The p38 mitogen-activated protein kinase regulates interleukin-1beta-induced IL-8 expression via an effect on the IL-8 promoter in intestinal epithelial cells

Several lines of evidence implicate the p38 mitogen-activated protein kinase (p38 MAPK) in the proinflammatory response to bacterial agents and cytokines. Equally, the transcription factor, nuclear factor (NF)-kappaB, is recognized to be a critical determinant of the inflammatory response in intestinal epithelial cells (IECs). However, the precise inter-relationship between the activation of p38 MAPK and activation of the transcription factor NF-kappaB in the intestinal epithelial cell (IEC) system, remains unknown. Here we show that interleukin (IL)-1beta activates all three MAPKs in Caco-2 cells. The production of IL-8 and monocyte chemotactic protein 1 (MCP-1) was attenuated by 50% when these cells were preincubated with the p38 MAPK inhibitor, SB 203580. Further investigation of the NF-kappaB signalling system revealed that the inhibitory effect was independent of the phosphorylation and degradation of IkappaBalpha, the binding partner of NF-kappaB. This effect was also independent of the DNA binding of the p65 Rel A subunit, as well as transactivation, determined by an NF-kappaB luciferase construct, using both SB 203580 and dominant-negative p38 MAPK. Evaluation of IL-8 and MCP-1 RNA messages by reverse transcription-polymerase chain reaction (RT-PCR) revealed that the inhibitory effect of SB 203580 was associated with a reduction in this parameter. Using an IL-8-luciferase promoter construct, an effect of p38 upon its activation by both pharmacological and dominant-negative p38 construct co-transfection was demonstrated. It is concluded that p38 MAPK influences the expression of chemokines in intestinal epithelial cells, through an effect upon the activation of the chemokine promoter, and does not directly involve the activation of the transcription factor NF-kappaB

Personal Injuries Proceedings Act 2002, s 32 - Duty to Disclose - consequences of non-disclosure - discretion to excuse non-compliance

In Newson v Aust Scan Pty Ltd t/a Ikea Springwood [2010] QSC 223 the Supreme Court examined the discretion under s 32(2) of the Personal Injuries Proceedings Act 2002 (Qld), to permit a document which has not been disclosed as required by the pre-court procedures under the PIPA to be used in a subsequent court proceeding. This appears to be the first time that the nature and parameters of the discretion have been judicially considered.

Serum YKL-40 and Interleukin 6 Levels in Hodgkin Lymphoma

Purpose Serum levels of the inflammatory markers YKL-40 and IL-6 are increased in many conditions, including cancers. We examined serum YKL-40 and IL-6 levels in patients with Hodgkin lymphoma (HL), a tumor with strong immunologic reaction to relatively few tumor cells, especially in nodular sclerosis HL. Experimental Design We analyzed Danish and Swedish patients with incident HL (N=470) and population controls from Denmark (N= 245 for YKL-40; N= 348 for IL-6). Serum YKL-40 and IL-6 levels were determined by ELISA, and log-transformed data were analysed by linear regression, adjusting for age and sex. Results Serum levels of YKL-40 and IL-6 were increased in HL patients compared to controls (YKL-40: 3.6-fold, IL-6: 8.3-fold; both p<0.0001). In samples from pre-treatment HL patients (N=176), levels were correlated with more advanced stages (ptrend 0.0001 for YKL-40 and 0.013 for IL-6) and in those with B symptoms, but levels were similar in nodular sclerosis and mixed cellularity subtypes, by EBV status, and in younger (<45 years old) and older patients. Patients tested soon after treatment onset had significantly lower levels than pre-treatment patients, but even >6 months after treatment onset, serum YKL-40 and IL-6 levels remained significantly increased, compared to controls. In patients who died (N=12), pre-treatment levels for both YKL-40 and IL-6 were higher than in survivors, although not statistically significantly. Conclusions Serum YKL-40 and IL-6 levels were increased in untreated HL patients and those with more advanced stages but did not differ significantly by HL histology. Following treatment, serum levels were significantly lower.

Ureaplasma parvum : understanding the complexities of intra-amniotic infection in an ovine model

The human Ureaplasma species are the most frequently isolated bacteria from the upper genital tract of pregnant women and can cause clinically asymptomatic, intra-uterine infections, which are difficult to treat with antimicrobials. Ureaplasma infection of the upper genital tract during pregnancy has been associated with numerous adverse outcomes including preterm birth, chorioamnionitis and neonatal respiratory diseases. The mechanisms by which ureaplasmas are able to chronically colonise the amniotic fluid and avoid eradication by (i) the host immune response and (ii) maternally-administered antimicrobials, remain virtually unexplored. To address this gap within the literature, this study investigated potential mechanisms by which ureaplasmas are able to cause chronic, intra-amniotic infections in an established ovine model. In this PhD program of research the effectiveness of standard, maternal erythromycin for the treatment of chronic, intra-amniotic ureaplasma infections was evaluated. At 55 days of gestation pregnant ewes received an intra-amniotic injection of either: a clinical Ureaplasma parvum serovar 3 isolate that was sensitive to macrolide antibiotics (n = 16); or 10B medium (n = 16). At 100 days of gestation, ewes were then randomised to receive either maternal erythromycin treatment (30 mg/kg/day for four days) or no treatment. Ureaplasmas were isolated from amniotic fluid, chorioamnion, umbilical cord and fetal lung specimens, which were collected at the time of preterm delivery of the fetus (125 days of gestation). Surprisingly, the numbers of ureaplasmas colonising the amniotic fluid and fetal tissues were not different between experimentally-infected animals that received erythromycin treatment or infected animals that did not receive treatment (p > 0.05), nor were there any differences in fetal inflammation and histological chorioamnionitis between these groups (p > 0.05). These data demonstrate the inability of maternal erythromycin to eradicate intra-uterine ureaplasma infections. Erythromycin was detected in the amniotic fluid of animals that received antimicrobial treatment (but not in those that did not receive treatment) by liquid chromatography-mass spectrometry; however, the concentrations were below therapeutic levels (<10 – 76 ng/mL). These findings indicate that the ineffectiveness of standard, maternal erythromycin treatment of intra-amniotic ureaplasma infections may be due to the poor placental transfer of this drug. Subsequently, the phenotypic and genotypic characteristics of ureaplasmas isolated from the amniotic fluid and chorioamnion of pregnant sheep after chronic, intra-amniotic infection and low-level exposure to erythromycin were investigated. At 55 days of gestation twelve pregnant ewes received an intra-amniotic injection of a clinical U. parvum serovar 3 isolate, which was sensitive to macrolide antibiotics. At 100 days of gestation, ewes received standard maternal erythromycin treatment (30 mg/kg/day for four days, n = 6) or saline (n = 6). Preterm fetuses were surgically delivered at 125 days of gestation and ureaplasmas were cultured from the amniotic fluid and the chorioamnion. The minimum inhibitory concentrations (MICs) of erythromycin, azithromycin and roxithromycin were determined for cultured ureaplasma isolates, and antimicrobial susceptibilities were different between ureaplasmas isolated from the amniotic fluid (MIC range = 0.08 – 1.0 mg/L) and chorioamnion (MIC range = 0.06 – 5.33 mg/L). However, the increased resistance to macrolide antibiotics observed in chorioamnion ureaplasma isolates occurred independently of exposure to erythromycin in vivo. Remarkably, domain V of the 23S ribosomal RNA gene (which is the target site of macrolide antimicrobials) of chorioamnion ureaplasmas demonstrated significant variability (125 polymorphisms out of 422 sequenced nucleotides, 29.6%) when compared to the amniotic fluid ureaplasma isolates and the inoculum strain. This sequence variability did not occur as a consequence of exposure to erythromycin, as the nucleotide substitutions were identical between chorioamnion ureaplasmas isolated from different animals, including those that did not receive erythromycin treatment. We propose that these mosaic-like 23S ribosomal RNA gene sequences may represent gene fragments transferred via horizontal gene transfer. The significant differences observed in (i) susceptibility to macrolide antimicrobials and (ii) 23S ribosomal RNA sequences of ureaplasmas isolated from the amniotic fluid and chorioamnion suggests that the anatomical site from which they were isolated may exert selective pressures that alter the socio-microbiological structure of the bacterial population, by selecting for genetic changes and altered antimicrobial susceptibility profiles. The final experiment for this PhD examined antigenic size variation of the multiple banded antigen (MBA, a surface-exposed lipoprotein and predicted ureaplasmal virulence factor) in chronic, intra-amniotic ureaplasma infections. Previously defined ‘virulent-derived’ and ‘avirulent-derived’ clonal U. parvum serovar 6 isolates (each expressing a single MBA protein) were injected into the amniotic fluid of pregnant ewes (n = 20) at 55 days of gestation, and amniotic fluid was collected by amniocentesis every two weeks until the time of near-term delivery of the fetus (at 140 days of gestation). Both the avirulent and virulent clonal ureaplasma strains generated MBA size variants (ranging in size from 32 – 170 kDa) within the amniotic fluid of pregnant ewes. The mean number of MBA size variants produced within the amniotic fluid was not different between the virulent (mean = 4.2 MBA variants) and avirulent (mean = 4.6 MBA variants) ureaplasma strains (p = 0.87). Intra-amniotic infection with the virulent strain was significantly associated with the presence of meconium-stained amniotic fluid (p = 0.01), which is an indicator of fetal distress in utero. However, the severity of histological chorioamnionitis was not different between the avirulent and virulent groups. We demonstrated that ureaplasmas were able to persist within the amniotic fluid of pregnant sheep for 85 days, despite the host mounting an innate and adaptive immune response. Pro-inflammatory cytokines (interleukin (IL)-1â, IL-6 and IL-8) were elevated within the chorioamnion tissue of pregnant sheep from both the avirulent and virulent treatment groups, and this was significantly associated with the production of anti-ureaplasma IgG antibodies within maternal sera (p < 0.05). These findings suggested that the inability of the host immune response to eradicate ureaplasmas from the amniotic cavity may be due to continual size variation of MBA surface-exposed epitopes. Taken together, these data confirm that ureaplasmas are able to cause long-term in utero infections in a sheep model, despite standard antimicrobial treatment and the development of a host immune response. The overall findings of this PhD project suggest that ureaplasmas are able to cause chronic, intra-amniotic infections due to (i) the limited placental transfer of erythromycin, which prevents the accumulation of therapeutic concentrations within the amniotic fluid; (ii) the ability of ureaplasmas to undergo rapid selection and genetic variation in vivo, resulting in ureaplasma isolates with variable MICs to macrolide antimicrobials colonising the amniotic fluid and chorioamnion; and (iii) antigenic size variation of the MBA, which may prevent eradication of ureaplasmas by the host immune response and account for differences in neonatal outcomes. The outcomes of this program of study have improved our understanding of the biology and pathogenesis of this highly adapted microorganism.

Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts

Chlamydiae are intracellular bacteria that commonly cause infections of the respiratory and genital tracts, which are major clinical problems. Infections are also linked to the aetiology of diseases such as asthma, emphysema and heart disease. The clinical management of infection is problematic and antibiotic resistance is emerging. Increased understanding of immune processes that are involved in both clearance and immunopathology of chlamydial infection is critical for the development of improved treatment strategies. Here, we show that IL-13 was produced in the lungs of mice rapidly after Chlamydia muridarum (Cmu) infection and promoted susceptibility to infection. Wild-type (WT) mice had increased disease severity, bacterial load and associated inflammation compared to IL-13 deficient (−/−) mice as early as 3 days post infection (p.i.). Intratracheal instillation of IL-13 enhanced bacterial load in IL-13−/− mice. There were no differences in early IFN-g and IL-10 expression between WT and IL-13−/− mice and depletion of CD4+ T cells did not affect infection in IL-13−/− mice. Collectively, these data demonstrate a lack of CD4+ T cell involvement and a novel role for IL-13 in innate responses to infection. We also showed that IL-13 deficiency increased macrophage uptake of Cmu in vitro and in vivo. Moreover, the depletion of IL-13 during infection of lung epithelial cells in vitro decreased the percentage of infected cells and reduced bacterial growth. Our results suggest that enhanced IL-13 responses in the airways, such as that found in asthmatics, may promote susceptibility to chlamydial lung infection. Importantly the role of IL-13 in regulating infection was not limited to the lung as we showed that IL-13 also promoted susceptibility to Cmu genital tract infection. Collectively our findings demonstrate that innate IL-13 release promotes infection that results in enhanced inflammation and have broad implications for the treatment of chlamydial infections and IL-13-associated diseases.

Carbohydrate gel ingestion and immunoendocrine responses to cycling in temperate and hot conditions

PURPOSE: Heat stress might attenuate the effects of carbohydrate on immunoendocrine responses to exercise by increasing endogenous glucose production and reducing the rate of exogenous carbohydrate oxidation. The authors compared the efficacy of carbohydrate consumption on immune responses to exercise in temperate vs. hot conditions. METHODS: Ten male cyclists exercised on 2 separate occasions in temperate (18.1 +/- 0.4 degrees C, 58% +/- 8% relative humidity) and on another 2 occasions in hot conditions (32.2 +/- 0.7 degrees C, 55% +/- 2% relative humidity). On each occasion, the cyclists exercised in a fed state for 90 min at approximately 60% VO2max and then completed a 16.1-km time trial. Every 15 min during the first 90 min of exercise, they consumed 0.24 g/kg body mass of a carbohydrate or placebo gel. RESULTS: Neutrophil counts increased during exercise in all trials (p < .05) and were significantly lower (40%, p = .006) after the carbohydrate than after the placebo trial in 32 degrees C. The concentrations of serum interleukin (IL)-6, IL-8, and IL-10 and plasma granulocyte-colony-stimulating factor, myeloperoxidase, and calprotectin also increased during exercise in all trials but did not differ significantly between the carbohydrate and placebo trials. Plasma norepinephrine concentration increased during exercise in all trials and was significantly higher (50%, p = .01) after the carbohydrate vs. the placebo trial in 32 degrees C. CONCLUSION: Carbohydrate ingestion attenuated neutrophil counts during exercise in hot conditions, whereas it had no effect on any other immune variables in either temperate or hot conditions.

Body temperature and its effect on leukocyte mobilization, cytokines and markers of neutrophil activation during and after exercise

We investigated the influence of rectal temperature on the immune system during and after exercise. Ten well-trained male cyclists completed exercise trials (90 min cycling at 60% VO(2max) + 16.1 - km time trial) on three separate occasions: once in 18 degrees C and twice in 32 degrees C. Twenty minutes after the trials in 32 degrees C, the cyclists sat for approximately 20 min in cold water (14 degrees C) on one occasion, whereas on another occasion they sat at room temperature. Rectal temperature increased significantly during cycling in both conditions, and was significantly higher after cycling in 32 degrees C than in 18 degrees C (P < 0.05). Leukocyte counts increased significantly during cycling but did not differ between the conditions. The concentrations of serum interleukin (IL)-6, IL-8 and IL-10, plasma catecholamines, granulocyte-colony stimulating factor, myeloperoxidase and calprotectin increased significantly following cycling in both conditions. The concentrations of serum IL-8 (25%), IL-10 (120%), IL-1 receptor antagonist (70%), tumour necrosis factor-alpha (17%), plasma myeloperoxidase (26%) and norepinephrine (130%) were significantly higher after cycling in 32 degrees C than in 18 degrees C. During recovery from exercise in 32 degrees C, rectal temperature was significantly lower in response to sitting in cold water than at room temperature. However, immune changes during 90 min of recovery did not differ significantly between sitting in cold water and at room temperature. The greater rise in rectal temperature during exercise in 32 degrees C increased the concentrations of serum IL-8, IL-10, IL-1ra, TNF-alpha and plasma myeloperoxidase, whereas the greater decline in rectal temperature during cold water immersion after exercise did not affect immune responses.

Exercise-induced muscle damage, plasma cytokines and markers of neutrophil activation

Introduction: Unaccustomed eccentric exercise often results in muscle damage and neutrophil activation. We examined changes in plasma cytokines stress hormones, creatine kinase activity and myoglobin concentration, neutrophil surface receptor expression, degranulation, and the capacity of neutrophils to generate reactive oxygen species in response to in vitro stimulation after downhill running. Methods: Ten well-trained male runners ran downhill on a treadmill at a gradient of -10% for 45 min at 60% V̇O2max. Blood was sampled immediately before (PRE) and after (POST), 1 h (1 h POST), and 24 h (24 h POST) after exercise. Results: At POST, there were significant increases (P < 0.01) in neutrophil count (32%), plasma interleukin (IL)-6 concentration (460%), myoglobin (Mb) concentration (1100%), and creatine kinase (CK) activity (40%). At 1 h POST, there were further increases above preexercise values for neutrophil count (85%), plasma Mb levels (1800%), and CK activity (56%), and plasma IL-6 concentration remained above preexercise values (410%) (P < 0.01). At 24 h POST, neutrophil counts and plasma IL-6 levels had returned to baseline, whereas plasma Mb concentration (100%) and CK activity (420%) were elevated above preexercise values (P < 0.01). There were no significant changes in neutrophil receptor expression, degranulation and respiratory burst activity, and plasma IL-8 and granulocyte-colony stimulating factor concentrations at any time after exercise. Neutrophil count correlated with plasma Mb concentration at POST (r = 0.64, P < 0.05), and with plasma CK activity at POST (r = 0.83, P < 0.01) and 1 h POST (r = 0.78, P < 0.01). Conclusion: Neutrophil activation remains unchanged after downhill running in well-trained runners, despite increases in plasma markers of muscle damage.

Cerebral cortex activation mapping upon electrical muscle stimulation by 32-channel time-domain functional near-infrared spectroscopy

The application of different EMS current thresholds on muscle activates not only the muscle but also peripheral sensory axons that send proprioceptive and pain signals to the cerebral cortex. A 32-channel time-domain fNIRS instrument was employed to map regional cortical activities under varied EMS current intensities applied on the right wrist extensor muscle. Eight healthy volunteers underwent four EMS at different current thresholds based on their individual maximal tolerated intensity (MTI), i.e., 10 % < 50 % < 100 % < over 100 % MTI. Time courses of the absolute oxygenated and deoxygenated hemoglobin concentrations primarily over the bilateral sensorimotor cortical (SMC) regions were extrapolated, and cortical activation maps were determined by general linear model using the NIRS-SPM software. The stimulation-induced wrist extension paradigm significantly increased activation of the contralateral SMC region according to the EMS intensities, while the ipsilateral SMC region showed no significant changes. This could be due in part to a nociceptive response to the higher EMS current intensities and result also from increased sensorimotor integration in these cortical regions.

A novel immunodeficiency disorder characterized by genetic amplification of interleukin 25

Many primary immunodeficiency disorders of differing etiologies have been well characterized, and much understanding of immunological processes has been gained by investigating the mechanisms of disease. Here, we have used a whole-genome approach, employing single-nucleotide polymorphism and gene expression microarrays, to provide insight into the molecular etiology of a novel immunodeficiency disorder. Using DNA copy number profiling, we define a hyperploid region on 14q11.2 in the immunodeficiency case associated with the interleukin (IL)-25 locus. This alteration was associated with significantly heightened expression of IL25 following T-cell activation. An associated dominant type 2 helper T cell bias in the immunodeficiency case provides a mechanistic explanation for recurrence of infections by pathogens met by Th1-driven responses. Furthermore, this highlights the capacity of IL25 to alter normal human immune responses.