Peanut allergens are rapidly transferred in human breast milk and can prevent sensitization in mice.

BACKGROUND: Food allergens have been evidenced in breast milk under physiological conditions, but the kinetic and the role of this passage in food allergies are still unclear. We then aimed to analyze the passage of peanut allergens in human breast milk and their allergenicity/immunomodulatory properties. METHODS: Human breast milk was collected from two non-atopic peanut-tolerant mothers before and at different time points after ingestion of 30 g of commercial roasted peanut. Ara h 6, Ara h 6 immune complexes, and the IgE binding capacity of breast milk samples were measured using specific immunoassays. Their allergenic functionality was then assessed using cell-based assay. Finally, human breast milk obtained before or after peanut ingestion was administered intragastrically to BALB/c mice at different ages, and mice were further experimentally sensitized to peanut using cholera toxin. RESULTS: Ara h 6 is detected as soon as 10 min after peanut ingestion, with peak values observed within the first hour after ingestion. The transfer is long-lasting, small quantities of peanut allergens being detected over a 24-h period. IgG-Ara h 6 and IgA-Ara h 6 immune complexes are evidenced, following a different kinetic of excretion than free allergens. Peanut allergens transferred in milk are IgE reactive and can induce an allergic reaction in vitro. However, administration of human breast milk to young mice, notably before weaning, does not lead to sensitization, but instead to partial oral tolerance. CONCLUSION: The low quantities of immunologically active allergens transferred through breast milk may prevent instead of priming allergic sensitization to peanut.

Peanut- and cow's milk-specific IgE, Th2 cells and local anaphylactic reaction are induced in Balb/c mice orally sensitized with cholera toxin.

BACKGROUND: The development of animal models developing specific immunoglobulin (Ig)E presenting the same specificity as human IgE and similar clinical symptoms as those observed in allergic patients are of great interest for the understanding of mechanisms involved in the induction and regulation of food allergy. METHODS: Balb/c female mice were sensitized with whole peanut protein extract (WPPE) by means of intraperitoneal (i.p.) injections with alum or gavages with cholera toxin (CT). The WPPE specific IgE, IgG1 and IgG2a were monitored. Th2 cells activation was analysed assaying interleukin (IL)-4 and IL-5 vs IFNgamma on reactivated splenocytes. Local anaphylactic reaction was evaluated by assaying histamine in faecal samples. The oral sensitization protocol was further extended to cow's milk proteins (CMP). RESULTS: Balb/c mice developed high peanut-specific IgE and IgG1 responses either after i.p. or oral sensitizations. In both cases, antibodies were specific to polymer of glycinin fragments, containing polypeptides from Ara h3/4, and to a lesser extent to Ara h1 and Ara h2. Interleukin-4 and IL-5 production were evidenced. Balb/c mice could also be sensitized to CMP, as demonstrated by CMP-specific IL-4 and IL-5 secretions and induction of IgE specific for whole caseins, beta-lactoglobulin, serum bovine albumin and lactoferrin. Of interest was the occurrence of a local anaphylactic reaction in the peanut and CM models. CONCLUSIONS: In contrast with previous authors, Balb/c mice were sensitized and evidenced an allergic reaction after oral administrations of peanut or CMP plus CT, providing an interesting model for further studies on immunopathogenic mechanisms.

Oral administration of recombinant Lactococcus lactis expressing bovine beta-lactoglobulin partially prevents mice from sensitization.

BACKGROUND: The use of probiotics such as Lactococcus lactis and other lactic acid bacteria (LAB) has been proposed for the management of food allergy. However, no experimental study has clearly demonstrated any preventive or therapeutic inhibition of an allergen-specific IgE response. OBJECTIVE: We aimed to study the immunomodulatory effect of recombinant L. lactis expressing bovine beta-lactoglobulin (BLG), a major cow's milk allergen, in a validated mouse model of allergy. METHODS: Six-week-old female Balb/c mice received five repeated doses of BLG, of L. lactis plus BLG, or of recombinant L. lactis by gavage. Different recombinant strains were inoculated, which corresponded to BLG doses ranging from 4 to 70 microg/mice. Mice were then sensitized by intra-peritoneal injection of BLG emulsified in incomplete Freund's adjuvant to induce high IgE concentrations. RESULTS: Pre-treatment with natural L. lactis plus BLG allowed induction of BLG-specific T-helper type 1 (Th1) response, and abrogated the oral tolerance induced by BLG alone, demonstrating the adjuvant effect of this non-colonizing LAB. Moreover, pre-treatment with some of the recombinant strains favoured the development of a Th1 response inhibiting the Th2 one: it induced a significant decrease of specific IgE response, and an intense increase of specific IgG2a and IFN-gamma productions. The most efficient strains that inhibited the IgE response were those producing the highest amounts of the BLG protein. CONCLUSION: Oral administration of some recombinant L. lactis was demonstrated to induce a specific Th1 response down-regulating a further Th2 one. Prophylaxis protocols will thus be evaluated using the most efficient strains.

A general method allowing the design of oligonucleotide primers to amplify the variable regions from immunoglobulin cDNA.

The amplification of variable regions of immunoglobulins by reverse transcription polymerase chain reaction (RT-PCR) has become an invaluable technique either for the cloning of monoclonal antibodies (mAbs), or for the building of single-chain fragment variable (ScFv) libraries. Numerous applications have been described either for studying the antigen-antibody interactions or for medical purposes, with the recent development of recombinant antibodies for therapeutic use. Several publications by different groups have reported primer sequences to perform such amplification, but the strategy used to design these primers, and particularly the way of performing the necessary alignments, generally appear poorly detailed. In the present work, we propose a rational method of designing primers in order to amplify the variable region of heavy chain (VH) and variable region of light chain (VL) domains for framework 1 (FR1) of immunoglobulins. The described sets of primers have been designed to hybridize with the entire VH and VL mouse repertory without modification of amino acids since amino acids of framework 1 play a role in the folding, and thus in the functionality, of recombinant antibody. These primers have been applied to the cloning of monoclonal antibodies previously produced in the laboratory. This approach can be extended to other species or members of the immunoglobulin superfamily.

Depletion of KIN17, a human DNA replication protein, increases the radiosensitivity of RKO cells.

The human KIN17 protein is a chromatin-associated protein involved in DNA replication. Certain tumor cell lines overproduce KIN17 protein. Among 16 cell lines, the highest KIN17 protein level was observed in H1299 non-small cell lung cancer cells, whereas the lowest was detected in MeWo melanoma cells. Cells displaying higher KIN17 protein levels exhibited elevated RPA70 protein contents. High KIN17 protein levels may be a consequence of the tumorigenic phenotype or a prerequisite for tumor progression. Twenty-four hours after exposure to ionizing radiation, after the completion of DNA repair, a co-induction of chromatin-bound KIN17 and RPA70 proteins was detected. Etoposide, an inhibitor of topoisomerase II generating double-strand breaks, triggered the concentration of KIN17 into punctuate intranuclear foci. KIN17 may be associated with unrepaired DNA sites. Flow cytometry analysis revealed that 48 h after transfection the uppermost KIN17-positive RKO cells shifted in the cell cycle toward higher DNA content, suggesting that KIN17 protein induced defects in chromatin conformation. Cells displaying reduced levels of KIN17 transcript exhibited a sixfold increased radiosensitivity at 2 Gy. The KIN17 protein may be a component of the DNA replication machinery that participates in the cellular response to unrepaired DSBs, and an impaired KIN17 pathway leads to an increased sensitivity to ionizing radiation.

Elicitation of the allergic reaction in beta-lactoglobulin-sensitized Balb/c mice: biochemical and clinical manifestations differ according to the structure of the allergen used for challenge.

BACKGROUND: Mouse models of allergy are used to study the mechanisms of induction and perpetuation of bronchopulmonary hyper-reactivity (BHR) as related to eosinophils and specific IgE. OBJECTIVE: Our aim was to adapt the current model for the study of bovine beta-lactoglobulin (BLG), a major cow's milk allergen, and to further analyse the mechanisms of the acute and late allergic reaction. METHODS: Female Balb/c mice were sensitized intraperitoneally with BLG and the influence of the adjuvant and of the BLG dose on the IgE response was analysed, IgE and IgG1 epitopes being characterized. Once optimized, this model was applied to the study of the active phase of allergy in the respiratory tract after a single airway challenge using native or denatured BLG, which contains only linear epitopes. RESULTS: An immediate allergic reaction was characterized by the rapid release of histamine into the bronchoalveolar lavage fluids. Prostaglandin (PG)D2 was only present when the standard histamine-releasing agent compound 48/80 or denatured BLG were used as triggers, whereas native BLG induced leukotriene release. Twenty-four hours after challenge, BHR, eosinophil influx, IL-4 and IL-5 production, plasma exudation and mucus production were very much increased, differently depending on the allergen structure, and indicated the occurrence of the late allergic reaction. Our results show that the murine model can be used to study the mechanisms of allergy to clinically relevant antigens, such as those contained in cow's milk. The acute allergic reaction, which depends on the structural feature of the allergen, is composed of two distinct pathways characterized by peptido-leukotrienes or PGD2 production, which may result from distinct activation intensities of mast cells, leading to distinct late reactions. CONCLUSION: This study thus demonstrates a clear link between the structural feature of a protein, and the physiopathology of the experimental asthmatic reaction.

Monoaminergic control of vasopressin and VIP expression in the mouse suprachiasmatic nucleus.

We studied the effects of serotonin and noradrenaline on the expression of arginine-vasopressin (AVP) and vasoactive intestinal peptide (VIP) in the suprachiasmatic nucleus (SCN). We used transgenic Tg8 mice knockout for the MAO-A (monoamine oxidase A) gene, which are characterized by increased amounts of serotonin and noradrenaline in brain compared to wild-type mice (C3H). The MAO-A deficiency caused an increase in AVP and VIP expression (determined by immunohistochemistry, enzyme immunoassay, and in situ hybridization) compared to C3H mice. The number of peptidergic neurons was also increased. Inhibiting serotonin or noradrenaline synthesis in Tg8 mice by the administration of parachlorophenylalanine or alpha-methylparatyrosine, respectively, the amounts of AVP, VIP and their mRNAs were decreased, but not the number of peptidergic neurons. This study indicates that serotonin and noradrenaline stimulate AVP and VIP expression, and could participate in the differentiation of the neurochemical phenotype in the mouse SCN.

Prion-like protein Doppel expression is not modified in scrapie-infected cells and in the brains of patients with Creutzfeldt-Jakob disease.

Doppel protein has been discovered in prnp knock-out mouse lines, with overproduction of this protein in the brain causing ataxia and neurodegeneration. We investigated whether Doppel expression (i) affected or was affected by the course of prion propagation in neuroblastoma cells, or (ii) modulated Creutzfeldt-Jakob disease pathogenesis. No change in Doppel production was detected in N2a cells, before or after infection. Transient murine Doppel gene expression had no effect on N2a viability or PrP(Sc) production. A sensitive immunometric assay revealed low levels of Doppel in human brain, reflecting weak transcription of the corresponding gene. No difference in brain Doppel levels was observed between Creutzfeldt-Jakob disease patients and controls, adding further evidence that Doppel is unlikely to be involved in prion disease pathogenesis.

Characterization of human NOV in biological fluids: an enzyme immunoassay for the quantification of human NOV in sera from patients with diseases of the adrenal gland and of the nervous system.

Immunochromatography has shown that human NOV (NOVH), a member of the CCN (CTGF/CYR61/NOV) family, forms a physiological complex with fibulin-1 in blood. We developed an enzyme immunoassay specific for NOVH and showed for the first time that the concentration of NOVH differs in each of these biological fluids. The normal concentration of NOVH circulating in the blood is 350-400 ng/ml, but this concentration varies with age. By using sera from patients with adrenal gland diseases we found that in vivo ACTH or glucocorticoids are not responsible for the high concentration of NOVH in this endocrine gland. However, the NOVH concentration was significantly modified in malignant adrenocortical tumors, but not in benign adrenocortical tumors. The concentration of NOVH was significantly decreased in patients suffering from astrocytomas or multiple sclerosis, two diseases of the nervous system. Thus, NOVH is a potentially useful marker for the diagnosis of these diseases.

Expression of excitatory amino acid transporter-2 (EAAT-2) and glutamine synthetase (GS) in brain macrophages and microglia of SIVmac251-infected macaques.

Na+-dependent transporters for glutamate (excitatory amino acid transporters, EAATs) clear extracellular glutamate in the brain and prevent excitotoxic neuronal damage. Glutamine synthetase (GS) provides metabolic support for neurones by producing the neurotrophic amino acid glutamine. EAAT and GS expression has recently been demonstrated in macrophages and microglial cells in vitro, and in two models of acute inflammation in vivo. This observation might modify our current understanding of brain inflammation, which considers activated microglia and brain macrophages as the main neurotoxic cells through their production of a variety of neurotoxins, including glutamate. EAAT and GS expression by these cells would entail neuroprotective and neurotrophic properties, counterbalancing the deleterious consequences of microglial activation. Macaque infection by the simian immunodeficiency virus (SIV) is considered the most relevant model for human acquired immunodeficiency syndrome (AIDS), including chronic inflammation of the brain at the early asymptomatic stage of the infection, followed by an AIDS-like disease where neuronal death occurs. We studied the expression of EAAT-2 and GS in the brains of three SIVmac251-infected and two noninfected cynomolgus macaques. We found that both microglia and brain macrophages expressed EAAT-2 and GS in infected primates, suggesting that these cells might, like astrocytes, clear extracellular glutamate and provide glutamine to neurones. Microglia and macrophages could thus have neuroprotective and neurotrophic properties in addition to their production of neurotoxins. This finding might explain the contrast between early intense microglial activation and the late occurrence of neuronal apoptotic cell death, which is mainly observed at the terminal stage of the disease.

Alteration of C-MYB DNA binding to cognate responsive elements in HL-60 variant cells.

AIMS: To establish whether the MYB protein expressed in HL-60 variant cells, which are cells resistant to 12-O-tetradecanoylphorbol-13-acetate (TPA) induced differentiation, is able to bind MYB recognition elements (MREs) involved in the transcriptional regulation of myb target genes. In addition, to determine whether alterations in the binding of the MYB protein to MREs affects HL-60 cell proliferation and differentiation. METHODS: Nuclear extracts of HL-60 variant cells exhibiting different degrees of resistance to TPA induced monocytic differentiation were used in electrophoretic mobility shift experiments (EMSAs), bandshift experiments performed with labelled oliogonucleotides containing the MYB consensus binding sequences. RESULTS: The MYB protein contained in nuclear extracts from HL-60 variant cells did not bind efficiently to the MYB recognition elements identified in the mim-1 and PR264 promoters. Molecular cloning of the myb gene and analysis of the MYB protein expressed in the HL-60 variant cells established that the lack of binding did not result from a structural alteration of MYB in these cells. The lack of MRE binding did not abrogate the ability of variant HL-60s to proliferate and to undergo differentiation. Furthermore, the expression of the PR264/SC35 splicing factor was not affected as a result of the altered MYB DNA binding activity. CONCLUSIONS: Because the MYB protein expressed in HL-60 variant cells did not appear to be structurally different from the MYB protein expressed in parental HL-60 cells, it is possible that the HL-60 variant cells contain a MYB binding inhibitory factor (MBIF) that interferes with MYB binding on MREs. The increased proliferation rate of HL-60 variant cells and their reduced serum requirement argues against the need for direct MYB binding in the regulation of cell growth.

Rapid test for the preclinical postmortem diagnosis of BSE in central nervous system tissue.

The efficacy of a rapid test for detecting PrP(Sc) in central nervous system tissue was evaluated for the postmortem diagnosis of BSE at different times during the course of the disease. One hundred and six samples of brain, at the level of the medulla oblongata, and spinal cord, derived from the experimental study of the pathogenesis of BSE carried out in Great Britain between 1991 and 1995, were examined. PrP(Sc) was detected in the samples from most of the exposed animals killed 32 months or more after they had been exposed to the agent, and before the onset of clinical signs which were first recorded at 35 months. Comparisons with the results of histology, fibril detection, PrP immunohistochemistry and mouse bioassay indicated that the rapid test is at least as sensitive as these conventional confirmatory diagnostic methods and its result can be obtained more quickly.

Genetic immunisation with bovine beta-lactoglobulin cDNA induces a preventive and persistent inhibition of specific anti-BLG IgE response in mice.

BACKGROUND: Various studies have shown that DNA immunisation with gene allergen induces a non-allergic response. METHODS: We applied this new type of vaccination to bovine beta-lactoglobulin (BLG), a major cow's milk allergen, using a plasmid that allows the production of a partially secreted protein. Specific antibodies and cytokines were quantified in different immunisation protocols. RESULTS: The primary response in mice immunised with BLG-encoding plasmid (pBLG) is of the Th1 type. Restricted recognition of a native form of BLG in pBLG mice contrasted with a broader range of recognition in BLG-in-alum-immunised mice, notwithstanding the fact that alum favours the presentation of a native form of the antigen. We also demonstrated an inhibitory effect of pDNA immunisation on the Th2 response induced by a subsequent immunisation using BLG adsorbed on alum. However, this preventive effect is highly dependent on the time of pre-administration of the pBLG, with an optimal effect when pDNA immunisation occurred at least 21 days before protein administration. This preventive effect resulted concomitantly in the inhibition of BLG-specific IgE, in the induction of specific IgG2a, and in the decrease of the specific IgG1/IgG2 ratio. It is accompanied by an increase in IFNgamma and IL-10 secretion. Moreover, the preventive effect was shown to be persistent even after a booster immunisation with alum-adsorbed BLG. The Th1 orientation of the response is very likely due to the presentation of the protein in the Th1 environment due to plasmid immunostimulatory sequences, as intramuscular injection of BLG itself leads to a weak Th2 response and had no preventive effect on a subsequent sensitisation. CONCLUSION: This study further demonstrates the potential use of DNA immunisation for prevention of IgE response, but the window of action seems to be very restricted if we are to inhibit an established Th2 response efficiently.

Modulation of COX-2 expression by statins in human aortic smooth muscle cells. Involvement of geranylgeranylated proteins.

Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis. Recently, cholesterol-independent effects of statins have been described. In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells. Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production. Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect. GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2. Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction. These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Direct determination of phosphorylated intracellular anabolites of stavudine (d4T) by liquid chromatography/tandem mass spectrometry.

The objective was to develop and validate a routine assay for active intracellular anabolites of stavudine (d4T), a nucleoside reverse transcriptase inhibitor in human PBMC, applicable to pharmacokinetic studies and treatment monitoring. This was achieved using liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS), which theoretically allies optimum sensitivity, specificity and high sample throughput. After cellular lysis in a Tris/methanol buffer, the extract spiked with 2[H(8)]-ATP (internal standard) is directly injected into the LC/MS/MS system. Phosphorylated metabolites of d4T as well as deoxythymidine-triphosphate, the competitor on the reverse transcriptase, are separated from d4T on a reverse-phase microbore column with ion pairing. The detection is performed in the multiple reaction monitoring (MRM) mode after drug ionisation in negative mode electrospray. The limit of quantitation for d4T-TP was 138 fmol per 7 mL blood (9.8 fmol per 10(6) cells) and CV% for repeatability and intermediate precision were lower than 15%. Stability of compounds was checked before and during the process of isolation of PBMC. Cellular samples from several d4T-treated patients were successfully analysed using this method and d4T-triphosphate and deoxythymidine triphosphate were recovered. In conclusion, we have developed and validated a routine LC/MS/MS method that allows the simultaneous determination of mono-, di- and triphosphorylated anabolites of d4T in PBMC as well as the natural corresponding triphosphate in one analysis. For the first time, the chain terminator ratio (d4T-TP/dT-TP) could be directly measured. This method can be used simply and routinely on more than 35 samples per day. Extension to other nucleoside analogues is under development.

Induction of cyclo-oxygenase-2 in human endothelial cells by SIN-1 in the absence of prostaglandin production.

Nitric oxide (NO) regulates cyclo-oxygenase (COX) activity in various cell systems and reports conflict in regard to its stimulatory versus inhibitory role. Incubation of human umbilical vein endothelial cells (HUVEC) with SIN-1 (3-morpholinosydnonimine), a donor of NO, resulted in a rapid and dose-dependent increase in the expression of COX-2 as analysed by Western and Northern blotting. Incubation of HUVEC with SIN-1 and interleukine (IL)-1alpha resulted in increased induction of COX-2 compared with IL-1alpha alone and corresponded to an additive effect. The COX-2 induction was dependent on a de novo synthesis since cycloheximide, an inhibitor of protein synthesis, blocked the enzyme expression. The increase in COX-2 expression was not accompanied by a corresponding change in prostaglandin (PG) production. However, the COX activity was partially recovered when immunoprecipitated COX-2 was incubated with arachidonic acid and haematin. Peroxynitrite, a highly reactive nitrogen molecule derived from the interaction of NO and superoxide anion, significantly increased COX-2 expression. Under these conditions and within the limit of detection of the antibody, selective antibody for nitrotyrosine failed to detect nitrated COX-2 in immunoprecipitated COX-2 when cells where incubated with SIN-1 or SIN-1+IL-1alpha. Ro 31-8220, a specific inhibitor of protein kinase (PK) C, blocked the induction of COX-2. Also, SB203580, the selective inhibitor of p38 MAP kinase, strongly blocked the induction of COX-2 by SIN-1 in the presence or absence of IL-1alpha, whereas the MEK-1 inhibitor, PD 98059, affected it to a lesser extent. These data demonstrate that SIN-1 induces COX-2 in HUVEC in the absence of PG formation and suggest a complex regulation of COX-2 expression and PG formation by NO in endothelial cells.

The vanilloid receptor (VR1)-mediated effects of anandamide are potently enhanced by the cAMP-dependent protein kinase.

The endogenous cannabinoid receptor ligand, anandamide (AEA), is a full agonist of the vanilloid receptor type 1 (VR1) for capsaicin. Here, we demonstrate that the potency and efficacy of AEA at VR1 receptors can be significantly increased by the concomitant activation of protein kinase A (PKA). In human embryonic kidney (HEK) cells over-expressing human VR1, AEA induces a rise in cytosolic Ca(2+) concentration that is mediated by this receptor. The EC(50) for this effect was decreased five-fold in the presence of forskolin (FRSK, 1-5 microM) or the cAMP analogue, 8-Br-cAMP (10-100 microM). The effects of 8-Br-cAMP and FRSK were blocked by a selective PKA inhibitor. The FRSK (10 nM) also potently enhanced the sensory neurone- and VR1-mediated constriction by AEA of isolated guinea-pig bronchi, and this effect was abolished by a PKA inhibitor. In rat dorsal root ganglia slices, AEA-induced release of substance P, an effect mediated by VR1 activation, was enhanced three-fold by FRSK (10 nM). Thus, the ability of AEA to stimulate sensory VR1, with subsequent neuropeptide release, appears to be regulated by the state of activation of PKA. This observation supports the hypothesis that endogenous AEA might stimulate VR1 under certain pathophysiological conditions.

The production and evaluation of antibodies for enzyme immunoassay of AZTTP.

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.

Role of peripheral N-methyl-D-aspartate (NMDA) receptors in visceral nociception in rats.

BACKGROUND & AIMS: N-methyl-D-aspartate (NMDA) receptors are ligand-gated ion channels that have an important role in long-term potentiation and memory processing in the central nervous system. The aims in this study were to determine whether NMDA receptors are expressed in the peripheral nervous system and identify their role in mediating behavioral pain responses to colonic distention in the normal gut. METHODS AND RESULTS: Immunohistochemical localization of the NR1 subunit showed that NMDA receptors are expressed on the cell bodies and peripheral terminals of primary afferent nerves innervating the colon. Dorsal root ganglia neurons retrogradely labeled from the colon in short-term culture responded to addition of NMDA with increased intracellular [Ca2+]. Activation of peripheral NMDA receptors in colonic tissue sections caused Ca2+-dependent release of the proinflammatory neuropeptides, calcitonin gene-related peptide and substance P. Behavioral pain responses to noxious mechanical stimulation were inhibited in a reversible, dose-dependent manner by intravenous administration of memantine, a noncompetitive antagonist of the NMDA receptor. Single fiber recordings of decentralized pelvic nerves showed that colorectal distention responsive afferent nerve activity was inhibited by memantine. CONCLUSIONS: Peripheral NMDA receptors are important in normal visceral pain transmission, and may provide a novel mechanism for development of peripheral sensitization and visceral hyperalgesia.

Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment.

BACKGROUND: Nitric oxide (NO) and prostanoids are mediators of vascular and bronchial tone that are postulated to be involved in asthma. Increased levels of both are found in asthmatic subjects and are synthesised by enzymes that have cytokine inducible forms: inducible NO synthase (iNOS) and cyclo-oxygenase-2 (COX-2), respectively. We hypothesised that the in vivo expression of iNOS and COX-2 in the airways would be increased in asthma, and that these cytokine inducible enzymes may represent targets for regulation by corticosteroid treatment. METHODS: Bronchial biopsy specimens were obtained from three groups of subjects: atopic asthmatics treated with beta(2) agonists alone (n=7), atopic asthmatics additionally receiving regular treatment with corticosteroids (n=8), and non-asthmatic control subjects (n=10). Expression of iNOS and COX-2 mRNA and immunoreactive protein was studied using in situ hybridisation and quantitative immunohistochemistry. RESULTS: Immunoreactivity and the hybridisation signal for iNOS and COX-2 were mainly localised in the airway epithelium. The proportion of epithelium immunostained was significantly greater in the non-steroid treated asthmatic subjects (iNOS 8.6 (1.8)%; COX-2 26.3 (4.6)%) than either the steroid treated asthmatics (iNOS 3.4 (1.0)%, p=0.009; COX-2 13.0 (0.6)%, p=0.0015) or the non-asthmatic controls (iNOS 4.2 (0.9)%, p=0.018; COX-2 11.6 (0.6)%, p=0.0003). Similarly, the hybridisation signal was stronger in the non-steroid treated group of asthmatic subjects than in the other two groups. CONCLUSIONS: These findings highlight the potential role of the airway epithelium both as a contributor to the inflammatory process in asthma and as a target for inhaled corticosteroid treatment in this disease.