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.

Unraveling the Impact of Blood Transfusion on Transcription Factors Regulating T Helper 1, 2, 17 and Regulatory T cells.

T helper 1 (TH1) and TH2 lymphocytes are the most important components of the immune system affected by blood transfusion. This study aimed`` to evaluate the effect of blood transfusion on gene expression of transcription factors related to the development of TH1, TH2, TH17 and regulatory T cells (Tregs). In this cross-sectional study, 20 patients diagnosed with abdominal aortic aneurysms requiring surgical repair were studied from January 2018 to August 2020. We utilized real-time PCR to evaluate the expression of transcription factor genes associated with TH1, TH2, TH17, and Treg, namely T-box-expressed-in-T-cells (T-bet), GATA-binding protein 3 (GATA-3), retinoid-related orphan receptor (RORγt), and fork head box protein 3 (Foxp3), respectively. The sampling occurred before anesthesia, 24- and 72 hours post-transfusion, and at the time of discharge. The results showed that the T-bet gene expression, compared to the time before transfusion, was significantly decreased 24 hours after blood transfusion and upon discharge while GATA3 genes exhibited a significant reduction both 24 and 72 hours after the transfusion, as compared to the pre-transfusion levels and the time of patient discharge. The Foxp3 gene demonstrated an increase at all study stages, with a notable surge, particularly 72 hours after red blood cell (RBC) transfusion. Conversely, the expression of RORγt gene, consistently decreased throughout all stages of the study. RBC transfusion in abdominal aortic aneurysm patients altered the balance of transcription gene expression of TH1, TH2, TH17, and Treg cells.

Red Blood Cell Immunization and Contributing Factors in 685 Thalassemia Patients.

Background: An analysis of red blood cell alloimmunization in patients with thalassemia can help to devise specific strategies to decrease the alloimmunization rate. This study explored the frequency and specificity of alloantibodies and autoantibodies against red blood cell (RBC) antigens in patients with thalassemia referring to the Iranian Blood Transfusion Organization (IBTO) Immunohematology Reference Laboratory (IRL) in Tehran. Materials and Methods: This study first examined the laboratory records of 23,113 patients suffering from different diseases referring to IBTO's IRL for pretransfusion testing in the 2008-2015 period. ABO and Rh(D) typing and antibody screening tests were performed for all 23,113 patient records and 685 (2.97%) beta-thalassemia patients with positive pre-transfusion test results (antibody screening and/or DAT) were selected for further investigation. Results: The antibody screening test was positive in 640 out of 685 thalassemic patients (93.4%). DAT was performed for 529 patients, 226 (33%) of which showed positive results. Meanwhile, 161 out of 685 beta-thalassemia patients (23.5%) had positive auto control test results, reflecting the possible presence of allo- and/or autoantibodies. The most common antigen-specific alloantibodies were directed against K and E RBC antigens with a frequency of 25% (Anti-K) and 11.91% (Anti-E), respectively. The development of two antibodies (double antibodies) in one patient was observed in 80 individuals (11.46%). Conclusion: Age, gender, history of pregnancy, and splenectomy were not contributing factors to the antibody presence in the patient population under study. Extended red blood cell phenotyping should be considered as an essential procedure for expected multi-transfused thalassemia patients before blood transfusion. Considering the high frequency of anti-K and anti-E observed in this study, it is recommended that thalassemia patients in Iran are tested through phenotyping of RBC units for K and E antigens before transfusion.

Viral safety of recovered plasma for contract fractionation; an Iranian experience, 2006-2015.

OBJECTIVE: The current study analysed the viral safety among Iranian blood donors. BACKGROUND: Plasma products demand is increasing in the world. With contract plasma fractionation, the plasma wastage decreases and the access of patients to plasma-derived medicines (PDM) improves. STUDY AND DESIGN METHOD: Screening results including hepatitis B surface antigen (HBsAg), anti-hepatitis C virus (HCV), and human immunodeficiency virus (HIV) Ag/Ab of 19 054 036 donations from 2006 to 2015 were analysed. The plasma for fractionation was tested for HBV DNA, HCV RNA, HIV RNA, HAV RNA, and Parvovirus B19 DNA by fractionators. New samples were collected from the positive donors and retested. The prevalence of serological and nucleic acid testing (NAT) markers per 105 donations, 95% confidential interval (CI), and p-values were calculated. RESULTS: The prevalence of markers was as follows: 250/105 donations for HBsAg from 516 in 2006 to 116/105 donations in 2015; 74/105 donations for HCV, decreasing from 127 to 41/105 and 3.6/105 for HIV during current study. During 10 years, 5 713 641 units of recovered plasma were shipped for contract fractionation to produce PDM; 0.26/105 donations for HBV DNA and 0.14/105 for HCV RNA were reported positive. The results of five retested samples for HBV and one sample for HCV were negative. CONCLUSION: The prevalence of HBV, HCV, and HIV in blood donations was extremely low. Thanks to the availability, high quality and safety of recovered plasma as a result of the improvements in the quality system at IBTO, this plasma could be used for the production of PDMPs.

Analysis of hospital blood components wastage in Iran (2005-2015).

BACK GROUND: Although blood components are precious resources, their wastage is still a problem in hospitals all over the world. As no comprehensive study has evaluated hospital wastage in Iran, the main aim of the study was to identify the wastage as a percentage of issue during 2005-2015 and the secondary objective was to focus on the reasons of the blood components wastage. STUDY DESIGNS AND METHODS: Wastage as a percentage of issues was calculated for red blood cells, plasma and platelet concentrate separately. Also, for each product, the percentage of wastage was calculated as the number of units wasted for each reason divided by the total number of units wasted. RESULTS: The wastage rate of red blood cells, plasma and platelet concentrate was 5.7 ± 0.7, 1.4 ± 0.4, and 3.2 ± 0.5, respectively. The main cause of red blood cells, plasma, and platelet concentrate wastage was date expiry and reserved/returned units of operating room and or ward. In 2015 compared to 2005, despite a significant decrease (p value<0.0001) in red blood cells and plasma expired units, there was a remarkable increase in expired PC units (p value<0.0001). In contrast to expired units, there was a significant increase (p value<0.0001) in reserved/returned units of operating room and or ward for red blood cells and plasma. CONCLUSION: Time expiry and reserved/returned from operating room were the most important reasons of blood component wastage. The percentage of wastage could be decreased by implementing MSBOS program and designing a software application for efficient management of reserved hospital inventories.

A successful experience of the Iranian blood transfusion organization in improving accessibility and affordability of plasma derived medicine.

Plasma is the liquid part of blood. It is estimated 21.6 million liters of plasma collect from Whole blood annually. From these plasma, 4.2 million liters transfuse, 8.1 million liters fractionate, 9.3 million liters waste. Nowadays, blood products and PDM (plasma derived medicine) consider as essential medicine in modern health care and transfusion medicine. Iranian blood transfusion organization as a non-profit organization was established in 1974 in order to centralize all blood transfusion activities from donor recruitment to distribution of blood components to hospitals. Iran is the only country in EMR region with the rate of 20-29.9 blood donations per 1000 population and reached 100% voluntary non-remunerated blood donation in 2007. RBCs and platelets demand are much more than FFPs so the IBTO was faced the surplus plasma that could cause surplus plasma wastage. Simultaneously, hospitals need more plasma derived medicine especially albumin, IVIG, factor VIII, factor IX. IBTO was faced the challenges such as Fractionators selection, Plasma volume shipment, Contract duration, Product profile, Multiple External audits, Cold chain maintenance, Transporting plasma across international borders, NAT test. To overcome plasma wastage and storage of PDM. IBTO involved toll manufacturing in 2005 and not only prevents plasma wastage but also save MOH (ministry of health) budget.

Blood collection, components preparation and distribution in Iran, 2008-2012.

BACKGROUND: The information about the dynamics of blood collection, components preparation and distribution in Iran was measured and compared during 2008-2012. STUDY DESIGNS AND METHODS: The survey instruments were based on collecting data from all 220 blood collections and blood processing centers over the country, registering them in the validated data base and reporting them to headquarter of Iranian Blood Transfusion Organization. RESULTS: Total blood collection increased during this period, and in 2012 represented a 12.6 percent increase compared to that in 2008. On average, red blood cells, fresh frozen plasma and platelet concentrate were prepared from 95.5 ± 2.4, 81 ± 3.8 and 47 ± 8.8 percent of all whole blood collection. From 2008 to 2011, the distribution of whole blood and fresh frozen plasma revealed different patterns. For whole blood, declines were noted, while for fresh frozen plasma increases were reported. In addition the distribution of red blood cells and platelet concentrate did not change considerably. Also between 2008 and 2012, the mean percentage of outdated and discarded units was 3.6 ± 1 and 5.2 ± 4.6. CONCLUSION: This study as a first national survey provides comprehensive information about the blood supply, components preparation and distribution, and helps to define strategy for the future.

The Effect of IL-22 and IL-28 in Induction of Type 1 Regulatory T (Tr1) Cells.

Cytokines have been introduced as critical inducers in the development of Th subpopulations.Cytokines like IL-10 are involved in inducing regulatory T cells such as Type 1 regulatory T (Tr1) cells cells. IL-22 is a member of IL-10 family of cytokines, and IL-28A is a member of IFN-γ family. In this study, cord blood mononuclear cells (CBMC) from normal healthy individuals were isolated by Ficoll and then naïve T cells were purified by CD4+CD25+ Regulatory T cell Isolation kit. The effect of these two cytokines on production of IL-5, TGF-ß, IL-10, IL-4 and IFN-γ cytokines from cord blood T cells was investigated to identify Tr1 cells as well as Th1 and Th2 polarization. Flow cytometric analysis showed that IL-28A and IL-22 were not effective in expression of IL-5 and TGF-ß either alone or in synergy, but in view of IL-10, IL-4 and IFN-γ, the results showed that IL-22 increased IL-10 and IL-4 but had a decreasing effect on IFN-γ. The results showed that IL-28A was not effective in increasing or decreasing the level of IL-10, IL-4 and IFN-γ. Therefore, according to these results, IL-22 and IL-28A were not effective in inducing Tr1 cells.

Expression and Purification of a Novel Computationally Designed Antigen for Simultaneously Detection of HTLV-1 and HBV Antibodies.

Computational tools are reliable alternatives to laborious work in chimeric protein design. In this study, a chimeric antigen was designed using computational techniques for simultaneous detection of anti-HTLV-I and anti-HBV in infected sera. Databases were searched for amino acid sequences of HBV/HLV-I diagnostic antigens. The immunodominant fragments were selected based on propensity scales. The diagnostic antigen was designed using these fragments. Secondary and tertiary structures were predicted and the B-cell epitopes were mapped on the surface of built model. The synthetic DNA coding antigen was sub-cloned into pGS21a expression vector. SDS-PAGE analysis showed that glutathione fused antigen was highly expressed in E. coli BL21 (DE3) cells. The recombinant antigen was purified by nickel affinity chromatography. ELISA results showed that soluble antigen could specifically react with the HTLV-I and HBV infected sera. This specific antigen could be used as suitable agent for antibody-antigen based screening tests and can help clinicians in order to perform quick and precise screening of the HBV and HTLV-I infections.

The effect of IL-28A on human cord blood CD4+ T cells.

BACKGROUND AND AIM: The utilization of umbilical cord blood transplantation (UCBT) has been increasing because of the potential advantage of rapid accessibility and the lesser risk of graft-versus-host disease (GVHD), thus allowing less strict HLA matching. IL-28A, also known as IFN-lambda2, has been regarded as a member of a new cytokine family that shares some features with type I interferon (IFN) and was shown to have antiviral activity. The aim of this study was to identify biological activity of IL-28 on cord blood CD4(+) T cells. MATERIALS AND METHODS: In this study, we cultured CD4(+) T cells with IL-28A (20 ng/ml), IL-2 (20 ng/ml) and 5microg/ml MACS Anti-Biotin MACSiBead Particles (bead-to-cell ratio 1:2) for 2 weeks. RESULTS: Flow cytometry analyses showed that IL-28A cannot be effective on CD25 and Foxp3 expression on cord blood CD4(+) T cells, and it is not involved in proliferation of these cells. Treg suppression assay also showed that this cytokine cannot induce production of regulatory T cells. CONCLUSION: We showed that IL-28A is not involved in expression of CD25 and Foxp3 markers and proliferation of CD4(+)CD25(-) T cells, and that our findings also suggest that induction of Foxp3 in T cells activated by anti-CD3/anti-CD28 does not result in the regulatory activity in these cells.

Generation of IL-8 and TNF-alpha in platelet concentrates during storage.

BACKGROUND: Platelet transfusion is accompanied by febrile nonhemolytic transfusion reactions. The generation of cytokines (like IL-1 beta, IL-6, IL-8, and TNF-alpha) in platelet concentrates by white cells is suggested to be responsible for febrile nonhemolytic transfusion reactions. The number of white cells in the platelet concentrates is crucial to cytokine generation. METHODS: This study was performed to determine whether WBC reduction in platelet concentrates by prestorage leukodepletion filters or inactivation by gamma radiation reduced the levels of these cytokines during storage for 3 days. Each of the platelet concentrates (n = 54) was prepared from a single random donor by platelet-rich plasma. This was then divided into four groups: 1) unfiltered, nonirradiated random-donner platelet concentrates (n = 13); 2) unfiltered, gamma-irradiated random-donner platelet concentrates (n = 16); 3) filtered, nonirradiated random-donner platelet concentrates (n = 14); and 4) filtered, gamma-irradiated random-donner platelet concentrates (n = 11). Cytokine levels in platelet concentrates supernatants were measured by ELISA kits according to the manufacturer's recommendations. RESULTS: Our results showed that IL-8 was detected in unfiltered, nonirradiated, and gamma-irradiated random-donner platelet concentrates but not in the filtered random-donner platelet concentrates. TNF-alpha was only detected in unfiltered, nonirradiated units. Compared with unfiltered platelet concentrates, prestorage filtration prevented a rise in the IL-8 and TNF-alpha on day 3 of storage. The concentration of IL-1 beta was lower than the minimum concentration value of the kit used for this purpose. IL-6 was detected only in 7 units of all filtered platelet concentrates on day 3. CONCLUSION: These data indicate that gamma irradiation can not prevent generation of IL-8 in platelet concentrates during storage, but prestorage leukoreduction of platelet concentrates can prevent accumulation of IL-6, IL-8, and TNF-alpha during storage.