Advanced wastewater treatment with ozonation and granular activated carbon filtration: Inactivation of antibiotic resistance targets in a long-term pilot study.

The inactivation of antibiotic resistant bacteria (ARB) and genes (ARGs) in an advanced plant combining ozonation and granular activated carbon (GAC) filtration applied for effluent after conventional activated sludge treatment at a full-scale urban wastewater treatment plant was investigated for over 13 consecutive months. The nitrite compensated specific ozone dose ranged between 0.4 and 0.7 g O3/g DOC with short-time sampling campaigns (0.2-0.9 g O3/g DOC). Samples were analysed with culture-dependent methods for bacterial targets and with qPCR for genes. The log removal values were correlated with a decrease of the matrix UV absorption at 254 nm (ΔUV254) and indicated a range of ΔUV254 that corresponds to a sufficient membrane damage to affect DNA. For trimethoprim/sulfamethoxazole resistant E. coli, sul1, ermB and tetW, this phase was observed at ΔUV254 of ~30 % (~0.5 g O3/g DOC). For ampicillin resistant E. coli and blaTEM-1, it was observed around 35-40 % (~0.7 g O3/g DOC), which can be linked to mechanisms related to oxidative damages in bacteria resistant to bactericidal antibiotics. GAC treatment resulted in a further abatement for trimethoprim/sulfamethoxazole E. coli, sul1 and tetW, and in increase in absolute and relative abundance of ermB and blaTEM-1.

[Implementation of the "Digital Pathology in Diagnostics" guideline : Support systems and their functionality]. / Umsetzung des Leitfadens "Digitale Pathologie in der Diagnostik" : Unterstützende Systeme und ihre Funktionalität.

BACKGROUND: The "Digital Pathology in Diagnostics - Assessment of Digital Images" guideline describes the technical and legal framework under which the use of this digital technology is justifiable for the individual pathologist. The focus is on conducting a validation study, defining minimum requirements for the generation and management of whole slide images, and ensuring the functionality and quality of the virtual microscopy solution used. By establishing a special web-based service, supportive services can be provided to assist the pathologist in the introduction of virtual microscopy and quality assurance. AIM: Presentation of the Digitale-Pathologie.de server and description of its services in the context of the guideline. RESULTS AND DISCUSSION: In the context of several scanner contests at the Charité in Berlin, as well as with the introduction of virtual microscopy in practice, many experiences were collected, which will be presented systematically. Essentially, this will provide support for the application of the guideline in practice. The following fields are discussed: implementation of the guideline recommendations, planning and evaluation of the validation study, and ensuring the correct color calibration of the slide scanner being used. Via the Digitale-Pathologie.de server, the possibility for self-monitoring and anonymously benchmarking will be offered. For example, errors in setup can be detected quickly and optimal settings can be identified.

["Digital Pathology in Diagnostics" guideline : Reporting on digital images]. / Leitfaden "Digitale Pathologie in der Diagnostik" : Befunderstellung an digitalen Bildern.

BACKGROUND: The digitization of medicine is gaining momentum in pathology. Long-known technologies have reached such a degree of maturity that their use in primary diagnostics in routine pathology will be possible. In spite of the complexity of technological solutions and the far-reaching consequences in terms of diagnostic reliability, as well as due to the high investments, the decision for a specific product may become highly sophisticated for a pathologist. AIM: An implementation guide for Digital Diagnostics in Pathology is presented to describe technical and legal conditions for making this new technology feasible for the single pathologist. RESULTS AND DISCUSSION: For more than two years, the Digital Pathology Commission of the Federal Association of German Pathologists developed and discussed this implementation guide for digital diagnostics, especially for the use of virtual microscopy in the daily pathology routine in Germany. The key is the principal comparability of diagnostic reliability between conventional stained microscopic slides and their digital images, which have to be shown by the potential user. In eight chapters, the validation procedure as well as technical minimum requirements in slide scanners, the visualization pipeline, archiving, and integration in the pathology workflow are described.

Cancer beyond organ and tissue specificity: next-generation-sequencing gene mutation data reveal complex genetic similarities across major cancers.

Cancer medicine relies on the paradigm that cancer is an organ- and tissue-specific disease, which is the basis for classifying tumors. With the extensive genomic information now available on tumors it is possible to conduct analyses to reveal common genetic features across cancer types and to explore whether the established anatomy-based tumor classification is actually reflected on the genetic level, which might provide important guides to new therapeutic directions. Here, we have conducted an extensive analysis of the genetic similarity of tumors from 14 major cancer entities using somatic mutation data from 4,796 cases available through The Cancer Genome Atlas (TCGA) based on all available genes as well as different cancer-related gene sets. Our analysis provides a systematic account of the genetic similarity network for major cancer types and shows that in about 43% of the cases on average, tumors of a particular anatomic site are genetically more similar to tumors from different organs and tissues (trans-similarity) than to tumors of the same origin (self-similarity). The observed similarities exist not only for carcinomas from different sites but are also present among neoplasms from different tissue origin, such as melanoma, acute myeloid leukemia, and glioblastoma. The current WHO cancer classification is therefore reflected on the genetic level by only about 57% of the tumors. These results provide a rationale to reconsider organ- and tissue-specificity in cancer and contribute to the discussion about whether personalized therapies targeting specific genetic alterations may be transferred to cancers from other anatomic sites with similar genetic properties.

Impact of filaggrin mutations on Raman spectra and biophysical properties of the stratum corneum in mild to moderate atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is associated with null mutations in the filaggrin (FLG) gene. OBJECTIVE: To assess the impact of FLG null mutations on biophysical properties and the molecular composition of the stratum corneum (SC) in healthy individuals and AD patients. METHODS: A total of 196 French adults, including 97 with a history of mild to moderate AD, were genotyped for the three major European FLG mutations. Components of the natural moisturizing factor (NMF), lipids and water content in the SC were determined using Raman spectroscopy. In addition, trans-epidermal water loss, capacitance and pH of the SC were measured. RESULTS: Stratum corneum concentrations of total NMF, water, ornithine and urocanic acid (UCA) were significantly lower in AD patients than in healthy controls. Null mutations of FLG were detected in 4% of controls and 10% of AD patients. FLG mutations were associated with increased SC levels of lactate, reduced concentrations of most other NMF components and higher disease severity in AD patients. In AD patients without FLG mutations, the content of NMF constituents decreased with increasing disease severity. The concomittant presence of low concentrations of histidine, alanine and either glycine or pyrrolidone-5-carboxylic acid (PCA) in the SC was associated with FLG mutations with 92% specificity. CONCLUSIONS: Our findings suggest a low prevalence of FLG mutations in mild AD and support an important role for filaggrin in determining the physicochemical parameters of the SC. The combined measurement of several filaggrin breakdown products in the SC may be useful to specifically predict the presence of FLG mutations.

[Virtual microscopy and routine diagnostics. A discussion paper]. / Virtuelle Mikroskopie und Routinediagnostik. Ein Diskussionspapier.

There are several areas of application for virtual microscopy in pathology. After broad use in education and research, we are now seeing its initial application in health care. We can predict that, on the basis of experience gained in digital radiology and early experience in pathology, VM will be established as routine. However, its introduction will follow a different course to that taken in digital radiology, which required that the computer be accepted as a "necessary evil" to record and display computer tomograms. Virtual microscopy needs to ensure that it supports the pathologist significantly in terms of access to archives, quantification of markers, display of biopsy stacks, cooperation with colleagues, etc. Consequently, the question of "When will virtual microscopy enter daily pathology practice?" may not only be answered on the basis of technical features. Of course, it is necessary that scanning speed goes below 1 min/cm(2) and that it remains financially viable, but more important is an optimal integration of virtual microscopy in daily pathology routine. This process will extend over several decades, as past developments in digital radiology have shown.

Airway inflammation induced after allergic poly-sensitization can be prevented by mucosal but not by systemic administration of poly-peptides.

BACKGROUND: Patients with multiple sensitizations require alternative forms of treatment, as the efficacy of conventional immunotherapy is unsatisfactory. OBJECTIVE: In the present study, we sought to compare the efficacy of a subcutaneously (s.c.) and a mucosally applied polyvalent vaccine to reduce allergic immune responses within airway and lung tissues. METHODS: Female BALB/c mice were intraperitoneally immunized with recombinant (r)Bet v 1, rPhl p 1 and rPhl p 5, followed by an aerosol challenge of birch and phleum pollen extract. For tolerance induction, either a mixture of the immunodominant peptides or a hybrid peptide of the respective antigens was s.c. injected or intranasally applied before poly-sensitization. RESULTS: Mucosal but not systemic pre-treatment with poly-peptides led to significant suppression of eosinophils and IL-5 production in bronchoalveolar lavages, as well as IL-5, IL-4, IL-13 and eotaxin levels in lung cell cultures. Lung histology showed a clear reduction of cellular infiltration and mucus production only in intranasally pre-treated mice. In accordance, also the systemic immune response, characterized by IgE-dependent basophil degranulation and IL-4 levels in vitro, was significantly reduced by mucosal antigen application, but only marginally influenced by subcutaneous pre-treatment. Both treatment routes led to up-regulated CTLA4 expression in splenocytes, whereas only after mucosal pre-treatment Foxp3 expression levels were enhanced in lung CD3(+) T cells. Furthermore, intranasal but not subcutaneous application of the peptides enhanced IL-10 levels in the lungs, indicating regulatory mechanisms operating in local tolerance induction. CONCLUSION: Mucosal application of peptides is superior to systemic application in preventing both local and systemic poly-allergic T helper2 immune responses, suggesting mucosal tolerance induction as an attractive strategy for the primary and secondary prevention of allergic multi-sensitization and lung pathology.

Evaluation of a novel internally controlled real-time PCR assay targeting the 16S rRNA gene for confirmation of Neisseria gonorrhoeae infections.

A novel HybProbe real-time LightCycler PCR assay was developed for confirmation of Neisseria gonorrhoeae in samples positive according to the COBAS AMPLICOR Chlamydia trachomatis/Neisseria gonorrhoeae PCR assay. The new assay amplifies 375 bp of the N. gonorrhoeae 16S rRNA gene and includes an internal amplification control introduced during DNA purification. The assay had 100% specificity because of the high specificity of the HybProbes and primers. Other Neisseria spp. failed to generate positive crossing-point values and melting peaks. The analytical sensitivity for N. gonorrhoeae DNA was 0.5 fg/PCR, corresponding to 0.3 CFU/PCR. Sensitivity was not impaired in the presence of higher DNA concentrations (>or=1000-fold) from Neisseria spp. other than N. gonorrhoeae. The sensitivity was similar to that reported for the COBAS AMPLICOR assay with cervical swab samples. To assess its clinical applicability as a confirmatory test, 38 (2.9%) of 1313 swabs that were positive according to the COBAS AMPLICOR assay were tested using the new in-house assay and the commercially available GenFlow Neisseria test. Twenty-one samples negative according to COBAS AMPLICOR also underwent confirmatory testing. Both confirmatory tests yielded identical results; the 21 negative samples remained negative, and only 11 (28.9%) of the samples positive according to COBAS AMPLICOR were positive after retesting, suggesting a low prevalence (0.84%) of N. gonorrhoeae infection in the study population. These data suggest that the novel real-time PCR assay is an excellent and easy to interpret confirmatory test for the existing COBAS AMPLICOR assay for N. gonorrhoeae.

[Study of efficiancy of teleconsultation: the Telepathology Consultation Service of the Professional Assoziation of German Pathologists for the screening program of breast carcinoma]. / Studie zur Effektivität der Telekonsultation: Der Telepathologie-konsultationsservice des Berufsverbandes Deutscher Pathologen im Rahmen des Mamma-Screening-Programms.

AIMS: In the autumn a German screening program was started for detecting breast cancer in the population of women fifty and above. For the first time in this program, quality assurance rules were established: All statements of the radiologists and pathologists have to be confirmed by a second opinion. This improvement in quality is combined with a delay in time and additional expence. A new Telepathology Consultation Service was developed based on the experiences of the Telepathology Consultation Center of the UICC to speed up the second opinion process. METHODS AND MATERIAL: The complete web-based service is operated under MS Windows 2003 Server, as web server the Internet Information Server, and the SQL-Server (both Microsoft) as the database. The websites, forms and control mechanism have been coded in by ASP scripts and JavaScript. A study to evaluate the effectiveness of telepathological consultation in comparison to conventional consultation has been carried out. Pathologists of the Professional Association of German Pathologists took part as well as requesting pathologists and as consultants for other participants. RESULTS AND CONCLUSION: The quality of telepathological diagnosis was comparable to the conventional diagnosis. Telepathology allows a faster respond of 1 to 2 day (conventional postal delay). The time to prepare a telepathology request is about twice as conventional. This ratio may be inverted by an interface between the Pathology Information System and the Telepathology Server and the use of virtual microscopy. The Telepathology Consultation Service of the Professional Association of German Pathologists is a fast and effective German-language, internet-based service for obtaining a second opinion.

Fetal autopsy: the most important contribution of pathology in a center for perinatal medicine.

OBJECTIVE: The aim of the current paper is to analyze the significant changes in fetal autopsy made by pathologists in a center for perinatal medicine in response to the recent rapid developments in prenatal medicine. METHODS: We use our technical equipment (interdisciplinary databank, Intranet, fine instruments, imaging techniques) for prenatal diagnostics and fetal pathology in the Center for Perinatal Medicine at the Charité University Medical Center in Berlin, Germany. RESULTS: Our technical setup has made it possible for us to plan autopsies in such a way that rare malformations in special cases can be documented in sectional planes corresponding to the prenatal sonographic images. Before the postmortem examination the pathologist receives all the prenatal findings obtained for the fetus and is able to examine the prenatal images on a monitor. The final diagnosis in the postmortem report includes the results of modern molecular pathology, clinical-genetic examination of the fetus, cytogenetic and molecular-cytogenetic findings and postmortem x-rays. CONCLUSIONS: To be able to incorporate these various results into a final report in routine work the use of an interdisciplinary database is essential. Such a database also ensures faster and more productive interdisciplinary communication in a center of perinatal medicine.

[Electronic communication in medicine]. / Elektronische Kommunikation in der Medizin.

Rapid developments in information and communication technology allow a fast transfer of extensive data. Geographical distances do not longer hinder an intense cooperation in diagnostics. The review shows the variety of possibilities for medical applications. Technique, routine application, experiences and the legal conditions are presented for telepathology as an example.

Fetal autopsy: a review of recent developments.

OBJECTIVE: To analyze recent changes in fetal autopsy in response to developments in prenatal medicine. METHOD: During the period 1988 through 1997, 783 fetuses (75% induced abortions, 18% spontaneous abortions, and 7% stillbirths, all between the 12th and 40th week of gestation) with prenatally diagnosed congenital malformations and chromosomal aberrations were analyzed. We divided the autopsies into two periods: period A (1988-92, n=370) and period B (1993-97, n=413). All fetuses were analyzed before completion of 20 weeks of gestation. The malformations of the organ systems were presented according to their frequency for fetuses independent of the weeks of gestation. RESULTS: An autopsy was performed prior to the completion of 20 weeks of gestation for 24% of the fetuses in period A and 45% in period B (P<0.0001). The number of diagnosed congenital heart malformations increased from 16% in period A to 23% in period B. The number of congenital heart malformations before completion of 20 weeks of gestation was only 21% in period A as compared to 42% in period B. CONCLUSIONS: In period B, clinical questions were raised in a more concrete form and ultrasound images gave more detailed information than in period A. Access to prenatal findings prior to postmortem examination has helped investigators to develop a specific, clinically oriented, autopsy strategy based on the prenatal findings which makes it possible to diagnose very small malformations and to select sectional planes of special interest. The use of an interdisciplinary database is required for communication.

Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1.

An estimated 100 million individuals suffer from birch pollen allergy. Specific immunotherapy, the only curative allergy treatment, can cause life-threatening anaphylactic side effects. Here, we report the genetic engineering of a recombinant trimer consisting of three covalently linked copies of the major birch pollen allergen, Bet v 1. The trimer exhibited profoundly reduced allergenic activity but contained similar secondary structures such as Bet v 1 wild type, Bet v 1-specific B cell and T-cell epitopes, and induced Th1 cytokine release. As immunogen, rBet v 1 trimer induced IgG antibodies, which blocked patients' IgE binding to Bet v 1 and related allergens. Thus, rBet v 1 trimer represents a novel hypoallergenic vaccine prototype for treatment of one of the most frequent allergy forms.

Improvement of breast cancer prognostication using cell kinetic-based silver-stainable nucleolar organizer region quantification of the MIB-1 positive tumor cell compartment.

Recently, it was stated that the proliferative activity (P) of a cell population could be indirectly calculated by multiplying the MIB-1 immunopositivity and silver-stainable nucleolar organizer region (AgNOR) features extracted exclusively in MIB-1 positive (pos.) nuclei: P=MIB-1 x AgNOR(MIB-1pos.). To study the prognostic significance of this hypothesis, MIB- immunohistochemistry and AgNOR staining were applied on a series of 89 cases of breast cancer with an 8-year follow-up period. The mean MIB-1 immunopositivity (MIB-1mean) was evaluated immunohistometrically on paraffin sections using a TV image analysis system CM-2 (Hund, Wetzlar, Germany). Later, a combined MIB-1/AgNOR staining was applied and evaluated using a TV image analysis system AMBA (IBSB, Berlin, Germany). The AgNOR features of 150 randomly chosen tumor nuclei were investigated, irrespective of their MIB-1 status (AgNOR count, AgNOR area). Later, a second measurement was performed on 100 MIB-1 positive tumor nuclei exclusively (AgNOR countMIB-1pos., AgNOR areaMIB-1pos.). AgNOR count and AgNOR countMIB-1pos. showed a different data distribution [2.7+/-0.7 (mean+/-SD) vs 3.9+/-1.1; r=0.315, P=0.014]. Similar results were obtained for AgNOR area and AgNOR areaMIB-1pos. (5.1+/-2.1 microm2 vs 7.5+/-2.4 microm2; r=0.501, P<0.001). Kaplan-Meier survival curves revealed significant differences for MIB-1mean (P=0.0018) and AgNOR areaMIB-1pos. (P=0.0340). In Cox models, both parameters provided independent prognostic information. Using their combination, the P, three groups of patients with statistically different survival could be separated (P=0.0014). Thus, the combination of MIB-1-immunopositivity and AgNOR measurements in MIB-1 positive nuclei appears to be more useful in breast cancer prognosis than the exclusive application of one of the two methods. By this combined application, probably effects of tumor biology are represented more precisely.

Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells: contribution of the NF-kappa b pathway.

In this study we aimed to investigate whether the therapeutic efficacy of anisodamine in the treatment of bacteraemic shock could--at least in part--be brought about by its direct interference with the lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of anisodamine on LPS-induced expression of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial activation. PAI-1 was measured in the conditioned media of human umbilical vein endothelial cells (HUVEC) by a specific enzyme-linked immunosorbent assay (ELISA) whereas TF activity was measured in the lysates of these cells by using a single step clotting assay. Results obtained in these assays were confirmed on the level of specific mRNA expression by Northern blotting using specific probes for human PAI-1 or TF. In order to evaluate a possible contribution of the NF-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B-binding oligonucleotides. When HUVEC were treated with 1 microg/ml LPS a significant increase in PAI-1 and TF activity was observed compared with cells incubated without LPS. Anisodamine dose-dependently inhibited this LPS-induced upregulation of PAI-1 and TF. Anisodamine alone had no effect on the constitutive expression of PAI-1 and TF in these cells. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. Furthermore, we could show by EMSA that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thus, we provide evidence that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-kappa B pathway might - at least in part - contribute to this effect. The ability of anisodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic shock.

The UICC Telepathology Consultation Center. International Union Against Cancer. A global approach to improving consultation for pathologists in cancer diagnosis.

BACKGROUND: The morphologic diagnosis of tumor specimens with precise tumor typing, staging, and grading remains the basis of almost all cancer treatments. Thus, in each tumor case, a histologic diagnosis of the highest quality should be the physician's priority. In approximately 10-20% of tumor cases, diagnostic uncertainty remains to some degree, requiring a second opinion in determining the biologic behavior, the histogenesis, the grade of dedifferentiation, or any other parameter. Facilitating the communication between pathologists and the exchange of cases, telepathology gains more and more importance. To benefit from this technical development, the International Union Against Cancer (UICC) has decided to establish a Telepathology Consultation Center (UICC-TPCC) for interested pathologists around the world. METHODS: The communication and exchange of histologic images works via the Internet. To ensure constant documentation, a case-based data base and image archive is used. Special TPCC software handles all requests to the TPCC and controls the interaction among requesting pathologists, TPCC, and UICC experts (transferring, reading, answering, logging, storing, etc.). All necessary data for controlling the telepathology service are stored in a customized SQL data base. The necessary equipment for the requesting pathologist is a personal computer; a digital or television camera/frame grabber, which is attached to a microscope; and access to the Internet. The requesting party contacts the TPCC's World Wide Web server and uploads the images and the clinical data of their case. To ensure uninterrupted functioning, the hardware will be part of a high-level communication center, which is connected via ATM (asynchronous transfer mode, 155 megabits per second) to the Internet. RESULTS: The UICC has decided to establish the TPCC at the Institute of Pathology at the Charité, Humboldt-University, Berlin, Germany. The TPCC will not make the diagnoses itself but will involve an affiliated specialized expert pathologist. He or she will be on the panel of UICC experts who will constitute the "diagnostic backbone" of the TPCC. The center will function as follows: If a pathologist anywhere on the globe is confronted with the diagnosis of a difficult tumor case, he takes digitized histologic images (5-40 in number) and sends them along with sufficient clinical data to the server of the UICC-TPCC, asking for a second opinion. The center checks the case and transfers it to one of the UICC experts. This expert makes his or her diagnostic suggestion, which is then transferred back to the requesting pathologist via the UICC-TPCC. CONCLUSIONS: The UICC-TPCC will be able to provide rapid and inexpensive diagnostic aid to pathologists all over the world, offering the possibility of a second opinion in accordance with the UICC-TNM and World Health Organization (WHO) standards. During the first and second year, the UICC-TPCC will be financed by sponsors. Telepathology makes the distribution of new developments of diagnostic standards, e.g., of the TNM system, WHO terminology, new tumor classifications, and updated information on actual technologies, globally accessible in a direct and rapid way. It also enables a high quality of education and teaching.

[Autopsy techniques in congenital heart defects. Influence of prenatal diagnosis on the planning and carrying out of autopsies]. / Sektionstechniken bei kongenitalen Herzfehlbildungen. Auswirkungen der Ergebnisse der pränatalen Diagnostik auf Sektionsplanung und -durchführung.

As a result of the quality of prenatal ultrasound and the expanded experience of prenatal diagnosticians, it is possible to observe congenital heart malformations in increasingly greater detail and at an ever earlier stage of gestation [4]. Since it is on the basis of ultrasound findings that decisions to terminate pregnancies are made, it is of cardinal importance that after termination monitoring and confirmation of the prenatal diagnosis be carried out. This need can only be adequately met by autopsy. There are different methods for carrying out autopsies when there is suspicion of a congenital heart defect: a) the Anderson sequential segmental analysis as modified according to the Berlin method; b) use of a special autoptic method corresponding to the ultrasound findings, based on defining a preferred sectional plane; c) stereomicroscopically; or d) microscopically after embedding and preparation of serial microscopic sections. For the pathologist the consequence is that he has to adapt his autopsy method to the ultrasound findings and the age of the fetus. This enables him to determine an optimal, case-based autopsy strategy for each type of cardiac defect, which is essential for monitoring of the prenatal diagnosis. The present paper discusses the various autoptic methods used in cases of congenital heart malformations and the consequences for the pathologist of the continuing improvements in prenatal diagnostics.