Novel stable analogues of the neurotensin C-terminal hexapeptide containing unnatural amino acids.

Neurotensin (NT) (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) exerts a dual function as a neurotransmitter/neuromodulator in the central nervous system and as a hormone/cellular mediator in periphery. This dual function of NT establishes a connection between brain and peripheral tissues that renders this peptide a central player in energy homeostasis. Many biological actions of NT are mediated through its interaction with three types of NT receptors (NTS receptors). Despite its role in energy homeostasis, NT has a short half-life that hampers further determination of the biological actions of this peptide and its receptors in brain and periphery. The short half-life of NT is due to the proteolytic degradation of its C-terminal side by several endopeptidases. Therefore, it is important to synthesize NT analogues with resistant bonds against metabolic deactivation. Based on these findings, we herein report the synthesis of ten linear, two cyclic and two dimeric analogues of NT with modifications in its structure that improve their metabolic stability, while retaining the ability to bind to NTS receptors. Modifications at position 11 (introduction of D-Tyrosine (OEthyl) [D-Tyr(Et)] or D-1-naphtylalanine [D-1-Nal] were combined with introduction of a L-Lysine or a D-Arginine at positions 8 or 9, and 1-[2-(aminophenyl)-2-oxoethyl]-1H-pyrrole-2-carboxylic acid (AOPC) at positions 7 or 8, resulting in compounds NT4-NT21. AOPC is an unnatural amino acid with promise in applications as a building block for the synthesis of peptidomimetic compounds. To biologically evaluate these analogues, we determined their plasma stability and their binding affinities to type 1 NT receptor (NTS1), endogenously expressed in HT-29 cells, Among the fourteen NT analogues, compounds, NT5, NT6, and NT8, which have D-Tyr(Et) at position 11, bound to NTS1 in a dose-response manner and with relatively high affinity but still lower than that of the natural peptide. Despite their lower binding affinities compared to NT, the NT5, NT6, and NT8 exhibited a remarkably higher stability, as a result of their chemistry, which provides protection from enzymatic activity. These results will set the basis for the rational design of novel NT molecules with improved pharmacological properties and enhanced enzymatic stability.

Effect of a 6-week supervised detraining period on bone metabolism markers and their association with ergometrics and components of the hypothalamic-pituitary-gonadal (HPG) axis in professional male soccer players.

The aim of this study was to examine the effect of a supervised 6-week detraining period on bone metabolism markers, and their association with ergometrics, and components of the hypothalamic-pituitary-gonadal (HPG) axis in elite male professional soccer players. Sixty-seven soccer players (mean age ± SD 23.4 ± 5.2 years) that were following a supervised training program participated in this study. Players were tested twice: immediately after the conclusion of the competition period, and following the detraining period, for the determination of bone-turnover rates, ergometrics, and components of the HPG-axis. The detraining period resulted in significant reduction in osteocalcin [OC] (p < 0.001), C-terminal propeptide of collagen type-I [CICP] (p = 0.002), and bone-alkaline-phosphatase [b-ALP] (p < 0.001) values, while C-terminal telopeptide [CTX] was increased (p < 0.001). No significant relationships were apparent between bone biomarkers and body weight, body-fat %, total testosterone, free testosterone, estradiol, follicle-stimulating hormone, and luteinizing hormone in both experimental sessions (p > 0.05). Similarly, despite the deterioration in ergometrics after detraining (all p < 0.001), no significant correlations were evident (p > 0.05) between bone biomarkers and maximal oxygen consumption, squat jump, countermovement jump, and 20 m sprint performance, and also between % change of bone biomarkers and ergometrics, apart from a weak relationship (p = 0.041) between OC and VO2max of questionable value. Our results suggest that the 6-week soccer off-season detraining period in our study negatively affected bone physiology as reflected by the suppression of bone-formation rate and a parallel induction of bone resorption. The cause of this acute alteration of bone-turnover rates is not related to the examined components of the HPG-axis, although parallels is not associated with the changes in ergometrics.

Serum chemerin levels are negatively associated with male fertility and reproductive hormones.

STUDY QUESTION: Are chemerin levels different in subfertile men compared to men from the general population, and how does chemerin relate to reproductive hormonal status? SUMMARY ANSWER: Chemerin is negatively associated to LH, SHBG and estradiol and lower levels of chemerin are detected among subfertile men compared to controls. WHAT IS KNOWN ALREADY: Adipokines have pleiotropic effects on tissue homeostasis and have been shown to affect both sex steroid production and action. Among adipokines the newly characterized chemokine chemerin is suggested to influence testosterone production in males, but whether serum levels associate with testosterone or male subfertility has not yet been reported. STUDY DESIGN, SIZE, DURATION: Case control study comprising a consecutive group of men from infertile couples referred to Reproductive Medicine Centre at Skane University Hospital from 2006 through 2012, and age-matched controls. Participants were enrolled in years 2011-2013. PARTICIPANTS/MATERIALS, SETTING, METHODS: Males from infertile couples (n = 180) aged 18-50 years with sperm concentration <20 × 106/ml and age-matched controls (n = 139) from the general population were enrolled. Serum concentrations of total testosterone (TT), calculated free testosterone (cFT), luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2) and sex-hormone binding globuline (SHBG) as well as the adipokines chemerin, adiponectin and leptin were measured. Anthropometrics and biochemical parameters of glucose and lipid metabolism were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Chemerin levels were lower in subfertile men compared to controls (mean diff. 7.1 ng/ml; 95% CI, 3.7; 11 ng/ml; P < 0.001) even after adjustment for BMI. After adjustment for age, BMI, smoking, leptin and adiponectin, chemerin associated negatively with LH (ß = -4.2; P = 0.02), E2 (ß = -10; P = 0.004) and SHBG (ß = -7.4, P = 0.003). Men with elevated LH levels had lower chemerin levels compared to those with LH levels within the normal range (mean diff. 4.8 ng/ml; 95% CI, 0.16; 9.4 ng/ml; P = 0.04). LIMITATIONS, REASONS FOR CAUTION: Single sample blood test with immunoassays for determination of hormone levels. Heterogeneous group of subfertile subjects. WIDER IMPLICATIONS OF THE FINDINGS: Even though chemerin has been positively associated with BMI, inverse association with subfertility suggests that it is independently linked to reproductive function, a hypothesis that warrants further assessment. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by grants from EU Interreg V (ReproUnion) program as well as Swedish Governmental Fund for Clinical Research. The authors have no conflicts of interest.

The kinase Akt1 controls macrophage response to lipopolysaccharide by regulating microRNAs.

MicroRNAs regulated by lipopolysaccharide (LPS) target genes that contribute to the inflammatory phenotype. Here, we showed that the protein kinase Akt1, which is activated by LPS, positively regulated miRNAs let-7e and miR-181c but negatively regulated miR-155 and miR-125b. In silico analyses and transfection studies revealed that let-7e repressed Toll-like receptor 4 (TLR4), whereas miR-155 repressed SOCS1, two proteins critical for LPS-driven TLR signaling, which regulate endotoxin sensitivity and tolerance. As a result, Akt1(-/-) macrophages exhibited increased responsiveness to LPS in culture and Akt1(-/-) mice did not develop endotoxin tolerance in vivo. Overexpression of let-7e and suppression of miR-155 in Akt1(-/-) macrophages restored sensitivity and tolerance to LPS in culture and in animals. These results indicate that Akt1 regulates the response of macrophages to LPS by controlling miRNA expression.

Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice.

Acute respiratory distress syndrome (ARDS) is a major cause of respiratory failure, with limited effective treatments available. Alveolar macrophages participate in the pathogenesis of ARDS. To investigate the role of macrophage activation in aseptic lung injury and identify molecular mediators with therapeutic potential, lung injury was induced in wild-type (WT) and Akt2(-/-) mice by hydrochloric acid aspiration. Acid-induced lung injury in WT mice was characterized by decreased lung compliance and increased protein and cytokine concentration in bronchoalveolar lavage fluid. Alveolar macrophages acquired a classical activation (M1) phenotype. Acid-induced lung injury was less severe in Akt2(-/-) mice compared with WT mice. Alveolar macrophages from acid-injured Akt2(-/-) mice demonstrated the alternative activation phenotype (M2). Although M2 polarization suppressed aseptic lung injury, it resulted in increased lung bacterial load when Akt2(-/-) mice were infected with Pseudomonas aeruginosa. miR-146a, an anti-inflammatory microRNA targeting TLR4 signaling, was induced during the late phase of lung injury in WT mice, whereas it was increased early in Akt2(-/-) mice. Indeed, miR-146a overexpression in WT macrophages suppressed LPS-induced inducible NO synthase (iNOS) and promoted M2 polarization, whereas miR-146a inhibition in Akt2(-/-) macrophages restored iNOS expression. Furthermore, miR-146a delivery or Akt2 silencing in WT mice exposed to acid resulted in suppression of iNOS in alveolar macrophages. In conclusion, Akt2 suppression and miR-146a induction promote the M2 macrophage phenotype, resulting in amelioration of acid-induced lung injury. In vivo modulation of macrophage phenotype through Akt2 or miR-146a could provide a potential therapeutic approach for aseptic ARDS; however, it may be deleterious in septic ARDS because of impaired bacterial clearance.

Structural-functional analysis of the third transmembrane domain of the corticotropin-releasing factor type 1 receptor: role in activation and allosteric antagonism.

The corticotropin-releasing factor (CRF) type 1 receptor (CRF1R) for the 41-amino acid peptide CRF is a class B G protein-coupled receptor, which plays a key role in the response of our body to stressful stimuli and the maintenance of homeostasis by regulating neural and endocrine functions. CRF and related peptides, such as sauvagine, bind to the extracellular regions of CRF1R and activate the receptor. In contrast, small nonpeptide antagonists, which are effective against stress-related disorders, such as depression and anxiety, have been proposed to interact with the helical transmembrane domains (TMs) of CRF1R and allosterically antagonize peptide binding and receptor activation. Here, we aimed to elucidate the role of the third TM (TM3) in the molecular mechanisms underlying activation of CRF1R. TM3 was selected because its tilted orientation, relative to the membrane, allows its residues to establish key interactions with ligands, other TM helices, and the G protein. Using a combination of pharmacological, biochemical, and computational approaches, we found that Phe-203(3.40) and Gly-210(3.47) in TM3 play an important role in receptor activation. Our experimental findings also suggest that Phe-203(3.40) interacts with nonpeptide antagonists.

Serum miR-155 as a potential biomarker of male fertility.

STUDY QUESTION: Are serum levels of micro-RNAs miR-155 and miR-146a associated with male fertility, low-grade systemic inflammation (LGSI) and androgens? SUMMARY ANSWER: miR-155 was associated with male subfertility independent of LGSI or androgens while miR-146a was only weakly associated with subfertility and LGSI. WHAT IS KNOWN ALREADY: Male subfertility has been associated with LGSI as well as with androgen deficiency. miR-155 and miR-146a are central regulators of inflammation and their level in cells and in the serum has been associated with several inflammatory conditions. STUDY DESIGN, SIZE, DURATION: In this case-control study, two independent groups of 60 subjects each (exploratory and confirmatory cohort) were randomly selected from an ongoing study on subfertile men (in total: hypogonadal; n = 40, eugonadal; n = 40 and control group n = 39) at a University Hospital Reproductive Medicine Centre. Individuals were matched for age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was isolated from cell-free serum. As internal control a synthetic miRNA, UniSp6, was added to each sample prior to extraction. miRNA expression levels were measured by real-time RT-PCR and presented as fold difference (arbitrary units, U) from control. Sera from these individuals had been previously analyzed for hormone and cytokine levels. MAIN RESULTS AND THE ROLE OF CHANCE: Serum levels of miR-155 were associated with levels of miR-146a (P < 0.0001), but only miR-146a was associated with inflammatory markers. miR-155 was strongly associated with subfertility (for subfertile group 1.88 U, 95% confidence interval (CI) 1.6-2.1 U versus 1.15, 95% CI 1.0-1.2 U in controls; P = 0.001). Receiver operating characteristic curve analysis indicated that miR-155 but not miR-146a can be used as a marker of subfertility. MiR-155 with a cutoff value of 1.77 had 47% sensitivity and 95% specificity for identifying subfertility and a positive predictive value (PPV) and negative predictive value (NPV) of 95 and 47%, respectively. When used in combination with FSH, sensitivity and specificity were 80 and 100%, respectively, while PPV and NPV were 100 and 71%, respectively, those values being higher than for the FSH alone. Repeating the results obtained in the exploratory cohort in an independent confirmatory cohort reduced the risk of a chance finding. LIMITATIONS, REASONS FOR CAUTION: Although the results from the exploratory cohort were confirmed in the confirmatory cohort, studies from other centers are needed to establish the role of miR-155 as a new biomarker of male fertility. Furthermore, the role of this marker in distinguishing between different groups of male subfertility is to be elucidated. WIDER IMPLICATIONS OF THE FINDINGS: Association of the inflammatory miRNA miR-155 with male fertility contributes to our understanding of the pathophysiology of subfertility and suggests a novel biomarker. Serum miR-155 in combination with FSH has higher diagnostic specificity and sensitivity compared with FSH alone. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from Swedish Governmental Grant (ALF), Skane county council research and development foundation, Skane University Hospital Fonds and by the EU and Greek funds under the action 'Education and lifelong learning' program THALIS-FAT-VESSEL (No 379527). The authors have no competing interests to disclose.

In prostate cancer, low adiponectin levels are not associated with insulin resistance.

PURPOSE: Adiponectin, an adipose tissue-derived hormone with insulin-sensitizing effect, has been inversely associated with several hormonally dependent malignancies. Prostate cancer is associated with low levels of adiponectin, which have been proposed as an independent risk factor for this malignancy. Aim of this study was to examine whether hypoadiponectinaemia in prostate is associated with insulin resistance. EXPERIMENTAL DESIGN: Plasma samples and covariate data in the context of a case-control study of 300 Greek men were evaluated including 75 patients with prostate cancer, 75 patients with benign prostatic hyperplasia (BPH) and 150 age-matched healthy controls. RESULTS: Patients with prostate cancer had significantly lower plasma adiponectin levels compared with the other two groups, that is BPH patients and healthy controls (7.4 ± 5 ng/mL vs. 11.5 ± 6.4 ng/mL and 12.8 ± 8 ng/mL, respectively). On the other hand, no statistically significant differences were found between patients with prostate cancer and the other two groups for both HOMA-IR and QUICKI (P-value = 0.551). As expected, in all three groups, the levels of adiponectin correlated negatively with HOMA-IR (rho = -0.214, P-value = 0.006), QUICKI (rho = 0.214, P-value = 0.006) and insulin levels (rho = 0.942, P-value < 0.001). CONCLUSION: In spite of what would have been expected from the relevant literature, our data suggest that the hypoadiponectinaemia in prostatic cancer does not appear to be associated with insulin resistance.

Akt1 and Akt2 protein kinases differentially contribute to macrophage polarization.

Activated macrophages are described as classically activated or M1 type and alternatively activated or M2 type, depending on their response to proinflammatory stimuli and the expression of genetic markers including iNOS, arginase1, Ym1, and Fizz1. Here we report that Akt kinases differentially contribute to macrophage polarization, with Akt1 ablation giving rise to an M1 and Akt2 ablation resulting in an M2 phenotype. Accordingly, Akt2(-/-) mice were more resistant to LPS-induced endotoxin shock and to dextran sulfate sodium (DSS)-induced colitis than wild-type mice, whereas Akt1(-/-) mice were more sensitive. Cell depletion and reconstitution experiments in a DSS-induced colitis model confirmed that the effect was macrophage-dependent. Gene-silencing studies showed that the M2 phenotype of Akt2(-/-) macrophages was cell autonomous. The microRNA miR-155, whose expression was repressed in naive and in LPS-stimulated Akt2(-/-) macrophages, and its target C/EBPß appear to play a key role in this process. C/EBPß, a hallmark of M2 macrophages that regulates Arg1, was up-regulated upon Akt2 ablation or silencing. Overexpression or silencing of miR-155 confirmed its central role in Akt isoform-dependent M1/M2 polarization of macrophages.

The association between obesity and fluid intelligence impairment is mediated by chronic low-grade inflammation.

Published evidence suggests that obesity impairs cognition. Development of chronic low-grade inflammation (CLGI) represents the earliest consequence of obesity. The present study investigated the association between obesity and fluid intelligence impairment and assessed the potential mediating role of CLGI and psychological (depression/anxiety symptoms), lifestyle (exercise) and physiological (metabolic dysfunction indices) factors in this association. Clinically healthy participants (n 188), grouped as per BMI, underwent cognitive (General Ability Measure for Adults), psychological (Beck Depression Inventory-II and State-Trait Anxiety Inventory) and activity (Godin leisure-time physical activity) measurements. Biochemical parameters included the following: (a) indices of CLGI (high-sensitivity C-reactive protein, erythrocyte sedimentation rate and fibrinogen); (b) insulin resistance (Homeostasis Model Assessment of Insulin Resistance index); (c) adiposity (plasma adiponectin). An inverse association between elevated BMI and fluid intelligence was observed, with obese participants displaying significantly poorer performance compared with age-matched normal-weight peers. Structural equation modelling results were consistent with a negative impact of obesity on cognition that was mediated by CLGI. The results of the present study support the hypothesis that reduced general cognitive ability is associated with obesity, an adverse effect mainly mediated by obesity-associated activation of innate immunity.

Corticotropin-releasing hormone deficiency results in impaired analgesic response during CFA-induced inflammation.

PURPOSE: Corticotropin-releasing hormone (CRH) plays an important role in relief of pain by releasing analgesia-associated molecules in several inflammatory states. During inflammation, peripheral CRH acts on cells of the immune system to stimulate the local expression of proopiomelanocortin (POMC) and the production of ß-endorphin, which in turn binds to opioid receptors on sensory neurons to produce antinociception. In the present study, we further investigated the role of endogenous CRH in inflammatory pain by determining the effects of Crh-deficiency on this process. METHODS: For this purpose, we used Crh-deficient (Crh-/-) mice and their wildtype (Crh + / +) littermates in the CFA (Complete Freund's Adjuvant)-induced inflammatory pain model. Pain thresholds were evaluated with the Hargreaves apparatus. RESULTS: Our experiments showed that Crh deficiency led to increased pain response, which was associated with decreased POMC mRNA levels in locally inflamed paws of these mice. Furthermore, Crh-/- mice had higher paw edema than Crh + / + mice. Histological evaluation of inflamed paw tissues revealed increased inflammatory response in Crh-/- mice. Protein levels of proinflammatory cytokines, such as IL-6, TNF-α, and IL-1ß, were higher in inflamed tissue of Crh-/- mice compared to wildtype mice. Corticosterone replacement increased the pain threshold of Crh-/- mice, restored their paw volume to the levels of wildtype mice, and significantly reduced their proinflammatory cytokine levels. Furthermore, glucocorticoid administration significantly increased POMC mRNA expression in the inflamed paw. CONCLUSION: Our data suggest that genetic deficiency of CRH is associated with increased pain. This effect is likely attributable to the accompanying glucocorticoid insufficiency and is in part mediated by opioids expressed locally.

The calcineurin-nuclear factor of activated T cells signaling pathway mediates the effect of corticotropin releasing factor and urocortins on catecholamine synthesis.

The biological effects of the Corticotropin-releasing factor (CRF) family of neuropeptides are mediated by mobilization of [Ca(2+)]. Aim of the current work was to examine if the calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the effect of CRF peptides in catecholamine synthesis and secretion from PC12 rat pheochromocytona cells, a model for the study of adrenal catecholamine production. PC12 cells express both types of CRF receptors. Our data are as follows: (a) The calcineurin inhibitor cyclosporine A (CsA) blocked norepinephrine secretion induced by ligands of either CRF type 1 (CRF(1)) or 2 (CRF(2)) receptors on PC12 cells. (b) Silencing NFAT2 expression using a selective NFAT2 siRNA blocked CRF(1) and CRF(2) -induced NE production. (c) CRF ligands induced NFAT transcriptional activity in cells transfected with a luciferase reporter construct controlled by NFAT binding elements (NFAT-Luc). (d) CsA completely blocked the stimulatory effect of CRF(1) and CRF(2) ligands on NFAT activity in NFAT-Luc transfected cells. (e) PKA, PKC, p38-MAPK, Tpl2, Ha-Ras, and AKT1 were crucial intermediates for both CRF(1) and CRF(2)-induced NFAT activation. Interestingly, MEK1/2 and ERK1/2 were crucial only for the CRF(2)-induced NFAT activation. (f) p38-MAPK and Tpl2 were crucial intermediates for both CRF(1) and CRF(2)-induced norepinephrine production, while AKT1 affected only CRF(2)-induced norepinephrine production. In conclusion, our data suggest that CRF(1) and CRF(2) ligands activate the transcription factor NFAT and its activation is prerequisite for CRF-induced catecholamine production from chromaffin cells.

Baseline plasma levels of interleukin-8 in stage IV non-small-cell lung cancer patients: relationship with nutritional status and prognosis.

Interleukin (IL)-8 promotes cellular proliferation and angiogenesis in patients with non-small-cell lung cancer (NSCLC) and may be related to cachexia. Our aim was to investigate the relationship of IL-8 levels with nutritional status, and clinical outcome of patients with NSCLC. Patients with metastatic NSCLC referred for first-line therapy were eligible. Baseline IL-8 levels were measured in plasma. The Mini Nutritional Assessment (MNA) was used for the evaluation of the nutritional status, and patients were classified into 3 groups: A (score 24-30) "well nourished," B (score 17-23.5) "risk of malnutrition," and C (0-16.5) "malnourishment." Response to first-line chemotherapy, time-to-tumor progression (TTP), and overall survival (OS) were also recorded. In total, 114 patients (101 males, 88.5%; mean age = 67.5 yr) were evaluated. Performance status was 0-1 in 62% of the patients. According to the MNA, the majority of patients (71%) was either at nutritional risk or malnourished. IL-8 levels were significantly different between MNA groups (P = 0.023) and correlated with TTP (P = 0.013) and OS (P = 0.001) in univariate analysis. Baseline IL-8 levels correlate with the nutritional status of patients with metastatic NSCLC, suggesting that this cytokine may be related with cachexia.

Immunosuppressed non-responders to two doses of mRNA SARS-CoV-2 vaccines achieve an immune response comparable to those of immunocompetent individuals after a third dose.

The SARS-CoV-2 vaccines trigger the production of neutralizing antibodies to the SARS-CoV-2 spike (S) protein and induce a T cell-mediated immune response. However, the antibody titers that confer protection against the SARS-CoV-2 virus are currently not well-established. While immunocompetent individuals achieve a high level of immune response after SARS-CoV-2 vaccination, it now appears that a high proportion of immunosuppressed or immunocompromised, patients exhibit low or no response to two doses of the vaccines. Most non-responders are on treatment with either glucocorticoids, mycophenolate-mofetil (MMF), the anti-CD20 monoclonal antibody rituximab, calcineurin inhibitors like cyclosporine and tacrolimus, rapamycin (mTOR) signaling cascade inhibitors (i.e., sirolimus and everolimus), azathioprine, or methotrexate given for a variety of diseases including autoimmune disorders, hematological malignancies, and solid cancers, while recipients of solid organ transplants also fall within this category. Recently, several published reports have suggested that a third dose of these vaccines induces an elevated antibody response against the SARS-CoV-2 S protein.

Adiponectin promotes endotoxin tolerance in macrophages by inducing IRAK-M expression.

High levels of plasma adiponectin are associated with low levels of inflammatory markers and cardioprotection. The mechanism via which adiponectin exerts its anti-inflammatory effect is yet unknown. In the present study, we demonstrate that globular adiponectin (gAd) induces the expression of the inactive isoform of IL-1R-associated kinases (IRAK), IRAK-M. Homologous deletion of IRAK-M in IRAK-M(-/-) mice abolished the tolerogenic properties of gAd because pretreatment of IRAK-M(-/-) macrophages with gAd did not suppress LPS-induced proinflammatory cytokine production. GAd activated the MAPKs MEK1/2 and ERK1/2 in macrophages via their upstream regulator Tpl2. Activation of ERK1/2 via Tpl2 appeared necessary for the induction of IRAK-M because gAd did not induce IRAK-M in Tpl2(-/-) macrophages or in macrophages pretreated with the MEK1/2 inhibitor UO126. In addition, activation of PI3K and Akt1 also appeared necessary for the induction of IRAK-M by gAd, because treatment of Akt1(-/-) macrophages or pretreatment of macrophages with the PI3K inhibitor wortmannin abolished gAd-induced IRAK-M expression. Analysis of IRAK-M expression in human peripheral blood cells confirmed that serum adiponectin was negatively associated with IRAK-M and responsiveness to LPS. In conclusion, our data demonstrate that IRAK-M is a major mediator of gAd-induced endotoxin tolerance in primary macrophages, expression of which depends on the activation of Tpl2/ERK and PI3K/Akt1 signaling pathways.

Tpl2 and ERK transduce antiproliferative T cell receptor signals and inhibit transformation of chronically stimulated T cells.

The protein kinase encoded by the Tpl2 protooncogene plays an obligatory role in the transduction of Toll-like receptor and death receptor signals in macrophages, B cells, mouse embryo fibroblasts, and epithelial cells in culture and promotes inflammatory responses in animals. To address its role in T cell activation, we crossed the T cell receptor (TCR) transgene 2C, which recognizes class I MHC presented peptides, into the Tpl2(-/-) genetic background. Surprisingly, the TCR2C(tg/tg)/Tpl2(-/-) mice developed T cell lymphomas with a latency of 4-6 months. The tumor cells were consistently TCR2C(+)CD8(+)CD4(-), suggesting that they were derived either from chronically stimulated mature T cells or from immature single positive (ISP) cells. Further studies showed that the population of CD8(+) ISP cells was not expanded in the thymus of TCR2C(tg/tg)/Tpl2(-/-) mice, making the latter hypothesis unlikely. Mature peripheral T cells of Tpl2(-/-) mice were defective in ERK activation and exhibited enhanced proliferation after TCR stimulation. The same cells were defective in the induction of CTLA4, a negative regulator of the T cell response, which is induced by TCR signals via ERK. These findings suggest that Tpl2 functions normally in a feedback loop that switches off the T cell response to TCR stimulation. As a result, Tpl2, a potent oncogene, functions as a tumor suppressor gene in chronically stimulated T cells.

The impact of stress on tumor growth: peripheral CRF mediates tumor-promoting effects of stress.

INTRODUCTION: Stress has been shown to be a tumor promoting factor. Both clinical and laboratory studies have shown that chronic stress is associated with tumor growth in several types of cancer. Corticotropin Releasing Factor (CRF) is the major hypothalamic mediator of stress, but is also expressed in peripheral tissues. Earlier studies have shown that peripheral CRF affects breast cancer cell proliferation and motility. The aim of the present study was to assess the significance of peripheral CRF on tumor growth as a mediator of the response to stress in vivo. METHODS: For this purpose we used the 4T1 breast cancer cell line in cell culture and in vivo. Cells were treated with CRF in culture and gene specific arrays were performed to identify genes directly affected by CRF and involved in breast cancer cell growth. To assess the impact of peripheral CRF as a stress mediator in tumor growth, Balb/c mice were orthotopically injected with 4T1 cells in the mammary fat pad to induce breast tumors. Mice were subjected to repetitive immobilization stress as a model of chronic stress. To inhibit the action of CRF, the CRF antagonist antalarmin was injected intraperitoneally. Breast tissue samples were histologically analyzed and assessed for neoangiogenesis. RESULTS: Array analysis revealed among other genes that CRF induced the expression of SMAD2 and ß-catenin, genes involved in breast cancer cell proliferation and cytoskeletal changes associated with metastasis. Cell transfection and luciferase assays confirmed the role of CRF in WNT- ß-catenin signaling. CRF induced 4T1 cell proliferation and augmented the TGF-ß action on proliferation confirming its impact on TGFß/SMAD2 signaling. In addition, CRF promoted actin reorganization and cell migration, suggesting a direct tumor-promoting action. Chronic stress augmented tumor growth in 4T1 breast tumor bearing mice and peripheral administration of the CRF antagonist antalarmin suppressed this effect. Moreover, antalarmin suppressed neoangiogenesis in 4T1 tumors in vivo. CONCLUSION: This is the first report demonstrating that peripheral CRF, at least in part, mediates the tumor-promoting effects of stress and implicates CRF in SMAD2 and ß-catenin expression.

Corticotropin Releasing Factor promotes breast cancer cell motility and invasiveness.

INTRODUCTION: Cancer cells secrete bioactive peptides that act in an autocrine or paracrine fashion affecting tumor growth and metastasis. Corticotropin-releasing factor (CRF), a hypothalamic neuropeptide that controls the response to stress, has been detected in breast cancer tissues and cell lines. CRF can affect breast cancer cells in an autocrine or paracrine manner via its production from innervating sympathetic neurons or immune cells. METHODS: In the present study we report our findings regarding the impact of CRF on breast cancer cell motility and invasiveness. For this purpose we used the MCF7 breast cancer cell line and evaluated the effect of CRF on motility and invasiveness using the wound-healing and boyden-chamber assays. In addition, we measured the effect of CRF on molecules that mediate motility by western blot, immunofluorescence, ELISA and RT-PCR. RESULTS: Our findings show that: 1. CRF transiently inhibited the apoptosis of MCF7 cells. 2. CRF enhanced MCF7 cell motility in a wound healing assay and their invasiveness through extracellular matrix. 3. CRF increased actin polymerization, phosphorylation of Focal Adhesion Kinase (FAK), providing a potential mechanism for the observed induction of MCF7 motility. 4. CRF induced the expression of Cox-1 but not Cox-2 in MCF7 cells as well as the production of prostaglandins, factors known to promote invasiveness and metastasis. CONCLUSION: Overall, our data suggest that CRF stimulates cell motility and invasiveness of MCF7 cells most probably via induction of FAK phosphorylation and actin filament reorganization and production of prostaglandins via Cox1. Based on these findings we postulate that the stress neuropeptide CRF present in the vicinity of tumors (either produced locally by the tumor cells themselves or by nearby normal cells or secreted from the innervations of surrounding tissues) may play an important role on breast tumor growth and metastatic capacity, providing a potential link between stress and tumor progression.

Fatty acids and postprandial inflammation.

PURPOSE OF REVIEW: Postprandial inflammation is an independent factor in evaluating food quality in addition to the well known parameters of nutritional value, caloric content and amount of carbohydrates, fats, proteins, minerals and vitamins. Among the latter, the quality and quantity of fatty acids in a meal is a major determinant of the magnitude of postprandial inflammation. Purpose of this review is to describe this exciting new area of research and its repercussions in the way we, the consumers, and the food industry evaluate the type and quantity of fat in food. RECENT FINDINGS: A number of, by now classical, epidemiologic studies have documented a strong association between the type of fatty acids consumed and the incidence of cardiovascular diseases. Recently published reports suggest that the adverse effect of dietary fatty acids on cardiovascular health depends on their postprandial modification of innate immunity ending in the so-called 'postprandial metabolic inflammation'. SUMMARY: The quantity of fat and its qualitative characteristics such as the percentage of saturated fatty acids and the ratio of n-3 to n-6 polyunsaturated fatty acids (PUFAs) in a meal have emerged as major determinants of the magnitude of postprandial inflammatory response. In this review, we will summarize all experimental evidence suggesting that the two families of PUFA appear to have antagonistic effects on postprandial inflammation, n-3 PUFA being anti-inflammatory while n-6 PUFA proinflammatory.

Neurosteroids as modulators of neurogenesis and neuronal survival.

Neurons and glia in the central nervous system express the necessary enzymes for the synthesis of neurosteroids that are produced in concentrations high enough to exert paracrine effects. Synthesis of brain neurosteroids declines with age, during stressful conditions (including major depression, chronic psychological stress), and in chronic inflammatory and neurodegenerative diseases. Recent reports associate the decrease of brain neurosteroids to neuronal dysfunction and degeneration. This review summarizes the recent findings on how the most studied neurosteroids (dehydroepiandrosterone, pregnenolone and their sulphate esters, progesterone and allopregnanolone) affect neuronal survival, neurite outgrowth and neurogenesis; furthermore, this review discusses potential applications of these neurosteroids in the therapeutic management of neurodegenerative conditions, including that of age-related brain atrophy.