The clinical utility of comprehensive measurement of autoimmune disease-related antibodies in patients with advanced solid tumors receiving immune checkpoint inhibitors: a retrospective study.

BACKGROUND: The comprehensive measurement of autoimmune disease-related antibodies (Abs) before immune checkpoint inhibitor (ICI) treatment may be useful for predicting the development of immune-related adverse events (irAEs); however, the clinical utility is not well known. MATERIALS AND METHODS: We retrospectively analyzed patients with advanced solid tumors treated with ICI monotherapy or doublet combination therapy between July 2014 and December 2020 at single institute. Anti-nuclear antibody (ANA), anti-thyroglobulin (Tg) Ab, anti-thyroid peroxidase (TPO) Ab, anti-glutamic acid decarboxylase (GAD) Ab, anti-acetylcholine esterase receptor (AchR) Ab, and platelet-associated immunoglobulin G (PA-IgG) Ab were comprehensively measured for the screening before ICI therapy. RESULTS: Of 275 registered patients (median age, 70 years; male, 64.4%; Eastern Cooperative Oncology Group performance status of 0 or 1, 88.7%; and prior regimen of 0-1/≥2, 88.7%/11.3%), 128 non-small-cell lung cancer, 35 gastric cancer, 33 head and neck cancer, 24 melanoma, 19 renal cell carcinoma, 13 urothelial carcinoma, 12 esophageal cancer, 5 malignant mesothelioma of pleura, 2 endometrial cancer, and 4 other cancer were included. The number of patients with positive ANA, Tg, TPO, PA-IgG, GAD, and AchR Abs was 52 (24.9%), 38 (14.5%), 11 (10.1%), 6 (3.5%), 5 (2.0%), and 1 (0.5%), respectively. There was no association between the development of any irAEs and Abs positivity, while thyroid dysfunction developed more frequently among patients with than without Tg Ab or TPO Ab (39.5% versus 12.5%, P < 0.01; 45.5% versus 14.3%, P = 0.02). CONCLUSIONS: The clinical utility of comprehensive measurement of autoimmune disease-related Abs before introduction of ICI therapy was limited for predicting irAE. However, Tg and TPO Abs were risk factors as regards the development of ICI-induced thyroid dysfunction.

Comparing the risk of hepatitis B virus reactivation between direct-acting antiviral therapies and interferon-based therapies for hepatitis C.

Hepatitis B virus (HBV) reactivation has been reported during antihepatitis C treatment in patients with hepatitis C virus (HCV) and HBV co-infection. We aimed to evaluate the frequency and risk factors of HBV reactivation during anti-HCV therapy and compared those between interferon (IFN)-free direct-acting antiviral (DAA) therapies and IFN-based therapies. Three hundred and twenty-two patients with HCV infection receiving anti-HCV therapy were retrospectively screened. The baseline HBV infection statuses of all eligible patients and the HBV-DNA level of all patients with current or previous HBV infection were examined at the end of treatment. In patients with baseline anti-HBs positivity, changes in anti-HBs titre were evaluated. Of 287 patients who met the inclusion criteria, 157 had current (n=4) or previous (n=153) HBV infection; 85 were treated with IFN-free DAA therapies and 72 were treated with IFN-based therapies. Six patients experienced HBV reactivation (n=2) or HBV reappearance (n=4) after IFN-free DAA therapies, while no patient developed HBV reactivation after IFN-based therapies. The risk factors of HBV reactivation or reappearance were DAA therapies and a reduction in anti-HBs titre to <12 mIU mL-1 by the end of treatment. The decline changes of anti-HBs titre were significantly higher in patients treated with DAA therapies. Although HBV reactivation hepatitis was not observed, three of four patients with HBV reactivation or reappearance after achieving HCV eradication had viremia 8 weeks after completion of therapy. A significant proportion of patients develop HBV reactivation or reappearance without hepatitis after IFN-free DAA therapies. Low levels of anti-HBs and their decrease to <12 mIU mL-1 after treatment are significant risk factors for HBV reactivation or reappearance.

Micro/extended-nano sampling interface from a living single cell.

Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells. Indeed, analysis of a specific analyte might require the analysis of a single molecule or several molecules. Analytical processes usually include sampling, chemical processing, and detection. Although several papers have reported chemical processing and detection methods for single cells, a sampling method compatible with maintaining the viability of a single cell during sampling has yet to be developed. Here, we propose a femtoliter sampling method from a living single cell using micro/nanofluidic device technology. The sampling of 39 fL of cytoplasm from a single human aortic endothelial cell was demonstrated and its viability after sampling was confirmed.

Simultaneous treatment with azelnidipine and olmesartan inhibits apoptosis of Hl-1 cardiac myocytes expressing E334k cMyBPC.

BACKGROUND: Apoptosis appears to play an important role in the pathogenesis of hypertrophic cardiomyopathy (HCM). We have previously reported 3 HCM patients carrying the E334K MYBPC3, and that heterologous expression of E334K cMyBPC in cultured cells induced apoptosis. The purpose of this study was to identify pharmacological agents that would inhibit apoptosis in HL-1 cardiomyocytes expressing E334K cMyBPC. METHODS AND RESULTS: E334K cMyBPC expression in cells increased levels of pro-apoptosis (p53, Bax and cytochrome c) and decreased levels of anti-apoptosis (Bcl-2 and Bcl-XL). While the beta blocker carvedilol (1 µM) normalized the level of p53 and Bcl-2 and the calcium channel blocker (CCB) bepridil (0.5 µM) normalized that of Bcl-2, both the CCB azelnidipine (1 µM) and the angiotensin receptor blocker (ARB) olmesartan (10 µM) normalized those of p53, Bax, cytochrome c, and Bcl-XL. Among those proteins, cytochrome c was the one which showed the highest degree of change. Both azelnidipine (0.1 µM) and olmesartan (1 µM) reduced the level of cytochrome c by 40.2 ± 4.3% and 31.3 ± 5.1%, respectively. The CCB amlodipine and the ARB valsartan reduced it only by 19.1 ± 2.1% and 20.1 ± 5.2%, respectively. Flow cytometric analysis and annexin V staining showed that treatment of cells with azelnidipine (0.1 µM) plus olmesartan (0.3 µM) or that with amlodipine (0.1 µM) plus valsartan (0.3 µM) reduced the number of apoptotic cells by 35.8 ± 10.5% and 18.4 ± 3.2%, respectively. CONCLUSION: Azelnidipine plus olmesartan or amlodipine plus valsartan inhibited apoptosis of HL-1 cells expressing E334K cMyBPC, and the former combination was more effective than the latter.

Lymphotropic hepatitis C virus has an interferon-resistant phenotype.

Hepatitis C virus (HCV) infects and associates with B cells, leading to abnormal B-cell activation and development of lymphoproliferative and autoimmune disorders. This immune perturbation may in turn be associated with the resistance of HCV against the host immune system. The objective of this study was to analyse the effects of HCV infection of B cells on the efficacy of interferon (IFN)-based therapy. The study enrolled 102 patients with chronic hepatitis C who were treated with pegylated IFN plus ribavirin. HCV RNA titres in B cells were compared in patients with rapid viral responder (RVR) vs non-RVR, sustained viral responder (SVR) vs non-SVR and null viral responder (NVR) vs VR. The levels of HCV RNA in B cells were significantly higher in non-RVR, non-SVR and NVR groups. Association between the therapy outcome and the positive B-cell HCV RNA was also investigated in relation to other known viral and host factors. Multivariable analyses showed that the positive B-cell HCV RNA and the minor single-nucleotide polymorphism near the IL28B gene (rs8099917) were independent factors associated with NVR in patients infected with HCV genotype 1. When these two factors were combined, the sensitivity, specificity, positive and negative predictive values for NVR were 92.3%, 98.2%, 92.3% and 98.2%, respectively. Genotype 1 and the presence of one or no mutations in the IFN-sensitivity determining region were associated with higher levels of B-cell HCV RNA. B-cell-tropic HCV appears to have an IFN-resistant phenotype. B-cell HCV RNA positivity is a predictive factor for resistance to IFN-based therapy.

Thin-section CT findings of patients with acute Streptococcus pneumoniae pneumonia with and without concurrent infection.

OBJECTIVES: The aim of this study was to compare the pulmonary thin-section CT findings of patients with acute Streptococcus pneumoniae pneumonia with and without concurrent infection. METHODS: The study group comprised 86 patients with acute S. pneumoniae pneumonia, 36 patients with S. pneumoniae pneumonia combined with Haemophilus influenzae infection, 26 patients with S. pneumoniae pneumonia combined with Pseudomonas aeruginosa infection and 22 patients with S. pneumoniae pneumonia combined with methicillin-susceptible Staphylococcus aureus (MSSA) infection. We compared the thin-section CT findings among the groups. RESULTS: Centrilobular nodules and bronchial wall thickening were significantly more frequent in patients with pneumonia caused by concurrent infection (H. influenzae: p<0.001 and p<0.001, P. aeruginosa: p<0.001 and p<0.001, MSSA: p<0.001 and p<0.001, respectively) than in those infected with S. pneumoniae alone. Cavity and bilateral pleural effusions were significantly more frequent in cases of S. pneumoniae pneumonia with concurrent P. aeruginosa infection than in cases of S. pneumoniae pneumonia alone (p<0.001 and p<0.001, respectively) or with concurrent H. influenzae (p<0.05 and p<0.001, respectively) or MSSA infection (p<0.05 and p<0.05, respectively). CONCLUSIONS: When a patient with S. pneumoniae pneumonia has centrilobular nodules, bronchial wall thickening, cavity or bilateral pleural effusions on CT images, concurrent infection should be considered.

Meticillin-resistant Staphylococcus aureus and meticillin-susceptible S. aureus pneumonia: comparison of clinical and thin-section CT findings.

OBJECTIVES: The purpose of this study was to compare the clinical and thin-section CT findings in patients with meticillin-resistant Staphylococcus aureus (MRSA) and meticillin-susceptible S. aureus (MSSA). METHODS: We retrospectively identified 201 patients with acute MRSA pneumonia and 164 patients with acute MSSA pneumonia who had undergone chest thin-section CT examinations between January 2004 and March 2009. Patients with concurrent infectious disease were excluded from our study. Consequently, our study group comprised 68 patients with MRSA pneumonia (37 male, 31 female) and 83 patients with MSSA pneumonia (32 male, 51 female). Clinical findings in the patients were assessed. Parenchymal abnormalities, lymph node enlargement and pleural effusion were assessed. RESULTS: Underlying diseases such as cardiovascular were significantly more frequent in the patients with MRSA pneumonia than in those with MSSA pneumonia. CT findings of centrilobular nodules, centrilobular nodules with a tree-in-bud pattern, and bronchial wall thickening were significantly more frequent in the patients with MSSA pneumonia than those with MRSA pneumonia (p = 0.038, p = 0.007 and p = 0.039, respectively). In the group with MRSA, parenchymal abnormalities were observed to be mainly peripherally distributed and the frequency was significantly higher than in the MSSA group (p = 0.028). Pleural effusion was significantly more frequent in the patients with MRSA pneumonia than those with MSSA pneumonia (p = 0.002). CONCLUSIONS: Findings from the evaluation of thin-section CT manifestations of pneumonia may be useful to distinguish between patients with acute MRSA pneumonia and those with MSSA pneumonia.

Mineral trioxide aggregate inhibits osteoclastic bone resorption.

Mineral trioxide aggregate (MTA), a commonly used endodontic repair material, is useful for both basic and clinical research, and the effect of MTA on osteoblast differentiation has been well-defined. However, the effects of MTA on osteoclastic bone resorption are not fully understood. Hence, the aim of this study is to examine the effect of MTA solution in the regulation of osteoclast bone-resorbing activity using osteoclasts formed in co-cultures of primary osteoblasts and bone marrow cells. MTA solution dose-dependently reduced the total area of pits formed by osteoclasts. The reduction of resorption induced by 20% MTA treatment was due to inhibition of osteoclastic bone-resorbing activity and had no effect on osteoclast number. A 20% MTA solution disrupted actin ring formation, a marker of osteoclastic bone resorption, by reducing phosphorylation and kinase activity of c-Src, and mRNA expressions of cathepsin K and mmp-9. A high concentration of MTA solution (50%) induced apoptosis of osteoclasts by increasing the expression of Bim, a member of the BH3-only (Bcl-2 homology) family of pro-apoptotic proteins. Taken together, our results suggest that MTA is a useful retrofilling material for several clinical situations because it both stimulates osteoblast differentiation and inhibits bone resorption.

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus.

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.

Transcriptional activation of the anchoring protein SAP97 by heat shock factor (HSF)-1 stabilizes K(v) 1.5 channels in HL-1 cells.

BACKGROUND AND PURPOSE: The expression of voltage-dependent K(+) channels (K(v) ) 1.5 is regulated by members of the heat shock protein (Hsp) family. We examined whether the heat shock transcription factor 1 (HSF-1) and its inducer geranylgeranylacetone (GGA) could affect the expression of K(v) 1.5 channels and its anchoring protein, synapse associated protein 97 (SAP97). EXPERIMENTAL APPROACH: Transfected mouse atrial cardiomyocytes (HL-1 cells) and COS7 cells were subjected to luciferase reporter gene assay and whole-cell patch clamp. Protein and mRNA extracts were subjected to Western blot and quantitative real-time polymerase chain reaction. KEY RESULTS: Heat shock of HL-1 cells induced expression of Hsp70, HSF-1, SAP97 and K(v) 1.5 proteins. These effects were reproduced by wild-type HSF-1. Both heat shock and expression of HSF-1, but not the R71G mutant, increased the SAP97 mRNA level. Small interfering RNA (siRNA) against SAP97 abolished HSF-1-induced increase of K(v) 1.5 and SAP97 proteins. A luciferase reporter gene assay revealed that the SAP97 promoter region (from -919 to -740) that contains heat shock elements (HSEs) was required for this induction. Suppression of SIRT1 function either by nicotinamide or siRNA decreased the level of SAP97 mRNA. SIRT1 activation by resveratrol had opposing effects. A treatment of the cells with GGA increased the level of SAP97 mRNA, K(v) 1.5 proteins and I(Kur) current, which could be modified with either resveratrol or nicotinamide. CONCLUSIONS AND IMPLICATIONS: HSF-1 induced transcription of SAP97 through SIRT1-dependent interaction with HSEs; the increase in SAP97 resulted in stabilization of K(v)1.5 channels. These effects were mimicked by GGA.

Experiment study on fracture fixation with low rigidity titanium alloy: plate fixation of tibia fracture model in rabbit.

In order to investigate bone tissue reaction to the low rigidity titanium alloy of TNTZ in bone plate fixation, animal experiment with rabbit was performed with X-ray follow-up and histological observation. Experimental fractures were made in rabbit tibiae, and fixed by different bone plates of SUS316L, Ti-6Al-4V and TNTZ. Although there was no significant difference in fracture healing, bone atrophy was observed in cortical bone especially under the bone plate, which was different in time course among three materials. The bone atrophy under the bone plate was confirmed as porous or poor bone tissue in histological observation. In addition, the diameter of the tibia bone was increased in TNTZ as the result of bone remodeling with a new cortical bone. It is confirmed that the elastic modulus of the bone plate will naturally influence bone tissue reaction to the bone plate fixation according to the Wolff's law of functional restoration.

Arsenic in various foods: cumulative data.

Data for the arsenic content in various foods were collated. The number of collected values was about 2500 columns, which enables an estimation of the range of arsenic contents in each food group. Data were categorized into six groups (crops, milk/meat/egg, fish, algae, seafood, others) and expressed as a percentile graph. In addition, the inorganic arsenic ratio of each food group was estimated. This approach enabled the authors to understand the arsenic contents of some food groups at a glance. The intake of inorganic arsenic seems to be mostly from seafood. The contribution from other categories of food is small.

Atomic force microscopy dissects the hierarchy of genome architectures in eukaryote, prokaryote, and chloroplast.

Because of its applicability to biological specimens (nonconductors), a single-molecule-imaging technique, atomic force microscopy (AFM), has been particularly powerful for visualizing and analyzing complex biological processes. Comparative analyses based on AFM observation revealed that the bacterial nucleoids and human chromatin were constituted by a detergent/salt-resistant 30-40-nm fiber that turned into thicker fibers with beads of 70-80 nm diameter. AFM observations of the 14-kbp plasmid and 110-kbp F plasmid purified from Escherichia coli demonstrated that the 70-80-nm fiber did not contain a eukaryotic nucleosome-like "beads-on-a-string" structure. Chloroplast nucleoid (that lacks bacterial-type nucleoid proteins and eukaryotic histones) also exhibited the 70-80-nm structural units. Interestingly, naked DNA appeared when the nucleoids from E. coli and chloroplast were treated with RNase, whereas only 30-nm chromatin fiber was released from the human nucleus with the same treatment. These observations suggest that the 30-40-nm nucleoid fiber is formed with a help of nucleoid proteins and RNA in E. coli and chroloplast, and that the eukaryotic 30-nm chromatin fiber is formed without RNA. On the other hand, the 70-80-nm beaded structures in both E. coli and human are dependent on RNA.

Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding.

The proper function of the genome largely depends on the higher order architecture of the chromosome. Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004). In this study, the hierarchy of genome organization was compared among E. coli, Staphylococcus aureus and Clostridium perfringens. A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E. coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber. An application of this on-substrate procedure to the S. aureus and the C. perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution. The E. coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps. However, the S. aureus and the C. perfringens nucleoids never underwent such tight compaction when they reached stationary phase. Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species. AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers. Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes.

Modulatory effect of fosfomycin on acute inflammation in the rat air pouch model.

We examined the effect of fosfomycin (FOM) on the inflammatory response induced by carrageenan in the rat. Air pouches were induced subcutaneously on the backs of rats and injected with carrageenan. The rats were treated with either vehicle or FOM at a dose of 100 mg/kg 1 h before carrageenan challenge. After carrageenan challenge (48 h), the air pouches were removed and analyzed. The volume, protein amounts and cell counts in the exudate obtained from FOM-treated animals were significantly reduced compared with that from vehicle-treated animals. The contents of PGE(2) and TNF-alpha, and mRNA for cyclooxygenase-2 were also markedly suppressed in FOM-treated rats. Histological examination showed suppression of the inflammatory response in the pouch tissues from FOM-treated rats.

Modulation of IgD and CD20 by ligation of CD5 on tonsillar B cells.

The CD5 molecule, pan T cell marker, has been known to be expressed on a minor population of B cells, termed B-1 cells. However, the physiological function and pathological role of CD5+B (B-1) cells remain to be fully elucidated in humans. In the present study, we aimed to clarify the significance of CD5 expression on the B lymphocytes in human tonsil. Using flow cytometric analysis by three-colour immunofluorescence staining, we observed a majority of the cell surface CD5-positive (sCD5+) B cells among the sIgD+ B-cell population, as previously described. Contrary to our expectation, approximately half of the sIgD+/sCD5+ B cells expressed CD38 on their cell surface. Furthermore, a small number of sCD5+ were observed in the sIgD- B cell population. The addition of anti-CD5 monoclonal antibody (MoAb) to the culture induced downmodulation of sCD20 and sIgD of the tonsillar B cells, resulting in an increase of sCD38-/sIgD- (memory) B cells during the 10 day culture periods in the CD40/l cell culture system. Our findings suggest that ligation of CD5 might transduce the signal to regulate B cell maturation.

Modulatory effect of macrolide antibiotics on the Th1- and Th2-type cytokine production.

The effect of the macrolide antibiotics, clarithromycin, midecamycin acetate and josamycin, on the generation of Th1- and Th2-type cytokines by mitogen-stimulated human T lymphocytes was compared with that of fosfomycin. The following results were obtained. These drugs demonstrated potent inhibitory activity on the release and gene expression of TNF-alpha and IL-2. Their inhibitory effect on IFN-alpha, IL-4, IL-5, IL-6 was less marked. The release of IL-10 was poorly suppressed. Clarithromycin had the most potent inhibitory effect of the drugs used. The present results suggested that anti-bacterial agents might modify the host's immunological response by interfering with the activity of T helper cells.

Synthetic activity of Sso DNA polymerase Y1, an archaeal DinB-like DNA polymerase, is stimulated by processivity factors proliferating cell nuclear antigen and replication factor C.

DNA replication efficiency is dictated by DNA polymerases (pol) and their associated proteins. The recent discovery of DNA polymerase Y family (DinB/UmuC/RAD30/REV1 superfamily) raises a question of whether the DNA polymerase activities are modified by accessory proteins such as proliferating cell nuclear antigen (PCNA). In fact, the activity of DNA pol IV (DinB) of Escherichia coli is enhanced upon interaction with the beta subunit, the processivity factor of DNA pol III. Here, we report the activity of Sso DNA pol Y1 encoded by the dbh gene of the archaeon Sulfolobus solfataricus is greatly enhanced by the presence of PCNA and replication factor C (RFC). Sso pol Y1 per se was a distributive enzyme but a substantial increase in the processivity was observed on poly(dA)-oligo(dT) in the presence of PCNA (039p or 048p) and RFC. The length of the synthesized DNA product reached at least 200 nucleotides. Sso pol Y1 displayed a higher affinity for DNA compared with pol IV of E. coli, suggesting that the two DNA polymerases have distinct reason(s) to require the processivity factors for efficient DNA synthesis. The abilities of pol Y1 and pol IV to bypass DNA lesions and their sensitive sites to protease are also discussed.

Overexpression of sigma factor, sigma(B), urges Staphylococcus aureus to thicken the cell wall and to resist beta-lactams.

Whole genome sequence analysis revealed that Staphylococcus aureus is provided with only a few sigma factors, including the alternative sigma factor, sigma(B), which is thought to regulate some stress responses. Since the sigB knock-out mutant did not show remarkable phenotypic difference, we constructed the over expressed mutant to examine the role of the sigB. Electron microscopic observation revealed that the mutant showed a variety of cell sizes compared with the parent strain which showed almost homogeneous cell sizes. The mutant delivered a thicker cell wall, about 20% thicker than the parent strain. It became resistant to the lytic activity of lysostaphin and also raised MICs to the cell-wall-affecting antibiotics. The yield of carotenoids and transcripts of pbps were also increased in the mutant. The result suggests that sigB plays some important roles in cell wall synthesis and in resistance to antibiotics that perturb the cell wall synthesis.