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Human mesenchymal stem cells (hMSCs) are multipotent cells that can differentiate into many cell types. Chondrogenesis is induced in hMSCs cultured as a micromass pellet to mimic cellular condensation during cartilage development, and exposed to transforming growth factor beta (TGFbeta). Interestingly, TGFbeta can also induce hMSC differentiation to smooth-muscle-like cell types, but it remains unclear what directs commitment between these two lineages. Our previous work revealed that cell shape regulates hMSC commitment between osteoblasts and adipocytes through RhoA signaling. Here we show that cell shape also confers a switch between chondrogenic and smooth muscle cell (SMC) fates. Adherent and well-spread hMSCs stimulated with TGF beta 3 upregulated SMC genes, whereas cells allowed to attach onto micropatterned substrates, but prevented from spreading and flattening, upregulated chondrogenic genes. Interestingly, cells undergoing SMC differentiation exhibited little change in RhoA, but significantly higher Rac1 activity than chondrogenic cells. Rac1 activation inhibited chondrogenesis and was necessary and sufficient for inducing SMC differentiation. Furthermore, TGF beta 3 and Rac1 signaling upregulated N-cadherin, which was required for SMC differentiation. These results demonstrate a chondrogenic-SMC fate decision mediated by cell shape, Rac1, and N-cadherin, and highlight the tight coupling between lineage commitment and the many changes in cell shape, cell-matrix adhesion, and cell-cell adhesion that occur during morphogenesis.

Background: Human leukocyte activation induced by specific and non-specific stimuli is characterized by the formation of lipid rafts defined as lipid-ordered domains that are more tightly packed than the surrounding non-raft phase of the bilayer. These lipid rafts are formed in parallel with profound membrane reorganization., Objectives: Analyse the rafting and non-rafting proteins present on the activated and resting basophil membrane and study their interest for the flow cytometric analysis of basophil activation., Methods: Human basophils obtained from samples used for diagnostic cellular tests such as basophil or lymphocyte activation tests were stimulated either by the formyl-methionyl-leucyl-phenylalanine peptide (fMLP), by an anti-IgE or by an allergen. After 40 min at 37 degrees C, they were labelled by different antibodies conjugated to fluorescent dyes as an anti-IgE FITC, an anti-CCR3 PE, an anti-CD63, an anti-CD203c PE, an anti-11b, annexin V FITC or by cholera toxin FITC. Moreover, several experiments were analysed using an Amnis cytometer, allowing one to obtain the picture of the analysed cells., Results: Anti-IgE or specific allergen elicits a membrane neo expression of CD63 at a high density and is poorly represented on resting basophil membrane. Upon an IgE-dependant activation some of the markers already present on resting basophil membrane, as CD203c, are up regulated and others, such as the IgE/IgE FcepsilonRI receptor and CCR3 are down regulated and submitted to the formation of clusters demonstrated by the pictures taken with the Amnis cytometer. For non-IgE dependant activators, such as fMLP, the picture was different since IgE was not down regulated, whereas CCR3 was down regulated. As demonstrated using annexin V or the cholera toxin used for analysing apoptosis, these phenomenon were paralleled by the formation of lipid rafts, gangliosides domains, such as GM1, which is accessible from the extra cellular medium., Conclusions: Basophil activation leads to membrane events close to the apoptosis phenomenon. The flow cytometric analysis of these membrane events may lead to protocols for allergen-induced activation and, may significantly increase cellular test sensitivity, particularly for drugs allergy diagnosis for which the usual protocols, such as those using CD63 alone, are insufficiently sensitive.

Aims: Transforming growth factor-beta1 (TGF-beta1) and endoglin play a causal role in promoting cardiac fibrosis. Atorvastatin has been shown to have an inhibitory effect on cardiac fibroblasts in vitro. However, the effects of statins on TGF-beta1 and endoglin are poorly understood. We therefore sought to investigate the molecular mechanisms of atorvastatin on endoglin expression after TGF-beta1 stimulation in cardiac fibroblasts., Methods and Results: Cultured cardiac fibroblasts were obtained from adult male Sprague-Dawley rat hearts. TGF-beta1 stimulation increased endoglin and collagen I expression and atorvastatin inhibited the induction of endoglin and collagen I by TGF-beta1. Phosphatidylinositol-3 kinase (PI-3) and Akt inhibitors (wortmannin and Akt inhibitor X) completely attenuated the endoglin protein expression induced by TGF-beta1. TGF-beta1 induced phosphorylation of PI-3 kinase and Akt, while atorvastatin and wortmannin and Akt inhibitor X inhibited the phosphorylation of PI-3 kinase and Akt induced by TGF-beta1. The gel shift and promoter activity assay showed that TGF-beta1 increased Smad3/4-binding activity and endoglin promoter activity, while wortmannin and atorvastatin inhibited the Smad3/4-binding activity and endoglin promoter activity induced by TGF-beta1. TGF-beta1 increased collagen I protein expression, while endoglin siRNA attenuated collagen I protein expression induced by TGF-beta1. Atorvastatin decreased left ventricular TGF-beta1, endoglin, and collagen I protein expression and fibrotic area in a rat model of volume overload heart failure., Conclusion: Atorvastatin inhibits endoglin expression through the inhibition of PI-3 kinase, Akt, and Smad3 phosphorylation, and reduced Smad3/4 binding activity and endoglin promoter activity in cardiac fibroblasts.

Purpose: Cytotoxic T lymphocyte antigen 4 (CTLA4), a key negative regulator of T-cell activation, is targeted by the antibody tremelimumab to release potentially useful antitumor activity., Experimental Design: This phase II trial investigated tremelimumab as a second-line treatment for patients with metastatic gastric and esophageal adenocarcinomas. Tremelimumab was given every 3 months until symptomatic disease progression. Safety, clinical efficacy, and immunologic activity were evaluated., Results: Eighteen patients received tremelimumab. Most drug-related toxicity was mild; however, there was a single death due to bowel perforation that complicated colitis. Four patients had stable disease with clinical benefit; one patient achieved a partial response after eight cycles (25.4 months) and remains well on study at 32.7 months. Markers of regulatory phenotype, forkhead box protein 3 and CTLA4, doubled transiently in CD4(+)CD25(high) lymphocytes in the first month after tremelimumab before returning to baseline. In contrast, CTLA4 increased in CD4(+)CD25(low/negative) lymphocytes throughout the cycle of treatment. De novo proliferative responses to tumor-associated antigens 5T4 (8 of 18 patients) and carcinoembryonic antigen (5 of 13) were detected. Patients with a posttreatment carcinoembryonic antigen proliferative response had median survival of 17.1 months compared with 4.7 months for nonresponders (P = 0.004). Baseline interleukin-2 release after T-cell activation was higher in patients with clinical benefit and toxicity., Conclusion: Despite the disappointing response rate of tremelimumab, one patient had a remarkably durable benefit for this poor-prognosis disease. In vitro evidence of enhanced proliferative responses to relevant tumor-associated antigens suggests that combining CTLA4 blockade with antigen-targeted therapy may warrant further investigation.

Hereditary haemorrhagic telangiectasia (HHT), or Rendu-Osler-Weber syndrome, is an autosomal dominant vascular disease. The clinical manifestations are epistaxis, mucocutaneous and gastrointestinal telangiectases, and arteriovenous malformations. There are two predominant types of HHT caused by mutations in Endoglin (ENG) and activin receptor-like kinase 1 (ALK1) (ACVRL1) genes, HHT1 and HHT2, respectively. No cure for HHT has been found and there is a current need to find new effective drug treatments for the disease. Some patients show severe epistaxis which interferes with their quality of life. We report preliminary results obtained with Raloxifene to treat epistaxis in postmenopausal HHT women diagnosed with osteoporosis. We tried to unravel the molecular mechanisms involved in the therapeutic effects of raloxifene. ENG and ACVRL1 genes code for proteins involved in the transforming growth factor beta pathway and it is widely accepted that haploinsufficiency is the origin for the pathogenicity of HHT. Therefore, identification of drugs able to increase the expression of those genes is essential to propose new therapies for HHT. In vitro results show that raloxifene increases the protein and mRNA expression of ENG and ALK1 in cultured endothelial cells. Raloxifene also stimulates the promoter activity of these genes, suggesting a transcriptional regulation of ENG and ALK1. Furthermore, Raloxifene improved endothelial cell functions like tubulogenesis and migration in agreement with the reported functional roles of Endoglin and ALK1. Our pilot study provides a further hint that oral administration of raloxifene may be beneficial for epistaxis treatment in HHT menopausal women. The molecular mechanisms of raloxifene involve counteracting the haploinsufficiency of ENG and ALK1.

Age-related cognitive deficits in hippocampus are correlated with neuroinflammatory changes, typified by increased pro-inflammatory cytokine production and microglial activation. We provide evidence that the neural cell adhesion molecule (NCAM)-derived mimetic peptide, FG loop (FGL), acts as a novel anti-inflammatory agent. Administration of FGL to aged rats attenuated the increased expression of markers of activated microglia, the increase in pro-inflammatory interleukin-1beta (IL-1beta) and the impairment in long-term potentiation (LTP). We report that the age-related increase in microglial activation was accompanied by decreased expression of neuronal CD200, and suggest that the proclivity of FGL to suppress microglial activation is due to its stimulatory effect on neuronal CD200. We demonstrate that FGL enhanced interleukin-4 (IL-4) release from glial cells and IL-4 in turn enhanced neuronal CD200 in vitro. We provide evidence that the increase in CD200 is reliant on IL-4-induced extracellular signal-regulated kinase (ERK) signal transduction. These findings provide the first evidence of a role for FGL as an anti-inflammatory agent and identify a mechanism by which FGL controls microglial activation.

Reports indicate that myeloid and plasmacytoid dendritic cells (mDCs and pDCs), which are key effector cells in host innate immune responses, can be infected with HIV-1 and are reduced in number and function during the chronic phase of HIV disease. Furthermore, it was recently demonstrated that a sustained loss of mDCs and pDCs occurs in SIV-infected macaques. Since loss of functional DC populations might impair innate immune responses to opportunistic microorganisms and neoplastic cells, we explored whether inoculation of naive and SIV- or SHIV-infected pigtailed macaques with the hematopoietic cytokine FLT3-ligand (FLT3-L) would expand the number of mDCs and pDCs in vivo. After the macaques received supraphysiologic doses of FLT3-L, mDCs, pDCs, and monocytes increased up to 45-fold in blood, lymph nodes, and bone marrow (BM), with DC expansion in the BM preceding mobilization in blood and lymphoid tissues. FLT3-L also increased serum levels of IL-12, at least transiently, and elicited higher surface expression of HLA-DR and the activation markers CD25 and CD69 on NK and T cells. During and after treatment of infected animals, APCs increased in number and were activated; however, CD4(+) T cell numbers, virion RNA, and anti-SIV/SHIV antibody titers remained relatively stable, suggesting that FLT3-L might be a safe modality to expand DC populations and provide therapeutic benefit during chronic lentivirus infections.

Objective: PD0325901, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), was associated with the occurrence of ocular retinal vein occlusion (RVO) during clinical trials in patients with solid tumors. As previous animal safety studies in rats and dogs did not identify the eye as a target organ of toxicity, this work was conducted to develop a rabbit model of ocular toxicity with PD0325901., Methods: Dutch-Belted rabbits were administered a single intravitreal injection of PD0325901 (0.5 or 1 mg/eye) or saline control, and ophthalmic examinations and retinal angiography were conducted over a 2-week period post-dose. In addition, mechanism of ocular toxicity was further explored in rat with microarray analysis., Results: PD0325901 treatment produced RVO with retinal vasculature leakage and hemorrhage within 48-h postinjection in Dutch-Belted rabbits. Subsequent retinal detachment and degeneration were also detected on day 8 postinjection. To evaluate the potential mechanism(s) of PD0325901-mediated RVO, male Brown Norway rats were orally administered PD0325901 (45 mg/kg/day) up to 5 days and retinal tissue was collected for gene array analysis. Although PD0325901 did not produce clinical evidence of RVO in rats, retinal gene expression suggested an increased oxidative stress and inflammatory response, endothelium and blood-retinal barrier damage, and prothrombotic effects. Moreover, soluble endothelial protein C receptor (sEPCR), a biomarker for RVO, was elevated in human umbilical vascular endothelial cells (HUVECs) cultured with PD0325901., Conclusions: This work has developed a rabbit model of PD0325901-induced RVO that may be used to characterize the cellular and molecular mechanisms of this effect in humans.

Background: Diabetes mellitus is characterized by hyperglycemia that plays an important role in the pathogenesis of diabetic complications including cardiovascular diseases. Moreover, hyperglycemia induces increased generation of advanced glycation end products (AGEs). The activation of platelets is associated with the development of cardiovascular diseases., Aim of the Study: The question whether AGEs acutely induce platelet activation as a response to exogenous stimulus is addressed., Materials and Methods: The effect of AGEs derived from food and human serum being purified by lysozyme affinity chromatography was examined by incubating in vitro freshly isolated blood platelets from fasted subjects at various concentrations and different time points. Platelet activation, determined as expression of surface markers CD62 and CD63, and the presence of the receptor for AGEs (RAGE) in platelet membranes was measured by flow cytometric analysis using specific antibodies., Results: Incubation with food-derived as well as serum-derived AGEs stimulated significantly the expression of CD62 up to 7.1-fold and CD63 up to 2.2-fold at the platelet surface membrane as a function of concentration and time. Incubation with thrombin or AGEs significantly increased RAGE expression twofold at the platelet surface membrane., Conclusions: The increase in surface activation marker and RAGE expression in platelets, resulting from concentrations of AGEs that occur in vivo after a meal or a drink as a source of exogenous AGEs, points to signaling mechanisms for food AGEs that could favor the precipitation of acute postprandial ischemic events.

MicroRNAs (miRs) are 21- to 23-nucleotide RNA molecules that regulate the stability or translational efficiency of target messenger RNAs of proteins involved in cell growth and apoptosis. miR-92 is part of the mir-17-92 cluster, which comprises members with an effect on cell proliferation. However, the role of miR-92 is unknown, and its targets have not been identified. Here, we describe a mechanism through which miR-92 contributes to regulate cell proliferation. Using a miR-92 synthetic double-strand oligonucleotide, we demonstrate that miR-92 increases 32D myeloid cell proliferation and 5-bromo-2-deoxyuridine (BrdU) incorporation and inhibits cell death. The effect is miR-92 specific since the miR-92 antagomir inhibits cell proliferation. Moreover, we show that miR-92 acts by modulating p63-isoform abundance through down-regulatation of endogenous DeltaNp63beta. Using luciferase reporters containing p63 3'UTR fragments with wild-type or mutant miR-92 complementary sites, we demonstrate that the wild-type 3'UTR is a direct target of miR-92. Finally, we observed that a miR-92-resistant DeltaNp63beta isoform (without 3'UTR) inhibits cell proliferation and parallels the effect of the antagomir. We conclude that one of the molecular mechanisms through which miR-92 increases cell proliferation is by negative regulation of an isoform of the cell-cycle regulator p63.

Human immunodeficiency virus infection leads to T-cell exhaustion and involution of lymphoid tissue. Recently, the programmed death-1 pathway was found to be crucial for virus-specific T-cell exhaustion during human immunodeficiency virus infection. Programmed death-1 expression was elevated on human immunodeficiency virus-specific peripheral blood CD8+ and CD4+ T cells and correlated with disease severity. During human immunodeficiency infection, lymphoid tissue acts as a major viral reservoir and is an important site for viral replication, but it is also essential for regulatory processes important for immune recovery. We compared programmed death-1 expression in 2 consecutive inguinal lymph nodes of 14 patients, excised before antiretroviral therapy (antiretroviral therapy as of 1997-1999) and 16 to 20 months under antiretroviral therapy. In analogy to lymph nodes of human immunodeficiency virus-negative individuals, in all treated patients, the germinal center area decreased, whereas the number of germinal centers did not significantly change. Programmed death-1 expression was mostly found in germinal centers. The absolute extent of programmed death 1 expression per section was not significantly altered after antiretroviral therapy resulting in a significant-relative increase of programmed death 1 per shrunken germinal center. In colocalization studies, CD45R0+ cells that include helper/inducer T cells strongly expressed programmed death-1 before and during therapy, whereas CD8+ T cells, fewer in numbers, showed a weak expression for programmed death-1. Thus, although antiretroviral therapy seems to reduce the number of programmed death-1-positive CD8+ T lymphocytes within germinal centers, it does not down-regulate programmed death-1 expression on the helper/inducer T-cell subset that may remain exhausted and therefore unable to trigger immune recovery.

YT-CB5, which had been continuously cultured with chrysotile B (CB)asbestos, showed impaired cytotoxicity with decreased expression of NKG2D and 2B4 NK cell-activating receptors. In the present study, the phosphorylation of extracellular signal-regulated kinase (ERK), which is known to induce degranulation downstream of many NK cell-activating receptors, was examined in YT-CB5 by flow cytometry and compared with the control line YT-Org. YT-CB5 exhibited impaired phosphorylation of ERK1/2 induced by the recognition of K562 cells, downstream of a process mediated by Src family kinase and phosphoinositide 3-kinase. YT-CB5 also exhibited impaired phosphorylation of ERK1/2 following incubation with K562 cells in the presence of anti-2B4 antibodies, where co-stimulation by 2B4 augmented the phosphorylation of ERK1/2 in YT-CB5 to a similar degree as in YT-Org. The phosphorylation of ERK1/2 induced by an inhibitor against phosphatase (PP) 1 and PP2A was also lower in YT-CB5 compared with YT-Org. Moreover, bead-bound antibodies to NKG2D, which contribute to cytotoxicity against K562 cells, induced negligible phosphorylation of ERK1/2 in YT-CB5, although antibodies to 2B4 induced a comparatively greater level of phosphorylation. Additionally, peripheral blood (PB-) NK cells with low expression of NKG2D showed lower phosphorylation of ERK1/2 mediated by anti-NKG2D antibodies compared with PB-NK cells with high expression of NKG2D. These results indicate that signal transduction events leading to the phosphorylation of ERK is impaired in YT-CB5 due to decreased expression of NKG2D. Further studies are required to clarify whether this suppressive effect of asbestos exposure on NK cells might promote lung cancer and mesothelioma in people who have inhaled asbestos.

LPS activates microglia, which are normally maintained in a quiescent state by CD200-CD200 receptor (CD200R) interaction. MAC-1 (a microglia marker) mRNA expression was increased in mice brains up to 1 year post LPS administration (i.p.). Minocycline treatment did not prevent LPS (5 mg/kg)-induced increase in MAC-1 mRNA but reduced that induced by 0.1 mg/kg LPS. CD200R mRNA decreased starting at 4 h, whereas CD200 mRNA increased at 4 h and decreased at 1 year post LPS inoculation. Thus, LPS-induced changes in CD200-CD200R equilibrium might keep microglia chronically activated. Minocycline does not effectively inhibit microglia activation induced by high-dose LPS.

Background: Vasculogenic mimicry (VM) was increasingly recognized as a form of aggressive melanoma acquiring blood supply. Genistein had attracted much attention as a potential anticancer agent. Therefore, we examined the effect of Genistein on VM in human uveal melanoma cells., Methods: VM structure was detected by periodic acid-Schiff (PAS) staining for uveal melanoma C918 cells cultured on the three-dimensional type I collagen gels after exposed to Genistein. We used reverse transcription polymerase chain reaction (RT-PCR) and Western Blot analysis to examine the effect of Genistein on vascular endothelial cadherin (VE-cadherin) mRNA and protein expression. The nude mice models of human uveal melanoma C918 cells were established to assess the number of VM using immunohistochemical and PAS double-staining., Results: Genistein inhibited the survival of C918 cells in vitro. The ectopic model study showed that VM in tumor tissue sections were significantly reduced by Genistein in vivo. In vitro, the VM structure was found in control, 25 and 50 microM Genistein-treatment groups but not in 100 and 200 microM. RT-PCR and Western Blot showed that 100 and 200 microM concentration of Genistein could significantly decrease VE-cadherin mRNA and protein expression of C918 cells compared with control (P < 0.05). However, the 25 and 50 microM Genistein slightly decreased the VE-cadherin level in vitro (P > 0.05)., Conclusion: Genistein inhibits VM formation of uveal melanoma cells in vivo and in vitro. One possible underlying molecular mechanism by which Genistein could inhibit VM formation of uveal melanoma is related to down-regulation of VE-cadherin.

Cytotoxic T-lymphocyte antigen 4 (CTLA-4) uniformly suppresses antigen-specific T cells during chronic infection with bacterial, parasitic or viral pathogens. However, the importance of CTLA-4 in controlling the T-cell response during acute infection or after priming with live attenuated vaccine vectors has not been well characterized. Since strategies aimed at blocking CTLA-4 are being actively developed to therapeutically augment T-cell-mediated immunity, the effects of CTLA-4 blockade on T-cell activation during these conditions need to be more clearly defined. We have examined the role of CTLA-4 in a prime-challenge model of acute bacterial infection using both attenuated and virulent strains of the intracellular bacterium Listeria monocytogenes. Although Foxp3(+) CD4(+) T cells are the predominant CTLA-4-expressing cell type in naïve mice, antigen-specific Foxp3(-) CD4(+) cells upregulate CTLA-4 expression after primary L. monocytogenes infection. Blockade of CTLA-4 results in increased numbers of L. monocytogenes-specific CD4 and CD8 T cells after primary infection with attenuated L. monocytogenes, and confers more rapid bacterial clearance after secondary challenge with virulent L. monocytogenes. Accordingly, CTLA-4 plays an important suppressive role in T-cell priming and protective immunity in a prime-challenge model of acute bacterial infection.

Helenalin is a naturally occuring sesquiterpene lactone extracted from Arnica montana and Arnica chamissonis ssp. foliosa. Helenalin and its derivatives are known for anti-cancer and anti-inflammatory effects via inhibiting NF-kappaB and telomerase activity and impairing protein and DNA synthesis, suggesting that helenalin is a potential candidate for the treatment of deregulated and unwanted T cell-mediated immune responses. Here we show that helenalin induces apoptosis in activated CD4+ T cells by triggering the mitochondrial pathway of apoptosis. Induction of apoptosis is accompanied by rapid stabilization of p53, nuclear localization of p53 and AIF, and an increase in ROS production that results in loss of mitochondrial membrane potential (DeltaPsim). Activated CD4+ T cells which survive exposure to helenalin undergo inhibition of proliferation by induction of G2/M cell cycle arrest. Cell cycle arrest is accompanied by the accumulation of cell cycle regulator proteins p21(WAF/CIP1), p2(KIP1) and cyclin D2, whereas abundance of cyclin A and B(1) is decreased. Cell surface expression of the activation-associated receptors CD25, CD27, CD28, CD120b as well as production of IL-2 are impaired. Transcriptional activation of genes encoding for CD25, IL-2 and IFN-gamma is mediated by transcription factors of the NFAT family, and we demonstrate that helenalin suppresses nuclear translocation of NFATc2 in activated CD4+ T cells. Thus, helenalin can be defined as a new immunosuppressive compound suited for the treatment of deregulated and unwanted T cell-mediated immune responses.

Metal toxicants which affect the immune system may contribute to an increased incidence of autoimmune diseases, infectious diseases and cancer. In the recent past, there has been a growing concern among health and environmental scientists on the impact of environmental exposure to heavy metal lead on human health. In some instances the immune system appears to be exquisitely sensitive to the toxic heavy metal lead as compared to other toxicological parameters. Susceptibility to autoimmunity is determined by both heritable traits and environmental factors, and in this context there has been considerable interest in the influences that exposure to heavy metals may have on the initiation and/or progression of autoimmune diseases. This review discussed the immunomodulatory role of toxic heavy metal lead, on autoimmunity, phenotypic expression of the immune cells and hypersensitivity.

Both NK cells and IL-15 play crucial roles in innate immunity against viral infections and cancer. Cigarette smoke is known to increase susceptibility to infections and certain cancers. Interleukin (IL)-15 plays an important role in immune responses by regulating proliferation, survival and functions of NK cells. Here, we examined the impact of cigarette smoke on IL-15 production and IL-15 mediated NK cell functions in human PBMCs. We report that cigarette smoke significantly suppresses the induction of IL-15 by poly I:C in human PBMCs. Serum IL-15 levels among smokers was significantly lower than non-smokers. In contrast to a profound increases in intracellular IL-15/IL-15Ralpha in poly I:C-treated PBMCs, exposure of PBMCs to smoke-conditioned media (SCM) diminished the IL-15/IL-15Ralpha production. We examined if inhibition of IL-15 production could lead to less NK cell activation. Interestingly, SCM-treated PBMCs had diminished up-regulation of NK cell activation marker, CD69, but not NKG2D compared with controls after poly I:C stimulation. We then confirmed by using IL-15 neutralizing antibody as well as exogenous IL-15 that the ploy I:C-induced NK cells activation was IL-15 mediated. More importantly, cigarette smoke significantly impaired NK cell cytolytic potential to kill K562 cancer cells which was found to be IL-15 mediated. The inhibition of IL-15 and its regulatory NK cell activities were linked to attenuated STAT3 and STAT5, but not ERK1/2 phosphorylations. We demonstrate, for the first time, that cigarette smoke compromises IL-15 production and as a result NK cell function which could link to the higher incidence of cancers or viral infections observed among smokers.

To develop a tumor vaccine directly targeting tumor antigen to dendritic cells in situ, we engineered human mesothelin (MSLN) into an antibody specific for mouse DEC-205, a receptor for antigen presentation. We then characterized both T cell and humoral responses to human MSLN and compared immunizing efficacy of DEC-205-targeted MSLN to nontargeted protein after a single-dose immunization. Targeting human MSLN to DEC-205 receptor induced stronger CD4(+) T-cell responses compared to high doses of mesothelin protein. Approximately 0.5% CD4(+) T cells were primed to produce IFN-gamma, tumor necrosis factor-alpha, and IL-2 via intracellular cytokine staining, and the T cells also could proliferate rapidly. The immune response exhibited breadth because the primed CD4(+) T cells responded to at least three epitopes in the H-2(b) background. Targeting MSLN protein to DEC-205 receptor also resulted in cross-presentation to CD8(+) T cells. Antibody responses against human MSLN were also detected in serum from primed mice by ELISA assays. In summary, targeting of MSLN to DEC-205 improves the induction of CD4(+) and CD8(+) T-cell immunity accompanied by an antibody response. DEC-205-targeting could be valuable for enhancing immunity to MSLN in cancers where this nonmutated protein is expressed.

Deoxynivalenol (DON) is the most prevalent trichothecene mycotoxin in crops in Europe and North America. It exhibits several toxic effects including impaired growth and immune dysregulation. Macrophages play pivotal role in the host defense; upon activation, they express several specific cell surface receptors that are important in adhesion and cell signaling. Several studies have demonstrated that DON can affect macrophages, however, very few data are available concerning the effect of DON on human macrophages, and the effect on macrophage cell surface receptors is unknown. In the present study, human blood monocytes, differentiated in vitro into macrophages, were activated with IFN-gamma, in the presence or absence of low concentrations of DON. The expression of CD11c, CD13, CD14, CD18, CD33, CD35, CD54, CD119 and HLA-DP/DQ/DR was analyzed by flow cytometry. As expected, macrophage activation by IFN-gamma upregulated the expression of CD54, CD14, CD119 and HLA-DP/DQ/DR. Incubation with DON decrease the cell surface expression of these activation markers in a dose-dependent manner. When cells were treated with 5muM DON, the mean fluorescence intensity measured for the expression of these receptors was the same as that observed in non-activated macrophages. This inhibitory effect of DON was only observed when the mycotoxin was applied before the activation signal. Taken together, our results suggest that low concentration of DON alter macrophage activation as measured by the expression of cell surface markers. This may have implications for human health when consuming DON contaminated feed.

Background: CD163 is a monocyte/macrophage specific receptor whose soluble form (sCD163) is elevated in inflammatory diseases. Obesity is associated with chronic inflammation and low adiponectin, an anti-inflammatory adipokine. Adiponectin, 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR) and metformin activate the AMP-kinase that exerts anti-inflammatory effects, and the influence of adiponectin and these drugs on monocytic CD163 was analysed, and cellular and sCD163 were determined in obesity and type 2 diabetes., Materials and Methods: Monocytes were incubated with adiponectin, AICAR or metformin. Furthermore, monocytes and serum were obtained from type 2 diabetic patients (T2D), overweight (defined as a body mass index > or = 25 kg m(-2)) and normal-weight (NW) controls. CD163 was analysed by immunoblot and sCD163 was measured by enzyme-linked immunosorbent assay in the supernatants of the monocytes and in serum., Results: In monocytes, adiponectin reduced cellular and surface CD163, whereas sCD163 was not altered in the corresponding supernatants. Further, metformin and AICAR downregulated CD163. Monocytic CD163 was higher in T2D and obesity, whereas sCD163 in the supernatants was not elevated and neither correlated with serum sCD163 nor systemic adiponectin. There was a positive correlation of monocytic sCD163 with serum but not with monocytic IL-6. In the serum of obese controls and T2D patients, sCD163 was significantly higher compared to NW donors and was positively associated with systemic IL-6., Conclusions: This study indicates that monocytic CD163 and systemic sCD163 are elevated in T2D and obesity. Adiponectin reduces CD163 in vitro, but additional factors related to obesity like IL-6 may be more relevant in vivo.

Purpose: The aims of the present study were to evaluate the clinical activity and the pharmacodynamic profile of the novel schedule of a single i.v. standard dose of cyclophosphamide (CTX) immediately followed by an oral metronomic CTX regimen with celecoxib (CXB) and dexamethasone (DEX) in advanced hormone-refractory prostate cancer patients., Experimental Design: Twenty-eight patients (68% docetaxel-resistant) received 500 mg/m2 CTX i.v. bolus on day 1 and, from day 2, 50 mg/day CTX p.o. plus 200 mg/twice a day CXB p.o. and 1 mg/day DEX p.o. until disease progression. Plasma vascular endothelial growth factor (VEGF) and thrombospondin-1 were detected by ELISA, and real-time reverse transcription-PCR of VEGF and thrombospondin-1 gene expression on peripheral blood mononuclear cell and of VE-cadherin (VE-C) in blood samples was done., Results: A confirmed prostate-specific antigen decrease of > or =50% from baseline was observed in 9 of 28 patients (32%). Median progression-free survival and overall survival were 3 months (95% confidence interval, 2.2-4.2 months) and 21 months (95% confidence interval, 12.4-29.4 months), respectively. Toxicity was mild and no grade 3 to 4 toxicities occurred. A significant relationship was found between plasma VEGF and prostate-specific antigen values (r = 0.4223; P < 0.001). VEGF levels significantly increased in nonresponders, whereas the responder patients maintained significantly lower levels of VE-C gene expression after the beginning of the treatment if compared with nonresponder ones., Conclusion: Metronomic CTX plus CXB and DEX showed favorable toxicity and activity profile in patients. VE-C gene expression and VEGF levels represent potentially useful pharmacodynamic markers for the clinical response.

Recent data have shown the presence of immunological alterations in adult patients suffering from obsessive-compulsive disorder (OCD). The objective of this study was to examine the possible effects of 12 months of treatment with different serotonergic drugs, such as clomipramine and selective serotonin reuptake inhibitors (SSRIs) on peripheral immunological cells of 18 OCD patients. Both the absolute number and percent of CD4+, CD8+, CD3+, CD19+ and CD56+ cells were measured in peripheral blood before and after treatment by means of a Facstar Flow Sorter apparatus. At baseline, all patients showed a significant increase of CD8+ and decrease of CD4+ lymphocytes when compared with a similar group of healthy control subjects; after the treatment, CD8+ and CD4+ cells, respectively, decreased and increased significantly, and the CD4+/CD8+ ratio increased, when compared with baseline values, in parallel with the clinical improvement. These data suggest that the alterations of immune cells reported in patients with OCD at baseline may be reverted by treatment with SRIs and should be considered a state-dependent marker, perhaps related to a condition of stress.

Background: FK778, a malononitrilamide analogue of lefunomide, is currently a promising immunosuppressive drug. Because the cellular and molecular mechanisms underlying the effects of FK778 are not entirely clarified. We studied its effects on human peripheral dendritic cells., Methods: Peripheral blood mononuclear cells (PBMC) from 12 healthy volunteers were isolated by density separation over Ficoll solution. After resuspension in adaptive immunotherapy medium (AIM)-V medium, they were cultured without exogenous growth factors. The study group was treated with FK 778 (50 microg/mL) or Rapamycin (10 ng/mL). The phenotype of dendritic cell was ascertained by indirect immunoflurescence for analysis by flow cytometry., Results: Compared with the Rapamycin-treated controls, the expressions of CD80, CD83, CD86, HLA-DA, CD54, CD62, CCR5, and CCR7 in the FK778-treated myeloid dendritic cells and the expression of CD80, CD83, CD86, HLA-DA, and CD54 in the FK778-treated plasmacytoid dendritic cells were significantly down-regulated., Conclusion: FK778 inhibited the differentiation and maturation of dendritic cells.

The enzyme indoleamine 2,3-dioxygenase (IDO) regulates immune responses through the capacity to degrade the essential amino acid tryptophan into kynurenine and other downstream metabolites that suppress effector T-cell function and favour the differentiation of regulatory T cells. Considerable experimental evidence indicates that IDO can be expressed by dendritic cells, by tumour cells or by surrounding stromal cells, either within proximity of the tumour or at distal sites. Recent advances in the biochemistry of IDO and in our understanding of the biological relevance of IDO-mediated tryptophan consumption to the establishment of dominant immune tolerance to cancer will be summarised and discussed. Within the wider context of cancer immunotherapy, this Review also delineates how IDO could be exploited as a molecular target for therapeutic intervention in order to boost anti-cancer immunity.

Inflammatory stimuli result in vascular leakage with potentially life threatening consequences. As a key barrier component, loss of vascular endothelial (VE-) cadherin-mediated adhesion often precedes endothelial breakdown. This study aimed to stabilize VE-cadherin transinteraction and endothelial barrier function using peptides targeting the VE-cadherin adhesive interface. After modelling the transinteracting VE-cadherin structure, an inhibiting single peptide (SP) against a VE-cadherin binding pocket was selected, which specifically blocked VE-cadherin transinteraction as analyzed by single molecule atomic force microscopy (AFM). The tandem peptide (TP) consisting of two SP sequences in tandem was designed to strengthen VE-cadherin adhesion by simultaneously binding and cross-bridging two interacting cadherin molecules. Indeed, in AFM experiments TP specifically rendered VE-cadherin transinteraction resistant against an inhibitory monoclonal antibody. Moreover, TP reduced VE-cadherin lateral mobility and enhanced binding of VE-cadherin-coated microbeads to cultured endothelial cells, but acted independently of the actin cytoskeleton. TP also stabilized endothelial barrier properties against the Ca(2+) ionophore A23187 and the inhibitory antibody. Finally, TP abolished endothelial permeability increase induced by tumour necrosis factor-alpha in microperfused venules in vivo. Stabilization of VE-cadherin adhesion by cross-bridging peptides may therefore be a novel therapeutic approach for the treatment of vascular hyperpermeability.

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor alpha (TNF- alpha)-induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-alpha stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-alpha-induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68-positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Summary: As the recognition that costimulatory signals are critical for optimal T-cell activation, proliferation, and differentiation, there has been an explosion in the study of costimulatory molecules and their roles in enhancing anti-donor T-cell responses following transplantation. Here, we focus on the bench-to-beside translation of blocking agents designed to target three critical costimulatory pathways: the CD28/CD80/CD86 pathway, the CD154/CD40 pathway, and the lymphocyte function associated antigen-1/intercellular adhesion molecule pathway. While blockade of each of these pathways proved promising in inhibiting donor-reactive T-cell responses and promoting long-term graft survival in murine models of transplantation, the progression of development of therapeutic agents to block these pathways has each taken a slightly different course. Both logistical and biological pitfalls have accompanied the translation of blockers of all three pathways into clinically applicable therapies, and the development of costimulatory blockade as a substitute for current standard-of-care calcineurin inhibitors has by no means reached completion. Collaboration between both the basic and clinical arenas will further propel the development of costimulation blockers currently in the pipeline, as well as of novel methods to target these critical pathways during transplantation.

It is now well established that increased programmed death-1-ligand 1 (PD-L1) surface expression in cancer cells and the resultant T cell suppression contribute to cancer cell immune evasion. Blockade of PD-L1 function has been shown to stimulate anti-cancer immunity. Therefore, compounds that can down-regulate PD-L1 surface expression in cancer cells may serve as novel immune modulators to promote cancer cell-reactive immune responses. In the present study, we examined the effects of nimesulide, a selective COX-2 inhibitor, on PD-L1 surface expression in breast cancer cells by flow cytometry. We demonstrated that nimesulide was able to inhibit IFN-gamma-induced PD-L1 surface expression in breast cancer cells. However, our data indicate that the inhibitory effects of nimesulide appear to be independent of COX-2/PGE2 signaling. Since nimesulide also exhibits anti-tumor activities by inducing cancer cell apoptosis and inhibiting cancer cell proliferation, our findings suggest that nimesulide may represent a new class of chemotherapeutic agents that possess dual functions to inhibit cancer cell growth and promote cancer cell immune responses.

There is currently no cure for aseptic loosening (AL) of total joint replacement (TJR) except surgical revision. The purpose of this study was to determine whether oral EM could improve the periprosthetic tissue profiles and reduce serum cytokine production in AL patients who are candidates for surgical revision. We recruited 32 AL patients. AL patients were treated with either EM (600 mg/day, n=18) or placebo (n=14) daily, started one month before surgery and ending on the day of surgery. Blood samples were obtained before EM treatment and during surgery. Periprosthetic tissues and joint fluids were collected during surgery. Our results demonstrate that oral EM reduces the inflammation of periprosthetic tissues, as manifested by the reduction of the numbers of infiltrating cells, CD68+ macrophages, RANKL+ cells, and TRAP+ cells. Remarkable decreases of TNFalpha (9.6-fold), IL-1beta (21.2-fold), and RANKL (76-fold) gene transcripts were observed in periprosthetic tissues of patients treated with oral EM. Serum levels of both TNFalpha and (to a lesser extent) IL-1beta were significantly reduced following EM treatment (p<0.05). Our results suggest that EM represents a biological cure or prevention for those patients who might need repeated revision surgeries and/or show the early signs of progressive osteolysis after TJR.

Cytotoxic lymphocyte antigen-4 (CTLA-4) blockade is an active immunotherapeutic strategy that is currently in clinical trials in cancer. There are several ongoing trials of anti-CTLA-4 in the metastatic setting of prostate cancer patients with reported clinical responses consisting of decreases in the prostate specific antigen (PSA) tumor marker for some patients. Immunologic markers that correlate with these clinical responses are necessary to guide further development of anti-CTLA-4 therapy in the treatment of cancer patients. We recently reported that CD4(+) inducible co-stimulator (ICOS)(hi) T cells that produce interferon-gamma (IFN-gamma) are increased in the peripheral blood and tumor tissues of bladder cancer patients treated with anti-CTLA-4 antibody. Here we present data from the same clinical trial in bladder cancer patients demonstrating a higher frequency of CD4(+)ICOS(hi) T cells and IFN-gamma mRNA levels in nonmalignant prostate tissues and incidental prostate tumor tissues removed at the time of radical cystoprostatectomy. Our data suggest immunologic markers that can be used to monitor prostate cancer patients who receive anti-CTLA-4 therapy and indicate that the immunologic impact of anti-CTLA-4 antibody can occur in both tumor and nonmalignant tissues. These data should be taken into consideration for evaluation of efficacy as well as immune-related adverse events associated with anti-CTLA-4 therapy.

Prostaglandin D(2) (PGD(2)) and its receptor chemoattractant receptor homologous molecule expressed on T(h)2 cells (CRTH2) have been implicated in the pathogenesis of numerous allergic diseases. We investigated the role of PGD(2) and CRTH2 in allergic cutaneous inflammation by using a highly potent and specific antagonist of CRTH2. Administration of this antagonist ameliorated cutaneous inflammation caused by either repeated epicutaneous ovalbumin or FITC sensitization. Gene expression and ELISA analysis revealed that there was reduced pro-inflammatory cytokine mRNA or protein produced. Importantly, the CRTH2 antagonist reduced total IgE, as well as antigen-specific IgE, IgG1 and IgG2a antibody levels. This reduction in antibody production correlated to reduced cytokines produced by splenocytes following in vitro antigen challenge. An examination of skin CD11c(+) dendritic cells (DC) showed that in mice treated with the CRTH2 antagonist, there was a decrease in the number of these cells that migrated to the draining lymph nodes in response to FITC application to the skin. Additionally, naive CD4(+) T lymphocytes co-cultured with skin-derived DC from CRTH2 antagonist-treated mice showed a reduced ability to produce a number of cytokines compared with DC from vehicle-treated mice. Collectively, these findings suggest that CRTH2 has a pivotal role in mediating the inflammation and the underlying immune response following epicutaneous sensitization.

In a recent study we have explored TfR2 expression in a panel of cancer cell lines and we observed that about 40% of these cell lines clearly express TfR2. Taking advantage of this observation and considering the frequent overexpression of c-Myc in cancer cells we have explored the existence of a possible relationship between c-Myc and TfR2 in these cell lines. Our results provided evidence that TfR2(+) cell lines express low c-Myc levels and low TfR1 levels, while TfR2(-) cell lines express high c-Myc and TfR1 levels. Using the erythroleukemic K562 TfR2(+) cells as a model, we observed that agents that enhance c-Myc expression, such as iron, determine a decrease of TfR2 expression, while molecules that induce a decreased c-Myc expression, such as the iron chelator desferoxamine or the kinase inhibitor ST 1571, induce an enhanced TfR2 expression. On the other hand, we have evaluated a possible effect of hypoxia and nitric oxide on TfR2 expression in erythroleukemia K526 and hepatoma HepG2 cells, providing evidence that: (i) agents inducing cellular hypoxia, such as CoCl(2), elicited a marked upmodulation of TfR1, but a downmodulation of TfR2 expression; (ii) NO(+) donors, such as sodium nitroprusside (SNP), induced a moderate decrease of TfR1, associated with a marked decline of TfR2 expression; (iii) NO donors, such as S-Nitroso-N-Acetylpenicillamine (SNAP), induced a clear increase of TfR1, associated with a moderate upmodulation of TfR2 expression. The ensemble of these observations suggests that in cancer cell lines TfR2 expression can be modulated through stimuli similar to those known to act on TfR1 and these findings may have important implications for our understanding of the role of TfR2 in the regulation of iron homeostasis.

Aims: Atherosclerosis is a chronic inflammatory disease regulated by immune mechanisms. CD69 is a cell surface receptor rapidly induced after leukocyte activation at sites of chronic inflammation. Genetic disruption of CD69 in the mouse aggravates collagen-induced arthritis (CIA), and partial depletion of CD69-expressing cells with anti-CD69 monoclonal antibody (mAb) prevents CIA development in wild-type mice, suggesting that this receptor negatively modulates immune and inflammatory responses. It has been recently reported that CD69 is upregulated in a large subset of T cells in atherosclerosis-prone apolipoprotein E-null mice (apoE(-/-)). In this study, we investigated whether altering CD69 function affects atherosclerosis development., Methods and Results: We studied native and diet-induced atherosclerosis in apoE(-/-) and doubly deficient apoE(-/-)CD69(-/-) mice and performed expression studies in tissues and primary cells derived from these animals. Plasma cholesterol level was unaffected by CD69 genetic inactivation. Although this genetic manipulation led to an elevated production of interferon gamma and interleukin 10 by activated T cells, apoE(-/-) and apoE(-/-)CD69(-/-) mice fed control and high-fat diet exhibited atheromas of similar size and composition when analysed at different stages of the disease. Likewise, anti-CD69 mAb treatment had no effect on plasma cholesterol and atherosclerosis burden in fat-fed apoE(-/-) mice., Conclusion: In contrast to previous studies highlighting the protective function of CD69 against CIA, an autoimmune inflammatory disease, our results rule out a significant role for CD69 against atherosclerosis in apoE(-/-) mice, an experimental disease model featuring a local inflammatory response triggered and sustained by alterations in lipid homeostasis.

Thrombus formation at sites of disrupted atherosclerotic plaques is a leading cause of death and disability worldwide. Although the platelet is now recognized to be a central regulator of thrombus formation, development of antiplatelet reagents that selectively target thrombosis over hemostasis represents a challenge. Existing prophylactic antiplatelet therapies are centered on the use of aspirin, an irreversible cyclooxygenase inhibitor, and a thienopyridine such as clopidogrel, which inactivates the adenosine diphosphate-stimulated P2Y(12) receptor. Although these compounds are widely used and have beneficial effects for patients, their antithrombotic benefit is complicated by an elevated bleeding risk and substantial or partial "resistance." Moreover, combination therapy with these two drugs increases the hemorrhagic risk even further. This review explores the possibility of inhibiting the platelet-surface ionotropic P2X(1) receptor and/or elevating CD39/NTPDase1 activity as new therapeutic approaches to reduce overall platelet reactivity and recruitment of surrounding platelets at prothrombotic locations. Because both proteins affect platelet activation at an early stage in the events leading to thrombosis but are less crucial in hemostasis, they provide new strategies to widen the cardiovascular therapeutic window without compromising safety.

Fully mature DC and, to a lesser extent, activated T and B cells express CD83, a surface molecule that appears to fulfil an important role in efficient T-cell activation. Recently, it has been shown that CD83 mRNA is transported from the nucleus to the cytoplasm by an uncommon route, involving the cellular RNA-binding protein HuR and the nuclear export receptor CRM1. Moreover, the shuttle phosphoprotein APRIL (ANP32B) has been shown to be required for HuR-mediated nucleocytoplasmic translocation of the CD83 mRNA by acting as an adaptor that links HuR and CRM1. Here, we are able to report that casein kinase 2 (CK2) phosphorylates APRIL on residue threonine244 (Thr(244)) and demonstrate that the CK2-specific inhibitor 4,5,6,7-tetrabromo-2-azabenzimidazole abolishes CD83 expression in activated Jurkat T cells by interfering with the nucleocytoplasmic translocation of CD83 mRNA. Depletion and knockdown studies demonstrate that the CK2 alpha' subunit is necessary for this regulation, whereas the CK2 alpha subunit seems to be dispensable. Taken together, the data presented significantly extend our knowledge of the complex regulation of CD83 mRNA processing and provides a novel strategy to interfere with CD83 expression.

As the overall prognosis and treatment response rate to standard chemotherapy for acute myeloid leukemia (AML) remains poor in the older adult population, there is a need for more effective therapeutic agents with lower toxicity profiles that can be offered to these patients. Gemtuzumab ozogamicin (GO) is an anti-CD33 monoclonal antibody that was approved by the US Food and Drug Administration for use as monotherapy in patients 60 years of age and older with relapsed AML. GO consists of a humanized anti-CD33 antibody (hP67.6) which is linked to N-acetyl-gamma calicheamicin 1,2-dimethyl hydrazine dichloride. Once the antibody attaches to the surface antigen, it is rapidly internalized. Calicheamicin, a potent enediyne, is subsequently released and acts as a cytotoxic anti-tumor agent. In this population, GO has an acceptable toxicity and yields response rates approaching 30%. The efficacy of GO as monotherapy and in combination therapy for treatment of both de novo and relapsed AML continues to be investigated.

Our investigation was carried out on an assumption that end results among patients radically-treated for colorectal cancer might be improved by use of enteroabsorption. The study group included 17, controls--13 patients with diagnostically verified stage I-III tumors. Mixed sorbent (microcellulose + polysorb) (6g) was administered, once a week, on the average of 20 days after operation. Immunological vigor was assayed 3 weeks after surgery: immunoglobulin levels--by turbodimetric method, cellular profile of lymphocytes--monoclonal antibodies to cell markers CD3, CD4, CD8, CD16 and CD22. As a result of adjuvant treatment CD22 (B-lymphocytes) concentration increased significantly--from 17.70 to 21.66 (22%), while CD16 (innate killers) both in absolute numbers (19%) and by percentage points (9%). Circulating immunocomplex levels in the sorbent-treatment group were significantly lower (37.44 ths units) than in control (48 ths units) (average 28%). No relapse or metastases were reported in either group.

Objective: The pathogenesis of symptoms of uncomplicated diverticular disease (UDD) is unclear, but changes in gut microflora and physiologic inflammation may be implicated. The objective of the study was to investigate the distribution of gut homing lymphocytes in peripheral blood and intestinal mucosa of UDD patients, and the effects of luminal antibiotic treatment., Material and Methods: Ten UDD patients and 10 age- and gender-matched healthy subjects underwent peripheral blood sampling, and colonoscopy with biopsies taken from the transverse and sigmoid colon. Treatment consisted of a 2-month course of rifaximin 1.2 g/day for 15 days/month. Blood sample and mucosal biopsies were repeated in UDD patients at the end of treatment. Flow cytometry was performed using monoclonal antibodies (CD3, CD4, CD8, CD25, CD19, CD45, CD62L, CD103)., Results: In peripheral blood, both CD4+ and CD8+/CD103+ were significantly higher in patients at baseline than in controls (0.95% versus 0.36%, and 0.5% versus 0.09%, respectively). After treatment, peripheral CD4+/CD103+ decreased (0.27%), while CD8+/CD103+ did not change (0.35%); on the contrary, peripheral CD25+ increased, the CD4+ subpopulation showing significantly higher levels than those in controls. No difference was found between lymphocytes in the diverticular sigmoid mucosa of patients at baseline and those in controls, but there was a significant decrease in CD8+/CD62L+ after treatment. In the normal transverse colon, CD4+/CD62L+ of patient at baseline were significantly lower than in controls. After treatment, CD4+/CD103+ levels significantly increased, while CD8+/CD62L+ levels significantly decreased., Conclusions: Both central and mucosal immunity may be modified in UDD patients, with an increased recruitment of CD103+ lymphocytes. A 2-month course of rifaximin appears to reduce CD103+ levels, suggesting a decrease in mobilization of mucosal homing.

Suppression of T-cell responses by host-derived regulatory factors is a key event leading to viral persistence. Antibody blockade of either IL-10 or programmed death-ligand 1 (PD-L1) during viral persistence enhances T-cell function and reduces viral titers. Because blockade of these immunoregulatory networks represents a powerful approach to establish immune control during persistent infection, it is important to determine whether these immunoinhibitory factors act independently or jointly and if combined blockade of these factors further enhances T-cell immunity and viral clearance. Herein, we demonstrate that the IL-10 and PD-L1 immunosuppressive pathways are mechanistically distinct. As a result, simultaneous blockade of IL-10 and PD-L1 was significantly more effective in restoring antiviral T-cell responses than blockade of either alone, and led to substantially enhanced control of an established persistent viral infection. Thus, combinatorial blockade of multiple immune-regulatory molecules may ultimately restore the T-cell responses required to tip the balance from viral persistence to immune-mediated control or elimination of persistent infection.

The shift to the production of a Th1 cytokine profile during an intracellular infection has been shown to depend on antigen presenting cells-derived IL-12 and T-cell-derived IFN-gamma production. IL-18 facilitates Th1 priming in synergy with IL-12 through the stimulation of IFN-gamma production by T cells, B cells, NK cells, macrophages and DCs. A low level of IFN-gamma production in PBMC cultures from lepromatous leprosy patients (LL) has been previously reported by several groups. We evaluated the synthesis of this cytokine after exogenous addition of recombinant IL-12 and IL-18 (IL12/IL18) in order to induce recovery of the IFN-gamma levels with Mycobacterium leprae antigenic stimulation. The aim of this study was to investigate if exogenous addition of IL12/IL18 to PBMC cell cultures in the presence of M. leprae antigens could induce recovery of IFN-gamma levels. We found that IFN-gamma levels in PBMCs cultured from LL patients were reestablished after exogenous addition of exogenous IL12/IL18 and we also observed a diminished IL-18R expression. Although the molecular mechanisms of IL12/IL18 synergy have not been clearly elucidated, we assume that recombinant cytokines can activate several transcription factors that induce IFN-gamma synthesis.

Regulatory T (Treg) cells are emerging as key players in the regulation of different immune responses, thereby representing potential candidates for therapeutic interventions in a broad variety of immunological disorders. While the reduction or loss in function would be of benefit during the treatment of cancer, induction and/or expansion of Treg cell function might be helpful to interfere with unwanted immune responses in transplantation medicine, during autoimmunity, allergy and inflammation. However, a better understanding of Treg cell biology is a prerequisite to specifically modulate its function during immune responses in vivo. In the present review we will discuss current concepts on different cell types, components and some novel surface receptors expressed by Treg cells, namely Neuropilin-1, CD83 and G protein-coupled receptor 83 which might represent promising targets for the modulation of Treg cell function in human disease.

Emergence of resistance to Imatinib complicates the treatment of chronic myeloid leukemia (CML). Second-generation Bcr-Abl inhibitors are capable to overcome resistance mediated by most mutations except T315I. As this mutation is causative for approximately 20% of clinically observed resistances, the need for novel treatment strategies becomes obvious. Here, we report on a novel kinase inhibitor PHA-680626 exhibiting strong inhibitory activity on both Bcr-Abl and Aurora kinases. Significant anti-proliferative and pro-apoptotic effects were observed in human BCR-ABL positive cell lines and murine BaF3 cells ectopically expressing wt BCR-ABL or the Imatinib-resistant BCR-ABL mutants M351T, E255K and, T315I. Treatment with PHA-680626 decreased phosphorylation of CrkL and histone H3. As CrkL represents a typical downstream target of Bcr-Abl while histone H3 phosphorylation is an indicator for Aurora kinase B activity, these findings indicate that effects of PHA-680626 are mediated via inhibition of both pathways. Moreover, high anti-proliferative activity of PHA-680626 was observed in primary CD34+ cells derived from CML patients at diagnosis or in blast crisis as well as from an individual harbouring the T315I mutation. Thus, both Bcr-Abl and Aurora kinase inhibition contribute to the efficacy of PHA-680626 against Imatinib-resistant BCR-ABL positive leukemias, particularly those harbouring the T315I mutation.

Background: This study evaluated the immunologic effects of thymoglobulin in modulating human CD4+ cells., Methods: Human CD4+ cells were purified from peripheral blood mononuclear cells by negative selection method. CD4+ cells were pretreated with thymoglobulin and incubated for 72 hr. Cells and culture supernatants were collected and studied by real-time quantitative polymerase chain reaction, fluorescence activated cell scanning, multiplex cytokine assay, and mixed lymphocyte reaction (MLR)., Results: Thymoglobulin pretreated CD4+ cells demonstrated up-regulation of gene transcripts for CTLA-4, OX40, forkhead box P3 (Foxp3), CD25, IFN-gamma, IL-10, and IL-2 as determined by real-time quantitative polymerase chain reaction. Fluorescence-activated cell scanning analysis demonstrated that CD4+ cells, pretreated with thymoglobulin, up-regulated CD25 expression on their surface, and the surface expression of CTLA-4 and OX40 and the expression of intracellular Foxp3 were observed in these CD4+CD25+ cells. Additionally, MLR demonstrated that thymoglobulin-pretreated cells partially inhibited proliferation of untreated autologous CD4+ cells in response to allogeneic cells. The high levels of IFN-gamma, IL-10, IL-2, and IL-4 were detected by multiplex cytokine assay in supernatants collected from cultures of thymoglobulin-pretreated CD4+ cells. The lymphocyte proliferation of allogeneic MLR was also partially blocked in the presence of supernatants from cultures of thymoglobulin-pretreated CD4+ cells., Conclusions: This study demonstrates that the unique effects of thymoglobulin in modulating CD4+ cells may be an important mechanism for its action in inducing immunosuppression and transplant tolerance.

Aim of Study: This study was performed to elicit the effectiveness of bee venom (BV), a traditional immunosuppressive Korean acupuncture agent, on the maturation of dendrtic cells (DCs)., Materials and Methods: Immature dendritic cells (iDCs) were generated from mouse bone marrow cells with GM-CSF. After 10 days of initial differentiation, DCs were activated with lipopolysaccharides (LPS) for another 48h in the presence or absence of BV. Surface molecule analysis, intracytoplasmic staining of cytokines, FITC-conjugated antigen uptake, and transwell migration assays were conducted with iDCs and activated DCs., Results: Up-regulation of costimulatory molecules, typical of mature DCs (mDCs) was inhibited by addition of BV. Pro-inflammatory cytokines were also found to be reduced with BV treatment in LPS-stimulated DC. A decrease in antigen uptake upon the maturation of DC was reversed in low dose BV treated mDC. In addition, BV treated mDC demonstrated reduced directional migration in response to CCL21, a lymphoid chemokine which directs mDC., Conclusions: BV may have a therapeutic effect an on abnormally activated immune status, such as autoimmune rheumatoid arthritis, through an immune-modulatory effect on DC.

Background & Aims: Hepatitis C virus (HCV) is a leading cause of chronic hepatitis worldwide. Viral attachment and entry, representing the first steps of virus-host cell interactions, are major targets of adaptive host cell defenses. The mechanisms of antibody-mediated neutralization by host neutralizing responses in HCV infection are only poorly understood. Retroviral HCV pseudotypes (HCVpp) and recombinant cell culture-derived HCV (HCVcc) have been successfully used to study viral entry and antibody-mediated neutralization., Methods: In this study, we used these model systems to investigate the mechanism of antibody-mediated neutralization by monoclonal antienvelope antibodies and polyclonal anti-HCV immunoglobulins purified from HCV-infected patients., Results: Using a panel of monoclonal antienvelope antibodies, we identified an epitope within the E1 glycoprotein targeted by human neutralizing antibodies during postbinding events. Interestingly, we observed that host neutralizing responses in the majority of HCV-infected individuals include antibodies targeting HCV entry after binding of the virus to the target cell membrane. Using a kinetic assay based on HCVpp and HCVcc entry, we demonstrate that purified antiviral immunoglobulins derived from individual HCV-infected patients appear to inhibit HCV infection at an entry step closely linked to CD81 and scavenger receptor BI (SR-BI)., Conclusions: Our results indicate that host neutralizing responses in HCV-infected patients target viral entry after HCV binding most likely related to HCV-CD81, and HCV-SR-BI interactions, as well as membrane fusion. These findings have implications not only for the understanding of the pathogenesis of HCV infection but also for the design of novel immunotherapeutic and preventive strategies.

Our laboratory and others have documented in some detail the immunological consequences which follow from interaction of the ubiquitously expressed molecule CD200 with its receptor(s) CD200R (expressed predominantly on cells of myeloid and lymphoid origin). In particular, there is evidence that these interactions lead to immunosuppressive signals which modulate graft rejection responses; decrease the manifestations of arthritis in rodent models; diminish mast cell mediator release in models of allergic disease; and favour the growth of tumors in both mice and humans. The development of small molecular weight agonists (and/or antagonists) of these interactions would thus likely have significant clinical importance. The data reported herein characterizes several such molecules in a number of rodent models.

Background and Objective: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) signalling of regulatory T cells regulates mucosal lymphocyte tolerance and differentiation, and may therefore have a beneficial effect in allergic diseases such as asthma. The aim of this study was to evaluate the effects of fluticasone propionate (FP) on CD4+CD25+ T cell co-expression of CTLA-4 in the sputum of mild asthmatic subjects., Methods: Eleven mild, stable asthmatic subjects completed a double-blind, randomized, crossover, placebo-controlled study to compare the effects of 14 days 200 microg twice daily FP and placebo. Before and after treatment, airway hyperresponsiveness was measured, and sputum was induced for measurements of CTLA-4+CD4+CD25+ cells, eosinophils and levels of IL-10, IL-13 and transforming growth factor (TGF)-beta., Results: FP treatment increased co-expression of CTLA-4 on sputum CD4+CD25+ cells from a mean (SEM) of 7.9% (1.8) to 12.7% (3.3) after 14 days treatment (P < 0.05) compared with placebo. FP treatment also significantly increased IL-10 levels, reduced per cent sputum eosinophils, and reduced airway hyperresponsiveness (P < 0.05). There was a significant negative correlation between the change in airway hyperresponsiveness and per cent sputum eosinophils (P < 0.01), but no correlation with changes in CTLA-4+CD4+CD25+ cells (P > 0.05). There was no change in the levels of sputum IL-13 or TGF-beta., Conclusions: The percentage of airway CTLA-4+CD4+CD25+ cells increased after FP treatment, coincident with improvements in airway inflammation and hyperresponsiveness.Whether improved asthma assessments are related to the increase in CTLA-4+CD4+CD25+ cells and thus improved regulation of T-cell tolerance and differentiation will require a larger sample size to determine. The normalization of CTLA-4+CD4+CD25+ cells in asthma may contribute to the management of this disease.

Yeast culture is widely used in animal feed and has been linked to beneficial effects on animal health and production. This study examined the anti-oxidant and immunomodulating effects of a consumable yeast culture, XP, in vitro. An aqueous extract of XP contained anti-oxidants able to enter living cells and quench free radicals. The XP extract induced an increased expression of CD69 and CD25 on NK and NKT cells, and an increased cytotoxic response to K562 tumor cells. The XP extract amplified ProteinA-induced B cell activation in vitro, as measured by induction of the CD86 antigen on B lymphoblasts in 7-day cultures. The data show an anti-inflammatory effect of the XP extract in conjunction with activation of NK cells and B lymphocytes in vitro. Further in vivo studies are needed to examine the impact of XP in animals with bacterial and viral infections, as well as around the time of vaccination.