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Candida spp. Are the most common fungal pathogens of systemic candidiasis. As there are pathognomonic signs or symptoms of systemic candidiasis, the diagnosis of invasive candidiasis remains a laboratory and clinical challenge. Thus, diagnosis assays to detect systemic candidiasis and to identify Candida virulence factors and associated pathogenesis through immunohistochemistry using specific monoclonals and polyclonals will be useful. Balb/c mice were immunized with C. albicans and C. parapsilosis and blood were checked for the presence of reactive antibodies using ELISA. Fusions were performed using the harvested spleen cells and NSI mouse myeloma cells and the clones were screened for the presence of antibodies producing hybrid cells by dot-blot. A total of forty and fifty clones producing monoclonal antibodies against C. albicans and C. parapsilosis respectively were obtained. Western blot analyses showed that the monoclonal antibodies against non heat-shocked (IgM) and heat-shocked (IgG3) C. albicans were reactive against the antigens with MW at 30 and 59 kDa. Experimental systemic candidiasis in mice and rats were induced through intravenous injection of C.albicans and C. parapsilosis. The liver, spleen, kidneys, brain, heart and lungs were collected for histopathological examination. Cystic lesions with large clumps of fungus consisting mainly of hyphae and some yeast were observed in the kidneys. Clusters of yeast cells were detected in multiple organs. The female mice and rats were more resistant to the C. albicans infection than the male counterparts. The rats were found to be more susceptible to C. albicans infection than Balb/c mice. C. parapsilosis was found to be less virulent than C. albicans. The animals infected with C. parapsilosis had mild infection in spleen and lungs only. However, all the majority of the organs in animals that succumbed to the C. albicans or C. parapsilosis infection had fungal invasion. Histopathhological examination revealed that all dead but no those surviving or control animals had mild to serve fungal colonization in brains. Fungal colonization in the brain may be a determinant factor for mortality in experimental systemic. Candidiasis. Two Isolates of fungal were obtained from blood culture on Sabouraud dextrose agar and were found to be of the same species with those used for the infection, based on restrictive length polymorphism and DNA alignment analysis. The monoclonal antibodies were purified, characterized and used in the immunoperoxidase and immunofluorescence techniques. The monoclonal antibodies reacted to surface epitopes on the yeast cells, germ tubes, hyphae and to immune complexes. The monoclonal antibodies obtained were used for the detection of antigens, together with the polyclonal antibody, in the experimental model and were found able to detect antigens present in the sera at 0.2µg/µL. Antibody levels were also determined using the ELISA method. The antibody levels against C. albicans or C. parapsilosis in infected mice or rats were increased compared with uninfected animals. The Circulating antigen level in the infected animals increased initially but declined subsequently. The cytokine levels (IL-4, IL-6, IL-10, IFNƴ and TNF) induced in the models were measured using flow cytometry. C. albicans and C.parapsilosis elicited different cytokine expression in mice and rats. The groups infected with C. albicans had higher IL-6 and TNF, but unchanged IL-4, IL-10 and IFNƴ levels when compared with uninfected mice. Elevated levels of TNF were seen in both mice and rats infected with C. albicans but not in those infected with C. parapsilosis. The level of IL-10 was lower for all the C. albicans and C. parapsilosis infected rodents compared with unifected rodents except for rats infected with C. albicans at the later stage of infection. In conclusion, monoclonal antibodies were produced and user for the detection of antibody, circulating antigen and immune complexes in the experimental systemic candidiasis model. These monoclonal antibodies may serve as potential primary capture antibodies for the development of rapid diagnostic test for human systemic Candida infection Fungal colonization in different organs and differential host immune responses against C. albicans and C. parapsilosis were shown in the present study.

Gestational diabetes mellitus (GDM) is a major public health concern affecting maternal and foetal health. It is defined as carbohydrate intolerance of variable severity first recognised during pregnancy that reflects a risk for adverse outcomes. It is still not known why GDM develops in some pregnant women. The progressive increase of insulin resistance during pregnancy and decrease in insulin secretion may lead to GDM. Recently, factors such as cytokines were found to play a role in insulin resistance. The possession of specific genetic polymorphisms have been implicated as predisposing factors and if such alterations are operational in this maternal condition, then one could hypothesise that genetic polymorphisms might be useful as a marker to diagnose susceptibility to GDM. Based on this concept, the aim of this study was to investigate the association between single nucleotide polymorphism (SNP) in the human promoter of the tumour necrosis factor-alpha (TNF-α) gene as well as interleukin-10 (IL-10) gene with the development of GDM. The study also involved quantification of plasma levels of TNF-α, IL-10, and insulin to determine associations with gestational age. The SNPs were genotyped using polymerase chain reaction and restriction fragment length polymorphism. Cytokines as well as insulin were quantified using ELISA. Demographic data derived from the selected population studied showed that pregnant women with GDM were significantly older and of higher parity and BMI than mothers with normal pregnancy (p<0.05). Ethnic distribution did not significantly differ in the two study groups (p>0.05), hence subsequent genetic studies did not attempt to look at ethnic group differences. Majority (75%) of the GDM mothers had HBA1c < 6.5 %, indicating good glycaemic control. Contrary to what is reported elsewhere, in this study, we found no significant differences in the incidence of antenatal complications as well as pregnancy outcomes between controls and study population. Neonatal and maternal outcomes were almost similar in the two groups. We think that this is due to the tight glycemic control, close monitoring of the GDM mother as well as timely intervention at 38 weeks of gestation that is practised at the tertiary hospital that this study was conducted. The difference of SNP genotype and allele frequencies observed in TNF-αpromoter gene at position -308 between the two groups were found to be not significant (p>0.05). This finding did not support our hypothesis that a SNP in the TNF- gene can be used as a predictive factor for GDM. Similarly, there were no significant (p>0.05) difference in the genotype and allele frequencies of SNP at positions – 824 and -1082 SNP in the promoter of the IL -10 gene between GDM and control groups. However, we found a significant (p<0.05) difference in the genotype and allele frequencies of a SNP at position -597 in the promoter of the IL-10 gene between the two study groups. Subjects who were carried the mutant alleles at position -597 in the promoter region of the human IL-10 gene were found to be 2.2 times more likely to develop GDM compared to those who do not have. We also found eight different theoretically possible allele combinations in our study groups. We showed that there were some significant (p<0.05) differences observed in haplotype frequencies between the GDM and control groups. In addition, we also detected two rare haplotypes i.e. ATG and ACA haplotype. Plasma levels of TNF-α as well as IL-10 were compared in the two study subjects in different stages of pregnancy and six weeks post partum. There were no significant (p>0.05) differences in the levels of these two cytokines observed at different stages of pregnancy as well as between control and GDM groups. The plasma levels of IL-10 were lower in GDM subjects compared to controls. However, this difference was found to be statistically not significant (P>0.05). This was not an expected finding but we postulate that this pattern was observed may be a reflection of the good glycaemic control in the GDM patients included in this study. It would be interesting to evaluate the IL-10 levels in GDM subjects with poor glycaemic control to see if there could be significant differences in the plasma IL-10 levels. The plasma levels of insulin increased with increasing gestational age and by third trimester and then decreased, with the GDM patients showing a consistently lower level. However, the difference was once again not statistically significant (P>0.05). Considering that our GDM patients were older, of higher parity and higher BMI, we postulate the impact of apoptosis on the islets cells or alternate impacts that may operate in insulin sensitisation when IL-10 levels didn’t reach that of normal patients. The implications of such findings are far and wide. The control subjects had significant differences between insulin levels of during pregnancy and post partum but in the GDM subjects, insulin levels at 6 weeks post partum were significantly different from insulin levels at 32 weeks of pregnancy, which may reflect lower insulin response and β-cells defect in GDM subjects. Although others have looked at other cytokines including adipokines in GDM, on the whole this study is a step towards understanding the role and genetics of cytokines as well as their plasma levels (of TNF-αand IL-10) in pregnancy. Our findings showed that SNP at position -597 was significantly associated with development of GDM and shows potential for use as a predictive factor. Although we could not establish any cause-effect relationship, as is often the case when biochemical and genetic markers are used to predict disease, we have tried to draw some valuable correlated data for these cytokines which may prove valuable in future studies. Taken together and with regard to other studies, it seems that there is no single underlying factor mediating the pathogenesis of diabetes in pregnancy. Therefore considering other variables in the formula could give us better predictive value. Perhaps a mathematical model needs to be developed that will include epidemiologic factors, plasma biomarkers and polymorphisms of single nucleotide in the affected population.

Epstein-Barr virus (EBV) is a class I carcinogen human herpes virus which has infected 90% of humanity and most prevalent among Asians, especially Chinese. After primary infection, EBV establishes the lifelong virus carrier state. EBV can be detected in two different tissues namely, B lymphocytes and epithelial cells. EBV is linked to the pathogenesis of a variety of human tumors and disorders, such as Burkitt’s lymphoma, and nasopharyngeal carcinoma. Algae are a potential source of antiviral compounds; however, there have been very few reports on the antiviral activity of microalgae extracts against EBV. The objective of this study was to investigate the antiviral activity of extracts from three microalgae, namely Ankistrodesmus convolutus UMACC 101, Synechococcus elongatus UMACC 105 and Spirulina platensis UMACC 161 against EBV in Burkitt’s lymphoma (BL) cell lines. Three EBV-positive BL cell lines, namely Akata, B95-8 and P3HR-1 were used as in vitro study model. A bioassay-guided fractionation approach was used for the screening of antiviral activity. The antiviral activity of the microalgae extracts was elucidated based on their inhibition efficacy in reducing number of cell-free viral particles being released by chemically induced lytic BL cells. This was assessed by quantifying the cell-free DNA using real-time PCR technique. In addition, the inhibition activity of microalgae extracts against the expression of the viral proteins LMP1, EBNA1 and ZEBRA in BL cells was assessed using immunocytochemistry technique. Two antiviral drugs namely acyclovir and foscarnet were chosen as positive controls. Methanol extracts from Ankistrodesmus convolutus and Synechococcus elongatus displayed low cytotoxicity (IC50 >200 µg/mL) and reduced the cell-free EBV viral load most effectively (EC50 <0.01 µg/mL) and thus, displayed high therapeutic index (>28000). The extracts decreased the expression of EBNA1 (>45%), LMP1 (>38%) and ZEBRA (>67%) effectively in P3HR-1 cells. After column chromatography fractionation, the non-polar fraction of the extract from Synechococcus elongatus (SEF1) reduced the amount of cell-free EBV DNA most effectively (EC50= 2.9μg/mL; therapeutic index >69) with low cytotoxicity (IC50 >200 μg/mL). SEF1 inhibited the expression of EBNA1 and ZEBRA (>40%) effectively in P3HR-1 cells. When SEF1 was further fractionated using HPLC, the sub-fraction SEF1’a was most active in reducing the cell-free EBV DNA (EC50= 1.38µ/mL; therapeutic index >14.5). It inhibited the expression of LMP1 moderately (25%) in P3HR-1 cells. The microalgae extracts did not interact with the cytoskeleton components (actin and tubulin) of BL cells during the release of cell-free EBV particles as revealed by the immunofluorescence study. The active constituents in the microalgae extracts tested might consist of pigments such as chlorophylls, carotenoids, phaeophytins and phycobilins. In conclusion, methanol extracts from Ankistrodesmus convolutus, Synechococcus elongatus and Spirulina platensis showed antiviral activity by inhibiting the release of EBV from the BL cells and the expression of the viral proteins LMP1, EBNA1 and ZEBRA in the host cells. The potential of the microalgae as a source of antiviral drugs against EBV is worth exploring.

Acanthamoeba parasites as aetiological agents for the sight threatening Acanthamoeba keratitis and the rare granulomatous amoebic encephalitis in humans were officially recognised in the 1970s. Since then, increasing numbers of Acanthamoeba-associated keratitis and encephalitis cases have been reported worldwide. In Malaysia, the first Acanthamoeba keratitis case was reported in 1995, in Hospital Kuala Lumpur. The first case was a female patient contact lens wearer. Subsequently, several more keratitis cases have been diagnosed in patients at the Hospital Kuala Lumpur and Hospital Universiti Kebangsaan Malaysia. However, reports of these cases have not been officially published. Granulomatous amoebic encephalitis has also been diagnosed in Hospital Universiti Sains, Kubang Kerian, Malaysia, but the report has not been officially published. Although Acanthamoeba organisms are ubiquitously distributed in the nature, only a few species are pathogenic to human. Many research groups have carried out studies on parasite characteristics which contribute to Acanthamoeba pathogenicity. Characteristics such as tolerance to high temperature, high osmotic pressure, extreme pH, ability to kill target cells in vitro and to cause pathological lesions in experimental animals, are usually evaluated for clinical isolates of Acanthamoeba. These properties are also useful indicators for predicting the pathogenicity of environmental isolates of Acanthamoeba. In Malaysia, thus far, no pathogenicity study has been conducted on any of the local isolates. The current study is based on isolates of Acanthamoeba spp. from dust samples of air-conditioners in Malaysia. These isolates were assessed for their pathogenic potential based on their physical tolerance to harsh growth condition, presence of molecular markers associated with pathogenicity, the ability to cause glial cell death in vitro and to infect and cause lesions in Balb/c mice. Prior to the pathogenicity characterisation, theidentities of these isolates were determined morphologically and molecularly. The associated mechanism for pathogenesis of Acanthamoeba was investigated using electron microscopy. Acanthamoeba organisms were cultured from dust samples using NNA culture plates incubated at ambient temperature (26C 2C) for up to a month. Twenty-four primary cultures were tested positive for Acanthamoeba spp. based on microscopy and PCR detection. Selected environmental isolates were axenised using HCl acid and gentamicin treatment to eliminate bacteria contaminants. Established axenic Acanthamoeba strains were determined for their clonality using PCR and sequencing. Twenty-one pure-cloned isolates designated as IMU1 to IMU21, were characterised morphologically and molecularly. Two keratitis isolates designated HTH136 and HKL55 were also included in the study. Both clinical isolates were similarly axenised and characterised morphologically and molecularly. Five species were identified according to the morphological criteria of Pussard and Pons (1977) and Page (1988) keys. These species were A. castellanii (IMU1 to IMU3, IMU6, IMU7, IMU9 and IMU18); A. culbertsoni (IMU10 to IMU13); A. griffini (IMU14); A.lenticulata (IMU16 and IMU17), and A. polyphaga (IMU8, IMU19 and HTH136). Species identities for the remaining six isolates (IMU4, IMU5, IMU15, IMU20, IMU21 and HKL55), however, could not be determined morphologically. At molecular characterisation, IMU14 was placed into T3 genotype; IMU16 and IMU17 were clustered in T5 genotype whereas the two clinical isolates and other environmental isolates were placed into T4 genotype. To predict the potential pathogenicity of the Acanthamoeba isolates used in the study, PCR primer pairs which could differentiate the pathogenic from non-pathogenic strains, temperature tolerance tests and osmotic tolerance tests were employed. Many isolates were predicted as potential pathogens based on the results of these tests. The virulence of the potential pathogenic strains was further confirmed by their ability to cause glial cell death in in vitro cytopathogenic assays. Seven environmental isolates (IMU9, IMU10, IMU14, IMU16, IMU17, IMU18 and IMU19) and the two clinical isolates (HTH136 and HKL55) were selected for pathogenicity studies in Balb/c mice. At 30 days post-infection, none of the mice succumbed to the infection with any of the Acanthamoeba isolates tested. However, pathological changes were detected in the liver and lung of mice infected with all the tested Acanthamoeba isolates. The lung was the main organ affected by Acanthamoeba. Infection caused by the more virulent Acanthamoeba isolates resulted in bronchiolitis, bronchopneumonia, and interstitial pneumonia whereas infection by the less virulent isolates provoked mild interstitial lung inflammation. Trophozoites were only occasionally seen within lung tissues. Often, Acanthamoeba organisms were not detected in these lesions; immune complex deposition was probably the predominant cause for the chronic inflammation in affected organ. It has been reported that the clinical and histopathological features of Acanthamoeba infection in humans and mice are essentially the same. Since there are similar pathological changes seen in mice infected with clinical isolates and the environmental isolates tested in the present study, it is concluded that these seven environmental strains can be considered as potential human pathogenic isolates. Electron microscopy was employed to gain some insight into the mechanism of glial cell death caused by one of the experimentally pathogenic strain - IMU17. Electron microscopy showed that the physical contact of Acanthamoeba trophozoites to glial cells could trigger apoptosis and necrosis of the target cells. Phagocytosis of glial cells by trophozoites was also observed. To our knowledge, this is the first report presenting the isolation and molecular characterisation of potential pathogenic Acanthamoeba spp. in indoor air-conditioners in Malaysia. Building occupants therefore should be aware of the risk of acquiring air-borne Acanthamoeba infection through the use of poorly maintained air-conditioners.

Herbal products are widely consumed all around the world. Since they are natural products, they are perceived as safe. However, because they are plant extracts, they contain phytochemicals with capabilities to inhibit or induce drug-metabolising enzymes. Cytochrome P450 (CYP) enzymes are fixed-function monooxygenases, which are expressed abundantly in human liver and play central roles in drug metabolism. Four isoforms, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 were selected in this study as they are expressed at high levels in human liver and collectively they metabolise more than 80% of drugs known to be CYP substrates. Andrographis paniculata (hempedu bumi) (AP), Centella asiatica (pegaga) (CA) and Orthosiphon stamineus (misai kucing) (OS) are three popular local herbs traditionally used for various conditions, including hypertension, diabetes, skin disorders, ailments of the kidneys, liver and bladder. Current research on these herbs mostly concentrates on the chemical and botanical analyses, standardisation of extract preparations and pharmacological elucidation of the extracts and bioactive constituents of the plants. To date, limited studies have been carried out to investigate CYP modulating effects of these herbs, hence their potential to cause pharmacokinetic interaction with CYP substrates. Our objective was to develop specific in vitro assays for screening of the modulatory effects of these commonly used Malaysian herbs on activities of major cytochrome P450 enzymes. Four in vitro assays, CYP2C9-mediated tolbutamide methylhydroxylase assay, CYP2C19-mediated S-mephenytoin 4-hydroxylase assay, CYP2D6-mediated dextromethorphan O-demethylase assay and CYP3A4-mediated testosterone 6-hydroxylase assay, were selected and established using published high performance liquid chromatography (HPLC) methods. Herbal extracts together with important known active compounds of the herbs were evaluated by acquiring relevant pharmacokinetic parameters including IC50 and Ki to predict their potential to cause clinically significant interactions. Generally, all the herbal preparations and constituents were found to cause differential modulatory effects on the four CYP isoforms investigated. AP ethanol and methanol extracts inhibited CYP isoforms activities more potently compared with aqueous and hexane extracts. Similarly, CYP2C9 and CYP3A4 were more susceptible to AP inhibitory effects compared to other two isoforms. Potent inhibition observed for CYP2C9 and CYP3A4 (Ki values below 20 μg/ml) implying potential risk of drug-herb interactions for common drug substrates of the isoforms especially when patients are consuming large amount of AP preparations. As for CA, two active constituents, asiatic acid and madecassic acid, were found to selectively inhibited CYP isoforms activities to different extents, with CYP2C9 and CYP2D6 inhibited more than the rest. Of particular interesting is the potent inhibitory effect of asiatic acid on CYP2C9 (Ki below 20 μM). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Only CA ethanol and dichloromethane extracts potently to moderately inhibited CYP2C9 and CYP2C19 activities among all the CA extracts, indicating the involvement of semi-polar constituents including asiatic acid or madecassic acid in the inhibitory effect. While CA showed propensity to inhibit CYP2C9, OS appeared to selectively inhibit CYP3A4 activity. OS dichloromethane extract inhibited CYP3A4 activity with moderate effect whereas petroleum ether extract moderately inhibited both CYP3A4 and CYP2C19. Eupatorin was the most potent inhibitor among the four OS active constituents investigated, showing moderate to strong inhibitory effects on CYP2C19, CYP2D6 and CYP3A4. Of particular clinical relevant was the potent inhibition of eupatorin on CYP2D6 and CYP3A4, with both Ki values fell below 11 µM, implying good potential for eupatorin interaction with substrates of these two isoforms. Caffeic acid, on the other hand, showed significant inductive effect on CYP2C9 and CYP3A4. Hence, the net effect of intake of OS products would depend on the relative concentrations of various constituents in the products, the CYP isoforms involved and the drug substrates taken by the patients. In conclusion, studies carried out in the present project have provided more insight into the guideline of rational administration and precaution to be taken for using A. paniculata, C. asiatica and O. stamineous. The four in vitro enzyme assays developed in this project have served as convenient and robust models for screening and predicting herb–drug interactions in humans. The in vitro inhibition data generated indicate that certain extracts and constituents of the three herbs are sufficiently inhibitory for important CYP isoforms to suggest the need for additional in vitro and in vivo studies to evaluate the possibility of clinically significant drug interactions.

Bacillus thuringiensis (Bt) parasporal proteins with anti-cancer activity have gained interest due to its selectivity towards cancer cells. This study determines the efficacy and mechanism of action of parasporal proteins harvested from a Malaysian Bt strain (Bt 18) against three leukaemic cell lines (CEM-SS, CCRF-SB and CCRF-HSB-2) compared to normal T-lymphocytes. Results from the present study shows that Bt 18 parasporal proteins consist of two subunits of polypeptide (68 kDa and 28 kDa) which co-elute during ion-exchange chromatography. The N-terminal sequence of Bt 18 parasporal proteins shows that 68-kDa polypeptide has high similarity with Cry25Aa and Cry24Aa, parasporal proteins found in a mosquitocidal isolate from Malaysia, Bt serovar jegathesan. However, the 68-kDa polypeptide of Bt 18 parasporal protein is non-insecticidal. The alignment results of N-terminal sequence of 68-kDa Bt 18 parasporal protein with various parasporin groups showed that it could be a new group of parasporins. The solubilised and activated parasporal proteins of Bt 18 exhibited moderate anti-cancer potential by lowering the percentage cell viability for CEM-SS, CCRF-SB and CCRF-HSB-2 cells, whilst being non-cytotoxic to normal human T lymphocytes. Interestingly further purification of the parasporal proteins from Bt 18 lowers the concentration of 28 kDa subunit and changes in inhibition selectivity for the three cell lines was observed. Phosphatidylserine externalisation, active caspase 3 and TUNEL assays detect and confirm apoptotic activity in the leukaemic cells treated with Bt 18 parasporal proteins. Western blot analysis suggests that Bt 18 binds to Glyceraldehyde 3-phosphate dehyrogenase (GAPDH) in all three cell lines. The present study suggests that Bt 18 parasporal proteins caused cell cycle arrest at S-phase and induce apoptosis that leads to the reduction of percentage cell viability, although more than one cellular pathway may be involved. When combined with the all the results obtained, the present study suggests that Bt 18 parasporal proteins would bind to cancer cells (CEM-SS, CCRF-SB and CCRF-HSB-2) on the surface membrane binding protein GAPDH that may lead to S-phase cell cycle arrest, initiating apoptosis and reducing cell viability.

Tocotrienols, isoforms of vitamin E, are not only known for their antioxidant, lipid-lowering properties and as anti-proliferating agents but also for their inhibitory effects on the growth of human breast cancer cells in vitro and in vivo. In this study, the effects of tocotrienol-rich fraction (TRF) from palm oil and its isomers (α, δ and γ-tocotrienol) were examined in 4T1 mouse mammary cancer cells. The 4T1 cells were cultured and grown in RPMI medium supplemented with different concentrations of tocotrienols. Cell numbers were determined at the end of twelve days of culture. Results showed that TRF and the individual fractions of palm tocotrienols inhibited the growth of 4T1 cells in vitro at lower concentrations (6-20 μg/ml) compared to tocopherols (> 20 μg/ml). Deltatocotrienol was found to be most inhibitory followed by γ-tocotrienol. Complete inhibition of 4T1 proliferation was achieved with 6 μg/ml δ-tocotrienol and 10 μg/ml γ-tocotrienol. The effects of TRF and tocotrienol isomers were investigated for proapoptotic activity by expression of FasL gene. This study showed that γ- and δ- tocotrienol (8 μg/ml) treated 4T1 cells expressed significantly (P < 0.01) higher levels of FasL compared to untreated control cells. The TRF showed pro-apoptotic effects at higher concentrations from 20 – 80 μg/ml). Tumourigenesis was examined and compared against control in both nude and BALB/c mice models. The mice were injected with MDA-MB-231 and 4T1 cells respectively for the different models, and were fed with TRF by oral gavage. Tumour incidence was reduced by 33% and 57% in nude mice and BALB/c mice respectively. In addition, the tumour load was significantly (P < 0.05) reduced by 72% and 93.6% respectively in tocotrienol-supplemented nude and BALB/c mice, when compared to the vehicle-fed control group. This study shows that palm tocotrienols have strong inhibitory effects on the growth of both MDA-MB-231 and 4T1 cells both in vitro and in vivo. The immune modulatory effects of tocotrienols were investigated and it was found that TRF enhanced production of NK cells (P < 0.05) as well as IFN-γ (P < 0.05), which in turn regulate the immune protection against cancer cells. These observations were recorded in both mice models. In this study, adipose tissue from control (vehicle-fed) and experimental (TRF-fed) animals were analysed for the presence of tocopherols and tocotrienols using HPLC. All the tocotrienol isomers increased significantly (P < 0.05) in the TRF supplemented mice compared to control whilst only α-T and γ-T showed significant increase in the supplemented nude mice and BALB/c mice. The anti-angiogenic activity of TRF and two tocotrienol isomers (γ and δ) were investigated using the human umbilical vein endothelial cells (HUVEC). Results showed that the level of Interleukin-8 (IL-8), an angiogenic activator was reduced in the HUVEC treated with TRF (P < 0.05) and the two tocotrienol isomers compared to α-tocopherol and control cells. The level of IL-8 was lowest in the δ-tocotrienol treated cells followed by γ-tocotrienol and TRF. In addition, TRF also down-regulated the expression of various angiogenic activators such as VEGF-C, KDR and FLT1 in tumours from mice fed with TRF as compared to control mice. Moreover, immunohistochemical expression of pro-angiogenic such as VEGF, VEGF-R2 and CD31 were found to be reduced in tumour and lung tissues of mice supplemented with TRF. This study shows that tocotrienols exhibited anti-angiogenic activity, which may play a key role in inhibiting the growth and metastasis of tumour. Microarray analysis was carried out on 4T1 cells treated with TRF, δ- tocotrienol, α-tocopherol and differential gene expression compared to untreated control cells were investigated. The outcome from this study revealed the identity of several interesting genes including Interleukin-24 (IL-24), death-associated protein 3 (Dap3), rat sarcoma oncogene (Rras) and platelet-derived growth factor- B (Pdgfb) that possess important functional role in cancer mechanisms. In this study, the microarray analysis was further subjected to investigate the effect of TRF on regulation of genes from tumour tissues of mice that were supplemented with TRF compared to that of control mice. This analysis again revealed the up-regulation of IL-24 in vivo with TRF-supplementation. In this study, we also found that tocotrienols up-regulated (P < 0.05) the expression of a novel-cytokine, IL-24 in the tumour and 4T1 cells. The IL-24 cytokine has been reported to play a significant role in tumour regression. In addition, while tocotrienols up-regulated the expression of IL-24, they also downregulated pro-angiogenic markers; VEGF and IL-8 at the same time. Studies have previously shown that IL-24 possess anti-angiogenic properties. Thus, the tumour protective mechanism exhibited by tocotrienols may be explained via the IL-24 mechanistic pathway.

Isolation of Kampar virus (KamV) and Melaka virus (MelV) from patients with acute respiratory syndrome suggest new pathogenic viruses emerging. KamV and MelV had been shown to be new members closely related to the Pulau virus (PulV) and Nelson Bay virus (NBV). PulV and NBV are mammalian fusogenic orthoreovirus which are isolated from fruit bats, Pteropus hypomelanus (Malaysia) and Pteropus poliocephalus (Australia), respectively. All four viruses are Orthoreovirus from the Reoviridae family, which contains a ten-segmented double-stranded RNA genome within an icosahedral, non-enveloped, double layer protein capsid. So far, only the S-class segments and corresponding -proteins of these mammalian fusogenic orthoreoviruses have been sequenced and analyzed. Single primer amplification technique was utilized to characterize the L- and M- class segments of KamV, PulV and MelV at the sequence level. The complete nucleotide sequence of the L- and M- class gene segments and their respective deduced amino acid sequences of KamV, MelV and PulV were studied in detail. L1, L2 and L3 segments (3896, 3832, 3954 base pairs, respectively) encode C, B and A proteins (1286, 1259, 1291 amino acids, respectively). M1, M2 and M3 segments (2295, 2028, 1896 base pairs, respectively) encode A, B and NS proteins (730, 675, 631 amino acids, respectively). Conserved motifs of avian reoviruses and mammalian reoviruses were found in KamV, PulV, MelV, indicating similar protein functionality. Phylogenic tree analysis showed possible segment reassortment in S- class segments among KamV, MelV and PulV. The M2 segments of the three viruses, which encode viral outer capsid protein, produce phylogenetic trees of different topology from M1 and M3 segments. This may be due to high selective mutation pressure acting on the M2 segment in order for the viruses to adapt to a different host. Similar topology of phylogenetic tree was observed in the L- class segments, indicating that no reassortment had occurred. Based on S- segments and - proteins of the three mammalian fusogenic orthoreoviruses, it can be concluded that a new genetic lineage of Nelson Bay Orthoreovirus species is co-circulating in the population. KamV, PulV and MelV are novel orthoreoviruses that diverged away from NBV. Expression of C proteins of these three fusogenic mammalian orthoreoviruses show cross reactivity with each other, but not with NBV.

In this study the risk factors that were associated with gross congenital abnormalities in the state of Penang were studied. The study constitutes various parts: a trend analysis, land use pattern and its association with congenital anomalies, qualitative, retrospective case control and a prospective cohort study. The participants for the case control and cohort study were recruited from the antenatal mothers residing in Penang. There was a high incidence of congenital anomalies among the Malay population although the majority of the population in Penang is Chinese. There was a cluster of cases among the fishermen in a village. Three groups of congenital anomalies were studied and they were hydrocephalus, Down’s syndrome, and cleft lip (CL) and cleft palate (CP). The risk factors identified were smoking, advanced age, low socio- economic status, diabetes, exposure to pesticides, and maternal and paternal occupational exposure to chemicals. Most of the cases were concentrated in the industrialized areas. The results that are presented here suggest that further attention needs to be focused on the possible association of exposure to chemicals and in-depth study on fishermen. The State of Penang is an industrialized state and a majority of them are manufacturing and electronic industries. There is an increased potential for hazardous exposure to chemicals if proper protection has not been carried out. Besides the higher incidence among the Malay population, a further in-depth study is needed to find the causes. In view of the public health concern, further studies and investigations with regards to the increased rate of incidence of congenital anomalies are justified to evaluate the role of exposure to chemicals and pesticides in relation to occupation; in addition, there needs to be a surveillance programme and a National registry for congenital anomalies in Malaysia.

Dietary fat type is known to modulate plasma lipid profile but its effects on plasma homocysteine, inflammatory markers and other lipid mediators are unclear. The main objective of the study is to investigate the effects of high-protein Malaysian diets prepared with palm olein, coconut oil or virgin olive oil on plasma homocysteine and selected markers of cardiovascular disease (CVD) in healthy Malaysian adults. A randomised; single-blind, crossover intervention with 3 dietary sequences of 5wk each and a 2 wk wash-out in between were conducted on 45 healthy subjects (36 females, 9 males; aged 30-55 years). The three different test fats, namely palmitic acid(16:O)-rich palm olein (PO), lauric + myristic acids(12:0 + 14:0)-rich coconut oil (CO), and oleic acid(18:1)-rich virgin olive oil (OO), were incorporated at 30% kcal into high-protein (20% kcal) Malaysian diets and provided as breakfast, lunch and dinner to the subjects according to a Latin-square design. During each dietary period, 2-hr postprandial blood samples were collected at the end of the 4th week and 12-hour fasting samples at the end of the 5th week, and analysed for plasma total homocysteine (tHcy) and selected markers of cardiovascular disease. No significant differences were observed on the effects of the three experimental diets on plasma tHcy and the inflammatory markers- high sensitive C-reactive protein (hsCRP), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-1β (IL-1 β), tumor necrosis factor-α (TNF- α) and interferon-γ (IFN- γ) as well as cells adhesion molecules - soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1) and member of the selectin family (E-selectin) in the postprandial and fasting states. Compared to PO and CO diets, OO diet lowered fasting plasma leukotriene B4 (LTB4). No significant difference was also found lowered in postprandial plasma LTB4 comparing OO vs CO diet but not PO. Compared to OO diet, PO diet increased fasting serum 6-keto prostaglandin F-1α (PGFlα) but not different from CO diet. However, the ratio of the serum PGFlα to 11-dehydro thromboxane B2(TXB2) was not altered between the three diets. The diets prepared with PO and OO had comparable non-hypercholesterolemic effects; postprandial TC for both diets and all fasting lipid indices for the OO diet were significantly lower (p < 0.05) compared with the CO diet. CO diet was found to decrease postprandial lipoprotein (a) (Lp(a)) unlike PO and OO. No significant differences were found in serum triglycerides (TAG), total-/HDL cholesterol, apolipoprotein A-1 (apoA-1) and apolipoprotein B (apoB) between the diets. Saturated fatty acid-rich diets prepared with either PO and CO, or a virgin OO diet high in oleic acid did not alter postprandial nor fasting plasma levels of tHcy and the selected inflammatory markers measured. However, OO diet was suggested to have beneficial effect in lipid mediators by lowering serum LTB4 compare to the other two diets. PO diet was suggested to have favourable effect on thrombosis tendency by raising PGF1α compared to OO diet but not CO. The experimental diets prepared with PO and OO had comparable non-hypercholesterolemic effects, while the 12:0+14:0-rich CO diet was more hypercholesterolemic compared with the 18:1-rich OO diet in the human volunteers.

Background: Women with previous Gestational Diabetes Mellitus (GDM) have increased risk for type 2 diabetes mellitus. Conventional diets for risk prevention have limited success in achieving or sustaining weight loss in them. Lowering dietary glycaemic index (GI) has been shown to facilitate greater weight loss in subjects with insulin resistance. Hence, we evaluated the effects of adding low GI nutrition education to conventional healthy dietary recommendations on glucose homeostasis, cardiovascular risks and body weight of women with previous GDM. Methodology: Seventy seven, non-diabetic, women with previous GDM (aged 20-40y, mean BMI: 26.4±4.6kg/m2) were randomised into two groups. Thirty-eight subjects received only conventional dietary recommendations (CHDR). Thirty-nine subjects received in addition to CHDR, low GI education (LGI). Changes in fasting (FBG) and 2h-postprandial blood glucose (2HPP), fasting insulin, blood pressure, fasting serum lipids, high sensitivity C-reactive protein, endothelial dysfunction, anthropometric measures, body composition and dietary intakes were measured before and after intervention. Results: After 12 months of intervention, CHDR subjects had significant increase in FBG, total body fat (both p<0.01) and trunk fat (p<0.05). CHDR subjects also had significant reductions in LDL Cholesterol (p<0.001). WHR significantly reduced in LGI group (p<0.05). Subjects in both groups had significant reductions in lean body mass (p<0.001), total: HDL Cholesterol and LDL: HDL Cholesterol ratios (p<0.05). However, changes in none of the outcomes measured were statistically different between groups (p≥0.05). Nevertheless, the magnitude of FBG increase was two-fold higher in the CHDR group (0.2±0.3 vs.0.3±1.0mmol/L, p=0.967, ES 0.6 vs. 0.3). Similarly, the magnitude of total body fat increase was also higher in the CHDR group, compared with the LGI group (1.2±2.4 vs. 0.87±2.7kg, p=0.580, ES 0.5 vs. 0.3). After 12 months, LGI group had a mean weight loss four times in magnitude to that observed in CHDR group (mean ± SD): 1.03±4.1 and 0.16±2.8kg, p=0.280, effect size 0.25 vs. 0.06). More subjects in LGI group attained 7% (p<0.01) and 10% (p<0.05) weight loss. After intervention, calculated dietary GI of LGI subjects were significantly lower (58 vs.64, p<0.001). Estimated intakes of dietary fibre was also significantly higher among LGI compared to CHDR subjects (17 vs. 13g, p<0.001). Interestingly, sub-group analysis showed LGI intervention had favourable outcomes in terms of managing FBG and triglycerides especially among subjects with higher baseline fasting insulin levels (p<0.05). Conclusion: LGI and CHDR nutrition-education interventions were comparable in terms of managing glucose homeostasis, cardiovascular risks and body weight among women with previous GDM one year after intervention. However, more subjects in LGI group achieved a clinically significant weight loss. Also, LGI nutrition education improved dietary fibre intakes as compared to CHDR. Low GI diets are a viable alternative to iso-caloric low-fat diets in managing post-GDM metabolic risks.

Breast cancer has become an escalating disease that has highly significant ramifications for future global health. In Malaysia, breast cancer is the number one cancer killer and affects the health of Malaysian Women from all Walks of life. Novel approaches on diagnosis and treatment of breast cancer are urgently needed to improve the clinical outcome of these patients. Recent studies have suggested that mesenchymal stem cells (MSCs) can migrate to and incorporate Within the tumour tissues This finding offers a novel Way to deliver therapeutic agents to the tumour sites. In the study, We propose to generate genetically engineered MSCs (GE-MSCs) that express and secrete two anti-tumour cytokines, IL-12 and IL-I8 The efficacy of these GE-MSCs Will then be tested in vitro on a panel of breast carcinoma cell lines (MCF-7, MDA-MB-231, and T-47D) In the present study, bone marrow-derived MSCs were characterized by immunophenotyping analysis and differentiation studies The plastic-adherent cells can be referred as MSCs as set by the International Society for Cellular Therapy. MSCs were also found to selectively migrate towards all breast carcinoma cells tested in a dose- dependent manner (p<0.05) in vitro. pBLAST42/hlL-12 and pFUSE/hIL-18 recombinant plasmid were successfully constructed and GE-MSCs Were generated by nucleofection with these plasmids and antibiotic selection. IL-12 and IL-18 secreted by GE-MSCs were able to induce IFN-7 production by peripheral blood mononuclear cells (PBMCs). A marked increase was observed When both cytokines were used, demonstrating their synergistic effects. Apart from that, these GE-MSCs retained the basic properties of MSCs such as differential plasticity and migration abilities. The GE-MSCs appear to possess potent antiproliferative and cytotoxic effects against breast carcinoma cells in the T-cell mediated and NK-cell mediated cytotoxicity assays. Further experiments have shown that the cytotoxicity observed may be partially owing to the induction of apoptosis by T cells and the induction of apoptosis and necrosis by NK cells, and the utility of GE-MSC to initiate apoptosis or necrosis Was specifically aimed at breast carcinoma cells, but not at non-tumourigenic breast cells. MSCs also do not prevent the priming of T cell activation as GE-MSCs Were capable of activating unprimed T lymphocytes by up-regulating the activation marker CD69. Increased expression of the surface molecule in T lymphocytes may in part enhance the treatment efficacy.

Pesticides are widely used in agricultural activities in Malaysia. Some of the common pesticides used include organophosphate compounds such as chlorpyrifos and diazinon, and organochlorine chemicals such as lindane, heptachlor, endosulfan, DDT, chlordane, aldrin, dieldrin and endrin. Pesticides can cause adverse effects on the ecosystems due to their long half-life, and ultimately may end up in the sea affecting the marine ecosystems. Humans are inevitably exposed to pesticides through environmental contamination or occupational use. The primary aim of this study was to assess the toxicity of five pesticides, namely chlorpyrifos, lindane, endosulfan, dichlorvos and malathion using an integrated approach based on microalgae and animal cells. In addition, the mutagenicity of the pesticides was assessed by Ames test using Salmonella typhimurium. Results showed that lindane was the only pesticide that could cause mutation after liver enzyme activation. The toxic effect of the pesticides on normal human liver (THLE-2) and mouse embryo fibroblast (3T3) cells was assessed based on 72 h MTT (3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazoliumbromide) assay. Of the pesticides tested, endosulfan was most toxic (EC50 = 20.4 μg/mL) against THLE-2 cells while chlorpyrifos was most toxic (EC50 = 2.5 μg/mL) against 3T3 cells. Toxicity testing of the pesticides on three marine microalgae, namely Pavlova gyrans, Isochrysis sp. (clone T-ISO), and Dunaliella tertiolecta was conducted based on their five-day growth response assessed by optical density at 620 nm (OD620). Of the five pesticides tested, endosulfan was most toxic against Pavlova gyrans (EC50 = 0.17 μg/mL) and Dunaliella tertiolecta (EC50 = 0.35 μg/mL). In comparison, Dunaliella tertiolecta was most resistant to all the pesticides tested (EC50 = 0.35 - 28.09 μg/mL). Random Amplified Polymorphic DNA (RAPD) technique was used to detect DNA damage caused by the pesticides in animal and algae cells. A total of 31 sets of primers were used and appearance/disappearance of bands in the pesticides-treated cells were compared with the control. The change in RAPD band patterns was more obvious in animal cells compared to microalgae. Most of the changes in PCR banding patterns were random, but the frequencies were higher when the DNA was amplified with primer OPB 5 series, OPB 7 series and OPB 8. Based on the RAPD analysis, Pavlova gyrans showed the least changes in terms of DNA band pattern compared to the other species of microalgae. The induction of apoptosis by the pesticides in animal and algae cells was assessed based on oxidative DNA damage, nucleus condensation, and caspase-3 activity. Endosulfan and dichlorvos caused oxidative DNA damage in both mouse embryo fibroblast and human liver cells, while chlorpyrifos only caused oxidative DNA damage in mouse embryo fibroblast cells. In nucleus and DNA condensation study by Hoechst stain, apoptosis was detected in malathion-treated mouse embryo fibroblast cells with obvious DNA condensation after exposure to the pesticides for 4 h. Results from the caspase-3 activation study showed that human liver cells were more susceptible to caspase-3 induction by pesticides than mouse embryo fibroblast cells. Endosulfan and lindane showed the ability to increase the caspase-3 activity in liver cells. The findings suggest that the pesticides tested could adversely affect the growth, and may cause DNA damage in both marine microalgae and animal cells. The pesticides may impact on the marine ecosystem as well as human health.

Global gene expression profiling has uncovered previously unrecognised subsets of human breast cancer, including the so-called “triple-negative” or “basal-like” tumours (BLBC) characterised by oestrogen/progesterone receptor negativity, lack of HER2/Neu amplification and high frequency of p53 mutation. These refractory tumours therefore are insensitive to hormonal or Herceptin-based therapy, and thus confer a markedly poor prognosis relative to other subtypes. To date, little progress has been made to identify specific molecular pathways associated with these refractory cancers that may be effectively targeted for therapeutic purposes. In the present study, I have established a robust high-throughput RNAi screening assay. Through a human kinome RNAi library screen, 37 candidate genes which mediate the proliferation and survival of basal-like MDA-MB-468 breast cancer cells have been identified. Subsequent meta-analyses via Oncomine and public database, and validation by RNAi approach have established the significance of five candidate kinases – AURKB, FGFR4, PIK3CD, RIPK1 and SPHK1 in the proliferation and survival of breast cancers. In particular, knock-down of endogenous FGFR4 induces tumour-specific cell death in basal-like MDA-MB-468 and HCC1937 but not in luminal-like breast cancers (SKBR3, T47D and MCF7). The FGFR4-dependent survival of these BLBC cells is attributed to the constitutive activation of FGFR4 in these cancer cells, as marked by the presence of phosphorylated-FGFR4. Further analysis reveals that FGFR4 mediates the BLBC cells survival through activation of AKT and STAT3 as knock-down of endogenous FGFR4 in MDA-MB-468 and HCC1937 cells significantly reduced AKT and STAT3 phosphorylation but not the ERK1/2 phosphorylation. Ectopic expression of a constitutively active myristoylated AKT completely abrogates the apoptosis induced by FGFR4 knock-down suggesting that AKT is required for the pro-survival effects of FGFR4. Furthermore, we demonstrated that FGF19, a ligand specific for FGFR4, was secreted in the basal-like MDA-MB-468, MDA-MB-231 and HCC1937 but not in luminal-like breast cancers (SKBR3, T47D and MCF-7) and the non-transformed myoepithelial cells (MCF-10A and HMEC). Interestingly, depletion of FGF19 in MDA-MB-468 and HCC1937 also suppresses the tumour growth and reduced AKT and STAT3 phosphorylation. Together, our results demonstrated the existence of a FGFR4-FGF19 autocrine loop that mediates the survival of BLBC cells and could potentially be developed as a therapeutic target for future treatment of BLBC.

Mites are eight-legged acari that are classified under the class of Arachnida. Mites are diverse groups of invertebrate that can survive in different habitats. Domestic mites include the house dust mites (HDM) and storage mites that are associated with the home environment. These are the mites of medical and economic importance. Inhalation of dead or live mites, their faecal matter and other byproducts from our living environment can trigger asthma, rhinitis and contact dermatitis. The two species of mites under this study, Dermatophagoides pteronyssinus and Tyrophagus putrescentiae, belong to the group of HDM and storage mites respectively. HDM infestation occurs at homes, including in carpets, mattresses and bedding, clothing, stored food products, and house hold pets. HDM is a major causative agent for asthma and rhinitis in Malaysia. Storage mites represent one of the occupational hazards for farmers, field hands, mill workers, warehouse operators and other food industrial workers. Group 10 allergen of mite is a muscle protein, tropomyosin, which has received attention due to its cross-reactivity with some allergens from invertebrates, particularly in seafood, responsible for severe systemic anaphylaxis. The objectives of this study were to produce recombinant Group 10 allergens of D. pteronyssinus and T. putrescentiae and to assess their IgE binding reactivity with human sera; to produce monoclonal antibodies against these recombinant allergens and to apply them in the localisation of inhaled mite Group 10 allergens in respiratory system of animal models. Two species of mites, D. pteronyssinus and T. putrescentiae were cultured and harvested. The mites were homogenised and the crude mite proteins were used to immunise the mice for the production of anti-mite polyclonal antibodies. Total RNA of mites was isolated and cDNA was constructed. Specific primers were designed for the amplification of group 10 allergen genes of both species of mite (Der p 10 and Tyr p 10) through polymerase chain reaction (PCR). The PCR produced a 855 bp band, which is in accordance with the reported molecular weight of mite tropomyosin in previous studies. DNA sequencing of the PCR products further confirmed that they are the group 10 allergen genes. The PCR products were purified and digested with restriction enzymes before they were cloned into the expression vector pRSET C. The recombinant plasmids (pRSET C/Der p 10 and pRSET C/Tyr p 10) were purified and transformed into the expression host E. coli, BL21. Colony PCR was carried out to screen and to select the colonies of BL21 that carried the recombinant plasmids. The selected positive BL21 clones were subjected to recombinant allergen protein expression under the induction of IPTG. The recombinant allergens were screened in western blot by using anti-His antibody and sera of immunised mice. A protein band of about 38 kDa which represent the group 10 mite allergen was detected. The recombinant allergens were purified with nickel-nitrilotriacetic acid (Ni-NTA) resin affinity column. Most of the unwanted bacterial proteins were removed throughout the purification process leaving mainly the recombinant allergen protein (38 kDa) in the elution fraction. A panel of 44 human sera from allergic and normal human subjects was used to assess the IgE binding reactivity of the recombinant mite tropomyosin by dot blot immunoassay. The results showed 4.5% (2 / 44) and 2.3% (1 / 44) positive reaction to recombinant Der p 10 and recombinant Tyr p 10 respectively. The purified recombinant mite allergens were used to immunise the mice for the production of monoclonal antibodies against group 10 allergen. The spleens of the mice were removed and splenocytes were fused with NS1 myeloma cells. The hybridoma cells were screened for the presence of anti-mite tropomyosin specific antibody using dot blot immunoassay and western blot. One positive hybridoma clone for each species of mites was selected for the study. The selected positive hybridoma clones were DP10-F11 (D. pteronyssinus) and TP10-C5 (T. putrescentiae). The result of antibody isotyping showed that both of them were IgG1 with κ light chains. DP10-F11 antibody was found to be cross-reactive against Tyrophagus putrescentiae (68%), Glycycometus malaysiensis (50%), Aleuroglyphus ovatus (51%), Blomia tropicalis (62%), D. farinae (102%) and shrimp (56%). TP10-C5 antibody demonstrated cross-reactivity with Dermatophagoides pteronyssinus (63%), Blomia tropicalis (59%), Dermatophagoides farinae (82%) and shrimp (58%). However, both of these antibodies did not cross-react with E. coli (< 50%). The hybridoma cells of DP10-F11 and TP10-C5 were intraperitoneally injected into the pristine-primed BALB/c mice and allowed to propagate inside them for the production of ascites fluid. The ascites fluids collected from mice were purified using the HiTrap Protein G HP column and fractioned protein liquid chromatography (FPLC). Both the purified monoclonal antibodies from ascites fluids showed positive reactivity to the recombinant mite tropomyosin in dot blot immunoassay and indirect ELISA. Female inbred Balb/c mice (n=6) were intranasally challenged with purified recombinant Der p 10 and recombinant Tyr p 10 for 10 consecutive days. Positive and negative control groups (n=6 each) were challenged with ovalbumin and PBS respectively. The mice from each group were re-challenged on day 17 for 3 consecutive days. The mice were sacrificed via cardiac puncture on the next day after re-challenge. Anti-mite tropomyosin polyclonal antibodies were detected in the sera of mice intranasally challenged with recombinant allergens. Histopathological changes such as infiltration of inflammatory cells and hypertrophy of airway epithelial cells were observed in the lung and trachea of mice challenged with recombinant allergens. The results of immunohistochemistry staining showed that both DP10-F11 and TP10-C5 antibodies demonstrated positive immunoreactivity at the alveolar epithelium in the lung tissues and fibrocartilaginous areas of the trachea. The recombinant group 10 allergens (tropomyosin) of D. pteronyssinus and T. putrescentiae were successfully produced in this study. Both of these recombinant allergens demonstrated their allergenicity through IgE binding reactivity with human sera. Monoclonal antibodies against recombinant group 10 allergens of D. pteronyssinus and T. putrescentiae were produced and were able to localize the inhaled allergens of D. pteronyssinus and T. putrescentiae in the respiratory system of mice. Both the research hypotheses of this study were accepted and all the objectives were achieved.

There are several types of vaccines used for prevention of infectious diseases such as attenuated microorganisms, recombinant proteins and DNA vaccines. Currently, studies are being carried out in developing vaccines for certain tumours. The cell-mediated arm of the immune system is the main arm involved in providing the host with the ability to defend, recover from viral infections and to prevent recurrent infections by the same virus. This type of immune response is also crucial in protecting the host against the onset, development and spread of tumour. Dendritic cells (DC) vaccine is a potent form of cancer immunotherapy that is currently being explored as a form of immunotherapy for various types of cancer. The potency of the DC vaccines is due to the ability of the DC to process and present antigens to T-lymphocytesas well as to induce antigen-specific immune responses. Development of DC vaccines against cancers has been hampered by poor immunogenicity of some of the tumour peptides. It has been proposed that induction of tumour-specific immune responses by DC vaccines could be boosted with the use ofasuitable adjuvants, which can help to induce a higher immune response to tumours as well as to confer protection against the said tumours. In addition, a few key elements of immune system such as metastatic tumour biology, cytokines productions, and antigen-specific lymphocyte activation need further understanding in order to develop an effective therapeutic agent to combat cancer diseases. The aim of this study is to examine the effectiveness of daily supplementation of tocotrienol-rich fraction (TRF) in enhancing the immunisation using DC vaccines against tumour antigens. Tocotrienol-rich fraction is a non-toxic natural micronutrient isolated from palm oil. There are several studies on the immune-enhancing effects of TRF and in this study we evaluate the use of TRF as an adjuvant to enhance tumour-specific immune response induced by DC-based cancer vaccines to prevent tumour growth and metastasis in a mouse model of breast cancer. In this model, 4T1 cells, which are mouse mammary cancer cells derived from BALB/c mice was used to induce breast cancer in female BALB/c mice. The results from thein-vitro work show that TRF markedly (p<0.05) inhibited proliferation of the 4T1 mouse mammary cancer cells. The 4T1 cells were more susceptible to TRF when compared toDC or mitogen-stimulated murine splenocytes. The IC50 value of TRF on 4T1 cells following 72 hours of culturewas found to be 8g/ml. In contrast, there was an overall increase in the viability of DCs and murine splenocytes treated with 20 to 25g/ml TRF for 72 hours. In thein-vivostudy, we found that the combination treatment of using DC pulsed with tumour lysate (TL) from 4T1 cells and supplemented daily with TRF (DC+TL+TRF) markedly (p<0.05) inhibited tumour growth and metastasis in mice with pre-established tumours. Systemic administration of 1 mg TRF daily was found to be capable of mediating significant increases in DC-based immunisations. In addition, TRF supplementation also was also efficacious in increasing cell-mediated immunity to tumour challenge as evident by the increased cytotoxic T-lymphocyte (CTL) activity observed in mice from the DC+TL+TRF group. Furthermore, interferon-gamma (IFN-) and interleukin-12 (IL-12) production was increased (p<0.05) in mice from the DC+TL+TRFtreated group compared to the other groups. From the analysis of the microarray data, we found several genes that were differentially regulated in the DC exposed to different treatments. From this list of genes,we chose to carry out further work on one interesting gene, which is known as the “Special AT rich binding protein-1” (SATB1) gene. The SATB1 gene was reported to have dual functions in different cells. It addition, it was reported to have the ability to induce growth of aggressive breast cancer cells. The expression of the SATB1 gene was stably silenced in the 4T1 cells (4T1SATB1-). When the SATB1-silenced 4T1 cells were used to induce tumour in female BALB/c mice, we observed that tumour growth and incidence was lower in mice injected with the 4T1SATB1-cells and treated with DC+TL and supplemented with TRF (DC+TL+TRF) when compared to corresponding group where tumour was induced using the wild-type 4T1 (4T1-WT) cells. In conclusion, in this study we show that TRF can be used as an adjuvant to enhance tumour-specific immune response induced by DC-based vaccines in a syngeneic mouse model of breast cancer. In addition, we also show that TRF may exert its anti-tumour activity by down regulating the expression of the SATB1 gene in the tumour cells as well as by activating tumour-specific cell-mediated (TH1) immune responses in the animal as evident by increased production of IFN- and IL-12 and increased CTL activity. Hence, DC-based vaccines together with TRF as adjuvant is useful in controlling growth and metastasis of pre-established tumours and therefore may be clinically useful as a new immunotherapeutic approach towards In conclusion, in this study we show that TRF can be used as an adjuvant to enhance tumour-specific immune response induced by DC-based vaccines in a syngeneic mouse model of breast cancer. In addition, we also show that TRF may exert its anti-tumour activity by down regulating the expression of the SATB1 gene in the tumour cells as well as by activating tumour-specific cell-mediated (TH1) immune responses in the animal as evident by increased production of IFN- and IL-12 and increased CTL activity. Hence, DC-based vaccines together with TRF as adjuvant is useful in controlling growth and metastasis of pre-established tumours and therefore may be clinically useful as a new immunotherapeutic approach towards cancers.

Acanthamoeba is ubiquitous free living amoebae in the soil and water. It is recognized as opportunistic pathogens that can cause ulcerative keratitis in contact lens wearer and granulomatous amoebic encephalitis in immune-compromised patients. The present work shows the detection of Acanthamoeba cyst in the mechanical ventilation and air conditioning system could give an indication of the ventilation system faulty. Dust swab on the supplying diffuser is a better approach when assessing the organism in the indoor ambient compared to single stage impactor sampler and dust particulates sampler. A logistic regression model was developed based on field data and human experiences obtained from 107 sampling points from nine industrial indoor air quality assessments. The organism is statistically correlated with total fungi count and respirable particulates, PM10 found within the ambient. It is also positively correlated with the sick building syndrome experienced by the indoor occupants. Efforts should be taken to consider Acanthamoeba as one of the ambient indicator when practicing indoor air quality assessment.

Toll-like receptor 4 (TLR-4) is recognised for its role in host innate immunity. Studies on TLR-4 over-activation had linked it to cancer survival and progression. Nevertheless, there is no TLR-4 inhibitor available for cancer treatment. Therefore, the aim of this project is to identify small molecules as TLR-4 inhibitors for cancer treatment. Computational screening methods were used to identify potential TLR-4 ligands (hits). In silico model was found to be in agreement with the results from the in vitro TLR-4 signalling assay. Lead compounds were then designed. Molecular docking reported that a potent TLR-4 inhibitor forms strong binding with both TLR-4 and myeloid differentiation 2 (MD-2). NF-κB reporter assay and Griess assay studies further confirmed these lead compounds induced potent and selective TLR-4 inhibitory effects. Immunoblotting on macrophages and HEK-BlueTM hTLR4 cells further confirmed that the most potent lead compounds, BZD3 and EP13, inhibited lipopolysaccharide (LPSEc) induced MyD88 dependent pathway, but not MyD88 independent pathway in macrophages and HEK-BlueTM hTLR-4 cells. These two lead compounds also induced potent cancer specific anti-proliferative effects and III apoptotic cell death in cancer cells. TLR-4 and MD-2 ELISA also confirmed that BZD3 and EP13 exhibited TLR-4 inhibitory effects on cancer cells by lowering the expression of TLR-4 and MD-2 levels in the cancer cells. Immunoblotting experiments supported that BZD3 and EP13 inhibited the phosphorylation of downstream MyD88 dependent and MyD88 independent pathways in cancer cells. In conclusion, this study has identified a panel of TLR-4 inhibitors that may play a role in cancer survival and progression.

The p53 tumour suppressor is the most frequently mutated gene in human cancers. Unlike other tumour-suppressor genes, p53 mutations mainly occur as missense mutations within the DNA-binding domain, leading to the stable expression of full-length mutant p53 proteins. A wide range of studies shows that mutant p53 proteins not only lose their tumour suppressor function, but may also actively promote tumourigenesis through gain-of-function (GOF) mechanisms. As such, expression of mutant p53 proteins is commonly associated with poor clinical prognosis and high metastatic rate. Anoikis is a form of apoptosis in response to loss of cell adhesion or inappropriate cell adhesion. Gaining resistance to anoikis may be a general necessity for the tumour cells to metastasise. Here, we showed that p53 R273H contact mutant, but not p53 R175H conformation mutant, suppressed anoikis by down-regulation of BCL2 modifying factor (BMF) expression. Ectopic expression of the activated form of AKT in MDA-MB-468 completely „rescued‟ the apoptotic effects following depletion of endogenous mutant p53, suggesting that p53 R273H mutant promotes anoikis resistance in breast cancer cells through suppression of BMF expression by activating AKT signalling pathway. In addition, our findings also demonstrated that p53 R273H contact mutant regulate the expression of miR-19 and miR-200 family member, suggesting that mutant p53 R273H might promote tumourigenesis through modulation of microRNAs expression. Finally, we showed that ectopic expression of wild-type and mutant p53 in p53-null cells confers differential regulation of metabolic genes, suggesting that wild-type and mutant p53 play a distinct role in regulating cellular metabolism in tumour cells in a cell type dependent context. In summary, our findings suggest a mechanism by which p53 mutants might promote tumorigenesis through 1) regulation of cancer cell survival via AKT-dependent suppression of BMF in a wide range of tumour types; 2) regulation of miRNA expression; and 3) modulation of cancer cell metabolism.