The Influence of Time of Day of Vaccination with BNT162b2 on the Adverse Drug Reactions and Efficacy of Humoral Response against SARS-CoV-2 in an Observational Study of Young Adults.

An increasing body of evidence from both academic and clinical studies shows that time-of-day exposure to antigens might significantly alter and modulate the development of adaptive immune responses. Considering the immense impact of the COVID-19 pandemic on global health and the diminished efficacy of vaccination in selected populations, such as older and immunocompromised patients, it is critical to search for the most optimal conditions for mounting immune responses against SARS-CoV-2. Hence, we conducted an observational study on 435 healthy young adults vaccinated with two doses of BNT162b2 (Pfizer-BioNTech) vaccine to determine whether time-of-day of vaccination influences either the magnitude of humoral response or number of adverse drug reactions (ADR) being reported. We found no significant differences between morning and afternoon vaccination in terms of both titers of anti-Spike antibodies and frequency of ADR in the studied population. In addition, our analysis of data on the occurrence of ADR in 1324 subjects demonstrated that the second administration of vaccine in those with previous SARS-CoV-2 infection was associated with lower incidence of ADR. In aggregate, vaccination against COVID-19 with two doses of BNT162b2 mRNA vaccine is presumed to generate an equally efficient anti-Spike humoral response.

Inhibition of arginase modulates T-cell response in the tumor microenvironment of lung carcinoma.

Immunotherapy has demonstrated significant activity in a broad range of cancer types, but still the majority of patients receiving it do not maintain durable therapeutic responses. Amino acid metabolism has been proposed to be involved in the regulation of immune response. Here, we investigated in detail the role of arginase 1 (Arg1) in the modulation of antitumor immune response against poorly immunogenic Lewis lung carcinoma. We observed that tumor progression is associated with an incremental increase in the number of Arg1+ myeloid cells that accumulate in the tumor microenvironment and cause systemic depletion of ʟ-arginine. In advanced tumors, the systemic concentrations of ʟ-arginine are decreased to levels that impair the proliferation of antigen-specific T-cells. Systemic or myeloid-specific Arg1 deletion improves antigen-induced proliferation of adoptively transferred T-cells and leads to inhibition of tumor growth. Arginase inhibitor was demonstrated to modestly inhibit tumor growth when used alone, and to potentiate antitumor effects of anti-PD-1 monoclonal antibodies and STING agonist. The effectiveness of the combination immunotherapy was insufficient to induce complete antitumor responses, but was significantly better than treatment with the checkpoint inhibitor alone. Together, these results indicate that arginase inhibition alone is of modest therapeutic benefit in poorly immunogenic tumors; however, in combination with other treatment strategies it may significantly improve survival outcomes.

Can Developments in Tissue Optical Clearing Aid Super-Resolution Microscopy Imaging?

The rapid development of super-resolution microscopy (SRM) techniques opens new avenues to examine cell and tissue details at a nanometer scale. Due to compatibility with specific labelling approaches, in vivo imaging and the relative ease of sample preparation, SRM appears to be a valuable alternative to laborious electron microscopy techniques. SRM, however, is not free from drawbacks, with the rapid quenching of the fluorescence signal, sensitivity to spherical aberrations and light scattering that typically limits imaging depth up to few micrometers being the most pronounced ones. Recently presented and robustly optimized sets of tissue optical clearing (TOC) techniques turn biological specimens transparent, which greatly increases the tissue thickness that is available for imaging without loss of resolution. Hence, SRM and TOC are naturally synergistic techniques, and a proper combination of these might promptly reveal the three-dimensional structure of entire organs with nanometer resolution. As such, an effort to introduce large-scale volumetric SRM has already started; in this review, we discuss TOC approaches that might be favorable during the preparation of SRM samples. Thus, special emphasis is put on TOC methods that enhance the preservation of fluorescence intensity, offer the homogenous distribution of molecular probes, and vastly decrease spherical aberrations. Finally, we review examples of studies in which both SRM and TOC were successfully applied to study biological systems.

Assessing functional status of cardiac lymphatics: From macroscopic imaging to molecular profiling.

Here we describe various techniques for visualization of the lymphatic vasculature, particularly in the heart. Addressing macro-, microscopic, and molecular levels of lymphatic organization, we give examples of how to explore the roles of specific antigens/markers expressed in lymphatic vessels and their extracellular matrix as structural and functional elements involved in various biological functions of lymphatics. Some obstacles and technical challenges related to lymphatic visualization are also discussed.

Myeloid Cell-Derived Arginase in Cancer Immune Response.

Amino acid metabolism is a critical regulator of the immune response, and its modulating becomes a promising approach in various forms of immunotherapy. Insufficient concentrations of essential amino acids restrict T-cells activation and proliferation. However, only arginases, that degrade L-arginine, as well as enzymes that hydrolyze L-tryptophan are substantially increased in cancer. Two arginase isoforms, ARG1 and ARG2, have been found to be present in tumors and their increased activity usually correlates with more advanced disease and worse clinical prognosis. Nearly all types of myeloid cells were reported to produce arginases and the increased numbers of various populations of myeloid-derived suppressor cells and macrophages correlate with inferior clinical outcomes of cancer patients. Here, we describe the role of arginases produced by myeloid cells in regulating various populations of immune cells, discuss molecular mechanisms of immunoregulatory processes involving L-arginine metabolism and outline therapeutic approaches to mitigate the negative effects of arginases on antitumor immune response. Development of potent arginase inhibitors, with improved pharmacokinetic properties, may lead to the elaboration of novel therapeutic strategies based on targeting immunoregulatory pathways controlled by L-arginine degradation.

Tissue clearing-based method for unobstructed three-dimensional imaging of mouse penis with subcellular resolution.

Although mice are widely used to elucidate factors contributing to penile disorders and develop treatment options, quantification of tissue changes upon intervention is either limited to minuscule tissue volume (histology) or acquired with limited spatial resolution (MRI/CT). Thus, imaging method suitable for expeditious acquisition of the entire mouse penis with subcellular resolution is described that relies on both aqueous- (clear, unobstructed brain imaging cocktails and computational analysis) and solvent-based (fluorescence-preserving capability imaging of solvent-cleared organs) tissue optical clearing (TOC). The combined TOC approach allows to image mouse penis innervation and vasculature with unprecedented detail and, for the first time, reveals the three-dimensional structure of murine penis fibrocartilage.

Systematic Evaluation of Chemically Distinct Tissue Optical Clearing Techniques in Murine Lymph Nodes.

Activation of adaptive immunity is a complex process coordinated at multiple levels in both time and the three-dimensional context of reactive lymph nodes (LNs). Although microscopy-based visualization of its spatiotemporal dynamics unravels complexities of developing immune response, such approach is highly limited by light-obstructing nature of tissue components. Recently, tissue optical clearing (TOC) techniques were established to bypass this obstacle and now allow to image and quantify the entire murine organs with cellular resolution. However, the spectrum of TOC is represented by wide variety of chemically distinct methods, each having certain advantages and disadvantages that were unsatisfactorily compared for suitability to LNs clearing. In this study, we have systematically tested 13 typical TOC techniques and assessed their impact on a number of critical factors such as LN transparency, imaging depth, change in size, compatibility with proteinaceous fluorophores, immunostaining, H&E staining, and light-sheet fluorescence microscopy. Based on the detailed data specific to TOC process of murine LNs, we provide a reliable reference for most suitable methods in an application-dependent manner.

Total Absence of Dystrophin Expression Exacerbates Ectopic Myofiber Calcification and Fibrosis and Alters Macrophage Infiltration Patterns.

Duchenne muscular dystrophy (DMD) causes severe disability and death of young men because of progressive muscle degeneration aggravated by sterile inflammation. DMD is also associated with cognitive and bone-function impairments. This complex phenotype results from the cumulative loss of a spectrum of dystrophin isoforms expressed from the largest human gene. Although there is evidence for the loss of shorter isoforms having impact in the central nervous system, their role in muscle is unclear. We found that at 8 weeks, the active phase of pathology in dystrophic mice, dystrophin-null mice (mdxßgeo) presented with a mildly exacerbated phenotype but without an earlier onset, increased serum creatine kinase levels, or decreased muscle strength. However, at 12 months, mdxßgeo diaphragm strength was lower, whereas fibrosis increased, compared with mdx. The most striking features of the dystrophin-null phenotype were increased ectopic myofiber calcification and altered macrophage infiltration patterns, particularly the close association of macrophages with calcified fibers. Ectopic calcification had the same temporal pattern of presentation and resolution in mdxßgeo and mdx muscles, despite significant intensity differences across muscle groups. Comparison of the rare dystrophin-null patients against those with mutations affecting full-length dystrophins may provide mechanistic insights for developing more effective treatments for DMD.

Whole-body clearing, staining and screening of calcium deposits in the mdx mouse model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal, X-linked genetic disorder. Although DMD is the most common form of muscular dystrophy, only two FDA-approved drugs were developed to delay its progression. In order to assess therapies for treating DMD, several murine models have recently been introduced. As the wide variety of murine models enlighten mechanisms underlying DMD pathology, the question on how to monitor the progression of the disease within the entire musculoskeletal system still remains to be answered. One considerable approach to monitor such progression is histological evaluation of calcium deposits within muscle biopsies. Although accurate, histology is limited to small tissue area and cannot be utilized to evaluate systemic progression of DMD. Therefore, we aimed to develop a methodology suitable for rapid and high-resolution screening of calcium deposits within the entire murine organism. METHODS: Procedures were performed on adult male C57BL/10-mdx and adult male C57BL mice. Animals were sacrificed, perfused, paraformaldehyde-fixed, and subjected to whole-body clearing using optimized perfusion-based CUBIC protocol. Next, cleared organisms were stained with alizarin red S to visualize calcium deposits and subjected to imaging. RESULTS: Study revealed presence of calcium deposits within degenerated muscles of the entire C57BL/10-mdx mouse organism. Calcified deposits were observed within skeletal muscles of the forelimb, diaphragm, lumbar region, pelvic region, and hindlimb. Calcified deposits found in quadriceps femoris, triceps brachii, and spinalis pars lumborum were characterized. Analysis of cumulative frequency distribution showed different distribution characteristics of calcified deposits in quadriceps femoris muscle in comparison to triceps brachii and spinalis pars lumborum muscles (p < 0.001) and quadriceps femoris vs spinalis pars lumborum (p < 0.001). Differences between the number of calcified deposits in selected muscles, their volume, and average volume were statistically significant. CONCLUSIONS: In aggregate, we present new methodology to monitor calcium deposits in situ in the mouse model of Duchenne muscular dystrophy. Sample imaging with the presented setup is feasible and applicable for whole-organ/body imaging. Accompanied by the development of custom-made LSFM apparatus, it allows targeted and precise characterization of calcium deposits in cleared muscles. Hence, presented approach might be broadly utilized to monitor degree to which muscles of the entire organism are affected by the necrosis and how is it altered by the treatment or physical activity of the animal. We believe that this would be a valuable tool for studying organs alternations in a wide group of animal models of muscle dystrophy and bone-oriented diseases.

Optimized perfusion-based CUBIC protocol for the efficient whole-body clearing and imaging of rat organs.

Whole-organ and whole-body optical tissue clearing methods allowing imaging in 3 dimensions are an area of profound research interest. Originally developed to study nervous tissue, they have been successfully applied to all murine organs, yet clearing and imaging of rat peripheral organs is less advanced. Here, a modification of CUBIC clearing protocol is presented. It provides a rapid and simple approach to clear the entire adult rat organism and thus all organs within as little as 4 days. Upgraded perfusion-based rat CUBIC protocol preserves both anatomical structure of organs and signal from proteinaceous fluorophores, and furthermore is compatible with antibody staining. Finally, it enables also volumetric cells analyses and is tailored for staining of calcium deposits within unsectioned soft tissues.

Shrinkage-mediated imaging of entire organs and organisms using uDISCO.

Recent tissue-clearing approaches have become important alternatives to standard histology approaches. However, light scattering in thick tissues and the size restrictions on samples that can be imaged with standard light-sheet microscopy pose limitations for analyzing large samples such as an entire rodent body. We developed 'ultimate DISCO' (uDISCO) clearing to overcome these limitations in volumetric imaging. uDISCO preserves fluorescent proteins over months and renders intact organs and rodent bodies transparent while reducing their size up to 65%. We used uDISCO to image neuronal connections and vasculature from head to toe over 7 cm and to perform unbiased screening of transplanted stem cells within the entire body of adult mice. uDISCO is compatible with diverse labeling methods and archival human tissue, and it can readily be used in various biomedical applications to study organization of large organ systems throughout entire organisms.

Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.

Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy.