Fluoxetine Induces Morphological Rearrangements of Serotonergic Fibers in the Hippocampus.

Serotonin (5-HT)-releasing fibers show substantial structural plasticity in response to genetically induced changes in 5-HT content. However, whether 5-HT fibers appear malleable also following clinically relevant variations in 5-HT levels that may occur throughout an individual's life has not been investigated. Here, using confocal imaging and 3D modeling analysis in Tph2GFP knock-in mice, we show that chronic administration of the antidepressant fluoxetine dramatically affects the morphology of 5-HT fibers innervating the dorsal and ventral hippocampus resulting in a reduced density of fibers. Importantly, GFP fluorescence levels appeared unaffected in the somata of both dorsal and median raphe 5-HT neurons, arguing against potential fluoxetine-mediated down-regulation of the Tph2 promoter driving GFP expression in the Tph2GFP mouse model. In keeping with this notion, mice bearing the pan-serotonergic driver Pet1-Cre partnered with a Cre-responsive tdTomato allele also showed similar morphological alterations in hippocampal 5-HT circuitry following chronic fluoxetine treatment. Moreover 5-HT fibers innervating the cortex showed proper density and no overt morphological disorganization, indicating that the reported fluoxetine-induced rearrangements were hippocampus specific. On the whole, these data suggest that 5-HT fibers are shaped in response to subtle changes of 5-HT homeostasis and may provide a structural basis by which antidepressants exert their therapeutic effect

IL-28 Supplants Requirement for Treg Cells in Protein σ1-Mediated Protection against Murine Experimental Autoimmune Encephalomyelitis (EAE)

Conventional methods to induce tolerance in humans have met with limited success. Hence, efforts to redirect tolerogen uptake using reovirus adhesin, protein sigma 1 (pσ1), may circumvent these shortcomings based upon the recent finding that when reovirus pσ1 is engineered to deliver chicken ovalbumin (OVA) mucosally, tolerance is obtained, even with a single dose. To test whether single-dose tolerance can be induced to treat EAE, proteolipid protein (PLP130–151) was genetically fused to OVA to pσ1 (PLP:OVA-pσ1) and shown to significantly ameliorate EAE, suppressing proinflammatory cytokines by IL-10+ forkhead box P3 (FoxP3)+ CD25+CD4+ Treg and IL-4+CD25−CD4+ Th2 cells. IL-10R or IL-4 neutralization reversed protection to EAE conferred by PLP:OVA-pσ1, and adoptive transfer of Ag-specific Treg or Th2 cells restored protection against EAE in recipients. Upon assessment of each relative participant, functional inactivation of CD25 impaired PLP:OVA-pσ1's protective capacity, triggering TGF-β-mediated inflammation; however, concomitant inactivation of TGF-β and CD25 reestablished PLP:OVA-pσ1-mediated protection by IL-28-producing FoxP3+CD25−CD4+ T cells. Thus, pσ1-based therapy can resolve EAE independently of or dependently upon CD25 and assigns IL-28 as an alternative therapy for autoimmunity

The maize (Zea mays) b-32 protein shows RIP activity in yeast cells

Ribosome-inactivating proteins (RIPs) are either single-chain (type 1) or two-chain (type 2) toxins. They are toxic to eukaryotic cells by cleaving a N-glycosidic bond in an extremely conserved loop located in the 28S RNA. This releases a specific adenine and inactivates the ribosome, ultimately inhibiting protein synthesis. Plant RIPs have been intensely investigated because of their projected antiviral, antifungal and insecticidal activity. RIPs also have biomedical applications as the toxic mojety of immunotoxins. Given their biotechnological potentials, it is strategic to develop platforms to rapidly evaluate the activity of recombinant RIPs. This investigation fills this need in that it reports that the yeast Saccharomyces cerevisiae is a model system to assess the impact of genetic manipulations on the functionality of a recombinant Zea mays RIP named b-32

Delivery of IL-35 by Lactococcus lactis Ameliorates Collagen-Induced Arthritis in Mice

IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3+ and Foxp3− CD39+ CD4+ T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6+ and CCR6− Foxp3+or− CD39+ CD4+ T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases

Comb-assisted cavity ring-down spectroscopy of a buffer-gas-cooled molecular beam

We demonstrate continuous-wave cavity ring-down spectroscopy of a partially hydrodynamic molecular beam emerging from a buffer-gas-cooling source. Specifically, the (ν1 + ν3) vibrational overtone band of acetylene (C2H2) around 1.5 μm is accessed using a narrow-linewidth diode laser stabilized against a GPS-disciplined rubidium clock via an optical frequency comb synthesizer. As an example, the absolute frequency of the R(1) component is measured with a fractional accuracy of ∼1 × 10(-9). Our approach represents the first step towards the extension of more sophisticated cavity-enhanced interrogation schemes, including saturated absorption cavity ring-down or two-photon excitation, to buffer-gas-cooled molecular beams

Serotonin drives striatal synaptic plasticity in a sex-related manner.

Abstract Introduction Plasticity at corticostriatal synapses is a key substrate for a variety of brain functions – including motor control, learning and reward processing – and is often disrupted in disease conditions. Despite intense research pointing toward a dynamic interplay between glutamate, dopamine (DA), and serotonin (5-HT) neurotransmission, their precise circuit and synaptic mechanisms regulating their role in striatal plasticity are still unclear. Here, we analyze the role of serotonergic raphe-striatal innervation in the regulation of DA-dependent corticostriatal plasticity. Methods Mice (males and females, 2–6 months of age) were housed in standard plexiglass cages at constant temperature (22 ± 1 °C) and maintained on a 12/12 h light/dark cycle with food and demineralized water ad libitum. In the present study, we used a knock-in mouse line in which the green fluorescent protein reporter gene (GFP) replaced the I Tph2 exon (Tph2GFP mice), allowing selective expression of GFP in the whole 5-HT system, highlighting both somata and neuritis of serotonergic neurons. Heterozygous, Tph2+/GFP, mice were intercrossed to obtain experimental cohorts, which included Wild-type (Tph2+/+), Heterozygous (Tph2+/GFP), and Mutant serotonin-depleted (Tph2GFP/GFP) animals. Results Using male and female mice, carrying on different Tph2 gene dosages, we show that Tph2 gene modulation results in sex-specific corticostriatal abnormalities, encompassing the abnormal amplitude of spontaneous glutamatergic transmission and the loss of Long Term Potentiation (LTP) in Tph2GFP/GFP mice of both sexes, while this form of plasticity is normally expressed in control mice (Tph2+/+). Once LTP is induced, only the Tph2+/GFP female mice present a loss of synaptic depotentiation. Conclusion We showed a relevant role of the interaction between dopaminergic and serotonergic systems in controlling striatal synaptic plasticity. Overall, our data unveil that 5-HT plays a primary role in regulating DA-dependent corticostriatal plasticity in a sex-related manner and propose altered 5-HT levels as a critical determinant of disease-associated plasticity defects

Mucosal Targeting of a BoNT/A Subunit Vaccine Adjuvanted with a Mast Cell Activator Enhances Induction of BoNT/A Neutralizing Antibodies in Rabbits

We previously reported that the immunogenicity of Hcβtre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hcβtre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice.New Zealand White or Dutch Belted rabbits were nasally immunized with Hcβtre or Hcβtre-Ad2F alone or combined with CT or C48/80, and serum samples were tested for the presence of Hcβtre-specific binding (ELISA) or BoNT/A neutralizing antibodies.Hcβtre-Ad2F nasally administered with CT induced serum anti-Hcβtre IgG ELISA and BoNT/A neutralizing antibody titers greater than those induced by Hcβtre + CT. C48/80 provided significant nasal adjuvant activity and induced BoNT/A-neutralizing antibodies similar to those induced by CT.Ad2F enhanced the nasal immunogenicity of Hcβtre, and the mast cell activator C48/80 was an effective adjuvant for nasal immunization in rabbits, an animal model with a nasal cavity anatomy similar to that in humans

Loss of Sialic Acid Binding Domain Redirects Protein σ1 to Enhance M Cell-Directed Vaccination

Ovalbumin (OVA) genetically fused to protein sigma 1 (pσ1) results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD) for immunization, modified OVA-pσ1, termed OVA-pσ1(short), was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4+ and CD8+ T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT) adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s) was more efficient for immunization than native OVA+CT. The immune antibodies (Abs) were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s) can be fused to vaccines to effectively elicit improved SIgA responses

LA PRESENZA DI COCAINA NELLE ACQUE SUPERFICIALI:STUDIO DELLE POSSIBILI RIPERCUSSIONI SULLA FAUNA ACQUATICA UTILIZZANDO IL BIOINDICATORE Anguilla anguilla

In Europa, nel corso dell’ultimo decennio, si è osservato un incremento globale del consumo di sostanze stupefacenti. Si stima che circa 14 milioni di europei abbiano fatto uso di sostanze stupefacenti almeno una volta nella vita, pari ad una media del 4,1 % di adulti della fascia dei 15-64 anni di età (OE DT, 2010). Le droghe e i loro metaboliti, eliminati per via urinaria, finiscono nella rete fognaria dalla quale vengono però solo parzialmente rimossi grazie agli impianti di trattamento (Heberer, 2002; Castiglioni et al., 2006 a,b). Perciò, sia le droghe che i loro metaboliti riescono a raggiungere le acque superficiali riceventi già contaminate da elevati livelli di inquinanti, quali pesticidi, metalli pesanti, alchilfenoli (Giordano et al., 2005; Mariager et al., 2005; Capaldo et al., 2006; Zuccato et al., 2008; Adamo et al., 2009; Maselli et al., 2010; Vörösmarty et al., 2010). Numerosi studi dimostrano, infatti, come le sostanze stupefacenti e i loro metaboliti rappresentino un nuovo gruppo di “inquinanti emergenti” del nostro pianeta (Binelli et al., 2012; Pal et al., 2012; Parolini et al., 2013; Pedriali et al., 2012). La loro determinazione (qualitativa e quantitativa) e monitoraggio in diversi substrati quali le acque, il terreno, le sedimentazioni e l’aria, rappresentano strumenti indiretti per valutare la diffusione dell’uso delle droghe nelle zone di rilevamento. Questi dati possono inoltre consentire di determinare il potenziale impatto tossicologico di tali sostanze, seppur presenti a basse concentrazioni ma ripetute nel tempo, nei vari habitat naturali. Indagini condotte sui fiumi Po, Olona, Lambro, Arno, Sarno, Tamigi, hanno rivelato la presenza di cocaina, morfina, amfetamine, tetraidrocannabinolo (principio attivo della cannabis) e codeina (Zuccato et al., 2008). In particolare, studi condotti da Zuccato et al. (2008) hanno evidenziato la presenza di concentrazioni variabili di cocaina, una delle droghe più diffuse, e del suo principale metabolita, la benzoilecgonina (BE), in diversi fiumi italiani e stranieri (44 e 15 ng/l rispettivamente, nei fiumi Olona e Lambro; circa 0.5 ng/l nel Po; circa 1,7 ng/l nel fiume Arno; da 0.13 a 6 ng/l nel Tamigi). Per quanto concerne la cocaina e il suo metabolita principale, BE, per il collettore in ingresso del depuratore di Nola, sono stati osservati livelli di cocaina compresi tra 233 e 252 ng/l; di BE compresi tra 669 e 770 ng/l. Per quanto riguarda il fiume Sarno, i livelli di cocaina sono pari a 13 ng/l; quelli di BE a 91 ng/l. (Istituto M. Negri, Milano 2008.) La presenza di sostanze stupefacenti nelle acque superficiali rappresenta un duplice problema, costituendo una potenziale minaccia sia per la fauna acquatica presente in tali ambienti, che per l’uomo, che si alimenta di specie ittiche esposte a tale contaminazione. Infatti, tenendo conto dei potenti effetti farmacologici delle droghe ritrovate e della loro presenza sotto forma di miscele complesse, anche con gli altri contaminanti, sono prevedibili effetti tossici ancora sconosciuti sia sugli organismi acquatici che sull’uomo, attraverso la catena alimentare. Non è possibile escludere, infatti, che queste droghe possano accumularsi nei pesci o alterarne il comportamento riproduttivo, come è stato dimostrato per gli antibatterici e i farmaci umani (Mason, 2003; Lovett et al., 2010). È quindi interessante intraprendere uno studio volto a verificare gli effetti delle droghe presenti nelle acque superficiali sulla fauna acquatica e, indirettamente, sulla salute umana. A tal fine è stata scelta la cocaina, una delle droghe più largamente utilizzate e presente in ambiente acquatico, e si è deciso di verificarne gli effetti sull’anguilla (Anguilla anguilla). La scelta di questo modello animale deriva da una serie di considerazioni. In primo luogo l’anguilla rappresenta un ottimo bioindicatore della contaminazione ambientale, vista la sua presenza sia nelle acque dolci che in quelle marine; in più è sedentaria, ricca di grasso e capace di accumulare molte sostanze lipofiliche, bio-concentrando in maniera significativa anche contaminanti presenti in quantità ridottissime (Oliveiro-Ribeiro et al., 2005; Belpaire, 2008; Bettinetti et al., 2010). Recenti studi condotti da Capaldo et al. (2012), proprio sull’anguilla europea, hanno infatti dimostrato che la cocaina tende ad accumularsi in numerosi tessuti di questo animale, con una maggiore affinità per il tessuto nervoso. Inoltre, l’anguilla è una specie di largo consumo nella popolazione. Infatti il suo allevamento fornisce all’incirca 45.000 tonnellate annue, più dell’80% del consumo mondiale di questa specie. Pertanto, il monitoraggio della contaminazione dell’anguilla è utile per la salvaguardia della salute umana, dal momento che è noto che la cocaina si accumula a livello dei tessuti periferici dell’organismo, e in particolare a livello del muscolo e del tessuto adiposo, che rappresentano la parte edibile dell’animale (Levisky et al., 2000; Bettinetti et al., 2010; Colucci et al., 2010). Infine l’anguilla è una specie a rischio di estinzione (Ciccotti, 2007), classificata come gravemente minacciata nella lista rossa dell’Unione Internazionale per la Conservazione della Natura (IUCN) ed oggetto, dal 2007, di un piano europeo di ricostituzione (nota IP/03/1332 del 2/10/2003 dell’UE). Il monitoraggio della contaminazione dell’anguilla è importante, perciò, anche ai fini della conservazione di questa specie, dal momento che proprio la contaminazione ambientale potrebbe aver contribuito al declino dello stock europeo dell’anguilla. E’ noto infatti che la cocaina aumenta la concentrazione di dopamina a livello della fessura sinaptica (Nestler e Malenka, 2004), e studi su Danio rerio (Lòpez-Patiño et al., 2008a,b) hanno evidenziato che la cocaina altera i livelli cerebrali di dopamina. Dal momento che questa ammina esplica un ruolo chiave nei processi riproduttivi dell’anguilla (Vidal et al., 2004; Sébert et al., 2008) è ipotizzabile che la cocaina, interferendo con essi, possa compromettere la sopravvivenza della specie