Mental health complaints among healthcare workers engaged in the care of COVID-19 patients: A prospective cohort study from Japan.

Background: Healthcare workers (HCWs) caring for patients with coronavirus disease-2019 (COVID-19) can experience physical and mental health burdens. It is imperative that hospitals reduce such burdens on frontline HCWs, protect them, and support their healthcare. This study aimed to investigate the association between occupation and the manifestation of physical or psychological symptoms among HCWs during the current COVID-19 pandemic. Methods: A twice-weekly survey using questionnaires targeting HCWs who care for COVID-19 patients was performed at Osaka Metropolitan University Hospital (tertiary hospital). The demographic characteristics of the participants, exposure level, and physical and psychological complaints were evaluated. Results: Seventy-one HCWs participated in this study, of whom 27 (38.0%) were doctors, 25 (35.2%) were nurses, and 19 (26.8%) were technicians. Among the HCWs, the proportions of those who experienced any physical or psychological symptoms were 28.2% and 31.0%, respectively. The frequency of depression and anxiety was obviously higher among the nurses than that among the doctors (both p < 0.01). Multivariate analysis revealed that being a nurse (odds ratio 4.90; p = 0.04) and having physical complaints (odds ratio 4.66; p = 0.02) might be independent predictors of the manifestation of psychological symptoms. Conclusion: Our results indicate that the follow-up of HCWs experiencing physical symptoms, especially nurses engaged in the care of COVID-19 patients, may require more careful management to improve the psychological outcomes. We believe that this study is the first step toward establishing a psychological health management strategy for HCWs caring for COVID-19 patients.

Impact of antimicrobial stewardship with the Xpert MRSA/SA BC assay at a tertiary hospital in Japan.

INTRODUCTION: Genetic testing is gaining increasing importance as a part of antimicrobial stewardship (AS). Rapid identification and determination of methicillin susceptibility using the Xpert MRSA/SA BC assay can improve the management of Staphylococcus aureus bacteremia (SAB) and reduce inappropriate antibiotic use. However, few reports have described the effectiveness of this approach. METHODS: The present study aimed to assess the influence of AS using the Xpert MRSA/SA BC assay. Cases were classified into the pre-intervention group (n = 98 patients), in which SAB was identified by traditional culture (November 2017 to November 2019), and the post-intervention group (n = 97 patients), in which the Xpert MRSA/SA BC assay was performed when necessary (December 2019 to December 2021). RESULTS: Patient characteristics, prognosis, duration of antimicrobial use, and length of hospital stay were compared between the groups. The Xpert assay was performed in 66 patients in the post-intervention group (68.0%). The two groups showed no significant differences in severity and mortality. The rate of cases treated with anti-MRSA agents reduced following the intervention (65.3% vs. 40.4%, p = 0.008). The number of cases involving definitive therapy within 24 h was higher in the post-intervention group (9.2% vs. 24.7%, p = 0.007). The hospitalization rate at >60 days was lower in Xpert implementation cases among MRSA bacteremia cases (28.6% vs. 0%, p = 0.01). CONCLUSIONS: Thus, the Xpert MRSA/SA BC assay has potential as an AS tool, especially for early definitive treatment to SAB and reduction of long-term hospitalization in MRSA bacteremia cases.

Fine-tuned cholesterol solubilizer, mono-6-O-α-D-maltosyl-γ-cyclodextrin, ameliorates experimental Niemann-Pick disease type C without hearing loss.

Niemann-Pick disease type C (NPC) is a fatal disorder with abnormal intracellular cholesterol trafficking resulting in neurodegeneration and hepatosplenomegaly. A cyclic heptasaccharide with different degrees of substitution of 2-hydroxypropyl groups, 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD), acts as a strong cholesterol solubilizer and is under investigation for treating this disease in clinical trials, but its physicochemical properties and ototoxicity remain a concern. Here, we evaluated the potential of mono-6-O-α-maltosyl-γ-CD (G2-γ-CD), a single-maltose-branched cyclic octasaccharide with a larger cavity than HP-ß-CD, for treating NPC. We identified that G2-γ-CD ameliorated NPC manifestations in model mice and showed lower ototoxicity in mice than HP-ß-CD. To investigate the molecular mechanisms of action behind the differential ototoxicity of these CDs, we performed cholesterol solubility analysis, proton nuclear magnetic resonance spectroscopy, and molecular modeling, and estimated that the cholesterol inclusion mode of G2-γ-CD maintained solely the 1:1 inclusion complex, whereas that of HP-ß-CD shifted to the highly-soluble 2:1 complex at higher concentrations. We predicted the associations of these differential complexations of CDs with cholesterol with the profile of disease attenuation and of the auditory cell toxicity using specific cell models. We proposed that G2-γ-CD can serve as a fine-tuned cholesterol solubilizer for treating NPC, being highly biocompatible and physicochemically suitable for clinical application.

Impact of coronavirus disease 2019 on infectious disease treatment and infection control at a tertiary hospital in Japan.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) has greatly impacted medical care practices. Although the effects on infectious disease treatment and infection control, such as antimicrobial resistance, have been specified, very few reports exist on the specific effects of COVID-19. METHODS: We investigated the effects of COVID-19 on daily medical practices at a tertiary hospital in Japan by comparing the use of hand sanitizers, the detection of bacteria from blood cultures, and the amount dose of antibacterial drugs used for one year before (April 2019 to March 2020, fiscal year 2019.) and after COVID-19 admissions began (April 2020 to March 2021, fiscal year 2020). RESULTS: The use of hand sanitizers increased by 1.4-3 times during the year after COVID-19 admissions began; the incidence of methicillin-susceptible Staphylococcus aureus and all S. aureus detected in blood cultures reduced in all departments. No decrease was observed in the usage of all antibacterial drugs; rather, the usage of all antibacterial drugs tended to increase in all departments. Therefore, no significant change was observed in the detection of drug-resistant bacteria and the trends of antibacterial drug use based on the acceptance of COVID-19 patients. CONCLUSIONS: The prevalence of drug-resistant bacteria and trends of antibacterial drug use remained unchanged despite the increased use of hand sanitizers due to the admission of patients with COVID-19.

Physical health complaints among healthcare workers engaged in the care of critically ill COVID-19 patients: A single tertiary-care center prospective study from Japan.

BACKGROUND: Healthcare workers (HCWs) who manage patients with the novel coronavirus disease 2019 (COVID-19) are at an increased risk and fear of contracting the infection themselves. Hospitals must reduce both the physical and mental burden of HCWs on the front lines and ensure their safety. No prospective study has focused on the physical health complaints among HCWs engaged in the care of critically ill COVID-19 patients. This study aimed to evaluate the prevalence of various physical symptoms experienced by HCWs following their exposure to COVID-19 patients and investigate the association between occupation and the manifestation of physical symptoms among HCWs at a tertiary hospital in Japan during the current ongoing COVID-19 pandemic. METHODS: A twice-weekly questionnaire targeting HCWs who care for COVID-19 patients was performed at Osaka City University Hospital from April 30 to May 31, 2020. The demographic characteristics of the participants, frequency of exposure to at-risk care, and physical complaints were evaluated. RESULTS: Seventy-six HCWs participated in this study, of whom 24 (31.6%) were doctors, 43 (56.6%) were nurses, and 9 (11.8%) were technicians. The frequency of experiencing any physical symptom was 25.0% among HCWs. Exposure to at-risk care was significantly higher among nurses than among doctors (p < 0.001). Notably, the frequency of physical symptoms among the nurses was very high at 39.5% and obviously higher than that of physical symptoms among the doctors (p < 0.01). CONCLUSIONS: Our results indicate that hospital occupational health care must be provided to HCWs who are engaged in the care of COVID-19 patients and are thus highly exposed to at-risk care.

Three-year epidemiological analysis of Clostridioides difficile infection and the value of hospital-infection control measures in a tertiary-care teaching hospital in Japan using PCR-based open-reading frame typing.

INTRODUCTION: Clostridioides difficile is an important causative pathogen in antibiotic-associated colitis and nosocomial infections. This study aimed to assess immunochromatographic test results for C. difficile infection and the utility of PCR-based open-reading frame typing (POT) for potentially controlling the intra-ward transmission of C. difficile. METHODS: We conducted a molecular epidemiological analysis using POT to investigate 102 inpatients who tested positive for the C. difficile toxin using immunochromatography in a tertiary-care teaching hospital in Japan between 2016 and 2018; isolates from the patients were obtained and cultured. RESULTS: The number of POT numbers detected in 2016, 2017, and 2018 were 27 (among 34 patients), 20 (among 31 patients), and 28 (among 37 patients), respectively. During this three-year period, there were seven cases whose bacterial strains with the same POT number was identified in the same ward within 6 months. The intra-ward transmission rate was the highest in 2017 (16.1%). Intra-ward transmission was identified at a higher rate in patients whose sample cultures tested toxin-positive than in patients whose sample cultures tested toxin- and glutamate-dehydrogenase-positive via immunochromatography (16% vs. 3%, p < 0.05). CONCLUSIONS: We conclude that the use of immunochromatographic tests for C. difficile diagnosis and epidemiological analyses via POT may be helpful for evaluating intra-ward transmission of C. difficile.

Blood culture reports by infectious disease physicians can improve prognosis of bacteremia, including weekend-onset cases.

OBJECTIVES: Cases of positive blood cultures were previously reported by a microbiological technologist (MT) to an attending physician (AP), and the Antimicrobial Stewardship team provided medical assistance by grasping the situation at the morning meeting the next day. Since April 2018, MTs have reported positive blood cultures to an infectious disease physician (IDP), who proposes the management approach to the AP and provides weekend support. This study assessed the effectiveness of blood culture reports provided by IDPs to APs on outcomes of bacteremia, including weekend-onset cases. METHODS: Patient characteristics and prognoses before (October 2017 to March 2018) and after intervention (April to September 2018) were compared. RESULTS: The pre-intervention and post-intervention groups comprised 134 and 161 patients, respectively. Patients were more likely to be older (>65 years) in the post-intervention group (p < 0.05). There were no significant between-group differences in infection severity. The rate of de-escalation significantly increased from 38.1%-57.8% (p = 0.001). The rates of 28-day and in-hospital mortality reduced following the intervention (21.3% vs. 8.2% and 32.8% vs. 10.6%; p = 0.004 and p < 0.001, respectively). In-hospital mortality for weekend-onset cases also reduced following the intervention (33.3% vs. 12.9%, p = 0.01). Sepsis was a poor prognostic factor (OR 8.070, 95% CI 3.320-19.600, p < 0.001) and intervention was a good prognostic factor (OR 0.311, 95% CI 0.142-0.680, p = 0.003) affecting 28-day mortality in multivariate analysis. CONCLUSIONS: Changes to blood culture result reporting protocols can improve outcomes of bacteremia, including weekend-onset cases.

The effect of intervention by an antimicrobial stewardship team on anaerobic bacteremia.

The effect of antimicrobial stewardship (AS) on anaerobic bacteremia is uncertain. This study aimed to assess the effect of interventions by the AS team (AST) on clinical and microbiological outcomes and antimicrobial use. An AS program was introduced at Osaka City University Hospital in January 2014; an interdisciplinary AST was established. We enrolled patients with anaerobic bacteremia between January 2009 and December 2018. Patients were classified into the pre-intervention group (from January 2009 to December 2013) and the post-intervention group (from January 2014 to December 2018). A significant decrease in definitive carbapenem use (P = 0.0242) and an increase in empiric tazobactam/piperacillin use (P = 0.0262) were observed in the post-intervention group. The de-escalation rate increased significantly from 9.38% to 32.7% (P = 0.0316) in the post-intervention group. The susceptibility of Bacteroides species and 30-day mortality did not worsen in the post-intervention group. These results showed that interventions by an AST can reduce carbapenem use and increase the de-escalation rate without worsening patient outcomes.

The intervention by an antimicrobial stewardship team can improve clinical and microbiological outcomes of resistant gram-negative bacteria.

Antibiotic stewardship (AS) improves patient outcomes and rates of antibiotic susceptibilities. However, the long-term effect of AS programs (ASPs) on mortality is unclear. This study aimed to assess the impact of bedside interventions by an AS team (AST) on clinical and microbiological outcomes. This retrospective study enrolled patients with bloodstream infections (BSI) and long-term use of broad-spectrum antibiotics (more than 7 days). The main outcomes were 30-day and in-hospital mortality of patients with BSI. The secondary outcomes were the day of therapy (DOT) and susceptibility of antipseudomonal agents. Cases were classified into two groups: the pre-ASP group comprised cases between 2011 and 2013 and the post-ASP group, between 2014 and 2016. The outcomes were then compared between the two groups. Among the patients with all BSI (n = 1187), no significant differences in 30-day mortality were observed between those in the pre-ASP and post-ASP groups. However, in-hospital mortality was significantly lower in the post-ASP group than that in the pre-ASP group (24.8% vs. 18.0%; P = 0.004). Furthermore, the 30-day and in-hospital mortality of resistant gram-negative bacteraemia was significantly lower (20.4% vs.10.5%; P = 0.04 and 28.0% vs.16.1%; P = 0.03). The DOT of broad-spectrum antibiotics decreased except that of tazobactam/piperacillin. The susceptibilities of tazobactam/piperacillin, ceftazidime, cefepime, sulbactam/cefoperazone, gentamicin, ciprofloxacin levofloxacin, imipenem and meropenem were significantly better. Interventions by the AST can improve the clinical and microbiological outcomes, especially resistant gram-negative bacteria. Furthermore, this effect of our ASP can continue for a long term.

In Vitro and In Vivo Evaluation of 6-O-α-Maltosyl-ß-Cyclodextrin as a Potential Therapeutic Agent Against Niemann-Pick Disease Type C.

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-ß-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-ß-CD (21.4 µmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.

The effects of antifungal stewardship programs at a tertiary-care teaching hospital in Japan.

Patients with invasive fungal diseases (IFDs) generally have a high mortality rate, and resistance to antifungal drugs and the high costs associated with it have led to recent problems, necessitating the appropriate use of antifungals. To this end, we launched Antifungal Stewardship Programs (AFSPs) in our hospital. Patients who were systemically administered antifungals from January 2011 to December 2016 were enrolled this study and divided into pre-intervention and intervention groups. No significant difference was observed in defined daily doses per 1000 patient-days (23.3 ± 8.0 vs 20.4 ± 10.8, p = 0.251) between the groups. The monthly average for the days of therapy per 1000 patient-days was significantly lower in the intervention group (15.1 ± 3.1 vs 12.7 ± 4.3, p = 0.009). The cost of the antifungals reduced over the 3-year period by $260,520 (13.5%). Furthermore, a decreasing trend was observed in both the 30-day mortality (40.9% vs 30.0%, p = 0.414) and in-hospital mortality (63.6% vs 36.7%, p = 0.054) in patients with candidemia. Our results indicate that AFSPs are efficacious and cost-effective approaches.

Role of 6-O-α-maltosyl-ß-cyclodextrin in lysosomal cholesterol deprivation in Npc1-deficient Chinese hamster ovary cells.

We aimed to investigate whether 6-O-α-maltosyl-ß-cyclodextrin (Mal-ßCD) is incorporated into cells and lysosomes during the release of unesterified cholesterol in Npc1-deficient Chinese hamster ovary (CHO) cells (Npc1 KO cells) and CHO-JP17 cells (JP17 cells). Internalization of Mal-ßCD in cells and lysosomes and extracellular release of lysosomal unesterified cholesterol were demonstrated by LC/MS/MS and LC/MS, respectively. Internalization of Mal-ßCD was greater in Npc1 KO cells than in JP17 cells. The majority of internalized Mal-ßCD in both cell types was metabolized by lysosomal α-glucosidase to 6-O-α-D-glucosyl-ß-cyclodextrin (Glc-ßCD). However, Mal-ßCD did not directly enter the lysosomes prepared from cell homogenates. Mal-ßCD-treated Npc1 KO cells and JP17 cells both released Mal-ßCD and Glc-ßCD, together with unesterified cholesterol, out of cells. The release of unesterified cholesterol by Mal-ßCD in Npc1 KO cells was much greater than that in JP17 cells. This study is the first to report the influx of Mal-ßCD into the lysosomes of Npc1 KO cells and JP17 cells, followed by generation of Glc-ßCD, and the efflux of Mal-ßCD/Glc-ßCD and unesterified cholesterol to the extracellular fluid, based on the quantitative LC/MS analysis.

Clinical Characteristics of Methicillin-resistant Coagulase-negative Staphylococcal Bacteremia in a Tertiary Hospital.

Objective Coagulase-negative staphylococci are among the most frequently isolated microorganisms in blood cultures. The aim of this study was to assess [1] the clinical characteristics of methicillin-resistant, coagulase-negative staphylococci bacteremia and [2] the susceptibility of the isolated bacteria to glycopeptides. Methods We retrospectively reviewed the medical records of 70 patients from whom methicillin-resistant coagulase-negative staphylococci had been isolated at Osaka City University Hospital between January 2010 and December 2013. We evaluated the patients' background, severity and prognosis of the disease, and the susceptibility of the isolated methicillin-resistant coagulase-negative staphylococci to glycopeptides. Results Out of the 70 patients tested, 28 (40.0%) had leukemia, and 36 (51.4%) had been treated for febrile neutropenia. Infection with Staphylococcus epidermidis accounted for 78.6% of patients. Thirty-nine cases (55.7%) were related to intravascular catheters, and 39 (55.7%) were treated using teicoplanin as a first-line therapy. The 30-day mortality rate was 4.3%. Regarding susceptibility, 20% of all isolates were non-susceptible to teicoplanin. According to multivariate analyses, it was observed that premedication using glycopeptides was independently associated with teicoplanin non-susceptibility (p=0.03; hazard ratio = 5.64; 95% confidence interval, 1.16-26.76). Conclusion Our results suggest that clinicians must use glycopeptides appropriately to prevent the development of further antibiotic resistance in methicillin-resistant coagulase-negative staphylococci.

Effectiveness of weekly polymerase chain reaction-based open reading frame typing analysis of all newly isolated methicillin-resistant Staphylococcus aureus strains for controlling nosocomial infections.

Polymerase chain reaction (PCR)-based open reading frame typing (POT) helps differentiate between bacterial strains based on the open reading frames (ORFs) of the prophage-encoding genes; multiplex PCR screening is performed to identify strains based on keeping patterns. At our hospital, surveillance of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) transmission is undertaken using POT to conduct molecular epidemiological analysis for all newly detected MRSA strains. In 2014, we performed POT only once a month; however, in 2015, we increased the frequency of POT to once a week, which helped us detect nosocomial transmission that would normally be difficult to detect, and thus achieve 40% reduction in nosocomial transmission, compared to that in 2014. This suggests that weekly POT screening for all MRSA strains is one of the effective methods available for minimizing nosocomial transmission of MRSA.

[A comparison of susceptibility of Pseudomonas aeruginosa clinical isolates to carbapenem antibiotics in our hospital].

We investigated the susceptibility of 400 Pseudomonas aeruginosa (P. aeruginosa) clinical isolates to 3 antipseudomonal carbapenems, namely, doripenem (DRPM), meropenem (MEPM), and imipenem (IPM). The test strains were isolated from the following specimens: respiratory (n = 194), urinary (n = 61), digestive (n = 38), pus (n = 36), skin (n = 21), blood (n = 9), upper respiratory tract and oral cavity (n = 8), and others (n = 33) at Osaka City University Hospital from July to October 2013. Test strains were categorized as susceptible, ≤ 2 µg/mL; intermediate, 4 µg/mL; and resistant, ≥ 8 µg/mL according to Clinical and Laboratory Standards Institute criteria (M100-S22), updated on January 2012. To compare the antimicrobial activities of these 3 carbapenems, the susceptibility rate for each agent was analyzed. Susceptibility to DRPM, MEPM, and IPM was 78.3%, 74.3%, and 64.8%, respectively, whereas resistance was 12.5%, 22.8%, and 28.5%, respectively. The frequency of strains resistant to DRPM was significantly lower than that for MEPM (p < 0.001) and IPM (p < 0.001). To compare the activities of the 3 carbapenems against the P. aeruginosa clinical isolates, we plotted the numbers of strains against each minimum inhibitory concentration (MIC) level. The MICs of DRPM were lower than those of MEPM in 19.8% of strains, and lower than those of IPM in 41.8% of strains, and the MICs of MEPM were lower than those of IPM in 33.0% of strains. Further, we found that 7.7% of the MEPM-resistant strains were susceptible to DRPM, 23.7% of the IPM-resistant strains were susceptible to DRPM, and 9.6% of the IPM-resistant strains were susceptible to MEPM; however, none of the MEPM-resistant strains was susceptible to IPM, and none of the DRPM-resistant strains was susceptible to MEPM or IPM. In conclusion, the in vitro activity of DRPM against the P. aeruginosa clinical isolates was superior to those of MEPM and IPM.

Role of lipid raft components and actin cytoskeleton in fibronectin-binding, surface expression, and de novo synthesis of integrin subunits in PGE2- or 8-Br-cAMP-stimulated mastocytoma P-815 cells.

Integrins are heterodimeric adhesion receptors essential for adhesion of non-adherent cells to extracellular ligands such as extracellular matrix components. The affinity of integrins for ligands is regulated through a process termed integrin activation and de novo synthesis. Integrin activation is regulated by lipid raft components and the actin structure. However, there is little information on the relationship between integrin activation and its de novo synthesis. Cancerous mouse mast cells, mastocytoma P-815 cells (P-815 cells) are known to bind to fibronectin through de novo synthesis of integrin subtypes by prostaglandin (PG) E2 stimulation. The purpose of this study was to clarify the relationship between lipid raft components and the actin cytoskeleton, and PGE2-induced P-815 cells adhesion to fibronectin and the increase in surface expression and mRNA and protein levels of αvß3 and αIIbß3 integrins. Cholesterol inhibitor 6-O-α-maltosyl-ß cyclodextrin, glycosylphosphatidylinositol-anchored proteins inhibitor phosphatidylinositol-specific phospholipase C and actin inhibitor cytochalasin D inhibited PGE2-induced cell adhesion to fibronectin, but did not regulate the surface expression and mRNA and protein levels of αv and αIIb, and ß3 integrin subunits. In addition, inhibitor of integrin modulate protein CD47 had no effect on PGE2- and 8-Br-cAMP-induced cell adhesion. These results suggest that lipid raft components and the actin cytoskeleton are directly involved in increasing of adhesion activity of integrin αIIb, αv and ß3 subunits to fibronectin but not in stimulating of de novo synthesis of them in PGE2-stimulated P-815 cells. The modulation of lipid rafts and the actin structure is essential for P-815 cells adhesion to fibronectin.

Effect of 6-O-α-maltosyl-ß cyclodextrin and its cholesterol inclusion complex on cellular cholesterol levels and ABCA1 and ABCG1 expression in mouse mastocytoma P-815 cells.

We have previously described 6-O-α-maltosyl-ß cyclodextrin (Mal-ßCD), which forms soluble inclusion complex with cholesterol. Here we further investigated the effect of Mal-ßCD and cholesterol/Mal-ßCD inclusion complex (CLM) on cellular cholesterol levels in a mouse mast cell line, mastocytoma P-815 cells (P-815 cells). Mal-ßCD removes cellular cholesterol forming inclusion complexes, while Mal-ßCD-induced lack of cellular cholesterol was replenished by the addition of CLM without cytotoxicity. Reduction and replenishment of cellular cholesterol in Mal-ßCD- and/or CLM-treated P-815 cells, respectively, were demonstrated by LC/MS and fluorescence microscopy with filipin III. CLM rather than free Mal-ßCD and free cholesterol was efficiently incorporated into P-815 cells and its incorporation was inhibited by incubation at low temperature, or with sodium azide and cytochalasin D. P-815 cells have been confirmed to express ATP-binding cassette (ABC) transporters, ABCA1, ABCG1, and P-glycoprotein (P-gp), by Western blot and mRNA analysis. Cholesterol reduction by Mal-ßCD abolishes the mRNA and protein expression of ABCA1 and ABCG1, but not of P-gp. Cholesterol loading by CLM restores the diminished ABCA1 and ABCG1 mRNA expression in Mal-ßCD-treated P-815 cells. However, both Mal-ßCD and CLM had no effect on P-gp activity measured by the rhodamine 123 efflux assay. These results indicate that alteration of cholesterol levels with Mal-ßCD or CLM led to down- or up-regulation of ABCA1 and ABCG1 expression in P-815 cells.

Physicochemical and biological properties of 2-O-α-D-galactosyl-cyclomaltohexaose (α-cyclodexterin) and -cyclomaltoheptaose (ß-cyclodextrin).

The physicochemical and biological properties of the new branched cyclomaltooligosaccharides (cyclodextrins; CDs), 2-O-α-D-galactosyl-cyclomaltohexaose (2-O-α-D-galactosyl-α-cyclodextrin, 2-Gal-αCD) and 2-O-α-D-galactosyl-cyclomaltoheptaose (2-O-α-D-galactosyl-ß-cyclodextrin, 2-Gal-ßCD), were investigated. The formation of inclusion complexes of 2-Gal-CDs with various kinds of guest compounds (clofibrate, cholesterol, cholecalciferol, digitoxin, digitoxigenin, and prostaglandin A(1)) was examined by a solubility method, and the results were compared with those of non-branched CDs and other 6-O-glycosyl-CDs such as 6-O-α-D-galactosyl-CDs, 6-O-α-D-glucosyl-CDs, and 6-O-α-maltosyl-CDs. The inclusion abilities of 2-Gal-αCD for clofibrate and prostaglandin A(1), and 2-Gal-ßCD for clofibrate, cholecalciferol, cholesterol, and digitoxigenin were markedly weaker than those of non-branched CD and other 6-O-glycosyl-CDs in each series, probably because of a steric hindrance caused by the α-(1→2)-galactoside linkage. The hemolytic activities of 2-Gal-CDs on human erythrocytes were the lowest among each CD series, and the compounds showed negligible cytotoxicity towards Caco-2 cells up to at least 100mM.

Induction of integrin ß3 in PGE2-stimulated adhesion of mastocytoma P-815 cells to the Arg-Gly-Asp-enriched fragment of fibronectin.

We previously demonstrated that prostaglandin (PG) E2 stimulates adhesion of mastocytoma P-815 cells (P-815 cells) to the Arg-Gly-Asp (RGD)-enriched matrix via the PGE2 receptor subtype EP4 [Hatae N, Kita A, Tanaka S, Sugimoto Y, Ichikawa A. Induction of adherent activity in mastocytoma P-815 cells by the cooperation of two prostaglandin E2 receptor subtypes, EP3 and EP4. J Biol Chem 2003;278:17977-81]. Here we investigated the role of various integrin subtypes in the induction of adherent activity in PGE(2)-stimulated P-815 cells. FACS analysis showed that P-815 cells express high levels of integrin α4, α5, ß1 and ß2 subunits and moderate levels of integrin αIIb, αv, ß3 and ß7 subunits. When treated with PGE2, the EP4 agonist ONO-AE1-329 or the cell permeable cAMP analogue, 8-Br-cAMP, P-815 cells showed markedly increased cell surface expression of integrin αIIb, αv and ß3 subunits, and these expressions were significantly reduced by addition of the protein synthesis inhibitor cycloheximide. Along with increased cell surface expression, mRNA and protein levels of the integrin ß3 subunit, but not of integrin αIIb and αv subunits, were simultaneously elevated. On the other hand, adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP was abolished by antibodies specific for integrin αv and ß3 subunits, but not by antibodies for integrin α4, α5, ß1, ß2 and ß7 subunits. Moreover, treatment with tirofiban, an integrin αIIbß3 antagonist, or eptifibatide, an integrin αvß3/αIIbß3 antagonist resulted in a decrease in adhesion of P-815 cells in response to PGE2 or 8-Br-cAMP. These results suggest that de novo synthesis of the integrin ß3 subunit plays a pivotal role in PGE2-induced adhesion of P-815 cells to the RGD-enriched matrix through EP4-mediated cAMP signaling.

Negative and positive ion mode LC/MS/MS for simple, sensitive analysis of sorbic acid.

Sorbic acid (SA: CH(3)-CH=CH-CH=CH-COOH) is one of the widely used food preservatives, although there have been some reports of its toxic activity, for example, on DNA and skin cells. In order to examine the effects of SA on mammalian tissues, we have developed a highly sensitive analytical method using LC/MS/MS with positive and negative ion mode electrospray ionization (ESI). In a previous study, we found that a nonacidic eluent offers better ionization efficiency than acids or their ammoniun salts. However, optimal results could not be obtained because the anion form of SA is poorly retained on a conventional reversed phase column. To resolve this problem, we chose a new type of column and used high-resolution mass spectrometry and positive ion mode analysis. There have only been a few reports using these methods in the positive mode, for example derivatized SA, because acid compounds such as SA are usually used in the negative ion mode. However, a new type of low-carbon-content and polar-endcapped C18 phase column was developed for better separation of SA from the matrix. High-resolution selected reaction monitoring (SRM) gave the best signal to noise ratio in normal-resolution SRM. In the positive ion mode, the CH(3)OH-0.05% HCOOH/0.1% CH(3)COOH eluent system yielded the best ionization efficiency. We propose a highly sensitive and simple analysis using a two-ion-mode ESI SRM method. Such systems should allow quantification of the amount of SA in or around the cells, without the need for pretreatment such as solid phase extraction.