Brain endothelial PPARgamma controls inflammation-induced CD4+ T cell adhesion and transmigration in vitro

An important step in the pathogenesis of multiple sclerosis is adhesion and transmigration of encephalitogenic T cells across brain endothelial cells (EC) which strongly relies on interaction with EC-expressed adhesion molecules. We provide molecular evidence that the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) is a negative regulator of brain EC inflammation. The PPARgamma agonist pioglitazone reduces transendothelial migration of encephalitogenic T cells across TNFalpha-stimulated brain EC. This effect is clearly PPARgamma mediated, as lentiviral PPARgamma overexpression in brain EC results in selective abrogation of inflammation-induced ICAM-1 and VCAM-1 upregulation and subsequent adhesion and transmigration of T cells. We therefore propose that PPARgamma in brain EC may be exploited to target detrimental EC-T cell interactions under inflammatory conditions.

Gamma-tocopherol supplementation inhibits protein nitration and ascorbate oxidation in rats with inflammation

Gamma-tocopherol (gammaT) complements alpha-tocopherol (alphaT) by trapping reactive nitrogen oxides to form a stable adduct, 5-nitro-gammaT [Christen et al., PNAS 94:3217-3222; 1997]. This observation led to the current investigation in which we studied the effects of gammaT supplementation on plasma and tissue vitamin C, vitamin E, and protein nitration before and after zymosan-induced acute peritonitis. Male Fischer 344 rats were fed for 4 weeks with either a normal chow diet with basal 32 mg alphaT/kg, or the same diet supplemented with approximately 90 mg d-gammaT/kg. Supplementation resulted in significantly higher levels of gammaT in plasma, liver, and kidney of control animals without affecting alphaT, total alphaT+gammaT or vitamin C. Intraperitoneal injection of zymosan caused a marked increase in 3-nitrotyrosine and a profound decline in vitamin C in all tissues examined. Supplementation with gammaT significantly inhibited protein nitration and ascorbate oxidation in the kidney, as indicated by the 29% and 56% reduction of kidney 3-nitrotyrosine and dehydroascorbate, respectively. Supplementation significantly attenuated inflammation-induced loss of vitamin C in the plasma (38%) and kidney (20%). Zymosan-treated animals had significantly higher plasma and tissue gammaT than nontreated pair-fed controls, and the elevation of gammaT was strongly accentuated by the supplementation. In contrast, alphaT did not significantly change in response to zymosan treatment. In untreated control animals, gammaT supplementation lowered basal levels of 3-nitrotyrosine in the kidney and buffered the starvation-induced changes in vitamin C in all tissues examined. Our study provides the first in vivo evidence that in rats with high basal amounts of alphaT, a moderate gammaT supplementation attenuates inflammation-mediated damage, and spares vitamin C during starvation-induced stress without affecting alphaT.

Analysis of plasma tocopherols alpha, gamma, and 5-nitro-gamma in rats with inflammation by HPLC coulometric detection

Reactive nitrogen oxide species (RNOS) have been implicated as effector molecules in inflammatory diseases. There is emerging evidence that gamma-tocopherol (gammaT), the major form of vitamin E in the North American diet, may play an important role in these diseases. GammaT scavenges RNOS such as peroxynitrite by forming a stable adduct, 5-nitro-gammaT (NGT). Here we describe a convenient HPLC method for the simultaneous determination of NGT, alphaT, and gammaT in blood plasma and other tissues. Coulometric detection of NGT separated on a deactivated reversed-phase column was linear over a wide range of concentrations and highly sensitive (approximately 10 fmol detection limit). NGT extracted from blood plasma of 15-week-old Fischer 344 rats was in the low nM range, representing approximately 4% of gammaT. Twenty-four h after intraperitoneal injection of zymosan, plasma NGT levels were 2-fold higher compared to fasted control animals when adjusted to gammaT or corrected for total neutral lipids, while alpha- and gammaT levels remained unchanged. These results demonstrate that nitration of gammaT is increased under inflammatory conditions and highlight the importance of RNOS reactions in the lipid phase. The present HPLC method should be helpful in clarifying the precise physiological role of gammaT.

Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) on cerebrospinal fluid inflammation induced by endotoxin

Endotoxin triggers the subarachnoid inflammation of gram-negative meningitis. This study examined the ability of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein (rBPI23) to block endotoxin-induced meningitis in rabbits. Intracisternal (ic) injection of 10-20 ng of meningococcal endotoxin induced high cerebrospinal fluid (CSF) concentrations of tumor necrosis factor (TNF) and CSF pleocytosis and increased CSF lactate concentrations. ic administration of rBPI23 significantly reduced meningococcal endotoxin-induced TNF release into CSF (P < .005), lactate concentrations (P < .001), and CSF white blood cell counts (P < .01). No such effect was observed in animals receiving intravenous rBPI23. Concentrations of rBPI23 in CSF were high after ic administration but low or undetectable after systemic administration. Thus, high concentrations of rBPI23 can effectively neutralize meningococcal endotoxin in CSF, but low CSF concentrations after systemic administration currently limit its potential usefulness as adjunctive drug treatment in gram-negative meningitis.

Hemoglobin vesicles reduce hypoxia-related inflammation in critically ischemic hamster flap tissue

OBJECTIVES: The aim of this study was to investigate the effect of a highly viscous, left-shifted hemoglobin vesicle solution (HbV) on the hypoxia-related inflammation and the microcirculation in critically ischemic peripheral tissue. DESIGN: Randomized prospective study. SETTING: University laboratory. SUBJECTS: Twenty-four male golden Syrian hamsters. INTERVENTIONS: Island flaps were dissected from the back skin of anesthetized hamsters for assessment with intravital microscopy. The flap included a critically ischemic, hypoxic area that was perfused via a collateralized vasculature. One hour after completion of the preparation, the animals received an injection of 25% of total blood volume of 0.9% NaCl or NaCl suspended with HbVs at a concentration of 5 g/dL (HbV5) or 10 g/dL (HbV10). MEASUREMENTS AND MAIN RESULTS: Plasma viscosity was increased from 1.32 cP to 1.61 cP and 2.14 cP after the administration of HbV5 and HbV10, respectively (both p < .01). Both HbV solutions raised partial oxygen tension (Clark-type microprobes) in the ischemic tissue from approximately 10 torr to 17 torr (p < .01), which was paralleled by an increase in capillary perfusion by > 200% (p < .01). The 50% increase in macromolecular capillary leakage found over time in the control animals was completely abolished by the HbV solutions (p < .01), which was accompanied by a > 50% (p < .01) reduction in cells immunohistochemically stained for tumor necrosis factor-alpha and interleukin-6 and in leukocyte counts, whereas no such changes were observed in the anatomically perfused, normoxic tissue. CONCLUSIONS: Our study suggests that in critically ischemic, hypoxic peripheral tissue, hypoxia-related inflammation may be reduced by a top-load infusion of HbV solutions. We attributed this effect to a restoration of tissue oxygenation and an increase in plasma viscosity, both of which may have resulted in attenuation of secondary microcirculatory impairments.

IL-3 induces a Pim1-dependent antiapoptotic pathway in primary human basophils

The contribution of basophils in allergic disease and other Th2-type immune responses depends on their persistence at sites of inflammation, but the ligands and molecular pathways supporting basophil survival are largely unknown. The comparison of rates of apoptosis and of the expression of antiapoptotic proteins in different human granulocyte types revealed that basophils have a considerably longer spontaneous life span than neutrophils and eosinophils consistent with high levels of constitutive Bcl-2 expression. Interleukin-3 (IL-3) is the only ligand that efficiently protects basophils from apoptosis as evidenced by screening a large number of stimuli. IL-3 up-regulates the expression of the antiapoptotic proteins cIAP2, Mcl-1, and Bcl-X(L) and induces a rapid and sustained de novo expression of the serine/threonine kinase Pim1 that closely correlates with cytokine-enhanced survival. Inhibitor studies and protein transduction of primary basophils using wild-type and kinase-dead Pim1-Tat fusion-proteins demonstrate the functional importance of Pim1 induction in the IL-3-enhanced survival. Our data further indicate that the antiapoptotic Pim1-mediated pathway operates independently of PI3-kinase but involves the activation of p38 MAPK. The induction of Pim1 leading to PI3-kinase-independent survival as described here for basophils may also be a relevant antiapoptotic mechanism in other terminally differentiated leukocyte types.

Allergic bronchopulmonary aspergillosis: the hunt for a diagnostic serological marker in cystic fibrosis patients

The diagnosis of allergic bronchopulmonary aspergillosis (ABPA) in cystic fibrosis patients remains challenging, mainly owing to overlapping symptoms of the underlying lung disease with clinical symptoms of ABPA. In addition, a varying mixture of diagnostic criteria, including clinical status, radiological findings and immunological measurements, has led to confusion and differing recommendations. In order to help simplify as well as standardize the diagnostic criteria for ABPA, different serological markers have been evaluated in the last 20 years and their usefulness has been assessed in many clinical studies. This review presents current diagnostic criteria of ABPA, with a special focus on serum markers supporting the diagnosis and explains why the hunt for a serological marker for ABPA is still ongoing.

Important research questions in allergy and related diseases: nonallergic rhinitis: a GA2LEN paper

Nonallergic rhinitis (NAR) can be defined as a chronic nasal inflammation which is not caused by systemic IgE-dependent mechanisms. It is common and probably affects far more than 200 million people worldwide. Both children and adults are affected. However, its exact prevalence is unknown and its phenotypes need to be evaluated using appropriate methods to better understand its pathophysiology, diagnosis and management. It is important to differentiate between infectious rhinitis, allergic/NAR and chronic rhinosinusitis, as management differs for each of these cases. Characterization of the phenotype, mechanisms and management of NAR represents one of the major unmet needs in allergic and nonallergic diseases. Studies on children and adults are required in order to appreciate the prevalence, phenotype, severity and co-morbidities of NAR. These studies should compare allergic and NAR and consider different age group populations including elderly subjects. Mechanistic studies should be carried out to better understand the disease(s) and risk factors and to guide towards an improved diagnosis and therapy. These studies need to take the heterogeneity of NAR into account. It is likely that neuronal mechanisms, T cells, innate immunity and possibly auto-immune responses all play a role in NAR and may also contribute to the symptoms of allergic rhinitis.

Soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 are produced at sites of inflammation and are markers of arthritis activity in Behçet's disease

OBJECTIVE: We analysed the production of soluble tumour necrosis factor receptors sTNFR1 and sTNFR2 at sites of inflammation and measured their plasma concentrations to evaluate them as biological markers of disease activity. METHODS: Plasma samples of 35 patients with Behçet's disease (BD) were collected prospectively at monthly intervals and grouped for inactive disease, active BD without arthritis, and active BD with arthritis. sTNFR1 and sTNFR2 concentrations were measured using immunoassays and compared with other biological disease activity parameters. Plasma sTNFR levels were compared to synovial fluid (SF) levels in seven patients. Sixteen tissue samples of mucocutaneous lesions were stained for TNFR2 expression by immunohistochemistry. RESULTS: sTNFR1 and sTNFR2 were found at increased plasma concentrations in active BD, with the highest concentration in active BD with arthritis (p<0.001). Concentrations of both sTNFRs were at least three times higher in SF of arthritic joints than in the corresponding plasma samples (p = 0.025). A change of more than 1 ng/mL of sTNFR2 plasma concentrations correlated with a concordant change in arthritic activity (96% confidence interval). Sensitivity to change was superior to that of sTNFR1, and other biological disease activity parameters such as erythrocyte sedimentation rate (ESR), immunoglobulin (Ig)G, IgA, and interleukin (IL)-10 plasma concentrations. A strong staining for TNFR2 was found in mucocutaneous lesions, where mast cells were identified as the major source for this receptor. CONCLUSIONS: This longitudinal study demonstrates that sTNFR2 plasma concentrations are closely linked with active BD, and especially with arthritis. Taken together with the expression of TNFR molecules in mast cells of mucocutaneous lesions, our results indicate a fundamental role for the TNF/TNFR pathway in BD.