Expression of Killer Cell Immunoglobulin-like Receptors (KIR) in Sex-associated Malignancies

@#Introduction: Sex shapes immune response with possible consequence on tumor immune escape. Acute lymphoblastic leukemia (ALL) predominates in males while ovarian cancer (OC) occurs in females. NK cells essential for tumor killing may have male preponderance. Association of sex, NK cell activity and malignancies is unclear. We hypothesize that sex differentially affects KIR expressions in sex-biased cancers. Method: Expression of inhibitory (KIR2DL1-5 and KIR3DL1-3) and activating (KIR2DS1-2 and 4-5 and KIR3DS1) genes in B-, T-cell ALL, OC and normal controls were determined by reverse-transcription polymerase-chain-reaction. Result: All normal males (but not females) expressed the framework genes and generally maintained haplotype A, except KIR3DL1. Normal females expressed more activating KIRs. Frequencies of KIR2DL1, 2DL4 and 2DS2 were significantly reduced among ovarian cancer patients. Sex difference in frequencies of KIR expression was not detected in ALL as majority were undetectable except framework gene KIR3DL2, was more frequent among T-ALL. Conclusion: Cancers may be associated with reduced KIR expression and influence of sex requires investigation.

Selection of Acid Salts: A Critical Step in Creating an Acidic Condition for Plasma Iron Release and Measurement

@#Introduction: Iron deficiency anaemia (IDA) is the most common cause of anaemia worldwide. Determination of body iron status is necessary to diagnose IDA. This can be measured using a biochemistry assessment of the serum/ plasma. Plasma/serum iron quantitation is also important in diagnosing iron overload disorders. However, iron studies are limited due to high cost and lack of access to biochemical analysers. Therefore, a cost- and technical-effective method is needed to measure human plasma iron concentration. Plasma iron is mainly transferrin-bound and an acidic plasmic condition is necessary to release the iron. This study investigated various candidate acid salts to achieve the acidic condition needed for plasma iron release. Method: Ten powdered or crystallised acid salts were studied for their water solubility as well as their pH reduction capability in revised simulated body fluid (r-SBF) and commercially available human plasma without any change in colour or form. Results: Six acid salts studied were discontinued from further investigation because they were insoluble in water. Another two candidates were unsuitable as they precipitated in r-SBF and human plasma. Maleic acid formed a jelly-like texture after a certain amount of time in human plasma. Only citric acid met all the criteria of a suitable acid salt to be investigated further as part of the reagent for a spontaneous plasma iron measurement. Conclusion: Citric acid, which is a colourless and odourless acid salt, was selected to lower the human plasma pH to an acidic condition for transferrin-bound iron release.

Plasma and Cell Lysate Proteins Associated With Treatment Outcome in Acute Myeloid Leukaemia

@#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.

Traumatic Injuries of the Dorso-lumbar Spine, Review of Surgical Approaches

In this review we supply information on medical methods for thoraco-lumbar spine fracture management, their efficiency and complication rates, based on previously published researches and also give background information on epidemiology and classification of thoraco-lumbar fractures. We conducted a narrative review over the literature using electronic databases as; MEDLINE, and EMBASE for studies involving data on Dorso-lumbar Spine traumatic injuries, published in September 2019. Spine fractures account for a large portion of musculoskeletal injuries worldwide. A classification of back cracks is essential in order to establish a typical language for therapy indicators and results. Clinical exam, mechanism of injury, and imaging are heavily trusted to choose regarding medical versus non-surgical management.

Evaluation of Apically Extruded Debris and Irrigants during Root Canal Preparation using Different Rotary Instrumentation Systems: An In-vitro Comparative Study

Background: Different instrumentation systems and techniques are used in the instrumentation of the root canal system which can result in debris formation that may be extruded beyond the apical foramen and cause post-operative pain. Aim of the study: Aim of the current study was to compare the amount of apically extruded debris and irrigants during instrumentation using 2 reciprocating single file systems (WaveOne Gold, Reciproc blue) and 2 continuous rotation file systems (ProTaper Gold, 2Shape) and comparing them to the control group (ProTaper Universal). Materials and Methods: Total 50 palatal roots of freshly extracted human maxillary first molars were collected for this study. Teeth were decoronated to a unified length of 15 mm, and then pushed through a pre-perforated rubber cap of pre-weighed glass vial then the root-cap complex was fitted on a glass vial and rubber dam ligated with dental floss was used to cover the glass vial for preventing the coronally extruded debris and irrigants from contaminating the external surface, gauge 25 needle was inserted parallel to the root surface through the rubber dam and cup. Samples were then randomly divided into 5 groups. Results: Data obtained were statistically analyzed using One Way ANOVA and LSD tests. The result showed that all groups resulted in apical extrusion of debris and irrigants, as it showed that the 2Shape Group B, Wave One Gold Group C and Reciproc blue Group D are statistically comparable, while ProTaper Gold Group A and ProTaper Universal Groups E showed statistically significant difference (p<0.05). Conclusions: All of the systems resulted in apical extrusion. There was no influence of kinematic movements on apical extrusion. The 2shape file system produced the least amount of apical extrusion while the ProTaper Universal showed the greatest amount.

Latent tuberculosis infection among medical students in Malaysia

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.

Latent tuberculosis infection among medical students in Malaysia

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.

Treatment of iron deficiency anaemia in patients with inflammatory bowel disease

In spite of the fact that anemia is the most widely recognized systemic sign of inflammatory bowel disease [IBD], among the expansive range of extraintestinal malady complexities experienced in IBD, including joint inflammation and osteopathy, it has for the most part gotten little thought. In any case, as far as recurrence, as well as to its potential impact on hospitalization rates and on the personal satisfaction and work, sickliness is, in fact, a huge and expensive intricacy of IBD

Frailty is multifactorial in nature, the most predominant etiological structures being iron deficiency anemia [IDA] and anemia of a chronic disease. In a condition related to irritation, for example, IBD, the assurance of iron status utilizing normal biochemical parameters alone is insufficient. A more exact evaluation might be achieved utilizing new iron lists including reticulocyte hemoglobin content, the rate of hypochromic red cells or zinc protoporphyrin. While oral iron supplementation has generally been a backbone of IDA treatment, it has likewise been connected to a broad gastrointestinal reactions and conceivable infection compounding. Be that as it may, numerous doctors are as yet hesitant to administer iron intravenously, in spite of the wide accessibility of an assortment of new IV arrangements with enhanced safety profiles, and in spite of the proposals of worldwide master rules. We present a review of the pathophysiologic mechanisms of IDA in IBD, improved diagnostic and therapeutic strategies, efficacy, and safety of iron replacement in IBD

Palm Tocotrienols Reduce Lipopolysaccharide-Stimulated Inflammatory Responses of Microglia

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).

Differential Expression Patterns of Leukaemia Associated Genes in Leukaemia Cell Lines Compared to Healthy Controls

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.

Subtractive Hybridization Identifies Stem Cell-Associated Genes in an Acute Myeloid Leukemia with Poor Prognosis

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, DFS12 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.

Increased soluble HLA-DRB1 in B-cell acute lymphoblastic leukaemia

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.

Establishing a Culture System that Supports in Vitro Expansion of Adult Microglia

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

Molecular responses during chemotherapy in acute myeloid leukemias in predicting poor-response to standard chemotherapy

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.