Insomnia and PTSD one month after wildfires: evidence for an independent role of the "fear of imminent death".

OBJECTIVE: The relationship of insomnia with Post-Traumatic Stress Disorder (PTSD) one month after wildfires and more specifically with the experience of 'fear of imminent death' were investigated. METHODS: Ninety-two randomly chosen victims of wildfires in the Greek province of Ilia, were assessed through a specifically designed semi-structured psychiatric interview comprising of questionnaires and scales to measure psychopathology, as well as psychosocial and environmental parameters. PTSD was set according to ICD-10 research diagnostic criteria, while insomnia was assessed with the Athens Insomnia Scale (AIS). RESULTS: The presence of insomnia was identified in 63.0% of the victims. 46.7% of the participants were diagnosed with PTSD in the first post-disaster month, while 51.1% of the total sample experienced 'fear of imminent death'. The majority of sleep complaints were significantly more frequent in subjects with PTSD. Female gender, PTSD, older age, and 'fear of imminent death' were independently associated with insomnia. CONCLUSIONS: The findings of the present study indicate that the diagnosis of insomnia, as well as, certain specific insomnia complaints were more frequent in female victims of wildfires who have experienced 'fear of imminent death' and have developed PTSD.

Glutamatergic Agents as Add-On Medication for the Treatment of Obsessive-Compulsive Disorder: A Systematic Review and Meta-Analysis.

OBJECTIVE: The aim of the present study was to review the existing literature on clinical trials with glutamatergic agents in adults with obsessive-compulsive disorder (OCD) and to perform a meta-analysis to estimate the overall effect size. DATA SOURCES: We searched in MEDLINE, Embase, and the Cochrane Library for eligible studies, using the following search terms: (glutamate OR glutaminergic OR glutamatergic OR NMDA OR AMPA OR kainate) AND (obsessive-compulsive disorder OR obsessive OR compulsive OR OCD). A separate search was performed for generally known glutamatergic agents. The databases were searched for articles published by May 31, 2015. STUDY SELECTION: Eligible studies were double-blind, randomized controlled trials that tested the efficacy of add-on treatment with a glutamatergic agent in patients with OCD. DATA EXTRACTION: Data were extracted independently by 2 reviewers. We extracted dichotomous data (number of patients with response and remission) to estimate relative risk ratios (RRs), as well as continuous data (scores in Yale-Brown Obsessive Compulsive Scale and Clinical Global Impressions-Severity of Illness and -Improvement scales), which were used to estimate standardized mean differences. Effect sizes were estimated using a random-effects model. RESULTS: Eight randomized controlled trials were identified. The overall ratio for response was RR = 3.71 (95% CI, 2.35-5.83; P < .001). When limited to the studies with treatment-resistant patients, the effect size remained significant (RR = 4.30; 95% CI, 2.19-8.43; P < .001). Secondary outcomes, such as the standardized mean differences for continuous data, showed the statistically significant superiority (P < .001) of glutamatergic agents over placebo. The risk of dropouts was RR = 1.18 (95% CI, 0.83-1.69; P = .361) and the risk of dropouts due to adverse effects was RR = 3.04 (95% CI, 1.57-5.89; P = .001). CONCLUSIONS: Glutamatergic agents are effective as add-on treatment for OCD in general and especially for treatment-refractory OCD.

Genome wide transcriptomic analysis identifies pathways affected by the infusion of Clostridium perfringens culture supernatant in the duodenum of broilers in situ.

Clostridium perfringens type A is the main etiological factor for necrotic enteritis, a multifactorial enteric disease that penalizes performance, health, and welfare of poultry. Lack of knowledge of host responses and disease pathogenesis is slowing down progress on developing therapies for disease control. A combined genomewide and targeted gene approach was used to investigate pathways and biological functions affected by the infusion of C. perfringens culture supernatant in the duodenum of broilers in 2 experiments. An in situ isolated loop of duodenum was prepared in anesthetized broilers of 3 wk of age (Exp. 1) and was infused either with crude C. perfringens culture supernatant (n = 7; treated), positive for necrotic enteritis B-like toxin (NetB) as determined by a cytotoxicity assay, or with a control preparation (n = 6; control). Birds were maintained alive for 1 h and then euthanized for tissue recovery. The use of the Affymetrix chicken genome array on RNA samples from loop tissue showed top biological functions affected by culture supernatant infusion included cell morphology, immune cell trafficking, and cell death; pathways affected included death receptor signaling, inflammatory response, and nuclear factor (NF)-κB signaling. In a second in situ study (Exp. 2), broilers were maintained alive for 4 h to monitor temporal expression patterns of targeted genes. Duodenal tissue was removed at 0.5, 1, 2, and 4 h post-infusion with culture supernatant (n = 9) or a control preparation (n = 5) for histology and gene expression analysis. Genes encoding proinflammatory cytokines, such as interferon γ (IFNγ), cell trafficking, such as neuroblastoma 1 (NBL1) and B cell CLL/Lymphoma 6 (BCL6), and cell death, such as Fas cell surface death receptor (FAS) and GTPase IMAP family member 8 (GIMAP8), were differentially expressed in the duodenum of treated and control broilers (P < 0.05). We have demonstrated that C. perfringens culture supernatant (NetB positive) infusion resulted in histological and gene expression changes consistent with necrotic enteritis in the duodenum of broilers. In the absence of live bacteria, crude culture supernatant resulted in early immunomodulation, inflammation, and cell death in the duodenum. The pathways identified here can be targeted for the development of new drugs, vaccines, and novel therapies for necrotic enteritis in broilers.

Functional characterisation of a TLR accessory protein, UNC93B1, in Atlantic salmon (Salmo salar).

Toll-like receptors (TLRs) are indispensable components of the innate immune system, which recognise conserved pathogen associated molecular patterns (PAMPs) and induce a series of defensive immune responses to protect the host. Biosynthesis, localisation and activation of TLRs are dependent on TLR accessory proteins. In this study, we identified the accessory protein, UNC93B1, from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs aided by the conserved gene synteny of genes flanking UNC93B1 in fish, birds and mammals. Phylogenetic analysis showed that salmon UNC93B1 grouped with other vertebrate UNC93B1 molecules, and had highest amino acid identity and similarity to zebrafish UNC93B1. The salmon UNC93B1 gene organisation was also similar in structure to mammalian UNC93B1. Our gene expression studies revealed that salmon UNC93B1 was more highly expressed in spleen, liver and gill tissues but was expressed at a lower level in head kidney tissue in post-smolts relative to parr. Moreover, salmon UNC93B1 mRNA transcripts were up-regulated in vivo in spleen tissue from polyI:C treated salmon and in vitro in polyI:C or IFNγ stimulated Salmon Head Kidney-1 (SHK-1) cells. Initial studies into the functional role of salmon UNC93B1 in fish TLR signalling found that both wild type salmon UNC93B1 and a molecule with a site-directed mutation (H424R) co-immunoprecipitated with salmon TLR19, TLR20a and TLR20d. Overall, these data illustrate the potential importance of UNC93B1 as an accessory protein in fish TLR signalling.

Identification and characterisation of TLR18-21 genes in Atlantic salmon (Salmo salar).

Teleost fish possess many types of toll-like receptor (TLR) some of which exist in other vertebrate groups and some that do not (ie so-called "fish-specific" TLRs). In this study, we identified in Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs seven TLRs that are not found in mammals, including six types of fish-specific TLRs (one TLR18, one TLR19, and four TLR20 members (two of which are putative soluble forms (s)) and one TLR21. Phylogenetic analysis revealed that teleost TLR19-21 are closely related with murine TLR11-TLR13, whilst teleost TLR18 groups with mammalian TLR1, 2, 6 and 10. A typical TLR protein domain structure was found in all these TLRs with the exception of TLR20b(s) and TLR20c(s). TLR-GFP expression plasmids transfected into SHK-1 cells showed that salmon TLR19, TLR20a and TLR20d were preferentially localised to the intracellular compartment. Real time PCR analysis suggested that salmon TLR19-TLR21 are mainly expressed in immune related organs, such as spleen, head kidney and gills, while TLR18 transcripts are more abundant in muscle. In vitro stimulation of primary head kidney cells with type I IFN, IFNγ and IL-1ß had no impact on TLR expression. Infectious salmon anaemia virus (ISAV) infection, in vivo, down-regulated TLR20a, TLR20b(s), TLR20d and TLR21 in infected salmon kidney tissue. In contrast, up-regulation of TLR19 and TLR20a expression was found in posterior kidney in rainbow trout with clinical proliferative kidney disease (PKD).

Antipsychotic-induced elevation of creatine kinase: a systematic review of the literature and recommendations for the clinical practice.

RATIONALE: The primary antipsychotic-induced creatine kinase elevation (i.e., not due to neuroleptic malignant syndrome, extrapyramidal symptoms, etc.) is a poorly studied condition. OBJECTIVES: The aims of the present study were to provide an overview of published cases with antipsychotic-induced creatine kinase elevation and give recommendations for the clinical practice. METHODS: PubMed and EMBASE were searched for eligible trials, case series, and case reports. We set a threshold at ten times the upper normal limit of the creatine kinase value in order to define an elevation as significant. RESULTS: The prevalence of significant creatine kinase elevation ranged between 2 and 7%. We found a total of 42 eligible cases. Men were overrepresented in our sample (81%). Patients with myoglobinuria were more likely to be symptomatic (Fisher's exact test, p = 0.006), whereas neither myoglobinuria (Mann-Whitney test, p > 0.10) nor symptoms (Mann-Whitney test, p = 0.64) were related to the magnitude of the creatine kinase (CK) elevation. In the majority of the cases, the antipsychotic medication was discontinued (86%). Forced diuresis was given in 36% of the patients. Eighty-three percent of the patients had no further complications. Only one case was found with a de novo acute renal failure. CONCLUSIONS: The discontinuation of the antipsychotic medication was a sufficient measure for the CK elevation to subside in the majority of the cases. Cases with myoglobinuria should eventually be treated more aggressively. Further recommendations for the clinical practice are presented.

Identification and characterization of TLR7, TLR8a2, TLR8b1 and TLR8b2 genes in Atlantic salmon (Salmo salar).

Mammalian Toll-like receptor (TLR) 7 and 8 are responsible for recognizing viral single-stranded RNA (ssRNA) and are activated by anti-viral imidazoquinoline compounds, leading to a series of defensive mechanisms being launched to protect the host against viruses. In this study, we identified two TLR7 (with one probably a pseudogene) and three TLR8 genes, namely TLR8a2, TLR8b1 and TLR8b2 from Atlantic salmon (Salmo salar) whole-genome shotgun (WGS) contigs. Bioinformatics analysis showed that salmon TLR7 and TLR8a2 are closely related to the corresponding trout orthologs, however, salmon TLR8b1 and TLR8b2 share the highest amino acid sequence similarity to zebrafish TLR8b and formed a subfamily of the piscine TLR8 molecules in phylogenetic tree analysis. A conserved gene synteny was found with the salmon TLR7/8a members as seen in other vertebrate loci. Deduced domain organisation of salmon TLR7 and TLR8 molecules showed similar structural features, with equal numbers of leucine-rich repeats (LRRs) and insertion motifs. Individual TLR molecules were expressed in a similar pattern between parr and post-smolts, with a high expression level in immune tissues. Promoter analysis predicted several transcription factor binding sites in the TLR8a1/2 and TLR8b1 5' flanking regions, namely C/EBP, AP-1, STAT, NFκB, and IRF family, suggesting cytokine regulation of the genes. Hence, three recombinant cytokines, type I IFN, IFNγ and IL-1ß were used to study the regulation of the salmon TLR gene expression levels in primary head kidney cells and the Salmon Head Kidney-1 (SHK-1) cell line. Salmon TLR7 and TLR8a1 gene expression was more sensitive to type I IFN and IFNγ treatment in primary head kidney cells and SHK-1 cells respectively, with no significant up-regulation of TLR8a2 and TLR8b2 by any of the treatments. On the other hand, salmon TLR8a1 and TLR8b1 were most sensitive to IL-1ß treatment in SHK-1 cells and primary head kidney cells, respectively. TLR8b2 was undetectable in SHK-1 cells under these same conditions.

Flagellin expression enhances Salmonella accumulation in TLR5-positive macrophages.

Recently, it has been reported that Salmonella secrete flagellin in response to host produced lysophospholipids. However, this monomer of the bacterial flagella activates Toll-like receptor 5 (TLR5) in the innate immune system. The objective of this study was to examine the role of flagellin expression during infection of species-specific macrophages (MPhi) which either expressed or lacked TLR5. Initially, TLR5-activity was confirmed in bovine MPhi using Salmonella typhimurium derived-flagellin. Within these cells, recombinant FliC induced a potent CXCL8 response when compared to the heterogeneous (FliC/FljB) form of purified flagellin. Furthermore, neither form of flagellin induced nitrite secretion which was subsequently detected after exposing bovine MPhi to LPS in the presence of IFN-gamma. Flagellin enhanced the accumulation of Salmonella enteritidis in TLR5-positive bovine and human MPhi which was independent of adhesion in bovine MPhi. In contrast, murine MPhis which lacked TLR5 were equally susceptible to hosting S. enteritidis, with or without flagellin. However, lack of flagellin in S. typhimurium marginally inhibited bacterial accumulation in bovine MPhi, where FljB and FliC compensated for the lack of each other. This study suggests that flagellin may be inducing TLR5-dependent internalisation mechanisms in Mcapital EF, Cyrillic which vary qualitatively between different species and Salmonella serotypes.

Production and novel quantification of haemolysins produced by coagulase-negative staphylococci isolated from subclinical mastitis in sheep.

Coagulase-negative staphylococci producing cell-damaging toxins were isolated from the milk of sheep with subclinical mastitis. The haemolytic activity of Coagulase-negative staphylococcal strains was assessed on solid and liquid culture media. More than 61% and 76% of the tested strains on solid media produced evidence of alpha- and delta- haemolysins and more than 78% produced synergistic haemolysis. However almost all isolates producing haemolysin in liquid culture media produced only very few units of haemolysin compared to the positive control of five Coagulase-positive strains of staphylococci. It was concluded that solid media are better for classifying Coagulase-negative staphylococci as producers or not of haemolysins, and liquid media for measuring the size of this activity within the first few hours of intramammary infection.

Naked DNA when co-administered intranasally with heat-labile enterotoxin of Escherichia coli primes effectively for systemic B- and T-cell responses to the encoded antigen.

In this study a novel prime-boost immunisation strategy was evaluated. Priming of BALB/c mice by the intranasal route with plasmid DNA encoding beta-galactosidase (LacZ) with or without heat-labile enterotoxin (LT) of Escherichia coli as a mucosal adjuvant, resulted in the induction of weak serum antibody and proliferative T-cell responses. However, following an intraperitoneal booster injection with the beta-galactosidase protein (beta-gal), strong antibody and proliferative T-cell responses were induced in all the mice. These responses were highest in mice primed intranasally with a mixture of LacZ+LT as compared to those mice primed with DNA (LacZ) or protein (beta-gal) alone. Moreover, LacZ+LT primed mice produced high avidity antibodies and the subclasses of serum antibodies were IgG1 and IgG2a, suggesting a mixed Th1/Th2-type response. Priming of mice with either protein (beta-gal) or DNA (LacZ) alone, produced predominantly IgG1 antibodies, suggesting a Th2-type response. These findings suggest that the use of a heterologous DNA-prime, protein-boost immunisation scheme combining different routes of administration, might be an advantageous strategy for the induction of accelerated immune responses.

Antibody responses of goldfish (Carassius auratus L.) to DNA-immunisation at different temperatures and feeding levels.

DNA vaccines which work in winter are needed for fish. At 18 weeks after intramuscular injection of a plasmid containing lacZ goldfish at 15 degrees C had five-fold more antibody to betagalactosidase than those at 25 degrees C or those at

The safety and longevity of DNA vaccines for fish.

A plasmid that contained the cytomegalovirus (CMV)-promoter-driven lacZ reporter gene (pCMV-lacZ) remained in the epaxial muscle of five of eight goldfish as covalently closed circles, the most functional form of plasmid, for at least 70 days at 22 degrees. It was not present in the gills or elsewhere by polymerase chain reaction and was not integrated. Its expressed protein, Escherichia coli beta-galactosidase (beta-gal), which was in the injected myofibres, was detected in all the fish at 4-21 days and in about half the fish from 28 days until the end of the experiment at 70 days. The numbers of cells that secreted antibody to beta-gal in the kidney peaked at 14 days. Serum antibody and proliferating kidney cells to beta-gal were in all fish from 14 days with a plateau of the responses from 21 days onwards. The plasmid did not induce autoimmune-like antibodies to itself or to single- or double-stranded salmon testis DNA. Plasmids can therefore induce long-term foreign protein expression whilst inducing humoral and cell-mediated immunity without autoimmunity or integration in goldfish.

Mammalian granulocyte-macrophage colony-stimulating factor and some CpG motifs have an effect on the immunogenicity of DNA and subunit vaccines in fish.

A eukaryotic plasmid DNA carrying the AACGTT CpG motif in its ampR gene is a 'danger' signal for mice and caused an increase in the specific antibody titres of fish and mice after immunization with beta-galactosidase (beta-gal). A second pUC-based plasmid, which is inactive in mice and contains the GACGTC CpG motif in its cytomegalovirus (CMV) promoter, had no effect on antibody responses to beta-gal in either fish or mice. A synthetic oligonucleotide, which contains the GACGTT motif, potentiated antibody responses to co-administered beta-gal protein in mice, but not in fish. This is early evidence that lower and higher vertebrates recognize different unmethylated CpG motifs as 'danger' signals. In addition, plasmid DNA expressing mouse granulocyte-macrophage colony-stimulating factor (GM-CSF) had a marked effect on cytotoxic T-cell-like activity in fish by reducing the average number of myofibres that expressed beta-gal, 28 days after co-injection with plasmid DNA expressing beta-gal. Although the mechanism by which the mouse GM-CSF exerted its biological effects in fish is unknown, this finding might have important implications for fish vaccination, particularly when cytotoxic T cells may play a critical role.

DNA is as effective as protein at inducing antibody in fish.

Antiviral vaccines are needed for fish. 50 microg plasmid DNA in saline by the intramuscular route and 10 microg beta-gal protein in a commercial oil adjuvant by the peritoneal route induced serum antibody of the same titre and avidity in goldfish. The DNA expressed beta-gal under control of the immediate early promoter/enhancer gene of human cytomegalovirus. Commercial bacterin vaccines are administered to fish by the intraperitoneal route with oil and this route for DNA induced only 2-fold less antibody than DNA by the intramuscular route. Bacterin vaccines and antiviral plasmid DNA could therefore be co-injected into the peritoneum of fish in an oil adjuvant as a single dose.