CRH-R2 signalling modulates feeding and circadian gene expression in hypothalamic mHypoA-2/30 neurons.

The hypothalamic type 2 corticotropin releasing hormone receptor (CRH-R2) plays critical roles in homeostatic regulation, particularly in fine tuning stress recovery. During acute stress, the CRH-R2 ligands CRH and urocortins promote adaptive responses and feeding inhibition. However, in rodent models of chronic stress, over-exposure of hypothalamic CRH-R2 to its cognate agonists is associated with urocortin 2 (Ucn2) resistance; attenuated cAMP-response element binding protein (CREB) phosphorylation and increased food intake. The molecular mechanisms involved in these altered CRH-R2 signalling responses are not well described. In the present study, we used the adult mouse hypothalamus-derived cell line mHypoA-2/30 to investigate CRH-R2 signalling characteristics focusing on gene expression of molecules involved in feeding and circadian regulation given the role of clock genes in metabolic control. We identified functional CRH-R2 receptors expressed in mHypoA-2/30 cells that differentially regulate CREB and AMP-activated protein kinase (AMPK) phosphorylation and downstream expression of the appetite-regulatory genes proopiomelanocortin (Pomc) and neuropeptide Y (Npy) in accordance with an anorexigenic effect. We studied for the first time the effects of Ucn2 on clock genes in native and in a circadian bioluminescence reporter expressing mHypoA-2/30 cells, detecting enhancing effects of Ucn2 on mRNA levels and rhythm amplitude of the circadian regulator Aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), which could facilitate anorexic responses in the activity circadian phase. These data uncover novel aspects of CRH-R2 hypothalamic signalling that might be important in regulation of circadian feeding during stress responses.

Impact of treatment variability and clinicopathological characteristics on survival in patients with Epstein-Barr-Virus positive diffuse large B cell lymphoma.

Patients with EBV-positive diffuse large B cell lymphoma not otherwise specified (EBV+ DLBCL (NOS)) recurrently present with advanced age and reduced performance status. They are therefore insufficiently represented in clinical trials and treatment is likely to differ. Here we assess clinicopathological characteristics, therapeutic variability and clinical outcome in the largest consecutively diagnosed EBV+ DLBCL (NOS) cohort published to date (n = 80; median age 70 years; range 19-90). Centralized and systematic haematopathological panel review was performed. By immunohistochemistry 60/80 patients were CD30-positive. Further, we identified nine EBV+ DLBCL (NOS) patients with associated or composite peripheral T cell lymphoma at diagnosis or relapse (preceded by clonal T cell populations within the initial DLBCL biopsy in 4/5 cases). Most patients (80%) were treated with R-CHOP-type therapy and 16 patients received none or less intensiveprotocols. Upon univariate analysis both R-CHOP-type therapy (OS: P < 0.0001; PFS: P = 0.0617) and negativity for CD30 (OS: P = 0.0002; PFS: P = 0.0002) showed a protective 66 effect, maintained upon multivariate analysis. In a propensity-score matched analysis with a cohort of non-EBV+ DLBCL (NOS) patients, balanced for all revised-international prognostic index factors, we found an EBV-association to hold no significant impact on progression-free and overall survival whilst exhibiting a trend favouring EBV-negativity (OS: P = 0.116; PFS: P = 0.269). Our findings provide insight into the clinical course of EBV+ DLBCL (NOS), highlight the ramifications of CD30-expression and underline the superior therapeutic efficacy of R-CHOP immunochemotherapy. Alternative therapies, incorporating tumour biology (e.g. CD30 directed therapies) need to be explored in EBV+ DLBCL (NOS) patients. Moreover our data advert to the close relationship between EBV+ DLBCL (NOS) and peripheral T cell lymphomas.

The thermogenic effect of nesfatin-1 requires recruitment of the melanocortin system.

Nesfatin-1 is a bioactive polypeptide expressed both in the brain and peripheral tissues and involved in the control of energy balance by reducing food intake. Central administration of nesfatin-1 significantly increases energy expenditure, as demonstrated by a higher dry heat loss; yet, the mechanisms underlying the thermogenic effect of central nesfatin-1 remain unknown. Therefore, in this study, we sought to investigate whether the increase in energy expenditure induced by nesfatin-1 is mediated by the central melanocortin pathway, which was previously reported to mediate central nesfatin-1´s effects on feeding and numerous other physiological functions. With the application of direct calorimetry, we found that intracerebroventricular nesfatin-1 (25 pmol) treatment increased dry heat loss and that this effect was fully blocked by simultaneous administration of an equimolar dose of the melanocortin 3/4 receptor antagonist, SHU9119. Interestingly, the nesfatin-1-induced increase in dry heat loss was positively correlated with body weight loss. In addition, as assessed with thermal imaging, intracerebroventricular nesfatin-1 (100 pmol) increased interscapular brown adipose tissue (iBAT) as well as tail temperature, suggesting increased heat production in the iBAT and heat dissipation over the tail surface. Finally, nesfatin-1 upregulated pro-opiomelanocortin and melanocortin 3 receptor mRNA expression in the hypothalamus, accompanied by a significant increase in iodothyronine deiodinase 2 and by a nonsignificant increase in uncoupling protein 1 and peroxisome proliferator-activated receptor gamma coactivator-1 alpha mRNA in the iBAT. Overall, we clearly demonstrate that nesfatin-1 requires the activation of the central melanocortin system to increase iBAT thermogenesis and, in turn, overall energy expenditure.

Transforming Growth Factor-ß1/Activin Receptor-like Kinase 5-Mediated Cell Migration is Dependent on the Protein Proteinase-Activated Receptor 2 but not on Proteinase-Activated Receptor 2-Stimulated Gq-Calcium Signaling.

Transforming growth factor-ß (TGF-ß), serine proteinases such as trypsin, and proteinase-activated receptor 2 (PAR2) promote tumor development by stimulating invasion and metastasis. Previously, we found that in cancer cells derived from pancreatic ductal adenocarcinoma (PDAC) PAR2 protein is necessary for TGF-ß1-dependent cell motility. Here, we show in the same cells that, conversely, the type I TGF-ß receptor activin receptor-like kinase 5 is dispensable for trypsin and PAR2 activating peptide (PAR2-AP)-induced migration. To reveal whether Gq-calcium signaling is a prerequisite for PAR2 to enhance TGF-ß signaling, we investigated the effects of PAR2-APs, PAR2 mutation and PAR2 inhibitors on TGF-ß1-induced migration, reporter gene activity, and Smad activation. Stimulation of cells with PAR2-AP alone failed to enhance basal or TGF-ß1-induced C-terminal phosphorylation of Smad3, Smad-dependent activity of a luciferase reporter gene, and cell migration. Consistently, in complementary loss of function studies, abrogation of the PAR2-Gq-calcium signaling arm failed to suppress TGF-ß1-induced cell migration, reporter gene activity, and Smad3 activation. Together, our findings suggest that the calcium-regulating motif is not required for PAR2 to synergize with TGF-ß1 to promote cell motility. Additional experiments in PDAC cells revealed that PAR2 and TGF-ß1 synergy may involve TGF-ß1 induction of enzymes that cause autocrine cleavage/activation of PAR2, possibly through a biased signaling function. Our results suggest that although reducing PAR2 protein expression may potentially block TGF-ß's prooncogenic function, inhibiting PAR2-Gq-calcium signaling alone would not be sufficient to achieve this effect.

Tissue-Specific Dissociation of Diurnal Transcriptome Rhythms During Sleep Restriction in Mice.

Study objectives: Shortened or mistimed sleep affects metabolic homeostasis, which may in part be mediated by dysregulation of endogenous circadian clocks. In this study, we assessed the contribution of sleep disruption to metabolic dysregulation by analysing diurnal transcriptome regulation in metabolic tissues of mice subjected to a sleep restriction (SR) paradigm. Methods: Male mice were subjected to 2 × 5 days of SR with enforced waking during the first 6 hours of the light phase. SR and control mice were sacrificed at different time points of the day and RNA preparations from the mediobasal hypothalamus (MBH), liver, and epididymal white adipose tissue (eWAT) were subjected to whole-genome microarray hybridization. Transcriptional rhythms were associated with changes in behavioral and physiological parameters such as sleep, body temperature, and food intake. Rhythm detection was performed with CircWave and transcription profiles were compared by 2-way analysis of variance and t-tests with Benjamini-Hochberg corrections. Results: Clock gene rhythms were blunted in all tissues, while transcriptome regulation was associated with either clock gene expression, sleep patterns, or food intake in a tissue-specific manner. Clock gene expression was associated with apoptosis pathways in the MBH and with tumor necrosis factor alpha signalling in liver. Food intake-associated genes included cilium movement genes in the MBH and lipid metabolism-associated transcripts in liver. Conclusions: In mice, repeated SR profoundly alters behavioral and molecular diurnal rhythms, disrupting essential signalling pathways in MBH, liver, and eWAT, which may underlie the metabolic and cognitive disturbances observed in sleep-restricted humans such as shift workers.

Nesfatin-1: functions and physiology of a novel regulatory peptide.

Nesfatin-1 was identified in 2006 as a potent anorexigenic peptide involved in the regulation of homeostatic feeding. It is processed from the precursor-peptide NEFA/nucleobindin 2 (NUCB2), which is expressed both in the central nervous system as well as in the periphery, from where it can access the brain via non-saturable transmembrane diffusion. In hypothalamus and brainstem, nesfatin-1 recruits the oxytocin, the melancortin and other systems to relay its anorexigenic properties. NUCB2/nesfatin-1 peptide expression in reward-related areas suggests that nesfatin-1 might also be involved in hedonic feeding. Besides its initially discovered anorexigenic properties, over the last years, other important functions of nesfatin-1 have been discovered, many of them related to energy homeostasis, e.g. energy expenditure and glucose homeostasis. Nesfatin-1 is not only affecting these physiological processes but also the alterations of the metabolic state (e.g. fat mass, glycemic state) have an impact on the synthesis and release of NUCB2 and/or nesfatin-1. Furthermore, nesfatin-1 exerts pleiotropic actions at the level of cardiovascular and digestive systems, as well as plays a role in stress response, behavior, sleep and reproduction. Despite the recent advances in nesfatin-1 research, a putative receptor has not been identified and furthermore potentially distinct functions of nesfatin-1 and its precursor NUCB2 have not been dissected yet. To tackle these open questions will be the major objectives of future research to broaden our knowledge on NUCB2/nesfatin-1.

Intranasal leptin reduces appetite and induces weight loss in rats with diet-induced obesity (DIO).

Resistance to brain-mediated effects of leptin is a characteristic feature of obesity, resulting from alterations in leptin receptor signaling in hypothalamic neurons and/or transport across the blood-brain-barrier. We have shown previously, that the latter can be circumvented by intranasal (i.n.) application of leptin in lean rats. This prompted us to test i.n. leptin in animals with diet-induced obesity (DIO) as a basis for future human administration. DIO was induced in male Wistar rats by feeding a cafeteria diet for 25 or 32 wk, respectively. Consecutively, these DIO animals (seven to eight per treatment) and standard diet rats (lean) (14-15 per treatment, matched for age and diet duration) were treated with 0.1, 0.2 mg/kg leptin, or control solution i.n. daily for 4 wk before onset of dark period. Energy intake and body weight were measured daily; blood glucose, serum insulin, and leptin were measured before and after treatment. Expression of hypothalamic neuropeptides was assessed by quantitative real-time PCR. We demonstrate, for the first time, that i.n. leptin reduces appetite and induces weight loss in DIO to the same extent as in lean rats. Our findings are supported accordingly by an altered expression pattern of anorexigenic and orexigenic neuropeptides in the hypothalamus, e.g. proopiomelanocortin, cocaine and amphetamine-related transcript, neuropeptide Y, agouti-related protein. It now appears clear that i.n. leptin is effectively acting in obese animals in the same fashion as in their lean counterparts. These findings now clearly warrant studies in humans and may open new perspectives in the treatment of obesity.

Endogenous ACTH, not only alpha-melanocyte-stimulating hormone, reduces food intake mediated by hypothalamic mechanisms.

ACTH and alpha-melanocyte-stimulating hormone (alpha-MSH) are both consecutively processed from proopiomelanocortin (POMC), which is synthesized in hypothalamic arcuate neurons innervating the paraventricular nuclei (PVN). POMC secretion/synthesis is regulated by energy availability. ACTH and alpha-MSH bind with equal affinity to melanocortin-4 receptors and elicit similar effects on signal transduction in-vitro. Endogenous alpha-MSH thus far is believed to be the major physiological agonist and to act in an anorexigenic manner. Until now, it was fully unknown whether endogenous ACTH is also involved in the regulation of appetite and food intake. In this study in rats, we now show that icv ACTH as well as alpha-MSH possess anorexigenic effects in the PVN or areas in close proximity in vivo and that the effect of ACTH is direct and not mediated via alpha-MSH. We investigated the roles of endogenous ACTH and alpha-MSH by PVN application of the respective antibodies under different physiological conditions. In satiated rats with high levels of ACTH and alpha-MSH in the PVN, antibody administration increased food intake and body weight gain; hungry animals were unaffected. Finally, repeated injections of ACTH antibodies into PVN resulted in persistently increased food intake during the light period. These data now provide robust evidence that endogenous ACTH without further processing acts in the PVN or areas in close proximity to reduce food intake under conditions of feeding-induced satiety.

Brain uptake of intranasally applied radioiodinated leptin in Wistar rats.

Leptin is mainly synthesized and secreted by fat cells in proportion to adipose tissue mass. Under physiological conditions, this hormone reduces food intake and increases thermogenesis through interactions with neurons in the central nervous system. However, transport of leptin into the central nervous system via the blood-brain barrier is saturable, and in obesity the feedback signal to the brain is markedly insufficient. In recent experiments we have shown, that intranasal (i.n.) delivery of leptin reduces food intake in rats. The aim of the present study was to explore the distribution of i.n. delivered leptin within brain, blood, and peripheral tissues. Application of [(125)I]leptin (0.03, 0.1, and 0.2 mg/kg) into male Wistar rats' nares (n = 8 per group) leads to supraphysiological brain leptin concentrations 30 min after application, with contents in the hypothalamus (7.3 +/- 2.6, 5.9 +/- 1.6, and 13.8 +/- 5.7 ng/g; P = 0.023; F = 6.157) being significantly higher than the brain average (1.2 +/- 0.2, 3.9 +/- 1.0, and 6.0 +/- 1.9 ng/g). In contrast, contents in the occipital/entorhinal cortex were lower than the brain average, indicating a minor participation of the transport via cerebrospinal fluid, which would have favored cerebrospinal fluid-exposed surfaces. In experiments employing the application of unlabeled leptin administered iv, we were able to show that excess blood leptin does not diminish brain uptake of i.n. leptin (as indicated by [(125)I]leptin), supporting a direct transport from nose to brain by circumvention of the blood-brain barrier. This study thus clearly demonstrates a rapid and highly effective transport of leptin from nose to brain.

Central nervous and metabolic effects of intranasally applied leptin.

In obesity, due to the resistance of leptin receptors at the blood brain barrier, increased peripheral leptin levels cannot act appropriately at brain sites relevant for appetite regulation. In this study, we focused on the intranasal application of leptin. This mode of administration provides a promising tool for a direct access of peptides to the brain by circumventing the blood brain barrier. Male Wistar rats were treated daily with 0.1 or 0.2 mg/kg leptin intranasally for 4 wk. Compared with controls, leptin-treated animals gained significantly less weight and exhibited significantly reduced food and water intake. Corticotropin-releasing factor mRNA expression in the paraventricular nucleus showed a tendency for up-regulation by leptin; neuropeptide Y mRNA expression in the arcuate nucleus was decreased. In the central nucleus of the amygdala, corticotropin-releasing factor mRNA was significantly elevated in leptin-treated animals, suggesting a role in affective and/or emotional aspects of food intake. Serum leptin levels were unchanged, indicating a direct action of leptin in the central nervous system without prior access to the periphery. The intranasal application thus represents a useful tool to administer leptin in a noninvasive way with rapid permeation into the central nervous system.

[Microbiological aspects and antibiotic therapy of diabetic foot infections]. / Mikrobiologische Aspekte und rationelle antibiotische Therapie des diabetischen Fusssyndroms.

IMMUNOLOGICAL AND MICROBIOLOGICAL ASPECTS OF DIABETIC FOOT INFECTIONS: Diabetic patients are at increased risk of severe skin and bone infections. Immunological disturbances are reasonable and due to altered specific and unspecific cellular immune responses. Analysis of epidemiology and microbial pathogenicity shows that staphylococci seem to be predestined to induce such infections. Staphylococcus aureus and coagulase-negative staphylococci are able to adhere to the wound ground by a sequela of mechanisms. Initial bacterial adherence is due to hydrophobicity, ion exchanges, and specific binding of bacterial adhesion molecules to cellular receptors. Moreover, staphylococci secrete polysaccharides which form a biofilm together with multilayer cell clusters. The highly structured communities within a biofilm are resistant to distinct immunoeffectors and have a decreased susceptibility to antibiotics in vivo. ASPECTS OF ANTIBIOTIC THERAPY: Assessing the severity of an infection is essential to selecting an antibiotic regimen, the mode of drug administration, and the duration of therapy. Regimens for severe and chronic infections are broader spectrum and often intravenously to obtain high drug concentrations immediately. Infections of the bone often require an antibiotic therapy for > 4 weeks, while a 1- to 2-week therapy for mild to moderate infections has been found to be effective. CONCLUSIONS: Because of the tremendous progress in diagnostics and therapy of diabetic foot infections, infectious complications can be successfully treated by appropriate wound care, metabolic control, and early surgical and antibiotic intervention. Bacterial biofilms involved into chronic infections are new aspects currently not visualized by clinical therapy. Besides the classic antimicrobial therapy, new concepts of an enzymatic therapy or the inhibition of bacterial "communication" (quorum sensing) are in progress and the hope for the future.