effect of Mir-451 upregulation on erythroid lineage differentiation of murine embryonic stem cells

Objective: MicroRNAs [miRNAs] are small endogenous non-coding regulatory RNAs that control mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role in hematopoietic lineage differentiation. Recent studies have shown that specific miRNAs such as Mir-451 have key roles in erythropoiesis

Materials and Methods: In this experimental study, murine embryonic stem cells [mESCs] were infected with lentiviruses containing pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors [Gata-1, Klf-1, Epor] and hemoglobin chains [alpha, beta, gamma, epsilon and zeta] genes using quantitative reverse transcriptase-polymerase chain reaction [qRT-PCR] and presence of erythroid surface antigens [TER-119 and CD235a] using flow cytometery. Colony-forming unit [CFU] assay was also on days 14thand 21thafter transduction

Results: Mature Mir-451 expression level increased by 3.434-fold relative to the untreated mESCs on day 4 after transduction [P<0.001]. Mir-451 up-regulation correlated with the induction of transcriptional factor [Gata-1, Klf-1, Epor] and hemoglobin chain [alpha, beta, gamma, epsilon and zeta] genes in mESCs [P<0.001] and also showed a strong correlation with presence of CD235a and Ter- 119 markers in these cells [13.084and 13.327-fold increse, respectively] [P<0.05]. Moreover, mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid [CFU-E] colonies [5.2-fold] compared with untreated control group [P<0.05]

Conclusion: Our results showed that Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produce artificial red blood cells [RBCs] without the presence of any stimulatory cytokines

[Chitosan nanopolymers effect in activating of mouse bone marrow derived dendritic cells]

Background and Aim: Regarding to various problems in the activation of dendritic cells and immune system's responses, finding of a safe, effective and applicable agent is highly desirable. Chitosan is an effective gene delivery agent and also a part of nanoscaffolds. In the present study, chitosan nanopolymers effect on dendritic immune cells were assessed

Materials and Methods: In this experimental-laboratory study, chitosan [150 KD] in acetic acid 1% solution was depolymerized to 10 KD oligomers using NaNO2. The oligomers particles were obtained by means of 2 normal NAOH molecules. Denderitic cells were derived from the rats' bone marrow using GM-SCF. On the treated denderitic cells CD40, CD86 and MHC-II maturation markers were evaluated by flowcytometery and TNF-alpha release was evaluated using ELISA method and T cell proliferation

Results: It was observed that dendrtic cells purity on the 8th day was more than 90%. Flowcytometery analysis showed an increase in all evaluated CD40, CD86 and MHC-II maturation markers [p<0.05]. TNF-alpha release and T cell proliferation significantly increased by chitosan treated denderitic cells compared to unstimulated or lipofectamin treated cells [P<0.05]

Conclusion: Results showed that chitosan nanopolymers significantly increased dendertic cell maturation phenotype, proinflamatory cytokine production, and induction of T cell proliferation. Therefore, chitosan nanocomplexes and scaffolds can induce and accelerate immune responses

Long acting propranolol and HSP-70 rich tumor lysate reduce tumor growth and enhance immune response against fibrosarcoma in Balb/c mice

Noradrenaline [NA], the principal neurotransmitter released from sympathetic nerve terminals, influences T-cell maturation, not only directly in developing T cells, but also indirectly, by acting on the thymic nonlymphoid cells. In vitro and in vivo studies have demonstrated the anti-proliferative, anti-migratory, antiangiogenic and cytotoxic properties of propranolol, beta-AR blocker, against various cancers. To evaluate the effect of propranolol on efficacy of HSP-70 rich lysate vaccine in immunotherapy of fibrosarcoma. Mouse fibrosarcoma WEHI-164 cells were used to immunize tumor-bearing mice with or without propranolol and HSP-70. Splenocytes proliferation, cytotoxic activity of the splenocytes, naturally occurring CD4+ CD25[high] T-reg cells and IFN-gamma and IL-4 secretion as well as tumor size, were assessed to describe the anti-tumor immune response. A significant increase in the level of IFN- gamma in the mice vaccinated with WEHI-164 cells enriched with HSP-70 and co-treated with propranolol was observed compared to controls. However, HSP enrichment or propranolol treatment alone did not enhance the immune response as measured by the level of IFN-gamma. Likewise, a decrease in tumor growth in the test group [p<0.01] and a significant increase in CTL activity [p<0.05] was observed. HSP enriched vaccine shows anti-tumor activity, probably due to the modulation of immune responses

Polymorphisms within exon 9, but not intron 8, of the vitamin d receptor gene are associated with Asthma

Deregulation of the immune system through allied factors and cytokine responses are thought to be important contributors to the pathogenesis of asthma. Vitamin D3 and its nuclear receptor appear to be factors that maybe involved in regulating i mmune responses during the progression of asthma. The aim of this study was to investigate the association between polymorphisms in intron 8 and exon 9 of the vitamin D receptor [VDR] and this disease. This study was performed on 100 asthmatic patients and 100 healthy controls. PCR-RFLP was performed to examine polymorphisms in intron 8 and exon 9 of VDR gene. Our results showed a statistically significant difference in the Taq-1 evaluated genotypes of exon 9 of the VDR gene when comparing healthy patients to asthmatic patients. Based on our results, it can be concluded that VDR and its functional polymorphisms may play an important role in the pathogenesis of asthma

miR-155 down Regulation by LNA inhibitor can reduce cell growth and proliferation in PC12 cell line

MicroRNAs [miRNAs] are a class of small non coding regulatory RNAs that have key functions in multiple cell processes. Deregulation of these tiny miRNAs are involved in various human diseases. MiR-155 is one of the multifunctional miRNA that its over-expression has been found to be associated with different kinds of cancer such as leukemia, breast and colon cancers. It is thought that deregulation and over-expression of this microRNA may be associated with PC12 cell proliferation. So, the aim of this study was to investigate the role of miR-155 expression on PC12 cell growth. For this reason, PC12 cells were cultured and transfected by 3 different concentration [25, 50 and 75 nmol] of either LNA anti-miR-155 or scramble antisense in 24-well plate. Then, total RNA was extracted from transfected cells. miRNA cDNAs were synthesized from isolated total RNA. In the second step, miR-155 expression level was analyzed using the quantitative real-time polymerase chain reaction [QRT-PCR]. MTT test was performed to evaluate cell viability. In the next step, apoptosis assay was assessed to investigate anti miR-155 effect on PC12 cells death. Obtained results were analyzed with t-test. MTT test revealed that cell viability of transfected cells with 75 nM of anti-miR- 155 to be reduced by half of the control and scramble groups [0.5 vs. 0.97 and 0.94]. Our data suggest that miR-155 over-expression is associated with PC12 cell growth. So, miR-155 down regulation by anti-miR-155 could open up new ways to restrain brain tumor growth, as anti-miR-155 causes PC12 cells to repress

Characterization of immune responses induced by combined clade-A HIV-1 recombinant adenovectors in mice

Numerous evidences indicate that in some HIV-1 positive patients, the humoral and cellular immune responses are induced against HIV-1 proteins and this is inversely related to the progress of infection. The aim of this study was the evaluation of the Adenovectors containing HIV genes in induction of immune responses in mice. The HIV-1 genes including gag p24, rev, nef and exon-1 of tat were amplified from HIV-1 RNA [clade-A]. The cDNA of each gene was cloned into a transfer vector. The transfer vector was then co-transformed into E. coli strain BJ5183 together with pAdenovector ?E1/E3. The recombinant adenoviral construct was transfected into QBI-293A cells. Recombinant viruses were purified and titrated on 293 cell plates. Expression of transgenes was evaluated using western blotting. Then 1012 viral particles were injected into 15 groups of 5 mice and all patterns of combination of these 4 HIV-1 genes were evaluated. After 2 weeks, humoral and cellular immune responses were evaluated using ELISA, cell proliferation and ELISpot [IL-2, IL-4 and IFN- gamma] assays, consecutively. It was demonstrated that each gene was expressed. The response targets were mostly toward Th1, though several Th2 responses were also observed. Single injection in our study induced a good cellular response but the humoral responses were not as strong as the cellular ones. Considering and comparing all results and evaluating the various possible interactions revealed that simultaneous injection of tat and gag has enhanced the humoral and cellular responses

[ effect of blood storage on apoptosis induction of Jurkat cells: an in vitro model]

It is a firm belief that blood transfusion is life-saving in many situations, but at the same time transfusion complications could be life-threatening. The possible effects of blood Transfusion Related Immunomodulatory [TRIM] and its related mechanisms is one of the important debatable subjects in the field of blood transfusion medicine. One of the mechanisms through which transfusion can induced TRIM effects in recipient is apoptosis induction. Aim of this study was to investigate the apoptotic effects of stored blood in an in vitro model. To evaluate the apoptotic effects of blood storage, we studied the effect of the plasma [from whole blood] during storage on days 3, 10, 21 and 35 on Jurkat cells, which are sensitive to apoptosis .The plasma of whole blood was separated by centrifugation on different days. Then, Jurkat cells were cultured with plasma for 24 hours. Finally apoptosis level was studied by using flowcytometry for analyzing Annexin V on Jurkat cells [by SeroTec Annexin V:FITC Assay Kit]. The percentages of apoptosis on days 3, 10, 21 and 35 were 3.85 +/- 1.52, 5.27 +/- 2.12, 8.44 +/- 1.90, 12.01 +/- 2.32, respectively. The percentages of apoptotic cells in negative and positive control group was 3.85 +/- 1.94 and 65.80 +/- 2.28, respectively. The results of this study showed that plasma of whole blood have the apoptotic effects which enhanced during the storage of plasma. This in vitro model is also suitable for studying other TRIM related mechanisms

Tolerance induction by CD40 silenced dendritic cells through antisense

One of the valuable tools for inhibiting the specific gene expression is antisense technique. To determine T cell responses, co-stimulatory molecule expression on the antigen presenting cells is important. In the present study, the effects of high affinity antisense against CD40 mRNA on the function and phenotype of DCs [dendritic cells] were investigated. The DCs were separated from the mice spleens and then cultured in vitro. By means of lipofectamine 2000, the antisense was delivered into the cells and the efficacy of transfection was estimated by flow cytometry. Also, the mRNA expression and protein synthesis were assessed by real time PCR and flow cytometry, respectively. The DCs were transfected with 6 M antisense and 2 l lipofectamine 2000. The percentage of CD40 expression in DCs was 38%. The results showed that CD40 expression is reduced in DCs to 22% and 24%. By annexine V and propidium iodine staining, we could evaluate the viability of the transfected cells. The inhibition of CD40 gene expression was associated with the increase in IL-4 secretion. This shifted the DCs to stimulate Th2 cytokine production from the allogenic T cells. In addition, in the MLR, the DCs without CD40 expression showed poor allostimulatory effects. This finding is valuable in the study of the costimulatory molecules of DCs. These data demonstrate that direct interference of the cell surface expression of CD40 at transcriptional level by antisense confers tolerogenecity potential of DCs. This approach is a useful tool through which DCs become tolerogenic and can be studied as a potential therapeutic option for the autoimmune diseases and allograft rejection

Soluble form of fasl [sFasL] in adult asthma

sFasL is the soluble form of FasL inducing apoptosis by binding to Fas. Fas/sFasL could be the most important mechanisms in inflammatory conditions such as asthma by controlling inflammatory responses. This study was undertaken to determine the level of sFasL in allergic and non- allergic asthmatic patients with different stages of asthma control. Twenty asthmatic patients were enrolled and divided into controlled and uncontrolled patient groups. They were divided into 4 subgroups including controlled/allergic, controlled/non-allergic, uncontrolled/allergic and uncontrolled/non-allergic subgroups. Five normal subjects were selected as a control group. From all subjects, 3 ml of blood was obtained and sFasL and IgE serum levels were evaluated by a specific ELISA kit. sFasL in the controlled and uncontrolled patient groups did not have any significant difference; but in the uncontrolled/allergic subgroup, it was significantly lower than that in the control group and also higher in the uncontrolled/non-allergic subgroup insignificantly. In patients with acute inflammatory conditions, sFasL had an increasing effect to control inflammation observed in uncontrolled/non-allergic subgroup, but unexpectedly not in the uncontrolled/allergic subgroup. Probably in allergic patients, there are factors or mechanisms that inhibit sFasL production or expression

Evaluation of relation between IL-4 and IFN-gamma polymorphisms and type 2 diabetes

Although, type 2 diabetes is the most frequent type of diabetes, its main cause is yet to be clarified. Several environmental and genetic parameters are believed to be involved in diabetes. It has also been established that cytokines play key roles in pathogenesis of diabetes. Expression of cytokines is different from person to person and in different societies. Several studies showed that polymorphisms of +874 of interferon-gamma [IFN-gamma] and -590 of interleukin-4 [IL-4] are associated with the regulation of expression of these genes. This study was aimed to find polymorphisms of these regions in type 2 diabetes patients. In this experimental study peripheral blood samples were collected from 160 type 2 diabetic patients and 160 healthy controls. DNA was extracted by salting out method. Polymorphisms of +874 of 1FN-gamma and -590 of IL-4 were analyzed by ARMS-PCR and RFLP-PCR. Our findings indicated that TT genotype of IFN-gamma was increased in type 2 diabetic patients compared to the control but difference was not significant. Our results didn't show any significant difference between IL-4 genotype in diabetic and healthy controls either. Our results suggested that TT genotype of IFN-gamma can be associated with diabetes. This association can be described by the fact that over expression of IFN-gamma shifts immune system to Th 1; therefore, pancreatic cells can be miscarried by immune cells

[Evaluation of expression rate of chemokines receptor CCR5 on peripheral blood CD8[+] T cells of occult hepatitis B infected patients]

Occult hepatitis B infection [OBI] is defined as a form of hepatitis B that despite absence of detectable HBsAg, HBV-DNA is present in patient's peripheral blood. Genetic and immunological differences appear to play important roles in producing OBI. Therefore, this project was aimed to examine the expression of a chemokine receptor [CCR5] on CD8[+] T cells of OBI patients. In this experimental study, 3,700 HBsAg- plasma samples were collected. Samples were tested for anti-HBc antibody and all of HBsAg-/anti-HBc[+] samples were screened for HBV-DNA by PCR. HBV-DNA positive samples were assigned as OBI cases. Also, flow cytometry analysis was performed to examine the expression of CCR5 on CD8[+] T cells of OBI patients. Results of current study showed that 352 [9.5%] cases of samples were positive for anti-HBc. Examination of HBsAg/anti-HBc[+] samples for HBV-DNA by PCR showed that 57 [16.1%] cases had HBV-DNA. Flow cytometric studies indicated lymphocytosis in these patients; however, the number of cells which expressed CD8[+] and CCR5 is decreased significantly in patients, compared to healthy control. In addition to CD8[+] T cells, the expression of CCR5 is also decreased on all immune cells. One of the chemokine receptors which are expressed by CD8[+] T cells is CCR5 and these cells are recruited to infected tissues, including liver by CCR5. Therefore, based on results of this investigation, one may conclude that due to the decreased expression of CCR5, the CD8[+] T cells are unable to respond to the chemokines [CCR5 ligands] and, hence, can not immigrate to the infected liver and incorporate in clearance of hepatitis B virus

Heat-treated tumor lysate loaded lymphoide dendritic cells: Evaluation of cytotoxic effect in the tumor bearing mice

Dendritic cells [DCs] are essential for the activation and polarization of T cells during an adaptive immune response. In this research we investigated the effect of the Lymphoide DCs pulsed with heat-treated tumor lysate [HTL] as a vaccine in tumor immunotherapy. The Balb/c mice were injected subcutaneously in the right flank with Wehi-164 fibrosarcoma cells 10 days before immunization with the DCs. Then hsp70 expression in the HTL was detected by using western blot analysis. The mice Lymphoide DCs subset were isolated by magnetic cell sorting [MACS], Then the HTL pulsed Lymphoide DCs, TL pulsed Lymphoide DCs and unpulsed Lymphoide DCs were subcutaneously injected. Tumor growth rate, survival, cytotoxic assay measured. The results showed that HTL-Lymphoide DCs vaccine significantly induced the tumor growth suppression and longer survival than the other immunized mice. Immunotherapy with HTL-Lymphoide DCs led to a significant increase in the activity of cytotoxic T cells in the tumor tissue. The current study suggests that specific anti-tumor immune responses against the fibrosarcoma can be induced by HTL-Lymphoide DCs and may provide a useful therapeutic approach for cancer treatment

efficiency of CD40 down regulation by siRNA and antisense ODN: comparison of lipofectamine and FuGENE6

Dendritic cells [DCs] are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs [siRNAs] and antisense oligodeoxynucleotides [ODN]s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs

Selective immunoglobulin a deficiency in Iranian blood donors: prevalence, laboratory and clinical findings

Selective deficiency of immunoglobulin A [IgA] is the most frequent primary hypogammaglobulinemia. As some IgA-deficient patients have IgA antibodies in their plasma which may cause anaphylactic reactions, blood centers usually maintain a list of IgA-deficient blood donors to prepare compatible blood components. In this study we determined the incidence of selective IgA deficiency [SIgAD] in normal adult Iranian population. 13022 normal Iranian blood donors were included in this study. The assay which we used was adapted to the manual pipetting system and ELISA reader was used for screening. Other classes of immunoglobulins [G, M], as well as secretory IgA and IgG subclasses were tested in IgA deficient cases by ELISA. SPSS was used for statistical analysis. Among 13022 studied cases, 11608 blood donors were males [89.14%] and 1414 were females [10.86%]. Their mean [ +/- SD] age and weight were 38.5 +/- 11 years and 82 +/- 12 Kg respectively. Twenty of the screened samples were found by means of ELISA to be IgA-deficient [less than 5mg/dl], [frequency; 1:651]. The data could indicate a compensation for IgA deficiency by serum IgM in one of our IgA deficient cases [Patient 5]. We observed a correlation between IgG3 and serum IgA in deficient cases [r=0.498, P=0.025]. Our results indicate that in present study the prevalence of S IgA D is in agreement with data from other Caucasians populations [from 1:300 to 1:700]. In conclusion, Selective IgA Deficiency could be almost asymptomatic in most cases in general population. Our study suggests that; due to high frequency of IgA deficiency in Iran, it seems necessary to measure IgA levels for every blood donor and blood recipient to find IgA deficient cases

Human leukocyte antigens [HLA] associated with selective IgA deficiency in Iran and Sweden

Selective IgA deficiency [IgAD] [serum IgA concentration of <0.07 g/l] is the most common primary immunodeficiency in Caucasians, with an estimated prevalence of 1/600. There are strong indications for involvement of genetic factors in development of the disease and the frequency of several extended major histocompatibility complex haplotypes [including HLA-A1, B8, DR3, DQ2] have previously been shown to be increased among Caucasian patients with IgAD. PCR was used to type HLA B, DR, and DQ alleles in 29 Iranian individuals with IgAD and 299 Swedish individuals with IgAD. The results indicate a strong association with the HLA B14, DR1 alleles in Iranian subjects and HLA B8, B12, B13, B14, B40, DR1, DR3, DR7, DQ2 and DQ5 alleles in Swedish subjects. Differences in HLA association of IgAD in Iran and Sweden confirm the notion of a genetic background of the disease and that multiple, potentially different genes within the MHC region might be involved in the pathogenesis of IgAD in different ethnic groups

Designing of relative quantitative Real-Time PCR for detection of CD40 gene expression in dendritic cell after suppression with antisense

Dendritic cells have a critical role in control and regulation of immune responses. It is believed that these cells can be used for the treatment of many diseases. One of the methods used in immunotherapy is based on generating of tolerogenic dendritic cells through inhibition of expression costimulatory molecules. CD40 is one of the costimulatory molecules, and inhibition of expression by antisense or siRNA techniques, can generate tolerogenic dendritic cells. Generation of tolerogenic dendritic cells will be useful in the treatment of many diseases. By developing a quantitive RT-PCR for evaluation of gene expression, generation of these cells could be possible. Using proper software we designed an Antisense and transfection of dendritic cells by lipofectamine 2000 [Invitrogen] could lead us to generate tolerogenic dendritic cells. In this study dendritic cells were extracted from of Balb/c mice Spleen and the purity of this extraction was determined by flow cytometry. BCL 1 cell line as a CD40 expressing control group and Wehi-164 cell line were cultured in RPMI-1640+10%FCS. Primer design for CD40 gene and house keeping gene [GADPH] was done by bioinformatic soft wares such as Beacon designer, mfold and Blast. RNasy plus mini kit [Qiagen] was used for RNA extraction and the Purity and Integrity were determined by O.D at 260/280 and agarose gel electrophoresis. In the next step cDNA synthesized and quantitative RT-PCR for CD 40 using IQ sybergreen [Biorad] were setup. Finally, standard curve for CD40 and internal control in different RNA concentrations were performed. After transfection with lipofectamin 2000 the amount of gene suppression were quantified by qualitative RT-PCR. Using gradient real time PCR, optimum annealing temperature, C[t] and delta Rn for CD40 and GADPH were determined, annealing temperature was 59.5 degree sign c and melting temperature was 84 degree sign c. Slope of the curve and the efficacy of PCR for CD40 and GADPH genes were quantified by serial dilution method. CD 40 Standard curve efficiency is 96.5 and the slope is -3.408 and the efficacy of GADPH standard curve is 94.1 and its slope is -3.471. The amount of CD40 gene suppression by antisense in dendritic cells was 1/32 and in BCL1 cell line was 1/64. Also the transfection reagent had no effect on the gene expression. The best time for CD40 gene expression is 48h after transfection. Semi quantitative PCR, absolute quantitative PCR and relative quantitative PCR are used to evaluative the expression of different genes such as CD40. In this study we found that for making CD40 and GADPH standard curves, Biorad two steps PCR kit [IQ -sybergreen] and Oligo dt for cDNA synthesis were very suitable. In this case, GADPH is a good internal control. Also for evaluation of gene suppression relative quantitative RT-PCR was more efficient compare to other methods such as northern blotting. The CD40 gene Suppression after 48 hours in dendritic cells was 1/32 and in BCL1 cells was 1/64

Increase of CXCR4 expression on expanded non-enriched cord blood CD34+cells using MSCs

A number of potential cell adhesion molecules, which mediate essential cell-to-cell or cell-to-matrix interactions, are expressed on the surface of CD34+ hematopoietic progenitor cells [HPCs], including integrins, CD44, and CXCR4. These molecules are essential for homing process. In this study, we compared the changes of expression of CD44 and CXCR4 on the CD34+ hematopoietic progenitor cells expanded on MSCs in the presence of cytokines. Cord blood CD34+ cells were expanded using human bone marrow mesenchymal stem cells and cytokines [TPO, SCF, FLt-3, IL-6, and IL-3], and then expression of CD44 and CXCR4 on CD34+ cells were evaluated by flow cytometric analysis. After 2 weeks of serum free culture of CD34+ cells in the presence of cytokines, the expression of CXCR4 on CD34+ cells was decreased 3.4 fold [p<0.05]. In contrast, the expression of CXCR4 on CD34+ cells expanded on hMSCs was increased [p<0.05]. The expression of CD44 on expanded CD34+ cells in both methods did not differ significantly. Our results indicated that co-culture of cord blood stem cells on hMSCs significantly increased CXCR4 expression on cord blood CD34+ cells

Establishment of a doxorubicin-Resistant subline from acute myeloid leukemia cell line

In order to determine the multidrug resistance [MDR] phenotype due to P-glycoprotein expression in haematological malignancies including acute myeloblastic leukemia [AML], a well-characterized P-gp expressing cell line was required to validate and standardize flow cytometric assays and to calibrate instruments. Therefore, this resistant subline of K562 was established for the first time in Iran in order to study the MDR phenotype due to P-gp expression in some cancers. A resistant subline of K562 [KDI/20] to Doxorubicin from the same parental K562 was derived by stepwise increasing the concentration of Doxorubicin up to 20 ng/ml as a gold standard. For flow cytometric assessment of P-gp expression, 4E3 anti-P-gp was used. The resistant cell line was studied by rhodamine 123 for functional assay of P-gp. MDR1 gene expression was also confirmed using RT-PCR. P-glycoprotein was expressed in final concentration of 20 ng/ml of Doxorubicin on 70% of K562 cells after 120 passages. The Rhodamine 123 influx was 37%. The over-expression of MDR1 gene was observed in a 30-cycle PCR. P-glycoprotein is expressed in human K562 cell line [K562] by continuous exposure to anticancer drug. P-glycoprotein expression is detected by several methods including flow cytometry and RT-PCR, and the number of PCR cycles is very important

Comparison of functional competence of umbilical cord and adult peripheral blood dendritic cells in allogenic mixed leukocyte reaction

Dendritic cells [DCs] are the most potent stimulators of primary T cell responses and play a key role in immune reactions after stem cell transplantation. Very little is known about the cord blood [CB] dendritic cells and their potential involvement in the low incidence and lower severity of acute graft-versus-host disease after CB transplantation. The aim of this study was the isolation of cord blood and peripheral blood dendritic cells and comparison of their functional competence and determination of their probable role in graft versus host disease after stem cell transplantation. In this study, fresh peripheral blood DCs [PBDCs] wereenriched as HLA-DR[+] cells, lacking the CDS, CDllb, CD14, CD16, CD19 and CD56, using immunomagnetic bead depletion. For cord blood dendritic cells [CBDCs] enrichment CD34[+] and CD66b[+] cells were needed to be depleted too. Immunomagnetically enriched PB/CB dendritic cells were co-cultured with adult Tlymphocytes and cell proliferation was measured by 3H-thymidine incorporation. Results showed that CBDCs were significantly poor stimulators of the mixed leukocyte reaction as compared with PBDCs [P < 0.05]. The demonstrated impairment of CBDCs function could be of importance in interpretation of the low incidence and milder severity of graft-versus-host disease [GVHD] in umbilical CB transplantation compared with peripheral blood or bone marrow stem cell transplantation

Detection of lactoferrin in the neutrophils and plasma of the patients suffering from hepatitis C

Lactoferrin [LF] has antimicrobial properties against bacteria, fungi and several viruses including herpes virus, HIV and hepatitis C virus. The aim of this study was to detect LF in PMNs and plasma of the patients suffering from hepatitis C and the healthy persons. The sonicated solutions of PMNs of two groups were evaluated by SDS-PAGE [10%], isoelectric focusing [30%] and dot blotting .The level of LF in plasma was measured by ELISA. The results confirmed the presence of LF in PMNs of the two groups. ELISA showed that the level of LF in plasma of patients was higher than normal persons. Based on these findings we conclude that not only the production of LF was not reduced in the patients but also its level was significantly increased compared with the normal persons [P<0.0001]