Leukocytes Of Children Research Articles

Профили экспрессии микроРНК в лейкоцитах крови как маркеры тяжести расстройств аутистического спектра у детей

Autism spectrum disorders are a heterogeneous group of neurodevelopmental disorders with unknown etiology. Immune dysfunction may be involved in the etiology and pathogenesis of autism. One of the regulators of interactions between the nervous and immune systems are microRNAs, which can be considered as key players in pathogenesis and diagnostic biomarkers. The present study is devoted to a search for the relationship between leukocyte expression of microRNAs: microRNA-155, microRNA-146a, microRNA-124, microRNA-21 and microRNA-9, and the concentration of cytokines in the blood plasma of autistic children, which was carried out to detect biological markers of the disease and its severity. It was found that in children with mild autism, the expression levels of the studied microRNAs in blood leukocytes did not differ from similar values in children with normotypical development. In leukocytes of children with severe autism spectrum disorders, the expression of microRNA-124 and microRNA-146a is reduced compared to normotypical children, and microRNA-146a is reduced compared to children with mild autism. Two significant positive correlations between microRNA-9/IFNg and microRNA-124/TNFa against the background of a negative interaction between microRNA-155 and TNFa were identified in the group of children with mild autism. Four significant negative relationships between microRNA-9/IFNg; microRNA-146a/IFNg; miR-146a/IL-6 and miR-155/IL-10 were found in children with severe autism spectrum disorders. The expression level of microRNA-146a in leukocytes less than 0.0035 с.u. with 86.7% sensitivity and 89.6% specificity may indicate severe autism in children. Thus, we have identified disturbances in the expression levels of important negative regulators of inflammation and participants in neuro-immune interactions – microRNA-146a and microRNA-124.

Diagnostic value of Epstein – Barr virus DNA quantification in blood leukocytes in children with infectious mononucleosis

Aim. To determine the threshold value of the Epstein-Barr virus (EBV) viral load (VL) in blood leukocytes to improve the laboratory diagnostics of infectious mononucleosis in children.Materials and methods. EBV DNA quantification in blood leukocytes in children aged 1-17 years (n=163) was determined by real-time polymerase chain reaction. VL were compared in groups of EBV mononucleosis (n=67), non-EBV mononucleosis (n=25) and healthy donors (n=25). Threshold was determined based on VL data from children with active and latent EBV infection. The R program and the RStudio environment were used for satistic analysis.Results. EBV DNA is found in blood leukocytes in infectious mononucleosis not associated with EBV and in healthy virus carriers, however VL in these groups is significantly lower than in patients with EBV mononucleosis (p<0.001). The threshold value was determined – 41 copies/105 cells (or 1.6 lg of EBV DNA/105 cells), which was characterized by acceptable values of specificity and sensitivity (0.90 and 0.85, respectively) of laboratory diagnostics. High EBV VL (equal to or above the set threshold) is associated with an 8.5-fold increased risk of detecting active EBV infection compared to children who have a low VL (below a set threshold) (RR 8.5; 95% CI: 3.7–19.7, p<0.001).Conclusion. In general, the results obtained create prerequisites for more intensive implementation of quantitative studies of EBV DNA in blood leukocytes, both in the context of improving the early diagnosis of infectious mononucleosis and its etiological interpretation, and in terms of detailing the features of the course of EBV infection.

Time course of altered DNA methylation evoked by critical illness and by early administration of parenteral nutrition in the paediatric ICU

BackgroundA genome-wide study identified de novo DNA methylation alterations in leukocytes of children at paediatric intensive care unit (PICU) discharge, offering a biological basis for their impaired long-term development. Early parenteral nutrition (early-PN) in PICU, compared with omitting PN in the first week (late-PN), explained differential methylation of 23% of the affected CpG-sites. We documented the time course of altered DNA methylation in PICU and the impact hereon of early nutritional management.ResultsWe selected 36 early-PN and 36 late-PN matched patients, and 42 matched healthy children. We quantified DNA methylation on days 3, 5 and 7 for the 147 CpG-sites of which methylation was normal upon PICU admission in this subset and altered by critical illness at PICU discharge. Methylation in patients differed from healthy children for 64.6% of the 147 CpG-sites on day 3, for 72.8% on day 5 and for 90.5% on day 7 as revealed by ANOVA at each time point. Within-patients methylation time course analyses for each CpG-site identified different patterns based on paired t test p value and direction of change. Rapid demethylation from admission to day 3 occurred for 76.2% of the CpG-sites, of which 67.9% remained equally demethylated or partially remethylated and 32.1% further demethylated beyond day 3. From admission to day 3, 19.7% of the CpG-sites became hypermethylated, of which, beyond day 3, 34.5% remained equally hypermethylated or partially demethylated again and 65.5% further hypermethylated. For 4.1% of the CpG-sites, changes only appeared beyond day 3. Finally, for the CpG-sites affected by early-PN on the last PICU day, earlier changes in DNA methylation were compared for early-PN and late-PN patients, revealing that 38.9% were already differentially methylated by day 3, another 25.0% by day 5 and another 13.9% by day 7.ConclusionsCritical illness- and early-PN-induced changes in DNA methylation occurred mainly within 3 days. Most abnormalities were at least partially maintained or got worse with longer time in PICU. Interventions targeting aberrant DNA methylation changes should be initiated early.

Expression analysis of apoptotic and survival genes in blood leukocytes of children with various forms of HHV-6 infection

Despite that human herpes virus type 6 (HHV-6) is extremely spread worldwide, molecular mechanisms of behind HHV-6 infection pathogenesis remain largely unexplored. No molecular markers were found linked to unfavorable course of HHV-6 infection which could allow to ease up selecting proper therapy and preventing development of complications. The aim of the study was to analyze expression of apoptosis and survival-related genes in blood leukocytes from 7–17-year-old children upon various forms of HHV-6 infection. The analysis was carried out by using DNA microarrays developed by us allowing to assess changes in expression level both of individual mRNAs and total gene set (-Σ). It was shown that during the acute phase of HHV-6 infection mRNA level was shifted toward pro-apoptotic factors. In the convalescence phase, most altered mRNA levels returned to normal. We have identified a set of mRNAs and genes whose expression level was significantly changed in acute disease phase. According to available data, these factors play an important role in regulation of studied signaling pathways. In order to search for HHV-6-associated factors, which markedly affect disease pattern of severe herpesvirus mixed infection, we analyzed significant changes of mRNA and genes expression levels in patients with severe HHV-6+EBV+CMV mixed infection and EBV+CMV mixed infection of moderate severity compared with healthy donors. The levels of 5 mRNAs (FAF1-NM_007051, DAPK2-NM_014326, CASP8AP2-NM_001137667, CASP8-NM_033356, BTK-NM_001287345) and 3 genes (FAS-Σ, Puma/BBC3-Σ, ITCH-Σ) were significantly increased in severe mixed infection comorbid with HHV-6 (EBV+CMV+HHV-6) but without HHV-6 (EBV+CMV) compared with healthy donors. Most of detected factors belong to Fas-mediated apoptosis pathway, and may be considered as candidate prognostic development factors of severe herpes virus infection involving HHV-6. This study profoundly extends existing understanding on molecular pathogenesis of HHV-6 infection involving apoptosis and pro-survival signaling pathways. Marked changes of mRNA and gene levels most likely contributed to the pathogenesis of HHV-6 as well as severe HHV-6+EBV+CMV mixed infection.

Apoptosis- and survival-related gene mRNA profile in peripheral blood leukocytes in children with acute EBV infectious mononucleosis

Acute EBV-associated mononucleosis develops mainly in children and in patients with functionally impaired immune system. Consequently, it may result in developing secondary immunodeficiency, neoplasms as well as diverse alterations in cell-mediated immune reaction. Despite extensively examining molecular mechanisms of EBV infection, it is also necessary seek for new molecular and genetic factors underlying pathogenesis of EBV-mediated mononucleosis and EBV-associated malignant cell transformation is necessary, which might be used in clinical practice to monitor clinical score as well as predictive parameters for EBV-associated complications such as immunocompromised conditions and neoplasms. Here, we proposed to use our splicing sensitive DNA microarrays to perform a comprehensive semi-quantitative mRNA expression analysis for major apoptosis- and survival-related signaling components in peripheral blood leukocytes collected from children with acute EBV infectious mononucleosis as well as during recovery period. Using such DNA microchips allowed to assess both total (denoted by Σ) and separate transcript expression resulting from alternative splicing. It was shown that the balance of mRNA levels in acute phase of EBV-infectious mononucleosis was shifted towards upregulated expression of anti-apoptotic factors and components of of NF-κB-linked pro-survival signaling able to profoundly augment apoptosis resistance. Moreover, some EBV-associated changes (BIM/BCL2L11-Σ, PUMA/ BBC3-NM_001127241, BID-Σ, CASP3-Σ, NFKB1-Σ, RELA-Σ) were in agreement with the data published before. In addition, we also found previously unknown changes in level of EBV-associated coding and noncoding transcripts (DCR1/ TNFRSF10C-NM_003841, DR5/TNFRSF10B-NR_027140, CASP6 beta/CASP6-NM_032992, CASP7-NM_033338). Analyzing their properties allowed to suggest that they play an important role in the pathogenesis of EBV-associated mononucleosis. However, at asymptomatic recovery stage, level of some mRNA expression was kept altered compared to healthy volunteers (DCR2/TNFRSF10D-NM_003840, CASP8-Σ, CASP3-Σ, BIM/BCL2L11-Σ, BCL2-NM_000633, MCL1-Σ, BCL-W/BCL2L2-Σ, BCL-XL/BCL2L1-NM_138578, BIRC2-NM_001166, XIAP-NM_001167, TRAF2-NM_021138, MAP3K14-Σ, NFKB1-Σ), which may point at postponed EBV-associated molecular consequences. On one hand, such changes may be due to long-lasting anti-EBV immune response, whereas, on the other hand, they might be influenced by EBV-associated factors facilitating virus persistence. Overall, we identified the molecular features predisposing to chronic course of EBV-infection. The data obtained further expand our understanding about the molecular pathogenetic mechanisms for EBV infectious mononucleosis.

Peculiarities of leukocyte apoptosis modulation in children with pyelonephritis

One of the leading places among inflammatory diseases of the urinary tract of children belongs to pyelonephritis, the course of which presents in most cases as a severe infectious disease threatening the patient’s life, which is the main reason for development of chronic kidney failure. This study was conducted to compare apoptosis stages in peripheral blood of children of different age categories with pyelonephritis depending on etiological factor and complications. The problem of mechanisms underlying immune system misregulation, especially functional activity of leukocytes in children with pyelonephritis, have not been explored in recent years. Assessment of leukocytes (neutrophils) apoptosis stages in peripheral blood of children of different age categories with pyelonephritis depending on complications and etiological factor was the aim of present study. The children's peripheral blood samples were analysed and assessed using a flow cytofluorimeter. The present study demonstrates an increase of the level of apoptotic cells at an early stage of apoptosis in children of all age categories with chronic pyelonephritis, which can be explained by associations of a wide range of pathogens and the presence of sequelae. An increase in the number of apoptotic cells in the late stage of apoptosis is observed in children aged 1 month – 8 years, in children 8–18 years, the amount of apoptotic cells is reduced by 1.5 times. The study of apoptosis stages allows complete characterization of the dynamics of the apoptotic process and supplementation of the pathogenesis of pyelonephritis in children. Such studies will make it possible to affect apoptosis modulation to regulate or correct it and encourage the finding of innovative solutions in the treatment related to influence on the immune response. We conclude that enhancement of peripheral blood leukocyte apoptosis in chronic form of pyelonephritis especially in young children is due to the polyetiology of this form of pyelonephritis and the development of complications.

Scintigraphy with 99mTc-HMPAO labeled leukocytes is still an accurate and convenient tool to rule out suspected inflammatory bowel disease in children.

Abdominal pain is a common complaint in children and its differential diagnosis includes inflammatory bowel disease (IBD). The aim of the study was to assess the diagnostic accuracy of scintigraphy with 99mTechnetium Hexamethylpropyleneamine Oxime (99mTc-HMPAO) labeled leukocytes in children with suspected IBD. Eighty-five children (age 12.4 ± 4.3 years, 47% boys) with suspected IBD based on clinical presentation, laboratory and ultrasound findings underwent scintigraphy with 99mTc-HMPAO labeled leukocytes. Abdominal scintigrams were acquired 40 min and 90 min post injection, and whole body scintigrams at 180 min. Scintigraphy was evaluated by two specialists in nuclear medicine. The results were compared with the final diagnosis established by endoscopy, histology, other imaging methods, and follow-up evaluated by an expert in pediatric gastroenterology. Scintigraphy results corresponded with the final diagnosis in 78 (91%) patients resulting in a sensitivity of 89% (95%CI 72 to 98%), specificity of 91% (95% CI 82 to 98%), and accuracy of 91% (95% CI 83 to 96%). The interobserver agreement was 0.82 (95% CI 0.75 to 0.88) and the radiation dose estimate was 4.2 ± 1.5 mSv. In 28 children (25 positives and 3 negatives on scintigraphy), the diagnosis of IBD was established by endoscopy, histology, MR enterography, or fluoroscopy. Five positive findings on scintigraphy were not confirmed by other methods or during follow-up. Scintigraphy with 99mTc-HMPAO labeled leukocytes in children with suspected IBD has high accuracy and offers a non-invasive option for detecting the presence of gastrointestinal inflammation. Scintigraphy is a powerful non-invasive decision-making tool in the management of suspected IBD that may spare a greater proportion of children of more invasive and demanding examinations.

Immunogenetic Status of Children with Mild Iodine Deficiency, Latent Iron Deficiency and Their Combination

Due to the prevalence of microelementosis (including iodine and iron deficiencies), cytogenetic abnormalities in children with microelement imbalance were studied.The objective of the research was to assess the abnormalities in the immunogenetic status of the organism by the frequency and spectrum of chromosomal aberrations, associations of acrocentric chromosomes and to determine the frequency of micronuclei in peripheral blood leukocytes in children with mild iodine deficiency, latent iron deficiency and their combination.Materials and methods. There were examined 68 boys and 65 girls at the age of 6 to 18 years. In the analysis of indicators, the main attention was paid to the age- (6-11 and 12-18 years) and gender-related peculiarities.Results and discussion. In all the children, associations of acrocentric chromosomes of two chromosomes were most commonly observed: in the control group, this indicator was 73.74%; in iodine deficiency, it was 67.72%; in iron deficiency, it was 67.68%; in combined microelementosis, the indictor was 68.68%. Chromosomal abnormalities were recorded in 56.03% of children. However, in the control group, this indicator was 40.94%, while in microelement imbalance, it was 71.13%. The most significant changes in the spectrum of chromosomal aberrations were identified in iodine and iron deficiencies (increase in the frequency of paired fragments, dicentrics, translocations, and the presence of a ring chromosome).Conclusions. Changes in the frequency and characteristics of the number of chromosomes in associations of acrocentric chromosomes, the frequency and spectrum of chromosomal aberrations, and the number of micronuclei indicated genotype instability, especially in iodine deficiency and combined microelement imbalance.

Primary DNA damage in salivary leukocytes of children exposed to air pollutants. MAPEC_LIFE project

Primary DNA damage in salivary leukocytes of children exposed to air pollutants. MAPEC_LIFE project

Dynamics of immunological features of oral liquid in children with chronic catarrhal gingivitis living on polluted territories with fluoride and iodine deficiency

The aim of the study was to assess the state of oral liquid (OL) immunity in children with chronic catarrhal gingivitis (CCG) living in adverse environmental conditions. 120 children aged 7-15 residing in ecologically unfavorable areas of Lviv region were examined, while 75 children living in 'relatively clean' region were involved in the control group. Chronic catarrhal gingivitis was diagnosed according to Danilevskiy classification (1994). The level of cytokines in oral liquid of 7-years-old children living in ecologically polluted region (EPR), was (198.19±4.11)·106/l, which was 1.4 times more than in the conditionally clean region (CCR): (141.09±4.10)·106/l (p<0.01). Analysis of cytokine profile in 7-years-old from EPR showed increased levels of IL-6 proinflammatory cytokines by 11.22% (13.78±0.38 pg/ml vs 12.39±0.50 pg/ml in controls, p<0.05) and the decrease of IL-4 anti-inflammatory cytokine by 26.9% (7.12±0.62 pg/ml vs 9.74±0.58 pg/ml, p<0.01). In 12 years-old from EPR quantity of leukocytes in OL was 1.3 times higher than in controls ((246.81±4.16)·106/l vs (190.02±4.11)·106/l, p<0.01), the increase of the IL-6 content of 27.1% (p<0.01) and reduce of the IL-4 of 21.5% (p<0.05) compared to controls was also seen. At the age of 15 further increase of leucocytes in children from EPR was revealed: (297.53±4.15)·106/l, which was 1.2 times higher than in controls (p<0.01). Changes of cytokine profile in this age group were characterized by increased content of IL-6 of 26.41% (p<0.05) and IL-4 drop of 28.53% (p>0.05). Thus the age-dependent trend for the increase of leukocytes count in OL and pro-inflammatory cytokine IL-6 with the decrease of anti-inflammatory cytokine IL-4 is noted in children with CCG living in EPR.

IL10 Single Nucleotide Polymorphisms are Related to Upregulation of Constitutive IL‐10 Production and Susceptibility to Helicobacter pylori Infection

Helicobacter pylori infection is a strong risk factor for gastric cancer, likely due to the extensive inflammation in the stomach mucosa caused by these bacteria. Many studies have reported an association between IL10 polymorphisms, the risk of gastric cancer, and IL-10 production. The aim of the study was to evaluate the association between IL10 genetic variants, Helicobacter pylori infection, and IL-10 production by peripheral blood leukocytes in children. We genotyped a total of 12 single nucleotide polymorphisms in IL10 in 1259 children aged 4-11years living in a poor urban area in Salvador, Brazil, using TaqMan probe based, 5' nuclease assay minor groove binder chemistry. Association tests were performed by logistic regression for Helicobacter pylori infection and linear regression for IL-10 spontaneous production (whole-blood cultures) including sex, age, and principal components for informative ancestry markers as covariates, using PLINK. Our results shown that IL10 single nucleotide polymorphisms rs1800896 (OR=1.63; 95% CI=1.11-2.39), rs3024491 (OR=1.71; 95% CI=1.14-2.57), rs1878672 (OR=1.79; 95% CI=1.19-2.68), and rs3024496 (OR=1.48; 95% CI=1.05-2.08) were positively associated with Helicobacter pylori infection. Eight single nucleotide polymorphisms were associated with spontaneous production of IL-10 in culture, of which three (rs1800896 and rs1878672, p=.04; rs3024491, p=.01) were strongly associated with infection by Helicobacter pylori. Our results indicate that IL10 variants rs1800896, rs3024491, rs1878672, and rs3024496 are more consistently associated with the presence of anti-H.pylori IgG by inducing increased production of IL-10. Further studies are underway to elucidate the role of additional genetic variants and to investigate their impact on the occurrence of gastric cancer.

Effect of Remote Ischemic Preconditioning on Phosphorylated Protein Signaling in Children Undergoing Tetralogy of Fallot Repair: A Randomized Controlled Trial

BackgroundOur previous randomized controlled trial demonstrated cardiorespiratory protection by remote ischemic preconditioning (RIPC) in children before cardiac surgery. However, the impact of RIPC on myocardial prosurvival intracellular signaling remains unknown in cyanosis. RIPC may augment phosphorylated protein signaling in myocardium and circulating leukocytes during tetralogy of Fallot (ToF) repair.Methods and ResultsChildren (n=40) undergoing ToF repair were double‐blind randomized to RIPC (n=11 boys, 9 girls) or control (sham RIPC: n=9 boys, 11 girls). Blood samples were taken before, immediately after, and 24 hours after cardiopulmonary bypass. Resected right ventricular outflow tract muscle and leukocytes were processed for protein expression and mitochondrial respiration. There was no difference in age (7.1±3.4 versus 7.1±3.4 months), weight (7.7±1.8 versus 7.5±1.9 kg), or bypass or aortic cross‐clamp times between the groups (control versus RIPC, mean±SD). No differences were seen between the groups for an increase in the ratio of phosphorylated to total protein for protein kinase B, p38 mitogen activated protein kinase, signal transducer and activator of transcription 3, glycogen synthase kinase 3β, heat shock protein 27, Connexin43, or markers associated with promotion of necrosis (serum cardiac troponin I), apoptosis (Bax, Bcl‐2), and autophagy (Parkin, Beclin‐1, LC3B). A high proportion of total proteins were in phosphorylated form in control and RIPC myocardium. In leukocytes, mitochondrial respiration and assessed protein levels did not differ between groups.ConclusionsIn patients with cyanotic heart disease, a high proportion of proteins are in phosphorylated form. RIPC does not further enhance phosphorylated protein signaling in myocardium or circulating leukocytes in children undergoing ToF repair.Clinical Trial RegistrationURL: (http://www.anzctr.org.au/trial_view.aspx?id=335613. Unique identifier: Australian New Zealand Clinical Trials Registry number ACTRN12610000496011.

Altered Genes Profile of Renin–Angiotensin System, Immune System, and Adipokines Receptors in Leukocytes of Children With Primary Hypertension

Renin-angiotensin system, metabolic abnormalities, and immune activity have a role in the pathogenesis of primary hypertension. We assessed the leukocyte mRNA expression of angiotensinogen, angiotensin converting enzyme, renin, angiotensin 2 type 1 receptor, CD14 molecule, adiponectin type 1 receptor, and leptin receptor in hypertensive children before and after nonpharmacological treatment. Leukocyte mRNA expression was measured by means of quantitative real-time reverse transcriptase-polymerase chain reaction in 23 hypertensive children before and after 6 months of nonpharmacological treatment based on dietary advice and physical activities. Twenty-three normotensive children matched for age, sex, and body mass index served as a control group. Before treatment patients had elevated expression of angiotensin converting enzyme and CD14 mRNA, decreased expression of angiotensinogen and angiotensin type 1 receptor mRNA, and unchanged expression of renin, adiponectin, and leptin receptors mRNA as compared with controls. Renin mRNA negatively correlated with 24-hour mean arterial pressure and carotid intima-media thickness. Six months of nonpharmacological treatment caused decrease of blood pressure and normalization of metabolic abnormalities. Renin, adiponectin, and leptin receptors mRNA expression decreased and were lower than in control group. Changes in blood pressure, left ventricular mass, carotid intima-media thickness, body mass index, and waist circumference did not correlate with changes in the expression of renin-angiotensin system genes, CD14, leptin, and adiponectin receptors mRNA. We conclude that leukocytes of hypertensive children displayed alterations in the expression of renin-angiotensin system genes as well as those of CD14. Nonpharmacological treatment resulted in downregulation of genes involved in renin-angiotensin activation and those engaged in leukocyte responses to adipokines.

Зміни експресії мРНК і білків антиоксидантних ферментів у лейкоцитах крові дітей, хворих на бронхіальну астму, при інтервальному гіпоксичному тренуванні

The effect of 10 days of interval hypoxic training (IHT) on the mRNA expression and protein content of antioxidant enzymes (superoxide dismutase, catalase and glutathione-S-transferase) in leukocytes of children with bronchial asthma (BA) was investigated. It was shown that after sessions of IHT the mRNA expression of superoxide dismutase decreased by 32.5% (P < 0,05), but the level of protein was unchanged. The level of catalase gene mRNA and protein content in leukocytes after IHT increased by 67% and 13% accordingly. We detected a 90% increase in glutathione S-transferase level following IHT.

Changes in mRNA and Protein Expression of Antioxidant Enzymes in Leukocytes of Children with Bronchial Asthma after Interval Hypoxic Training

Changes in mRNA and Protein Expression of Antioxidant Enzymes in Leukocytes of Children with Bronchial Asthma after Interval Hypoxic Training

Fecal Leukocytes in Children Infected with Diarrheagenic Escherichia coli

The purpose of this study was to determine the presence and quantity of fecal leukocytes in children infected with diarrheagenic Escherichia coli and to compare these levels between diarrhea and control cases. We analyzed 1,474 stool samples from 935 diarrhea episodes and 539 from healthy controls of a cohort study of children younger than 2 years of age in Lima, Peru. Stools were analyzed for common enteric pathogens, and diarrheagenic E. coli isolates were studied by a multiplex real-time PCR. Stool smears were stained with methylene blue and read by a blinded observer to determine the number of polymorphonuclear leukocytes per high-power field (L/hpf). Fecal leukocytes at >10 L/hpf were present in 11.8% (110/935) of all diarrheal episodes versus 1.1% (6/539) in controls (P < 0.001). Among stool samples with diarrheagenic E. coli as the only pathogen isolated (excluding coinfection), fecal leukocytes at >10 L/hpf were present in 8.5% (18/212) of diarrhea versus 1.3% (2/157) of control samples (P < 0.01). Ninety-five percent of 99 diarrheagenic E. coli diarrhea samples were positive for fecal lactoferrin. Adjusting for the presence of blood in stools, age, sex, undernutrition, and breastfeeding, enterotoxigenic E. coli (ETEC) isolation as a single pathogen, excluding coinfections, was highly associated with the presence of fecal leukocytes (>10 L/hpf) with an odds ratio (OR) of 4.1 (95% confidence interval [CI], 1.08 to 15.51; P < 0.05). Although diarrheagenic E. coli was isolated with similar frequencies in diarrhea and control samples, clearly it was associated with a more inflammatory response during symptomatic infection; however, in general, these pathogens elicited a mild inflammatory response.

Evidence for a modulatory effect of IL-10 on both Th1 and Th2 cytokine production: The role of the environment

Allergic and other immune-mediated diseases are complex disease states determined by interplay between host genetics and environmental factors. Environmental changes such as fewer infections and reduced exposure to microbial products have been suggested to have led to insufficient regulation of Th1 and Th2 immune responses, causing an increased incidence of inflammatory diseases. The objective of the present study was to investigate the effect of poor living environmental conditions on mitogen-induced production of cytokines (Th1 and Th2) by peripheral blood leukocytes in children living in urban Brazil and investigate the role of IL-10 in modifying this effect. Our data showed that the proportion of children producing Th1 and Th2 cytokines was lower among those with poor living conditions and that this finding was stronger in children producing IL-10. These results provide a possible biologic explanation for the temporal trends of increasing risk of inflammatory diseases observed in populations living in affluent countries.

Informativeness of Some Hematological Indices for the Evaluation of Influence of Low-Degree Atmospheric Pollution on Health

ISEE-0347 Background and Objective: Considering that blood tissue is one of the most sensitive to changes in levels of atmospheric pollution, we set ourselves the aim of making comparative analysis of some hematological indices in clinically healthy children exposed to the influence of low-degree but of different levels atmospheric pollution. Methods: In 1998 we investigated the first group–100 children at the age of 12.68 ± 0.56 years (X ± SD) from Dimitrovgrad living at average annual levels of TSPM between 0.312–0.089 mg/m3 and SO2 between 166.90–3.30 μg/m3; The second group (94 children 12.8 ± 0.7 y from the same schools), living at levels of pollution respectively 0.206–0.066 mg/m3 (TSPM) and 146.16–1.44 (SO2), were investigated in 2003. For both groups we studied also the inactive forms of hemoglobin (methemoglobin, carboxyhemoglobin, sulphhemoglobin). All children were studied in the first decade of May in specialized laboratories of Medical University–Plovdiv. Results: There were reliably lower levels of the erythrocytes (X ± SD: 4.52 ± 0.37 vs. 4.77 ± 0.41 × 1012/l, P = 0.000), hemoglobin (127.87 ± 7,87 vs. 132.02 ± 8.43 g/l, P = 0.000), thrombocytes (268.59 ± 58.60 vs. 312.37 ± 73.56 × 109/l) and higher number of leucocytes in children living at higher levels of atmospheric pollution (6.97 ± 1.61 vs. 6.50 ± 1.59 × 109/l, P = 0.042). No statistically significant differences were found in the levels of inactive forms of hemoglobin between the two groups of children. Conclusion: Prolonged exposure to low-degree atmospheric pollution suppresses hemopoesis. Standard hematological tests are good indicator for health evaluation of the influence of low-degree atmospheric pollution but the inactive forms of hemoglobin as indices are not informative enough.

Body fat and circulating leukocytes in children

To determine the effects of obesity on baseline levels of circulating granulocytes, monocytes, and lymphocyte subtypes in otherwise healthy children. Two group comparison of leukocytes in normal weight control and overweight children. In total, 38 boys and girls, ages 6-18 years, divided in two groups: normal weight, (NW, BMI<85th %tile, n=15) and overweight (OW, body mass index (BMI)>85th %tile, n=23). BMI obtained from direct measures of height and body mass. Body fat was assessed by DEXA. Complete blood counts (CBC) were obtained by standard clinical hematology methods and surface antigen staining by flow cytometry. The OW group compared to the NW group had increased total leukocytes counts (P=0.011), neutrophils (P=0.006), monocytes (P=0.008), total T (CD3) lymphocytes (P=0.022), and Helper T (CD4(+)) cells (P=0.003). Significant correlations were evident between leukocytes, and BMI percentile, BMI, or percent body fat. Neither lean body mass nor VO(2peak) per unit lean body mass were significantly related to any of the leukocytes. Percent body fat and BMI percentile were positively correlated (P<0.05) to total T cells (CD3) and/or helper T cells (CD4(+)). A group of 23 overweight children displayed elevated counts in most types of circulating immune cells, suggesting the presence of low-grade systemic inflammation, a known pathogenetic mechanism underlying most long-term complications of obesity. Our data provide an additional rationale for the importance of avoiding or correcting pediatric obesity.

Intracellular cytokines in peripheral blood leucocytes in children with chronic renal failure

We have previously shown that children with mild renal impairment show significant changes in leucocyte subsets and circulating cytokines, indicating that these patients show an increased inflammatory state. We hypothesised that measurement of intracellular cytokine production by lymphocytes and monocytes would more precisely define the immunological mechanism associated with the inflammatory state in children with pre-dialytic chronic renal failure. Blood was collected from children with chronic renal failure (CRF) who were not yet on dialysis and an age-matched control group. Leucocyte subsets and intracellular cytokine production were determined using flow cytometry. Children with CRF showed increased production of interleukin (IL)-12 by monocytes accompanied by decreased production of interferon (IFN)-gamma and increased production of IL-4 by T cells. There were no significant changes in the production of IL-8, IL-10, IL-6, IL-1alpha or tumour necrosis factor (TNF)-alpha by monocytes or in IL-2 or TNF-alpha production by T cells. There were no significant differences in total white cell count or lymphocyte count. There was a significant decrease in both B and NK cells. This study examines intracellular cytokine production in children with CRF in detail. It is the first to show that children with relatively mild renal failure display significant immunological changes of lymphocyte subsets and leucocyte cytokine production. These data provide a more accurate understanding of the immunological changes that may contribute to the clinical manifestations and progression of the disease.