Diffusion behavior of cerebral metabolites of congenital portal systemic shunt mice assessed noninvasively by diffusion‐weighted 1H magnetic resonance spectroscopy

Diffusion‐weighted ¹H‐MRS (DW‐MRS) allows for noninvasive investigation of the cellular compartmentalization of cerebral metabolites. DW‐MRS applied to the congenital portal systemic shunt (PSS) mouse brain may provide specific insight into alterations of cellular restrictions associated with PSS in humans. At 14.1 T, adult male PSS and their age‐matched healthy (Ctrl) mice were studied using DW‐MRS covering b‐values ranging from 0 to 45 ms/μm² to determine the diffusion behavior of abundant metabolites. The remarkable sensitivity and spectral resolution, in combination with very high diffusion weighting, allowed for precise measurement of the diffusion properties of endogenous N‐acetyl‐aspartate, total creatine, myo‐inositol, total choline with extension to glutamine and glutamate in mouse brains, in vivo. Most metabolites had comparable diffusion properties in PSS and Ctrl mice, suggesting that intracellular distribution space for these metabolites was not affected in the model. The slightly different diffusivity of the slow decaying component of taurine (0.015 ± 0.003 μm²/ms in PSS vs 0.021 ± 0.002 μm²/ms in Ctrl, P < 0.05) might support a cellular redistribution of taurine in the PSS mouse brain.     

Sexual dimorphism in hepatic lipids is associated with the evolution of metabolic status in mice

Ectopic lipid accumulation in the liver is implicated in metabolic disease in an age‐ and sex‐dependent manner. The role of hepatic lipids has been well established within the scope of metabolic insults in mice, but has been insufficiently characterized under standard housing conditions, where age‐related metabolic alterations are known to occur. We studied a total of 10 male and 10 female mice longitudinally. At 3, 7 and 11 months of age, non‐invasive ¹H‐magnetic resonance spectroscopy (¹H‐MRS) was used to monitor hepatic lipid content (HLC) and fatty acid composition in vivo, and glucose homeostasis was assessed with glucose and insulin challenges. At the end of the study, hepatic lipids were comprehensively characterized by nuclear magnetic resonance (NMR) and liquid chromatography‐mass spectrometric analyses of liver tissue samples. In males, HLC increased from 1.4 ± 0.1% at 3 months to 2.9 ± 0.3% at 7 months (p < 0.01) and 2.7 ± 0.3% at 11 months (p < 0.05), in correlation with fasting insulin levels (p < 0.01, r = 0.51) and parameters from the insulin tolerance test (ITT; p < 0.001, r = –0.69 versus area under the curve; p < 0.01, r = –0.57 versus blood glucose drop at 1 h post‐ITT; p < 0.01, r = 0.55 versus blood glucose at 3 h post‐ITT). The metabolic performance of females remained the same throughout the study, and HLC was higher than that of males at 3 months (2.7 ± 0.2%, p < 0.01), but comparable at 7 months (2.2 ± 0.2%) and 11 months (2.2 ± 0.1%). Strong sexual dimorphism in bioactive lipid species, including diacylglycerols (higher in males, p < 0.0001), phosphatidylinositols (higher in females, p < 0.001) and omega‐3 polyunsaturated fatty acids (higher in females, p < 0.01), was found to be in good correlation with metabolic scores at 11 months. Therefore, in mice housed under standard conditions, sex‐specific composition of bioactive lipids is associated with metabolic protection in females, whose metabolic performance was independent of hepatic cytosolic lipid content.     

MRI reveals increased tumorigenesis following high fat feeding in a mouse model of triple‐negative breast cancer

High animal fat consumption is associated with an increase in triple‐negative breast cancer (TNBC) risk. Based on previous MRI studies demonstrating the feasibility of detecting very early non‐palpable mammary cancers in simian virus 40 large T antigen (SV40TAg) mice, we examined the effect of dietary fat fed from weaning to young adulthood in this model of TNBC. Virgin female C3(1)SV40TAg mice (n = 16) were weaned at 3–4 weeks of age and then fed either a low fat diet (LFD) (n = 8, 3.7 kcal/g; 17.2% kcal from vegetable oil) or a high animal fat diet (HAFD) (n = 8, 5.3 kcal/g; 60% kcal from lard). After 8 weeks on the diet (12 weeks of age), fast spin echo MR images of inguinal mammary glands were acquired at 9.4 T. Following in vivo MRI, mice were sacrificed and inguinal mammary glands were excised and formalin fixed for ex vivo MRI. 3D volume‐rendered MR images were then correlated with mammary gland histology to assess the glandular parenchyma and tumor burden. Using in vivo MRI, an average of 3.88 ± 1.03 tumors were detected per HAFD‐fed mouse compared with an average of 1.25 ± 1.16 per LFD‐fed mouse (p < 0.007). Additionally, the average tumor volume was significantly higher following HAFD feeding (0.53 ± 0.45 mm³) compared with LFD feeding (0.20 ± 0.08 mm³, p < 0.02). Analysis of ex vivo MR and histology images demonstrated that HAFD mouse mammary glands had denser parenchyma, irregular and enlarged ducts, dilated blood vessels, increased white adipose tissue, and increased tumor invasion. MRI and histological studies of the SV40TAg mice demonstrated that HAFD feeding also resulted in higher cancer incidence and larger mammary tumors. Unlike other imaging methods for assessing environmental effects on mammary cancer growth, MRI allows routine serial measurements and reliable detection of small cancers as well as accurate tumor volume measurements and assessment of the three‐dimensional distribution of tumors over time.     

Prediction of pathologic response to neoadjuvant chemotherapy in patients with breast cancer using diffusion‐weighted imaging and MRS

The aim of this study was to determine whether tumor size, MRS parameters and apparent diffusion coefficient (ADC) measurements could be applied to predict pathologic complete response (pCR) after neoadjuvant chemotherapy (NAC). Ninety patients with breast cancer (median size, 4.5 cm; range, 1.6–9.5 cm) were evaluated with single‐voxel ¹H MRS and dynamic contrast‐enhanced MRI. Diffusion‐weighted imaging was performed in 41 of these patients using a 1.5‐T scanner before and after completion of NAC. Pre‐ and post‐treatment measurements and changes in tumor size, MRS parameters [absolute and normalized total choline‐containing compound (tCho) integral and tCho signal‐to‐noise ratio (SNR)] and ADCs in pCR versus non‐pCR were compared using the nonparametric Mann–Whitney test. Receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic performance of each parameter. After NAC, 30 patients (33%) showed pCR and 60 (67%) showed non‐pCR. At pretreatment, ADC was the only significant parameter in differentiating between pCR and non‐pCR [(0.83 ± 0.05) × 10^–3 versus (0.97 ± 0.14) × 10^–3 mm²/s] (p = 0.014). Post‐treatment measurements after completion of NAC and changes in tumor size (both p < 0.001), MRS parameters (p = 0.027 and p = 0.020 for absolute tCho integral, p = 0.036 and p = 0.023 for normalized tCho integral, and p = 0.032 and p = 0.061 for tCho SNR) and ADC (p = 0.003 and p < 0.001) were significantly different between the pCR and non‐pCR groups, except for changes in tCho SNR. In ROC analysis, the areas under the ROC curve (AUCs) of 0.63–0.73 were obtained for tumor size and MRS parameters. AUCs for pre‐ and post‐treatment ADC and changes in ADC were 0.75, 0.80 and 0.96, respectively. The optimal cut‐off of the percentage change in ADC for predicting pCR was 40.7%, yielding 100% sensitivity and 91% specificity. Patients with pCR showed significantly lower pretreatment ADCs than those with non‐pCR. The change in ADC after NAC was the most accurate predictor of pCR. Copyright © 2012 John Wiley & Sons, Ltd.     

Bone mineral 31P and matrix‐bound water densities measured by solid‐state 31P and 1H MRI

Bone is a composite material consisting of mineral and hydrated collagen fractions. MRI of bone is challenging because of extremely short transverse relaxation times, but solid‐state imaging sequences exist that can acquire the short‐lived signal from bone tissue. Previous work to quantify bone density via MRI used powerful experimental scanners. This work seeks to establish the feasibility of MRI‐based measurement on clinical scanners of bone mineral and collagen‐bound water densities, the latter as a surrogate of matrix density, and to examine the associations of these parameters with porosity and donors’ age. Mineral and matrix‐bound water images of reference phantoms and cortical bone from 16 human donors, aged 27–97 years, were acquired by zero‐echo‐time 31‐phosphorus (³¹P) and 1‐hydrogen (¹H) MRI on whole body 7T and 3T scanners, respectively. Images were corrected for relaxation and RF inhomogeneity to obtain density maps. Cortical porosity was measured by micro‐computed tomography (μCT), and apparent mineral density by peripheral quantitative CT (pQCT). MRI‐derived densities were compared to X‐ray‐based measurements by least‐squares regression. Mean bone mineral ³¹P density was 6.74 ± 1.22 mol/l (corresponding to 1129 ± 204 mg/cc mineral), and mean bound water ¹H density was 31.3 ± 4.2 mol/l (corresponding to 28.3 ± 3.7 %v/v). Both ³¹P and bound water (BW) densities were correlated negatively with porosity (³¹P: R² = 0.32, p < 0.005; BW: R² = 0.63, p < 0.0005) and age (³¹P: R² = 0.39, p < 0.05; BW: R² = 0.70, p < 0.0001), and positively with pQCT density (³¹P: R² = 0.46, p < 0.05; BW: R² = 0.50, p < 0.005). In contrast, the bone mineralization ratio (expressed here as the ratio of ³¹P density to bound water density), which is proportional to true bone mineralization, was found to be uncorrelated with porosity, age or pQCT density. This work establishes the feasibility of image‐based quantification of bone mineral and bound water densities using clinical hardware. Copyright © 2014 John Wiley & Sons, Ltd.     

The Effect of Aspirin on Recurrent Fetal Loss in Experimental Antiphospholipid Syndrome

PURPOSE: To evaluate the effect of aspirin treatment upon fetal loss in mice with experimental antiphospholipid syndrome (APLS). MATERIALS AND METHODS: Experimental APLS was induced in pregnant mice by passive transfer of mouse monoclonal anticardiolipin antibody. The mice were treated with high (100μg/d) or low (10μg/d) does of aspirin, using vitaminC(100μg/d or 10μg/d)as a control. The mice were assessed for the presence of lupus anticoagulants (prolonged aPTT), thrombocytopenia, degree of fetal resorption rate and mean embryo and placental weights. RESULTS: The mice with APLS had a higher fetal resorption rate(45.7± 12.2% vs 2.5 ± 0.4%, P<0.001), reduced placenta mean weight(104 ± 8 mgvs 169 ±7mg, P<0.001), prolonged aPTT (94± 14sec vs 39±4sec, P<0.001), and reduced mean platelet count(597± 186 ± 10³/mm³vs 847±51 ± 10³/mm³,P<0.001). The groupof mice with APLS, who were treated with low-dose aspirin, had a lower resorption rate (11.1 ±9.3% vs 45.7±12.2%, P<0.001), a higher placenta mean weight (178 ± 8 mg vs 104 ± 8 mg, P<0.001), a higher mean embryo weight (1042 ± 134 mg vs 721±91 mg, P<0.001), and a lower aPTT (58±15 sec vs 94±14 sec, P, <0.001). Micewho were treated with high-dose aspirin also had a lower resorption rate, although not as much as in the low-dose aspirin group (34.2 ± 12.7% vs 45.7 ± 12.2%, P<0.001). CONCLUSION: Aspirin, especially in low dose, has a protective effect against obstetrical complications associated with experimental APLS.     

Neurometabolic profiles of the substantia nigra and striatum of MPTP‐intoxicated common marmosets: An in vivo proton MRS study at 9.4 T

Given the strong coupling between the substantia nigra (SN) and striatum (STR) in the early stage of Parkinson's disease (PD), yet only a few studies reported to date that have simultaneously investigated the neurochemistry of these two brain regions in vivo, we performed longitudinal metabolic profiling in the SN and STR of 1‐methyl‐1,2,3,6‐tetrahydropyridine (MPTP)‐intoxicated common marmoset monkey models of PD (n = 10) by using proton MRS (¹H–MRS) at 9.4 T. T₂ relaxometry was also performed in the SN by using MRI. Data were classified into control, MPTP_2weeks, and MPTP_6‐10 weeks groups according to the treatment duration. In the SN, T₂ of the MPTP_6‐10 weeks group was lower than that of the control group (44.33 ± 1.75 versus 47.21 ± 2.47 ms, p < 0.05). The N‐acetylaspartate to total creatine ratio (NAA/tCr) and γ‐aminobutyric acid to tCr ratio (GABA/tCr) of the MPTP_6‐10 weeks group were lower than those of the control group (0.41 ± 0.04 versus 0.54 ± 0.08 (p < 0.01) and 0.19 ± 0.03 versus 0.30 ± 0.09 (p < 0.05), respectively). The glutathione to tCr ratio (GSH/tCr) was correlated with T₂ for the MPTP_6‐10 weeks group (r = 0.83, p = 0.04). In the STR, however, GABA/tCr of the MPTP_6‐10 weeks group was higher than that of the control group (0.25 ± 0.10 versus 0.16 ± 0.05, p < 0.05). These findings may be an in vivo depiction of the altered basal ganglion circuit in PD brain resulting from the degeneration of nigral dopaminergic neurons and disruption of nigrostriatal dopaminergic projections. Given the important role of non‐human primates in translational studies, our findings provide better understanding of the complicated evolution of PD.     

Diffusion‐weighted MRI monitoring of pancreatic cancer response to radiofrequency heat‐enhanced intratumor chemotherapy

The aim of this study was to evaluate the feasibility of using diffusion‐weighted MRI to monitor the early response of pancreatic cancers to radiofrequency heat (RFH)‐enhanced chemotherapy. Human pancreatic carcinoma cells (PANC‐1) in different groups and 24 mice with pancreatic cancer xenografts in four groups were treated with phosphate‐buffered saline (PBS) as a control, RFH at 42 °C, gemcitabine and gemcitabine plus RFH at 42 °C. One day before and 1, 7 and 14 days after treatment, diffusion‐weighted MRI and T₂‐weighted imaging were applied to monitor the apparent diffusion coefficients (ADCs) of tumors and tumor growth. MRI findings were correlated with the results of tumor apoptosis analysis. In the in vitro experiments, the quantitative viability assay showed lower relative cell viabilities for treatment with gemcitabine plus RFH at 42 °C relative to treatment with RFH only and gemcitabine only (37 ± 5% versus 65 ± 4% and 58 ± 8%, respectively, p < 0.05). In the in vivo experiments, the combination therapy resulted in smaller relative tumor volumes than RFH only and chemotherapy only (0.82 ± 0.17 versus 2.23 ± 0.90 and 1.64 ± 0.44, respectively, p = 0.003). In vivo, 14‐T MRI demonstrated a remarkable decrease in ADCs at day 1 and increased ADCs at days 7 and 14 in the combination therapy group. The apoptosis index in the combination therapy group was significantly higher than those in the chemotherapy‐only, RFH‐only and PBS treatment groups (37 ± 6% versus 20 ± 5%, 8 ± 2% and 3 ± 1%, respectively, p < 0.05). This study confirms that it is feasible to use MRI to monitor RFH‐enhanced chemotherapy in pancreatic cancers, which may present new options for the efficient treatment of pancreatic malignancies using MRI/RFH‐integrated local chemotherapy. Copyright © 2013 John Wiley & Sons, Ltd.     

Initial investigation of glucose metabolism in mouse brain using enriched 17O‐glucose and dynamic 17O‐MRS

In this initial work, the in vivo degradation of ¹⁷O‐labeled glucose was studied during cellular glycolysis. To monitor cellular glucose metabolism, direct ¹⁷O‐magnetic resonance spectroscopy (MRS) was used in the mouse brain at 9.4 T. Non‐localized spectra were acquired with a custom‐built transmit/receive (Tx/Rx) two‐turn surface coil and a free induction decay (FID) sequence with a short TR of 5.4 ms. The dynamics of labeled oxygen in the anomeric 1‐OH and 6‐CH₂OH groups was detected using a Hankel–Lanczos singular value decomposition (HLSVD) algorithm for water suppression. Time‐resolved ¹⁷O‐MRS (temporal resolution, 42/10.5 s) was performed in 10 anesthetized (1.25% isoflurane) mice after injection of a 2.2 M solution containing 2.5 mg/g body weight of differently labeled ¹⁷O‐glucose dissolved in 0.9% physiological saline. From a pharmacokinetic model fit of the H₂¹⁷O concentration–time course, a mean apparent cerebral metabolic rate of ¹⁷O‐labeled glucose in mouse brain of CMR_Glc = 0.07 ± 0.02 μmol/g/min was extracted, which is of the same order of magnitude as a literature value of 0.26 ± 0.06 μmol/g/min reported by ¹⁸F‐fluorodeoxyglucose (¹⁸F‐FDG) positron emission tomography (PET). In addition, we studied the chemical exchange kinetics of aqueous solutions of ¹⁷O‐labeled glucose at the C1 and C6 positions with dynamic ¹⁷O‐MRS. In conclusion, the results of the exchange and in vivo experiments demonstrate that the C6‐¹⁷OH label in the 6‐CH₂OH group is transformed only glycolytically by the enzyme enolase into the metabolic end‐product H₂¹⁷O, whereas C1‐¹⁷OH ends up in water via direct hydrolysis as well as glycolysis. Therefore, dynamic ¹⁷O‐MRS of highly labeled ¹⁷O‐glucose could provide a valuable non‐radioactive alternative to FDG PET in order to investigate glucose metabolism.     

Cerebral hemodynamics and pseudo‐continuous arterial spin labeling considerations in adults with sickle cell anemia

Sickle cell anemia (SCA) is a genetic disorder resulting in reduced oxygen carrying capacity and elevated stroke risk. Pseudo‐continuous arterial spin labeling (pCASL) measures of cerebral blood flow (CBF) may have relevance for stroke risk assessment; however, the effects of elevated flow velocity and reduced bolus arrival time (BAT) on CBF quantification in SCA patients have not been thoroughly characterized, and pCASL model parameters used in healthy adults are often applied to patients with SCA. Here, cervical arterial flow velocities and pCASL labeling efficiencies were computed in adults with SCA (n = 19) and age‐ and race‐matched controls without sickle trait (n = 7) using pCASL in sequence with phase contrast MR angiography (MRA). Controls (n = 7) and a subgroup of patients (n = 8) also underwent multi‐post‐labeling‐delay pCASL for BAT assessment. Mean flow velocities were elevated in SCA adults (velocity = 28.3 ± 4.1 cm/s) compared with controls (velocity = 24.5 ± 3.8 cm/s), and mean pCASL labeling efficiency (α) was reduced in SCA adults (α = 0.72) relative to controls (α = 0.91). In patients, mean whole‐brain CBF from phase contrast MRA was 91.8 ± 18.1 ml/100 g/min, while mean pCASL CBF when assuming a constant labeling efficiency of 0.86 was 75.2 ± 17.3 ml/100 g/min (p < 0.01), resulting in a mean absolute quantification error of 23% when a labeling efficiency appropriate for controls was assumed. This difference cannot be accounted for by BAT (whole‐brain BAT: control, 1.13 ± 0.06 s; SCA, 1.02 ± 0.09 s) or tissue T₁ variation. In conclusion, BAT variation influences pCASL quantification less than elevated cervical arterial velocity and labeling efficiency variation in SCA adults; thus, a lower labeling efficiency (α = 0.72) or subject‐specific labeling efficiency should be incorporated for SCA patients.     

Rituximab‐induced interleukin‐15 reduction associated with clinical improvement in rheumatoid arthritis

Rituximab therapy alters all aspects of B‐cell participation in the disturbed immune response of rheumatoid arthritis patients. To determine the impact of B‐cell depletion on other immune compartments, we analysed levels of soluble and surface interleukin‐15 (IL‐15) along with the frequency of IL‐15‐related subsets after rituximab treatment. We then studied the correlation of observed changes with clinical activity. Heparinized blood samples from 33 rheumatoid arthritis patients were collected on days 0, 30, 90 and 180 after each of three rituximab cycles. Serum cytokine levels were determined by ELISA. Interleukin‐15 trans‐presentation was analysed by cytometry. Flow cytometry with monoclonal antibodies was performed to analyse circulating cell subsets. Interleukin‐15 was detected in the serum of 25 patients before initiating the treatment. Rituximab then progressively reduced serum IL‐15 (138 ± 21 pg/ml at baseline, 48 ± 18 pg/ml after third cycle, P = 0·03) along with IL‐17 (1197 ± 203 pg/ml at baseline, 623 ± 213 pg/ml after third cycle, P = 0·03) and tended to increase the frequency of circulating regulatory T cells (3·1 ± 1 cells/μl at baseline, 7·7 ± 2 cells/μl after third cycle). Rituximab also significantly decreased IL‐15 trans‐presentation on surface monocytes of patients negative for IL‐15 serum (mean fluorescence intensity: 4·82 ± 1·30 at baseline, 1·42 ± 0·69 after third cycle P = 0·05). Reduction of serum IL‐15 was associated with decrease in CD8⁺ CD45RO⁺/RA⁺ ratio (1·17 ± 0·21 at baseline, 0·36 ± 0·06 at third cycle, P = 0·02). DAS28, erythrocyte sedimentation rate and C‐reactive protein correlated significantly with CD8⁺ CD45RO⁺/RA⁺ ratio (R = 0·323, R = 0·357, R = 0·369 respectively, P < 0·001). Our results suggest that sustained clinical improvement after rituximab treatment is associated with IL‐15/memory T‐cell‐related mechanisms beyond circulating B cells.     

Diversity and association of HLA/KIR receptors with type 2 diabetes in South India

The present study was undertaken to delineate the association(s) of KIR–HLA combination in South Indian Type 2 diabetes mellitus (T2DM) patients. The T2DM patients (n = 343) and healthy controls (n = 309) were genotyped for KIR/HLA ligands by PCR‐SSP method. The increased frequency of activatory KIR (aKIR) 2DS2 (OR = 1.91; p < 2.91 × 10^?4) was observed in patients suggesting a susceptible association. The frequencies of iKIR 2DL2 (OR = 0.38; p < 1.55 × 10^?5) and aKIRs 2DS1 (OR = 0.60; p < 0.001) and 3DS1 (OR = 0.52; p < 5.83 × 10^?5) were decreased in patients suggesting protective associations. The C1/C2 combinatorial analysis has revealed an increased frequency of C1⁺/C2^? in T2DM patients (OR = 1.62; p < 0.014). The KIR “AB” genotype (OR = 2.41; p < 3.87 × 10^?5) was observed to be higher in patients. However, the “BB” genotype (OR = 0.32; p < 4.71 × 10^?7) was increased in controls. The KIR motifs, “Tel‐B/B” (OR = 1.84; p < 0.007), were observed higher among patients. However, the frequency of “Tel‐A/B” motif genotype was decreased in patients (OR = 0.56; p < 3.13 × 10^?4). The iKIR/HLA combinations such as 2DL2/3 +C1 and 3DL2+A3/A11 were increased in patients (OR = 3.90; p < 7.5 × 10^?5) suggesting susceptible associations. On the contrary, the aKIR+HLA combinations such as 2DS2+C1, 2DS1+C2 and 3DS1+Bw4 were less frequent in patients (OR = 0.32; p < 4.2 × 10^?4) suggesting protective associations. Thus, the present study clearly establishes the positive and negative associations of different KIR–HLA receptor combinations with T2DM in South India.     

Lingo‐1 Inhibited by RNA Interference Promotes Functional Recovery of Experimental Autoimmune Encephalomyelitis

Lingo‐1 is a negative regulator of myelination. Repairment of demyelinating diseases, such as multiple sclerosis (MS)/experimental autoimmune encephalomyelitis (EAE), requires activation of the myelination program. In this study, we observed the effect of RNA interference on Lingo‐1 expression, and the impact of Lingo‐1 suppression on functional recovery and myelination/remyelination in EAE mice. Lentiviral vectors encoding Lingo‐1 short hairpin RNA (LV/Lingo‐1‐shRNA) were constructed to inhibit Lingo‐1 expression. LV/Lingo‐1‐shRNA of different titers were transferred into myelin oligodendrocyte glycoprotein‐induced EAE mice by intracerebroventricular (ICV) injection. Meanwhile, lentiviral vectors carrying nonsense gene sequence (LVCON053) were used as negative control. The Lingo‐1 expression was detected and locomotor function was evaluated at different time points (on days 1,3,7,14,21, and 30 after ICV injection). Myelination was investigated by luxol fast blue (LFB) staining.LV/Lingo‐1‐shRNA administration via ICV injection could efficiently down‐regulate the Lingo‐1 mRNA and protein expression in EAE mice on days 7,14,21, and 30 (P < 0.01), especially in the 5 × 10⁸ TU/mL and 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups. The locomotor function score in the LV/Lingo‐1‐shRNA treated groups were significantly lower than the untreated or LVCON053 group from day 7 on. The 5 × 10⁸ TU/mL LV/Lingo‐1‐shRNA group achieved the best functional improvement (0.87 ± 0.11 vs. 3.05 ± 0.13, P < 0.001). Enhanced myelination/remyelination was observed in the 5 × 10⁷, 5 × 10⁸, 5 × 10⁹ TU/mL LV/Lingo‐1‐shRNA groups by LFB staining (P < 0.05, P < 0.01, and P < 0.05).The data showed that administering LV/Lingo‐1‐shRNA by ICV injection could efficiently knockdown Lingo‐1 expression in vivo, improve functional recovery and enhance myelination/remyelination. Antagonism of Lingo‐1 by RNA interference is, therefore, a promising approach for the treatment of demyelinating diseases, such as MS/EAE. Anat Rec, 297:2356–2363, 2014. © 2014 Wiley Periodicals, Inc.     

Additive hypothermic effects of the 5‐HT1A receptor agonist 8‐OH‐DPAT and the dopamine D2/3 receptor agonist 7‐OH‐DPAT in the rat

The objective of this study was to examine possible interactions between serotonergic and dopaminergic agents lowering core temperature via stimulation of 5‐HT_1A and dopamine (DA) D₂ receptors, respectively. The effects of the 5‐HT_1A receptor agonist (±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin HBr (8‐OH‐DPAT) and the DA D_2/3 receptor agonist 7‐OH‐DPAT on core temperature was monitored in adult male Wistar rats, approximately 300 g body weight. The temperature probe was connected to a PC‐assisted temperature instrument, and an automated printer device was activated when the temperature reading had stabilized (±0.1 °C) for 10 s. As expected, 7‐OH‐DPAT [0.5 and 2.0 μmol kg^–1 subcutaneous (s.c.)] as well as 8‐OH‐DPAT (0.15–2.4 μmol kg^–1 s.c.), produced a dose‐dependent hypothermia. When combined, there were additive effects of the two compounds, although the effects of 7‐OH‐DPAT were attenuated by 8‐OH‐DPAT at the higher doses (0.6–2.4 μmol kg^–1), in all probability because of emerging DA D₂ receptor blocking properties of the latter compound.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

C‐Myc participates in β‐catenin–mediated drug resistance in A549/DDP lung adenocarcinoma cells

The aim of this study was to investigate c‐Myc and β‐catenin–mediated drug resistance in A549/DDP lung adenocarcinoma cells. Cisplatin sensitivity was determined by the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) toxicity assay. β‐Catenin and c‐Myc protein expression following cisplatin treatment were determined using western blotting and immunofluorescence. Flow cytometry was performed to detect cell cycle and apoptosis in A549, A549/DDP, and c‐Myc small interfering RNA (siRNA)–transfected A549/DDP cells before and after treatment with different doses of cisplatin. The median inhibitory concentration (IC₅₀) in cisplatin‐treated A549 and A549/DDP cells was 5.769 ± 0.24 μmol/L and 28.373 ± 0.96 μmol/L, respectively; the cisplatin resistance of A549 cells was about five times that of A549/DDP cells. Endogenous β‐catenin and c‐Myc expression in A549/DDP cells were higher than that in A549 cells, and were upregulated in A549/DDP cells (p < 0.05) and downregulated in A549 cells after 48 h cisplatin treatment (p < 0.05). β‐catenin localization transferred from membrane/cytoplasmic/nuclear to cytoplasmic/nuclear, and c‐Myc localization transferred from cytoplasmic/nuclear to nuclear in both cell lines following cisplatin treatment. The rate of apoptosis increased in a dose‐dependent manner with cisplatin. After 48‐h transfection with c‐myc siRNA, A549/DDP cells were blocked in the S phase, and G0/G1‐phase cells increased. Simultaneously, the apoptotic rate was increased (p < 0.05) and the IC₅₀ decreased significantly (p < 0.05). C‐myc, the downstream target gene of β‐catenin, plays an important role in regulating cisplatin resistance in A549/DDP cells. C‐Myc siRNA improved the sensitivity of A549/DDP cells to cisplatin.     

In vivo mouse myocardial 31P MRS using three‐dimensional image‐selected in vivo spectroscopy (3D ISIS): technical considerations and biochemical validations

³¹P MRS provides a unique non‐invasive window into myocardial energy homeostasis. Mouse models of cardiac disease are widely used in preclinical studies, but the application of ³¹P MRS in the in vivo mouse heart has been limited. The small‐sized, fast‐beating mouse heart imposes challenges regarding localized signal acquisition devoid of contamination with signal originating from surrounding tissues. Here, we report the implementation and validation of three‐dimensional image‐selected in vivo spectroscopy (3D ISIS) for localized ³¹P MRS of the in vivo mouse heart at 9.4 T. Cardiac ³¹P MR spectra were acquired in vivo in healthy mice (n = 9) and in transverse aortic constricted (TAC) mice (n = 8) using respiratory‐gated, cardiac‐triggered 3D ISIS. Localization and potential signal contamination were assessed with ³¹P MRS experiments in the anterior myocardial wall, liver, skeletal muscle and blood. For healthy hearts, results were validated against ex vivo biochemical assays. Effects of isoflurane anesthesia were assessed by measuring in vivo hemodynamics and blood gases. The myocardial energy status, assessed via the phosphocreatine (PCr) to adenosine 5′‐triphosphate (ATP) ratio, was approximately 25% lower in TAC mice compared with controls (0.76 ± 0.13 versus 1.00 ± 0.15; P < 0.01). Localization with one‐dimensional (1D) ISIS resulted in two‐fold higher PCr/ATP ratios than measured with 3D ISIS, because of the high PCr levels of chest skeletal muscle that contaminate the 1D ISIS measurements. Ex vivo determinations of the myocardial PCr/ATP ratio (0.94 ± 0.24; n = 8) confirmed the in vivo observations in control mice. Heart rate (497 ± 76 beats/min), mean arterial pressure (90 ± 3.3 mmHg) and blood oxygen saturation (96.2 ± 0.6%) during the experimental conditions of in vivo ³¹P MRS were within the normal physiological range. Our results show that respiratory‐gated, cardiac‐triggered 3D ISIS allows for non‐invasive assessments of in vivo mouse myocardial energy homeostasis with ³¹P MRS under physiological conditions. Copyright © 2015 John Wiley & Sons, Ltd.     

In vitro and in vivo efficacies of amlodipine against <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

Listeria monocytogenes causes suppurative gastritis in BALB/c mice. We investigated the effect of the antihypertensive drug amlodipine (Aml) on the growth of L. monocytogenes in vitro and in vivo. Aml showed noteworthy inhibitory action (minimum inhibitory concentration, MIC₉₀ 32 μg/ml) against Listeria strains and demonstrated cidal (minimum bactericidal concentration, MBC 64 μg/ml) activity. Aml administered orally at 2.5 μg/g in female BALB/c mice for 7 days, commencing 4 days before oral challenge (1 × 10⁸ CFU/ml with L. monocytogenes ATCC 51774), significantly reduced bacterial counts in the stomach (P < 0.01), liver (P < 0.01), and spleen (P < 0.05), and decreased (P < 0.05) gastric lesions, neutrophilic infiltration, edema, vascular degeneration, and necrosis of gastric tissues. It caused the down-regulation of expression of inflammatory cytokines (IFN-γ, IL-1β, and TNF-α) compared to drug-free control. Aml may be used in the presence of an antibiotic as adjunct therapy that boosts the host immunity against Listeria. Further, QSAR studies might contribute in manipulating it as a lead compound for the synthesis of new, more effective non-antibiotics (helper compounds), perhaps devoid of side-effects, that could be recommended as compassionate therapy for listeriosis.     

Carbohydrate supplementation delays DNA damage in elite runners during intensive microcycle training

The aim of this study was to evaluate the effect of carbohydrate supplementation on free plasma DNA and conventional markers of training and tissue damage in long-distance runners undergoing an overload training program. Twenty-four male runners were randomly assigned to two groups (CHO group and control group). The participants were submitted to an overload training program (days 1–8), followed by a high-intensity intermittent running protocol (10 × 800 m) on day 9. The runners received maltodextrin solution (CHO group) or zero energy placebo solution as the control equivalent before, during, and after this protocol. After 8 days of intensive training, baseline LDH levels remained constant in the CHO group (before: 449.1 ± 18.2, after: 474.3 ± 22.8 U/L) and increased in the control group (from 413.5 ± 23.0 to 501.8 ± 24.1 U/L, p < 0.05). On day 9, LDH concentrations were lower in the CHO group (509.2 ± 23.1 U/L) than in the control group (643.3 ± 32.9 U/L, p < 0.01) post-intermittent running. Carbohydrate ingestion attenuated the increase of free plasma DNA post-intermittent running (48,240.3 ± 5,431.8 alleles/mL) when compared to the control group (73,751.8 ± 11,546.6 alleles/mL, p < 0.01). Leukocyte counts were lower in the CHO group than in the control group post-intermittent running (9.1 ± 0.1 vs. 12.2 ± 0.7 cells/μL; p < 0.01) and at 80 min of recovery (10.6 ± 0.1 vs. 13.9 ± 1.1 cells/μL; p < 0.01). Cortisol levels were positively correlated with free plasma DNA, leukocytes, and LDH (all r > 0.4 and p < 0.001). The results showed that ingestion of a carbohydrate beverage resulted in less DNA damage and attenuated the acute post-exercise inflammation response, providing better recovery during intense training.     

Increased killer B cells in chronic HCV infection may lead to autoimmunity and increased viral load

Regulatory B (B_reg) cells are characterized by various membrane markers and the secretion of different inhibitory cytokines. A new subset of B_reg cells was identified as CD5^hi Fas‐ligand (FasL)^hi. Their main reported role is to suppress anti‐viral and anti‐tumour immune responses, and, hence they have been dubbed ‘killer’ B cells. In this study, we aim to assess the role of these cells in chronic hepatitis C virus (HCV) infection, and determine if they contribute to the increased viral load and persistence of HCV and its related autoimmunity. (i) FasL expression on CD5^hi B cells is increased significantly in HCV‐infected patients compared to healthy individuals [28·06 ± 6·71 mean fluorescence intensity (MFI) ± standard error of the mean (s.e.m.), median = 27·9 versus 10·87 ± 3·97 MFI ± s.e.m., median = 10·3, respectively, P <  0·0001]. (ii) Killer B cells from HCV patients increased autologous CD4⁺ T cell apoptosis compared to the apoptosis in healthy individuals [39·17% ± 7·18% mean ± standard deviation (s.d.), median = 39·6 versus 25·92 ± 8·65%, mean ± s.d., median = 24·1%, P <  0·0001, respectively]. A similar increase was observed in CD8⁺ T cell apoptosis (54·67 ± 15·49% mean ± s.d., median = 57·3 versus 21·07% ± 7·4%, mean ± s.d., median = 20%, P = 0·0006, respectively). (iii) By neutralizing FasL with monoclonal anti‐FasL antibodies, we have shown that the induction of apoptosis by killer B cells is FasL‐dependent. (iv) Increased expression of FasL on CD5^hi B cells is correlated positively with an increased viral load and the presence of anti‐nuclear antibodies and rheumatoid factor in HCV. This is the first study in which killer B cells have been suggested to play a pathogenic role in HCV. They seem to be involved in HCV's ability to escape efficient immune responses.