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Background: Allergic rhinitis (AR) is strongly associated with a type 2 response, characterized by the cytokines IL-5, IL-4 and IL-13. Several studies have implicated ILC2 and TH2A (CD161+ TH2) but it is not yet entirely clear which subsets are driving the common allergic reactions underlying AR. The objective of this study aims to identify critical pathogenic cell populations associated with AR and to determine their phenotype and functional contribution to disease progression. Methods: We identified integral allergic-specific cell types by transcriptomic sequencing. Whole blood, PBMCs and plasma from a cross-sectional cohort of 216 individuals were analysed by 9-colour flow cytometry and ultra-sensitive cytokine bead arrays using unsupervised clustering algorithms. Clinically active AR cases were further analysed by functional mass cytometry to define phenotype and cytokine secretion (IL-2, IL-3, IL-4, IL-5, IL-9, IL13, IL-17A and IL-22) as well as the expression of the hematopoietic prostaglandin D synthetase (HPGDS). Results: The unbiased analysis revealed that atopy and AR manifestation corelated only with eosinophils, plasma IL-5 and CD161+ TH2 cells. In-depth characterization further revealed that the CD45RB CD161+ TH2 subset were most closely associated with severity. This subset is able to concomitantly secrete multiple cytokines including IL-5, IL-13 and IL-4 and has been previously reported to be associated with other eosinophilic allergies. Conclusion: CD45RB CD161+ TH2 have key roles in driving the allergic response in AR. Neutralizing the CD45RB CD161+ TH2 subset should disrupt the allergic response pathway, thus providing a target for lasting therapeutic interventions.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host–pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection. Supplementary Information The online version contains supplementary material available at 10.1007/s10875-021-01142-z.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that have become dominant as the pandemic progresses bear the ORF8 mutation together with multiple spike mutations. A 382-nucleotide deletion (Δ382) in the ORF7b and ORF8 regions has been associated with milder disease phenotype and less systemic inflammation in COVID-19 patients. However, its impact on host immunity against SARS-CoV-2 remains undefined. Here, RNA-sequencing was performed to elucidate whole blood transcriptomic profiles and identify contrasting immune signatures between patients infected with either wildtype or Δ382 SARS-CoV-2 variant. Interestingly, the immune landscape of Δ382 SARS-CoV-2 infected patients featured an increased adaptive immune response, evidenced by enrichment of genes related to T cell functionality, a more robust SARS-CoV-2-specific T cell immunity, as well as a more rapid antibody response. At the molecular level, eukaryotic initiation factor 2 signaling was found to be upregulated in patients bearing Δ382, and its associated genes were correlated with systemic levels of T cell-associated and pro-inflammatory cytokines. This study provides more in-depth insight into the host-pathogen interactions of ORF8 with great promise as a therapeutic target to combat SARS-CoV-2 infection.

INTRODUCTION Resident tissue macrophages (RTMs) are a heterogeneous population of immune cells occupying multiple tissue niches and exhibiting microenvironment-specific phenotypes and functions. In certain tissues such as the brain, lung, and liver, embryonically derived RTMs maintain themselves by self-renewal, whereas others, including those in the gut, dermis, and pancreas, are replaced by monocytes, at levels that are tissue specific. Once they arrive in their tissue of residence, monocytes undergo extensive differentiation according to molecular cues provided by their distinct tissue-specific niches, enabling their development into specialized RTMs that support local tissue function. RATIONALE As a result of this ontogenetic and tissue niche heterogeneity, each tissue contains multiple populations of macrophages. For example, in the murine lung, alveolar macrophages are the major embryonically derived population in the alveolar spaces, whereas a minor population named interstitial macrophages (IMs) resides within the lung parenchyma. Previous results reported several phenotypically distinct IM subpopulations, whose relationship remained unknown. Do they represent independent populations or, rather, different points on the spectrum of maturation and activation states? How do these differences relate to their localization in tissue or roles in tissue function in health and disease? Does such macrophage heterogeneity also exist in other tissues? RESULTS Here, using single-cell mRNA sequencing, we unbiasedly identified two independent populations exhibiting distinct gene expression profiles and phenotypes: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) RTMs. We uncovered evidence of parallel populations in multiple others tissues, including the heart, fat, and dermis, as well as in human lung and omental and subcutaneous fat tissues, suggesting that a similar dichotomy is observed in human tissues. We further demonstrated that both populations are slowly replaced by Ly6Chi monocytes. Importantly, using complementary fate-mapping models, we showed that monocyte-derived RTMs (MRTMs) are two separate lineages, rather than representing points along a developmental or maturation continuum. Notably, these distinct MRTM populations preferentially reside within different, but conserved, subtissular niches, located either adjacent to nerve bundles and fibers (Lyve1loMHCIIhi) or blood vessels (Lyve1hiMHCIIlo) across tissues. Finally, by acutely depleting Lyve1hiMHCIIlo MRTMs using a mouse model of inducible macrophage depletion during the induction of fibrosis, we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung and heart fibrosis, demonstrating their critical role in tissue inflammation. CONCLUSION Two independent MRTMs populations exist across tissues with specific niche-dependent phenotype and functional programming. Their different roles in homeostasis, immune regulation, and fibrosis renders them attractive and separate cellular targets for the therapeutic exploitation of RTM subsets.

Studies in human aging mainly focus on αβ T cells while γδ T cells have been neglected despite their role in immunosurveillance. In this study, we investigated γδ T cells and its subsets (i.e. Vδ1 , Vδ2 and DN γδ (γδ Vδ1-Vδ2-)) by deep immunophenotyping. Our data shows that peripheral Vδ2 are differentially susceptible to life-long stresses against all other αβ and γδ T cells. Using aging and cytomegalovirus history (the main confounding factor in immune-aging studies) as immunological models and via functional assays, telomere length determination, epigenetic methylome profiling and DNA damage response capacity, we show that Vδ2 T cells are resistant from cellular senescence. Our results highlight the differential impact of stress on γδ T cells subsets, reveal surface markers that are functionally relevant for γδ T cells subsets and highlight possible mechanisms that enable Vδ2 to be resistant to cellular aging. This new finding reinforces the concept that Vδ2 have an “innate-like” behavior and are more resilient to the environment as compared to “adaptive-like” Vδ1 T cells. Funding Statement: Weili Xu is funded by A*STAR Graduate Academy (AGA). The work was funded by the Singapore Immunology Network, the Agency for Science Technology and Research (JCO grant #1434m00115) and the Skin Research Institute of Singapore (SRG grant #14018). Declaration of Interests: The authors have no conflict of interests. Ethics Approval Statement: Participants of the study were recruited under National University of Singapore Institutional Review Board (IRB code NUS-IRB 01-256). Cone blood was obtained from Health Science Authority (HSA) Singapore, approved under NUS-IRB 10-250.

Summary Microglia are embryonically seeded macrophages that contribute to brain development, homeostasis, and pathologies. It is thus essential to decipher how microglial properties are temporally regulated by intrinsic and extrinsic factors, such as sexual identity and the microbiome. Here, we found that microglia undergo differentiation phases, discernable by transcriptomic signatures and chromatin accessibility landscapes, which can diverge in adult males and females. Remarkably, the absence of microbiome in germ-free mice had a time and sexually dimorphic impact both prenatally and postnatally: microglia were more profoundly perturbed in male embryos and female adults. Antibiotic treatment of adult mice triggered sexually biased microglial responses revealing both acute and long-term effects of microbiota depletion. Finally, human fetal microglia exhibited significant overlap with the murine transcriptomic signature. Our study shows that microglia respond to environmental challenges in a sex- and time-dependent manner from prenatal stages, with major implications for our understanding of microglial contributions to health and disease., Graphical Abstract, Highlights • Microglia undergo sequential phases of differentiation during development • The maternal microbiome influences microglial properties during prenatal stages • The absence of the microbiome has a sex- and time-specific impact on microglia • Microbiome depletions have acute and long-term effects on microglial properties, Microglia respond to environmental challenges, such as signals from the gut microbiome, in a sex- and time-dependent manner.

DCs are crucial for sensing pathogens and triggering immune response. Upon activation by pathogen-associated molecular pattern (PAMP) ligands, GM-CSF myeloid DCs (GM-DCs) secrete several cytokines, including IL-2. DC IL-2 has been shown to be important for innate and adaptive immune responses; however, IL-2 importance in DC physiology has never been demonstrated. Here, we show that autocrine IL-2 signaling is functional in murine GM-DCs in an early time window after PAMPs stimulation. IL-2 signaling selectively activates the JAK/STAT5 pathway by assembling holo-receptor complexes at the cell surface. Using the sensitivity of targeted mass spectrometry, we show conclusively that GM-DCs express CD122, the IL-2 receptor β-chain, at steady state. In myeloid DCs, this cytokine pathway inhibits survival of PAMP-matured GM-DCs which is crucial for maintaining immune tolerance and preventing autoimmunity. Our findings suggest that immune regulation by this novel autocrine signaling pathway can potentially be used in DC immunotherapy.

Circulating CCR2+ monocytes are crucial for maintaining the adult tissue-resident F4/80hiMHCIIhi macrophage pool in the intestinal lamina propria. Here we show that a subpopulation of CCR2-independent F4/80hiMHCIIlow macrophages, which are the most abundant F4/80hi cells in neonates, gradually decline in number in adulthood; these macrophages likely represent the fetal contribution to F4/80hi cells. In colon adenomas of ApcMin/+ mice, F4/80hiMHCIIlow macrophages are not only preserved, but become the dominant subpopulation among tumour-resident macrophages during tumour progression. Furthermore, these pro-tumoural F4/80hiMHCIIlow and F4/80hiMHCIIhi macrophages can self-renew in the tumour and maintain their numbers mostly independent from bone marrow contribution. Analyses of colon adenomas indicate that CSF1 may be a key facilitator of macrophage self-renewal. In summary, the tumour microenvironment creates an isolated niche for tissue-resident macrophages that favours macrophage survival and self-renewal., Tissue-resident F4/80hi macrophages can be found both in normal gut as well as in intestinal tumours. Here the authors show that in the colon these macrophages are CCR2-dependent, while in tumours they gain the ability to self-renew, relying on CSF1 and promoting cancer progression.

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils and mature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. In summary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses. We thank all members of L.G.N laboratory, the SIgN (Singapore Immunology Network) flow-cytometry team, the SIgN functional genomics team for their assistance with transcriptomics, and the SIgN mouse core facility for their technical help and support. This research was funded by SIgN core funding, A*STAR (Agency for Science, Technology and Research), Singapore. Sí

Background Expression quantitative trait loci (eQTL) databases represent a valuable resource to link disease-associated SNPs to specific candidate genes whose gene expression is significantly modulated by the SNP under investigation. We previously identified signal inhibitory receptor on leukocytes-1 (SIRL-1) as a powerful regulator of human innate immune cell function. While it is constitutively high expressed on neutrophils, on monocytes the SIRL-1 surface expression varies strongly between individuals. The underlying mechanism of regulation, its genetic control as well as potential clinical implications had not been explored yet. Methods Whole blood eQTL data of a Chinese cohort was used to identify SNPs regulating the expression of VSTM1, the gene encoding SIRL-1. The genotype effect was validated by flow cytometry (cell surface expression), correlated with electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP) and bisulfite sequencing (C-methylation) and its functional impact studied the inhibition of reactive oxygen species (ROS). Results We found a significant association of a single CpG-SNP, rs612529T/C, located in the promoter of VSTM1. Through flow cytometry analysis we confirmed that primarily in the monocytes the protein level of SIRL-1 is strongly associated with genotype of this SNP. In monocytes, the T allele of this SNP facilitates binding of the transcription factors YY1 and PU.1, of which the latter has been recently shown to act as docking site for modifiers of DNA methylation. In line with this notion rs612529T associates with a complete demethylation of the VSTM1 promoter correlating with the allele-specific upregulation of SIRL-1 expression. In monocytes, this upregulation strongly impacts the IgA-induced production of ROS by these cells. Through targeted association analysis we found a significant Meta P value of 1.14 × 10–6 for rs612529 for association to atopic dermatitis (AD). Conclusion Low expression of SIRL-1 on monocytes is associated with an increased risk for the manifestation of an inflammatory skin disease. It thus underlines the role of both the cell subset and this inhibitory immune receptor in maintaining immune homeostasis in the skin. Notably, the genetic regulation is achieved by a single CpG-SNP, which controls the overall methylation state of the promoter gene segment. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0404-6) contains supplementary material, which is available to authorized users.

EMSA competition of PU.1 (ETS family) and YY1 consensus probes with rs612529 probes. EMSA experiments were performed with radiolabeled probes representing the consensus binding sequence of PU.1 and YY1. A EMSA competition experiment with consensus PU.1-probe. Nuclear extracts from primary monocytes were exposed to radiolabeled PU.1 consensus alone (lane 1) or together with excess of unlabeled PU.1 consensus probes (lane 2), PU.1 mutant probes (lane 3), or increasing amounts of rs612529 C (lanes 4–6) or T probes (lanes 7–9). The competitors were added to the binding reactions at increasing concentrations prior to the incubation with probe. B EMSA competition experiment with consensus YY1-probe. Radiolabeled YY1 probe was used alone (lane 1) or with excess of unlabeled YY1 probe (lane 2), YY1 mutant probe (lane 3), or increasing amounts of rs612529 C (lanes 4–7) or T probes (lanes 8–11). (PDF 243 kb)

Oligonucleotide sequences of probes used in EMSA experiments. The table lists the sequence of oligonucleotides used as radiolabeled probes or as cold competitors in EMSA. VSTM1 C and T probe sequences represent the two allelic variants of a 25-bp region in the VSTM1 promoter containing the rs612529 SNP. Consensus and mutant oligonucleotides are based on consensus binding sites of the respective transcription factor. (PDF 51Â kb)

Gating strategy for the flow cytometry analysis of SIRL-1 expression on whole blood samples. Whole blood samples stained with antibodies specific for CCR3, CD16, CD123, CD14, FcÎľRIÎą, and HLA-DR were counterstained with either anti-SIRL-1 or isotype-matched control antibody. Gates allowing the simultaneous analysis of the SIRL-1 staining on neutrophils, eosinophils, basophils, monocytes, mDC, pDC, B cells, and NK & T cells are indicated. (PDF 210Â kb)

In silico prediction of transcription factors with allele-specific binding to rs612529. The web based prediction tools JASPAR (A) and TRANSFAC (B) were used to identify potential transcription factor binding sites in the VSTM1 promoter region affected by the allelic variations of rs612529 T/C. Only sites predicted with a binding score greater than 5.0 (JASPAR) and 0.9 (TRANSFAC) were selected for further analysis. The nucleotide underlined in red indicates the location of rs612529 within binding motif of the respective TF. Yellow shaded rows indicate transcription factors predicted to be highly specific for T allele.5. (PDF 55Â kb)

Model on the potentiating effect of a regulatory CpG-SNP. A Conventional vs. regulatory CpG-SNPs. The methylation of CpG pairs results in the condensation of chromatin due to a tighter packaging of the histone-DNA complexes (open circle: non-methylated CpG, closed circle: methylated CpG). While a conventional CpG-SNP can assist this process by providing only one additional allele-dependent methylation site (middle panel), a regulatory CpG-SNP (such as rs612529) modulates the recruiting of CpG-modifying enzymes (right panel). In the case of rs612529, this seems to be mediated by the allele-specific binding of PU.1 to the T allele of the CpG-SNP. In monocytes PU.1 was reported to bind the demethylase Tet-2. B Potentiating effect. Recruitment of Tet-2 mediates the chromatin-opening by facilitating the demethylation of all CpG pairs in the vicinity. The epigenetic effect is allele-specific (T allele) and direction of the direction probably cell-typeâ dependent. (PDF 415Â kb)

VSTM1 promoter sequence. The sequence of the region 411 bases upstream of the TSS and 282 bases downstream (untranslated region, Exon1, and part of Intron 1 of VSTM1 transcript) was obtained from UCSC genome browser ( https://genome.ucsc.edu/ ). The location of rs612529 T/C (underlined) as well as of each CpG site is indicted (red bold font). The transcription start site is indicated by â TSS,â the translational start by the start codon ATG. The transcribed region is indicated by small letters. Primers for bisulfite sequencing (F1/F2 and B1/B2) as well as the primer pair used for rs612529 genotyping and ChiP analysis are indicated by empty arrows. (PDF 161Â kb)

Treatment of post-transplant patients with immunosuppressive drugs targeting the calcineurin-nuclear factor of activated T cells (NFAT) pathway, including cyclosporine A or tacrolimus, is commonly associated with a higher incidence of opportunistic infections, such as Aspergillus fumigatus, which can lead to severe life-threatening conditions. A component of the A. fumigatus cell wall, β-glucan, is recognized by dendritic cells (DCs) via the Dectin-1 receptor, triggering downstream signaling that leads to calcineurin-NFAT binding, NFAT translocation, and transcription of NFAT-regulated genes. Here, we address the question of whether calcineurin signaling in CD11c-expressing cells, such as DCs, has a specific role in the innate control of A. fumigatus. Impairment of calcineurin in CD11c-expressing cells (CD11ccrecnb1loxP) significantly increased susceptibility to systemic A. fumigatus infection and to intranasal infection in irradiated mice undergoing bone marrow transplant. Global expression profiling of bone marrow-derived DCs identified calcineurin-regulated processes in the immune response to infection, including expression of pentraxin-3, an important antifungal defense protein. These results suggest that calcineurin inhibition directly impairs important immunoprotective functions of myeloid cells, as shown by the higher susceptibility of CD11ccrecnbloxP mice in models of systemic and invasive pulmonary aspergillosis, including after allogeneic bone marrow transplantation. These findings are relevant to the clinical management of transplant patients with severe Aspergillus infections.

Neutrophils are an abundant immune cell type involved in both antimicrobial defence and autoimmunity. The regulation of their gene expression, however, is still largely unknown. Here we report an eQTL study on isolated neutrophils from 114 healthy individuals of Chinese ethnicity, identifying 21,210 eQTLs on 832 unique genes. Unsupervised clustering analysis of these eQTLs confirms their role in inflammatory responses and immunological diseases but also indicates strong involvement in dermatological pathologies. One of the strongest eQTL identified (rs2058660) is also the tagSNP of a linkage block reported to affect leprosy and Crohn's disease in opposite directions. In a functional study, we can link the C allele with low expression of the β-chain of IL18-receptor (IL18RAP). In neutrophils, this results in a reduced responsiveness to IL-18, detected both on the RNA and protein level. Thus, the polymorphic regulation of human neutrophils can impact beneficial as well as pathological inflammatory responses., Neutrophils are abundant immune cells important for antimicrobial defence and in autoimmunity. Here, by mapping expression quantitative trait loci (eQTL) in neutrophils of Chinese ethnicity from Singapore, Andiappan et al. provide a resource for understanding immune-related trait associated genetic variants.

DCs are crucial for sensing pathogens and triggering immune response. Upon activation by pathogen-associated molecular pattern (PAMP) ligands, GM-CSF myeloid DCs (GM-DCs) secrete several cytokines, including IL-2. DC IL-2 has been shown to be important for innate and adaptive immune responses; however, IL-2 importance in DC physiology has never been demonstrated. Here, we show that autocrine IL-2 signaling is functional in murine GM-DCs in an early time window after PAMPs stimulation. IL-2 signaling selectively activates the JAK/STAT5 pathway by assembling holo-receptor complexes at the cell surface. Using the sensitivity of targeted mass spectrometry, we show conclusively that GM-DCs express CD122, the IL-2 receptor β-chain, at steady state. In myeloid DCs, this cytokine pathway inhibits survival of PAMP-matured GM-DCs which is crucial for maintaining immune tolerance and preventing autoimmunity. Our findings suggest that immune regulation by this novel autocrine signaling pathway can potentially be used in DC immunotherapy.

Background Allergic rhinitis (AR) and asthma are common allergic conditions with a shared genetic component to their cause. The 17q12-21 locus includes several genes that have been linked to asthma susceptibility, but the role of this locus in AR is unclear. Asthma and AR in adults of Chinese ethnicity in Singapore are predominately caused by sensitization against house dust mites with a nearly complete penetrance of the allergen, which presents a unique opportunity for accurately identifying genetic associations with allergic diseases. Objective We sought to define the functional role of 17q12-21 in patients with AR and allergic asthma. Methods We asked whether single nucleotide polymorphisms (SNPs) in the 17q12-21 locus were associated with AR or asthma in a cohort of 3460 ethnic Chinese subjects residing in Singapore (1435 in the discovery phase and 2025 in the validation phase). Full-blood mRNA gene expression data, plasma IgE levels, and immune cell frequencies in peripheral blood were tested against the tag SNP genotypes. Luciferase assays were used to measure the effect of putative promoter SNPs on expression of the asthma-associated orosomucoid-like 3 gene (ORMDL3) . Results Within 17q12-21, only the tag SNP rs8076131 was significantly associated with asthma ( P = 8.53 × 10 −10 ; odds ratio, 0.6715), and AR status was independent of SNPs in this region. C-A alleles at rs8076131 resulted in significantly increased ORMDL3 expression in HEK293 cells in vitro relative to T-G alleles. Moreover, subjects with the risk genotype AA exhibited significantly higher total IgE levels and higher blood eosinophil counts than those with the lower-risk genotypes. Conclusion The 17q12-21 locus has a strong genetic association with allergic asthma but not with AR. The polymorphic effect of this locus is attributed to the linkage set tagged by rs8076131, which affects the expression of ORMDL3 , protein phosphatase 1, regulatory inhibitor subunit 1B ( PPP1R1B ), zona pellucida binding protein 2 ( ZPBP2 ), and gasdermin B (GSDMB) and is correlated with high IgE levels and eosinophil counts in subjects bearing the risk genotype.

Pyrosequencing is a DNA sequencing technique based on sequencing-by-synthesis enabling rapid and real-time sequence determination. Although ample genomic research has been undertaken using pyrosequencing, the requirement of relatively high amount of DNA template and the difficulty in sequencing the homopolymeric regions limit its key advantages in the applications directing towards clinical research. In this study, we demonstrate that pyrosequencing on homopolymeric regions with 10 identical nucleotides can be successfully performed with optimal amount of DNA (0.3125-5 pmol) immobilized on conventional non-porous Sepharose beads. We also validate that by using porous silica beads, the sequencing signal increased 3.5-folds as compared to that produced from same amount of DNA immobilized on solid Sepharose beads. Our results strongly indicate that with optimized quantity of DNA and suitable solid support, the performance of pyrosequencing on homopolymeric regions and its detection limit has been significantly improved.

The design and development of an inexpensive and customizable platform, for ultra-high throughput pyrosequenicng is presented. The platform comprises a micro-chip containing millions of reaction wells on a fiber optic platform, integrated with fluidics and a CMOS image sensor for chemiluminescence detection. Proof of concept experiments were performed to demonstrate the viability of the system for sequencing. Synthetic DNA, with various sequences, were designed and sequenced in the Pyrome sequencer. An experiment designed to test the loss of beads during long reads demonstrated that there is no appreciable loss of beads after sequencing 25 base pairs.

Gating strategy for monocytes expressing FcγRIII, FcεRIα, or FcαR. PBMCs were stained with antibodies specific for FcγRIII (CD16), FceRIα, FcαR (CD89), and CD14 together with anti-SIRL-1, or isotype-matched control antibody. Monocytes were gated based on scatter properties (SSC-A and FSC-A) and live/dead-staining (AmCyan). Gates for FcγRIII+ monocytes, FcεRIα + monocytes, and FcαR+ monocytes are indicated in plots displaying the CD14 staining vs. the respective Fc-receptor. (PDF 297 kb)

Cohorts used in the study. The data for this study were collected from seven different cohorts. Four of these cohorts were established for the functional association studies (eQTL, FACS analysis, ROS assay, DNA methylation, and ChiP assay), while the three other cohorts were used for the genetic association studies with atopic dermatitis (AD). Size of the cohorts as well as ethnicity and appropriate reference are provided for each cohort. (PPTX 80Â kb)

Cohorts used in the study. The data for this study were collected from seven different cohorts. Four of these cohorts were established for the functional association studies (eQTL, FACS analysis, ROS assay, DNA methylation, and ChiP assay), while the three other cohorts were used for the genetic association studies with atopic dermatitis (AD). Size of the cohorts as well as ethnicity and appropriate reference are provided for each cohort. (PPTX 80Â kb)

Gating strategy for monocytes expressing FcγRIII, FcεRIα, or FcαR. PBMCs were stained with antibodies specific for FcγRIII (CD16), FceRIα, FcαR (CD89), and CD14 together with anti-SIRL-1, or isotype-matched control antibody. Monocytes were gated based on scatter properties (SSC-A and FSC-A) and live/dead-staining (AmCyan). Gates for FcγRIII+ monocytes, FcεRIα + monocytes, and FcαR+ monocytes are indicated in plots displaying the CD14 staining vs. the respective Fc-receptor. (PDF 297 kb)