Calcium currents in interstitial cells from the guinea-pig bladder.

OBJECTIVE: To determine whether there are inward currents in interstitial cells (IC) isolated from the guinea-pig detrusor and if so, to characterise them using the patch-clamp technique and pharmacological agents. MATERIALS AND METHODS: Using the whole-cell patch-clamp technique, inward currents were studied in IC enzymatically isolated from the detrusor of the guinea-pig bladder. Currents were evoked by stepping positively from a holding potential of - 80 mV. RESULTS: Outward K+ currents were blocked by Cs+ internal solution to reveal inward currents, which activated at voltages more positive than - 50 mV, peaked at 0 mV, reversed near + 50 mV and were half-maximally activated at - 27 mV. The inward currents showed voltage-dependent inactivation and were half-maximally inactivated at - 36 mV. Fitting the activation and inactivation data with a Boltzmann function revealed a window current between - 40 mV and + 20 mV. The decay of the current evoked at 0 mV could be fitted with a single exponential with a mean time-constant of 88 ms. Replacing external Ca2+ with Ba2+ significantly increased this to 344 ms. The current amplitude was augmented by Ba2+, and by Bay K 8644. Inward currents were significantly reduced by 1 microm nifedipine, across the voltage range, but the blockade was more effective on the current evoked at 0 mV than that evoked by a step to - 20 mV, perhaps indicating voltage-dependence of the action of nifedipine or another component of inward current. Increasing the concentration of the drug to 10 microm caused no further significant reduction either at 0 mV or at -20 mV. However, in the presence of 1 microm nifedipine the latter current was significantly reduced by 100 microm Ni2+. Both currents were significantly reduced in Ca2+-free solution. CONCLUSIONS: IC from the guinea-pig detrusor possess inward currents with typical characteristics of L-type Ca2+ current. They also have a component of inward Ca2+ current, which was resistant to nifedipine, but sensitive to Ni2+. Further work is needed to characterise the latter conductance. PMID: 16686735 [PubMed - indexed for MEDLINE]

Characterization of outward K(+) currents in isolated smooth muscle cells from sheep urethra.

The perforated-patch technique was used to measure membrane currents in smooth muscle cells from sheep urethra. Depolarizing pulses evoked large transient outward currents and several components of sustained current. The transient current and a component of sustained current were blocked by iberiotoxin, penitrem A, and nifedipine but were unaffected by apamin or 4-aminopyridine, suggesting that they were mediated by large-conductance Ca(2+)-activated K(+) (BK) channels. When the BK current was blocked by exposure to penitrem A (100 nM) and Ca(2+)-free bath solution, there remained a voltage-sensitive K(+) current that was moderately sensitive to blockade with tetraethylammonium (TEA; half-maximal effective dose = 3.0 +/- 0.8 mM) but not 4-aminopyridine. Penitrem A (100 nM) increased the spike amplitude and plateau potential in slow waves evoked in single cells, whereas addition of TEA (10 mM) further increased the plateau potential and duration. In conclusion, both Ca(2+)-activated and voltage-dependent K(+) currents were found in urethral myocytes. Both of these currents are capable of contributing to the slow wave in these cells, suggesting that they are likely to influence urethral tone under certain conditions.

Hyperpolarisation-activated inward current in isolated sheep mesenteric lymphatic smooth muscle.

1. Freshly isolated sheep lymphatic smooth muscle cells were studied using the perforated patch-clamp technique. Hyperpolarisation with constant-current pulses caused a time-dependent rectification evident as a depolarising 'sag' followed by an anode-break overshoot at the end of the pulse. Both sag and overshoot were blocked with 1 mM Cs+. 2. Cells were voltage clamped at -30 mV and stepped to -120 mV in 10 mV steps of 2 s duration. Steps negative to -60 mV evoked a slowly activating, non-inactivating inward current which increased in size and rate of activation with increasing hyperpolarisation. 3. The slowly activating current was reduced in Na+-free bathing solution but enhanced when the extracellular K+ concentration was increased to 60 mM. The current was significantly reduced by 1 mM Cs+ and 1 microM ZD7288 but not by 1.8 mM Ba2+. 4. The steady-state activation curve of the underlying conductance showed a threshold at -50 mV and half-maximal activation at -81 mV. Neither threshold nor half-maximal activation was significantly affected by increasing the external K+ concentration to 60 mM. 5. The frequency of spontaneous contractions and fluid propulsion in isolated cannulated segments of sheep mesenteric lymphatics were decreased by 1 mM Cs+ and by 1 microM ZD7288. 6. We conclude that sheep lymphatics have a hyperpolarisation-activated inward current similar to the If seen in sinoatrial node cells of the heart. Blockade of this current slows spontaneous pumping in intact lymphatic vessels suggesting that it is important in normal pacemaking.

Ca2+ current and Ca(2+)-activated chloride current in isolated smooth muscle cells of the sheep urethra.

1. Isolated sheep urethral cells were studied using the perforated patch clamp technique (T = 37 degrees C). Depolarizing steps ranging from -40 to -10 mV evoked an inward current that peaked within 10 ms and a slower inward current. Stepping back to the holding potential of -80 mV evoked large inward tail currents. All three currents were abolished by nifedipine (1 microM). Substitution of external Ca2+ with Ba2+ resulted in potentiation of the fast inward current and blockade of the slow current and tails. 2. Changing the chloride equilibrium potential (ECl) from 0 to +27 mV shifted the reversal potential of the tail currents from 1 +/- 1 to 27 +/- 1 mV (number of cells, n = 5). Chloride channel blockers, niflumic acid (10 microM) and anthracene-9-carboxylic acid (9AC, 1 mM), reduced the slow current and tails suggesting that these were Ca(2+)-activated Cl- currents, ICl(Ca). 4. Caffeine (10 mM) induced currents that reversed at ECl and were blocked by niflumic acid (10 microM). 5. In current clamp mode, some cells developed spontaneous transient depolarizations (STDs) and action potentials. Short exposure to nifedipine blocked the action potentials and unmasked STDs. In contrast, 9AC and niflumic acid reduced the amplitude of the STDs and blocked the action potentials. 6. In conclusion, these cells have both L-type ICa and ICl(Ca). The former appears to be responsible for the upstroke of the action potential, while the latter may act as a pacemaker current.

Perspectives for quantum state engineering via high nonlinearity in a double-EIT regime

We analyse the possibilities for quantum state engineering offered by a model for Kerr-type nonlinearity enhanced by electromagnetically induced transparency (EIT), which was recently proposed by Petrosyan and Kurizki [2002, Phys. Rev. A, 65, 33833]. We go beyond the semiclassical treatment and derive a quantum version of the model with both a full Hamiltonian approach and an analysis in terms of dressed states. The preparation of an entangled coherent state via a cross-phase modulation effect is demonstrated. We briefly show that the violation of locality for such an entangled coherent state is robust against low detection efficiency. Finally, we investigate the possibility of a bi-chromatic photon blockade realized via the interaction of a low density beam of atoms with a bi-modal electromagnetic cavity which is externally driven. We show the effectiveness of the blockade effect even when more than a single atom is inside the cavity. The possibility to control two different cavity modes allows some insights into the generation of an entangled state of cavity modes.

Single-photon tunneling via localized surface plasmons

Strong evidence of a single-photon tunneling effect, a direct analog of single-electron tunneling, has been obtained in the measurements of light tunneling through individual subwavelength pinholes in a gold film covered with a layer of polydiacetylene. The transmission of some pinholes reached saturation because of the optical nonlinearity of polydiacetylene at a very low light intensity of a few thousand photons per second. This result is explained theoretically in terms of a "photon blockade," similar to the Coulomb blockade phenomenon observed in single-electron tunneling experiments. Single-photon tunneling may find applications in the fields of quantum communication and information processing.

Survival of Swiss-Webster mouse cerebellar granule neurons is promoted by a combination of potassium channel blockers

Cultured cerebellar granule neurons (CGN) are commonly used to assess neurotoxicity, but are routinely maintained in supraphysiological (25 mM) extracellular K⁺ concentrations [K⁺]_o. We investigated the effect of potassium channel blockade on survival of CGN derived from Swiss-Webster mice in supraphysiological (25 mM) and physiological (5.6 mM) [K⁺]_o. CGN were cultured for 5 days in 25 mM K⁺, then in 5.6 mM K⁺ or 25 mM K⁺ (control). Viability, assayed 24 h later by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) reduction and by lactate dehydrogenase (LDH) release, was ∼50% in 5.6 mM K⁺ versus 25 mM K⁺ (p < .001). Potassium channel blockers, 2 mM 4-aminopyridine (4-AP), 2 mM tetraethylammonium (TEA) or 1 mM Ba²⁺, individually afforded limited protection in 5.6 mM K⁺. However, survival in 5.6 mM K⁺ with a combination of 4-AP, TEA and Ba²⁺ was similar to survival in 25 mM K⁺ without blockers (p < .001 versus 5.6 mM K⁺ alone). CGN survival in 25 mM K⁺ was attenuated 25% by 2 μM nifedipine (p > .001), but nifedipine did not attenuate neuroprotection by K⁺ channel blockers. Together, these results suggest that the survival of CGN depends on the K⁺ permeability of the membrane rather than the activity of a particular type of K⁺ channel, and that the mechanism of neuroprotection by K⁺ channel blockers is different from that of elevated [K⁺]_o.

The PD-1/PD-L1 Axis: An Immune-Mediated Mechanism of Chemoresistance in Cancer

The ability of tumour cells to avoid immune destruction (immune escape) and their acquired resistance to anti-cancer drugs constitute important barriers to the successful management of cancer. The interaction between specific molecules on the surface of tumour cells with their corresponding receptors on immune effector cells can result in inhibition of these effector cells, consequently allowing tumour cells to evade the host’s anti-tumour immune response. The interaction of the Programmed Death Ligand 1 (PD-L1) on the surface of tumour cells with the Programmed Death-1 (PD-1) receptor on cytotoxic T lymphocytes leads to inactivation of these immune effectors, and is a specific example of an immune escape mechanism tumour cells use to avoid immune destruction. Clinically, antibodies capable of blocking the PD-1/PD-L1 interaction have demonstrated significant therapeutic benefit, and are currently being used to help bolster patients’ immune response against malignant cells in a variety of cancer types. Here we show that the PD-1/PD-L1 interaction also leads to tumour cell resistance to conventional chemotherapeutic agents. Incubation of PD-L1-expressing human and mouse tumour cells with PD-1-expressing Jurkat T cells or purified recombinant PD-1 resulted in tumour cell resistance to doxorubicin and docetaxel. Interference with the PD-1/PD-L1 interaction using blocking anti-PD-1 or anti-PD-L1 antibody or shRNA-mediated gene silencing resulted in attenuation of PD-1/PD-L1-mediated drug resistance. Moreover, inhibition of the PD-1/PD-L1 signalling axis using anti-PD-1 antibody enhanced the effect of doxorubicin chemotherapy to inhibit 4T1 tumour cell metastasis in an in vivo mouse model of mammary carcinoma. These findings indicate that blockade of the PD-1/PD-L1 axis may be a useful approach to immunosensitize and chemosensitize tumours in cancer patients and provide a rationale for the use of anti-PD-1/PD-L1 antibodies as adjuvants to chemotherapy.

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

CCL11 blocks IL-4 and GM-CSF signaling in hematopoietic cells and hinders dendritic cell differentiation via suppressor of cytokine signaling expression

The chemokine eotaxin/CCL11 is an important mediator of leukocyte migration, but its effect on inflammatory cytokine signaling has not been explored. In this study, we find that CCL11 induces suppressor of cytokine signaling (SOCS) 1 and SOCS3 expression in murine macrophages, human monocytes, and dendritic cells (DCs). We also discover that CCL11 inhibits GM-CSF-mediated STAT5 activation and IL-4-induced STAT6 activation in a range of hematopoietic cells. This blockade of cytokine signaling by CCL11 results in reduced differentiation and endocytic ability of DCs, implicating CCL11-induced SOCS as mediators of chemotactic inflammatory control. These findings demonstrate cross-talk between chemokine and cytokine responses, suggesting that myeloid cells tracking to the inflammatory site do not differentiate in the presence of this chemokine, revealing another role for SOCS in inflammatory regulation. J. Leukoc. Biol. 85: 289-297; 2009.

A randomised, controlled trial of conventional versus automated weaning from mechanical ventilation using SmartcareTM/PS

Objective
Preliminary assessment of an automated weaning system (SmartCare™/PS) compared to usual management of weaning from mechanical ventilation performed in the absence of formal protocols.


Design and setting
A randomised, controlled pilot study in one Australian intensive care unit.


Patients
A total of 102 patients were equally divided between SmartCare/PS and Control.

Interventions
The automated system titrated pressure support, conducted a spontaneous breathing trial and provided notification of success (“separation potential”).

Measurements and results
The median time from the first identified point of suitability for weaning commencement to the state of “separation potential” using SmartCare/PS was 20 h (interquartile range, IQR, 2–40) compared to 8 h (IQR 2–43) with Control (log-rank P = 0.3). The median time to successful extubation was 43 h (IQR 6–169) using SmartCare/PS and 40 (14–87) with Control (log-rank P = 0.6). Unadjusted, the estimated probability of reaching “separation potential” was 21% lower (95% CI, 48% lower to 20% greater) with SmartCare/PS compared to Control. Adjusted for other covariates (age, gender, APACHE II, SOFAmax, neuromuscular blockade, corticosteroids, coma and elevated blood glucose), these estimates were 31% lower (95% CI, 56% lower to 9% greater) with SmartCare/PS. The study groups showed comparable rates of reintubation, non-invasive ventilation post-extubation, tracheostomy, sedation, neuromuscular blockade and use of corticosteroids.

Conclusions
Substantial reductions in weaning duration previously demonstrated were not confirmed when the SmartCare/PS system was compared to weaning managed by experienced critical care specialty nurses, using a 1:1 nurse-to-patient ratio. The effect of SmartCare/PS may be influenced by the local clinical organisational context.

Reliability of blood pressure determination with the Finapres with altered physiological states or pharmacodynamic conditions

The blood pressure waveform is modified on distal propagation by phenomena such as dispersion, reflection and the state of the arterial compliance. The consequent effects are amplification and narrowing of the wave, with an increased systolic, reduced diastolic and essentially unaltered mean blood pressure. The Finapres measures the peripheral pressure using the volume clamp principle; it has not been validated under altered physiological conditions and during pharmacodynamic interventions. We studied simultaneous Finapres and brachial blood pressures (using a conventional oscillometric sphygmomanometer—Vitalmap) in ten normal volunteers at rest, and during dynamic exercise and a cold pressor test. The effects of pharmacodynamic intervention were examined following beta-adrenoceptor blockade with propranolol (160 mg) or beta-adrenoceptor modulation with the beta-adrenoceptor partial agonist celiprolol (400 mg). The Finapres systolic pressure was significantly higher than the brachial value during all three test states. The difference between the systolic pressures measured by the two devices was shown to increase significantly during the cold pressor test, but not during dynamic (supine bicycle) exercise. The Finapres diastolic pressure was significantly higher than the Vitalmap value during exercise and the cold pressor test. The differences between the two methods increased significantly over time. Beta-adrenergic blockade with propranolol or modulation with celiprolol had no significant interaction with the pressure differences between the Finapres and Vitalmap techniques. The results would support the view that the Finapres can provide blood pressure information which is robust under most circumstances. Although this pharmacodynamic intervention did not alter the relationship between the peripheral and central blood pressure, it is important to note that this dynamic relationship is sensitive to circulatory loading conditions and wave transmission characteristics; it is possible that the Finapres could be less reliable in clinical settings where potent vasoactive agents were being administered.

Estimation of in situ stresses using anisotropic elasticity and suction measurements

Sampling and specimen preparation produce changes in mean effective stresses and pore water pressures, even with ‘perfect sampling’. The paper takes an existing simplified three-parameter cross-anisotropic elastic model and uses it to model these changes. The required ratio of cross-anisotropic parameters J/3G* can be obtained from standard CIU triaxial tests. If measurements are also made of suctions in unloaded specimens in the laboratory, then a combination of J/3G*, the measured suction, and the effective overburden pressure permits an estimation of the horizontal effective pressure and the K 0 ‘at rest’ coefficient. This can be helpful in numerical modelling that needs to start from in situ conditions, and in planning pressure levels for reconsolidation of clay specimens in the laboratory. Tests were done on Belfast Upper Boulder Clay from a depth of 28 m. Values of horizontal in situ effective stress estimated from these measurements compare favorably with conventional estimates of the ‘at rest’ coefficient K 0 and the overconsolidation ratio. Estimates of horizontal stress in London Clay were made using published data and the results compared with actual measurements. Again reasonable agreement was obtained.

Identifying additional patients with diabetic nephropathy using the UK primary care initiative

AIMS:
The aim of this study was to use general practice data to estimate the prevalence of diabetic nephropathy within the registered diabetes patients and examine variation in practice prevalence and management performance since introduction of this initiative.
METHODS:
Reported quality indicators from the Northern Ireland General Practice Quality and Outcomes Framework were analysed for diabetes and diabetic nephropathy prevalence and management in the period 2004-2008. Variation in prevalence at practice level was assessed using multiple linear regression adjusting for age, practice size, deprivation and glycaemic control.
RESULTS:
In 2006-2007, 57,454 (4.1%) adult diabetic patients were registered in the denominator population of 1.4 million compared with 51,923 (3.8%) in 2004-2005 (mean practice range 0.5-7.7%). Diabetic nephropathy prevalence was 15.1 and 11.5%, respectively (8688 and 5955 patients). Documented diabetic nephropathy prevalence showed marked variation across practices (range 0-100%) and was significantly negatively correlated with diabetes list size, albumin creatinine ratio testing rates and renin-angiotensin-aldosterone system blockade use and positively correlated with exception reporting rates. Specifically, for every increase in 100 diabetic patients to a register, documented diabetic nephropathy prevalence reduced by 40% (P=0.003). On the positive side, median albumin-creatinine ratio testing rates doubled to 82% compared with figures in the pre-Framework era.
CONCLUSIONS:
Implementation of the Northern Ireland General Practice Quality and Outcomes Framework has positively benefitted testing for diabetic nephropathy and increased numbers of detected patients in a short space of time. Large variation in diabetic nephropathy prevalence remains and is associated with diabetes registry size, screening and treatment practices, suggesting that understanding this variation may help detect and better manage diabetic nephropathy.

Hyperglycaemia-induced pro-inflammatory responses by retinal Müller glia are regulated by the receptor for advanced glycation end-products (RAGE)

Aims/hypothesis: Up-regulation of the receptor for AGEs (RAGE) and its ligands in diabetes has been observed in various tissues. Here, we sought to determine levels of RAGE and one of its most important ligands, S100B, in diabetic retina, and to investigate the regulatory role of S100B and RAGE in Müller glia.

Methods: Streptozotocin-diabetes was induced in Sprague-Dawley rats. RAGE, S100B and glial fibrillary acidic protein (GFAP) were detected in retinal cryosections. In parallel, the human retinal Müller cell line, MIO-M1, was maintained in normal glucose (5.5 mmol/l) or high glucose (25 mmol/l). RAGE knockdown was achieved using small interfering RNA (siRNA), while soluble RAGE was used as a competitive inhibitor of RAGE ligand binding. RAGE, S100B and cytokines were detected using quantitative RT-PCR, western blotting, cytokine protein arrays or ELISA. Activation of mitogen-activated protein kinase (MAPK) by RAGE was determined by western blotting.

Results: Compared with non-diabetic controls, RAGE and S100B were significantly elevated in the diabetic retina with apparent localisation in the Müller glia, occurring concomitantly with upregulation of GFAP. Exposure of MIO-M1 cells to high glucose induced increased production of RAGE and S100B. RAGE signalling via MAPK pathway was linked to cytokine production. Blockade of RAGE prevented cytokine responses induced by high glucose and S100B in Müller glia.

Conclusions/interpretation: Hyperglycaemia in vivo and in vitro exposure to high glucose induce upregulation of RAGE and its ligands, leading to RAGE signalling, which links to pro-inflammatory responses by retinal Müller glia. These data shed light on the potential clinical application of RAGE blockade to inhibit the progression of diabetic retinopathy.