Improving gene expression analysis efficacy from formalin-fixed paraffin embedded tissues.

INTRODUCTION: Improving RNA isolation and cDNA synthesis techniques has emerged due to advancements in the knowledge of molecular basis of most diseases. This in turn increased the need of higher quantity and quality of the extracted genetic material to be used for a variety of diagnostic tests and experiments. AIM: The aim of the study was to compare three modified methods for RNA extraction from formalin-fixed paraffin embedded (FFPE) biopsied tissue and different cDNA synthesis strategies to facilitate study of gene expression. MATERIALS AND METHODS: Compared RNA extraction methods were: lysis buffer, phenol-based extraction, and combination of both with concomitant use of silica-based spin columns. RNA quantity and purity were estimated spectrophotometrically. Different priming strategies for cDNA synthesis were applied: oligo dT, combination of oligo dT and random hexamer, and gene specific primer. Two-step RT-qPCR of ribosomal protein L37A on preamplified and non-preamplified cDNA templates was performed. RESULTS: The combination of lysis buffer with phenol based extraction gave higher RNA yield. By doing cDNA preamplification, the confidence of detection by qPCR was raised, and efficiency was improved. The preamplified template increased the sensitivity of analysis. CONCLUSIONS: Together, the combination of approaches improved substantially the reproducibility and validity of quantitative gene expression analyses from FFPE tissues.

Radiation-induced low grade fibromyxoid sarcoma of the larynx: a case report and literature review.

Low grade fibromyxoid sarcoma (LGFMS) is an uncommon variant of fibrosarcoma with high risk of local recurrence, immense metastatic potential and frequently protracted period between tumour presentation and metastasis. This unusual malignancy rarely affects the region of the head and neck which makes cases of laryngeal LGFMS extremely infrequent. To date, LGFMS of the larynx has been scatteredly mentioned in the literature. Neither incidence nor causes and risk factors for laryngeal LGFMS have been clarified so far. To the authors' knowledge, this is the first case report that discusses the clinical course, imaging diagnosis, histopathological evaluation and surgical approach to radiation-induced laryngeal LGFMS.We present a case of a 70-year-old man who developed a LGFMS after previous radiotherapy (RT) for squamous cell carcinoma (SCC) of the larynx. The latency period between the time of radiation exposure and the diagnosis of LGFMS was twenty-seven months. After re-confirming the diagnosis with second biopsy and extensive imaging evaluation the patient was subjected to an open partial resection of the larynx. Owing to the rarity of the tumour, there is no established protocol with follow-up recommendations.This case highlights the importance of considering the RT history of the patient in order to monitor radiotherapy-related complications, including the occurrence of LGFMS.

Thrombophlebitis Migrans (Trousseau Syndrome) in Pancreatic Adenocarcinoma: An Autopsy Report.

First described by a French physician Armand Trousseau, the Trousseau sign of malignancy is a classic example of paraneoplastic syndrome, caused by adenocarcinomas predominantly of the stomach, pancreas, and lung. The condition presents as recurring and migrating episodes of thrombophlebitis that can involve the upper and lower limbs, thoracic and abdominal wall, and the major blood vessels of the abdomen. These recurring episodes may lead to a detachment of a thrombus and the formation of pulmonary thromboembolism (PTE), often proving fatal for the patient. Herein we present a case of a 60-year-old male patient referred for autopsy. The patient was admitted with acute onset of gastrointestinal tract symptoms and after admission, his condition deteriorated rapidly, with new-onset neurological symptoms and an acute massive fatal episode of PTE. Previous medical history was uneventful, apart from several episodes of recurring lower limb thrombophlebitis for the past six months, resulting in two prior episodes of PTE. The autopsy revealed a massive PTE, with multiple thrombi in the venous vessels, including the two common iliac veins and the inferior vena cava. Histological evaluation revealed pancreatic adenocarcinoma with distant metastasis to a number of organs and abdominal thrombophlebitis with embolization of the pulmonary arteries.

Protective effects of Aronia melanocarpa juices either alone or combined with extracts from Rosa canina or Alchemilla vulgaris in a rat model of indomethacin-induced gastric ulcers.

The aim of the study was to investigate the effects of four Aronia melanocarpa-based juices in a rat model of indomethacin-induced gastric ulceration. THE JUICES WERE: AM1 and AM2 (produced from aronia fruits at 20 °C and 60 °C, respectively), AMRC (a mixture of AM2 with Rosa canina extract) and AMAV (aronia juice with Alchemilla vulgaris). Male Wistar rats were used. Each of the juices (10 ml/kg) was administered for 10 days. Indomethacin (30 mg/kg) was injected subcutaneously and after 4 h, the effects were estimated. Indomethacin caused heavy destructions of the gastric mucosa, increased the expression of Bax and decreased the expression of Bcl-2, induced a certain increase in lipid peroxidation and a slight decrease in gastric PGE2 content. The pretreatment with the juices reduced the severity of indomethacin-induced gastric lesions and antagonized the effects of indomethacin on apoptosis and lipid peroxidation. The highest was the protective effect of AMAV, the juice with the highest polyphenolic content. The protective effect of Aronia melanocarpa-based juices against indomethacin-induced gastric lesions could be attributed to their polyphenolic contents. The mechanism involved to the highest extent in the protective effect of the juices was the inhibition of apoptosis.

Molecular Mechanisms of Melatonin Protection from Gastric Mucosal Apoptotic Injury in Experimental Burns.

Melatonin, a basic secretory pineal gland product, is a nontoxic, multifunctional molecule. It has antioxidant and anti-apoptotic activities and protects tissues from injury. The objective of the present study was to determine the molecular mechanism of melatonin anti-apoptotic effect on gastric injury in a rat burn model. We hypothesized that melatonin gastric protection may be related to the activation of transcription erythroid 2-related factor 2 (Nrf2). Using a 30% total body surface area (TBSA) rat burn model, melatonin (10 mg/kg, i.p.) was injected immediately and 12 h after thermal skin injury. Via light immunohistochemistry, we determined the tissue level of 4-hydroxy-2-nonenal (4-HNE) as a marker of lipid peroxidation, Bcl-2 and Bax as apoptosis-related proteins, and Nrf2. Results are presented as medians (interquartile range (IQR)). Thermal trauma in burned animals, compared with the controls, increased the expression of pro-apoptotic Bax protein (1.37 (0.94-1.47)), decreased anti-apoptotic Bcl-2 protein (1.16 (1.06-1.23), p < 0.001) in epithelial cells, and elevated Bax/Bcl-2 ratios (p < 0.05). Tissue 4-HNE and Nrf2 levels were increased following severe burns (1.55 (0.98-1.61) and 1.16 (1.01-1.25), p < 0.05, respectively). Melatonin significantly decreased 4-HNE (0.87 (0.74-0.96), p < 0.01) and upregulated Nrf2 (1.55 (1.52-1.65), p < 0.001) levels. It also augmented Bax (1.68 (1.5-1.8), p < 0.001) and Bcl-2 expressions (1.96 (1.89-2.01), p < 0.0001), but reduced Bax/Bcl-2 ratios (p < 0.05). Our results suggest that experimental thermal trauma induces oxidative gastric mucosal injury. Melatonin manifests a gastroprotective effect through Nrf2 activation, lipid peroxidation attenuation, and Bax/Bcl-2 ratio modification as well.

Aronia melanocarpa fruit juice ameliorates the symptoms of inflammatory bowel disease in TNBS-induced colitis in rats.

Trinitrobenzensulfonic acid (TNBS) is commonly used to induce an experimental inflammatory bowel disease (IBD) model. Oxidative stress and inflammation have been proposed as mechanisms underlying the pathophysiology of IBD. Aronia melanocarpa fruit juice (AMFJ) is extremely rich in polyphenolic substances, mainly proanthocyanidins, flavonoids and phenolic acids. The aim of this study was to evaluate the effect of AMFJ in a rat TNBSinduced colitis model and to compare the effect of the juice with that of sulfasalazine. Colitis was induced by TNBS in male Wistar rats. After the induction of colitis, AMFJ at three doses (2.5, 5 and 10 mL/kg) and sulfasalazine (400 mg/kg) were administered orally till the 14th experimental day. Severity of colitis was assessed by macroscopic and histopathological criteria. Oxidative stress was evaluated by the concentration of thiobarbituric acid reactive substances (TBARS). TNBS caused severe colonic damage. AMFJ dose-dependently ameliorated TNBS-induced colitis. It improved the macroscopic and microscopic signs of colitis, and prevented the increase of colonic TBARS concentrations. Regarding different indices, the effect of AMFJ was comparable or even higher than that of sulfasalazine. In conclusion, the ameliorative effects of AMFJ in the experimental TNBSinduced colitis might be the result of its potent antioxidant and antiinflammatory properties.

Effects of duodenogastric reflux on gastrin cells, somatostatin cells and serotonin cells in human antral gastric mucosa.

Duodenogastric reflux (DGR) has been found to give rise to a hypochlorhydria secondary to alkaline reflux. We investigated whether there is a link between DGR and the gastrin, somatostatin, and serotonin cell numbers and the granular content of gastrin, somatostatin, and serotonin in endocrine cells in human antral mucosa. We investigated 38 selected Helicobacter pylori-negative patients with visual primary excessive DGR in upper endoscopy and symptoms of epigastric pain and bile vomiting. Ten control patients were included in this study. None of the patients had peptic ulcer or had received any medication. Antrum (10 biopsies from five different zones: the lesser and major curvature, the anterior and posterior wall, and the pylorus) and corpus (two biopsies from major curvature about 10 cm below the cardia) biopsy specimens were collected for routine histology, as well as for light and electron immunohistochemistry. In patients without atrophy or intestinal metaplasia and in patients with mild atrophy or mild intestinal metaplasia, the number of gastrin and somatostatin cells was not different from that in controls. In moderate atrophy or moderate intestinal metaplasia, however, the number of gastrin and somatostatin cells decreased. Serotonin cell number was significantly higher in all patients with DGR as compared with controls. The mean somatostatin granular content was increased (3.6+/-0.2 vs. 3.2+/-0.1). In addition, lysosomes with engulfed somatostatin granules were found. The mean serotonin granular content was decreased (2.3+/-0.3 vs. 2.9+/-0.3), while the mean gastrin granular content remained unchanged (2.5+/-0.3 vs. 2.4+/-0.2). Ultrastructurally, the granules in serotonin-positive cells corresponded to the gastric variant or to the intestinal variant of serotonin cells. The endocrine cells were found to have few granules positive for serotonin. It is concluded that DGR inhibits somatostatin granular release, but stimulates both serotonin granular release and serotonin cell growth.

Ultrastructural immunohistochemical localization of gastrin, somatostatin and serotonin in endocrine cells of human antral gastric mucosa.

Five types of endocrine cells are found in the human antral gastric mucosa: gastrin (G) cells, somatostatin (D) cells, enterochromaffin (EC) cells and cells with an unknown secretory product (D1 cells and P cells). The content of secretory granules, gastrin, somatostatin and serotonin, was evaluated using electron microscopic immunohistochemistry and was compared with the granular content in G cells, D cells and EC cells as determined by routine electron microscopy. Semi-quantitative scoring of the granular content was performed on a scale 1-4 (empty-full). The content of gastrin (2.5 +/- 0.2) and somatostatin (3.3 +/- 0.2) in the granules was not different from the granular content in G cells (2.5 +/- 0.3; p > 0.05) and D cells (3.5 +/- 0.2; p > 0.05). Gastrin was also found in G cells in a nongranular form. The content of serotonin in granules (2.8 +/- 0.3) was smaller than the granular content in EC cells (3.7 +/- 0.2; p < 0.05). In intermediate-full and intermediate-empty granules, serotonin was localized in the periphery of granules whereas the granular content in EC cells was localized in an eccentric or central pattern. The granular content of D1 cells and P cells was 3.8 +/- 0.2, and 3.4 +/- 0.2, respectively. It is concluded that gastrin and somatostatin immunostaining in granules of G cells and D cells reflects the granular content in G cells and D cells, respectively, whereas serotonin immunostaining does not agree with the granular content of EC cells.

Endocrine cells in gastric carcinoma and adjacent mucosa. An immunohistochemical and ultrastructural study.

Endocrine cells are often found in human gastric carcinoma and may be recognized by the immunoreactivity of their chromogranin A, peptides and biogenic amines content. Anti-chromogranin A was used to investigate the morphology of endocrine cells using light and electron microscope immunohistochemical techniques. The hormone content of endocrine cells was examined in both tumour tissue and tumour-adjacent mucosa. It was found that the endocrine cells in tumour tissue were malignant, often had amphocrine differentiation and did not resemble a normal cell type. The hormone content of endocrine cells in tumour tissue seldom corresponded to the hormonal content of endocrine cells in tumour-adjacent mucosa. In intestinal-type carcinoma and in some parts of diffuse-type gastric carcinomas, endocrine cell hyperplasia and an alteration of the differentiation in the tumour-adjacent mucosa were discovered. The distribution of endocrine cells in the tumour tissue was different in both types of gastric carcinoma. The results reported here suggest that endocrine cell differentiation of malignant endocrine cells in human gastric carcinoma develops in a different way from that of endocrine cells in tumour-adjacent mucosa, and as a result, diverse hormonal products may appear in tumour tissue.