High amounts of glutamate are found in the brains of people with multiple sclerosis, an inflammatory disease marked by progressive demyelination. Glutamate might affect neuroinflammation via effects on immune cells. Knockout mice lacking metabotropic glutamate receptor-4 (mGluR4) were markedly vulnerable to experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis) and developed responses dominated by interleukin-17-producing T helper (T(H)17) cells. In dendritic cells (DCs) from those mice, defective mGluR4 signaling-which would normally decrease intracellular cAMP formation-biased T(H) cell commitment to the T(H)17 phenotype. In wild-type mice, mGluR4 was constitutively expressed in all peripheral DCs, and this expression increased after cell activation. Treatment of wild-type mice with a selective mGluR4 enhancer increased EAE resistance via regulatory T (T(reg)) cells. The high amounts of glutamate in neuroinflammation might reflect a counterregulatory mechanism that is protective in nature and might be harnessed therapeutically for restricting immunopathology in multiple sclerosis.

A greater ethical conscience, new global rules and a modified perception of ethical consciousness entail a more rigorous control on utilizations of vertebrates for in vivo studies. To cope with this new scenario, numerous alternatives to rodents have been proposed. Among these, the greater wax moth Galleria mellonella had a preponderant role, especially in the microbiological field, as demonstrated by the growing number of recent scientific publications. The reasons for its success must be sought in its peculiar characteristics such as the innate immune response mechanisms and the ability to grow at a temperature of 37°C. This review aims to describe the most relevant features of G. mellonella in microbiology, highlighting the most recent and relevant research on antibacterial strategies, novel drug tests and toxicological studies. Although solutions for some limitations are required, G. mellonella has all the necessary host features to be a consolidated in vivo model host.

Cinnabarinic acid is an endogenous metabolite of the kynurenine pathway that meets the structural requirements to interact with glutamate receptors. We found that cinnabarinic acid acts as a partial agonist of type 4 metabotropic glutamate (mGlu4) receptors, with no activity at other mGlu receptor subtypes. We also tested the activity of cinnabarinic acid on native mGlu4 receptors by examining 1) the inhibition of cAMP formation in cultured cerebellar granule cells; 2) protection against excitotoxic neuronal death in mixed cultures of cortical cells; and 3) protection against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice after local infusion into the external globus pallidus. In all these models, cinnabarinic acid behaved similarly to conventional mGlu4 receptor agonists, and, at least in cultured neurons, the action of low concentrations of cinnabarinic acid was largely attenuated by genetic deletion of mGlu4 receptors. However, high concentrations of cinnabarinic acid were still active in the absence of mGlu4 receptors, suggesting that the compound may have off-target effects. Mutagenesis and molecular modeling experiments showed that cinnabarinic acid acts as an orthosteric agonist interacting with residues of the glutamate binding pocket of mGlu4. Accordingly, cinnabarinic acid did not activate truncated mGlu4 receptors lacking the N-terminal Venus-flytrap domain, as opposed to the mGlu4 receptor enhancer, N-phenyl-7-(hydroxyimino) cyclopropa[b-]chromen-1a-carboxamide (PHCCC). Finally, we could detect endogenous cinnabarinic acid in brain tissue and peripheral organs by high-performance liquid chromatography-tandem mass spectrometry analysis. Levels increased substantially during inflammation induced by lipopolysaccharide. We conclude that cinnabarinic acid is a novel endogenous orthosteric agonist of mGlu4 receptors endowed with neuroprotective activity.

The chemokine RANTES is critically involved in neuroinflammation and has been implicated in the pathophysiology of multiple sclerosis. We examined the possibility that activation of G-protein-coupled metabotropic glutamate (mGlu) receptors regulates the formation of RANTES in glial cells. A 15 hr exposure of cultured astrocytes to tumor necrosis factor-alpha and interferon-gamma induced a substantial increase in both RANTES mRNA and extracellular RANTES levels. These increases were markedly reduced when astrocytes were coincubated with l-2-amino-4-phosphonobutanoate (l-AP-4), 4-phosphonophenylglycine, or l-serine-O-phosphate, which selectively activate group III mGlu receptor subtypes (i.e., mGlu4, -6, -7, and -8 receptors). Agonists of mGlu1/5 or mGlu2/3 receptors were virtually inactive. Inhibition of RANTES release produced by l-AP-4 was attenuated by the selective group III mGlu receptor antagonist (R,S)-alpha-methylserine-O-phosphate or by pretreatment of the cultures with pertussis toxin. Cultured astrocytes expressed mGlu4 receptors, and the ability of l-AP-4 to inhibit RANTES release was markedly reduced in cultures prepared from mGlu4 knock-out mice. This suggests that activation of mGlu4 receptors negatively modulates the production of RANTES in glial cells. We also examined the effect of l-AP-4 on the development of experimental allergic encephalomyelitis (EAE) in Lewis rats. l-AP-4 was subcutaneously infused for 28 d by an osmotic minipump that released 250 nl/hr of a solution of 250 mm of the drug. Detectable levels of l-AP-4 ( approximately 100 nm) were found in the brain dialysate of EAE rats. Infusion of l-AP-4 did not affect the time at onset and the severity of neurological symptoms but significantly increased the rate of recovery from EAE. In addition, lower levels of RANTES mRNA were found in the cerebellum and spinal cord of EAE rats infused with l-AP-4. These results suggest that pharmacological activation of group III mGlu receptors may be useful in the experimental treatment of neuroinflammatory CNS disorders.

Interleukin-12 (IL-12) is a heterodimeric cytokine produced by macrophages and dendritic cells that has a central role in cell-mediated immune responses through its ability to polarize T helper (Th)0 lymphocytes into the Th1 subset by stimulating interferon gamma (IFN-␥) production by the T lymphocytes and natural killer cells. 1 Dysregulated production of IL-12 is thought to be pathogenically involved in the development of Th1-dependent immunoinflammatory diseases such as insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis, autoimmune thyroiditis, Crohn's disease, and multiple sclerosis (reviewed in Trembleau et al. 2 ). Hence, studies conducted by ourselves and others in rodent models of these diseases show that blockade of endogenous IL-12 with specific antagonists such as neutralizing, monoclonal antibody (mAb) to IL-12 or the IL-12p40 homodimer ameliorates the course of the diseases. 3-9 Conversely, exogenous IL-12 accelerates the onset and increases the incidence of IDDM in NOD mice and augments the severity of the other disease models. 8,[10][11][12] Under certain conditions, however, IL-12 has had beneficial effects in mice suffering from Th1-mediated autoimmune diseases. 8,[13][14][15][16] This is thought to be mediated by the ability of IL-12 to stimulate the production of Th2-derived anti-inflammatory cytokines such as IL-4 and IL-10 17-20 (reviewed in Muraille and Leo 21 ).Few and contradictory results have also been obtained regarding the role of IL-12 in immunoinflammatory hepatitis. Although IL-12 does not induce liver damage by itself, 22 its synthesis is up-regulated in vivo in murine hepatitis virus infection, 23 and IL-12 induces a cytolytic response against regenerating hepatocytes primarily through its enhancement of tumor necrosis factor (TNF) production. 24 Using a model of hepatitis that can be induced in Propionibacterium acnesprimed mice by low doses of lipopolysaccharide (LPS), Tanaka et al. 25 observed that anti-IL-12 mAb given after challenge with P. acnes protected the mice from the hepatitisinducing effect of LPS. Conversely, exogenous IL-12 rendered otherwise resistant BALB/c mice susceptible to P. acnes/LPSinduced hepatitis. 25 Recently, Tsutsui et al. 26 found that anti-IL-18 mAb, but not anti-IL-12 mAb prevented P. acnes/LPSinduced hepatitis when these antibodies were injected immediately before LPS challenge. 26 Hepatitis can also be induced in mice given a single intravenous injection of 20 mg/kg of Concanavalin A (ConA) 27,28 (reviewed in Lohse and Meyer zum Bushenfelde 29 ). This disease is characterized by a markedly increased plasma level of alanine transaminase (ALT) 8 to 24 hours after injection and simultaneous infiltration of the liver with neutrophils, macrophages, and T cells, followed by apoptosis and necrosis of the hepatocytes. [27][28][29] The contribution of T cells in this model is underscored by the resistance of nude athymic mice to the hepatitis-inducing effect of ConA and by the preventive effect