A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging

DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy

Principles and Fundamentals of Optical Imaging

In this chapter I will give a brief general introduction to optical imaging and then discuss in more detail some of the methods specifically used for imaging cortical dynamics today. Absorption and fluorescence microscopy can be used to form direct, diffraction-limited images but standard methods are often only applicable to superficial layers of cortical tissue. Two-photon microscopy takes an intermediate role since the illumination pathway is diffraction-limited but the detection pathway is not. Losses in the illumination path can be compensated using higher laser power. Since the detection pathway does not require image formation, the method can substantially increase the imaging depth. Understanding the role of scattering is important in this case since non-descanned detection can substantially enhance the imaging performance. Finally, I will discuss some of the most widely used imaging methods that all rely on diffuse scattering such as diffuse optical tomography, laser speckle imaging, and intrinsic optical imaging. These purely scattering-based methods offer a much higher imaging depth, although at a substantially reduced spatial resolution

Risk factors for infections caused by carbapenem-resistant Enterobacterales: an international matched case-control-control study (EURECA)

Cases were patients with complicated urinary tract infection (cUTI), complicated intraabdominal (cIAI), pneumonia or bacteraemia from other sources (BSI-OS) due to CRE; control groups were patients with infection caused by carbapenem-susceptible Enterobacterales (CSE), and by non-infected patients, respectively. Matching criteria included type of infection for CSE group, ward and duration of hospital admission. Conditional logistic regression was used to identify risk factors. Findings Overall, 235 CRE case patients, 235 CSE controls and 705 non-infected controls were included. The CRE infections were cUTI (133, 56.7%), pneumonia (44, 18.7%), cIAI and BSI-OS (29, 12.3% each). Carbapenemase genes were found in 228 isolates: OXA-48/like, 112 (47.6%), KPC, 84 (35.7%), and metallo-beta-lactamases, 44 (18.7%); 13 produced two. The risk factors for CRE infection in both type of controls were (adjusted OR for CSE controls; 95% CI; p value) previous colonisation/infection by CRE (6.94; 2.74-15.53; <0.001), urinary catheter (1.78; 1.03-3.07; 0.038) and exposure to broad spectrum antibiotics, as categorical (2.20; 1.25-3.88; 0.006) and time-dependent (1.04 per day; 1.00-1.07; 0.014); chronic renal failure (2.81; 1.40-5.64; 0.004) and admission from home (0.44; 0.23-0.85; 0.014) were significant only for CSE controls. Subgroup analyses provided similar results. Interpretation The main risk factors for CRE infections in hospitals with high incidence included previous coloni-zation, urinary catheter and exposure to broad spectrum antibiotics

Deep tissue optical and optoacoustic molecular imaging technologies for small animal research and drug discovery.

For centuries, biological discoveries were based on optical imaging, in particular microscopy but also several chromophoric assays and photographic approaches. With the recent emergence of methods appropriate for bio-marker in vivo staining, such as bioluminescence, fluorescent molecular probes and proteins, as well as nanoparticle-based targeted agents, significant attention has been shifted toward in vivo interrogations of different dynamic biological processes at the molecular level. This progress has been largely supported by the development of advanced tomographic imaging technologies suitable for obtaining volumetric visualization of bio-marker distributions in small animals at a whole-body or whole-organ scale, an imaging frontier that is not accessible by the existing tissue-sectioning microscopic techniques due to intensive light scattering beyond the depth of a few hundred microns. Major examples of such recently developed optical imaging modalities are reviewed here, including bioluminescence tomography (BLT), fluorescence molecular tomography (FMT), and optical projection tomography (OPT). The pharmaceutical imaging community has quickly appropriated itself of these novel forms of optical imaging, since they come with very compelling advantages, such as quantitative three-dimensional capabilities, direct correlation to the biological cultures, easiness and cost-effectiveness of use, and the use of safe non-ionizing radiation. Some multi-modality approaches, combining light with other imaging modalities such as X-Ray CT or MRI, giving the ability to acquire both an optical contrast reconstruction along with a hi-fidelity anatomical images, are also reviewed. A separate section is devoted to the hybrid imaging techniques based on the optoacoustic phenomenon, such as multispectral optoacoustic tomography (MSOT), which are poised to leverage the traditional contrast and specificity advantages of optical spectrum by delivering an ever powerful set of capabilities, including real-time operation and high spatial resolution, not affected by the scattering nature of biological tissues

Un-mixing molecular agents from absorbing tissue in multispectral optoacoustic tomography.

Detection of intrinsic or extrinsically administered chromophores and photo-absorbing nanoparticles has been achieved by Multi-Spectral Optoacoustic Tomography (MSOT). The detection sensitivity of MSOT depends not only on the signal to noise ratio considerations, as in conventional optoacoustic (photoacoustic) tomography implementations, but also on the ability to resolve the molecular targets of interest from the absorbing tissue background by means of spectral un-mixing or sub-pixel detection methods. However, it is not known which un-mixing methods are optimally suited for the characteristics of multispectral optoacoustic images. In this work we investigated the performance of different sub-pixel detection methods, typically used in remote sensing hyperspectral imaging, within the context of MSOT. A quantitative comparison of the different algorithmic approaches was carried out in an effort to identify methods that operate optimally under the particulars of MSOT applications. We find that statistical sub-pixel detection methods can demonstrate a unique detection performance with up to five times enhanced sensitivity as compared to linear un-mixing approximations, under the condition that the optical agent of interest is sparsely present within the tissue volume, as common when using targeted agents and reporter genes