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Transmission of bluetongue (BT) virus serotype 8 (BTV-8) via artificial insemination of contaminated frozen semen from naturally infected bulls was investigated in two independent experiments. Healthy, BT negative heifers were hormonally synchronized and artificially inseminated at oestrus. In total, six groups of three heifers received semen from four batches derived from three bulls naturally infected with BTV-8. Each experiment included one control heifer that was not inseminated and that remained BT negative throughout. BTV viraemia and seroconversion were determined in 8 out of 18 inseminated heifers, and BTV was isolated from five of these animals. These eight heifers only displayed mild clinical signs of BT, if any at all, but six of them experienced pregnancy loss between weeks four and eight of gestation, and five of them became BT PCR and antibody positive. The other two infected heifers gave birth at term to two healthy and BT negative calves. The BT viral load varied among the semen batches used and this had a significant impact on the infection rate, the time of onset of viraemia post artificial insemination, and the gestational stage at which pregnancy loss occurred. These results, which confirm unusual features of BTV-8 infection, should not be extrapolated to infection with other BTV strains without thorough evaluation. This study also adds weight to the hypothesis that the re-emergence of BTV-8 in France in 2015 may be attributable to the use of contaminated bovine semen.

Recently, concerns have been raised about potential adverse effects of synthetic amorphous silica, commonly used as food additive (E551), since silica nanoparticles have been detected in food containing E551. We examined the biodistribution and excretion in female Sprague-Dawley rats of NM-200, a well characterized nanostructured silica representative for food applications. A single intravenous injection of NM-200 was applied at a dose of 20 mg/kg bw , followed by autopsy after 6 and 24 h. The main organs where silicon accumulated were liver and spleen. The silicon concentration significantly decreased in spleen between 6 and 24 h. In liver the tendency was the same but the effect was not significant. This could be due to clearance of the spleen to the liver via the splenic vein, while liver clearance takes more time due to hepatic processing and biliary excretion. In treated animals the liver showed in addition a prominent increase of macrophages between both evaluation moments. Within the first 24 h, silicon was mainly excreted through urine. Further studies are necessary to evaluate the toxicokinetics of different types of silica nanomaterials at lower exposure doses in order to be able to predict kinetics and toxicity of silica nanoparticles depending on their physicochemical characteristics.

This study reports on the diagnostic potential of IFN-γ release assays and serology for Mycobacterium bovis in six naturally M. avium subsp. paratuberculosis (Map) exposed bulls of which four were intratracheally infected with a Belgian field strain of M. bovis. Heparinized blood, serum and fecal samples were collected at regular time intervals for mycobacteria-specific IFN-γ release assays, antibody analysis and for Map culture respectively. Single intradermal skin test (SIT) with bovine tuberculin (PPD-B) was performed on day 115 and animals were sacrificed on day 133 after M. bovis infection. Organs were collected and stored for histopathological examination, modified Ziehl-Neelsen staining and bacteriological analysis of M. bovis and Map by culture and RT-PCR. Prior to infection five animals showed positive IFN-γ responses to avian PPD (PPD-A) and four were positive in Map PCR (IS900) on faeces. Three M. bovis infected animals reacted as early as day 14 with sustained higher PPD-B than PPD-A specific IFN-γ responses, whereas the fourth animal (with the strongest PPD-A response prior to infection) showed sustained higher PPD-B specific IFN-γ levels only a day 56 after infection. Two of the infected animals had a sustained positive IFN-γ response to the ESAT-6/CFP-10/TB7.7 (QuantiFERON ® -TB Gold) peptide cocktail as early as day 14, among which the animal with the initial high PPD-A response. Later during infection, positive responses were found to ESAT-6 peptides in three infected bulls and to CFP-10 peptides in all four infected bulls. One of the control animals reacted intermittently to the ESAT-6/CFP10/TB7.7 cocktail. Prior to SIT, weak but positive MPB83/MBP70 specific antibody responses were detected in two of the infected bulls. All four M. bovis infected bulls reacted with a positive skin test and showed, as reported by others, increased mycobacteria specific IFN-γ production and increased positive responses in MPB83/MBP70 specific serology after SIT. At autopsy, M. bovis lesions were detected in all four experimentally infected bulls. Our results indicate that in Map exposed cattle, M. bovis diagnosis using IFN-γ assays needs a combination of PPD-B/A and ESAT-6/CFP10 for early and optimal sensitivity and that sensitivity of MPB83/MBP70 serodiagnosis is dramatically increased by prior skin testing. Map exposure did not interfere with the development of SIT in M. bovis infected animals, but resulted in a false positive M. bovis specific IFN-γ and antibody response after SIT in one of the two control animals (which remained negative in skin-test)., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The risk of tick-borne encephalitis virus (TBEV) introduction into Belgium remains high, and the presence of infected wildlife in Belgium is suspected. Domestic animals can serve as excellent sentinels for TBEV surveillance to install an early warning surveillance component for this emerging zoonotic disease of public health importance. In a targeted, risk-based and cross-sectional sampling design, serological screening was performed on Belgian cattle (n=650), selected from the 2010 Belgian national cattle surveillance serum bank. All samples were subjected to a gold standard TBEV seroneutralization test (SNT), based on the rapid fluorescent focus inhibition test (RFFIT) protocol. Seventeen bovines were seropositive (titer >1/15) and six had borderline results (1/10 < titer < 1/15). The accuracy of the RFFIT-SNT was confirmed in a mouse inoculation test. The overall bovine TBEV seroprevalence in the targeted area was estimated between 2.61% and 4.29%. This confirms for the first time the presence of infected foci in Belgium. Further surveillance in cattle, other sentinels, ticks, and humans at risk is recommended to further determine the location and size of endemic foci and the risk for public health.

Cholesterol granuloma in the middle ear is a pathological condition often associated with otitis media in humans. Cholesterol granulomas in cats are rarely described. To our knowledge, this is the first report of middle ear cholesterol granuloma in a cat, associated with otitis media and leptomeningitis due to a Streptococcus canis septicemia.

In sheep, susceptibility to scrapie is mainly determined by codons 136, 154, and 171 of the PRNP gene. Five haplotypes are usually present (ARR, ARQ, ARH, AHQ, and VRQ). The ARR haplotype confers the greatest resistance to classical scrapie while VRQ renders animals most susceptible. In 2004, the European Union implemented a breeding program that promotes selection of the ARR haplotype while reducing the incidence of VRQ. From 2006 to 2011 in Belgium, frequency for the ARR/ARR genotypes increased from 38.3% to 63.8% (n = 6,437), the ARQ haplotype diminished from 21.1% to 12.9%, and the VRQ haplotype decreased from 2.0% to 1.7%. The status of codon 141, a determinant for atypical scrapie, was also evaluated. Out of 27 different breeds (n = 5,163), nine were abundant. The ARR/ARR frequency increased in eight of these nine major breeds. The selection program has had a major impact on the ARR haplotype frequency in Belgium. However, the occurrence of atypical scrapie represents a critical point for this program that warrants the continuous monitoring of scrapie. Additionally, genotype frequencies among the breeds varied greatly. Texel, a breed that is common in Belgium, can still be selected for due to its average ARR frequency.

Objective: To evaluate the impact of modulation of the membrane-bound efflux pump P-glycoprotein (P-gp) on plasma concentrations of orally administered prednisolone in dogs., Animals: 7 healthy adult Beagles., Procedures: Each dog received 3 treatments (control [no treatment], rifampicin [100 mg/d, PO, for 21 days, as an inducer of P-gp], and ketoconazole [100 mg/d, PO, for 21 days, as an inhibitor of P-gp]). A single dose of prednisolone (1 mg/kg, PO) was administered on day 8 of each treatment period. There was a 7-day washout period between subsequent treatments. Plasma concentrations of prednisolone were determined by use of a validated liquid chromatography-tandem mass spectrometry method. Duodenum and colon biopsy specimens were obtained endoscopically from anesthetized dogs and assessed for P-gp protein labeling via immunohistochemical analysis and mRNA quantification via real-time PCR assay. Total fecal collection was performed for evaluation of effects of P-gp modulation on digestion of nutrients., Results: Rifampicin treatment upregulated duodenal P-gp in dogs and significantly reduced the area under the plasma concentration-time curve of prednisolone. Ketoconazole typically downregulated expression of duodenal P-gp, with a subsequent increase in the area under the plasma concentration-time curve of prednisolone. There was a noticeable interindividual difference in response. Digestion of nutrients was not affected., Conclusions and Clinical Relevance: Modulation of P-gp expression influenced plasma concentrations of prednisolone after oral administration in dogs. Thus, treatment response to prednisolone may be influenced by coadministration of P-gp-modulating medications or feed ingredients.

Background: Automated peritoneal dialysis (APD) provides the opportunity for home-based dialysis, enabling the patient to optimize their lifestyle by maintaining their normal daily routine. Use of a larger bag size and biocompatible solution is desirable. In an effort to further improve patient convenience and reduce the probability of infusing only the buffer contents from the outflow chamber, we designed a 5-L dual-chamber container system with a dual-seal system consisting of a long seal between the dextrose chamber and buffer chambers and a short SafetyMoon™ seal between the buffer chamber and container outflow connector., Methods: The safety and effectiveness of this new container system was assessed in a non-interventional, prospective, open-label, multi-centre, uncontrolled, Baxter-sponsored post-authorization safety study in 249 patients from 7 countries in Europe., Results: No mis-infusion events were noted throughout the study where 68 519 Physioneal™ 5-L bags in Clear-Flex™ were used for an average (SD) of 4.3 (1.9) months per patient. Overall, the percentage of patients and/or care providers rating the 5-L bag preparation as very easy or easy at baseline (0-8 weeks), 9-16, 17-24 and 25-32 weeks ranged from 94 to 97%. Assuming a Poisson distribution for the bag count data, the estimated change in number of bicarbonate/lactate dialysis fluid bags (5 or 2.5 L) as a percent of prior bag use was -36%, while the estimated change in number of bags for ALL solutions as a percent of prior bag use was -31%. The predominant reasons given by the investigators for prescribing 5-L PD solutions at study onset were biocompatibility, easier and convenient for their patients to use, physiological pH and less bag connections. None of the 92 serious adverse events were suspected to be related to the Physioneal 5-L PD solution., Conclusions: Use of a larger, Physioneal 5-L bag mitigates the concern regarding the possibility of mis-infusing the buffer chamber solution, is convenient to use by the patient/health care provider and is associated with more than a 30% reduction in the weekly number of dialysis solution bags required per patient for their APD therapy.

Background: Icodextrin is a glucose polymer derived by hydrolysis of cornstarch. The different biocompatibility profile of icodextrin-containing peritoneal dialysis (PD) solutions may have a positive influence on peritoneal host defence. Furthermore, cases of sterile peritonitis potentially associated with icodextrin have been reported., Methods: The primary objective of this multicentre, longitudinal, observational, non-interventional, prospective cohort study, which included 722 PD patients, was to evaluate the incidence of overall peritonitis in patients treated with icodextrin-containing PD solutions (Extraneal) used during one long-dwell exchange/day compared with those treated with non-icodextrin-containing PD solutions. The secondary objective was to determine if culture-negative peritonitis rates differed between patients treated with icodextrin from two independent manufacturers. All peritonitis episodes were assessed by a Steering Committee in a blind manner., Results: There was no significant difference between icodextrin-treated and control patients in the adjusted overall, culture-positive or culture-negative peritonitis rates. When stratified by the icodextrin supplier, there was no significant difference in the adjusted rate of culture-negative peritonitis episodes between groups., Conclusion: Subjects receiving icodextrin as part of their PD regimen experienced neither a higher rate of culture-negative peritonitis nor a lower rate of infectious peritonitis compared with non-icodextrin users. There was no significant influence of the icodextrin raw material supplier on peritonitis rates.

The detection of multidrug resistance (MDR) in clinical samples is still a topic for discussion. One method, proven extremely useful for detection of membrane proteins in patients with hematological malignancies is the flow cytometrical analysis of individual tumor cells. Recently an assay was described based on the labeling of the P-glycoprotein (P-gp) with the monoclonal antibody MRK16, combined with detection of active daunorubicin (DNR) extrusion. In order to improve the specificity of the assay, on line with the results obtained by Wall et al., we exploited staining with Fluo-3. Both assays prove to be able to discriminate between drug-resistant and drug-sensitive cells. A major drawback of labeling with Fluo-3 in combination with the monoclonal antibody MRK16 is the important overlap of emission spectra of both fluorochromes. Moreover, using Fluo-3 for the detection of MDR might be complicated by the fact that differences in fluorescence intensities are not solely dependent on the presence of P-gp, but also on the activity of cytosolic esterases and the intracellular calcium concentration. Combination of the detection of structural and functional aspects of the MDR-associated protein may lead to a more precise detection of the MDR-positive patient.

This paper describes the adaptation of the MTT assay to hypoxic conditions in order to test the in vitro effect of piracetam on hypoxic cells and particularly on the radiosensitivity of hypoxic cells since this drug has shown clinical effect on acute and chronic hypoxia. The V79 cell line was selected by reference to preliminary hypoxic experiments using clonogenic assay and euoxic experiments using clonogenic and MTT assays. Cell growth and survival in our hypoxic conditions were assessed using MTT assay with an enclosure and special 48-well plates both made of glass. Growth curves on glass versus reference polystyrene plates were comparable and confirm the validity of using special glass plates. Growth curves on glass plates after 1-hour exposure to nitrogen versus air were comparable, so there is no bias effect due to gas composition. Survival curves using MTT versus reference clonogenic assay were comparable after radiation exposure in eu- and hypoxic conditions, and confirm the validity of our original technique for creating hypoxia. The Oxygen Enhancement Ratio was of about 3 for 1-hour hypoxic exposure. Piracetam gave no cytotoxic effect up to 10 mM of piracetam. Growth curves after continuous drug exposure and 1-hour euoxic versus hypoxic exposure gave no cytotoxic effect up to 10 mM of piracetam. Survival curves after continuous drug exposure to 10 mM of piracetam gave no significant effect on the radiosensitivity of hypoxic V79 cells using MTT or clonogenic assay. However, this does not preclude a potential in vivo effect of piracetam on the radiosensitivity owing to its action on microcirculation and its rheologic properties. The adaptation of the MTT assay to hypoxic irradiation conditions yields the easy screening of radiosensitizing drugs: shorter incubation, semi-automatic method and simultaneous analysis with different serial concentrations thanks to the special 48-well glass plates.

Multidrug resistance (MDR) is responsible for a decrease in sensitivity of tumor cells tumor cells to unrelated, naturally occurring anticancer drugs. This resistance is correlated with expression and activity of a membrane protein, P-gp 170, functioning as a drug-extruding pump. It has been well described in in vitro situations; however, the clinical detection and implications are not yet clear. Multiple detection assays have been developed based on the discovery of the MDR gene family and the corresponding protein. Southern, Northern, or Western blot analysis, S1 nuclease protection or PCR-based assays, immunohistochemical detection or functionality tests by flow cytometry have been used extensively. However, by use of these techniques on clinical material, both normal and malignant, contradictory results have emerged. The sensitivity and specificity of a certain technique are always limited by unavoidable parameters, for example, skill of the technician. Moreover, the complexity of the development of resistance against anticancer agents (external determinants), such as the diversity of tumor tissues, the simultaneous presence of other resistance mechanisms, and the low expression level, make MDR detection equivocal and can lead to contradictory results. Previous treatment influencing the MDR profile and inappropriate timing of the test make a possible correlation between MDR expression and chemotherapeutic resistance difficult to establish and can lead to discordant results. In this review, the need for proper criteria is stressed. No single detection technique provides the ideal test to detect MDR. Tandem testing could give more certainty, although small sample size limit this application. Formulation of a standard assay with better definition of a positivity is essential before clinical trials are started.

In this study the activity of KW-2149 and two of its metabolites, M-16 and M-18, was measured against cell lines with different types of resistance. The influence of these metabolites and of the exposure time on the cytotoxic efficacy of KW-2149 was investigated. Against the human ovarian carcinoma cell lines, AOvC and A2780, KW-2149 was more active than mitomycin C (MMC), with an IC50 of, respectively, 3.4 nM and 9.82 microM for KW-2149 and 18.2 nM and 67.71 microM for MMC. Activity of M-18 was significant against both cell lines and was comparable with that of KW-2149. Against an MMC-resistant cell line, AOvCMMC, the resistance factor (RF) for KW-2149 was 3.1 versus 8.0 for MMC. Tested against a cisplatin-resistant cell line, AOvCCDDP, KW-2149 had a RF of 7.7 versus 2.4 for MMC. Increasing the exposure time from 1 to 8 h decreased the RF for KW-2149 from 7.7 to 3.0. In an MDR mediated resistant cell line, A2780mdr+, prolongation of exposure time increased RF for KW-2149 and MMC but decreased RF for M-18 from 7.0 at 1 h to 5.3 at 8 h. Tested against a rat colon carcinoma cell line CC531, KW-2149 and M-18 again demonstrated superior or equal activity compared with MMC, IC50 being, respectively, 0.6, 2.1 and 2.6. Here again M-18 showed an aberrant sensitivity pattern, as its activity decreased with mdr-1 expression in contrast to the other mitomycins. Our data confirm the activity of KW-2149 as an agent with equal or superior activity as compared with MMC. It is concluded that the metabolite M-18 can contribute to the activity of KW-2149. Efficacy of both KW-2149 and its metabolites increases with increasing exposure times. The increments of exposure time appeared even as a means to overcome resistance in some instances.

Most anticancer agents fail to induce clear responses in the treatment of colorectal cancer. This can be explained by involvement of overexpression of the membrane glycoprotein, P-gp 170, which is associated with multidrug resistance (MDR), and/or with involvement of ras. Fluoropyrimidines are amongst the few options in the chemotherapeutic treatment of colorectal cancers. 5'-Deoxy-5-fluorouridine (dFUrd) needs intracellular activation via 5-fluorouracil into 5-fluoro-2'-deoxyuridine-5'-monophosphate and 5-fluorouridine-5'-triphosphate. In the present study, the cytotoxic activity of dFUrd in vitro and dFUrd combined with daunorubicin (DNR) was assessed with the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium) bromide assay in cells with increased P-gp 170 expression versus controls. Surprisingly, dFUrd was most active in cells with high P-gp 170 expression, a finding which can not be explained by intracellular metabolic activity only. The results also show that dFUrd improves the DNR uptake in MDR-positive cells, and this is related with increased cytotoxicity of the anthracycline.

A rat colon carcinoma cell line, CC531, was exposed to stepwise increasing concentrations of colchicine. A cell line, CC531mdr+, which grows in the presence of 0.2 microM of colchicine was obtained. A revertant cell line, CC531rev was isolated upon colchicine withdrawal. The CC531mdr+ displayed a multidrug-resistant phenotype. Marked resistance to the selecting agent colchicine, was found (RF = 37.5) as well as to vinblastine (RF = 11.3) and actinomycin D (RF = 2.6). Cross resistance to doxorubicin (RF = 8) and daunorubicin (RF = 13.3) was demonstrated. Verapamil was able to reverse drug resistance to colchicine and daunorubicin. The revertant cell line, CC531rev, showed increased sensitivity to colchicine (RF = 0.43), vinblastine (RF = 0.13), doxorubicin (RF = 0.28) and daunorubicin (RF = 0.56). Marked cross resistance to cis-platinum (RF = 6.9) was also induced in CC531mdr+ and was maintained in CC531rev. We conclude that CC531 displays an intrinsic low-level multidrug-resistant phenotype, which was amplified in the CC531mdr+ variant. This correlates with a higher level of expression of P-glycoprotein. CC531rev lacks the multidrug-resistant phenotype and can be used as the drug-sensitive counterpart of the latter two cell lines. Furthermore, it has been shown that in these cell lines cis-platinum resistance is mediated through a mechanism independent of the multidrug-resistant phenotype, since the revertant cell line CC531rev has lost the multidrug-resistant phenotype while retaining the concomitantly induced cis-platinum resistance of the multidrug-resistant variant CC531mdr+.

The development of resistance to anticancer drugs urges the search for different treatment modalities. Several investigators have reported the concomitant development of drug resistance and resistance to natural killer (NK), lymphokine-activated killer (LAK) or monocyte/macrophage cell lysis, while others described unchanged or even increased susceptibility. We investigated this subject in the rat colon carcinoma cell line, CC531-PAR, which is intrinsically multidrug-resistant (MDR), and in three sublines derived from this parental cell line: a cell line with an increased MDR phenotype (CC531-COL), a revertant line from CC531-COL (CC531-REV), which demonstrates enhanced sensitivity to anticancer drugs of the MDR phenotype, and an independently developed cisplatin-resistant line (CC531-CIS). In a 4-h 51Cr-release assay we found no difference in susceptibility to NK cell lysis. No significant differences in lysability by adherent LAK (aLAK) cells were observed in a 4-h assay. In a prolonged 20-h 51Cr-release assay an enhanced sensitivity to aLAK-cell-mediated lysis was observed in the revertant, P-glycoprotein-negative cell line and in the cisplatin-resistant cell line (CC531-CIS). None of the cell lines was completely resistant to lysis by aLAK cells. Therefore, a role for immunotherapy in the treatment of drug-resistant tumors remains a realistic option.

The CMF-regimen is amongst the most effective chemotherapeutic approaches in the treatment of breast cancer. It is generally accepted that the efficacy of the combination of the three agents used in the regimen, i.e., cyclophosphamide (CY), methotrexate (MTX) and fluorouracil (FUra), is based on interactions between the drugs at the intratumoral level. In WAG/Rij rats we previously demonstrated that change of FUra clearance at the first day of the CMF-regimen occurs owing to concomitant CY + MTX. In the present study clearance of FUra and the first product of FUra catabolism, FUraH2, were monitored at day 1 and day 8 of the regimen upon treatment with single agent FUra (F), MTX + FUra (MF), CY + FUra (CF), and CY + MTX + FUra (CMF). At the first day of treatment, FUra and FUraH2 systemic exposure was demonstrated to be increased in CMF-treated rats owing to concomitant CY+MTX. At the eighth day of treatment it was found that repeated CY administration during the previous seven days in CF-treated rats resulted in increased FUra and FUraH2 systemic exposure and therefore increased the dose of FUra artificially. It is concluded that altered FUra clearance owing to extratumoral interactions by concomitant CY and MTX contributes to the efficacy of the CMF-regimen.