Discovery of positive allosteric modulators for the metabotropic glutamate receptor subtype 5 from a series of N-(1,3-diphenyl-1H- pyrazol-5-yl)benzamides that potentiate receptor function in vivo

This report describes the discovery of the first centrally active allosteric modulators of the metabotropic glutamate receptor subtype 5 (mGluR5). Appropriately substituted N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamides (e.g., 8) have been identified as a novel class of potent positive allosteric modulators of mGluR5 that potentiate the response to glutamate. An iterative analogue library synthesis approach provided potentiators with excellent potency and selectivity for mGluR5 (vs mGluRs 1-4, 7, 8). Compound 8q demonstrated in vivo proof of concept in an animal behavior model where known antipsychotics are active, supporting the development of new antipsychotics based on the NMDA hypofunction model for schizophrenia.     

The contribution of CD4+ CD25+ T-regulatory-cells to immune suppression in sepsis

Studies have indicated that there is a development of generalized immune dysfunction after septic insult. However, the mechanisms responsible for these changes remain unclear. Recently, accumulating evidence shows that several lymphocyte subpopulations such as NKT-, CD4(+)-Th2-T-, CD8(+)-T-, gammadelta-T-, and CD4+ CD25+ T regulatory cells are capable of actively contributing to the induction of septic immune suppression. Thus, our aim was to investigate the contribution of CD4+ CD25+ cells to the immune dysfunction seen in sepsis. To study this, C57BL/6J, C57BL/6-Il6(tm1Kopf) (interleukin [IL] 6 -/-), and C57BL/6-Il10(tm1Cgn) (IL-10 -/-) mice were subjected to cecal ligation and puncture (CLP) or sham operations. Twenty-four hours later, blood was collected, and splenocytes were isolated. Phenotypic expression of CD4/CD25 (by fluorescence-activated cell sorter), cell proliferation (presented as proliferation index = [with anti-CD3]/[without anti-CD3]), and immune suppressive capacity (by in vitro add-back experiments) were assessed. The results indicate a marked elevation in CD4+ CD25+ cell levels and their proliferation index after sepsis in background mice. CD4+ CD25- cells from sham and CLP mice proliferated equally. However, coculture of CD4+ CD25- with CD4+ CD25+ cells suppressed their proliferation in both sham and CLP mice. Depletion of CD25+ cells in vivo before CLP markedly restored CD4+ CD25- proliferative capacity and Th1 cytokine release while not altering plasma proinflammatory cytokine levels. Subsequently, IL-6 -/- and IL-10 -/- mice were used to elucidate the possible mediator(s) regulating the changes seen after sepsis. Although CD4+ CD25+ cells increased after septic insult in both C57BL/6J and IL-6 -/- mice, this was not observed in IL-10 -/- mice. Similarly, in vitro proliferation studies showed that proliferation index increased in CD4+ CD25+ cells from septic C57BL/6J and IL6 -/- mice, but it remained the same in IL-10 -/- mice. Surprisingly, depletion of CD25+ cells before inducing sepsis did not alter septic mortality. Together, these findings suggest that although CD4+ CD25+ T regulatory cells induced by IL-10 seem to contribute to aspects of sepsis-induced lymphoid immune suppression, the oblation of CD25+ cells does not provide a survival advantage or disadvantage.     

A novel selective positive allosteric modulator of metabotropic glutamate receptor subtype 5 has in vivo activity and antipsychotic-like effects in rat behavioral models

We found that 3-cyano-N-(1,3-diphenyl-1H-pyrazol-5-yl)benzamide (CDPPB) is a potent and selective positive allosteric modulator of the metabotropic glutamate receptor subtype 5 (mGluR5). In Chinese hamster ovary cells expressing human mGluR5, CDPPB potentiated threshold responses to glutamate in fluorometric Ca2+ assays more than 7-fold with an EC50 value of approximately 27 nM. At 1 microM, CDPPB shifted mGluR5 agonist concentration response curves to glutamate, quisqualate, and (R,S)-3,5-dihydroxyphenylglycine 3- to 9-fold to the left. At higher concentrations, CDPPB exhibited agonist-like activity on cells expressing mGluR5. No other activity was observed on any other mGluR or cell type at concentrations up to 10 microM. CDPPB had no effect on [3H]quisqualate binding to mGluR5 but did compete for binding of [3H]methoxyPEPy, an analog of the selective mGluR5 negative allosteric modulator MPEP. CDPPB was found to be brain penetrant and reversed amphetamine-induced locomotor activity and amphetamine-induced deficits in prepulse inhibition in rats, two models sensitive to antipsychotic drug treatment. These results demonstrate that positive allosteric modulation of mGluR5 produces behavioral effects, suggesting that such modulation serves as a viable approach to increasing mGluR5 activity in vivo. These effects are consistent with the hypothesis that allosteric potentiation of mGluR5 may provide a novel approach for development of antipsychotic agents.     

The effect of prestorage irradiation on posttransfusion red cell survival

Transfusion-associated graft-versus-host disease (TA-GVHD) may occur whenever immunologically competent allogeneic lymphocytes are transfused to an immunocompromised recipient. Irradiation of blood components eliminates the risk of TA-GVHD but may damage the cellular elements in the transfused component, particularly if the cells are stored for prolonged periods in the irradiated state. To study the effect of irradiation on long-term storage of red cells, AS-1 red cells from eight normal subjects were prepared on two occasions. On one occasion, the units were stored as standard AS-1 red cells for 42 days at 4 degrees C; on the other, they were exposed to 3000 cGy radiation within 4 hours of collection and then were stored as AS-1 red cells for 42 days at 4 degrees C. The donations were at least 12 weeks apart. Irradiated units demonstrated significant elevations in poststorage plasma hemoglobin (Hb) (623 +/- 206 vs. 429 +/- 194 g/dL [6230 +/- 2060 vs. 4290 +/- 1940 g/L], p less than 0.02) and plasma potassium (78 +/- 4 vs. 43 +/- 9 mEq/L [78 +/- 4 vs. 43 +/- 9 mmol/L], p less than 0.01) and significant decreases in red cell ATP (1.9 +/- 0.2 vs. 2.1 +/- 0.3 microM/g Hb, p less than 0.04) and 24-hour posttransfusion red cell recovery (68.5 vs. 78.4%, p less than 0.02), as compared to nonirradiated units. It can be concluded that irradiation with 3000 cGy damages red cells and that long-term storage in the irradiated state may enhance this damage. Red cells should not be stored for 42 days after irradiation with 3000 cGy.     

Fractionation of Spatial Memory in GRM2/3 (mGlu2/mGlu3) Double Knockout Mice Reveals a Role for Group II Metabotropic Glutamate Receptors at the Interface Between Arousal and Cognition

Group II metabotropic glutamate receptors (mGluR2 and mGluR3, encoded by GRM2 and GRM3) are implicated in hippocampal function and cognition, and in the pathophysiology and treatment of schizophrenia and other psychiatric disorders. However, pharmacological and behavioral studies with group II mGluR agonists and antagonists have produced complex results. Here, we studied hippocampus-dependent memory in GRM2/3 double knockout (GRM2/3^−/−) mice in an iterative sequence of experiments. We found that they were impaired on appetitively motivated spatial reference and working memory tasks, and on a spatial novelty preference task that relies on animals'' exploratory drive, but were unimpaired on aversively motivated spatial memory paradigms. GRM2/3^−/− mice also performed normally on an appetitively motivated, non-spatial, visual discrimination task. These results likely reflect an interaction between GRM2/3 genotype and the arousal-inducing properties of the experimental paradigm. The deficit seen on appetitive and exploratory spatial memory tasks may be absent in aversive tasks because the latter induce higher levels of arousal, which rescue spatial learning. Consistent with an altered arousal–cognition relationship in GRM2/3^−/− mice, injection stress worsened appetitively motivated, spatial working memory in wild-types, but enhanced performance in GRM2/3^−/− mice. GRM2/3^−/− mice were also hypoactive in response to amphetamine. This fractionation of hippocampus-dependent memory depending on the appetitive-aversive context is to our knowledge unique, and suggests a role for group II mGluRs at the interface of arousal and cognition. These arousal-dependent effects may explain apparently conflicting data from previous studies, and have translational relevance for the involvement of these receptors in schizophrenia and other disorders.     

Allosteric modulators of NR2B-containing NMDA receptors: molecular mechanisms and therapeutic potential

N-methyl-D-aspartate receptors (NMDARs) are ion channels gated by glutamate, the major excitatory neurotransmitter in the mammalian central nervous system (CNS). They are widespread in the CNS and are involved in numerous physiological and pathological processes including synaptic plasticity, chronic pain and psychosis. Aberrant NMDAR activity also plays an important role in the neuronal loss associated with ischaemic insults and major degenerative disorders including Parkinson''s and Alzheimer''s disease. Agents that target and alter NMDAR function may, thus, have therapeutic benefit. Interestingly, NMDARs are endowed with multiple extracellular regulatory sites that recognize ions or small molecule ligands, some of which are likely to regulate receptor function in vivo. These allosteric sites, which differ from agonist-binding and channel-permeation sites, provide means to modulate, either positively or negatively, NMDAR activity. The present review focuses on allosteric modulation of NMDARs containing the NR2B subunit. Indeed, the NR2B subunit confers a particularly rich pharmacology with distinct recognition sites for exogenous and endogenous allosteric ligands. Moreover, NR2B-containing receptors, compared with other NMDAR subtypes, appear to contribute preferentially to pathological processes linked to overexcitation of glutamatergic pathways. The actions of extracellular H⁺, Mg²⁺, Zn²⁺, of polyamines and neurosteroids, and of the synthetic compounds ifenprodil and derivatives (‘prodils’) are presented. Particular emphasis is put upon the structural determinants and molecular mechanisms that underlie the effects exerted by these agents. A better understanding of how NR2B-containing NMDARs (and NMDARs in general) operate and how they can be modulated should help define new strategies to counteract the deleterious effects of dysregulated NMDAR activity.     

Ponseti technique of correcting idiopathic clubfoot deformity

The efficacy of the Ponseti method of clubfoot treatment at Queen Elizabeth Central Hospital (QECH) was analysed from December 2000 to December 2001. Ninety one patients, 60 boys and 31 girls were prospectively and consecutively enrolled. 31 patients had a unilateral clubfoot and 60 had bilateral clubfeet. 77 patients had primary idiopathic clubfoot and 14 patients had clubfeet associated with other congenital anomalies such as arthrogryposis. 32 patients (35%) were lost to follow up; records were inadequate for 6 patients leaving 54 patients (59%) available for analysis. Three main groups were assessed. Group 1 (24 patients): virgin previously untreated primary idiopathic clubfeet: Ponseti method used from outset. Group 2 (19 patients): complex, primary idiopathic clubfeet: Ponseti method introduced after other manipulation techniques. Group 3 (11 patients): clubfeet associated with other congenital anomalies. In group 1, the mean age at start of treatment was 9.7 weeks and the mean time to correction of deformity was 7.4 weeks. 20 out of 24 patients (84%) had correction of deformity and remained corrected. 4 patients had recurrence of deformity mainly due to non compliance with treatment and correction was achieved once treatment restarted. In group 2, 19 patients had been on treatment for a mean period of 32 weeks prior to commencement of Ponseti treatment. In 17 of these patients the deformity was still uncorrected. Ponseti treatment was commenced at a mean age of 36 weeks and correction was achieved in all 17 patients after a mean treatment duration of 7.1 weeks. In group 3, correction of deformity was initially achieved in only 60%. The period to achieve correction was long and incidence of recurrence of deformity was high.The success of conservative treatment of clubfeet using the Ponseti method has resulted in large decrease in the number of surgical procedures performed under general anaesthaesia such as posteromedial releases in the treatment of clubfeet at QECH. This method has now been adopted as the Standard treatment of clubfoot and is being advocated nationwide.     

Gene Probe Analysis in an Informative Family with Multiple Endocrine Neoplasia Syndrome Type 2A (MEN 2A). Improvement in Carrier Risk Estimation

Gene probe analysis of the MEN 2A locus on chromosome 10 hasbeen undertaken using the markers TB10.163, RBP 3 and TB14.34in a large kindred with familial medullary thyroid carcinomas,with or without phaeochromocytomas or primary hyperparathyroidism.A maximum LOD score of 2.97 gave strong evidence of close linkagewith zero recombination. For 12 members of the family so far not known to be affectedby any form of the disease the estimated risk of carrying thegene has been considerably decreased in all but one, whose riskhas been greatly increased.     

Risk factors for infection in Malaysian patients with systemic lupus erythematosus

To determine the incidence, types and risk factors for infection in systemic lupus erythematosus (SLE) patients in Kuala Lumpur, Malaysia, we retrospectively reviewed the medical records of 102 patients with definite SLE attending a specialist clinic. Details of major infections (pneumonia or severe infection requiring intravenous therapy) and minor infections, and their time of onset in relation to immunosuppressive therapy and disease flares were recorded. There were 77 major and 163 minor infections during 564 patient-years of follow-up. In the month following a course of pulse methylprednisolone, the incidence of major infection was 20 times higher and the incidence of minor infection was 10 times higher than at other periods (p<0.0001). In the month after disease flare, the incidence of major infection was 10 times higher and the incidence of minor infection six times higher than at other times (p<0.0001). After allowing for methylprednisolone therapy and disease flares, there was no increase in the rate of infections during treatment with azathioprine, oral or intravenous cyclophosphamide. There was no effect of renal involvement on infection rate.