Specific delineation of BK polyomavirus in kidney tissue with a digoxigenin-labeled DNA probe.

The detection of BK polyomavirus (BK virus, BKV) in kidney tissue is hampered by nonspecificity of antibodies suited to immunohistochemistry, and nonspecific background with in situ hybridization. The biotin-labeled DNA probe that is commercially available (Enzo Life Sciences, Inc.) shows good signal, but the intrinsic background in kidney tissue is high. We determined that the intrinsic background is due to endogenous biotin or biotin-binding activity in the renal tubular epithelium. Neither antibody blocking procedures nor an avidin/biotin block were entirely satisfactory for eliminating this background staining. We developed a digoxigenin-labeled DNA probe, and protocol, for detecting BK virus in formalin-fixed, paraffin embedded, kidney tissue obtained at autopsy. The hybridization signal is strong and there is no perceptible background staining. Eleven negative control kidneys all failed to hybridize. Conditions for low stringency hybridization may be employed, detecting both the related JC polyomavirus and BKV. Alternatively, high stringency hybridization conditions may be utilized, detecting BKV only. BK associated tubular necrosis is clearly demonstrated in two cases of BK nephritis.

p53 status correlates with histopathological response in patients with soft tissue sarcomas treated using isolated limb perfusion with TNF-alpha and melphalan.

BACKGROUND: Recombinant tumor necrosis factor-alpha (TNF-alpha) combined to melphalan is clinically administered through isolated limb perfusion (ILP) for regionally advanced soft tissue sarcomas of the limbs. In preclinical studies, wild-type p53 gene is involved in the regulation of cytotoxic action of TNF-alpha and loss of p53 function contributes to the resistance of tumour cells to TNF-alpha. The relationship between p53 status and response to TNF-alpha and melphalan in patients undergoing ILP is unknown. PATIENTS AND METHODS: We studied 110 cases of unresectable limbs sarcomas treated by ILP. Immunohistochemistry was carried out using DO7mAb, which reacts with an antigenic determinant from the N-terminal region of both the wild-type and mutant forms of the p53 protein, and PAb1620mAb, which reacts with the 1620 epitope characteristic of the wild-type native conformation of the p53 protein. The immunohistochemistry data were then correlated with various clinical parameters. RESULTS: P53DO7 was found expressed at high levels in 28 patients, whereas PAb1620 was negative in 20. The tumours with poor histological response to ILP with TNF-alpha and melphalan showed significantly higher levels of p53-mutated protein. CONCLUSIONS: Our results might be a clue to a role of p53 protein status in TNF-alpha and melphalan response in clinical use.

Assessment of vascular invasion by bone and soft tissue tumours of the limbs: usefulness of MDCT angiography.

OBJECTIVE: To evaluate the accuracy of computed tomography angiography (CTA) in predicting arterial encasement by limb tumours, by comparing CTA with surgical findings (gold standard). METHODS: Preoperative CTA images of 55 arteries in 48 patients were assessed for arterial status: cross-sectional CTA images were scored as showing a fat plane between artery and tumour (score 0), slight contact between artery and tumour (score 1), partial arterial encasement (score 2) or total arterial encasement (score 3). Reformatted CTA images were assessed for arterial displacement, rigid wall, stenosis or occlusion. At surgery, arteries were classified as free or surgically encased; 45 arteries were free and 10 were surgically encased. RESULTS: Multivariate logistic regression identified the axial CTA score as a relevant predictor for arterial encasement and subsequent vascular intervention during surgery. All sites where CTA showed a fat plane between the tumour and the artery were classified as free at surgery (n = 28/28). The sensitivity of total arterial encasement on CTA (score 3) was 90%, specificity 93%, accuracy 93% and positive likelihood ratio 13.5. CONCLUSION: CTA evidence of total arterial encasement is a highly specific indication of arterial encasement. The presence of fat between the tumour and the artery on CTA rules out arterial involvement at surgery.

The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.

Embryonic stem cells (ESCs) offer attractive prospective as potential source of neurons for cell replacement therapy in human neurodegenerative diseases. Besides, ESCs neural differentiation enables in vitro tissue engineering for fundamental research and drug discovery aimed at the nervous system. We have established stable and long-term three-dimensional (3D) culture conditions which can be used to model long latency and complex neurodegenerative diseases. Mouse ESCs-derived neural progenitor cells generated by MS5 stromal cells induction, result in strictly neural 3D cultures of about 120-mum thick, whose cells expressed mature neuronal, astrocytes and myelin markers. Neurons were from the glutamatergic and gabaergic lineages. This nervous tissue was spatially organized in specific layers resembling brain sub-ependymal (SE) nervous tissue, and was maintained in vitro for at least 3.5 months with great stability. Electron microscopy showed the presence of mature synapses and myelinated axons, suggesting functional maturation. Electrophysiological activity revealed biological signals involving action potential propagation along neuronal fibres and synaptic-like release of neurotransmitters. The rapid development and stabilization of this 3D cultures model result in an abundant and long-lasting production that is compatible with multiple and productive investigations for neurodegenerative diseases modeling, drug and toxicology screening, stress and aging research.

Tissue distribution of the Ankara strain of vaccinia virus (MVA) after mucosal or systemic administration.

MVA is a candidate vector for vaccination against pathogens and tumors. Little is known about its behaviour in mucosal tissues. We have investigated the fate and biosafety of MVA, when inoculated by different routes in C57BL/6 mice. Intranasal inoculation targeted the virus to the nasal associated lymphoid tissue and the lungs, whereas systemic inoculation led to distribution of MVA in almost all lymphoid organs, lungs and ovaries. Intravaginal, intrarectal and intragastric inoculations failed to induce efficient infection. After 48 h no virus was detectable any more in the organs analyzed. Upon intranasal inoculation, no inflammatory reactions were detected in the central nervous system as well as the upper and lower airways. These results show the tropism of MVA and indicate that high doses of recombinant MVA are safe when nasally administered, a vaccination route known to elicit strong cellular and humoral immune responses in the female genital tract.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Prediction of Recanalization Trumps Prediction of Tissue Fate: The Penumbra: A Dual-edged Sword.

BACKGROUND AND PURPOSE: To determine whether infarct core or penumbra is the more significant predictor of outcome in acute ischemic stroke, and whether the results are affected by the statistical method used. METHODS: Clinical and imaging data were collected in 165 patients with acute ischemic stroke. We reviewed the noncontrast head computed tomography (CT) to determine the Alberta Score Program Early CT score and assess for hyperdense middle cerebral artery. We reviewed CT-angiogram for site of occlusion and collateral flow score. From perfusion-CT, we calculated the volumes of infarct core and ischemic penumbra. Recanalization status was assessed on early follow-up imaging. Clinical data included age, several time points, National Institutes of Health Stroke Scale at admission, treatment type, and modified Rankin score at 90 days. Two multivariate regression analyses were conducted to determine which variables predicted outcome best. In the first analysis, we did not include recanalization status among the potential predicting variables. In the second, we included recanalization status and its interaction between perfusion-CT variables. RESULTS: Among the 165 study patients, 76 had a good outcome (modified Rankin score ≤2) and 89 had a poor outcome (modified Rankin score >2). In our first analysis, the most important predictors were age (P<0.001) and National Institutes of Health Stroke Scale at admission (P=0.001). The imaging variables were not important predictors of outcome (P>0.05). In the second analysis, when the recanalization status and its interaction with perfusion-CT variables were included, recanalization status and perfusion-CT penumbra volume became the significant predictors (P<0.001). CONCLUSIONS: Imaging prediction of tissue fate, more specifically imaging of the ischemic penumbra, matters only if recanalization can also be predicted.

Tissue-specific FLAGELLIN-SENSING 2 (FLS2) expression in roots restores immune responses in Arabidopsis fls2 mutants.

The flagellin receptor of Arabidopsis, At-FLAGELLIN SENSING 2 (FLS2), has become a model for mechanistic and functional studies on plant immune receptors. Responses to flagellin or its active epitope flagellin 22 (flg22) have been extensively studied in Arabidopsis leaves. However, the perception of microbe-associated molecular patterns (MAMPs) and the immune responses in roots are poorly understood. Here, we show that isolated root tissue is able to induce pattern-triggered immunity (PTI) responses upon flg22 perception, in contrast to elf18 (the active epitope of elongation factor thermo unstable (EF-Tu)). Making use of fls2 mutant plants and tissue-specific promoters, we generated transgenic Arabidopsis lines expressing FLS2 only in certain root tissues. This allowed us to study the spatial requirements for flg22 responses in the root. Remarkably, the intensity of the immune responses did not always correlate with the expression level of the FLS2 receptor, but depended on the expressing tissue, supporting the idea that MAMP perception and sensitivity in different tissues contribute to a proper balance of defense responses according to the expected exposure to elicitors. In summary, we conclude that each investigated root tissue is able to perceive flg22 if FLS2 is present and that tissue identity is a major element of MAMP perception in roots.

Body fat distribution in white and black women: different patterns of intraabdominal and subcutaneous abdominal adipose tissue utilization with weight loss.

BACKGROUND: Intraabdominal adipose tissue (IAAT) is the body fat depot most strongly related to disease risk. Weight reduction is advocated for overweight people to reduce total body fat and IAAT, although little is known about the effect of weight loss on abdominal fat distribution in different races. OBJECTIVE: We compared the effects of diet-induced weight loss on changes in abdominal fat distribution in white and black women. DESIGN: We studied 23 white and 23 black women, similar in age and body composition, in the overweight state [mean body mass index (BMI; in kg/m(2)): 28.8] and the normal-weight state (mean BMI: 24.0) and 38 never-overweight control women (mean BMI: 23.4). We measured total body fat by using a 4-compartment model, trunk fat by using dual-energy X-ray absorptiometry, and cross-sectional areas of IAAT (at the fourth and fifth lumbar vertebrae) and subcutaneous abdominal adipose tissue (SAAT) by using computed tomography. RESULTS: Weight loss was similar in white and black women (13.1 and 12.6 kg, respectively), as were losses of total fat, trunk fat, and waist circumference. However, white women lost more IAAT (P < 0.001) and less SAAT (P < 0.03) than did black women. Fat patterns regressed toward those of their respective control groups. Changes in waist circumference correlated with changes in IAAT in white women (r = 0.54, P < 0.05) but not in black women (r = 0.19, NS). CONCLUSIONS: Despite comparable decreases in total and trunk fat, white women lost more IAAT and less SAAT than did black women. Waist circumference was not a suitable surrogate marker for tracking changes in the visceral fat compartment in black women.

Advanced MRI unravels the nature of tissue alterations in early multiple sclerosis.

INTRODUCTION: In patients with multiple sclerosis (MS), conventional magnetic resonance imaging (MRI) provides only limited insights into the nature of brain damage with modest clinic-radiological correlation. In this study, we applied recent advances in MRI techniques to study brain microstructural alterations in early relapsing-remitting MS (RRMS) patients with minor deficits. Further, we investigated the potential use of advanced MRI to predict functional performances in these patients. METHODS: Brain relaxometry (T1, T2, T2*) and magnetization transfer MRI were performed at 3T in 36 RRMS patients and 18 healthy controls (HC). Multicontrast analysis was used to assess for microstructural alterations in normal-appearing (NA) tissue and lesions. A generalized linear model was computed to predict clinical performance in patients using multicontrast MRI data, conventional MRI measures as well as demographic and behavioral data as covariates. RESULTS: Quantitative T2 and T2* relaxometry were significantly increased in temporal normal-appearing white matter (NAWM) of patients compared to HC, indicating subtle microedema (P = 0.03 and 0.004). Furthermore, significant T1 and magnetization transfer ratio (MTR) variations in lesions (mean T1 z-score: 4.42 and mean MTR z-score: -4.09) suggested substantial tissue loss. Combinations of multicontrast and conventional MRI data significantly predicted cognitive fatigue (P = 0.01, Adj-R (2) = 0.4), attention (P = 0.0005, Adj-R (2) = 0.6), and disability (P = 0.03, Adj-R (2) = 0.4). CONCLUSION: Advanced MRI techniques at 3T, unraveled the nature of brain tissue damage in early MS and substantially improved clinical-radiological correlations in patients with minor deficits, as compared to conventional measures of disease.

The zinc transporter SLC39A13/ZIP13 is required for connective tissue development; its involvement in BMP/TGF-beta signaling pathways.

BACKGROUND: Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-beta signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. CONCLUSIONS/SIGNIFICANCE: Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-beta signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-beta signaling and connective tissue dysfunction.

Effect of dark chocolate on renal tissue oxygenation as measured by BOLD-MRI in healthy volunteers.

Background: Cocoa is rich in flavonoids, has anti-oxidative properties and increases the bioavailability of nitric oxide (NO). Adequate renal tissue oxygenation is crucial for the maintenance of renal function. The goal of this study was to investigate the effect of cocoa-rich dark chocolate (DC) on renal tissue oxygenation in humans, as compared to flavonoid-poor white chocolate (WC). Methods: Ten healthy volunteers with preserved kidney function (mean age ± SD 35 ± 12 years, 70% women, BMI 21 ± 3 kg/m2) underwent blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) before and 2 hours after the ingestion of 1 g/kg of DC (70% cocoa). Renal tissue oxygenation was determined by the measurement of R2* maps on 4 coronal slices covering both kidneys. The mean R2* (= 1/T2*) values in the medulla and cortex were calculated, a low R2* indicating high tissue oxygenation. Eight participants also underwent BOLD-MRI at least 1 week later, before and 2 hours after the intake of 1 g/kg WC. Results: The mean medullary R2* was lower after DC intake compared to baseline (28.2 ± 1.3 s-1 vs. 29.6 ± 1.3 s-1, p = 0.04), whereas cortical and medullary R2* values did not change after WC intake. The change in medullary R2* correlated with the level of circulating (epi)catechines, metabolites of flavonoids (r = 0.74, p = 0.037), and was independent of plasma renin activity. Conclusion: This study suggests for the first time an increase of renal medullary oxygenation after intake of dark chocolate. Whether this is linked to flavonoid-induced changes in renal perfusion or oxygen consumption, and whether cocoa has potentially renoprotective properties, merits further study.

Monoclonal antibodies identify a CEA crossreacting antigen of 95 kD (NCA-95) distinct in antigenicity and tissue distribution from the previously described NCA of 55 kD.

During the selection of monoclonal antibodies (MAb) raised against purified carcinoembryonic antigen (CEA), two MAbs were identified which immunoprecipitated a glycoprotein of 95 kD present both in perchloric acid extracts of normal lung and on the surface of normal granulocytes. This antigen was distinct from the previously reported normal glycoprotein crossreacting with CEA (NCA) which had a molecular weight of 55 kD. The difference between the smaller and the larger crossreacting antigens termed NCA-55 and NCA-95, respectively, was demonstrated by SDS-polyacrylamide gel electrophoresis, by elution from Sephadex-G200 and by selective binding to a series of anti-CEA MAb. Out of six MAb which all bound CEA purified from colon carcinoma, three did not react with these two crossreacting antigens, one bound only NCA-95, one reacted only with NCA-55 and one reacted with both NCA-55 and NCA-95. Immunoadsorbent purified preparations of 125I labelled NCA-95 and NCA-55 were found useful for the screening of new anti-CEA MAb. In addition, when tested on frozen sections of colon carcinoma, normal spleen, normal lung and pancreas, each type of MAb gave a clearly different pattern of reactivity. The three anti-CEA MAb which did not bind any of the crossreacting antigens stained only the colon carcinoma cells; the MAb binding to either one of the two types of NCA gave a similar pattern of reactivity both on colon carcinoma cells and on granulocytes. However, on normal lung and pancreas, the MAb binding NCA-55 stained granulocytes as well as bronchiolar and alveolar epithelial cells in lung and inter- and intra-lobular duct epithelial cells in pancreas, whereas the MAb binding only NCA-95 stained only the granulocytes. Thus, the newly identified NCA-95 appears to differ from NCA-55 not only in terms of molecular size and antigenicity but also by the fact that in normal lung and pancreas it is found in granulocytes but not in epithelial cells.

Comparative assessement of intimal hyperplasia development after 14 days in two different experimental settings : tissue culture versus ex vivo continuous perfusion of human saphenous vein

Résumé de l'article : L'hyperplasie intimale est un processus de remodelage vasculaire ubiquitaire après une lésion, pouvant menacer la perméabilité de tout type de reconstruction vasculaire. Les mécanismes physiopathologiques impliqués dans le développement de l'hyperplasie intimale ne sont que partiellement élucidés. Il est par conséquent nécessaire d'effectuer des recherches complémentaires afin d'en améliorer la compréhension et ainsi permettre l'élaboration de nouvelles stratégies thérapeutiques médicamenteuses. La culture de veines en milieu statique permet le développement de l'hyperplasie intimale. Ce modèle maintient la viabilité tissulaire, comme décrit précédemment dans d'autres études, mais empêche l'analyse des paramètres hémodynamiques. La mise au point d'un modèle de perfusion in vitro permettant la perfusion de segments vasculaires représente une approche expérimentale intégrant les différents facteurs hémodynamiques. Le système de perfusion (Ex Vivo Vein Support System) que nous avons élaboré conserve l'intégrité pariétale ainsi que les propriétés vasomotrices des veines pour une durée de 14 jours. Cette étude démontre que les deux modèles permettent le développement de l'hyperplasie intimale. Toutefois, les propriétés vasomotrices ainsi que l'influence des paramètres hémodynamiques ne peuvent être analysées que par l'utilisation du système de perfusion. Ce dernier a permis de perfuser des vaisseaux humains sans contamination bactérienne tout en maintenant l'intégrité cellulaire. Ce modèle de perfusion se rapproche plus des conditions hémodynamiques rencontrées in vivo que le modèle statique. Abstract : Background. Intimal hyperplasia (IH) is a vascular remodeling process which often leads to failure of arterial bypass or hemodialysis access. Experimental and clinical work have provided insight in IH development; however, further studies under precise con-trolled conditions are required to improve therapeutic strategies to inhibit IH development. Ex vivo perfusion of human vessel segments under standardized hemodynamic conditions may provide an adequate experimental approach for this purpose. Therefore, chronically perfused venous segments were studied and compared to traditional static culture procedures with regard to functional and histomorphologic characteristics as well as gene expression. Materials and methods. Static vein culture allowing high tissue viability was performed as previously described. Ex vivo vein support system (EVVSS) was performed using a vein support system consisting of an incubator with a perfusion chamber and a pump. EVVSS allows vessel perfusion under continuous flow while maintaining controlled hemodynamic conditions. Each human saphenous vein was divided in two parts, one cultured in a Pyrex dish and the other part perfused in EVVSS for 14 days. Testing of vasomotion, histomorphometry, expression of CD 31, Factor VIII, MIB 1, α-actin, and PAI-1 were determined before and after 14 days of either experimental conditions. Results, Human venous segments cultured under traditional or perfused conditions exhibited similar IH after 14 days as shown by histomorphometry. Smooth-muscle cell ( SMC) was preserved after chronic perfusion. Although integrity of both endothelial and smooth-muscle cells appears to be maintained in both culture conditions as confirmed by CD31, factor VIII and α-actin expression, a few smooth-muscle cells in the media stained positive for factor VIII. Cell-proliferation marker MIB-1 was also detected in the two settings and PAI-1 mRNA expression and activity increased significantly after 14 days of culture and perfusion. Conclusion. This study demonstrates the feasibility to chronically perfuse human vessels under sterile conditions with preservation of cellular integrity and vascular contractility. To gain insights into the mechanisms leading to IH, it will now be possible to study vascular remodeling not only under static conditions but also in hemodynamic environment mimicking as closely as possible the flow conditions encountered in reconstructive vascular surgery.

Deoxyribonucleic acid content as an indicator of progression of squamous cell carcinogenesis in the esophagus: comparative analysis on imprint-cytospin and tissue section preparation.

BACKGROUND: The purpose of this study was to explore the potential use of image analysis on tissue sections preparation as a predictive marker of early malignant changes during squamous cell (SC) carcinogenesis in the esophagus. Results of DNA ploidy quantification on formalin-fixed, paraffin-embedded tissue using two different techniques were compared: imprint-cytospin and 6 microm thick tissue sections preparation. METHODS: This retrospective study included 26 surgical specimens of squamous cell carcinoma (SCC) from patients who underwent surgery alone at the Department of Surgery in CHUV Hospital in Lausanne between January 1993 and December 2000. We analyzed 53 samples of healthy tissue, 43 tumors and 7 lymph node metastases. RESULTS: Diploid DNA histogram patterns were observed in all histologically healthy tissues, either distant or proximal to the lesion. Aneuploidy was observed in 34 (79%) of 43 carcinomas, namely 24 (75%) of 32 early squamous cell carcinomas and 10 (91%) of 11 advanced carcinomas. DNA content was similar in the different tumor stages, whether patients presented with single or multiple synchronous tumors. All lymph node metastases had similar DNA content as their primary tumor. CONCLUSIONS: Early malignant changes in the esophagus are associated with alteration in DNA content, and aneuploidy tends to correlate with progression of invasive SCC. A very good correlation between imprint-cytospin and tissue section analysis was observed. Although each method used here showed advantages and disadvantages; tissue sections preparation provided useful information on aberrant cell-cycle regulation and helped select the optimal treatment for the individual patient along with consideration of other clinical parameters.