Holger Schultz

The TGF-beta-Pseudoreceptor BAMBI is strongly expressed in COPD lungs and regulated by nontypeable Haemophilus influenzae

BackgroundNontypeable 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)-β.MethodsNTHI 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-β 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)-α and TGF-β expression were evaluated in lung tissue and cell culture using ELISA.ResultsIn 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-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 to a lesser degree on alveolar epithelial cells (AEC). Measurement of cytokine concentrations in lung tissue supernatants revealed a decreased expression of TGF-β (p < 0.05) in combination with a strong proinflammatory response (p < 0.01).ConclusionsWe 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-β expression may influence inflammation induced tissue remodeling.

TKTL1 is overexpressed in a large portion of non-small cell lung cancer specimens

In several tumors the transketolase activity, controlled inter alia by enzymes of the pentose phosphate pathway which is an alternative, energy generating reaction-cascade to glycolysis, has been correlated with proliferation. The increase of thiamine-dependant transketolase enzyme reactions is induced especially through upregulated transketolase-like enzyme 1 (TKTL1)-activity; that shows TKTL1 to be a causative enzyme for tumors enhanced, anaerobic glucose degradation. We investigated TKTL1-expression in 88 human, formalin-fixed non-small cell lung cancer tissues and 24 carcinomas of the breast by immunohistochemical stainings applying a 0 to 3 staining-score system (3 = strongest expression). For means of validation we additionally stained 40 NSCLC fixed and paraffin-embedded utilizing the HOPE-technique; showing comparable results to the formalin-fixed, paraffin-embedded specimens (not shown). Potential correlations with age, sex, TNM-classification parameters and tumor grading as well as tumor transcription factor 1 (TTF1) and surfactant protein A (SPA) expression were investigated. 40.9% of the analyzed lung tumors expressed TKTL1 weakly (Score 1), 38.6% moderately (score 2) and 17.1% strongly (score 3). 3 tumors were diagnosed TKTL1-negative (3.4%; score 0). All Breast cancer specimen stainings were positive and scored 1: 32%; scored 2: 36%; scored 3: 32%. Alveolar macrophages and Alveolar Epithelial Cells Type II were also found to be TKTL1-positive.None of the listed clinical parameters could be found to show a significant correlation to TKTL1 signal appearance.Although we describe the expression of TKTL1 in lung cancers, we need to state that up till now there is no scientific indication for any treatment regimens based upon these findings.

A model of the isolated perfused rat small intestine

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.

Expression of the acute phase protein haptoglobin in human lung cancer and tumor-free lung tissues.

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.

On the significance of Surfactant Protein-A within the human lungs

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.

Proteomics Out of the Archive: Two-dimensional Electrophoresis and Mass Spectrometry Using HOPE-fixed, Paraffin-embedded Tissues

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.

CXCR7 transcription in human non-small cell lung cancer and tumor-free lung tissues; possible regulation upon chemotherapy

Various chemokine receptors are expressed by malignant tumors and have been associated with tumor progression and metastasis [4]. A recent study demonstrated the promotion of tumor growth in breast and lung cancer by CXCR7, formerly known as orphan receptor CMKOR1/RDC1 utilizing cell lines and animal models. CXCR7 expression in human breast and lung cancer specimens, as well as in the tumor vasculature was shown on the protein level by immunohistochemistry. Here, we employed human lung tissues treated with the novel hepes–glutamic acid buffer-mediated organic solvent protection effect (HOPE) technique [5] to analyze the overall expression of CRCX7 on the mRNA level in non-small cell lung cancer (NSCLC) and tumor-free lung tissues. Additionally, the effect of chemotherapy upon the transcription of CRXR7 was addressed, employing a currently established human model system [1, 3]. Applying RT-PCR with a specific primer set spanning an amplicon of 156 bp (5′–TGCTGGACATCTTCTCCATC-3′ forward, 5′-CTTCATCAGCTCCTACCT-3′ reverse), we found transcription of CXCR7 in all of the 30 NSCLC samples analyzed. A set of ten examples is shown in Fig. 1a. Specificity of the reverse-transcription polymerase chain reaction (RTPCR) was verified by sequencing of the PCR products. The results of RT-PCR were then validated by in situ hybridization (N=21) using a double stranded DNA probe labeled with digoxigenin, which revealed transcripts of CXCR7 within the tumor cells of 17 samples (Fig. 1b) with only some occasional signals in the tumor-associated vasculature [2]. Moreover, 20 specimens of tumor-free human lungs were also subjected to in situ hybridization and all revealed transcripts localized mainly in alveolar macrophages (Fig. 1c) and in cells, which reflect the morphology of alveolar epithelial cells type II (Fig. 1d). Stimulation of tumor tissues with physiological concentrations of carboplat and gemcitabine (N=30) was performed within the human model system designated short-term stimulation of tissues in comparison to corresponding tissues, which received cultivation without stimulation [3]. Here, we found an upregulation of CXCR7 transcription upon chemotherapy in 13.4% of the cases, downregulation in 33.3%, and no regulation in 53.3%, as determined by semiquantitative RT-PCR in relation to glyceraldehyde-3-phosphate dehydrogenase (exemplified in the lower panel of Fig. 1a; note upregulation of CXCR7 in sample 4 upon chemotherapy). Taken together, our study verifies the recently reported results on the mRNA level in a comparably large amount of specimens [4]. However, although some signals were occasionally observable, we did not detect strong expression within the tumor-associated vasculature on the RNA level. The effects of chemotherapy onto CXCR7 are heterogeneous. Nevertheless, the same holds true for the overall characteristics of the tumors themselves and their Virchows Arch (2008) 452:347–348 DOI 10.1007/s00428-008-0579-8

A novel human ex vivo model for the analysis of molecular events during lung cancer chemotherapy

BackgroundNon-small cell lung cancer (NSCLC) causes most of cancer related deaths in humans and is characterized by poor prognosis regarding efficiency of chemotherapeutical treatment and long-term survival of the patients. The purpose of the present study was the development of a human ex vivo tissue culture model and the analysis of the effects of conventional chemotherapy, which then can serve as a tool to test new chemotherapeutical regimens in NSCLC.MethodsIn a short-term tissue culture model designated STST (Short-Term Stimulation of Tissues) in combination with the novel *HOPE-fixation and paraffin embedding method we examined the responsiveness of 41 human NSCLC tissue specimens to the individual cytotoxic drugs carboplatin, vinorelbine or gemcitabine. Viability was analyzed by LIFE/DEAD assay, TUNEL-staining and colorimetric MTT assay. Expression of Ki-67 protein and of BrdU (bromodeoxyuridine) uptake as markers for proliferation and of cleaved (activated) effector caspase-3 as indicator of late phase apoptosis were assessed by immunohistochemistry. Transcription of caspase-3 was analyzed by RT-PCR. Flow cytometry was utilized to determine caspase-3 in human cancer cell lines.ResultsViability, proliferation and apoptosis of the tissues were moderately affected by cultivation. In human breast cancer, small-cell lung cancer (SCLC) and human cell lines (CPC-N, HEK) proliferative capacity was clearly reduced by all 3 chemotherapeutic agents in a very similar manner. Cleavage of caspase-3 was induced in the chemo-sensitive types of cancer (breast cancer, SCLC). Drug-induced effects in human NSCLC tissues were less evident than in the chemo-sensitive tumors with more pronounced effects in adenocarcinomas as compared to squamous cell carcinomas.ConclusionAlthough there was high heterogeneity among the individual tumor tissue responses as expected, we clearly demonstrate specific multiple drug-induced effects simultaneously. Thus, STST provides a useful human model to study numerous aspects of mechanisms underlying tumor responsiveness towards improved anticancer treatment. The results presented here shall serve as a base for multiple functional tests of novel chemotherapeutic approaches to NSCLC in the future.*Hepes – Glutamic acid buffer mediated Organic solvent Protection Effect

LED-FISH: Fluorescence microscopy based on light emitting diodes for the molecular analysis of Her-2/neu oncogene amplification

Light emitting diodes (LED), which are available as small monochromatic light sources with characteristic features such as maximum illumination power combined with minimum energy consumption and extremely long lifespan have already proved as a highly potential low-cost alternative for specific diagnostic applications in clinical medicine such as tuberculosis fluorescence microscopy. Likewise, the most reliable evaluation of Her-2/neu (c-erbB2) gene amplification, which has been established in the last few years for routine diagnosis in clinical pathology as determinant towards Herceptin-based treatment of patients with breast cancer, is based on fluorescence in situ hybridization (FISH) and corresponding high priced fluorescence equipment. In order to test the possibility to utilize the advantages of low-cost LED technology on FISH analysis of c-erbB2 gene expression for routine diagnostic purposes, the applicability of a standard bright field Carl Zeiss Axiostar Plus microscope equipped with a Fraen AFTER* LED Fluorescence Microscope Kit for the detection of Her-2/neu gene signals was compared to an advanced Nikon Eclipse 80i fluorescence microscope in combination with a conventional 100W mercury vapor lamp. Both microscopes were fitted with the same Quicam FAST CCD digital camera to unequivocally compare the quality of the captured images. C-erbB2 gene expression was analyzed in 30 different human tissue samples of primary invasive breast cancer, following formalin fixation and subsequent paraffin-embedding. The Her2/neu gene signals (green) were identifiable in the tumor cells in all cases and images of equal quality were captured under almost identical conditions by 480 nm (blue) LED module equipped standard Axiostar microscope as compared to conventional fluorescence microscopy. In this first attempt, these monochromatic LED elements proved in principle to be suitable for the detection of Her-2/neu gene expression by FISH. Thus, our own experiences emphasize the high potential of this technology to provide a serious alternative to conventional fluorescence microscopy in routine pathology; representing a sustainable technological progress, this low-cost technology will clearly give direction also to the growing field of molecular pathology.* AFTER = Amplified Fluorescence by transmitted Excitation of Radiation

Diagnostic outcome of two different CT-guided fine needle biopsy procedures

BackgroundCT-guided fine needle bioptic procedures (CTFNP) are characterised by low invasiveness, precise sample collection, a high diagnostic efficiency and support a rapid diagnostic process. A number of different fine needles and bioptic procedures are mainly used for tumour diagnostics today.The aim of the present study was to characterise the most important technical issues of fine needle bioptic procedures. In addition, we directly compared the diagnostic outcome and reliability of the most commonly used Rotex Screw Needle – (RSN) and Yale Needle – (YN) bioptic procedure.MethodsIn an experimental part of the study, using pig spleen, we measured the maximum number of sampled cells using different needles and aspiration volumes.For the clinical questions we analysed all consecutive 340 patients in which CTFNP were performed between 1/97–12/05 in the hospital Grosshansdorf. We evaluated the number of adverse events based on all clinical available information and compared the cytological findings with the respective final diagnosis (confirmed: clinically n = 192, histologically n = 148).ResultsUsing the YN with at least some negative pressure we found a proportional increase of cell and tissue recovery with increasing number of needle movements.A sensitivity of 78% and a specificity 98% indicate a high diagnostic outcome of CTFNP. We found no statistical significant difference in terms of sensitivity (80 vs. 68%) as well as complication rates (5.9 vs. 4.4%) between RSN or YN.ConclusionAs fine needle basically works like a cutting instrument, it is possible to raise the cell/tissue recovery. Keeping this in mind we found a high diagnostic outcome of CTFNP, which was largely independent of needle type and bioptic technique, and comparable with other conventional bioptic procedures.