Dlk1 Up Regulated Might Be an Important Event in Human MDS.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4842-4842
Author(s):  
Q.F. Xiao ◽  
Zi X. Chen ◽  
Dan D. Liu ◽  
Jian N. Cen ◽  
Jun He ◽  
...  
Keyword(s):  
Experimental System ◽  
K562 Cells ◽  
In Vitro Transcription ◽  
Clinical Samples ◽  
Mrna Levels ◽  
Amplification Protocol ◽  
Cd34 Cells ◽  
Normal Controls

Abstract The diagnosis of myelodysplastic syndrome (MDS) is made largely on the dysplastic morphology of BM cells from aspiration or biopsies. Prognosis scored by IPSS is depending on the percentage of marrow myeloblasts and the clonal cytogenetic abnormalities. To expand the understanding of genetic defects in hematopoietic cells of MDS in hope of finding novel genes correlated to pathogenesis and provide possible diagnostic marker for MDS, we have applied microarray to analyze the clinical samples from MDS patients. Total RNAs of CD34+ cells from 8 patients ( 2 RAEBt,2 RAEB,2 RA,1 RAS,1 CAA ) and one healthy people were extracted followed by a double in vitro transcription to circumvent the limited number of CD34+ cells. Following a modified Affymetrix target amplification protocol. Biotinylated cRNA was synthesized from 50 ng total RNA by double-round amplification and hybridized to an Human Genome U133 Plus 2.0 Array (Affymetrix). From the expression profile of 18404 different genes, we revealed that DNTT,MLL3,IL1R2,MAPK1,IGLL1 were down regulated while EGR-1, Rap1GAP or MAF were up regulated compared with normal controls. Most notably, Dlk1 was up regulated in MDS, while down regulated in AML and normal. By real-time RT-PCR we confirmed that in BMNCs the median levels of Dlk1 transcript in patients with RA and RAS were 2.55 (range, 0.00–23.7), RAEB and RAEBt were 8.24(range, 2.01–18.44), AML were 1.88 (range, 0.12–5.13), and other patients were 0.37(range, 0.00–1.79), respectively. The abundance of Dlk1 mRNA in MNCs from most MDS patients was markedly greater than that in the MNCs from others (P <0.05 ). Dlk1 expression in RAEB and RAEBt is markedly higher than AML (P <0.05 ) Forced expression of Dlk1 in transfected K562 cells resulted in faster growth than control cells, affected apoptosis induced by As2O3. and reduced the G2 arrested cells induced by TPA. By using the same experimental system we found that forced expression of Dlk1 can increase the mRNA levels of HES1 and p21WAF1 transcript variant 1. To elucidate the mechanisms we analyzed the levels of phosphorylated-p38 and p38 in Dlk1 transfected K562 cells treated with TPA. Dlk1 inhibited p38 phosphorylation while expression of p38 kept no change. These results support further investigation on the role of Dlk1 in abnormal hematopoiesis in MDSheterogeneous cell component. Diagnosis is currently depending on the dysplastic morphology of.

Plitidepsin Inhibits the Growth of Cells Harboring JAK2V617F Mutation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3907-3907
Author(s):  
Costanza Bogani ◽  
Paola Guglielmelli ◽  
Niccolò Bartalucci ◽  
Miguel Aracil ◽  
Maria Fe Paz ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Lines ◽  
Murine Model ◽  
Myeloproliferative Neoplasms ◽  
K562 Cells ◽  
Jak2v617f Mutation ◽  
Mrna Levels ◽  
Significance Level ◽  
Cd34 Cells

Abstract Abstract 3907 Poster Board III-843 Plitidepsin (Aplidin®) is a novel cyclic depsipeptide derived from the marine tunicate Aplidium albicans, currently obtained by chemical synthesis, that is under Phase II clinical development. Plitidepsin is effective against a large panel of tumor cells, and although precise action mechanisms have still to be ascertained the drug induces an oxidative stress, activation of Rac1 GTPase and inhibition of protein phosphatases, overall leading to sustained activation of JNK and p38MAPK. In a previous report (Verrucci M et al, ASH 2008, 2787A) we evaluated Plitidepsin activity in the GATA-1low murine model of myelofibrosis. Plitidepsin corrected thrombocytopenia of myelofibrotic mice, reduced the frequency of megakaryocytes (Mk) and normalized angiogenesis in the bone marrow, and prevented extramedullary hematopoiesis. In the present study, we assessed the effects of Plitidepsin on cell lines harboring homozygous (HEL and UKE-1, a gift of W. Fiedler) or heterozygous (SET2) JAK2V617F mutation and on cells from patients (pts) with myeloproliferative neoplasms (MPN). In a short-term (3 days) proliferation assay we found that Plitidepsin prevented cell growth with IC50 values of 1.0±0.3 nM for HEL, 0.5±0.03 nM for UKE-1, and 0.8±0.02 nM for SET2, that were all lower than 1.5±0.1 nM for the BCR/ABL mutated K562 cell line (P<.001 in case of UKE-1 cells). Also Ba/F3 cells transduced with the V617F allele (a gift of R. Skoda) were found more sensitive to Plitidepsin (IC50= 0.03±0.01 nM) than the wild-type counterpart (IC50= 0.4±0.03 nM; P<0.02). Similar results were obtained using a 14-day clonogenic assay in agar cultures. These data indicated that Plitidepsin was active at very low nanomolar concentrations against cell lines harboring JAK2V617F mutation. We then evaluated the effects of Plitidepsin on the growth of BFU-E, CFU-GM and CFU-Mk from MPN pts; all five Polycythemia Vera (PV) and 4/5 Primary Myelofibrosis (PMF) pts analyzed were JAK2V617F mutated. As shown in the Table, PMF pts presented significantly lower IC50 value than controls (Ctrl; P<.002) for all type of clonogenic progenitors; cells from PMF pts resulted also significantly more sensitive to Plitidepsin than those from PV patients (P<.02), while the difference between PV and Ctrl did not reach the significance level. To evaluate whether Plitidepsin also affected the latest stages of differentiation and maturation of MKs, that is the most overtly affected cell lineage in PMF, we added Plitidepsin on day +7 of a two-stage liquid culture system initiated with CD34+ cells purified from the PB of PMF patients; the generation of CD61+ Mks was measured 5 days later by FACS analysis. However, we found that the number of CD61+ cells was no different between cultures containing or not Plitidepsin, overall suggesting that the drug mainly affected early proliferation of Mk progenitors rather than influencing their differentiation. We then performed single colony genotyping to quantify the proportion of hematopoietic colonies harboring the JAK2V617F mutation which grew in growth factor-supplemented methylcellulose cultures initiated with purified CD34+ cells from PMF patients in the presence of 1 nM Plitidepsin. Initial data in 3 pts were available; in one, the proportion of JAK2-mutated BFU-E colonies decreased from 51% to 27% while no changes were observed in the other two pts. Finally, since a correlation between levels of p27(Kip1) and the response of tumor cells to Plitidepsin has been described, we measured p27 levels in different cell lines after exposure to Plitidepsin. We observed that p27 mRNA levels increased 15-fold and 30-fold in UKE1 and HEL cells, respectively, compared to K562 cells after 24 hr with 1nM Plitidepsin; such an increase was mirrored by a protein content 1.9- to 3.5-fold greater than baseline in UKE-1 cells at 1 and 10 nM Plitidepsin, suggesting that JAK2V617F mutated cells responded to the drug by modulating their p27 levels. Collectively, we provided evidence that Plitidepsin has in-vitro activity against MPN cells, particularly from PMF pts. These results, as well as those which were previously described in the GATA1low murine model, provided the rationale for a clinical trial in patients with myelofibrosis that is being developed within the Myeloproliferative Disorders Research Consortium (MPD-MRC). Plitidepsin IC50 (nM) BFU-E CFU-GM CFU-Mk Ctrl (n=5) 8.7 ± 2.3 8.2 ± 3.5 1.7 ± 0.9 PV (n=5) 5.2 ± 2.0 7.4 ± 4.0 not done PMF (n=5) 1.1 ± 0.6 1.6 ± 0.4 0.4 ± 0.06 Disclosures: Aracil: PharmaMar: Employment. Fe Paz:PharmaMar: Employment. Vannucchi:PharmaMar: Research Funding.

scholarly journals Renal miR-148b is associated with megalin down-regulation in IgA nephropathy

2018 ◽  
Vol 38 (6) ◽  
Author(s):  
Lu Wen ◽  
Zhanzheng Zhao ◽  
Jing Xiao ◽  
Zheng Wang ◽  
Xiangfei He ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Mrna Expression ◽  
Iga Nephropathy ◽  
Linear Regression Analysis ◽  
Multiple Linear Regression Analysis ◽  
Mrna Levels ◽  
Immunofluorescence Labeling ◽  
Normal Controls

Megalin is essential for proximal tubule reabsorption of filtered proteins, hormones, and vitamins, and its dysfunction has been reported in IgA nephropathy (IgAN). miR-148b has been shown to regulate renal megalin expression in vitro and in animal models of kidney disease. We examined a potential role of miR-148b and other miRNAs in regulating megalin expression in IgAN by analyzing the association between megalin and miR-148b, miR-21, miR-146a, and miR-192 expression. Quantitative PCR (qPCR) analysis identified a marked increase in renal levels of several miRNAs, including miR-148b, miR-21, miR-146a, and a significant decrease in megalin mRNA levels in IgAN patients when compared with normal controls. By multiple linear regression analysis, however, only renal miR-148b was independently associated with megalin mRNA levels in IgAN. Proximal tubule megalin expression was further evaluated by immunofluorescence labeling of biopsies from the patients. The megalin expression was significantly lower in patients with highest levels of renal miR-148b compared with patients with lowest levels. To examine the direct effects of the miRNAs on megalin and other membrane proteins expression, proximal tubule LLC-PK1 cells were transfected with miR-148b, miR-21, miR-146a, or miR-192 mimics. Transfection with miR-148b mimic, but not the other three miRNA mimics inhibited endogenous megalin mRNA expression. No significant effect of any of the four miRNA mimics was observed on cubilin or aquaporin 1 (AQP1) mRNA expression. The findings suggest that miR-148b negatively regulates megalin expression in IgAN, which may affect renal uptake and metabolism of essential substances.

scholarly journals The ABCC6 Transporter Plays a Significant Role in the Efflux of Nilotinib and Dasatinib, and May Contribute to Tyrosine Kinase Inhibitor Resistance

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4237-4237
Author(s):  
Laura N Eadie ◽  
Jarrad M Goyne ◽  
Timothy P. Hughes ◽  
Deborah L White
Keyword(s):  
Mrna Expression ◽  
Board Of Directors ◽  
Cell Lines ◽  
Research Funding ◽  
K562 Cells ◽  
Mrna Levels ◽  
Advisory Committees ◽  
Ku812 Cells

Abstract Efflux transporters ABCB1 and ABCG2 interact with tyrosine kinase inhibitors (TKIs) and mediate drug resistance, however, evidence of the interaction of other potentially relevant drug transporters with TKIs is lacking. We investigated the involvement of the closely related transporter ABCC6, in imatinib (IM), nilotinib (NIL) and dasatinib (DAS) transport and also the role of ABCC6 in NIL resistance. The impact of short-term (overnight) exposure to NIL on mRNA expression of ABC transporters in three BCR-ABL1+ cell lines was assessed by Taqman transporter array: K562, K562-Dox and KU812 cells. Several transporters of interest were identified, including ABCC6, based on alterations in mRNA expression. In order to elucidate the importance of ABCC6 in the development of NIL resistance, ABCC6 mRNA levels were determined by RT-PCR in K562 and K562-Dox NIL-resistant lines generated in vitro and compared with ABCC6 mRNA levels in respective parental control cells. ABCC6 protein expression was confirmed by western blot. p-Crkl dependent IC50 experiments in the absence and presence of three ABCC6 inhibitors (indomethacin, INDO; probenecid, PRO; pantoprazole, PP) were performed in patient mononuclear cells (MNCs) and BCR-ABL1+ cell lines to assess the role of ABCC6 in NIL, IM and DAS transport. A marked increase in ABCC6 mRNA expression in response to short-term in vitro NIL exposure occurred: in K562 and KU812 cells ABCC6 mRNA levels increased 9.5- and 9.7-fold in response to overnight NIL exposure respectively. Increased expression of ABCC6 was also observed in cells subjected to long-term NIL exposure during development of NIL resistance in vitro. NIL-resistant K562 cells demonstrated up to 57-fold higher levels of ABCC6 mRNA compared with control cells (p=0.002). Analogous results were observed in NIL-resistant K562-Dox cells (up to 33-fold higher levels of ABCC6 mRNA p=0.002). In order to determine the relevance of ABCC6 in patient cells, p-Crkl dependent IC50 experiments were performed in MNCs from de novo CML patients in the absence and presence of ABCC6 inhibition. Results demonstrated a significant reduction in IC50NIL in the presence of all three ABCC6 inhibitors compared with IC50NIL in the absence of inhibitors. Similar results were observed for IC50DAS but not IC50IM. Experiments in three parental BCR-ABL1+ cell lines confirmed these findings (Table 1). Notably, comparison of IC50 values in the absence of ABCC6 inhibition in KU812 vs. K562 cells revealed that KU812 cells demonstrated increased IC50NIL (307 vs. 257 nM, p=0.0493) and IC50DAS (14 vs 8 nM, p=0.0005). This was unexpected given both cell lines demonstrate negligible expression of ABCB1 (a transporter known to interact with both NIL and DAS). However, assessment of ABCC6 protein levels by western blotting revealed KU812 cells have greater levels of ABCC6 when compared with K562 cells: 53% in KU812 vs. 24% in K562 (ABCC6 normalised to β-actin). A greater %reduction in IC50NIL and IC50DAS in the presence of ABCC6 inhibition was also observed in KU812 cells compared with K562 cells confirming the role of ABCC6 in the transport of NIL and DAS. Combined, these studies highlight the importance of ABCC6 in the export of NIL and DAS from patient MNCs and BCR-ABL1+ cell lines. This is the first report of ABCC6 involvement in TKI transport and results suggest ABCC6 overexpression may also contribute to NIL resistance. The addition of ABCC6 inhibitors to NIL and DAS therapy may enhance the efficacy of these TKIs in the treatment of CML. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Ariad: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals Splicing factor SRSF1 promotes breast cancer progression via oncogenic splice switching of PTPMT1

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Jun-Xian Du ◽  
Yi-Hong Luo ◽  
Si-Jia Zhang ◽  
Biao Wang ◽  
Cong Chen ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Progression ◽  
High Risk Group ◽  
Clinical Samples ◽  
Binding Motif ◽  
Ki 67 ◽  
Tissue Samples

Abstract Background Intensive evidence has highlighted the effect of aberrant alternative splicing (AS) events on cancer progression when triggered by dysregulation of the SR protein family. Nonetheless, the underlying mechanism in breast cancer (BRCA) remains elusive. Here we sought to explore the molecular function of SRSF1 and identify the key AS events regulated by SRSF1 in BRCA. Methods We conducted a comprehensive analysis of the expression and clinical correlation of SRSF1 in BRCA based on the TCGA dataset, Metabric database and clinical tissue samples. Functional analysis of SRSF1 in BRCA was conducted in vitro and in vivo. SRSF1-mediated AS events and their binding motifs were identified by RNA-seq, RNA immunoprecipitation-PCR (RIP-PCR) and in vivo crosslinking followed by immunoprecipitation (CLIP), which was further validated by the minigene reporter assay. PTPMT1 exon 3 (E3) AS was identified to partially mediate the oncogenic role of SRSF1 by the P-AKT/C-MYC axis. Finally, the expression and clinical significance of these AS events were validated in clinical samples and using the TCGA database. Results SRSF1 expression was consistently upregulated in BRCA samples, positively associated with tumor grade and the Ki-67 index, and correlated with poor prognosis in a hormone receptor-positive (HR+) cohort, which facilitated proliferation, cell migration and inhibited apoptosis in vitro and in vivo. We identified SRSF1-mediated AS events and discovered the SRSF1 binding motif in the regulation of splice switching of PTPMT1. Furthermore, PTPMT1 splice switching was regulated by SRSF1 by binding directly to its motif in E3 which partially mediated the oncogenic role of SRSF1 by the AKT/C-MYC axis. Additionally, PTPMT1 splice switching was validated in tissue samples of BRCA patients and using the TCGA database. The high-risk group, identified by AS of PTPMT1 and expression of SRSF1, possessed poorer prognosis in the stage I/II TCGA BRCA cohort. Conclusions SRSF1 exerts oncogenic roles in BRCA partially by regulating the AS of PTPMT1, which could be a therapeutic target candidate in BRCA and a prognostic factor in HR+ BRCA patient.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals The Role of the FOXO1/β2-AR/p-NF-κB p65 Pathway in the Development of Endometrial Stromal Cells in Pregnant Mice under Restraint Stress

2021 ◽  
Vol 22 (3) ◽  
pp. 1478
Author(s):  
Jiayin Lu ◽  
Yaoxing Chen ◽  
Zixu Wang ◽  
Jing Cao ◽  
Yulan Dong
Keyword(s):  
Oxidative Stress ◽  
Stromal Cells ◽  
Restraint Stress ◽  
Target Genes ◽  
Mrna Levels ◽  
Endometrial Stromal Cells ◽  
Protein Levels ◽  
Pregnant Mice

Restraint stress causes various maternal diseases during pregnancy. β2-Adrenergic receptor (β2-AR) and Forkhead transcription factor class O 1 (FOXO1) are critical factors not only in stress, but also in reproduction. However, the role of FOXO1 in restraint stress, causing changes in the β2-AR pathway in pregnant mice, has been unclear. The aim of this research was to investigate the β2-AR pathway of restraint stress and its impact on the oxidative stress of the maternal uterus. In the study, maternal mice were treated with restraint stress by being restrained in a transparent and ventilated device before sacrifice on Pregnancy Day 5 (P5), Pregnancy Day 10 (P10), Pregnancy Day 15 (P15), and Pregnancy Day 20 (P20) as well as on Non-Pregnancy Day 5 (NP5). Restraint stress augmented blood corticosterone (CORT), norepinephrine (NE), and blood glucose levels, while oestradiol (E2) levels decreased. Moreover, restraint stress increased the mRNA levels of the FOXO family, β2-AR, and even the protein levels of FOXO1 and β2-AR in the uterus and ovaries. Furthermore, restraint stress increased uterine oxidative stress level. In vitro, the protein levels of FOXO1 were also obviously increased when β2-AR was activated in endometrial stromal cells (ESCs). In addition, phosphorylated-nuclear factor kappa-B p65 (p-NF-κB p65) and its target genes decreased significantly when FOXO1 was inhibited. Overall, it can be said that the β2-AR/FOXO1/p-NF-κB p65 pathway was activated when pregnant mice were under restraint stress. This study provides a scientific basis for the origin of psychological stress in pregnant women.

Continuous Cultures of Plasmodium Falciparum Established in Tanzania From Patients With Acute Malaria

2021 ◽  
Author(s):  
Florence Humphrey Urio ◽  
Matilda Mkombachepa ◽  
Gration Rwegasira ◽  
Twilumba Makene ◽  
Billy Ngasala ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Continuous Culture ◽  
Drug Sensitivity ◽  
Culture Media ◽  
Experimental System ◽  
Dar Es Salaam ◽  
Clinical Samples ◽  
Host Cell Interactions ◽  
Set Up

Abstract BackgroundMalaria morbidity and mortality, almost entirely from Plasmodium falciparum, are still rampant in Africa: therefore, it is important to study the biology of the parasite and the parasite-host cell interactions. In vitro cultivation of Plasmodium falciparum is most useful for this purpose, as well as for investigating drug resistance and possible new therapies. Here we report that the Trager & Jensen continuous culture of P. falciparum can be established in a laboratory in Tanzania with minimal facilities and with modest expenditure.MethodsAn in vitro set-up of continuous culture of P. falciparum was carried out in 2016 to 2020 at Muhimbili university of health and allied sciences, Dar-es salaam. Parasite samples were obtained from patients with acute malaria, frozen parasites and live cultures. Data was collected and analyzed using GraphPad Prism version 8.ResultsWe have successfully achieved exponential growth of existing strains that are used worldwide, as well as of parasites in clinical samples from patients with acute malaria. In the aim to optimize growth we have compared human serum and bovine serum albumin as components of the culture media. In addition, culture synchronization has been achieved using sorbitol.ConclusionThis experimental system is now available to our institution and to researchers aiming at investigating drug sensitivity and mechanisms of protection against Plasmodium falciparum that accrue from various genes expressed in red cells.

Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

scholarly journals High CD169 Monocyte/Lymphocyte Ratio Reflects the Immunophenotyping Disruption and Predicts Oxygen Need in COVID-19 Patients

2021 ◽  
Author(s):  
Antonella Minutolo ◽  
Vita Petrone ◽  
Marialaura Fanelli ◽  
Marco Iannetta ◽  
Martina Giudice ◽  
...  
Keyword(s):  
Disease Progression ◽  
Inflammatory Markers ◽  
Predictive Marker ◽  
Pulmonary Involvement ◽  
Mrna Levels ◽  
Healthy Donors ◽  
Respiratory Outcome ◽  
Early Biomarker

Background: CD169 has been found overexpressed in the blood of COVID-19 patients and identified as a biomarker in the early disease. We have analysed CD169 in blood cells of COVID-19 patients to assess its role as predictive marker of the disease. Methods : The ratio of the CD169 Median median Fluorescence fluorescence Intensity intensity of CD169 between monocytes and lymphocytes (CD169 RMFI ) was analysed by flow cytometry in blood samples of COVID-19 patients (COV) and healthy donors (HD ) and correlated with immunophenotyping, inflammatory markers, cytokines mRNA expression, pulmonary involvement and disease progression. Results: CD169 RMFI increased in COV but not in HD. CD169 RMFI correlated with T-cell differentiation and exhaustion markers as well as with B cells maturation and differentiation. In vitro stimulation of PBMCs of HD with SARS-CoV-2 Spike spike protein induced CD169 RMFI together with IL-6 and IL-10 gene expression. Likewise, CD169 RMFI correlated with blood cytokine mRNA levels, inflammatory markers, and pneumonia severity in patients which that had not received any treatment at sampling. Notably, in untreated patients, CD169 RMFI reflected the respiratory outcome during hospitalization. Conclusion : Considering the immunological role of CD169 and its involvement during the infection and the progression of COVID-19, it could be considered as an early biomarker to evaluate disease progression and clinical outcome.

scholarly journals BCAT1 Activates PI3K/AKT/mTOR Pathway and Contributes to the Angiogenesis and Tumorigenicity of Gastric Cancer

2021 ◽  
Vol 9 ◽  
Author(s):  
Xiong Shu ◽  
Pan-Pan Zhan ◽  
Li-Xin Sun ◽  
Long Yu ◽  
Jun Liu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Tumor Growth ◽  
Western Blotting ◽  
Mtor Pathway ◽  
Clinical Samples ◽  
Xenograft Models ◽  
Cell Phenotypes

BackgroundFocusing on antiangiogenesis may provide promising choices for treatment of gastric cancer (GC). This study aimed to investigate the mechanistic role of BCAT1 in the pathogenesis of GC, particularly in angiogenesis.MethodsBioinformatics and clinical samples analysis were used to investigate the expression and potential mechanism of BCAT1 in GC. BGC823 cells with BCAT1 overexpression or silencing were induced by lentiviral transduction. Cell phenotypes and angiogenesis were evaluated. The relevant proteins were quantized by Western blotting, immunohistochemistry, or immunofluorescence. Xenograft models were constructed to confirm the role of BCAT1 in vivo.ResultsBCAT1 was overexpressed in GC patients and associated with lower survival. BCAT1 expression was correlated with proliferation-, invasion-, or angiogenesis-related markers expression and pathways. Silencing BCAT1 expression suppressed cell viability, colony formation, cycle progression, invasion, and angiogenesis of BGC823 cells, as well as the tumor growth of xenograft models, whereas overexpressing BCAT1 had the opposite results both in vitro and in vivo. Bioinformatics analysis and Western blotting demonstrated that BCAT1 activated the PI3K/AKT/mTOR pathway. The addition of LY294002 reversed the tumor growth induced by BCAT1 overexpression, further verifying this mechanism.ConclusionBCAT1 might act as an oncogene by facilitating proliferation, invasion, and angiogenesis through activation of the PI3K/AKT/mTOR pathway. This finding could aid the optimization of antiangiogenesis strategies.

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