The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
The human genome contains approximately 20 thousand protein-coding genes, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown-although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% of λ or κ light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease.
The role that broadly neutralizing antibodies (bNAbs) play in natural clearance of human hepatitis C virus (HCV) infection and the underlying mechanisms remain unknown. Here, we investigate the mechanism by which bNAbs, isolated from two humans who spontaneously cleared HCV infection, contribute to HCV control. Using viral gene sequences amplified from longitudinal plasma of the two subjects, we found that these bNAbs, which target the front layer of the HCV envelope protein E2, neutralized most autologous HCV strains. Acquisition of resistance to bNAbs by some autologous strains was accompanied by progressive loss of E2 protein function, and temporally associated with HCV clearance. These data demonstrate that bNAbs can mediate clearance of human HCV infection by neutralizing infecting strains and driving escaped viruses to an unfit state. These immunopathologic events distinguish HCV from HIV-1 and suggest that development of an HCV vaccine may be achievable.
Copyright © 2018 Elsevier Inc. All rights reserved.
Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its antihyperglycemic action primarily through lowering hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5'-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P), and knock-in (KI) of this mutant in mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The antihyperglycemic effect of metformin in high-fat diet-fed diabetic FBP1-KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action for metformin and provide further evidence that molecular targeting of FBP1 can have antihyperglycemic effects.
Lung alveoli, which are unique to air-breathing organisms, have been challenging to generate from pluripotent stem cells (PSCs) in part because there are limited model systems available to provide the necessary developmental roadmaps for in vitro differentiation. Here we report the generation of alveolar epithelial type 2 cells (AEC2s), the facultative progenitors of lung alveoli, from human PSCs. Using multicolored fluorescent reporter lines, we track and purify human SFTPC+ alveolar progenitors as they emerge from endodermal precursors in response to stimulation of Wnt and FGF signaling. Purified PSC-derived SFTPC+ cells form monolayered epithelial "alveolospheres" in 3D cultures without the need for mesenchymal support, exhibit self-renewal capacity, and display additional AEC2 functional capacities. Footprint-free CRISPR-based gene correction of PSCs derived from patients carrying a homozygous surfactant mutation (SFTPB) restores surfactant processing in AEC2s. Thus, PSC-derived AEC2s provide a platform for disease modeling and future functional regeneration of the distal lung.
Copyright © 2017 Elsevier Inc. All rights reserved.
Susceptibility and protection against human autoimmune diseases, including type I diabetes, multiple sclerosis, and Goodpasture disease, is associated with particular human leukocyte antigen (HLA) alleles. However, the mechanisms underpinning such HLA-mediated effects on self-tolerance remain unclear. Here we investigate the molecular mechanism of Goodpasture disease, an HLA-linked autoimmune renal disorder characterized by an immunodominant CD4 T-cell self-epitope derived from the α3 chain of type IV collagen (α3). While HLA-DR15 confers a markedly increased disease risk, the protective HLA-DR1 allele is dominantly protective in trans with HLA-DR15 (ref. 2). We show that autoreactive α3-specific T cells expand in patients with Goodpasture disease and, in α3-immunized HLA-DR15 transgenic mice, α3-specific T cells infiltrate the kidney and mice develop Goodpasture disease. HLA-DR15 and HLA-DR1 exhibit distinct peptide repertoires and binding preferences and present the α3 epitope in different binding registers. HLA-DR15-α3 tetramer T cells in HLA-DR15 transgenic mice exhibit a conventional T-cell phenotype (T) that secretes pro-inflammatory cytokines. In contrast, HLA-DR1-α3 tetramer T cells in HLA-DR1 and HLA-DR15/DR1 transgenic mice are predominantly CD4Foxp3 regulatory T cells (T cells) expressing tolerogenic cytokines. HLA-DR1-induced T cells confer resistance to disease in HLA-DR15/DR1 transgenic mice. HLA-DR15 and HLA-DR1 healthy human donors display altered α3-specific T-cell antigen receptor usage, HLA-DR15-α3 tetramer Foxp3 T and HLA-DR1-α3 tetramer Foxp3CD25CD127 T dominant phenotypes. Moreover, patients with Goodpasture disease display a clonally expanded α3-specific CD4 T-cell repertoire. Accordingly, we provide a mechanistic basis for the dominantly protective effect of HLA in autoimmune disease, whereby HLA polymorphism shapes the relative abundance of self-epitope specific T cells that leads to protection or causation of autoimmunity.
To assess the effect of chemotherapy on mitochondrial genome mutations in cancer survivors and their offspring, a study sequenced the full mitochondrial genome and determined the mitochondrial DNA heteroplasmic (mtDNA) mutation rate. To build a model for counts of heteroplasmic mutations in mothers and their offspring, bivariate Poisson regression was used to examine the relationship between mutation count and clinical information while accounting for the paired correlation. However, if the sequencing depth is not adequate, a limited fraction of the mtDNA will be available for variant calling. The classical bivariate Poisson regression model treats the offset term as equal within pairs; thus, it cannot be applied directly. In this research, we propose an extended bivariate Poisson regression model that has a more general offset term to adjust the length of the accessible genome for each observation. We evaluate the performance of the proposed method with comprehensive simulations, and the results show that the regression model provides unbiased parameter estimations. The use of the model is also demonstrated using the paired mtDNA dataset.
Wilms tumor (WT) is the most common renal neoplasm of childhood and affects 1 in 10 000 children aged less than 15 years. These embryonal tumors are thought to arise from primitive nephrogenic rests that derive from the metanephric mesenchyme during kidney development and are characterized partly by increased Wnt/β-catenin signaling. We previously showed that coordinate activation of Ras and β-catenin accelerates the growth and metastatic progression of a murine WT model. Here, we show that activating KRAS mutations can be found in human WT. In addition, high levels of phosphorylated AKT are present in the majority of WT. We further show in a mouse model and in renal epithelial cells that Ras cooperates with β-catenin to drive metastatic disease progression and promotes in vitro tumor cell growth, migration, and colony formation in soft agar. Cellular transformation and metastatic disease progression of WT cells are in part dependent on PI3K/AKT activation and are inhibited via pharmacological inhibition of this pathway. Our studies suggest both KRAS mutations and AKT activation are present in WT and may represent novel therapeutic targets for this disease.
© 2017 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.
In familial pulmonary arterial hypertension (FPAH), the autosomal dominant disease-causing BMPR2 mutation is only 20% penetrant, suggesting that genetic variation provides modifiers that alleviate the disease. Here, we used comparison of induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) from three families with unaffected mutation carriers (UMCs), FPAH patients, and gender-matched controls to investigate this variation. Our analysis identified features of UMC iPSC-ECs related to modifiers of BMPR2 signaling or to differentially expressed genes. FPAH-iPSC-ECs showed reduced adhesion, survival, migration, and angiogenesis compared to UMC-iPSC-ECs and control cells. The "rescued" phenotype of UMC cells was related to an increase in specific BMPR2 activators and/or a reduction in inhibitors, and the improved cell adhesion could be attributed to preservation of related signaling. The improved survival was related to increased BIRC3 and was independent of BMPR2. Our findings therefore highlight protective modifiers for FPAH that could help inform development of future treatment strategies.
Copyright © 2017 Elsevier Inc. All rights reserved.
DNA-protein cross-links (DPCs) are bulky DNA lesions that form both endogenously and following exposure to bis-electrophiles such as common antitumor agents. The structural and biological consequences of DPCs have not been fully elucidated due to the complexity of these adducts. The most common site of DPC formation in DNA following treatment with bis-electrophiles such as nitrogen mustards and cisplatin is the N7 position of guanine, but the resulting conjugates are hydrolytically labile and thus are not suitable for structural and biological studies. In this report, hydrolytically stable structural mimics of N7-guanine-conjugated DPCs were generated by reductive amination reactions between the Lys and Arg side chains of proteins/peptides and aldehyde groups linked to 7-deazaguanine residues in DNA. These model DPCs were subjected to in vitro replication in the presence of human translesion synthesis DNA polymerases. DPCs containing full-length proteins (11-28 kDa) or a 23-mer peptide blocked human polymerases η and κ. DPC conjugates to a 10-mer peptide were bypassed with nucleotide insertion efficiency 50-100-fold lower than for native G. Both human polymerase (hPol) κ and hPol η inserted the correct base (C) opposite the 10-mer peptide cross-link, although small amounts of T were added by hPol η. Molecular dynamics simulation of an hPol κ ternary complex containing a template-primer DNA with dCTP opposite the 10-mer peptide DPC revealed that this bulky lesion can be accommodated in the polymerase active site by aligning with the major groove of the adducted DNA within the ternary complex of polymerase and dCTP.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
We analyzed 3,872 common genetic variants across the ESR1 locus (encoding estrogen receptor α) in 118,816 subjects from three international consortia. We found evidence for at least five independent causal variants, each associated with different phenotype sets, including estrogen receptor (ER(+) or ER(-)) and human ERBB2 (HER2(+) or HER2(-)) tumor subtypes, mammographic density and tumor grade. The best candidate causal variants for ER(-) tumors lie in four separate enhancer elements, and their risk alleles reduce expression of ESR1, RMND1 and CCDC170, whereas the risk alleles of the strongest candidates for the remaining independent causal variant disrupt a silencer element and putatively increase ESR1 and RMND1 expression.