High-content phenotypic screening and triaging strategy to identify small molecules driving oligodendrocyte progenitor cell differentiation.

Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation.

Compound screening platform using human induced pluripotent stem cells to identify small molecules that promote chondrogenesis.

Articular cartilage, which is mainly composed of collagen II, enables smooth skeletal movement. Degeneration of collagen II can be caused by various events, such as injury, but degeneration especially increases over the course of normal aging. Unfortunately, the body does not fully repair itself from this type of degeneration, resulting in impaired movement. Microfracture, an articular cartilage repair surgical technique, has been commonly used in the clinic to induce the repair of tissue at damage sites. Mesenchymal stem cells (MSC) have also been used as cell therapy to repair degenerated cartilage. However, the therapeutic outcomes of all these techniques vary in different patients depending on their age, health, lesion size and the extent of damage to the cartilage. The repairing tissues either form fibrocartilage or go into a hypertrophic stage, both of which do not reproduce the equivalent functionality of endogenous hyaline cartilage. One of the reasons for this is inefficient chondrogenesis by endogenous and exogenous MSC. Drugs that promote chondrogenesis could be used to induce self-repair of damaged cartilage as a non-invasive approach alone, or combined with other techniques to greatly assist the therapeutic outcomes. The recent development of human induced pluripotent stem cell (iPSCs), which are able to self-renew and differentiate into multiple cell types, provides a potentially valuable cell resource for drug screening in a "more relevant" cell type. Here we report a screening platform using human iPSCs in a multi-well plate format to identify compounds that could promote chondrogenesis.

The interaction of various liquids with long-term denture soft lining materials.

OBJECTIVES: To study the uptake of liquids, representative of those encountered orally, by long-term denture soft lining materials, and analyze the data in terms of appropriate theories. METHODS: Four proprietary and one experimental soft lining material were investigated, and the weight change presented as a function of time in both aqueous and organic fluids over the course of a year. A separate experiment determined the equilibrium swelling in ethanol of poly(ethyl methacrylate) and poly(methyl methacrylate). RESULTS: Uptake date for the five soft lining materials in various aqueous solution, coconut oil and HB307 are reported. The experimental value for the equilibrium swelling of poly(ethyl methacrylate) and poly(methyl methacrylate) in ethanol was reported to indicate the solubility parameter of the system. SIGNIFICANCE: The results have been analyzed by relevant theoretical models, which have been shown to explain the experimental data.

Intracellular insulin-like growth factor-1 induces Bcl-2 expression in airway epithelial cells.

Bcl-2, a prosurvival protein, regulates programmed cell death during development and repair processes, and it can be oncogenic when cell proliferation is deregulated. The present study investigated what factors modulate Bcl-2 expression in airway epithelial cells and identified the pathways involved. Microarray analysis of mRNA from airway epithelial cells captured by laser microdissection showed that increased expression of IL-1ß and insulin-like growth factor-1 (IGF-1) coincided with induced Bcl-2 expression compared with controls. Treatment of cultured airway epithelial cells with IL-1ß and IGF-1 induced Bcl-2 expression by increasing Bcl-2 mRNA stability with no discernible changes in promoter activity. Silencing the IGF-1 expression using short hairpin RNA showed that intracellular IGF-1 (IC-IGF-1) was increasing Bcl-2 expression. Blocking epidermal growth factor receptor or IGF-1R activation also suppressed IC-IGF-1 and abolished the Bcl-2 induction. Induced expression and colocalization of IC-IGF-1 and Bcl-2 were observed in airway epithelial cells of mice exposed to LPS or cigarette smoke and of patients with cystic fibrosis and chronic bronchitis but not in the respective controls. These studies demonstrate that IC-IGF-1 induces Bcl-2 expression in epithelial cells via IGF-1R and epidermal growth factor receptor pathways, and targeting IC-IGF-1 could be beneficial to treat chronic airway diseases.

A cell-based protein-protein interaction method using a permuted luciferase reporter.

We have developed a novel cell-based protein-protein interaction assay method. The method relies on conversion of an inactive permuted luciferase containing a Tobacco Etch Virus protease (TEV) cleavage sequence fused onto protein (A) to an active luciferase upon interaction and cleavage by another protein (B) fused with the TEV protease. We demonstrate assay applicability for ligand-induced protein-protein interactions including G-protein coupled receptors, receptor tyrosine kinases and nuclear hormone receptors.

Posterior mandibular residual ridge resorption in patients with overdentures supported by two or four endosseous implants in a 10-year prospective comparative study.

PURPOSE: The aim of this study was to evaluate the effect of treatment with either two or four mandibular endosseous implants with an overdenture on mandibular posterior residual ridge resorption over a 10-year period. MATERIALS AND METHODS: Sixty edentulous patients with residual mandibular height between 12 and 18 mm participated. Thirty patients were treated with an overdenture supported by two IMZ implants (group A) and 30 patients were treated with an overdenture with four IMZ implants (group B). Before treatment and 10 years after treatment, panoramic radiographs were taken and compared to ascertain possible bone loss. Proportional area measurements were used to determine changes in the mandibular posterior residual ridge bilaterally. RESULTS: There was a statistically significant difference in mandibular posterior residual ridge resorption between the two treatment protocols. The posterior bone area index was reduced by a mean of 10% for group A and 6% for group B over 10 years. CONCLUSIONS: There was a significant difference in mandibular posterior residual ridge resorption between patients treated with either two or four implants to stabilize an overdenture. No correlation was shown between mandibular posterior residual ridge resorption and peri-implant marginal bone loss. The confounding factors of marginal bone loss around the implants, age, gender, initial mandibular height, and the number of years the patient had been edentulous failed to show a significant effect on posterior ridge resorption.

Effect of antifungal gels incorporated into a tissue conditioning material on the growth of Candida albicans.

OBJECTIVE: The aim of this study was to examine the effectiveness of antifungal gels incorporated into a tissue conditioner which inhibits the growth of Candida albicans in vitro. BACKGROUND: The release of drugs from relining materials has been demonstrated earlier. However, the incorporation of antifungal agents in gel form has not yet been studied. MATERIALS AND METHODS: Visco-gel(®) tissue conditioner was prepared with chlorhexidine digluconate and miconazole in gel form in a concentration of 5, 10, 15, 20 and 25% by volume. Sample discs were prepared and placed on Sabouraud Dextrose Agar (SDA) plates which had been previously inoculated with C. albicans, and incubated aerobically at 37 °C. To investigate antifungal activity over time, Visco-gel discs containing 20%v/v miconazole were prepared and immersed in water for different time periods before being placed on SDA plates inoculated with C. albicans. RESULTS: Chlorhexidine digluconate gel added to tissue conditioner had no inhibition effect on the growth of C. albicans. Incorporation of miconazole gave a dose-related inhibitory effect on candidal growth. Immersion of the discs in water showed an inverse relationship between time of immersion and degree of inhibition. CONCLUSION: Miconazole added in gel form to Visco-gel(®) had an inhibitory effect on the growth of C. albicans in vitro.

The death domain-containing kinase RIP1 regulates p27(Kip1) levels through the PI3K-Akt-forkhead pathway.

Elucidating the cross-talk between inflammatory and cell proliferation pathways might provide important insights into the pathogenesis of inflammation-induced cancer. Here, we show that the receptor-interacting protein 1 (RIP1)-an essential mediator of inflammation-induced nuclear factor-kappaB (NF-kappaB) activation-regulates p27(Kip1) levels and cell-cycle progression. RIP1 regulates p27(Kip1) levels by an NF-kappaB-independent signal that involves activation of the phosphatidylinositol 3-kinase (PI3K)-Akt-forkhead pathway. Mouse embryonic fibroblasts (MEFs) from RIP1-knockout mice express high levels of p27(Kip1). Reconstitution of MEFs with RIP1 downregulates p27(Kip1) levels in a PI3K-dependent manner. RIP1 regulates p27(Kip1) at the messenger RNA level by regulating the p27(Kip1) promoter through the forkhead transcription factors. RIP1 expression blocks accumulation of cells in G(1) in response to serum starvation and favours cell-cycle progression. Finally, we show that overexpression of p27(Kip1) blocks the effects of RIP1 on the cell cycle. Thus, our study provides a new insight into how components of inflammatory and immune signalling pathways regulate cell-cycle progression.

Human T helper (Th) cell lineage commitment is not directly linked to the secretion of IFN-gamma or IL-4: characterization of Th cells isolated by FACS based on IFN-gamma and IL-4 secretion.

Upon activation in vitro, only a fraction of the bulk human T helper cell cultures secret the hallmark Th1/2 cytokines (IFN-gamma for Th1 and IL-4 for Th2, respectively). It is uncertain whether these IFN-gamma-/IL-4- cells are differentiated Th1 or Th2 cells. Here, we have characterized live IFN-gamma+, IL-4+ and IFN-gamma-/IL-4- cells isolated from Th cell cultures treated under Th1 or Th2 polarizing conditions by employing affinity matrix capture technology. RNA samples from the sorted cells were analyzed by real time RT-PCR and microarrays. The double negative cells from either Th1 or Th2 cultures expressed lower levels of Th1/Th2 marker cytokine genes (IFNgamma, IL4, and IL5). However, they were comparable with the IFN-gamma+ or IL-4+ cells in the expression levels of other Th1/Th2 marker genes (GATA3, Tbet, and IL12Rbeta2). Most importantly, these double negative cells were already committed in their Th1/Th2 lineages. Gene expression profiling analysis showed that very few previously identified Th1/Th2 marker genes were differentially expressed between the IFN-gamma or IL-4 producers and the non-producers, further underscoring the similarity between these two groups.

Flavopiridol sensitivity of cancer cells isolated from ascites and pleural fluids.

PURPOSE: We examined the efficacy of flavopiridol, a cyclin-dependent kinase inhibitor that is undergoing clinical trials, on primary cancer cells isolated from the ascites or pleural fluids of patients with metastatic cancers. EXPERIMENTAL DESIGN: Metastasized cancer cells were isolated from the pleural fluids (n = 20) or ascites (n = 15) of patients, most of whom were refractory to chemotherapy. These primary cancer cells were used within 2 weeks of isolation without selecting for proliferative capacities. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide viability assay was used to characterize the response of these cancer cells to commonly used chemotherapeutic agents, and their response to flavopiridol was compared with rapidly dividing cultured cell lines. RESULTS: The primary cancer cells displayed phenotypes that were different from established cell lines; they had very low replication rates, dividing every 1 to 2 weeks, and underwent replicative senescence within five passages. These primary tumor cells retained their resistance to chemotherapeutic drugs exhibited by the respective patients but did not show cross-resistance to other agents. However, these cancer cells showed sensitivity to flavopiridol with an average LD50 of 50 nmol/L (range, 21.5-69 nmol/L), similar to the LD50 in established cell lines. Because senescent cells also showed similar sensitivity to flavopiridol, it suggests that the mechanism of action is not dependent on the activity of cyclin-dependent kinases that regulate the progression of the cell cycle. CONCLUSION: Using cancer cells isolated from the ascites or pleural fluids, this study shows the potential of flavopiridol against cancer cells that have developed resistance to conventional chemotherapeutic agents.

Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.

BACKGROUND: Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology. RESULTS: The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results. CONCLUSIONS: This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.

SPRR1B overexpression enhances entry of cells into the G0 phase of the cell cycle.

Many studies have established the role of SPRR1B during squamous differentiation of skin and respiratory epithelial cells. However, its role in nonsquamous cells is largely unknown. We reported that expression of SPRR1B in Chinese hamster ovary (CHO) cells is increased as they enter the G0 phase of the cell cycle. The purpose of this study was to further investigate the SPRR1B expression pattern in nonsquamous tumors and to study its role in these cells. Expression of SPRR1B was detected by Northern blotting in a higher percentage of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced compared with beryllium metal-induced rat lung adenocarcinomas. In situ hybridizations confirmed that SPRR1B is expressed in individual or clusters of cells of nonsquamous cells from mouse, rat, and human adenocarcinomas. The same pattern of expression was observed in adenocarcinomas formed in nude mice from cell lines established from adenocarcinomas. SPRR1B expression was downregulated in the cell lines derived from adenocarcinoma when cells were enriched in G0 at low confluence. Tetraploidy was induced in CHO, mouse, and human tumor cell lines by stably overexpressing SPRR1B, whereas control cells showed no change in ploidy. Inducible expression of this protein for shorter periods using the ecdyson system did not affect growth rate or the ploidy of CHO cells but accelerated entry into G0/G1 compared with controls. These findings indicate that SPRR1B is likely coupled primarily to signals responsible for withdrawal from the proliferative state rather than the final stages of cellular quiescence and that its overexpression for prolonged periods may disrupt the normal progression of mitosis.

Posterior mandibular residual ridge resorption in patients with conventional dentures and implant overdentures.

PURPOSE: This study investigated the effects of certain systemic and local factors on resorption of the posterior mandibular residual ridge under conventional dentures and overdentures supported by 2 implants. MATERIALS AND METHODS: Proportional area measurements of the posterior mandible were made on rotational tomograms taken immediately before and 5 years after treatment. The area was bounded by a line joining gonion to the lowest point of the mental foramen and the crest of the residual ridge and was expressed as a proportion of an area that was not dependent on the ridge. The use of proportions rather than actual measurements minimized errors related to magnification and distortion. RESULTS: The estimated average reduction in height was 1.25 mm in 5 years (1.63 mm for conventional denture groups and 0.69 mm for implant overdenture groups, ie, almost 1 mm less in the overdenture group). DISCUSSION AND CONCLUSION: Female gender was a risk factor for greater resorption. Other factors, such as the number of years a patient had been edentulous, initial height of the mandible, and the number of dentures used, failed to show an association with resorption of the residual posterior mandibular ridge, while the statistically significant effect of age was unlikely to be clinically significant.

Case report: removal of fractured endodontic posts with a sonic instrument.

Fracture of endodontic posts within the root canal system is one of the causes of failure to restore endodontically treated teeth. Various techniques, with varying degrees of success, have been proposed in the literature for the removal of fractured posts prior to re-restoring the tooth. This case report describes the use of a sonic device to dislodge and remove two fractured prefabricated metal endodontic posts from teeth UR1 and UL2. The reader is also introduced to a variety of post removal techniques available.

The effects of fixed and removable implant-stabilised prostheses on posterior mandibular residual ridge resorption.

This study investigated the change over time in the area of the posterior mandibular residual ridge in patients wearing either i) mandibular overdentures stabilised by two implants (Brånemark System; Nobel Biocare, Göteborg, Sweden) connected by a bar, or ii) mandibular fixed cantilever prostheses stabilised on five or six implants. Proportional measurements were made in order to compare the area of the residual ridge with an area of bone uninfluenced by resorption. Measurements were made by digitising tracings of panoramic radiographs that were taken shortly after implant insertion and up to seven years later. With the use of overdentures, the posterior bone area index reduced by a mean of 1.1% per annum, while a mean bone area index increase of 1.6% per annum was demonstrated in association with fixed prostheses. A multiple linear regression model was fitted to predict the change in posterior area from type of prosthesis, gender, age, years of edentulism and initial height of the mandible. The model was only significant for initial height of mandible (P = 0.04) and type of prosthesis (P = 0.0001). In conclusion, patients rehabilitated with implant-stabilised mandibular overdentures demonstrated low rates of posterior mandibular residual ridge resorption, while patients rehabilitated with implant-stabilised mandibular fixed cantilever prostheses demonstrated bone apposition in the same area.

Disinfection procedures: their effect on the dimensional accuracy and surface quality of irreversible hydrocolloid impression materials and gypsum casts.

OBJECTIVES: This study primarily investigated the effect of disinfection procedures (Perform and sodium hypochlorite) on the dimensional accuracy and surface quality of four irreversible hydrocolloid impression materials and the resultant gypsum casts. The antibacterial efficacy of the procedures was also studied. METHODS: Dimensional accuracy was determined from the mean percentage deviation of six measurements taken from casts made from disinfected impressions compared with corresponding measurements from the master model and controls. Statistical analysis of data was determined by analysis of variance. Surface quality was determined using a stainless steel test block in accordance with ISO 1563. RESULTS: The dimensional accuracy of the impression materials tested were of a comparable standard following disinfection. The surface quality of casts taken from Blueprint Cremix impressions were unaffected by the disinfection procedures. The remaining impression materials studied showed greater surface deterioration on casts following disinfection with sodium hypochlorite than immersion in Perform. All disinfection procedures selected proved appropriate for antibacterial purposes. SIGNIFICANCE: Individual analysis of impression materials is required to determine their suitability to a given disinfection protocol.