Proteasome inhibition reduces proliferation, collagen expression, and inflammatory cytokine production in nasal mucosa and polyp fibroblasts

Proteasome inhibitors, used in cancer treatment for their proapoptotic effects, have anti-inflammatory and antifibrotic effects on animal models of various inflammatory and fibrotic diseases. Their effects in cells from patients affected by either inflammatory or fibrotic diseases have been poorly investigated. Nasal polyposis is a chronic inflammatory disease of the sinus mucosa characterized by tissue inflammation and remodeling. We tested the hypothesis that proteasome inhibition of nasal polyp fibroblasts might reduce their proliferation and inflammatory and fibrotic response. Accordingly, we investigated the effect of the proteasome inhibitor Z-Leu-Leu-Leu-B(OH)2 (MG262) on cell viability and proliferation and on the production of collagen and inflammatory cytokines in nasal polyp and nasal mucosa fibroblasts obtained from surgery specimens. MG262 reduced the viability of nasal mucosa and polyp fibroblasts concentration- and time-dependently, with marked effects after 48 h of treatment. The proteasome inhibitor bortezomib provoked a similar effect. MG262-induced cell death involved loss of mitochondrial membrane potential, caspase-3 and poly(ADP-ribose) polymerase activation, induction of c-Jun phosphorylation, and mitogen-activated protein kinase phosphatase-1 expression. Low concentrations of MG262 provoked growth arrest, inhibited DNA replication and retinoblastoma phosphorylation, and increased expression of the cell cycle inhibitors p21 and p27. MG262 concentration-dependently inhibited basal and transforming growth factor-β-induced collagen mRNA expression and interleukin (IL)-1β-induced production of IL-6, IL-8, monocyte chemoattractant protein-1, regulated on activation normal T cell expressed and secreted, and granulocyte/macrophage colony-stimulating factor in both fibroblast types. MG262 inhibited IL-1β/tumor necrosis factor-α-induced activation of nuclear factor-κB. We conclude that noncytotoxic treatment with MG262 reduces the proliferative, fibrotic, and inflammatory response of nasal fibroblasts, whereas high MG262 concentrations induce apoptosis.

ADHD candidate gene (DRD4 exon III) affects inhibitory control in a healthy sample.

Background Dopamine is believed to be a key neurotransmitter in the development of attention-deficit/ hyperactivity disorder (ADHD). Several recent studies point to an association of the dopamine D4 receptor (DRD4) gene and this condition. More specifically, the 7 repeat variant of a variable number of tandem repeats (VNTR) polymorphism in exon III of this gene is suggested to bear a higher risk for ADHD. In the present study, we investigated the role of this polymorphism in the modulation of neurophysiological correlates of response inhibition (Go/Nogo task) in a healthy, high-functioning sample. Results Homozygous 7 repeat carriers showed a tendency for more accurate behavior in the Go/Nogo task compared to homozygous 4 repeat carriers. Moreover, 7 repeat carriers presented an increased nogo-related theta band response together with a reduced go-related beta decrease. Conclusions These data point to improved cognitive functions and prefrontal control in the 7 repeat carriers, probably due to the D4 receptor's modulatory role in prefrontal areas. The results are discussed with respect to previous behavioral data on this polymorphism and animal studies on the impact of the D4 receptor on cognitive functions.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

CD4 expression decrease by antisense oligonucleotides. Inhibition of rat T CD4+ cell reactivity

In previous studies, we have demonstrated the inhibition of CD4 expression in rat lymphocytes treated with phorbol myristate acetate (PMA) by antisense oligonucleotides (AS-ODNs) directed against the AUG start region of the cd4 gene. The aim of the present study was to inhibit CD4 expression in lymphocytes without promoting CD4 synthesis and to determine the effect of this inhibition on CD4+ T cell function. Four 21-mer ODNs against the rat cd4 gene (AS-CD4-1 to AS-CD4-4) were used. Surface CD4 expression was measured by immunofluorescence staining and flow cytometry, and mRNA CD4 expression was measured by RT-PCR. T CD4+ cell function was determined by specific and unspecific proliferative response of rat-primed lymphocytes. After 24 hours of incubation, AS-CD4-2 and AS-CD4-4 reduced lymphocyte surface CD4 expression by 40%. This effect remained for 72 hours and was not observed on other surface molecules, such as CD3, CD5, or CD8. CD4 mRNA expression was reduced up to 40% at 24 hours with AS-CD4-2 and AS-CD4-4. After 48 hours treatment, CD4 mRNA decreased up to 27% and 29% for AS-CD4-2 and AS-CD4-4, respectively. AS-CD4-2 and AS-CD4-4 inhibited T CD4+ cell proliferative response upon antigen-specific and unspecific stimuli. Therefore, AS-ODNs against CD4 molecules inhibited surface and mRNA CD4 expression, under physiologic turnover and, consequently, modulate T CD4+ cell reactivity.

SMRT-mediated co-shuttling enables export of class IIa HDACs independent of their CaM kinase phosphorylation sites

The Class IIa histone deacetylases (HDAC)4 and HDAC5 play a role in neuronal survival and behavioral adaptation in the CNS. Phosphorylation at 2/3 N-terminal sites promote their nuclear export. We investigated whether non-canonical signaling routes to Class IIa HDAC export exist because of their association with the co-repressor Silencing Mediator Of Retinoic And Thyroid Hormone Receptors (SMRT). We found that, while HDAC5 and HDAC4 mutants lacking their N-terminal phosphorylation sites (HDAC4(MUT), HDAC5(MUT)) are constitutively nuclear, co-expression with SMRT renders them exportable by signals that trigger SMRT export, such as synaptic activity, HDAC inhibition, and Brain Derived Neurotrophic Factor (BDNF) signaling. We found that SMRT's repression domain 3 (RD3) is critical for co-shuttling of HDAC5(MUT), consistent with the role for this domain in Class IIa HDAC association. In the context of BDNF signaling, we found that HDAC5(WT), which was more cytoplasmic than HDAC5(MUT), accumulated in the nucleus after BDNF treatment. However, co-expression of SMRT blocked BDNF-induced HDAC5(WT) import in a RD3-dependent manner. In effect, SMRT-mediated HDAC5(WT) export was opposing the BDNF-induced HDAC5 nuclear accumulation observed in SMRT's absence. Thus, SMRT's presence may render Class IIa HDACs exportable by a wider range of signals than those which simply

Inhibition of nociceptive responses after systemic administration of amidated kyotorphin

BACKGROUND AND PURPOSE Kyotorphin (KTP; L-Tyr-L-Arg), an endogenous neuropeptide, is potently analgesic when delivered directly to the central nervous system. Its weak analgesic effects after systemic administration have been explained by inability to cross the blood-brain barrier (BBB) and detract from the possible clinical use of KTP as an analgesic. In this study, we aimed to increase the lipophilicity of KTP by amidation and to evaluate the analgesic efficacy of a new KTP derivative (KTP-amide - KTP-NH 2). EXPERIMENTAL APPROACH We synthesized KTP-NH 2. This peptide was given systemically to assess its ability to cross the BBB. A wide range of pain models, including acute, sustained and chronic inflammatory and neuropathic pain, were used to characterize analgesic efficacies of KTP-NH 2. Binding to opioid receptors and toxicity were also measured. KEY RESULTS KTP-NH 2, unlike its precursor KTP, was lipophilic and highly analgesic following systemic administration in several acute and chronic pain models, without inducing toxic effects or affecting motor responses and blood pressure. Binding to opioid receptors was minimal. KTP-NH 2 inhibited nociceptive responses of spinal neurons. Its analgesic effects were prevented by intrathecal or i.p. administration of naloxone. CONCLUSIONS AND IMPLICATIONS Amidation allowed KTP to show good analgesic ability after systemic delivery in acute and chronic pain models. The indirect opioid-mediated actions of KTP-NH 2 may explain why this compound retained its analgesic effects although the usual side effects of opioids were absent, which is a desired feature in next-generation pain medications

The role of executive functions in the control of aggressive behavior

An extensive literature suggests a link between executive functions and aggressive behavior in humans, pointing mostly to an inverse relationship, i.e., increased tendencies toward aggression in individuals scoring low on executive function tests. This literature is limited, though, in terms of the groups studied and the measures of executive functions. In this paper, we present data from two studies addressing these issues. In a first behavioral study, we asked whether high trait aggressiveness is related to reduced executive functions. A sample of over 600 students performed in an extensive behavioral test battery including paradigms addressing executive functions such as the Eriksen Flanker task, Stroop task, n-back task, and Tower of London (TOL). High trait aggressive participants were found to have a significantly reduced latency score in the TOL, indicating more impulsive behavior compared to low trait aggressive participants. No other differences were detected. In an EEG-study, we assessed neural and behavioral correlates of error monitoring and response inhibition in participants who were characterized based on their laboratory-induced aggressive behavior in a competitive reaction time task. Participants who retaliated more in the aggression paradigm and had reduced frontal activity when being provoked did not, however, show any reduction in behavioral or neural correlates of executive control compared to the less aggressive participants. Our results question a strong relationship between aggression and executive functions at least for healthy, high-functioning people.

The use of mixed methods research in the behavioral sciences field

Mixed methods research involves the combined use of quantitative and qualitative methods in the same research study, and it is becoming increasingly important in several scientific areas. The aim of this paper is to review and compare through a mixed methods multiple-case study the application of this methodology in three reputable behavioural science journals: the Journal of Organizational Behavior, Addictive Behaviors and Psicothema. A quantitative analysis was carried out to review all the papers published in these journals during the period 2003-2008 and classify them into two blocks: theoretical and empirical, with the latter being further subdivided into three subtypes (quantitative, qualitative and mixed). A qualitative analysis determined the main characteristics of the mixed methods studies identified, in order to describe in more detail the ways in which the two methods are combined based on their purpose, priority, implementation and research design. From the journals selected, a total of 1.958 articles were analysed, the majority of which corresponded to empirical studies, with only a small number referring to research that used mixed methods. Nonetheless, mixed methods research does appear in all the behavioural science journals studied within the period selected, showing a range of designs, where the sequential equal weight mixed methods research design seems to stand out.