Attenuation of experimental arthritis in TRPV1R knockout mice.

The Transient Receptor Potential Vanilloid 1 (TRPV1R) is a ligand-gated, non-selective cation channel expressed predominantly by sensory neurons. TRPV1Rs respond to a variety of noxious stimuli including capsaicin, intense heat and acid. These factors, combined with behavioral studies, show that TRPV1Rs are involved in nociception. The aim of our study was to determine whether TRPV1Rs play a role in the development and maintenance of inflammation and mechanical hyperalgesia by studying the development of unilateral joint inflammation in TRPV1R-/- mice. Knee joints of TRPV1R-/- or wild-type (WT) mice were injected with FCA (200 microg) under temporary anesthesia, and the resulting inflammation and hyperalgesia measured for 35 days. Histological analysis was performed on joints at the end of the study. TRPV1R-/- mice developed mild joint swelling which was significantly less than that obtained in WT mice (P < 0.05, Mann-Whitney). The ratio of the weight distribution between the hind limbs in TRPV1R-/- mice was also significantly less than in WT mice (P < 0.05, Mann-Whitney). Neither swelling nor hypersensitivity was completely absent in the knockout mice, indicating either that other mechanisms are involved or that a compensatory mechanism operates in TRPV1R-/- mice. These results suggest that TRPV1 receptors are important for the development of joint inflammation and the associated mechanical hypersensitivity observed in this model.

Neurosteroid hydroxylase CYP7B: vivid reporter activity in dentate gyrus of gene-targeted mice and abolition of a widespread pathway of steroid and oxysterol hydroxylation.

The major adrenal steroid dehydroepiandrosterone (DHEA) enhances memory and immune function but has no known dedicated receptor; local metabolism may govern its activity. We described a cytochrome P450 expressed in brain and other tissues, CYP7B, that catalyzes the 7alpha-hydroxylation of oxysterols and 3beta-hydroxysteroids including DHEA. We report here that CYP7B mRNA and 7alpha-hydroxylation activity are widespread in rat tissues. However, steroids related to DHEA are reported to be modified at positions other than 7alpha, exemplified by prominent 6alpha-hydroxylation of 5alpha-androstane-3beta,17beta-diol (A/anediol) in some rodent tissues including brain. To determine whether CYP7B is responsible for these and other activities we disrupted the mouse Cyp7b gene by targeted insertion of an IRES-lacZ reporter cassette, placing reporter enzyme activity (beta-galactosidase) under Cyp7b promoter control. In heterozygous mouse brain, chromogenic detection of reporter activity was strikingly restricted to the dentate gyrus. Staining did not exactly reproduce the in situ hybridization expression pattern; post-transcriptional control is inferred. Lower level staining was detected in cerebellum, liver, and kidney, and which largely paralleled mRNA distribution. Liver and kidney expression was sexually dimorphic. Mice homozygous for the insertion are viable and superficially normal, but ex vivo metabolism of DHEA to 7alpha-hydroxy-DHEA was abolished in brain, spleen, thymus, heart, lung, prostate, uterus, and mammary gland; lower abundance metabolites were also eliminated. 7alpha-Hydroxylation of 25-hydroxycholesterol and related substrates was also abolished, as was presumed 6alpha-hydroxylation of A/anediol. These different enzyme activities therefore derive from the Cyp7b gene. CYP7B is thus a major extrahepatic steroid and oxysterol hydroxylase and provides the predominant route for local metabolism of DHEA and related molecules in brain and other tissues.

Anandamide activates peripheral nociceptors in normal and arthritic rat knee joints.

The effects of the endogenous cannabinoid anandamide were studied on peripheral, polymodal nociceptors recorded from normal and chronically inflamed (Freund's adjuvant) knee joint afferents in rats anaesthetized with pentobarbitone. Anandamide (860 nmol) caused a rapid, short lasting excitation of a sub-population of capsaicin-sensitive nociceptive afferents in normal knee joints (7.2+/-2.3 impulses s(-1); n=15 units from five animals). In arthritic joints there were 9.7+/-3.0 impulses s(-1) (n=11 from six animals), which was not significantly different from normal joints. The excitation was dose dependent (8.6 - 2900 nmol) and mediated by activation of the vanilloid receptor (VR(1)) as it was abolished by the VR1 antagonist capsazepine (1 mg kg(-1)). Our results show that anandamide, at high doses, can activate nociceptive afferents innervating the rat knee joints, in contrast with its widely described analgesic actions.

Immunoglobulin E suppression and cytokine modulation in mice orally tolerized to beta-lactoglobulin.

This study was designed to confirm the tolerogenic properties of beta-lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed beta-lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non-specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP-fed, versus saline-fed mice, by measuring specific seric and intestinal antibody responses, delayed-type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three-week-old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti-BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP-fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) versus an increase of IL-10 and transforming growth factor-beta (TGF-beta) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows' milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.

Detection of the M(r) 190,000 multidrug resistance protein, MRP, with monoclonal antibodies.

MRP is a M(r) 190,000 integral membrane phosphoglycoprotein that is overexpressed in some drug-selected resistant cell lines and has been shown to cause multidrug resistance in transfected cells. Five murine hybridoma cell lines (QCRL-1, QCRL-2, QCRL-3, QCRL-4, and QCRL-6) have been generated which secrete monoclonal antibodies (MAbs) that react specifically with membrane proteins of MRP-overexpressing, multidrug-resistant, drug-selected H69AR cells and MRP-transfected HeLa cells (T5) but not the respective parental (H69) and vector-transfected (C1) cells. The ability of three of these MAbs (QCRL-1, QCRL-2, and QCRL-3) to selectively immunoprecipitate a M(r) 190,000 protein from 35S-labeled H69AR and T5 membranes indicates that these MAbs are specific for MRP. MAb QCRL-1 is also capable of detecting the low levels of MRP present in revertant H69PR cells by immunoblot analysis. Indirect immunofluorescence analyses show that MAbs QCRL-1, QCRL-2, and QCRL-3) strongly and differentially react with fixed T5 and H69AR cells but not with unfixed cells, suggesting that these MAbs recognize intracellular MRP epitopes. The availability of reagents for the specific and sensitive immunodetection of MRP should greatly facilitate biological and clinical studies of this novel drug resistance protein.