Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments

Meta-analysis of shared genetic architecture across ten pediatric autoimmune diseases

Genome-wide association studies (GWASs) have identified hundreds of susceptibility genes, including shared associations across clinically distinct autoimmune diseases. We performed an inverse χ(2) meta-analysis across ten pediatric-age-of-onset autoimmune diseases (pAIDs) in a case-control study including more than 6,035 cases and 10,718 shared population-based controls. We identified 27 genome-wide significant loci associated with one or more pAIDs, mapping to in silico-replicated autoimmune-associated genes (including IL2RA) and new candidate loci with established immunoregulatory functions such as ADGRL2, TENM3, ANKRD30A, ADCY7 and CD40LG. The pAID-associated single-nucleotide polymorphisms (SNPs) were functionally enriched for deoxyribonuclease (DNase)-hypersensitivity sites, expression quantitative trait loci (eQTLs), microRNA (miRNA)-binding sites and coding variants. We also identified biologically correlated, pAID-associated candidate gene sets on the basis of immune cell expression profiling and found evidence of genetic sharing. Network and protein-interaction analyses demonstrated converging roles for the signaling pathways of type 1, 2 and 17 helper T cells (TH1, TH2 and TH17), JAK-STAT, interferon and interleukin in multiple autoimmune diseases

Secondary immunologic consequences in chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome).

Clinical evidence suggests that patients with Chromosome 22q11.2 deletion (Ch22q11.2D) have an increased prevalence of atopic and autoimmune disease and this has been without explanation. We hypothesized that the increase in atopy was due to homeostatic proliferation of T cells leading to a Th2 skew. We performed intracellular cytokine staining to define Th1/Th2 phenotypes in toddlers (early homeostatic proliferation) and adults (post homeostatic proliferation) with this syndrome. To attempt to understand the predisposition to autoimmunity we performed immunophenotyping analyses to define Th17 cells and B cell subsets. Adult Ch22q11.2D patients had a higher percentage of IL-4+CD4+ T cells than controls. Th17 cells were no different in patients and controls. In addition, adult Ch22q11.2D syndrome patients had significantly lower switched memory B cells, suggesting a dysregulated B cell compartment. These studies demonstrate that the decrement in T cell production has secondary consequences in the immune system, which could mold the patients\u27 clinical picture

Analysis of Catalytic Hydrothermal Conversion Jet Fuel and Surrogate Mixture Formulation: Components, Properties, and Combustion

Chemical analysis and property measurements of a catalytic hydrothermal conversion jet (CHCJ) fuel were used to formulate hydrocarbon mixtures for use as fuel surrogates. Using conventional gas chromatography/(electron ionization) quadrupole mass spectrometry (GC/(EI)­Q MS) and advanced two-dimensional gas chromatography/(electron ionization) high resolution time-of-flight mass spectrometry (GC×GC/(EI)­TOF MS), CHCJ was found to differ from Jet-A fuel and to contain mostly linear alkanes, alkylcyclohexanes, and alkylbenzenes, with small amounts of branched alkanes and multiring aromatic compounds. Various surrogates were prepared containing <i>n</i>-dodecane, <i>n</i>-butyl­cyclo­hexane, and <i>n</i>-butyl­benzene, and their density, viscosity, speed of sound, surface tension, and derived cetane number (DCN) were measured to determine the compositions that most closely matched that of the CHCJ. The optimal surrogates were (1) <i>n</i>-butylcyclo­hexane, (2) 0.64 mole fraction of <i>n</i>-butyl­benzene in <i>n</i>-dodecane, and (3) three three-component blends of <i>n</i>-butylcyclo­hexane, <i>n</i>-butyl­benzene, and <i>n</i>-dodecane with ratios of <i>n</i>-dodecane to <i>n</i>-butylcyclo­hexane of 0.25, 0.50, and 0.75 corresponding to a lower, medium, and higher <i>n</i>-butyl­benzene concentration. Since fuel logistics in the military could be greatly simplified by use of a single fuel for both jet and diesel engines, this study examined this alternative jet fuel and its potential surrogates with respect to combustion in a diesel engine. Combustion experiments using a Waukesha diesel Cooperative Fuels Research (CFR) engine showed that all surrogate mixtures emulated the combustion engine performance of CHCJ in the areas of thermal efficiency, ignition delay, relative rate of heat release, crank angle degree 50% fuel burned location, and burn duration. All the surrogate mixtures operated in the Waukesha engine all showed statistically similar performance to the CHCJ fuel; however, the midaromatic (<i>n</i>-butyl­benzene) three-component surrogate was marginally closer than either the higher or lower aromatic blends. These results show that DCN and other physical property measurements of a jet fuel can be used in conjunction with chemical composition to design surrogate fuel mixtures that match jet fuel performance in a diesel engine. These surrogate mixtures can be used in modeling studies to help determine the aspects of jet fuels that would enable them to have acceptable performance in a military diesel engine