فهرست مطالب mahmood jeddi

Despite the significant progress in the treatment of Acute Lymphoblastic Leukemia (ALL) in children, it still remains as one of the most challenging malignancies in adults. Identification of new biomarkers may improve the management of adult ALL. Proteins expressed on the cell surface can be considered as disease-associated biomarkers with potential for diagnosis and targeted therapies. Thus, membrane proteome studies give essential information about the disease-related biomarkers.

We applied 2-dimensional blue-native SDS-PAGE technique followed by MALDI-TOF/TOF-mass spectrometry to study the cell membrane proteome of peripheral blood mononuclear cells of adult B-ALL patients in comparison to that of the healthy controls.

Sixty seven differentially expressed protein spots were detected, among them 52 proteins were found to be up-regulated but the other 15 proteins were downregulated in B-ALL. Five differentially expressed proteins, involved in energy metabolism pathways, were detected in B-ALL patients compared to the healthy control group.

Differentially expressed proteins provide an insight into the molecular biology of B-ALL. Further studies must be done to confirm our data to be considered as potential targets for detection and treatment of B-ALL.

We have recently produced an inhibitory mouse anti-human HER2 mAb (2A8) which displayed potent anti-tumor activity in combination with trastuzumab. To describe chimerization and functional characterization of 2A8 mAb. The VH and VL genes of 2A8 mAb were amplified from cDNA of the mouse hybridoma, ligated to constant regions of human immunoglobulin, and expressed in CHO cell line. Reactivity with four members of human HER family, the inhibitory effects and antibody-dependent cell cytotoxicity (ADCC) of purified chimeric mAb (c2A8) were assessed by ELISA, XTT, H3-tymidine incorporation and lactate dehydrogenase assays. Inhibition of ERK and AKT downstream signaling pathways by the chimeric antibody were analyzed by Western blotting. Chimeric 2A8 mAb bound to recombinant human HER2 and did not cross-react with the other members of HER family. Moreover, c2A8 was able to recognize HER2-overexpressing cancer cell line and inhibited growth and proliferation of these cells. The binding affinity of c2A8 was comparable to the mouse parental mAb. ADCC and Western blotting results showed that the mouse 2A8 mAb was successfully chimerized and could significantly inhibit phosphorylation of AKT in combination with trastuzumab. The c2A8 mAb is potentially a valuable tool for targeted immunotherapy of HER2 positive cancers.

Prostate cancer is the second most common cancer in men. Prostate-Specific Antigen (PSA) is a tumor-associated glycoprotein with enzymatic activity which is secreted by the prostate gland. Following entry to the blood, 70-90% of PSA forms complexes with protease inhibitors and its enzymatic activity is inhibited. The serum level of PSA is increased and the rate of free PSA (fPSA) to total PSA is decreased in prostate cancer patients. Therefore, measurement of PSA and fPSA in serum is very valuable for diagnosis and prognosis of prostate cancer. In the present study, five anti PSA monoclonal Antibodies (mAb) were characterized by Enzyme-Linked Immunosorbent Assay (ELISA) and immunoblotting. For designing a sandwich ELISA, epitope specificity of these antibodies was studied by a competition ELISA. Free PSA was purified by electroelution technique from seminal plasma and used to produce polyclonal anti-fPSA antibody in rabbit. Purified polyclonal antibody (pAb) and mAbs were conjugated with HRP enzyme and Biotin (Bio) to set up the sandwich ELISA. Three of the mAbs were found to recognize PSA similarly. One of these mAbs (2G3) was paired with anti-fPSA pAb to detect fPSA in serum. Eventually, serum fPSA concentration of 356 subjects was measured and compared by our designed ELISA and a commercial ELISA kit. Our results demonstrated a significant correlation (r=0.68; p<0.001) between the two assays. Sensitivity and specificity of our designed ELISA was 72.4 and 82.8%, respectively. These results imply suitability of our designed ELISA for detection of fPSA in patients with prostate cancer.

Among the many pneumococcal antigens, choline-binding proteins (CPBs) display high immunogenicity in animal models. This study aims to determine the immunogenicity of CbpM, CbpG and CbpL proteins in a mice model. The genes were cloned into pET21a expression vector and the recombinant proteins were produced. Mice and rabbits were immunized with the purified recombinant proteins. Subsequently, the mice were challenged with S. pneumoniae and their survival as well as bacterial clearances were followed. The antibody responses of the mice immunized with recombinant proteins were determined by ELISA assay. The opsonophagocytosis assay was performed using rabbit’s sera. Passive immunization was also carried out in another group of mice using two doses of anti-CbPs antibodies. Finally, these mice were challenged and their survival as well as bacterial clearance were determined. The mice actively immunized with CbpM, CbpG and CbpL recombinant proteins showed survival rate of 100%, 85% and 75%, respectively. The survival rates among passively immunized mice groups which received100µg/ml dose of anti-CbpM, anti-CbpG and anti- CbpL were 50%, 50% and 25%, respectively. For all groups, however, only 25% of mice which received 10µg/ml dose of antibody survived after bacterial challenge. The rates of opsonization with anti-CbpM, anti-CbpG, and anti-CbpL antibodies at 100 and 10µg/ml doses, were found to be 45.6 and 14.7%, 82.3 and 12.9% and 12.2 and 9.35%, respectively. Our findings suggest that the recombinant proteins particularly CbpM and CbpG can protect the mice against pneumococcus19F serotype and effectively induce a protective antibody response. Thus, CbpG and CbpM proteins might be used as suitable vaccine candidate in pneumococcal vaccine formulations.

One of the promising methods in fertility preservation among women with cancer is cryopreservation of ovarian cortex but there are many drawbacks such as apoptosis and considerable reduction of follicular density in the transplanted ovary. One solution to reduce ischemic damage is enhancing angiogenesis after transplantation of ovarian cortex tissue.

The aim of this study was to investigate the effect of Setarud, on angiogenesis in transplanted human ovarian tissue.

In this case-control study, twenty-four nude mice were implanted subcutaneously, with human ovarian tissues, from four women. The mice were randomly divided into two groups (n=12): the experimental group was treated with Setarud, while control group received only vehicle. Each group was divided into three subgroups (n=4) based on the graft recovery days post transplantation (PT). The transplanted fragments were removed on days 2, 7 and 30 PT and the expression of Angiopoietin-1, Angiopoietin-2, and VEGF at both gene and protein levels and vascular density were studied in the grafted ovarian tissues.

On the 2nd and 7th day PT, the level of Angiopoietin-1 gene expression in case group was significantly lower than that in control group, while the opposite results were obtained for Angiopoietin-2 and VEGF. These results were also confirmed at the protein level. The density of vessels in Setarud group elevated significantly on day 7 PT compared to pre-treatment state.

Our results showed that administration of Setarud may stimulates angiogenesis in transplanted human ovarian tissues, although further researches are needed before a clear judgment is made.

Methylenetetrahydrofolate reductase (MTHFR) single-nucleotide polymorphisms (SNPs) C677T and A1298C have been described as strong risk factors for idiopathic recurrent miscarriage (RM). However, very few studies have investigated the association of paternal MTHFR SNPs with RM. The aim of the present study was to evaluate the prevalence of paternal C677T and A1298C SNPs among Iranian RM couples. The study subjects comprised 225 couples with more than three consecutive pregnancy losses, and 100 control couples with no history of pregnancy complications. All females in the case group had MTHFR polymorphisms; and genotype SNPs were analyzed by PCR-RFLP. Groups were statistically compared using Mann Whitney U-test and Chi-square statistical tests. The p<0.05 were considered significant. Statistically significant difference was detected in the frequency of MTHFR SNPs in male partners of the two groups (p=0.019). Combined heterozygosity of MTHFR polymorphisms was a common phenomenon in the males; 52 (23.1%) and 14 14%) of males in RM and control groups, respectively. Absence of combined homozygosity for both SNPs in all studied groups/genders was observed. The MTHFR gene composition of male partners of RM couples may contribute to increased risk of miscarriage.

GnRH agonist administration in the luteal phase has been suggested to beneficially affect the outcome of intracytoplasmic sperm injection (ICSI) and embryo transfer (ET) cycles. This blind randomized controlled study evaluates the effect of GnRH (Gonadotropine Releasing Hormone) agonist administration on ICSI outcome in GnRH antagonist ovarian stimulation protocol in women with 2 or more previous IVF/ICSI-ET failures. One hundred IVF failure women who underwent ICSI cycles and stimulated with GnRH antagonist ovarian stimulation protocol, were included in the study. Women were randomly assigned to intervention (received a single dose injection of GnRH agonist (0.1 mg of Decapeptil) subcutaneously 6 days after oocyte retrieval) and control (did not receive GnRH agonist) groups. Implantation and clinical pregnancy rates were the primary outcome measures. Although the age of women, the number of embryos transferred in the current cycle and the quality of the transferred embryos were similar in the two groups, there was a significantly higher rate of implantation (Mann Whitney test, p=0.041) and pregnancy (32.6% vs. 12.5%, p=0.030, OR=3.3, 95%CI, 1.08 to 10.4) in the intervention group. Our results suggested that, in addition to routine luteal phase support using progesterone, administration of 0.1 mg of Decapeptil 6 days after oocyte retrieval in women with previous history of 2 or more IVF/ICSI failures led to a significant improvement in implantation and pregnancy rates after ICSI following ovarian stimulation with GnRH antagonist protocol.

Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and pro-duction of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene ex-pression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p<0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p<0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dose-dependent manner (p<0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p<0.05). Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus.

Recurrent pregnancy loss (RPL) is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes.

Conflicting data suggest an association between estrogen receptor alpha (ESR1) gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL.

In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects.

The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups (p=0.20 and p=0.09, respectively). A significantly negative correlation was observed between -397C/T and -351A/G (r=-0.852, p<0.001) in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found (D’: 0.959; r-square= 0.758, p<0.001).

This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population.

Our preliminary data on the protein expression of SORT1 in ovarian carcinoma tissues showed that sortilin was overexpressed in ovarian carcinoma patients and cell lines, while non-malignant ovaries expressed comparably lower amount of this protein. In spite of diverse ligands and also different putative functions of sortilin (NTR3), the function of overexpressed sortilin in ovarian carcinoma cells is an intriguing subject of inquiry. The aim of this study was, therefore, to investigate the functional role of sortilin in survival of ovarian carcinoma cell line. Expression of sortilin was knocked down using RNAi technology in the ovarian carcinoma cell line, Caov-4. Silencing of SORT1 expression was assessed using real-time qPCR and Western blot analyses. Apoptosis induction was evaluated using flow cytometry by considering annexin-V FITC binding. [3H]-thymidine incorporation assay was also used to evaluate cell proliferation capacity. Real-time qPCR and Western blot analyses showed that expression of sortilin was reduced by nearly 70-80% in the siRNA transfected cells. Knocking down of sortilin expression resulted in increased apoptosis (27.5±0.48%) in siRNA-treated ovarian carcinoma cell line. Sortilin silencing led to significant inhibition of proliferation (40.1%) in siRNA-transfected Caov-4 cells as compared to mock control-transfected counterpart (p<0.05). As it was suspected from overexpression of sortilin in ovarian tumor cells, a cell survival role for sortilin can be deduced from these results. In conclusion, the potency of apoptosis induction via silencing of sortilin expression in tumor cells may introduce sortilin as a potential candidate for developing a novel targeted therapy in patients with ovarian carcinoma.

Pro-inflammatory and anti-inflammatory cytokines and polymorphisms of their genes have been described to be involved in the pathogenesis of recurrent miscarriage (RM). To investigate the association between RM and five polymorphisms of cytokine genes, interleukin 10 (IL-10), (-592 A/C, -819 C/T, -1082 A/G), IL-6 (-174 C/G) and IL-17 (-197 G/A) in Iranian women. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to determine the frequencies of the IL-6, IL-10 and IL-17 gene polymorphisms in 85 women with RM compared with 104 healthy controls. The frequencies of IL- 10 promoter gene polymorphisms (-592 A/C and -819 C/T) were significantly higher in RM women than those in controls (p=0.003). However, no statistically significant differences were observed in the frequencies of IL-6 (-174 C/G), IL-10 (-1082 A/G) and IL-17 (-197 G/A) polymorphisms between RM women and controls. These results suggest that IL-10 gene polymorphism screening might have some relevance in patients with RM, a suggestion which requires further studies.

Antibodies against actin, as one of the most widely studied structural and multifunctional housekeeping proteins in eukaryotic cells, are used as internal loading controls in western blot analyses. The aim of this study was to produce polyclonal antibody against a synthetic peptide derived from N-terminal region of β-actin protein to be used as a protein loading control in western blot and other assay systems. A synthetic peptide derived from β-actin protein was designed and conjugated to Keyhole limpet hemocyanin (KLH (and used to immunize a white New Zealand rabbit. The antibody was purified from serum by affinity chromatography column. The purity of the antibody was determined by SDS-PAGE and its ability to recognize the immunizing peptide was measured by ELISA. The reactivity of the antibody with β-actin protein in a panel of different cell lysates was then evaluated by western blot. In addition, the reactivity of the antibody with the corresponding protein was also evaluated by Immunocytochemistry and Immunohistochemistry in different samples. The antibody could recognize the immunizing peptide in ELISA. It could also recognize β-actin protein in western blot as well as in immunocytochemistry and immunohistochemistry. Our data suggest that this antibody may be used as an internal control in western blot analyses as well as in other immunological applications such as ELISA,immunocytochemistry and immunohistochemistry.

Human CD34 is a transmembrane glycoprotein which is expressed in human hematopoietic stem cells (HSCs) and the small- vessel endothelial cells of a variety of tissues. CD34 plays a critical role as a marker for diagnosis and classification of leukemia. Anti CD34 antibodies are used for isolation and purification of HSCs from bone marrow, peripheral blood and cord blood. To characterize a newly produced monoclonal antibody against a human CD34 peptide. Anti CD34 monoclonal antibody (Clone 2C10-D3) was purified from mouse ascitic fluid and hybridoma cell culture supernatants by affinity chromatography and its immune reactivity was examined by ELISA. The purified antibody was further characterized using Western blot and flow cytometry on TF1 (Human Erythroblast) cell line. ELISA experiment revealed that the antibody recognized CD34 peptide. Western blot analysis on TF1 cell lysate confirmed the reactivity of the antibody with a 42 KDa protein. Blocking the antibody with a saturating concentration of specific CD34 peptide resulted in loss of its activity with TF1 lysate in Western blot. The 2C10-D3 antibody reacted with TF1 cells in flow cytometry in a similar manner to a commercial anti CD34 monoclonal antibody. Our data suggest that the anti CD34 monoclonal antibody (Clone 2C10-D3) is an appropriate antibody to study the CD34+ cells by flow cytometry and Western blot.

The antibody response to hepatitis B surface antigen (HBsAg) controls hepatitis B virus infection. The "a" determinant of HBsAg is the most important target for protective antibody response, diagnosis and immunoprophylaxis. Mutations in this area may induce immune escape mutants and affect the performance of HBsAg assays. To construct clinically relevant recombinant mutant forms of HBsAg and assessment of their reactivity with anti-HBs monoclonal antibodies (MAbs). Wild type (wt) and mutant (mt) HBsAg genes were constructed by site directed mutagenesis and SEOing PCR. The amplified genes were inserted into pCMV6-neo plasmid and transfected in CHO cell line. The expression of wt- and mtHBsAg was assessed by commercial ELISA assays and stable cells were established and cloned by limiting dilution. The recombinant mutants were further characterized using a panel of anti-HBs monoclonal antibodies (MAbs) and the pattern of their reactivity was assessed by ELISA. Ten HBsAg mutants having single mutation within the "a" determinant including P120E, T123N, Q129H, M133L, K141E, P142S, D144A, G145R, N146S and C147S together with a wt form were successfully constructed and expressed in CHO cells. Reactivity of anti-HBs MAbs with mtHBsAgs displayed different patterns. The effect of mutations on antibody binding differed depending on the amino acid involved and its location within the ‘‘a’’ determinant. Mutation at amino acids 123 and 145 resulted in either complete loss or significant reduction of binding to all anti-HBs MAbs. Our panel of mtHBsAgs is a valuable tool for assessment of the antibody response to HBV escape mutants and may have substantial implications in HBV immunological diagnostics.

Spermatogonial stem cells (SSCs), a subset of undifferentiated type A spermatogonia, are the foundation of complex process of spermatogenesis and could be propagated in vitro culture conditions for long time for germ cell transplantation and fertility preservation. The aim of this study was in vitro propagation of human spermatogonial stem cells (SSCs) and improvement of presence of human Germ Stem Cells (hGSCs) were assessed by specific markers POU domain, class 5, transcription factor 1 (POU5F1), also known as Octamer-binding transcription factor 4 (Oct-4) and PLZF (Promyelocytic leukaemia zinc finger protein). Human testicular cells were isolated by enzymatic digestion (Collagenase IV and Trypsin). Germ cells were cultured in Stem-Pro 34 media supplemented by growth factors such as glial cell line-derived neurotrophic factor, basic fibroblast growth factor, epidermal growth factor and leukemia inhibitory factor to support self-renewal divisions. Germline stem cell clusters were passaged and expanded every week. Immunofluorecent study was accomplished by Anti-Oct4 antibody through the culture. The spermatogonial stem cells genes expression, PLZF, was studied in testis tissue and germ stem cells entire the culture. hGSCs clusters from a brain dead patient developed in testicular cell culture and then cultured and propagated up to 6 weeks. During the culture Oct4 were a specific marker for identification of hGSCs in testis tissue. Expression of PLZF was applied on RNA level in germ stem cells. hGSCs indicated by SSCs specific marker can be cultured and propagated for long-term in vitro conditions.

The development of an effective subunit vaccine against brucellosis is a research area of intense. But optimization of recombinant proteins production in Escherichia coli and content of endotoxins associated with final recombinant proteins are very important.. In the present study, expression and purification of Brucella melitensis rHSP and rTF were optimized to reduce endotoxin contaminants.. pDEST-tf and pDEST-hsp were transformed into E. coli BL21 (DE3), and then B. melitensis recombinant HSPA and TF proteins were overexpressed. Purification of these proteins was optimized to remove most of endotoxin contaminants from the end product using 0.1% Triton X-114 in washing buffers.. An endotoxin reduction of less than 0.05 EUmg/1 was achieved with protein recovery close to an 80% yield.. As this new protocol requires only one step to simultaneously purify tagged proteins and eliminate endotoxins, it represents a substantial advantage in time, effort, and expense..

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˚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..

Nowadays, Chlamydia trachomatis is known as a causative agent of infertility. Because of, asymptomatic nature of infection, many may suffer from its lasting complications such as infertility. This study was performed in Tehran during April 2007 to April 2008 to compare the prevalence of Chlamydia trachomatis infection in fertile and infertile women using ELISA and PCR methods. Overall, 234 infertile and 223 pregnant women, as the fertile group, participated in this hospital-based case-control study. After completing an informed consent form and the questionnaire, first catch urine and blood sample were obtained for PCR and ELISA (IgG, IgM) tests, respectively. Logistic regression analysis was used to control possible confounding factors, and determine adjusted odds ratio of infertility due to the infection. PCR results revealed that 29 (12.4%) of the infertile and 19 (8.5%) of the fertile women were positive for C. trachomatis infection (p=0.440). IgG was positive in 21 (9.0%) of the infertile and 11 (5.0%) in the fertile group (p=0.093). IgM assays identified that 2 (0.9%) of the infertile and 4 (1.8%) of the fertile women were positive for the micro-organism (p=0.375). We found no significant differences among fertile and infertile women for Chlamydia trachomatis infection. Nevertheless, molecular techniques which are more sensitive, more specific and non-invasive can be used to detect C. trachomatis infection.

Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East. In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein (DnaK) and an outer membrane protein (Omp31) of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis.Results and It was proved that immunized serum contains antibodies against recombinant Omp31 (rOmp31) and DnaK (rDnaK) by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy.

Spermatogonial stem cells are subpopulation of spermatogonial cells in testis tissue that support beginning and maintenance of spermatogenesis. Ubiquitin carboxy-terminal hydrolase L1 (UCHL1) could be a specific marker for identification of spermatogonial stem cells including spermatogonial sperm cells (SSCs) in testis tissue and during the culture; therefore we undertook this study to culture these human testicular stem cells (hTSCs) in vitro and approved the presence of human testicular stem cells (hTSCs) by UCHL1, also known as PGP9.5. Enzymatic digestion of human testicular biopsies was done by collagenase IV (4 mg/ml) and trypsin (0.25%). Differential plating of testicular cells in DMEM/F12 and 10% FBS was applied for 16 hr. Floating cells were collected and transferred onto laminin-coated plates with Stem-Pro 34 media supplemented with growth factors of GDNF, bFGF, EGF and LIF to support self-renewal divisions; testicular stem cell clusters were passaged every 14 days for two months. Spermatogonial cells propagation was studied through Expression of UCHL1 in testis tissue and the entire testicular stem cell culture. Testicular stem cell clusters from 10 patients with obstructive azoospermia were cultured on laminin-coated plates and subsequently propagated for two months. The average of harvested viable cells was approximately 89.6%. UCHL1 was expressed as specific marker in testicular stem cells entire the culture. Human testicular stem cells could be obtained from human testicular tissue by a simple digestion, culturing and propagation method for long-term in vitro conditions. Propagation of these cells approved by specific marker UCHL1, during the culture period.

We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

R-Phycoerythrin (R-PE), a fluorescent protein from phycobiliprotein family, is isolated from red algae. Conjugation of antibodies to R-PE facilitates multiple fluorescent staining methods. In the present study polyclonal antibodies and polyclonal F(ab'')2 fragment antibodies were conjugated to R-PE by two different methods. The efficiency of the methods was evaluated using Immunocytochemistry (ICC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE). In the first conjugation method, PE was attached to SMCC linker followed by conjugation of antibody to PE-SMCC. In the second method, SH groups were added onto R-PE molecule, while the antibody was attached to SPDP linker. Then, the antibody-SPDP molecule was conjugated to R-PE. Our results showed that the two conjugation methods did not have any abrogative effects on the antibody binding activity.

In many acute leukemias, normal differentiation does not occur. However, in many cell lines derived from hematologic malignancies, differentiation or programmed cell death (apoptosis) can be induced by variety of agents including: Vitamin analogs, demethylating agents, cyclic AMP analogs and anti-proliferative agents. To the best of our knowledge there has been not any study specifically to analyze apoptotic and anti-proliferative effects of 4-HPR (a vitamin analog) in NB-4 cell line. To test whether this drug has activity in acute myeloid leukemia (AML), we first analyzed the anti-proliferative effect of 4-HPR in one AML cell line (NB-4) using MTT Assay. Next we tested whether this drug induced apoptotic cell death. The ability of this compound to induce apoptosis of cancer cells was examined by Annexin V-FITC Assay using Flow cytometry. We also analyzed the cell cycle progression by PI staining using flow cytometry. Using MTT assay, NB-4 cells exhibited increased inhibition of proliferation at micromolar concentrations of 4-HPR at 24, 48 and 72 hrs post treatment. Flow cytometry analysis indicates that 4-HPR is a potent inducer of in vitro apoptotic cell death, and cell cycle analysis revealed an increase in S phase population. In total, the results indicate that 4-HPR is a strong inhibitor of AML cell proliferation and a potent inducer of in vitro apoptotic cell death. Further studies are required to evaluate the in vitro effects of 4-HPR in AML blasts derived from AML patients.

Spermatogonia are the male germ line stem cells whose life long expansion is needed for permanent production of spermatozoa. The present study was designed to examine the effect of hCG treatment on germ cell proliferation following stem cell transplantation in mice. Spermatogonial stem cells were isolated from neonatal mice testes and characterized by alkaline phosphatase, immunoreactivity and morphological analysis. hCG was injected into normal and cell transplanted mice. We then evaluated the testosterone levels and cell number in normal mice. After that, cyclin B1 gene expression was investigated in transplanted mice. Different doses of busulfan were injected to investigate the effects of chemotherapy on morphological criteria and preparation of recipient mice for transplantation. In this report we show proliferative potential of spermatogonial stem cells after cytotoxic treatment, transplantation efficiency by semi-quantitative RT-PCR, and hCG effect on stem cell regeneration in normal mice and following cell transplantation. The results indicate that spermatogonial stem cells can proliferate after transplantation, and the efficiency of their transplantation depends on hormonal treatment. Therefore, hormonal treatment after stem cell transplantation will be a powerful avenue for increasing the efficiency of transplantation and fertility restoration.