Menstrual blood-derived stromal stem cells augment cd4+ t cells proliferation

Background: It is more than sixty years that the concept of the fetal allograft and immunological paradox of pregnancy was proposed and in this context, several regulatory networks and mechanisms have been introduced so far. It is now generally recognized that mesenchymal stem cells exert potent immunoregulatory activity. In this study, for the first time, the potential impact of Menstrual blood Stem Cells [MenSCs], as surrogate for endometrial stem cells, on proliferative capacity of CD4+ T cells was tested

Methods: MenSCs and Bone marrow Mesenchymal Stem Cells [BMSCs] were isolated and assessed for their immunophenotypic features and multi-lineage differentiation capability. BMSCs and MenSCs with or without IFNGamma pre-stimulation were co-cultured with purified anti-CD3/CD28-activated CD4+ T cells and the extent of T cell proliferation at different MenSCs: T cell ratios were investigated by CSFE flow cytometry. IDO activity of both cell types was measured after stimulation with IFNGamma by a colorimetric assay

Results: MenSCs exhibited dual mesenchymal and embryonic markers and multilineage differentiation capacity. MenSCs significantly increased proliferation of CD4+ cells at ratios 1:2, 1:4 and 1:8. IFNGamma pre-treated BMSCs but not MenSCs significantly suppressed CD4+ T cells proliferation. Such proliferation promoting capacity of MenSCs was not correlated with IDO activity as these cells showed the high IDO activity following IFNGamma treatment

Conclusion: Although augmentation of T cell proliferation by MenSCs can be a basis for maintenance of endometrial homeostasis to cope with ascending infections, this may not fulfill the requirement for immunological tolerance to a semi-allogeneic fetus. However, more investigation is needed to examine whether or not the immunomodulatory properties of these cells are affected by endometrial microenvironment during pregnancy

Preliminary assessment of various additives on the specific reactivity of anti- rHBsAg monoclonal antibodies

Antibodies have a wide application in diagnosis and treatment. In order to maintain optimal stability of various functional parts of antibodies such as antigen binding sites, several approaches have been suggested. Using additives such as polysaccharides and polyols is one of the main methods in protecting antibodies against aggregation or degradation in the formulation. The aim of this study was to evaluate the protective effect of various additives on the specific reactivity of monoclonal antibodies [mAbs] against recombinant HBsAg [rHBsAg] epitopes. To estimate the protective effect of different additives on the stability of antibody against conformational epitopes [S3 antibody] and linear epitopes [S7 and S11 antibodies] of rHBsAg, heat shock at 37[degree]C was performed in liquid and solid phases. Environmental factors were considered to be constant. The specific reactivity of antibodies was evaluated using ELISA method. The data were analyzed using SPSS software by Mann-Whitney nonparametric test with the confidence interval of 95%. Our results showed that 0.25 M sucrose, 0.04 M trehalose and 0.5% BSA had the most protective effect on maintaining the reactivity of mAbs [S3] against conformational epitopes of rHBsAg. Results obtained from S7 and S11 mAbs against linear characteristics showed minor differences. The most efficient protective additives were 0.04 M trehalose and 1 M sucrose. Nowadays, application of appropriate additives is important for increasing the stability of antibodies. It was concluded that sucrose, trehalose and BSA have considerable effects on the specific reactivity of anti rHBsAg mAbs during long storage

Targeted delivery of 5-fluorouracil with monoclonal antibody modified bovine serum albumin nanoparticles

Herein, 1F2, an anti-HER2 monoclonal antibody [mAb], was covalently coupled to the surface of 5-Fluorouracil [5-FU] loaded bovine serum albumin [BSA] nanoparticles. Concerning two different crosslinkers for conjugation of 1F2, Maleimide-poly [ethylene glycol]-Succinimidyl carbonate [Mal-PEG5000-NHS] was selected due to its higher conjugation efficiency [23 +/- 4%] obtained in comparison to N-succinimidyl 3-[2-Pyridyl Dithio] Propionate [SPDP] [8 +/- 2%]. A slight increase in the average particle size with a negligible prolongation of the 5-FU release was observed after 1F2 coupling. The 1F2-coupled 5-FU-loaded BSA nanoparticles interacted with nearly all HER2 receptors available on the surface of HER2-positive SKBR3 cells. No cellular uptake was observed for HER2-negative MCF7 cells. Physicochemical and biological properties of the mAb-modified nanoparticles did not significantly alter after three months of storage at room temperature. The in-vitro cytotoxicity evaluation by MTT assay, demonstrated lower SKBR3 viability [50.7 +/- 9 %] after 5 hours contact with 1F2-coupled 5-FU-loaded BSA nanoparticles in comparison with the other control systems due to their cell attachment and internalization after washing. In addition, no significant toxicity was observed on MCF7 cells. This novel system can efficiently be employed for targeted delivery of 5-FU to HER2-positive cancerous cells

Genetic profile variation in vaccine strains and clinical isolates of bordetella pertussis recovered from Iranian patients

Re-emergence of pertussis has been reported in Iran despite a high rate of vaccination coverage. Low efficacy of the vaccine might be due to the genetic divergence between Clinical versus vaccine strains. In the current study, the genetic profiles of Clinical isolates and vaccine strains of Bordetella pertussis [B. pertussis] were assessed by using Pulsed Field Gel Electrophoresis [PFGE]. Following phenotypic and molecular identification of isolates, XbaIdigested genomic DNA of 5 Clinical isolates, 2 vaccine strains and a Tohama I strain were analyzed by PFGE along with B. parapertussis as a control. Seven distinct PFGE profiles were found among all examined isolates/strains. In 5 Clinical isolates, 4 profiles were identified whereas the vaccine strains displayed 2 distinct profiles. The reference strain, Tohama I had a distinct profile. Vaccine and Clinical profiles had low similarity, with relatedness of approximately 40%. The genetic profiles of B. pertussis were different between circulating isolates and vaccine strains used in the national vaccination programs. Since new genetic profiles of B. pertussis can be disseminated periodically, the profiles of isolates circulating in the population should be monitored over the course of the re-emergence

Cloning, expression and characterization of recombinant human Fc receptor like 1, 2 and 4 molecules

The Fc receptor like [FCRL] molecules belong to the immunoglobulin [Ig] superfamily with potentially immunoregulatory function. Among the FCRL family FCRL2 and 4 are predominantly expressed on memory B cells and FCRL1 is a pan- B cell marker. To date, no ligand has been identified for the human FCRL1, 2 and 4 molecules. Cloning, expression, purification and structural analysis of the extracellular domain of human FCRL1, 2 and 4 proteins. In this study, the extracellular part of human FCRL1, 2 and 4 were subcloned into prokaryotic expression vectors pET-28b [+] and transformed into BL21-DE3 E.coli strain. Protein expression was optimized by fine adjustments such as induction time, incubation temperature and expression hosts. Recombinant FCRL proteins were purified by metal affinity chromatography using Ni-NTA resin. Purified FCRL proteins were further characterized by SDS-PAGE and immunoblotting using His-tag and FCRL specific polyclonal antibodies. Our results demonstrated that FCRL1, 2 and 4 were successfully expressed in pET-28b [+] vector. Optimization of the expression procedure showed that IPTG induction at OD600 = 0.9 and overnight incubation at 37[degree]C resulted in the highest expression levels of FCRL proteins ranging from approximately 15% [FCRL1] to 25% [FCRL2 and 4] of the total bacterial lysate proteins. These purified recombinant proteins are potentially a valuable tool for investigating the immunoregulatory function of FCRL molecules and the production of specific mAbs for immunotherapeutic interventions.

Expression, purification and characterization of three overlapping immunodominant recombinant fragments from Bordetella pertussis filamentous hemagglutinin

Filamentous hemagglutinin [FHA] is one of the most important immunoprotective antigens of Bordetella pertussis [B. pertussis] and a major component of the acellular pertussis vaccine. In the present study, three overlapping recombinant fragments from the immunodominant region of FHA were produced and their immunogenicity was investigated. Three overlapping coding sequences of FHA antigen were amplified from B. pertussis genomic DNA by PCR. Amplified fragments were expressed in Escherichia coli [E. coli] BL21[DE3] strain and purified through His-tag using Nickel-based chromatography. Purified fragments were characterized by SDS-PAGE and Western blotting techniques. In vitro peripheral blood mononuclear cells [PBMC] proliferation and IFN- gamma production were assessed in a limited number of healthy adults vaccinated with a commercial acellular pertussis vaccine in response to all purified FHA fragments by H3-Thymidine incorporation and ELISA, respectively. Recombinant FHA segments were successfully cloned and produced at high levels in E. coli BL21[DE3]. SDS-PAGE and Western blot analyses confirmed their purity and reactivity. All three recombinant fragments together with a commercial native FHA were able to induce in vitro PBMC proliferation and IFN- gamma production. Our preliminary results suggest that these overlapping recombinant FHA fragments are immunogenic and may prove to be immunoprotective

Production and characterization of mouse monoclonal antibodies recognizing human Pan-IgG specific conformational or linear epitopes

Pan-IgG specific monoclonal antibodies [MAbs] are essential tools for assessment of humoral immunity, immune deficiency and gammopathy. In this study, four hybridoma clones producing MAbs with different specificities for human IgG isotypes were established. Splenocytes from Balb/c mice immunized with Fc fractions of human IgG were fused with SP2/0 myeloma cells. Hybridoma cells were selected in HAT selective medium and cloned by limiting dilution assay. Antibody-secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed using a panel of purified myeloma IgG proteins by ELISA and immunoblotting. Cross-reactivity to immunoglobulins [Igs] of other species was studied by indirect ELISA using serum samples collected from 9 animals. Immunoblotting studies revealed recognition of either linear or conformational epitopes by these MAbs. The most abundant cross-reactivity [100%] was observed with monkey Igs, while no cross-reactivity was detected with rabbit, guinea pig, sheep, goat, cat and hen sera. Two of the MAbs crossreacted with either dog or horse sera. The affinity constant of two MAbs was measured by ELISA and found to be 0.74×10[8] M[-1] and 0.96×10[7] M[-1]. Our results indicate that these pan-IgG specific MAbs recognize restricted linear or conformational epitopes located on all human IgG subclasses with no cross-reactivity to IgG from most species studied. These MAbs are potentially useful tools for quantification of total or antigen-specific IgG levels

Association of HLA-DRB1, DQA1 and DQB1 alleles and haplotypes with common variable immunodeficiency in iranian patients

Common Variable Immunodeficiency [CVID] is an antibody deficiency syndrome that often co-occurs in families with selective IgA deficiency [IgAD]. This study was designed to investigate the frequency of DR and DQ loci of HLA class II region in common variable immunodeficiency [CVID] patients. Fifteen Iranian patients with CVID or IgAD [mean age 14.6 +/- 5.4, range 4-25 years; 9 male and 6 female] and 63 healthy controls were studied. Establishment of B-lymphoblastoid cell lines was performed using Epstein-Barr-virus [EBV] immortalization technique and HLA alleles were typed using polymerase chain reaction based on sequence specific primers [PCR-SSP]. DRB1 alleles including DRB1 *04 [p=0.03] and DRB1 *11 [p=0.01] significantly showed higher frequency in the studied subjects. In contrast, DRB1 *301 [p=0.04] and DRB1 *07 [p=0.02] alleles were negatively associated with CVID. For DQB1 and DQA1 loci, DQB1 *0302 [p=0.047] and DQA1 *03011 [p=0.001] demon-strated high frequency in cases, while DQB1 *0201 [p=0.02] and DQA1 *0201 [p=0.01] were detected to be low when compared to controls. Haplotype analysis indicated that frequency of DRB1*04-DQB1*03011-DQA1 *03011 [p=0.02], DRB1 *11-DQB1 *03011-DQA1 *0505 [p=0.047], DRB1 *11-DQA1 *0505 [p=0.04] and DRB1*04-DQA1*03011 [p=0.02] haplotypes were significantly higher in patient group, while only the frequency of the DRB1 *07-DQA1 *0201 haplotype gene was statistically lower in control group [p=0.02]. According to the results, it could be deduced that the HLA-DR and DQ loci may contribute to the pathogenesis of CVID or they might be considered as suitable markers for the possibility of the occurrence of this genetic defect

Differential WNT gene expression in various subtypes of acute lymphoblastic leukemia

Dysregulation of WNT signaling has been reported in many malignancies. This study was conducted to investigate the expression pattern of 14 members of the WNT gene family in different immunophenotypic subtypes of ALL. Semi-quantitative RT-PCR was performed on samples from 71 ALL patients and 36 age-matched healthy individuals. The ALL patients were categorized into B-ALL [76%], T-ALL [22.6%] and mixed lineage [1.4%] and the B-ALL cases were further classified into pro-B, pre-BI, pre-BII and immature/mature-B based on immunophenotypic results. Among the WNT genes, WNT-7B [p=0.026], WNT-9A [p=0.020] and WNT-16B [p=0.023] were significantly over-expressed, whereas WNT-2B [p=0.033], WNT-5A [p=0.016], WNT-7A [p<0.0001] and WNT-10A [p<0.0001] were down-regulated in B-ALL. Among the T-ALL subtype, however, significant down-regulation of WNT-2B, WNT-5B, WNT-7A, WNT-10A and WNT-11 was evident. Comparison between B-ALL subtypes showed significant over-expression of WNT-7B, WNT-9A and WNT-5B in certain subtypes. Our results suggest contribution of the WNT genes in leukemogenesis of ALL

Comparative expression profile of orphan receptor tyrosine kinase ROR1 in Iranian patients with lymphoid and myeloid leukemias

It has recently been shown that ROR1, a member of the receptor tyrosine kinase family, is overexpressed in leukemic B cells of Chronic Lymphocytic Leukemia [CLL] and a subset of Acute Lymphoblastic Leukemia [ALL]. In this comparative study the expression profile of ROR1 mRNA was investigated in Iranian patients with CLL and Acute Myelogenous Leukemia [AML] and the results were compared with those previously reported in our Iranian ALL patients. RT-PCR was performed on bone marrow and/or peripheral blood samples of 84 CLL and 12 AML patients. CLL samples were classified into immunoglobulin heavy chain variable region [IGHV] gene mutated [n=55] and unmutated [n=29] and also indolent [n=42] and progressive [n=39] subtypes. ROR1 expression was identified in 94% of our CLL patients, but none of the AML patients expressed ROR1. No significant differences were observed between different CLL subtypes for ROR1 expression. Taken together the present data and our previous results on ROR1 expression in ALL, our findings propose ROR1 as a tumor-associated antigen overexpressed in a large proportion of lymphoid [CLL and ALL], but not myeloid [AML] leukemias. Expression of ROR1 seems to be associated to lineage and differentiation stages of leukemic cells with a potential implication for immunotherapy

Lymphocyte cytotoxicity of oxLDL in patients with atherosclerosis

Atherosclerosis, a chronic inflammatory disease of the vessel wall is characterized by local and systemic immune responses to a variety of antigens. Oxidized low-density lipoprotein [oxLDL] is considered as an important determining factor in the pathogenesis of atherosclerosis. The purpose of this study was to investigate the degree of peripheral blood mononuclear cells [PBMC] vulnerability to in vitro oxLDL-induced cytotoxicity from atherosclerotic patients in comparison to healthy individuals. Thirty patients with atherosclerotic lesions, confirmed by angiography, and 30 matched healthy individuals were investigated. PBMC was prepared from individuals' blood samples which were further stimulated with low dose [1 micro g/mL] and high dose [50 micro g/mL] of extensively oxidized LDL. MTT assay was utilized to measure cell viability and proliferation. Stimulation index [SI] was calculated as mean ratio of optical density [OD] of the stimulated cells divided by OD of untreated cells. Low dose oxLDL treatment caused no significant proliferative or cytotoxic effect in the control group; however, similar treatment caused significant cytotoxic effect in the patient group compared to the controls [p=0.026]. High dose oxLDL treatment induced more significant cytotoxicity in the patient compared to the control group [p=0.006]. Comparison of the SI between the two groups of patients and controls showed significantly lower index by either the low [p=0.03] or the high dose [p<0.001] oxLDL in the patients compared to the controls. PBMC from patients with atherosclerosis showed increased susceptibility to oxLDL-induced cytotoxicity. Our results imply that prolonged exposure to elevated levels of circulating oxLDL could weaken the cellular defense mechanisms by progressive depletion of the pool of antiapoptotic proteins, rendering the cells more vulnerable to oxLDL-induced cell death

Production and characterization of mouse monoclonal antibodies recognizing multiple subclasses of human IgG

Different IgG subclass profiles are produced in response to different antigenic stimuli in a variety of diseases. IgG subclass levels may reflect disease severity. Quantification of IgG subclasses depends on the availability if specific Monoclonal antibodies [MAbs]. In the present study seven hybridoma clones producing MAbs reactive with multiple subclasses of human IgG were established. Splenocytes form Balb/c mice immunized with Fc fractions of human IgG1 or IgG2 myeloma proteins were fused with mouse myeloma cells. Fused cells were selected and cloned by limiting dilution assay. Antibody secreting cells were screened by Enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified human myeloma paraproteins of different IgG subclasses by ELISA and immunoblotting. Cross-reactivity to immunoglobulins [Igs] of other species was studied by indirect ELISA using serum samples collected from 9 animals. The MAbs were found to react with triple IgG subclasses, including IgG1,2,4 [n=4] and IgG1,2,3 [n=3]. Immunoblotting studies revealed recognition of linear [n=4] or conformational [n=3] epitopes by these MAbs. The most abundant cross-reactivity [71.4%] was observed with monkey Ig while no cross-reactivity was detected with hen and cat sera. The MAbs mostly displayed a restricted pattern of cross-reactivity and one of them did not bind to any of the animal sera tested. The affinity constant of 3 MABs was measured by ELISA. Based on the data obtained from this study, mouse MAbs reactive with multiple human IgG subclasses are directed to a variety of immunogenic epitopes, mostly shared with IgG of other species. Thes MAbs are valuable tools for purification of non-reactive IgG subclasses through negative affinity chromatography. These Mabs could also provide an opportunity for epitope mapping of the Fc region of IgG, as well as serological phylogenetic studies

Development of a sensitive enzyme-linked immunosorbent assay for detection of hepatitis B surface antigen using novel monoclonal antibodies

Hepatitis B virus [HBV] infection is the 10th leading cause of death worldwide. The most important diagnostic and screening marker for HBV infection is Hepatitis B surface antigen [HBsAg], and the most widely used HBsAg screening test is Enzyme-linked Immunosorbent Assay [ELISA]. In this study, an ELISA assay has been developed for detection of HBsAg using two novel monoclonal antibodies [mAb] as capture layer and a polyclonal biotinylated Ab as detector phase. We evaluated the sensitivity, specificity, detection limit, seroconversion time, positive and negative predictive values and reproducibility of our assay with standard panels and different serum samples. The results were compared with a well established commercial kit. Both assays showed similar detection limit values of 0.5 to 0.7 ng/ml and the same seroconversion periods of 42 and 65 days for [ad] and [ay] serotypes of HBsAg, respectively. Sensitivity and specificity of the assay were 98.98% and 99.6%, respectively. The positive and negative predictive values of our assay were also calculated as 99.49% and 99.2%, respectively. Analysis of reproducibility of the present assay demonstrated 3.96% and 5.85% intra-and inter-assay coefficient of variations, respectively, which were less than those obtained by the commercial kit. There was a highly significant correlation between our designed assay and the commercial ELISA kit [p < 0.0001, r = 0.957]. Altogether, our results indicate that the designed assay is comparable to the commercial kit in terms of sensitivity, specificity, positive and negative predictive values and reproducibility and could be employed for diagnosis of HBV infection in blood samples

Optimization of gene transfection in murine myeloma cell lines using different transfection reagents

Purification and isolation of cellular target proteins for monoclonal antibody [MAb] production is a difficult and time-consuming process. Immunization of mice with murine cell lines stably transfected with genes coding for xenogenic target molecules is an alternative method for mouse immunization and MAb production. Here we present data on transfection efficiency of some commercial reagents used for transfection of murine myeloma cell lines. Little is known about transfectability of murine myeloma cell lines by different transfection reagents. Mouse myeloma cell lines [SP[2]/O, NSO, NS1, Ag8, and P3U1] were transfected with pEGFP-N1 vector using Lipofectamine 2000, jetPEI and LyoVec commercial transfection reagents in different combinations. The transfection permissible HEK293-FT cell line was used as a control in transfection procedure. Transfected cells, expressing the Enhanced Green Fluorescent Protein [EGFP], were analyzed by flow cytometry 48 hrs post transfection. Our results showed transfection efficiency of 71%, 57% and 22% for HEK293-FT, 5.5%, 3.4% and 1% for SP[2]/O, 55.7%, 21.1% and 9.3% for NSO, 8.2%, 6% and 5.5% for NS1, 22%, 49.2% and 5.5% for Ag8 and 6.3%, 21.5% and 4.6% for P3U1 cell lines after transfection with Lipofectamine 2000, jetPEI and LyoVec reagents, respectively. Our data indicate that NSO and Ag8 are efficiently transfected by Lipofectamine 2000 and jetPEI reagents. Finally, we propose Ag8 and NSO cell lines as suitable host cells for efficient expression of target genes which can be used for mouse immunization and MAb production

Generation and characterization of mouse hybridomas secreting monoclonal antibodies specific for human lgG3

Mammalians express several subclasses of the IgG molecule. In human being there are four homologous IgG subclasses, each of which is structurally unique and has different functions. Quantification of IgG subclasses is fundamental to clinical assessment and diagnosis of many diseases as such assessments depends on the availability of subclassspecific antibodies [Abs], particularly monoclonal antibodies [MAbs]. In the present study, we produced and characterized two murine MAbs specific for human lgG3 molecule. These MAbs were obtained by the fusion of myeloma cells with splenocytes from Balb/c mice immunized with heavy chain of a human IgG3 myeloma protein. Fused cells were selected in, hypoxanthine, aminopterine and thymidine [HAT] medium and cloned by limiting dilution assay. Ab-secreting cells were screened by enzyme-linked immunosorbent assay [ELISA] and the specificity of secreted MAbs was further analyzed, using a panel of purified myeloma proteins by ELISA and immunoblotting. Two stable hybridomas designated 1F18G7 and 1F18A11 were obtained secreting MAbs specific for Fc fragment of human IgG3. None of these MAbs showed cross-reactivity with other immunoglobulin isotypes derived from human and nine other animals, except 1F18A11 which displayed a weak cross-reactivity with only dog serum. Immunoblotting results indicate that these MAbs react with linear epitope [s] located in the heavy chain of human IgG3 molecules. The affinity constant of 1F18G7 and 1F18A11 MAbs was found to be 0.81x10[9] Mol[-1] and 0.7x10[9] Mol[-1], respectively, as measured by ELISA. These two MAbs with relatively high affinity can be useful tools for quantification of IgG3 subclass levels in human serum

Modeling breast acini in tissue culture for detection of malignant phenotype reversion to non-malignant phenotype

Evidence is accumulating to support disruption of tissue architecture as a powerful event in tumor formation. For the past four decades, intensive cancer research with the premise of "cancer as a cell based-disease" focused on finding oncogenes or tumor suppressor genes. However, the role of the tissue architecture was neglected. Three dimensional [3D] cell cultures which can recapitulate major aspects of the microenvironment are appropriate models for exploring cancer. For the first time in Iran, we have launched Matrigel based non-malignant, tumorigenic and reverted breast 3D cell cultures. Non-tumorigenic MCF-10A and tumorigenic MCF-7 breast cell lines were cultured on plastic and Matrigel. MCF-7 cell lines were reverted to normal phenotype via AIIB2 and LY 294002 inhibitors against beta1 integrin and class I phosphatidylinositol 3-kinase, respectively. MCF-10A acini were distinguishably different from MCF-7 on Matrigel. MCF-10A formed organized hollow spherical structures which were in stark contrast to the MCF-7 disorganized cluster of cells. Matrigel allowed visual monitoring of MCF-7 cells treated with inhibitors. After treatment of MCF-7 cells, we observed reversion of MCF-7 phenotype toward normal, comparable to MCF-10A acini. The 3D culture provides a microenvironment which allows malignant and non-malignant cells to demonstrate near physiological behavior and this can distinguish nonmalignant from malignant cells. The 3D culture also allows visual monitoring of malignant phenotype reversion to organized spheres

Immunophenotypic characterization of the leukemic B-cells from Iranian patients with chronic lymphocytic leukemia: association between CD38 expression and disease progression

Patients with B-cell chronic lymphocytic leukemia [B-CLL] have heterogeneous clinical courses, thus several biological parameters need to be added to the current clinical staging systems to predict disease outcome. Recent immunophenotypic studies performed mainly in Western populations have demonstrated the prognostic value of CD38 and ZAP-70 expression in B-CLL. To investigate the expression pattern of a variety of membrane antigens on leukemic cells from Iranian patients with CLL and to find out if there are any differences in the expression of these markers between indolent and progressive groups. In the present study, peripheral blood samples from 87 Iranian patients with B-CLL were analysed by flow cytometry. In all cases, the neoplastic cells displayed B-CLL phenotype [CD5[+]/CD19[+]/sIg[+]]. The vast majority of the cases expressed CD23, but failed to stain for CD3 or CD14. The leukemic cells of most patients expressed CD27 [84/87, 95.4%] and CD45RO [74/87, 83.9%] molecules, suggesting a memory B-cell phenotype. Comparison between the indolent [n=42] and progressive [n=37] patients revealed significantly higher frequency and intensity of CD38 expression in progressive group [40.5%] compared to indolent [11.9%] patients [p<0.05]. None of the other membrane antigens were differentially expressed in these two groups of patients. Our results obtained in an Asian ethnic population confirm and extend previous findings obtained from Western populations regarding the association of CD38 expression and disease progression in B-CLL

HLA-DRB1, DQA1 and DQB1 alleles and haplotypes frequencies in Iranian healthy adult responders and non-responders to recombinant hepatitis B vaccine

Different studies have demonstrated that a small proportion of healthy individuals receiving the hepatitis B [HB] vaccine do not produce protective levels of anti-HB antibody, a phenomenon which could be linked to certain human leukocyte antigen [HLA] class-II alleles or haplotypes. The present study was undertaken to determine the frequency of HLA class-II alleles in Iranian healthy adult responders and non-responders to HB vaccine. Twelve non-responders [anti-HBs antibody < 10 IU/L] and 46 responders [anti-HBs antibody > 100 IU/L] were tissue typed for HLA class-II. HLA-DRB1, DQB1 and DQA1 alleles were determined using polymerase chain reaction based on sequence specific primers [PCR-SSP] technique. Accessibility to excess amount of genomic DNA was possible using Epstein-Barr virus [EBV]-transformed B-cells established from all vaccinees. Our results demonstrated increased frequencies of HLA- DRB1*07, DRB1*03, DRB1*04, DQB1*0201, DQA1*0201 alleles and HLA- DRB1*07/DQB1*0201/DQA1*0201 and DRB1*04/DQB1*0302/DQA1*03011 haplotypes in the non-responder group. Comparison between responders and non-responders revealed only a significant difference for DQB1*0201 allele [p < 0.05]. These findings confirm the association of certain HLA alleles and haplotypes with the lack of antibody response to HB vaccine in an Iranian population

Immunologic basis and immunoprophylaxis of RhD induced hemolytic disease of the newborn [HDN]

RhD antigen is the most immunogenic and clinically significant antigen of red blood cells after ABO system. It has historically been associated with hemolytic disease of the newborn [HDN] which is now routinely prevented by the administration of polyclonal anti-D immunoglobulin. This management of HDN has proven to be one of the most successful cases of prophylactic treatment based on antibody mediated immune suppression [AMIS]. Despite the increasing efficiency of treatment, the mechanism of action of anti-D is not completely defined. There is a widespread interest in obtaining a reliable therapeutic monoclonal anti-D, due to difficulty of maintaining a pool of high titer volunteer donors for plasma collection and also increasing demand for antenatal prophylaxis and safety issues with plasma derived products. Candidate monoclonal anti-D preparations should demonstrate appropriate functionality in both in vitro and in vivo assays comparable to polyclonal anti-D immunoglobulin. These criteria are reviewed in addition to the factors regulating development of D specific immune response in D negative individuals and its suppression in HDN prophylaxis

IL-1, IL-1R and TNFalpha gene polymorphisms in Iranian patients with multiple sclerosis

Different research groups have extensively studied the associations of cytokine gene polymorphisms in different diseases. The role of cytokines gene polymorphisms in multiple sclerosis [MS], as a chronic Immune-mediated neurodegenerative disease, has been previously reported in the various populations. For determining pro-inflammatory cytokine gene polymorphisms, 100 relapsing remitting multiple sclerosis [RRMS] Iranian patients and 140 normal individuals as control enrolled in this study. DNA of each sample was extracted by a modified salting out method. Cytokine single gene nucleotide polymorphisms including IL-1alpha -889, IL-1beta [-511 and +3962], IL-1R pst1 1970, IL-1RA mspal 11100, and TNF-alpha [-308 and -238] were determined by using the PCR-SSP method. The results of our data indicate the decrease in frequency of IL-1alpha TC-889 genotype [p=0.002], IL-1beta TC +3962 genotype [p=0.004], IL-1R T pst1 1970 allele [p= 0.0001], IL-1 RA TC Mspa1 11100 genotype [p=0.009], TNF-alpha A-308 allele [p=0.0002] and AG genotype [p=0.00001] in the patients group versus normal subjects. On the other hand the frequency of IL-1alpha TT -889 genotype [p=0.028], IL-1R C pst1 1970 allele [p=0.0001] and CC genotype [p=0.00006], TNFalpha G -308 allele [p=0.0002] and GG genotype [p=0.000001] decreased significantly in the patients versus normal subjects. These results suggest that polymorphic variations of these pro-inflammatory cytokines may play an important role in susceptibility of Iranian multiple sclerosis patients