Academic literature on the topic 'In toto immunofluorescence'

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.

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.

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.

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.

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]

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.

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.

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.

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.

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.

Les modèles d'étude in vitro 3D permettent de prendre en compte le rôle du microenvironnement en mimant l'organisation et l'hétérogénéité d'une microtumeur avasculaire. Ils permettent ainsi d'explorer les réponses fonctionnelles et pharmacologiques du contrôle de la prolifération dans des conditions plus proches de la réalité physiopathologique. Mes travaux de thèse se sont intéressés à l'évaluation de la prolifération dans le modèle 3D de sphéroïdes tumoraux et à sa régulation, en particulier par l'oxygène. Ils ont reposé sur la mise en œuvre d'approches expérimentales d'ingénierie et de biologie cellulaire, de microscopie et de traitement et analyse d'image. Une première étude réalisée sur des coupes de sphéroïdes nous a permis de montrer que la pression partielle en oxygène est un paramètre clé qui influence la répartition de la prolifération dans le temps et dans l'espace. Nous avons poursuivi cette étude dans les sphéroïdes in toto, en mettant en place une méthodologie de marquage par immunofluorescence en 3D, associée à l'imagerie par feuille de lumière et à la quantification par traitement d'image. L'application de cette procédure nous a permis d'explorer la prolifération au cours de la croissance de sphéroïdes MCF-7 cultivés en physioxie vs normoxie et de confirmer le rôle limitant de l'oxygène pour la prolifération en 3D. Nous avons également montré la réversibilité de l'arrêt de prolifération induit par l'anoxie et la possibilité de l'utiliser pour conditionner et conserver les sphéroïdes. Enfin, le développement d'une stratégie d'imagerie en 3D par illumination structurée nous a permis d'évaluer l'efficacité de composés par analyse d'image automatisé sur des sphéroïdes cultivés en matrice. L'ensemble de nos travaux ont permis de développer des méthodes et des outils d'intérêt dans l'étude de la biologie du cancer et de la réponse en pharmacologie anti-tumorale. Leur application a notamment permis de souligner l'importance de la physioxie pour l'étude de la prolifération et l'évaluation la réponse aux drogues. Ces travaux ouvrent également des perspectives applicatives pour la société IMACTIV-3D
In vitro 3D models allow the role of the microenvironment to be taken into account by mimicking the organization and heterogeneity of an avascular microtumor. They thus make it possible to explore proliferation and pharmacological responses under conditions closer to the pathophysiological reality. My thesis work focused on the evaluation of proliferation in the 3D model of tumor spheroids and its regulation, particularly by oxygen. They were based on the implementation of experimental approaches in engineering and cell biology, microscopy and image processing and analysis. A first study carried out on spheroid sections has allowed us to show that partial oxygen pressure is a key parameter that influences the distribution of proliferation in time and space. We continued this study in whole spheroids in toto, by implementing a 3D immunofluorescence labelling methodology, combined with light sheet imaging and image processing quantification. The application of this procedure allowed us to explore the proliferation during the growth of MCF-7 spheroids grown in physioxia vs normoxia and to confirm the limiting role of oxygen for 3D proliferation. We have also shown the reversibility of anoxia-induced proliferation arrest and the possibility of using it to condition and preserve spheroids. Finally, the development of a 3D imaging strategy using structured illumination allowed us to evaluate the effectiveness of compounds by automated image analysis on spheroids grown in a matrix. All of our work has made it possible to develop methods and tools of interest in the study of cancer biology and response in anti-tumor pharmacology. Their application has made it possible to highlight the importance of physioxia for the study of proliferation and the evaluation of drug response. This work also opens up application perspectives for the IMACTIV-3D company

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Toxoplasma gondii is an obligatory intracellular parasite, capable of infecting a large variety of intermediate hosts including humans. In the domiciliary environment, the infected final hosts (cats) spread the oocists by their excrements, which quickly reach maturity in the ground and become infectants. Therefore, toxoplasmosis is acquired by humans through the ingestion of oocists found in the domiciliary environment or by eating insufficiently cooked meat, infected with cysts. The antitoxoplasma antibodies presence can be determined by several serologic reactions, using different serologic markers to distinguish recent infection from toxoplasma-illness. In this work, we carried out serologic assays, defined a curve by the digital recognition of color using the Toxo IgG Digital Microscopical Immunofluorescence. The curve was related with the results in UI/ml read by the quantitative determination of IgG antibodies for the Toxoplasma gondii of Toxo in the G VIDAS® system. The existing correlation between sera in UI/ml and their values found through the digitalized green intensity curve was evidenced. In the analysis between the predetermined sera in UI/ml and its read values, did not show differences that could affect the laboratorial diagnosis. The analysis of the behavior of the curves defined by this works, shows that the approach adopted by this work is promising. for the development of a novel method for Immunofluorescence analysis with significant gains in precision and price reduction. The rational use of technologies, even so the rationality is not neutral, implies the election of technologies to be financed and the identification of the conditions or sub-groups where they will have to be used, in the direction to become the system of more efficient health for the objective to protect and to recoup the health of the population. Correlation to the unemployment level social impact in companies affected by the technological modernization progress. The expectation is that it has, through public politics in set with private initiative, action for creation of courses that prepare the workers for the use of these new technologies.
O Toxoplasma gondii é um parasito intracelular obrigatório, capaz de infectar uma grande variedade de hospedeiros intermediários, incluindo o homem. No ambiente domiciliar, os hospedeiros definitivos infectados (gatos) eliminam os oocistos pelas fezes, os quais, rapidamente, atingem maturidade no solo e se tornam infectantes. Portanto, a toxoplasmose é adquirida pelo homem por meio da ingestão de oocistos encontrados no meio ambiente domiciliar ou de carnes infectadas com cistos e insuficientemente cozidas. A ocorrência de anticorpos antitoxoplasma tem sido determinada por diversas reações sorológicas, utilizando-se diferentes marcadores sorológicos que possibilitam distinguir infecção recente de toxoplasma-doença. Neste projeto de pesquisa, realizaram-se ensaios sorológicos em que a curva obtida pelo reconhecimento digital de cor, pela técnica de Imunofluorescência Microscópica Digital para Toxo IgG, foi relacionada com os resultados em UI/mL obtidos pela determinação quantitativa de anticorpos IgG para o Toxoplasma gondii do sistema VIDAS® Toxo G. Ficou evidenciada a correlação existente entre os soros em UI/mL predeterminados e seus valores encontrados através da equação da curva digitalizada da intensidade de verde. Na comparação entre os soros em UI/mL predeterminados e seus valores encontrados, não foram observadas diferenças que pudessem alterar o diagnóstico clínico-laboratorial. A utilização do conhecimento sobre o comportamento das curvas resultantes das interações das leituras visual e digital demonstra que a proposta deste trabalho é promissora para o desenvolvimento de uma nova forma de análise de imunoflourescência, com significativos ganhos de precisão e redução de preços. A racionalidade não é neutra, portanto o uso racional de tecnologias implica a seleção daquelas que devem ser financiadas e a identificação das condições ou subgrupos em que deverão ser utilizadas com o fim de tornar o sistema de saúde mais eficiente para proteger e recuperar a saúde da população. Com relação ao possível desemprego em empresas envolvidas no processo de modernização tecnológica, o que se espera é que, por meio de políticas públicas em conjunto com a iniciativa privada, sejam desenvolvidas ações no sentido de preparar os trabalhadores para o uso dessas novas tecnologias.

In platelets the presence of basic subunit proteins of microtubules as well as microfilaments has been verified a long time ago and it was also shown that both of these cytoskeletal systems go through a tremendous reorganization during the activation process. Surprisingly, none of the components of intermediate filaments has so far been identified in these cells, perhaps because platelets were considered too motile to have intermediate filaments, the most static structures among the three major cytoskeletal systems. By using two different monoclonal antibodies (II C4 and II D8) here we attempted to establish if vimen-tin, an intermediate filament subunit protein present in most differentiating cells, in cells grown in tissue culture and in certain fully differentiated cells also exists in human platelets The IgM type antibodies were characterized by various immuno-morphological and immunobiochemical techniques. They labelled selectively colcemid-sensitive filamentous structures in fibroblasts, in endothelial cells and in vascular but not myometrium smooth muscle and were shown to be monospecific against epitopes on vimentin in fibroblast homogenate. When whole platelet homogenate was submitted to high resolution gradient SDS PAGE and then electroblotted to nitrocellulose sheet, both antibodies reacted with a single protein band of 55 kD that comigrated with fibroblast vimentin. By immunofluorescence microscopy an annular ring-like structure was stained for vimentin that suggests a membran skeletal role for this protein. During pseu-dopode formation a redistribution of vimentin could be observed. Parallel with the disappearance of ring-like structures vimentin appears in pseudopodia suggesting that like in the case of other filamentous systems platelet activation induces a structural reorganization of the intermediate filaments, as well.