Redirection of CAR-T Cell Cytotoxicity Using Metabolic Glycan Labeling with Unnatural Sugars.

T cells expressing chimeric antigen receptors (CAR-T cells) have shown unprecedented clinical responses against hematological malignancies. However, some patients relapse after CAR-T cell therapy due to antigen-negative escape variants. Additionally, CAR-T cell therapies showed limited clinical efficacy in solid tumors with high antigen heterogeneity. To overcome this, we metabolically labeled the glycans on cancer cells to redirect CAR-T cell cytotoxicity regardless of the endogenous antigen expression status of the cancer cells. We found that modifying cancer cells with N-azidoacetylmannosamine and bicyclo[6.1.0]non-4-yne-fluorescein isothiocyanate can elicit selective and durable cytotoxicity of anti-FITC CAR-T cells. Furthermore, we demonstrated that dinitrophenyl-conjugated sialic acid (Sia-DNP) generated DNP-modified glycans on cancer cells in situ that could be effectively targeted by anti-DNP CAR-T cells to eradicate established tumors in xenograft models. Our study illustrates that metabolic glycan labeling using unnatural sugars can be combined with CAR-T cell therapy to provide novel cancer immunotherapy for solid tumors that lack viable target antigens.

A Rare Case of Well-Differentiated Parapharyngeal Liposarcoma.

Parapharyngeal liposarcoma is a rare malignant tumor, the pathogenesis of which is uncertain. Parapharyngeal liposarcoma often causes nonspecific clinical features, including dysphagia, globus sensation, and/or respiratory disturbances. The parapharyngeal space is a rare location for head and neck liposarcoma, and these lesions may therefore be confused with a variety of other tumors that can originate in this area. Here, we report a case of liposarcoma originating from the prestyloid parapharyngeal space and discuss the possible differential diagnosis on radiographic findings.

Single-Molecule Sensing of an Anticancer Therapeutic Protein-Protein Interaction Using the Chemically Modified OmpG Nanopore.

Nanopore sensors are a highly attractive platform for single-molecule sensing for sequencing, disease diagnostics, and drug screening. Outer membrane protein G (OmpG) nanopores have advantages for single-molecule sensing owing to their rigid monomeric structure, which comprises seven flexible loops, providing distinct gating patterns upon analyte binding. Blocking of the protein-protein interaction between B-cell lymphoma-extra-large (Bcl-xL) and the BH3 domain of Bcl-2 homologous antagonist/killer (Bak-BH3) has been reported as a promising strategy for anticancer therapy. Here, we characterized the interaction between Bcl-xL and Bak-BH3 as well as its inhibition by a small-molecule inhibitor using click chemistry-based Bak-BH3 peptide-conjugated OmpG nanopores. The binding of Bcl-xL to Bak-BH3 generated characteristic gating signals involving significant changes in the amplitudes of noise and gating parameters such as gating frequency, open probability, and durations of open and closed states. Notably, specific inhibition of Bcl-xL by the small-molecule antagonist, ABT-737, led to the recovery of the noise and gating parameters. Collectively, these results revealed that the chemically modified OmpG nanopore can serve as a valuable sensor platform for ultrasensitive, rapid, and single-molecule-based drug screening against protein-protein interactions, which are therapeutic targets for various diseases.

A Recombinant Ig Fragment (IgCw-γεκ) Comprising the Cγ1-Cε2-4 and Cκ Domains Is an Alternative Reagent to Human IgE.

Human IgE is useful for immunological assays, such as sensitization of FcεRI-positive cells and IgE measurement. In this study, we report the development of a recombinant Ig fragment, designated IgCw-γεκ, as an alternative reagent to human IgE. IgCw-γεκ (∼130 kDa) comprises two hybrid constant H chain regions (Cγ1-Cε2-4, each ∼53 kDa) and two constant κ L chains (Cκ, each ∼12 kDa) and lacks a V domain. The presence of Cγ1 instead of Cε1 within the H chain increased the production yield and facilitated assembly of the H and L chains. IgCw-γεκ was produced in cultured human embryonic kidney 293F cells, with a yield of ∼27 mg/l. IgCw-γεκ bound to human FcεRIαRs expressed on the surface of rat basophilic leukemia-2H3 cells. A ß-hexosaminidase release assay revealed that the biological activity of IgCw-γεκ was comparable with that of IgE. The IgE concentration measured using IgCw-γεκ as a standard was similar to that measured using IgE as a standard. These results suggest that the IgCw-γεκ molecule retains the basic characteristics of IgE, but does not cross-react with Ags, making it an alternative to the IgE isotype references used in a variety of immunological assays.

Progressive Transformation of Germinal Centers in Axillary Lymph Nodes Mimicking Metastatic Lymphadenopathy after Breast Cancer Surgery: A Case Report.

Progressive transformation of germinal centers (PTGC) is a rarely diagnosed, benign disease of the lymph nodes that commonly manifests as chronic lymphadenopathy. PTGC may be characterized by single or multiple non-tender lymph nodes, and it commonly involves the cervical, axillary, and inguinal areas. Although PTGC is identified with concurrent lymphoma in some patients, it is not considered as a premalignant entity. Histopathologic diagnosis of PTGC is rarely made, and imaging findings have been reported in very few studies. We present a case of PTGC that occurred at the contralateral axillary lymph nodes and mimicked metastatic lymphadenopathy after breast cancer surgery. We also discuss its imaging findings.

Mammographic, Sonographic, and MRI Features of Primary Neuroendocrine Carcinoma of the Breast: A Case Report.

Primary neuroendocrine carcinomas of the breast are a rare, distinct category of breast carcinomas that require immunohistochemical staining for diagnosis. Currently, there is not enough evidence on the clinical pattern, prognosis, and proper management of the disease. Only few case series have described the imaging findings of neuroendocrine carcinomas of the breast. We herein present a case of a primary neuroendocrine carcinoma of the breast (small cell) presenting as a locally aggressive tumor with metastatic disease, and describe the radiologic findings.

GSK3 Restrains Germinal Center B Cells to Form Plasma Cells.

B cells in the germinal center (GC) are programmed to form plasma cells (PCs) or memory B cells according to signals received by receptors that are translated to carry out appropriate activities of transcription factors. However, the precise mechanism underlying this process to complete the GC reaction is unclear. In this study, we show that both genetic ablation and pharmacological inhibition of glycogen synthase kinase 3 (GSK3) in GC B cells of mice facilitate the cell fate decision toward PC formation, accompanied by acquisition of dark zone B cell properties. Mechanistically, under stimulation with CD40L and IL-21, GSK3 inactivation synergistically induced the transcription factors Foxo1 and c-Myc, leading to increased levels of key transcription factors required for PC differentiation, including IRF4. This GSK3-mediated alteration of transcriptional factors in turn facilitated the dark zone transition and consequent PC fate commitment. Our study thus reveals the upstream master regulator responsible for interpreting external cues in GC B cells to form PCs mediated by key transcription factors.

The 'classic stromatolite' Cryptozoön is a keratose sponge-microbial consortium.

Animal evolution transformed microbial mat development. Canonically inferred negative effects include grazing, disturbance and competition for space. In contrast, ancient examples of cooperation between microbial mats and invertebrates have rarely been reported. Late Cambrian (~485 million years) Cryptozoön is widely regarded as the first stromatolite to have received a taxonomic name and has been compared with present-day examples at Shark Bay, Australia. Here, we show that Cryptozoön is an interlayered consortium of keratose ('horny') sponge and microbial carbonate in roughly equal proportions. Cryptozoön's well-defined layering reflects repeated alternation of sponge and microbial mat. Its distinctive lateral growth is due to the ability of keratosans to colonize steep and overhanging surfaces. Contrary to the perception of Phanerozoic stromatolites as anachronistic survivors in a eukaryotic world, Cryptozoön suggests mutualistic behaviour in which sponges and microbial mats cooperated to gain support, stability and relief, while sharing substrates, bacteria and metabolites. Keratosan-microbial consortia may have been mistaken for stromatolites throughout the record of the past 500 million years, and possibly longer.

Three-dimensional CBCT based evaluation of the inferior part of the maxillary sinus: Retrospective Study.

The maxillary sinus is the largest of the four paranasal sinuses in humans, and its close proximity to the teeth means that caution is required during dental treatment. In particular, implant surgeries involving the maxillary posterior teeth should include evaluating the inferior part of the maxillary sinus. The purpose of this study is to evaluate the differences by comparing the inferior part of the maxillary sinus based on the nasal cavity floor (NCF) between patients (male 30, female 30) genders through the use of the three-dimensional (3-D) program that can facilitate 3-D visualizations. The present study results obtained from 3-D visualizations using cone beam computed tomography (CBCT) data showed that the inferior part of the maxillary sinus was mostly larger in males than in females. In addition, the utilization of 3-D visualization data was more likely to assure accuracy than when using data obtained by two-dimensional (2-D) imaging. Therefore, 3-D visualizations of the inferior part of the maxillary sinus will contribute to accurate analyses of its anatomical structure during implant surgery and other operations. Further studies utilizing 3-D visualization will yield useful fundamental data and guidelines for future research.

Cathelicidin-Related Antimicrobial Peptide Regulates CD73 Expression in Mouse Th17 Cells via p38.

The effector function of tumor-infiltrated CD4+ T cells is readily suppressed by many types of immune regulators in the tumor microenvironment, which is one of the major mechanisms of immune tolerance against cancer. Cathelicidin-related antimicrobial peptide (CRAMP), the mouse analog of LL-37 peptide in humans, is a cationic antimicrobial peptide belonging to the cathelicidin family; however, its secretion by cancer cells and role in the tumor microenvironment (TME) remain unclear. In this study, we explored the possibility of an interaction between effector CD4+ T cells and CRAMP using in vitro-generated mouse Th17 cells. We found that CRAMP stimulates Th17 cells to express the ectonucleotidase CD73, while simultaneously inducing cell death. This finding suggested that CD73-expressing Th17 cells may function as immune suppressor cells instead of effector cells. In addition, treatment of pharmacological inhibitors of the transforming growth factor-beta (TGF-ß) signaling pathway showed that induction of CD73 expression is mediated by the p38 signaling pathway. Overall, our findings suggest that tumor-derived LL-37 likely functions as an immune suppressor that induces immune tolerance against tumors through shaping effector Th17 cells into suppressor Th17 cells, suggesting a new intervention target to improve cancer immunotherapy.

Obovatol inhibits NLRP3, AIM2, and non-canonical inflammasome activation.

BACKGROUND: Obovatol, a biphenolic chemical originating from Magnolia obovata, has been utilized as a traditional medicine for the treatment of inflammatory diseases. Inflammasome induces maturation of inflammatory cytokines in response to intracellular danger signals, and its dysregulation induces inflammatory diseases. PURPOSE: The effect of obovatol on inflammasome activation has not been reported, although its anti-inflammatory properties have been studied. STUDY DESIGN/METHODS: Obovatol was treated to macrophages with inflammasome triggers, and secretions of interleukin (IL)-1ß, IL-18, and caspase-1 were measured as readouts of inflammasome activation. In addition, Asc pyroptosome formation, caspase-1 activity, and mitochondrial reactive oxygen species (ROS) production were analyzed in mechanical studies. Anti-inflammasome properties of obovatol were confirmed in an animal model. RESULTS: Obovatol inhibited NLRP3, AIM2, and non-canonical inflammasomes through inhibition of Asc pyroptosome formation and mitochondrial ROS generation. In addition, obovatol disrupted the priming step of inflammasome activation and inhibited transcription of inflammatory cytokines. In mice, obovatol attenuated serum IL-1ß elevation in response to monosodium urate crystals. CONCLUSION: Obovatol is suggested as an inhibitor of NLRP3, AIM2, and non-canonical inflammasomes.

Anti-Oxidative and Anti-Inflammatory Activity of Kenya Grade AA Green Coffee Bean Extracts.

BACKGROUND: Kenya AA green coffee bean extracts were tested for natural ingredients used for anti-oxidative and anti-inflammatory purposes in cosmetic products. METHODS: Anti-oxidative activities were measured by total polyphenol, 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. Anti-inflammatory activities were evaluated via nitric oxide (NO) assays, and through quantification of inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) protein expression by western blotting. Data analyses were performed using independent Student's t-tests, with statistical significance set at P < 0.05. RESULTS: Total polyphenol content of water and ethanol extract was 169.0 ± 3.1 mg and 300.34 ± 16.6 mg tannic acid/g dry weight, respectively. The DPPH and ABTS radical scavenging activities of all the extracts were significantly increased in a concentration-dependent manner. Kenya AA green coffee bean extracts were toxic at a concentration of 1,000 µg/mL in RAW 264.7 cells. Anti-inflammatory activity as determined by NO assay showed that lipopolysaccharide (LPS)-induced NO was significantly inhibited following treatment with Kenya AA green coffee bean extracts in a concentration-dependent manner. iNOS and COX-2 protein expression was also significantly inhibited following treatment. CONCLUSION: These results highlight the potential of Kenya AA green coffee bean extracts as a naturally active anti-inflammatory agent in cosmetic products.

Differential regulation of senescence and in vitro differentiation by 17ß-estradiol between mesenchymal stem cells derived from male and female mini-pigs.

The characterization and potential of mesenchymal stem cells (MSCs) are gender dependent and estrogen influences these properties. This study demonstrated that supplementation with 17ß-estradiol (E2) increases the proliferation of bone marrow-MSCs derived from male and female mini-pigs (Mp- and Fp-BMSCs) in a concentration-dependent manner, with 10(-12) M E2 suggested as the optimal dose of E2 that led to the greatest improvement in BMSCs proliferation. Supplementation of 10(-12) M E2 resulted in down-regulation of ß-galactosidase activity and pro-apoptotic activity in both BMSCs, while anti-apoptotic activity was up-regulated in only Fp-BMSCs. Further, E2 increased the osteogenic ability of Fp-BMSCs. Based on these findings, optimal utilization of E2 can improve cellular senescence and apoptosis, as well as in vitro osteogenesis of BMSCs, and could therefore be useful in stem cell therapy, particularly in bone regeneration for adult females.

Intraductal Ultrasonography without Radiocontrast Cholangiogramin Patients with Extrahepatic Biliary Disease.

BACKGROUND/AIMS: Intraductal ultrasonography (IDUS) has been performed as an adjunct to endoscopic retrograde cholangiography (ERC) during radiocontrast cholangiography (RC). Radiation exposure during RC poses a health risk to both patients and examiners. We evaluated the feasibility of IDUS without RC in various extrahepatic biliary diseases. METHODS: IDUS was performed with the insertion of an IDUS probe from the papilla of Vater to the confluent portion of the common hepatic duct without fluoroscopy. The technical success rate and procedure-related complications were evaluated retrospectively. RESULTS: Wire-guided IDUS without RC was performed in 105 patients. The mean age was 66.5 years, and 50 (47.6%) were male. The IDUS diagnoses included choledocholithiasis (73, 69.5%), benign biliary stricture (11, 10.5%), choledocholithiasis with biliary pancreatitis (9, 8.6%), bile duct cancer (5, 4.8%), pancreatic cancer (1, 0.9%), and others (6, 5.7%). After IDUS, 66 (62.8%) underwent stone removal, 19 (18.1%) underwent biliary drainage, and 7 (6.6%) underwent brush cytology and biopsy. No significant complications such as perforation or severe pancreatitis occurred. CONCLUSIONS: IDUS without RC was a feasible and safe approach in patients with various extrahepatic biliary diseases. We anticipate a potentially important role of IDUS in various ERC procedures because it lacks the hazards of RC.

Promotion of full-thickness wound healing using epigallocatechin-3-O-gallate/poly (lactic-co-glycolic acid) membrane as temporary wound dressing.

Epigallocatechin-3-O-gallate (EGCG) is a major polyphenolic compound in green tea. It has been known that EGCG regulates the secretion of cytokines and the activation of skin cells during wound healing. In this study, various concentrations of EGCG were added to the electrospun membranes composed of poly (lactic-co-glycolic acid) (PLGA), and its healing effects on full-thickness wounds created in nude mice were investigated. The electrospun membranes containing 5 wt% EGCG (5EGCG/PLGA membrane) exhibited cytotoxicity in human dermal fibroblasts (HDFs) as HDF morphologies were transformed on them. In the animal study, cell infiltration of mice treated with electrospun membranes containing 1 wt% EGCG (1EGCG/PLGA membrane) significantly increased after 2 weeks. The immunoreactivity of Ki-67 (re-epithelialization at the wound site) and CD 31 (formation of blood vessels) also increased in the mice treated with 1EGCG/PLGA membranes in comparison with the mice treated with PLGA membranes. These results suggest that 1EGCG/PLGA can enhance wound healing in full thickness by accelerating cell infiltration, re-epithelialization, and angiogenesis.

Comparison of spread of subarachnoid sensory block and incidence of hypotension in early and late second trimester of pregnancy.

BACKGROUND: Obstetric or non-obstetric surgery can be performed in pregnant women during the second trimester. We evaluated maximal sensory block level (MSBL), and other sensory block characteristics after spinal anesthesia in early and late second trimester pregnant women. METHODS: Forty-four pregnant women scheduled for cervical cerclage under spinal anesthesia in the early second trimester (Group E) or in the late second trimester (Group L) were enrolled in this study. Spinal anesthesia was performed at the L3/4 and 7 mg of 0.5% hyperbaric bupivacaine was injected into the subarachnoid space. Hemodynamic variables, incidence of nausea/vomiting, ephedrine dose, and sensory block were recorded every 2.5 min during the first 15 min and 20 min after the injection. The MSBL, the time at which MSBL was achieved, and the time to the two-segment regression of the sensory level were also recorded. RESULTS: The maximum number of segments blocked was significantly greater in the Group L than in the Group E. The incidence of nausea/vomiting and hypotension, and dose of administrated ephedrine were significantly higher in the Group L than in the Group E. The mean arterial pressure during the 15 min after subarachnoid injection was significantly lower compared to the baseline value in the Group L. CONCLUSIONS: The MSBL of spinal anesthesia with hyperbaric bupivacaine 7 mg were T9 in the early and T5 in the late second trimester groups. Pregnant women in the late second trimester exhibited increased incidence of hypotension and need for ephedrine than women in the early second trimester.

Comparison of cerebrospinal fluid biomarkers between idiopathic normal pressure hydrocephalus and subarachnoid hemorrhage-induced chronic hydrocephalus: a pilot study.

BACKGROUND: We examined the cerebrospinal fluid (CSF) markers of subarachnoid hemorrhage (SAH)-induced and idiopathic normal pressure hydrocephalus (INPH) to investigate the pathophysiology and mechanism of communicating hydrocephalus compared to obstructive hydrocephalus. MATERIAL/METHODS: We obtained CSF samples from 8 INPH, 10 SAH-induced hydrocephalus, and 6 unmatched patients with non-hemorrhagic obstructive hydrocephalus during their ventriculoperitoneal shunt operations. Transforming growth factor (TGF)-ß1, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), and total tau in the CSF were analyzed via enzyme-linked immunosorbent assay. RESULTS: The mean VEGF levels in the CSF of patients with SAH-induced hydrocephalus, INPH, and obstructive hydrocephalus were 239 ± 131, 239 ± 75, and 163 ± 122 pg/mL, respectively. The total tau concentrations in the CSF of the groups were 1139 ± 1900, 325 ± 325, and 1550 ± 2886 pg/mL, respectively. TNF-α values were 114 ± 34, 134 ± 38, and 55 ± 16 pg/mL, respectively. TGF-ß1 values were 953 ± 430, 869 ± 447, and 136 ± 63 pg/mL, respectively. A significant difference in TNF-α and TGF-ß1 levels was observed only between SAH-induced and chronic obstructive hydrocephalus, and between INPH and chronic obstructive hydrocephalus (p<0.01). CONCLUSIONS: No significant differences in the 4 CSF biomarker levels were observed between INPH and SAH-induced hydrocephalus, whereas CSF TNF-α and TGF-ß1 levels were increased compared to those in patients with chronic obstructive hydrocephalus. Post-SAH hydrocephalus and INPH are probably more destructive to neural tissues, and then stimulate the inflammatory reaction and healing process, compared with obstructive hydrocephalus.

Direct interactions between the coiled-coil tip of DksA and the trigger loop of RNA polymerase mediate transcriptional regulation.

Escherichia coli DksA is a transcription factor that binds to RNA polymerase (RNAP) without binding to DNA, destabilizing RNAP-promoter interactions, sensitizing RNAP to the global regulator ppGpp, and regulating transcription of several hundred target genes, including those encoding rRNA. Previously, we described promoter sequences and kinetic properties that account for DksA's promoter specificity, but how DksA exerts its effects on RNAP has remained unclear. To better understand DksA's mechanism of action, we incorporated benzoyl-phenylalanine at specific positions in DksA and mapped its cross-links to RNAP, constraining computational docking of the two proteins. The resulting evidence-based model of the DksA-RNAP complex as well as additional genetic and biochemical approaches confirmed that DksA binds to the RNAP secondary channel, defined the orientation of DksA in the channel, and predicted a network of DksA interactions with RNAP that includes the rim helices and the mobile trigger loop (TL) domain. Engineered cysteine substitutions in the TL and DksA coiled-coil tip generated a disulfide bond between them, and the interacting residues were absolutely required for DksA function. We suggest that DksA traps the TL in a conformation that destabilizes promoter complexes, an interaction explaining the requirement for the DksA tip and its effects on transcription.

Increased Vascular Endothelial Growth Factor in the Ventricular Cerebrospinal Fluid as a Predictive Marker for Subsequent Ventriculoperitoneal Shunt Infection : A Comparison Study among Hydrocephalic Patients.

OBJECTIVE: The aim of this study is to determine the association between the cerebrospinal fluid (CSF) biomarkers and inflammation, and the predictive value of these CSF biomarkers for subsequent shunt associated infection. METHODS: We obtained CSF samples from the patients with hydrocephalus during ventriculoperitoneal (VP) shunt operations. Twenty-two patients were enrolled for this study and divided into 3 groups: subarachnoid hemorrhage (SAH)-induced hydrocephalus, idiopathic normal pressure hydrocephalus (INPH) and hydrocephalus with a subsequent shunt infection. We analyzed the transforming growth factor-ß1, tumor necrosis factor-α, vascular endothelial growth factor (VEGF) and total tau in the CSF by performing enzyme-linked immunosorbent assay. The subsequent development of shunt infection was confirmed by the clinical presentations, the CSF parameters and CSF culture from the shunt devices. RESULTS: The mean VEGF concentration (±standard deviation) in the CSF of the SAH-induced hydrocephalus, INPH and shunt infection groups was 236±138, 237±80 and 627±391 pg/mL, respectively. There was a significant difference among the three groups (p=0.01). Between the SAH-induced hydrocephalus and infection groups and between the INPH and infection groups, there was a significant difference of the VEGF levels (p<0.01). However, the other marker levels did not differ among them. CONCLUSION: The present study showed that only the CSF VEGF levels are associated with the subsequent development of shunt infection. Our results suggest that increased CSF VEGF could provide a good condition for bacteria that are introduced at the time of surgery to grow in the brain, rather than reflecting a sequel of bacterial infection before VP shunt.