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1.
Rev. esp. med. nucl. imagen mol. (Ed. impr.) ; 37(5): 285-289, sept.-oct. 2018. ilus, tab
Article in Spanish | IBECS | ID: ibc-178220

ABSTRACT

Objetivo: Después de la inyección del radiofármaco se observa con frecuencia una concentración incrementada de 123I-MIBG en el lóbulo hepático izquierdo en comparación con el derecho, pero la razón de este hallazgo sigue siendo desconocida. Nuestro objetivo fue analizar retrospectivamente el patrón de absorción de 123I-MIBG entre los 2 lóbulos hepáticos y correlacionar nuestros resultados con algunas características epidemiológicas/clínicas. Material y métodos: Se seleccionaron 94 gammagrafías 123I-MIBG de 71 pacientes. Las regiones de interés se dibujaron en los lóbulos derecho e izquierdo utilizando secciones tomográficas transversales y se calcularon relaciones de actividad de izquierda a derecha (relación I/D) a las 6 y 24horas de la administración del radiofármaco. Resultados: Veintisiete exámenes fueron positivos para lesiones hipermetabólicas, mientras que los 67 restantes fueron negativos. En todos los casos las relaciones medias de I/D temprana y tardía fueron superiores a 1; la relación I/D temprana media fue de 1,37 y la relación I/D tardía de 1,52. La relación I/D tardía fue significativamente mayor que la precoz. No hubo diferencias en la relación I/D con respecto a la edad, sexo, enfermedad primaria y resultado de la gammagrafía. Conclusiones: La absorción de 123I-MIBG fue mayor en el lóbulo hepático izquierdo en comparación con el derecho, y esta relación no se correlacionó con ninguna característica epidemiológica o clínica. La razón de este metabolismo todavía no se ha explicado, y algunas hipótesis biomoleculares podrían ser probadas en modelos 3D dinámicos in vitro


Purpose: After radiopharmaceutical injection, a heightened 123I-MIBG concentration is frequently observed in the left hepatic lobe compared to the right one, but the reason of this finding remains unknown. Our aim was to retrospectively analyze the different 123I-MIBG uptake pattern between the two hepatic lobes and correlate our results with some epidemiological/clinical features. Material and methods: Ninety-four 123I-MIBG scintigraphies from 71 patients were selected. Regions of interest were drawn in the right and left lobes using transverse tomographic sections and left to right activity ratios (L/R ratio) were calculated at 6 and 24h after radiotracer administration. Results: Twenty-seven examinations were positive for hypermetabolic lesions while the remaining 67 were negative. In all cases mean early and delayed L/R ratios were greater than 1.00; average early L/R ratio was 1.37 and delayed L/R ratio 1.52. The delayed L/R ratio was significantly higher than the early one. There was no difference in the L/R ratios with regard to age, gender, primary disease and result of scintigraphy. Conclusions: 123I-MIBG uptake was higher in left hepatic lobe compared to right and this ratio did not correlate with any epidemiological or clinical feature. The reason of this metabolic is not yet explained and some biomolecular hypotheses could be tested in 3D dynamic in vitro models


Subject(s)
Humans , Male , Female , Infant , Child, Preschool , Child , Adolescent , Young Adult , Adult , Middle Aged , Aged , Aged, 80 and over , 3-Iodobenzylguanidine/pharmacokinetics , Liver , Single Photon Emission Computed Tomography Computed Tomography/methods , Radiopharmaceuticals/pharmacokinetics , Retrospective Studies
2.
Article in English, Spanish | MEDLINE | ID: mdl-29754941

ABSTRACT

PURPOSE: After radiopharmaceutical injection, a heightened 123I-MIBG concentration is frequently observed in the left hepatic lobe compared to the right one, but the reason of this finding remains unknown. Our aim was to retrospectively analyze the different 123I-MIBG uptake pattern between the two hepatic lobes and correlate our results with some epidemiological/clinical features. MATERIAL AND METHODS: Ninety-four 123I-MIBG scintigraphies from 71 patients were selected. Regions of interest were drawn in the right and left lobes using transverse tomographic sections and left to right activity ratios (L/R ratio) were calculated at 6 and 24h after radiotracer administration. RESULTS: Twenty-seven examinations were positive for hypermetabolic lesions while the remaining 67 were negative. In all cases mean early and delayed L/R ratios were greater than 1.00; average early L/R ratio was 1.37 and delayed L/R ratio 1.52. The delayed L/R ratio was significantly higher than the early one. There was no difference in the L/R ratios with regard to age, gender, primary disease and result of scintigraphy. CONCLUSIONS: 123I-MIBG uptake was higher in left hepatic lobe compared to right and this ratio did not correlate with any epidemiological or clinical feature. The reason of this metabolic is not yet explained and some biomolecular hypotheses could be tested in 3D dynamic in vitro models.


Subject(s)
3-Iodobenzylguanidine/pharmacokinetics , Liver Neoplasms/diagnostic imaging , Liver Neoplasms/metabolism , Liver/metabolism , Radiopharmaceuticals/pharmacokinetics , Adolescent , Adult , Aged , Aged, 80 and over , Child , Child, Preschool , Female , Humans , Infant , Male , Middle Aged , Retrospective Studies , Tissue Distribution , Young Adult
3.
Water Res ; 102: 211-220, 2016 10 01.
Article in English | MEDLINE | ID: mdl-27344252

ABSTRACT

The occurrence of emerging organic micropollutants (OMPs) in sewage sludge has been widely reported; nevertheless, their fate during sludge treatment remains unclear. The objective of this work was to study the fate of OMPs during mesophilic and thermophilic anaerobic digestion (AD), the most common processes used for sludge stabilization, by using raw sewage sludge without spiking OMPs. Moreover, the results of analytical chemistry were complemented with biological assays in order to verify the possible adverse effects (estrogenic and genotoxic) on the environment and human health in view of an agricultural (re)use of digested sludge. Musk fragrances (AHTN, HHCB), ibuprofen (IBP) and triclosan (TCS) were the most abundant compounds detected in sewage sludge. In general, the efficiency of the AD process was not dependent on operational parameters but compound-specific: some OMPs were highly biotransformed (e.g. sulfamethoxazole and naproxen), while others were only slightly affected (e.g. IBP and TCS) or even unaltered (e.g. AHTN and HHCB). The MCF-7 assay evidenced that estrogenicity removal was driven by temperature. The Ames test did not show point mutation in Salmonella typhimurium while the Comet test exhibited a genotoxic effect on human leukocytes attenuated by AD. This study highlights the importance of combining chemical analysis and biological activities in order to establish appropriate operational strategies for a safer disposal of sewage sludge. Actually, it was demonstrated that temperature has an insignificant effect on the disappearance of the parent compounds while it is crucial to decrease estrogenicity.


Subject(s)
Sewage/chemistry , Waste Disposal, Fluid , Anaerobiosis , Humans , Perfume , Sulfamethoxazole , Temperature
4.
Sci Total Environ ; 458-460: 160-8, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23648445

ABSTRACT

WWTP (wastewater treatment plant) effluents are considered to be a major source for the release in the aquatic environment of EDCs (Endocrine-Disrupting Compounds), a group of anthropogenic substances able to alter the normal function of the endocrine system. The application of conventional processes (e.g. activated sludge with biological nitrogen removal) does not provide complete elimination of all these micropollutants and, consequently, an advanced treatment should be implemented. This experimental work was conducted on the tertiary ozonation stage of a 140,000 p.e. activated sludge WWTP, treating a mixed domestic and textile wastewater: an integrated monitoring, including both chemical (nonylphenol, together with the parent compounds mono- and di-ethoxylated, and bisphenol A were chosen as model EDCs) and biological (estrogenic and genotoxic activities) analyses, was carried out. Removal efficiencies of measured EDCs varied from 20% to 70%, depending on flow conditions (ozone dosage being 0.5 gO3/gTOC). Biological tests, furthermore, displayed that the oxidation stage did not significantly reduce (only by 20%) the estrogenicity of the effluent and revealed the presence and/or formation of genotoxic compounds. These results highlight the importance of the application of an integrated (biological+chemical) analytical procedure for a global evaluation of treatment suitability; poor performances recorded in this study have been attributed to the presence of a significant industrial component in the influent wastewater.


Subject(s)
Endocrine Disruptors/analysis , Endocrine Disruptors/chemistry , Environmental Restoration and Remediation/methods , Ozone/chemistry , Water Pollutants, Chemical/analysis , Water Purification/methods , Comet Assay , Estrogens/chemistry , Gas Chromatography-Mass Spectrometry , Italy , Micronucleus Tests , Mutagens/chemistry , Solid Phase Extraction
5.
J Mech Behav Biomed Mater ; 2(2): 164-72, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19627820

ABSTRACT

Skeletal abnormalities reported in humans and laboratory animals after spaceflight, include cancellous osteopenia, decreased cortical and cancellous bone formation, aberrant matrix ultrastructure, decreased mineralization and reduced bone strength. Although considerable effort has been made up to now to understand the skeletal effects of spaceflight, in order to estimate health risk, our knowledge in this area is still largely incomplete. It is widely accepted that the mechanical strength of cancellous bone is related not only to the mineral content, but also to the trabecular micro-architecture arrangement. Three-dimensional numerical analysis of bone volumes has been shown to be an important tool in this field. The Cell Method, a recently introduced numerical method, has been applied to static analysis of structures obtained from 3D reconstruction of micro-computed tomography scans performed at the Elettra Synchrotron facility (Trieste, Italy) in order to quantify changes in trabecular bone architecture. In the present study, the Cell Method model is used to compare the micro-tomographed structure of fragments of rats bone explants (tibial proximal epiphyses) harvested after 3 days and after 1, 2, 3 and 4 weeks of culture in the RCCS bioreactor, which represents the unique existing bioreactor, operating on the Earth's surface, capable of successfully reproducing, in vitro, optimal conditions in order to simulate a microgravity environment. Although preliminary, our results seem to suggest that the exposure of tibial bone explants to simulated microgravity conditions obtained by the RCCS bioreactor, are consistent with skeletal changes observed after spaceflight.


Subject(s)
Bone and Bones/anatomy & histology , Bone and Bones/cytology , Weightlessness Simulation , Animals , Biomechanical Phenomena , Bone and Bones/diagnostic imaging , Cells, Cultured , Elastic Modulus , Image Processing, Computer-Assisted , Male , Models, Biological , Rats , Rats, Sprague-Dawley , X-Ray Microtomography
6.
Genes Nutr ; 4(1): 13-22, 2009 Mar.
Article in English | MEDLINE | ID: mdl-19104883

ABSTRACT

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.

7.
Toxicol In Vitro ; 21(2): 284-92, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17055213

ABSTRACT

Manganese (Mn) is both an essential nutrient and a toxicant, with specific effects on liver and kidney (acute exposure) and on central nervous system (CNS) (chronic exposure). Mn neurotoxicity includes neurobehavioral disorders and extra-pyramidal motor dysfunctions (manganism), possibly due to focal injuries to the basal ganglia. Even if widely investigated, the molecular mechanisms responsible for Mn toxicity remain to be clarified. Aim of this study was to identify suitable in vitro models to investigate these molecular pathways. To this purpose we compared the effect of manganese chloride on four cell lines, representative of the main target organs of Mn toxicity in vivo. HepG2 and MDCK cell lines were selected for liver and kidney, respectively; glial GL15 and neuronal SHSY5Y cells were used as models of CNS components. To complete the "motor system" model, skeletal muscle C2C12 cells were also included. Our results demonstrate that hepatic, renal, glial and neuronal cell types differently react to Mn, mirroring the specific in vivo response of the tissue they represent. This confirms their value as suitable in vitro models to study Mn-related toxic events. Interestingly, also muscle C2C12 cells showed a noticeable sensitivity to Mn, preferential targets being differentiated myotubes.


Subject(s)
Chlorides/toxicity , Animals , Cell Count , Cell Survival/drug effects , Cells, Cultured , Dogs , Dose-Response Relationship, Drug , Humans , Kidney/drug effects , Liver/drug effects , Manganese Compounds , Mice , Neurons/drug effects , Organ Specificity , Time Factors
8.
Toxicol In Vitro ; 20(6): 874-81, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16503110

ABSTRACT

Relative insolubility of inorganic Pb compounds is one of the major problems in the evaluation of the toxicological profile of this metal. Different characteristics of Pb-containing solutions may, in fact, alter the biological properties of Pb compounds and influence their toxic potency. To investigate these aspects, we used selected experimental conditions to evaluate and compare the specific biological effects of five inorganic Pb compounds (soluble salts and oxide) on the viability and proliferation rate of a rat liver-derived cell line (REL cells). The study was performed according to classical toxicological criteria (dose- and time-response, reversibility/transience of the effect). Each Pb compound was accurately solubilised and the quantification of the real concentration of Pb(II) ions was performed either on the culture media used for each treatment, or on the extracts of exposed cells. Our study shows that four, out of the five Pb compounds we tested, induce the same dose- and time-related anti-proliferative effects on REL cells, being these effects also reversible, transient and directly related to the intracellular content of the metal. Since the intracellular concentration of the metal and, consequently, its biological effects on REL cells, directly depends on the bioavailability of the Pb(II) cation present in the treatment solutions, our results indicate that, in the experimental procedures aimed to assess the toxic potency of this metal, the solubility of each Pb compound should be carefully evaluated and taken into account.


Subject(s)
Lead/toxicity , Liver/drug effects , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Cells, Cultured , Epithelial Cells/drug effects , Lead/metabolism , Liver/pathology , Rats
9.
Cell Death Differ ; 12(7): 761-72, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15818410

ABSTRACT

Opposite effects of nuclear factor-kappaB (NF-kappaB) on neuron survival rely on activation of diverse NF-kappaB factors. While p65 is necessary for glutamate-induced cell death, c-Rel mediates prosurvival effects of interleukin-1beta. However, it is unknown whether activation of c-Rel-dependent pathways reduces neuron vulnerability to amyloid-beta (Abeta), a peptide implicated in Alzheimer's disease pathogenesis. We show that neuroprotection elicited by activation of metabotropic glutamate receptors type 5 (mGlu5) against Abeta toxicity depends on c-Rel activation. Abeta peptide induced NF-kappaB factors p50 and p65. The mGlu5 agonists activated c-Rel, besides p50 and p65, and the expression of manganese superoxide dismutase (MnSOD) and Bcl-X(L). Targeting c-Rel expression by RNA interference suppressed the induction of both antiapoptotic genes. Targeting c-Rel or Bcl-X(L) prevented the prosurvival effect of mGlu5 agonists. Conversely, c-Rel overexpression or TAT-Bcl-X(L) addition rescued neurons from Abeta toxicity. These data demonstrate that mGlu5 receptor activation promotes a c-Rel-dependent antiapoptotic pathway responsible for neuroprotection against Abeta peptide.


Subject(s)
Amyloid beta-Peptides/toxicity , NF-kappa B/metabolism , Neurons/drug effects , Neuroprotective Agents/pharmacology , Proto-Oncogene Proteins c-rel/metabolism , Receptors, Metabotropic Glutamate/agonists , Receptors, Metabotropic Glutamate/metabolism , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cells, Cultured , Cerebral Cortex/cytology , Enzyme Inhibitors/pharmacology , Excitatory Amino Acid Agonists/pharmacology , Gene Deletion , Gene Silencing , Glycine/analogs & derivatives , Glycine/pharmacology , Humans , Mice , Mice, Inbred C57BL , Neurons/cytology , Neurons/metabolism , Neurons/pathology , Neuroprotective Agents/metabolism , Phenylacetates/pharmacology , Proto-Oncogene Proteins c-rel/deficiency , Proto-Oncogene Proteins c-rel/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Metabotropic Glutamate/genetics , Superoxide Dismutase/metabolism
10.
Med Lav ; 93(3): 267-78, 2002.
Article in Italian | MEDLINE | ID: mdl-12197277

ABSTRACT

OBJECTIVES: Mercury (Hg), one of the most diffused and hazardous organ-specific environmental contaminants, exists in a wide variety of physical and chemical states, each of which with unique characteristics of target organ specificity. Exposure to Hg vapour and to organic mercurials specifically affects the CNS, while the kidney is the target organ for inorganic Hg compounds. Despite the increasing number of studies, the molecular bases of the nephrotoxic potential of Hg has not, up to now, been clarified, even if there is evidence suggesting that the ability of the metal to interact with proteins (thiol groups) or to generate oxygen radicals may play a major role. Within this context, the aim of the present study was to investigate, in vitro, the mechanism(s) of the early nephrotoxic potential of mercury chloride (HgCl2), one of the most diffused and biologically active mercury (Hg2+) compounds. For this purpose, two kidney-derived in vitro systems (the MDCK and the LLC-PK1 cell lines) were tested for their sensitivity to the salt, and MDCK was chosen as the most suitable in vitro model for our study. As possible biological markers of the organ-specific toxicity of the metal we analysed: i) critical biochemical parameters related to oxidative stress conditions (effect of Hg2+ on the anti-oxidant status of the cell), and ii) gap-junctional function (GJIC). METHODS: Classical toxicity tests (MTT and NR) were used for assessing the sensitivity (IC50) of LLP-CK1 and MDCK cell lines to the mercuric salt. Complete solubilisation of the salt in the culture media was verified by inductively coupled plasma mass spectrometry (ICP-MS). The influence of the metal on cell growth rate and viability were evaluated by conventional proliferation assays. For the following mechanistic studies, cells were exposed for different time periods (4 to 72 hours) to non-cytotoxic (0.1-50 microM) HgCl2 concentrations. The biochemical analysis of the pro-oxidant properties of the mercuric compound was performed by the measurement of anti-oxidant cellular defences against H2O2 [catalase (Cat), glutathione peroxidase (Gpx), and total glutathione (GSH)]. The influence of the metal on the GJIC capacity of MDCK cells was assessed by the "microinjection/dye-coupling" assay. RESULTS: Among the two kidney-derived in vitro systems, MDCK cell line was the most specifically sensitive to the toxic effect of HgCl2: it was, consequently, chosen as a "tubular cell model" for the following experimental steps. Tested for various time periods at increasing concentrations, the HgCl2 effect on MDCK cell proliferation and viability was found to be time- and dose-related. For concentrations < or = 50 microM, HgCl2 inhibits MDCK cell growth rate, being this effect significant (> 50% in respect to untreated controls) from the 24th from the beginning of the treatment, while, for concentrations > 50 microM, the metal causes cell death. Concerning the influence of HgCl2 on MDCK anti-oxidant defences, the most interesting results were obtained by analysing the influence of the mercury salt on the GSH cell content and Gpx activity. Both were, in fact, significantly affected by the presence of the mercury ion. HgCl2 also induced a rapid, dose- and time-related inhibitory effect on the GJIC capacity of the cells. CONCLUSIONS: Even if further investigations are needed to better clarify the possible causal relationship between our findings, they indicate that: a) MDCK cells represent a suitable in vitro model for the study of Hg nephrotoxicity; b) GJIC function is, among those considered in our study, one of the most sensitive biological endpoints for investigating the mechanism(s) of Hg2+ specific toxicity.


Subject(s)
Kidney/drug effects , Mercuric Chloride/pharmacology , Animals , Catalase/metabolism , Cell Communication/drug effects , Cell Division/drug effects , Cell Line/drug effects , Dogs , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Fluorescent Dyes/analysis , Glutathione/metabolism , Glutathione Peroxidase/metabolism , Isoquinolines/analysis , Kidney/cytology , Mercuric Chloride/administration & dosage , Mercuric Chloride/toxicity , Oxidation-Reduction , Oxidative Stress , Sensitivity and Specificity , Solubility , Swine
11.
Toxicol In Vitro ; 16(4): 457-65, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12110286

ABSTRACT

In this study, the early nephrotoxic potential of mercuric chloride (HgCl(2)) has been evaluated in vitro, by exposing a renal-derived cell system, the tubular epithelial Madin-Darby canine kidney (MDCK) cell line, to the presence of increasing HgCl(2) concentrations (0.1-100 microM) for different periods of time (from 4 to 72 h). As possible biological markers of the tubular-specific toxicity of HgCl(2) in exposed-MDCK cultures we analysed: (i) critical biochemical parameters related to oxidative stress conditions and (ii) gap-junctional function (GJIC). HgCl(2) cytotoxicity was evaluated by cell-density assay. The biochemical analysis of the pro-oxidant properties of the mercuric ion (Hg(2+)) was performed by evaluating the effect of the metal salt on the antioxidant status of the MDCK cells. The cell glutathione (GSH) content and the activity of glutathione peroxidase (Gpx) and catalase (Cat), two enzymes engaged in the H(2)O(2) degradation, were quantified. HgCl(2) influence on MDCK GJIC was analysed by the microinjection/dye-transfer assay. HgCl(2)-induced morphological changes in MDCK cells were also taken into account. Our results, proving that subcytotoxic (0.1-10 microM) HgCl(2) concentrations affect either the antioxidant defences of MDCK cells or their GJIC, indicate these critical functions as suitable biological targets of early mercury-induced tubular cell injury.


Subject(s)
Cell Communication/drug effects , Disinfectants/toxicity , Gap Junctions/physiology , Kidney Tubules/cytology , Mercuric Chloride/toxicity , Oxidative Stress , Animals , Antioxidants , Cell Line , Dogs , Dose-Response Relationship, Drug , Gap Junctions/drug effects , Glutathione Peroxidase/pharmacology
12.
BMC Physiol ; 1: 4, 2001.
Article in English | MEDLINE | ID: mdl-11384510

ABSTRACT

BACKGROUND: It has been proposed that GL15, a human cell line derived from glioblastoma multiforme, is a possible astroglial-like cell model, based on the presence of cytoplasmic glial fibrillary acidic protein. RESULTS: The aim of this work was to delineate the functional characteristics of GL15 cells using various experimental approaches, including the study of morphology, mechanism of induction of intracellular Ca2+ increase by different physiological agonists, and the presence and permeability of the gap-junction system during cell differentiation. Immunostaining experiments showed the presence and localization of specific glial markers, such as glial fibrillary acidic protein and S100B, and the lack of the neuronal marker S100A. Notably, all the Ca2+ pathways present in astrocytes were detected in GL15 cells. In particular, oscillations in intracellular Ca2+ levels were recorded either spontaneously, or in the presence of ATP or glutamate (but not KCl). Immunolabelling assays and confocal microscopy, substantiated by Western blot analyses, revealed the presence of connexin43, a subunit of astrocyte gap-junction channels. The protein is organised in characteristic spots on the plasma membrane at cell-cell contact regions, and its presence and distribution depends on the differentiative status of the cell. Finally, a microinjection/dye-transfer assay, employed to determine gap-junction functionality, clearly demonstrated that the cells were functionally coupled, albeit to varying degrees, in differentiated and undifferentiated phenotypes. CONCLUSIONS: In conclusion, results from this study support the use of the GL15 cell line as a suitable in vitro astrocyte model, which provides a valuable guide for studying glial physiological features at various differentiation phases.


Subject(s)
Astrocytes/physiology , Calcium/metabolism , Cell Line, Tumor , Gap Junctions/physiology , Astrocytes/chemistry , Astrocytes/cytology , Cell Communication , Cell Differentiation , Connexin 43/analysis , Connexin 43/immunology , Humans , Immunoblotting , Immunohistochemistry , Phenotype
13.
Exp Cell Res ; 249(2): 248-59, 1999 Jun 15.
Article in English | MEDLINE | ID: mdl-10366424

ABSTRACT

Immortalization of chondrocytes by SV40 T Ag has often been reported to trigger the loss of expression of type II collagen, one of the main differentiation markers, although some immortalized chondrocyte lines maintaining a differentiated phenotype have also been described. Here, we show using transient cotransfections in differentiated chondrocytes that, in contrast to c-src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activity. Then, we report the possibility of immortalizing rabbit articular chondrocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enhancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional regulation of the col2a1 gene through long-term culture. In precrisis pCol2SV-transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2a1 regulatory sequences. Some pCol2SV-transfected chondrocytes were definitively immortalized, after a short crisis period. However, type II collagen synthesis was restricted to a small proportion of cells, which went on to decrease with subculture, while the proportion of cells expressing T Ag was not affected. In these postcrisis cells, T Ag remained at least partially under the control of functional col2a1 regulatory elements as assessed by all-trans-retinoic acid down-regulation.


Subject(s)
Antigens, Polyomavirus Transforming/biosynthesis , Chondrocytes/metabolism , Collagen/biosynthesis , Collagen/genetics , Gene Expression Regulation , Simian virus 40/physiology , Animals , Antigens, Polyomavirus Transforming/genetics , Apoptosis/drug effects , Cell Differentiation/genetics , Cell Line, Transformed , Cell Transformation, Viral/genetics , Chondrocytes/chemistry , Chondrocytes/drug effects , Collagen/metabolism , Down-Regulation/drug effects , Enhancer Elements, Genetic , Fluorescent Antibody Technique, Indirect , Gene Expression Regulation/drug effects , Plasmids , Promoter Regions, Genetic , Rabbits , Rats , Transcription, Genetic , Transfection , Tretinoin/pharmacology
14.
J Cell Physiol ; 176(2): 303-13, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9648917

ABSTRACT

Chondrocytes cultivated in monolayer rapidly divide and lose their morphological and biochemical characteristics, whereas they maintain their phenotype for long periods of time when they are cultivated in alginate beads. Because cartilage has a low cellularity and is difficult to obtain in large quantities, the number of available cells often becomes a limiting factor in studies of chondrocyte biology. Therefore, we explored the possibility of restoring the differentiated properties of chondrocytes by cultivating them in alginate beads after two multiplication passages in monolayer. This resulted in the reexpression of the two main markers of differentiated chondrocytes: Aggrecan and type II collagen gene expression was strongly reinduced from day 4 after alginate inclusion and paralleled protein expression. However, 2 weeks were necessary for total suppression of type I and III collagen synthesis, indicators of a modulated phenotype. Interleukin-1beta, a cytokine that is present in the synovial fluid of rheumatoid arthritis patients, induces many metabolic changes on the chondrocyte biology. Compared with cells in primary culture, the production of nitric oxide and 92-kDa gelatinase in response to interleukin-1beta was impaired in cells at passage 2 in monolayer but was fully recovered after their culture in alginate beads for 2 weeks. This suggests that the effects of interleukin-1beta on cartilage depend on the differentiation state of chondrocytes. This makes the culture in alginate beads a relevant model for the study of chondrocyte biology in the presence of interleukin-1beta and other mediators of cartilage destruction in rheumatoid arthritis and osteoarthrosis.


Subject(s)
Cell Culture Techniques/methods , Chondrocytes/cytology , Chondrocytes/metabolism , Extracellular Matrix Proteins , Interleukin-1/pharmacology , Aggrecans , Alcian Blue , Alginates , Animals , Cartilage, Articular/cytology , Cell Differentiation/physiology , Chondrocytes/drug effects , Chondroitin Sulfate Proteoglycans/genetics , Collagen/biosynthesis , Collagen/genetics , Coloring Agents , Gene Expression/physiology , Immunohistochemistry , Lectins, C-Type , Microspheres , Phenotype , Proteoglycans/biosynthesis , Proteoglycans/genetics , RNA/metabolism , RNA, Messenger/analysis , Rabbits , Staining and Labeling
16.
Cytotechnology ; 19(1): 89-94, 1995 Jan.
Article in English | MEDLINE | ID: mdl-22358908

ABSTRACT

The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding ß-galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (ß-galactosidase assay).Results revealed that addition of 4 U ml(-1) of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a "giant" calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.

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