Metabolomics of Duodenal Juice for Biliary Tract Cancer Diagnosis.

The poor prognosis of malignant biliary diseases is partially caused by their difficult early diagnosis. Therefore, many patients are only diagnosed at advanced stages. This study aimed to improve diagnosis by clarifying the differences in the duodenal juice metabolomes of benign and malignant biliary diseases. From October 2021 to January 2023, duodenal juice was obtained from 67 patients with suspected biliary diseases who required endoscopic ultrasonography and endoscopic retrograde cholangiography for diagnosis/treatment. The samples metabolomes were analyzed via nuclear magnet resonance spectroscopy using an 800-MHz spectrometer. Metabolomes of malignant and benign diseases were then compared, and multivariate analysis was performed to determine the relevant factors for malignancy/benignancy. For benignancy, no significant predictors were observed. For malignancy, acetone was a significant predictor, with higher concentrations in the malignant group than in the benign group. Regarding the receiver operating characteristic curve analysis for biliary tract carcinoma diagnosis, the predictive value of acetone in duodenal juice was comparable with serum CA19-9 levels (area under the curve: 0.7330 vs. 0.691, p = 0.697). In conclusion, duodenal juice metabolomics is a feasible method that is available for differential diagnosis in the biliary disease field.

Clinical significance of dispersion imaging by shear wave elastography in the treatment and diagnosis of pancreatic cancer.

BACKGROUND AND AIM: Recently, dispersion imaging by shear wave elastography has been developed to visualize a tissue viscosity-related factor by measuring the dispersion slope. However, clinical significance of dispersion imaging in the field of pancreatic cancer is unknown. This study aimed to investigate the clinical significance of dispersion imaging in the treatment and diagnosis of pancreatic cancer. METHODS: We measured shear wave dispersion slope (SWD) (m/s/kHz) and shear wave elasticity (SWE) (kPa) in patients with pancreatic ductal adenocarcinoma (PDA). The primary endpoint was the relationship between the changes in SWD and SWE values before and after chemotherapy and the response to chemotherapy. Secondary endpoints included SWD and SWE values in relation to differences between PDA and non-PDA sites and histopathological scores of stroma, inflammation, fibrosis, and necrosis in endoscopic ultrasound-guided fine-needle aspiration specimens. RESULTS: Fifty-six patients were included, 30 of whom underwent chemotherapy. There was no relationship between the changes of SWD and SWE values and chemotherapy responses. In 56 patients, the median SWD value was 12.20 m/s/kHz (interquartile range [IQR]: 10.88-13.61) at PDA sites and 13.57 m/s/kHz (IQR: 12.28-16.20) at non-PDA sites (P = 0.005). The median SWE value was 8.18 kPa (IQR: 7.00-9.74) at PDA sites and 6.14 kPa (IQR: 5.40-6.77) at non-PDA sites (P < 0.001). Histopathological evaluation revealed that inflammation scores were correlated with SWD values (rs  = 0.42, P < 0.001). CONCLUSIONS: Dispersion imaging in pancreatic cancer would be useful for diagnosis and assessing inflammation.

Lumbar Spinal Canal Stenosis in Acromegaly: A Case Report and Literature Review.

A 60-year-old Japanese man diagnosed with acromegaly at 28 years old had difficulty walking due to worsening back pain. He had been treated with somatostatin analog since 57 years old, but his pain and numbness continued to worsen. Lumbar magnetic resonance imaging showed disc bulging at L3/4 and L4/5, and he was diagnosed with lumbar spinal canal stenosis due to hypertrophy of the yellow ligament. Patients with acromegaly may complain of osteoarthropathy, so we must pay attention to the symptoms of spinal canal stenosis in collaboration with orthopedic specialists.

Genetic Analyses of Cell-Free DNA in Pancreatic Juice or Bile for Diagnosing Pancreatic Duct and Biliary Tract Strictures.

Poor prognosis of pancreaticobiliary malignancies is attributed to intrinsic biological aggressiveness and the lack of reliable methods for early diagnosis. This study aimed to evaluate the feasibility and availability of pancreatic juice- and bile-derived cell-free DNA (cfDNA) for diagnosing pancreaticobiliary strictures. From October 2020 to February 2022, pancreatic juice or bile was obtained from 50 patients with pancreaticobiliary strictures during endoscopic retrograde cholangiopancreatography. cfDNAs extracted from the samples were analyzed using next-generation sequencing and a cancer gene panel. The obtained cfDNAs, genetic data and clinical information were analyzed for diagnosis. cfDNA concentrations in pancreatic juice were higher in the intraductal papillary mucinous neoplasm group than in the other groups, whereas those in bile were similar in all groups. In pancreatic juice, the sensitivity, specificity and positive and negative predictive values of cfDNA analyses were 33%, 100%, 100% and 71.4%, respectively, whereas those of cytological analyses were 0%, 100%, 0% and 62.5%, respectively. In bile, those of cell-free DNA analyses were 53%, 75%, 89.5% and 28.6%, respectively, whereas those of cytological analyses were 19%, 100%, 100% and 16%, respectively. In conclusion, pancreatic juice- and bile-derived cfDNA is a novel liquid biopsy tool that can diagnose pancreaticobiliary strictures.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Treatment of severe pneumonia by hinokitiol in a murine antimicrobial-resistant pneumococcal pneumonia model.

Streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. Antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. Hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against S. pneumoniae in vitro regardless of antimicrobial resistance. In this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. Male 8-week-old BALB/c mice were intratracheally infected with S. pneumoniae strains D39 (antimicrobial susceptible) and NU4471 (macrolide resistant). After 1 h, hinokitiol was injected via the tracheal route. Hinokitiol significantly decreased the number of S. pneumoniae in the bronchoalveolar lavage fluid (BALF) and the concentration of pneumococcal DNA in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. In addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the BALF and serum. Repeated hinokitiol injection at 18 h intervals showed downward trend in the number of S. pneumoniae in the BALF and the concentration of S. pneumoniae DNA in the serum with the number of hinokitiol administrations. These findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. Accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia.

An injectable hydrogel-formulated inhibitor of prolyl-4-hydroxylase promotes T regulatory cell recruitment and enhances alveolar bone regeneration during resolution of experimental periodontitis.

The hypoxia-inducible factor 1α (HIF-1α) is critically involved in tissue regeneration. Hence, the pharmacological prevention of HIF-1α degradation by prolyl hydroxylase (PHD) under normoxic conditions is emerging as a promising option in regenerative medicine. Using a mouse model of ligature-induced periodontitis and resolution, we tested the ability of an injectable hydrogel-formulated PHD inhibitor, 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid (1,4-DPCA/hydrogel), to promote regeneration of alveolar bone lost owing to experimental periodontitis. Mice injected subcutaneously with 1,4-DPCA/hydrogel at the onset of periodontitis resolution displayed significantly increased gingival HIF-1α protein levels and bone regeneration, as compared to mice treated with vehicle control. The 1,4-DPCA/hydrogel-induced increase in bone regeneration was associated with elevated expression of osteogenic genes, decreased expression of pro-inflammatory cytokine genes, and increased abundance of FOXP3+ T regulatory (Treg) cells in the periodontal tissue. The enhancing effect of 1,4-DPCA/hydrogel on Treg cell accumulation and bone regeneration was reversed by AMD3100, an antagonist of the chemokine receptor CXCR4 that mediates Treg cell recruitment. In conclusion, the administration of 1,4-DPCA/hydrogel at the onset of periodontitis resolution promotes CXCR4-dependent accumulation of Treg cells and alveolar bone regeneration, suggesting a novel approach for regaining bone lost due to periodontitis.

Aggregatibacter actinomycetemcomitans induces detachment and death of human gingival epithelial cells and fibroblasts via elastase release following leukotoxin-dependent neutrophil lysis.

Aggregatibacter actinomycetemcomitans is considered to be associated with periodontitis. Leukotoxin (LtxA), which destroys leukocytes in humans, is one of this bacterium's major virulence factors. Amounts of neutrophil elastase (NE), which is normally localized in the cytoplasm of neutrophils, are reportedly increased in the saliva of patients with periodontitis. However, the mechanism by which NE is released from human neutrophils and the role of NE in periodontitis is unclear. In the present study, it was hypothesized that LtxA induces NE release from human neutrophils, which subsequently causes the breakdown of periodontal tissues. LtxA-treatment did not induce significant cytotoxicity against human gingival epithelial cells (HGECs) or human gingival fibroblasts (HGFs). However, it did induce significant cytotoxicity against human neutrophils, leading to NE release. Furthermore, NE and the supernatant from LtxA-treated human neutrophils induced detachment and death of HGECs and HGFs, these effects being inhibited by administration of an NE inhibitor, sivelestat. The present results suggest that LtxA mediates human neutrophil lysis and induces the subsequent release of NE, which eventually results in detachment and death of HGECs and HGFs. Thus, LtxA-induced release of NE could cause breakdown of periodontal tissue and thereby exacerbate periodontitis.

Immunization with pneumococcal elongation factor Tu enhances serotype-independent protection against Streptococcus pneumoniae infection.

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

Antimicrobial susceptibility of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from children with acute otitis media in Japan from 2014 to 2017.

Increase in antimicrobial resistance (AMR) among pathogenic bacteria is a serious threat to public health. Surveillance studies to monitor shifting trends in resistance are important and guide the selection of appropriate antimicrobial agents for a particular organism. Furthermore, these studies help in dissemination of accurate information regarding AMR to the public. In this study, we investigated the antimicrobial susceptibility patterns of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis clinical isolates from outpatient children with acute otitis media in Japan from 2014 to 2017. A total of 8693 strains (2415 of S. pneumoniae, 3657 of H. influenzae, and 2621 of M. catarrhalis) were clinically isolated, and their antimicrobial susceptibilities to benzylpenicillin (PCG), ampicillin (ABPC), amoxicillin-clavulanic (AMPC/CVA), azithromycin (AZM), ceftriaxone (CTRX), and levofloxacin (LVFX) were investigated. Based on the minimum inhibitory concentration (MIC) breakpoints, the average proportion of S. pneumoniae isolates non-susceptible to PCG and AZM was 38.2% and 82.0% respectively. The average proportion of H. influenzae isolates non-susceptible to ABPC, CVA/AMPC, and CTRX was 61.9%, 43.5%, and 49.4%, respectively. The high prevalence of these resistant organisms is attributed to frequent use of antibiotic agents in Japan. Moreover, the proportion of LVFX-non-susceptible H. influenzae isolates increased in this four-year study. Here, we report updates regarding the AMR trends amongst the major pathogens that cause acute otitis media in Japan. Continuing surveillance of antimicrobial susceptibility and application of control measures against further transmission are required to decrease the emergence of resistant strains.

Peptides from rice endosperm protein restrain periodontal bone loss in mouse model of periodontitis.

OBJECTIVE: Food-derived peptides have been reported to exhibit antibacterial activity against periodontal pathogenic bacteria. However, no effect has been shown on inflammation and bone resorption in periodontal pathology. The overall objective of the current study was to investigate how rice peptides influence biological defense mechanisms against periodontitis-induced inflammatory bone loss, and identify their novel functions as a potential anti-inflammatory drug. DESIGN: The expression of inflammatory and osteoclast-related molecules was examined in mouse macrophage-derived RAW 264.7 cell cultures using qPCR. Subsequently, the effect of these peptides on inflammatory bone loss in mouse periodontitis was examined using a mouse model of tooth ligation. Briefly, periodontal bone loss was induced for 7 days in mice by ligating the maxillary second molar and leaving the contralateral tooth un-ligated (baseline control). The mice were microinjected daily with the peptide in the gingiva until the day before euthanization. One week after the ligation, TRAP-positive multinucleated cells (MNCs) were enumerated from five random coronal sections of the ligated sites in each mouse. RESULTS: Rice peptides REP9 and REP11 significantly inhibited transcription activity of inflammatory and osteoclast-related molecules. Local treatment with the rice peptides, in mice subjected to ligature-induced periodontitis, inhibited inflammatory bone loss, explaining the decreased numbers of osteoclasts in bone tissue sections. CONCLUSION: Therefore, these data suggested that the rice peptides possess a protective effect against periodontitis.

Mechanism of Macrolide-Induced Inhibition of Pneumolysin Release Involves Impairment of Autolysin Release in Macrolide-Resistant Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of community-acquired pneumonia. Over the past 2 decades, macrolide resistance among S. pneumoniae organisms has been increasing steadily and has escalated at an alarming rate worldwide. However, the use of macrolides in the treatment of community-acquired pneumonia has been reported to be effective regardless of the antibiotic susceptibility of the causative pneumococci. Although previous studies suggested that sub-MICs of macrolides inhibit the production of the pneumococcal pore-forming toxin pneumolysin by macrolide-resistant S. pneumoniae (MRSP), the underlying mechanisms of the inhibitory effect have not been fully elucidated. Here, we show that the release of pneumococcal autolysin, which promotes cell lysis and the release of pneumolysin, was inhibited by treatment with azithromycin and erythromycin, whereas replenishing with recombinant autolysin restored the release of pneumolysin from MRSP. Additionally, macrolides significantly downregulated ply transcription followed by a slight decrease of the intracellular pneumolysin level. These findings suggest the mechanisms involved in the inhibition of pneumolysin in MRSP, which may provide an additional explanation for the benefits of macrolides on the outcome of treatment for pneumococcal diseases.

Refractive-index-sensing optical comb based on photonic radio-frequency conversion with intracavity multi-mode interference fiber sensor.

Optical frequency combs (OFCs) have attracted attention as optical frequency rulers due to their tooth-like discrete spectra together with their inherent mode-locking nature and phase-locking control to a frequency standard. Based on this concept, their applications until now have been demonstrated in the fields of optical frequency metrology. However, if the utility of OFCs can be further expanded beyond their application by exploiting new aspects of OFCs, this will lead to new developments in optical metrology and instrumentation. Here, we report a fiber sensing application of OFCs based on a coherent link between the optical and radio frequencies, enabling high-precision refractive index measurement based on frequency measurement in radio-frequency (RF) region. Our technique encodes a refractive index change of a liquid sample into a repetition frequency of OFC by a combination of an intracavity multi-mode-interference fiber sensor and wavelength dispersion of a cavity fiber. Then, the change in refractive index is read out by measuring the repetition frequency in RF region based on a frequency standard. Use of an OFC as a photonic RF converter will lead to the development of new applications in high-precision fiber sensing with the help of functional fiber sensors and precise RF measurement.

Neutrophil Elastase Subverts the Immune Response by Cleaving Toll-Like Receptors and Cytokines in Pneumococcal Pneumonia.

Excessive activation of neutrophils results in the release of neutrophil elastase (NE), which leads to lung injury in severe pneumonia. Previously, we demonstrated a novel immune subversion mechanism involving microbial exploitation of this NE ability, which eventually promotes disruption of the pulmonary epithelial barrier. In the present study, we investigated the effect of NE on host innate immune response. THP-1-derived macrophages were stimulated with heat-killed Streptococcus pneumoniae or lipopolysaccharide in the presence or absence of NE followed by analysis of toll-like receptor (TLR) and cytokine expression. Additionally, the biological significance of NE was confirmed in an in vivo mouse intratracheal infection model. NE downregulated the gene transcription of multiple cytokines in THP-1-derived macrophages through the cleavage of TLRs and myeloid differentiation factor 2. Additionally, NE cleaved inflammatory cytokines and chemokines. In a mouse model of intratracheal pneumococcal challenge, administration of an NE inhibitor significantly increased proinflammatory cytokine levels in bronchoalveolar lavage fluid, enhanced bacterial clearance, and improved survival rates. Our work indicates that NE subverts the innate immune response and that inhibition of this enzyme may constitute a novel therapeutic option for the treatment of pneumococcal pneumonia.

Pneumococcal DNA-binding proteins released through autolysis induce the production of proinflammatory cytokines via toll-like receptor 4.

Streptococcus pneumoniae is a leading cause of bacterial pneumonia. Our previous study suggested that S. pneumoniae autolysis-dependently releases intracellular pneumolysin, which subsequently leads to lung injury. In this study, we hypothesized that pneumococcal autolysis induces the leakage of additional intracellular molecules that could increase the pathogenicity of S. pneumoniae. Liquid chromatography tandem-mass spectrometry analysis identified that chaperone protein DnaK, elongation factor Tu (EF-Tu), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were released with pneumococcal DNA by autolysis. We demonstrated that recombinant (r) DnaK, rEF-Tu, and rGAPDH induced significantly higher levels of interleukin-6 and tumor necrosis factor production in peritoneal macrophages and THP-1-derived macrophage-like cells via toll-like receptor 4. Furthermore, the DNA-binding activity of these proteins was confirmed by surface plasmon resonance assay. We demonstrated that pneumococcal DnaK, EF-Tu, and GAPDH induced the production of proinflammatory cytokines in macrophages, and might cause host tissue damage and affect the development of pneumococcal diseases.

IgG4-related Lung Pseudotumor and Pleural Inflammation with Autoimmune Hepatitis.

A 63-year-old man was admitted to our department following a secondary medical examination. Blood tests showed high levels of liver enzymes, IgG, IgG4, and antinuclear antibody. Computed tomography showed tumors in the bilateral lower lobes of the lungs and pleural thickening. After pleural and liver biopsy procedures, he was conclusively diagnosed with IgG4-related lung pseudotumor and pleural inflammation with autoimmune hepatitis. We started treatment with prednisolone 40 mg/day, and chest radiograph and blood tests showed signs of improvement. This was a rare case that suggested an association between IgG4-related disease and autoimmune hepatitis.

Antimicrobial activity of ethylene-vinyl acetate containing bioactive filler against oral bacteria.

We developed an ethylene vinyl acetate (EVA) containing surface pre-reacted glass-ionomer (S-PRG) filler, as a new mouthguard material for preventing intraoral bacterial infection. We examined its physical properties, antimicrobial activity against a major cariogenic bacterium Streptococcus mutans and a periodontopathogen Porphyromonas gingivalis, and its cytotoxicity toward human gingival epithelial cells. S-PRG filler was added to EVA copolymer at 5, 10, 20, or 40 wt% and was processed into disc-shaped test specimens. Only minor differences between the Shore hardness and rebound resilience properties of EVA materials with and without the S-PRG filler were observed. The specimens with S-PRG filler showed bacteriostatic activity toward S. mutans and P. gingivalis and inhibited S. mutans biofilm formation. No cytotoxicity against the gingival epithelial cells was observed. Our findings show that EVA containing S-PRG filler has antimicrobial activity toward pathogenic oral bacteria and may be an effective material for maintaining the oral health of athletes.