Vesico-urethral anastomotic stenosis following radical prostatectomy: a multi-institutional outcome analysis with a focus on endoscopic approach, surgical sequence, and the impact of radiation therapy.

OBJECTIVES: To investigate the predictors of recurrence and of de novo incontinence in patients treated by transurethral incision or resection for vesico-urethral anastomotic stenosis (VUAS) after radical prostatectomy. MATERIAL AND METHODS: All patients undergoing endoscopic treatment for VUAS between March 2009 and October 2016 were identified in our multi-institutional database. Digital chart reviews were performed and patients contacted for follow-up. Recurrence was defined as any need for further instrumentation or surgery, and de-novo-incontinence as patient-reported outcome. RESULTS: Of 103 patients undergoing endoscopic VUAS treatment, 67 (65%) underwent transurethral resection (TR) and 36 (35%) transurethral incision (TI). TI was performed more frequently as primary treatment compared to TR (58% vs. 37%; p = 0.041). Primary and repeated treatment was performed in 46 (45%) and 57 patients (55%), respectively. Overall, 38 patients (37%) had a history of radiation therapy. There was no difference in time to recurrence for primary vs repeat VUAS treatment, previous vs no radiation, TR compared to TI (all p > 0.08). Regarding treatment success, no difference was found for primary vs. repeat VUAS treatment (50% vs. 37%), previous radiation vs. no radiation (42% vs. 43%), and TR vs. TI (37% vs. 53%; all p ≥ 0.1). Postoperative de novo incontinence was more common after TI vs. TR (31% vs. 12%; p = 0.032), no difference was observed for previous radiation therapy vs. no radiation therapy (18% vs. 18%; p > 0.9) or primary vs. repeat VUAS treatment (22% vs. 16%; p = 0.5). CONCLUSION: VUAS recurrence after endoscopic treatment is not predictable. Endoscopic treatment with TI showed a higher risk for de novo incontinence than TR, and previous irradiation and the number of treatments do not influence incontinence.

The T-plasty as therapy for recurrent bladder neck stenosis: success rate, functional outcome, and patient satisfaction.

PURPOSE: To determine success rate (SR), functional outcome, and patient satisfaction of a modified YV-plasty for reconstruction of the bladder neck in case of recurrent bladder neck stenosis (BNS) after transurethral surgery of the prostate: the T-plasty. PATIENTS AND METHODS: We identified all patients who underwent T-plasty at our center between December 2008 and July 2016. Patients' charts were reviewed. Patients were queried by telephone and by mail at time of follow-up (FU). Primary endpoint was SR. Secondary endpoints were complications, continence, satisfaction, and changes in quality of life measured by validated questionnaires. RESULTS: Thirty patients underwent the T-plasty. Median age at surgery was 69 (IQR 62-73) years. Most patients had BNS due to TUR-P [n = 25 (83.3%)]. No severe blood loss or severe complications occurred perioperatively. Median FU was 45 (IQR 18-64) months. Three patients were lost to FU. Success rate was 100%. Compared to pre-OP Q max, mean Q max post-OP improved significantly [pre-OP 6.79 (SD ± 4.76) ml/s vs post-OP was 24.42 (SD ± 12.61) ml/s; (t(5) = 4.12, p = 0.009)]. Mean post-void residual urine decreased significantly [pre-OP 140.77 (SD ± 105.41) ml vs post-OP 14.5 (SD ± 22.42) ml; (t(9) = -3.86, p = 0.004)]. One patient developed a de-novo-incontinence post-OP. Mean ICIQ-SF Score was 1.2 (SD ± 2.27). 88.5% of patients were pleased or delighted by surgery. 75% of patients claimed their quality of life has been (strongly) improved. CONCLUSIONS: The T-plasty is a valuable option as treatment of recurrent BNS. SR, rates of continence, and high patient satisfaction are very encouraging.

Management of Anterior Urethral Strictures in Adults: A Survey of Contemporary Practice in Germany.

INTRODUCTION: Treatment methods of anterior urethral strictures in adults have undergone considerable changes in the recent past. Our goal was to determine national practice patterns among German urologists and to compare results with the results of prior international surveys. METHODS: We conducted a survey on the management of urethral strictures among German urologists. RESULTS: Eight hundred forty-five urologists, representing about 14.6% of German urologists, answered the survey. Most common procedures were direct vision internal urethrotomy (DVIU; 87.2%), blind internal urethrotomy (57.5%), dilatation (56.3%), ventral buccal mucosa graft urethroplasty (31.6%) and excision and primary anastomosis (28.9%). In case of a 3.5-cm bulbar stricture and in the case of a 1-cm bulbar stricture after 2 failed DVIUs, a consecutive urethroplasty was significantly more often favoured compared to transurethral treatment options (44.9 vs. 21.3% and 59.4 vs. 8.3%, both p < 0.001). CONCLUSION: Open urethral reconstruction reveals to be a more common method in practice nowadays. Adherence to recommended treatment algorithms improved in comparison to prior surveys.

Direct Vision Internal Urethrotomy for Short Anterior Urethral Strictures and Beyond: Success Rates, Predictors of Treatment Failure, and Recurrence Management.

OBJECTIVE: To determine success rates, predictors of recurrence, and recurrence management of patients treated for short anterior urethral strictures by direct vision internal urethrotomy (DVIU). MATERIALS AND METHODS: We identified 128 patients who underwent DVIU of the anterior urethra between December 2009 and March 2016. Follow-up was conducted by telephone interviews. Success rates were assessed by Kaplan-Meier estimators. Predictors of stricture recurrence and different further therapy strategies were identified by uni- and multivariable Cox regression analyses. RESULTS: The mean age was 63.8 years (standard deviation: 16.3) and the overall success rate was 51.6% (N = 66) at a median follow-up of 16 months (interquartile range: 6-43). Median time to stricture recurrence was six months (interquartile range: 2-12). In uni- and multivariable analyses, only repeat DVIU (hazard ratio [HR] = 1.87, 95% confidence interval (CI) = 1.13-3.11, P= .015; and HR=1.78, 95% CI = 1.05-3.03, P = .032, respectively) was a risk factor for recurrence. Of 62 patients with recurrence, 35.5% underwent urethroplasty, 29% underwent further endoscopic treatment, and 33.9% did not undergo further interventional therapy. Age (HR = 1.05, 95% CI = 1.01-1.09, P = .019) and diabetes (HR = 2.90, 95% CI = 1.02-8.26, P = .047) were predictors of no further interventional therapy. CONCLUSION: DVIU seems justifiable in short urethral strictures as a primary treatment. Prior DVIU was a risk factor for recurrence. In case of recurrence, about one-third of the patients did not undergo any further therapy. Higher age and diabetes predicted the denial of any further treatment.

The T-plasty: a modified YV-plasty for highly recurrent bladder neck contracture after transurethral surgery for benign hyperplasia of the prostate: clinical outcome and patient satisfaction.

OBJECTIVE: To describe a modified surgical technique for treatment of highly recurrent bladder neck contracture (BNC) after transurethral surgery for benign hyperplasia and to evaluate success rate and patient satisfaction of this novel technique. METHODS: Ten patients with highly recurrent BNC and multiple prior attempts of endoscopic treatment underwent the T-plasty. Perioperative complications were recorded and classified according to the Clavien classification. Patient reported functional outcomes were retrospectively analysed using a standardized questionnaire assessing recurrence of stenosis, incontinence, satisfaction and changes in quality of life (QoL). The questionnaires included validated IPSS and SF-8-health survey items. RESULTS: Mean age at the time of surgery was 69.2 years (range 61-79), and the mean follow-up was 26 months (range 3-46). No complications grade 3 or higher according to the Clavien classification occurred. Success rate was 100 %. No de novo stress incontinence occurred. Urinary stream was described as very strong to moderate by 80 % of the patients, mean post-operative IPSS-score was 11.3 (range 4-29), and mean post-operative IPSS-QoL was 2.4 (range 1-5). Patients satisfaction was very high or high in 90 %, and QoL improved in 90 %. The SF-8-health survey showed values comparable to the reference population. CONCLUSION: The T-plasty represents a safe and valuable option in treating highly recurrent BNC after surgery for benign hyperplasia. It offers multiple advantages compared to other techniques such as a single-staged approach and the opportunity for reconstruction of a reliable wide bladder neck by usage of two well-vascularized flaps. Success rate, low rate of complications and preservation of continence are highly encouraging.

Cancer cell invasion and EMT marker expression: a three-dimensional study of the human cancer-host interface.

Cancer cell invasion takes place at the cancer-host interface and is a prerequisite for distant metastasis. The relationships between current biological and clinical concepts such as cell migration modes, tumour budding and epithelial-mesenchymal transition (EMT) remains unclear in several aspects, especially for the 'real' situation in human cancer. We developed a novel method that provides exact three-dimensional (3D) information on both microscopic morphology and gene expression, over a virtually unlimited spatial range, by reconstruction from serial immunostained tissue slices. Quantitative 3D assessment of tumour budding at the cancer-host interface in human pancreatic, colorectal, lung and breast adenocarcinoma suggests collective cell migration as the mechanism of cancer cell invasion, while single cancer cell migration seems to be virtually absent. Budding tumour cells display a shift towards spindle-like as well as a rounded morphology. This is associated with decreased E-cadherin staining intensity and a shift from membranous to cytoplasmic staining, as well as increased nuclear ZEB1 expression.

Panurethral and complex urethral strictures. Reconstruction in several steps: current techniques and indications.

Patients with panurethral and complex urethral strictures after failed urethral reconstruction due to strictures and hypospadias repair is a rare but challenging condition. Contemporary surgical techniques include one and two staged urethroplasties using different graft substitutes (i.e., buccal mucosa) or full thickness skin grafts (i.e., from the inner thigh(, thereby providing satisfactory results with reducing the re-stricture rate in these patients. However, all current techniques do so at the expense of higher revision rates and thus requiring multiple procedures. Studies investigating the outcomes of reconstruction in panurethral and complex urethral strictures often have heterogeneous patient cohorts including children and adults, different underlying causes, and different techniques, thus allowing only limited interpretation of the published data. In the field of urethral reconstruction, where personal experience and expertise presents an accepted necessity, however, leading to rather small single center studies,only well-designed randomized clinical trials can truly answer the question of which technique will be advantageous in these patients.

Nuclear import of LASP-1 is regulated by phosphorylation and dynamic protein-protein interactions.

LASP-1 is a multidomain protein predominantly localized at focal contacts, where it regulates cytoskeleton dynamics and cell migration. However, in different tumor entities, a nuclear LASP-1 accumulation is observed, thought to have an important role in cancer progression. Until now, the molecular mechanisms that control LASP-1 nuclear import were not elucidated. Here, we identified a novel LASP-1-binding partner, zona occludens protein 2 (ZO-2), and established its role in the signal transduction pathway of LASP-1 nucleo-cytoplasmatic shuttling. Phosphorylation of LASP-1 by PKA at serine 146 induces translocation of the LASP-1/ZO-2 complex from the cytoplasm to the nucleus. Interaction occurs within the carboxyterminal proline-rich motif of ZO-2 and the SH3 domain in LASP-1. In situ proximity ligation assay confirmed the direct binding between LASP-1 and ZO-2 and visualized the shuttling. Nuclear export is mediated by Crm-1 and a newly identified nuclear export signal in LASP-1. Finally, dephosphorylation of LASP-1 by phosphatase PP2B is suggested to relocalize the protein back to focal contacts. In summary, we define a new pathway for LASP-1 in tumor progression.

The role of interleukin-18 in vesicular stomatitis virus infection of the CNS.

Intranasal application of vesicular stomatitis virus (VSV) results in the initial infection of the olfactory receptor neurons and a rapid progression of the virus through the mouse central nervous system (CNS). Interleukin-18 (IL-18) is an 18.3-kd cytokine that induces interferon gamma (IFN-gamma) production in mice. IL-18 is synthesized as an inactive precursor that is cleaved and activated by caspase-1/interleukin-1beta converting enzyme (ICE). IL-18 shares several biological properties with IL-12, including the ability to induce IFN-gamma production in T lymphocytes and natural killer (NK) cells. In the CNS, microglia and astrocytes produce IL-18 and IL-12. We have previously shown that IL-12 promotes recovery from VSV encephalitis. This led us to examine the potential role of IL-18 in the pathogenesis of VSV encephalitis. We show that both IL-18 and caspase-1 mRNA are consistently present in the CNS of mice. The addition of exogenous IL-18 to cell cultures does not affect the production of VSV, and addition of exogenous IL-18 at the time of infection does not alter the morbidity or mortality of BALB/c mice. In vitro studies with neutralizing monoclonal antibody to IL-18 had no effect. From these results we conclude that in this system and under the experimental conditions used, unlike IL-12 and IFN-gamma, IL-18 does not play a significant role in the host response to VSV infection.

Mechanisms of cytokine-mediated inhibition of viral replication.

In this report, the role of nitric oxide synthase (NOS) and IL-12 administration in inhibition of vesicular stomatitis virus (VSV) from infected neuroblastoma cells was examined. We previously have shown that cytokine treatment of cells results in the induction of NOS-1, and this is associated with a 2 log inhibition of VSV production. We performed these studies to examine the mechanism by which viral replication is suppressed. Neuroblastoma cells (NB41A3) were treated with either IL-12 or medium and subsequently infected with VSV. Viral protein and mRNA were isolated from these cells, and their levels were measured by Western or Northern blots, respectively. mRNA levels were decreased modestly, but viral proteins were decreased substantially in cells pretreated with IL-12, suggesting that the inhibitory effect of NO is working at the translational level. Cytokine treatment of cells was not associated with oxidative stress. The viral proteins also were nitrosylated. These data suggest that the mechanism of NO inhibition of viral replication occurs through translational interference and posttranslational modifications of viral components.

[3H]MDL 105,519 binds with equal high affinity to both assembled and unassembled NR1 subunits of the NMDA receptor.

[3H]MDL 105,519 (((E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1[3H]-indole-2-ca rboxylic acid) is a novel radioligand which binds with high affinity, Kd = 2.5 nM, to the glycine site of adult rodent forebrain, N-methyl-D-aspartate subtype of glutamate receptors. As with other glycine site antagonists, the major determinants for high-affinity binding of [3H]MDL 105,519 resides upon the NRI subunit, and not the NR2 subunits. [3H]MDL 105,519 binds with equal affinity, Kd = 3 nM, to both NR1-1a or NR1-4b splice variants, as well as the NRI-1a/NR2A receptor expressed in human embryonic kidney (HEK) 293 cells. One percent Triton X-100/1 M NaCl solubilises with a recovery of 15+/-3%, a mixed pool of assembled and unassembled forebrain NR1 subunit polypeptides. In this preparation, the recovery of [3H]MK801 ((+)-5-[3H]methyl-10,11-dihydrodibenzo[alpha,d]cyclohepten-5 ,10-imine binding activity (7+/-1%) reflects the amount of assembled NR1 subunits whereas [3H]MDL 105,519 binds quantitatively, with a recovery of 19+/-4% and Kd = 3 nM, to both assembled and unassembled NRI subunits. Therefore, [3H]MDL 105,519 should prove a useful ligand, in conjunction with immunopurification approaches, to address the question of NMDA receptor subunit stoichiometry.

IFN-gamma is not required in the IL-12 response to vesicular stomatitis virus infection of the olfactory bulb.

In this report, the role of IFN-gamma in host defense to exogenous IL-12 and clearance of vesicular stomatitis virus (VSV) from the central nervous system was examined. Wild-type and IFN-gamma knockout mice infected with VSV were treated with IL-12 or medium. In both groups, IL-12 treatment resulted in 1) substantially decreased VSV titers in brain homogenates and diminished immunohistochemical detection of VSV Ags in tissue sections; 2) induction of types 1, 2, and 3 nitric oxide synthase; and 3) induction of MHC molecules and rapid infiltration of both T cells and NK cells. These results suggest that IFN-gamma production, both systemically and in the olfactory bulb, contributes to but is not essential for clearance of VSV from the brain. Neutralization of TNF-alpha in IFN-gamma knockout mice mice treated with IL-12 was accompanied by the same immunohistochemical changes, implying that neither IFN-gamma nor TNF-alpha was required. In vitro studies using purified IL-12 or IFN-gamma in culture medium induced nitric oxide synthase isoforms in neurons, glia, and macrophages, and MHC II on glia and macrophages. These data suggest that IL-12 directly activates neurons to promote viral clearance in vivo.

Interleukin-12 promotes recovery from viral encephalitis.

Infusion of interleukin-12 (IL-12) enhances recovery from lethal experimental vesicular stomatitis virus (VSV) infection of the central nervous system (CNS). Interleukin-12 treatment resulted in: 1) increased survival frequency; 2) faster recovery from weight loss; 3) substantially decreased VSV titers in brain homogenates and diminished immunohistochemical detection of VSV antigens in tissue sections; 4) earlier and increased CNS expression of types 1, 2, and 3 nitric oxide synthase (NOS) and both major histocompatibility complex (MHC) class I and class II antigens; 5) earlier and increased blood and CNS levels of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). These results suggest that IL-12 enhances recovery from VSV infection of the CNS.

Activation of type III nitric oxide synthase in astrocytes following a neurotropic viral infection.

Type III nitric oxide synthase (type III NOS), also known as endothelial cell nitric oxide synthase (eNOS or ecNOS or NOS-3), is a constitutively expressed, calcium- and calmodulin-dependent, isoform of NOS. Its expression has been localized to endothelial cells and a subset of neurons in the brain. We report here that resident astrocytes of the central nervous system (CNS) of mice express type III NOS. Following an experimental neurotropic viral infection, the expression of type III NOS on reactive astrocytes increases substantially, predominantly in virally infected regions of the brain. This upregulation of type III NOS expression is also evident following cytokine treatment in vitro. The intraperitoneal (i.p.) administration of IL-12, a potent activator of IFN-gamma and TNF-alpha production, results in a substantial increase in type III NOS immunoreactivity in astrocytes. Cytokine-mediated activation of type III NOS is observed in vitro following exposure of a C6 glioma cells, which constitutively express type III NOS, to IL-12, IFN-gamma, and TNF-alpha treatment. We conclude that astrocytes of the murine CNS express type III NOS, which may be positively regulated by a number of cytokines following viral infection. Type III NOS expression by astrocytes represents a novel source of nitric oxide in the brain. It may be important in regulating perfusion and maintaining the blood-brain barrier. Given the intimate association of astrocytes with endothelial cells and neurons, increased activity of type III NOS following viral infection may be beneficial in inhibition of viral infection in neighboring cells.

Interferon-gamma induced type I nitric oxide synthase activity inhibits viral replication in neurons.

Type I NOS expression increases in OB neurons during VSV infection. Immunocytochemical staining of NB41A3 cells indicates constitutive expression of interferon (IFN)-gamma receptor and type I NOS. IFN-gamma treatment of NB41A3 cells increased NO production and type I NOS protein. In vitro replication of VSV, polio virus type I, and Herpes Simplex virus type I (HSV-1) is significantly inhibited by IFN-gamma induced type I NOS and antagonized by NOS inhibitors. In contrast, while IFN-gamma treatment inhibited influenza and Sindbis virus replication, a different pathway(s) was involved. The isoform-selective NOS inhibitor. 7-nitroindazole (7NI) was used to treat mice, resulting in a 10-fold higher titer of virus in brain homogenates, and abrogated the recovery-promoting effect of interleukin-12 treatment. Thus, IFN-gamma induced type I NOS activity may play an important role in host immunity against neurotropic viral infections.

Processing of a viral glycoprotein in the endoplasmic reticulum for class II presentation.

Endogenous processing of viral glycoproteins for presentation to CD4+T cells is a poorly investigated aspect of antigen processing and presentation. This pathway may involve not only pathogens, but also self proteins, and may thus be involved in self-tolerance. We have characterized the processing of the endoplasmic reticulum-restricted glycoprotein (G) of vesicular stomatitis virus, termed poison tail (Gpt), biochemically and enzymatically, and by T cell recognition assays. Expressed with a vaccinia vector, Gpt remains endoglycosidase H-sensitive and does not mature to endoglycosidase D sensitivity. The protein is degraded in the ER with a T1/2 of 4 h. Gpt peptides are not secreted since Gpt-infected cells are unable to sensitize uninfected antigen-presenting cells in an innocent bystander assay. Using flow cytometry, Gpt is undetectable on the plasma membrane; in contrast, wild-type G is readily found on the surface or secreted into the milieu as soluble G following infection of A20 cells with a vaccinia recombinant expressing G. The degradation of Gpt is sensitive to the thiol reagent diamide and occurs optimally at physiological pH. A series of proteolytic inhibitors were tested: 3,4-dichloroisocoumarin and 1-chloro-3-tosylamido-7-amino-2-heptanone inhibited degradation, which suggests the involvement of a serine protease. The degradation does not require transport to the Golgi complex, and is not sensitive to a variety of lysosomotropic agents. We show that the degradation products include the immunogenic epitopes recognized by a panel of T cell clones and hybridomas.

Pathogenesis of murine encephalitis limited by defective interfering particles. An immunohistochemical study.

To determine whether defective interfering (DI) particles alter viral encephalitis BALB/c mice were inoculated intranasally with standard vesicular stomatitis virus (VSV) and its DI particles. Addition of 10(7) PFU equivalents of DI particles to 10(5) PFU of VSV reduced morbidity but did not delay disease onset. Less mortality was also observed. When 10(3) PFU equivalents of DI particles or UV-irradiated DI particles were substituted, these effects were absent. Attempts to correlate mortality with virus recovered from the brain could not be made due to considerable variations in the few surviving mice. Immunohistochemical analysis obtained from 121 mice showed that inoculation of DI particles limited the specific pathways of VSV antigen dissemination within the central nervous system, and new pathways were not substituted. In the group of mice with reduced mortality due to DI particles, at day 4 post inoculation VSV antigen was limited to the outer layers of the glomeruli of the olfactory bulb and to the accessory olfactory bulb, whereas there was deeper invasion of the olfactory bulb and olfactory ventricular system with mice infected with standard VSV alone. Correlation between mortality and extent of invasion became more difficult to make from 8 days on, when VSV antigens were found in discrete areas of the brain. By 12 days, few surviving mice contained any detectable VSV antigen in their brains. These results demonstrate that DI particles have potential as therapeutic agents. Also, mortality resulting from VSV-induced encephalitis, although poorly understood, may be determined very early, possibly while the virus is replicating at the site of inoculation.

The earliest events in vesicular stomatitis virus infection of the murine olfactory neuroepithelium and entry of the central nervous system.

After intranasal instillation of mice with vesicular stomatitis virus (VSV), olfactory receptor neurons are infected. By 12 to 24 hr postinfection, VSV antigens are observed in adjoining supporting and basal cells and in other structures of the olfactory epithelium and lamina propria. Peripheral deafferentation of the olfactory epithelium with Triton X-100 or bilateral surgical bulbectomy does not prevent spread of VSV to the central nervous system (CNS); the route of spread differs considerably from the route taken when the olfactory nerve is intact. In contrast to rabies virus and HSV-1, VSV does not use the trigeminal nerve for entry into the brain, as the trigeminal ganglion remains virus-free following intranasal infection. These results indicate that VSV has a strong tropism for olfactory receptor cells, using them for entry into the CNS. Both retrograde and anterograde transneuronal and nonneuronal (ependymal cells and cerebrospinal fluid) pathways are utilized by VSV within the CNS.

Inhibition of vesicular stomatitis virus infection by nitric oxide.

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.

The collision of cotranscribing E. coli RNA polymerases studied in vitro.

The collision of cotranscribing E. coli ternary complexes was brought about in vitro by forcing the first "leading" complex to stall before first template C, due to the absence of CTP, and then permitting initiation of a second "following" complex. Following collision, the transcript of the leading complex was increased in length by as much as 7 nt., despite the absence of CTP, but did not dissociate. Upon addition of CTP, the leading complex aborted at exactly the positions requiring incorporation of the next Cs', the following complex continued transcription. The observations point to the importance of linking appropriate promoter efficiency with transcriptional pausing times, and the role of transcriptional collisions in termination events.