scholarly journals Evaluation of the Correlation between the mRNA Expression Levels of ystA and ymoA Genes in Y. enterocolitica Strains with Different Enterotoxic Properties

Pathogens ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1136
Author(s):  
Agata Bancerz-Kisiel ◽  
Karolina Lipczyńska-Ilczuk
Keyword(s):  
Mrna Expression ◽  
Clinical Symptoms ◽  
Expression Level ◽  
Common Source ◽  
Expression Levels ◽  
Relative Expression ◽  
Source Of Infection ◽  
Relative Expression Level ◽  
Causative Agents ◽  
Mrna Expression Levels

Yersinia enterocolitica is one of the main causative agents of human diarrhea. Pigs are a reservoir and the most common source of infection for humans. The aim of this study was to analyze the expression of ystA and ymoA genes in Y. enterocolitica strains with different enterotoxic properties, isolated from humans and pigs. The experiment involved two groups of Y. enterocolitica strains producing and not producing enterotoxin YstA, which were isolated from humans and pigs. All strains were ystA- and ymoA-positive. The expression of ystA and ymoA genes was analyzed by quantitative real-time PCR (qPCR). The relative expression level of the ystA gene was significantly higher than the expression level of the ymoA gene in Y. enterocolitica strains isolated from humans with clinical symptoms of yersiniosis. In other strains, a significant decrease in ystA gene transcription was observed, and the relative expression level of the ymoA gene was significantly higher than the expression level of the ystA gene. Statistically significant differences were not observed in either group of strains isolated from pigs. The results of our study revealed a correlation between mRNA expression levels of ystA and ymoA genes in Y. enterocolitica strains isolated from humans.

The Impact of ymoA Gene Expression on Enterotoxin YstA Production by Yersinia Enterocolitica Strains With Different Enterotoxic Properties

2021 ◽  
Author(s):  
Agata Bancerz-Kisiel ◽  
Karolina Lipczyńska-Ilczuk
Keyword(s):  
Yersinia Enterocolitica ◽  
Clinical Signs ◽  
Expression Level ◽  
Relative Expression ◽  
Source Of Infection ◽  
Relative Expression Level ◽  
Genes Expression ◽  
Causative Agents ◽  
The Impact ◽  
Mrna Expression Levels

Abstract Yersinia enterocolitica is one of the main causative agents of human diarrhoea and the reservoir and source of infection for humans are pigs. Strains isolated from humans with clinical yersiniosis and diarrhoea are able to produce Yersinia stable toxins – Yst. However, enterotoxin-producing capabilities have been attributed to the ymoA gene which encodes the production of the Yersinia modulator protein – YmoA. The aim of this study was to analyse ystA and ymoA genes expression in Y. enterocolitica strains with different enterotoxic properties, isolated from humans and pigs. The experiment involved two groups of Y. enterocolitica strains producing and not producing enterotoxin YstA, which were isolated from humans and pigs. All these strains were ystA- and ymoA-positive. The relative expression level of the ystA gene was significantly higher than the expression level of the ymoA gene in Y. enterocolitica strains isolated from humans with clinical signs characteristic for yersiniosis. In others, a significant decrease in ystA gene transcription was observed, and the relative expression level of the ymoA gene was significantly higher than the expression level of the ystA gene. Statistically significant differences were not observed in either group of strains isolated from pigs. The results of our study revealed a correlation between the mRNA expression levels of ystA and ymoA genes in Y. enterocolitica strains isolated from humans.

scholarly journals Expression and clinical significance of PTPN12 in clear cell renal cell carcinoma

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052093604
Author(s):  
Yi Jin ◽  
Tian-xi Wang ◽  
Hao Li ◽  
Peng Guo ◽  
Qing-qing Wang
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Mrna Expression ◽  
Renal Cell ◽  
Clear Cell ◽  
Cell Renal Cell Carcinoma ◽  
Expression Levels ◽  
Relative Expression ◽  
Normal Tissues ◽  
Mrna Expression Levels

Background Clear cell renal cell carcinoma (ccRCC) is a common urological disease. Expression of the protein tyrosine phosphatase 12 gene ( PTPN12) is decreased in many cancers; however, the relationship between PTPN12 gene function and renal cancer remains unclear. Methods We detected PTPN12 protein expression in ccRCC and corresponding normal tissues from 64 patients with ccRCC by immunohistochemistry, and relative PTPN12 mRNA levels by real-time quantitative polymerase chain reaction. The relationships between the relative expression levels of PTPN12 mRNA and the patients’ clinical data were analyzed. Results PTPN12 protein and mRNA expression levels were significantly lower in ccRCC compared with the corresponding normal tissues. The mRNA expression levels in the ccRCC and corresponding normal tissues from the 64 patients with ccRCC were 0.459±0.445 and 1.001±0.128, respectively, compared with the control (glyceraldehyde 3-phosphate dehydrogenase). There was a significant correlation between relative expression of PTPN12 mRNA in ccRCC tissues and tumor diameter and clinical stage. Conclusion The expression levels of PTPN12 protein and mRNA were significantly lower in ccRCC tissues compared with normal tissues. The role of PTPN12 may provide new insights and evidence to aid the diagnosis and targeted therapy of ccRCC.

scholarly journals Th2 cytokine bias induced by silver nanoparticles in peripheral blood mononuclear cells of common bottlenose dolphins (Tursiops truncatus)

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5432 ◽  
Author(s):  
Wen-Ta Li ◽  
Lei-Ya Wang ◽  
Hui-Wen Chang ◽  
Wei-Cheng Yang ◽  
Chieh Lo ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Mononuclear Cells ◽  
Bottlenose Dolphins ◽  
Expression Level ◽  
Mrna Expression Level ◽  
Expression Levels ◽  
Th2 Cytokine ◽  
Tnf Α ◽  
Ifn Γ ◽  
Mrna Expression Levels

Background Silver nanoparticles (AgNPs) have been widely used in many commercial products due to their excellent antibacterial ability. The AgNPs are released into the environment, gradually accumulate in the ocean, and may affect animals at high trophic levels, such as cetaceans and humans, via the food chain. Hence, the negative health impacts caused by AgNPs in cetaceans are of concern. Cytokines play a major role in the modulation of immune system and can be classified into two types: Th1 and Th2. Th1/Th2 balance can be evaluated by the ratios of their polarizing cytokines (i.e., interferon [IFN]-γ/Interleukin [IL]-4), and animals with imbalanced Th1/Th2 response may become more susceptible to certain kinds of infection. Therefore, the present study evaluated the in vitro cytokine responses of cetacean peripheral blood mononuclear cells (cPBMCs) to 20 nm citrate-AgNPs (C-AgNP20) by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Methods Blood samples were collected from six captive common bottlenose dolphins (Tursiops truncatus). The cPBMCs were isolated and utilized for evaluating the in vitro cytokine responses. The cytokines evaluated included IL-2, IL-4, IL-10, IL-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α. The geometric means of two housekeeping genes (HKGs), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M), of each sample were determined and used to normalize the mRNA expression levels of target genes. Results The ratio of late apoptotic/necrotic cells of cPBMCs significantly increased with or without concanavalin A (ConA) stimulation after 24 h of 10 µg/ml C-AgNP20 treatment. At 4 h of culture, the mRNA expression level of IL-10 was significantly decreased with 1 µg/ml C-AgNP20 treatment. At 24 h of culture with 1 µg/ml C-AgNP20, the mRNA expression levels of all cytokines were significantly decreased, with the exceptions of IL-4 and IL-10. The IFN-γ/IL-4 ratio was significantly decreased at 24 h of culture with 1 µg/ml C-AgNP20 treatment, and the IL-12/IL-4 ratio was significantly decreased at 4 or 24 h of culture with 0.1 or 1 µg/ml C-AgNP20 treatment, respectively. Furthermore, the mRNA expression level of TNF-α was significantly decreased by 1 µg/ml C-AgNP20 after 24 h of culture. Discussion The present study demonstrated that the sublethal dose of C-AgNP20 (≤1 µg/ml) had an inhibitory effect on the cytokine mRNA expression levels of cPBMCs with the evidence of Th2 cytokine bias and significantly decreased the mRNA expression level of TNF-α. Th2 cytokine bias is associated with enhanced immunity against parasites but decreased immunity to intracellular microorganisms. TNF-α is a contributing factor for the inflammatory response against the infection of intracellular pathogens. In summary, our data indicate that C-AgNP20 suppresses the cellular immune response and thereby increases the susceptibility of cetaceans to infection by intracellular microorganisms.

scholarly journals Relationship between the mRNA expression levels of calpains 1/2 and proteins involved in cytoskeleton remodeling

Acta Naturae ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 110-113
Author(s):  
Gelena V. Kakurina ◽  
Elena S. Kolegova ◽  
Elena E. Shashova ◽  
Olga V. Cheremisina ◽  
Evgeniy L. Choynzonov ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Cancer Progression ◽  
Expression Level ◽  
Actin Binding ◽  
Published Data ◽  
Cytoskeletal Reorganization ◽  
Expression Levels ◽  
Cytoskeleton Remodeling ◽  
The Relationship ◽  
Mrna Expression Levels

Remodeling of the cytoskeleton underlies various cellular processes, including those associated with metastasis. The role of the proteases and proteins involved in cytoskeletal reorganization is being actively studied. However, there are no published data on the relationship between the mRNA expression levels of calpains 1/2 (CAPN 1/2) and the proteins associated with cytoskeleton remodeling. Therefore, the purpose of our study was to establish the relationship between the mRNA expression levels of CAPN 1/2 and the proteins involved in cytoskeletal reorganization, such as cell motility markers (SNAI1, VIM, and RND3) and actin-binding proteins (CFN1, PFN1, EZR, FSCN1, and CAP1) using the model of laryngeal/laryngopharyngeal squamous cell carcinoma (LC). The gene expression level was determined by reverse transcriptase real-time PCR and calculated using the 2-Ct method in paired tissue samples of 44 patients with LC (T1-4N0-2M0). The patients were divided into two groups: those with low and those with high CAPN 1/2 expression levels. It was found that metastasis in LC patients was associated with decreased expression levels of VIM and CAP1, and increased levels of CAPN1. A high level of CAPN2 was accompanied by a high expression level of EZR, indicating the activation of invasion processes. The results obtained need to be confirmed in further studies using a larger sample of patients and target genes. Our study is important in elucidating the mechanisms that underlie cancer progression and metastasis, a development that could subsequently open the way to a search for new prognostic and predictive markers of laryngeal/laryngopharyngeal cancer progression.

scholarly journals Expression analysis and single-nucleotide polymorphisms of MLPH and PMEL17 genes associated with melanin deposition in Xuefeng black-bone chickens

2021 ◽  
Author(s):  
Xiangyong Qu ◽  
Yuying Deng
Keyword(s):  
Mrna Expression ◽  
Pectoral Muscle ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Expression Levels ◽  
Relative Expression Level ◽  
L Value ◽  
Breeding Selection ◽  
Mrna Expression Levels ◽  
Selection Of

Melanin deposition related genes such as MLPH and PMEL17 play an important role in black-bone chicken. This study was aimed to identify and associate SNPs in the MLPH and PMEL17 genes with melanin content of pectoral muscle (MCPM) in Xuefeng black-bone chicken. A total of 120 Xuefeng black-bone chickens at 120-day-old were randomly selected to measure blackness of pectoral muscle (BPM), according to the degree of BPM selected 22 high blackness (HB) and 22 low blackness (LB) chickens to determine the MCPM, and extract DNA and mRNA. The results indicated that the MCPM in the HB group was higher than in the LB group (P < 0.01), and the L value in the HB group was lower than in the LB group (P < 0.01). And we measured the mRNA expression levels of MLPH and PMEL17 genes in pectoral muscle by quantitative real-time PCR. The results revealed that the mRNA expression levels of MLPH gene (P < 0.05) and PMEL17 gene (P < 0.01) in the HB group was higher than in the LB group, and the mRNA relative expression level of MLPH and PMEL17 genes with MCPM was positive correlation (P < 0.01). And the sequencing results found that a total of 17 SNPs were found in MLPH gene, the C-1411T was associated with MCPM (P< 0.05), there was no difference in MCPM among other locus (P> > 0.05). And there were 10 SNPs in PMEL17 gene, the G-1843C, C-2812T, and G-2794A were associated with MCPM (P < 0.05), there was no difference in the MCPM among other locus (P > 0.05). These SNPs could be molecular markers for breeding selection of blackness traits.

Synergic Effects of Immunosuppressive Agents on T Lymphocyte Function and Cytokine Expression Pattern.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5213-5213
Author(s):  
Mao Fang Lin ◽  
Yan Ling Jiang
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
T Lymphocyte ◽  
Immunosuppressive Agents ◽  
Reaction System ◽  
Expression Level ◽  
Lymphocyte Function ◽  
Relative Expression ◽  
Relative Expression Level ◽  
Ifn Γ

Abstract The regimen including mycophenolate mofetil (MMF) and cyclosporine A (CsA) has been successfully used as an effective method in the prophylaxis of acute graft versus host disease. Herewith we investigated the effect of CsA, mycophenolic acid (MPA) alone or in combination with anti B7–1 mAb on proliferation of T lymphocytes and their possible mechanisms. The T lymphocyte reaction system was established in vitro and was detected by 5-bromo-2-deoxyuridine incorporation method; the expression of IL-2, IFN-γ and IL-10 on mRNA and protein level were detected by RT-PCR and ELISA respectively. Experimental concentration of MPA (50μmol/L) and CsA(0.33μmol/L) inhibited the T lymphocyte proliferation significantly. CsA in combination with anti B7–1 mAb(10mg/L) had a much stronger effect on the inhibition(p<0.01). There was no difference between groups treated either with MPA or with the combination of MPA and anti B7–1 mAb. Both of CsA and MPA could inhibit the protein expression of IL-2 and IFN-γ. The protein expression of IL-2 were 99.70±9.15 pg/ml, 48.19±8.67pg/ml and 42.73±14.64 pg/ml in groups of control, treated with CsA or MPA respectively. The protein expression of IFN-γ in the group treated with CsA or MPA decreased significantly than the control (6.81±5.24 pg/ml, 7.87±4.22 pg/ml vs 82.42±25.55 pg/ml, p<0.05). Compared with that treated with CsA alone, The protein expression of IFN-γ in the group treated with CsA in combination with anti B7–1 mAb decreased significantly (0.30±0.52 pg/ml vs 6.81±5.24 pg/ml, p<0.05). Both of CsA and MPA increased the protein expression of IL-10. Compared with MPA alone, MPA in combination with anti B7–1 mAb increased significantly the expression of IL-10 (941.90±56.61 pg/ml vs 770.95±126.85 pg/ml, p<0.05). The relative expression level of IL-2 mRNA (IL-2/GAPDH) and IFN-γ mRNA (IFN-γ /GAPDH) in the CsA or MPA group was significantly lower than the control (p<0.01). The relative expression level of IFN-γ mRNA in combination group of CsA with anti B7–1 mAb decreased even more significantly (0.38±0.05 vs 0.59±0.02, p<0.01). On the other hand, this combination could up-regulate IL-10 mRNA expression (1.80±0.13 vs 1.38±0.06, p<0.01). Combination of MPA with anti-B7–1 mAb showed a statistically significant increase in IL-10 mRNA expression (1.28±0.06 vs 0.84±0.09, p<0.01) as compared with MPA alone. In conclusion, MPA and CsA induced the changes of cytokine expressive spectrum and the Th1 and Th2 shift might be involved in the immunosuppressive effect. The combination of immunosuppressive agents, especially with anti B7–1 mAb had a synergic effect.

WT-1 Expression Level In BM Is The Great Prognostic Marker In Three Of Classification IPSS, WPSS, and Latest Revised IPSS(IPSS-R)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2795-2795
Author(s):  
Sumiko Kobayashi ◽  
Yasunori Ueda ◽  
Mineo Kurokawa ◽  
Kiyoyuki Ogata ◽  
Hirohiko Shibayama ◽  
...  
Keyword(s):  
Regression Analysis ◽  
Mrna Expression ◽  
Survival Time ◽  
Research Funding ◽  
Mrna Level ◽  
Expression Level ◽  
Mrna Expression Level ◽  
Expression Levels ◽  
Wbc Count ◽  
Mrna Expression Levels

Abstract Objective/material/method It is known new classification named revised IPSS(IPSS-R) as a prognosis in MDS patients in 2012. To investigate the clinical utility of WT1 mRNA expression level including IPSS-R, we studied of MDS patients who provided consent at 17 medical institutions nationwide from Dec. 2008 to Sep. 2009 were enrolled in Japan. A total of 172 subjects including 115 MDS patients by IPSS (RA:69, RARS:9, RAEB:24, RAEB-t :13 ). 13 patients who developed AML from MDS, and 44 patients with non malignant hematological disorders who provided consent. This study was designed to follow up 82 patients, who gave secondary consent, among 115 MDS patients registered in study ODK-0801 for 5 years up to 2014 to analyze in detail the relationship between the WT1 mRNA expression level and prognosis. WT1 mRNA level in PB and BM were measured using the WT1 mRNA assay kit “OTSUKA” (OTSUKA PHARMACEUTICAL CO., LTD). WT1 mRNA expression levels in peripheral blood (and in bone marrow, if possible) were measured periodically to evaluate the usefulness of the WT1 mRNA expression level as a monitoring marker of MDS and analyze changes in pathology in individual patients by central review and changes in WT1 mRNA expression levels. In this study, value of 50 copies/μgRNA was set as the cut off value for WT-1mRNA expression. The differences in clinical and demographic data were assessed in the chi-square test and logistic regression analysis. The survival data was analyzed using Kaplan-Meier method and compared by the log-rank test. This study presents the results of interim analysis 3 years after the start of the investigation. Results The comparison study between the patients categorized into three groups by the WT1 mRNA level　in BM ,GroupI:less than 102 copies/μgRNA(n=35), Group II: 102 to 104 copies/μgRNA(n=30), and GroupIII: more than 104 copies/μgRNA(n=17) resulted that the survival rates decreased significantly as the WT1 mRNA level increased(GI vs GII P<0.01, GI vs GIII P<0.01, GII vs GIII p<0.067), respectively. In multivariate Cox proportional hazard regression analysis of IPSS, five out of fifteen parameters, which are WBC count (P=0.0001), IPSS score (P=0.0003), blast in PB(P=0.0011), WT1 mRNA level in BM(P=0.0055), sex(P=0.093) were independently associated with survival time, respectively. And we studied same analysis used WPSS. WBC count (P=0.0001), WPSS socre(P=0.0001), blast in PB(P=0.0037), WT1 mRNA level in BM(P=0.0029), sex(P=0.012) were independently associated with survival time, respectively. Also we analyzed using IPSS-R. In multivariate Cox proportional hazard regression analysis, five out of fifteen parameters, WBC count (P=0.0001), IPSS-R score(P=0.0001), blast in PB(P=0.0012), WT1 mRNA level in BM(P=0.01), sex(P=0.01) were independently associated with survival time, but not ANC(both score and absolute number), Hb, platelet, karyotype or other parameters. Using these selected five valiables, we provided the level of WT-1mRNA with 4 risk groups which classified :100, 50100, 100,100, 150,100copies/μRNA). According to increasing of WT-1mRNA level, survival duration shortened the increase with the increase in each risk group. Discussion The WT-1 mRNA level in BM was positively correlated with the prognosis of MDS stage and tended to be correlated with advanced risk of IPSS, WPSS, and IPSS-R. Therefore it was considered to be a useful prognostic marker for MDS. Conclusion We found that the survival time was shortened significantly with an increase in WT1 mRNA expression levels in both peripheral blood and bone marrow, demonstrating that WT1 mRNA expression levels are a highly useful prognostic factor in MDS even if we use any of classification of IPSS, WPSS, IPSS-R. This assay has great possibility to contribute to more appropriate therapeutic decisions in MDS patients as well as diagnosis and to evaluate the timing of allogeneic transplantation. Disclosures: Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding. Usuki:Alexion Pharmaceuticals, Inc.: Speakers Bureau. Ohyashiki:Novartis: Honoraria, Research Funding.

scholarly journals Attenuation of the Apoptosis Stimulating Protein of TP53-1 (ASPP1) Is a Predictive Marker for Therapy Resistance and Overall Survival in Acute Myeloid Leukemia (AML)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1298-1298
Author(s):  
Marcus M. Schittenhelm ◽  
Max Kaiser ◽  
Vasileia Tsintari ◽  
Gunnar Blumenstock ◽  
Balazs Györffy ◽  
...  
Keyword(s):  
Overall Survival ◽  
Risk Stratification ◽  
Genetic Risk ◽  
Risk Group ◽  
Advisory Board ◽  
Expression Level ◽  
Expression Levels ◽  
Relative Expression ◽  
Patient Cohort ◽  
Relative Expression Level

Abstract ASPP1 (PPP1R13B) belongs to a family of p53-binding proteins and enhances apoptosis by stimulation of p53-transactivation of selected proapoptotic target genes. It is preferentially expressed in hematopoietic stem cells (HSC) and together with p53 preserves the genomic integrity of the HSC pool. We have previously demonstrated that ASPP1 is attenuated in AML, which is linked to methylation of the promoter region. ASPP1-interferference models suggest a functional role in leukemogenesis and therapy response. We now provide evidence that ASPP1 is highly altered in AML and attenuated expression levels associate with inferior outcomes: mRNA expression patterns of ASPP1 were assessed in an unselected patient cohort with newly diagnosed AML (n=39) - and were found to be significantly lower compared to bone marrow aspirates of 12 healthy donors (p &lt; 0.0001, Mann-Whitney test) with a median relative expression level of 0.33 (ASPP1 AML) vs. 0.95 (ASPP1 donor). Subcohort analysis by genetic risk profile according to the European LeukemiaNet (ELN) 2017 genetic risk stratification revealed significantly lower expression levels of the intermediate/adverse risk group (n=27) compared to the favorable risk group (n=10, p=0,013) with a median relative expression level of 0.21 (ASPP1 int/adv) vs. 0.46 (ASPP1 good). In addition, analysis of patients undergoing induction chemotherapy demonstrated a significantly lower complete remission (CR) rate in patients with attenuated ASPP1 levels (p=0.0015) with a median relative expression level of 0.13 (ASPP1 nonCR) vs. 0.51 (ASPP1 CR). Of note, patients with a &gt;10x fold decrease of ASPP2 expression were exclusively found in the patient cohort failing induction therapy. To validate these observations in an independent patient set, we next performed a database RNAseq screen on a defined, unselected AML cohort (n=142, FAB M3 were excluded). Together, ASPP1 was independently confirmed as an adverse prognostic factor: Overall survival (OS) was longer in the ASPP1 high cohort vs. the ASPP1 low group with a hazard ratio for death of 0.63. Even though this analysis closely failed significance (0.082) due to lack of power, probability to be alive after 70 months was 45% (ASPP1 high) vs. 10% (ASPP1 low) with a median survival of 19.23 months (ASPP1 low) vs. 26.4 months (ASPP1 high). Most interestingly, looking at the prognostic good risk population: attenuation of ASPP1 resulted in a dramatic decrease of survival with a hazard ratio of 0.29 and a probability to be alive after 70 months of 25% (ASPP1 low) vs. 85% (ASPP1 high). Consequently, screening for ASPP1 expression may define a patient cohort likely to fail (induction) therapy - which might be a target population for hypomethylating agents (HMA) to reconstitute ASPP1 and sensitize towards (chemo)therapy. To address this hypothesis, MOLM-14 acute leukemia cells and patient derived freshly-isolated AML samples were treated with decitabine - resulting in upregulation of ASPP1 mRNA expression levels in all assessed samples. As expected, cells were more chemo-susceptible when exposed to decitabine followed by daunorubicin and cytarabine in an ASPP1-dependent manner: MOLM-14.ASPP1i cell line strains did not reveal a significant change of proapoptotic efficacy when compared to mock strains which significantly benefitted from this approach with more than doubled proapoptotic rates (**** p &lt; 0.0001, t-test), again confirming ASPP1 target specificity. Our results demonstrate that dysfunctional regulation of ASPP1 expression is frequently observed in AML, which associates with therapy responses and survival outcome. Prospective clinical studies are warranted to evaluate the role as a biomarker for risk stratification. Further, we provide a rationale to re-sensitize high-risk patients (defined as ASPP1 low-expressors) towards chemotherapy using HMA priming upfront - and this strategy should be followed in future trials. Disclosures Schittenhelm: BMS: Other: advisory board; Takeda: Other: advisory board; Astellas: Other: advisory board; University of Tuebingen: Patents & Royalties: patent for ASPP2k. Kampa-Schittenhelm: University of Tuebingen: Patents & Royalties: patent related to ASPP2k.

scholarly journals The Role of TLR4 Inducing Macrophage Pyroptosis in the Pathogenesis of Severe Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1114-1114
Author(s):  
Rong Fu ◽  
Tian Zhang ◽  
Ling Deng ◽  
Yang Zhao ◽  
Xiaoyu Zhao ◽  
...  
Keyword(s):  
T Cells ◽  
Cd8 T Cells ◽  
Culture Supernatant ◽  
Expression Level ◽  
Healthy Controls ◽  
Expression Levels ◽  
Relative Expression ◽  
Caspase 1 ◽  
Relative Expression Level ◽  
Mrna And Protein Expression

Abstract Introduction：To investigate the expression level of macrophage pyroptosis in patients with severe aplastic anemia (SAA) and its effect on downstream effector T cells, the study explored the mechanism of Toll like receptor 4 (TLR4) inducing macrophage pyroptosis and maintaining immune homeostasis of SAA through pyruvate kinase M2 (PKM2) at cellular and molecular levels, which provided a theoretical basis for the targeted therapy of macrophages in SAA. Three groups of bone marrow macrophages of new diagnosed SAA patients, remission patients and healthy controls were induced and cultured in vitro. qRT-PCR and Western Blot were used to detect pyroptosis-related indicators IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD expression level; ELISA to detect the concentration of IL-1β and IL-18 in the culture supernatant. Results: 1. The mRNA and protein levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed SAA group were significantly higher than those in healthy controls, and the IL-1β and IL-18 concentration in the macrophage culture supernatant increased significantly. The relative mRNA expression levels of macrophages IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD in the new diagnosed group were significantly negatively correlated with WBC, RBC, Hb and PLT. 2. The relative expression level of TLR4 mRNA in macrophages in the new diagnosed group was significantly higher than that in the remission group and healthy controls, and the protein level of TLR4 was significantly increased; at the same time, the relative expression level of TLR4 mRNA was positively correlated with IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD in macrophages. After knocking down TLR4 or adding TLR4 inhibitors, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1 and GSDMD were significantly lower than those in healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant reduced significantly. 3. The expression rate of PKM2 in macrophages of new diagnosed group by flow cytometry was significantly higher than that in the remission group and heathy controls, and the levels of PKM2 mRNA and protein were significantly increased. At the same time, the relative expression level of PKM2 mRNA was positively correlated with the expression levels of IL-1β, IL-18, NLRP3, Caspase-1, and GSDMD. After knocking down PKM2, the mRNA and protein expression levels of IL-1β, IL-18, NLRP3, Caspase-1, GSDMD were significantly lower than those healthy controls, and the concentration of IL-1β and IL-18 in the culture supernatant was also significantly reduced. After knocking down TLR4 or adding TLR4 inhibitor, PKM2 mRNA and protein expression levels in macrophages were significantly lower than those in healthy controls. 4. When CD8+ T cells were co-cultured with macrophages which were knocked down TLR4 or added with inhibitors, the expressions of perforin and granzyme B in CD8+ T cells were significantly reduced. CD8+ T cells were further co-cultured with K562 and the apoptosis level of K562 was reduced. Summary：The level of macrophage pyroptosis in SAA patients increased, and the expression levels of macrophages TLR4 and PKM2 increased and positively correlated with the level of pyroptosis. Inhibiting the expression of TLR4 and PKM2, respectively, reduced the level of macrophage pyroptosis, and at the same time inhibiting TLR4 expression reduced the expression of PKM2. Macrophages that inhibited TLR4 expression reduced the killing function of CD8+ T cells. Therefore, TLR4 may induce pyroptosis of macrophages in SAA patients through PKM2 and enhance the killing function of CD8+ T cells. Disclosures No relevant conflicts of interest to declare.

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