Cloning, protein expression and immunogenicity of HBs-murine IL-18 fusion DNA vaccine

Hepatitis B is a global serious disease caused by hepatitis B virus (HBV). There is no known cure for hepatitis B. The best way to deal with the disease is by preventing with hepatitis B vaccine. However, the current protein-based vaccines made up of recombinant hepatitis B surface antigen (HBsAg) are ineffective in chronic HBV carriers and a significant number of the vaccinees do not mount the protective immune response. Novel DNA-based immunization may overcome the deficits of the protein-based immunization and may provide more effective prophylactic and therapeutic outcomes. In this study, we constructed a recombinant plasmid carrying gene encoding the HBV surface antigen (HBs) linked to DNA segment encoding full-length murine interleukin-18, i.e. pcDNA-HBs-IL-18. Immunogenicity of the DNA construct was carried out in BALB/c mice in comparison with mock, i.e. pcDNA3.1+ and vaccines comprised of pRc/CMV-HBs and pRc/CMV-HBs plus pcDNA-IL-18. All vaccinated mice revealed significant serum anti-HBs IgG response after two intramuscular injections of the vaccines at 28 day interval as compared to the level of mock. Co-administration of pRc/CMV-HBs and pcDNA-IL-18 elicited arbitrarily higher levels of anti-HBs IgG than the levels in mice immunized with pRc/CMV-HBs alone and mice that received pcDNA-HBs-IL-18 although not statistically different. Further experiments are needed to investigate the subisotypes of the IgG antibody, the kinetics of cytokine and the cell-mediated immune response. For this communication, the prototype HBs-IL-18 DNA vaccine was successfully constructed and the gene encoding murine IL-18 was successfully cloned. The latter can be co-injected with the antigen coding DNA or used as a fusion partner to the DNA for priming the immune response. The recombinant HBs and full-length IL-18 proteins have potential for other research purposes. They may be used also as standard proteins in the protein quantification assay.     

The Response of Anesthetic Agent Monitors to Trifluoromethane Warns of the Presence of Carbon Monoxide from Anesthetic Breakdown

Objective. Trifluoromethane and CO are produced simultaneously duringthe breakdown of isoflurane and desflurane by dry CO2absorbents. Trifluoromethane interferes with anesthetic agent monitoring, andthe interference can be used as a marker to indicate anesthetic breakdown withCO production. This study tests representative types of gas monitors todetermine their ability to provide a clinically useful warning of COproduction in circle breathing systems. Methods. Isoflurane anddesflurane were reacted with dry Baralyme® at 45 °C. Standardizedsamples of breakdown products were created from mixtures of reacted andunreacted gases to simulate the partial degrees of reaction which might resultduring clinical episodes of anesthetic breakdown using 1% or 2% isoflurane and 6% or 12% desflurane. These mixtures were measured by the monitors tested, andthe indication of the wrong agent or a mixture of agents due to the presenceof trifluoromethane was recorded and related to the CO concentration in thegas mixtures. Results. When presented with trifluoromethane fromanesthetic breakdown, monochromatic infrared monitors displayedinappropriately large amounts of isoflurane or desflurane. Agent identifyinginfrared and Raman scattering monitors varied in their sensitivity totrifluoromethane. Mass spectrometers measuring enflurane at mass to charge= 69 were most sensitive to trifluoromethane. Conclusions. Monochromaticinfrared monitors were unable to indicate anesthetic breakdown viainterference by trifluoromethane, but did indicate falsely elevated anestheticconcentrations. Agent identifying infrared and Raman monitors provided warningof desflurane breakdown via the interference of trifluoromethane by displayingthe wrong agent or mixed agents, but may not be sensitive enough to warn ofisoflurane breakdown. Some mass spectrometers provided the most sensitivewarnings to anesthetic breakdown via trifluoromethane, but additional dataprocessing by some patient monitor units reduced their overall effectiveness.     

Systematic review of appropriate cognitive assessment instruments used in clinical trials of schizophrenia,major depressive disorder and bipolar disorder

Cognitive dysfunction is increasingly recognized as a symptom in mental conditions including schizophrenia, major depressive disorder (MDD), and bipolar disorder (BPD). Despite the many available cognitive assessment instruments, consensus is lacking on their appropriate use in clinical trials. We conducted a systematic literature review in Embase, PubMed/Medline and PsychINFO to identify appropriate cognitive function instruments for use in clinical trials of schizophrenia, MDD, and BPD. Instruments were identified from the articles. Instruments and articles were excluded if they did not address schizophrenia, MDD, or BPD. Instrument appropriateness was further assessed by the criteria of the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) initiative: test–retest reliability, utility, relationship to functional status, potential changeability to pharmacological agents, and tolerability and practicality for clinical trials. The database search yielded 173 articles describing 150 instruments used to assess cognitive function. Seventeen additional instruments were identified through Google and clinicaltrials.gov. Among all these, only 30 (18%) were deemed appropriate for use in the diseases of interest. Of these, 27 were studied in schizophrenia, one in MDD and two in BPD. These findings suggest the need for careful selection of appropriate cognitive assessment instruments, as not all may be valid in these disorders.     

Abdominal Surgical Incision Induces Remote Preconditioning of Trauma (RPCT) via Activation of Bradykinin Receptors (BK2R) and the Cytochrome P450 Epoxygenase Pathway in Canine Hearts

Objective  

Recently, a novel observation was made in which nonischemic trauma at a site remote from the heart produced by a transverse abdominal incision resulted in a marked reduction of infarct size (IS) in the mouse heart via activation of sensory nerve fibers in the skin and subsequent activation of bradykinin 2 receptors (BK2R). This phenomenon was termed remote preconditioning of trauma (RPCT). Since RPCT may have potential clinical implications we attempted to confirm these findings in a large animal model, the dog. The epoxyeicosatrienoic acids (EETs) have also recently been shown to be antinociceptive and have been shown to mimic ischemic preconditioning (IPC) and postconditioning (POC) in dogs, therefore, we tested the role of the EETs in RPCT.     

Schistosomal myeloradiculopathy due to Schistosoma mansoni: Report on 17 cases from an endemic area

Background:

After malaria, schistosomiasis is the second most prevalent tropical disease. The prevalence of oviposition in CNS of infected persons varies from 0.3 to 30%. The conus medullaris is a primary site of schistosomiasis, either granulomatous or acute necrotizing myelitis.

Objective:

To report the clinical, radiological, and laboratory results of spinal cord schistosomiasis (SCS) and to design proper therapeutic regimens.

Materials and Methods:

Seventeen patients (13 males and four females) with SCS were enrolled between 1994 and 2009 at Mansoura University Hospitals. Their median age at diagnosis was 19 years (13-30 years). Independent neurological, radiological, and laboratory assessments were performed for both groups, excluding pathological confirmation that was done earlier in eight patients (Group 1). In the group 2 (nine patients), indirect hemagglutination (IHA) test for bilharziasis in blood and cerebrospinal fluid (CSF) was performed. Higher positive titer in CSF than serum indicated SCS plus induction of antibilharzial and corticosteroid protocols for 12 months with a three-year follow-up.

Results:

Rate of neurological symptoms of granulomatous intramedullary cord lesion was assessed independently in 16 cases and acute paraparesis in one case. All patients in group 2 had positive IHA against Schistosoma mansoni with median CSF and serum ranges 1/640 and 1/320, respectively. Seven patients (41.18%) had complete recovery, eight patients (47.06%) showed partial recovery, and no response was reported in two patients (11.76%) (P = 0.005). There was no recorded mortality in the current registry.

Conclusions:

Rapid diagnosis of SCS with early medical therapies for 12 months is a crucial tool to complete recovery.     

APOA-1 is a Novel Marker of Erythroid Cell Maturation from Hematopoietic Stem Cells in Mice and Humans

The mechanism that regulates the terminal maturation of hematopoietic stem cells into erythroid cells is poorly understood. Therefore, identifying genes and surface markers that are restricted to specific stages of erythroid maturation will further our understanding of erythropoiesis. To identify genes expressed at discrete stages of erythroid development, we screened for genes that contributed to the proliferation and maturation of erythropoietin (EPO)-dependent UT-7/EPO cells. After transducing erythroid cells with a human fetal liver (FL)-derived lentiviral cDNA library and culturing the cells in the absence of EPO, we identified 17 candidate genes that supported erythroid colony formation. In addition, the mouse homologues of these candidate genes were identified and their expression was examined in E12.5 erythroid populations by qRT-PCR. The expression of candidate erythroid marker was also assessed at the protein level by immunohistochemistry and ELISA. Our study demonstrated that expression of the Apoa-1 gene, an apolipoprotein family member, significantly increased as hematopoietic stem cells differentiated into mature erythroid cells in the mouse FL. The Apoa-1 protein was more abundant in mature erythroid cells than hematopoietic stem and progenitor cells in the mouse FL by ELISA. Moreover, APOA-1 gene expression was detected in mature erythroid cells from human peripheral blood. We conclude that APOA-1 is a novel marker of the terminal erythroid maturation of hematopoietic stem cells in both mice and humans.     

Variation in Mesodermal and Hematopoietic Potential of Adult Skin-derived Induced Pluripotent Stem Cell Lines in Mice

Induced pluripotent stem cells (iPSCs) are a promising tool for regenerative medicine. Use of iPSC lines for future hematotherapy will require examination of their hematopoietic potential. Adult skin fibroblast somatic cells constitute a source of iPSCs that can be accessed clinically without ethical issues. Here, we used different methods to compare mesodermal and hematopoietic potential by embryoid body formation of five iPSC lines established from adult mouse tail-tip fibroblasts (TTFs). We observed variation in proliferation and in expression of genes (Brachyury, Tbx1, Gata1, Klf1, Csf1r) and proteins (Flk1, Ter119 and CD45) among TTF-derived lines. 256H18 iPSCs showed highest proliferation and most efficient differentiation into mesodermal and hematopoietic cells, while expression levels of the pluripotency genes Oct3/4, Sox2, Klf4 and Nanog were lowest among lines analyzed. By contrast, the 212B2 line, transduced with c-Myc, showed lowest proliferation and differentiation potential, although expression levels of Oct3/4, Sox2 and Klf4 were highest. Overall, we find that mesodermal and hematopoietic potential varies among iPSCs from an identical tissue source and that c-Myc expression likely underlies these differences.     

(1aR,5aR)1a,3,5,5a-Tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalene-4-carboxylic acid (MK-1903): a potent GPR109a agonist that lowers free fatty acids in humans

G-protein coupled receptor (GPCR) GPR109a is a molecular target for nicotinic acid and is expressed in adipocytes, spleen, and immune cells. Nicotinic acid has long been used for the treatment of dyslipidemia due to its capacity to positively affect serum lipids to a greater extent than other currently marketed drugs. We report a series of tricyclic pyrazole carboxylic acids that are potent and selective agonists of GPR109a. Compound R,R-19a (MK-1903) was advanced through preclinical studies, was well tolerated, and presented no apparent safety concerns. Compound R,R-19a was advanced into a phase 1 clinical trial and produced a robust decrease in plasma free fatty acids. On the basis of these results, R,R-19a was evaluated in a phase 2 study in humans. Because R,R-19a produced only a weak effect on serum lipids as compared with niacin, we conclude that the beneficial effects of niacin are most likely the result of an undefined GPR109a independent pathway.     

Kinetics and pathogenicity of equine herpesvirus-9 infection following intraperitoneal inoculation in hamsters

The kinetics of infection and pathogenicity of equine herpesvirus-9 (EHV-9) was studied in a hamster model. Five-week-old Syrian hamsters and 5-day-old suckling hamsters were inoculated intraperitoneally with 10(5) and 4×10(4) plaque-forming units of EHV-9, respectively. EHV-9 antigens were detected by immunocytochemistry in the peritoneal macrophages, which may be the primary site of virus attachment and propagation at 6h post inoculation (hpi). At 12 hpi, viral antigen was observed in the abdominal nerves and ganglia (mainly the coeliac ganglia). Virus antigen was detected in the dorsal root (spinal) ganglia, in parts of the spinal cord (particularly the mid-lumbar area) and in the myenteric plexuses at 36, 48 and 72 hpi, respectively. At 96 hpi, virus antigen was detected in the most caudal part of the brain. Polymerase chain reaction conducted on samples of the blood, spinal cord and brain revealed EHV-9 DNA in the spinal cord at 36 hpi and in the blood at 48 hpi and for 4 days after this initial detection. It is suggested that after initial propagation in the abdominal macrophages, EHV-9 infected the abdominal ganglia or myenteric plexuses and then travelled to the brain via the peripheral nerves and spinal cord. Examination of other organs also revealed the presence of EHV-9, suggesting that the virus might infect tissues other than those of the nervous system.