Childhood allergy is preceded by an absence of gut lactobacilli species and higher levels of atopy-related plasma chemokines.

Alterations in the composition and reduced diversity of the infant microbiome are associated with allergic disease in children. Further, an altered microbiota is linked to immune dysregulation, including skewing of different T helper (Th) subsets, which is also seen in atopic individuals. The aim of this study was, therefore, to investigate the associations between gut lactobacilli and Th-related plasma factors in allergy development during childhood. A total of 194 children with known allergy status at 1 year of age were followed to 10 years of age. We used real-time polymerase chain reaction (PCR) to investigate the presence of three lactobacilli species (Lactobacillus casei, L. paracasei, L. rhamnosus) in infant fecal samples (collected between 1 week and 2 months of age) from a subgroup of children. Plasma chemokines and cytokines were quantified at 6 months and at 1, 2, 5 and 10 years of age with Luminex or enzyme-linked immunosorbent assay (ELISA). Fractional exhaled nitrogen oxide (FeNO) was measured and spirometry performed at 10 years of age. The data were analysed by non-parametric testing and a logistic regression model adjusted for parental allergy. An absence of these lactobacilli and higher levels of the chemokines BCA-1/CXCL13, CCL17/TARC, MIP-3α/CCL20 and MDC/CCL22 in plasma at 6 months of age preceded allergy development. The presence of lactobacilli associated with lower levels of atopy-related chemokines during infancy, together with higher levels of interferon (IFN)-γ and lower FeNO during later childhood. The results indicate that the presence of certain lactobacilli species in the infant gut may influence allergy-related parameters in the peripheral immune system, and thereby contribute to allergy protection.

CD161(int)CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut.

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161(int)CD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Developments in the field of allergy in 2014 through the eyes of Clinical and Experimental Allergy.

The pathogenesis of asthma continues to be a major topic of interest to our authors with reviews and original papers on the role of viruses, mechanisms of inflammation, biomarkers, and phenotypes of asthma being major topics. A number of papers described new treatments for asthma focusing on blocking the Th2 response reflecting the fact that two decades of work in this area is finally bearing fruit. The pathogenesis of chronic rhinosinusitis is a growing area of interest, but there has been less on the genetics of airways disease than in previous years possibly reflecting the degree of rigour (and therefore a smaller body of work), with which these sorts of studies are now being undertaken. There continues to be a wide range of papers dealing with mechanisms of allergic disease ranging from clinical-based studies to basic research and the use of in vivo animal models especially mice. As before, mechanisms and new approaches to immunotherapy are common themes. Several were published in the allergens section investigating modification of allergens to increase their effectiveness and reduce the risk of adverse events. Risk factors for allergic disease was a common theme in the epidemiology section and food allergy a common theme in clinical allergy with papers on the development of protocols to induce tolerance and attempts to find biomarkers to distinguish sensitization from allergic disease. This was another exciting year for the editors, and we hope the readers of the journal.

Pregnancy-associated inflammatory markers are elevated in pregnant women with systemic lupus erythematosus.

During normal pregnancy a dampening in T cell-mediated immunity is compensated by an increased pro-inflammatory activity. Likewise, the autoimmune disease systemic lupus erythematosus (SLE) is associated with inflammatory activity and pregnancy complications occur frequently in women with SLE. The aim of this study was to elucidate how SLE influences the chemokine and cytokine balance during and after pregnancy. Blood samples were taken from pregnant women with or without SLE at second and third trimester and 8-12 weeks after pregnancy. Cytokines (interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A, TNF, IFN-γ and IFN-α), chemokines (CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, CCL2/MCP-1, CCL5/RANTES and CCL17/TARC), soluble IL-6 receptor (sIL-6R) and soluble glycoprotein 130 (gp130) were measured in serum using cytometric bead array (CBA) or enzyme-linked immunosorbent assay (ELISA). Women with SLE had increased serum concentrations of CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10 and IL-10 compared to controls both during and after pregnancy. Further, when dividing the patients based on disease activity, the women with active disease had the highest levels. Importantly, women with SLE seemed to respond to pregnancy in a similar way as controls, since the changes of cytokines and chemokines over the course of pregnancy were similar but with overall higher levels in the patient group. In conclusion, changes in pro- and anti-inflammatory serum components during pregnancy in women with SLE, occurring on top of already more pro-inflammatory levels, might increase their risk for pregnancy complications and flares. How their children are affected by this heightened inflammatory milieu during pregnancy needs further investigation.

Peanut sensitization during the first 5 yr of life is associated with elevated levels of peanut-specific IgG.

BACKGROUND: Allergen-specific IgE antibodies are implicated in allergic diseases while allergen-specific IgG antibodies have been proposed to prevent allergic reactions. The objective for this study was to study whether the immune response (IgG and IgG4) to peanut differs in IgE-sensitized and non-sensitized young children. METHODS: A total of 239 children have been followed prospectively from birth to 5 yr of age. The levels of IgG and IgG4 to peanut, Ara h 2, and Ara h 8 were analyzed at 2 and 5 yr of age and related to IgE sensitization and peanut consumption. RESULTS: The levels of peanut-specific IgG and IgG4 were significantly higher in peanut-sensitized children at 2 and 5 yr of age when compared with non-sensitized children and children sensitized to other food/inhalant allergens. A strong correlation was seen between levels of peanut-specific IgG/IgG4-ratios and peanut-specific IgE at 5 yr of age. Children avoiding peanuts, a subgroup of the peanut sensitized, had statistically significant higher levels of IgE to peanut and a tendency of higher IgG and IgG4 levels to peanut. In the avoidance group, significant correlations between IgE and IgG/IgG4 to peanut were found compared with children eating peanuts. CONCLUSION: Peanut-specific IgG or IgG4 levels were elevated in peanut-sensitized children especially those avoiding peanuts. In our study, IgG and IgG4 do not seem to indicate tolerance or protection from sensitization.

Maternal country of origin, breast milk characteristics and potential influences on immunity in offspring.

Breast milk contains pro- and anti-inflammatory cytokines and chemokines with potential to influence immunological maturation in the child. We have shown previously that country of birth is associated with the cytokine/chemokine profile of breast milk. In this study we have investigated how these differences in breast milk affect the cellular response of cord blood mononuclear cells (CBMCs) and intestinal epithelial cells (IECs, cell line HT-29) to microbial challenge. Ninety-five women were included: 30 from Mali in West Africa, 32 Swedish immigrants and 33 native Swedish women. CBMCs or IECs were stimulated in vitro with breast milk, alone or in combination with lipopolysaccharide (LPS) or peptidoglycan (PGN). Breast milk in general abrogated the LPS-induced down-regulation of surface CD14 and Toll-like receptor (TLR)-4 expression on CB monocytes, while inhibiting the PGN-induced TLR-2 up-regulation. However, breast milk from immigrant women together with LPS induced a lower CBMC release of interleukin (IL)-6 (P = 0·034) and CXCL-8/IL-8 (P = 0·037) compared with breast milk from Swedish women, while breast milk from Swedish women and Mali women tended to increase the response. The same pattern of CXCL-8/IL-8 release could be seen after stimulation of IECs (HT-29). The lower CBMC and IEC (HT-29) responses to microbial compounds by breast milk from immigrant women could be explained by the fact that breast milk from the immigrant group showed a divergent pro- and anti-inflammatory content for CXCL-8/IL-8, transforming growth factor-ß1 and soluble CD14, compared to the other two groups of women. This may have implications for maturation of their children's immune responses.

Influence of early gut microbiota on the maturation of childhood mucosal and systemic immune responses.

INTRODUCTION: Among sensitized infants, those with high, as compared with low levels, of salivary secretory IgA (SIgA) are less likely to develop allergic symptoms. Also, early colonization with certain gut microbiota, e.g. Lactobacilli and Bifidobacterium species, might be associated with less allergy development. Although animal and in vitro studies emphasize the role of the commensal gut microbiota in the development of the immune system, the influence of the gut microbiota on immune development in infants is unclear. OBJECTIVE: To assess whether early colonization with certain gut microbiota species associates with mucosal and systemic immune responses i.e. salivary SIgA and the spontaneous Toll-like receptor (TLR) 2 and TLR4 mRNA expression and lipopolysaccharide (LPS)-induced cytokine/chemokine responses in peripheral blood mononuclear cells (PBMCs). METHODS: Fecal samples were collected at 1 week, 1 month and 2 months after birth from 64 Swedish infants, followed prospectively up to 5 years of age. Bacterial DNA was analysed with real-time PCR using primers binding to Clostridium difficile, four species of bifidobacteria, two lactobacilli groups and Bacteroides fragilis. Saliva was collected at age 6 and 12 months and at 2 and 5 years and SIgA was measured with ELISA. The PBMCs, collected 12 months after birth, were analysed for TLR2 and TLR4 mRNA expression with real-time PCR. Further, the PBMCs were stimulated with LPS, and cytokine/chemokine responses were measured with Luminex. RESULTS: The number of Bifidobacterium species in the early fecal samples correlated significantly with the total levels of salivary SIgA at 6 months. Early colonization with Bifidobacterium species, lactobacilli groups or C. difficile did not influence TLR2 and TLR4 expression in PBMCs. However, PBMCs from infants colonized early with high amounts of Bacteroides fragilis expressed lower levels of TLR4 mRNA spontaneously. Furthermore, LPS-induced production of inflammatory cytokines and chemokines, e.g. IL-6 and CCL4 (MIP-1 beta), was inversely correlated to the relative amounts of Bacteroides fragilis in the early fecal samples. CONCLUSION: Bifidobacterial diversity may enhance the maturation of the mucosal SIgA system and early intense colonization with Bacteroides fragilis might down-regulate LPS responsiveness in infancy.

Altered early infant gut microbiota in children developing allergy up to 5 years of age.

BACKGROUND: Early colonization with bifidobacteria and lactobacilli is postulated to protect children from allergy, while Clostridium (C.) difficile colonization might be associated with allergic disease. Previous studies of infant gut microbiota in relation to subsequent allergy development have mostly employed culture-dependent techniques, studied genera of bacteria and the follow-up period was limited to 2 years. OBJECTIVE: To relate gut microbiota in early infancy, notably bifidobacteria and lactobacilli at species level, to allergy development during the first 5 years of life and study if environmental factors influence the early infant gut microbiota. METHODS: Fecal samples were collected at 1 week, 1 month and 2 months after birth from 47 Swedish infants, followed prospectively to 5 years of age. Bacterial DNA was analysed with real-time PCR and related to allergy development, family size as well as endotoxin and Fel d 1 levels in house dust samples. Primers binding to C. difficile, four species of bifidobacteria, two lactobacilli groups and Bacteroides fragilis were used. Children regarded as allergic manifested allergic symptoms and were skin prick test positive during their first 5 years while non-allergic children were neither. RESULTS: Children who developed allergy were significantly less often colonized with lactobacilli group I (Lactobacillus (L.) rhamnosus, L. casei, L. paracasei), Bifidobacterium adolescentis and C. difficile during their first 2 months. Infants colonized with several Bifidobacterium species had been exposed to higher amounts of endotoxin and grew up in larger families than infants harbouring few species. CONCLUSION: A more diverse gut microbiota early in life might prevent allergy development and may be related to the previously suggested inverse relationship between allergy, family size and endotoxin exposure.

Impaired Toll-like receptor 2 signalling in monocytes from 5-year-old allergic children.

The relative composition of the two major monocytic subsets CD14(+)CD16(-) and CD14(+)CD16(+) is altered in some allergic diseases. These two subsets display different patterns of Toll-like receptor levels, which could have implications for activation of innate immunity leading to reduced immunoglobulin E-specific adaptive immune responses. This study aimed to investigate if allergic status at the age of 5 years is linked to differences in monocytic subset composition and their Toll-like receptor levels, and further, to determine if Toll-like receptor regulation and cytokine production upon microbial stimuli is influenced by the allergic phenotype. Peripheral blood mononuclear cells from 5-year-old allergic and non-allergic children were stimulated in vitro with lipopolysaccharide and peptidoglycan. Cells were analysed with flow cytometry for expression of CD14, Toll-like receptors 2 and 4 and p38-mitogen-activated protein kinase (MAPK). The release of cytokines and chemokines [tumour necrosis factor, interleukin (IL)-1 beta, IL-6, IL-8, IL-10, IL-12p70] into culture supernatants was measured with cytometric bead array. For unstimulated cells there were no differences in frequency of the monocytic subsets or their Toll-like receptor levels between allergic and non-allergic children. However, monocytes from allergic children had a significantly lower up-regulation of Toll-like receptor 2 upon peptidoglycan stimulation. Further, monocytes from allergic children had a higher spontaneous production of IL-6, but there were no differences between the two groups regarding p38-MAPK activity or cytokine and chemokine production upon stimulation. The allergic subjects in this study have a monocytic population that seems to display a hyporesponsive state as implicated by impaired regulation of Toll-like receptor 2 upon peptidoglycan stimulation.

Epstein-Barr virus and cytomegalovirus are differentially associated with numbers of cytokine-producing cells and early atopy.

BACKGROUND: We have previously shown that Epstein-Barr virus (EBV) seropositivity, at 2 years of age, was inversely related to IgE-sensitization and that this effect was enhanced when EBV is combined with cytomegalovirus (CMV) seropositivity. We hypothesize that early exposure to EBV or CMV will affect the cytokine balance in the individual. OBJECTIVE: The aim of this study was to relate the cytokine profile in peripheral blood mononuclear cells (PBMC) to the EBV and CMV serostatus and IgE-sensitization in children at 2 years of age. METHODS: Seventy-five children were followed prospectively from birth until 2 years of age. Their EBV and CMV serostatus was correlated to the numbers of IFN-gamma, IL-4, IL-10 and IL-12-producing PBMC following PHA stimulation in vitro. Skin prick tests and allergen-specific IgE antibodies were used to assess IgE-sensitization. RESULTS: In the study cohort, there was an inverse association between EBV seropositivity and IgE-sensitization but not with CMV seropositivity. Following linear regression analysis, we did not detect any statistically significant associations between children with IgG antibodies against EBV at 2 years of age and the investigated cytokines. However, there was a non-significant tendency to a positive association between high numbers of all individual cytokine-producing cells and EBV seropositivity. Children who were CMV seropositive had significantly higher numbers of IFN-gamma and lower numbers of IL-4-producing cells compared with CMV negative children. There was a significant, positive association between the number of IL-4-producing cells and IgE-sensitization. CONCLUSION: Taken together our results indicate that infections with EBV and CMV in different ways will interact with the immune system and may protect children from developing early atopy.

Maternal allergy influences p38-mitogen-activated protein kinase activity upon microbial challenge in CD14+ monocytes from 2-year-old children.

BACKGROUND: The development of allergic diseases is dependent on genetic and environmental factors. It has been shown previously that cord blood mononuclear cells (CBMCs) from infants with parental allergy have altered cytokine profiles upon bacterial encounter; it might be possible that such impairment persists during the early years of childhood. OBJECTIVE: The aim of this study was to investigate anti-microbial responses with regard to p38-mitogen-activated protein kinase (MAPK) activity in CD14(+) monocytes and IL-6 release from mononuclear cells in the same group of children at birth and at 2 years of age. Methods Paired samples of CBMCs and peripheral blood mononuclear cells (PBMCs) were stimulated with either lipopolysaccharide (LPS) or peptidoglycan in vitro. CD14(+) monocytes were analysed for p38-MAPK activity by flow cytometry, and soluble IL-6 receptor, soluble glycoprotein130 and IL-6 release from PBMC cultures were quantified by ELISA. RESULTS: CBMCs from newborns with allergic mothers tended to have a lower IL-6 response following an LPS (P=0.09) challenge compared with the group without maternal allergy while p38-MAPK activation levels did not differ between the groups. PBMCs from 2-year-olds with allergic mothers released significantly less (P<0.05) IL-6 upon peptidoglycan stimuli compared with age-matched infants with non-allergic mothers. Infants with allergic mothers displayed markedly reduced CD14(+) monocyte p38-MAPK phosphorylation after LPS (P<0.05) and peptidoglycan (P<0.01) challenge. This altered anti-microbial response was attributed to maternal allergy rather than to being IgE-sensitized at 2 years of age. CONCLUSION: Monocytes from children with allergic mothers are less responsive to bacterial challenge than monocytes from children with non-allergic mothers, and this impairment persists during the first 2 years of infancy.

Relationship between maternal and child cytokine responses to allergen and phytohaemagglutinin 2 years after delivery.

Little is known at present about the relation between parental and child cytokine profiles. In this study we aimed to investigate the cytokine profile of 2-year-old children and their corresponding mothers, 2 years after delivery. Peripheral blood mononuclear cells (PBMC) were isolated from IgE-sensitized (n=15) and non-esitized (n=58) 2-year-old children and their mothers. The responses to ovalbumin, cat and phytohaemagglutinin (PHA) were investigated using the enzyme-linked immunospot (ELISpot) technique. Interferon (IFN)-gamma-, interleukin (IL)-4-, IL-10- and IL-12-producing cells were enumerated. At 2 years of age, IgE-sensitized children exhibited increased numbers of IL-4-producing cells in response to PHA and also showed an increase in IL-10- and IL-12-producing cells to allergen that was more pronounced in response to ovalbumin than to cat. A statistically significant increase in the numbers of IFN-gamma-, IL-10- and IL-12-producing cells to the allergens, but not to PHA, was found in the mothers of IgE-sensitized children irrespective of their own atopic status. IgE levels and cytokine responses were correlated between the mothers and their children, indicating that cytokine responses to both allergen and PHA might be governed by genetic factors. We speculate that the increased cytokine response to allergen, as opposed to the allergic status of the mother, might be a better predictor and/or a risk factor for the child to develop IgE-sensitization in early life.

Levels of soluble CD30 in cord blood and peripheral blood during childhood are not correlated with the development of atopic disease or a family history of atopy.

BACKGROUND: The CD30 molecule has been linked to Th2 responses. Furthermore, elevated levels of the soluble form of CD30 (sCD30) in blood as well as of the expression of CD30 on the plasma membrane of T cells are associated with atopic disease. OBJECTIVE: To assess the potential usefulness of sCD30 levels as a prognostic indicator of and/or diagnostic marker for the development of atopic disease in children. METHODS: sCD30 levels in cord blood and peripheral blood from 36 2-year-old (10 atopic and 26 non-atopic) and 74 7-year-old (35 atopic and 39 non-atopic) children were determined employing an ELISA procedure. Atopy was diagnosed on the basis of clinical evaluation in combination with a positive skin prick test. RESULTS: No significant correlation between sCD30 levels in cord blood and the development of atopic disease at 2 or 7 years of age was observed. At 7 years of age, the circulating sCD30 levels in children with atopic disease (median 41 U/mL, range 6-503 U/mL) did not differ from the corresponding values for non-atopic subjects (median 41 U/mL, range 8-402 U/mL). The same was true for children at 2 years of age. Furthermore, the sCD30 levels of children who had developed atopic eczema/dermatitis syndrome by the age of 7 years (median 49 U/mL, range 14-503 U/mL) were not significantly elevated in comparison with those of the non-atopic children. Finally, neither sCD30 levels in cord blood nor peripheral blood at 2 or 7 years of age could be linked to a family history of atopy. CONCLUSION: These findings indicate that the sCD30 concentration in cord blood is not a reliable prognostic indicator of, nor a useful diagnostic marker for, atopic disease in children up to 7 years of age. If such correlations do exist, they might be masked by age-dependent variations in the circulating levels of sCD30, which may reflect individual differences in the maturation of children's immunological responses.

CD14 and development of atopic disease at 2 years of age in children with atopic or non-atopic mothers.

BACKGROUND: CD14, a myeloid cell marker and LPS receptor has been acclaimed to play a role in development and manifestation of atopic allergy, as the gene encoding CD14 is located in a chromosomal region linked to total IgE levels and atopic disease. OBJECTIVE: To investigate the levels of soluble (s) and membrane bound (m) CD14 in cord blood and at 2 years of age from children with atopic or non-atopic mothers and relate these parameters to atopy development at 2 years of age. METHODS: Blood samples were collected at delivery (cord blood) and at 2 years of age among infants with atopic (n = 41) and non-atopic (n = 32) mothers. Blood samples were also obtained from mothers at the same occasions. Levels of sCD14 and total IgE were measured in plasma, and percentages of CD14+ cells were measured in cord and peripheral blood mononuclear cells. RESULTS: We observed significant differences in sCD14 levels in cord blood, where children with atopic mothers had the highest levels. The same pattern could be observed in the mothers at delivery. At 2 years of age no significant differences in sCD14 levels were observed between children with atopic mothers and children with non-atopic mothers and no association between sCD14 and atopic disease was found. Further, we observed large differences in sCD14 and mCD14 with respect to age, where newborns displayed a higher frequency of CD14+ cells compared with the 2-year-olds and the mothers. The reverse was observed for sCD14, with significantly lower values in cord blood than those seen in the 2-year-olds and mothers. CONCLUSION: Based on our findings, we suggest that CD14 could be involved in the regulation of IgE production, but that it might also be important for the maturation and development of the neonatal immune system.

IgE is expressed on, but not produced by, fetal cells in the human placenta irrespective of maternal atopy.

The prevalence of atopic diseases in children has increased during the last decades. Atopic symptoms usually appear early in life. This implies an early priming for atopic disease, possibly even at the fetal level. We therefore compared the presence and production of IgE in the local in utero environment during pregnancy in atopic and non-atopic women. Eighty-six women were included in the study. Fifty women were demonstrated to be atopics, based on clinical symptoms of atopic disease together with a positive Phadiatop and/or skin prick test. Placentas from these term pregnancies were obtained. Slices covering the full thickness of the placenta were cut clockwise around the umbilical cord and were analysed with immunohistochemistry. Surprisingly, numerous IgE+ cells, located primarily in the fetal villous stroma, were detected in a majority of the investigated placentas irrespective of the atopy of the mother or maternal or fetal total serum IgE levels. The placental IgE could not be demonstrated to be bound to IgE receptors, but was shown to be bound to fetal macrophages, possibly via FcgammaRI. No evidence was found for local fetal IgE production, although cells producing epsilon transcripts were occasionally detected in the decidua. We describe here the novel finding of numerous IgE+ cells in the human placenta, suggesting an hitherto unknown role for IgE in a successful pregnancy outcome, irrespective of whether or not the mother is atopic.