Morphological Criteria for Comparing Effects of X-Rays and Neon Ions on Mouse Small Intestine

Several techniques have been used to assess changes in different parts of mouse small intestine three days after a single dose of either 16.5 Gy X-rays or 11 Gy neon beam. The doses were chosen to be approximately equivalent in terms of their effect on the number of microcolonies present. In qualitative terms, villous damage was seen after both types of radiation exposure: collared crypts, similar to those seen in biopsies taken from patients suffering from coeliac disease, were conspicuous after neon irradiation. In semi quantitative terms the doses used, although estimated from previous work to give biologically equivalent damage, produced a greater drop in microcolony numbers after X-irradiation, This makes all the more important the fact that significantly greater changes were seen after neon irradiation - a greater drop was seen in the number of villous profiles and the number of goblet cells per villus. There was also greater breakdown in the integrity of the villous basement membrane. Different responses after the two types of irradiation are therefore seen in the cryptal and villous compartment. Progress is being made towards identifying and quantitating radiation induced changes in different populations of cells or tissues in the small intestine

Validation and utilization of a TFE3 break-apart FISH assay for Xp11.2 translocation renal cell carcinoma and alveolar soft part sarcoma

Background: Xp11.2 or TFE3 translocation renal cell carcinomas (RCC) and alveolar soft part sarcoma (ASPS) are characterized by chromosome translocations involving the Xp11.2 breakpoint resulting in transcription factor TFE3 gene fusions. The most common translocations documented in TFE3 RCCs are t(X;1) (p11.2;q21) and t(X;17) (p11.2;q25) which leads to fusion of TFE3 gene on Xp11.2 with PRCC or ASPL respectively. TFE3 immunohistochemistry (IHC) has been inconsistent over time due to background staining problems in part related to fixation issues. Karyotyping to detect TFE3 gene rearrangement requires typically unavailable fresh tissue. Reverse transcriptase-polymerase chain reaction (RT-PCR) is generally very challenging due to degradation of RNA in archival material. The study objective was to develop and validate a TFE3 break-apart fluorescence in situ hybridization (FISH) assay to confirm Xp11 translocation RCCs and ASPS. Methods: Representative sections of formalin-fixed paraffin-embedded tissue blocks were selected in 40 possible cases. Approximately 60 tumor cells were analyzed in the targeted region. The validation of TFE3 FISH was done with 11 negative and two positive cases. Cut off for a positive result was validated as >7.15 % positive nuclei with any pattern of break-apart signals. FISH evaluation was done blinded of the immunohistochemical or karyotype data. Results: Three out of forty cases were positive for the TFE3 break-apart signals by FISH. The negative cases were reported as clear cell RCC with papillary features (10), clear cell RCC with sarcomatoid areas (2), Papillary RCC with clear cell areas (9), Chromophobe RCC (2), RCC, unclassified type (3) and renal medullary carcinoma (1). 3 of the negative cases were consultation cases for renal tumor with unknown histology. Seven negative cases were soft tissue tumor suspicious for ASPS. Conclusion: Our study validates the utility of TFE3 break-apart FISH on formalin-fixed paraffin-embedded tissue sections for diagnosis and confirmation of Xp11.2 translocation RCCs and ASPS

Conventional liquid-based techniques versus Cytyc Thinprep(® )processing of urinary samples: a qualitative approach

BACKGROUND: The aim of our study was to objectively compare Cytyc Thinprep(® )and other methods of obtaining thin layer cytologic preparations (cytocentrifugation, direct smearing and Millipore(® )filtration) in urine cytopathology. METHODS: Thinprep slides were compared to direct smears in 79 cases. Cytocentrifugation carried out with the Thermo Shandon Cytospin(® )4 was compared to Thinprep in 106 cases, and comparison with Millipore filtration followed by blotting was obtained in 22 cases. Quality was assessed by scoring cellularity, fixation, red blood cells, leukocytes and nuclear abnormalities. RESULTS: The data show that 1) smearing allows good overall results to be obtained, 2) Cytocentrifugation with reusable TPX(® )chambers should be avoided, 3) Cytocentrifugation using disposable chambers (Cytofunnels(® )or Megafunnel(® )chambers) gives excellent results equalling or surpassing Thinprep and 4) Millipore filtration should be avoided, owing to its poor global quality. Despite differences in quality, the techniques studied have no impact on the diagnostic accuracy as evaluated by the rate of abnormalities. CONCLUSION: We conclude that conventional methods such as cytocentrifugation remain the most appropriate ones for current treatment of urinary samples. Cytyc Thinprep processing, owing to its cost, could be used essentially for cytology-based molecular studies

Construction of tissue microarrays from prostate needle biopsy specimens

Needle biopsies are taken as standard diagnostic specimens for many cancers, but no technique exists for the high-throughput analysis of multiple individual immunohistochemical (IHC) markers using these samples. Here we present a simple and highly reliable technique for constructing tissue microarrays (TMAs) from prostatic needle biopsies. Serial sectioning of the TMAs, called ‘Checkerboard TMAs', facilitated expression analysis of multiple proteins using IHC markers. In total, 100% of the analysed biopsies within the TMA both preserved their antigenicity and maintained their morphology. Checkerboard TMAs will allow the use of needle biopsies (i) alongside other tissue specimens (trans-urethral resection of prostates and prostatectomies in the case of prostate cancer) in clinical correlation studies when searching for new prognostic markers, and (ii) in a diagnostic context for assessing expression of multiple proteins in cancers from patients prior to treatment

Oxidants, antioxidants, and respiratory tract lining fluids.

Respiratory tract lining fluids (RTLFs) are a heterogeneous group of substances covering the respiratory tract epithelial cells (RTECs) from nasal mucosa to alveoli. Antioxidant contained in the RTLFs can be expected to provide an initial defense against inhaled environmental toxins. The major antioxidants in RTLF include mucin, uric acid, protein (largely albumin), ascorbic acid, and reduced glutathione (GSH). RTLF antioxidants can be augmented by such processes as transudation/exudation of plasma constituents; RTEC secretory processes, including glandular mucus secretion; and cellular antioxidants derived from lysis of RTECs and of inflammatory cells. The antioxidant composition of RTLFs and their role in modulating normal and pathophysiologic RTEC functions under conditions of oxidative stress are yet to be fully characterized

Early detection of urothelial premalignant lesions using hexaminolevulinate fluorescence cystoscopy in high risk patients

<p>Abstract</p> <p>Background</p> <p>To evaluate fluorescence cystoscopy with hexaminolevulinate (HAL) in the early detection of dysplasia (DYS) and carcinoma in situ (CIS) in select high risk patients.</p> <p>Methods</p> <p>We selected 30 consecutive bladder cancer patients at high risk for progression. After endoscopic resection, all patients received (a) induction BCG schedule when needed, and (b) white light and fluorescence cystoscopy after 3 months. HAL at doses of 85 mg (GE Healthcare, Buckinghamshire, United Kingdom) dissolved in 50 ml of solvent to obtain an 8 mmol/L solution was instilled intravesically with a 12 Fr catheter into an empty bladder and left for 90 minutes. The solution was freshly prepared immediately before instillation. Cystoscopy was performed within 120 minutes of bladder emptying. Standard and fluorescence cystoscopy was performed using a double light system (Combilight PDD light source 5133, Wolf, Germany) which allowed an inspection under both white and blue light.</p> <p>Results</p> <p>The overall incidence was 43.3% dysplasia, 23.3% CIS, and 13.3% superficial transitional cell cancer. In 21 patients, HAL cystoscopy was positive with one or more fluorescent flat lesions. Of the positive cases, there were 4 CIS, 10 DYS, 2 association of CIS and DYS, 4 well-differentiated non-infiltrating bladder cancers, and 1 chronic cystitis. In 9 patients with negative HAL results, random biopsies showed 1 CIS and 1 DYS. HAL cystoscopy showed 90.1% sensitivity and 87.5% specificity with 95.2% positive predictive value and 77.8% negative predictive value.</p> <p>Conclusion</p> <p>Photodynamic diagnosis should be considered a very important tool in the diagnosis of potentially evolving flat lesions on the bladder mucosa such as DYS and CIS. Moreover, detection of dysplasic lesions that are considered precursors of CIS may play an important role in preventing disease progression. In our opinion, HAL cystoscopy should be recommended in the early follow-up of high risk patients.</p

The effect of titanium dioxide nanoparticles on pulmonary surfactant function and ultrastructure

<p>Abstract</p> <p>Background</p> <p>Pulmonary surfactant reduces surface tension and is present at the air-liquid interface in the alveoli where inhaled nanoparticles preferentially deposit. We investigated the effect of titanium dioxide (TiO₂) nanosized particles (NSP) and microsized particles (MSP) on biophysical surfactant function after direct particle contact and after surface area cycling <it>in vitro</it>. In addition, TiO₂effects on surfactant ultrastructure were visualized.</p> <p>Methods</p> <p>A natural porcine surfactant preparation was incubated with increasing concentrations (50-500 μg/ml) of TiO₂NSP or MSP, respectively. Biophysical surfactant function was measured in a pulsating bubble surfactometer before and after surface area cycling. Furthermore, surfactant ultrastructure was evaluated with a transmission electron microscope.</p> <p>Results</p> <p>TiO₂NSP, but not MSP, induced a surfactant dysfunction. For TiO₂NSP, adsorption surface tension (γ_ads) increased in a dose-dependent manner from 28.2 ± 2.3 mN/m to 33.2 ± 2.3 mN/m (p < 0.01), and surface tension at minimum bubble size (γ_min) slightly increased from 4.8 ± 0.5 mN/m up to 8.4 ± 1.3 mN/m (p < 0.01) at high TiO₂NSP concentrations. Presence of NSP during surface area cycling caused large and significant increases in both γ_ads(63.6 ± 0.4 mN/m) and γ_min(21.1 ± 0.4 mN/m). Interestingly, TiO₂NSP induced aberrations in the surfactant ultrastructure. Lamellar body like structures were deformed and decreased in size. In addition, unilamellar vesicles were formed. Particle aggregates were found between single lamellae.</p> <p>Conclusion</p> <p>TiO₂nanosized particles can alter the structure and function of pulmonary surfactant. Particle size and surface area respectively play a critical role for the biophysical surfactant response in the lung.</p

The androgen receptor can signal through Wnt/β-Catenin in prostate cancer cells as an adaptation mechanism to castration levels of androgens

<p>Abstract</p> <p>Background</p> <p>A crucial event in Prostate Cancer progression is the conversion from a hormone-sensitive to a hormone-refractory disease state. Correlating with this transition, androgen receptor (AR) amplification and mutations are often observed in patients failing hormonal ablation therapies. β-Catenin, an essential component of the canonical Wnt signaling pathway, was shown to be a coactivator of the AR signaling in the presence of androgens. However, it is not yet clear what effect the increased levels of the AR could have on the Wnt signaling pathway in these hormone-refractory prostate cells.</p> <p>Results</p> <p>Transient transfections of several human prostate cancer cell lines with the AR and multiple components of the Wnt signaling pathway demonstrate that the AR overexpression can potentiate the transcriptional activities of Wnt/β-Catenin signaling. In addition, the simultaneous activation of the Wnt signaling pathway and overexpression of the AR promote prostate cancer cell growth and transformation at castration levels of androgens. Interestingly, the presence of physiological levels of androgen or other AR agonists inhibits these effects. These observations are consistent with the nuclear co-localization of the AR and β-Catenin shown by immunohistochemistry in human prostate cancer samples. Furthermore, chromatin immunoprecipitation assays showed that Wnt3A can recruit the AR to the promoter regions of Myc and Cyclin D1, which are well-characterized downstream targets of the Wnt signalling pathway. The same assays demonstrated that the AR and β-Catenin can be recruited to the promoter and enhancer regions of a known AR target gene PSA upon Wnt signaling. These results suggest that the AR is promoting Wnt signaling at the chromatin level.</p> <p>Conclusion</p> <p>Our findings suggest that the AR signaling through the Wnt/β-Catenin pathway should be added to the well established functional interactions between both pathways. Moreover, our data show that via this interaction the AR could promote prostate cell malignancy in a ligand-independent manner.</p

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma

BACKGROUND Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2'antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renalcell carcinoma consisted of at least three subtypes based on molecular and phenotypic features

Corrigendum: Inflammation and Tissue Remodeling in the Bladder and Urethra in Feline Interstitial Cystitis

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating chronic disease of unknown etiology. A naturally occurring disease termed feline interstitial cystitis (FIC) reproduces many features of IC/BPS patients. To gain insights into mechanisms underlying IC/BPS, we investigated pathological changes in the lamina propria (LP) of the bladder and proximal urethra in cats with FIC, using histological and molecular methods. Compared to control cat tissue, we found an increased number of de-granulated mast cells, accumulation of leukocytes, increased cyclooxygenase (COX)-1 expression in the bladder LP, and increased COX-2 expression in the urethra LP from cats with FIC. We also found increased suburothelial proliferation, evidenced by mucosal von Brunn’s nests, neovascularization and alterations in elastin content. Scanning electron microscopy revealed normal appearance of the superficial urethral epithelium, including the neuroendocrine cells (termed paraneurons), in FIC urethrae. Together, these histological findings suggest the presence of chronic inflammation of unknown origin leading to tissue remodeling. Since the mucosa functions as part of a “sensory network” and urothelial cells, nerves and other cells in the LP are influenced by the composition of the underlying tissues including the vasculature, the changes observed in the present study may alter the communication of sensory information between different cellular components. This type of mucosal signaling can also extend to the urethra, where recent evidence has revealed that the urethral epithelium is likely to be part of a signaling system involving paraneurons and sensory nerves. Taken together, our data suggest a more prominent role for chronic inflammation and tissue remodeling than previously thought, which may result in alterations in mucosal signaling within the urinary bladder and proximal urethra that may contribute to altered sensations and pain in cats and humans with this syndrome