Antileukemia Activity of Human Natural Killer Cell-Derived Nanomagic Bullets against Acute Myeloid Leukemia (AML).

Background: Cancer is among the serious health problems of the medical world, for treatment of which severe treatments are used. However, the prognosis of cancer patients is still poor. The application of NK cell-derived exosomes (NK-Exo) is a new method for cancer immunotherapy. These nanoparticles with a size range of 30-120 nm are a small model of mother cells. In this study, the anti-tumor activity of NK-Exo and LAK-Exo (activated NK cell-derived exosome) against acute myeloid leukemia (AML) is investigated in vitro. Materials and Methods: The MACS method was performed for the separation of NK cells from the buffy coats of healthy donors, and an EXOCIBE kit was used for the isolation of NK-Exo. After treating the KG-1 cell line with different doses of NK-Exo, MTT assay, and annexin V-PE were done to evaluate cell proliferation and apoptosis, respectively, and for confirmation of involved proteins, Real-Time PCR and western blotting were performed. Results: Anti-tumor activity of NK-Exo and LAK-Exo was dose- and time-dependent. Their highest activities were observed following 48 hours of incubation with 50 µg/ml exosome (p<0.0001). However, this cytotoxic activity was also seen over a short period of time with low concentrations of NK-Exo (p<0.05) and LAK-Exo (p<0.001).The cytotoxic effect of LAK-Exo on target cells was significantly higher than NK-EXO. The induction of apoptosis by different pathways was time-point dependent. Total apoptosis was 34.56% and 51.6% after 48 hours of tumor cell coculture with 50µg/ml NK-Exo and LAK-Exo, respectively. Significant expression of CASPASE3, P38, and CYTOCHROME C genes was observed in the cells treated with 50 µg/ml NK-Exo and LAK-Exo. Conclusion: Our study confirmed the antileukemia activity of NK-Exo against AML tumor cells in vitro. Therefore, NK-Exo can be considered as a promising and effective treatment for leukemia therapy.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study

Abstract Introduction The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. Objective The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). Method For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). Results The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ± 1.28, 17.3 ± 0.96 and 21.2 ± 0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ± 4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. Conclusion According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

Optimizing the recovery of peripheral blood mononuclear cells trapped in leukoreduction filters - A comparison study.

INTRODUCTION: The isolation of captured peripheral blood mononuclear cells (PBMNCs) from leukoreduction filters (LRFs) can be of great importance in terms of bringing the lost cells back into use. OBJECTIVE: The aim of this study was to evaluate various methods based on their potential to recover the peripheral blood cells from LRFs with a focus on mononuclear cells (MNCs). METHOD: For cell isolation from LRFs, three distinct methods (back-flushing, direct and vacuum pump) were compared through the calculation of the yield of isolated MNCs. The viability of extracted cells was determined by the flow cytometry technique. Moreover, the recovered MNCs were characterized regarding the presence of blood stem cell purification. The cell culture, microscopic observation, and immunophenotyping were employed to characterize the blood stem cells (hematopoietic, mesenchymal and progenitor endothelial stem cells). RESULTS: The yield of isolation obtained in the back-flushing, direct and vacuum pump methods were 17.7 ±â€¯1.28, 17.3 ±â€¯0.96 and 21.2 ±â€¯0.90 percent, respectively. Although the highest potential for total blood cell recovery belonged to the vacuum pump method, the lowest cell viability (85.73 ±â€¯4.84%) was observed in this method. However, the isolation process of the back-flushing and direct methods had less effect on cell viability. The characterization of the isolated MNCs displayed that the dominant positive phenotype was for CD34/CD45, indicating hematopoietic stem cells. In addition, the endothelial stem/progenitor cells were significantly detected as CD31/CD133 positive cells. CONCLUSION: According to our results and considering the safety and efficiency potential of each of the applied methods, the back-flushing in comparison with the other methods can be considered a suitable procedure for MNC isolation from LRFs.

Expression and CpG island methylation pattern of MMP-2 and MMP-9 genes in patients with congenital factor XIII deficiency and intracranial hemorrhage.

OBJECTIVES: Congenital factor XIII (FXIII) deficiency is a rare severe bleeding disorder. Intracranial hemorrhage (ICH) is the leading cause of mortality and morbidity in FXIII deficiency. However, its pathogenesis is not well understood yet. In this study, we investigated the expression and CpG island methylation status of matrix metalloproteinase-2 (MMP-2) and MMP-9 in patients with FXIII deficiency and ICH. METHODS: Forty patients with FXIII deficiency including twenty patients with ICH, and twenty without ICH were recruited as case and control groups, respectively. Methylation status was determined by bisulfite sequencing polymerase chain reaction (PCR), and gene expression was assessed by quantitative real-time PCR. RESULTS AND DISCUSSION: We found an unmethylated pattern for both MMP-2 and MMP-9 genes in the case group. Both genes were partially methylated in the control group, while the percentage of methylated CpGs was significantly higher in MMP-9 than MMP-2 (P = 0.001). Furthermore, higher expression of MMP-9 (in both the mRNA and protein levels) was found in the case than control group (P = 0.008 and P = 0.009, respectively). On the other hand, there was no significant difference in MMP-2 expression level (neither mRNA nor protein) between the two groups (P = 0.12 and P = 0.25, respectively). CONCLUSION: Our findings indicated that MMP-9 over-expression might be related to ICH in FXIII deficiency, and gene methylation effectively regulates its expression. Future researches will expand our understanding of the pathogenesis of ICH in congenital FXIII deficiency.

Upregulation of miR-210 promotes differentiation of mesenchymal stem cells (MSCs) into osteoblasts.

Numerous studies indicated that microRNAs are critical in the regulation of cellular differentiation, by controlling the expression of underlying genes. The aim of this study was to investigate the effect of miR-210 upregulation on differentiation of human umbilical cord blood (HUCB)-derived mesenchymal stem cells (MSCs) into osteoblasts. MSCs were isolated from HUCB and confirmed by their adipogenic/osteogenic differentiation and flow cytometric analysis of surface markers. Pre-miR-210 was amplified from human DNA, digested and ligated with plenti-III-mir-green fluorescent protein (GFP) vector, and cloned in STBL4 bacteria. After confirmation with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the plenti-III-GFP segment bearing pre-miR-210 was transfected into MSCs by electroporation. Two control vectors, pmaxGFP and Scramble, were transfected separately into MSCs. The expression of miR-210 and genes related to osteoblast differentiation, i.e., runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and osteocalcin gene, in the three groups of transfected MSCs was analyzed 0, 7, 14, and 21 days of transfection by quantitative reverse transcription PCR (qRT-PCR). Overexpression of miR-210 was observed in MSCs transfected with miR-210-bearing plasmid, and this was significantly different compared to Scramble group (p < 0.05). Significantly increased expression of Runx2 (at day 7 and 14), ALP and osteocalcin genes (at all time points for both genes) was observed in MSCs with miR-210-bearing plasmid compared to controls. Overall, the overexpression of miR-210 in MSCs led to MSC differentiation into osteoblasts, most probably by upregulating the Runx2, ALP, and osteocalcin genes at different stages of cell differentiation. Our study confirms the potential of miRNAs in developing novel therapeutic strategies that could target regulatory mechanisms of cellular differentiation in various disease states.

Study of the Relationship between HPA-1 and HPA-5 Gene Polymorphisms and Refractory to Platelet Therapy and Recombinant Factor VII in Glanzmann Thrombasthenia Patients in Southeast of Iran.

Background: Glanzmann Thrombasthenia (GT) is a rare autosomal disease. HPA (Human Platelet Alloantigen) is a surface polymorphic alloantigen of platelets. This study was intended to investigate and compare the polymorphism of HPA-1 and HPA-5 genes in two groups of GT patients, with and without resistance to platelet and recombinant factor VII therapy. Materials and Methods: This case control study was performed on GT patients (n=16) with resistance to platelet therapy and recombinant factor VII and control group of GT patients (n=16) without resistance to platelet therapy and recombinant factor VII. The consent form was completed by each patient. Gene polymorphisms of HPA-1 and HPA-5 were investigated using SSP-PCR, and the obtained data were analyzed using statistical software SPSS16.0. Results: The results indicated no significant relationship between the studied genes and their resistance to platelet therapy and recombinant factor VII. The frequencies of HPA-1 genotype a/a were 98% and 94% in patient and control groups, respectively. The frequency of allele b was found to be less than allele a. The value of this allele was 4% in patient group and 1% in control group. In addition, the HPA-5a/a (98%) was the most frequent alloantigen?? (check it) in both groups. Seven percent (7%) of the patients had the HPA-5a/b genotype, and the HPA-5b/b was found to be absent in these individuals. Conclusion: According to the results obtained, it could be concluded that these genes play no role in resistance to platelet therapy.

The Role of MicroRNAs in Myeloproliferative Neoplasia.

MiRs are 17-25 nucleotide non-coding RNAs. These RNAs target approximately 80% of protein coding mRNAs. MiRs control gene expression and altered expression of them affects the development of cancer. MiRs can function as tumor suppressor via down-regulation of proto-oncogenes and may function as oncogenes by suppressing tumor suppressors. Myeloproliferative neoplasias (formerly known as chronic myeloproliferative disorders) form a class of hematologic malignancies demonstrating the expansion of stem cells in one or more hematopoietic cell lines. CML results from an acquired translocation known as BCR-ABL (Philadelphia chromosome). JAK2V617F mutation is present in over 95% of PV, 55% of ET and 65% of PMF cases. Aberrant expression of miR is associated with myeloproliferative neoplasias, pathogenesis, disease progress and response to treatment. MiRs can also be potential therapeutic targets. CML is mainly treated by tyrosine kinase inhibitors such as Imatinib. In addition, altered function of miRs may be used as a prognostic factor in treatment. Resistance to Imatinib is currently a major clinical problem. The role of a number of miRs has been demonstrated in this resistance. Changing expression pattern of miRs can be effective in response to treatment and inhibition of drug resistance. In this paper, we set out to evaluate the effect of miRs in pathogenesis and treatment of MPN.

Central nervous system bleeding in pediatric patients with factor XIII deficiency: a study on 23 new cases.

BACKGROUND: Factor XIII (FXIII) deficiency is an extremely rare bleeding disorder, which has the highest incidence in Sistan and Baluchistan Province in Iran, compared to its overall incidence around the world. This disorder has different clinical manifestations ranging from mild bleeding tendency to lethal bleeding episodes including central nervous system (CNS) hemorrhage. The aim of this study was to evaluate the demographic data, pattern of CNS bleeding, and the role of plasminogen activator inhibitor-1 (PAI-1) (PAI-1) 4G/5G and thrombin activatable fibrinolysis inhibitor (TAFI) Thr325Ile polymorphisms in intracranial and extracranial hemorrhages in 23 new cases of FXIII-deficient subjects. METHODS: This case-control study was conducted on 23 FXIII-deficient patients with CNS bleeding episodes and 23 patients as the control group with FXIII deficiency but without any history of CNS bleeding. Initially, to confirm the molecular defect, both groups were evaluated for the most frequently reported mutation of FXIII (Trp187Arg mutation) in a previous study in Sistan and Baluchistan Province. Then, demographic data, clinical manifestations, and pattern of CNS bleeding were determined. Eventually, the patients were assessed for PAI-14G/5G and TAFI Thr325Ile polymorphisms. RESULTS: The results of this study revealed that all the subjects (including the case and control groups) were homozygous for Trp187Arg mutation. Nineteen patients (82.6%) had intracranial hemorrhage (ICH) and four patients (17.4%) had extracranial hemorrhage (ECH). Intraparenchymal hemorrhage was the most common form of ICH (89.5%), and epidural hemorrhage was observed in two patients (10.5%). Anatomic regions in patients with intraparenchymal hemorrhage were temporal in six (35.3%), occipital in four (23.5%), diffused intraparenchymal in four (23.5%), temporal-occipital in two (11.8%), and subdural with temporal in one (5.9%) patient. We found that in the case group, 14 patients (60.8%) were homozygous for TAFI Thr325Ile polymorphism and 8 cases (34.7%) were heterozygous. In the control group, 4 (17.4%) and 13 (56.5%) patients were homozygous and heterozygous, respectively (P < 0.001 vs. P < 0.01).We also found that an equal number of patients (two individuals) in the case and control groups (8.7% in each group) were heterozygous for PAI-14G/5G polymorphism. CONCLUSION: It seems that PAI-14G/5G polymorphism does not have any effect on occurrence of ICH and ECH in patients with FXIII deficiency, while TAFI Thr325Ile is a strong genetic risk factor (odds ratio:14.9, 95% confidence interval: 7.4-31.1).

Frequency of beta-thalassemia or beta-hemoglobinopathy carriers simultaneously affected with alpha-thalassemia in Iran.

BACKGROUND: Beta-thalassemias are prevalent heritable single gene disorders affecting the quantity of the hemoglobin molecule. Rarely, a co-inheritance of these impairments with alpha-thalassemia and/or a hemoglobinopathy occurs and makes an important double heterozygote or homozygous state. Thus finding these cases is essential for genetic counseling. The present study aimed to identify the prevalence of coexistent alpha-thalassemia mutations, hemoglobinopathies, and beta-thalassemia determinants. METHODS: This descriptive study was performed on 5760 patients. We used complete blood cell count, Hb electrophoresis, and HbA2 measurement for thalassemia carrier identification. Increased HbA2 (> or = 3.5%) is the standard diagnostic marker for beta-thalassemia, while normal HbA2 with low MCH and MCV can indicate an alpha-thalassemia carrier or atypical beta-thalassemia minor. Individuals with MCV < 80 fL, MCH < 27 pg, and hemoglobin < or = 15.3 g/dL in men or < or = 14 g/dL in women, were candidates for molecular thalassemia investigations. Patients with abnormal hemoglobin varieties in hemoglobin electrophoresis were referred to a genetics laboratory for hemoglobinopathy detection. RESULTS: 141 subjects out of 5760 were affected by alpha and beta-thalassemia or a beta-hemoglobinopathy simultaneously, including: 13 (11.1%) fetuses, 55 (38.2%) male cases, and 73 (50.7%) females. Among these 141 alpha-thalassemia patients, 92 cases (65.24%) were beta-thalassemia carriers and 3 (2.12%) were beta-thalassemia major, 43(30.49%) had beta-hemoglobinopathies, and 3 cases (2.12%) had co-inherited beta-thalassemia and variant hemoglobins. 31 beta-gene mutations were observed in this population, the most common being HbS Cd6 (A > T) (24%). These thalassemia determinants account for about 46% of all detected mutations. As for alpha-gene mutations, -3.7 detection was the most prevalent. CONCLUSIONS: The relatively high prevalence of co-inherited alpha-thalassemia and hemoglobinopathies among beta-thalassemia carriers indicates the importance of molecular analysis to diagnose these double heterozygous or sole homozygous cases for prenatal diagnostic purposes and putting forth strategies to prevent more complicated and dangerous combinations.

The effect of exercise training on the level of tissue IL-6 and vascular endothelial growth factor in breast cancer bearing mice.

OBJECTIVES: The goal of this study was assessing the prophylactic effect of exercise and its role as an adjuvant therapy on level of cytokines involved in angiogenesis in estrogen-dependent breast cancer. MATERIALS AND METHODS: Forty female BALB/c mice were randomly assigned to exercise-tumor-exercise (ETE), exercise-tumor-rest (ETR), rest-tumor-exercise (RTE) and rest-tumor-rest (RTR) groups. After orientation in the environment, two groups of mice performed continuous endurance exercise for 8 weeks, and thereafter estrogen-dependent MC4L2 cancer cells were injected to them. Then, one group of each of trained and non-trained mice performed endurance exercise 5 days per week for 6 weeks. Tumor volume was measured by a digital caliper weekly. Finally, the mice were sacrificed; tumor tissue was removed, immediately frozen and kept in -70°C. Tumor sample was homogenized; levels of cytokines were measured and quantified using ELISA. RESULTS: There was significant reduction in the level of interlukin-6 (IL-6) (P=0.001), Vascular endothelial growth factor (VEGF) (P=0.0001) and tumor volume (P=0.0001) among the groups performing endurance exercise after malignancy (RTE and ETE) in comparison with groups not performing endurance exercise (ETR and RTR), and these results were in agreement with tumor growth rate. CONCLUSION: Exercise can cause reduction in levels of pro-inflammatory cytokines in tumor tissue. Decreased IL-6 production could reduce the generation of VEGF, resulting in reduced intra-tumor angiogenesis. Due to reduction of the level of these cytokines in groups doing exercise before and after malignancy, exercise is presumed to be an adjuvant therapy in estrogen-receptor dependent tumors in addition to its effective prophylactic role.

Evaluation of the Effect of miR-26b Up-Regulation on HbF Expression in Erythroleukemic K-562 Cell Line.

BACKGROUND: The major hemoglobin in the fetus is hemoglobin F (HbF) (α2γ2), whereas in adult humans, hemoglobin A (α2ß2) is predominately expressed. Several studies have indicated that expression of the HbF subunit γ-globin might be regulated post-transcriptionally. This could be done by small non-coding RNAs called microRNAs which target mRNAs in a sequence-specific manner and lead to translational repression or mRNA decay. The aim of this study is to evaluate the effect of miR-26b up-regulation on γ-globin gene expression in K-562 cell line. METHODS: These cells were grown in RPMI 1640 and pre miR-26b and were transfected within K-562 cell line using lentiviral vector. After RNA extraction and cDNA synthesis in selected days, miRNA up-regulation was confirmed by miRNA real time PCR and then γand ßchain and GATA-1 expression were investigated by RT and QRT-PCR. RESULTS: The viability of cells before transfection was 90%. Three and 7 days after transfection, through the use of relative Q-PCR, the γ chain expression increased 3.7, 6.8 and 3.8 folds and GATA-1 expression increased 2.1, 6.0 and 8.0 in comparison with untransfected cells. CONCLUSION: The data suggest that miR-26b can be involved in the increase of γ-globin gene expression in K-562 cell line. We suggest that miR-26b may be a significant therapeutic target for increasing HbF levels in patients with sickle cell disease and ß-thalassemia.

The Effect of miR-210 Up-regulation on Proliferation and Survival of Mouse Bone Marrow Derived Mesenchymal Stem Cell.

BACKGROUND: Bone marrow derived mesenchymal stem cells (MSCs) are a population of multipotent progenitors which have the capacity of proliferation and differentiation into mesenchymal lineage cells. Hypoxia could promote the proliferation of MSCs. Micro-RNAs are endogenous RNAs that can play an important role in some processes such as proliferation and differentiation. MiR-210 could help for better proliferation of MSCs since this miRNA could activate HIF pathway. In current study we investigated if MSCs can preserve their differentiation and proliferation ability under normoxic conditions by upregulation of miR-210. MATERIALS AND METHODS: MSCs isolated from C57 BL/6 mice by flushing it's femurs into the cell culture media. After 72 hours, MSCs which are plastic adherent cells were attached to the flask and non-adherent cells were removed. Subsequently, MSCs induced to differentiate into osteocytes and adipocytes with specific differentiation media in order to confirm their identity and multipotency. Then miR-210 was inserted in Lentiviruse vectors and affected MSCs. In each passage, the number and viability of cells were evaluated. RESULTS: Comparison between miR-210 infected MSCs with control cells showed that miR-210 has ability to increase proliferation of MSCs significantly. CONCLUSION: We showed that miR-210 has ability to induce proliferation of MSCs without any negative effect on their differentiation abilities. Further studies are needed for evaluation of probable effects of miR-210 mechanisms on MSCs proliferation.

Evaluation of liver and kidney function in favism patients.

BACKGROUND: G6PD deficiency is the most common enzymopathy of red blood cells. The clinical symptoms of favism are jaundice, hematuria and haemolytic anaemia that seem to affect liver and kidney in long term. Thus we evaluate kidney and liver function of favism patients in an endemic area of the disease with a high rate of fava beans cultivation. METHODS: This study was performed on favism patients and healthy controls referring to Iranshahr central hospital. Liver and kidney function tests were performed. RESULTS: The results showed a statistically significant difference between these two groups (p <0.05) for liver function tests, (AST, ALT and ALP), but not for renal tests (BUN and creatinine) (p >0.05). CONCLUSION: Due to abnormalities were seen in the liver function tests of these patients, we suggest that these tests be regularly performed for favism patients who are constantly exposed to oxidant agents.

Motor control as the control of perception.

This paper describes a test of Perceptual Control Theory (PCT), which views motor control as part of a process of controlling perceptual inputs rather than motor outputs. Sixteen undergraduate students (M age = 19.9 yr.) were asked to control one of three different perceptual aspects of an animated display--a shape, a motion or a sequence--using the same motor output, a key press. Animation rate was varied while quality of control was measured in terms of the proportion of time that the perception was maintained in the goal state. The results showed that increased animation rate made it hardest to control the more complex perceptions (motion and sequence) even though the same output was used to control all perceptions. This result is consistent with PCT, which predicts that the temporal constraints on control are ultimately a function of the type of perception controlled rather than the type of output used to control it.

Anemia and thrombocytopenia in acute and chronic renal failure.

BACKGROUND: Acute renal failure describes as a syndrome by rapid decline in the ability of the kidney to eliminate waste products, regulate acid-base balance, and manage water homeostasis. When this impairment is prolonged and entered chronic phase, erythropoietin secretion by this organ is decreasing and toxic metabolic accumulates and causes hematological changes include decrease of HCT, MCV and RBC and platelet counts. This study evaluates present of anemia and thrombocytopenia in patients with acute and chronic renal failure. MATERIALS AND METHODS: This study conducted on 132 patients with renal impairment and also 179 healthy individuals as two separated control groups. Initially patients with renal problem were tested and after confirmation of impairment, patients were divided in two groups, acute with less than 3 months and chronic with more than 3 months renal failure, based on duration of the disease. Then complete blood count performed for each patient and finally obtained data were analyzed by SPSS software. RESULTS: Comparison between 96 patients with acute and 36 patients with chronic renal failure revealed that severity of anemia (HCT, Hb and MCV) between these two groups were statistically high in comparison with control groups (P > 0.05) but thrombocytopenia in patients with chronic renal failure was statistically different from control and the acute ones (P < 0.001). CONCLUSION: It was recommended that in patients with chronic renal failure, to prevent the risk of bleeding, platelet count should be checked periodically.