Immunobiotic Ligilactobacillus salivarius FFIG58 Confers Long-Term Protection against Streptococcus pneumoniae.

Previously, we isolated potentially probiotic Ligilactobacillus salivarius strains from the intestines of wakame-fed pigs. The strains were characterized based on their ability to modulate the innate immune responses triggered by the activation of Toll-like receptor (TLR)-3 or TLR4 signaling pathways in intestinal mucosa. In this work, we aimed to evaluate whether nasally administered L. salivarius strains are capable of modulating the innate immune response in the respiratory tract and conferring long-term protection against the respiratory pathogen Streptococcus pneumoniae. Infant mice (3-weeks-old) were nasally primed with L. salivarius strains and then stimulated with the TLR3 agonist poly(I:C). Five or thirty days after the last poly(I:C) administration mice were infected with pneumococci. Among the strains evaluated, L. salivarius FFIG58 had a remarkable ability to enhance the protection against the secondary pneumococcal infection by modulating the respiratory immune response. L. salivarius FFIG58 improved the ability of alveolar macrophages to produce interleukin (IL)-6, interferon (IFN)-γ, IFN-ß, tumor necrosis factor (TNF)-α, IL-27, chemokine C-C motif ligand 2 (CCL2), chemokine C-X-C motif ligand 2 (CXCL2), and CXCL10 in response to pneumococcal challenge. Furthermore, results showed that the nasal priming of infant mice with the FFIG58 strain protected the animals against secondary infection until 30 days after stimulation with poly(I:C), raising the possibility of using nasally administered immunobiotics to stimulate trained immunity in the respiratory tract.

Theiler's Murine Encephalomyelitis Virus Replicates in Primary Neuron Cultures and Impairs Spine Density Formation.

In this study, we examined infection with the highly neurovirulent GDVII, the less neurovirulent DA strains, and with a mutant DA, which lacks the L* protein (L*-1) involved in viral persistence and demyelinating disease, to analyze the direct effects of Theiler's murine encephalomyelitis virus (TMEV) replication using primary cultures of mouse brain hippocampal neurons. All viruses replicate in cultured neurons, with GDVII having the highest titers and L*-1 the lowest. Accordingly, all were positive for viral antigen staining 3 days postinfection (dpi), and DA and L*-1 were also positive after 12 dpi. NeuN + immunostaining showed an early and almost complete absence of positive cells in cultures infected with GDVII, an approximately 50% reduction in cultures infected with DA, and fewer changes in L*-1 strains at 3 dpi. Accordingly, staining with chloromethyltetramethylrosamine orange (Mitotracker OrangeTM) as a parameter for cell viability showed similar results. Moreover, at 1 dpi, the strain DA induced higher transcript levels of neuroprotective genes such as IFN-Iß, IRF7, and IRF8. At 3 dpi, strains GDVII and DA, but not the L*-1 mutant, showed lower PKR expression. In addition, confocal analysis showed that L*-1-infected neurons exhibited a decrease in spine density. Treatment with poly (I:C), which is structurally related to dsRNA and is known to trigger IFN type I synthesis, reduced spine density even more. These results confirmed the use of mouse hippocampal neuron cultures as a model to study neuronal responses after TMEV infection, particularly in the formation of spine density.

Immune Transcriptional Response in Head Kidney Primary Cell Cultures Isolated from the Three Most Important Species in Chilean Salmonids Aquaculture.

Fish cell culture is a common in vitro tool for studies in different fields such as virology, toxicology, pathology and immunology of fish. Fish cell cultures are a promising help to study how to diagnose and control relevant viral and intracellular bacterial infections in aquaculture. They can also be used for developing vaccines and immunostimulants, especially with the ethical demand aiming to reduce and replace the number of fish used in research. This study aimed to isolate head kidney primary cell cultures from three Chilean salmonids: Salmo salar, Oncorhynchus kisutch, and Oncorhynchus mykiss, and characterize the response to bacterial and viral stimuli by evaluating various markers of the innate and adaptive immune response. Specifically, the primary cell cultures of the head kidney from the three salmonids studied were cultured and exposed to two substances that mimic molecular patterns of different pathogens, i.e., Lipopolysaccharide (LPS) (bacterial) and Polyinosinic: polycytidylic acid (POLY I:C). Subsequently, we determined the mRNA expression profiles of the TLR-1, TLR-8, IgM, TLR-5, and MHC II genes. Head kidney primary cell cultures from the three species grown in vitro responded differently to POLY I:C and LPS. This is the first study to demonstrate and characterize the expression of immune genes in head kidney primary cell culture isolated from three salmonid species. It also indicates their potential role in developing immune responses as defense response agents and targets of immunoregulatory factors.

Transcription factor EB (TFEB) participates in antiviral immune responses independent of mTORC1 in macrophage of large yellow croaker (Larimichthys crocea).

Transcription factor EB (TFEB) plays an integral role in the production of proinflammatory cytokines and chemokines in response to pathogen stimulation in mammals. However, the role of TFEB in antiviral immune responses and the potential regulatory mechanisms in fish remain poorly understood. Here, we cloned and characterized Larimichthys crocea TFEB (LcTFEB) with 524 amino acids and a typical basic helix-loop-helix-leucine zipper domain. LcTFEB could translocate into the nucleus upon starvation and had a comparatively high expression in immune tissues. Similar to the expression of antiviral immune genes, the transcriptional expression and activity of LcTFEB showed a trend of increasing and then decreasing with the prolongation of stimulation. Inhibition of LcTFEB using siRNA dramatically increased the polyinosinic-polycytidylic acid (poly (I:C))-induced interferon response and pro-inflammatory cytokines mRNA expression levels, whereas pharmacological activation and overexpression of LcTFEB exhibited the reverse effects. Mechanically, LcTFEB might promote the expression of IFNh as negative feedback to limit the virus-induced inflammatory responses. Notably, although inhibition of mTORC1 exacerbated poly (I:C)-triggered inflammatory responses, the effects of LcTFEB were independent of mTORC1. Overall, this study revealed an unidentified critical role of LcTFEB in the regulation of antiviral immune responses and promoted the understanding of TFEB in the antiviral immunity of fish macrophages.

Immune System Modulation by the Adjuvants Poly (I:C) and Montanide ISA 720.

Adjuvants are essential for vaccine development, especially subunit-based vaccines such as those containing recombinant proteins. Increasing the knowledge of the immune response mechanisms generated by adjuvants should facilitate the formulation of vaccines in the future. The present work describes the immune phenotypes induced by Poly (I:C) and Montanide ISA 720 in the context of mice immunization with a recombinant protein based on the Plasmodium vivax circumsporozoite protein (PvCSP) sequence. Mice immunized with the recombinant protein plus Montanide ISA 720 showed an overall more robust humoral response, inducing antibodies with greater avidity to the antigen. A general trend for mixed Th1/Th2 inflammatory cytokine profile was increased in Montanide-adjuvanted mice, while a balanced profile was observed in Poly (I:C)-adjuvanted mice. Montanide ISA 720 induced a gene signature in B lymphocytes characteristic of heme biosynthesis, suggesting increased differentiation to Plasma Cells. On the other hand, Poly (I:C) provoked more perturbations in T cell transcriptome. These results extend the understanding of the modulation of specific immune responses induced by different classes of adjuvants, and could support the optimization of subunit-based vaccines.

Immunogenicity of SARS-CoV-2 Trimeric Spike Protein Associated to Poly(I:C) Plus Alum.

The SARS-CoV-2 pandemic has had a social and economic impact worldwide, and vaccination is an efficient strategy for diminishing those damages. New adjuvant formulations are required for the high vaccine demands, especially adjuvant formulations that induce a Th1 phenotype. Herein we assess a vaccination strategy using a combination of Alum and polyinosinic:polycytidylic acid [Poly(I:C)] adjuvants plus the SARS-CoV-2 spike protein in a prefusion trimeric conformation by an intradermal (ID) route. We found high levels of IgG anti-spike antibodies in the serum by enzyme linked immunosorbent assay (ELISA) and high neutralizing titers against SARS-CoV-2 in vitro by neutralization assay, after two or three immunizations. By evaluating the production of IgG subtypes, as expected, we found that formulations containing Poly(I:C) induced IgG2a whereas Alum did not. The combination of these two adjuvants induced high levels of both IgG1 and IgG2a. In addition, cellular immune responses of CD4+ and CD8+ T cells producing interferon-gamma were equivalent, demonstrating that the Alum + Poly(I:C) combination supported a Th1 profile. Based on the high neutralizing titers, we evaluated B cells in the germinal centers, which are specific for receptor-binding domain (RBD) and spike, and observed that more positive B cells were induced upon the Alum + Poly(I:C) combination. Moreover, these B cells produced antibodies against both RBD and non-RBD sites. We also studied the impact of this vaccination preparation [spike protein with Alum + Poly(I:C)] in the lungs of mice challenged with inactivated SARS-CoV-2 virus. We found a production of IgG, but not IgA, and a reduction in neutrophil recruitment in the bronchoalveolar lavage fluid (BALF) of mice, suggesting that our immunization scheme reduced lung inflammation. Altogether, our data suggest that Alum and Poly(I:C) together is a possible adjuvant combination for vaccines against SARS-CoV-2 by the intradermal route.

Nasal administration of a non-viable Lactobacillus casei to infant mice modulates lung damage induced by Poly I:C and hyperreactivity in airways.

Viral respiratory infections caused by RNA viruses are one of the most important diseases around the world. The aim of this work was to study whether the nasal administration of non-viable Lactobacillus casei (LcM) was able to enhance respiratory antiviral defenses in young mice challenged with Poly I:C. Three-week-old BALB/c mice were nasally challenged with Poly I:C, used to mimic the pro-inflammatory state of lung infections caused by RNA viruses. LcM was nasally administered 2 days before Poly I:C challenge. Lactate dehydrogenase (LDH) activity, albumin concentration in broncho-alveolar lavages (BAL), wet-to-dry lung weight ratio, and total and differential leukocytes counts in blood were evaluated. Also, α, λ, γ interferons, IL-10, TNF-α, IL-4 in BAL and nasal lavages and total IgE in BAL and serum, were evaluated by ELISA. Poly I:C induced pulmonary injuries while alteration of bronchoalveolar-capillary barrier was reduced by nasal administration of LcM. Moreover, alterations in leukocyte counts induced by Poly I:C were regulated. LcM favorably modulated the cytokines responses triggered by Poly I:C challenge in nasal and lung mucosal compartments. Also, LcM decreased IgE levels in BAL and plasma compared with the Poly I:C group. LcM nasally administered reduced the lung damage induced by Poly I:C and prevented airway hyperreactivity.

Human prostate epithelial cells and prostate-derived stem cells malignantly transformed in vitro with sodium arsenite show impaired Toll like receptor -3 (TLR3)-associated anti-tumor pathway.

A therapeutic strategy for prostate cancer (PCa) involves the use of 9-cis-retinoic acid (9cRA) to induce cancer stem cells (CSCs) differentiation and apoptosis. Polyinosinic:polycytidylic acid (PIC) is a Toll-like receptor 3 (TLR3) agonist that induces tumor cells apoptosis after activation. PIC+9cRA combination activates retinoic acid receptor ß (RARß) re-expression, leading to CSC differentiation and growth arrest. Since inorganic arsenic (iAs) targets prostatic stem cells (SCs), we hypothesized that arsenic-transformed SCs (As-CSCs) show an impaired TLR3-associated anti-tumor pathway and, therefore, are unresponsive to PIC activation. We evaluated TLR3-mediated activation of anti-tumor pathway based in RARß expression, on As-CSC and iAs-transformed epithelial cells (CAsE-PE). As-CSCs and CAsE-PE showed lower TLR3 and RARß basal expression compared to their respective isogenic controls WPE-Stem and RWPE-1. Also, iAs transformants showed reduced expression of mediators in TLR3 pathway. Importantly, As-CSCs were irresponsive to PIC+9cRA in terms of increased RARß and decreased SC-markers expression, while CAsE-PE, a heterogeneous cell line having a small SC population, were partially responsive. These observations indicate that iAs can impair TLR3 expression and anti-tumor pathway activated by PIC+9cRA in SCs and prostatic epithelial cells. These findings suggest that TLR3-activation based therapy may be an ineffective therapeutic alternative for iAs-associated PCa.

Low-dose candesartan prevents schizophrenia-like behavioral alterations in a neurodevelopmental two-hit model of schizophrenia.

Schizophrenia is a severe mental disorder with complex etiopathogenesis. Based on its neurodevelopmental features, an animal model induced by "two-hit" based on perinatal immune activation followed by peripubertal unpredictable stress was proposed. Sex influences the immune response, and concerning schizophrenia, it impacts the age of onset and symptoms severity. The neurobiological mechanisms underlying the influence of sex in schizophrenia is poorly understood. Our study aimed to evaluate sex influence on proinflammatory and oxidant alterations in male and female mice exposed to the two-hit model of schizophrenia, and its prevention by candesartan, an angiotensin II type 1 receptor (AT1R) blocker with neuroprotective properties. The two-hit model induced schizophrenia-like behavioral changes in animals of both sexes. Hippocampal microglial activation alongside the increased expression of NF-κB, and proinflammatory cytokines, namely interleukin (IL)-1ß and TNF-α, were observed in male animals. Conversely, females presented increased hippocampal and plasma levels of nitrite and plasma lipid peroxidation. Peripubertal administration of low-dose candesartan (0.3 mg/kg PO) prevented behavioral, hippocampal, and systemic changes in male and female mice. While these results indicate the influence of sex on inflammatory and oxidative changes induced by the two-hit model, candesartan was effective in both males and females. The present study advances the neurobiological mechanisms underlying sex influence in schizophrenia and opens new avenues to prevent this devasting mental disorder.

Quantification of neurotransmitters in microdialysate samples following quetiapine dosing to schizophrenia phenotyped rats using a validated LC-MS/MS method.

A versatile method was developed and validated for simultaneous determination of the monoamine neurotransmitters (MNT) dopamine (DA), 3-4-dyhydroxyphenilacetic acid (DOPAC), homovanilic acid (HVA), serotonin (5-HT) and 5-hydroxyindolacetic acid (5-HIAA) in rat brain microdialysate samples using high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS). The method allowed for small sample volume, using positive and negative ionization mode in a single run analysis without any derivatization or cleanup steps. Analytes were quantified at concentrations ranging from 100 ng/mL to 0.05, 10, 0.5, 0.1 or 1 ng/mL (lower limit of quantification, LLOQ) of DA, DOPAC, HVA, 5-HT and 5-HIAA, respectively, showing linearity (r > 0.98), accuracy, and precision (R.S.D ± 15%) according to validation limits accepted by international guidelines. The method was successfully applied for monitoring the concentration changes of MNT in microdialysate samples from medium prefrontal cortex of Wistar rats in a neurodevelopmental model of schizophrenia before and after quetiapine 5 mg/kg i.v. bolus dose administration. No alterations in MNTs were observed in schizophrenia phenotyped rats (SPR) in comparison to the baseline shading a light on the limited response rate to antipsychotic drugs observed in chronic schizophrenic patients.

Sex influences in the preventive effects of N-acetylcysteine in a two-hit animal model of schizophrenia.

BACKGROUND: Schizophrenia (SCZ) is a neurodevelopmental disorder influenced by patient sex. Mechanisms underlying sex differences in SCZ remain unknown. A two-hit model of SCZ combines the exposure to perinatal infection (first-hit) with peripubertal unpredictable stress (PUS, second-hit). N-acetylcysteine (NAC) has been tested in SCZ because of the involvement of glutathione mechanisms in its neurobiology. AIMS: We aim to investigate whether NAC administration to peripubertal rats of both sexes could prevent behavioral and neurochemical changes induced by the two-hit model. METHODS: Wistar rats were exposed to polyinosinic:polycytidylic acid (a viral mimetic) or saline on postnatal days (PND) 5-7. On PND30-59 they received saline or NAC 220 mg/kg and between PND40-48 were subjected to PUS or left undisturbed. On PND60 behavioral and oxidative alterations were evaluated in the prefrontal cortex (PFC) and striatum. Mechanisms of hippocampal memory regulation such as immune expression of G protein-coupled estrogen receptor 1 (GPER), α7-nAChR and parvalbumin were also evaluated. RESULTS: NAC prevented sensorimotor gating deficits only in females, while it prevented alterations in social interaction, working memory and locomotor activity in both sexes. Again, in rats of both sexes, NAC prevented the following neurochemical alterations: glutathione (GSH) and nitrite levels in the PFC and lipid peroxidation in the PFC and striatum. Striatal oxidative alterations in GSH and nitrite were observed in females and prevented by NAC. Two-hit induced hippocampal alterations in females, namely expression of GPER-1, α7-nAChR and parvalbumin, were prevented by NAC. CONCLUSION: Our results highlights the influences of sex in NAC preventive effects in rats exposed to a two-hit schizophrenia model.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Bacterial and viral pathogen-associated molecular patterns induce divergent early transcriptomic landscapes in a bovine macrophage cell line.

BACKGROUND: Pathogens stimulate immune functions of macrophages. Macrophages are a key sentinel cell regulating the response to pathogenic ligands and orchestrating the direction of the immune response. Our study aimed at investigating the early transcriptomic changes of bovine macrophages (Bomacs) in response to stimulation with CpG DNA or polyI:C, representing bacterial and viral ligands respectively, and performed transcriptomics by RNA sequencing (RNASeq). KEGG, GO and IPA analytical tools were used to reconstruct pathways, networks and to map out molecular and cellular functions of differentially expressed genes (DE) in stimulated cells. RESULTS: A one-way ANOVA analysis of RNASeq data revealed significant differences between the CpG DNA and polyI:C-stimulated Bomac. Of the 13,740 genes mapped to the bovine genome, 2245 had p-value ≤0.05, deemed as DE. At 6 h post stimulation of Bomac, poly(I:C) induced a very different transcriptomic profile from that induced by CpG DNA. Whereas, 347 genes were upregulated and 210 downregulated in response to CpG DNA, poly(I:C) upregulated 761 genes and downregulated 414 genes. The topmost DE genes in poly(I:C)-stimulated cells had thousand-fold changes with highly significant p-values, whereas in CpG DNA stimulated cells had 2-5-fold changes with less stringent p-values. The highest DE genes in both stimulations belonged to the TNF superfamily, TNFSF18 (CpG) and TNFSF10 (poly(I:C)) and in both cases the lowest downregulated gene was CYP1A1. CpG DNA highly induced canonical pathways that are unrelated to immune response in Bomac. CpG DNA influenced expression of genes involved in molecular and cellular functions in free radical scavenging. By contrast, poly(I:C) highly induced exclusively canonical pathways directly related to antiviral immune functions mediated by interferon signalling genes. The transcriptomic profile after poly(I:C)-stimulation was consistent with induction of TLR3 signalling. CONCLUSION: CpG DNA and poly(I:C) induce different early transcriptional landscapes in Bomac, but each is suited to a specific function of macrophages during interaction with pathogens. Poly(I:C) influenced antiviral response genes, whereas CpG DNA influenced genes important for phagocytic processes. Poly(I:C) was more potent in setting the inflammatory landscape desirable for an efficient immune response against virus infection.

N-3 polyunsaturated fatty acids and clozapine abrogates poly I: C-induced immune alterations in primary hippocampal neurons.

The viral mimetic polyinosinic:polycytidylic acid (poly I:C) is an important tool to study the consequences of viral infection to the development of neuropsychiatric disorders. Here, based on the premise of omega-3 polyunsaturated fatty acids (n3 PUFAs) as supplemental treatment to antipsychotics in schizophrenia, we investigated the involvement of NFkB pathway in the effects of n3 PUFAs or of the atypical antipsychotic clozapine in hippocampal poly I:C-challenged neurons. Primary hippocampal neuronal cultures were exposed to n3 PUFAs (DHA4.35 µM/EPA7.10 µM, DHA 8.7 µM/EPA14.21 µM or DHA17.4 µM/EPA28.42 µM) or clozapine (1.5 or 3 µM) in the presence or absence of poly I:C. MTT assay revealed that poly I:C-induced reduction in cell viability was prevented by n3 PUFAs or clozapine. N3 PUFAs (DHA 8.7 µM/EPA14.21 µM) or clozapine (3 µM) significantly reduced poly I:C-induced increase in iNOS, NFkB (p50/p65), IL-6 and nitrite when compared to non-treated cells. Only n3 PUFAs prevented poly I:C-induced deficits in BDNF. On the other hand, poly I:C caused a marked reduction in DCX immunoexpression, which was prevented only by clozapine. Thus, n3 PUFAs and clozapine exert in vitro neuroprotective effects against poly I:C immune challenge in hippocampal neurons, by mechanisms possibly involving the inhibition of canonical NFkB pathway. The present study adds further evidences to the mechanisms underlying n3 PUFAs and clozapine neuroprotective effects against viral immune challenges. Since n3 PUFAs is a safe strategy for use during pregnancy, our results also add further evidence for the use of this supplement in order to prevent alterations induced by viral hits during this developmental period.

Identification of Secreted Proteins Involved in Nonspecific dsRNA-Mediated Lutzomyia longipalpis LL5 Cell Antiviral Response.

Hematophagous insects transmit infectious diseases. Sand flies are vectors of leishmaniasis, but can also transmit viruses. We have been studying immune responses of Lutzomyia longipalpis, the main vector of visceral leishmaniasis in the Americas. We identified a non-specific antiviral response in L. longipalpis LL5 embryonic cells when treated with non-specific double-stranded RNAs (dsRNAs). This response is reminiscent of interferon response in mammals. We are investigating putative effectors for this antiviral response. Secreted molecules have been implicated in immune responses, including interferon-related responses. We conducted a mass spectrometry analysis of conditioned medium from LL5 cells 24 and 48 h after dsRNA or mock treatment. We identified 304 proteins. At 24 h, 19 proteins had an abundance equal or greater than 2-fold change, while the levels of 17 proteins were reduced when compared to control cells. At the 48 h time point, these numbers were 33 and 71, respectively. The two most abundant secreted peptides at 24 h in the dsRNA-transfected group were phospholipid scramblase, an interferon-inducible protein that mediates antiviral activity, and forskolin-binding protein (FKBP), a member of the immunophilin family, which mediates the effect of immunosuppressive drugs. The transcription profile of most candidates did not follow the pattern of secreted protein abundance.

Novel IFNγ homologue identified in Nile tilapia (Oreochromis niloticus) links with immune response in gills under different stimuli.

Interferon gamma (IFN-γ) has important roles in both innate and adaptive immune responses. This cytokine plays a very important role in defining Th1 immune response in all vertebrates. In the present study, we identified and isolated for the first time the gene coding for Nile tilapia (Oreochromis niloticus) IFNγ from spleen lymphocytes. The isolated tilapia IFNγ has between 24 and 62% of amino acid identity as compared to reported sequences for other teleost fishes. It has close phylogenetic relationships with IFNγ molecules belonging to the group of Perciforms and presents the typical structural characteristics of gamma interferon molecules. The tissue expression analysis showed that IFNγ is expressed constitutively in head kidney, skin, intestine, muscle and brain. Its expression was not detected in gills by conventional RT-PCR. However, under conditions of stimulation with Poly I:C and LPS, IFNγ expression was up-regulated in gills after 24 h post-stimulation. IFNγ expression was also induced in gills 24 h after Edwardsiella tarda infection suggesting its important role in immunity against intracellular bacteria. The recombinant protein produced in Escherichia coli induced Mx gene transcription in head kidney primary culture cells. These results are the first steps to characterize the role of tilapia IFNγ in the defense against pathogens in tilapia. Furthermore, the isolation of this molecule provides a new tool to characterize the cellular immune response to various stimuli in this organism.

Effects of early or late prenatal immune activation in mice on behavioral and neuroanatomical abnormalities relevant to schizophrenia in the adulthood.

Maternal immune activation (MIA) during pregnancy in rodents increases the risk of the offspring to develop schizophrenia-related behaviors, suggesting a relationship between the immune system and the brain development. Here we tested the hypothesis that MIA induced by the viral mimetic polyinosinic-polycytidylic acid (poly I:C) in early or late gestation of mice leads to behavioral and neuroanatomical disorders in the adulthood. On gestational days (GDs) 9 or 17 pregnant dams were treated with poly I:C or saline via intravenous route and the offspring behaviors were measured during adulthood. Considering the progressive structural neuroanatomical alterations in the brain of individuals with schizophrenia, we used magnetic resonance imaging (MRI) to perform brain morphometric analysis of the offspring aged one year. MIA on GD9 or GD17 led to increased basal locomotor activity, enhanced motor responses to ketamine, a psychotomimetic drug, and reduced time spent in the center of the arena, suggesting an increased anxiety-like behavior. In addition, MIA on GD17 reduced glucose preference in the offspring. None of the treatments altered the relative volume of the lateral ventricles. However, a decrease in brain volume, especially for posterior structures, was observed for one-year-old animals treated with poly I:C compared with control groups. Thus, activation of the maternal immune system at different GDs lead to neuroanatomical and behavioral alterations possibly related to the positive and negative symptoms of schizophrenia. These results provide insights on neuroimmunonological and neurodevelopmental aspects of certain psychopathologies, such as schizophrenia.

Immunobiotic Lactobacillus strains reduce small intestinal injury induced by intraepithelial lymphocytes after Toll-like receptor 3 activation.

OBJECTIVE: Intestinal intraepithelial lymphocytes (IELs) play critical roles in disrupting epithelial homeostasis after Toll-like receptor (TLR)-3 activation with genomic rotavirus dsRNA or the synthetic dsRNA analog poly(I:C). The capacity of immunobiotic Lactobacillus rhamnosus CRL1505 (Lr1505) or Lactobacillus plantarum CRL1506 (Lp1506) to beneficially modulate IELs response after TLR3 activation was investigated in vivo using a mice model. RESULTS: Intraperitoneal administration of poly(I:C) induced inflammatory-mediated intestinal tissue damage through the increase of inflammatory cells (CD3(+)NK1.1(+), CD3(+)CD8αα(+), CD8αα(+)NKG2D(+)) and pro-inflammatory mediators (TNF-α, IL-1ß, IFN-γ, IL-15, RAE1, IL-8). Increased expression of intestinal TLR3, MDA5, and RIG-I was also observed after poly(I:C) challenge. Treatment with Lr1505 or Lp1506 prior to TLR3 activation significantly reduced the levels of TNF-α, IL-15, RAE1, and increased serum and intestinal IL-10. Moreover, CD3(+)NK1.1(+), CD3(+)CD8αα(+), and CD8αα(+)NKG2D(+) cells were lower in lactobacilli-treated mice when compared to controls. The immunomodulatory capacities of lactobacilli allowed a significant reduction of intestinal tissue damage. CONCLUSIONS: This work demonstrates the reduction of TLR3-mediated intestinal tissue injury by immunobiotic lactobacilli through the modulation of intraepithelial lymphocytes response. It is a step forward in the understanding of the cellular mechanisms involved in the antiviral capabilities of immunobiotic strains.

Short-term effects of Poly(I:C) on gut permeability.

The intestinal barrier function depends on an adequate response to pathogens by the epithelium. Toll-like receptor 3 (TLR-3) recognizes double-stranded RNA, a virus-associated molecular pattern. Activation of TLR-3 with Poly(I:C), a synthetic agonist, modulates tissue repair and permeability in other epithelia; however, the effects of local luminal TLR-3 agonists on gut barrier function are unknown. The aim of this investigation was to evaluate short-term effects of Poly(I:C) on rat ileal and colonic permeability ex vivo. We also studied the acute effects of intrarectal administration of Poly(I:C) on colonic barrier function. Ileum tissues displayed decreased transepithelial electrical resistance (TEER) 1h after incubation with 200µg/mL Poly(I:C); however, the mucosa-to-serosa transit of macromolecules (4.4 and 40kDa dextrans - TD4.4 and FD40, respectively) remained unchanged. Conversely, colon tissue preparations stimulated with 200µg/mL Poly(I:C) showed a decreased thinning of the mucosal layer after 2h and a decreased transit of FD40 after 3h, in comparison to controls. There was no change in colonic TEER after 3h of treatment. In addition, colon tissue taken from rats 6h after an intrarectal administration of 100µg Poly(I:C) also showed decreased permeability to FD40 in the everted gut sac assay at 3h post-extraction. Tissue morphology remained unchanged. Our results suggest that an acute exposure to Poly(I:C) reduces colon permeability to macromolecules but increases ileum permeability to electrolytes/small molecules ex vivo. Although the mechanism associated to these effects needs further investigation, to our knowledge this is the first report of a direct effect of a TLR-3 ligand in intestinal barrier function and may be of significance to understand region-specific interactions between gut mucosa and microbiota.

Effects of prenatal immune activation on amphetamine-induced addictive behaviors: Contributions from animal models.

BACKGROUND: Prenatal environmental adversities may affect brain development and are associated with increased risk for schizophrenia, an illness with 50% comorbidity with addiction. Maternal immune activation by poly-inosinic-citidilic acid (Poly(I:C)) exposure can promote behavioral alterations consistent with schizophrenia symptoms in rodents. OBJECTIVES: Considering the vulnerability to addiction in patients with schizophrenia, we evaluated the interactions between prenatal Poly(I:C) administration and addiction in two animal models (behavioral sensitization and conditioned place preference - CPP) in mice repeatedly treated with amphetamine (AMP). Additionally, stereotyped behavior and cross-sensitization with cocaine (COC) were also investigated. METHODS: Swiss male mice offspring were submitted to prenatal administration of 5mg/kg Poly(I:C) in the 9(th) day of pregnancy. At the age of 90days, mice were treated with 2.5mg/kg AMP for 9days to evaluate behavioral sensitization or stereotyped behavior. Cross-sensitization with 10mg/kg COC was evaluated 24h after the last treatment day. For AMP-induced CPP evaluation, mice were treated during 8 consecutive days. RESULTS: Prenatal Poly(I:C) administration potentiated both AMP-induced behavioral sensitization and CPP. Furthermore, Poly(I:C) increased cross-sensitization with COC. CONCLUSIONS: Prenatal administration of Poly(I:C) is able to potentiate vulnerability to addiction in two animal models, without however modulating stereotyped behavior.