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High occurrence of in vitro apoptosis of lymphocytes induced by serum from systemic lupus erythematosus patients is associated with increased serum levels of anti-C1q autoantibodies

Hasan SI, Mohd Ashari NS, Mohd Daud K, Che Husin CM

Int J Rheum Dis, 2013 Aug;16(4):430-6.
PMID: 23992264 DOI: 10.1111/1756-185X.12062

BACKGROUND: The ethiopathogenesis of increased apoptosis of lymphocytes in systemic lupus erythematosus (SLE) is still incompletely understood but anti-C1q autoantibodies have been shown to induce apoptosis in lymphocytes from healthy donors and certain cell lines.
AIM: This study was undertaken to investigate the relationship between peripheral lymphocyte apoptosis and serum levels of anti-C1q autoantibodies in SLE patients.
METHODS: The sera of 124 patients with SLE involving 62 active SLE and 62 inactive SLE, fulfilling America College of Rheumatology (ACR) classification criteria for SLE (1997) were incubated with peripheral blood lymphocytes of healthy donors. The results were compared with 124 sex- and age-matched normal controls. Apoptotic lymphocytes (AL) were detected by flow cytometry using annexin V and propidium iodide binding. Anti-C1q autoantibodies were detected by an enzyme-linked immunoassay kit for all SLE patients.
RESULTS: Results demonstrated that the percentage of AL in the peripheral blood of active SLE patients was significantly higher (n = 62, 34.95 ± 12.78%) than that of the inactive SLE patients (n = 62, 30.69 ± 10.13%, P = 0.042, 95%CI = 0.16-8.36) and normal controls (n = 124, 27.92 ± 10.22%, P = 0.001, 95%CI = 3.33-10.73). The percentage of AL significantly correlated with serum levels of anti-C1q autoantibodies in the active SLE patients (r = 0.263, P = 0.039) but not in the inactive SLE patients (r = 0.170, P = 0.185).
CONCLUSION: The results of this study suggest that increased serum levels of anti-C1q autoantibodies are responsible for apoptosis and may play a pathogenic role in SLE patients, especially in active disease.
KEYWORDS: anti-C1q; apoptosis; flowcytometry; systemic lupus erythematosus
Study site: Medical outpatient clinic and medical wards, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia

Matched MeSH terms: Cells, Cultured
Fulltext Cancer-associated fibroblasts promote proliferation of endometrial cancer cells

Subramaniam KS, Tham ST, Mohamed Z, Woo YL, Mat Adenan NA, Chung I

PLoS One, 2013;8(7):e68923.
PMID: 23922669 DOI: 10.1371/journal.pone.0068923

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.

Matched MeSH terms: Tumor Cells, Cultured
Suppressive effect of maslinic acid on PMA-induced protein kinase C in human B-lymphoblastoid cells

Mooi LY, Yew WT, Hsum YW, Soo KK, Hoon LS, Chieng YC

Asian Pac J Cancer Prev, 2012;13(4):1177-82.
PMID: 22799301

Protein kinase C (PKC) has been implicated in carcinogenesis and displays variable expression profiles during cancer progression. Studies of dietary phytochemicals on cancer signalling pathway regulation have been conducted to search for potent signalling regulatory agents. The present study was designed to evaluate any suppressive effect of maslinic acid on PKC expression in human B-lymphoblastoid cells (Raji cells), and to identify the PKC isoforms expressed. Effects of maslinic acid on PKC activity were determined using a PepTag assay for non-radioactive detection of PKC. The highest expression in Raji cells was obtained at 20 nM PMA induced for 6 hours. Suppressive effects of maslinic acid were compared with those of four PKC inhibitors (H- 7, rottlerin, sphingosine, staurosporine) and two triterpenes (oleanolic acid and ursolic acid). The IC₅₀ values achieved for maslinic acid, staurosporine, H-7, sphingosine, rottlerin, ursolic acid and oleanolic acid were 11.52, 0.011, 0.767, 2.45, 5.46, 27.93 and 39.29 μM, respectively. Four PKC isoforms, PKC βI, βII, δ, and ζ, were identified in Raji cells via western blotting. Maslinic acid suppressed the expression of PKC βI, δ, and ζ in a concentration-dependent manner. These preliminary results suggest promising suppressive effects of maslinic acid on PKC activity in Raji cells. Maslinic acid could be a potent cancer chemopreventive agent that may be involved in regulating many downstream signalling pathways that are activated through PKC receptors.

Matched MeSH terms: Cells, Cultured
Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

Zawawi MS, Dharmapatni AA, Cantley MD, McHugh KP, Haynes DR, Crotti TN

Biochem Biophys Res Commun, 2012 Oct 19;427(2):404-9.
PMID: 23000414 DOI: 10.1016/j.bbrc.2012.09.077

Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway in osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcRγ) and DNAX-activating protein 12kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin (β3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p<0.05) reduced the expression of NFATc1, CathK, OSCAR, FcRγ, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p<0.05) decreased CathK, OSCAR, FcRγ, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.

Matched MeSH terms: Cells, Cultured
Fulltext Growth inhibition of human gynecologic and colon cancer cells by Phyllanthus watsonii through apoptosis induction

Ramasamy S, Abdul Wahab N, Zainal Abidin N, Manickam S, Zakaria Z

PLoS One, 2012;7(4):e34793.
PMID: 22536331 DOI: 10.1371/journal.pone.0034793

Phyllanthus watsonii Airy Shaw is an endemic plant found in Peninsular Malaysia. Although there are numerous reports on the anti cancer properties of other Phyllanthus species, published information on the cytotoxicity of P. watsonii are very limited. The present study was carried out with bioassay-guided fractionation approach to evaluate the cytotoxicity and apoptosis induction capability of the P. watsonii extracts and fractions on human gynecologic (SKOV-3 and Ca Ski) and colon (HT-29) cancer cells. P. watsonii extracts exhibited strong cytotoxicity on all the cancer cells studied with IC(50) values of ≤ 20.0 µg/mL. Hexane extract of P. watsonii was further subjected to bioassay-guided fractionation and yielded 10 fractions (PW-1→PW-10). PW-4→PW-8 portrayed stronger cytotoxic activity and was further subjected to bioassay-guided fractionation and resulted with 8 sub-fractions (PPWH-1→PPWH-8). PPWH-7 possessed greatest cytotoxicity (IC(50) values ranged from 0.66-0.83 µg/mL) and was selective on the cancer cells studied. LC-MS/MS analysis of PPWH-7 revealed the presence of ellagic acid, geranic acid, glochidone, betulin, phyllanthin and sterol glucoside. Marked morphological changes, ladder-like appearance of DNA and increment in caspase-3 activity indicating apoptosis were clearly observed in both human gynecologic and colon cancer cells treated with P. watsonii especially with PPWH-7. The study also indicated that P. watsonii extracts arrested cell cycle at different growth phases in SKOV-3, Ca Ski and HT-29 cells. Cytotoxic and apoptotic potential of the endemic P. watsonii was investigated for the first time by bioassay-guided approach. These results demonstrated that P. watsonii selectively inhibits the growth of SKOV-3, Ca Ski and HT-29 cells through apoptosis induction and cell cycle modulation. Hence, P. watsonii has the potential to be further exploited for the discovery and development of new anti cancer drugs.

Matched MeSH terms: Cells, Cultured
Fulltext The modulation of PPARγ1 and PPARγ2 mRNA expression by ciglitazone in CD3/CD28-activated naïve and memory CD4+ T cells

Norazmi MN, Mohamed R, Nurul AA, Yaacob NS

Clin. Dev. Immunol., 2012;2012:849195.
PMID: 22548115 DOI: 10.1155/2012/849195

Given their roles in immune regulation, the expression of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) 1 and 2 isoforms was investigated in human naïve (CD45RA+) and memory (CD45RO+) CD4+ T cells. Stimulation of both types of cells via the CD3/CD28 pathway resulted in high expression of both PPARγ receptors as measured by real-time PCR. Treatment with the PPARγ agonist, ciglitazone, increased PPARγ1 expression but decreased PPARγ2 expression in stimulated naïve and memory cells. Furthermore, when present, the magnitude of both PPARγ receptors expression was lower in naïve cells, perhaps suggesting a lower regulatory control of these cells. Similar profiles of selected proinflammatory cytokines were expressed by the two cell types following stimulation. The induction of PPARγ1 and suppression of PPARγ2 expressions in naïve and memory CD4+ T cells in the presence of ciglitazone suggest that the PPARγ subtypes may have different roles in the regulation of T-cell function.

Matched MeSH terms: Cells, Cultured
Fulltext Piper sarmentosum inhibits ICAM-1 and Nox4 gene expression in oxidative stress-induced human umbilical vein endothelial cells

Ugusman A, Zakaria Z, Hui CK, Nordin NA

BMC Complement Altern Med, 2011;11:31.
PMID: 21496279 DOI: 10.1186/1472-6882-11-31

Aqueous extract of Piper sarmentosum (AEPS) is known to possess antioxidant and anti-atherosclerotic activities but the mechanism responsible for it remains unclear. In early part of atherosclerosis, nuclear factor-kappa B (NF-κB) induces the expression of cellular adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1) and E-selectin. NADPH oxidase 4 (Nox4) is the predominant source of superoxide in the endothelial cells whereas superoxide dismutase 1 (SOD1), catalase (CAT) and glutathione peroxidase (GPx) are the antioxidant enzymes responsible for inactivating reactive oxygen species. The present study aimed to investigate the effects of AEPS on the gene expression of NF-κB, VCAM-1, ICAM-1, E-selectin, Nox4, SOD1, CAT and GPx in cultured human umbilical vein endothelial cells (HUVECs).

Matched MeSH terms: Cells, Cultured
Expansion of human articular chondrocytes and formation of tissue-engineered cartilage: a step towards exploring a potential use of matrix-induced cell therapy

Munirah S, Samsudin OC, Aminuddin BS, Ruszymah BH

Tissue Cell, 2010 Oct;42(5):282-92.
PMID: 20810142 DOI: 10.1016/j.tice.2010.07.002

Monolayer culture expansion remains as a fundamental step to acquire sufficient number of cells for 3D constructs formation. It has been well-documented that cell expansion is however accompanied by cellular dedifferentiation. In order to promote cell growth and circumvent cellular dedifferentiation, we evaluated the effects of Transforming Growth Factor Beta-2 (TGF-β2), Insulin-like Growth Factor-I (IGF-I) and basic Fibroblast Growth Factor (bFGF) combination on articular chondrocytes culture and 'chondrocytes-fibrin' construct formation. Chondrocytes were serially cultured in: (1) F12:DMEM+10% Foetal Bovine Serum (FBS) with growth factors (FD10GFs), (2) F12:DMEM+2%FBS with the growth factors (FD2GFs) and, (3) F12:DMEM+10%FBS without growth factors (FD) as control. Cultured chondrocytes were evaluated by means of growth kinetics parameters, cell cycle analysis, quantitative phenotypic expression of collagen type II, aggrecan core protein sox-9 and collagen type I and, immunochemistry technique. Harvested chondrocytes were incorporated with plasma-derived fibrin and were polymerized to form the 3D constructs and implanted subcutaneously at the dorsum of athymic nude mice for eight (8) weeks. Resulted constructs were assigned for gross inspections and microscopic evaluation using standard histochemicals staining, immunochemistry technique and, quantitative phenotypic expression of cartilage markers to reassure cartilaginous tissue formation. Growth kinetics performance of chondrocytes cultured in three (3) types of culture media from the most to least was in the following order: FD10GFs>FD2GFs>FD. Following growth kinetics analysis, we decided to use FD10GFs and FD (control) for further evaluation and 'chondrocytes-fibrin' constructs formation. Chondrocytes cultured in FD10GFs preserved the normal diploid state (2c) with no evidence of aneuploidy, haploidy or tetraploidy. Expression of cartilage-specific markers namely collagen type II, aggrecan core protein and sox-9 were significantly higher in FD10GFs when compared to control. After implantation, 'chondrocytes-fibrin' constructs exhibited firm, white, smooth and glistening cartilage-like properties. FD10GFs constructs formed better quality cartilage-like tissue than FD constructs in term of overall cartilaginous tissue formation, cells organization and extracellular matrix distribution in the specimens. Cartilaginous tissue formation was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan was confirmed by positive Safranin O staining. Collagen type II exhibited immunopositivity at the pericellular and inter-territorial matrix area. Chondrogenic properties of the construct were further confirmed by the expression of genes encoding collagen type II, aggrecan core protein and sox9. In conclusion, FD10GFs promotes the proliferation of chondrocytes and formation of good quality 'chondrocytes-fibrin' constructs which may have potential use of matrix-induced cell implantation.

Matched MeSH terms: Cells, Cultured
Fulltext Goniothalamin induces coronary artery smooth muscle cells apoptosis: the p53-dependent caspase-2 activation pathway

Chan KM, Rajab NF, Siegel D, Din LB, Ross D, Inayat-Hussain SH

Toxicol. Sci., 2010 Aug;116(2):533-48.
PMID: 20498002 DOI: 10.1093/toxsci/kfq151

Goniothalamin (GN), a styryl-lactone isolated from Goniothalamus andersonii, has been demonstrated to possess antirestenostic properties by inducing apoptosis on coronary artery smooth muscle cells (CASMCs). In this study, the molecular mechanisms of GN-induced CASMCs apoptosis were further elucidated. Apoptosis assessment based on the externalization of phosphatidylserine demonstrated that GN induces CASMCs apoptosis in a concentration-dependent manner. The GN-induced DNA damage occurred with concomitant elevation of p53 as early as 2 h, demonstrating an upstream signal for apoptosis. However, the p53 elevation in GN-treated CASMCs was independent of NAD(P)H: quinone oxidoreductase 1 and Mdm-2 expression. An increase in hydrogen peroxide and reduction in free thiols confirmed the role for oxidative stress in GN treatment. Pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-FMK) that significantly abrogated GN-induced CASMCs apoptosis suggested the involvement of caspase(s). The role of apical caspase-2, -8, and -9 was then investigated, and sequential activation of caspase-2 and -9 but not caspase-8 leading to downstream caspase-3 cleavage was observed in GN-treated CASMCs. Reduction of ATP level and decrease in oxygen consumption further confirmed the role of mitochondria in GN-induced apoptosis in CASMCs. The mitochondrial release of cytochrome c was seen without mitochondrial membrane potential loss and was independent of cardiolipin. These data provide insight into the mechanisms of GN-induced apoptosis, which may have important implications in the development of drug-eluting stents.

Matched MeSH terms: Cells, Cultured
Apoptotic effects of Physalis minima L. chloroform extract in human breast carcinoma T-47D cells mediated by c-myc-, p53-, and caspase-3-dependent pathways

Ooi KL, Tengku Muhammad TS, Lim CH, Sulaiman SF

Integr Cancer Ther, 2010 Mar;9(1):73-83.
PMID: 20150224 DOI: 10.1177/1534735409356443

The chloroform extract of Physalis minima produced a significant growth inhibition against human T-47D breast carcinoma cells as compared with other extracts with an EC(50) value of 3.8 microg/mL. An analysis of cell death mechanisms indicated that the extract elicited an apoptotic cell death. mRNA expression analysis revealed the coregulation of apoptotic genes, that is, c-myc , p53, and caspase-3. The c-myc was significantly induced by the chloroform extract at the earlier phase of treatment, followed by p53 and caspase-3. Biochemical assay and ultrastructural observation displayed typical apoptotic features in the treated cells, including DNA fragmentation, blebbing and convolution of cell membrane, clumping and margination of chromatin, and production of membrane-bound apoptotic bodies. The presence of different stages of apoptotic cell death and phosphatidylserine externalization were further reconfirmed by annexin V and propidium iodide staining. Thus, the results from this study strongly suggest that the chloroform extract of P. minima induced apoptotic cell death via p53-, caspase-3-, and c-myc-dependent pathways.

Matched MeSH terms: Tumor Cells, Cultured
Cryopreserved mesenchymal stromal cell treatment is safe and feasible for severe dilated ischemic cardiomyopathy

Chin SP, Poey AC, Wong CY, Chang SK, Teh W, Mohr TJ, et al.

Cytotherapy, 2010;12(1):31-7.
PMID: 19878080 DOI: 10.3109/14653240903313966

Bone marrow (BM) mesenchymal stromal cells (MSC) represent a novel therapy for severe heart failure with extensive myocardial scarring, especially when performed concurrently with conventional revascularization. However, stem cells are difficult to transport in culture media without risk of contamination, infection and reduced viability. We tested the feasibility and safety of off-site MSC culture and expansion with freeze-controlled cryopreservation and subsequent rapid thawing of cells immediately prior to implantation to treat severe dilated ischemic cardiomyopathy.

Matched MeSH terms: Cells, Cultured
Modification of palm oil for anti-inflammatory nutraceutical properties

Zainal Z, Longman AJ, Hurst S, Duggan K, Hughes CE, Caterson B, et al.

Lipids, 2009 Jul;44(7):581-92.
PMID: 19449050 DOI: 10.1007/s11745-009-3304-8

Palm oil is one of the most important edible oils in the world. Its composition (rich in palmitate and oleate) make it suitable for general food uses but its utility could be increased if its fatty acid quality could be varied. In this study, we have modified a palm olein fraction by transesterification with the n-3 polyunsaturated fatty acids, alpha-linolenate or eicosapentaenoic acid (EPA). Evaluation of the potential nutritional efficacy of the oils was made using chondrocyte culture systems which can be used to mimic many of the degenerative and inflammatory pathways involved in arthritis. On stimulation of such cultures with interleukin-1alpha, they showed increased expression of cyclooxygenase-2, the inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha), IL-1alpha and IL-1beta and the proteinase ADAMTS-4. This increased expression was not affected by challenge of the cultures with palm olein alone but showed concentration-dependent reduction by the modified oil in a manner similar to EPA. These results show clearly that it is possible to modify palm oil conveniently to produce a nutraceutical with effective anti-inflammatory properties.

Matched MeSH terms: Cells, Cultured
Protective effect of different parts of Cassia fistula on human umbilical vein endothelial cells against glycated protein-induced toxicity in vitro

Einstein JW, Mustafa MR, Nishigaki I, Rajkapoor B, Moh MA

Methods Find Exp Clin Pharmacol, 2008 Oct;30(8):599-605.
PMID: 19088944 DOI: 10.1358/mf.2008.30.8.1268401

The protective effect of methanol extracts of Cassia fistula (flowers, leaves and bark) was examined in vitro in human umbilical vein endothelial cells (HUVEC) against toxicity induced by glycated protein (GFBS) in vitro. The experiments consisted of eight groups of HUVEC with five flasks in each group. Group I was treated with 15% FBS, group II with GFBS (70 microM) alone, and the other six groups were treated with GFBS plus 25 and 50 microg of each of the three types of C. fistula extracts. After 72 h of incubation, cells were collected and tested for lipid peroxidation, antioxidant enzyme activities and glutathione S-transferase (GST). The protective effect of C. fistula extracts against GFBS-induced cytotoxicity was examined in HUVEC by using trypan blue exclusion and MTT assays. Results showed that HUVEC incubated with GFBS alone showed a significant (P < 0.001) elevation of lipid peroxidation accompanied by depletion of superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR), in addition to decreased cytosolic GST. Treatment of HUVEC with C. fistula extracts at a concentration of 25 and 50 microg significantly decreased lipid peroxidation and normalized the activities of the antioxidant enzymes and GST levels in a concentration-dependent manner. Morphological changes of HUVEC were compared with respective controls; in addition, the C. fistula extracts increased the viability of HUVEC damaged by GFBS. A protective effect of C. fistula extracts on HUVEC against GFBS-induced toxicity suggested a potential beneficial effect of the extract in preventing diabetic angiopathies.

Matched MeSH terms: Cells, Cultured
Upregulation of SOX-2, FZD9, Nestin, OCT-4 and FGF-4 expression in human chorion derived-stem cells after angiogenic induction

Abdul Rahman H, Manzor NF, Tan GC, Tan AE, Chua KH

Med J Malaysia, 2008 Jul;63 Suppl A:57-8.
PMID: 19024982

Angiogenic induction was made to promote angiogenesis by differentiating stem cells towards endothelial cells. However, the stemness property of induced cells has not been revealed yet. Hence, we aim to evaluate the differential mRNA expression of stemness genes in human chorion-derived stem cells (CDSC) after being cultured in EDM50 comprised bFGF and VEGF. Results indicated that CDSC cultured in EMD50 expressed significantly higher mRNA level of Sox-2, FZD9, BST-1 and Nestin. In addition Oct-4, FGF-4 and ABCG-2 were also upregulated. Our finding suggested that CDSC after angiogenic induction enhanced its stem cell properties. This could be contributed for the mechanism of stem cell therapy in ischemic problem.

Matched MeSH terms: Cells, Cultured
The oxidative stress of hyperglycemia and the inflammatory process in endothelial cells

Muniandy S, Qvist R, Yan GO, Bee CJ, Chu YK, Rayappan AV

J. Med. Invest., 2009 Feb;56(1-2):6-10.
PMID: 19262007

Hyperglycemia and insulin resistance are common in many critically ill patients. Hyperglycemia increases the production of reactive oxygen species in cells, stimulates the production of the potent proinflammatory cytokines IL-8 and TNF-alpha, and enhances the expression of haem oxygenase-1, an inducible stress protein. It has been shown that administration of insulin and the semi-essential amino acid glutamine have been beneficial to the septic patient. The aim of our study is to test whether these two molecules, glutamine and insulin used in combination attenuate the proinflammatory responses in endothelial cells which have been triggered by hyperglycaemia. Our results demonstrate that a combination of insulin and glutamine are significantly more effective in reducing the expression of IL-8, TNF-alpha and HO-1 than insulin or glutamine alone.

Matched MeSH terms: Cells, Cultured
Human nasal turbinates as a viable source of respiratory epithelial cells using co-culture system versus dispase-dissociation technique

Noruddin NA, Saim AB, Chua KH, Idrus R

Laryngoscope, 2007 Dec;117(12):2139-45.
PMID: 17891046

OBJECTIVE: To compare a co-culture system with a conventional dispase-dissociation method for obtaining functional human respiratory epithelial cells from the nasal turbinates for tissue engineering application.
METHODS: Human respiratory epithelial cells were serially passaged using a co-culture system and a conventional dispase-dissociation technique. The growth kinetics and gene expression levels of the cultured respiratory epithelial cells were compared. Four genes were investigated, namely cytokeratin-18, a marker for ciliated and secretory epithelial cells; cytokeratin-14, a marker for basal epithelial cells; MKI67, a proliferation marker; and MUC5B, a marker for mucin secretion. Immunocytochemical analysis was performed using monoclonal antibodies against the high molecular-weight cytokeratin 34 beta E12, cytokeratin 18, and MUC5A to investigate the protein expression from cultured respiratory epithelial cells.
RESULTS: Respiratory epithelial cells cultured using both methods maintained polygonal morphology throughout the passages. At passage 1, co-cultured respiratory epithelial showed a 2.6-times higher growth rate compared to conventional dispase dissociation technique, and 7.8 times higher at passage 2. Better basal gene expression was observed by co-cultured respiratory epithelial cells compared to dispase dissociated cells. Immunocytochemical analyses were positive for the respiratory epithelial cells cultured using both techniques.
CONCLUSION: Co-culture system produced superior quality of cultured human respiratory epithelial cells from the nasal turbinates as compared to dispase dissociation technique.

Matched MeSH terms: Cells, Cultured
Interleukin-6 inhibits human peroxisome proliferator activated receptor alpha gene expression via CCAAT/enhancer-binding proteins in hepatocytes

Chew CH, Chew GS, Najimudin N, Tengku-Muhammad TS

Int J Biochem Cell Biol, 2007;39(10):1975-86.
PMID: 17616429

Peroxisome proliferator activated receptor alpha has been implicated as a regulator of acute phase response genes in hepatocytes. Interleukin-6 is widely known as a major cytokine responsible in the regulation of acute phase proteins and, therefore, acute phase response. Unfortunately, to date, very little is understood about the molecular mechanisms by which interleukin-6 regulates the gene expression of peroxisome proliferator activated receptor alpha. Here, we report the molecular mechanisms by which peroxisome proliferator activated receptor alpha was regulated by interleukin-6 in human HepG2 cells. Interleukin-6 was shown to down-regulate the peroxisome proliferator activated receptor alpha gene expression at the level of gene transcription. Functional dissection of human peroxisome proliferator activated receptor alpha promoter B revealed the role of predicted CCAAT/enhancer-binding protein binding site (-164/+34) in mediating the interleukin-6 inhibitory effects on peroxisome proliferator activated receptor alpha mRNA expression and electrophoretic mobility shift assay showed the binding of CCAAT/enhancer-binding protein isoforms to this cis-acting elements was increased in interleukin-6-treated HepG2 cells. Co-transfection experiments, then, demonstrated that CCAAT/enhancer-binding protein beta either in homodimer or heterodimer with CCAAT/enhancer-binding protein alpha and CCAAT/enhancer-binding protein delta plays a predominant role in inhibiting the transcriptional activity of peroxisome proliferator activated receptor alpha promoter B, thus, reducing the peroxisome proliferator activated receptor alpha mRNA expression. These studies, therefore, suggest a novel mechanism for interleukin-6-mediated inhibition of peroxisome proliferator activated receptor alpha gene expression that involves the activation of CCAAT/enhancer-binding protein isoforms with CCAAT/enhancer-binding protein beta may play a major role.

Matched MeSH terms: Tumor Cells, Cultured
Cord blood CD34+ cells cultured with FLT3L, stem cell factor, interleukin-6, and IL-3 produce CD11c+CD1a-/c- myeloid dendritic cells

Fadilah SA, Vuckovic S, Khalil D, Hart DN

Stem Cells Dev, 2007 Oct;16(5):849-55.
PMID: 17999605

Methods that allow expansion of myeloid dendritic cells (MDCs) from CD34(+) cells are potentially important for boosting anti-leukemic responses after cord blood (CB) hematopoietic stem cell transplantation (HSCT). We showed that the combination of early-acting cytokines FLT3-ligand (FL), stem cell factor (SCF), interleukin (IL)-3, and IL-6 supported the generation of CD11c(+)CD16() CD1a()/c() MDCs from CB CD34(+) cells or CB myeloid precursors. Early-acting cytokine-derived MDCs were maintained within the myeloid CD33(+)CD14()CD15() precursors with a mean of 4 x 10(6) cells generated from 1-4 x 10(4) CB CD34(+) cells or myeloid precursors after 2 weeks. After 8-12 days of culture the MDCs expressed higher levels of HLA-DR antigen but lower levels of CD40 and CD86 antigen, compared to adult blood MDCs. At this stage of differentiation, the early-acting cytokine-derived MDCs had acquired the ability to induce greater allogeneic T cell proliferation than monocytes or granulocytes derived from same culture. Early-acting cytokine-derived MDCs exposed to the cytokine cocktail (CC) comprising IL-1beta, IL-6, tumor necrosis factor (TNF)-alpha, and prostaglandin E (PGE)-2, upregulated the surface co-stimulatory molecules CD40 and CD86 and enhanced allogeneic T cell proliferation, as is characteristic of MDCs maturation. The reliable production of MDCs from CB CD34(+) cells provides a novel way to study their lineage commitment pathway(s) and also a potential means of enriching CB with MDCs to improve prospects for DC immunotherapy following CB HSCT.

Matched MeSH terms: Cells, Cultured
Fulltext Establishment of an in vitro system representing the chicken gut-associated lymphoid tissue

Alitheen NB, McClure SJ, Yeap SK, Kristeen-Teo YW, Tan SW, McCullagh P

PLoS One, 2012;7(11):e49188.
PMID: 23185307 DOI: 10.1371/journal.pone.0049188

The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a(+), a majority of those emigrating onto the supporting membrane were Bu-1a(-) and IgM(+). Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.

Matched MeSH terms: Cells, Cultured
Keloid pathogenesis via Drosophila similar to mothers against decapentaplegic (SMAD) signaling in a primary epithelial-mesenchymal in vitro model treated with biomedical-grade chitosan porous skin regenerating template

Lim CK, Halim AS, Yaacob NS, Zainol I, Noorsal K

J Biosci Bioeng, 2013 Apr;115(4):453-8.
PMID: 23177217 DOI: 10.1016/j.jbiosc.2012.10.010

The effects of locally produced chitosan (CPSRT-NC-bicarbonate) in the intervention of keloid pathogenesis were investigated in vitro. A human keratinocyte-fibroblast co-culture model was established to investigate the protein levels of human collagen type-I, III and V in a western blotting analysis, the secreted transforming growth factor-β1 (TGF-β1) in an enzyme-linked immunosorbent assay (ELISA) and the mRNA levels of TGF-β1's intracellular signaling molecules (SMAD2, 3, 4 and 7) in a real-time PCR analysis. Keratinocyte-fibroblast co-cultures were maintained in DKSFM:DMEM:F12 (2:2:1) medium. Collagen type-I was found to be the dominant form in primary normal human dermal fibroblast (pNHDF) co-cultures, whereas collagen type-III was more abundant in primary keloid-derived human dermal fibroblast (pKHDF) co-cultures. Collagen type-V was present as a minor component in the skin. TGF-β1, SMAD2 and SMAD4 were expressed more in the pKHDF than the pNHDF co-cultures. Co-cultures with normal keratinocytes suppressed collagen type-III, SMAD2, SMAD4 and TGF-β1 expressions and CPSRT-NC-bicarbonate enhanced this effect. In conclusion, the CPSRT-NC-bicarbonate in association with normal-derived keratinocytes demonstrated an ability to reduce TGF-β1, SMAD2 and SMAD4 expressions in keloid-derived fibroblast cultures, which may be useful in keloid intervention.

Matched MeSH terms: Cells, Cultured

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