Diazepam and ethanol differently modulate neuronal activity in organotypic cortical cultures.

BACKGROUND: The pharmacodynamic results of diazepam and ethanol administration are similar, in that each can mediate amnestic and sedative-hypnotic effects. Although each of these molecules effectively reduce the activity of central neurons, diazepam does so through modulation of a more specific set of receptor targets (GABAA receptors containing a γ-subunit), while alcohol is less selective in its receptor bioactivity. Our investigation focuses on divergent actions of diazepam and ethanol on the firing patterns of cultured cortical neurons. METHOD: We used electrophysiological recordings from organotypic slice cultures derived from Sprague-Dawley rat neocortex. We exposed these cultures to either diazepam (15 and 30 µM, n = 7) or ethanol (30 and 60 mM, n = 11) and recorded the electrical activity at baseline and experimental conditions. For analysis, we extracted the episodes of spontaneous activity, i.e., cortical up-states. After separation of action potential and local field potential (LFP) activity, we looked at differences in the number of action potentials, in the spectral power of the LFP, as well as in the coupling between action potential and LFP phase. RESULTS: While both substances seem to decrease neocortical action potential firing in a not significantly different (p = 0.659, Mann-Whitney U) fashion, diazepam increases the spectral power of the up-state without significantly impacting the spectral composition, whereas ethanol does not significantly change the spectral power but the oscillatory architecture of the up-state as revealed by the Friedman test with Bonferroni correction (p < 0.05). Further, the action potential to LFP-phase coupling reveals a synchronizing effect of diazepam for a wide frequency range and a narrow-band de-synchronizing effect for ethanol (p < 0.05, Kolmogorov-Smirnov test). CONCLUSION: Diazepam and ethanol, induce specific patterns of network depressant actions. Diazepam induces cortical network inhibition and increased synchronicity via gamma subunit containing GABAA receptors. Ethanol also induces cortical network inhibition, but without an increase in synchronicity via a wider span of molecular targets.

Tbet or Continued RORγt Expression Is Not Required for Th17-Associated Immunopathology.

The discovery of Th17 cell plasticity, in which CD4(+) IL-17-producing Th17 cells give rise to IL-17/IFN-γ double-producing cells and Th1-like IFNγ(+) ex-Th17 lymphocytes, has raised questions regarding which of these cell types contribute to immunopathology during inflammatory diseases. In this study, we show using Helicobacter hepaticus-induced intestinal inflammation that IL-17A(Cre)- or Rag1(Cre)-mediated deletion of Tbx21 has no effect on the generation of IL-17/IFN-γ double-producing cells, but leads to a marked absence of Th1-like IFNγ(+) ex-Th17 cells. Despite the lack of Th1-like ex-Th17 cells, the degree of H. hepaticus-triggered intestinal inflammation in mice in which Tbx21 was excised in IL-17-producing or Rag1-expressing cells is indistinguishable from that observed in control mice. In stark contrast, using experimental autoimmune encephalomyelitis, we show that IL-17A(Cre)-mediated deletion of Tbx21 prevents the conversion of Th17 cells to IL-17A/IFN-γ double-producing cells as well as Th1-like IFN-γ(+) ex-Th17 cells. However, IL-17A(Cre)-mediated deletion of Tbx21 has only limited effects on disease course in this model and is not compensated by Ag-specific Th1 cells. IL-17A(Cre)-mediated deletion of Rorc reveals that RORγt is essential for the maintenance of the Th17 cell lineage, but not immunopathology during experimental autoimmune encephalomyelitis. These results show that neither the single Th17 subset, nor its progeny, is solely responsible for immunopathology or autoimmunity.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

PURPOSE: CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL DESIGN: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Phenotypical characterization of human Th17 cells unambiguously identified by surface IL-17A expression.

Th17 cells are involved in the defense against bacteria and fungi and play a prominent role in the pathogenesis of autoimmune diseases, but research on human Th17 cells is hindered due to the lack of a surface marker. In this study, we report that a subset of human and mouse CD4(+) T cells as well as human Th17 T cell clones express IL-17A on their surface upon stimulation. Correlation of surface IL-17A expression with intracellular IL-17A production and with RORgammat mRNA expression identified surface IL-17A as a specific marker for human and mouse Th17 cells. Phenotype characterization of ex vivo CD4(+) IL-17A(+) cells showed that the chemokines CCR6 and CCR4, costimulatory molecules, as well as CD2 and CD49d were more prominently expressed on these cells than in surface IL-17A(-) cells, supporting the concept of Th17 cells as a potent inflammatory effector subtype. In addition, we generated human Th1, Th1/17 (producing both IFN-gamma and IL-17A), and Th17 T cell clones based on single cell sorting of surface IL-17A(-), IL-17A(int), and IL-17A(high) CD4(+) T cells, respectively, and showed the plasticity of the double producing clones to the cytokine milieu. The identification of surface IL-17A as a marker for Th17 cells should facilitate research on this subset.

Prednisolone treatment induces tolerogenic dendritic cells and a regulatory milieu in myasthenia gravis patients.

FOXP3-expressing naturally occurring CD4(+)CD25(high) T regulatory cells (Treg) are relevant in the control of autoimmunity, and a defect in this cell population has been observed in several human autoimmune diseases. We hypothesized that altered functions of peripheral Treg cells might play a role in the immunopathogenesis of myasthenia gravis, a T cell-dependent autoimmune disease characterized by the presence of pathogenic autoantibodies specific for the nicotinic acetylcholine receptor. We report in this study a significant decrease in the in vitro suppressive function of peripheral Treg cells isolated from myasthenia patients in comparison to those from healthy donors. Interestingly, Treg cells from prednisolone-treated myasthenia gravis patients showed an improved suppressive function compared with untreated patients, suggesting that prednisolone may play a role in the control of the peripheral regulatory network. Indeed, prednisolone treatment prevents LPS-induced maturation of monocyte-derived dendritic cells by hampering the up-regulation of costimulatory molecules and by limiting secretion of IL-12 and IL-23, and enhancing IL-10. In addition, CD4(+) T cells cultured in the presence of such tolerogenic dendritic cells are hyporesponsive and can suppress autologous CD4(+) T cell proliferation. The results shown in this study indicate that prednisolone treatment promotes an environment that favors immune regulation rather than inflammation.

Phenotypical and functional characterization of T helper 17 cells in multiple sclerosis.

Multiple sclerosis is a T cell-mediated demyelinating disease of the central nervous system. Interleukin-17-producing T helper cells, named Th17 cells, represent a novel CD4+ T cell effector subset involved in the response against extracellular pathogens. In addition, Th17 cells are pathogenic in several animal models of autoimmune disease, including the animal model for multiple sclerosis, but their function in multiple sclerosis remains to be elucidated. In this study, we analysed the frequency and the phenotype of Th17 cells in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients. We show that the frequency of Th17 cells is significantly higher in the cerebrospinal fluid of patients with relapsing-remitting multiple sclerosis during relapse, in comparison to relapsing-remitting patients in remission or to patients with other non-inflammatory neurological diseases. Similarly, in patients with clinically isolated syndrome during their first neurological episode, Th17 cells are more abundant than in clinically isolated syndrome patients with no acute symptoms. Patients with inflammatory neurological diseases other than multiple sclerosis also showed increased frequency of Th17 cells compared to patients with no inflammatory diseases. To assess a potential pathological impact of Th17 cells in disease, we generated T cell clones from the cerebrospinal fluid and peripheral blood of patients with multiple sclerosis. We found that Th17 clones expressed higher basal levels of the activation markers CD5, CD69, CD2 and human leukocyte antigen-DR as well as of the CD28-related family of co-stimulatory molecules, when compared to Th1 clones, and confirmed these findings with ex vivo human T cells. Molecules involved in T cell adhesion to endothelium, such as CD49d, CD6 and the melanoma cell adhesion molecule, were also more abundant on the Th17 than on the Th1 cells. Furthermore, functional assays showed that Th17 clones were more prone than Th1 clones to melanoma cell adhesion molecule-mediated adhesion to endothelial cells, and that Th17 cells had a higher proliferative capacity and were less susceptible to suppression than Th1 cells. Altogether our data suggest that Th17 cells display a high pathogenic potential and may constitute a relevant pathogenic subset in multiple sclerosis.

Cellular Stress in the Context of an Inflammatory Environment Supports TGF-ß-Independent T Helper-17 Differentiation.

T helper-17 (Th17) cells are associated with inflammatory disorders and cancer. We report that environmental conditions resulting in cellular stress, such as low oxygen, glucose, and isotonic stress, particularly enhance the generation of Th17 cells. Pharmacological inhibition of cell stress reduces Th17 cell differentiation while stress inducers enhance the development of Th17 cells. The cellular stress response results in Th17 cell development via sustained cytoplasmic calcium levels and, in part, XBP1 activity. Furthermore, in an inflammatory environment, conditions resulting in cell stress can bring about de novo Th17 cell differentiation, even in the absence of transforming growth factor ß (TGF-ß) signaling. In vivo, cell stress inhibition enhances resistance to Th17-mediated autoimmunity while stress-exposed T cells enhance disease severity. Adverse metabolic environments during inflammation provide a link between adaptive immunity and inflammation and may represent a risk factor for the development of chronic inflammatory conditions by facilitating Th17 cell differentiation.

Transient inhibition of ROR-γt therapeutically limits intestinal inflammation by reducing TH17 cells and preserving group 3 innate lymphoid cells.

RAR-related orphan receptor-γt (ROR-γt) directs differentiation of proinflammatory T helper 17 (TH17) cells and is a potential therapeutic target in chronic autoimmune and inflammatory diseases. However, ROR-γt-dependent group 3 innate lymphoid cells ILC3s provide essential immunity and tissue protection in the intestine, suggesting that targeting ROR-γt could also result in impaired host defense after infection or enhanced tissue damage. Here, we demonstrate that transient chemical inhibition of ROR-γt in mice selectively reduces cytokine production from TH17 but not ILCs in the context of intestinal infection with Citrobacter rodentium, resulting in preserved innate immunity. Temporal deletion of Rorc (encoding ROR-γt) in mature ILCs also did not impair cytokine response in the steady state or during infection. Finally, pharmacologic inhibition of ROR-γt provided therapeutic benefit in mouse models of intestinal inflammation and reduced the frequency of TH17 cells but not ILCs isolated from primary intestinal samples of individuals with inflammatory bowel disease (IBD). Collectively, these results reveal differential requirements for ROR-γt in the maintenance of TH17 cell and ILC3 responses and suggest that transient inhibition of ROR-γt is a safe and effective therapeutic approach during intestinal inflammation.

Cellular Plasticity of CD4+ T Cells in the Intestine.

Barrier sites such as the gastrointestinal tract are in constant contact with the environment, which contains both beneficial and harmful components. The immune system at the epithelia must make the distinction between these components to balance tolerance, protection, and immunopathology. This is achieved via multifaceted immune recognition, highly organized lymphoid structures, and the interaction of many types of immune cells. The adaptive immune response in the gut is orchestrated by CD4(+) helper T (Th) cells, which are integral to gut immunity. In recent years, it has become apparent that the functional identity of these Th cells is not as fixed as initially thought. Plasticity in differentiated T cell subsets has now been firmly established, in both health and disease. The gut, in particular, utilizes CD4(+) T cell plasticity to mold CD4(+) T cell phenotypes to maintain its finely poised balance of tolerance and inflammation and to encourage biodiversity within the enteric microbiome. In this review, we will discuss intestinal helper T cell plasticity and our current understanding of its mechanisms, including our growing knowledge of an evolutionarily ancient symbiosis between microbiota and malleable CD4(+) T cell effectors.

Dietary influences on intestinal immunity.

The function of the gastrointestinal tract relies on a monolayer of epithelial cells, which are essential for the uptake of nutrients. The fragile lining requires protection against insults by a diverse array of antigens. This is accomplished by the mucosa-associated lymphoid tissues of the gastrointestinal tract, which constitute a highly organized immune organ. In this Review, we discuss several recent findings that provide a compelling link between dietary compounds and the organization and maintenance of immune tissues and lymphocytes in the intestine. We highlight some of the molecular players involved, in particular ligand-activated nuclear receptors in lymphoid cells.