Interleukin 2 (IL2) was injected peritumorally and intranodally in 36 patients with unresectable squamous cell carcinoma of the head and neck enrolled in an Eastern Cooperative Oncology Group-sponsored phase Ib trial (EST P-Z388). Groups of 6 patients received escalating doses(200, 2 x 10(3), 2 x 10(4), 2 x 10(5), 2 x 10(6), and 4 x 10(6) units) of IL2 daily 5 times/week for 2 weeks. Tumor biopsies were obtained before and after IL2 therapy. Tumor tissue was provided for histology, and the remaining fresh tissue was divided for snap-freezing in -75 degrees C and for separation of tumor-infiltrating lymphocytes (TIL) and tumor cells. Immunophenotyping of TIL performed on cryostat sections of paired pre- and post-IL2 biopsy tissues showed increases after IL2 therapy in the number of T-cells (P = 0.005), natural killer (NK; CD16+) cells (P = 0.0001), CD25+ cells (P = 0.004), and HLA-DR+ cells (P = 0.001) accumulating in the tumor stroma. In the tumor parenchyma, NK cells (P = 0.0001) and HLA-DR+ cells (P = 0.003) were increased after IL2 therapy. The T:NK cell ratios in the tumor stroma and parenchyma were decreased after therapy, suggesting selective accumulation of NK cells. By flow cytometry, TIL recovered from post-IL2 biopsy tissues were enriched (P < 0.05) in CD3-CD56+ (NK) cells. In situ hybridization with [35S] cDNA probes for cytokines and IL2 receptors indicated that the numbers of cells expressing mRNA for IL2, tumor necrosis factor alpha, IL1-beta, gamma-interferon, transforming growth factor beta, and IL2 receptor p55 or p70 were increased in post-IL2 biopsy tissues as compared to pre-IL2 tissues. Cytolytic activity of TIL isolated from post-IL2 tissues was also increased, as determined in 4-h 51Cr release assays against K562 targets (12 +/- 3 mean lytic units/10(7) cells +/- SEM pre-IL2 versus 46 +/- 13 post-IL2; n = 16) and against autologous tumor (13 +/- 8 versus 68 +/- 26; n = 9). Fresh TIL of one clinical responder showed relatively high levels (195 lytic units) of autotumor cytotoxicity after IL2 therapy versus no activity prior to therapy. In the blood, NK and lymphokine-activated killer cell activity, and percentages of CD3-CD56+ NK cells and of activated (CD25+) T-lymphocytes were increased for all doses of IL2.(ABSTRACT TRUNCATED AT 400 WORDS)

The role of fever in host defense, if indeed it has one, is poorly understood. Fever in response to exogenous agents is mediated by a host macrophage product called endogenous pyrogen (EP). Recently it has been shown that EP is probably identical to interleukin 1 (IL1), an immunostimulatory macrophage product that induces T-cell proliferation. We postulated that the pyrogenic and immunostimulatory actions of this host mediator might be interrelated and tested T-cell proliferation induced by IL1 at a temperature characteristic of fever. The T-cell proliferative response to IL1 (and to the lymphokine, interleukin 2) was greatly increased at 39 degrees C compared to 37 degrees C, while B-cell mitogenesis in response to lipopolysaccharide was not. These findings suggest that, if similar events occur in vivo, fever may have important immunoregulatory significance and call into question the current indiscriminate use of antipyretic agents.

Although glucocorticoids (GC) represent the most frequently used immunosuppressive drugs, their effects are still not well understood. In our previous studies, we have shown that treatment of monocytes with GC does not cause a global suppression of monocytic effector functions, but rather induces differentiation of a specific anti-inflammatory phenotype. The anti-inflammatory role of peroxisome proliferator-activated receptor (PPAR)-γ has been extensively studied during recent years. However, a relationship between GC treatment and PPAR-γ expression in macrophages has not been investigated so far. Studies using PPAR-γ-deficient mice have frequently provided controversial results. A potential reason is the use of primary cells, which commonly represent inhomogeneous populations burdened with side effects and influenced by bystander cells. To overcome this constraint, we established ER-Hoxb8-immortalized bone marrow-derived macrophages from Ppargfl/fl and LysM-Cre Ppargfl/fl mice in this study. In contrast to primary macrophages, the ER-Hoxb8 system allows the generation of a homogeneous and well-defined population of resting macrophages. We could show that the loss of PPAR-γ resulted in delayed kinetic of differentiation of monocytes into macrophages as assessed by reduced F4/80, but increased Ly6C expression in early phases of differentiation. As expected, PPAR-γ-deficient macrophages displayed an increased pro-inflammatory phenotype upon long-term LPS stimulation characterized by an elevated production of pro-inflammatory cytokines TNF-α, IL1-β, IL-6, IL-12 and a reduced production of anti-inflammatory cytokine IL-10 compared to PPAR-γ WT cells. Moreover, PPAR-γ-deficient macrophages showed impaired phagocytosis. GC treatment of macrophages led to the upregulation of PPAR-γ expression. However, there were no differences in GC-induced suppression of cytokines between both cell types, implicating a PPAR-γ-independent mechanism. Intriguingly, GC treatment resulted in an increased in vitro migration only in PPAR-γ-deficient macrophages. Performing a newly developed in vivo cell-tracking experiment, we could confirm that GC induces an increased recruitment of PPAR-γ KO, but not PPAR-γ WT macrophages to the site of inflammation. Our findings suggest a specific effect of PPAR-γ on GC-induced migration in macrophages. In conclusion, we could demonstrate that PPAR-γ exerts anti-inflammatory activities and shapes macrophage functions. Moreover, we identified a molecular link between GC and PPAR-γ and could show for the first time that PPAR-γ modulates GC-induced migration in macrophages.

Sézary syndrome is a cutaneous T cell lymphoma characterized by infiltration of the skin by CD4+ cells. These cells generally respond poorly to mitogens and T cell activators. We have studied the action of IL1 to IL4, IL6, and IL7 on the proliferation of Sézary cells from 12 patients. With the exception of IL2 and IL7, the cytokines studied had no proliferative effect on these cells. Whereas IL2 had only a low proliferative capacity (two- to threefold increase) on peripheral blood mononuclear cells, recombinant IL7 constantly induced a very significant (3-40-fold increase) proliferative response, and was used successfully to generate cell lines in three out of eight cases. Growth of Sézary cell lines was shown to be strictly dependent on IL7, and after 2-5 wk of culture presented a switch to a homogeneous phenotype CD3+4+8-7- (except for one line that remained CD7+), with a typical morphology of Sézary cells. Their tumoral origin was demonstrated by the expression of the same T cell receptor-beta gene rearrangement as the patients' T cells. Importantly, cultured normal epidermal keratinocyte supernatants could support the growth of our Sézary lines. Furthermore, the proliferative activity contained in these supernatants was completely blocked by a monoclonal anti-IL7 antibody. These results suggest that IL7 may, therefore, represent an important cytokine in the physiopathology of cutaneous T cell lymphoma.

OBJECTIVE

Intestinal manipulation triggers an inflammatory cascade within the muscularis causing postoperative ileus (POI). The aim of this study was to investigate the recovery and therapeutic potential of interleukin 10 (<em>IL1</em>0) for POI.

METHODS

POI was induced by bowel surgical manipulation (SM) in wild-type, <em>IL1</em>0(-/-) and recombinant murine <em>IL1</em>0 (rm<em>IL1</em>0)-treated mice. Immunohistochemistry localised <em>IL1</em>0 in the muscularis externa, histochemistry quantified neutrophil recruitment, and quantitative PCR quantified alterations in mRNA. Luminex multiplex analysis, Griess reaction and ELISA measured proteins, nitric oxide (NO) and prostanoid release from the muscularis externa, respectively, in 24 h organ culture. Gastrointestinal transit and jejunal circular muscle organ bath techniques assessed gastrointestinal function.

RESULTS

In <em>IL1</em>0 knockouts compared with the wild type, the expression of numerous proinflammatory mRNAs (IL6, <em>IL1</em> <em>beta</em>, chemokine C-C motif ligand 2 (CCL2) and haem oxygenase-1) and proteins (IL6, <em>IL1</em> alpha, <em>IL1</em>2, <em>IL1</em>7, interferon gamma, tumour necrosis factor alpha, CCL2, interferon-inducible protein-10 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) were accentuated, and release of muscle inhibitors NO and prostanoids was increased; motility never recovered from manipulation and mortality rate was 87.5%. In wild types, complete functional recovery occurred in 7 days with no mortality. SM delay in transit and suppression in jejunal circular muscle contractions were significantly improved by rm<em>IL1</em>0 treatment. Upregulation in <em>IL1</em> <em>beta</em>, IL6 and CCL2 mRNAs and inflammatory mediators (<em>IL1</em> alpha, IL6, CCL2, macrophage inflammatory protein-1 alpha, GM-CSF, NO and prostaglandin) after SM were significantly less with rm<em>IL1</em>0 treatment, which resulted in a decrease in neutrophil recruitment compared with SM controls.

CONCLUSIONS

<em>IL1</em>0 plays an obligatory role in postoperative intestinal recovery, and exogenous <em>IL1</em>0 prevents its development. Pre-emptive exogenous recombinant human <em>IL1</em>0 could be a treatment for the prevention of clinical POI.

Genetic studies suggest that Zn transporters such as ZnT8 play a role in insulin secretion by pancreatic beta-cells; however, little is known about the dynamic roles of Zn trafficking pathways on beta-cell physiology. To test the acute effects of the inflammatory cytokines interleukin 1 beta (IL1 beta) and tumor necrosis factor alpha (TNFalpha) on Zn homeostasis, the mRNA expression profile of Zn transporters of the ZnT and ZIP families was examined. Exposure of MIN6 cells or primary murine islets to IL1 beta or TNFalpha altered the mRNA expression profile of Zn transporters; most notable was decreased ZnT8 mRNA levels. siRNA-mediated gene knockdown was used to examine the effects of decreased ZnT8 expression in primary dispersed murine islet cells from C57/BL6 mice and MIN6 cells. ZnT8 knockdown in these murine islets led to reduced glucose stimulated insulin secretion without altering the total cellular insulin content or cell viability at normal or supraphysiological Zn concentrations. The labile Zn content determined by flow cytometry after loading with the Zn-specific sensor FluoZin-3 AM was decreased in MIN6 cells following ZnT8 knockdown or IL1 beta treatment. These results suggest that an acute decrease in ZnT8 levels impairs beta-cell function and Zn homeostasis, and may contribute to inflammatory cytokine-induced alterations in beta-cell function.

OBJECTIVE

Mixed bacterial vaccine (MBV, Coley's toxins) is a historical, vaguely defined preparation of heat-inactivated Streptococcus pyogenes and Serratia marcescens used as nonspecific immunotherapy in the treatment of cancer. The mechanism of action is suspected to have an immunologic basis, yet it is poorly defined up to now. We developed a new, biochemically well defined and current good manufacturing practice-compliant MBV preparation, which has been investigated in patients with NY-ESO-1 expressing cancers.

METHODS

Patients received MBV subcutaneously at a starting dose of 250 EU (endotoxin units) twice a week. The MBV dose was escalated in each patient until a body temperature of 38°C to 39.5°C was induced or up to the maximum dose of 547.000 EU. Changes in serum cytokine levels were determined and immune responses to NY-ESO-1 were evaluated. Tumor response was assessed according to RECIST.

RESULTS

Twelve patients were enrolled and 11 of them developed fever after the administration of MBV. Ten of 12 patients showed a consistent increase in serum IL-6 levels with the highest levels coinciding with the highest body temperature. A subgroup of patients showed increasing levels of TNF-α, IFN-γ, and IL1-β. A patient with metastatic bladder cancer showed a partial tumor response strongly correlated with MBV-induced fever and highly elevated levels of several cytokines.

CONCLUSIONS

MBV at fever-inducing dose levels can lead to a massive induction of immunoregulatory cytokines that may be involved in inducing tumor regressions. We propose to further explore the role of MBV as a potent immune modulator at higher dose levels and in conjunction with antigen-specific cancer vaccines.

OBJECTIVE

Childhood trauma has been associated adult stress-related disorders. However, little is known about physiologic alterations in adults with a history of early life trauma that do not have current psychiatric or medical diagnoses. In this study, the relationships between childhood adversity and cytokine and C-reactive protein (CRP) levels in healthy adults were examined.

METHODS

Participants included men (n = 18) and women (n = 20) who did not meet DSM-IV criteria for Axis I psychiatric disorders or any major medical illness. Cytokine and CRP levels were obtained from baseline blood samples. Subjects completed the Early Trauma Inventory Self Report (ETI-SR). The primary outcomes included serum interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL1-β), and CRP levels. In addition, the mean numbers of traumatic experiences (sexual, physical, emotional, general, and the summed total) were measured.

RESULTS

Significant positive associations were found between the total ETI score and IL-6 (p = 0.05), IL1-β (p < 0.05), and TNF-α (p = 0.01). Significant positive correlations were found between the number of general traumas and IL1-β (p < 0.05), TNF-α (p < 0.05), and IL-6 (p < 0.01). Neither the total number of traumas nor any of the trauma subscales were significantly associated with CRP levels.

CONCLUSIONS

The positive association between childhood trauma and basal cytokine levels supports the extant literature demonstrating the long-term impact of childhood trauma and stress on homeostatic systems. Importantly, this association was found in healthy adults, suggesting that these alterations may precede the development of significant stress-related psychiatric disorder or disease.

Systemic or intratesticular release of TNF alpha and IL1 beta have been implicated in the reduced testosterone biosynthesis and impaired production of competent spermatozoa found in human patients suffering from sepsis or chronic inflammation. Although in vitro and in vivo studies have demonstrated that TNF alpha and IL1 beta intercept the hypothalamic-pituitary testis axis at different levels, the site(s) of action and relative contribution of each cytokine to the overall testicular failure associated to systemic inflammatory processes remains poorly defined. In this study we show that intratesticular delivery of TNF alpha induced a rapid (4 h) and sustained (up to 24 h) reduction in steroidogenic acute regulatory (StAR) protein expression and testosterone biosynthesis in nonstimulated or human chorionic gonadotropin-treated intact or hypophysectomized rats. Bilateral treatment with cell-permeant short-chain ceramides (C2-cer or C6-cer) reproduced the early (4 h) inhibitory action of TNFalpha on testosterone biosynthesis and testicular StAR expression. The inhibitory action of C2-cer or C6-cer was not observed in animals treated with inactive analogs (dihydroceramide), phosphorylcholine, sphingosine, or sphingosine-1P. In sharp contrast to the previously described ability of IL1 beta to prevent human chorionic gonadotropin-stimulated Leydig cell steroidogenesis in vitro, serum testosterone and testicular StAR protein expression remained unchanged in animals bilaterally injected with this cytokine. These data support the concept that TNF alpha triggers different effector mechanisms to directly inhibit Leydig cell StAR expression and steroidogenesis, which ultimately contribute to the global reproductive failure associated with chronic inflammation and sepsis.

We examined whether hypoxia alone could produce changes in the permeability of brain capillary endothelial cells (EC) and whether a stimulation of hypoxic status alters the gene expression of occludin and glucose transporter 1 (GLUT1). Exposure of EC to hypoxia resulted in increased permeability, with the greatest decrease in transendothelial electrical resistance (TER) at 40 h. Moreover, hypoxia alone induced the expression of both mRNA in EC. Furthermore, we found that interleukin-1 (IL-1)beta, glutamate, hydrogen peroxide (H2O2), and sodium nitroprusside (SNP) induced the expression of mRNA for occludin and GULT1 under normoxic condition. The decrease in TER due to hypoxia was inhibited on addition of an anti-IL1 antibody and nitric oxide synthase (NOS) inhibitor in EC. These results indicate that the expression of occludin and GLUT1 mRNA is sensitive to exposure to hypoxia and that the changes of permeability in EC are associated with IL-1beta and NO.

Opioids have been found to modulate the immune system by regulating the function of immunocompetent cells. Several studies suggest that the interaction between immune and opioid systems is not unidirectional, but rather reciprocal, in nature. In the CNS, one cellular target of immune system activation is the astrocytes. These glial cells have been shown to produce the opioid peptide, proenkephalin, to express the mu-, delta-, and kappa-opioid receptors, and to respond to the immune factor interleukin-1 beta (IL1 beta) with an increased proenkephalin synthesis. To characterize more completely the astrocytic opioid response to immune factor stimulation, we examined the effect of IL1 beta (1 ng/ml) on the mu-receptor mRNA expression in primary astrocyte-enriched cultures derived from rat (postnatal day 1-2) cortex, striatum, cerebellum, hippocampus, and hypothalamus. A 24-h treatment with IL1 beta produced a 70-80% increase in the mu-receptor mRNA expression in the striatal, cerebellar, and hippocampal cultures but had no effect on this expression in the cortical and hypothalamic cultures. This observation represents one of the few demonstrated increases in levels of the mu-receptor mRNA in vitro or in vivo, since the cloning of the receptor. The enhanced mu-receptor mRNA expression, together with the previous observation that IL1 beta stimulates proenkephalin synthesis in astrocytes, supports the IL1 beta-mediated regulation of an astroglial opioid peptide and receptor in vitro, a phenomenon that may be significant in the modulation of the gliotic response to neuronal damage. Therefore, the astroglial opioid "system" may be important in the IL1 beta-initiated, coordinated response to CNS infection, trauma, or injury.

The conjunctiva is a moist mucosal membrane that is constantly exposed to an array of potential pathogens and triggers of inflammation. The NACHT, leucine rich repeat (LRR), and pyrin domain-containing protein 3 (NLRP3) is a Nod-like receptor that can sense pathogens or other triggers, and is highly expressed in wet mucosal membranes. NLRP3 is a member of the multi-protein complex termed the NLRP3 inflammasome that activates the caspase 1 pathway, inducing the secretion of biologically active IL-1β, a major initiator and promoter of inflammation. The purpose of this study was to: (1) determine whether NLRP3 is expressed in the conjunctiva and (2) determine whether goblet cells specifically contribute to innate mediated inflammation via secretion of IL-1β. We report that the receptors known to be involved in the priming and activation of the NLRP3 inflammasome, the purinergic receptors P2X4 and P2X7 and the bacterial Toll-like receptor 2 are present and functional in conjunctival goblet cells. Toxin-containing Staphylococcus aureus (S. aureus), which activates the NLRP3 inflammasome, increased the expression of the inflammasome proteins NLRP3, ASC and pro- and mature caspase 1 in conjunctival goblet cells. The biologically active form of IL-1β was detected in goblet cell culture supernatants in response to S. aureus, which was reduced when the cells were treated with the caspase 1 inhibitor Z-YVAD. We conclude that the NLRP3 inflammasome components are present in conjunctival goblet cells. The NRLP3 inflammasome appears to be activated in conjunctival goblet cells by toxin-containing S. aureus via the caspase 1 pathway to secrete mature IL1-β. Thus goblet cells contribute to the innate immune response in the conjunctiva by activation of the NLRP3 inflammasome.

Currently, MVA virus vectors carrying HIV-1 genes are being developed as HIV-1/AIDS prophylactic/therapeutic vaccines. Nevertheless, little is known about the impact of these vectors on human dendritic cells (DC) and their capacity to present HIV-1 antigens to human HIV-specific T cells. This study aimed to characterize the interaction of MVA and MVA expressing the HIV-1 genes Env-Gag-Pol-Nef of clade B (referred to as MVA-B) in human monocyte-derived dendritic cells (MDDC) and the subsequent processes of HIV-1 antigen presentation and activation of memory HIV-1-specific T lymphocytes. For these purposes, we performed ex vivo assays with MDDC and autologous lymphocytes from asymptomatic HIV-infected patients. Infection of MDDC with MVA-B or MVA, at the optimal dose of 0.3 PFU/MDDC, induced by itself a moderate degree of maturation of MDDC, involving secretion of cytokines and chemokines (IL1-ra, IL-7, TNF-α, IL-6, IL-12, IL-15, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, IP-10, MIG, and IFN-α). MDDC infected with MVA or MVA-B and following a period of 48 h or 72 h of maturation were able to migrate toward CCL19 or CCL21 chemokine gradients. MVA-B infection induced apoptosis of the infected cells and the resulting apoptotic bodies were engulfed by the uninfected MDDC, which cross-presented HIV-1 antigens to autologous CD8(+) T lymphocytes. MVA-B-infected MDDC co-cultured with autologous T lymphocytes induced a highly functional HIV-specific CD8(+) T cell response including proliferation, secretion of IFN-γ, IL-2, TNF-α, MIP-1β, MIP-1α, RANTES and IL-6, and strong cytotoxic activity against autologous HIV-1-infected CD4(+) T lymphocytes. These results evidence the adjuvant role of the vector itself (MVA) and support the clinical development of prophylactic and therapeutic anti-HIV vaccines based on MVA-B.

The level of adhesion molecules expressed at the endothelial cell surface is critical in the control of inflammation. Adenylate cyclase (AC) activity allowing cyclic adenosine monophosphate (cAMP) production can modulate the inflammatory process. We investigated the AC-dependent modulation of ICAM-1 surface expression in human umbilical venous endothelial cells (HUVEC). Pretreatment of HUVEC with forskolin significantly upregulated tumor necrosis factor alpha (TNF-alpha)- and interleukin-1 alpha (IL1-alpha)-induced ICAM-1 surface expression exclusively after a prolonged time of incubation with forskolin (at least 7-8 h). A poorly metabolizable analog of cAMP, dibutyryl-cAMP, mimicked forskolin effect on ICAM-1 surface expression. Protein kinase A (PKA) inhibitor H89 prevented forskolin effect on ICAM-1 surface expression. Upregulation of ICAM-1 surface level occurred through the increase in its mRNA levels and also to a subsequent activation of ICAM-1 intracellular trafficking towards cell surface. Stimulation by agonists of beta-adrenergic receptors did not alter the TNF-alpha-induced ICAM-1 surface expression. Pretreatment of HUVEC with pertussis toxin, which is known to activate AC through Gialpha inhibition, upregulated mRNA levels and surface expression of ICAM-1 induced by TNF-alpha. This effect was serum-dependent, since ICAM-1 expression was no more upregulated by pertussis toxin in cells cultured in 1% instead of 20% serum-enriched medium. However, forskolin treatment of HUVEC did not modify their overall adhesive properties. In conclusion, a persistent cAMP level elevation activating PKA is able to enhance ICAM-1 expression at the cell surface of endothelial cells placed under pro-inflammatory conditions. Combination of activation of gene transcription and membrane targeting may account for this augmentation.

OBJECTIVE

In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils.

METHODS

Isolated human neutrophils from healthy volunteers.

METHODS

Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies.

METHODS

Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction.

CONCLUSIONS

The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Receptor activator of nuclear factor κ B ligand (RANKL) plays a crucial role in the bone erosion of rheumatoid arthritis (RA) by prompting osteoclastogenesis. Considering that 1,25(OH)2D3 has been suggested as a potent inducer of RANKL expression, it should clarify whether vitamin D supplement could result in RANKL overexpression and thereby facilitate excessive osteoclastogenesis and bone resorption in RA. Here, we investigated modulatory effect of 1,25(OH)2D3 on the expression of RANKL and its decoy receptor osteoprotegerin (OPG) in an inflammatory condition of human rheumatoid synoviocyte MH7A. MH7A cells were stimulated with IL1 β and then treated with different concentrations of 1,25(OH)2D3 for 48 h. A significantly elevated OPG/RANKL ratio and markedly decreased levels of IL-6 and TNF β mRNA expression in cells and IL-6 protein in supernatants were observed in IL1 β -induced MH7A in the presence of 1,25(OH)2D3 compared with those in the absence of it. Osteoclast formation was obviously decreased when RAW264.7 cells were treated with both 1,25(OH)2D3 and IL1 β . In summary, although it has a biological function to induce RANKL expression, 1,25(OH)2D3 could upregulate OPG/RANKL ratio and mediate anti-inflammatory action in an inflammatory milieu of synoviocyte, contributing to the inhibition of inflammation-induced osteoclastogenesis in RA.

Fibroblasts from normal human adult skin were cultured in vitro in the presence and absence of different concentrations of pentoxifylline or a pentoxifylline analog, A81-3138 (10(-1)-10(3) micrograms/ml). Similar concentration dependent reductions in normal proliferation of fibroblasts in fetal calf serum-driven subconfluent cultures were detected following treatment with pentoxifylline or A81-3138. Fibroblasts assayed as confluent cultures produced sub-normal amounts of collagen, glycosaminoglycans (GAGs), and fibronectin in a fashion dependent upon the concentration of pentoxifylline. In contrast, fibroblasts exposed to pentoxifylline elaborated double the collagenase activity produced by normal, untreated fibroblasts. The reduced proliferation and reduced synthetic activities were not due to a lethal toxic effect on fibroblasts by pentoxifylline and A81-3138, nor was the reduction in collagen synthesis simply due to an inability to secrete newly synthesized intracellular collagen. Unlike pentoxifylline-induced inhibition of collagen and fibronectin production, which was detected only in cultures supplemented with serum, pentoxifylline inhibits, to a similar degree, both constitutive and serum-driven production of GAGs. The addition of IL1 beta (2.5 and 10.0 U/ml) to serum-driven fibroblast cultures resulted in greater proliferation, which was inhibitable by the presence of pentoxifylline and A81-3138 as anti-fibrotic agents in certain disorders of fibrosis.

As the commonest complication of diabetes mellitus (DM), diabetic retinopathy (DR) is a neuro-vascular disease with chronic inflammatory. Methane could exert potential therapeutic interest in inflammatory pathologies in previous studies. Our study aims to evaluate the protective effects of methane-rich saline on DR and investigate the potential role of related MicroRNA (miRNA) in diabetic rats. Streptozotocin-induced diabetic Sprague-Dawley rats were injected intraperitoneally with methane-rich or normal saline (5 ml/kg) daily for eight weeks. Morphology changes and blood-retinal barrier (BRB) permeability were assessed by hematoxylin eosin staining and Evans blue leakage. Retinal inflammatory cytokines levels of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL1-β) were evaluated by immunohistochemistry. Retinal protein expressions of glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) were determined by western blotting. Retinal miRNA expressions were examined by miRNA-specific microarray, verified by quantitative RT-PCR and predicted by GO enrichment and KEGG pathway analysis. There was no significant changes in blood glucose level and body weight of diabetic rats with methane-rich or normal saline treatment, but the decreased retinal thickness, retinal ganglial cell loss and BRB breakdown were all significantly suppressed by methane treatment. DM-induced retinal overexpressions of TNF-α, IL-1β, GFAP and VEGF were also significantly ameliorated. Moreover, the methane treatment significantly up-regulated retinal levels of miR-192-5p (related to apoptosis and tyrosine kinase signaling pathway) and miR-335 (related to proliferation, oxidative stress and leukocyte). Methane exerts protective effect on DR via anti-inflammation, which may be related to the regulatory mechanism of miRNAs.

Superficial-zone protein (SZP), also known as lubricin, is a key mediator of boundary lubrication and plays an important role in the functional integrity of the diarthrodial joint. The aim of this investigation was to examine the role of transforming growth factor beta (TGF-beta) and interleukin-1 beta (IL-1beta) on the expression of SZP in various compartments of the bovine knee joint: the superficial zone of articular cartilage, synovium, meniscus, and anterior and posterior cruciate ligaments. The effects of TGF-betabeta on SZP expression were examined in explants and cells from the different tissue compartments. TGF-betabeta down-regulated it. Quantitative analysis of secreted proteins in the medium of the cells demonstrated significant stimulation by TGF-betaIL1-beta of the accumulation of SZP protein in all four tissues. Real-time polymerase chain reaction analysis revealed that TGF-betabeta down-regulated it. These results revealed the modulation of SZP expression in various compartments of the knee joint by TGF-betabeta. In addition, SZP was found to be immunolocalized at the surface layer of cells in histological sections of all four tissue compartments. Collectively, results of the current study on regulation of SZP expression by TGF-beta and IL-1 help provide new insights, into tissue engineering strategies to repair and regenerate the different tissue compartments in the articular joint with optimal lubrication.

Lithium is the gold-standard treatment for bipolar disorder, a severe mental illness. A large body of evidence suggests that inflammation plays a role in the pathogenesis of bipolar disorder and that mood stabilizers exhibit anti-inflammatory properties. However, contradicting findings have also been reported. In this study, we examined the effects of lithium on LPS-induced inflammation in rat primary glia cells. Cells were pre-treated with lithium (1 or 10 mM) for 6 or 24 h, after which, inflammation was induced by the addition of LPS (for another 18 h) to the culture medium. Thereafter, medium was collected and cells were harvested for further analyses. Levels of TNF-α, IL1-β and PGE(2) were determined by ELISA and NO levels by the Griess reaction assay. Expression levels of cyclooxygenase-2 (COX-2) and inducible NO synthase (iNOS) were examined by Western blot analysis. We found that pre-treatment with lithium 10 mM (but not 1 mM) significantly reduced LPS-induced secretion of TNF-α, IL1-β, PGE(2) and NO. In addition, lithium significantly reduced the expression of COX-2 and iNOS. These findings indicate that lithium exhibits a potent anti-inflammatory effect. However, it's important to emphasize that this effect was obtained mainly under treatment with an extra-therapeutic concentration of the drug.

In this study, the effects of sublethal dose of Androctonus australis hector (Aah) venom on the enzymatic activities (creatine phospho-kinase and lactate dehydrogenase) and histopathological changes of heart and lungs' organs were determined 24h following envenoming NMRI mice. The effects of Aah venom on the lytic activity of the complement system, plasma cytokine rates (IL1-beta, IL-6, TNF-alpha, IL-4 and IL-10) and the peripheral blood cell infiltration were also studied. Microscopically, treated animals showed severe myocardial edema, hemorrhages and necroses and severe acute bronchopneumonia with alveolar edema and hemorrhages. High serum levels of lactate dehydrogenase and creatine kinase correlate to the tissue lesions. The results showed fast kinetics of production of pro-inflammatory (IL1-beta, IL-6, TNF-alpha) and anti-inflammatory (IL-4 and IL-10) cytokines at 30min in blood sera. An increase in serum lytic activity of envenomed animals and leucocytosis in peripheral blood with predominance of mononuclear and neutrophil cells were also observed. In conclusion, the results reported in the present study suggest that pathophysiological manifestations of Aah envenomation may be mediated sequentially or simultaneously by cytokines and the complement system, which in turn activate leukocyte to produce tissue damage.

Depression represents a common comorbidity of epilepsy and is frequently resistant to selective serotonin reuptake inhibitors (SSRI). We tested the hypothesis that the SSRI resistance in epilepsy associated depression may be a result of a pathologically enhanced interleukin-1β (IL1-β) signaling, and consequently that the blockade of IL1-β may restore the effectiveness of SSRI. Epilepsy and concurrent depression-like impairments were induced in Wistar rats by pilocarpine status epilepticus (SE). The effects of the 2-week long treatment with fluoxetine, interleukin-1 receptor antagonist (IL-1ra), and their combination were examined using behavioral, biochemical, neuroendocrine, and autoradiographic assays. In post-SE rats, depression-like impairments included behavioral deficits indicative of hopelessness and anhedonia; the hyperactivity of the hypothalamo-pituitary-adrenocortical axis; the diminished serotonin output from raphe nucleus; and the upregulation of presynaptic serotonin 1-A (5-HT1A) receptors. Fluoxetine monotherapy exerted no antidepressant effects, whereas the treatment with IL-1ra led to the complete reversal of anhedonia and to a partial improvement of all other depressive impairments. Combined administration of fluoxetine and IL-1ra completely abolished all hallmarks of epilepsy-associated depressive abnormalities, with the exception of the hyperactivity of the hypothalamo-pituitary-adrenocortical axis, the latter remaining only partially improved. We propose that in certain forms of depression, including but not limited to depression associated with epilepsy, the resistance to SSRI may be driven by the pathologically enhanced interleukin-1β signaling and by the subsequent upregulation of presynaptic 5-HT1A receptors. In such forms of depression, the use of interleukin-1β blockers in conjunction with SSRI may represent an effective therapeutic approach.

BACKGROUND

Anecdotal animal and human studies have implicated the symptomatic and neuroprotective roles of niacin in Parkinson's disease (PD). Niacin has a high affinity for GPR109A, an anti-inflammatory receptor. Niacin is also thought to be involved in the regulation of circadian rhythm. Here we evaluated the relationships among the receptor, niacin levels and EEG night-sleep in individuals with PD.

RESULTS

GPR109A expression (blood and brain), niacin index (NAD-NADP ratio) and cytokine markers (blood) were analyzed. Measures of night-sleep function (EEG) and perceived sleep quality (questionnaire) were assessed. We observed significant up-regulation of GPR109A expression in the blood as well as in the substantia nigra (SN) in the PD group compared to age-matched controls. Confocal microscopy demonstrated co-localization of GPR109A staining with microglia in PD SN. Pro and anti-inflammatory cytokines did not show significant differences between the groups; however IL1-β, IL-4 and IL-7 showed an upward trend in PD. Time to sleep (sleep latency), EEG REM and sleep efficiency were different between PD and age-matched controls. Niacin levels were lower in PD and were associated with increased frequency of experiencing body pain and decreased duration of deep sleep.

CONCLUSIONS

The findings of associations among the GPR109A receptor, niacin levels and night-sleep function in individuals with PD are novel. Further studies are needed to understand the pathophysiological mechanisms of action of niacin, GPR109A expression and their associations with night-sleep function. It would be also crucial to study GPR109A expression in neurons, astrocytes, and microglia in PD. A clinical trial to determine the symptomatic and/or neuroprotective effect of niacin supplementation is warranted.

Mid-life hypertension and cerebrovascular dysfunction are associated with increased risk of later life dementia, including Alzheimer's disease (AD). The classical renin-angiotensin system (cRAS), a physiological regulator of blood pressure, functions independently within the brain and is overactive in AD. cRAS-targeting anti-hypertensive drugs are associated with reduced incidence of AD, delayed onset of cognitive decline, and reduced levels of Aβ and tau in both animal models and human pathological studies. cRAS activity is moderated by a downstream regulatory RAS pathway (rRAS), which is underactive in AD and is strongly associated with pathological hallmarks in human AD, and cognitive decline in animal models of CNS disease. We now show that enhancement of brain ACE2 activity, a major effector of rRAS, by intraperitoneal administration of diminazene aceturate (DIZE), an established activator of ACE2, lowered hippocampal Aβ and restored cognition in mid-aged (13-14-month-old) symptomatic Tg2576 mice. We confirmed that the protective effects of DIZE were directly mediated through ACE2 and were associated with reduced hippocampal soluble Aβ₄₂ and IL1-β levels. DIZE restored hippocampal MasR levels in conjunction with increased NMDA NR2B and downstream ERK signalling expression in hippocampal synaptosomes from Tg2576 mice. Chronic (10 weeks) administration of DIZE to pre-symptomatic 9-10-month-old Tg2576 mice, and acute (10 days) treatment in cognitively impaired 12-13-month-old mice, prevented the development of cognitive impairment. Together these data demonstrate that ACE2 enhancement protects against and reverses amyloid-related hippocampal pathology and cognitive impairment in a preclinical model of AD.