Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

OBJECTIVES: The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. METHODS: The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. KEY FINDINGS: Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. CONCLUSIONS: This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

Renal cell carcinoma in children and adolescence: our experience.

BACKGROUND: Literature on renal cell carcinoma (RCC) in children is lacking. Occasional case report has been mentioned. Aims and objective of our study are to evaluate the clinical presentation and outcome in children with RCC. PATIENTS AND METHODS: Records of 11 children and adolescence, from January 2007 to June 2011, who were treated for RCC were retrospectively analysed. Age, clinical presentation, any paraneoplastic symptom or sign, haematological, bio-chemical investigations, radiological imaging's, operative details, pathological reports and treatment details were taken from hospital records and results were analysed. All patients were followed-up with complete haemogram, biochemical investigations, ultrasonography - whole abdomen and chest X-ray at 6 months interval and patients with stages 2 and 3 were also followed-up with contrast enhanced computed tomography - whole abdomen. They were followed-up for 2-5 years. RESULTS: All had undergone open radical nephrectomy with eight hilar lymph node dissection and three formal lymphadenectomy. None had received adjuvant therapy. Four patients with stage 1 were well at 5, 4, 2.5 and 2 years. One patient with stage 1 was lost to follow-up. Three patients with stage 2 were well at 4, 3 and 2 years of follow-up while three with stage 3 were well at 5, 4 and 2 years of follow-up. CONCLUSIONS: Lymph node dissection not only improves the survival, but it guides one the exact pathological staging and one can adopt the more aggressive follow-up in advanced pathological staging and strict follow-up is mandatory.

Emphysematous pyelonephritis: Our experience with conservative management in 14 cases.

CONTEXT: Emphysematous pyelonephritis (EPN) is a rare, severe, acute, necrotizing infection of the kidney. In this study, we present the clinical details, the management strategies, and the outcome of fourteen patients of EPN managed at our center. MATERIALS AND METHODS: A retrospective analysis of the hospital records was done. A total of fourteen patients with EPN were admitted in our hospital from August 2007 to February 2011. All the patients were managed conservatively. Follow-up ranged from six months to one year. RESULTS: Of the fourteen patients, four belonged to class I, five to class II, four to class IIIA and one to class IIIB. All the patients had history of fever, 43% had localized flank pain while 36% had vague abdominal discomfort. Renal angle tenderness was the most common sign, seen in 86% of the patients. E. coli was the most common bacteria, which was isolated from urine in 57% of the patients. On the risk factor stratification, three patients had simultaneous presence of 2 or more risk factors (thrombocytopenia-2 patients; renal function impairment-7 patients; shock-1 patient). All the patients were initially managed with aggressive fluid and electrolyte resuscitation, control of blood sugar levels, and broad spectrum antibiotics. Intervention, in the form of percutaneous drainage or DJ stenting, was done in six patients. One patient failed to respond to this minimally invasive modality of treatment and had to undergo an open drainage. Thus, the acute episode was managed with conservative management strategies in all the patients; however, three patients underwent nephrectomy due to poorly-functioning kidney during follow-up. CONCLUSIONS: EPN is now being more readily diagnosed, at an early stage, making conservative management of EPN a safe, effective, and feasible option.

Novel nanosomes for gene delivery to Plasmodium falciparum-infected red blood cells.

Malaria threatens millions of people annually and is a burden to human health and economic development. Unfortunately in terms of disease control, no effective vaccines are available and the efficacy of treatment is limited by drug resistance. Genetic manipulation in Plasmodium falciparum is hampered due to the absence of robust methods for genetic analyses. Electroporation-based transfection methods have allowed the study of gene function in P. falciparum, with low efficiency. A lipid nanoparticle was developed that allowed nuclear targeting of pDNA with increased efficiency in reporter assay, compared to traditional electroporation method. This method has for the first time, facilitated transfection using both circular and linear DNA in P. falciparum thereby serving as an alternative to electroporation with an increase in transfection efficiency. Availability of a robust method for functional genomic studies in these organisms may be a catalyst for discovery of novel targets for developing drugs and vaccines.

Buccal mucosal graft urethroplasty in long segment anterior urethral stricture - is it gold standard?

To assess the success of dorsal onlay buccal mucosal graft (BMG) urethroplasty in long segment anterior urethral stricture extending from external meatus to bulbar urethra). We studied 40 patients with long segment anterior urethral stricture, who underwent substitution urethroplasty using dorsal onlay BMG from January 2002 to December 2007. The patients were in the age range of 15-65 years (mean 35 years) in the LS group and 16-63 years (mean 34 years) in the non-lichen sclerosus (NLS) group. The cause of stricture was LS in 20 and NLS (inflammatory and idiopathic) in the other 20 patients. The mean stricture length was 14.5 cm (range 12-17 cm) in the LS group while it was 14.0 cm (range 12-16 cm) in the NLS group. The patients were evaluated with antegrade, retrograde urethrograms and sono-urethrograms and they were followed- up with uroflometery at three months for one year, then six- monthly for two years and then annually. The contrast studies were repeated at six-monthly intervals for one year and then annually for one year. Success was defined as normal voiding pattern without any intervention post-operatively. Median follow-up was 48 months (18-72 months) in the LS group, while it was 42 months (12-72 months) in the NLS group. Among the NLS group patients, three patients developed restricture on follow-up, while seven patients among the LS group developed restricture. We conclude that the high percentage of recurrence of strictures (35%) among the LS group renders BMG urethroplasty in long segment anterior urethral stricture an unacceptable solution, and it needs further study.

Successful management of auto knotted intravesical infant feeding tube by minimal invasive technique.

We report a case of acute urinary retention due to intravesical auto knotting of infant feeding tube in a child treated successfully by endoscopic approach.

Renal pleomorphic undifferentiated sarcoma: a rarity.

A 70-year-old male presented with progressive weight loss for eight months. Radiological imaging showed a large tumor in the right kidney. The patient underwent right open radical nephrectomy and histopathology revealed pleomorphic undifferentiated sarcoma (PUS) earlier known as malignant fibrous histiocytoma (MFH). One year after surgery, the patient developed pulmonary metastasis. Unfortunately, the patient died after six months.

Collecting duct carcinoma: a rare renal tumor.

The most common primary malignant renal tumor is renal cell carcinoma (RCC), which accounts for 3% of all adult malignancies. Bellini duct carcinoma or collecting duct carcinoma is an unusual rare variant of RCC. This histologically distinct tumor is very rare, with less than 100 cases reported in the literature, and accounts for approximately 1% of all malignant renal epithelial tumors. We report two cases of collecting duct carcinoma and highlight the rarity of these tumors and their similarity to RCC.

Inhibition of hepatitis C virus replication by intracellular delivery of multiple siRNAs by nanosomes.

Sustained antiviral responses of chronic hepatitis C virus (HCV) infection have improved recently by the use of direct-acting antiviral agents along with interferon (IFN)-α and ribavirin. However, the emergence of drug-resistant variants is expected to be a major problem. We describe here a novel combinatorial small interfering RNA (siRNA) nanosome-based antiviral approach to clear HCV infection. Multiple siRNAs targeted to the highly conserved 5'-untranslated region (UTR) of the HCV genome were synthesized and encapsulated into lipid nanoparticles called nanosomes. We show that siRNA can be repeatedly delivered to 100% of cells in culture using nanosomes without toxicity. Six siRNAs dramatically reduced HCV replication in both the replicon and infectious cell culture model. Repeated treatments with two siRNAs were better than a single siRNA treatment in minimizing the development of an escape mutant, resulting in rapid inhibition of viral replication. Systemic administration of combinatorial siRNA-nanosomes is well tolerated in BALB/c mice without liver injury or histological toxicity. As a proof-of-principle, we showed that systemic injections of siRNA nanosomes significantly reduced HCV replication in a liver tumor-xenotransplant mouse model of HCV. Our results indicate that systemic delivery of combinatorial siRNA nanosomes can be used to minimize the development of escape mutants and inhibition of HCV infection.

Urinary tract endometriosis: Review of 19 cases.

AIM: The aim of our study was to evaluate the treatment outcomes of medical and surgical management of urinary tract endometriosis. MATERIALS AND METHODS: Urinary tract endometriosis patients enrolled between Jan 2006 and May 2010 were retrospectively reviewed. Preoperative datas (mode of presentation, diagnosis, imaging), intraoperative findings (location and size of lesion), postoperative histopathology and follow-up were recorded and results were analyzed and the success rate of different modalities of treatment was calculated. RESULTS: In our study, of nineteen patients, nine had vesical involvement and ten had ureteric involvement. Among the vesical group, the success rate of transurethral resection followed by injection leuproide was 60% (3/5), while among the partial cystectomy group, the success rate was 100%. Among patients with ureteric involvement, success rate of distal ureterectomy and reimplantation was 100%, laparoscopic ureterolysis with Double J stenting followed by injection leuprolide was 75% while that of Gonadotropin- releasing hormone (GnRh) analogue alone was 67%. CONCLUSION: One should have a high index of suspicion with irritative voiding symptoms with or without hematuria, with negative urine culture, in all premenopausal women to diagnose urinary tract endometriosis. Partial cystectomy is a better alternative to transurethral resection followed by GnRh analogue in vesical endometriosis. Approach to the ureter must be individualised depending upon the severity of disease and dilatation of the upper tract to maximise the preservation of renal function.

Development and optimization of nanosomal formulations for siRNA delivery to the liver.

The objective of this study is to develop an effective siRNA delivery system for successful delivery to the liver for the treatment of HCV. Nanosize liposomes (nanosomes) have been prepared using a mixture of cholesterol and DOTAP. A functional siRNA was encapsulated into nanosomes following condensation with protamine sulfate. The delivery of siRNA was optimized in an in vitro cell culture system. The efficacy of the formulations was evaluated by measuring functional gene silencing and cytotoxicity. Encapsulation of siRNA ≥ 7.4 nM resulted in successful delivery of siRNA to nearly 100% of cells. The formulations containing lipid-to-siRNA ratio ≥ 10.56:1 instantly cleared approximately 85% of HCV while maintaining cell viability at about 90%. The formulations were sonicated to further reduce the particle size. The size of these formulations was decreased up to 100 nm. However, there were no significant changes observed in zeta potential, or in siRNA encapsulation and integrity following sonication. The sonicated formulations also showed higher liver hepatocytes deposition and gene silencing properties. This study therefore provides a novel approach of siRNA delivery to liver hepatocytes, which can also be applied to treat HCV in chronic liver diseases.

Stability of lyophilized siRNA nanosome formulations.

The goal of this study is to evaluate the stability of lyophilized siRNA formulations. The gene silencing efficiency of a stored lyophilized siRNA formulation (i.e. siRNA nanosomes) was evaluated in interferon-α (IFN-α) resistant hepatitis C virus (HCV) at different time points up to three months in an in vitro cell culture model and compared with freshly prepared siRNA formulations. Novel siRNA sequences were encapsulated within nanosize liposomes following condensation with protamine sulfate. The siRNA encapsulated nanosomes were lyophilized and stored at 4 °C for 3 months, along with liquid liposomes (L) and lyophilized liposome powder (P) which were subsequently used to prepare siRNA nanosomes (L) and siRNA nanosomes (P), respectively at different time points. Physiochemical and biological properties of all three formulations were compared at different time points up to 3 months. The particle size of the stored siRNA nanosomes (642 ± 25 nm) was considerably larger initially in comparison with the liquid liposomes (134 ± 5 nm) and lyophilized liposomes (118 ± 3). However, the particle size gradually became smaller over time (413 ± 128 nm by the third month). The zeta potential of all three formulations was initially very high (> +40 mV), followed by a gradual decrease over time. The amount of siRNA in the stored siRNA nanosomes decreased ∼18 % during the 3 month storage period (1.16 ± 0.03 nmol initially on day 1 vs. 0.95 ± 0.04 nmol after 3 months). With respect to biological potency, all three formulations were significantly effective to knock-down HCV throughout the storage time. The cell viability was well-maintained throughout this period. Thus, this study indicates that the stored lyophilized siRNA formulation is as effective as the fresh preparation and that long-term storage could be a viable option to treat deadly diseases such as cancer and viral infection.

Rhabdomyolysis and myogloginuric acute renal failure in the lithotomy/exaggerated lithotomy position of urogenital surgeries.

OBJECTIVE: To evaluate rhabdomyolysis and it's management in lithotomy and the exaggerated lithotomy positions during urogenital surgeries. DESIGN: Retrospective study SETTING: Institute of Post Graduate Medical Education and Research (IPGME & R), Kolkata, India. MATERIALS AND METHODS: Patients undergoing urogenital surgeries (lithotomy and the exaggerated lithotomy positions). INTERVENTION(S): All four cases of rhabdomyolysis which occurred after such positional urogenital surgeries were treated with conservative management for prolonged period with hemodialysis. One case which developed compartment syndrome underwent fasciotomy and also managed with conservative approach as other cases. MAIN OUTCOME MEASURE: Rhabdomylysis is now a rare complication in any open or laparoscopic surgery. But prolonged lithotomy or exaggerated lithotomy position surgeries have been shown to expose patients to the risk of rhabdomylysis and acute renal failure. RESULTS: In our institute patients undergoing urogenital surgeries in lithotomy and the exaggerated lithotomy positions only developed rhabdomyolysis and myogloginuric acute renal failure. All procedures were of prolonged duration (mean five hours and ten minutes). Three patients developed rhabdomyolysis and acute renal failure without compartmental syndrome and one with compartmental syndrome. Rhabdomyolysis with the appearance of acute renal failure is discussed. CONCLUSION: Overall, our cases showed that rhabdomyolysis and acute renal failure can develop in such operative positions even in the absence of compartmental syndrome, and that duration of surgery is the most important risk factor for such complications. So we should be careful regarding duration of surgery in lithotomy procedure to prevent such morbid complications.

Prostatic abscess: diagnosis and management in the modern antibiotic era.

This retrospective study was aimed at analyzing the clinical findings and therapeutic strategies in 24 patients who were admitted with prostatic abscess, during the period from 1999 to 2008. The diagnosis of prostatic abscesses was made clinically by digital rectal palpation based on the presence of positive fluctuation with tenderness. All cases were confirmed by trans-rectal ultrasound (TRUS), and only positive cases were included in this study. The diagnostic work-up included analysis of midstream urine and abscess fluid culture for pathogens. Therapeutic options included endoscopic trans-urethral incision or trans-perineal aspiration under ultrasound guidance, or conservative therapy. Of the 24 patients studied, 45.83% of the cases had a pre-disposing factor, and diabetes mellitus (37.50%) was the most common. Digital rectal palpation revealed fluctuation in 70.83% of the cases. Trans-abdominal ultrasonography missed the condition in 29.16% of the cases. On TRUS, all the study patients showed hypo-echoic zones, while nine others showed internal septations. In most of the cases, the lesion was peripheral. A causative pathogen could be identified in 70.83% of the cases. Surgical drainage of the abscess by trans-urethral deroofing was performed in 17 cases (including one with failed aspiration), trans-perineal aspiration under TRUS guidance was performed in three cases and conservative therapy was followed in five cases. Our data confirms the importance of predisposing factors in the pathogenesis of prostatic abscess. In most of the cases, the clue to diagnosis is obtained by digital rectal palpation. TRUS gives the definite diagnosis and also helps in follow-up of patients. Trans-urethral deroofing is the ideal therapy where the abscess cavity is more than 1 cm, although in some selected cases, TRUS-guided aspiration or conservative therapy does have a role in treatment.

Composite thin film and electrospun biomaterials for urologic tissue reconstruction.

A replacement material for autologous grafts for urinary tract reconstruction would dramatically reduce the complications of surgery for these procedures. However, acellular materials have not proven to work sufficiently well, and cell-seeded materials are technically challenging and time consuming to generate. An important function of the urinary tract is to prevent urine leakage into the surrounding tissue--a function usually performed by the urothelium. We hypothesize that by providing an impermeable barrier in the acellular graft material, urine leakage would be minimized, as the urothelium forms in vivo. However, since urothelial cells require access to nutrients from the supporting vasculature, the impermeable barrier must degrade over time. Here we present the development of a novel biomaterial composed of the common degradable polymers, poly(ε-caprolactone) and poly(L-lactic acid) and generated by electrospinning directly onto spin-coated thin films. The composite scaffolds with thin films on the luminal surface were compared to their electrospun counterparts and commercially available small intestinal submucosa by surface analysis using scanning electron microscopy and by analysis of permeability to small molecules. In addition, the materials were examined for their ability to support urothelial cell adhesion, proliferation, and multilayered urothelium formation. We provide evidence that these unique composite scaffolds provide significant benefit over commonly used acellular materials in vitro and suggest that they be further examined in vivo.

Sutureless circumcision - An Indian experience.

CONTEXT: Traditionally, circumcision wounds are closed by absorbable sutures. However, certain alternative methods are also being utilized to overcome the shortcomings of the conventionally used method for circumcision wound closuring. In the current study, the use of tissue glue ((iso amyl 2-cyanoacrylate) has been compared with traditional suturing for the approximation of circumcision wounds. In our study, both the methods were found to be comparable with significantly less time consumed in glue group. AIMS: The purpose of the present study has been to compare directly cyanoacrylate as a better alternative to conventional suture material in terms of cosmetic result, time consumed and incidence of infection (comparative study). MATERIALS AND METHODS: The results of all the patients seen in outpatient department for circumcision were included in this study. The study was conducted from Aug 2009 to May 2010. The tissue glue (iso amyl 2-cyanoacrylate) was compared to sutures as a method of wound approximation in circumcision. RESULTS: Tissue glue group has been observed to have less wound inflammation, bleeding or hematoma rate and was cosmetically superior as compared to suture group; however, none of these findings could reach statistically significant level. The mean time taken for circumcision was 14.2 min (SD 2.42), when tissue glue was used for wound approximation. However, it was 24.4 min (SD 5.06) in case of the use of sutures. This difference was found as highly significant (P value < 0.001). However, regarding postoperative pain no significant reduction was observed when glue was used. CONCLUSIONS: This study showed that the use of tissue glue in comparison to sutures is having the following advantages: Cosmetically superiorLess time consuming All other parameters measured were nearly the same and statically insignificant.

Laparoscopic versus open pyeloplasty: comparison of two surgical approaches- a single centre experience of three years.

UPJO causes hydronephrosis and progressive renal impairment may ensue if left uncorrected. Open pyeloplasty remains the standard against which new technique must be compared. We analyzed the comparison of Laparoscopic and open pyeloplasty in a randomized prospective trial. A prospective randomized study was done from January 2004 to January 2007 in which a total of 28 Laparoscopic and 34 open pyeloplasty were done. All laparoscopic pyeloplasties were performed transperitoneally. Standard open Anderson Hynes pyeloplasty, spiral flap or VY plasty was done depending on anatomic consideration. Patients were followed with DTPA scan at 3 months and IVP at 6 months. Perioperative parameters including operative time, analgesic use, hospital stay, and complication and success rates were compared. Mean total operative time with stent placement in LP group was 244.2 min (188-300 min) compared to 122 min (100-140 min) in open group. Compared to open pyeloplasty the post operative diclofenac requirement was significantly less in LP group (mean107.14 mg) and open group required mean of (682.35 mg) The duration of analgesic requirement was also significantly less in LP group. The post operative hospital stay in LP was mean 8.29 days (7-11) and was significantly less than open group (mean 3.14 Days (2-7 days). Open pyeloplasty has been the gold standard for UPJO repair and achieves success rates exceeding 90%. Laparoscopic pyeloplasty provides a minimally invasive alternative to repair UPJO and has developed world wide as the first minimally option to match success rate of open pyeloplasty. Its potential advantages including less post op pain, shorter hospital stay an improved cosmesis has been proved in some comparative series. The only disadvantage seems to be longer operative time. LP has a minimal level of morbidity and short hospital stay compared to open approach Although Laparoscopic pyeloplasty has the disadvantages of longer operative time and requires significant skill of intracorporeal knotting but it is here to stay and represents an emerging standard of care.

Laparoscopic adrenalectomy: A single center experience.

AIMS: To evaluate the efficacy and safety of laparoscopic adrenalectomy in benign adrenal disorders. METHODS AND MATERIAL: Since July 2007, twenty patients have undergone laparoscopic adrenalectomy for various benign adrenal disorders at our institution. Every patient underwent contrast enhanced CT-abdomen. Serum corticosteroid levels were conducted in all, and urinary metanephrines, normetanephrines and VMA levels were performed in suspected pheochromocytoma. All the patients underwent laparoscopic adrenalectomy via the transperitoneal approach. RESULTS: The patients were in the age range of 18-57 years, eleven males and nine females, seven right, eleven left, two bilateral. The mean operative time was 150 minutes (120-180), mean hospital stay four days (3-5), mean intraoperative blood loss 150 ml and mean post-operative analgesic need was for 36 (24-72) hours. One out of twenty-two laparoscopic operations had to be converted into open adrenalectomy due to intra-operative complications. CONCLUSIONS: Laparoscopic adrenalectomy is a safe, effective and useful procedure without any major post-operative complication and is the gold standard for all benign adrenal disorders.