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1.  The Possible Potential Therapeutic Targets for Drug Induced Gingival Overgrowth 
Mediators of Inflammation  2013;2013:639468.
Gingival overgrowth is a side effect of certain medications. The most fibrotic drug-induced lesions develop in response to therapy with phenytoin, the least fibrotic lesions are caused by cyclosporin A, and the intermediate fibrosis occurs in nifedipine-induced gingival overgrowth. Fibrosis is one of the largest groups of diseases for which there is no therapy but is believed to occur because of a persistent tissue repair program. During connective tissue repair, activated gingival fibroblasts synthesize and remodel newly created extracellular matrix. Proteins such as transforming growth factor (TGF), endothelin-1 (ET-1), angiotensin II (Ang II), connective tissue growth factor (CCN2/CTGF), insulin-like growth factor (IGF), and platelet-derived growth factor (PDGF) appear to act in a network that contributes to the development of gingival fibrosis. Since inflammation is the prerequisite for gingival overgrowth, mast cells and its protease enzymes also play a vital role in the pathogenesis of gingival fibrosis. Drugs targeting these proteins are currently under consideration as antifibrotic treatments. This review summarizes recent observations concerning the contribution of TGF-β, CTGF, IGF, PDGF, ET-1, Ang II, and mast cell chymase and tryptase enzymes to fibroblast activation in gingival fibrosis and the potential utility of agents blocking these proteins in affecting the outcome of drug-induced gingival overgrowth.
doi:10.1155/2013/639468
PMCID: PMC3652200  PMID: 23690667
2.  Influence of Mast Cells in Drug-Induced Gingival Overgrowth 
Mediators of Inflammation  2013;2013:275172.
Mast cells (MCs) are multifunctional effector cells that were originally thought to be involved in allergic disorders. Now it is known that they contain an array of mediators with a multitude of effects on many other cells. MCs have become a recent concern in drug-induced gingival overgrowth (DIGO), an unwanted outcome of systemic medication. Most of the studies have confirmed the significant presence of inflammation as a prerequisite for the overgrowth to occur. The inflammatory changes within the gingival tissue appear to influence the interaction between the inducing drug and the fibroblast activity. The development of antibodies to MC-specific enzymes, tryptase and chymase, has facilitated the study of mast cells in DIGO. Many immunohistochemical studies involving MCs have been conducted; as a result, DIGO tissues are found to have increased the number of MCs in the gingiva, especially in the area of fibrosis. At the cellular level, gingival fibrogenesis is initiated by several mediators which induce the recruitment of a large number of inflammatory cells, including MCs. The purpose of this paper is to access the roles played by MCs in gingival overgrowth to hypothesize a relationship between these highly specialized cells in the pathogenesis of DIGO.
doi:10.1155/2013/275172
PMCID: PMC3569901  PMID: 23431239
3.  Hepatoprotective Activity of Elephantopus scaber on Alcohol-Induced Liver Damage in Mice 
Elephantopus scaber has been traditionally used as liver tonic. However, the protective effect of E. scaber on ethanol-induced liver damage is still unclear. In this study, we have compared the in vivo hepatoprotective effect of E. scaber with Phyllanthus niruri on the ethanol-induced liver damage in mice. The total phenolic and total flavanoid content of E. scaber ethanol extract were determined in this study. Accelerating serum biochemical profiles (including AST, ALT, ALP, triglyceride, and total bilirubin) associated with fat drop and necrotic body in the liver section were observed in the mice treated with ethanol. Low concentration of E. scaber was able to reduce serum biochemical profiles and the fat accumulation in the liver. Furthermore, high concentration of E. scaber and positive control P. niruri were able to revert the liver damage, which is comparable to the normal control. Added to this, E. scaber did not possess any oral acute toxicity on mice. These results suggest the potential effect of this extract as a hepatoprotective agent towards-ethanol induced liver damage without any oral acute toxicity effect. These activities might be contributed, or at least in part, by its high total phenolic and flavonoid contents.
doi:10.1155/2012/417953
PMCID: PMC3437691  PMID: 22973401
4.  Crude ethyl acetate extract of marine microalga, Chaetoceros calcitrans, induces Apoptosis in MDA-MB-231 breast cancer cells 
Pharmacognosy Magazine  2014;10(37):1-8.
Background:
Marine brown diatom Chaetoceros calcitrans and green microalga Nannochloropsis oculata are beneficial materials for various applications in the food, nutraceutical, pharmaceutical and cosmeceutical industries.
Objective:
This study investigated cytotoxicity of different crude solvent extracts from C. calcitrans and N. oculata against various cancer cell lines.
Materials and Methods:
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was carried out to screen the cytotoxic effects of hexane (Hex), dichloromethane (DCM), ethyl acetate, and methanol extract from C. calcitrans and N. oculata toward various cancer cell lines. Flow cytometry cell cycle was used to determine the cell cycle arrest while the mode of cell death was investigated through acridine orange/propidium iodide (AOPI) staining, Annexin V-Fluorescein Isothiocyanate (FITC) and Terminal deoxynucleotidyl transferase-mediated d-UTP Nick End Labeling (TUNEL) assays. Expression profile of apoptotic and proliferative-related genes was then determined using the multiplex gene expression profiler (GeXP).
Results:
Crude ethyl acetate (CEA) extract of C. calcitrans inhibited growth of MDA-MB-231 cells, with IC50 of 60 μg/mL after 72 h of treatment. Further studies were conducted to determine the mode of cell death at various concentrations of this extract: 30, 60 and 120 μg/mL. The mode of cell death was mainly apoptosis as shown through apoptosis determination test. The expression data from GeXP showed that caspase-4 was upregulated while B-cell leukemia/lymphoma 2(Bcl-2) was down regulated. Thus, caspase-4 induction endoplasmic reticulum death pathway is believed to be one of the mechanisms underlying the induction of apoptosis while Bcl-2 induced S and G2/M cell cycle phase arrest in MDA-MB-231 cells.
Conclusion:
CEA extract of C. calcitrans showed the highest cytotoxicity on MDA-MB-231 via apoptosis.
doi:10.4103/0973-1296.126650
PMCID: PMC3969653
Apoptosis; Chaetoceros calcitrans; crude ethyl acetate extract; gene expression profiler; MDA-MB-231
5.  Hepatoprotective Effect of Fermented Soybean (Nutrient Enriched Soybean Tempeh) against Alcohol-Induced Liver Damage in Mice 
Recently, soybean tempeh has received great attention due to many advantages such as higher nutritional value, lower production cost, and shorter fermentation time. In this study, the in vivo hepatoprotective and antioxidant effects of nutrient enriched soybean tempeh (NESTE) were determined. NESTE fermentation process which involved anaerobic incubation was previously proclaimed to increase the content of amino acids and antioxidant properties remarkably. The evaluation of histological sections, serum biochemical markers (aspartate aminotransferase (AST), alanine aminotransferase (ALT), and cholesterol and triglycerides (TG)), liver immune response level (nitric oxide (NO)) and liver antioxidant level (superoxide dismutase (SOD), ferric reducing antioxidant power (FRAP), and malondialdehyde (MDA)) was conducted in order to compare the effects of nonfermented soybean extract (SBE) and fermented soybean extract (NESTE) on alcohol-induced liver damage in mice. Results demonstrated that 1000 mg/kg of NESTE can significantly reduce the levels of AST, ALT, cholesterol, TG, MDA, and NO. On the other hand, it also raised the level of SOD and FRAP. Furthermore, the histological examination on 1000 mg/kg NESTE treatment group showed that this extract was capable of recovering the damaged hepatocytes to their normal structures. Thus, it can be concluded that NESTE produced through fermentation process was able to enhance hepatoprotective and antioxidant effects in vivo.
doi:10.1155/2013/274274
PMCID: PMC3766609  PMID: 24058369
6.  Rhaphidophora korthalsii modulates peripheral blood natural killer cell proliferation, cytokine secretion and cytotoxicity 
Background
Rhaphidophora korthalsii (Araceae) is a root-climber plant which has been widely used in Chinese traditional medicine for cancer and skin disease treatment. Previous reports have recorded its immunomodulatory effects on mice splenocyte and human peripheral blood. This study investigated the potential immunostimulatory effect of Rhaphidophora korthalsii on human PBMC enriched NK cell.
Methods
PBMC was exposed to various concentrations of R. korthalsii extract and the T and NK cell population in the control and extract treated PBMC were identified by immunophenotyping. Intracellular perforin and granzyme B expressions were detected by flow cytometry and extra-cellular Granzyme B, IFN-γ and TNF-α production in the isolated NK cells were determined by ELISA. The cytotoxicity of effector NK cell towards target K562 cell was assessed by CytoTox 96 assay.
Results
Rhaphidophora korthalsii methanol extract significantly increased PBMC NK cell population and intracellular perforin and granzyme B expressions. Moreover, the extract also enhanced the secretion of IFN-γ and TNF-α which subsequently enhanced the cytotoxicity of NK cell against the NK sensitive target K562 cell line. NK cell enriched with extract treated PBMC showed better activation than NK cell directly treated with the extract.
Conclusion
Our findings indicated a potential IL-2 free immunotherapy through direct and indirect R. korthalsii stimulation on NK cell activation.
doi:10.1186/1472-6882-13-145
PMCID: PMC3701493  PMID: 23800124
Rhaphidophora korthalsii; NK cell; Immunomodulation
7.  In Vivo Immunomodulation and Lipid Peroxidation Activities Contributed to Chemoprevention Effects of Fermented Mung Bean against Breast Cancer 
Mung bean has been reported to have antioxidant, cytotoxic, and immunomodulatory effects in vitro. Fermented products are reported to have enhanced immunomodulation and cancer chemopreventive effects. In this study, fermented mung bean treatments in vivo were studied by monitoring tumor development, spleen immunity, serum cytokine (interleukin 2 and interferon gamma) levels, and spleen/tumor antioxidant levels after injection with low and high risk 4T1 breast cancer cells. Pretreatment with fermented mung bean was associated with delayed tumor formation in low risk mice. Furthermore, this treatment was connected with higher serum anticancer cytokine levels, spleen T cell populations, splenocyte cytotoxicity, and spleen/tumor antioxidant levels. Histopathological evaluation of fermented mung bean treated tumor revealed lower event of mitotic division. On the other hand, antioxidant and nitric oxide levels that were significantly increased in the untreated mice were inhibited in the fermented mung bean treated groups. These results suggested that fermented mung bean has potential cancer chemoprevention effects through the stimulation of immunity, lipid peroxidation, and anti-inflammation.
doi:10.1155/2013/708464
PMCID: PMC3654717  PMID: 23710232
8.  Display of the Viral Epitopes on Lactococcus lactis: A Model for Food Grade Vaccine against EV71 
In this study, we have developed a system for display of antigens of Enterovirus type 71 (EV71) on the cell surface of L. lactis. The viral capsid protein (VP1) gene from a local viral isolate was utilized as the candidate vaccine for the development of oral live vaccines against EV71 using L. lactis as a carrier. We expressed fusion proteins in E. coli and purified fusion proteins were incubated with L. lactis. We confirmed that mice orally fed with L. lactis displaying these fusion proteins on its surface were able to mount an immune response against the epitopes of EV71. This is the first example of an EV71 antigen displayed on the surface of a food grade organism and opens a new perspective for alternative vaccine strategies against the EV71. We believe that the method of protein docking utilized in this study will allow for more flexible presentations of short peptides and proteins on the surface of L. lactis to be useful as a delivery vehicle.
doi:10.1155/2013/431315
PMCID: PMC3586462  PMID: 23476790
9.  Cytotoxic Effect of Ethanol Extract of Microalga, Chaetoceros calcitrans, and Its Mechanisms in Inducing Apoptosis in Human Breast Cancer Cell Line 
BioMed Research International  2012;2013:783690.
Marine microalgae have been prominently featured in cancer research. Here, we examined cytotoxic effect and apoptosis mechanism of crude ethanol extracts of an indigenous microalga, Chaetoceros calcitrans (UPMAAHU10) on human breast cell lines. MCF-7 was more sensitive than MCF-10A with IC50 value of 3.00 ± 0.65, whilst the IC50 value of Tamoxifen against MCF-7 was 12.00 ± 0.52 μg/mL after 24 hour incubation. Based on Annexin V/Propidium iodide and cell cycle flow cytometry analysis, it was found that inhibition of cell growth by EEC on MCF-7 cells was through the induction of apoptosis without cell cycle arrest. The apoptotic cells at subG0/G1 phase in treated MCF-7 cells at 48 and 72 hours showed 34 and 16 folds increased compared to extract treated MCF-10A cells which showed only 6 and 7 folds increased at the same time points, respectively. Based on GeXP study, EEC induced apoptosis on MCF-7 cells via modulation of CDK2, MDM2, p21Cip1, Cyclin A2, Bax and Bcl-2. The EEC treated MCF-7 cells also showed an increase in Bax/Bcl-2 ratio that in turn activated the caspase-dependent pathways by activating caspase 7. Thus, marine microalga, Chaetoceros calcitrans may be considered a good candidate to be developed as a new anti-breast cancer drug.
doi:10.1155/2013/783690
PMCID: PMC3591159  PMID: 23509778
10.  Antioxidant and Hepatoprotective Effect of Aqueous Extract of Germinated and Fermented Mung Bean on Ethanol-Mediated Liver Damage 
BioMed Research International  2012;2013:693613.
Mung bean is a hepatoprotective agent in dietary supplements. Fermentation and germination processes are well recognized to enhance the nutritional values especially the concentration of active compounds such as amino acids and GABA of various foods. In this study, antioxidant and hepatoprotective effects of freeze-dried mung bean and amino-acid- and GABA-enriched germinated and fermented mung bean aqueous extracts were compared. Liver superoxide dismutase (SOD), malondialdehyde (MDA), ferric reducing antioxidant power (FRAP), nitric oxide (NO) levels, and serum biochemical profile such as aspartate transaminase (AST), alanine transaminase (ALT), triglycerides (TG), and cholesterol and histopathological changes were examined for the antioxidant and hepatoprotective effects of these treatments. Germinated and fermented mung bean have recorded an increase of 27.9 and 7.3 times of GABA and 8.7 and 13.2 times of amino acid improvement, respectively, as compared to normal mung bean. Besides, improvement of antioxidant levels, serum markers, and NO level associated with better histopathological evaluation indicated that these extracts could promote effective recovery from hepatocyte damage. These results suggested that freeze-dried, germinated, and fermented mung bean aqueous extracts enriched with amino acids and GABA possessed better hepatoprotective effect as compared to normal mung bean.
doi:10.1155/2013/693613
PMCID: PMC3591205  PMID: 23484140
11.  Establishment of an In Vitro System Representing the Chicken Gut-Associated Lymphoid Tissue 
PLoS ONE  2012;7(11):e49188.
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.
doi:10.1371/journal.pone.0049188
PMCID: PMC3501491  PMID: 23185307
12.  Antihyperglycemic Effects of Fermented and Nonfermented Mung Bean Extracts on Alloxan-Induced-Diabetic Mice 
Mung bean was reported as a potential antidiabetic agent while fermented food has been proposed as one of the major contributors that can reduce the risk of diabetes in Asian populations. In this study, we have compared the normoglycemic effect, glucose-induced hyperglycemic effect, and alloxan-induced hyperglycemic effect of fermented and nonfermented mung bean extracts. Our results showed that fermented mung bean extracts did not induce hypoglycemic effect on normal mice but significantly reduced the blood sugar levels of glucose- and alloxan-induced hyperglycemic mice. The serum levels of cholesterol, triglyceride (TG), and low-density lipoprotein (LDL) were also lowered while insulin secretion and antioxidant level as measured by malonaldehyde (MDA) assays were significantly improved in the plasma of the fermented mung bean-treated group in alloxan-induced hyperglycemic mouse. These results indicated that fermentation using Mardi Rhizopus sp. strain 5351 inoculums could enhance the antihyperglycemic and the antioxidant effects of mung bean in alloxan-treated mice. The improvement in the antihyperglycemic effect may also be contributed by the increased content of GABA and the free amino acid that are present in the fermented mung bean extracts.
doi:10.1155/2012/285430
PMCID: PMC3469204  PMID: 23091343
13.  Betulinic Acid Inhibits Growth of Cultured Vascular Smooth Muscle Cells In Vitro by Inducing G1 Arrest and Apoptosis 
Betulinic acid is a widely available plant-derived triterpene which is reported to possess selective cytotoxic activity against cancer cells of neuroectodermal origin and leukemia. However, the potential of betulinic acid as an antiproliferative and cytotoxic agent on vascular smooth muscle (VSMC) is still unclear. This study was carried out to demonstrate the antiproliferative and cytotoxic effect of betulinic acid on VSMCs using 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry cell cycle assay, BrdU proliferation assay, acridine orange/propidium iodide staining, and comet assay. Result from MTT and BrdU assays indicated that betulinic acid was able to inhibit the growth and proliferation of VSMCs in a dose-dependent manner with IC50 of 3.8 μg/mL significantly (P < 0.05). Nevertheless, betulinic acid exhibited G1 cell cycle arrest in flow cytometry cell cycle profiling and low level of DNA damage against VSMC in acridine orange/propidium iodide and comet assay after 24 h of treatment. In conclusion, betulinic acid induced G1 cell cycle arrest and dose-dependent DNA damage on VSMC.
doi:10.1155/2012/251362
PMCID: PMC3463985  PMID: 23056140
14.  In vitro evaluation of Pandanus amaryllifolius ethanol extract for induction of cell death on non-hormone dependent human breast adenocarcinoma MDA-MB-231 cell via apoptosis 
Background
Our previous study had shown that P. amaryllifolius was able to selectively inhibit cell proliferation of hormone independent breast cancer cell line MDA-MB-231. To understand the mode of killing and mechanism of action for P. amaryllifolius, the ethanol extract was evaluated for their alteration of cell cycle progression, PS externalization, DNA fragmentation and expression of anti/pro-apoptotic related protein.
Results
Cell cycle progression analysis, Annexin V and Tunel assays suggested that IC50 of P. amaryllifolius ethanol extract induced G0/G1 cell cycle arrest, PS externalization and DNA fragmentation. On the other hand, ELISA for cytochrome c, caspase-3/7, 8 and 9 indicated that apoptosis was contributed by mitochondrial cytochrome c release via induction of caspase 3/7, 9, and p53 was associated with the suppression of XIAP in P. amaryllifolius treated MDA-MB-231 cells.
Conclusion
Our findings suggest that P. amaryllifolius ethanol extract induced apoptosis on hormone independent breast cancer cell line MDA-MB-231.
doi:10.1186/1472-6882-12-134
PMCID: PMC3479033  PMID: 22909149
Pandanus amaryllifolius; MDA-MB-231; Apoptosis; Cytochrome c; Caspase
15.  In Vitro and In Vivo Toxicity of Garcinia or Hydroxycitric Acid: A Review 
Obesity is one of the pandemic chronic diseases commonly associated with health disorders such as heart attack, high blood pressure, diabetes or even cancer. Among the current natural products for obesity and weight control, Garcinia or more specifically hydroxycitric acid (HCA) extracted from Garcinia has been widely used. The evaluation of the potential toxicity of weight control supplement is of the utmost importance as it requires long term continuous consumption in order to maintain its effects. Majority of reports demonstrated the efficacy of Garcinia/HCA without any toxicity found. However, a few clinical toxicity reports on weight-loss diet supplements of which some were combinations that included Garcinia/HCA as an active ingredient showed potential toxicity towards spermatogenesis. Nonetheless, it cannot be concluded that Garcinia/HCA is unsafe. Those products which have been reported to possess adverse effects are either polyherbal or multi-component in nature. To date, there is no case study or report showing the direct adverse effect of HCA. The structure, mechanism of action, long history of the use of Garcinia/HCA and comprehensive scientific evidence had shown “no observed adverse effect level (NOAEL)” at levels up to 2800 mg/day, suggesting its safety for use.
doi:10.1155/2012/197920
PMCID: PMC3424601  PMID: 22924054
16.  Co-administration of avian influenza virus H5 plasmid DNA with chicken IL-15 and IL-18 enhanced chickens immune responses 
Background
DNA vaccines offer several advantages over conventional vaccines in the development of effective vaccines against avian influenza virus (AIV). However, one of the limitations of the DNA vaccine in poultry is that it induces poor immune responses. In this study, chicken interleukin (IL) -15 and IL-18 were used as genetic adjuvants to improve the immune responses induced from the H5 DNA vaccination in chickens. The immunogenicity of the recombinant plasmid DNA was analyzed based on the antibody production, T cell responses and cytokine production, following inoculation in 1-day-old (Trial 1) and 14-day-old (Trial 2) specific-pathogen-free chickens. Hence, the purpose of the present study was to explore the role of chicken IL-15 and IL-18 as adjuvants following the vaccination of chickens with the H5 DNA vaccine.
Results
The overall HI antibody titer in chickens immunized with pDis/H5 + pDis/IL-15 was higher compared to chickens immunized with pDis/H5 (p < 0.05). The findings revealed that the inoculation of the 14-day-old chickens exhibited a shorter time to achieve the highest HI titer in comparison to the inoculation of the 1-day-old chickens. The cellular immunity was assessed by the flow cytometry analysis to enumerate CD4+ and CD8 + T cells in the peripheral blood. The chickens inoculated with pDis/H5 + pDis/IL-15 demonstrated the highest increase in CD4+ T cells population relative to the control chickens. However, this study revealed that pDis/H5 + pDis/IL-15 was not significant (P > 0.05) in inducing CD8+ T cells. Meanwhile, with the exception of Trial 1, the flow cytometry results for Trial 2 demonstrated that the pDis/H5 + pDis/IL-18 inoculated group was able to trigger a higher increase in CD4+ T cells than the pDis/H5 group (P < 0.05). On the other hand, the pDis/H5 + pDis/IL-18 group was not significant (P > 0.05) in modulating CD8+ T cells population in both trials. The pDis/H5 + pDis/IL-15 inoculated group showed the highest IL-15 gene expression in both trials compared to other inoculated groups (P < 0.05). Similar results were obtained for the IL-18 expression where the pDis/H5 + pDis/IL-18 groups in both trials (Table 8) were significantly higher compared to the control group (P < 0.05). However, the expressions of other cytokines remained low or undetected by GeXP assay.
Conclusions
This study shows the diverse immunogenicity of pDis/H5 co-administered with chicken IL-15 and IL-18,with pDis/H5 + pDis/IL-15 being a better vaccine candidate compared to other groups.
doi:10.1186/1746-6148-8-132
PMCID: PMC3511295  PMID: 22866758
Avian influenza virus; DNA vaccine; H5; IL-15; IL-18; CD4+ T cells; CD8+ T cells
17.  In vitro cytotoxicity of Strobilanthes crispus ethanol extract on hormone dependent human breast adenocarcinoma MCF-7 cell 
Background
Strobilanthes crispus has been traditionally used as antidiabetic, anticancer, diuretic, antilytic and laxative agent. However, cytotoxicity and antiproliferative effect of S. crispus is still unclear.
Results
Strobilanthes cripus was able to reduce cell viability and proliferation in MTT and BrdU assays. Both cell cycle progression and Tunel assay suggested that IC50 of S. crispus ethanol extract induced sub-G1 cell cycle phase, and DNA fragmentation. On the other hand, translocation of mitochondria cytochrome c release, induction of caspase 3/7 and p53 while suppress XIAP on treated MCF-7 cell were also observed in this study.
Conclusion
Our findings suggest that S. crispus ethanol extract induced apoptosis and DNA fragmentation on hormone dependent breast cancer cell line MCF-7 via mitochondria dependent p53 apoptosis pathway.
doi:10.1186/1472-6882-12-35
PMCID: PMC3377542  PMID: 22471785
Strobilanthes crispus; MCF-7; Apoptosis; p53
18.  scFv Antibody: Principles and Clinical Application 
To date, generation of single-chain fragment variable (scFv) has become an established technique used to produce a completely functional antigen-binding fragment in bacterial systems. The advances in antibody engineering have now facilitated a more efficient and generally applicable method to produce Fv fragments. Basically, scFv antibodies produced from phage display can be genetically fused to the marker proteins, such as fluorescent proteins or alkaline phosphatase. These bifunctional proteins having both antigen-binding capacity and marker activity can be obtained from transformed bacteria and used for one-step immunodetection of biological agents. Alternatively, antibody fragments could also be applied in the construction of immunotoxins, therapeutic gene delivery, and anticancer intrabodies for therapeutic purposes. This paper provides an overview of the current studies on the principle, generation, and application of scFv. The potential of scFv in breast cancer research is also discussed in this paper.
doi:10.1155/2012/980250
PMCID: PMC3312285  PMID: 22474489
19.  BDMC33, A Curcumin Derivative Suppresses Inflammatory Responses in Macrophage-Like Cellular System: Role of Inhibition in NF-κB and MAPK Signaling Pathways 
Our preliminary screening has shown that curcumin derivative BDMC33 [2,6-bis(2,5-dimethoxybenzylidene)cyclohexanone] exerted promising nitric oxide inhibitory activity in activated macrophages. However, the molecular basis and mechanism for its pharmacological action is yet to be elucidated. The aim of this study was to investigate the anti-inflammatory properties of BDMC33 and elucidate its underlying mechanism action in macrophage cells. Our current study demonstrated that BDMC33 inhibits the secretion of major pro-inflammatory mediators in stimulated macrophages, and includes NO, TNF-α and IL-1β through interference in both nuclear factor kappaB (NF-κB) and mitogen activator protein kinase (MAPK) signaling cascade in IFN-γ/LPS-stimulated macrophages. Moreover, BDMC33 also interrupted LPS signaling through inhibiting the surface expression of CD-14 accessory molecules. In addition, the inhibitory action of BDMC33 not only restricted the macrophages cell (RAW264.7), but also inhibited the secretion of NO and TNF-α in IFN-γ/LPS-challenged microglial cells (BV-2). The experimental data suggests the inflammatory action of BDMC33 on activated macrophage-like cellular systems, which could be used as a future therapeutic agent in the management of chronic inflammatory diseases.
doi:10.3390/ijms13032985
PMCID: PMC3317699  PMID: 22489138
anti-inflammatory; BV-2; curcumin; MAPK; NF-κB; RAW 264.7
20.  Immunomodulatory Effect of Rhaphidophora korthalsii on Natural Killer Cell Cytotoxicity 
The in vivo immunomodulatory effect of ethanolic extracts from leaves of Rhaphidophora korthalsii was determined via immune cell proliferation, T/NK cell phenotyping, and splenocyte cytotoxicity of BALB/c mice after 5 consecutive days of i.p. administration at various concentrations. Splenocyte proliferation index, cytotoxicity, peripheral blood T/NK cell population, and plasma cytokine (IL-2 and IFN-γ) in mice were assessed on day 5 and day 15. High concentration of extract (350 μg/mice/day for 5 consecutive days) was able to stimulate immune cell proliferation, peripheral blood NK cell population, IL-2, and IFN- γ cytokines, as well as splenocyte cytotoxicity against Yac-1 cell line. Unlike rIL-2 which degraded rapidly, the stimulatory effect from the extract managed to last until day 15. These results suggested the potential of this extract as an alternative immunostimulator, and they encourage further study on guided fractionation and purification to identify the active ingredients that contribute to this in vitro and in vivo immunomodulatory activity.
doi:10.1155/2012/786487
PMCID: PMC3175394  PMID: 21941589
21.  Development of avian influenza virus H5 DNA vaccine and MDP-1 gene of Mycobacterium bovis as genetic adjuvant 
Background
Studies have shown that DNA vaccines can induce protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines. Vaccines are frequently formulated with adjuvants to improve their release, delivery and presentation to the host immune system.
Methods
The H5 gene of H5N1 virus (A/Ck/Malaysia/5858/04) was cloned separately into pcDNA3.1 + vector. The immunogenicity of the cloned H5 DNA vaccine was tested on SPF chickens using two different approaches. First approach was using H5 DNA vaccine (pcDNA3.1/H5) and the second was using H5 DNA vaccine in addition to the pcDNA3.1/MDP1 vaccine. Ten days old chickens inoculated three times with two weeks intervals. The spleen and muscle samples from chickens immunized with H5 (pcDNA3.1/H5) and H5 + MDP1 (pcDNA3.1/H5 + pcDNA3.1/MDP1) vaccines were collected after sacrificing the chickens and successfully expressed H5 and MDP1 RNA transcripts. The sera of immunized chickens were collected prior to first immunization and every week after immunization; and analyzed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test.
Results
Results of competitive ELISA showed successful antibody responses two weeks post immunization. The HI test showed an increased in antibody titers during the course of experiment in group immunized with H5 and H5 + MDP1 vaccines. The result showed that the constructed DNA vaccines were able to produce detectable antibody titer in which the group immunized with H5 + MDP1 vaccine produced higher antibody comparing to H5 vaccine alone.
Conclusions
This study shows for the first time the usefulness of MDP1 as a genetic adjuvant for H5 DNA vaccine.
doi:10.1186/1479-0556-8-4
PMCID: PMC2887864  PMID: 20497569
22.  Development of Multicellular Tumor Spheroid (MCTS) Culture from Breast Cancer Cell and a High Throughput Screening Method Using the MTT Assay 
PLoS ONE  2012;7(9):e44640.
In comparison to monolayer cells, MCTS has been claimed as more suitable candidate for studying drug penetration due to the high resemblance to solid tumors. However, the cultivation of MCTS is cumbersome, time consuming, and most technique fail to generate spheroids with uniform sizes. Therefore, the application of spheroid cultures in high throughput screening has been rather limiting. Besides, the lack of a well established screening protocol method that is applicable to spheroid could also be attributed to this limitation. Here we report a simple way of cultivating homogenous MCTS cultures with compact and rigid structure from the MCF-7 cells. Besides, we had also made some modifications to the standard MTT assay to realize high throughput screening of these spheroids. Using the modified protocol, tamoxifen showed cytotoxicity effect towards MCTS cultures from MCF-7 with high consistency. The results correlated well with the cultures’ response assessed by LDH release assay but the latter assay was not ideal for detecting a wide range of cytotoxicity due to high basal background reading. The MTT assay emerged as a better indicator to apoptosis event in comparison to the LDH release assay. Therefore, the method for spheroid generation and the modified MTT assay we reported here could be potentially applied to high throughput screening for response of spheroid cultures generated from MCF-7 as well as other cancer cell lines towards cytotoxic stimuli.
doi:10.1371/journal.pone.0044640
PMCID: PMC3435305  PMID: 22970274

Results 1-22 (22)