Germline Transcription Research Articles

Central nervous system infiltrates are characterized by features of ongoing B cell-related immune activity in MP4-induced experimental autoimmune encephalomyelitis

In multiple sclerosis (MS) lymphoid follicle-like aggregates have been reported in the meninges of patients. Here we investigated the functional relevance of B cell infiltration into the central nervous system (CNS) in MP4-induced experimental autoimmune encephalomyelitis (EAE), a B cell-dependent mouse model of MS. In chronic EAE, B cell aggregates were characterized by the presence of CXCL13+ and germinal center CD10+ B cells. Germline transcripts were expressed in the CNS and particularly related to TH17-associated isotypes. We also observed B cells with restricted VH gene usage that differed from clones found in the spleen. Finally, we detected CNS-restricted spreading of the antigen-specific B cell response towards a myelin and a neuronal autoantigen. These data imply the development of autonomous B cell-mediated autoimmunity in the CNS in EAE — a concept that might also apply to MS itself.

Expression of TCR-Vβ peptides by murine bone marrow cells does not identify T-cell progenitors.

Germline transcription has been described for both immunoglobulin and T-cell receptor (TCR) genes, raising questions of their functional significance during haematopoiesis. Previously, an immature murine T-cell line was shown to bind antibody to TCR-Vβ8.2 in absence of anti-Cβ antibody binding, and an equivalent cell subset was also identified in the mesenteric lymph node. Here, we investigate whether germline transcription and cell surface Vβ8.2 expression could therefore represent a potential marker of T-cell progenitors. Cells with the TCR phenotype of Vβ8.2+Cβ− are found in several lymphoid sites, and among the lineage-negative (Lin−) fraction of hematopoietic progenitors in bone marrow (BM). Cell surface marker analysis of these cells identified subsets reflecting common lymphoid progenitors, common myeloid progenitors and multipotential progenitors. To assess whether the Lin−Vβ8.2+Cβ− BM subset contains hematopoietic progenitors, cells were sorted and adoptively transferred into sub-lethally irradiated recipients. No T-cell or myeloid progeny were detected following introduction of cells via the intrathymic or intravenous routes. However, B-cell development was detected in spleen. This pattern of restricted in vivo reconstitution disputes Lin−Vβ8.2+Cβ− BM cells as committed T-cell progenitors, but raises the possibility of progenitors with potential for B-cell development.

Constraints contributed by chromatin looping limit recombination targeting during Ig class switch recombination.

Engagement of promoters with distal elements in long-range looping interactions has been implicated in regulation of Ig class switch recombination (CSR). The principles determining the spatial and regulatory relationships among Igh transcriptional elements remain poorly defined. We examined the chromosome conformation of C region (CH) loci that are targeted for CSR in a cytokine-dependent fashion in mature B lymphocytes. Germline transcription (GLT) of the γ1 and ε CH loci is controlled by two transcription factors, IL-4-inducible STAT6 and LPS-activated NF-κB. We showed that although STAT6 deficiency triggered loss of GLT, deletion of NF-κB p50 abolished both GLT and γ1 locus:enhancer looping. Thus, chromatin looping between CH loci and Igh enhancers is independent of GLT production and STAT6, whereas the establishment and maintenance of these chromatin contacts requires NF-κB p50. Comparative analysis of the endogenous γ1 locus and a knock-in heterologous promoter in mice identified the promoter per se as the interactive looping element and showed that transcription elongation is dispensable for promoter/enhancer interactions. Interposition of the LPS-responsive heterologous promoter between the LPS-inducible γ3 and γ2b loci altered GLT expression and essentially abolished direct IgG2b switching while maintaining a sequential μ→γ3→γ2b format. Our study provides evidence that promoter/enhancer looping interactions can introduce negative constraints on distal promoters and affect their ability to engage in germline transcription and determine CSR targeting.

Specification of Vδ and Vα Usage by Tcra/Tcrd Locus V Gene Segment Promoters

The Tcra/Tcrd locus undergoes V-Dδ-Jδ rearrangement in CD4(-)CD8(-) thymocytes to form the TCRδ chain of the γδ TCR and V-Jα rearrangement in CD4(+)CD8(+) thymocytes to form the TCRα-chain of the αβ TCR. Most V segments in the locus participate in V-Jα rearrangement, but only a small and partially overlapping subset participates in V-Dδ-Jδ rearrangement. What specifies any particular Tcra/Tcrd locus V gene segment as a Vδ, a Vα, or both is currently unknown. We tested the hypothesis that V segment usage is specified by V segment promoter-dependent chromatin accessibility in developing thymocytes. TRAV15/DV6 family V gene segments contribute to both the Tcrd and the Tcra repertoires, whereas TRAV12 family V gene segments contribute almost exclusively to the Tcra repertoire. To understand whether the TRAV15/DV6 promoter region specifies TRAV15/DV6 as a Vδ, we used gene targeting to replace the promoter region of a TRAV12 family member with one from a TRAV15/DV6 family member. The TRAV15/DV6 promoter region conferred increased germline transcription and histone modifications to TRAV12 in double-negative thymocytes and caused a substantial increase in usage of TRAV12 in Tcrd recombination events. Our results demonstrate that usage of TRAV15/DV6 family V gene segments for Tcrd recombination in double-negative thymocytes is regulated, at least in part, by intrinsic features of TRAV15/DV6 promoters, and argue that Tcra/Tcrd locus Vδ gene segments are defined by their local chromatin accessibility in CD4(-)CD8(-) thymocytes.

The proximal J kappa germline-transcript promoter facilitates receptor editing through control of ordered recombination.

V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal—in contrast to the distal—GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.

Ginsenoside Rg1 and 20(S)-Rg3 Induce IgA Production by Mouse B Cells.

Ginsenosides are the major components of ginseng, which is known to modulate blood pressure, metabolism, and immune function, and has been used to treat various diseases. It has been reported that ginseng and several ginsenosides have immunoregulatory effects on the innate and T cell-mediated immune response. However, their effects on the humoral immune response have not been fully explored. The present study examined the direct effects of red ginseng extract (RGE) and ginsenosides on mouse B cell proliferation and on antibody production and the expression of germline transcripts (GLT) by mouse B cells in vitro. RGE slightly reduced B cell proliferation, but increased IgA production by LPS-stimulated B cells. Furthermore, ginsenoside Rg1 and 20(S)-Rg3 selectively induced IgA production and expression of GLTα transcripts by LPS-stimulated B cells. Collectively, these results suggest that ginsenoside Rg1 and 20(S)-Rg3 can drive the differentiation of B cells into IgA-producing cells through the selective induction of GLTα expression.

T-cell internal antigen 1 counteracts somatic RNA degradation during early Xenopus embryogenesis.

In Xenopus laevis, maternal transcripts that localize to the vegetal cortex of the oocyte are specifically inherited by prospective germ cells during cleavage stages. While a large fraction of maternal transcripts is degraded during the maternal to zygotic transition (MZT), transcripts associated with the germ-line are stable. A sequence in the dead end 1 3'UTR mediates vegetal localization in the oocyte as well as miR mediated clearance in somatic cells and germ cell specific stabilization during the MZT in embryos. We could identify Tia1 to co-precipitate with known components of vegetal localization RNPs in X. laevis oocytes. Tia1 interacts and co-localizes with various localization elements from vegetally localizing RNAs. In X. laevis embryos, ectopic expression of Tia1 counteracts somatic degradation of dnd1 localization element reporter RNAs and it can synergize with Dnd1 protein in reporter RNA stabilization. Ectopic Tia1 also protects several endogenous localizing and germ cell specific mRNAs from somatic degradation. Thus, proteins that protect germ-line transcripts from miR mediated decay during the MZT in embryos might bind these RNAs already in the oocyte.

Increased humoral immunity in the jejunum of diarrhoea-predominant irritable bowel syndrome associated with clinical manifestations

Background and aimsAltered intestinal barrier is associated with immune activation and clinical symptoms in diarrhoea-predominant IBS (IBS-D). Increased mucosal antigen load may induce specific responses; however, local antibody production and...

Local Immunoglobulin Class Switch Recombination in Chronic Rhinosinusitis with Nasal Polyposis

Objectives: Chronic rhinosinusitis with nasal polyposis (CRSwNP) is characterized by B cell inflammation. We sought to determine whether immunoglobulin class switch recombination (CSR) occurred locally within polyps and to quantify the expression of proteins involved in regulating CSR. Germline transcripts (GLTs) are produced during CSR, and activation-induced cytidine deaminase (AICD) is an enzyme that is required for CSR. Thus, GLT and AICD expression can be used as markers for CSR. Methods: Polyp (n = 12-37), tonsil (n = 7), and uncinate tissue (UT) from patients with CRSwNP (n = 9), CRSsNP (n = 10), and normal controls (n = 11-12) were collected. Quantitative reverse transcriptase polymerase chain reaction for GAPDH, GUSB (housekeeping genes), AICD, IL-15, iNOS, BAFF, and A1, A2, G1, G4, and E GLT was performed using cDNA from tissue. Relative expression was calculated in relation to a housekeeping gene. Results: AICD expression was significantly increased in tonsils compared with all other groups ( P < .05), and polyp AICD expression was significantly elevated compared with UTwNP and UTsNP ( P < .01). Tonsils expressed significantly more A2 GLT than all other groups ( P < .05). E GLT was significantly increased in polyps versus UTnl ( P < .05). G1 GLT was increased in polyps compared with all other groups ( P < .05 vs UTwNP). G4 GLT was also increased in polyps compared with all other tissues, but this did not reach significance. Only IL-15 showed significantly increased expression in polyps versus UTnl and UTwNP ( P < .01). Conclusions: These results suggest that local CSR is occurring in polyps of patients with CRSwNP.

Berberine and limonin suppress IgE production by human B cells and peripheral blood mononuclear cells from food-allergic patients

BackgroundCurrently, there is no satisfactory treatment for IgE-mediated food allergy. Food Allergy Herbal Formula 2 (FAHF-2) and butanol-purified FAHF-2 (B-FAHF-2) have been shown to protect against peanut-induced anaphylaxis and inhibit IgE synthesis in a murine model. ObjectiveTo determine which herbs and compounds in FAHF-2 and B-FAHF-2 suppress IgE production. MethodsThe effect of FAHF-2 and B-FAHF-2 on IgE production was determined using a human B-cell line (U266). Individual compounds were isolated and identified using column chromatography, liquid chromatographic mass spectrometry, and nuclear magnetic resonance techniques. The potency of compounds on IgE suppression were investigated using U266 cells and verified using human peripheral blood mononuclear cells (n = 25) from peanut-allergic patients. Epsilon germline transcript expression was determined. Phosphorylated IκBα level was analyzed using the In-Cell Western assay. The mRNA expression of signal transducer and activator of transcription-3, T-box transcription factor TBX21, interferon-γ, forkhead box P3, GATA-binding protein 3, interleukin-10, and interleukin-5 also were analyzed using real-time polymerase chain reaction. ResultsFAHF-2 and B-FAHF-2 inhibited IgE production by U266 cells. B-FAHF-2 was 9 times more effective than FAHF-2. Two compounds that inhibited IgE production were isolated from Philodendron chinensis and identified as berberine and limonin. Berberine was more potent and inhibited IgE production by peripheral blood mononuclear cells by 80% at 0.62 μg/mL. Berberine significantly inhibited ε-germline transcript expression by peripheral blood mononuclear cells. Phosphorylated IκBα level was significantly suppressed and mRNA expressions of T-box transcription factor TBX21 and signal transducer and activator of transcription-3 were significantly increased by berberine. ConclusionBerberine and limonin mediated IgE suppression. The mechanism by which berberine modulates ε-germline transcript expression might be through regulating the phosphorylated IκBα level and the expressions of signal transducer and activator of transcription-3 and T-box transcription factor TBX21. Trial RegistrationClinicaltrials.gov identifier NCT00602160.

Exosomes derived from Burkitt's lymphoma cell lines induce proliferation, differentiation, and class-switch recombination in B cells.

Exosomes, nano-sized membrane vesicles, are released by various cells and are found in many human body fluids. They are active players in intercellular communication and have immune-suppressive, immune-regulatory, and immune-stimulatory functions. EBV is a ubiquitous human herpesvirus that is associated with various lymphoid and epithelial malignancies. EBV infection of B cells in vitro induces the release of exosomes that harbor the viral latent membrane protein 1 (LMP1). LMP1 per se mimics CD40 signaling and induces proliferation of B lymphocytes and T cell-independent class-switch recombination. Constitutive LMP1 signaling within B cells is blunted through the shedding of LMP1 via exosomes. In this study, we investigated the functional effect of exosomes derived from the DG75 Burkitt's lymphoma cell line and its sublines (LMP1 transfected and EBV infected), with the hypothesis that they might mimic exosomes released during EBV-associated diseases. We show that exosomes released during primary EBV infection of B cells harbored LMP1, and similar levels were detected in exosomes from LMP1-transfected DG75 cells. DG75 exosomes efficiently bound to human B cells within PBMCs and were internalized by isolated B cells. In turn, this led to proliferation, induction of activation-induced cytidine deaminase, and the production of circle and germline transcripts for IgG1 in B cells. Finally, exosomes harboring LMP1 enhanced proliferation and drove B cell differentiation toward a plasmablast-like phenotype. In conclusion, our results suggest that exosomes released from EBV-infected B cells have a stimulatory capacity and interfere with the fate of human B cells.

TRIF signaling is essential for TLR4-driven IgE class switching (IRM5P.707)

Abstract The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and TRAM, which recruits TRIF. Following stimulation with LPS+IL-4, Tram-/- and Trif-/- B cells completely failed to express Cε germ line transcripts (GLT) and secrete IgE. In contrast, Myd88-/- B cells had normal expression of Cγ1 GLT, but reduced IgE secretion in response to LPS+IL-4. Following LPS+IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram-/- and Trif-/- B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram-/-,Trif-/-, and Myd88-/- B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40+IL-4. Following stimulation with LPS+IL-4, Trif-/- B cells failed to sustain NFκB p65 nuclear translocation beyond 3 hours and had reduced binding of p65 to the Iε promoter. Addition of the NFκB inhibitor, JSH-23, to wild-type B cells 15 hours after LPS+IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion, but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NFκB driven by TRIF is essential for LPS+IL-4 driven activation of the Cε locus and class switching to IgE.

Rab7 plays a B cell-intrinsic and critical role in T-dependent and T-independent antibody responses by mediating AID expression and class switch DNA recombination (IRM8P.706)

Abstract Rab7 is a small GTPase that mediates endosomal functions and regulates T cell homeostasis. Here, we have addressed the B cell-inherent role of Rab7 in antibody responses and underlying mechanisms by constructing Igh+/Cγ1-cre Rab7fl/fl mice. These mice displayed normal B cell and T cell development and survival, and were knocked-out of the Rab7 gene specifically in B cells activated to undergo IgH germline Iγ1-Sγ1-Cγ1 transcription. This transcription process is induced by IL-4 plus a primary CSR-inducing stimulus, and is required for antibody switching from IgM to IgG1, but not to other Ig isotypes. Igh+/Cγ1-cre Rab7fl/fl mice had virtually abrogated levels of IgG1, but normal levels of IgM and other switched Ig isotypes, and failed to mount specific IgG1 responses to T-dependent or T-independent antigens. They showed markedly reduced IgG1+ B cells and antibody-forming cells, with unaltered B cell proliferation, survival and differentiation into germinal center B cells and plasma cells. Purified naïve B cells from these mice were defective in CSR to IgG1, despite normal proliferation and plasma cell differentiation. These together with the unaltered germline transcription but significantly decreased expression of AID (essential for CSR and SHM) demonstrate that B cell Rab7 mediates CSR and, therefore, the antibody response by upregulating AID, likely by regulating intracellular membranes that facilitate signal transduction for activation of specific transcription factors.

Alkylglycerols modulate the proliferation and differentiation of non-specific agonist and specific antigen-stimulated splenic lymphocytes.

Alkylglycerols (AKGs) are ether-linked glycerols derived from shark liver oil and found in small amounts in human milk. Previous studies showed that oral AKGs administration significantly increased the immune response in mice. The aim of the present study was to investigate the in vitro immunomodulatory effect of AKGs on stimulating splenic lymphocyte responses. C57BL/6 mice were immunized with hepatitis B surface antigen (HBsAg). Splenic B cells were purified and stimulated with anti-BCR and anti-CD38. Meanwhile, splenic CD4+ T cells were purified and stimulated with anti-CD3 and anti-CD28. For antigen specific stimulation, the purified CD4+ T cells were cocultured with HBsAg -pulsed dendritic cells. The stimulated lymphocytes were treated with different concentrations of AKGs. The cell proliferation was assessed by [3H]-thymidine incorporation assay. The maturation of B cells was assessed by examining the germline (GL) transcription of IgG (γ1) mRNA expression, and the surface expressions of CD80/CD86 markers were examined by flow cytometry analysis. Th1/Th2 polarity was assessed by T-BET (Th1)/GATA-3 (Th2) flow cytometry assay and by characteristic cytokines ELISA assay (TNF-α and IFN-γ for Th1; IL-4 and IL-10 for Th2). It was found that AKGs significantly increased the BCR/CD38 -stimulated B cell proliferation. The T cell proliferation in response to CD3/CD28 or specific antigen stimulation was also increased by AKGs. The transcriptional level of IgG (γ1) and the expressions of CD80/CD86 molecules were markedly increased by AKGs in BCR/CD38 -stimulated B cells. Meanwhile, the results showed that AKGs increased the expression of T-BET transcriptional factor and the production of Th1 cytokines (TNF-α and IFN-γ) upon CD3/CD28 stimulation; whereas, levels of Th2 cytokines (IL-4 and IL-10) were decreased by AKGs. Our study demonstrated that AKGs can modulate immune responses by boosting the proliferation and maturation of murine lymphocytes in vitro.

Silent no more: Endogenous small RNA pathways promote gene expression.

Endogenous small RNA pathways related to RNA interference (RNAi) play a well-documented role in protecting host genomes from the invasion of foreign nucleic acids. In C. elegans, the PIWI type Argonaute, PRG-1, through an association with 21U-RNAs, mediates a genome surveillance process by constantly scanning the genome for potentially deleterious invading elements. Upon recognition of foreign nucleic acids, PRG-1 initiates a cascade of cytoplasmic and nuclear events that results in heritable epigenetic silencing of these transcripts and their coding genomic loci. If the PRG-1/21U-RNA genome surveillance pathway has the capacity to target most of the C. elegans transcriptome, what mechanisms exist to protect endogenous transcripts from being silenced by this pathway? In this commentary, we discuss three recent publications that implicate the CSR-1 small RNA pathway in the heritable activation of germline transcripts, propose a model as to why not all epialleles behave similarly, and touch on the practical implications of these findings.

Global effects of the CSR-1 RNA interference pathway on the transcriptional landscape

Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions.

TRIF Signaling Is Essential for TLR4-Driven IgE Class Switching

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-β (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.

Expression of Genes Related to Anti-Inflammatory Pathways Are Modified Among Farmers’ Children

BackgroundThe hygiene hypothesis states that children exposed to higher loads of microbes such as farmers’ children suffer less from allergies later in life. Several immunological mechanisms underpinning the hygiene hypothesis have been proposed such as a shift in T helper cell balance, T regulatory cell activity, or immune regulatory mechanisms induced by the innate immunity.ObjectiveTo investigate whether the proposed immunological mechanisms for the hygiene hypotheses are found in farmers’ children.MethodsWe assessed gene expression levels of 64 essential markers of the innate and adaptive immunity by quantitative real-time PCR in white blood cells in 316 Swiss children of the PARSIFAL study to compare farmers’ to non-farmers’ expressions and to associate them to the prevalence of asthma and rhinoconjunctivitis, total and allergen-specific IgE in serum, and expression of Cε germ-line transcripts.ResultsWe found enhanced expression of genes of the innate immunity such as IRAK-4 and RIPK1 and enhanced expression of regulatory molecules such as IL-10, TGF-β, SOCS4, and IRAK-2 in farmers’ children. Furthermore, farmers’ children expressed less of the TH1 associated cytokine IFN-γ while TH2 associated transcription factor GATA3 was enhanced. No significant associations between the assessed immunological markers and allergic diseases or sensitization to allergens were observed.ConclusionFarmers’ children express multiple increased innate immune response and immune regulatory molecules, which may contribute to the mechanisms of action of the hygiene hypothesis.

Increased food diversity in the first year of life is inversely associated with allergic diseases

The role of dietary factors in the development of allergies is a topic of debate, especially the potential associations between infant feeding practices and allergic diseases. Previously, we reported that increased food diversity introduced during the first year of life reduced the risk of atopic dermatitis. In this study we investigated the association between the introduction of food during the first year of life and the development of asthma, allergic rhinitis, food allergy, or atopic sensitization, taking precautions to address reverse causality. We further analyzed the association between food diversity and gene expression of T-cell markers and of Cε germline transcript, reflecting antibody isotype switching to IgE, measured at 6 years of age. Eight hundred fifty-six children who participated in a birth cohort study, Protection Against Allergy Study in Rural Environments/EFRAIM, were included. Feeding practices were reported by parents in monthly diaries during the first year of life. Data on environmental factors and allergic diseases were collected from questionnaires administered from birth up to 6 years of age. An increased diversity of complementary food introduced in the first year of life was inversely associated with asthma with a dose-response effect (adjusted odds ratio with each additional food item introduced, 0.74 [95% CI, 0.61-0.89]). A similar effect was observed for food allergy and food sensitization. Furthermore, increased food diversity was significantly associated with an increased expression of forkhead box protein 3 and a decreased expression of Cε germline transcript. An increased diversity of food within the first year of life might have a protective effect on asthma, food allergy, and food sensitization and is associated with increased expression of a marker for regulatory T cells.

Midazolam inhibits IgE production in mice via suppression of class switch recombination

Anaphylactic shock is characterized by increased capillary permeability and a decline in blood pressure due to excessive production of IgE. Midazolam (MDZ) is reported to have immunomodulatory properties. However, little is known about the effect of MDZ on the production of IgE antibody. We examined whether MDZ can suppress antigen-specific and total IgE production followed by IgE class switch recombination (CSR). MDZ was administered intraperitoneally to mice prior to ovalbumin (OVA) plus native cholera toxin (nCT) immunization. Serum OVA-specific and total IgE responses, and surface IgE-positive B cells were analyzed by ELISA and flow cytometry. Furthermore, expression levels of CSR-associated molecules such as germ-line transcript ε (εGLT), germ-circle tanscript ε (εCT), AID, and Id2 in the spleen were compared. The levels of interferon-gamma (IFN-γ) and interleukin (IL)-4 mRNA and protein were also examined in the spleen and serum. MDZ significantly suppressed OVA-specific and total IgE levels in plasma and surface IgE-positive B cells in the spleen. Moreover, MDZ-treated mice had significantly reduced levels of εGLT and εCT. Furthermore, although the levels of IFN-γ mRNA and protein were significantly elevated, those of IL-4 were reduced in MDZ-treated mice. Therefore, MDZ may be an important modulator of allergic responses through its ability to downregulate IgE production.