Study on genotyping and coinfection rate of human parvovirus 4 among the HTLV-I/II infected blood donors in Khorasan Razavi, Iran.

Human Parvovirus 4 (PARV4) is an emerging virus infecting individuals with other blood-borne diseases. This study aimed to determine the prevalence of PARV4 in confirmed HTLVI/II positive samples from blood donors, assessing PARV4 viral load (DNA) and genotyping. METHODS: A novel qReal-Time PCR, based on a plasmid construct, was developed to simultaneously detect all three PARV4 genotypes using in-house primers and probes. Positive qPCR samples were subjected to nested PCR amplification and subsequent sequencing. Phylogenetic trees were constructed using the Neighbor-joining (N.J.) method. RESULTS: The coinfection rate of PARV4-DNA in HTLVI/II confirmed infected donors, who were previously deferred, was 14.4 % (13 out of 90), with no observed association with donation status (p = 1.0). Phylogenetic analysis indicated that PARV4-positive samples closely resembled genotype 2 in Iran.qPCR quantification demonstrated significant PARV4 viral loads in positive samples, ranging between 104 and 106 DNA copies/mL of serum. CONCLUSION: This study presents the first evaluation of HTLVI/II and PARV4coinfection rates among blood donors. Notably, elevated PARV4-DNA titers were detected in HTLVI/II-positive donors. Given PARV's resistance to standard plasma refinery inactivation methods and the absence of its targeted inactivation, its potential impact remains a concern.

Changes in BMD T-score from pre-to post-treatment with biosimilar teriparatide: A single-arm, multi-center study.

Introduction: Teriparatide is a recombinant analog of the parathyroid hormone and an anabolic treatment modality for osteoporosis. This study aimed to evaluate the effectiveness of biosimilar teriparatide (CinnoPar®, CinnaGen Co., Iran) in osteoporotic patients after at least one year of treatment. Methods: In this multi-center, single-arm study, 239 eligible patients received subcutaneous injections of biosimilar teriparatide 20 µg once daily for at least one year. The main outcome measure was the change in bone mineral density (BMD) T-score from baseline (pre-treatment) to end of the study (post-treatment). In addition, the change in the fracture risk assessment tool (FRAX) score was calculated to estimate the 10-year probability of major and hip fractures pre-and post-treatment. Results: A total of 239 patients (age, 63 ± 12.14 years; female, 88.28 %) were included, of which 27.62 % (66/239), 14.64 % (35/239), and 57.74 % (138/239) received biosimilar teriparatide for 12-16 months, 17-20 months, and 21-24 months, respectively. From baseline to end of the study, the T-score at the lumbar spine increased from -2.67 ± 1.04 to -2.26 ± 1.11 (mean percent change, 13.07 ± 62.89; p-value<0.001). Similarly, the T-score at femoral neck increased from -2.18 ± 0.87 to -2.09 ± 0.93 (mean percent change, 3.81 ± 31.52; p-value = 0.006). The proportions of patients with maintained or improved BMD T-score at the lumbar spine and femoral neck sites were 85.36 % (204/239) and 69.04 % (165/239), respectively. Similar results were obtained in subgroups of patients with rheumatoid arthritis and those with a history of a previous fracture or parental hip fracture. FRAX scores did not change significantly during the study (p-values of 0.551 and 0.973 at the lumbar spine and femoral neck, respectively). Conclusion: We observed considerable improvements in BMD following treatment with the biosimilar teriparatide for one year or more. The biosimilar teriparatide can be considered as an effective treatment option in female and male patients with osteoporosis.

Directed targeting of B-cell maturation antigen-specific CAR T cells by bioinformatic approaches: From in-silico to in-vitro.

AIMS: Chimeric Antigen Receptor (CAR) T-cell is a breakthrough in cancer immunotherapy. The primary step of successful CAR T cell therapy is designing a specific single-chain fragment variable (scFv). This study aims to verify the designed anti-BCMA (B cell maturation antigen) CAR using bioinformatic techniques with the following experimental evaluations. MAIN METHODS: Following the second generation of anti-BCMA CAR designing, the protein structure, function prediction, physicochemical complementarity at the ligand-receptor interface, and biding sites analysis of anti-BCMA CAR construct were confirmed using different modeling and docking server, including Expasy, I-TASSER, HDock, and PyMOL software. To generate CAR T-cells, isolated T cells were transduced. Then, anti-BCMA CAR mRNA and its surface expression were confirmed by real-time -PCR and flow cytometry methods, respectively. To evaluate the surface expression of anti-BCMA CAR, anti-(Fab')2 and anti-CD8 antibodies were employed. Finally, anti-BCMA CAR T cells were co-cultured with BCMA+/- cell lines to assess the expression of CD69 and CD107a as activation and cytotoxicity markers. KEY FINDINGS: In-silico results approved the suitable protein folding, perfect orientation, and correct locating of functional domains at the receptor-ligand binding site. The in-vitro results confirmed high expression of scFv (89 ± 1.15% (and CD8α (54 ± 2.88%). The expression of CD69 (91.97 ± 1.7%) and CD107a (92.05 ± 1.29%) were significantly increased, indicating appropriate activation and cytotoxicity. SIGNIFICANCE: In-silico studies before experimental assessments are crucial for state-of-art CAR designing. Highly activation and cytotoxicity of anti-BCMA CAR T-cell revealed that our CAR construct methodology would be applicable to define the road map of CAR T cell therapy.

The upregulation of Gata transcription factors family and FOG-1 in expanded and differentiated cord blood-derived CD34+ hematopoietic stem cells to megakaryocyte lineage during co-culture with cord blood mesenchymal stem cells.

BACKGROUND: Umbilical cord blood (UCB) has improved into an attractive and alternative source of allogeneic hematopoietic stem cells (all-HSCs) in clinics and, research for three decades. Recently, it has been shown that the limited cell dose of, this valuable source can be enhanced by the ex vivo expansion of cells in many, ways. We evaluated the expression of the Gata transcription factors family and FOG-1, in expanded and differentiated cord blood-derived CD34 + hematopoietic stem cells to, megakaryocytes lineage., Methods: Separated mononuclear cells were cultured in DMEM complete medium., Harvested cells as a mesenchymal stem cell at 85 % confluency were cultured with, trypsin/EDTA and in 24-well plates. The characteristic analyses of isolated UCB- MSCs, were done by flow cytometry and adipogenic, chondrogenic, and osteogenic, differentiation assays. MACS purified UCB-CD34 + hematopoietic cells cultivated and, differentiated to megakaryocyte progenitor cells in the presence of cytokine cocktail, with UCB-MSCs. Then, the GATA1, GATA2, GATA3, and FOG-1 genes expression, after differentiation to megakaryocyte progenitor cells were performed by quantitative, real-time polymerase chain reaction (PCR)., Results: In this study, the results of real-time-PCR showed that the fold change, expression of GATA-1, FOG-1, and GATA-2 genes after co-culturing with UCB-MSCs, significantly increased to 7.3, 4.7, and 3.3-fold in comparison with control groups;respectively., Conclusion: UCB-MSCs can increase the expansion and differentiation of UCBCD34 + , to megakaryocyte progenitor cells through upregulation of GATA-1, GATA-2, and FOG-1 gene expression.

Investigation of human parvovirus B19 prevalence in a large healthy umbilical cord blood donors.

Background and Objectives: Umbilical cord blood (UCB) was used to source hematopoietic stem cells in the past. Despite the apparent advantages of UCB transplantation, virus reactivation poses a considerable danger in allogeneic hematopoietic stem cell transplantation (HSCT). Human Parvovirus B19 is regarded as a potential threat to UCB contamination. This study aimed to evaluate the prevalence of parvovirus B19 in cord blood donors by Semi-Nested PCR. This study is the first largescale report of the B19 DNA in cord blood donors in Iran. Materials and Methods: A total of 691 umbilical cord blood were collected under standard procedure. Then, DNA from buffy coat and plasma were extracted, and semi-nested PCR was performed for all samples. Results: Two out of 691 samples (0.29%) indicated viremia in plasma and buffy coat. Conclusion: In this line, designing and validating a quantitative PCR assay for detection, quantification, and discrimination of Human B19 DNA genotypes of cord blood donors is necessary to enhance the safety of this source of stem cells.

Integrating landmark modeling framework and machine learning algorithms for dynamic prediction of tuberculosis treatment outcomes.

OBJECTIVE: This study aims to establish an informative dynamic prediction model of treatment outcomes using follow-up records of tuberculosis (TB) patients, which can timely detect cases when the current treatment plan may not be effective. MATERIALS AND METHODS: We used 122 267 follow-up records from 17 958 new cases of pulmonary TB in the Republic of Moldova. A dynamic prediction framework integrating landmark modeling and machine learning algorithms was designed to predict patient outcomes during the course of treatment. Sensitivity and positive predictive value (PPV) were calculated to evaluate performance of the model at critical time points. New measures were defined to determine when follow-up laboratory tests should be conducted to obtain most informative results. RESULTS: The random-forest algorithm performed better than support vector machine and penalized multinomial logistic regression models for predicting TB treatment outcomes. For all 3 outcome classes (ie, cured, not cured, and died after 24 months following treatment initiation), sensitivity and PPV of prediction models improved as more follow-up information was collected. Specifically, sensitivity and PPV increased from 0.55 to 0.84 and from 0.32 to 0.88, respectively, for the not cured class. CONCLUSION: The dynamic prediction framework utilizes longitudinal laboratory test results to predict patient outcomes at various landmarks. Sputum culture and smear results are among the important variables for prediction; however, the most recent sputum result is not always the most informative one. This framework can potentially facilitate a more effective treatment monitoring program and provide insights for policymakers toward improved guidelines on follow-up tests.

Human cord blood-derived viral pathogens as the potential threats to the hematopoietic stem cell transplantation safety: A mini review.

Umbilical cord blood (UCB) is a valuable source of hematopoietic stem cells (HSCs) and potential alternative for bone marrow transplantation for patients who lack human leukocyte antigen (HLA)-matched donors. The main practical advantages of UCB over other HSC sources are the immediate availability, lower incidence of graft-versus-host disease, minimal risk to the donor, and lower requirement for HLA compatibility. However, the use of UCB is limited by delayed engraftment and poor immune reconstitution, leading to a high rate of infection-related mortality. Therefore, severe infectious complications, especially due to viral pathogens remain the leading cause of morbidity and mortality during the post-UCB transplantation (UCBT) period. In this context, careful screening and excluding the viral-contaminated UCB units might be an effective policy to reduce the rate of UCBT-related infection and mortality. Taken together, complete prevention of the transmission of donor-derived viral pathogens in stem cell transplantation is not possible. However, having the knowledge of the transmission route and prevalence of viruses will improve the safety of transplantation. To the best of our knowledge, there are few studies that focused on the risk of virus transmission through the UCB transplant compared to other HSC sources. This review summarizes the general aspects concerning the prevalence, characteristics, and risk factors of viral infections with a focus on the impact of viral pathogens on cord blood transplantation safety.

The potential therapeutic effect of melatonin on human ovarian cancer by inhibition of invasion and migration of cancer stem cells.

There is an urgent need to identify targeting molecules to control invasion and metastasis in cancer patients. We first isolated cancer stem cells (CSCs) from SKOV3 ovarian cancer cells and then investigated the role of melatonin in invasiveness and migration of CSCs compared to SKOV3 cells. The proportion of CSCs in SKOV3 cells was as low as 1.28% with overexpression of both CD133 and CD44. The ability of spheroid formation along with SOX2 overexpression revealed a high self-renewal potential in isolated cells. Melatonin (3.4 mM) inhibited proliferation of CSCs by 23% which was confirmed by a marked decrease in protein expression of Ki67, as a proliferation marker. Applying luzindole, a melatonin receptor 1, 2 inhibitor, partially abolished anti-proliferative effect of melatonin. Melatonin also decreased Epithelial mesenchymal transition (EMT) related gene expressions including ZEB1, ZEB2, snail and vimentin with increase in E-cadherin as a negative EMT regulator. Incubation of CSCs with melatonin showed a marked decrease in matrix metalloproteinase 9 (MMP9) expression and activity. Melatonin also inhibited CSCs migration in a partially receptor dependent and PI3k and MAPK independent manner. Melatonin can be considered as an important adjuvant to control invasion and metastasis especially in patients with high melatonin receptor expression.

The effect of hypoxia preconditioning on the neural and stemness genes expression profiling in human umbilical cord blood mesenchymal stem cells.

In recent years, human umbilical cord blood-derived mesenchymal stem cell (hUB-MSCs) has been regarded as an alternative source for stem cell therapy. In this study, we evaluated the effect of hypoxia preconditioning (HPC) on the expression of Nt-3, GFAP, Nestin, Oct-4 and Nanog genes and proliferative capacity of hUB-MSCs in comparison with normoxic conditions. HPC+Hypoxia protocol includes cultured hUB-MSCs for 15min at 2.5% O2 and after that reoxygenation for 30min at 21% O2 (HPC), and then hypoxia preconditioned hUB-MSCs subjected to 2.5% O2 for 72h (Hypoxia). Conclusively, the results showed that hypoxic preconditioning is an effective strategy for enhancing proliferation capacity of hUB-MSCs, and also can trigger expression of some of the neural genes. In addition, the concept of involvement of oxygen tension in the expression of some of the neural genes of hUB-MSCs would be a good sign of enhanced neural differentiation potential in vitro.

Kynurenic acid downregulates IL-17/1L-23 axis in vitro.

Exploring the function of interleukin (IL) 17 and related cytokine interactions have been proven useful toward understanding the role of inflammation in autoimmune diseases. Production of the inflammatory cytokine IL-23 by dendritic cells (DC's) has been shown to promote IL-17 expression by Th17 cells. It is well established that Th17 cells play an important role in several autoimmune diseases including psoriasis and alopecia. Our recent investigations have suggested that Kynurenine-rich environment can shift a pro-inflammatory response to an anti-inflammatory response, as is the case in the presence of the enzyme Indoleamine 2,3 dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation and Kynurenine (Kyn) production. In this study, we sought to explore the potential role of kynurenic acid (KynA), in modulating the expression of IL-23 and IL-17 by DCs and CD4+ cells, respectively. The result of flow cytometry demonstrated that the frequency of IL-23-producing DCs is reduced with 100 µg/ml of KynA as compared with that of LPS-stimulated DCs. KynA (100 µg/ml) addition to activated T cells significantly decreased the level of IL-17 mRNA and frequency of IL-17+ T cells as compared to that of concanavalin (Con) A-activated T cells. To examine the mechanism of the suppressive role of KynA on IL-23/IL-17 in these cells, cells were treated with 3 µM G-protein-coupled receptor35 (GPCR35) inhibitor (CID), for 60 min. The result showed that the reduction of both adenylate cyclase (AC) and cyclic adenosine monophosphate (cAMP) by KynA is involved in suppression of LPS-induced IL-23p19 expression. Since GPCR35 is also detected on T cells; therefore, it is concluded that KynA plays an important role in modulating the expression of IL-23 and IL-17 in DCs and Th17 cells through inhibiting GPCR35 and downregulation of both AC and cAMP.

Microvesicles preparation from mesenchymal stem cells.

BACKGROUND: Extracellular vesicles are particles ranged from 30 nm to 5µm and subcategorized into three groups; exosomes, microvesicles and apoptotic bodies, each of which have different biological impact. Lack of a standard method for the detection and isolation of MVs has led to a challenging issue that is a worth considering. In this study, we isolated MVs from the conditioned medium of UC-MSCs by four different schemes of ultracentrifugation. METHODS: We examined the efficacy of differential centrifugation ranging from 10,000×g to 60,000×g on UCMSCs- derived microvesicles yield and purity. The fractions were evaluated by Dynamic Light Scattering (DLS) method, total protein quantification and flow cytometry. RESULTS: UC-MSCs were spindle cells that adhered to plastic culture flasks. These cells expressed MSC markers such as CD44 and CD73, whereas were negative for hematopoietic markers CD45 and CD34. UC-MSCparticles were successfully isolated. Particles were heterogeneous vesicles of approximately 50 to 1250 nm in diameter that bear the surface-expressed molecules UC-MSCs such as; CD90, CD106, CD166 and CD44, and negative for CD34, CD63, and CD9. According to the results of DLS method, centrifugation at 10,000, 20,000, 40,000 and 60,000 ×g, all gave MVs of less than 1000 nm. It is of notion that only at the centrifugation rates of 40,000 and 60,000×g, particles of less than 100 nm in diameter were also obtained. CONCLUSION: The choice of exact speed greatly influences the purity of MVs and their yield. Our findings indicate that centrifugation at 20,000×g is appropriate for the purification of UC-MSC-MVs.

The effect of methylprednisolone treatment on fibrinolysis, thecoagulation system, and blood loss in cardiac surgery.

BACKGROUND/AIM: The purpose of this study was to examine steroid pretreatment in order to decrease postoperative coagulopathy disorders and bleeding. MATERIALS AND METHODS: In this randomized double-blinded study, the efficacy of low versus high doses of methylprednisolone on the coagulation system and postoperative bleeding was compared in patients who were undergoing cardiac surgery with cardiopulmonary bypass (CPB). The platelet response to agonists, D-dimer concentration, tissue plasminogen activator (tPA), plasminogen activator inhibitor (PAI-1) antigens, and platelet receptors CD42b, CD62P, and CD41a were evaluated. RESULTS: The platelet response to agonists was reduced. The mean concentrations of D-dimer and tPA antigen increased although PAI-1 concentration did not show any significant changes following heparin neutralization. Postoperative expression of CD42b showed no changes in comparison with preoperation values in both groups. There was a significant increase in the expression of CD62P with a methylprednisolone dose of 15 mg/kg, while there was just a slight increase with a dose of 5 mg/kg. CD41a, as a fibrinogen receptor, was increased significantly after CPB in both groups. Significant data were shown in decreasing blood loss with a high dose of methylprednisolone. CONCLUSION: Methylprednisolone at a dose of 15 mg/kg reduced bleeding, probably by increasing CD62P after heparin neutralization, which can activate platelet activation in favor of better hemostasis.

Quantification of viral genome in cord blood donors by real time PCR to investigate human herpesvirus type 8 active infection.

Umbilical cord blood (UCB) is one of the most important sources of hematopoietic stem cells which can be used for transplantation. The transplanted CB stem cells might cause infections in recipients. The aim of this study is to evaluate Human Herpes Virus8 (HHV8) as a Rhadinovirus among CB samples in order to assess safety of cord blood stem cells transplantation. To assess this aim, we surveyed 800 cord blood specimens by Real Time PCR.The overall HHV8 incidence in cord blood mononuclear cells was 1.38% and none of them was in lytic phase of HHV8. The authors suggest further HHV8 study on CB samples for transplantation.

Improving the neuronal differentiation efficiency of umbilical cord blood-derived mesenchymal stem cells cultivated under appropriate conditions.

OBJECTIVES: Umbilical cord blood-derived mesenchymal stromal cells (UCB-MSCs) are ideally suited for use in various cell-based therapies. We investigated a novel induction protocol (NIP) to improve the neuronal differentiation of human UCB-MSCs under appropriate conditions. MATERIALS AND METHODS: This experimental study was performed in Iranian Blood Transfusion Organization (IBTO), Tehran, Iran. UCB-MSCs were cultured in DMEM medium supplemented with 10% FBS in a humidified incubator in equilibration with 5% CO2 at 37°C. For neuronal differentiation of UCB-MSCs, DMEM was removed and replaced with pre-induction medium containing RA, bFGF, EGF, and basal medium for two days. Then, NGF, IBMX, AsA, and Neurobasal medium were used for six days for this purpose. Real-time PCR was performed to analyze the neuronal differentiation of UCB-MSCs for the first time in Iran. RESULTS: We found that the maximum and minimum levels of gene expression were related to GFAP and nestin, respectively. In addition, our study showed that compared to other neuronal inducers, RA might play the main role in neuronal differentiation and fate of MSCs compared to other neuronal inducers. CONCLUSION: Our data showed that the combination of chemical (RA, IBMX, AsA) and growth factors (NGF, EGF, bFGF) in NIP may improve the efficiency of neuronal differentiation of UCB-MSCs and may provide a new method for easy and quick application of UCB-MSCs in regenerative medicine in the future. However, the functionality of neuron-like cells must be carefully assessed in animal experiments prior to use in clinical applications.

Detection of interleukin-19 mRNA in C57BL/6 mice astroglial cells and brain cortex.

INTRODUCTION: Astrocytes are the most abundant glial cell type. In addition to their neurological roles, astrocytes also have immune functions. They have been involved in antigen presentation in the central nervous system (CNS). Activated astrocytes express adhesion molecules, chemokines and release several inflammatory mediators, pro-inflammatory cytokines, neurotrophic and neuroprotective factors, thus these cells have a dual role within the CNS: neuroinflammation and repair processes. IL-19, IL-20, IL-22, IL-24, IL-26, IL-28A, IL-28B, and IL-29 are members of the IL-10 family of cytokines. These cytokines have different biological functions in spite of partial amino acid sequences homology. Signal transduction of the IL-10 family of cytokines is through R1-type and R2-type receptors. METHODS: No information has been available about the expression and regulation of IL-19 in mice astrocytes and brain. To investigate the expression of IL-19, we examined its expression in C57BL/6 mice astroglial cells in response to lipopolysaccharide (LPS), using reverse-transcription polymerase chain reaction (RT-PCR) method. RESULTS: We provide for the first time, evidence that astrocytes can express IL-19 mRNA following LPS stimulation. Furthermore, we have found the expression of IL-19 mRNA in the cortex of adult C57BL/6 mice following intraperitoneal (i.p.) administration of LPS. DISCUSSION: This finding will contribute to current knowledge on the function and behavior of cells and mediators during inflammatory conditions in the brain.

Detection of IL-20R1 and IL-20R2 mRNA in C57BL/6 mice astroglial cells and brain cortex following LPS stimulation.

BACKGROUND: Astrocytes, which comprise ~90% of overall brain mass, are involved in brain immunity. These cells represent the non-professional class of CNS-resident APCs and may promote or inhibit CNS inflammation depending on the cytokines they secrete. IL-10 family of cytokines and their receptors, IL-20R1 and IL-20R2, may have a role in shifting astrocytes to a neuroprotective or neurodegenerative function. OBJECTIVE: To address the expression of IL-20R1 and IL-20R2 cytokine receptors in astrocytes and brain cortex of C57BL/6 mice. METHODS: We investigated the expression of IL-20R1 and IL-20R2 in C57BL/6 mice astroglial cells and brain cortex in response to lipopolysaccharide (LPS), using reverse-transcription polymerase chain reaction (RT-PCR) method. RESULTS: Astrocytes were able to express IL-20R1 and IL-20R2 mRNA not only in response to LPS stimulation but also in the absence of LPS. Furthermore, we found the expression of IL-20R1 and IL-20R2 mRNA in the cortex of adult C57BL/6 mice. CONCLUSIONS: IL-20R1 and IL-20R2 are constitutively express in the brain. Since most neuropathological processes involve astrocytes and inflammatory cytokines, these findings have important implications for future therapeutic strategies.

Strong immune responses induced by a DNA vaccine containing HPV16 truncated E7 C-terminal linked to HSP70 gene.

BACKGROUND: Vaccines capable of controlling tumor virus based infections are found difficult to develop due to the consistence latent infection in the host. DNA vaccines are attractive tools for the development of HPV vaccines and inducing antigen-specific immunity owing to the stability, simplicity of delivery, safety and cost effectiveness. However, there is a need to increase their potency by procedures such as using HSP70 gene as an adjuvant. OBJECTIVE: To evaluate a DNA vaccine containing HPV16 truncated E7 C-terminal cytotoxic T-lymphocyte epitopes linked to HSP70 gene (HSP70-tE7) in an animal model. METHODS: Mice were immunized with the plasmid DNA after pre-treatment with cardiotoxin. The splenocytes of immunized mice were then tested for CTL activity by detecting the apoptosis and necrosis in target cells, cytokine production by ELISA, CD4 and CD8 frequencies by flow cytometry, and lymphocyte stimulation by MTT assay. RESULTS: The recombinant expression vector was able to elicit immune responses close to that of full length E7 complete gene. Although the use of a small part of a target antigen can induce immune responses equivalent to the full length antigen, it fails to elicit statistically significant stronger immune responses when fused with HSP70 compared to the complete E7 gene alone. CONCLUSION: The potent immunogenicity of HPV16 E7 was preserved in the HSP70-tE7 vaccine and may represent a target of choice for the therapeutic vaccination strategies. However, to improve the immunogenicity polytope DNA vaccines which elicit multiple effector and memory CTL responses should be considered in future studies of DNA-based cancer vaccines.

Effects of CXCR1 and CXCR2 inhibition on expansion and differentiation of umbilical cord blood CD133(+) cells into megakaryocyte progenitor cells.

OBJECTIVE: There have been various reports on the roles of CXC receptors (CXCR) in modulation of hematopoiesis. In the present study, we investigated the effects of CXCR1 and/or CXCR2 inhibition on expansion and differentiation of umbilical cord blood (UCB) CD133(+) cells into megakaryocytic progenitors. MATERIALS AND METHODS: Purified UCB CD133(+) cells were cultured in a serum-free liquid culture either in the presence or absence of neutralizing anti-CXCR1 and/or anti-CXCR2 antibodies in combination with a conventional cytokine cocktail for up to 14days. Expression of megakaryocytic lineage markers (CD41 and CD61) and determination of ploidy level were determined by flowcytometry. In addition, colony-forming unit assay was performed using CD133(+) cultures in serum-free collagen-based medium containing the cytokine cocktail plus neutralizing CXCR1 and -R2 antibodies. Colony forming unit-megakaryocyte (CFU-MKs) and non-MKs were counted after immunocytochemistry staining on day 12. RESULTS: We show that while simultaneous inhibition of both CXCR1 and -R2 causes a significant reduction in the fold expansion of UCB CD133(+) cells, it also leads to an increase in percentages of CD61(+), CD41(+), and CFU-MK populations. CONCLUSION: CXCR1 and CXCR2 play significant roles in the suppression of megakaryopoiesis. We demonstrate that blocking of this suppressive effect by a simultaneous inhibition of both receptors can enhance the differentiation of UCB CD133(+) cells into megakayocytic progenitors.

Platelet growth factors suppress ex vivo expansion and enhance differentiation of umbilical cord blood CD133+ stem cells to megakaryocyte progenitor cells.

BACKGROUND AND OBJECTIVES: Umbilical cord blood (UCB) is a rich source of hematopoietic cells. Here, for the first time, we surveyed the effects of different concentrations of platelet growth factors and cytokine cocktail (CC) on the expansion and differentiation of UCB CD133(+) stem cells into megakaryocyte progenitors. MATERIALS AND METHODS: UCB CD133(+) cells were separated by magnetic cell sorting and cultured in different concentrations of platelet growth factors in combination with a CC containing interleukins 3 and 6, stem cell factor, and thrombopoietin. Cell expansion and differentiation were assessed using mononuclear cell count and flow cytometry. RESULTS: The results show that either activated platelet-rich plasma or the platelet supernatant, when added in the first day of culture, significantly suppress the expansion of CD133(+) cells after 7 days in culture (p < 0.05). By contrast, the expression of CD41, CD61, and CD42b markers in the presence of all platelet growth factors increased compared with that of the control (p < 0.05). CONCLUSION: Taken together, platelet growth factors in the presence of CC suppress ex vivo expansion of UCB CD133(+) cells and enhance their differentiation into megakaryocytic progenitor cells in a dose- and time-dependent manner.

Cytotoxicity of human cord blood natural killer cells is enhanced by recombinant interleukin-15.

Hematopoietic cord blood (CB) stem cell transplantation has more advantages to other cell sources because of lower Graft Versus Host Disease (GVHD). Interleukin-15(IL-15) is an immunoregulatory cytokine, known to enhance cytolytic function of cord Natural Killer (NK) cells. The aim of this study was to investigate the effect of IL-15 on NK cytotoxicity simultaneously in different cell death stages. We compared the ability of IL-15 to enhance the NK cytotoxicity of CB in comparison to adult blood Mononuclear Cells (MNCs) against K562 target cells by co-staining with AnnexinV-FITC and Propidium Iodide after 3.5 h incubation at 37 °C and 5% CO2 by using flow cytometric method. We also evaluated phenotypic changes after treatment by IL-15 in both cell sources. Our results indicated that CB samples had lower level of apoptosis, while necrosis was negligible; also by escalating Effector: Target (E: T), we got higher level of apoptosis and necrosis in peripheral blood (PB). NK activity of cord and adult MNCs was enhanced by incubation with IL-15 (10 ng/ml) for 72 h with significantly higher results of PB in comparison to CB (p<0.0001). Moreover, IL-15 increased the percentage of CD3-/CD56+ and CD25+ cells after 72 h incubation. Results showed incubation with human recombinant (hr) IL-15 for 3 days increased NK activity. Taken together, these results indicated that NK cytotoxicity of CB MNCs could be augmented by human recombinant (hr) IL-15, but this activity did not reach to same level of PB counterparts. We established that CD25 expression on CB MNCs could be increased with IL-15, in 72-hour cultures, but to a lesser degree compared to that on corresponding adult PB MNCs.