Multiscale quantification of morphological heterogeneity with creation of a predictor of longer survival in glioblastoma

Intratumor heterogeneity is a main cause of the dismal prognosis of glioblastoma (GBM). Yet, there remains a lack of a uniform assessment of the degree of heterogeneity. With a multiscale approach, we addressed the hypothesis that intratumor heterogeneity exists on different levels comprising traditional regional analyses, but also innovative methods including computer-assisted analysis of tumor morphology combined with epigenomic data. With this aim, 157 biopsies of 37 patients with therapy-naive IDH-wildtype GBM were analyzed regarding the intratumor variance of protein expression of glial marker GFAP, microglia marker Iba1 and proliferation marker Mib1. Hematoxylin and eosin stained slides were evaluated for tumor vascularization. For the estimation of pixel intensity and nuclear profiling, automated analysis was used. Additionally, DNA methylation profiling was conducted separately for the single biopsies. Scoring systems were established to integrate several parameters into one score for the four examined modalities of heterogeneity (regional, cellular, pixel-level and epigenomic). As a result, we could show that heterogeneity was detected in all four modalities. Furthermore, for the regional, cellular and epigenomic level, we confirmed the results of earlier studies stating that a higher degree of heterogeneity is associated with poorer overall survival. To integrate all modalities into one score, we designed a predictor of longer survival, which showed a highly significant separation regarding the OS. In conclusion, multiscale intratumor heterogeneity exists in glioblastoma and its degree has an impact on overall survival. In future studies, the implementation of a broadly feasible heterogeneity index should be considered.     

The vagal nerve as a link between the nervous and immune system in the instance of polymicrobial sepsis

Background The role of the vagal nerve in the autonomic nervous system is widely well known. Recently, an additional function was revealed serving as a connector between the nervous and immune system. This connection is called the “cholinergic inflammatory pathway.” Through stimulation of the acetylcholine receptors located upon the macrophages, the “unspecific” immune system can be directly influenced. Methods The vagal nerve was completely transected directly posterior to its passage through the diaphragm. The effect of complete vagotomy was analyzed using a murine model of polymicrobial peritonitis (colon ascendens stent peritonitis, CASP). Survival and clinical course of vagotomized or sham-operated mice were analyzed in the CASP model. Results After CASP surgery, vagotomy led to a significantly increased mortality (64.7%) in comparison to sham-vagotomized animals (34%). No difference in the bacterial load of various tissues (lung, liver, spleen, blood, lavage fluid, and kidney) from septic animals with or without vagotomy was observed. Vagotomized animals reveal elevated serum cytokine levels (TNF, IL-6, IL-10, and MCP-1) 20 h after the induction of polymicrobial peritonitis. Conclusion The vagal nerve is therefore an important modulator of the immune system. W. Kessler and T. Traeger contributed equally to this work Best of Forum Papers presented at the Annual Meeting of the German Society of Surgery, 2–5 May 2006, Berlin, Germany     

Receptive field scatter,topography and map variability in different layers of the hindpaw representation of rat somatosensory cortex

We recorded neurons extracellularly in layers II/III, IV, and V of the hindpaw representation of primary somatosensory cortex in anesthetized rats and studied laminar features of receptive fields (RFs) and representational maps. On average, RFs were smallest in layer IV and largest in layer V; however, for individual penetrations we found substantial deviations from this rule. Within the hindpaw representation, a distinct rostrocaudal gradient of RF size was present in all layers. While layer V RFs were generally largest independent of this gradient, layer IV RFs recorded caudally representing the proximal portions of the paw were larger than layer II/III RFs recorded rostrally representing the digits. The individual scatter of the locations of RFs across laminar groups was in the range of several millimeters, corresponding to about 25% of the average RF diameter. The cutaneous representations of the hindpaw in extragranular layers were confined to the areal extent defined by responsive sites in layer IV. Comparison between RFs determined quantitatively and by handplotting showed a reliable correspondence. Repeated measurements of RFs revealed spontaneous fluctuations of RF size of no more than 5% of the initial condition over an observation period of several hours. The topography and variability of cortical maps of the hindpaw representation were studied with a quantitative interpolation method taking into account the geometric centers of RFs and the corresponding cortical recording sites. On average, the overall topography in terms of preservation of neighborhood relations was present in all layers, although some individual maps showed severe distortions of topography. Factors contributing to map variability were overall position of the representation on the cortical surface, internal topography and spatial extent. Interindividual variability of map layout was always highest in the digit representations. Local topographic orderliness was lowest in layer V, but comparable in layers II/III and IV. Within layer IV, the lowest orderliness was observed in the digit representations. Our data emphasize a substantial variability of RF size, overlap and position across layers and within layers. At the level of representational maps, we found a similar degree of variability that often co-varied across layers, with little evidence for significant layer specificity. Laminar differences are likely to arise from the specific input-output pattern, layer-specific cell types and the connectivity between different layers. Our findings emphasizing similarities in the variability across layers support the notion of tightly coupled columnar interactions between different layers.     

Voronoi Polyhedra Analysis of Optimized Arterial Tree Models

Topological and metric properties of Voronoi polyhedra (VP) generated by the distal end points of terminal segments in arterial tree models grown by the method of constrained constructive optimization (CCO) are analyzed with the aim to characterize the spatial distribution of their supply sites relative to randomly distributed points as a reference model. The distributions of the number N _f of Voronoi cell faces, cell volume V, surface area S, area A of individual cell faces, and asphericity parameter of the CCO models are all significantly different from the ones of random points, whereas the distributions of V, S, and are also significantly different among CCO models optimized for minimum intravascular volume and minimum segment length (p < 0.0001). The distributions of N _f , V, and S of the CCO models are reasonably well approximated by two-parameter gamma distributions. We study scaling of intravascular blood volume and arterial cross-sectional area with the volume of supplied tissue, the latter being represented by the VP of the respective terminal segments. We observe scaling exponents from 1.20 ± 0.007 to 1.08 ± 0.005 for intravascular blood volume and 0.77 ± 0.01 for arterial cross-sectional area. Setting terminal flows proportional to the associated VP volumes during tree construction yields a relative dispersion of terminal flows of 37% and a coefficient of skewness of 1.12. © 2003 Biomedical Engineering Society. PAC2003: 8719Uv, 8710+e, 4720Ky, 0260Pn, 0230Oz     

High diversity of ankA sequences of Anaplasma phagocytophilum among Ixodes ricinus ticks in Germany

In Germany humans with acute granulocytic ehrlichiosis have not yet been described. Here, we characterized three different genes of Anaplasma phagocytophilum strains infecting German Ixodes ricinus ticks in order to test whether they differ from strains in other European countries and the United States. A total of 1,022 I. ricinus ticks were investigated for infection with A. phagocytophilum by nested PCR and sequence analysis. Forty-two (4.1%) ticks were infected. For all positive ticks, parts of the 16S rRNA and groESL genes were sequenced. The complete coding sequence of the ankA gene could be determined in 24 samples. The 16S rRNA and groESL gene sequences were as much as 100% identical to known sequences. Fifteen ankA sequences were >/=99.37% identical to sequences derived from humans with granulocytic ehrlichiosis in Europe and from a horse with granulocytic ehrlichiosis in Germany. Thus, German I. ricinus ticks most likely harbor A. phagocytophilum strains that can cause disease in humans. Nine additional sequences were clearly different from known ankA sequences. Because these newly described sequences have never been obtained from diseased humans or animals, their biological significance is currently unknown. Based on this unexpected sequence heterogeneity, we propose to use the ankA gene for further phylogenetic analyses of A. phagocytophilum and to investigate the biology and pathogenicity of strains that differ in the ankA gene.     

The use of a double-marker shuttle vector to study DNA double-strand break repair in wild-type and radiation-sensitive mutants of the yeast Saccharomyces cerevisiae

An episomal DNA vector (YpJA18), encoding two selectable recombinant yeast genes (TRP1, URA3), was constructed to assess the fidelity of DNA repair in haploid repair-competent (RAD) wild-type yeast and several radiation-sensitive mutants. Either a DNA double-strand break (DSB) or a double-strand gap of 169 bp (DSG) was introduced by restriction enzymes in-vitro within the coding sequence of the URA3 gene of this vector. To eliminate transfer artefacts, selection was first applied for the undamaged TRP1 gene followed by counter selection for URA3 gene activity, which indicated correct repair of the DSB and DSG. Correct repair of the damaged URA3 gene was found to be about 90% in RAD cells (normalized for the expression of undamaged URA3 in TRP ⁺ transformants). Plasmids isolated from the transformants (URA ⁺ TRP ⁺) carry both unique sites (ApaI and NcoI) within the URA3 gene indicating the precise restitution of the 169-bp gap. An excision-repair-defective rad4-4 mutant repaired these lesions as correctly as RAD cells, whereas the mutants rad50-1, rad51-1 and rad54-1, proven to be defective in DSB repair and mitotic recombination, showed less than 5% correct repair of such lesions. In contrast, a representative of the RAD6 epistasis group of genes, the rev2-1 mutant which is sensitive towards UV and ionizing radiation, had a significantly reduced ability (about 20%) for the correct repair of both DSBs and DSGs.     

Marginalzonenlymphome: extranodale vom MALT-Typ, nodale und splenische

Marginal zone B-cell lymphomas (MCL) of extranodal, nodal and splenic origin appear to be different lymphoma entities with a similar growth pattern in the marginal zone of the B-follicles. Decisive for the detection of MCL as a distinct lymphoma entity was the "MALT concept" for lymphoid infiltrates in the gastric and intestinal mucosa as described by Isaacson et al. in the 1980's. Immunohistological stainings for the immunoglobulin light and heavy chains and molecular pathological studies of the immunoglobulin heavy chain gene configuration have subsequently confirmed the neoplastic nature of the extranodal infiltrates and differentiated marginal zone cells from mantle zone cells. In 1994, the MCL of MALT type as well as of nodal and splenic origin were included in the REAL classification and in 1998 in the new WHO classification for lymphomas. Meanwhile extranodal MCL of MALT-type have been observed in almost every organ and site of the body, by far most frequently in the gastric mucosa. Beside the typical growth pattern, lymphoepithelial lesions are a distinct diagnostic feature of extranodal MCL. Clinically, the small cell extranodal MCL show a very good prognosis with regression after treatment. As for nodal and splenic MCL, we need further studies to evaluate the prognostic aspects and to compare them with other B-cell lymphomas. The same is true for primary extranodal large B-cell lymphomas or blastic transformation to a large cell lymphoma; in these tumors the diagnosis of a MALT type lymphoma should only be made if a small cell component with MALT-specific criteria can be proved.     

Diffuse lymphocyte-predominant Hodgkin''s disease (diffuse paragranuloma). A variant of the B-cell-derived nodular type.

Lymph node sections from 10 cases of mixed nodular/diffuse and 10 cases of completely diffuse lymphocyte-predominant Hodgkin's disease (LPHD) were immunophenotyped. The results obtained were compared with those of nodular LPHD (nodular paragranuloma). In conventional stains, nodular/diffuse LPHD differed from diffuse LPHD in the presence of nodularity, which can be best demonstrated with silver impregnation. Immunohistologic analysis showed a correlation of the difference in nodularity with the presence or absence and pattern of follicular dendritic cell (FDC) meshwork, ie, a relatively sharply defined and large spherical meshwork was present in nodular areas of nodular/diffuse LPHD, whereas FDCs were either absent or present in a diffuse, ill-defined meshwork, usually of small size, in the diffuse zones of nodular/diffuse LPHD and in diffuse LPHD. The amount of FDC meshwork corresponded roughly to the number of reactive B cells and T cells, meaning that in diffuse areas significantly fewer B cells and more T cells were observed than in nodular areas. The immunohistologic analysis also showed that the antigen profile (positivity with the monoclonal B-cell marker L26 in the majority [14/20] of cases and negativity for CD15 in all but one of 20 cases) of the tumor cells in both nodular/diffuse LPHD and diffuse LPHD were comparable while it was different from the antigen profile (L26- and CD15+) in most cases of nodular sclerosis and mixed cellularity types of HD. This suggests that the considered subtypes of LPHD differ mainly in FDC pattern, but not in origin and nature of the tumor cells. This further justifies assignment of the above-mentioned LPHD subtypes to the category paragranuloma (LPHD).     

Irreversible shock of rats after acute renal vein thrombosis

Summary After occlusion of the renal veins rats die quickly in progressive shock (within 4.5 h), but after ligating the renal hilum of both Kidneys they survive 27 h. To learn why renal vein occlusion is so rapidly lethal, and what substances are given off and by what method from the hemorrhagically infarcted kidneys, we studied eight groups of rats, each containing at least seven animals. The groups differed in the combination of hilar structures (renal veins, ureters, lymphatics) ligated. We compared: survival times, changes in blood pressure, blood volume, levels of plasma kinins, adenosine, and lactate, changes of blood pH, responses to Indomethacin, Trasylol^®, and plasma expanders, tubular and capillary flow rates, histopathological changes in organs and cerebral blood flow and changes in the blood coagulation system. Our results suggest that the venous stasis, anoxia, and hemorrhagic necrosis caused by bilateral venous occlusion release into renal lymphatics toxic substances which reach the systemic circulation and induce irreversible shock. We have excluded prostaglandins and adenosine as the toxic substances inducing shock but could not rule out an action of the kallikrein-kinin-system. We postulate that the striking degenerative changes occurring in the arterioles of the brain after bilateral venous occlusion may mean these vessels are especially susceptible to high levels of lactic acid and that this may explain why these animals die so quickly. Our conclusions should help not only in understanding why high levels of lactate in shock portend a poor prognosis but also help in formulating appropriate therapy for circulatory failure of renal origin and for protracted hypotension after extensive tissue injury.The studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres SystemPresented in part: Jäckh and Steinhausen, 1976; Dallenbach et al., 1978; Zimmerhackl et al., 1979We dedicate this paper to Wilhelm Doerr, Dr. med., Professor of Pathology, University of Heidelberg on the occasion of his 65th birthday (August 25th, 1979)