No abstract

We characterised the behavioural phenotype of mice heterozygous (Oxtr(+/-)) for the oxytocin receptor gene (Oxtr) and compared it with that of Oxtr null mice (Oxtr(-/-)), which display autistic-like behaviours, including impaired sociability and preference for social novelty, impaired cognitive flexibility, and increased aggression. Similar to Oxtr(-/-) mice, the Oxtr(+/-) showed impaired sociability and preference for social novelty but, unlike the null genotype, their cognitive flexibility and aggression were normal. By autoradiography, Oxtr(+/-) mice were found to have approximately 50% fewer oxytocin receptors (OXTRs) in all of the examined brain regions. Thus, because a partial reduction in Oxtr gene expression is sufficient to compromise social behaviour, the Oxtr acts as a haploinsufficient gene. Furthermore, the inactivation of the Oxtr gene affects specific behaviours in a dose-dependent manner: social behaviour is sensitive to even a partial reduction in Oxtr gene expression, whereas defects in aggression and cognitive flexibility require the complete inactivation of the Oxtr gene to emerge. We then investigated the rescue of the Oxtr(+/-) social deficits by oxytocin (OT) and Thr(4)Gly(7)OT (TGOT) administered i.c.v. at different doses. TGOT was more potent than OT in rescuing sociability and social novelty in both genotypes. Furthermore, the TGOT doses that reverted impaired sociability and preference for social novelty in Oxtr(+/-) were lower than those required in Oxtr(-/-), thus suggesting that the rescue effect is mediated by OXTR in Oxtr(+/-) and by other receptors (presumably vasopressin V1a receptors) in Oxtr(-/-). In line with this, a low dose of the selective oxytocin antagonist desGlyDTyrOVT blocks the rescue effect of TGOT only in the Oxtr(+/-) genotype, whereas the less selective antagonist SR49059 blocks rescue in both genotypes. In conclusion, the Oxtr(+/-) mouse is a unique animal model for investigating how partial loss of the Oxtr gene impair social interactions, and for designing pharmacological rescue strategies.

Activated lymphocytes release nano-sized vesicles (exosomes) containing microRNAs that can be monitored in the bloodstream. We asked whether elicitation of immune responses is followed by release of lymphocyte-specific microRNAs. We found that, upon activation in vitro, human and mouse lymphocytes down-modulate intracellular miR-150 and accumulate it in exosomes. In vivo, miR-150 levels increased significantly in serum of humans immunized with flu vaccines and in mice immunized with ovalbumin, and this increase correlated with elevation of antibody titers. Immunization of immune-deficient mice, lacking MHCII, resulted neither in antibody production nor in elevation of circulating miR-150. This study provides proof of concept that serum microRNAs can be detected, with minimally invasive procedure, as biomarkers of vaccination and more in general of adaptive immune responses. Furthermore, the prompt reduction of intracellular level of miR-150, a key regulator of mRNAs critical for lymphocyte differentiation and functions, linked to its release in the external milieu suggests that the selective extracellular disposal of microRNAs can be a rapid way to regulate gene expression during lymphocyte activation.

The neuropeptides oxytocin (OT) and vasopressin (AVP) have been shown to play a central role in social behaviors; as a consequence, they have been recognized as potential drugs to treat neurodevelopmental and psychiatric disorders characterized by impaired social interactions. However, despite the basic and preclinical relevance of mouse strains carrying genetic alterations in the OT/AVP systems to basic and preclinical translational neuroscience, the pharmacological profile of mouse OT/AVP receptor subtypes has not been fully characterized. To fill in this gap, we have characterized a number of OT and AVP agonists and antagonists at three murine OT/AVP receptors expressed in the nervous system as follows: the oxytocin (mOTR) and vasopressin V1a (mV1aR) and V1b (mV1bR) subtypes. These three receptors were transiently expressed in vitro for binding and intracellular signaling assays, and then a homology model of the mOTR structure was constructed to investigate how its molecular features compare with human and rat OTR orthologs. Our data indicate that the selectivity profile of the natural ligands, OT and AVP, is conserved in humans, rats, and mice. Furthermore, we found that the synthetic peptide [Thr 4 Gly 7 ]OT (TGOT) is remarkably selective for the mOTR and, like the endogenous OT ligand, activates G q and G i and recruits b-arrestins. Finally, we report three antagonists that exhibit remarkably high affinities and selectivities at mOTRs. These highly selective pharmacological tools will contribute to the investigation of the specific physiologic and pathologic roles of mOTR for the development of selective OT-based therapeutics.

Upon T cell receptor stimulation, CD4 T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4 T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4 T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4 T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells and their biological effect in cell to cell communication during the adaptive immune response.

Oxytocin (OT) is a small nonapeptide that regulates multiple peripheral (i.e. parturition and lactation) as well as central biological functions. Within the brain, OT is especially involved in the regulation of social behavior hence promoting maternal care and aggression, pair bonding, sexual activity, social memory processes and recognition of conspecific in various animal species (Donaldson and Young 2008). The role of OT in processing social stimuli and modulating pro-social responses has also been confirmed in humans, where the peptide has been shown to reduce stress and anxiety, to increase trust and generosity, and to improve the ability of interpreting mental states (Heinrichs et al. 2009). Accordingly, a link between autism spectrum disorders and the OT system has been proposed.Oxytocin is mainly synthesized in the magnocellular neurons of the supraoptic nuclei (SON) and paraventricular nuclei of the hypothalamus, projecting to the posterior pituitary gland, where OT is released into the bloodstream to exert its peripheral effects. Moreover, the dendrites of magnocellular neurons are densely packed with OT-containing neurosecretory granules that, upon exocytosis, regulate Address correspondence and reprint requests to Bice Chini, CNR, Institute of Neuroscience, Milan, Italy. E-mail: b.chini@in.cnr.itAbbreviations used: AVP, arginine-vasopressin; GnRH, gonadotropin releasing hormone; IR, inward rectifier; IRK, inwardly-rectifying potassium channel; GIRK, G protein-coupled inwardly-rectifying potassium channel; GPCR, G protein-coupled receptor; GTPcS, guanosine 5¢-O-[gamma-thio]triphosphate; OTR, oxytocin receptor; PLCb, phospholipase C b; PTX, pertussis toxin; SON, supraoptic nucleus; Thr-OT, Thr 4 Gly 7 -oxytocin; V 1a R, V 1b R, V 2 R, AVP receptors of the V 1a , V 1b and V 2 subtype. AbstractOxytocin receptor is a seven transmembrane receptor widely expressed in the CNS that triggers G i or G q protein-mediated signaling cascades leading to the regulation of a variety of neuroendocrine and cognitive functions. We decided to investigate whether and how the promiscuous receptor/G protein coupling affects neuronal excitability. As an experimental model, we used the immortalized gonadotropinreleasing hormone-positive GN11 cell line displaying the features of immature, migrating olfactory neurons. Using RT-PCR analysis, we detected the presence of oxytocin receptors whose stimulation by oxytocin led to the accumulation of inositol phosphates and to the inhibition of cell proliferation, and the expression of several inward rectifier (IR) K + channel subtypes. Moreover, electrophysiological and pharmacological inspections using whole-cell patchclamp recordings evidenced that in GN11 cells, IR channel subtypes are responsive to oxytocin. In particular, we found that: (i) peptide activation of receptor either inhibited or stimulated IR conductances, and (ii) IR current inhibition was mediated by a pertussis toxin-resistant G protein presumably of the G q/11 subtype, and by phospholipase C, whereas IR current ac...

We studied the combined anti-human immunodeficiency virus type 1 (HIV-1) effects of a derivative of stromaderived factor 1␤ (SDF-1␤), Met-SDF-1␤, and a modified form of RANTES, aminooxypentane (AOP)-RANTES. The antiviral agents were tested singly or in combination at 95 and 99% virus inhibitory concentrations. Clinical R5 and X4 HIV-1 isolates were used. AOP-RANTES inhibited R5 but not X4 viruses, whereas Met-SDF-1␤ had the opposite effect. Combinations of these compounds inhibited mixed infections with R5 and X4 viruses (95 to 99%), whereas single drugs were less inhibitory (32 to 61%). Combinations of R5 and X4 inhibitors are promising and deserve further evaluation.In 1995, Cocchi et al. reported potent in vitro human immunodeficiency virus type 1 (HIV-1) inhibition by three chemokines secreted by CD8 ϩ T lymphocytes (4) and focused attention on this class of molecules with low molecular masses (8 to 12 kDa). The chemokines described, RANTES, macrophage inflammatory protein-1␣ (MIP-1␣), and MIP-1␤, belong to the group of C-C chemokines that block HIV-1 entry into cells (5).Relationships among membrane coreceptors, chemokines, and cellular tropism were further defined in 1996 by Feng et al., who described a novel molecule which acted as a cofactor for T-cell-tropic HIV-1 isolates but not for macrophage-tropic isolates (14). This receptor, which was already known but did not have an identified natural ligand, belongs to the C-X-C chemokine receptor superfamily and was named "fusin," or CXCR4. The receptor for HIV-1 macrophage-tropic isolates was subsequently identified and named C-C chemokine receptor 5 (CCR5). CCR5 reacts with the chemokines RANTES, MIP-1␣, and MIP-1␤ (9, 12). The natural ligand for CXCR4 is stroma derived factor-1 (SDF-1), an ␣-chemokine with chemotactic properties for T lymphocytes and a developmental role in B lymphocyte maturation (2,25).During the early phases of HIV-1 infection, R5 viral strains usually predominate, whereas X4 strains frequently emerge in the late stages of HIV-1 infection, accompanied by a decline in peripheral blood CD4 lymphocytes and a clinical progression toward AIDS (7,34). Viral isolates with a dual tropism could represent a transition phase between viral R5 and X4 phenotypes, or they may represent X4 viruses that have maintained an ability to infect macrophages (31, 33).The aim of our study was to evaluate the interactions between attachment and entry inhibitors of HIV-1 infection. Our experiments suggest that the use of combined inhibitors of R5 and X4 viruses may be useful in inhibiting mixed infections. Analysis of cellular tropism of the different viral isolates on transformed cell lines. The two viral isolates examined in this study, RM and DK, were derived from two patients with primary HIV-1 infection acute syndrome (29), and the isolates were used to infect U87MG-transformed CD4 ϩ cells transfected with CCR5 or CXCR4 coreceptors (8), provided by Dan R. Littman (The Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New Yo...

A young woman received a diagnosis of abdominal, sporadic lymphangioleiomyomatosis (LAM) and multiple abdominal lymphangioleiomyomas and was referred for recurrent chylous ascites responding only to a fat-free diet. On admission, pulmonary function test (PFT) results showed a moderate reduction in the transfer factor for carbon monoxide with normal exercise performance. The serum vascular endothelial growth factor D (VEGF-D) level was 2,209 pg/mL. DNA sequences, amplified at loci kg8, D16S3395, D16S3024, D16S521, and D16S291 on chromosome 16p13.3, showed a loss of heterozygosity (LOH) only for kg8. Fat-free total parenteral nutrition in association with sirolimus (2 mg po daily) was initiated. Serum sirolimus levels were maintained at concentrations between 5 and 15 ng/mL. After 1 month, reintroduction of a low-fat oral feeding was achieved without recurrence of ascites. PFT results were stable. Interestingly, clinical improvement was associated with a reduction in the VEGF-D serum level (1,558 pg/mL). LOH at the kg8 biomarker in blood LAM cells was no longer detected.