Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Differential control of two forms of glutamate release by group III metabotropic glutamate receptors at rat entorhinal synapses

Neurotransmitter release at CNS synapses occurs via both action potential-dependent and independent mechanisms, and it has generally been accepted that these two forms of release are regulated in parallel. We examined the effects of activation of group III metabotropic glutamate receptors (mGluRs) on stimulus-evoked and spontaneous glutamate release onto entorhinal cortical neurones in rats, and found a differential regulation of action potential-dependent and independent forms of release. Activation of presynaptic mGluRs depressed the amplitude of stimulus-evoked excitatory postsynaptic currents, but concurrently enhanced the frequency of spontaneous excitatory currents. Moreover, these differential effects on glutamate release were mediated by pharmacologically separable mechanisms. Application of the specific activator of adenylyl cyclase, forskolin, mimicked the effect of mGluR activation on spontaneous, but not evoked release, and inhibition of adenylyl cyclase with 9-tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536) blocked mGluR-mediated enhancement of spontaneous release, but not depression of evoked release. Occlusion studies with calcium channel blockers suggested that the group III mGluRs might depress evoked release through inhibition of both N and P/Q, but not R-type calcium channels. We suggest that the concurrent depression of action potential-evoked, and enhancement of action potential-independent glutamate release operate through discrete second messenger/effector systems at excitatory entorhinal terminals in rat brain. © 2007 IBRO.

Depression of glutamate and GABA release by presynaptic GABAB receptors in the entorhinal cortex in normal and chronically epileptic rats

Presynaptic GABAB receptors (GABABR) control glutamate and GABA release at many synapses in the nervous system. In the present study we used whole-cell patch-clamp recordings of spontaneous excitatory and inhibitory synaptic currents in the presence of TTX to monitor glutamate and GABA release from synapses in layer II and V of the rat entorhinal cortex (EC)in vitro. In both layers the release of both transmitters was reduced by application of GABABR agonists. Quantitatively, the depression of GABA release in layer II and layer V, and of glutamate release in layer V was similar, but glutamate release in layer II was depressed to a greater extent. The data suggest that the same GABABR may be present on both GABA and glutamate terminals in the EC, but that the heteroreceptor may show a greater level of expression in layer II. Studies with GABABR antagonists suggested that neither the auto- nor the heteroreceptor was consistently tonically activated by ambient GABA in the presence of TTX. Studies in EC slices from rats made chronically epileptic using a pilocarpine model of temporal lobe epilepsy revealed a reduced effectiveness of both auto- and heteroreceptor function in both layers. This could suggest that enhanced glutamate and GABA release in the EC may be associated with the development of the epileptic condition. Copyright © 2006 S. Karger AG.

Codon-precise, synthetic, antibody fragment libraries built using automated hexamer codon additions and validated through next generation sequencing

We have previously described ProxiMAX, a technology that enables the fabrication of precise, combinatorial gene libraries via codon-by-codon saturation mutagenesis. ProxiMAX was originally performed using manual, enzymatic transfer of codons via blunt-end ligation. Here we present Colibra™: an automated, proprietary version of ProxiMAX used specifically for antibody library generation, in which double-codon hexamers are transferred during the saturation cycling process. The reduction in process complexity, resulting library quality and an unprecedented saturation of up to 24 contiguous codons are described. Utility of the method is demonstrated via fabrication of complementarity determining regions (CDR) in antibody fragment libraries and next generation sequencing (NGS) analysis of their quality and diversity.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm – with or without anchoring them in the plasma membrane –, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised neither by adding a C-terminal stabilisation signal nor by anchoring the protein at the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Effects of Endothelin 3 on the Tumor-Angiogenic Response in a Novel Melanoma Mouse Model

Skin cancer is the most common form of cancer in the United States. Melanoma is a particular type of skin cancer, which arises from the malignant transformation of melanocytes and generally exhibits a high propensity to metastasize. Melanoma progression is dependent on angiogenesis to deliver the oxygen and nutrients required to maintain the altered metabolism of rapidly proliferating tumorigenic cells. Recent studies have implicated the growth factor Endothelin 3 (Edn3) in melanoma progression and metastasis. The aim of this study was to examine the role that Edn3 plays in the angiogenesis of melanocytic lesions. For this purpose, Dct-Grm1 transgenic mice, which spontaneously acquire melanocytic lesions through the aberrant expression of the metabotropic glutamate receptor 1 (mGluR1), were crossed with K5-Edn3 transgenic mice that overexpress Edn3. Tumors in the Dct-Grm1/K5-Edn3 experimental population were examined and compared to the control Dct-Grm1 population using immuno-fluorescent staining targeted against the vascular endothelial cell marker CD31. Proteomic arrays were also used and identified changes in the expression of specific angiogenic factors. CD31 antibody staining results revealed an increased vascular density in Dct-Grm1/K5-Edn3 tumors compared with tumors from the Dct-Grm1 controls. Analysis of the relative expression of angiogenic proteins showed an upregulation of various vascular factors in tumors from the Dct-Grm1/K5-Edn3 population, including VEGF-B, MMP-8, MMP-9, and Angiogenin. These results suggest that endothelin signaling promotes angiogenesis in melanocytic lesions. Targeting the factors upregulated by Edn3 signaling may prove effective in hindering melanoma progression.

The palatability of monosodium glutamate-enhanced vegetables as rated by Cuban-American older adults at a congregate meal site

The purpose of this study was to investigate the efficacy of using monosodium glutamate (MSG) as a means to increase palatability and prospective consumption of vegetables at a congregate meal site with Cuban-American clients. Thirty to 32 subjects participated in hedonic testing each day. MSG-enhanced (2 g MSG/500 g vegetable) and non-enhanced beets, string beans, carrots and peas were evaluated for palatability, preference and prospective consumption. Results showed that MSG significantly increased both palatability and prospective consumption of string beans (ps < .05) but not of the other 3 vegetables tested. These findings provide some evidence that MSG can be used to increase the palatability of vegetables served at congregate meal sites. However, these results suggest that older adults may not find the palatability of some vegetables to be improved by MSG and that optimal flavor enhancement cannot be achieved by adding the same amount of MSG to every vegetable.

Effets du cuivre (II) sur le transport à haute affinité du glutamate dans des astrocytes en culture : proposition d'un mécanisme physiopathologique de la maladie de Wilson

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Rôle du glutamate dans l'effet de l'amphétamine sur la récompense et l'activité locomotrice chez le rongeur

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Les mécanismes excitotoxiques et le rôle de transporteurs de glutamate dans la physiopathologie des traumatismes crâniens = Excitotoxic mechanisms and the role of glutamate transporters in the pathophysiology of traumatic brain injury

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Contribution relative des sous-types 1 et 5 des récepteurs métabotropes du glutamate dans la potentialisation à long terme des interneurones hippocampiques chez la souris

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Implication du glutamate 346 de NHE1 dans le transport du Na⁺ et l'interaction avec les inhibiteurs

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

A Therapeutic Antibody for Cancer, Derived from Single Human B Cells.

Some patients with cancer never develop metastasis, and their host response might provide cues for innovative treatment strategies. We previously reported an association between autoantibodies against complement factor H (CFH) and early-stage lung cancer. CFH prevents complement-mediated cytotoxicity (CDC) by inhibiting formation of cell-lytic membrane attack complexes on self-surfaces. In an effort to translate these findings into a biologic therapy for cancer, we isolated and expressed DNA sequences encoding high-affinity human CFH antibodies directly from single, sorted B cells obtained from patients with the antibody. The co-crystal structure of a CFH antibody-target complex shows a conformational change in the target relative to the native structure. This recombinant CFH antibody causes complement activation and release of anaphylatoxins, promotes CDC of tumor cell lines, and inhibits tumor growth in vivo. The isolation of anti-tumor antibodies derived from single human B cells represents an alternative paradigm in antibody drug discovery.

Maternal HIV-1 envelope-specific antibody responses and reduced risk of perinatal transmission.

Despite the wide availability of antiretroviral drugs, more than 250,000 infants are vertically infected with HIV-1 annually, emphasizing the need for additional interventions to eliminate pediatric HIV-1 infections. Here, we aimed to define humoral immune correlates of risk of mother-to-child transmission (MTCT) of HIV-1, including responses associated with protection in the RV144 vaccine trial. Eighty-three untreated, HIV-1-transmitting mothers and 165 propensity score-matched nontransmitting mothers were selected from the Women and Infants Transmission Study (WITS) of US nonbreastfeeding, HIV-1-infected mothers. In a multivariable logistic regression model, the magnitude of the maternal IgG responses specific for the third variable loop (V3) of the HIV-1 envelope was predictive of a reduced risk of MTCT. Neutralizing Ab responses against easy-to-neutralize (tier 1) HIV-1 strains also predicted a reduced risk of peripartum transmission in secondary analyses. Moreover, recombinant maternal V3-specific IgG mAbs mediated neutralization of autologous HIV-1 isolates. Thus, common V3-specific Ab responses in maternal plasma predicted a reduced risk of MTCT and mediated autologous virus neutralization, suggesting that boosting these maternal Ab responses may further reduce HIV-1 MTCT.