LPS- and LTA-induced expression of IL-6 and TNF-α in neonatal and adult blood: role of MAPKs and NF-κB.

As nuclear factor kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) seem to be critical mediators in the inflammatory response, we studied the effects of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) on (a) the activation of NF-κB and MAPKs and (b) the expression of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) with or without the specific inhibitors of these intracellular signal transduction pathways in neonatal cord and adult blood. TNF-α and IL-6 concentrations showed a sharp increase in the supernatants of cord and adult whole blood after stimulation. TNF-α concentrations were significantly higher, whereas IL-6 concentrations were tendentially lower in adult blood after stimulation. Stimulation with LPS or LTA resulted in a significantly decreased activation of p38 MAPK in neonatal compared with adult blood. Although LTA failed to induce additional ERK1/2 phosphorylation, LPS stimulation mediated the moderately increased levels of activated ERK1/2 in neonatal monocytes. The addition of the p38 MAPK inhibitor SB202190 significantly decreased IL-6 and TNF-α production upon LPS or LTA stimulation. Furthermore, the inhibition of ERK1/2 was able to reduce LPS-stimulated TNF-α production in neonatal blood. We conclude that p38 MAPK as well as ERK1/2 phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Anti-inflammatory functions of protein C require RAGE and ICAM-1 in a stimulus-dependent manner.

By binding ß 2-integrins both ICAM-1 and the receptor for advanced glycation end products (RAGE) mediate leukocyte recruitment in a stimulus-dependent manner. Using different inflammatory mouse models we investigated how RAGE and ICAM-1 are involved in anti-inflammatory functions of protein C (PC; Ceprotin, 100 U/kg). We found that, depending on the stimulus, RAGE and ICAM-1 are cooperatively involved in PC-induced inhibition of leukocyte recruitment in cremaster models of inflammation. During short-term proinflammatory stimulation (trauma, fMLP, and CXCL1), ICAM-1 is more important for mediation of anti-inflammatory effects of PC, whereas RAGE plays a major role after longer proinflammatory stimulation (TNF α ). In contrast to WT and Icam-1(-/-) mice, PC had no effect on bronchoalveolar neutrophil emigration in RAGE(-/-) mice during LPS-induced acute lung injury, suggesting that RAGE critically mediates PC effects during acute lung inflammation. In parallel, PC treatment effectively blocked leukocyte recruitment and improved survival of WT mice and Icam-1-deficient mice in LPS-induced endotoxemia, but failed to do so in RAGE-deficient mice. Exploring underlying mechanisms, we found that PC is capable of downregulating intracellular RAGE and extracellular ICAM-1 in endothelial cells. Taken together, our data show that RAGE and ICAM-1 are required for the anti-inflammatory functions of PC.

Is discontinuation of clopidogrel necessary for intracapsular hip fracture surgery? Analysis of 102 hemiarthroplasties.

BACKGROUND: An increasing number of elderly patients are managed with long-term antiplatelet therapy. Such patients often present with hip fracture requiring surgical intervention and may be at increased risk of perioperative bleeding and complications. The aim of this study was to ascertain whether it is necessary to stop clopidogrel preoperatively to avoid postoperative complications following hip hemiarthroplasty surgery in patients with intracapsular hip fracture. MATERIALS AND METHODS: A retrospective review of 102 patients with intracapsular hip fracture with either perioperative clopidogrel therapy [clopidogrel group (CG)] or no previous clopidogrel exposure [no clopidogrel group (NCG)] who underwent hip hemiarthroplasty surgery was undertaken. Statistical comparison on pre- and postoperative haemoglobin, American Society of Anesthesiologists (ASA) grade, comorbidities, operative time, transfusion requirements, hospital length of stay (LOS), wound infection, haematoma and reoperation rate between the two groups was undertaken. Regression analysis was undertaken to ascertain the risk ratios (RR) of complications and transfusion associated with clopidogrel. RESULTS: There was no difference with respect to ASA grade, comorbidities (except cardiac comorbidities), pre- and postoperative haemoglobin levels, operation time, age or gender between the two groups. Four and two patients, respectively, required transfusion postoperatively in the CG and NCG (p = 0.37). There was no difference with respect to LOS, wound infection, haematoma or reoperation rate between the two groups postoperatively. The covariate-adjusted RR for complications and transfusion while being on clopidogrel were 0.43 [95% confidence interval (CI) 0.07-2.60] and 3.96 (95% CI 0.40-39.68), respectively. CONCLUSION: Continuing clopidogrel therapy throughout the perioperative period in patients with intracapsular hip fracture is not associated with an increased risk of complications following hip hemiarthroplasty surgery.

Protein C concentrate controls leukocyte recruitment during inflammation and improves survival during endotoxemia after efficient in vivo activation.

Anti-inflammatory properties of protein C (PC) concentrate are poorly studied compared to activated protein C, although PC is suggested to be safer in clinical use. We investigated how PC interferes with the leukocyte recruitment cascade during acute inflammation and its efficacy during murine endotoxemia. We found that similar to activated protein infusion, intravenous PC application reduced leukocyte recruitment in inflamed tissues in a dose- and time-dependent manner. During both tumor necrosis factor-α induced and trauma-induced inflammation of the cremaster muscle, intravital microscopy revealed that leukocyte adhesion and transmigration, but not rolling, were profoundly inhibited by 100 U/kg PC. Moreover, PC blocked leukocyte emigration into the bronchoalveolar space during lipopolysaccharide (LPS) induced acute lung injury. PC was efficiently activated in a murine endotoxemia model, which reduced leukocyte infiltration of organs and strongly improved survival (75% versus 25% of control mice). Dependent on the inflammatory model, PC provoked a significant inhibition of leukocyte recruitment as early as 1 hour after administration. PC-induced inhibition of leukocyte recruitment during acute inflammation critically involves thrombomodulin-mediated PC activation, subsequent endothelial PC receptor and protease-activated receptor-1-dependent signaling, and down-regulation of intercellular adhesion molecule 1 leading to reduced endothelial inflammatory response. We conclude that during acute inflammation and sepsis, PC is a fast acting and effective therapeutic approach to block leukocyte recruitment and improve survival.

Delayed detection of cytomegalovirus-specific T-helper cells in a preterm infant following intrauterine exposure to tacrolimus.

After exposure to tacrolimus and CMV reactivation in utero, a preterm infant had a positive CMV polymerase chain reaction (PCR) in urine on day 2 of life. Naive T-cells were reduced, but stimulation with CMV antigen and a polyclonal T-cell activator in vitro yielded no measurable CD4+ T-cell response. When repeated on day 30, polyclonal activation was in the range of healthy adults, while the immune response to CMV was incomplete with a lack of IFNgamma+ CD4+ T-cells and no anti-CMV IgM. Thus, exposure to tacrolimus in utero caused a transitional T-cell activation defect which did not compromise viral clearance.

High DMBT1 concentrations in breast milk correlate with increased risk of infection in preterm and term neonates.

BACKGROUND: Human milk contains immune molecules involved in the protection of newborns against infections. We analyzed the concentration of Deleted in Malignant Brain Tumors 1 (DMBT1), a protein with functions in innate immunity, in breast milk. METHODS: DMBT1 was detected in breast milk by Western blotting and its concentration was quantified by ELISA in 95 breast milk samples collected from mothers of preterm and term neonates during the first four weeks after delivery. Possible effects of maternal or neonatal parameters were analyzed by different statistical tests. RESULTS: The mean DMBT1 concentration (± standard error of the mean) in the tested milk samples was 2.48 ± 0.26 µg/mL (range: 0.112 µg/mL to 17.984 µg/mL) and represented 0.0087% of the total protein content. The comparison between the newborns with infection and the newborns without infection revealed significantly higher DMBT1 concentrations in breast milk in the group with infection (6.72 ± 2.53 µg/mL versus 2.20 ± 0.35 µg/mL (P = 0.031)). Neither maternal nor neonatal parameters showed a correlation with the milk DMBT1 levels. CONCLUSIONS: DMBT1 is a component of breast milk after birth and is up-regulated in the breast milk from mothers with newborns suffering from neonatal infection. Thus, breast milk DMBT1 may be part of the innate immunity similar to secretory IgA.

CXCL1-triggered interaction of LFA1 and ICAM1 control glucose-induced leukocyte recruitment during inflammation in vivo.

It is well acknowledged that proinflammatory stimulation during acute hyperglycemia is able to aggravate inflammatory diseases. However, the mechanisms of proinflammatory effects of glucose are controversially discussed. We investigated leukocyte recruitment after intravenous injection of glucose in different inflammatory models using intravital microscopy. Flow chamber experiments, expression analysis, functional depletion, and knockout of key adhesion molecules gave mechanistic insight in involved pathways. We demonstrated that a single injection of glucose rapidly increased blood glucose levels in a dose-dependent manner. Notably, during tumor necrosis factor (TNF) α-induced inflammation leukocyte recruitment was not further enhanced by glucose administration, whereas glucose injection profoundly augmented leukocyte adhesion and transmigration into inflamed tissue in the trauma model, indicating that proinflammatory properties of glucose are stimulus dependent. Experiments with functional or genetic inhibition of the chemokine receptor CXCR2, intercellular adhesion molecule 1 (ICAM1), and lymphocyte function antigen 1 (LFA1) suggest that keratino-derived-chemokine CXCL1-triggered interactions of ICAM1 and LFA1 are crucially involved in the trauma model of inflammation. The lacking effect of glucose on ß(2) integrin expression and on leukocyte adhesion in dynamic flow chamber experiments argues against leukocyte-driven underlying mechanisms and favours an endothelial pathway since endothelial ICAM1 expression was significantly upregulated in response to glucose.

Increased resistance of tumor cells to daunorubicin after transfection of cDNAs coding for anthracycline inactivating enzymes.

Carbonyl reduction is a main but undesired metabolic pathway of the anti-cancer drug daunorubicin (DRC). The resulting alcohol metabolite daunorubicinol has a far less anti-tumor potency and, in addition, is responsible for the life-threatening cardiac toxicity that limits the clinical use of DRC. Elevated levels of carbonyl-reducing enzymes in cancer cells may therefore contribute to the development of DRC chemoresistance and affect the clinical outcome. In the present investigation, human pancreas carcinoma cells were transfected with three important DRC reductases, namely carbonyl reductase (CBR1), aldehyde reductase (AKR1A1) and aldose reductase (AKR1B1), and levels of resistance towards DCR determined. Overexpression of all three reductases lead to a higher DRC inactivation and to an elevation of chemoresistance (7-fold for CBR1, 4.5-fold for AKR1A1 and 3.7-fold for AKR1B1), when IC(50)-values were considered. Coadministration of DRC reductase inhibitors in DRC chemotherapy may be desirable since this would reduce the formation of the cardiotoxic alcohol metabolite and prevent drug resistance.

Orofacial myofunctional disorders in children with asymmetry of the posture and locomotion apparatus.

352 children radiologically identified with asymmetry in the occipito-cervical region were assessed on a number of myofunctional measures. In all children an orthopedic examination was conducted including a functional test of the upper cervical spine and the iliac joint, the postural test by Matthiass, as well as gait analysis. During a second examination the orofacial myofunctional status was recorded. In general, about 70% of the children revealed orofacial myofunctional disorders. Correlational analysis was conducted in order to determine whether specific myofunctional variables were associated with postural and orthopedic alterations. A weak body posture correlated statistically significantly with all assessed myofunctional variables. On the other hand, all orthopedic items correlated significantly with a reclined head position. A blockade of the iliac spine correlated significantly with persistent habits, articulation disorders and tongue dysfunction, whereas functional asymmetry of the upper cervical spine correlated significantly with incompetent lips. A finding of at least five statistically significant correlations within each orofacial variable underlined the complex symptomatology of myofunctional disorders, so that consideration needs to be given to adequate treatment approaches. The data generated by the present study stress the importance of early interdisciplinary screening in children to ensure a physiological development of the orofacial region and the still growing vertebral column. To help understand the complexity of symptoms influencing orofacial development, an explanatory model of the "interactive functional box system" is given.

Neutrophil chemotaxis in cord blood of term and preterm neonates is reduced in preterm neonates and influenced by the mode of delivery and anaesthesia.

Bacterial infections, even without any perinatal risk factors, are common in newborns, especially in preterm neonates. The aim of this study was to evaluate possible impairment of neutrophil chemotaxis in term and preterm neonates compared with adults as well as neonates with different modes of delivery and anaesthesia. We analysed the expression of the adhesion molecule L-Selectin as well as shape change, spontaneous and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced transmigration of neutrophils in a flow cytometric assay of chemotaxis after spontaneous delivery with Cesarian Section (CS) under spinal anaesthesia (mepivacaine, sufentanil), epidural anaesthesia (ropivacaine or bupivacaine, sufentanil) or general anaesthesia (ketamine, thiopental, succinylcholine). Chemokinesis was higher (p=0.008) in cord blood neutrophils than in the adult ones, whereas those could be more stimulated by fMLP (p=0.02). After vaginal delivery neutrophils showed a higher spontaneous and fMLP-stimulated chemotactic response compared to neonates after CS without labor. Comparing different types of anaesthesia for CS, spinal anaesthesia resulted in less impairment on chemotaxis than general anaesthesia or epidural anaesthesia. The new flow cytometric assay of neutrophil chemotaxis is an appropriate and objective method to analyse functional differences even in very small volumes of blood, essential in neonatology. Term neonates do not show reduced chemotaxis compared to adults. Preterm neonates present with reduced chemotaxis and chemokinesis, confirming the well known deficits in their neutrophil function. The side effects of maternal drugs on the neonatal immune system have to be considered especially when the immune response is already impaired, as in preterm infants.

Correlations between dentition anomalies and diseases of the of the postural and movement apparatus--a literature review.

From anatomic and functional aspects the stomatognathic system and the upper cervical spine are closely interlinked. Together with complex neuromuscular relationships, this gives rise to an important field of cooperation between orthodontics and orthopedics. The literature appeals for close interdisciplinary cooperation for patients with syndromes and for those with torticollis and scoliosis. Since orthopedic points of contact are obvious in these special cases, orthopedic aspects are now being taken increasingly into account in farther-reaching studies. With the rising popularity of manual medicine, these aspects are being recognized from the orthopedic point of view too in terms of functional correlations and are being increasingly debated at international congresses and in the literature. Although relevant publications were initially confined to studies of moderate scientific interest or case reports, potential correlations have undergone scientific investigation in recent interdisciplinary studies. Despite the many clinical studies, no unequivocal recommendation can be given for basic conditions under which an orthopedist is bound to be consulted on patients with orthodontic findings. This literature review is aimed at providing an introduction to this still hotly debated issue.

RAGE controls activation and anti-inflammatory signalling of protein C.

AIMS: The receptor for advanced glycation endproducts, RAGE, is a multiligand receptor and NF-κB activator leading to perpetuation of inflammation. We investigated whether and how RAGE is involved in mediation of anti-inflammatory properties of protein C. METHODS AND RESULTS: We analyzed the effect of protein C on leukocyte adhesion and transmigration in WT- and RAGE-deficient mice using intravital microscopy of cremaster muscle venules during trauma- and TNFα-induced inflammation. Both, protein C (PC, Ceprotin, 100 U/kg) and activated protein C (aPC, 24 µg/kg/h) treatment significantly inhibited leukocyte adhesion in WT mice in these inflammation models. The impaired leukocyte adhesion after trauma-induced inflammation in RAGE knockout mice could not be further reduced by PC and aPC. After TNFα-stimulation, however, aPC but not PC treatment effectively blocked leukocyte adhesion in these mice. Consequently, we asked whether RAGE is involved in PC activation. Since RAGE-deficient mice and endothelial cells showed insufficient PC activation, and since thrombomodulin (TM) and endothelial protein C receptor (EPCR) are reduced on the mRNA and protein level in RAGE deficient endothelial cells, an involvement of RAGE in TM-EPCR-dependent PC activation is likely. Moreover, TNFα-induced activation of MAPK and upregulation of ICAM-1 and VCAM-1 are reduced both in response to aPC treatment and in the absence of RAGE. Thus, there seems to be interplay of the RAGE and the PC pathway in inflammation. CONCLUSION: RAGE controls anti-inflammatory properties and activation of PC, which might involve EPCR and TM.

Titanium elastic stable intramedullary nailing of displaced midshaft clavicle fractures: A review of 38 cases.

INTRODUCTION: Clavicle fractures accounting for 3 to 5% of all adult fractures are usually treated non-operatively. There is an increasing trend toward their surgical fixation. The aim of our study was to investigate the outcome following titanium elastic stable intramedullary nailing (ESIN) for midshaft non-comminuted clavicle fractures with >20 mm shortening/displacement. MATERIALS AND METHODS: A total of 38 patients, which met inclusion criteria, were reviewed retrospectively. There were 32 males and six females. The mean age was 27.6 years. The patients were assessed for clinical/radiological union and by Oxford Shoulder and QuickDASH scores. 71% patients required open reduction. RESULTS: 100% union was achieved at average of 11.3 weeks. The average follow-up was 12 months. The average Oxford Shoulder and QuickDASH scores were 45.6 and 6.7, respectively. 47% patients had nail removal. One patient had lateral nail protrusion while other required its medial trimming. CONCLUSION: In our hands, ESIN is safe and minimally invasive with good patient satisfaction, cosmetic appearance, and overall outcome.

Inhibition of LPS-Induced Activation of Coagulation by p38 MAPK Inhibitor.

During Gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and coagulation. However, the underlying LPS signalling mechanism on coagulation activation remains complex. To determine the role of the intracellular signalling factors p38 mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and c-Jun N-terminal kinase (JNK) in the procoagulant response to LPS, coagulation process of human whole blood exposed to specific inhibitors was measured by thrombelastography. Samples were stimulated with LPS (100 µg/mL) after preincubation with BAY117082 (specific NF-κB inhibitor), SP600125 (specific JNK inhibitor), SB203580 (specific p38 MAPK inhibitor), or vehicle. SB203580 strongly inhibited LPS-induced coagulation activation, whereas BAY117082 and SP600125 showed no significant effect. Activation of p38 MAPK, NF-κB, and JNK and respective inhibitory effects were confirmed by Multi-Target Sandwich ELISA. In conclusion, activation of p38 MAPK is crucial for early LPS-induced activation of coagulation.

Lipopolysaccharide-induced expression of Th1/Th2 cytokines in whole neonatal cord and adult blood: role of nuclear factor-kappa B and p38 MAPK.

BACKGROUND: Sepsis continues to be a leading cause of morbidity and mortality in newborns. OBJECTIVE: As both nuclear factor-kappa B (NF-κB) and p38 mitogen-activated protein kinase (MAPK) appear to be critical mediators in inflammatory response, we studied the effects of lipopolysaccharide (LPS) on expression and function of NF-κB and p38 MAPK in whole neonatal cord and adult blood. METHODS: Th1/Th2 cytokine concentrations and phosphorylation of NF-κB and p38 MAPK were determined by flow-cytometric analysis. RESULTS: Tumor necrosis factor-alpha (TNF-α), IL-6, and IL-10 concentrations were significantly elevated in supernatants of neonatal and adult blood after LPS stimulation for 4 h. IFN-γ, IL-4, and IL-2 showed no significant alterations. Furthermore, TNF-α concentrations were significantly higher in adult compared to neonatal blood after LPS stimulation. Stimulation with LPS resulted in significantly decreased activation of p38 MAPK in neonatal blood, whereas NF-κB showed no difference. Following inhibition of p38 MAPK with the specific inhibitor SB-202190, levels of TNF-α and IL-6 significantly decreased in neonatal and adult blood, whereas pharmacological inhibition of NF-κB with SC-514 showed no significant effect on cytokine expression. CONCLUSIONS: We conclude that p38 MAPK phosphorylation is crucially involved in LPS activation and could explain the differences in early cytokine response between neonatal and adult blood.

Prophylactic administration of surfactant in extremely premature infants.

Objective. To investigate whether prophylactic surfactant administration is superior over selective treatment in preterm infants with respiratory distress syndrome (RDS). Methods. In our retrospective analysis, we compared premature infants (23 + 0 to 26 + 6 weeks) receiving 200 mg/kg surfactant (curosurf(®)) within five minutes after birth (prophylactic group, N = 31) with those infants who received surfactant therapy for established RDS (selective group, N = 34). Results. Prophylactic therapy significantly decreased the need for mechanical ventilation (74 hours per patient versus 171 hours per patient, resp.). We observed a reduced incidence of interstitial emphysema (0% versus 9%, resp.), pneumothoraces (3% versus 9%, resp.), chronic lung disease (26% versus 38%, resp.), and surfactant doses per patient (1.3 versus 1.8, resp.), although those variables did not reach significance. Conclusion. We conclude that infants under 27 weeks' gestation profit from prophylactic surfactant administration by reducing the time of mechanical ventilation. This in turn could contribute to reduce the risk for mechanical ventilation associated complications, without any detrimental short-term side effects.

Lipopolysaccharide-induced activation of coagulation in neonatal cord and adult blood monitored by thrombelastography.

INTRODUCTION: Although precisely balanced hemostasis in newborns is rapidly changing during early development. During gram-negative sepsis, lipopolysaccharide (LPS) activates toll-like receptor (TLR) 4 and induces complex responses of immune system and hemostasis. In the present study we compared LPS-induced activation of coagulation in cord blood (CB) samples of healthy newborns to whole blood (WB) samples from healthy adult volunteers. MATERIALS AND METHODS: Samples were incubated with LPS for various incubation periods (0-2-3-4 hrs), and coagulation was measured by rotation thrombelastography (TEG). RESULTS: All parameters (clotting time (CT), clot formation time (CFT), maximal clot formation (MCF), and angle alpha) were affected with increasing incubation period with LPS (100 ng/ml) in adult and cord WB. In the absence of LPS, CTs and CFTs were significantly shorter and MCFs and angels alpha were significantly longer in cord than in adult WB samples. Reduction of CT due to LPS incubation was significantly stronger in CB than in adult WB samples. Pyrrolidine dithiocarbamate, a specific inhibitor of the NFkappaB pathway, inhibited the effects of LPS on CT in adult and cord WB. CONCLUSION: In summary, TEG proved to be a sensitive and reliable tool for the determination of LPS-induced tissue factor mediated activation of hemostasis in whole blood samples from adults and neonates.

Gene expression profiles of adult peripheral and cord blood mononuclear cells altered by lipopolysaccharide.

BACKGROUND: Neonatal Gram-negative sepsis is often characterized by a fulminant clinical course, compared to adults, resulting in higher morbidity and mortality. Genome-wide gene expression analysis can provide insights into the molecular alterations in sepsis. OBJECTIVES: To evaluate in vitro activation of the neonatal and adult immune system, gene expression patterns were compared in mononuclear cells from cord (CBMNC) and adult peripheral blood (APBMNC). METHODS: To better understand the influence of early molecular signals on the effects of sepsis, Affymetrix gene profiling (8,475 genes) was done on RNA isolated from CBMNC and APBMNC without and after incubation with 100 ng/ml lipopolysaccharide (LPS). RESULTS: We demonstrated significant alterations in the expression of 108 CBMNC and APBMNC genes compared with basal levels, 188 significant changes in CBMNC and 97 in APBMNC, including cytokines, chemokines and immunoregulatory genes. Furthermore, we found 5 genes showing a significant interaction effect between cell type and LPS stimulation, including tumor necrosis factor receptor superfamily, member 6 (FAS), absent in melanoma 2, malic enzyme 1, hemoglobin epsilon 1, and trans-prenyltransferase. CONCLUSIONS: These results provide further support for a marked difference in the pathogenesis of neonatal and adult sepsis and may stimulate additional studies to investigate some of the altered genes as potential new targets for diagnostic tools and therapeutic strategies.