Structure-Activity Studies of 3,9-Diazaspiro[5.5]undecane-Based γ-Aminobutyric Acid Type A Receptor Antagonists with Immunomodulatory Effect.

The 3,9-diazaspiro[5.5]undecane-based compounds 2027 and 018 have previously been reported to be potent competitive γ-aminobutyric acid type A receptor (GABAAR) antagonists showing low cellular membrane permeability. Given the emerging peripheral application of GABAAR ligands, we hypothesize 2027 analogs as promising lead structures for peripheral GABAAR inhibition. We herein report a study on the structural determinants of 2027 in order to suggest a potential binding mode as a basis for rational design. The study identified the importance of the spirocyclic benzamide, compensating for the conventional acidic moiety, for GABAAR ligands. The structurally simplified m-methylphenyl analog 1e displayed binding affinity in the high-nanomolar range (Ki = 180 nM) and was superior to 2027 and 018 regarding selectivity for the extrasynaptic α4ßδ subtype versus the α1- and α2- containing subtypes. Importantly, 1e was shown to efficiently rescue inhibition of T cell proliferation, providing a platform to explore the immunomodulatory potential for this class of compounds.

Activation of GABA(A) receptors inhibits T cell proliferation.

BACKGROUND: The major sites for fast synaptic inhibition in the central nervous system (CNS) are ion channels activated by γ-aminobutyric acid (GABA). These receptors are referred as GABA(A) receptors (GABA(A)R). Recent evidence indicates a role of GABA(A)R in modulating the immune response. This work aimed to discern the role of GABA and GABA(A)Rs in human and mouse T cell activity. METHODS: Mouse splenocytes or human peripheral blood mononuclear cells (PBMCs) were activated with anti-CD3 antibodies and the proliferation of both CD8+ and CD4+ T cells assessed through flow cytometry. Subsequently, the effects on T cell proliferation of either GABA(A)R modulation by diazepam that is also capable of activating mitochondrial based translocator protein (TSPO), alprazolam and allopregnanolone or inhibition by bicucculine methiodide (BMI) and (1,2,5,6-Tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) were assessed. RESULTS: Positive modulation of GABA(A)Rs either by benzodiazepines or the neurosteroid allopregnanolone inhibits both mouse and human T cell proliferation. GABAergic inhibition of T cell proliferation by benzodiazepines could be rescued by GABA(A)R blocking. Our data suggest that benzodiazepines influence T cell proliferation through both TSPO and GABA(A)Rs activation. CONCLUSIONS: We conclude that activation of GABA(A)Rs provides immunosuppression by inhibiting T cell proliferation.

The cytotoxic molecule granulysin is capable of inducing either chemotaxis or fugetaxis in dendritic cells depending on maturation: a role for Vδ2+ γδ T cells in the modulation of immune response to tumour?

Release of granulysin by γδ T cells contributes to tumour cell killing. A cytolytic 9000 MW isoform of granulysin kills tumour cells directly, whereas a 15 000 MW precursor has been hypothesized to cause both the maturation and migration of dendritic cell (DC) populations. Recruiting DC to a tumour is beneficial as these cells initiate adaptive immune responses, which contribute to the eradication of malignancies. In this study, Vδ2+ γδ T cells were activated by stimulation of peripheral blood mononuclear cells with zoledronic acid or Bacillus Calmette-Guérin (BCG), or were isolated and cultured with tumour targets. Although a large proportion of resting Vδ2+ γδ T cells expressed 15 000 MW granulysin, 9000 MW granulysin expression was induced only after stimulation with BCG. Increased levels of activation and granulysin secretion were also observed when Vδ2+ γδ T cells were cultured with the human B-cell lymphoma line Daudi. High concentrations of recombinant 15 000 MW granulysin caused migration and maturation of immature DC, and also initiated fugetaxis in mature DC. Conversely, low concentrations of recombinant 15 000 MW granulysin resulted in migration of mature DC, but not immature DC. Our data therefore support the hypothesis that Vδ2+ γδ T cells can release granulysin, which may modulate recruitment of DC, initiating adaptive immune responses.

Public and patient involvement in child health research and service improvements: a survey of hospital doctors.

OBJECTIVES: To determine whether paediatricians are supported by their organisations to encourage patient and public involvement (PPI) in research activities and clinical improvement work, the challenges they face and how they think these could be addressed by the Royal College of Paediatrics and Child Health (RCPCH). DESIGN: A survey. SETTING: UK consultant paediatricians and staff associate specialist and specialty (SAS) doctors who are members of RCPCH. MAIN OUTCOME MEASURES: The proportion of respondents who said that PPI was central to research and service improvements in their organisation, the type of local support for PPI activity, challenges in undertaking PPI and the support members wanted from RCPCH. RESULTS: There was a response rate of 44.4% (n=1924). In their organisation, 29.1% of respondents stated PPI was central to research and 36.1% to service improvement; 46% were unaware of support for PPI and 15% said there was no support. The main challenges for PPI activity were a lack of clinician time, local support and funding. Respondents wanted RCPCH to advocate for protected time for PPI, provide access to PPI groups and deliver guidance and training. CONCLUSIONS: The majority of paediatricians feel unsupported to undertake PPI activity by their local organisation. The RCPCH has a key role to enable all paediatricians to work with children, young people and their carers to improve the quality of research and clinical services as demonstrated by RCPCH's ongoing activity in these crucial and important areas.