A number of studies reported that the MTS in vitro cytotoxicity assay is a convenient method for assessing cell viability. The main features found with this assay are its ease of use, accuracy and ...rapid indication of toxicity. It might well be a useful tool in human health risk assessment if it can be shown that this assay also has an acceptable sensitivity and specificity. This is of interest particularly when exposure to unknown chemical substances requires the rapid detection and evaluation of toxic effects. In this study, the cytotoxicity of 20 chemicals selected from the MEIC priority list was determined with the MTS assay. Since it could be shown that interactions between detection reagents and test chemicals might influence the results of this assay, preliminary experiments were carried out such that artifactual results due to test chemical interference could be excluded from this study. IC
50 (50% inhibitory concentration) were established for each test chemical in two human cell lines (F1-73 and HeLa) and later compared with published toxicity data of the same chemicals established with in vitro and in vivo toxicological test systems. Direct comparisons of the data showed a generally lower sensitivity of the MTS assay, which is influenced by biological test organisms, cell type and exposure time. In terms of the specificity of the MTS assay, the results showed a good correlation between data obtained with the MTS assay and published data. The lowest correlation was found when the MTS assay was compared with in vivo studies, however, this finding corresponds well with other published in vitro–in vivo correlations. The highest correlation was found when the MTS assay was compared with test systems using human cell lines or exposure times of 3–24 h. Since the sensitivity of the MTS assay might be increased using different cell types or by extended incubation, this assay is found to provide ideal features of a good measurement system that might also be used for on site toxicological assessments.
Calcium phosphate ceramics with different hydroxyapatite (HA) and tricalcium phosphate (TCP) ratios have different chemical properties. Does the difference in phase composition affect osteoblast ...behavior? In this study, osteoblasts were cultured on 4 kinds of calcium phosphate ceramics, i.e. pure (HA), HT1 (HA/TCP, 70/30), HT2 (HA/TCP, 35/65), and pure TCP. Cell proliferation of SaOS-2 cells together with bone-related genes’ mRNA expression and protein production in osteoblasts cultured on different calcium phosphate ceramics were detected at different time points. Data suggested that cell proliferation rate on TCP ceramics was lower than that on the other substrates tested. Generally, mRNA expressions for ostenectin and osteocalcin were similar among the four kinds of ceramics in most circumstances, whereas at six days, alkaline phosphatase mRNA expression was higher on HA and HT1 surfaces than on the other two materials. Collagen I mRNA expression was also affected by the phase composition of substrates. Osteocalcin and bone sialoprotein production in SaOS-2 cells was very similar no matter which ceramic surface the cells were grown upon. This study revealed that calcium phosphate ceramics substrate could support osteoblast growth and bone-related gene expression and its gene expression pattern explained the basis of the biocompatibility and bioactivity for calcium phosphate ceramics.
Human osteoblast-like cells SaOS-2 (ATCC HTB85) were seeded onto three kinds of hydroxyapatite (HA) ceramics sintered at different temperature (1200°C, 1000°C and 800°C). Scanning electron microscopy ...(SEM) was conducted to detect the surface microstructure. Cells were cultured on these substrates for 6 and 12 days and cell proliferation rate and mRNA expression for osteocalcin, osteonectin, type I collagen and alkaline phosphatase and protein production for osteocalcin, bone sialoprotein and osteonectin were detected with quantitative in situ hybridization and immunocytochemistry techniques. SEM revealed that crystal particle size was affected by sintering temperature. Result showed that cell proliferation rate on HA ceramics sintered at 1200°C was the highest. Osteonectin and type I collagen mRNA expression was not altered by sintering temperature. After 12 days in culture, bone sialoprotein, osteocalcin and osteonectin proteins levels were significantly (
p<0.05) higher when SaOS-2 cells were cultured on HA sintered at 1200°C, compared to the other two surfaces, suggesting that HA sintered at high temperature may be a better candidate for in vivo implantation. This result provides valuable information concerning the clinic application of HA ceramics sintered at different temperature.
Summary
Background Ascorbic acid has numerous essential and beneficial functions in normal and photoaged skin. Ionisation of ascorbic acid in aqueous topical formulations leads to oxidative ...degradation. Ascorbic acid in an anhydrous vehicle would inherently have greater stability.
Objective The objective of this study was to observe the effects of two anhydrous formulations containing microfine particles of ascorbic acid on neocollagenesis and cytokeratin production in ex vivo human skin.
Methods Vitamin C preparations were applied topically onto the surface of freshly excised human abdominal skin. Following an exposure time of 48 h with appropriate controls, skin discs were cut into sections, placed on slides and assessed using immunohistochemical (antibodies: collagen type I, III, cytokeratin) staining. Analysis was performed using microscopy and descriptive rating.
Results Both formulations resulted in increased production of collagen types I and III and cytokeratin.
Conclusion The application of anhydrous formulations containing microfine particles of ascorbic acid to ex vivo human skin in this study resulted in neocollagenesis and increased production of cytokeratin. This approach appears to enable biological effects of ascorbic acid in the skin using a vehicle which would provide it greater stability than an aqueous vehicle.
Thrombopoietin (TPO), the specific cytokine that regulates platelet production, is expressed in human bone marrow (BM), kidney, and liver. There appears to be no regulation of TPO in the kidney and ...liver, but TPO messenger RNA (mRNA) expression can be modulated in the stromal cells of the BM. In this study, we used primary human BM stromal cells as a model to study the regulation of TPO mRNA expression in response to various platelet alpha-granular proteins. We showed that platelet-derived growth factor (PDGF) BB and fibroblast growth factor (FGF) 2 stimulated TPO mRNA expression in both a dose-dependent and time-dependent manner. The addition of 50 ng/mL of PDGF and 20 ng/mL of FGF resulted in maximal induction of TPO mRNA expression in 4 hours. We also found that platelet factor 4 (PF4), thrombospondin (TSP), and transforming growth factor-beta (TGF-beta) are negative modulators of megakaryocytopoiesis. We observed suppression in TPO mRNA expression with 1 microg/mL of both PF4 and TSP and 50 ng/mL of TGF-beta, with maximal suppression occurring 4 hours after the addition of these proteins. Finally, the addition of whole-platelet lysate produced a dose-dependent inhibition of TPO expression. On the basis of these findings, we propose that the platelet alpha-granular proteins studied may regulate TPO gene expression in BM stromal cells by means of a feedback mechanism.
The molecular mechanisms responsible for metastasis are not fully understood. Recently, expression of the KAI1 gene on human chromosome 11p11.2 was found to be down-regulated in metastatic prostate ...cancer cell lines compared with normal human prostate, suggesting that KAI1 may be a metastasis suppressor gene. The aim of this study was to investigate whether there is reduced expression of KAI1 in late-stage bladder cancer. Sixty-six paraffin-embedded bladder tissue sections were analyzed for KAI1 mRNA by in situ hybridization. Nineteen of these were from patients with no histological evidence of bladder cancer, and 47 were from papillary transitional cell carcinomas (TCCs); of these, 16 were highly invasive. KAI1 mRNA was highly expressed in the specimens of normal bladder (11 of 11; 100%), inflammatory bladder (5 of 8; 63%), and noninvasive papillary TCCs of grades 1 and 2 (15 of 24; 63%), compared to grade 3 papillary TCCs (1 of 7; 14%) or invasive TCCs (1 of 16; 6%). The differences in expression between local and invasive disease were statistically significant (P </= 0.01, chi2 test). Our results suggest that down-regulation of KAI1 mRNA is significantly associated with invasive bladder cancer and that KAI1 may represent an invasion/metastasis suppressor gene in bladder cancer.
Cytotoxicity of Australian tea tree oil (oil of Melaleuca alternifolia) and its major oxygenated monoterpenes: terpinen-4-ol, 1,8-cineole and alpha-terpineol were investigated using the MTS ...(3-(4,5-dimethylthiazol-2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfophenyl) 2H-tetrazolium) assay at two exposure times: 4 and 24 h on five different human cell lines. These cell lines included: Hep G2, a hepatocellular carcinomic human cell line; HeLa, an epithelioid carcinomic cell line; MOLT-4, a human lymphoblastic leukaemic T-cell line; K-562, a human chronic myelogenous leukaemia cell line; and CTVR-1, an early B-cell line from the bone marrow cells of a patient with acute myeloid leukaemia. The overall rating for cytotoxicity of tea tree oil and its components was alpha-terpineoltea tree oilterpinen-4-ol1,8-cineole and with comparison with the controls used mercuric chloridetea tree oilaspirin. Antimicrobial activity (MICs) displayed a similar pattern where alpha-terpineolterpinen-4-oltea tree oil1,8-cineole. The IC50 (a concentration that causes a reduction by half of the activity of mitochondrial dehydrogenase) value of tea tree oil ranged from 0.02 g/L for the Hep G2 cell line to 2.8 g/L for the HeLa cell line
The importance of KAI1 to colorectal cancer (CRC) progression is unclear, with conflicting data regarding changes in KAI1 expression during tumour progression.
In situ hybridisation and ...immunohistochemistry, with a semiquantitative scoring system, were used to assess KAI1 mRNA and protein levels, respectively, in normal colon samples (43), and non-metastatic (24) or metastatic (33) primary cancers, and liver metastases (48).
There was significant loss of KAI1 mRNA and protein staining in non-metastatic primary tumours versus normal tissues (Bonferroni, p < 0.05) but levels recovered to near normal in metastatic primary tumours and liver metastases. Increased KAI1 expression significantly correlated with distant metastases (Mann-Whitney, p = 0.0001, mRNA; p = 0.033, protein), cancer-specific survival (Cox regression analysis p = 0.0002, mRNA; 0.0493, protein) and overall patient survival (p = 0.0001, mRNA). Multivariate analysis (Cox proportional hazards model) confirmed high KAI1 mRNA expression was an independent prognostic indicator of distant metastasis (p < 0.0001), cancer-specific survival (p < 0.0001) and overall patient survival (p < 0.0001).
These data indicate a complex relationship between KAI1 and progression of human CRC, in which expression is reduced in localised primary tumours, but regained in disease associated with metastasis.
