Detection of cancer at an early stage is pivotal for successful treatment and long term survival, yet early diagnosis requires sensitive and specific markers that can be easily detected by screening procedures. Differences in the surface structure of tumor and healthy cells, if sufficiently pronounced and discernible, may serve that purpose. We analyzed the luminal surface of healthy and neoplastic human colorectal tissues for the presence and architecture of the glycocalyx a dense network of highly glycosylated proteins using transmission electron microscopy. The ultrastructural analyses showed that 93% of healthy mucosae were covered by an intact glycocalyx. Contrarily, on over 90% of the surface of neoplastic cells the glycocalyx was absent. The sensitivity and specificity of our marker "absence of a glycocalyx" are excellent, being 91% (83-96%) and 96% (89-99%) for adenocarcinomas and 94% (73-100%) and 92% (85-97%) for precancerous polyps (means and 95% confidence intervals). Using a cell culture model we could demonstrate that a particulate probe targeting a cell surface receptor usually concealed beneath the glycocalyx can bind selectively to glycocalyx-free areas of a tumor cell layer. We propose that the absence of a glycocalyx may serve as novel type of tumor marker. If the absence of the glycocalyx can be detected e.g. via binding of imaging probes to non-shielded surface receptors of anomalously differentiated cells, this tumor marker could be used to enable early diagnosis of colorectal cancer.

With regard to the growing number of targeted therapies in lung cancer, a specific and reliable sub-differentiation is of crucial importance. Without discriminating adenocarcinomas from squamous cell carcinomas, molecular based diagnostics that e.g. target certain mutations in the EGFR gene are not applicable and hence, the patients cannot profit from the new therapies. To generate an antibody that, reliably detects adenocarcinomas of the lung in addition to the established markers, we immunized mice with primary human alveolar cells type II. Hybridomas were produced to obtain cell culture supernatants that were screened on tissue micro arrays. Among others, we identified one clone that strongly binds human adenocarcinomas of the lung. Since most of patient material are Formalin-fixed and paraffin-embedded, we established an antigen retrieval protocol that works on FFPE tissues. Here we present a monoclonal antibody, designated MAdL as a new specific marker for adenocarcinomas of the lung.

TGF-β signaling in non-malignant pulmonary diseases to date is still controversially discussed. Nontypeable Haemophilus influenzae (NTHI) may play a role in the pathogenesis of chronic obstructive pulmonary disease (COPD) as an infectious trigger. Comparably few data are available regarding the influence of acute and persistent infection on tissue remodeling and repair factors such as transforming growth factor (TGF)-β. Here we show that the TGF-β pseudoreceptor BAMBI is expressed in the human lung. NTHI infection was analyzed in lung tissues obtained from COPD patients and controls utilizing a human ex vivo and in vitro models. Detection of NTHI was achieved by in situ hybridization (ISH). For characterization of TGF-β] signaling molecules, transcriptome array analyses were conducted. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and RT-PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-alpha and TGF-β expression were evaluated in lung tissue and cell culture using ELISA. An infection with NTHI was detected in vivo in 38% of the COPD patients in contrast to 0% of controls. Transcriptome arrays showed no significant changes of TGF-β receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and on alveolar epithelial cells (AEC). We show for the first time a considerable expression of the TGF pseudoreceptor BAMBI in the human lung. BAMBI is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro.

Loss of intestinal barrier function and subsequent edema formation remains a serious clinical problem leading to hypoperfusion, anastomotic leakage, bacterial translocation, and inflammatory mediator liberation. The inflammatory mediator platelet activating factor (PAF) promotes eicosanoid-mediated edema formation and vasoconstriction. Fish oil-derived (n-3) fatty acids (FA) favor the production of less injurious eicosanoids but may also increase intestinal paracellular permeability. We hypothesized that dietary (n-3) FA would ameliorate PAF-induced vasoconstriction and enhance vascular leakage of dextran tracers. Rats were fed either an (n-3) FA-rich diet (EPA-rich diet; 4.0 g/kg EPA, 2.8 g/kg DHA) or a control diet (CON diet; 0.0 g/kg EPA and DHA) for 3 wk. Subsequently, isolated and perfused small intestines were stimulated with PAF and arterial pressure and the translocation of fluid and macromolecules from the vasculature to lumen and lymphatics were analyzed. In intestines of rats fed the EPA-rich diet, intestinal phospholipids contained up to 470% more EPA and DHA at the expense of arachidonic acid (AA). The PAF-induced increase in arterial pressure was not affected by the EPA-rich diet. However, PAF-induced fluid loss from the vascular perfusate was higher in intestines of rats fed the EPA-rich diet. This was accompanied by a greater basal loss of dextran from the vascular perfusate and a higher PAF-induced transfer of dextran from the vasculature to the lumen (P = 0.058) and lymphatics. Our data suggest that augmented intestinal barrier permeability to fluid and macromolecules is a possible side effect of (n-3) FA-rich diet supplementation.

Background: Different therapy regimens in non-small-cell lung cancer (NSCLC) are of rising clinical importance, and therefore a clear-cut subdifferentiation is mandatory. The common immunohistochemical markers available today are well applicable for subdifferentiation, but a fraction of indistinct cases still remains, demanding upgrades of the panel by new markers. Methods: We report here the generation and evaluation of a new monoclonal antibody carrying the MAdL designation, which was raised against primary isolated human alveolar epithelial cells type 2. Results: Upon screening, one clone (MAdL) was identified as a marker for alveolar epithelial cell type II, alveolar macrophages and adenocarcinomas of the lung. In a large-scale study, this antibody, with an optimised staining procedure for formalin-fixed tissues, was then evaluated together with the established markers thyroid transcription factor-1, surfactant protein-A, pro-surfactant protein-B and napsin A in a series of 362 lung cancer specimens. The MAdL displays a high specificity (>99%) for adenocarcinomas of the lung, together with a sensitivity of 76.5%, and is capable of delivering independent additional diagnostic information to the established markers. Conclusion: We conclude that MAdL is a new specific marker for adenocarcinomas of the lung, which helps to clarify subdifferentiation in a considerable portion of NSCLCs.

HISTORY:A 56-year-old woman had two years previously undergone a neck dissection and subsequent adjuvant radiotherapy for an adenocarcinoma at the base of the tongue (pT2NOMOG2) when a percutaneous endoscopic gastrostomy (PEG) catheter had been placed. She was now admitted for chemotherapy, recent onset of severe pain in the left hip and knee having been caused by metastasis of a non-small-cell lung carcinoma (NSCLC). She was cachectic and in a reduced general condition (Karnofsky index 80), but had recently only occasionally used the PEG catheter. There were no inflammatory changes of the skin at the site of the PEG.TREATMENT, COURSE AND OUTCOME:The first chemotherapy cycle was initially without complication, but after a week the patient's general condition deteriorated and she developed nausea, fever and pain around the markedly inflamed site of the PEG catheter insertion. Laboratory tests indicated severe neutropenia. Intensive antibiotic and antimycotic treatment at first brought about some improvement, but she died 11 days after admission. Necropsy revealed invasive aspergillosis, with the PEG as the portal of entry and spreading to the stomach and intestines, where numerous hyphae were identified. There had also been a disseminated intravascular coagulopathy.CONCLUSION:Bacterial infections (and occasionally, but difficult to diagnose, fungal infection) are quite common as a result of neutropenia during chemotherapy of solid tumors. Various risk factors, including reduced general condition and weight loss, must be individually assessed in the prevention or treatment of associated infectious complications in such cases.

In the last decade, pathologic approaches concerning diagnosis and treatment of lung carcinomas have increasingly moved towards the implementation of molecular methods into the process of decision. In this study, an overview is given referring to the variety of tumors in the lung including common primary lung neoplasms and secondary tumors, and a modus operandi is presented which integrates immunology as well as molecular pathology within the process of finding correct diagnoses. Besides the conventional and approved methods and techniques leading to appropriate treatment including so-called targeted therapies, pathologist's work meanwhile depends on both histologic and molecular results. Since molecular techniques have increasingly entered the field of routine diagnostics, challenges and possibilities have changed and are still rapidly developing. The proceeding integration of molecular-biologic investigations into the process of diagnosing has changed the nature of diagnostics and will continuously grow in the near future. Only by obtaining a proper diagnosis, the optimal treatment of a patient can be assured, whereupon the knowledge of gene mutations and/or altered protein expression is crucial. By identifying those novel molecular target structures, the therapeutic spectrum is tremendously enlarged and will finally improve the patient's prognosis by personalized targeted therapies.

Einleitung: Untersuchungen am Proteom humaner Lungengewebe setzen eine adäquate Bearbeitung der Gewebe voraus. Essentiell für das Ergebnis aller Proteinuntersuchungen an Archivmaterial ist die Qualität der Gewebefixierung mit wenig molekularen Alterationen auch nach längerer Lagerungszeit sowie die Möglichkeit, die fixierten Gewebe über Jahre archivieren zu können. Die dafür zur Verfügung stehenden Standard-Techniken sind das Einfrieren der Gewebe in flüssigem Stickstoff (und Lagerung bei -80°C) oder die Formalinfixierung mit folgender Paraffineinbettung. Beide Methoden gehen mit der Entstehung von Artefakten einher: Formalin ist für Denaturierung und Quervernetzung von Proteinen verantwortlich und wird vornehmlich bei histologischen Untersuchungen verwendet, Gefriermaterial als Standardfixierung für Proteomstudien besitzt eine schlechte Morphologie und ist eingeschränkt in der Dauer seiner Lagerfähigkeit (ab 3 Jahre). Methoden: In dieser Studie wurden Proteomuntersuchungen mithilfe von 2-D-Elektrophorese und Massenspektrometrie auf Basis der HOPE (Hepes-glutamic acid buffer mediated organic solvent protection effect)-Technik etabliert. Dies bietet Vorteile in Fixierungs- und Archivierungsqualität und gewährleistet guten morphologischen wie molekularen Erhalt, da keine Quervernetzung der Proteine und kaum Denaturierung stattfindet. Die HOPE-Technik wird mit einer Paraffineinbettung abgeschlossen. Zur Anwendung kamen humane Lungenkarzinomgewebe und tumorfreie Lungengewebe. Ergebnisse: Mit HOPE-Material konnten reproduzierbare Spotmuster sowie eine erfolgreiche Proteinidentifikation über Massenspektrometrie erreicht werden. Die hohe Auflösung der Muster und die Anzahl detektierbarer Proteine sind mit denen von Gefriermaterial vergleichbar. Diskussion: Diese Methode bietet neue Möglichkeiten bei Studien an Paraffin-Gewebearchiven mit dem Ziel der Identifikation krankheitsspezifischer Proteinexpressionsmuster und einzelner krankheitsrelevanter Moleküle.

Background: Nontypeable Haemophilus influenzae (NTHI) may play a role as an infectious trigger in the pathogenesis of chronic obstructive pulmonary disease (COPD). Few data are available regarding the influence of acute and persistent infection on tissue remodelling and repair factors such as transforming growth factor (TGF)-beta.Methods: NTHI infection in lung tissues obtained from COPD patients and controls was studied in vivo and using an in vitro model. Infection experiments were performed with two different clinical isolates. Detection of NTHI was done using in situ hybridization (ISH) in unstimulated and in in vitro infected lung tissue. For characterization of TGF-beta signaling molecules a transcriptome array was performed. Expression of the TGF-pseudoreceptor BMP and Activin Membrane-bound Inhibitor (BAMBI) was analyzed using immunohistochemistry (IHC), ISH and PCR. CXC chemokine ligand (CXCL)-8, tumor necrosis factor (TNF)-alpha and TGF-beta expression were evaluated in lung tissue and cell culture using ELISA.Results: In 38% of COPD patients infection with NTHI was detected in vivo in contrast to 0% of controls (p < 0.05). Transcriptome arrays showed no significant changes of TGF-beta receptors 1 and 2 and Smad-3 expression, whereas a strong expression of BAMBI with upregulation after in vitro infection of COPD lung tissue was demonstrated. BAMBI was expressed ubiquitously on alveolar macrophages (AM) and to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-beta (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).Conclusions: We show for the first time the expression of the TGF pseudoreceptor BAMBI in the human lung, which is upregulated in response to NTHI infection in COPD lung tissue in vivo and in vitro. The combination of NTHI-mediated induction of proinflammatory cytokines and inhibition of TGF-beta expression may influence inflammation induced tissue remodeling.

Th e novel HOPE technique has proven to be an excellent tool for both research as well as diagnostic aspects. Th is presentation will focus on the advantages provided by this unique fi xation technique as compared to formalin fi xation as the current standard with regard to integrity as well as long-term preservation of both nucleic acids and antigenic structures. A complete panel of modern molecular analyses are demonstrated, including DNA extraction, RT-PCR analysis, in situ hybridization, Northern-blot and WesternBlot that has been successfully applied on human tissues of diff erent organ origin. Clinical applications with regard to mechanisms of human lung infection (COPD) and Her-2 diagnostics for human breast cancer are also included. Furthermore, recent results are presented using an ex vivo short-term tissue culture model (STST) that has been established in combination with the HOPEfi xation method. Th is STST model is suitable not only as a promising model of the initial phase of lung infection, but has also considerably improved the possibilities to identify new clinically relevant molecular targets for anticancer treatment of human lung cancer. In conclusion, the HOPE-technique represents a valuable tool for extensive molecular analyses in human tissues, whereas STST represents a multifunctional ex vivo model for various aspects in clinical research and modern diagnostic pathology. In combination, a solid base is provided for the development of effi cient High Troughput assays to further enhance the diagnostics in human severe disease.

Proteome analyses provide diagnostic information which can be essential for therapeutic predictions. The application of such techniques for analyzing paraffin-embedded tissue samples is widely hampered by the use of formalin fixation requiring antigen retrieval procedures in molecular pathology. In prior studies, the HEPES-glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique of tissue fixation has been shown to provide a broad array of biochemical investigations with excellent preservation of morphological structures, DNA, RNA, and proteins, thus supporting the multimethod analysis of archived specimens. Here we show that HOPE fixation is also useful in proteomic investigations by allowing two-dimensional electrophoresis (2DE) and mass spectrometry, using lung cancer tissues. Two-dimensional gels of two-protein extraction protocols derived from HOPE-fixed material displayed characteristic spot patterns with high reproducibility. For comparison, 2DE analysis of ethanol-fixed, formalin-fixed, and frozen samples from the same tissues was performed. Western blotting confirmed immunoreactivity of 2DE-separated proteins from HOPE-fixed tissue samples. Additionally, distinct spots were excised from HOPE-derived 2D gels and successfully subjected to peptide mass fingerprinting. In conclusion, paraffin archives containing HOPE-fixed tissues are applicable to a wide spectrum of molecular investigations including common biochemical methods for proteome analyses and therefore represent a unique source for molecular investigations in the rapidly growing field of molecular pathology. This manuscript contains online supplemental material at http://www.jhc.org . Please visit this article online to view these materials.

Despite considerable progress in the development of individualized targeted therapies of tumor diseases, identification of additional reliable target molecules is still mandatory. One of the most recent targets is microtubule-associated human EML4 generating a fusion-type oncogene with ALK demonstrating marked transforming activity in lung cancer. Since EML4 is a poorly characterized protein with regard to expression, function and regulation in human tissue, specimens of human tumor and tumor-free tissues obtained from patients with NSCLC were analyzed to determine the cellular localization. All tissue samples have been previously fixed with the novel HOPE-technique and paraffin embedded. Determination of both gene expression and protein levels of EML4 were performed using RT-PCR, in situ hybridization as well as immunohistochemistry, respectively. In human NSCLC tissue samples, possible regulation of EML4 transcription upon chemotherapy with combinations of most established cytotoxic drugs for NSCLC treatment was also studied employing the recently established ex vivo tissue culture model STST. In normal lung, both marked mRNA and protein levels of EML4 were localized in alveolar macrophages. In contrast, lung tumor tissues always showed consistent transcriptional expression in situ and by RT-PCR. Stimulation of NSCLC tissues with chemotherapeutics revealed heterogeneous effects on EML4 mRNA levels. Based on its expression patterns in both tumor-free lung and NSCLC tissues, human EML4 is likely to be closely associated with processes involved in local inflammation of the lung as well as with tumor behavior. Thus, our results suggest that EML4 may have the potential as a therapeutic target molecule in NSCLC chemotherapy.

Introduction: The acute phase protein Haptoglobin (Hp) consists of two different polypeptide chains. Originating from the liver; the biological function is binding free hemoglobin (Hb) and preventing oxidative stress. Elevated amounts of Hp in plasma were observed were infection, inflammation and malignant diseases. Purpose of this study was to investigate protein and mRNA expression within human lung tissues and tumors. Methods: We performed immunohistochemistry, in situ hybridization and RT-PCR analyzing 115 tissue samples. 47 adenocarcinomas, 42 squamous cell carcinomas, 13 small cell lung cancers and 13 tumor-free lungs were investigated. After lobectomy/pneumonectomy tissues were fixed and paraffin-embedded using formalin (immunohistochemistry) or the HOPE-solution (other methods). Results: Immunohistochemistry and in situ hybridization revealed a high level of Hp expression in adenocarcinomas (40.4%) in contrast to squamous cell carcinomas (4.8%, expression in alveolar epithelial cells type II surrounding the tumor). One small cell carcinoma showed Hp expression. In tumor-free lungs we located Hp in alveolar macrophages; alveolar epithelial cells type II and bronchi. RT-PCR results confirmed elevated expression. Discussion: We characterized that Hp protein expressed in lung cancers and tumor-free lungs showing an extra hepatic function. Due to the known immunosuppressive properties of Hp, its broad synthesis is strongly suggestive for a function as a fundamental, pulmonary, local defense element and confirms recent reports that haptoglobin plays an important role in protection processes and repairing mechanisms of injured tissues.

High throughput equipment, like tissue arrayer and autostainer, has meanwhile been integrated in pathology and research on a daily-use basis and provides comparability, reproducibility and celerity of processing. Since molecular investigations possessing high throughput characteristics (transcription arrays and 2-D-electrophoresis) can be combined with this equipment, effi cient strategies to fi nd disease-relevant molecules can be developed. In this study transcription array data derived from human lungs, tumor and tumor-free specimens, was evaluated to disclose strong gene regulations. Candidate molecules, one of which was haptoglobin, were found and studied intensively employing high throughput immunohistochemistry. For evaluation of signals from immunohistochemistry, an automated stand-alone tissue microarray detection system (“Spot Browser”) was used to analyze stained tissue microarrays. We describe diff erent technical and molecular high throughput tools whose combination represents an effi cient strategy for the identifi cation of most important candidate molecules with high importance in the fi eld of lung diseases including cancer, infl ammation, allergy, and infection. All tissue conservation was further based on the formalin-, ethanoland xylolfree HOPE-fi xation which provides advantageous molecular conservation due to its gentle fi xation mechanism. Th is technique for paraffi n-embedded material maintains morphology equal to formalin and avoids protein crosslinking and degradation of RNA and DNA in a long-term manner; moreover, all molecular-biologic and biochemical methods are compatible. In conclusion, HOPE-fi xed tissues processed with high throughput equipment and analyzed using molecular high throughput techniques provide a powerful combination as the basis for multimethodical investigations of archived tissue specimens and therefore for the fast detection of disease-associated molecules.

Surfactant Protein-A (SP-A) is the most prominent among four proteins in the pulmonary surfactant-system. SP-A is expressed by alveolar epithelial cells type II as well as by a portion of non small cell lung carcinomas (NSCLC).The expression of SP-A is complexly regulated on the transcriptional and the chromosomal level. SP-A is a major player in the pulmonary cytokine-network and moreover has been described to act in the pulmonary host defense.By the use of cell culture or animal models the functional properties have been repeatedly shown in many aspects, often bearing surprising properties which strongly indicate the physiological importance of SP-A. To date SP-A is recognized as a molecule essential for pulmonary development, structure and function. An upcoming number of reports deals with the role of SP-A for pulmonary pathology. This article gives an overview about the state of knowledge on SP-A focused in applications for human pulmonary disorders and points out the importance for pathology-orientated research approaches using immunohistochemistry or in situ hybridization as promising methods to further elucidate the role of this molecule in adult lung diseases.

Intestinal edema remains a serious clinical problem, and novel approaches to study its pathophysiology are needed. It was our aim to develop a long-term stable isolated perfused rat small bowel preparation permitting analysis of vascular, luminal, interstitial, and lymphatic compartments and to demonstrate the utility of this model by studying the effects of the proinflammatory mediator platelet-activating factor (PAF). A temperature-controlled chamber with an integrated balance was designed to perfuse isolated intestines through the mesenteric artery and the gut lumen. Steroids or oxygen carriers were not needed. Functional and morphological integrity of the tissue was preserved for several hours as confirmed by oxygen consumption, venous lactate-to-pyruvate ratio, arterial and venous pH, lactose digestion and galactose uptake, intravascular and luminal pressures, maintained fluid homeostasis, gut motility, and quantitative light microscopic analysis. Administration of PAF caused typical effects such as vasoconstriction, gut atony, and loss of galactose uptake. PAF also elicited a transient loss of 20% of the perfusate liquid from the mesenteric vascular bed, two-thirds of which were transferred to the lumen. All these responses were entirely reversible. This new model provides detailed insights into the physiology of the small intestine and will allow to study fundamental processes such as fluid homeostasis, barrier functions, transport mechanisms, and immune responses in this organ. Using this model, here we show a dramatic and yet reversible response of the rat small bowel to PAF, suggesting luminal water clearance as a novel safety factor in the intestine that may be of clinical relevance.

Besides its main function, i.e., the binding of free hemoglobin and prevention of oxidative stress, the acute phase protein haptoglobin acts as a potent immunoreactive modulator. As part of an investigation that aimed at illuminating the role of acute phase proteins in the local defense of the lungs, this study is the first to describe the expression and synthesis of haptoglobin in human lung tissues and lung tumors. Prompted by the results obtained from a transcription array study, we analyzed 115 lung (cancer) specimens using immunohistochemistry. Thirty-seven specimens were subjected to mRNA-in situ hybridization. 40.4% of the adenocarcinomas showed distinct granular and perinuclear staining of the tumor cells. By contrast, only 4.8% of the squamous cell carcinomas showed haptoglobin within tumor cells, but 19% displayed haptoglobin expressing alveolar epithelial cells type II surrounding the tumor. One small cell lung cancer displayed haptoglobin expression. In tumor-free lungs, we located haptoglobin in alveolar macrophages, alveolar epithelial cells type II, and bronchiolar cells. In situ hybridization verified the results of immunohistochemistry. The results were further verified by RT-PCR and Western blot compared to liver tissues, which both showed comparable amounts of haptoglobin mRNA and protein in NSCLC and in liver, while tumor-free lung tissues showed lower expression. Due to the known immunomodulatory effects of haptoglobin, its broad expression and synthesis within human lung tissues strongly suggests a function as a fundamental pulmonary local defense element.