MHC Class I Enables MSCs to Evade NK-Cell-Mediated Cytotoxicity and Exert Immunosuppressive Activity.

Allogeneic mesenchymal stem/stromal cells (MSCs) are frequently used in clinical trials due to their low expression of major histocompatibility complex (MHC) class I and lack of MHC class II. However, the levels of MHC classes I and II in MSCs are increased by inflammatory stimuli, raising concerns over potential adverse effects associated with allogeneic cell therapy. Also, it is unclear how the host immune response to MHC-mismatched MSCs affects the therapeutic efficacy of the cells. Herein, using strategies to manipulate MHC genes in human bone marrow-derived MSCs via the CRISPR-Cas9 system, plasmids, or siRNAs, we found that inhibition of MHC class I-not MHC class II-in MSCs lowered the survival rate of MSCs and their immunosuppressive potency in mice with experimental autoimmune uveoretinitis, specifically by increasing MSC vulnerability to natural killer (NK)-cell-mediated cytotoxicity. A subsequent survey of MSC batches derived from 6 human donors confirmed a significant correlation between MSC survival rate and susceptibility to NK cells with the potency of MSCs to increase MHC class I level upon stimulation. Our overall results demonstrate that MHC class I enables MSCs to evade NK-cell-mediated cytotoxicity and exert immunosuppressive activity.

Multi-Threaded Sound Propagation Algorithm to Improve Performance on Mobile Devices.

We propose a multi-threaded algorithm that can improve the performance of geometric acoustic (GA)-based sound propagation algorithms in mobile devices. In general, sound propagation algorithms require high computational cost because they perform based on ray tracing algorithms. For this reason, it is difficult to operate sound propagation algorithms in mobile environments. To solve this problem, we processed the early reflection and late reverberation steps in parallel and verified the performance in four scenes based on eight sound sources. The experimental results showed that the performance of the proposed method was on average 1.77 times better than that of the single-threaded method, demonstrating that our algorithm can improve the performance of mobile devices.

Flow Characteristics of the Conjugate of Anti-CD3 Monoclonal Antibodies and Magnetic Nanoparticle in PBS and Blood Vessels.

Previously, anti-CD3 antibodies delivered intravenously have been known for their negative side effects. The experimental conditions for optimal liquid production are derived from the Fc-directed conjugation of anti-CD3 foralumab antibodies and magnetic nanoparticles (Ab-MNPs). The anti-CD3 antibodies are prepared for conjugation with MNPs using SiteClick antibody labelling kits. The successful conjugation of the Ab-MNPs is confirmed using a transmission electron microscopy (TEM) image and an energy dispersive spectroscopy (EDS) analysis. The average values ​​of the moving speed of MNPs and Ab-MNPs in phosphate buffer saline (PBS) were + 3.16 pix/frame and + 6.70 pix/frame in the x-axis, respectively. This implies that MNPs with CD3 antibodies attached to the surface through biocompatible ligand functional groups has better fluidity in PBS. Afterwards, a non-clinical animal testing for the flow characteristics of Ab-MNPs inside a blood vessel is carried out to observe the effects of Ab-MNP delivery through intravenous injection.

Double-Edged Network Effects on Disclosing Traumatic Experiences Among Korean "Comfort Women".

This study examines the effects of social networks on the disclosure of stigmatizing and traumatic sexual assault experiences. We analyzed publicly archived oral histories of Korean "comfort women" from World War II, employing an innovative method combining word embedding analysis, word frequency comparison, and grounded theory. By extracting their significant social relationships from narrated survivor stories, we parsed two distinctive disclosure patterns according to timing of disclosure: early disclosers and late disclosers. The latter were more socially embedded than the former, indicating the constraining aspect of social networks, in which the size of social networks was positively associated with delayed disclosure. Qualitative findings further elaborated that social networks have double-edged effects. Survivors' familial networks functioned as both social constraints and social support for public disclosure. Yet, the late disclosers tend to exploit it more as constraints for the fear of transgenerational transmission of social scorn and stigma. The findings contribute to enhancing a culturally relevant understanding of trauma and the repercussions of human trafficking.

The change of antibiotic susceptibility in febrile urinary tract infection in childhood and adolescence during the last decade.

PURPOSE: The purpose of this study was to clarify the pattern of antibiotic resistance in pediatric urinary tract infections (UTIs). MATERIALS AND METHODS: We analyzed the data of entire urine culture tests and antibiotic susceptibility tests performed on hospitalized patients for febrile UTI at the Uijeongbu St. Mary's Hospital during 2010-2020. A retrospective analysis was performed using medical records of urine culture results and antibiotic susceptibility results in patients with UTIs. RESULTS: We performed urine cultures from 2,491 patients, and identified bacterial types in 1,651 cases. We found that the resistance rates to ampicillin, ampicillin/sulbactam, cefazolin, gentamicin, piperacillin, tobramycin, and trimethoprim/sulfamethoxazole were already over 20% in 2010. The resistance rates to many other antibiotics also steadily increased over time. Among the antibiotics tested in 2020, only amikacin, cefoxitin, imipenem, piperacillin/tazobactam, and tigecycline showed the resistance rates below 20%. Noticeably, ciprofloxacin also showed an increase in the resistance rate from 7.3% in 2010 (S 139 vs. R 11) to 27.78% in 2019 (S 104 vs. R 40) and even over 30% (33.96%) in 2020 (S 35 vs. R 18). CONCLUSIONS: Antibiotic resistance is a serious problem in pediatric UTIs. In the treatment of pediatric UTIs, more caution is needed in the use of antibiotics. It may be necessary to apply appropriate antibiotic management programs such as antibiotics steward program for pediatric patients. Failure of a proper response strategy coping with antibiotic resistance may accelerate the resistance crisis.

Mesenchymal stromal cells for corneal transplantation: Literature review and suggestions for successful clinical trials.

Corneal transplantation is a routine procedure for patients with corneal blindness. Despite the streamlining of surgical techniques and deeper understanding of the cellular and molecular pathways mediating rejection, corticosteroids are still the main immunosuppressive regimen in corneal transplantation, and the 15-year survival of corneal transplants remains as low as 50%, which is poorer than that for most solid organ transplants. Recently, mesenchymal stromal cells (MSCs) with unique regenerative and immune-modulating properties have emerged as a promising cell therapy to promote transplant tolerance, minimize the use of immunosuppressants, and prevent chronic rejection. Here, we review the literature on preclinical studies of MSCs for corneal transplantation and summarize the key findings from clinical trials with MSCs in solid organ transplantation. Finally, we highlight current issues and challenges regarding MSC therapies and suggest strategies for safe and effective MSC-based therapies in clinical transplantation.

Multiscale Assay of Unlabeled Neurite Dynamics Using Phase Imaging with Computational Specificity.

Primary neuronal cultures have been widely used to study neuronal morphology, neurophysiology, neurodegenerative processes, and molecular mechanism of synaptic plasticity underlying learning and memory. However, the unique behavioral properties of neurons make them challenging to study, with phenotypic differences expressed as subtle changes in neuronal arborization rather than easy-to-assay features such as cell count. The need to analyze morphology, growth, and intracellular transport has motivated the development of increasingly sophisticated microscopes and image analysis techniques. Due to its high-contrast, high-specificity output, many assays rely on confocal fluorescence microscopy, genetic methods, or antibody staining techniques. These approaches often limit the ability to measure quantitatively dynamic activity such as intracellular transport and growth. In this work, we describe a method for label-free live-cell cell imaging with antibody staining specificity by estimating the associated fluorescence signals via quantitative phase imaging and deep convolutional neural networks. This computationally inferred fluorescence image is then used to generate a semantic segmentation map, annotating subcellular compartments of live unlabeled neural cultures. These synthetic fluorescence maps were further applied to study the time-lapse development of hippocampal neurons, highlighting the relationships between the cellular dry mass production and the dynamic transport activity within the nucleus and neurites. Our implementation provides a high-throughput strategy to analyze neural network arborization dynamically, with high specificity and without the typical phototoxicity and photobleaching limitations associated with fluorescent markers.

Bacterially Delivered miRNA-Mediated Toll-like Receptor 8 Gene Silencing for Combined Therapy in a Murine Model of Atopic Dermatitis: Therapeutic Effect of miRTLR8 in AD.

In atopic dermatitis (AD), skin inflammation is caused by complex interactions between genetic disposition and aberrant innate/adaptive immune responses. Toll-like receptors (TLRs) are key molecules in the innate/adaptive immune response as they recognize various molecular motifs associated with pathogens. Among them, TLR8 is implicated in eczematous skin reactions. We investigated the combined therapeutic effects of TLR8 gene silencing by the bacterial delivery of miRNA. We used Salmonella as a vector to deliver TLR8 miRNA. The recombinant strain of Salmonella enterica subsp. enterica serovar Typhimurium (ST) expressing TLR8 miRNA (ST-miRTLR8) was prepared for knockdown of TLR8. After oral administration of ST-miRTLR8 into mice, we observed the cytokine levels, skin pathology and scratching behaviors in an AD-like mouse model. TLR8 down-regulation decreased macrophage-derived chemokine concentrations in activated human mast cells. Serum IgE and interleukin-4 production were suppressed whereas IFN-γ was induced after oral administration of ST-miRTLR8. Scratching behaviors and skin inflammation were also improved. In addition, attenuated S. typhimurium safely accumulated in mouse macrophages and showed adjuvant effects. This study shows that the recombinant miRNA that expresses the TLR8 miRNA has therapeutic effects by suppressing Th2 inflammation. TLR gene modulation using miRNA via Salmonella vectors will thus have a double-protective effect in the treatment of AD.

Metabolic Evidence Rather Than Amounts of Red or Processed Meat as a Risk on Korean Colorectal Cancer.

The incidence of colorectal cancer (CRC) has increased in Korea, a newly-industrialized Asian country, with the dramatic increase of meat intake. To assess the risks of red or processed meat consumption on CRC, we performed a case-control study with biological monitoring of urinary1-OHP, PhIP, and MeIQx for the meat exposure; dG-C8 MeIQx and dG-C8 PhIP for HCA-induced DNA adducts; and homocysteine and C-reactive protein (CRP) in blood as well as malondialdehyde (MDA) and 31fatty acids in urine for inflammation and lipid alteration. We further analyzed global DNA methylation and expression of 15 CRC-related genes. As a result, the consumption of red or processed meat was not higher in the cases than in the controls. However, urinary MeIQx and PhIP were associated with the intake of red meat and urinary 1-OHP. MDA and multiple fatty acids were related to the exposure biomarkers. Most of the 31 fatty acids and multiple saturated fatty acids were higher in the cases than in the controls. Finally, the cases showed upregulation of PTGS2, which is related to pro-inflammatory fatty acids. This study describes indirect mechanisms of CRC via lipid alteration with a series of processes including exposure to red meat, alteration of fatty acids, and relevant gene expression.

Corneal nerve healing after in situ laser nerve transection.

PURPOSE: We have previously reported that lamellar dissection of the cornea transects stromal nerves, and that regenerating neurites form a dense net along the surgical plane. In these experiments, we have disrupted the stromal nerve trunks in situ, without incising the cornea, to determine the regeneration events in the absence of a surgical plane. METHODS: Thy1-YFP mice were anesthetized and in vivo images of the corneal nerves were obtained with a wide-field stereofluorescent microscope. A far infrared XYRCOS Laser attached to 20X objective of an upright microscope was used to perform in situ transection of the stromal nerves. 3 types of laser transections were performed (n = 5/group): (i) point transection (a single cut); (ii) segmental transection (two cuts enclosing a segment of nerve trunk); and (iii) annular transection (cuts on all nerve trunks crossing the perimeter of a 0.8 mm diameter circular area centered on the corneal apex). Mice were imaged sequentially for 4 weeks thereafter to assess nerve degeneration (disappearance or weakening of original fluorescence intensity) or regeneration (appearance of new fluorescent fronds). Beta-3-tubulin immunostaining was performed on corneal whole-mounts to demonstrate nerve disruption. RESULTS: The pattern of stromal nerves in corneas of the same mouse and in corneas of littermates was dissimilar. Two distinct patterns were observed, often within the same cornea: (i) interconnected trunks that spanned limbus to limbus; or (ii) dichotomously branching trunks that terminate at the corneal apex. Point transections did not cause degeneration of proximal or distal segment in interconnected trunks, but resulted in degeneration of distal segment of branching trunks. In segmental transections, the nerve segment enclosed within the two laser cuts degenerated. Lack of beta-3 tubulin staining at transection site confirmed nerve transection. In interconnected trunks, at 4 weeks, a hyperfluorescent plaque filled the gap created by the transection. In annular transections, some nerve trunks degenerated, while others regained or retained fluorescence. CONCLUSIONS: Interconnected stromal nerves in murine corneas do not degenerate after in situ point transection and show evidence of healing at the site of disruption. Presence or absence of a surgical plane influences corneal nerve regeneration after transection.

Hyperosmolar stress induces neutrophil extracellular trap formation: implications for dry eye disease.

PURPOSE: To determine if hyperosmolar stress can stimulate human neutrophils to form neutrophil extracellular traps (NETs) and to investigate potential strategies to reduce formation of NETs (NETosis) in a hyperosmolar environment. METHODS: Neutrophils were isolated from peripheral venous blood of healthy subjects and incubated in iso-osmolar (280 mOsM) or hyperosmolar (420 mOsM) media for 4 hours. Neutrophil extracellular traps were quantified using a PicoGreen dye assay to measure extracellular DNA. Two known inhibitors of NETosis, staurosporine and anti-ß2 integrin blocking antibody, and two proresolution formyl peptide receptor 2 (FPR2) agonists, annexin/lipocortin-1 mimetic peptide and 15-epi-lipoxin A4, were evaluated as possible strategies to reduce hyperosmolarity-induced NETosis. RESULTS: The amount of NETs induced by hyperosmolar medium (420 mOsM) increased linearly over time to 3.2 ± 0.3 times that induced by iso-osmolar medium at 4 hours (P < 0.05). NETosis increased exponentially with increasing osmolarity and was independent of the stimulus used to increase osmolarity. Upon neutrophil exposure to hyperosmolar stress, restoration of iso-osmolar conditions decreased NET formation by 52.7% ± 5% (P < 0.05) but did not completely abrogate it. Among the strategies tested to reduce NETosis in a hyperosmolar environment, annexin-1 peptide was the most efficacious. CONCLUSIONS: Hyperosmolarity induces formation of NETs by neutrophils. This NETosis mechanism may explain the presence of excessive NETs on the ocular surface of patients with dry eye disease. Because they reduce hyperosmolarity-induced NETosis, FPR2 agonists may have therapeutic potential in these patients.

Keratocytes derived from spheroid culture of corneal stromal cells resemble tissue resident keratocytes.

PURPOSE: Corneal stromal cells transform to precursor cells in spheroid culture. We determined whether keratocytes derived from spheroid culture of murine corneal stromal cells resemble tissue resident keratocytes. METHODS: Spheroid culture was performed by seeding dissociated stromal cells onto ultra-low attachment plates containing serum-free mesenchymal stem cell culture medium. Spheroids were characterized with phenotype specific markers and stemness transcription factor genes. Spheroids and adherent cells in culture were induced to differentiate to keratocytes using keratocyte induction medium (KIM) and compared with tissue resident keratocytes. RESULTS: Stromal cells formed spheroids in ultra-low attachment plates, but not in polystyrene tissue culture dishes. Keratocan expression and abundance was significantly higher in spheroids as compared to adherent cells whereas alpha-smooth muscle actin (α-SMA) was significantly lower. As compared to adherent culture-derived cells, the expressions of keratocan, aldehyde dehydrogenase (ALDH3A1) and α-SMA in spheroid-derived cells approximated much more closely the levels of these genes in tissue resident keratocytes. Of the stemness genes, Nanog and Oct4 were upregulated in the spheroids. CONCLUSION: Stemness transcription factor genes are upregulated in spheroids. Keratocytes derived from spheroids resemble tissue resident keratocytes, thus increasing manifolds the quantity of these cells for in-vitro experiments.