Aceruloplasminemia is an autosomal recessive disorder of iron metabolism. Affected individuals evidence iron accumulation in tissue parenchyma in association with absent serum ceruloplasmin. Genetic studies of such patients reveal inherited mutations in the ceruloplasmin gene. To elucidate the role of ceruloplasmin in iron homeostasis, we created an animal model of aceruloplasminemia by disrupting the murine ceruloplasmin ( Cp ) gene. Although normal at birth, Cp ^−/− mice demonstrate progressive accumulation of iron such that by one year of age all animals have a prominent elevation in serum ferritin and a 3- to 6-fold increase in the iron content of the liver and spleen. Histological analysis of affected tissues in these mice shows abundant iron stores within reticuloendothelial cells and hepatocytes. Ferrokinetic studies in Cp ^+/+ and Cp ^−/− mice reveal equivalent rates of iron absorption and plasma iron turnover, suggesting that iron accumulation results from altered compartmentalization within the iron cycle. Consistent with this concept, Cp ^−/− mice showed no abnormalities in cellular iron uptake but a striking impairment in the movement of iron out of reticuloendothelial cells and hepatocytes. Our findings reveal an essential physiologic role for ceruloplasmin in determining the rate of iron efflux from cells with mobilizable iron stores.

Glioblastoma (GBM) is the most common primary malignant brain cancer, and is currently incurable. Chimeric Antigen Receptor (CAR) T-cells have shown promise in GBM treatment. While we have shown that combinatorial targeting of two glioma antigens offsets antigen escape and enhances T-cell effector functions, the inter-patient variability in surface antigen expression between patients hinders the clinical impact of targeting two antigen pairs. This study addresses targeting 3 antigens using a single CAR T-cell product for broader application. We analyzed the surface expression of 3 targetable glioma antigens (HER2, IL13Rα2 and EphA2) in 15 primary GBM samples. Accordingly, we created a trivalent T-cell product armed with three CAR molecules specific for these validated targets encoded by a single universal (U) tricistronic transgene (UCAR T-cells). Our data showed that co-targeting HER2, IL13Rα2 and EphA2 could overcome inter-patient variability by a tendency to capture near 100% of tumor cells in most tumors tested in this cohort. UCAR T-cells made from GBM patients’ blood uniformly expressed all three CAR molecules with distinct antigen specificity. UCAR T-cells mediated robust immune synapses with tumor targets forming more polarized microtubule organizing centers (MTOC) and exhibited improved cytotoxicity and cytokine release over best monospecific and bispecific CAR T-cells per patient tumor profile. Lastly, low doses of UCAR T-cells controlled established autologous GBM patient derived xenografts (PDX) and improved survival of treated animals. UCAR T-cells can overcome antigenic heterogeneity in GBM and lead to improved treatment outcomes.

Langerhans cell histiocytosis (LCH) is a rare disease characterized by heterogeneous lesions containing CD207⁺ Langerhans cells (LCs) and lymphocytes that can arise in almost any tissue and cause significant morbidity and mortality. After decades of research, the cause of LCH remains speculative. A prevailing model suggests that LCH arises from malignant transformation and metastasis of epidermal LCs. In this study, CD207⁺ cells and CD3⁺ T cells were isolated from LCH lesions to determine cell-specific gene expression. Compared with control epidermal CD207⁺ cells, the LCH CD207⁺ cells yielded 2113 differentially expressed genes (false discovery rate < 0.01). Surprisingly, the expression of many genes previously associated with LCH, including cell-cycle regulators, proinflammatory cytokines, and chemokines, were not significantly different from control LCs in our study. However, several novel genes whose products activate and recruit T cells to sites of inflammation, including SPP1 (osteopontin), were highly overexpressed in LCH CD207⁺ cells. Furthermore, several genes associated with immature myeloid dendritic cells were overexpressed in LCH CD207⁺ cells. Compared with the peripheral CD3⁺ cells from LCH patients, the LCH lesion CD3⁺ cells yielded only 162 differentially regulated genes (false discovery rate < 0.01), and the expression profile of the LCH lesion CD3⁺ cells was consistent with an activated regulatory T cell phenotype with increased expression of FOXP3, CTLA4, and SPP1. Results from this study support a model of LCH pathogenesis in which lesions do not arise from epidermal LCs but from accumulation of bone marrow-derived immature myeloid dendritic cells that recruit activated lymphocytes.

Subtypes are widely found in cancer. They are characterized with different behaviors in clinical and molecular profiles, such as survival rates, gene signature and copy number aberrations (CNAs). While cancer is generally believed to have been caused by genetic aberrations, the number of such events is tremendous in the cancer tissue and only a small subset of them may be tumorigenic. On the other hand, gene expression signature of a subtype represents residuals of the subtype-specific cancer mechanisms. Using high-throughput data to link these factors to define subtype boundaries and identify subtype-specific drivers, is a promising yet largely unexplored topic.

Salmonella bacteriophage repressor Mnt belongs to the ribbon-helix-helix class of transcription factors. Previous SELEX results suggested that interactions of Mnt with positions 16 and 17 of the operator DNA are not independent. Using a newly developed high-throughput quantitative multiple fluorescence relative affinity (QuMFRA) assay, we directly quantified the relative equilibrium binding constants (K(ref)) of Mnt to operators carrying all the possible dinucleotide combinations at these two positions. Results show that Mnt prefers binding to C, instead of wild-type A, at position 16 when wild-type C at position 17 is changed to other bases. The measured K(ref) values of double mutants were also higher than the values predicted from single mutants, demonstrating the non-independence of these two positions. The ability to produce a large number of quantitative binding data simultaneously and the potential to scale up makes QuMFRA a valuable tool for the large-scale study of macromolecular interaction.

BACKGROUND: Central nervous system Langerhans cell histiocytosis (CNS‐LCH) brain involvement may include mass lesions and/or a neurodegenerative disease (LCH‐ND) of unknown etiology. The goal of this study was to define the mechanisms of pathogenesis that drive CNS‐LCH.METHODS: Cerebrospinal fluid (CSF) biomarkers including CSF proteins and extracellular BRAFV600E DNA were analyzed in CSF from patients with CNS‐LCH lesions compared with patients with brain tumors and other neurodegenerative conditions. Additionally, the presence of BRAFV600E was tested in peripheral mononuclear blood cells (PBMCs) as well as brain biopsies from LCH‐ND patients, and the response to BRAF‐V600E inhibitor was evaluated in 4 patients with progressive disease.RESULTS: Osteopontin was the only consistently elevated CSF protein in patients with CNS‐LCH compared with patients with other brain pathologies. BRAFV600E DNA was detected in CSF of only 2/20 (10%) cases, both with LCH‐ND and active lesions outside the CNS. However, BRAFV600E⁺ PBMCs were detected with significantly higher frequency at all stages of therapy in LCH patients who developed LCH‐ND. Brain biopsies of patients with LCH‐ND demonstrated diffuse perivascular infiltration by BRAFV600E⁺ cells with monocyte phenotype (CD14⁺CD33⁺CD163⁺P2RY12^‐) and associated osteopontin expression. Three of 4 patients with LCH‐ND treated with BRAF‐V600E inhibitor experienced significant clinical and radiologic improvement.CONCLUSION: In LCH‐ND patients, BRAFV600E⁺ cells in PBMCs and infiltrating myeloid/monocytic cells in the brain is consistent with LCH‐ND as an active demyelinating process arising from a mutated hematopoietic precursor from which LCH lesion CD207⁺ cells are also derived. Therapy directed against myeloid precursors with activated MAPK signaling may be effective for LCH‐ND. Cancer 2018;124:2607‐20. © 2018 American Cancer Society.