Vesico-intestinal fistulas, vesico-sigmoid fistula in the course of sigmoid cancer.
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Adenocarcinoma↗
Biomedical subjects
G Wallner
Publications and source records attributed to G Wallner.
Nucleolar organizer regions (NORs) as a prognostic factor in patients with squamous-cell oesophageal cancer.
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Adult↗
Flow cytometric analysis of the in situ accessibility of Escherichia coli 16S rRNA for fluorescently labeled oligonucleotide probes.
In situ identification of whole fixed bacterial cells by hybridization with fluorescently labeled, rRNA-targeted oligonucleotide probes is often limited by low signal intensities. In addition to an impermeability of the cell periphery and a low cellular rRNA content, the three-dimensional structure of the ribosome may hinder the access of oligonucleotides to their target sites. Until now, a systematic study on the accessibility of 16S rRNA target sites had not been done. Here, we report fluorescence intensities obtained with more than 200 oligonucleotide probes (mostly 18-mers) used with whole fixed cells of Escherichia coli DSM 30083(T). Two overlapping sets of adjacent oligonucleotides, 171 in total, were designed to cover the full length of the 16S rRNA. The two sets are shifted by 5 to 13 nucleotides. The probes were labeled with carboxyfluorescein, and signal intensities of hybridized cells were quantified by flow cytometry. Care was taken that the signal intensity of cells was dependent solely on the in situ accessibility of probe target sites. The brightest signal resulted from probe Eco1482, complementary to positions 1482 to 1499. With this probe, the fluorescence was 1.7 times brighter than that of the standard bacterial probe EUB338 and 44 times brighter than that of the worst probe, Eco468. The distribution of probe-conferred cell fluorescence in six arbitrarily set brightness classes (classes I to VI; 100 to 81%, 80 to 61%, 60 to 41%, 40 to 21%, 20 to 6%, and 5 to 0% of the brightness with Eco1482, respectively) was as follows: I, 4%; II, 14%; III, 21%; IV, 29%, V, 19%; and VI, 13%. A more detailed analysis of helices 6, 18, and 23 with additional probes demonstrated that a shift of the target region by only a few bases could result in a decline of cell fluorescence from >80 to <10%. Considering the high evolutionary conservation of 16S rRNA, the in situ accessibility map of E. coli should facilitate a more rational selection of probe target sites for other species as well.
Base Sequence↗
Cytokine gene-modified tumor cells for prophylactic and therapeutic vaccination: IL-2, IFN-gamma, or combination IL-2 + IFN-gamma.
Murine melanoma cells were engineered to express interleukin-2 (IL-2), interferon-gamma (IFN-gamma) or both cytokines at various dose levels by means of the adenovirus-enhanced transferrinfection (AVET) method. The gene-modified cells were tested for their potency to induce an antitumor immune response in two experimental settings with different tumor load. In a prophylactic vaccination model, both IL-2 and IFN-gamma showed a dose-dependent protection against tumor cell challenge in two melanoma models. In the therapeutic vaccination model, where mice with measurable tumors were treated, immunization with IL-2 or IFN-gamma gene-modified cells led to complete tumor regression in 30% or 20% of the tumor-bearing animals respectively. The combination of IL-2 + IFN-gamma resulted in complete tumor regression in up to 50% of the tumor-bearing mice.
Animals↗
Sustained cytokine delivery for anticancer vaccination: liposomes as alternative for gene-transfected tumor cells.
Vaccination with tumor cells genetically engineered to produce interleukin (IL)-2 is an attractive strategy to enhance antitumor immune responses. The improved antitumor immunity upon vaccination with IL-2 gene-modified tumor cells may be due to the prolonged presence of the cytokine at the vaccination site. Because liposomes have been used for sustained delivery of a variety of agents, we compared the protective effect of vaccines consisting of IL-2 gene-modified B16 melanoma cells to that of vaccines composed of IL-2 liposomes and irradiated melanoma cells. The results indicate that both approaches equally protect against a lethal challenge with B16 melanoma cells. More than 20% of the protected animals developed vitiligo at the vaccination and/or tumor challenge site.
Animals↗
Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery.
Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.
Animals↗
Flow sorting of microorganisms for molecular analysis.
Not only classical cultivation-based methods but also the new molecular approaches may result in incomplete and selective information on the natural diversity of microbial communities. Flow sorting of microorganisms from environmental samples allows the deliberate selection of cell populations of interest from highly diverse systems for molecular analysis. Several cellular parameters that can be measured by flow cytometry are useful as sort criteria. Here, we report sorting of bacteria from activated sludge, lake water, and lake sediment according to differences in light scattering, DNA content, and/or affiliation to certain phylogenetic groups as assessed by fluorescein-labeled, rRNA-targeted oligonucleotide probes. Microscopy of the sorted cells showed that populations of originally low abundance could be strongly enriched by flow sorting (up to 280-fold), depending on the original abundance of the cells of interest and the type of sample sorted. The purity of the cells of interest could be further increased by repeated sorting, but this increase was limited by cell aggregation in the case of activated-sludge samples. It was possible to amplify almost full-length 16S ribosomal DNA (rDNA) fragments from sorted microbial cells by PCR, even after fixation with paraformaldehyde and in situ hybridization. Dot blot hybridization and sequencing demonstrated that most of the amplified rDNA originated from those cells that had been selected for by flow sorting. Comparative analysis of 16S rDNA sequences revealed previously unknown species of magnetotactic or activated-sludge bacteria.
Bacteria↗
[Stomach and esophageal neoplasm. Changes over 16 years in clinical materials].
Increasing prevalence of adenocarcinoma of the esophagus and esophago-gastric junction has been reported. The aim of the study was to determine whether this phenomenon is reflected in the cohort of patients referred for surgery to our institution. Clinical and pathological records of patients with adenocarcinoma of the stomach (n = 433) or gastro-esophageal junction (n = 302), and squamous cell carcinoma of the esophagus (n = 266) were reviewed from 1981 to 1996. Yearly prevalence of carcinoma of the gastric cardia in comparison to carcinoma of (a more distal) stomach has not changed, ranging 19-46%. From 1981 to 1984 out of 58 gastric resections, 14 (24%) total gastrectomies were done, whereas from 1993 to 1996 total gastrectomies were performed in 104 out of 138 (75%) patients with gastric cancer (p < 0.001). In the first 4 years of the study period adenocarcinoma of the cardia and/or esophagus was found in 19% of all patients with esophageal and junctional tumors, while in the last 4 years,-in 30%. Resection rates for gastric and cardiac cancers have not changed significantly, 75-100% and 21-65% respectively. Resection rate for carcinoma of the esophagus increased from 50% (17/34) to 79% (53/67) (p = 0.006, test chi2). Increasing rate of total gastrectomies can be explained by a trend towards more proximal localisation of the primary gastric tumors and/or clinical application of Laurén classification for the choice of operative procedure. Higher resection rate for carcinoma of the esophagus is a result of increasing experience of the surgical team, improvement in preoperative staging, new palliative modalities, and application of preoperative chemo-/radiotherapy.
Carcinoma, Squamous Cell↗
Topography of Helicobacter pylori infection in gastric cancer patients.
The role of Helicobacter pylori in gastric carcinogenesis is a subject of an increasing interest. In this report we describe a prospective study on the resected stomachs to establish the prevalence of H. pylori in different types of gastric carcinoma. The material consisted of 62 consecutive patients operated on stomach adenocarcinomas Fifty six percent of the patients were intestinal type, 34%--diffuse type and 10%--mixed type. The presence of H. pylori was studied in specimens from surgically removed stomachs. The conformation of the bacterial infection was done by means of rapid urease test, microbiological culture, Warthin-Starry and immunohistochemical staining. The overall prevalence of H. pylori infection was 69% (43/62). There was a statistically significant difference in the infection rates between the types of carcinoma--75% in the intestinal type and 62% in the diffuse type. The most sensitive was immunohistochemical staining. The bacterial colonies were cumulated far from the tumor tissue. In cardiac cancer the most intense of infection was an antrum and lower part of gastric body. In opposite; in antrum and pylorus cancer the scope of colonisation increased in fundus and subcardiac region with statistical signification. We could not detect H. pylori in the tumor tissue itself as in the normal mucosa of the stomach. In gastric antrum the most intense colonisation was detected on mucosal atrophy, but in the upper part of the stomach--on the mucosal metaplasia.
Adult↗
[Correlations between risk factors for metastasis in early tumors of the large intestine].
UNLABELLED: Intramucosal colorectal cancers (Tis) are treated by local excision. Controversies remain as to the management of patients with the submucosally invasive colorectal cancers (EIC). The decision for radical surgery or local excision depends on an estimate of the risk of metastases. AIM: To estimate correlations between the risk factors for metastases in EIC and to evaluate the tests that identify the metastasising EICs. In the study, we investigated the relationships between selected prognostic factors (the gross appearance, depth of submucosal invasion, vessel invasion, grade, presence of adenoma component, and p53 overexpression) and the frequency of metastases in 64 EICs. STATISTICAL ANALYSIS: Fisher's exact test and log-linear analysis. Tests' evaluation: sensitivity, specificity, predictive values. The metastases were detected in 11 tumours (17%). The metastases were found with the significantly (p < 0.05) higher frequency in moderately differentiated adenocarcinomas, in tumours with massive invasion, with vessel invasion, and with the p53 overexpression. The only interaction was between the grade and the depth of invasion. No other interaction between the risk factors was found with the multivariate analysis. The evaluation of the depth of invasion was characterised by the highest sensitivity in detecting metastases, and by the highest negative predictive value. The highest specificity and the positive predictive value were found for the evaluation of vessel invasion. The massive submucosal invasion, vessel invasion, moderately differentiated characteristics, and the p53 overexpression seem to be the independent risk factors for metastases in EIC. The evaluation of these factors should be helpful as prognostic tests and for the proper choice of the treatment modality for the patients with EIC.
Adenocarcinoma↗
[Postoperative evaluation of esophageal mucosal change dynamics in patients with reflux diseases].
Comparative value of esophagoscopy and biopsy in correlation with 24 hour esophageal pH metry parameters were assessed in 53 consecutive patients operated on due to GERD. The special attention was paid to the type and intensity of esophageal mucosa injury and the quantity of the pathological gastroesophageal reflux. The therapeutic effect of the Nissen Rossetti fundoplication on postoperative changes in esophageal mucosa was also estimated on the average 18 months after the antireflux procedure. Pre and postoperative efficiency of the antireflux mechanism and patients conditions were evaluated on the grounds of anamnesis, physical examination, x-ray examination, endoscopy with biopsy, 24 hours pH metry, LES and body of the esophagus manometry. The results were shown as mean and standard deviation. Statistical significance was determined using SPSS/PC-packet, Smirnow-Kolmogorow and Wilcoxon test, with p values < 0.05 considered as significant. After the operation complete and durable relief of reflux symptoms was obtained in 83% patients, with high significant decrease of DeMeester score from 21.15 +/- 12.19 to 3.43 +/- 5.22, p < 0.0002. Endoscopy following the operation confirmed significant regression of the GERD pathology, mainly in II and III degree according to Savary Miller classification, from 98.1% to 18.9%, p < 0.005. In comparison with other types of the pathology, Barrett's esophagus was always connected with significant higher quantity of the pathological gastroesophageal reflux before as well as after the operation. Biopsy following the operation revealed not significant regression of mucosal GERD lesions in 54.7%. The healing changes were observed first of all in the most severe esophageal injuries-chronic squamous esophagitis (64%), CLE except intestinal type of metaplasia (86.&%) and low grade dysplasia of squamous epithelium (100%). The absence of regression after the operation was always combined with unsatisfactory decrease of 24 hour pH metry parameters: DeMeester score, percentage of total time pH < 4.0 and time pH < 4.0 in supine position. Postoperative progression of GERD pathology was noticed in 12.6% patients. One third patients had the same biopsy outcome before and after the operation, even in those without any GERD complaints. Biopsy in correlation with 24 hour pH metry analysis similarly like endoscopy revealed in CLE twice higher DeMeester score than squamous esophagitis, hyperplasia or normal esophageal mucosa. The study proved that more advanced stages of GERD pathology were connected with higher quantity of the gastroesophageal reflux. The results presented in this study confirmed high efficacy of the Nissen Rossetti fundoplication in surgical treatment of GERD. We would to emphasize that with unsatisfactory biopsy results following the operation should be the reason for permanent follow up endoscopic and histopathologic verification.
Adult↗
[The effect of Nissen-Rossetti fundoplication on the motor function of swallowing in patients with reflux diseases].
This prospective study was undertaken to assess the motility of the middle and lower esophagus and LES resting pressure changes following the N-R fundoplication for GER. The study was carried out in 53 consecutive patients (37 women, 16 men, median age 49.1 +/- 6.2 years), operated due to antireflux mechanism insufficiency. Threefold solid state for manometry and double channel for pH metry catheters in the body of the esophagus were used before and after the operation (median follow up time was 18 months). The LES length and resting pressure were evaluated with Synectics On-line interface. The objective results obtained during 24-h pH metry and manometry were shown as median and standard deviation, with statistical significances determined using the SPSS/PC+ packet (p < 0.05 considered as significant). The individual patients' data were analyzed by Synectics software. 24-h pH metry confirmed that Nissen-Rossetti fundoplication efficaciously reduced pathological GER (significant decrease of DeMeester score from 105.6 +/- 12.2 to 5.2 +/- 3.4, p < 0.0002). Manometry proved the increase of the LES resting pressure from 7.6 +/- 3.2 to 18.2 +/- 4.2, p < 0.0005, and its total length from 3.1 +/- 0.8 to 4.2 +/- 1.0, p < 0.01. The improvement of the esophageal body peristalsis was found first of all in total period. Motility changes following N-R fundoplication during the reflux period were unsatisfactory. The frequency of peristalic contractions with higher amplitude and longer duration significantly increased after the operation in total period in all levels. The increase of the complete peristaltic contractions following N-R fundoplication was high significant from 22.15% to 60.21%, p < 0.0001. Although significant improvement of the efficacy of esophageal peristalis was observed, however it was still under 50% of normal level all the time (15.4% v. 41.7%, p < 0.002). The most impressive improvement of esophageal peristalis was noticed in the lower part of the esophagus. The increase of the complete peristaltic contractions during the reflux period was observed only in upright position. Least profitable changes in esophageal body motility were noticed during the reflux period in supine position. In comparison with the total period manometry results the efficacy of the esophageal perystalsis during the reflux period was defected before as well as after the operation, (11.2% v. 22.5%, p = NS).
Adult↗
[Prognostic significance of cellular immunity in surgical treatment of esophageal cancer].
The aim of the study was to investigate the influence of perioperative changes of the cellular immunity parameters on long-term survival following esophagectomy for carcinoma of the esophagus. In 39 patients several immunological parameters have been assessed before and 2 weeks after the operation: WBC, lymphocyte count, lymphocyte T% (E-rosette test), helper/suppressor lymphocyte T subpopulations (teophylline test), NBT test, and in vivo Multitest CMI (skin delayed hypersensitivity for recall antigens). Survival was analyzed using Kaplan-Meier method according to the stage (pTNM, UICC'92) and to the grade of perioperative cellular immunity (pCI) deterioration. Three groups of patients were identified with: 1) improvement or stabilization of pCI (n = 5); 2) deterioration of 1 or 2 pCI parameters (n = 21); and 3) deterioration of 3 or more pCI parameters (n = 13). Follow-up time ranged from 4 to 65 months. Eight patients died in hospital due to postoperative complications, 3 patients are alive (1 of them with Tis alive at 52 months of follow-up), and the remaining patients died during follow-up. Mean survival time of patients in stage II (n = 7), III (n = 17), and IV (n = 14) was 18, 17, and 6 months respectively (p = 0.017, log rank test). Mean survival time of patients in group 1, 2, and 3 was 21, 20, and 5 months respectively (p < 0.001, log rank test). On bivariate survival analysis encountering both factors, stage was found to be the only independent prognostic factor (p = 0.008, Cox regression model). Deterioration of the cellular immunity in the perioperative period is entirely related to the stage of the disease, which is the only independent prognostic factor in carcinoma of the esophagus.
Adenocarcinoma↗
Characterization and complete genome sequences of high- and low- virulence variants of tick-borne encephalitis virus.
The entire genomic sequences of two strains (Hypr and 263) of the flavivirus tick-borne encephalitis (TBE) virus differing in virulence from the prototypic strain Neudoerfl were determined. Strain Hypr is a human isolate of TBE virus with a high laboratory passage history which exhibits a significantly higher neuro-invasiveness in mice compared to the prototype strain. Strain 263 is a low-passage tick-isolate with a temperature-sensitive and attenuated phenotype. Except for the heterogeneous 3' non-coding regions strains Hypr and 263 share, respectively, 97.2% and 97.6% nucleotide sequence identity with strain Neudoerfl, and differ by a total of 42 and 36 amino acids from the prototypic strain. Of these, only 12 amino acids for each of the two strains represent non-conservative differences unique to an individual strain and some of these are located at positions highly conserved among flaviviruses. Based on these observations, the potential biological significance of particular sequence differences is discussed in the context of the current knowledge about molecular determinants of flavivirus virulence.
Animals↗
The flavivirus 3'-noncoding region: extensive size heterogeneity independent of evolutionary relationships among strains of tick-borne encephalitis virus.
The sequences of the 3'-noncoding regions (NCR) of 12 strains of tick-borne encephalitis (TBE) virus were analyzed and found to vary in length from 350 to approximately 750 nucleotides. The size heterogeneity is restricted to a variable region following the stop codon, whereas the most 3'-terminal 350 nucleotides form a highly conserved core element containing several potentially important sequence motifs and secondary structure elements. A homoadenosine tract previously thought to form the 3'-terminus of some TBE virus strains was now shown to be an internal part of the variable region of certain strains. The strains included in this study were isolated from both humans and ticks over a time period of more than 40 years at various locations throughout the entire endemic area of TBE virus, but there was no correlation between these parameters and the observed lengths of the 3'-NCRs. Identity data calculated from common 3'-NCR sequences and also from short sections of the open reading frame indicated that coding and noncoding sequences were linked during evolution, but the lengths of the 3'-NCRs were independent of these relationships. These observations together with detailed analyses and alignments of the sequences suggest that the variable region was originally acquired through duplication and recombination events, but--much more recently during evolution--various portions of this region were lost again, resulting in the now observed heterogeneous 3'-NCRs.
Animals↗
Flow cytometric analysis of activated sludge with rRNA-targeted probes.
Samples from a wastewater treatment plant were hybridized with fluorescein-labeled oligonucleotide probes specific for members of the domains Bacteria and Eucarya; the alpha, beta, and gamma subclasses of the class Proteobacteria; or the genus Acinetobacter. Subsequently, they were counterstained with the DNA-specific dye Hoechst 33342 and analyzed by flow cytometry. By quantifying forward angle light scatter and Hoechst- and probe-conferred fluorescence as measures for cell size, DNA content, and rRNA content, respectively, not only relative abundances but also assessments of general metabolic activity for each of these groups were obtained. Hybridizations with a positive control probe binding to all bacteria showed that in the activated-sludge samples examined, 70 to 80% of the Hoechst-stained cells could unambiguously be identified by this method. The majority of the detected cells (approximately 40%) were beta-subclass Proteobacteria. Flow cytometric and microscopic counts were in general agreement. Discrepancies were found in particular for those populations that occurred predominantly in flocs (alpha subclass of the Proteobacteria) or chains (Acinetobacter spp.). Although the dispersal of aggregates needs to be improved, flow cytometry combined with rRNA-based in situ probing appears to be a powerful tool for the rapid and highly automated analysis of the microbial communities in activated sludge.
Acinetobacter↗
Is gastric cancer heterogeneity the clue to HLA-DR associated susceptibility?
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Adult↗
Optimizing fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms.
A combination of fluorescent rRNA-targeted oligonucleotide probes ("phylogenetic stains") and flow cytometry was used for a high resolution automated analysis of mixed microbial populations. Fixed cells of bacteria and yeasts were hybridized in suspension with fluorescein- or tetramethylrhodamine-labeled oligonucleotide probes complementary to group-specific regions of the 16S ribosomal RNA (rRNA) molecules. Quantifying probe-conferred cell fluorescence by flow cytometry, we could discriminate between target and nontarget cell populations. We critically examined changes of the hybridization conditions, kinetics of the hybridization, and posthybridization treatments. Intermediate probe concentrations, addition of detergent to the hybridization buffer, and a posthybridization washing step were found to increase the signal to noise ratio. We could demonstrate a linear correlation between growth rate and probe-conferred fluorescence of Escherichia coli and Pseudomonas cepacia cells. Oligonucleotides labeled with multiple fluorochromes showed elevated levels of nonspecific binding and therefore could not be used to lower the detection limits, which still restrict studies with fluorescing rRNA-targeted oligonucleotide probes to well-growing microbial cells. Two probes of different specificities--one labeled with fluorescein, the other with tetramethylrhodamine--could be applied simultaneously for dual color analysis.
Bacteria↗