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BackgroundAdenovirus-vectored (Ad-vectored) vaccines are typically administered via i.m. injection to humans and are incapable of inducing respiratory mucosal immunity. However, aerosol delivery of Ad-vectored vaccines remains poorly characterized, and its ability to induce mucosal immunity in humans is unknown. This phase Ib trial evaluated the safety and immunogenicity of human serotype-5 Ad-vectored tuberculosis (TB) vaccine (AdHu5Ag85A) delivered to humans via inhaled aerosol or i.m. injection.MethodsThirty-one healthy, previously BCG-vaccinated adults were enrolled. AdHu5Ag85A was administered by single-dose aerosol using Aeroneb Solo Nebulizer or by i.m. injection. The study consisted of the low-dose (LD) aerosol, high-dose (HD) aerosol, and i.m. groups. The adverse events were assessed at various times after vaccination. Immunogenicity data were collected from the peripheral blood and bronchoalveolar lavage samples at baseline, as well as at select time points after vaccination.ResultsThe nebulized aerosol droplets were < 5.39 μm in size. Both LD and HD of AdHu5Ag85A administered by aerosol inhalation and i.m. injection were safe and well tolerated. Both aerosol doses, particularly LD, but not i.m., vaccination markedly induced airway tissue-resident memory CD4+ and CD8+ T cells of polyfunctionality. While as expected, i.m. vaccination induced Ag85A-specific T cell responses in the blood, the LD aerosol vaccination also elicited such T cells in the blood. Furthermore, the LD aerosol vaccination induced persisting transcriptional changes in alveolar macrophages.ConclusionInhaled aerosol delivery of Ad-vectored vaccine is a safe and superior way to elicit respiratory mucosal immunity. This study warrants further development of aerosol vaccine strategies against respiratory pathogens, including TB and COVID-19.Trial registrationClinicalTrial.gov, NCT02337270.FundingThe Canadian Institutes for Health Research (CIHR) and the Natural Sciences and Engineering Research Council of Canada funded this work.

Effective vaccine delivery and coverage to rural and resource-poor countries is hindered by the dependence on cold chain storage. As such, developments of cold chain-free technologies are highly sought. Although spray dried adenoviral vectors have shown long term stability at ambient temperatures and relatively low humidity, it remains to be determined whether similar excipient formulations are applicable to other viral vectors. To address this, we have spray dried vesicular stomatitis virus (VSV)-vectors with a panel of well-characterized sugar excipients to determine the optimal formulation for vector stabilization. Upon reconstitution, we show that trehalose conferred superior stability of VSV both in vitro and in vivo. Importantly, following cold chain-free storage at elevated temperatures at 37 °C for 15 days, we show that a VSV-vectored vaccine retains its in vivo immunogenicity, whereas a liquid control completely lost its immune-stimulating ability. Our results provide foundational evidence that spray drying with properly tested excipients can stabilize viral vectors such as VSV, allowing them to be stored long-term at elevated temperatures without dependency on cold chain conditions.

Considering the substantive potential benefits of thermally stable dry powder vaccines to public health, causes for inactivation of their sensitive viral vectors during preparation require intensive study. The focus of this work was atomization of suspensions containing encapsulating excipients and a human type 5 adenovirus, involving a detailed investigation of shear stresses in the nozzle of a spray dryer. Samples were sprayed at 25 °C into falcon tubes and immediately evaluated for viral activity by in vitro testing, minimizing the confounding of thermal effects on the deactivation of the virus, although interfacial stresses could not be decoupled from shear stresses. Despite the expectations of only virus deactivation with ever-increasing shear stresses in the spray nozzle, some conditions were found to show better activity than the positive control, leading to investigations of viral aggregation. It was found that the adenovirus experienced minor aggregation when mixed with the excipient solutions, which was reversed by subjecting samples to moderate shear conditions in the spray nozzle. At very high shear rates, the activity diminished again because of damage to the viral capsid fibers, which also led to the production of new aggregates after atomization. Despite these findings, activity losses caused by shear were small compared to the overall spray drying process loss. However, formulation composition, solution viscosity, and process conditions should be considered carefully for optimization because of their impact on aggregation. This is the first known report comparing shear, aggregation, and biological activity loss during the atomization step of spray drying viral vaccines.

A cadmium germanium arsenide compound, Cd 3 Ge 2 As 4 , was synthesized using a double-containment fused quartz ampoule method within a rocking furnace and a melt-quench technique. The crystal structure was determined from single-crystal X-ray diffraction (SC-XRD), scanning and transmission electron microscopies ( i.e. SEM, STEM, and TEM), and selected area diffraction (SAD) and confirmed with electron backscatter diffraction (EBSD). The chemistry was verified with electron energy loss spectroscopy (EELS).

Spray drying is emerging as a promising technique to produce thermally stable powder vaccines containing viral vectors. One of the most important factors in developing dry powder vaccines is the selection of the excipient carrier, however this process is time intensive, and uses large amounts of costly viral material. In this work, an acoustic levitator modified with a hot air delivery system was evaluated for its ability to mimic spray drying and acts as a screening method for excipient selection. The ability of three binary excipient blends to stabilize a human type 5 adenovirus was evaluated through an in vitro activity assay based on the expression of green fluorescent protein. Dried particle size and shape, glass transition temperature, moisture content, and crystallinity of powders produced by levitation and spray drying were compared. The particles created in the acoustic levitator under moderate heat can be considered representative of the powders that would be produced via spray drying. Viral vector titre losses differ between the methods, however, the trends with respect to excipient performance remain the same. Key material characteristics such as particle morphology and thermal properties are conserved when using the levitator. The acoustic levitator is a good starting point for dry powder vaccine development, and can be used to identify promising excipients while consuming minimal amounts of the viral vector., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Two enveloped viral vectors, vesicular stomatitis virus and influenza virus, and a non-enveloped viral vector, human adenovirus type 5, were encapsulated by spray drying to enhance thermal stability.Results with these candidates led to the hypothesis that stability performance of chosen excipients may be less virus-specific, as previously postulated in the literature, and more differentiated based on whether the virus has a lipid envelope. Spray dried samples were characterized for their thermal properties, RNA viability and in vitro viral activity after storage at 37 °C for up to 30 days or at 45 °C for up to 3 days. The enveloped viral vectors, as a group, were more thermally stable in trehalose while the non-enveloped viral vector showed higher activity with mannitol as the primary excipient in blends. Trehalose shows strong hydrogen bonds with the envelope's lipid membrane than the other carbohydrates, more effectively replacing water molecules while maintaining the fluidity of the membrane. Conversely, the small size of mannitol molecules was attributed to the more effective hydrogen bonding between water and the protein capsid of non-enveloped viral vectors. In all cases, a matrix with high glass transition temperature contributed to thermal stabilization through vitrification. This work suggests that carbohydrate stabilizer selection may be more dependent on the envelope rather than the specific viral vector, which, if universally true, will provide a guideline for future formulation development., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Patients with suspected colorectal cancer (CRC) usually undergo colonoscopy. Flexible sigmoidoscopy (FS) may be preferred if proximal cancer risk is low. We investigated which patients could undergo FS alone., Methods: Cohort study of 7375 patients (≥55 years) referred with suspected CRC to 21 English hospitals (2004-2007), followed using hospital records and cancer registries. We calculated yields and number of needed whole-colon examinations (NNE) to diagnose one cancer by symptoms/signs and subsite. We considered narrow (haemoglobin <11 g/dL men; <10 g/dL women) and broad (<13 g/dL men; <12 g/dL women) anaemia definitions and iron-deficiency anaemia (IDA)., Results: One hundred and twenty-seven proximal and 429 distal CRCs were diagnosed. A broad anaemia definition identified 80% of proximal cancers; a narrow definition with IDA identified 39%. In patients with broad definition anaemia and/or abdominal mass, proximal cancer yield and NNE were 4.8% (97/2022) and 21. In patients without broad definition anaemia and/or abdominal mass, with rectal bleeding or increased stool frequency (41% of cohort), proximal cancer yield and NNE were 0.4% (13/3031) and 234., Conclusion: Most proximal cancers are accompanied by broad definition anaemia. In patients without broad definition anaemia and/or abdominal mass, with rectal bleeding or increased stool frequency, proximal cancer is rare and FS should suffice.

Current global vaccination programs are challenged by costs associated with vaccine cold chain storage and administration. A solid, thermally stable oral dosage form for vaccines would alleviate these costs but is difficult to produce due to general vaccine instability and the complication of bypassing the gastric barrier. We developed a novel consecutive spray drying method that is suitable for use with biologics and employs Eudragit L100 polymer as the enteric coating. More specifically, in step 1, recombinant replication deficient human type-5 adenovirus and vesicular stomatitis virus were encapsulated by spray drying with sugars from a water solution, and in step 2, the microparticles from step 1 were suspended in ethanol with Eudragit and spray dried again. Up to 25% of the starting material was fully encapsulated within the enteric coating, and encapsulation efficiency was largely dependent on spray gas flow rate and the solids concentration in the feed. After step 2, the coated vaccine-sugar particles maintained their thermostability and were slightly larger in size with a rugous surface morphology compared to the particles produced in step 1. The coated particles retained viral vector activity in vitro after 15 min incubation in 1 M HCl (simulating the stomach environment) and anhydrous ethanol (to dissolve the Eudragit outer shell). The production of dry, orally administered vaccine particles from consecutive spray drying offers the potential to remedy a number of vaccine storage, transportation, and administration limitations.

Cold chain-free vaccine technologies are needed to ensure effective vaccine delivery and coverage, particularly in resource-poor countries. However, the immunogenicity and thermostability of spray dried live viral vector-based vaccines such as recombinant adenoviral-vectored vaccines remain to be investigated. To address this issue, we have spray dried human adenoviral (AdHu5)- and chimpanzee adenoviral (AdCh68)-vectored tuberculosis vaccines in a mannitol and dextran matrix. Spray dried powders containing these two vaccines display the morphologic and chemical properties desired for long-term thermostability and vaccination. Upon reconstitution, they effectively transfected the cells in vitro with relatively small losses in viral infectivity related to the spray drying process. Following in vivo vaccination, AdHu5- and AdCh68-vectored vaccines were as immunogenic as the conventional fresh, cryopreserved liquid vaccine samples. Of importance, even after cold chain-free storage, at ambient temperatures and relatively low humidity for 30 and 90days, the vaccines retained their in vivo immunogenicity, while the liquid vaccine samples stored under the same conditions lost their immune-activating capability almost entirely. Our results support further development of our spray drying technologies for generating thermally stable adenoviral-vectored and other viral-vectored vaccines., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Purpose: This work examines the relevance of viral activity in the optimization of spray drying process parameters for the development of thermally stable vaccine powders. In some instances, the actual active pharmaceutical ingredient (API) is not included in the process optimization as it is deemed too costly to use until the final selection of operating conditions, however, that approach is inappropriate for highly labile biopharmaceutics. We investigate the effects of spray drying parameters on i) yield, ii) particle size and iii) viral vector activity of a mannitol/dextran encapsulated recombinant human type 5 adenoviral vector vaccine, to demonstrate the effects and magnitude of each effect on the three responses, and further show that the API must be included earlier in the optimization., Methods: A design of experiments approach was used with response surface methodology (RSM) to optimize parameters including inlet temperature, spray gas flow rate, liquid feed rate and solute concentration in the feed., Results: In general, good conditions for maintaining viral activity led to reduced yield and fewer particles of the desired size. Within the range of parameters tested, the yield varied from 50 to 90%, the percentage of ideally size particles was 10-50%, and the viral vector titre loss was 0.25-4.0 log loss., Conclusions: RSM indicates that the most significant spray drying parameters are the inlet temperature and spray gas flow rate. It was not possible to optimize all three output variables with one set of parameters, indicating that there will only be one dominant criteria for processing which in the case of viral vaccines will likely be viral vector activity.

We have produced a thermally stable recombinant human type 5 adenoviral vector (AdHu5) through spray drying with three excipient formulations (l-leucine, lactose/trehalose and mannitol/dextran). Spray drying leads to immobilization of the viral vector which is believed to prevent viral protein unfolding, aggregation and inactivation. The spray dried powders were characterized by scanning electron microscopy, differential scanning calorimetry, Karl Fischer titrations, and X-ray diffraction to identify the effects of temperature and atmospheric moisture on the immobilizing matrix. Thermal stability of the viral vector was confirmed in vitro by infection of A549 lung epithelial cells. Mannitol/dextran powders showed the greatest improvement in thermal stability with almost no viral activity loss after storage at 20°C for 90days (0.7±0.3 log TCID50) which is a significant improvement over the current -80°C storage protocol. Furthermore, viral activity was retained over short term exposure (72h) to temperatures as high as 55°C. Conversely, all powders exhibited activity loss when subjected to moisture due to amplified molecular motion of the matrix. Overall, a straightforward method ideal for the production of thermally stable vaccines has been demonstrated through spray drying AdHu5 with a blend of mannitol and dextran and storing the powder under low humidity conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Hot-melt granulation (HMG) by twin screw extrusion is a novel technology for the continuous processing of pharmaceuticals but confidence must still be gained regarding whether the environment affects drug properties. In this preliminary study, granulation was studied for a model product containing lactose monohydrate and active ingredients of differing water solubility, namely ibuprofen versus caffeine. The formulations were granulated at 220 rpm and 100°C with polyethylene glycol binders of differing molecular weights and at concentrations between 6.5% and 20%. In terms of granule properties, the low melting point of ibuprofen had a dominant influence by producing larger, stronger granules, whereas the caffeine products were more comparable to a blank containing no active ingredient. Drug degradation was study by differential scanning calorimetry, X-ray diffraction, and high-pressure liquid chromatography. The only detected change was the dehydration of lactose monohydrate for the caffeine and blank products, whereas the lubricating influence of the ibuprofen protected its granules. The short residence time (∼60 s) was consider to be influential in minimizing damage of the drug despite the high temperature and shear attributed to HMG inside a twin screw extruder., (© 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Understanding changes in microbial structure due to biodiesel storage is important both for protecting integrity of storage systems and fuel quality management. In this work a simulated storage system was used to study the effect of biodiesel (0%, 25%, 50%, 75% and 100%) on a microbial population, which was followed by community level physiological profiling (CLPP), 16s rDNA analysis and plating in selective media. Results proved that structure and functionality were affected by biodiesel. CLPP showed at least three populations: one corresponding to diesel, one to biodiesel and one to blends of diesel and biodiesel. Analysis of 16s rDNA revealed that microbial composition was different for populations growing in diesel and biodiesel. Genera identified are known for degradation of hydrocarbons and emulsifier production. Maximum growth was obtained in biodiesel; however, microbial counts in standard media were lower for this samples. Acidification of culture media was observed at high biodiesel concentration., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Purpose: This study was designed to develop a mathematical model for predicting the number of lymph nodes harvested in bowel cancer resection specimens based on the current clinical practice in the United Kingdom., Methods: Prospective clinical data were collected from 8,409 newly diagnosed bowel cancer patients presenting to 79 hospitals in Great Britain and Ireland during a variable 12-month period from 2000 to 2002. A two-level hierarchical regression model was used to identify predictors for lymph node harvest. The model was internally validated by comparing observed and model predicted lymph node harvest for patient subgroups., Results: Inclusion criteria were satisfied by 5,164 patients. The average lymph node harvest was 11.7 nodes with significant between-center variability in lymph node harvest (range, 5.5-21.3 nodes). Increasing age, American Society of Anesthesiology grade, and preoperative radiotherapy were associated with a reduction of lymph node harvest (P < 0.001). Abdominoperineal resection of the rectum and transverse colectomy were the lowest yield procedures for lymph node harvest. Independent predictors of lymph node harvest were age, American Society of Anesthesiology grade, Dukes stage, operative urgency, type of resection, and preoperative radiotherapy. When tested, the model was found to accurately predict lymph node harvest for group statistics (comparison of observed and model predicted lymph node harvest F(1,5154) = 0.63; P = 0.427)., Conclusions: The results of the study suggest that the minimum number of lymph nodes harvested in colorectal cancer surgery cannot be set at a fixed value. The lymph node harvest model provides a simple tool to the frontline clinician for comparing standards between multidisciplinary bowel cancer teams.

Purpose: This study was designed to develop a model for predicting postoperative mortality in elderly patients undergoing surgery for colorectal cancer., Methods: This multicenter study was conducted by using routinely collected clinical data, assessing patients older than aged 80 years, with 30-day operative mortality as the primary end point. Data were collected from The Association of Coloproctology of Great Britain and Ireland database, encompassing 8,077 newly diagnosed colorectal cancer patients undergoing resectional surgery in 79 hospitals between April 2000 to March 2002, The Association of Coloproctology Malignant Bowel Obstruction Study, encompassing 1,046 patients with malignant bowel obstruction in 148 hospitals, between April 1998 to March 1999, and The Wales-Trent audit, encompassing 3,522 newly diagnosed colorectal cancer patients, between July 1992 to June 1993. A multilevel logistic regression model was developed to adjust for case-mix and to accommodate the variability of outcomes between the three study populations. The model was internally validated using a Bayesian resampling technique and tested using measures of discrimination, calibration, and subgroup analysis., Results: A total of 2,533 patients satisfied the inclusion criteria, with a 30-day mortality of 15.6 percent. Multivariate analysis identified the following independent risk factors: age (odds ratio for 85-90, 90-95, >95 vs. 80-85 = 1.1, 1.8, 2.9), American Society of Anesthesiology grade (odds ratio for Grade III, IV vs. I-II = 2.7, 6.1), operative urgency (odds ratio for emergency vs. elective = 1.9), no cancer excision vs. resection (odds ratio = 1.2), and metastatic disease (odds ratio for metastases vs. no metastases = 1.9). The model offered adequate discrimination (area under receiver operator curve = 0.732) and excellent agreement between observed and predicted outcomes during eight colorectal procedures (P = 0.885)., Conclusions: The elderly colorectal cancer model can accurately estimate 30-day mortality in patients older than aged 80 years undergoing surgery for colorectal cancer. Because the mortality can be considerable, this may have important implications when determining management for this group of patients.

Bowel cancer is a major cause of morbidity and death and is a high cost to health care systems. Screening currently offers the best chance of improving outcomes from bowel cancer. When introducing screening, the problems encountered in other cancers need to be avoided to maximize benefits and minimize harms.

Objective: To develop a mathematical model that will predict the probability of death after surgery for colorectal cancer., Design: Descriptive study using routinely collected clinical data., Data Source: The database of the Association of Coloproctology of Great Britain and Ireland (ACPGBI), encompassing 8077 patients with a new diagnosis of colorectal cancer in 73 hospitals during a 12 month period., Statistical Analysis: A three level hierarchical logistic regression model was used to identify independent predictors of operative mortality. The model was developed on 60% of the patient population and its validity tested on the remaining 40%., Results: Overall postoperative mortality was 7.5% (95% confidence interval 6.9% to 8.1%). Independent predictors of death were age, American Society of Anesthesiology (ASA) grade, Dukes's stage, urgency of the operation, and cancer excision. When tested the predictive model showed good discrimination (area under the receiver operating characteristic curve = (0.775) and calibration (comparison of observed with expected mortality across different procedures; Hosmer-Lemeshow statistic = 6.34, 8 df, P = 0.610)., Conclusions: Clinicians can predict postoperative death by using a simple numerical table derived from the statistical model of the ACPGBI. The model can be used in everyday practice for preoperative counselling of patients and their carers as a part of multidisciplinary care. It may also be used to compare the outcomes between multidisciplinary teams for colorectal cancer.

An improved functional approach for designing MRI (magnetic resonance imaging) main magnets with active shielding is presented. By nulling one or two external moments as well as a certain series of internal moments of the magnetic field, new designs with improved shielding in combination with or without shorter magnet lengths are obtained. The improved method can be employed to design short and practical superconducting magnets at any given field strength. The resulting designs yield the desired field homogeneity inside the region of interest without using superconducting shim coils. This approach requires only a modest amount of computing power. One of the design steps, a contour plot of the continuous current solutions, can be utilized to study stretch goals for favorable design parameters.