Costunolide inhibits production of tumor necrosis factor-α and interleukin-6 by inducing heme oxygenase-1 in RAW264.7 macrophages

Objectives: Heme oxygenase (HO)-1 expression via nuclear factor-erythroid 2-related factor 2 (Nrf2) activation has an ability to inhibit tumor necrosis factor (TNF)-α and interleukin (IL)-6 production. Costunolide has been reported to inhibit IL-1 production, but whether other cytokines could be inhibited remains to be confirmed. We investigated the effects of costunolide and its components (α-methylene-γ-butyrolactone; CH2-BL, α-methyl-γ-butyrolactone; CH3-BL, and γ-butyrolactone; BL) on HO-1 expression as well as TNF-α and IL-6 production in RAW264.7 macrophages. Methods: HO-1 expression and Nrf2 nuclear accumulation were analyzed by Western blot analysis. The production of TNF-α and IL-6 in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS) was assayed by ELISA. Results: Costunolide and CH2-BL induced HO-1 expression and Nrf2 nuclear accumulation, whereas CH3-BL and BL did not. Pre-incubation with costunolide inhibited LPS-induced production of TNF-α and IL-6. The inhibitory effects of costunolide on TNF-α and IL-6 production were abrogated by tin protoporphyrin, an HO inhibitor. Conclusions: Costunolide is an effective HO-1 inducer capable of inhibiting macrophage-derived pro-inflammatory cytokines. CH2-BL moiety of costunolide is essential for Nrf2 activation leading to HO-1 expression. Received 28 January 2007; returned for revision 4 April 2007; returned for final revision 25 June 2007; accepted by A. Falus 14 July 2007     

The effect of various inflammatory agents on the phagocytosis and cytokine profile of mouse and rat macrophages

Objective and design: The effects of various inflammatory stimuli on the cytokine profile and phagocytic capacity of mouse and rat peritoneal macrophages were investigated in vitro. The correlations between cytokine concentrations and the expressions of NOS II and arginase were also studied. Methods: Mice and rats were injected intraperitoneally with various inflammatory agents. Peritoneal macrophages were isolated. The levels of eight cytokines were determined in macrophage cultures by ELISA test. Phagocytic capacity of macrophages was measured by the ingestion of M. Luteus. Results: The most marked changes caused by i. p. treatments were observed in the levels of IL-1 and IL-6 in mice and of IL-12 in rats. IFN-γ level were increased mainly in rat cells while TNF-α production was rather enhanced in mice. Phagocytic capacity of macrophages was higher in rat samples and it increased with all treatments, except BCG, without marked differences between different treatments. Conclusions: Each inflammatory agent caused an increase in cytokine productions in both species, with marked differences among cytokines. Correlations were found in mouse between IL-6 level and NOS II expression, and IL-10 level with arginase expression. In rat macrophages, IFN-γ, TNF-α and MIP-2 productions were in good correlation with NOS II expression. Received 26 March 2007; returned for revision 18 April 2007; returned for final revision 6 July 2007; accepted by A. Falus 7 September 2007     

A selective H4R antagonist prevents antigen-induced asthma-like reaction and airway inflammation in guinea pigs

Objective:  Curdlan, an extracellular bacterial polysaccharide, is a linear β-1,3-glucan. Previously, we developed Curdlan-oligo (CRDO). We investigated its effect on the production of cytokines in leukocytes from mice, and compared its activity with that of SCG, a 6-branched 1,3-β-glucan. Methods:  Splenocytes from DBA/2 mice were cultured with CRDO or SCG (0, 1, 10 or 100 μg/ml) in vitro, and then the supernatants were collected to measure cytokines. Bone marrow-derived dendritic cells (BMDCs) were cultured with CRDO (0, 1, 10 or 100 ng/ml) in vitro, and then the supernatant was collected to measure cytokines. Results:  SCG stimulated splenocytes in DBA/2 mice to produce GM-CSF, IFN-γ and TNF-α. CRDO induced production of GM-CSF and IFN-γ, but not TNF-α. The amounts of GM-CSF and IFN-γ were small compared with those produced in response to SCG. The effect of SCG on TNF-α production was partially inhibited by CRDO. In bone marrow-derived dendritic cells, CRDO induced production of TNF-α and IL-6. Conclusion:  Taken together, these results suggest that CRDO stimulated mouse leukocytes to induce the production of cytokines, and the mechanism of the effect of CRDO on leukocytes is different from that of SCG. Received 30 June 2008; returned for revision 5 August 2008; received from final revision 3 September 2008; accepted by A. Bauhofer 22 September 2008     

Isoliquiritigenin, from Dalbergia odorifera, up-regulates anti-inflammatory heme oxygenase-1 expression in RAW264.7 macrophages

Objectives:  Isoliquiritigenin (ISL), one of the major constituents of Dalbergia odorifera T. Chen (Leguminosae), is reported to exert anti-inflammatory effects, but the relevant anti-inflammatory mechanisms are not completely understood. Heme oxygenase-1 (HO-1) has been proven to be involved in the resolution of inflammatory responses. In this study, we investigated whether ISL could induce HO-1 expression in RAW264.7 macrophages, and if so, whether HO-1 could mediate the anti-inflammatory effects of ISL. Methods:  The protein expression of inducible nitric oxide synthase and HO-1 was analyzed by western blot analysis. The production of nitric oxide (NO) and interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) was assayed by Griess and ELISA, respectively. The TNF-α and HO-1 mRNA expression was analyzed by northern blot analysis. Results:  ISL markedly suppressed LPS-induced NO, IL-1β, and TNF-α production. ISL induced HO-1 expression through the extracellular signal-regulated kinase1/2 pathway in RAW264.7 macrophages. The effects of ISL on LPS-induced NO and TNF-α production were reversed by the HO-1 inhibitor, tin protoporphyrin. Conclusions:  ISL is an effective HO-1 inducer capable of inhibiting macrophage-derived inflammation. Received 13 August 2008; returned for revision 15 September 2008; returned for final revision 28 October 2008; accepted by G. Wallace 24 November 2008     

Infection of non-encapsulated species of Trichinella ameliorates experimental autoimmune encephalomyelitis involving suppression of Th17 and Th1 response

Epidemiological and experimental studies have indicated that helminth infections can ameliorate autoimmune diseases. The present study investigated the amelioration effect of the Trichinella pseudospiralis infection on experimental autoimmune encephalomyelitis (EAE), a T-cell-mediated autoimmune disease of central nervous system (CNS), and expression kinetics of Th17 and Th1 cytokine which play a crucial role in the pathogenesis of EAE. The results indicated that the infection of helminth T. pseudospiralis obviously ameliorated clinical severity and greatly delayed the onset of EAE induced by myelin oligodendrocyte glycoprotein (MOG) immunization. Infection caused much lesser inflammatory infiltration and demyilination in the CNS of infected EAE mice than uninfected EAE mice. The reduced infiltration was also suggested by the expressions of the inflammation cytokines, IL-17, IL-6, IL-1β, IFN-γ, and TNF-α, which were high in the spinal cords of the uninfected EAE mice, but was nearly normal or low in the infected EAE mice. The increased production of MOG-induced IL-17 and IFN-γ and the expression of IL-6, IL-1β, TGF-β in splenocytes after restimulation with MOG was inhibited in the infected EAE mice. On the other hand, the greatly induced Th2 response was observed in the splenocytes of the infected EAE mice. The present study showed that T. pseudospiralis infection can suppresses EAE by reducing the inflammatory infiltration in CNS, likely associated with the suppression of Th17 and Th1 responses by the infection.     

Differential characteristics of the early stage of lung inflammation induced by SARS-CoV Nucleocapsid protein related to age in the mouse

Objective:  Severe acute respiratory syndrome (SARS) is an acute infectious disease of the respiratory system which has newly emerged. Interestingly, it appears to be a disease that predominantly affects adults while the mortality in children is extremely low. However, the pathogenesis of SARS in relation to different characteristics relevant to age remains unclear. Material and Methods:  To better understand the role of cytokines in the immunopathological processes of SARS, weanling (4 weeks old), young (6 weeks old) and adult (10 weeks old) male BALB/C mice were inoculated intranasally with N-protein of SARS-CoV in this study. Serum or lung homogenate levels of some cytokines such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) along with acute injury lung index and histology were also analyzed. Results:  Histopathological analysis of adult male BALB/C mice after N-protein infection showed progressive inflammatory reactions, especially pulmonary edema, in accordance with a moderately (~13%) elevated level of W/D ratio at 24 h. Although adult groups underwent a progressive lung inflammation in the acute phase accompanied by raised levels of TNF-α in serum, no significant changes in lung TNF-α level were reported simultaneously. Moreover, adult SARS infected BALB/c mice showed elevated levels of IFN-γ while IFN-γ levels in weanling and young groups had no obvious association with lung inflammation. Conclusion:  Our study supports the observation that adult mice do have progressively greater immune reactions than weanling and adolescent ones over time. The relative immaturity of the immune system in weanlings may confer benefit leading to less impairment of lung function. However, the measurement of TNF-α and IFN-γ levels was not indicative of the severity of lung injury at the early stage of disease. Received 25 March 2008; returned for revision 2 July 2008; received from final revision 24 November 2008; accepted by Graham R. Wallace 23 December 2008     

Expression profiles of cytokines and chemokines in murine MDR1a-/- colitis

Objective: MDR1a-/- mice spontaneously develop colitis as the result of imperfect epithelial barrier derived from MDR1a deficiency in the large intestine; however, the pathogenesis is not well understood. This study investigated the expression profiles of cytokines and chemokines in murine MDR1a-/- colitis. Methods: MDR1a-/- and wild-type FVB mice were monitored from the 6^th to the 16^th week of age. Production of various cytokines and chemokines in the large intestine and mesenteric lymph node (MLN) cells was examined. Results: Inflammatory cell infiltration, IL-1β production, and MPO activity were aberrantly enhanced in the tissue of MDR1a-/- mice. Under various stimuli, MLN cells produced higher levels of Th1-type (IFN-γ, IL-2, and IL-12) and proinflammatory (IL-1β and TNF-α) cytokines. Inflammatory chemokines MIP-2/CXCL2, KC/CXCL1, MIP-1α/CCL3, MCP-1/CCL2, and RANTES/CCL5 were also markedly upregulated in the tissue. Conclusion: Since the expression profiles of cytokines and chemokines correspond well with those in human IBD, MDR1a-/- mouse is a useful model for the analysis of IBD pathophysiology. Received 12 May 2006; returned for revision vision 15 July 2006; accepted by I. Ahnfelt-R?nne 18 January 2007     

Changes in cell culture temperature alter release of inflammatory mediators in murine macrophagic RAW264.7 cells

Objectives: To examine whether moderate changes in cell culture temperature influence the production of various cytokines and associated mediators of inflammation. Methods: We performed lipopolysaccharide (LPS) stimulation of the murine macrophagic RAW264.7 cell line under hyperthermic (40 °C), normothermic (37 °C) and hypothermic (34 °C) conditions. We then measured the levels of heat shock protein 70 (HSP70), heat shock factor protein (HSF) and nuclear factor–kB (NF-kB) dimers (p50 and p65) in the cells, and the levels of high mobility group box 1 (HMGB1) and the cytokines tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β) and interleukin 6 (IL-6) in the culture supernatants. Results: Levels of HMGB1, IL-1β, IL-6, and TNF-α, as well as NF-kB dimers (p50 and p65), were all reduced following LPS stimulation at 40 °C and 34 °C compared with those at 37 °C. Levels of HSP70 and HSF increased at 40 °C and 34 °C. Conclusions: The application of moderate hyperthermia and hypothermia after LPS-induced cell activation attenuated the inflammatory response and reduced the likelihood of cell damage. These findings suggest that moderate temperature changes modulate the inflammatory response and could be a useful therapy against sepsis. Received 1 October 2006; returned for revision 16 November 2006; returned for final revision 23 January 2007; accepted by M. Katori 14 March 2007     

A Leaf Methanolic Extract of Wercklea insignis Attenuates the Lipopolysaccharide-Induced Inflammatory Response by Blocking the NF-κB Signaling Pathway in RAW 264.7 Macrophages

The biological activity of Wercklea insignis (WI) in inflammation and the underlying mechanisms of action of extracts of this plant are largely unknown. In the present study, we investigated the effects of a WI methanolic extract on lipopolysaccharide-stimulated inflammation in the mouse macrophage cell line, RAW 264.7. A WI methanolic extract significantly inhibited NO, PGE₂, IL-6, IL-1β, and TNF-α production in LPS-stimulated RAW 264.7 cells. Expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α were suppressed by the extract at both the mRNA and protein levels in lipopolysaccharide (LPS)-stimulated cells. Additionally, the attenuation of inflammatory responses in RAW 264.7 cells by the WI extract was closely associated with suppression of phosphorylation of mitogen-activated protein kinase (MAPK) molecules, including ERK, JNK1/2, and p38 MAPK and translocation of the nuclear factor (NF)-κB p65 subunit into the nucleus. The effect of WI extract was investigated against carrageenan-induced paw edema in female (20–25 g). Our results collectively indicate that the WI extract inhibits LPS-induced inflammatory responses by blocking the NF-κB signaling pathway in macrophages, supporting use of the extract as a therapeutic anti-inflammatory treatment.     

TNF-α mRNA Expression Correlates with TGF-β mRNA Expression <Emphasis Type="Italic">In Vivo</Emphasis>

TNF-α is postulated to play a significant role in regulating TGF-β₁ expression. In lung fibroblasts, for example, TNF-α is supposed to induce TGF-β₁ via AP-1 activation. TNF-α receptor, knock-out mice are resistant to induced fibrosis and over-expression of TNF-α causes increased TGF-β₁ production in mice. Therefore, we investigated whether TNF-α mRNA levels are associated with the TGF-β₁ mRNA levels of blood leucocytes in humans. Quantitative real-time PCR of TNF-α and TGF-β₁ was performed in 118 Germans. Calculations of expression were made with the 2^−ΔΔCT method. When the investigated population was divided in two groups (TNF-α low and TNF-α high) by the median of the determined TNF-α expression, highly significant (p < 0.0001) differences of TGF-β1 mRNA expression were revealed. Additionally, dividing the investigated population into quartiles of the determined TNF-α expression showed significantly different TGF-β1 mRNA expressions. Comparing the determined CT-values of TNF-α in context with these of TGF-β1, a coefficient of determination R ² = 0.4635 was calculated. In this study we demonstrated in vivo a significant association of the relative TNF-α/B2M mRNA expression and the relative TGF-β₁/B2M mRNA expression in 118 Germans.     

Inhibitory effect of ZPDC glycoprotein on the expression of inflammation-related cytokines through p38 MAP kinase and JNK in lipopolysaccharide-stimulated RAW 264.7 cells

Objective:  This study was carried out to investigate the anti-inflammatory potentials of 24 kDa glycoprotein isolated from Zanthoxylum piperitum DC fruit (ZPDC glycoprotein) in lipopolysaccharide (LPS)-stimulated murine macrophage cell line (RAW 264.7 cells). Material and Methods:  RAW 264.7 cells were treated with ZPDC glycoprotein (50–200 μg/ml) in presence of LPS (2 μg/ml). The changes of the levels of inflammation-related factors were determined by using Western blot, EMSA, and RT-PCR. Results:  ZPDC glycoprotein has inhibitory effects on the phosphorylation of p38 mitogen-activated protein (MAP) kinase and c-Jun N-terminal kinase (JNK), on the DNA binding activity of activator protein-1 (AP-1), and on the expression of c-Jun and c-Fos in LPS-stimulated RAW 264.7 cells. Interestingly, the DNA binding activity of AP-1 was attenuated by treatment with inhibitors of p38 MAP kinase and JNK. In addition, ZPDC glycoprotein (200 μg/ml) not only diminished the production of superoxide anion, hydrogen peroxide, and nitric oxide, but also suppressed the expression of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and proteins (iNOS, COX-2, and MMP-9) in LPS- stimulated RAW 264.7 cells. Conclusions:  The present study demonstrates that ZPDC glycoprotein is a natural anti-oxidant and one of the modulators of pro-inflammatory signal transduction pathways in RAW 264.7 cells. Received 2 June 2008; returned for revision 12 August 2008; received from final revision 15 August 2008; accepted by G. Wallace 18 September 2008     

Retinoic Acid Enhances the Production of IL-10 While Reducing the Synthesis of IL-12 and TNF-α from LPS-Stimulated Monocytes/Macrophages

Vitamin A and its metabolites, e.g., all trans-retinoic acid (atRA) and 9-cis-retinoic acid have attracted considerable attention as compounds that have a broad range of immune modulating effects on both humoral and cellular immune responses. The cellular and molecular mechanisms that underlie the effects of retinoids on the immune system remain to be more clearly defined. These immune modulating effects of atRA may be mediated by cytokines elaborated by monocytes and other cell types. To further understand the mechanism(s) by which retinoids affect the immune response, we examined the effects of atRA on several proinflammatory and immune modulating cytokines produced by monocytes. The effects of atRA on LPS-induced mRNA expression of IL-10, IL-12p40, TNF-α, IL-18, and TGF-β in the THP-1 monocyte/macrophage cell line and in cord blood mononuclear cells were measured by competitive RT-PCR. The ELISPOT was employed to evaluate IL-10 and TNF-α protein production enumerating the number of IL-10 and TNF-α producing cells. The addition of atRA to cell cultures potentiated the LPS-induced IL-10 mRNA expression and the number of IL-10 secreting cells from THP-1 cells and cord blood mononuclear cells. In contrast, the addition of atRA inhibited the LPS-induced TNF-α and IL-12p40 mRNA expression, and the number of ELISPOT positive cells for TNF-α. atRA did not change the LPS-induced mRNA expression of IL-18 and TGF-β. These results suggest that atRA may have multiple effects on LPS-induced monocyte/macrophage derived cytokines. While atRA downregulated the proinflammatory cytokines, e.g., IL-12 and TNF-α, the production of an immune modulating cytokine, IL-10 was enhanced by atRA. The effects of atRA on these cytokines may play an important role in the modulation of the immune and inflammatory responses.     

Ratio between the number of Th1 and Th2 lymphocytes in the peripheral blood and concentration of proinflammatory cytokines in lochia of women with postpartum endometritis

We estimated the percentage of CD4⁺ lymphocytes synthesizing IFN-γ and IL-4 in the peripheral blood and measured the concentrations of IL-1, IL-6, and TNF-α in lochia of patients with postpartum endometritis. T lymphocytes with intracellular IFN-γ prevailed in women with physiological course of the postpartum period. The number of cells containing IL-4 increased in patients with postpartum endometritis. On days 5–7 after childbirth the concentrations of proinflammatory cytokines IL-1β and TNF-α in lochia of these patients were higher than in healthy postpartum women (by 1.4 and 2.7 times, respectively). The concentrations of cytokines IL-1β, TNF-α, and IL-6 increased most significantly in patients with postpartum endometritis and active viral infection (by 3.4, 4.2, and 1.6 times, respectively). __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 140, No. 12, pp. 622–624, December, 2005     

Ivermectin inhibits LPS-induced production of inflammatory cytokines and improves LPS-induced survival in mice

Objective and Design:  To investigate whether ivermectin, a semi-synthetic derivative of a family of macrocyclic lactones could inhibit lipopolysaccharide (LPS)-induced inflammation in vivo and in vitro. Materials and Methods:  C57BL/6 mice were administered ivermectin (or saline) orally and challenged intraperitoneally with LPS at a lethal dose of 32 mg/kg. RAW 264.7 murine macrophages were stimulated with LPS at 1 μg/ml, with or without ivermectin for 6, 12 and 24 h. The production of tumor necrosis factor-α (TNF-α), interleukin-1? (IL-1?) and interleukin-6 (IL-6) in serum from mice and supernatants from cells were measured by ELISA. Nuclear factor-kB (NF-kB) translocation with subunit p65 was evaluated by immunocytochemical analysis. Results:  Ivermectin improved mouse survival rate induced by a lethal dose of LPS. In addition, ivermectin significantly decreased the production of TNF-α, IL-1? and IL-6 in vivo and in vitro. Furthermore, ivermectin suppressed NF-kB translocation induced by LPS. Conclusions:  The results indicate that ivermectin may inhibit LPS-induced production of inflammatory cytokines by blocking NF-kB pathway and improve LPS-induced survival in mice. This finding might provide a new strategy for the treatment of endotoxemia and associated inflammation. Xuemei Zhang and Yu Song contributed equally to this work Received 13 January 2008; returned for revision 7 February 2008; received from final revision 3 April 2008; accepted by M. Parnham 4 April 2008 Xuemei Zhang and Yu Song contributed equally to this work     

An insufficient anti-inflammatory cytokine response in mouse brain is associated with increased tissue pathology and viral load during Japanese encephalitis virus infection

Summary Infection of the central nervous system with Japanese encephalitis virus (JEV) results in fatal encephalitis in humans. No reports exist describing the sequence of pathological changes and their correlation to the immune response in the brain following infection with JEV. In this report, we analyzed inducible nitric oxide synthase (iNOS) mRNA, proinflammatory (IFN-γ, TNF-α) and anti-inflammatory (IL-4, IL-10) cytokine expression, viral load, and the correlation of these factors with the major histopathological changes in brain of JEV challenged mice at different time points during infection. We report for the first time that in JE, there is a progressive decline in the level of IL-4. The extent of progressive decrease in IL-4 and IL-10 level following viral infection is inversely correlated to the increased level of proinflammatory cytokines and histopathological changes with negative consequences following viral infection. In contrast, proinflammatory mediators like IFN-γ and TNF-α were significantly upregulated (P < 0.05). A negative correlation between IFN-γ and iNOS indicates their independent actions during JEV infection. To conclude, an insufficient anti-inflammatory cytokine response indicated by IL-4 and IL-10 in the brain is associated with increased tissue pathology and viral load, which regulates inflammatory responses driven by IFN-γ in concert with TNF-α to cause brain tissue damage. Correspondence: Dr. Tapan N. Dhole, Professor and Head, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow 226 014, India     

Type I Interferons Attenuate T Cell Activating Functions of Human Mast Cells by Decreasing TNF-α Production and OX40 Ligand Expression While Increasing IL-10 Production

Recent studies have demonstrated that mast cells not only mediate inflammatory reactions in type I allergy but also play an important role in adaptive immunity. In the present study, we investigated the effects of interferon-α, which shares the same receptor as IFN-β, on human cord blood-derived mast cells. Mast cells produced TNF-α, and IL-10, and expressed OX40 ligand upon activation by crosslinking of FcɛRI. When treated with interferon-α, TNF-α production was decreased while IL-10 and TGF-β productions were increased. Furthermore, flow cytometric analysis revealed that interferon-α downregulated expression OX40 ligand on mast cells which is crucial for mast cell-T cell interaction. We confirmed that the viability of mast cells was not affected by interferon-α treatment. Accordingly, interferon-α-treated mast cells induced lower levels of CD4⁺ T cell proliferation compared with those without interferon-α treatment. These results suggest that type I interferons suppress T cell immune responses through their regulatory effects on mast cells.     

Nitric oxide and pro-inflammatory cytokines correlate with pain intensity in chronic pain patients

Objective: Inflammatory cytokines as well as nitric oxide (NO) play a key role in the pathogenesis of persistent and exaggerated pain states. To document this, we investigated whether a range of cytokines and NO were detectable in the plasma of chronic pain patients and whether cytokine and NO levels correlated with pain severity. Methods: Plasma samples of 94 chronic pain patients and 6 healthy volunteers were obtained. Average pain intensity during the last 24h was assessed on a 11-point numeric rating scale and patients were distributed to three groups: light, moderate and severe pain. The concentrations of TNF-α, GM-CSF, interleukin (IL)-1β, IL-6, IL-8, interferon (IFN)-γ, IL-2, IL-4, IL-5, IL-10 and nitrate/nitrite were determined. Results: Patients with light pain demonstrated significantly increased levels of IL-6 compared to controls. In the severe pain group IL-6 and nitrate/nitrite were significantly increased. Serum concentrations of IL-1β, TNF-α, IL-2 and IL-4 were increased but as we adjusted the level of significance at p = 0.0045, most cytokine plasma levels failed to reach statistical significance. Conclusions: Pro-inflammatory cytokines (IL-1β, IL-2, IL-6, IFN-γ, TNF-α) in the plasma correlate with increasing pain intensity. Chronic pain patients show a significant increase in plasma levels of NO in comparison to healthy controls. Received 30 May 2006; returned for revision 17 July 2006; accepted by G. Geisslinger 7 August 2006     

<Emphasis Type="Italic">Cryptosporidium parvum</Emphasis> infection stimulates the secretion of TGF-β, IL-8 and RANTES by Caco-2 cell line

Cryptosporidium parvum is a common cause of diarrhea in humans. Although mild inflammatory mucosal infiltrate is usually observed, limited information is currently available on the pathogenic mechanisms involved in this phenomenon. The aim of this work was to investigate in vitro the influence of C. parvum infection on the secretion of lymphocyte-targeted chemokines (RANTES, MIP-1α, MIP-1β, IL-8), proinflammatory cytokines (TNF-α, GM-CSF and IL-6) and TGF-β by human enterocytic Caco-2 cells. C. parvum infection stimulates IL-8, RANTES and TGF-β secretion by both the basal and apical side of caco-2 cells. A slight increase in TNF-α production by infected cells was observed in the apical compartment. Data suggest that enterocytic chemokines and/or TGF-β are involved in the initiation and regulation of the mucosal response to C. parvum infection.     

Kakkalide and Its Metabolite Irisolidone Ameliorate Carrageenan-Induced Inflammation in Mice by Inhibiting NF-κB Pathway

The anti-inflammatory activities of kakkalide, a major constituent of the flower of Pueraria thunbergiana, and irisolidone, a metabolite of kakkalide produced by intestinal microflora, against carrageenan-induced inflammation in air pouches on the backs of mice and in lipopolysaccharide (LPS)-stimulated peritoneal macrophages were investigated. Kakkalide and irisolidone down-regulated the gene expression of cytokines [tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β)] and cyclooxygenase-2 (COX-2) and the production of pro-inflammatory cytokines, TNF-α and IL-1β, and inflammatory mediators, NO and prostaglandin E₂ (PGE₂), in LPS-stimulated peritoneal macrophages. These agents also inhibited the phosphorylation of IκB-α and the nuclear translocation of nuclear factor-kappa B (NF-κB). Orally administered kakkalide and irisolidone significantly reduced carrageenan-induced inflammatory markers, leukocyte number, and protein amount in the exudates of the air pouch. These constituents also inhibited PGE₂ production and COX-2 inducible nitric oxide synthase, IL-1β, and TNF-α expression. These agents also inhibited NF-κB activation. The anti-inflammatory effects of irisolidone were more potent than those of kakkalide. Based on these findings, kakkalide and irisolidone may inhibit inflammatory reactions via NF-κB pathway, and irisolidone, a metabolite of kakkalide, may more potently inhibit these inflammatory reactions.