Toll-like receptor-induced expression of epithelial cytokine receptors on haemopoietic progenitors is altered in allergic asthma.

BACKGROUND: Haemopoietic progenitor cells (HPC) migrate to sites of allergic inflammation where, upon stimulation with epithelial cytokines, they produce Th2 cytokines and differentiate into mature eosinophils and basophils. They also express Toll-like receptors (TLR) involved in antimicrobial responses. OBJECTIVE: The objective of this study was to compare TLR expression on peripheral blood HPC and TLR-induced responses, in particular changes in epithelial cytokine receptors, in healthy and asthmatic subjects at baseline and following allergen challenge. METHODS: Ten healthy and 11 allergic asthmatic subjects were studied. HPC-enriched cell populations were stimulated with TLR-2, TLR-4 or TLR-9 ligands. TLR expression by circulating HPC and interleukin (IL)-25 (IL-17RB), IL-33 (ST2) and thymic stromal lymphopoietin receptor (TSLPR) expression after TLR ligation were examined by flow cytometry at baseline and, in asthmatics, following allergen challenge. The effects of dexamethasone (Dex) on TLR-induced responses were also assessed. RESULTS: Asthmatics had significantly lower circulating HPC expressing TLR-2 and TLR-9 with a similar trend for TLR-4. TLR-4 stimulation of HPC yielded higher numbers of TSLPR+ cells in asthmatics compared with healthy subjects. A similar trend was seen for TLR-9 ligation, an effect further augmented by allergen inhalation. Allergen challenge also enhanced TLR-induced ST2 expression on HPC. Treatment with Dex in vitro increased TLR-4-induced TSLPR expression but had no effect on other epithelial cytokine receptors. CONCLUSIONS AND CLINICAL RELEVANCE: These data demonstrate an interaction between allergen and TLR ligand exposure in asthmatics. Allergen inhalation augments the TLR-induced inflammatory response by HPC, possibly leading to increased "in situ haemopoiesis" through up-regulation of TSLPR. These findings show that HPC may be a part of the pro-inflammatory cascade in pathogen-induced asthma exacerbation through their increased responsiveness to TLR stimulation.

T cell-mediated induction of thymic stromal lymphopoietin in differentiated human primary bronchial epithelial cells.

BACKGROUND: Inhaled peptide challenge has been shown to induce T cell-mediated, isolated late asthmatic reaction (LAR), characterized by recruitment of CD4(+) T cells and increased levels of thymus and activation-regulated chemokine (TARC; CCL17). Epithelial-derived thymic stromal lymphopoietin (TSLP) has been shown to modulate dendritic cell function to promote TH 2 responses via CCL17 production. OBJECTIVES: To elucidate the mechanisms involved in allergen-specific T cell-induced LAR and recruitment of CD4(+) T cells by examining the effects of T cell-derived factors on the induction of TSLP in primary bronchial epithelial cells (PBEC). METHODS: PBEC grown at air-liquid interface from healthy individuals and patients with asthma were stimulated with double-stranded RNA (dsRNA) or supernatants from activated allergen-specific T cells. TSLP was measured in PBEC culture supernatants. Neutralizing antibodies and signalling inhibitors were used to examine the mechanisms responsible for the induction of epithelial-derived TSLP. The functional activity of PBEC-derived TSLP was measured using a bioassay involving the induction of CCL17 production from monocyte-derived dendritic cells (moDC). RESULTS: Both dsRNA and allergen-specific T cells induced enhanced TSLP secretion from asthmatic PBEC compared to healthy PBEC. Activated PBEC culture supernatant induced TSLP-dependent CCL17 production from moDC in a manner related to clinical asthmatic status. IL-1ß, IL-6, and CXCL8, rather than TH 2 cytokines (IL-4/5/13), appeared to be the principle mediators of allergen-specific T cell-dependent induction of epithelial-derived TSLP, which was regulated by the MEK, MAPK, and NFκB pathways. CONCLUSION AND CLINICAL RELEVANCE: Our data reveal a novel effect of allergen-specific T cells as a positive regulator of TSLP production by epithelial cells, suggesting T cell-airway epithelium interactions that may lead to maintenance and amplification of allergic inflammation. TSLP is currently a candidate for therapeutic intervention in asthma, but the factors that drive TSLP expression (T cell-derived factors) may be equally relevant in the treatment of allergic inflammation.

Magnetic resonance enterography is feasible and reliable in multicenter clinical trials in patients with Crohn's disease, and may help select subjects with active inflammation.

BACKGROUND: Reliable tools for patient selection are critical for clinical drug trials. AIM: To evaluate a consensus-based, standardised magnetic resonance enterography (MRE) protocol for selecting patients for inclusion in Crohn's disease (CD) multicenter clinical trials. METHODS: This study recruited 20 patients [Crohn's Disease Activity Index (CDAI) scores: <150 (n = 8); 150-220 (n = 4); 220-450 (n = 8)], to undergo ileocolonoscopy and two MREs (with and without colonic contrast) within a 14-day period. Procedures were scored centrally using, Magnetic Resonance Index of Activity (MaRIA), and both Crohn's Disease Endoscopic Index of Severity (CDEIS) and Simplified Endoscopic Score (SES-CD). RESULTS: 37 MREs were acquired. Both MREs were evaluable in 16 patients for calculation of test-retest and inter-reader reliability scores. The MaRIA scores for the terminal ileum had excellent test-retest and inter-reader reliability, with correlations >0.9. The proximal ileum showed strong within-reader agreement (0.90-0.96), and fair between-reader agreement (0.59-0.72). MRE procedures were tolerable. MaRIA scores correlated with CDEIS and SES-CD (0.63 and 0.71), but not with CDAI (0.34). MRE identified 3 patients with intra-abdominal complications, who would otherwise have been included in clinical trials. Furthermore, both MRE and ileocolonoscopy identified active bowel wall inflammation in 2 patients with CDAI <150, and none in 1 patient with CDAI > 220. Data quality was good/excellent in 85% of scans, and fair or better in 96%. CONCLUSIONS: Magnetic resonance enterography of high-quality and reproducibility was feasible in a global multi- centre setting, with evidence for improved selectivity over CDAI and ileocolonoscopy in identifying appropriate CD patients for inclusion in therapeutic intervention trials.

Nitric oxide synthesis and isoprostane production in subjects with type 1 diabetes and normal urinary albumin excretion.

The role of nitric oxide (NO) and free radicals in the development of microvascular disease in type 1 diabetes remains unclear. We have measured NO and isoprostane (a stable marker of in vivo lipid peroxidation) production in 13 type 1 diabetic subjects with normal urinary albumin excretion and 13 healthy volunteers. Whole-body NO synthesis was quantified by measuring the urinary excretion of 15N-nitrate after the intravenous administration of L-[15N]2-arginine. The urinary excretion of the major urinary metabolite of 15-F2t-isoprostane (8-iso-prostaglandin-F2alpha), 2,3-dinor-5,6-dihydro-F2t-IsoP, was quantified as a marker of in vivo lipid peroxidation. Whole-body NO synthesis was significantly higher in diabetic subjects compared with control subjects (342 vs. 216 nmol 15N-nitrate/mmol creatinine [95% CI of the difference 45-207], P = 0.005). This increase was not explained by a difference in renal function between the 2 groups. There was no difference in 2,3-dinor-5,6-dihydro-F2t-IsoP excretion between diabetic subjects and control subjects (44.8+/-7.8 vs. 41.4+/-10.0 ng/mmol creatinine, mean +/- 95% CI). However, there was an inverse correlation between NO synthesis and free radical activity in subjects with diabetes (r = -0.62, P = 0.012) that was not observed in control subjects (r = 0.37, P = 0.107). We conclude that whole-body NO synthesis is higher in type 1 diabetic subjects with normal urinary albumin excretion than in control subjects. The inverse correlation between isoprostane production and NO synthesis in diabetic subjects is consistent with the hypothesis that NO is being inactivated by reactive oxygen species.

Plasma protein binding of lidocaine and warfarin in insulin-dependent and non-insulin-dependent diabetes mellitus.

We examined the plasma protein binding of an acidic drug (warfarin bound to albumin) and a basic drug [lidocaine (lignocaine) bound to alpha 1-acid glycoprotein] in 15 patients with insulin-dependent diabetes mellitus (IDDM) and 15 matched controls. We also examined protein binding of warfarin and lidocaine in 30 patients with non-insulin-dependent diabetes (NIDDM) and 25 controls. Compared with control, the binding of both warfarin (98.81 +/- 0.02 vs 98.57 +/- 0.03%, mean +/- SEM) and of lidocaine (69 +/- 2 vs 58 +/- 2%) was significantly reduced in IDDM. This group had lower concentrations of both albumin and alpha 1-acid glycoprotein (AAG), achieving statistical significance vs control for albumin only. In the patients with NIDDM, who had a similar level of glycosylated haemoglobin, while there was no significant difference in the binding of lidocaine there was a significant increase in warfarin binding compared with the control population (99.01 +/- 0.03 vs 98.82 +/- 0.04%). This study suggests that binding of both acidic and basic drugs is altered in both IDDM and NIDDM.

Genetics of hypertension. Therapeutic implications.

Essential hypertension affects approximately 20% of the adult population, and has a multifactorial origin arising from an interaction between susceptibility genes and environmental factors. The understanding of the molecular basis of essential hypertension may provide us with new and more specific pharmacological agents, and perhaps the ability to individualise treatment and maximise the reduction in risk of morbidity and mortality from cardiovascular disease. Hypertension due to single gene abnormalities is very rare; however, it follows a Mendelian model of inheritance and therefore can be identified successfully using family linkage studies. Since clear Mendelian models of inheritance cannot readily be assigned in essential hypertension as there may be variable penetrance of susceptibility genes, other studies with designs based on affected sibling pairs, family-based association studies and case-control studies have been performed. The renin-angiotensin system (RAS) plays an integral part in the control of blood pressure, and genetic polymorphisms within this system and their effect on the response to antihypertensive therapy are now being studied. Polymorphisms of the angiotensin converting enzyme (ACE) gene, although associated with left ventricular hypertrophy, do not appear to have a clear association with hypertension. Studies on the association of genotype with response to antihypertensive therapy are less consistent for genetic polymorphisms of the RAS. Although some of the results are positive, patient numbers have been small in the studies completed to date. Genetic polymorphisms of the adrenergic receptors have been associated with blood pressure variation in African-Americans, White Americans and African-Caribbeans. A beta 2-adrenoceptor polymorphisms exhibits agonist-mediated receptor downregulation which may lead to enhanced peripheral vasoconstriction. Therapeutic studies have not yet been completed on patients with this genotype. A further polymorphism of the alpha-adducin gene has been associated with essential hypertension. This may influence blood pressure response to sodium loading/depletion and response to long term treatment with a thiazide diuretic, but further studies are needed to clarify this. Antisense oligonucleotides targeted against genes of the RAS, e.g. angiotensinogen and the angiotensin type 1 receptor, are being modified to improve targeting and thereby reduce toxicity. However, gene therapy is unlikely to replace pharmacological therapy in the foreseeable future. The immediate goal should be to enhance our understanding of the genetic nature of essential hypertension based on the interaction of genetic makeup with the environment, with a view to individualising antihypertensive therapy.

Effects of drugs on glucose tolerance in non-insulin-dependent diabetics (Part I).

Non-insulin-dependent diabetes mellitus (NIDDM) is being increasingly diagnosed as its importance as a risk factor for the development of cardiovascular disease continues to be recognised. Good metabolic control remains a major goal of drug therapy as it decreases the severity and incidence of diabetic complications. Many drugs have been known to interfere with glucose control, either in a beneficial or, more commonly, in a deleterious fashion. Unfortunately in many instances drug-induced effects have not been looked at specifically in NIDDM. Thiazide diuretics have been shown to cause a deterioration in glucose control not only in the general population but especially in patients who have impaired glucose tolerance. While the effect appears less with potassium supplementation and the lower dosage employed nowadays, thiazide diuretics are best avoided in diabetic patients. Loop diuretics have been reported to reduce glucose control to a lesser extent than thiazides. Although indapamide would appear not to interfere with blood sugar control in NIDDM, higher doses that cause potassium loss may cause a deterioration. beta-Adrenoceptor antagonists have been reported to cause a rise in blood sugar and glycosylated haemoglobin in NIDDM. The effect may be more marked in patients on oral hypoglycaemic agents as opposed to diet alone and in those on concomitant thiazide diuretics. The greatest effect was seen with propranolol, and the least with cardioselective and the less lipophilic beta-blockers. It is of interest that alpha-blockade with prazosin seems to antagonise beta-adrenoceptor blocker-induced deterioration in glucose control. The calcium antagonists have differing effects which may be structure related. In some, but not all, studies use of the dihydropyridines such as nifedipine has been associated with a deterioration in glucose control in NIDDM. Long term studies are needed to assess definitively their effect on glucose control. Verapamil, on the other hand, has in 1 small study been found to have a beneficial effect on glucose control in NIDDM. Centrally acting alpha-agonists such as the antihypertensive drug clonidine have not been shown to result in a deterioration in glucose control when used in NIDDM, although there are isolated case reports. Long term therapy with the more specific agonist guanfacine was reported in 1 uncontrolled study to have a beneficial effect on glucose tolerance in NIDDM. Uncontrolled studies suggest that phenothiazines may aggravate diabetic control. The significance of a number of recent observations is not fully clear.(ABSTRACT TRUNCATED AT 400 WORDS)

The genetic basis of hypertension: progress and opportunities.

Essential hypertension results from the interaction of genetic and environmental factors. To date progress has mainly concerned rare genetic forms of hypertension, but molecular advances and current large collaborative studies make it likely that common genetic factors in hypertension will soon be determined.

A humanized monoclonal antibody targeting the ß7 integrin selectively blocks intestinal homing of T lymphocytes.

BACKGROUND AND PURPOSE: rhuMAb Beta7 is a humanized anti-human ß7 monoclonal antibody currently in phase I in inflammatory bowel disease. rhuMAb Beta7 binds the ß7 subunit of the integrins α4ß7 and αEß7, blocking interaction with their ligands. These integrins play key roles in immune cell homing to and retention in mucosal sites, and are associated with chronic inflammatory diseases of the gastrointestinal tract. The goal of this study was to evaluate the mucosal specificity of rhuMAb Beta7. EXPERIMENTAL APPROACH: We assessed the effect of murine anti-Beta7 on lymphocyte homing in mouse models of autoimmune disease. We also compared the effect of rhuMAb Beta7 on circulating mucosal-homing versus peripheral-homing T cells in naïve non-human primates. KEY RESULTS: In cynomolgus monkeys, occupancy of ß7 integrin receptors by rhuMAb Beta7 correlated with an increase in circulating ß7(+) mucosal-homing lymphocytes, with no apparent effect on levels of circulating ß7(-) peripheral-homing lymphocytes. rhuMAb Beta7 also inhibited lymphocyte homing to the inflamed colons of severe combined immunodeficient mice in CD45RB(high) CD4(+) T-cell transfer models. Consistent with a lack of effect on peripheral homing, in a mouse model of experimental autoimmune encephalomyelitis, anti-ß7 treatment resulted in no amelioration of CNS inflammation. CONCLUSIONS AND IMPLICATIONS: The results presented here suggest that rhuMAb Beta7 selectively blocks lymphocyte homing to the gastrointestinal tract without affecting lymphocyte trafficking to non-mucosal tissues. rhuMAb Beta7 provides a targeted therapeutic approach with the potential for a more attractive benefit:risk ratio than currently available inflammatory bowel disease therapies.

Human hepatic stellate cell lines, LX-1 and LX-2: new tools for analysis of hepatic fibrosis.

BACKGROUND: Hepatic stellate cells (HSCs) are a major fibrogenic cell type that contributes to collagen accumulation during chronic liver disease. With increasing interest in developing antifibrotic therapies, there is a need for cell lines that preserve the in vivo phenotype of human HSCs to elucidate pathways of human hepatic fibrosis. We established and characterised two human HSC cell lines termed LX-1 and LX-2, and compared their features with those of primary human stellate cells. METHODS AND RESULTS: LX-1 and LX-2 were generated by either SV40 T antigen immortalisation (LX-1) or spontaneous immortalisation in low serum conditions (LX-2). Both lines express alpha smooth muscle actin, vimentin, and glial fibrillary acid protein, as visualised by immunocytochemistry. Similar to primary HSCs, both lines express key receptors regulating hepatic fibrosis, including platelet derived growth factor receptor beta (betaPDGF-R), obese receptor long form (Ob-RL), and discoidin domain receptor 2 (DDR2), and also proteins involved in matrix remodelling; matrix metalloproteinase (MMP)-2, tissue inhibitor of matrix metalloproteinase (TIMP)-2, and MT1-MMP, as determined by western analyses. LX-2 have reduced expression of TIMP-1. LX-2, but not LX-1, proliferate in response to PDGF. Both lines express mRNAs for alpha1(I) procollagen and HSP47. Transforming growth factor beta1 stimulation increased their alpha1(I) procollagen mRNA expression, as determined by quantitative reverse transcription-polymerase chain reaction. LX-2, but not LX-1, cells are highly transfectable. Both lines had a retinoid phenotype typical of stellate cells. Microarray analyses showed strong similarity in gene expression between primary HSCs and either LX-1 (98.4%) or LX-2 (98.7%), with expression of multiple neuronal genes. CONCLUSIONS: LX-1 and LX-2 human HSC lines provide valuable new tools in the study of liver disease. Both lines retain key features of HSCs. Two unique advantages of LX-2 are their viability in serum free media and high transfectability.

ACE inhibition: postsynaptic adrenergic sympatholytic action in men.

BACKGROUND: The mechanisms by which ACE inhibitors produce a sustained clinical benefit are not entirely clear but may involve the sympathetic nervous system. We compared the effect of local brachial artery infusions of an ACE inhibitor (perindoprilat) with the effect of placebo (0.9% NaCl) on endogenously mediated (lower body negative pressure [LBNP]) and exogenously mediated (brachial artery infusions of norepinephrine) sympathetic vasoconstriction. METHODS AND RESULTS: Eight healthy, normotensive male volunteers (20 to 32 years) were studied on one occasion. Forearm blood flow (FABF; mL x dL forearm(-1) x min(-1)) responses to LBNP (-20 cm H2O) and increasing increments of norepinephrine (60, 120, and 240 pmol/min) were compared when coinfused with placebo and perindoprilat (5 nmol/mL). FABF was measured simultaneously in both arms by venous occlusion plethysmography with mercury-in-Silastic strain gauges with drugs infused locally at the left brachial artery. The right arm served as a control. Baseline FABFs did not differ between the infused and control arms (3.04+/-0.52 versus 3.05+/-0.42 mL x dL forearm(-1) x min(-1); P=.98). Perindoprilat did not alter FABF when infused alone, but the FABF response to LBNP in the infused arm was attenuated during the perindoprilat infusion compared with placebo (-17.8+/-4.3% versus -33.8+/-3.1%, respectively; P=.015). The FABF response to the maximum dose of norepinephrine was also attenuated during the perindoprilat infusion compared with placebo (-28.3+/-1.4% versus -36.9+/-2.8%, respectively; P=.015). The mean slope of the FABF (log transformed) versus norepinephrine dose-response curve was significantly attenuated by perindoprilat compared with placebo (-0.11+/-0.019 versus -0.02+/-0.02; P=.001). CONCLUSIONS: We conclude that ACE inhibition has a significant postsynaptic sympatholytic effect in the forearm circulation of men.

Inhibition of platelet aggregation with glyceryl trinitrate and xanthine oxidoreductase.

Xanthine oxidoreductase (XOR) is a mammalian enzyme that possesses a series of redox centers, which use either NAD(+) or molecular oxygen for oxidation of the purines xanthine and hypoxanthine to uric acid. The ability of XOR to act as an NADH oxidase is a less well recognized function of the enzyme, and it is this function that we used to explore the metabolism of glyceryl trinitrate. The antiplatelet effect of nitric oxide (NO) on platelet aggregation was used as a bioassay to assess the bioconversion of glyceryl trinitrate to NO by XOR. The thromboxane mimetic U46619, 2 microM, was used to stimulate platelet aggregation in platelet-rich plasma prepared from healthy drug-free human volunteers. All incubations were carried out at 37 degrees C for 2 min after the addition of U46619. XOR produced a dose-dependent antiaggregant effect when incubated with glyceryl trinitrate (GTN), 220 microM. This did not occur when GTN or XOR was incubated with platelet-rich plasma independently. The antiaggregant effect of XOR plus GTN was dose dependently inhibited by allopurinol, with an IC(50) of 100 microM. The addition of superoxide dismutase (SOD), 100 U/ml produced a shift to the left in the antiaggregant dose-response curve for XOR. The IC(50) for XOR at 200 U/l without SOD was decreased to 80 U/l with SOD. Oxyhemoglobin, an extracellular NO scavenger, produced a dose-dependent, noncompetitive inhibition of the antiaggregant effect of XOR plus GTN. These findings suggest that GTN may be reduced to NO in vitro by the enzyme XOR in sufficient amounts to inhibit platelet aggregation.

The ingestion of inorganic nitrate increases gastric S-nitrosothiol levels and inhibits platelet function in humans.

Platelets play an important role in the development of vascular disease, while vegetarian diets, which are rich in inorganic nitrate, protect against it. This study was performed to assess the effect of potassium nitrate (KNO(3)) ingestion on platelet function in humans. Oral KNO(3) (2 mmol) was given to healthy volunteers and its effect on platelet function assessed by measuring the aggregant effect of collagen. Blood samples were taken for measurement of plasma S-nitrosothiols (RSNO) and platelet cyclic GMP and nitrotyrosine levels. Gastric juice samples were taken for measurement of RSNO. In a separate study, the effect of oral KNO(3) on portal RSNO levels in patients with intrahepatic porto-systemic shunts was assessed. KNO(3) caused a significant increase in gastric RSNO levels, from 0.46 +/- 0.06 to 3.62 +/- 2.82 microM (t(max) 45 min; P < 0.001), and significantly inhibited platelet function (t(max) 60 min; P < 0.001). There was no effect on systemic or portal RSNO, platelet cGMP or platelet nitrotyrosine levels. Oral KNO(3) inhibits platelet aggregation. The time course suggests that gastric RSNO production may be involved in this effect. The protection against vascular events associated with a high intake of vegetables may be due to their high nitrate content.