Effects of thyroid hormones and aldosterone on mineralocorticoid binding sites in the toad bladder.

In the urinary bladder of the toad Bufo marinus triiodothyronine selectively inhibits the late effect of aldosterone on Na+ transport. We have investigated whether T3 might mediate its antimineralocorticoid action by controlling: i) the level of aldosterone binding sites in the soluble (cytosolic) pool isolated from tissues treated with T3 (60 nM) for up to 20 hr of incubation; ii) the kinetics of uptake of 3H-aldosterone into cytoplasmic and nuclear fractions after 2 or 20 hr of exposure to T3. The number and the affinity of Type I (high affinity, low capacity) and Type II (low affinity, high capacity) cytosolic binding sites (measured at 0 degrees C) did not vary significantly after 18 hr of exposure to T3, while aldosterone-dependent Na+ transport was significantly inhibited. In addition, T3 did not modify the kinetics of uptake (90 min) of 3H-aldosterone into cytoplasmic and nuclear fractions of toad bladder incubated in vitro at 25 degrees C. By contrast, aldosterone itself was able to down-regulate its cytosolic and nuclear binding sites after an 18-hr exposure to the steroid hormone (10 or 80 nM). T3 slightly (20%) but significantly potentiated the down regulation of nuclear binding sites. In conclusion, T3 does not appear to have major effects on the regulation of the aldosterone receptor, which could explain in a simple manner its antimineralocorticoid action.

Whole-body 3D T1-weighted MR imaging in patients with prostate cancer: feasibility and evaluation in screening for metastatic disease.

PURPOSE: To develop and assess the diagnostic performance of a three-dimensional (3D) whole-body T1-weighted magnetic resonance (MR) imaging pulse sequence at 3.0 T for bone and node staging in patients with prostate cancer. MATERIALS AND METHODS This prospective study was approved by the institutional ethics committee; informed consent was obtained from all patients. Thirty patients with prostate cancer at high risk for metastases underwent whole-body 3D T1-weighted imaging in addition to the routine MR imaging protocol for node and/or bone metastasis screening, which included coronal two-dimensional (2D) whole-body T1-weighted MR imaging, sagittal proton-density fat-saturated (PDFS) imaging of the spine, and whole-body diffusion-weighted MR imaging. Two observers read the 2D and 3D images separately in a blinded manner for bone and node screening. Images were read in random order. The consensus review of MR images and the findings at prospective clinical and MR imaging follow-up at 6 months were used as the standard of reference. The interobserver agreement and diagnostic performance of each sequence were assessed on per-patient and per-lesion bases. RESULTS: The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were significantly higher with whole-body 3D T1-weighted imaging than with whole-body 2D T1-weighted imaging regardless of the reference region (bone or fat) and lesion location (bone or node) (P < .003 for all). For node metastasis, diagnostic performance (area under the receiver operating characteristic curve) was higher for whole-body 3D T1-weighted imaging (per-patient analysis; observer 1: P < .001 for 2D T1-weighted imaging vs 3D T1-weighted imaging, P = .006 for 2D T1-weighted imaging + PDFS imaging vs 3D T1-weighted imaging; observer 2: P = .006 for 2D T1-weighted imaging vs 3D T1-weighted imaging, P = .006 for 2D T1-weighted imaging + PDFS imaging vs 3D T1-weighted imaging), as was sensitivity (per-lesion analysis; observer 1: P < .001 for 2D T1-weighted imaging vs 3D T1-weighted imaging, P < .001 for 2D T1-weighted imaging + PDFS imaging vs 3D T1-weighted imaging; observer 2: P < .001 for 2D T1-weighted imaging vs 3D T1-weighted imaging, P < .001 for 2D T1-weighted imaging + PDFS imaging vs 3D T1-weighted imaging). CONCLUSION: Whole-body MR imaging is feasible with a 3D T1-weighted sequence and provides better SNR and CNR compared with 2D sequences, with a diagnostic performance that is as good or better for the detection of bone metastases and better for the detection of lymph node metastases.

Diagnostic performance of CT-arthrography and 1.5T MR-arthrography for the assessment of glenohumeral joint cartilage: a comparative study with arthroscopic correlation.

PURPOSE: To compare the diagnostic performance of multi-detector CT arthrography (CTA) and 1.5-T MR arthrography (MRA) in detecting hyaline cartilage lesions of the shoulder, with arthroscopic correlation. PATIENTS AND METHODS: CTA and MRA prospectively obtained in 56 consecutive patients following the same arthrographic procedure were independently evaluated for glenohumeral cartilage lesions (modified Outerbridge grade ≥2 and grade 4) by two musculoskeletal radiologists. The cartilage surface was divided in 18 anatomical areas. Arthroscopy was taken as the reference standard. Diagnostic performance of CTA and MRA was compared using ROC analysis. Interobserver and intraobserver agreement was determined by κ statistics. RESULTS: Sensitivity and specificity of CTA varied from 46.4 to 82.4 % and from 89.0 to 95.9 % respectively; sensitivity and specificity of MRA varied from 31.9 to 66.2 % and from 91.1 to 97.5 % respectively. Diagnostic performance of CTA was statistically significantly better than MRA for both readers (all p ≤ 0.04). Interobserver agreement for the evaluation of cartilage lesions was substantial with CTA (κ = 0.63) and moderate with MRA (κ = 0.54). Intraobserver agreement was almost perfect with both CTA (κ = 0.94-0.95) and MRA (κ = 0.83-0.87). CONCLUSION: The diagnostic performance of CTA and MRA for the detection of glenohumeral cartilage lesions is moderate, although statistically significantly better with CTA. KEY POINTS: ? CTA has moderate diagnostic performance for detecting glenohumeral cartilage substance loss. ? MRA has moderate diagnostic performance for detecting glenohumeral cartilage substance loss. ? CTA is more accurate than MRA for detecting cartilage substance loss.

Mineralogical, K-Ar, stable and Sr isotope systematics of K-white micas during very low-grade metamorphism of limestones (Helvetic nappes, Western Switzerland)

Mineralogical, K-Ar, Rb-Sr and stable isotope analyses have been carried out on K-white micas from Helvetic Malm limestones in order to examine their evolution during very low- to low-grade Alpine metamorphism, associated with intense ductile deformation. Metamorphic temperatures were estimated al approximately 300-degrees-C from stable isotopes (quartz-calcite thermometry), occurrence of chloritoid, and `'epizonal'' illite crystallinity index. K-white micas consist of variable mixtures of 2M, phengite and muscovite, as revealed by detailed X-ray diffraction analyses using peak decomposition of the (060, 331) spectra. K-Ar apparent ages display a strong grain-size dependence in which mainly fine-grained size fractions (< 2 mum) record Alpine ages (37-15 Ma). However, these ages provide a relative rather than an absolute chronology of the diachronous Alpine metamorphic evolution of the Helvetic nappes. The resetting of the K-Ar isotopic system of K-white micas to Alpine metamorphic conditions reflects an apparent combination of crystallization/recrystallization and radiogenic Ar-40 diffusion loss. The oxygen isotope compositions of micas (+ 15 to + 22 parts per thousand) are intermediate between detrital and O-18-enriched values expected for micas neoformed within an abundant marine carbonate matrix. No isotopic equilibrium has been reached between calcite and micas. The variable depletion of hydrogen isotope compositions (- 126 to - 82 parts per thousand) is influenced by the interaction with organic matter under closed-system conditions. Organic matter, if not removed, may also represent a serious source of error in K-Ar age determination, by introducing radiogenic Ar-40 contamination. Sr-87/Sr-86 isotope ratios of micas range from 0.70879 to 0.70902 with one outlier at 0.71794. The low values reflect Sr exchange with calcite occurring during crystallization/recrystallization of micas under closed-system conditions.

Opposing roles for calcineurin and ATF3 in squamous skin cancer.

Calcineurin inhibitors such as cyclosporin A (CsA) are the mainstay of immunosuppressive treatment for organ transplant recipients. Squamous cell carcinoma (SCC) of the skin is a major complication of treatment with these drugs, with a 65 to 100-fold higher risk than in the normal population. By contrast, the incidence of basal cell carcinoma (BCC), the other major keratinocyte-derived tumour of the skin, of melanoma and of internal malignancies increases to a significantly lesser extent. Here we report that genetic and pharmacological suppression of calcineurin/nuclear factor of activated T cells (NFAT) function promotes tumour formation in mouse skin and in xenografts, in immune compromised mice, of H-ras(V12) (also known as Hras1)-expressing primary human keratinocytes or keratinocyte-derived SCC cells. Calcineurin/NFAT inhibition counteracts p53 (also known as TRP53)-dependent cancer cell senescence, thereby increasing tumorigenic potential. ATF3, a member of the 'enlarged' AP-1 family, is selectively induced by calcineurin/NFAT inhibition, both under experimental conditions and in clinically occurring tumours, and increased ATF3 expression accounts for suppression of p53-dependent senescence and enhanced tumorigenic potential. Thus, intact calcineurin/NFAT signalling is critically required for p53 and senescence-associated mechanisms that protect against skin squamous cancer development.

Cooperation between the transcription factors p63 and IRF6 is essential to prevent cleft palate in mice.

Cleft palate is a common congenital disorder that affects up to 1 in 2,500 live human births and results in considerable morbidity to affected individuals and their families. The etiology of cleft palate is complex, with both genetic and environmental factors implicated. Mutations in the transcription factor-encoding genes p63 and interferon regulatory factor 6 (IRF6) have individually been identified as causes of cleft palate; however, a relationship between the key transcription factors p63 and IRF6 has not been determined. Here, we used both mouse models and human primary keratinocytes from patients with cleft palate to demonstrate that IRF6 and p63 interact epistatically during development of the secondary palate. Mice simultaneously carrying a heterozygous deletion of p63 and the Irf6 knockin mutation R84C, which causes cleft palate in humans, displayed ectodermal abnormalities that led to cleft palate. Furthermore, we showed that p63 transactivated IRF6 by binding to an upstream enhancer element; genetic variation within this enhancer element is associated with increased susceptibility to cleft lip. Our findings therefore identify p63 as a key regulatory molecule during palate development and provide a mechanism for the cooperative role of p63 and IRF6 in orofacial development in mice and humans.

Increased renal responsiveness to vasopressin and enhanced V2 receptor signaling in RGS2-/- mice.

The antidiuretic effect of vasopressin is mediated by V2 receptors (V2R) that are located in kidney connecting tubules and collecting ducts. This study provides evidence that V2R signaling is negatively regulated by regulator of G protein signaling 2 (RGS2), a member of the family of RGS proteins. This study demonstrates that (1) RGS2 expression in the kidney is restricted to the vasopressin-sensitive part of the nephron (thick ascending limb, connecting tubule, and collecting duct); (2) expression of RGS2 is rapidly upregulated by vasopressin; (3) the vasopressin-dependent accumulation of cAMP, the principal messenger of V2R signaling, is significantly higher in collecting ducts that are microdissected from the RGS2(-/-) mice compared with their wild-type littermates; and (4) analysis of urine output of mice that were exposed to water restriction followed by acute water loading revealed that RGS2(-/-) mice exhibit an increased renal responsiveness to vasopressin. It is proposed that RGS2 is involved in negative feedback regulation of V2R signaling.

Mutations causing Liddle syndrome reduce sodium-dependent downregulation of the epithelial sodium channel in the Xenopus oocyte expression system.

Liddle syndrome is an autosomal dominant form of hypertension resulting from deletion or missense mutations of a PPPxY motif in the cytoplasmic COOH terminus of either the beta or gamma subunit of the epithelial Na channel (ENaC). These mutations lead to increased channel activity. In this study we show that wild-type ENaC is downregulated by intracellular Na+, and that Liddle mutants decrease the channel sensitivity to inhibition by intracellular Na+. This event results at high intracellular Na+ activity in 1.2-2.4-fold higher cell surface expression, and 2.8-3.5-fold higher average current per channel in Liddle mutants compared with the wild type. In addition, we show that a rapid increase in the intracellular Na+ activity induced downregulation of the activity of wild-type ENaC, but not Liddle mutants, on a time scale of minutes, which was directly correlated to the magnitude of the Na+ influx into the oocytes. Feedback inhibition of ENaC by intracellular Na+ likely represents an important cellular mechanism for controlling Na+ reabsorption in the distal nephron that has important implications for the pathogenesis of hypertension.

Control of transepithelial Na+ transport and Na-K-ATPase by oxytocin and aldosterone.

Short- and long-term effect of oxytocin on Na+ transport and Na-K-ATPase biosynthesis in the toad bladder, and the potential interaction of this hormone with aldosterone have been studied, leading to the following observations. An early Na+ transport response (oxytocin, 50 mU/ml) peaked at 10-15 min of hormone addition. At maximal stimulation a three- to fourfold increase in Na+ transport was observed, a sustained Na+ transport response (about two-fold control base line) was observed as long as the hormone was present in the medium and for up to 20 h of incubation. Pretreatment for 30 min with actinomycin D (2 micrograms/ml) did not inhibit the early response, but significantly impaired the sustained response, suggesting that de novo protein synthesis was required. The simultaneous addition of the two hormones led within 60 min to a marked potentiation of the action on Na+ transport. This synergism could be mimicked by exogenous cyclic adenosine monophosphate (cAMP). Oxytocin alone (18 h exposure, 50 mU/ml) increased the relative rate of synthesis of both alpha and beta subunits of Na-K-ATPase (1.9- and 1.6-fold, respectively; P less than 0.05), whereas aldosterone (80 nM) increased the relative rate of synthesis of the same subunits (2.6- and 2.2-fold, respectively; P less than 0.02). Finally, in contrast to what was observed at the physiological level, the interaction of oxytocin and aldosterone did not lead to a similar potentiation at the biochemical level, i.e., induction of Na-K-ATPase biosynthesis (2.7- and 2.9-fold, for alpha and beta subunits, respectively; P less than 0.025).

U.S.-Norway Comparison of Interval Breast Cancers

Background: Publications from the International Breast Screening Network (IBSN) have shown that varying definitions create hurdles for comparison of screening performance. Interval breast cancer rates are particularly affected. Objective: to test whether variations in definition of interval cancer rates (ICR) affect comparisons of international ICR, specific to a comparison of ICR in Norway and North Carolina (NC). Methods: An interval cancer (IC) was defined as a cancer diagnosed following a negative screening mammogram in a defined follow-up period. ICR was calculated for women ages 50-69, at subsequent screening in Norway and NC, during the time period 1996 - 2002. ICR was defined using three different denominators (negative screens, negative final assessments and all screens) and three different numerators (DCIS, invasive cancer and all cancers). ICR was then calculated with two methods: 1) number of ICs divided by the number of screens, and ICs divided by the number of women-years at risk for IC. Results: There were no differences in ICR depending on the definition used. In the 1-12 month follow up period ICR (based on number of screens) were: 0.53, 0.54, and 0.54 for Norway; and 1.20, 1.25 and 1.17 for NC, for negative screens, negative final assessment and all screens, respectively: The same trend was seen for 13-24 and 1-24 months follow-up. Using women-years for the analysis did not change the trend. ICR was higher in NC compared to Norway under all definitions and in all follow-up time periods, regardless of calculation method. Conclusion: The ICR within or between Norway and NC did not differ by definition used. ICR were higher in NC than Norway. There are many potential explanations for the difference.

Clinical benefit and quality of life in patients with advanced pancreatic cancer receiving gemcitabine plus capecitabine versus gemcitabine alone: a randomized multicenter phase III clinical trial--SAKK 44/00-CECOG/PAN.1.3.001.

PURPOSE: To compare clinical benefit response (CBR) and quality of life (QOL) in patients receiving gemcitabine (Gem) plus capecitabine (Cap) versus single-agent Gem for advanced/metastatic pancreatic cancer. PATIENTS AND METHODS: Patients were randomly assigned to receive GemCap (oral Cap 650 mg/m(2) twice daily on days 1 through 14 plus Gem 1,000 mg/m(2) in a 30-minute infusion on days 1 and 8 every 3 weeks) or Gem (1,000 mg/m(2) in a 30-minute infusion weekly for 7 weeks, followed by a 1-week break, and then weekly for 3 weeks every 4 weeks) for 24 weeks or until progression. CBR criteria and QOL indicators were assessed over this period. CBR was defined as improvement from baseline for >or= 4 consecutive weeks in pain (pain intensity or analgesic consumption) and Karnofsky performance status, stability in one but improvement in the other, or stability in pain and performance status but improvement in weight. RESULTS: Of 319 patients, 19% treated with GemCap and 20% treated with Gem experienced a CBR, with a median duration of 9.5 and 6.5 weeks, respectively (P < .02); 54% of patients treated with GemCap and 60% treated with Gem had no CBR (remaining patients were not assessable). There was no treatment difference in QOL (n = 311). QOL indicators were improving under chemotherapy (P < .05). These changes differed by the time to failure, with a worsening 1 to 2 months before treatment failure (all P < .05). CONCLUSION: There is no indication of a difference in CBR or QOL between GemCap and Gem. Regardless of their initial condition, some patients experience an improvement in QOL on chemotherapy, followed by a worsening before treatment failure.

Contribution of (sub)domains of Staphylococcus aureus fibronectin-binding protein to the proinflammatory and procoagulant response of human vascular endothelial cells.

The Staphylococcus aureus fibronectin (Fn) -binding protein A (FnBPA) is involved in bacterium-endothelium interactions which is one of the crucial events leading to infective endocarditis (IE). We previously showed that the sole expression of S. aureus FnBPA was sufficient to confer to non-invasive Lactococcus lactis bacteria the capacity to invade human endothelial cells (ECs) and to launch the typical endothelial proinflammatory and procoagulant responses that characterize IE. In the present study we further questioned whether these bacterium-EC interactions could be reproduced by single or combined FnBPA sub-domains (A, B, C or D) using a large library of truncated FnBPA constructs expressed in L. lactis. Significant invasion of cultured ECs was found for L. lactis expressing the FnBPA subdomains CD (aa 604-877) or A4(+16) (aa 432-559). Moreover, this correlates with the capacity of these fragments to elicit in vitro a marked increase in EC surface expression of both ICAM-1 and VCAM-1 and secretion of the CXCL8 chemokine and finally to induce a tissue factor-dependent endothelial coagulation response. We thus conclude that (sub)domains of the staphylococcal FnBPA molecule that express Fn-binding modules, alone or in combination, are sufficient to evoke an endothelial proinflammatory as well as a procoagulant response and thus account for IE severity.

ENaC-mediated alveolar fluid clearance and lung fluid balance depend on the channel-activating protease 1.

Sodium transport via epithelial sodium channels (ENaC) expressed in alveolar epithelial cells (AEC) provides the driving force for removal of fluid from the alveolar space. The membrane-bound channel-activating protease 1 (CAP1/Prss8) activates ENaC in vitro in various expression systems. To study the role of CAP1/Prss8 in alveolar sodium transport and lung fluid balance in vivo, we generated mice lacking CAP1/Prss8 in the alveolar epithelium using conditional Cre-loxP-mediated recombination. Deficiency of CAP1/Prss8 in AEC induced in vitro a 40% decrease in ENaC-mediated sodium currents. Sodium-driven alveolar fluid clearance (AFC) was reduced in CAP1/Prss8-deficient mice, due to a 48% decrease in amiloride-sensitive clearance, and was less sensitive to beta(2)-agonist treatment. Intra-alveolar treatment with neutrophil elastase, a soluble serine protease activating ENaC at the cell surface, fully restored basal AFC and the stimulation by beta(2)-agonists. Finally, acute volume-overload increased alveolar lining fluid volume in CAP1/Prss8-deficient mice. This study reveals that CAP1 plays a crucial role in the regulation of ENaC-mediated alveolar sodium and water transport and in mouse lung fluid balance.

Stability and robustness of blood variables in an antidoping context.

Introduction:  With the setting up of the newly Athlete's Biological Passport antidoping programme, novel guidelines have been introduced to guarantee results beyond reproach. We investigated in this context, the effect of storage time on the variables commonly measured for the haematological passport. We also wanted to assess for these variables, the within and between analyzer variations. Methods:  Blood samples were obtained from top level male professional cyclists (27 samples for the first part of the study and 102 for the second part) taking part to major stage races. After collection, they were transported under refrigerated conditions (2 °C < T < 12 °C), delivered to the antidoping laboratory, analysed and then stored at approximately 4 °C to conduct analysis at different time points up to 72 h after delivery. A mixed-model procedure was used to determine the stability of the different variables. Results:  As expected haemoglobin concentration was not affected by storage and showed stability for at least 72 h. Under the conditions of our investigation, the reticulocytes percentage showed a much better stability than previous published data (> 48 h) and the technical comparison of the haematology analyzer demonstrated excellent results. Conclusion:  In conclusion, our data clearly demonstrate that as long as the World Anti-Doping Agency's guidelines are followed rigorously, all blood results reach the quality level required in the antidoping context.

Necrotizing fasciitis: contribution and limitations of diagnostic imaging.

Necrotizing fasciitis is a rare, rapidly spreading, deep-seated infection causing thrombosis of the blood vessels located in the fascia. Necrotizing fasciitis is a surgical emergency. The diagnosis typically relies on clinical findings of severe sepsis and intense pain, although subacute forms may be difficult to recognize. Imaging studies can help to differentiate necrotizing fasciitis from infections located more superficially (dermohypodermitis). The presence of gas within the necrotized fasciae is characteristic but may be lacking. The main finding is thickening of the deep fasciae due to fluid accumulation and reactive hyperemia, which can be visualized using computed tomography and, above all, magnetic resonance imaging (high signal on contrast-enhanced T1 images and T2 images, best seen with fat saturation). These findings lack specificity, as they can be seen in non-necrotizing fasciitis and even in non-inflammatory conditions. Signs that support a diagnosis of necrotizing fasciitis include extensive involvement of the deep intermuscular fascias (high sensitivity but low specificity), thickening to more than 3mm, and partial or complete absence on post-gadolinium images of signal enhancement of the thickened fasciae (fairly high sensitivity and specificity). Ultrasonography is not recommended in adults, as the infiltration of the hypodermis blocks ultrasound transmission. Thus, imaging studies in patients with necrotizing fasciitis may be challenging to interpret. Although imaging may help to confirm deep tissue involvement and to evaluate lesion spread, it should never delay emergency surgical treatment in patients with established necrotizing fasciitis.