Susceptibility of dopamine D5 receptor targeted mice to cysteamine

BACKGROUND: Recently we demonstrated that gastric mucosa of rats can synthesize, store and release dopamine. Out of five different subtypes, mRNA of D5 (=D1b) dopamine receptor is very abundant in the gastric epithelium. D1 receptor selective dopamine agonists have been shown to protect against experimental gastro-duodenal lesions. AIMS: To test the hypothesis that protective effects of dopamine involve D5 receptors, mucosal lesions were induced in D5 receptor deficient (KO) and wild-type (WT) mice using cysteamine. Morphology and gastric acid secretion of D5 KO mice were also studied. METHODS: Single doses of 600 mg/kg, 300 mg/kg cysteamine or vehicle were administered subcutaneously to fasted animals. After 24 h, number and severity of gastro-duodenal lesions were analyzed. Basal and histamine-induced maximal gastric acid output were measured by a stomach-sac wash-through method. RESULTS: All the KOs in the 600 mg/kg cysteamine group died within 4 h showing symptoms of toxicity while three out of four WTs survived (P<0.05). Mortality after 300 mg/kg cysteamine was significantly higher in KOs versus the WTs: 6/14 versus 2/11, P<0.05. Gastric lesion-index was also significantly higher in KOs (median, middle quartile): four (3-9) versus 0 (0-0), P<0.05. Duodenal lesions did not develop from this single dose of cysteamine in either genotype. Basal and histamine-induced maximal gastric acid output were comparable in the two genotypes. CONCLUSIONS: This study demonstrates that loss of D5 receptor causes mucosal vulnerability and increased toxicity of cysteamine in genetically manipulated mice. Thus, D5 receptor subtype is indeed likely to be involved in protective effects of dopamine in the stomach.     

A Single-Dose Influenza A (H5N1) Vaccine Safe and Immunogenic in Adult and Elderly Patients: an Approach to Pandemic Vaccine Development

With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic vaccines. Here we report a randomized multicenter dose-finding clinical trial of a whole-virion, inactivated, adjuvanted H5N1 vaccine in adult and elderly volunteers. Four hundred eighty patients were randomly assigned to receive one or two doses of 3.5 μg of the vaccine or one dose of 6 or 12 μg. The subjects were monitored for safety analysis, and serum samples were obtained to assess immunogenicity by hemagglutination inhibition and microneutralization tests. The subjects developed antibody responses against the influenza A (H5N1) virus. Single doses of ≥6 μg fulfilled EU and U.S. licensing criteria for interpandemic and pandemic influenza vaccines. Except for occasional injection site pain, malaise, and fever, no adverse events were observed. We found that the present vaccine is safe and immunogenic in healthy adult and elderly subjects and requires low doses and, unlike any other H5N1 vaccines, only one injection to trigger immune responses which comply with licensing criteria. A vaccine using the same methods as those described in this report, but based on a wild-type swine-origin 2009 (H1N1) influenza A virus isolate from the United States (supplied by the CDC), has been developed and is currently being tested by our group.With the ongoing pandemic of influenza A (H1N1) virus infection and the threat of high fatality rates for recent human cases of infection with highly pathogenic H5N1 strains, there has been considerable interest in developing pandemic influenza vaccines.With new cases continuing to emerge, as of June 2009, the avian influenza A (H5N1) virus subtype has caused 433 human infections in 15 countries, as confirmed by the World Health Organization (WHO), resulting in severe illness with a high fatality rate (30). Human-to-human spread has been strongly suspected and even evidenced by statistical methods (22, 33). With new human infections continuing to develop, this subtype continues to represent a potential source of an influenza pandemic (33).Mass vaccination is the most effective approach to reduce illness and death from pandemic influenza. Therefore, vaccine producers are currently developing and assessing vaccines against H5N1 viruses (2, 14, 31). The effects of split, subvirion, and whole-virion H5N1 vaccines have been tested, with various immunogenicity results (31). Three whole-virion vaccines have been tested so far, two of which required two-dose regimens (4, 14), while a one-dose regimen with the present vaccine was found to be immunogenic in 146 adult subjects (24).The objective of the present study was to determine the safety and immunogenicity of an inactivated whole-virion vaccine against influenza A/Vietnam/1194/2004, using multiple dosing and administration schedules, for adult and elderly subjects. To date, this is the only influenza pandemic prototype vaccine trial examining single-dose regimens in elderly patients.     

Influence of the blood concentration of prolactin on the length of the sensitive period for establishing maternal behavior in sheep at parturition

The fading of postpartum maternal interest for the neonate (sensitive period) in ewes separated from their young at lambing is delayed when parturition is induced with 20 mg of estradiol benzoate (EB). An experiment was carried out to investigate the role of prolactin in this phenomenon. The sensitive period was studied in three groups of parturient ewes. In all groups lambs were removed at birth and reintroduced to their mothers 24 hr later. Maternal acceptance was tested at this time. In group 1 (dexamethasone D), ewes were induced to lamb with dexamethasone (15 mg im). In group 2 (EB), ewes were treated with 20 mg of estradiol benzoate (im). In group 3 (EB + CB 154) ewes received 20 mg of EB as in group 2 and 1 mg of CB 154 (sc) every 12 hr to prevent the enhanced secretion of prolactin which normally occurs after EB injection. The concentration of prolactin was highest in group 2 (EB), lowest in group 3 (EB + CB), and intermediate in group 1 (D) (p ? 0.001 between groups). By contrast, the proportion of ewes showing maternal behavior was similar in groups 2 and 3 ($\text{15}\text{23}$ and $\text{17}\text{22}$), both of which differed from group 1 ($\text{3}\text{22}\text{; p ? 0.005}$). It is concluded that the lengthening of the sensitive period for establishing maternal behavior in sheep following EB induced parturition is not related with high levels of prolactin in the peripheral circulation.     

Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.     

Phosphatidylthreonine and Lipid-Mediated Control of Parasite Virulence

The major membrane phospholipid classes, described thus far, include phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylserine (PtdSer), and phosphatidylinositol (PtdIns). Here, we demonstrate the natural occurrence and genetic origin of an exclusive and rather abundant lipid, phosphatidylthreonine (PtdThr), in a common eukaryotic model parasite, Toxoplasma gondii. The parasite expresses a novel enzyme PtdThr synthase (TgPTS) to produce this lipid in its endoplasmic reticulum. Genetic disruption of TgPTS abrogates de novo synthesis of PtdThr and impairs the lytic cycle and virulence of T. gondii. The observed phenotype is caused by a reduced gliding motility, which blights the parasite egress and ensuing host cell invasion. Notably, the PTS mutant can prevent acute as well as yet-incurable chronic toxoplasmosis in a mouse model, which endorses its potential clinical utility as a metabolically attenuated vaccine. Together, the work also illustrates the functional speciation of two evolutionarily related membrane phospholipids, i.e., PtdThr and PtdSer.     

The Production of Reactive Oxygen Species in Intact Isolated Nerve Terminals Is Independent of the Mitochondrial Membrane Potential

Dependence on mitochondrial membrane potential (m) of hydrogen peroxide formation of in situ mitochondria in response to inhibition of complex I or III was studied in synaptosomes. Blockage of electron flow through complex I by rotenone or that through complex III by antimycin resulted in an increase in the rate of H₂O₂ generation as measured with the Amplex red assay. Membrane potential of mitochondria was dissipated by either FCCP (250nM) or DNP (50mM) and then the rate of H₂O₂ production was followed. Neither of the uncouplers had a significant effect on the rate of H₂O₂ production induced by rotenone or antimycin. Inhibition of the F₀F₁-ATPase by oligomycin, which also eliminates m in the presence of rotenone and antimycin, respectively, was also without effect on the ROS formation induced by rotenone and only slightly reduced the antimycin-induced H₂O₂ production. These results indicate that ROS generation of in situ mitochondria in nerve terminals in response to inhibition of complex I or complex III is independent of m. In addition, we detected a significant antimycin-induced H₂O₂ production when the flow of electrons through complex I was inhibited by rotenone, indicating that the respiratory chain of in situ mitochondria in synaptosomes has a substantial electron influx distal from the rotenone site, which could contribute to ROS generation when the complex III is inhibited.