The endogenous repertoire harbors self-reactive CD4+ T cell clones that adopt a follicular helper T cell-like phenotype at steady state.

The T cell repertoire of healthy mice and humans harbors self-reactive CD4+ conventional T (Tconv) cells capable of inducing autoimmunity. Using T cell receptor profiling paired with in vivo clonal analysis of T cell differentiation, we identified Tconv cell clones that are recurrently enriched in non-lymphoid organs following ablation of Foxp3+ regulatory T (Treg) cells. A subset of these clones was highly proliferative in the lymphoid organs at steady state and exhibited overt reactivity to self-ligands displayed by dendritic cells, yet were not purged by clonal deletion. These clones spontaneously adopted numerous hallmarks of follicular helper T (TFH) cells, including expression of Bcl6 and PD-1, exhibited an elevated propensity to localize within B cell follicles at steady state, and produced interferon-γ in non-lymphoid organs following sustained Treg cell depletion. Our work identifies a naturally occurring population of self-reactive TFH-like cells and delineates a previously unappreciated fate for self-specific Tconv cells.

Quantitative Time-of-Flight Head Magnetic Resonance Angiography of Cerebrovascular Disease.

BACKGROUND: Standard Cartesian time-of-flight (TOF) head magnetic resonance angiography (MRA) is routinely used to evaluate the intracranial arteries, but does not provide quantitative hemodynamic information that is useful for patient risk stratification as well as for monitoring treatment and tracking changes in blood flow over time. Quantitative TOF (qTOF) MRA represents a new and efficient method for simultaneous evaluating the intracranial arteries and quantifying blood flow velocity, but it has not yet been evaluated in patients with cerebrovascular disease. PURPOSE: To evaluate qTOF for simultaneously evaluating the intracranial arteries and quantifying intracranial blood flow velocity in patients with cerebrovascular disease, without the need for a phase contrast (PC) scan. STUDY TYPE: Prospective. SUBJECTS: Twenty-four patients (18 female, 6 male) with cerebrovascular disease. FIELD STRENGTH/SEQUENCES: Head MRA at 3 T using gradient-echo 3D qTOF, standard Cartesian TOF, and PC protocols. ASSESSMENT: Three independent readers assessed arterial image quality using a 4-point scale (1: non-diagnostic, 4: excellent) and artifact presence. Total and component flow velocities obtained with qTOF and PC were measured. STATISTICAL TESTS: Wilcoxon signed-rank tests, Gwet's AC2, intraclass correlation coefficients (ICC) for absolute agreement, Bland-Altman analyses, tests of equal proportions. P values <0.05 were considered statistically significant. RESULTS: Averaged across readers and compared to standard Cartesian TOF, qTOF significantly improved overall arterial image quality (3.8 ± 0.2 vs. 3.6 ± 0.5), image quality at locations of pathology (3.7 ± 0.5 vs. 3.4 ± 0.7), and increased the proportion of evaluations rated without artifacts (63.9% [46/72] vs. 37.5% [27/72]). qTOF significantly agreed with PC for total flow velocity (ICC = 0.71) and component flow velocity (ICC = 0.89). DATA CONCLUSION: qTOF angiography of the head matched or improved upon the image quality of standard Cartesian TOF, reduced image artifacts, and provided quantitative hemodynamic data, without the need for a PC scan. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 2.

Dark Blood Contrast-Enhanced Brain MRI Using Echo-uT1 RESS.

BACKGROUND: The widely used magnetization-prepared rapid gradient-echo (MPRAGE) sequence makes enhancing lesions and blood vessels appear bright after gadolinium administration. However, dark blood imaging using T1-weighted Sampling Perfection with Application optimized Contrast using different flip angle Evolution (T1 SPACE) can be advantageous since it improves the conspicuity of small metastases and leptomeningeal disease. As a potential alternative to T1 SPACE, we evaluated a new dark blood sequence called echo-uT1 RESS (unbalanced T1 Relaxation-Enhanced Steady-State). PURPOSE: We compared the performance of echo-uT1 RESS with Dixon fid-uT1 RESS, MPRAGE, and T1 SPACE. STUDY TYPE: Retrospective, IRB approved. SUBJECTS/PHANTOM: Phantom to assess flow properties of echo-uT1 RESS. Twenty-one patients (14 female, age range 35-82 years) with primary and secondary brain tumors. FIELD STRENGTH/SEQUENCES: 3 Tesla/MPRAGE, T1 SPACE, Dixon fid-uT1 RESS, echo-uT1 RESS. ASSESSMENT: Flow phantom signal vs. velocity as a function of flip angle and sequence. Qualitative image assessment on 4-point scale. Quantitative evaluation of tumor-to-brain contrast, apparent contrast-to-noise ratio (aCNR), and vessel-to-brain aCNR. STATISTICAL TESTS: Friedman and Mann-Whitney U tests. A P value <0.05 was considered statistically significant. RESULTS: In the phantom, echo-uT1 RESS showed greater flow-dependent signal loss than fid-uT1 RESS. In patients, blood vessels appeared bright with MPRAGE, gray with fid-uT1 RESS, and dark with T1 SPACE and echo-uT1 RESS. For MPRAGE, Dixon fid-uT1 RESS, echo-uT1 RESS, and T1 SPACE, respective tumor-to-brain contrast values were 0.6 ± 0.3, 1.3 ± 0.5, 1.0 ± 0.4, and 0.6 ± 0.4, while normalized aCNR values were 68.9 ± 50.9, 128.4 ± 59.2, 74.2 ± 42.1, and 99.4 ± 73.9. DATA CONCLUSION: Volumetric dark blood contrast-enhanced brain MRI is feasible using echo-uT1 RESS. The dark blood effect was improved vs. fid-uT1 RESS, while both uT1 RESS versions provided better tumor-to-brain contrast than MPRAGE. Whereas T1 SPACE provided better tumor aSNR, echo-uT1 RESS provided better Weber contrast, lesion sharpness and a more consistent dark blood effect. EVIDENCE LEVEL: 3 TECHNICAL EFFICACY: Stage 1.

Fibrinogen Is a Specific Trigger for Cytolytic Eosinophil Degranulation.

In inflamed human tissues, we often find intact eosinophilic granules, but not eosinophils themselves. Eosinophils, tissue-dwelling granulocytes with several homeostatic roles, have a surprising association with fibrinogen and tissue remodeling. Fibrinogen is a complex glycoprotein with regulatory roles in hemostasis, tumor development, wound healing, and atherogenesis. Despite its significance, the functional link between eosinophils and fibrinogen is not understood. We tested IL-5-primed mouse bone marrow-derived and human blood-sorted eosinophil activity against FITC-linked fibrinogen substrates. The interactions between these scaffolds and adhering eosinophils were quantified using three-dimensional laser spectral, confocal, and transmission electron microscopy. Eosinophils were labeled with major basic protein (MBP) Ab to visualize granules and assessed by flow cytometry. Both mouse and human eosinophils showed firm adhesion and degraded up to 27 ± 3.1% of the substrate area. This co-occurred with active MBP-positive granule release and the expression of integrin CD11b. Mass spectrometry analysis of fibrinogen proteolytic reactions detected the presence of eosinophil peroxidase, MBP, and fibrin α-, ß-, and γ-chains. Eosinophil activity was adhesion dependent, as a blocking Ab against CD11b significantly reduced adhesion, degranulation, and fibrinogenolysis. Although adhered, eosinophils exhibited no proteolytic activity on collagen matrices. Cytolytic degranulation was defined by loss of membrane integrity, cell death, and presence of cell-free granules. From transmission electron microscopy images, we observed only fibrinogen-exposed eosinophils undergoing this process. To our knowledge, this is the first report to show that fibrinogen is a specific trigger for cytolytic eosinophil degranulation with implications in human disease.

Super-resolution head and neck MRA using deep machine learning.

PURPOSE: To probe the feasibility of deep learning-based super-resolution (SR) reconstruction applied to nonenhanced MR angiography (MRA) of the head and neck. METHODS: High-resolution 3D thin-slab stack-of-stars quiescent interval slice-selective (QISS) MRA of the head and neck was obtained in eight subjects (seven healthy volunteers, one patient) at 3T. The spatial resolution of high-resolution ground-truth MRA data in the slice-encoding direction was reduced by factors of 2 to 6. Four deep neural network (DNN) SR reconstructions were applied, with two based on U-Net architectures (2D and 3D) and two (2D and 3D) consisting of serial convolutions with a residual connection. SR images were compared to ground-truth high-resolution data using Dice similarity coefficient (DSC), structural similarity index measure (SSIM), arterial diameter, and arterial sharpness measurements. Image review of the optimal DNN SR reconstruction was done by two experienced neuroradiologists. RESULTS: DNN SR of up to twofold and fourfold lower-resolution (LR) input volumes provided images that resembled those of the original high-resolution ground-truth volumes for intracranial and extracranial arterial segments, and improved DSC, SSIM, arterial diameters, and arterial sharpness relative to LR volumes (P < .001). The 3D DNN SR outperformed 2D DNN SR reconstruction. According to two neuroradiologists, 3D DNN SR reconstruction consistently improved image quality with respect to LR input volumes (P < .001). CONCLUSION: DNN-based SR reconstruction of 3D head and neck QISS MRA offers the potential for up to fourfold reduction in acquisition time for neck vessels without the need to commensurately sacrifice spatial resolution.

High spatial resolution whole-neck MR angiography using thin-slab stack-of-stars quiescent interval slice-selective acquisition.

PURPOSE: To report a 3D multi-echo thin-slab stack-of-stars (tsSOS) quiescent-interval slice-selective (QISS) strategy for high-resolution magnetic resonance angiography (MRA) of the entire neck in under seven minutes. METHODS: The neck arteries of eight subjects were imaged at 3 Tesla. Multi-echo 3D tsSOS QISS using a FLASH readout was compared with 3D tsSOS FLASH, 2D QISS, 2D TOF, and 3D TOF. A root-mean-square (RMS) combination of echo time images was tested. Evaluation metrics included arterial signal-to-noise ratio (SNR), arterial-to-muscle contrast-to-noise ratio (CNR), and image quality. RESULTS: 3D multi-echo tsSOS QISS using a RMS combination of echo time images increased SNR and CNR by 60% and 63% with respect to the reconstruction obtained with the shortest echo time. 3D tsSOS QISS showed superior CNR with respect to 3D tsSOS FLASH imaging, and more than 3-fold higher SNR and CNR with respect to 2D radial QISS when normalized for voxel size. 3D tsSOS QISS provided good to excellent image quality that exceeded the image quality of 2D QISS, 2D TOF, and 3D TOF (P < .05). CONCLUSION: Whole-neck high-resolution nonenhanced MRA is feasible using 3D tsSOS QISS, and produced image quality that exceeded those of competing nonenhanced MRA protocols at 3 Tesla.

Ungated nonenhanced radial quiescent interval slice-selective (QISS) magnetic resonance angiography of the neck: Evaluation of image quality.

BACKGROUND: Standard-of-care time-of-flight (TOF) techniques for nonenhanced magnetic resonance angiography (NEMRA) of the carotid bifurcation and other cervical arteries often provide nondiagnostic image quality due to motion and flow artifacts. PURPOSE: To perform an initial evaluation of an ungated radial quiescent-interval slice-selective (QISS) technique for NEMRA of the neck, in comparison with 2D TOF and contrast-enhanced magnetic resonance angiography (CEMRA). STUDY TYPE: Retrospective. POPULATION: Sixty patients referred for neck MR angiography. FIELD STRENGTH/SEQUENCE: Ungated radial QISS at 3T. ASSESSMENT: Three radiologists scored image quality of 18 arterial segments using a 4-point scale (1, nondiagnostic; 2, fair; 3, good; 4, excellent), and two radiologists graded proximal internal carotid stenosis using five categories (<50%, 50-69%, 70-99%, occlusion, nondiagnostic). STATISTICAL TESTS: Friedman tests with post-hoc Wilcoxon signed-rank tests; unweighted Gwet's AC1 statistic; tests for equality of proportions. RESULTS: Ungated radial QISS provided image quality that significantly exceeded 2D TOF (mean scores of 2.7 vs. 2.0, 2.7 vs. 2.2, and 2.9 vs. 2.3; P < 0.001, all comparisons), while CEMRA provided the best image quality (mean scores of 3.6, 3.7, and 3.5 for the three reviewers). Interrater agreement of image quality scores was substantial for CEMRA (AC1 = 0.70, P < 0.001), and moderate for QISS (AC1 = 0.43, P < 0.001) and TOF (AC1 = 0.41, P < 0.001). Compared with TOF, QISS NEMRA provided a significantly higher percentage of diagnostic segments for all three reviewers (91.0% vs. 71.7%, 93.5% vs. 72.9%, 95.5% vs. 85.2%; P < 0.0001) and demonstrated better agreement with CEMRA for grading of proximal internal carotid stenosis (AC1 = 0.94 vs. 0.73 for reviewer 1, P < 0.05; AC1 = 0.89 vs. 0.68 for reviewer 2, P < 0.05). DATA CONCLUSION: In this initial study, ungated radial QISS significantly outperformed 2D TOF for the evaluation of the neck arteries, with overall better image quality and more diagnostic arterial segments, and improved agreement with CEMRA for grading stenosis of the proximal internal carotid artery. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 1 J. Magn. Reson. Imaging 2019;50:1798-1807.

Mechanism for initiation of food allergy: Dependence on skin barrier mutations and environmental allergen costimulation.

BACKGROUND: Mechanisms for the development of food allergy in neonates are unknown but clearly linked in patient populations to a genetic predisposition to skin barrier defects. Whether skin barrier defects contribute functionally to development of food allergy is unknown. OBJECTIVE: The purpose of the study was to determine whether skin barrier mutations, which are primarily heterozygous in patient populations, contribute to the development of food allergy. METHODS: Mice heterozygous for the filaggrin (Flg)ft and Tmem79ma mutations were skin sensitized with environmental and food allergens. After sensitization, mice received oral challenge with food allergen, and then inflammation, inflammatory mediators, and anaphylaxis were measured. RESULTS: We define development of inflammation, inflammatory mediators, and food allergen-induced anaphylaxis in neonatal mice with skin barrier mutations after brief concurrent cutaneous exposure to food and environmental allergens. Moreover, neonates of allergic mothers have increased responses to suboptimal sensitization with food allergens. Importantly, responses to food allergens by these neonatal mice were dependent on genetic defects in skin barrier function and on exposure to environmental allergens. ST2 blockade during skin sensitization inhibited the development of anaphylaxis, antigen-specific IgE, and inflammatory mediators. Neonatal anaphylactic responses and antigen-specific IgE were also inhibited by oral pre-exposure to food allergen, but interestingly, this was blunted by concurrent pre-exposure of the skin to environmental allergen. CONCLUSION: These studies uncover mechanisms for food allergy sensitization and anaphylaxis in neonatal mice that are consistent with features of human early-life exposures and genetics in patients with clinical food allergy and demonstrate that changes in barrier function drive development of anaphylaxis to food allergen.

Nonenhanced hybridized arterial spin labeled magnetic resonance angiography of the extracranial carotid arteries using a fast low angle shot readout at 3 Tesla.

BACKGROUND: To evaluate ungated nonenhanced hybridized arterial spin labeling (hASL) magnetic resonance angiography (MRA) of the extracranial carotid arteries using a fast low angle shot (FLASH) readout at 3 Tesla. METHODS: In this retrospective, institutional review board-approved and HIPAA-compliant study, we evaluated the image quality (4-point scale) of nonenhanced hASL MRA using a FLASH readout with respect to contrast-enhanced MRA (CEMRA) in 37 patients presenting with neurologic symptoms. Two certified neuroradiologists independently evaluated 407 arterial segments (11 per patient) for image quality. The presence of vascular pathology was determined by consensus reading. Gwet's AC1 was used to assess inter-rater agreement in image quality scores, and image quality scores were correlated with age and body mass index. Objective measurements of arterial lumen area and sharpness in the carotid arteries were compared to values obtained with CEMRA. Comparisons were also made with conventional nonenhanced 2D time-of-flight (TOF) MRA. RESULTS: CEMRA provided the best image quality, while nonenhanced hASL FLASH MRA provided image quality that exceeded 2D TOF at the carotid bifurcation and in the internal and external carotid arteries. All nine vascular abnormalities of the carotid and intracranial arteries detected by CEMRA were depicted with hASL MRA, with no false positives. Inter-rater agreement of image quality scores was highest for CEMRA (AC1 = 0.87), followed by hASL (AC1 = 0.61) and TOF (AC1 = 0.43) (P < 0.001, all comparisons). With respect to CEMRA, agreement in cross-sectional lumen area was significantly better with hASL than TOF in the common carotid artery (intraclass correlation (ICC) = 0.90 versus 0.66; P < 0.05) and at the carotid bifurcation (ICC = 0.87 versus 0.54; P < 0.05). Nonenhanced hASL MRA provided superior arterial sharpness with respect to CEMRA and 2D TOF (P < 0.001). CONCLUSION: Although inferior to CEMRA in terms of image quality and inter-rater agreement, hASL FLASH MRA offers an alternative to 2D TOF for the nonenhanced evaluation of the extracranial carotid arteries at 3 Tesla. Compared with 2D TOF, nonenhanced hASL FLASH MRA provides improved quantification of arterial cross-sectional area, vessel sharpness, inter-rater agreement and image quality.

5-HTP inhibits eosinophilia via intracellular endothelial 5-HTRs; SNPs in 5-HTRs associate with asthmatic lung function.

Background: Previous research showed that 5-hydroxytryptophan (5HTP), a metabolic precursor of serotonin, reduces allergic lung inflammation by inhibiting eosinophil migration across endothelial monolayers. Objective: It is unknown if serotonin receptors are involved in mediating this 5HTP function or if serotonin receptor (HTR) single nucleotide polymorphisms (SNPs) associate with lung function in humans. Methods: Serotonin receptor subtypes were assessed by qPCR, western blot, confocal microscopy, pharmacological inhibitors and siRNA knockdown. HTR SNPs were assessed in two cohorts. Results: Pharmacological inhibition or siRNA knockdown of the serotonin receptors HTR1A or HTR1B in endothelial cells abrogated the inhibitory effects of 5HTP on eosinophil transendothelial migration. In contrast, eosinophil transendothelial migration was not inhibited by siRNA knockdown of HTR1A or HTR1B in eosinophils. Surprisingly, these HTRs were intracellular in endothelial cells and an extracellular supplementation with serotonin did not inhibit eosinophil transendothelial migration. This is consistent with the inability of serotonin to cross membranes, the lack of selective serotonin reuptake receptors on endothelial cells, and the studies showing minimal impact of selective serotonin reuptake inhibitors on asthma. To extend our HTR studies to humans with asthma, we examined the CHIRAH and GALA cohorts for HTR SNPs that affect HTR function or are associated with behavior disorders. A polygenic index of SNPs in HTRs was associated with lower lung function in asthmatics. Conclusions: Serotonin receptors mediate 5HTP inhibition of transendothelial migration and HTR SNPs associate with lower lung function. These results may serve to aid in design of novel interventions for allergic inflammation.

More than neutrophils: Lin(+)Ly6G(+)IL-5Rα(+) multipotent myeloid cells (MMCs) are dominant in normal murine bone marrow and retain capacity to differentiate into eosinophils and monocytes.

Bone marrow is a hematopoietic site harboring multiple populations of myeloid cells in different stages of differentiation. Murine bone marrow eosinophils are traditionally identified by Siglec-F(+) staining using flow cytometry, whereas neutrophils are characterized by Ly6G(+) expression. However, using flow cytometry to characterize bone marrow hematopoietic cells in wild-type mice, we found substantial gray areas in identification of these cells. Siglec-F(+) mature eosinophil population constituted only a minority of bone marrow Lin(+)CD45(+) pool (5%). A substantial population of Siglec-F(-) cells was double positive for neutrophil marker Ly6G and eosinophil lineage marker, IL-5Rα. This granulocyte population with mixed neutrophil and eosinophil characteristics is typically attributable to neutrophil pool based on neutral granule staining and expression of Ly6G and myeloid peroxidase. It is distinct from Lineage(-) myeloid progenitors or Siglec-F(+)Ly6G(+) maturing eosinophil precursors, and can be accurately identified by Lineage(+) staining and positive expression of markers IL-5Rα and Ly6G. At 15-50% of all CD45(+) hematopoietic cells in adult mice (percentage varies by sex and age), this is a surprisingly dominant population, which increases with age in both male and female mice. RNA-seq characterization of these cells revealed a complex immune profile and the capacity to secrete constituents of the extracellular matrix. When sorted from bone marrow, these resident cells had neutrophilic phenotype but readily acquired all characteristics of eosinophils when cultured with G-CSF or IL-5, including expression of Siglec-F and granular proteins (Epx, Mbp). Surprisingly, these cells were also able to differentiate into Ly6C(+) monocytes when cultured with M-CSF. Herein described is the discovery of an unexpected hematopoietic flexibility of a dominant population of multipotent myeloid cells, typically categorized as neutrophils, but with the previously unknown plasticity to contribute to mature pools of eosinophils and monocytes.

Beyond Il-5: Metabolic Reprogramming and Stromal Support Are Prerequisite for Generation and Survival of Long-Lived Eosinophil.

Eosinophils play surprisingly diverse roles in health and disease. Accordingly, we have now begun to appreciate the scope of the functional and phenotypic heterogeneity and plasticity of these cells. Along with tissue-recruited subsets during inflammation, there are tissue resident eosinophil phenotypes with potentially longer life spans and less dependency on IL-5 for survival. Current models to study murine eosinophils ex vivo rely on IL-5-sustained expansion of eosinophils from bone marrow hematopoietic progenitors. Although it does generate eosinophils (bmEos) in high purity, such systems are short-lived (14 days on average) and depend on IL-5. In this report, we present a novel method of differentiating large numbers of pure bone marrow-derived eosinophils with a long-lived phenotype (llEos) (40 days on average) that require IL-5 for initial differentiation, but not for subsequent survival. We identified two key factors in the development of llEos: metabolic adaptation and reprogramming induced by suppressed nutrient intake during active differentiation (from Day 7 of culture), and interaction with IL-5-primed stromal cells for the remainder of the protocol. This regimen results in a higher yield and viability of mature eosinophils. Phenotypically, llEos develop as Siglec-F(+)Ly6G(+) cells transitioning to Siglec-F(+) only, and exhibit typical eosinophil features with red eosin granular staining, as well as the ability to chemotax to eotaxin Ccl11 and process fibrinogen. This culture system requires less reagent input and allows us to study eosinophils long-term, which is a significant improvement over IL-5-driven differentiation protocols. Moreover, it provides important insights into factors governing eosinophil plasticity and the ability to assume long-lived IL-5-independent phenotypes.

ß-Glucosylceramide From Allergic Mothers Enhances Offspring Responsiveness to Allergen.

In animals and humans, offspring of allergic mothers have increased responsiveness to allergen and the allergen-specificity of the offspring can be different than that of the mother. In our preclinical models, the mother's allergic responses influence development of the fetus and offspring by elevating numbers of cells in dendritic cell subsets. A major question is the identity of maternal factors of allergic mothers that alter offspring development of responsiveness to allergen. Lipids are altered during allergic responses and lipids are transported to the fetus for growth and formation of fetal membranes. We hypothesized that pro-inflammatory lipids, that are elevated in allergic mothers, are transported to the fetus and regulate fetal immune development. We demonstrate in this report that there was a significant 2-fold increase in ß-glucosylceramides (ßGlcCer) in allergic mothers, the fetal liver and her offspring. The ßGlcCer were transported from mother's plasma, across the placenta, to the fetus and in breastmilk to the offspring. Administration of ßGlcCer to non-allergic mothers was sufficient for offspring responses to allergen. Importantly, maternal administration of a clinically relevant pharmacological inhibitor of ßGlcCer synthase returned ßGlcCer to normal levels in the allergic mothers and her offspring and blocked the offspring increase in dendritic cell subsets and offspring allergen responsiveness. In summary, allergic mothers had increased ßGlcCer that was transported to offspring and mediated increases in offspring DCs and responsiveness to allergen. These data have a significant impact on our understanding of mechanisms for development of allergies in offspring of allergic mothers and have the potential to lead to novel interventions that significantly impact risk for allergic disease early in life.

Altered selection on a single self-ligand promotes susceptibility to organ-specific T cell infiltration.

For the large array of self-peptide/MHC class II (pMHC-II) complexes displayed in the body, it is unclear whether CD4+ T cell tolerance must be imparted for each individual complex or whether pMHC-II-nonspecific bystander mechanisms are sufficient to confer tolerance by acting broadly on T cells reactive to multiple self-pMHC-II ligands. Here, via reconstitution of T cell-deficient mice, we demonstrate that altered T cell selection on a single prostate-specific self-pMHC-II ligand renders recipient mice susceptible to prostate-specific T cell infiltration. Mechanistically, this self-pMHC-II complex is required for directing antigen-specific cells into the Foxp3+ regulatory T cell lineage but does not induce clonal deletion to a measurable extent. Thus, our data demonstrate that polyclonal T reg cells are unable to functionally compensate for a breach in tolerance to a single self-pMHC-II complex in this setting, revealing vulnerabilities in antigen-nonspecific bystander mechanisms of immune tolerance.

Effector T cell responses unleashed by regulatory T cell ablation exacerbate oral squamous cell carcinoma.

Immune suppression by CD4+FOXP3+ regulatory T (Treg) cells and tumor infiltration by CD8+ effector T cells represent two major factors impacting response to cancer immunotherapy. Using deconvolution-based transcriptional profiling of human papilloma virus (HPV)-negative oral squamous cell carcinomas (OSCCs) and other solid cancers, we demonstrate that the density of Treg cells does not correlate with that of CD8+ T cells in many tumors, revealing polarized clusters enriched for either CD8+ T cells or CD4+ Treg and conventional T cells. In a mouse model of carcinogen-induced OSCC characterized by CD4+ T cell enrichment, late-stage Treg cell ablation triggers increased densities of both CD4+ and CD8+ effector T cells within oral lesions. Notably, this intervention does not induce tumor regression but instead induces rapid emergence of invasive OSCCs via an effector T cell-dependent process. Thus, induction of a T cell-inflamed phenotype via therapeutic manipulation of Treg cells may trigger unexpected tumor-promoting effects in OSCC.

Novel inhibitors complexed with glutamate dehydrogenase: allosteric regulation by control of protein dynamics.

Mammalian glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate to 2-oxoglutarate using NAD(P)(+) as coenzyme. Unlike its counterparts from other animal kingdoms, mammalian GDH is regulated by a host of ligands. The recently discovered hyperinsulinism/hyperammonemia disorder showed that the loss of allosteric inhibition of GDH by GTP causes excessive secretion of insulin. Subsequent studies demonstrated that wild-type and hyperinsulinemia/hyperammonemia forms of GDH are inhibited by the green tea polyphenols, epigallocatechin gallate and epicatechin gallate. This was followed by high throughput studies that identified more stable inhibitors, including hexachlorophene, GW5074, and bithionol. Shown here are the structures of GDH complexed with these three compounds. Hexachlorophene forms a ring around the internal cavity in GDH through aromatic stacking interactions between the drug and GDH as well as between the drug molecules themselves. In contrast, GW5074 and bithionol both bind as pairs of stacked compounds at hexameric 2-fold axes between the dimers of subunits. The internal core of GDH contracts when the catalytic cleft closes during enzymatic turnover. None of the drugs cause conformational changes in the contact residues, but all bind to key interfaces involved in this contraction process. Therefore, it seems likely that the drugs inhibit enzymatic turnover by inhibiting this transition. Indeed, this expansion/contraction process may play a major role in the inter-subunit communication and allosteric regulation observed in GDH.

Quantitative cerebral MR perfusion imaging: preliminary results in stroke.

PURPOSE: To evaluate quantitative cerebral blood flow (qCBF) with traditional time-based measurements or metrics of cerebral perfusion: time to peak (Tmax) and mean transit time (MTT) in stroke patients. MATERIALS AND METHODS: Nine ischemic stroke patients (four male, five female, 63 ± 16 years old) were included in the study which was Health Insurance Portability and Accountability Act compliant and institutional review board approved. Cerebral perfusion was quantified using the Bookend method. Mean values of qCBF, Tmax, and MTT were determined in regions of interest (ROIs). ROIs were drawn on diffusion weighted images in diffusion positive, critically ischemic (CI), in ipsilateral normal region immediately surrounding the critically ischemic region, the presumed penumbra (PP), and in contralateral diffusion negative control, presumed normal region (PN) of gray and white matter separately (GM and WM). RESULTS: In both GM and WM, qCBF measures distinguished the studied brain regions with the most markedly reduced values in regions corresponding to extent of likely ischemic injury. In planned comparisons, only qCBF measurements differed significantly between CI and PP tissues. ROC analysis supported the utility of qCBF for discriminating brain regions differing in the likely extent of ischemic injury (CI and PN regions - qCBF: area under the curve [AUC] = 0.96, Tmax: AUC = 0.96, MTT: AUC = 0.72). Importantly, qCBF afforded the best discrimination of CI and PP regions (qCBF: AUC = 0.82, Tmax: AUC = 0.65, MTT: AUC = 0.52). CONCLUSION: This initial evaluation indicates that quantitative MRI perfusion is feasible in ischemic stroke patients. qCBF derived with this strategy provide enhanced discrimination of CI and PP compared to time-based imaging metrics. This approach merits investigation in larger clinical studies.

Eosinophil accumulation in postnatal lung is specific to the primary septation phase of development.

Type 2 immune cells and eosinophils are transiently present in the lung tissue not only in pathology (allergic disease, parasite expulsion) but also during normal postnatal development. However, the lung developmental processes underlying airway recruitment of eosinophils after birth remain unexplored. We determined that in mice, mature eosinophils are transiently recruited to the lung during postnatal days 3-14, which specifically corresponds to the primary septation/alveolarization phase of lung development. Developmental eosinophils peaked during P10-14 and exhibited Siglec-Fmed/highCD11c-/low phenotypes, similar to allergic asthma models. By interrogating the lung transcriptome and proteome during peak eosinophil recruitment in postnatal development, we identified markers that functionally capture the establishment of the mesenchymal-epithelial interface (Nes, Smo, Wnt5a, Nog) and the deposition of the provisional extracellular matrix (ECM) (Tnc, Postn, Spon2, Thbs2) as a key lung morphogenetic event associating with eosinophils. Tenascin-C (TNC) was identified as one of the key ECM markers in the lung epithelial-mesenchymal interface both at the RNA and protein levels, consistently associating with eosinophils in development and disease in mice and humans. As determined by RNA-seq analysis, naïve murine eosinophils cultured with ECM enriched in TNC significantly induced expression of Siglec-F, CD11c, eosinophil peroxidase, and other markers typical for activated eosinophils in development and allergic inflammatory responses. TNC knockout mice had an altered eosinophil recruitment profile in development. Collectively, our results indicate that lung morphogenetic processes associated with heightened Type 2 immunity are not merely a tissue "background" but specifically guide immune cells both in development and pathology.

Acquired Chiari malformation Type I associated with a supratentorial arteriovenous malformation. Case report and review of the literature.

Chiari malformation Type I (CM-I), a condition defined by caudal descent of the cerebellar tonsils through the foramen magnum, is generally considered a congenital lesion. Several authors, however, have described an acquired form that appears identical to the congenital lesion on neuroimages. The most commonly reported cause of an acquired CM-I is cerebrospinal fluid diversion through a lumboperitoneal shunt. In this paper, the authors report the case of a patient in whom an acquired CM-I developed in association with a supratentorial arteriovenous malformation (AVM) of the brain. Development of the acquired CM was documented on serial magnetic resonance images. Moreover, the CM was seen to originate and worsen in concert with the clinicoradiological progression of the AVM. The underlying mechanism responsible for the acquired CM in this case is thought to be a high-flow venopathy of the transverse and sigmoid sinuses causing occlusion on the right and redirection of venous outflow into posterior fossa veins, with consequent venous congestion and swelling of the posterior fossa structures.

The role of computerized tomography-myelography in a patient with spontaneous intracranial hypotension from multiple cerebrospinal fluid leaks.

OBJECTIVES: To describe a patient with spontaneous intracranial hypotension (SIH) secondary to multiple sites of cerebrospinal fluid (CSF) leaks, a rarely described phenomenon. To compare computerized tomography-myelography with radionuclide cisternography as confirmatory diagnostic aids in SIH. METHODS: A patient with SIH had transient or no response to 2 thoracic epidural blood patches. A computerized tomography-myelography showed bilateral CSF leaks at T11-T12 and T12-L1 levels and on the left side of T10-T11. RESULTS: A left paramedian epidural blood patch was performed at T12-L1 under fluoroscopy guidance. Injection of 1 mL dye showed confinement of the dye at the left T11-L1 epidural space. The patient had 90% to 95% relief of her headache and complete relief at 4 months. She was asymptomatic 1 year after the last epidural blood patch. CONCLUSIONS: In a patient with SIH, a computerized tomography-myelography should be performed if an initial epidural blood patch is ineffective. This is to show the vertebral level and site(s) of CSF leak and to guide the physician to the ideal vertebral level for the epidural blood patch.