Morpho-functional analysis of patient-derived xenografts reveals differential impact of gastric cancer and chemotherapy on the tumor ecosystem, affecting immune check point, metabolism, and sarcopenia.

OBJECTIVES: Gastric cancer (GC) is an aggressive disease due to late diagnosis resulting from the lack of easy diagnostic tools, resistances toward immunotherapy (due to low PD-L1 expression), or chemotherapies (due to p53 mutations), and comorbidity factors, notably muscle atrophy. To improve our understanding of this complex pathology, we established patient-derived xenograft (PDX) models and characterized the tumor ecosystem using a morpho-functional approach combining high-resolution imaging with molecular analyses, regarding the expression of relevant therapeutic biomarkers and the presence of muscle atrophy. MATERIALS AND METHODS: GC tissues samples were implanted in nude mice. Established PDX, treated with cisplatin or not, were imaged by magnetic resonance imaging (MRI) and analyzed for the expression of relevant biomarkers (p53, PD-L1, PD-1, HER-2, CDX2, CAIX, CD31, a-SAM) and by transcriptomics. RESULTS: Three well-differentiated, one moderately and one poorly differentiated adenocarcinomas were established. All retained the architectural and histological features of their primary tumors. MRI allowed in-real-time evaluation of differences between PDX, in terms of substructure, post-therapeutic changes, and muscle atrophy. Immunohistochemistry showed differential expression of p53, HER-2, CDX2, a-SAM, PD-L1, PD-1, CAIX, and CD31 between models and upon cisplatin treatment. Transcriptomics revealed treatment-induced hypoxia and metabolic reprograming in the tumor microenvironment. CONCLUSION: Our PDX models are representative for the heterogeneity and complexity of human tumors, with differences in structure, histology, muscle atrophy, and the different biomarkers making them valuable for the analyses of the impact of platinum drugs or new therapies on the tumor and its microenvironment.

PO-33 - Heparin suppresses progression of small cell lung cancer (SCLC) in an orthotopic mouse model.

INTRODUCTION: Lung cancer is the leading cause of cancer-related death worldwide. Small cell lung cancer (SCLC) is thereby a highly aggressive neuroendocrine carcinoma representing about 15% of all lung cancer cases. Due to the highly metastatic behavior and multidrug resistance, the long-term survival of patients is very low. AIM: Current clinical studies revealed an increased survival of SCLC patients treated with heparin. Thus, the role of heparin in SCLC progression was analyzed with the focus on cell adhesion, cell survival and metastasis formation. MATERIALS AND METHODS: Heparins were tested for their capacities to alter migration, adhesion and viability of SCLC cells in vitro as well as tumor growth and metastasis formation in vivo. RESULTS: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) both strongly inhibited migration as well as adhesion of SCLC cells to fibronectin and stromal cells. In addition, Heparin induced cellular apoptosis and also increased apoptotic effects of conventional chemotherapeutics in vitro. To investigate the role of LMWH on metastasis formation in vivo, an orthotopic xenograft mouse model with spontaneous metastasis formation has been established. The primary tumors in this mouse model show a marked capacity to metastasize to characteristic distant organs, reflecting advanced steps of malignant progression. Treatment of tumor-bearing mice with LMWH suppressed progression of SCLC. CONCLUSIONS: Administration of LMWH in addition to the conventional treatment might reduce metastasis formation and development of chemoresistance, leading to an improved survival rate of patients suffering from SCLC.

Impact of milk fish farming in the tropics on potentially pathogenic vibrios.

Ratios of sucrose-negative to sucrose-positive vibrios on TCBS agar (suc-/suc+) indicate the abundance of potential human pathogenic non-cholera vibrios in coastal mariculture environments of the Lingayen Gulf (Philippines. In guts of adult maricultured milkfish (Chanos chanos) of suc- vibrios reached extreme peak values ranging between 2 and 545 million per g wet weight. Suc- vibrios outnumbered suc+ vibrios in anoxic sediments, too, and were rarely predominant in coastal waters or in oxidized sediments. Suc-/suc+ ratios in sediments increased toward the mariculture areas with distance from the open sea at decreasing redox potentials. There is circumstantial evidence that suc- vibrios can be dispersed from mariculture areas to adjacent environments including coral reefs. An immediate human health risk by pathogenic Vibrio species is discounted, since milkfish guts contained mainly members of the Enterovibrio group. A representative isolate of these contained proteolytic and other virulence factors, but no genes encoding toxins characteristic of clinical Vibrio species.

Electron-phonon coupling in the conventional superconductor YNi2B2C at high phonon energies studied by time-of-flight neutron spectroscopy.

We report an inelastic neutron scattering investigation of phonons with energies up to 159 meV in the conventional superconductor YNi(2)B(2)C. Using the sweep mode, a newly developed time-of-flight technique involving the continuous rotation of a single crystal specimen, allowed us to measure a four-dimensional volume in (Q, E) space and, thus, determine the dispersion surface and linewidths of the A(1g) (≈102 meV) and A(u) (≈159 meV) type phonon modes over the whole Brillouin zone. Despite of having linewidths of Γ=10 meV, A(1g) modes do not strongly contribute to the total electron-phonon coupling constant λ. However, experimental linewidths show a remarkable agreement with ab initio calculations over the complete phonon energy range, demonstrating the accuracy of such calculations in a rare comparison to a comprehensive experimental data set.

Spleen tyrosine kinase (Syk) is a potent target for GvHD prevention at different cellular levels.

Acute graft-versus-host disease (GvHD) limits the applicability of allogeneic hematopoietic cell transplantation for the treatment of leukemia. GvHD occurs as a consequence of multiple activating events in antigen-presenting cells (APCs) and T cells (Tcs). Spleen tyrosine kinase (Syk) is an intracellular non-receptor tyrosine kinase involved in multiple signaling events of immune cells. Therefore, we hypothesized that Syk may be a promising target to inhibit GvHD, which involves activation of different immune cell populations. In vivo expansion of luciferase(+) donor Tcs in mice developing GvHD was reduced by treatment with the Syk inhibitor Fostamatinib, which led to increased survival and reduced histologically confirmed GvHD severity. Importantly, in vivo and in vitro cytotoxicity against leukemia target cells and anti-murine cytomegalovirus immune responses were not impacted by Fostamatinib. In APCs Syk inhibition reduced the expression of costimulatory molecules and disrupted cytoskeletal organization with consecutive APC migratory defects in vitro and in vivo while phagocytic activity remained intact. On the basis of these immunomodulatory effects on different cell populations, we conclude that Syk targeting in alloantigen-activated Tcs and APCs with pharmacologic inhibitors, already applied successfully in anti-lymphoma therapy, has clinical potential to reduce GvHD, especially as anti-leukemia and anti-viral immunity were preserved.

Direct observation of the superconducting gap in phonon spectra.

We show that the superconducting energy gap 2Delta can be directly observed in phonon spectra, as predicted by recent theories. In addition, since each phonon probes the gap on only a small part of the Fermi surface, the gap anisotropy can be studied in detail. Our neutron scattering investigation of the anisotropic conventional superconductor YNi2B2C demonstrates this new application of phonon spectroscopy.

Aromatic compound degradation by iron reducing bacteria isolated from irrigated tropical paddy soils.

Forty-six candidate phenol/benzoate degrading-iron reducing bacteria were isolated from long-term irrigated tropical paddy soils by enrichment procedures. Pure cultures and some prepared mixed cultures were examined for ferric oxide reduction and phenol/benzoate degradation. All the isolates were iron reducers, but only 56.5% could couple iron reduction to phenol and/or benzoate degradation, as evidenced by depletion of phenol and benzoate after one week incubation. Analysis of degradative capability using Biolog MT plates revealed that most of them could degrade other aromatic compounds such as ferulic acid, vanillic acid, and hydroxybenzoate. Mixed-cultures and soil samples displayed greater capacity for aromatic degradation and iron reduction than pure bacterial isolates, suggesting that these reactions may be coupled via a consortia-based mechanism in paddy soils.

Pronounced in-plane anisotropy of phonon anomalies in YBa(2)Cu(3)O(6.6).

The dispersion of the Cu-O bond-stretching and bond-bending vibrations in YBa(2)Cu(3)O(6.6) has been studied by high resolution inelastic neutron scattering. While the behavior of the bond-bending vibrations can be well accounted for by a simple potential model, the bond-stretching vibrations show a highly anomalous behavior. The displacement pattern of the most anomalous phonons is in principle consistent with dynamic charge stripe formation. However, charge stripes would have to extend along the a axis and not the b axis as inferred from the magnetic fluctuations by Mook et al. [Nature (London) 404, 729 (2000)].

Iron content in musculus longissimus lumborum et thoracis (m.l.l.t.) of fattening pigs.

Pork is among the most essential dietary sources of iron in Germany, considering its beneficial absorption rate. During 1997 and 1998 meat samples from 52 pigs per year were collected at the Thuringian performance testing station in order to examine the total iron content of pork. Musculus longissismus lumborum et thoracis (13./14. thoracic vertebra) from the right side of the carcasses were prepared for analyses. The total iron content was measured by atomic emission spectroscopy with inductively coupled plasma. The iron content of the meat was 4.18 +/- 0.97 mg/kg fresh material. This result was significantly lower than corresponding data from literature. An influence of the genetic composition of the pigs examined on the total iron content could be observed. The daily gain (R = -0.26) and intramuscular fat content (R = -0.28) were inversely correlated to the iron content. However, the lean meat proportion (R = 0.26) as well as haem pigment content (R = 0.27) were directly correlated to the total iron content of the meat.

Purification and biochemical characterization of a basic superantigen (SPEX/SMEZ3) from Streptococcus pyogenes.

A potent basic superantigen (designated streptococcal pyrogenic exotoxin X, SPEX/SMEZ3) was purified to homogeneity from culture supernatants of a Streptococcus pyogenes scarlatina strain of type 12 (genotype speA(-), speC(-)) and characterized. Sequence alignments revealed SPEX to be an allele of the streptococcal mitogens type Z (SMEZ). The N-terminal amino acid sequence of SPEX was found with LEVDNNSLLR to be identical to the recently described acidic superantigen SMEZ. Although SPEX/SMEZ genes were present in all of the streptococcal strains tested, a toxin production could only be detected in a small number of strains. The produced toxin concentration in the culture supernatants of positive strains differed between 0 and 20 ng ml(-1). The purified SPEX stimulated human T-lymphocytes with Vbeta8 specificity at extremely low concentrations (lower than 100 pg ml(-1)).

Bronchoscopic aspects of renal cell carcinoma (RCC).

A symposium on RCC in July 1998 in Tübingen was where we to reviewed our patients with endobronchial metastatic RCC treated by bronchoscopy since 1981. 24 of 26 consecutive cases are the subject of the present study. Tumor history, X-ray findings, endobronchial appearance, treatment and the pathogenetic mechanisms of this special form of metastatic spread are demonstrated and discussed. In 5 patients > 10 years relapsed between nephrectomy and bronchoscopic tumor therapy. A thrombuslike tumor growth and bleeding were striking bronchoscopic features. In X-ray besides atelectasis, hilar or mediastinal masses were seen half of the patients, in 50% without parenchymal lung nodes, suggesting lymphogenic spread. In several patients bronchial recanalisation could be successfully repeated over long periods. Abnormal X-ray, haemoptysis or dyspnoa in patients with RCC demands bronchoscopy. In case of bronchial tumor obstruction bronchoscopic treatment offers excellent palliation.

Estimating microbial population counts by 'most probable number' using Microsoft Excel.

A computer-assisted method for determining population counts using the 'most probable number' (MPN) was developed. The Microsoft Excel spreadsheet and its Solver tool were used to generate MPNs, error estimates and confidence limits. Our method was flexible, allowing the use of unbalanced replication schemes and varying replication numbers and inoculation volumes. Furthermore, it required no programming skills and generated fast results, which were comparable to those of standard MPN tables and MPN software.

Extracellular superoxide dismutase from Streptococcus pyogenes type 12 strain is manganese-dependent.

Highly purified extracellular superoxide dismutase was obtained from Streptococcus pyogenes strain 12,714 (type 12) by adsorption of culture supernatant on phenyl-Sepharose following preparative isoelectric focusing of eluates and a final gel filtration purification on Superdex 200. The purified superoxide dismutase of S. pyogenes was found to be a homodimer. The monomeric protein had a molecular mass of 22,442 Da and an isoelectric point of 4.0. The enzymatic activity was strongly manganese-dependent. The N-terminal sequence of the purified mature protein was AIILPELPYAYDALEPQUFDA and corresponded to the first amino acids following the methionine initiation codon with no evidence of a leader sequence for the mature protein. The DNA sequence of the superoxide dismutase gene of strain 12,714 was found to be almost identical to the corresponding sequences reported in the gene bank data from other S. pyogenes serotypes and showed strong homology to superoxide dismutases from other Gram-positive bacteria.

Use of an oxygen-insensitive microscale biosensor for methane to measure methane concentration profiles in a rice paddy.

An oxygen-insensitive microscale biosensor for methane was constructed by furnishing a previously described biosensor with an oxygen guard. The guard consisted of a glass capillary containing heterotrophic bacteria, which consumed oxygen diffusing through the tip membrane, thus preventing it from diffusing into the methane-sensing unit. Oxygen microprofiles were measured through the oxygen guard capillary, demonstrating the principle and limitations of the method. When the tip of the guard capillary was exposed to 100% oxygen at 21 degrees C, heterotrophic oxygen consumption prevented oxygen from diffusing further than 170 mum into the capillary, whereas atmospheric levels of oxygen were consumed within 50 mum. The capacity of the oxygen guard for scavenging oxygen decreased with decreasing temperature, and atmospheric levels of oxygen caused oxygen penetration to 200 mum at 5 degrees C. The sensors could be manufactured with tip diameters as small as 25 mum, and response times were about 1 min at room temperature. Pore water profiles of methane concentrations in a rice paddy soil were measured, and a strong correlation between the depths of oxygen penetration and methane appearance was observed as a function of the light regimen; this finding confirmed the role of microbenthic photosynthesis in limiting methane emissions from surfaces of waterlogged sediments and soils.

C repeats of the streptococcal M1 protein achieve the human serum albumin binding ability by flanking regions which stabilize the coiled-coil conformation.

The M and M-like proteins of Streptococcus pyogenes are fibrous cell surface proteins. They have multiple binding sites for several human proteins and are composed of the C-terminal anchor domain, the alpha-helical coiled-coil domain, and the N-terminal non-coiled-coil domain. The coiled-coil domain of the M1 protein consists of repeat units called B, C, and D and a spacer unit S between B and C. Recombinant fragments A-B-S-C-D, A-B-S, B-S-C, S-C, S-C-D, C-D, and C of the coiled-coil domain were studied by analyzing their secondary structures and binding affinities to human serum albumin (HSA). As shown by circular dichroism, all fragments are in an alpha-helical conformation. C-D and S-C-D form coiled coils at room temperature and bind below 37 degrees C with high affinity to HSA. C-D and S-C-D unfold in two steps with Tm values of approximately 31 and approximately 65 degrees C; complex formation with HSA increases the unfolding temperatures. B-S-C has a lower alpha-helical content, a less pronounced coiled-coil conformation, and a reduced thermal stability, binds HSA weaker, and is only slightly stabilized by HSA binding in comparison to C-D and S-C-D. C and S-C are less stable than the other fragments and are not organized as coiled coils showing some features of alpha-helical single strands only below 20 degrees C, and binding of HSA was not observed. The results indicate that the formation of coiled-coil structures, supported by flanking D regions and, to a lesser extent also B regions, is essential for the binding of C repeat units to HSA.

Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.