CD28/CTLA4-B7 interaction is dispensable for T cell stimulation by mouse mammary tumor virus superantigen but not for B cell differentiation and virus dissemination.

B cells are the primary targets of infection for mouse mammary tumor virus (MMTV). However, for productive retroviral infection, T cell stimulation through the virally-encoded superantigen (SAG) is necessary. It activates B cells and leads to cell division and differentiation. To characterize the role of B cell differentiation for the MMTV life cycle, we studied the course of infection in transgenic mice deficient for CD28/CTLA4-B7 interactions (mCTLA4-H gamma 1 transgenic mice). B cell infection occurred in CTLA4-H gamma 1 transgenic mice as integrated proviral DNA could be detected in draining lymph node cells early after infection by polymerase chain reaction analysis. In mice expressing I-E, B cells were able to present the viral SAG efficiently to V beta 6+ T cells. These cells expanded specifically and were triggered to express the activation marker CD69. Further stages of progression of infection appeared to be defective. Kinetics experiments indicated that T and B cell stimulation stopped more rapidly than in control mice. B cells acquired an activated CD69+ phenotype, were induced to produce IgM but only partially switched to IgG secretion. Finally, the dissemination of infected cells to other lymph nodes and spleen was reduced and the peripheral deletion of V beta 6+ T cells was minimal. In contrast, in mice lacking I-E, T cell stimulation was also impaired and B cell activation undetectable. These data implicate B7-dependent cellular interactions for superantigenic T cell stimulation by low-affinity TCR ligands and suggest a role of B cell differentiation in viral dissemination and peripheral T cell deletion.

Controlled study of 50-Hz repetitive transcranial magnetic stimulation for the treatment of Parkinson disease.

OBJECTIVE: To investigate the safety and efficacy of 50-Hz repetitive transcranial magnetic stimulation (rTMS) in the treatment of motor symptoms in Parkinson disease (PD). BACKGROUND: Progression of PD is characterized by the emergence of motor deficits that gradually respond less to dopaminergic therapy. rTMS has shown promising results in improving gait, a major cause of disability, and may provide a therapeutic alternative. Prior controlled studies suggest that an increase in stimulation frequency might enhance therapeutic efficacy. METHODS: In this randomized, double blind, sham-controlled study, the authors investigated the safety and efficacy of 50-Hz rTMS of the motor cortices in 8 sessions over 2 weeks. Assessment of safety and clinical efficacy over a 1-month period included timed tests of gait and bradykinesia, Unified Parkinson's Disease Rating Scale (UPDRS), and additional clinical, neurophysiological, and neuropsychological parameters. In addition, the safety of 50-Hz rTMS was tested with electromyography-electroencephalogram (EMG-EEG) monitoring during and after stimulation. RESULTS: The authors investigated 26 patients with mild to moderate PD: 13 received 50-Hz rTMS and 13 sham stimulation. The 50-Hz rTMS did not improve gait, bradykinesia, and global and motor UPDRS, but there appeared a short-lived "on"-state improvement in activities of daily living (UPDRS II). The 50-Hz rTMS lengthened the cortical silent period, but other neurophysiological and neuropsychological measures remained unchanged. EMG/EEG recorded no pathological increase of cortical excitability or epileptic activity. There were no adverse effects. CONCLUSION: It appears that 50-Hz rTMS of the motor cortices is safe, but it fails to improve motor performance and functional status in PD. Prolonged stimulation or other techniques with rTMS might be more efficacious but need to be established in future research.

Microtubule-depolymerizing agents used in antibody-drug conjugates induce antitumor immunity by stimulation of dendritic cells.

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies. Cancer Immunol Res; 2(8); 741-55. ©2014 AACR.

Electrode location and clinical outcome in hippocampal electrical stimulation for mesial temporal lobe epilepsy.

PURPOSE: To study the clinical outcome in hippocampal deep brain stimulation (DBS) for the treatment of patients with refractory mesial temporal lobe epilepsy (MTLE) according to the electrode location. METHODS: Eight MTLE patients implanted in the hippocampus and stimulated with high-frequency DBS were included in this study. Five underwent invasive recordings with depth electrodes to localize ictal onset zone prior to chronic DBS. Position of the active contacts of the electrode was calculated on postoperative imaging. The distances to the ictal onset zone were measured as well as atlas-based hippocampus structures impacted by stimulation were identified. Both were correlated with seizure frequency reduction. RESULTS: The distances between active electrode location and estimated ictal onset zone were 11±4.3 or 9.1±2.3mm for patients with a >50% or <50% reduction in seizure frequency. In patients (N=6) showing a >50% seizure frequency reduction, 100% had the active contacts located <3mm from the subiculum (p<0.05). The 2 non-responders patients were stimulated on contacts located >3mm to the subiculum. CONCLUSION: Decrease of epileptogenic activity induced by hippocampal DBS in refractory MTLE: (1) seems not directly associated with the vicinity of active electrode to the ictal focus determined by invasive recordings; (2) might be obtained through the neuromodulation of the subiculum.

Direct electrical stimulation using a battery-operated device for induction and modulation of colonic contractions in pigs.

Direct electrical stimulation of the colon offers a promising approach for the induction of propulsive colonic contractions by using an implantable device. The objective of this study was to assess the feasibility to induce colonic contractions using a commercially available battery-operated stimulator (maximum pulse width of 1 ms and maximum amplitude of 10 V). Three pairs of pacing electrodes were inserted into the cecal seromuscular layer of anesthetized pigs. During a first set of in vivo experiments conducted on six animals, a pacing protocol leading to cecum contractions was determined: stimulation bursts with 1 ms pulse width, 10 V amplitude (7-15 mA), 120 Hz frequency, and 30-s burst duration, repeated every 2-5 min. In a second testing phase, an evaluation of the pacing protocol was performed in four animals (120 stimulation bursts in total). By using the battery-operated stimulator, contractions of the cecum and movement of contents could be induced in 92% of all stimulations. A cecal shortening of about 30% and an average intraluminal pressure increase of 10.0 +/- 6.0 mmHg were observed.

Adrenergic, dopaminergic, and muscarinic receptor stimulation leads to PKA phosphorylation of Na-K-ATPase.

Na-K-adenosinetriphosphatase (Na-K-ATPase) is a potential target for phosphorylation by protein kinase A (PKA) and C (PKC). We have investigated whether the Na-K-ATPase alpha-subunit becomes phosphorylated at its PKA or PKC phosphorylation sites upon stimulation of G protein-coupled receptors primarily linked either to the PKA or the PKC pathway. COS-7 cells, transiently or stably expressing Bufo marinus Na-K-ATPase wild-type alpha- or mutant alpha-subunits affected in its PKA or PKC phosphorylation site, were transfected with recombinant DNA encoding beta 2- or alpha 1-adrenergic (AR), dopaminergic (D1A-R), or muscarinic cholinergic (M1-AChR) receptor subspecies. Agonist stimulation of beta 2-AR or D1A-R led to phosphorylation of the wild-type alpha-subunit, as well as the PKC mutant, but not of the PKA mutant, indicating that these receptors can phosphorylate the Na-K-ATPase via PKA activation. Surprisingly, stimulation of the alpha 1B-AR, alpha 1C-AR, and M1-AChR also increased the phosphorylation of the wild-type alpha-subunit and its PKC mutant but not of its PKA mutant. Thus the phosphorylation induced by these primarily phospholipase C-linked receptors seems mainly mediated by PKA activation. These data indicate that the Na-K-ATPase alpha-subunit can act as an ultimate target for PKA phosphorylation in a cascade starting with agonist-receptor interaction and leading finally to a phosphorylation-mediated regulation of the enzyme.

Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.

PURPOSE: To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors. METHODS: Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells. CONCLUSIONS: The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.

Cerebellar transcranial direct current stimulation modulates verbal working memory.

BACKGROUND: Neuroimaging studies show cerebellar activations in a wide range of cognitive tasks and patients with cerebellar lesions often present cognitive deficits suggesting a cerebellar role in higher-order cognition. OBJECTIVE: We used cathodal transcranial direct current stimulation (tDCS), known to inhibit neuronal excitability, over the cerebellum to investigate if cathodal tDCS impairs verbal working memory, an important higher-order cognitive faculty. METHOD: We tested verbal working memory as measured by forward and backward digit spans in 40 healthy young participants before and after applying cathodal tDCS (2 mA, stimulation duration 25 min) to the right cerebellum using a randomized, sham-controlled, double-blind, cross-over design. In addition, we tested the effect of cerebellar tDCS on word reading, finger tapping and a visually cued sensorimotor task. RESULTS: In line with lower digit spans in patients with cerebellar lesions, cerebellar tDCS reduced forward digit spans and blocked the practice dependent increase in backward digit spans. No effects of tDCS on word reading, finger tapping or the visually cued sensorimotor task were found. CONCLUSION: Our results support the view that the cerebellum contributes to verbal working memory as measured by forward and backward digit spans. Moreover, the induction of reversible "virtual cerebellar lesions" in healthy individuals by means of tDCS may improve our understanding of the mechanistic basis of verbal working memory deficits in patients with cerebellar lesions.

Gonadotropin-releasing hormone secretion from hypothalamic neurons: stimulation by insulin and potentiation by leptin.

Insulin and leptin are peripheral metabolic factors signaling the body needs in energy to the central nervous system. Because energy homeostasis and reproductive function are closely related phenomena, we investigated the respective roles played by insulin and leptin in the hypothalamic control of GnRH secretion. We observed that increasing circulating insulin levels, by performing hyperinsulinemic clamp studies in male mice, was associated with a significant rise in LH secretion. This effect of insulin is likely mediated at the hypothalamic level, because it was also found to stimulate the secretion and the expression of GnRH by hypothalamic neurons in culture. Leptin was found to potentiate the effect of insulin on GnRH secretion in vitro but was devoid of any effect on its own. These data represent the first evidence of direct insulin sensing by hypothalamic neurons involved in activating the neuroendocrine gonadotrope axis. They also demonstrate that these neurons can integrate different hormonal signals to modulate net hypothalamic GnRH output. We propose that such integration is an essential mechanism for the adaptation of reproductive function to changes in the metabolic status of an individual.

Late outcome of spinal cord stimulation for unreconstructable and limb-threatening lower limb ischemia.

OBJECTIVES: To determine whether the initial benefits of spinal cord stimulation (SCS) treatment for critical limb ischemia (CLI) persist over years. DESIGN: Analysis of data prospectively collected for every CLI patient receiving permanent SCS. Follow-up range 12 to 98 months (mean 46+/-23, median 50 months). POPULATION: 87 patients (28% stage III, 72%stage IV) with unreconstructable CLI due (83%) or not (17%) to atherosclerosis and with an initial sitting/supine transcutaneous pO2 gradient >15 mmHg. METHODS: Assessment of actuarial patient survival (PS), limb salvage (LS) and amputation-free patient survival (AFPS). Analysis of the impact of 15 risk factors on long-term outcomes using the Fischer's exact test for categorical variables and the t test for continuous variables. RESULTS: Follow-up was complete for patient and limb survival. A single non-atherosclerotic patient died during follow-up. Among atherosclerotic patients PS decreased from 88% at 1y, to 76% at 3y, 64% at 5y and 57% at 7y. LS reached 84% at 1y, 78% at 2y, 75% at 3y and remained stable thereafter. Diabetes was found to affect LS (p<0.05) and heart disease to reduce PS (p<0.01). AFPS was reduced in heart patients (p<0.01), diabetics (p<0.05) and in patients with previous stroke (p<0.05). CONCLUSIONS: In CLI patients the beneficial effects of SCS persist far beyond the first year of treatment and major amputation becomes infrequent after the second year.

Evaluation of the early changes occurring in the draining lymph node upon subcutaneous stimulation

Adjuvants have been shown since many years to have an important role in enhancing the immune responses against the co-administered antigens used as vaccines. The continuous study of the mechanism of action of adjuvants is necessary to develop further safe and efficacious vaccines. Complete Freund's adjuvant (CFA) is currently in use as adjuvant to induce some autoimmune diseases in murine models, therefore the study of the mechanisms involved in the generation of the related immune responses could be instrumental for the understanding of the induction of inflammatory Thl7 responses. In the present work, we showed in C57B1/6 mice that CFA peripheral administration induces very early, at 6 h, a potent influx of CDllb+ cells, mainly neutrophils (CD11b+Ly6GhighLy6Cint) and monocytes (CD11b+Ly6GlowLy6Chigh), in the draining lymph node. By investigating the route by which neutrophils reach the lymph node we observed that, around 20% of them arrive from the afferent lymph and the majority stains positive for Mycobacterium tuberculosis. We also observed a correlation between the influx of neutrophils and an increase in IL-23 and IL-Ιβ, together with several inflammatory chemokines, in the draining lymph node. Concomitantly, we detected the expression of the IL-23 receptor on CDllc+ DCs. Moreover, we confirmed the ability of murine neutrophils to express IL-23 both, in vitro by stimulating bone-marrow extracted PMNs with Mycobacterium tuberculosis, and on total cells from draining lymph node by immunohistochemistry. We also observed by in vivo priming a reduction in the percentage of IFN-γ and CXCR3 expressing Τ cells upon depletion of neutrophils. Altogether, we show that upon stimulation from the periphery, the draining lymph node undergo changes in cytokine/chemokine production leading to the recruitment of different leukocytes subpopulations. Here we show that CFA induces a rapid influx of neutrophils which are responsible for the production of IL-23 that in turn influences the generation of Τ helper cells.

cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.

Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.

BOLD responses to trigeminal nerve stimulation.

The current study investigates a new model of barrel cortex activation using stimulation of the infraorbital branch of the trigeminal nerve. A robust and reproducible activation of the rat barrel cortex was obtained following trigeminal nerve stimulation. Blood oxygen level-dependent (BOLD) effects were obtained in the primary somatosensory barrel cortex (S1BF), the secondary somatosensory cortex (S2) and the motor cortex. These cortical areas were reached from afferent pathways from the trigeminal ganglion, the trigeminal nuclei and thalamic nuclei from which neurons project their axons upon whisker stimulation. The maximum BOLD responses were obtained for a stimulus frequency of 1 Hz, a stimulus pulse width of 100 μs and for current intensities between 1.5 and 3 mA. The BOLD response was nonlinear as a function of frequency and current intensity. Additionally, modeling BOLD responses in the rat barrel cortex from separate cerebral blood flow (CBF) and cerebral metabolic rate of oxygen (CMRO(2)) measurements showed good agreement with the shape and amplitude of measured BOLD responses as a function of stimulus frequency and will potentially allow to identify the sources of BOLD nonlinearities. Activation of the rat barrel cortex using trigeminal nerve stimulation will contribute to the interpretation of the BOLD signals from functional magnetic resonance imaging studies.