PD‐1 modulates steady‐state and infection‐induced IL‐10 production in vivo

Programmed death‐1 (PD‐1) plays an important role in mediating immune tolerance through mechanisms that remain unclear. Herein, we investigated whether PD‐1 prevents excessive host tissue damage during infection with the protozoan parasite, Toxoplasma gondii. Surprisingly, our results demonstrate that PD‐1‐deficient mice have increased susceptibility to T. gondii, with increased parasite cyst counts along with reduced type‐1 cytokine responses (IL‐12 and IFN‐γ). PD‐1^?/? DCs showed no cell intrinsic defect in IL‐12 production in vitro. Instead, PD‐1 neutralization via genetic or pharmacological approaches resulted in a striking increase in IL‐10 release, which impaired type‐1‐inflammation during infection. Our results indicate that the absence of PD‐1 increases IL‐10 production even in the absence of infection. Although the possibility that such increased IL‐10 protects against autoimmune damage is speculative, our results show that IL‐10 suppresses the development of protective Th1 immune response after T. gondii infection.     

Motion‐compensated b‐tensor encoding for in vivo cardiac diffusion‐weighted imaging

Motion is a major confound in diffusion‐weighted imaging (DWI) in the body, and it is a common cause of image artefacts. The effects are particularly severe in cardiac applications, due to the nonrigid cyclical deformation of the myocardium. Spin echo‐based DWI commonly employs gradient moment‐nulling techniques to desensitise the acquisition to velocity and acceleration, ie, nulling gradient moments up to the 2nd order (M2‐nulled). However, current M2‐nulled DWI scans are limited to encode diffusion along a single direction at a time. We propose a method for designing b‐tensors of arbitrary shapes, including planar, spherical, prolate and oblate tensors, while nulling gradient moments up to the 2nd order and beyond. The design strategy comprises initialising the diffusion encoding gradients in two encoding blocks about the refocusing pulse, followed by appropriate scaling and rotation, which further enables nulling undesired effects of concomitant gradients. Proof‐of‐concept assessment of in vivo mean diffusivity (MD) was performed using linear and spherical tensor encoding (LTE and STE, respectively) in the hearts of five healthy volunteers. The results of the M2‐nulled STE showed that (a) the sequence was robust to cardiac motion, and (b) MD was higher than that acquired using standard M2‐nulled LTE, where diffusion‐weighting was applied in three orthogonal directions, which may be attributed to the presence of restricted diffusion and microscopic diffusion anisotropy. Provided adequate signal‐to‐noise ratio, STE could significantly shorten estimation of MD compared with the conventional LTE approach. Importantly, our theoretical analysis and the proposed gradient waveform design may be useful in microstructure imaging beyond diffusion tensor imaging where the effects of motion must be suppressed.     

Localized singlet‐filtered MRS in vivo

MR is a prominent technology to investigate diseases, with millions of clinical procedures performed every year. Metabolic dysfunction is one common aspect associated with many diseases. Thus, understanding and monitoring metabolic changes is essential to develop cures for many illnesses, including for example cancer and neurodegeneration. MR methodologies are especially suited to study endogenous metabolites and processes within an organism in vivo, which has led to many insights about physiological functions. Advancing metabolic MR techniques is therefore key to further understand physiological processes. Here, we introduce an approach based on nuclear spin singlet states to specifically filter metabolic signals and particularly show that singlet‐filtered glutamate can be observed distinctly in the hippocampus of a living mouse in vivo. This development opens opportunities to make use of the singlet spin phenomenon in vivo and besides its use as a filter to provide scope for new contrast agents.     

IL‐23‐driven encephalo‐tropism and Th17 polarization during CNS‐inflammation in vivo

IL‐23 but not IL‐12 is essential for the development of autoimmune tissue inflammation in mice. Conversely, IL‐12 and IL‐23 impact on the polarization of Th1 and Th17 cells, respectively. While both polarized T helper populations can mediate autoimmune inflammation, their redundancy in the pathogenesis of EAE indicates that IL‐23 exerts its crucial influence on the disease independent of its T helper polarizing capacity. To study the impact of IL‐23 and IL‐12 on the behavior of encephalitogenic T cells in vivo, we generated BM‐chimeric mice in which we can trace individual populations of IL‐23 or IL‐12 responsive T helper cells during EAE. We observed that T cells, which lack IL‐12Rβ1 (no IL‐12 and IL‐23 signaling), fail to invade the CNS and do not acquire a Th17 phenotype. In contrast, loss of IL‐12 signaling prevents Th1 polarization but does not prevent T‐cell entry into the CNS. The loss of IL‐12R engagement does not appear to alter T‐cell expansion but leads to their accumulation in secondary lymphoid organs. We found that IL‐23 licenses T cells to invade the target tissue and to exert their effector function, whereas IL‐12 is critical for Th1 differentiation, but does not influence the pathogenic capacity of auto‐reactive T helper cells in vivo.     

B cell tolerance to epidermal ribonuclear‐associated neo‐autoantigen in vivo

Defining how self‐antigens are perceived by the immune system is pivotal to understand how tolerance is maintained under homeostatic conditions. Clinically relevant, natural autoantigens targeted by autoantibodies, in e.g. systemic lupus erythematosus (SLE), commonly have an intrinsic ability to engage not only the B cell receptor (BCR), but also a co‐stimulatory pathway in B cells, such as the Toll‐like receptor (TLR)‐7 pathway. Here we developed a novel mouse model displaying inducible expression of a fluorescent epidermal neo‐autoantigen carrying an OT‐II T cell epitope, B cell antigen and associated ribonucleic acids capable of stimulating TLR‐7. The neo‐autoantigen was expressed in skin, but did not drain in intact form into draining lymph nodes, even after ultraviolet B (UVB)‐stimulated induction of apoptosis in the basal layer. Adoptively transferred autoreactive B cells were excluded follicularly and perished at the T–B border in the spleen, preventing their recirculation and encounter with antigen peripherally. This transitional check‐point was bypassed by crossing the reporter to a BCR knock‐in line on a C4‐deficient background. Adoptively transferred OT‐II T cells homed rapidly into cutaneous lymph nodes and up‐regulated CD69. Surprisingly, however, tolerance was not broken, as the T cells subsequently down‐regulated activation markers and contracted. Our results highlight how sequestration of intracellular and peripheral antigen, the transitional B cell tolerance check‐point and T cell regulation co‐operate to maintain immunological tolerance in vivo.     

High‐resolution in vivo MR‐STAT using a matrix‐free and parallelized reconstruction algorithm

MR‐STAT is a recently proposed framework that allows the reconstruction of multiple quantitative parameter maps from a single short scan by performing spatial localisation and parameter estimation on the time‐domain data simultaneously, without relying on the fast Fourier transform (FFT). To do this at high resolution, specialized algorithms are required to solve the underlying large‐scale nonlinear optimisation problem. We propose a matrix‐free and parallelized inexact Gauss–Newton based reconstruction algorithm for this purpose. The proposed algorithm is implemented on a high‐performance computing cluster and is demonstrated to be able to generate high‐resolution (1 mm 1 mm in‐plane resolution) quantitative parameter maps in simulation, phantom, and in vivo brain experiments. Reconstructed and values for the gel phantoms are in agreement with results from gold standard measurements and, for the in vivo experiments, the quantitative values show good agreement with literature values. In all experiments, short pulse sequences with robust Cartesian sampling are used, for which MR fingerprinting reconstructions are shown to fail.     

Commensal microflora and interferon‐γ promote steady‐state interleukin‐7 production in vivo

IL‐7 is a major regulator of lymphocyte homeostasis; however, little is known about the mechanisms that regulate IL‐7 production. To study Il7 gene regulation in vivo, we generated a novel IL‐7‐reporter mouse, which allows the non‐invasive quantification of Il7 gene activity in live mice and, additionally, the simultaneous activation/inactivation of target genes in IL‐7‐producing cells. With these IL‐7‐reporter mice, we identify thymus, skin and intestine as major sources of IL‐7 in vivo. Importantly, we show that IFN‐γ and the commensal microflora promote steady‐state IL‐7 production in the intestine. Furthermore, we demonstrate that the blockade of IFN‐γ signaling in intestinal epithelial cells strongly reduces their IFN‐γ‐driven IL‐7 production. In summary, our data suggest a feedback loop in which commensal bacteria drive IFN‐γ production by lymphocytes, which in turn promotes epithelial cell IL‐7 production and the survival of IL‐7‐dependent lymphocytes.     

Non‐invasive detection of glycine as a biomarker of malignancy in childhood brain tumours using in‐vivo 1H MRS at 1.5 Tesla confirmed by ex‐vivo high‐resolution magic‐angle spinning NMR

Management of brain tumours in children would benefit from improved non‐invasive diagnosis, characterisation and prognostic biomarkers. Metabolite profiles derived from in‐vivo MRS have been shown to provide such information. Studies indicate that using optimum a priori information on metabolite contents in the construction of linear combination (LC) models of MR spectra leads to improved metabolite profile estimation. Glycine (Gly) is usually neglected in such models due to strong overlap with myo‐inositol (mI) and a low concentration in normal brain. However, biological studies indicate that Gly is abundant in high‐grade brain tumours. This study aimed to investigate the quantitation of Gly in paediatric brain tumours using MRS analysed by LCModel?, and its potential as a non‐invasive biomarker of malignancy. Single‐voxel MRS was performed using PRESS (TR 1500 ms, TE 30 ms/135 ms) on a 1.5 T scanner. Forty‐seven cases (18 high grade (HG), 17 low grade (LG), 12 ungraded) were retrospectively selected if both short‐TE and long‐TE MRS (n = 33) or short‐TE MRS and high‐resolution magic‐angle spinning (HRMAS) of matched surgical samples (n = 15) were available. The inclusion of Gly in LCModel? analyses led to significantly reduced fit residues for both short‐TE and long‐TE MRS (p < 0.05). The Gly concentrations estimated from short‐TE MRS were significantly correlated with the long‐TE values (R = 0.91, p < 0.001). The Gly concentration estimated by LCModel? was significantly higher in HG versus LG tumours for both short‐TE (p < 1e‐6) and long‐TE (p = 0.003) MRS. This was consistent with the HRMAS results, which showed a significantly higher normalised Gly concentration in HG tumours (p < 0.05) and a significant correlation with the normalised Gly concentration measured from short‐TE in‐vivo MRS (p < 0.05). This study suggests that glycine can be reliably detected in paediatric brain tumours using in‐vivo MRS on standard clinical scanners and that it is a promising biomarker of tumour aggressiveness. Copyright © 2009 John Wiley & Sons, Ltd.     

Endothelium‐dependent responses in the microcirculation observed in vivo

Endothelium‐dependent responses were first demonstrated 40 years ago in the aorta. Since then, extensive research has been conducted in vitro using conductance vessels and materials derived from them. However, the microcirculation controls blood flow to vital organs and has been the focus of in vivo studies of endothelium‐dependent dilation beginning immediately after the first in vitro report. Initial in vivo studies employed a light/dye technique for selectively damaging the endothelium to unequivocally prove, in vivo, the existence of endothelium‐dependent dilation and in the microvasculature. Endothelium‐dependent constriction was similarly proven. Endothelium‐dependent agonists include acetylcholine (ACh), bradykinin, arachidonic acid, calcium ionophore A‐23187, calcitonin gene‐related peptide (CGRP), serotonin, histamine and endothelin‐1. Normal and disease states have been studied. Endothelial nitric oxide synthase, cyclooxygenase and cytochrome P450 have been shown to generate the mediators of the responses. Some of the key enzyme systems generate reactive oxygen species (ROS) like superoxide which may prevent EDR. However, one ROS, namely H₂O₂, is one of a number of hyperpolarizing factors that cause dilation initiated by endothelium. Depending upon microvascular bed, a single agonist may use different pathways to elicit an endothelium‐dependent response. Interpretation of studies using inhibitors of eNOS is complicated by the fact that these inhibitors may also inhibit ATP‐sensitive potassium channels. Other in vivo observations of brain arterioles failed to establish nitric oxide as the mediator of responses elicited by CGRP or by ACh and suggest that a nitrosothiol may be a better fit for the latter.     

An Overhauser‐enhanced‐MRI platform for dynamic free radical imaging in vivo

Overhauser‐enhanced MRI (OMRI) is an electron‐proton double‐resonance imaging technique of interest for its ability to non‐invasively measure the concentration and distribution of free radicals. In vivo OMRI experiments are typically undertaken at ultra‐low magnetic field (ULF), as both RF power absorption and penetration issues—a consequence of the high resonance frequencies of electron spins—are mitigated. However, working at ULF causes a drastic reduction in MRI sensitivity. Here, we report on the design, construction and performance of an OMRI platform optimized for high NMR sensitivity and low RF power absorbance, exploring challenges unique to probe design in the ULF regime. We use this platform to demonstrate dynamic imaging of TEMPOL in a rat model. The work presented here demonstrates improved speed and sensitivity of in vivo OMRI, extending the scope of OMRI to the study of dynamic processes such as metabolism.     

Boosting of post‐exposure human T‐cell and B‐cell recall responses in vivo by Burkholderia pseudomallei‐related proteins

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease with high incidence and mortality in South East Asia and northern Australia. To date there is no protective vaccine and antibiotic treatment is prolonged and not always effective. Most people living in endemic areas have been exposed to the bacteria and have developed some immunity, which may have helped to prevent disease. Here, we used a humanized mouse model (hu‐PBL‐SCID), reconstituted with human peripheral blood mononuclear cells from seropositive donors, to illustrate the potential of three known antigens (FliC, OmpA and N‐PilO2) for boosting both T‐cell and B‐cell immune responses. All three antigens boosted the production of specific antibodies in vivo, and increased the number of antibody and interferon‐γ‐secreting cells, and induced antibody affinity maturation. Moreover, antigen‐specific antibodies isolated from either seropositive individuals or boosted mice, were found to enhance phagocytosis and oxidative burst activities from human polymorphonuclear cells. Our study demonstrates that FliC, OmpA and N‐PilO2 can stimulate human memory T and B cells and highlight the potential of the hu‐PBL‐SCID system for screening and evaluation of novel protein antigens for inclusion in future vaccine trials against melioidosis.     

Diploic venous anatomy studied in‐vivo by MRI

Calvarial diploic venous anatomy has been studied post‐mortem, but few studies have addressed these venous structures in‐vivo. Previous work in our laboratory has shown that intraosseous infusion through the skull diploic space near the diploic veins in animals and humans does access the superior sagittal sinus and the systemic venous system. We developed a volumetric method of imaging the diploic veins in‐vivo using MRI, intravenous gadolinium, and digital subtraction to provide for three‐dimensional depiction and exact localization of these veins. We hypothesized that this technique would allow for an assessment of the probability of existence, distribution, and concentration of diploic veins in the skull. We scanned 31 neurosurgical patients, and were able to create 3D diploic venous maps in 74% of them. These maps were processed using Adobe Photoshop CS2. Mathworks MatLab 6.5, once customized, counted the number of pixels occupied by the diploic veins in the processed image. The probability of veins was highest in the occipital regions (100%). The inferior occipital (4.1%) and posterior parietal (4.1%) regions had the highest concentrations of diploic veins. Digital subtraction venography using a volumetric MRI sequence can demonstrate the diploic veins in‐vivo. The inferior occipital region may be the best area for an intraosseous infusion device because it has the greatest likelihood of containing a vein and also has the highest concentration of veins. Clin. Anat. 22:296–301, 2009. © 2009 Wiley‐Liss, Inc.     

Assessment of 5‐fluorouracil and 4‐nitroquinoline‐1‐oxide in vivo genotoxicity with Pig‐a mutation and micronucleus endpoints

Genotoxicity assessments were conducted on male Sprague Dawley rats treated with 5‐fluorouracil (5‐FU) and 4‐nitroquinoline‐1–oxide (4NQO) as part of an international validation trial of the Pig‐a mutant phenotype assay. Rats were orally exposed to 0, 11.5, 23, or 46 mg/kg/day 5‐FU for three consecutive days (Days 1–3); blood was sampled on Days ?1, 4, 15, 29, and 45. Pig‐a mutant phenotype reticulocyte (RET^CD59?) and mutant phenotype erythrocyte (RBC^CD59?) frequencies were determined on Days ?1, 15, 29, and 45, and percent micronucleated reticulocytes (%MN‐RET) were measured on Day 4. Rats were treated with 4NQO for 28 consecutive days by oral gavage, at doses of 1.5, 3, or 6 mg/kg/day. RBC^CD59? and RET^CD59? frequencies were determined on Days ?1, 15, and 29, and MN‐RET were quantified on Day 29. Whereas 5‐FU was found to increase %MN‐RET, no significant increases were observed for RBC^CD59? or RET^CD59? at any of the time points studied. The high dose of 4NQO (6 mg/kg/day) was observed to markedly increase RBC^CD59? and RET^CD59? frequencies, and this same dose level caused a weak but significantly elevated increase in MN‐RET (approximately twofold). Collectively, the results provide additional support for the combination of Pig‐a mutation and MN‐RET into acute and 28‐day repeat‐dose studies. Environ. Mol. Mutagen. 55:735–740, 2014. © 2014 Wiley Periodicals, Inc.     

The in vivo J‐difference editing MEGA‐PRESS technique for the detection of n‐3 fatty acids

In this study, we present a method for the detection of n‐3 fatty acid (n‐3 FA) signals using MRS in adipose tissue in vivo. This method (called oMEGA‐PRESS) is based on the selective detection of the CH₃ signal of n‐3 FA using the MEGA‐PRESS (MEshcher–GArwood Point‐RESolved Spectroscopy) J‐difference editing technique. We optimized the envelope shape and frequency of spectral editing pulses to minimize the spurious co‐editing and incomplete subtraction of the CH₃ signal of other FAs, which normally obscure the n‐3 FA CH₃ signal in MR spectra acquired using standard PRESS techniques. The post‐processing of the individual data scans with the phase and frequency correction before data subtraction and averaging was implemented to further improve the quality of in vivo spectra. The technique was optimized in vitro on lipid phantoms using various concentrations of n‐3 FA and examined in vivo at 3 T on 15 healthy volunteers. The proportion of n‐3 FA estimated by the oMEGA‐PRESS method in phantoms showed a highly significant linear correlation with the n‐3 FA content determined by gas chromatography. The signal attributed to n‐3 FA was observed in all subjects. Comparisons with the standard PRESS technique revealed an enhanced identification of the n‐3 FA signal using oMEGA‐PRESS. The presented method may be useful for the non‐invasive quantification of n‐3 FA in adipose tissue, and could aid in obtaining a better understanding of various aspects of n‐3 FA metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo targeting of human DC‐SIGN drastically enhances CD8+ T‐cell‐mediated protective immunity

Vaccination is one of the oldest yet still most effective methods to prevent infectious diseases. However, eradication of intracellular pathogens and treatment of certain diseases like cancer requiring efficient cytotoxic immune responses remain a medical challenge. In mice, a successful approach to induce strong cytotoxic CD8⁺ T‐cell (CTL) reactions is to target antigens to DCs using specific antibodies against surface receptors in combination with adjuvants. A major drawback for translating this strategy into one for the clinic is the lack of analogous targets in human DCs. DC‐SIGN (DC‐specific‐ICAM3‐grabbing‐nonintegrin/CD209) is a C‐type lectin receptor with potent endocytic capacity and a highly restricted expression on human immature DCs. Therefore, DC‐SIGN represents an ideal candidate for DC targeting. Using transgenic mice that express human DC‐SIGN under the control of the murine CD11c promoter (hSIGN mice), we explored the efficacy of anti‐DC‐SIGN antibodies to target antigens to DCs and induce protective immune responses in vivo. We show that anti‐DC‐SIGN antibodies conjugated to OVA induced strong and persistent antigen‐specific CD4⁺ and CD8⁺ T‐cell responses, which efficiently protected from infection with OVA‐expressing Listeria monocytogenes. Thus, we propose DC targeting via DC‐SIGN as a promising strategy for novel vaccination protocols against intracellular pathogens.     

Radiation‐induced bystander effects in vivo are epigenetically regulated in a tissue‐specific manner

Exposure of animal body parts to ionizing radiation (IR) can lead to molecular changes in distant shielded “bystander” tissues and organs. Nevertheless, tissue specificity of bystander responses within the same organism has not been examined in detail. Studies on in vivo bystander effect conducted so far analyzed changes induced by single‐dose exposure. The potential of fractionated irradiation to induce bystander effects in vivo has never been studied. We analyzed changes in global DNA methylation and microRNAome in skin and spleen of animals subjected to single‐dose (acute or fractionated) whole‐body or cranial exposure to 0.5 Gy of X‐rays. We found that IR‐induced DNA methylation changes in bystander spleen and skin were distinct. Acute radiation exposure resulted in a significant loss of global DNA methylation in the exposed and bystander spleen 6 hr, 96 hr, and 14 days after irradiation. Fractionated irradiation led to hypomethylation in bystander spleen 6 hr after whole‐body exposure, and 6 hr, 96 hr, and 14 days after cranial irradiation. Contrarily, changes in the skin of the same animals were seen only 6 hr after acute whole‐body and head exposure. DNA hypomethylation observed in spleen was paralleled by a reduction of methyl‐binding protein MeCP2 expression. Irradiation also induced tissue‐specific microRNAome alterations in skin and spleen. For the first time, we have shown that IR‐induced epigenetic bystander effects that occur in the same organism are triggered by both acute and fractionated exposure and are very distinct in different bystander organs. Future studies are clearly needed to address organismal and carcinogenic repercussions of those changes. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.     

Non‐uniformly under‐sampled multi‐dimensional spectroscopic imaging in vivo: maximum entropy versus compressed sensing reconstruction

The four‐dimensional (4D) echo‐planar correlated spectroscopic imaging (EP‐COSI) sequence allows for the simultaneous acquisition of two spatial (k_y, k_x) and two spectral (t₂, t₁) dimensions in vivo in a single recording. However, its scan time is directly proportional to the number of increments in the k_y and t₁ dimensions, and a single scan can take 20–40 min using typical parameters, which is too long to be used for a routine clinical protocol. The present work describes efforts to accelerate EP‐COSI data acquisition by application of non‐uniform under‐sampling (NUS) to the k_y–t₁ plane of simulated and in vivo EP‐COSI datasets then reconstructing missing samples using maximum entropy (MaxEnt) and compressed sensing (CS). Both reconstruction problems were solved using the Cambridge algorithm, which offers many workflow improvements over other l₁‐norm solvers. Reconstructions of retrospectively under‐sampled simulated data demonstrate that the MaxEnt and CS reconstructions successfully restore data fidelity at signal‐to‐noise ratios (SNRs) from 4 to 20 and 5× to 1.25× NUS. Retrospectively and prospectively 4× under‐sampled 4D EP‐COSI in vivo datasets show that both reconstruction methods successfully remove NUS artifacts; however, MaxEnt provides reconstructions equal to or better than CS. Our results show that NUS combined with iterative reconstruction can reduce 4D EP‐COSI scan times by 75% to a clinically viable 5 min in vivo, with MaxEnt being the preferred method. Copyright © 2013 John Wiley & Sons, Ltd.     

Division‐linked differentiation can account for CD8+ T‐cell phenotype in vivo

The CD8⁺ T‐cell response to infection involves a large initial expansion in the numbers of responding cells, accompanied by differentiation of these cells. Expression of the adhesion molecule CD62L is high on naïve cells and rapidly downregulated on the surface of the majority (～90%) of cells during the ‘effector’ phase of acute infection. Adoptive transfer studies have been used to study differentiation in this system; however, relatively little work has investigated the phenotype of cells in the endogenous repertoire. We demonstrate that the extent of CD62L down‐regulation is positively correlated with clone size in vivo, consistent with division‐linked differentiation of responding cells. Other features of the endogenous CD62L^hi and CD62L^lo repertoire are that the CD62L^lo repertoire is less diverse than the CD62L^hi repertoire and represents a subset of clonotypes found in the CD62L^hi repertoire. To test whether these observations are compatible with a mechanism of division‐linked differentiation, we developed a mathematical model, where there is a probability of CD62L down‐regulation associated with cell division. Comparison of model results with experimental data suggests that division‐linked differentiation provides a simple mechanism to explain the relationship between clone size and phenotype of CD8⁺ T cells during acute infection.