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IL-12 is a pro-inflammatory cytokine that induces the production of interferon-γ (IFNγ) and favours the differentiation of T helper 1 (Th1) cells. In the presence of IL-12 human Treg cells acquire a Th1-like phenotype with reduced suppressive activity in vitro. Primary biliary cholangitis (PBC) is an autoimmune cholestatic liver disease characterised by high Th1 and Th17 infiltrating cells, reduced frequencies of Treg cells, and a genetic association with IL-12 signalling. Herein, we sought to evaluate the IL-12 signalling pathway in PBC pathology, by studying human samples from patients with PBC, alongside those with primary Sjögren's syndrome (pSS)(autoimmune disease with IL-12 signalling gene association), primary sclerosing cholangitis (PSC) (cholestatic liver disease without IL-12 gene association) and healthy individuals. Our data revealed that TLR stimulation of PBC (n = 17) and pSS monocytes (n = 6) resulted in significant induction of IL12A mRNA (p 0.05, p 0.01, respectively) compared to PSC monocytes (n = 13) and at similar levels to HC monocytes (n = 8). PSC monocytes expressed significantly less IL-12p70 (108 pg/ml, mean) and IL-23 (358 pg/ml) compared to HC (458 pg/ml and 951 pg/ml, respectively) (p 0.01, p 0.05). Treg cells from patients with PBC (n = 16) and pSS (n = 3) but not PSC (n = 10) and HC (n = 8) responded to low dose (10 ng/ml) IL-12 stimulation by significant upregulation of IFNγ (mean 277 and 254 pg/ml, respectively) compared to PSC and HC Treg cells (mean 22 and 77 pg/ml, respectively)(p 0.05). This effect was mediated by the rapid and strong phosphorylation of STAT4 on Treg cells from patients with PBC and pSS (p 0.05) but not PSC and HC. In the liver of patients with PBC (n = 7) a significantly higher proportion of IL-12Rβ2

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity.

Highlights • Curcumin + FOLFOX inhibits growth of primary cancer stem cell (CSC) spheroid models. • Curcumin + FOLFOX decreases expression of CSC markers in primary CSC spheroid models. • Curcumin enhances proapoptotic effects of chemotherapy in explant culture. • Curcumin is safe and tolerable in combination with FOLFOX chemotherapy. • Curcumin is perceived by patients as an acceptable daily adjunct to chemotherapy., In vitro and pre-clinical studies have suggested that addition of the diet-derived agent curcumin may provide a suitable adjunct to enhance efficacy of chemotherapy in models of colorectal cancer. However, the majority of evidence for this currently derives from established cell lines. Here, we utilised patient-derived colorectal liver metastases (CRLM) to assess whether curcumin may provide added benefit over 5-fluorouracil (5-FU) and oxaliplatin (FOLFOX) in cancer stem cell (CSC) models. Combination of curcumin with FOLFOX chemotherapy was then assessed clinically in a phase I dose escalation study. Curcumin alone and in combination significantly reduced spheroid number in CRLM CSC models, and decreased the number of cells with high aldehyde dehydrogenase activity (ALDHhigh/CD133−). Addition of curcumin to oxaliplatin/5-FU enhanced anti-proliferative and pro-apoptotic effects in a proportion of patient-derived explants, whilst reducing expression of stem cell-associated markers ALDH and CD133. The phase I dose escalation study revealed curcumin to be a safe and tolerable adjunct to FOLFOX chemotherapy in patients with CRLM (n = 12) at doses up to 2 grams daily. Curcumin may provide added benefit in subsets of patients when administered with FOLFOX, and is a well-tolerated chemotherapy adjunct.

The sympathetic nervous system is frequently activated in hypertension and may modify various aspects of renal function. Whether modulation of the sympathetic nervous system directly influences the development of renal fibrosis is yet to be established. The current study investigates the role of the alpha-1 adrenoceptor on human mesangial cell scarring.Human mesangial cells were injured with macrophage-conditioned medium (MPCM) and treated with doxazosin for 1 or 3 days.alpha-1 Adrenoceptor antagonist doxazosin of 2 micromol/l reduced fibronectin protein in MPCM-injured female mesangial cells by 31 +/- 1.03% (P0.001) and by 9.5 +/- 0.3% (P = 0.01) in male mesangial cells. The differential response between sexes was significant (P = 0.004). alpha-1B Adrenoceptors were detected in human mesangial cells by reverse transcription-polymerase chain reaction with expression in female cells being 87% higher than in males (P = 0.04). Injury with MPCM reduced alpha-1B adrenoceptor mRNA expression in both cell types. Doxazosin had no effect on the protein levels of transforming growth factor-beta (TGF-beta) or interleukin-1beta (IL-1beta), however, a small reduction in tumour necrosis factor-alpha (TNF-alpha) levels was observed. Doxazosin had no effect on the modulators of matrix turnover matrix metalloproteinases MMP3, MMP9 and tissue inhibitor of matrix metalloproteinases (TIMP-1), although a significant reduction in tissue plasminogen activator (tPA); (36.5 +/- 2.6%, P0.001) was observed. Doxazosin caused an up-regulation of kallikrein expression, both at mRNA and protein levels. Co-treatment with the bradykinin B2 receptor antagonist HOE140 was able to attenuate the effects of doxazosin treatment on fibronectin levels.These data suggest that inhibition of alpha-1B adrenoceptors in mesangial cells exerts an anti-fibrotic effect in a sex-specific manner via modulation of the kallikrein-kinin/plasminogen activator system.

The role of bradykinin in the antifibrotic actions of perindoprilat on human mesangial cells.BackgroundAngiotensin-converting enzyme inhibitors (ACE-I) protect against the development of glomerulosclerosis using mechanisms partly dissociated from their systemic antihypertensive action. The aim of the current study was to delineate the mechanism of action underlying the antifibrotic effects of the ACE-I perindoprilat in the context of macrophage-mediated scarring in human mesangial cells.MethodsMesangial cells were treated with macrophage-conditioned medium (MPCM) in the presence or absence of the ACE-I perindoprilat.ResultsForty μmol/L perindoprilat reduced MPCM-induced mesangial cell fibronectin levels by 19.4 ± 0.6% (P < 0.001). Immunoprecipitation of 35S-methionine biosynthetically labeled fibronectin and Northern analysis suggested that the decrease in fibronectin levels was not caused by reduced synthesis. MPCM stimulated the production of matrix metalloproteinases (MMP) 2, 3, and 9 in mesangial cells; however, these were not significantly altered by ACE-I treatment, and neither was production of their tissue inhibitor of metalloproteinases (TIMP-1). Addition of exogenous bradykinin to MPCM-treated mesangial cells resulted in a 22.5 ± 1.4% (P < 0.02) reduction in secreted fibronectin levels, while semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and Southern blotting demonstrated that bradykinin B2 receptor expression was up regulated by 71 ± 30% in MPCM-stimulated mesangial cells in response to ACE-I treatment (P = 0.032). Moreover, the bradykinin B2 receptor antagonist HOE 140 attenuated the beneficial effects of perindoprilat. MPCM-stimulated mesangial cell protein expression levels of plasminogen activator system components tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) were altered after treatment with ACE-I.ConclusionThese results suggest that ACE-I-induced renoprotection, in the context of macrophage-stimulated mesangial cell scarring, is mediated, at least in part, via the actions of bradykinin.

Shade avoidance in higher plants is regulated by the action of multiple phytochrome (phy) species that detect changes in the red/far-red ratio (R/FR) of incident light and initiate a redirection of growth and an acceleration of flowering. The phyB mutant of Arabidopsis is constitutively elongated and early flowering and displays attenuated responses to both reduced R/FR and end-of-day far-red light, conditions that induce strong shade-avoidance reactions in wild-type plants. This indicates that phyB plays an important role in the control of shade avoidance. In Arabidopsis phyB and phyD are the products of a recently duplicated gene and share approximately 80% identity. We investigated the role played by phyD in shade avoidance by analyzing the responses of phyD-deficient mutants. Compared with the monogenic phyB mutant, the phyB-phyDdouble mutant flowers early and has a smaller leaf area, phenotypes that are characteristic of shade avoidance. Furthermore, compared with the monogenic phyB mutant, the phyB-phyDdouble mutant shows a more attenuated response to a reduced R/FR for these responses. Compared with the phyA-phyB double mutant, the phyA-phyB-phyD triple mutant has elongated petioles and displays an enhanced elongation of internodes in response to end-of-day far-red light. These characteristics indicate that phyD acts in the shade-avoidance syndrome by controlling flowering time and leaf area and that phyC and/or phyE also play a role.

Background Albuminuria and elevated C-reactive protein (CRP) levels are common manifestations of many inflammatory diseases. Cardiovascular-based drugs, with secondary anti-inflammatory actions, such as angiotensin-converting enzyme-inhibitors are able to reduce both proteinuria and CRP levels, raising the question of whether CRP directly influences the processes that result in proteinuria. As proteinuria is thought to be induced as a result of podocyte dysfunction, we investigated whether there is a pathomechanistic link with CRP. Methods Podocytes were analysed for evidence of endogenous CRP production in response to inflammatory agents. In addition, they were incubated in the presence of various concentrations of exogenous CRP and analysed for evidence of a response to treatment. Results Our results demonstrated that inflammatory agents such as macrophage-conditioned medium and interleukin-1β induced the expression of CRP messenger RNA in podocytes. However, they were unable to induce CRP protein. Stimulation of podocytes with exogenous CRP demonstrated that 10 μg/mL CRP induced a low but significant level of interleukin-6 secretion. Tumour necrosis factor α, however, was not detected. CRP did up-regulate the expression of the slit diaphragm proteins nephrin and CD2AP, as well as the structural proteins ezrin and podocalyxin-like protein-1, proteins known to be involved in signalling via the phosphotidylinositol-3 (PI-3) kinase pathway. CRP exposure reduced caspase-3 enzyme activity and up-regulated the expression of the anti-apoptotic protein Bcl-2. In the presence of the PI-3 kinase inhibitor LY294002, the ability of CRP to suppress caspase-3 activity was significantly reduced. Conclusions Taken together, these data suggest that rather than inducing podocyte damage, CRP may be a survival factor for podocytes by maintaining their structural integrity and initiating a survival cascade, which may facilitate podocyte recovery from injury.

Low density lipoprotein receptor-related protein (LRP) is a multifunctional endocytic receptor implicated in the modulation of a number of cellular processes, including the turnover of proteases and the degradation of extracellular matrix proteins. As such, it can play a key role in the control of fibrosis. The aim of this investigation was to ascertain whether the anti-fibrotic effects exerted by the angiotensin-converting enzyme inhibitor (ACE-I) perindoprilat on macrophage-conditioned medium (MPCM)-injured human mesangial cells can be modulated by this receptor. Addition of receptor-associated protein to MPCM-injured mesangial cells with and without ACE-I increased the amount of tissue plasminogen activator protein detected in mesangial cell culture supernatants without affecting the protein levels of plasminogen activator inhibitor-1. The ability of ACE-I to reduce fibronectin was diminished in the presence of receptor-associated protein. ACE-I induced an increase in mesangial cell MMP9 mRNA, but reduced the MMP9 enzyme activity detected in mesangial cell supernatants. Mesangial cell lysates from ACE-I-treated cells were able to bind immobilized fibronectin at higher dilutions than cell lysates from untreated cells. Flow cytometry showed that MPCM induced an increase in LRP surface expression in mesangial cells over that in control cells and that this expression was further increased by ACE-I treatment. The increase in LRP expression in response to ACE-I was also observed by Western blotting. Northern blot analysis of RNA extracted from cells following a 24-h exposure to MPCM with and without ACE-I demonstrated that there was no change in LRP mRNA expression upon ACE-I treatment. In conclusion, we show that ACE-I treatment is able to modulate mesangial cell-surface expression of LRP, providing an additional mechanism whereby ACE-Is can mediate anti-fibrotic actions independent of their hemodynamic actions.

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40 micromol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treatment, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.

Several polymorphisms in genes of the reninangiotensin-aldosterone system have been found to have pleiotropic effects on cardiovascular disorders. Recently, a polymorphism (-344 C/T) in the promoter region of the aldosterone synthase gene (CYP11B2), which may influence plasma aldosterone levels, has been reported to strongly influence left ventricular diameters and mass in young adults and arterial stiffness in essential hypertensives. We investigated any association with risk of myocardial infarction (MI). CYP11B2 -344 polymorphism genotypes were determined by polymerase chain reaction (PCR) in 542 acute MI cases and 500 control subjects without history of coronary disease. All subjects were white and

Plants have evolved exquisite sensory systems for monitoring their light environment. The intensity, quality, direction and duration of light are continuously monitored by the plant and the information gained is used to modulate all aspects of plant development. Several classes of distinct photoreceptors, sensitive to different regions of the light spectrum, mediate the developmental responses of plants to light signals. The red–far–red light–absorbing, reversibly photochromic phytochromes are perhaps the best characterized of these. Higher plants possess a family of phytochromes, the apoproteins of which are encoded by a small, divergent gene family. Arabidopsis has five apophytochrome–encoding genes, PHYA–PHYE . Different phytochromes have discrete biochemical and physiological properties, are differentially expressed and are involved in the perception of different light signals. Photoreceptor and signal transduction mutants of Arabidopsis are proving to be valuable tools in the molecular dissection of photomorphogenesis. Mutants deficient in four of the five phytochromes have now been isolated. Their analysis indicates considerable overlap in the physiological functions of different phytochromes. In addition, mutants defining components acting downstream of the phytochromes have provided evidence that different members of the family use different signalling pathways.

Seven cytologically distinct stages during micro spore development were identified and used to define the activation of an Arabidopsis anther-specific gene (apg) in transgenic tobacco plants containing an apg promoter-gus fusion. Histochemical analysis of GUS activity showed that the apg promoter was activated in miduninucleate microspores prior to equatorial and polar nuclear migration. Quantitative analysis in isolated spores showed that GUS activity per milligram of protein decreased progressively during pollen development, but on a per spore basis showed a second peak of activity in mid-bicellular pollen. Activation of the apg promoter in isolated gametophytic cells during development was also investigated following DNA delivery by microprojectile bombardment. Levels of transient expression were detectable in uninucleate microspores, but peaked in mid-bicellular pollen, in contrast to the progressive increase in activity of the promoter of the ‘late’ pollen gene lat52. These data show that the apg promoter is activated in a biphasic pattern, and indicate that the activity of transcription factors which mediate apg promoter activity persist through microspore mitosis, but decrease during pollen maturation.

From a screen of M2 seedlings derived from gamma-mutagenesis of seeds doubly null for phytochromes phyA and phyB, we isolated a mutant lacking phyE. The PHYE gene of the selected mutant, phyE-1, was found to contain a 1-bp deletion at a position equivalent to codon 726, which is predicted to result in a premature stop at codon 739. Immunoblot analysis showed that the phyE protein was undetectable in the phyE-1 mutant. In the phyA- and phyB-deficient background, phyE deficiency led to early flowering, elongation of internodes between adjacent rosette leaves, and reduced petiole elongation. This is a phenocopy of the response of phyA phyB seedlings to end-of-day far-red light treatments. Furthermore, a phyE deficiency attenuated the responses of phyA phyB seedlings to end-of-day far-red light treatments. Monogenic phyE mutants were indistinguishable from wild-type seedlings. However, phyB phyE double mutants flowered earlier and had longer petioles than did phyB mutants. The elongation and flowering responses conferred by phyE deficiency are typical of shade avoidance responses to the low red/far-red ratio. We conclude that in conjunction with phyB and to a lesser extent with phyD, phyE functions in the regulation of the shade avoidance syndrome.

We describe a rapid transient assay system for analysing the efficacy of diphtheria toxin A chain (DT-A) derivatives as agents of genetic cell ablation in plant tissues. Because of the extreme cytotoxic effects of DT-A in eukaryotic cells we are investigating the use of temperature sensitive (DT-A tsm ) and attenuated (tox 176) derivatives of DT-A to generate more intermediate phenotypes. The relative toxicities of DT-A, DT-A tsm and tox 176 were measured by determining their effects on the expression of a co-transfected luciferase reporter gene construct, using microprojectile bombardment into plant tissues. In pollen and leaves incubated at 16°C, the expression of DT-A or DT-A tsm resulted in a significant reduction in luciferase (LUC) activity. However, at 30°C the expression of DT-A tsm had no effect on the level of LUC activity, in contrast to the DT-A gene, which abolished LUC activity. Furthermore, at the lower permissive temperature DT-A tsm was between 50- and 200-fold less toxic than DT-A. Similar co-bombardment experiments showed that, in contrast to the mammalian systems, the toxicity of tox 176 was not attenuated relative to DT-A.