Conventional type I dendritic cells maintain a reservoir of proliferative tumor-antigen specific TCF-1+ CD8+ T cells in tumor-draining lymph nodes.

In tumors, a subset of CD8+ T cells expressing the transcription factor TCF-1 drives the response to immune checkpoint blockade. We examined the mechanisms that maintain these cells in an autochthonous model of lung adenocarcinoma. Longitudinal sampling and single-cell sequencing of tumor-antigen specific TCF-1+ CD8+ T cells revealed that while intratumoral TCF-1+ CD8+ T cells acquired dysfunctional features and decreased in number as tumors progressed, TCF-1+ CD8+ T cell frequency in the tumor draining LN (dLN) remained stable. Two discrete intratumoral TCF-1+ CD8+ T cell subsets developed over time-a proliferative SlamF6+ subset and a non-cycling SlamF6- subset. Blocking dLN egress decreased the frequency of intratumoral SlamF6+ TCF-1+ CD8+ T cells. Conventional type I dendritic cell (cDC1) in dLN decreased in number with tumor progression, and Flt3L+anti-CD40 treatment recovered SlamF6+ T cell frequencies and decreased tumor burden. Thus, cDC1s in tumor dLN maintain a reservoir of TCF-1+ CD8+ T cells and their decrease contributes to failed anti-tumor immunity.

Development, inter-rater reliability and feasibility of a checklist to assess implementation (Ch-IMP) in systematic reviews: the case of provider-based prevention and treatment programs targeting children and youth.

BACKGROUND: Several papers report deficiencies in the reporting of information about the implementation of interventions in clinical trials. Information about implementation is also required in systematic reviews of complex interventions to facilitate the translation and uptake of evidence of provider-based prevention and treatment programs. To capture whether and how implementation is assessed within systematic effectiveness reviews, we developed a checklist for implementation (Ch-IMP) and piloted it in a cohort of reviews on provider-based prevention and treatment interventions for children and young people. This paper reports on the inter-rater reliability, feasibility and reasons for discrepant ratings. METHODS: Checklist domains were informed by a framework for program theory; items within domains were generated from a literature review. The checklist was pilot-tested on a cohort of 27 effectiveness reviews targeting children and youth. Two raters independently extracted information on 47 items. Inter-rater reliability was evaluated using percentage agreement and unweighted kappa coefficients. Reasons for discrepant ratings were content analysed. RESULTS: Kappa coefficients ranged from 0.37 to 1.00 and were not influenced by one-sided bias. Most kappa values were classified as excellent (n = 20) or good (n = 17) with a few items categorised as fair (n = 7) or poor (n = 1). Prevalence-adjusted kappa coefficients indicate good or excellent agreement for all but one item. Four areas contributed to scoring discrepancies: 1) clarity or sufficiency of information provided in the review; 2) information missed in the review; 3) issues encountered with the tool; and 4) issues encountered at the review level. Use of the tool demands time investment and it requires adjustment to improve its feasibility for wider use. CONCLUSIONS: The case of provider-based prevention and treatment interventions showed relevancy in developing and piloting the Ch-IMP as a useful tool for assessing the extent to which systematic reviews assess the quality of implementation. The checklist could be used by authors and editors to improve the quality of systematic reviews, and shows promise as a pedagogical tool to facilitate the extraction and reporting of implementation characteristics.

Purification of regulatory T cells with the use of a fully enclosed high-speed microfluidic system.

BACKGROUND AIMS: Despite promising advances in cellular therapies, it will be difficult to fully test or implement new therapies until advances are made in the processes for cell preparation. This study describes the use of an advanced prototype of a flow-cytometry cell purification system constructed for operation in a clinical environment to prepare regulatory T cells defined as CD4(+)/CD25(bright)/CD127(neg/low). METHODS: The sort performance of the Gigasort system was directly compared with available droplet sorters using mixtures of highly fluorescent and non-fluorescent 5-µm polystyrene particles. CD4(+)-enriched cell preparations were processed with the use of a sterile, disposable fluid handling unit with a chip containing parallel microfluidic-based sorters. RESULTS: Similar purity and sort efficiency as found with droplet sorters were obtained with the 24-channel chip sorter system. Starting with 450 million fresh peripheral blood mononuclear cells, 150,000 to 1.7 million cells that were, on average, 85% FoxP3-positive and 97% viable, were obtained in <4 h. CONCLUSIONS: This study presents a technology adapted to regulatory requirements for clinical cell purification and that achieves high throughput and cell-friendly conditions by use of a microfluidic chip with 24 parallel microsorters, providing a rapid, sterile method of purifying regulatory T cells accurately and with excellent viability.

Diversity of Mycoplasma hyopneumoniae strains from pigs across Ontario, Canada.

Mycoplasma hyopneumoniae causes highly contagious swine enzootic pneumonia worldwide. It has been reported that highly diversified M. hyopneumoniae strains exist in different parts of the world. We found p146 gene sequencing analysis, an affordable and simple-to-perform typing method, to be specific and highly sensitive when applied to the molecular typing of 113 M. hyopneumoniae-positive clinical samples directly without culture. The samples were submitted to the Animal Health Laboratory at the University of Guelph (Ontario, Canada) during 2009-2017 from 40 different geographic areas in Ontario. Using a previously described criterion of grouping strains with < 4-bp differences into the same molecular type (p146 type), the 113 clinical samples clustered into 19 p146 genotypes. Dominant types were found in 2016 and 2017 only, indicating that highly diversified M. hyopneumoniae strains existed in Ontario. Some strains from the same geographic location but different years had the same sequence types, indicating that the same strain types circulate persistently in the field. Different p146 genotypes were also identified from similar geographic locations, indicating that either M. hyopneumoniae strains are prone to mutation or that multiple strains can infect the same or nearby swine production units.

Projected sustainability of innovative social programs.

This study is an exploratory examination of the projected sustainability of more than 100 projects funded by the Australian government. Using data collected by the body that evaluated the projects and data from a government database, it examines the predictors of various forms of sustainability. Findings show that some two thirds of the project leaders who expected their programs to continue after the expiration of the initial funding expected them to continue with the same activities and target population; almost half envisioned them diversifying to new activities, target groups, or locations. Auspice organization involvement increased the expectation that the project would be continued, project effectiveness decreased that expectation, and diversity of initial funding became less important as other sources of support and sustainability were taken into consideration.

IL-33 Signaling Alters Regulatory T Cell Diversity in Support of Tumor Development.

Regulatory T cells (Tregs) can impair anti-tumor immune responses and are associated with poor prognosis in multiple cancer types. Tregs in human tumors span diverse transcriptional states distinct from those of peripheral Tregs, but their contribution to tumor development remains unknown. Here, we use single-cell RNA sequencing (RNA-seq) to longitudinally profile dynamic shifts in the distribution of Tregs in a genetically engineered mouse model of lung adenocarcinoma. In this model, interferon-responsive Tregs are more prevalent early in tumor development, whereas a specialized effector phenotype characterized by enhanced expression of the interleukin-33 receptor ST2 is predominant in advanced disease. Treg-specific deletion of ST2 alters the evolution of effector Treg diversity, increases infiltration of CD8+ T cells into tumors, and decreases tumor burden. Our study shows that ST2 plays a critical role in Treg-mediated immunosuppression in cancer, highlighting potential paths for therapeutic intervention.

Potential use of DNA barcodes in regulatory science: applications of the Regulatory Fish Encyclopedia.

The use of a DNA-based identification system (DNA barcoding) founded on the mitochondrial gene cytochrome c oxidase subunit I (COI) was investigated for updating the U.S. Food and Drug Administration Regulatory Fish Encyclopedia (RFE; http://www.cfsan.fda.gov/-frf/rfe0.html). The RFE is a compilation of data used to identify fish species. It was compiled to help regulators identify species substitution that could result in potential adverse health consequences or could be a source of economic fraud. For each of many aquatic species commonly sold in the United States, the RFE includes high-resolution photographs of whole fish and their marketed product forms and species-specific biochemical patterns for authenticated fish species. These patterns currently include data from isoelectric focusing studies. In this article, we describe the generation of DNA barcodes for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE. These barcode sequences can be used as an additional identification resource. In a blind study, 60 unknown fish muscle samples were barcoded, and the results were compared with the RFE barcode reference library. All 60 samples were correctly identified to species based on the barcoding data. Our study indicates that DNA barcoding can be a powerful tool for species identification and has broad potential applications.

Development and field validation of a Mycoplasma iowae real-time polymerase chain reaction assay.

A Mycoplasma iowae real-time polymerase chain reaction (PCR) assay using primers and probes targeting the 16S rRNA gene was developed and field-validated in this study. The assay specifically identified M. iowae with a detection limit of 80 colony-forming units (cfu) per turkey cloacal swab sample (3.2 cfu per PCR reaction). It was validated by testing 154 field turkey cloacal swab samples in parallel with culture isolation. The diagnostic sensitivity of the PCR was 97.6%, and the specificity was 95.5%. The real-time PCR developed in this study is a rapid, sensitive, and cost-effective alternative to culture isolation for detecting M. iowae from cloacal swab samples.

Using simulation to design a central sterilization department.

A simulation project was performed to assist with redesign of the surgery department of a large tertiary hospital and to help administrators make the best decisions about relocating, staffing, and equipping the central sterilization department. A simulation model was created to analyze department configurations, staff schedules, equipment capacities, and cart-washing requirements. Performance measures examined include tray turnaround time, surgery-delay rate, and work-in-process levels. The analysis provides significant insight into how the proposed system will perform, allowing planning for expected patient volume increases. This work illustrates how simulation can facilitate the design of a central sterilization department and improve surgical sterilization operations.

Multi-locus sequence types of Mycoplasma bovis isolated from Ontario, Canada in the past three decades have a temporal distribution.

A total of 217 Mycoplasma bovis isolates cultured from clinical cases in Ontario, Canada, over the past 30 y were selected to be characterized by a multi-locus sequence typing (MLST) method. Eleven housekeeping genes were evaluated for suitability for MLST; 2 loci that had been used in prior MLST schemes, dnaN and metS, along with hsp70 were chosen for further sequence analysis. The remaining loci- adk, efp, gmk, gyrB, polC, rpoB, tpiA, and uvrC genes-were not used because they had little to no sequence variation. The sequence data from the chosen loci ( dnaN, hsp70, metS) generated 28 sequence types (STs), with the 3 loci having 15, 5, and 7 alleles, respectively. These molecular typing results revealed that the STs had a temporal distribution; over the course of 3 decades, some STs disappeared and new STs appeared. Recent isolates had a greater variety of STs, which may indicate that new strains are emerging more rapidly now than in the past.

Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples.

A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively.

Saxitoxin puffer fish poisoning in the United States, with the first report of Pyrodinium bahamense as the putative toxin source.

BACKGROUND: From January 2002 to May 2004, 28 puffer fish poisoning (PFP) cases in Florida, New Jersey, Virginia, and New York were linked to the Indian River Lagoon (IRL) in Florida. Saxitoxins (STXs) of unknown source were first identified in fillet remnants from a New Jersey PFP case in 2002. METHODS: We used the standard mouse bioassay (MBA), receptor binding assay (RBA), mouse neuroblastoma cytotoxicity assay (MNCA), Ridascreen ELISA, MIST Alert assay, HPLC, and liquid chromatography-mass spectrometry (LC-MS) to determine the presence of STX, decarbamoyl STX (dc-STX), and N-sulfocarbamoyl (B1) toxin in puffer fish tissues, clonal cultures, and natural bloom samples of Pyrodinium bahamense from the IRL. RESULTS: We found STXs in 516 IRL southern (Sphoeroides nephelus), checkered (Sphoeroides testudineus), and bandtail (Sphoeroides spengleri) puffer fish. During 36 months of monitoring, we detected STXs in skin, muscle, and viscera, with concentrations up to 22,104 microg STX equivalents (eq)/100 g tissue (action level, 80 microg STX eq/100 g tissue) in ovaries. Puffer fish tissues, clonal cultures, and natural bloom samples of P. bahamense from the IRL tested toxic in the MBA, RBA, MNCA, Ridascreen ELISA, and MIST Alert assay and positive for STX, dc-STX, and B1 toxin by HPLC and LC-MS. Skin mucus of IRL southern puffer fish captive for 1-year was highly toxic compared to Florida Gulf coast puffer fish. Therefore, we confirm puffer fish to be a hazardous reservoir of STXs in Florida's marine waters and implicate the dinoflagellate P. bahamense as the putative toxin source. CONCLUSIONS: Associated with fatal paralytic shellfish poisoning (PSP) in the Pacific but not known to be toxic in the western Atlantic, P. bahamense is an emerging public health threat. We propose characterizing this food poisoning syndrome as saxitoxin puffer fish poisoning (SPFP) to distinguish it from PFP, which is traditionally associated with tetrodotoxin, and from PSP caused by STXs in shellfish.

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

Evaluation of methods for the identification of Salmonella enterica serotype Typhimurium DT104 from poultry environmental samples.

An increase in the prevalence of Salmonella enterica serotype Typhimurium DT104 has been reported worldwide. This study examined the prevalence of this microorganism in poultry environmental samples from commercial layer flocks and pullet environments as well as the sensitivity and specificity of a PCR-based method, and multiple antibiotic resistance profile of Salmonella serogroup B isolates in relation to the serotype and phagetype reference method for the identification of Salmonella Typhimurium DT104. A total of 435 Salmonella isolates were obtained from poultry house environmental samples tested during a 20-month period representing a prevalence of 5.5%. Of these, 313 (72%) isolates were identified as Salmonella serogroup B isolates. These isolates were tested by a PCR-based assay, and for resistance to five antibiotics: ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (ACSSuT) for the rapid identification of Salmonella Typhimurium DT104. Upon comparing the antibiotic resistance and PCR results with serotype and phage type data, the sensitivity and specificity for the identification of Salmonella Typhimurium DT104 of both methods were found to be 100%, and 99.6%, respectively. Both methods can be completed within 24 h after obtaining an isolate, while serotyping and phagetyping required more than 5 days to complete.

Larval fly activity on sunlit versus shaded raccoon carrion in southwestern West Virginia with special reference to the black blowfly (Diptera: Calliphoridae).

Larvae of Phormia regina (Meigen), Phaenicia sp., and Sarcophaga sp. were identified from raccoon carcasses placed in sunlit and shaded areas at a southwestern West Virginia site in May of 2000. Samples of larvae were taken from each carcass at 3-h intervals over a 153-h experimental period. Phormia regina was clearly the dominant species with large numbers of third instars observed at every 3-h collection period from 81 to 153 h on both carcasses. Mean lengths of third-instar P. regina larvae collected from the sunlit carcass were significantly greater than mean lengths of larvae collected from the shaded carcass. Third-instar Phaenicia sp. also appeared at 81 h on both carcasses, but relatively few (< or = 4) individuals were present in each 3-h collection sample from 81 through 126 h. Larvae of this species were not present in samples from either carcass in those 3-h intervals from 129 to 147 h. Sarcophaga sp. larvae were also collected, but only in samples taken from the sunlit carcass at 81 and 93 h. Ambient temperatures were recorded throughout the experimental period, whereas maggot mass temperatures were not recorded until the appearance of large numbers of second instars at 48 h. From 48 to 69 h, maggot mass temperatures were equivalent to ambient temperatures; but after 69 h, maggot mass temperatures were considerably elevated over ambient temperatures.

Effects of on-board and dockside handling on the formation of biogenic amines in mahimahi (Coryphaena hippurus), skipjack tuna (Katsuwonus pelamis), and yellowfin tuna (Thunnus albacares).

Consumer illnesses by scombroid poisonings have been a continuing problem for many years. The intoxications follow the ingestion of fish such as tuna and mahimahi that have undergone bacterial decomposition, leading to the formation of biogenic amines. Research studies have concluded that histamine is one of the indicators of scombrotoxic fish and that other amines, such as cadaverine, could be involved in the illnesses. Guidance for the handling of fish on board fishing vessels to prevent the production of scombrotoxic fish has been limited by a lack of data addressing changes that occur in fish from the water to delivery at dockside. In this study, the changes in selected biogenic amines were determined in mahimahi and tuna, which were captured and held in seawater at 25 to 35 degrees C for incubation times up to 18 h. The fillets from the treated fish were sectioned by transverse cuts and analyzed for histamine, cadaverine, and putrescine. Results showed that at 26 degrees C, more than 12 h of incubation were required before a histamine concentration of 50 ppm was reached in mahimahi. At 35 degrees C, 50 ppm histamine formed within 9 h. Similar results were found for skipjack and yellowfin tuna. Histamine concentrations exceeded 500 ppm within an additional 3 h of incubation in mahimahi. At both temperatures, an increase in the concentration of cadaverine preceded an increase in histamine levels. Changes in putrescine concentrations in the fish were less pronounced. The study also demonstrated that histidine decarboxylase activity was retained in some frozen samples of fish and could result in further increases in histamine on thawing.

Gas chromatographic method for putrescine and cadaverine in shrimp.

A gas-liquid chromatographic method developed for the determination of putrescine and cadaverine in fishery products was modified for application to the determination of diamines in shrimp. Addition of potassium chloride and hydrochloric acid to the methanol-water extraction solvent resulted in increased recovery of the diamines and minimized gel formation. The recovery of putrescine increased on average from 64 to 98%, and the recovery of cadaverine increased from 85 to 93%. The chromatographic separation of the derivatized diamines was significantly improved with a change from an OV-225 column (cyanopropyl methyl phenyl methyl silicone) to a more polar HP-Innowax column (crosslinked polyethylene glycol). Background levels of putrescine and cadaverine in known high-quality shrimp ranged from 0 to 0.7 ppm. Shrimp that failed sensory examination generally contained putrescine at levels >4.8 ppm and cadaverine at levels >1.3 ppm.

Prion protein genotypes of sheep as determined from 3343 samples submitted from Ontario and other provinces of Canada from 2005 to 2012.

This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had a higher frequency of the ARQ allele.

Dans la présente étude les génotypes de la protéine prion du mouton (PrP) d'échantillons en provenance de l'Ontario et d'autres provinces canadiennes soumis au Animal Health Laboratory de l'Université de Guelph, Ontario, entre 2005 et 2012 ont été analysés. En Ontario, la proportion de moutons résistants à la tremblante a augmentée entre 2005 et 2012 tel que démontré par une augmentation de l'haplotype ARR. Lorsque les provinces canadiennes (Alberta, Ontario, Québec, et Nouvelle-Écosse) ont été comparées de 2008 à 2012, des proportions élevées de moutons résistants à la tremblante ont été trouvés dans toutes les provinces. Les proportions de moutons résistants étaient plus faibles en Alberta et au Québec qu'en Ontario ou en Nouvelle-Écosse. L'Alberta avait une proportion plus élevée de moutons susceptibles et une fréquence plus élevée d'allèles VRQ, et le Québec une fréquence plus élevée de l'allèle ARQ.(Traduit par Docteur Serge Messier).

A randomized controlled trial of an internal family systems-based psychotherapeutic intervention on outcomes in rheumatoid arthritis: a proof-of-concept study.

OBJECTIVE: To conduct a proof-of-concept randomized trial of an Internal Family Systems (IFS) psychotherapeutic intervention on rheumatoid arthritis (RA) disease activity and psychological status. METHODS: Patients with RA were randomized to either an IFS group for 9 months (n = 39) or an education (control) group (n = 40) that received mailed materials on RA symptoms and management. The groups were evaluated every 3 months until intervention end and 1 year later. Self-assessed joint pain (RA Disease Activity Index joint score), Short Form-12 physical function score, visual analog scale for overall pain and mental health status (Beck Depression Inventory, and State Trait Anxiety Inventory) were assessed. The 28-joint Disease Activity Score-C-reactive Protein 4 was determined by rheumatologists blinded to group assignment. Treatment effects were estimated by between-group differences, and mixed model repeated measures compared trends between study arms at 9 months and 1 year after intervention end. RESULTS: Of 79 participants randomized, 68 completed the study assessments and 82% of the IFS group completed the protocol. Posttreatment improvements favoring the IFS group occurred in overall pain [mean treatment effects -14.9 (29.1 SD); p = 0.04], and physical function [14.6 (25.3); p = 0.04]. Posttreatment improvements were sustained 1 year later in self-assessed joint pain [-0.6 (1.1); p = 0.04], self-compassion [1.8 (2.8); p = 0.01], and depressive symptoms [-3.2 (5.0); p =0.01]. There were no sustained improvements in anxiety, self-efficacy, or disease activity. CONCLUSION: An IFS-based intervention is feasible and acceptable to patients with RA and may complement medical management of the disease. Future efficacy trials are warranted. ClinicalTrials.gov identifier: NCT00869349.