Objectification of evaluation criteria in microscopic agglutination test using deep learning.

We aim to objectify the evaluation criteria of agglutination rate estimation in the Microscopic Agglutination Test (MAT). This study proposes a deep learning method that extracts free leptospires from dark-field microscopic images and calculates the agglutination rate. The experiments show the effect of objectification with real pictures.

Insufficient anti-spike RBD IgA responses after triple vaccination with intramuscular mRNA BNT162b2 vaccine against SARS-CoV-2.

Objectives: This study aims to examine whether the parenterally administered mRNA-based COVID-19 vaccines can induce sufficient mucosal-type IgA responses to prevent SARS-CoV-2 transmission. Methods: We examined the longitudinal kinetics of SARS-CoV-2 spike RBD-specific IgA and IgG responses in sera of Japanese healthcare workers (HCWs) after receiving two doses and the third dose of BNT162b2 mRNA vaccines. During the prospective cohort study, Omicron breakthrough infections occurred in 62 participants among 370 HCWs who had received triple doses of the vaccine. Pre-breakthrough sera of infected HCWs and non-infected HCWs were examined for the levels of anti-RBD IgA and IgG titers. Results: The seropositivity of anti-RBD IgA at 1 M after the second vaccine (2D-1M) and after the third dose (3D-1M) was 65.4% and 87.4%, respectively, and wanes quickly. The boosting effect on anti-RBD Ab titers following breakthrough infections was more notable for anti-RBD IgA than for IgG. There were partial cause-relationships between the lower anti-RBD IgA or IgG at pre-breakthrough sera and the breakthrough infection. Conclusions: Parenterally administered COVID-19 vaccines do not generate sufficient mucosal-type IgA responses despite strong systemic IgG responses to SARS-CoV-2. These results demonstrate the necessity and importance of reevaluating vaccine design and scheduling to efficiently increase oral or respiratory mucosal immunity against SARS-CoV-2.

The appearance of anti-spike receptor binding domain immunoglobulin G4 responses after repetitive immunization with messenger RNA-based COVID-19 vaccines.

OBJECTIVES: It is crucial to analyze the consequences of repeated messenger RNA (mRNA)-based COVID-19 vaccinations on SARS-CoV-2 spike receptor binding domain (RBD)-specific immunoglobulin (Ig)G subclass and the possible causal relationship with breakthrough infection. METHODS: We examined the longitudinal kinetics of RBD-specific IgG subclass antibodies in sera after receiving the second, third, and fourth doses of mRNA-based COVID-19 vaccines in Japanese healthcare workers. Anti-RBD IgG subclass in sera of patients with COVID-19-infected who had not received the COVID-19 vaccine were also examined. We compared anti-RBD IgG subclass antibody titers in the serum of pre-breakthrough-infected vaccinees and non-infected vaccinees. RESULTS: The seropositivity of anti-RBD IgG4 after the vaccination was 6.76% at 1 month after the second dose, gradually increased to 50.5% at 6 months after the second dose, and reached 97.2% at 1 month after the third dose. The seropositivity and titers of anti-RBD IgG1/IgG3 quickly reached the maximum at 1 month after the second dose and declined afterward. The elevated anti-RBD IgG4 Ab levels observed after repeated vaccinations were unlikely to increase the risk of breakthrough infection. CONCLUSIONS: Repeated vaccinations induce delayed but drastic increases in anti-RBD IgG4 responses. Further functional investigations are needed to reveal the magnitude of the high contribution of spike-specific IgG4 subclasses after repeated mRNA-based COVID-19 vaccinations.

Chlamydia pneumoniae Lung Infection in Mice Induces Fatty Acid-Binding Protein 4-Dependent White Adipose Tissue Pathology.

Fatty acid-binding protein 4 (FABP4) is a critical immune-metabolic modulator, mainly expressed in adipocytes and macrophages, secreted from adipocytes in association with lipolysis, and plays essential pathogenic roles in cardiovascular and metabolic diseases. We previously reported Chlamydia pneumoniae infecting murine 3T3-L1 adipocytes and causing lipolysis and FABP4 secretion in vitro. However, it is still unknown whether C. pneumoniae intranasal lung infection targets white adipose tissues (WATs), induces lipolysis, and causes FABP4 secretion in vivo. In this study, we demonstrate that C. pneumoniae lung infection causes robust lipolysis in WAT. Infection-induced WAT lipolysis was diminished in FABP4-/- mice or FABP4 inhibitor-pretreated wild-type mice. Infection by C. pneumoniae in wild-type but not FABP4-/- mice induces the accumulation of TNF-α- and IL-6-producing M1-like adipose tissue macrophages in WAT. Infection-induced WAT pathology is augmented by endoplasmic reticulum (ER) stress/the unfolded protein response (UPR), which is abrogated by treatment with azoramide, a modulator of the UPR. C. pneumoniae lung infection is suggested to target WAT and induce lipolysis and FABP4 secretion in vivo via ER stress/UPR. FABP4 released from infected adipocytes may be taken up by other neighboring intact adipocytes or adipose tissue macrophages. This process can further induce ER stress activation and trigger lipolysis and inflammation, followed by FABP4 secretion, leading to WAT pathology. A better understanding of the role of FABP4 in C. pneumoniae infection-induced WAT pathology will provide the basis for rational intervention measures directed at C. pneumoniae infection and metabolic syndrome, such as atherosclerosis, for which robust epidemiologic evidence exists.

Epipharyngeal Abrasive Therapy Down-regulates the Expression of Cav1.2: A Key Molecule in Influenza Virus Entry.

BACKGROUND/AIM: Influenza A virus (IAV) infection causes an inflammatory response to the respiratory mucosa. The viral glycoprotein hemagglutinin (HA) binds to the sialylated voltage-dependent Ca2+ channel (Cav1.2) in ciliated epithelium. The binding of HA and sialylated Cav1.2 is considered essential to IAV infection, entry, and IAV-induced Ca2+ oscillation. The epipharynx comprises the ciliated epithelium, which is the initial target for viruses that cause upper respiratory tract infections. Previously, we showed that epipharyngeal abrasive therapy (EAT), a treatment for chronic epipharyngitis in Japan, which scratches the epipharyngeal mucosa with a cotton swab containing zinc chloride, induces squamous metaplasia. In this study, we evaluated whether squamous metaplasia by EAT affects the expression patterns of Cav1.2. PATIENTS AND METHODS: The study subjects were seven patients who had not been treated with EAT and 11 patients who had. For the immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of Cav1.2 was described using the immunohistochemical score (IHC score). RESULTS: The IHC scores for Cav1.2 in the EAT-treated group was 4.19-fold lower than those in the non-treated group (p=0.0034). CONCLUSION: EAT down-regulates the expression of Cav1.2, a key cell surface molecule in influenza virus entry via squamous metaplasia. Thus, EAT may be a simple method for preventing influenza infection.

Epipharyngeal Abrasive Therapy (EAT) Reduces the mRNA Expression of Major Proinflammatory Cytokine IL-6 in Chronic Epipharyngitis.

The epipharynx, located behind the nasal cavity, is responsible for upper respiratory tract immunity; however, it is also the site of frequent acute and chronic inflammation. Previous reports have suggested that chronic epipharyngitis is involved not only in local symptoms such as cough and postnasal drip, but also in systemic inflammatory diseases such as IgA nephropathy and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and Long COVID. Epipharyngeal Abrasive Therapy (EAT), which is an effective treatment for chronic epipharyngitis in Japan, is reported to be effective for these intractable diseases. The sedation of chronic epipharyngitis by EAT induces suppression of the inflammatory cytokines and improves systemic symptoms, which is considered to be one of the mechanisms, but there is no report that has proved this hypothesis. The purpose of this study was to clarify the anti-inflammatory effect of EAT histologically. The study subjects were 8 patients who were not treated with EAT and 11 patients who were treated with EAT for chronic epipharyngitis for 1 month or more. For immunohistochemical assessment, the expression pattern of IL-6 mRNA, which plays a central role in the human cytokine network, was analyzed using in situ hybridization. The expression of IL-6 in the EAT-treated group was significantly lower than those in the EAT nontreated group (p = 0.0015). In addition, EAT suppressed the expression of tumor necrosis factor alpha (TNFα), a crucial proinflammatory cytokine. As a result, continuous EAT suppressed submucosal cell aggregation and reduced inflammatory cytokines. Thus, EAT may contribute to the improvement of systemic inflammatory diseases through the suppression of IL-6 expression.

Bortezomib Eliminates Persistent Chlamydia trachomatis Infection through Rapid and Specific Host Cell Apoptosis.

Chlamydia trachomatis, a parasitic intracellular bacterium, is a major human pathogen that causes millions of trachoma, sexually transmitted infections, and pneumonia cases worldwide. Previously, peptidomimetic inhibitors consisting of a hydrophobic dipeptide derivative exhibited significant inhibitory effects against chlamydial growth. Based on this finding, this study showed that both bortezomib (BTZ) and ixazomib (IXA), anticancer drugs characterized by proteasome inhibitors, have intensive inhibitory activity against Chlamydia. Both BTZ and IXA consisted of hydrophobic dipeptide derivatives and strongly restricted the growth of Chlamydia (BTZ, IC50 = 24 nM). In contrast, no growth inhibitory effect was observed for other nonintracellular parasitic bacteria, such as Escherichia coli. BTZ and IXA appeared to inhibit chlamydial growth bacteriostatically via electron microscopy. Surprisingly, Chlamydia-infected cells that induced a persistent infection state were selectively eliminated by BTZ treatment, whereas uninfected cells survived. These results strongly suggested the potential of boron compounds based on hydrophobic dipeptides for treating chlamydial infections, including persistent infections, which may be useful for future therapeutic use in chlamydial infectious diseases.

Longitudinal Dynamics of SARS-CoV-2 IgG Antibody Responses after the Two-Dose Regimen of BNT162b2 Vaccination and the Effect of a Third Dose on Healthcare Workers in Japan.

Analysis of longitudinal dynamics of humoral immune responses to the BNT162b2 COVID-19 vaccine might provide useful information to predict the effectiveness of BNT162b2 in preventing SARS-CoV-2 infection. Herein, we measure anti-RBD IgG at 1, 3 and 6 months (M) after the second dose of BNT162b2, and at 1 M after a third dose of BNT162b2 vaccination in 431 COVID-19-naïve healthcare workers (HCWs) in Japan. All HCWs mounted high-anti-RBD IgG responses after the two-dose regimen of BNT162b2 vaccinations. Older persons and males presented lower anti-RBD IgG responses than younger adults and females, respectively. The decay in anti-RBD IgG started from 1 M after the second dose of BNT162b2 and anti-RBD IgG titers dropped to nearly one-tenth at 6 M after the second vaccination. Subsequently, the participants received a third dose of BNT162b2 at 8 M after the second dose of BNT162b2 vaccine. Anti-RBD antibody titers 1 M after the third dose of BNT162b2 increased seventeen times that of 6 M after the second dose, and was twice higher than the peak antibody titers at 1 M after the second dose of vaccination. The negative effect of age for the male gender on anti-RBD IgG antibody titers was not observed at 1 M after the third dose of BNT162b2 vaccine. There were no notable adverse events reported, which required hospitalization in these participants. These results suggest that the third dose of BNT162b2 safely improves humoral immunity against SARS-CoV-2 with no major adverse events.

Epipharyngeal Abrasive Therapy (EAT) Has Potential as a Novel Method for Long COVID Treatment.

COVID-19 often causes sequelae after initial recovery, referred to collectively as long COVID. Long COVID is considered to be caused by the persistence of chronic inflammation after acute COVID-19 infection. We found that all long COVID patients had residual inflammation in the epipharynx, an important site of coronavirus replication, and some long COVID symptoms are similar to those associated with chronic epipharyngitis. Epipharyngeal abrasive therapy (EAT) is a treatment for chronic epipharyngitis in Japan that involves applying zinc chloride as an anti-inflammatory agent to the epipharyngeal mucosa. In this study, we evaluated the efficacy of EAT for the treatment of long COVID. The subjects in this study were 58 patients with long COVID who were treated with EAT in the outpatient department once a week for one month (mean age = 38.4 ± 12.9 years). The intensities of fatigue, headache, and attention disorder, which are reported as frequent symptoms of long COVID, were assessed before and after EAT using the visual analog scale (VAS). EAT reduced inflammation in the epipharynx and significantly improved the intensity of fatigue, headache, and attention disorder, which may be related to myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). These results suggest that EAT has potential as a novel method for long COVID treatment.

Emerging resistance to beta-lactams in Pantoea ananatis isolated from an immunocompetent patient with bacteremia.

We observed an emerging resistance to ß-lactams in a P. ananatis bacteremia case. Whole genome sequence analysis detected two ß-lactamase genes as well as related genes that regulate the ß-lactamase genes in the chromosome. The induction experiment resulted in the expression of the class A ß-lactamase gene in the isolate.

Epipharyngeal Abrasive Therapy Down-regulates the Expression of SARS-CoV-2 Entry Factors ACE2 and TMPRSS2.

BACKGROUND: The epipharynx, with its high expression of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) entry factors angiotensin-converting enzyme 2 (ACE2) and transmembrane protease, serine 2 (TMPRSS2), is a primary target for SARS-CoV-2 replication in the early stage of Coronavirus Disease 19 (COVID-19). Epipharyngeal abrasive therapy (EAT) is a treatment for epipharyngitis in Japan which involves applying zinc chloride to the epipharyngeal mucosa. In this study, we evaluated the expression patterns of ACE2 and TMPRSS2 in tissue samples from patients before and after EAT. PATIENTS AND METHODS: The study subjects were seven patients that had not been treated with EAT and 11 patients that had. For immunohistochemical assessment of the epipharyngeal mucosa, the staining intensity of ACE2 and TMPRSS2 was described as an immunohistochemical score (IHC score). RESULTS: The IHC scores for ACE2 and TEMPRSS2 in the EAT-treated group were 3.40-fold and 1.81-fold lower, respectively, than those in the non-treated group (p=0.0208 and p=0.0244, respectively). CONCLUSION: EAT down-regulates the expression of SARS-CoV-2 entry factors ACE2 and TMPRSS2. Thus, EAT has potential as a novel COVID-19 preventative method.

Ephedrae Herba and Cinnamomi Cortex interactions with G glycoprotein inhibit respiratory syncytial virus infectivity.

Although respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children, no effective therapies are available. Recently, RSV G, the attachment glycoprotein, has become a major focus in the development of therapeutic strategies against RSV infection. Treatment of RSV-infected cultured cells with maoto, a traditional herbal medicine for acute febrile diseases, significantly reduced the viral RNA and titers. RSV attachment to the cell surface was inhibited both in the presence of maoto and when RSV particles were pre-treated with maoto. We demonstrated that maoto components, Ephedrae Herba (EH) and Cinnamomi Cortex (CC), specifically interacted with the central conserved domain (CCD) of G protein, and also found that this interaction blocked viral attachment to the cellular receptor CX3CR1. Genetic mutation of CX3C motif on the CCD, the epitope for CX3CR1, decreased the binding capacity to EH and CC, suggesting that CX3C motif was the target for EH and CC. Finally, oral administration of maoto for five days to RSV-infected mice significantly reduced the lung viral titers. These experiments clearly showed the anti-RSV activity of EH and CC mixed in maoto. Taken together, this study provides insights for the rational design of therapies against RSV infection.

MicroRNAs miR-4535 and miR-1915-5p in amniotic fluid as predictive biomarkers for chorioamnionitis.

BACKGROUND: This study was performed to investigate the clinical significance of miR-4535 and miR-1915-5p in severe chorioamnionitis. MATERIALS & METHODS: Amniotic fluid samples from 37 patients with severe chorioamnionitis were subjected to miRNA array analysis and ddPCR™. Diagnostic values were assessed using the receiver operating characteristic curve. The patients were separated into three groups according to Blanc's criteria. RESULTS: The expression of miR-4535 and miR-1915-5p was significantly correlated with the copy number of 16S rDNA, had extremely high diagnostic accuracy for severe chorioamnionitis, and was linked to maternal and fetal inflammation. CONCLUSION: miR-4535 and miR-1915-5p serve as promising biomarkers for the diagnosis of severe chorioamnionitis.

Chlamydia pneumoniae infection-induced endoplasmic reticulum stress causes fatty acid-binding protein 4 secretion in murine adipocytes.

Fatty acid-binding protein 4 (FABP4) is predominantly expressed in adipocytes and macrophages and regulates metabolic and inflammatory pathways. FABP4 is secreted from adipocytes during lipolysis, and elevated circulating FABP4 levels are associated with obesity, metabolic disease, and cardiac dysfunction. We previously reported that the bacterial respiratory pathogen Chlamydia pneumoniae infects murine adipocytes and exploits host FABP4 to mobilize fat and replicate within adipocytes. However, whether C. pneumoniae induces FABP4 secretion from adipocytes has not been determined. Here, we show that FABP4 is actively secreted by murine adipocytes upon C. pneumoniae infection. Chemical inhibition of lipase activity and genetic deficiency of hormone-sensitive lipase blocked FABP4 secretion from C. pneumoniae-infected adipocytes. Mechanistically, C. pneumoniae infection induced endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), resulting in elevated levels of mitochondrial reactive oxygen species and cytosolic Ca2+ Of note, exposure to a mitochondrial reactive oxygen species-specific scavenger, MitoTEMPO, reduced FABP4 release from C. pneumoniae-infected adipocytes. Furthermore, treatment with azoramide, which protects cells against ER stress, decreased FABP4 release from C. pneumoniae-infected adipocytes. Using gene silencing of CHOP (C/EBP homologous protein), a central regulator of ER stress, we further validated the role of C. pneumoniae infection-induced ER stress/UPR in promoting FABP4 secretion. Overall, these results indicate that C. pneumoniae infection robustly induces FABP4 secretion from adipocytes by stimulating ER stress/UPR. Our findings shed additional light on the etiological link between C. pneumoniae infection and metabolic syndrome.

Anti-chlamydial activities of cell-permeable hydrophobic dipeptide-containing derivatives.

The obligate intracellular bacteria chlamydia is major human pathogen that causes millions of trachoma, sexually transmitted infections and pneumonia worldwide. We serendipitously found that both calpain inhibitors z-Val-Phe-CHO and z-Leu-Nle-CHO showed marked inhibitory activity against chlamydial growth in human epithelial HeLa cells, whereas other calpain inhibitors not. These peptidomimetic inhibitors consist of N-benzyloxycarbonyl group and hydrophobic dipeptide derivatives. Both compounds strongly restrict the chlamydial growth even addition at the 12 h post infection. Notably, inhibitors-mediated growth inhibition of chlamydia was independent on host calpain activity. Electron microscopic analysis revealed that z-Val-Phe-CHO inhibited chlamydial growth by arresting bacterial cell division and RB-EB re-transition, but not by changing into persistent state. We searched and found that z-Leu-Leu-CHO and z-Phe-Ala-FMK also inhibited chlamydial growth. Neither biotin-hydrophobic dipeptide nor morpholinoureidyl-hydrophobic dipeptide shows inhibitory effects on chlamydial intracellular growth. Our results suggested the possibility of some chemical derivatives based on z-hydrophobic dipeptide group for future therapeutic usage to the chlamydial infectious disease.

Chlamydia trachomatis targets mitochondrial dynamics to promote intracellular survival and proliferation.

Chlamydia trachomatis is an obligate intracellular bacterium that scavenges host metabolic products for its replication. Mitochondria are the power plants of eukaryotic cells and provide most of the cellular ATP via oxidative phosphorylation. Several intracellular pathogens target mitochondria as part of their obligatory cellular reprogramming. This study was designed to analyse the mitochondrial morphological changes in response to C. trachomatis infection in HeLa cells. Mitochondrial elongation and fragmentation were found at the early stages and late stages of C. trachomatis infection, respectively. C. trachomatis infection-induced mitochondrial elongation was associated with the increase of mitochondrial respiratory activity, ATP production, and intracellular growth of C. trachomatis. Silencing mitochondrial fusion mediator proteins abrogated the C. trachomatis infection-induced elevation in the oxygen consumption rate and attenuated chlamydial proliferation. Mechanistically, C. trachomatis induced the elevation of intracellular cAMP at the early phase of infection, followed by the phosphorylation of fission-inactive serine residue 637 (S637) of Drp1, resulting in mitochondrial elongation. Accordingly, treatment with adenylate cyclase inhibitor diminished mitochondrial elongation and bacterial growth in infected cells. Collectively, these results strongly indicate that C. trachomatis promotes its intracellular growth by targeting mitochondrial dynamics to regulate ATP synthesis via inhibition of the fission mediator Drp1.

Loss of ATP2A2 Allows Herpes Simplex Virus 1 Infection of a Human Epidermis Model by Disrupting Innate Immunity and Barrier Function.

Destruction of epidermal barrier function associated with atopic dermatitis or Darier's disease often causes severe secondary skin infections. Patients with skin barrier disorders often repeatedly acquire Kaposi varicelliform eruption, which is caused by herpes simplex virus, but the underlying mechanisms and effective preventive methods have yet to be found. Viral infection through an impaired epidermal barrier can be prevented by enhancing innate immunity and/or inhibiting viral entry. In this study, we established a three-dimensional skin barrier dysfunction model by silencing ATP2A2, which is mutated in some Darier's disease patients. We confirmed the loss of desmosomes and presence of histopathological clefts in the suprabasal layer. Herpes simplex virus 1 applied to the stratum corneum infected the deep epidermis. An innate immune reaction was assessed by evaluating the expression of IFNB1 and related genes. Pretreatment with polyinosinic-polycytidylic acid alone or plus the antimicrobial peptide, LL37 enhanced IFN-ß production and suppressed viral replication. Furthermore, topical application of a white petrolatum ointment containing heparin, which binds viral glycoproteins related to virus entry, strongly inhibited viral replication, probably by inhibiting invasion. Our human barrier-dysfunctional model will have future application for identifying the mechanism of Kaposi varicelliform eruption onset, preventive methods, and therapies.

Chlamydia pneumoniae exploits adipocyte lipid chaperone FABP4 to facilitate fat mobilization and intracellular growth in murine adipocytes.

Fatty acid-binding protein 4 (FABP4), a cytosolic lipid chaperone predominantly expressed in adipocytes and macrophages, modulates lipid fluxes, trafficking, signaling, and metabolism. Recent studies have demonstrated that FABP4 regulates metabolic and inflammatory pathways, and in mouse models its inhibition can improve type 2 diabetes mellitus and atherosclerosis. However, the role of FABP4 in bacterial infection, metabolic crosstalk between host and pathogen, and bacterial pathogenesis have not been studied. As an obligate intracellular pathogen, Chlamydia pneumoniae needs to obtain nutrients such as ATP and lipids from host cells. Here, we show that C. pneumoniae successfully infects and proliferates in murine adipocytes by inducing hormone sensitive lipase (HSL)-mediated lipolysis. Chemical inhibition or genetic manipulation of HSL significantly abrogated the intracellular growth of C. pneumoniae in adipocytes. Liberated free fatty acids were utilized to generate ATP via ß-oxidation, which C. pneumoniae usurped for its replication. Strikingly, chemical inhibition or genetic silencing of FABP4 significantly abrogated C. pneumoniae infection-induced lipolysis and mobilization of liberated FFAs, resulting in reduced bacterial growth in adipocytes. Collectively, these results demonstrate that C. pneumoniae exploits host FABP4 to facilitate fat mobilization and intracellular replication in adipocytes. This work uncovers a novel strategy used by intracellular pathogens for acquiring energy via hijacking of the host lipid metabolism pathway.

Maoto, a Traditional Japanese Herbal Medicine, Inhibits Uncoating of Influenza Virus.

We previously reported in randomized controlled trials that maoto, a traditional herbal medicine, showed clinical and virological efficacy for seasonal influenza. In this study, a culturing system for influenza was used to test the effect of maoto. A549 cells in the culture were infected with influenza virus A (PR8) and followed after treatment with maoto; the virus titers in the culture supernatant, intracellular viral proteins, and viral RNA were determined. When infected cells were cultured with maoto for 24 hr, the virus titer and protein were significantly reduced compared with medium only. Other subtypes, A/H3N2, H1N1pdm, and B, were also inhibited by maoto. Proliferation of viral RNA in a 6 hr culture was inhibited by maoto in the early phase, especially in the first 30 min. Focusing on the entry step of the influenza virus, we found that endosomal pH, regulated by vacuolar-type H+ ATPase (V-ATPase) located in the membrane, was increased when treated with maoto. We also found that uncoating of influenza viruses was also inhibited by maoto, resulting in the increase of the number of virus particles in endosomes. These results strongly suggest that the inhibition of endosomal acidification by maoto results in blocking influenza virus entry to cytoplasm, probably through the inhibition of V-ATPase. The present study provides evidence that supports the clinical use of maoto for the treatment of influenza.

Pancreatic fistulae secondary to trypsinogen activation by Pseudomonas aeruginosa infection after pancreatoduodenectomy.

BACKGROUND: Pancreatic fistula after pancreatoduodenectomy (PD) is associated with high mortality and morbidity. Trypsinogen activation and bacteria, although hypothesized to be interrelated etiopathogenetically, have not had their relationship and pathogenic mechanisms elucidated. This study investigated bacterial involvement in pancreatic juice activation perioperatively after PD at sites of pancreatic fistula formation. METHODS: Fifty patients underwent PD; postoperative pancreatic fistulae were graded based on the International Study Group for Pancreatic Fistula grading criteria. Bacteria were isolated from cultures of drainage fluid. Digested peptides from trypsinogen and bacterial culture supernatants underwent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation and mass spectrometric analysis. Zymography was used to detect the trypsinogen activator. RESULTS: Pseudomonas aeruginosa and Enterobacter cloacae isolated from drainage fluid in patients with grades B and C pancreatic fistulae could cause trypsinogen activation. Trypsinogen activation by P. aeruginosa and E. cloacae were preventable by the use of a serine protease inhibitor in vitro. A protease in the supernatant from P. aeruginosa-positive cultures acted as the trypsinogen activator. CONCLUSIONS: Infection with P. aeruginosa perioperatively to PD entails secretion of a protease activator of trypsinogen to trypsin. Bacterial infection control in the perioperative PD period could be crucial to prevent development of pancreatic fistula.