[Show abstract][Hide abstract] ABSTRACT: Respiratory tract infections are common in patients suffering from pulmonary fibrosis. The interplay between bacterial infection and fibrosis is characterised poorly.
To assess the effect of Gram-positive bacterial infection on fibrosis exacerbation in mice.
Fibrosis progression in response to Streptococcus pneumoniae was examined in two different mouse models of pulmonary fibrosis.
We demonstrate that wild-type mice exposed to adenoviral vector delivery of active transforming growth factor-β1 (TGFß1) or diphteria toxin (DT) treatment of transgenic mice expressing the DT receptor (DTR) under control of the surfactant protein C (SPC) promoter (SPC-DTR) to induce pulmonary fibrosis developed progressive fibrosis following infection with Spn, without exhibiting impaired lung protective immunity against Spn. Antibiotic treatment abolished infection-induced fibrosis progression. The cytotoxin pneumolysin (Ply) of Spn caused this phenomenon in a TLR4-independent manner, as Spn lacking Ply (SpnΔply) failed to trigger progressive fibrogenesis, whereas purified recombinant Ply did. Progressive fibrogenesis was also observed in AdTGFβ1-exposed Ply-challenged TLR4 KO mice. Increased apoptotic cell death of alveolar epithelial cells along with an attenuated intrapulmonary release of antifibrogenic prostaglandin E2 was found to underlie progressive fibrogenesis in Ply-challenged AdTGFβ1-exposed mice. Importantly, vaccination of mice with the non-cytotoxic Ply derivative B (PdB) substantially attenuated Ply-induced progression of lung fibrosis in AdTGFβ1-exposed mice.
Our data unravel a novel mechanism by which infection with Spn through Ply release induces progression of established lung fibrosis, which can be attenuated by protein-based vaccination of mice.
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[Show abstract][Hide abstract] ABSTRACT: Within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation of hepatocyte polarization and maintenance of metabolic function. We here report the establishment of a liver organoid that integrates NPCs in a vascular layer composed of endothelial cells and tissue macrophages and a hepatic layer comprising stellate cells co-cultured with hepatocytes. The three-dimensional liver organoid is embedded in a microfluidically perfused biochip that enables sufficient nutrition supply and resembles morphological aspects of the human liver sinusoid. It utilizes a suspended membrane as a cell substrate mimicking the space of Disse. Luminescence-based sensor spots were integrated into the chip to allow online measurement of cellular oxygen consumption. Application of microfluidic flow induces defined expression of ZO-1, transferrin, ASGPR-1 along with an increased expression of MRP-2 transporter protein within the liver organoids. Moreover, perfusion was accompanied by an increased hepatobiliary secretion of 5(6)-carboxy-20,70-dichlorofluorescein and an enhanced formation of hepatocyte microvilli. From this we conclude that the perfused liver organoid shares relevant morphological and functional characteristics with the human liver and represents a new in vitro research tool to study human hepatocellular physiology at the cellular level under conditions close to the physiological situation.
[Show abstract][Hide abstract] ABSTRACT: Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.
[Show abstract][Hide abstract] ABSTRACT: Antigen presentation in chronic skin disorders is mediated through the interleukin (IL)-12/IL-23 pathway and, hence, through the IL-12 receptor. Recent evidence suggesting dysregulated antigen presentation in skin lesions of hidradenitis suppurativa (HS) led to investigate the role of single nucleotide polymorphisms (SNPs) of the gene IL-12RB1 coding for the IL12-Rβ1 receptor subunit. Genomic DNA was isolated from 139 patients and 113 healthy controls; nine SNPs in the transcribed region of IL12RB1 were genotyped. No significant differences of genotype and allele frequencies were found between the two groups. Two common haplotypes were recognized, namely h1 and h2. Carriage of h2 related with minor frequency alleles was associated with a greater risk for the acquisition of Hurley III disease stage and with the involvement of a greater number of skin areas. Patients with the h1 haplotype presented disease at an older age. This is the genetic study enrolling the largest number of patients with HS to date. Although SNPs of IL12RB1 do not seem to convey genetic predisposition, they are associated directly with the phenotype of the disease.
[Show abstract][Hide abstract] ABSTRACT: Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA), found abundantly in oily fish, may have diverse health-promoting effects, potentially protecting the immune, nervous, and cardiovascular systems. However, the mechanisms underlying the purported health-promoting effects of DHA remain largely unclear, in part because molecular signaling pathways and effectors of DHA are only beginning to be revealed. In vascular smooth muscle cells, large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels provide a critical vasodilatory influence. We report here that DHA with an EC50 of ∼500 nM rapidly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit β1, increasing currents by up to ∼20-fold. The DHA action is observed in cell-free patches and does not require voltage-sensor activation or Ca(2+) binding but involves destabilization of the closed conformation of the ion conduction gate. DHA lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester, contained in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids, and these fatty acids-unlike their ethyl ester derivatives-activate the channels and lower blood pressure. This finding has practical implications for the use of omega-3 fatty acids as nutraceuticals for the general public and also for the critically ill receiving omega-3-enriched formulas.
Proceedings of the National Academy of Sciences 03/2013; 110(12). DOI:10.1073/pnas.1221997110 · 9.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Monitoring hepatic blood flow and function might be crucial in treating critically ill patients. Intra-abdominal hypertension is associated with decreased abdominal blood flow, organ dysfunction, and increased mortality. The plasma disappearance rate (PDR) of indocyanine green (ICG) is considered to be a compound marker for hepatosplanchnic perfusion and hepatocellular membrane transport and correlates well with survival in critically ill patients. However, correlation between PDRICG and intra-abdominal pressure (IAP) remains poorly understood. The aim of this retrospective study was to investigate the correlation between PDRICG and classic liver laboratory parameters, IAP and abdominal perfusion pressure (APP). The secondary goal was to evaluate IAP, APP, and PDRICG as prognostic factors for mortality.
A total of 182 paired IAP and PDRICG measurements were performed in 40 critically ill patients. The mean values per patient were used for comparison. The IAP was measured using either a balloon-tipped stomach catheter connected to an IAP monitor (Spiegelberg, Hamburg, Germany, or CiMON, Pulsion Medical Systems, Munich, Germany) or a bladder FoleyManometer (Holtech Medical, Charlottenlund, Denmark). PDRICG was measured at the bedside using the LiMON device (Pulsion Medical Systems, Munich, Germany). Primary endpoint was hospital mortality.
There was no significant correlation between PDRICG and classic liver laboratory parameters, but PDRICG did correlate significantly with APP (R = 0.62) and was inversely correlated with IAP (R = -0.52). Changes in PDRICG were associated with significant concomitant changes in APP (R = 0.73) and opposite changes in IAP (R = 0.61). The IAP was significantly higher (14.6 ± 4.6 vs. 11.1 ± 5.3 mmHg, p = 0.03), and PDRICG (10 ± 8.3 vs. 15.9 ± 5.2%, p = 0.02) and APP (43.6 ± 9 vs. 57.9 ± 12.2 mmHg, p
< 0.0001) were significantly lower in non-survivors.
PDRICG is positively correlated to APP and inversely correlated to IAP. Changes in APP are associated with significant concomitant changes in PDRICG, while changes in IAP are associated with opposite changes in PDRICG, suggesting that an increase in IAP may compromise hepatosplanchnic perfusion. Both PDRICG and IAP are correlated with outcome. Measurement of PDRICG may be a useful additional clinical tool to assess the negative effects of increased IAP on liver perfusion and function.
Annals of Intensive Care 12/2012; 2 Suppl 1(Suppl 1):S19. DOI:10.1186/2110-5820-2-S1-S19 · 3.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heme and its breakdown products CO, Fe, and bilirubin are being recognized as signaling molecules or even therapeutic agents, but also exert adverse effects when released at high concentrations. Manipulating the pathway confers protection in rodent sepsis models via both control of free heme and formation of its first and higher-order products. Thus, regulatory elements present in human heme oxygenase 1 (HMOX1) and biliverdin reductases (BLVRA/B) genes might impact outcome. We tested whether a highly polymorphic (GT)n microsatellite and single-nucleotide polymorphisms in HMOX1 and BLVRA/B genes are associated with outcome of sepsis. Two cohorts (n = 430 and 398 patients) with severe sepsis were screened for single-nucleotide polymorphisms and/or the microsatellite by fragment length analysis and genotyping techniques. Heme oxygenase 1 plasma levels were determined in additional patients with severe sepsis (n = 92) by enzyme-linked immunosorbent assay. Based on mean Sepsis-related Organ Failure Assessment scores, patients homozygous for rs2071746 A allele or medium length (GT)n microsatellites of HMOX1 showed higher 28-day mortality (P = 0.047 and P = 0.033) in one cohort compared with other genotypes, whereas 90-day mortality rates showed no association. The T allele was less frequently observed in both cohorts than would be expected according to Hardy-Weinberg equilibrium. Heme oxygenase 1 plasma levels were elevated in septic patients, independent of the genotype. Single-nucleotide polymorphisms within BLVRA/B showed no association with outcome. Short (GT)n repeats that are in linkage disequilibrium with the T allele of rs2071746 in HMOX1 are associated with favorable outcome, whereas no association with gene variants of BLVRA/B, involved in the generation of higher-order metabolites, was noticed.
[Show abstract][Hide abstract] ABSTRACT: Myocardial function is depressed in sepsis and is an important prognosticator in the human condition. Using echocardiography in a long-term, fluid-resuscitated Wistar rat model of fecal peritonitis we investigated whether depressed myocardial function could be detected at an early stage of sepsis and, if so, whether the degree of depression could predict eventual outcome. At 6 hours' post-insult, a stroke volume <0.17ml prognosticated 3-day mortality with positive and negative predictive values of 93% and 80%, respectively. Subsequent fluid loading studies demonstrated intrinsic myocardial depression with poor prognosis animals tolerating less fluid than either good prognosis or sham-operated animals. Cardiac gene expression analysis at 6 hours detected 527 transcripts significantly up- or down-regulated by the septic process, including genes related to inflammatory and cell cycle pathways. Predicted mortality was associated with significant differences in transcripts of genes expressing proteins related to the TLR-2/MyD88 and JAK-STAT inflammatory pathways, b-adrenergic signaling and intracellular calcium cycling. Our findings highlight the presence of myocardial depression in early sepsis and its prognostic significance. Transcriptomic analysis in heart tissue identified changes in signaling pathways that correlated with clinical dysfunction. These pathways merit further study to both better understand and potentially modify the disease process.
[Show abstract][Hide abstract] ABSTRACT: Experimental models, mimicking physiology, and molecular dynamics of diseases in human, harbor the possibility to study the effect of interventions and transfer results from bench to bedside. Recent advances in high-throughput technologies, standardized protocols, and integration of knowledge from databases yielded rising consistency and usability of results for inter-species comparisons. Here, we explored similarities and dissimilarities in gene expression from blood samples of a murine sepsis model (peritoneal contamination and infection, PCI) and patients from the pediatric intensive care unit (PICU) measured by microarrays. Applying a consistent pre-processing and analysis workflow, differentially expressed genes (DEG) from PCI and PICU data significantly overlapped. A major fraction of DEG was commonly expressed and mapped to adaptive and innate immune response related pathways, whereas the minor fraction, including the chemokine (C-C motif) ligand 4, exhibited constant inter-species disparities. Reproducibility of transcriptomic observations was validated experimentally in PCI. These data underline, that inter-species comparison can obtain commonly expressed transcriptomic features despite missing homologs and different protocols. Our findings point toward a high suitability of an animal sepsis model and further experimental efforts in order to transfer results from animal experiments to the bedside.
Frontiers in Microbiology 09/2012; 3:284. DOI:10.3389/fmicb.2012.00284 · 3.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Patients with multiple injuries surviving the initial insult are highly susceptible to secondary pneumonia, frequently progressing into sepsis and multiorgan failure. However, the underlying mechanisms of posttraumatic immunosuppression are poorly understood. We hypothesized that dysregulated p38 mitogen-activated protein kinase (MAPK) signaling accounts for impaired lung protective immunity in a model of trauma/hemorrhage (T/H) and subsequent pneumococcal pneumonia in mice.
C57BL6/N mice were subjected to trauma by midline laparotomy, and T/H was induced by midline laparotomy followed by cannulation of femoral arteries and veins to induce hemorrhage. Subsequently, mice were infected with Streptococcus pneumoniae. In selected experiments, mice were treated with a p38 MAPK inhibitor or vehicle control immediately after induction of T/H.
Mice subjected to T/H showed significantly increased p38 MAPK activation in their lungs, which was accompanied by a reduced Escherichia coli phagocytosis by macrophages from T/H mice in vitro and an impaired pneumococcal killing activity of T/H mice in vivo, overall resulting in increased mortality of T/H mice after infection with S. pneumoniae. Application of p38 MAPK inhibitor BIRB796 immediately after T/H induction improved the bacterial phagocytosis activity of macrophages from T/H mice in vitro and lung pneumococcal killing in vivo but did not improve the survival of T/H mice challenged with S. pneumoniae.
T/H triggers sustained p38 MAPK activation in the lungs of mice, which attenuates lung macrophage antibacterial activities and renders mice more susceptible to pneumococcal pneumonia. However, no major role for dysregulated p38 MAPK to affect survival of T/H mice after pneumococcal challenge was detected, suggesting that dysregulated p38 MAPK activity may possibly play only a limited role in posttraumatic immunosuppression in mice.
[Show abstract][Hide abstract] ABSTRACT: Levels of circulating angiopoietin-2 (Ang-2) increase in sepsis, raising the possibility that Ang-2 acts as a modulator in the sepsis cascade. To investigate this, experimental sepsis was induced in male C57BL6 mice by a multidrug-resistant isolate of Pseudomonas aeruginosa; survival was determined along with neutrophil tissue infiltration and release of proinflammatory cytokines. Survival was significantly increased either by pretreatment with recombinant Ang-2 2 h before or treatment with recombinant Ang-2 30 min after bacterial challenge. Likewise, Ang-2 pretreatment protected against sepsis-related death elicited by Escherichia coli; however, Ang-2 failed to provide protection in lipopolysaccharide (LPS)-challenged mice. The survival advantage of Ang-2 in response to P. aeruginosa challenge was lost in tumor necrosis factor (TNF)-deficient mice or neutropenic mice. Infiltration of the liver by neutrophils was elevated in the Ang-2 group compared with saline-treated animals. Serum TNF-α levels were reduced by Ang-2, whereas those of interleukin (IL)-6 and IL-10 remained unchanged. This was accompanied by lower release of TNF-α by stimulated splenocytes. When applied to U937 cells in vitro, heat-killed P. aeruginosa induced the secretion of IL-6 and TNF-α; low levels of exogenous TNF-α synergized with P. aeruginosa. This synergistic effect was abolished after the addition of Ang-2. These results put in evidence a striking protective role of Ang-2 in experimental sepsis evoked by a multidrug-resistant isolate of P. aeruginosa attributed to modulation of TNF-α production and changes in neutrophil migration. The protective role of Ang-2 is shown when whole microorganisms are used and not LPS, suggesting complex interactions with the host immune response.
Journal of Pharmacology and Experimental Therapeutics 08/2012; 343(2):278-87. DOI:10.1124/jpet.112.195180 · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Applying intravital fluorescence microscopy, we assessed sinusoidal delivery and biliary clearance of two different polymethine dyes. DY635, a benzopyrylium-based hemocyanine dye with shorter excitation wavelength than indocyanine green (ICG), was validated for assessment of hepatic excretory function. Decrease of DY635 and ICG reflecting transcellular transport was 83 ± 4% (DY635) and 14 ± 2% (ICG; p < 0.05) over 35 minutes, respectively. In cholestasis, hepatobiliary excretion of DY635 was markedly impaired (control 3176 ± 148 pmol vs. cholestatic 1929 ± 179 pmol; p < 0.05). DY635 even enabled an analysis at high resolution suggesting 1.) hepatocyte uncoupling and 2.) failure of primarily the canalicular pole, allowing in vivo insights into molecular mechanisms of this critical facet of hepatobiliary function.
Journal of Biophotonics 07/2012; 5(7):571-81. DOI:10.1002/jbio.201100118 · 4.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The first response to infection in the blood is mediated by leukocytes. As a result crucial information can be gained from a hemogram. Conventional methods such as blood smears and automated sorting procedures are not capable of recording detailed biochemical information of the different leukocytes. In this study, Raman spectroscopy has been applied to investigate the differences between the leukocyte subtypes which have been obtained from healthy donors. Raman imaging was able to visualize the same morphological features as standard staining methods without the need of any label. Unsupervised statistical methods such as principal component analysis and hierarchical cluster analysis were able to separate Raman spectra of the two most abundant leukocytes, the neutrophils and lymphocytes (with a special focus on CD4(+) T-lymphocytes). For the same cells a classification model was built to allow an automated Raman-based differentiation of the cell type in the future. The classification model could achieve an accuracy of 94% in the validation step and could predict the identity of unknown cells from a completely different donor with an accuracy of 81% when using single spectra and with an accuracy of 97% when using the majority vote from all individual spectra of the cell. This marks a promising step toward automated Raman spectroscopic blood analysis which holds the potential not only to assign the numbers of the cells but also to yield important biochemical information.