Self-assembled adipose-derived mesenchymal stem cells as an extracellular matrix component- and growth factor-enriched filler.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

Comparison between qPCR, VIDAS immunoassays, and agar streaking for the detection of Listeria monocytogenes from food and environmental surfaces containing and not containing Listeria innocua.

Comparisons among a qPCR assay, VIDAS® assays and a conventional agar streaking method following the same enrichment for the detection of Listeria monocytogenes were performed under two challenging conditions. In the first comparison, L. innocua and L. monocytogenes were coinoculated into sausages at ratios (L. innocua-to-L. monocytogenes) of 10, 100, 1000, and 10 000. qPCR provided the most sensitive detection at all ratios after both 24-h and 48-h enrichments. A modified VIDAS® LMO2 assay (i.e., replacement of the kit-specified enrichment scheme with the enrichment scheme used in this study) and agar streaking yielded equivalent results when the ratio was 10 and 100; agar streaking was more sensitive when the ratio was 1000; neither method could detect L. monocytogenes at the ratio of 10 000. Enrichment duration of 48 h was needed for modified VIDAS® to detect L. monocytogenes when the ratio was 1000. Agar streaking after 24-h enrichment isolated L. monocytogenes better than after 48-h enrichment when the ratio was 100 and 1000. In the second comparison, we followed the validation guidelines of AOAC International and inoculated L. monocytogenes, without any L. innocua, onto lettuce and stainless-steel surfaces at low levels. The numbers of positive samples detected by qPCR, VIDAS® LIS assay, modified VIDAS® LMO2 assay, and agar streaking after 48-h enrichment were not statistically different. Our data showed that qPCR was the most sensitive method, while agar streaking and VIDAS® performed reasonably well. Streaking after 24-h enrichment was needed when background flora could overgrow L. monocytogenes during prolonged enrichment, and this is critical for confirming rapid screening assays. Appropriate selection of enrichment duration and rapid assays will enhance the testing of L. monocytogenes in food and environmental samples.

Generation of novel oncolytic vaccinia virus with improved intravenous efficacy through protection against complement-mediated lysis and evasion of neutralization by vaccinia virus-specific antibodies.

BACKGROUND: Oncolytic virus immunotherapy has revolutionized cancer immunotherapy by efficiently inducing both oncolysis and systemic immune activation. Locoregional administration has been used for oncolytic virus therapy, but its applications to deep-seated cancers have been limited. Although systemic delivery of the oncolytic virus would maximize viral immunotherapy's potential, this remains a hurdle due to the rapid removal of the administered virus by the complement and innate immune system. Infected cells produce some vaccinia viruses as extracellular enveloped virions, which evade complement attack and achieve longer survival by expressing host complement regulatory proteins (CRPs) on the host-derived envelope. Here, we generated SJ-600 series oncolytic vaccinia viruses that can mimic complement-resistant extracellular enveloped virions by incorporating human CRP CD55 on the intracellular mature virion (IMV) membrane. METHODS: The N-terminus of the human CD55 protein was fused to the transmembrane domains of the six type I membrane proteins of the IMV; the resulting recombinant viruses were named SJ-600 series viruses. The SJ-600 series viruses also expressed human granulocyte-macrophage colony-stimulating factor (GM-CSF) to activate dendritic cells. The viral thymidine kinase (J2R) gene was replaced by genes encoding the CD55 fusion proteins and GM-CSF. RESULTS: SJ-600 series viruses expressing human CD55 on the IMV membrane showed resistance to serum virus neutralization. SJ-607 virus, which showed the highest CD55 expression and the highest resistance to serum complement-mediated lysis, exhibited superior anticancer activity in three human cancer xenograft models, compared with the control Pexa-Vec (JX-594) virus, after single-dose intravenous administration. The SJ-607 virus administration elicited neutralizing antibody formation in two immunocompetent mouse strains like the control JX-594 virus. Remarkably, we found that the SJ-607 virus evades neutralization by vaccinia virus-specific antibodies. CONCLUSION: Our new oncolytic vaccinia virus platform, which expresses human CD55 protein on its membrane, prolonged viral survival by protecting against complement-mediated lysis and by evading neutralization by vaccinia virus-specific antibodies; this may provide a continuous antitumor efficacy until a complete remission has been achieved. Such a platform may expand the target cancer profile to include deep-seated cancers and widespread metastatic cancers.

Oncolytic Vaccinia Virus Augments T Cell Factor 1-Positive Stem-like CD8+ T Cells, Which Underlies the Efficacy of Anti-PD-1 Combination Immunotherapy.

Oncolytic virotherapy has garnered attention as an antigen-agnostic therapeutic cancer vaccine that induces cancer-specific T cell responses without additional antigen loading. As anticancer immune responses are compromised by a lack of antigenicity and chronic immunosuppressive microenvironments, an effective immuno-oncology modality that converts cold tumors into hot tumors is crucial. To evaluate the immune-activating characteristics of oncolytic vaccinia virus (VACV; JX-594, pexastimogene devacirepvec), diverse murine syngeneic cancer models with different tissue types and immune microenvironments were used. Intratumorally administered mJX-594, a murine variant of JX-594, potently increased CD8+ T cells, including antigen-specific cancer CD8+ T cells, and decreased immunosuppressive cells irrespective of tissue type or therapeutic efficacy. Remodeling of tumors into inflamed ones by mJX-594 led to a response to combined anti-PD-1 treatment, but not to mJX-594 or anti-PD-1 monotherapy. mJX-594 treatment increased T cell factor 1-positive stem-like T cells among cancer-specific CD8+ T cells, and anti-PD-1 combination treatment further increased proliferation of these cells, which was important for therapeutic efficacy. The presence of functional cancer-specific CD8+ T cells in the spleen and bone marrow for an extended period, which proliferated upon encountering cancer antigen-loaded splenic dendritic cells, further indicated that long-term durable anticancer immunity was elicited by oncolytic VACV.

A Nanostructured Phthalocyanine/Albumin Supramolecular Assembly for Fluorescence Turn-On Imaging and Photodynamic Immunotherapy.

Smart phototheranostic nanomaterials are of significant interest for high-quality imaging and targeted therapy in the precision medicine field. Herein, a nanoscale photosensitizer (NanoPcM) is constructed through the self-assembly of morpholine-substituted silicon phthalocyanine (PcM) and albumin. NanoPcM displays a turn-on fluorescence depending on the acid-induced abolition of the photoinduced electron transfer effect (change in molecular structure) and disassembly of the nanostructure (change in supramolecular structure), which enables low-background and tumor-targeted fluorescence imaging. In addition, its efficient type I photoreaction endows NanoPcM with a superior immunogenic photodynamic therapy (PDT) effect against solid tumors. The combination of NanoPcM-based PDT and αPD-1-based immunotherapy can efficiently inhibit tumor growth, reduce spontaneous lung metastasis, and trigger abscopal effects. This study should provide a perspective for the future design of nanomaterials as promising phototheranostics for cancer imaging and therapy.

High-resolution microscopic diffusion anisotropy imaging in the human hippocampus at 3T.

PURPOSE: Several neurological conditions are associated with microstructural changes in the hippocampus that can be observed using DWI. Imaging studies often use protocols with whole-brain coverage, imposing limits on image resolution and worsening partial-volume effects. Also, conventional single-diffusion-encoding methods confound microscopic diffusion anisotropy with size variance of microscopic diffusion environments. This study addresses these issues by implementing a multidimensional diffusion-encoding protocol for microstructural imaging of the hippocampus at high resolution. METHODS: The hippocampus of 8 healthy volunteers was imaged at 1.5-mm isotropic resolution with a multidimensional diffusion-encoding sequence developed in house. Microscopic fractional anisotropy (µFA) and normalized size variance (CMD ) were estimated using q-space trajectory imaging, and their values were compared with DTI metrics. The overall scan time was 1 hour. The reproducibility of the protocol was confirmed with scan-rescan experiments, and a shorter protocol (14 minutes) was defined for situations with time constraints. RESULTS: Mean µFA (0.47) was greater than mean FA (0.20), indicating orientation dispersion in hippocampal tissue microstructure. Mean CMD was 0.17. The reproducibility of q-space trajectory imaging metrics was comparable to DTI, and microstructural metrics in the healthy hippocampus are reported. CONCLUSION: This work shows the feasibility of high-resolution microscopic anisotropy imaging in the human hippocampus at 3 T and provides reference values for microstructural metrics in a healthy hippocampus.

Comparison of three neutralizing broths for environmental sampling of low levels of Listeria monocytogenes desiccated on stainless steel surfaces and exposed to quaternary ammonium compounds.

BACKGROUND: An effective environmental sampling method involves the use of a transport/neutralizing broth with the ability to neutralize sanitizer residues that are collected during sampling and to maintain viability of stressed Listeria monocytogenes (Lm) cells. RESULTS: We applied Lm onto stainless steel surfaces and then subjected Lm to desiccation stress for 16-18 h at room temperature (RT, 21-24 °C). This was followed by the subsequent application of Whisper™ V, a quaternary ammonium compound (QAC)-based sanitizer, diluted to 400 ppm and 8000 ppm of active quat, for 6 h. We then sampled Lm with sponges pre-moistened in three transport broths, Dey/Engley (D/E) broth, Letheen broth and HiCap™ broth, to generate environmental samples that contained sanitizer residues and low levels of stressed Lm, which were subsequently analyzed by an enrichment-based method. This scheme conformed with validation guidelines of AOAC International by using 20 environmental test portions per broth that contained low levels of Lm such that not all test portions were positive (i.e., fractional positive). We showed that D/E broth, Letheen broth and HiCap™ broth performed similarly when no quat or 400 ppm of quat was applied to the Lm contaminating stainless steel surfaces. However, when 8000 ppm of quat was applied, Letheen broth did not effectively neutralize the QAC in the samples. These comparisons were performed on samples stored under three conditions after collection to replicate scenarios of sample transport, RT for 2 h, 4 °C for 24 h and 4 °C for 72 h. Comparisons under the three different scenarios generally reached the same conclusions. In addition, we further demonstrated that storing Letheen and HiCap™ broths at RT for two months before sampling did not reduce their capacity to neutralize sanitizers. CONCLUSIONS: We developed a scheme to evaluate the ability of transport broths to neutralize QAC sanitizers. The three transport broths performed similarly with a commonly used concentration of quat, but Letheen broth could not effectively neutralize a very high concentration of QAC. The performance of transport broths was not significantly affected under the assessed pre-sampling and post-sampling storage conditions.

Ultrasonographic evaluation of sural nerve for nerve conduction study.

OBJECTIVE: To understand various morphologic types and locations of the sural nerve (SN) that are important for nerve conduction studies or nerve grafting procedures. The aim of this study was to describe the course and variations of the SN based on ultrasonographic findings for an adequate nerve conduction study. METHODS: A total of 112 SNs in 56 volunteers with no history of trauma or surgery were examined by ultrasonography. The location and formation of the SNs in relation to the medial and lateral sural cutaneous nerve were investigated. We measured the horizontal distance between the SNs and the midline of the calf at the level of 14 cm from the lateral malleolus, and the distance between the SNs and the most prominent part of the lateral malleolus. RESULTS: SN variants was classified into four types according to the medial and lateral sural cutaneous nerve; type 1 (73.2%), type 2 (17.9%), type 3 (8.0%), and type 4 (0.9%). The mean distance between the SN and the midline of the calf was 1.02±0.63 cm, the SN and the most prominent part of the lateral malleolus was 2.14±0.15 cm. CONCLUSION: Variations in the location and formation of the SN was examined by ultrasonography, and the results of this study would increase the accuracy of the SN conduction study.