Thyrotropin-releasing hormone-stimulated thyrotropin expression involves islet-brain-1/c-Jun N-terminal kinase interacting protein-1.

Islet-brain-1 (IB1)/c-Jun N-terminal kinase interacting protein 1 (JIP-1) is a scaffold protein that is expressed at high levels in neurons and the endocrine pancreas. IB1/JIP-1 interacts with the c-Jun N-terminal kinase and mediates the specific physiological stimuli (such as cytokines). However, the potential role of the protein in the pituitary has not been evaluated. Herein, we examined expression of the gene encoding IB1/JIP-1 and its translated product in the anterior pituitary gland and a pituitary cell line, GH3. We then examined the potential role of IB1/JIP-1 in controlling TSH-beta gene expression. Exposure of GH3 cells to TRH stimulated the expression of IB1/JIP-1 protein levels, mRNA, and transcription of the promoter. The increase of IB1/JIP-1 content by transient transfection study of a vector encoding IB1/JIP-1 or by the stimulation of TRH stimulates TSH-beta promoter activity. This effect is not found in the presence of a mutated nonfunctional (IB1S59N) IB1/JIP-1 protein. Together, these facts point to a central role of the IB1/JIP-1 protein in the control of TRH-mediated TSH-beta stimulation.

Cytokine mRNA profile of Epstein-Barr virus-stimulated highly differentiated T cells in multiple sclerosis: a pilot study.

The reason why EBV-specific cellular immune responses are abnormal in multiple sclerosis (MS) patients is still missing. In this exploratory pilot study, we assessed IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-17, IFN-gamma, TGF-beta1 and FOXP3 mRNA expression in EBV-stimulated highly differentiated T cells (T(HD)) of MS patients and healthy controls (HC). We found increased levels of IFN-gamma and IL-4 mRNA in CD8+ T(HD) cells of MS patients. All the other tested molecules were expressed similarly in MS patients and HC. Interestingly, increased IFN-gamma and IL-4 suggest that the control of EBV replication may be insufficient in MS patients.

Stimulated sweating as a therapy to reduce interdialytic weight gain and improve potassium balance in chronic hemodialysis patients: a pilot study.

Controlling the extracellular volume in hemodialysis patients is a difficult task. The aim of this study was to evaluate the capacity of different methods of stimulated sweating to reduce mean interdialytic weight gain (IWG), to improve blood pressure regulation, and potassium/urea balance. Two center, crossover pilot study. In Lausanne, hemodialysis patients took four hot-water baths a week, 30 minutes each, on nondialysis days during 1 month. In Sfax, patients visited the local Hammam Center four times a week. Hemodynamic parameters were recorded, and weekly laboratory analysis was performed. Results were compared with a preceding 1-month control period. In Lausanne, five patients (all men, median age 55 years) participated. Bathing temperature was (mean ± standard deviation) 41.2 ± 3°C and sweating-induced weight loss 600 ± 500 g. Mean IWG (control vs. intervention period) decreased from 2.3 ± 0.9 to 1.8 ± 1 kg (P = 0.004), Systolic blood pressure from 139 ± 21 to 136 ± 22 mmHg (P = 0.4), and diastolic blood pressure form 79 ± 12 to 75 ± 13 mmHg (P = 0.08); antihypertensive therapy could be reduced from 2.8 ± 0.4 to 1.9 ± 0.5 antihypertensive drugs per patient (P = 0.01). In Sfax (n = 9, median age 46 years), weight loss per Hammam session was 420 ± 100 g. No differences were found in IWG or BP, but predialysis serum potassium level decreased from 5.9 ± 0.8 to 5.5 ± 0.9 mmol/L (P = 0.04) and urea from 26.9 ± 6 to 23.1 ± 6 mmol/L (P = 0.02). Hot-water baths appear to be a safe way to reduce IWG in selected hemodialysis patients. Hammam visits reduce serum potassium and urea levels, but not IWG. More data in larger patient groups are necessary before definite conclusion can be drawn.

Improvement of saliva production in ENT cancer patients treated by IMRT

Intensity modulated radiotherapy (IMRT) is a conformal radiotherapy that produces concave and irregular target volume dose distributions. IMRT has a potential to reduce the volume of healthy tissue irradiated to a high dose, but this often at the price of an increased volume of normal tissue irradiated to a low dose. Clinical benefits of IMRT are expected to be most pronounced at the body sites where sensitive normal tissues surround or are located next to a target with a complex 3D shape. The irradiation doses needed for tumor control are often markedly higher than the tolerance of the radiation sensitive structures such as the spinal cord, the optic nerves, the eyes, or the salivary glands in the treatment of head and neck cancer. Parotid gland salivary flow is markedly reduced following a cumulative dose of 30 50 Gy given with conventional fractionation and xerostomia may be prevented in most patients using a conformal parotid-sparing radiotherapy technique. However, in cohort studies where IMRT was compared with conventional and conformal radiotherapy techniques in the treatment of laryngeal or oropharyngeal carcinoma, the dosimetric advantage of IMRT translated into a reduction of late salivary toxicity with no apparent adverse impact on the tumor control. IMRT might reduce the radiation dose to the major salivary glands and the risk of permanent xerostomia without compromizing the likelihood for cure. Alternatively, IMRT might allow the target dose escalation at a given level of normal tissue damage. We describe here the clinical results on postirradiation salivary gland function in head and neck cancer patients treated with IMRT, and the technical aspects of IMRT applied. The results suggest that the major salivary gland function can be maintained with IMRT without a need to compromise the clinical target volume dose, or the locoregional control.

Repeated exposure to Lutzomyia intermedia sand fly saliva induces local expression of interferon-inducible genes both at the site of injection in mice and in human blood.

During a blood meal, Lutzomyia intermedia sand flies transmit Leishmania braziliensis, a parasite causing tegumentary leishmaniasis. In experimental leishmaniasis, pre-exposure to saliva of most blood-feeding sand flies results in parasite establishment in absence of any skin damages in mice challenged with dermotropic Leishmania species together with saliva. In contrast, pre-immunization with Lu. intermedia salivary gland sonicate (SGS) results in enhanced skin inflammatory exacerbation upon co-inoculation of Lu. intermedia SGS and L. braziliensis. These data highlight potential unique features of both L. braziliensis and Lu. intermedia. In this study, we investigated the genes modulated by Lu. intermedia SGS immunization to understand their potential impact on the subsequent cutaneous immune response following inoculation of both SGS and L. braziliensis. The cellular recruitment and global gene expression profile was analyzed in mice repeatedly inoculated or not with Lu. intermedia. Microarray gene analysis revealed the upregulation of a distinct set of IFN-inducible genes, an immune signature not seen to the same extent in control animals. Of note this INF-inducible gene set was not induced in SGS pre-immunized mice subsequently co-inoculated with SGS and L. braziliensis. These data suggest the parasite prevented the upregulation of this Lu. intermedia saliva-related immune signature. The presence of these IFN-inducible genes was further analyzed in peripheral blood mononuclear cells (PBMCs) sampled from uninfected human individuals living in a L. braziliensis-endemic region of Brazil thus regularly exposed to Lu. intermedia bites. PBMCs were cultured in presence or absence of Lu. intermedia SGS. Using qRT-PCR we established that the IFN-inducible genes induced in the skin of SGS pre-immunized mice, were also upregulated by SGS in PBMCs from human individuals regularly exposed to Lu. intermedia bites, but not in PBMCs of control subjects. These data demonstrate that repeated exposure to Lu. intermedia SGS induces the expression of potentially host-protective IFN-inducible genes.

Alveolar epithelial CNGA1 channels mediate cGMP-stimulated, amiloride-insensitive, lung liquid absorption.

Impairment of lung liquid absorption can lead to severe respiratory symptoms, such as those observed in pulmonary oedema. In the adult lung, liquid absorption is driven by cation transport through two pathways: a well-established amiloride-sensitive Na(+) channel (ENaC) and, more controversially, an amiloride-insensitive channel that may belong to the cyclic nucleotide-gated (CNG) channel family. Here, we show robust CNGA1 (but not CNGA2 or CNGA3) channel expression principally in rat alveolar type I cells; CNGA3 was expressed in ciliated airway epithelial cells. Using a rat in situ lung liquid clearance assay, CNG channel activation with 1 mM 8Br-cGMP resulted in an approximate 1.8-fold stimulation of lung liquid absorption. There was no stimulation by 8Br-cGMP when applied in the presence of either 100 μM L: -cis-diltiazem or 100 nM pseudechetoxin (PsTx), a specific inhibitor of CNGA1 channels. Channel specificity of PsTx and amiloride was confirmed by patch clamp experiments showing that CNGA1 channels in HEK 293 cells were not inhibited by 100 μM amiloride and that recombinant αβγ-ENaC were not inhibited by 100 nM PsTx. Importantly, 8Br-cGMP stimulated lung liquid absorption in situ, even in the presence of 50 μM amiloride. Furthermore, neither L: -cis-diltiazem nor PsTx affected the β(2)-adrenoceptor agonist-stimulated lung liquid absorption, but, as expected, amiloride completely ablated it. Thus, transport through alveolar CNGA1 channels, located in type I cells, underlies the amiloride-insensitive component of lung liquid reabsorption. Furthermore, our in situ data highlight the potential of CNGA1 as a novel therapeutic target for the treatment of diseases characterised by lung liquid overload.

Traitement des epilepsies réfractaires: rôle de la stimulation electrique [Treatment of pharmacoresistant epilepsy: stimulated refractoriness].

Antiepileptic drugs allow controlling seizures in 70% of patients. For the others, a presurgical work-up should be undertaken, especially if a focal seizure origin is suspected; however, only a fraction of pharmacoresistant patients will be offered resective (curative) surgery. In the last 15 years, several palliative therapies using extra- or intracranial electrical stimulations have been developed. This article presents the vagal nerve stimulation, the deep brain stimulation (targeting the mesiotemporal region or the thalamus), and the cortical stimulation "on demand". All show an overall long-term responder rate between 30-50%, but less than 5% of patients becoming seizure free. It is to hope that a better understanding of epileptogenic mechanisms and of the implicated neuronal networks will lead to an improvement of these proportions.

Limitations of stimulated echo acquisition mode (STEAM) techniques in cardiac applications.

Stimulated echoes are widely used for imaging functional tissue parameters such as diffusion coefficient, perfusion, and flow rates. They are potentially interesting for the assessment of various cardiac functions. However, severe limitations of the stimulated echo acquisition mode occur, which are related to the special dynamic properties of the beating heart and flowing blood. To the well-known signal decay due to longitudinal relaxation and through-plane motion between the preparation and the read-out period of the stimulated echoes, additional signal loss is often observed. As the prepared magnetization is fixed with respect to the tissue, this signal loss is caused by the tissue deformation during the cardiac cycle, which leads to a modification of the modulation frequency of the magnetization. These effects are theoretically derived and corroborated by phantom and in vivo experiments.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Structure and function of a "yellow" protein from saliva of the sand fly Lutzomyia longipalpis that confers protective immunity against Leishmania major infection.

LJM11, an abundant salivary protein from the sand fly Lutzomyia longipalpis, belongs to the insect "yellow" family of proteins. In this study, we immunized mice with 17 plasmids encoding L. longiplapis salivary proteins and demonstrated that LJM11 confers protective immunity against Leishmania major infection. This protection correlates with a strong induction of a delayed type hypersensitivity (DTH) response following exposure to L. longipalpis saliva. Additionally, splenocytes of exposed mice produce IFN-γ upon stimulation with LJM11, demonstrating the systemic induction of Th1 immunity by this protein. In contrast to LJM11, LJM111, another yellow protein from L. longipalpis saliva, does not produce a DTH response in these mice, suggesting that structural or functional features specific to LJM11 are important for the induction of a robust DTH response. To examine these features, we used calorimetric analysis to probe a possible ligand binding function for the salivary yellow proteins. LJM11, LJM111, and LJM17 all acted as high affinity binders of prohemostatic and proinflammatory biogenic amines, particularly serotonin, catecholamines, and histamine. We also determined the crystal structure of LJM11, revealing a six-bladed β-propeller fold with a single ligand binding pocket located in the central part of the propeller structure on one face of the molecule. A hypothetical model of LJM11 suggests a positive electrostatic potential on the face containing entry to the ligand binding pocket, whereas LJM111 is negative to neutral over its entire surface. This may be the reason for differences in antigenicity between the two proteins.

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.

Stimulated-echo acquisition mode (STEAM) MRI for black-blood delayed hyperenhanced myocardial imaging.

PURPOSE: To develop a breathhold method for black-blood viability imaging of the heart that may facilitate identifying the endocardial border. MATERIALS AND METHODS: Three stimulated-echo acquisition mode (STEAM) images were obtained almost simultaneously during the same acquisition using three different demodulation values. Two of the three images were used to construct a black-blood image of the heart. The third image was a T(1)-weighted viability image that enabled detection of hyperintense infarcted myocardium after contrast agent administration. The three STEAM images were combined into one composite black-blood viability image of the heart. The composite STEAM images were compared to conventional inversion-recovery (IR) delayed hyperenhanced (DHE) images in nine human subjects studied on a 3T MRI scanner. RESULTS: STEAM images showed black-blood characteristics and a significant improvement in the blood-infarct signal-difference to noise ratio (SDNR) when compared to the IR-DHE images (34 +/- 4.1 vs. 10 +/- 2.9, mean +/- standard deviation (SD), P < 0.002). There was sufficient myocardium-infarct SDNR in the STEAM images to accurately delineate infarcted regions. The extracted infarcts demonstrated good agreement with the IR-DHE images. CONCLUSION: The STEAM black-blood property allows for better delineation of the blood-infarct border, which would enhance the fast and accurate measurement of infarct size.

Correction of through-plane deformation artifacts in stimulated echo acquisition mode cardiac imaging.

Attempts to use a stimulated echo acquisition mode (STEAM) in cardiac imaging are impeded by imaging artifacts that result in signal attenuation and nulling of the cardiac tissue. In this work, we present a method to reduce this artifact by acquiring two sets of stimulated echo images with two different demodulations. The resulting two images are combined to recover the signal loss and weighted to compensate for possible deformation-dependent intensity variation. Numerical simulations were used to validate the theory. Also, the proposed correction method was applied to in vivo imaging of normal volunteers (n = 6) and animal models with induced infarction (n = 3). The results show the ability of the method to recover the lost myocardial signal and generate artifact-free black-blood cardiac images.

Human Prominin-1 (CD133) Is Detected in Both Neoplastic and Non-Neoplastic Salivary Gland Diseases and Released into Saliva in a Ubiquitinated Form.

Prominin-1 (CD133) is physiologically expressed at the apical membranes of secretory (serous and mucous) and duct cells of major salivary glands. We investigated its expression in various human salivary gland lesions using two distinct anti-prominin-1 monoclonal antibodies (80B258 and AC133) applied on paraffin-embedded sections and characterized its occurrence in saliva. The 80B258 epitope was extensively expressed in adenoid cystic carcinoma, in lesser extent in acinic cell carcinoma and pleomorphic adenoma, and rarely in mucoepidermoid carcinoma. The 80B258 immunoreactivity was predominately detected at the apical membrane of tumor cells showing acinar or intercalated duct cell differentiation, which lined duct- or cyst-like structures, and in luminal secretions. It was observed on the whole cell membrane in non-luminal structures present in the vicinity of thin-walled blood vessels and hemorrhagic areas in adenoid cystic carcinoma. Of note, AC133 labeled only a subset of 80B258-positive structures. In peritumoral salivary gland tissues as well as in obstructive sialadenitis, an up-regulation of prominin-1 (both 80B258 and AC133 immunoreactivities) was observed in intercalated duct cells. In most tissues, prominin-1 was partially co-expressed with two cancer markers: carcinoembryonic antigen (CEA) and mucin-1 (MUC1). Differential centrifugation of saliva followed by immunoblotting indicated that all three markers were released in association with small membrane vesicles. Immuno-isolated prominin-1-positive vesicles contained CEA and MUC1, but also exosome-related proteins CD63, flotillin-1, flotillin-2 and the adaptor protein syntenin-1. The latter protein was shown to interact with prominin-1 as demonstrated by its co-immunoisolation. A fraction of saliva-associated prominin-1 appeared to be ubiquitinated. Collectively, our findings bring new insights into the biochemistry and trafficking of prominin-1 as well as its immunohistochemical profile in certain types of salivary gland tumors and inflammatory diseases.

Expression of the peroxisome proliferator-activated receptor alpha gene is stimulated by stress and follows a diurnal rhythm.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.