Apoptosis is a tightly programmed cell suicide which occurs in multiple physiologic and pathological conditions where it plays an important role in tissue development and homeostasis by eliminating unwanted and damaged cells. Appropriate apoptosis signalling is crucial in maintaining the fine balance between cell death and cell survival in cancer. In response to death stimuli the morphology of the cell undergoes unique changes. The aim of this study was to examine and compare the changes in the cell surface morphology using scanning electron microscopy in HCS-2 cells, following 24 hour treatment with components of highly active antiretroviral therapy (HAART) at their clinical plasma concentrations. The cells were fixed in 2.5% Glutaraldehyde and post-fixed in 1% osmium tetroxide. The cells were then dehydrated through a graded series of alcohol and treated with hexamethyl-disilazane, then coated with a double layer of carbon. The cells were viewed under a Zeiss Ultra FEG Scanning Electron Microscope and a one way ANOVA and Tukey Kramer Post Hoc test was conducted based on the scoring of surface morphology of the cells using JMP 11 statistical software. The drugs used in this study induced morphological features which are known to be characteristic of apoptotic cell death. The drug combinations (ATP and LPV/r) were seemingly more effective than individual treatments in inducing cell death because morphological features observed were more advanced than those observed in individual treatments. However, LPV/r was more potent than ATP. In conclusion, HAART showed anticancer properties by inducing cell death through apoptosis.
Background: Oral biofilms are the root cause of major oral diseases. As in vitro biofilms are not representative of the intraoral milieu, various devices have been manufactured over the years to develop Appliance Grown Oral Biofilm (AGOB). Objective: To review various intraoral appliances used to develop AGOB for microbiological analysis, and to judge the optimal means for such analyses. Design: Four databases (PubMed, Science Direct, Scopus and Medline) were searched by two independent reviewers, and articles featuring the key words 'device' OR 'splint' OR 'appliance'; 'Oral biofilm' OR 'dental plaque'; 'in vivo' OR 'in situ'; 'Microbiology' OR 'Bacteria' OR 'microbiome'; were included. The standard Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) were adopted for data gathering. Results: Of the 517 articles which met the initial inclusion criteria, 24 were deemed eligible for review. The age of the AGOB, sampled at various intervals, ranged from 30 min to 28 days. The most commonly used microbiome analytical methods were fluorescence microscopy, total cell count using conventional, and molecular tools including Next Generation Sequencing (NGS) platforms. Conclusions: No uniformly superior method for collecting AGOB could be discerned. NGS platforms are preferable for AGOB analyses.
This study evaluated the effect of pulsed low-intensity ultrasound on cell proliferation, collagen production and glycosaminoglycan deposition by human dermal fibroblasts encapsulated in alginate. Hoechst 33258 assay for cell number, hydroxyproline assay for collagen content, dimethylmethylene blue assay for glycosaminoglycan content and scanning electron microscopy were performed on the encapsulated cells treated with pulsed low-intensity ultrasound and a control group that remained untreated. Pulsed low-intensity ultrasound showed a significant effect on cell proliferation and collagen deposition but no consistent pattern for glycosaminoglycan content. Alcian blue staining showed that glycosaminoglycans were deposited around the cells in both treated and control groups. These results suggest that pulsed low-intensity ultrasound alone shows a positive effect on cell proliferation and collagen deposition even without growth factor supplements.
Development of an optogenetically controllable human neural network model in three-dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light-sensitive neurons were generated by transducing channelrhodopsin-2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell-type specific promoters. A mixed population of human iPSC-derived cortical neurons, astrocytes and progenitor cells were obtained (Axol-ChR2) upon neural differentiation. Pan-neuronal promoter synapsin-1 (SYN1) and excitatory neuron-specific promoter calcium-calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol-ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine-glycine-aspartate (RGD) and small molecules (Y-27632). Improved RGD-alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol-ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2- by both the CaMKII and SYN1 promoters. The RGD-alginate hydrogel system supports the growth of differentiated Axol-ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non-invasive control of human neural networks in 3D.
ABSTRACT
Clinacanthus nutans (C. nutans) leaf extracts have been widely used by cancer patients in Malaysia and local practice claims a cure to cancer. There were several studies done to determine the cytotoxicity potency of C. nutans extracts on various types of cells. However, there is still lacking on the knowledge regarding the combination effect of C. nutans with anticancer drugs. Thus, the study was carried out to determine the cytotoxicity potency of C. nutans extracts and paclitaxel (PTX) alone and, in combination on MDA-MB-231 cells. The cells were treated with 100% ethanol extract of C. nutans (CNE) and water extract of C. nutans (CNA), PTX and combination of both extracts and PTX for 72 hours and the cytotoxic activity was determined using SRB assay. Result showed that CNE had little cytotoxic activity, whereas CNA showed no cytotoxic activity on MDA-MB-231 cells. For combination treatment of C. nutans extracts and PTX, only CNE showed significant enhanced PTX-induced cytotoxicity (p < 0.05), meanwhile CNA inhibited PTX-induced cytotoxicity significantly (p < 0.05). As a conclusion, CNE was able to increase PTX potency to inhibit the viability of MDA-MB-231 cells.
Gold nanoparticles (AuNPs) have been extensively investigated as dose enhancement agent to increase the lethal dose to the tumours while minimizing dose to the normal tissue. Their intriguing properties and characteristics such as small size and shape provide favorable option in increasing radiotherapy therapeutic efficiency. In this study, the effects of AuNPs size on the dose enhancement effects irradiated under megavoltage photon beams were investigated. The study was conducted in-vitro on HeLa cells using AuNPs of 5 nm and 15 nm sizes. The cells samples were incubated with AuNPs and irradiated with photon beam of energy 6 MV and 10 MV at 100 cm SSD and 10 cm x 10 cm field size. Clonogenic assay were performed to observe the dose enhancement effects on cell survival. Dose enhancement factor (DEF) were extrapolated and evaluated from the cell survival curves. The results show that both sizes of AuNPs produce dose enhancement with the larger size AuNPs of 15 nm produce more dose enhancement compare to 5 nm AuNPs for 6 MV photon beam. Dose enhancements were observed for 10 MV photon beams but DEF for both sizes AuNPs shows no differences. In conclusion, larger size AuNPs produce higher dose enhancement compare to small size of AuNPs which conclude that nanoparticles size is important factor that need to be taken into account for AuNPs to be applied in radiotherapy.
Therapeutic application of metallic nanoparticles such as gold nanoparticles have been extensively investigated and intriguing finding have been reported. Superparamagnetic iron oxide nanoparticles (SPION) could also potentially have therapeutic properties that can be exploited to enhance radiotherapy outcome. In this study, investigations on the dose enhancement effects inflicted by SPIONs under irradiation with megavoltage photon beam radiotherapy were conducted. T24 human bladder cancer cell lines were pretreated with 1 mMol/L of SPION and irradiated with 6 MV and 10 MV photon beam at different doses.The non-treated cells irradiation was used as a control. Clonogenic assay was performed to determine the cell survival. Linear quadratic (LQ) model are used as fitting curve and does enhancement factors (DEF) were extrapolated from the curves. The cytotoxicity indicated cell growth normally after 72 hours and no long term cytotoxicity effects of SPIONs towards the cells were observed. The dose enhancement effects were observed for both 6 MV and 10 MV photon beam with DEF obtained 1.71 and 2.50, respectively. This reduction of cell colonies growth could be resulted from the interaction that induced free radical and reactive oxygen species (ROS) by megavoltage photon beams. The SPIONs were therefore act as multifunction nanoparticle both in diagnostic agent and radiotherapy as radiation dose enhancer, thus clearly qualified as future theranostic agents.
The purpose of the study was to determine the effect of out-of-field photon beams radiotherapy to the cancer cell survival. In this study, HeLa and T24 cancer cells were irradiated with 6 MV and 10 MV photon beams in two different conditions, one with intercellular communication with the in-field cell and one without the communication. Cells survival was determined by clonogenic assay. In the presence of intercellular communication, the cell death was increased which indicate the presence of radiation induced bystander effects (RIBE). The effects were also dependent on the cell types and photon energy where the HeLa cells exhibit less survival compares to T24 cells and the effects were prominent at higher photon energy. This study demonstrates that the out-of-field cells in conjunction with RIBE plays important roles in the cells response towards megavoltage photon beam radiation therapy.
Old intact male dogs are often predisposed to benign prostatic hyperplasia. Both hypertrophy and hyperplasia of prostate
gland occur due to hormonal influences. Clinical signs such as haematuria, stranguria and tenesmus are often associated with benign
prostatic hyperplasia. A seven year-old local breed intact male dog was presented with a primary complaint of haematuria and
stranguria. Diagnostic work-up conducted inclusive of complete blood count, serum biochemistry, urinalysis, abdominal radiograph,
cystourethrography and abdominal ultrasonography. The dog was diagnosed with cystic benign prostatic hyperplasia. Prostatic
omentalisation and castration were performed in this case.
Introduction: Filipino β°-deletion is predominant among the β-thalassaemia patients in the indigenous population of Sabah, Malaysia particularly among the Kadazandusun. Individuals who co-inherit with α- and β-thalassaemia will demonstrate milder clinical symptoms with modified complete blood count (CBC) and Hb subtype parameters. HBS1L-MYB variants act as one of the key regulator of haematopoiesis and erythropoiesis and display strong association
with variation of HbF levels. Therefore, this study aims to evaluate the association between genetic variants in HBS1L-MYB with Hb subtypes level among Filipino β°-deletion carriers co-inherited with -α3.7 deletion. Methods: Filipino β°-deletion and -α3.7 deletion were identified using gap-polymerase chain reaction (PCR). A total of 34 subjects found with coinheritance of Filipino β°-deletion and -α3.7 deletion were subjected for HBS1L-MYB intergenic polymorphisms (HMIP) analysis. Hb subtypes level were quantified using BioRad Variant II Hb analyser. Genotyping of HBS1L-MYB variants rs9399137 and rs11759553 was done using own designed tetra primer ARMS-PCR. Results: The minor allele frequencies (MAF) of the two HMIP is found more than 0.05 (rs11759553, MAF=0.18 and rs9399137, MAF=0.15), indicating the significance of these variants among the study subjects. Significant difference was found between HbF level and HBS1L-MYB variant rs11759553 with p-value less than 0.05 (p=0.001). Subjects with homozygous genotype for rs11759553 (T/T) was found with higher HbF, followed by heterozygous (A/T) and wild type (A/A). rs11759553 and rs9399137 was found did not influence the level of HbA and HbA2. HMIP of rs11759553 and rs9399137 are found significant among Filipino β°-deletion carriers co-inherited with -α3.7deletion with its high minor allelic frequency and high HbF level. Strong association with HbF level was demonstrated when
coinheritance of rs11759553. Conclusion: This study demonstrates that there are significant associations between certain genetic variants in HBS1L-MYB with Hb subtypes level among Filipino β°-deletion carriers co-inherited with -α3.7 deletion.
Introduction: Production of nitric oxide (NO) is one of the main responses elicited by a variety of
immune cells such as macrophages (e.g. microglia, resident macrophages of brain), during inflammation. Evaluation of NO levels in the inflammatory milieu is considered important to the understanding of the intensity of an immune response; and has been performed using different methods including the Griess assay. To assay NO in culture, an appropriate number of cells are stimulated into an inflammatory phenotype. Common stimuli include lipopolysaccharide (LPS), IFN-γ and TNF-α. However, overt stimulation could cause cell cytotoxicity therefore an ideal concentration of LPS should be used. Objective: To set-up a model of BV-2 cell activation that allows the assay of detectable levels of NO. Optimization of BV-2 microglia cell density and LPS concentrations after stimulation by bacterial lipopolysaccharide (LPS) for the Griess assay is demonstrated in this study. Methods: BV-2 microglia were cultured at different cell densities, and treated with LPS at three concentrations (1, 5, 10 μg/ml). NO production in culture supernatants were then measured at 18, 24, 48 and 72 hours. Moreover, methyl tetrazolium assay (MTT) was also performed to ensure that NO measurement is performed at no-cytotoxic concentrations of LPS. Results and Conclusions: NO production follows a temporal pattern. The density of 25000 cells/ well was the ideal seeding density for NO evaluation in BV-2 cells. BV-2 stimulation by LPS is dose dependent, and NO levels are increased proportional to the LPS concentration up to 1.0μg/ml, whereas the higher LPS concentrations are associated with decreased cell viability may be caused by the high toxic levels of LPS or NO. Although Griess assay has been commonly used by the scientists, however, optimization of its parameters on BV-2 cells will be useful for the experiments which will be performed on this particular cell line. The optimized pattern of Griess assay on BV-2 cells was achieved in this study, hence easier and more practical for the future scientists to perform Griess assay on BV-2 cells.
Peripheral blood (PB) CD34+ cells enumeration is currently the most reliable method to guide the timing of stem cell harvest. However, its usage is restricted by being technically challenging, costly, and time-consuming. Immature reticulocyte fraction (IRF) determination, which is simpler and cheaper and has a faster turn-around time, has been proposed for a similar purpose. The purpose of this study is to evaluate the value of IRF in guiding stem cell harvest and examine the correlation between IRF and PB CD34+ cells count. Daily pre-harvest tests, i.e. PB CD34+ cells and IRF from 21 patients scheduled for autologous PBSC transplant were assessed. Stem cells harvests were commenced when the PB CD34+ cell count were more than 10 cell/ul. A total of 205 pre-harvest tests were analysed. Following stem cell mobilisations, both the IRF and PB CD 34+ cell counts rose with a variable pattern. In this study, we observed that the IRF peaks preceded the PB CD34+ count by 2 days. On the day of stem cell harvest, all the peak IRF values were >0.3. The PB CD34+ cell counts correlated with the harvested stem cell yield, whereby r2 = 0.77, p < 0.021. In autologous stem cell mobilisation, we believe that IRF is a useful screening tool to predict the rise of the PB CD34+ cell counts as it is a simple, fast and less costly. An IRF of > 0.3 may be used as a cut-off value for the initiation of PB CD34+ quantification prior to stem cell harvest.
We report a case of factitious dermatitis in a 17 year female student who presented with recurrent pain .swelling and subcutaneous crepitations of the forearm. A thorough investigation was done. Full blood count, erythrocyte sedimentation rate was normal. Plain radiographs revealed the presence of subcutaneous emphysema. MRI showed similar findings and revealed normal muscles. Colonoscopy and OGDS were normal. except for a small polyp at the gastro-esophageal junction. Based on the clinical findings and lack of correlation with the investigations a diagnosis of factitious subcutaneous emphysema was made.
The transwell migration assay is commonly used for assessing cell migration. It involves the enumeration of cells that
have migrated across a pore-containing membrane. We describe a randomised approach to quantifying migrated
cells and compare it to a conventional full cell count. We used ATP as a chemoattractant and automatic cell quantification performed on all fields (Full count; FC) or 10 randomly selected fields (Randomised count; RC). The two
methods were compared by evaluating standard deviations (SD), coefficient of variation (CV) and using the Bland-Altman analysis. The dispersion of data is higher with the RC approach (3.77-6.66% CV for control; 3.89-4.48% CV
for ATP-treated wells) compared to FC (0.27-0.46% CV for control; 0.05-0.09% CV for ATP-treated wells), but are
acceptable considering that the number of migrated cells are in the thousands. Both methods verified that an ATP
migration assay for BV2 microglia was established, demonstrating that the RC approach is reliable and comparable
to a full count.
Introduction: Acute leukaemia in children accounts for 25-30% of malignant diagnosis. Survival from acute leukaemia continue to improve. Treatment outcome depends on factors like gender, age at diagnosis, parental education, the initial total white cell count (TWC), cerebrospinal fluids (CSF) infiltration, immunophenotype and treatment response. Objectives: The objectives were to evaluate the survival of children with acute leukaemia who received chemotherapy and identify relevant factors. Methodology: The study was a retrospective record review at the Paediatric Oncology Unit, Hospital Universiti Sains Malaysia (Hospital USM). The data collected depending on pre-set research proforma from the year 1990 to 2010. Survival analysis of each type of leukaemia was completed using multiple Cox regression model. Results: A total of 334 cases were identified, only 283 patients received treatment at Hospital USM. There were 224 patients with acute lymphoblastic leukaemia (ALL) and 59 with acute myeloid leukaemia (AML). Overall survival (OS) rate at 3 months for ALL and AML were 89.3% and 72.9% respectively. The event-free survival (EFS) rate for ALL at 1, 3, and 5 years were 69.6%, 54.1% and 47.8% respectively. For AML, the EFS rate at 1, 3, and 5 years were 52.0%, 42.4% and 38.1% respectively. Multiple Cox regression model showed children’s age at diagnosis and early response to steroid therapy were the most significant prognostic factors for ALL survival, whereas the spleen size and treatment protocol were the most significant prognostic factors for AML. Conclusion: Survival rate in this study was comparable to developing countries. ALL had better outcome compared to AML.
We report two cases of biphenotypic acute leukaemia diagnosed in Hospital Universiti Kebangsaan Malaysia (HUKM), their clinical, haematological characteristics and response to chemotherapy. Both patients are middle-aged ladies who presented with hepatosplenomegaly and high white cell count, mainly composed of blast cells. Their bone marrow aspirations were hypercellular comprising of more than 90% heterogenous blast cells. Cytochemical analyses show more than 3% positivity towards peroxidase, with smaller blasts showing block positivity towards PAS. Immunophenotypically, the blasts showed dual expression of CD 33 and CD 19, CD 19 and CD34, intra CD22, intra TdT and intraMPO. One of the patients showed presence of the Philadelphia chromosome on cytogenetic analysis which was confirmed by Fluorecsence In Situ Hybridisation (FISH). Molecular analysis also showed presence of the BCR-ABL fusion protein. Both patients were given combination chemotherapy consisting of daunorubicin and cytosine arabinoside.However, the patient with positive Philadelphia chromosome BCR-ABL did not achieve morphological remission after induction chemotherapy. In view of the poor prognosis of this disease, both the patients were planned for upfront peripheral blood stem cell transplantation.
Leukaemic stem cells have heterogenous differentiation potential. The immunophenotypes of blast cells are usually consistent throughout the disease course even at relapse. Rarely, blast cells may undergo a ‘lineage switch’ during the course of disease especially during relapse. We would like to highlight such a case in a 10- year old boy who presented with a two weeks history of lethargy, poor appetite, low grade fever, respiratory distress, cardiac failure, generalized oedema and hepatosplenomegaly. Full blood count showed a leucocyte count of 41.5x10 9 /L and platelet count of 37x10 9 /L. The peripheral blood film showed presence of numerous blast cells. Bone marrow aspiration revealed a hypercellular marrow, which consisted of mainly blast cells with high nuclear to cytoplasmic ratio and inconspicuous nucleoli. Immunophenotyping and cytochemistry results were consistent with the diagnosis of Tcell acute lymphoblastic leukaemia. The patient achieved remission after treatment with UK ALL 97 protocol, regime B chemotherapy. However, he relapsed seven months after the initial diagnosis with 26% blast cells in the bone marrow aspirate. The majority was L1 blast cells admixed with some L2 blast cells. Immunophenotyping was consistent with common precursor B acute lymphoblastic leukaemia. The treatment was changed to a more lineage specific chemotherapy. Nonetheless, the patient never achieved remission and was planned for palliative management. This case illustrated a unique and rare case of rapid lineage switch from T-cell acute lymphoblastic leukaemia to common precursor B-cell acute lymphoblastic leukaemia.