Fluorescence background quenching as a means to increase Signal to Background ratio - a proof of concept during Nerve Imaging

Introduction: Adequate signal to background ratios are critical for the implementation of fluorescence-guided surgery technologies. While local tracer administrations help to reduce the chance of systemic side effects, reduced spatial migration and non-specific tracer diffusion can impair the discrimination between the tissue of interest and the background. To combat background signals associated with local tracer administration, we explored a pretargeting concept aimed at quenching non-specific fluorescence signals. The efficacy of this concept was evaluated in an in vivo neuronal tracing set-up.Methods: Neuronal tracing was achieved using a wheat germ agglutinin (WGA) lectin. functionalized with an azide-containing Cy5 dye (N-3-Cy5-WGA). A Cy7 quencher dye (Cy7-DBCO) was subsequently used to yield Cy7-Cy5-WGA, a compound wherein the Cy5 emission is quenched by Forster resonance energy transfer to Cy7. The photophysical properties of N-3-Cy5-WGA and Cy7-Cy5-WGA were evaluated together with deactivation kinetics in situ, in vitro (Schwannoma cell culture), ex vivo (muscle tissue from mice; used for dose optimization), and in vivo (nervus ischiadicus in THY-1 YFP mice).Results: In situ, conjugation of Cy7-DBCO to N-3-Cy5-WGA resulted in >90% reduction of the Cy5 fluorescence signal intensity at 30 minutes after addition of the quencher. In cells, pretargeting with the N-3-Cy5-WGA lectin yielded membranous staining, which could efficiently be deactivated by Cy7-DBCO over the course of 30 minutes (91% Cy5 signal decrease). In ex vivo muscle tissue, administration of Cy7-DBCO at the site where N-3-Cy5-WGA was injected induced 80-90% quenching of the Cy5-related signal after 10-20 minutes, while the Cy7-related signal remained stable over time. In vivo, Cy7-DBCO effectively quenched the non-specific background signal up to 73% within 5 minutes, resulting in a 50% increase in the signal-to-background ratio between the nerve and injection site.Conclusion: The presented pretargeted fluorescence-quenching technology allowed fast and effective reduction of the background signal at the injection site, while preserving in vivo nerve visualization. While this proof-of-principle study was focused on imaging of nerves using a fluorescent WGA-lectin, the same concept could in the future also apply to applications such as sentinel node imaging

Health-related quality of life among persons with initial mild, moderate, and severe or critical COVID-19 at 1 and 12 months after infection: a prospective cohort study

BACKGROUND: Currently, there is limited evidence about the long-term impact on physical, social and emotional functioning, i.e. health-related quality of life (HRQL) after mild or moderate COVID-19 not requiring hospitalization. We compared HRQL among persons with initial mild, moderate or severe/critical COVID-19 at 1 and 12 months following illness onset with Dutch population norms and investigated the impact of restrictive public health control measures on HRQL. METHODS: RECoVERED, a prospective cohort study in Amsterdam, the Netherlands, enrolled adult participants after confirmed SARS-CoV-2 diagnosis. HRQL was assessed with the Medical Outcomes Study Short Form 36-item health survey (SF-36). SF-36 scores were converted to standard scores based on an age- and sex-matched representative reference sample of the Dutch population. Differences in HRQL over time were compared among persons with initial mild, moderate or severe/critical COVID-19 using mixed linear models adjusted for potential confounders. RESULTS: By December 2021, 349 persons were enrolled of whom 269 completed at least one SF-36 form (77%). One month after illness onset, HRQL was significantly below population norms on all SF-36 domains except general health and bodily pain among persons with mild COVID-19. After 12 months, persons with mild COVID-19 had HRQL within population norms, whereas persons with moderate or severe/critical COVID-19 had HRQL below population norms on more than half of the SF-36 domains. Dutch-origin participants had significantly better HRQL than participants with a migration background. Participants with three or more COVID-19 high-risk comorbidities had worse HRQL than part participants with fewer comorbidities. Participants who completed the SF-36 when restrictive public health control measures applied reported less limitations in social and physical functioning and less impaired mental health than participants who completed the SF-36 when no restrictive measures applied. CONCLUSIONS: Twelve months after illness onset, persons with initial mild COVID-19 had HRQL within population norms, whereas persons with initial moderate or severe/critical COVID-19 still had impaired HRQL. Having a migration background and a higher number of COVID-19 high-risk comorbidities were associated with worse HRQL. Interestingly, HRQL was less impaired during periods when restrictive public health control measures were in place compared to periods without. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s12916-022-02615-7

Inhibition of β2 Integrin–mediated Leukocyte Cell Adhesion

Many integrins mediate cell attachment to the extracellular matrix by recognizing short tripeptide sequences such as arginine–glycine–aspartic acid and leucine–aspartate–valine. Using phage display, we have now found that the leukocyte-specific b2 integrins bind sequences containing a leucine–leucine–glycine (LLG) tripeptide motif. An LLG motif is present on intercellular adhesion molecule (ICAM)-1, the major b2 integrin ligand, but also on several matrix proteins, including von Willebrand factor. We developed a novel b2 integrin antagonist peptide CPCFLLGCC (called LLG-C4), the structure of which was determined by nuclear magnetic resonance. The LLG-C4 peptide inhibited leukocyte adhesion to ICAM-1, and, interestingly, also to von Willebrand factor. When immobilized on plastic, the LLG-C4 sequence supported the b2 integrin–mediated leukocyte adhesion, but not b1 or b3 integrin–mediated cell adhesion. These results suggest that LLG sequences exposed on ICAM-1 and on von Willebrand factor at sites of vascular injury play a role in the binding of leukocytes, and LLG-C4 and peptidomimetics derived from it could provide a therapeutic approach to inflammatory reactions

Advancing intraoperative magnetic tracing using 3D freehand magnetic particle imaging

Purpose Sentinel lymph node biopsy is a routine procedure for nodal staging in penile cancer. Most commonly, this procedure is guided by radioactive tracers, providing various forms of preoperative and intraoperative guidance. This is further extended with fluorescence imaging using hybrid radioactive-fluorescence tracers. Alternatively, a magnetic-based approach has become available using superparamagnetic iron-oxide nanoparticles (SPIONs). This study investigates a novel freehand magnetic particle imaging and navigation modality (fhMPI) for intraoperative localization, along with a hybrid approach, combining magnetic and fluorescence guidance. Materials and methods The fhMPI set-up was built with a surgical navigation device, optical tracking system and magnetometer probe. A dedicated reconstruction software based on a look-up-table method was used to reconstruct a superficial 3D volume of the SPION distribution in tissue. For fluorescence guidance, indocyanine green (ICG) was added to the SPIONs. The fhMPI modality was characterized in phantoms, ex vivo human skin and in vivo porcine surgery. Results Phantom and human skin explants illustrated that the current fhMPI modality had a sensitivity of 2.2 x 10(-2) mg/mL SPIONs, a resolving power of at least 7 mm and a depth penetration up to 1.5 cm. Evaluation during porcine surgery showed that fhMPI allowed for an augmented reality image overlay of the tracer distribution in tissue, as well as 3D virtual navigation. Besides, using the hybrid approach, fluorescence imaging provided a visual confirmation of localized nodes. Conclusion fhMPI is feasible in vivo, providing 3D imaging and navigation for magnetic nanoparticles in the operating room, expanding the guidance possibilities during magnetic sentinel lymph node procedures. Furthermore, the integration of ICG provides the ability to visually refine and confirm correct localization. Further clinical evaluation should verify these findings in human patients as well.Imaging- and therapeutic targets in neoplastic and musculoskeletal inflammatory diseas

Middle Neolithic farming of open-air sites in SE France: new insights from archaeobotanical investigations of three wells found at Les Bagnoles (L'Isle-sur-la-Sorgue, Dépt. Vaucluse, France)

Previous reviews of Middle Neolithic agricultural practice (4400–3500 cal bc) in southern France have highlighted a change in crop assemblages after 4000 cal bc, with a reduction of naked wheat and an increase of emmer and partly of einkorn. The recent investigation of three wells from the site of Les Bagnoles (4250–3800 cal bc) in the periphery of the southern Rhône valley yielded an unprecedented amount of waterlogged uncharred and charred plant macro remains that offer new insights into crop diversity and its changes over time. The results from the wells at Les Bagnoles were compared with other dated sunken features from open-air sites (in contrast to caves and rock shelters), with the aim of identifying patterns sug-gesting changes in the crop spectra between the early (MN1) and late (MN2) Middle Neolithic phases from taphonomically comparable contexts. The results from Les Bagnoles demonstrate that oil crops and pulses are underrepresented in dry sites and that they were a significant part of Middle Neolithic agriculture. They also indicate an increase in the representation of einkorn (instead of emmer) during MN2 that is also visible in other open-air sites. The comparison of the archaeobotani-cal results with silo storage capacity values as a proxy for average production capacity per household leads us to propose a possible drop in naked wheat productivity and opens new questions in factors affecting crop choice at the beginning of the 4th millennium cal bc

Chemically augmented malaria sporozoites display an altered immunogenic profile

Despite promising results in malaria-naïve individuals, whole sporozoite (SPZ) vaccine efficacy in malaria-endemic settings has been suboptimal. Vaccine hypo-responsiveness due to previous malaria exposure has been posited as responsible, indicating the need for SPZ vaccines of increased immunogenicity. To this end, we here demonstrate a proof-of-concept for altering SPZ immunogenicity, where supramolecular chemistry enables chemical augmentation of the parasite surface with a TLR7 agonist-based adjuvant (SPZ-SAS(CL307)). In vitro, SPZ-SAS(CL307) remained well recognized by immune cells and induced a 35-fold increase in the production of pro-inflammatory IL-6 (p + cells 11.1 ± 1.8 vs. 9.4 ± 1.5%, p + T cells (4.7 ± 4.3 vs. 1.8 ± 0.7%, p + T cells (3.6 ± 1.4 vs. 2.5 ± 0.9%, p Host-parasite interactio

Response and survival of metastatic melanoma patients treated with immune checkpoint inhibition for recurrent disease on adjuvant dendritic cell vaccination

Vaccination with autologous dendritic cells (DC) loaded ex vivo with melanoma-associated antigens is currently being tested as an adjuvant treatment modality for resected locoregional metastatic (stage III) melanoma. Based on its mechanism of action, DC vaccination might potentiate the clinical efficacy of concurrent or sequential immune checkpoint inhibition (ICI). The purpose of this study was to determine the efficacy of ICI administered following recurrent disease during, or after, adjuvant DC vaccination. To this end, we retrospectively analyzed clinical responses of 51 melanoma patients with either irresectable stage III or stage IV disease treated with first- or second-line ICI following recurrence on adjuvant DC vaccination. Patients were analyzed according to the form of ICI administered: PD-1 inhibition monotherapy (nivolumab or pembrolizumab), ipilimumab monotherapy or combined treatment with ipilimumab and nivolumab. Treatment with first- or second-line PD-1 inhibition monotherapy after recurrence on adjuvant DC vaccination resulted in a response rate of 52%. In patients treated with ipilimumab monotherapy and ipilimumab-nivolumab response rates were 35% and 75%, respectively. In conclusion, ICI is effective in melanoma patients with recurrent disease on adjuvant DC vaccination