Hypothalamo-pituitary-interrenal responses to opioid substances in the trout. I. Effects of morphine on the release in vitro of corticotrophin-releasing activity from the hypothalamus and corticotrophin from the pituitary gland

The effects of morphine and naloxone on the secretion in vitro of corticotrophin (ACTH) and and corticotrophin-releasing factor (CRF) by the pars distalis and hypothalamus, respectively, have been studied in the trout. The spontaneous in vitro secretion of corticotrophin by the pars distalis is depressed significantly by the addition of high concentrations of morphine (10(-6)-10(-7) mol/litre) to the incubation medium. The effect is naloxone reversible. Morphine does not influence the response of the pituitary tissue to exogenous CRF41, suggesting that the inhibitory influence of the opiate is exerted primarily on the CRF nerve terminals within the pars distalis and not on the corticotrophs. At considerably lower concentrations (10(-10)-10(-8) mol/litre) morphine stimulates the release of CRF from the isolated trout hypothalamus in vitro. Its effects are dose-dependent and antagonized by naloxone. The results suggest that two anatomically and pharmacologically distinct populations of opioid receptors mediate opposing actions of morphine on the hypothalamo-pituitary-corticotrophic system in the trout.     

The induction of oral tolerance to Actinomyces viscosus in mice

OBJECTIVES: To determine whether oral tolerance with the oral bacterium Actinomyces viscosus was inducible in mice. MATERIALS AND METHODS: Mice were intragastrically (i.g.) and then intraperitoneally (i.p.) immunized with heat-killed A. viscosus. A control group of mice received only saline. A delayed type hypersensitivity (DTH) response and the levels of isotype specific antibodies were assessed. Spleen cells from mice that were i.g. immunized with A. viscosus were transferred to A. viscosus-primed mice in vivo and in vitro. Furthermore, mice were i.g. immunized with saline or A. viscosus and then challenged i.p. with saline, A. viscosus, or Porphyromonas gingivalis. RESULTS: Intragastric immunization with A. viscosus suppressed both DTH and serum specific antibodies to A. viscosus. DTH suppression lasted until week 4, while serum immunoglobulin (Ig)A and both IgG and IgM specific antibody levels remained suppressed up to week 8 and 12 respectively. IgG specific antibody suppression was transferable. The DTH response and serum antibodies specific to A. viscosus were suppressed in mice after i.g. challenged with A. viscosus but not P. gingivalis. CONCLUSION: Mucosal presentation of A. viscosus in mice led to the suppression of immune response to this bacterium in an antigen-specific fashion. Tolerance of DTH response was short lived, while suppression of antigen-specific IgG antibodies in mucosally tolerized mice was long-lasting.     

A New Assay for the Measurement of Total Progressive Antithrombin

A new rapid method for assaying total antithrombin activity has been developed based on the inactivation of thrombin incorporated into an agarose gel, during the radial diffusion of plasma in the gel. The area of thrombin inactivation is subsequently observed by the coagulation of fibrinogen in a separate agarose gel layer poured over the thrombin gel. The method is described in detail and its accuracy assessed with respect to other antithrombin assays. Using specific antisera to alpha2-globulin (antithrombin III), alpha2-macroglobin and alpha1-antitrypsin, total antithrombin activity measured by this assay consisted of 47% alpha2-globulin, 29% alpha2-macroglobulin and 26% alpha1-antitrypsin.     

Identification of blast cells in the peripheral blood of patients with acute leukaemia using the Technicon H-1

The Technicon H-1 counter represents a refinement of the cytochemistry-based technology of its predecessors, the H6000 and the Hemalog-D. It also has a new channel, the basophil-lobularity channel, which is said to enhance the sensitivity of leukaemic blast detection in comparison with previous instruments. To evaluate this facility, 35 adult patients with acute leukaemia at different phases of their disease were monitored for the presence of circulating leukaemic blasts during a 4-week period. The ability of the H-1 to detect blasts was compared to a careful manual review of a blood and bone marrow smear. Using the latter review as the standard, the sensitivity was 83.8% with a specificity of 78%. Exclusion of patients with severe leucopenia (less than 1.0 x 10(9)/l) increased the specificity to 89%, with little alteration in the sensitivity. We were unable to confirm the high degree of sensitivity claimed in previous reports. The H-1 blast flag, however, would appear useful for screening patients who are off therapy or on maintenance regimens.     

Microarray based comparative genomic hybridization testing in deletion bearing patients with Angelman syndrome: genotype-phenotype correlations

Background

Angelman syndrome (AS) is a neurodevelopmental disorder characterised by severe mental retardation, dysmorphic features, ataxia, seizures, and typical behavioural characteristics, including a happy sociable disposition. AS is caused by maternal deficiency of UBE3A (E6 associated protein ubiquitin protein ligase 3A gene), located in an imprinted region on chromosome 15q11‐q13. Although there are four different molecular types of AS, deletions of the 15q11‐q13 region account for approximately 70% of the AS patients. These deletions are usually detected by fluorescence in situ hybridisation studies. The deletions can also be subclassified based on their size into class I and class II, with the former being larger and encompassing the latter.

Methods

We studied 22 patients with AS due to microdeletions using a microarray based comparative genomic hybridisation (array CGH) assay to define the deletions and analysed their phenotypic severity, especially expression of the autism phenotype, in order to establish clinical correlations.

Results

Overall, children with larger, class I deletions were significantly more likely to meet criteria for autism, had lower cognitive scores, and lower expressive language scores compared with children with smaller, class II deletions. Children with class I deletions also required more medications to control their seizures than did those in the class II group.

Conclusions

There are four known genes (NIPA1, NIPA2, CYFIP1, & GCP5) that are affected by class I but not class II deletions, thus raising the possibility of a role for these genes in autism as well as the development of expressive language skills.     

Organ-specific inflammation following acute ethanol and burn injury

Clinical and experimental evidence demonstrates that ethanol exposure prior to injury alters local and systemic inflammatory responses, increasing morbidity and mortality. Moreover, the aberrant inflammatory responses can directly and indirectly lead to the poor prognosis after injury by altering leukocyte infiltration into the wound site and remote organs and by suppressing immunity leading to increased susceptibility to opportunistic infections. Recent studies from our laboratory have focused on inflammatory responses at the wound site and in other distal organs after exposure to acute ethanol and burn injury. This combined insult leads to increased mortality after dermal or intratracheal pseudomonas infection, relative to infected mice given ethanol or burn injury alone. The increased mortality in mice given ethanol and burn injury parallels elevated serum levels of proinflammatory cytokines, IL-6 and TNF-alpha, marked infiltration of leukocytes into the lung and gut, as well as immunosuppression at the sites of infection. Bacterial translocation from the gut is likely to be responsible, in part, for the aberrant accumulation of leukocytes in the lungs of ethanol-exposed, burn-injured mice. Additionally, other factors, such as expression of adhesion molecules, increased chemokine production, and leakiness of the vascular endothelium, may also be involved.     

Complex functions of phosphatidylinositol 4,5-bisphosphate in regulation of TRPC5 cation channels

The canonical transient receptor potential (TRPC) proteins have been recognized as key players in calcium entry pathways activated through phospholipase-C-coupled receptors. While it is clearly demonstrated that members of the TRPC3/6/7 subfamily are activated by diacylglycerol, the mechanism by which phospholipase C activates members of the TRPC1/4/5 subfamily remains a mystery. In this paper, we provide evidence for both negative and positive modulatory roles for membrane polyphosphoinositides in the regulation of TRPC5 channels. Depletion of polyphosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP₂) through inhibition of phosphatidylinositol 4-kinase activates calcium entry and membrane currents in TRPC5-expressing but not in TRPC3- or TRPC7-expressing cells. Inclusion of polyphosphatidylinositol 4-phosphate or PIP₂, but not phosphatidylinositol 3,4,5-trisphosphate, in the patch pipette inhibited TRPC5 currents. Paradoxically, depletion of PIP₂ with a directed 5-phosphatase strategy inhibited TRPC5. Furthermore, when the activity of single TRPC5 channels was examined in excised patches, the channels were robustly activated by PIP₂. These findings indicate complex functions for regulation of TRPC5 by PIP₂, and we propose that membrane polyphosphoinositides may have at least two distinct functions in regulating TRPC5 channel activity. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mohamed Trebak and Loic Lemonnier contributed equally to this work.