Toward A Regulatory Pathway for the Use of in Silico Trials in the CE Marking of Medical Devices.

In Silico Trials methodologies will play a growing and fundamental role in the development and de-risking of new medical devices in the future. While the regulatory pathway for Digital Patient and Personal Health Forecasting solutions is clear, it is more complex for In Silico Trials solutions, and therefore deserves a deeper analysis. In this position paper, we investigate the current state of the art towards the regulatory system for in silico trials applied to medical devices while exploring the European regulatory system toward this topic. We suggest that the European regulatory system should start a process of innovation: in principle to limit distorted quality by different internal processes within notified bodies, hence avoiding that the more innovative and competitive companies focus their attention on the needs of other large markets, like the USA, where the use of such radical innovations is already rapidly developing.

Psychedelic Psychotherapy: Building Wholeness Through Connection.

BACKGROUND: We are confronted with dire statistics that document our current mental health crisis. New treatment modalities are desperately needed to address escalating mental suffering and trauma. Psychedelic medicines are attracting increased attention in psychiatry as effective treatment for a range of conditions. The mechanisms of actions and context necessary to maximize their full healing potential represent a radical departure from current psychiatric frameworks and present an opportunity to reimagine psychiatry as a healing art. OBJECTIVE: Psychedelic psychotherapy leverages biological, psychological, and spiritual domains to harness innate healing potentials. A novel psychotherapeutic methodology utilizing psychedelic medicines as catalyzing agents is presented, one that provides a developmental model to promotes self-actualization. The paper outlines transformational psychotherapy, the therapeutic process and corresponding practice implications. CONCLUSION: Psychedelic psychotherapy represents a paradigm shift in healing, one that promotes self-integration and whole health. These shifts in internal health are correspondingly reflected in enhanced empathy, improved relatedness, and increased capacity for social connection. Much of human suffering and disregard for the planet is a reflection of our own collective inner impoverishment, fundamental disconnects, and unaddressed trauma. Psychedelic psychotherapy offers a healing approach to restore beauty and health to both the inner and outer worlds we inhabit.

Preferred product characteristics for therapeutic vaccines to improve tuberculosis treatment outcomes: Key considerations from World Health Organization consultations.

Treating tuberculosis (TB) requires a multidrug course of treatment lasting 6 months, or longer for drug-resistant TB, which is difficult to complete and often not well tolerated. Treatment failure and recurrence after end-of-treatment can have devastating consequences, including progressive debilitation, death, the transmission of Mycobacterium tuberculosis - the infectious agent responsible for causing TB - to others, and may be associated with the development of drug-resistant TB. The burden on health systems is important, with severe economic consequences. Vaccines have the potential to serve as immunotherapeutic adjuncts to antibiotic treatment regimens for TB. A therapeutic vaccine for TB patients, administered towards completion of a prescribed course of drug therapy or at certain time(s) during treatment, could improve outcomes through immune-mediated control and even clearance of bacteria, potentially prevent re-infection, and provide an opportunity to shorten and simplify drug treatment regimens. The preferred product characteristics (PPC) for therapeutic TB vaccines described in this document are intended to provide guidance to scientists, funding agencies, public and private sector organizations developing such vaccine candidates. This document presents potential clinical end-points for evidence generation and discusses key considerations about potential clinical development strategies.

Comparison of pellicle and shake flask-grown BCG strains by quality control assays and protection studies.

A global BCG vaccine shortage began in 2013 which impacted availability for infant vaccinations, as well as preclinical studies and clinical trials of new TB vaccines. Stakeholders met in 2015 at McGill University in Montreal to discuss the shortage and potential mitigation strategies. Manufacturing BCG through a more tractable liquid fermentation process instead of the traditional pellicle growth method was considered a potentially viable strategy. This pilot program compared pellicle-grown and shake flask-grown BCG strains (as a first step towards modeling fermenter-produced BCG vaccine) in selected quality control assays, as well as mouse and guinea pig protection studies. Conventional pellicle-grown, lyophilized BCG WHO Reference Reagents (Danish, Moreau, Russian, Tokyo strains) were obtained from the National Institute for Biological Standards and Control (NIBSC), UK. Strains were grown in shake flasks and glycerol stocks prepared. Shake flask-grown BCG culture preparations generally met the requirements of quality control testing at NIBSC. In mouse and guinea pig protection studies there were no significant differences in lung colony forming units (CFUs) between shake flask-grown and pellicle-grown preparations, with the exception of BCG Russian, where the shake flask-grown preparation protected better in mice (P = 0.0042), but the pellicle-grown preparation protected better in guinea pigs (P = 0.0015). Producing BCG vaccines by a more tractable liquid growth process could be a viable solution to the global BCG shortage.

A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.

Tuberculosis vaccines--state of the art, and novel approaches to vaccine development.

The quest for a vaccine that could have a major impact in reducing the current global burden of TB disease in humans continues to be extremely challenging. Significant gaps in our knowledge and understanding of the pathogenesis and immunology of tuberculosis continue to undermine efforts to break new ground, and traditional approaches to vaccine development have thus far met with limited success. Existing and novel candidate vaccines are being assessed in the context of their ability to impact the various stages that culminate in disease transmission and an increase in the global burden of disease. Innovative methods of vaccine administration and delivery have provided a fresh stimulus to the search for the elusive vaccine. Here we discuss the current status of preclinical vaccine development, providing insights into alternative approaches to vaccine delivery and promising candidate vaccines. The state of the art of clinical development also is reviewed.

Generation of bright isolated attosecond soft X-ray pulses driven by multicycle midinfrared lasers.

High harmonic generation driven by femtosecond lasers makes it possible to capture the fastest dynamics in molecules and materials. However, to date the shortest subfemtosecond (attosecond, 10(-18) s) pulses have been produced only in the extreme UV region of the spectrum below 100 eV, which limits the range of materials and molecular systems that can be explored. Here we experimentally demonstrate a remarkable convergence of physics: when midinfrared lasers are used to drive high harmonic generation, the conditions for optimal bright, soft X-ray generation naturally coincide with the generation of isolated attosecond pulses. The temporal window over which phase matching occurs shrinks rapidly with increasing driving laser wavelength, to the extent that bright isolated attosecond pulses are the norm for 2-µm driving lasers. Harnessing this realization, we experimentally demonstrate the generation of isolated soft X-ray attosecond pulses at photon energies up to 180 eV for the first time, to our knowledge, with a transform limit of 35 attoseconds (as), and a predicted linear chirp of 300 as. Most surprisingly, advanced theory shows that in contrast with as pulse generation in the extreme UV, long-duration, 10-cycle, driving laser pulses are required to generate isolated soft X-ray bursts efficiently, to mitigate group velocity walk-off between the laser and the X-ray fields that otherwise limit the conversion efficiency. Our work demonstrates a clear and straightforward approach for robustly generating bright isolated attosecond pulses of electromagnetic radiation throughout the soft X-ray region of the spectrum.

Rational approach to selection and clinical development of TB vaccine candidates.

A rational process is clearly needed and can be extremely helpful for selection, assessing and advancing TB vaccine candidates from entry into preclinical and clinical development and for advancing candidates from early safety and immunogenicity clinical trials to proof-of-concept and pivotal efficacy trials. A joint effort between Aeras and the Tuberculosis Vaccine Initiative has focused on the development of objective criteria for a number of key general vaccine characteristics which can be assessed at critical stages of development. In order to maximize development efficiency, increase likelihood of success, and optimize use of scarce resources, this process includes establishment of gates for moving TB vaccine candidates through progressive development stages based on meeting the established criteria for specific vaccine candidates.

OX40 ligand fusion protein delivered simultaneously with the BCG vaccine provides superior protection against murine Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis infection claims approximately 2 million lives per year, and improved efficacy of the BCG vaccine remains a World Health Organization priority. Successful vaccination against M. tuberculosis requires the induction and maintenance of T cells. Targeting molecules that promote T-cell survival may therefore provide an alternative strategy to classic adjuvants. We show that the interaction between T-cell-expressed OX40 and OX40L on antigen-presenting cells is critical for effective immunity to BCG. However, because OX40L is lost rapidly from antigen-presenting cells following BCG vaccination, maintenance of OX40-expressing vaccine-activated T cells may not be optimal. Delivering an OX40L:Ig fusion protein simultaneously with BCG provided superior immunity to intravenous and aerosol M. tuberculosis challenge even 6 months after vaccination, an effect that depends on natural killer 1.1(+) cells. Attenuated vaccines may therefore lack sufficient innate stimulation to maintain vaccine-specific T cells, which can be replaced by reagents binding inducible T-cell costimulators.

Adjuvant effects on antibody titre.

Antibody titre is a measure of the presence and amount of antibodies specific to an antigen that are present in the blood. In particular, the titre of an antibody sample is a measure of the antibody concentration determined under a defined set of conditions, with the antibody concentration being commonly established by enzyme-linked immunosorbent assay, also called ELISA, or a variation of this technology.Enzyme-linked immunosorbent assay, also called ELISA, enzyme immunoassay or EIA, is a biochemical technique used to detect the presence of an antibody or an antigen in a sample. The ELISA/EIA approach is an extremely robust technology and readily amenable to validation and quality control if adherence to ISO quality standards is required. This chapter describes in detail a specific ELISA protocol which has potential generic application.

Functional antibody assays.

Functional antibody assays can broadly be divided into three categories: neutralisation, serum bactericidal antibody (SBA) and opsonophagocytic assays (OPA). These biological assays are generally more complex than antibody-binding counterparts. They invariably involve multiple biological components, many of which are difficult or impossible to standardise. The aim of this chapter is to provide working examples of these assays and highlight the key issues to be addressed to ensure they are able to provide reliable data.

Time-resolved momentum imaging system for molecular dynamics studies using a tabletop ultrafast extreme-ultraviolet light source.

We describe a momentum imaging setup for direct time-resolved studies of ionization-induced molecular dynamics. This system uses a tabletop ultrafast extreme-ultraviolet (EUV) light source based on high harmonic upconversion of a femtosecond laser. The high photon energy (around 42 eV) allows access to inner-valence states of a variety of small molecules via single photon excitation, while the sub--10-fs pulse duration makes it possible to follow the resulting dynamics in real time. To obtain a complete picture of molecular dynamics following EUV induced photofragmentation, we apply the versatile cold target recoil ion momentum spectroscopy reaction microscope technique, which makes use of coincident three-dimensional momentum imaging of fragments resulting from photoexcitation. This system is capable of pump-probe spectroscopy by using a combination of EUV and IR laser pulses with either beam as a pump or probe pulse. We report several experiments performed using this system.

Comparison of the interaction of tomatine with mixed monolayers containing phospholipid, egg sphingomyelin, and sterols.

The interaction of the glycoalkaloid tomatine with monolayers of a phospholipid (dimyristoylphosphatidylcholine, DMPC), and sphingolipid (egg sphingomyelin), and cholesterol is compared. Using measurements of the surface pressure response as a function of the subphase concentration of tomatine, interfacial binding constants are estimated for mixed monolayers of DMPC and cholesterol and for those of egg sphingomyelin and cholesterol of mole ratio 7:3. The binding constants obtained suggest a stronger interaction of tomatine with DMPC and cholesterol mixed monolayers, reflecting easier displacement of cholesterol from its interaction with DMPC than from its interaction with egg sphingomyelin. Mixtures of tomatine and cholesterol are found to spread directly at the water-air interface and form stable monolayers, suggesting that cholesterol holds tomatine at the interface despite the absence of observed monolayer behavior for tomatine alone. The interaction of tomatine with DMPC and cholesterol monolayers is found to exhibit a pH dependence in agreement with previously reported results for its interaction with liposomes; in particular, the interaction is much less at pH 5 than at pH 7 or pH 9. It is found that while tomatine interacts strongly with monolayers containing sitosterol, it does not interact with monolayers containing sitosterol glucoside. The response of monolayers of varying composition of DMPC and cholesterol to tomatine is also examined. Brewster angle microscopy (BAM) reveals further evidence for formation of suspected islands of tomatine + cholesterol complexes upon interaction with mixed monolayers of lipid and sterol.

Interaction of the glycoalkaloid tomatine with DMPC and sterol monolayers studied by surface pressure measurements and Brewster angle microscopy.

The interaction of the glycoalkaloid tomatine with monolayers of dimyristoylphosphatidylcholine (DMPC) and cholesterol, as well as other selected sterols, has been investigated using surface pressure measurements at constant area and Brewster angle microscopy (BAM). The interaction of tomatine with sterol monolayers is found to vary with the structure of the sterol. The interaction of tomatine with cholesterol-containing monolayers results in a surface pressure increase accompanied by the appearance of a mottled texture. Morphological changes are observed that suggest the formation of tomatine-cholesterol complexes that aggregate at the water-air interface. No morphology change observable by BAM is observed for monolayers containing epicholesterol, suggesting that the stereochemistry of hydrogen bonding between the sterol and the sugar units on tomatine is of particular significance. Strong interactions are observed with cholestanol- and coprostanol-containing monolayers, and BAM reveals formation of spiked aggregates upon interaction with 7:3 mole ratio DMPC/coprostanol mixed monolayers. More modest surface pressure changes are observed for cholestenone- and epicoprostanol-containing monolayers. A much smaller surface pressure increase is observed when tomatine is injected beneath a pure DMPC monolayer.

Fringe-free, background-free, collinear third-harmonic generation frequency-resolved optical gating measurements for multiphoton microscopy.

A background-free, fringe-free form of frequency-resolved optical gating using the third-harmonic signal generated from a glass coverslip is used to characterize 100 fs pulses at the focus of a 0.65 NA objective.

High-order harmonic generation from ions in a capillary discharge.

We demonstrate a significant extension of the high-order harmonic cutoff by using a fully-ionized capillary discharge plasma as the generation medium. The preionized plasma dramatically reduces ionization-induced defocusing and energy loss of the driving laser due to ionization. This allows for significantly higher photon energies, up to 150 eV, to be generated from xenon ions, compared with the 70 eV observed previously. We also demonstrate enhancement of the harmonic flux of nearly 2 orders of magnitude at photon energies around 90 eV when the capillary discharge is used to ionize xenon, compared with harmonic generation in a hollow waveguide. The use of a plasma as a medium for high-order harmonic generation shows great promise for extending efficient harmonic generation to much shorter wavelengths using ions.

Laboratory investigation of immune responses to acellular pertussis vaccines when used for boosting adolescents after primary immunisation with whole cell pertussis vaccines: a comparison with data from clinical study.

The lack of unequivocal immunological correlates of human protection and an absence of a validated animal model for acellular pertussis vaccines, compounded by limited opportunity to undertake efficacy studies in humans and laboratory evaluation side by side, has made it difficult to compare vaccines and formulations. In the present study, the effect on the booster response to pertussis in adolescents primed in infancy with whole cell pertussis vaccine, of three low dose acellular pertussis/diphtheria/tetanus toxoid (TdPa) formulations with or without inactivated poliomyelitis vaccine (IPV) components, was investigated. To assess the relationship between laboratory vaccine evaluation and clinical trial performance, parallel evaluation of the same TdPa vaccines were carried out in a mouse booster model with whole cell pertussis vaccine priming. Prior to boosting, the clinical subjects had low cell mediated immune responses (CMI) responses to pertussis vaccine components. After boosting, all TdPa formulations stimulated CMI responses to the pertussis vaccine components assessed. The booster responses to the pertussis antigens remained skewed towards Th1 type even though acellular pertussis vaccines were used. In general the antibody and CMI responses to pertussis antigens in the mouse model followed the trend seen in the human subjects. Protection against aerosol challenge with virulent Bordetella pertussis was related to the magnitude of the antibody and CMI responses in the mouse model. As in the human subjects, the responses remained skewed towards Th1 type.

Therapeutic efficacy of high-dose intravenous immunoglobulin in Mycobacterium tuberculosis infection in mice.

Intravenous immunoglobulin (IVIg) is used to treat patients with primary antibody deficiencies and, at high doses, to treat a range of autoimmune and inflammatory disorders. With high-dose IVIg (hdIVIg), immunomodulatory mechanisms act on a range of cells, including T cells, B cells, and dendritic cells. Here, we demonstrate that the treatment of M. tuberculosis-infected mice with a single cycle of hdIVIg resulted in substantially reduced bacterial loads in the spleen and lungs when administered at either an early or late stage of infection. Titration of the IVIg showed a clear dose-response effect. There was no reduction in bacterial load when mice were given equimolar doses of another human protein, human serum albumin, or maltose, the stabilizing agent in the IVIg preparation. HdIVIg in vitro had no inhibitory effect on the growth of M. tuberculosis in murine bone marrow-derived macrophages. In addition, the effect of hdIVIg on bacterial loads was not observed in nude mice, suggesting the involvement of conventional T cells. Analysis of T cells infiltrating the lungs revealed only small increases in CD8(+) but not CD4(+) T-cell numbers in hdIVIg-treated mice. The mechanism of action of hdIVIg against tuberculosis in mice remains to be determined. Nevertheless, since hdIVIg is already widely used clinically, the magnitude and long duration of the therapeutic effect seen here suggest that IVIg, or components of it, may find ready application as an adjunct to therapy of human tuberculosis.

Vaccination with live attenuated simian immunodeficiency virus for 21 days protects against superinfection.

The identification of mechanisms that prevent infection with human immunodeficiency virus (HIV) or simian immunodeficiency virus (SIV) would facilitate the development of an effective AIDS vaccine. In time-course experiments, protection against detectable superinfection with homologous wild-type SIV was achieved within 21 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral immunity. Partial protection against superinfection was achieved within 10 days of inoculation with live attenuated SIV, prior to the development of detectable anti-SIV humoral and cellular immunity. Furthermore, co-inoculation of live attenuated SIV with wild-type SIV resulted in a significant reduction in peak virus loads compared to controls that received wild-type SIV alone. These findings imply that innate immunity or non-immune mechanisms are a significant component of early protection against superinfection conferred by inoculation with live attenuated SIV.