MAIT cell activation in adolescents is impacted by bile acid concentrations and body weight.

Bile acids (BAs) are produced by liver hepatocytes and were recently shown to exert functions additional to their well-known role in lipid digestion. As yet it is not known whether the mucosal-associated invariant T (MAIT) cells, which represent 10-15% of the hepatic T cell population, are affected by BAs. The focus of the present investigation was on the association of BA serum concentration with MAIT cell function and inflammatory parameters as well as on the relationship of these parameters to body weight. Blood samples from 41 normal weight and 41 overweight children of the Lifestyle Immune System Allergy (LISA) study were analyzed with respect to MAIT cell surface and activation markers [CD107a, CD137, CD69, interferon (IFN)-γ, tumor necrosis factor (TNF)-α] after Escherichia coli stimulation, mRNA expression of promyelocytic leukemia zinc finger protein (PLZF) and major histocompatibility complex class I-related gene protein (MR1), the inflammatory markers C-reactive protein (CRP), interleukin (IL)-8 and macrophage inflammatory protein (MIP)-1α as well as the concentrations of 13 conjugated and unconjugated BAs. Higher body weight was associated with reduced MAIT cell activation and expression of natural killer cell marker (NKp80) and chemokine receptor (CXCR3). BA concentrations were positively associated with the inflammatory parameters CRP, IL-8 and MIP-1α, but were negatively associated with the number of activated MAIT cells and the MAIT cell transcription factor PLZF. These relationships were exclusively found with conjugated BAs. BA-mediated inhibition of MAIT cell activation was confirmed in vitro. Thus, conjugated BAs have the capacity to modulate the balance between pro- and anti-inflammatory immune responses.

Maternal cytokine status may prime the metabolic profile and increase risk of obesity in children.

BACKGROUND: The maternal inflammation status during pregnancy has been associated with metabolic imprinting and obesity development in the child. However, the influence of the maternal Th2 cytokines, interleukin-4 (IL4), IL5 and IL13, has not been studied so far. METHODS: We investigated the relationship between maternal innate (IL6, IL8, IL10 and tumor necrosis factor-α (TNFa)) and adaptive (interferon-γ, IL4, IL5 and IL13) blood cytokine levels at 34 weeks of gestation and children's overweight development until the age of 3 years in 407 children of the German longitudinal LINA (Lifestyle and Environmental Factors and their Influence on Newborns Allergy risk) cohort. Children's body weight and height were measured during the annual clinical visits or acquired from questionnaires. Body mass index (BMI) Z-scores were calculated according to the WHO reference data to adjust for child's age and gender. Cytokine secretion was stimulated with phytohemagglutinin or lipopolysaccharide and measured by cytometric bead assay. Furthermore, we assessed metabolic parameter in blood of 318 children at age 1 using the AbsoluteIDQ p180 Kit (Biocrates LIFE Science AG). RESULTS: Applying logistic regression models, we found that an increase of maternal IL4 and IL13 was associated with a decreased risk for overweight development in 1- and 2-year-old children. This effect was consistent up to the age of 3 years for IL13 and mainly concerns children without maternal history of atopy. Children's acylcarnitine concentrations at 1 year were positively correlated with maternal IL13 levels and inversely associated with the BMI Z-score at age 1. CONCLUSIONS: We were able to show for the first time that the maternal Th2 status may be linked inversely to early childhood overweight development accompanied by an altered metabolic profile of the fetus. However, our data do not support a direct mediating role of acylcarnitines on maternal IL13-induced weight development.

The LINA cohort: indoor chemical exposure, circulating eosinophil/basophil (Eo/B) progenitors and early life skin manifestations.

BACKGROUND: Hematopoietic progenitor cells, especially those committed to the Eo/B lineage, are known to contribute to allergic inflammation. OBJECTIVE: The aim of the present study was to investigate whether environmental factors are associated with changes in numbers of circulating Eo/B progenitors at 1 year of age. METHODS: Peripheral blood from 60 1-year-old children enrolled in the LINA (Lifestyle and environmental factors and their Influence on Newborns Allergy risk) birth cohort was assessed for Eo/B progenitor cells (Eo/B CFU) using standardized and validated methylcellulose assays. Frozen peripheral blood mononuclear cells (PBMC) were cultured in the presence of IL-3, IL-5 or GM-CSF, and Eo/B CFUs enumerated. Clinical outcomes and exposure to environmental tobacco smoke (ETS) were documented by standardized questionnaires, and indoor volatile organic compound (VOC) concentrations were assessed by passive sampling. RESULTS: Children with skin manifestations (atopic dermatitis or cradle cap) within the first year of life had higher numbers of circulating IL-3-, IL-5- or GM-CSF-stimulated Eo/B CFUs (P < 0.05) at 1 year. In children with cradle cap, a positive correlation was found between Eo/B CFUs and exposure to ETS-related VOCs during pregnancy or at 1 year of age (P < 0.05). CONCLUSIONS AND CLINICAL RELEVANCE: This is the first demonstration that environmental exposures are positively associated with levels of circulating Eo/B progenitors. The recruitment and differentiation of Eo/B progenitors in response to environmental triggers may play a role in the development of skin manifestations during the first year of life.

Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year.

BACKGROUND: Regulatory T cells (Tregs) with stable FOXP3 expression are characterized by a specific demethylated region in the FOXP3 gene (Treg-specific demethylated region, TSDR). The aim of this study was to analyse the influence of prenatal factors on cord blood Treg numbers, as detected by changes in the TSDR demethylation, and the subsequent risk for allergic diseases. METHODS: Analyses were performed within the LINA study in blood samples from pregnant women (34th gestational week) and in cord blood (n = 346 mother-child pairs). Treg numbers were detected via DNA demethylation in the FOXP3 TSDR. At age 1, total and specific IgE was measured in children's blood. In addition, maternal cytokine production (Th1/Th2/Th17) was analysed. Exposure and disease outcomes were assessed by questionnaires. RESULTS: Boys had lower Treg numbers compared with girls (P < 0.001). Parental atopy history, particularly maternal hay fever and paternal asthma were related to lower Treg numbers in cord blood (adj. MR = 0.81, 95% CI = 0.68-0.97; adj. MR = 0.60, 95% CI = 0.45-0.81). Maternal cytokines (IL-13, IL-17E and IFN-γ) and maternal smoking/exposure to tobacco smoke during pregnancy were also associated with decreased cord blood Treg numbers (adj. MR = 0.89, 95% CI = 0.97-1.00). Children with lower Treg numbers at birth had a higher risk to develop atopic dermatitis (adj. OR = 1.55, 95% CI = 1.00-2.41) and sensitization to food allergens (adj. OR = 1.55, 95% CI = 1.06-2.25) during the first year of life. CONCLUSIONS: These results indicate that both genetic and environmental factors presumably influence the development of foetal Tregs. Low cord blood Treg numbers may predict early atopic dermatitis.

Association of neuropeptides with Th1/Th2 balance and allergic sensitization in children.

BACKGROUND: Among neurogenic factors, the neuropeptides have an important regulatory influence on immune system activity and may lead to allergic sensitization. OBJECTIVE: The aim of our study was to investigate the relationship of the neuropeptides vasoactive intestinal peptide (VIP), somatostatin (SOM) and substance P (SP) on modulation of Th1/Th2 balance and allergic sensitization in children. METHODS: Within the LISAplus (Life style-Immune system-Allergy) study, blood samples of 321 six-year-old children were analysed for concentration of neuropeptides, Th1 and Th2 cytokines, transcription factors for T cell regulation and suppressors of cytokine signalling. In addition, samples were screened for specific IgE against inhalant and food allergens. RESULTS: Children with high SOM values showed a Th2 polarization and a reduced expression of FOXP3, the marker for regulatory T cells. High (VIP) levels correlated inversely with the expression of T cell transcription factors (Tbet and SOCS3). In contrast, elevated levels of SP were associated with reduced GATA3 and SOCS3 expression and with increased IFN-gamma concentrations. Allergic sensitization was more prevalent in children with higher SOM and VIP concentrations but not associated with SP levels. CONCLUSION: Our data reveal an association between neuropeptides and modulatory effects on immune cells in vivo, especially on Th1/Th2 balance with a correlation to allergic sensitization in children. We suggest that elevated SOM and VIP concentrations and the inducing factors should be considered as allergy risk factors.

Electrophysiological studies on the hippocampus and prefrontal cortex assessing the effects of amyloidosis in amyloid precursor protein 23 transgenic mice.

In vitro and in vivo electrophysiological studies were done to investigate the neuronal function of the hippocampus and prefrontal cortex in the amyloid precursor protein (APP) 23 transgenic mouse model for amyloidosis developed by Sturchler-Pierrat et al. [Proc Natl Acad Sci USA 94 (1997) 13287]. Brain slices were taken from 3, 6, 9, 12, 18 and 24 month old wildtype and hemizygous type APP23 mice. Extracellular field potentials were recorded from the CA1 region of the hippocampus while stimulating the Schaffer collaterals. In addition, extracellular field potentials were elicited from areas within layer V/VI of the prefrontal cortex by stimulating the same layer V/VI. Basic synaptic function in the hippocampus was reduced in hemizygous APP23 mice compared with their wildtype littermates at 12 and 18 months of age, whereas, it was unaltered within the prefrontal cortex. Long-term potentiation in the hippocampus and the prefrontal cortex of hemizygous APP23 mice was similar compared with their wildtype littermates. In vivo electrophysiological experiments were done in 3, 9, 18 and 24 month old wildtype and hemizygous APP23 mice. No differences were observed in the number of single spontaneously active units recorded within the prefrontal cortex of hemizygous APP23 mice compared with their wildtype littermates. Field potentials elicited during stimulation of cortico-cortical pathways to assess synaptic transmission and short-term synaptic plasticity were also unchanged in hemizygous APP23 mice. Furthermore, presumable antidromic field potentials recorded in the prefrontal cortex during stimulation of the striatum were similar between the hemizygous APP23 and wildtype mice at each age. The present study shows that amyloidosis impairs basic synaptic function but not long-term potentiation in the hippocampus, however, does not alter any of the neurophysiological functions measured within the prefrontal cortex. These findings suggest that amyloidosis may be involved in altering some neurophysiological functions within only certain brain structures. Although APP23 mice have impaired cognitive performance, long-term plasticity, a cellular model for memory, is not affected, raising the question on the relationship between these processes.

STAT3 is constitutively active in some patients with Polycythemia rubra vera.

OBJECTIVE: Polycythemia vera is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. While it has been shown that progenitor cells of P. vera patients are hypersensitive to several growth factors including erythropoietin, insulin-like growth factor-1, thrombopoietin, interleukin-3, and granulocyte/monocyte colony-stimulating factor, the molecular pathogenesis of this disease remains unknown. Growth factor hypersensitivity could be mediated by changes in signal transduction pathways. We therefore investigated a common downstream effector of cytokines, the signal transducers and activators of transcription (STATs). A constitutive activation of STAT factors could explain the increased proliferation of P. vera cells even in the absence of growth factor stimulation. METHODS: Peripheral granulocytes from patients with P. vera and from healthy volunteers were assayed for STAT1, 3, and 5 DNA binding by electrophoretic mobility shift assay. RESULTS: Four of 14 P. vera patients analyzed showed constitutive STAT3 DNA binding in unstimulated peripheral granulocytes, while none of the 17 healthy volunteers tested did. None of the subjects showed constitutive STAT1 or STAT5 activity. Western blotting demonstrated that, in the three patients, STAT3 is constitutively phosphorylated on Tyr 705, whereas it is unphosphorylated in the other patients and in controls. Interestingly, constitutive STAT3 activity did not correlate with the duration of disease or the treatment regimen. It was observed in a recently diagnosed patient and in two patients treated only with phlebotomy. CONCLUSION: Our data suggest that constitutive phosphorylation and activation of STAT3 is not a secondary event induced by mutagenizing agents or by prolonged hyperproliferation of hematopoietic cells, but rather represents a primary molecular aberration. Constitutively active STAT3 may contribute to the growth factor hypersensitivity of P. vera cells.

Cloning of PRV-1, a novel member of the uPAR receptor superfamily, which is overexpressed in polycythemia rubra vera.

Polycythemia vera (PV) is a clonal stem cell disorder characterized by hyperproliferation of the erythroid, myeloid, and megakaryocytic lineages. Although it has been shown that progenitor cells of patients with PV are hypersensitive to several growth factors, the molecular pathogenesis of this disease remains unknown. To investigate the molecular defects underlying PV, we used subtractive hybridization to isolate complementary DNAs (cDNAs) differentially expressed in patients with PV versus normal controls. We isolated a novel gene, subsequently named PRV-1, which is highly expressed in granulocytes from patients with PV (n = 19), but not detectable in normal control granulocytes (n = 21). Moreover, PRV-1 is not expressed in mononuclear cells from patients with chronic myelogenous leukemia (n = 4) or acute myelogenous leukemia (n = 5) or in granulocytes from patients with essential thrombocythemia (n = 4) or secondary erythrocytosis (n = 4). Northern blot analysis showed that PRV-1 is highly expressed in normal human bone marrow and to a much lesser degree in fetal liver. It is not expressed in a variety of other tissues tested. Although PRV-1 is not expressed in resting granulocytes from normal controls, stimulation of these cells with granulocyte colony-stimulating factor induces PRV-1 expression. The PRV-1 cDNA encodes an open reading frame of 437 amino acids, which contains a signal peptide at the N-terminus and a hydrophobic segment at the C-terminus. In addition, PRV-1 contains 2 cysteine-rich domains homologous to those found in the uPAR/Ly6/CD59/snake toxin-receptor superfamily. We therefore propose that PRV-1 represents a novel hematopoietic receptor. (Blood. 2000;95:2569-2576)

Immunocytochemical localization of stanniocalcin cells in the rat kidney.

Stanniocalcin (STC) is a polypeptide hormone that was first discovered in fishes, where it functions as a regulator of calcium and phosphate homoeostasis. Recently, complementary DNAs encoding human STC (hSTC) have been characterized, and recombinant hSTC has been synthesized in a bacterial expression system. In preliminary studies, STC-immunoreactive cells have already been identified in human kidney tubules with antibodies to recombinant hSTC. The purpose of this study was to map the overall spatial distribution of STC cells in mammalian kidney, using the rat as a model system. Immunocytochemistry was performed on fixed sections of rat kidney tissue using hSTC antiserum in conjunction with fluorescein isothiocyanate-conjugated second antibodies. STC-immunoreactive cells were found in cortical thick ascending limb, in macula densa, in distal convoluted tubules, and in the cortical and medullary collecting ducts. All cortical thick ascending limb cells contained immunoreactive STC. Most distal convoluted tubules cells contained STC, and these were identified as principal cells. The distribution of STC cells in cortical and medullary collecting ducts also corresponded closely to the known frequently of principle cells in these segments, suggesting that principal cells are the site of STC storage and/or synthesis in both distal convoluted tubules and collecting ducts. Some collecting duct intercalated cells contained STC as well, and these were tentatively identified as alpha-type intercalated cells. As all tubular segments containing STC are known to be involved in regulated ion transport, renally derived STC may be acting in an autocrine, paracrine and/or endocrine fashion to regulate one or more of these transport processes.

Thin layer chromatographic detection and indirect gas chromatographic determination of three carbamate pesticides.

Carbamate pesticide residues are extracted from vegetables and fruits with methylene chloride. The extracts are spotted on silica gel plates and the pesticides are detected by an enzymatic inhibition technique. For quantitative determination, aliquots of the methylene chloride extracts are evaporated to dryness in a rotary evaporator. After the residues are dissolved in ethanol, 0.5N NaOH is added in the hydrolysis step. To remove a number of possible interferences the hydrolyzed phenols are steam-distilled and treated with 1-fluoro-2,4-dinitrobenzene and/or 4-chloro-alpha,alpha,alpha-trifluoro-3,5-dinitrotoluene to form the ether derivatives. Efficiency in the conversion of the phenolic moieties to the phenyl ethers is about 100%. The resulting electron-capturing derivatives enable the carbamate pesticides to be detected in vegetables and fruits at the 0.05 ppm level. Recoveries of 90-94% were obtained from vegetables and fruits fortified with 0.5-2.0 ppm carbaryl, Mesurol, and propoxur.