Potent antitumoral activity of TRAIL through generation of tumor-targeted single-chain fusion proteins.

In an attempt to improve TRAIL's (tumor necrosis factor-related apoptosis-inducing ligand) tumor selective activity a variant was designed, in which the three TRAIL protomers are expressed as a single polypeptide chain (scTRAIL). By genetic fusion with a single-chain antibody fragment (scFv) recognizing the extracellular domain of ErbB2, we further equipped scTRAIL with tumor-targeting properties. We studied tumor targeting and apoptosis induction of scFv-scTRAIL in comparison with non-targeted scTRAIL. Importantly, the tumor antigen-targeted scTRAIL fusion protein showed higher apoptotic activity in vitro, with a predominant action by TRAIL-R2 signaling. Pharmacokinetic studies revealed increased plasma half-life of the targeted scTRAIL fusion protein compared with scTRAIL. In vivo studies in a mouse tumor model with xenotransplanted Colo205 cells confirmed greater response to the ErbB2-specific scTRAIL fusion protein compared with non-targeted scTRAIL both under local and systemic application regimen. Together, in vitro and in vivo data give proof of concept of higher therapeutic activity of tumor-targeted scFv-scTRAIL molecules. Further, we envisage that through targeting of scTRAIL, potential side effects should be minimized. We propose that scFv-mediated tumor targeting of single-chain TRAIL represents a promising strategy to improve TRAIL's antitumoral action and to minimize potential unwanted actions on normal tissues.Cell Death and Disease (2010) 1, e68; doi:10.1038/cddis.2010.45; published online 26 August 2010.

Activation of CD95L fusion protein prodrugs by tumor-associated proteases.

To achieve tumor cell-restricted activation of CD95, we developed a CD95L fusion protein format, in which CD95L activity is only unmasked upon antibody-mediated binding to tumor cells and subsequent processing by tumor-associated proteases, such as matrix metalloproteases (MMPs) and urokinase plasminogen activator (uPA). On target-negative, but MMP- and uPA-expressing HT1080 tumor cells, the CD95L prodrugs were virtually inactive. On target antigen-expressing HT1080 cells, however, the CD95L prodrugs showed an apoptotic activity comparable to soluble CD95L artificially activated by crosslinking. CD95 activation by the CD95L prodrugs was preceded by prodrug processing. Apoptosis was blocked by inhibitors of MMPs or uPA and by neutralizing antibodies recognizing the targeted cell surface antigen or the CD95L moiety of the prodrugs. In a xenotransplantation tumor model, local application of the prodrug reduced the growth of target antigen-expressing, but not antigen-negative tumor cells, verifying targeted CD95L prodrug activation in vivo.

Restoration of membrane TNF-like activity by cell surface targeting and matrix metalloproteinase-mediated processing of a TNF prodrug.

Tumor necrosis factor (TNF) prodrugs are fusion proteins comprised of an N-terminal single-chain antibody variable fragment (scFv) targeting a TNF effector and a C-terminal TNF receptor (TNFR)1-derived inhibitor module. Introduction of matrix metalloproteinase (MMP)-2 recognition motifs between TNF and the TNFR1 fragment allowed activation by recombinant MMP-2 and MMP-expressing HT1080 cells. Processing by endogeneous MMPs required specific membrane binding of the TNF prodrug via the targeting scFv, ensuring strictly antigen-dependent activation. Interestingly, TNF bioactivity of the processed prodrug was approximately 1000-fold higher upon scFv-mediated targeting, and signaled juxtatropic cell death also to antigen-negative cells. Microscopical analyses of TNFR2 clustering and TNF receptor-associated factor 2 recruitment at contact sites to adjacent cells revealed the formation of stable TNFR complexes by target-bound, processed prodrug, resembling the increased signal capacity of natural, membrane-expressed TNF. MMP-2-sensitive TNF prodrugs represent novel cytokine-based reagents for targeted cancer therapy, which should be exploitable for MMP-overexpressing tumors.

Detection of membrane-bound tumor necrosis factor (TNF): an analysis of TNF-specific reagents.

Tumor necrosis factor (TNF) exists in two bioactive forms, the membrane integrated form and the proteolytically derived soluble cytokine. Both forms of TNF are involved in a variety of different physiological and pathophysiological situations. Here we analyzed different human and mouse TNF-specific reagents for their ability to determine the expression of membrane-expressed TNF. The data prove some antibodies to be very useful for the analysis of transmembrane TNF expression because these antibodies distinguish between the transmembrane form of TNF and soluble TNF bound to cellular TNF receptors. In addition, we found that recombinant human TNF receptor fusion proteins are advantageous tools to analyze both human and mouse transmembrane TNF expression.

Regulation of the murine NMDA-receptor-subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ-F1) homologues.

We have cloned the 5'-region of the murine N-methyl-d-aspartate (NMDA) receptor channel subunit NR2C (GluRepsilon3) gene and characterized the cis- and trans-activating regulatory elements responsible for its tissue specific activity. By using a native epsilon3-promoter/lacZ-construct & various 5'-deletion constructs, we compared beta-galactosidase expression in non-neuronal NIH3T3 cells and in neuronal epsilon3-gene-expressing HT-4 cells and show that large parts of the epsilon3 promoter are responsible for the repression of the epsilon3 gene in non-neuronal cells. Deletion of exon 1 sequences led to an enhancement of epsilon3 transcription, suggesting a role of the 5'-untranslated region in epsilon3 gene regulation. Sequence analysis of the promoter region revealed potential binding sites for the transcription factor Sp1, the murine fushi tarazu factor1 (FTZ-F1) homologues, embryonic LTR binding proteins (ELP1,2,3) and steroidogenic factor (SF-1), as well as for the chicken ovalbumin upstream promoter transcription-factor (COUP-TF). Electrophoretic mobility shift assays confirmed specific binding of Sp1, SF-1 and COUP-TFI. Whereas point mutation studies indicate that, in neuronal HT-4 cells, Sp1 is apparently not critically involved in basal epsilon3 gene transcription, SF1 is a positive regulator. This was evident from a selective enhancement of epsilon3-promoter-driven reporter gene expression upon cotransfection of an SF1-expression vector, which was reverted by deletion and point mutation of the SF1 binding site.

Evaluation of urinary IL-1alpha and IL-1beta in gravid females and patients with bacterial cystitis and microscopic hematuria.

OBJECTIVES: to determine IL-1alpha and IL-1beta levels in patients with bacterial cystitis, microscopic hematuria, and gravid females relative to a control group of normal subjects. METHODS: enzyme immunoassays were used to measure concomitantly urinary IL-1alpha and IL-1beta in clean catch urine samples from normal subjects (n = 31) and study patients (n = 46). All normal subjects and patients underwent urinalysis, urine culture, and urine creatinine level determination. Since the IL-1alpha assay was developed for serum, the utility of the assay for urine specimens was unknown. The key parameters of urine collection, processing and sample storage for IL-1alpha were evaluated in detail. RESULTS: mean values +/- SEM (pg/mg) for IL-1alpha/ Cr and IL-1beta/Cr were control group (0.25 +/- 0.10 and 0.17 +/- 0.06), bacterial cystitis (9.97 +/- 1.15 and 42.45 +/- 1.86), and microscopic hematuria (2.81 +/- 0.65 and 2.82 +/- 0.70). Differences in cytokine levels between the control group and patients with either bacterial cystitis or microscopic hematuria were statistically significant for both IL-1alpha/Cr (P < 0.026; P < 0.007, respectively) and IL-1beta /Cr (P < 0.0004; P < 0.014, respectively). IL-1beta/Cr correlates better with pyuria than IL-1alpha/ Cr (P = 0.02 vs P = 0.44). In gravid females, only IL-1alpha was significantly elevated relative to non-pregnant females (IL-1beta elevation approached statistical significance). Gravid females with positive urine cultures could not be distinguished from those with negative cultures based on either interleukin (P > 0.05). CONCLUSIONS: Significant elevations of IL-1alpha and IL-1beta occur in patients with bacterial cystitis and microscopic hematuria. Correlation between pyuria and cytokine elevation was stronger for IL-1beta than for IL-1alpha. Changes in IL-1alpha may reflect changes in the bladder epithelium rather than in the inflammatory leukocytes. The ability of IL-1alpha and IL-1beta to serve as markers for bacterial cystitis in gravid females is diminished due to high basal levels during pregnancy.

Conservative management of colon injury following percutaneous renal surgery.

OBJECTIVES: Colon injury during percutaneous renal surgery is rare and can result in significant morbidity. Our objective was threefold: (1) to identify risk factors for colon injuries; (2) to optimize prevention of such injuries; and (3) to devise a treatment strategy for optimal management of such colon injuries. METHODS: Between July 1990 and July 1995, all percutaneous renal procedures performed at three kidney stone centers were reviewed (Kaiser Permanente Medical Center, Los Angeles; Hospital of the Good Samaritan, Los Angeles; and University of California at San Francisco). In addition, a review of the pertinent literature was performed. RESULTS: Five patients who suffered colon injuries during percutaneous renal surgery were identified. All had undergone percutaneous nephrolithotomy, and all injuries were extraperitoneal. Mean age was 31 years (range 17 to 52). Three patients were considered lean, and the other two were of average body habitus. Four of 5 patients were male. Three injuries occurred on the left side and two on the right. Recognition of colon injury occurred postoperatively in 4 patients and intraoperatively in 1 patient. Presenting signs and symptoms included fever, fecaluria, abdominal pain, and leukocytosis. CONCLUSIONS: High risk patients for colon injuries are young, lean males with minimal retroperitoneal fat, in whom a retrorenal colon is more likely. High risk patients should be accessed with a more superior and medial puncture. Retroperitoneal colon injuries can be successfully managed conservatively with early recognition and appropriate drainage of the urinary and intestinal tracts. A treatment algorithm is presented.

Tubeless percutaneous renal surgery.

PURPOSE: We challenge the requirement for routine placement of a nephrostomy tube following percutaneous renal surgery. MATERIALS AND METHODS: A total of 50 patients underwent tubeless percutaneous renal procedures consisting of nephrolithotripsy, endopyelotomy, and stone extraction plus endopyelotomy performed during the same setting. In the initial 30 patients a Double-J* stent and a Councill nephrostomy tube were placed at the end of the procedure. The Councill catheter was removed 2 to 3 hours postoperatively. The subsequent 20 patients received only a Double-J stent with no Councill catheter. This study group was compared to a control group of 50 age, sex and procedure matched patients who had previously undergone standard percutaneous renal procedures with routine placement of postoperative nephrostomy tubes. The incidence of complications, analgesia requirements, length of hospitalization, interval to return to normal activities and cost of treatment were compared between the 2 groups. RESULTS: All 50 tubeless percutaneous procedures were performed successfully without significant complications. In the initial 15 patients postoperative renal ultrasound demonstrated no urinoma. Hospitalization was 0.6 days for the study group and 4.6 days for the controls (p = 0.0001). Average parenteral or intramuscular analgesia requirements were 11.58 and 36.06 mg. morphine sulfate, respectively (p = 0.0001), with patients requiring oral analgesia for 5.9 and 11.7 days, respectively (p = 0.0001). Patients in the study group returned to normal activities within 17.85 days versus 26.6 days for the controls (p = 0.0004). The costs of the procedures were $1,638 and $3,750 (129% greater), respectively, for a cost saving of $2,112 per case. CONCLUSIONS: Tubeless percutaneous renal surgery is a safe procedure and offers numerous advantages over routine placement of a nephrostomy tube. The hospitalization, analgesia requirements, return to normal activities as well as cost are significantly less with this new technique.

"Tubeless" percutaneous surgery: a new advance in the technique of percutaneous renal surgery.

We describe our modification of the technique of traditional percutaneous renal surgery called "tubeless" percutaneous renal surgery. Fifty patients have now undergone percutaneous renal procedures without the use of a postoperative nephrostomy tube consisting of percutaneous nephrolithotripsy, percutaneous endopyelotomy, and both percutaneous stone extraction and endopyelotomy in the same setting. Our current modification of standard percutaneous surgical technique includes the placement of an internal ureteral catheter with primary closure of the access site using hemostatic skin sutures. The study group was compared to a control group of 50 patients who were age, sex and procedure matched who had undergone standard percutaneous renal procedures previously with routine placement of postoperative nephrostomy tubes. The incidence of complications, analgesia requirements, length of hospitalization, time of return to normal activities, and cost of treatment were compared between the two groups. All tubeless percutaneous procedures were successfully performed without significant complications. The initial 15 patients had postoperative renal ultrasounds demonstrating no urinoma. Hospital stay, analgesia requirements, and the patient's ability to return to normal activities were statistically significantly decreased in the patient group studied. The cost of a "tubeless" procedure was $1,638 compared with $3,750 (129% greater) for traditional percutaneous surgery (cost saving of $2,112/case). Tubeless percutaneous renal surgery is a safe procedure and offers advantages over the routine placement of a nephrostomy tube. The hospitalization period, analgesia requirements, return to normal activities, and cost are significantly less with this new technique.

Primary bladder neck obstruction: urodynamic findings and treatment results in 36 men.

PURPOSE: We reviewed the urodynamic findings and treatment outcomes of a large series of men with primary bladder neck obstruction. MATERIALS AND METHODS: A retrospective review was done of the presenting symptoms and urodynamic findings of 36 men with primary bladder neck obstruction. Outcomes after treatment with alpha-blockers, transurethral incision of the bladder neck and prostate, or no long-term therapy were determined by chart review and patient survey in the majority of cases. RESULTS: Mean age of the men was 41 years. Patients had significant lower urinary tract symptoms, decreased peak urinary flow rates, elevated post-void residual, markedly elevated peak voiding pressures and poor funneling of the bladder neck during voiding. Although most patients initially chose alpha-blocker therapy, only 30% of those beginning alpha-blockers continued them long term, usually due to inadequate symptomatic improvement. A total of 18 men underwent transurethral incision, which resulted in significant improvements in symptom scores, peak urinary flow rates, post-void residual and peak voiding pressures. Patients reported a mean 87% overall improvement in symptoms after transurethral incision. CONCLUSIONS: Video urodynamics facilitate diagnosis of primary bladder neck obstruction. Transurethral incision is the most effective therapy for primary bladder neck obstruction.

Dimethyl sulfoxide does not trigger urine histamine release in interstitial cystitis.

OBJECTIVES: Dimethyl sulfoxide (DMSO), an agent that provides symptomatic relief in patients with interstitial cystitis (IC) works via an unknown mechanism. We investigated whether DMSO acts as a chemical stimulant of mast cell degranulation. METHODS: A radioimmunoassay (RIA) specific for histamine was used to test this hypothesis. Twelve women with strictly diagnosed IC were treated with intravesical instillations of DMSO. Treatments were repeated at varying intervals, and each patient received three to six treatments. Urine histamine levels were measured before and after each intravesical instillation of DMSO. Dilutional effects of DMSO were corrected for by conversion of urine histamine concentration to urine histamine:creatinine ratio. RESULTS: The RIA was unaffected by the addition of DMSO to urine. No consistent change in the urine histamine:creatinine ratio following DMSO instillation was found. Trend analysis revealed no trend in the histamine:creatinine ratio with time. CONCLUSIONS: The relief of symptoms reported in 50% to 77% of patients treated with intravesical DMSO is not related to detectable mast cell release of histamine. Other mechanisms of action must be investigated to explain the beneficial effect of this agent.