bluuebunny/crossref_metadata_2025 huggingface dataset:
HER2 evaluation using the novel rabbit monoclonal antibody SP3 and CISH in tissue microarrays of invasive breast carcinomas
Authors: Ricardo Sara Alexandra Vinhas , Milanezi Fernanda , Carvalho Sílvia Teresa , Leitão Dina Raquel Aguilera , Schmitt Fernando Carlos Lander
Published: December 2006

Background:Laboratory methods for HER2 assessment currently include immunohistochemical (IHC) methods (measuring protein overexpression) and fluorescence in situ hybridisation (FISH) (measuring gene amplification). The measure of HER2 protein by IHC is usually assessed by the mouse monoclonal antibody CB11, and polyclonal antibodies (Herceptest) directed against the internal portion of the receptor. Recently, chromogenic in situ hybridisation (CISH), in which HER2 is detected by a peroxidase reaction and the gene amplification can be determined by regular bright-field microscopy, has emerged as an alternative to FISH.

Aims:To evaluate the status of HER2 in tissue microarrays (TMAs) of invasive breast cancer using the novel rabbit monoclonal antibody SP3 directed against the external portion of HER2, and correlate the results with CB11 and CISH.

Methods:IHC was performed with two antibodies (CB11 and SP3) and CISH for HER2 in 10 TMA blocks with 190 formalin-fixed paraffin-embedded cases of invasive breast carcinomas.

Results:The correlation between SP3 and CB11 was significant (p<0.001) with an agreement rate of 86.9%. When the staining pattern of the two antibodies was compared, the majority of SP3 immunostainings were assessed more easily, with a strong complete membrane staining pattern without non-specific cytoplasmic staining. There was a good correlation between SP3 and CISH (p<0.001). 23/24 SP3 3+ cases showed gene amplification, 97.3% of the cases without gene amplification were SP3 negative, and 6/7 SP3 2+ were amplified.

Conclusion:The high level of agreement between SP3, a monoclonal antibody that recognises the extracellular domain of the HER2 receptor, and CB11 and CISH, shows that this novel antibody is a reliable candidate to evaluate the expression of HER2 in breast cancer.
Expression of EphA2 and EphrinA-1 in vulvar carcinomas and its relation to prognosis
Authors: Holm R , Knopp S , Suo Z , Tropè C , Nesland J M
Published: December 2006

Aims:

To examine the expression of EphA2 and EphrinA-1 in vulvar squamous cell carcinomas and investigate their prognostic relevance.

Methods:

Tumours from 224 patients with vulvar squamous cell carcinomas were investigated for expression of EphA2 and EphrinA-1 using single and double immunostaining methods.

Results:

High expression (strong/moderate staining intensity) of EphA2 and EphrinA-1 was observed in 114 (51%) and 126 (56%) vulvar carcinomas, respectively. In the three cases tested using the double immunostaining method, colocalisation of EphA2 and EphrinA-1 proteins was identified in the same neoplastic cells. High EphA2 expression was significantly correlated to high expression of EphrinA-1 (p<0.01) and cyclin A (p<0.01), large tumour size (p = 0.03), deep invasion (p<0.01) and higher FIGO stage (p = 0.05). A correlation between high EphrinA-1 expression and high levels of cyclin A (p<0.01) and p21 (p<0.01), deep invasion (p<0.01) and higher FIGO stage (p = 0.01) was also seen. In univariate analysis, high expression of EphrinA-1 was associated with poor survival (p = 0.03). However, in the multivariate analysis neither EphrinA-1 nor EphA2 were significantly correlated to survival.

Conclusions:

EphA2 and EphrinA-1 were overexpressed in 51% and 56% of the vulvar squamous cell carcinomas, respectively, and high levels of EphA2 and EphrinA-1 proteins were associated with deep tumour invasion and high FIGO stage. However, EphA2 and EphrinA-1 were not independently associated with clinical outcome in vulvar carcinomas.
Urine cytology screening for polyoma virus infection following renal transplantation: the Oxford experience
Authors: Thamboo Thomas P , Jeffery Katie J M , Friend Peter J , Turner Gareth D H , Roberts Ian S D
Published: December 2006

Objective:To review the first year of a monthly urine cytology screening service, introduced to identify renal transplant patients at risk of polyoma virus nephropathy (PVN), at an early, potentially treatable, stage.

Methods and results:Monthly urine samples (n = 392) were received from 97/108 transplant recipients in 2005. Of 56 patients with follow-up >6 months, 20% and 9% had significant (>10 decoy cells/cytospin) and non-significant positive cytology, respectively. The first positive urine samples occurred most commonly in the second and third month post-transplantation and patients with significantly positive samples had higher 3-month and 6-month serum creatinine levels than patients with negative urine cytology (p<0.01). Four patients with positive urine cytology had a subsequent positive plasma BK virus PCR; 3/97 patients had biopsy-proven PVN, all in the third month, 1–6 weeks after first positive urine samples.

Conclusions:Significant PV viruria is common following renal transplantation with onset usually within the first 3 months. Viruria is associated with worse graft function at 3 and 6 months. The time between urine positivity and clinical PVN is short. More frequent early urine screening would be required to achieve clinical benefit.
Salmonella typhimuriummeningitis in an adult patient with AIDS
Authors: Swe K Swe , Nagel G , Van der Westhuizen M , Hoosen A A
Published: December 2006

Salmonella meningitis is an unusual complication of Salmonella sepsis and occurs mainly in children. A rare case ofSalmonella typhimuriummeningitis occurring in an adult HIV positive man who presented with a history of fever and diarrhoea is reported. On examination he was dehydrated, and had oral thrush, weakness of lower limbs and neck stiffness. A septic diagnostic screen was performed and he was commenced on empiric intravenous cefotaxime therapy for meningitis.S typhimuriumwas cultured from cerebrospinal fluid and blood culture specimens. It was non-lactose fermenting, oxidase negative, H2S positive and motile. Cefotaxime was continued for 14 days and the patient responded without neurological sequelae.
MYCamplification in breast cancer: a chromogenic in situ hybridisation study
Authors: Rodriguez-Pinilla S Maria , Jones Robin L , Lambros Maryou B K , Arriola Edurne , Savage Kay , James Michelle , Pinder Sarah E , Reis-Filho Jorge S
Published: December 2006

Aims:To analyse the correlation betweenMYCamplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based chemotherapy. Given the high prevalence ofMYCamplification in tumours ofBRCA1mutation carriers and the similarities between these and sporadic “basal-like” carcinomas, the prevalence ofMYCamplification in “basal-like” breast carcinomas was investigated.

Methods:MYCgene copy number was assessed on tissue microarrays containing duplicate cores of 245 invasive breast carcinomas by means of chromogenic in situ hybridisation using SpotLight C-MYC amplification probe and chromosome 8 centromeric probe (CEP8). Signals were evaluated at 400× magnification; 30 morphologically unequivocal neoplastic cells in each core were counted for the presence of the gene and CEP8 probes.

Results:Amplification was defined as aMYC:CEP8 ratio >2. Signals for bothMYCand CEP8 were assessable in 196/245 (80%) tumours.MYCamplification was found in 19/196 cases (9.7%) and was not associated with tumour size, histological grade, positivity for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor, cytokeratins 14, 5/6 and 17, MIB1 or p53. Only 4% of basal-like carcinomas showedMYCamplification, compared to 8.75% and 10.7% of luminal and HER2 tumours respectively. On univariate analysis,MYCamplification displayed a significant association with shorter metastasis-free and overall survival and proved to be an independent prognostic factor on multivariate survival analysis.

Conclusion:MYCamplification is not associated with “basal-like” phenotype and proved to be an independent prognostic factor for breast cancer patients treated with anthracycline-based chemotherapy.
Endothelin-1 expression correlates with atypical histological features in mammary phyllodes tumours
Authors: Tse Gary M , Chaiwun Benjaporn , Lau Kin-Mang , Scolyer Richard , Lee C Soon , Karim Rooshdiya Z , Putti Thomas C , Law Bonita K , Lui Philip C , Tan Puay Hoon
Published: December 2006

Background and Aims:Endothelin-1 expression is increased in infiltrating duct carcinoma and is associated with larger tumour size, higher histological grade and lymphovascular permeation. This has not been evaluated in phyllodes tumours, which are uncommon fibroepithelial lesions with potential for local recurrences or distant metastasis. While the grading of phyllodes tumours depends on a combination of histological parameters, prediction of their behaviour remains difficult.

Method:A large series of 461 phyllodes tumours (291 benign, 115 borderline malignant and 55 frankly malignant) were evaluated for endothelin-1 expression in both the epithelial cells and stromal cells by immunohistochemistry; results were correlated with the tumour grade.

Results:For benign phyllodes tumours, the epithelial staining of endothelin was negative, weak, moderate and strong in 6%, 26%, 15% and 53% of cases respectively; results were 4%, 18%, 19% and 59% respectively for borderline and 6%, 18%, 6% and 70% respectively for frankly malignant tumours. For the stromal staining, the negative, weak, moderate and strong staining was 32%, 19%, 18% and 31% respectively for benign phyllodes, 24%, 13%, 10% and 53% respectively for borderline and 8%, 16%, 17% and 59% respectively for frankly malignant tumours. There was correlation between epithelial and stromal staining, and the stromal staining correlated with histological features of stromal cellularity, stromal cell nuclear pleomorphism, margin status and stromal overgrowth.

Conclusion:These observations suggest a close relationship between the epithelial and stromal elements in phyllodes tumours; endothelin may play a significant role in the malignant progression of phyllodes tumours.
INFORMATION GEOMETRY AND PHASE TRANSITIONS
Authors: PORTESI MARIELA , PLASTINO ANGEL L. , PENNINI FLAVIA
Published: December 2006

We present, from an information theoretic viewpoint, an analysis of phase transitions and critical phenomena in quantum systems. Our study is based on geometrical considerations within the Riemannian space of thermodynamic parameters that characterize the system. A metric for the space can be derived from an appropriate definition of distance between quantum states. For this purpose, we consider generalized α-divergences that include the standard Kullback–Leibler relative entropy. The use of other measures of information distance is taken into account, and the thermodynamic stability of the system is discussed from this geometric perspective.
THE DESIGN AND TESTING OF A FIRST-ORDER LOGIC-BASED STOCHASTIC MODELING LANGUAGE
Authors: PLESS DANIEL J. , CHAKRABARTI CHAYAN , RAMMOHAN ROSHAN , LUGER GEORGE F.
Published: December 2006

We have created a logic-based, Turing-complete language for stochastic modeling. Since the inference scheme for this language is based on a variant of Pearl's loopy belief propagation algorithm, we call it Loopy Logic. Traditional Bayesian networks have limited expressive power, basically constrained to finite domains as in the propositional calculus. Our language contains variables that can capture general classes of situations, events and relationships. A first-order language is also able to reason about potentially infinite classes and situations using constructs such as hidden Markov models(HMMs). Our language uses an Expectation-Maximization (EM) type learning of parameters. This has a natural fit with the Loopy Belief Propagation used for inference since both can be viewed as iterative message passing algorithms. We present the syntax and theoretical foundations for our Loopy Logic language. We then demonstrate three examples of stochastic modeling and diagnosis that explore the representational power of the language. A mechanical fault detection example displays how Loopy Logic can model time-series processes using an HMM variant. A digital circuit example exhibits the probabilistic modeling capabilities, and finally, a parameter fitting example demonstrates the power for learning unknown stochastic values.
SEMANTIC DERIVATION VERIFICATION: TECHNIQUES AND IMPLEMENTATION
Authors: SUTCLIFFE GEOFF
Published: December 2006

Automated Theorem Proving (ATP) systems are complex pieces of software, and thus may have bugs that make them unsound. In order to guard against unsoundness, the derivations output by an ATP system may be semantically verified by trusted ATP systems that check the required semantic properties of each inference step. Such verification needs to be augmented by structural verification that checks that inferences have been used correctly in the context of the overall derivation. This paper describes techniques for semantic verification of derivations, and reports on their implementation and testing in the GDV verifier.
THE ENDOTHELIUM AS A TARGET FOR INFECTIONS
Authors: Valbuena Gustavo , Walker David H.
Published: December 2006

The endothelial cells lining vascular and lymphatic vessels are targets of several infectious agents, including viruses and bacteria, that lead to dramatic changes in their functions. Understanding the pathophysiological mechanisms that cause the clinical manifestations of those infections has been advanced through the use of animal models and in vitro systems; however, there are also abundant studies that explore the consequences of endothelial infection in vitro without supporting evidence that endothelial cells are actual in vivo targets of infection in human diseases. This article defines criteria for considering an infection as truly endothelium-targeted and reviews the literature that offers insights into the pathogenesis of human endothelial-target infections.
INFLAMMATION AND ATHEROSCLEROSIS
Authors: Hansson Göran K. , Robertson Anna-Karin L. , Söderberg-Nauclér Cecilia
Published: December 2006

Atherosclerosis, the cause of myocardial infarction, stroke, and ischemic gangrene, is an inflammatory disease. The atherosclerotic process is initiated when cholesterol-containing low-density lipoproteins accumulate in the intima and activate the endothelium. Leukocyte adhesion molecules and chemokines promote recruitment of monocytes and T cells. Monocytes differentiate into macrophages and upregulate pattern recognition receptors, including scavenger receptors and toll-like receptors. Scavenger receptors mediate lipoprotein internalization, which leads to foam-cell formation. Toll-like receptors transmit activating signals that lead to the release of cytokines, proteases, and vasoactive molecules. T cells in lesions recognize local antigens and mount T helper-1 responses with secretion of pro-inflammatory cytokines that contribute to local inflammation and growth of the plaque. Intensified inflammatory activation may lead to local proteolysis, plaque rupture, and thrombus formation, which causes ischemia and infarction. Inflammatory markers are already used to monitor the disease process and anti-inflammatory therapy may be useful to control disease activity.
Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro
Authors: Amemori Sadahiro , Ootani Akifumi , Aoki Shigehisa , Fujise Takehiro , Shimoda Ryo , Kakimoto Takashi , Shiraishi Ryosuke , Sakata Yasuhisa , Tsunada Seiji , Iwakiri Ryuichi , Fujimoto Kazuma
Published: December 2006

Obesity, a risk factor for colon cancer, is associated with elevated serum levels of leptin, a protein produced by adipocytes. The aim of the present study was to clarify the effects of adipose tissue on colon cancer proliferation by using cultured cell lines. To achieve this, colon cancer cells (CACO-2, T84, and HT29) were cocultured with adipose tissue, isolated mature adipocytes, and isolated preadipocytes in a three-dimensional collagen gel culture system. The adipocytes and preadipocytes used were isolated from C57BL/6J and leptin-deficient ob/ob mice. Proliferation of the cancer cells was evaluated by nuclear bromodeoxyuridine uptake. The adipose tissue, mature adipocytes, and preadipocytes isolated from C57BL/6J mice significantly increased the proliferation of the colon cancer cells. This trophic effect of mature adipocytes on the cancer cell lines was observed only for cells from lean littermates and not for those from ob/ob mice. In contrast, the trophic effect of preadipocytes was not abolished in ob/ob mice, and this finding was supported by the result that leptin had a trophic effect on cancer cells. In conclusion, adipocytes were able to enhance the proliferation of colon cancer cells in vitro, partly via leptin, suggesting that adipose tissues, including mature adipocytes and preadipocytes, may promote the growth of colorectal cancer.
Lowering of interstitial fluid pressure after neurogenic inflammation in mouse skin is partly dependent on mast cells
Authors: Karlsen Tine V. , Bletsa Athanasia , Gjerde Eli-Anne B. , Reed Rolf K.
Published: December 2006

Neurogenic inflammation is known to induce lowering of interstitial fluid pressure (Pif) in mouse skin. This study examined the possible role of mast cell activation secondary to neuropeptide release in lowering of Pifby using KitW/ KitW-vmice, which are devoid of mast cells, including connective tissue mast cells (CTMCs). Pifwas measured in paw skin of anesthetized (fentanyl-fluanison and midazolam, 1:1) mice with glass capillaries connected to a servo-controlled counterpressure system. In contrast to wild-type mice, intravenous administration of mast cell-activating compound 48/80 induced no lowering of Pifin KitW/ KitW-vmice. Intravenous challenge with substance P (SP), calcitonin gene-related peptide (CGRP), or capsaicin induced a significant ( P < 0.05) lowering of Pifin wild-type mice to −2.16 ± 0.28, −1.96 ± 0.11, and −2.22 ± 0.19 mmHg, respectively, compared with vehicle (−0.49 ± 0.11 mmHg). In KitW/ KitW-vmice the Pifresponse to SP was completely abolished (−0.53 ± 0.32 mmHg) while the response to CGRP and capsaicin was attenuated (−1.33 ± 0.13 and −1.42 ± 0.13 mmHg, respectively) although significantly ( P < 0.05) lowered compared with vehicle. Immunohistochemical analysis revealed no difference in distribution or density of SP- and CGRP-immunoreactive fibers in paws of KitW/ KitW-vcompared with wild-type mice. We conclude that lowering of Pifnormally depends on mast cells. However, the sensory nerves can also elicit a lowering of Pifthat is independent of mast cells.
Chronic verapamil treatment remodelsICa,Lin mouse ventricle
Authors: Schroder Elizabeth , Magyar Janos , Burgess Don , Andres Douglas , Satin Jonathan
Published: December 2006

In this study we tested the hypothesis that ventricular homeostasis of L-type Ca2+current ( ICa,L) minimally involves regulation of the main pore-forming α-subunit (CaV1.2) and auxiliary proteins that serve as positive or negative regulators of ICa,L. We treated animals for 24 h with verapamil (Ver, 3.6 mg·kg−1·day−1), isoproterenol (Iso, 30 mg·kg−1·day−1), or Iso + Ver via osmotic minipumps. To test for alterations of Ca2+channel complex components we performed real-time PCR and Western blot analysis on ventricle. In addition, cardiac myocytes (CMs) were dispersed and current was recorded in the whole cell configuration to evaluate ICa,L. Surprisingly, 24- to 48-h Ver increased CaV1.2 mRNA and protein and ICa,Lcurrent (Ver 11 ± 1pA/pF vs. control 7 ± 0.5pA/pF; P < 0.01). ICa,Lfrom CMs in Ver mice showed no change in whole cell capacitance. To examine the in vivo effects of a physiologically relevant Ca2+channel agonist, we treated mice with Iso. Twenty-four-hour Iso infusion increased heart rate; CaV1.2- and CaVβ2mRNA levels were constant, but the Ca2+channel subunit mRNA Rem was increased twofold. Cells isolated from 24-h Iso hearts showed no change in basal ICa,Ldensity and diminished responsiveness to acute 1 μM Iso. To further examine the homeostatic regulation of the Ca2+channel, we treated animals for 24 h with Iso + Ver. The influence of Iso + Ver was similar that of to Iso alone on Ca2+channel mRNAs and ICa,L, with the exception that it prevented the increase in Rem seen with Iso treatment. Long-term Ca2+channel blockade induces an increase of CaV1.2 mRNA and protein and significantly increases ICa,L.
Atorvastatin inhibits angiotensin-converting enzyme induction in differentiating human macrophages
Authors: Saijonmaa Outi , Nyman Tuulikki , Fyhrquist Frej
Published: December 2006

Statins are effective drugs in the prevention of cardiovascular disease. Recent studies suggested that statins have additional beneficial effects on the vascular wall independent of their cholesterol-lowering effects. We investigated whether atorvastatin influences angiotensin-converting enzyme (ACE) production in differentiating human macrophages. Human peripheral blood monocytes (PBM) were isolated from fresh buffy coats. The cells were allowed to differentiate for 0–8 days in macrophage serum-free medium with 5 ng/ml granulocyte-macrophage colony-stimulating factor. Atorvastatin (0.005–0.5 μM), mevalonate (200–400 μM), geranylgeranyl pyrophosphate (1.25–2.5 μM), and/or farnesylpyrophosphate (FPP; 1.25–2.5 μM) was added on the second day of differentiation and then every other day. After incubation time, the ACE amount in intact macrophages was measured. ACE amount in PBM was low. A marked time-dependent ACE induction was noticed during differentiation of monocytes to macrophages. Atorvastatin treatment inhibited ACE induction during differentiation. In the presence of mevalonate, atorvastatin failed to downregulate ACE production. Cotreatment of the cells with atorvastatin and FPP reversed the suppressive effect of atorvastatin on ACE. In conclusion, atorvastatin inhibited ACE upregulation, normally occurring in differentiating human macrophages. This effect was mediated via the mevalonate pathway, and inhibition of FPP was probably involved. The finding that atorvastatin inhibited ACE upregulation may represent a novel pleiotropic action and an additional beneficial effect of statins in treatment of cardiovascular disease.
SP-D-deficient mice are resistant to hyperoxia
Authors: Jain Deepika , Atochina-Vasserman Elena , Kadire Helchem , Tomer Yaniv , Inch Adam , Scott Pamela , Savani Rashmin C. , Gow Andrew J. , Beers Michael F.
Published: December 2006

Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D−/−) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D−/−mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D−/−mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 ± 1.4 × 105in SP-D−/−vs. 4.04 ± 0.25 × 105in SP-D+/+mice) and neutrophils (1.2 ± 0.4 × 105vs. 0.03 ± 0.02 × 105in SP-D−/−and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D−/−mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D−/−mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D−/−phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.
Nerve growth factor acutely potentiates synaptic transmission in vitro and induces dendritic growth in vivo on adult neurons in airway parasympathetic ganglia
Authors: Hazari Mehdi S. , Pan Jenny H. , Myers Allen C.
Published: December 2006

Elevated levels of nerve growth factor (NGF) and NGF-mediated neural plasticity may have a role in airway diseases such as asthma and chronic obstructive pulmonary disease (COPD). Although NGF is known to affect sensory and sympathetic nerves, especially during development, little is known regarding its effect on parasympathetic nerves, especially on adult neurons. The purpose of this study was to analyze the acute and chronic effects of NGF on the electrophysiological and anatomical properties of neurons in airway parasympathetic ganglia from adult guinea pigs. Using single cell recording, direct application of NGF caused a lasting decrease in the cumulative action potential afterhyperpolarization (AHP) and increased the amplitude of vagus nerve-stimulated nicotinic fast excitatory postsynaptic potentials. Neuronal responsiveness to nicotinic receptor stimulation was increased by NGF, which was blocked by the tyrosine kinase inhibitor, K-252a, implicating neurotrophin-specific (Trk) receptors. Neurotrophin-3 and brain-derived neurotrophic factor had no effect on the synaptic potentials, AHP, or nicotinic response; inhibition of cyclooxygenase with indomethacin inhibited the effect of NGF on the cumulative AHP. Forty-eight hours after in vivo application of NGF to the trachealis muscle caused an increase in dendritic length on innervating neurons. These results are the first to demonstrate that NGF increases the excitability of lower airway parasympathetic neurons, primarily through enhanced synaptic efficacy and changes to intrinsic neuron properties. NGF also had dramatic effects on the growth of dendrites in vivo. Such effects may indicate a new role for NGF in the regulation of parasympathetic tone in the diseased or inflamed lower airways.
Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells
Authors: Resnik Ernesto R. , Keck Maggie , Sukovich David J. , Herron Jean M. , Cornfield David N.
Published: December 2006

Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O2) tension with a decrease in cytosolic calcium ([Ca2+]i), an acute increase in O2 tension has no net effect on [Ca2+]i in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O2 tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (≥4th generation) and maintained under hypoxic conditions (∼25 Torr) in primary culture. After fura 2 loading, apparent [Ca2+]i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca2+]i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca2+ and intracellular store depletion with CPA, followed by superfusion with Ca2+-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.
Polar head groups are important for barrier-protective effects of oxidized phospholipids on pulmonary endothelium
Authors: Birukova Anna A. , Fu Panfeng , Chatchavalvanich Santipongse , Burdette Dylan , Oskolkova Olga , Bochkov Valery N. , Birukov Konstantin G.
Published: December 2006

We have previously described protective effects of oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphocholine (OxPAPC) on pulmonary endothelial cell (EC) barrier function and demonstrated the critical role of cyclopentenone-containing modifications of arachidonoyl moiety in OxPAPC protective effects. In this study we used oxidized phosphocholine (OxPAPC), phosphoserine (OxPAPS), and glycerophosphate (OxPAPA) to investigate the role of polar head groups in EC barrier-protective responses to oxidized phospholipids (OxPLs). OxPAPC and OxPAPS induced sustained barrier enhancement in pulmonary EC, whereas OxPAPA caused a transient protective response as judged by measurements of transendothelial electrical resistance (TER). Non-OxPLs showed no effects on TER levels. All three OxPLs caused enhancement of peripheral EC actin cytoskeleton. OxPAPC and OxPAPS completely abolished LPS-induced EC hyperpermeability in vitro, whereas OxPAPA showed only a partial protective effect. In vivo, intravenous injection of OxPAPS or OxPAPC (1.5 mg/kg) markedly attenuated increases in the protein content, cell counts, and myeloperoxidase activities detected in bronchoalveolar lavage fluid upon intratracheal LPS instillation in mice, although OxPAPC showed less potency. All three OxPLs partially attenuated EC barrier dysfunction induced by IL-6 and thrombin. Their protective effects against thrombin-induced EC barrier dysfunction were linked to the attenuation of the thrombin-induced Rho pathway of EC hyperpermeability and stimulation of Rac-mediated mechanisms of EC barrier recovery. These results demonstrate for the first time the essential role of polar OxPL groups in blunting the LPS-induced EC dysfunction in vitro and in vivo and suggest the mechanism of agonist-induced hyperpermeability attenuation by OxPLs via reduction of Rho and stimulation of Rac signaling.
Glucose, insulin, and leptin signaling pathways modulate nitric oxide synthesis in glucose-inhibited neurons in the ventromedial hypothalamus
Authors: Canabal Debra D. , Song Zhentao , Potian Joseph G. , Beuve Annie , McArdle Joseph J. , Routh Vanessa H.
Published: December 2006

Glucose-sensing neurons in the ventromedial hypothalamus (VMH) are involved in the regulation of glucose homeostasis. Glucose-sensing neurons alter their action potential frequency in response to physiological changes in extracellular glucose, insulin, and leptin. Glucose-excited neurons decrease, whereas glucose-inhibited (GI) neurons increase, their action potential frequency when extracellular glucose is reduced. Central nitric oxide (NO) synthesis is regulated by changes in local fuel availability, as well as insulin and leptin. NO is involved in the regulation of food intake and is altered in obesity and diabetes. Thus this study tests the hypothesis that NO synthesis is a site of convergence for glucose, leptin, and insulin signaling in VMH glucose-sensing neurons. With the use of the NO-sensitive dye 4-amino-5-methylamino-2′,7′-difluorofluorescein in conjunction with the membrane potential-sensitive dye fluorometric imaging plate reader, we found that glucose and leptin suppress, whereas insulin stimulates neuronal nitric oxide synthase (nNOS)-dependent NO production in cultured VMH GI neurons. The effects of glucose and leptin were mediated by suppression of AMP-activated protein kinase (AMPK). The AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) increased both NO production and neuronal activity in GI neurons. In contrast, the effects of insulin on NO production were blocked by the phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Furthermore, decreased glucose, insulin, and AICAR increase the phosphorylation of VMH nNOS, whereas leptin decreases it. Finally, VMH neurons express soluble guanylyl cyclase, a downstream mediator of NO signaling. Thus NO may mediate, in part, glucose, leptin, and insulin signaling in VMH glucose-sensing neurons.
Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging
Authors: O'Riordan Tomás C. , Fitzgerald Kathleen , Ponomarev Gelii V. , Mackrill John , Hynes James , Taylor Cormac , Papkovsky Dmitri B.
Published: December 2006

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca2+ influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.
Involvement of adipose tissues in the early hypolipidemic action of PPARγ agonism in the rat
Authors: Laplante Mathieu , Festuccia William T. , Soucy Geneviève , Gélinas Yves , Lalonde Josée , Deshaies Yves
Published: December 2006

Agonists of the peroxisome proliferator-activated receptor γ (PPARγ) are insulin sensitizers that potently improve lipemia in rodents. This study aimed to determine the contribution of lipid secretion vs. clearance and the involvement of white adipose tissue (WAT) and brown adipose tissue (BAT) in the rapid hypolipidemic action of PPARγ agonism. Male rats were treated with rosiglitazone (RSG; 15 mg·kg−1·day−1) for 1 to 4 days, and determinants of lipid metabolism were assessed postprandially. Serum triglycerides (TG) were lowered (−54%) after 3 days of RSG treatment, due to accelerated clearance from blood without contribution of changes in secretion rates. Both BAT and WAT were the major sites of RSG action on TG clearance, the increase in TG-derived fatty acid (FA) uptake reaching threefold in BAT and 60–90% in WAT depots. Accelerated TG clearance was associated with increased lipoprotein lipase (LPL) activity mostly in BAT. Serum nonesterified FA were lowered (−20%) by a single dose of RSG, an effect associated with increased expression levels of FA binding/transport (fatty acid binding protein-4), esterification (diacylglycerol acyltransferase-1), and recycling glycerol kinase and phosphoenolpyruvate carboxykinase enzymes in BAT and WAT, suggesting FA trapping. After 4 days of RSG treatment, nonesterified fatty acid (NEFA) uptake was also stimulated in both BAT (2.5-fold) and WAT (40%). These findings demonstrate the causal involvement of increased efficiency of LPL-mediated TG clearance and reveal the important contribution of TG-derived and albumin-bound FA uptake by BAT in the rapid hypolipidemic action of PPARγ agonism in the rat.
Postnatal maturation of laryngeal chemoreflexes in the preterm lamb
Authors: St-Hilaire Marie , Samson Nathalie , Nsegbe Elise , Duvareille Charles , Moreau-Bussière François , Micheau Philippe , Lebon Johann , Praud Jean-Paul
Published: December 2006

Laryngeal chemoreflexes (LCR) are triggered by the contact of liquids with the laryngeal mucosa. In the mature organism, LCR trigger lower airway protective responses (coughing, effective swallowing, and arousal) to prevent aspiration. General belief holds that LCR are responsible for apnea and bradycardia in the newborn mammal, including humans. Our laboratory has recently shown that LCR in full-term lambs are consistently analogous to the mature LCR reported in adult mammals, without significant apneas and bradycardias (St-Hilaire M, Nsegbe E, Gagnon-Gervais K, Samson N, Moreau-Bussiere F, Fortier PH, and Praud J-P. J Appl Physiol 98: 2197–2203, 2005). The aim of the present study was to assess LCR in nonsedated, newborn preterm lambs born at 132 days of gestation (term = 147 days). The preterm lambs were instrumented for recording glottal adductor electromyogram, electroencephalogram, eye movements, heart rate, respiration, and oximetry. A chronic supraglottal catheter was used for injecting 0.5 ml of saline, distilled water, and HCl (pH 2) during quiet sleep, active sleep, and wakefulness on postnatal days 7 (D7) and 14 (D14). Laryngeal stimulation by water or HCl on D7 induced significant apneas, bradycardia, and desaturation, which, at times, appeared potentially life-threatening. No significant apneas, bradycardias, or desaturation were observed on D14. No consistent effects of sleep state could be shown in the present study. In conclusion, laryngeal stimulation by liquids triggers potentially dangerous LCR in preterm lambs on D7, but not on D14. It is proposed that maturation of the LCR between D7 and D14 is partly involved in the disappearance of apneas/bradycardias of prematurity with postnatal age.
The effects of training in hyperoxia vs. normoxia on skeletal muscle enzyme activities and exercise performance
Authors: Perry Christopher G. R. , Talanian Jason L. , Heigenhauser George J. F. , Spriet Lawrence L.
Published: December 2006

Inspiring a hyperoxic (H) gas permits subjects to exercise at higher power outputs while training, but there is controversy as to whether this improves skeletal muscle oxidative capacity, maximal O2 consumption (V̇o2 max), and endurance performance to a greater extent than training in normoxia (N). To determine whether the higher power output during H training leads to a greater increase in these parameters, nine recreationally active subjects were randomly assigned in a single-blind fashion to train in H (60% O2) or N for 6 wk (3 sessions/wk of 10 × 4 min at 90% V̇o2 max). Training heart rate (HR) was maintained during the study by increasing power output. After at least 6 wk of detraining, a second 6-wk training protocol was completed with the other breathing condition. V̇o2 max and cycle time to exhaustion at 90% of pretraining V̇o2 max were tested in room air pre- and posttraining. Muscle biopsies were sampled pre- and posttraining for citrate synthase (CS), β-hydroxyacyl-coenzyme A dehydrogenase (β-HAD), and mitochondrial aspartate aminotransferase (m-AsAT) activity measurements. Training power outputs were 8% higher (17 W) in H vs. N. However, both conditions produced similar improvements in V̇o2 max (11–12%); time to exhaustion (∼100%); and CS (H, 30%; N, 32%), β-HAD (H, 23%; N, 21%), and m-AsAT (H, 21%; N, 26%) activities. We conclude that the additional training stimulus provided by training in H was not sufficient to produce greater increases in the aerobic capacity of skeletal muscle and whole body V̇o2 max and exercise performance compared with training in N.
Bisphenol A facilitates bypass of androgen ablation therapy in prostate cancer
Authors: Wetherill Yelena B. , Hess-Wilson Janet K. , Comstock Clay E.S. , Shah Supriya A. , Buncher C. Ralph , Sallans Larry , Limbach Patrick A. , Schwemberger Sandy , Babcock George F. , Knudsen Karen E.
Published: December 2006

Prostatic adenocarcinomas depend on androgen for growth and survival. First line treatment of disseminated disease exploits this dependence by specifically targeting androgen receptor function. Clinical evidence has shown that androgen receptor is reactivated in recurrent tumors despite the continuance of androgen deprivation therapy. Several factors have been shown to restore androgen receptor activity under these conditions, including somatic mutation of the androgen receptor ligand-binding domain. We have shown previously that select tumor-derived mutants of the androgen receptor are receptive to activation by bisphenol A (BPA), an endocrine-disrupting compound that is leached from polycarbonate plastics and epoxy resins into the human food supply. Moreover, we have shown that BPA can promote cell cycle progression in cultured prostate cancer cells under conditions of androgen deprivation. Here, we challenged the effect of BPA on the therapeutic response in a xenograft model system of prostate cancer containing the endogenous BPA-responsive AR-T877A mutant protein. We show that after androgen deprivation, BPA enhanced both cellular proliferation rates and tumor growth. These effects were mediated, at least in part, through androgen receptor activity, as prostate-specific antigen levels rose with accelerated kinetics in BPA-exposed animals. Thus, at levels relevant to human exposure, BPA can modulate tumor cell growth and advance biochemical recurrence in tumors expressing the AR-T877A mutation. [Mol Cancer Ther 2006;5(12):3181–90]
Chemosensitization of carcinoma cells using epithelial cell adhesion molecule–targeted liposomal antisense against bcl-2/bcl-xL
Authors: Hussain Sajid , Plückthun Andreas , Allen Theresa M. , Zangemeister-Wittke Uwe
Published: December 2006

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy. [Mol Cancer Ther 2006;5(12):3170–80]
Tautomycetin inhibits growth of colorectal cancer cells through p21cip/WAF1 induction via the extracellular signal–regulated kinase pathway
Authors: Lee Joon-Hee , Lee Jung-Soo , Kim Sung-Eun , Moon Byoung-San , Kim Yong-Chul , Lee Seung-Kyou , Lee Sang-Kyou , Choi Kang-Yell
Published: December 2006

Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21Cip/WAF induction via the extracellular signal–regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21Cip/WAF by 150 nmol tautomycetin. The crucial role of p21Cip/WAF1 in the extracellular signal–regulated kinase pathway–dependent antiproliferative responses by tautomycetin was confirmed by using p21Cip/WAF1 gene–deleted HCT-116 cells. The growth inhibitory effect of tautomycetin was acquired by regulation of Raf-1 activity through inhibition of protein phosphatase type 1 and protein phosphatase type 2A with high preference toward protein phosphatase type 1. Tautomycetin could be a potential drug for colorectal cancer. [Mol Cancer Ther 2006;5(12):3222–31]
Topoisomerase II and tubulin inhibitors both induce the formation of apoptotic topoisomerase I cleavage complexes
Authors: Sordet Olivier , Goldman Abby , Pommier Yves
Published: December 2006

Topoisomerase I (Top1) is a ubiquitous enzyme that removes DNA supercoiling generated during transcription and replication. Top1 can be trapped on DNA as cleavage complexes by the anticancer drugs referred to as Top1 inhibitors as well as by alterations of the DNA structure. We reported recently that Top1 cleavage complexes (Top1cc) are trapped during apoptosis induced by arsenic trioxide and staurosporine. In the present study, we generalize the occurrence of apoptotic Top1cc in response to anticancer drugs, which by themselves do not directly interact with Top1: the topoisomerase II inhibitors etoposide, doxorubicin, and amsacrine, and the tubulin inhibitors vinblastine and Taxol. In all cases, the Top1cc form in the early phase of apoptosis and persist throughout the apoptotic process. Their formation is prevented by the caspase inhibitor benzyloxycarbonyl-Val-Ala-dl-Asp(OMe)-fluoromethylketone and the antioxidant N-acetyl-l-cysteine. We propose that the trapping of Top1cc is a general process of programmed cell death, which is caused by alterations of the DNA structure (oxidized bases and strand breaks) induced by caspases and reactive oxygen species. [Mol Cancer Ther 2006;5(12):3139–44]
Finances of the Independent Dialysis Facility
Authors: DeOreo Peter B.
Published: December 2006

Medicare pays 80% of the cost of dialysis treatment and associated medications. Congress directed the Centers for Medicare and Medicaid Services (CMS) to develop both a process of regular and more or less ‘automatic’ updates of composite rate setting and ‘bundling’ as much of the laboratory and ancillary medications as possible into the composite rate. In response to this mandate, CMS revised the wage indexing process, added an annual update, and removed the limits on the wage index range. CMS has moved the ‘margin’ from medication acquisition and administration to an annually revised ‘drug add-on’ to the composite rate and fixed reimbursement of separately billed medication (ancillary) to the average sales price +6%. CMS is funding a demonstration project on near 100% bundling to be completed by 2008 that will include metrics for automatically increasing the base composite rate.
Size Matters: Body Composition and Outcomes in Maintenance Hemodialysis Patients
Authors: Kotanko Peter , Thijssen Stephan , Kitzler Thomas , Wystrychowski Grzegorz , Sarkar Shubho R. , Zhu Fansan , Gotch Frank , Levin Nathan W.
Published: December 2006

In hemodialysis patients a low body mass index (BMI) is correlated with an unfavorable clinical outcome, a phenomenon known as ‘reverse epidemiology’. Mechanisms underlying this observation are unclear. We propose the following: uremic toxin generation occurs predominantly in visceral organs and the mass of key uremiogenic viscera (gut, liver) relative to body weight is higher in small people. Consequently, the rate of uremic toxin generation per unit of BMI is higher in patients with a low BMI. Body water, mainly determined by muscle mass, serves as a dilution compartment for uremic toxins. Therefore, the concentration of uremic toxins is higher in small subjects. Uremic toxins are taken up by adipose and muscle tissues, subsequently metabolized and stored. Thus, the larger the ratio of fat and muscle mass to visceral mass, the lower the concentration of uremic toxins and the better the survival. To test this hypothesis, studies on uremic toxin kinetics in relation to body composition are needed.