New insight into flavivirus maturation from structure/function studies of the yellow fever virus envelope protein complex.

IMPORTANCE: All enveloped viruses enter cells by fusing their envelope with a target cell membrane while avoiding premature fusion with membranes of the producer cell-the latter being particularly important for viruses that bud at internal membranes. Flaviviruses bud in the endoplasmic reticulum, are transported through the TGN to reach the external milieu, and enter other cells via receptor-mediated endocytosis. The trigger for membrane fusion is the acidic environment of early endosomes, which has a similar pH to the TGN of the producer cell. The viral particles therefore become activated to react to mildly acidic pH only after their release into the neutral pH extracellular environment. Our study shows that for yellow fever virus (YFV), the mechanism of activation involves actively knocking out the fusion brake (protein pr) through a localized conformational change of the envelope protein upon exposure to the neutral pH external environment. Our study has important implications for understanding the molecular mechanism of flavivirus fusion activation in general and points to an alternative way of interfering with this process as an antiviral treatment.

Long-term satisfaction of patients after laparoscopic and robotic-assisted hysterectomy.

INTRODUCTION: Da-Vinci-Xi is the most recent device used in gynecologic robotic surgery. The aim of the present study was to compare the long-term satisfaction of patients who had undergone conventional laparoscopic hysterectomy or robotic assisted laparoscopic hysterectomy using the Da-Vinci-Xi surgical system. METHODS: All hysterectomies performed at the University Hospital of Luebeck from 2018 to 2019 were reviewed. Postoperative outcomes were compared between women who had undergone total hysterectomy with da Vinci Xi (n = 42) or conventional laparoscopy (n = 97). Postoperative outcomes included pain, elimination of complaints after surgery, bladder function, sexual function, satisfaction with the cosmetic outcome, positive experiences after robotic surgery, and satisfaction with the surgeon's preoperative explanation. Obese patients were evaluated separately in a subgroup analysis. RESULTS: Both groups had similar baseline characteristics and complication rates. Preoperative complaints subsided after surgery in a little more than 90% of patients. No significant differences were noted between groups in this regard (p = 0.262), or with reference to postoperative pain after one week (p = 0.866) and one month (p = 0.580), stress incontinence (p = 0.343), sexual function (p = 0.766) and the cosmetic outcome of the abdominal incisions (p = 0.273). The majority of patients who had undergone robotic surgery (96.8%) would be willing to undergo the procedure again if necessary. The subgroup analysis of obese patients revealed no significant differences. CONCLUSION: The Da-Vinci-Xi device did not improve the long-term surgical satisfaction of normal-weight or obese patients who underwent hysterectomy compared with patients who underwent conventional laparoscopy performed by experienced laparoscopic surgeons.

Low-dose 2-deoxy glucose stabilises tolerogenic dendritic cells and generates potent in vivo immunosuppressive effects.

Cell therapies for autoimmune diseases using tolerogenic dendritic cells (tolDC) have been promisingly explored. A major stumbling block has been generating stable tolDC, with low risk of converting to mature immunogenic DC (mDC), exacerbating disease. mDC induction involves a metabolic shift to lactate production from oxidative phosphorylation (OXPHOS) and ß-oxidation, the homeostatic energy source for resting DC. Inhibition of glycolysis through the administration of 2-deoxy glucose (2-DG) has been shown to prevent autoimmune disease experimentally but is not clinically feasible. We show here that treatment of mouse bone marrow-derived tolDC ex vivo with low-dose 2-DG (2.5 mM) (2-DGtolDC) induces a stable tolerogenic phenotype demonstrated by their failure to engage lactate production when challenged with mycobacterial antigen (Mtb). ~ 15% of 2-DGtolDC express low levels of MHC class II and 30% express CD86, while they are negative for CD40. 2-DGtolDC also express increased immune checkpoint molecules PDL-1 and SIRP-1α. Antigen-specific T cell proliferation is reduced in response to 2-DGtolDC in vitro. Mtb-stimulated 2-DGtolDC do not engage aerobic glycolysis but respond to challenge via increased OXPHOS. They also have decreased levels of p65 phosphorylation, with increased phosphorylation of the non-canonical p100 pathway. A stable tolDC phenotype is associated with sustained SIRP-1α phosphorylation and p85-AKT and PI3K signalling inhibition. Further, 2-DGtolDC preferentially secrete IL-10 rather than IL-12 upon Mtb-stimulation. Importantly, a single subcutaneous administration of 2-DGtolDC prevented experimental autoimmune uveoretinitis (EAU) in vivo. Inhibiting glycolysis of autologous tolDC prior to transfer may be a useful approach to providing stable tolDC therapy for autoimmune/immune-mediated diseases.

Myeloid protein tyrosine phosphatase 1B (PTP1B) deficiency protects against atherosclerotic plaque formation in the ApoE-/- mouse model of atherosclerosis with alterations in IL10/AMPKα pathway.

OBJECTIVE: Cardiovascular disease (CVD) is the most prevalent cause of mortality among patients with Type 1 or Type 2 diabetes, due to accelerated atherosclerosis. Recent evidence suggests a strong link between atherosclerosis and insulin resistance due to impaired insulin receptor (IR) signaling. Moreover, inflammatory cells, in particular macrophages, play a key role in pathogenesis of atherosclerosis and insulin resistance in humans. We hypothesized that inhibiting the activity of protein tyrosine phosphatase 1B (PTP1B), the major negative regulator of the IR, specifically in macrophages, would have beneficial anti-inflammatory effects and lead to protection against atherosclerosis and CVD. METHODS: We generated novel macrophage-specific PTP1B knockout mice on atherogenic background (ApoE-/-/LysM-PTP1B). Mice were fed standard or pro-atherogenic diet, and body weight, adiposity (echoMRI), glucose homeostasis, atherosclerotic plaque development, and molecular, biochemical and targeted lipidomic eicosanoid analyses were performed. RESULTS: Myeloid-PTP1B knockout mice on atherogenic background (ApoE-/-/LysM-PTP1B) exhibited a striking improvement in glucose homeostasis, decreased circulating lipids and decreased atherosclerotic plaque lesions, in the absence of body weight/adiposity differences. This was associated with enhanced phosphorylation of aortic Akt, AMPKα and increased secretion of circulating anti-inflammatory cytokine interleukin-10 (IL-10) and prostaglandin E2 (PGE2), without measurable alterations in IR phosphorylation, suggesting a direct beneficial effect of myeloid-PTP1B targeting. CONCLUSIONS: Here we demonstrate that inhibiting the activity of PTP1B specifically in myeloid lineage cells protects against atherosclerotic plaque formation, under atherogenic conditions, in an ApoE-/- mouse model of atherosclerosis. Our findings suggest for the first time that macrophage PTP1B targeting could be a therapeutic target for atherosclerosis treatment and reduction of CVD risk.

Medical Liability and Patient Law in Germany: Main Features with Particular Focus on Treatments in the Field of Interventional Radiology.

UNLABELLED: On February 26th, 2013 the patient law became effective in Germany. Goal of the lawmakers was a most authoritative case law for liability of malpractice and to improve enforcement of the rights of the patients. The following article contains several examples detailing legal situation. By no means should these discourage those persons who treat patients. Rather should they be sensitized to to various aspects of this increasingly important field of law. To identify relevant sources according to judicial standard research was conducted including first- and second selection. Goal was the identification of jurisdiction, literature and other various analyses that all deal with liability of malpractice and patient law within the field of Interventional Radiology--with particular focus on transarterial chemoembolization of the liver and related procedures. In summary, 89 different sources were included and analyzed. The individual who treats a patient is liable for an error in treatment if it causes injury to life, the body or the patient's health. Independent of the error in treatment the individual providing medical care is liable for mistakes made in the context of obtaining informed consent. Prerequisite is the presence of an error made when obtaining informed consent and its causality for the patient's consent for the treatment. Without an effective consent the treatment is considered illegal whether it was free of treatment error or not. The new patient law does not cause material change of the German liablity of malpractice law. KEY POINTS: •On February 26th, 2013 the new patient law came into effect. Materially, there was no fundamental remodeling of the German liability for medical malpractice. •Regarding a physician's liability for medical malpractice two different elements of an offence come into consideration: for one the liability for malpractice and, in turn, liability for errors made during medical consultation in the process of obtaining informed consent. •Forensic practice shows that patients frequently enforce both offences concurrently.

[End-of-life decisions and practices in critically ill patients in the cardiac intensive care unit. A nationwide survey]. / Begrenzung und Abbruch lebenserhaltender Maßnahmen bei kritisch kranken kardiochirurgischen Patienten. Ergebnisse einer bundesweiten Umfrageaktion.

BACKGROUND: Ethical and medical criteria in the decision-making process of withholding or withdrawal of life support therapy in critically ill patients present a great challenge in intensive care medicine. OBJECTIVES: The purpose of this work was to assess medical and ethical criteria that influence the decision-making process for changing the aim of therapy in critically ill cardiac surgery patients. MATERIALS AND METHODS: A questionnaire was distributed to all German cardiac surgery centers (n = 79). All clinical directors, intensive care unit (ICU) consultants and ICU head nurses were asked to complete questionnaires (n = 237). RESULTS: In all, 86 of 237 (36.3 %) questionnaires were returned. Medical reasons which influence the decision-making process for changing the aim of therapy were cranial computed tomography (cCT) with poor prognosis (91.9 %), multi-organ failure (70.9 %), and failure of assist device therapy (69.8 %). Concerning ethical reasons, poor expected quality of life (48.8 %) and the presumed patient's wishes (40.7 %) were reported. There was a significant difference regarding the perception of the three different professional groups concerning medical and ethical criteria as well as the involvement in the decision-making process. CONCLUSION: In critically ill cardiac surgery patients, medical reasons which influence the decision-making process for changing the aim of therapy included cCT with poor prognosis, multi-organ failure, and failure of assist device therapy. Further studies are mandatory in order to be able to provide adequate answers to this difficult topic.

Realising the European network of biodosimetry: RENEB-status quo.

Creating a sustainable network in biological and retrospective dosimetry that involves a large number of experienced laboratories throughout the European Union (EU) will significantly improve the accident and emergency response capabilities in case of a large-scale radiological emergency. A well-organised cooperative action involving EU laboratories will offer the best chance for fast and trustworthy dose assessments that are urgently needed in an emergency situation. To this end, the EC supports the establishment of a European network in biological dosimetry (RENEB). The RENEB project started in January 2012 involving cooperation of 23 organisations from 16 European countries. The purpose of RENEB is to increase the biodosimetry capacities in case of large-scale radiological emergency scenarios. The progress of the project since its inception is presented, comprising the consolidation process of the network with its operational platform, intercomparison exercises, training activities, proceedings in quality assurance and horizon scanning for new methods and partners. Additionally, the benefit of the network for the radiation research community as a whole is addressed.

Web-based scoring of the dicentric assay, a collaborative biodosimetric scoring strategy for population triage in large scale radiation accidents.

In the case of a large scale radiation accident high throughput methods of biological dosimetry for population triage are needed to identify individuals requiring clinical treatment. The dicentric assay performed in web-based scoring mode may be a very suitable technique. Within the MULTIBIODOSE EU FP7 project a network is being established of 8 laboratories with expertise in dose estimations based on the dicentric assay. Here, the manual dicentric assay was tested in a web-based scoring mode. More than 23,000 high resolution images of metaphase spreads (only first mitosis) were captured by four laboratories and established as image galleries on the internet (cloud). The galleries included images of a complete dose effect curve (0-5.0 Gy) and three types of irradiation scenarios simulating acute whole body, partial body and protracted exposure. The blood samples had been irradiated in vitro with gamma rays at the University of Ghent, Belgium. Two laboratories provided image galleries from Fluorescence plus Giemsa stained slides (3 h colcemid) and the image galleries from the other two laboratories contained images from Giemsa stained preparations (24 h colcemid). Each of the 8 participating laboratories analysed 3 dose points of the dose effect curve (scoring 100 cells for each point) and 3 unknown dose points (50 cells) for each of the 3 simulated irradiation scenarios. At first all analyses were performed in a QuickScan Mode without scoring individual chromosomes, followed by conventional scoring (only complete cells, 46 centromeres). The calibration curves obtained using these two scoring methods were very similar, with no significant difference in the linear-quadratic curve coefficients. Analysis of variance showed a significant effect of dose on the yield of dicentrics, but no significant effect of the laboratories, different methods of slide preparation or different incubation times used for colcemid. The results obtained to date within the MULTIBIODOSE project by a network of 8 collaborating laboratories throughout Europe are very promising. The dicentric assay in the web based scoring mode as a high throughput scoring strategy is a useful application for biodosimetry in the case of a large scale radiation accident.

Influence of storage time and amount of red blood cell transfusion on postoperative renal function: an observational cohort study.

INTRODUCTION: To identify the impact of storage time and amount of transfused red blood cell units on renal function. METHODS: Consecutive transfused patients (n=492), undergoing cardiac surgery at a single centre and receiving at least one red blood cell unit, were pooled in different groups depending on storage time and amount of transfusion. RESULTS: Altogether 2,133 red blood cell units were transfused (mean age 21.87 days). Pre- and intraoperative data were similar between groups. Postoperative serum creatinine (p<0.01), glomerular filtration rate (p<0.01), and urea (p<0.01) showed a significant correlation with the amount of transfused red blood cell units, but not with storage time. Acute kidney insufficiency (creatinine values greater than 2.0 mg/dl or a duplication of the preoperative value) developed in 29% of patients and was associated with red blood cell mean age (p=0.042), absolute age (p=0.028), and amount of transfused (p<0.01) units. Acute kidney failure requiring renal replacement therapy occurred in 9.6% of patients and was associated with the amount of transfusion (p<0.01).  CONCLUSIONS: Worsening of renal function after cardiac surgery is associated with storage time and amount of transfused red blood cell units. Acute kidney insufficiency was defined as serum creatinine values greater than 2.0 mg/dl or a duplication of the preoperative value (baseline). Acute kidney failure was defined as becoming dependent upon dialysis.

Manure management for greenhouse gas mitigation.

Ongoing intensification and specialisation of livestock production lead to increasing volumes of manure to be managed, which are a source of the greenhouse gases (GHGs) methane (CH4) and nitrous oxide (N2O). Net emissions of CH4 and N2O result from a multitude of microbial activities in the manure environment. Their relative importance depends not only on manure composition and local management practices with respect to treatment, storage and field application, but also on ambient climatic conditions. The diversity of livestock production systems, and their associated manure management, is discussed on the basis of four regional cases (Sub-Saharan Africa, Southeast Asia, China and Europe) with increasing levels of intensification and priorities with respect to nutrient management and environmental regulation. GHG mitigation options for production systems based on solid and liquid manure management are then presented, and potentials for positive and negative interactions between pollutants, and between management practices, are discussed. The diversity of manure properties and environmental conditions necessitate a modelling approach for improving estimates of GHG emissions, and for predicting effects of management changes for GHG mitigation, and requirements for such a model are discussed. Finally, we briefly discuss drivers for, and barriers against, introduction of GHG mitigation measures for livestock production. There is no conflict between efforts to improve food and feed production, and efforts to reduce GHG emissions from manure management. Growth in livestock populations are projected to occur mainly in intensive production systems where, for this and other reasons, the largest potentials for GHG mitigation may be found.

Automatic scoring of dicentric chromosomes as a tool in large scale radiation accidents.

Mass casualty scenarios of radiation exposure require high throughput biological dosimetry techniques for population triage in order to rapidly identify individuals who require clinical treatment. The manual dicentric assay is a highly suitable technique, but it is also very time consuming and requires well trained scorers. In the framework of the MULTIBIODOSE EU FP7 project, semi-automated dicentric scoring has been established in six European biodosimetry laboratories. Whole blood was irradiated with a Co-60 gamma source resulting in 8 different doses between 0 and 4.5Gy and then shipped to the six participating laboratories. To investigate two different scoring strategies, cell cultures were set up with short term (2-3h) or long term (24h) colcemid treatment. Three classifiers for automatic dicentric detection were applied, two of which were developed specifically for these two different culture techniques. The automation procedure included metaphase finding, capture of cells at high resolution and detection of dicentric candidates. The automatically detected dicentric candidates were then evaluated by a trained human scorer, which led to the term 'semi-automated' being applied to the analysis. The six participating laboratories established at least one semi-automated calibration curve each, using the appropriate classifier for their colcemid treatment time. There was no significant difference between the calibration curves established, regardless of the classifier used. The ratio of false positive to true positive dicentric candidates was dose dependent. The total staff effort required for analysing 150 metaphases using the semi-automated approach was 2 min as opposed to 60 min for manual scoring of 50 metaphases. Semi-automated dicentric scoring is a useful tool in a large scale radiation accident as it enables high throughput screening of samples for fast triage of potentially exposed individuals. Furthermore, the results from the participating laboratories were comparable which supports networking between laboratories for this assay.

[Rectal goblet cell carcinoid. Primary tumor or metastasis?]. / Becherzellkarzinoid des Rektums. Primarius oder Metastase?

Goblet cell carcinoids are biphasic neoplasms of the gastrointestinal tract composed of a glandular and neuroendocrine differentiation. Typically, goblet cell carcinoids are localized in the vermiform appendix. We report the case of a 60-year-old female patient with the diagnosis of a 1.2-cm rectal goblet cell carcinoid tumor discovered during prophylactic proctocolonoscopy. Because of the known aggressive behavior of this entity, a rectosigmoidectomy was performed. The preoperative staging revealed neither local nor systemic spread. After 8 months, the patient is in good health. As a primary tumor of the extraappendiceal gastrointestinal tract, goblet cell carcinoids are a rarity. It is generally recommended to exclude metastasis of a primary appendiceal neoplasm. However, since the patient underwent an appendectomy in 1974, primary origin in the rectum is favored.

Realising the European Network of Biodosimetry (RENEB).

In Europe, a network for biological dosimetry has been created to strengthen the emergency preparedness and response capabilities in case of a large-scale nuclear accident or radiological emergency. Through the RENEB (Realising the European Network of Biodosimetry) project, 23 experienced laboratories from 16 European countries will establish a sustainable network for rapid, comprehensive and standardised biodosimetry provision that would be urgently required in an emergency situation on European ground. The foundation of the network is formed by five main pillars: (1) the ad hoc operational basis, (2) a basis of future developments, (3) an effective quality-management system, (4) arrangements to guarantee long-term sustainability and (5) awareness of the existence of RENEB. RENEB will thus provide a mechanism for quick, efficient and reliable support within the European radiation emergency management. The scientific basis of RENEB will concurrently contribute to increased safety in the field of radiation protection.

[Characterization of the EGF receptor status in penile cancer : retrospective analysis of the course of the disease in 45 patients]. / Charakterisierung des EGF-Rezeptorstatus beim Peniskarzinom : Retrospektive Auswertung der Krankheitsverläufe von 45 Patienten.

INTRODUCTION: Therapeutic success in metastasized squamous cell carcinoma is poor. Some entities, such as head-and-neck tumors or non-small-cell lung cancer, show an over expression of the EGF receptor. In latest studies target-specific substances against the EGF receptor have already been combined with chemotherapy or radiotherapy. Some studies showed a clear advantage of this combination concerning remission rates as well as survival. EGF receptor status has not yet been examined in penile cancer, therefore, a retrospective analysis of the receptor status was performed in patients treated over the last 14 years and correlations with the clinical course were investigated. PATIENTS AND METHODS: The analysis included 45 patients, who underwent primary or secondary treatment at the Department of Urology of the University of Essen during 1990 to 2004. Histological preparations existed for 44 patients. Using immunohistochemistry the expression of EGF receptors was determined. RESULTS: A total of 25 patients were primarily without positive lymph nodes (6 times cN0 and 19 times pN0), while 20 patients had pathologically proven lymph node metastases and 3 of them also had hematogeneous metastases. Out of 42 patients with follow-up 18 are still living of whom only 3 primarily had positive lymph nodes. These patients received adjuvant chemotherapy after resection. Out of the remaining 15 patients, 4 primarily N0 patients developed a lymphogenic recurrence, which was also resected and 3 patients also received adjuvant chemotherapy. Of the patients 24 died, 22 because of penile cancer. Of these 22 patients 16 primarily had positive lymph nodes and 5 of them also had an extensive primary tumor. Surgery was the treatment of choice in these cases and 10 patients also received chemotherapy. Nevertheless, 15 patients developed several recurrences. Distinguishing primarily node-negative and node-positive patients, the Kaplan-Meyer survival curves showed a significant difference (p<0.001). Median overall survival was 55.5 compared to 34 months and median 5-year survival was 76.9% compared to 15.8%. Of the tumors 40 out of 44 (91%) showed a positive or strong positive EGF receptor expression of the primary tumor as well as of the metastases. A correlation between EGF receptor expression and survival could not be shown. CONCLUSION: Clinical data underline the prognostic value of the primary lymph node status as well as the therapeutic value of an ileoinguinal lymphadenectomy and adjuvant chemotherapy. It could also be shown that inductive chemotherapy is not very successful. EGF receptor expression was high and comparable to other squamous cell carcinomas, but there was no correlation to survival.

High frequency of de novo mutations in Li-Fraumeni syndrome.

BACKGROUND: Li-Fraumeni syndrome is an autosomal dominant cancer predisposition syndrome caused by germline mutations in the TP53 gene. The frequency of germline de novo TP53 mutations is largely unknown; few unequivocal de novo mutations have been reported. METHODS AND RESULTS: Of 341 patients with early onset cancer sent for clinical testing to a national reference laboratory, 75 patients had TP53 germline mutations. Five (7%) de novo mutations were identified, as well as an additional 10 TP53 germline mutations likely to be de novo by family history. The frequency of de novo TP53 mutations in this patient sample is at least 7% and may be as high as 20%. CONCLUSIONS: The possibility that de novo germline TP53 mutations are relatively common has implications for testing and the identification of potential Li-Fraumeni syndrome in patients with little or no family history of cancer.

Management of poststernotomy mediastinitis: experience and results of different therapy modalities.

BACKGROUND: Different primary treatment modalities have been utilized to treat poststernotomy mediastinitis (PM) following cardiac surgery. METHODS: A literature survey using the key phrases "treatment of deep sternal wound infection" and "poststernotomy-mediastinitis" was performed. Furthermore, a questionnaire regarding the primary treatment of PM was distributed to all 79 German heart surgery centers. RESULTS: The review of the literature shows that the current understanding is based purely on retrospective studies, not on evidence-based medicine. All 79 German heart centers replied to the questionnaire. Vacuum-assisted closure therapy (V. A. C.(R)) is used in 28/79 (35 %) heart centers as the "first-line" treatment, 22/79 (28 %) perform primary reclosure in conjunction with a double-tube irrigation/suction system, and in 29/79 (37 %) German heart centers both treatment options were used according to the intraoperative conditions. CONCLUSIONS: As a primary treatment for PM two treatment modalities are currently in use: primary reclosure coupled with a double-tube suction/irrigation system versus V. A. C.(R) therapy. Since prospective randomized studies have not yet been performed, controlled clinical trials comparing both treatment modalities are pivotal to define the evidence for patients presenting with PM.

EGFR somatic doublets in lung cancer are frequent and generally arise from a pair of driver mutations uncommonly seen as singlet mutations: one-third of doublets occur at five pairs of amino acids.

Doublet mutations in cancer are not well studied. We find that allelic somatic doublet mutations are present at high frequency in the epidermal growth factor receptor (EGFR) tyrosine kinase (TK) domain in lung cancers. When doublets from the literature are added, a total of 96 doublets are available for analysis. The frequency of doublets overall is 6%, which is sevenfold greater than that observed in normal tissue in mouse. All characterized doublets are allelic, and silent mutations occur rarely. About half of all doublets contain one or two of 12 distinct missense mutations at five amino acids: E709, G719, S768, T790 and L861. The mutations at these five amino acids are seldom reported as singlets. Moreover, when the common L858 target is included, more than one-third of EGFR doublets are one of five specific missense pairs: G719/E709, G719/S768, G719/L861, L858/E709 and L858/T790. Structure suggests function: The data imply that most EGFR doublets are NOT consistent with a 'driver and passenger' mutation mechanism. EGFR doublets are highly skewed relative to singlets, consistent with functional selection of two individually suboptimal mutations that, in combination, have enhanced oncogenic potential.

PAP-LMPCR for improved, allele-specific footprinting and automated chromatin fine structure analysis.

The analysis of chromatin fine structure and transcription factor occupancy of differentially expressed genes by in vivo footprinting and ligation-mediated-PCR (LMPCR) is a powerful tool to understand the impact of chromatin on gene expression. However, as with all PCR-based techniques, the accuracy of the experiments has often been reduced by sequence similarities and the presence of GC-rich or repeat sequences, and some sequences are completely refractory to analysis. Here we describe a novel method, pyrophosphorolysis activated polymerization LMPCR or PAP-LMPCR, which is capable of generating accurate and reproducible footprints specific for individual alleles and can read through sequences previously not accessible for analysis. In addition, we have adapted this technique for automation, thus enabling the simultaneous and rapid analysis of chromatin structure at many different genes.

[Can preoperative fine-needle aspiration of parotid tumors pose problems for a definitive histological diagnosis?]. / Kann die präoperative Feinnadelpunktion bei Parotistumoren die definitive histologische Diagnose erschweren?

INTRODUCTION: Fine-needle aspiration cytology (FNAC) is a minimally invasive procedure that offers high specifity and acceptable sensitivity in the preoperative diagnosis of parotid tumors. FNAC provides information that can be particularly valuable in surgical planning and patient education. In some cases, it may even help prevent unnecessary surgery. After an FNA procedure, on rare occasions, morphological changes in the tumor may be observed and can make a definitive histological examination difficult or even impossible. METHODS: Histopathological changes in parotid tumors after preoperative FNAC are described on the basis of a case report and a survey of the literature. RESULTS: In rare cases, puncture-induced histopathological changes, such as hyalinization of the subepithelial stroma, necrosis and squamous metaplasia, so extensive that a definitive histological diagnosis is difficult, are observed. In the present case report, fine-needle aspiration of a 3 x 2 cm tumor, which had been classified as malignant on the basis of cytological findings, resulted in complete necrosis and made a definitive histological diagnosis impossible. Similar cases have been described by other authors in recent years. DISCUSSION: In rare cases, histological changes in the tumor tissue can occur after an FNA procedure, making a definitive histological examination difficult or even impossible. When a preoperative FNAC is performed, the pathologist in charge should be advised accordingly. In addition, patients should be informed about this infrequent risk before undergoing the procedure.

Molecular studies on suspect very virulent infectious bursal disease virus genomic RNA samples.

Infectious bursal disease (IBD) associated with high mortality was first observed in Europe in the mid-1980s. The viruses identified in those outbreaks were described as being very virulent infectious bursal disease virus (vvIBDV) strains. These viruses have spread to nearly every continent but have not yet been identified in North America, Australia, and New Zealand. There is a real and immediate concern that the very virulent form of IBDV will continue to spread until it is present on every continent. Genomic RNA samples from IBDV strains suspected of being very virulent were submitted to our laboratory for molecular analysis. Nucleotide sequences of the VP2 gene hypervariable sequence region were determined for 18 of these viruses. A comparison with published vvIBDV sequences indicated that all but one sample (Thai 4) had nucleotide and predicted amino acid sequences consistent with vvIBDV strains. Published sequences and the nucleotide sequences of our 17 putative vvIBDV strains were used to identify unique nucleotides in the VP2 gene. Probe pairs for a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay were designed based on these unique sequences and then used to test the 17 genomic samples that were identified by nucleotide sequencing to be consistent with vvIBDV, plus the one Thai 4 sample that was not consistent with vvIBDV. Using melting temperature (Tm) analysis following real-time RT-PCR, two probe pairs (vv232 and vv256) successfully identified the 17 putative vvIBDV strains and distinguished them from the Thai 4 sample. An additional 26 genomic RNA samples submitted as suspect vvIBDV strains were then tested using the vv232 and vv256 probes. Based on the melting point analysis of these two probes, all 26 samples contained nucleotide sequences consistent with vvIBDV strains. The specificity of the vv232 and vv256 probe pairs was evaluated using 19 non-vvIBDV strains. In every case, the probes distinguished the 19 classic and variant (non-vvIBDV) strains from the putative vvIBDV strains. Diagnostic assays that can reliably identify vvIBDV strains are needed for surveillance programs designed to monitor the spread of these viruses.