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Objective This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue. Methods Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages. Results In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P < 0.05). The expression of several KP enzyme genes (indoleamine 2,3-dioxygenase 1 [IDO1], kynureninase [KYNU], kynurenine 3-monooxygenase [KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P < 0.05), and their expression was induced by proinflammatory cytokines in human primary adipocytes (P < 0.05), except for KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P < 0.05). Conclusions In the context of obesity, the presence of macrophages in adipose tissue may contribute to diverting KP toward KMO activation.

BACKGROUND In recent years, there has been an increasing interest in targeting human prostate tumor-associated antigens (TAAs) for prostate cancer immunotherapy as an alternative to other therapeutic modalities. However, immunologic tolerance to TAA poses a significant obstacle to effective, TAA-targeted immunotherapy. We sought to investigate whether androgen deprivation would result in circumventing immune tolerance to prostate TAA by impacting CD8 cell responses. METHODS To this end, we generated a transgenic mouse that expresses the human prostate-specific antigen (PSA) specifically in the prostate, and crossed it to the HLA-A2.1 transgenic mouse to evaluate how androgen deprivation affects human HLA A2.1-resticted T cell responses following immunization of PSA-expressing mice by vaccinia-PSA (PROSTVAC). RESULTS Our PSA transgenic mouse showed restricted expression of PSA in the prostate and detectable circulating PSA levels. Additionally, PSA expression was androgen-dependent with reduced PSA expression in the prostate within 1 week of castration, and undetectable PSA by day 42 after castration as evaluated by ELISA. Castration of the PSA/A2.1 hybrid mouse prior to immunization with a PSA-expressing recombinant vaccinia virus resulted in a significant augmentation of PSA-specific cytotoxic lymphocytes. CONCLUSIONS This humanized hybrid mouse model provides a well-defined system to gain additional insight into the mechanisms of immune tolerance to PSA and to test novel strategies aiming at circumventing immune tolerance to PSA and other TAA for targeted prostate cancer immunotherapy. Prostate © 2010 Wiley-Liss, Inc.

Haptoglobin (HP) is an acute phase protein synthesized by liver cells in response to IL-6. HP has been demonstrated to modulate the immune response and to have anti-inflammatory activities. To analyze HP's effect on autoimmune inflammation, we here studied the course of EAE induced by immunization of Hp knockout (Hp(-/-)) and syngeneic WT mice with myelin oligodendrocyte glycoprotein peptide (MOG(35-55)). Hp(-/-)mice suffered from a more severe disease that was associated with increased expression of IL-17A, IL-6, and IFN-gamma mRNA in the CNS and with a denser cellular infiltrate in the spinal cord. During the recovery phase, a significantly higher number of myeloid DC, CD8+ cells, IL-17+ CD4+ and IFN-gamma+ CD4+ cells persisted in the CNS of Hp(-/-) mice. Absence of HP affected the priming and differentiation of T cells after MOG(35-55) immunization, as levels of Th2 cytokines produced in response to MOG stimulation by Hp(-/-) T cells were reduced. These results suggest that HP plays a modulatory and protective role on autoimmune inflammation of the CNS.

BACKGROUND. Failure of cancer immunotherapy is essentially due to immunological tolerance to tumor-associated antigens (TAAs), as these antigens are also expressed in healthy tissues. METHODS. Here, we used transgenic adenocarcinoma of mouse prostate (TRAMP) mice, which develop lethal prostate cancer due to prostate-specific expression of SV40 T antigen (Tag), to evaluate effects of prostatic transformation on oncogene TAA-specific tolerance and to test the possibility of breaking such tolerance using a modified recombinant vaccinia virus. RESULTS. We showed that Tag expression in TRAMP mice is uniquely extra-thymic, and levels of prostatic Tag expression positively correlate with malignant transformation of the prostate. Yet, young tumor-free TRAMP mice were tolerant to Tag antigen. We therefore attempted overcoming such peripheral oncogene-specific T cell tolerance through immunization with a vaccinia construct encoding Tag immunogenic epitopes. This vaccination modality showed that oncogene-specific tolerance was successfully overcome by effective in vivo priming of Tag-specific cytotoxic T cells (CTLs). However, this was restricted to young TRAMP

We report that haptoglobin, an acute-phase protein produced by liver cells in response to interleukin-6 (IL-6), can modulate the inflammatory response induced by endotoxins. We provide evidence that haptoglobin has the ability to selectively antagonize lipopolysaccharide (LPS) effects in vitro by suppressing monocyte production of tumour necrosis factor-alpha, IL-10 and IL-12, while it fails to inhibit the production of IL-6, IL-8 and IL-1 receptor antagonist. In two animal models of LPS-induced bronchopulmonary hyperreactivity and endotoxic shock, haptoglobin knockout mice were more sensitive to LPS effects compared to their wild-type counterparts. The present data suggest that haptoglobin regulates monocyte activation following LPS stimulation. The increase in haptoglobin levels during an acute-phase reaction may generate a feedback effect which dampens the severity of cytokine release and protects against endotoxin-induced effects.

The contribution of Ca(2+) release from intracellular stores to the rise in the free cytosolic Ca(2+) concentration ([Ca(2+)](c)) triggered by Ca(2+) influx was investigated in mouse pancreatic beta-cells. Depolarization of beta-cells by 45 mm K(+) (in the presence of 15 mm glucose and 0.1 mm diazoxide) evoked two types of [Ca(2+)](c) responses: a monotonic and sustained elevation; or a sustained elevation superimposed by a transient [Ca(2+)](c) peak (TCP) (40-120 s after the onset of depolarization). Simultaneous measurements of [Ca(2+)](c) and voltage-dependent Ca(2+) current established that the TCP did not result from a larger Ca(2+) current. Abolition of the TCP by thapsigargin and its absence in sarco-endoplasmic reticulum Ca(2+)-ATPase 3 (SERCA3) knockout mice show that it is caused by Ca(2+) mobilization from the endoplasmic reticulum. A TCP could not be evoked by the sole depolarization of beta-cells but required a rise in [Ca(2+)](c) pointing to a Ca(2+)-induced Ca(2+) release (CICR). This CICR did not involve inositol 1,4,5-trisphosphate (IP(3)) receptors (IP(3)Rs) because it was resistant to heparin. Nor did it involve ryanodine receptors (RyRs) because it persisted after blockade of RyRs with ryanodine, and was not mimicked by caffeine, a RyR agonist. Moreover, RyR1 and RyR2 mRNA were not found and RyR3 mRNA was only slightly expressed in purified beta-cells. A CICR could also be detected in a limited number of cells in response to glucose. Our data demonstrate, for the first time in living cells, the existence of an atypical CICR that is independent from the IP(3)R and the RyR. This CICR is prominent in response to a supraphysiological stimulation with high K(+), but plays little role in response to glucose in non-obese mouse pancreatic beta-cells.

Summary T helper cell type 1 (Th1) and type 2 (Th2) immune responses are characterized by a different pattern of cytokine expression following T-cell activation. Alterations of the ratio of Th1 to Th2 cells are important determinants of susceptibility to viral and parasitic infections, allergies, anti-tumour responses, and autoimmunity. In this work we bring new evidence for an effect of haptoglobin (Hp), a positive acute-phase protein, on T-lymphocyte functions. We show that Hp specifically interacts with both resting and activated CD4+ and CD8+ T cells. This specific binding results in a strong suppression of induced T-cell proliferation. In addition, Hp exhibits a strong in vitro inhibitory effect on Th2 cytokine release, while the production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) is only slightly inhibited at high Hp doses. As a result, the presence of Hp promotes Th1 activation over Th2 activation in vivo as evidenced in Hp-deficient mice. Anti-CD3 monoclonal antibody injection indeed resulted in predominant IL-4 production in Hp−/− mice, in contrast to predominant IFN-γ production in Hp+/+ mice. We conclude that Hp plays a modulating role on the Th1/Th2 balance by promoting a dominant Th1 cellular response. This points to a role of acute-phase proteins in balancing immune responses.

Haptoglobin (Hp) is an acute phase reactant produced by hepatocytes. There is evidence for an immunomodulatory potential of Hp, though there is no clear evidence yet about the mechanisms of this action. We have previously shown that Hp interacts with the β2-integrin CD11b/CD18. In addition, other investigators reported the binding of Hp to B lymphocytes through the CD22 receptor, and to neutrophils through two different receptors. In the present study, we investigated the interaction of haptoglobin with the human mast cell line HMC-1. We report that fluorescein isothiocyanate (FITC)-labelled haptoglobin binds to this cell line and that binding is increased by calcium in a dose- and time-dependent manner. Hp binding sites on HMC-1 were upregulated upon stimulation with phorbol myristate acetate (PMA)/A23187 and after treatment with anti-CD43 and anti-CD44 monoclonal antibodies (MoAbs). HMC-1 cells do not express either CD11b/CD18 or CD22 receptors, indicating that the haptoglobin-binding receptor on this cell line is different from the known receptors. Assessment of cell function showed that Hp inhibits the spontaneous growth of HMC-1 up till 40% at higher Hp concentrations, but it did not exhibit any effect on the expression of CD54 on the release of either tryptase or IL-1ra. In conclusion, haptoglobin binds specifically to human mast cells via a receptor different from CD11b/CD18 and CD22, and may play a role in the modulation of mast cell functions. Exploration of Hp effects in mast cell-dependent diseases such as allergic rhinitis and urticaria seems warranted.

During airway inflammation, airway surface liquid volume (ASLV) expansion may result from the movement of plasma proteins and excess liquid into the airway lumen due to extravasation and elevation of subepithelial hydrostatic pressure. We previously demonstrated that elevation of submucosal hydrostatic pressure increases airway epithelium permeability resulting in ASLV expansion by 500 μL cm(-2) h(-1). Liquid reabsorption by healthy airway epithelium is regulated by active Na(+) transport at a rate of 5 μL cm(-2) h(-1). Thus, during inflammation the airway epithelium may be submerged by a large volume of luminal liquid. Here, we have investigated the mechanism by which ASLV expansion alters active epithelial Na(+) transport, and we have characterized the time course of the change. We used primary cultures of tracheal airway epithelium maintained under air interface (basal ASLV, depth is 7 ± 0.5 μm). To mimic airway flooding, ASLV was expanded to a depth of 5 mm. On switching from basal to expanded ASLV conditions, short-circuit current (Isc, a measure of total transepithelial active ion transport) declined by 90% with a half-time (t1/2) of 1 h. 24 h after the switch, there was no significant change in ATP concentration nor in the number of functional sodium pumps as revealed by [(3)H]-ouabain binding. However, amiloride-sensitive uptake of (22)Na(+) was reduced by 70% upon ASLV expansion. This process is reversible since after returning cells back to air interface, Isc recovered with a t1/2 of 5-10 h. These results may have important clinical implications concerning the development of Na(+) channels activators and resolution of pulmonary edema.