Transcriptional analysis of nasal polyps fibroblasts reveals a new source of pro-inflammatory signaling in CRSwNP.

BACKGROUND: Fibroblasts and others mesenchymal cells have recently been identified as critical cells triggering tissue-specific inflammatory responses. Persistent activation of fibroblasts inflammatory program has been suggested as an underlying cause of chronic inflammation in a wide range of tissues and pathologies. Nevertheless, the role of fibroblasts in the emergence of chronic inflammation in the upper airway has not been previously addressed. We aimed to elucidate whether fibroblasts could have a role in the inflammatory response in chronic rhinosinusitis with nasal polyps (CRSwNP). METHODOLOGY: We performed whole-transcriptome microarray in fibroblast cultured from CRSwNP samples and confirmed our results by qRT-PCR. We selected patients without other associated diseases in upper airway. To investigate shifts in transcriptional profile we used fibroblasts from nasal polyps and uncinate mucosae from patient with CRSwNP, and fibroblasts from uncinate mucosae from healthy subjects as controls. RESULTS: This study exposes activation of a pro-inflammatory and pro-fibrotic transcriptional program in nasal polyps and CRSwNP fibroblasts when compared to controls. Our Gene-set Enrichment Analysis (GSEA) pointed to common up-regulation of several pro-inflammatory pathways in patients-derived fibroblasts, along with higher mRNA expression levels of cytokines, growth factors and extracellular matrix components. CONCLUSIONS: Our work reveals a potential new source of inflammatory signaling in CRSwNP. Furthermore, our results suggest that deregulated inflammatory signaling in tissue-resident fibroblasts could support a Type-2 inflammatory response. Further investigations will be necessary to demonstrate the functionality of these novel results.

Pembrolizumab in combination with gemcitabine for patients with HER2-negative advanced breast cancer: GEICAM/2015-04 (PANGEA-Breast) study.

BACKGROUND: We evaluated a new chemoimmunotherapy combination based on the anti-PD1 monoclonal antibody pembrolizumab and the pyrimidine antimetabolite gemcitabine in HER2- advanced breast cancer (ABC) patients previously treated in the advanced setting, in order to explore a potential synergism that could eventually obtain long term benefit in these patients. METHODS: HER2-negative ABC patients received 21-day cycles of pembrolizumab 200 mg (day 1) and gemcitabine (days 1 and 8). A run-in-phase (6 + 6 design) was planned with two dose levels (DL) of gemcitabine (1,250 mg/m2 [DL0]; 1,000 mg/m2 [DL1]) to determine the recommended phase II dose (RP2D). The primary objective was objective response rate (ORR). Tumor infiltrating lymphocytes (TILs) density and PD-L1 expression in tumors and myeloid-derived suppressor cells (MDSCs) levels in peripheral blood were analyzed. RESULTS: Fourteen patients were treated with DL0, resulting in RP2D. Thirty-six patients were evaluated during the first stage of Simon's design. Recruitment was stopped as statistical assumptions were not met. The median age was 52; 21 (58%) patients had triple-negative disease, 28 (78%) visceral involvement, and 27 (75%) ≥ 2 metastatic locations. Progression disease was observed in 29 patients. ORR was 15% (95% CI, 5-32). Eight patients were treated ≥ 6 months before progression. Fourteen patients reported grade ≥ 3 treatment-related adverse events. Due to the small sample size, we did not find any clear association between immune tumor biomarkers and treatment efficacy that could identify a subgroup with higher probability of response or better survival. However, patients that experienced a clinical benefit showed decreased MDSCs levels in peripheral blood along the treatment. CONCLUSION: Pembrolizumab 200 mg and gemcitabine 1,250 mg/m2 were considered as RP2D. The objective of ORR was not met; however, 22% patients were on treatment for ≥ 6 months. ABC patients that could benefit of chemoimmunotherapy strategies must be carefully selected by robust and validated biomarkers. In our heavily pretreated population, TILs, PD-L1 expression and MDSCs levels could not identify a subgroup of patients for whom the combination of gemcitabine and pembrolizumab would induce long term benefit. TRIAL REGISTRATION: ClinicalTrials.gov and EudraCT (NCT03025880 and 2016-001,779-54, respectively). Registration dates: 20/01/2017 and 18/11/2016, respectively.

New horizons in breast cancer: the promise of immunotherapy.

Immunology and immunotherapy of cancer is an expanding field in oncology, with recent great achievements obtained through the new successful approaches implemented to circumvent immune evasion, which is undoubtedly considered a novel hallmark of cancer. Translational research in this topic has revealed targets that can be modulated in the clinical setting with new compounds and strategies. Like most of the tumors, breast cancer is considered a complex and heterogeneous disease in which host immune responses have been also recently demonstrated of critical relevance. T infiltrating lymphocyte measurement is suggested as a powerful new tool necessary to predict early breast cancer evolution, especially for the her2-positive and triple-negative subtypes. Other biomarkers in tissue and peripheral blood are under intense scrutiny to ascertain their eventual role as prognostic and/or predictive factors. This background has fueled the interest in developing clinical research strategies to test activity of modern immunotherapy in breast cancer, which constitutes the main focus of this review.

Breast Cancer Immunology and Immunotherapy: Current Status and Future Perspectives.

Cancer immunology has gained renewed interest in the past few years due to emerging findings on mechanisms involved in tumoral immune evasion. Indisputably, immune edition is currently considered a critical hallmark of cancer. Basic research has revealed new targets which can be modulated in the clinical setting with new compounds and strategies. As recent evidence confirms, breast cancer (BC) is a complex and heterogeneous disease in which host immune responses play a substantial role. T-infiltrating lymphocytes measurement is suggested as a powerful new tool necessary to predict early BC evolution, especially in HER2-positive and triple negative subtypes. However, T-infiltrating lymphocytes, genomic platforms, and many other biomarkers in tissue and peripheral blood (e.g., regulatory T cells and myeloid-derived suppressor cells) are not the only factors being evaluated regarding their potential role as prognostic and/or predictive factors. Many ongoing clinical trials are exploring the activity of immune checkpoint modulators in BC treatment, both in the advanced and neoadjuvant setting. Although this field is expanding with exciting new discoveries and promising clinical results-and creating great expectations-there remain many uncertainties yet to be addressed satisfactorily before this long awaited therapeutic promise can come to fruition.

Insulin and Leptin Signaling in Placenta from Gestational Diabetic Subjects.

Insulin and leptin receptors are known to share signaling pathways, such as JAK2/STAT-3 (Janus kinase2/signal transduction and activator of transcription3), MAPK (Mitogen activated protein kinase), and PI3K (phosphoinositide 3-kinase). Both positive and negative cross-talk have been previously found in different cellular systems. Gestational diabetes (GDM) is a pathophysiological state with high circulating levels of both insulin and leptin. We have previously found that these 3 signaling pathways are activated in placenta from GDM patients to promote translation, involving the activation of leptin receptor. Now, we have tested the hypothesis that both leptin and insulin receptors might contribute to this activation in a positive way that may become negative when the system is overactivated. We studied the activation of leptin and insulin receptors in placenta from GDM and healthy pregnancies. We have also performed in vitro studies with insulin and leptin stimulation of trophoblast explants from healthy placenta. We have found that both leptin and insulin receptors are activated in placenta from GDM. In vitro stimulation of trophoblast explants with both leptin and insulin at submaximal doses (0.1 nM) potentiated the activation of signaling, whereas preincubation with maximal concentrations of insulin (10 nM) and further stimulation with leptin showed negative effect. Trophoblastic explants from GDM placenta, which presented high signaling levels, had a negative signaling effect when further incubated in vitro with leptin. In conclusion, insulin and leptin receptors have positive effects on signaling, contributing to high signaling levels in GDM placenta, but insulin and leptin have negative effects upon overstimulation.

Expression and immunohistochemical localization of leptin receptor in human periapical granuloma.

AIM: To investigate the expression and immunohistochemical localization of leptin receptor (LEPR) in human periapical granulomas. METHODOLOGY: Periapical inflammatory lesions were obtained from extracted human teeth and teeth which underwent periapical surgery. After their histopathological categorization as periapical granulomas (n = 20), they were examined by immunohistochemistry using human LEPR monoclonal antibodies. LEPR mRNA expression was also determined by quantitative real-time PCR (qRT-PCR), and the amount of LEPR protein was analysed by immunoblot. RESULTS: All granuloma samples expressed LEPR. Amongst inflammatory cells, only macrophages showed expression of LEPR. Western blot analysis revealed the presence in the samples of a protein with apparent molecular weight of ~120 kDa, corresponding to the estimated molecular weight of LEPR. The qRT-PCR analysis demonstrated the expression of LEPR mRNA, corresponding the size of the amplified fragment (338 bp), assessed by agarose gel electrophoresis, to that of LEPR mRNA. CONCLUSIONS: Human periapical granulomas express LEPR. In periapical granulomas, only macrophages showed expression of LEPR. This finding suggests that leptin can play a role in inflammatory and immune periapical responses.

Activated translation signaling in placenta from pregnant women with gestational diabetes mellitus: possible role of leptin.

Placentas from gestational diabetes (GDM) suffer from structural and functional changes including overgrowth. That is why we aimed to study [³H]-leucine incorporation into protein in addition to translation signaling in placenta from GDM. Thus, we investigated the expression of leptin and leptin receptor (LEPR), as well as the activation state of signaling proteins regulating protein synthesis, such as mTOR, S6 Kinase, EIF4E-BP1, EIF4E, and eEF2 by measuring protein phosphorylation by immunoblot. [³H]-Leucine incorporation into protein also was determined in trophoblastic placenta explants from GDM and control pregnancy. We found that leptin and LEPR expression are increased in placentas from GDM and the translation machinery activity as well as [³H]-leucine incorporation into protein were higher in placentas from GDM compared with placentas from control pregnancy. In conclusion, protein synthesis rate is increased in placenta from GDM patients, and this may be due, at least in part, by the activation of translation signaling. The increased expression of leptin and LEPR may contribute to these effects. These results may provide a possible mechanism for the previously observed increase in placenta growth in GDM.

Elsevier Trophoblast Research Award lecture: Molecular mechanisms underlying estrogen functions in trophoblastic cells--focus on leptin expression.

The steroid hormone 17ß-estradiol is an estrogen that influences multiple aspects of placental function and fetal development in humans. During early pregnancy it plays a role in the regulation of blastocyst implantation, trophoblast differentiation and invasiveness, remodeling of uterine arteries, immunology and trophoblast production of hormones such as leptin. Estradiol exerts some effects through the action of classical estrogen receptors ERα and ERß, which act as ligand-activated transcription factors and regulate gene expression. In addition, estradiol can elicit rapid responses from membrane-associated receptors, like activation of protein-kinase pathways. Thus, the cellular effects of estradiol will depend on the specific receptors expressed and the integration of their signaling events. Leptin, the 16,000MW protein product of the obese gene, was originally considered an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblastic cells. Expression of leptin in placenta is highly regulated by key pregnancy molecules as hCG and estradiol. The aim of this paper is to review the molecular mechanisms underlying estrogen functions in trophoblastic cells; focusing on mechanisms involved in estradiol regulation of placental leptin expression.

Review: Leptin gene expression in the placenta--regulation of a key hormone in trophoblast proliferation and survival.

Leptin is a 16000 MW protein originally described as an adipocyte-derived signaling molecule for the central control of metabolism. However, pleiotropic effects of leptin have been identified in reproduction and pregnancy. The leptin gene is expressed in placenta, where leptin promotes proliferation and survival of trophoblast cells. Study of the major signaling pathways known to be triggered by leptin receptor has revealed that leptin stimulates JAK/STAT, MAPK and PI3K pathways in placental cells. Leptin also exerts an antiapoptotic action in placenta and this effect is mediated by the MAPK pathway. Moreover, leptin stimulates protein synthesis by activating the translational machinery via both PI3K and MAPK pathways. Expression of leptin in placenta is highly regulated, suggesting that certain key pregnancy molecules participate in such regulation. An important hormone in reproduction, hCG, induces leptin expression in trophoblast cells and this effect involves the MAPK signal transduction pathway. Moreover, the cyclic nucleotide cAMP, which has profound actions upon human trophoblast function, also stimulates leptin expression and this effect seems to be mediated by crosstalk between the PKA and MAPK signaling pathways. Estrogens play a central role in reproduction. 17ß-estradiol upregulates leptin expression in placental cells through genomic and non-genomic actions, probably via crosstalk between estrogen receptor-α and the MAPK and PI3K signal transduction pathways. Taken together these findings give a better understanding of the function of leptin and the regulatory mechanisms of leptin expression in human placental trophoblast and further support the importance of leptin in the biology of reproduction.

Profile of patients triply infected with HIV and the hepatitis B and C viruses in the HAART era.

HIV-HCV-HBV-coinfected patients were assessed to characterize the viral interactions in the setting of HIV coinfection and in the HAART era. All positive anti-HCV antibody and HBs antigen-positive HIV-infected patients were identified at five HIV clinics. Antihepatitis delta (HDV) antibody, serum HIV RNA, HCV RNA, and HBV DNA quantification and genotype determinations were performed. Out of 67 patients identified 47 (70%) were receiving anti-HBV therapy. HCV RNA and HBV DNA were detectable in 52.5% and 37% of patients, respectively. All possible patterns were found, regardless of anti-HBV therapy. HDV coinfection was associated with undetectable HCV RNA [RR 9.52 (95% CI 1.85-49.01); p = 0.007]. Independent factors predicting undetectable HBV DNA lacked HBeAg [RR 13.94 (95% CI 3.05-63.72); p = 0.001] and use of anti-HBV therapy [RR 11.42 (95% CI 2.43-53.54); p = 0.002]. Replication and genotypes of HCV or HBV had no impact on the replication of the other virus. In conclusion, in this cohort of triple infection (HBV/HCV/HIV) various viral patterns were identified. Spontaneous HCV clearance was frequent, and it was independently associated with HDV coinfection. In the absence of HBV therapy, HBV most often actively replicates. HBV/HCV replication or genotypes were not related to the replication of the other virus.

Leptin promotes cell survival and activates Jurkat T lymphocytes by stimulation of mitogen-activated protein kinase.

Leptin (Ob) is a non-glycosylated peptide hormone that regulates energy homeostasis centrally, but also has systemic effects including the regulation of the immune function. We have reported previously that leptin activates human peripheral blood lymphocytes co-stimulated with phytohaemagglutinin (PHA) (4 microg/ml), which prevented the employment of pharmacological inhibitors of signalling pathways. In the present study, we used Jurkat T cells that responded to leptin with minimal PHA co-stimulation (0.25 microg/ml). The long isoform of leptin receptor is expressed on Jurkat T cells and upon leptin stimulation, the expression of early activation marker CD69 increases in a dose-dependent manner (0.1-10 nM). We have also found that leptin activates receptor-associated kinases of the Janus family-signal transucers and activators of transcription (JAK-STAT), mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 kinase (PI3K) signalling pathways. Moreover, we sought to study the possible effect of leptin on cell survival and apoptosis of Jurkat T cells by culture in serum-free conditions. We have assayed the early phases of apoptosis by flow cytometric detection of fluorescein isothiocyanate (FITC)-labelled annexin V simultaneously with dye exclusion of propidium iodide (PI). As well, we have assayed the activation level of caspase-3 by inmunoblot with a specific antibody that recognizes active caspase-3. We have found that leptin inhibits the apoptotic process dose-dependently. By using pharmacological inhibitors, we have found that the stimulatory and anti-apoptotic effects of leptin in Jurkat T cells are dependent on MAPK activation, rather than the PI3K pathway, providing new data regarding the mechanism of action of leptin in T cells, which may be useful to understand more clearly the association between nutritional status and the immune function.

Human leptin signaling in human peripheral blood mononuclear cells: activation of the JAK-STAT pathway.

Leptin is an adipocyte-secreted hormone that centrally regulates weight control. However, the leptin receptor is expressed not only in the central nervous system, but also in other systems, such as reproductive, hematopoietic, and immune tissues, suggesting various roles in addition to the regulation of food intake and energy expenditure. The leptin receptor bears homology to members of the class I cytokine receptor family. Leptin has previously been shown to enhance cytokine production by murine peritoneal macrophages and human circulating monocytes, where human leptin promotes activation and proliferation. We have recently found that the leptin receptor is expressed not only in monocytes but also in both CD4(+) and CD8(+) T lymphocytes. Besides, leptin enhances proliferation and activation of T lymphocytes when they are costimulated by PHA or Con A. In this paper, we have studied the signal transduction of the leptin receptor in peripheral blood mononuclear cells. We found that leptin stimulation activates the JAK-STAT signaling pathway. More specifically, we found that JAK-2/3 and STAT-3 are activated by tyrosine phosphorylation upon leptin stimulation. Moreover, leptin stimulated tyrosine phosphorylation of the RNA binding protein Sam68 and its association with STAT-3. These effects were dose-dependent (0.1-10 nM) and transient (5-30 min). We also observed the leptin stimulated translocation of activated STAT-3 from the cytoplasm to the nucleus. These results indicate that human leptin receptor in circulating mononuclear cells has the signaling capacity to activate JAK-STAT cascade. This pathway may mediate, at least in part, the action of human leptin in human peripheral blood mononuclear cells.

Sam68 is a docking protein linking GAP and PI3K in insulin receptor signaling.

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K. In the present work, using HTC-IR cells, we have found that insulin stimulation promotes the relocalization of Sam68 from the nucleus to the cytoplasm, and we have further studied the role of Sam68 in insulin receptor signaling complexes, by co-precipitating experiments. Thus, Sam68 is co-precipitated with p85 PI3K, IRS-1 and IR. The association of Sam68 with these complexes is mediated by the SH2 domains of PI3K. Moreover, we have found that Sam68 is a p120GAP associated protein after Tyr-phosphorylation by the IR. This association is mediated by the SH2 domains of GAP (preferentially the C-terminal SH2). Thus, Sam68 is linking p120GAP to PI3K signaling pathway. In fact, PI3K activity was increased in both anti-Sam68 and anti-GAP immmunoprecipitates upon insulin stimulation. We propose that the recruitment of the docking protein Sam68 to the PI3K pathway may serve to allow the association of other signaling molecules, i.e. p120GAP. In this way, these signaling complexes may modulate other signaling cascades of IR, such as p21Ras pathway.

Pancreastatin, a chromogranin A-derived peptide, inhibits DNA and protein synthesis by producing nitric oxide in HTC rat hepatoma cells.

BACKGROUND/AIMS: Pancreastatin, a chromogranin A-derived peptide, has a counter-regulatory effect on insulin action. We have previously characterized pancreastatin receptor and signalling in rat liver and HTC hepatoma cells. A G alpha(q/11)-PLC-beta pathway leads to an increase in [Ca2+]i, PKC and mitogen activated protein kinase (MAPK) activation. These data suggested that pancreastatin might have a role in growth and proliferation, similar to other calcium-mobilizing hormones. METHODS: DNA and protein synthesis were measured as [3H]-thymidine and [3H]-leucine incorporation. Nitric oxide (NO) was determined by the Griess method and cGMP production was quantified by enzyme-linked immunoassay. RESULTS: Contrary to the expected results, we have found that pancreastatin inhibits protein and DNA synthesis in HTC hepatoma cells. On the other hand, when the activity of NO synthase was inhibited by N-monomethyl-L-arginine (NMLA), the inhibitory effect of pancreastatin on DNA and protein synthesis was not only reverted, but a dose-dependent stimulatory effect was observed, probably due to MAPK activation, since it was prevented by PD98059. These data strongly suggested the role of NO in the inhibitory effect of pancreastatin on protein and DNA synthesis, which is overcoming the effect on MAPK activation. Moreover, pancreastatin dose-dependently increased NO production in parallel to cyclic guanosine monophosphate (cGMP). Both effects were prevented by NMLA. Finally, an indirect effect of pancreastatin through the induction of apoptosis was ruled out. CONCLUSIONS: Therefore, the NO and the cGMP produced by the NO-activated guanylate cyclase may mediate the dose-dependent inhibitory effect of pancreastatin on growth and proliferation in HTC hepatoma cells.

Homocysteine thiolactone inhibits insulin signaling, and glutathione has a protective effect.

Hyperhomocysteinemia and insulin resistance are independent factors for cardiovascular disease. Most of the angiotoxic effects of homocysteine are related to the formation of homocysteine thiolactone and the consequent increase in oxidative stress. The oxidative stress has also been shown to impair insulin action, therefore leading to insulin resistance. In order to study a putative direct effect of homocysteine on insulin signaling, we have characterized the molecular counter-regulation of the early events in the signal transduction of the insulin receptor, and the metabolic end-point of glycogen synthesis. We employed HTC rat hepatoma cells transfected with the human insulin receptor. A 10 min exposure to homocysteine thiolactone (50 microM) resulted in a significant inhibition of insulin-stimulated tyrosine phosphorylation of the insulin receptor beta-subunit and its substrates IRS-1 and p60-70, as well as their association with the p85 regulatory subunit of phosphatidylinositol 3-kinase. These effects led to impairment of the insulin-stimulated phosphatidylinositol 3-kinase activity, which plays a central role in regulating insulin action. Thus, insulin-stimulated glycogen synthesis was also inhibited by homocysteine thiolactone. To investigate whether oxidative stress was mediating the counter-regulatory effect of homocysteine thiolactone on insulin signaling, we preincubated the cells (5 min) with 250 microM glutathione prior to the incubation with homocysteine (10 min) and subsequent insulin challenge. Glutathione completely abolished the effects of homocysteine thiolactone on insulin-receptor signaling and restored the insulin-stimulated glycogen synthesis. In conclusion, these data suggest that homocysteine thiolactone impairs insulin signaling by a mechanism involving oxidative stress, leading to a defect in insulin action.

Characterization of pancreastatin receptor and signaling in rat HTC hepatoma cells.

Pancreastatin, a chromogranin A-derived peptide widely distributed throughout the neuroendocrine system, has a general inhibitory effect on endocrine secretion and a counterregulatory effect on insulin action. We have recently described the cross-talk of pancreastatin with insulin signaling in rat hepatoma cells (HTC), where it inhibits insulin action and signaling through the serine phosphorylation of the insulin receptor, thereby impairing tyrosine kinase activity. Here, we have characterized pancreastatin receptors and signaling in HTC cells. The pancreastatin effector systems were studied by determining phospholipase C activity in HTC membranes and mitogen-activated protein kinase (MAPK) phosphorylation activity in HTC cells. Binding studies with radiolabeled pancreastatin showed a population of high affinity binding sites, with a B(max) of 8 fmol/mg protein and a K(d) of 0.6 nM. Moreover, we assessed the coupling of the receptor with a G protein system by inhibiting the binding with guanine nucleotide and by measuring the GTP binding to HTC membranes. We found that pancreastatin receptor was coupled with a G alpha(q/11) protein which activates phospholipase C-beta(1) and phospholipase C-beta(3), in addition to MAPK via both beta gamma and alpha(q/11).

Affinity purification of pancreastatin receptor-Gq/11 protein complex from rat liver membranes.

Pancreastatin, a chromogranin A derived peptide, exerts a glycogenolytic effect on the hepatocyte. This effect is initiated by binding to membrane receptors which are coupled to pertussis toxin insensitive G proteins belonging to the Gq/11 family. We have recently solubilized active pancreastatin receptors from rat liver membranes still functionally coupled to G proteins. Here, we have purified pancreastatin receptors by a two-step procedure. First, pancreastatin receptors with their associated Gq/11 regulatory proteins were purified from liver membranes by lectin absorption chromatography on wheat germ agglutinin immobilized on agarose. A biotinylated rat pancreastatin analog was tested for binding to liver membranes before using it for affinity purification. Unlabeled biotinylated rat pancreastatin competed for 125I-labeled [Tyr0]PST binding to solubilized receptors with a Kd = 0.27 nM, comparable to that of native pancreastatin. The biotinylated analog was immobilized on streptavidin-coated Sepharose beads and used to further affinity purify wheat germ agglutinin eluted receptor material. Specific elution at low pH showed that the receptor protein was purified as an 80-kDa protein in association with a G protein of the q/11 family, as demonstrated by specific immunoblot analysis. The specificity of the receptor band was assessed by chemical cross-linking of the purified material followed by SDS-PAGE and autoradiography. In conclusion, we have purified pancreastatin receptor as a glycoprotein of 80 kDa physically associated with a Gq/11 protein.

Human leptin enhances activation and proliferation of human circulating T lymphocytes.

Leptin is an adipocyte-secreted hormone that centrally regulates weight control. However, leptin receptor is expressed not only in the central nervous system, but also in other systems such as reproductive and hematopoietic tissues. Human leptin has previously been shown to enhance cytokine production by murine peritoneal macrophages and human circulating monocytes. In this paper we have assessed the presence of leptin receptors in peripheral human T lymphocytes and we have studied their functional role. Both CD4(+) and CD8(+) T lymphocytes express leptin receptors. Moreover, we show that human leptin dose-dependently enhances proliferation and activation of human circulating T lymphocytes when they are costimulated by PHA or Con A. Leptin alone was not able to activate T lymphocytes. To confirm a direct effect of leptin on T lymphocytes, monocytes were extracted by adhesion to culture flasks. The early activation surface marker CD69 was then induced in both CD4(+) and CD8(+) T lymphocytes after 8 h stimulation with PHA or Con A. Leptin dose-dependently enhanced stimulated CD69 expression. Moreover, leptin dose-dependently enhanced the expression of the late activation markers CD25 and CD71 in both CD4(+) and CD8(+) T lymphocytes after 48 h stimulation with PHA or Con A. Finally, we have found that leptin modulates CD4(+) T lymphocyte activation toward Th1 phenotype by stimulating the synthesis of IL-2 and IFN-gamma. These results demonstrate the presence of the leptin receptor in human circulating CD4(+) and CD8(+) T lymphocytes and a functional role of leptin as a modulator (enhancer) of lymphocyte stimulation with a shift toward Th1 cytokine-production profile. This function of leptin may have some relevance in the pathophysiology of immunologic alterations related to obesity.

Stimulation of glycogen synthesis by insulin requires S6 kinase and phosphatidylinositol-3-kinase in HTC-IR cells.

In order to study the role of phosphatidylinositol-3-kinase (PI3K), PKB, FRAP, S6 kinase, and MAP kinase in insulin-stimulated glycogen synthesis, we used a specific inhibitor of PI3K, LY294002, the immunosuppressant inhibitor of FRAP, rapamycin, and the inhibitor of MAPK kinase (MEK)/MAPK, PD98059, in rat HTC hepatoma cells overexpressing human insulin receptors. The PI3K inhibitor LY294002 completely blocks insulin-stimulated glycogen synthesis by inhibiting glycogen synthase, PKB (Akt-1), and FRAP (RAFT) autophosphorylation, as well as p70 S6 kinase activation, whereas insulin receptor substrates tyrosine phosphorylation and MEK activity were not affected. However, rapamycin only partially blocks insulin-stimulated glycogen synthesis by partial inhibition of glycogen synthase, whereas it completely blocks S6 kinase activation and FRAP autophosphorylation, but does not affect either PKB autophosphorylation, MEK activity, or insulin receptor tyrosine phosphorylation. Insulin-stimulated glycogen synthesis and glycogen synthase were not affected by the MEK/MAPK inhibitor PD98059. These data suggest that the PI3K, and not the MAPK pathway plays an important role in the insulin-stimulated glycogen synthesis in the hepatocyte, partly mediated by FRAP and S6 kinase activation. However, the inhibition of FRAP and S6 kinase activation is not sufficient to block insulin-stimulated glycogen synthesis, suggesting an important role of a branching pathway upstream of S6 kinase and downstream of PI3K, which is probably mediated by PKB in the signaling of the insulin receptor in hepatoma HTC cells.