The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

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.

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction.

CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via ß2-adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by ß2-adrenergic agonists and TNF-α.

Procoagulant Activity of Red Blood Cell-Derived Microvesicles during Red Cell Storage.

BACKGROUND: Red blood cells (RBCs) undergo structural and biochemical alterations during storage which are collectively called RBC storage lesion and cause a decrease in RBC recovery and survival. During storage, erythrocytes release an increasing number of microvesicles (MVs) that have key roles in biological processes. We aimed to investigate the procoagulant activity (PCA) of RBC-derived MVs during storage. METHODS: 20 packed RBCs were stored for up to 42 days. Samples were taken at seven different times and evaluated for the presence of RBC-MVs. MVs were separated, and following filtration flow cytometry was used to characterize RBC-MVs based on the expression of glycophorin A (Gly.A) and annexin V (AnnV) antigens. The coagulant activity of RBC-MVs was tested by clotting time (CT) and PCA assays. Results were compared before and after filtration. RESULTS: Flow cytometry revealed a 17.6-fold increase in RBC-MVs after 6 weeks of storage. Significant correlations were found between AnnV+ MVs and PCA (r = 0.96; p < 0.001), and CT (r = -0.77; p < 0.001) which was associated with increased PCA and shortened CT with RBC aging. Filtration of samples efficiently removed MVs (p < 0.001) and also reduced in vitro PCA of MVs (p < 0.001). CONCLUSION: RBC-MVs are procoagulant (particularly AnnV+ MVs) Reduction of MVs from RBC concentrates may reduce the risk of transfusion-induced thrombotic complications.

Meta-analysis of cytomegalovirus seroprevalence in volunteer blood donors and healthy subjects in Iran from 1992 to 2013.

OBJECTIVES: Human cytomegalovirus (CMV), a double-strand DNA herpesvirus, can be transmitted via blood transfusion which is especially important for immunocompromised recipients and can cause a fatal infection. CMV seroprevalence in Iran was studied on blood donors, healthy subjects, and some patients. Highly variable rates were detected. The purpose of this study was to review CMV seroprevalence in blood donors and apparently healthy individuals, in Iran. MATERIALS AND METHODS: One hundred and fifty-eight electronic and paper-based resources and databases including published articles in internal and external journals, seminars, dissertations, and theses available in the database and different websites were used to be systematically reviewed as a meta-analysis. Less related articles to the issue, papers of specific high risk population, and articles with not enough information, were excluded. Eventually 22 articles that satisfied our selection criteria were systematically reviewed and analyzed. To explore heterogeneity between studies the I square (I(2)) index was used. Data were analyzed using the statistical software package (STATA) 11. RESULTS: The heterogeneity between selected studies was 97% with an I(2) statistic. In this study a random effects model was used for meta-analysis. The prevalence of CMV IgG and CMV IgM antibodies in the country were estimated to be 92% (95% CI: 90-94) and 2.6% (95% CI: 1.7-3.6), respectively. CONCLUSION: Given high rate of CMV seropositivity in Iran, it seems that CMVAbs screening would not be a reasonable and affordable approach to prevent CMV infection via transfusion especially for immune compromised recipients, so alternative strategies should be considered.

Prevalence and trends of transfusion-transmitted infections among blood donors in Tehran, Iran from 2008 to 2013.

BACKGROUND: Evaluation of trends in the rate of transfusion-transmitted infections (TTIs) in blood donors is essential for monitoring blood supply safety and donor screening effectiveness. The aim of this study was to determine the trends and prevalence of hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and syphilis seromarkers among blood donors referred to Tehran Blood Transfusion Center (TBTC) from 2008 to 2013. MATERIALS AND METHODS: The data of all blood donors referred to TBTC between 2008 and 2013 were collected. The prevalence of HBV, HCV, HIV, and syphilis infections were expressed by donation year and donors' characteristics (age, gender, educational level and donor status). RESULTS: Among 1,796,090 individuals who donated blood at TBTC from 2008 to 2013, analysis of trend for the prevalence of HBV showed a significant decrease from 423 to 153 per 10(5) donors. The similar pattern of decrease was observed for the prevalence of HCV from 139 to 69 per 10(5) donors, however the rate of decrease in HCV prevalence was slower than the rate of decrease in HBV prevalence. The prevalence of HIV was constant while the prevalence of syphilis showed a sharp decrease in 2009 and a constant prevalence from 2010 to 2013. The top three parameters influenced the rate of TTIs were donor status, age, and educational level. CONCLUSION: The decreasing prevalence and trends of TTIs among the studied donors demonstrated that the safety measures which were employed in recent years in Iranian Blood Transfusion Organization have been effective.

RBC alloimmunization and double alloantibodies in thalassemic patients.

PURPOSE: Alloimmunization is a common consequence of chronic blood transfusion. Double alloantibody production may complicate the condition of such patients especially for finding matched blood. In this study, we evaluated the frequency of alloantibodies in thalassemic patients with previous history of transfusion reactions. SAMPLES AND METHODS: This study was performed on 441 multiply transfused thalassemia patients Antibody screening test was carried out using three cell-panel by gel method. Positive patients were followed up for antibody identification using 11-cell panel. Direct combs' test was performed to detect auto antibodies. RESULTS: In a total of 441 cases (362 thalassemia major and 79 intermedia), 234 were males (53.1%) and 207 females (46.9%); mean age 22 years, range 3-61 years. Alloimmunization was detected in 50(11.3%) patients, including 37(74%) patients with one alloantibody, 8(16%) with two antibodies, 4(8%) patients with unknown antibodies and one patient (2%) with autoantibody. The most common alloantibodies were anti-Rh antibodies (-E/e/C/c/Cw) (26%), anti-K (28%), anti-D (16%), and anti-Colton (4%). Double antibodies were detected in eight out of 50 patients, including: Anti-D+anti-C (8%), anti-D+anti-E (2%), anti-Kell+anti-D (2%), and anti-Kell+KPa (2%). A significant association was observed between the transfusion reaction history and the alloantibody detection results (p < 0.05). CONCLUSION: Antibody production against RBC antigens makes hard condition in regular blood transfusion. Double antibodies production may more complicate this situation. Thus, it is advisable to phenotype patients and matches the red cells in multiply transfused thalassemia patients.

In vitro platelet quality in storage containers used for pediatric transfusions.

BACKGROUND: The in vitro quality of small-volume platelet (PLT) aliquots for pediatric transfusions was assessed to determine the best practice approach. STUDY DESIGN AND METHODS: Small volumes (50 mL) of single apheresis PLT components (APCs), collected on either CaridianBCT Trima or Haemonetics MCS+ instruments, were aliquoted on Days 2, 3, 4, and 5 postcollection into Fenwal PL1240 or 4R2014 bags or 60-mL polypropylene syringes. Samples were tested for in vitro quality at their recommended expiry times (4 hr for 4R2014 bags and syringes or Day 5 for PL1240 bags). Assays included pH, CD62P expression, and metabolic measures. RESULTS: CD62P expression increased throughout storage in all containers. Among the small-volume containers, pH, pCO(2) , lactate, and bicarbonate varied considerably. Regardless of the day of aliquoting, pCO(2) was significantly higher and pO(2) was significantly lower in gas-impermeable syringes than other containers. No bacterial growth was detected in any sample. CONCLUSION: The quality of APCs aliquoted into small-volume containers meets regulatory requirements and is generally equivalent to that of full-volume APCs at expiry.