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Fulltext Multiple gene targeting siRNAs for down regulation of Immediate Early-2 (Ie2) and DNA polymerase genes mediated inhibition of novel rat Cytomegalovirus (strain All-03)

Balakrishnan KN, Abdullah AA, Bala JA, Jesse FFA, Abdullah CAC, Noordin MM, et al.

Virol J, 2020 Oct 27;17(1):164.
PMID: 33109247 DOI: 10.1186/s12985-020-01436-5

BACKGROUND: Cytomegalovirus (CMV) is an opportunistic pathogen that causes severe complications in congenitally infected newborns and non-immunocompetent individuals. Developing an effective vaccine is a major public health priority and current drugs are fronting resistance and side effects on recipients. In the present study, with the aim of exploring new strategies to counteract CMV replication, several anti-CMV siRNAs targeting IE2 and DNA polymerase gene regions were characterized and used as in combinations for antiviral therapy.
METHODS: The rat embryo fibroblast (REF) cells were transfected with multi siRNA before infecting with CMV strain ALL-03. Viral growth inhibition was measured by tissue culture infectious dose (TCID50), cytopathic effect (CPE) and droplet digital PCR (ddPCR) while IE2 and DNA polymerase gene knockdown was determined by real-time PCR. Ganciclovir was deployed as a control to benchmark the efficacy of antiviral activities of respective individual siRNAs.
RESULTS: There was no significant cytotoxicity encountered for all the combinations of siRNAs on REF cells analyzed by MTT colorimetric assay (P > 0.05). Cytopathic effects (CPE) in cells infected by RCMV ALL-03 had developed significantly less and at much slower rate compared to control group. The expression of targeted genes was downregulated successfully resulted in significant reduction (P Flow cytometry analysis showed a greater percentage of viable and early apoptosis of combined siRNAs-treated cells compared to control group. Notably, the siRNAs targeting gene regions were sequenced and mutations were not encountered, thereby avoiding the formation of mutant with potential resistant viruses.
CONCLUSIONS: In conclusion. The study demonstrated a tremendous promise of innovative approach with the deployment of combined siRNAs targeting at several genes simultaneously with the aim to control CMV replication in host cells.

Matched MeSH terms: Flow Cytometry
Retropatellar fat pad-derived stem cells from older osteoarthritic patients have lesser differentiation capacity and expression of stemness genes

Chua KH, Zaman Wan Safwani WK, Hamid AA, Shuhup SK, Mohd Haflah NH, Mohd Yahaya NH

Cytotherapy, 2014 May;16(5):599-611.
PMID: 24290076 DOI: 10.1016/j.jcyt.2013.08.013

The use of retropatellar fat pad-derived mesenchymal stromal cells (RFMSCs) for cell-based therapy, particularly for cartilage repair, has been reported by several investigators in recent years. However, the effects of the donor's age and medical condition on the characteristics of RFMSCs have not been well established. The aim of this study was to determine whether age and medical condition can reduce the multipotential of stem cells isolated from the retropatellar fat pad.

Matched MeSH terms: Flow Cytometry
Fulltext Monobenzyltin Complex C1 Induces Apoptosis in MCF-7 Breast Cancer Cells through the Intrinsic Signaling Pathway and through the Targeting of MCF-7-Derived Breast Cancer Stem Cells via the Wnt/β-Catenin Signaling Pathway

Fani S, Dehghan F, Karimian H, Mun Lo K, Ebrahimi Nigjeh S, Swee Keong Y, et al.

PLoS One, 2016 Aug 16;11(8):e0160836.
PMID: 27529753 DOI: 10.1371/journal.pone.0160836

Monobenzyltin Schiff base complex, [N-(3,5-dichloro-2-oxidobenzylidene)-4-chlorobenzyhydrazidato](o-methylbenzyl)aquatin(IV) chloride, C1, is an organotin non-platinum metal-based agent. The present study was conducted to investigate its effects on MCF-7 cells with respect to the induction of apoptosis and its inhibitory effect against MCF-7 breast cancer stem cells. As determined in a previous study, compound C1 revealed strong antiproliferative activity on MCF-7 cells with an IC50 value of 2.5 μg/mL. Annexin V/propidium iodide staining coupled with flow cytometry indicated the induction of apoptosis in treated cells. Compound C1 induced apoptosis in MCF-7 cells and was mediated through the intrinsic pathway with a reduction in mitochondrial membrane potential and mitochondrial cytochrome c release to cytosol. Complex C1 activated caspase 9 as a result of cytochrome c release. Subsequently, western blot and real time PCR revealed a significant increase in Bax and Bad expression and a significant decrease in the expression levels of Bcl2 and HSP70. Furthermore, a flow cytometric analysis showed that treatment with compound C1 caused a significant arrest of MCF-7 cells in G0/G1 phase. The inhibitory analysis of compound C1 against derived MCF-7 stem cells showed a significant reduction in the aldehyde dehydrogenase-positive cell population and a significant reduction in the population of MCF-7 cancer stem cells in primary, secondary, and tertiary mammospheres. Moreover, treatment with C1 down-regulated the Wnt/β-catenin self-renewal pathway. These findings indicate that complex C1 is a suppressive agent of MCF-7 cells that functions through the induction of apoptosis, cell cycle arrest, and the targeting of MCF-7-derived cancer stem cells. This work may lead to a better treatment strategy for the reduction of breast cancer recurrence.

Matched MeSH terms: Flow Cytometry
Fulltext A novel Bruton's tyrosine kinase gene (BTK) invariant splice site mutation in a Malaysian family with X-linked agammaglobulinemia

Chear CT, Gill HK, Ramly NH, Dhaliwal JS, Bujang N, Ripen AM, et al.

Asian Pac J Allergy Immunol, 2013 Dec;31(4):320-4.
PMID: 24383975 DOI: 10.12932/AP0304.31.4.2013

X-linked agammaglobulinemia (XLA) is a rare genetic disorder caused by mutations in the Bruton's tyrosine kinase (BTK) gene. These mutations cause defects in early B cell development. A patient with no circulating B cells and low serum immunoglobulin isotypes was studied as were his mother and sister. Monocyte BTK protein expression was evaluated by flow cytometry. The mutation was determined using PCR and followed by sequencing. Flow cytometry showed the patient lacked BTK protein expression in his monocytes while the mother and sister had 62% and 40% of the monocytes showing BTK protein expressions respectively. The patient had a novel base substitution in the first nucleotide of intron 9 in the BTK gene, and the mutation was IVS9+1Gflow cytometry and genetic study is necessary in the diagnosis of X-linked agammaglobulinemia and may be used for subsequent genetic counseling, carrier detection and prenatal diagnosis.

Matched MeSH terms: Flow Cytometry
Fulltext Eco-Friendly Formulated Zinc Oxide Nanoparticles: Induction of Cell Cycle Arrest and Apoptosis in the MCF-7 Cancer Cell Line

Boroumand Moghaddam A, Moniri M, Azizi S, Abdul Rahim R, Bin Ariff A, Navaderi M, et al.

Genes (Basel), 2017 Oct 20;8(10).
PMID: 29053567 DOI: 10.3390/genes8100281

Green products have strong potential in the discovery and development of unique drugs. Zinc oxide nanoparticles (ZnO NPs) have been observed to have powerful cytotoxicity against cells that cause breast cancer. The present study aims to examine the cell cycle profile, status of cell death, and pathways of apoptosis in breast cancer cells (MCF-7) treated with biosynthesized ZnO NPs. The anti-proliferative activity of ZnO NPs was determined using MTT assay. Cell cycle analysis and the mode of cell death were evaluated using a flow cytometry instrument. Quantitative real-time-PCR (qRT-PCR) was employed to investigate the expression of apoptosis in MCF-7 cells. ZnO NPs were cytotoxic to the MCF-7 cells in a dose-dependent manner. The 50% growth inhibition concentration (IC50) of ZnO NPs at 24 h was 121 µg/mL. Cell cycle analysis revealed that ZnO NPs induced sub-G₁ phase (apoptosis), with values of 1.87% at 0 μg/mL (control), 71.49% at IC25, 98.91% at IC50, and 99.44% at IC75. Annexin V/propidium iodide (PI) flow cytometry analysis confirmed that ZnO NPs induce apoptosis in MCF-7 cells. The pro-apoptotic genes p53, p21, Bax, and JNK were upregulated, whereas anti-apoptotic genes Bcl-2, AKT1, and ERK1/2 were downregulated in a dose-dependent manner. The arrest and apoptosis of MCF-7 cells were induced by ZnO NPs through several signalling pathways.

Matched MeSH terms: Flow Cytometry
Detection and quantification of IgM(+) lymphocytes in fetal lamb spleen, liver and lymph nodes by flow cytometry

Alitheen N, McClure S, McCullagh P

Immunol Cell Biol, 2007 Jul;85(5):391-3.
PMID: 17515929

The first stage in Peyer's patch development in the fetal lamb is characterized by the colonization of the rudimentary Peyer's patches by precursor cells expressing the IgM surface receptor. In the fetal lamb, the spleen has been implicated as the source of gene-rearranged IgM(+) B lymphocytes. This study was intended to quantitate IgM(+) lymphocytes in the spleen, lymph nodes and liver of fetal lambs at various gestational ages between 63 and 110 days using flow cytometry. Flow-cytometric analysis revealed that IgM(+) lymphocytes were rare in the liver being consistently less than 1% at every gestational age examined. IgM(+) lymphocytes were detected in the spleen (mean 9.18%) and prescapular lymph nodes (mean 11.89%) as early as 63 days. In both spleen and lymph nodes, the highest representation of IgM(+) lymphocytes occurred between 70 and 86 days gestation. The highest mean percentage of IgM(+) lymphocytes was observed in the spleen (22.63%) and lymph nodes (17.02%) at 75 days gestation. From 98 days onwards, B-lymphocyte density gradually decreased in both spleen and prescapular lymph nodes. This study indicates that substantial populations of IgM(+) lymphocytes were present in both the spleen and prescapular lymph nodes from 70 days gestation and implies that both of these locations could be potential sources for the normal colonization of the ileal Peyer's patches.

Matched MeSH terms: Flow Cytometry*
Effect of extracts from Phyllanthus watsonii Airy Shaw on cell apoptosis in cultured human breast cancer MCF-7 cells

Ramasamy S, Abdul Wahab N, Zainal Abidin N, Manickam S

Exp. Toxicol. Pathol., 2013 Mar;65(3):341-9.
PMID: 22217449 DOI: 10.1016/j.etp.2011.11.005

Species of Phyllanthus have traditionally been used for hundreds of years for treating many ailments including diabetes, anemia, bronchitis and hepatitis. The present study aims to investigate the cytotoxic and apoptotic effects of methanol (PWM), hexane (PWH) and ethyl acetate (PWE) extracts from the leaves of the endemic plant Phyllanthus watsonii Airy Shaw (Phyllanthaceae) on MCF-7 human breast cancer cells. We observed that the PWM, PWH and PWE extracts were cytotoxic and selectively inhibited the growth and proliferation of MCF-7 cells compared to untreated control in a dose dependent manner with an IC(50) of 12.7 ± 4.65, 7.9 ± 0.60 and 7.7 ± 0.29 μg/ml, respectively. However, the extracts were not toxic at these concentrations to normal human lung fibroblast MRC-5 cells. Cell death induced by PWM, PWH and PWE extracts were mainly due to apoptosis which was characterized by apoptotic morphological changes and a nuclear DNA fragmentation. Caspase-3 activation following P. watsonii extracts treatment was also evident for apoptotic cell death which was preceded by an S phase cell cycle perturbation. The results suggested that the cytotoxic activity of P. watsonii extracts was related to an early event of cell cycle perturbation and a later event of apoptosis. Hence, P. watsonii displays potential to be further exploited in the discovery and development of new anticancer agents.

Matched MeSH terms: Flow Cytometry
Fulltext Correlation between corresponding protein and MRNA in acute myeloid leukaemia (AML)

Siti Zuleha Idris, Stephnie Yiau Kang Xian, Lee CinDee, Eusni Rahayu Mohd. Tohit, Chang Kian Meng, Maha Abdullah

Malaysian Journal of Medicine and Health Sciences, 2019;15(108):43-43.
MyJurnal

Introduction: Protein and gene expressions are intensively profiled for potential biomarkers in diagnosis or prognosis of diseases. The correlation between corresponding protein and mRNA of a gene is important to establish whether transcript levels of a given gene can be used as proxies for the corresponding protein levels. mRNA profiling is more commonly utilised as this method is cheaper and the technology more advanced. Acute myeloid leukaemia (AML) is a heterogeneous group of malignant precursors of the myeloid lineage that leads to death if not treated. Cytokines and death receptors are commonly evaluated in this disease in search of potential biomarkers; however, the mRNA/protein correlations of these biomarkers are still unclear. Methods: Semi-quantitative expression of mRNA expression and protein levels of IL-1β, IL-18Rα, IL-6, TNF-α and DR5 were measured by conventional polymerase reaction (PCR) and flow cytometry in 11 cases of AML at diagnosis. Correlation in the intensity of the PCR amplicon and corre-sponding mean fluorescence intensity of protein was determined by Spearman’s rank correlation test. Results: None of the cytokines/death receptor was significantly correlated except IL-6 (Rs= -0.6287, p=0.038). Unexpectedly, this was also a significant negative correlation. Conclusion: For the majority of selected biomarkers in AML, whether secreted or surface-expressed, mRNA and protein expressions were not significantly correlated. The strong negative correlation for IL-6 is worth further investigation.

Matched MeSH terms: Flow Cytometry
Fulltext Differential expression patterns of leukaemia associated genes in leukaemia cell lines compared to healthy controls

Ang Pei-Shen, Rajesh Ramasamy, Noor Hamidah Hussin, Cheong Soon-Keng, Seow Heng-Fong, Maha Abdullah

Malaysian Journal of Medicine and Health Sciences, 2016;12(1):0-0.
MyJurnal

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.

Matched MeSH terms: Flow Cytometry
One-Year Outcomes of Living Related Kidney Transplant in Patients With Preformed HLA Donor-Specific Antibodies: A Single-Center Experience in Malaysia

Jalalonmuhali M, Ng KP, Mohd Shariff NH, Lee YW, Wong AH, Gan CC, et al.

Transplant Proc, 2020 05 21;52(6):1718-1722.
PMID: 32448671 DOI: 10.1016/j.transproceed.2020.02.140

The shortage of deceased donors led to an increase of living related renal transplant performed in the presence of donor-specific antibodies (DSAs) or ABO incompatibilities. There are various desensitization protocols that have been proposed. Here, we describe the outcome of these sensitized patients. This is a prospective cohort study recruiting all kidney transplant recipients from August 2016 until June 2018. Deceased donations, ABO incompatible patients, and sensitized patients who were not prescribed on our desensitization protocol were excluded. Recipients were screened for the presence of HLA-antibodies 1 month before transplant. Those with positive DSA will undergo flow cytometry (risk stratification). We are using a protocol that consisted of intravenous rituximab 200 mg (day -14), intravenous antithymocyte globulin 5mg/kg (day 0-4), plasma exchange post transplant for patients with mean fluorescent intensity (MFI) < 3000, and negative flow cytometry. Those patients with MFI ≥ 3000 or positive flow cytometry need extra cycles pretransplant. A total of 40 patients were recruited, and 20 were sensitized patients. Among the sensitized group 4 of 20 had flow cytometry crossmatch positive, while all had preformed HLA-DSA. A total of 8 of 20 had class I HLA-DSA, 11 of 20 had class II HLA-DSA, and 1of 20 was positive for both class I and II HLA-DSA. Mean immunodominant MFI was 2133.4 (standard deviation [SD], 4451.24) and 1383.7 (SD, 2979.02) for class I and class II, respectively. At 1 year, mean serum creatinine was 108.90 (SD, 25.95) and 118.42 (SD, 31.68) in sensitized and unsensitized patients, respectively. One of 20 unsensitized patients had Banff 1B rejection at 3 months, and there was no significant rejection in sensitized patients at 6 months and 1 year. There was no difference in the occurrence of de novo HLA-DSA between the groups. Desensitization protocols may help to overcome incompatibility barriers in living donor renal transplant. The combination of low-dose rituximab, antithymocyte globulin, and judicious use of plasma exchange has worked well for our cohort.

Matched MeSH terms: Flow Cytometry
Fulltext In vitro assessment of anti-proliferative effect induced by α-mangostin from Cratoxylum arborescens on HeLa cells

El Habbash AI, Mohd Hashim N, Ibrahim MY, Yahayu M, Omer FAE, Abd Rahman M, et al.

PeerJ, 2017;5:e3460.
PMID: 28740747 DOI: 10.7717/peerj.3460

Natural medicinal products possess diverse chemical structures and have been an essential source for drug discovery. Therefore, in this study, α-mangostin (AM) is a plant-derived compound was investigated for the apoptotic effect on human cervical cancer cells (HeLa). The cytotoxic effects of AM on the viability of HeLa and human normal ovarian cell line (SV40) were evaluated by using MTT assay. Results showed that AM inhibited HeLa cells viability at concentration- and time-dependent manner with IC50 value of 24.53 ± 1.48 µM at 24 h. The apoptogenic effects of AM on HeLa were assessed using fluorescence microscopy analysis. The effect of AM on cell proliferation was also studied through clonogenic assay. ROS production evaluation, flow cytometry (cell cycle) analysis, caspases 3/7, 8, and 9 assessment and multiple cytotoxicity assays were conducted to determine the mechanism of cell apoptosis. This was associated with G2/M phase cell cycle arrest and elevation in ROS production. AM induced mitochondrial apoptosis which was confirmed based on the significant increase in the levels of caspases 3/7 and 9 in a dose-dependent manner. Furthermore, the MMP disruption and increased cell permeability, concurrent with cytochrome c release from the mitochondria to the cytosol provided evidence that AM can induce apoptosis via mitochondrial-dependent pathway. AM exerted a remarkable antitumor effect and induced characteristic apoptogenic morphological changes on HeLa cells, which indicates the occurrence of cell death. This study reveals that AM could be a potential antitumor compound on cervical cancer in vitro and can be considered for further cervical cancer preclinical and in vivo testing.

Matched MeSH terms: Flow Cytometry
CXCL 9 and CXCL 10 as Sensitive markers of disease activity in patients with rheumatoid arthritis

Kuan WP, Tam LS, Wong CK, Ko FW, Li T, Zhu T, et al.

J Rheumatol, 2010 Feb;37(2):257-64.
PMID: 20032101 DOI: 10.3899/jrheum.090769

OBJECTIVE:
To assess whether serum levels of CC and CXC chemokines correlate with disease activity in patients with rheumatoid arthritis (RA), and to determine whether these effects predict clinical response.

METHODS:
Serum levels of the chemokines CC (CCL2, CCL5) and CXC (CXCL8, CXCL9, CXCL10) were quantified at baseline and after 12 weeks of treatment with disease-modifying antirheumatic drugs or biologic agents in 28 patients using flow cytometry. Serum from 40 healthy individuals was collected for comparison at baseline. Response to treatment was classified according to the European League Against Rheumatism (EULAR) response criteria. Remission of disease was defined as a Disease Activity Score < 2.6.

RESULTS:
The baseline serum concentrations of CC and CXC chemokines were significantly elevated in patients with active RA compared to healthy controls (p < 0.05) except for CCL2. Significant improvement in all disease activity measurements was observed after 12 weeks of treatment. Seventeen (60.7%) patients achieved good to moderate response based on the EULAR response criteria, and 5 (17.9%) patients achieved remission. The improvement in clinical activity in patients with RA was accompanied by a significant reduction in the serum concentration of CXCL9 and CXCL10 (p < 0.001). A significant reduction in the serum level of CXCL10 was also observed in the group that achieved EULAR response. Serum concentration of CCL5 remained significantly elevated in patients with RA (n = 5) who achieved remission compared to the healthy controls (p < 0.05).

CONCLUSION:
Serum concentration of CXCL9 and CXCL10 may serve as sensitive biomarkers for disease activity in patients with RA.
Study done in Hong Kong

Matched MeSH terms: Flow Cytometry
Fulltext The estimation of foetomaternal haemorrhage by flowcytometry

Nurasyikin, Y., Suria, A.A., Ng, Soon Peng, Leong, C.F.

Medicine & Health, 2015;10(2):112-122.
MyJurnal

Fetomaternal haemorrhage (FMH) may occur following a sensitizing event, during pregnancy or at delivery. In cases of rhesus (Rh) incompatibility between mother and the fetus, it can thus subject to the haemolytic disease of the newborn. The Kleihauer test for quantification of FMH lacks standardization and results are less accurate. Furthermore, it cannot differentiate the foetal cell from the adult HbF. Flowcytometry analysis using monoclonal antibodies, is a new technique for the quantification of FMH and it allows larger number of cells to be analysed. It is also able to differentiate the foetal cell from maternal HbF, and thus is more sensitive and accurate. The objective of our study was to determine the FMH using the flowcytometric analysis of anti-HbF antibody and to correlate the FMH using flow cytometry and the standard Kleihauer test. Ninety eight peripheral blood samples from pregnant women at more than 20 weeks of pregnancy and post delivery were analyzed by both methods. The percentage of the foetal cells were recorded and the FMH were calculated. We found a fair correlation between the two methods with the correlation coefficient r = 0.633 (p

Matched MeSH terms: Flow Cytometry
Fulltext Aggressive variant large granular lymphocytic leukaemia: a case report

Sabariah, M. N., Zainina, S., Faridah, I., Leong, C. F.

Malaysian Journal of Medicine and Health Sciences, 2011;7(1):57-60.
MyJurnal

Clonal disorders of LGL may either be CD3+ CD56- or CD3- CD56+ phenotype and these have been designated as T-cell leukaemia (T-LGL) or natural killer cell (NK)-LGL leukaemia respectively. Clonality is usually demonstrated by clonal rearrangement of T-cell receptor gene rearrangement or identified by flowcytometry analysis. Most patients with T-LGL will have an indolent course. In this report we described an aggressiveness of disease in a patient with clonal CD3+ LGL leukaemia whose cells also co-expressed CD56 diagnosed by flowcytometry. The patient responded well to interrupt ALL standard risk protocol however succumbed to her disease while waiting for upfront stem cell transplant. This case highlights on both the classical laboratory findings of rare entity of disease as well as a review of the literature pertaining particularly on its management.

Matched MeSH terms: Flow Cytometry
Fulltext Transfected human mesenchymal stem cells do not lose their surface markers and differentiation properties

Yap FL, Cheong SK, Ammu R, Leong CF

Malays J Pathol, 2009 Dec;31(2):113-20.
PMID: 20514854 MyJurnal

In this study, we evaluated the biological properties of human mesenchymal stem cells transfected (hMSC) with a plasmid vector expressing human cytokine interleukin-12 (IL-12). Surface markers were analysed by immunophenotyping using flow cytometry. Differentiation capability was evaluated towards adipogenesis and osteogenesis. We demonstrated that successfully transfected hMSC retained their surface immunophenotypes and differentiation potential into adipocytes and osteocytes. These results indicate that hMSC may be a suitable vehicle for gene transduction.

Matched MeSH terms: Flow Cytometry
Fulltext The Immunomodulatory CEA Cell Adhesion Molecule 6 (CEACAM6/CD66c) Is a Protein Receptor for the Influenza a Virus

Rahman SK, Ansari MA, Gaur P, Ahmad I, Chakravarty C, Verma DK, et al.

Viruses, 2021 04 21;13(5).
PMID: 33919410 DOI: 10.3390/v13050726

To establish a productive infection in host cells, viruses often use one or multiple host membrane glycoproteins as their receptors. For Influenza A virus (IAV) such a glycoprotein receptor has not been described, to date. Here we show that IAV is using the host membrane glycoprotein CD66c as a receptor for entry into human epithelial lung cells. Neuraminidase (NA), a viral spike protein, binds to CD66c on the cell surface during IAV entry into the host cells. Lung cells overexpressing CD66c showed an increase in virus binding and subsequent entry into the cell. Upon comparison, CD66c demonstrated higher binding capacity than other membrane glycoproteins (EGFR and DC-SIGN) reported earlier to facilitate IAV entry into host cells. siRNA mediated knockdown of CD66c from lung cells inhibited virus binding on cell surface and entry into cells. Blocking CD66c by antibody on the cell surface resulted in decreased virus entry. We found that CD66c is a specific glycoprotein receptor for influenza A virus that did not affect entry of non-IAV RNA virus (Hepatitis C virus). Finally, IAV pre-incubated with recombinant CD66c protein when administered intranasally in mice showed decreased cytopathic effects in mice lungs. This publication is the first to report CD66c (Carcinoembryonic cell adhesion molecule 6 or CEACAM6) as a glycoprotein receptor for Influenza A virus.

Matched MeSH terms: Flow Cytometry
Fulltext Rosiglitazone diminishes the high-glucose-induced modulation of 5-fluorouracil cytotoxicity in colorectal cancer cells

Lau MF, Vellasamy S, Chua KH, Sabaratnam V, Kuppusamy UR

EXCLI J, 2018;17:186-199.
PMID: 29743857 DOI: 10.17179/excli2018-1011

Colorectal cancer (CRC) is the third most leading cause of morbidity and mortality throughout the world. 5-fluorouracil (5-FU), which is often administrated to disrupt carcinogenesis, was found to elevate blood glucose level among CRC patients. Thus, this study was conducted to evaluate the influence of rosiglitazone on antiproliferative effect of 5-FU using cellular model. Two human colonic carcinoma cell lines (HCT 116 and HT 29) were cultured in the presence of 5-FU, rosiglitazone or in combination under normal and high glucose concentration. The drug cytotoxicity was evaluated using the MTT assay whereas the assessment of cell cycle was carried out using the flow cytometry technique. Combination index (CI) method was used to determine the drug interaction between rosiglitazone and 5-FU. High glucose diminished the cytotoxic effect of 5-FU but at a high drug dosage, this effect could be overcome. Cell cycle analysis demonstrated that 5-FU and rosiglitazone caused G1-phase arrest and S-phase arrest, respectively. CI values indicated that rosiglitazone exerted synergistic effect on 5-FU regardless of glucose levels. This study is the first to demonstrate the influence of rosiglitazone on cytotoxicity of 5-FU under normal or high glucose level. Rosiglitazone may be a promising drug for enhancing the efficacy of 5-FU in the treatment of CRC associated with hyperglycemia.

Matched MeSH terms: Flow Cytometry
Fulltext Lymphocyte subsets in systemic lupus erythematosus

Cheong SK, Chin SF, Kong NC

Malays J Pathol, 1997 Dec;19(2):121-5.
PMID: 10879252

Systemic lupus erythematosus (SLE) is an autoimmune disease characterised by increased B cell activity and depressed T cell function. However, the contribution of the immunoregulatory system to its pathogenesis is still unclear. The recent development in the production of monoclonal antibodies and the availability of bench-top flow cytometers have allowed rapid quantitation of peripheral blood lymphocyte subsets. We analysed the distribution of the lymphocyte subsets in 24 patients with active SLE and 18 with inactive SLE. The distribution of immunoregulatory cells in 72 normal volunteers was used as control. Statistical analysis showed that there were significant differences between both the SLE groups and the normal controls, for total lymphocytes, T cells, B cells, T helper cells, T suppressor cells, T helper/suppressor ratio and natural killer cells. There was a significant difference for T helper cells between active and inactive SLE. T helper cells levels were found to be low in inactive SLE and lower in active SLE. It appears that treatment-induced remissions did not restore the levels of immunoregulatory cells to normal. Thus, T helper cell levels reflect disease activity and longitudinal assays of T helper cells may serve as an indicator of disease reactivation.

Matched MeSH terms: Flow Cytometry
Fulltext Mucosal-associated invariant T cell alterations during the development of human type 1 diabetes

Gazali AM, Schroderus AM, Näntö-Salonen K, Rintamäki R, Pihlajamäki J, Knip M, et al.

Diabetologia, 2020 11;63(11):2396-2409.
PMID: 32880687 DOI: 10.1007/s00125-020-05257-7

AIMS/HYPOTHESIS: Mucosal-associated invariant T (MAIT) cells are innate-like T cells that recognise derivatives of bacterial riboflavin metabolites presented by MHC-Ib-related protein 1 (MR1) molecules and are important effector cells for mucosal immunity. Their development can be influenced by the intestinal microbiome. Since the development of type 1 diabetes has been associated with changes in the gut microbiome, this can be hypothesised to lead to alterations in circulating MAIT cells. Accordingly, peripheral blood MAIT cell alterations have been reported previously in patients with type 1 diabetes. However, a comprehensive analysis of the frequency and phenotype of circulating MAIT cells at different stages of type 1 diabetes progression is currently lacking.
METHODS: We analysed the frequency, phenotype and functionality of peripheral blood MAIT cells, as well as γδ T cells, invariant natural killer T (iNKT) cells and natural killer (NK) cells with flow cytometry in a cross-sectional paediatric cohort (aged 2-15) consisting of 51 children with newly diagnosed type 1 diabetes, 27 autoantibody-positive (AAb+) at-risk children, and 113 healthy control children of similar age and HLA class II background. The frequency of MAIT cells was also assessed in a separate cross-sectional adult cohort (aged 19-39) of 33 adults with established type 1 diabetes and 37 healthy individuals of similar age.
RESULTS: Children with newly diagnosed type 1 diabetes displayed a proportional increase of CD8-CD27- MAIT cells compared with healthy control children (median 4.6% vs 3.1% of MAIT cells, respectively, p = 0.004), which was associated with reduced expression of C-C chemokine receptor (CCR)5 (median 90.0% vs 94.3% of MAIT cells, p = 0.02) and β7 integrin (median 73.5% vs 81.7% of MAIT cells, p = 0.004), as well as decreased production of IFN-γ (median 57.1% vs 69.3% of MAIT cells, p = 0.04) by the MAIT cells. The frequency of MAIT cells was also decreased in AAb+ children who later progressed to type 1 diabetes compared with healthy control children (median 0.44% vs 0.96% of CD3+ T cells, p = 0.04), as well as in adult patients with a short duration of type 1 diabetes (less than 6 years after diagnosis) compared with control individuals (median 0.87% vs 2.19% of CD3+ T cells, p = 0.007). No alterations in γδ T cell, iNKT cell or NK cell frequencies were observed in children with type 1 diabetes or in AAb+ children, with the exception of an increased frequency of IL-17A+ γδ T cells in children with newly diagnosed diabetes compared with healthy control children (median 1.58% vs 1.09% of γδ T cells, p = 0.002).
CONCLUSIONS/INTERPRETATION: Changes in the frequency and phenotype of circulating MAIT cells were detectable before, at the onset and after diagnosis of type 1 diabetes in cross-sectional cohorts. Our results suggest a possible temporal association between peripheral blood MAIT cell alterations and the clinical onset of type 1 diabetes. Graphical abstract.

Matched MeSH terms: Flow Cytometry
Different effects of two newly-isolated probiotic Lactobacillus plantarum 15HN and Lactococcus lactis subsp. Lactis 44Lac strains from traditional dairy products on cancer cell lines

Haghshenas B, Abdullah N, Nami Y, Radiah D, Rosli R, Khosroushahi AY

Anaerobe, 2014 Dec;30:51-9.
PMID: 25168457 DOI: 10.1016/j.anaerobe.2014.08.009

Lactobacillus and Lactococcus strains isolated from food products can be introduced as probiotics because of their health-promoting characteristics and non-pathogenic nature. This study aims to perform the isolation, molecular identification, and probiotic characterization of Lactobacillus and Lactococcus strains from traditional Iranian dairy products. Primary probiotic assessments indicated high tolerance to low pH and high bile salt conditions, high anti-pathogenic activities, and susceptibility to high consumption antibiotics, thus proving that both strains possess probiotic potential. Cytotoxicity assessments were used to analyze the effects of the secreted metabolite on different cancer cell lines, including HT29, AGS, MCF-7, and HeLa, as well as a normal human cell line (HUVEC). Results showed acceptable cytotoxic properties for secreted metabolites (40 μg/ml dry weight) of Lactococcus lactis subsp. Lactis 44Lac. Such performance was similar to that of Taxol against all of the treated cancer cell lines; however, the strain exhibited no toxicity on the normal cell line. Cytotoxic assessments through flow cytometry and fluorescent microscopy demonstrated that apoptosis is the main cytotoxic mechanism for secreted metabolites of L. lactis subsp. Lactis 44Lac. By contrast, the effects of protease-treated metabolites on the AGS cell line verified the protein nature of anti-cancer metabolites. However, precise characterizations and in vitro/in vivo investigations on purified proteins should be conducted before these metabolites are introduced as potential anti-cancer therapeutics.

Matched MeSH terms: Flow Cytometry

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