1.Association between MDM2-SNP309 polymorphism and bladder cancer in Mongolians
Baasansuren S ; Po-Shen WANG ; Ulziisaikhan E ; Amarsaikhan S ; Yi-Jang LEE ; Shiirevnyamba A
Mongolian Medical Sciences 2015;171(1):4-8
BACKGROUND:The mouse double minute 2 (MDM2) is a negative regulator of the p53 tumor suppressor protein.Overexpression of MDM2 is associated with poor survival and is a useful predictive factor for poor prognosisin various cancers in human. Studies revealed a genetic polymorphism located in intron 1 of the MDM2gene, MDM2-SNP309, (a change from T to G) is main functional polymorphism and important to developtumors. However, inconsistent associations between the MDM2-SNP309 and the risk or early onset ageof human different cancers have been reported worldwide. These conflicting results may have dependedon different patient subgroups and ethnicities studies. We studied the association of the MDM2-SNP309polymorphism andbladder cancer in Mongolian patients for the first time.OBJECTIVE:To investigate association between MDM2-SNP309 and the risk bladder cancer or early onset age of thecancer in Mongolian patients.MATERIALS AND METHODS:We genotyped MDM2-SNP309 in 44 patients with bladder cancer and 44 age and gender matched healthycontrols among Mongolian people.Genomic DNA was extracted from whole blood samples by the standardmethod of Qiagen mini blood DNA extraction kit (Qiagen Inc., Valencia, CA) and PCR amplification wasperformed using 100 ng genomic DNA template according to manufacturer’s protocol (Invitrogen, Carlsbad,CA). MDM2 SNP309 genotyping was carried out by restriction fragment length polymorphism assay.RESULTS: The allele frequencies of MDM2 SNP309 in the 44 bladder cancer patients were wild-type (T/T) 27.3%,homozygous (G/G) 34.1% and heterozygous (T/G) 38.6% whereas in the control cases were wild-type(T/T) 29.5%, homozygous (G/G) 20.5% and heterozygous (T/G) 50.0%. The proportion of homozygous(G/G) genotype was higher for bladder cancer cases than for healthy controls. Compared to the low-risk(wild type) genotype, an increased risk association with bladder cancer was shown for the GG genotype(OR=2.0, 95% CI=1.03-1.84). There is also a significant difference in median age onset of bladder cancerbetween GG low and high risk genotypes T/T and T/G (p=0,003)( p=0.0001), respectively (Figure2).CONCLUTION: The current sample data suggests that MDM2 SNP309 GG genotype may be associated withthe risk of bladder cancer as well as an earlier age onset in Mongolian patients with bladder cancer.
2.Gene mutation associated with drug resistance in M.tuberculosis strains isolated from national TB prevalence survey in Mongolia
Tsetsegtuya B ; Baasansuren E ; Oyuntuya T ; Tserelmaa B ; Gundsuren Sh ; Tsolmon B ; Naranbat N ; Tsatsralt-Od B ; Buyankhishig B ; Naranzul D
Mongolian Medical Sciences 2019;187(1):3-10
Background:
According to the First National Tuberculosis (TB) Prevalence Survey in Mongolia the prevalence of
bacteriologically-confirmed pulmonary TB among adults was 559.6 (95% CI: 454.5–664.7) per 100000
population in 2014–2015. This was three times as high as previously estimated. Nationwide anti-tuberculosis (TB) drug resistance survey was conducted in 1999 and 2007 in Mongolia. Share of multidrug resistant TB (MDR-TB) cases among newly notified TB cases increased from 1.0% in 1999 to 1.4% in 2007. Accordingly, we aimed to perform drug susceptibility test on strains isolated from TB Prevalence Survey and to determine the prevalence of drug resistant TB.
Material and Methods:
All 242 MTB strains isolated from the survey TB cases were tested GenoTypeMTBDRplus test and conventional 1st line DST on solid medium.
Result:
Conventional DST and GenoTypeMTBDRplus tests done for 93.8% (227/242) of them and 6.2% (15/242) were tested by GenoTypeMTBDRplus only. A 61.6% (95%CI 55.3-67.4) of all cases were susceptible to first line anti-TB drugs, any drug resistance and MDR-TBdetected as 38.4% (95% CI 32.5-44.7)and 9.5% (95% CI 6.4-13.9), respectively. Prevalence of MDR-TB was7.8% (95% CI 4.9-12.4) among new and 17.9% (95% CI 9.0-32.7) among previously treated cases. The 64 strains were identified as a resistant to isoniazid, 32.8% (42/64) and 65.6% (21/64) were katG, and inhAmutation, respectively. One isolate (1.6%) was mutations in both the inhAand katGgenes.The predominant mutations detected in therpoB were S531L (91.3%) among rifampicin resistant isolates and the mutation in inhAwas C–15T (100%) and katG mutation was S315T1 (100%) among isoniazid-resistant isolates.
Conclusion
Prevalence of cases with DR-TB is high among prevalent TB cases, especially prevalence of MDR-TB among new cases. In comparison to previous studies, DR-TB cases seem to be increased. Rifampicin resistant strains have a mutation of the rpoBand resistance to isoniazid is predominantly associated with the inhA mutation.
3.The effect of TLR9 ligand on IFN-ү signaling
Erkhembayar Sh ; Battsetseg Ts ; Baljinnyam T ; Altai E ; Baasansuren E ; Javkhlan B ; Batkhishig M ; Dolgorsuren S ; Ulziisaikhan J ; Enkhsaikhan L ; Tsendmaa Ts ; Galindev B ; Khongorzul B ; Baigalmaa B ; Nyambayar D ; Munkhbat B ; Bilegtsaikhan Ts
Health Laboratory 2017;6(1):15-23
Introduction:
The aim of this research project is to elucidate the crosstalk of innate and adaptive immune reactions against the DNA containing bacteria.
:
This study held in the Core laboratory, Science Technology Center, Mongolian National University of
Medical Sciences (MNUMS). Murine aortal endothelial cells, END-D cultured and the cell viability checked by MTT assay. In addition, the NO production, protein and gene expression studied by Griess Reagent
assay, R.T-PCR and immunoblotting, respectively.
Results:
0.1µM, 1µM and 10µM of TLR9 ligand exhibited no cytotoxic action against the cells by MTT assay. IFN-ү alone induced NO production in END-D cells. In the other hand, TLR9 ligand at 0.1µM, 1µM and 10µM up-regulated IFN-ү induced NO production in dose dependent manner. RTPCR results exhibit that TLR9 ligand up regulates iNOS mRNA. Immunoblotting analysis showed the enhanced iNOS protein expression and phosphorylation of STAT1 in cells pre-treated with TLR9 ligand.
Discussion:
We have demonstrated CpG DNA, TLR9 ligand, up-regulates IFN-ү induced NO via enhanced IFN-ү signaling. The result of Western Blotting and RT-PCR support the up-regulation of NO. CpG DNA can be used as agent against virus and bacteria. Further research need to be conducted.
Conclusion
TLR9 ligand, CpG DNA up-regulates IFN-ү induced NO production in time and dose dependent manner. TLR9 ligand augments the expression of iNOS mRNA and STAT1 phosphorylation in response to IFN-ү.
4.EFFECT OF TLR7 LIGAND ON SIGNAL TRANSDUCTION OF INTERFERON GAMMA
Baasansuren E ; Javkhlan B ; Baljinnyam T ; Erkhembayar Sh ; Batkhishig M ; Dolgorsuren S ; Enkhsaikhan L ; Ulziisaikhan J ; Khongorzul B ; Baigalmaa B ; Galindev B ; Sodnomtsogt L ; Nyambayar D ; Nyamdorj D ; Munkhtuvshin N ; Munkhbat B ; Bilegtsaikhan Ts
Innovation 2017;11(4):14-17
BACKGROUND: Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. TLR7 is expressed on monocytes, macrophages and dendritic cells, T cell, B cell and eosinophiles. TLR7, originally identified as recognizing imidaquinoline, loxibrine, broprimine and ssRNA, ssRNA viruses such as vesicular stomatitis virus, influenza A virus and human immunodefiency virus. It is known that virus ssRNA affects signaling molecule of IFN-y. Objective: To determine gene and protein activation of IFN-y signal transduction by TLR7 ligand in the endothelial cells.
MATERIAL: In study we used mouse aortic linear endothelial cell which is cultured (END-D) in 5% heat- inactivated fetal calf serum (FCS), medium (DMEM) containing antibiotic mix(penicillin G, streptomycin, amphotericin B) at 37°C (5% CO2). Endothelial cells treated with synthetic IFN-γ and imiquimodligands, then the NO (nitric oxide) concentration in the supernatant is determined by Griess reagent. Endothelial cells are cultured in 6 well cell culture plate and in each well 2*104cells are expected to be grown for 24 hours of culture. Then, the cells are treated with synthetic IFN-γ and имиквимод ligand for 6 hours and the NO signaling gene activation iNOS mRNA expression which is induced by IFN-γ is determined by RT-qPCR. Endothelial cells are cultured in 12 well cell culture plate and in each well 2*104 cells are expected to be grown for 18 hours of culture. Then, the cells are treated with synthetic IFN-γ and imiquimodligands for 24 hours and the NO signaling protein iNOS expression which is induced by IFN-γ is determined by western blotting. The experiment was conducted as representation mean of at least three test results. The difference between statistical probabilities is determined by the “Students” t test. The p<0.01 value is assumed to be statistically different.
RESULTS: TLR7 ligand imiquimodaugmented interferon gamma induced nitric oxide production TLR7 ligand imiquimodincreased interferon gamma induced iNOS mRNA gene expression. TLR7 ilgand imiquimodup-regulated interferon gamma induced iNOS protein expression.
CONCLUSIONS: TLR7 ligand imiquimod augments IFN-γ signaling in the endothelial cells. This synergistic effect has revealed in the levels of gene and protein expression.
5.The effect of regulator proteins on the IFN-γ/TLR9 synergistic signal transduction
Baljinnyam T ; Khulan O ; Erkhembayar Sh ; Baasansuren E ; Jawkhlan B ; Batkhishig ; Enkhsaikhan L ; Galindew B ; Tsewelmaa N ; Baigalmaa B ; Hongorzul B ; Sodnomtsogt L ; Nyambayar D ; Batbaatar G ; Monhbat B ; Munkhtuwshin N ; Bilegtsaikhan Ts
Health Laboratory 2018;8(1):8-13
Introduction:
When human body encounters external pathogens primary/innate immunity cells are activated by recognizing them and secondary/adaptive immunity is activated consecutively. Immune cell surface receptors, called Toll-like receptors (TLRs) recognize and bind pathogens. In our previous study, we revealed that there is a synergistic action between TLR9 and IFN-γ signaling in the endothelial cells.
Purpose:
To determine the role of negative and positive regulatory proteins on the IFN-γ/TLR9 synergistic signaling pathway
Materials and Methods:
This study was held in the Core Laboratory, Science Technology Center, Mongolian National University of Medical Sciences (MNUMS). In this study, murine endothelial cell (END-D) culture was used. The negative and positive regulator protein expression was detected by Western blotting.
Result:
Result of immunoblotting assay indicated that CpG DNA enhanced IFN-γ positive regulator protein p38 phosphorylation in the endothelial cells. Treatment by TLR9 ligand CpG DNA and IFN-γ increased p38
activation in 0.5 hour and 1 hour. CpG DNA inhibited IFN-γ negative regulator SOCS1 protein expression in 4 hr and 8 hr. Therefore, TLR9 ligand CpG DNA increased IFN-γ signal transduction in the endothelial cell line.
Conclusion
TLR9 ligand CpG DNA has decreased IFN-γ negative regulator protein SOCS1 expression. CpG DNA has increased IFN-γ positive regulator protein p38 phosphorylation.
6.Role of negative regulators on the TLR7 ligand/IFN-γ signaling in the endothelial cells
Baasansuren E ; Javkhlan B ; Baljinnyam T ; Khulan U ; Batkhishig M ; Enkhsaikhan L ; Ulziisaikhan J ; Khongorzul B ; Baigalmaa B ; Galindev B ; Tsevelmaa N ; Sodnomtsogt L ; Nyambayar D ; Munkhtuvshin N ; Munkhbat B ; Bilegtsaikhan Ts
Health Laboratory 2018;8(1):14-18
Introduction:
Toll like receptors (TLRs) are a class of proteins that key role in the innate immune system. The SOCS1 and SHP2 proteins are negative-feed loop inhibitors of signaling of JAK/STAT and TLRs pathways.
Purpose:
To determine negative regulator protein activation which is activated through TLR7 ligand/IFN-γ signal transduction in endothelial cells.
Methods:
We used mouse aortic linear endothelial cell (END-D); protein expressio was detected by western blotting
Results:
We analyzed a time dependent stimulation effects of negative regulator proteins stimulated by TLR7 ligand/IFN-γ in endothelial cell cultures. Imiquimod of 10 μg/ml treatment of 1 hr was followed by 100 ng/ml IFN-γ stimulation for 1-8hr to analysis of negative regulator SOCS1 and SHP2 protein expression.
In untreated cells, there was low activations of negative regulator SOCS1 and SHP2 proteins. IFN-γ stimulation alone had increased SOCS1 and SHP2 protein expressions, also imiquimod treatment highly elevated SOCS1 and SHP2 expressions. However imiquimod and IFN-γ doubled treatment have decreased activation of negative regulator SOCS1 and SHP2 proteins. These findings suggest SOCS1 and SHP2 proteins are inhibitors in the TLR7 ligand/IFN-γ signaling.
Conclusion
Negative regulators, SOCS1 and SHP2 strongly suppressed activations of TLR7 ligand/IFN-γ signaling
7.Study on influence of the CpG DNA on activation of IFN-γ signaling transduction regulatory proteins
Baljinnyam T ; Khulan U ; Erkhembayar Sh ; Baasansuren E ; Javkhlan B ; Batkhishig M ; Enkhsaikhan L ; Ulziisaikhan J ; Baigalmaa B ; Galindev B ; Tsevelmaa N ; Khongorzul B ; Sodnomtsogt L ; Munkhbat B ; Munkhtuvshin N ; Bilegtsaikhan Ts
Mongolian Medical Sciences 2018;186(4):10-13
Introduction:
When human body encounters external pathogens primary/innate immunity cells are activated by
recognizing them and secondary/adaptive immunity is activated consecutively. In our previous study,
we revealed that there is a synergistic action between TLR9 and IFN-γ signaling in the endothelial cells.
Purpose:
To determine the role of negative and positive regulator proteins on the IFN-γ/TLR9 signaling pathway.
Methods:
In this study, murine endothelial cell (END-D) culture was used. END-D cells pre-treated with TLR9
ligand CpG DNA and then stimulated with IFN-γ. The negative (SHP-2, SOCS1, PIAS1) and positive
(p38) regulator protein expression was detected by Western blotting.
Results and Conclusion
Treatment by TLR9 ligand CpG DNA and IFN-γ increased positive regulator p38 phosphorylation in 0.5
hour. CpG DNA inhibited IFN-γ negative regulator PIAS1 protein expression in 6 hour and SOCS1 and
SHP-2 expression could not affect in 4 hour.