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The objective of this study was to identify a panel of microRNAs (miRNAs) differentially expressed in high-grade non-muscle invasive (NMI; TaG3-T1G3) urothelial carcinoma that progress to muscle-invasive disease compared to those that remain non-muscle invasive, whether recurrence happens or not. Eighty-nine high-grade NMI urothelial carcinoma lesions were identified and total RNA was extracted from paraffin-embedded tissue. Patients were categorized as either having a non-muscle invasive lesion with no evidence of progression over a 3-year period or as having a similar lesion showing progression to muscle invasion over the same period. In addition, comparison of miRNA expression levels between patients with and without prior intravesical therapy was performed. Total RNA was pooled for microarray analysis in each group (non-progressors and progressors), and qRT-PCR of individual samples validated differential expression between non-progressive and progressive lesions. MiR-32-5p, -224-5p, and -412-3p were associated with cancer-specific survival. Downregulation of miR-203a-3p and miR-205-5p were significantly linked to progression in non-muscle invasive bladder tumors. These miRNAs include those implicated in epithelial mesenchymal transition, previously identified as members of a panel characterizing transition from the non-invasive to invasive phenotype in bladder tumors. Furthermore, we were able to identify specific miRNAs that are linked to postoperative outcome in patients with high grade NMI urothelial carcinoma of the bladder (UCB) that progressed to muscle-invasive (MI) disease.

RNA from cell-free urine was analyzed in an attempt to identify a microRNA (miRNA) profile that could be used as a non-invasive diagnostic assay to detect the presence of urothelial carcinoma of the bladder (UCB) and provide a discriminatory signature for different stages of progression. In addition, the presence of specific miRNAs co-isolating with urinary extracellular vesicles/exosomes was investigated. RNA was isolated from cell-free urine of patients diagnosed with UCB (TaG1, T1G3, ≥T2, CIS) and control patients (healthy control and UCB patients with no evidence of disease). MiRNAs were profiled by qRT-PCR array on pooled samples within each group. Validation of the miRNAs was performed on individual samples using qRT-PCR. Extracellular vesicles were isolated via ultracentrifugation. 236 miRNAs were detected in at least one of the pooled samples. Seven of the miRNAs validated on individual samples had significantly higher levels in the cancer group. A panel of miRNAs discriminated between cancer and cancer-free patients with a sensitivity of 88% and specificity of 78%, (AUC=88.8%). We recorded a sensitivity of 80% for TaG1, 95% for T1G3, 90% for ≥T2 with specificity of 77% for healthy controls and 80% for no evidence of disease. Select miRNAs were detected in extracellular vesicles of UCB patients and healthy controls, albeit at different levels. Utilizing this non-invasive assay, we identified miRNA capable of detecting UCB and distinguishing different stages of progression, providing evidence that miRNA profiling in cell-free urine holds promise for the development of valuable clinical diagnostic tools.

MicroRNAs (miRNA) are small, noncoding RNAs with important regulatory roles in development, differentiation, cell proliferation, and death as well as the complex process of acquired drug resistance. The goal of this study was to identify specific miRNAs and their potential protein targets that confer acquired resistance to gemcitabine in urothelial carcinoma of the bladder (UCB) cell lines. Gemcitabine-resistant cells were established from 6 cell lines following exposure to escalating concentrations of the drug and by passaging cells in the presence of the drug over a 2- to 3-month period. Differential miRNA expression was identified in a microarray format comparing untreated controls with resistant cell lines, representing the maximum tolerated concentration, and results were validated via qRT-PCR. The involvement of specific miRNAs in chemoresistance was confirmed with transfection experiments, followed by clonogenic assays and Western blot analysis. Gemcitabine resistance was generated in 6 UCB cell lines. Microarray analysis comparing miRNA expression between gemcitabine-resistant and parental cells identified the differential expression of 66 miRNAs. Confirmation of differential expression was recorded via qRT-PCR in a subset of these miRNAs. Within this group, let-7b and let-7i exhibited decreased expression, while miR-1290 and miR-138 displayed increased expression levels in gemcitabine-resistant cells. Transfection of pre-miR-138 and pre-miR-1290 into parental cells attenuated cell death after exposure to gemcitabine, while transfection of pre-miR-let-7b and pre-miR-let-7i into the resistant cells augmented cell death. Mucin-4 was up-regulated in gemcitabine-resistant cells. Ectopic expression of let-7i and let-7b in the resistant cells resulted in the down-regulation of mucin-4. These results suggest a role for miRNAs 1290, 138, let-7i, and let-7b in imparting resistance to gemcitabine in UCB cell lines in part through the modulation of mucin-4. Alterations in these miRNAs and/or mucin-4 may constitute a potential therapeutic strategy for improving the efficacy of gemcitabine in UCB.

Background: Protein expression profiles throughout 28 days of peripheral nerve regeneration were characterized using an established rat sciatic nerve transection injury model. Reverse phase protein microarrays were used to identify the spatial and temporal expression profile of multiple proteins implicated in peripheral nerve regeneration including growth factors, extracellular matrix proteins, and proteins involved in adhesion and migration. This high-throughput approach enabled the simultaneous analysis of 3,360 samples on a nitrocellulose-coated slide., Results: The extracellular matrix proteins collagen I and III, laminin gamma-1, fibronectin, nidogen and versican displayed an early increase in protein levels in the guide and proximal sections of the regenerating nerve with levels at or above the baseline expression of intact nerve by the end of the 28 day experimental course. The 28 day protein levels were also at or above baseline in the distal segment however an early increase was only noted for laminin, nidogen, and fibronectin. While the level of epidermal growth factor, ciliary neurotrophic factor and fibroblast growth factor-1 and -2 increased throughout the experimental course in the proximal and distal segments, nerve growth factor only increased in the distal segment and fibroblast growth factor-1 and -2 and nerve growth factor were the only proteins in that group to show an early increase in the guide contents. As expected, several proteins involved in cell adhesion and motility; namely focal adhesion kinase, N-cadherin and β-catenin increased earlier in the proximal and distal segments than in the guide contents reflecting the relatively acellular matrix of the early regenerate., Conclusions: In this study we identified changes in expression of multiple proteins over time linked to regeneration of the rat sciatic nerve both demonstrating the utility of reverse phase protein arrays in nerve regeneration research and revealing a detailed, composite spatiotemporal expression profile of peripheral nerve regeneration.

Unlabelled: What's known on the subject? and What does the study add? Epithelial-mesenchymal transition (EMT) is involved in tumor progression where the underlying cellular changes associated with EMT have been identified in in vitro models and confirmed in a limited number of in vivo studies. ZEB1, which targets E-cadherin repression, is a transcriptional regulator that has been implicated in EMT, and is associated with uterine and colorectal cancers. Regulation of ZEB1 expression has been shown to involve different microRNAs (miRNAs), identifying a potential role for miRNA in EMT. In the present study we have identified novel expression of ZEB1 in bladder tumours and shown a role for ZEB1 in enhanced migration and invasion potential in in vitro assays. Confirmation of ZEB1 expression in bladder tumours was shown in tissue microarrays (TMAs)., Objective: To evaluate ZEB1 expression in bladder tumorigenesis and define a possible role for this transcription factor in urothelial carcinomas of the bladder (UCBs)., Materials and Methods: Five hundred and fifty-eight samples were assembled in 10 tissue microarrays (TMAs; 263 non-muscle-invasive Ta/T1/Tis, 295 muscle-invasive T2-T4). All tumours were transitional cell carcinomas (TCCs) and processed for immunohistochemistry to assess nuclear ZEB1 expression. Expression levels of ZEB1 were modulated in bladder carcinoma cell lines CUBIII or UM-UC-3 after forced expression or shRNA knockdown, respectively. Protein expression levels were determined using western blot analysis and transfectants were assessed for migration and invasion potential in standard in vitro assays., Results: Nuclear ZEB1 expression was recorded in 22.8% of non-muscle-invasive UCBs and 21.7% of muscle-invasive UCBs, including 24.1% grade I/II and 21.1% grade III tumours, and absent in normal bladder mucosa. No significant correlation was observed for tumour stage and grade, nodal involvement, vascular invasion, metastasis and overall or cancer-specific survival. The introduction or knockdown of ZEB1 expression in bladder carcinoma cell lines showed enhanced or reduced migration and invasive potential, respectively. Changes in ZEB1 expression were accompanied by altered microRNA (miRNA) expression underlying events linked to epithelial-mesenchymal transition (EMT)., Conclusion: The results in the present study showed novel expression of ZEB1 in bladder cancer in the absence of a link to clinical variables of change, including metastasis and survival. However, in vitro assays showed enhanced or reduced migration and invasion after the introduction or reduction of ZEB1, respectively, in transfected bladder cell lines. Modulation in expression of ZEB1 was closely linked to changes in the miR-200 family along with alternative known prognostic indicators of bladder tumour progression., (© 2010 THE AUTHORS. JOURNAL COMPILATION © 2010 BJU INTERNATIONAL.)

Purpose: We assessed the ability of different classes of histone deacetylase inhibitors to target tumor and invasive suppressor genes in a panel of bladder carcinoma cell lines using reverse phase protein arrays., Materials and Methods: Three poorly, moderately and highly invasive cell lines were exposed to histone deacetylase inhibitors, trichostatin A, apicidin, valproic acid (Sigma) and MS-275 (AXXORA) for 0 to 36 hours. Lysates were harvested and arrayed in a 10-fold dilution series in duplicate. Data points were collected and analyzed using a concentration interpolation methodology after normalization., Results: Protein expression profiles revealed up-regulation of gamma-catenin in highly invasive lines, and alpha-catenin in moderately and highly invasive lines after exposure to all histone deacetylase inhibitors, apicidin and MS-275, respectively. Gelsolin was up-regulated in poorly and moderately invasive lines after exposure to all histone deacetylase inhibitors. Desmoglein was down-regulated in poorly and moderately invasive cell lines by all 4 histone deacetylase inhibitors, in addition to decreased FAK (Transduction Laboratories) expression in moderately and highly invasive lines exposed to valproic acid and MS-275., Conclusions: Different histone deacetylase inhibitor classes have the potential to modulate tumor and invasive suppressor gene expression, identifying histone deacetylase inhibitors as potential therapeutic agents for bladder cancer. Reverse phase protein arrays enable high throughput screening of multiple compounds to assess the expression profile of specific protein groups targeted for therapy., (Copyright 2010 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objective: The goal of this study was to identify a microRNA (miRNA) signature in bladder cancer capable of differentiating superficial from invasive disease., Methods: Expression profiling of 343 miRNAs was performed in a microarray format using noninvasive and invasive bladder carcinoma cell lines with differential expression confirmed using a single molecule detection platform assay. miR-21 and miR-205 expression levels were determined in 53 bladder tumors (28 superficial and 25 invasive). Sensitivity, specificity, and a ROC curve were calculated to determine the discriminatory power of the miRNA ratio to predict invasion. Knockdown and forced expression of miRNAs was performed to evaluate their role in invasion., Results: Expression profiling of 343 miRNAs, using noninvasive and invasive bladder cell lines, revealed significant differential expression of 9 miRNAs. Cell lines characterized as invasive showed a miR-21:miR-205 ratio at least 10-fold higher than the quantitative ratio obtained from non-invasive cell lines. The same expression ratio was determined in 53 bladder tumors. From these results, we recorded a sensitivity and specificity of 100% and 78%, respectively, using a cutoff of 1.79 to predict an invasive lesion. The area under the receiver operator characteristic curve was 0.89. Using in vitro invasion assays, we have demonstrated a role for miR-21 in establishing the invasive phenotype of bladder carcinoma cells., Conclusion: In this study, we identified a miR-21:miR-205 expression ratio that has the ability to distinguish between invasive and noninvasive bladder tumors with high sensitivity and specificity, with the potential to identify superficial lesions at high risk to progress., (Copyright 2010 Elsevier Inc. All rights reserved.)

Purpose: Given the steadily growing cancer survivor population, increasing pressure has been placed on more effective clinical approaches and biomarker assays to manage care. For bladder cancer despite the high probability of recurrence the number of patients with recurrent disease is significantly lower than the number that remains cancer free at any monitoring interval. We developed a noninvasive urine assay using a novel approach to identify patients without recurrent cancer with extremely high confidence., Materials and Methods: Previous studies show that matrix metalloproteinases are increased in the urine of patients with cancer compared to that in disease-free individuals. To determine the clinical usefulness of these markers as monitors for bladder cancer recurrence we measured and compared metalloproteinase-2, metalloproteinase-9 and metalloproteinase-9/neutrophil gelatinase-associated lipocalin by enzyme-linked immunosorbent assay and zymography in a set of 530 samples, including 84 samples from patients with bladder cancer., Results: Initial studies using urine metalloproteinase to discriminate disease-free patients from those with bladder cancer resulted in 80% sensitivity (67 of 84) and 71% specificity (318 of 446) for metalloproteinase-9. By applying our novel Clinical Intervention Determining Diagnostic() clinical approach to metalloproteinase-9 we correctly identified 42% of cases that were cystoscopy negative with 98% negative predictive value., Conclusions: A noninvasive urine diagnostic assay that uses metalloproteinases with the Clinical Intervention Determining Diagnostic could lead to more efficient treatment in bladder cancer survivors by decreasing the number of negative cystoscopies (42%), allowing physicians to more selectively monitor those at high risk.

Necrosis of surgically transferred flaps is a major problem in reconstructive surgery. We investigated efficacy of a new vector system-adeno-associated viral 2 (AAV2)-mediated bFGF gene transfer to enhance survival of the ischemic flap. Thirty-eight Sprague-Dawley rats were divided into 3 gene therapy groups and 1 nontreated control of 9 or 10 each. 7.5 x 10(10) AAV2-bFGF viral particles were injected to the dorsum of each of the 29 rats; these rats were divided into 3 groups according to the timing of flap elevation. At the time of surgery, 1 week, and 2 weeks after surgery, flaps of 3 x 7 cm were raised. One week after surgery, flap viability was measured. Vascularization and immunohistochemical staining of the bFGF were evaluated of histologic sections. Flap viability was significantly improved by the AAV2-bFGF gene therapy at the time of surgery, and the flaps with the greatest survival area were found in the rats injected with AAV2-bFGF, 2 weeks before surgery. However, flap viability was significantly decreased by the gene therapy 1 week before surgery. Histologically, vascularity was increased in the groups with AAV2-bFGF injection and immunohistochemical staining showed greatly enhanced bFGF expression by gene transfer. The novel approach of AAV2-bFGF gene therapy shows encouraging manifestations in improving survival of flaps when the flaps are prefabricated during or 2 weeks before surgery.

Objective: To identify changes associated with P-cadherin expression in bladder cancer and evaluate the potential role of such events in determining the clinical outcome and cell behaviour, as the function of P-cadherin in normal epithelium is unknown, as is its potential role in neoplastic progression in different cancers., Materials and Methods: In all, 536 bladder tumour specimens from 408 patients were assembled in seven tissue microarrays. Paraffin sections from each array were processed for immunohistochemistry to assess the expression of P-cadherin. The expression of P-cadherin was forced using lipofectin, followed by an assessment of migration and invasion potential using standard in vitro assays., Results: The absence of P-cadherin staining was associated with muscle-invasive disease, grade 3 (P < 0.001) and nodal disease (P = 0.009). Similar results were obtained when considering cytoplasmic and unrestricted localization of P-cadherin (P < 0.001), except for nodal involvement. The group with cytoplasmic location of P-cadherin showed a shorter cancer-specific survival than the group with membrane location of P-cadherin (P = 0.03). Forced expression of P-cadherin in EJ and UM-UC-3 cells, that constitutively lack P-cadherin expression, resulted in modulation of catenin expression and enhanced migration of EJ and UM-UC-3/P-cadherin transfectants (>200%)., Conclusions: These results showed that loss of expression, cytoplasmic relocation or unrestricted tissue location of P-cadherin was associated with a poor clinical outcome and prognosis in bladder cancer. From the in vitro work it is evident that P-cadherin plays a role in regulating the migration potential of bladder carcinoma cells.

Objective: To identify the frequency of change in the expression and localization of p120(ctn) in bladder tumours and its association with clinical outcomes, and to investigate the potential role of p120(ctn) in the migratory and invasive behaviour of bladder carcinoma cells., Materials and Methods: In all, 425 superficial tumour specimens (Ta, Tis and T1) and 305 invasive (T2-T4) tumour specimens from 534 patients were assembled in 10 tissue microarrays. P120(ctn) immunostaining was scored for intensity and cellular localization and correlated with clinical variables and survival analysis. Knockdown of p120(ctn) was achieved using small-interference RNA (siRNA) followed by the assessment of migration and invasion behaviour in standard in vitro assays., Results: The expression levels of p120 catenin inversely correlated with pathological tumour stage (P < 0.001), histological grade (P < 0.001), presence of lymphovascular invasion (P = 0.02) but not lymph node (LN) involvement (P = 0.17). Non-membranous localization of p120(ctn) correlated with stage (P < 0.001), grade (P < 0.001), lymphovascular invasion (P = 0.04) and LN-positive disease (P = 0.02). A low expression level of p120(ctn) was linked to a poor outcome in cancer-specific survival analysis. Knockdown of p120(ctn) using siRNA resulted in a significant reduction in the migration and invasive potential of bladder carcinoma cells., Conclusions: Our findings suggest that p120(ctn) acts as a prognostic factor in bladder tumours and has a primary role to play in the migratory and invasive behaviour of bladder carcinoma cells.

Purpose: We established the frequency of mutation of the epidermal growth factor receptor in bladder cancer and determined whether the activation status of epidermal growth factor receptor confers sensitivity to erlotinib., Materials and Methods: The identification of mutations in the kinase domain (exons 18-21) of epidermal growth factor receptor was performed using single strand conformation polymorphism. The action of erlotinib was established within a bladder carcinoma cell panel using clonogenic assays and Western blot analysis., Results: In 112 invasive bladder tumors a total of 6 mutations in 4 patients (3.6%) were identified in exon 21. Erlotinib demonstrated concentration dependent inhibition of growth where three cell lines showed high and 2 showed low sensitivity to the drug. Erlotinib inhibited activation of epidermal growth factor receptor, mitogen activated protein kinase, Akt and STAT3. However, the activation status of Akt was maintained in cell lines that were insensitive to the inhibitory action of erlotinib and were characterized as having undergone epithelial-to-mesenchymal transition., Conclusions: Although mutations in the coding region of epidermal growth factor receptor are rare in invasive bladder tumors, differential sensitivity to erlotinib was recorded within a panel of cell lines. Maintenance of the phosphorylation status of Akt in the presence of erlotinib along with epithelial-to-mesenchymal transition correlates with insensitivity to growth inhibition in bladder carcinoma cell lines. Even in the absence of epidermal growth factor receptor mutations erlotinib shows potential as a therapeutic agent for the treatment of bladder cancer.

Green tea has been reported as potential dietary protection against numerous cancers and has been shown to have activity in bladder tumor inhibition in different animal models. The goal of this study was to examine the effects of (-)-epigallocatechin gallate (EGCG-the major phytochemical in green tea) on growth inhibition and behavior of human bladder carcinoma cells and to identify the altered signaling pathway(s) underlying the response to EGCG exposure. EGCG inhibited the in vitro growth of invasive bladder carcinoma cells with an IC(50) range of 70-87 microM. At a concentration of 20 microM, EGCG decreased the migratory potential of bladder carcinoma cells with concomitant activation of p42/44 MAPK and STAT3 and inactivation of Akt. Using biochemical inhibitors of MAPK/ERK, and siRNA to knockdown STAT3 and Akt, inhibition of migration was recorded associated with Akt but not MAPK/ERK or STAT3 signaling in bladder cells. In addition, EGCG downregulated N-cadherin in a dose-dependent manner where reduction in N-cadherin expression paralleled declining migratory potential. Continuous feeding of EGCG to mice prior to and during the establishment of bladder carcinoma xenografts in vivo revealed >50% reduction in mean final tumor volume (P

Invasive breast cancer has a high risk of recurrence to incurable disease and needs improved prognostic and therapeutic tools. Our work combines clinical and molecular analyses to show that the transcriptional repressor HBP1 may be a new target for invasive breast cancer. Previous work indicated that HBP1 regulated proliferation and senescence and inhibited Wnt signaling. Two of these functions have been associated with invasive breast cancer. In 76 breast tumors, we identified 10 HBP1 mutations/variants that were associated with fully invasive breast cancer. In a separate analysis, we found that a subset of invasive breast cancer specimens also had reduced HBP1 mRNA levels. These clinical correlations suggested that mutation or reduction of HBP1 occurs in invasive breast cancer and that HBP1 might regulate the proliferation and invasiveness of this breast cancer type. Analysis of the HBP1 mutants showed they were functionally defective for suppressing Wnt signaling. To test the consequences of reduced HBP1 levels, we used RNA interference to knock down HBP1 and observed increased Wnt signaling, tumorigenic proliferation, and invasiveness in cell and animal breast cancer models. Lastly, statistical analysis of a breast cancer patient database linked reduced HBP1 expression to breast cancer recurrence. In considering two-gene criteria for relapse potential, reduced expression of HBP1 and SFRP1, which is another Wnt inhibitor that was recently linked to invasive breast cancer, strikingly correlated with recurrence. Together, these data indicate that HBP1 may be a molecularly and clinically relevant regulator of breast cancer transitions that eventually lead to poor prognosis.

Background and Purpose: One million nosocomial infections occur each year in patients with prosthetic devices. We analyzed a polypropylene mesh coated with nanocrystalline silver particles (NCSP) as a means of preventing such infections., Methods: Nanocrystalline silver was applied to polypropylene mesh using physical vapor deposition in three doses: low (0.31 mg/cm(2)), medium (0.64 mg/cm(2)), and high (1.13 mg/cm(2)). The zone of inhibition (ZOI) test was conducted by incubating either uncoated polypropylene mesh (UM) or silver-coated mesh (CM) with and without various amounts of bovine serum albumin (BSA) and then on agar plates with Staphylococcus aureus and calculating the ZOI. The bactericidal effects of the NCSP-coated meshes were assessed by incubating either UM or CM in medium with S. aureus and performing serial dilutions at 4 and 8 h. Scanning electron microscopy (SEM) was used to examine the surface of UM and CM with and without bacterial incubation., Results: There was an increasing ZOI for low-, medium-, and high-dose CM and no ZOI for UM (p < 0.001 for all CM compared with UM). Incubating the mesh with various amounts of BSA produced persistent ZOIs with the medium- and high-dose CM; however, the low-dose CM became attenuated by such treatment, with no ZOI being seen with meshes incubated in 10% BSA. All concentrations of CM were bactericidal, as no bacteria grew at 8 h of incubation. The SEM images showed clusters of S. aureus on the surface of UM and no clusters on CM., Conclusions: The CM demonstrated significant bactericidal activity compared with UM. Nanocrystalline silver particles may decrease the incidence of postoperative prosthetic mesh infections and be useful as a coating for other prosthetic materials.

Purpose: Epithelial to mesenchymal transition (EMT) is reportedly an important transition in cancer progression in which the underlying cellular changes have been identified mainly using in vitro models. In this study, we examined the expression pattern of EMT markers in vivo and determined the occurrence and clinical significance of these events in a series of bladder carcinomas., Experimental Design: Eight hundred and twenty-five tumor samples from 572 bladder cancer patients were assembled in 10 tissue microarrays. Paraffin sections from each tissue microarray were subjected to antigen retrieval and processed by immunohistochemistry for the expression of E-cadherin, plakoglobin, beta-catenin, N-cadherin, and vimentin., Results: Pathologic expression of E-cadherin, beta-catenin, plakoglobin, and vimentin were associated with the clinicopathologic variables of grade and stage with only the cytoplasmic localization of plakoglobin found associated with lymph node status. Associations between the aforementioned markers were found significant as determined by the Spearman correlation coefficient with N-cadherin showing no associations in this analysis. In univariate survival analysis involving patients who underwent cystectomy, the reduction or loss of plakoglobin significantly influenced overall survival (P = 0.02) in which the median time to death was 2 years compared with 4 years when a normal level of plakoglobin was recorded. When the analysis was done for cancer-specific survival, low levels of both plakoglobin (P = 0.02) and beta-catenin (P = 0.02) significantly influenced survival., Conclusion: The putative markers of EMT defined within a panel of bladder carcinoma cell lines were recorded in vivo, frequently associated with tumors of high grade and stage. Although multivariate analysis showed no significant influence of the EMT biomarkers on survival, alterations associated with plakoglobin were identified as significant prognostic features in these tumors.

Purpose: Delivery of growth factor genes that may substantially increase the healing rate of injured digital flexor tendons is a new application of gene therapy. Adenoviral, adeno-associated viral (AAV), and liposome-plasmid vectors have been used to deliver genes to tendons, but the tendon reactions to these vectors--particularly in contrast to the healing responses in the injured tendons--were unknown. This study was designed to compare the tissue reactions of the earlier-mentioned vectors in tendons with the healing responses of injured flexor tendons., Methods: Forty-two flexor digitorum profundus tendons of 6 New Zealand white rabbits were used. Eighteen tendons were divided into 3 groups of 6 each and injected with different vectors: adenoviral vector, AAV2-luciferase vector, or pCMV-beta vector with liposome. Another 12 tendons were cut and repaired. At 3, 7, and 14 days, the tendons were harvested and stained with hematoxylin and eosin. Normal flexor tendons were harvested as controls., Results: The tissue reactions of the liposome-plasmid vector in tendons were the most prominent among the 3 vectors tested. The adenoviral vector elicited a moderate degree of tissue reaction. The AAV2 vector caused remarkable reactions in epitenon but almost no reactions in endotenon. Early-stage tissue reactions were more robust in the injured tendons. Compared with early-stage inflammatory and healing responses, the reactions elicited by these vectors were less severe., Conclusions: The 3 gene delivery systems tested elicit less severe tissue reactions in flexor tendons compared with early-stage inflammatory changes in injured tendons. Adenoviral and AAV vectors elicit less severe tissue reactions than liposome-plasmid vectors. The AAV2 vector appears to cause almost no reaction in endotenon. In terms of tissue reactions, the adenoviral and AAV2 vectors, in particular AAV2, are suitable gene delivery systems for future gene transfer to the tendon in vivo.

Purpose: The aim of this study was to evaluate the utility of the DNA integrity assay (DIA) as a plasma-based screening tool for the detection of prostate cancer., Experimental Design: Blood samples were collected from patients with biopsy-proven prostate cancer prior to prostatectomy (n = 123) and processed as two-spin plasma preparations. The three control groups included: males <40 years old with no history of cancer (group 1, n = 20); cancer-free postprostatectomy patients (group 2, n = 25), and patients with a negative prostate biopsy (group 3, n = 22). DNA in plasma preparations were isolated, hybrid-captured, and DNA fragments (200 bp, 1.3, 1.8, and 2.4 kb) were multiplexed in real-time PCR. A baseline cutoff was determined for individual fragment lengths to establish a DIA score for each patient sample., Results: Patients with prostate cancer (86 of 123; 69.9%) were determined to have a positive DIA score of >or=7. The DIA results from control groups 1, 2, and 3 showed specificities of 90%, 92%, and 68.2%, respectively. Of the patients with negative age-adjusted prostate-specific antigen (PSA) and prostate cancer, 19 of 30 (63%) had a positive DIA score. The area under the receiver operating characteristic curve for DIA was 0.788., Conclusion: While detecting 69.9% of those with prostate cancer, DIA maintained an overall specificity of 68.2% to 92%, a range favorably comparable to that currently accepted for PSA (60-70%). The variability in specificity between control groups is likely explained by the established 19% to 30% detection of prostate cancer on subsequent biopsies associated with control group 3. DIA detected 63% of the prostate cancers undetected by currently accepted PSA ranges.

Genetic and biochemical de-regulation of Wnt signaling is correlated with breast and other cancers. Our goal was to identify compounds that block Wnt signaling as a first step toward investigating new strategies for suppression of invasive and other breast cancers. In a limited phytonutrient screen, EGCG ((-)-epigallocatechin 3-gallate), the major phytochemical in green tea, emerged as an intriguing candidate. Epidemiological studies have associated green tea consumption with reduced recurrence of invasive and other breast cancers. Wnt signaling was inhibited by EGCG in a dose-dependent manner in breast cancer cells. The apparent mechanism targeted the HBP1 transcriptional repressor, which we had previously characterized as a suppressor of Wnt signaling. EGCG treatment induced HBP1 transcriptional repressor levels through an increase in HBP1 mRNA stability, but not transcriptional initiation. To test functionality, DNA-based short hairpin RNA (shRNA) was used to knockdown the endogenous HBP1 gene. Consistently, the HBP1 knockdown lines had reduced sensitivity to EGCG in the suppression of Wnt signaling and of a target gene (c-MYC). Because our ongoing studies clinically link abrogation of HBP1 with invasive breast cancer, we tested if EGCG also regulated biological functions associated with de-regulated Wnt signaling and with invasive breast cancer. EGCG reduced both breast cancer cell tumorigenic proliferation and invasiveness in an HBP1-dependent manner. Together, the emerging mechanism is that EGCG blocks Wnt signaling by inducing the HBP1 transcriptional repressor and inhibits aspects of invasive breast cancer. These studies provide a framework for considering future studies in breast cancer treatment and prevention.

The objective of this study was to investigate the efficacy of combination gene therapy with multiple angiogenic growth factor cDNAs to enhance survival of ischemic skin flaps in a rat model. Sixty Sprague-Dawley rats were divided into six groups. Varying combinations of VEGF165, PDGF-B, and bFGF-plasmids were injected to prefabricate the flaps. Random skin flaps were raised on the dorsal aspect of rats following prefabrication with growth factor cDNAs. Flap viability was determined by measurement of percentage area of survival. The efficacy of gene therapy was evaluated by flap survival and neovascularization of representative histologic sections stained immunohistologically. The VEGF165 plus bFGF cDNAs enhanced the viability of the flap and neovascularization most effectively; the flap survival area was 64.3 +/- 8.7% after transfer of these two growth factor genes. Addition of PDGF-B cDNA is deleterious to the effects of combined VEGF165 and bFGF, leading to a significant decrease in flap viability (44.9 +/- 2.7%). Viability of the flaps with combined VEGF165 and bFGF cDNA transfer was significantly greater than that of the flaps with VEGF165 transfer alone (57.6 +/- 5.2%) or sham plasmid control (52.3 +/- 5.0%). Combined transfer of VEGF165 and bFGF cDNA is the most effective combination of multiple growth factor genes to improve flap viability in this model. Simultaneous transfer of three growth factor genes (VEGF165, PDGF-B, and bFGF) is deleterious to flap survival, at least for the ratio of lipofectin:transgene employed.

Objective: To evaluate PP2 as a modulator of the cadherin/catenin complex in late-stage bladder carcinoma cells, and to assess its potential invasion-suppressor activity in this model., Materials and Methods: A panel of five human bladder carcinoma cells, characterizing late-stage disease, was used to determine the concentration for 50% inhibition of PP2 in cell-proliferation assays. Modulation of cadherin/catenin expression by PP2 was determined in Western blot analysis, with an assessment of the activation status of mitogen-activated protein kinase and Akt signalling pathways. Altered invasive capacity linked to these variables was determined in standard in vitro invasion assays., Results: PP2 elicited concentration-dependent growth inhibition in all bladder cell lines within the panel, with growth suppression recorded at 10-35 micromol/L PP2. Distinct morphological changes were recorded in cell lines exposed to PP2, accompanied by up-regulation of plakoglobin expression in a subset of lines. Exposure of cells to PP2 resulted in inactivation of Akt in all cells and a concomitant reduction in in vitro invasive capacity., Conclusions: These results show that PP2 inhibits bladder carcinoma cell growth and can modulate plakoglobin expression in a subset of cell lines. In addition, PP2 can suppress the in vitro invasive capacity of bladder carcinoma cells by modulating the activation status of Akt.

The reduction or loss of plakoglobin expression in late-stage bladder cancer has been correlated with poor survival where upregulation of this catenin member by histone deacetylase inhibitors has been shown to accompany tumour suppression in an in vivo model. In this study, we directly addressed the question of the role of plakoglobin in bladder tumorigenesis following restoration, or knockdown of expression in bladder carcinoma cell lines. Restoration of plakoglobin expression resulted in a reduction in migration and suppression of tumorigenic potential in vivo. Immunocytochemistry revealed cytoplasmic and membranous localisation of plakoglobin in transfectants with < 1% of cells displaying detectable nuclear localisation of plakoglobin. siRNA knockdown experiments targeting plakoglobin, revealed enhanced migration in all cell lines in the presence and absence of E-cadherin expression. In bladder cell lines expressing low levels of plakoglobin and desmoglein-2, elevated levels of desmoglein-2 were detected following restoration of plakoglobin expression in transfected cell lines. Analysis of wnt signalling revealed no activation event associated with plakoglobin expression in the bladder model. These results show that plakoglobin acts as a tumour suppressor gene in bladder carcinoma cells and the silencing of plakoglobin gene expression in late-stage bladder cancer is a primary event in tumour progression.

Histone deacetylase inhibitors (HDACis) are emerging as a promising new class of anticancer agents displaying growth-inhibitory activity and low toxicity in vivo. In this study, we examined the effect of sodium butyrate (NaB) and trichostatin A (TSA) on the growth of human bladder carcinoma cell lines in culture and TSA on the growth of EJ and UM-UC-3 human bladder xenografts in nude mice. NaB and TSA suppressed the growth of bladder cell lines at millimolar (1.5-4.3 mM) and micromolar (0.03-0.33 microM) concentrations, respectively, inducing concentration-dependent cell death. Bladder carcinoma cells within the experimental panel displayed the phenotype of late-stage bladder lesions expressing N-cadherin in the absence of E-cadherin accompanied by low levels of plakoglobin expression. Exposure of these cells to HDACis resulted in upregulation of plakoglobin with no change in E-cadherin expression. A 2-hr exposure to TSA was the minimal time required to upregulate plakoglobin in cells with downregulation to baseline levels occurring within 24 hr following drug removal. In mice bearing EJ and UM-UC-3 bladder xenografts, TSA (500 microg/kg/day) caused suppression of tumor growth compared with mice receiving vehicle alone. A > 70% reduction in mean final tumor volume was recorded in both bladder xenograft models with no detectable toxicity. The results suggest that TSA inhibits bladder carcinoma cell growth and may be a useful, relatively nontoxic agent for consideration in the treatment of late-stage bladder tumors., ((c) 2004 Wiley-Liss, Inc.)

Novel N-cadherin expression has been linked to the invasive phenotype in bladder tumors yet a primary role for N-cadherin in invasion has not been defined in this model. To address this, N-cadherin was stably transfected into E-cadherin expressing bladder carcinoma cells. This resulted in an enhanced invasive capacity in in vitro assays that was blocked by incubation with an N-cadherin function-blocking antibody in a dose-dependent manner. Analysis of the signaling pathway(s) implicated in N-cadherin-mediated invasion in bladder carcinoma cell lines revealed no correlation between MAPK signaling and invasion, in the presence or absence of fibroblast growth factor 2. Also, while MAPK and p38 kinase inhibitors did not alter the invasive behavior of these cells, an increase in the phosphorylation of Akt at serine-473 was detected in N-cadherin transfectants, suggestive of N-cadherin-mediated Akt activation in bladder cell invasion. Incubation of N-cadherin transfectants with either PI3 kinase or Akt inhibitors resulted in a significant decrease in the invasive capacity of these cells. Exposure of cells to PP2, a src family kinase inhibitor, also decreased the invasive potential of N-cadherin transfectants and resulted in reduced phosphorylation of Akt. The involvement of Akt signaling in bladder cell invasion was also supported by the inhibition of bladder cell invasion by cells constitutively expressing an activated Akt kinase, using the PI3 kinase and Akt inhibitors and PP2. These results suggest that activation of PI3/AKT kinase following N-cadherin expression contributes to the increased invasive potential of bladder carcinoma cells.

In this study we investigated the effects of materials prepared with electrical poling on neurite outgrowth in vitro and nerve regeneration in vivo. Neuro-2a cells were seeded on poled and unpoled poly(lactic-co-glycolic) (PLGA) films and observed at time periods 24, 48 and 72 h post-seeding. The percentage of cells with neurites and the neurites per cell were quantified using light microscopy. At 48 and 72 h post-seeding, both the number of cells with neurites and the neurites per cell were significantly increased on the poled films compared to those on unpoled films. An established rat sciatic nerve model was used for in vivo studies to assess the effects of PLGA guides, poled for two different periods, on peripheral nerve regeneration. Guides were inserted in rats to bridge a 1.0 cm gap created in the right sciatic nerve. After four weeks, nerves regenerated through poled guides displayed a significant increase in conduction velocity and significantly increased numbers of axons across the guides, as compared to nerves regenerating through an unpoled guidance channel. Electrical poling was shown to promote neurite growth, axon regeneration and the conduction rate of the repaired nerve. We concluded that guides prepared with electrical poling enhance peripheral nerve regeneration.

Background: Mutations in fibroblast growth factor 3 receptor (FGFR3) are frequent events in low-grade bladder tumors. To assess the potential utility of the detection of FGFR3 mutations in a screening modality, the authors analyzed urine sediment DNA samples from 192 patients in a retrospective study., Methods: Urine sediment DNA samples from 192 patients were prepared. Seventy-two patients had undergone transurethral resection (TURBT group) of mainly Ta lesions and 120 patients had undergone cystectomy (cystectomy group). The majority of patients in the cystectomy group had more advanced tumors compared with patients in the TURBT group. DNA preparations were screened for FGFR3 mutations in exons 7, 10, and 15 using single-strand conformation polymorphism (SSCP) and DNA sequencing., Results: Using SSCP, 67% of patients in the TURBT group and 28% in the cystectomy group displayed FGFR3 mutations. Comparative analysis of cytology results and FGFR3 mutational analysis were performed in 122 cases. Within the TURBT group, FGFR3 mutation analysis outperformed cytology. FGFR3 mutation analysis identified change in 68% of urine sediment DNA samples whereas cytology recorded the presence of tumor cells in 32% of the DNA samples. In the cystectomy group, cytology outperformed FGFR3 mutation analysis. Cytology recorded tumor detection in 90% of patients, while SSCP identified mutational change in 24%., Conclusions: Combining FGFR3 mutation results with cytology in both groups correctly identified tumor presence in 105 of 122 (86%) of patients. The greater sensitivity of FGFR3 mutation detection over cytology in identifying the presence of low-grade, superficial bladder tumors represents a potential new tool to complement standard cytology in screening patients for bladder tumors and recurrent disease., (Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11536)

Background: Molecular genetic analysis of DNA in patient stools has been proposed for screening of colorectal cancer (CRC). Because nonapoptotic cells shed from tumors may contain DNA that is less degraded than DNA fragments from healthy colonic mucosa, our aim was to show that DNA fragments isolated from stools of patients with CRC had higher integrity than DNA isolated from stools of patients with healthy colonic mucosa., Methods: We purified DNA from the stools of a colonoscopy-negative control group and patients with CRC and examined the relationship between long DNA fragments and clinical status by determining stool DNA integrity, using oligonucleotide-based hybrid captures with specific target sequences in increasingly long PCR reactions (200 bp, 400 bp, 800 bp, 1.3 kb, 1.8 kb, 24 kb). DNA fragments obtained from CRC patients were compared with fragments obtained from colonoscopy-negative individuals for length and/or integrity., Results: DNA fragments isolated from CRC patients were of higher molecular weight (>18 bands detected of a total of 24 possible bands) than fragments isolated from fecal DNA of the colonoscopy-negative control group., Conclusions: The presence of long DNA fragments in stool is associated with CRC and may be related to disease-associated differences in the regulation of proliferation and apoptosis. An assay of fecal DNA integrity may be a useful biomarker for the detection of CRC.

Using an established rat peripheral-nerve regeneration model, the authors have demonstrated enhancement of regeneration following subcutaneous priming of bioresorbable poly(lactic-co-glycolic)acid (PLGA) guides in vivo. Four weeks after nerve reconstruction, regeneration of the peripheral nerve through the cell-infiltrated guides displayed a significant increase in the total axon number and myelination status recorded in primed over unprimed guides, demonstrating the importance of cell-mediated events in the regeneration process. To define the different components enhancing nerve regeneration in this model, they have focused on identifying factors capable of eliciting Schwann-cell migration, since this has been identified as an early and necessary event in nerve regeneration. Using an in vitro migration assay, screening of a limited number of cellular and extracellular factors has demonstrated differential promotion of Schwann-cell migration. Of interest, combining fibronectin and bFGF resulted in a two-fold enhancement in Schwann-cell migration over that recorded with either alone. These results describe a rapid screening process for identifying various molecules and combinations thereof, with potential involvement in Schwann-cell migration. Coupling these findings to the use of the PLGA guide as an in vivo delivery system provides a rationale for the selection of exogenous factors to test for the enhancement of peripheral-nerve regeneration.

Purpose: We evaluate the efficacy of temperature based radio frequency ablation as a potential treatment modality for small (less than 3.5 cm.) renal tumors., Materials and Methods: We treated 15 patients with a total of 20 tumors with radio frequency ablation through an open surgical approach immediately before partial nephrectomy. All tumors were biopsied before radio frequency ablation treatment. Tumors were heated to 90 to 110C for 6 to 16 minutes (mean 9.1). Tumor ablation was monitored by direct vision and ultrasound. Partial nephrectomy was performed in standard fashion. All specimens were stained with hematoxylin and eosin, and 5 specimens were stained for nicotinamide adenine dinucleotide (NADH) diaphorase activity., Results: Tumors ranged from 1.5 to 3.5 cm. (mean 2.4) in greatest dimension. All 20 specimens had evidence of morphologically unchanged tumor and normal renal parenchyma on standard hematoxylin and eosin staining. Of the 5 specimens 4 stained positively for NADH in areas confirmed to be tumor in hematoxylin and eosin stained neighboring sections. There was 1 intraoperative renal pelvic thermal injury requiring pyeloplasty and 2 postoperative caliceal leaks requiring stent placement., Conclusions: In our series radio frequency therapy did not result in total tumor destruction when specimens were examined with hematoxylin and eosin or NADH staining. We believe that radio frequency interstitial tumor ablation of renal cell carcinoma without subsequent tissue resection should continue to be an investigational treatment modality for those who would otherwise undergo partial or radical nephrectomy.

Purpose: We investigated the incidence of loss of heterozygosity (LOH) and microsatellite instability in sporadic prostate cancer and surrounding tissue at loci encompassing the HPC1 and PTEN genes., Materials and Methods: Surgical specimens from 63 patients with sporadic stage T3 or T4 prostatic adenocarcinoma were analyzed for LOH and microsatellite instability. Microdissected tissue included morphologically normal foci, benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma. LOH analysis was performed using 4 microsatellite markers that map in the region of the 1q24 to 25 locus of the putative prostate cancer susceptibility gene HPC1 and 4 that map in the region of the 10q23 locus of the PTEN gene., Results: The incidence of LOH on 10q was consistent with that previously reported in prostatic tumors. LOH associated with the PTEN locus was recorded in morphologically normal foci, BPH and adenocarcinoma. Sequence analysis of PTEN in a limited number of lesions revealed mutations in nontumor and tumor tissue. Analysis of the DS10215 locus showed significant LOH in tumor but not in benign tissue, suggestive of a tumor suppressor gene in this region associated with prostatic neoplastic progression. In contrast, no significant LOH was observed in the same tissues at 4 loci on chromosome 1q. In this study we recorded elevated levels of microsatellite instability in benign prostatic tissue with an additional increase associated with prostatic adenocarcinoma., Conclusions: The low incidence of LOH in the region of the HPC1 locus in all prostate lesions studied suggests that this putative hereditary prostate cancer susceptibility locus does not appear to have a role in sporadic prostate cancer, at least not in the context of LOH. In contrast, analysis of the same tissues for LOH at chromosome 10q confirmed frequent alterations in this region linked to late stage prostate cancer. PTEN mutations in microdissected morphologically normal and BPH tissue showed alterations in nontumor tissue surrounding adenocarcinoma. Microsatellite instability was increased in adenocarcinomas over an elevated background recorded in surrounding tissues.

Aims: The relation between lobular carcinoma in situ (LCIS) and invasive breast cancer is unresolved. In an attempt to establish whether LCIS is a precursor of invasive cancer the mutational status and the expression of E-cadherin was analysed in LCIS and associated invasive breast carcinoma in 23 patients., Methods: Foci of LCIS and associated invasive carcinoma were individually microdissected from tissue from 23 patients. Exons 4-16 of the E-cadherin gene were analysed using single strand conformation polymorphism (SSCP); protein expression and the localisation of E-cadherin and beta-catenin were assessed with the use of immunohistochemistry., Results: Immunohistochemistry revealed a lack of expression of E-cadherin and beta-catenin in most LCIS samples and invasive foci. In all but four cases, the staining pattern was identical in the LCIS and associated invasive areas. When E-cadherin was absent, beta-catenin was also undetected, suggesting a lack of expression of alternative classic cadherin members in these lesions. Coincident E-cadherin mutations in LCIS and associated invasive carcinoma were not identified in this series of patients. However, mutational analysis of E-cadherin in multiple foci of carcinoma in situ surrounding an invasive lesion provided evidence to support ductal carcinoma in situ as a precursor of invasive ductal carcinoma., Conclusion: These data support the hypothesis that LCIS is not a precursor of invasive breast carcinoma but a marker of increased risk of developing invasive disease.

Loss or reduced expression of E-cadherin has been shown to be associated with poor survival in patients with bladder cancer. In numerous cases, loss of E-cadherin expression in bladder tumors has been accompanied by continued association of catenins with the membrane, suggestive of the expression of an alternative cadherin member. In this study we examined 75 bladder tumors using immunohistochemistry for the expression of E-, P-cadherin, and alpha-, beta-, and gamma-catenins. As reported previously, loss or reduced E-cadherin expression is a frequent event in late stage bladder cancer, accompanied by less frequent alterations associated with different catenin family members. Analysis of 51 tumors for expression of E-, P-, and N-cadherin showed P-cadherin localized to the basal cell layers of normal urothelium, with retention of expression in the majority of tumors. In low-grade tumors P-cadherin was found localized to an expanded basal cell compartment, contrasting with the more extensive staining observed in late stage tumors. Membranous P-cadherin staining was often found in the absence of E-cadherin staining. N-cadherin is not expressed in normal bladder mucosa, but detection of this cadherin member was recorded in 39% (20/51) of bladder tumors. Unlike P-cadherin, membranous N-cadherin was detected in focal regions within tumors, representing novel expression in urothelial neoplastic progression. Although focal N-cadherin staining was observed in 3 noninvasive lesions, the majority of tumors expressing N-cadherin were invasive (17/20). Coexpression of E-, P-, and N-cadherin was recorded in 5 grade 2 bladder tumors. Expression of P-cadherin is maintained throughout bladder tumorigenesis, accompanied by aberrant expression of N-cadherin. Clearly, neither P- nor N-cadherin act in an invasive-suppressor mode in bladder cancer, but whether they have a primary role to play in urothelial neoplastic progression has yet to be established.

Loss of heterozygosity (LOH) on 10q is associated with late-stage events in urothelial neoplastic progression. The tumor suppressor gene PTEN, which is mutated or homozygously deleted in numerous cancers, maps to a region of 10q within the reported region of minimal loss in bladder tumors. In two recent studies alterations in the PTEN gene occur at a low frequency in bladder tumors displaying 10q LOH. We have screened 35 late-stage bladder tumors for mutations in PTEN and MXI1, both genes mapping to chromosome 10q. Using single-strand conformation polymorphism analysis, we identified 6 tumors harboring mutations in PTEN and 2 additional tumors displaying homozygous deletion at this locus. No MXI1 mutations were identified within the same tumor panel. Of 16 bladder tumor cell lines analyzed, 2 showed homozygous deletion of PTEN and 3 harbored point mutations resulting in an amino acid change. Two cell lines harbored missense mutations in MXI1. We report a significantly higher frequency of PTEN alterations in bladder carcinoma (23%) than was previously recorded, with no accompanying mutations in the MXI1 gene., (Copyright 2000 Wiley-Liss, Inc.)

PTEN, a candidate tumor suppressor gene located at chromosome 10q23.3, has been shown to be mutated in approximately 40% of endometrial cancers. Such mutations have also been identified in endometrial hyperplasia, indicating that inactivation of the PTEN tumor suppressor gene is an early event in the genesis of some endometrial cancers. In this study, we have extended the analysis of PTEN in gynecological cancer to include adenocarcinoma of the cervix and vulvar carcinomas. Microdissected tissue (including normal tissues), preneoplastic, and neoplastic lesions were analyzed from 9 patients with cervical cancer and 10 patients with vulvar cancer. Only 1 cervical adenocarcinoma displayed a PTEN mutation. In contrast, five of eight vulvar carcinomas studied harbored PTEN mutations. Alterations were identified in carcinoma in situ as well as squamous cell carcinoma of the vulva. In two patients, PTEN mutations were identified in mucosal regions with mild or focal dysplasia. These results suggest that PTEN is frequently altered in vulvar carcinomas and can be found associated with early dysplastic changes in vulvar mucosa.

Using an established rat peripheral nerve regeneration model, we investigated the role of glial growth factor (GGF) in nerve regeneration in combination with a novel bioresorbable poly(lactic-co-glycolic) acid (PLGA) guide in vivo. Schwann cells, established from a 1-cm segment of excised rat sciatic nerve, were isolated and seeded onto nerve guides with or without GGF (n = 24/group). Living nerve guides were re-established in these animals, and nerve regeneration was assessed over a period of 12 weeks. Histological studies revealed a reduction in the total axon count and the number of myelinated axons in the presence of exogenously added Schwann cells compared to saline controls. In contrast, the addition of GGF alone enhanced the total number of axons and significantly increased the number of blood vessels. Although combining GGF with Schwann cells negated the enhanced numbers of axons and blood vessels seen with GGF alone, this combination resulted in the highest myelination index and the fastest conduction velocities recorded. The PLGA guide material did not trigger any histologically detectable host response and was permissive for nerve regeneration in this animal model. The results from this study demonstrate the potential utility of this guide in vivo and establish a promotional role for GGF in nerve regeneration.

Purpose: We present preliminary clinical, histochemical and molecular findings for 5 patients with micropapillary transitional cell carcinoma of the bladder, a rare histological variant not widely recognized in the urological literature., Materials and Methods: The 5 patients were prospectively identified. In 3 cases immunohistochemical staining for expression of CD31, p53, E-cadherin, and alpha, beta and gamma-catenin was performed on paraffin embedded tissue. Sequencing was used to identify point mutations in exons 5 to 9 of p53, and exons 1 and 2 of H-ras., Results: Of the patients 2 died within 1 year of presentation to our institution with rapid local extension along the bladder serosal surface and ureteral sheaths. Another patient had progression to invasive disease within 22 months. In the 3 cases with immunohistochemical staining p53 was negative, despite positive staining of nonmicropapillary transitional cell carcinoma within the same specimen. Stains for the angiotrophic marker CD31 were negative. In all 3 cases normal membrane associated alpha, beta and gamma-catenin expression was present. Examination of p53 sequences revealed a single point mutation in exon 8 of 1 case. In 2 cases different mutations in exon 1 of H-ras were noted., Conclusions: Micropapillary transitional cell carcinoma is a rare and highly aggressive variant. Paradoxically, our study demonstrated no significant p53 abnormalities. The lacunar histological pattern did not appear to represent invasion of vascular spaces. Rather, these tumors seemed to have the ability to disrupt and replace the normal stromal matrix to achieve rapid nonendothelial extension. Thus, micropapillary histology may predict a lesser likelihood of surgical cure.

Background: Sucrase-isomaltase (SI) is a tissue-based phenotypic marker that is an independent prognostic factor in colorectal cancer (CRC). DF3 and galectin 3 are two other tissue-based markers that are upregulated during neoplastic transformation. Because p53 mutations are acquired during neoplastic progression, we reasoned that alterations in SI and p53 may be associated despite an apparent lack of biological interaction., Methods: Paraffin sections from 183 patients who underwent surgery at New England Deaconess Hospital (NEDH) between 1965 and 1977 were analyzed first by immunohistochemistry (IHC) for the expression of the markers SI, DF3, and galectin 3, which were scored as absent or present. Paraffin sections from a second group of 59 patients who underwent surgery at NEDH between 1985 and 1992 were analyzed by IHC for the expression of p53 as well as SI, DF3, and galectin 3. p53 nuclear staining was scored as absent or present. Previous work has shown that p53 is mutated in all cells with nuclear staining and in 10% of tumors that are unstained., Results: SI expression was not associated with the expression of either DF3 or galectin 3, and neither DF3 nor galectin 3 were prognostic factors in CRC. None of the phenotypic markers were associated with any of the clinicopathologic variables. However, 21 of 24 p53-positive cases (88%) expressed SI, whereas 15 of 35 p53-negative cases (43%) were also SI negative (p = 0.02, Fisher exact test). p53 expression was not associated with expression of DF3 or galectin 3., Conclusions: SI expression and p53 mutation are associated significantly in CRC. Although the mechanism underlying such an association in presently unknown, the association may define a subset of patients with a worse prognosis.