Assessment of immunostimulatory responses to the antimiR-22 oligonucleotide compound RES-010 in human peripheral blood mononuclear cells.

microRNA-22 (miR-22) is a key regulator of lipid and energy homeostasis and represents a promising therapeutic target for NAFLD and obesity. We have previously identified a locked nucleic acid (LNA)-modified antisense oligonucleotide compound complementary to miR-22, designated as RES-010 that mediated robust inhibition of miR-22 function in cultured cells and in vivo. In this study we investigated the immune potential of RES-010 in human peripheral blood mononuclear cells (PBMCs). We treated fresh human peripheral blood mononuclear cells isolated from six healthy volunteers with different concentrations of the RES-010 compound and assessed its proinflammatory effects by quantifying IL-1ß, IL-6, IFN-γ, TNF-α, IFN-α2a, IFN-ß, IL-10, and IL-17A in the supernatants collected 24 h of treatment with RES-010. The T-cell activation markers, CD69, HLA-DR, and CD25 were evaluated by flow cytometry after 24 and 144 h of treatment, respectively, whereas cell viability was assessed after 24 h of treatment with RES-010. Our results show that RES-010 compound does not induce any significant immunostimulatory responses in human PBMCs in vitro compared to controls, implying that the proinflammatory potential of RES-010 is low.

Differential effects of glycogen synthase kinase 3 (GSK3) inhibition by lithium or selective inhibitors in the central nervous system.

Glycogen synthase kinase (GSK3) is a constitutively active serine-threonine kinase associated to neurological and psychiatric disorders. GSK3 inhibition is considered a mediator of the efficacy of the mood-stabiliser lithium. This study aimed at comparing the central nervous system effect of lithium with the selective GSK3 inhibitors AZ1080 and compound A in biochemical, cellular, and behavioural tests. Collapsin response mediator protein 2 is a neuron-specific GSK3 substrate. Lithium, AZ1080, and compound A inhibited its phosphorylation in rat primary neurons with different pIC50. After systemic treatments with lithium or GSK3 inhibitors to assess specific functional responses, phosphorylation was unchanged in adult rat brain, while it was strongly inhibited by GSK3 inhibitors in pups, differently from lithium. Lithium may exert neurotrophic effect by increasing brain-derived neurotrophic factor (BDNF) levels: in the present experimental conditions, lithium exerted opposite effects on plasma BDNF levels compared to GSK3 inhibitors, suggesting this effect might not be necessarily mediated by GSK3 inhibition alone. While plasma thyroid-stimulating hormone and luteinising hormone were not affected by lithium, they were decreased by selective inhibitors. GH and prolactin displayed similar responses towards reduction. Follicle-stimulating hormone levels were not altered by treatments, whereas melatonin was specifically increased by AZ1080. Lithium impaired mouse spontaneous locomotion and decreased amphetamine-induced hyper-locomotion. AZ1080 had no effects on locomotion, while compound A reduced spontaneous locomotor activity without effects on amphetamine-induced hyper-locomotion. The present results indicate that a broad correlation between the effects of lithium and selective GSK3 inhibitors could not be devised, suggesting alternative mechanisms, whereas overlapping results could be obtained in specific assays.

Adenoviral-mediated Cre expression effectively suppresses GlyT1 binding in the thalamic area of GlyT1 conditional knock-out mice.

To properly understand the function of genes of neurological interest, in vivo manipulation in the adult is essential, particularly when the target gene is involved in brain development. Moreover, since the physiological effects of target protein may be region-specific, targeting a distinct brain region could be required to dissect these effects in specific brain locations. Infection of somatic tissues of transgenic mice bearing loxP-flanked gene sequences with a viral vector expressing Cre recombinase provides a means of allowing flexible spatio-temporal control of target gene expression. Viral vector-mediated Cre expression could be used to mediate localized gene modulation in a specific brain region. In the present study this technology was applied to the glycine transporter type-1 (GlyT1) protein which is responsible for the uptake of synaptic glycine in the forebrain and has been implicated as a therapeutic target for the treatment of schizophrenia. Since GlyT1 is widely expressed in glial cells, we employed an adenoviral-based vector (Ad5) to deliver Cre protein, due to the preferentially transduction of glial cells by adenoviral vectors in rodent brain. We show significant reduced GlyT1 binding specifically in the thalamic area of conditional GlyT1 (GlyT1c) transgenic mice injected with Ad5-Cre virus, as measured by GlyT1 autoradiography. In conclusion, we demonstrated the validity of viral vector-mediated delivery of Cre to loxP targeted transgenic mice as a novel strategy to investigate target gene function in selected subregions of the adult brain, which provides a valuable technique to investigate gene function both in normal physiology and in disease models.

Lipopolysaccharide primes neutrophils for a rapid response to IL-10.

Responsiveness of human neutrophils to IL-10 was recently shown to be strictly dependent on the levels of IL-10R1 expression. Activation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and induction of suppressor of cytokine signaling (SOCS)-3 protein by IL-10 are in fact negligible in circulating or freshly isolated ("time 0") neutrophils, but become readily measurable in neutrophils cultured for 4 h in the presence or absence of LPS. In this study, we show that modulation by IL-10 of LPS-induced TNF-alpha, CXCL8/IL-8 and IL-1 receptor antagonist (IL-1ra) mRNA accumulation in neutrophils already expressing a functional IL-10R and antigenic SOCS-3 (i.e. in "4-h-cultured" neutrophils) occurs with kinetics that are similar to those observed in "time 0" neutrophils, depends on de novo protein synthesis, but does not require SOCS-1, SOCS-3, heme oxygenase and Bcl-3 induction. By contrast, we show that IL-10 alone rapidly modulates the expression of TNF-alpha, CXCL8/IL-8 and IL-1ra mRNA, without any new protein synthesis requirement, if neutrophils have been previously exposed to LPS for at least 4 h. These findings suggest that LPS prepares neutrophils to optimally respond to IL-10 in terms of rapid gene modulation via mechanisms that, presumably, depend on specific LPS-induced protein(s).