The immunoexpression of aquaporin 1, PAX2, PAX8, connexin 36, connexin 43 in human fetal kidney.

INTRODUCTION: The kidney develops from two mesodermal primordia. Aquaporin 1 (AQP1) is a membrane protein characteristic to epithelial and endothelial cell of the human body. The Pax family of genes encodes transcription factors with important role in intrauterine development. Connexins are transmembrane proteins found in gap junctions. We monitored the changes in the expression of AQP1, paired box gene 2 (PAX2), paired box gene 8 (PAX8), connexin 36 (Cx36) and connexin 43 (Cx43) proteins in fetal renal tissue. MATERIALS AND METHODS: We studied 34 post mortem fetuses of 9 to 24 weeks from the Laboratory of Pathology, Emergency County Hospital of Târgu Mures, Romania, using immunohistochemistry. RESULTS: AQP1 expression appeared in the apical and basolateral parts of cells, lining the proximal convoluted tubules and the descending limb of Henle's loop, then in the tubule pole of Bowman's capsule also. Nuclear expression of PAX2 was observed in structures developed both from the ureteric bud and the metanephric mesenchyme, and of PAX8 was observed in the proximal convoluted tubule's epithelium, Henle's loop, and collecting ducts. Cytoplasmic expression of Cx36 was localized to nephrons in different developmental stages, glomerular vessels and collecting ducts, and of Cx43 was localized to the endothelium of glomerular and peritubular vessels, as well as to the epithelium of the proximal tubules. DISCUSSIONS AND CONCLUSIONS: Nephrogenesis begins in the embryonic period, and continues into the fetal period as well. It is regulated by a wide array of markers. The current study supplements literature data regarding immunoexpression of these markers during renal development in the fetal period.

Allele-specific PCR method for identification of EGFR mutations in non-small cell lung cancer: formalin-fixed paraffin-embedded tissue versus fresh tissue.

UNLABELLED: The study of epidermal growth factor receptor (EGFR) gene mutations in lung adenocarcinoma patients has a special clinical significance in the selection of patients for tyrosine-kinase inhibitor therapy. The aim of this study was to identify patients with EGFR mutations using allele-specific polymerase chain reaction (PCR), from formalin-fixed paraffin-embedded (FFPE) tissue and fresh tissue (FT). MATERIALS AND METHODS: We performed a retrospective study using 13 cases of FFPE lung adenocarcinoma, and a prospective study using seven fresh samples of lung carcinomas (FT), collected by intraoperative dissection of the tumors. Using the DNA extracted from the FFPE tissue and FT, we attempted to identify deletions of exon 19 and point mutations of exon 21, according to the allele-specific PCR method described by Dahse et al. (2008). RESULTS: In all seven cases of FT (three adenocarcinomas, three squamous carcinomas, one large-cell carcinoma), we identified the wild type allele and the internal control in case of exon 19, and the wild type allele for exon 21, but not the mutated alleles. Considering that no standard method for formalin fixation and paraffin embedding has been implemented at the Laboratory of Pathology, the DNA extracted from these samples became fragmented and damaged, which compromised the results of PCR testing aimed at the detection of EGFR mutations. CONCLUSIONS: The presented method can be implemented at our laboratory to identify these mutations from fresh tissue collected during surgical resection. Additionally, standardization of formalin fixation and paraffin embedding of surgical samples is required, in order the enable subsequent processing using molecular biology methods.

Quantitative morphometric and immunohistochemical analysis and their correlates in cirrhosis--A study on explant livers.

BACKGROUND: Reproducible structural analysis was made on cirrhotic human liver samples in order to reveal potential connections between morphological and laboratory parameters. MATERIAL AND METHODS: Large histological samples were taken from segment VII of 56 cirrhotic livers removed in connection with liver transplantation. Picro Sirius red and immunohistochemically (smooth muscle actin [SMA], cytokeratin 7 [CK7], Ki-67) stained sections were digitalized and morphometric evaluation was performed. RESULTS: The Picro Sirius-stained fibrotic area correlated with the average thickness of the three broadest septa, extent of SMA positivity, alkaline phosphatase (ALP) values and it was lower in the viral hepatitis related cirrhoses than in samples with non-viral etiology. The extent of SMA staining increased with the CK7-positive ductular reaction. The proliferative activity of the hepatocytes correlated positively with the Ki-67 labeling of the ductular cells and inversely with the septum thickness. These data support the potential functional connection among different structural components, for example, myofibroblasts, ductular reaction and fibrogenesis but challenges the widely proposed role of ductular cells in regeneration. CONCLUSION: Unbiased morphological characterization of cirrhotic livers can provide valuable, clinically relevant information. Similar evaluation of routine core biopsies may increase the significance of this 'Gold Standard' examination.

Functional Heart Valve Scaffolds Obtained by Complete Decellularization of Porcine Aortic Roots in a Novel Differential Pressure Gradient Perfusion System.

There is a great need for living valve replacements for patients of all ages. Such constructs could be built by tissue engineering, with perspective of the unique structure and biology of the aortic root. The aortic valve root is composed of several different tissues, and careful structural and functional consideration has to be given to each segment and component. Previous work has shown that immersion techniques are inadequate for whole-root decellularization, with the aortic wall segment being particularly resistant to decellularization. The aim of this study was to develop a differential pressure gradient perfusion system capable of being rigorous enough to decellularize the aortic root wall while gentle enough to preserve the integrity of the cusps. Fresh porcine aortic roots have been subjected to various regimens of perfusion decellularization using detergents and enzymes and results compared to immersion decellularized roots. Success criteria for evaluation of each root segment (cusp, muscle, sinus, wall) for decellularization completeness, tissue integrity, and valve functionality were defined using complementary methods of cell analysis (histology with nuclear and matrix stains and DNA analysis), biomechanics (biaxial and bending tests), and physiologic heart valve bioreactor testing (with advanced image analysis of open-close cycles and geometric orifice area measurement). Fully acellular porcine roots treated with the optimized method exhibited preserved macroscopic structures and microscopic matrix components, which translated into conserved anisotropic mechanical properties, including bending and excellent valve functionality when tested in aortic flow and pressure conditions. This study highlighted the importance of (1) adapting decellularization methods to specific target tissues, (2) combining several methods of cell analysis compared to relying solely on histology, (3) developing relevant valve-specific mechanical tests, and (4) in vitro testing of valve functionality.

Real-time quantitative PCR detection of WT1 and M-BCR-ABL expressions in chronic myeloid leukemia.

UNLABELLED: The Philadelphia chromosome and the resulting BCR-ABL fusion gene represent the hallmark event in chronic myeloid leukemia (CML) and their discoveries radically changed the management of these patients. Currently Wilms tumor 1 gene (WT1) is intensively investigated as high WT1 expression levels have been demonstrated in case of multiple solid tumors and malignant hematological syndromes (acute myeloid and lymphoid leukemia, myelodysplastic syndromes and chronic myeloid leukemia). The aim of our study was to investigate the WT1 expression in CML patients and its possible contribution to disease evolution. PATIENTS AND METHODS: In the Laboratory of Molecular Biology, University of Medicine and Pharmacy of Tirgu Mures, Romania, we regularly determined the M-BCR-ABL and WT1 expression levels by RQ-PCR (real-time quantitative polymerase chain reaction) testing in case of 19 CML patients: six patients monitorized from the diagnosis and 13 patients first tested during therapy. RESULTS: Eight CML (four advanced stage and four CP) patients showed high WT1 expression level, and in case of 11 patients the WT1 expression levels were undetectable or lower than 0.02%. The only significant difference between the high and low WT1 expression groups was represented by the clinical stage. In the majority of pretreated patients (10 out of 13 patients), the WT1 expression levels were low or undetectable. CONCLUSIONS: High WT1 expression in CML patients is detected especially in the advanced stages of the disease. Efficient Imatinib therapy may contribute to low WT1 levels in CP patients.

Ovariectomized rats' femur treated with fibrates and statins. Assessment of pore-size distribution by ¹H-NMR relaxometry.

The effects of two wonder drugs, simvastatins and fenofibrates on the proximal part of the femoris of a series of ovariectomized and non-ovariectomized Wistar albino rats was estimated qualitatively and semi-quantitatively by the modern method of 1D 1H-NMR T2-distribution. The 72 rats subjected to this study were divided in six groups and were sacrificed at two, four, six and eight weeks after ovariectomy and the proximal part of femoris was harvested. The CPMG (Carr-Purcell-Meiboom-Gill) echoes train curves were measured for the bones fully saturated with water during two months after two months of natural drying. These decays were analyzed by Laplace inversion and an average of normalized T2-distributions was considered for all rat's groups. The 1D averaged T2-distributions present four peaks, which were associated with protons in four major environments, from which the free water protons are used as spy molecules to explore the boundaries of cavities. In the approximation of spherical pores, the averaged T2-distributions were transformed in distributions of pores diameters. These were found in the range from 2 µm up to 2 mm. The relative amplitudes, widths and position of deconvoluted distributions of small, medium and large cavities are used for a qualitatively analysis of the effect of our lipid-lowering drugs. For a semi-quantitatively analysis, we chose the diameter d of proximal part of femoris' trabecular cavities. We show that the positive or negative effects of treatments with simvastatins and fenofibrates are strongly dependent on the duration of treatment. Moreover, the treatment of healthy bone is generally counter-indicated.

Comparative study of HER2, EGFR, p53 and PTEN expression in the human gastrointestinal tract during fetal period.

INTRODUCTION: HER2, EGFR, p53 and PTEN are important in organization of the germ layers, in embryonic development and morphogenesis, in the development and differentiation of certain organ systems and in embryonic morphogenesis. Our goal is the comparative examination of the expression of these markers in the digestive tract of 9-24-week-old fetuses. MATERIALS AND METHODS: We studied using immunohistochemical techniques esophagus, stomach, small and large intestine tissue samples collected from 18 post mortem fetuses of 9-24 weeks. RESULTS: HER2 and PTEN expression appears as early as the 9-12 weeks period in the digestive tract, but HER2 expression decreases in the 21-24 weeks period and then disappears. EGFR expression appears only during the 13-16 weeks period. The expression of p53 is strong until week 21, and then it is restricted to the deeper layers of the epithelium. CONCLUSIONS: Our findings suggest that these markers have role also in the fetal period and complete the scarce data found in literature about the expression of the studied markers in the development of the digestive tract.

Changes in immunoexpression of p53, Ki-67, Ets-1, APAF-1 and PTEN in serrated and conventional colon adenomas.

UNLABELLED: The balance between apoptosis and proliferation is tipped towards a decrease of apoptosis as the colonocyte progresses in the adenoma to carcinoma sequence of colon carcinogenesis. According to literature data, proteins like p53, Ki-67, APAF-1, Ets-1, PTEN contribute to inhibition of apoptosis and stimulation of proliferation. AIM: Considering the complex interference among colorectal carcinogenetic mechanisms, our aim was to study the markers Ets-1 and APAF-1 relative to p53, Ki-67 and PTEN expression in colon adenomas/polyps (A/P). MATERIALS AND METHODS: We performed immunohistochemistry on 99 colon A/P cases from the material of the Department of Pathology, Emergency County Hospital of Tirgu Mures, Romania. Secondary EnVision Flex/HRP (Horseradish peroxidase) (20 minutes) was used for signal amplification. RESULTS: The majority of A/P show increased Ki-67, p53, Ets-1 expression, decreased APAF-1 expression and preserved PTEN expression. p53, Ki-67, Ets-1 and APAF-1 demonstrated statistically significant correlations with histological type and grade of dysplasia. We also observed that expression of these proteins in the intestinal crypts has a typical distribution according to histological type and grade of dysplasia. CONCLUSIONS: In case of hyperplastic polyps APAF-1 expression decreases as p53 and Ki-67 expression increases, followed by a decrease in PTEN expression in serrated adenomas, and an increase of Ets-1 expression in conventional adenomas.

Immunohistochemical study of Ki67, CD34 and p53 expression in human tooth buds.

AIM OF THE STUDY: Establishment of Ki67, p53 and CD34 expression in human tooth buds of different stages of odontogenetic development. MATERIALS AND METHODS: Tissue samples containing tooth buds were removed from the incisor areas of human fetuses in different stages of development (weeks 9-10, 12-13, 13-16, 21-24), and from the canine and molar areas of 21-24 weeks fetuses. The tissue fragments were fixed using formalin and were processed using common histological techniques with paraffin embedding. Immunostaining for Ki67, p53 and CD34 has been performed using the dextran method and moist heat antigen retrieval (except for CD34). The resulting slides were photographed and quantitatively evaluated. RESULTS: Ki67 immunoexpression decreases with advancement of the developmental stage of the tooth bud: in the inner enamel epithelium, between weeks 9 and 16 (IEE), in the preameloblasts (PB) between weeks 13 and 16, in the ameloblasts (AB) between weeks 21 and 24; outer enamel epithelium (OEE); stratum intermedium (SI); in the dental papilla: between weeks 9 and 10 in the dental papilla (DP), between weeks 13 and 16 in the outer layer of the dental papilla (DP1) and in the central layer of the dental papilla (DP2). Likewise, we noted Ki67 expression in the odontoblast layer (O) and pulp (P), between weeks 21 and 24. Concerning CD34 expression, we observed a decrease from weeks 9-10 until weeks 13-16, followed by an increase until weeks 21-24 of intrauterine life. From weeks 9-10, we observed a constant decrease of expression until weeks 13-16, followed by an increase during weeks 21-24. CONCLUSIONS: All Ki67, p53 and CD34 have been identified in the tooth bud. Ki67 expression gradually decreases with the embryonic development of the tooth, while p53 and CD34 expression decreases from weeks 9-10 to weeks 13-16 of intrauterine life, followed by an increase until weeks 21-24.

Expansion of hepatic stem cell compartment boosts liver regeneration.

The hepatic stem cells reside periportally forming the canals of Hering in normal liver. They can be identified by their unique immunophenotype in rat. The oval cells, the progenies of stem cells invade deep the liver parenchyma after activation and differentiate into focally arranged small-and eventually trabecularly ordered regular hepatocytes. We have observed that upon the completion of intense oval cell reactions narrow ductular structures are present in the parenchyma, we propose to call them parenchymal ductules. These parenchymal ductules have the same immunophenotype [cytokeratin (CK)7-/CK19+/alpha-fetoprotein (AFP)-/delta-like protein (DLK)-] as the resting stem cells of the canals of Hering, but different from them reside scattered in the parenchyma. In our present experiments, we have investigated in an in vivo functional assay if the presence of these parenchymal ductules has any impact on a progenitor cell driven regeneration process. Parenchymal ductules were induced either by an established model of oval cell induction consisting of the administration of necrogenic dose of carbontetrachloride to 2-acetaminofluorene pretreated rats (AAF/CCl4) or a large necrogenic dose of diethylnitrosamine (DEN). The oval cells expanded faster and the foci evolved earlier after repeated injury in the livers with preexistent parenchymal ductules. When the animals were left to survive for one more year increased liver tumor formation was observed exclusively in the DEN treated rats. Thus, repeated oval cell reactions are not necessarily carcinogenic. We conclude that the expansion of hepatic stem cell compartment conceptually can be used to facilitate liver regeneration without an increased risk of tumorigenesis.

Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors.

BACKGROUND: The immunohistochemical demonstration of Enhancer of zeste homologue 2 (EZH2) proved to be a useful marker in several tumor types. It has been described to distinguish reliably hepatocellular carcinomas from liver adenomas and other benign hepatocellular lesions. However, no other types of malignant liver tumors were studied so far. METHODS: To evaluate the diagnostic value of this protein in hepatic tumors we have investigated the presence of EZH2 by immunohistochemistry in hepatocellular carcinomas and other common hepatic tumors.EZH2 expression was examined in 44 hepatocellular carcinomas, 23 cholangiocarcinomas, 31 hepatoblastomas, 16 other childhood tumor types (rhabdomyosarcoma, neuroblastoma, Wilms' tumor and rhabdoid tumor), 17 metastatic liver tumors 24 hepatocellular adenomas, 15 high grade dysplastic nodules, 3 biliary cystadenomas, 3 biliary hamartomas and 3 Caroli's diseases. RESULTS: Most of the malignant liver tumors were positive for EZH2, but neither of the adenomas, cirrhotic/dysplastic nodules, reactive and hamartomatous biliary ductules stained positively. CONCLUSIONS: Our immunostainings confirm that EZH2 is a sensitive marker of hepatocellular carcinoma, but its specificity is very low, since almost all the investigated malignant liver tumors were positive regardless of their histogenesis. Based on these results EZH2 is a sensitive marker of malignancy in hepatic tumors. In routine surgical pathology EZH2 could be most helpful to diagnose cholangiocarcinomas, because as far as we know this is the first marker to distinguish transformed and reactive biliary structures. Although hepatoblastomas also express EZH2, the diagnostic significance of this observation seems to be quite limited whereas, the structurally similar, other blastic childhood tumors are also positive. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1173195902735693.

An immunohistochemical study of colon adenomas and carcinomas: E-cadherin, Syndecan-1, Ets-1.

It is thought that dysregulation of E-cadherin, syndecan-1 (CD138) and Ets-1 is involved in carcinoma development. E-cadherin is an important epithelial cell adhesion molecule; syndecan-1 (CD138) is a regulatory proteoglycan in both cell-cell and cell-matrix adhesion and Ets-1 is a proto-oncogene and transcription factor, which takes part in extracellular matrix remodeling. Our goal was to study the changes in the expression of these molecules during colon carcinoma development and progression. We tested 117 colon adenomas and 149 de novo and ex adenoma carcinomas of the colon, using the Ultravision Polymer system. The positive reaction rate was 100% for E-cadherin, 98.3% for syndecan-1 and 22.4% for Ets-1 in adenomas, while in carcinomas it was 88.5%, 62.4% and 56.3% respectively. We found decreasing expression of E-cadherin and syndecan-1 throughout colon carcinoma progression and an opposite regulation for the Ets-1 protein. Decrease in expression of syndecan-1 is more pronounced in carcinomas compared to E-cadherin. De novo carcinomas have lower E-cadherin and syndecan-1 expression, and higher Ets-1 expression compared to ex adenoma carcinomas. These findings support the hypothesis that there are differences in the carcinogenesis of these tumors.