TPI ASM8 reduces eosinophil progenitors in sputum after allergen challenge

Background TPI ASM8 contains two modified phosphorothioate antisense oligonucleotides (AON), one targeting the common beta chain (βc) of the IL‐3/IL‐5/GM‐CSF receptors and the other targeting the chemokine receptor CCR3. Inhalation of TPI ASM8 significantly improves lung function and sputum eosinophilia after allergen inhalation challenge in asthmatics. Objective This study assessed whether TPI ASM8 reduces airway levels of haemopoietic progenitor cells. Methods This open‐label study was conducted in 14 stable, allergic mild asthmatic subjects with early‐ and late‐phase allergen‐induced bronchoconstriction. Subjects underwent allergen challenges after 4‐day treatment with placebo, 4 mg b.i.d. and 8 mg o.d. of TPI ASM8. Sputum was induced before, 7 and 24 h after allergen challenges for progenitor measurements. Treatments were separated by 2–3 weeks. Results TPI ASM8 reduced allergen‐induced sputum eosinophils, and the early and late asthmatic responses (P<0.05). TPI ASM8 also reduced the number of CD34⁺CCR3⁺ cells (P=0.004) and CD34⁺IL‐5Rα⁺ cells (P=0.016), and the proportion of CD34⁺ cells expressing IL‐5Rα (P=0.036). Conclusions and Clinical Relevance TPI ASM8 was safe and well tolerated. The results of this study demonstrate blocking of CCR3 and βc expression by TPI ASM8 significantly inhibits the accumulation of eosinophils and eosinophil progenitors in the airways after allergen challenge. Inhibition of airway progenitor cell accumulation presents a novel therapeutic target. Cite this as: H. Imaoka, H. Campbell, I. Babirad, R. M. Watson, M. Mistry, R. Sehmi and G. M. Gauvreau, Clinical & Experimental Allergy, 2011 (41) 1740–1746.     

Combined Ex Vivo 9.4T MRI and Quantitative Histopathological Study in Normal and Pathological Neocortical Resections in Focal Epilepsy

High‐resolution magnetic resonance imaging (MRI) may improve the preoperative diagnosis of focal cortical dysplasia (FCD) in epilepsy. Quantitative 9.4T MRI was carried out (T1, T2, T2* and magnetization transfer ratio) on 13 cortical resections, representing pathologically confirmed FCD (five cases) and normal cortex. Quantitative immunohistochemistry for myelination (myelin basic protein/SMI94), neuronal populations [microtubule‐associated protein 2 (MAP2), neurofilament (SMI31, SMI32), synaptophysin, NeuN, calbindin], reactive glia (GFAP), microglia (CD68) and blood–brain barrier permeability (albumin) was carried out in 43 regions of interest (ROI) from normal and abnormal white matter and cortex. MRI was spatially aligned and quantitative analysis carried out on corresponding ROI. Line profile analysis (LPA) of intensity gradients through the cortex was carried out on MRI and immunostained sections. An inverse correlation was noted between myelin/SMI94 and T1, T2 (P < 0.005) and T2* (P < 0.05; Spearman's correlation) and a positive correlation between neuronal MAP2 and T1 (P < 0.005) and T2* (P < 0.05) over all ROI. Similar pathology–MRI correlations were observed for histologically unremarkable white matter ROI only. LPA showed altered gradient contours in regions of FCD, reflecting abnormal cortical lamination and myelo‐architecture, including a preoperatively undetected FCD case. This study demonstrates the ability of quantitative 9.4T MRI to detect subtle differences in neuronal numbers and myelination in histologically normal appearing white matter and LPA in the evaluation of cortical dyslamination. These methods may be translatable to the in vivo detection of mild cortical malformations.     

Expanded neurochemical profile in the early stage of Huntington disease using proton magnetic resonance spectroscopy

The striatum is a well‐known region affected in Huntington disease (HD). However, other regions, including the visual cortex, are implicated. We have identified previously an abnormal energy response in the visual cortex of patients at an early stage of HD using ³¹P magnetic resonance spectroscopy (³¹P MRS). We therefore sought to further characterize these metabolic alterations with ¹H MRS using a well‐validated semi‐localized by adiabatic selective refocusing (semi‐LASER) sequence that allows the measurement of an expanded number of neurometabolites. Ten early affected patients [Unified Huntington Disease Rating Scale (UHDRS), total motor score = 13.6 ± 10.8] and 10 healthy volunteers of similar age and body mass index (BMI) were recruited for the study. We performed ¹H MRS in the striatum – the region that is primarily affected in HD – and the visual cortex. The protocol allowed a reliable quantification of 10 metabolites in the visual cortex and eight in the striatum, compared with three to five metabolites in previous ¹H MRS studies performed in HD. We identified higher total creatine (p < 0.05) in the visual cortex and lower glutamate (p < 0.001) and total creatine (p < 0.05) in the striatum of patients with HD compared with controls. Less abundant neurometabolites [glutamine, γ‐aminobutyric acid (GABA), glutathione, aspartate] showed similar concentrations in both groups. The protocol allowed the measurement of several additional metabolites compared with standard vendor protocols. Our study points to early changes in metabolites involved in energy metabolism in the visual cortex and striatum of patients with HD. Decreased striatal glutamate could reflect early neuronal dysfunction or impaired glutamatergic neurotransmission.     

The effect of AVP‐V1 receptor stimulation on local GFR in the rat kidney

Aim: Arginine vasopressin (AVP) might influence urinary concentration ability by altering the intrarenal distribution of glomerular filtration rate (GFR). Methods: To study this possibility we have measured the intracortical distribution of GFR following acute AVP‐V₁ receptor stimulation in anaesthetized female Sprague–Dawley (SPD) rats during euvolemia and water diuresis by the aprotinin method, allowing two consecutive measurements of zonal GFR in the same kidney. Results: Acute i.v. bolus injection of 50 ng V₁ receptor agonist ([Phe², Ile³, Orn⁸]‐vasopressin) followed by a continuous infusion of 5 ng min^?1 in euvolemic rats reduced GFR by 25% in outer cortex (OC), 20% in middle cortex (MC) and 19% in inner cortex (IC) relative to vehicle infusion (all P < 0.05). In water diuretic rats V₁ receptor agonist reduced GFR by 22% in OC, 10% in MC and 11% in IC relative to vehicle infusion (P < 0.05). GFR decreased slightly more in OC than in MC and IC in both euvolemic and water diuretic rats (P < 0.05) indicating a distribution of GFR towards MC and IC. Acute infusion of the selective non‐peptide V₁ receptor antagonist OPC‐21268 in euvolemic rats reduced GFR by 14% in OC, 13% in MC and 11% in IC relative to vehicle infusion (P < 0.05), with no significant difference between the layers. Conclusions: The change in distribution of GFR not only between OC and IC, but also between OC and MC suggests that the afferent/efferent arterioles and not the medullary vasa recta is the main site of resistance change. We conclude that acute i.v. infusion of V₁ receptor agonist in high doses reduces GFR more in superficial than in deep cortex in both euvolemic and water diuretic rats and that this may be of some importance for water conservation, adding to the V₂‐ receptor effect on water permeability of the collecting ducts.     

Effect of intensive exercise‐induced testicular gametogenic and steroidogenic disorders in mature male Wistar strain rats: a correlative approach to oxidative stress

Aims: In order to investigate the effects of intensive exercise on reproductive dysfunctions in relation to oxidative stress, a total of 12 male rats (age: 3 months, weight: 127 ± 2.86 g) were randomly divided into: (1) control group (CG, n = 6) and (2) experimental group (Exp. G, n = 6). Methods: An exercise protocol of 3 h swimming day^?1, 5 days week^?1 was followed for 4 weeks in Exp. G, with no exercise in CG. All the animals were killed; blood, testes and the accessory sex organs were collected for estimation of different parameters. Results: A significant diminution (P < 0.001) was noted in testicular Δ⁵, 3β‐hydroxy‐steroid dehydrogenase (Δ⁵, 3β‐HSD), 17β‐hydroxy steroid dehydrogenase (17β‐HSD); plasma levels of testosterone, luteinizing hormone (LH); preleptotine spermatocytes (pLSc), midpachytene spermatocytes (mPSc) and stage 7 spermatids (7Sd); with no significant alteration in follicle stimulating hormone (FSH) and spermatogoia A (Asg) after intensive exercise. A significant elevation (P < 0.001) in malondialdehyde (MDA) and conjugated dienes (CD) along with significant reduction (P < 0.001) in glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione‐s‐transferase (GST) and peroxidase were found in testes of Exp. G. Moreover, the somatic index of testes and accessory sex organs were also decreased significantly (P < 0.001) after exercise. High correlations have been found in 17 β‐HSD with CAT (r = 0.90, P < 0.05) and peroxidase (r = 0.83, P < 0.05), epididymal somatic index with CD (r = ?0.91; P < 0.05) and GSH (r = 0.84, P < 0.05). Conclusion: The present study focused an chronic intensive exercise‐induced oxidative stress that may cause dysfunctions in male reproductive system including steroidogenesis and spermatogenesis.     

Glutathione deficiency intensifies ischaemia‐reperfusion induced cardiac dysfunction and oxidative stress

The efficacy of glutathione (GSH) in protecting ischaemia‐reperfusion (I‐R) induced cardiac dysfunction and myocardial oxidative stress was studied in open‐chest, stunned rat heart model. Female Sprague–Dawley rats were randomly divided into three experimental groups: (1) GSH‐depletion, by injection of buthionine sulphoxamine (BSO, 4 mmol kg^–1, i.p.) 24 h prior to I‐R, (2) BSO injection (4 mmol kg^–1, i.p.) in conjunction with acivicin (AT125, 0.05 mmol kg^–1, i.v.) infusion 1 h prior to I‐R, and (3) control (C), receiving saline treatment. Each group was further divided into I‐R, with surgical occlusion of the main left coronary artery (LCA) for 30 min followed by 20 min reperfusion, and sham. Myocardial GSH content and GSH : glutathione disulphide (GSSG) ratio were decreased by ?50% (P < 0.01) in both BSO and BSO + AT125 vs. C. Ischaemia‐reperfusion suppressed GSH in both left and right ventricles of C (P < 0.01) and left ventricles of BSO and BSO + AT125 (P < 0.05). Contractility (+dP/dt and –dP/dt) in C heart decreased 55% (P < 0.01) after I and recovered 90% after I‐R, whereas ±dP/dt in BSO decreased 57% (P < 0.01) with ischaemia and recovered 76 and 84% (P < 0.05), respectively, after I‐R. For BSO + AT125, ±dP/dt were 64 and 76% (P < 0.01) lower after ischaemia, and recovered only 67 and 61% (P < 0.01) after I‐R. Left ventricular systolic pressure in C, BSO and BSO + AT125 reached 95 (P > 0.05) 87 and 82% (P < 0.05) of their respective sham values after I‐R. Rate‐pressure double product was 11% (P > 0.05) and 25% (P < 0.05) lower in BSO and BSO + AT125, compared with Saline, respectively. BSO and BSO + AT125 rats demonstrated significantly lower liver GSH and heart Mn superoxide dismutase activity than C rats after I‐R. These data indicate that GSH depletion by inhibition of its synthesis and transport can exacerbate cardiac dysfunction inflicted by in vivo I‐R. Part of the aetiology may involve impaired myocardial antioxidant defenses and whole‐body GSH homeostasis.     

Diminished CXCR5 expression in peripheral blood of patients with Sjögren's syndrome may relate to both genotype and salivary gland homing

Genetic investigations of Sjögren's syndrome (SS) have identified a susceptibility locus at p23.3 of chromosome 11, which contains the CXCR5 gene. C‐X‐C motif chemokine receptor 5 (CXCR5) is a chemokine receptor expressed on B and T cell subsets, and binds the chemotactic ligand C‐X‐C motif chemokine ligand 13 (CXCL13). In this study we aimed to link the genetic association with functional effects and explore the CXCR5/CXCL13 axis in SS. Expression quantitative trait loci analysis of the 11q23.3 locus was performed using B cell mRNA expression data from genotyped individuals. Lymphocyte surface markers were assessed by flow cytometry, and CXCL13 levels by a proximity extension assay. CXCR5⁺ and CXCL13⁺ cells in minor salivary glands were detected using immunohistochemistry. Our results demonstrated that SS‐associated genetic polymorphisms affected the expression of CXCR5 (P < 0·01). Notably, a decreased percentage of CXCR5⁺ cells, with lower CXCR5 expression, was observed for most circulating B and T cell subsets in SS patients, reaching statistical significance in CD19⁺CD27⁺immunoglobulin (Ig)D⁺ marginal zone (P < 0·001), CD19⁺CD27⁺IgD^– memory (P < 0·05) and CD27‐IgD double‐negative (P < 0·01) B cells and CD4⁺CXCR3^–CCR6⁺ Th17 cells (P < 0·05). CXCL13 levels were increased in patient plasma (P < 0·001), and immunohistochemical staining revealed expression of CXCL13 and higher numbers of CXCR5⁺ cells (P < 0·0001) within focal infiltrates and interstitially in salivary glands of SS patients. In conclusion, we link a genetic susceptibility allele for SS to a functional phenotype in terms of decreased CXCR5 expression. The decrease of CXCR5⁺ cells in circulation was also related to homing of B and T cells to the autoimmune target organ. Therapeutic drugs targeting the CXCR5/CXCL13 axis may be useful in SS.     

Folate‐Conjugated Poly(N‐(2‐hydroxypropyl) methacrylamide)‐block‐Poly(benzyl methacrylate): Synthesis,Self‐Assembly,and Drug Release

Well‐defined amphiphilic diblock copolymers of poly(N‐(2‐hydroxypropyl)methacrylamide)‐block‐poly(benzyl methacrylate) (PHPMA‐b‐PBnMA) are synthesized using reversible addition–fragmentation chain transfer polymerization. The terminal dithiobenzoate groups are converted into carboxylic acids. The copolymers self‐assemble into micelles with a PBnMA core and PHPMA shell. Their mean size is <30 nm, and can be regulated by the length of the hydrophilic chain. The compatibility between the hydrophobic segment and the drug doxorubicin (DOX) affords more interaction of the cores with DOX. Fluorescence spectra are used to determine the critical micelle concentration of the folate‐conjugated amphiphilic block copolymer. Dynamic light scattering measurements reveal the stability of the micelles with or without DOX. Drug release experiments show that the DOX‐loaded micelles are stable under simulated circulation conditions and the DOX can be quickly released under acidic endosome pH.     

Accumulation of neurofibrillary tangles and activated microglia is associated with lower neuron densities in the aphasic variant of Alzheimer’s disease

The neurofibrillary tangles (NFT) and amyloid‐ß plaques (AP) that comprise Alzheimer’s disease (AD) neuropathology are associated with neurodegeneration and microglial activation. Activated microglia exist on a dynamic spectrum of morphologic subtypes that include resting, surveillant microglia capable of converting to activated, hypertrophic microglia closely linked to neuroinflammatory processes and AD neuropathology in amnestic AD. However, quantitative analyses of microglial subtypes and neurons are lacking in non‐amnestic clinical AD variants, including primary progressive aphasia (PPA‐AD). PPA‐AD is a language disorder characterized by cortical atrophy and NFT densities concentrated to the language‐dominant hemisphere. Here, a stereologic investigation of five PPA‐AD participants determined the densities and distributions of neurons and microglial subtypes to examine how cellular changes relate to AD neuropathology and may contribute to cortical atrophy. Adjacent series of sections were immunostained for neurons (NeuN) and microglia (HLA‐DR) from bilateral language and non‐language regions where in vivo cortical atrophy and Thioflavin‐S‐positive APs and NFTs were previously quantified. NeuN‐positive neurons and morphologic subtypes of HLA‐DR‐positive microglia (i.e., resting [ramified] microglia and activated [hypertrophic] microglia) were quantified using unbiased stereology. Relationships between neurons, microglia, AD neuropathology, and cortical atrophy were determined using linear mixed models. NFT densities were positively associated with hypertrophic microglia densities (P < 0.01) and inversely related to neuron densities (P = 0.01). Hypertrophic microglia densities were inversely related to densities of neurons (P < 0.01) and ramified microglia (P < 0.01). Ramified microglia densities were positively associated with neuron densities (P = 0.02) and inversely related to cortical atrophy (P = 0.03). Our findings provide converging evidence of divergent roles for microglial subtypes in patterns of neurodegeneration, which includes hypertrophic microglia likely driving a neuroinflammatory response more sensitive to NFTs than APs in PPA‐AD. Moreover, the accumulation of both NFTs and activated hypertrophic microglia in association with low neuron densities suggest they may collectively contribute to focal neurodegeneration characteristic of PPA‐AD.     

Noise-induced cell death in the mouse medial geniculate body and primary auditory cortex

Noise-induced effects within the inner ear have been well investigated for several years. However, this peripheral damage cannot fully explain the audiological symptoms in noise-induced hearing loss (NIHL), e.g. tinnitus, recruitment, reduced speech intelligibility, hyperacusis. There are few reports on central noise effects. Noise can induce an apoptosis of neuronal tissue within the lower auditory pathway. Higher auditory structures (e.g. medial geniculate body, auditory cortex) are characterized by metabolic changes after noise exposure. However, little is known about the microstructural changes of the higher auditory pathway after noise exposure. The present paper was therefore aimed at investigating the cell density in the medial geniculate body (MGB) and the primary auditory cortex (AI) after noise exposure. Normal hearing mice were exposed to noise (10 kHz center frequency at 115 dB SPL for 3 h) at the age of 21 days under anesthesia (Ketamin/Rompun, 10:1). After 1 week, auditory brainstem response recordings (ABR) were performed in noise exposed and normal hearing animals. After fixation, the brain was microdissected and stained (Kluever-Barrera). The cell density in the MGB subdivisions and the AI were determined by counting the cells within a grid. Noise-exposed animals showed a significant ABR threshold shift over the whole frequency range. Cell density was significantly reduced in all subdivisions of the MGB and in layers IV-VI of AI. The present findings demonstrate a significant noise-induced change of the neuronal cytoarchitecture in central key areas of auditory processing. These changes could contribute to the complex psychoacoustic symptoms after NIHL.     

Separation of plasma‐derived exosomes into CD3(+) and CD3(–) fractions allows for association of immune cell and tumour cell markers with disease activity in HNSCC patients

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour‐ or T cell‐derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3^(–) from CD3⁽⁺⁾ exosomes by immunocapture using anti‐CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on‐bead flow cytometry. Tumour protein‐enriched CD3^(–) exosomes were CD44v3⁽⁺⁾. Surprisingly, mean levels of programmed death ligand 1 (PD‐L1), cytotoxic T lymphocyte antigen 4 (CTLA‐4) and cyclooxygenase‐2 (COX‐2) were similar in CD3⁽⁺⁾ and CD3^(–) exosomes, although the latter induced higher (P < 0·0025) ex‐vivo apoptosis of CD8⁽⁺⁾ T cells and greater (P < 0·005) conversion of CD4⁺ T cells to CD4⁽⁺⁾CD39⁽⁺⁾ regulatory T cells (T_reg). CD3⁽⁺⁾ and CD3^(–) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD‐L1 and COX‐2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3⁽⁺⁾ and CD3^(–) exosomes of HNSCC patients had higher PD‐L1, COX‐2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3^(–)/CD44v3‐enriched and CD3⁽⁺⁾ exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers.     

Growth hormone and prolactin responses during partial and whole body warm‐water immersions

Aim: To elucidate the role of core and skin thermoreceptors in the release of growth hormone (GH) and prolactin (PRL), a sequence of two experiments using whole‐body (head‐out) and partial (one forearm) hot water immersions was performed. Methods: Experiment 1: Nine healthy men were exposed to head‐out and partial water immersions (25 min, 38–39 °C). Results: Head‐out immersion increased the core temperature (38.0 ± 0.1 vs. 36.7 ± 0.1 °C, P < 0.001) and plasma concentration of the hormones (GH, 16.1 ± 4.5 vs. 1.2 ± 0.4 ng mL^?1, P < 0.01; PRL, 9.1 ± 1.0 vs. 6.4 ± 0.4 ng mL^?1, P < 0.05). During the partial immersion the core temperature was slightly elevated (36.8 ± 0.1 vs. 36.6 ± 0.1, P < 0.001), the concentration of GH increased (4.8 ± 1.7 vs. 0.6 ± 0.3, P < 0.05), while plasma PRL decreased (7.6 ± 0.8, 6.0 ± 0.6, 5.2 ± 0.6, P < 0.01). Experiment 2: Seven volunteers immersed one forearm once in 39 °C and once in 38 °C water. The measurements were performed in 5‐min intervals. The GH concentration increased gradually from the beginning of the immersions (min 10; 39 °C: 1.9 ± 1.0 vs. 0.6 ± 0.3 ng mL^?1, P < 0.01; 38 °C: 0.19 ± 0.03 vs. 0.14 ± 0.03, P < 0.05) and peaked after their completion (39 °C: +10 min, 3.7 ± 2.0, P < 0.001; 38 °C: +15 min, 0.86 ± 0.61, P < 0.01). The core temperature was unchanged until min 15 of the 39 °C bath. Thereafter, it increased about 0.15 °C above the baseline (P < 0.01). Immersion in 38 °C water did not induce core temperature changes. Conclusions: Peripheral thermoreceptors are involved in GH release when the body is exposed to elevated environmental temperature while a substantial elevation of core temperature is a precondition of PRL release.     

Reliable estimation of brain intravoxel incoherent motion parameters using denoised diffusion‐weighted MRI

In this study, we evaluate whether diffusion‐weighted magnetic resonance imaging (DW‐MRI) data after denoising can provide a reliable estimation of brain intravoxel incoherent motion (IVIM) perfusion parameters. Brain DW‐MRI was performed in five healthy volunteers on a 3 T clinical scanner with 12 different b‐values ranging from 0 to 1000 s/mm². DW‐MRI data denoised using the proposed method were fitted with a biexponential model to extract perfusion fraction (PF), diffusion coefficient (D) and pseudo‐diffusion coefficient (D*). To further evaluate the accuracy and precision of parameter estimation, IVIM parametric images obtained from one volunteer were used to resimulate the DW‐MRI data using the biexponential model with the same b‐values. Rician noise was added to generate DW‐MRI data with various signal‐to‐noise ratio (SNR) levels. The experimental results showed that the denoised DW‐MRI data yielded precise estimates for all IVIM parameters. We also found that IVIM parameters were significantly different between gray matter and white matter (P < 0.05), except for D* (P = 0.6). Our simulation results show that the proposed image denoising method displays good performance in estimating IVIM parameters (both bias and coefficient of variation were <12% for PF, D and D*) in the presence of different levels of simulated Rician noise (SNR_b=0 = 20‐40). Simulations and experiments show that brain DW‐MRI data after denoising can provide a reliable estimation of IVIM parameters.     

Neuropathologic basis of in vivo cortical atrophy in the aphasic variant of Alzheimer's disease

The neuropathologic basis of in vivo cortical atrophy in clinical dementia syndromes remains poorly understood. This includes primary progressive aphasia (PPA), a language‐based dementia syndrome characterized by asymmetric cortical atrophy. The neurofibrillary tangles (NFTs) and amyloid‐ß plaques (APs) of Alzheimer's disease (AD) can cause PPA, but a quantitative investigation of the relationships between NFTs, APs and in vivo cortical atrophy in PPA‐AD is lacking. The present study measured cortical atrophy from corresponding bilateral regions in five PPA‐AD participants with in vivo magnetic resonance imaging scans 7–30 months before death and acquired stereologic estimates of NFTs and dense‐core APs visualized with the Thioflavin‐S stain. Linear mixed models accounting for repeated measures and stratified by hemisphere and region (language vs. non‐language) were used to determine the relationships between cortical atrophy and AD neuropathology and their regional selectivity. Consistent with the aphasic profile of PPA, left language regions displayed more cortical atrophy (P = 0.01) and NFT densities (P = 0.02) compared to right language homologues. Left language regions also showed more cortical atrophy (P < 0.01) and NFT densities (P = 0.02) than left non‐language regions. A subset of data was analyzed to determine the predilection of AD neuropathology for neocortical regions compared to entorhinal cortex in the left hemisphere, which showed that the three most atrophied language regions had greater NFT (P = 0.04) and AP densities (P < 0.01) than the entorhinal cortex. These results provide quantitative evidence that NFT accumulation in PPA selectively targets the language network and may not follow the Braak staging of neurofibrillary degeneration characteristic of amnestic AD. Only NFT densities, not AP densities, were positively associated with cortical atrophy within left language regions (P < 0.01) and right language homologues (P < 0.01). Given previous findings from amnestic AD, the current study of PPA‐AD provides converging evidence that NFTs are the principal determinants of atrophy and clinical phenotypes associated with AD.     

Role of adenosine in exercise‐induced human skeletal muscle vasodilatation

The role of adenosine in exercise‐induced human skeletal muscle vasodilatation remains unknown. We therefore evaluated the effect of theophylline‐induced adenosine receptor blockade in six subjects and the vasodilator potency of adenosine infused in the femoral artery of seven subjects. During one‐legged, knee‐extensor exercise at ～48% of peak power output, intravenous (i.v.) theophylline decreased (P < 0.003) femoral artery blood flow (F_aBF) by ～20%, i.e. from 3.6 ± 0.5 to 2.9 ± 0.5 L min^?1, and leg vascular conductance (VC) from 33.4 ± 9.1 to 27.7 ± 8.5 mL min^?1 mmHg^?1, whereas heart rate (HR), mean arterial pressure (MAP), leg oxygen uptake and lactate release remained unaltered (P = n.s.). Bolus injections of adenosine (2.5 mg) at rest rapidly increased (P < 0.05) F_aBF from 0.3 ± 0.03 L min^?1 to a 15‐fold peak elevation (P < 0.05) at 4.1 ± 0.5 L min^?1. Continuous infusion of adenosine at rest and during one‐legged exercise at ～62% of peak power output increased (P < 0.05) F_aBF dose‐dependently to level off (P = ns) at 8.3 ± 1.0 and 8.2 ± 1.4 L min^?1, respectively. One‐legged exercise alone increased (P < 0.05) F_aBF to 4.7 ± 1.7 L min^?1. Leg oxygen uptake was unaltered (P = n.s.) with adenosine infusion during both rest and exercise. The present findings demonstrate that endogenous adenosine controls at least ～20% of the hyperaemic response to submaximal exercise in skeletal muscle of humans. The results also clearly show that arterial infusion of exogenous adenosine has the potential to evoke a vasodilator response that mimics the increase in blood flow observed in response to exercise.     

Effect of activated human polymorphonuclear leucocytes on T lymphocyte proliferation and viability

Human polymorphonuclear leucocytes (PMN) are thought to be immunosuppressive. The suppressive mechanism(s) used by PMN are, however, not well defined and in this study they were analysed using T‐cell responses to CD3⁺ CD28 monoclonal antibodies (mAb) as a readout. We demonstrate that in vitro activated PMN (PMN^act) can, without any T‐cell interaction, induce apparent T‐cell suppression by inhibiting the stimulatory capacity of the CD3 mAb. However, a cell‐directed suppression of T‐cell proliferation was observed when PMN^act were added to pre‐activated T cells that are already committed to polyclonal proliferation. This suppression was partially reversed by catalase addition (P < 0·01) and largely reversed by addition of exogenous interleukin‐2 (P < 0·001) but was not significantly reduced by nitric oxide synthase inhibition, myeloperoxidase inhibition or addition of excess arginine. Following removal of PMN^act, suppressed T cells could respond normally to further stimulation. In addition to suppressing proliferation, co‐culture with PMN^act also induced a significant decrease in T‐cell viability that was reversed by catalase addition (P < 0·05). The addition of the arginase inhibitor N‐hydroxy‐nor‐l ‐arginine induced both a further significant, catalase‐sensitive, loss in T‐cell viability and increased nitrite release (P < 0·001). These data demonstrate that PMN, when activated, can both induce T‐cell death and reversibly inhibit proliferation of activated T cells. The mechanisms underlying these distinct processes and the effects of arginase inhibitors on PMN induced cytotoxicity merit further investigation.     

RORγt antagonist suppresses M3 muscarinic acetylcholine receptor‐induced Sjögren's syndrome‐like sialadenitis

We showed recently that M3 muscarinic acetylcholine receptor (M3R)‐reactive CD3⁺ T cells play a pathogenic role in the development of murine autoimmune sialadenitis (MIS), which mimics Sjögren's syndrome (SS). The aim of this study was to determine the effectiveness and mechanism of action of retinoic acid‐related orphan receptor‐gamma t (RORγt) antagonist (A213) in MIS. Splenocytes from M3R knockout (M3R^–/–) mice immunized with murine M3R peptide mixture were inoculated into recombination‐activating gene 1 knockout (Rag‐1^–/–) mice (M3R^–/–→Rag‐1^–/–) with MIS. Immunized M3R^–/– mice (pretransfer treatment) and M3R^–/–→Rag‐1^–/– mice (post‐transfer treatment) were treated with A213 every 3 days. Salivary volume, severity of sialadenitis and cytokine production from M3R peptide‐stimulated splenocytes and lymph node cells were examined. Effects of A213 on cytokine production were analysed by enzyme‐linked immunosorbent assay (ELISA) and on T helper type 1 (Th1), Th17 and Th2 differentiation from CD4⁺ T cells by flow cytometry. Pretransfer A213 treatment maintained salivary volume, improved MIS and reduced interferon (IFN)‐γ and interleukin (IL)‐17 production significantly compared with phosphate‐buffered saline (PBS) (P < 0·05). These suppressive effects involved CD4⁺ T cells rather than CD11c⁺ cells. Post‐transfer treatment with A213 increased salivary volume (P < 0·05), suppressed MIS (P < 0·005) and reduced IFN‐γ and IL‐17 production (P < 0·05). In vitro, A213 suppressed IFN‐γ and IL‐17 production from M3R‐stimulated splenocytes and CD4⁺ T cells of immunized M3R^–/– mice (P < 0·05). In contrast with M3R specific responses, A213 suppressed only IL‐17 production from Th17 differentiated CD4⁺ T cells without any effect on Th1 and Th2 differentiation in vitro. Our findings suggested that RORγt antagonism is potentially suitable treatment strategy for SS‐like sialadenitis through suppression of IL‐17 and IFN‐γ production by M3R‐specific T cells.     

Size and number of binucleate and mononucleate superior cervical ganglion neurons in young capybaras

The total number of neurons in the superior cervical ganglion (SCG) of adult capybaras is known from a previous study, where a marked occurrence of binucleate neurons (13%) was also noted. Here, distribution, number and fate of binucleate neurons were examined in younger, developing capybaras, aged 3 months. The mean neuronal cross-sectional area was 575.2 μm² for mononucleate neurons and 806.8 μm² in binucleate neurons. Frequency of binucleate neurons was about 36%. The mean ganglion volume was about 190 mm³ in young capybaras and the mean neuronal density was about 9,517 neurons/mm³. The total number of neurons per ganglion was about 1.81 mill. Neuronal cell bodies constituted 22.5% of the ganglion volume and the average neuronal volume was 23,600 μm³. By comparing the present data with those previously published the conclusion is drawn that the maturation period was characterized by the following points: a 26% remarkable decrease in neuronal density which was significant (P < 0.05) and a significant 16% (P < 0.05) decrease in the total number of SCG neurons accompanied by a 23% decrease in the total number of SCG binucleate neurons.     

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.