The effect of prior exercise on ex vivo induction of heme oxygenase-1 in human lymphocytes

It was postulated that prior demanding exercise would suppress the induction of the oxidant-responsive protein Heme Oxygenase-1 (HO-1) in mononuclear cells following subsequent ex vivo H(2)O(2) treatment. Eight male subjects completed two trials in a randomized order (one rest and one exercise) and ex vivo HO-1 protein induction was determined following H(2)O(2) treatment in lymphocytes and monocytes before and after each trial using a newly developed and reproducible assay. Lymphocytes obtained 2 h post-exercise showed a modest reduction in HO-1 protein expression in response to ex vivo treatment with H(2)O(2) (p<0.05). The plasma concentration of the HO-1 suppressor alpha1-antitrypsin increased immediately post-exercise (p<0.05) and it is tentatively suggested that this may explain the modest transient reduction in ex vivo HO-1 protein induction in lymphocytes in response to an independent oxidant challenge following a prior bout of demanding exercise.     

Is MR Spectroscopy Really the Best MR-Based Method for the Evaluation of Fatty Liver in Diabetic Patients in Clinical Practice?

Objective

To investigate if magnetic resonance spectroscopy (MRS) is the best Magnetic Resonance (MR)-based method when compared to gradient-echo magnetic resonance imaging (MRI) for the detection and quantification of liver steatosis in diabetic patients in the clinical practice using liver biopsy as the reference standard, and to assess the influence of steatohepatitis and fibrosis on liver fat quantification.

Methods

Institutional approval and patient consent were obtained for this prospective study. Seventy-three patients with type 2 diabetes (60 women and 13 men; mean age, 54±9 years) underwent MRI and MRS at 3.0 T. The liver fat fraction was calculated from triple- and multi-echo gradient-echo sequences, and MRS data. Liver specimens were obtained in all patients. The accuracy for liver fat detection was estimated by receiver operator characteristic (ROC) analysis, and the correlation between fat quantification by imaging and histolopathology was analyzed by Spearman''s correlation coefficients.

Results

The prevalence of hepatic steatosis was 92%. All gradient-echo MRI and MRS findings strongly correlated with biopsy findings (triple-echo, rho = 0.819; multi-echo, rho = 0.773; MRS, rho = 0.767). Areas under the ROC curves to detect mild, moderate, and severe steatosis were: triple-echo sequences, 0.961, 0.975, and 0.962; multi-echo sequences, 0.878, 0.979, and 0.961; and MRS, 0.981, 0.980, and 0.954. The thresholds for mild, moderate, and severe steatosis were: triple-echo sequences, 4.09, 9.34, and 12.34, multi-echo sequences, 7.53, 11.75, and 15.08, and MRS, 1.71, 11.69, and 14.91. Quantification was not significantly influenced by steatohepatitis or fibrosis.

Conclusions

Liver fat quantification by MR methods strongly correlates with histopathology. Due to the wide availability and easier post-processing, gradient-echo sequences may represent the best imaging method for the detection and quantification of liver fat fraction in diabetic patients in the clinical practice.     

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).     

Early intervention with gene-modified mesenchymal stem cells overexpressing interleukin-4 enhances anti-inflammatory responses and functional recovery in experimental autoimmune demyelination

Mesenchymal stem/stromal cells (MSCs) can be isolated from most adult tissues and hold considerable promise for tissue regenerative therapies. Some of the potential advantages that MSCs have over other adult stem cell types include: (1) their relative ease of isolation, culture and expansion; (2) their immunomodulatory properties; (3) they can provide trophic support to injured tissues; (4) they can be transduced by retroviral vectors at a high efficiency; (5) they have an ability to home to sites of inflammation and injury. Collectively these characteristics suggest that MSCs are attractive vehicles for cell and gene therapy applications. In the current study, we investigated whether transplantation of human adipose-derived MSCs (Ad-MSCs) engineered to overexpress the anti-inflammatory cytokine interleukin (IL)-4 was efficacious in experimental autoimmune encephalomyelitis (EAE). Ad-MSCs transduced with a bicistronic lentiviral vector encoding mouse IL-4 and enhanced green fluorescent protein (Ad-IL4-MSCs) stably expressed, relatively high levels of both transgenes. Importantly the phenotypic and functional attributes of Ad-IL4-MSCs, such as the expression of homing molecules and differentiation capacity, was not altered by the transduction process. Notably, the early administration of Ad-IL4-MSCs in mice with EAE at the time of T-cell priming attenuated clinical disease. This protective effect was associated with a reduction in peripheral MOG-specific T-cell responses and a shift from a pro- to an anti-inflammatory cytokine response. These data suggest that the delivery of Ad-MSCs genetically engineered to express anti-inflammatory cytokines may provide a rational approach to promote immunomodulation and tissue protection in a number of inflammatory and degenerative diseases including multiple sclerosis.     

Multivectorial abnormal cell migration in the NOD mouse thymus

We previously described a fibronectin/VLA-5-dependent impairment of NOD thymocyte migration, correlated with partial thymocyte arrest within thymic perivascular spaces. Yet, NOD thymocytes still emigrate, suggesting the involvement of other cell migration-related alterations. In this context, the aim of this work was to study the role of extracellular matrix ligands, alone or in combination with the chemokine CXCL12, in NOD thymocyte migration. Intrathymic contents of CXCL12, fibronectin, and laminin were evaluated by immunohistochemistry while the expression of corresponding receptors was ascertained by flow cytometry. Thymocyte migration was measured using Transwell chambers and transendothelial migration was evaluated in the same system, but using an endothelial cell monolayer within the chambers. NOD thymocytes express much lower VLA-5 than C57BL/6 thymocytes. This defect was particularly severe in CD4(+) thymocytes expressing Foxp3, thus in keeping with the arrest of Foxp3(+) cells within the NOD giant perivascular spaces. We observed an enhancement in CXCL12, laminin, and fibronectin deposition and colocalization in the NOD thymus. Furthermore, we detected altered expression of the CXCL12 receptor CXCR4 and the laminin receptor VLA-6, as well as enhanced migratory capacity of NOD thymocytes toward these molecules, combined or alone. Moreover, transendothelial migration of NOD thymocytes was diminished in the presence of exogenous fibronectin. Our data unravel the existence of multiple cell migration-related abnormalities in NOD thymocytes, comprising both down- and up-regulation of specific responses. Although remaining to be experimentally demonstrated, these events may have consequences on the appearance of autoimmunity in NOD mice.     

Genetic profiling of Trypanosoma cruzi directly in infected tissues using nested PCR of polymorphic microsatellites

The investigation of the importance of the genetics of Trypanosoma cruzi in determining the clinical course of Chagas disease will depend on precise characterisation of the parasites present in the tissue lesions. This can be adequately accomplished by the use of hypervariable nuclear markers such as microsatellites. However the unilocal nature of these loci and the scarcity of parasites in chronic lesions make it necessary to use high sensitivity PCR with nested primers, whose design depends on the availability of long flanking regions, a feature not hitherto available for any known T. cruzi microsatellites. Herein, making use of the extensive T. cruzi genome sequence now available and using the Tandem Repeats Finder software, it was possible to identify and characterise seven new microsatellite loci – six composed of trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed of tetranucleotide (TcAAAT6) motifs. All except the TcCAA10 locus were physically mapped onto distinct intergenic regions of chromosome III of the CL Brener clone contigs. The TcCAA10 locus was localised within a hypothetical protein gene in the T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Using the TcTAC15 locus it was possible to separate the strains belonging to the T. cruzi I lineage (DTU I) from those belonging to T. cruzi II (DTU IIb), T. cruzi III (DTU IIc) and a hybrid group (DTU IId, IIe). The long flanking regions of these novel microsatellites allowed construction of nested primers and the use of full nested PCR protocols. This strategy enabled us to detect and differentiate T. cruzi strains directly in clinical specimens including heart, blood, CSF and skin tissues from patients in the acute and chronic phases of Chagas disease.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Establishment and impact of insect agents deployed for the biological control of invasive Asteraceae: prospects for the control of <Emphasis Type="Italic">Senecio madagascariensis</Emphasis>

Several invasive Asteraceae have been targeted for biological control worldwide, with variable success. Senecio madagascariensis Poiret, which invades agricultural lands in Australia and Hawaii, is a recent target. Since several potential insect agents were recorded in the plant’s native range in South Africa, we assessed biocontrol efforts against asteraceous weeds to determine those most likely to deliver success. Some 108 insect species, from five orders and 23 families, were deployed against 38 weed taxa, mostly in the mainland USA, Canada, Australia and New Zealand. Coleoptera (mainly Curculionidae and Chrysomelidae), Diptera (Tephritidae) and Lepidoptera (Tortricidae) featured the most. Despite high establishment success (73% of releases across countries), only 37% of successful releases achieved meaningful impact. Although root-feeding and stem-feeding insects appeared to be the best candidates, neither insect family nor feeding guild significantly influenced the probability of success. This synthesis of the global contribution of different guilds of specialist herbivores to the management of invasive Asteraceae is guiding the selection of candidate agents for the biocontrol of S. madagascariensis in Australia.