Toll-like receptor 2 is highly expressed in lesions of acne inversa and colocalizes with C-type lectin receptor

BACKGROUND: Acne inversa (hidradenitis suppurativa) is a chronic inflammatory and cicatricial disorder that affects skin areas rich in apocrine glands and terminal hairs, such as perineum and axillae. The exact pathogenesis of the disease is not well understood and the mechanisms by which bacterial superinfection contributes to the disease progression are not clear. Toll-like receptors (TLRs) expressed by inflammatory cells play a crucial role in the innate immune response to bacteria. OBJECTIVES: We sought to investigate the role of TLR2 in the pathogenesis of acne inversa. METHODS: We investigated the expression of TLR2 using real-time polymerase chain reaction analysis and immunohistochemical stainings of tissue samples from patients with acne inversa. Furthermore, we phenotypically characterized the infiltrating cells and their expression of TLR2. RESULTS: Compared with normal skin, a highly increased in situ expression of TLR2 in acne inversa skin lesions was found at both the mRNA and the protein level. The most abundant cells in the dermal infiltrate of acne inversa were CD68+ macrophages, CD209+ dendritic cells (DCs) and CD3+ T cells. CD19+ B cells and CD56+ natural killer cells were found only in small numbers. Double staining with fluorescence-labelled antibodies showed that TLR2 was expressed by infiltrating macrophages (CD68+) and DCs (CD209+). Flow cytometric analysis of isolated infiltrating cells further confirmed surface expression of TLR2 by macrophages and DCs. CONCLUSIONS: These data indicate that the enhanced expression of TLR2 by infiltrating macrophages and DCs may contribute to the pathogenesis of inflammatory lesions of acne inversa.

Adenosine A2A receptor gene expression in the normal striatum and after 6-OH-dopamine lesion

Adenosine A2A receptors are present on enkephalinergic medium sized striatal neurons in the rat and have an important function in the modulation of striatal output. In order to establish more accurately whether adenosine transmission is a generalized phenomenon in mammalian striatum we compared the A2A R expression in the mouse, rat, cat and human striatum. Secondly we compared the modulation of enkephalin gene expression and A2A receptor gene expression in rat striatal neurons after 6-OH-dopamine lesion of the substantia nigra. Hybridization histochemistry was performed with a 35S-labelled radioactive oligonucleotide probe. The results showed high expression of A2A adenosine receptor genes only in the medium-sized cells of the striatum in all examined species. In the rat striatum, expression of A2A receptors was not significantly altered after lesion of the dopaminergic pathways with 6-OH-dopamine even though enkephalin gene expression was up-regulated. The absence of a change in A2A receptor gene expression after 6-OH-dopamine treatment speaks against a dependency on dopaminergic innervation. The maintained inhibitory function of A2A R on motor activity in spite of dopamine depletion could be partly responsible for the depression of locomotor activity observed in basal ganglia disorders such as Parkinson's disease.

A 5-year longitudinal study of the relationships between stress, coping, and immune cell beta(2)-adrenergic receptor sensitivity

Caring for a spouse with Alzheimer's disease (AD) is associated with overall health decline and impaired cardiovascular functioning. This morbidity may be related to the effects of caregiving stress and impaired coping on beta(2)-adrenergic receptors, which mediate hemodynamic and vascular responses and are important for peripheral blood mononuclear cell (PBMC) trafficking and cytokine production. This study investigated the longitudinal relationship between stress, personal mastery, and beta(2)-adrenergic receptor sensitivity assessed in vitro on PBMC. Over a 5-year study, 115 spousal AD caregivers completed annual assessments of caregiving stress, mastery, and PBMC beta(2)-adrenergic receptor sensitivity, as assessed by in vitro isoproterenol stimulation. Heightened caregiving stress was associated with significantly decreased receptor sensitivity, whereas greater sense of personal mastery was associated with significantly increased receptor sensitivity. These results suggest that increased stress may be associated with a desensitization of beta(2)-receptors, which may contribute to the development of illness among caregivers. However, increased mastery is associated with increased receptor sensitivity, and may therefore serve as a resource factor for improved health in this population.

Highly potent 4-amino-indolo[2,3-c]azepin-3-one-containing somatostatin mimetics with a range of sst receptor selectivities

The synthesis, biological evaluation, and conformational analysis of 4-amino-indolo[2,3-c]azepin-3-one (Aia)-containing SRIF mimetics are reported. Different subtype selectivities are observed depending on the N- and C-terminal substituents of the D-Aia-Lys dipeptide mimetic. An sst(5)-selective analogue with subnanomolar binding affinity was obtained that is the most potent agonist reported to date. A nonselective mimetic with high potency was also identified. This study allows a better definition of the bioactive conformation of the essential D-Trp side chain in the somatostatin pharmacophore.

Effects of Toll-like receptor 2 agonist Pam(3)CysSK(4) on inflammation and brain damage in experimental pneumococcal meningitis

TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam(3)CysSK(4) was applied intracisternally (0.5 microg in 10 microl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam(3)CysSK(4) on apoptosis was investigated by intracisternal application of 0.5 microg in 10 microl saline and 40 microg in 20 microl saline. Pam(3)CysSK(4) neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam(3)CysSK(4) induced pleocytosis, TNF-alpha and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam(3)CysSK(4) at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.

Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway

Studies from our lab have shown that decreasing myocardial G protein-coupled receptor kinase 2 (GRK2) activity and expression can prevent heart failure progression after myocardial infarction. Since GRK2 appears to also act as a pro-death kinase in myocytes, we investigated the effect of cardiomyocyte-specific GRK2 ablation on the acute response to cardiac ischemia/reperfusion (I/R) injury. To do this we utilized two independent lines of GRK2 knockout (KO) mice where the GRK2 gene was deleted in only cardiomyocytes either constitutively at birth or in an inducible manner that occurred in adult mice prior to I/R. These GRK2 KO mice and appropriate control mice were subjected to a sham procedure or 30 min of myocardial ischemia via coronary artery ligation followed by 24 hrs reperfusion. Echocardiography and hemodynamic measurements showed significantly improved post-I/R cardiac function in both GRK2 KO lines, which correlated with smaller infarct sizes in GRK2 KO mice compared to controls. Moreover, there was significantly less TUNEL positive myocytes, less caspase-3, and -9 but not caspase-8 activities in GRK2 KO mice compared to control mice after I/R injury. Of note, we found that lowering cardiac GRK2 expression was associated with significantly lower cytosolic cytochrome C levels in both lines of GRK2 KO mice after I/R compared to corresponding control animals. Mechanistically, the anti-apoptotic effects of lowering GRK2 expression were accompanied by increased levels of Bcl-2, Bcl-xl, and increased activation of Akt after I/R injury. These findings were reproduced in vitro in cultured cardiomyocytes and GRK2 mRNA silencing. Therefore, lowering GRK2 expression in cardiomyocytes limits I/R-induced injury and improves post-ischemia recovery by decreasing myocyte apoptosis at least partially via Akt/Bcl-2 mediated mitochondrial protection and implicates mitochondrial-dependent actions, solidifying GRK2 as a pro-death kinase in the heart.

Sphingosine 1-phosphate (S1P) induces COX-2 expression and PGE2 formation via S1P receptor 2 in renal mesangial cells

Understanding the mechanisms of sphingosine 1-phosphate (S1P)-induced cyclooxygenase (COX)-2 expression and prostaglandin E2 (PGE2) formation in renal mesangial cells may provide potential therapeutic targets to treat inflammatory glomerular diseases. Thus, we evaluated the S1P-dependent signaling mechanisms which are responsible for enhanced COX-2 expression and PGE2 formation in rat mesangial cells under basal conditions. Furthermore, we investigated whether these mechanisms are operative in the presence of angiotensin II (Ang II) and of the pro-inflammatory cytokine interleukin-1β (IL-1β). Treatment of rat and human mesangial cells with S1P led to concentration-dependent enhanced expression of COX-2. Pharmacological and molecular biology approaches revealed that the S1P-dependent increase of COX-2 mRNA and protein expression was mediated via activation of S1P receptor 2 (S1P2). Further, inhibition of Gi and p42/p44 MAPK signaling, both downstream of S1P2, abolished the S1P-induced COX-2 expression. In addition, S1P/S1P2-dependent upregulation of COX-2 led to significantly elevated PGE2 levels, which were further potentiated in the presence of Ang II and IL-1β. A functional consequence downstream of S1P/S1P2 signaling is mesangial cell migration that is stimulated by S1P. Interestingly, inhibition of COX-2 by celecoxib and SC-236 completely abolished the migratory response. Overall, our results demonstrate that extracellular S1P induces COX-2 expression via activation of S1P2 and subsequent Gi and p42/p44 MAPK-dependent signaling in renal mesangial cells leading to enhanced PGE2 formation and cell migration that essentially requires COX-2. Thus, targeting S1P/S1P2 signaling pathways might be a novel strategy to treat renal inflammatory diseases.

Randomised trial of clazosentan, an endothelin receptor antagonist, in patients with aneurysmal subarachnoid hemorrhage undergoing surgical clipping (CONSCIOUS-2)

We report here results of a randomized, double-blind, placebo-controlled study ( http://www.ClinicalTrials.gov , NCT00558311) that investigated the effect of clazosentan (5 mg/h, n = 768) or placebo (n = 389) administered for up to 14 days in patients with aneurysmal subarachnoid hemorrhage (SAH) repaired by surgical clipping. The primary endpoint was a composite of all-cause mortality, new cerebral infarction or delayed ischemic neurological deficit due to vasospasm, and rescue therapy for vasospasm. The main secondary endpoint was the Glasgow Outcome Scale Extended (GOSE), which was dichotomized. Twenty-one percent of clazosentan- compared to 25% of placebo-treated patients met the primary endpoint (relative risk reduction [RRR] [95% CI]: 17% [-4% to 33%]; p = 0.10). Poor outcome (GOSE score ≤ 4) occurred in 29% of clazosentan- and 25% of placebo-treated patients (RRR: -18% [-45% to 4%]; p = 0.10). In prespecified subgroups, mortality/vasospasm-related morbidity was reduced in clazosentan-treated patients by 33% (8-51%) in poor WFNS (World Federation of Neurological Surgeons) grade (≥III) and 25% (5-41%) in patients with diffuse, thick SAH. Lung complications, anemia and hypotension occurred more frequently with clazosentan. Mortality (week 12) was 6% in both groups. The results showed that clazosentan nonsignificantly decreased mortality/vasospasm-related morbidity and nonsignificantly increased poor functional outcome in patients with aneurysmal SAH undergoing surgical clipping.

Toll-like receptor 2-deficient mice are highly susceptible to Streptococcus pneumoniae meningitis because of reduced bacterial clearing and enhanced inflammation

Toll-like receptor-2 (TLR2) mediates host responses to gram-positive bacterial wall components. TLR2 function was investigated in a murine Streptococcus pneumoniae meningitis model in wild-type (wt) and TLR2-deficient (TLR2(-/-)) mice. TLR2(-/-) mice showed earlier time of death than wt mice (P<.02). Plasma interleukin-6 levels and bacterial numbers in blood and peripheral organs were similar for both strains. With ceftriaxone therapy, none of the wt but 27% of the TLR2(-/-) mice died (P<.04). Beyond 3 hours after infection, TLR2(-/-) mice had higher bacterial loads in brain than did wt mice, as assessed with luciferase-tagged S. pneumoniae by means of a Xenogen-CCD (charge-coupled device) camera. After 24 h, tumor necrosis factor activity was higher in cerebrospinal fluid of TLR2(-/-) than wt mice (P<.05) and was related to increased blood-brain barrier permeability (Evans blue staining, P<.02). In conclusion, the lack of TLR2 was associated with earlier death from meningitis, which was not due to sepsis but to reduced brain bacterial clearing, followed by increased intrathecal inflammation.

Dopamine receptor D4 polymorphism predicts the effect of L-DOPA on gambling behavior

BACKGROUND There is ample evidence that a subgroup of Parkinson's disease patients who are treated with dopaminergic drugs develop certain behavioral addictions such as pathological gambling. The fact that only a subgroup of these patients develops pathological gambling suggests an interaction between dopaminergic drug treatment and individual susceptibility factors. These are potentially of genetic origin, since research in healthy subjects suggests that vulnerability for pathological gambling may be linked to variation in the dopamine receptor D4 (DRD4) gene. Using a pharmacogenetic approach, we investigated how variation in this gene modulates the impact of dopaminergic stimulation on gambling behavior in healthy subjects. METHODS We administered 300 mg of L-dihydroxyphenylalanine (L-DOPA) or placebo to 200 healthy male subjects who were all genotyped for their DRD4 polymorphism. Subjects played a gambling task 60 minutes after L-DOPA administration. RESULTS Without considering genetic information, L-DOPA administration did not lead to an increase in gambling propensity compared with placebo. As expected, however, an individual's DRD4 polymorphism accounted for variation in gambling behavior after the administration of L-DOPA. Subjects who carry at least one copy of the 7-repeat allele showed an increased gambling propensity after dopaminergic stimulation. CONCLUSIONS These findings demonstrate that genetic variation in the DRD4 gene determines an individual's gambling behavior in response to a dopaminergic drug challenge. They may have implications for the treatment of Parkinson's disease patients by offering a genotype approach for determining individual susceptibilities for pathological gambling and may also afford insights into the vulnerability mechanisms underlying addictive behavior.

T-cell reprogramming through targeted CD4-coreceptor and T-cell receptor expression on maturing thymocytes by latent Circoviridae family member porcine circovirus type 2 cell infections in the thymus

Although porcine circovirus type 2 (PCV2)-associated diseases have been evaluated for known immune evasion strategies, the pathogenicity of these viruses remained concealed for decades. Surprisingly, the same viruses that cause panzootics in livestock are widespread in young, unaffected animals. Recently, evidence has emerged that circovirus-like viruses are also linked to complex diseases in humans, including children. We detected PCV2 genome-carrying cells in fetal pig thymi. To elucidate virus pathogenicity, we developed a new pig infection model by in vivo transfection of recombinant PCV2 and the immunosuppressant cofactor cyclosporine A. Using flow cytometry, immunofluorescence and fluorescence in situ hybridization, we found evidence that PCV2 dictates positive and negative selection of maturing T cells in the thymus. We show for the first time that PCV2-infected cells reside at the corticomedullary junction of the thymus. In diseased animals, we found polyclonal deletion of single positive cells (SPs) that may result from a loss of major histocompatibility complex class-II expression at the corticomedullary junction. The percentage of PCV2 antigen-presenting cells correlated with the degree of viremia and, in turn, the severity of the defect in thymocyte maturation. Moreover, the reversed T-cell receptor/CD4-coreceptor expression dichotomy on thymocytes at the CD4(+)CD8(interm) and CD4SP cell stage is viremia-dependent, resulting in a specific hypo-responsiveness of T-helper cells. We compare our results with the only other better-studied member of Circoviridae, chicken anemia virus. Our data show that PCV2 infection leads to thymocyte selection dysregulation, adding a valuable dimension to our understanding of virus pathogenicity.