). In- vitro matrigel adhesion and invasion assays were used to determine the malignant potential of the cells. Cells were treated with HGF at 50 ng/ml for TUR and invasion assay. Immunofluorescence was used to localise Cl-11 within the cell. Results: Successful transfection of T24 cells was confirmed by endogenous GFP fluorescence and by an enhanced Cl-11 mRNA signal in T24Cl-11 compared to wild- type (T24WT) or T24GFP. There was no difference in TUR between the three cell types. Adhesion was significantly higher in T24Cl-11 than in T24GFP [median (IQR); 0.11 (0.096–0.137) vs 0.095 (0.089- 0.115), p=0.045]. T24Cl-11 cells had significantly reduced invasiveness in response to HGF, compared to T24GFP [mean (95% CI); 2.4 (1.8–3.01) vs 3.31 (2.89–3.71), p=0.032]. Immunoflourescence staining with Cl-11 showed redistribution of Cl-11 protein to the cytoplasm and perinuclear regions. Conclusion: Cl-11does not appear to be involved in the TJ function in T24 bladder cancer cells; however, its cytoplasmic location and its effect on the metastatic potential of these cells suggests that it may have a role in modulating the cytoskeletal or signalling network of the cell. Further studies are required to elucidate the exact mechanisms involved. No significant financial relationships to disclose.
30
Yoon, S. J., E. H. Jun, Y. H. Kim, H. Y. Lee, N. H. Kim, and K. A. Lee. "257DIFFERENTIAL GENE EXPRESSION IN GERMINAL VESICLE AND METAPHASE II MOUSE OOCYTES." Reproduction, Fertility and Development 16, no. 2 (2004): 248. http://dx.doi.org/10.1071/rdv16n1ab257.
Full textAbstract:
The present study was conducted to identify oocyte-specific genes according to developmental stage. We used an accurate Annealing Control Primer (ACP;; SeeGene, Seoul, Korea)-based GeneFishing technology. This new and innovative method has developed 120 ACPs so far to locate differentially expressed genes. This method is more specific and sensitive than differential display-PCR that is labor-intensive and results in a high degree of false positives. To identify genes differentially expressed in germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, fully grown GV-intact and MII oocytes were obtained. The addition of 0.2mM IBMX in the collection medium inhibited GV breakdown. The mRNA samples were prepared from the same number of GV and MII oocytes using a Dynabeads mRNA DIRECT Kit. GeneFishing system utilizes a two-stage PCR to fish out authentic differentially expressed genes by regulating binding of each portion of ACP at a different stage of PCR. The differentially expressed bands were extracted and cloned into a TOPO TA cloning vector (Invitrogen, Carlsbad, CA, USA), sequenced, and analyzed by BLAST search. To confirm the differential expression of selected transcripts, RT-PCR and immunofluorescence staining were conducted. Using 12 different ACPs, we isolated 27 differentially expressed bands from GV and MII oocytes. BLAST results indicated that most of these bands had strong homology with known genes. We confirmed that pleckstrin homology, protein kinase D2 (PKD), and COP9 (constitutive photomorphogenic) homologs were GV-specific, while minichromosome maintenance deficient 2 mitotin (MCM2), malate dehydrogenase, soluble Mor2, growth arrest specific 6 (Gas-6), Bcl-2 homolog (Diva), and suppressor of cytokine signaling 4 were expressed highly in MII. Immunofluoresence staining results confirmed higher expression of Gas-6 protein in MII oocytes compared to GV oocytes. Immunohistochemistry results with ovarian tissues demonstrated that MCM2 protein was highly expressed in the nucleus of developing oocytes and granulosa cells, and Diva was highly expressed in the oocytes of primordial follicles. Immunofluoresence staining for these proteins is under investigation. The functions of many of these genes have been determined in systems other than gamete systems. Those data provide insight about their function in oocyte maturation. For example, PKD, COP9, and pleckstrin homology are genes in the axis of a newly identified PKD signaling system. Therefore, further investigation of the functions of each gene identified in this study will provide a basis for understanding the mechanism of oocyte maturation.
31
Marins, Fernanda R., Jennifer A. Iddings, Marco A. Fontes, and Jessica A. Filosa. "Abstract 019: Evidence that Remodeling of Insular Cortex Neurovascular Unit Contributes to Hypertension-related Sympathoexcitation." Hypertension 68, suppl_1 (September 2016). http://dx.doi.org/10.1161/hyp.68.suppl_1.019.
Full textAbstract:
Introduction and Hypothesis: The intermediate region of the posterior insular cortex (intermediate IC) mediates sympathoexcitatory responses to the heart and kidneys. Previous evidence indicates that hypertension alters both structure and function of neurons, blood vessels, astrocytes and microglia, disrupting the architecture of the neurovascular unit (NVU) in specific brain regions. Thus, the goal of this study is to evaluate the functional and anatomical integrity of the NVU in the intermediate IC during hypertension using in vivo and in situ experiments in male hypertensive (SHR) and normotensive (WKY) rats. Methods: Under urethane anesthesia, NMDA microinjection (0.2mM/100nL) was performed in the intermediate IC with simultaneous recording of renal sympathetic nerve activity (RSNA), heart rate (HR) and mean arterial pressure (MAP). NVU structure was investigated by immunofluorescence for NMDA receptors (NR1, NeuN and TOTO), blood vessels (perfused with 70kDa FITC-dextran), astrocytes (GFAP) and microglia (Iba1). Results: NMDA injections into intermediate IC of SHR (n=4) evoked higher amplitude responses of RSNA (Δ= WKY 26 ± 1.5 vs. SHR 44 ± 4.1 % of baseline, P =0.006), MAP (Δ= WKY 9 ± 1.8 vs. SHR 19 ± 2.2 mmHg, P =0.017) and HR (Δ=WKY 40 ± 2.5 vs. SHR 54 ± 4.9 bpm, P =0.044). Immunofluorescence data of the intermediate IC of SHR showed increased NMDA receptor density (WKY 16.67± 1.05% vs. SHR 24.17± 1.68%, n=6, P =0.003). Vascular density (WKY 1.73± 0.13% vs. SHR 2.52± 0.27%, n=10, P =0.015), branch and end-point number were also increased suggesting angiogenesis at the IC of SHR. Additionally, IC of SHR presented greater GFAP immunoreactivity (WKY 9.37± 2.28% vs. SHR 19.51± 3.52%, n=13, P =0.023) and increased contact between astrocyte processes and the vasculature (Δ= 12.8%, n=13, P =0.015). Skeleton analysis indicated enhanced microglia activation in IC of SHR (reduced number of branches, junctions, end-points and process length, n=13), suggesting an inflammatory process in this region. Conclusions: These findings suggest that the neurogenic origin of hypertension in SHR is associated with marked alterations to NVU structure within the IC contributing to enhanced NMDA-mediated sympathoexcitatory responses and maintenance of hypertension.
32
Mohamed, Tamer M., Bernhard Ellinger, Vera Stankovikj, Adriana Wiesinger, Kaomei Guan, Ludwig Neyses, Delvac Oceandy, Sheraz Gul, and Elizabeth J. Cartwright. "Abstract 12279: Novel and Reliable Method to Screen for Drug Cardiac Toxicity Using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (hiPS-CM)." Circulation 130, suppl_2 (November 25, 2014). http://dx.doi.org/10.1161/circ.130.suppl_2.12279.
Full textAbstract:
Unexpected cardiotoxicity underlies high rates of attrition during drug development, posing a multi-billion dollar burden on the pharmaceutical industry. Over reliance on the use of animals and materials derived from animals in preclinical assays to predict the cardiotoxic effect of new drugs in humans has contributed to this problem. The drug responses in human cardiomyocytes compared to animal-derived primary cardiomyocytes (CM) for in vitro assays is not always the same. Here, we describe a combination of human pluripotent stem cell-derived cardiomyocytes (hiPS-CM) and high content analysis confocal microscopy as a potential solution for this major setback in drug development. Initially human skin fibroblasts were reprogrammed into human induced pluripotent stem cells (hiPSCs) by lentiviral transduction and utilization of the following combination of transcription factors: Oct3/4, Sox2, c-Myc and Klf4. The human pluripotent stem cell phenotype of the generated hIPSCs was confirmed. hiPSCs were differentiated into cardiomyocytes and the cardiac cell phenotype was confirmed by immunofluorescence and RT-PCR analysis of cardiac markers i.e. αMHC, cTNT, NKX2.5 and connexin 43. Seventeen known cardiotoxic compounds, as well as controls, were applied to the cells in 384 well format at a dose of 10μM for 48 hours. Then hiPSC-CM underwent confocal microscopy high content analysis to simultaneously evaluate the cell mitochondrial transmembrane potential (using TMRM dye), and plasma membrane permeability (using TOTO-3 dye). All known cardiotoxins showed a significant decrease in mitochondrial transmembrane potential ranging from 74% to 95% and an increase in plasma membrane permeability ranging from 67-327 fold in comparison to the controls. These results showed 100% prediction rate of cardiotoxicity of known cardiotoxins by hiPS-CM. This was compared to only 12% general cytotoxicity prediction rate when these compounds tested on A549 and ACHN cancer cell lines. In conclusion, combining two state of the art technologies 1) hiPSC-CM and 2) confocal microscopy high content analysis, we were able to provide a reliable high throughput method to assess cardiotoxicity of compounds.
33
Apetse, Kossivi, Komlan Kouassi, Nyinèvi Komla Anayo, Kokouvi Panabalo Waklatsi, Mensah Kokou Guinhouya, Léhleng Agba, Vinyo Kodzo Kumako, et al. "Neuromyelitis Optica Spectrum Disorders in Black African: Experience of Togo (2015–2020)." Journal of Neurosciences in Rural Practice, July 11, 2022. http://dx.doi.org/10.1055/s-0042-1750081.
Full textAbstract:
Abstract Introduction Neuromyelitis optica spectrum disorders (NMOSD) would disproportionately affect blacks within mixed populations. However, they are rarely reported in black African. The objective of this work was to report the experience of Togo, a West African country in terms of NMOSD. Methods This is a series of six cases diagnosed between 2015 and 2020 in the only three neurology departments in Togo. The diagnosis of NMOSD was made according to the criteria of the International Panel for NMO Diagnosis (2015) and the patients had a minimum clinical follow-up of 6 months after the diagnosis. The search for anti-aquaporin 4 (AQP4) antibodies was performed by immunofluorescence on transfected cells. Results The mean age was 25.33 years and the sex ratio female/male was 5/1. The average time between the first attack and the diagnosis was 122.83 days. Clinically, there was isolated medullary involvement (2/6), simultaneous opticomedullary involvement (3/6), and area postrema syndrome (1/6). Five patients were anti-AQP4 positive. All six patients had extensive longitudinal myelitis. At 6 months of follow-up, there was one case of death and one case of blindness. Conclusion The rarity of NMOSD cases in Togo could be linked to an underestimation. To better characterize the NMOSDs of the black African population, multicenter and multidisciplinary studies are necessary.