MMP-9 mediated Syndecan-4 shedding correlates with osteoarthritis severity.

OBJECTIVE: Osteoarthritis (OA) is a degenerative joint disease inducing the degradation of the articular cartilage. Syndecan-4 (Sdc4) is a heparan sulfate proteoglycan, expressed under inflammatory conditions and by chondrocytes during OA. Little is known about Sdc4 shedding and its regulation in OA. Therefore, we investigated the regulation of Sdc4 shedding and underlying shedding mechanisms under OA conditions. DESIGN: Articular cartilage, serum, synovial fluid and synovial membrane from OA patients with different radiological severity were analyzed. ELISA, RT-qPCR and IHC for Sdc4, MMP-2 and -9 were performed. MMP inhibitors and siRNA were evaluated for their effect on Sdc4 shedding by ELISA and on IL-1 signaling by western blot (pERK/ERK). RESULTS: Shed Sdc4 was increased in synovial fluid of OA patients, but not in the serum and is a good predictor (AUC = 0.72) for OA severity with a sensitivity of 67.5% and specificity 65.2%. MMP-9, but not MMP-2, was increased in cartilage and synovial membrane at mRNA levels and in the synovial fluid at protein levels. Shed Sdc4 correlated with the amount of MMP-9 in synovial fluid. Further, the inhibition and knock-down of MMP-9 decreased the amount of shed Sdc4 in vitro. Increased Sdc4 shedding resulted in less phosphorylation of ERK upon IL-1ß stimulation. CONCLUSION: Shed Sdc4 might be a good prognostic biomarker for OA mediated cartilage degradation. MMP-9 seems to be the relevant sheddase for Sdc4 under OA conditions, desensitizing chondrocytes towards IL-1 signaling.

Targeting ß-catenin dependent Wnt signaling via peptidomimetic inhibitors in murine chondrocytes and OA cartilage.

OBJECTIVE: The canonical Wnt signaling pathway has been shown to be involved in regulating chondrocyte hypertrophic differentiation during Osteoarthritis (OA). The aim of this study was to test the therapeutic potential of two stapled peptide canonical Wnt inhibitors - SAH-Bcl9 and StAx-35R - in preventing Wnt induced cartilage changes in OA. METHODS: Primary neonatal murine chondrocytes and cartilage explants from OA patients undergoing total joint replacement for knee OA, were used for microscopy to determine matrix and cell penetrating capacity of fluorescein isothiocyanate FITC-tagged SAH-Bcl9 and StAx-35R peptides. T cell factor/lymphoid enhancer-binding factor (TCF/LEF) reporter assays were used to monitor the inhibition of Wnt3a induced ß-catenin signaling by each peptide. Changes in chondrocyte phenotypic marker gene expression were analyzed by qRT PCR. RESULTS: Both peptides localized intercellular in primary murine chondrocytes and cartilage explants. They inhibited Wnt3a induced TCF/LEF promoter activity in primary murine chondrocytes. Both inhibitors did not rescue Wnt3a altered expression of chondrocyte phenotypic genes (Sox9, Col2a1, Acan) and hypertrophy marker gene (Col10a1) at high doses (100 ng/ml). Upon application of 10 ng/ml Wnt3a, StAx-35R partially reversed the Wnt effect on Sox9 and Col2a1 gene expression. Both peptides, however, reversed the downregulation of SOX9 and aggrecan (ACAN), and decrease of COL10A1 gene expression in preserved human OA cartilage explants. CONCLUSION: These data indicate that blockade of canonical Wnt signaling might be a therapeutic strategy to treat early OA cases and protect further cartilage degradation by preventing chondrocyte hypertrophic differentiation.

[Surgical interventions in patients with mast cell activation disease. Aspects relevant for surgery using the example of a cholecystectomy]. / Chirurgische Eingriffe an Patienten mit Mastzellüberaktivitätserkrankung. Operationsrelevante Aspekte am Beispiel einer Cholezystektomie.

BACKGROUND: Systemic mast cell activation disease (MCAD) is characterized by an increased and unregulated release of mast cell mediators which can evoke a multifaceted clinical picture often resembling irritable bowel syndrome or fibromyalgia. Because of the considerable prevalence (~ 17 %) of MCAD surgeons are frequently unwittingly confronted with MCAD patients in whom unexpected intraoperative and postoperative complications may occur. Therefore, knowledge of the particular requirements is of relevance for surgical treatment of MCAD patients. OBJECTIVE: The present paper outlines a concept of surgical treatment of MCAD patients based on the literature which is illustrated by a case report on emergency laparoscopic cholecystectomy. CONCLUSIONS: Due to the high prevalence of MCAD in the general population it can be assumed that the frequency in the surgical patient population is similar. If a patient has MCAD, specific characteristics should be taken into account in the surgical procedure to avoid increased operative and complication risks resulting from MCAD.

[Risk-adapted multimodal laboratory cervical screening---Pap test of the future?]. / Risikoadaptierte multimodale gynäkozytologische Krebsvorsorge. Pap-Test der Zukunft?

The objective of screening for cervical cancer is to reduce mortality and incidence of the disease. To date there is extensive and strong evidence that this can be achieved by cytology-based screening programs, which continue to be the mainstay of cervical prevention worldwide despite their inherent methodological limitations. This article presents a review on the utility of conventional, ancillary and experimental methods for cervical screening both as single tests and test combinations, and describes possible future directions for enhanced screening accuracy using risk-adapted protocols.

An oncogenetic tree model in gastrointestinal stromal tumours (GISTs) identifies different pathways of cytogenetic evolution with prognostic implications.

To model the cytogenetic evolution in gastrointestinal stromal tumour (GIST), an oncogenetic tree model was reconstructed using comparative genomic hybridization data from 203 primary GISTs (116 gastric and 87 intestinal GISTs, including 151 newly analysed cases), with follow-up available in 173 cases (mean 40 months; maximum 133 months). The oncogenetic tree model identified three major cytogenetic pathways: one initiated by -14q, one by -1p, and another by -22q. The -14q pathway mainly characterized gastric tumours with predominantly stable karyotypes and more favourable clinical course. On the other hand, the -1p pathway was more characteristic of intestinal GISTs, with an increased capacity for cytogenetic complexity and more aggressive clinical course. Loss of 22q, more closely associated with -1p than -14q, appeared to initiate the critical transition to an unfavourable cytogenetic subpathway. This -22q pathway included accumulation of +8q, -9p, and -9q, which could all predict disease-free survival in addition to tumour site. Thus, insights into the cytogenetic evolution obtained from oncogenetic tree models may eventually help to gain a better understanding of the heterogeneous site-dependent biological behaviour of GISTs.

Frequency of hereditary non-polyposis colorectal cancer among unselected patients with colorectal cancer in Germany.

INTRODUCTION: Hereditary non-polyposis colorectal cancer (HNPCC) is a major form of familial colorectal cancer (CRC). It is diagnosed when either the Amsterdam criteria (AC) are fulfilled or mutations in one of the mismatch repair (MMR) genes have been identified. This project aims at estimating the proportion of HNPCC among unselected patients with CRC. PATIENTS AND METHODS: During a period of 2 years, a total of 351 non-selected patients with CRC were registered prospectively. 92 patients met the Bethesda criteria (9 of them fulfilled the AC) and 259 did not. 348 tumours were examined for microsatellite instability (MSI) and expression of MMR proteins. RESULTS: MSI-H and MSI-L were identified in 17 and 6%, respectively. Loss of MSH2 or MLH1 was found in 1.5 and 8.8%, respectively. Based on the results of tumour tissue analyses, 80 patients with MSI and/or loss of MSH2 or MLH1 expression were identified as candidates for germline mutation screening. DNA of 40/80 patients was available. These patients were screened for MSH2 and MLH1 mutations; 19/40 patients with MSI and normal MSH2 or MLH1 expression were screened for mutations in MSH6. Three patients had relevant MMR gene mutations and six variants of unknown functional relevance were detected. CONCLUSIONS: After adjusting for the cases not evaluable for germline mutations, 1.7% of the CRC patients had HNPCC proven by molecular genetics.

Detection of lymphatic invasion in early stage primary colorectal cancer with the monoclonal antibody D2-40.

PURPOSE: The purpose of this study was to investigate the presence of lymphatic invasion detected by D2-40 immunostaining compared to conventional hematoxylin-eosin (HE) staining in primary colorectal cancer (CRC) and the development of focal new lymphangiogenesis and peritumoral lymphatic proliferation in relation to the tumor stages. Additionally, we analyzed the relation of peritumoral inflammatory reaction (PIR) to tumor stages in CRC. The identification of new categories of patients with high-risk CRC would be very helpful in improving treatment strategies and patient outcome especially in early CRC. PATIENTS AND METHOD: Biopsies were taken from 41 patients with colorectal adenocarcinomas at different stages of disease. Immunohistochemistry was performed on paraffin-embedded sections. First, the whole section was screened for the presence of lymphatic invasion and PIR with routine HE staining. After analysis of the HE-stained slides, the slides were destained and reused for immunohistochemistry with the D2-40 monoclonal antibody. D2-40-immunostained sections were screened for the presence of lymphatic invasion, the proliferation of lymphatic vessels and focally newly developed lymph vessels. RESULTS: Using the D2-40 antibody for immunostaining, our results demonstrate a significantly higher detection (p < 0.05) of lymphatic vessel invasion compared to routine HE staining in primary CRC. 22% more patients with lymphatic vessel invasion could be identified compared to routine HE staining, especially in node-negative tumor stage (UICC II). The positive predictive value of lymphatic invasion evaluated by D2-40 immunostaining to predict lymph node metastasis is 92% (negative predictive value 81%). High PIR was shown in UICC stage I and II. These infiltrations were rarely seen in UICC stage III and were absent in UICC stage IV. Higher UICC tumor stage is associated with a higher rate of focally newly developed lymphatic vessels. In UICC stage I we found peritumoral lymphatic vessel proliferation only in one case (14%) and in UICC stage II no case was found. 47% of the cases in UICC stage III and 50% of the cases in UICC stage IV showed focal peritumoral lymphatic vessel proliferation. CONCLUSIONS: Immunostaining with D2-40 significantly increased the detection rate of lymphatic invasion compared to conventional HE staining in primary CRC. The D2-40 antibody specific for lymphatic endothelium cells has the potential for a prognostic marker in early stage CRC. Further prospective studies are necessary to evaluate the prognostic value of lymphatic invasion and the induction of tumor lymphangiogenesis and its role in human cancer progression.

Swiss DVI at the tsunami disaster: expect the unexpected.

The conclusions reached while considering various aspects of the implemented strategy in the identification procedures in the wake of the tsunami disaster of December 26, 2004 are outlined. The lessons to be learned are discussed.

HPV in anal squamous cell carcinoma and anal intraepithelial neoplasia (AIN). Impact of HPV analysis of anal lesions on diagnosis and prognosis.

BACKGROUND AND AIMS: Majority of cases of anal squamous cell carcinoma are human papilloma virus (HPV)-induced and result from anal intraepithelial neoplasia (AIN). This study was conducted to examine methods which may enable the routine diagnosis of HPV-induced changes in the anal rim and the consequences of such detection especially in view of a more sensitive diagnosis of AIN. Results were clinically correlated. METHODS: The study included biopsy samples from 87 patients who had been diagnosed with the following disease patterns: 47 invasive anal carcinoma, 33 AIN of varying severity and seven condylomatous lesions. In 52 of these cases, a tumour was clinically suspected. All biopsies were retrospectively examined for microscopic indications of HPV infection. After microdissection, additional HPV analysis via PCR was carried out. RESULTS: In 38 of 47 cases of anal carcinoma, HPV DNA could be detected via PCR (80.9%), the majority of which were HPV 16 (33/38=86.8%). In 29 of the 33 cases of AIN, HPV DNA was detected (87.9%), most of these in AIN III (15/16=93.8%). Histological markers of HPV infection were detected in all 87 cases. DISCUSSION: In our series, the clinical diagnosis of the invasive anal carcinoma had a high sensitivity of 93.6%, with a specificity of 80%. The positive predictive value was 84.6%, and the negative predictive value 91.4%. In contrast, AIN had been detected clinically in none of the cases. In this situation, especially with high-risk patients, our findings recommend anal HPV screening in combination with anal cytology and anoscopy. CONCLUSION: Based on our results, we urgently recommend for any histological report on excision of anal lesions to include a statement whether histological markers of HPV infection were detected. In individual cases, validation via HPV PCR must be considered.

Can we detect cervical human papillomavirus (HPV) infection by cytomorphology alone? Diagnostic value of non-classic cytological signs of HPV effect in minimally abnormal Pap tests.

OBJECTIVE: Our aim was to assess the validity of non-classical cytological signs in minimally abnormal cervical smears for the prediction of HPV infection. METHODS: 164 ThinPrep monolayers were re-screened for mild nuclear changes, disorders of keratinisation, abortive koilocytes and 'measles cells', as well as degenerative changes. HPV DNA was detected by GP5+/6+ and MY09/MY11 consensus primer PCR assays. RESULTS: Seventy six of 164 cases (46.3%) had HPV positivity by PCR. All cytomorphological features studied were significantly associated with the presence of HPV. Mild nuclear changes had 100% sensitivity and 100% negative predictive value for HPV infection. CONCLUSIONS: Our results indicate that non-classic cytomorphological signs can improve the sensitivity of cytology for detecting HPV. Minimally abnormal Pap smears lacking mild nuclear changes (16%) in the present study--do not require further molecular HPV testing.

Population-based registration of unselected colorectal cancer patients: five-year survival in the region of Bonn/Rhine-Sieg, Germany.

INTRODUCTION: Epidemiological data of colorectal cancer are sparse and often incomplete. Therefore, we initiated a population-based examination of five-year survival of colorectal cancer patients. METHODS: For complete registration, diagnosis and tumour stage of all patients in the region of Bonn/Rhine-Sieg were assessed independently according to reports of medical practitioners and pathologists. Each patient was followed by a standardised questionnaire during a period of five years. RESULTS: Between June and November, 1994 348 patients were registered. Median age at diagnosis was 69 years for males (n = 160) and 72 years for females (n = 188). According to the UICC classification 18, 26, 23 and 26 % had stage I-IV tumours, respectively; the tumour stage remained unclear in 7 %. Adjuvant (radio)-chemotherapy was indicated in 89 patients, but only 49 % of these were treated. Five-year overall survival (OS) and relative overall survival were 41 and 54 %, respectively. Although disease-free survival (DFS) was significantly better for early stage colorectal cancer, OS did not differ significantly between stage I and stage III tumours. Young patients diagnosed before the age of 50 had a significantly lower DFS. These data were comparable with other European countries but were lower than data reported in the USA. DISCUSSION: The high rate of patients with stage IV colorectal cancer and the low proportion of patients receiving adjuvant (radio)-chemotherapy according to international or national consensus recommendations were disappointing. Although data were comparable with other European countries more efforts are necessary to establish effective screening programs for asymptomatic patients and to increase the willingness for standardised adjuvant treatment.

DNA cytometry confirms the utility of the Bethesda system for the classification of Papanicolaou smears.

BACKGROUND: Developed in 1989, the Bethesda System has largely replaced previous classifications of Papanicolaou (Pap) smears from the uterine cervix. The system is binary, dividing smears into two groups - low-grade, squamous, epithelial lesions (LSIL) or high-grade, squamous, epithelial lesions (HSIL). A third category, atypical squamous cells of undetermined significance (ASCUS), is used to classify minimal cellular changes that do not satisfy the criteria for the low- or high-grade categories. This study was designed to confirm the utility of this binary division and to compare the results with another classification system (the Munich II Nomenclature) that is not binary but contains three divisions or grades for dysplasia - low, intermediate, and high. METHODS: Pap smears were obtained from 593 women with a cytologic diagnosis of dysplasia based on the Munich System. Smears were then classified by the Bethesda System into LSIL or HSIL. Patients were followed for 2 years either with biopsy or repeat cytology. The initial smears were restained by the Feulgen method, and ploidy was evaluated by interactive DNA cytometry. RESULTS: Of 241 cases of LSIL, 39% were diploid, 57% polyploid, and 4% aneuploid. Of 352 cases classified HSIL, 4% were diploid, 17% polyploid, and 79% aneuploid. After 2 years of follow-up, 2 of 108 patients who were biopsied and who were originally classified as diploid progressed to cervical intraepithelial neoplasia/carcinoma in situ (CIN/CIS) whereas 109 of 217 patients who were aneuploid and biopsied were found to have CINIII/CIS. CONCLUSIONS: The two divisions of the Bethesda System, LSIL and HSIL, correlated with ploidy as evaluated by cytometry. Aneuploidy was found to be useful to separate cases of HSIL from those of LSIL as defined in the Bethesda System. Because of the binary division, use of a system with three divisions for dysplasia, such as the Munich II Nomenclature, creates a therapeutic dilemma because a single diagnostic category (usually the intermediate grade) may contain both self-limiting and progressive lesions. DNA cytometry of Pap smears was found to be useful as a routine procedure.

Gibberellin A(4/7) and the Promotion of Flowering in Pinus radiata: Effects on Partitioning of Photoassimilate within the Bud during Primordia Diferentiation.

Gibberellin A(4/7) mixture (GA(4/7)), a highly effective promoter of early and enhanced flowering in the Pinaceae, caused a significant reallocation of dry matter and (14)C-photosynthate within terminal buds of Pinus radiata D. Don within 8 days of hormone treatment. Treatment with GA(4/7) to terminal shoots of vigorous, potentially flowering mature grafted propagules reduced the flow of photoassimilated (14)C and dry matter into the terminal bud as a whole, but significantly increased the dry matter and (14)C allocated within the bud to developing long-shoot primordia (potential seed-cone buds). This was accomplished at the expense of the structural tissues, the apical dome region, and the vegetative branch buds. Although GA(3) caused a similar reallocation of dry matter within the terminal bud, it was significantly less effective than GA(4/7) thus appears to have, in addition to any nutrient diversion abilities, a distinct morphogenic function in sexual differentiation.