Kerstin Röser

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule.

The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

Qualitative and quantitative observations of bone tissue reactions to anodised implants.

Research projects focusing on biomaterials related factors; the bulk implant material, the macro-design of the implant and the microsurface roughness are routinely being conducted at our laboratories. In this study, we have investigated the bone tissue reactions to turned commercially pure (c.p.) titanium implants with various thicknesses of the oxide films after 6 weeks of insertion in rabbit bone. The control c.p. titanium implants had an oxide thickness of 17-200 nm while the test implants revealed an oxide thickness between 600 and 1000 nm. Routine histological investigations of the tissue reactions around the implants and enzyme histochemical detections of alkaline and acid phosphatase activities demonstrated similar findings around both the control and test implants. In general, the histomorphometrical parameters (bone to implant contact and newly formed bone) revealed significant quantitative differences between the control and test implants. The test implants demonstrated a greater bone response histomorphometrically than control implants and the osteoconductivity was more pronounced around the test implant surfaces. The parameters that differed between the implant surfaces, i.e. the oxide thickness, the pore size distribution, the porosity and the crystallinity of the surface oxides may represent factors that have an influence on the histomorphometrical results indicated by a stronger bone tissue response to the test implant surfaces, with an oxide thickness of more than 600 nm.

Oxidized implants and their influence on the bone response

Surface oxide properties are regarded to be of great importance in establishing successful osseointegration of titanium implants. Despite a large number of theoretical questions on the precise role of oxide properties of titanium implants, current knowledge obtained from in vivo studies is lacking. The present study is designed to address two aspects. The first is to verify whether oxide properties of titanium implants indeed influence the in vivo bone tissue responses. The second, is to investigate what oxide properties underline such bone tissue responses. For these purposes, screw-shaped/turned implants have been prepared by electrochemical oxidation methods, resulting in a wide range of oxide properties in terms of: (i) oxide thickness ranging from 200 to 1000 nm, (ii) the surface morphology of barrier and porous oxide film structures, (iii) micro pore configuration – pore sizes<8 μm by length, about 1.27 μ2 to 2.1 μm2 by area and porosity of about 12.7–24.4%, (iv) the crystal structures of amorphous, anatase and mixtures of anatase and rutile type, (v) the chemical compositions of TiO2 and finally, (vi) surface roughness of 0.96–1.03 μm (Sa). These implant oxide properties were divided into test implant samples of Group II, III, IV and V. Control samples (Group I) were turned commercially pure titanium implants. Quantitative bone tissue responses were evaluated biomechanically by resonance frequency analysis (RFA) and removal torque (RT) test. Quantitative histomorphometric analyses and qualitative enzyme histochemical detection of alkaline (ALP) and acidic phosphatase (ACP) activities were investigated on cut and ground sections after six weeks of implant insertion in rabbit tibia. In essence, from the biomechanical and quantitative histomorphometric measurements we concluded that oxide properties of titanium implants, i.e. the oxide thickness, the microporous structure, and the crystallinity significantly influence the bone tissue response. At this stage, however, it is not clear whether oxide properties influence the bone tissue response separately or synergistically.© 2001 Kluwer Academic Publishers

Enzyme and immunohistochemistry on undecalcified bone and bone marrow biopsies after embedding in plastic: A new embedding method for routine application

A simplified method of low temperature methyl and butyl methacrylate embedding (up −20° to −15° C) is demonstrated using a proper redox system of benzoyl peroxide and aromatic amine. This method combines the morphological superiority of plastic-embedded bone tissue and bone marrow sections with the advantages of specific enzyme histochemical and immunochemical markers. The method permits good preservation of morphological details, the survival of antigenic determinants and the retention of enzyme activities. The specimens were fixed in 1.6% formaldehyde and 5% sucrose in 0.02 M phosphate buffer at pH 7.4, washed in 0.02 M phosphate buffer and 5% sucrose, dehydrated with acetone and impregnated with monomers of embedding medium. All these steps were carried out at +4° C. The method presented is especially suitable for enzyme histological and immunohistological diagnosis of primary and secondary bone tumours, soft tissue tumours, as well as myelo- and lymphoproliferative disorders of bone marrow biopsies. Examples are demonstrated with mono- and polyclonal antibodies and reaction products of hydrolytic enzymes.

Translocations t(11;18)(q21;q21) and t(14;18)(q32;q21) are the main chromosomal abnormalities involving MLT/MALT1 in MALT lymphomas.

The recently discovered MLT/MALT1 gene is fused with the API2 gene in the t(11;18)(q21;q21), which characterizes about one-third of MALT lymphomas. In order to screen for variant translocations and amplifications of MLT/MALT1, we have developed a novel, undirected two-color interphase fluorescence in situ hybridization (FISH) assay with two PAC clones flanking MLT/MALT1. This assay was applied to 108 marginal zone B-cell lymphomas (MZBCLs), including 72 extranodal MALT lymphomas, 17 nodal, and 19 splenic MZBCL. In 19 MALT lymphomas (26%), but in none of the nodal or splenic MZBCL, separated hybridization signals of the MLT/MALT1 flanking probes, were found. Further FISH analyses showed that 12 of these 19 cases displayed the classical t(11;18) and the remaining seven cases revealed the novel t(14;18)(q32;q21), involving the MLT/MALT1 and IGH genes. The frequency at which these translocations occurred varied significantly with the primary location of disease. The t(11;18) was mainly detected in gastrointestinal MALT lymphomas, whereas the t(14;18) occurred in MALT lymphomas of the parotid gland and the conjunctiva. Amplification of MLT/MALT1 was not observed in any of the lymphomas analyzed. We conclude that the translocations t(11;18)(q21;q21) and t(14;18)(q21;q32) represent the main structural aberrations involving MLT/MALT1 in MALT lymphomas, whereas true amplifications of MLT/MALT1 occur rarely in MZBCL.

A new type of MAML2 fusion in mucoepidermoid carcinoma.

The present study reports for the first time a CRTC3‐MAML2 fusion gene in a mucoepidermoid carcinoma, as determined by RT‐PCR and sequencing. We screened a total of 67 formalin‐fixed, paraffin‐embedded mucoepidermoid carcinomas for the presence of chimeric genes. In one of these samples, a CRTC3‐MAML2 fusion gene was detected. Thus, this report demonstrates the existence of a fusion of MAML2 with CREB regulated transcriptional coactivator CRTC3 additional to the already known fusion of MAML2 and CRTC1. Both gene fusions seem to result in an identical tumor phenotype and the fusion genes CRTC1‐MAML2 and CRTC3‐MAML2 may play a similar role in the development of mucoepidermoid carcinomas.

A closer look at Warthin tumors and the t(11;19)

The translocation t(11;19)(q21;p13) has been described in mucoepidermoid carcinoma (MEC) and rarely in Warthin tumors (WT), both tumors of the salivary gland. The translocation creates a fusion gene in which exon 1 of CRTC1 is linked to exons 2-5 of MAML2. To verify the translocation in WT, we performed nested reverse transcriptase-polymerase chain reaction using RNA from 48 WTs. This revealed the t(11;19)(q21;p13) translocation and expression of the chimeric gene in two metaplastic WT samples, but in none of the remaining ordinary 46 WTs. On review, the two positive cases were classified as tumors highly suspect for MEC. Indeed, our experience and published observations of the t(11;19)(q21;p13) translocation in WT reveal that only a small subset of WTs are positive, and that these tumors are often classified as infarcted or metaplastic WT, known to overlap considerably with MEC on purely morphological grounds. We therefore conclude that the presence of the t(11;19)(q21;p13) rearrangement favors a diagnosis of MEC.

A new approach to demonstrate cellular activity in bone formation adjacent to implants.

Bone tissue repeatedly formed in titanium 6-aluminum 4-vanadium rabbit bone harvest implants was collected in vivo at various times between 12 days and 5 weeks. Qualitative and quantitative examinations on undecalcified thin sections were performed in the light microscope. The amount of bone tissue was calculated on routinely stained sections. Alkaline (ALP) and acid phosphatase (ACP) enzyme activities were investigated. We also performed immunohistological detection of bone matrix proteins. Increasing bone density as well as an increasing mineralization of the tissue was observed in the biopsies with increasing time. The ALP and ACP activities were similar at short times (12 days and 2 weeks). With increasing time the ALP activity was stronger than that of ACP. The results from the immunohistochemical detection of osteonectin, osteopontin, bone sialoprotein, and collagen I and II demonstrated changes in the tissue differentiation with time. The tissue formation in the canal became more mature with time of ingrowth, as observed with the various techniques used in this study. Owing to these methodical developments, undecalcified ground sections may be used for detailed analysis of various phases of tissue formation in close proximity to implants.

Selenium deficiency and fulvic acid supplementation induces fibrosis of cartilage and disturbs subchondral ossification in knee joints of mice: An animal model study of Kashin-Beck disease

Kashin-Beck disease is an acquired, chronic and degenerative osteoarticular disorder. Selenium deficiency and fulvic acid in drinking water have been implicated in the cause of this disease. Pathologically, chondronecrosis of the growth plate and articular cartilage and subconsequent disturbance of ossification were observed in the joints. In this animal model study, mice were fed with a selenium deficient diet and fulvic acid supplemented drinking water for two generations. In undecalcified histological preparations of bone we carried out histological staining to detect mineralized and unmineralized bone and cartilage. The results revealed that selenium deficiency and fulvic acid supplementation induced degeneration of the articular cartilage in the knee joints of mice. Dynamic fluorescent labelling of ossification, enzyme histochemical detection of alkaline phosphatase activity in osteoblasts and a typical immunohistochemical localization of collagens type I and II indicated the development of fibrocartilage at the articular surface of knee joints, resembling the early stages of osteoarthrosis. This became obvious by disturbed development of the articular space and meniscus, markedly impaired formation of subchondral bone and early differentiation failure during enchondral ossification. This animal model provides an approach to study the molecular pathogenesis of Kashin-Beck disease.

Subclones of C6 rat glioma cells differing in intermediate filament protein expression

The C6 rat glioma cell line is shown to consist of a mixed population of cells which either contain vimentin (80% of the cells) or completely lack any cytoplasmic intermediate filament (IF) proteins. Subclones could be established with both phenotypes, indicating that these IF protein expression patterns represent stable phenotypic markers. Absence of IF proteins in C6 subclones could consistently be correlated with an altered cell morphology and a pronounced increase in the number of actin stress fibers. In vitro translation and hybridization assays suggest the absence of vimentin to result from a block at the transcriptional level. The data indicate that subcloning of the C6 cell line on the basis of IF protein expression seems to be a reasonable approach for obtaining homogeneous C6 cell populations which may represent suitable experimental models for studies on vimentin expression and glioma cell differentiation.