The combined use of non-radioactive in situ hybridization and real-time RT-PCR to assess gene expression in cryosections

Gene expression changes in pathophysiological states can be spatiotemporally monitored by in situ hybridization and reliably quantified by real-time RT-PCR. Here we developed a new method whereby adjacent slides of frozen sections can be used for gene expression analysis by in situ hybridization and real-time RT-PCR. We applied this method to assess the mRNA expression of connexin 43 (Cx43), the major astrocytic connexin, after kainate-induced seizures in rat hippocampus. Gap junction-building connexins play a role in the pathogenesis of several diseases of the brain, including epilepsy. The number of Cx43 mRNA-positive cells in the hippocampus of kainate-treated and control rats was automatically quantified by computerized image analysis of brain sections hybridized with DIG-labeled RNA probes. In parallel, real-time RT-PCR was used to examine the relative Cx43 mRNA levels in hippocampal tissue from adjacent brain sections. Applying these two very sensitive methods we showed that kainate induced seizures do not affect hippocampal connexin 43 mRNA expression.     

Intraductal papillomas of the breast: diagnosis and management of 151 patients

Introduction

The assessment of papillary lesions continues to be a challenging area in breast radiology and pathology. The management of intraductal papillomas without atypia of the breast remains controversial. The purpose of the present study was to determine diagnostic accuracy of radiographical diagnosis, core biopsy, and surgical excision in papillary breast lesions.

Material and methods

By using files from 1995 to 2010, 151 cases of intraductal papilloma with or without atypia were identified. Patients were stratified as follows: core biopsy followed by surgical excision (n = 61), core biopsy alone (n = 19), and surgical excision alone (n = 71).

Results

The upstage rate of intraductal papillomas without atypia on core biopsy to atypia or malignancy on excision was 8.9%. Excision specimens revealed intraductal papillomas without atypia in 68 out of 71 cases, and atypical papillomas in 3 cases.

Conclusion

Our findings suggest that radiographic and histopathological diagnosis of intraductal papillomas show high accuracy and good concordance. In cases where the radiographic diagnosis reveals suspicious lesions core biopsy represents the first choice.     

Glibenclamide effects on renal function and histology after acute hemorrhage in rats under sevoflurane anesthesia

INTRODUCTION: Hypovolemia from hemorrhage evokes protective compensatory reactions, such as the renin-angiotensin system, which interferes in the clearance function and can lead to ischemia. This study was designed to evaluate the effects of glibenclamide, a K(+)(ATP) channel blocker, on renal function and histology in rats in a state of hemorrhagic shock under sevoflurane anesthesia. MATERIAL AND METHODS: Twenty Wistar rats were randomized into two groups of 10 animals each (G1 and G2), only one of which (G2) received intravenous glibenclamide (1 microg.g(-1)), 60 min before bleeding was begun. Both groups were anesthetized with sevoflurane and kept on spontaneous respiration with oxygen-air, while being bled of 30% of volemia in three stages with 10 min intervals. There was an evaluation of renal function - sodium para-aminohippurate and iothalamate clearances, filtration fraction, renal blood flow, renal vascular resistance - and renal histology. Renal function attributes were evaluated at three moments: M1 and M2, coinciding with the first and third stages of bleeding; and M3, 30 min after M2, when the animals were subjected to bilateral nephrectomy before being sacrificed. RESULTS: Significant differences were found in para-aminohippurate clearance, G1 < G2, and higher renal vascular resistance values were observed in G1. Histological examination showed the greater vulnerability of kidneys exposed to sevoflurane alone (G1) with higher scores of vascular and tubular dilatation. There were vascular congestion and tubular vacuolization only in G1. Necrosis and signs of tubular regeneration did not differ in both groups. CONCLUSION: Treatment with glibenclamide attenuated acutely the renal histological changes after hemorrhage in rats under sevoflurane anesthesia.     

Increased oxidative stress in the mouse adriamycin model of glomerulosclerosis is accompanied by deposition of ferric iron and altered GGT activity in renal cortex

Chronic renal failure evolves inevitable towards glomerular and tubulo-interstitial sclerosis. This pathological process involves a disturbed redox status of the kidney tissue, leading to irreversible damage. In this study we investigate in an adriamycin model of chronic renal failure in mice the evolution of in vivo hydrogen peroxide production, and the possible role of gamma-glutamyl transpeptidase and ferric iron in the process. Histological changes and ferric iron deposits are evaluated by histochemical staining. To evaluate oxidative stress residual catalase activity, TBARS formation and gamma-glutamyl transpeptidase activity are measured spectrophotometrically. While catalase activity remains the same, a decreased residual catalase activity indicates an increased formation of hydrogen peroxide. Both the activity of gamma-glutamyl transpeptidase and TBARS formation is increased at early stages of the disease. Ferric iron is clearly present in the proximal tubule. Twenty days after adriamycin injection all parameters decrease, probably due to the destruction of the tissue. Our data show the involvement of oxidative stress in the progression of adriamycin induced renal failure in mice. Both radical production and oxidative damage are measurable, while the altered activity of gamma-glutamyl transpeptidase and the deposition of ferric iron suggest the involvement of these factors in the development of a disturbed redox status in the kidney cortex.     

3-[123I]Iodo-L-alpha-methyl tyrosine transport into human fibroblasts and comparison with Ewing's sarcoma cells

The cellular transport systems and the transport kinetics of [123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [123I]IMT is exclusively transported into fibroblasts via the sodium independent system L. An apparent Michaelis constant K(m) = 116.2 +/- 18.9 microM and a maximum transport velocity V(max) = 191.6 +/- 13.9 pmol x (10(6) cells)(-1) x min(-1) were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing's sarcoma cell lines VH-64 and CADO-ES-1.     

Diagnostic impact of 18F-FDG PET–CT evaluating solid pancreatic lesions versus endosonography, endoscopic retrograde cholangio-pancreatography with intraductal ultrasonography and abdominal ultrasound

Purpose  This prospective single-centre phase II trial assessed the diagnostic impact of ¹⁸F-FDG PET–CT in the evaluation of solid pancreatic lesions (∅ ≥10 mm) compared to endosonography (EUS), endoscopic retrograde cholangio-pancreatography (ERCP) with intraductal ultrasound (IDUS), abdominal ultrasound (US) and histopathological reference. Methods  Forty-six patients (32 men/14 women, ∅ 61.7 years) with suspected pancreatic neoplasms underwent PET–CT with contrast-enhanced biphasic multi-detector CT of the upper abdomen followed by a diagnostic work-up with EUS, ERCP with IDUS and US within 3 weeks. PET–CT data sets were analysed by two expert readers in a consensus reading. Histology from surgery, biopsy/fine-needle aspiration and/or clinical follow-up ≥12 months served as standard of reference. Results  Twenty-seven pancreatic malignancies were histopathologically proven; 19 patients had benign diseases: 36/46 lesions (78%) were detected in the head of the pancreas, 7/46 and 3/46 in the body and tail region, respectively. Sensitivity and specificity of PET–CT were 89% and 74%, respectively; positive predictive value (PPV) and negative predictive value (NPV) were 83% and 82%, respectively. Sensitivity (81–89%), specificity (74–88%), PPV (83–90%) and NPV (77–82%) achieved by EUS, ERCP and US were not significantly different. PET analysis revealed significantly higher maximum mean standardised uptake values (SUV_max 6.5 ± 4.6) in patients with pancreatic malignancy (benign lesions: SUV_max 4.2 ± 1.5; p < 0.05). PET–CT revealed cervical lymphonodal metastasis from occult bronchogenic carcinoma and a tubular colon adenoma with intermediate dysplasia on polypectomy, respectively. Conclusions   ¹⁸F-FDG PET–CT achieves a comparably high diagnostic impact evaluating small solid pancreatic lesions versus conventional reference imaging modalities. Additional clinical diagnoses are derived from concomitant whole-body PET–CT imaging. Verena Schick and Christiane Franzius contributed equally to this work.     

Comparison of 1.0 M gadobutrol and 0.5 M gadopentate dimeglumine-enhanced MRI in 471 patients with known or suspected renal lesions: results of a multicenter, single-blind, interindividual, randomized clinical phase III trial

The purpose of this phase III clinical trial was to compare two different extracellular contrast agents, 1.0 M gadobutrol and 0.5 M gadopentate dimeglumine, for magnetic resonance imaging (MRI) in patients with known or suspected focal renal lesions. Using a multicenter, single-blind, interindividual, randomized study design, both contrast agents were compared in a total of 471 patients regarding their diagnostic accuracy, sensitivity, and specificity to correctly classify focal lesions of the kidney. To test for noninferiority the diagnostic accuracy rates for both contrast agents were compared with CT results based on a blinded reading. The average diagnostic accuracy across the three blinded readers (‘average reader’) was 83.7% for gadobutrol and 87.3% for gadopentate dimeglumine. The increase in accuracy from precontrast to combined precontrast and postcontrast MRI was 8.0% for gadobutrol and 6.9% for gadopentate dimeglumine. Sensitivity of the average reader was 85.2% for gadobutrol and 88.7% for gadopentate dimeglumine. Specificity of the average reader was 82.1% for gadobutrol and 86.1% for gadopentate dimeglumine. In conclusion, this study documents evidence for the noninferiority of a single i.v. bolus injection of 1.0 M gadobutrol compared with 0.5 M gadopentate dimeglumine in the diagnostic assessment of renal lesions with CE-MRI.

The role of the kallikrein-kinin system,matrix metalloproteinases,and tissue inhibitors of metalloproteinases in the early restenosis of covered stents in the femoropopliteal arterial segment

Objective

The purpose of this study was to investigate the roles of the kallikrein-kinin system and matrix metalloproteinases (MMPs) in the development of arterial restenosis attributable to intimal hyperplasia in the femoropopliteal arteries.