Cell Squeeze: driving more effective CD8 T-cell activation through cytosolic antigen delivery.

Cell Squeeze is a novel technology that relies on temporarily disrupting the cell membrane to deliver cargo directly into the cytosol. This approach is applicable to a broad range of cell types (peripheral blood mononuclear cells, red blood cells, hematopoietic stem cells, etc.) and cargos (peptides, proteins, small molecules, nucleic acids, and gene-editing complexes) while minimally disrupting normal cell function. By enabling direct cytosolic delivery, one can use this technology to dramatically enhance major histocompatibility complex (MHC) class I presentation of antigens (Ags) for CD8+ T-cell activation-a longstanding challenge for the therapeutic cancer vaccine field that has generally relied on cross-presentation of endocytosed Ags. In addition, by coupling improved MHC class I presentation with coexpression of additional stimulatory factors or systemic immune modulators, one can further enhance the potential impact of an antitumor CD8 response. Pursuing a more direct cellular engineering strategy, which is independent of viral transduction, genetic manipulation, and expansion steps, enables <24 h manufacturing of autologous cell therapies. Through generation of more sophisticated, multifunctional, cell-based vaccines, clinical testing of this technology will elucidate its potential for impact across multiple tumor types.

New opportunities at the next-generation neutrino experiments I: BSM neutrino physics and dark matter.

The combination of the high intensity proton beam facilities and massive detectors for precision measurements of neutrino oscillation parameters including the charge-parity violating (CPV) phase will open the door to help make beyond the standard model (BSM) physics reachable even in low energy regimes in the accelerator-based experiments. Large-mass detectors with highly precise tracking and energy measurements, excellent timing resolution, and low energy thresholds will enable the searches for BSM phenomena from cosmogenic origin, as well. Therefore, it is also conceivable that BSM topics in the next-generation neutrino experiments could be the dominant physics topics in the foreseeable future, as the precision of the neutrino oscillation parameter and CPV measurements continue to improve.This paper provides a review of the current landscape of BSM theory in neutrino experiments in two selected areas of the BSM topics-dark matter and neutrino related BSM-and summarizes the current results from existing neutrino experiments to set benchmarks for both theory and experiment. This paper then provides a review of upcoming neutrino experiments throughout the next 10 to 15 year time scale and their capabilities to set the foundation for potential reach in BSM physics in the two aforementioned themes. An important outcome of this paper is to ensure theoretical and simulation tools exist to carry out studies of these new areas of physics, from the first day of the experiments, such as Deep Underground Neutrino Experiment in the U.S. and Hyper-Kamiokande Experiment in Japan.

Long-term clinical outcomes of endoscopic submucosal dissection for colorectal neoplasia with or without the hybrid technique.

AIM: The main aim of this study was to compare the long-term outcome of a conventional colorectal endoscopic submucosal dissection (ESD) in which submucosal dissection was continued throughout until the completion of resection (ESD-T) to hybrid endoscopic submucosal dissection (ESD-H) in the colorectum. METHOD: Medical records of 836 colorectal neoplasia patients treated by ESD-T or ESD-H were reviewed. ESD-H was defined as colorectal ESD with additional snaring in the final stage of the procedure. Primary outcomes were the overall and metastatic recurrence rates. Secondary outcomes were short-term outcomes such as the en bloc resection rate, procedure time and adverse events. RESULTS: The overall recurrence rate was higher in the ESD-H than in the ESD-T group (5.7% vs 0.7%, P = 0.001). The metastatic recurrence rate showed no significant difference between these groups (1.4% vs 1.4%, P = 1.000). Multivariate analysis revealed that a failed en bloc resection (hazard ratio 24.097; 95% CI 5.446-106.237; P < 0.001) and larger tumour size (hazard ratio 1.042; 95% CI 1.014-1.070; P = 0.003) were independently associated with overall recurrence. The ESD-H group showed a lower en bloc resection rate (56.8% vs 96.5%, P < 0.001), shorter procedure time (45.6 vs 54.3 min, P < 0.001) and higher perforation rate (10.3% vs 6.0%, P = 0.029). CONCLUSION: Although long-term outcomes in terms of overall recurrence are inferior following ESD-H, a failed en bloc resection and large tumour size are the only independent risk factors for recurrence. Further investigations are warranted to improve the long-term outcomes of ESD-H.

Midkine Promotes Odontoblast-like Differentiation and Tertiary Dentin Formation.

Autophagy is an intracellular self-degradation process that is essential for tissue development, cell differentiation, and survival. Nevertheless, the role of autophagy in tooth development has not been definitively identified. The goal of this study was to investigate how autophagy is involved in midkine (MK)-mediated odontoblast-like differentiation, mineralization, and tertiary dentin formation in a mouse tooth pulp exposure model. In vitro studies show that MK and LC3 have similar expression patterns during odontoblast-like cell differentiation. Odontoblast-like cell differentiation is promoted through MK-mediated autophagy, which leads to increased mineralized nodule formation. Subcutaneous transplantation of hydroxyapatite/tricalcium phosphate with rMK-treated human dental pulp cells led to dentin pulp-like tissue formation through MK-mediated autophagy. Furthermore, MK-mediated autophagy induces differentiation of dental pulp cells into odontoblast-like cells that form DSP-positive tertiary dentin in vivo. Our findings may provide 1) novel insight into the role of MK in regulating odontoblast-like differentiation and dentin formation in particular via autophagy and 2) potential application of MK in vital pulp therapy.

CPNE7 Induces Biological Dentin Sealing in a Dentin Hypersensitivity Model.

Dentin hypersensitivity commonly occurs due to opened dentinal tubules for many reasons. In our previous study, copine 7 (CPNE7) could induce dentin formation for an indirect pulp-capping model in vivo. This study aims to investigate the formation of tertiary dentin when CPNE7 is applied to intentionally exposed dentin with nothing over it in vivo, whether it affects microleakage of the teeth, and the penetration ability of CPNE7 molecules through dentinal tubules in vitro. Cervical dentin areas of 6 maxillary incisors of 5 beagles were exposed to a class V-like lesion, and 1 side of 3 maxillary incisors was adapted with recombinant CPNE7 protein for 5 min as the experimental group. The other side was the control group, and there was no treatment of ethylenediaminetetraacetic acid (EDTA) and CPNE7 after preparation. The defects were exposed without any restorations, and all beagles were sacrificed after 4 wk. The fluid penetration of exposed dentin areas was investigated by a microleakage-testing device and confocal laser scanning microscope. Tertiary dentin formation was confirmed with histological scanning electronic microscopic analysis. Tertiary dentin formation reduces dentinal fluid flow due to occluded tubules or discontinuity with primary or secondary dentin. The in vivo hypersensitivity model with the anterior teeth of beagle dogs showed newly formed tertiary dentin at the dentin-pulp boundary in recombinant CPNE7-treated teeth when compared with the untreated control group in histologic analysis. Scanning electronic microscopic analysis revealed occluded sites with mineral deposition of intratubular dentin. In the permeability test, the mean microleakage value of the CPNE7-treated group was significantly lower than that of the control group (P < 0.05). The tubular penetration of rhodamine B-combined CPNE7 was confirmed under confocal laser scanning microscope. CPNE7 induces formation of tertiary dentin through shallowly exposed dentinal tubules, which decreases dentin permeability.

Locally delivered ethyl-2,5-dihydroxybenzoate using 3D printed bone implant for promotion of bone regeneration in a osteoporotic animal model.

Osteoporosis is a disease characterized by low bone mass, most commonly caused by an increase in bone resorption that is not matched by sufficient bone formation. The most common complications of postmenopausal osteoporosis are bone-related defects and fractures. Fracture healing is a multifactorial bone regeneration process, influenced by both biological and mechanical factors related to age, osteoporosis and stability of the osteosynthesis. During the treatment of bone defects in osteoporotic conditions, imbalanced bone remodeling is the leading cause for implant failure. To overcome these problems, ethyl-2,5-dihydroxybenzoate (E-2,5-DHB), a drug that promotes bone formation and inhibits bone resorption, was used. E-2,5-DHB-incorporating titanium (Ti) implants using poly(lactic-co-glycolic acid) (PLGA) coating for local delivery of E-2,5-DHB were developed and the effects on bone healing of femoral defects were evaluated in an osteoporotic model. The release of E-2,5-DHB resulted in decreased bone resorption and increased bone formation around the implant. Thus, it was confirmed that, in the osteoporotic model, bone healing was increased and implant fixation was enhanced. These results suggested that E-2,5-DHB-coated Ti implants have great potential as an ultimate local drug delivery system for bone tissue scaffolds.

Genetic driver mutations define the expression signature and microenvironmental composition of high-grade gliomas.

High-grade gliomas (HGG), including glioblastomas, are characterized by invasive growth, resistance to therapy, and high inter- and intra-tumoral heterogeneity. The key histological hallmarks of glioblastoma are pseudopalisading necrosis and microvascular proliferation, which allow pathologists to distinguish glioblastoma from lower-grade gliomas. In addition to being genetically and molecularly heterogeneous, HGG are also heterogeneous with respect to the composition of their microenvironment. The question of whether this microenvironmental heterogeneity is driven by the molecular identity of the tumor remains controversial. However, this question is of utmost importance since microenvironmental, non-neoplastic cells are key components of the most radiotherapy- and chemotherapy-resistant niches of the tumor. Our work demonstrates a versatile, reliable, and reproducible adult HGG mouse model with NF1-silencing as a driver mutation. This model shows significant differences in tumor microenvironment, expression of subtype-specific markers, and response to standard therapy when compared to our established PDGFB-overexpressing HGG mouse model. PDGFB-overexpressing and NF1-silenced murine tumors closely cluster with human proneural and mesenchymal subtypes, as well as PDGFRA-amplified and NF1-deleted/mutant human tumors, respectively, at both the RNA and protein expression levels. These models can be generated in fully immunocompetent mixed or C57BL/6 genetic background mice, and therefore can easily be incorporated into preclinical studies for cancer cell-specific or immune cell-targeting drug discovery studies.

Tertiary Dentin Formation after Indirect Pulp Capping Using Protein CPNE7.

If there is a partial loss of dentin, the exposed dentinal surface should be protected by an indirect pulp capping (IPC) procedure to preserve pulp vitality and prevent symptoms of dentin hypersensitivity. In our previous study, copine7 (CPNE7) induced odontoblast differentiation in vitro and promoted dentin formation in vivo. The aim of this study was to investigate the possibility of IPC therapy using the CPNE7 protein at the exposed dentinal surface and the resulting effects on tertiary dentin formation in a beagle model. CPNE7 promoted mineralization of odontoblasts and had high calcium ion-binding capacity. The in vivo IPC model with canine teeth showed that regeneration of physiologic reactionary dentin with dentinal tubule structures was clearly observed beneath the remaining dentin in the CPNE7 group, whereas irregular features of reparative dentin were generated in the mineral trioxide aggregate (MTA) group. The CPNE7+MTA group also showed typical reactionary dentin without reparative dentin, showing synergistic effects of CPNE7 with MTA. A scanning electron microscopy analysis showed that dentinal tubules beneath the original dentin were occluded by the deposition of peritubular dentin in the CPNE7 and CPNE7+MTA groups, whereas those in the control group were opened. Therefore, CPNE7 may be able to serve as a novel IPC material and improve symptoms of dentin hypersensitivity.

Periodontal healing with a preameloblast-conditioned medium in dogs.

BACKGROUND AND OBJECTIVE: The predictability of conventional periodontal treatments for damaged periodontal tissue is limited, particularly on the regeneration of new cementum. As signaling molecules, a range of growth factors has been used to promote periodontal regeneration on periodontal ligament (PDL) and cementum defects. A preameloblast-conditioned medium (PA-CM) was prepared from cultured murine apical bud cells, which can differentiate into ameloblasts. We examined the effect of PA-CM on PDL cells and cementoblasts in vitro and evaluated histologically the effects of PA-CM on the regeneration of experimentally induced periodontal defects in vivo. MATERIAL AND METHODS: In vitro, the effects of PA-CM on the migration of human PDL cells were examined using a scratch wound healing assay and a transwell assay. The differentiation and mineralization potential of PA-CM-treated human PDL cells and murine cementoblastic OCCM-30 cells was examined by real-time polymerase chain reaction and Alizarin red-S staining. In vivo, six mongrel dogs (12-16 kg; 6-8 mo old) were used. Twenty-four roots were replanted with either, (i) only periodontal defects (n = 12; control group), or (ii) periodontal defects and PA-CM treatment (n = 12; experimental group). In the experimental group, the PDL and cementum between notches was removed using a Gracey curette and soaked in 0.08 mL water containing 80 µg of a PA-CM for 2 min. The dogs were killed at 4 and 8 wk post-surgery. RESULTS: The in vitro results showed that PA-CM stimulated the migration of PDL cells and promoted the differentiation and mineralization of PDL cells and cementoblasts. Real-time polymerase chain reaction analysis revealed stronger expression of Runx2, Osx, OC, Bsp and Cap mRNAs in the PA-CM-treated PDL cells and cementoblasts than those in the control cells. In vivo, newly formed PDL-like tissue and cementum-like tissue were observed partially between the root surfaces and newly formed bone in the experimental group. The regenerated PDL-like tissue in the experimental group was significantly higher than that in the control group at 8 wk (p < 0.05). The replacement resorption on the experimental group was significantly lower than that in the control group at 8 wk (p < 0.05). In addition, the amount of newly formed cementum-like tissue in the experimental group was significantly higher than that in the control group at 4 and 8 wk (p < 0.05). CONCLUSION: These results suggest that PA-CM has the potential to regenerate periodontal tissues in PDL and cementum defects.

Self-expandable metal stents for malignant esophageal obstruction: a comparative study between extrinsic and intrinsic compression.

Self-expandable metal stents (SEMSs) are effective for malignant esophageal obstruction, but usefulness of SEMSs in extrinsic lesions is yet to be elucidated. This study is aimed at evaluating the clinical usefulness of SEMSs in the extrinsic compression compared with intrinsic. A retrospective review was conducted for 105 patients (intrinsic, 85; extrinsic, 20) with malignant esophageal obstruction who underwent endoscopic SEMSs placement. Technical and clinical success rates were evaluated and clinical outcomes were compared between extrinsic and intrinsic group. Extrinsic group was mostly pulmonary origin. Overall technical and clinical success rate was 100% and 91%, respectively, without immediate complications. Extrinsic and intrinsic group did not differ significantly in clinical success rate. The median stent patency time was 131.3 ± 85.8 days in intrinsic group while that of extrinsic was 54.6 ± 45.1 due to shorter survival after stent insertion. The 4-, 8-, and 12-week patency rates were 90.5%, 78.8%, and 64.9% respectively in intrinsic group, while stents of extrinsic group remained patent until death. Uncovered, fully covered, and double-layered stent were used evenly and the types did not influence patency in both groups. In conclusion, esophageal SEMSs can safely and effectively be used for malignant extrinsic compression as well as intrinsic.

Novel MMP20 and KLK4 Mutations in Amelogenesis Imperfecta.

In order to achieve highly mineralized tooth enamel, enamel proteinases serve the important function of removing the remaining organic matrix in the mineralization and maturation of the enamel matrix. Mutations in the kallikrein 4 (KLK4), enamelysin (MMP20), and WDR72 genes have been identified as causing hypomaturation enamel defects in an autosomal-recessive hereditary pattern. In this report, 2 consanguineous families with a hypomaturation-type enamel defect were recruited, and mutational analysis was performed to determine the molecular genetic etiology of the disease. Whole exome sequencing and autozygosity mapping identified novel homozygous mutations in the KLK4 (c.620_621delCT, p.Ser207Trpfs*38) and MMP20 (c.1054G>A, p.Glu352Lys) genes. Further analysis on the effect of the mutations on the translation, secretion, and function of KLK4 and MMP20 revealed that mutant KLK4 was degraded intracellularly and became inactive while mutant MMP20 was expressed at a normal level but secreted only minimally with proteolytic function.

Panoramic measures for oral bone mass in detecting osteoporosis: a systematic review and meta-analysis.

Different quantitative and qualitative indices calculated on oral panoramic radiographs have been proposed as useful tools to screen for reduced skeletal bone mineral density (BMD). Our aim was to systematically review the literature on linear and qualitative panoramic measures and to assess the accuracy of these indices by performing a meta-analysis of their sensitivity and specificity. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) statement was followed. Fifty studies were included in the qualitative appraisal and 19 were considered for meta-analysis. The methodological quality of the retrieved studies, assessed with the QUADAS-2 tool, was on average low. Three indices were reported by most of the studies: mandibular cortical width, panoramic mandibular index, and the Klemetti index. Mandibular cortical width presented with a better accuracy in excluding osteopenia/osteoporosis (specificity), since patients with a cortical width more than 4 mm had a normal BMD in 90% of the cases. Almost all studies used a cutoff of 0.3 for the panoramic mandibular index, resulting in an estimated sensitivity and specificity in detecting reduced BMD, respectively, of 0.723 (SE 0.160; 95% confidence interval [CI], 0.352-0.926) and 0.733 (SE 0.066; 95% CI, 0.587-0.841). The presence of any kind of mandibular cortical erosion gave an estimated sensitivity and specificity in detecting reduced BMD, respectively, of 0.789 (SE 0.031; 95% CI, 0.721-0.843) and 0.562 (SE 0.047; 95% CI, 0.47-0.651) and a sensitivity and specificity in detecting osteoporosis, respectively, of 0.806 (SE 0.105; 95% CI, 0.528-0.9200) and 0.643 (SE 0.109; 95% CI, 0.417-0.820). The mandibular cortical width, panoramic mandibular index, and Klemetti index are overall useful tools that potentially could be used by dentists to screen for low BMD. Their limitations are mainly related to the experience/agreement between different operators and the different image quality and magnification of the panoramic radiographs.

Esophageal mucosal mast cell infiltration and changes in segmental smooth muscle contraction in noncardiac chest pain.

Mast cells release potent mediators that alter enteric nerve and smooth muscle functions and may contribute to the pathogenesis of functional gastrointestinal disorders. The goal of this study was to determine if mucosal mast cell infiltration was associated with smooth muscle segmental changes in esophageal contraction. All patients with noncardiac chest pain (NCCP) were divided into two groups consisting of patients with non-erosive reflux disease or functional chest pain (FCP) according to the results of ambulatory 24 hours esophageal pH monitoring and high-resolution manometry. Pressure-volume (PV) was calculated by multiplying the length of the esophageal segment, duration of the contraction, and mean pressure over the entire space-time box (P mean). Quantification of mast cells was performed in five consecutive nonoverlapping immunostained sections. Spearman correlation analysis showed that the distal segment PV correlated with the mast cell count in all of the patients combined and in patients with FCP with correlation coefficients of 0.509 and 0.436, respectively (P = 0.004 and P = 0.042). Similar findings were observed for the segmental ratio of distal to proximal smooth muscle PV in all patients and in patients with FCP (correlation coefficients 0.566; P = 0.001 and correlation coefficients 0.525; P = 0.012, respectively). Mucosal mast cell infiltration was associated with distal esophageal contraction as a key pathophysiologic factor of NCCP.

Effect of dietary supplementation of procyanidin on growth performance and immune response in pigs.

This study was performed to determine the effect of dietary supplementation of procyanidin on growth performance, blood characteristics, and immune function in growing pigs. In experiment 1 (Exp. 1), thirty-two crossbred pigs with an initial BW of 19.2±0.3 kg were allocated into 4 treatments for an 8-wk experiment: i) CON (basal diet), ii) MOS 0.1 (basal diet+0.1% mannanoligosaccharide), iii) Pro-1 (basal diet+0.01% procyanidin), and iv) Pro-2 (basal diet+0.02% procyanidin). Pigs fed Pro-1 and Pro-2 diets had greater (p<0.05) gain:feed ratio compared with those fed CON or MOS 0.1 diets. Serum creatinine concentration was less (p<0.05) in Pro-2 treatment than those in CON, MOS 0.1 and Pro-1 treatments. In Exp. 2, twelve pigs (BW 13.4±1.3 kg) received basal diet with i) 0 (CON), ii) 0.02% (Pro-0.02%), and iii) 0.04% procyanidin (Pro-0.04%) for 4 wk. Concentration of platelets was lower (p<0.05) in the Pro-0.04% group compared to CON at 24 h after lipopolysaccharide (LPS) challenge. In addition, secretion of cytokines from cultured peripheral blood mononuclear cells (PBMC) in the presence or absence of procyanidin was examined. The levels of interleukin (IL)-1ß, IL-6 and tumor necrosis factor (TNF)-α were lower (p<0.05) in Pro (LPS-stimulated PBMCs+procyanidin) than those in CON (LPS-stimulated PBMCs+PBS) at 4 h after LPS challenge. These data suggest that dietary addition of procyanidin improves feed efficiency and anti-inflammatory cytokines of pigs.

Aß1-42 reduces P-glycoprotein in the blood-brain barrier through RAGE-NF-κB signaling.

The reduced clearance of amyloid-ß peptide (Aß) from the brain partly accounts for the neurotoxic accumulation of Aß in Alzheimer's disease (AD). Recently, it has been suggested that P-glycoprotein (P-gp), which is an efflux transporter expressed on the luminal membrane of the brain capillary endothelium, is capable of transporting Aß out of the brain. Although evidence has shown that restoring P-gp reduces brain Aß in a mouse model of AD, the molecular mechanisms underlying the decrease in P-gp expression in AD is largely unknown. We found that Aß1-42 reduced P-gp expression in the murine brain endothelial cell line bEnd.3, which was consistent with our in vivo data that P-gp expression was significantly reduced, especially near amyloid plaques in the brains of five familial AD mutations (5XFAD) mice that are used as an animal model for AD. A neutralizing antibody against the receptor for advanced glycation end products (RAGE) and an inhibitor of nuclear factor-kappa B (NF-κB) signaling prevented the decrease in Aß1-42-induced P-gp expression, suggesting that Aß reduced P-gp expression through NF-κB signaling by interacting with RAGE. In addition, we observed that the P-gp reduction by Aß was rescued in bEnd.3 cells receiving inductive signals or factors from astrocytes making contacts with endothelial cells (ECs). These results support that alterations of astrocyte-EC contacts were closely associated with P-gp expression. This suggestion was further supported by the observation of a loss of astrocyte polarity in the brains of 5XFAD mice. Taken together, we found that P-gp downregulation by Aß was mediated through RAGE-NF-κB signaling pathway in ECs and that the contact between astrocytes and ECs was an important factor in the regulation of P-gp expression.

Heterogeneity of mucosal mast cell infiltration in subgroups of patients with esophageal chest pain.

BACKGROUND: Although there is growing evidence that an increase in mucosal mast cells (MMCs) in the small and large intestine is associated with visceral hypersensitivity, few studies have evaluated MMCs in humans with esophageal symptoms. The aim of this study was to investigate esophageal MMC distribution in patients with non-cardiac chest pain (NCCP) and to examine the association between the number of gut MMCs and other functional gastrointestinal disorders. METHODS: Forty-two consecutive NCCP patients and 10 healthy controls completed a questionnaire for bowel symptoms, chest pain intensity score, and psychologic depression. Esophageal, duodenal, and rectal MMCs were identified immunohistochemically and quantified by image analysis. KEY RESULTS: Numbers of MMCs were significantly higher in NCCP patients vs healthy controls (11.8 ± 5.6 vs 7.6 ± 3.7 MMCs/high-power field, p = 0.026). In comparison of subgroups classified by 24-h impedance-pH monitoring, esophageal MMC counts were highest in the hypersensitive esophagus group (p < 0.01) and were also significantly increased in the functional chest pain group (p < 0.05). A positive correlation between esophageal and duodenal MMC counts was observed in patients with functional dyspepsia (FD; Spearman ρ = 0.604, p = 0.037). In particular, patients with clinical overlap with irritable bowel syndrome showed a strong positive correlation between esophageal and rectal MMC numbers (Spearman ρ = 0.857, p = 0.010). CONCLUSIONS & INFERENCES: Among NCCP patients, increased MMC infiltration occurs in subgroups with hypersensitive esophagus and functional chest pain. In subpopulations with overlap with FD or irritable bowel syndrome, esophageal MMC counts demonstrated significant positive correlations with duodenal or rectal MMC counts.