فهرست مطالب fatemeh ghaemimanesh

Cell surface expression of sortilin in different types of cancer signifies it as a therapeutic target for cancer therapy. The aim of this study was to detect sortilin expression in bladder cancer cells using an anti-sortilin monoclonal antibody (mAb) to evaluate sortilin as a target for developing diagnostic and therapeutic agents against bladder carcinoma.

The protein expression of sortilin in bladder cancer tissues and cell lines (5637 and EJ138) was investigated by immunohistochemistry (IHC), immune-cytochemistry (ICC), and flow cytometry. Furthermore, the capability of anti-sortilin mAb in apoptosis induction in bladder cancer cells was evaluated.

A high expression level was observed in bladder carcinoma tissues (P≤0.001) and cell lines, using IHC and ICC, respectively. Flow cytometry results showed cell surface expression of 27.5±3% (P≤0.01), 74.4±7.8% (P≤0.001), and 4.2±0.4% of sortilin in EJ138, 5637, and HFFF cells, respectively. In EJ138 anti-sortilin mAb induced apoptosis in 25.2±11.5% (P≤0.05) (early) and 4.5±1.1% (P>0.05) (late) after 6 h incubation, while for 12 h, the values of 11.6±3.8% (P>0.05) and 20.7±4.4% (P≤0.05) were achieved. In 5637 cells, 6 h incubation resulted in 10.2±0.3% (P>0.05) and 6.6±1.4% (P>0.05) apoptosis induction, while these values were 12.1±0.8% (P>0.05) and 27.4±4.5% (P≤0.01) after 12 h. The HFFF cells did not show significant apoptosis.

The overexpression of sortilin in bladder tumor cells and its potential in inducing apoptosis via directed targeting with the specific monoclonal antibody may represent this protein as a potential candidate of targeted therapy in bladder carcinoma.

The overexpression of sortilin/neurotensin receptor 3 has previously been reported in various human solid tumors but not in hematological malignancies. Here, we report the overexpression of sortilin in leukemic cells from patients with Chronic Lymphocytic Leukemia (CLL).

Flow cytometry was used to compare the expression of sortilin in CLL pa-tients (n=52) and healthy individuals (n=26). Also, in vitro apoptosis induction was assessed in CLL Peripheral Blood Mononuclear Cell (PBMCs) following directly targeting of sortilin.

The results showed a significant expression of sortilin on the surface of CLL PBMCs (range from 2.2 to 71.5%) in comparison to healthy individuals (range from 0.03 to 7.4%) (p≤0.0001). The optimal cut-off value of sortilin expression was deter-mined at 7.2% with high sensitivity and specificity. Treatment of leukemic cells with anti-sortilin antibody could induce apoptosis without any effect on normal cells.

Apoptosis induction in CLL cells together with a significant correlation between the expression of sortilin and CD23 represent a possible functional role of sortilin in leukemogenesis of CLL cells. Therefore, sortilin might be considered as a promising novel biomarker in diagnosis, monitoring, and therapy of patients with CLL.

We have previously reported the aberrant expression of Fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL). Although FMOD has been considered as a cytoplasmic or secretory protein, we discovered the cell surface expression of FMOD in leukemic B cells via anchoring with glycosylphosphatidylinositol (GPI). To evaluate FMOD as a new biomarker in CLL patients in comparison with healthy individuals. A monoclonal antibody was generated against human FMOD. The cell surface expression of FMOD in 52 CLL patients and 45 healthy individuals were compared by flow cytometry. A bacterial phosphatidylinositol-specific phospholipase C (PI-PLC) was used to determine the cell surface localization of FMOD using ELISA and flow cytometry techniques. Annexin V-FITC and propidium iodide (PI) was used to detect apoptosis induction in CLL PBMCs following in vitro incubation with anti-FMOD mAb. The results demonstrated the widespread cell surface expression of GPI-anchored FMOD in CLL patients (median: 79.9 %), although healthy individuals had low FMOD expression (median: 6.2 %) (p≤0.0001). The cut-off value of FMOD expression was estimated with high sensitivity and specificity at 17.9%. Furthermore, in vitro apoptosis induction of leukemic cells following incubation with anti-FMOD mAb showed a direct apoptosis of CLL cells (27.9%) with very low effect on healthy PBMCs (6%). The membrane-anchoring of FMOD by means of a GPI moiety in leukemic cells supports FMOD as a highly potential diagnostic and therapeutic target in CLL patients.

The unique expression of fibromodulin (FMOD) in patients with chronic lymphocytic leukemia (CLL) has been previously reported. Detecting FMOD in CLL patients using specific anti-FMOD mAbs might provide a promising method in detection, monitoring, and prognosis of CLL. In this study, we aimed for producing specific antibodies against FMOD to facilitate further cohort study of CLL, thus addressing FMOD as a potential target of detection. Human FMOD gene (1087 bp) was extracted from genome of the CLL patients, and was cloned into the expression vector of pET-22b (+). The recombinant FMOD protein (rFMOD) was expressed in Escherichia coli. The purified rFMOD protein was used as an immunogen in rabbit and mice. Hybridoma technology was used to develop the monoclonal antibodies (mAbs). Polyclonal antibody (pAb) was purified from the rabbit sera using affinity column. The reactivity of anti-FMOD antibodies was assessed in ELISA, immunocytochemistry (ICC) and Western blot. ICC results showed that the anti-FMOD antibodies specifically detected FMOD in CLL PBMCs and cell lines. The developed anti-FMOD pAb detected FMOD in CLL lysates, compared to healthy PBMCs, in Western blot and ELISA. The developed anti-FMOD mAbs, and pAb specifically detect FMOD in CLL samples and might be used as research tools for further investigations in CLL.

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.

Varicella zoster virus (VZV) is a member of herpes family viruses, which causes varicella (chickenpox) after primary infection and herpes zoster (shingles) because of latent virus reactivation from dorsal root ganglia. Generally, prevalence of varicella antibodies increases with age. We aimed to compare the prevalence of anti-VZV antibody in children under seven years old, in order to obtain a preliminarily picture of general presence of these antibodies to design an immunization plan. In this cross-sectional study, performed from September 2011 to September 2012 in Tehran, Iran, 267 serum samples including sera from 7 month old infants, n= 87; 18 month old children, n= 86; and 6 year old children, n= 94 were assessed for the presence of specific IgG antibodies against VZV, using ELISA technique. 4.6% of 7 month, 12.8% of 18 month and 21.3% of 6-year-old children were seropositive. No relation was found between demographic variables (e.g. age and birth weight) and seropositivity in these age groups. VZV antibodies increased with age. Serum levels of varicella antibodies were elevated in 18 months old compared to 7 months old children, significantly (P < 0.001). In view of the significant elevation of VZV antibodies in children from 7 months to 18 months of age and rate of seronegative children, our results support the necessity of varicella immunization between 7 and 18 months of age in order to prevent viral infection.

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.