Extra cellular matrix remodelling after heterotopic rat heart transplantation: gene expression profiling and involvement of ED-A+ fibronectin, alpha-smooth muscle actin and B+ tenascin-C in chronic cardiac allograft rejection.

Chronic cardiac rejection is represented by cardiac allograft vasculopathy (CAV) and cardiac interstitial fibrosis (CIF) known to cause severe complications. These processes are accompanied by remarkable changes in the cardiac extra cellular matrix (cECM). The aim of our study was to analyse the cECM remodelling in chronic rejection and to elucidate a potential role of ED-A domain containing fibronectin (ED-A(+) Fn), alpha smooth muscle actin (ASMA) and B domain containing tenascin-C (B(+) Tn-C). A model of chronic rejection after heterotopic rat heart transplantation was used. Allografts, recipient and control hearts were subjected to histological assessment of rejection grade, to real-time PCR based analysis of 84 genes of ECM and adhesion molecules and to immunofluorescence labelling procedures, including ED-A(+) Fn, ASMA and B(+) Tn-C antibodies. Histological analysis revealed different grades of chronic rejection. By gene expression analysis, a relevant up-regulation of the majority of ECM genes in association with chronic rejection could be shown. For 8 genes, there was a relevant up-regulation in allografts as well as in the corresponding recipient hearts. Association of ASMA positive cells with the grade of chronic rejection could be proven. In CAV and also in CIF there were extensive co-depositions of ED-A(+) Fn, ASMA and B(+) Tn-C. In conclusion, chronic cardiac allograft rejection is associated with a cECM remodelling. ASMA protein deposition in CAV, and CIF is a valuable marker to detect chronic rejection. Interactions of VSMCs and Fibro-/Myofibroblasts with ED-A(+) Fn and B(+) Tn-C might functionally contribute to the development of chronic cardiac rejection.

Epidermal growth factor receptor kinase domain mutations are rare in salivary gland carcinomas.

Activating mutations within the epidermal growth factor (EGFR) tyrosine kinase domain identify non-small cell lung cancer patients with improved clinical response to tyrosine kinase inhibitor therapy. Recently, we identified two EGFR mutations in a cohort of 25 salivary gland carcinomas (SGCs) by screening the tumour samples for the both most common hotspot mutations in exons 19 and 21 by allele-specific PCR. Here, we present a comprehensive sequencing analysis of the entire critical EGFR tyrosine kinase domain in 65 SGC of the main histopathological types. We found EGFR mutations in the tyrosine kinase domain to be a rare event in SGCs. No additional mutations other than the two known exon 19 deletions (c.2235_2249del15) in a mucoepidermoid carcinoma and an adenoid cystic carcinoma have been detected. Other putative predictive markers for EGFR-targeted therapy in SGCs might be relevant and should be investigated.

Negative regulation of Ros receptor tyrosine kinase signaling. An epithelial function of the SH2 domain protein tyrosine phosphatase SHP-1.

Male "viable motheaten" (me(v)) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of me(v) mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH(2)-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

Preliminary results of small arterial substitute performed with a new cylindrical biomaterial composed of bacterial cellulose.

OBJECTIVE: Tissue-engineered blood vessels (TEBVs) represent an innovative approach for overcoming reconstructive problems associated with extended vascular diseases by providing small-calibre vascular grafts. This study aimed to evaluate a novel biomaterial of bacterially synthesised cellulose (BC) as a potential scaffold for TEBV. METHODS: Highly crystalline cellulose was produced by a bacterium (Acetobacter xylinum) using glucose as a source of carbon. Using a patented process, hollow-shaped segments of BC were created with a length of 10mm, an inner diameter of 3.0-3.7mm and a wall thickness of 0.6-1.0mm. These grafts were used to replace the carotid arteries of eight pigs, and after a follow-up period of 3 months, the grafts were removed and analysed, both macro- and microscopically. RESULTS: Seven grafts (87.5%) remained patent, whereas one graft was found to be occluded. Scanning electron microscopic examination revealed rapid re-cellularisation by recipient endothelial cells. Light microscopic examination showed a three-layered wall structure of the BC segments, with cellulose still being present in the media. CONCLUSION: These data indicate that the innovative BC-engineering technique results in the production of stable vascular conduits, which exhibit attractive properties for their use in future TEBV programmes for vascular surgery.

Detection of drug-sensitizing EGFR exon 19 deletion mutations in salivary gland carcinoma.

Activating mutations within the epidermal growth factor receptor (EGFR) identify lung adenocarcinoma patients with improved clinical responses to tyrosine kinase inhibitors gefitinib and erlotinib. By screening salivary gland carcinoma, two drug-sensitizing EGFR exon 19 delE746-A750 mutations were identified in an adenocystic and in a mucoepidermoid carcinoma of the parotid gland.

Consequences of one-lung flooding: a histological and immunological investigation.

BACKGROUND: Videothoracoscopic lung sonography after partial fluid instillation could be a new method for endoscopic detection of lung lesions. Histopathological consequences of unilateral diagnostic or therapeutic lung flooding under bronchoalveolar lavage has yet to be defined. The aim of the study was to investigate histological and immunohistological alterations induced by one-lung flooding (OLF). METHODS: 13 female pigs were subjected to OLF (15 ml isotonic electrolyte solution per kg for 60 minutes), and lung tissue was collected 30 minutes, 2 hours, 24 hours, 48 hours, 6 days, 8 days, and 10 weeks after flooding. Histological examinations and immunohistochemical labeling for surfactant protein A (SP-A) were performed. Cellular proliferation was measured by Ki67 immunohistochemical labeling. Apoptosis was detected through enzymatic in-situ labeling of apoptosis-induced DNA strand breaks by means of the TUNEL (TdT-mediated dUTP nick end labeling) method. RESULTS: Histological analyses revealed the presence of inflammatory cell infiltrates in the interstitium at 24 hours after OLF. However, no destruction of the alveolar wall and no pulmonary oedema were observed. In addition, OLF was not associated with any decrease in surfactant protein A immunoreactivity. Two hours after OLF, the number of apoptotic cells was increased (OLF: 7% vs. CONTROL: 0.6%, p < 0.05), but cellular proliferation was unchanged. Conversely, at 48 h after OLF, the number of apoptotic cells had returned to control levels, but cellular proliferation had increased (OLF: 5% vs. CONTROL: 1.1%, p < 0.05). Cellular proliferation returned to baseline levels eight days after OLF. CONCLUSIONS: These data demonstrate that OLF is not associated with destruction of the alveolar texture, atelectasis-provoking surfactant loss, or any irreversible damage to the pulmonary parenchyma. Lung flooding for the purpose of videothoracoscopic lung sonography is safe and justifiable. But repeated lung flooding under bronchoalveolar lavage involving the same lung area within 1 week is not to be recommended.

[Performance of conventional oral brush biopsies]. / Wertigkeit der konventionellen oralen Bürstenbiopsie.

BACKGROUND: This study evaluated the performance of oral brush biopsies using standard morphological analysis and haematoxylin and eosin (HE) staining for detecting oral squamous cell carcinomas and their respective precursor lesions PATIENTS AND METHODS: Brush biopsies were obtained in 169 consecutive patients who underwent routine biopsies and histological examination for clinically suspicious oral lesions. Air-dried smears were processed by acetone fixation and HE staining. Cytological assessment used well-established criteria of atypia to classify the specimen as either "tumor negative" (no signs of atypia, no malignant cells) or "tumor positive" (malignant cells, any sign of atypia or doubtful cells). RESULTS: Despite a sufficient number of cells, a definite cytological diagnosis could not be established in six cases. According to the criteria specified above, these specimens were classified as "tumor positive." The cytological analysis identified 49 out of 62 oral malignancies (sensitivity 79%). Seven out of 107 benign lesions were classified as false positive (specificity 93%). The positive and negative predictive values were each 88%. CONCLUSION: Oral brush biopsies will identify only about 80% of oral malignancies when the smears are processed by routine HE stains and are analysed via standard morphological criteria. Thus, this technique should not be used for diagnostic proof or to exclude malignant cells in a lesion suspicious for cancer. However, oral brush biopsy provides a versatile back-up strategy to uncover the true nature of the disease if a lesion is clinically considered benign by mistake.

Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature.

Interleukin-12 (IL-12) is a heterodimeric cytokine with potent immunostimulatory activity and anti-angiogenic properties. Its clinical applications are limited, however, by severe side-effects. Here we report that an IL-12 fusion protein, consisting of IL-12 fused to a human antibody fragment specific to the oncofetal ED-B domain of fibronectin, markedly enhances the antitumor activity of this cytokine, as demonstrated in a mouse lung-metastasis model and in two models of mice bearing different aggressive murine tumors. The residual small tumor masses seen in the treated mice were infiltrated with lymphocytes, macrophages, and natural killer cells and had elevated interferon gamma (IFN-gamma). These results are of therapeutic relevance as the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is not detectable in normal vessels and tissues.

[Urethroplasty with tissue matrix. Microsurgical animal experiments with small intestinal submucosa]. / Urethroplastik mit Gewebematrix. Mikrochirurgische Tierexperimente mit "small intestinal submucosa".

BACKGROUND: The purpose of this study was to examine whether use of small intestinal submucosa in microsurgical urethroplasty in Wistar rats represents an alternative. METHODS: In 20 Wistar rats microsurgical urethroplasty with small intestinal submucosa was done to repair distal or proximal urethral defects. Four weeks later urethral calibration and urethrography were performed in addition to histological studies. RESULTS AND CONCLUSIONS: Of the 20 animals, 19 survived the observation period without evidence of fistulas, strictures, stenoses, or necroses. The histological results confirmed an intact tissue layer.

[Urothelium cancer with trophoblastic differentiation. An unusual case of a 77 year old patient]. / Das Urothelkarzinom mit trophoblastischer Differenzierung. Eine seltene Tumorvariante am Beispiel einer 77-jährigen Patientin.

Urothelium carcinomas with beta HCG positive markers are a rarity in tumour differentiation. Syncytiotrophoblastic and, in a few cases, cytotrophoblastic giant cells are typical for this carcinoma. Such differentiation has an intensified potential for invasiveness and is accompanied by increased angiogenesis. In the present case, the mixture of trophoblastic cells indicates a common stem cell. In comparison with beta HCG negative transitional cell carcinoma, the prognosis is bad for beta HCG positive carcinoma. For this reason, a radical operation should be taken into consideration as early as possible.

Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice.

The selective thrombosis of tumor blood vessels, leading to the starvation and subsequent death of tumor cells, is an attractive anticancer strategy. Here we report that a fusion protein, consisting of an antibody fragment specific for the oncofoetal ED-B domain of fibronectin fused to the extracellular domain of tissue factor, selectively targets tumor blood vessels in vivo. Furthermore, this fusion protein mediates the complete and selective infarction of three different types of solid tumors in mice. At the highest doses administered, complete tumor eradication was observed in 30% of the mice treated without apparent side effects. These results are of therapeutic relevance because the ED-B domain of fibronectin, a naturally occurring marker of angiogenesis identical in mouse and man, is expressed in the majority of aggressive solid tumors but is undetectable in normal vessels and tissues.

Renal cell carcinomas produce IL-6, IL-10, IL-11, and TGF-beta 1 in primary cultures and modulate T lymphocyte blast transformation.

We investigated the immunomodulatory capacity of primary cultures of renal cell carcinomas (RCC) by assessing production of cytokines and modulation of mitogen-induced T lymphocyte blast transformation. The results clearly show that immunomodulatory capacity is a common feature of RCC and that in vitro these tumors can produce interleukin-10 (IL-10) up to 20 ng/ml, IL-6 up to 35 micrograms/ml (> 250 kU/ml in the B9 system), IL-11 up to 15 micrograms/ml, and transforming growth factor-beta 1 (TGF-beta 1) up to 22 ng/ml. Furthermore, these tumors have the capacity to modulate T cell blast transformation over two orders of magnitude in each direction. The correlations of the immunologic properties of tumor cell cultures with the conventional classification of tumors (histology, cytology, staging, grading, presence of metastases, and secondary tumors) are analyzed. The significance of these findings for modulation of local immunity by RCC as well as for patient outcome is discussed.

Expression of EDA+ and EDB+ fibronectin splice variants in bone.

The extracellular matrix component fibronectin (fn) has fundamental functions in cell attachment, differentiation, proliferation, and migration. Isoforms of cellular fibronectin, named EDA+ fibronectin or embryonal EDB+ fibronectin, are generated by alternative splicing of its mRNA precursors. Little is known about the expression of EDA+ and EDB+ fibronectin splice variants in human bone. The aim of this study was to investigate the expression pattern of fibronectin splice variants in bone cell lines and in different human bone tissue samples (mature bone, early stages of fracture healing, hypotrophic nonunion, osteosarcoma). Analysis was done by immunostaining with recombinant and monoclonal antibodies, qualitative RT-PCR and LightCycler-based real-time quantitative RT-PCR assay. In osteoblast and osteosarcoma cell lines, abundant expression of EDA+ and EDB+ fibronectin was found in immunocytochemistry. High transcription levels of both splice variants mRNA were seen in quantitative RT-PCR in osteosarcoma cell lines. In mature bone, EDA+ and EDB+ were not detectable in immunohistochemistry. Transcription of mRNA in both splice variants was absent in these samples. Early stages of fracture healing and osteosarcoma cell samples exhibited extensive staining for EDA+ and EDB+ fibronectin, and high mRNA levels were found. Both osteosarcoma and bone fracture healing tissue expressed high mRNA levels of the fibronectin splice variants independent of benign or malignant behavior. Low level of EDA+ fibronectin mRNA transcription and focal immunohistochemical staining of EDA+ fibronectin was found in hypotrophic nonunions, whereas EDB+ fibronectin was not detected by immunohistochemistry and qualitative or quantitative PCR. EDA+ fibronectin was found in granulation tissue-forming processes in bone independent from bone-forming activity. EDB+ fibronectin was seen only in high-turnover new osteoid-forming processes like early stages of fracture healing and osteosarcoma and was absent in low-turnover processes like mature bone and hypotrophic nonunion. Both EDA+ and EDB+ fibronectin mark active processes in bone without differentiation between malignant or benign activity. In conclusion, EDA+ and EDB+ fibronectin splice variants are strong markers for active fibrogenetic and osteoid-forming processes in human bones.

Immunolocalization of tenascin-C, alpha9 integrin subunit, and alphavbeta6 integrin during wound healing in human oral mucosa.

Tenascin-C (TN-C) and its isoforms are multidomain extracellular matrix (ECM) proteins that are believed to be involved in the regulation of stromal-epithelial interactions. Some of the interactions between TN-C and cells are mediated by integrins. In this study we analyzed the expression of TN-C and its large molecular weight splice isoform (TN-C(L)) and the putative TN-C-binding alpha9 and alphavbeta6 integrins during human wound repair. In 3-day-old oral mucosal wounds, immunoreactivity for alpha9 integrin localized abundantly at the migrating basal wound epithelial cells. TN-C and TN-C(L) were localized in the matrix between and underneath alpha9-expressing epithelial cells. In parallel with gradual downregulation of alpha9 integrin immunoreactivity in 7-day and older wounds, the expression of alphavbeta6 integrin was temporarily induced. Integrin alphavbeta6 co-localized in the same area as TN-C and TN-C(L) immunoreactivity at the cell-cell contacts of the basal and suprabasal cell layers of the wound epithelium. During granulation tissue formation and reorganization from 7 to 28 days after wounding, TN-C and TN-C(L) were abundantly localized in the granulation tissue. The findings show that TN-C(L) is expressed under the migrating epithelial front and in the granulation tissue during matrix deposition in wound repair. Preferential localization of alpha9 integrin in migrating epithelial cells and of alphavbeta6 integrin in epithelium after wound closure suggests different functions for these integrins in wound repair.

A comparative quantitative analysis of laminin-5 in the basement membrane of normal, hyperplastic, and malignant oral mucosa by confocal immunofluorescence imaging.

Laminin-5 (Ln-5) is a heterotrimeric basement membrane (BM) molecule (alpha3beta3gamma2). It is a principal protein constituent of the anchoring filaments, which connect the BM with the hemidesmosomes of the basal keratinocytes and possess a crucial function in keratinocyte adhesion. Confocal immunofluorescence imaging is introduced for a quantitative evaluation of the Ln-5 content in the BM of oral squamous epithelium. The BM of normal oral mucosa was used as a reference (100%) for comparative analysis and showed a nearly uniform Ln-5 immunofluorescence intensity (99-100%). In all hyperplastic lesions of oral mucosa, the Ln-5 immunofluorescence intensity was increased (107-141%). The increased Ln-5 content in the BM of hyperplastic lesions suggests an increased keratinocyte-BM adhesion, possibly resulting in a higher stability of the oral mucosa. In contrast, in the oral squamous cell carcinoma (OSCC) invasive front, the remaining BM segments were characterized by a decrease in Ln-5 immunofluorescence intensity (35-74%). A stronger decrease of Ln-5-linked kerationocyte-BM adhesion correlates with a higher tumor grade. Because in central areas of carcinoma BM segments with a normal Ln-5 content could be demonstrated, the fundamental Ln-5 diminution in BM segments of the invasive front should be considered as an invasion-associated phenomenon.

Telomerase activity and telomere length in different areas of renal cell carcinoma.

Telomerase activity and telomere length were analyzed in a total of 59 surgically removed primary renal cell carcinoma (RCC). The study includes tissue from the centre of the tumor, several different peripheral tumor areas, metastases and secondary tumors. None of the normal renal cortex tissues used as control exhibited telomerase activity. In contrast, telomerase activity was detected in 55 out df 59 (=93%) tested primary RCC. There was no case with intratumoral heterogeneity concerning the telomerase activity status. All metastases and secondary tumors were telomerase-positive. In the four telomerase deficient tumors all measured telomeric repeat fragments were shortened in comparison to the normal tissue. As these patients exhibit no metastases or secondary tumors a less malignant variant of RCC is supposed. There was no correlation between telomerase activity and specific histopathological subtypes of RCC or specific chromosomal aberrations. As telomerase activity is not associated with advanced stages of tumors it may be an important early event in the development of RCC. Thus, telomerase activity may be a prevalent marker for early and late stages of all subtypes of RCC.

First experiences in using a new ultrasound mode and ultrasound contrast agent in the diagnosis of blunt renal trauma: a feasibility study in an animal model.

RATIONALE AND OBJECTIVES: To evaluate the diagnostic value of the new ultrasound mode "wide-band harmonic" (WBH) using an ultrasound contrast agent in blunt renal trauma in an animal model. METHODS: A defined blunt renal trauma was induced in 10 rabbits according to published standards. Ultrasound (B-mode, color and power Doppler, WBH) was performed before and after trauma, with and without using a contrast agent (Levovist). Ultrasound features were compared with histologic findings. RESULTS: In 2 of the 10 rabbits, three focal renal intraparenchymal lesions with diameters ranging from 1.0 to 1.8 mm were found that could be identified only using WBH with contrast. Six of the 10 rabbits developed a subcapsular hematoma with a thickness of up to 1.5 mm, which was identified by conventional B-mode as well as WBH. Histologic workup confirmed these findings of intraparenchymal hematomas and did not reveal further lesions. CONCLUSIONS: Only 20% of the experimental subjects developed parenchymal lesions with diameters of 1.0 mm or larger. All these lesions were identified only using WBH. These results indicate the potential to use WBH plus contrast for the diagnosis of blunt renal trauma.

Cyclosporin A-induced graft-versus-host disease following autologous bone marrow and stem cell transplantation in hematological malignancies of childhood.

Cyclosporin A (CsA) can induce graft-versus-host disease (GVHD) following autologous bone marrow transplantation (ABMT) and autologous peripheral blood stem cell transplantation (APBSCT) in adults. We investigated whether GVHD can be induced following ABMT and APBSCT in childhood, and which cells are involved in the pathogenesis of this syndrome. We conducted a prospective study of 20 children and adolescents with hematological malignancies receiving CsA after ABMT and APBSCT. Skin biopsies were obtained on day 21 after transplantation or in the event of a rash. Immunophenotypic analysis of peripheral blood lymphocytes was performed on days 14, 21, 28 and 60 after transplantation. Clinical GVHD of the skin, confirmed by histological criteria, occurred in five patients. Five patients had no clinical GVHD but had acute GVHD alterations on routine skin biopsy. In all 10 patients with a positive skin biopsy for GVHD, CD4+ lymphocytes were the predominant cells in the epidermis. Immunophenotypic analysis of peripheral blood lymphocytes revealed a significantly increased CD4/CD8 ratio in patients with a positive skin biopsy (P < 0.01). Our findings indicate that it is possible to induce acute GVHD following ABMT and APBSCT in childhood. In addition, CD4+ lymphocytes play an important role in the pathogenesis of CsA-induced GVHD.

Primary cutaneous marginal center lymphoma - complete remission induced by interferon alpha2a.

Marginal zone lymphoma (MZL) is a distinct entity among B-cell lymphomas. We report on a 53-year-old woman who developed disseminated primary cutaneous MZL with secondary lymph node involvement and perinodular spreading. The tumor cell phenotype was characterized as CD20/CD79a/kappa/lambda+/ bcl-2-positive, CD3/5/15/39/bcl-1-negative. Ki-67 was expressed by 20-35% of tumor cells. There was no evidence of systemic (including bone marrow) involvement. The diagnosis of MZL with plasmacellular differentiation (Stage IVa) was made. The patient was treated with interferon alpha2a injected s.c. at 9x10(6) U 3 days a week for 1 year. During this time the skin lesions completely disappeared. No evidence of lymph node or extracutaneous disease was found. The patient remains in complete remission. Side effects were only of grade I (WHO); the Karnovsky index was 90%. As shown for other types of primary cutaneous B-cell lymphoma, prolonged interferon alpha monotherapy may be effective in controlling the disease and/or inducing complete remission in MZL.

Fibrillary co-deposition of laminin-5 and large unspliced tenascin-C in the invasive front of oral squamous cell carcinoma in vivo and in vitro.

PURPOSE: Invasion of oral squamous cell carcinoma (OSCC) is associated with laminin-5 (Ln-5) synthesis, focal Ln-5 loss from the basement membrane (BM), and Ln-5 depositions in the stroma beneath invading carcinoma cell complexes. METHODS: The study is focused on the laminin-5 matrix reorganisation within the stroma of the OSCC invasive front outside the basement membrane region as well as in OSCC-fibroblast co-culture in relation to unspliced tenascin-C (Tn-CL) and ED-B+ fibronectin (ED-B+ fn) using confocal laser scanning microscopy. RESULTS: In vivo, Ln-5 was demonstrated as fibrillary deposition in the invasive front. It was co-localised to Tn-CL. In pure OSCC cultures, Ln-5 was synthesised and deposited as a spot-like matrix. Fibrillary structures were not found. In contrast, in the OSCC-fibroblast co-culture, a fibrillary Ln-5 matrix organisation was revealed within the interface of OSCC cell-fibroblast complexes exclusively in co-distribution with Tn-CL and ED-B+ fn. CONCLUSION: At least in vitro, a carcinoma cell-stroma fibroblast interaction is indispensable for fibrillary Ln-5/Tn-CL matrix organisation. Behind the parallels to the initial basement membrane formation in organotypic cultures, the fibrillary multiprotein complexes at the OSCC cell-fibroblast interface are suggested as provisional basement membrane fragments with a possible supportive role for invasive tumour behaviour.