5-Lipoxygenase gene expression in the thymus.

Eicosanoids are arachidonic acid metabolites issued both the cyclooxygenase and the lipoxygenase pathways. Many of these products were reported to modulate the immune response. Since most of eicosanoids have a short half life they are considered as local immunomodulators. Interactions between eicosanoids and thymocytes appear to be complex within the thymus. It was reported that cyclooxegenase derivatives of arachidonic acid are produced in this primary lymphoid organ mostly by cells of the thymic microenvironment. On the other hand it is not yet clearly established (1) what is the location of the lipoxygenase-positive cells within the gland and (2) what is the ratio of cells producing lipoxygenase metabolites of arachidonic acid when compared to the whole thymocyte population. Using two oligonucleotides complementary to the rat 5-lipoxygenase mRNA we demonstrated (by both hybridization on Northern blots and in situ hybridization) the expression of the 5-lipoxygenase gene in the thymus. 5-lipoxygenase positive cells appear to be associated in "clusters" and are mostly located in the thymic cortex. It is likely that they belong to the thymic microenvironment.     

Phenylalanine hydroxylase expression in primary rat hepatocytes is modulated by oxygen concentration

In this work we have investigated the regulation of rat phenylalanine hydroxylase (rPAH) expression by oxygen in primary cultures of rat hepatocytes. We show that rPAH is negatively modulated at the mRNA, protein and activity levels by pO₂ of 16% (periportal hepatic levels) compared to 8% (perivenous hepatic levels). Our results suggest that PAH might be metabolically zonated in vivo, and preferentially found in perivenous hepatocytes with high glucose consumption and largely influenced by insulin levels.     

CXCR4 expression on monocytes is up-regulated by dexamethasone and is modulated by autologous CD3+ T cells

Chemokines and their receptors regulate cell migration to sites of inflammation. The glucocorticoid dexamethasone has potent anti-inflammatory effects, yet paradoxically up-regulates expression of some cytokine receptors. We have examined the effects of dexamethasone on chemokine receptor expression. Using an RNase protection assay, we show that dexamethasone up-regulates human peripheral blood mononuclear cell (PBMC) expression of CXCR4 mRNA. Flow cytometric analysis demonstrated that increased expression of CXCR4, but not CXCR1 and CXCR2, occurred on both monocytes and CD3+ T cells in PBMC mixed cultures. A stromal-derived factor (SDF)-1alpha-mediated calcium influx was detected on monocytes. Basal levels of CXCR4 expression on purified monocytes were lower when compared with monocytes in mixed PBMC cultures. Co-culture of monocytes with purified CD3+ T cells led to enhanced basal expression of CXCR4 on monocytes. The use of transwells to partition CD3+ T cells resulted in increased CXCR4 expression on monocytes, suggesting that CD3+ T-cell derived soluble factors regulate CXCR4 expression.     

Long-term depression is not modulated by ATP receptors in the rat CA1 hippocampal region

ATP is an important extracellular messenger in the CNS. In the hippocampus, a brain structure relevant for learning and memory processes, it acts both as a modulator and as a mediator of synaptic transmission, with implications for synaptic plasticity phenomena. Recent evidence suggests that ATP modulates activity-dependent long-term potentiation (LTP) of Schaffer collateral-CA1 synapses. However, it remains unclear if ATP also modulates LTP counterpart's phenomenon, long-term depression (LTD), in the rat hippocampus. This study investigated the effect of ATP analogues on homosynaptic LTD, induced by low-frequency stimulation of the Schaffer collaterals (1 Hz; 900 pulses) in the CA1 region of young rat hippocampal slices. The metabolically stable ATP analogues beta,gamma-ImATP (20 microM), a P2 receptor agonist, and alpha,beta-MeATP (20 microM), a preferential P2X(1,3) receptor agonist, did not modify LTD (LTD values of 14.7+/-0.5% and 14.1+/-3% for aCSF controls and of 15.1+/-4% and 19.0+/-5.2% for beta,gamma-ImATP and alpha,beta-MeATP, respectively). The ATP analogue beta,gamma-ImATP (20 microM) did not modify LTD also in the presence of the adenosine A1 receptor antagonist DPCPX (50 nM) (21.5+/-4.2% for DPCPX only and of 23.8+/-8.9% for DPCPX plus beta,gamma-ImATP). Finally, the preferential P2X(1,3) receptor antagonist NF023 (10 microM) had also no effect on LTD (18.6+/-5.2% for aCSF and of 18.7+/-5.2% for NF023). The present results suggest that ATP does not modulate activity-dependent homosynaptic LTD in the rat CA1 hippocampal region by activating P2 receptors.     

Inhibition of interleukin-5 gene expression by dexamethasone.

The effect of glucocorticoids on interleukin-5 (IL-5) gene expression was assessed in human peripheral blood mononuclear cells. IL-5 expression was stimulated by phytohaemagglutinin (PHA), IL-2, phorbol myristate acetate (PMA) or Ionomycin. A semi-quantitative assay for IL-5 gene expression was developed, based on RNA extraction and the polymerase chain reaction. IL-5 expression in response to PHA was profoundly inhibited by 10(-6) M dexamethasone, and significant inhibition was detected at doses of dexamethasone as low as 10(-9) M. When dexamethasone was added to the cells at the same time as PHA, the inhibitory effect could be detected as early as 3 hr. Dexamethasone at 10(-6) M also profoundly inhibited the IL-5 response to PMA and to IL-2, but the IL-5 response to Ionomycin was not significantly affected. These results suggest that dexamethasone may be capable of interfering with a pathway involving protein kinase C. There is increasing evidence that IL-5 may play a pathogenic role in asthma and other manifestations of acute hypersensitivity. The present findings indicate that inhibition of IL-5 expression may be one of the mechanisms whereby glucocorticoids exert their beneficial effects in diseases such as asthma.     

Patterns of gene expression in the developing chick thymus.

Gene expression in thymic T cells during late embryogenesis and early growth in chicks was examined using cDNA microarrays. Gene expression patterns were profiled into nine clusters by using self-organizing maps (SOM) clustering analysis. The expression patterns for a set of genes confirmed current information on the development of immune response. Expression of cell surface markers (MHC class I alpha chain, MHC class II associated invariant chain, CD8 beta chain, CD18, and beta2-microglobulin), and genes involved in the innate immune response (NK lysin-like) increased with age, and these patterns were consistent with an increase in the immune responsiveness of the young chick. The expression of cytokine receptor common gamma chain (gammac), death receptor-3 (DR3), and TCR alpha chain increased up to 1 day of age and then decreased. DR3 could play a role in the apoptosis during T-cell maturation, while the differential expression of TCR genes could reflect regulation of the rearrangement of TCR genes and TCR-mediated signal transduction during T cell development. Three genes coding for previously uncharacterized proteins are included in the clusters. These gene expression profiling studies provide background information on the developing chick immune system and provide preliminary functional information on unknown proteins.     

Cyclosporine-A treatment inhibits the expression of metabotropic glutamate receptors in rat thymus

We have evaluated the expression of metabotropic glutamate receptors (mGluR subtypes 2/3, 4 and 5) in rat thymus under normal and experimental conditions after 2 and 21 days of cyclosporine-A treatment. In normal rats, immunohistochemical analysis showed that expression of mGluRs was high in dendritic cells and lymphocytes of the medulla whereas it was weak in lymphocytes of the cortex. However, there were some differences in the expression of mGluRs subtypes. mGluR5 showed strong expression in lymphocytes of medulla and dendritic cells. mGluR2/3 and mGluR4 were moderately expressed in lymphocytes and dendritic cells of the medulla and weakly in cortical lymphocytes. Immunoblotting showed moderate levels of mGluR2/3 and mGluR4 and strong levels of mGluRS. After 2 days of cyclosporine-A treatment, we observed by immunohistochemistry and immunoblotting a distinct decrease in all mGluRs and their expression had almost completely disappeared after 21 days of treatment. The results clearly indicate that: 1) mGluR2/3, 4 and 5 are widely expressed in thymic cells; 2) the mGluR5 subtype is expressed most strongly in medullary cells; and 3) cyclosporine-A rapidly inhibits expression of all mGluR subtypes after 2 days of treatment and their complete disappearance after prolonged treatment. These findings may indicate a possible mechanism by which cyclosporine-A produces its immunosupressive effects.     

Opioid gene expression in rat striatum is modulated via opioid receptors: evidence from localized receptor inactivation

The irreversible opiate antagonists beta-funaltrexamine (beta-FNA) and beta-chlornaltrexamine (beta-CNA) were injected into rat striatum. One day later, mu-opioid receptor binding, as assessed by receptor autoradiography, was abolished in the injected striatum, while [3H]spiperone binding remained largely unaffected. In situ hybridization was used to examine possible effects on striatal proenkephalin mRNA and prodynorphin mRNA content. The levels of proenkephalin mRNA were increased ipsilaterally following beta-CNA injection, but were slightly decreased following beta-FNA injection. Prodynorphin mRNA content was also increased by beta-CNA administration. The results provide evidence for a differential modulation of striatal opioid peptide gene expression via distinct opioid receptors.     

Endometrial electrogenic ion secretion in the rat is modulated by prostaglandins

In the isolated rat uterus, maximal increases in endometrial electrogenic ion secretion produced by luminal prostaglandins (PGs) E1, E2 and F1, and measured as the short-circuit current (Isc), displayed bell-shaped dose-response curves. PGE1 was the most potent and PGF1 the least. The dose-response relationships for the decrease in resistance were curvilinear for PGE1 and PGF1 but bell shaped for PGE2. PGF1 was the most potent at inhibiting the increase in Isc induced by serosal adrenaline but not in inhibiting the fall in tissue resistance. The electrogenic ion secretion of rat endometrium is modulated by prostaglandins via a mechanism also influenced by adrenaline.     

Startle during backward evaluative conditioning is not modulated by instructions

Instructions highlighting that backward conditional stimuli (CSs) stop unconditional stimuli (USs) result in their acquiring valence opposite to that of the US on explicit measures of valence. We assessed whether such instructions would influence startle blink modulation in the same way. Two groups were presented with concurrent forward and backward evaluative conditioning (CS-US-CS) using cartoon aliens as CSs, and pleasant, neutral, and unpleasant sounds as USs. Startle magnitude was measured during conditioning and valence ratings were assessed after conditioning. Participants in the “start-stop” instructions group (n = 41) were instructed to learn whether CSs started or stopped US presentations, while participants in the “observe” instructions group (n = 41) were told to pay attention to the stimuli as they would be asked questions about them after the experiment. In the start-stop instructions group backward CSs paired with positive USs were rated as less pleasant than backward CSs paired with neutral and negative USs (contrast effect), whereas ratings of backward CSs did not differ in the observe instructions group. Startle magnitude was larger during backward CSs paired with positive USs in comparison to CSs paired with neutral or negative USs in both instruction groups. Startle blink modulation was unaffected by instructions, suggesting that startle indexes the emotional state at the time of probe presentation rather than CS valence based on propositional information about the function of the CS.     

Differential thymus dependence of rat CD8 isoform expression.

Expression of the rat CD8 molecule was studied using five novel monoclonal antibodies (mAb), four of which are specific for the V-like domain of CD8 alpha, whereas one reacts either with the beta chain or with a determinant only expressed on the CD8 alpha/beta heterodimer. mAb to both chains effectively blocked purified lymph node CD8 T cells in mixed lymphocyte reaction and in cell-mediated cytotoxicity. Flow cytometric analysis showed that CD8 T cells from lymph nodes or spleen of normal rats almost exclusively express the alpha/beta isoform, regardless of the T cell receptor isotype (alpha/beta or gamma/delta). In contrast, natural killer (NK) cells carry only CD8 alpha chains. This CD8 alpha + beta - phenotype was also prominent among CD8 T cells from athymic rats and from intestinal epithelium of normal rats. CD8 alpha homodimers can also be expressed as a result of activation, as shown by analysis of CD4 CD8 double-positive T cells obtained from highly purified lymph node CD4 T cells by in vitrok stimulation. Such CD4+CD8 alpha + beta - cells also represent a major subset among adult intestinal intraepithelial lymphocytes (IEL), suggesting local activation. Taken together, the difference in CD8 isoform expression among T cells from athymic rats, NK cells, and gut IEL versus CD8 T cells from peripheral lymphatic organs of euthymic animals suggests that like in mice, expression of the CD8 heterodimer is more dependent on intrathymic maturation than that of the homodimer. Since the more stringent thymus dependence of CD8 alpha + beta + T cells may be due to a requirement for thymic selection on self major histocompatibility complex class I antigens, the virtually exclusive CD8 alpha + beta + phenotype of peripheral rat gamma/delta T cells could mean that antigen recognition by this subset is also restricted by MHC class I molecules.     

Neutrophil generation of inflammatory precursors is not modulated by docosahexaenoic acid

Objective and design  

It is believed that correction of membrane fatty acid deficiency in cystic fibrosis (CF) downregulates the synthesis of proinflammatory mediators. We tested the hypothesis that an increase of the proportion of docosahexaenoic acid (DHA) in the membrane in vitro changes the neutrophil response to Pseudomonas aeruginosa lipopolysaccharide (LPS).     

Colonic nociception via nucleus submedius is modulated by pontine centres in the rat

The rat thalamic nucleus submedius responds to noxious pressure stimuli in the colon. Some neurons in and near Barrington's nucleus, a pontine center related to bladder function, also respond to colon distension. We hypothesized that colonic nociception may be relayed via Barrington's nucleus to the nucleus submedius. Experiments were carried out in 22 urethane-anesthetized male rats. Noxious stimuli were applied to the toes using standardized clips and to the colon by inflation of the balloon to 80 mmHg for 30 s using a barostat. The brain was exposed to allow recording from the nucleus submedius with a monopolar tungsten electrode and the activity of rectus muscle was assessed via silver wire electrodes. A glass pipette was inserted into Barrington's nucleus for injection of 5 mM CoCl2, a temporary neural blocker. The site of CoCl2 injection was confirmed by the presence of FluoroGold which was incorporated into the CoCl2 solution. We recorded 51 units in submedius that were excited by noxious toe pinch, 4 were inhibited. Colon distension to 80 mmHg produced visceromotor responses, excited 23 units in submedius and inhibited 13 units. Injection of CoCl2 into the region of Barrington's nucleus blocked the response to colon distension in 10 of 12 Sm units tested, but had no influence on the accompanying visceromotor response. These data point to a previously unrecognized relationship between Barrington's nucleus and submedius that may subserve colon nociception.     

Clusterin is highly expressed in pancreatic endocrine tumours but not in solid pseudopapillary tumours

AIMS: Clusterin is a sulphated glycoprotein, implicated in many processes, including tumorigenesis. Several studies have reported its overexpression in many human neoplasms, including prostatic and pancreatic adenocarcinoma, but its expression has not been described previously in other pancreatic tumours. Our aim was to investigate the expression of clusterin by immunohistochemistry in 30 endocrine pancreatic tumours (ENTs) and 22 solid pseudopapillary tumours (SPPTs) to document its potential in differential diagnosis, and the possible correlation between this expression and clinicopathological parameters. METHODS AND RESULTS: Cytoplasmic positivity was scored qualitatively (weak, moderate or strong immunoreactivity) and quantitatively on a four-tiered scale. The pattern of immunoreactivity (cytoplasmic, secretory or Golgi pattern) was also assessed. Except for scattered tumour cells in five cases, all SPPTs were negative, while all ENTs showed strong immunoreactivity in a variable proportion of tumour cells. Neither the reactivity score nor the pattern of immunoreactivity was correlated with tumour size, vascular permeation, perineural invasion or lymph node metastasis. DISCUSSION: The expression of clusterin in all ENTs is of interest and could be an additional useful marker in the differential diagnosis with SPPTs. However, the lack of correlation between clusterin expression and clinicopathological parameters rules out a role as a predictive marker for endocrine tumour aggressiveness.     

Isolation-induced vocalization in Wistar rat pups is not increased by naltrexone.

Rat pups, when removed from dam and littermates and isolated in an unfamiliar milieu, emit a characteristic ultrasonic vocalization that can be quieted by the administration of a nonsedating dose of morphine. Wistar pups, aged 7, 10, 12, 14, or 16 days, were tested after receiving intraperitoneal injections of the opiate antagonist naltrexone (0.0, 0.5, 1.0, or 5.0 mg/kg). The rate of isolation-induced ultrasonic vocalizations was unaffected by naltrexone at any dose, and there were no significant naltrexone-related changes on other behavioral measures. The complexity of the opioid system is discussed, as it may be involved in the vocal reaction to isolation.     

Luminol-independent chemiluminescence by phagocytes is markedly enhanced by dexamethasone,not by other glucocorticosteroids

The effect of several glucocorticosteroids on the generation of reactive oxygen species (ROS) was examined. The ROS assessed were O ₂ ^– , H₂O₂, OH·, and chemiluminescence (CL) (determined in the presence or absence of luminol), generated by both opsonized zymosan-stimulated neutrophils or monocytes and by the xanthine-xanthine oxidase system. Except for luminol-independent CL, only high concentrations (10^–4 M) of steroids could decrease each ROS. In contrast, luminol-independent CL generation in the phagocyte system was increased in a dose-dependent manner by the addition of dexamethasone, but not by any other steroid. Further, in lymphocyte cultures stimulated with Con A for four days, luminol-independent CL generation was demonstrated and enhanced by the addition of dexamethasone, although CL generation was not detected in the absence of dexamethasone. These findings provide evidence that CL does not always represent light specific to ROS, and they suggest the possibility that dexamethasone induces emission of light at sites of inflammation.