Quantifying cerebellum grey matter and white matter perfusion using pulsed arterial spin labeling.

To facilitate quantification of cerebellum cerebral blood flow (CBF), studies were performed to systematically optimize arterial spin labeling (ASL) parameters for measuring cerebellum perfusion, segment cerebellum to obtain separate CBF values for grey matter (GM) and white matter (WM), and compare FAIR ASST to PICORE. Cerebellum GM and WM CBF were measured with optimized ASL parameters using FAIR ASST and PICORE in five subjects. Influence of volume averaging in voxels on cerebellar grey and white matter boundaries was minimized by high-probability threshold masks. Cerebellar CBF values determined by FAIR ASST were 43.8 ± 5.1 mL/100 g/min for GM and 27.6 ± 4.5 mL/100 g/min for WM. Quantitative perfusion studies indicated that CBF in cerebellum GM is 1.6 times greater than that in cerebellum WM. Compared to PICORE, FAIR ASST produced similar CBF estimations but less subtraction error and lower temporal, spatial, and intersubject variability. These are important advantages for detecting group and/or condition differences in CBF values.

Advancing the high throughput identification of liver fibrosis protein signatures using multiplexed ion mobility spectrometry.

Rapid diagnosis of disease states using less invasive, safer, and more clinically acceptable approaches than presently employed is a crucial direction for the field of medicine. While MS-based proteomics approaches have attempted to meet these objectives, challenges such as the enormous dynamic range of protein concentrations in clinically relevant biofluid samples coupled with the need to address human biodiversity have slowed their employment. Herein, we report on the use of a new instrumental platform that addresses these challenges by coupling technical advances in rapid gas phase multiplexed ion mobility spectrometry separations with liquid chromatography and MS to dramatically increase measurement sensitivity and throughput, further enabling future high throughput MS-based clinical applications. An initial application of the liquid chromatography--ion mobility spectrometry-MS platform analyzing blood serum samples from 60 postliver transplant patients with recurrent fibrosis progression and 60 nontransplant patients illustrates its potential utility for disease characterization.

Evaluation of the WARP-turbo spin echo sequence for 3 Tesla magnetic resonance imaging of stifle joints in dogs with stainless steel tibial plateau leveling osteotomy implants.

Susceptibility artifacts caused by ferromagnetic implants compromise magnetic resonance imaging (MRI) of the canine stifle after tibial plateau leveling osteotomy (TPLO) procedures. The WARP-turbo spin echo sequence is being developed to mitigate artifacts and utilizes slice encoding for metal artifact reduction. The aim of the current study was to evaluate the WARP-turbo spin echo sequence for imaging post TPLO canine stifle joints. Proton density weighted images of 19 canine cadaver limbs were made post TPLO using a 3 Tesla MRI scanner. Susceptibility artifact sizes were recorded and compared for WARP vs. conventional turbo spin echo sequences. Three evaluators graded depiction quality for the tibial tuberosity, medial and lateral menisci, tibial osteotomy, and caudal cruciate ligament as sufficient or insufficient to make a diagnosis. Artifacts were subjectively smaller and local structures were better depicted in WARP-turbo spin echo images. Signal void area was also reduced by 75% (sagittal) and 49% (dorsal) in WARP vs. conventional turbo spin echo images. Evaluators were significantly more likely to grade local anatomy depiction as adequate for making a diagnosis in WARP-turbo spin echo images in the sagittal but not dorsal plane. The proportion of image sets with anatomic structure depiction graded adequate to make a diagnosis ranged from 28 to 68% in sagittal WARP-turbo spin echo images compared to 0-19% in turbo spin echo images. Findings indicated that the WARP-turbo spin echo sequence reduces the severity of susceptibility artifacts in canine stifle joints post TPLO. However, variable depiction of local anatomy warrants further refinement of the technique.

Magnetic resonance imaging in cadaver dogs with metallic vertebral implants at 3 Tesla: evaluation of the WARP-turbo spin echo sequence.

STUDY DESIGN: Laboratory investigation, ex vivo. OBJECTIVE: Postoperative complications are common after spinal implantation procedures, and magnetic resonance imaging (MRI) would be the ideal modality to image these patients. Unfortunately, the implants cause artifacts that can render MRI nondiagnostic. The WARP-turbo spin echo (TSE) sequence has been developed to mitigate artifacts caused by metal. The objective of this investigation was to evaluate the performance of the WARP-TSE sequence in canine cadaver specimens after implantation with metallic vertebral implants. SUMMARY OF BACKGROUND DATA: Magnetic field strength, implant type, and MRI acquisition technique all play a role in the severity of susceptibility artifacts. The WARP-TSE sequence uses increased bandwidth, view angle tilting, and SEMAC (slice-encoding metal artifact correction) to correct for susceptibility artifact. The WARP-TSE technique has outperformed conventional techniques in patients, after total hip arthroplasty. However, published reports of its application in subjects with vertebral column implants are lacking. METHODS: Ex vivo anterior stabilization of the atlantoaxial joint was performed on 6 adult small breed (<8 kg) cadaver dogs using stainless steel screws and polymethylmethacrylate. Axial and sagittal T2-weighted and short tau inversion recovery MRI was performed using conventional pulse sequences and WARP-TSE sequences at 3 T. Images were assessed qualitatively and quantitatively. RESULTS: Images made with the WARP-TSE sequence had smaller susceptibility artifacts and superior spinal cord margin depiction. WARP-TSE sequences reduced the length over which susceptibility artifacts caused spinal cord margin depiction interference by 24.9% to 71.5% with scan times of approximately 12 to 16 minutes. CONCLUSION: The WARP-TSE sequence is a viable option for evaluating the vertebral column after implantation with stainless steel implants. LEVEL OF EVIDENCE: N/A.

Anteroposterior perfusion heterogeneity in human hippocampus measured by arterial spin labeling MRI.

Measurements of blood flow in the human hippocampus are complicated by its relatively small size, unusual anatomy and patterns of blood supply. Only a handful of arterial spin labeling (ASL) MRI articles have reported regional cerebral blood flow (rCBF) values for the human hippocampus. Numerous reports have found heterogeneity in a number of other physiological and biochemical parameters along the longitudinal hippocampal axis. There is, however, only one ASL study of perfusion properties as a function of anteroposterior location in the hippocampus, reporting that rCBF is lower and the arterial transit time (ATT) is longer in the anterior hippocampus than in the posterior hippocampus of the rat brain. The purpose of this article was to measure ATT and rCBF in anterior, middle and posterior normal adult human hippocampus. To better distinguish anteroposterior perfusion heterogeneity in the hippocampus, a modified ASL method, called Orthogonally Positioned Tagging Imaging Method for Arterial Labeling with Flow-sensitive Alternating Inversion Recovery (OPTIMAL FAIR), was developed that provides high in-plane resolution with oblique coronal imaging slices perpendicular to the long axis of the hippocampus to minimize partial volume effects. Perfusion studies performed with this modified FAIR method at 3 T indicated that anterior, middle and posterior human hippocampus segments have unique transit time and rCBF values. Of these three longitudinal hippocampal regions, the middle hippocampus has the highest perfusion and the shortest transit time and the anterior hippocampus has the lowest perfusion and the longest transit time. Copyright © 2013 John Wiley & Sons, Ltd.

Thoracic and lumbar spinal cord diffusion tensor imaging in dogs.

PURPOSE: To analyze four clinically applicable diffusion tensor imaging (DTI) protocols (two each in the transverse and sagittal planes) in the normal dog. MATERIALS AND METHODS: Seven healthy Dachshund dogs were scanned with four DTI protocols. Within each plane, identical spatial resolution was used while the number of diffusion-encoding directions and averages varied. Agreement of measured fractional anisotropy (FA) and apparent diffusion coefficient (ADC) was analyzed with Bland-Altman methods, subjective image quality within each plane was compared, and FA and ADC were explored as a function of anatomic location. RESULTS: There was good agreement in FA and ADC values within each plane. FA had the smallest bias and most precision. No difference was detected in subjective image quality within each plane. FA and ADC were slightly higher cranial to the lumbar intumescence compared to within it. CONCLUSION: DTI is a promising tool in the assessment of spinal cord injury (SCI) in the study of dogs with intervertebral disk herniation as a preclinical model of human SCI.

H5N1 virus causes significant perturbations in host proteome very early in influenza virus-infected primary human monocyte-derived macrophages.

H5N1 influenza viruses, which cause disease in humans, have unusually high pathogenicity. The temporal response of primary human monocyte-derived macrophages infected with highly pathogenic H5N1 and seasonal H1N1 influenza viruses was evaluated using mass spectrometry-based quantitative proteomic profiling. This was done in order to demonstrate significant perturbation of the host proteome upon viral infection, as early as 1 hour after infection. This early host response distinguished H5N1 infection from H1N1 infection, the latter inducing less of a response. The most pronounced effect was observed on the translational machinery, suggesting that H5N1 might gain advantage in replication by using the cell protein synthesis machinery early in the infection.

Quantitative proteomic analysis of HIV-1 infected CD4+ T cells reveals an early host response in important biological pathways: protein synthesis, cell proliferation, and T-cell activation.

Human immunodeficiency virus (HIV-1) depends upon host-encoded proteins to facilitate its replication while at the same time inhibiting critical components of innate and/or intrinsic immune response pathways. To characterize the host cell response on protein levels in CD4+ lymphoblastoid SUP-T1 cells after infection with HIV-1 strain LAI, we used mass spectrometry (MS)-based global quantitation with iTRAQ (isobaric tag for relative and absolute quantification). We found 266, 60 and 22 proteins differentially expressed (DE) (P-value ≤ 0.05) at 4, 8, and 20 hours post-infection (hpi), respectively, compared to time-matched mock-infected samples. The majority of changes in protein abundance occurred at an early stage of infection well before the de novo production of viral proteins. Functional analyses of these DE proteins showed enrichment in several biological pathways including protein synthesis, cell proliferation, and T-cell activation. Importantly, these early changes before the time of robust viral production have not been described before.

Morphine produces immunosuppressive effects in nonhuman primates at the proteomic and cellular levels.

Morphine has long been known to have immunosuppressive properties in vivo, but the molecular and immunologic changes induced by it are incompletely understood. To explore how these changes interact with lentiviral infections in vivo, animals from two nonhuman primate species (African green monkeys and pigtailed macaques) were provided morphine and studied using a systems biology approach. Biological specimens were obtained from multiple sources (e.g. lymph node, colon, cerebrospinal fluid, and peripheral blood) before and after the administration of morphine (titrated up to a maximum dose of 5 mg/kg over a period of 20 days). Cellular immune, plasma cytokine, and proteome changes were measured and morphine-induced changes in these parameters were assessed on an interorgan, interindividual, and interspecies basis. In both species, morphine was associated with decreased levels of Ki-67(+) T-cell activation but with only minimal changes in overall T-cell counts, neutrophil counts, and NK cell counts. Although changes in T-cell maturation were observed, these varied across the various tissue/fluid compartments studied. Proteomic analysis revealed a morphine-induced suppressive effect in lymph nodes, with decreased abundance of protein mediators involved in the functional categories of energy metabolism, signaling, and maintenance of cell structure. These findings have direct relevance for understanding the impact of heroin addiction and the opioids used to treat addiction as well as on the potential interplay between opioid abuse and the immunological response to an infective agent.

Proteome and computational analyses reveal new insights into the mechanisms of hepatitis C virus-mediated liver disease posttransplantation.

UNLABELLED: Liver transplant tissues offer the unique opportunity to model the longitudinal protein abundance changes occurring during hepatitis C virus (HCV)-associated liver disease progression in vivo. In this study, our goal was to identify molecular signatures, and potential key regulatory proteins, representative of the processes influencing early progression to fibrosis. We performed global protein profiling analyses on 24 liver biopsy specimens obtained from 15 HCV(+) liver transplant recipients at 6 and/or 12 months posttransplantation. Differentially regulated proteins associated with early progression to fibrosis were identified by analysis of the area under the receiver operating characteristic curve. Analysis of serum metabolites was performed on samples obtained from an independent cohort of 60 HCV(+) liver transplant patients. Computational modeling approaches were applied to identify potential key regulatory proteins of liver fibrogenesis. Among 4,324 proteins identified, 250 exhibited significant differential regulation in patients with rapidly progressive fibrosis. Patients with rapid fibrosis progression exhibited enrichment in differentially regulated proteins associated with various immune, hepatoprotective, and fibrogenic processes. The observed increase in proinflammatory activity and impairment in antioxidant defenses suggests that patients who develop significant liver injury experience elevated oxidative stresses. This was supported by an independent study demonstrating the altered abundance of oxidative stress-associated serum metabolites in patients who develop severe liver injury. Computational modeling approaches further highlight a potentially important link between HCV-associated oxidative stress and epigenetic regulatory mechanisms impacting on liver fibrogenesis. CONCLUSION: Our proteome and metabolome analyses provide new insights into the role for increased oxidative stress in the rapid fibrosis progression observed in HCV(+) liver transplant recipients. These findings may prove useful in prognostic applications for predicting early progression to fibrosis.

Improved quantification of brain perfusion using FAIR with active suppression of superior tagging (FAIR ASST).

PURPOSE: To address two problems for perfusion studies in the middle or inferior brain regions: (1) to reduce venous artifacts due to the intrinsic superior labeling of FAIR; (2) to alleviate the discrepancy of the existence of both superior and inferior boluses, but with only the inferior bolus having a temporally defined bolus width with Q2TIPs or QUIPSS. MATERIALS AND METHODS: Superior tagging suppression methods for FAIR with different combinations of pre- and postinversion superior saturation pulses were evaluated and compared with FAIR with Q2TIPS for producing perfusion maps of superior, middle, and inferior brain regions. RESULTS: One preinversion plus two postinversion superior saturation radio frequency pulses effectively suppressed the superior tagging of FAIR and sufficiently eliminated venous artifacts without negative effects, avoiding the overestimations of cerebral blood flow that can occur in FAIR. CONCLUSION: FAIR ASST improves FAIR with Q2TIPS and provides more reliable and accurate blood flow estimations for perfusion studies of middle and lower brain regions. FAIR ASST confers the advantages of asymmetric PASL techniques, such as PICORE, in which only the inferiorly labeled blood is used for perfusion quantification, to the symmetric PASL technique FAIR, while preserving the robustness of FAIR against MT effects.

Comprehensive proteomic analysis of influenza virus polymerase complex reveals a novel association with mitochondrial proteins and RNA polymerase accessory factors.

The trimeric RNA polymerase complex (3P, for PA-PB1-PB2) of influenza A virus (IAV) is an important viral determinant of pathogenicity and host range restriction. Specific interactions of the polymerase complex with host proteins may be determining factors in both of these characteristics and play important roles in the viral life cycle. To investigate this question, we performed a comprehensive proteomic analysis of human host proteins associated with the polymerase of the well-characterized H5N1 Vietnam/1203/04 isolate. We identified over 400 proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS), of which over 300 were found to bind to the PA subunit alone. The most intriguing and novel finding was the large number of mitochondrial proteins (∼20%) that associated with the PA subunit. These proteins mediate molecular transport across the mitochondrial membrane or regulate membrane potential and may in concert with the identified mitochondrion-associated apoptosis inducing factor (AIFM1) have roles in the induction of apoptosis upon association with PA. Additionally, we identified host factors that associated with the PA-PB1 (68 proteins) and/or the 3P complex (34 proteins) including proteins that have roles in innate antiviral signaling (e.g., ZAPS or HaxI) or are cellular RNA polymerase accessory factors (e.g., polymerase I transcript release factor [PTRF] or Supt5H). IAV strain-specific host factor binding to the polymerase was not observed in our analysis. Overall, this study has shed light into the complex contributions of the IAV polymerase to host cell pathogenicity and allows for direct investigations into the biological significance of these newly described interactions.

Mapping primary gyrogenesis during fetal development in primate brains: high-resolution in utero structural MRI of fetal brain development in pregnant baboons.

The global and regional changes in the fetal cerebral cortex in primates were mapped during primary gyrification (PG; weeks 17-25 of 26 weeks total gestation). Studying pregnant baboons using high-resolution MRI in utero, measurements included cerebral volume, cortical surface area, gyrification index and length and depth of 10 primary cortical sulci. Seven normally developing fetuses were imaged in two animals longitudinally and sequentially. We compared these results to those on PG that from the ferret studies and analyzed them in the context of our recent studies of phylogenetics of cerebral gyrification. We observed that in both primates and non-primates, the cerebrum undergoes a very rapid transformation into the gyrencephalic state, subsequently accompanied by an accelerated growth in brain volume and cortical surface area. However, PG trends in baboons exhibited some critical differences from those observed in ferrets. For example, in baboons, the growth along the long (length) axis of cortical sulci was unrelated to the growth along the short (depth) axis and far outpaced it. Additionally, the correlation between the rate of growth along the short sulcal axis and heritability of sulcal depth was negative and approached significance (r = -0.60; p < 0.10), while the same trend for long axis was positive and not significant (p = 0.3; p = 0.40). These findings, in an animal that shares a highly orchestrated pattern of PG with humans, suggest that ontogenic processes that influence changes in sulcal length and depth are diverse and possibly driven by different factors in primates than in non-primates.

Fetal brain during a binge drinking episode: a dynamic susceptibility contrast MRI fetal brain perfusion study.

We assessed the effects of a single episode of maternal alcohol intoxication on fetal brain blood perfusion in three pregnant dams (baboons) at the 24th week of pregnancy using dynamic susceptibility contrast magnetic resonance imaging. After the oral administration of alcohol, there was a four-fold increase in the peak contrast concentrations in the fetal brain. In addition, we observed a two- to three-fold increase in the contrast uptake and washout rates in the fetal brain. The underlying mechanisms of these changes are unknown, but we hypothesized that these could include the alcohol-mediated changes in placental permeability and fetal cerebral blood flow. Our findings indicate that alcohol intoxication produces profound changes, which may detrimentally influence neurodevelopmental processes in the brain.

Th17 cytokines stimulate CCL20 expression in keratinocytes in vitro and in vivo: implications for psoriasis pathogenesis.

T helper (Th) 17 cells have recently been implicated in psoriasis pathogenesis, but mechanisms of how these cells traffic into inflamed skin are unknown. By immunostaining for interleukin (IL)-17A and IL-22, we show numerous cells present in psoriasis lesions that produce these cytokines. We next found that Th17 cytokines (IL-17A, IL-22, and tumor necrosis factor (TNF)-alpha) markedly increased the expression of CC chemokine ligand (CCL) 20, a CC chemokine receptor (CCR)6 ligand, in human keratinocyte monolayer and raft cultures in a dose- and time-dependent manner. Lastly, we showed in mice that subcutaneous injection with recombinant IL-17A, IL-22, or TNF-alpha led to the upregulation of both CCL20 and CCR6 expression in skin as well as cutaneous T-cell infiltration. Taken together, these data show that Th17 cytokines stimulate CCL20 production in vitro and in vivo, and thus provide a potential explanation of how CCR6-positive Th17 cells maintain their continual presence in psoriasis through a positive chemotactic feedback loop.

Fas ligand-induced proinflammatory transcriptional responses in reconstructed human epidermis. Recruitment of the epidermal growth factor receptor and activation of MAP kinases.

Fas ligand (FasL) exerts potent proapoptotic and proinflammatory actions on epidermal keratinocytes and has been implicated in the pathogenesis of eczema, toxic epidermal necrolysis, and drug-induced skin eruptions. We used reconstructed human epidermis to investigate the mechanisms of FasL-induced inflammatory responses and their relationships with FasL-triggered caspase activity. Caspase activity was a potent antagonist of the pro-inflammatory gene expression triggered by FasL prior to the onset of cell death. Furthermore, we found that FasL-stimulated autocrine production of epidermal growth factor receptor (EGFR) ligands, and the subsequent activation of EGFR and ERK1 and ERK2 mitogen-activated protein kinases, were obligatory extracellular steps for the FasL-induced expression of a subset of inflammatory mediators, including CXCL8/interleukin (IL)-8, ICAM-1, IL-1alpha, IL-1beta, CCL20/MIP-3alpha, and thymic stromal lymphopoietin. These results expand the known physiological role of EGFR and its ligands from promoting keratinocyte mitogenesis and survival to mediating FasL-induced epidermal inflammation.

Fas ligand elicits a caspase-independent proinflammatory response in human keratinocytes: implications for dermatitis.

Fas ligand (FasL) causes apoptosis of epidermal keratinocytes and triggers the appearance of spongiosis in eczematous dermatitis. We demonstrate here that FasL also aggravates inflammation by triggering the expression of proinflammatory cytokines, chemokines, and adhesion molecules in keratinocytes. In HaCaT cells and in reconstructed human epidermis (RHE), FasL triggered a NF-kappaB-dependent mRNA accumulation of inflammatory cytokines (tumor necrosis factor-alpha, IL-6, and IL-1beta), chemokines (CCL2/MCP-1, CXCL1/GROalpha, CXCL3/GROgamma, and CXCL8/IL-8), and the adhesion molecule ICAM-1. Oligomerization of Fas was required both for apoptosis and for gene expression. Inhibition of caspase activity abolished FasL-dependent apoptosis; however, it failed to suppress the expression of FasL-induced genes. Additionally, in the presence of caspase inhibitors, but not in their absence, FasL triggered the accumulation of CCL5/RANTES (regulated on activation normal T cell expressed and secreted) mRNA. Our findings identify a novel proinflammatory role of FasL in keratinocytes that is independent of caspase activity and is separable from apoptosis. Thus, in addition to causing spongiosis, FasL may play a direct role in triggering and/or sustaining inflammation in eczemas.

Retrospective motion correction protocol for high-resolution anatomical MRI.

Modern computational brain morphology methods require that anatomical images be acquired at high resolution and with a high signal-to-noise ratio. This often translates into long acquisition times (>20 minutes) and images susceptible to head motion. In this study we tested retrospective motion correction (RMC), common for functional MRI (fMRI) and PET image motion correction, as a means to improve the quality of high-resolution 3-D anatomical MR images. RMC methods are known to be effective for correcting interscan motion; therefore, a single high-resolution 3-D MRI brain study was divided into six shorter acquisition segments to help shift intrascan motion into interscan motion. To help reduce intrascan head motion, each segment image was reviewed for motion artifacts and repeated if necessary. Interscan motion correction was done by spatially registering images to the third image and forming a single average motion-corrected image. RMC was tested on 35 subjects who were considered at high risk for head motion. Our results show that RMC provided better contrast-to-noise ratio and boundary detail when compared to nonmotion-corrected averaged images.