Theses (MSc. Medical and Health Sciences)

Many in vivo and in vitro studies have demonstrated the crucial role of dendritic cells (DC) in activating T-lymphocytes. In this study, we aimed to investigate the therapeutic roles of DC in breast cancer using in vitro and in vivo models. We also compared the therapeutic efficacy between cryopreserved and freshly generated DC using the same approaches. Human DC were generated from peripheral blood mobilised CD34+ human haematopoietic stem cells (HSC) using appropriate cytokines cocktail. The resulting DC were characterised based on morphology, phenotype and functional properties such as their ability to induce tumour-specific cytotoxic T-lymphocytes (CTL) against human breast cancer cell lines in vitro. Murine DC were generated from bone marrow (BM) cells harvested from female BALB/c mice and were similarly characterised. The mouse DC were found to be CD11C+CD11b+CD4-CD8- i.e. double negative dendritic cells (DNDC). An animal model using murine breast cancer cells was established for in vivo studies. The cryopreserved and freshly generated DNDC showed similar morphology and phenotype. These DC were pulsed with tumour lysate and injected into experimental animals with established tumours. Both cryopreserved and freshly generated DC were able inhibit the growth of established tumours in a similar manner. The mean time for tumour volume to reach 500 mm3 was significantly delayed (P<0.05) in mice treated with either freshly generated (33.9 days) or cryopreserved (35 days) BM-DC as compared to control mice (25.7 days). In another model, mice were vaccinated with tumour lysate-pulsed dendritic cells (TPDC) prior to the induction of breast cancer. Tumour growth in vaccinated mice were found to be significantly (P<0.05) slower as compared to control untreated mice. The mean time for tumour volume of control and DC-vaccinated mice group to reach the 500 mm3 was 24.3 days and 48.2 days respectively. Tumour lysate-pulsed DNDC (TPDC) and unpulsed DNDC produced no IFN- even in the presence of IL-12 or both IL-12 and IL-2. T-lymphocytes cultured with TPDC produced higher IFN- and lower IL-4 and IL-10 level, indicating that a Th1 immune response was elicited by DNDC. Our data show that DC generated from CD34+ HSC are capable of eliciting immune response towards human breast cancer in vitro. Murine bone-marrow-derived DNDC were shown to have different functional properties from previously described DNDC isolated from mouse spleen. These findings offer new insights into the functional properties of DNDC from different sources. We also showed that cryopreserved and freshly generated murine DC share similar therapeutic efficacy and causes an initial retardation in tumour growth shortly after they are administered.

Rheumatoid arthritis (RA) is an autoimmune disease characterised by chronic inflammation and synovitis, which will often lead to the destruction of cartilage and bones. A large number of cytokines are detected in rheumatoid synovium and they have been classified into two groups according to their inflammatory properties i.e. pro- and anti-inflammatory cytokines. Although the effect of pro- and anti-inflammatory cytokines are more clearly defined in animal models, their role in the pathogenesis of RA in humans is still poorly understood. Interleukin-10 (IL-10) is an anti-inflammatory cytokine and has been reported to exert a protective role in animal models of RA. The human IL-10 gene promoter region is polymorphic and three proximal single nucleotide polymorphisms (SNPs) at positions -1082G>A, -824C>T and -597C>A have been reported. These SNPs are found in the putative regulatory region, which could affect the transcription of the human IL-10 gene and this in turn could affect the production of IL-10. We compared the frequencies of the SNPs in the human IL-10 gene promoter between RA patients and control subjects. The SNPs in the human IL-10 gene promoter were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. The results from the PCR-RFLP were validated by DNA sequencing. The differences of the SNP genotype, allele or haplotype frequencies observed between RA patients and control subjects were found to be statistically not significant. The SNPs in the human IL-10 gene promoter have also been shown to be statistically correlated with the clinical measures of RA such as deformity, production of rheumatoid factor and early age of RA onset. The levels of IL-10 produced by Concanavalin A (Con-A)-stimulated peripheral blood leucocytes (PBL) were obtained from RA patients and control subjects using ELISA. The -1082G wild-type allele was associated with higher IL-10 protein production compared to the -1082A mutant allele. Interleukin-10 production was significantly lower in RA patients compared to control subjects. We also measured the levels of interferon-gamma (IFN-γ) and Interleukin-4 (IL-4) production in conjunction with IL-10 production. There was significantly higher production of IFN-γ in RA patients than control subjects. In contrast, significantly lower production of IL-4 was observed in these patients. The results confirm that RA is a TH1-driven clinical condition and that TH2 cytokines are important in the control of RA. The selective expression of Interleukin-12 beta-2 receptor (IL-12β2R) mRNA and soluble CD30 (sCD30) protein were analysed to study the basis of TH1/TH2 immune response in RA. The IL-12β2R mRNA expression was analysed using reverse-transcription-polymerase chain reaction (RT-PCR) method while the sCD30 levels were determined by ELISA. There was no significant difference observed on the expression of the IL-12β2R mRNA between RA patients and control subjects. However, mean sCD30 level was significantly higher in RA patients than control subjects. Soluble CD30 level was significantly lower in RA patients with active disease than those with their disease under control.

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in Malaysia. Progression of the CRC and its invasiveness involve several possible mechanisms such as defects in angiogenesis, cell cycle, Wnt signalling pathway and most recently, by Stat3 signalling pathway. The current study was carried out to evaluate proteins involved in these pathways and to relate their expression with tumour (histological grading and Dukes’ staging) and patient characteristics (ethnicity, age, and gender), with a view to identify potential biomarkers for use in patient management. A total of 93 CRC specimens were studied histologically using Haematoxylin and Eosin (H&E) staining as well as immunohistochemical staining for vascular endothelial growth factor (VEGF), matrix metalloproteinase 7 (MMP-7), cyclin D1, and cyclin E. A total of 90 specimens were evaluated for CD31 and 80 for -catenin, and 75 for Stat3 and pStat3 expression. The histological findings were correlated with morphological characteristics including tumour differentiation, Dukes’ staging, and patient characteristics. Positive expression rates for -catenin, VEGF, CD31, cyclin E, MMP-7, Stat3, cyclin D1, and pStat3 were 68.8%, 59.1%, 55.6%, 55.1%, 38.8%, 26.8%, 22.6%, and 13.3% respectively. Significant association was found between overexpression of VEGF, MMP-7, β-catenin (cytoplasmic and nuclear expression), and Stat3 with the histological grading of CRC. However, no significant association was found between the overexpression of these markers and Dukes’ staging or other patient characteristics. No significant association was found between cyclin E, cyclin D1, pStat3, and CD31 with tumour or patient characteristics. Co-expression analyses showed that VEGF expression was significantly associated with MMP-7, -catenin, Stat3 and pStat3 (P < 0.05). A positive VEGF has a high positive predictive value of > 85% for β-catenin. Significant association of cyclin D1 was shown for β-catenin, and the positive predictive value of the former for the latter was 90%. The study has shown that VEGF, MMP-7, β-catenin and Stat3 correlate well with the histological grading but not the staging of CRC. There is a high positive predictive value between VEGF and β-catenin, and also between cyclin D1 and β-catenin. However, -catenin and VEGF have the highest positive expression rates. It is concluded that these two biomarkers would be most useful for evaluating the histological grade of these tumours.

The Jehai is one of the group of “original people” (Aborigines) of Peninsular Malaysia, who live in the Belum and the Temenggor forest that straddles Upper Perak and in West Kelantan. Our study population are the Jehai who live in 4 villages. One Jehai village is located in Sungai Tekam river and the other three Jehai villages are located along the lakeside of Lake Temenggor. No health research has been done so far on this Jehai community. They live without safe water supply, electricity, latrines and proper garbage disposal. Their incomes are low and unfixed; in fact their survival still depends on their ability to live creatively with their natural environment. A total of 175 stool samples, preserved in 10% formalin or PVA (polyvinyl alcohol) were processed and stained using Trichrome, modified Ziehl-Neelsen and iodine stains and examined under microscope to identify the eggs of intestinal helminths and intestinal protozoa. The prevalence rates of Trichuris trichiura, Ascaris lumbricoides and hookworm among the Jehai were 70.8%, 24.0%, and 10.9% respectively. The prevalence of Entamoeba coli, Entamoeba histolytica, Giardia lamblia, Blastocystis hominis, and microsporidium were 40.6%, 33.7%, 25.7%, 91.4%, and 27.4% respectively . The difference in prevalence among the gender, age and ethnic groups were found to be not significant. The age group 0-9 years old had the highest prevalence rate of all the intestinal parasites. A total of 157 finger-prick blood samples were made into thin and thick blood smears, stained with 10% Giemsa and examined with microscope. We found the overall infection rate of Brugia malayi and Plasmodium vivax were 1.27% and 12.1 % respectively. The overall prevalence of Schistosoma spp. specific antibody positive by ELISA test was 20.4%. Seventy three point two (73.2) percent were found to be anaemic with an overall mean haemoglobin concentration of 9.6 g/dl. We also found positive associations between anaemia with the hookworm and malaria infections. In conclusion, high prevalence rate of intestinal parasite infections and Schistosoma sp. were found in these Jehai. Poor sanitation and poor living conditions probably contribute to these findings. Relatively low prevalence rates of filariasis and malaria could have resulted from the Jehai unknown immunity systems or their genetic variability. High prevalence of anaemia was due to their poor nutrition and high intestinal parasite infections. The present study represents the first investigation on parasitic infections among such a large group of Jehai after their change from a nomadic existence to a government sponsored resettlement.

Vitamin E is a major lipid-soluble anti-oxidant, which scavenges free radicals in biological membrane and protects the cellular structure against oxidative damage. Several studies have shown that vitamin E, both tocopherol and tocotrienol supplementation induces a favourable effect on animal and human immune system and has been implicated in the reduced risk for several immune and inflammatory diseases. In this study, We first examined the immunoinodulatory effects of orally administered tocotrienols and tocopherols in the mouse model upon an immunogenic challenge, and then we investigated the effect of tocotrienol rich fraction (TRF) supplementation on immune modulation in normal healthy volunteers Whose immune system was challenged With a booster tetanus toxoid (TT) vaccine. In the mouse model, 20 female BALB/c mice were divided into four groups of five mice each and were orally gavaged with on-tocopherol (α-T), TRF and δ-tocotiienol (δ-T3) respectively while the control animals were not given any supplements. Two Weeks after the supplementation, mice from all four groups received a single dose of TT vaccine (4 flocculation units (Lf)) injected intramuscularly in the left hind leg quadriceps of the mice. This was followed by a second and third booster doses at intervals of two-weeks. Sera from mice were collected via retro-orbital bleeding on day 0, 28 and 56. All experimental animals were orally-gavaged with the respective supplements for two months until they were sacrificed on day 56. Blood, spleen and adipose tissue were harvested from the sacrificed mice. At autopsy, splenocytes from control and experimental mice were isolated and cultured in vitro in the presence of Concanavalin-A (ConA), TT or Lipopolysaccharide (LPS). The cytokine produced by the cultured splenocytes and the amount of anti-TT antibodies generated were quantified using the enzyme linked immunosorbent assay (ELISA) while the proliferation of splenic lymphocytes was measured using the methyl thiazole tetrazolium (MTT) assay. Results from the animal study demonstrate that vitamin E-treated mice showed a significant (P < 0. 05) increase in splenocytes proliferation as compared to untreated animals after TT vaccination. However, no differences in splenocytes proliferation were observed among groups treated with the different types of vitamin E. Interferon-gamma (IFN-ƴ) and Interleukin-4 (IL-4) productions by Con A or TT-stimulated splenocytes also significantly (P < 0. 05) increased in the vitamin E supplemented animals compared to control animals. The level of IFN-ƴ was highest in δ-T3 supplemented animals and this was followed by TRF and α-T fed mice. However, there was no significant (P > 0.05) difference observed in the levels of IL-4 produced within the vitamin E-treated groups. Secretion of Tumour Necrosis Factor-alpha (INF-α) was suppressed in the vitamin E-treated group compared to the untreated animals. The production of anti-TT antibodies was significantly (P < 0.05) augmented in δ-T3 and TRF supplemented animals compared to α-T fed mice. In the human study, 100 healthy female volunteers aged between 18-25 years were recruited to participate ill a randomised, placebo-controlled, double- blinded clinical trial. The volunteers were given either 400 mg vitamin E (TRF) or placebo daily for 2 months. On day 28, all volunteers were vaccinated with the TT vaccine (20 Lt) intramuscularly. Blood samples were obtained from all volunteers on day 0, 28, and 56 for flow cytometry analysis, blood leucocyte culture and analysis of plasma vitamin E as well as anti-TT antibody levels. Plasma vitamin E was determined by high performance liquid chromatography (HPLC) while plasma levels of anti-TT antibody i.e. Immunoglobulin G (IgG) was quantified by ELISA. The amount of IFN-ƴ, IL-4, Interleukin-10 (IL-10) and Interleukin-6 (IL-6) produced by mitogen or TT-stimulated leucocyte culture was determined using ELISA. The results from the clinical trial showed that TRF supplementation significantly increased total vitamin E levels ill the plasma of the TRF supplemented group as compared to placebo, indicating overall compliance. Volunteers supplemented with TRF showed a significant (P < 0.05) enhanced production of IFN-ƴ and IL-4 by the mitogen or TT-stimulated leucocytes as compared to control group. However, no differences were observed in the levels of IL-10 produced between the TRF and placebo supplemented groups. Volunteers supplemented with TRF produced significantly (P < 0.05) lower amounts of IL-6 as compared to the control group. Anti-TT IgG production was also significantly (P < 0.05) augmented in the TRF supplemented group compared to placebo group. In contrast, no significant changes were obse1ved in the proportion of total T-lymphocytes, B-lymphocytes as well as NK cells between volunteers who received TRF and placebo supplementation. The results from the clinical trial concur with the findings form the animal study. These results demonstrate the efficacy of TRF and 8-T3 for modulating an immune response towards a mixed TH1/TH2 type upon an immunogenic challenge. Therefore; we conclude that TRF and 8-T3 from palm oil have immunostimulatory effects and potential clinical benefits to enhance immune response to vaccines and probably, infectious agents.

Hypertensive disorders in Pregnancy contributes to about 12% of maternal deaths in Malaysia and similarly worldwide. Early detection and adequate management are preventable strategies. Biochemical markers of abnormal placentation would be more specific in early detection than the traditional clinical tools used. The aim of this study is to estimate maternal PlGF and sFlt-1 in PIH and to compare these values with normotensive pregnancies. As a corollary, the study also aimed to determine correlation between these biomarkers and morphometry and histopathology. The study examined three core modalities in relation to the investigation of PIH, viz. plasma concentrations of PlGF and sFlt-1 using commercially available test kits, morphometric evaluation and histopathology of the placenta. Statistical comparisan were performed using ANOVA, Spearmans Rank Correlation and Pearson Correlation tests (SPSS version 15.0). PlGF levels were found to be lower in PIH women in comparison to normotensive during antepartum and intrapartum period whereas sFlt-1 was elevated in PIH when compared to normotensives during the same stages of pregnancy. An inverse relationship between these two biomarkers was observed and strengthened by correlation analysis. It was noted that sFlt-1 remained significantly higher in PIH when compared to normotensives in the postpartum period. There were no differences among ethnic group, parity and maternal age. Unlike normotensive women where sFlt-1 level peaks at delivery and yet remain lower than PIH women, sFlt-1 levels in PIH women were much higher than normotensive women and continued to remain high during both antepartum and intrapartum period. PlGF was observed to have significant inverse correlation with total villous surface area of the placental periphery (TCsa – C) and villous capillarisation of the placental periphery (VC – C). Villous capillarisation and intervillous space it was generally observed that PIH women had higher villous capillarisation in comparison to normotensive women in all groups but statistically was not significant. Histopathological scorings by two independent pathologists revealed no significant difference between PIH, normotensive and HPT placentas. When correlating biomarker and histopathology scoring it was observed that when sFlt-1 intraprtum level increases, placental histopathological scoring becomes worse From the findings in this study, several conclusions can be drawn. Firstly, some of the biomarkers (sFlt-1 in particular) are useful and reliable in differentiating normotensive and PIH women. This also directly indicates that sFlt-1 is associated with the occurrence of PIH in women and it is possible that the longer one is exposed to sFlt-1 influence, the higher the likelihood of PIH development. Second, it is likely that the development of PIH in women assessed in this study is related to defective capillarisation since we have shown that there is an inverse relationship between the pro-angiogenic factor PlGF and the anti-angiogenic factor sFlt-1. Third, while morphometric and histopathologic changes were identified in this study, the assessment done using these two techniques showed that these two modalities were unable to distinguish between normotensive, PIH and HPT women, as no significant correlations were observed. However the correlation observed with TCsa – C and VC – C and PlGF indicate possible capillarisation defect, and thus warrants further investigation with a larger sample size in a multi-centric study. This study definitely provides evidence that plasma biomarkers and placental morphometry should remain a mainstay in PIH research and large scale clinical trials using these biomarkers should be conducted to help elucidate the complex mechanism of pregnancy induced hypertension.

To achieve the objectives, the following was performed and analysed. Bt18 parasporal proteins were separated through anion exchange chromatography and a 68-kDa parasporal protein with cytotoxic activity was purified. Polyclonal IgG (anti-Bt18) for the 68-kDa parasporal protein was successfully raised and purified. The purified anti-Bt-18 IgG showed interaction with parasporal proteins of Bt18, using immunoblot analysis. The purified anti-Bt18 IgG was tested for cross-reactivity against other Bt and Bacillus isolates using Indirect ELISA (quantitative assay). It was observed that Bt isolates share similar antigenic properties with the 68-kDa protein of Bt18 whereas very low cross-reactivity was observed with Bacillus sphaericus, Bacillus subtilis and Bacillus cereus isolates (less than 17%). Immunoblot analysis (qualitative assay) for cross-reactivity also showed that there was a 68- kDa protein in Bt isolates. Cross-reactivity studies via quantitative and qualitative analysis was repeated using purified Bt parasporal protein. Indirect ELISA assay showed the percentage of cross-reactivity was reduced up to 60% after purification for some Bt isolates; Bt2, Bt10, Bt15, Bt19 and Bt20. Meanwhile other Bt isolates still showed more than 90% cross-reactivity. Qualitative assay for cross-reactivity via immunoblot assay detected bands for all the Bt isolates. Similar band profiles were observed in immunoblots using either purified or unpurified Bt parasporal proteins. Receptor binding assay revealed that Bt18 parasporal proteins bound to a 34-kDa protein from the CEM-SS cells lysate. N-terminal amino acid sequence of the 34-kDa protein was GKVKVGVNGFGRIGG. NCBI protein BLAST revealed that the binding protein was Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a protein with multiple functions, including a vital role in mitochondrial apoptosis. It is interesting to note that Bt18 produces parasporal proteins that act like parasporins. Binding of Bt18 parasporal proteins to GAPDH is a significant finding in this study as literature shows no studies reports on identification of a putative receptor for parasporins. Immuno-staining to detect the binding of Bt18 parasporal protein on CEM-SS cells showed brownish ring formation around the cells, suggesting that the protein may bind to a receptor on the plasma membrane of cells. The specificity of Bt18 parasporal proteins for different monosaccharides showed preferential binding (decreasing order) to D-mannose, D-Lactose and D-glucose. Glycoprotein staining showed that Bt18 parasporal proteins contain three distinct glycoprotein bands about; 200, 100 and 68-kDa. Therefore, two key conclusions can be made; that there is a target protein for Bt18 parasporal proteins in CEM-SS cells and that Bt18 parasporal proteins are lectins that specifically recognise monosaccharides.

Vibrio parahaemolyticus is a gram-negative halophilic bacteria found mainly in the marine and estuarine environment. Cases of V.parahaemolyticus were mostly sporadic and associated with diverse serovars. The emergence of a pandemic serovar O3:K6 in 1996 however changed the epidemiology abruptly and has since accounted for V.parahaemolyticus outbreaks worldwide. In Malaysia, little information concerning the molecular epidemiology of this organism is known in spite of cases reported regionally. Therefore this study elucidates the serotype distribution and molecular characteristics of V.parahaemolyticus isolates in Malaysia. The strains studied were a collection of 17 clinical and 42 environmental isolates obtained from 1997 to 2007. From this study, O3:K6 was the dominant serotype and was found only in clinical isolates. Other serotypes found in clinical isolates include O4:K68, O1:KUT, O3:K5 and O4:K8. The environmental isolates were different with O12:KUT, O3:KUT and O6:KUT as the major serotypes. Serotype O4:K68, O4:K8, O1:KUT, and O4:K12 were found in both clinical and environmental isolates. One environmental isolate was untypable by the O and K antisera. PCR results showed all clinical and environmental isolates were positive for the toxR gene. Based on its genotypic traits, the V.parahaemolyticus isolates were divided into four groups. Group I (tdh+, trh-, ORF8+, GS-PCR+), Group II (tdh+, trh-, ORF8-, GS-PCR-), Group III (tdh+, trh+, ORF8-, GS-PCR-) and Group IV (tdh-, trh-, ORF8-, GS-PCR-). Of this, 13 clinical isolates with serotype O3:K6 and O4:K68 falls into Group 1, thus belonging to the pandemic clone. Two clinical isolates were in Group II whereas one clinical isolate was in Group III. Interestingly one clinical isolate falls into Group IV which is similar for all environmental isolates. The DNA sequences of the PCR products when analysed using Basic Local Alignment Search Tool (BLAST) showed high sequence similarity when compared to published sequence in the GenBank. PFGE was performed to determine the genetic relatedness of the isolates. All clinical isolates in Group I belonged to PFGE Type 1 with 9 subtypes, making it the dominant pattern. These isolates exhibits the same molecular characteristics as the pandemic strains found in other countries. The environmental isolates produce more diverse patterns and were clearly different form the clinical isolates. In conclusion, this study showed that the serotype distribution among the clinical isolates in Malaysia is stable within the last 10 years from 1997 to 2007, with O3:K6 as the dominant serotype. This serotype has been circulating in Malaysia as early as 1997. Although some serotypes were found in both clinical and environmental isolates, their molecular characteristics were totally different. None of the environmental isolates were positive for the toxin genes, ORF8 and GS-PCR. Finally, PFGE was shown to be more discriminative than serotyping and the two methods were not correlated.

The work described in this study aimed to clone and express CYP2A6 wild type and mutant proteins in bacterial system, to use the expressed proteins to investigate the structural and functional consequences of reported allele CYP2A6*15 (Lys194Glu), CYP2A6*16 (Arg203Ser), CYP2A6*21 (Lys476Arg) and CYP2A6*22 (Asp158Glu and Leu160Ile) and to characterize potency and mechanism of modulation of naturally occurring flavonoids on wild type CYP2A6 activity. Human wild type CYP2A6 was initially isolated from the human liver total RNA and four mutant alleles were successfully generated. Wild type and mutant CYP2A6 proteins were expressed at comparable level as revealed by Western blotting analyses. Interestingly, except for CYP2A6*22, all mutant CYP2A6 proteins exhibited catalytically active enzyme activities as evidenced from the high Soret peak at 450 nm in the absorbance spectral analyses and comparable activities from fluorescence-based coumarin 7-hydroxylase assay. Such observations indicated that amino acid mutations in the primary sequence of the respective CYP2A6 alleles did not affect the structural stability and functional catalytic activity in coumarin 7-hydroxylation. CYP2A6*22 exhibited only half the activity of the wild-type indicating that Asp158Glu and Leu160Ile substitutions had caused compromised activity in this allele. Screening of flavonoid compounds on coumarin 7-hydroxylase assay indicated different potency of inhibition on the wild type CYP2A6 activity. Among the twenty-three compounds tested, members were from flavonol group of hydroxylated flavonoids were the most inhibitory, with myricetin being the most potent inhibitor followed by quercetin, galangin and kaempferol. Further exploration of the inhibition mechanism of these compounds revealed that myricetin, galangin and kaempferol exhibited mixed-type of inhibition pattern while quercetin was observed to portrait competitive mode of inhibition in coumarin 7-hydroxylation. Structure-function analyses revealed that degree of inhibition was closely related to the number and location of hydroxyl groups, glycosylation of these free hydroxyl groups, degree of saturation of the flavane nucleus as well as the presence of the alkoxylated function.

Morinda citrifolia, better known as Mengkudu in Malaysia, or Noni in the western world has been used traditionally to treat many ailments including asthma, diabetes, high blood pressure, irregular menstruation and is believed to have anticoagulant, analgesic, anti-inflammatory, antioxidant, antiseptic, antibiotic, anticancer and immunomodulating activities. Research findings with the fruit juice (Tahitian Noni Juice) supported some of these claims. This study determines the effect of the hot water extract of the fruits of Morinda citrofolia (McHWE) on human blood coagulation, in vitro. Various concentrations of the lyophilized extract was used to study the effect on clotting time, coagulation profile and platelet aggregation with PBS (phosphate buffer) and EDTA used as the negative and the positive controls respectively. The data obtained, was analyzed using the ‘paired T-test’ and the ‘Wilcoxon Singed Ranks test’ (non-parametric test). All tested concentrations of McHWE (10mg/ml, to 150mg/ml) significantly increased clotting time compared to control (p<0.001). Prolongation in clotting time between the subsequent concentrations tested was also shown to be statistically significant (p<0.001) where a concentration dependent effect was demonstrated. The increase in concentration produces increase in response. Blood did not clot even after 24 hours with concentrations of McHWE higher than 150mg/ml. The various concentrations of McHWE (100mg/ml to 250mg/ml) affected the coagulation profile assay, with significant prolongation of the activated partial thromboplastin time (APTT), prothrombin time (PT), and the thrombin time (TT) respectively (p<0.001). A concentration dependent pattern was exhibited with all coagulation profile assays tested. The platelet aggregation assay, which showed a statistical significant decrease in platelet aggregation (p<0.001) with McHWE (concentrations 5mg/ml and 25mg/ml) for agonist, collagen, ristocetin and arachidonic acid, suggested that McHWE was most sensitive to the collagen test where a statistical significant decrease in platelet aggregation (p<0.001) was seen with concentration as low as 0.5mg/ml. Partial fractionation of crude McHWE was carried out with various solvents of n-butano1 and ethyl acetate. Both soluble and insoluble in ethyl acetate (which showed anticoagulant activity) was fractionated using silica gel column chromatography, with 3 different solvent systems: dichloromethane-acetic acid-methanol-water (64:32:12:8) {solvent system 1}, methanol-ethyl acetate (8:2) {solvent system 2} and Methanol-water (8:2) {Solvent system 3} with fractions of solvent system 1 showing highest anticoagulant activity (determined with the clotting time assay). Determination of the active compound(s) present was carried out using TLC and phytochemical analysis which suggested the presence of alkaloids, anthraquinones and saponins.

Bacillus thuringiensis (Bt) has long been used in the agriculture fields as insecticides. Recent studies showed that Bt toxins exhibit cytotoxicity against human cancer cells. This study looked at the cytotoxic effect of the toxin of a locally isolated Bt, namely Bt 18, and the mechanism of cell death it induced on a leukaemic cell line, CEM-SS. Previous works demonstrated that Bt 18 toxin preferentially killed CEM-SS cells but exerted little or no cytotoxicity on other human cancer cell lines and human T lymphocytes. Bt 18 was cultured, its toxin was harvested and purified by FPLC using Resource Q® column. Cell viability assays were carried out using the standard MTT calorimetric method. The mode of cell death was determined using the Cell Death Detection ELISAPLUS kit (Roche). Morphological analysis was carried out using light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bt 18 toxin was biotinylated for homologous competitive binding study using three leukaemic cell lines, CEM-SS, CCRF-SB, CCR-HSB-2 and a totally different class of cells, MCF-7 (breast cancer cells) for the determination of the dissociation constant (Kd) for each cell line. Heterologous competitive binding study using cisplatin, doxorubicin, etoposide, methotrexate and navelbine (commercial anticancer drugs), Btj toxin and Bt 22 toxin was carried out to determine if competition for binding sites existed between Bt 18 toxin and these test compounds on CEM-SS. To localise the binding site, the biotinylated toxin was tagged with FITC-conjugated avidin and visualised under the confocal microscope. At 72 hours, Bt 18 toxin and Btj toxin significantly reduced cell viability of CEM-SS to 72.45% and 63.62% at 20μg/ml respectively (p<0.001). Normal human T lymphocytes remained relatively unharmed with cell viability of 86.66% for Bt 18 toxin at 160μg/ml (p>0.05) but was significantly reduced to 55.77% for Btj toxin at 160μg/ml (p<0.05). The mode of cell death was found to be apoptosis for Bt 18 toxin and Btj toxin using the Cell Death ELISAPLUS kit. At 72 hours, the highest percentage of apoptotic cell death was achieved at 34.32% and 41.84% for Bt 18 toxin and Btj toxin respectively (p<0.001). Light microscopy, SEM and TEM demonstrated early apoptotic features at 3, 6 and 12 hours, followed by late secondary necrotic changes at 24 and 72 hours for both toxins. Homologous competitive binding study revealed decreasing binding affinity of Bt 18 toxin for CEM-SS, CCRF-SB and CCRF-HSB-2, with Kd of 8.44nM, 14.98nM and 17.71nM respectively. Kd for MCF-7 could not be determined due to insufficient displacement of the biotinylated toxin. Heterologous competitive study showed little competition between biotinylated Bt 18 toxin and Btj toxin, Bt 22 toxin (<30%) and all five anticancer drugs (<45%). Confocal microscopy revealed binding of biotinylated Bt 18 toxin at the periphery of the cell. In conclusion, Bt 18 toxin selectively exhibited cytotoxicity on CEM-SS but not normal human T lymphocytes. The mode of cell death was apoptosis. It most likely bound to binding sites on the cell surface of CEM-SS and its mechanism of cell death most likely differed from that of Btj toxin, Bt 22 toxin and all five anticancer drugs used in this study.

Nasopharyngeal carcinoma (NPC) is a type of neoplasm that is highly prevalent in East Asia and Africa with Epstein-Barr virus (EBV), genetic, and dietary factors being implicated as possible aetiologic factors. There have been studies conducted to examine the association of cytokines with the metastasis and invasion properties of cancer cells but the effect is still unclear yet in NPC. In the present study, the roles of EBV latent membrane protein-1 (LMP1) with Interleukin-6 (IL-6), Interleukin-10 (IL-10) and transforming growth factor-beta 1 (TGF-β1) in the metastasis of NPC were investigated. TW01 cells expressing LMP1 (TW01-LMP1) were established via lipofection of LMP1 gene into TW01 cells. These cells were treated with 100 pg/ml IL-6, 1000 pg/ml IL-10 and 100 pg/ml TGF-β1, separately and also in combination at their respective concentration for 48 hours. The cells were then subjected to laminin adhesion assay and later on enzyme-linked immunosorbent assay (ELISA) for the study of matrix metalloproteinase-3 (MMP-3), MMP-9 and vascular endothelial growth factor (VEGF) protein expression. The migration and apoptotic properties of the treated cells were also analysed using scratch test and caspase-3 apoptosis assay. Expression of bmi-1 and ngx6 gene was investigated using real time reverse transcriptase polymerase chain reaction. TW01-LMP1 cells treated with IL-6 (100 pg/ml) showed significant decreases in laminin attachment (p <0.05). The expression of MMP-3 and VEGF was also up-regulated with the presence of LMP1 (p <0.05). Besides that, IL-6 was also found to significantly up-regulated the expression of MMP-9 in NPC cells (p <0.05). In contrast, the presence of IL-10 significantly enhanced the adherence of TW01-LMP1 cells towards laminin (p <0.05). Treatment of TW01-LMP1 with IL-6, IL-10 and TGF-β1 resulted in increased migration by 3.3, 2.5 and 3-fold respectively. TW01 cells were able to evade apoptosis when LMP1 was being expressed (p < 0.05). In conclusion, IL-6 was suggested to promote metastasis in the presence of LMP1 and laminin by up-regulating the expression of MMP-9, bmi-1 gene, enhancing the cell migration and down-regulating the expression of ngx6 gene.

Chronic myeloid leukaemia (CML), is the most common myeloproliferative disease. Despite recent advances in combination chemotherapy and also stem cell transplantation, only about 7-12% of patients achieve molecular remission. Leukaemic cells use various mechanisms to avoid recognition by the immune system. Dendritic cells (DC) are professional antigen presenting cells of the immune system. These cells play a crucial role in the induction of anti—tumour immune responses. DC can be generated in-vitro from CD34⁺ stem cells or more mature CD14⁺ monocytes. The use of DC is an attractive immunotherapeutic strategy against cancer. In this study, we attempted to generate DC vaccines and investigate their activities against chronic myeloid leukaemia cells. DC were generated from monocytes isolated and enriched from peripheral blood. The isolated monocytes were subsequently cultured in RPMI medium supplemented with GM-CSF and IL-4. On day 7, tumour lysate obtained from K562 cells, a CML cell line, was used to pulse and to initiate further maturation of the DC in a culture medium supplemented with GM-CSF, IL-4 and TNF alpha. The DC-based leukaemia vaccines were ready on Day 15. On Day 15, the cultured cells showed matured DC morphology. Flow cytometry analysis showed strong expression of CD86 and HLA-DR. These cells did not express CD14, a monocyte marker. This showed that monocytes have been successfully differentiated to DC. Mixed leucocytes reaction (MLR) indicated that the generated DC vaccines were capable of inducing proliferative responses in allogeneic lymphocytes. The DC vaccines also exhibited considerable cytolytic activity against the K562 CML cells. In conclusion, we have successfully generated DC vaccines which are active against CML cells in vitro.

Tocotrienol is a sub-class in the vitamin E family. Tocotrienols have been shown to play a key role in multiple cellular processes that are considered to be major anti-cancer mechanisms. These include antioxidant activity, inhibiting proliferation of cancer cells, inducing apoptosis in cancer cells and inhibiting metastasis. Tocotrienols have been shown to inhibit proliferation and growth of several types of cancer cells including breast and prostate cancer cells. The present work was on determining the dose and time-dependent effects of tocotrienol rich fractions (TRF), tocotrienol isomers (e.g. αT3, δT3 and γT3) and alpha tocopherol on the proliferation and growth of oestrogen-positive MCF-7 human breast cancer cell lines in vitro, gene expression profiling in MCF-7 cells co-cultured with the various vitamin E isomers at half maximal inhibitory concentration (IC50) using microarray human bead chips as well as use of in silico approach such as molecular modelling via computer simulation to understand the molecular mechanism(s) of tocotrienols on the functions of various important biological molecules such as NF-κB, ESR1, EGFR and MIG6 and TTK. The results from this study showed that tocotrienols significantly (p<0.05) inhibit the proliferation of MCF-7 cells when compared to control i.e. untreated cells. The inhibition observed was found to be dose and time dependent. In contrast, α-tocopherol can only significantly (p<0.05) inhibit proliferation of the MCF-7 cells when a higher concentration of this vitamin E isomer was used. The IC50 and the order of potency for the compounds tested for Day 3, 6 and 10 was found to be δT3 (Day 3: 7.6, Day 6: 5.0, Day 10: 3.0) < γT3 (Day 3: 7.71, Day 6: 3.94 , Day 10: 3.0) < TRF (Day 3: 10.86, Day 6: 5.2, Day 10: 3.2) < αT3 (Day 3: 12.4, Day 6: 7.2, Day 10: 6.06) < αT (Day 3: 50.2) and the trend maintained at all time interval studied. Our data suggest that tocotrienols act continuously over a period of time, with low doses being as effective as higher doses when the treatment period is inceased. Gene expression study of vitamin E (TRF, α-tocopherol and α-, δ-, and γ-tocotrienol) treated on MCF-7 cells revealed expression of genes that can serve as potential targets for the treatment of breast cancer. Expression levels of selected differentially expressed genes were verified by quantitative real-time polymerase chain reaction (qRT-PCR). The results showed that tocotrienols alter the expression of genes that encode for various immune responses, tumour and metastasis suppressors, apoptotic signalling proteins, transcription factors, protein biosynthesis regulators, and many others. In particular, our results showed that treatment with tocotrienols downregulated the expression of API5 and upregulated the expression MIG6 at higher fold-change difference compared with control and the same reconfirmed using qRT-PCR. These genes regulate the expression of other genes that are pertinent to breast cancer. A molecular modelling was performed using a docking program Autodock version 3.0.5, which employs Lamarkian Genetic Algorithm and Arguslab 4.0 to predict the binding affinities of four isomers of tocotrienol (α-T3, β-T3, δ-T3 and γ-T3), against the active sites of various proteins that have been described in the literature to play key roles in cancer (NF-κB, ESR1, EGFR and MIG6 and TTK). The approach helped to putatively identify the potential target(s) of the tocotrienol isomers. The surfaces of the proteins were explored using this approach to identify fields that are most favourable in energy wise for interaction with the tocotrienols. The efficacy of binding was quantified in terms of binding energy and co-efficient constant, Ki. The results indicated that isomers of tocotrienols have the high predicted binding affinities against NF-κB, ESR1, EGFR and MIG6 and TTK proteins, while δ-tocotrienol and γ-tocotrienol interacts the most with the essential amino acid residues of the proteins studied.

Advances in the understanding of tumour biology have allowed targeted therapies such as immunotherapy against specific molecular targets to develop. Interferon gamma (IFN-ƴ), the sole Type II interferon cytokine, is a pleiotropic cytokine, is apleiotropic cytokine endowed with potent immunomodulatory effects on a variety of immune cells in vitro and in vivo. IFN-ƴ has been used to treat several diseases and malignancies in severely immunocompromised patients including chronic granulomatous disease and osteopetrosis and leukemia. However, there are a few key elements of the immune system of a tumour microenvironment such as metastatic tumour biology, cytokine and antigen-specific lymphocyte production, and the role of T-regulatory (Treg) cells, that need further understanding in order to develop an effective therapeutic agent to combat diseases such as breast cancer. The work described in the study aimed to develop a functional BALB/c murine model of breast cancer and to approximate the time of metastatic onset, to investigate the therapeutic effects of IFN-ƴ in the murine breast cancer model, to elucidate the various immune responses and immunomodulatory effects following metastasis in the breast cancer model and therapeutic administration of IFN-ƴ, to study the roles of various cytokines and phenotypic cell surface markers in the growth and spread of breast cancer of breast of breast cancer in model and to determine the ratio of T-regulatory cells to helper cells (Treg:Th) in a metastatic breast cancer murine model and following therapeutic administration of IFN-ƴ. For the in vitro work, effect of IFN-ƴ and tumour necrosis factor alpha (TNF-α)treatment on 4T1 cells was assessed using MTT assay to elucidate the viability of the cells in the presence of the cytokines, which may shed light on their anti-tumoural and anti-angiogenic properties. Production of IFN-ƴ, TNF-α and GM-CSF were assessed using ELISA to understand their role in the growth and progression of 4T1 Tumour cells. Cell surface markers such as MHC Class II, CD95 and CD95l were analysed using flow cytometry to evaluate their role of immune response induction in the microenvironment of tumour cells. IFN-ƴ and TNF-α proved to be efficient therapeutic agents when administered to 4T1 cells, with 37.42% and 51.68% of cells inhibited when exposed to highest concentration of IFN- ƴ at 20pg/mL and highest concentration of TNF-α 20pg/mL respectively. 4T1 cells elicited highest production of TNF-α at 176.95pg/mL, IFN-ƴ at 86.98pg/mL and the least with GM-CSF at 64.36pg/mL. No expression of MHC Class II, CD95 and CD95L were detected in 4t1 Cells. Our study has successfully characterized various immune responses elicited in the tumour microenvironment, induced by the tumour cells alone and also with prior stimulation of certain cytokines. For the metastasis and IFN-ƴ treatment study, six-week-old female BALB/c mice were inoculated with 4T1 murine breast cancer cells. The treatment group were administered with 40pg/mL. IFN-ƴ every other day following the day the tumour was palpable. Mice weight and primary tumour mass volume were regularly recorded to study the physical effects of a vigorously growing and spreading of cancer cell line. Gross and histological studies were carried out to determine the approximate day of metastatic onset. Production of IFN-ƴ and TNF-α was assessed by ELISA to understand its role in tumour growth and metastasis. Production of antigen-specific T Cells were analysed through mixed lymphocyte reactions, and this was to elucidate immunological response following tumour invasion. Lymphocyte markers such as CD4⁺,CD8⁺,CD16⁺and CD19 were analysed to elucidate its role in tumour growth and progression. Role of Treg cells were envisaged to understand the immune escape mechanisms employed by these cells and their immunosuppressive effects in a tumour model. Our study showed that the metastatic onset occurs approximately 14 days after the mice were inoculated with 4T1 murine breast cancer cells. Gross studies showed hepatosplenomegaly and slight congestion in the spleen. The breast cancer cells from primary tumour were found to spread rapidly to the liver on day 14. Following IFN-ƴ treatment, the size of primary tumour was reduced by 76% and there was less invasive metastatic clusters seen in liver sections. IFN-ƴ production and TNF-α was slightly higher in inoculated mice serum and splenocytes compared to the treatment group. Production of antigen-specific T Cells in the treatment group was higher than the untreated breast cancer-laden group. CD4⁺and CD16 were seen to be higher in the breast cancer-laden mice compared to control mice and CD8 and CD19 production were regressed in the experimental group CD4⁺,CD8⁺,CD16⁺and CD19 were slightly elevated in the treatment group than the untreated breast cancer-laden group, but the numbers towered those of control group. Elucidation of the factors underlying breast cancer progression and metastasis has been tremendously fruitful from mouse models in which multiple stages of tumour progression are further confirmed. We have successfully envisaged that the 4T1 murine breast cancer cells can migrate and metastasise rapidly to the liver., eliciting various immune responses, and the therapeutic administration of IFN-ƴ was effective in arresting the growth of primary tumour and a retardation of metastasis, also eliciting potent immune responses.

Background: Toxoplasma gondii is an obligate intracellular parasite. Its infective tachyzoites can evade the healthy host’s immune system by forming cysts in the host’s tissues, particularly the brain. The bradyzoites in the cysts remain dormant in brain, but may re-activate as tachyzoites if the host’s immunity is compromised. Re- activated cysts lead to acute toxoplasmic encephalitis, a deadly complication in these patients. The current treatment regime of toxoplasmosis still fail to eliminate T. gondii from the infected host, mainly due to low drug concentration attained in the brain. Spiramycin is a macrolide antibiotic [P-glycoprotein substrate, P-gp], tested effective against acute and chronic murine toxoplasmosis. Metronidazole is a nitro-imidazole antibiotic which inhibits the P450 [CYP] 3A4 & P-glycoprotein. Objectives: The aim of this study is to see the effectiveness of spiramycin in combination with a P-gp inhibitor (metronidazole) to enhance the drug delivery and uptake in brain of an animal model and to determine its efficacy in an animal model with chronic toxoplasmosis. Methods: A HPLC method was developed to determine and quantify spiramycin and metronidazole simultaneously. A Phenomenex C-18 [150 x 3.8 mm, 5 μm] column and acetonitrile-phosphate buffer [pH 2.5] with a gradient elution [20/ 80 to 30/ 70 in 3 min] at 1 mL/ min flow, 29°C and 232 nm was used for the study. In order to study the in vivo interactions between both the drugs and to establish the pharmacokinetic profile of spiramycin in plasma and brain, three groups of male Balb/c mice were dosed with only metronidazole 500 mg/ kg, only spiramycin 400 mg/ kg and spiramycin 400 mg/ kg co-administered with metronidazole 500 mg/ kg respectively. All the drugs were given orally. The animals were euthanized at pre-determined time points; 0.5, 1.0, 2.0, 2.5, 4.0, 6.0, 8.0, 10.0 and 12.0 hours. Brain and blood samples were obtained, processed and subjected to HPLC analysis. The pharmacokinetic parameters were calculated using non-compartmental techniques. In order to determine the onset and development of chronic toxoplasmosis, the male Balb/c mice were challenged with tachyzoites of T. gondii [ME 49 Strain]. The survival and disease state conversion were analyzed. At selected time-point based on the survival study, the mice were treated either with spiramycin 400 mg/ kg or metronidazole 500 mg/ kg or spiramycin 400 mg/ kg co-administered with metronidazole 500 mg/ kg, including infected untreated and positive control groups. The treatment efficacy was determined by microscopic counting of brain cysts and cysts cultured in vitro. Results: The HPLC method was linear [0.25– 50.0 μg/ mL], the LLOQ was 0.25 μg/ mL, intra- and inter-day variability, precision and accuracy were within 15%. Recoveries were above 75% and there was no matrix interference. Metronidazole eluted at 3 min and spiramycin at 5 min. In plasma, metronidazole did not affect the pharmacokinetics of spiramycin. In brain, metronidazole had more profound effects on the bio-distribution of spiramycin to the brain with a 1.7-fold increase in CMAX [p<0.05] and the AUC0–, was 64% greater [p<0.001]. Brain cysts developed two weeks post-acute infection [865 ± 86 cysts] and reached plateau phase at four weeks [1448 ± 81 cysts]. Reactivation of cysts in vitro was seen after one week of incubation and the bradyzoites-tachyzoites conversion was not influenced by the age of the brain cysts. In the study on treatment efficacy, the co- administration of spiramycin with metronidazole, showed a 15-fold and 10-fold reduction of brain cysts [79  23] with reference to the control group and spiramycin treated group respectively. The enhanced brain spiramycin concentration is probably due to the inhibition of P-glycoprotein transporter at the blood brain barrier by metronidazole, which facilitates the uptake of spiramycin into the brain. Conclusion: The combination of spiramycin and metronidazole was more effective than spiramycin alone in reducing brain cysts and cysts in vitro. This synergistic effect of drug interaction may have clinical translatability in the treatment and prevention of chronic toxoplasmosis.

Psoriasis vulgaris is a common disease with unknown pathogenesis. The treatment of the disease remains a challenge internationally due to the remission and recurrence nature of the disease. Psoriasis vulgaris was recently reported as an independent risk factor for ischaemic heart diseases, thromboembolic events and cerebrovascular accidents due to the higher level of plasma homocysteine in these patients, the result of defective methylenetetrahydrofolate reductase (MTHFR) enzymes. If these factors are strongly associated, homocysteinaemia could be lowered by the supplementations of folic acid and vitamin B₁₂ therefore reducing the risk of ischaemic heart diseases, thromboembolic events and cerebrovascular accidents in the psoriasis vulgaris patients. Though previous studies in the Far-East (China) supported the strong association of the MTHFR 677 C > T gene polymorphism and psoriasis vulgaris, there were some contradicting studies reported in the West (Austria and the Czech Republic). Therefore, the investigation on the association of the MTHFR 677 C > T gene polymorphism (NM_005957) and psoriasis vulgaris patients amongst the Malaysian population i.e. the Chinese, Indian and Malay ethnic groups of the Asian origin would be useful. This study was designed according to the Declaration of Helsinki and was approved by the ethics committee of the International Medical University, Malaysia. Cases (n = 200) were patients with psoriasis vulgaris and controls (n = 167) were subjects that do not have any history of psoriasis vulgaris, were age- (± 5 years), gender- and race-matched with the cases. DNA was extracted from the whole blood of both the cases and controls. Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) analysis of the MTHFR gene were performed in the presence of Taqαl restrictive enzyme. There was no significant increase (p = 0.392) of the MTHFR gene polymorphism (677 C > T) between cases and controls in the Malaysian population. Two homozygous mutations were seen in the controls and none in the cases. Subsequently, subsets of these cases (n = 41) and controls (n = 43) were age- (± 5 years), gender- and race-matched and selected for the determination of serum homocysteine, vitamin B₁₂ and folic acid levels. Though the highest homocysteine level was seen in the cases with heterozygous mutation to the MTHFR gene, the association was not statistically significant (p = 0.27). Homocysteine levels in cases were negatively correlated with vitamin B₁₂(r = -0.173) and folic acid (r = -0.345) levels. Skin biopsies were obtained from psoriasis vulgaris patients (n = 17) and non-psoriatic voluntary subjects who were undergoing their orthopaedic surgeries (n = 6). These biopsies were immuno-stained with antibodies against vascular endothelial growth factor (VEGF) subfamily- A, B, C, D and placenta growth factor (PIGF); nerve growth factor (NGF) and von Willebrand factor (vWFr). The VEGF-C (p = 0.016) and NGF (p = 0.027) were significantly expressed in the skin of psoriatic cases when compared with the controls. The NGF was the only marker that was solely expressed in the cases and absent in all the controls. No significant correlations between the angiogenesis markers staining intensities and PASI score or homocysteine levels were seen. In conclusion, no significant association between MTHFR gene polymorphism and psoriasis vulgaris amongst the Malaysian population i.e. the Chinese, Indian and Malay ethnic groups was seen. There was no association between the homocysteine level and psoriasis vulgaris in the Malaysian population. Significant expressions of NGF and VEGF-C in the cases may imply the importance of angiogenesis and lymphangiogenesis in the pathogenesis of the disease.

Palm oil contains biologically active phytonutrients that are important antioxidants used in various ailments and chemoprevention. This study was conducted to compare the antiproliferative effect of various tocotrienol isomers [tocotrienol-rich fraction (TRF), tocotrienol-enrich fraction (TEF), and (α-,δ-,γ-) T3 fractions] with other palm phytonutrients such as carotenoids, squalene and Coenzyme-Q10 on two human breast cancer cell lines, namely the MDA-MB-231 (oestrogen-independent) and MCF-7 (oestrogen-dependent) cells. Alpha-tocopherol was also included in this study as this was a phytonutrient that is present in palm oil. We were able to obtain a time dependent and dose dependent partial inhibition concentrations (IC50) for all the phytonutients studied with the exception of α-tocopherol. The range of concentrations used to determine the IC50 value was 0-20 g/ml. None of the palm phytonutrients with the exception of the tocotrienol isomers could completely suppress the growth of the two human breast cancer cells. Cellular uptake of tocotrienols was found to be better than -tocopherol; hence this could explain its biopotency. Next, we looked at strategies involving combinations of tocotrienols at IC50 with the other palm phytonutrients as this could provide a much wider range of anti-proliferative mechanisms, which could result in higher efficacy than a single agent alone. Although the combination treatments reduced the growth of these cells in the exponential stage, the treatments could not completely suppress cell proliferation. With most combinations, it was not possible to determine the IC50 values as cell growth reached a plateau. In addition, no additive or synergistic effects were observed. In order to assess the underlying cell death mechanisms, the respective IC50 concentrations were used. Based on DNA fragmentation analysis, the results showed that tocotrienols selectively enhanced induction of apoptosis as compared to Paclitaxel, which was used as a positive control. This activity was also associated with the time-dependent cleavage of the poly-(ADP-ribose) polymerase (PARP), a DNA repair protein, demonstrating an apoptotic cell death signalling pathway. Programmed cell death has emerged as a potential target for cancer treatment at various stages of cancer progression. The cell lines studied are aggressive human breast cancer cell lines that have been shown to have high levels of activated transcription factor nuclear factor-kappa-B (NF-B) and PARP expression. The ability of these compounds to inhibit NF-κB increases the sensitivity of cancer cells to apoptotic action. The levels of cyclooxygenase-2 (COX-2) are reported to be elevated in aggressive breast cancers. Elevated levels of COX-2 could result in certain kinds of cancer cells becoming resistant to programmed cell death mostly due to the impaired ability of the cell to undergo intrinsic cell death. In this study, COX-2, an NF-B regulated gene product associated with proliferation was found to be down-regulated with all treatments. Our current observation indicates tocotrienols, carotenoids, squalene and Coenzyme-Q10 possesses anti-proliferative effect on MDA-MB-231 and MCF-7 human breast cancer cells, and the mechanism could be through inhibition of inflammatory factors COX-2 and NF-B. Besides, the evidence of the significant anti-proliferative and apoptotic effects of tocotrienols not only reveals but also reinforces the notion that tocotrienols are indeed the hallmark nutrient in palm oil as potent anti-tumour agent in inhibiting breast cancer.

Hyposalivation is an excessive, objectively determinable reduction in salivation which affects primarily menopausal women and geriatric patients above 65 years of age. Ageing per se as a cause of hyposalivation has been controversial. Strong support for ageing as a cause of hyposalivation exists. For instance, Yamamura and co-workers observed an age-related drop in salivation in ageing C57BL/6 female mice. The present study aims to determine whether the age-related hyposalivation observed in ageing mice can be reversed by upregulating the water channel molecule Aquaporin 5 (AQP5) with edible bird’s nest (EBN) and ginger extracts (6-shogaol, 6-gingerol, 8-gingerol, and 10-gingerol). To evaluate whether the treatments upregulate AQP5 expression, a cell line assay had to be developed. Another aim of the present study was to determine the suitability of the human salivary gland (HSG) and A-253 cell lines for a cell line assay that studies the modulation of AQP5 expression. 26 female, C57BL/6 mice were arranged into four groups (8 control mice, 6 Decitabine-treated mice, 6 6-shogaol-treated mice, and 6 EBN- treated mice). Stimulated saliva was collected from the mice from weeks 6 to 16 to observe whether there was an age-related hyposalivation. The mice were treated from weeks 17 to 19, and their saliva collected to determine whether the treatments were able to reverse the age-related hyposalivation. HSG, A-253, and MCF7 cells were cultured and studied for endogenous AQP5 mRNA and protein expression. RNA was extracted from the cells, and reverse transcription polymerase chain reaction (RT-PCR) was conducted to identify whether these cells expressed AQP5 mRNA. Protein was extracted from the cells, and western blot was conducted to identify whether they expressed AQP5 protein. A-253 and MCF7 cells were treated with the natural substances. To evaluate whether their expressions were downregulated or upregulated from expression levels in the untreated cells, reverse transcription quantitative polymerase chain reaction (RT-QPCR) was conducted. An age-related hyposalivation was observed in the mice as they aged from weeks 6 to 16, validating the age-related hyposalivation observed by Yamamura and co-workers, and lending further support to the position that ageing per se is a cause of hyposalivation. EBN was unable to reverse this age-related drop in salivation, but 6-shogaol was able to, indicating that 6-shogaol is a potential therapeutic agent for age-related hyposalivation. Furthermore, the success of the mouse experiments indicate that the mouse experimental design is suitable for the screening of future natural substances for hyposalivation therapy. Neither HSG nor A-253 cells met the criteria required for the cell line assay, as neither expressed a basal level of AQP5 mRNA and protein. MCF7, due to its reliability in expressing AQP5, was proposed as a proxy cell line until a suitable cell line is discovered. QPCR results from the cell treatment studies indicate that the gingerols and EBN have an upregulation effect on AQP5 mRNA at the earlier durations of treatment (48 hours); however, these findings must be taken with a grain of salt, as A-253 cells are not suitably ideal for the type of cell line required.

Enteric fever is a severe multi-systemic illness that is endemic in most tropical and subtropical countries with limited sanitation. It is propagated by asymptomatic biliary carriers who intermittently shed Salmonella serovar Typhi (Salmonella ser. Typhi) and/or Salmonella serovar Paratyphi A (Salmonella ser. Paratyphi A) in their stool for years. The aim of this study was to establish if the presence of Salmonella in bile as determined by PCR and culture, correlated with the detection by ELISA of immunoglobulin G (IgG) and immunoglobulin A (IgA) to the 50KDa outer membrane protein (OMP) of typhoidal Salmonella, in the sera of gastroenterology patients. Bile was artificially spiked with 1-107cfu/mL of pure Salmonella ser. Typhi and Salmonella ser. Paratyphi A cultures. Various DNA extraction methods were compared for the ability to yield PCR inhibitor-free DNA extract from bile. There was a 5-fold increase in DNA yield for either Salmonella species when commercial kits were used for DNA extraction compared to conventional methods (p<0.01). PCR sensitivity was higher for both Salmonella ser. Typhi (2.46 x 10 ◦cfu/mL) and Salmonella ser. Paratyphi A (2.66 x 10 ◦ cfu/mL) DNA extracted by the commercial kits. To keep costs minimal, a low-cost DNA extraction method utilizing Dowex ion-exchange resin was developed. The Dowex based protocol successfully overcame PCR inhibition. The resin selectively bound to inhibitors not to target DNA (P=0.669). The PCR sensitivity of the DNA extract was comparable to that of commercial DNA extraction kits (2.6 x 10² for Salmonella ser. Typhi and 2.8 x 10◦ for Salmonella ser. Paratyphi A). The method was shown to be repeatable (P=0.73), and adopted in subsequent DNA extractions. About 320 bile and corresponding blood specimens were collected from patients undergoing endoscopic retrograde cholangiopancreotography (ERCP) and biliary surgery at the Asian Institute of Gastroenterology, Hyderabad, India; and at hospital Tuanku Ja’afar (Seremban), Negeri Sembilan, Malaysia. Bile was cultured for Salmonella using standard protocols. Of the 150 isolates recovered, there was only 1 Salmonella ser. Typhi and 1 Salmonella spp. Lactose fermenters (124), Pseudomonas (12), Shigella (2), Enterococcus (1) and Proteus spp. (9) were also isolated. DNA was extracted from bile using the established Dowex method, and amplified in multiplex Salmonella PCR. Culture and PCR were both 100% sensitive and specific for Salmonella. A sandwich ELISA kit (TyphiLiza, Malaysian Bio-Diagnostics Research Sdn Bhd, Selangor, Malaysia), was standardized to enable detection of serum immunoglobulin IgG (IgG) and immunoglobulin A (IgA) to the 50KDa outer membrane protein (OMP) of Salmonella ser. Typhi in carriers. Cut-off values of OD 0.8 and 0.3 at 205nm for IgG and IgA respectively, were established and adopted in the interpretation of serological data. The 50KDa OMP was non-specific, and performed poorly as a sub-marker for IgG and IgA in carriers. No relationship could be established between isolation and positive Salmonella PCR; with positive IgG and IgA levels to the 50KDa OMP of Typhi. Isolation rates of Salmonella were below expected (<1%). It is possible that the incidence of Salmonella carrier state has declined since the last reports in literature, most probably due to the increase in use of fluoroquinolones in the treatment of acute typhoid cases. Key words: enteric fever; chronic carrier; bile; PCR; Salmonella serovar Typhi; Salmonella serovar Paratyphi A; 50KDa OMP