Association of Circulating Procoagulant Microvesicles with Painful Vaso-Occlusive Crisis in Sickle Cell Disease.

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.

The significance of surface neutrophilic MPO expression level in NETosis and NETosis-associated coagulopathies in covid-19 infected patients.

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.

COVID-19 restrictions and greenhouse gas savings in selected Islamic and MENA countries: An environmental input-output approach for climate policies.

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.

Phosflow assessment of PDGFRA phosphorylation state: A guide for tyrosine kinase inhibitor targeted therapy in hypereosinophilia patients.

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.

Skewed intracellular cytokine production of iNKT cells toward Th2-related responses in alloimmunized thalassemia patients.

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.

GDF-15 negatively regulates excess erythropoiesis and its overexpression is involved in erythroid hyperplasia.

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.

Evaluation of regulatory T cells frequency and FoxP3/GDF-15 gene expression in ß-thalassemia major patients with and without alloantibody; correlation with serum ferritin and folate levels.

ß-thalassemia major is one of the most common hematologic disorders in the world. It causes severe anemia and patients require regular blood transfusions, which causes different complications such as iron overload and alloimmunization. Regulatory T cells (Tregs) have an important role in regulation of immune responses. FoxP3 is the major marker of Tregs and its expression can be influenced by different factors. GDF-15 is another gene that plays a role in iron homeostasis and regulation of immune system in different diseases. The aim of this study was to assess the frequency of Tregs and FoxP3/GDF-15 gene expression in ß-thalassemia major patients with and without alloantibody as well as its correlation with different factors such as serum ferritin and folate levels. This study was conducted on 68 ß-thalassemia major patients with and without alloantibodies in comparison with 20 healthy individuals with matched age and sex as control group. Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and real-time PCR were performed in order to evaluate serum ferritin and folate levels, frequency of Tregs, and the expression of FoxP3 and GDF-15 genes, respectively. The percentage and absolute count of Tregs were increased in patients compared with controls (P = 0.0003), but there was no difference between responders and non-responders (P > 0.05). The Tregs count correlated positively with serum ferritin. No correlation was observed between target genes and serum ferritin and folate, but there was a positive significant correlation between the expression of FoxP3 and GDF-15 genes, which shows the immunosuppressive role of GDF-15.

G22A Polymorphism of Adenosine Deaminase and its Association with Biochemical Characteristics of Gestational Diabetes Mellitus in an Iranian Population.

Adenosine deaminase (ADA) is an important regulator of insulin action. The single nucleotide polymorphism (SNP) G22A in the ADA gene decreases enzymatic activity of ADA. The aim of this study was to investigate the relationship between the SNP G22A and blood glycemic control, insulin resistance, and obesity of gestational diabetes mellitus (GDM) patients in an Iranian population. SNP G22A was determined in women with GDM (N=70) and healthy pregnant women (control, N=70) using polymerase chain reaction-restriction fragment length polymorphism. Fasting plasma glucose (FPG), hemoglobin A1C (HbA1c), plasma insulin levels and plasma lipids were measured using commercial kits. Homeostasis model of assessment for insulin resistance (HOMA-IR) was calculated. The distribution of genotypes and alleles among GDM patients was similar to that of the control group. FPG and HbA1c were significantly higher in GDM patients with GG genotype compared with GDM patients with GA+AA genotype and non-GDM patients. The frequency of GG genotype was significantly higher in obese GDM patients compared to lean GDM patients. The SNP G22A in the ADA gene was not associated with the risk of GDM in our population. GG genotype was associated with poor glycemic control and obesity in GDM patients.

Evaluation of red cell membrane cytoskeletal disorders using a flow cytometric method in South iran.

OBJECTIVE: The diagnosis of hereditary red blood cell (RBC) membrane disorders, and in particular hereditary spherocytosis (HS) and Southeast Asian ovalocytosis (SAO), is based on clinical history, RBC morphology, and other conventional tests such as osmotic fragility. However, there are some milder cases of these disorders that are difficult to diagnose. The application of eosin-5'-maleimide (EMA) was evaluated for screening of RBC membrane defects along with some other anemias. We used EMA dye, which binds mostly to band 3 protein and to a lesser extent some other membrane proteins, for screening of some membrane defects such as HS. MATERIALS AND METHODS: Fresh RBCs from hematologically normal controls and patients with HS, SAO, hereditary elliptocytosis, hereditary spherocytosis with pincered cells, severe iron deficiency, thalassemia minor, and autoimmune hemolytic anemia were stained with EMA dye and analyzed for mean fluorescent intensity (MFI) using a flow cytometer. RESULTS: RBCs from patients with HS and iron deficiency showed a significant reduction in MFI compared to those from normal controls (p<0.0001 and p<0.001, respectively), while macrocytic RBCs showed a significant increase in MFI (p<0.01). A significant correlation was shown between mean corpuscular volume and MFI, with the exceptions of HS and thalassemia minor. CONCLUSION: Our results showed that the flow cytometric method could be a reliable diagnostic method for screening and confirmation, with higher sensitivity and specificity (95% and 93%, respectively) than conventional routine tests for HS patients prior to further specific membrane protein molecular tests.

Bioinformatics-Guided Discovery of miRNAs Involved in Apoptosis Modulated by Parthenolide Combined with Vincristine in The NALM6 Cell Line.

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.

Analysis of ß/α globin ratio by using relative qRT-PCR for diagnosis of beta-thalassemia carriers.

BACKGROUND: Current routine tests for premarital screening of ß-thalassemia carriers are not applicable for diagnosis of rare atypical minor ß-thalassemia cases. A more specialized laboratory evaluation for them is the measurement of ß/α chain synthesis ratio with the assistance of radioactive amino acids. This method is also no longer routinely accessible. Consequently it is required to establish a rapid, trouble-free, and reliable method that encompasses all the cases of ß-thalassemia carriers. Therefore we have determined ß/α-globin mRNA ratio by applying relative qRT-PCR in various ß-thalassemia patients. METHODS: Reticulocytes RNA extraction and subsequent cDNA synthesis were performed, followed by relative qRT-PCR for α- and ß-globin chain genes and ß-actin gene as an endogenous reference. ß/α-Globin gene ratio was then evaluated with the Pfaffl method. RESULTS: The mean of ß/α ratio was 0.99, 0.81, 0.69, and 0.69 for normal population, minor, intermediate, and major ß-thalassemia, respectively. Approximately 6% of cases with minor thalassemia RBC index and normal HbA2 and having a decreased ß/α ratio were located in the minor ß-thalassemia group. The mean of ß/α mRNA ratio in normal individuals and minor ß-thalassemia was significantly different with all other groups (P-value < 0.05). Nevertheless, there was no such association between ß/α mRNA ratio in major and intermediate ß-thalassemia. CONCLUSION: According to the significant differences achieved, no overlapping between minor ß-thalassemia and normal group, capability of diagnosing atypical minor ß-thalassemia, and accessibility of this technique, we can declare that this method could be suggested as a routine premarital screening test for ß-thalassemia carriers.

Comparison of Interleukin-33 Serum Levels in Patients with Breast Cancer and Idiopathic Granulomatous Mastitis.

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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The mRNA Expression of PTEN, LEF1, JAK3, LC3 and p62/SQSTM1 Genes in Patients with Chronic Myeloid Leukemia.

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.

Molecular Mechanisms of Glaucoma Pathogenesis with Implications to Caveolin Adaptor Protein and Caveolin-Shp2 Axis.

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.

Expression of the Hepatitis C Virus core-NS3 Fusion Protein on the Surface of Bacterial Ghosts: Prospects for Vaccine Production.

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.

Trehalose An Additive Solution for Platelet Concentrate to Protect Platelets from Apoptosis and Clearance during Their Storage at 4°C.

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.

Decreased expression of autophagy-related genes in the complete remission phase of acute myeloid leukemia.

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.

The Pathogenic Aspects of Human Parvovirus B19 NS1 Protein in Chronic and Inflammatory Diseases.

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.

Diverse Effect of Vitamin C and N-Acetylcysteine on Aluminum-Induced Eryptosis.

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.