Ranid Herpesvirus 3 and Proliferative Dermatitis in Free-Ranging Wild Common Frogs (Rana Temporaria).

Amphibian pathogens are of current interest as contributors to the global decline of amphibians. However, compared with chytrid fungi and ranaviruses, herpesviruses have received relatively little attention. Two ranid herpesviruses have been described: namely, Ranid herpesvirus 1 (RHV1) and Ranid herpesvirus 2 (RHV2). This article describes the discovery and partial characterization of a novel virus tentatively named Ranid herpesvirus 3 (RHV3), a candidate member of the genus Batrachovirus in the family Alloherpesviridae. RHV3 infection in wild common frogs (Rana temporaria) was associated with severe multifocal epidermal hyperplasia, dermal edema, a minor inflammatory response, and variable mucous gland degeneration. Intranuclear inclusions were numerous in the affected epidermis together with unique extracellular aggregates of herpesvirus-like particles. The RHV3-associated skin disease has features similar to those of a condition recognized in European frogs for the last 20 years and whose cause has remained elusive. The genome of RHV3 shares most of the features of the Alloherpesviruses. The characterization of this presumptive pathogen may be of value for amphibian conservation and for a better understanding of the biology of Alloherpesviruses.

Aeromonas salmonicida type III secretion system-effectors-mediated immune suppression in rainbow trout (Oncorhynchus mykiss).

Aeromonas salmonicida subsp. salmonicida, the etiologic agent of furunculosis, is a major pathogen in aquaculture. Together with other pathogens, it is characterized by the presence of a type 3 secretion system (T3SS). The T3SS is the main virulence mechanism of A. salmonicida. It is used by the bacterium to secrete and translocate several toxins and effector proteins into the host cell. Some of these factors have a detrimental impact on the integrity of the cell cytoskeleton, likely contributing to impair phagocytosis. Furthermore, it has been suggested that effectors of the T3SS are able to modulate the host's immune response. Here we present the first partial characterization of the immune response in rainbow trout (Oncorhynchus mykiss) infected with distinct strains of A. salmonicida either carrying (i) a fully functional T3SS or (ii) a functionally impaired T3SS or (iii) devoid of T3SS ("cured" strain). Infection with an A. salmonicida strain either carrying a fully functional or a secretion-impaired T3SS was associated with a strong and persistent immune suppression. However, the infection appeared to be fatal only in the presence of a fully functional T3SS. In contrast, the absence of T3SS was neither associated with immune suppression nor fish death. These findings suggest that the T3SS and T3SS-delivered effector molecules and toxins of A. salmonicida do not only impair the host cells' cytoskeleton thus damaging cell physiology and phagocytosis, but also heavily affect the transcription of critical immune mediators including the shut-down of important warning signals to recognize infection and induce immune defense.

Factors associated with sun protection compliance: results from a nationwide cross-sectional evaluation of 2215 patients from a dermatological consultation.

BACKGROUND: Campaigns designed to promote sun protection often fail to induce long-term changes in behaviour. There is limited information on patients with low compliance to sun protection recommendations from dermatologists. OBJECTIVES: To characterize dermatology patients at higher risk of low compliance to sun protection measures, and to investigate the relationship between sun protection behaviour, knowledge about accurate sun protection recommendations, ultraviolet (UV)-associated risks and level of UV exposure. METHODS: An anonymous self-administered multiple-choice questionnaire was distributed by dermatologists to patients receiving a sunscreen prescription. Four domains were explored: sun protection behaviour, sun protection knowledge, level of UV exposure and knowledge about UV-associated risks. We modelled sun protection behaviour and determined factors associated with low compliance to sun protection measures. RESULTS: In total 2215 questionnaires were analysed. Patients stratified by risk who better complied with sun protection measures had a better knowledge of UV-associated risks (mean score 14·45 ± 3·20 vs. 12·75 ± 3·29 and 11·20 ± 3·80, P < 0·0001) and sun protection measures (mean score 12·08 ± 2·79 vs. 10·68 ± 3·11 and 9·00 ± 3·63, P < 0·0001). Patients who better complied with sun protection measures also reported higher levels of sun exposure (mean score 4·24 ± 2·26 vs. 4·02 ± 2·05 and 3·34 ± 2·14, P < 0·0001). Factors associated with low adherence to sun protection behaviour were age below 20 or over 64 years, male sex, lower knowledge about accurate sun protection recommendations and UV-associated risks, and low UV exposure. CONCLUSIONS: This study shows the complex relationship between UV exposure, knowledge about UV-associated risks, and knowledge about sun protection recommendations and behaviour. Future skin cancer prevention programmes should focus on specific populations with low sun protection behaviour and high UV exposure.

Fatal combined infection with canine distemper virus and orthopoxvirus in a group of Asian marmots (Marmota caudata).

A fatal combined infection with canine distemper virus (CDV) and orthopoxvirus (OPXV) in Asian marmots (Marmota caudata) is reported in this article. A total of 7 Asian marmots from a small zoological garden in Switzerland were found dead in hibernation during a routine check in the winter of 2011. The marmots died in February 2011. No clinical signs of disease were observed at any time. The viruses were detected in all individuals for which the tissues were available (n = 3). Detection of the viruses was performed by reverse transcription polymerase chain reaction. The most consistent gross lesion was a neck and thorax edema. A necrotizing pharyngitis and a multifocal necrotizing pneumonia were observed histologically. Numerous large intracytoplasmic eosinophilic inclusions were seen in the epithelial cells of the pharynx, of the airways, and in the skin keratinocytes. Brain lesions were limited to mild multifocal gliosis. Phylogenetic analysis revealed that the marmot CDV strain was closely related to the clusters of CDVs detected in Switzerland in wild carnivores during a local outbreak in 2002 and the 2009-2010 nationwide epidemic, suggesting a spillover of this virus from wildlife. The OPXV was most closely related to a strain of cowpoxvirus, a poxvirus species considered endemic in Europe. This is the first reported instance of CDV infection in a rodent species and of a combined CDV and OPXV infection.

Emergence of canine distemper virus strains with modified molecular signature and enhanced neuronal tropism leading to high mortality in wild carnivores.

An ongoing canine distemper epidemic was first detected in Switzerland in the spring of 2009. Compared to previous local canine distemper outbreaks, it was characterized by unusually high morbidity and mortality, rapid spread over the country, and susceptibility of several wild carnivore species. Here, the authors describe the associated pathologic changes and phylogenetic and biological features of a multiple highly virulent canine distemper virus (CDV) strain detected in and/or isolated from red foxes (Vulpes vulpes), Eurasian badgers (Meles meles), stone (Martes foina) and pine (Martes martes) martens, from a Eurasian lynx (Lynx lynx), and a domestic dog. The main lesions included interstitial to bronchointerstitial pneumonia and meningopolioencephalitis, whereas demyelination--the classic presentation of CDV infection--was observed in few cases only. In the brain lesions, viral inclusions were mainly in the nuclei of the neurons. Some significant differences in brain and lung lesions were observed between foxes and mustelids. Swiss CDV isolates shared together with a Hungarian CDV strain detected in 2004. In vitro analysis of the hemagglutinin protein from one of the Swiss CDV strains revealed functional and structural differences from that of the reference strain A75/17, with the Swiss strain showing increased surface expression and binding efficiency to the signaling lymphocyte activation molecule (SLAM). These features might be part of a novel molecular signature, which might have contributed to an increase in virus pathogenicity, partially explaining the high morbidity and mortality, the rapid spread, and the large host spectrum observed in this outbreak.

Effects of three modifiers of glycolysis on ATP, lactate, hypoxia, and growth in human tumor cell lines in vivo.

BACKGROUND: High pretreatment tumor lactate content is associated with poor outcome after fractionated irradiation in human squamous cell carcinoma (hSCC) xenografts. Therefore, decreasing lactate content might be a promising approach for increasing tumor radiosensitivity. As the basis for such experiments, the effects of the biochemical inhibitors pyruvate dehydrogenase kinase dichloroacetate (DCA), lactate dehydrogenase oxamate, and monocarboxylic acid transporter-1 α-cyano-4-hydroxycinnamate (CHC) on tumor micromilieu and growth were investigated. MATERIALS AND METHODS: Oxygen consumption (OCR) and extracellular acidification rates (ECAR) were measured in FaDu and UT-SCC-5 hSCC in response to DCA in vitro. Mice bearing FaDu, UT-SCC-5, and WiDr colorectal adenocarcinoma received either DCA in drinking water or DCA injected twice a day, or CHC injected daily. WiDr was also treated daily with oxamate. FaDu and UT-SCC-5 were either excised 8 days after treatment for histology or tumor growth was monitored. WiDr tumors were excised at 8 mm. Effect of inhibitors on ATP, lactate, hypoxia, and Ki67 labeling index (LI) was evaluated. RESULTS: DCA increased OCR and decreased ECAR in vitro. None of the treatments with inhibitors significantly changed lactate content, hypoxia levels, and Ki67 LI in the three tumor lines in vivo. ATP concentration significantly decreased after only daily twice injections of DCA in FaDu accompanied by a significant increase in necrotic fraction. Tumor growth was not affected by any of the treatments. CONCLUSION: Overall, tumor micromilieu and tumor growth could not be changed by glycolysis modifiers in the three tumor cell lines in vivo. Further studies are necessary to explore the impact of metabolic targets on radiation response.

Manipulation of glycolysis in malignant tumors: fantasy or therapy?

After Warburg stated his hypothesis on tumor cell metabolism about 80 years ago, the field of carbohydrate metabolism of cancer cells and solid tumors is experiencing a boom for the past few years. Numerous studies have been focused on the characteristics of cancer metabolism and its accessibility to novel therapeutic interventions. Malignant transformation is associated with an increase in glycolytic flux, mainly caused by an upregulation of numerous glycolysis-related genes in the majority of human cancers. As a consequence of these alterations, tumor cells are producing lactate at higher levels compared to non-malignant tissue, even in the presence of oxygen, a phenomenon termed "aerobic glycolysis" or "Warburg effect". A correlation between alterations in glycolytic pathways and therapy resistance in tumors is partially due to radical scavenger properties of specific metabolites which may decrease therapy-induced radical formation. Glycolytic activity and glycolysis-linked metabolic milieu are often variable between individual tumors resulting in variations in treatment response and aggressiveness. The peculiarities of tumor cell metabolism can be utilized for cancer diagnostics, such as metabolic imaging techniques and metabolic tumor markers. An emerging field of research is the manipulation of tumor cell metabolism for therapeutic purposes; respective studies include approaches of glycolysis inhibition or forcing mitochondrial respiration, respectively, based on biochemical and molecular principles. Up to now, such approach could eliminate tumors in patients without side effects when applied as single drug. Nevertheless, several agents that manipulate tumor metabolism may become a supportive therapy in combination with established cancer treatments.

[Metabolit-induced bioluminescence for tumor prediction: detection of metabolites in tumors of the head and neck region]. / Metabolitinduzierte Biolumineszenz zur Tumorprädiktion: Detektion von Metaboliten in Tumoren des Kopf-Hals-Bereiches.

Solid tumors show an altered metabolism with respect to glycolysis in comparison to normal tissue. Recently, the determination of different glycolytic metabolites for tumor diagnosis and therapeutic decision-making became the focus of interest for various research groups. In particular an increased lactate concentration in tumor tissue appears to be a predictor of an adverse prognosis. Imaging of induced bioluminescence in rapidly frozen tumor biopsies is an established technique for the detection of selected substances. In this method the metabolites of interest are biochemically linked to luciferases. A microscopic photon counting system registers the light intensity and after calibration reflects the concentration distribution of metabolites. In contrast to other methods it is possible to detect different metabolites from one specific area of tissue. Preliminary results of a pilot study on oral cancer patients suggest a prognostic impact in terms of high lactate concentrations being associated with poor survival. This technique could increase the validity and significance of tumor grading and might be supportive decision guidance for tumor therapy in the future.

[Lactate and redox status in malignant tumors]. / Laktat und Redoxstatus in malignen Tumoren.

This article summarizes data from experimental and clinical oncology which are indicative of a pivotal role of tumor carbohydrate metabolism in malignant behavior and outcome of treatment. In primary tumors, such as cervix carcinomas, head and neck squamous cell carcinomas or rectum adenocarcinomas, elevated lactate levels as a mirror of a high glycolytic activity, are correlated even at the initial diagnosis with a high level of malignancy as indicated by increased formation of metastases or an elevated radiotherapy resistance. The relationship between therapeutic resistance and glycolysis may at least partially be due to the radical scavenging potential of glycolytic intermediates, mainly pyruvate and lactate and to the link between these metabolites and the cellular redox status. On the basis of these data and other considerations, a novel technique has been developed for imaging the lactate/pyruvate ratio in tumor biopsies using quantitative bioluminescence. More research effort should, therefore, be focussed on the redox status of tumors in oncological studies in the future.

Comparative quantitative assessment of GnRH- and LH-receptor mRNA expression in the urinary tract of sexually intact and spayed female dogs.

Ovariectomy interrupts the regulatory loop in the hypothalamus-pituitary-gonad axis, leading to a several-fold increase in gonadotropin levels. This rise in hormonal secretion may play a causal role in ovariectomy-related urinary incontinence. The purpose of this study was to examine the effect of ovariectomy in bitches on the expression of GnRH- and LH-receptors in the lower urinary tract, and assess the relationship between receptor expression and plasma gonadotropin concentrations. Plasma gonadotropins were measured in 37 client-owned bitches. Biopsies were harvested from the mid-ventral bladder wall in all dogs, and from nine further locations within the lower urinary tract in 17 of the 37 animals. Messenger RNA of the LH and GnRH receptors was quantified using RT-PCR with the TaqMan Universal PCR Master Mix. Gonadotropins were measured with a canine-specific FSH-immunoradiometric assay and LH-radioimmunoassay. The hierarchical mixed ANOVA model using MINITAB, Mann-Whitney U-test, unpaired means comparison and linear regressions using StatView were applied for statistical analyses. Messenger RNA for both receptors was detected in all biopsy samples. Age was negatively correlated to mRNA expression of the LH and the GnRH receptors. A relationship between the mRNA values and the plasma gonadotropin concentrations was not established. Evaluation of results within each of the biopsy locations revealed greater LH-receptor expression in the proximal second quarter of the urethra in spayed bitches than in intact bitches (P=0.0481). Increased mRNA expression of LH receptors in this location could possibly play a role in the decrease in closing pressure of the urethra following ovariectomy.

Immune dysregulation in flea allergy dermatitis--a model for the immunopathogenesis of allergic dermatitis.

BACKGROUND: Flea allergy dermatitis (FAD) is a common skin disease in dogs and can be induced experimentally. It often coexists with other allergic conditions. So far no studies have investigated the quantitative production of cytokine mRNA in skin biopsies and peripheral blood mononuclear cells (PBMC) in flea allergic dogs. OBJECTIVE: The aim of our study was to improve the understanding of the immunopathogenesis of allergic dermatitis as a response to fleabites. MATERIAL AND METHODS: Allergic and non-allergic dogs were exposed to fleas. Before and after 4 days of flea exposure mRNA was isolated from biopsies and PBMC. Production of chymase, tryptase, IL-4, IL-5, IL-13, TNF-alpha and IFN-gamma mRNA was measured by real-time RT-PCR. The inflammatory infiltrate in the skin was scored semi-quantitatively. The number of eosinophils, mast cells (MC) and IgE+ cells/mm2 was evaluated to complete the picture. RESULTS: FAD was associated with a higher number of MC before flea exposure and with a significant increase of eosinophils after flea exposure as compared to non-allergic dogs. The number of IgE+ cells was higher in allergic dogs before and after flea exposure. In allergic dogs mRNA for most cytokines and proteases tested was higher before flea exposure than after flea exposure. After exposure to fleas an increased mRNA production was only observed in non-allergic dogs. In vitro stimulation with flea antigen resulted in a decreased expression of most cytokines in allergic dogs before flea exposure. In contrast, in PBMC, only increased levels of IL-4 and IL-5 mRNA were observed in allergic dogs before flea exposure. However, after flea exposure and additional stimulation with flea antigen the production of mRNA for all cytokines tested was significantly increased in allergic dogs. CONCLUSION: We demonstrated that the response in biopsies and PBMC is different and that FAD is associated with a TH2 response.

Cutaneous leishmaniasis in naturally infected dogs is associated with a T helper-2-biased immune response.

Skin lesions are a frequent manifestation of Leishmania infantum infections in Mediterranean countries. This study demonstrates by real-time reverse transcriptase-polymerase chain reaction the local cytokine response in skin biopsies from Leishmania-infected dogs (n=10). As controls, we investigated skin biopsies from healthy (n=10) and fleabite hypersensitive dogs (n=10). We established a quantitative PCR to determine the parasite burden in biopsies. The objective was to elucidate whether a correlation exists between parasite number, histologic response, and T helper-1 (TH1)/T helper-2 (TH2) cytokine expression in lesional skin of naturally infected dogs. In Leishmania-infected dogs, interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma) messenger RNA production was significantly higher than controls. Furthermore, dogs with a high Leishmania burden had a significantly higher IL-4 expression, whereas no difference was noted with regard to expression of other cytokines. By comparing the pattern of inflammation and cytokine expression, a clear trend became evident in that levels of IL-4, TNF-alpha, and IFN-gamma were elevated in biopsies with a periadnexal nodular pattern and in biopsies where the severity of the periadnexal infiltrate was equal to the perivascular to interstitial infiltrate. Expression of IL-4, IL-13, and TNF-alpha was slightly increased in biopsies where plasma cells prevailed on lymphocytes, whereas expression of IFN-gamma was moderately higher when lymphocytes were predominating. In summary, the present study demonstrates that the local immune response in naturally occurring leishmaniasis includes TH1 as well as TH2 cytokine subsets. Furthermore, respective data suggest that increased expression of the TH2-type cytokine IL-4 is associated with both severe clinical signs and a high parasite burden in the skin lesions.

Detection of oxidative base DNA damage by a new biochemical assay.

Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers.

Repair of oxidative DNA damage in vitro: a tool for screening antioxidative compounds.

Reactive oxygen species (ROS) provoke the formation of base DNA alterations that are processed by an excision step of the lesion followed by a repair synthesis and ligation step to restore the strand continuity. We have reported previously the detection of DNA adducts by an in vitro chemiluminescence DNA repair synthesis assay (Salles et al., 1995) which allows the measurement of repair synthesis by cell-free extracts in damaged plasmid DNA adsorbed on sensitized microplate wells. The 3D (DNA damage detection) assay was performed in the presence of biotin-dUTP which was incorporated during the repair synthesis step. The extent of repair synthesis was measured in an ELISA reaction with ExtrAvidin-horse radish peroxidase and chemiluminescence detection. The 3D assay allows detection of any type of base alterations including base oxidation. Interestingly, under controlled production of ROS a screening procedure of antioxidants might be carried out with the 3D assay. By taking advantage of plasmid DNA adsorption, oxidative base damage can be recognized by the Escherichia coli Fpg protein which was detected in an ELISA reaction with specific antibody and chemiluminescence measurement (4D assay). With the sceening procedure of antioxidative compounds in mind, the development of such assays and their drawbacks are discussed.