The Determination of Carbohydrates by High-Performance Anion-Exchange Chromatography Coupled with Pulsed Amperometric Detection (HPAEC-PAD).

Chromatography techniques are widely used to separate, identify, and quantify molecules depending on their physicochemical properties. Standard methods range from simple size exclusion to separation based on affinity or ion exchange. Here, we present a method for the direct analysis of carbohydrates in Magnaporthe oryzae using high-performance anion-exchange chromatography (HPAEC) coupled with pulsed amperometric detection (PAD). The combination of HPAEC with PAD provides the highest selectivity and sensitivity with minimal sample preparation and cleanup time. Utilizing our HPAEC-PAD approach, we obtain reliable and highly reproducible determination of carbohydrates produced as osmotic stress response by M. oryzae. Thus, the method described provides a fast, precise, and comprehensive analysis of stress-dependent metabolic adjustments of carbohydrates not only relevant for M. oryzae but also applicable in other systems.

[Robot-assisted Mediastinal Mass Resection]. / Roboterassistierte Thoraxchirurgie bei Mediastinaltumoren.

In recent years, robot-assisted thoracic surgery is gaining more and widespread interest in Europe. Due to the narrow space and the complexity of anatomical structures, conventional minimally invasive mediastinal surgery may be challenging for the thoracic surgeon. Robot-assisted mediastinal surgery opens up new possibilities for minimally invasive surgery, as it permits greater dexterity, a three-dimensional view, and tremor adjustment, which allows the surgeon to perform complex procedures in small thoracic spaces. As robotic platforms continue to evolve, more complex mediastinal thoracic surgical interventions will be facilitated, translating to improved outcomes for patients. This article provides an overview of the current status of robot-assisted mediastinal surgery and summarises general aspects of the indication, set-up and steps of robot-assisted thoracoscopic surgery in mediastinal mass resections.

Rapid adaptation of signaling networks in the fungal pathogen Magnaporthe oryzae.

BACKGROUND: One fundamental question in biology is how the evolution of eukaryotic signaling networks has taken place. "Loss of function" (lof) mutants from components of the high osmolarity glycerol (HOG) signaling pathway in the filamentous fungus Magnaporthe oryzae are viable, but impaired in osmoregulation. RESULTS: After long-term cultivation upon high osmolarity, stable individuals with reestablished osmoregulation capacity arise independently from each of the mutants with inactivated HOG pathway. This phenomenon is extremely reproducible and occurs only in osmosensitive mutants related to the HOG pathway - not in other osmosensitive Magnaporthe mutants. The major compatible solute produced by these adapted strains to cope with high osmolarity is glycerol, whereas it is arabitol in the wildtype strain. Genome and transcriptome analysis resulted in candidate genes related to glycerol metabolism, perhaps responsible for an epigenetic induced reestablishment of osmoregulation, since these genes do not show structural variations within the coding or promotor sequences. CONCLUSION: This is the first report of a stable adaptation in eukaryotes by producing different metabolites and opens a door for the scientific community since the HOG pathway is worked on intensively in many eukaryotic model organisms.

Dectin-1 is expressed in human lung and mediates the proinflammatory immune response to nontypeable Haemophilus influenzae.

UNLABELLED: The C-type lectin receptor Dectin-1 is expressed mainly on myeloid cells mediating the immune response targeting respiratory pathogens such as Aspergillus fumigatus and Mycobacterium tuberculosis. The pulmonary epithelium serves as an important interface for interactions between these pathogens and the respiratory tract. Therefore, we analyzed the expression pattern of Dectin-1 in the human lung. Immunohistochemically stained human lung sections from 17 out of 19 individuals were positive for Dectin-1, which was expressed mainly apically on bronchial and alveolar epithelium. Our results showed no correlation with chronic obstructive pulmonary disease (COPD) or the smoking habits of the patients. Nontypeable Haemophilus influenzae (NTHI), an important bacterial pathogen of the respiratory tract with significant importance in COPD, has also been proposed to be recognized by Dectin-1, suggesting a possible impact on the NTHI-dependent immune response in human airways. Therefore, the involvement of Dectin-1 in NTHI-triggered cytokine responses was investigated in primary normal human bronchial epithelial (NHBE) cells and in the A549 cell line stably transfected with Dectin-1. The presence of Dectin-1 significantly increased cytokine release in response to NTHI in NHBE and A549 cells. In addition, phosphorylation of the Dectin-1 hem-immunoreceptor tyrosine-based activation motif (hemITAM) was essential for the Dectin-1-triggered response to NTHI in A549 cells. In conclusion, in human airways, epithelium-expressed Dectin-1 may play a significant role in generating an NTHI-mediated, proinflammatory immune response. IMPORTANCE: In this study, we demonstrated, for the first time, the expression of Dectin-1 on human lung tissues and, in particular, pulmonary epithelium by making use of immunohistochemical staining. The epithelial lining of the human airways is an important interface for host-pathogen interactions. Therefore, our data suggest that epithelium-expressed Dectin-1 is of considerable importance for the interaction of the human airways with pathogens detected by this receptor, such as A. fumigatus and M. tuberculosis. Moreover, we further demonstrated that, in pulmonary epithelial cells, Dectin-1 enhances the proinflammatory immune response to NTHI. In COPD patients, NTHI is a major cause of respiratory tract infections and is associated with proinflammatory immune responses in the lower airways. Therefore, our data suggest that the functional interaction of Dectin-1 with NTHI in human airways may have an important impact on the pathogenesis of COPD.

Carcinoembryonic antigen (CEA)-related cell adhesion molecules are co-expressed in the human lung and their expression can be modulated in bronchial epithelial cells by non-typable Haemophilus influenzae, Moraxella catarrhalis, TLR3, and type I and II interferons.

BACKGROUND: The carcinoembryonic antigen (CEA)-related cell adhesion molecules CEACAM1 (BGP, CD66a), CEACAM5 (CEA, CD66e) and CEACAM6 (NCA, CD66c) are expressed in human lung. They play a role in innate and adaptive immunity and are targets for various bacterial and viral adhesins. Two pathogens that colonize the normally sterile lower respiratory tract in patients with chronic obstructive pulmonary disease (COPD) are non-typable Haemophilus influenzae (NTHI) and Moraxella catarrhalis. Both pathogens bind to CEACAMs and elicit a variety of cellular reactions, including bacterial internalization, cell adhesion and apoptosis. METHODS: To analyze the (co-) expression of CEACAM1, CEACAM5 and CEACAM6 in different lung tissues with respect to COPD, smoking status and granulocyte infiltration, immunohistochemically stained paraffin sections of 19 donors were studied. To address short-term effects of cigarette smoke and acute inflammation, transcriptional regulation of CEACAM5, CEACAM6 and different CEACAM1 isoforms by cigarette smoke extract, interferons, Toll-like receptor agonists, and bacteria was tested in normal human bronchial epithelial (NHBE) cells by quantitative PCR. Corresponding CEACAM protein levels were determined by flow cytometry. RESULTS: Immunohistochemical analysis of lung sections showed the most frequent and intense staining for CEACAM1, CEACAM5 and CEACAM6 in bronchial and alveolar epithelium, but revealed no significant differences in connection with COPD, smoking status and granulocyte infiltration. In NHBE cells, mRNA expression of CEACAM1 isoforms CEACAM1-4L, CEACAM1-4S, CEACAM1-3L and CEACAM1-3S were up-regulated by interferons alpha, beta and gamma, as well as the TLR3 agonist polyinosinic:polycytidylic acid (poly I:C). Interferon-gamma also increased CEACAM5 expression. These results were confirmed on protein level by FACS analysis. Importantly, also NTHI and M. catarrhalis increased CEACAM1 mRNA levels. This effect was independent of the ability to bind to CEACAM1. The expression of CEACAM6 was not affected by any treatment or bacterial infection. CONCLUSIONS: While we did not find a direct correlation between CEACAM1 expression and COPD, the COPD-associated bacteria NTHi and M. catarrhalis were able to increase the expression of their own receptor on host cells. Further, the data suggest a role for CEACAM1 and CEACAM5 in the phenomenon of increased host susceptibility to bacterial infection upon viral challenge in the human respiratory tract.

Analysis of the airway microbiota of healthy individuals and patients with chronic obstructive pulmonary disease by T-RFLP and clone sequencing.

UNLABELLED: Chronic obstructive pulmonary disease (COPD) is a progressive, inflammatory lung disease that affects a large number of patients and has significant impact. One hallmark of the disease is the presence of bacteria in the lower airways. OBJECTIVE: The aim of this study was to analyze the detailed structure of microbial communities found in the lungs of healthy individuals and patients with COPD. Nine COPD patients as compared and 9 healthy individuals underwent flexible bronchoscopy and BAL was performed. Bacterial nucleic acids were subjected to terminal restriction fragment (TRF) length polymorphism and clone library analysis. Overall, we identified 326 T-RFLP band, 159 in patients and 167 in healthy controls. The results of the TRF analysis correlated partly with the data obtained from clone sequencing. Although the results of the sequencing showed high diversity, the genera Prevotella, Sphingomonas, Pseudomonas, Acinetobacter, Fusobacterium, Megasphaera, Veillonella, Staphylococcus, and Streptococcus constituted the major part of the core microbiome found in both groups. A TRF band possibly representing Pseudomonas sp. monoinfection was associated with a reduction of the microbial diversity. Non-cultural methods reveal the complexity of the pulmonary microbiome in healthy individuals and in patients with COPD. Alterations of the microbiome in pulmonary diseases are correlated with disease.

Metastatic adrenocortical carcinoma: results of 56 pulmonary metastasectomies in 24 patients.

BACKGROUND: Surgical resection is an important form of treatment for metastatic disease in patients with adrenocortical carcinoma (ACC). However, data about the results of this treatment are sparse. We reviewed our experience with the resection of pulmonary lesions metastatic from ACC as a means of evaluating such results. METHODS: A retrospective review of the database at a German national registry for ACC identified 24 patients (9 men and 15 women; median age, 41 years) who underwent pulmonary metastasectomy for primary ACC during the study period of 1989 through 2009. Only patients who met the criteria for potentially curative surgery, defined as the presumed feasibility of resecting all visualized tumorous lesions, were included. RESULTS: No perioperative deaths occurred in 56 pulmonary metastasectomies done on the patients in the study. The overall cumulative rate of 5-year survival, calculated from the time of first pulmonary surgery, was 24.5%, and the median survival was 50.2 months. Age younger than 41 years at the time of first pulmonary metastasectomy and repeated pulmonary metastasectomy were associated with longer survival in a univariate analysis. In accord with this, we observed a median survival of 31.9 months in patients 41 years of age or older as compared with a median survival of 59.3 months in younger patients (p = 0.004). In patients with repeated pulmonary metastasectomies, median survival after the first resection was significantly longer, at 59.3 months than in patients who had only one pulmonary resection, whose median survival was 31.9 months (p = 0.001). CONCLUSIONS: We conclude that surgical resection of pulmonary metastases for ACC should be regarded as safe, with the potential for producing long-term survival in a highly selected group of patients. Younger patients may benefit more than older ones from such resection, and the recurrence of pulmonary metastases should not preclude repeated surgical resections of these lesions.