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Thomas Tschernig

Publications and source records attributed to Thomas Tschernig.

At least 19 recordsLinked to original sources
PubMed2006

The alveolar epithelial type I-like cell line as an adequate model for leukocyte migration studies in vitro.

The lung is unique as leukocytes not only migrate into the bronchoalveolar space but also return to the parenchyma and then via the lymphatics to the draining lymph node. The aim of this study was to investigate the migration of leukocytes via an epithelial monolayer in a Transwell system against a chemokine gradient. Rat type I-like R3/1 alveolar epithelial cells were cultivated on a Transwell polyester membrane (pore diameter 3 microm) for 3 days until a monolayer was formed. The tightness of the monolayer was determined by transepithelial transport of horseradish peroxidase. Isolated human and rat peripheral blood mononuclear cells (PBMC) were placed in the upper chamber, and different concentrations of monocyte chemotactic protein-1 (MCP-1) in the lower chamber. The transmigration of PBMC was quantified and investigated by light and transmission electron microscopy. PBMC migrated through the epithelial cell barrier intercellularly as well as transcellularly. The migration of PBMC against the MCP-1 gradient was dose dependent. The results indicate that this model could help in the study of key events involved in chemokine-induced cell migration from the airways into tissue.

Animals↗
PubMed2006

Injection of IL-12- and IL-18-encoding plasmids ameliorates the autoimmune pathology of MRL/Mp-Tnfrsf6lpr mice: synergistic effect on autoimmune symptoms.

IL-12 and IL-18 are mediators involved in the onset and progression of the autoimmune disease developing in MRL/Mp-Tnfrsf6(lpr) (lpr) mice, which display symptoms similar to the human systemic lupus erythematosus (SLE). The pathology is characterized by progressive lymphadenopathy and auto-antibody-mediated multiple organ failure, e.g. glomerulonephritis, or pneumonitis and a concomitant increase in serum levels for IFNgamma and tumor necrosis factor-alpha (TNFalpha). In this study, we intramuscularly injected lpr mice with plasmids encoding IL-12 and IL-18, either alone or in combination, in order to affect the development of the autoimmune disease. Five biweekly injections of the combined plasmids starting at 4-5 weeks of age diminished serum levels of TNFalpha and reduced the ability of lymphocytes from treated mice to produce IFNgamma in vitro. Injection of both plasmids synergistically attenuated the development of autoimmune syndromes, lymphoproliferation in secondary lymphoid organs, proteinuria and kidney damage, and pneumonitis. We conclude that IL-12 and IL-18 synergistically affect the pathogenesis of the T(h)1-dependent autoimmune syndrome of lpr mice and that approaches that target both IL-12 and IL-18 may be a therapeutic option in the treatment of autoimmune SLE.

Animals↗
PubMed2006

Role of inducible nitric oxide synthase and NADPH oxidase in early control of Burkholderia pseudomallei infection in mice.

Infection with the soil bacterium Burkholderia pseudomallei can result in a variety of clinical outcomes, including asymptomatic infection. The initial immune defense mechanisms which might contribute to the various outcomes after environmental contact with B. pseudomallei are largely unknown. We have previously shown that relatively resistant C57BL/6 mice can restrict bacterial B. pseudomallei growth more efficiently within 1 day after infection than highly susceptible BALB/c mice. By using this model, our study aimed to investigate the role of macrophage-mediated effector mechanisms during early B. pseudomallei infection. Depletion of macrophages revealed an essential role of these cells in the early control of infection in BALB/c and C57BL/6 mice. Strikingly, the comparison of the anti-B. pseudomallei activity of bone marrow-derived macrophages (BMM) from C57BL/6 and BALB/c mice revealed an enhanced bactericidal activity of C57BL/6 BMM, particularly after gamma interferon (IFN-gamma) stimulation. In vitro experiments with C57BL/6 gp91phox-/- BMM showed an impaired intracellular killing of B. pseudomallei compared to experiments with wild-type cells, although C57BL/6 gp91phox-/- cells still exhibited substantial killing activity. The anti-B. pseudomallei activity of C57BL/6 iNOS-/- BMM was not impaired. C57BL/6 gp91phox-/- mice lacking a functional NADPH oxidase were more susceptible to infection, whereas C57BL/6 mice lacking inducible nitric oxide synthase (iNOS) did not show increased susceptibility but were slightly more resistant during the early phase of infection. Thus, our data suggest that IFN-gamma-mediated but iNOS-independent anti-B. pseudomallei mechanisms of macrophages might contribute to the enhanced resistance of C57BL/6 mice compared to that of BALB/c mice in the early phase of infection.

Animals↗
PubMed2006

Critical role for p47phox in renin-angiotensin system activation and blood pressure regulation.

OBJECTIVE: Renin-angiotensin system (RAS) activation leads to increased production of NAD(P)H oxidase-derived reactive oxygen species (ROS), and both have been implicated in the initiation and progression of arterial hypertension, atherosclerosis, and cardiac hypertrophy. The cytosolic subunit p47phox is critically involved in agonist-induced NAD(P)H oxidase activation. Here, we investigated the role of p47phox in blood pressure control, endothelium-dependent relaxation, cardiac hypertrophy, RAS activation, and renal oxidative stress under resting conditions. METHODS AND RESULTS: Mice deficient in p47phox (on C57BL/6 background) developed significantly higher systolic blood pressure levels compared to C57BL/6 wild-type animals (136.0+/-3.0 mmHg vs. 112.2+/-2.6, P<0.01, n=16) as measured by the tail cuff method from week 6 up to week 12 post partum. The increase in blood pressure in p47phox-/- mice was associated with an impaired endothelium-dependent relaxation (P<0.005 vs. wild-type, n=11). At the age of 12 weeks p47phox-/- mice showed increased plasma renin activity as analyzed by radioimmunoassay (14.5+/-1.8 ng/mL/h vs. 9.6+/-1.7 ng/mL/h, P<0.05, n=10) and enhanced angiotensin converting enzyme (ACE) activity in the kidney and aorta as measured by Hip-His-Leu cleavage (7.6+/-0.8 vs. 4.8+/-0.9 nmol/L His-Leu/mg protein, P<0.05, n=5) compared to wild-type mice. No differences in oxygen radical formation was determined in kidney samples by lucigenin- and luminol-enhanced chemiluminescence or by electron spin resonance spectroscopy. Consistently, treatment with the radical scavenger tempol did not lower blood pressure in p47phox-/- mice, whereas ACE and angiotensin II type I receptor inhibition normalized blood pressure. CONCLUSION: Deficiency of the NAD(P)H oxidase subunit p47phox leads to RAS activation, which subsequently contributes to blood pressure increase in a ROS-independent manner.

Angiotensin II Type 1 Receptor Blockers↗
PubMed2006

Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells.

Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer's patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2+/-3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.

Animals↗
PubMed2006

Animal models of asthma: innovative methods of lung research and new pharmacological targets.

Allergic diseases like bronchial asthma are increasing in societies with western lifestyle. In the last years substantial progress was made in the understanding of the underlying mechanisms and explanations like the hygiene hypothesis were developed. However the exact mechanisms of the physiological and immunological events in the lung leading to bronchial asthma are still not fully understood. Therefore, animal models of asthma have been established and improved to study the complex cellular interactions in vivo. Since mice became the most frequently used animal species the methods for detecting lung physiology, e.g. lung function measurements were adapted to the small size of the murine lung. Laser-dissection and precision cut lung slices have become common techniques to get a view into distinct lung compartments and cells. In addition genomic and proteomic approaches are now used widely. On the other hand a major conclusion of the workshop stated that more than one species is necessary in research and for pharmacological screening in asthma and COPD. The resulting new understanding in the mechanisms of asthma pathogenesis has lead to a rapid identification of novel pharmaceutical targets for treatment of the disease.

Animals↗
PubMed2006

Molecular and functional changes of pulmonary surfactant in response to hyperoxia.

Surfactant comprises phosphatidylcholine (PC) together with anionic phospholipids, neutral lipids, and surfactant proteins SP-A to-D. Its composition is highly specific, with dipalmitoyl-PC, palmitoyl-myristoyl-PC, and palmitoyl-palmitoleoyl-PC as its predominant PC species, but with low polyunsaturated phospholipids. Changes in pulmonary metabolism and function in response to injuries depend on their duration and whether adaptation can occur. We examined in rats prolonged (7 days) versus acute (2 days) exposure to non-lethal oxygen concentrations (85%) with respect to the composition and metabolism of individual lung phospholipid molecular species. Progressive inflammation, structural alteration, and involvement of type II pneumocytes were confirmed by augmented bromodeoxyuridine incorporation, broadening of alveolar septa, and increased granulocyte, macrophage, SP-A, and SP-D concentrations. Surfactant function was impaired after 2 days, but normalized with duration of hyperoxia, which was attributable to inhibition but not to alteration in SP-B/C concentrations. Phospholipid pool sizes and PC synthesis by lung tissue, as assessed by [methyl-(3)H]-choline incorporation, were unchanged after 2 days, although after 7 days they were elevated 1.7-fold. By contrast, incorporation of labeled PC into tissue pools of surfactant and lung lavage fluid decreased progressively. Moreover, concentrations of arachidonic acid containing phospholipids were augmented at the expense of saturated palmitoyl-myristoyl-PC and dipalmitoyl-PC. We conclude a persisting impairment in the intracellular trafficking and secretion of newly synthesized PC, accompanied by a progressive increase in alveolar arachidonic acid containing phospholipids in spite of recovery of acutely impaired surfactant function and adaptive increase of overall PC synthesis.

Animals↗
PubMed2006

Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus.

The primary target of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is epithelial cells in the respiratory and intestinal tract. The cellular receptor for SARS-CoV, angiotensin-converting enzyme 2 (ACE2), has been shown to be localized on the apical plasma membrane of polarized respiratory epithelial cells and to mediate infection from the apical side of these cells. Here, these results were confirmed and extended by including a colon carcinoma cell line (Caco-2), a lung carcinoma cell line (Calu-3) and Vero E6 cells in our analysis. All three cell types expressed human ACE2 on the apical membrane domain and were infected via this route, as determined with vesicular stomatitis virus pseudotypes containing the S protein of SARS-CoV. In a histological analysis of the respiratory tract, ACE2 was detected in the trachea, main bronchus and alveoli, and occasionally also in the small bronchi. These data will help us to understand the pathogenesis of SARS-CoV infection.

Angiotensin-Converting Enzyme 2↗
PubMed2005

Improved intranasal immunization with live-attenuated measles virus after co-inoculation of the lipopeptide MALP-2.

The macrophage-activating lipopeptide with a molecular weight of 2kDa (MALP-2) activates antigen presenting cells of human, mouse and rat origin in vitro and in vivo. Here, we demonstrate that MALP-2 induces MIP1alpha and beta, MIP-2, Gro, TNFalpha, IL1alpha and IL6 in cells of cotton rats (Sigmodon hispidus) in vitro. Intranasal inoculation into cotton rats leads to migration of neutrophils and other leucocytes into the lung lumen and lung tissue. After intranasal co-inoculation of MALP-2 with live-attenuated measles vaccine virus, higher titers of neutralizing antibodies are induced but the proliferative T cell response did not increase. Immunization leads to protective immunity in the absence, but not in the presence of passively transferred measles virus (MV) specific antibodies.

Adjuvants, Immunologic↗
PubMed2005

IL-13 regulates the immune response to inhaled antigens.

The large inhibitory effect of IL-13 blockers on the asthma phenotype prompted us to ask whether IL-13 would play a role in regulating the allergic immune response in addition to its documented effects on structural pulmonary cells. Because IL-13 does not interact with murine T or B cells, but with monocytes, macrophages, and dendritic cells (DCs), we examined the role of IL-13 in the activation of pulmonary macrophages and DCs and in the priming of an immune response to a harmless, inhaled Ag. We found that a majority of cells called "alveolar or interstitial macrophages" express CD11c at high levels (CD11c(high)) and are a mixture of at least two cell types as follows: 1) cells of a mixed phenotype expressing DC and macrophage markers (CD11c, CD205, and F4/80) but little MHC class II (MHC II); and 2) DC-like cells expressing CD11c, CD205, MHC II, and costimulatory molecules. Endogenous IL-13 was necessary to induce and sustain the increase in MHC II and CD40 expression by pulmonary CD11c(high) cells, demonstrated by giving an IL-13 inhibitor as a measure of prevention or reversal to allergen-primed and -challenged mice. Conversely, IL-13 given by inhalation to naive mice increased the expression of MHC II and costimulatory molecules by CD11c(high) cells in an IL-4Ralpha-dependent manner. We found that exogenous IL-13 exaggerated the immune and inflammatory responses to an inhaled, harmless Ag, whereas endogenous IL-13 was necessary for the priming of naive mice with an inhaled, harmless Ag. These data indicate that blockade of IL-13 may have therapeutic potential for controlling the immune response to inhaled Ags.

Administration, Inhalation↗
PubMed2005

Efficacy of macrophage-activating lipopeptide-2 combined with interferon-gamma in a murine asthma model.

RATIONALE: The incidence and prevalence of allergic asthma, caused by Th2-mediated inflammation in response to environmental antigens, is increasing. Epidemiologic data suggest that a lack of Th1-inducing factors may play a pivotal role in the development of this disease. We have previously shown that dendritic cells treated with macrophage-activating lipopeptide-2 (MALP-2) combined with IFN-gamma modulate the Th2 response toward Th1 in an in vitro allergy model. OBJECTIVE: To test in vivo efficacy of this regime, the effects of the substances were evaluated in a mouse model of allergic airway inflammation. METHODS: Female Balb/c mice were sensitized to ovalbumin, whereas control animals were sham-sensitized with adjuvant only. After 4 weeks, MALP-2 and IFN-gamma or NaCl, respectively, were intratracheally instillated. After inhalational ovalbumin challenge, airway hyperreactivity (AHR) to inhaled methacholine was measured by head-out body plethysmography. The animals were subsequently killed to sample bronchoalveolar lavage fluid and lungs. RESULTS: Sensitized NaCl-treated mice developed marked AHR compared with sham-sensitized animals. This coincided with eosinophilia as well as the amplification of eotaxin and the Th2 cytokines interleukin (IL)-5 and IL-13 in the bronchoalveolar lavage fluid. Treatment of sensitized mice with MALP-2 and IFN-gamma significantly reduced AHR compared with the sensitized, NaCl-treated positive control. Eosinophilia as well as Th2 cytokines were reduced to the levels of unsensitized animals. In contrast, IL-12p70 and neutrophils were markedly increased by treatment with both substances. CONCLUSION: These data demonstrate the in vivo efficacy of MALP-2 and IFN-gamma to reduce allergic inflammation and AHR in allergic asthma.

Administration, Inhalation↗
PubMed2005

Murine pulmonary infection with Listeria monocytogenes: differential susceptibility of BALB/c, C57BL/6 and DBA/2 mice.

Murine listeriosis is a paradigm to understand host pathogen interactions. Airway infections with Listeria monocytogenes, although representing a serious problem in early onset neonatal listeriosis, has not been investigated in detail in animal models so far. Here, the susceptibility of BALB/c, DBA/2 and C57BL/6 mice towards an intratracheal (i.t.) infection with virulent L. monocytogenes EGDe and the attenuated variant L. monocytogenes EGD hlyW491A(pERL3-CMVGFP) is reported. The course of infection was characterized by determination of bacterial numbers in the organs and assessment of the health condition of the mice. The distribution and cellular localization of Listeria in the airways was assessed by immunocytochemistry and confocal and electron microscopy. The differential susceptibility of inbred mouse strains to airway infections with L. monocytogenes could be assigned to the major virulence factor listeriolysin O. Resistant C57BL/6 mice were not affected by the two listerial strains. In contrast, BALB/c and DBA/2 mice showed differential susceptibility towards L. monocytogenes EGDe and attenuated bacteria, with all the mice being killed by the wild-type bacteria but rarely by the variant that secretes a listeriolysin of only 10% activity of that of the wild-type toxin. Thus, listeriolysin is a decisive factor for differential susceptibility against Listeria. After i.t. application, bacteria were predominantly localized in the peribronchiolar space and invaded alveolar macrophages but rarely lung epithelial cells. Dissemination from the lung into the deep organs started almost immediately after application, although a pulmonary bacterial reservoir remained during the first 4 days.

Animals↗
PubMed2005

Decrease of the surface fraction of surfactant proteins containing clara cells and type II pneumocytes in a rat asthma model.

In asthma surfactant proteins (SP) might differ in distribution and composition and thus play a role in pathophysiology of this disease. Therefore, the well-established animal model of ovalbumin sensitized and challenged rats were used to study the distribution of surfactant proteins in Clara cells and type II pneumocytes. Serial sections of paraffin embedded lung tissue were sequentially immunostained by the avidin-biotin-peroxidase complex (ABC) technique. Antisera against SP-A, SP-B and Clara cell specific protein (CC10) were used. We determined stereologically' the surface fraction of immunolabelled cells and semiquantitatively the percentage of test fields containing labelled alveolar macrophages. In allergen sensitized and provocated rat lungs: (1) the surface fraction of SP-A and SP-B positive Clara cells was significantly reduced, (2) the surface fraction of Clara cells stained with CC10 was coincided with controls, (3) the surface fraction of SP-A and not of SP-B possitive type II pneumocytes decreased significantly, (4) a significantly higher percentage of test fields with SP-A labelled alveolar macrophages was evaluated. Thus, in this animal model of asthma the inflammatory process after allergen challenge is accompanied by alterations in the distribution patterns of SP in Clara cells and type II pneumocytes.

Animals↗
PubMed2005

Expression of the anaphylatoxin receptors C3aR and C5aR is increased in fatal asthma.

BACKGROUND: The mechanisms leading to death from asthma are not completely understood. Recent studies suggest the involvement of the anaphylatoxins C3a and C5a, generated during complement activation, and their receptors C3aR and C5aR in the pathogenesis of asthma. OBJECTIVE: The aim of our study was to investigate the expression of C3aR and C5aR in fatal asthma. METHODS: We analyzed lung tissue from 14 subjects who died of asthma (fatal asthma; FA) and 14 subjects who died of nonpulmonary causes (controls) and bronchial biopsy specimens from 16 subjects with mild intermittent asthma (MIA). C3aR and C5aR expression was evaluated by immunohistochemistry, and a semiquantitative analysis of the intensity of staining was performed according to a visual analogue scale (score, 0-3). RESULTS: C3aR was expressed on airway epithelium, smooth muscle, submucosal, and parenchymal vessels. C5aR was expressed on myeloid cells infiltrating the submucosa and on airway epithelium. Statistical analysis demonstrated higher expression of C3aR on submucosal vessels in FA compared with controls and MIA (median [minimum-maximum], controls, 0.24 [0-1.48]; MIA, 0.0 [0-1.00]; FA, 1.56 [0.13-3]; P = .002). C3aR was also increased on parenchymal vessels in FA (controls, 0.56 [0-2.00]; FA, 1.81 [0.5-3]; P = .0004). C5aR expression on airway epithelium was increased in FA compared with controls and MIA (controls, 1.25 [0.25-3]; MIA, 1.00 [0-2.00]; FA, 3.00 [1.13-3.00]; P = .001). CONCLUSION: The results of our study suggest a role of complement in FA.

Adult↗
PubMed2005

Frequency and potential cause of bronchus-associated lymphoid tissue in fetal lungs.

Bronchus-associated lymphoid tissue consists of lymphoid follicles with or without a germinal center within the bronchial wall. Bronchus-associated lymphoid tissue is part of the integrated mucosal immune system and present in about 50% of healthy infants. We examined a series of 141 fetal and neonatal lungs and detected bronchus-associated lymphoid tissue in 100% of cases with amniotic infection while postpartum perinatal pneumonia did not elicit bronchus-associated lymphoid tissue formation. Only rarely and in low density, bronchus-associated lymphoid tissue was present in non-infected fetuses. The in utero formation of bronchus-associated lymphoid tissue seems to be a reactive phenomenon and - as has been shown in another study - does not portend an adverse prognosis.

Autopsy↗
PubMed2005

Leukocyte infiltration of the periarterial space of the lung after allergen provocation in a rat asthma model.

The periarterial space has recently been described and its physiological and pathophysiological role during inflammatory and allergic reactions has been reviewed. The present studies used a light-/electron-microscopic approach to characterize the periarterial space in an asthma model in Brown Norway rats. After repeated sensitization with ovalbumin and heat-killed Bordetella pertussis bacilli, airway challenge was carried out after 1 further week. Four or 24 h after challenge, rats were fixed by perfusion or instillation and processed for microscopy. Several periarterial capillaries and connective tissue characterized the tissue between small pulmonary arteries, bronchioles and alveolar septa. Additionally, a partly pronounced interstitial edema was seen independent of the kind of fixation. Not only small arteries but also arterioles and venules were partly surrounded by edematous fluid already visible by light microscopy. Within the connective tissue and within the periarterial fluid, numerous leukocytes, predominantly eosinophils, were found. However, leukocytes were detected only rarely in the vascular lumen. Only sporadically were eosinophils seen in the wall of small arteries or venules. Eosinophils transmigrating the endothelium of capillaries or arterioles were not visible 4 or 24 h after challenge. Thus, granulocytes transmigrate in the periarterial space very rapidly or even earlier than 4 h after challenge. The location of transmigration in the periarterial space needs further investigation.

Allergens↗
PubMed2004

A new rat type I-like alveolar epithelial cell line R3/1: bleomycin effects on caveolin expression.

The study of function and regulation of the phenotype of alveolar type I (AT I) epithelial cells is limited by the rareness of suitable cell lines or primary cultures of this cell type. We describe in the present study the type I-like rat epithelial cell line R3/1. This cell line displays in vitro a phenotype with several characteristic features of AT I cells. R3/1 cells were analysed for mRNA and protein content of markers related to the AT I cell type (T1alpha, ICAM-1, connexin-43, caveolins-1 and -2) and AT II phenotypes [surfactant proteins (SPs) A, B, C and D]. The mRNAs for SPs were found to be at a low level. Moderate protein levels for SP-A and SP-B were found, and SP-C and SP-D proteins were not detectable. R3/1 cells are positive for CD44s, E-cadherin, cytokeratin, vimentin and RAGE, and bind the lectins BPA and SBA. For demonstration of the suitability of R3/1 cells for in vitro studies on epithelial injury, the cells were treated with bleomycin. As shown by real-time RT-PCR and immunoblotting, bleomycin-treatment of R3/1 cells resulted in a decrease in mRNA and protein for both caveolin-1 and caveolin-2 in comparison with controls. The AT I-like cell line R3/1 may serve as a promising tool for the study of lung cell biology.

Animals↗