Roles of the ubiquitin peptidase USP18 in multiple sclerosis and the response to interferon-ß treatment.

BACKGROUND AND PURPOSE: Ubiquitin specific peptidase 18 (USP18) is a deubiquitinating enzyme that functions as a negative regulator of the type I interferon (IFN) signalling pathway and is specifically induced by type I IFNs. In the present study, previous observations by our group were expanded suggesting an implication of USP18 in multiple sclerosis (MS) based on the finding of a deficient expression of the gene in peripheral blood mononuclear cells from MS patients compared with healthy controls. METHODS: Two polymorphisms, rs2542109 (intronic) and rs9618216 (promoter), were genotyped in a cohort of 691 relapse-onset MS patients and 1028 healthy controls and in 225 MS patients treated with IFNß and classified into responders and non-responders after 2 years of treatment according to clinical criteria. Correlations between genotypes and expression levels for USP18 and its target ISG15 were performed by real-time polymerase chain reaction. RESULTS: Two USP18 haplotypes were significantly associated with MS, TG and CG. Additional experiments revealed that CG carriers were characterized by lower USP18 gene expression levels in peripheral blood mononuclear cells and higher clinical disease activity. Finally, AA homozygosis for the intronic polymorphism rs2542109 was associated with the responder phenotype; however, USP18 expression levels induced by IFNß did not differ amongst MS patients carrying different rs2542109 genotypes. CONCLUSIONS: Altogether, these results point to a role of USP18 in MS pathogenesis and the therapeutic response to IFNß.

Aberrant development of intrinsic brain activity in a rat model of caregiver maltreatment of offspring.

Caregiver maltreatment induces vulnerability to later-life psychopathology. Clinical and preclinical evidence suggest changes in prefrontal and limbic circuitry underlie this susceptibility. We examined this question using a rat model of maternal maltreatment and methods translated from humans, resting-state functional magnetic resonance imaging (R-fMRI). Rat pups were reared by mothers provided with insufficient or abundant bedding for nest building from postnatal (PN) days 8 to 12 and underwent behavioral assessments of affect-related behaviors (forced swim, sucrose preference and social interaction) in adolescence (PN45) and early adulthood (PN60). R-fMRI sessions were conducted under light anesthesia at both ages. Offspring reared with insufficient bedding (that is, maltreated) displayed enduring negative affective behaviors. Amygdala-prefrontal cortex (PFC) functional connectivity increased significantly from adolescence to adulthood in controls, but not in maltreated animals. We computed the fractional amplitude of low-frequency fluctuations (fALFF), an index of intrinsic brain activity, and found that fALFF in medial prefrontal cortex and anterior cingulate cortex (MPFC/ACC) increased significantly with age in controls but remained unchanged in maltreated animals during adolescence and adulthood. We used a seed-based analysis to explore changes in functional connectivity between this region and the whole brain. Compared with controls, maltreated animals demonstrated reduced functional connectivity between MPFC/ACC and left caudate/putamen across both ages. Functional connectivity between MPFC/ACC and right caudate/putamen showed a group by age interaction: decreased in controls but increased in maltreated animals. These data suggest that maltreatment induces vulnerability to psychopathology and is associated with differential developmental trajectories of prefrontal and subcortical circuits underlying affect regulation.

Optic aphasia, optic apraxia, and loss of dreaming.

A 47-year-old man with a left temporo-occipital infarct in the area of the posterior cerebral artery is presented. The neuropsychological examination did not reveal aphasia or gross mental deficits. The patient presented with alexia without agraphia, color agnosia, but few visual perceptual deficits. The main impairment was in confrontation naming; he was incapable of naming objects and pictures, not from lack of recognition (excluding visual agnosia) but from lack of access to the appropriate word (optic aphasia). The patient also exhibited a deficit in the evocation of gesture from the visual presentation of an object (optic apraxia) and a difficulty in "conjuring up" visual images of objects (impaired visual imagery) and loss of dreams. The fundamental deficit of this patient is tentatively explained in terms of visuoverbal and visuogestural disconnection and a deficit of mental imagery.

Unpredictable neonatal stress enhances adult anxiety and alters amygdala gene expression related to serotonin and GABA.

Anxiety-related disorders are among the most common psychiatric illnesses, thought to have both genetic and environmental causes. Early-life trauma, such as abuse from a caregiver, can be predictable or unpredictable, each resulting in increased prevalence and severity of a unique set of disorders. In this study, we examined the influence of early unpredictable trauma on both the behavioral expression of adult anxiety and gene expression within the amygdala. Neonatal rats were exposed to unpaired odor-shock conditioning for 5 days, which produces deficits in adult behavior and amygdala dysfunction. In adulthood, we used the Light/Dark box test to measure anxiety-related behaviors, measuring the latency to enter the lit area and quantified urination and defecation. The amygdala was then dissected and a microarray analysis was performed to examine changes in gene expression. Animals that had received early unpredictable trauma displayed significantly longer latencies to enter the lit area and more defecation and urination. The microarray analysis revealed over-represented genes related to learning and memory, synaptic transmission and trans-membrane transport. Gene ontology and pathway analysis identified highly represented disease states related to anxiety phenotypes, including social anxiety, obsessive-compulsive disorders, post-traumatic stress disorder and bipolar disorder. Addiction-related genes were also overrepresented in this analysis. Unpredictable shock during early development increased anxiety-like behaviors in adulthood with concomitant changes in genes related to neurotransmission, resulting in gene expression patterns similar to anxiety-related psychiatric disorders.

Electrical impedance spectroscopy measurements using a four-electrode configuration improve on-line monitoring of cell concentration in adherent animal cell cultures.

This paper describes the improvement in the use of electrical impedance spectroscopy (EIS) for animal cell concentration monitoring of adherent cultures by using a four-electrode configuration instead of the commonly used two-electrode configuration. This four-electrode configuration prevents cell concentration measurements from external masking effects such as the electrode covering ratio, the degree of cellular adherence to the electrodes and the impedance of the measuring electrodes. Cell concentration was monitored using both four-electrode and two-electrode configurations in vero cell and human mesenchymal stem cell cultures in order to analyze the attained improvement in two cell lines with opposite growth characteristics. The experiments performed with vero cell cultures evidenced that the four-electrode configuration enables cell concentration measurements along all culture phases, even once the culture reached cell confluence (over 2×10(5) cells/cm(2)), confirming that this configuration is less effected by all the external influences. The experiments performed with human mesenchymal stem cells demonstrated good sensitivity of the measurement at very low cell concentrations, as well as a very good robustness all over the 12-days experiment. Finally, off-line cell measurements during cell cultures proved good accuracy of impedance measurements carried out with a four-electrode configuration along all cell growth phases, enabling determination of relevant cell growth parameters.

Phosphoinositide 3-kinase inhibitors protect mouse kidney cells from cyclosporine-induced cell death.

The use of cyclosporine has been restricted by its nephrotoxic effects mediated, in part, by reactive oxygen species (ROS). Phosphoinositide 3-kinase, protein kinase B, and extracellular regulated kinase (ERK) pathways are related to survival and cell death and are activated after ROS generation. In this study, we evaluated the effects of cyclosporine on these pathways and their contribution to cyclosporine-induced toxicity. Viability of cells derived from the proximal tubule of transgenic mice was measured with Trypan Blue, ROS generation by a fluorescent probe, while ERK and phosphoinositide 3-kinase/protein kinase B activation were monitored with phospho-specific antibodies. Cyclosporine decreased cell viability and induced ROS generation and ERK and phosphoinositide 3-kinase activation. Both pathways were activated by the epidermal growth factor receptor (EGFR). Antioxidants blocked ERK activation but failed to inhibit protein kinase B phosphorylation or prevent cyclosporine toxicity. ERK inhibition did not protect from cyclosporine-induced cell death. EGFR or phosphoinositide 3-kinase inhibitors protected from cyclosporine-triggered cell death without decreasing ROS. Small interfering RNA against the catalytic subunit of phosphoinositide 3-kinase decreased protein kinase B phosphorylation but did not prevent cyclosporine-mediated cell death. Our results show that EGFR mediates the cytotoxic effects of cyclosporine through an ROS-independent mechanism. Cyclosporine-induced cell death is triggered by a non-classical phosphoinositide 3-kinase and does not require ERK activation.

Four versus two-electrode measurement strategies for cell growing and differentiation monitoring using electrical impedance spectroscopy.

The aim of this work is to provide optimization tools for cell and tissue engineering processes through continuous monitoring of cell cultures. Structural cell properties can be obtained from non-destructive electrical measurements by using electrical impedance spectroscopy (EIS). EIS measurements on monolayer animal cell cultures are usually performed using a two-electrode strategy. Because of this, the measurement is very sensitive to the electrode covering ratio and to the degree of adherence of cells. Of course, these parameters give useful information but can mask the behaviour of the cell layer above the electrodes. In a previous work, preliminary measurements with commercial microelectrode structures were performed with simulated grow processes using the settlement of cell suspensions with two and four microelectrode strategies to validate the technique. In this work, real cell growths of Vero cells are described and the resulting EIS biomass density estimators are compared to cell counts. The four-electrode impedance spectra are fitted to the Cole-Cole impedance model and the time course of their parameters are extracted and studied.

Multiple automated minibioreactor system for multifunctional screening in biotechnology.

The current techniques applied in biotechnology allow to obtain many types of molecules that must be tested on cell cultures (high throughput screening HTS). Although such tests are usually carried out automatically on mini or microwell plates, the procedures in the preindustrial stage are performed almost manually on higher volume recipients known as bioreactors. The growth conditions in both stages are completely different. The screening system presented in this work is based on the multiwell test plates philosophy, a disposable multiple minibioreactor that allows reproduction of industrial bioreactor culture conditions: aeration, stirring, temperature, O2, pH and visible range optical absorbance measurements. It is possible to reproduce the growth conditions for both suspended and adherent animal cell types using 1 to 10 ml vol. bioreactors. In the case of bacteria or yeast, it is not possible to achieve a high biomass concentration, due to the reduced head volume air supply.