Kidney and liver are the main organs of expression of a key metabolic enzyme alanine:glyoxylate aminotransferase 2 in humans.

BACKGROUND: The metabolic syndrome is a cluster of cardiovascular risk factors and is highly predictive for development of cardiovascular diseases. An association between elevated plasma levels of the endogenous inhibitor of nitric oxide synthases asymmetric dimethylarginine (ADMA) and risk of cardiovascular diseases has been demonstrated in numerous epidemiological studies. ADMA can be catabolized by dimethylarginine dimethylaminohydrolase (DDAH) or metabolized through a much less understood alternative pathway by alanine:glyoxylate aminotransferase 2 (AGXT2) with the formation of α-keto-δ-(N,N-dimethylguanidino)valeric acid (ADGV). Previous RT-PCR and Western Blot studies suggested that Agxt2 is expressed in the mouse kidney and liver at comparable levels, while Northern Blot and in-situ RNA-hybridisation experiments demonstrated that the kidney is the main organ of Agxt2 expression in rats. Given this discrepancy, the goal of the current study was to analyse the expression of AGXT2 in human tissues. MATERIAL AND METHODS: We analyzed AGXT2 expression in human tissues from a normal tissue bank by RT-PCR and further validated the results by Western Blot. We also performed immunohistochemical staining for AGXT2 and double fluorescent staining with an anti-AGXT2 antibody and a monoclonal anti-mitochondrial antibody. RESULTS: We saw the strongest expression of AGXT2 in the kidney and liver and confirmed this results on protein level. By IHC staining we were able to show that AGXT2 is present in the convoluted tubule in the kidney and in the liver hepatocytes. The double fluorescent staining revealed mitochondrial localization of AGXT2. CONCLUSIONS: Our current data suggest that both hepatocytes and kidney tubular epithelial cells are the major sources of AGXT2 in humans. We also demonstrated the mitochondrial localization of human AGXT2 enzyme.

RNA quality in fresh-frozen gastrointestinal tumor specimens-experiences from the tumor and healthy tissue bank TU Dresden.

The term "pre-analytics" summarizes all procedures concerned with specimen collection or processing as well as logistical aspects like transport or storage of tissue specimens. All or these variables as well as tissue-specific characteristics affect sample quality. While certain parameters like warm ischemia or tissue-specific characteristics cannot be changed, other parameters can be assessed and optimized. The aim of this study was to determine RNA quality by assessing the RIN values of specimens from different organs and to assess the influence of vacuum preservation. Samples from the GI tract, in general, appear to have lower RNA quality when compared to samples from other organ sites. This may be due to the digestive enzymes or bacterial colonization. Processing time in pathology does not significantly influence RNA quality. Tissue preservation with a vacuum sealer leads to preserved RNA quality over an extended period of time and offers a feasible alternative to minimize the influence of transport time into pathology.

Participation in and support of clinical studies and other scientific investigations - Statement of the German Society for Pathology.

Clinical studies and preclinical investigations are essential in order to test new therapies and diagnostic techniques aimed at sustainable improvements in the treatment of patients. Fortunately, the number of clinical studies is continuously increasing and pathology and tissue-based research are more frequently involved. Pathologists are essential in this process and committed to support it by joining forces with our clinical partners. The investigative diagnostic technologies we apply to human cell and tissue samples and our specific expertise are essential contributions to the quality and success of preclinical investigations, clinical studies, and the implementation of results into clinical diagnostic pathology. In order to support this process, the German Society of Pathology has formulated a statement on the participation in and support of clinical studies and other scientific investigations with a special focus on tissue-based research.

Clonal evolution including partial loss of human leukocyte antigen genes favoring extramedullary acute myeloid leukemia relapse after matched related allogeneic hematopoietic stem cell transplantation.

BACKGROUND: Relapse of acute myeloid leukemia (AML) after allogeneic hematopoietic stem cell transplantation (HSCT) leaves few therapeutic options, and mechanisms of immune escape of recurring leukemic cells remain poorly understood. Recently, acquired loss of mismatched human leukocyte antigen (HLA) was demonstrated in patients with AML undergoing haploidentical allogeneic HSCT and was suggested not to occur in HLA-matched HSCT. We hypothesized that this mechanism applies to extramedullary AML relapse which occurs frequently after allogeneic HSCT and might also not be restricted to haploidentical HSCT. METHODS: DNA from extramedullary AML relapse after HSCT was compared with bone marrow at diagnosis with array comparative genomic hybridization to investigate relapse-specific genomic aberrations in relapsing AML after allogeneic HSCT. Formalin-fixed, paraffin-embedded tissues from the same points of time were assessed for HLA, major histocompatibility complex class I chain-related gene A, and TAP2 immunohistochemistry staining to assess cell surface expression of deleted loci encoded on chromosome 6p. RESULTS: Array comparative genomic hybridization revealed a partial loss of chromosome 6p in extramedullary myeloid sarcoma relapse of AML after sustained complete remission was achieved through matched related allogeneic HSCT. Among others, a deleted region 6p21.32-p21.33, which included several HLA class I genes, was detected. CONCLUSIONS: These results suggest that the loss of HLA class I haplotype also occurs in AML relapse after HLA-matched related HSCT. Partial loss of several HLA class I genes and subsequent reduced presentation of minor histocompatibility antigens and reduced ligation of activating natural killer-cell receptors may explain the loss of graft-versus-leukemia response and extramedullary AML relapse in tissue with reduced immunologic surveillance.

Prognostic relevance of tumour cell-associated uPAR expression in invasive ductal breast carcinoma.

AIMS: The urokinase-type plasminogen activator receptor (uPAR) is a key molecule for pericellular proteolysis in tumour cell invasion and metastasis. The aim was to evaluate the prognostic impact of uPAR in invasive breast cancer dependent on which cell types within the tumour express uPAR. METHODS AND RESULTS: uPAR expression was analysed by immunohistochemistry in 270 tumour tissue specimens of invasive ductal breast carcinomas using tissue microarrays. For evaluation of uPAR immunoexpression we used the epitope-mapped, uPAR domain II-specific monoclonal antibody IID7. High uPAR score values in both tumour cells (uPAR-Tc) and stromal cells were significantly related to high tumour grade (G3), and inversely correlated with oestrogen receptor status. On multivariate analysis, high uPAR-Tc values contributed independent prognostic information for disease-free survival (hazard ratio 1.93, P = 0.007) when adjusted for prognostically relevant clinicopathological parameters, whereas uPAR expression in stromal cells was not related to prognosis. In addition, elevated uPAR-Tc values were found to be prognostic indicators in clinically relevant subgroups of patients with invasive breast cancer. CONCLUSIONS: In invasive breast cancer uPAR expression in invasive carcinoma cells, but not in stromal cells, has a significant impact on patients' prognosis, and contributes to a more aggressive tumour phenotype.

Prognostic significance of immunohistochemical RhoA expression on survival in pancreatic ductal adenocarcinoma: a high-throughput analysis.

Among all human carcinomas, pancreatic cancer has one of the worst survival rates. Most patients will die of this cancer shortly after diagnosis, and currently, surgery is the only potential cure. Ductal adenocarcinoma is the most common histologic type. The search for prognostic parameters has progressed from mere physical or histomorphological tumor properties to molecular parameters. These, in turn, might point toward new therapeutic strategies. The K-ras oncogene is known to play a role in early stages of ductal adenocarcinoma carcinogenesis, and ras homologues are differentially expressed in cancerous versus normal ductal cells. RhoA belongs to a family of ras homologues comprising RhoA, RhoB, and RhoC. It is a guanosine triphosphatase associated with the cytoskeleton that seems to be involved in epithelial mesenchymal transition, a process of dedifferentiation. Immunohistologic RhoA expression was studied in a tissue microarray of 94 pancreatic ductal adenocarcinomas and correlated with clinicopathologic parameters and follow-up. RhoA protein expression, measured as labeling intensity or evaluated as percentage of reactive tumor cells, correlated with overall survival. A multivariate analysis demonstrated that RhoA protein expression is independent from other known prognostic parameters such as tumor size or grade. Moreover, a score combining RhoA expression with tumor size and grade resulted in a highly significant increase in the prognostic value for the overall survival of patients with pancreatic ductal adenocarcinoma.

Human herpesvirus 8 and angiosarcoma: analysis of 40 cases and review of the literature.

AIMS: To prove a possible involvement of the endotheliotropic human herpesvirus 8 (HHV-8) in the pathogenesis of angiosarcoma in samples from patients in a low HHV-8 seroprevalence area. METHODS: A comprehensive series of angiosarcomas (n = 40) as well as positive and negative control tissues from patients with Kaposi's sarcoma, human immunodeficiency virus (HIV)-associated multicentric Castleman's disease or juvenile haemangioma, respectively, was analysed with two sensitive methods: immunohistochemical staining for the HHV-8 latency-associated nuclear antigen 1 (LANA-1); and polymerase chain reaction (PCR) for HHV-8 VP23 DNA sequences. RESULTS: None of the angiosarcoma cases and none of the negative control samples (juvenile haemangiomas) revealed positive immunohistochemical staining with the LANA-1 antibody. In contrast, HHV-8 LANA-1 was clearly detected in all analysed cases of Kaposi's sarcoma and multicentric Castleman's disease. These results were confirmed by PCR assay at the DNA level. CONCLUSION: In conclusion, the great majority of angiosarcomas investigated to date, including the series of 40 angiosarcomas analysed here, does not contain HHV-8 DNA sequences or protein. This argues against a relevant role of the endotheliotropic HHV-8 in the pathogenesis of angiosarcoma and, for vascular diseases, speaks in favour of a relatively restricted pathogenic role of HHV-8 to Kaposi's sarcoma and multicentric Castleman's disease.

Guanylate-binding protein-1 expression is selectively induced by inflammatory cytokines and is an activation marker of endothelial cells during inflammatory diseases.

During angiogenesis and inflammatory processes, endothelial cells acquire different activation phenotypes, whose identification may help in understanding the complex network of angiogenic and inflammatory interactions in vivo. To this goal we investigated the expression of the human guanylate-binding protein (GBP)-1 that is highly induced by inflammatory cytokines (ICs) and, therefore, may characterize IC-activated cells. Using a new rat monoclonal antibody raised against GBP-1, we show that GBP-1 is a cytoplasmic protein and that its expression in endothelial cells is selectively induced by interferon-gamma, interleukin-1alpha, interleukin-1beta, or tumor necrosis factor-alpha, but not by other cytokines, chemokines, or growth factors. Moreover, we found that GBP-1 expression is highly associated with vascular endothelial cells as confirmed by the simultaneous detection of GBP-1 and the endothelial cell-associated marker CD31 in a broad range of human tissues. Notably, GBP-1 expression was undetectable in the skin, but it was highly induced in vessels of skin diseases with a high-inflammatory component including psoriasis, adverse drug reactions, and Kaposi's sarcoma. These results indicate that GBP-1 is a novel cellular activation marker that characterizes the IC-activated phenotype of endothelial cells.

A morphogenetic concept of salivary duct regeneration and metaplasia.

The exact mechanisms of physiological regeneration and of metaplastic processes of the salivary duct have not been definitely established, although regeneration from a putative uncommitted stem cell population has long been favored. In the present study, double immunohistochemical labeling for Ki 67 and alpha-actin or different cytokeratin subtypes, respectively, made possible an exact localization and quantification of cellular proliferation in the regular salivary duct and in different types of metaplasia. Our data demonstrate a baseline proliferative capacity in all five cell types of the salivary duct. Luminal secretory cells of the acinus and intercalated duct regenerate independently from myoepithelial or basal cells. In contrast, the renewal of oxyphilic cells in the striated and excretory duct is maintained by proliferation and differentiation of basal cells. The great majority of metaplasias develops from uncommitted, Bcl-2 positive basal cells of striated/excretory ducts which possess an enormous capacity for pluridirectional morphogenetic differentiation. Despite this important role of basal cells, our findings demonstrate that all cell types principally have to be considered as potential progenitor cells for salivary gland tumors. The improved insight into regenerative and metaplastic processes of the salivary duct may contribute to a better understanding of the complex formal carcinogenesis.

Differential diagnosis of salivary acinic cell carcinoma and adenocarcinoma (NOS). A comparison of (immuno-)histochemical markers.

A correct histologic differential diagnosis between salivary acinic cell carcinoma (ACC) and adenocarcinoma not otherwise specified (AC-NOS) is highly relevant because of the strikingly different biologic behavior and related therapeutical strategies. The distinction between both tumor types can be difficult because of an enormous variation in histologic appearance, with either type showing partially overlapping morphologic features. Owing to a lack of approved markers, the expression of PAS-staining, alpha-Amylase, alpha-1 Anti-trypsin, cytokeratin (CK)-subtypes 7/18 and Ki-67 was evaluated in 16 cases of ACC and 16 cases of AC-NOS. CK 7 is identified as the most reliable marker with strong positivity in AC-NOS, and complete or preponderant negativity in ACC. The characteristic membranous staining pattern of CK 18 in ACC, in contrast to a diffuse cytoplasmic pattern in AC-NOS, proved to be an additional valuable criterion. PAS and alpha-Amylase are only of little value when ACC is diagnosed, as many cases are only faintly positive or completely negative. The proliferation index (Ki-67) proved to be significantly higher in AC-NOS; however, the diagnostic usefulness is limited by a relevant overlap. In conclusion, we recommend CK 7 and 18 as the most valuable markers in cases with difficult differential diagnosis between ACC and AC-NOS.