Interferon subverts an AHR-JUN axis to promote CXCL13+ T cells in lupus.

Systemic lupus erythematosus (SLE) is prototypical autoimmune disease driven by pathological T cell-B cell interactions1,2. Expansion of T follicular helper (TFH) and T peripheral helper (TPH) cells, two T cell populations that provide help to B cells, is a prominent feature of SLE3,4. Human TFH and TPH cells characteristically produce high levels of the B cell chemoattractant CXCL13 (refs. 5,6), yet regulation of T cell CXCL13 production and the relationship between CXCL13+ T cells and other T cell states remains unclear. Here, we identify an imbalance in CD4+ T cell phenotypes in patients with SLE, with expansion of PD-1+/ICOS+ CXCL13+ T cells and reduction of CD96hi IL-22+ T cells. Using CRISPR screens, we identify the aryl hydrocarbon receptor (AHR) as a potent negative regulator of CXCL13 production by human CD4+ T cells. Transcriptomic, epigenetic and functional studies demonstrate that AHR coordinates with AP-1 family member JUN to prevent CXCL13+ TPH/TFH cell differentiation and promote an IL-22+ phenotype. Type I interferon, a pathogenic driver of SLE7, opposes AHR and JUN to promote T cell production of CXCL13. These results place CXCL13+ TPH/TFH cells on a polarization axis opposite from T helper 22 (TH22) cells and reveal AHR, JUN and interferon as key regulators of these divergent T cell states.

A Microengineered Airway Lung Chip Models Key Features of Viral-induced Exacerbation of Asthma.

Viral-induced exacerbation of asthma remains a major cause of hospitalization and mortality. New human-relevant models of the airways are urgently needed to understand how respiratory infections may trigger asthma attacks and to advance treatment development. Here, we describe a new human-relevant model of rhinovirus-induced asthma exacerbation that recapitulates viral infection of asthmatic airway epithelium and neutrophil transepithelial migration, and enables evaluation of immunomodulatory therapy. Specifically, a microengineered model of fully differentiated human mucociliary airway epithelium was stimulated with IL-13 to induce a T-helper cell type 2 asthmatic phenotype and infected with live human rhinovirus 16 (HRV16) to reproduce key features of viral-induced asthma exacerbation. We observed that the infection with HRV16 replicated key hallmarks of the cytopathology and inflammatory responses observed in human airways. Generation of a T-helper cell type 2 microenvironment through exogenous IL-13 stimulation induced features of asthmatic airways, including goblet cell hyperplasia, reduction of cilia beating frequency, and endothelial activation, but did not alter rhinovirus infectivity or replication. High-resolution kinetic analysis of secreted inflammatory markers revealed that IL-13 treatment altered IL-6, IFN-λ1, and CXCL10 secretion in response to HRV16. Neutrophil transepithelial migration was greatest when viral infection was combined with IL-13 treatment, whereas treatment with MK-7123, a CXCR2 antagonist, reduced neutrophil diapedesis in all conditions. In conclusion, our microengineered Airway Lung-Chip provides a novel human-relevant platform for exploring the complex mechanisms underlying viral-induced asthma exacerbation. Our data suggest that IL-13 may impair the hosts' ability to mount an appropriate and coordinated immune response to rhinovirus infection. We also show that the Airway Lung-Chip can be used to assess the efficacy of modulators of the immune response.

Characterizing Pharmacokinetic-Pharmacodynamic Relationships and Efficacy of PI3Kδ Inhibitors in Respiratory Models of TH2 and TH1 Inflammation.

We leveraged a clinical pharmacokinetic (PK)/pharmacodynamics (PD)/efficacy relationship established with an oral phosphatidylinositol 3-kinase (PI3K)δ inhibitor (Idelalisib) in a nasal allergen challenge study to determine whether a comparable PK/PD/efficacy relationship with PI3Kδ inhibitors was observed in preclinical respiratory models of type 2 T helper cell (TH2) and type 1 T helper cell (TH1) inflammation. Results from an in vitro rat blood basophil (CD63) activation assay were used as a PD biomarker. IC50 values for PI3Kδ inhibitors, MSD-496486311, MSD-126796721, Idelalisib, and Duvelisib, were 1.2, 4.8, 0.8, and 0.5 µM. In the ovalbumin Brown Norway TH2 pulmonary inflammation model, all PI3Kδ inhibitors produced a dose-dependent inhibition of bronchoalveolar lavage eosinophils (maximum effect between 80% and 99%). In a follow-up experiment designed to investigate PK attributes [maximum (or peak) plasma concentration (Cmax), area under the curve (AUC), time on target (ToT)] that govern PI3Kδ efficacy, MSD-496486311 [3 mg/kg every day (QD) and 100 mg/kg QD] produced 16% and 93% inhibition of eosinophils, whereas doses (20 mg/kg QD, 10 mg/kg twice per day, and 3 mg/kg three times per day) produced 54% to 66% inhibition. Our profiling suggests that impact of PI3Kδ inhibitors on eosinophils is supported by a PK target with a ToT over the course of treatment close to the PD IC50 rather than strictly driven by AUC, Cmax, or Cmin (minimum blood plasma concentration) coverage. Additional studies in an Altenaria alternata rat model, a sheep Ascaris-sensitive sheep model, and a TH1-driven rat ozone exposure model did not challenge our hypothesis, suggesting that an IC50 level of TE (target engagement) sustained for 24 hours is required to produce efficacy in these traditional models. We conclude that the PK/PD observations in our animal models appear to align with clinical results associated with a TH2 airway disease.

Small airway-on-a-chip enables analysis of human lung inflammation and drug responses in vitro.

Here we describe the development of a human lung 'small airway-on-a-chip' containing a differentiated, mucociliary bronchiolar epithelium and an underlying microvascular endothelium that experiences fluid flow, which allows for analysis of organ-level lung pathophysiology in vitro. Exposure of the epithelium to interleukin-13 (IL-13) reconstituted the goblet cell hyperplasia, cytokine hypersecretion and decreased ciliary function of asthmatics. Small airway chips lined with epithelial cells from individuals with chronic obstructive pulmonary disease recapitulated features of the disease such as selective cytokine hypersecretion, increased neutrophil recruitment and clinical exacerbation by exposure to viral and bacterial infections. With this robust in vitro method for modeling human lung inflammatory disorders, it is possible to detect synergistic effects of lung endothelium and epithelium on cytokine secretion, identify new biomarkers of disease exacerbation and measure responses to anti-inflammatory compounds that inhibit cytokine-induced recruitment of circulating neutrophils under flow.

Establishment of a 96-well transwell system using primary human gut organoids to capture multiple quantitative pathway readouts.

Disruptions in the gut epithelial barrier can lead to the development of chronic indications such as inflammatory bowel disease (IBD). Historically, barrier function has been assessed in cancer cell lines, which do not contain all human intestinal cell types, leading to poor translatability. To bridge this gap, we adapted human primary gut organoids grown as monolayers to quantify transcription factor phosphorylation, gene expression, cytokine production, and barrier function. In this work we describe and characterize a novel 96-well human gut organoid-derived monolayer system that enables quantitative assessment of candidate therapeutics. Normal human intestine differentiation patterns and barrier function were characterized and confirmed to recapitulate key aspects of in vivo biology. Next, cellular response to TNF-α (a central driver of IBD) was determined using a diverse cadre of quantitative readouts. We showed that TNF-α pathway antagonists rescued damage caused by TNF-α in a dose-dependent manner, indicating that this system is suitable for quantitative assessment of barrier modulating factors. Taken together, we have established a robust primary cell-based 96-well system capable of interrogating questions around mucosal response. This system is well suited to provide pivotal functional data to support translational target and drug discovery efforts.

Identification and characterization of lipopolysaccharide binding protein (LBP) as an estrogen receptor α specific serum biomarker.

Estrogen Receptor α (ERα) and Estrogen Receptor ß (ERß) are steroid nuclear receptors that transduce estrogen signaling to control diverse physiological processes linked to reproduction, bone remodeling, behavior, immune response and endocrine-related diseases. In order to differentiate between ERα and ERß mediated effects in vivo, ER subtype selective biomarkers are essential. We utilized ERα knockout (AERKO) and ERß knockout (BERKO) mouse liver RNA and genome wide profiling to identify novel ERα selective serum biomarker candidates. Results from the gene array experiments were validated using real-time RT-PCR and subsequent ELISA's to demonstrate changes in serum proteins. Here we present data that Lipopolysacharide Binding Protein (LBP) is a novel liver-derived ERα selective biomarker that can be measured in serum.

Longitudinal Immune Cell Profiling in Patients With Early Systemic Lupus Erythematosus.

OBJECTIVES: To investigate the immune cell profiles of patients with systemic lupus erythematosus (SLE), and to identify longitudinal changes in those profiles over time. METHODS: We employed mass cytometry with 3 different panels of 38-39 markers (an immunophenotyping panel, a T cell/monocyte panel, and a B cell panel) in cryopreserved peripheral blood mononuclear cells (PBMCs) from 9 patients with early SLE, 15 patients with established SLE, and 14 controls without autoimmune disease. We used machine learning-driven clustering, flow self-organizing maps, and dimensional reduction with t-distributed stochastic neighbor embedding to identify unique cell populations in early SLE and established SLE. We used mass cytometry data of PBMCs from 19 patients with early rheumatoid arthritis (RA) and 23 controls to compare levels of specific cell populations in early RA and SLE. For the 9 patients with early SLE, longitudinal mass cytometry analysis was applied to PBMCs at enrollment, 6 months after enrollment, and 1 year after enrollment. Serum samples were also assayed for 65 cytokines using Luminex multiplex assay, and associations between cell types and cytokines/chemokines were assessed. RESULTS: Levels of peripheral helper T cells, follicular helper T (Tfh) cells, and several Ki-67+ proliferating subsets (ICOS+Ki-67+ CD8 T cells, Ki-67+ regulatory T cells, CD19intermediate Ki-67high plasmablasts, and PU.1high Ki-67high monocytes) were increased in patients with early SLE, with more prominent alterations than were seen in patients with early RA. Longitudinal mass cytometry and multiplex serum cytokine assays of samples from patients with early SLE revealed that levels of Tfh cells and CXCL10 had decreased 1 year after enrollment. Levels of CXCL13 were positively correlated with levels of several of the expanded cell populations in early SLE. CONCLUSION: Two major helper T cell subsets and unique Ki-67+ proliferating immune cell subsets were expanded in patients in the early phase of SLE, and the immunologic features characteristic of early SLE evolved over time.

Comparative analysis of the effects of antimuscarinic agents on bladder functions in both nonhuman primates and rodents.

Both the physiological role of muscarinic receptors for bladder function and the therapeutic efficacy of antimuscarinic agents for overactive bladder syndrome are well documented. We investigated the effect of antimuscarinic agents with different subtype selectivity on urodynamic parameters in nonhuman primates and rodents and compared plasma levels of these agents between species. Anesthetized rhesus monkeys were transurethrally catheterized, and the bladder was infused with saline. Urodynamic parameters were measured before and after intravenous drug administration. Tolterodine (nonselective) and oxybutynin (moderately M(3)-selective) increased bladder capacity at lower doses than those required to decrease micturition pressure. However, higher doses of darifenacin (M(3)-selective) were needed to increase the bladder capacity than those needed to decrease the micturition pressure. In rats, tolterodine had no effect on the bladder capacity but decreased the micturition pressure at all of the doses administered. Oxybutynin also decreased micturition pressure and increased bladder capacity at the highest dose. Plasma levels of these drugs overlap in both species. These results suggest that, in addition to the M(3) receptor, other muscarinic receptor subtypes contribute to regulate bladder storage function in nonhuman primates, since less subtype-selective tolterodine and oxybutynin showed higher specificity to the bladder capacity effect than the effect on micturition pressure compared with M(3)-selective darifenacin. In addition, the role of muscarinic receptors in bladder storage function varies between primates and rodents. Compared with rodents, muscarinic receptors may play a more active role during the storage phase to regulate the functional bladder capacity in primates.

Non-invasive muscle contraction assay to study rodent models of sarcopenia.

BACKGROUND: Age-related sarcopenia is a disease state of loss of muscle mass and strength that affects physical function and mobility leading to falls, fractures, and disability. The need for therapies to treat age-related sarcopenia has attracted intensive preclinical research. To facilitate the discovery of these therapies, we have developed a non-invasive rat muscle functional assay system to efficiently measure muscle force and evaluate the efficacy of drug candidates. METHODS: The lower leg muscles of anesthetized rats are artificially stimulated with surface electrodes on the knee holders and the heel support, causing the lower leg muscles to push isometric pedals that are attached to force transducers. We developed a stimulation protocol to perform a fatigability test that reveals functional muscle parameters like maximal force, the rate of fatigue, fatigue-resistant force, as well as a fatigable muscle force index. The system is evaluated in a rat aging model and a rat glucocorticoid-induced muscle loss model RESULTS: The aged rats were generally weaker than adult rats and showed a greater reduction in their fatigable force when compared to their fatigue-resistant force. Glucocorticoid treated rats mostly lost fatigable force and fatigued at a higher rate, indicating reduced force from glycolytic fibers with reduced energy reserves. CONCLUSIONS: The involuntary contraction assay is a reliable system to assess muscle function in rodents and can be applied in preclinical research, including age-related sarcopenia and other myopathy.

Projected Dose Optimization of Amino- and Hydroxypyrrolidine Purine PI3Kδ Immunomodulators.

The approvals of idelalisib and duvelisib have validated PI3Kδ inhibitors for the treatment for hematological malignancies driven by the PI3K/AKT pathway. Our program led to the identification of structurally distinct heterocycloalkyl purine inhibitors with excellent isoform and kinome selectivity; however, they had high projected human doses. Improved ligand contacts gave potency enhancements, while replacement of metabolic liabilities led to extended half-lives in preclinical species, affording PI3Kδ inhibitors with low once-daily predicted human doses. Treatment of C57BL/6-Foxp3-GDL reporter mice with 30 and 100 mg/kg/day of 3c (MSD-496486311) led to a 70% reduction in Foxp3-expressing regulatory T cells as observed through bioluminescence imaging with luciferin, consistent with the role of PI3K/AKT signaling in Treg cell proliferation. As a model for allergic rhinitis and asthma, treatment of ovalbumin-challenged Brown Norway rats with 0.3 to 30 mg/kg/day of 3c gave a dose-dependent reduction in pulmonary bronchoalveolar lavage inflammation eosinophil cell count.

Estradiol and ERß agonists enhance recognition memory, and DPN, an ERß agonist, alters brain monoamines.

Effects of estradiol benzoate (EB), ERα-selective agonist, propyl pyrazole triol (PPT) and ERß-selective agonists, diarylpropionitrile (DPN) and Compound 19 (C-19) on memory were investigated in OVX rats using object recognition (OR) and placement (OP) memory tasks. Treatments were acute (behavior 4h later) or sub chronic (daily injections for 2 days with behavior 48 h later). Objects were explored in sample trials (T1), and discrimination between sample (old) and new object/location in recognition trials (T2) was examined after 2-4h inter-trial delays. Subjects treated sub chronically with EB, DPN, and C-19, but not PPT, discriminated between old and new objects and objects in old and new locations, suggesting that, at these doses and duration of treatments, estrogenic interactions with ERß contribute to enhancements in recognition memory. Acute injections of DPN, but not PPT, immediately after T1, also enhanced discrimination for both tasks (C19 was not investigated). Effects of EB, DPN and PPT on anxiety and locomotion, measured on elevated plus maze and open field, did not appear to account for the mnemonic enhancements. Monoamines and metabolites were measured following DPN treatment in subjects that did not receive behavioral testing. DPN was associated with alterations in monoamines in several brain areas: indexed by the metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), or the MHPG/norepinephrine (NE) ratio, NE activity was increased by 60-130% in the prefrontal cortex (PFC) and ventral hippocampus, and NE activity was decreased by 40-80% in the v. diagonal bands and CA1. Levels of the dopamine (DA) metabolite, homovanillic acid (HVA), increased 100% in the PFC and decreased by 50% in the dentate gyrus following DPN treatment. The metabolite of serotonin, 5-hydroxyindole acetic acid (5-HIAA), was increased in the PFC and CA3, by approximately 20%. No monoaminergic changes were noted in striatum or medial septum. Results suggest that ERß mediates sub chronic and acute effects of estrogens on recognition memory and that memory enhancements by DPN may occur, in part, through alterations in monoaminergic containing systems primarily in PFC and hippocampus.

The NLRP3 inflammasome mediates DSS-induced intestinal inflammation in Nod2 knockout mice.

Crohn's disease (CD) is a chronic disorder of the gastrointestinal tract characterized by inflammation and intestinal epithelial injury. Loss of function mutations in the intracellular bacterial sensor NOD2 are major risk factors for the development of CD. In the absence of robust bacterial recognition by NOD2 an inflammatory cascade is initiated through alternative PRRs leading to CD. In the present study, MCC950, a specific small molecule inhibitor of NLR pyrin domain-containing protein 3 (NLRP3), abrogated dextran sodium sulfate (DSS)-induced intestinal inflammation in Nod2-/- mice. NLRP3 inflammasome formation was observed at a higher rate in NOD2-deficient small intestinal lamina propria cells after insult by DSS. NLRP3 complex formation led to an increase in IL-1ß secretion in both the small intestine and colon of Nod2ko mice. This increase in IL-1ß secretion in the intestine was attenuated by MCC950 leading to decreased disease severity in Nod2ko mice. Our work suggests that NLRP3 inflammasome activation may be a key driver of intestinal inflammation in the absence of functional NOD2. NLRP3 pathway inhibition can prevent intestinal inflammation in the absence of robust NOD2 signaling.

PD-1hiCXCR5- T peripheral helper cells promote B cell responses in lupus via MAF and IL-21.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by pathologic T cell-B cell interactions and autoantibody production. Defining the T cell populations that drive B cell responses in SLE may enable design of therapies that specifically target pathologic cell subsets. Here, we evaluated the phenotypes of CD4+ T cells in the circulation of 52 SLE patients drawn from multiple cohorts and identified a highly expanded PD-1hiCXCR5-CD4+ T cell population. Cytometric, transcriptomic, and functional assays demonstrated that PD-1hiCXCR5-CD4+ T cells from SLE patients are T peripheral helper (Tph) cells, a CXCR5- T cell population that stimulates B cell responses via IL-21. The frequency of Tph cells, but not T follicular helper (Tfh) cells, correlated with both clinical disease activity and the frequency of CD11c+ B cells in SLE patients. PD-1hiCD4+ T cells were found within lupus nephritis kidneys and correlated with B cell numbers in the kidney. Both IL-21 neutralization and CRISPR-mediated deletion of MAF abrogated the ability of Tph cells to induce memory B cell differentiation into plasmablasts in vitro. These findings identify Tph cells as a highly expanded T cell population in SLE and suggest a key role for Tph cells in stimulating pathologic B cell responses.

Nuclear and extranuclear estrogen binding sites in the rat forebrain and autonomic medullary areas.

Immunocytochemical studies have shown that nuclear and extranuclear estrogen receptors (ERs) are present in several extrahypothalamic brain regions. The goal of this study was to determine the subcellular location of functional ERs, particularly extranuclear ERs, by demonstrating (125)I-estradiol binding in the rat forebrain and medullary sections prepared for light and electron microscopic autoradiography. Some sections were immunocytochemically labeled with the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), prior to the autoradiographic procedure. By light microscopy, dense accumulations of silver grains denoting (125)I-estradiol binding were observed over cells in the ventromedial and arcuate hypothalamic nuclei, amygdala, and nucleus of the solitary tract. In sections labeled for TH, large accumulations of silver grains were admixed with TH-labeled processes in the medial nucleus of the amygdala and over TH-labeled perikarya in the medial and commissural nucleus of the solitary tract. Electron microscopic analyses were focused on the rostral ventrolateral medulla and the hippocampal CA1 region, two regions previously shown to have extranuclear ERs. In the rostral ventrolateral medulla, silver grains indicative of (125)I-estradiol binding were found within a few large terminals, affiliated with mitochondria. In the hippocampus, autoradiographic silver grains denoting (125)I-estradiol binding were associated with mitochondria in dendritic shafts or were near synaptic specializations on dendritic spines. These patterns of silver grain labeling were not seen in sections from rats that received (125)I-estradiol combined with cold estradiol. The association of (125)I-estradiol binding with pre- and postsynaptic profiles supports a functional role for nonnuclear ERs in brain.

A human tissue-based functional assay platform to evaluate the immune function impact of small molecule inhibitors that target the immune system.

While the immune system is essential for the maintenance of the homeostasis, health and survival of humans, aberrant immune responses can lead to chronic inflammatory and autoimmune disorders. Pharmacological modulation of drug targets in the immune system to ameliorate disease also carry a risk of immunosuppression that could lead to adverse outcomes. Therefore, it is important to understand the 'immune fingerprint' of novel therapeutics as they relate to current and, clinically used immunological therapies to better understand their potential therapeutic benefit as well as immunosuppressive ability that might lead to adverse events such as infection risks and cancer. Since the mechanistic investigation of pharmacological modulators in a drug discovery setting is largely compound- and mechanism-centric but not comprehensive in terms of immune system impact, we developed a human tissue based functional assay platform to evaluate the impact of pharmacological modulators on a range of innate and adaptive immune functions. Here, we demonstrate that it is possible to generate a qualitative and quantitative immune system impact of pharmacological modulators, which might help better understand and predict the benefit-risk profiles of these compounds in the treatment of immune disorders.

Evidence that estrogen directly and indirectly modulates C1 adrenergic bulbospinal neurons in the rostral ventrolateral medulla.

Blood pressure in women increases after menopause, and sympathetic tone in female rats decreases with estrogen injections in the rostral ventrolateral medulla (RVLM) region that contains bulbospinal C1 adrenergic neurons and is involved in blood pressure control. We investigated the anatomical and physiological basis for estrogen effects in the RVLM. Neurons with alpha- or beta-subtypes of estrogen receptor (ER) immunoreactivity (-ir) overlapped in distribution with tyrosine hydroxylase (TH)-containing C1 neurons. Immunoelectron microscopy revealed that ERalpha- and ERbeta-ir had distinct cellular and subcellular distributions. ERalpha-ir was most commonly in TH-lacking profiles, many of which were axons and peptide-containing afferents that contacted TH-containing dendrites. ERalpha-ir was also in some TH-containing dendrites. ERbeta-ir was most frequently in TH-containing somata and dendrites, particularly on endoplasmic reticula, mitochondria, and plasma membranes. In whole-cell patch clamp recordings from isolated bulbospinal RVLM neurons, 17beta-estradiol dose-dependently reduced voltage-gated Ca(++) currents, especially the long-lasting (L-type) component. This inhibition was reversed by washing or prevented by adding the non-subtype-selective ER antagonist ICI182780. An ERbeta-selective agonist, but not an ERalpha-selective agonist, reproduced the Ca(++) current inhibition. The data indicate that estrogens can modulate the function of RVLM C1 bulbospinal neurons either directly, through extranuclear ERbeta, or indirectly through extranuclear ERalpha in selected afferents. Moreover, Ca(++) current inhibition may underlie the decrease in sympathetic tone evoked by local 17beta-estradiol application. These findings provide a structural and functional basis for the effects of estrogens on blood pressure control and suggest a mechanism for the modulation of cardiovascular function by estrogen in women.

A novel selective androgen receptor modulator (SARM) MK-4541 exerts anti-androgenic activity in the prostate cancer xenograft R-3327G and anabolic activity on skeletal muscle mass & function in castrated mice.

The androgen receptor (AR) is a member of the nuclear hormone receptor super family of transcription factors. Androgens play an essential role in the development, growth, and maintenance of male sex organs, as well as the musculoskeletal and central nervous systems. Yet with advancing age, androgens can drive the onset of prostate cancer, the second leading cause of cancer death in males within the United States. Androgen deprivation therapy (ADT) by pharmacologic and/or surgical castration induces apoptosis of prostate cells and subsequent shrinkage of the prostate and prostate tumors. However, ADT is associated with significant musculoskeletal and behavioral adverse effects. The unique pharmacological activity of selective androgen receptor modulator (SARM) MK-4541 recently has been reported as an AR antagonist with 5α-reductase inhibitor function. The molecule inhibits proliferation and induces apoptosis in AR positive, androgen dependent prostate cancer cells. Importantly, MK-4541 inhibited androgen-dependent prostate growth in male rats yet maintained lean body mass and bone formation following ovariectomy in female rats. In the present study, we evaluated the effects of SARM MK-4541 in the androgen-dependent Dunning R3327-G prostate carcinoma xenograft mouse model as well as on skeletal muscle mass and function, and AR-regulated behavior in mice. MK-4541 significantly inhibited the growth of R3327-G prostate tumors, exhibited anti-androgen effects on the seminal vesicles, reduced plasma testosterone concentrations in intact males, and inhibited Ki67 expression. MK-4541 treated xenografts appeared similar to xenografts in castrated mice. Importantly, we demonstrate that MK-4541 exhibited anabolic activity in androgen deficient conditions, increasing lean body mass and muscle function in adult castrated mice. Moreover, MK-4541 treatment restored general activity levels in castrated mice. Thus, MK-4541 exhibits an optimum profile as an adjuvant therapy to ADT which may provide potent anti-androgenic activity at the prostate yet protective activity on skeletal muscle and behavior in patients.

Inhibition of spleen tyrosine kinase attenuates IgE-mediated airway contraction and mediator release in human precision cut lung slices.

BACKGROUND AND PURPOSE: Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. EXPERIMENTAL APPROACH: Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. KEY RESULTS: While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. CONCLUSIONS AND IMPLICATIONS: A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe.

BACKGROUND: Distinct expression patterns of estrogen receptor (ER)-alpha and ER-beta are displayed in the murine central nervous system. ER-beta is the predominant form of the receptor expressed in the murine midbrain dorsal raphe nucleus (DRN). Tryptophan hydroxylase (TPH) is abundantly expressed in the serotonergic neurons of the DRN and is regulated by estrogen in both the monkey and the guinea pig. METHODS: In this study we used immunocytochemistry to show that ER-beta and TPH are colocalized in the serotonergic cells of the murine DRN. We utilized the ER-alpha and ER-beta gene deletion mouse models and in situ hybridization to demonstrate that ER-beta is responsible for regulating TPH1 mRNA expression. RESULTS: Estrogen increased TPH1 mRNA expression in the DRN of wild type and ER-alpha knockout mice (alpha-ERKO) but not ER-beta knockouts (beta-ERKO). CONCLUSIONS: These data indicate that ER-beta is responsible for mediating estrogen regulated TPH1 expression in the murine DRN.

Ultrastructural localization of estrogen receptor beta immunoreactivity in the rat hippocampal formation.

Several lines of evidence indicate that estrogen affects hippocampal synaptic plasticity through rapid nongenomic mechanisms, possibly by binding to plasma membrane estrogen receptors (ERs). We have previously shown that ERalpha immunoreactivity (ir) is in select interneuron nuclei and in several extranuclear locations, including dendritic spines and axon terminals, within the rat hippocampal formation (Milner et al., [2001] J Comp Neurol 429:355). The present study sought to determine the cellular and subcellular locations of ERbeta-ir. Coronal hippocampal sections from diestrus rats were immunolabeled with antibodies to ERbeta and examined by light and electron microscopy. By light microscopy, ERbeta-ir was primarily in the perikarya and proximal dendrites of pyramidal and granule cells. ERbeta-ir was also in a few nonprincipal cells and scattered nuclei in the ventral subiculum and CA3 region. Ultrastructural analysis revealed ERbeta-ir at several extranuclear sites in all hippocampal subregions. ERbeta-ir was affiliated with cytoplasmic organelles, especially endomembranes and mitochondria, and with plasma membranes primarily of principal cell perikarya and proximal dendrites. ERbeta-ir was in dendritic spines, many arising from pyramidal and granule cell dendrites. In both dendritic shafts and spines, ERbeta-ir was near the perisynaptic zone adjacent to synapses formed by unlabeled terminals. ERbeta-ir was in preterminal axons and axon terminals, associated with clusters of small, synaptic vesicles. ERbeta-labeled terminals formed both asymmetric and symmetric synapses with dendrites. ERbeta-ir also was detected in glial profiles. The cellular and subcellular localization of ERbeta-ir was generally similar to that of ERalpha, except that ERbeta was more extensively found at extranuclear sites. These results suggest that ERbeta may serve primarily as a nongenomic transducer of estrogen actions in the hippocampal formation.