Citations

Abstract Thymic stromal lymphopoietin (TSLP) is produced by epithelial cells and triggers dendritic cell-mediated Th2-type inflammation. Although TSLP is up-regulated in epithelium of patients with asthma, the factors that control TSLP production have not been studied extensively. Because mouse models suggest roles for protease(s) in Th2-type immune responses, we hypothesized that proteases from airborne allergens may induce TSLP production in a human airway epithelial cell line, BEAS-2B. TSLP mRNA and protein were induced when BEAS-2B cells were exposed to prototypic proteases, namely, trypsin and papain. TSLP induction by trypsin required intact protease activity and also a protease-sensing G protein-coupled receptor, protease-activated receptor (PAR)-2; TSLP induction by papain was partially dependent on PAR-2. In humans, exposure to ubiquitous airborne fungi, such as Alternaria, is implicated in the development and exacerbation of asthma. When BEAS-2B cells or normal human bronchial epithelial cells were exposed to Alternaria extract, TSLP was potently induced. The TSLP-inducing activity of Alternaria was partially blocked by treating the extract with a cysteine protease inhibitor, E-64, or by infecting BEAS-2B cells with small interfering RNA for PAR-2. Protease-induced TSLP production by BEAS-2B cells was enhanced synergistically by IL-4 and abolished by IFN-γ. These findings demonstrate that TSLP expression is induced in airway epithelial cells by exposure to allergen-derived proteases and that PAR-2 is involved in the process. By promoting TSLP production in the airways, proteases associated with airborne allergens may facilitate the development and/or exacerbation of Th2-type airway inflammation, particularly in allergic individuals.

Abstract The invasion of malignant glioma cells into the surrounding normal brain precludes effective clinical treatment. In this report, we investigated the role of the NH2-terminal FERM domain in the regulation of the promigratory function of Pyk2. We report that the substitution of residues that constitute a small cleft on the surface of the F3 module of the FERM domain do not significantly alter Pyk2 expression but result in the loss of Pyk2 phosphorylation. A monoclonal antibody, designated 12A10, specifically targeting the Pyk2 FERM domain was generated and recognizes an epitope located on the β5C-α1C surface of the F3 module of the FERM domain. Amino acid substitutions in the F3 module that resulted in the loss of Pyk2 phosphorylation also inhibited the binding of 12A10, suggesting that the 12A10 epitope overlaps a site that plays a role in Pyk2 activity. Conjugation of 12A10 to a membrane transport peptide led to intracellular accumulation and inhibition of glioma cell migration in a concentration-dependent manner. A single chain Fv fragment of 12A10 was stable when expressed in the intracellular environment, interacted directly with Pyk2, reduced Pyk2 phosphorylation, and inhibited glioma cell migration in vitro. Stable intracellular expression of the 12A10 scFv significantly extended survival in a glioma xenograft model. Together, these data substantiate a central role for the FERM domain in regulation of Pyk2 activity and identify the F3 module as a novel target to inhibit Pyk2 activity and inhibit glioma progression. [Mol Cancer Ther 2009;8(6):1505–14]

As humans age, the tonic level of activity in sympathetic vasoconstrictor nerves increases and may contribute to age-related increases in blood pressure. In previous studies in normotensive young men with varying levels of resting sympathetic nerve activity, we observed a balance among factors contributing to blood pressure regulation, such that higher sympathetic activity was associated with lower cardiac output and lesser vascular responsiveness to α-adrenergic agonists, which limited the impact of high sympathetic activity on blood pressure. In the present study, we tested the hypothesis that older normotensive men would exhibit a similar balance among these variables (sympathetic nerve activity, cardiac output, and α-adrenergic responsiveness) but that this balance would be shifted toward higher sympathetic nerve activity values. We measured muscle sympathetic nerve activity, cardiac output, arterial pressure, and forearm vasoconstrictor responses in 17 older men and compared these with previous data collected in 14 younger men. Muscle sympathetic activity (burst incidence) was positively related to diastolic blood pressure in the older men ( r =0.49; P =0.05); this relationship was not observed in young men. In addition, there was no relationship between cardiac output and muscle sympathetic activity ( r =0.29; P >0.05) or between muscle sympathetic activity and vasoconstrictor responses in the older men (eg, norepinephrine: r =−0.21; P >0.05). Although our older subjects were normotensive, the relationship between muscle sympathetic nerve activity and diastolic blood pressure and the lack of “balance” among the other variables suggest that these changes with aging may contribute to the risk of sympathetically mediated hypertension in older humans.

Abstract The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release.

Natural orifice translumenal endoscopic surgery (NOTES) is a novel surgical concept that may provide a multitasking platform for minimally invasive surgery in the 21st century. With NOTES, natural hollow organs are used to gain access to body cavities via the mouth, anus, vagina, and urethra, thereby eliminating skin incisions. Access to the body cavity through the entry portal for the NOTES procedure and closure of the portal after surgery are important treatment considerations. In this review, access and closure techniques for NOTES-related procedures are discussed. Technical descriptions for access are also described. Closure techniques using experimental and commercially available devices are also reviewed. Based on the experimental and early clinical experiences to date, the future of NOTES appears quite promising. Nonetheless, ongoing fundamental research is necessary to determine the safety and benefit of these procedures. While technical challenges are currently present, the issues are being resolved with the development of optimal instrumentation and techniques. We believe that once standardized safe and reliable access and closure techniques are established, a major shift in minimally invasive surgery will likely be inevitable.

Object In a companion study, the authors describe the development of a new instrument named the Wireless Instantaneous Neurotransmitter Concentration System (WINCS), which couples digital telemetry with fast-scan cyclic voltammetry (FSCV) to measure extracellular concentrations of dopamine. In the present study, the authors describe the extended capability of the WINCS to use fixed potential amperometry (FPA) to measure extracellular concentrations of dopamine, as well as glutamate and adenosine. Compared with other electrochemical techniques such as FSCV or high-speed chronoamperometry, FPA offers superior temporal resolution and, in combination with enzyme-linked biosensors, the potential to monitor nonelectroactive analytes in real time. Methods The WINCS design incorporated a transimpedance amplifier with associated analog circuitry for FPA; a microprocessor; a Bluetooth transceiver; and a single, battery-powered, multilayer, printed circuit board. The WINCS was tested with 3 distinct recording electrodes: 1) a carbon-fiber microelectrode (CFM) to measure dopamine; 2) a glutamate oxidase enzyme–linked electrode to measure glutamate; and 3) a multiple enzyme–linked electrode (adenosine deaminase, nucleoside phosphorylase, and xanthine oxidase) to measure adenosine. Proof-of-principle analyses included noise assessments and in vitro and in vivo measurements that were compared with similar analyses by using a commercial hardwired electrochemical system (EA161 Picostat, eDAQ; Pty Ltd). In urethane-anesthetized rats, dopamine release was monitored in the striatum following deep brain stimulation (DBS) of ascending dopaminergic fibers in the medial forebrain bundle (MFB). In separate rat experiments, DBS-evoked adenosine release was monitored in the ventrolateral thalamus. To test the WINCS in an operating room setting resembling human neurosurgery, cortical glutamate release in response to motor cortex stimulation (MCS) was monitored using a large-mammal animal model, the pig. Results The WINCS, which is designed in compliance with FDA-recognized consensus standards for medical electrical device safety, successfully measured dopamine, glutamate, and adenosine, both in vitro and in vivo. The WINCS detected striatal dopamine release at the implanted CFM during DBS of the MFB. The DBS-evoked adenosine release in the rat thalamus and MCS-evoked glutamate release in the pig cortex were also successfully measured. Overall, in vitro and in vivo testing demonstrated signals comparable to a commercial hardwired electrochemical system for FPA. Conclusions By incorporating FPA, the chemical repertoire of WINCS-measurable neurotransmitters is expanded to include glutamate and other nonelectroactive species for which the evolving field of enzyme-linked biosensors exists. Because many neurotransmitters are not electrochemically active, FPA in combination with enzyme-linked microelectrodes represents a powerful intraoperative tool for rapid and selective neurochemical sampling in important anatomical targets during functional neurosurgery.

Object Emerging evidence supports the hypothesis that modulation of specific central neuronal systems contributes to the clinical efficacy of deep brain stimulation (DBS) and motor cortex stimulation (MCS). Real-time monitoring of the neurochemical output of targeted regions may therefore advance functional neurosurgery by, among other goals, providing a strategy for investigation of mechanisms, identification of new candidate neurotransmitters, and chemically guided placement of the stimulating electrode. The authors report the development of a device called the Wireless Instantaneous Neurotransmitter Concentration System (WINCS) for intraoperative neurochemical monitoring during functional neurosurgery. This device supports fast-scan cyclic voltammetry (FSCV) at a carbon-fiber microelectrode (CFM) for real-time, spatially and chemically resolved neurotransmitter measurements in the brain. Methods The FSCV study consisted of a triangle wave scanned between −0.4 and 1 V at a rate of 300 V/second and applied at 10 Hz. All voltages were compared with an Ag/AgCl reference electrode. The CFM was constructed by aspirating a single carbon fiber (r = 2.5 μm) into a glass capillary and pulling the capillary to a microscopic tip by using a pipette puller. The exposed carbon fiber (that is, the sensing region) extended beyond the glass insulation by ~ 100 μm. The neurotransmitter dopamine was selected as the analyte for most trials. Proof-of-principle tests included in vitro flow injection and noise analysis, and in vivo measurements in urethane-anesthetized rats by monitoring dopamine release in the striatum following high-frequency electrical stimulation of the medial forebrain bundle. Direct comparisons were made to a conventional hardwired system. Results The WINCS, designed in compliance with FDA-recognized consensus standards for medical electrical device safety, consisted of 4 modules: 1) front-end analog circuit for FSCV (that is, current-to-voltage transducer); 2) Bluetooth transceiver; 3) microprocessor; and 4) direct-current battery. A Windows-XP laptop computer running custom software and equipped with a Universal Serial Bus–connected Bluetooth transceiver served as the base station. Computer software directed wireless data acquisition at 100 kilosamples/second and remote control of FSCV operation and adjustable waveform parameters. The WINCS provided reliable, high-fidelity measurements of dopamine and other neurochemicals such as serotonin, norepinephrine, and ascorbic acid by using FSCV at CFM and by flow injection analysis. In rats, the WINCS detected subsecond striatal dopamine release at the implanted sensor during high-frequency stimulation of ascending dopaminergic fibers. Overall, in vitro and in vivo testing demonstrated comparable signals to a conventional hardwired electrochemical system for FSCV. Importantly, the WINCS reduced susceptibility to electromagnetic noise typically found in an operating room setting. Conclusions Taken together, these results demonstrate that the WINCS is well suited for intraoperative neurochemical monitoring. It is anticipated that neurotransmitter measurements at an implanted chemical sensor will prove useful for advancing functional neurosurgery.

Abstract Previous studies have suggested that minor alleles for ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 may influence breast cancer risk, but the evidence is inconclusive due to their small sample size. These polymorphisms were genotyped in more than 30,000 breast cancer cases and 30,000 controls, primarily of European descent, from 30 studies in the Breast Cancer Association Consortium. We calculated odds ratios (OR) and 95% confidence intervals (95% CI) as a measure of association. We found that the minor alleles for these polymorphisms were not related to invasive breast cancer risk overall in women of European descent: ECCR4 per-allele OR (95% CI) = 0.99 (0.97-1.02), minor allele frequency = 27.5%; TNF 1.00 (0.95-1.06), 5.0%; CASP10 1.02 (0.98-1.07), 6.5%; PGR 1.02 (0.99-1.06), 15.3%; and BID 0.98 (0.86-1.12), 1.7%. However, we observed significant between-study heterogeneity for associations with risk for single-nucleotide polymorphisms (SNP) in CASP10, PGR, and BID. Estimates were imprecise for women of Asian and African descent due to small numbers and lower minor allele frequencies (with the exception of BID SNP). The ORs for each copy of the minor allele were not significantly different by estrogen or progesterone receptor status, nor were any significant interactions found between the polymorphisms and age or family history of breast cancer. In conclusion, our data provide persuasive evidence against an overall association between invasive breast cancer risk and ERCC4 rs744154, TNF rs361525, CASP10 rs13010627, PGR rs1042838, and BID rs8190315 genotypes among women of European descent. (Cancer Epidemiol Biomarkers Prev 2009;18(5):1610–6)