Introduction: The vast majority of in vitro research on microglia are based on cells isolated from neonatal animals (3-5 days of age). Studying microglia of adults has been limited by the lack of a suitable culture system that supports their growth. In this study, we describe a protocol for growing microglia of adults based on modifications of the technique for culturing microglia isolated from neonatal rats. Methods: Mixed glia isolated from adult rats (age range of 1 month to 3 years old) were seeded in culture flasks coated with poly-L-lysine. Cells were maintained in DMEM media supplemented with insulin-transferrin-selenium (ITS) and recombinant human macrophage colony-stimulating factor (M-CSF). Mild trypsinisation was carried out to isolate microglia from mixed glia culture. Results: Microglia cells of adult rats were successfully grown in vitro. For the expansion of adult microglia, it was observed that coating the cell culture flasks with poly-L-lysine was crucial to encourage cell adherence. The substitution of insulin in culture media with ITS was found to improve cell yield and reduced the number of days required for culture from 28 days to 14 days. Addition of M-CSF to cell culture medium, along with the improvisations described above provided the best adult microglia cell yield (2.91 ± 0.56 x 10⁶ cells) compared to the technique of replating cells (0.91 ± 0.65 x 10⁶ cells; p<0.05). Conclusion: Optimisation of primary cell culture technique by coating culture flasks with poly-L-lysine,supplementation of culture medium with ITS and M-CSF allowed microglia of adult rats to be successfully cultured in vitro

Introduction: Current prognostic markers have improved survival prediction, however, it has not advanced treatment strategies. Gene expression profiling may identify biological markers suitable as therapeutic targets. Leukaemia stem cell is associated with adverse outcome, however, its biological characteristics are still being investigated. We observed higher in vitro cell viability in acute myeloid leukaemia (AML) samples with poor prognosis, which may be stem cell related. Objective: The objective of this study was to profile highly expressed genes in an AML sample of poor prognosis/high viability and compare with a sample of good prognosis/low viability. Method: Subtractive hybridization was performed on two AML samples with high blast counts (>80%), a poor prognosis, PP (disease free survival, DFS<12 months) and a good prognosis, GP (DFS>12 months) sample. The PP sample had higher CD34+ counts (73% vs 46%) and higher cell viability than the GP sample. cDNA libraries were subsequently cloned and sequenced. Results: cDNA subtracted from the PP samples was identified as genes active during fetal/embryonic development (LCOR, CNOT1, ORMDL1), HOX- related genes (HOXA3, PBX3, SF3B1), hematopoiesis (SELL, IL-3RA) and aerobic glycolysis/hypoxia (PGK1, HIGD1A) -associated genes. Majority of GP clones isolated contained genes involved in oxidative phosphorylation, OXPHOS (COXs, ATPs, MTND4 and MTRNR2), protein synthesis (including ribosomal proteins, initiating and elongation factors), chromatin remodeling (H2AFZ, PTMA), cell motility (MALAT1, CALM2, TMSB4X), and mitochondria (HSPA9, MPO) genes. Conclusion: Thus, the PP sample exhibited stem cell-like features while the GP sample showed cells at a high level of cell activity. These genes are potential prognostic markers and targets for therapy.
Leukemia, Myeloid, Acute

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/ hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages.
Neoplasms

Introduction: The potential immunoregulatory effects of tocotrienols, the less studied form of vitamin E, had not been determined for microglia until our last publication showcased primary evidence of palm tocotrienols limiting microglia activation, explicitly by inhibiting nitric oxide (NO) production. Here we further explored the nitrite scavenging activity of the two most potent NO-reducing tocotrienol isoforms - δ- tocotrienol and Tocomin50% (contains a spectrum of tocotrienols and α-tocopherol) based on their inhibitory effects on NO production and also their effects on CD40 (a microglial co-stimulator molecule) expression of BV2 microglia. Methods: BV2 cells were treated with two different doses of tocotrienols (δ-tocotrienol: 3.96 μg/mL and 19.80 μg/mL; Tocomin50%: 47.50 μg/mL and 237.50 μg/mL) followed by stimulation with 1 μg/mL of lipopolysaccharide (LPS). A chemical scavenging assay was conducted to study the nitrite scavenging activity of δ- tocotrienol. Together with Tocomin50%, we also determined their effects on CD40 expression of BV2 microglia via flow cytometry. Results: We demonstrate that the inhibitory effect of tocotrienols on NO production by microglia is not attributed to their nitrite scavenging activity. Additionally, tocotrienols also reduced the expression of the microglial co-stimulator molecule, CD40. Conclusions: Our data aids the further characterisation of the actions of tocotrienols on microglia, offering insight into the potential modulatory properties of palm tocotrienols on microglial inflammatory responses within the central nervous system (CNS).

Signal transduction pathways are constitutively expressed in leukaemic cells resulting in aberrant survival of the cells. It is postulated that in cells of chemo-sensitive patients, chemotherapy induces apoptotic signals leading to cell death while survival signals are maintained in cells of chemo-resistant patients. There is very little information currently, on the expression of these mediators in patients immediately after chemotherapy initiation. We examined the expression pattern of proinflammatory cytokines, signaling molecules of the PI3K and MAPK pathways molecules and death receptor, DR5 on paired samples at diagnosis and during chemotherapy in acute myeloid leukaemia patients treated with cytosine arabinoside and daunorubicin. The results were correlated with remission status one month after chemotherapy. We found that in chemo-sensitive patients, chemotherapy significantly increased the percentage of cases expressing TNF-alpha (p = 0.025, n = 9) and IL-6 (p = 0.002, n = 11) compared to chemo-resistant cases. We also observed an increased percentage of chemo-sensitive cases expressing DR5 and phosphorylated p38, and Jnk. Thus, expression of TNF-alpha, IL-6, DR5, phospho-p38 and phospho-Jnk may regulate cell death in chemo-sensitive cases. In contrast, a significantly higher percentage of chemo-resistant cases expressed phospho-Bad (p = 0.027, n = 9). IL-beta and IL-18 were also found to be higher in chemo-resistant cases at diagnosis and during chemotherapy. Thus, expression of various cellular molecules in leukaemic blasts during chemotherapy may be useful in predicting treatment outcome. These cellular molecules may also be potential targets for alternative therapy.

Soluble HLA (sHLA) are potential tumour markers released in order to counter immune surveillance. sHLA-class II is less known especially in acute lymphoblastic leukaemia (ALL). This study aimed to investigate soluble, surface and allelic expression of HLA Class II (sHLA-DR) in B-cell ALL patients and compare with soluble expression in normal individuals. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to measure soluble HLA-DRB1 in plasma. Flow cytometric analysis was performed to determine median fluorescence intensity in HLA-DR surface expression. HLA-DNA typing by polymerase chain reaction, sequence specific oligonucleotides, PCRSSO was performed to determine HLA-DRB1 type in ALL samples. Results showed sHLA-DRB1 (mean+SEM) was significantly increased (p=0.001) in plasma of ALL patients (0.260±0.057 μg/mL; n=30) compared to healthy controls (0.051±0.007μg/mL; n=31) of Malay ethnicity. However, these levels did not correlate with percentage or median fluorescence intensity of HLA-DR expressed on leukemia blasts (CD19+CD34+/-CD45loHLA-DR+) or in the normal B cell population (CD19+CD34- CD45hiHLA-DR+) of patients. No significant difference was observed in gender (male/female) or age (paediatric/adult). Only a trend in reduced sHLA was observed in patients carrying HLA-DR04. These results have to be validated with a larger number of samples.

Soluble HLA (sHLA) are potential tumour markers released in order to counter immune surveillance. sHLA-class II is less known especially in acute lymphoblastic leukaemia (ALL). This study aimed to investigate soluble, surface and allelic expression of HLA Class II (sHLA-DR) in B-cell ALL patients and compare with soluble expression in normal individuals. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to measure soluble HLA-DRB1 in plasma. Flow cytometric analysis was performed to determine median fluorescence intensity in HLA-DR surface expression. HLA-DNA typing by polymerase chain reaction, sequence specific oligonucleotides, PCRSSO was performed to determine HLA-DRB1 type in ALL samples. Results showed sHLA-DRB1 (mean±SEM) was significantly increased (p=0.001) in plasma of ALL patients (0.260 ±0.057 μg/mL; n=30) compared to healthy controls (0.051 ± 0.007µg/mL; n=31) of Malay ethnicity. However, these levels did not correlate with percentage or median fluorescence intensity of HLA-DR expressed on leukemia blasts (CD19+CD34 ± CD45(lo)HLA-DR+) or in the normal B cell population (CD19+CD34- CD45(hi)HLA-DR+) of patients. No significant difference was observed in gender (male/female) or age (paediatric/adult). Only a trend in reduced sHLA was observed in patients carrying HLA-DR04. These results have to be validated with a larger number of samples.

Introduction: Estimation of gestational age (GA) is clinically crucial for managing pregnancy and assessing the foetal anatomy, growth and development. Transverse cerebellar diameter (TCD) has been reported as an accurate tool for dating the pregnancy. This study aimed to determine the accuracy of foetal TCD for dating the pregnancy and to construct a reference chart for GA of Yemeni foetuses. Methods: We conducted this prospective cross-sectional study among 400 Yemeni pregnant women between 18 and 40 weeks of gestation provided that they were with known last menstrual period and singleton normal pregnancies. Sonographic TCDs were measured for each foetus. The mean TCD was measured for gestational weeks separately, and a polynomial regression model was then used to predict the GA by TCD. Results: There was a robust correlation between GA and TCD (r = 0.995, p <0.001). The coefficient of determination was 0.989, which indicates that the TCD explains 98.9% of GA change. Conclusion: TCD is an accurate tool to estimate the GA in second and third trimesters, including final weeks, among Yemeni pregnant women as in developed countries. The narrow range between the 5% and 95% percentiles is a significant indication of the accuracy of the TCD in estimating the GA. Therefore, clinicians are recommended to use TCD for estimating the GA and should be trained on measuring it accurately.

Objective: This study aimed to determine prevalence of latent tuberculosis infection among medical students and tuberculosis exposure at the health facilities. Methods: A cross-section of study year 1 (n=68) and year 5 (n=75) medical students in a local university were recruited for latent tuberculosis infection testing using QuantiFERON-TB Gold Plus and a questionnaire analyzed for multivariate risk. Results: The majority of the study were vaccinated with BCG. None of year 1 medical students were positive for latent tuberculosis infection, however, six (8.0%) year 5 students were tested positive for latent tuberculosis infection. A higher incidence of year 5 medical students claimed to be exposed to tuberculosis at health facility (65.3% vs. 4.4%) and a higher percentage reported contact with tuberculosis case over the preceding year compared to year 1 students (30.7% vs. 8.8%). Conclusion: We observed a higher incidence of latent tuberculosis infection and higher exposure to tuberculosis in health facilities among year 5 medical students. Baseline screening and monitoring for progression to tuberculosis infection may benefit tuberculosis management programs.

Introduction: Drug-resistance is a major hindrance to successful treatment of AML. Current predictive biomarkers are mainly genetic aberrations and insufficient in foretelling treatment outcome in all acute myeloid leukaemia (AML) due to its heterogeneous and aggressive nature. Proteins are stable and reliable. Secreted proteins in AML may have predictive or prognostic values for early intervention. Proteomic studies on AML are few and further investigations will benefit in selection of best markers. The aim of the study was to identify differentially expressed plasma proteins in AML with different treatment outcome. Methods: Two-dimensional electrophoresis (2-DE) technique was utilised to identify proteins differentially expressed in chemo-sensitive/chemo-resistant AML. Plasma and peripheral blood mononuclear cell (PBMC) lysate proteome analysis were performed on six chemo-resistant, four chemo-sensitive and six healthy controls and seven chemo-resistant, three chemo-sensitive and six healthy controls, respectively. Each experiment was conducted in duplicate or triplicate. Images were captured and protein spots detected by software. Differentially expressed protein spots were excised from gel and proteins were identified using LC/MS/MS. Proteins spots that were also detected in healthy controls were excluded. Results: Comparing mean % volume of each spot demonstrated significantly enhanced expression of apoliprotein-E (APO-E) and haptoglobin (HP) (p<0.05) in plasma and HNRNP H1 (p=0.049) in cell lysate of chemo-sensitive group. Serotransferrin (STF) from plasma and DNA-PK from cell lysate (p=0.01) were associated with chemo-resistance. Conclusion: This preliminary study identified several potential predictive biomarkers associated with chemo-resistance/chemo-sensitivity to treatment in AML. Further studies with a larger number of samples are required to validate the results.