Cathepsin D specifically cleaves the chemokines macrophage inflammatory protein-1 alpha, macrophage inflammatory protein-1 beta, and SLC that are expressed in human breast cancer

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1 alpha (CCL3), MIP-1 beta (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1 alpha and MIP-1 beta degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu(58) to Trp(59) bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1 beta hybrids indicated that processing of MIP-1 beta might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1 alpha, MIP-1 beta, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes.

Spatio-temporal dynamics of alpha brain electric fields, and cognitive modes

Brian electric activity is viewed as sequences of momentary maps of potential distribution. Frequency-domain source modeling, estimation of the complexity of the trajectory of the mapped brain field distributions in state space, and microstate parsing were used as analysis tools. Input-presentation as well as task-free (spontaneous thought) data collection paradigms were employed. We found: Alpha EEG field strength is more affected by visualizing mentation than by abstract mentation, both input-driven as well as self-generated. There are different neuronal populations and brain locations of the electric generators for different temporal frequencies of the brain field. Different alpha frequencies execute different brain functions as revealed by canonical correlations with mentation profiles. Different modes of mentation engage the same temporal frequencies at different brain locations. The basic structure of alpha electric fields implies inhomogeneity over time — alpha consists of concatenated global microstates in the sub-second range, characterized by quasi-stable field topographies, and rapid transitions between the microstates. In general, brain activity is strongly discontinuous, indicating that parsing into field landscape-defined microstates is appropriate. Different modes of spontaneous and induced mentation are associated with different brain electric microstates; these are proposed as candidates for psychophysiological ``atoms of thought''.

Quantitative comparison of 3 enamel-stripping devices in vitro: how precisely can we strip teeth?

INTRODUCTION In this in-vitro study, we aimed to investigate the predictability of the expected amount of stripping using 3 common stripping devices on premolars. METHODS One hundred eighty extracted premolars were mounted and aligned in silicone. Tooth mobility was tested with Periotest (Medizintechnik Gulden, Modautal, Germany) (8.3 ± 2.8 units). The selected methods for interproximal enamel reduction were hand-pulled strips (Horico, Hapf Ringleb & Company, Berlin, Germany), oscillating segmental disks (O-drive-OD 30; KaVo Dental, Biberach, Germany), and motor-driven abrasive strips (Orthofile; SDC Switzerland, Lugano-Grancia, Switzerland). With each device, the operator intended to strip 0.1, 0.2, 0.3, or 0.4 mm on the mesial side of 15 teeth. The teeth were scanned before and after stripping with a 3-dimensional laser scanner. Superposition and measurement of stripped enamel on the most mesial point of the tooth were conducted with Viewbox software (dHal Software, Kifissia, Greece). The Wilcoxon signed rank test and the Kruskal-Wallis test were applied; statistical significance was set at alpha ≤ 0.05. RESULTS Large variations between the intended and the actual amounts of stripped enamel, and between stripping procedures, were observed. Significant differences were found at 0.1 mm of intended stripping (P ≤ 0.05) for the hand-pulled method and at 0.4 mm of intended stripping (P ≤ 0.001 to P = 0.05) for all methods. For all scenarios of enamel reduction, the actual amount of stripping was less than the predetermined and expected amount of stripping. The Kruskal-Wallis analysis showed no significant differences between the 3 methods. CONCLUSIONS There were variations in the stripped amounts of enamel, and the stripping technique did not appear to be a significant predictor of the actual amount of enamel reduction. In most cases, actual stripping was less than the intended amount of enamel reduction.

Distinct roles of estrogen receptors alpha and beta mediating acute vasodilation of epicardial coronary arteries.

This study investigated the contribution of estrogen receptors (ERs) alpha and beta for epicardial coronary artery function, vascular NO bioactivity, and superoxide (O(2)(-)) formation. Porcine coronary rings were suspended in organ chambers and precontracted with prostaglandin F(2alpha) to determine direct effects of the selective ER agonists 4,4',4''-(4-propyl-[(1)H]pyrazole-1,3,5-triyl)tris-phenol (PPT) or 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) or the nonselective ER agonist 17beta-estradiol. Indirect effects on contractility to U46619 and relaxation to bradykinin were assessed and effects on NO, nitrite, and O(2)(-) formation were measured in cultured cells. Within 5 minutes, selective ERalpha activation by PPT, but not 17beta-estradiol or the ERbeta agonist DPN, caused rapid, NO-dependent, and endothelium-dependent relaxation (49+/-5%; P<0.001 versus ethanol). PPT also caused sustained endothelium- and NO-independent vasodilation similar to 17beta-estradiol after 60 minutes (72+/-3%; P<0.001 versus ethanol). DPN induced endothelium-dependent NO-independent relaxation via endothelium-dependent hyperpolarization (40+/-4%; P<0.01 versus ethanol). 17beta-Estradiol and PPT, but not DPN, attenuated the responses to U46619 and bradykinin. All of the ER agonists increased NO and nitrite formation in vascular endothelial but not smooth muscle cells and attenuated vascular smooth muscle cell O(2)(-) formation (P<0.001). ERalpha activation had the most potent effects on both nitrite formation and inhibiting O(2)(-) (P<0.05). These data demonstrate novel and differential mechanisms by which ERalpha and ERbeta activation control coronary artery vasoreactivity in males and females and regulate vascular NO and O(2)(-) formation. The findings indicate that coronary vascular effects of sex hormones differ with regard to affinity to ERalpha and ERbeta, which will contribute to beneficial and adverse effects of hormone replacement therapy.

Reduced bone breakage and increased bone strength in free range laying hens fed omega-3 polyunsaturated fatty acid supplemented diets

INTRODUCTION The omega-3 and omega-6 polyunsaturated fatty acids (PUFAs) are the immediate precursors to a number of important mediators of immunity, inflammation and bone function, with products of omega-6 generally thought to promote inflammation and favour bone resorption. Western diets generally provide a 10 to 20-fold deficit in omega-3 PUFAs compared with omega-6, and this is thought to have contributed to the marked rise in incidence of disorders of modern human societies, such as heart disease, colitis and perhaps osteoporosis. Many of our food production animals, fed on grains rich in omega-6, are also exposed to a dietary deficit in omega-3, with perhaps similar health consequences. Bone fragility due to osteoporotic changes in laying hens is a major economic and welfare problem, with our recent estimates of breakage rates indicating up to 95% of free range hens suffer breaks during lay. METHODS Free range hens housed in full scale commercial systems were provided diets supplemented with omega-3 alpha linolenic acid, and the skeletal benefits were investigated by comparison to standard diets rich in omega-6. RESULTS There was a significant 40-60% reduction in keel bone breakage rate, and a corresponding reduction in breakage severity in the omega-3 supplemented hens. There was significantly greater bone density and bone mineral content, alongside increases in total bone and trabecular volumes. The mechanical properties of the omega-3 supplemented hens were improved, with strength, energy to break and stiffness demonstrating significant increases. Alkaline phosphatase (an osteoblast marker) and tartrate-resistant acid phosphatase (an osteoclast marker) both showed significant increases with the omega-3 diets, indicating enhanced bone turnover. This was corroborated by the significantly lower levels of the mature collagen crosslinks, hydroxylysyl pyridinoline, lysyl pyridinoline and histidinohydroxy-lysinonorleucine, with a corresponding significant shift in the mature:immature crosslink ratio. CONCLUSIONS The improved skeletal health in laying hens corresponds to as many as 68million fewer hens suffering keel fractures in the EU each year. The biomechanical and biochemical evidence suggests that increased bone turnover has enhanced the bone mechanical properties, and that this may suggest potential benefits for human osteoporosis.

Caspase-3 mediates hippocampal apoptosis in pneumococcal meningitis

Bacterial meningitis causes neuronal apoptosis in the hippocampal dentate gyrus, which is associated with learning and memory impairments after cured disease. The execution of the apoptotic program involves pathways that converge on activation of caspase-3, which is required for morphological changes associated with apoptosis. Here, the time course and the role of caspase-3 in neuronal apoptosis was assessed in an infant rat model of pneumococcal meningitis. During clinically asymptotic meningitis (0-12 h after infection), only minor apoptotic damage to the dentate gyrus was observed, while the acute phase (18-24 h) was characterized by a massive increase of apoptotic cells, which peaked at 36 h. In the subacute phase of the disease (36-72 h), the number of apoptotic cells decreased to control levels. Enzymatic caspase-3 activity was significantly increased in hippocampal tissue of infected animals compared to controls at 22 h. The activated enzyme was localized to immature cells of the dentate gyrus, and in vivo activity was evidenced by cleavage of the amyloid-beta precursor protein. Intracisternal administration of the caspase-3-specific inhibitor Ac-DEVD-CHO significantly reduced apoptosis in the hippocampal dentate gyrus. In contrast to a study where the decrease of hippocampal apoptosis after administration of a pan-caspase inhibitor was due to downmodulation of the inflammatory response, our data demonstrate that specific inhibition of caspase-3 did not affect inflammation assessed by TNF-alpha and IL-1beta concentrations in the cerebrospinal fluid space. Taken together, the present results identify caspase-3 as a key effector of neuronal apoptosis in pneumococcal meningitis.

Assignment of the porcine tumour necrosis factor alpha and beta genes to the chromosome region 7p11-q11 by in situ hybridization

The loci of the porcine tumour necrosis factor genes, alpha (TNFA) and beta (TNFB), have been chromosomally assigned by radioactive in situ hybridization. The genomic probes for TNFA and TNFB yielded signals above 7p11-q11, a region that has been shown earlier to carry the porcine major histocompatibility locus (SLA). These mapping data along with preliminary molecular studies suggest a genomic organization of the SLA that is similar to that of human and murine major histocompatibility complexes.

Folate Receptor Targeted Alpha-Therapy Using Terbium-149

Terbium-149 is among the most interesting therapeutic nuclides for medical applications. It decays by emission of short-range α-particles (Eα = 3.967 MeV) with a half-life of 4.12 h. The goal of this study was to investigate the anticancer efficacy of a 149Tb-labeled DOTA-folate conjugate (cm09) using folate receptor (FR)-positive cancer cells in vitro and in tumor-bearing mice. 149Tb was produced at the ISOLDE facility at CERN. Radiolabeling of cm09 with purified 149Tb resulted in a specific activity of ~1.2 MBq/nmol. In vitro assays performed with 149Tb-cm09 revealed a reduced KB cell viability in a FR-specific and activity concentration-dependent manner. Tumor-bearing mice were injected with saline only (group A) or with 149Tb-cm09 (group B: 2.2 MBq; group C: 3.0 MBq). A significant tumor growth delay was found in treated animals resulting in an increased average survival time of mice which received 149Tb-cm09 (B: 30.5 d; C: 43 d) compared to untreated controls (A: 21 d). Analysis of blood parameters revealed no signs of acute toxicity to the kidneys or liver in treated mice over the time of investigation. These results demonstrated the potential of folate-based α-radionuclide therapy in tumor-bearing mice.

Norepinephrine infusion with and without alpha-adrenergic blockade by phentolamine increases salivary alpha amylase in healthy men

BACKGROUND: Mental stress reliably induces increases in salivary alpha amylase (sAA), a suggested surrogate marker for sympathetic nervous system (SNS) reactivity. While stress-induced sAA increases correlate with norepinephrine (NE) secretion, a potential mediating role of noradrenergic mechanisms remains unclear. In this study, we investigated for the first time in humans whether a NE-stress-reactivity mimicking NE-infusion with and without alpha-adrenergic blockade by phentolamine would induce changes in sAA. METHODS: In a single-blind placebo-controlled within-subjects design, 21 healthy men (29-66 years) took part in three different experimental trials varying in terms of substance infusion with a 1-min first infusion followed by a 15-min second infusion: saline-infusion (trial-1), NE-infusion (5 μg/min) without alpha-adrenergic blockade (trial-2), and with phentolamine-induced non-selective blockade of alpha1- and alpha2-adrenergic receptors (trial-3). Saliva samples were collected immediately before, during, and several times after substance infusion in addition to blood pressure and heart rate readings. RESULTS: Experimental trials significantly differed in sAA reactivity to substance-infusion (p=.001) with higher sAA reactivity following NE-infusion with (trial-3; p=.001) and without alpha-adrenergic-blockade (trial-2; p=.004) as compared to placebo-infusion (trial-1); sAA infusion reactivity did not differ between trial-2 and trial-3 (p=.29). Effective phentolamine application was verified by blood pressure and heart rate infusion reactivity. Salivary cortisol was not affected by NE, either with or without alpha-adrenergic-blockade. CONCLUSIONS: We found that NE-infusion stimulates sAA secretion, regardless of co-administered non-selective alpha-adrenergic blockade by phentolamine, suggesting that the mechanism underlying stress-induced sAA increases may involve NE.

CD62L (L-selectin) shedding for assessment of functional blockade of TNF alpha in anti-TNF treated IBD patients: clinical feasibility and perspectives (DOP060)

Background Tumor necrosis factor (TNF) inhibition is central to the therapy of inflammatory bowel diseases (IBD). However, loss of response (LOR) is frequent and additional tests to help decision making with costly anti-TNF Therapy are needed. Methods Consecutive IBD Patients receiving anti-TNF therapy (Infliximab (IFX) or Adalimumab (after IFX LOR) from Bern University Hospital were identified and followed prospectively. Patient whole blood was stimulated with a dose-titration of two triggers of TLR receptors human: TNF and LPS. Median fluorescence intensity of CD62L on the surface of granulocytes was quantified by surface staining with specific antibodies (CD33, CD62L) and flow cytometry and logistic curves to these data permits the calculation of EC50 or the half maximal effective concentration TNF concentration to induce shedding [1]. A shift in the concentration were CD62L shedding occurred was seen before and after the anti-TNF agent administraion which permits to predict the response to the drug. This predicted response was correlated to the clinical evolution of the patients in order to analyze the ability of this test to identify LOR to IFX. Results We collected prospective clinical data and blood samples, before and after anti-TNF agent administration, on 33 IBD patients, 25 Crohn's disease and 8 ulcerative colitis patients (45% females) between June 2012 and November 2013. The assay showed a functional blockade of IFX (PFR) for 22 patients (17 CD and 5 UC) whereas 11 (8 CD and 3 UC) had no functional response (NR) to IFX. Clinical characteristics (e.g. diagnosis, disease location, smoking status, BMI and number of infusions) were no significantly different between predicted PFR and NR. Among the 22 Patients with PRF, only 1 patient was a clinical non responder (LOR to IFX), based on clinical prospective evaluation by IBD gastroenterologists (PJ, AM), and among the 11 predicted NR, 3 had no clinical LOR. Sensitivity of this test was 95% and specificity 73% and AUC adjusted for age and gender was 0.81 (Figure 1). During follow up (median 10 mo, 3–15) 8 “hard” outcomes occured (3 medic. flares, 4 resections and 1 new fistula) 2 in the PFR and 6 in the NR group (25% vs. 75%; p < 0.01). Correlation with clinical response is presented in Figure 2. Figure 1. Figure 2. Correlation clinical response - log EC50 changes: 1 No, 2 partial, 3 complete clinical response. Conclusion CD62L (L-Selectin) shedding is the first validated test of functional blockade of TNF alpha in anti-TNF treated IBD patients and will be a useful tool to guide medical decision on the use of anti-TNF agents. Comparative studies with ATI and trough level of IFX are ongoing. 1. Nicola Patuto, Emma Slack, Frank Seibold and Andrew J. Macpherson, (2011), Quantitating Anti-TNF Functionality to Inform Dosing and Choice of Therapy, Gastroenterology, 140 (5, Suppl. I), S689.

Total Tumor Volume and Alpha Fetoprotein for selection of transplant candidates with hepatocellular carcinoma: A prospective validation.

The selection of liver transplant candidates with hepatocellular carcinoma (HCC) is currently validated based on Milan criteria. The use of extended criteria has remained a matter of debate, mainly because of the absence of prospective validation. The present prospective study recruited patients according to the previously proposed Total Tumor Volume (TTV ≤115 cm(3) )/alpha fetoprotein (AFP ≤400 ng/ml) score. Patients with AFP >400 ng/ml were excluded, and as such the Milan group was modified to include only patients with AFP <400 ng/ml; these patients were compared to patients beyond Milan, but within TTV/AFP. From January 2007 to March 2013, 233 patients with HCC were listed for liver transplantation. Of them, 195 patients were within Milan, and 38 beyond Milan but within TTV/AFP. The average follow-up from listing was 33,9 ±24,9 months. The risk of drop-out was higher for patients beyond Milan but within TTV/AFP (16/38, 42,1%), than for patients within Milan (49/195, 25,1%, p=0,033). In parallel, intent-to-treat survival from listing was lower in the patients beyond Milan (53,8% vs. 71,6% at four years, p<0,001). After a median waiting time of 8 months, 166 patients were transplanted, 134 patients within Milan criteria, and 32 beyond Milan but within TTV/AFP. They demonstrated acceptable and similar recurrence rates (4,5% vs. 9,4%, p=0,138) and post-transplant survivals (78,7% vs. 74,6% at four years, p=0,932). CONCLUSION Based on the present prospective study, HCC liver transplant candidate selection could be expanded to the TTV (≤115 cm(3) )/AFP (≤400 ng/ml) criteria in centers with at least 8-month waiting time. An increased risk of drop-out on the waiting list can be expected but with equivalent and satisfactory post-transplant survival. This article is protected by copyright. All rights reserved.

Synthesis and incorporation into a-DNA of a novel conformationally constrained alpha-nucleoside analogue

We describe the synthesis and incorporation into alpha-DNA of a novel conformationally constrained alpha-nucleoside analogue. The carbohydrate part of this analogue was prepared in 4 steps from the known bicyclic precursor 1 via a stereospecific, intramolecular, Et 3B mediated radical addition to a keto-function as the key step. The thus obtained intermediate 4 was transformed stereoselectively into the corresponding alpha-nucleoside analogues 7 and 8 containing the bases adenine and thymine, and were further elaborated into the phosphoramidite building blocks 11 and 12 . Both building blocks were incorporated into alpha-oligodeoxynucleotides and their pairing behavior to parallel complementary DNA studied by UV-melting experiments. Single substitutions of alpha-deoxyribnucleoside units by the new analogues in the center of duplexes were found to be thermally destabilizing by only -0.8 to -3.1›C.