Identifying sources of measurement error in assessing dietary intakes - Results of a multi-country ring-trial.

BACKGROUND AND AIMS: Epidemiological investigations include dietary intakes as primary exposures or potential confounders. To reduce bias, data collection protocols include the administration of questionnaires together with measurements of biomarkers. Some error, however, remains and needs to be considered in the analysis and interpretation of results. The European Food Safety Authority supported a ring-trial to compare the precision and reproducibility of dietary assessment methods applied in Europe. METHODS AND RESULTS: Software applications used to collect 24-hour recalls and food records in six countries (Estonia, Italy, Latvia, Portugal, Spain, and Sweden) were assessed. The intake of 256 foods was identically reported to each method. Experienced interviewers participated and were instructed to repeat national protocols closely. The error in recording quantities, compared with reference values, was variable but in about 60% of recorded quantities was in the range of ±20%. Errors were however unsystematic and independent of the food type or quantification method used - although food pictures performed better. The reproducibility of some tools was limited. The methods generally captured additional ingredients (usually flavoring agents), but not sweetening agents or fortification and failed to record packaging information in about 60% of the cases. CONCLUSION: In a design that eliminated respondent bias, this study indicates that softwares, supporting databases and interviewers generally introduce random error in dietary assessments. The inclusion of large sample sizes and food pictures to quantify portions, together with enhanced attention on interviewers' training, standardisation of procedures and regular tool upgrades are essential in assuring a study's quality and comparability.

Effect of sex hormones on plasma phospholipid fatty acid composition in intact rats and rats with bilaterally occluded carotid arteries.

The effects of 8-days treatment with 17alpha-estradiol (33.3 microg/kg) and progesterone (1.7 mg/kg) on plasma lipids and fatty acid composition of plasma phospholipids were examined in intact (INT) and bilaterally common carotid arteries occluded (BCO) male Wistar rats. Significant decrease of triglyceride level was found in BCO rats after the estradiol treatment. Both hormones elevated proportion of 18:1n-7 fatty acid in INT, but they failed to have such an effect in BCO. Estradiol increased 22:5n-3 and total n-3 polyunsaturated fatty acids (PUFA) in intact, and decreased 18:2n-6 in BCO rats. Significantly lower level of total n-3 was found in progesterone-treated than in estradiol-treated BCO rats. Given that n-3 PUFA have many beneficial effects on cell and tissue function, while n-6 PUFA have mostly the opposite effects, estradiol, rather than progesterone, was seen to improve plasma lipids and phospholipids FA profiles in INT and BCO animals. Estradiol significantly elevated the estimated activity of delta9-desaturases and progesterone of delta5-desaturase in BCO group, with no effects in INT rats.

Different fatty acid composition of serum phospholipids of small and appropriate for gestational age preterm infants and of milk from their mothers.

BACKGROUND: Placental supply of fatty acids (FA) is essential for normal foetal development but in premature infants this supply is interrupted. To investigate the association of intrautrine growth restriction with serum phospholipid and breast milk FA composition, we compared preterm infants small for gestational age (SGA) and matched appropriate for gestational age (AGA), and their mothers' milk during the first 4 weeks of postnatal life. METHODS: Sera from 11 SGA and 12 AGA infants born 34-36 weeks of gestation were collected at birth, 14(th) and 28(th) day, and breast milk on 14(th) and 28(th) day after birth. FA composition was analyzed by gas chromatography. RESULTS: Preterm SGA infants had significantly lower oleic, total monounsaturated FA (MUFA), docosahexaenoic acid (DHA) and n-3 polyunsaturated FA (PUFA) and higher levels of stearic and linoleic acid at birth than AGA infants (p<0.05). DHA was significantly lower, whereas docosatetraenoic and docosapentaenoic acids were higher in SGA infants after 28 days. Mothers of AGA infants had markedly lower levels of MUFA and higher levels of total and n-6 PUFA in their breast milk. CONCLUSION: SGA infants have altered serum phospholipid FA composition at birth and during their first month of life, probably due to inadequate transplacental supply and activity of desaturase system. RESULTS on human milk suggest that pregnancies with AGA or SGA would later influence breast milk FA composition.

Systemic alterations in concentrations and distribution of plasma phospholipids in prostate cancer patients.

The relationship between plasma levels of total phospholipids (PL) and/or PL fractions and neoplastic diseases are not fully understood. Therefore, the aim of this study was to analyze concentrations and distribution of plasma phospholipids in patients with prostate cancer (PCa) related to the Gleason score, clinical stage and pathologic grade of prostate cancer. We analyzed plasma phospholipids in 57 newly diagnosed, untreated PCa patients and in 43 age-matched healthy male subjects. Significantly lower (P < 0.01) levels of total plasma PL and all PL classes were found in PCa patients when compared with healthy subjects. The relative concentrations of PL fractions were also changed. Further decrease of total PL and PL fractions was found related to an increase of clinical stadium, pathologic grade, and Gleason score, with phosphatidylethanolamine as the most sensitive plasma PL, the level of which significantly decreased even at the first stage of PCa. Our results showed an altered plasma PL profile in PCa patients, which may contribute to monitoring of the disease progression.

Fish oil supplementation improved liver phospholipids fatty acid composition and parameters of oxidative stress in male Wistar rats.

In the present study, we examined the effects of fish oil supplementation in 3 months old male Wistar rats on changes in plasma and liver lipid metabolism and oxidative stress parameters. Twenty Wistar rats were randomly divided into two groups of ten animals: control group and intervention group, treated for 6 weeks with fish oil capsules containing 45 mg eicosapentanoic acid and 30 mg docosahexanoic acid. After intervention, biochemical parameters in plasma [triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL) and total cholesterol, urea, creatinine and uric acid], fatty acid (FAs) profile of liver phospholipids and parameters of oxidative stress in liver [activity of catalase, superoxide dismutase and paraoxonase (PON1), concentration of nitrites, lipid peroxidation (LPO), free thiol (SH) groups and lactate dehydrogenase (LDH) izoenzymes were determined. Treatment with fish oil improved FAs profile of liver phospholipids, increasing n-3 FAs and decreasing n-6/n-3 ratio. Significant decrease in plasma TG and LDL concentration, and increase in the level of HDL and uric acid were found in intervention group at the end of the study. Catalase activity, LPO, and nitrites concentration in liver were significantly decreased, after the supplementation, together with elevated PON1 activity. Applied treatment significantly improved plasma lipid profile, liver FAs composition and parameters of oxidative stress in male Wistar rats.

Capacity development in food composition database management and nutritional research and education in Central and Eastern European, Middle Eastern and North African countries.

BACKGROUND/OBJECTIVES: Capacity development (CD) in food and nutrition is much more than formal training and includes human resource development, and organisational, institutional and legal framework development with the aim of enhancing nutrition-relevant knowledge and skills to support infrastructural development. The goal of the European Food Information Resource (EuroFIR) Network of Excellence has been to develop and integrate food composition data throughout Europe. EuroFIR joined forces in CD with the United Nations (UN) University and UN System Standing Committee on Nutrition, the Network for Capacity Development in Nutrition in Central and Eastern Europe, the Central and Eastern European Countries Food Data Systems network and with the Middle East and North African Capacity Building Initiative. The aim of this paper is to discuss an inventory of the status of food composition databases (FCDBs) and the training needs of compilers in non-EuroFIR countries in Central and Eastern Europe (CEE) and in the Middle East and North Africa (MENA), and to present the CD achieved through EuroFIR and other network collaborations. SUBJECTS/METHODS: Two online questionnaires were created addressing the FCDB status and specific training needs in countries of the targeted regions. Data were collected during 2006-2008 and then analysed. Subsequently, CD activities were organised. RESULTS: Contacts were established in 19 CEE and 7 MENA countries, of which several had national food composition tables, but no electronic versions. Education, training, workshops, networking and the sharing of experiences were uniformly requested. Subsequently, CD activities in EuroFIR were organised focussing on food composition courses, exchange visits, workshops and individual training for PhD students, junior scientists and other staff categories, as well as conferences linked to food composition research and food information. To facilitate CD activities, EuroFIR has signed a Memorandum of Understanding with the Czech Republic, Hungary, Slovenia, Croatia and Estonia. CONCLUSIONS: EuroFIR has created training activities that complement national activities. Collaboration with other networks has provided an overview of FCDB status and training needs, providing directions for CD activities in those countries. This provides a platform for new funding and further development and networking for CD, which would be conducive to European Commission objectives and public health strategies for CD.

EURRECA nutritional planning and dietary assessment software tool: NutPlan.

BACKGROUND/OBJECTIVES: 'NutPlan' is developed within the EURRECA Network of Excellence (EURopean micronutrient RECommendations Aligned (http://www.eurreca.org). It is a user-friendly software programme with multiple functions: individual and group nutrition planning, recipe calculation, creating food labels, diet planning and nutrient intake assessment. This paper describes the newly developed software and its features. SUBJECTS/METHODS: 'NutPlan' contains the following databases: foods, dish recipes, meals, menus, average menus and glossary. These databases enable diet planning and diet analysis by comparing foods, dishes, meals or menus with currently available nutritional recommendations accessible by a link to EURRECA tool Nutri-RecQuest to meet individual/group nutritional needs.The software is upgraded by inserting new items (for example, foods, dishes, meals) and for a connection to other software programmes, thus allowing more advanced calculations to be completed. CONCLUSION: 'NutPlan' might be the software of choice for individual and group diet planning. It is aimed particularly at Eastern European and West Balkan countries, which currently lack dietary software. It is envisaged for use by small and medium enterprises in the food industry, as well as by health professionals, researchers and policy makers, and can be recommended for educational purposes. Given its characteristics of being upgraded to include new country-specific food data/database, it can be recognized as an important tool in nutritional capacity development in the Central Eastern European and other regions.

Nutri-RecQuest: a web-based search engine on current micronutrient recommendations.

BACKGROUND: The EURRECA (EURopean micronutrient RECommendations Aligned) Network of Excellence collated current micronutrient recommendations. A user-friendly tool, Nutri-RecQuest, was developed to allow access to the collated data and to create a database source for use in other nutritional software tools. METHODS: Recommendations, that is, intakes of micronutrients sufficient to meet the requirements of the majority of healthy individuals of that population, from 37 European countries/organizations and eight key non-European countries/regions comprising 29 micronutrients were entered into a database. General information on the source of the recommendations, as well scientific background information, was added. RESULTS: A user-friendly web-based interface was developed to provide efficient search, comparison, display, print and export functions. CONCLUSION: Easy access to existing recommendations through the web-based tool may be valuable for bodies responsible for setting recommendations, as well as for users of recommendations including scientists, policy makers, health professionals and industry. Adding related dietary reference values such as average nutrient requirements and upper limits may extend the utility of the tool.

Lipid profile as a prognostic factor in cancer patients.

PURPOSE: The relationship between plasma lipid levels and neoplastic diseases is still unclear. The aim of this study was to analyse the lipid profile of individuals with non-Hodgkin's lymphoma (NHL) or prostate carcinoma (CaP) and to follow serum lipid levels changes in NHL patients according to their response to chemotherapy. PATIENTS AND METHODS: Forty-seven patients with NHL, 57 patients with CaP, two control groups composed of 29 and 43 age- and sex-matched healthy adults, related to NHL and CaP patients, respectively, were included in the study. Follow-up studies of NHL patients were carried out after the 3rd and 6th cycle of chemotherapy. RESULTS: Initial plasma cholesterol (Chol), HDL-cholesterol (HDL-Chol) and phospholipids (PL) values were significantly lower in patients with NHL or CaP than in controls. Following chemotherapy, we noticed a progressive increase in lipid levels in NHL patients with complete remission (CR) and stable disease (SD), and further decrease in patients with the disease progression. CONCLUSION: Decreased plasma Chol, HDL-Chol and PL levels of patients with NHL or CaP can be considered as non-specific prognostic parameters in patients with these malignancies.

[Biochemical and antropometric monitoring of growth in preterm infants].

Enriched human milk may stimulate gain weight in preterm infants during neonatal period. Aim is biochemical assessment of preterm infants, feed by mother's milk fortificated with special domestic formula (pre)Impamil during the first month of life. 80 preterm infants (45 male and 35 female), up to 36 weeks of gestation. BW less than 2500 g, which started enteral intake in the first three day of life. Total volume intake was in range from 70 ml/kg first day, to 170-200 ml/kg, after 10th day of life. Mother's milk fortification was prepared as 5% mixture solution of (pre)Impamil. The dynamics of biochemical analyses started on the first day of study and was repeated once weekly. We analysed levels of: total protein, albumin, prealbumin, transferin, urea, Ca ionised, P and alkaline phosphatase using standard biochemical methods. Statistical analyses completed by ANOVA test, one factorial analyses of variance. During monitoring total protein level increased in the second week of life (p < 0.03), as well as albumin (p < 0.03). Prealbumin level increased, as well as transferin (p-NS). Initial level of ionised fraction of Ca was significantly lower (p < 0.03) at the beginning of the study, compared to the rest. Serum level of P increased, as well as level of alkaline phosphatase at the end of first and second week (p < 0.01). Conclusion is that biochemical parameters as level of protein and albumin are important at the assessement growth in preterm infants on special feeding regimes.

PGE2/TXB2 imbalance in neonatal hypoxemic respiratory failure.

BACKGROUND: An imbalance of vaso-constrictor and -dilator mediators has been implicated in the pathogenesis of the pulmonary hypertension accompanying neonatal hypoxemic respiratory failure (NHRF). AIM: To characterize plasma PGE2, TXB2 and their ratio in normal newborns and in those with NHRF. METHODS: Twenty newborns with NHRF received inhaled PGE1 (IPGE1) by jet nebulizer in doses of 25, 50, 150 and 300 ng/kg/min followed by weaning. Blood for PGE2 and TXB2 assay using EIA was available in 8 neonates with NHRF prior to IPGE1. Umbilical cord arterial samples were also obtained at delivery from 10 normal newborns to serve as controls. RESULTS: Compared to normal newborns, those with NHRF had significantly lower PGE2/TXB2 ratios after controlling for preterm gestation (< 37 weeks) and postnatal age (p < 0.05). Notably, all subjects except one in the NHRF group had a value of < 1.0 (range 0.1-1.2) compared to a value of > 1.0 in all subjects in the Control group (range 1.1-5.2). CONCLUSIONS: Lower PGE2/TXB2 ratio in subjects with NHRF compared with controls reflects a predominance of vaso-constrictor activity in these patients as the basis of pulmonary hypertension. Plasma PGE2/TXB2 ratio may have important implications for the diagnosis and treatment of NHRF.

Ibuprofen attenuates early lung injury in endotoxemic, neutropenic rats.

The objective of our study was to determine the role of ibuprofen in protecting neutropenic rats from cardiopulmonary injury due to endotoxemia. We hypothesized that ibuprofen would offer pulmonary protection by altering cytokine production. Neutropenic rats received E. coli lipopolysaccharide (LPS) alone or ibuprofen and LPS. After 4 h, arterial blood gases, heart rate and blood pressure were measured. Blood and bronchoalveolar lavage fluid (BALF) were collected for TNF- alpha and MIP-2 concentrations. Lung tissue for iNOS mRNA and myeloperoxidase were obtained. The ibuprofen group had decreased heart rate and better oxygenation. Ibuprofen suppressed TNF- alpha and MIP-2 production in blood and MIP-2 concentrations in BALF. Lung mRNA for iNOS was higher in the ibuprofen group. Neutrophil infiltration in the lung was similar in both groups. Ibuprofen attenuated cardiopulmonary dysfunction by decreasing the early cytokine response. The balance of vasodilator to vasoconstrictor production in the lung may favor vasodilation as shown by increased iNOS mRNA and suppression of thromboxane.

Group B Streptococci and inducible nitric oxide synthase: modulation by nuclear factor kappa B and ibuprofen.

Group B Streptococci (GBS) neonatal infections cause a complex inflammatory process involving numerous biochemical mediators. Nitric Oxide (NO) is generated by many cell types in response to different inflammatory signals. Nuclear factor kappa B (NFkappaB) plays an important role in the inflammatory process and may induce a number of biochemical mediator genes, including the inducible nitric oxide synthase (iNOS). We tested the hypothesis that GBS induces iNOS gene expression through activation of NFkappaB. We also tested whether ibuprofen (IBU) will suppress iNOS expression by blocking NFkappaB activation. Cerebral microvascular endothelial cells isolated from newborn piglets were harvested for the determination of iNOS gene expression and activation of NFkappaB. GBS significantly induced iNOS mRNA expression (5- to 6-fold, P < .005) and iNOS protein (3- to 4-fold, P < .01) at 24 hours. DNA-NFkappaB binding activity was detected within 15 minutes of GBS treatment and reached a maximal effect at 3 hours. Treatment with IBU significantly suppressed GBS-induced iNOS mRNA expression at 24 hours, and NFkappaB activity at 3 hours, suggesting that suppression of GBS-induced iNOS mRNA expression by IBU occurs by blocking of NFkappaB activation. These data show that NFkappaB activation is an early step in the induction of iNOS gene expression by GBS and that this interaction may play a vital role in the pathogenesis of GBS neonatal infections.

Nitric oxide, prostaglandins, and impaired cerebral blood flow autoregulation in group B streptococcal neonatal meningitis.

Impaired autoregulation of cerebral blood flow (CBF) contributes to CNS damage during neonatal meningitis. We tested (i) the hypothesis that cerebrovascular autoregulation is impaired during early onset group B streptococcal (GBS) meningitis, (ii) whether this impairment is regulated by vasoactive mediators such as prostaglandins and (or) nitric oxide (NO), and (iii) whether this impairment is preventable by specific and (or) nonspecific inhibitors: dexamethasone, ibuprofen, and Nomega-nitro-L-arginine, a NO inhibitor. Sterile saline or 10(9) colony-forming units (cfu) of heat-killed GBS was injected into the cerebral ventricle of newborn piglets. CBF autoregulation was determined by altering cerebral perfusion pressure (CPP) with balloon-tipped catheters placed in the aorta. GBS produced a narrow range of CBF autoregulation due to an impairment at the upper limit of CPP. We report that in vivo in the early stages (first 2 h) of induced GBS inflammation (i) GBS impairs the upper limit of cerebrovascular autoregulation; (ii) ibuprofen, dexamethasone, and Nomega-nitro-L-arginine not only prevent this GBS-induced autoregulatory impairment but improve the range of cerebrovascular autoregulation; (iii) these autoregulatory changes do not involve circulating cerebral prostanoids; and (iv) the observed changes correlate with the induction of NO synthase gene expression. Thus, acute early onset GBS-induced impairment of the upper limit of CBF autoregulation can be correlated with increases of NO synthase production, suggesting that NO is a vasoactive mediator of CBF.

Deregulation of cyclooxygenase and nitric oxide synthase gene expression in the inflammatory cascade triggered by experimental group B streptococcal meningitis in the newborn brain and cerebral microvessels.

Group B Streptococcus (GBS) is the most common cause of neonatal sepsis and meningitis. Despite antibiotics, GBS in the newborn initiates a cascade of molecular and biological events leading to altered cerebral perfusion, blood-brain barrier disruption, cerebral edema, intracranial hypertension, neurological damage, and even death. Having previously shown that GBS infection impairs cerebral blood flow autoregulation and increases prostaglandin (PG) levels, we examined the regulation of some crucial inflammatory mediators (PGs, nitric oxide (NO), tumor necrosis factor-a) in the brain and cerebral microvessels (MVs) from newborn piglets. Cyclooxygenase (COX), the key enzyme in PG biosynthesis, exists in two isoforms, COX-1 and COX-2. Both may be directly induced by NO in a model of renal inflammation. Besides its neurotransmitter role, NO is a potent vasorelaxant whose production is catalyzed by at least three distinct nitric oxide synthases (NOS) (bNOS, ecNOS, iNOS). Western blot analyses showed that the newborn (4 day old) brain expressed lower levels of COX-1 (8-fold), COX-2 (20-fold), bNOS (12-fold), and ecNOS (5-fold) than in the 1 day old. MV showed approximately equal levels of COX-2, lower levels of COX-1 (4-fold), bNOS (5-fold), and higher levels of ecNOS (20-fold) in comparison to 4-day-old cerebral MV. A 4-day-old brain expressed lower levels of bNOS (5-fold), ecNOS (10-fold), and COX-1 (2-fold) than the 6-week-old pig. COX-2 protein was undetected in a 4-day-old pig brain, but present in great excess in MV. Purified MV showed lower ecNOS (14-fold), COX-1 (2-fold), and about equal levels of bNOS and COX-2 in comparison with MV from 6-week-old pigs. Reverse transcription polymerase chain reaction analyses confirmed these results. Treatment with noo-nitro-L-arginine (LNA), a NOS inhibitor, downregulated COX-1 expression in the newborn brain and both COX-1 and COX-2 cerebral MV expression. GBS infection (10(9) colony-forming units, 0.5 mL intracerebroventricular) of sedated newborn piglets induced the expression of tumor necrosis factor-alpha in the cerebrospinal fluid after 2 hours, upregulated bNOS expression in both brain and MVs, upregulated ecNOS in MVs, and downregulated COX-1, COX-2, and ecNOS in the brain. GBS did not trigger the expression of iNOS. Our data suggest that there is a net deficiency of NOS isoforms in the immature brain and microvasculature of the 4-day-old piglet and that the differences in expression lead to the immature control of NO and PG production, rendering newborns particularly susceptible to neurological damage because of the undeveloped nature of their response mechanisms. Moreover, the GBS-induced cascade deregulates the gene expression of interacting inflammatory mediators and may cause a net vasoconstrictor/vasodilator imbalance, leading to cerebral hypertension and edema in the early stages of infection. Pharmacological manipulations of the inflammatory cascade could lead to novel therapeutic approaches for the treatment of GBS meningitis.

GA-binding protein is involved in altered expression of ribosomal protein L32 gene.

Differentiation of BC3H1 myoblasts to myocytes is accompanied by a 67% drop in the rate of rpL32 gene transcription. Addition of high concentrations of serum to resting myocyte populations stimulates cell growth and subsequent dedifferentiation to proliferating myoblasts with a return to the normal rate of rpL32 gene transcription. During these growth rate changes the binding activities of previously identified factors (beta, gamma, delta) which interact with the rpL32 gene promoter were examined by mobility shift assays. Binding of the beta factor (an Ets related protein) to an oligonucleotide containing the beta element was reduced significantly in myocyte nuclear extracts, but subsequent dedifferentiation increased binding within 30 min in either the presence or absence of the cycloheximide. Binding of the gamma and delta factors to their respective elements changed only slightly during these processes. Dephosphorylation of either myoblast or myocyte extracts resulted in increased binding of the beta factor suggesting that binding activity of the beta factor is modulated by phosphorylation during the changes in BC3H1 myoblasts growth rate. In addition, mobility shift assays with recombinant GABP alpha and beta proteins and their specific antibodies revealed that GABP proteins bind to the rpL32 gene promoter in a sequence dependent manner, and that similar proteins are present in BC3H1 myoblast/myocyte extracts. These results support the premise that the GABP heterodimer is the rpL32 beta factor. Furthermore, during BC3H1 myoblast differentiation and dedifferentiation neither the levels of the GABP alpha and beta proteins nor their respective mRNAs change. These results suggest that GABP is a constitutively expressed protein and is involved in regulating rpL32 gene by post-transcriptional modifications.

Structure of recombinant rat UBF by electron image analysis and homology modelling.

We have studied the structure of recombinant rat UBF (rrUBF), an RNA polymerase I transcription factor, by electron microscopy and image analysis of single particles contrasted with methylamine tungstate. Recombinant rat UBF appeared to be a flat, U-shaped protein with a central region of low density. In the dominant projections, 2-fold mirror symmetry was seen, consistent with the dimerization properties of this molecule, and of dimensions in agreement with the length of DNA that rat UBF protects in footprinting studies. Electron microscopy of various rrUBF-DNA complexes confirmed that our recombinant protein was fully able to bind the 45S rDNA promoter, and that it caused substantial bends in the DNA. Upon extended incubation in a droplet covered by a lipid monolayer at the liquid-air interface, rrUBF formed long filamentous arrays with a railway track appearance. This structure was interpreted to consist of overlapping rrUBF dimers 3.5 nm apart, which value would represent the thickness of the protein. Our results show rrUBF to interact with and bend the promoter DNA into a roughly 10 nm diameter superhelix. Based on all these electron microscopical results, an atomic structure was predicted by homology modelling of the HMG fingers, and connected by energy minimized intervening segments.

CD40 expression by human peripheral blood eosinophils.

In this study, we have investigated CD40 expression in human peripheral blood eosinophils and in human chronically inflamed nasal tissues, i.e., nasal polyps. We show by both reverse transcriptase-PCR and Northern blot analysis that eosinophils from allergic subjects express human CD40 mRNA. We also show that constitutive CD40 mRNA expression in eosinophils could be upregulated by exposure to IgA immune complexes and downregulated by IL-10 and the synthetic steroid budesonide. In addition, we demonstrate that eosinophils express CD40 protein by flow cytometry. Such expression is biologically functional as cross-linking CD40 with CD40 mAbs enhances eosinophil survival in a dose-dependent fashion; in addition, CD40 ligation stimulates eosinophils to release GM-CSF. CD40-mediated eosinophil survival was largely inhibited by an anti-GM-CSF neutralizing antibody suggesting GM-CSF involvement in the survival enhancing mechanism. CD40 mRNA was also detected in total RNA extracted from nasal polyp tissues but not in RNA isolated from normal nasal mucosa (inferior turbinate); by immunohistochemistry, we were able to detect immunoreactive CD40 protein in a variety of cell types in the polyp stroma, but primarily in eosinophils. These observations suggest previously unforeseen interactions between eosinophils and cells expressing the CD40 ligand and, thus, novel pathways by which eosinophils may contribute to the regulation of airway inflammation.

Interleukin (IL)-10 inhibits long-term IL-6 production but not preformed mediator release from rat peritoneal mast cells.

Mast cells have been implicated in a number of diseases involving chronic inflammation including asthma, rheumatoid arthritis, and inflammatory bowel diseases. They are a potent source of several cytokines, including IL-6 and TNF-alpha. Freshly isolated rat peritoneal mast cells will produce IL-6 in response to anti-IgE, LPS, PGE1, or PGE2; however, the mechanisms by which such cytokine production is regulated are poorly understood. IL-10 is recognized as an important immunoregulatory cytokine with effects on T cell development and the production of inflammatory cytokines. IL-10 has previously been described to enhance mast cell development in the context of IL-3 and IL-4. In the current study, we have examined the ability of IL-10 to modulate rat peritoneal mast cell IL-6 and TNF-alpha production in response to a variety of stimuli. We have observed that recombinant murine IL-10 can inhibit the production of both IL-6 and TNF-alpha by mast cells without altering the degree of histamine release in response to anti-IgE. Concentrations of IL-10 as low as 0.2 ng/ml were sufficient to inhibit IL-6 production by LPS- or anti-IgE-activated cells significantly. IL-10 also inhibited PGE1- and PGE2-induced IL-6 production. The relative potency of IL-10 as an inhibitor of mast cell IL-6 production was highly dependent upon the stimulus used, with a 10-fold difference in the IC50 for LPS- or anti-IgE-activated cells (0.21 ng/ml) and cells activated with a combination of LPS and PGE2 (2.29 ng/ml). This suggests that prostanoids may limit the ability of IL-10 to modulate mast cell IL-6 production in the context of inflammation. These data have important implications for the regulation of mast cell IL-6 in inflammatory diseases involving prostanoid production and the effects of treatment with cyclooxygenase inhibitors. Our results also demonstrate a dual role for IL-10 on mast cells as a growth factor and inhibitor of cytokine production.

Distinct immunohistochemical localization of IL-4 in human inflamed airway tissues. IL-4 is localized to eosinophils in vivo and is released by peripheral blood eosinophils.

Nasal polyposis and asthma are inflammatory conditions of the airways characterized by infiltration of activated inflammatory cells, particularly eosinophils. IL-4 is a multifunctional cytokine considered to play an important role in eosinophilic inflammation. We examined the cellular distribution of immunoreactive IL-4 in nasal polyps, as well as in the bronchial mucosa of both nonasthmatic control subjects (n = 6) and patients with well-characterized mild asthma (n = 6) subjected to a diluent or an allergen challenge. To determine eosinophilic contribution, tissue sections were counterstained with FITC after IL-4 immunostaining. No eosinophils were observed in the bronchial mucosa of nonasthmatic subjects. Nasal polyp tissues contained approximately 15 times more eosinophils per mm2 compared with bronchial tissues from asthmatics after a diluent challenge. Allergen challenge resulted in a marked increase in eosinophil density in bronchial tissues. A negligible number of cells immunostaining IL-4 was observed in bronchial tissues from nonasthmatic control subjects. The density of IL-4-positive cells in nasal polyp tissues was almost three times greater compared with asthmatics bronchial tissues after a diluent challenge. Approximately 90% of the IL-4-positive cells in bronchial tissues did not exhibit fluorescence after FITC counterstaining; in contrast, about 80% of the IL-4-positive cells in nasal polyp tissues did. We also show that peripheral blood eosinophils from allergic subjects express IL-4 mRNA by Northern blot analysis, particularly on stimulation with secretory IgA immune complexes. Finally, the supernatant of stimulated eosinophils contained approximately 50 pg/10(6) cells of IL-4 as determined by ELISA. These data demonstrate that eosinophils express the message and release IL-4 in vitro, and that these cells are the primary source of immunoreactive IL-4 in tissues undergoing chronic severe mucosal inflammation.