Comparison of ectoparasite communities of sylvatic and urban wild mesomammals and unowned community cats in north-central Florida.

The adaptation of wildlife species to urban environments can drive changes in the ecology of ectoparasites and vector-borne disease. To better understand ectoparasite dynamics in an urban environment, we investigated the ectoparasite communities of 183 sylvatic and urban opossums and raccoons captured across four seasons at a rural research station and within the city of Gainesville, FL, and of 115 community cats from the Gainesville, FL area. Amblyomma americanum (L.) (Acari: Ixodidae), Dermacentor variabilis (Say), and Ixodes texanus Banks were collected from raccoons, A. americanum, D. variabilis, and Ixodes scapularis Say from opossums, and A. americanum from cats. Few ticks were collected from urban animals, although species richness of ectoparasites was similar between urban and sylvatic habitats. Ctenocephalides felis (Bouché) (Siphonaptera: Pulicidae) was collected from all sampled host species, but was particularly abundant on opossums. Additionally, Orchopeas howardi (Baker) (Siphonaptera: Ceratophyllidae) was collected from raccoons, and O. howardi and Xenopsylla cheopis (Rothschild) (Siphonaptera: Pulicidae) from opossums. Only raccoons were infested with raccoon lice, and only cats were infested with cat lice. Primarily opossums were infested with mites. Ectoparasite community composition varied by habitat, host species, and season; seasonal variation in ectoparasite communities differed between the sylvatic and urban habitats. While urban mesomammals did not appear to play an important role in supporting tick populations in an urban habitat, urban opossums appear to serve as an alternate host for large numbers of cat fleas, which may be an important consideration for treatment and control efforts against ectoparasites of companion animals.

SARS-CoV-2 surface and air contamination in an acute healthcare setting during the first and second pandemic waves.

BACKGROUND: Surfaces and air in healthcare facilities can be contaminated with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Previously, the authors identified SARS-CoV-2 RNA on surfaces and air in their hospital during the first wave of the coronavirus disease 2019 pandemic (April 2020). AIM: To explore whether the profile of SARS-CoV-2 surface and air contamination had changed between April 2020 and January 2021. METHODS: This was a prospective, cross-sectional, observational study in a multi-site London hospital. In January 2021, surface and air samples were collected from comparable areas to those sampled in April 2020, comprising six clinical areas and a public area. SARS-CoV-2 was detected using reverse transcription polymerase chain reaction and viral culture. Sampling was also undertaken in two wards with natural ventilation alone. The ability of the prevalent variants at the time of the study to survive on dry surfaces was evaluated. FINDINGS: No viable virus was recovered from surfaces or air. Five percent (N=14) of 270 surface samples and 4% (N=1) of 27 air samples were positive for SARS-CoV-2, which was significantly lower than in April 2020 [52% (N=114) of 218 surface samples and 48% (N=13) of 27 air samples (P<0.001, Fisher's exact test)]. There was no clear difference in the proportion of surface and air samples positive for SARS-CoV-2 RNA based on the type of ventilation in the ward. All variants tested survived on dry surfaces for >72 h, with a <3-log10 reduction in viable count. CONCLUSION: This study suggests that enhanced infection prevention measures have reduced the burden of SARS-CoV-2 RNA on surfaces and air in healthcare facilities.

Assessment of the Microbiological Quality and Safety of Unpasteurized Milk Cheese for Sale in England between 2019 and 2020.

ABSTRACT: Cheese made with unpasteurized milk has been associated with outbreaks of illness. However, there are limited data on the prevalence of Shiga toxin-producing Escherichia coli (STEC) in these products and a lack of clarity over the significance of E. coli as a general indicator of hygiene in raw milk cheeses. The aim of this study was to provide further data to address both of these issues, as well as assessing the overall microbiological quality of raw milk cheeses available to consumers in England. A total of 629 samples of cheese were collected from retailers, catering premises, and manufacturers throughout England. The majority (80%) were made using cow's milk, with 14% made from sheep's milk and 5% from goat's milk. Samples were from 18 different countries of origin, with the majority originating from either the United Kingdom (40%) or France (35%). When interpreted against European Union microbiological criteria and United Kingdom guidance, 82% were considered to be of satisfactory microbiological quality, 5% were borderline, and 12% were unsatisfactory. Four samples (0.6%) were potentially injurious to health due to the isolation of STEC from one, >104 CFU/g of coagulase-positive staphylococci in two, and >100 CFU/g of Listeria monocytogenes in the fourth sample. Indicator E. coli and Listeria species were detected more frequently in soft compared with hard cheese. Higher levels of indicator E. coli were significantly associated with a greater likelihood of detecting Shiga toxin genes (stx1 and/or stx2).

Imported edible leaves collected at retail sale in England during 2017 with an emphasis on betel and curry leaves: microbiological quality with respect to Salmonella, Shiga-toxin-producing E. coli (STEC) and levels of Escherichia coli.

AIMS: To investigate the microbiological quality of imported fresh leaves on retail sale during 2017 with respect to Salmonella, Shiga-toxin-producing Escherichia coli (STEC) and levels of E. coli. METHODS AND RESULTS: Two hundred and seventy-nine samples of imported edible leaves (69 banana, 77 betel, 118 curry and 15 other types) were tested. Salmonella spp. were confirmed by whole-genome sequencing and isolated from 44 samples, 26% from curry leaves, 14% from betel and 2·4% from all other leaf types: 80% of all samples contained ≥102 , 44% ≥103 and 22% ≥104 CFU of E. coli CFU per g. All samples where Salmonella were detected also yielded ≥20 CFU of E. coli/g. 54 samples were tested for STEC which was detected in six samples and isolated from three: one was identified as STEC O157:H7. CONCLUSIONS: This report further highlights an ongoing problem of Salmonella contamination of imported fresh edible leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: Among all food tested by Public Health England (approximately 11 000 per annum), curry leaves were the herb most commonly contaminated with Salmonella, and betel leaves were the most commonly contaminated ready-to-eat food. The high proportion with unsatisfactory E. coli levels and the detection of STEC suggests risks of contamination by multiple enteric pathogens.

An assessment of the microbiological quality and safety of raw drinking milk on retail sale in England.

AIMS: This study aimed to review the microbiological results for raw drinking milk (RDM) samples submitted to Public Health England laboratories between 2014 and 2016 in order to produce up-to-date data on the microbiological safety of RDM and inform future risk assessments on its sale. METHODS AND RESULTS: A total of 902 samples of RDM were collected from retail sale in England for microbiological examination. Overall, 454 of 770 samples (59·0%) taken for routine monitoring were of a satisfactory quality, whilst eight (1·0%) were 'unsatisfactory and potentially injurious to health' due to the presence of Shiga toxin-producing Escherichia coli, Campylobacter or elevated levels of Listeria monocytogenes or coagulase-positive staphylococci. In contrast, 16 of 114 (14·0%) of samples taken in follow-up to a previous unsatisfactory result and 5 of 18 (27·8%) of samples related to illness were potentially injurious. A total of 229 of 902 samples (25·4%) gave unsatisfactory results due to elevated aerobic colony counts and/or coliforms, whilst 139 of 902 samples (15·4%) were of borderline quality due to coagulase-positive staphylococci. Listeria monocytogenes was detected at levels of <100 CFU per ml in 66 of 902 samples (7·3%) and other Listeria species in 44 of 902 samples (4·9%). CONCLUSIONS: Pathogens and/or indicators of poor hygiene were present in almost half of samples examined. Cows' milk samples gave a significantly greater proportion of unsatisfactory results compared to milk from other species (i.e. goat, sheep, buffalo, camel). SIGNIFICANCE AND IMPACT OF THE STUDY: These results demonstrate the importance of maintaining strict controls on the production and sale of this product.

'DrinkThink' alcohol screening and brief intervention for young people: a qualitative evaluation of training and implementation.

Background: Alcohol Screening and Brief Intervention (ASBI) helps reduce risky drinking in adults, but less is known about its effectiveness with young people. This article explores implementation of DrinkThink, an ASBI co-produced with young people, by health, youth and social care professionals trained in its delivery. Methods: A qualitative evaluation was conducted using focus groups with 33 staff trained to deliver DrinkThink, and eight interviews with trained participants and service managers. These were recorded, transcribed and a thematic analysis undertaken. Results: DrinkThink was not delivered fully by health, youth or social care agencies. The reasons for this varied by setting but included: the training staff received, a working culture that was ill-suited to the intervention, staff attitudes towards alcohol which prioritized other health problems presented by young people, over alcohol use. Conclusions: Implementation was limited because staff had not been involved in the design and planning of DrinkThink. Staffs' perceptions of alcohol problems in young people and the diverse cultures in which they work were subsequently not accounted for in the design. Co-producing youth focused ASBIs with the professionals expected to deliver them, and the young people whom they target, may ensure greater success in integrating them into working practice.

An assessment of the microbiological quality of lightly cooked food (including sous-vide) at the point of consumption in England.

This observational study aims to investigate the microbiological quality of commercially prepared lightly cooked foods with a major component of food of animal origin and collected as would be served to a consumer. A total of 356 samples were collected from catering (92%), retail (7%) or producers (1%) and all were independent of known incidents of foodborne illness. Using standard methods, all samples were tested for: the presence of Campylobacter spp. and Salmonella spp. and enumerated for levels of, Bacillus spp. including B. cereus, Clostridium perfringens, Listeria spp. including L. monocytogenes, Staphylococcus aureus, Escherichia coli, Enterobacteriacea and aerobic colony count (ACC). Results were interpreted as unsatisfactory, borderline or satisfactory according to the Health Protection Agency guidelines for assessing the microbiological safety of ready-to-eat foods placed on the market. Amongst all samples, 70% were classified as satisfactory, 18% were borderline and 12% were of unsatisfactory microbiological quality. Amongst the unsatisfactory samples, six (2%) were potentially injurious to health due to the presence of: Salmonella spp. (one duck breast); Campylobacter spp. (two duck breast and one chicken liver pâté); L. monocytogenes at 4·3 × 103 cfu (colony-forming units)/g (one duck confit with foie gras ballotin) and C. perfringens at 2·5 × 105 cfu/g (one chicken liver pâté). The remaining unsatisfactory samples were due to high levels of indicator E. coli, Enterobacteriaceae or ACC.

British Dietetic Association systematic review and evidence-based practice guidelines for the dietary management of irritable bowel syndrome in adults (2016 update).

BACKGROUND: The first British Dietetic Association (BDA) guidelines for the dietary management of irritable bowel syndrome (IBS) in adults were published in 2012. Subsequently, there has been a wealth of new research. The aim of this work was to systematically review the evidence for the role of diet in the management of IBS and to update the guidelines. METHODS: Twelve questions relating to diet and IBS were defined based on review of the previous guideline questions, current evidence and clinical practice. Chosen topics were on healthy eating and lifestyle (alcohol, caffeine, spicy food, elimination diets, fat and fluid intakes and dietary habits), milk and dairy, dietary fibre, fermentable carbohydrates, gluten, probiotics and elimination diets/food hypersensitivity. Data sources were CINAHL, Cochrane Register of Controlled Trials, Embase, Medline, Scopus and Web of Science up to October 2015. Studies were assessed independently in duplicate using risk of bias tools specific to each included study based on inclusion and exclusion criteria for each question. National Health and Medical Research Council grading evidence levels were used to develop evidence statements and recommendations, in accordance with Practice-based Evidence in Nutrition Global protocol used by the BDA. RESULTS: Eighty-six studies were critically appraised to generate 46 evidence statements, 15 clinical recommendations and four research recommendations. The IBS dietary algorithm was simplified to first-line (healthy eating, provided by any healthcare professional) and second-line [low FODMAP (fermentable oligosaccharides, disaccharides, monosaccharides and polyols) to be provided by dietitian] dietary advice. CONCLUSIONS: These guidelines provide updated comprehensive evidence-based details to achieve the successful dietary management of IBS in adults.

The occurrence of Salmonella in raw and ready-to-eat bean sprouts and sprouted seeds on retail sale in England and Northern Ireland.

UNLABELLED: A total of 554 samples of bean sprouts or other sprouted seeds were collected at retail sale and submitted to nine Official Control Laboratories in England and Northern Ireland during January to March 2011. Samples (100 g) were tested for the presence of Salmonella using the EN ISO 6579:2002 method. Products labelled as ready-to-eat comprised 23% of the samples and 61% were labelled as raw or to-cook: the remaining 12% had no indication if the food was intended as ready-to-eat or ready-to-cook, and 4% were not recorded. Salmonella spp. were detected from four samples of mung-bean sprouts (0·7% of all the 554 samples) and all four isolates were confirmed as Salmonella enterica serovar Abaetetuba (11 : k : 1,5). Two of the samples where Salmonella was detected were sold as ready-to-eat (labelled 'rinse and serve' only): The remaining two were from samples labelled as ready-to-cook. SIGNIFICANCE AND IMPACT OF THE STUDY: Consumption of sprouted seeds have been associated with infections from a range of foodborne pathogens, particularly Salmonella and shigatoxin producing Escherichia coli (STEC). However, there is limited data (including that from EU monitoring) on foodborne pathogens in samples of this food type which are not associated with outbreaks of infection. Out of 554 raw and ready-to-eat bean sprouts and sprouted seeds sampled at retail, Salmonella spp. was detected from four samples. This study illustrated the potential of this product to be contaminated with a human pathogen and the importance of considering the intended use and preparation of specific food in assessing microbiological risks.

Sensitivity to food additives, vaso-active amines and salicylates: a review of the evidence.

Although there is considerable literature pertaining to IgE and non IgE-mediated food allergy, there is a paucity of information on non-immune mediated reactions to foods, other than metabolic disorders such as lactose intolerance. Food additives and naturally occurring 'food chemicals' have long been reported as having the potential to provoke symptoms in those who are more sensitive to their effects. Diets low in 'food chemicals' gained prominence in the 1970s and 1980s, and their popularity remains, although the evidence of their efficacy is very limited. This review focuses on the available evidence for the role and likely adverse effects of both added and natural 'food chemicals' including benzoate, sulphite, monosodium glutamate, vaso-active or biogenic amines and salicylate. Studies assessing the efficacy of the restriction of these substances in the diet have mainly been undertaken in adults, but the paper will also touch on the use of such diets in children. The difficulty of reviewing the available evidence is that few of the studies have been controlled and, for many, considerable time has elapsed since their publication. Meanwhile dietary patterns and habits have changed hugely in the interim, so the conclusions may not be relevant for our current dietary norms. The conclusion of the review is that there may be some benefit in the removal of an additive or a group of foods high in natural food chemicals from the diet for a limited period for certain individuals, providing the diagnostic pathway is followed and the foods are reintroduced back into the diet to assess for the efficacy of removal. However diets involving the removal of multiple additives and food chemicals have the very great potential to lead to nutritional deficiency especially in the paediatric population. Any dietary intervention, whether for the purposes of diagnosis or management of food allergy or food intolerance, should be adapted to the individual's dietary habits and a suitably trained dietitian should ensure nutritional needs are met. Ultimately a healthy diet should be the aim for all patients presenting in the allergy clinic.

An assessment of the microbiological safety of fresh whole-leaf herbs from retail premises in the United Kingdom with a focus on Salmonella spp.

AIMS: Fresh herbs have been associated with a number of outbreaks in recent years, in the United Kingdom and elsewhere. A study of fresh herbs was carried out to assess their microbiological safety in relation to Salmonella contamination and levels of Escherichia coli. METHODS AND RESULTS: Between January and March 2014, 774 samples of ready-to-eat, fresh, whole-leaf herbs were collected from retail premises in the United Kingdom. Overall, Salmonella was detected in nine samples (1·2%). Of these, five were curry leaves. Other herbs contaminated with Salmonella were basil (two samples), walleria (1) and coriander (1). Escherichia coli was detected in 13% of samples, with 11% containing unsatisfactory levels (≥10(2) g(-1)). CONCLUSIONS: Whilst 88% of samples in this study were of an acceptable microbiological quality, the presence of Salmonella and/or elevated E. coli levels in 12% is a cause for concern. Curry leaves, in particular, had significantly higher rates of contamination with both Salmonella and E. coli than other herbs. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights the potential public health risk associated with the consumption of certain ready-to-eat fresh herbs, and the need for good hygiene practices and effective decontamination procedures during the growth, harvesting and subsequent handling of these products.

Effects of linseeds on the symptoms of irritable bowel syndrome: a pilot randomised controlled trial.

BACKGROUND: Manipulation of dietary fibre intake represents a longstanding treatment for patients with irritable bowel syndrome (IBS), particularly for those with constipation. Linseeds are often recommended by both clinicians and dietitians as a source of dietary fibre to alleviate symptoms. Recent guidance on the management of irritable bowel syndrome (IBS) advises that linseeds may reduce wind and bloating, although there is limited clinical evidence to support this recommendation. The present pilot study aimed to compare the clinical effectiveness of: (i) whole linseeds versus ground linseeds; (ii) whole linseeds versus no linseeds; and (iii) ground linseeds versus no linseeds in the management of IBS symptoms. METHODS: In an open randomised controlled trial, subjects with IBS (n = 40) were allocated to one of three intervention groups: two tablespoons of whole linseeds per day (n = 14), two tablespoons of ground linseeds per day (n = 13) and no linseeds as controls (n = 13). Symptom severity (primary outcome) and bowel habit were assessed before and after a 4-week intervention and statistical differences between the groups were compared. RESULTS: Thirty-one subjects completed the present study. Between-group analysis comparing the improvement in symptom severity did not reach statistical significance for whole linseeds (n = 11) versus ground linseeds (n = 11; P = 0.62), whole linseeds versus controls (n = 9; P = 0.12) and ground linseeds versus controls (P = 0.10). There were no significant changes in stool frequency or stool consistency for any of the groups. CONCLUSIONS: Linseeds may be useful in relief of IBS symptoms. Further research is needed to detect clear differences between the effects of whole and ground linseeds.

Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

British Dietetic Association evidence-based guidelines for the dietary management of irritable bowel syndrome in adults.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic debilitating functional gastrointestinal disorder. Diet and lifestyle changes are important management strategies. The aim of these guidelines is to systematically review key aspects of the dietary management of IBS, with the aim of providing evidence-based guidelines for use by registered dietitians. METHODS: Questions relating to diet and IBS symptom management were developed by a guideline development group. These included the role of milk and lactose, nonstarch polysaccharides (NSP), fermentable carbohydrates in abdominal bloating, probiotics and empirical or elimination diets. A comprehensive literature search was conducted and relevant studies from January 1985 to November 2009 were identified using the electronic database search engines: Cinahl, Cochrane Library, Embase, Medline, Scopus and Web of Science. Evidence statements, recommendations, good practice points and research recommendations were developed. RESULTS: Thirty studies were critically appraised. A dietetic care pathway was produced following a logical sequence of treatment and formed the basis of these guidelines. Three lines of dietary management were identified. first line: Clinical and dietary assessment, healthy eating and lifestyle management with some general advice on lactose and NSP. Second line: Advanced dietary interventions to improve symptoms based on NSP, fermentable carbohydrates and probiotics. Third line: Elimination and empirical diets. Research recommendations were also identified relating to the need for adequately powered and well designed randomised controlled trials. CONCLUSIONS: These guidelines provide evidence-based details of how to achieve the successful dietary management of IBS.

School toilets: facilitating hand hygiene? A review of primary school hygiene facilities in a developed country.

BACKGROUND: Clean hands play an important role in preventing infectious disease transmission. The physical quality of any toilet and handwashing facilities is an important determinant of whether and how it is used, especially for school children. METHODS: This study assessed the physical quality of toilet and handwashing facilities used by 9 year olds at 68 primary schools in three cities in the South Island of New Zealand. The facilities were assessed for availability, functionality and provision of hand basins, hygiene products and drying facilities. RESULTS: Nineteen schools (28%) followed the New Zealand Ministry of Education Code of Practice for toilet and bathroom facilities in schools, by providing warm water, liquid soap at every basin and functioning hand drying facilities. A further 25 schools (37%) would have met the standards except they provided only cold water (21 schools) or the cloth roller towels were unusable (4 schools). The other 24 schools' toilet facilities were deficient in some way, including one with no soap and six that provided no drying facilities. School socioeconomic position and toilet facility quality were not related. CONCLUSIONS: These results suggest that a significant number of New Zealand children do not currently have access to high quality hygiene facilities at school.

Scale-up and manufacturing of clinical-grade self-inactivating γ-retroviral vectors by transient transfection.

The need for γ-retroviral (gRV) vectors with a self-inactivating (SIN) design for clinical application has prompted a shift in methodology of vector manufacturing from the traditional use of stable producer lines to transient transfection-based techniques. Herein, we set out to define and optimize a scalable manufacturing process for the production of gRV vectors using transfection in a closed-system bioreactor in compliance with current good manufacturing practices (cGMP). The process was based on transient transfection of 293T cells on Fibra-Cel disks in the Wave Bioreactor. Cells were harvested from tissue culture flasks and transferred to the bioreactor containing Fibra-Cel in the presence of vector plasmid, packaging plasmids and calcium-phosphate in Dulbecco's modified Eagle's medium and 10% fetal bovine serum. Virus supernatant was harvested at 10-14 h intervals. Using optimized procedures, a total of five ecotropic cGMP-grade gRV vectors were produced (9 liters each) with titers up to 3.6 × 10(7) infectious units per milliliter on 3T3 cells. One GMP preparation of vector-like particles was also produced. These results describe an optimized process for the generation of SIN viral vectors by transfection using a disposable platform that allows for the generation of clinical-grade viral vectors without the need for cleaning validation in a cost-effective manner.

A pilot study of dose-intensified procarbazine, CCNU, vincristine for poor prognosis brain tumors utilizing fibronectin-assisted, retroviral-mediated modification of CD34+ peripheral blood cells with O6-methylguanine DNA methyltransferase.

Administration of chemotherapy is often limited by myelosuppression. Expression of drug-resistance genes in hematopoietic cells has been proposed as a means to decrease the toxicity of cytotoxic agents. In this pilot study, we utilized a retroviral vector expressing methylguanine DNA methyltransferase (MGMT) to transduce hematopoietic progenitors, which were subsequently used in the setting of alkylator therapy (procarbazine, CCNU, vincristine (PCV)) for poor prognosis brain tumors. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells were collected by apheresis and enriched for CD34+ expression. Nine subjects were infused with CD34+-enriched cells treated in a transduction procedure involving a 4-day exposure to cytokines with vector exposure on days 3 and 4. No major adverse event was related to the gene therapy procedure. Importantly, the engraftment kinetics of the treated product was similar to unmanipulated peripheral blood stem cells, suggesting that the ex vivo manipulation did not significantly reduce engrafting progenitor cell function. Gene-transduced cells were detected in all subjects. Although the level and duration was limited, patients receiving cells transduced using fibronectin 'preloaded' with virus supernatant appeared to show improved in vivo marking frequency. These findings demonstrate the feasibility and safety of utilizing MGMT-transduced CD34+ peripheral blood progenitor cells in the setting of chemotherapy.

Design and synthesis of 2-oxo-imidazolidine-4-carboxylic acid hydroxyamides as potent matrix metalloproteinase-13 inhibitors.

A novel series of imidazolidinone-based matrix metalloproteinase (MMP) inhibitors was discovered by structural modification of pyrrolidinone la. Potent inhibition of MMP-13 was exhibited by the analogues having 4-(4-fluorophenoxy)phenyl (4a, IC50 = 3 nM) and 4-(naphth-2-yloxy)phenyl (4h, IC50 = 4 nM) as P1' groups.

Packaging cell line DNA contamination of vector supernatants: implication for laboratory and clinical research.

Investigators conducting retroviral gene therapy trials are required to monitor for the presence of replication-competent retrovirus (RCR). The required testing utilizes a combination of biologic assays and molecular tests using PCR. In the course of a human clinical gene therapy trial, we detected 4070A viral envelope sequences in CD34(+) peripheral blood stem cells 2 days after transduction using a PCR-based assay, suggesting the presence of RCR. The supernatant and producer cells used for vector generation had been negative in extensive screening using the extended S(+)/L(-) assay. The presence of a replication-competent virus was subsequently excluded by a combination of biologic and PCR analyses. The source of the 4070A viral envelope sequences was determined to be packaging cell line DNA in the vector supernatant. The analysis of a variety of vector supernatants by quantitative real-time PCR revealed 4070A envelope DNA sequences from the packaging cell line in concentrations equivalent to approximately 50-500 focus-forming units per milliliter of wild-type 4070A virus. When PCR was performed after reverse transcriptase treatment of supernatant (i.e., assessing both RNA and DNA content), 4070A envelope sequence concentrations ranged from 10(2) to 3.5 x 10(3) focus-forming units per milliliter of wild-type 4070A virus. Our data indicate that PCR should not be used to analyze transduced cells for RCR within the first 2 weeks of vector exposure. Furthermore, investigators using PCR to analyze transduction efficiency shortly after vector exposure may experience false-positive findings.

Safety testing for replication-competent retrovirus associated with gibbon ape leukemia virus-pseudotyped retroviral vectors.

The potential pathogenicity of replication-competent retroviruses (RCR) requires vigilant testing to exclude inadvertent contamination of clinical gene therapy vector products with RCR. Pseudotyped vectors using the gibbon ape leukemia virus (GALV) envelope have entered into clinical trials but specific recommendations regarding methods for screening of vector product and analysis of clinical samples have not been set forth. Unfortunately, current screening assays used for detecting amphotropic RCR are not suitable for GALV-pseudotyped RCR. We modified the extended S+/L- assay for RCR detection by using human 293 cells for virus amplification. Of five cell lines tested, 293 cells were selected because they combined a high transduction efficiency and an ability to generate RCR at high titer. After optimizing the amplification assay, a dilution of GALV virus could consistently be detected at a dilution of 10(-6). In coculture experiments, one GALV-infected cell could be consistently detected in 10(6) uninfected cells. A PCR-based assay was developed that was capable of detecting 100 copies of a GALV envelope containing plasmid diluted in 1 microg of DNA obtained from uninfected cells. PCR was also able to detect one GALV-infected cell in 10(6) uninfected cells. These assays will be suitable for testing of vector preparations and for monitoring of clinical samples from patients treated in clinical gene therapy protocols. The assays developed are similar in methodology and sensitivity to those currently used for certification of amphotropic retroviral vectors.