Inhibition of vascular adhesion protein-1 enhances the anti-tumor effects of immune checkpoint inhibitors.

Modulation of the immunosuppressive tumor microenvironment (TME) is essential for enhancing the anti-tumor effects of immune checkpoint inhibitors (ICIs). Adhesion molecules and enzymes such as vascular adhesion protein-1 (VAP-1), which are expressed in some cancers and tumor vascular endothelial cells, may be involved in the generation of an immunosuppressive TME. In this study, the role of VAP-1 in TME was investigated in 2 murine colon cancer models and human cancer cells. Intraperitoneal administration of the VAP-1-specific inhibitor U-V296 inhibited murine tumor growth by enhancing IFN-γ-producing tumor antigen-specific CD8+ T cells. U-V296 exhibited significant synergistic anti-tumor effects with ICIs. In the TME of mice treated with U-V296, the expression of genes associated with M2-like macrophages, Th2 cells (Il4, Retnla, and Irf4), angiogenesis (Pecam1), and fibrosis (Acta2, Loxl2) were significantly decreased, and the Th1/Th2 balance was increased. H2 O2 , an enzymatic product of VAP-1, which promoted the production of IL-4 by mouse Th2 and inhibited IFN-γ by mouse Th1 and human tumor-infiltrating lymphocytes, was decreased in tumors and CD31+ tumor vascular endothelial cells in the TMEs of mice treated with VAP-1 inhibitor. TCGA database analysis showed that VAP-1 expression was a negative prognostic factor in human cancers, exhibiting a significant positive correlation with IL-4, IL4R, and IL-13 expression and a negative correlation with IFN-γ expression. These results indicated that VAP-1 is involved in the immunosuppressive TMEs through H2 O2 -associated Th2/M2 conditions and may be an attractive target for the development of combination cancer immunotherapy with ICIs.

Mapping of the binding sites of human diamine oxidase (DAO) monoclonal antibodies.

OBJECTIVE: Recently we characterized five mouse monoclonal antibodies that allow the specific and sensitive detection of human diamine oxidase (DAO). To understand differences in binding characteristics and recognition of enzyme variants, we mapped the antibody binding sites. METHODS: Fragments of human DAO were expressed as glutathione-S-transferase fusion proteins that were used for testing antibody binding on immunoblots. Combined information from species cross-reactivity, sequence comparison and binding site-prediction software were used to localize the epitope recognized by each antibody. RESULTS: All five monoclonal DAO antibodies bound to linear epitopes between the N3 and enzymatic domains of the 732 amino acid protein. The binding sites could be mapped onto amino acid regions V262-E278 and P279-R288, respectively, which exhibit considerable sequence variation in mammals explaining the fact that the human DAO antibodies do not cross-react with DAO from other species. The antibodies efficiently bind only denatured human DAO but not the native protein. CONCLUSIONS: Characterization of the binding sites of the DAO antibodies revealed that the antibodies bind two adjacent epitopes and exhibit similar binding characteristics and species cross-reactivity. As the epitopes do not overlap any of the amino acid substitutions described for clinically significant DAO gene polymorphisms, our antibodies will also be useful for analyses of the mutant DAO proteins.

Endothelial amine oxidase AOC3 transiently contributes to adaptive immune responses in the airways.

Amine oxidase, copper containing 3 (AOC3, also known as vascular adhesion protein-1 (VAP-1)) is an endothelial adhesion molecule that contributes to the extravasation of neutrophils, macrophages, and lymphocytes to sites of inflammation. However, the role of AOC3/VAP-1 in allergic responses remains unknown. Here, we studied eosinophil and CD4+ T-cell recruitment to the airways using AOC3/VAP-1-deficient mice. In an OVA-triggered asthma model, AOC3/VAP-1 slightly contributed to the accumulation of leukocytes in lungs in an age-dependent manner. We then established a new model to kinetically measure recruitment of OVA-specific CD4+ T cells to different airway immune compartments during the priming and effector phases of an adaptive immune response. The results showed that in the absence of AOC3/VAP-1, recruitment of antigen-specific CD4+ T cells to draining bronchial lymph nodes is reduced by 89% on day 3 after tracheal allergen exposure, but this difference was not observed on day 6. The dispersal of effector cells to lung and tracheal mucosa is AOC3/VAP-1 independent. Thus, in allergic airway reactions, AOC3/VAP-1 transiently contributes to the antigen-specific, CD4+ T-cell traffic to secondary lymphatic tissues, but not to airway mucosa or lung parenchyma. Our results suggest a largely redundant function for AOC3/VAP-1 in allergic inflammatory responses of the airways.

VCAM-1 and VAP-1 recruit myeloid cells that promote pulmonary metastasis in mice.

Pulmonary metastasis is a frequent cause of poor outcome in cancer patients. The formation of pulmonary metastasis is greatly facilitated by recruitment of myeloid cells, which are crucial for tumor cell survival and extravasation. During inflammation, homing of myeloid cells is mediated by endothelial activation, raising the question of a potential role for endothelial activation in myeloid cell recruitment during pulmonary metastasis. Here, we show that metastatic tumor cell attachment causes the induction of the endothelial activation markers vascular cell adhesion molecule-1 (VCAM-1) and vascular adhesion protein-1 (VAP-1). Induction of VCAM-1 is dependent on tumor cell-clot formation, decreasing upon induction of tissue factor pathway inhibitor or treatment with hirudin. Furthermore, inhibition of endothelial activation with a VCAM-1 blocking antibody or a VAP-1 small molecule inhibitor leads to reduced myeloid cell recruitment and diminished tumor cell survival and metastasis without affecting tumor cell adhesion. Simultaneous inhibition of VCAM-1 and VAP-1 does not result in further reduction in myeloid cell recruitment and tumor cell survival, suggesting that both act through closely related mechanisms. These results establish VCAM-1 and VAP-1 as mediators of myeloid cell recruitment in metastasis and identify VAP-1 as a potential target for therapeutic intervention to combat early metastasis.

Therapeutic advantage of anti-VAP-1 over anti-α4 integrin antibody in concanavalin a-induced hepatitis.

UNLABELLED: Hepatitis induced by concanavalin A (Con A) in mice is well known to be a T-lymphocyte-mediated injury. It has been reported that T helper (Th)1 and Th2 lymphocytes use α4 integrin and vascular adhesion protein (VAP)-1, respectively, to adhere within the hepatic sinusoids. Therefore, we investigated whether inhibition of these molecules ameliorates or worsens the Con A-induced hepatic injury in vivo. Vehicle or antibody to α4 integrin or VAP-1 was intravenously administered 30 minutes before Con A administration. In control mice Con A markedly increased the serum alanine aminotransferase (ALT) level in a dose-dependent manner, and induced a massive infiltration of CD3, particularly interleukin (IL)-4 producing CD4 T cells and liver injury. Both parameters were reduced by anti-VAP-1 antibody despite antibody only blocking the adhesion, not the amine oxidase activity of VAP-1. Both activities of VAP-1 were eliminated in VAP-1-deficient mice and both Con A-induced liver injury and CD4 T-cell infiltration were eradicated. In contrast to anti-VAP-1, anti-α4 integrin antibody reduced interferon-gamma (IFN-γ)-producing CD3 T cells but this worsened Con A hepatitis, suggesting inhibition of a suppressor cell. Con A induced the recruitment of CD49d(+) monocytic myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs) into the liver. Anti-α4 integrin dramatically blocked the influx of MDSCs but not Tregs. CONCLUSION: Our findings show that VAP-1 and α4 integrin have opposing effects in Con A-induced hepatic injury, which is associated with blocking the recruitment of CD4 lymphocytes and monocytic MDSCs, respectively. Moreover, these data provide the rationale for a potential therapeutic approach to target adhesion molecules in autoimmune hepatitis.

New tools for studying old questions: antibodies for human diamine oxidase.

Diamine oxidase (DAO) oxidatively deaminates histamine and other diamines. Due to the lack of antibodies for human DAO, many findings on this enzyme had not been confirmed in man. Therefore, we produced a series of monoclonal antibodies by immunizing mice with human DAO protein fragments expressed in vitro. Five different monoclonal antibodies specific for human DAO were obtained that do not recognize any other human protein and can detect DAO with 100-fold greater sensitivity than the most sensitive enzymatic assays currently available. Using these antibodies allowed confirming the expression and cellular localization of DAO in various human tissues such as kidney, intestine and placenta where the presence of the enzyme had previously been deduced from activity measurement and DAO mRNA analysis. Due to the high sensitivity of the novel monoclonal antibodies, DAO was also detected at sites that previously evaded unequivocal proof of DAO enzymatic activity such as the urine. On the other hand, with these antibodies it was possible to show that DAO is normally not present in human liver and blood serum. The new monoclonal antibodies not only allow a comprehensive quantitative evaluation of the expression of DAO at the cellular level in man but will also facilitate sensitive analyses of disease-associated alterations of this enzyme.

Small-molecule inhibitors of vascular adhesion protein-1 reduce the accumulation of myeloid cells into tumors and attenuate tumor growth in mice.

Vascular adhesion protein-1 (VAP-1) is an endothelial, cell surface-expressed oxidase involved in leukocyte traffic. The adhesive function of VAP-1 can be blocked by anti-VAP-1 Abs and small-molecule inhibitors. However, the effects of VAP-1 blockade on antitumor immunity and tumor progression are unknown. In this paper, we used anti-VAP-1 mAbs and small-molecule inhibitors of VAP-1 in B16 melanoma and EL-4 lymphoma tumor models in C57BL/6 mice. Leukocyte accumulation into tumors and neoangiogenesis were evaluated by immunohistochemistry, flow cytometry, and intravital videomicroscopy. We found that both anti-VAP-1 Abs and VAP-1 inhibitors reduced the number of leukocytes in the tumors, but they targeted partially different leukocyte subpopulations. Anti-VAP-1 Abs selectively inhibited infiltration of CD8-positive lymphocytes into tumors and had no effect on accumulation of myeloid cells into tumors. In contrast, the VAP-1 inhibitors significantly reduced only the number of proangiogenic Gr-1(+)CD11b(+) myeloid cells in melanomas and lymphomas. Blocking of VAP-1 by either means left tumor homing of regulatory T cells and type 2 immune-suppressing monocytes/macrophages intact. Notably, VAP-1 inhibitors, but not anti-VAP-1 Abs, retarded the growth of melanomas and lymphomas and reduced tumor neoangiogenesis. The VAP-1 inhibitors also reduced the binding of Gr-1(+) myeloid cells to the tumor vasculature. We conclude that tumors use the catalytic activity of VAP-1 to recruit myeloid cells into tumors and to support tumor progression. Small-molecule VAP-1 inhibitors therefore might be a potential new tool for immunotherapy of tumors.

Human Siglec-10 can bind to vascular adhesion protein-1 and serves as its substrate.

Leukocytes migrate from the blood into areas of inflammation by interacting with various adhesion molecules on endothelial cells. Vascular adhesion protein-1 (VAP-1) is a glycoprotein expressed on inflamed endothelium where it plays a dual role: it is both an enzyme that oxidizes primary amines and an adhesin that is involved in leukocyte trafficking to sites of inflammation. Although VAP-1 was identified more than 15 years ago, the counterreceptor(s) for VAP-1 on leukocytes has remained unknown. Here we have identified Siglec-10 as a leukocyte ligand for VAP-1 using phage display screenings. The binding between Siglec-10 and VAP-1 was verified by different adhesion assays, and this interaction was also consistent with molecular modeling. Moreover, the interaction between Siglec-10 and VAP-1 led to increased hydrogen peroxide production, indicating that Siglec-10 serves as a substrate for VAP-1. Thus, the Siglec-10-VAP-1 interaction seems to mediate lymphocyte adhesion to endothelium and has the potential to modify the inflammatory microenvironment via the enzymatic end products.

Investigating the safety and activity of the use of BTT1023 (Timolumab), in the treatment of patients with primary sclerosing cholangitis (BUTEO): A single-arm, two-stage, open-label, multi-centre, phase II clinical trial protocol.

INTRODUCTION: Primary sclerosing cholangitis (PSC) is a progressive inflammatory liver disease characterised by relentless liver fibrosis and a high unmet need for new therapies. Preventing fibrosis represents an important area of interest in the development of vital new drugs. Vascular adhesion protein-1 (VAP-1) drives inflammation in liver disease, and provision of an antibody against VAP-1 blunts fibrosis in murine models of liver injury. METHODS AND ANALYSIS: BUTEO is a single-arm, two-stage, open-label, multi-centre, phase II clinical trial. Up to 59 patients will receive treatment with anti-VAP monoclonal antibody, BTT1023, over a 78-day treatment period. Adults with PSC and a serum alkaline phosphatase (ALP) of at least 1.5 times the upper limit of normal will be included. Our primary outcome measure is a reduction in ALP by >25% from baseline to Day 99. Secondary outcome measures include safety and tolerability, changes pre therapy/post therapy in circulating serum VAP-1 as well as imaging findings. The first patient participant was recruited on 08 September 2015. ETHICS AND DISSEMINATION: This protocol has been approved by the Research Ethics Committee (REC, reference 14/EM/1272). The first REC approval date was 06 January 2015 with three subsequent approved amendments. This article refers to protocol V3.0, dated 16 March 2016. Results will be disseminated via peer-reviewed publication and presentation at international conferences. TRIAL REGISTRATION: The trial is registered with the European Medicines agency (EudraCT: 2014-002393-37), the National Institute for Health Research (Portfolio ID: 18051) and ISRCTN: 11233255. The clinicaltrials.gov identifier is NCT02239211. Pre-results.

Elevated serum soluble vascular adhesion protein-1 (VAP-1) in patients with active relapsing remitting multiple sclerosis.

Vascular adhesion protein-1 (VAP-1) is an endothelial cell molecule which controls leukocyte infiltration into tissues. Elevated serum soluble VAP-1-levels have been described in certain diseases with an inflammatory component. VAP-1 expression or function has not previously been studied in multiple sclerosis (MS). We report here that the concentration of soluble VAP-1 in serum is significantly higher in multiple sclerosis patients with ongoing inflammatory activity, as demonstrated by gadolinium-enhancing MRI lesions, when compared to patients with no gadolinium-enhancing lesions (555+/-195 vs. 388+/-102 ng/ml, p=0.0068). We propose that VAP-1 might participate in controlling leukocyte entry into inflamed brain.

Human vascular adhesion protein-1 (VAP-1) plays a critical role in lymphocyte-endothelial cell adhesion cascade under shear.

Lymphocyte binding to vascular endothelium is a prerequisite for the movement of immune cells from the blood into lymphoid tissues and into sites of inflammation. Human vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein involved in this interaction. It also displays an enzymatic (monoamine oxidase) activity. Here we examined how recombinant human VAP-1 mediates lymphocyte binding using rotatory and flow chamber binding assays. VAP-1 cDNA transfected into an endothelial cell line, which does not bind lymphocytes, renders the cell line capable of binding lymphocytes in a shear-dependent manner. VAP-1 transfectants bound lymphocytes 5 times better than monocytes with a preference for T killer cells, and no specific granulocyte adherence was detectable. The binding is partially inhibited by anti-VAP-1 monoclonal antibodies or by blocking lymphocyte L-selectin and CD18 integrins, but not by inhibition of several other homing-associated molecules. In contrast, CD44 ligation on lymphocytes markedly upregulates their VAP-1-dependent adhesion, suggesting that the VAP-1 counterreceptor can be activated via CD44. The transfectant model also allowed us to perform detailed structure-function analyses of VAP-1. We show that the exposed integrin-binding motif RGD or the enzymatic activity is not indispensable for VAP-1-dependent adhesion. Together, these data show that VAP-1 can reconstitute the lymphocyte-endothelial adhesion cascade under shear and propose a critical role for VAP-1 in lymphocyte emigration from the blood.

Immunochemical reactivity of native and modified preparations of pig kidney diamine oxidase.

This paper describes the antigenicity of pig kidney diamine oxidase [EC 1.4.3.6] and the possible role of constituent amino acids in the epitope structure of the enzyme. The loss of 62% of the biological activity after DAO-anti-DAO antibodies interaction was attributed to the steric hindrance caused by binding of antibody to the enzyme molecule. A gradual loss in antigenicity during ultraviolet (UV) irradiation was observed without any significant conformational change, demonstrating the destruction of antigenic determinants. However, ethoxyformylation of nine histidyl residues with complete inactivation caused no change in immunoreactivity. The results indicate that the antigenic sites and catalytic sites are located at different positions along the polypeptide chain. Moreover, the results of lysine residue modification were suggestive of possible involvement of lysine in the antigenic determinants of DAO.

Semicarbazide-sensitive amine oxidase in pig heart.

A semicarbazide-sensitive amine oxidase (SSAO) (E.C.1.4.3.6) has been purified from pig heart. Western blot analysis showed that the enzyme reacts with a polyclonal antibody raised against homogeneous crystalline pig plasma benzylamine oxidase (BAO). A subunit molecular mass of 97 KDa obtained by SDS electrophoresis is identical to the plasma enzyme. The purification procedure consisted of sequential DEAE cellulose, octyl-Sepharose, Con A-Sepharose and hydroxyapatite columns. Two peaks of activity were obtained on octyl-Sepharose which were found to be kinetically and immunologically indistinguishable. The specific activity of the purified enzyme was 0.045 mumol/min/mg of protein at 37 degrees C and the Km for benzylamine was estimated to be 63 microM. The enzyme was inhibited by carbonyl reagents such as semicarbazide but was insensitive to the effect of pargyline.

[Effect of the active immunization to plasma amine oxidase on the passive avoidance conditioned reflex]. / Vliianie aktivnoi immunizatsii k aminoksidaze plazmy krovi na formirovanie uslovnoi reaktsii passivnogo izbeganiia u belykh krys.

The influence of immune suppression of blood serum amine oxidase on mnemonic processes was studied during passive avoidance conditioning in white rats. The monoaminergic system is known to participate in conditioning. Our previous studies showed that the active immunization against serum amine oxidase results in suppression of endogenous serum amine oxidase and brain mitochondrial monoamine oxidase activities. We also revealed the specific changes in catecholamine concentrations. In this study, we observed a positive effect of immune suppression of serum amine oxidase on the passive avoidance conditioning during its reproduction on the next day and 45 days later. Thus, the obtained results suggest the possibility of using the active immunization against endogenous amine oxidase for regulation of mnestic processes.

Preclinical evaluation of a radioiodinated fully human antibody for in vivo imaging of vascular adhesion protein-1-positive vasculature in inflammation.

UNLABELLED: Vascular adhesion protein-1 (VAP-1) is an endothelial glycoprotein mediating leukocyte trafficking from blood to sites of inflammation. BTT-1023 is a fully human monoclonal anti-VAP-1 antibody developed to treat inflammatory diseases. In this study, we preclinically evaluated radioiodinated BTT-1023 for inflammation imaging. METHODS: Rabbits were intravenously injected with radioiodinated BTT-1023. Distribution and pharmacokinetics were assessed by PET/CT up to 72 h after injection. Human radiation dose estimates for (124)I-BTT-1023 were extrapolated. Additionally, rabbits with chemically induced synovitis were imaged with (123)I-BTT-1023 SPECT/CT. RESULTS: Radioiodinated BTT-1023 cleared rapidly from blood circulation and distributed to liver and thyroid. Inflamed joints were delineated by SPECT/CT. The estimated human effective dose due to (124)I-BTT-1023 was 0.55 mSv/MBq, if blockage of thyroid uptake is assumed. CONCLUSION: The radioiodinated BTT-1023 was able to detect mild inflammation in vivo. Clinical (124)I-BTT-1023 PET studies with injected radioactivity of 0.5-0.7 MBq/kg may be justified.

The diamine oxidase gene is associated with hypersensitivity response to non-steroidal anti-inflammatory drugs.

UNLABELLED: Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3-2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively). CONCLUSIONS AND IMPLICATIONS: The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.

Soluble intercellular adhesion molecule-1, D-lactate and diamine oxidase in patients with inflammatory bowel disease.

AIM: To study the levels of serum soluble intercellular adhesion molecule-1 (sICAM-1), plasma D-lactate and diamine oxidase (DAO) in patients with inflammatory bowel disease (IBD), and the potential clinical significance. METHODS: Sixty-nine patients with IBD and 30 healthy controls were included in this study. The concentration of sICAM-1 was detected with enzyme-linked immunosorbent assay, the level of D-lactate and DAO was measured by spectroscopic analysis, and the number of white blood cells (WBC) was determined by routine procedure. RESULTS: The levels of sICAM-l, DAO, and WBC in IBD patients were significantly higher than those in the control group (P < 0.01). sICAM-l in IBD patients was found to be closely related to the levels of DAO and D-lactate (212.94 +/- 69.89 vs 6.35 +/- 2.35, P = 0.000), DAO 212.94 +/- 69.89 vs 8.65 +/- 3.54, P = 0.000) and WBC (212.94 +/- 69.89 vs 7.40 +/- 2.61, P = 0.000), but no significant difference was observed between patients with ulcerative colitis and patients with Crohn's disease. The post-treatment levels of sICAM-l, D-lactate and WBC were significantly lower than before treatment (sICAM-l 206.57 +/- 79.21 vs 146.21 +/- 64.43, P = 0.000), (D-lactate 1.46 +/- 0.94 vs 0.52 +/- 0.32, P = 0.000) and (WBC 7.24 +/- 0.2.33 vs 5.21 +/- 3.21, P = 0.000). CONCLUSION: sICAM-1, D-lactate and DAO are closely related to the specific conditions of IBD, and thus could be used as a major diagnostic index.

Vascular adhesion protein-1 enhances tumor growth by supporting recruitment of Gr-1+CD11b+ myeloid cells into tumors.

Cancer growth is regulated by several nonmalignant cell types, such as leukocytes and endothelial cells, which reside in the stroma of the tumor. Vascular adhesion protein-1 (VAP-1) is an amine oxidase enzyme that is expressed on the surface of endothelial cells. It supports leukocyte traffic into inflamed tissues, but nothing is known about its possible role in cancer biology in vivo. Here, we report that B16 melanoma and EL-4 lymphoma remain smaller in VAP-1-deficient mice than in wild-type controls. We found an unexpected defect in tumor angiogenesis in the absence of VAP-1. VAP-1 also selectively enhanced the recruitment of Gr-1+CD11b+ myeloid cells into the tumors. Generation of mice expressing enzymatically inactive VAP-1 showed that the oxidase activity of VAP-1 was necessary to support neoangiogenesis, myeloid cell recruitment, and tumor growth in vivo. These data describe VAP-1 as the first adhesion molecule known to be involved in the recruitment of Gr-1+CD11b+ myeloid cells into tumors. They also suggest that VAP-1 is a potential new tool for immunotherapy of tumors that could be exploited to reduce tumor burden by controlling the traffic of Gr-1+CD11b+ myeloid cells.

The effects of 40 hours of total sleep deprivation on inflammatory markers in healthy young adults.

Inflammatory cytokines are released in response to stress, tissue damage, and infection. Acutely, this response is adaptive; however, chronic elevation of inflammatory proteins can contribute to health problems including cardiovascular, endocrine, mood, and sleep disorders. Few studies have examined how sleep deprivation acutely affects inflammatory markers, which was the aim of the current study. Nineteen healthy men and women aged 28.05+/-8.56 (mean+/-SD) were totally sleep deprived for 40 h under constant routine conditions. Pro-inflammatory markers: intracellular adhesion molecule-1 (ICAM-1), E-selectin, vascular adhesion molecule-1 (VCAM-1), c-reactive protein (CRP), interleukin-6 (IL-6), and interleukin-1beta (IL-1beta), and the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1ra) were assayed in plasma. Daytime levels during baseline (hours 1-15 of scheduled wakefulness) were compared to daytime levels during sleep deprivation (hours 25-39 of scheduled wakefulness), thus controlling for circadian phase within an individual. Repeated measures ANOVA with planned comparisons showed that 40 h of total sleep deprivation induced a significant increase in E-selectin, ICAM-1, IL-1beta, and IL-1ra, a significant decrease in CRP and IL-6, and no significant change in VCAM-1. Alterations in circulating levels of pro- and anti-inflammatory cytokines and cell adhesion molecules during sleep deprivation were consistent with both increased and decreased inflammation. These findings suggest that one night of sleep loss triggers a stress response that includes stimulation of both pro- and anti-inflammatory proteins in the healthy young subjects tested under our experimental conditions.