No abstract available

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.

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.

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.

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) 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.

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.

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.