Influence of Mid-Trimester Amniotic Fluid on Endogenous and Lipopolysaccharide-Mediated Responses of Mononuclear Lymphoid Cells

Problem We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. Method of study Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) -6, 10, 12, 23, tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein (MCP)-1. Results Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-alpha release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eightto ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-alpha production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. Conclusion Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

Quercetin decreases inflammatory response and increases insulin action in skeletal muscle of ob/ob mice and in L6 myotubes

Quercetin is a potent anti-inflammatory flavonoid, but its capacity to modulate insulin sensitivity in obese insulin resistant conditions is unknown. This study investigated the effect of quercetin treatment upon insulin sensitivity of ob/ob mice and its potential molecular mechanisms. Obese ob/ob mice were treated with quercetin for 10 weeks, and L6 myotubes were treated with either palmitate or tumor necrosis factor-alpha (TNF alpha) plus quercetin. Cells and muscles were processed for analysis of glucose transporter 4 (GLUT4), TNF alpha and inducible nitric oxide synthase (iNOS) expression, and c-Jun N-terminal kinase (JNK) and inhibitor of nuclear factor-kappa B (NF-kappa B) kinase (I kappa K) phosphorylation. Myotubes were assayed for glucose uptake and NF-kappa B translocation. Chromatin immunoprecipitation assessed NF-kappa B binding to GLUT4 promoter. Quercetin treatment improved whole body insulin sensitivity by increasing GLUT4 expression and decreasing JNK phosphorylation, and TNF alpha and iNOS expression in skeletal muscle. Quercetin suppressed palmitate-induced upregulation of TNF alpha and iNOS and restored normal levels of GLUT4 in myotubes. In parallel, quercetin suppressed TNF alpha-induced reduction of glucose uptake in myotubes. Nuclear accumulation of NF-kappa B in myotubes and binding of NF-kappa B to GLUT4 promoter in muscles of ob/ob mice were also reduced by quercetin. We demonstrated that quercetin decreased the inflammatory status in skeletal muscle of obese mice and in L6 myotubes. This effect was followed by increased muscle GLUT4, with parallel improvement of insulin sensitivity. These results point out quercetin as a putative strategy to manage inflammatory-related insulin resistance. (C) 2012 Elsevier B.V. All rights reserved.

The effect of CCL3 and CCR1 in bone remodeling induced by mechanical loading during orthodontic tooth movement in mice

Bone remodeling is affected by mechanical loading and inflammatory mediators, including chemokines. The chemokine (C–C motif) ligand 3 (CCL3) is involved in bone remodeling by binding to C–C chemokine receptors 1 and 5 (CCR1 and CCR5) expressed on osteoclasts and osteoblasts. Our group has previously demonstrated that CCR5 down-regulates mechanical loading-induced bone resorption. Thus, the present study aimed to investigate the role of CCR1 and CCL3 in bone remodeling induced by mechanical loading during orthodontic tooth movement in mice. Our results showed that bone remodeling was significantly decreased in CCL3−/− and CCR1−/− mice and in animals treated with Met-RANTES (an antagonist of CCR5 and CCR1). mRNA levels of receptor activator of nuclear factor kappa-B (RANK), its ligand RANKL, tumor necrosis factor alpha (TNF-α) and RANKL/osteoprotegerin (OPG) ratio were diminished in the periodontium of CCL3−/− mice and in the group treated with Met-RANTES. Met-RANTES treatment also reduced the levels of cathepsin K and metalloproteinase 13 (MMP13). The expression of the osteoblast markers runt-related transcription factor 2 (RUNX2) and periostin was decreased, while osteocalcin (OCN) was augmented in CCL3−/− and Met-RANTES-treated mice. Altogether, these findings show that CCR1 is pivotal for bone remodeling induced by mechanical loading during orthodontic tooth movement and these actions depend, at least in part, on CCL3.

MyD88 signaling is directly involved in the development of murine placental Malaria

Malaria is a widespread infectious disease caused by the parasite Plasmodium. During pregnancy, malaria infection leads to a range of complications that can affect both the mother and fetus, including stillbirth, infant mortality, and low birth weight. In this study, we utilized a mouse model of placental malaria (PM) infection to determine the importance of the protein MyD88 in the host immune response to Plasmodium during pregnancy. Initially, we demonstrated that Plasmodium berghei NK65GFP adhered to placental tissue via chondroitin sulfate A and induced PM in mice with a C57BL/6 genetic background. To evaluate the involvement of MyD88 in the pathology of PM, we performed a histopathological analysis of placentas obtained from MyD88(-/-) and wild-type (WT) mice following infection on the 19th gestational day. Our data demonstrated that the detrimental placental alterations observed in the infected mice were correlated with the expression of MyD88. Moreover, in the absence of this protein, production of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) was significantly reduced in the infected mice. More importantly, in contrast to fetuses from infected WT mice, which exhibited a reduction in body weight, the fetuses from infected MyD88(-/-) mice did not display significant weight loss compared to their noninfected littermates. In addition, we observed a decrement of maternal care associated with malaria infection, which was attenuated in the MyD88-deficient mice. Collectively, the results of this study illustrate the pivotal importance of the MyD88 signaling pathway in the pathogenesis of placental malaria, thus presenting new possibilities for targeting MyD88 in therapeutic interventions.

Regulation der BDNF-Expression in Endothelzellen

Das Neurotrophin BDNF ist ein protektiver Faktor, der das Wachstum, die Differenzierung und das Überleben neuronaler Zellen fördert. Neben der neuronalen Expression wird BDNF auch peripher exprimiert, so auch in Endothelzellen. Dort stimuliert BDNF die Angiogenese und fördert das Endothelzellüberleben. Eine Regulation der BDNF-Expression unter pathologischen Bedingungen wie Epilepsie, M. Alzheimer, M. Parkinson, Depression und Ischämie ist bereits mehrfach beschrieben worden. Literaturdaten zeigen veränderte BDNF-Expressionen unter pathologischen Bedingungen zeitgleich mit einem erhöhten Spiegel des Tumornekrosefaktors (TNF-a) bzw. einer Aktivierung der Proteinkinase C (PKC). Ob ein erhöhter TNF-a-Spiegel bzw. die Aktivierung der PKC Ursache der veränderten BDNF-Expression ist, ist bisher noch nicht bekannt. In der vorliegenden Arbeit konnte gezeigt werden, dass sowohl TNF-a als auch eine Aktivierung der PKC in peripheren Endothelzellen die BDNF-Expression konzentrations- und zeitabhängig reduziert. Im Fall von TNF-a wird diese Reduktion über den TNF-a-Rezeptor 1 (TNFR1) vermittelt und auf dem Niveau der Transkription reguliert. Weiterhin konnte gezeigt werden, dass BDNF die Angiogenese-Aktivität von humanen Umbilikalvenen-Endothelzellen (HUVEC) in Abhängigkeit der BDNF-Rezeptoren TrkB und p75NTR stimuliert. TNF-a hingegen reduziert die Angiogenese in HUVEC. Bei der Regulation der BDNF-Expression durch den PKC-aktivierenden Phorbolester Phorbol-12-Myristat-13-Acetat (PMA) konnte eine Beteiligung der PKC-Isoformen d gezeigt werden. Die Verminderung der BDNF-Expression durch PKC-Aktivierung konnte durch Inhibitoren der PKC d aufgehoben werden. PMA hatte keine destabilisierende Wirkung auf die BDNF-mRNA. Auch hier wird BDNF durch PMA auf dem Niveau der Transkription reguliert. Weiterhin ist bisher eine pharmakologische Regulation der BDNF-Expression noch nicht näher untersucht worden. Erstmalig konnte eine Wirkung des b1-Adrenorezeptorblockers Nebivolol auf die BDNF-mRNA-Expression beobachtet werden. Nebivolol erhöht die BDNF-Expression in zerebralen Endothelzellen in vitro und im Mäuseherzen in vivo. Hierbei handelt es sich um eine substanzspezifische Wirkung von Nebivolol, die NO-unabhängig verläuft und nicht über den b3-Adrenozeptor vermittelt wird. Teile der klinisch beobachteten protektiven Wirkungen von Nebivolol könnten auf eine erhöhte BDNF-Expression zurückgeführt werden.

Role of Toll interleukin-1 receptor (IL-1R) 8, a negative regulator of IL-1R/Toll-like receptor signaling, in resistance to acute Pseudomonas aeruginosa lung infection

Toll interleukin-1 receptor (IL-1R) 8 (TIR8), also known as single Ig IL-1 receptor (IL-R)-related molecule, or SIGIRR, is a member of the IL-1R-like family, primarily expressed by epithelial cells. Current evidence suggests that TIR8 plays a nonredundant role as a negative regulator in vivo under different inflammatory conditions that are dependent on IL-R and Toll-like receptor (TLR) activation. In the present study, we examined the role of TIR8 in innate resistance to acute lung infections caused by Pseudomonas aeruginosa, a Gram-negative pathogen responsible for life-threatening infections in immunocompromised individuals and cystic fibrosis patients. We show that Tir8 deficiency in mice was associated with increased susceptibility to acute P. aeruginosa infection, in terms of mortality and bacterial load, and to exacerbated local and systemic production of proinflammatory cytokines (gamma interferon [IFN-γ], tumor necrosis factor alpha [TNF-α], IL-1β, and IL-6) and chemokines (CXCL1, CXCL2, and CCL2). It has been reported that host defense against P. aeruginosa acute lung infection can be improved by blocking IL-1 since exaggerated IL-1β production may be harmful for the host in this infection. In agreement with these data, IL-1RI deficiency rescues the phenotype observed in Tir8-deficient mice: in Tir8-/- IL-1RI-/- double knockout mice we observed higher survival rates, enhanced bacterial clearance, and reduced levels of local and systemic cytokine and chemokine levels than in Tir8-deficient mice. These results suggest that TIR8 has a nonredundant effect in modulating the inflammation caused by P. aeruginosa, in particular, by negatively regulating IL-1RI signaling, which plays a major role in the pathogenesis of this infectious disease.

Tumefactive demyelinating lesions during etanercept treatment requiring decompressive hemicraniectomy

Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory and immunoregulatory cytokine involved in the pathogenesis of several autoimmune disorders. Etanercept, a TNF-α antagonist (anti-TNF-α) acting as a soluble TNF-α receptor, has been associated with neurological demyelinating disorders. This paper aims to report an unusual case showing tumefactive central nervous system (CNS) inflammatory demyelination in a patient in the course of TNF -α antagonist therapy, requiring decompressive hemicraniectomy. This report is based on magnetic resonance imaging (MRI) findings and histology. A biopsy confirmed the inflammatory demyelinating nature of the lesions. The clinical presentation is unusual due to the severity of the disease process, requiring decompressive hemicraniotomy with a clinically favorable outcome.

Methylprednisolone Fails to Preserve Pulmonary Surfactant and Blood-Air Barrier Integrity in a Porcine Cardiopulmonary Bypass Model

BACKGROUND: Pulmonary inflammation after cardiac surgery with cardiopulmonary bypass (CPB) has been linked to respiratory dysfunction and ultrastructural injury. Whether pretreatment with methylprednisolone (MP) can preserve pulmonary surfactant and blood-air barrier, thereby improving pulmonary function, was tested in a porcine CPB-model. MATERIALS AND METHODS: After randomizing pigs to placebo (PLA; n = 5) or MP (30 mg/kg, MP; n = 5), animals were subjected to 3 h of CPB with 1 h of cardioplegic cardiac arrest. Hemodynamic data, plasma tumor necrosis factor-alpha (TNF-alpha, ELISA), and pulmonary function parameters were assessed before, 15 min after CPB, and 8 h after CPB. Lung biopsies were analyzed for TNF-alpha (Western blot) or blood-air barrier and surfactant morphology (electron microscopy, stereology). RESULTS: Systemic TNF-alpha increased and cardiac index decreased at 8 h after CPB in PLA (P < 0.05 versus pre-CPB), but not in MP (P < 0.05 versus PLA). In both groups, at 8 h after CPB, PaO(2) and PaO(2)/FiO(2) were decreased and arterio-alveolar oxygen difference and pulmonary vascular resistance were increased (P < 0.05 versus baseline). Postoperative pulmonary TNF-alpha remained unchanged in both groups, but tended to be higher in PLA (P = 0.06 versus MP). The volume fraction of inactivated intra-alveolar surfactant was increased in PLA (58 +/- 17% versus 83 +/- 6%) and MP (55 +/- 18% versus 80 +/- 17%) after CPB (P < 0.05 versus baseline for both groups). Profound blood-air barrier injury was present in both groups at 8 h as indicated by an increased blood-air barrier integrity score (PLA: 1.28 +/- 0.03 versus 1.70 +/- 0.1; MP: 1.27 +/- 0.08 versus 1.81 +/- 0.1; P < 0.05). CONCLUSION: Despite reduction of the systemic inflammatory response and pulmonary TNF-alpha generation, methylprednisolone fails to decrease pulmonary TNF-alpha and to preserve pulmonary surfactant morphology, blood-air barrier integrity, and pulmonary function after CPB.

The stereochemistry of the amino acid side chain influences the inflammatory potential of muramyl dipeptide in experimental meningitis

Intrathecal injections of 50 to 100 micro g of (N-acetylmuramyl-L-alanyl-D-isoglutamine) muramyl dipeptide (MDP)/rabbit dose-dependently triggered tumor necrosis factor alpha (TNF-alpha) secretion (12 to 40,000 pg/ml) preceding the influx of leukocytes in the subarachnoid space of rabbits. Intrathecal instillation of heat-killed unencapsulated R6 pneumococci produced a comparable leukocyte influx but only a minimal level of preceding TNF-alpha secretion. The stereochemistry of the first amino acid (L-alanine) of the MDP played a crucial role with regard to its inflammatory potential. Isomers harboring D-alanine in first position did not induce TNF-alpha secretion and influx of leukocytes. This stereospecificity of MDPs was also confirmed by measuring TNF-alpha release from human peripheral mononuclear blood cells stimulated in vitro. These data show that the inflammatory potential of MDPs depends on the stereochemistry of the first amino acid of the peptide side chain and suggest that intact pneumococci and MDPs induce inflammation by different pathways.

Matrix metalloproteinases contribute to brain damage in experimental pneumococcal meningitis

The present study was performed to evaluate the role of matrix metalloproteinases (MMP) in the pathogenesis of the inflammatory reaction and the development of neuronal injury in a rat model of bacterial meningitis. mRNA encoding specific MMPs (MMP-3, MMP-7, MMP-8, and MMP-9) and the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) were significantly (P < 0.04) upregulated, compared to the beta-actin housekeeping gene, in cortical homogenates at 20 h after infection. In parallel, concentrations of MMP-9 and TNF-alpha in cerebrospinal fluid (CSF) were significantly increased in rats with bacterial meningitis compared to uninfected animals (P = 0.002) and showed a close correlation (r = 0.76; P < 0. 001). Treatment with a hydroxamic acid-type MMP inhibitor (GM6001; 65 mg/kg intraperitoneally every 12 h) beginning at the time of infection significantly lowered the MMP-9 (P < 0.02) and TNF-alpha (P < 0.02) levels in CSF. Histopathology at 25.5 +/- 5.7 h after infection showed neuronal injury (median [range], 3.5% [0 to 17.5%] of the cortex), which was significantly (P < 0.01) reduced to 0% (0 to 10.8%) by GM6001. This is the first report to demonstrate that MMPs contribute to the development of neuronal injury in bacterial meningitis and that inhibition of MMPs may be an effective approach to prevent brain damage as a consequence of the disease.

Predictors of inflammation in response to anthracycline-based chemotherapy for breast cancer

Although chemotherapy for breast cancer can increase inflammation, few studies have examined predictors of this phenomenon. This study examined potential contributions of demographics, disease characteristics, and treatment regimens to markers of inflammation in response to chemotherapy for breast cancer. Thirty-five women with stage I-III-A breast cancer (mean age 50 years) were studied prior to cycle 1 and prior to cycle 4 of anthracycline-based chemotherapy. Circulating levels of inflammatory markers with high relevance to breast cancer were examined, including C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-alpha), Interleukin-1 receptor antagonist (IL1-RA), vascular endothelial growth factor (VEGF), soluble intercellular adhesion molecule-1 (sICAM-1), Interleukin- (IL-6), soluble P-selectin (sP-selectin), and von Willebrand factor (vWf). Chemotherapy was associated with elevations in VEGF (p < or = 0.01), sICAM-1 (p < or = 0.01), sP-selectin (p < or = 0.02) and vWf (p < or = 0.05). Multiple regression analysis controlling for age and body mass index (BMI) showed that higher post-chemotherapy levels of inflammation were consistently related to higher pre-chemotherapy levels of inflammation (ps < or =0.05) as well as to certain disease characteristics. Post-chemotherapy IL-6 levels were higher in patients who had larger tumors (p < or = 0.05) while post-chemotherapy VEGF levels were higher in patients who had smaller tumors (p < or = 0.05). Post-chemotherapy sP-selectin levels were highest in women who had received epirubicin, cytoxan, 5-fluorouracil chemotherapy (p < or = 0.01). These findings indicate that chemotherapy treatment can be associated with elevations in certain markers of inflammation, particularly markers of endothelial and platelet activation. Inflammation in response to chemotherapy is most significantly related to inflammation that existed prior to chemotherapy but also potentially to treatment regimen and to certain disease characteristics.

Do common genetic variants in endotoxin signaling pathway contribute to predisposition to alcoholic liver cirrhosis?

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), produced by endotoxin-activated Kupffer cells, play a key role in the pathogenesis of alcoholic liver cirrhosis (ALC). Alleles TNFA -238A, IL1B -31T and variant IL1RN*2 of repeat polymorphism in the gene encoding the IL-1 receptor antagonist increase production of TNF-alpha and IL-1beta, respectively. Alleles CD14 -159T, TLR4 c.896G and TLR4 c.1196T modify activation of Kupffer cells by endotoxin. We confirmed the published associations between these common variants and genetic predisposition to ALC by means of a large case-control association study conducted on two Central European populations. METHODS: The study population comprised a Czech sample of 198 ALC patients and 370 controls (MONICA project), and a German sample of 173 ALC patients and 331 controls (KORA-Augsburg), and 109 heavy drinkers without liver disease. RESULTS: Single locus analysis revealed no significant difference between patients and controls in all tested loci. Diplotype [IL1RN 2/ 2; IL1B -31T+] was associated with increased risk of ALC in the pilot study, but not in the validation samples. CONCLUSIONS: Although cytokine mediated immune reactions play a role in the pathogenesis of ALC, hereditary susceptibility caused by variants in the corresponding genes is low in Central European populations.

Endothelial cell activation leads to neutrophil transmigration as supported by the sequential roles of ICAM-2, JAM-A, and PECAM-1

Leukocyte transmigration is mediated by endothelial cell (EC) junctional molecules, but the associated mechanisms remain unclear. Here we investigate how intercellular adhesion molecule-2 (ICAM-2), junctional adhesion molecule-A (JAM-A), and platelet endothelial cell adhesion molecule (PECAM-1) mediate neutrophil transmigration in a stimulus-dependent manner (eg, as induced by interleukin-1beta [IL-1beta] but not tumor necrosis factor-alpha [TNF-alpha]), and demonstrate their ability to act in sequence. Using a cell-transfer technique, transmigration responses of wild-type and TNF-alpha p55/p75 receptor-deficient leukocytes (TNFR(-/-)) through mouse cremasteric venules were quantified by fluorescence intravital microscopy. Whereas wild-type leukocytes showed a normal transmigration response to TNF-alpha in ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) recipient mice, TNFR(-/-) leukocytes exhibited a reduced transmigration response. Hence, when the ability of TNF-alpha to directly stimulate neutrophils is blocked, TNF-alpha-induced neutrophil transmigration is rendered dependent on ICAM-2, JAM-A, and PECAM-1, suggesting that the stimulus-dependent role of these molecules is governed by the target cell being activated. Furthermore, analysis of the site of arrest of neutrophils in inflamed tissues from ICAM-2(-/-), JAM-A(-/-), and PECAM-1(-/-) mice demonstrated that these molecules act sequentially to mediate transmigration. Collectively, the findings provide novel insights into the mechanisms of action of key molecules implicated in leukocyte transmigration.

Relationship between inflammation and cognitive function in obstructive sleep apnea

OBJECTIVES: Obstructive sleep apnea (OSA) can have adverse effects on cognitive functioning, mood, and cardiovascular functioning. OSA brings with it disturbances in sleep architecture, oxygenation, sympathetic nervous system function, and inflammatory processes. It is not clear which of these mechanisms is linked to the decrease in cognitive functioning. This study examined the effect of inflammatory parameters on cognitive dysfunction. MATERIALS AND METHODS: Thirty-nine patients with untreated sleep apnea were evaluated by polysomnography and completed a battery of neuropsychological tests. After the first night of evaluation in the sleep laboratory, blood samples were taken for analysis of interleukin 6, tumor necrosis factor-alpha (TNF-alpha), and soluble TNF receptor 1 (sTNF-R1). RESULTS: sTNF-R1 significantly correlated with cognitive dysfunction. In hierarchical linear regression analysis, measures of obstructive sleep apnea severity explained 5.5% of the variance in cognitive dysfunction (n.s.). After including sTNF-R1, percentage of variance explained by the full model increased more than threefold to 19.6% (F = 2.84, df = 3, 36, p = 0.05). Only sTNF-R1 had a significant individual relationship with cognitive dysfunction (beta = 0.376 t = 2.48, p = 0.02). CONCLUSIONS: sTNF-R1 as a marker of chronic inflammation may be associated with diminished neuropsychological functioning in patients with OSA.

Depressed type 1 cytokine synthesis by superantigen-activated CD4+ T cells of women with human papillomavirus-related high-grade squamous intraepithelial lesions.

Carcinoma of the cervix is causally related to infection with the human papillomavirus (HPV), and T cells play a pivotal role in the immune response of the host to rid itself of HPV infection. Therefore, we assessed the T-cell function of women with HPV-related cervical neoplasia against a superantigen, Staphylococcus enterotoxin B (SEB). Each woman provided a cervical brush specimen for HPV DNA testing and Papanicolaou (Pap) smears for the staging of cervical lesions. They also provided a blood specimen for determination of the ability of CD4(+) T and CD8(+) T cells to synthesize Th1 (interleukin-2 [IL-2], gamma interferon [IFN-gamma], and tumor necrosis factor alpha [TNF-alpha]) and Th2 (IL-10) cytokines in response to activation with SEB. Compared with control subjects with self-attested negative Pap smears, women with high-grade squamous intraepithelial lesions (HSIL) had significantly lower percentages of activated CD4(+) T cells that produced IL-2 (P = 0.045), IFN-gamma (P = 0.040), and TNF-alpha (P = 0.015) and a significantly lower percentage of activated CD8(+) T cells that produced IL-2 (P < 0.01). These data indicate that women with HPV-related cervical HSIL show a decrease in Th1 cytokine production by activated CD4(+) T cells and suggested that compromised T-helper functions may negatively impact the function of cytotoxic CD8(+) T cells.