Comparison of Three Different Methods of Transfection for the Production of Recombinant Adenovirus Expressing Human Carcinoembryonic Antigen Gene.

Adenoviral vectors (AdVs) are widely used as a gene delivery vehicle and vaccine design due to their genetic stability, transfer capacity of large genes, production at high titers, and remarkable efficacy of transduction. One of the most important applications of AdVs is in cancer immunotherapy. Tumor-associated antigens are overexpressed in cancer cells; however, they cannot induce immune responses sufficiently. Therefore, the immune system must be stimulated against these antigens to kill the cancer cells. This study described the construction steps of a recombinant AdV expressing human carcinoembryonic antigen (CEA) gene. Furthermore, in order to achieve a high titer of the virus, an efficient transfection was required. Three various transfection reagents were compared to achieve the best method of transfection. Carcinoembryonic antigen was cloned into the pAdV and transfected into the A293 cells using three different reagents, including polyethylenimine (PEI), calcium phosphate, and DMRIE-C. The PEI had the highest transfection efficiency, which was selected for the transfection of the recombinant plasmid. It has low toxicity for cells and is suitable for large-scale transfection. The virus produced in this study can be applied as a vaccine in cancer immunotherapy for stimulating the immune system against CEA-expressing tumors.

Synergistic Effects of NDRG2 Overexpression and Radiotherapy on Cell Death of Human Prostate LNCaP Cells.

BACKGROUND: Radiation therapy is among the most conventional cancer therapeutic modalities with effective local tumor control. However, due to the development of radio-resistance, tumor recurrence and metastasis often occur following radiation therapy. In recent years, combination of radiotherapy and gene therapy has been suggested to overcome this problem. The aim of the current study was to explore the potential synergistic effects of N-Myc Downstream-Regulated Gene 2 (NDRG2) overexpression, a newly identified candidate tumor suppressor gene, with radiotherapy against proliferation of prostate LNCaP cell line. MATERIALS AND METHODS: In this study, LNCaP cells were exposed to X-ray radiation in the presence or absence of NDRG2 overexpression using plasmid PSES- pAdenoVator-PSA-NDRG2-IRES-GFP. The effects of NDRG2 overexpression, X-ray radiation or combination of both on the cell proliferation and apoptosis of LNCaP cells were then analyzed using MTT assay and flow cytometery, respectively. RESULTS: Results of MTT assay showed that NDRG2 overexpression and X-ray radiation had a synergistic effect against proliferation of LNCaP cells. Moreover, NDRG2 overexpression increased apoptotic effect of X-ray radiation in LNCaP cells synergistically. CONCLUSION: Our findings suggested that NDRG2 overexpression in combination with radiotherapy may be an effective therapeutic option against prostate cancer.

The possible role of NS3 protease activity of hepatitis C virus on fibrogenesis and miR-122 expression in hepatic stellate cells.

The various roles of hepatitis C virus (HCV) NS3 protein in viral pathogenesis are emphasized, especially in the progression of fibrosis and tumors. The levels of miR-122 have been widely accepted as a critical factor in viral pathogenesis and disease progression. However, the possible correlation between miR-122 levels and fibrosis state has been less investigated. Therefore, in this study, plasmids expressing protease competent and protease mutated non-structural proteins 3 (NS3) were transfected into LX-2 cell line. Subsequently, the total RNA was extracted and real-time PCR was performed to measure the expression level of miR-122, collagen type 1 alpha 1 (COL1A1), alpha smooth muscle actin (α-SMA), and tissue inhibitor of metaloproteinase 1 (TIMP-1). Moreover, the transforming growth factor beta (TGF-ß) levels in the supernatants of transfected cells were evaluated by ELISA. The gene expression analysis of fibrotic genes and TGF-ß cytokine in LX-2 cells showed that protease competent NS3 had a significant fibrogenic impact when compared to protease defective NS3 or GFP control plasmids (P <0.001). The results also demonstrated that the expression of miR-122 was downregulated in both versions of the cells transfected with NS3 plasmids (P <0.01) irrespective of protease function. These results suggested that the protease function of NS3 protein is a crucial factor for the induction of hepatic fibrosis but it doesn't play a complete role in the expression of miR-122.

Seroepidemiology of human T-cell lymphotropic virus among Iranian adult thalassemic patients.

BACKGROUND: A large number of transfusion-dependent thalassemic patients is at a substantial risk for transfusion-transmitted infections. Human T-cell lymphotropic virus (HTLV) is a blood-borne pathogen and can be transmitted via cellular products. We aimed to evaluate the seroprevalence of HTLV in transfusion-dependent thalassemic patients referred to Tehran Adult Thalassemia Clinic. METHODS: From 2008 to 2010, 257 transfusion-dependent thalassemic patients who referred to Tehran Adult Thalassemia Clinic were enrolled. The seroprevalence of HTLV, hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV were assessed using enzyme-linked immunosorbant assay (ELISA). Also, the samples with positive result for anti-HTLVAb (by ELISA) were reassessed using Western blot for HTLV. RESULTS: Among the 257 transfusion-dependent thalassemic patients who were tested for anti-HTLVAb, 29 (11.3%, 95% CI = 7.8-15.6%) were found to be anti-HTLVAb positive by ELISA and Western blot. No case was detected to be HBsAg positive, whereas 16% had HBV seroconversion criteria, and more than 95% had anti-HBsAb in their sera. Also, 103 (40.1%) patients were HCV seropositive, 13 (5.1%) patients of which were co-infected with HCV/HTLV. Among the HTLV-infected patients, 44.8% were co-infected with HCV, whereas 39.5% of HTLV-seronegative individuals were HCV mono-infected (P > 0.05). CONCLUSION: This study showed that transfusion-dependent thalassemic patients were in higher risk for transmission of different blood-borne pathogens such as HTLV. The screening of HTLV in Iranian blood donors is recommended.

Laboratory effect of HBV infection in kidney transplant recipients and donors.

BACKGROUND: Viral infections are the most common cause of opportunistic infections after kidney transplantation. Among hepatotropic viruses that induce kidney graft failure and rejection, hepatitis B virus (HBV) has an important and critical role. Extrahepatic HBV-related disorders increase morbidity and mortality in kidney transplant recipients. OBJECTIVE: To analyze the molecular prevalence of HBV infection in kidney transplant recipients and donors before and after transplantation. PATIENTS AND METHODS: This cross-sectional study included 273 serum samples collected between 2005 and 2008 in 96 kidney transplant recipients and 59 donors. Detection of HBV DNA was via amplification of the S gene fragment of HBV genome using a qualitative simple polymerase chain reaction assay. Also analyzed were statistical relationships between HBV infection and laboratory and clinical demographic data in all kidney transplant donors and recipients. RESULTS: The HBV genome was detected in 102 of 273 serum samples. Molecular HBV infection was demonstrated in 2 of 13 serum samples (15.4%) from recipients tested before transplantation. HBV DNA was detected in 42 of 96 patients (43.7%) after kidney transplantation. The HBV genome was demonstrated in 21 of 59 donors (35.6%).Significant relationships were observed between HBV infections and hematologic and biochemical indices after kidney transplantation. CONCLUSION: Detection of a high molecular prevalence of HBV infection in kidney recipients enforces the importance of HBV infection in clinical outcome.

Atorvastatin improves the response to sildenafil in hypercholesterolemic men with erectile dysfunction not initially responsive to sildenafil.

Despite the initial enthusiasm, the significant number of patients in whom sildenafil is contraindicated or ineffective is a major challenge to all urologists. Our aim was to determine the safety and efficacy of adjunctive atorvastatin in restoring normal erectile function in hypercholesterolemic (low-density lipoprotein (LDL) cholesterol >120 mg per 100 ml) sildenafil nonresponders. The study comprised 131 men with ED not responding to sildenafil citrate. They were randomized either to 40 mg atorvastatin daily (n=66, group 1) or matching placebo (n=65, group 2) for 12 weeks while they were taking on-demand 100 mg sildenafil. Erectile function was subjectively assessed using the 5-item version of the International Index of Erectile Function (IIEF-5) questionnaire and response to the global efficacy question (GEQ). Serum biochemical and lipid profile (total cholesterol, triglycerides, LDL cholesterol and high-density lipoprotein cholesterol) analyses were performed at baseline and repeated at post-treatment weeks 6 and 12. Compared with the placebo group (59 patients, mean age+/-s.d. 61.9+/-6.1, mean years ED 3.9+/-1.8), the atorvastatin group (59 patients, mean age+/-s.d. 63.9+/-6.9, mean years ED 3.7+/-1.6) had significantly greater improvements in all IIEF-5 questions (P=0.01) and GEQ (P=0.001). Subgroup analyses did reveal trends in the atorvastatin group to indicate that a change in the IIEF-5 score is affected by age, severity of ED and baseline serum levels of LDL. Patients with moderate (r=0.28, P=0.01) and severe (r=0.20, P=0.01) ED had better positive response rates to adjunctive atorvastatin than patients with mild to moderate ED. None of the patients taking atorvastatin achieved a response of 5 to the IIEF-5 questions and none of the patients regained normal erectile function as defined by the IIEF-5 score >21. Subjects experienced a statistically significant but modest improvement in erectile function. Further investigation is needed to test the usefulness of long-term atorvastatin administration to restore erectile function in sildenafil nonresponders.

Incidental prostatic adenocarcinoma in patients with PSA less than 4 ng/mL undergoing radical cystoprostatectomy for bladder cancer in Iranian men.

OBJECTIVE: To assess the incidence of prostate adenocarcinoma in patients undergoing radical cystoprostatectomy due to bladder cancer in Iranian men. MATERIALS AND METHODS: Fifty cystoprostatectomy specimens removed due to bladder malignancy (2004-2005) at two referral centers (Shaheed Modarress and Shaheed Labbafinejad Hospitals, Tehran, Iran) were examined for the coincidental finding of prostate cancer (PCa). At the time of surgery the patient's serum PSA was less than 4 ng/mL and there were no suspicious lesions by digital rectal examination. Pathologic grade, stage, morphometric volume, number of tumor foci and association with areas of high grade prostatic intraepithelial neoplasia (HGPIN) were assessed by light microscopy. All specimens were totally embedded and whole-mounted. Clinically significant cancers were defined as tumors with >or= 0.5 mL volume, Gleason pattern 4 or 5, pT3, positive surgical margin, and multifocality > 3. RESULTS: Incidentally detected cancer was found in 7 (14%) of cystoprostatectomy specimens. HGPIN was present in 1 (14.3%) of the cystoprostatectomies with incidentally detected prostate cancer. None of cystoprostatectomies without prostate cancer had HGPIN. Four (57%) of the detected cancers were significant. CONCLUSION: We conclude that incidentally detected prostate cancer in Iran is lower than the rates reported in other countries. Further studies are warranted for better declaration of variability of prostate cancer between different ethnic groups.

Incidental prostatic adenocarcinoma in patients with PSA less than 4 ng/ml undergoing radical cystoprostatectomy for bladder cancer in iranian men

OBJECTIVE:To assess the incidence of prostate adenocarcinoma in patients undergoing radical cystoprostatectomy due to bladder cancer in Iranian men. MATERIALS AND METHODS: Fifty cystoprostatectomy specimens removed due to bladder malignancy (2004-2005) at two referral centers (Shaheed Modarress and Shaheed Labbafinejad Hospitals, Tehran, Iran) were examined for the coincidental finding of prostate cancer (PCa). At the time of surgery the patient's serum PSA was less than 4 ng/mL and there were no suspicious lesions by digital rectal examination. Pathologic grade, stage, morphometric volume, number of tumor foci and association with areas of high grade prostatic intraepithelial neoplasia (HGPIN) were assessed by light microscopy. All specimens were totally embedded and whole-mounted. Clinically significant cancers were defined as tumors with 0.5 mL volume, Gleason pattern 4 or 5, pT3, positive surgical margin, and multifocality > 3. RESULTS: Incidentally detected cancer was found in 7 (14 percent) of cystoprostatectomy specimens. HGPIN was present in 1 (14.3 percent) of the cystoprostatectomies with incidentally detected prostate cancer. None of cystoprostatectomies without prostate cancer had HGPIN. Four (57 percent) of the detected cancers were significant. CONCLUSION: We conclude that incidentally detected prostate cancer in Iran is lower than the rates reported in other countries. Further studies are warranted for better declaration of variability of prostate cancer between different ethnic groups.

Safety and efficacy of tramadol in the treatment of idiopathic detrusor overactivity: a double-blind, placebo-controlled, randomized study.

AIM: To evaluate the efficacy and safety of tramadol in patients with idiopathic detrusor overactivity (IDO). METHODS: A total of 76 patients 18 years or older with IDO were randomly assigned to receive 100 mg tramadol sustained release (group 1, n = 38) or placebo (group 2, n = 38) every 12 h for 12 weeks. Clinical evaluation was performed at baseline and every 2 weeks during treatment. All patients underwent urodynamics and ice water test at baseline and 12-week treatment. Main outcome measures were number of voids per 24 h, urine volume per void and episodes of urge incontinence per 24 h on a frequency volume chart and detailed recording of adverse effect. RESULTS: After 12 weeks of treatment mean number of voids per 24 h +/- SD decreased from 9.3 +/- 3.2 to 5.1 +/- 2.1 (P < 0.001 vs. placebo) [95% confidence interval (CI) -5.1--0.4]. At that time mean urine volume per void increased from 158 +/- 32 to 198 +/- 76 ml (P < 0.001 vs. placebo) (95% CI 8-22), while mean number of incontinence episodes per 24 h decreased from 3.2 +/- 3.3 to 1.6 +/- 2.8 (P < 0.001 vs. placebo) (95% CI -2-0.3). Tramadol induced significant improvements in urodynamic parameters. More adverse effects were associated with tramadol treatment than with placebo (P < 0.05). The main adverse event with tramadol was nausea. CONCLUSIONS: In patients with non-neurogenic IDO tramadol provided beneficial clinical and urodynamic results. Further studies are required to draw final conclusions on the efficacy of this drug in IDO.

Safety and efficacy of citalopram in the treatment of premature ejaculation: a double-blind placebo-controlled, fixed dose, randomized study.

We evaluate the efficacy and safety of citalopram, a potent and highly selective serotonin reuptake inhibitor (SSRI) antidepressant, in patients with premature ejaculation (PE). In total, 58 potent men with PE were included in the study. Patients were randomly assigned to receive 20 mg oral daily citalopram (group 1, n = 29) or placebo (group 2, n = 29), during a 12-week period for each agent. Pretreatment evaluation included history and physical examination, intravaginal ejaculatory latency time (IVELT) evaluation, International Index of Erectile Function (IIEF) and Meares-Stamey test. The efficacy of two treatments was assessed every 2 weeks during treatment, at the end of study and in 3- and 6-month follow-up after cessation of treatment, using responses to IIEF, IVELT evaluation, mean intercourse satisfaction domain, mean weekly coitus episodes and adverse drug effects. The trial was completed by 51 (88%) men. Analysis revealed a difference in the evolution of IVELT delay over time (P < 0.001). The IVELT after citalopram and placebo gradually increased from 32 and 28 s to approximately 268 and 38 s, respectively. The mean weekly intercourse episodes increased from pretreatment values of 1.3 and 1.2 to 2.4 and 1.4, for citalopram and placebo, respectively (P < 0.05). Baseline mean intercourse satisfaction domain values of IIEF 10 and 11 reached to 16 and 10 at 12-week treatment in groups 1 and 2, respectively (P < 0.05). Mean IVELT in group 1 was 210 and 198 s at 3- and 6-month follow-up, while in group 2 it was 27 and 25 s (P < 0.001), respectively. At 3- and six-month intercourse satisfaction domain values of IIEF were 15 and 14 in group 1 and 10 and 10 (P < 0.05) in group 2, respectively. Group 1 patients reported a significantly higher number of intercourse episodes per week (P < 0.05). Mean number of adverse events was 12 for citalopram and 4 for placebo (P < 0.05). In conclusion, these results indicate that citalopram has significantly better results in terms of IVELT and intercourse satisfaction versus placebo. Further studies with different dosages and treatment regimens are necessary to draw final conclusions on the efficacy of this drug in PE and to prolong the efficacy.

Erectile dysfunction: clinical guidelines (2).

PURPOSE: According to a survey, the Massachusetts Male Aging Study, 52% of men beyond 40 years of age may have some degrees of erectile failure, and it is projected to affect 322 million men worldwide by 2025. We present a framework for the evaluation, treatment, and follow-up of the male patient who presents with erectile dysfunction. MATERIALS AND METHODS: A comprehensive review of the literature was conducted using the MEDLINE database for all articles from 1975 through 2004 on male sexual dysfunction and the most pertinent articles are discussed. RESULTS: Remarkable progress has been made in the treatment of erectile dysfunction (ED). Erectile dysfunction is a common condition associated with aging, chronic illnesses and various modifiable risk factors. Erectile dysfunction can be due to vasculogenic, neurogenic, hormonal, and/or psychogenic factors as well as alterations in the nitric oxide/cyclic guanosine monophosphate pathway or other regulatory mechanisms. The number of consultations from new patients presenting with erectile dysfunction and resulting costs for health care systems are increasing. Urologist should be the evaluating physician who supervises the surgical, medical, and hormonal treatment and who refers the patient, as necessary, to other members of the multidisciplinary team. CONCLUSION: Erectile dysfunction has a significant negative impact on quality of life. Male sexual dysfunction, especially erectile dysfunction, necessitates a comprehensive medical and psychologic evaluation involving both partners. All possible risk factors should be outlined and corrected, when feasible.

Extracorporeal shock wave lithotripsy of renal calculi during early pregnancy.

OBJECTIVE: To evaluate the safety of renal stone treatment by extracorporeal shock wave lithotripsy (ESWL) during pregnancy. PATIENTS AND METHODS: Data from 824 women of reproductive age who were treated by ESWL were analysed and the patients surveyed using a questionnaire about pregnancies, to which 636 (76%) responded. Of these patients, six had inadvertently undergone ESWL for renal stones during the first month of pregnancy. The stones were detected and located during ESWL using ultrasonography. RESULTS: The six patients gave birth to six children who had no detectable malformations or chromosomal anomalies. CONCLUSION: Although these results suggest that there were no adverse effects of ESWL during early pregnancy, a larger series should be assessed to confirm the safety and long-term effects of ultrasound-guided ESWL in the treatment of renal calculi during pregnancy. We do not advocate lithotripsy as a treatment for renal calculi in pregnancy, but inadvertent lithotripsy in a pregnant woman is not a cause for concern.

Accuracy of residual urine measurement in men: comparison between real-time ultrasonography and catheterization.

PURPOSE: The practical value of ultrasonography as a rapid means to determine accurately residual urine volume was assessed. MATERIALS AND METHODS: Transverse and sagittal bladder diameters, as well as areas from longitudinal and transverse images, were measured with real-time ultrasonography in 324 men immediately after voiding. Calculated bladder volumes using measured diameters and areas for each of 11 formulas in the literature were compared to the corresponding measured total residual urine volumes. RESULTS: The lower limit of ultrasonographic visualization of urine in the bladder was approximately 48 ml. No correlation existed between ultrasound calculated bladder volumes and measured residual urine for any of the 11 formulas. CONCLUSIONS: Ultrasonography cannot rapidly measure bladder volumes accurately to date. Catheterization remains the most accurate method of assessing post-void residuals but in many cases it may not be the best approach to patient care.

Penile fractures: evaluation, therapeutic approaches and long-term results.

PURPOSE: Traumatic rupture of the corpus cavernosum of the penis is rare and has been reported infrequently. We demonstrated the moderate frequency and most common etiologies of this condition. MATERIALS AND METHODS: Between February 1990 and June 1993, 68 patients underwent an emergency operation 3 hours to 4 days after penile fracture. RESULTS: Among 32 patients who completed a questionnaire functional results were excellent except for 3 with penile curvature and pain during coitus who underwent surgery more than 48 hours after injury. CONCLUSIONS: Fracture of the penis is a common urological problem. In our series the most common cause was manipulation. To avoid serious complications immediate surgical intervention is recommended.