Fractional ventilation mapping using inert fluorinated gas MRI in rat models of inflammation and fibrosis

The purpose of this study was to extend established methods for fractional ventilation mapping using ¹⁹F MRI of inert fluorinated gases to rat models of pulmonary inflammation and fibrosis. In this study, five rats were instilled with lipopolysaccharide (LPS) in the lungs two days prior to imaging, six rats were instilled with bleomycin in the lungs two weeks prior to imaging and an additional four rats were used as controls. ¹⁹F MR lung imaging was performed at 3 T with rats continuously breathing a mixture of sulfur hexafluoride and O₂. Fractional ventilation maps were obtained using a wash‐out approach, by switching the breathing mixture to pure O₂, and acquiring images following each successive wash‐out breath. The mean fractional ventilation (r) was 0.29 ± 0.05 for control rats, 0.23 ± 0.10 for LPS‐instilled rats and 0.19 ± 0.03 for bleomycin‐instilled rats. Bleomycin‐instilled rats had a significantly decreased mean r value compared with controls (P = 0.010). Although LPS‐instilled rats had a slightly reduced mean r value, this trend was not statistically significant (P = 0.556). Fractional ventilation gradients were calculated in the anterior/posterior (A/P) direction, and the mean A/P gradient was ?0.005 ± 0.008 cm^?1 for control rats, 0.013 ± 0.005 cm^?1 for LPS‐instilled rats and 0.009 ± 0.018 cm^?1 for bleomycin‐instilled rats. Fractional ventilation gradients were significantly different for control rats compared with LPS‐instilled rats only (P = 0.016). The ventilation gradients calculated from control rats showed the expected gravitational relationship, while ventilation gradients calculated from LPS‐ and bleomycin‐instilled rats showed the opposite trend. Histology confirmed that LPS‐instilled rats had a significantly elevated alveolar wall thickness, while bleomycin‐instilled rats showed signs of substantial fibrosis. Overall, ¹⁹F MRI may be able to detect the effects of pulmonary inflammation and fibrosis using a simple and inexpensive imaging approach that can potentially be translated to humans. Copyright © 2016 John Wiley & Sons, Ltd.     

CEST MRI of sepsis‐induced acute kidney injury

Sepsis‐induced acute kidney injury (SAKI) is a major complication of kidney disease associated with increased mortality and faster progression. Therefore, the development of imaging biomarkers to detect septic AKI is of great clinical interest. In this study, we aimed to characterize the endogenous chemical exchange saturation transfer (CEST) MRI contrast in the lipopolysaccharide (LPS)‐induced SAKI mouse model and to investigate the use of CEST MRI for detecting such injury. We used a SAKI mouse model that was generated by i.p. injection of 10 mg/kg LPS. The resulting kidney injury was confirmed by the elevation of serum creatinine and histology. MRI assessments were performed 24 h after LPS injection, including CEST MRI at different B₁ strengths (1, 1.8 and 3 μT), T₁ mapping, T₂ mapping and conventional magnetization transfer contrast (MTC) MRI. The CEST MRI results were analyzed using Z‐spectra, in which the normalized water signal saturation (S_sat/S₀) is measured as a function of saturation frequency. Substantial decreases in CEST contrast were observed at both 3.5 and ? 3.5 ppm frequency offset from water at all B₁ powers, with the most significant difference obtained at a B₁ of 1.8 μT. The average S_sat/S₀ differences between injured and normal kidneys were 0.07 (0.55 ± 0.04 versus 0.62 ± 0.04, P = 0.0028) and 0.07 (0.50 ± 0.04 versus 0.57 ± 0.03, P = 0.0008) for 3.5 and ? 3.5 ppm, respectively. In contrast, the T₁ and T₂ relaxation times and MTC contrast in the injured kidneys did not show a significant change compared with the normal control. Our results showed that CEST MRI is more sensitive to the pathological changes in injured kidneys than the changes in T₁, T₂ and MTC effect, indicating its potential clinical utility for molecular imaging of renal diseases.     

Down syndrome, autoimmunity and T regulatory cells

Autoimmune diseases are more represented in Down syndrome (DS) individuals compared to chromosomally normal people. Natural T regulatory cells (nT_reg) have been considered to be primary in the role of controlling the intensity and targets of the immune response. We have investigated the phenotypical and functional alteration of nT_reg in a group of DS people. The phenotypical characteristic of T_reg cells of 29 DS was analysed and compared with an age‐matched healthy control group. The inhibitory potential of CD4⁺CD25^highCD127^low T regulatory cells was evaluated on autologous CD4⁺CD25^‐ T cell proliferation in response to activation with a mytogenic pan‐stimulus (anti‐CD2, anti‐CD3 and anti‐CD28 antibodies). The CD4⁺CD25^high cells in the DS and control groups were 2·692 ± 0·3808%, n = 29 and 1·246 ± 0·119, n = 29%, respectively (P = 0.0007), with a percentage of forkhead box protein 3 (FoxP3)‐expressing cells of 79·21 ± 3·376%, n = 29 and 59·75 ± 4·496%, respectively (P = 0.0015). CD4⁺CD25⁺FoxP3⁺ cells were increased in peripheral blood from DS subjects (DS mean 5·231 ± 0·6065% n = 29, control mean 3·076 ± 0·3140% n = 29). The majority of CD4⁺CD25^high were CD127^low and expressed a high percentage of FoxP3 (natural T_reg phenotype). While the proliferative capacity of DS T cells was not altered significantly compared to normal individuals, a reduced inhibitory potential of T_reg compared to healthy controls was clearly observed (mean healthy control inhibition in T_eff : T_reg 1:1 co‐culture: 58·9% ± 4·157%, n = 10 versus mean DS inhibition in T_eff : T_reg 1:1 co‐culture: 39·8 ± 4·788%, n = 10, P = 0.0075; mean healthy control inhibition in T_eff : T_reg 1:0·5 co‐culture: 45·10 ± 5·858%, n = 10 versus DS inhibition in T_eff : T_reg 1:0·5 co‐culture: 24·10 ± 5·517%, n = 10, P = 0.0177). DS people present an over‐expressed peripheral nT_reg population with a defective inhibitory activity that may partially explain the increased frequency of autoimmune disease.     

In vivo MRI analysis of depth‐dependent ultrastructure in human knee cartilage at 7 T

Signal intensities of T₂‐weighted magnetic resonance images depend on the local fiber arrangement in hyaline cartilage. The aims of this study were to determine whether angle‐sensitive MRI at 7 T can be used to quantify the cartilage ultrastructure of the knee in vivo and to assess potential differences with age. Ten younger (21–30) and ten older (55–76 years old) healthy volunteers were imaged with a T₂‐weighted spin‐echo sequence in a 7 T whole‐body MRI. A “fascicle” model was assumed to describe the depth‐dependent fiber arrangement of cartilage. The R/T boundary positions between radial and transitional zones were assessed from intensity profiles in small regions of interest in the femur and tibia, and normalized to cartilage thickness using logistic curve fits. The quality of our highly resolved (0.3 × 0.3 × 1.0 mm³) MR cartilage images were high enough for quantitative analysis (goodness of fit R² = 0.91 ± 0.09). Between younger and older subjects, normalized positions of the R/T boundary, with value 0 at the bone–cartilage interface and 1 at the cartilage surface, were significantly (p < 0.05) different in femoral (0.51 ± 0.12 versus 0.41 ± 0.10), but not in tibial cartilage (0.65 ± 0.11 versus 0.57 ± 0.09, p = 0.119). Within both age groups, differences between femoral and tibial R/T boundaries were significant. Using a fascicle model and angle‐sensitive MRI, the depth‐dependent anisotropic fiber arrangement of knee cartilage could be assessed in vivo from a single 7 T MR image. The derived quantitative parameter, thickness of the radial zone, may serve as an indicator of the structural integrity of cartilage. This method may potentially be suitable to detect and monitor early osteoarthritis because the progressive disintegration of the anisotropic network is also indicative of arthritic changes in cartilage. Copyright © 2013 John Wiley & Sons, Ltd.     

Immunoregulatory Effects of Carvedilol on Rat Experimental Autoimmune Myocarditis

The cardioprotective effects of carvedilol were studied in a rat model of experimental autoimmune myocarditis (EAM). After immunization, rats were orally administered 10 mg/kg/day carvedilol (group C, n = 15) or a vehicle control (Group V, n = 12) for 3 weeks. On day 21, echocardiography and haemodynamic parameters, myocarditis areas, and cytokine concentrations were measured. Serum carvedilol concentrations ranged from 10.5 ± 2.6 to 31.7 ± 9.3 ng/ml over a 24 h period on day 20. Carvedilol decreased the myocarditis areas (23.07 ± 1.6 versus 33.65 ± 2.71%, P < 0.0001). The left ventricular fractional shortening and the absolute value of +dP/dt or –dP/dt were significantly higher in Group C. Heart rate, systolic blood pressure, left ventricular end‐diastolic pressure and central venous pressure were significantly lower in Group C. The serum and mRNA levels of interleukin (IL)‐1β (53.58 ± 6.42 versus 98.75 ± 6.53 pg/ml, P < 0.0001 and 0.298 ± 0.04 versus 0.818 ± 0.252, P < 0.0001, respectively) and TNF‐α (14.82 ± 1.95 versus 29.52 ± 3.7 pg/ml, P = 0.0008 and 0.088 ± 0.006 versus 0.168 ± 0.072, P = 0.0051, respectively) were markedly decreased, whereas IL‐10 (24.92 ± 2.94 versus 15.25 ± 3.13 pg/ml, P = 0.015 and 0.302 ± 0.022 versus 0.107 ± 0.02, P < 0.0001, respectively) and IL‐1 receptor antagonist (1.95 ± 0.28 versus 0.52 ± 0.10 pg/ml, P < 0.0001 and 0.112 ± 0.009 versus 0.051 ± 0.002, P < 0.0001, respectively) were markedly increased in the Group C. These results indicate that in rats carvedilol protects against EAM.     

Fast volumetric imaging of bound and pore water in cortical bone using three‐dimensional ultrashort‐TE (UTE) and inversion recovery UTE sequences

We report the three‐dimensional ultrashort‐TE (3D UTE) and adiabatic inversion recovery UTE (IR‐UTE) sequences employing a radial trajectory with conical view ordering for bi‐component T₂* analysis of bound water (T₂*^BW) and pore water (T₂*^PW) in cortical bone. An interleaved dual‐echo 3D UTE acquisition scheme was developed for fast bi‐component analysis of bound and pore water in cortical bone. A 3D IR‐UTE acquisition scheme employing multiple spokes per IR was developed for bound water imaging. Two‐dimensional UTE (2D UTE) and IR‐UTE sequences were employed for comparison. The sequences were applied to bovine bone samples (n = 6) and volunteers (n = 6) using a 3‐T scanner. Bi‐component fitting of 3D UTE images of bovine samples showed a mean T₂*^BW of 0.26 ± 0.04 ms and T₂*^PW of 4.16 ± 0.35 ms, with fractions of 21.5 ± 3.6% and 78.5 ± 3.6%, respectively. The 3D IR‐UTE signal showed a single‐component decay with a mean T₂*^BW of 0.29 ± 0.05 ms, suggesting selective imaging of bound water. Similar results were achieved with the 2D UTE and IR‐UTE sequences. Bi‐component fitting of 3D UTE images of the tibial midshafts of healthy volunteers showed a mean T₂*^BW of 0.32 ± 0.08 ms and T₂*^PW of 5.78 ± 1.24 ms, with fractions of 34.2 ± 7.4% and 65.8 ± 7.4%, respectively. Single‐component fitting of 3D IR‐UTE images showed a mean T₂*^BW of 0.35 ± 0.09 ms. The 3D UTE and 3D IR‐UTE techniques allow fast volumetric mapping of bound and pore water in cortical bone. Copyright © 2016 John Wiley & Sons, Ltd.     

Quantification of MRI signal of transgenic grafts overexpressing ferritin in murine myocardial infarcts

The noninvasive detection of transplanted cells in damaged organs and the longitudinal follow‐up of cell fate and graft size are important for the evaluation of cell therapy. We have shown previously that the overexpression of the natural iron storage protein, ferritin, permits the detection of engrafted cells in mouse heart by MRI, but further imaging optimization is required. Here, we report a systematic evaluation of ferritin‐based stem cell imaging in infarcted mouse hearts in vivo using three cardiac‐gated pulse sequences in a 3‐T scanner: black‐blood proton‐density‐weighted turbo spin echo (PD TSE BB), bright‐blood T₂*‐weighted gradient echo (GRE) and black‐blood T₂*‐weighted GRE with improved motion‐sensitized‐driven equilibrium (iMSDE) preparation. Transgenic C2C12 myoblast grafts overexpressing ferritin did not change MRI contrast in the PD TSE BB images, but showed a 20% reduction in signal intensity ratio in black‐blood T₂*‐weighted iMSDE (p < 0.05) and a 30% reduction in bright‐blood T₂*‐weighted GRE (p < 0.0001). Graft size measurements by T₂* iMSDE and T₂* GRE were highly correlated with histological assessments (r = 0.79 and r = 0.89, respectively). Unlabeled wild‐type C2C12 cells transplanted to mouse heart did not change the MRI signal intensity, although endogenous hemosiderin was seen in some infarcts. These data support the use of ferritin to track the survival, growth and migration of stem cells transplanted into the injured heart. Copyright © 2012 John Wiley & Sons, Ltd.     

Monocyte implication in renal allograft dysfunction

Macrophages are involved in the development and progression of kidney fibrosis. The aim of this study was to analyse the phenotype of circulating monocytes and their ability to predict kidney allograft dysfunction in living kidney transplant recipients. Whole blood samples from 25 kidney recipients and 17 donors were collected at five time‐points. Monocyte phenotype was analysed by flow cytometry, and interleukin (IL)‐10 and soluble CD163 by enzyme‐linked immunosorbent assay. One week after transplantation, surface CD163 and IL‐10 levels increased significantly from baseline [2·99 ± 1·38 mean fluorescence intensity (MFI) to 5·18 ± 2·42 MFI for CD163; 4·5 ± 1·46 pg/ml to 6·7 ± 2·5 pg/ml for IL‐10]. This CD163 increase correlated with 4‐month creatinine levels (r = 0·4394, P = 0·04). However, soluble CD163 decreased significantly from baseline at 1 week (797·11 ± 340·45 ng/ml to 576·50 ± 293·60 ng/ml). CD14⁺CD16^– monocytes increased at 4 months and correlated positively with creatinine levels at 12 and 24 months (r = 0·6348, P = 0·002 and r = 0·467, P = 0·028, respectively) and negatively with Modification of Diet in Renal Disease (MDRD) at 12 months (r = 0·6056, P = 0·003). At 4 months, IL‐10 decreased significantly (P = 0·008) and correlated positively with creatinine at 2 years (r = 0·68, P = 0·010) and with CD14⁺CD16^– monocytes at 4 months (r = 0·732, P = 0·004). At 24 h, levels of human leucocyte antigen D‐related declined from 12·12 ± 5·99 to 5·21 ± 3·84 and CD86 expression decreased from 2·76 ± 1·08 to 1·87 ± 0·95. Both markers recovered progressively until 12 months, when they decreased again. These results indicate that monitoring monocytes could be a promising new prognostic tool of graft dysfunction in renal transplant patients.     

Evidence of multiexponential T2 in rat glioblastoma

The aim of this study was to characterize multiexponential T₂ (MET₂) relaxation in a rat C6 glioblastoma tumor model. To do this, rats (n = 11) were inoculated with the C6 cells via stereotaxic injection into the brain. Ten days later, MET₂ measurements were performed in vivo using a single‐slice, multi‐echo spin‐echo sequence at 7.0 T. Tumor signal was biexponential in eight animals with a short‐lived T₂ component (T₂ = 20.7 ± 5.4 ms across samples) representing 6.8 ± 6.2% of the total signal and a long‐lived T₂ component (T₂ = 76.4 ± 9.3 ms) representing the remaining signal fraction. In contrast, signal from contralateral grey matter was consistently monoexponential (T₂ = 48.8 ± 2.3 ms). Additional ex vivo studies (n = 3) and Monte Carlo simulations showed that the in vivo results were not significantly corrupted by partial volume averaging or noise. The underlying physiological origin of the observed MET₂ components is unknown; however, MET₂ analysis may hold promise as a non‐invasive tool for characterizing tumor microenvironment in vivo on a sub‐voxel scale. Copyright © 2009 John Wiley & Sons, Ltd.     

Increased systolic blood pressure after mild cold and rewarming: relation to cold-induced thermogenesis and age

Aim: Higher winter mortality in elderly has been associated with augmented systolic blood pressure (SBP) response and with impaired defense of core temperature. Here we investigated whether the augmented SBP upon mild cold exposure remains after a rewarming period, and whether SBP changes are linked to thermoregulation. Therefore, we tested the following hypotheses: cold‐induced increase in SBP (1) remains augmented after rewarming in elderly compared to young adults (2) is related to non‐shivering thermogenesis (NST) upon mild cold (3) is related to vasoconstriction upon mild cold. Methods: Blood pressure, energy expenditure (EE), skin and core temperature, skin perfusion (abdomen, forearm, both sides of hand) and % body fat were measured in 12 young adults (Y) and 12 elderly (E). Supine subjects were exposed to a thermoneutral baseline 0.5 h (T_air = 30.1 °C), 1 h mild cold (T_air = 20.7 °C), 1 h rewarming (T_air = 34.8 °C) and 1 h baseline (T_air = 30.5 °C). Results: Upon mild cold only the young adults showed significant NST (Y: +2.5 ± 0.6 W m^?2, P < 0.05). No significant age effects in vasoconstriction were observed. After rewarming per cent change in SBP (%ΔSBP) remained significantly increased in both age groups and was augmented in elderly (Y: +5.0% ± 1.2% vs. E: +14.7% ± 3.1%, P < 0.05). Regression analysis revealed that %ΔSBP significantly related to ΔEE upon mild cold (P < 0.01, r² = 0.35) and in elderly also to %body fat (P < 0.02, r² = 0.57). Conclusion: Individual changes in SBP after rewarming correlate negatively to NST. Elderly did not show NST, which explains the greater SBP increase in this group. In elderly a relatively large %body fat protected against the adverse effects of mild cold.     

Stability and sensitivity of water T2 obtained with IDEAL‐CPMG in healthy and fat‐infiltrated skeletal muscle

Quantifying muscle water T₂ (T₂‐water) independently of intramuscular fat content is essential in establishing T₂‐water as an outcome measure for imminent new therapy trials in neuromuscular diseases. IDEAL‐CPMG combines chemical shift fat–water separation with T₂ relaxometry to obtain such a measure. Here we evaluate the reproducibility and B₁ sensitivity of IDEAL‐CPMG T₂‐water and fat fraction (f.f.) values in healthy subjects, and demonstrate the potential of the method to quantify T₂‐water variation in diseased muscle displaying varying degrees of fatty infiltration. The calf muscles of 11 healthy individuals (40.5 ± 10.2 years) were scanned twice at 3 T with an inter‐scan interval of 4 weeks using IDEAL‐CPMG, and 12 patients with hypokalemic periodic paralysis (HypoPP) (42.3 ± 11.5 years) were also imaged. An exponential was fitted to the signal decay of the separated water and fat components to determine T₂‐water and the fat signal amplitude muscle regions manually segmented. Overall mean calf‐level muscle T₂‐water in healthy subjects was 31.2 ± 2.0 ms, without significant inter‐muscle differences (p = 0.37). Inter‐subject and inter‐scan coefficients of variation were 5.7% and 3.2% respectively for T₂‐water and 41.1% and 15.4% for f.f. Bland–Altman mean bias and ±95% coefficients of repeatability were for T₂‐water (0.15, ?2.65, 2.95) ms and f.f. (?0.02, ?1.99, 2.03)%. There was no relationship between T₂‐water (ρ = 0.16, p = 0.07) or f.f. (ρ = 0.03, p = 0.7761) and B₁ error or any correlation between T₂‐water and f.f. in the healthy subjects (ρ = 0.07, p = 0.40). In HypoPP there was a measurable relationship between T₂‐water and f.f. (ρ = 0.59, p < 0.001). IDEAL‐CPMG provides a feasible way to quantify T₂‐water in muscle that is reproducible and sensitive to meaningful physiological changes without post hoc modeling of the fat contribution. In patients, IDEAL‐CPMG measured elevations in T₂‐water and f.f. while showing a weak relationship between these parameters, thus showing promise as a practical means of quantifying muscle water in patient populations.     

Losartan decreases vasopressin-mediated cAMP accumulation in the thick ascending limb of the loop of Henle in rats with congestive heart failure

Introduction: Vasopressin (AVP) stimulates sodium reabsorption and Na,K,2Cl‐cotransporter (NKCC2) protein level in the thick ascending limb (TAL) of Henle's loop in rats. Rats with congestive heart failure (CHF) have increased protein level of NKCC2, which can be normalized by angiotensin II receptor type‐1 (AT₁) blockade with losartan. Aim: In this study, we investigated whether CHF rats displayed changes in AVP stimulated cAMP formation in the TAL and examined the role of AT₁ receptor blockade on this system. Method: CHF was induced by ligation of the left anterior descending coronary artery (LAD). SHAM‐operated rats were used as controls. Half of the rats were treated with losartan (10 mg kg day^?1 i.p.). Results: CHF rats were characterized by increased left ventricular end diastolic pressure. Measurement of cAMP in isolated outer medullary TAL showed that both basal and AVP (10^?6 m ) stimulated cAMP levels were significantly increased in CHF rats (25.52 ± 4.49 pmol cAMP μg^?1 protein, P < 0.05) compared to Sham rats (8.13 ± 1.14 pmol cAMP μg^?1 protein), P < 0.05). Losartan significantly reduced the basal level of cAMP in CHF rats (CHF: 12.56 ± 1.93 fmol μg^?1 protein vs. Los‐CHF: 7.49 ± 1.08, P < 0.05), but not in Sham rats (SHAM: 4.66 ± 0.59 vs. Los‐SHAM: 4.75 ± 0.71). AVP‐mediated cAMP accumulation was absent in both groups treated with losartan (Los‐SHAM: 4.75 ± 0.71 and Los‐CHF: 7.49 ± 1.08). Conclusion: The results indicate that the increased NKCC2 protein level in the mTAL from CHF rats is associated with increased cAMP accumulation in this segment. Furthermore, the finding that AT₁ receptor blockade prevents AVP‐mediated cAMP accumulation in both SHAM and CHF rats suggests an interaction between angiotensin II and AVP in regulation of mTAL Na reabsorption.     

Novel therapeutic compound tuftsin–phosphorylcholine attenuates collagen‐induced arthritis

Treatment with helminthes and helminthes ova improved the clinical symptoms of several autoimmune diseases in patients and in animal models. Phosphorylcholine (PC) proved to be the immunomodulatory molecule. We aimed to decipher the tolerogenic potential of tuftsin–PC (TPC), a novel helminth‐based compound in collagen‐induced arthritis (CIA) a mouse model of rheumatoid arthritis (RA). CIA DBA/1 mice were treated with TPC subcutaneously (5 µg/0.1 ml) or orally (250 µg/0.1 ml), starting prior to disease induction. The control groups were treated with PBS. Collagen antibodies were tested by enzyme‐linked immunosorbent assay (ELISA), cytokine protein levels by ELISA kits and regulatory T (T_reg) and regulatory B (B_reg) cell phenotypes by fluorescence‐activated cell sorter (FACS). TPC‐treated mice had a significantly lower arthritis score of 1.5 in comparison with control mice 11.8 (P < 0.0001) in both subcutaneous and orally treated groups at day 31. Moreover, histology analysis demonstrated highly inflamed joints in control mice, whereas TPC‐treated mice maintained normal joint structure. Furthermore, TPC decreased the titres of circulating collagen II antibodies in mice sera (P < 0.0001), enhanced expression of IL‐10 (P < 0.0001) and inhibited production of tumour necrosis factor (TNF)‐α, interleukin (IL)?17 and IL‐1β (P < 0.0001). TPC significantly expanded the CD4⁺CD25⁺ forkhead box protein 3 (FoxP3⁺) T_reg cells and CD19⁺IL‐10⁺CD5^highCD1d^highT cell immunoglobulin mucin‐1 (TIM‐1⁺) B_reg cell phenotypes (P < 0.0001) in treated mice. Our data indicate that treatment with TPC attenuates CIA in mice demonstrated by low arthritic score and normal joints histology. TPC treatment reduced proinflammatory cytokines and increased anti‐inflammatory cytokine expression, as well as expansion of T_reg and B_reg cells. Our results may lead to a new approach for a natural therapy for early rheumatoid arthritis onset.     

T2 measurement of J-coupled metabolites in the human brain at 3T

Proton T₂ relaxation times of metabolites in the human brain were measured using point resolved spectroscopy at 3T in vivo. Four echo times (54, 112, 246 and 374 ms) were selected from numerical and phantom analyses for effective detection of the glutamate multiplet at ~ 2.35 ppm. In vivo data were obtained from medial and left occipital cortices of five healthy volunteers. The cortices contained predominantly gray and white matter, respectively. Spectra were analyzed with LCModel software using volume‐localized calculated spectra of brain metabolites. The estimate of the signal strength vs. TE was fitted to a monoexponential function for estimation of apparent T₂ (T₂^?). T₂^? was estimated to be similar between the brain regions for creatine, choline, glutamate and myo‐inositol, but significantly different for N‐acetylaspartate singlet and multiplet. T₂^?s of glutamate and myo‐inositol were measured as 181 ± 16 and 197 ± 14 ms (mean ± SD, N = 5) for medial occipital cortices, and 180 ± 12 and 196 ± 17 ms for left occipital cortices, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Structure and mineralisation density of antler and pedicle bone in red deer (Cervus elaphus L.) exposed to different levels of environmental fluoride: a quantitative backscattered electron imaging study

The structure and relative degree of mineralisation of antler and pedicle bone of yearling red deer stags exposed either to low or high levels of environmental fluoride were determined by digital quantitative backscattered electron (BSE) imaging. Bone fluoride content (BFC) in antlers (845±86 mg F^?/kg ash, arithmetic mean±S.E.M. ) and pedicles (1448±154 mg F^?/kg ash) of deer from a highly fluoride polluted area in North Bohemia (Czech Republic) were significantly higher (P < 0.001) than those of controls from uncontaminated regions in West Germany (antlers: 206±41, pedicles: 322±52 mg F^?/kg ash). Mean (56.5±4.5%) and maximum (84.9±2.1%) mineralised bone area of the control antlers significantly (P < 0.05 and P < 0.001, respectively) exceeded the corresponding values for the N. Bohemian deer (43.3±1.3 and 73.3±1.9%, respectively), while the pedicles from the 2 groups did not differ significantly. In the pooled antler samples (n = 18), negative correlations existed between BFC and mean (r_s = ?0.62, P < 0.01) as well as maximum (r_s = ?0.69, P < 0.01) mineralised bone area. Morphological imaging revealed a decreased width and an increased porosity of the antler cortex in the N. Bohemian specimens. Mean (148.5±1.7) and maximum (154.2±1.7) BSE‐signal intensities (= grey levels; range between a monobrominated (grey level 0) and a monoiodinated (grey level 255) dimethacrylate resin standard) of the antlers from the controls were significantly higher than those of the N. Bohemian deer (140.7±2.1 and 145.7±2.2, respectively; P < 0.05 for both comparisons). In the pooled antler samples, negative correlations between BFC and mean (r_s = ?0.51, P < 0.05) as well as maximum (r_s = ?0.52, P < 0.05) BSE‐signal intensities were observed. No significant differences in mineralisation density parameters were found for the 2 pedicle samples, and BFC and mineralisation density of the pooled pedicles were uncorrelated. Morphological imaging revealed bone mottling (denoting increased remodelling activity) and frequent occurrence of apparently increased osteocyte lacunae in some of the pedicles from the N. Bohemian deer. It is concluded that the reduced amount of mineralised bone in, and the lower mineralisation density of, the N. Bohemian antlers resulted from a fluoride induced disturbance of bone mineralisation. The rapid growth of antlers leads both to a high mineral demand and a high rate of fluoride uptake during antlerogenesis. This, and the limited lifespan of antlers, which does not allow for a compensation of a delay in the onset or progression of the mineralisation process, renders antler bone particularly susceptible to fluoride. Antlers are therefore considered a useful model for studying fluoride effects on bone formation. Furthermore, analysis of cast antlers enables a noninvasive monitoring of environmental pollution by fluorides.     

Low numbers of CD8+ T lymphocytes in hereditary haemochromatosis are explained by a decrease of the most mature CD8+ effector memory T cells

Low CD8⁺ T lymphocyte numbers have long been described in hereditary haemochromatosis (HH). Recently, two conserved haplotypes localized near the microsatellite D6S105 at the major histocompatibility complex (MHC) class I region were described predicting the clinical expression of HH and the CD8⁺ T lymphocyte numbers. The A‐A‐T haplotype was associated with a severe clinical expression of HH and low CD8⁺ T lymphocyte numbers, while the G‐G‐G haplotype was associated with a milder clinical expression of HH and high CD8⁺ T lymphocyte numbers. As CD8⁺ T lymphocytes are a very heterogeneous population, in this study we analysed the CD8⁺ subpopulations of naive, central memory (T_CM) and effector memory (T_EM), and further subsets of CD8⁺ T_EM cells in 47 HH patients and 68 controls. In addition, association studies were conducted between the conserved haplotypes and the CD8⁺ T cell subpopulations in HH. Variations of the numbers of naive and central memory cells with age were similar between HH patients and controls. For T_EM cells and the T_EM CD27^‐CD28^‐ subset no effect of age was observed in HH [R² = 0·001, not significant (n.s.) and R² = 0·01, n.s., respectively] contrasting with the increasing of these subpopulations with age in controls (R² = 0·09, P = 0·017 and R² = 0·22, P = 0·0005, respectively). Interestingly, patients homozygous for the A‐A‐T haplotype have lower numbers of CD8⁺ T_EM cells due especially to lower numbers of T_EM CD27^‐CD28^‐ (0·206 ± 0·119 and 0·066 ± 0·067 × 10⁶ cells/ml, respectively) than patients carrying the G‐G‐G haplotype (0·358 ± 0·195 and 0·246 ± 0·202 × 10⁶ cells/ml, respectively). This may suggest an inability of HH patients to differentiate the CD8⁺ T cells into the most mature phenotype.     

In vivo 31P magnetic resonance spectroscopy of the human liver at 7 T: an initial experience

Phosphorus (³¹P) MRS is a powerful tool for the non‐invasive investigation of human liver metabolism. Four in vivo ³¹P localization approaches (single voxel image selected in vivo spectroscopy (3D‐ISIS), slab selective 1D‐ISIS, 2D chemical shift imaging (CSI), and 3D‐CSI) with different voxel volumes and acquisition times were demonstrated in nine healthy volunteers. Localization techniques provided comparable signal‐to‐noise ratios normalized for voxel volume and acquisition time differences, Cramer–Rao lower bounds (8.7 ± 3.3%_1D‐ISIS, 7.6 ± 2.5%_3D‐ISIS, 8.6 ± 4.2%_2D‐CSI, 10.3 ± 2.7%_3D‐CSI), and linewidths (50 ± 24 Hz_1D‐ISIS, 34 ± 10 Hz_3D‐ISIS, 33 ± 10 Hz_2D‐CSI, 34 ± 11 Hz_3D‐CSI). Longitudinal (T₁) relaxation times of human liver metabolites at 7 T were assessed by 1D‐ISIS inversion recovery in the same volunteers (n = 9). T₁ relaxation times of hepatic ³¹P metabolites at 7 T were the following: phosphorylethanolamine – 4.41 ± 1.55 s; phosphorylcholine – 3.74 ± 1.31 s; inorganic phosphate – 0.70 ± 0.33 s; glycerol 3‐phosphorylethanolamine – 6.19 ± 0.91 s; glycerol 3‐phosphorylcholine – 5.94 ± 0.73 s; γ‐adenosine triphosphate (ATP) – 0.50 ± 0.08 s; α‐ATP – 0.46 ± 0.07 s; β‐ATP – 0.56 ± 0.07 s. The improved spectral resolution at 7 T enabled separation of resonances in the phosphomonoester and phosphodiester spectral region as well as nicotinamide adenine dinucleotide and uridine diphosphoglucose signals. An additional resonance at 2.06 ppm previously assigned to phosphoenolpyruvate or phosphatidylcholine is also detectable. These are the first ³¹P metabolite relaxation time measurements at 7 T in human liver, and they will help in the exploration of new, exciting questions in metabolic research with 7 T MR. Copyright © 2014 John Wiley & Sons, Ltd.     

Adiabatic multi‐echo 31P spectroscopic imaging (AMESING) at 7 T for the measurement of transverse relaxation times and regaining of sensitivity in tissues with short T2* values

An adiabatic multi‐echo spectroscopic imaging (AMESING) sequence, used for ³¹P MRSI, with spherical k‐space sampling and compensated phase‐encoding gradients, was implemented on a whole‐body 7‐T MR system. One free induction decay (FID) and up to five symmetric echoes can be acquired with this sequence. In tissues with low T₂* and high T₂, this can theoretically lead to a potential maximum signal‐to‐noise ratio (SNR) increase of almost a factor of three, compared with a conventional FID acquisition with Ernst‐angle excitation. However, with T₂ values being, in practice, ≤400 ms, a maximum enhancement of approximately two compared with low flip Ernst‐angle excitation should be feasible. The multi‐echo sequence enables the determination of localized T₂ values, and was validated with ³¹P three‐dimensional MRSI on the calf muscle and breast of a healthy volunteer, and subsequently applied in a patient with breast cancer. The T₂ values of phosphocreatine, phosphodiesters (PDE) and inorganic phosphate in calf muscle were 193 ± 5 ms, 375 ± 44 ms and 96 ± 10 ms, respectively, and the apparent T₂ value of γ‐ATP was 25 ± 6 ms. A T₂ value of 136 ± 15 ms for inorganic phosphate was measured in glandular breast tissue of a healthy volunteer. The T₂ values of phosphomonoesters (PME) and PDE in breast cancer tissue (ductulolobular carcinoma) ranged between 170 and 210 ms, and the PME to PDE ratios were calculated to be phosphoethanolamine/glycerophosphoethanolamine = 2.7, phosphocholine/glycerophosphocholine = 1.8 and PME/PDE = 2.3. Considering the relatively short T₂* values of the metabolites in breast tissue at 7 T, the echo spacing can be short without compromising spectral resolution, whilst maximizing the sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.