Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Serum cytokines elevated during gluten-mediated cytokine release in coeliac disease.

Cytokines have been extensively studied in coeliac disease, but cytokine release related to exposure to gluten and associated symptoms has only recently been described. Prominent, early elevations in serum interleukin (IL)-2 after gluten support a central role for T cell activation in the clinical reactions to gluten in coeliac disease. The aim of this study was to establish a quantitative hierarchy of serum cytokines and their relation to symptoms in patients with coeliac disease during gluten-mediated cytokine release reactions. Sera were analyzed from coeliac disease patients on a gluten free-diet (n = 25) and from a parallel cohort of healthy volunteers (n = 25) who underwent an unmasked gluten challenge. Sera were collected at baseline and 2, 4 and 6 h after consuming 10 g vital wheat gluten flour; 187 cytokines were assessed. Confirmatory analyses were performed by high-sensitivity electrochemiluminescence immunoassay. Cytokine elevations were correlated with symptoms. Cytokine release following gluten challenge in coeliac disease patients included significant elevations of IL-2, chemokine (C-C motif) ligand 20 (CCL20), IL-6, chemokine (C-X-C motif) ligand (CXCL)9, CXCL8, interferon (IFN)-γ, IL-10, IL-22, IL-17A, tumour necrosis factor (TNF)-α, CCL2 and amphiregulin. IL-2 and IL-17A were earliest to rise. Peak levels of cytokines were generally at 4 h. IL-2 increased most (median 57-fold), then CCL20 (median 10-fold). Cytokine changes were strongly correlated with one another, and the most severely symptomatic patients had the highest elevations. Early elevations of IL-2, IL-17A, IL-22 and IFN-γ after gluten in patients with coeliac disease implicates rapidly activated T cells as their probable source. Cytokine release after gluten could aid in monitoring experimental treatments and support diagnosis.

[Selected independent risk factors of atherosclerosis in children as the basis of early detection of premature ischemic heart disease]. / Wybrane niezalezne czynniki ryzyka miazdzycy u dzieci jako podstawa wczesnego wykrywania zagrozenia choroba niedokrwienna serca.

This review briefly summarises the scientific evidence for the child's future risk of ischemic heart diseases (IHD). The conventional risk factors such as dyslipidemia, hypertension, diabetes and smoking can not account for all the cases of cardiovascular diseases. Therefore other risk factors such as fibrinogen, homocysteine, paraoxonaze and abnormality in antioxidant defence systems are included. Among the lipids parameters the level of lipoprotein (a) and increased plasma cholesterol, specifically LDL-cholesterol may be used as a marker of family history of IHD and hypercholesterolemia. Additionally, low level of HDL-cholesterol is also related with endothelial dysfunction and increased oxidative stress. It has been hypothesised that free radicals mediate in the development of IHD and that antioxidants play a protective role in prevention of this pathology. Many of the major risk factors can be modified through diet, body mass control, exercise and (if necessary) through pharmacological intervention. Therefore, the efficacious prevention should be related with the early detection of risk factors particularly in children with familial dyslipidemia, hypertension and IHD.

[Homocysteine and some lipid parameters in hypercholesterolemic children]. / Homocysteina i wybrane parametry przemian lipidowych u dzieci z hipercholesterolemia.

High serum homocysteine (tHcy) concentration is increasingly recognised as independent risk factor for atherosclerosis, early coronary heart disease (CHD) and other vascular diseases. It has been proved that adult cardiovascular disease begins in childhood. In the presented studies we determined concentrations of homocysteine, lipids and lipoproteins in plasma of hypercholesterolemic and normocholesterolemic children. In hypercholesterolemic children total cholesterol, LDL cholesterol, apolipoprotein B and triglycerides were significantly higher, whereas HDL cholesterol and apolipoprotein A-I were lower in comparison to the control group. Total serum homocysteine in children with positive family history for cardiovascular disease CHD(+) was significantly higher than in the control groups, and in CHD(-) group. It was respectively 7.3 micromol/1 versus 5.45 micromol/l versus 5.21 micromol/l. The results obtained in our study indicate that in hypercholesterolemic children with positive family history for CHD, the concentration of tHcy can be considered as a separate predictive risk factor for premature cardiovascular disease.

Serum homocysteine and lipoprotein (a) concentrations in hypercholesterolemic and normocholesterolemic children.

The relationship between lipids, lipoproteins, total homocysteine, and lipoprotein (a) was studied in hypercholesterolemic and normocholesterolemic children. In hypercholesterolemic children, concentrations of total cholesterol, low-density lipoprotein (LDL) cholesterol, apolipoprotein B, and triglycerides were significantly higher compared to levels in controls, whereas concentrations of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I were lower compared to those in the control group. Total serum homocysteine concentrations in children with a positive family history for cardiovascular disease CHD(+) (7.28 micromol/L) were significantly higher than those in the control group (5.45 micromol/L), and in the group of CHD(-) children (5.25 micromol/L). The median value of lipoprotein (a) in patients was 31.5 mg/dL (range, 11-209 mg/dL) and in the control group, 19 mg/dL (range, 11-95 mg/dL). Concentrations of Lp (a), exceeding 30 mg/dL, were present in 45% of CHD(+) children, in 29% of CHD(-) children, and in only 11% of the control group.

[The role of leptin in human obesity].

The mechanism involved in body mass regulation in humans includes genetic, environmental, and behavioural factors. Human obesity is usually associated with a positive energy balance. Genetic studies in obese mice have revealed the Ob. gene, its products leptin and the leptin receptor to be important factors in the regulation of both appetite and energy expenditure. Leptin is a 16-kilodaltons adipocyte-derived hormone -which circulates in the serum as the free and bound forms. The leptin serum level reflects the amount of energy stored in adipose tissue. Leptin acts through the leptin receptor, -which belongs to the cytokine - receptor family. In rodents as well as in humans, homozygous mutations in genes encoding leptin or the leptin receptor cause early-onset morbid obesity, hyperphagia, and reduced energy expenditure. Recent studies have demonstrated that Ob. gene expression is increased in human obesity. However, mutations of Ob. gene present in the mouse are rare in the human population.

[Evaluation of iron status in women with pregnancy complicated by tobacco smoking]. / Ocena statusu zelaza u kobiet w ciazy powiklanej paleniem tytoniu.

Relatively common iron deficiency in pregnant women is assumed to be enhanced by cigarette smoking. In the presented studies we determined cotinine in serum and urine of 75 pregnant women in order to select groups of smoking women and tobacco abstinence. In the smoking group, a mean concentration of cotinine 1039 +/- 560 mg/L in serum and 1025 +/- 540 mg/L in urine were observed. For assessment of iron status we determined in serum: iron, iron-binding capacity (TIBC), transferrin, transferrin saturation, soluble transferrin receptor and ferritin. In serum of smoking woman in comparison to non smoking, the level of ferritin and transferrin was higher but non significantly. Significant increase of TIBC (p < 0.05) and decrease of transferrin saturation (p < 0.05) was observed. Therefore iron deficiency in transport compartment can not be excluded. The concentration of soluble transferrin receptor was the same in both groups studied. However, in late pregnancy (above 27 week of gestation) ferritin concentration less than 20 mg/L of serum was observed in 70% of smoking and only in 39% of non smoking women (p < 0.05). We concluded that cigarette smoking during pregnancy did not have any effect on the entry of iron-bearing transferrin to cells mediated by soluble transferrin receptor, but affected the level of iron-storage ferritin, which leads to iron deficiency in the storage compartment (ID I).

Differentiation of Bacteroides ovatus and Bacteroides thetaiotaomicron by means of bacteriophage.

Two members of the Bacteroides fragilis group, B. ovatus and B. thetaiotaomicron, are difficult to distinguish by biochemical methods. They are currently identified on the basis of their variable ability to ferment salicin. We studied a method of identification for these two species by using cell lysis by bacteriophages. A total of 38 bacteriophages were used to distinguish the two species. Identification by bacteriophages was compared with species identification by prereduced anaerobically sterilized biochemical testing with salicin as the differentiating test. A total of 215 clinical isolates biochemically identified as B. ovatus or B. thetaiotaomicron were tested. A total of 100% of the strains identified as B. ovatus by bacteriophages produced strong acid in salicin (pH less than or equal to 5.4). However, 40% of the strains identified as B. thetaiotaomicron by bacteriophages also produced strong acid in salicin, and an additional 39% produced weak acid (pH 5.5 to 5.7). This study demonstrates that salicin fermentation is an inadequate test for the differentiation of B. ovatus and B. thetaiotaomicron.

In vitro activity of penicillin and rifampin against group B streptococci.

Eighty-eight strains of group B streptococci were studied to determine their in vitro susceptibility to penicillin and rifampin alone and in combination. Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) were determined by broth microdilution; studies of synergy were performed by microdilution checkerboards and time-kill curves. The MIC90 and MBC90 for penicillin were both less than or equal to 0.06 microgram/ml. The MIC90 and MBC90 for rifampin were less than or equal to 0.25 microgram/ml and greater than 32.0 microgram/ml, respectively. Checkerboard MIC determinations revealed synergy for 46 strains (52%), an additive or indifferent effect for 42 strains (48%), and no antagonism. MBC checkerboards showed antagonism for 62 strains (70%); in 45 (73%) of these 62 strains, high concentrations of rifampin increased the MBC of penicillin, whereas low concentrations decreased the MBC of penicillin. Time-kill studies performed on selected strains supported the results of MBC checkerboards, although concentrations of antibiotics that were antagonistic in the checkerboards had an indifferent effect in time-kill experiments. When concentrations of antibiotics were used that are clinically achievable in cerebrospinal fluid, delayed killing was seen even though synergy had been observed in both MIC and MBC checkerboards.

Evaluation of blood culture procedures in a pediatric hospital.

To determine optimal clinical laboratory techniques for detecting pediatric bacteremia, we studied 7,768 consecutive blood cultures in a 1-year period. Blood was inoculated into one vented 50-ml bottle of brucella broth with 0.05% sodium polyanetholsulfonate and one unvented 50-ml bottle of Columbia broth with 0.05% sodium polyanetholsulfonate and 0.05% cysteine. Bottles were visually examined for growth on days 1 through 7 and blindly subcultured aerobically and anaerobically on days 1, 2, and 7. There were 724 (9.3%) positive cultures, and 484 (6.2%) were clinically significant. The most frequent isolates from bacteremic patients were Haemophilus influenzae (24%) and Streptococcus pneumoniae (17%). Growth was noted in only one bottle in 25% of clinically significant isolates. Bottles inoculated with greater than or equal to 1 ml of blood became positive earlier than bottles inoculated with less than 1 ml. After 1 day of incubation, 48% of the clinically significant cultures showed growth on visual examination, whereas 85% showed growth on subculture. Only 19% of Haemophilus isolates were detected visually on day 1, whereas 88% were recovered on subculture. By day 7, 3.5% of all isolates (including 18% of pneumococcal isolates and 1% of Haemophilus isolates) could no longer be recovered on subculture. We conclude that a two-bottle blood culture system and blind subculture within 24 h will optimize detection of pediatric bacteremia.