Lacking cytokine production in ES cells and ES-cell-derived vascular cells stimulated by TNF-alpha is rescued by HDAC inhibitor trichostatin A

Inflammation and TNF-alpha signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-alpha receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-alpha stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c-fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c-jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c-fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-alpha stimulation due to low levels of c-fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.

Studies of alpha-adrenoceptor antagonists on sympathetic mydriasis in rabbits

This study was undertaken to identify the alpha-adrenergic receptor type responsible for sympathetically evoked mydriasis in pentobarbital-anesthetized rabbits. Frequency-response curves of pupillary dilation were generated by stimulation of the preganglionic cervical sympathetic nerve (1-64 Hz). Evoked mydriatic responses were inhibited by systemic administration of nonselective alpha-adrenergic antagonists, phentolamine (0.3-10 mg/kg) and phenoxybenzamine (0.03-0.3 mg/kg), as well as the selective alpha(1)-adrenergic antagonist, prazosin (0.1-1 mg/kg). The alpha(2)-adrenergic antagonist, RS 79948 (0.3 mg/kg, i.v.) was without inhibitory effect, but potentiated the mydriatic response. In addition, the selective alpha(1A)-adrenoceptor antagonist, 5-methylurapidil (0.1-1 mg/kg, i.v.), antagonized the elicited mydriasis in a dose-dependent fashion. Unlike previous observations that prazosin does not block the adrenoceptor in rabbit iris dilator muscle, our results suggest that prazosin is effective in inhibiting neuronally elicited mydriasis in this species, and that alpha(1A)-adrenoceptors appear to mediate the response.

Histamine H3 receptor-mediated inhibition of sympathetically evoked mydriasis in rats

This study was designed to determine if the histamine H3 receptor agonist R-alpha-methylhistamine would play a role in modulation of sympathetically evoked mydriasis in anesthetized rats, and if so, to ascertain the specific receptor subtype(s) involved. Reproducible frequency-response curves of pupillary dilation were generated by stimulation of the cervical preganglionic sympathetic nerve (1-32 Hz). Systemic administration of R-alpha-methylhistamine (0.3-3.0 mg kg(-1)) produced a dose-related inhibition of the evoked mydriasis. The greatest inhibition was seen at lower frequency levels, with about 43% depression observed at 2 Hz. The specific histamine H3 receptor antagonist, clobenpropit (3.0 mg kg(-1), i.v.), blocked the inhibitory effect of R-alpha-methylhistamine, whereas neither the histamine H2 receptor antagonist, cimetidine (5.0 mg kg(-1), i.v.), nor the histamine H1 receptor antagonist, chlorpheniramine (0.5 mg kg(-1), i.v.), was effective. The histamine H2 receptor agonist, dimaprit (10 mg kg(-1), i.v.), was also without effect on the evoked mydriasis. R-alpha-methylhistamine (3.0 mg kg(-1)) did not inhibit phenylephrine-induced mydriasis. These results support the conclusion that R-alpha-methylhistamine produces inhibition of sympathetically evoked mydriasis via histamine H3 receptor stimulation, presumably by an action on presynaptic histamine H3 receptors.

Characterization of protein C receptor expression in monocytes

Many sequelae associated with endotoxaemic-induced shock result from excessive production of the cytokine mediators, tumour necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1) and IL-6 from lipopolysaccharide (LPS)-activated monocytes. Protein C (PC)/activated protein C (APC) has potent cytokine-modifying properties and is protective in animal models and human clinical trials of sepsis. The precise mechanism by which this anti-inflammatory response is achieved remains unknown; however, the recently described endothelial protein C receptor (EPCR) appears to be essential for this function. The pivotal role that monocytes play in the pathophysiology of septic shock led us to investigate the possible expression of a protein C receptor on the monocyte membrane. We used similarity algorithms to screen human sequence databases for paralogues of the EPCR but found none. However, using reverse transcription-polymerase chain reaction (RT-PCR), we detected an mRNA transcribed in primary human monocytes and THP1 cells that was identical to human EPCR mRNA. We also used immunocytochemical analysis to demonstrate the expression of a protein C receptor on the surface of monocytes encoded by the same gene as EPCR. These results confirm a new member of the protein C pathway involving primary monocytes. Further characterization will be necessary to compare and contrast its biological properties with those of EPCR.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

Tumor necrosis factor receptor expression and signaling in renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC), a tubular epithelial cell (TEC) malignancy, frequently secretes tumor necrosis factor (TNF). TNF signals via two distinct receptors (TNFRs). TNFR1, expressed in normal kidney primarily on endothelial cells, activates apoptotic signaling kinase 1 and nuclear factor-kappaB (NF-kappaB) and induces cell death, whereas TNFR2, inducibly expressed on endothelial cells and on TECs by injury, activates endothelial/epithelial tyrosine kinase (Etk), which trans-activates vascular endothelial growth factor receptor 2 (VEGFR2) to promote cell proliferation. We investigated TNFR expression in clinical samples and function in short-term organ cultures of ccRCC tissue treated with wild-type TNF or specific muteins selective for TNFR1 (R1-TNF) or TNFR2 (R2-TNF). There is a significant increase in TNFR2 but not TNFR1 expression on malignant TECs that correlates with increasing malignant grade. In ccRCC organ cultures, R1-TNF increases TNFR1, activates apoptotic signaling kinase and NF-kappaB, and promotes apoptosis in malignant TECs. R2-TNF increases TNFR2, activates NF-kappaB, Etk, and VEGFR2 and increases entry into the cell cycle. Wild-type TNF induces both sets of responses. R2-TNF actions are blocked by pretreatment with a VEGFR2 kinase inhibitor. We conclude that TNF, acting through TNFR2, is an autocrine growth factor for ccRCC acting via Etk-VEGFR2 cross-talk, insights that may provide a more effective therapeutic approach to this disease.

Structural basis for the nuclear import of the human androgen receptor

Ligand-dependent nuclear import is crucial for the function of the androgen receptor (AR) in both health and disease. The unliganded AR is retained in the cytoplasm but, on binding 5alpha-dihydrotestosterone, it translocates into the nucleus and alters transcription of its target genes. Nuclear import of AR is mediated by the nuclear import factor importin-alpha, which functions as a receptor that recognises and binds to specific nuclear localisation signal (NLS) motifs on cargo proteins. We show here that the AR binds to importin-alpha directly, albeit more weakly than the NLS of SV40 or nucleoplasmin. We describe the 2.6-angstroms-resolution crystal structure of the importin-alpha-AR-NLS complex, and show that the AR binds to the major NLS-binding site on importin-alpha in a manner different from most other NLSs. Finally, we have shown that pathological mutations within the NLS of AR that are associated with prostate cancer and androgen-insensitivity syndrome reduce the binding affinity to importin-alpha and, subsequently, retard nuclear import; surprisingly, however, the transcriptional activity of these mutants varies widely. Thus, in addition to its function in the nuclear import of AR, the NLS in the hinge region of AR has a separate, quite distinct role on transactivation, which becomes apparent once nuclear import has been achieved.

TNF alpha-Induced p38MAPK Activation Regulates TRPA1 and TRPV4 Activity in Odontoblast-Like Cells

The transient receptor potential (TRP) channels are unique cellular sensors that are widely expressed in many neuronal and nonneuronal cells. Among the TRP family members, TRPA1 and TRPV4 are emerging as candidate mechanosensitive channels that play a pivotal role in inflammatory pain and mechanical hyperalgesia. Odontoblasts are nonneuronal cells that possess many of the features of mechanosensitive cells and mediate important defense and sensory functions. However, the effect of inflammation on the activity of the odontoblast's mechanosensitive channels remains unknown. By using immunohistochemistry and calcium microfluorimetry, we showed that odontoblast-like cells express TRPA1 and TRPV4 and that these channels were activated by hypotonicity-induced membrane stretch. Short treatment of odontoblast-like cells with tumor necrosis factor (TNF)-α enhanced TRPA1 and TRPV4 responses to their chemical agonists and membrane stretch. This enhanced channel activity was accompanied by phospho-p38 mitogen-activated protein kinase (MAPK) expression. Treatment of cells with the p38 inhibitor SB202190 reduced TNF-α effects, suggesting modulation of channel activity via p38 MAPK. In addition, TNF-α treatment also resulted in an up-regulation of TRPA1 expression but down-regulation of TRPV4. Unlike TRPV4, enhanced TRPA1 expression was also evident in dental pulp of carious compared with noncarious teeth. SB202190 treatment significantly reduced TNF-α-induced TRPA1 expression, suggesting a role for p38 MAPK signaling in modulating both the transcriptional and non-transcriptional regulation of TRP channels in odontoblasts.

Transient receptor potential (TRP) channel expression in pulpal fibroblasts

Introduction: Transient receptor potential (TRP) channels are widely, but not uniformly, distributed in tissues. To date the dominant focus of attention has been on TRP expression and functionality in neurons. However, their expression and activation in selected non-neuronal cells suggest TRPs have a potential role in coordinating cross-talk during the inflammatory process. Fibroblasts comprise the major cell type in the dental pulp and play an important role in pulpal inflammation. Objectives: The aim of this study was to investigate the expression and functionality of the TRP channels TRPA1, TRPM8, TRPV4 and TRPV1 in human dental pulp fibroblasts. Methods: Dental pulp fibroblasts were derived by explant culture of pulps removed from extracted healthy teeth. Fibroblasts were cultured in DMEM supplemented with 10% FCS, 100U/ml penicillin and 100µg/ml streptomycin. Protein expression of TRP channels was investigated by SDS- polyacrylamide gel electrophoresis and Western blotting of cell lysates from fibroblast cells in culture. TRPA1, TRPM8, TRPV4 and TRPV1 expression was determined by specific antibodies, detected using appropriate anti-species antibodies and chemiluminescence. Functionality of TRP channels was determined by Ca2+ microfluorimetry. Cells were grown on cover slips and incubated with Fura 2AM prior to stimulation with icilin (TRPA1 agonist), menthol (TRPM8 agonist), 4 alpha-phorbol 12,13-didecanoate (4alphaPDD) (TRPV4 agonist) or capsaicin (TRPV1 agonist). Emitted fluorescence (F340/F380) was used to determine intracellular [Ca2+] levels. Results: Fibroblast expression of TRPA1, TRPM8, TRPV4 and TRPV1 was confirmed at the protein level by Western blotting. Increased intracellular [Ca2+] levels in response to icillin, methanol, 4alphaPDD and capsacin, indicated functional expression of TRPA1, TRPM8, TRPV4 and TRPV respectively. Conclusions: The presence and functionality of TRP channels on dental pulp fibroblasts suggests a potential role for these cells in the pulpal neurogenic inflammatory response. (Supported by a research grant from the Royal College of Surgeons of Edinburgh).

Alpha-2 agonists for sedation of mechanically ventilated adults in intensive care units: a systematic review

BACKGROUND: Care of critically ill patients in intensive care units (ICUs) often requires potentially invasive or uncomfortable procedures, such as mechanical ventilation (MV). Sedation can alleviate pain and discomfort, provide protection from stressful or harmful events, prevent anxiety and promote sleep. Various sedative agents are available for use in ICUs. In the UK, the most commonly used sedatives are propofol (Diprivan(®), AstraZeneca), benzodiazepines [e.g. midazolam (Hypnovel(®), Roche) and lorazepam (Ativan(®), Pfizer)] and alpha-2 adrenergic receptor agonists [e.g. dexmedetomidine (Dexdor(®), Orion Corporation) and clonidine (Catapres(®), Boehringer Ingelheim)]. Sedative agents vary in onset/duration of effects and in their side effects. The pattern of sedation of alpha-2 agonists is quite different from that of other sedatives in that patients can be aroused readily and their cognitive performance on psychometric tests is usually preserved. Moreover, respiratory depression is less frequent after alpha-2 agonists than after other sedative agents.

OBJECTIVES: To conduct a systematic review to evaluate the comparative effects of alpha-2 agonists (dexmedetomidine and clonidine) and propofol or benzodiazepines (midazolam and lorazepam) in mechanically ventilated adults admitted to ICUs.

DATA SOURCES: We searched major electronic databases (e.g. MEDLINE without revisions, MEDLINE In-Process & Other Non-Indexed Citations, EMBASE and Cochrane Central Register of Controlled Trials) from 1999 to 2014.

METHODS: Evidence was considered from randomised controlled trials (RCTs) comparing dexmedetomidine with clonidine or dexmedetomidine or clonidine with propofol or benzodiazepines such as midazolam, lorazepam and diazepam (Diazemuls(®), Actavis UK Limited). Primary outcomes included mortality, duration of MV, length of ICU stay and adverse events. One reviewer extracted data and assessed the risk of bias of included trials. A second reviewer cross-checked all the data extracted. Random-effects meta-analyses were used for data synthesis.

RESULTS: Eighteen RCTs (2489 adult patients) were included. One trial at unclear risk of bias compared dexmedetomidine with clonidine and found that target sedation was achieved in a higher number of patients treated with dexmedetomidine with lesser need for additional sedation. The remaining 17 trials compared dexmedetomidine with propofol or benzodiazepines (midazolam or lorazepam). Trials varied considerably with regard to clinical population, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded outcome assessors. Compared with propofol or benzodiazepines (midazolam or lorazepam), dexmedetomidine had no significant effects on mortality [risk ratio (RR) 1.03, 95% confidence interval (CI) 0.85 to 1.24, I (2) = 0%; p = 0.78]. Length of ICU stay (mean difference -1.26 days, 95% CI -1.96 to -0.55 days, I (2) = 31%; p = 0.0004) and time to extubation (mean difference -1.85 days, 95% CI -2.61 to -1.09 days, I (2) = 0%; p < 0.00001) were significantly shorter among patients who received dexmedetomidine. No difference in time to target sedation range was observed between sedative interventions (I (2) = 0%; p = 0.14). Dexmedetomidine was associated with a higher risk of bradycardia (RR 1.88, 95% CI 1.28 to 2.77, I (2) = 46%; p = 0.001).

LIMITATIONS: Trials varied considerably with regard to participants, type of comparators, dose of sedative agents, outcome measures and length of follow-up. Overall, risk of bias was generally high or unclear. In particular, few trials blinded assessors.

CONCLUSIONS: Evidence on the use of clonidine in ICUs is very limited. Dexmedetomidine may be effective in reducing ICU length of stay and time to extubation in critically ill ICU patients. Risk of bradycardia but not of overall mortality is higher among patients treated with dexmedetomidine. Well-designed RCTs are needed to assess the use of clonidine in ICUs and identify subgroups of patients that are more likely to benefit from the use of dexmedetomidine.

STUDY REGISTRATION: This study is registered as PROSPERO CRD42014014101.

FUNDING: The National Institute for Health Research Health Technology Assessment programme. The Health Services Research Unit is core funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorates.

Melanoma patients respond to a cytotoxic T lymphocyte-defined self-peptide with diverse and nonoverlapping T-cell receptor repertoires.

HLA-A2+ melanoma patients develop naturally a strong CD8+ T cell response to a self-peptide derived from Melan-A. Here, we have used HLA-A2/peptide tetramers to isolate Melan-A-specific T cells from tumor-infiltrated lymph nodes of two HLA-A2+ melanoma patients and analyzed their TCR beta chain V segment and complementarity determining region 3 length and sequence. We found a broad diversity in Melan-A-specific immune T-cell receptor (TCR) repertoires in terms of both TCR beta chain variable gene segment usage and clonal composition. In addition, immune TCR repertoires selected in the patients were not overlapping. In contrast to previously characterized CD8+ T-cell responses to viral infections, this study provides evidence against usage of highly restricted TCR repertoire in the natural response to a self-differentiation tumor antigen.

Peroxisome proliferator-activated receptor structures: ligand specificity, molecular switch and interactions with regulators.

Peroxisome proliferator-activated receptors (PPARs) compose a family of nuclear receptors that mediate the effects of lipidic ligands at the transcriptional level. In this review, we highlight advances in the understanding of the PPAR ligand binding domain (LBD) structure at the atomic level. The overall structure of PPARs LBD is described, and important protein ligand interactions are presented. Structure-activity relationships between isotypes structures and ligand specificity are addressed. It is shown that the numerous experimental three-dimensional structures available, together with in silico simulations, help understanding the role played by the activating function-2 (AF-2) in PPARs activation and its underlying molecular mechanism. The relation between the PPARs constitutive activity and the intrinsic stability of the active conformation is discussed. Finally, the interactions of PPARs LBD with co-activators or co-repressors, as well as with the retinoid X receptor (RXR) are described and considered in relation to PPARs activation.

D-amphetamine fails to increase extracellular dopamine levels in mice lacking alpha 1b-adrenergic receptors: relationship between functional and nonfunctional dopamine release.

It was found recently that locomotor and rewarding effects of psychostimulants and opiates were dramatically decreased or suppressed in mice lacking alpha1b-adrenergic receptors [alpha1b-adrenergic receptor knock-outs (alpha1bAR-KOs)] (Drouin et al., 2002). Here we show that blunted locomotor responses induced by 3 and 6 mg/kg d-amphetamine in alpha1bAR-KO mice [-84 and -74%, respectively, when compared with wild-type (WT) mice] are correlated with an absence of d-amphetamine-induced increase in extracellular dopamine (DA) levels in the nucleus accumbens of alpha1bAR-KO mice. Moreover, basal extracellular DA levels in the nucleus accumbens are lower in alpha1bAR-KO than in WT littermates (-28%; p < 0.001). In rats however, prazosin, an alpha1-adrenergic antagonist, decreases d-amphetamine-induced locomotor hyperactivity without affecting extracellular DA levels in the nucleus accumbens, a finding related to the presence of an important nonfunctional release of DA (Darracq et al., 1998). We show here that local d-amphetamine releases nonfunctional DA with the same affinity but a more than threefold lower amplitude in C57BL6/J mice than in Sprague Dawley rats. Altogether, this suggests that a trans-synaptic mechanism amplifies functional DA into nonfunctional DA release. Our data confirm the presence of a powerful coupling between noradrenergic and dopaminergic neurons through the stimulation of alpha1b-adrenergic receptors and indicate that nonfunctional DA release is critical in the interpretation of changes in extracellular DA levels. These results suggest that alpha1b-adrenergic receptors may be important therapeutic pharmacological targets not only in addiction but also in psychosis because most neuroleptics possess anti-alpha1-adrenergic properties.

CD14 expression on monocytes and TNF alpha production in patients with septic shock, cardiogenic shock or bacterial pneumonia.

OBJECTIVES: In patients with septic shock, circulating monocytes become refractory to stimulation with microbial products. Whether this hyporesponsive state is induced by infection or is related to shock is unknown. To address this question, we measured TNF alpha production by monocytes or by whole blood obtained from healthy volunteers (controls), from patients with septic shock, from patients with severe infection (bacterial pneumonia) without shock, and from patients with cardiogenic shock without infection. MEASUREMENTS: The numbers of circulating monocytes, of CD14+ monocytes, and the expression of monocyte CD14 and the LPS receptor, were assessed by flow cytometry. Monocytes or whole blood were stimulated with lipopolysaccharide endotoxin (LPS), heat-killed Escherichia coli or Staphylococcus aureus, and TNF alpha production was measured by bioassay. RESULTS: The number of circulating monocytes, of CD14+ monocytes, and the monocyte CD14 expression were significantly lower in patients with septic shock than in controls, in patients with bacterial pneumonia or in those with cardiogenic shock (p < 0.001). Monocytes or whole blood of patients with septic shock exhibited a profound deficiency of TNF alpha production in response to all stimuli (p < 0.05 compared to controls). Whole blood of patients with cardiogenic shock also exhibited this defect (p < 0.05 compared to controls), although to a lesser extent, despite normal monocyte counts and normal CD14 expression. CONCLUSIONS: Unlike patients with bacterial pneumonia, patients with septic or cardiogenic shock display profoundly defective TNF alpha production in response to a broad range of infectious stimuli. Thus, down-regulation of cytokine production appears to occur in patients with systemic, but not localised, albeit severe, infections and also in patients with non-infectious circulatory failure. Whilst depletion of monocytes and reduced monocyte CD14 expression are likely to be critical components of the hyporesponsiveness observed in patients with septic shock, other as yet unidentified factors are at work in this group and in patients with cardiogenic shock.

Molecular and therapeutic characterization of anti-ectodysplasin A receptor (EDAR) agonist monoclonal antibodies.

The TNF family ligand ectodysplasin A (EDA) and its receptor EDAR are required for proper development of skin appendages such as hair, teeth, and eccrine sweat glands. Loss of function mutations in the Eda gene cause X-linked hypohidrotic ectodermal dysplasia (XLHED), a condition that can be ameliorated in mice and dogs by timely administration of recombinant EDA. In this study, several agonist anti-EDAR monoclonal antibodies were generated that cross-react with the extracellular domains of human, dog, rat, mouse, and chicken EDAR. Their half-life in adult mice was about 11 days. They induced tail hair and sweat gland formation when administered to newborn EDA-deficient Tabby mice, with an EC(50) of 0.1 to 0.7 mg/kg. Divalency was necessary and sufficient for this therapeutic activity. Only some antibodies were also agonists in an in vitro surrogate activity assay based on the activation of the apoptotic Fas pathway. Activity in this assay correlated with small dissociation constants. When administered in utero in mice or at birth in dogs, agonist antibodies reverted several ectodermal dysplasia features, including tooth morphology. These antibodies are therefore predicted to efficiently trigger EDAR signaling in many vertebrate species and will be particularly suited for long term treatments.