CTC-5: A novel digital pathology approach to characterise circulating tumour cell biodiversity.

Metastatic progression and tumor evolution complicates the clinical management of cancer patients. Circulating tumor cell (CTC) characterization is a growing discipline that aims to elucidate tumor metastasis and evolution processes. CTCs offer the clinical potential to monitor cancer patients for therapy response, disease relapse, and screen 'at risk' groups for the onset of malignancy. However, such clinical utility is currently limited to breast, prostate, and colorectal cancer patients. Further understanding of the basic CTC biology of other malignancies is required to progress them towards clinical utility. Unfortunately, such basic clinical research is often limited by restrictive characterization methods and high-cost barrier to entry for CTC isolation and imaging infrastructure. As experimental clinical results on applications of CTC are accumulating, it is becoming clear that a two-tier system of CTC isolation and characterization is required. The first tier is to facilitate basic research into CTC characterization. This basic research then informs a second tier specialised in clinical prognostic and diagnostic testing. This study presented in this manuscript describes the development and application of a low-cost, CTC isolation and characterization pipeline; CTC-5. This approach uses an established 'isolation by size' approach (ScreenCell Cyto) and combines histochemical morphology stains and multiparametric immunofluorescence on the same isolated CTCs. This enables capture and characterization of CTCs independent of biomarker-based pre-selection and accommodates both single CTCs and clusters of CTCs. Additionally, the developed open-source software is provided to facilitate the synchronization of microscopy data from multiple sources (https://github.com/CTC5/). This enables high parameter histochemical and immunofluorescent analysis of CTCs with existing microscopy infrastructure without investment in CTC specific imaging hardware. Our approach confirmed by the number of successful tests represents a potential major advance towards highly accessible low-cost technology aiming at the basic research tier of CTC isolation and characterization. The biomarker independent approach facilitates closing the gap between malignancies with poorly, and well-defined CTC phenotypes. As is currently the case for some of the most commonly occurring breast, prostate and colorectal cancers, such advances will ultimately benefit the patient, as early detection of relapse or onset of malignancy strongly correlates with their prognosis.

Apocynin and ebselen reduce influenza A virus-induced lung inflammation in cigarette smoke-exposed mice.

Influenza A virus (IAV) infections are a common cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). Oxidative stress is increased in COPD, IAV-induced lung inflammation and AECOPD. Therefore, we investigated whether targeting oxidative stress with the Nox2 oxidase inhibitors and ROS scavengers, apocynin and ebselen could ameliorate lung inflammation in a mouse model of AECOPD. Male BALB/c mice were exposed to cigarette smoke (CS) generated from 9 cigarettes per day for 4 days. On day 5, mice were infected with 1 × 10(4.5) PFUs of the IAV Mem71 (H3N1). BALF inflammation, viral titers, superoxide production and whole lung cytokine, chemokine and protease mRNA expression were assessed 3 and 7 days post infection. IAV infection resulted in a greater increase in BALF inflammation in mice that had been exposed to CS compared to non-smoking mice. This increase in BALF inflammation in CS-exposed mice caused by IAV infection was associated with elevated gene expression of pro-inflammatory cytokines, chemokines and proteases, compared to CS alone mice. Apocynin and ebselen significantly reduced the exacerbated BALF inflammation and pro-inflammatory cytokine, chemokine and protease expression caused by IAV infection in CS mice. Targeting oxidative stress using apocynin and ebselen reduces IAV-induced lung inflammation in CS-exposed mice and may be therapeutically exploited to alleviate AECOPD.

NOX1 deficiency in apolipoprotein E-knockout mice is associated with elevated plasma lipids and enhanced atherosclerosis.

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.

Influenza A virus and TLR7 activation potentiate NOX2 oxidase-dependent ROS production in macrophages.

Influenza A virus infects resident alveolar macrophages in the respiratory tract resulting in Toll like receptor 7 (TLR7) activation that triggers an inflammatory response to resolve the infection. Macrophages are also major sources of reactive oxygen species (ROS) via the NOX2-containing NADPH oxidase. Although ROS are crucial for pathogen clearance, in response to influenza A virus, ROS are touted as being culprit mediators of the lung tissue injury. The aim of the present study was to determine whether influenza A virus infection and TLR7 activation of macrophages, results in alterations in their ROS production. Here we demonstrate using immunofluorescence that influenza A virus (Hong Kong X-31 strain; H3N2) internalizes in RAW264.7 cells and mouse alveolar macrophages within 1 h, resulting in a significant enhancement in the stimulated NOX2 oxidase-dependent oxidative burst, although virus had no effect on basal ROS. The specific TLR7 agonist imiquimod (10 µg/ml) elevated basal superoxide production and, in a similar fashion to influenza A virus, enhanced NOX2 oxidase-dependent oxidative burst. By contrast, the TLR3 agonist, poly I:C (1-100 µg/ml) failed to influence the oxidative burst to NOX2 oxidase. A peptide corresponding to the region 337-348 on p47phox conjugated to a HIV-tat, designed to inhibit the phosphorylation of Ser346 on p47phox suppressed the influenza A virus- and imiquimod-induced enhancement in the oxidative burst. In conclusion, this study demonstrates for the first time that influenza A virus and TLR7 activation enhance the NOX2 oxidase-dependent oxidative burst in macrophages, which might underpin the acute lung injury to influenza A virus infection.

Nitrergic relaxation of the mouse gastric fundus is mediated by cyclic GMP-dependent and ryanodine-sensitive mechanisms.

1. Ryanodine-sensitive, Ca(2+) release ('Ca(2+) sparks') from the sarcoplasmic reticulum (SR) can activate plasmalemmal Ca(2+)-activated K(+) channels (K(Ca)) to cause membrane hyperpolarization and smooth muscle relaxation. Since cyclic guanosine monophosphate (cyclic GMP) can modulate Ca(2+) spark activity, the aim of the present study was to determine if Ca(2+) spark-like events are involved in NO-dependent, NANC relaxations to electrical field stimulation (EFS) of mouse, longitudinal smooth muscle of the gastric fundus in isolated strips contracted to approximately 40% of their maximum contraction. 2. NANC relaxations to EFS were almost abolished by both the NO synthase inhibitor, N(G)-nitro-L-arginine (L-NOARG; 100 microM) and the guanylate cyclase inhibitor, 1-H-oxodiazol-[1,2,4]-[4,3-alpha] quinoxaline-1-one (ODQ; 10 microM). Also, ODQ abolished relaxations to the NO donor, sodium nitroprusside (SNP; 1 nM - 30 microM). NANC relaxations and SNP-evoked relaxations were both partly ryanodine (10 microM)- and nifedipine (0.3 microM)-sensitive, but in each case, the inhibitory effects of ryanodine and nifedipine were additive. 3. Apamin (1 microM), charybdotoxin (0.1 microM), iberiotoxin (0.1 microM), tetraethylammonium (TEA; 1 mM), glibenclamide (10 microM) and 4-aminopyridine (1 mM) had no effect on either NANC- or SNP-evoked relaxations, the latter of which were also unaffected by high extracellular K(+) (68 mM). 4. Caffeine (0.1 - 1 mM) caused concentration-dependent relaxations of gastric fundus which were inhibited by ryanodine but unaffected by L-NOARG. 5. Relaxation to ATP (30 microM) was abolished by nifedipine, partly inhibited by apamin and ryanodine, but was unaffected by L-NOARG. 6. In conclusion, the results of the present study show that nitrergic relaxations in the mouse longitudinal gastric fundus occur via a cyclic GMP-activated ryanodine-sensitive mechanism, which does not appear to involve activation of K(+) channels.

Apamin-sensitive, non-nitric oxide (NO) endothelium-dependent relaxations to bradykinin in the bovine isolated coronary artery: no role for cytochrome P450 and K+.

Since cytochrome P(450)-derived metabolites of arachidonic acid and K(+) have been implicated in endothelium-derived hyperpolarizing factor (EDHF)-dependent responses, the aim of this study was to determine whether such factors contribute to non-nitric oxide (NO), endothelium-dependent relaxation to bradykinin (BK) in bovine isolated coronary artery. In rings of artery contracted with U46619 and treated with indomethacin (3 microM) and N(G)-nitro-L-arginine (L-NOARG; 100 microM), relaxation to BK (0.01 nM-0.3 microM) was blocked by approximately 60% after inhibition of K(+) channels with either high extracellular K(+) (high [K(+)](o); 15 - 67 mM) or apamin (0.3 microM). Ouabain (1 microM), an inhibitor of Na(+)/K(+)-ATPase, decreased the sensitivity to BK without affecting the maximum response. In L-NOARG-treated rings, ouabain had no further effect on the relaxation to BK. An inhibitor of inward-rectifying K(+) channels, Ba(2+) (30 microM), had no effect on relaxations to BK in the absence or presence of either L-NOARG or ouabain. KCl (2.5 - 10 mM) elicited small relaxations ( approximately 20%) that were abolished by nifedipine (0.3 microM) and ouabain. Both the high [K(+)](o)/apamin-sensitive relaxation to BK, and the relaxation to the K(ATP) channel-opener, levcromakalim (0.6 microM), were unaffected by the cytochrome P(450) inhibitor, 7-ethoxyresorufin (10 microM), or by co-treatment with a phospholipase A(2) inhibitor, arachidonyl trifluoromethyl ketone (AACOCF(3); 3 microM) and a diacylglycerol (DAG)-lipase inhibitor, 1, 6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC 80267; 30 microM). The non-NO/high [K(+)](o)-insensitive, approximately 40% relaxation to BK was, however, abolished by these treatments. Therefore, neither cytochrome P(450)-derived metabolites of arachidonic acid nor K(+) appear to mediate the EDHF-like relaxation to BK (i.e the non-NO, high [K(+)](o)/apamin-sensitive component) in bovine coronary arteries. Cytochrome P(450)-derived metabolites may be released at higher BK concentrations to act in parallel with NO and the high [K(+)](o)/apamin-sensitive mechanism.

Protease-activated receptors mediate apamin-sensitive relaxation of mouse and guinea pig gastrointestinal smooth muscle.

BACKGROUND & AIMS: Protease-activated receptor (PAR)-1 and PAR-2 are expressed on gastrointestinal smooth muscle, but knowledge of their functionality is limited. The aim of this study was to determine if PAR-1 and PAR-2 mediate gastrointestinal smooth muscle relaxation and to clarify the underlying mechanisms. METHODS: Responses to PAR activation using the serine proteases thrombin and trypsin and the peptide agonists for PAR-1 and PAR-2, SFLLRN-NH2 and SLIGRL-NH2, respectively, were investigated in submaximally contracted longitudinal strips of mouse gastric fundus and guinea pig taenia coli. RESULTS: In mouse gastric fundus, both thrombin and trypsin caused relaxations followed by contractions. SFLLRN-NH2 and SLIGRL-NH2 caused similar biphasic responses, the relaxation components of which were eliminated by apamin or ryanodine. For SFLLRN-NH2, apamin and ryanodine revealed contractions. Nifedipine inhibited both relaxations and contractions to each peptide. In guinea-pig taenia coli, thrombin but not trypsin caused relaxation, whereas SFLLRN-NH2 and SLIGRL-NH2 caused concentration-dependent relaxations that were eliminated by apamin but were unaffected by ryanodine. CONCLUSIONS: The mouse gastric fundus and guinea pig taenia coli contain functional PAR-1 and PAR-2 that mediate relaxations via ryanodine-sensitive and -insensitive activation of small-conductance, Ca2+-activated K+ channels. We propose that smooth muscle PARs act as sensors for inflammatory signals in gut and respond by inhibiting gut motility during peritoneal infections or tissue damage.

Apamin- and nitric oxide-sensitive biphasic non-adrenergic non-cholinergic inhibitory junction potentials in the rat anococcygeus muscle.

1. Changes in membrane potential following electrical field stimulation (EFS; 1, 2 and 5 pulses at 5 Hz, 0.5 ms duration, 60-80 V) of non-adrenergic non-cholinergic (NANC) inhibitory nerves in the rat isolated anococcygeus muscle were measured using standard intracellular recording techniques. Resting membrane potential ranged between -60 and -70 mV. 2. In the presence of guanethidine (30 microM), atropine (1 microM), propranolol (1 microM) and phentolamine (0.05 microM) to establish NANC conditions, the membrane potential depolarized to between -40 and -50 mV. Under these conditions, EFS caused pulse-dependent, tetrodotoxin (1 microM)-sensitive biphasic inhibitory junction potentials (IJPs) comprising a fast onset and time-to-peak phase followed by a second, slower phase that delayed repolarization. The duration of NANC IJPs ranged between 10 and 20 s. 3. Inhibition of small-conductance Ca2+-activated K+ channels with apamin (0.1 microM) selectively blocked the first fast phase of the NANC IJP, whereas inhibitors of large-conductance Ca2+-activated K+ channels (charybdotoxin and iberiotoxin) and ATP-sensitive K+ channels (glibenclamide) all had no effect on NANC IJPs. 4. Both the nitric oxide synthase inhibitor N G-nitro-L-arginine (L-NOARG; 100 microM) and the inhibitor of soluble guanylate cyclase 1-H-oxodiazol-[1,2,4]-[4,3-a] quinoxaline-1-one (ODQ; 10 microM) had no effect on the first fast phase of the NANC IJP. Each treatment, however, markedly inhibited the slow phase with the duration of the IJP reduced to between 1 and 3 s. The L-NOARG-resistant fast phase of the NANC IJP was almost abolished by the subsequent addition of apamin (0.1 microM). 5. In conclusion, the present study demonstrates unequivocal NANC nerve-mediated biphasic IJPs in the rat isolated anococcygeus. We propose that nitric oxide (NO), via activation of cGMP-dependent K+ channels, and a non-NO inhibitory factor which activates apamin-sensitive K+ channels contribute to NANC nerve-evoked IJPs in the rat anococcygeus.

Evidence that NO acts as a redundant NANC inhibitory neurotransmitter in the guinea-pig isolated taenia coli.

1. The relative contribution of the putative transmitters, nitric oxide (NO) and an apamin-sensitive factor, possibly ATP, to inhibitory responses evoked by electrical field stimulation (EFS; 0.2-5 Hz, 0.2 ms duration, supra-maximal voltage for 10 s) of non-adrenergic, non-cholinergic (NANC) nerves was investigated in the guinea-pig isolated taenia coli contracted with histamine (1 microM). 2. Peak relaxations to EFS (0.2-5 Hz) were tetrodotoxin (1 microM)-sensitive, maximal at 0.2 Hz and completely resistant to the nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG; 100 microM) in either the presence or absence of atropine (1 microM). Furthermore, the specific inhibitor of soluble guanylyl cyclase, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxaline-1-one (ODQ; 10 microM), the cytochrome P450 inhibitor and free radical generator, 7-ethoxyresorufin (7-ER; 10 microM) and the NO scavenger, oxyhaemoglobin (HbO; 30 microM) had no effect on EFS-induced relaxations alone and in combination with L-NOARG (100 microM). 3. Maximum relaxation to the NO donor, sodium nitroprusside (SNP; 1 microM) was significantly reduced by HbO (30 microM), abolished by 7-ER (10 microM) and ODQ (10 microM) but was unaffected by apamin (0.1 microM), an inhibitor of small conductance Ca(2+)-activated K+ channels. 4. The relaxation to EFS at 0.2 Hz was resistant to apamin but those to 0.5 and 5 Hz were significantly reduced. EFS (0.2-5 Hz)-evoked relaxations that persisted in the presence of apamin were further significantly inhibited by L-NOARG (100 microM) or ODQ (10 microM), but not by HbO (30 microM) or 7-ER (10 microM). 5. ATP (1-30 microM) produced concentration-dependent relaxations that were abolished by apamin (0.1 microM), unaffected by ODQ (10 microM) but only significantly reduced by L-NOARG (100 microM) at the lowest concentration of ATP (1 microM) used. 6. Nifedipine (0.3 microM), abolished contractions to 67 mM KCl, histamine (10 microM), endothelin-1 (0.03 microM), 5-hydroxytryptamine (5-HT; 10 microM) and the thromboxane-mimetic, 9-11-dideoxy-9 alpha, 11 alpha-methano-epoxy-prostaglandin F2 alpha (U46619; 0.1 microM). 7. The findings of the present study suggest that NO is released during NANC nerve stimulation, but plays no role in NANC relaxations in the guinea-pig taenia coli unless the effects of another apamin-sensitive, nerve-derived hyperpolarizing factor (NDHF) are blocked. Thus, we propose that in this tissue, NO acts as a 'backup' or redundant NANC nerve inhibitory transmitter and like NDHF mediates relaxation via hyperpolarization.

Evidence that both nitric oxide (NO) and a non-NO hyperpolarizing factor elicit NANC nerve-mediated relaxation in the rat isolated anocococcygeus.

1. Responses to electrical field stimulation (EFS; 0.5-10 Hz, 0.2 ms duration, supramaximal voltage for 20 s) of non-adrenergic, non-cholinergic, (NANC) nerves were obtained in preparations of rat anococcygeus pre-contracted with titrated concentrations of phenylephrine (0.1-1 microM) to approximately 40% of their maximum contraction to phenylephrine (Fmax) regardless of drug treatment. 2. With this set level of active force, NANC nerve stimulation resulted in relaxations that were maximal (peak relaxation) at 0.5-1 Hz, abolished by tetrodotoxin (1 microM) but only minimally blocked by the nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine, (L-NOARG; 100 microM). Furthermore, the nitric oxide (NO) scavenger, oxyhaemoglobin (HbO; 30 microM) gave no further block alone or in combination with L-NOARG (100 microM). By comparison, in preparations contracted with phenylephrine to approximately 70% Fmax, relaxations to NANC nerve stimulation were markedly reduced or abolished by combined treatment with L-NOARG (100 microM) and HbO (30 microM). 3. Nifedipine (0.3 microM) significantly inhibited NANC nerve-mediated relaxations, which became frequency-dependent and abolished those resistant to L-NOARG (100 microM) and HbO (30 microM). 4. These data suggest that a non-NO, hyperpolarizing factor and NO both contribute to NANC nerve-mediated inhibitory responses in the rat anococcygeus. However, responses to the non-NO factor were observed only in preparations contracted sub-maximally by a nifedipine-sensitive mechanism.