ABSTRACT
Sleeve resections of the lungs have affected the oncologic radicality, parenchyma and lung function-saving resections and extended the indications for operations in thoracic surgery. Whenever lung amputations can be avoided by bronchoplastic and/or angioplastic procedures with the same radicality, sleeve resection should be performed. In centrally located distinct malignomas, intraluminal tumor growth (T3) infiltrations of peribronchial or extrabronchial areas, the lobular ostia and the pulmonary artery (T2/T3) as well as lymph node involvement (N1/N2), these procedures give a better qualitative survival and lower morbidity and mortality rates. Broncoscope-guided localization of a double lumen tube and routine anesthesia monitoring are mandatory. Before performing sleeve resections a complete lymph node dissection should be done without denuding the area of the anastomosis and sparing the bronchial arteries. Preoperative endoscopic biopsies, knowledge of the topography and mobilization of the vascular and bronchial tree, subtile operation techniques, perioperative and postoperative videobronchoscopic guidance as well as intraoperative frozen sections and a tension-free and smooth anastomosis, avoid postoperative complications. Depending on the blood supply of the bronchial tree a vascularized flap is indicated. Operability can therefore be achieved in elderly patients with limited pulmonary function, particularly those under adjuvant or neoadjuvant therapy who are no longer suitable for pneumonectomy.
Subject(s)
Bronchi/surgery , Lung Neoplasms/surgery , Organ Sparing Treatments/methods , Pneumonectomy/methods , Pulmonary Artery/surgery , Pulmonary Veins/surgery , Age Factors , Aged , Anastomosis, Surgical/methods , Biopsy , Bronchi/blood supply , Bronchi/pathology , Bronchoscopy/methods , Humans , Lung/pathology , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Lymphatic Metastasis/pathology , Monitoring, Intraoperative/methods , Neoplasm Invasiveness , Neoplasm Staging , Organ Sparing Treatments/mortality , Pulmonary Artery/pathology , Pulmonary Veins/pathology , Respiratory Function Tests , Survival RateSubject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/therapy , Carcinoma, Small Cell/diagnosis , Carcinoma, Small Cell/therapy , Cooperative Behavior , Interdisciplinary Communication , Lung Neoplasms/diagnosis , Lung Neoplasms/therapy , Pancoast Syndrome/diagnosis , Pancoast Syndrome/therapy , Carcinoma, Non-Small-Cell Lung/pathology , Carcinoma, Non-Small-Cell Lung/prevention & control , Carcinoma, Small Cell/pathology , Carcinoma, Small Cell/prevention & control , Combined Modality Therapy , Early Diagnosis , Follow-Up Studies , Germany , Humans , Lung Neoplasms/pathology , Lung Neoplasms/prevention & control , Neoplasm Staging , Pancoast Syndrome/pathology , Pancoast Syndrome/prevention & control , Societies, MedicalABSTRACT
BACKGROUND: Management of tracheal ruptures in critically ill patients is challenging. Conservative treatment has been described, but in mechanically ventilated patients with distal tracheal ruptures surgical repair might be inevitable. Strategies for diagnosis and treatment of tracheal ruptures and handling of mechanical ventilation remain to be clarified. Our aim was to comprise a structured diagnostic and treatment protocol for patients suspicious of tracheal injury, including detailed principles of mechanical ventilation and specific indications for conservative or surgical treatment. METHODS: Patients with tracheal ruptures were compared in accordance to the need of mechanical ventilation and to indication for surgical repair. In patients suffering from tracheal ruptures affecting the whole tracheal wall and with protrusion of mediastinal structures into the lumen surgery was indicated. We compared ventilatory, hemodynamic and clinical parameters between the different patient groups. We report our structured approach in diagnostics and treatment of tracheal ruptures and place special emphasis on respiratory management. RESULTS: Seventeen patients with tracheal rupture were identified. In 8 patients surgical repair was performed 1.8±1.5 days after diagnosis. Previous to surgery, ventilation parameters improved significantly: plateau pressure decreased, percentage of assisted spontaneous breathing increased and compliance improved. Conservative treatment was successful in long-term ventilated patients (13.7±8 days) even when suffering from distal lesions. CONCLUSION: Invasiveness of mechanical ventilation and obstruction of tracheal lumen might indicate conservative or surgical treatment strategies in long-term ventilated patients suffering from iatrogenic tracheal rupture. Indications for surgical repair remain to be further clarified.
Subject(s)
Trachea/injuries , Wounds and Injuries/diagnosis , Wounds and Injuries/therapy , Adult , Aged , Aged, 80 and over , Critical Illness , Extracorporeal Membrane Oxygenation , Female , Hemodynamics/physiology , Humans , Iatrogenic Disease , Male , Middle Aged , Respiration, Artificial , Respiratory Distress Syndrome/therapy , Retrospective Studies , Rupture , Trachea/surgery , Young AdultSubject(s)
Aftercare , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/prevention & control , Carcinoma, Non-Small-Cell Lung/therapy , Evidence-Based Medicine , Lung Neoplasms/diagnosis , Lung Neoplasms/prevention & control , Lung Neoplasms/therapy , Carcinoma, Non-Small-Cell Lung/etiology , Combined Modality Therapy , Cooperative Behavior , Cross-Sectional Studies , Early Diagnosis , Follow-Up Studies , Germany , Humans , Incidence , Interdisciplinary Communication , Lung Neoplasms/etiology , Neoplasm Staging , Palliative Care , Patient Care Team , Prognosis , Risk Factors , Survival RateABSTRACT
The development of surgery in Germany was correct and to the point. General surgery was the field from which all specialization grew. Surgical fields today must comply with scientific demands and medical progress. They can remain successful only if the various branches respond to developments in respect to minimum capacities, procedural and structural conditions, and specialized education and training. "General surgery" as a basic foundation provides an important cross-section of the eight-pronged model of the various specializations. As such it is able to deliver effectively a broad spectrum of good patient care. The network of thoracal surgical centers guarantees sufficient and efficient emergent care, because the more beams, the stronger is the roof. At the same time we must remain responsive to developments in the European Union as a whole. Determination and a progressive view by all surgical societies allow the kind of structuring that will guarantee the future of general surgery. We must inspire our new trainees!
Subject(s)
General Surgery/trends , Thoracic Surgery/trends , Curriculum/trends , Education, Medical, Continuing/trends , Education, Medical, Graduate/trends , European Union , Forecasting , General Surgery/education , Germany , Health Services Needs and Demand/trends , Humans , Societies, Medical/trends , Surgicenters/trends , Thoracic Surgery/educationABSTRACT
Even though the lung represents a special immune compartment with the capacity of a high inflammatory response, ineffective anti-tumour immunity is common in lung-associated malignancies. We asked whether a differential composition of the immune cell infiltrate in malignant (MLTAs) and non-malignant lung tissue areas (N-MLTAs) exists and might potentially contribute to this effect. We performed a comparative analysis of immune cells residing in MLTAs and N-MLTAs of non-small cell lung cancer (NSCLC) patients. To this end, we used immunophenotyping and functional analyses on directly isolated immune cells and tissue arrays on archived paraffin-embedded specimens. A strong T cell infiltration was prominent in both tissue compartments whereas CD4(+)CD25(+)CD127(-) T regulatory cells were present in MLTAs only. Nonetheless, concurrent functional ex vivo T cell analyses revealed no significant difference between T cells of MLTA and N-MLTA, suggesting that tumour-infiltrating T cells were not functionally impaired. Interestingly, T cell infiltration was less pronounced in specimens with a high neutrophilic infiltrate. NK cell infiltration was strikingly heterogenous between MLTA and N-MLTA. While NK cells were almost absent in the malignant tissue regions, non-malignant counterparts were selectively populated by NK cells and those NK cells showed strong cytotoxic activity ex vivo. We report that malignant and non-malignant tissue areas in NSCLC are selectively infiltrated by certain immune cell types with NK cells being displaced from the tumour tissue. These phenomena have important implications for tumour immunology of NSCLC and should be considered for the development of future immunologic intervention therapies.
Subject(s)
Carcinoma, Non-Small-Cell Lung/immunology , Killer Cells, Natural/physiology , Lung Neoplasms/immunology , Lung/immunology , Lymphocytes, Tumor-Infiltrating/physiology , T-Lymphocytes, Regulatory/physiology , Aged , Carcinoma, Non-Small-Cell Lung/pathology , Female , Humans , Lung/pathology , Lung Neoplasms/pathology , Male , Middle Aged , Receptors, Immunologic/physiology , Receptors, Natural Killer CellABSTRACT
Intensive care in thoracic surgery requires highly specialised abilities and knowledge in addition to usual intensive care standards. The operative chest intensivist must be experienced in special surgical methods, interventional bronchology, and tube management. Cooperating with chest physicians and anaesthesiologists, he takes the central position in intensive therapy. This is described in detail and explained by examples. Furthermore, aspects of education in this specialty will be emphasised.
Subject(s)
Critical Care , Postoperative Complications/therapy , Thoracic Surgical Procedures , Bronchoscopy , Drainage , Empyema, Pleural/diagnosis , Empyema, Pleural/etiology , Empyema, Pleural/therapy , Humans , Patient Care Team , Pneumonectomy , Postoperative Complications/diagnosis , Postoperative Hemorrhage/diagnosis , Postoperative Hemorrhage/etiology , Postoperative Hemorrhage/therapy , Shock, Septic/diagnosis , Shock, Septic/etiology , Shock, Septic/therapy , Specialization , Surgical Wound Infection/diagnosis , Surgical Wound Infection/etiology , Surgical Wound Infection/therapyABSTRACT
BACKGROUND: The loss of alveolar walls is a hallmark of emphysema. As fibroblasts play an important role in the maintenance of alveolar structure, a change in fibroblast phenotype could be involved in the pathogenesis of this disease. In a previous study we found a reduced in vitro proliferation rate and number of population doublings of parenchymal lung fibroblasts from patients with emphysema and we hypothesized that these findings could be related to a premature cellular aging of these cells. In this study, we therefore compared cellular senescence markers and expression of respective genes between lung fibroblasts from patients with emphysema and control patients without COPD. METHODS: Primary lung fibroblasts were obtained from 13 patients with moderate to severe lung emphysema (E) and 15 controls (C) undergoing surgery for lung tumor resection or volume reduction (n = 2). Fibroblasts (8E/9C) were stained for senescence-associated beta-galactosidase (SA-beta-Gal). In independent cultures, DNA from lung fibroblasts (7E/8C) was assessed for mean telomere length. Two exploratory 12 k cDNA microarrays were used to assess gene expression in pooled fibroblasts (3E/3C). Subsequently, expression of selected genes was evaluated by quantitative PCR (qPCR) in fibroblasts of individual patients (10E/9C) and protein concentration was analyzed in the cell culture supernatant. RESULTS: The median (quartiles) percentage of fibroblasts positive for SA-beta-Gal was 4.4 (3.2;4.7) % in controls and 16.0 (10.0;24.8) % in emphysema (p = 0.001), while telomere length was not different. Among the candidates for differentially expressed genes in the array (factor > or = 3), 15 were upregulated and 121 downregulated in emphysema. qPCR confirmed the upregulation of insulin-like growth factor-binding protein (IGFBP)-3 and IGFBP-rP1 (p = 0.029, p = 0.0002), while expression of IGFBP-5, -rP2 (CTGF), -rP4 (Cyr61), FOSL1, LOXL2, OAZ1 and CDK4 was not different between groups. In line with the gene expression we found increased cell culture supernatant concentrations of IGFBP-3 (p = 0.006) in emphysema. CONCLUSION: These data support the hypothesis that premature aging of lung fibroblasts occurs in emphysema, via a telomere-independent mechanism. The upregulation of the senescence-associated IGFBP-3 and -rP1 in emphysema suggests that inhibition of the action of insulin and insulin-like growth factors could be involved in the reduced in vitro-proliferation rate.
Subject(s)
Cellular Senescence , Emphysema/pathology , Fibroblasts/pathology , Lung/pathology , Biomarkers , Emphysema/genetics , Emphysema/physiopathology , Emphysema/surgery , Gene Expression Regulation , Humans , In Vitro Techniques , Lung/cytology , Lung/physiology , Lung/physiopathology , Lung Neoplasms/pathology , Lung Neoplasms/surgery , Polymerase Chain Reaction , Pulmonary Alveoli/pathology , Reference Values , Respiratory Function Tests , Telomere/pathology , Telomere/ultrastructure , beta-Galactosidase/metabolismABSTRACT
Invasive pulmonary aspergillosis is a life-threatening disease, developing from infection mostly by Aspergillus fumigatus. It is an opportunistic infection and occurs mostly in immunosuppressed patients, after tuberculosis, in patients with AIDS or malignomas. The most common symptom and complication is hemoptysis up to massive bleeding. Surgical treatment is obligatory on patients with a pulmonary aspergilloma. Time and form of intervention should be discussed between surgeon and pulmonologist. Resection should follow anatomical borders like lobectomy or pneumectomy. Any remaining intrathoracic cavities should be filled either with remaining lung tissue or with muscle flaps of latissimus or pectoralis muscle or omentum majus. Following these rules, intervention can be performed with reasonable mortality and morbidity rate.
Subject(s)
Aspergillosis/surgery , Aspergillus fumigatus , Lung Diseases, Fungal/surgery , Adolescent , Adult , Aged , Aspergillosis/diagnosis , Aspergillosis/microbiology , Aspergillosis/mortality , Female , Humans , Lung Diseases, Fungal/diagnosis , Lung Diseases, Fungal/microbiology , Lung Diseases, Fungal/mortality , Male , Middle AgedABSTRACT
Aspiration of foreign bodies is extremely rare in adults, but it may occur during the course of dental treatment and become a serious problem for the patient. A case of a 77-year old man with chronic cough after dental treatment and unrecognised aspiration of an implant is presented. The implant remained unidentified for three years as an endobronchial foreign body of the lower lobe bronchus. A bronchoscopic removal failed and surgery was indicated for lobectomy of the right lower lobe because of chronic pneumonia. A review of prevention, signs and symptoms, management and documentation of the complications is presented.
Subject(s)
Dental Implants , Foreign Bodies , Pneumonia, Aspiration/surgery , Aged , Humans , MaleABSTRACT
Emphysema is characterised by a loss of alveolar structure, as reflected in elastic recoil and gas exchange. As fibroblasts play a key role in the maintenance of structure, the current authors hypothesised that their proliferation might be constitutively impaired in lung emphysema. Using explant cultures, lung fibroblasts were obtained from resected lungs of 10 patients with emphysema (median forced expiratory volume in one second (FEV1) 40% predicted) and 10 control patients (FEV1, 95% pred). The doubling time (DT) was measured over 4 days under standard conditions (10% foetal calf serum) prior and after cryopreservation. Additionally, in seven samples per group the total population doubling level (PDL) was determined. In emphysema, mean+/-sem DT was 33.6+/-2.8 h compared with 24.8+/-1.4 h in controls. The differences in DT were preserved after cryopreservation. Groups also differed in the initial slope of the PDL plot during long-term culture (up to 35 days). However, the median (range) maximum PDL did not differ significantly between groups (13.8 (7.4-22.6) versus 20.2 (11.2-25.5)). The current authors, therefore, suggest that the reduced proliferation rate in vitro of lung fibroblasts from patients with emphysema reflects a persistent, intrinsic failure of cellular replacement and maintenance in this disease, possibly in relation to pre-term aging.
Subject(s)
Fibroblasts/physiology , Pulmonary Emphysema/physiopathology , Aged , Cell Culture Techniques , Cell Proliferation , Female , Humans , Lung/physiopathology , Male , Middle AgedABSTRACT
In contrast to the spectrum of biochemical analyses of fresh material, that of archived specimens is widely restricted. Fixation of specimens with formalin, the most commonly used fixative, usually prevents further molecular analysis, since it leads to degradation of nucleic acids and denaturation of the antigenic determinants of proteins. To overcome these problems, the Hepes-glutamic acid buffer mediated Organic solvent Protection Effect (HOPE)-fixation technique has been developed, which preserves nucleic acids and antigenic determinants of proteins, thus expanding the applicability of immunohistochemical methods. In this study, we investigated whether HOPE-fixed tissue can be analyzed by Western blotting. Furthermore, a comparison with conventionally fixed and frozen material was made. The specimens used were tumor-free and obtained from lobectomies for lung cancer. All four antibodies tested, i.e., antibodies specific for focal adhesion kinase, surfactant protein A, PI-3-kinase, and IKKalpha, worked well if used for immunoblotting of HOPE-fixed and frozen tissue. By contrast, these antibodies showed no or only very weak specific binding if formalin-fixed specimens were analyzed. Our findings show that HOPE fixation maintains the antigenicity of proteins better than formalin fixation. The possibility for performing Western blotting with archived paraffin-embedded specimens extends the options for diagnostic and scientific analyses of fixed tissues.
Subject(s)
Blotting, Western/methods , Fixatives , Paraffin Embedding , Tissue Fixation/methods , Cryopreservation , Humans , Lung/chemistry , Lung/metabolism , Proteins/analysis , Proteins/metabolism , Sensitivity and Specificity , Time FactorsABSTRACT
Indirect assessments have shown a superior lung deposition of HFA-BDP (Ventolair/Qvar) compared to CFC-BDP (Aerobec). The aim of this study was to assess the concentrations of BDP and its metabolite 17-BMP in airways and peripheral tissue from resected lung specimens after inhalation of these BDP formulations. Immediately prior to surgery for lung cancer, 10 patients inhaled 1000 microg of either CFC-BDP (n = 5) or HFA-BDP (n = 5) Mouthwash was collected after inhalation, and serum before, during, and after surgery. There was no significant difference between CFC and HFA in the concentration of 17-BMP in bronchi (median, 4365 vs 4121 pg/g tissue). After CFC, concentrations of 17-BMP were lower in peripheral tissue (1424 vs 2089 pg/g; ANCOVA, p = 0.001) and in serum taken immediately after inhalation (688 vs 1219 pg/ml, p < 0.01). Furthermore, the CFC group showed a higher concentration of BDP in the mouthwash (17,660 vs 1320 ng/ml, p < 0.05), but the concentration of 17-BMP was lower (452 vs 1028 ng/ml, n.s.). These findings indicate a predominantly peripheral deposition of HFA-BDP, in line with previous data. They also provide evidence for a faster uptake and metabolism of HFA-BDP, probably because BDP is dissolved in HFA and has a smaller particle size distribution than the CFC suspensions.
Subject(s)
Aerosol Propellants/chemistry , Beclomethasone/analogs & derivatives , Beclomethasone/metabolism , Beclomethasone/pharmacokinetics , Chlorofluorocarbons/chemistry , Glucocorticoids/pharmacology , Hydrocarbons, Fluorinated/chemistry , Administration, Inhalation , Adult , Aged , Beclomethasone/administration & dosage , Beclomethasone/analysis , Bronchi/chemistry , Drug Carriers , Female , Glucocorticoids/administration & dosage , Glucocorticoids/analysis , Humans , Lung/chemistry , Male , Middle Aged , Particle Size , Time Factors , Tissue DistributionABSTRACT
UNLABELLED: Objective of this study was to investigate the balance of surfactant-apoprotein A1 and A2 (SP-A1 and SP-A2) at transcriptional level in tumor and tumor-free lung-tissues of patients with non-small-cell lung-carcinomas. MATERIALS AND METHODS: Nonfixed snap-frozen tumor and tumor-free sections of lungs, excised from 21 patients with adenocarcinomas of the lung and 6 patients with squamous cell carcinomas (control group) were analyzed. This was accomplished by RT-PCR using a SP-A1/SP-A2 consensus sequence, followed by enzymatic restriction. RESULTS: Tumor-containing tissues of adenocarcinomas showed higher SP-A1/SP-A2 ratios than the tumor free tissues. There was a significant difference in expression of SP-A1/SP-A2-mRNA in intra-individual tumor and tumor-free lung-tissues of adenocarcinoma patients (p = 0,05). In cases of squamous cell carcinomas this was not observed. CONCLUSIONS: A novel method to investigate the transcriptional activity of surfactant-apoproteins A1 and A2 in snap-frozen lung tissues was established. It was shown, that adenocarcinoma-tissues display higher SP-A1/SP-A2 than the corresponding tumor-free tissues and that the variation of SP-A-mRNA expression rises in cases of higher tumor-grading.
Subject(s)
Carcinoma, Non-Small-Cell Lung/genetics , Lung Neoplasms/genetics , Lung/metabolism , Pulmonary Surfactant-Associated Protein A/analogs & derivatives , Pulmonary Surfactant-Associated Protein A/genetics , Transcription, Genetic , Adenocarcinoma/genetics , Gene Expression Regulation, Neoplastic/genetics , HumansABSTRACT
In individual cases of tuberculosis surgical therapy is part of an interdisciplinary therapeutic concept. Identifying the optimal point of time for surgery has a strong impact on the further course of the healing process and thus on the prognosis. Indications for surgical therapy are: 1. multi-drug-resistant pulmonary tuberculosis (MDR-TB), 2. late-onset complications (apergilloma, tuberculosis of the tracheobronchial tree or of mediastinal or hilar lymph nodes), 3. tuberculosis of the pleura. In MDR-TB an individual concept for therapy based on all chemotherapeutic options as well as thorough planning of the surgical procedure must be provided. Resections in tuberculotic late-onset complications should be performed in patients with persisting cavernous pulmonary tuberculosis as well as in patients with post tuberculosis complex; main goal is the prevention of tuberculotic relapse. Surgical strategies for tuberculosis of the pleura include the debridement of the pleural cavity in video assisted surgical technique, decortication, partial thoracoplasty with musculoplasty or chest fenestration.
Subject(s)
Patient Selection , Preoperative Care/methods , Risk Assessment/methods , Tuberculosis, Lymph Node/surgery , Tuberculosis, Multidrug-Resistant/surgery , Tuberculosis, Pleural/surgery , Tuberculosis, Pulmonary/surgery , Antitubercular Agents/therapeutic use , Humans , Practice Patterns, Physicians' , Tuberculosis, Lymph Node/drug therapy , Tuberculosis, Lymph Node/etiology , Tuberculosis, Multidrug-Resistant/complications , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Pleural/complications , Tuberculosis, Pleural/drug therapy , Tuberculosis, Pulmonary/complications , Tuberculosis, Pulmonary/drug therapyABSTRACT
To study the role of small airways in the early allergic response (EAR), the method of human precision-cut lung slices (PCLS) was developed and used to examine the bronchoconstriction elicited by passive sensitisation and allergen provocation. Viable human PCLS of 250-microm thickness containing airways <1.5 mm in outer diameter were prepared from lung lobes obtained from lung resection and taken into culture. According to the low release of lactate dehydrogenase and the constant ciliary beat frequency, human PCLS were viable for at least 3 days. Following overnight passive sensitisation with serum from allergic individuals, administration of grass-pollen extract or activating immunoglobulin E antibody resulted in immediate airway contraction that was quantified by videomicroscopy. The extent of the EAR increased with decreasing airway size (outer airway diameter), with the strongest response occurring in the terminal bronchioles. Histamine receptor antagonism was ineffective, and leukotriene or thromboxane receptor antagonism attenuated the early allergic response only in some cases. However, simultaneous blockade of leukotriene and thromboxane receptors almost completely prevented the early allergic response in the precision-cut lung slices from all individuals, suggesting such a dual treatment as a potential future asthma therapy.
Subject(s)
Bronchi/immunology , Bronchial Provocation Tests , Bronchoconstriction/immunology , Culture Techniques/methods , Hypersensitivity/immunology , Immunization, Passive , Lung/immunology , Acetates/pharmacology , Anti-Allergic Agents , Anti-Asthmatic Agents/pharmacology , Bridged Bicyclo Compounds, Heterocyclic , Bronchi/drug effects , Bronchi/ultrastructure , Bronchoconstriction/drug effects , Cilia/drug effects , Cilia/immunology , Cilia/ultrastructure , Cyclopropanes , Fatty Acids, Unsaturated , Humans , Hydrazines/pharmacology , Lung/drug effects , Lung/ultrastructure , Quinolines/pharmacology , Reaction Time/drug effects , Reaction Time/immunology , Sulfides , Time Factors , Triprolidine/pharmacologyABSTRACT
We report the use of HOPE-fixation (HOPE = Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) for specimens utilized for in situ hybridization targeting mRNA. For this purpose, an optimized protocol was developed and repeatedly tested on HOPE-fixed lung specimens. We observed that neither pretreatment, permeabilizing the cells, nor prehybridization is necessary to generate signals. After deparaffinizing, the random primed digoxigenin-labeled probes are directly hybridized together with yeast tRNA for blocking unspecific signals. Detection was performed using anti digoxigenin antibodies conjugated with alkaline phosphatase and new-fuchsine or NBT/BCIP as substrates. The results were verified by RT-PCR and adequate negative controls. Signals for human surfactant protein-A and interferon-gamma-inducible protein-10 developed rapidly within 10 min, accompanied by high signal intensities comparable to those observed in immunohistochemistry. Signal enhancement by biotinyl-tyramide, although giving suitable results as well, did not lead to higher signal intensities, and thus was not necessary in conjunction with the probes tested so far. These experiments were performed with material stored under appropriate conditions (at +4 degrees C) up to five years. To sum up, these initial results, obtained with the novel HOPE-fixative, are promising as regards the enhancement of the capabilities of in situ hybridization in the future.
Subject(s)
In Situ Hybridization/methods , Lung/metabolism , Proteolipids/metabolism , Pulmonary Surfactants/metabolism , Tissue Fixation/methods , Cells, Cultured , Cross-Linking Reagents/chemistry , Humans , Paraffin Embedding , Proteolipids/genetics , Pulmonary Surfactant-Associated Proteins , Pulmonary Surfactants/genetics , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Transcription, GeneticABSTRACT
Ro 25-1553 is a metabolically stable analogue of endogenous vasoactive intestinal polypeptide (VIP). This compound is a potent bronchodilator in vitro as well as in vivo. Moreover, Ro 25-1553 has been shown to be highly selective of the VPAC2 receptor. We assessed the effect of Ro 25-1553 on isolated human bronchi and pulmonary arteries in vitro. Macroscopically normal human airways and pulmonary arteries were obtained from patients undergoing surgery for lung cancer. The relaxing capability of Ro 25-1553 on bronchial and pulmonary artery tone was measured using standard techniques. Bronchial rings were pre-contracted with 0.1 mM histamine, and tone in pulmonary artery rings was induced with 10 microM PGF2alpha. Increasing concentrations of Ro 25-1553 within a range of 1 pM to 10 microM were added and isometric tension changes were recorded. Ro 25-1553 caused a concentration-dependent relaxation of airway and pulmonary artery preparations, with an EC50 of approximately 10 nM and a maximal relaxation of 70%-75% of the induced tone. The presence of VPAC2 receptors in the two tissues, though low in density, was confirmed by in situ hybridization, immunocytochemistry and ligand binding. These findings indicate that the VIP analogue Ro 25-1553 may be useful in the treatment of asthma and/or chronic obstructive pulmonary diseases.