Matrix metalloproteinase­1 and microRNA­486­5p in urinary exosomes can be used to detect early lung cancer: A preliminary report.

The present study describes a novel molecular-genetic method suitable for lung cancer (LC) screening in the work-place and at community health centers. Using urinary-isolated exosomes from 35 patients with LC and 40 healthy volunteers, the expression ratio of MMP-1/CD63, and the relative expression levels of both microRNA (miRNA)-21 and miRNA-486-5p were measured. MMP-1/CD63 expression ratio was significantly higher in patients with LC than in the healthy controls {1.342 [95% confidence interval (CI): 0.890-1.974] vs. 0.600 (0.490-0.900); P<0.0001}. The relative expression of miRNA-486-5p in male healthy controls was significantly different from that in female healthy controls, whereas there was no significant difference in miRNA-21. Receiver operating characteristic curve (ROC) analysis of MMP-1/CD63 showed 92.5% sensitivity and 54.3% specificity, whereas miRNA-486-5p showed 85% sensitivity and 70.8% specificity for men, and 70.0% sensitivity and 72.7% specificity for women. The logistic regression model used to evaluate the association of LC with the combination of MMP-1/CD63 and miRNA-486-5p revealed that the area under the ROC curve was 0.954 (95% CI: 0.908-1.000), and the model had 89% sensitivity and 88% specificity after adjusting for age, sex and smoking status. These data suggested that the combined analysis of MMP-1/CD63 and miRNA-486-5p in urinary exosomes may be used to detect patients with early-stage LC in the work-place and at community health centers, although confirmational studies are warranted.

Smoking cessation in the elderly as a sign of susceptibility to symptomatic COVID-19 reinfection in the United States.

Background: We aimed to clarify the relationship between coronavirus disease 2019 (COVID-19) reinfection and basic disease and smoking status. Methods: The electronic health records of 165,320 patients with COVID-19 from January 1, 2020, to August 27, 2021, were analyzed. Data on age, race, sex, smoking status (never, current, former), and basic disease were analyzed using Cox proportional hazard models. Results: In total, 6,133 patients (3.7%) were reinfected. The overall reinfection rate for never, current, and former smokers was 4.2, 3.5, and 5.7%, respectively. Although the risk of reinfection was highest among former smokers aged ≥65 years (7.7% [422/5,460]), the reinfection rate among current smokers aged ≥65 years was 6.2% (341/5,543). Among reinfected patients, the number of basic diseases was higher in former smokers (2.41 ± 1.16) than in current (2.28 ± 1.07, P = 0.07) and never smokers (2.07 ± 1.05, P < 0.001). Former smokers who are older may have been exposed to factors that increase their risk of symptomatic COVID-19 reinfection.

Mint3 depletion-mediated glycolytic and oxidative alterations promote pyroptosis and prevent the spread of Listeria monocytogenes infection in macrophages.

Listeria monocytogenes (LM) infection induces pyroptosis, a form of regulated necrosis, in host macrophages via inflammasome activation. Here, we examined the role of Mint3 in macrophages, which promotes glycolysis via hypoxia-inducible factor-1 activation, during the initiation of pyroptosis following LM infection. Our results showed that Mint3-deficient mice were more resistant to lethal listeriosis than wild-type (WT) mice. Additionally, the mutant mice showed higher levels of IL-1ß/IL-18 in the peritoneal fluid during LM infection than WT mice. Moreover, ablation of Mint3 markedly increased the activation of caspase-1, maturation of gasdermin D, and pyroptosis in macrophages infected with LM in vitro, suggesting that Mint3 depletion promotes pyroptosis. Further analyses revealed that Mint3 depletion upregulates inflammasome assembly preceding pyroptosis via glycolysis reduction and reactive oxygen species production. Pharmacological inhibition of glycolysis conferred resistance to listeriosis in a Mint3-dependent manner. Moreover, Mint3-deficient mice treated with the caspase-1 inhibitor VX-765 were as susceptible to LM infection as WT mice. Taken together, these results suggest that Mint3 depletion promotes pyroptosis in host macrophages, thereby preventing the spread of LM infection. Mint3 may serve as a target for treating severe listeriosis by inducing pyroptosis in LM-infected macrophages.

TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation.

Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

Novel breast cancer screening: combined expression of miR-21 and MMP-1 in urinary exosomes detects 95% of breast cancer without metastasis.

Serum and tissue miR-21 expression in patients with breast cancer (BC) is a useful biomarker for cancer diagnosis, progression, and treatment. Matrix metalloproteinase-1 (MMP-1) is also important in breast cancer carcinogenesis. However, miR-21 and MMP-1/CD63 in urine exosomes in these patients have not been examined. Urine samples were collected from patients with BC and 26 healthy females. Urinary exosomes were isolated and confirmed by western blotting with anti-CD63 antibody and electron microscopy observation. MiR-21 and MMP-1/CD63 expression was examined by quantitative RT-PCR and western blotting, respectively. Patients with very early stage breast cancer were evaluated. MiR-21 expression in the patients was 0.26 [95% CI: 0.20-0.78], which was significant lower than in the 26 controls (1.00 [95% CI: 1.01-3.37], p = 0.0947). MMP-1/CD63 expression in patients was significantly higher than in controls (1.74 [95% CI: 0.86-5.08] vs 0.535 [95% CI: -0.01-2.81], p = 0.0001). Sensitivity and specificity were 0.708 and 0.783 for miR-21 and 0.792 and 0.840 for MMP-1/CD63, respectively. Sensitivity and specificity of combined expression were 95% and 79%, respectively. The sensitivity of MMP-1/CD63 expression in urinary exosomes was better than that of miR-21 expression. Thus, miR-21 and MMP/CD63 may be useful markers for BC screening.

Acute onset of autoimmune hepatitis with sinusoidal and central vein endotheliitis, and marked involvement of activated dendritic cells: A case report.

RATIONALE: Autoimmune hepatitis (AIH) is characterised by interface hepatitis. However, some acute cases exhibit atypical centrilobular necrosis with mild portal inflammation. Detailed histological and ultrastructural analyses of acute AIH are limited. PATIENT CONCERNS: A 44-year-old female was admitted to our hospital with jaundice, general fatigue and liver dysfunction. Her transaminase levels were elevated, her immunoglobulin G level was 1735 mg/dL and her anti-nuclear titres were ×80. DIAGNOSIS: AIH was diagnosed, and histochemical examination of a liver biopsy showed the presence of atypical histological features of prominent centrilobular necrosis and central vein and hepatic sinusoidal endotheliitis. Electron microscopy showed that dendritic cells (DCs) and lymphocytes were attached to disrupted liver sinusoidal endothelial cells (LSECs) in hepatic sinusoids and that DCs attached to LSECs via pseudopods in the central vein. INTERVENTIONS AND OUTCOMES: The patient was started on 40 mg/day prednisolone to control the hepatic inflammation. Her aspartate and alanine aminotransferase levels started declining after prednisolone was initiated. Three weeks later, these levels had normalised. The dosage of prednisolone was gradually decreased as liver function improved. The patient remains under observation and continues to receive 2.5 mg prednisolone. LESSONS: An important marker of acute AIH may be the presence of activated DCs in the hepatic sinusoids and central vein.

Mint3/Apba3 depletion ameliorates severe murine influenza pneumonia and macrophage cytokine production in response to the influenza virus.

Influenza virus (IFV) infection is a common cause of severe pneumonia. Studies have suggested that excessive activation of the host immune system including macrophages is responsible for the severe pathologies mediated by IFV infection. Here, we focused on the X11 protein family member Mint3/Apba3, known to promote ATP production via glycolysis by activating hypoxia inducible factor-1 (HIF-1) in macrophages, and examined its roles in lung pathogenesis and anti-viral defence upon IFV infection. Mint3-deficient mice exhibited improved influenza pneumonia with reduced inflammatory cytokines/chemokine levels and neutrophil infiltration in the IFV-infected lungs without alteration in viral burden, type-I interferon production, or acquired immunity. In macrophages, Mint3 depletion attenuated NF-κB signalling and the resultant cytokine/chemokine production in response to IFV infection by increasing IκBα and activating the cellular energy sensor AMPK, respectively. Thus, Mint3 might represent one of the likely therapeutic targets for the treatment of severe influenza pneumonia without affecting host anti-viral defence through suppressing macrophage cytokine/chemokine production.

Reevaluation of erythropoietin production by the nephron.

Erythropoietin production has been reported to occur in the peritubular interstitial fibroblasts in the kidney. Since the erythropoietin production in the nephron is controversial, we reevaluated the erythropoietin production in the kidney. We examined mRNA expressions of erythropoietin and HIF PHD2 using high-sensitive in situ hybridization system (ISH) and protein expression of HIF PHD2 using immunohistochemistry in the kidney. We further investigated the mechanism of erythropoietin production by hypoxia in vitro using human liver hepatocell (HepG2) and rat intercalated cell line (IN-IC cells). ISH in mice showed mRNA expression of erythropoietin in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs) and cortical collecting ducts (CCDs) but not in the peritubular cells under normal conditions. Hypoxia induced mRNA expression of erythropoietin largely in peritubular cells and slightly in PCTs, DCTs, and CCDs. Double staining with AQP3 or AE1 indicated that erythropoietin mRNA expresses mainly in ß-intercalated or non α/non ß-intercalated cells of the collecting ducts. Immunohistochemistry in rat showed the expression of HIF PHD2 in the collecting ducts and peritubular cells and its increase by anemia in peritubular cells. In IN-IC cells, hypoxia increased mRNA expression of erythropoietin, erythropoietin concentration in the medium and protein expression of HIF PHD2. These data suggest that erythropoietin is produced by the cortical nephrons mainly in the intercalated cells, but not in the peritubular cells, in normal hematopoietic condition and by mainly peritubular cells in hypoxia, suggesting the different regulation mechanism between the nephrons and peritubular cells.

Helicobacter pylori, a carcinogen, induces the expression of melanoma antigen-encoding gene (Mage)-A3, a cancer/testis antigen.

Cancer/testis antigens (CTAs) are known to be expressed in various cancer types but are minimally or not expressed in normal tissues except for germline tissues. CTAs are attractive targets for cancer immunotherapy and diagnosis because of their restricted expression. The mechanisms of CTAs expression are unclear because the inducers of CTAs expression remain to be elucidated. We hypothesized that carcinogens may induce the cellular expression of CTAs. To prove this, we attempted to inoculate Helicobacter pylori, a known carcinogen, in Meth-A cells, normal gastric cells, and normal splenocytes and induce the expression of a CTA. Melanoma antigen-encoding gene (Mage)-A3, one of the CTAs, was not expressed in both normal cells but in Meth-A cells inoculated with H. pylori. Furthermore, we performed limiting dilution using Meth-A cells inoculated with H. pylori and established derivative clone from Meth-A designated as Meth-A/pylori/3C3 which permanently express Mage-A3 after excluding H. pylori. We herein report the first successful induction of a CTA in a cell line via exposure to a carcinogenic agent. Furthermore, the establishment of Meth-A/pylori/3C3, which is Meth-A expressing Mage-A3, is considered to contribute to the resolution of the mechanism of CTAs expression.

Redox signal-mediated sensitization of transient receptor potential melastatin 2 (TRPM2) to temperature affects macrophage functions.

The ability to sense temperature is essential for organism survival and efficient metabolism. Body temperatures profoundly affect many physiological functions, including immunity. Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca(2+)-permeable cation channel expressed in a wide range of immunocytes. TRPM2 is activated by adenosine diphosphate ribose and hydrogen peroxide (H(2)O(2)), although the activation mechanism by H(2)O(2) is not well understood. Here we report a unique activation mechanism in which H(2)O(2) lowers the temperature threshold for TRPM2 activation, termed "sensitization," through Met oxidation and adenosine diphosphate ribose production. This sensitization is completely abolished by a single mutation at Met-214, indicating that the temperature threshold of TRPM2 activation is regulated by redox signals that enable channel activity at physiological body temperatures. Loss of TRPM2 attenuates zymosan-evoked macrophage functions, including cytokine release and fever-enhanced phagocytic activity. These findings suggest that redox signals sensitize TRPM2 downstream of NADPH oxidase activity and make TRPM2 active at physiological body temperature, leading to increased cytosolic Ca(2+) concentrations. Our results suggest that TRPM2 sensitization plays important roles in macrophage functions.

Identification of proteins that associate with integrin α2 by proteomic analysis in human fibrosarcoma HT-1080 cells.

Integrins are adhesion receptors for components of the extracellular matrix (ECMs) that regulate multiple cellular functions, such as migration, invasion, proliferation, and survival by mediating bidirectional signal transmission. Even though many proteins have been reported to associate with integrins both on and in cells, systemic analyses of the adhesome have not been carried out. In previous studies, we identified proteins associating with a membrane-type protease, MT1-MMP, using nano-flow liquid chromatography/tandem mass spectrometry (nano-LC/MS/MS) of associated proteins prepared by optimized conditions for cell lysis and purification. Since integrins were identified as MT1-MMP-associated proteins, we next applied this method to analyze integrin-associated proteins. In this study, we expressed integrin α2 fused at the C terminus to a FLAG peptide in HT1080 cells. Cells stably expressing the chimeric protein were lysed with 1% Brij-98 and affinity purified using anti-FLAG antibody. Integrin ß1 co-purified with integrin α2 confirming the specificity of the purification procedure. Analysis of the purified mixture by nano-LC/MS/MS identified 70 proteins. Nineteen of these were membrane proteins, including adhesion proteins, receptors, transporters, proteinases, and ion-channel receptors, and the balance were cytoplasmic. Interestingly, eight of the proteins had previously been shown to associate with MT1-MMP. We believe the present study provides a platform to facilitate the study of the mechanisms of cell adhesion, migration, and invasion.

Oral administration of heat-killed Lactobacillus pentosus strain b240 augments protection against influenza virus infection in mice.

Host-defense mechanisms against influenza virus (IFV) infection involve both innate and acquired immunities. Among other components, secretory immunoglobulin A (SIgA) in the airway mucosa plays a particularly pivotal role in preventing IFV infection. Among 150 strains of lactic acid bacteria, Lactobacillus pentosus strain b240 (b240) has the highest IgA-inducing potency in mouse Peyer's patch cells. We previously reported a practical new finding that oral ingestion of nonviable heat-killed b240 elevates salivary IgA secretion in humans. The present study aimed to determine if nonviable b240 can prevent IFV infection in mice. In a BALB/c mouse model infected with lethal levels of IFV A/PR8/34 (H1N1), oral administration of b240 for 3 weeks by gavage prior to IFV infection significantly prolonged the survival period. For IFV infection at nonlethal levels, the infectious titers of IFV in the lungs 7 days after infection were significantly reduced after similar b240 administration. Both anti-IFV IgA and immunoglobulin G titers in bronchoalveolar lavage fluid and plasma on day 7 were significantly higher in the b240-treated group than the control group. The augmentation of the anti-IFV immune response by b240 application was preliminarily confirmed by the elevated production of IFV-driven T-cell factors during mixed lymphocyte reactions with b240-primed splenocytes. These results suggest that oral nonviable heat-killed b240 intake can facilitate protection against IFV infection.

High throughput analysis of proteins associating with a proinvasive MT1-MMP in human malignant melanoma A375 cells.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), a powerful modulator of the pericellular environment, promotes migration, invasion, and proliferation of cells. To perform its potent proteolytic activity in a controlled manner, MT1-MMP has to be regulated precisely. However, our knowledge about substrates and regulatory proteins is still very limited. In this study we identify a catalog of proteins that directly or indirectly interact with MT1-MMP. We expressed a FLAG-tagged MT1-MMP stably in human malignant melanoma A375 cells. We prepared cell lysate using Brij98 and MT1-MMP was affinity purified together with associating proteins using an anti-FLAG antibody. A distinct set of membrane proteins was found to copurify with MT1-MMP when biotin-labeled proteins were monitored. The proteins were analyzed with an integrated system composed of nano-flow liquid chromatography and tandem mass spectrometry. We identified 158 proteins including several previously reported to bind MT1-MMP, although most had not previously been identified. Six of these membrane proteins, including one previously shown to interact with MT1-MMP, were co-expressed with MT1-MMP in HT1080 cells. Five of the latter were found to associate with MT1-MMP in an immunoprecipitation assay. Immunostaining of cells expressing each of these test proteins revealed that one colocalized with MT1-MMP at the ruffling membrane and the other at the perinuclear vesicles. In contrast, another protein which did not coprecipitate with MT1-MMP showed no colocalization. Recombinant MT1-MMP cleaved two of the tested proteins at least in vitro. Thus, we provide a valuable resource to identify substrates and regulators of MT1-MMP in tumor cells.