Development and optimization of a differentiated airway epithelial cell model of the bovine respiratory tract

Cattle are subject to economically-important respiratory tract infections by various bacterial and viral pathogens and there is an urgent need for the development of more realistic in vitro models of the bovine respiratory tract to improve our knowledge of disease pathogenesis. In the present study, we have optimized the culture conditions in serum-free medium that allow bovine bronchial epithelial cells (BBECs) grown at an air-liquid interface to differentiate into a three-dimensional epithelium that is highly representative of the bovine airway. Epidermal growth factor was required to trigger both proliferation and differentiation of BBECs whilst retinoic acid was also essential for mucociliary differentiation. Triiodothyronine was demonstrated not to be important for the differentiation of BBECs. Oxygen concentration had a minimal effect although optimal ciliation was achieved when BBECs were cultured at 14% oxygen tension. Insert pore-density had a significant effect on the growth and differentiation of BBECs; a high-pore-density was required to trigger optimum differentiation. The established BBEC model will have wide-ranging applications for the study of bacterial and viral infections of the bovine respiratory tract; it will contribute to the development of improved vaccines and therapeutics and will reduce the use of cattle in in vivo experimentation

Comparative bioinformatic and proteomic approaches to evaluate the outer membrane proteome of the fish pathogen Yersinia ruckeri

Yersinia ruckeri is the aetiological agent of enteric redmouth (ERM) disease and is responsible for significant economic losses in farmed salmonids. Enteric redmouth disease is associated primarily with rainbow trout (Oncorhynchus mykiss, Walbaum) but its incidence in Atlantic salmon (Salmo salar) is increasing. Outer membrane proteins (OMPs) of Gram-negative bacteria are located at the host-pathogen interface and play important roles in virulence. The outer membrane of Y. ruckeri is poorly characterised and little is known about its composition and the roles of individual OMPs in virulence. Here, we employed a bioinformatic pipeline to first predict the OMP composition of Y. ruckeri. Comparative proteomic approaches were subsequently used to identify those proteins expressed in vitro in eight representative isolates recovered from Atlantic salmon and rainbow trout. One hundred and forty-one OMPs were predicted from four Y. ruckeri genomes and 77 of these were identified in three or more genomes and were considered as “core” proteins. Gel-free and gel-based proteomic approaches together identified 65 OMPs in a single reference isolate and subsequent gel-free analysis identified 64 OMPs in the eight Atlantic salmon and rainbow trout isolates. Together, our gel-free and gel-based proteomic analyses identified 84 unique OMPs in Y. ruckeri. Significance: Yersinia ruckeri is an important pathogen of Atlantic salmon and rainbow trout and is of major economic significance to the aquaculture industry worldwide. Disease outbreaks are becoming more problematic in Atlantic salmon and there is an urgent need to investigate in further detail the cell-surface (outer membrane) composition of strains infecting each of these host species. Currently, the outer membrane of Y. ruckeri is poorly characterised and very little is known about the OMP composition of strains infecting each of these salmonid species. This study represents the most comprehensive comparative outer membrane proteomic analysis of Y. ruckeri to date, encompassing isolates of different biotypes, serotypes, OMP-types and hosts of origin and provides insights into the potential roles of these diverse proteins in host-pathogen interactions. The study has identified key OMPs likely to be involved in disease pathogenesis and makes a significant contribution to furthering our understanding of the cell-surface composition of this important fish pathogen that will be relevant to the development of improved vaccines and therapeutics

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

Functional and quality of life outcomes of localised prostate cancer treatments (prostate testing for cancer and treatment [ProtecT] study)

Objective To investigate the functional and quality of life (QoL) outcomes of treatments for localised prostate cancer and inform treatment decision-making. Patients and Methods Men aged 50–69 years diagnosed with localised prostate cancer by prostate-specific antigen testing and biopsies at nine UK centres in the Prostate Testing for Cancer and Treatment (ProtecT) trial were randomised to, or chose one of, three treatments. Of 2565 participants, 1135 men received active monitoring (AM), 750 a radical prostatectomy (RP), 603 external-beam radiotherapy (EBRT) with concurrent androgen-deprivation therapy (ADT) and 77 low-dose-rate brachytherapy (BT, not a randomised treatment). Patient-reported outcome measures (PROMs) completed annually for 6 years were analysed by initial treatment and censored for subsequent treatments. Mixed effects models were adjusted for baseline characteristics using propensity scores. Results Treatment-received analyses revealed different impacts of treatments over 6 years. Men remaining on AM experienced gradual declines in sexual and urinary function with age (e.g., increases in erectile dysfunction from 35% of men at baseline to 53% at 6 years and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6 years. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6 years, and after EBRT this was reported by 69% and 74%, respectively (P < 0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1 pad/day, persisting for 20% at 6 years, compared with no change in men receiving EBRT or AM (P < 0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6 years and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (P < 0.001) with lesser effects after BT. No treatment affected mental or physical QoL. Conclusion Treatment decision-making for localised prostate cancer can be informed by these 6-year functional and QoL outcomes

Development and use of an in vitro air-liquid interface model of the bovine respiratory tract and multifaceted proteomic approaches to investigate host-pathogen interactions of Mannheimia haemolytica

In the present study, an air-liquid interface culture system was optimised for the use of bovine airway epithelial cells. This culture system has been adopted for other species, where a pseudostratified epithelium comprising multiple cell types and resembling the in vivo environment is produced. Several aspects of the epithelial culture process were optimised, including (i) the cell harvesting process and the expansion of cells in submerged culture, (ii) the substrate material used for air-liquid interface culture, the effects of collagen-coating of the substrate, and the substrate porosity, (iii) the oxygen tension (iv) growth factor (retinoic acid and epidermal growth factor) concentration used during culture and (v) the anatomical region of the respiratory tract from which the epithelial cells were harvested. The optimisation of the aforementioned conditions resulted in the production of an in vitro respiratory tract model that was more than 40% ciliated, pseudostratified with a layer of P63-expressing basal cells and capable of mucus and CC10 (Clara cell secretory protein) production. Thus, an in vitro model of the bovine respiratory tract was established to determine the host-pathogen interactions of M. haemolytica with the respiratory epithelium. A rigorous proteomic characterisation was carried out on the outer membrane of M. haemolytica. The Sarkosyl insoluble fractions of seven M. haemolytica and one M. glucosida isolates, representing a range of serotypes and host species, were prepared in triplicate. Tryptic digests of each sample were prepared using gel-based and gel-free proteomic digests before analysis by LC-MS/MS. Data was searched against the NCBI Uniprot database using MSCOT (Matrix Science). Assembly of the data revealed that a total of 83 unique outer membrane proteins were distributed amongst the eight Mannheimia spp. isolates. Fifty seven of the 83 OMPs were identified using both gel-free and gel-based methodologies with a further 18 and eight identified with a single methodology alone. Several proteins were identified in an isolate specific manner; two OMPs exhibited a strong pattern of serotype-specific identification. The YadA-like trimeric autotransporter was identified with full reproducibility in A2 serotype strains yet not in strains of different serotype. Conversely, the lipoprotein, plpD was identified in at least two replicates of all isolates except those of serotype A2 where it was not identified at all. The air-liquid interface culture system was used to study the adherence and colonisation of Mannheimia haemolytica to the bovine respiratory tract. In a pilot study, pathogenic bovine and ovine isolates M. haemolytica isolates were studied with bovine and ovine air-liquid interface airway epithelial cells. Bovine and ovine M. haemolytica isolates were found to adhere to the epithelial cells, six hours post-infection in the case of an ovine isolate and 24 hours in the case of a bovine isolate. Bovine isolates of M. haemolytica were predominantly found to invade and adhere to the sub-apical regions of the epithelium with relatively few bacteria found on the apical face. Adherence to the sub-apical epithelium represents the first description of M. haemoyltica causing invasive disease. Microscopic evidence of outer membrane vesicle production and exopolymeric material were also noted for one of the ovine isolates during infection. Following optimisation of the bovine air-liquid interface culture system, an experiment was conducted that examined the ability of M. haemolytica to adhere and colonise air-liquid interface cultures with epithelial cells derived from different anatomical regions of the respiratory tract. Cells derived from the nasopharynx, the mid-trachea and the secondary/tertiary bronchi were differentiated at ALI and infected with pathogenic and non-pathogenic bovine isolates of M. haemolytica. The non-pathogenic M. haemolytica isolate was found to adhere and colonise the nasopharyngeal derived cells better than either the bronchial or tracheal derived cells. Conversely, the pathogenic M. haemolytica isolate was found to have significantly greater adherence to the bronchial derived epithelial cultures and significantly higher adherence (P>0.0001) than the non-pathogenic isolate. The pathogenic M. haemolytica isolate was also found to colonise the nasopharyngeal-derived epithelial culture albeit with lower frequency than was observed for the bronchial-derived epithelial cells. The results presented herein demonstrate the development of a novel in vitro tool for studying the infection process of respiratory pathogens. The use of this tool to study the infection properties of M. haemolytica in combination with characterisation of the outer membrane proteome provides new insight into the process of colonisation and highlights the protein machinery that likely contributes to the process

HELICOPTER FLIGHT TESTING and REAL TIME ANALYSIS with DATA FLOW ARRAY PROCESSORS

International Telemetering Conference Proceedings / October 13-16, 1986 / Riviera Hotel, Las Vegas, NevadaWhen flight testing helicopters, it is essential to process and analyze many parameters spontaneously and accurately for instantaneous feedback in order to make spot decisions on the safety and integrity of the aircraft. As various maneuvers stress the airframe or load oscillatory components, the absolute limits as well as interrelated limits including average and cumulative cycle loading must be continuously monitored. This paper presents a complete acquisition and analysis system (LDF/ADS) that contains modularly expandable array processors which provide real time acquisition, processing and analysis of multiple concurrent data streams and parameters. Simple limits checking and engineering units conversions are performed as well as more complex spectrum analyses, correlations and other high level interprocessing interactively with the operator. An example configuration is presented herein which illustrates how the system interacts with the operator during an actual flight test. The processed and derived parameters are discussed and the part they play in decision making is demonstrated. The LDF/ADS system may perform vibration analyses on many structural components during flight. Potential problems may also be isolated and reported during flight. Signatures or frequency domain representations of past problems or failures may be stored in nonvolatile memory and the LDF/ADS system will perform real time convolutions to determine the degrees of correlation of a present problem with all known past problems and reply instantly. This real time fault isolation is an indispensable tool for potential savings in lives and aircraft as well as eliminating unnecessary down time.International Foundation for TelemeteringProceedings from the International Telemetering Conference are made available by the International Foundation for Telemetering and the University of Arizona Libraries. Visit http://www.telemetry.org/index.php/contact-us if you have questions about items in this collection