Microvesicles released from pneumolysin-stimulated lung epithelial cells carry mitochondrial cargo and suppress neutrophil oxidative burst.

Microvesicles (MVs) are cell-derived extracellular vesicles that have emerged as markers and mediators of acute lung injury (ALI). One of the most common pathogens in pneumonia-induced ALI is Streptococcus pneumoniae (Spn), but the role of MVs during Spn lung infection is largely unknown. In the first line of defense against Spn and its major virulence factor, pneumolysin (PLY), are the alveolar epithelial cells (AEC). In this study, we aim to characterize MVs shed from PLY-stimulated AEC and explore their contribution in mediating crosstalk with neutrophils. Using in vitro cell and ex vivo (human lung tissue) models, we demonstrated that Spn in a PLY-dependent manner stimulates AEC to release increased numbers of MVs. Spn infected mice also had higher levels of epithelial-derived MVs in their alveolar compartment compared to control. Furthermore, MVs released from PLY-stimulated AEC contain mitochondrial content and can be taken up by neutrophils. These MVs then suppress the ability of neutrophils to produce reactive oxygen species, a critical host-defense mechanism. Taken together, our results demonstrate that AEC in response to pneumococcal PLY release MVs that carry mitochondrial cargo and suggest that these MVs regulate innate immune responses during lung injury.

Spleen tyrosine kinase inhibition blocks airway constriction and protects from Th2-induced airway inflammation and remodeling.

BACKGROUND: Spleen tyrosine kinase (Syk) is an intracellular nonreceptor tyrosine kinase, which has been implicated as central immune modulator promoting allergic airway inflammation. Syk inhibition has been proposed as a new therapeutic approach in asthma. However, the direct effects of Syk inhibition on airway constriction independent of allergen sensitization remain elusive. METHODS: Spectral confocal microscopy of human and murine lung tissue was performed to localize Syk expression. The effects of prophylactic or therapeutic Syk inhibition on allergic airway inflammation, hyperresponsiveness, and airway remodeling were analyzed in allergen-sensitized and airway-challenged mice. The effects of Syk inhibitors BAY 61-3606 or BI 1002494 on airway function were investigated in isolated lungs of wild-type, PKCα-deficient, mast cell-deficient, or eNOS-deficient mice. RESULTS: Spleen tyrosine kinase expression was found in human and murine airway smooth muscle cells. Syk inhibition reduced allergic airway inflammation, airway hyperresponsiveness, and pulmonary collagen deposition. In naïve mice, Syk inhibition diminished airway responsiveness independently of mast cells, or PKCα or eNOS expression and rapidly reversed established bronchoconstriction independently of NO. Simultaneous inhibition of Syk and PKC revealed additive dilatory effects, whereas combined inhibition of Syk and rho kinase or Syk and p38 MAPK did not cause additive bronchodilation. CONCLUSIONS: Spleen tyrosine kinase inhibition directly attenuates airway smooth muscle cell contraction independent of its protective immunomodulatory effects on allergic airway inflammation, hyperresponsiveness, and airway remodeling. Syk mediates bronchoconstriction in a NO-independent manner, presumably via rho kinase and p38 MAPK, and Syk inhibition might present a promising therapeutic approach in chronic asthma as well as acute asthma attacks.

[Peripartum management plan for patients with mental illnesses : Strategies to reduce the risk of postpartum relapse]. / Peripartales Management bei psychischer Vorerkrankung : Strategien zur Rezidivprophylaxe nach der Entbindung.

BACKGROUND: Transition to parenthood is challenging but for women with a history of recurrent psychiatric disorders becoming a mother has a number of additional issues. Women with a history of mood disorders or psychoses are at increased risk for exacerbation in the vulnerable postpartum period and fear the potential risk of medication during pregnancy for the unborn child as much as they fear a relapse. In these difficult situations women and their families seek advice and support from mental health providers and obstetricians. METHODS AND RESULTS: Based on the treatment of 420 mentally ill women with a desire to have children and pregnancy and prospective documentation of 196 pregnancies over the last 10 years (2006-2016) the authors developed the Bonn concept of peripartum management (BKPM). The plan was designed to reduce the incidence and severity of postpartum relapses in women suffering from psychiatric disorders. Factors to be considered include antenatal and postpartum medication as well as reduction of stress and stimuli, sleep preservation, social support and help from the partner in caring for the baby. Of the 196 women in the BKPM only 4.6 % (n = 9) experienced a severe postpartum relapse with hospitalization. Additionally, the informed consent discussion with patient and partner as part of the peripartum management plan showed positive effects on how women and their families experienced autonomy and safety during pregnancy and postpartum. DISCUSSION: Careful planning and monitoring with a structured perinatal management plan can reduce the risk of relapse in the perinatal period and thus support women with a history of mental disorder in the transition to motherhood. Therefore, the management concept employed in Bonn contributes to the major goal of current peripartum psychiatric care in developing effective prevention strategies for women at high risk.

Moxifloxacin is not anti-inflammatory in experimental pneumococcal pneumonia.

OBJECTIVES: Anti-inflammatory functions of antibiotics may counteract deleterious hyperinflammation in pneumonia. Moxifloxacin reportedly exhibits immunomodulatory properties, but experimental evidence in pneumonia is lacking. Therefore, we investigated moxifloxacin in comparison with ampicillin regarding pneumonia-associated pulmonary and systemic inflammation and lung injury. METHODS: Ex vivo infected human lung tissue and mice with pneumococcal pneumonia were examined regarding local inflammatory response and bacterial growth. In vivo, clinical course of the disease, leucocyte dynamics, pulmonary vascular permeability, lung pathology and systemic inflammation were investigated. In addition, transcellular electrical resistance of thrombin-stimulated endothelial cell monolayers was quantified. RESULTS: Moxifloxacin reduced cytokine production in TNF-α-stimulated, but not in pneumococci-infected, human lung tissue. In vivo, moxifloxacin treatment resulted in reduced bacterial load as compared with ampicillin, whereas inflammatory parameters and lung pathology were not different. Moxifloxacin-treated mice developed less pulmonary vascular permeability during pneumonia, but neither combination therapy with moxifloxacin and ampicillin in vivo nor examination of endothelial monolayer integrity in vitro supported direct barrier-stabilizing effects of moxifloxacin. CONCLUSIONS: The current experimental data do not support the hypothesis that moxifloxacin exhibits potent anti-inflammatory properties in pneumococcal pneumonia.

Induced apoptosis of chondrocytes by Porphyromonas gingivalis as a possible pathway for cartilage loss in rheumatoid arthritis.

The role of bacterial infections in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Patients with RA often exhibit periodontal disease, which is associated with pathogens like Porphyromonas gingivalis. The present study examines the direct effects of P. gingivalis on apoptosis of human chondrocytes (a feature of inflammatory joint diseases) as one can assume an interrelation of pathogenesis of RA and P. gingivalis infections. Primary chondrocytes were infected with P. gingivalis. Early apoptotic and dead cell analysis was performed using Annexin-V, 7AAD, and propidium iodide and examined by flow cytometry and fluorescence microscopy. Caspase activation and DNA fragmentation were determined by western blot analysis and TUNEL reaction. Flow cytometry and fluorescence microscopy demonstrated an increase of Annexin-V-positive early apoptotic chondrocytes after infection. Western blot showed upregulation of activated caspase-3 expression, and TUNEL reaction revealed considerable DNA fragmentation following infection. The data show that P. gingivalis promotes early and later stages of apoptosis of primary human chondrocytes, which might contribute to the joint damage seen in the pathogenesis of RA.

PKC(alpha) and PKC(epsilon) differentially regulate Legionella pneumophila-induced GM-CSF.

Legionella pneumophila is an important causative agent of severe pneumonia in humans. The human alveolar epithelium is an effective barrier for inhaled microorganisms and actively participates in the initiation of innate host defense. Although secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) is essential for the elimination of invading Legionella spp., mechanisms of Legionella pneumophila-induced release of this cytokine are widely unknown. In this study, we have demonstrated a toll-like receptor (TLR)2- and TLR5-dependent release of GM-CSF in L. pneumophila-infected human alveolar epithelial cells. GM-CSF secretion was not dependent on the bacteria type II or type IV secretion system. Furthermore, an increase in protein kinase C (PKC) activity, particularly PKC(alpha) and PKC(epsilon), was noted. Blocking of PKC(alpha) and PKC(epsilon) activity or expression, but not of PKC(beta), PKC(delta), PKC(eta), PKC(theta), and PKC(zeta), significantly reduced the synthesis of GM-CSF in infected cells. While PKC(alpha) was critical for the initiation of a nuclear factor-kappaB-mediated GM-CSF expression, PKC(epsilon) regulated GM-CSF production via activator protein 1. Thus, differential regulation of GM-CSF, production by PKC isoforms, contributes to the host response in Legionnaires' disease.

Effects of Porphyromonas gingivalis on cell cycle progression and apoptosis of primary human chondrocytes.

BACKGROUND: It has been suggested that bacterial infections have a role in the pathogenesis of rheumatoid arthritis (RA). P gingivalis, a Gram-negative, anaerobic rod, is one of the major pathogens associated with periodontal disease. OBJECTIVE: To examine P gingivalis infection and its effects on cell cycle progression and apoptosis of human articular chondrocytes. METHODS: Primary human chondrocytes cultured in monolayers were challenged with P gingivalis. Infection and invasion of P gingivalis into chondrocytes was analysed by scanning electron microscopy, double immunofluorescence and by antibiotic protection and invasion assay. Cell cycle progression of infected chondrocytes was evaluated by flow cytometry. Also, cell apoptosis was visualised by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) of DNA strand breaks and by western blot analysis. RESULTS: Data showed that P gingivalis could adhere and infect primary human chondrocytes. After chondrocyte infection, intracellular localisation of P gingivalis was noted. Flow cytometry analyses demonstrated affected cell cycle progression, with an increase of the G(1) phase and a significant decrease of the G(2) phase after infection. In addition, increased apoptosis of P gingivalis-infected chondrocytes was visualised by TUNEL assay and by upregulation of caspase-3 protein expression. CONCLUSION: These data demonstrate that P gingivalis infects primary human chondrocytes and affects cellular responses, which might contribute to the tissue damage seen in the pathogenesis of rheumatoid arthritis.

Differential regulation of Moraxella catarrhalis-induced interleukin-8 response by protein kinase C isoforms.

Moraxella catarrhalis is a major cause of infectious exacerbations of chronic obstructive lung disease. In pulmonary epithelial cells, M. catarrhalis induces release of the pro-inflammatory cytokine interleukin (IL)-8, which plays a pivotal role in orchestrating airway inflammation. The present study demonstrated that protein kinase (PK)C was activated by Moraxella infection and positively regulated M. catarrhalis-triggered nuclear factor (NF)-kappaB activation and subsequent IL-8 release. Activation of the PKC/NF-kappaB signalling pathway was found to be dependent on expression of the Moraxella-specific ubiquitous surface protein A2. In addition, it was shown that specific isoforms of PKC play differential roles in the fine-tuning of the M. catarrhalis-induced NF-kappaB-dependent gene expression through controlling il8 promoter activity. Inhibition of PKCalpha and epsilon with chemical inhibitors or using short interfering RNA-mediated gene silencing significantly suppressed, whereas inhibition of PKCtheta increased, the M. catarrhalis-induced IL-8 transcription and cytokine release. In conclusion, it was shown that Moraxella catarrhalis infection activates protein kinase C and its isoforms alpha, epsilon and theta, which differentially regulate interleukin-8 transcription in human pulmonary epithelial cells.

Allergic lung inflammation induces pulmonary vascular hyperresponsiveness.

Pulmonary arterial vasoconstriction is an important early component of pulmonary hypertension. Inflammatory mechanisms play a prominent role in the pathogenesis of pulmonary hypertension. The present authors investigated the potential role of acute allergic lung inflammation for alterations in pulmonary haemodynamics. BALB/c mice were intraperitoneally sensitised to ovalbumin and challenged by ovalbumin inhalation. Subsequently, lungs were ventilated and perfused ex vivo, and pulmonary arterial pressure (P(pa)) was continuously monitored. Isolated perfused lungs of allergen-sensitised and -challenged mice showed five-fold enhanced P(pa) responses to serotonin, which is reported to be a significant contributor to pulmonary hypertension in humans. This increase in P(pa) was abolished by the serotonin receptor-2A antagonist ketanserin, but not the serotonin receptor-1B antagonist GR127935. Intracellular signalling to serotonin involved phosphatidylcholine-specific phospholipase C and protein kinase C, as well as Rho-kinase, as assessed by employing the specific inhibitors D609, bisindolylmaleimide and Y27632, respectively. In addition to serotonin, impressively enhanced P(pa) increases in allergic lungs were also evoked by the thromboxane receptor agonist U46619, angiotensin II and endothelin-1. In conclusion, allergic lung inflammation was accompanied by impressive pulmonary vascular hyperresponsiveness. These results suggest a possible role for allergic inflammation in the development of pulmonary arterial hypertension.

Differences in cell activation by Chlamydophila pneumoniae and Chlamydia trachomatis infection in human endothelial cells.

Seroepidemiological studies and demonstration of viable bacteria in atherosclerotic plaques have linked Chlamydophila pneumoniae infection to the development of chronic vascular lesions and coronary heart disease. In this study, we characterized C. pneumoniae-mediated effects on human endothelial cells and demonstrated enhanced phosphorylation and activation of the endothelial mitogen-activated protein kinase (MAPK) family members extracellular receptor kinase (ERK1/2), p38-MAPK, and c-Jun-NH2 kinase (JNK). Subsequent interleukin-8 (IL-8) expression was dependent on p38-MAPK and ERK1/2 activation as demonstrated by preincubation of endothelial cells with specific inhibitors for the p38-MAPK (SB202190) or ERK (U0126) pathway. Inhibition of either MAPK had almost no effect on intercellular cell adhesion molecule 1 (ICAM-1) expression. While Chlamydia trachomatis was also able to infect endothelial cells, it did not induce the expression of endothelial IL-8 or ICAM-1. These effects were specific for a direct stimulation with viable C. pneumoniae and independent of paracrine release of endothelial cell-derived mediators like platelet-activating factor, NO, prostaglandins, or leukotrienes. Thus, C. pneumoniae triggers an early signal transduction cascade in target cells that could lead to endothelial cell activation, inflammation, and thrombosis, which in turn may result in or promote atherosclerosis.

Comparison of different detection methods in quantitative microdensitometry.

Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.

Breast MR imaging screening in 192 women proved or suspected to be carriers of a breast cancer susceptibility gene: preliminary results.

PURPOSE: To compare magnetic resonance (MR) imaging with conventional imaging in screening high-risk women. MATERIALS AND METHODS: This prospective trial included 192 asymptomatic and six symptomatic women who, on the basis of personal or family history or genetic analysis, were suspected or proved to carry a breast cancer susceptibility gene. RESULTS: Fifteen breast cancers were identified: nine in the 192 asymptomatic women (six in the first and three in the second screening round) and six in the symptomatic patients. Concerning the asymptomatic women, four of the nine breast cancers were detected and correctly classified with mammography and ultrasonography (US) combined; another two cancers were visible as well-circumscribed masses and were diagnosed as fibroadenomas. MR imaging allowed the correct classification and local staging of all nine cancers. In 105 asymptomatic women with validation of the 1st-year screening results, the sensitivities of mammography, US, and MR imaging were 33%, 33% (mammography and US combined, 44%), and 100%, respectively; the positive predictive values were 30%, 12%, and 64%, respectively. CONCLUSION: The accuracy of MR imaging is significantly higher than that of conventional imaging in screening high-risk women. Difficulties can be caused by an atypical manifestation of hereditary breast cancers at both conventional and MR imaging and by contrast material enhancement associated with hormonal stimulation.

[Catamenial seizures--an analysis]. / Katameniale Anfälle--eine Analyse.

Catamenial seizures are defined as epileptic seizures occurring during distinct phases of the menstrual cycle (i.e., periovulatory, premenstrually and during menstruation). The cyclic changes of the gonadotropic hormones are thought to be the main cause of the seizures. This hypothesis is supported by results of animal experiments, which have shown oestrogens to increase neuronal excitability whereas progesterone lowered it. We investigated 21 women with epilepsy who reported a catamenial increase in seizure frequency. Only 24% of these women actually exhibited a catamenial manifestation in more than 75% of seizures. The incidence of catamenial seizures is reported in the literature to be between 10% and 72%. Catamenial seizures are treated with anticonvulsant drugs. However, when anticonvulsants have failed to suppress seizures, progesterone or progesterone-derivates have been administered with success. We treated 16 patients with a synthetic GnRH-analogue to suppress the hormonal release of gonadotropins. Four of the six patients with only catamenial seizure manifestations and no other seizures became seizure free. Ten patients had catamenial seizures as well as seizures not related to the menstrual cycle. A decrease in seizure frequency by more than 50% was achieved in 7 of these 10 patients.