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Differences were examined among 24 distinct elements mediating VanA-type glycopeptide resistance in enterococci isolated from hospital patients and non-human sources in the UK. The methods used included long-PCR restriction fragment length polymorphism (L-PCR RFLP) analysis and DNA hybridization. All elements had conserved vanRSHAX genes, but variation occurred upstream of vanR and downstream of vanX. Twenty-one VanA elements had significant alterations upstream of vanR in the transportation genes orf1 and orf2: either parts of these genes were absent or they were disrupted by IS1216V or IS3-like insertion sequences. Among VanA elements with alterations downstream of vanX, seven lacked vanY, one lacked boy vanY and vanZ, and ten had copies of insertion sequence IS1216V between vanX and vanY. All VanA elements of group D (from geographically and temporally diverse enterococci) were characterized by the presence of an IS1216V/IS3-like/orf1 complex and a point mutation in vanX, both of which were absent from the other 23 groups of VanA elements. This finding is consistent with the dissemination of a stable resistance element. We conclude that L-PCR RFLP analysis, combined with DNA hybridization, merits further development for studying the evolution and epidemiology of VanA resistance elements in enterococci. [ABSTRACT FROM PUBLISHER]

During 2001-2002, high-level gentamicin-resistant (HLGR) Enterococcus faecalis isolates were detected in 2 patients in Denmark who had infective endocarditis and in pigs and pork. Our results demonstrate that these isolates belong to the same clonal group, which suggests that pigs are a source of HLGR E. faecalis infection in humans. [ABSTRACT FROM AUTHOR]

Inflammation and autoimmunity are known as central processes in many skin diseases, including psoriasis. It is therefore important to develop pre-clinical models that describe disease-related aspects to enable testing of pharmaceutical drug candidates and formulations. A widely accepted pre-clinical model of psoriasis is the imiquimod (IMQ)-induced skin inflammation mouse model, where topically applied IMQ provokes local skin inflammation. In this study, we investigated the abundance of a subset of matrix metalloproteinases (MMPs) in skin from mice with IMQ-induced skin inflammation and skin from naïve mice using targeted proteomics. Our findings reveal a significant increase in the abundance of MMP-2, MMP-7, MMP-8, and MMP-13 after treatment with IMQ compared to the control skin, while MMP-3, MMP-9, and MMP-10 were exclusively detected in the IMQ-treated skin. The increased abundance and broader representation of MMPs in the IMQ-treated skin provide valuable insight into the pathophysiology of skin inflammation in the IMQ model, adding to previous studies on cytokine levels using conventional immunochemical methods. Specifically, the changes in the MMP profiles observed in the IMQ-treated skin resemble the MMP patterns found in skin lesions of individuals with psoriasis. Ultimately, the differences in MMP abundance under IMQ-induced inflammation as compared to non-inflamed control skin can be exploited as a model to investigate drug efficacy or performance of drug delivery systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Knowledge of COVID-19 and the pandemic's effects on Danish children's body weight is limited., Objective: Objectives were to investigate (I) risk of weight changes among Danish children with and without SARS-CoV-2, (II) associations between weight changes, psychological symptoms, and long COVID symptoms, and (III) weight distribution pre- and post-pandemic., Methods: A national survey was administered to all Danish children aged 0-18 years, with prior COVID-19 (cases) and matched references including questions on weight, weight changes during the pandemic and long COVID-related symptoms. Descriptive statistics and logistic regression were used. Weight distribution was compared with a pre-pandemic database., Results: In all, 17 627 cases and 54 656 references were included. The 4-18-year-old cases had lower odds of unintended weight gain. The 2-3-year-old cases had higher odds and the 15-18-year-old cases lower odds of weight loss compared to references. Regardless of COVID-19 status, any reported long COVID-related symptom was associated with a change in body weight. No sign of increasing obesity rates was found among Danish children post-pandemic., Conclusion: COVID-19 was associated with higher odds of weight loss in 2-3-year-olds and lower odds of unintended weight gain in 4-18-year-olds. Any long COVID-related symptom was associated with higher odds of weight changes regardless of COVID-19 status., (© 2023 The Authors. Pediatric Obesity published by John Wiley & Sons Ltd on behalf of World Obesity Federation.)

Enzyme-responsive hydrogels, formed by step growth photopolymerization of biscysteine peptide linkers with alkene functionalized polyethylene glycol, provide interesting opportunities as biomaterials and drug delivery systems. In this study, we developed stimuli-responsive, specific, and cytocompatible hydrogels for delivery of anti-inflammatory drugs for the treatment of inflammatory skin diseases. We designed peptide linkers with optimized sensitivity towards matrix metalloproteinases, a family of proteolytic enzymes overexpressed in the extracellular matrix of the skin during inflammation. The peptide linkers were crosslinked with branched 4-arm and 8-arm polyethylene glycols by thiol-norbornene photopolymerization, leading to the formation of a hydrogel network, in which the anti-inflammatory Janus kinase inhibitor tofacitinib citrate was incorporated. The hydrogels were extensively characterized by physical properties, in vitro release studies, cytocompatibility with fibroblasts, and anti-inflammatory efficacy testing in both an atopic dermatitis-like keratinocyte assay and an activated T-cell assay. The drug release was studied after single and multiple-time exposure to matrix metalloproteinase 9 to mimic inflammatory flare-ups. Drug release was found to be triggered by matrix metalloproteinase 9 and to depend on type of crosslinker and of the polyethylene glycol polymer, due to differences in architecture and swelling behavior. Moreover, swollen hydrogels showed elastic properties similar to those of extracellular matrix proteins in the dermis. Cell studies revealed limited cytotoxicity when fibroblasts and keratinocytes were exposed to the hydrogels or their enzymatic cleavage products. Taken together, our results suggest multi-arm polyethylene glycol hydrogels as promising matrix metalloproteinase-responsive drug delivery systems, with potential in the treatment of inflammatory skin disease. STATEMENT OF SIGNIFICANCE: Smart responsive drug delivery systems such as matrix metalloproteinase-responsive hydrogels are excellent candidates for the treatment of inflammatory skin diseases including psoriasis. Their release profile can be optimized to correspond to the patient's individual disease state by tuning formulation parameters and disease-related stimuli, providing personalized treatment solutions. However, insufficient cross-linking efficiency, low matrix metalloproteinase sensitivity, and undesirable drug release kinetics remain major challenges in the development of such drug delivery systems. In this study, we address shortcomings of previous work by designing peptide linkers with optimized sensitivity towards matrix metalloproteinases and high cross-linking efficiencies. We further provide a proof-of-concept for the usability of the hydrogels in inflammatory skin conditions by employing a drug release set-up simulating inflammatory flare-ups., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Introduction: Low anterior resection syndrome (LARS) is defined as disordered bowel function following rectal resection, which is detrimental to quality of life (QoL). A recent international consensus definition of LARS stresses the importance of focusing on both the symptoms and the consequences that the symptoms have for the individual patient as studies indicate that LARS has a negative impact on patients' QoL. However, an ongoing PROM study investigating late sequelae after rectal cancer finds that a minor proportion of patients scoring major LARS experience none or only little impact on quality of life., Aim: The aim of this study was to identify patients' considerations and coping strategies to establish why the burden caused by major LARS had little or no influence on their QoL., Materials and Methods: This was a qualitative interview study based on 21 semi-structured individual telephone interviews with patients treated for rectal cancer. Data were analysed using a hermeneutic inspired thematic analysis., Results and Conclusion: Three themes emerged from the analysis; Adapting new life situation, Altering life perception and the Importance of relationships. Major LARS and its consequences following rectal cancer may be managed or altered by adopting problem-focused and emotion-focused coping strategies. Maintaining a positive attitude and having a good network of family and friends constitute a surplus, allowing patients to cope with the need for changed behaviour and appreciate the life that they have been given. Accepting that major LARS and its consequences cause limitations in life allowed patients to change their normality threshold over time., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer CV declared a past collaboration with the author PC to the handling editor., (Copyright © 2022 Laursen, Sørensen, Majgaard, Jensen, Jacobsen, Kjær, Juul, Christensen and Mikkelsen.)

Antibiotic consumption is considered to be a main driver of antibiotic resistant bacteria. Mink breeding follows a distinctive seasonal reproduction cycle, and all of the mink produced in the northern hemisphere are bred, born, and pelted around the same time of year. Some of the diseases are age-related, which is reflected in the seasonal variation of antibiotic consumption. The seasonality makes mink a good model for the investigation of the association between antibiotic consumption and resistance. The objectives of this study were (1) to monitor the farm level of antibiotic resistance during one production cycle and (2) to assess the potential associations between antibiotic consumption and resistance. Twenty-four farms were included in this study (Denmark n = 20, Iceland n = 2, and The Netherlands n = 2), following a cohort of animals born in 2018. Staphylococcus delphini and Escherichia coli were isolated from samples of the carcasses and faeces and were collected randomly. The isolates were susceptibility tested and subsequently divided into the sensitive wildtype (WT) and the resistant non-wildtype (NWT) populations. The antibiotic consumption relative to the sampling periods was assessed as having a short-term or a long-term impact, i.e., in two explanatory factors. For both S. delphini and E. coli , a large between-farm variation of NWT profiles was detected. In the final multivariable, generalized linear mixed models, significant associations between NWT isolates and the consumption of specific antibiotics were found: the short-term use of tetracyclines in the growth period was associated with the occurrence of tetracycline NWT E. coli in the growth period (OR: 11.94 [1.78; 89.28]), and the long-term use of macrolide and tetracyclines was associated with the occurrence of erythromycin NWT S. delphini in the weaning period (OR: 18.2 [2.26; 321.36]) and tetracycline NWT S. delphini in the growth period (OR: 8.2 [1.27; 63.31]), respectively. Farms with zero consumption in the study years prior to sampling also had a substantial proportion of NWT isolates, indicating that NWT isolates are persistent and/or widely spread in the environment. Generally, a high occurrence of tetracycline NWTs was observed. NWT isolates with resistance against the most commonly used antibiotics were found on all the farms, stressing the need for routine surveillance and the prudent use of antibiotics. The results offer a preview of the complex relationship between consumption and resistance, demonstrating some significant associations between use and resistance. Moreover, antibiotic-resistant bacteria are present even on farms with no antibiotic consumption over extended periods, and theoretical explanations supported by the data are offered.

Objective: To investigate the risk of venous thromboembolism (VTE) in Danish women operated within a day surgery setting and to evaluate whether the current use of thromboprophylaxis without using graduated elastic compression stockings (GCS) is an appropriate treatment to prevent VTE., Study Design: A retrospective cohort study including women who underwent laparoscopic hysterectomy or vaginal prolapse operation for benign disease from January 2014 to December 2017 at the Gynecology Day Surgery Unit, Regional Hospital of Randers, Denmark. The primary outcome was VTE diagnosed within three months postoperatively. Only one dose of pharmacological thromboprophylaxis (PTP) was given to women stratified at high risk of VTE. None of the women used GCS., Results: A total of 671 women were included. Vaginal prolapse operations were performed on 626 women, and laparoscopic hysterectomy on 45 women. PTP was used for only 220 (32.8%) of these women. A total of 346 (51.5%) women were stratified as at high risk of VTE according to the national recommendations. Only 218 (63%) of these women received PTP, while 128 women (37%) did not receive PTP. The incidence of VTE within three months postoperatively was 0%. Only 13 (1.9%) of the women were readmitted within 14 days postoperatively due to hemorrhaging or hematoma; six out of these 13 women (46%) received PTP postoperatively. Re-operation was performed in seven (1%) women due to hemorrhaging, and three out of the seven (42.9%) had PTP postoperatively., Conclusion: The risk of VTE in Danish women operated within a day surgery setting is probably very low since we found no cases of VTE in our setup. The beneficial effect of routine use of GCS and one dose of PTP postoperatively given to all women who had undergone MIS in a day surgery setting are questioned. One dose of PTP postoperatively without GCS can be considered to only women stratified as high-risk of VTE until there is more evidence whether these women actually need thromboprophylaxis postoperatively at all., Precis: The incidence of VTE in women undergoing laparoscopic hysterectomy or vaginal prolapse operation in a day surgery setting without using graduated elastic compression stockings is very low., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Streptococcus phocae infection has been described in salmon, sea otters, and several families of pinnipeds. The pathology of the infected animals has mainly been located in the respiratory tract and reproductive system, and with indications of septicemia. In this study, we report the finding of S. phocae in diagnostic material from three unrelated cases of farmed mink. Since S. phocae initially has been described in pinnipeds, two isolates from wild harbor seals were included. All isolates originated from Denmark. To our knowledge, this is the first report of S. phocae infection in mink. The animals (three mink, two seals) were necropsied, and samples were collected for bacteriology, virology, and histopathology. Additionally, the S. phocae isolates were whole genome sequenced and compared to sequences of previously reported isolates from other host species. S. phocae was isolated from the lungs of one mink and one seal with bacteremia, and from one seal with pneumonia. The two remaining mink had dermal infections on the paws and S. phocae was isolated from the lesions. The analysis of the sequence data showed that the three mink isolates and one seal isolate were closely related. Further investigation is needed to conclude whether S. phocae is establishing as commensal in farmed mink and to uncover the infection related pathology in mink. Streptococcus phocae has been described as an emerging pathogen in other species, therefore future awareness and surveillance of this pathogen is crucial., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We caught stable- and house flies on a Danish LA-MRSA positive pig farm. Stable- and house flies were housed together and culled over time to test for the presence of live LA-MRSA bacteria at 24 h intervals to establish the length of time for which LA-MRSA can persist on flies. On average, 7% of stable flies and 27% of house flies tested positive for LA-MRSA immediately upon removal from the farm. LA-MRSA prevalence decreased over time and estimates based on a Kaplan-Meier time-to-event analysis indicated that the probability of a stable- or house fly testing positive for LA-MRSA was 5.4% and 7.8% after 24 h, 3.5% and 4.3% after 48 h, 3.1% and 2.2% after 72 h and 0.4% and 0% after 96 h of removal from the pig farm, respectively. Simultaneously, we found that caged cultivated house flies became carriers of LA-MRSA, without direct contact with pigs, in the same proportions as wild flies inside the farm. We provide distance distributions of Danish pig farms and residential addresses as well as the calculated maximum dispersal potentials of stable- and house flies, which suggest that there is a potential for stable- and house flies dispersing live LA-MRSA bacteria into the surrounding environment of a pig farm. This potential should therefore be considered when modelling the spread between farms or the risk posed to humans living in close proximity to LA-MRSA pig farm sources.

Study Question: Can antioxidant treatment with N-acetylcysteine (NAC) protect ovarian follicles from ischemia-reperfusion injury in xenotransplanted human ovarian tissue?, Summary Answer: Daily administration of NAC for 7-12 days post-transplantation reduced ischemia-reperfusion injury and increased follicle survival in human ovarian xenografts by upregulating the antioxidant defense system and exerting anti-inflammatory and antiapoptotic effects., What Is Known Already: Freezing of human ovarian tissue is performed with high follicular survival rates but up to 70% of follicles appear to be lost due to hypoxia and ischemia-reperfusion injury during ovarian tissue transplantation (OTT). NAC has been demonstrated to possess antioxidant and antiapoptotic properties, and studies in rodents have shown that intraperitoneal administration of NAC reduces ischemia-reperfusion injury and increases follicle survival in autotransplanted murine ovaries., Study Design, Size, Duration: Pieces of frozen-thawed human ovarian tissue from 28 women aged 23-36 years were transplanted to immunodeficient mice in short- and long-term xenograft studies or cultured in vitro. Three short-term xenograft studies (1-week duration) were performed, in which saline or 150 mg/kg NAC was administered for 7 days post-transplantation (n = 12 patients per group). Two long-term xenograft studies (4 weeks of duration) were performed. In one of these studies, saline or 150 mg/kg NAC was administered for 12 days (n = 12 patients per group), while in the other study 50, 150 or 300 mg/kg NAC was administered for 7 days (n = 8 patients per group). In addition, human ovarian tissue (n = 12 pieces from three patients per group) was cultured with increasing concentrations of NAC (0, 5, 25 and 75 mM) for 4 days in vitro., Participants/materials, Setting, Methods: Donated ovarian tissue was obtained from women who had undergone ovarian tissue cryopreservation for fertility preservation at the University Hospital of Copenhagen. Cortical tissue pieces (5 × 5 × 1 mm) were transplanted subcutaneously to immunodeficient mice and NAC or saline was injected intraperitoneally. Grafts were retrieved after 1 or 4 weeks and follicle density was assessed. Gene expression analysis of antioxidant defense markers (superoxide dismutase; Sod1/SOD1, heme oxygenase-1; Hmox1/HMOX1, catalase; Cat/CAT), proinflammatory cytokines (tumor necrosis factor-alpha; Tnf-α, interleukin-1-beta; Il1-β, interleukin 6; Il6), apoptotic factors (B-cell lymphoma 2; Bcl2/BCL2, Bcl-2-associated X protein; Bax/BAX) and angiogenic factors (vascular endothelial growth factor A; Vegfa/VEGFA, angiopoietin-like 4; Angptl4/ANGPTL4) was performed in 1-week-old human ovarian xenografts and in cultured human ovarian tissue. Grafts retrieved after 4 weeks were histologically processed and analyzed for vascularization by CD31 immunohistochemical staining, fibrosis by Masson's Trichrome staining and apoptosis by immunofluorescence using cleaved caspase-3., Main Results and the Role of Chance: After 1-week grafting, the relative expression of Sod1, Hmox1 and Cat was significantly higher in the group receiving 150 mg/kg NAC (NAC150-treated group) compared to controls (P = 0.04, P = 0.03, and P = 0.01, respectively), whereas the expression levels of Tnf-α, Il1-β and Il6 were reduced. The Bax/Bcl2 ratio was also significantly reduced in the NAC150-treated group (P < 0.005). In vitro, the relative gene expression of SOD1, HMOX1 and CAT increased significantly in the human ovarian tissue with increasing concentrations of NAC (P < 0.001 for all genes). However, the expression of VEGFA and ANGPTL4 as well as the BAX/BCL2 ratio decreased significantly with increasing concentrations of NAC (P < 0.02, P < 0.001 and P < 0.001, respectively). After 4-week grafting, fibrosis measured by collagen content was similar in the NAC150-treated group compared to controls (control: 56.6% ± 2.2; NAC150: 57.6% ± 1.8), whereas a statistically significant reduction in the CD31-positive vessel area was found (control: 0.69% ± 0.08; NAC150: 0.51% ± 0.07; P < 0.02). Furthermore, a reduced immunoreactivity of cleaved caspase-3 was observed in follicles of the NAC150-treated xenografts compared to controls. Follicle density (follicles/mm3, mean ± SD) was higher in the NAC150-treated group compared to the control group in the 1-week xenografts (control: 19.5 ± 26.3; NAC150: 34.2 ± 53.5) and 4-week xenografts (control: 9.3 ± 11.0; NAC150: 14.4 ± 15.0). Overall, a 2-fold increase in follicle density was observed in the NAC150-group after 1-week grafting where fold changes in follicle density were calculated in relation to grafts from the same patient. Around a 5-fold increase in follicle density was observed in the NAC150 and NAC300 groups after 4-week grafting., Large Scale Data: N/A., Limitations, Reasons for Caution: Follicle density in the human ovarian cortex is highly heterogeneous and can vary 100-fold between cortex pieces from the same woman. A high variability in follicle density within and between treatment groups and patients was found in the current study. Thus, solid conclusions cannot be made. While intraperitoneal injections of NAC appeared to reduce ischemia-reperfusion injury in human ovarian xenografts, different administration routes should be investigated in order to optimize NAC for potential clinical use., Wider Implications of the Findings: This is the first study to demonstrate the antioxidant, anti-inflammatory and antiapoptotic properties of NAC in xenotransplanted human ovarian tissue. Therefore, NAC appears to be a promising candidate for protecting ovarian follicles from ischemia-reperfusion injury. This provides the initial steps toward clinical application of NAC, which could potentially reduce the loss of ovarian follicles following OTT., Study Funding/competing Interest(s): We are grateful to the Danish Childhood Cancer Foundation, Hørslev Foundation, Aase and Einar Danielsen's Foundation (grant number: 10-001999), Dagmar Marshalls Foundation, Else and Mogens Wedell-Wedellsborgs Foundation, Knud and Edith Eriksens Mindefond, and Fabrikant Einar Willumsens Mindelegat for funding this study. None of the authors have any competing interests to declare., (© The Author(s) 2020. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Optimizing antimicrobial dosage regimens and development of breakpoints for antimicrobial susceptibility testing are important prerequisites for rational antimicrobial use. The objectives of the study were (1) to produce MIC data for four mink pathogens and (2) to employ these MIC data to support the development of tentative epidemiological cut-off values (TECOFFs), which may be used for future development of mink-specific antimicrobial dosages and breakpoints. Broth microdilution was used to establish MIC distributions for 322 mink bacterial isolates of clinical origin from six European mink-producing countries. The included species were E. coli ( n = 162), S. delphini ( n = 63), S. canis ( n = 42), and P. aeruginosa ( n = 55). Sixty-four E. coli isolates and 34 S. delphini isolates were whole-genome sequenced and analyzed for antimicrobial resistance genes. No EUCAST MIC data are available on S. delphini and S. canis , hence tentative ECOFFs were suggested for the majority of the tested antimicrobials. For E. coli and P. aeruginosa , the wildtype distributions were in accordance with EUCAST data. Overall, the genotypes of the sequenced isolates were in concordance with the phenotypes. These data constitute an important piece in the puzzle of developing antimicrobial dosages and clinical breakpoints for mink. Until pharmacokinetic and clinical data become available, the (tentative) ECOFFs can be used for monitoring resistance development and as surrogates for clinical breakpoints., (Copyright © 2020 Nikolaisen, Ronaghinia, Lassen, Chehabi, Lindegaard, Struve, Chriél, Damborg, Kahlmeter, Jensen and Pedersen.)

The transmission of antimicrobial resistance genes from enteric bacteria from the animal reservoir to indigenous bacteria in meat is a serious concern, as it can contribute to human exposure to antimicrobial resistance genes. The aim of this study was to investigate plasmid-mediated horizontal transfer of antimicrobial resistance genes from Escherichia coli to indigenous environmental bacteria in minced pork stored at 10 and 37 °C. E. coli MG1555 containing a gfp-tagged plasmid carrying tetracycline, kanamycin and streptomycin resistance genes was used as the donor with the indigenous bacteria in minced pork acting as potential recipients. The results demonstrated that enteric members of the pork meat microbiota were able to receive gfp-plasmids from the E. coli donor strain at both 10 and 37 °C. The majority of transconjugants were identified as Serratia spp. through sequencing of their 16S rRNA genes. This indicates that environmental Serratia spp. and other Enterobacteriaceae may play a role as carrier of antimicrobial resistance genes through the meat production chain to the consumer., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In vitro activation of resting ovarian follicles, with the use of mechanical stress and/or pharmacological compounds, is an emerging and novel approach for infertility treatment. The aim of this study was to assess the sphingolipid, sphingosine-1-phosphate (S1P), as a potential in vitro activation agent in murine and human ovarian tissues and isolated follicles. Juvenile murine ovaries and donated human ovarian tissues, from 10 women undergoing ovarian tissue cryopreservation for fertility preservation, were incubated with or without 12 μM S1P for 3 h for quantitative PCR analysis, and 12 h for xenotransplantation or culture studies. Gene expression analyses were performed for genes downstream of the Hippo signaling pathway. Murine ovaries and isolated murine and human preantral follicles showed significantly increased mRNA expression levels of Ccn2/CCN2 following S1P treatment compared to controls. This increase was shown to be specific for the Hippo signaling pathway and for the S1P2 receptor, as co-treatment with Hippo-inhibitor, verteporfin and S1PR2 antagonist, JTE-013, reduced the S1P-induced Ccn2 gene expression in murine ovaries. Histological evaluation of human cortical tissues (5 × 5 × 1 mm; n = 30; three pieces per patient) xenografted for 6 weeks and juvenile murine ovaries cultured for 4 days (n = 9) or allografted for 2 weeks (n = 48) showed no differences in the distribution of resting or growing follicles in S1P-treated ovarian tissues compared to controls. Collectively, S1P increased Ccn2/CCN2 gene expression in isolated preantral follicles and ovarian tissue from mice and human, but it did not promote follicle activation or growth in vivo. Thus, S1P does not appear to be a potent in vitro activation agent under these experimental conditions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Cross and co-resistance to antimicrobials are presented for 765 Danish Escherichia coli isolates of porcine origin from 2009 to 2013. All isolates and data originate from the DANMAP surveillance but have not previously been used to describe the occurrence of cross and co- resistance. Data presented here clearly indicate the ability of low classified antimicrobials as ampicillin to uphold resistance to critical important antimicrobials for human treatment., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Introduction: Responsible conduct of research is the basis for the credibility of all research. Research misconduct is defined as the fabrication, falsification or plagiarism committed willfully or grossly negligently in the planning, performing or reporting of research. We undertook a survey of knowledge of the attitudes towards and experiences with research misconduct among PhD students in clinical research., Methods: A questionnaire previously used in Swedish and Norwegian studies was distributed to PhD students (n = 330) affiliated with the Department of Clinical Research or Department of Regional Health Research, University of Southern Denmark., Results: A total of 165 PhD students completed the questionnaire in full or in part, yielding an overall response rate of 50%. 18-34% reported to have heard (within the past year) about researchers who had plagiarised, falsified or fabricated data, or plagiarised publications. None reported this to occur in their own department. Few stated that they had felt under pressure to either falsify data (1%) or present results in a misleading way (3%). However, 22% stated to have felt an unethical pressure (within the past year) regarding the inclusion or order of authors., Conclusions: Results indicate that, albeit at a low frequency, research misconduct involving PhD students is taking place. Likewise, a high fraction of respondents reported to have been under pressure regarding authorships, which points to questionable research practices in clinical research., Funding: not relevant., Trial Registration: not relevant., (Articles published in the DMJ are “open access”. This means that the articles are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits any non-commercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.)

Background: To investigate whether neonatal hyperglycaemia in the first postnatal week is associated with treatment-demanding retinopathy of prematurity (ROP)., Methods: This is a Danish national, retrospective, case-control study of premature infants (birth period 2003-2006). Three national registers were searched, and data were linked through a unique civil registration number. The study sample consisted of 106 cases each matched with two comparison infants. Matching criteria were gestational age (GA) at birth, ROP not registered and born at the same neonatal intensive care unit. Potential 'new' risk factors were analysed in a multivariate logistic regression model, while adjusted for previously recognised risk factors (ie, GA at birth, small for gestational age, multiple birth and male sex)., Results: Hospital records of 310 preterm infants (106 treated; 204 comparison infants) were available. Nutrition in terms of energy (kcal/kg/week) and protein (g/kg/week) given to the preterm infants during the first postnatal week were statistically insignificant between the study groups (Mann-Whitney U test; p=0.165/p=0.163). Early postnatal weight gain between the two study groups was borderline significant (t-test; p=0.047). Hyperglycaemic events (indexed value) were statistically significantly different between the two study groups (Mann-Whitney U test; p<0.001). Hyperglycaemia was a statistically independent risk factor (OR: 1.022; 95% CI 1.002 to 1.042; p=0.031)., Conclusion: An independent association was found between the occurrence of hyperglycaemic events during the first postnatal week and later development of treatment-demanding ROP, when adjusted for known risk factors., Competing Interests: Competing interests: None declared., (© Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.)

Background: To study the long-term development (104 weeks) of insulin antibodies during treatment with insulin detemir (IDet) and insulin aspart (IAsp) in children with type 1 diabetes aged 2-16 years., Methods: A 52-week, two-arm, randomized trial comparing IDet and neutral protamine Hagedorn insulin, both in combination with IAsp, was followed by a one-arm, 52-week extension trial of the IDet + IAsp arm. The present analysis was conducted in children who completed the randomized trial and entered into the extension trial., Results: Of the 177 children randomized to IDet treatment, 146 entered the extension trial. IDet-IAsp cross-reacting antibodies peaked within the first 39 weeks of treatment before gradually declining. A similar pattern was seen for IDet-specific and IAsp-specific antibodies. At end of trial (EOT), no correlation was observed between the level of IDet-specific or IAsp-specific antibodies or IDet-IAsp cross-reacting antibodies and either glycated hemoglobin (HbA1c) or basal insulin dose. Mean HbA1c was stable during the treatment period, with a slight increase over time from 8.41% (68.4 mmol/mol) at baseline to 8.74% (72 mmol/mol) at EOT. Mean IDet dose increased from 0.43 U/kg at baseline to 0.66 U/kg at EOT. Mean IAsp dose increased from 0.46 U/kg to 0.51 U/kg at EOT., Conclusion: Although treatment with IDet and IAsp is associated with development of specific and cross-reacting antibodies, no correlation between insulin antibodies and basal insulin dose or HbA1c was found., Funding: Novo Nordisk A/S. ClinicalTrials.gov identifiers: NCT00435019 and NCT00623194.

Objectives: Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units., Methods: From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed., Results: VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA&#95;N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids)., Conclusions: Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

In the development of transdermal and topical products it is important to understand how formulation ingredients interact with the molecular components of the upper layer of the skin, the stratum corneum (SC), and thereby influence its macroscopic barrier properties. The aim here was to investigate the effect of two commonly used excipients, transcutol and dexpanthenol, on the molecular as well as the macroscopic properties of the skin membrane. Polarization transfer solid-state NMR methods were combined with steady-state flux and impedance spectroscopy measurements to investigate how these common excipients influence the molecular components of SC and its barrier function at strictly controlled hydration conditions in vitro with excised porcine skin. The NMR results provide completely new molecular insight into how transcutol and dexpanthenol affect specific molecular segments of both SC lipids and proteins. The presence of transcutol or dexpanthenol in the formulation at fixed water activity results in increased effective skin permeability of the model drug metronidazole. Finally, impedance spectroscopy data show clear changes of the effective skin capacitance after treatment with transcutol or dexpanthenol. Based on the complementary data, we are able to draw direct links between effects on the molecular properties and on the macroscopic barrier function of the skin barrier under treatment with formulations containing transcutol or dexpanthenol., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background and Aim: Gastroesophageal junction cancer is one of the leading causes to cancer-related death and the prognosis is poor. However, progress has been made over the last couple of decades with the introduction of multimodality treatment and optimized surgery. Three-year survival rates have improved to 50% in patients receiving neoadjuvant therapy. Only a few studies have focused on the difference of postoperative complications in patients receiving neoadjuvant therapy in relation to a comparative surgery-only group. The aim of this study was to compare the prevalence of postoperative complications of patients with cancer at the gastroesophageal junction treated with either neoadjuvant chemotherapy or surgery alone in patients from "The Danish Clinical Registry of Carcinomas of the Esophagus, the Gastro-Esophageal Junction and the Stomach.", Materials and Methods: A historical follow-up study, comparing postoperative complications between two cohorts before and after implementation of chemotherapy was completed., Results: In all, 180 consecutive patients treated with perioperative chemotherapy and a comparative surgery-only group of patients were identified from The Danish Clinical Registry of Carcinomas of the Esophagus, the Gastro-Esophageal Junction and the Stomach. No difference was found in demographics between the two groups, except for alcohol consumption and a lower T and N stage in the surgery-only group, and no difference in complication rates was found. Furthermore, no variable in the multivariate analysis was significantly associated with anastomotic leakage which was considered the most severe complication., Conclusion: Since perioperative chemotherapy does not appear to increase surgical complications, the future challenges include defining the optimal combination of chemo- and/or radiotherapy, but more importantly also to select the patients who will benefit the most from the different neoadjuvant strategies., (© The Finnish Surgical Society 2015.)

Compromised skin integrity of farmed Atlantic salmon, commonly occurring under low temperature and stressful conditions, has major impacts on animal welfare and economic productivity. Even fish with minimal scale loss and minor wounds can suffer from secondary infections, causing downgrading and mortalities. Wound healing is a complex process, where water temperature and nutrition play key roles. In this study, Atlantic salmon (260 g) were held at different water temperatures (4 or 12 °C) and fed three different diets for 10 weeks, before artificial wounds were inflicted and the wound healing process monitored for 2 weeks. The fish were fed either a control diet, a diet supplemented with zinc (Zn) or a diet containing a combination of functional ingredients in addition to Zn. The effect of diet was assessed through subjective and quantitative skin histology and the transcription of skin-associated chemokines. Histology confirmed that wound healing was faster at 12 °C. The epidermis was more organised, and image analyses of digitised skin slides showed that fish fed diets with added Zn had a significantly larger area of the epidermis covered by mucous cells in the deeper layers after 2 weeks, representing more advanced healing progression. Constitutive levels of the newly described chemokines, herein named CK 11A, B and C, confirmed their preferential expression in skin compared to other tissues. Contrasting modulation profiles at 4 and 12 °C were seen for all three chemokines during the wound healing time course, while the Zn-supplemented diets significantly increased the expression of CK 11A and B during the first 24 h of the healing phase.

Skin integrity is recognized as of vital consideration for both animal welfare and final product quality of farmed fish. This study examines the effects of three different rearing temperatures (4, 10 and 16 °C) on the skin of healthy Atlantic salmon post-smolts. Changes in skin condition were assessed by the means of skin composition analyses, quantitative histology assessments and transcriptome analysis. Level of protein, vitamin C and vitamin E was significantly higher at 16 °C compared with 4 °C. Quantitative histology measurements showed that the epidermal thickness decreased from low to high temperature, whereas the epidermal area comprising mucous cells increased. The difference was only significant between 4 and 16 °C. Both high and low temperature exhibited significant changes in the skin transcriptome. A number of immune-related transcripts responded at both temperatures. Contrary to well-described immunosuppressive effects of low water temperature on systemic immunity, a subtle increase in skin-mediated immunity was observed, suggesting a pre-activation of the mucosal system at 4 °C. Upregulation of a number of heat-shock proteins correlating with a decrease in epidermal thickness suggested a stress response in the skin at high temperature. The results demonstrate distinctive temperature-related effects on the skin of Atlantic salmon., (© 2014 John Wiley & Sons Ltd.)

The occurrence and diversity of vancomycin-resistant Enterococcus faecium (VREF) were investigated in 100 Danish broiler flocks 15 years after the avoparcin ban. VREF occurred in 47 flocks at low fecal concentrations detectable only by selective enrichment. Vancomycin resistance was prevalently associated with a transferable nontypeable plasmid lineage occurring in multiple E. faecium clones. Coselection of sequence type 842 by tetracycline use only partly explained the persistence of vancomycin resistance in the absence of detectable plasmid coresistance and toxin-antitoxin systems., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including antimicrobial resistance genes encoded by mobile genetic elements (MGEs). Here, we investigate this mobilome in successful hospital associated genetic lineages, E. faecium sequence type (ST)17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) by DNA microarray analyses., Results: The hybridization patterns of 272 representative targets including plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29) and clustered regularly interspaced short palindromic repeats (CRISPR)-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. RCR-, Rep&#95;3-, RepA&#95;N- and Inc18-family plasmids were highly prevalent and with the exception of Rep&#95;3, evenly distributed between the species. There was a considerable difference in the replicon profile, with rep 17/pRUM , rep 2/pRE25 , rep 14/EFNP1 and rep 20/pLG1 dominating in E. faecium and rep 9/pCF10 , rep 2/pRE25 and rep 7 in E. faecalis strains. We observed an overall high correlation between the presence and absence of genes coding for resistance towards antibiotics, metals, biocides and their corresponding MGEs as well as their phenotypic antimicrobial susceptibility pattern. Although most IS families were represented in both E. faecalis and E. faecium, specific IS elements within these families were distributed in only one species. The prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982- and IS4-transposases was significantly higher in E. faecium than E. faecalis, and that of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 that have only been reported in few enterococcal isolates were well represented in the E. faecium strains. E. faecalis ST40 strains harboured possible functional CRISPR-Cas systems, and still resistance and prophage sequences were generally well represented., Conclusions: The targeted MGEs were highly prevalent among the selected STs, underlining their potential importance in the evolution of hospital-adapted lineages of enterococci. Although the propensity of inter-species horizontal gene transfer (HGT) must be emphasized, the considerable species-specificity of these MGEs indicates a separate vertical evolution of MGEs within each species, and for E. faecalis within each ST.

Background: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type., Results: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro)., Conclusion: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

A method was developed for simultaneous determination of the mycotoxins: ochratoxin A (OTA) and fumonisins B2 (FB2), B4 (FB4), and B6 (FB6) in green, roasted, and instant coffee. Extraction was performed by QuEChERS (quick, easy, cheap, effective, rugged, and safe) under acidic conditions followed by mixed-mode reversed phase-anion exchange solid phase extraction. OTA and FB2 were detected at levels down to 0.5 and 2 μg/kg by UHPLC-MS/MS and quantitated via isotope dilution using U-(13)C-labeled FB2 and OTA as internal standards. Mixing 20% isopropanol in the acetonitrile of the acidic UHPLC gradient system increased the signal intensity by 50% and decreased the ion-suppression with 50-75% in roasted coffee samples. About half of the roasted coffee samples (n = 57, from 9 countries) contained detectable levels of OTA, however, with only 5 samples above the EU regulatory limit of 5 μg/kg and the highest with 21 μg/kg. None of the 25 instant coffee samples contained OTA above the EU regulatory level of 10 μg/kg. Nonetheless, the toxin could be detected in 56% of the analyzed instant coffee samples. Fumonisins were not detected in any of the roasted or instant coffee samples (n = 82). However, in the green coffee samples (n = 18) almost half of the samples were positive with a maximum value of 164 μg/kg (sum of FB2, FB4, and FB6). This discrepancy between green coffee and processed coffees indicated that the fumonisins decompose during the roasting process, which was confirmed in roasting experiments. Here fumonisins could not be detected after roasting of the green, 164 μg/kg coffee, sample. Under the same conditions, OTA was reduced from 2.4 to 0.5 μg/kg.

Aim. Developing a theoretical framework explaining patients' behaviour and actions related to unmet needs during interactions with health care professionals in hospital-based outpatient respiratory medical clinics. Background. The outpatient respiratory medical clinic plays a prominent role in many patients' lives regarding treatment and counselling increasing the need for a better understanding of patients' perspective to the counselling of the health care professionals. Design. The study is exploratory and based on Charmaz's interpretation of grounded theory. Methods. The study included 65 field observations with a sample of 43 patients, 11 doctors, and 11 nurses, as well as 30 interviews with patients, conducted through theoretical sampling from three outpatient respiratory medical clinics in Denmark. Findings. The patients' efforts to share their significant stories triggered predominantly an adaptation or resistance behaviour, conceptualized as "fitting in" and "fighting back" behaviour, explaining the patients' counterreactions to unrecognized needs during the medical encounter. Conclusion. Firstly this study allows for a better understanding of patients' counterreactions in the time-pressured and, simultaneously, tight structured guidance program in the outpatient clinic. Secondly the study offers practical and ethical implications as to how health care professionals' attitudes towards patients can increase their ability to support emotional suffering and increase patient participation and responsiveness to guidance in the lifestyle changes.

The immune response of the adipose tissue (AT) has been neglected in most animal models until recently, when the observations made in human and mice linking obesity to chronic inflammation and diabetes highlighted an important immune component of this tissue. In the current study, we have immunologically characterized the AT for the first time in teleosts. We have analyzed the capacity of rainbow trout (Oncorhynchus mykiss) AT to produce different immune mediators and we have identified the presence of local populations of B lymphocytes expressing IgM, IgD or IgT, CD8α+ cells and cells expressing major histocompatibility complex II (MHC-II). Because trout AT retained antigens from the peritoneal cavity, we analyzed the effects of intraperitoneal infection with viral hemorrhagic septicemia virus (VHSV) on AT functionality. A wide range of secreted immune factors were modulated within the AT in response to VHSV. Furthermore, the viral infection provoked a significant decrease in the number of IgM+ cells which, along with an increased secretion of IgM in the tissue, suggested a differentiation of B cells into plasmablasts. The virus also increased the number of CD8α+ cells in the AT. Finally, when a fat-enriched diet was fed to the fish, a significant modulation of immune gene expression in the AT was also observed. Thus, we have demonstrated for the first time in teleost that the AT functions as a relevant immune tissue; responsive to peritoneal viral infections and that this immune response can be modulated by the fat-content in the diet.

Liraglutide is a human glucagon-like peptide-1 (GLP-1) analogue approved for the treatment of type 2 diabetes. It is based on human GLP-1 with the addition of a 16-carbon fatty acid, which facilitates binding to plasma proteins, thus prolonging the elimination half-life and allowing once-daily administration. It has not been possible to quantify liraglutide protein binding by ultrafiltration (the usual method of choice), as the lipophilic molecule becomes trapped in the filter membrane. Therefore, the aim of this study was to develop a methodology that could determine the extent of liraglutide binding to plasma proteins in vitro. We report here the details of a novel reiterated stepwise equilibrium dialysis assay that has successfully been used to quantify liraglutide plasma protein binding. The assay allowed quantification of liraglutide binding to proteins in purified plasma protein solutions and human plasma samples and was effective at plasma dilutions as low as 5%. At a clinically relevant liraglutide concentration (10(4) pM), greater than 98.9% of liraglutide was bound to protein. Specific binding to human serum albumin and α1-acid glycoprotein was 99.4% and 99.3%, respectively. The novel methodology described herein could have an application in the quantification of plasma protein binding of other highly lipophilic drug molecules., (Copyright © 2013 Wiley Periodicals, Inc.)

Health diets that contain immunostimulants and other functional ingredients can strengthen the immune response in Atlantic salmon, Salmo salar, and thereby reduce sea lice, Lepeophtheirus salmonis, infection levels. Such diets can be used to supplement other treatments and will potentially reduce the need for delousing and medication. A sea lice infection trial was conducted on fish with an average weight of 215 g. One control diet and four experimental diets containing functional ingredients were produced. The diets were fed to salmon for 4 weeks before infection with sea lice copepodids. When lice had developed to chalimus III/IV, 88 fish per diet were examined for lice loads. Mucus samples from fish fed the different diets were taken before and after lice infection. Mass spectrometry-based proteomics was used to characterize the protein composition in the epidermal mucus of Atlantic salmon and to identify quantitative alterations in protein expression. Multivariate analysis of the generated data sets was performed to identify protein biomarkers. Putative biomarkers associated with functional feed intake and with sea lice infection have been identified and can form the basis for strategic validation experiments with selected functional feeds., (© 2013 Blackwell Publishing Ltd.)

The complete and annotated genome sequence of Enterococcus faecalis D32, a commensal strain isolated from a Danish pig, suggests putative adaptation to the porcine host and absence of distinct virulence-associated traits.

Rheumatoid arthritis is characterized by systemic inflammation of synovial joints leading to erosion and cartilage destruction. Although efficacious anti-tumor necrosis factor α (TNF-α) biologic therapies exist, there is an unmet medical need for safe and more efficient treatment regimens for disease remission. We evaluated the anti-inflammatory effects of poly(dl-lactide-co-glycolide acid) (PLGA) nanoparticles loaded with small interfering RNA (siRNA) directed against TNF-α in vitro and in vivo. The siRNA-loaded PLGA nanoparticles mediated a dose-dependent TNF-α silencing in lipopolysaccharide-activated RAW 264.7 cells in vitro. The severity of collagen antibody-induced arthritis in DBA/1J mice was assessed by paw scoring and compared to the degree of magnetic resonance imaging (MRI)-quantified joint effusion and bone marrow edema. Two intra-articular treatments per joint with nanoparticles loaded with TNF-α siRNA (1 μg) resulted in a reduction in disease activity, evident by a significant decrease of the paw scores and joint effusions, as compared to treatment with PLGA nanoparticles loaded with non-specific control siRNA, whereas the degree of bone marrow edema in the tibial and femoral head remained unchanged. When the siRNA dose was 5 or 10 μg, there was no difference between the specific and the non-specific siRNA treatment groups. These findings suggest that MRI is a promising method for evaluation of early disease progression and treatment in murine arthritis models. In addition, proper siRNA dosing seems to be important for a positive therapeutic outcome in vivo. However, further studies are needed to fully clarify the mechanism(s) underlying the observed anti-inflammatory effects of the siRNA-loaded nanoparticles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: Tumor necrosis factor α (TNF-α) plays a key role in the progression of rheumatoid arthritis and is an important target for anti-rheumatic therapies. TNF-α expression can be silenced with small interfering RNA (siRNA), but efficacy is dependent on efficient and safe siRNA delivery vehicles. We aimed to identify polymeric nanocarriers for anti-TNF-α siRNA with optimal efficacy and minimal off-target effects in vitro., Methods: TNF-α silencing with polymeric siRNA nanocarriers was compared in lipopolysaccharide-activated RAW 264.7 macrophages by real-time reverse transcription (RT)-PCR. Expression of non-target genes involved in inflammation, apoptosis, and cell cycle progression was determined by RT-PCR, toxicity evaluated by propidium iodide and annexin V staining., Results: PAMAM dendrimers (G4 and G7) and dextran nanogels mediated remarkably high concentration-dependent gene silencing and low toxicity; dioleoyltrimethylammoniumpropane-modified poly(DL-lactide-co-glycolide acid) nanoparticles, thiolated, trimethylated chitosan and poly[(2-hydroxypropyl)methacrylamide 1-methyl-2-piperidine methanol] polyplexes were less efficient transfectants. There were minor changes in the regulation of off-target genes, mainly dependent on nanocarrier and siRNA concentration., Conclusions: Dextran nanogels and PAMAM dendrimers mediated high gene silencing with minor toxicity and off-target transcriptional changes and are therefore expected to be suitable siRNA delivery systems in vivo.

Matrix systems based on biocompatible and biodegradable polymers like the United States Food and Drug Administration (FDA)-approved polymer poly(DL-lactide-co-glycolide acid) (PLGA) are promising for the delivery of small interfering RNA (siRNA) due to favorable safety profiles, sustained release properties and improved colloidal stability, as compared to polyplexes. The purpose of this study was to design a dry powder formulation based on cationic lipid-modified PLGA nanoparticles intended for treatment of severe lung diseases by pulmonary delivery of siRNA. The cationic lipid dioleoyltrimethylammoniumpropane (DOTAP) was incorporated into the PLGA matrix to potentiate the gene silencing efficiency. The gene knock-down level in vitro was positively correlated to the weight ratio of DOTAP in the particles, and 73% silencing was achieved in the presence of 10% (v/v) serum at 25% (w/w) DOTAP. Optimal properties were found for nanoparticles modified with 15% (w/w) DOTAP, which reduced the gene expression with 54%. This formulation was spray-dried with mannitol into nanocomposite microparticles of an aerodynamic size appropriate for lung deposition. The spray-drying process did not affect the physicochemical properties of the readily re-dispersible nanoparticles, and most importantly, the in vitro gene silencing activity was preserved during spray-drying. The siRNA content in the powder was similar to the theoretical loading and the siRNA was intact, suggesting that the siRNA is preserved during the spray-drying process. Finally, X-ray powder diffraction analysis demonstrated that mannitol remained in a crystalline state upon spray-drying with PLGA nanoparticles suggesting that the sugar excipient might exert its stabilizing effect by sterical inhibition of the interactions between adjacent nanoparticles. This study demonstrates that spray-drying is an excellent technique for engineering dry powder formulations of siRNA nanoparticles, which might enable the local delivery of biologically active siRNA directly to the lung tissue., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Thirteen plant species were tested for their suitability as hosts for Abagrotis orbis (Grote), a climbing cutworm pest of grapevines in British Columbia. Choice tests were also conducted to investigate larval feeding preferences for the Brassicaceae species joi choi, Brassica rapa variety. Chinensis L., spring draba; Draba verna L.; and shepherd's purse, Capsella bursa-pastoris (L.) Medik; compared with postdormant buds of grape, Vitis vinifera L. (Vitaceae), and leaves of nine other plant species from several families. Results showed that tah tsai, Brassica rapa L. variety rosularis (M. Tsen & S. H. Lee) Hanelt (Brassicaceae), is a superior host for A. orbis based on shorter time to adult eclosion, heavier pupae, and higher rates of survival. Later-instar larvae died when fed draba, whereas those reared on shepherd's purse did not survive beyond the third instar. White clover, Trifolium repens L. (Fabaceae), and grape leaves were unsuitable hosts throughout development. Fifth-instar A. orbis preferred plants of the Brassicaceae family, dandelion, Taraxacum officinale Weber (Asteraceae), and strawberry, Fragaria sp. L. (Rosaceae), compared with postdormant grape buds. The results of this study suggest that the winter annual mustards draba and shepherd's purse that often grow abundantly in vine rows might help reduce climbing cutworm damage to the buds of grapevines.