Hours Of Culture Research Articles

The application of fluorescence in situ hybridization in detecting chronic myeloid leukemia

To evaluate the clinical significance of the application of fluorescence in situ hybridization (FISH) in detecting chronic myeloid leukemia (CML). Chromosome preparation was made by using 24-hour culture. FISH technique using dual color dual fusion (DC-DF) BCR/ABL probe was performed in all 158 cases and R-banding was also employed for karyotyping in some patients. Among the 158 cases, 98 cases were Ph positive, of which 69 cases (70.4%) were typical FISH pattern (1R1G2F), the other 29 cases (29.6%) showed 12 different types of atypical FISH pattern. The most frequent atypical patterns found were 1R1G1F in 7 cases (7.1%), 2R1G1F in 5 cases (5.1%), 1R1G2F and 1R1G3F in 4 cases (4.1%), 2R2G1F in 3 cases (3.1%). Karyotype analysis on 18 CML cases with atypical FISH patterns demonstrated that the atypical FISH patterns were due to variant translocation in 3 cases; the additional third signal was because of a supernumerary Ph chromosome. The karyotyping results did not conform to FISH results in four cases suggesting the conceivable mistakes in karyotyping. The 1R1G1F signal pattern seen in 3 cases with classical t(9;22) resulted from the deletion of derivative chromosome 9. The 1R1G2F signal pattern detected in 40% to 64% of interphase cells of 3 cases without Ph chromosome by conventional cytogenetic analysis suggested a submicroscopic translocation. Three cases treated with Glivec or bone marrow transplantation showed normal karyotypes with a small amount of BCR/ABL positive cells by FISH detection. FISH technique is of great value for the diagnosis of CML and confirmation of variant translocation, occult Ph translocation, derivative chromosome 9 deletion, therapeutic effect of interferon and Glivec as well as detection of minimal residual disease after bone marrow transplantation.

Higher Placental Anti-Inflammatory IL-10 Cytokine Expression in HIV-1 Infected Women Receiving Longer Zidovudine Prophylaxis Associated with Nevirapine

Placental cytokine balance may be critical for the control of mother-to-child transmission (MTCT) of HIV. We assessed whether the type and duration of antiretrovirals used for prevention of HIV-1-MTCT modified the inflammatory cytokine profile. We investigated the levels of cytokine expression in the placentas of 61 HIV-1-infected women who received zidovudine (ZDV) plus single dose nevirapine (SD-NVP) or ZDV only for prevention of MTCT. Placentas of 38 HIV-1-uninfected women were included as controls. All placentas were obtained after vaginal delivery. Levels of mRNA and cytokine expression were quantified using real-time PCR and ELISA, respectively, in placental explants and 24-hour culture supernatants and analyzed in relation to the women's characteristics and the type and duration of antiretroviral prophylaxis. HIV-1-infected and uninfected women did not show any differences in the expression of placental cytokine secretion except for a trend toward lower TNF-alpha mRNA levels in HIV-1-infected women. Within the HIV-1-infected group, women who were exposed to a long duration of ZDV (>72 days) or received SD-NVP less than 5h prior to delivery, more frequently expressed detectable levels of IL-10 in their placentas (32% versus 7% (p = 0.01) and 32% versus 5% (p = 0.02), respectively). No infant was found to be HIV-1-infected. Our results showed a normalization of the placental cytokine balance in HIV-1-infected women receiving antiretroviral prophylaxis. Furthermore, the type and duration of antiretroviral prophylaxis have an impact on the placental anti-inflammatory IL-10 expression level, which may contribute to controlling HIV replication at the placental level, thus reducing MTCT of HIV-1.

Osteoblasts interact with MTA surfaces and express Runx2

Periradicular healing involves osteoblasts that are dependent on the Runt-related transcription factor 2 (Runx2). The purpose of this study was to determine if mineral trioxide aggregate (MTA) root-end filling materials support Runx2 expression in osteoblasts. Human alveolar bone cells were grown on alternative formulations of MTA. Cell-surface interactions were visualized by scanning electron microscopy. Gene expression was examined by reverse-transcription polymerase chain reaction and Western blot analysis. Cells attached to and spread out on MTA surfaces within 24 hours and formed a collagenous matrix overlay within 1 week of growth. Runx2 expression increased from low levels in the 24-hour cultures to an abundance during 2 weeks of growth and differentiation on MTA surfaces and on tissue culture plastic controls. The cells responded similarly to ProRoot, Tooth-Colored MTA, and MTA mixed with local anesthetic solution. Mineral trioxide aggregate materials support cell attachment and Runx2 expression in osteoblasts.

Cuscuta chinensisExtract Promotes Osteoblast Differentiation and Mineralization in Human Osteoblast-Like MG-63 Cells

The aim of the present study was to investigate whether the aqueous extract of To-Sa-Za (TSZ-AE), the seed of Cuscuta chinensis Lam., which is a traditional medicinal herb commonly used in Korea and other oriental countries, could induce osteogenic activity in human osteoblast-like MG-63 cells. TSZ-AE treatment mildly promoted the proliferation of MG-63 cells at doses of 500 and 1,000 microg/mL in the 24-hour culture period. Dose-dependent increases in alkaline phosphatase (ALP) activity and collagen synthesis were shown at 48 and 72 hours of incubation. The release of bone morphogenetic protein (BMP)-2 but not osteocalcin in the MG-63 cells was induced by TSZ-AE at 72 hours (100-1,000 microg/mL). In addition, TSZ-AE markedly increased mRNA expression of ALP, collagen, and BMP-2 in the MG-63 cells in a dose-dependent manner. Mineralization in the culture of MG-63 cells was significantly induced at 500 and 1,000 microg/mL TSZ-AE treatment. In conclusion, this study shows that TSZ-AE enhanced ALP activity, collagen synthesis, BMP-2 expression, and mineralization in MG-63 cells. These results strongly suggest that C. chinensis can play an important role in osteoblastic bone formation and may possibly lead to the development of bone-forming drugs.

Interventional effects of emodin on transforming growth factor-beta1/integrin-linked kinase signal way in interleukin-1β-induced transdifferen-tiation of rat tubular epithelial-myofibroblasts

To study the effects of transforming growth factor-beta1/integrin-linked kinase (TGF-beta1/ILK) signal way in interleukin-1beta (IL-1beta)-induced rat tubular epithelial-myofibroblast transdifferentiation (TEMT), and to investigate whether emodin inhibits IL-1beta-induced TEMT through the TGF-beta1/ILK signal way-dependent mechanism. Normal rat kidney epithelial cell line (NRK52E) was used in this study. NRK52E cells were divided into blank control group, emodin control group, IL-1beta-induced group, emodin-inhibited group, SB431542 (TGF-beta 1 type I receptor blocker)-inhibited group, emodin plus SB431542-inhibited group, emodin-pretreated group and emodin-reversed group. After 48-hour culture, morphological changes of the NRK52E cells were observed by an inverted phase contrast microscope. The expressions of alpha-smooth muscle actin (alpha-SMA) and E-cadherin were detected by two-color immunohistochemical staining, while the expressions of TGF-beta1 and ILK were detected by one-color immunohistochemical staining. We also performed the imaging analysis to quantitatively analyze the result of the immunohistochemical staining. The secretion of fibronectin (FN) was analyzed by enzyme-linked immunosorbent assay. Compared with the blank control group, IL-1beta might induce TEMT, which was showed in increasing expression of alpha-SMA, increasing secreting of FN and decreasing expression of E-cadherin, and at the same time the expressions of TGF-beta1 and ILK were enhanced (P<0.05). Emodin might inhibit all of those changes induced by IL-1beta (P<0.05). When TGF-beta1 signal way was intercepted, IL-1beta induced-TEMT was suppressed and the expression of ILK was decreased, however, there was no significant difference in expression of TGF-beta1 between the SB431542 group and the IL-1beta-induced group. Compared with emodin-inhibited group, emodin-pretreatment could not prevent IL-1beta induced-TEMT in a certain extent, but emodin could not revert IL-1beta-induced TEMT. Spearman correlation analysis showed that TGF-beta1 expression had positive correlation with expressions of alpha-SMA, FN, ILK and negative correlation with E-cadherin expression, and the expression of ILK was positively correlated with the expressions of alpha-SMA and FN and negatively correlated with E-cadherin expression. IL-1beta induces TEMT partly depending on TGF-beta1/ILK signal way, partly via which emodin inhibits the TEMT induced by IL-1beta.

Changes in population of S. enterica serotype Enteritidis in experimentally contaminated milk depending on temperature and acidity

Salmonella bacteria are among the most frequent causes of food poisoning, and milk and milk products are food items through which these bacteria can be transmitted. More recent data indicate that the infective dose of Salmonella is very small - up to 101/g/ml food. The preservation of Salmonella in milk depends on the acidity, temperature and the duration of storage. The objective of the work was to examine the survival of Salmonella in milk depending on the changes of the total number of bacteria, pH and temperature. In the study, 0.1 ml of 24-hour bouillon culture of S. enteritidis was inoculated in raw collective milk (104 cfu/ml) and in marketed pasteurized milk (102 cfu/ml) each. The examined milk samples were maintained at room temperature and at a temperature of 7oC. At the beginning of the examinations, after 24h, 48h, 72h, after 7, 14 and 21 days, decimal solutions were made from the milk, and, following incubation, the number of S. enteritidis was determined (Rambach agar), the total number of bacteria (base for total number of bacteria) by multiplying the number of grown colonies and the corresponding solution, and the milk pH was measured. There was a bigger reduction of S. enteritidis at room temperature because the pH value dropped more swiftly. The lowest pH at which S. enteritidis was isolated from experimentally contaminated milk was 3.88. The results of the examinations show that Salmonella can adapt to biochemical processes during milk acid fermentation of soft cheeses. No Salmonella bacteria were isolated from experimentally contaminated pasterurized milk at both maintenance temperatures after seven days of investigations.

Chromosome Aberrations in Peripheral Blood Lymphocytes of Croatian Hospital Staff Occupationally Exposed to Low Levels of Ionising Radiation

Medical staff is an occupational group exposed to different agents suspected to induce genetic damage. Among them ionising radiation is the most studied. Cytogenetic analysis of human chromosomes in peripheral lymphocytes allows direct detection of mutation in somatic cells. This study investigated the cytogenetic effects of low-level ionising x-radiation in 48-hour peripheral blood lymphocyte cultures sampled from 765 hospital staff occupationally exposed to several agents known or suspected to induce chromosome damage and compared them with 200 control subjects. The exposed subjects were divided in eight (8) groups according to their specialties and job titles. The exposed groups manifested an increase in all types of chromosome aberrations. Acentric fragments were the most frequent chromosome-type aberration. Dicentric chromosomes were statistically significant only in urologists/gynaecologists. Age and smoking significantly influenced the incidence of dicentrics in the exposed groups. The frequency of ring chromosomes was low in all exposed groups (range: 0-2), and none were found in the control group. These findings indicate the importance of periodic medical checkups of hospital staff occupationally exposed to low doses of ionising radiation. The purpose is to create an individual cytogenetic register, where changes could evidence individual risks.

Alcohol Withdrawal‐Induced Hippocampal Neurotoxicity In Vitro and Seizures In Vivo are Both Reduced by Memantine

The ethanol withdrawal (EWD) syndrome is typically treated using benzodiazepines such as diazepam. However there is concern that benzodiazepines may not prevent neurotoxicity associated with EWD. Antagonists of glutamate/N-Methyl-D-Aspartate receptors (NMDARs) such as MK801 have been shown to be effective against both EWD-induced neurotoxicity in vitro and seizures in vivo. However, most of these agents have adverse side effects. An exception is the moderate affinity NMDAR channel blocker memantine, used in Alzheimer's dementia. The present studies examined the ability of memantine to protect against EWD-related toxicity in vitro and seizures in vivo. Organotypic hippocampal slice cultures from neonatal rat pups were treated starting at 15 days in vitro with 100 mM ethanol for 10 days followed by a 24-hour EWD period. During the 24-hour EWD period cultures were treated with memantine (15 or 30 microM). MK801 (10 microM) was utilized as a positive control. For the in vivo studies, the ability of memantine (2, 5, 10, and 15 mg/kg) to reduce convulsions was analyzed in Swiss-Webster mice using the handling induced convulsion test paradigm. In vitro studies demonstrated that memantine is effective at blocking EWD-induced neurotoxicity. In vivo experiments showed that memantine also significantly reduced convulsions induced by EWD in mice. Memantine may be of therapeutic value during alcohol detoxification by virtue of its having neuroprotective effects in addition to anti-seizure activity. The potential role of memantine in treatment of alcoholism is deserving of further study.

Effects of simulated digests of Biota orientalis and a dietary nutraceutical on interleukin-1– induced inflammatory responses in cartilage explants

To test the hypothesis that simulated digests of Biota orientalis (BO) and a dietary nutraceutical (DN; composed of mussel, shark cartilage, abalone, and BO seed lipid extract) inhibit prostaglandin E2 (PGE2), nitric oxide (NO), and glycosaminoglycan (GAG) production in interleukin (IL)-1-stimulated cartilage explants. Cartilage tissue from 12 pigs. Articular cartilage explants were conditioned with a simulated digest of BO (BOsim) or DN (DNsim) at concentrations of 0, 0.06, or 0.18 mg/mL or indomethacin (INDOsim; 0 or 0.02 mg/mL) for 72 hours. Control explants received digest vehicle only. Explants were or were not stimulated with recombinant human-IL-1beta (10 or 0 ng/mL) during the final 48 hours of culture. Concentrations of PGE2, GAG, and NO in media samples (mPGE2,mGAG, and mNO concentrations, respectively) were analyzed, and explant tissue was stained fluorochromatically to determine chondrocyte viability. Treatment effects during the final 48-hour culture period were analyzed. IL-1 increased mPGE2, mGAG, and mNO concentrations in control explants without adversely affecting cell viability. Treatment with INDOsim blocked PGE2 production and increased mNO concentration in IL-1-stimulated and unstimulated explants and increased mGAG concentration in unstimulated explants. Treatment with DNsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration in IL-1-stimulated and unstimulated explants, reduced mNO concentration in IL-1-stimulated explants, and increased mNO concentration in unstimulated explants. Treatment with 0.18 mg of DNsim/mL increased cell viability in the presence of IL-1. In IL-1-stimulated explants, BOsim (0.06 and 0.18 mg/mL) reduced mPGE2 concentration, but 0.18 mg of BOsim/mL increased cell viability. Effects of IL-1 on cartilage explants in vitro were modulated by DNsim and BOsim.

An investigation of the initial attachment and orientation of osteoblast-like cells on laser grooved Ti-6Al-4V surfaces

This paper presents the results of an experimental study of the initial cell spreading and adhesion on longitudinally- and transversally-oriented micro-grooves produced by the laser irradiation of laser grooved Ti-6Al-4V surfaces. The initial spreading and orientations of human osteosarcoma (HOS) cells were observed and quantified after 15-min, 1-hour, 4-hour and 24-hour cell culture periods. Immuno-fluorescence staining of adhesion proteins (actin and vinculin) was then used to study the spreading and adhesion of HOS cells in 1 hour and 4 hour culture experiments. The initial cell adhesion was also quantified using enzymatic detachment tests. The results showed that cell spreading and adhesion were enhanced by longitudinally- and transversally-oriented micro-grooves. The effects, which increase with time, were not remarkable after 1 hour, but obvious after 4 hours. Contact guidance was found to promote cell adhesion due to the increase in interactions between the focal adhesions and the patterned extra-cellular matrix (ECM) proteins on the laser micro-grooved surfaces.

Alkylator-Induced DNA Damage Response in Multiple Myeloma (MM) Cells Is Amplified by Histone Deacetylase (HDAC) Inhibition, Resulting in Significant Synergism in Cell Death

One of the main mechanisms of action of HDAC inhibitors is the transcriptional reactivation of dormant tumor-suppressor genes through acetylation of histones, thereby inducing apoptosis. Treatment with HDACI has also been shown to induce chromatin destabilization in a transcription independent way. In the current study, we sought to determine whether HDAC inhibition induces DNA damage and amplifies alkylator-induced mitotic cell death in both melphalan sensitive- and resistant-MM cell lines (RPMI8226, 8226/LR5). The IC50 values of SNDX275, a class I HDACI agent, and melphalan on the 72-hour MTT assay were 268.05 nM and 245.94 nM in the RPMI8226, and 309.91 nM and 8657.46 nM in the 8226/LR5, respectively. When combined together at clinically attainable concentrations, the combination index by the Chou-Talalay method ranged from 0.27 to 0.75 for the RPMI8226 and from 0.33 to 0.7 for the 8226/LR5, indicating a powerful synergism. For elucidation of molecular mechanisms, MM1S and RPMI8226 cell lines were investigated for apoptosis, histone acetylation, cell cycle analysis, DNA double strand break and DNA damage response serially in 48-hour culture with SNDX-275 at 500 nM and melphalan at 10 μM, alone and in combination. Cleavage of PARP was seen following treatment with each SNDX275 and melphalan, but was highest at 48 hours with the combination of both. Apoptosis was associated with cleavage of caspases of 8, 3 and 9, which was most intense on combination. Melphalan amplified SNDX275-induced acetylation of H3. In cell cycle analysis by flow cytometry, SNDX275 caused an increase in G0-G1 and a decrease in S and G2-M. Cyclin D1, E2F-1 and p53 on western blot were not affected but expression of p21 increased. Melphalan arrested the cell cycle at G2, increased expression of p53 in the RPMI8226 and of p21 in the MM1S. The combination intensified the increase in p21 in both cell lines and in p53 only in the RPMI8226. Phosphorylation of H2AX, a marker of DNA double strand break, increased in a time dependent manner following each drug, along with an increase in phosphorylation of CHK1 and CHK2, indicative of initiation of DNA damage response. The increase in γH2AX and pCHK1 & 2, however, was considerably higher on combination than each drug alone. Furthermore, morphologic assessment of dead cells by the 48 hours of culture revealed a significant increase in mitotic catastrophe on combination in the MM1S: 0% on SNDX275 alone; 10% on melphalan alone; 43.4% on combination. The current study suggests that HDAC inhibition synergizes with melphalan in MM cells and that intensification of DNA damage is one of the mechanisms. Further studies are necessary to understand the role of HDAC inhibition for induction of mitotic catastrophe.

Rapamycin Causes Reversible Cell Cycle Slowing, Rather Than Cell Cycle Arrest, in Myeloid and Lymphoid Cell Lines and in Acute Leukemia Cells

The mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates cell survival and proliferation, is constitutively activated in diverse malignancies, and constitutive mTOR activation has been implicated in both malignant transformation and resistance to chemotherapy. The mTOR inhibitor rapamycin, which inhibits mTOR signaling and has immunosuppressive effects at nanomolar concentrations, also has antineoplastic activity, but its reported effects on cell cycle and on apoptosis have been variable. Several studies evaluated lymphoid and myeloid cell lines after 24 or 48 hours of exposure to rapamycin and reported cell cycle arrest in G1, while others reported apoptosis. Rapamycin is being incorporated into treatment regimens in acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), and other hematologic malignancies, and knowledge of its growth inhibitory and cytotoxic effects will be useful in designing combination regimens. To this end, the concentration-dependent effects of rapamycin on survival, proliferation, cell cycle and apoptosis of myeloid and lymphoid cell lines and acute leukemia cells were studied. Assays were performed at serial time points extending to 96 hours. Cells studied included HL60 myeloid and Jurkat T-lymphoblastic leukemia, 8226 myeloma and U937 histiocytic lymphoma cells and ML-1 myeloid leukemia cells, which have intact p53, as well as multidrug resistant HL60/VCR, HL60/ADR and 8226/MR20 cells overexpressing the ATP-binding cassette (ABC) proteins ABCB1, ABCC1 and ABCG2, respectively. Pretreatment blasts from 9 patients with AML and 4 with ALL were also studied. Cell survival was measured following 96-hour (cell lines) or 48-hour (patient samples) culture with rapamycin at picomolar to micromolar concentrations using the WST-1 colorimetric assay. Cells grown in suspension culture without rapamycin and with 1 nanomolar to 100 micromolar rapamycin were counted at serial time points. Cell cycle was analyzed by propidium iodide labeling and bromodeoxyuridine incorporation, cell division by carboxyfluorescein succinimidyl ester (CFSE) staining and apoptosis by Annexin V labeling, all measured by flow cytometry. Concentration-dependent effects of rapamycin on cell lines and acute leukemia cells in the WST-1 assay were biphasic, occurring at nanomolar and again at micromolar concentrations, with a plateau from approximately 10 nanomolar to 10 micromolar. At nanomolar concentrations rapamycin prolonged cell doubling times by 20–40%, as evidenced by CFSE staining at serial time points, but it did not cause cell cycle arrest. Examined at serial time points, cells continuously exposed to rapamycin at nanomolar concentrations did not accumulate in any single phase of the cell cycle, nor did they become apoptotic. Doubling times increased, resulting in decreased cell numbers at each time point in relation to control cultures without rapamycin. When cells cultured with rapamycin were transferred to rapamycin-free medium, doubling times reverted to those in rapamycin-free cultures, demonstrating that cell cycle slowing resulted from continuous exposure to rapamycin and was fully reversible in the absence of rapamycin. In contrast to rapamycin at nanomolar concentrations, rapamycin at micromolar concentrations caused apoptosis of cell lines and acute leukemia cells. Thus rapamycin at nanomolar concentrations increases the doubling time of myeloid and lymphoid cell lines and acute leukemia cells, while rapamycin at micromolar concentrations causes apoptosis. Rapamycin doses used in immunosuppressive regimens are well tolerated and yield nanomolar plasma concentrations. Our data suggest that continuous oral administration of rapamycin or its analogs at the doses used in immunosuppressive regimens may slow growth of leukemia cells and might have utility as maintenance therapy in the minimal residual disease setting, given the known lack of effect on normal cells. Higher doses of rapamycin are being studied in phase I clinical trials. Finally, increased understanding of rapamycin's effect on leukemia cell growth will help direct its incorporation into combination therapy.

Activator protein-1 mRNA expression, cell proliferation and collagen synthesis induced by tumor necrosis factor-α in cardiac fibroblasts from rats and the effect of Qiangxin Decoction

To investigate the function of activator protein-1 (AP-1) in proliferation and collagen synthesis of cardiac fibroblasts (CFs) in neonatal rats induced by tumor necrosis factor-alpha (TNF-alpha), and to explore the mechanism of Qiangxin Decoction (QXD), a compound traditional Chinese herbal medicine, in reversing cardiac fibrosis. CFs derived from neonatal rats were cultured with enzymatic dissociation, and fibrosis of the CFs was induced by TNF-alpha. The CFs were divided into normal control group, untreated group, 5% QXD-containing serum group, 10% QXD-containing serum group and 20% QXD-containing serum group. After 24-hour culture of QXD-containing serum, AP-1 mRNA was detected by real-time fluorescent quantitative polymerase chain reaction; proliferation and collagen synthesis of CFs were assayed by thiazolyl blue assay (MTT) and measured by hydroxyproline respectively, in order to determine the effect of different dosage decoction on the proliferation and collagen synthesis of CFs. After 24-hour stimulation of TNF-alpha in CFs, compared with those in normal control group, the expression of AP-1 mRNA and cardiac fibroblast proliferation and collagen synthesis increased significantly (P<0.05). However, Qiangxin Decoction could reduce the expression of AP-1 mRNA and decreased the proliferation and collagen synthesis of CFs significantly (P<0.05). Qiangxin Decoction can inhibit proliferation and collagen synthesis of CFs induced by TNF-alpha, and reverse cardiac fibrosis, which may be related to its down-regulation of the expression of AP-1.

Double Deficiency of Tetraspanins CD9 and CD81 Alters Cell Motility and Protease Production of Macrophages and Causes Chronic Obstructive Pulmonary Disease-like Phenotype in Mice

CD9 and CD81 are closely related tetraspanins that regulate cell motility and signaling by facilitating the organization of multimolecular membrane complexes, including integrins. We show that CD9 and CD81 are down-regulated in smoking-related inflammatory response of a macrophage line, RAW264.7. When functions of CD9 and CD81 were ablated with monoclonal antibody treatment, small interfering RNA transfection, or gene knock-out, macrophages were less motile and produced larger amounts of matrix metalloproteinase (MMP)-2 and MMP-9 than control cells in vitro. In line with this, CD9/CD81 double-knock-out mice spontaneously developed pulmonary emphysema, a major pathological component of chronic obstructive pulmonary disease (COPD). The mutant lung contained an increased number of alveolar macrophages with elevated activities of MMP-2 and MMP-9 and progressively displayed enlarged airspace and disruption of elastic fibers in the alveoli. Secretory cell metaplasia, a finding similar to goblet cell metaplasia in cigarette smokers, was also observed in the epithelium of terminal bronchioles. With aging, the double-knockout mice showed extrapulmonary phenotypes, including weight loss, kyphosis, and osteopenia. These results suggest that the tetraspanins CD9 and CD81 regulate cell motility and protease production of macrophages and that their dysfunction may underlie the progression of COPD.

Effects of curcumin on secretion of adiponectin and interleukin-6 in human adipose tissues: an in vitro study

To investigate the effects of different concentrations of curcumin on secretion of adiponectin (APN) and interleukin-6 (IL-6) in human adipose tissues cultivated in vitro. Seven male patients with kidney stones were admitted. Abdominal subcutaneous adipose tissue and perirenal adipose tissue were collected from the operating-patients, and were cultivated with different concentrations of curcumin (10 and 100 microg/ml) in vitro. The contents of APN and IL-6 in the culture medium of adipose tissue cells were measured by enzyme-linked immunosorbent assay (ELISA) after they were cultured for 6 and 24 hours. Compared with the blank control group, the content of APN in the adipose tissue culture medium was increased by 100 microg/ml curcumin (P<0.05) after 6-hour culture, and the content of IL-6 was significantly decreased by 100 microg/ml curcumin after 6- and 24-hour culture (P<0.05). 100 microg/ml curcumin can increase APN secretion and decrease IL-6 secretion in human adipose tissues cultivated in vitro.

Effect of eltrombopag on proliferation of solid and hematologic tumor cell lines in vitro

14628 Background: Eltrombopag is an oral platelet growth factor in clinical studies to treat thrombocytopenia. It increases platelet production by interacting with the transmembrane domain of the receptor, thereby inducing proliferation and differentiation of cells in the megakaryocytic lineage. TPOR is expressed on the surface of some leukemia and stem cells. The following experiments were conducted to evaluate the effects of eltrombopag on the proliferation of tumor cell lines in vitro. Methods: 10 solid tumor cell lines were examined: non-small cell lung carcinoma (NSCLC) cell lines (A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H322M, NCI-H460, NCI-H522) and the hepatocellular carcinoma cell line, HepG2. 13 cells lines from various AML, CML, and lymphoblastic leukemias included: U937, ML-2, OCI- AML2, OCI-AML3, RPMI-8226, HEL 92.1.7, HL-60, K562, SR, CCRF-CEM, MOLT-4, NOMO-1, OCI-M1. Proliferation was measured by 3H-thymidine incorporation and CellTiter-Glo in the hematologic and solid tumors, respectively. Doxorubicin (Dox) was used as + control in the solid tumors. Myeloid differentiation was assayed by flow cytometry and apoptosis by Annexin V. TPOR mRNA expression was determined by Taqman in 7 cell lines. Results: All solid tumor lines demonstrated decreased proliferation after 72-hour culture w/eltrombopag (IC50=5.7 to 14 ug/mL). However, 3 NSCLC lines were less-responsive to both Dox and eltrombopag. Eltrombopag did not increase proliferation of any of the hematologic cell lines and decreased the proliferation of 11 of the 13 hematologic cell lines, (IC50=5.6 to 15.4 ug/mL). Two cell lines, NOMO-1 and OCI-M1, demonstrated an IC50 >40 ug/mL. Further examination of K562, U937 and OCI-AML3 demonstrated a decrease in total cell number as well as decreased proliferation. Annexin V staining suggests that the cells die by a method other than apoptosis. No change in differentiation was observed after eltrombopag treatment. In the cell lines examined, no relationship between the expression of TPOR mRNA and proliferation IC50 was observed. Conclusion: Eltrombopag did not increase proliferation or differentiation of hematologic tumor cell lines. Eltrombopag caused a dose-dependent decrease in proliferation in most of the 23 solid and hematologic tumor cell lines tested. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration GlaxoSmithKline GlaxoSmithKline GlaxoSmithKline

Expression of c-met in human kidney fibroblasts induced by high glucose in vitro and the regulation of Radix Astragali

To investigate the effect of high glucose on the expression of c-met in human kidney fibroblasts in vitro, and to explore the regulation of Radix Astragali. A cell culture system of human kidney fibroblasts was developed in vitro. The human kidney fibroblasts were divided into normal control group, high glucose group and mannitol group. Expressions of c-met and transforming growth factor-beta1 (TGF-beta1) mRNAs were detected by reverse transcription polymerase chain reaction (RT-PCR) and the expressions of c-met protein were analyzed by Western blot method after 6-, 12-, 24-, 48- and 96-hour culture. The human kidney fibroblasts were also cultured with 10% Radix Astragali containing serum; the expressions of c-met mRNA and protein were detected after 24- and 48-hour culture. Compared with the normal control group, expression of c-met mRNA in the high glucose group was significantly increased after 12-hour culture (P<0.05), arriving at the peak after 24-hour culture (P<0.01). The level of TGF-beta1 mRNA was higher in the high glucose group than that in the normal control group after 24-hour culture (P<0.05), arriving at the peak after 96-hour culture (P<0.01). Forty-eight hours after treating with 10% Radix Astragali containing serum, the levels of c-met mRNA and protein in fibroblasts were increased, and were higher than those in the high glucose group (P<0.01, P<0.05). High glucose can induce the expressions of c-met mRNA and protein in earlier period, and then inhibit the expressions. Radix Astragali can up-regulate the expressions of c-met mRNA and protein of human kidney fibroblasts, which may be one of its action mechanisms in delaying the progression of diabetic nephropathy.

Integration of conventional cytogenetics, comparative genomic hybridisation and interphase fluorescence in situ hybridisation for the detection of genomic rearrangements in acute leukaemia

Aims:To screen for genomic imbalances in patients with acute leukaemia using conventional (G-banding) and molecular (comparative genomic hybridisation (CGH) and fluorescence in situ hybridisation (FISH)) methods to determine whether an...

Nucleophosmin ( NPM1) Mutations in Acute Myeloid Leukemia: An Ongoing (Cytoplasmic) Tale of Dueling Mutations and Duality of Molecular Genetic Testing Methodologies

This Commentary highlights two articles that focus on molecular techniques to identify mutations in nucleophosmin (NPM1), which is the most frequently mutated gene in cytogenetically normal acute myeloid leukemia (CN-AML)

Neurturin suppresses injury‐induced neuronal activating transcription factor 3 expression in cultured guinea pig cardiac ganglia

Cultured guinea pig atrial whole mounts containing the intrinsic cardiac ganglia were used as an in vitro model to investigate the induction of the stress/injury marker activating transcription factor 3 (ATF-3). ATF-3 expression was quantified by using immunocytochemical labeling and real-time PCR. In freshly isolated ganglia, no neuronal or Schwann cell nuclei exhibited ATF-3 immunoreactivity. In 2-hour cultures, the induction of ATF-3 expression was evident in many Schwann cell nuclei, whereas no neuronal nuclei were ATF-3 immunoreactive. Beginning at 4 hours, the percentage of neurons with ATF-3-immunoreactive nuclei increased progressively, and, by 48 hours in culture, approximately 95% of the cardiac neurons had ATF-3-immunoreactive nuclei. Neurturin significantly suppressed ATF-3 expression in 48-hour-cultured neurons without effect on ATF-3 expression in Schwann cell nuclei. Neuturin also could reverse neuronal ATF-3 expression after its induction. The suppression of ATF-3 induction by neurturin was mediated by activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Glial-derived neurotrophic factor (GDNF) also suppressed neuronal ATF-3 induction during culture. However, culture in serum-free media, presence of nerve growth factor, or addition of pituitary adenylate cyclase-activating polypeptide had no effect on ATF-3 induction in the 48-hour-cultured cardiac neurons. By 4 hours in culture, there was a significant increase in ATF-3 transcript levels, and neurturin partially suppressed ATF-3 transcript levels in 48-hour cultures. It is proposed that the loss of target-derived neurturin is a potential mechanism stimulating injury-induced expression of ATF-3 in cardiac neurons.