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At least 793 records · Page 44Linked to original sources
PubMed2002

Evaluation of Supermix as an in vitro model of human liver microsomal drug metabolism.

SUPERMIX is a commercially available formulation of insect cell-expressed human drug-metabolizing cytochrome P450 (CYP) isoforms, mixed in proportions that are optimized to parallel their relative activities in human liver microsomes. We have evaluated the apparent functional affinity and capacity of individual CYP isoforms in SUPERMIX in comparison with microsomes from a panel of 12 human livers, using enzyme kinetic studies of isoform-selective index reactions. In addition, we have measured the concentration of NADPH cytochrome P450 oxidoreductase (OR) in SUPERMIX and compared it with the concentrations of this accessory electron transfer protein in human liver microsomes. No important differences were evident in the catalytic activities of CYPs 1A2, 2C8, 2C9, 2C19, 2D6 and 3A4 between SUPERMIX and human liver microsomes. However, SUPERMIX lacks CYP2B6 activity and did not hydroxylate the antidepressant bupropion, a clinically relevant substrate of this enzyme. In addition, the concentration of OR in SUPERMIX (1198 pmol mg protein(-1)) is 17-fold higher than the mean value in human liver microsomes (70 pmol mg protein(-1)). In conclusion, SUPERMIX lacks CYP2B6 activity and contains supraphysiological concentrations of the accessory electron transfer protein OR. These factors should be considered when this formulation is used as an in vitro model in human liver microsomal drug metabolism studies.

Algorithms↗
PubMed1998

Efficient in vitro model for immunotoxicologic assessment of mammary silicone implants.

In clinical and experimental studies, silicone gel has been assumed to cause immune alterations that may be related to macrophage activation of silicone implants. However, it has not been proven that the immunotoxicities are caused by the direct contact of macrophages and silicone gel because there has not been an adequate experimental model. In the present study, silicone gel was loaded directly onto Petri dishes and was distributed uniformly to the bottom by centrifugation. Peritoneal macrophages and splenic lymphocytes were cultured either on the silicone-coated plates or on the conventional, normal plates, and their functions were compared with each other. The experiments were repeated three times. The cytotoxic activities of peritoneal macrophages on cancer cells were markedly augmented by cultivation on silicone gel, and the primary T-dependent immunoglobulin M response in which macrophages participated as antigen presenting cells was also enhanced by incubation on silicone gel. However, macrophage-unrelated functions mediated by B and T lymphocytes were not affected by the silicone gel treatment. It was proven that the direct contact of macrophages with silicone gel was a primary cause of acute immune activation that was related to foreign body reaction. In addition, the present in vitro model exhibited similar silicone-induced immunotoxicities in previous animal and clinical studies.

Animals↗
PubMed2004

Transforming growth factor-beta-induced stimulation of formation of collagen fiber network and anti-fibrotic effect of taurine in an in vitro model of hepatic fibrosis.

The cell strain M, which was established from normal rat liver cells, is characterized by the active formation of a collagen fiber network. In this study, we investigated the characterization of M cells and evaluated the anti-fibrogenic effects of taurine using this culture system. M cells expressed cytokeratin (CK)8, CK18, vimentin, and alpha-smooth muscle actin, whereas expression of CK-19 or desmin was not detected. Also, M cells expressed transforming growth factor (TGF)-beta1, -beta2, and TGF-beta type I and II receptors, and treatment with TGF-beta1 (1ng/ml) for 6 days markedly stimulated the formation of a collagen fiber network and expression of procollagen alpha1(I) mRNA. When M cells were treated with various concentrations of taurine (10-50mM), network formation and procollagen alpha1(I) expression were significantly suppressed in a dose dependent manner. Additionally, even in the presence of TGF-beta1, taurine treatment effectively reduced the formation of a collagen fiber network. These results suggest that M cells exhibit features of not only hepatocytes but also myofibroblasts, and TGF-beta1 plays an important role in the formation of collagen fiber networks in this culture system. Additionally, this M cell culture system is appropriate for use as an in vitro model of hepatic fibrosis in the evaluation of the anti-fibrogenic effects of various agents.

Journal Article↗
PubMed1984

Effect of glomerular basement membrane on the initiation of chemiluminescence and lysosomal enzyme release in human polymorphonuclear leucocytes: an in vitro model of glomerular disease.

The effect of glomerular basement membrane (GBM) on the stimulation of human polymorphonuclear leucocytes (PMNL) was investigated using the technique of chemiluminescence (CL). GBM, in the absence of serum, did not initiate CL of PMNL and neither did GBM preincubated with fresh human or rabbit serum. In contrast, GBM preincubated with rabbit anti-human GBM serum (anti-GBM), and then thoroughly washed, initiated an immediate and marked CL response. Heat inactivation of the anti-GBM only partially reduced the CL, suggesting that both Fc and C3b receptors on the PMNL were involved. The addition of fresh, but not heat-inactivated, human serum to PMNL incubated with untreated GBM resulted in a CL response after a short lag. Subsequent analysis of the serum by electrophoresis and immunofixation indicated that the GBM had activated complement. GBM preincubated with anti-GBM was found to elicit the selective release of lysosomal enzymes from PMNL, one of these enzymes degrading the collagen moiety of the GBM. Preincubation of GBM with the serum from a patient with Goodpasture's syndrome also initiated a CL response and a selective release of lysosomal enzymes from PMNL. These results provide additional evidence for imputing a role for PMNL in the pathogenesis of glomerulonephritis. Furthermore, the technique offers a useful in vitro model for the study of immunologically mediated glomerular disease.

Anti-Glomerular Basement Membrane Disease↗
PubMed1996

Epstein-Barr-virus-associated lymphoproliferative syndrome in severe combined immunodeficiency: establishment of a lymphoblastoid cell line as an in vitro model for biological and therapeutic studies.

Patients with primary or secondary immunodeficiency are at high risk for B cell lymphoproliferative syndromes (LPS) that are generally Epstein-Barr virus (EBV)-associated. We established a cell line, termed JuWa, from an immunoblastic lymphoma that developed in a child with severe combined immunodeficiency. JuWa cells were representative of the original lymph node as shown by a similar IgH gene rearrangement pattern. The cell line exhibited the typical features of a lymphoblastoid cell line (LCL): (1) growth pattern in large clumps, (2) lack of structural chromosome abnormalities, (3) type III latency with expression of EBV-associated EBNA2 and LMP, as well as B cell activation markers CD23 and CD30, thereby showing characteristics of an EBV producer cell line, i.e. a latent infection with a small subpopulation of cells spontaneously entering the lytic cycle, (4) inducibility of the lytic cycle by IdU and TPA, leading to an increase of early antigen and viral capsid antigen-positive cells from 1 to 15-20%, and (5) elimination of the linear viral genomes by treatment with acyclovir (ACV), without affecting the circular episomal genomes. After withdrawal of ACV, viral replication resumed within 7 days. Thus, JuWa cells support the concept of the LCL-like features of LPS and lymphomas occurring in the setting of immunodeficiency. In our in vitro model, ACV treatment could effectively suppress the viral replication but not cure EBV infection of B cells.

Acyclovir↗
PubMed2005

Immunoglobulin a modulates inflammatory responses in an in vitro model of pneumonia.

BACKGROUND: Preservation of mucosal immunity has been shown to affect the risk and outcome of pneumonia in severely injured patients. Secretory immunoglobulin A (SIgA) is the principle humoral defense of mucosal surfaces in the body and has several antiinflammatory properties. Polymorphonuclear neutrophils (PMN) function to kill invading microorganisms, but their exaggerated inflammatory responses may cause tissue injury to the host. The purpose of this study was to compare the ability of different immunoglobulin (Ig) isotypes to modulate PMN cytotoxic potential cocultured with respiratory epithelial cells challenged with bacteria. METHODS: Calu-3 cell monolayers were established on membranes (0.1-microm pore) in a two-chamber culture system. Escherichia coli (EC) incubated with either polyclonal SIgA or IgG was inoculated into the apical chamber and PMNs (10/mL) added to the basal chamber. PMN cytotoxic potential was indexed by % CD11b expression, superoxide anion (O2) production, and elastase release. Dextran flux was used to index Calu-3 monolayer permeability. RESULTS: Addition of EC to PMN-Calu-3 cell coculture increased % CD11b expression, O2 production, and elastase release. IgG had no effect on PMN activation after EC challenge. SIgA abrogated PMN activation and the increase in Calu-3 cell monolayer permeability noted with EC or EC + IgG treatment groups. CONCLUSION: PMN cytotoxic potential was decreased by the presence of SIgA but not IgG in an in vitro model to simulate pneumonia in vivo. SIgA may not only function to protect against microbial invasion of mucosal surfaces, but may also protect against tissue injury from an exaggerated inflammatory response.

Apoptosis↗
PubMed1998

An in vitro model for cytogenetic conversion in CML. Interferon-alpha preferentially inhibits the outgrowth of malignant stem cells preserved in long-term culture.

IFN-alpha has been shown to prolong survival in chronic myeloid leukemia patients, but its mechanism of action is still not understood. The human cobblestone area-forming cell (CAFC) assay allows for the measurement of the concentration of normal as well as malignant stem cells, while their progeny can be measured in parallel long-term culture (LTC) in flasks. Using CAFC and LTC assays, we have examined direct effects of IFN-alpha (500; 5,000 IU/ml) on the maintenance and outgrowth of CD34-enriched normal and malignant stem cells, obtained from six patients with an established major cytogenetic response to IFN-alpha and from four nonresponding patients. CAFC concentrations were not affected by IFN-alpha. In contrast, IFN-alpha strongly inhibited the clonogenic output in flask LTC. Nucleated cells (NC) produced in LTC were evaluated by fluorescent in situ hybridization (FISH) for the presence of the Philadelphia (Ph) translocation. After 8 wk of LTC, the percentage of Ph+ NCs produced was significantly more inhibited by IFN-alpha in responding patients than in nonresponders. Control LTC without IFN-alpha showed no significant differences of Ph+ NC production between responders and nonresponders. These findings provide the first in vitro model for cytogenetic conversion and suggest that direct antiproliferative effects of IFN-alpha account for the cytogenetic response observed clinically.

Adult↗
PubMed1992

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.

Animals↗
PubMed1998

Retinol uptake and metabolism, and cellular retinol binding protein expression in an in vitro model of hepatic stellate cells.

Liver is a major site of retinoid metabolism and storage, and more than 80% of the liver retinoids are stored in hepatic stellate cells. These cells represent less than 1% of the total liver protein, reaching a very high relative intracellular retinoid concentration. The plasma level of retinol is maintained close to 2 microM, and hepatic stellate cells have to be able both to uptake or to release retinol depending upon the extracellular retinol status. In view of their paucity in the liver tissue, stellate cells have been studied in primary cultures, in which they loose rapidly the stored lipids and retinol, and convert spontaneously into the activated myofibroblast phenotype, turning a long-term study of their retinol metabolism impossible. We have analyzed the retinol metabolism in the established GRX cell line, representative of stellate cells. We showed that this cell line behaves very similarly, with respect the retinol uptake and release, to primary cultures of hepatic stellate cells. Moreover, we showed that the cellular retinol binding protein (CRBP-I) expression in these cells, relevant for both uptake and esterification of retinol, responds to the extracellular retinol status, and is correlated to the retinol binding capacity of the cytosol. Its expression is not associated with the overall induction of the lipocyte phenotype by other agents. We conclude that the GRX cell line represents an in vitro model of hepatic stellate cells, and responds very efficiently to wide variations of the extracellular retinol status by autonomous controls of its uptake, storage or release.

Adipocytes↗
PubMed1998

Response of the human peritoneal mesothelial cell to injury: an in vitro model of peritoneal wound healing.

BACKGROUND: The denudation of the peritoneal mesothelium and damage to the underlying interstitium is a frequent finding in patients receiving continuous ambulatory peritoneal dialysis as a treatment for end-stage renal failure. The response of the mesothelium to injury from repeated episodes of infection or from exposure to dialysis fluids has not been extensively studied. The present study describes a simple and reproducible method with which to investigate the response of human mesothelial cells to injury. METHODS: The model of peritoneal injury consists of mechanically wounding a monolayer of human peritoneal mesothelial cells with a glass probe and following the repopulation of the denuded area by time-lapse photomicroscopy. In addition immunohistochemistry was used to follow the response of marker proteins for stress fibers and focal adhesions as well as macromolecules associated with the extracellular matrix. RESULTS: Under serum-free conditions the wound (0.58 +/- 0.094 mm; mean +/- SD; N = 20) closed within 72 +/- 5 hours (N = 8). This rate of healing was enhanced by fetal calf serum, by human serum (10%) and by undiluted spent non-infected dialysate. The repair process over the first 48 hours was the result of cell migration, was independent of cell proliferation and involved the de novo synthesis of several different extracellular matrix components. An early event in the healing process was the rapid reorganization of intracellular stress fibers together with the formation of associated focal adhesions in cells at the wound edge. CONCLUSION: This in vitro model should prove invaluable in characterizing the process of wound healing within the peritoneal cavity, thus allowing a better understanding of the response to infection as well as any effect of dialysis fluids in this pattern of cell behavior.

Blood Physiological Phenomena↗
PubMed1992

The rat external urethral sphincter an in vitro model to evaluate the activity of drugs on the smooth and striated components of the urinary bladder outlet.

Electrical field stimulation (EFS, 2.5 Hz, 60 V, 1 ms, trains of 5 sec every 5 min) of the rat external urethral sphincter (EUS) produced contractile responses characterized by a "slow" tonic contraction on which was superimposed a series of phasic "twitch-like" contractions. Both responses were abolished by tetrodotoxin (0.6 microM), and their amplitude was significantly lower in samples taken from denervated (15 days before) sphincters. The tonic contraction showed duration, voltage, and frequency dependency, whereas the twitches were markedly duration dependent. No correlation was found between the amplitude of the tonic and that of the twitch-like contractions. Phentolamine (3 microM) reduced by 95% the amplitude of the tonic contraction produced by maximal parameters, whereas it did not affect the twitches. On the other hand, hexamethonium (10 microM) was ineffective on both components. Dantrolene (10 microM) inhibited the twitch response, whereas it did not influence the tonic component. Preincubation with d-tubocurarine (0.2 mM) or succinylcholine (2 mM) significantly inhibited the amplitude of twitches produced by EFS (0.1 Hz, 60 V) up to a duration of 50 microseconds. Stimulus width higher than 50 microseconds, resulted in twitches that were resistant to neuromuscular blocking agents but sensitive to dantrolene (10 microM). Our results indicate that the rat external urethral sphincter is a reliable and easy "in vitro" model for studying the activity of drugs capable of interfering with the nerve-mediated activity of the striated and smooth muscle portion of the urinary bladder outlet.

Animals↗
PubMed1992

Molecular characterization of the stretch-induced adaptation of cultured cardiac cells. An in vitro model of load-induced cardiac hypertrophy.

Although it is a well-known fact that hemodynamic load is a major determinant of cardiac muscle mass and its phenotype, little is known as to how mechanical load is converted into intracellular signals of gene regulation. To address this question, we characterized the stretch-induced adaptation of cultured neonatal cardiocytes grown on a stretchable substrate in a serum-free medium. Static stretch (20%) of the cells was applied without cell injury. Stretch caused hypertrophy in myocytes and hyperplasia in non-myocytes. Stretch caused an induction of immediate-early genes such as c-fos, c-jun, c-myc, JE, and Egr-1, but not Hsp70. Immunostaining showed that the stretch-induced Fos protein localized in the nucleus of both myocytes and non-myocytes. Nuclear extracts from stretched myocytes contained DNA binding activity to the AP-1 and Egr-1 consensus sequences. In myocytes, the induction of immediate-early genes was followed by expression of "fetal" genes such as skeletal alpha-actin, atrial natriuretic factor, and beta-myosin heavy chain. DNA transfection experiments showed that the "stretch-response element" of the c-fos gene promoter is present within 356 base pairs of the 5'-flanking region, whereas that of the atrial natriuretic factor and the beta-myosin heavy chain genes is probably located outside of 3412 and 628 base pairs of the 5'-flanking region, respectively. These results demonstrate that the phenotype of stretched cardiocytes in this in vitro model closely mimics that of hemodynamic load-induced hypertrophy in vivo. This model seems to be a suitable system with which to dissect the molecular mechanisms of load-induced hypertrophy of cardiac muscle.

Adaptation, Physiological↗
PubMed1997

Development of immortalized human cerebromicrovascular endothelial cell line as an in vitro model of the human blood-brain barrier.

The objective of this study was to generate an immortal cell line representative of specialized human brain microvascular endothelia forming the blood-brain barrier (BBB) in vivo. Human capillary and microvascular endothelial cells (HCEC) were transfected with the plasmid pSV3-neo coding for the SV40 large T antigen and the neomycin gene. The neomycin-resistant transfected cells overcame proliferative senescence, and after a 6-8 wk period of crisis produced immortalization-competent cell colonies. Single-cell clones of near-diploid genotype were isolated from these colonies, propagated, and characterized. Immortalized HCEC (SV-HCEC) exhibited accelerated proliferation rates, but remained serum and anchorage dependent and retained the characteristic cobblestone morphology at confluence. SV-HCEC displayed a stable nuclear expression of SV40 large T antigen, lacked the invasiveness of transformed cells, and maintained major phenotypic properties of early passage control cells including expression of factor VIII-related antigen, uptake of acetylated low-density lipoprotein, binding of fluorescently labeled lectins, expression of transferrin receptor and transferrin receptor-mediated endocytosis, and high activities of the BBB-specific enzymes alkaline phosphatase and gamma-glutamyl transpeptidase. The diffusion of radiolabeled sucrose across SV-HCEC monolayers was fivefold lower than that observed with human lung microvascular endothelial cells. Furthermore, media conditioned by fetal human astrocytes increased the transendothelial electrical resistance of SV-HCEC monolayers by 2.5-fold. Therefore, this newly established human cell line expressing the specialized phenotype of BBB endothelium may serve as a readily available in vitro model for studying the properties of the human BBB.

Alkaline Phosphatase↗
PubMed2005

Immature rat brain slices exposed to oxygen-glucose deprivation as an in vitro model of neonatal hypoxic-ischemic encephalopathy.

To analyze whether exposure to oxygen-glucose deprivation (OGD) of immature rat brain slices might reproduce the main pathophysiologic events leading to neuronal death in neonatal hypoxic-ischemic encephalopathy (NHIE), 500 microm-thick brain slices were obtained from 7-day-old Wistar rats, and incubated in oxygenated physiological solution. In OGD group, oxygen and glucose were removed from the medium for 10-30 min (n = 25); then, slices were re-incubated in normal medium. In control group the medium composition remained unchanged (CG, n = 30). Medium samples were obtained every 30 min for 3 h. To analyze neuronal damage, slices were stained with Nissl and CA1 area of hippocampus and cortex were observed under microscopy. In addition, neuronal death was quantified as LDH released to the medium determined by spectrophotometry. Additionally, medium glutamate (Glu) levels were determined by HPLC and those of TNFalpha by ELISA, whereas inducible nitric oxide synthase expression was determined by Western blot performed on slices homogenate. Optimal OGD time was established in 20 min. After OGD, a significant decrease in the number of neurones in hippocampus and cortex was observed. LDH release was maximal at 30 min, when it was five-fold greater than in CG. Furthermore, medium Glu concentrations were 200 times greater than CG levels at the end of OGD period. A linear relationship between Glu and LDH release was demonstrated. Finally, 3 h after OGD a significant induction of iNOS as well as an increase in TNFalpha release were observed. In conclusion, OGD appears as a feasible and reproducible in vitro model, leading to a neuronal damage, which is physiopathologically similar to that found in NHIE.

Animals↗
PubMed2006

Activation by IL-1 of bovine articular chondrocytes in culture within a 3D collagen-based scaffold. An in vitro model to address the effect of compounds with therapeutic potential in osteoarthritis.

OBJECTIVE: To determine the best protocol for the preparation of a tissue-engineered cartilage to investigate the potential anti-arthritic and/or anti-osteoarthritic effects of drugs. METHODS: Calf articular chondrocytes, seeded in collagen sponges were grown in culture for up to 1 month. At day 14 cultures received interleukin (IL)-1beta (ranging from 0.1 to 20 ng/ml) for 1 to 3 days. Analyses of gene expression for extracellular matrix proteins, collagen-binding integrins, matrix metalloproteinases (MMPs), aggrecanases, TIMPs, IL-1Ra and Ikappa-Balpha were carried out using real-time polymerase chain reaction (PCR). Metalloproteinase activities were analysed in the culture medium using both zymography and fluorogenic peptide substrates. RESULTS: We selected a culture for 15 or 17 days with collagen sponges seeded with 10(7) chondrocytes showing a minimal cell proliferation, a maximal sulphated glycosaminoglycan (sGAG) deposition and a high expression of COL2A1, aggrecan and the alpha10 integrin sub-unit and low expression of COL1A2 and the alpha11 integrin sub-unit. In the presence of 1 ng/ml IL-1beta, we observed at day 15 up-regulations of 450-fold for MMP-1, 60-fold for MMP-13, 54-fold for ADAMTS-4 and MMP-3 and 10-fold for ADAMTS-5 and IL-1Ra. Down-regulations of 2.5-fold for COL2A1 and aggrecan were observed only at day 17. At the protein level a dose-dependent increase of total MMP-1 and MMP-13 was noted with less than 15% in the active form. CONCLUSIONS: This in vitro model of chondrocyte culture in three dimensional (3D) seems well adapted to investigate the responses of these cells to inflammatory cytokines and to evaluate the potential anti-inflammatory effects of drugs.

ADAM Proteins↗
PubMed2005

In vitro modeling of human pancreatic duct epithelial cell transformation defines gene expression changes induced by K-ras oncogenic activation in pancreatic carcinogenesis.

Genetic analysis of pancreatic ductal adenocarcinomas and their putative precursor lesions, pancreatic intraepithelial neoplasias (PanIN), has shown a multistep molecular paradigm for duct cell carcinogenesis. Mutational activation or inactivation of the K-ras, p16(INK4A), Smad4, and p53 genes occur at progressive and high frequencies in these lesions. Oncogenic activation of the K-ras gene occurs in >90% of pancreatic ductal carcinoma and is found early in the PanIN-carcinoma sequence, but its functional roles remain poorly understood. We show here that the expression of K-ras(G12V) oncogene in a near diploid HPV16-E6E7 gene immortalized human pancreatic duct epithelial cell line originally derived from normal pancreas induced the formation of carcinoma in 50% of severe combined immunodeficient mice implanted with these cells. A tumor cell line established from one of these tumors formed ductal cancer when implanted orthotopically. These cells also showed increased activation of the mitogen-activated protein kinase, AKT, and nuclear factor-kappaB pathways. Microarray expression profiling studies identified 584 genes whose expression seemed specifically up-regulated by the K-ras oncogene expression. Forty-two of these genes have been reported previously as differentially overexpressed in pancreatic cancer cell lines or primary tumors. Real-time PCR confirmed the overexpression of a large number of these genes. Immunohistochemistry done on tissue microarrays constructed from PanIN and pancreatic cancer samples showed laminin beta3 overexpression starting in high-grade PanINs and occurring in >90% of pancreatic ductal carcinoma. The in vitro modeling of human pancreatic duct epithelial cell transformation may provide mechanistic insights on gene expression changes that occur during multistage pancreatic duct cell carcinogenesis.

Carcinoma, Pancreatic Ductal↗
PubMed1996

Malignant transformation of human ectocervical cells immortalized by HPV 18: in vitro model of carcinogenesis by cigarette smoke.

In addition to the established role of human papillomaviruses (HPVs) in cervical cancer, smoking has been suggested to be an important cofactor. Previously, primary human ectocervical cells immortalized by HPV types 16 and 18 DNA did not form tumors on nude mice. Here, we derived a new line of HPV 18-immortalized ectocervical cells (HEC-18-1), which was also non-tumorigenic. To examine the role of cigarette smoking in the progression of cervical cancer initiated by HPV 18, we adapted these cells to growth in serum and high calcium and treated the cells with cigarette smoke condensate until tumorigenic cells (HEC-18-1C) were produced. Moderate and late passage serum-adapted untreated HEC-18-1 (HEC-18-1S) remained non-tumorigenic. A typical HEC-18-1C tumor was an invasive squamous cell carcinoma, from which we established a clonal line of cells (HEC-18-1CT). Although the physical state of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was not affected by malignant transformation and the gene expression of HPV 18 was affected little, the differentiation of the epithelium derived in organotypic (raft) culture from HEC-18-1CT was altered dramatically. Moderate and late passage HEC-18-1 and HEC-18-1S were reconstructed into mild dysplasia in organotypic (raft) culture. On the other hand, the moderate passage malignantly transformed HEC-18-1CT displayed severe dysplasia/carcinoma in situ in raft culture. We describe here the first direct evidence of the role of cigarette smoke in the progression of HPV-initiated carcinogenesis using an in vitro model system.

Animals↗
PubMedDate not supplied

Pig chondrocyte xenoimplants for human chondral defect repair: an in vitro model.

The objective of this study was to evaluate the use of cultured porcine chondrocyte xenotransplantation for the repair of human chondral defects. Two-millimeter-diameter defects were drilled into explants of femoral cartilage from healthy adult donors. No cells were implanted in the chondral defects of the control group, while pig chondrocytes from normal femoral cartilage were deposited into the treated chondral defects. Cartilage explants were cultured for 4, 8, and 12 weeks. Tissue sections were processed for standard histologic staining and immunostaining with monoclonal antibodies against types I and II collagen, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. The porcine origin of chondrocytes was confirmed using a specific pig monoclonal anti-CD46. Repair was only observed in the cell-treated defects. Mono- or bilayers of cells were detected after 4 culture weeks on the bottom of the defects, while after 8-12 weeks a repair tissue filled near 30-40 percent of the defect. At 8 weeks, the newly synthesized tissue was composed of a fibrous mesh including some cells. However, at 12 weeks it showed a hypercellular hyaline-like region. This hypercellular region showed excellent bonding with the native cartilage, cells were located in numerous lacunae, and a high content of proteoglycans as indicated by an intense toluidine blue stain was observed. The repaired tissue showed positive immunostaining for both type I and II collagen, as well as chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, and integrin subunit beta1. Positive staining for porcine anti-CD46 was localized exclusively in the neo-synthesized tissue. We conclude that xenotransplantation of pig chondrocytes can repair, in an in vitro model, defects in human articular cartilage.

Aged↗