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OBJECTIVE: Acute lymphoblastic leukemia (ALL) is a highly heterogeneous leukemia. Despite the current improvement in conventional chemotherapy and high survival rates, the outcomes remain challenging. Sesquiterpen extracted from the Tanacetum parthenium, parthenolide, is a potential anticancer agent that can modulate the expression of miRNAs and induce apoptosis. The objective of this study was to investigate the effect of parthenolide in combination with vincristine and alone on the apoptosis rate and expression of miR-125b-5p, miR-181b-5p, and miR-17-5p in the NALM6 cell line. MATERIALS AND METHODS: In this experimental study, cell viability and metabolic activity were determined through MTT assay and PI staining. Flow cytometry was applied to evaluate the rate of apoptosis. The expression of miRNAs was assessed using real-time polymerase chain reaction. Bioinformatic analyses, including Cytoscape, RNAhybrid, and signaling pathway analysis were employed to investigate the association of miR-17-5p, miR-181b-5p and miR-125b- 5p with apoptosis. Further, molecular docking served to validate the modulation of these miRNAs by parthenolide and vincristine treatment. RESULTS: The MTT assay indicated that 7.7 µM of parthenolide decreased the metabolic activity to 50% after 48 hours. PI staining analysis indicated that at concentrations below the half maximal inhibitory concentration, parthenolide caused 50% cell death. Flow cytometric analysis indicated that parthenolide (1.925 µM) in combination with vincristine (1.2 nM) induced apoptosis in 83.2% of the cells. Real-time quantitative reverse transcription polymerase chain reaction (qRTPCR) analysis showed significant changes in the expression levels of miR-17-5p, miR-125b-5p, and miR-181b-5p. Moreover, the combination therapy downregulated the expression of miRNAs significantly. This was consistent with our bioinformatic analysis demonstrating that the studied miRNAs are regulators of apoptosis. Finally, molecular docking validated the modulation of the miRNAs by parthenolide and vincristine. CONCLUSION: Parthenolide in combination with vincristine triggers apoptosis at a high rate in the NALM6 cell line. Moreover, this combination therapy can decrease the expression of miR-17-5p, miR-181b-5p, and miR-125b-5p.
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Introduction: Thrombotic complication is one of the features of sickle cell disease (SCD), characterized by appearance of phosphatidylserine on the outer membrane of sickle-shaped red blood cells and most abundantly on membrane protrusions called microvesicles (MVs). However, the exact mechanism by which MVs may enhance coagulant activity in SCD patients has not been fully addressed. The aim of this study was to further investigate the procoagulant activity of circulating MVs in sickle cell crises. Materials and Methods: Subjects included in this cross-sectional study were 47 patients with SCD and 25 normal subjects with written informed consent obtained from all the participants. MV analysis was conducted by using CD61, CD235α, and Annexin-V monoclonal antibodies. The coagulant activity of MVs was determined by an ELISA-based procoagulant activity assay. Results: The majority of MVs were originated from platelets (CD61+) and erythrocytes (CD235+). These MVs demonstrated significantly enhanced levels during the painful crisis when compared with the steady-state period (p < 0.001) and controls (p < 0.001). Also, the procoagulant activity of MVs was significantly higher in crisis compared to those of steady state (p < 0.001) and positively correlated with the number of Annexin-V+ MVs (p < 0.001). Significant correlations were found between erythrocyte-derived MVs with hemolysis marker (r = 0.51, p < 0.001) and the hemoglobin level (r = -0.63, p < 0.001). Conclusion: The numbers of platelet- and erythrocyte-derived MVs are related to painful crisis, and their quantification in SCD may be helpful for identifying cases at increased risk of thrombotic complications.
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Glaucoma is a common retinal disorder characterized by progressive optic nerve damage, resulting in visual impairment and potential blindness. Elevated intraocular pressure (IOP) is a major risk factor, but some patients still experience disease progression despite IOP-lowering treatments. Genome-wide association studies have linked variations in the Caveolin1/2 (CAV-1/2) gene loci to glaucoma risk. Cav-1, a key protein in caveolae membrane invaginations, is involved in signaling pathways and its absence impairs retinal function. Recent research suggests that Cav-1 is implicated in modulating the BDNF/TrkB signaling pathway in retinal ganglion cells, which plays a critical role in retinal ganglion cell (RGC) health and protection against apoptosis. Understanding the interplay between these proteins could shed light on glaucoma pathogenesis and provide potential therapeutic targets.
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Background: Antigen presentation using bacterial surface display systems, on one hand, has the benefits of bacterial carriers, including low-cost production and ease of manipulation. On the other hand, the bacteria can help in stimulating the immune system as an adjuvant. For example, using bacterial surface display technology, we developed a hepatitis C virus (HCV) multiple antigens displaying bacteria's surface and then turned it into a bacterial ghost. Methods: The HCV core and NS3 proteins' conserved epitopes were cloned into the AIDA gene plasmid as an auto transporter. The recombinant plasmid was then transformed into Escherichia coli (E. coli) Bl21 (DE3). Recombinant bacteria were then turned into a bacterial ghost, an empty cell envelope. Whole-cell ELISA, flow cytometry, and Western blot techniques were used for monitoring the expression of proteins on the surface of bacteria. Results: A fusion protein of HCV core-NS3-AIDA was successfully expressed on the E. coli Bl21 (DE3) surface and confirmed by western blotting, Enzyme-Linked Immunosorbent Assay (ELISA), and flow cytometry detection techniques. Conclusion: The presence of HCV antigens on non-pathogen bacteria surfaces holds promise for developing safe and cost-benefit-accessible vaccines with optimal intrinsic adjuvant effects and exposure of heterologous antigens to the immune system.
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BACKGROUND: Breast cancer (BC) is the main cause of cancer death in women. Idiopathic granulomatous mastitis (IGM), a rare chronic disease that clinically mimics breast carcinoma, and is associated with high mortality and morbidity, but an immediate and accurate diagnosis can substantially decrease these rates. Expressed by numerous human tissues, interleukin-33 (IL-33) has an inductive role in the network of pro-inflammatory cytokines. The aim of this study was to investigate the serum levels of IL-33 in BC and IGM patients in comparison with healthy women. MATERIALS AND METHODS: This descriptive-analytical study was carried out on 28 patients with BC and 25 patients with IGM as the patient groups and 25 healthy volunteers with normal screening reports as the control group. Histopathological pattern of BC and IGM were confirmed by specialized pathologists. The serum concentration of IL-33 was measured using enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions. RESULTS: The mean age of the patients with BC and IGM and the control group was 49.1, 37.1, and 36.8 years, respectively. There was no significant difference in IL-33 expression among the participants with regard to age, marital status, body mass index (BMI), and menopausal status. IL-33 assay indicated a significant difference between the BC (P=0.011) and IGM (P=0.031) groups compared to the controls, although no substantial differences were observed between the IGM and BC groups. CONCLUSION: IL-33 can be considered a significant factor distinguishing IGM and BC patients from controls, although it cannot be applied to diagnose and differentiate BC from IGM patients.
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Subject(s)
Breast Neoplasms , Granulomatous Mastitis , Female , Humans , Breast Neoplasms/pathology , Diagnosis, Differential , Granulomatous Mastitis/diagnosis , Granulomatous Mastitis/pathology , Immunoglobulin M , Interleukin-33ABSTRACT
INTRODUCTION: Chronic myeloid leukemia (CML) is a progressive myeloproliferative disorder resulting from forming a chimeric BCR-ABL gene. The proteins derived from this gene can affect some genes from various signaling pathways such as PI3K/AKT/Wnt/catenin/JAK/Stat involved in proliferation, differentiation, cell death, and genes related to autophagy. Imatinib is the first-line treatment for CML patients, with durable and proper responses in Iranian children and adult CML patients. Hence, we aimed to evaluate the mRNA expression of some selected key genes from those pathways in patients with CML before and under treatment. METHODS: In the case-control study, the mRNA expression of PTEN, LEF1, JAK3, LC3 and p62 genes were measured in 51 CML patients (6 patients before treatment and 45 patients under treatment with imatinib mesylate) and 40 healthy controls using the Real-time PCR method. RESULTS: The mRNA expression of PTEN and P62 were significantly higher in newly diagnosed patients than in controls (P<0.0001 and P = 0.0183, respectively), while the expression of the LC3 gene was significantly lower in the untreated newly diagnosed group than in control subjects (P = 0.0191). The expression level of PTEN, LEF1, JAK3 and P62 genes were significantly decreased in patients under treatment than in the group before treatment (P = 0.0172, P = 0.0002, P = 0.0047 and P = 0.0038, respectively). A positive correlation was seen between the gene expression of P62 and BCR-ABL in the patients under treatment (r 0529, P = 0.016). CONCLUSION: Our findings showed that the changes in expression of these genes were related to the patient's treatment. Due to the key role of these genes in proliferation, differentiation and tumor suppression, it is proposed that these genes may be helpful for follow-up of treatment in CML patients.
Subject(s)
Antineoplastic Agents , Leukemia, Myelogenous, Chronic, BCR-ABL Positive , Adult , Child , Humans , Sequestosome-1 Protein/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Phosphatidylinositol 3-Kinases/pharmacology , Phosphatidylinositol 3-Kinases/therapeutic use , Case-Control Studies , Iran , Imatinib Mesylate/therapeutic use , Imatinib Mesylate/pharmacology , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , RNA, Messenger/genetics , RNA, Messenger/pharmacology , RNA, Messenger/therapeutic use , Antineoplastic Agents/pharmacology , Apoptosis , Janus Kinase 3/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , PTEN Phosphohydrolase/pharmacologyABSTRACT
INTRODUCTION: Inflammatory response-induced coagulopathy is a common complication associated with severe form of covid-19 infection. Evidences suggest that neutrophil extracellular traps (NETs) play a significant role in triggering the immunothrombosis in this condition. We aimed to evaluate the diagnostic value of surface neutrophilic myeloperoxidase (MPO) as NETosis biomarker for predicting the risk of covid-19-associated coagulopathies. METHODS: Covid-19 infection was assessed by real-time-PCR and plasma d-dimer levels were measured by ELFA. Based on the covid-19 infection and d-dimer level outcomes, patients were categorized into four groups. Any alteration in the serum level of IL-6, H3Cit and neutrophilic surface MPO were analyzed by CLIA, ELISA, and flow cytometry, respectively. RESULTS: H3Cit variations and different d-dimer values confirmed the association between NETosis and coagulopathies. Findings showed that the expression of neutrophilic MPO reduced in cases with NETosis, which was correlated with increased levels of H3Cit. ANC/MPO ratio was signified as a valuable marker to discriminate the covid-19 and non covid-19-associated coagulopathies and could be considered as a prognostic factor due to its noteworthy correlation with serum IL-6 concentration. CONCLUSION: Declined levels of surface neutrophilic MPO in NETosis correlate with covid-19-associated coagulopathies and increased IL-6 levels, as a potential biomarker of covid-19 disease severity.
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Background: The nonstructural protein (NS1) of human parvovirus B19 (hPVB19) is considered to be a double-edged sword in its pathogenesis. NS1 protein promotes cell death by apoptosis in erythroid-lineage cells and is also implicated in triggering and the progression of various inflammation and autoimmune disorders. Objectives: We investigated the possible role of hPVB19 NS1 in the modulation of proinflammatory cytokines in nonpermissive HEK-293T cells. Methods: A plasmid containing the fully sequenced NS1 gene (pCMV6-AC-GFP-NS1) was transfected into HEK-293T cells. Transfection efficiency was assessed by fluorescent microscopy over time. Mock (pCMV6-AC-GFP) transfected cells were used as controls. The percentage of apoptotic cells was measured by flow cytometry at 24, 48, and 72 h posttransfection. Interleukin 6 (IL-6) mRNA, as a pleiotropic cytokine, was measured by real-time PCR. Furthermore, cellular supernatants were collected to determine the type and quantity of cytokines produced by mock- and NS1-transfected cells using flow cytometry. Results: Fold change in the expression level of IL-6 mRNA in transfected cells after 72 hr of incubation was found to be 3.01 when compared with mock-transfected cells; however, cell apoptosis did not happen over time. Also, the concentration of cytokines such as IL-2, IL-6, IL-9, IL-17A, IL-21, IL-22, interferon (IFN)-γ, and tumor necrosis factor α (TNF-α) increased in NS1-transfected cells. Conclusions: Overall, our results indicated that proinflammatory cytokine levels had increased following the expression of hPVB19 NS1 in HEK-293T cells, consistent with a role for NS1 expression facilitating the upregulation of inflammatory reactions. Therefore, hPVB19 NS1 function may play a role in the progression of some chronic and inflammatory diseases.
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Objective: Although cold storage of platelets (PLTs) could decrease the risk of bacterial growth, it could affect on the PLTs viability and hemostatic function. At cold temperatures, trehalose can be used to substitute water, inhibit the solid-liquid transition phase of the PLT membrane, and stop Glycoprotein Ibα (GPIbα) polymerization. In this study, we evaluated the potential of trehalose for reducing the negative effects of cold storage on the apoptosis and the clearance rates of PLTs after long-term storage at cold. Materials and Methods: In this experimental study, PLT concentrates (PCs) were maintained for five days in the different circumstances. PLTs were subsequently counted by using an automated hematology analyzer. Also water-soluble tetrazolium salt (WST-1) assay was performed to estimate the viability of PLTs. The activity of lactate dehydrogenase enzyme (LDH) was determined by a biochemical analyzer. And human active caspase-3 levels were measured by using enzyme-linked immunosorbent assay (ELISA) method. Also, we applied flow cytometry technique. Results: PLTs count and viability were higher, while LDH amount was lower in trehalose-treated PLTs when compared with two other groups (P=0.03). The highest increase in the amount of caspase-3 levels in the PLTs was observed at 4°C. However, trehalose-treated and 4°C PLTs had a lower amount of active caspase-3 in comparison with 4°C PLTs. The level of PS expression on PLTs was lower in the trehalose-treated PLTs in compared with the two other groups (P=0.03). PLTs ingestion by HepG2 cells was enhanced in the 4°C-stored PLTs. However, the ingestion rate was significantly reduced in the trehalose-treated PLTs on day 5 of storage (P=0.03). Conclusion: Trehalose can moderate the effects of cold temperature on the apoptosis, viability, and the survival rate of PLTs. It also decreases the ingestion rate of refrigerated PLTs in vitro.
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BACKGROUND: Autophagy is a conserved recycling process in cells. However, the effects of autophagy on the remission and treatment response of acute myeloid leukemia (AML) patients have not been clarified. METHODS: The expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes was assessed in 32 newly diagnosed AML patients, 18 complete remission (CR) patients, and seven relapsed patients, as well as 15 controls, by real-time polymerase chain reaction (PCR). RESULTS: The expression of all five genes was significantly higher in the newly diagnosed AML patients as compared to the controls (p < 0.0001). The MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 gene expression significantly reduced in CR patients compared to newly diagnosed AML patients (p = 0.006, 0.003, 0.0002, 0.006, and 0.004, respectively). The AMBRA1 gene expression was significantly higher in the relapsed cases as compared to both newly diagnosed (p = 0.01) and CR patients (p = 0.03). Moreover, a significant positive correlation was observed between the expression of MAP1LC3B (r = 0.739, p = 0.000001), ATG5 (r = 0.682, p = 0.00001), and ATG10 (r = 0.586, p = 0.0004) genes and white blood cell (WBC) count in patients at diagnosis. CONCLUSION: The expression of MAP1LC3B, ATG5, ATG10, RB1CC1, and AMBRA1 genes can be examined to follow-up the remission of AML and the patient's response to treatment.
Subject(s)
Leukemia, Myeloid, Acute , Adaptor Proteins, Signal Transducing/genetics , Autophagy/genetics , Humans , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/metabolism , Real-Time Polymerase Chain Reaction , Recurrence , Remission InductionABSTRACT
As addressed by many studies, greenhouse gas has a significant impact on the different aspects of life and more importantly on the whole environment. The excessive emission of green gas leads to climate change which is regarded as one of the most significant challenges of 21 century. Hence, in this regard, this paper has addressed the changing greenhouse gas (GHG) emissions in 18 countries of the MENA region. For this purpose, ten different scenarios of this disease's future status and its restrictions were considered in an input-output modelling framework. The empirical results indicated that the emission of greenhouse gas is reduced under all scenarios. However, some countries experience more reduction due to the restriction because of COVID-19 like Syria, Iran, Yemen and Lebenon. Based on the ninth scenario, Iran and Syria have the highest reduction in emission of greenhouse gas by 13.1 and 13.8 per cent, and based on the tenth scenario, Lebenan and Syria will experience the highest reduction in emission by about 13.1 and 17.9 per cent. The results show that according to scenario 10 (explosive intensification of the pandemic without the wave subsiding over a while) and scenario 9 (the pandemic worsens step by step without subsiding over a while), Syria and Iran have the highest reduction in greenhouse gas emissions, respectively. According to scenario 1 (rapid and complete control of disease), Bahrain, Qatar, and Kuwait have the lowest reduction in GHG emissions. Besides, the study draws several fruitful implications regarding environmental concerns as sectoral analysis such as Hotels and Restaurants, Retail Trade, Fishing, Wholesale Trade, and Transport sectors. Moreover, policymakers should be alert that notwithstanding all limitations, Private Households and Public Administration develop their emissions during the pandemic since quarantine intensifies the supply of these services. Surprisingly, none of the policy restrictions have a significant impact on GHG emissions from Education, Health, and Other Services, Petroleum , Chemical, and Non-Metallic Mineral Products, Textiles and Wearing Apparel, and Re-export & Re-import, demonstrating the robust and established nature of these sectors' activities. To control the emissions of the quarantine-neutral sectors, long- and mid-term structural and environmental policies should be considered. The researchers are guided by the novel implications in terms of how various industries might reduce emissions in different ways. Supplementary Information: The online version contains supplementary material available at 10.1007/s10668-021-02018-3.
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BACKGROUND: Short hairpin RNA (shRNA) has proven to be a powerful tool to study genes' function through RNA interference mechanism. Three different methods have been used in previous studies to produce shRNA expression vectors including oligonucleotide-based cloning, polymerase chain reaction (PCR)-based cloning, and primer extension PCR approaches. The aim of this study was designing a reliable and simple method according to the primer extension strategy for constructing four shRNA vectors in order to target different regions of Metadherin (MTDH) mRNA in human leukemic cell line Jurkat. METHODS: Oligonucleotides for construction of four shRNA vectors were designed, synthesized and fused to U6 promoter. Each U6-shRNA cassette was cloned into a pGFP-V-RS vector. MTDH shRNAs were transfected into the Jurkat cell line by using the electroporation method. The ability of shRNAs to knock down MTDH mRNA was analyzed through qRT-PCR. Apoptosis assay was used to evaluate the effect of down regulation of MTDH expression on cell integrity. RESULTS: A significant reduction (about 80%) in the expression levels of MTDH mRNA and an increase in the percentages of apoptotic cells (about 20%) were observed in the test group in comparison with control. CONCLUSION: MTDH shRNA constructs effectively inhibited gene expression. However, simplicity and inexpensiveness of the method were additional advantages for its application.
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SH2 domain containing tyrosine phosphatase 2 (Shp2; PTPN11) regulates several intracellular pathways downstream of multiple growth factor receptors. Our studies implicate that Shp2 interacts with Caveolin-1 (Cav-1) protein in retinal ganglion cells (RGCs) and negatively regulates BDNF/TrkB signaling. This study aimed to investigate the mechanisms underlying the protective effects of shp2 silencing in the RGCs in glaucomatous conditions. Methods: Shp2 was silenced in the Cav-1 deficient mice and the age matched wildtype littermates using adeno-associated viral (AAV) constructs. Shp2 expression modulation was performed in an acute and a chronic mouse model of experimental glaucoma. AAV2 expressing Shp2 eGFP-shRNA under a strong synthetic CAG promoter was administered intravitreally in the animals' eyes. The contralateral eye received AAV-eGFP-scramble-shRNA as control. Animals with Shp2 downregulation were subjected to either microbead injections or acute ocular hypertension experimental paradigm. Changes in inner retinal function were evaluated by measuring positive scotopic threshold response (pSTR) while structural and biochemical alterations were evaluated through H&E staining, western blotting and immunohistochemical analysis of the retinal tissues. Results: A greater loss of pSTR amplitudes was observed in the WT mice compared to Cav-1-/- retinas in both the models. Silencing of Shp2 phosphatase imparted protection against inner retinal function loss in chronic glaucoma model in WT mice. The functional rescue also translated to structural preservation of ganglion cell layer in the chronic glaucoma condition in WT mice which was not evident in Cav-1-/- mice retinas. Conclusions: This study indicates that protective effects of Shp2 ablation under chronic experimental glaucoma conditions are dependent on Cav-1 in the retina, suggesting in vivo interactions between the two proteins.
Subject(s)
Caveolin 1/physiology , Genetic Therapy , Genetic Vectors/therapeutic use , Glaucoma/therapy , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Retina/pathology , Alpha-Globulins/genetics , Animals , Apoptosis , Brain-Derived Neurotrophic Factor/physiology , Caveolin 1/deficiency , Caveolin 1/genetics , DNA, Complementary/genetics , Dependovirus/genetics , Focal Adhesion Kinase 1/physiology , Gene Knockdown Techniques , Genes, Reporter , Genes, Synthetic , Glaucoma/metabolism , Glaucoma/pathology , Integrin beta1/physiology , Intraocular Pressure , Intravitreal Injections , Membrane Glycoproteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Promoter Regions, Genetic , Protein Tyrosine Phosphatase, Non-Receptor Type 11/biosynthesis , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein-Tyrosine Kinases/physiology , Up-RegulationABSTRACT
BACKGROUND: Ionizing radiation plays a significant role in cancer treatment. Despite recent advances in radiotherapy approaches, the existence of irradiation-resistant cancer cells is still a noteworthy challenge. Therefore, developing novel therapeutic approaches are still warranted in order to increase the sensitivity of tumor cells to radiation. Many types of research rely on the role of mitochondria in radiation protection. OBJECTIVE: Here, we aimed to target the mitochondria of monocyticleukemia (THP-1) radio-resistant cell line cells by a mitochondrial disrupting peptide, D (KLAKLAK)2, and investigate the synergistic effect of Gamma-irradiation and KLA for tumor cells inhibition in vitro. MATERIAL AND METHODS: In this experimental study, KLA was delivered into THP-1 cells using a Cell-Penetrating Peptide (CPP).The cells were then exposed to gamma-ray radiation both in the presence and absence of KLA conjugated with CPP. The impacts of KLA, ionizing radiation or combination of both were then evaluated on the cell proliferation and apoptosis of THP-1 cells using MTT assay and flow cytometry, respectively. RESULTS: The MTT assay indicated the anti-proliferative effects of combined D (KLAKLAK)2 peptide with ionizing radiation on THP-1cells. Moreover, synergetic effects of KLA and ionizing radiation reduced cell viability and consequently enhanced cell apoptosis. CONCLUSION: Using KLA peptide in combination with ionizing irradiation increases the anticancer effects of radio-resistant THP-1 cells. Therefore, the combinational therapy of (KLAKLAK)2 and radiation is a promising strategy for cancer treatment the in future.
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PURPOSE: The role of oxidative stress in Aluminum (Al)-induced apoptotic effects has been investigated and suicidal death of erythrocytes, eryptosis, is characterized by cell shrinkage and phosphatidylserine externalization (PSE) at the surface of the erythrocyte cell membrane. Eryptosis is stimulated by an increase in cytosolic Ca2+ concentration and reactive oxygen species (ROS). This ex vivo study was conducted to evaluate the effect of well-known antioxidants including vitamin C (vit C) and N-acetylcysteine (NAC), against Al-induced hemolysis and eryptosis. METHODS: Isolated erythrocytes from the healthy volunteers were partitioned into various groups (6 replicates/group) and treated by various concentrations of Al (3-100 µM) in the presence and absence of vit C (0.6 mM) and NAC (1 mM). After 24 hours of treatment, hemolysis was determined from hemoglobin levels in the supernatant. Flowcytometric methods were applied to measure PSE, cell shrinkage, Ca2+ content, and ROS abundance using annexin V-binding, forward scatter, Fluo3-fluorescence, and DCFDA dependent fluorescence, respectively. Reduced glutathione (GSH) was measured by the ELISA method. RESULTS: The results showed that a 24 hours' exposure of the erythrocytes to Al (10-100 µM) significantly increased hemolysis in a dose and Ca2+dependent manner. Al also dramatically decreased forward scatter. The percentage of PSE cells, Fluo3-fluorescence, and DCFDA fluorescence were increased by Al. Furthermore, cotreatment with NAC inhibited the effect of Al on hemolysis, eryptosis, and ROS production. Vit C decreased Al-induced ROS production. However, increased Al-induced eryptosis. There were no significant changes in glutathione after the ALCL3 treatment. CONCLUSIONS: Al-induced eryptosis and hemolysis through triggering oxidative stress, while NAC could diverse this effect. In contrast, vit C might intensify Al-induced eryptosis at particular doses through a less known mechanism.
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INTRODUCTION: Red blood cell alloimmunization is a challenging issue in thalassemia patients. Several studies have investigated the role of different immune system compartment in alloimmunization, but the exact mechanism remains unclear. Considering the immunoregulatory function of iNKT cells and their subsets, in this study, we evaluated the possible role of these cells in alloimmunization status of thalassemia patients. METHODS: 78 ß-thalassemia major patients (41 alloimmunized and 37 non-alloimmunized) and 17 healthy controls were engaged in this study. Mononuclear cells were isolated from peripheral blood samples and stimulated for cytokine production. Samples were subjected to flow cytometry for enumeration of iNKT cells and characterized based on their cytokine production pattern. Finally, the results correlated with alloimmunization status, clinical and laboratory data. RESULTS: Results demonstrated that the number of iNKT, iNKT+IFN-ɤ+, and iNKT+IL-4+ cells in thalassemia group was significantly higher than healthy controls while no significant change was observed in the number of these cells between alloimmunized and non-alloimmunized thalassemia patients. Interestingly, the ratio of iNKT+IL-4+: iNKT+IFN-γ+ cells in alloimmunized thalassemia group represent a considerable increase in comparison to both non-alloimmunized thalassemia group and healthy controls. However, evaluating this value in non-alloimmunized group represents an approximately equal ratio of 0.94, which was almost similar to this ratio in the control group (0.99). CONCLUSION: Our results illustrated a noteworthy imbalance in the ratio of iNKT cell subsets in favour of IL-4 producing iNKT cells in alloimmunized thalassemia patients. Regarding the role of IL-4 in stimulating the Th2-related immune responses, this imbalance could consider as a possible mechanism in alloantibody responses of thalassemia patients.
Subject(s)
Interferon-gamma/immunology , Interleukin-4/immunology , Natural Killer T-Cells/immunology , Th2 Cells/immunology , Thalassemia/immunology , Adult , Cells, Cultured , Female , Humans , Immunity/immunology , Isoantibodies/immunology , Leukocytes, Mononuclear/immunology , MaleABSTRACT
Clonal eosinophilia is a hematologic disorder caused by translocation in growth factor receptor (GFR) genes. Despite the identified molecular mechanisms underlying clonal hypereosinophilia, the distinction between clonal and reactive eosinophilia has remained challenging due to the diversity of partner genes for translocated GFRs. This study aimed to examine the feasibility of phosphoflow cytometry in the diagnosis of clonal hypereosinophilia through evaluating the level of platelet-derived growth factor receptor alpha (PDGFRA) phosphorylation and its correlation with PDGFRA genetic aberration. Blood samples were collected from 45 hypereosinophilia patients and 10 healthy controls. Using phosphoflow cytometry method, the phosphorylation state of PDGFRA was assessed. The specificity of phosflow results was confirmed by western blotting and eventually compared with qRT-PCR expression analysis of 3'-region of PDGFRA. To detect the genetic aberration of PDGFRA, 5'-rapid amplification of cDNA ends (5'-RACE) was performed. Phosflow analysis illustrated that 9 of 45 hypereosinophilic patients had higher level of PDGFRA phosphorylation while sequence analysis of 5'-RACE-PCR fragments confirmed that in seven cases of them, there was a PDGFRA-FIP1L1 fusion. We also verified that two of nine patients with hyperposphorylated PDGFRA hold ETV6-PDGFRA and STRN-PDGFRA rearrangements. Importantly, nine cases also had significantly higher levels of PDGFRA mRNA expression when compared with healthy controls, and cases with no PDGFRA rearrangement. These findings highlight a robust correlation between hyperphosphorylation state of PDGFRA and aberrant PDGFRA gene fusions. This implicates phosflow as an efficient and reliable technique raising an intriguing possibility that it could replace other genomic and cDNA-amplification-based diagnostic approaches with limited effectiveness.
Subject(s)
Hypereosinophilic Syndrome , mRNA Cleavage and Polyadenylation Factors , Humans , Hypereosinophilic Syndrome/diagnosis , Hypereosinophilic Syndrome/drug therapy , Hypereosinophilic Syndrome/genetics , Oncogene Proteins, Fusion/metabolism , Phosphorylation , Protein Kinase Inhibitors , Receptor, Platelet-Derived Growth Factor alpha/genetics , Receptor, Platelet-Derived Growth Factor alpha/metabolismABSTRACT
Growth differentiation factor-15 (GDF-15) is a member of TGF-ß superfamily. Among hematopoietic cells, this factor is mainly produced by erythroid series and is recently considered a biomarker of ineffective erythropoiesis (IE). Whether IE induces enhanced GDF-15 expression or is prompted by it, has remained elusive. In this study we investigated how high levels of GDF-15 contribute to IE-associated erythroid dysplasia. We assessed mRNA levels of GDF-15 during erythroid maturation as well as in patients with IE using qRT-PCR. Later, the erythroid colony-forming capacity of GDF-15-treated hematopoietic stem cells (HSCs) was evaluated by CFC assay. Any effect of elevated levels of GDF-15 on erythroid maturation was ultimately examined by expression analysis of erythroid-associated transcription factors and flow cytometry analysis of CD235a expression. GDF-15 mRNA expression increased during erythroid differentiation and also in ß-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Treating the cells with high GDF-15 concentration (50 ng/ml) resulted in an approximate 30% decline in the capacity of erythroid colony formation of HSCs and CD235a positive cells. Additionally, erythroid-specific transcription factors showed significant down-regulation in the early stages of erythroid differentiation. According to the expression level of GDF-15 and the role it plays in the erythroid system, high-levels of this factor could be an auto-modulatory mechanism to control the excessive production of erythroid cells.
Subject(s)
Erythroid Precursor Cells/pathology , Erythropoiesis , Growth Differentiation Factor 15/metabolism , Hematopoietic Stem Cells/pathology , Hyperplasia/pathology , beta-Thalassemia/pathology , Case-Control Studies , Cell Differentiation , Erythroid Precursor Cells/metabolism , Hematopoietic Stem Cells/metabolism , Humans , Hyperplasia/metabolism , Stem Cell Factor/metabolism , Transforming Growth Factor beta/metabolism , beta-Thalassemia/metabolismABSTRACT
Gold nanoparticles (AuNPs) are commonly used in biosensors of various kinds. However, its application to extract DNA from cancer tissues has not been extensively studied. The purification of DNA from cancer tissues is an important step in diagnostic and therapeutic development. Almost, all cervical cancer cases are associated with high-risk human papillomavirus (HR-HPV) infection. Accurate viral diagnosis has so far relied on the extraction of adequate amounts of DNA from formalin-fixed, paraffin-embedded (FFPE) tissue samples. Till now, no specific and sensitive DNA purification method has been introduced for the extraction of HR-HPV from FFPE tissue. Since the commercially available purification kits are not sensitive and specific enough for HR-HPV DNA targets, in this study, a DNA purification method was designed based on AuNPs to purify sufficient amounts of HR-HPV DNA from cervical cancer tissue samples. AuNPs were coated with a series of oligonucleotide probes to hybridize to specific DNA sequences of HR-HPV genotypes. Results showed that 733 out of 800 copies of type-specific HPV DNA were recovered with complete specificity, compared to 36 copies with a standard commercial kit (Qiagen FFPE). The high yield of DNA (91.6%) is the main advantage of the AuNPs-probe purification method.