Five-year trends in bacterial contamination of dialysis equipment.

Bedside dialysis monitoring equipment for hemodialysis are located in the bioburden section upstream of the endotoxin-retentive filter for dialysis fluid sterilization. We observed 26 equipment at our institution for bacterial contamination at least once every 4 weeks for 5 years with another ultrafiltration membrane upstream to prevent bacterial contamination. Bacterial contamination levels were highest and most diverse at the time of the first flush. During subsequent initial cleanng, the contamination level decreased, and bacterial species converged almost exclusively to one genus, namely Methylobacterium spp. During clinical use, the equipment were cleaned and disinfected daily after dialysis, and daily operations and maintenance were performed using aseptic techniques. Although the frequency of bacterial detection decreased annually, the same bacterial genotypes observed at the first flush were isolated even after long time periods and were thought to persist in the equipment possibly by forming biofilm. Pseudomonas aeruginosa was newly detected after the replacement of parts during breakdown maintenance, indicating the need to sterilize replacement parts. Thus, the bioburden should be assessed regularly as part of the management of in-house-produced dialysis fluid.

Sodium Hypochlorite is Effective against Biofilms in Dialysis Equipment.

To test the efficacy of chemical disinfectants against bacterial biofilms in hemodialysis equipment, a Center for Disease Control and Prevention (CDC)-Biofilm Reactor was used to create biofilms. Methylobacterium radiotolerance was isolated from the hemodialysis fluid and used as the test organism. We examined the efficacy of sodium hypochlorite (NaOCl) in elimination of planktonic cells compared to that in the case of biofilms. Planktonic bacteria were completely eliminated at 50 parts per million (ppm) of NaOCl, which is the lowest concentration for clinical use. The viable cell count in the biofilm reached its minimum value around a logarithmic reduction value (LRV) of 6, when the concentration was raised to 1000 ppm and the reaction time was extended by 1 hour or more. Furthermore, at 200 ppm, the LRV was elevated depending on the time. And the LRV while maintaining static conditions for 6 hours at 200 ppm was similar to that of short time at 1000 ppm. These results suggest that NaOCl has sufficient bactericidal activity even for biofilms at a practical concentration and reaction time, and that the CDC-Biofilm Reactor is an effective tool for finding useful disinfection conditions.

Change during an 8-Year Period in Streptococcus Pyogenes emm Types in Pharyngeal Isolates from Children with Noninvasive Infections.

BACKGROUND: Streptococcus pyogenes, or group A streptococcus (GAS), is one of the most common bacterial pathogens in children. GAS can cause such nonserious and noninvasive diseases as pharyngitis and skin infection, as well as serious, invasive diseases like streptococcal toxic shock syndrome. One factor that makes GAS pathogenic is the type-specific M protein on its cell surface. To identify emm types and their characteristics, we previously examined GAS strains isolated from children with noninvasive infections at our hospital. The present study was conducted 8 years later, for comparison. METHODS: The 23 participants were inpatients and outpatients at Nippon Medical School Tama Nagayama Hospital during 2016 and 2017. A pharyngeal swab specimen was obtained from each child, and genes encoding M proteins were amplified by polymerase chain reaction. RESULTS: emm type analysis identified emm1 in 11 of the 23 strains and emm12 in 4. Three group G streptococcus (GGS) strains carried M-like protein genes. CONCLUSIONS: The predominant emm type was emm12 in our previous report and emm1 in this study. This study also identified 3 GGS strains among the isolates, which carried either the stg245, stg6795, or stg840 M-like protein gene. One GAS strain carried stg485, a gene associated with GGS rather than GAS.

Neutralizing antibodies for Helicobacter pylori urease inhibit bacterial colonization in the murine stomach in vivo.

Helicobacter pylori (H. pylori) urease is a key protein for persistent infection of the bacteria in the stomach. Although H. pylori generally induce anti-H. pylori-specific antibodies (Abs), these Abs do not usually work for eradication or prevention of the H. pylori infection. In our previous study, we identified a linear epitope composed of 19-mer peptides termed UB-33, CHHLDKSIKEDVQFADSRI, within the large subunit of H. pylori urease. Anti-UB-33-specific Abs neutralized the enzymatic activity of H. pylori urease in vitro. In the present study, we evaluated the effect of immunization of BALB/c mice with H. pylori UB-33 peptide. After confirming the production of anti-UB-33-specific Abs, mice were challenged orally with H. pylori Sydney Strain-1 (SS-1). Mice producing anti-UB-33-specific Abs were not infected with SS-1, and the amount of SS-1 isolate in their stomach was significantly reduced. Also, the urease-negative mutant of H. pylori, HPP1801, did not colonize in the stomach, indicating that H. pylori urease was a critical element for infection of H. pylori in the gastric mucosa. Moreover, mice producing UB-33-specific Abs apparently suppressed H. pylori infection in the stomach where anti-UB-33 Abs were secreted in the gastric juice, indicating that H. pylori colonization was inhibited in the presence of anti-UB-33 Abs. In addition, the neutralization activity of sera from mice immunized with purified urease was less potent than that in the sera from mice immunized with UB-33. Furthermore, the recognition of epitope UB-33 was mediated through Toll-like receptor 2 (TLR2) on the B-1 cells using TLR2-knockout BALB/c mice in vivo. These results indicate that liner peptide UB-33 should be used for immunization to induce neutralizing Abs instead of purified H. pylori urease to prevent H. pylori infection and their colonization in the stomach.

Detection Method for Aquatic Bacteria of the Fingers, as a Potential Origin of the Aqueous Solution Contamination.

　Aquatic bacteria were isolated from the hands of working staffs by an adapted culture protocol. When the sample solution obtained by the" glove juice method" was incubated for 3 days at room temperature, viable cell counts increased up to 105-fold, and the majority of the isolated colonies were shown to be Gram-negative aquatic bacteria, which carry the risk of contaminating water. Using R2A medium, coagulase-negative staphylococci were the dominant microbes immediately after recovery from the hands. Here it was revealed that bacteria of the phylum Proteobacteria isolated from the hand can be the causative bacteria of aqueous contamination. This modification in the GJ method may be useful as an effective training protocol to demonstrate the importance of hand hygiene and clean operation for aseptic manufacturing.

Rapid detection of microbes in the dialysis solution by the microcolony fluorescence staining method (Millflex quantum).

A chemiluminescence system, Milliflex Quantum (MFQ), to detect microcolonies, has been used in the pharmaceutical field. In this study, we investigated aquatic bacteria in hemodialysis solutions sampled from bioburden areas in 4 dialysis faculties. Using MFQ, microcolonies could be detected after a short incubation period. The colony count detected with MFQ after a 48-hour incubation was 92% ± 39%, compared to that after the conventionally used 7-14-day incubation period; in addition, the results also showed a linear correlation. Moreover, MFQ-based analysis allowed the visualization of damaged cells and of the high density due to the excessive amount of bacteria. These results suggested that MFQ had adequate sensitivity to detect microbacteria in dialysis solutions, and it was useful for validating the conditions of conventional culture methods.

Increased mitochondrial functions in human glioblastoma cells persistently infected with measles virus.

Measles virus (MV) is known for its ability to cause an acute infection with a potential of development of persistent infection. However, knowledge of how viral genes and cellular factors interact to cause or maintain the persistent infection has remained unclear. We have previously reported the possible involvement of mitochondrial short chain enoyl-CoA hydratase (ECHS), which is localized at mitochondria, in the regulation of MV replication. In this study we found increased functions of mitochondria in MV-persistently infected cells compared with uninfected or acutely infected cells. Furthermore, impairment of mitochondrial functions by treatment with mitochondrial inhibitors such as ethidium bromide (EtBr) or carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) induced the cytopathic effects of extensive syncytial formation in persistently infected cells. These findings suggest that mitochondria are one of the subcellular organelles contributing to regulate persistent infection of MV. Recent studies showed mitochondria provide an integral platform for retinoic acid-inducible protein (RIG-I)-like cytosolic receptors (RLRs) signaling and participate in cellular innate antiviral immunity. Our findings not only reveal a role of mitochondria in RLR mediated antiviral signaling but also suggest that mitochondria contribute to the regulation of persistent viral infection.

E-cadherin interactions are required for Langerhans cell differentiation.

Human skin contains the following two distinct DC subsets: (i) Langerhans cells (LCs), expressing Langerin but not DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), are predominantly localized in the epidermis; and (ii) dermal DCs, expressing DC-SIGN but not Langerin, are observed mainly in the dermis. It is not known whether localization in the epidermis provides cues for LC differentiation. Here, we show that E-cadherin expressed by epidermal keratinocytes (KCs) is crucial for differentiation of LCs. Monocytes differentiated into LC-like cells in presence of IL-4, GM-CSF, and TGF-ß1. However, these LC-like cells expressed not only Langerin but also DC-SIGN. Notably, co-culturing of these LC-like cells with KCs expressing E-cadherin or recombinant E-cadherin strongly decreased expression of DC-SIGN and further induced a phenotype similar to purified epidermal LCs. Moreover, pretreatment of LC-like cells with anti-E-cadherin-specific antibody completely abolished their Langerin expression, indicating the requirement of E-cadherin-E-cadherin interactions for the differentiation into Langerin(+) cells. These findings suggest that E-cadherin expressed by KCs provide environmental cues that induce differentiation of LCs in the epidermis.

Induction of rapid apoptosis for class I MHC molecule-restricted CD8(+) HIV-1 gp160-specific murine activated CTLs by free antigenic peptide in vivo.

We have previously reported that the cytotoxic activity of murine CD8(+) CTLs specific for HIV-1 gp160 envelope protein was markedly inhibited in vitro by brief exposure to a free epitope peptide P18-I10 (aa: RGPGRAFVTI) using the epitope-specific CTL line (LINE-IIIB) or a clone (RT-1). We have also shown that recently stimulated P18-I10-specific murine CTLs rapidly fell into apoptosis in vitro after brief exposure to the free epitope peptide. In the present study, we examined whether similar inactivation or apoptosis of recently stimulated CTLs occurred in vivo by exposure to the free epitope peptide using TCR transgenic (Tg-RT-1) mice expressing TCRαß genes of CTL clone RT-1. When the Tg mice were inoculated with recombinant vaccinia virus expressing HIV-1-IIIB gp160 genes followed by injection of P18-I10 epitope peptide, apparent reduction in the number of CTLs determined by flow cytometry using H-2D(d)/P18-I10 pentamer was observed within a few hours after the injection. Most of the H-2D(d)/P18-I10 pentamer-stained cells were positive for Annexin V and apoptosis was confirmed by microscopic analyses. Moreover, when mice were pretreated with immunosuppressive agents, such as cyclosporin A and tacrolimus (FK506), induction of apoptosis by P18-I10 was significantly inhibited and CTL cytotoxicity was maintained. These results suggest that the rapid loss of virus-specific CD8(+) CTLs might occur in vivo through apoptosis in the early stages of viral infection when activated CTLs may encounter viral epitope(s) released from virus-infected cells attacked by CTLs and we can prevent the loss by pretreatment with immunosuppressive agents.

A case of streptococcal toxic shock syndrome due to Group G streptococci identified as Streptococcus dysgalactiae subsp. equisimilis.

A 79-year-old man with a 3-month history of lymphedema of the lower limbs, and diabetes mellitus, was admitted to our hospital for suspected deep venous thrombosis. Several hours after admission, leg pain and purpura-like skin color appeared. On the 2nd hospital day, he was referred to our department for possible acute occlusive peripheral artery disease (PAD) and skin necrosis with blisters; however, computed tomography with contrast showed no occlusive lesions. He had already developed shock and necrotizing deep soft-tissue infections of the left lower leg. Laboratory findings revealed renal dysfunction and coagulation system collapse. Soon after PAD was ruled out, clinical findings suggested necrotizing deep soft-tissue infections, shock state, disseminated intravascular coagulation, and multiple organ failure. These symptoms led to a high suspicion of the well-recognized streptococcal toxic shock syndrome (STSS). With a high suspicion of STSS, we detected Group G ß-hemolytic streptococci (GGS) from samples aspirated from the leg bullae, and the species was identified as Streptococcus dysgalactiae subsp. equisimilis (SDSE) by 16S-ribosomal RNA sequencing. However, unfortunately, surgical debridement was impossible due to the broad area of skin change. Despite adequate antimicrobial therapy and intensive care, the patient died on the 3rd hospital day. The M-protein gene (emm) typing of the isolated SDSE was revealed to be stG6792. This type of SDSE is the most frequent cause of STSS due to GGS in Japan. We consider it to be crucial to rapidly distinguish STSS from acute occlusive PAD to achieve life-saving interventions in patients with severe soft-tissue infections.

Production of autoantibodies by murine B-1a cells stimulated with Helicobacter pylori urease through toll-like receptor 2 signaling.

Helicobacter pylori infection is associated with several autoimmune diseases, in which autoantibody-producing B cells must be activated. Among these B cells, CD5-positive B-1a cells from BALB/c mice were confirmed to secrete autoantibodies when cocultured with purified H. pylori urease in the absence of T cells. To determine the mechanisms for autoantibody production, CD5-positive B-1a cells were sorted from murine spleen cells and stimulated with either purified H. pylori urease or H. pylori coated onto plates (referred to hereafter as plate-coated H. pylori), and autoantibody production was measured by enzyme-linked immunosorbent assay (ELISA). Complete urease was not secreted from H. pylori but was visually expressed over the bacterium-like endotoxin. Urease-positive plated-coated H. pylori stimulated B-1a cells to produce autoantibodies, although urease-deficient isotype-matched H. pylori did not. Autoantibody secretion by B-1a cells was inhibited when bacteria were pretreated with anti-H. pylori urease-specific antibody having neutralizing ability against urease enzymatic activity but not with anti-H. pylori urease-specific antibody without neutralizing capacity. The B-1a cells externally express various Toll-like receptors (TLRs): TLR1, TLR2, TLR4, and TLR6. Among the TLRs, blocking of TLR2 on B-1a cells with a specific monoclonal antibody (MAb), T2.5, inhibited autoantibody secretion when B-1a cells were stimulated with plate-coated H. pylori or H. pylori urease. Moreover, B-1a cells from TLR2-knockout mice did not produce those autoantibodies. The present study provides evidence that functional urease expressed on the surface of H. pylori will directly stimulate B-1a cells via innate TLR2 to produce various autoantibodies and may induce autoimmune disorders.

Transformation of breast milk macrophages by HTLV-I: implications for HTLV-I transmission via breastfeeding.

Human T cell leukemia virus type I (HTLV-I), a causative agent of adult T-cell leukemia (ATL), is transmitted from mother to child predominantly by breastfeeding. The source of HTLV-I-infected cells in breast milk has been thought to be T cells, however, the majority of cells in breast milk are CD14(+) macrophages but not CD3(+) T lymphocytes, and no data are available regarding HTLV-I transmission through breast milk macrophages (BrMMpsi). To explore the potential of BrMMpsi as a possible source of infection in mother to child transmission (MTCT) of HTLV-I, an immortalized cell line (HTLV-BrMMpsi) has been established from BrMMpsi by infection with HTLV-I. HTLV-BrMMpsi retained macrophage characteristics and did not express a complete dendritic cell (DC) phenotype; nevertheless, HTLV-BrMMpsi efficiently promoted T cell proliferation in primary allogeneic mixed lymphocyte reaction (MLR) like DC. Moreover, HTLV-I infection could be transmitted from HTLV-BrMMpsi to activated T cells in the peripheral blood. These findings suggested that BrMMpsi might be an appropriate HTLV-I reservoir involved in MTCT transmission via breastfeeding.

Quick method of multimeric protein production for biologically active substances such as human GM-CSF (hGM-CSF).

The C-terminal fragment of C4b-binding protein (C4BP)-based multimerizing system was applied to hGM-CSF to induce dendritic cells (DCs) from peripheral blood monocytes (PBMCs), to see whether the C4BP could stimulate immature DCs, since DCs, equipped with pattern recognition receptors such as toll-like receptors (TLRs), are hypersensitive to various immunologically active molecules like LPS. hGM-CSF gene was merged to the 3'-terminal region of the C4BPalpha-chain gene, and the transfected human 293FT cells produced sufficient amount of octameric hGM-CSF, which resulted in iDCs with the same phenotype and the same response to a TRL4 ligand, LPS and a TLR3 ligand, poly I:C, as those induced with authentic monomeric hGM-CSF. These results suggest that the C4BP-based multimerizing system could facilitate the design of self-associating multimeric recombinant proteins without stimulating iDCs, which might be seen with the other multimerizing systems such as that using Fc fragment of IgM.

Relation of leukocytosis in prostatic fluid and inflamed prostatic tissue.

In our continuing investigation of the significance of leukocytosis in prostatic fluid (PF), the relation of leukocytosis in PF to that in selected sections of prostate with significant inflammation was studies with whole-mount specimens obtained at radical prostatectomy from 12 patients with prostate cancer. Although leukocytosis was observed both in PF and in prostate tissue in all 12 patients, there was no correlation between the leukocyte count in PF and the intensity of inflammation. However, the ratio of macrophages among leukocytes in PF correlated with the number of ducts filled with macrophages in prostate tissue (p=0.0481). This finding was consistent with our previous finding that activation of macrophages in PF reflects active inflammation in prostate tissue. Further studies are needed to clarify the roles of macrophages and whole leukocytes in PF and prostate tissue.

BCG vaccine elicits both T-cell mediated and humoral immune responses directed against mycobacterial lipid components.

The universe of antigens recognized by alphabeta T cells has recently been expanded to include not only major histocompatibility complex (MHC)-presented protein antigens but also CD1-presented lipid antigens. The significance of lipid-reactive T cells in host defense has been appreciated, using the guinea pig model of human tuberculosis. Here, we show that immunization with Mycobacterium bovis bacillus Calmette-Guerin (BCG), the commonly used anti-tuberculosis vaccine, induces activation of guinea pig cytotoxic T cells recognizing BCG lipids in the context of CD1 molecules. Further, BCG-immunized, but not mock-immunized, guinea pigs mount IgG antibody responses directed against lipoarabinomannnan, an essential cell wall lipid component of mycobacteria. These observations emphasize the ability of BCG to activate the host adaptive immunity to mycobacteria-derived lipids, which could potentially contribute to protection against tuberculosis.

Study of prostatic fluid from patients with elevated levels of prostate-specific antigen.

INTRODUCTION: Characteristics of prostatic fluid (PF), which can be obtained in large amounts during screening transrectal ultrasound just before prostate biopsy to detect prostate cancer, were investigated. These characteristics include the amount of PF obtained and the number of leukocytes in PF, which would be useful for planning cell-biological or immunological studies of leukocytes in PF and for increasing the understanding of prostatitis in elderly men. PATIENTS AND METHODS: The volume of PF and the number of leukocytes in PF were measured in 50 patients suspected of having prostate cancer because of elevated levels of serum prostate-specific antigen (PSA). Correlations of the volume of PF, the number of leukocytes per milliliter, the total leukocyte number with age and prostate volume and correlation of PSA levels with the number of leukocytes per milliliter and total leukocyte number were also investigated. RESULTS: The average patient age was 67.2 years, and PF specimens were obtained from 43 of the 50 patients (86%). The mean +/- SD of PF volume, number of leukocytes in PF, and total leukocyte number were 347.65 +/- 305.76 microl, 4.84 +/- 6.07 x 10(6) /ml, and 1.47 +/- 2.10 x 10(6), respectively. A correlation was observed only between the total leukocyte number and the volume of the transitional zone (P=0.039). CONCLUSIONS: These data provide information for investigators to plan cell-biological or immunological studies of leukocytes in PF and for understanding prostatitis in elderly men.

Temperature-dependent biosynthesis of glucose monomycolate and its recognition by CD1-restricted T cells.

Mycolic acids are long chain fatty acids that constitute the lipid-rich cell wall framework of mycobacteria. Upon infection, mycobacteria begin to synthesize glucose monomycolate (GMM), a glucosylated species of mycolic acids, by utilizing host-derived glucose as sugar source. Accordingly, GMM production serves as a good indicator for local invasion of mycobacteria, and its detection by the host immune system would favor efficient monitoring of mycobacterial infection. Here, we found that GMM was produced abundantly at 30 degrees C rather than at 37 degrees C and recognized by a GMM-specific, CD1-restricted T cell line that was isolated from mycobacteria-infected human skin. Since the common portal sites for mycobacterial infection include ventilating alveoli of the lung and the externally exposed skin that often render invading microbes survive at reduced temperature, sampling GMM by CD1 lipid antigen-presenting molecules may allow the host to detect mycobacterial infection at its early phases.

Resistance to viral infection by intraepithelial lymphocytes in HIV-1 P18-I10-specific T-cell receptor transgenic mice.

For the analysis of mucosal immunity to HIV-1, we have recently established a line of transgenic (Tg) mice expressing the TCRalpha and TCRbeta genes of the murine CTL clone RT1 specific for P18-I10 (RGPGRAFVTI), an immunodominant gp160 envelope-derived epitope of IIIB isolate, restricted by the H-2D(d) MHC-I molecule. Here we examine those cells bearing specific TCR among the intraepithelial lymphocytes (IELs), with flow cytometric analysis using H-2D(d)/P18-I10 tetramers. We observed three distinct CD3(+), tetramer positive populations among the IELs: extra-thymic CD8alphabeta(+), alphabetaTCR T-cells; CD8 alphaalpha+, gammadeltaTCR T-cells; and thymus-derived CD8alphabeta+, alphabetaTCR T-cells. Challenge of these Tg mice with P18-I10 encoded by a vaccinia virus vector, either intrarectally (i.r.) or intraperitoneally (i.p.), revealed that the intraepithelial compartment seems to be a major site for prevention of the spread of viral infection. Such immunity appears due to the thymus-derived, CD8alphabeta+ antigen-specific CTLs together with CD8alphaalpha+ gammadelta cells, which regulate virus spread. This model system for studying CTL based immunity at mucosal sites should prove helpful in developing rational approaches for HIV control.

Age-dependent decrease of polymeric Ig receptor expression and IgA elevation in ddY mice: a possible cause of IgA nephropathy.

Individual animals in the closed colony population of ddY mice were analyzed to clarify the major cause of age-dependent elevation of serum IgA and the appearance of human IgA nephropathy (IgAN)-like symptoms. Based on the serum IgA levels, the mice were classified into two subgroups. One was a high serum IgA group with some manifestations of IgAN through aging (ddY(High)), and the other was a normal serum IgA group without IgAN (ddY(Norm)). The ratio of urinary IgA to serum IgA was significantly reduced in ddY(High) mice, suggesting an impaired IgA clearance via secretion through the epithelial barrier. The actual clearance rate of the intravenously injected dimeric IgA in ddY(High) mice was found to be slower than that in ddY(Norm) mice. Furthermore, we found that the polymeric Ig receptors (pIgRs) that mediate transcytosis of IgA were poorly expressed in the glomeruli as well as in the intestine of ddY(High) mice, whereas the pIgRs were more abundantly expressed in ddY(Norm) mice. In addition, the comparative study using polymerase chain reaction showed that decreased pIgR expression occurred at the transcriptional level in the ddY(High) population. Taken together, these results suggest that a systemic defect in pIgR expression may result in impaired IgA secretion and accumulation of IgA in the serum of ddY(High) mice. The age-dependent changes of pIgR expression in the dimeric IgA secretion sites of ddY(High) mice suggest a possible cause for the elevation of serum IgA level and the pathogenesis of IgAN-like disease.

Frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae in Tokyo in 2002.

The frequency of alterations in the GyrA subunit of DNA gyrase and the ParC subunit of topoisomerase IV in 19 clinical isolates of Neisseria gonorrhoeae obtained in Tokyo in 2002 was studied. The frequencies of GyrA and ParC mutations in these 19 isolates were 100% (19 of 19) and 84.2% (16 of 19), respectively, and these results were 1.48-fold (100%/67.6%) and 3.58-fold (84.2%/23.5%) higher, respectively, than the frequencies reported in 1998 in 68 isolates obtained in Fukuoka during the period from 1992 to 1996. Isolates with increasing numbers of mutations were more resistant not only to levofloxacin but also to other antibiotics. The 50% and 90% minimum inhibitory concentrations (MICs) to levofloxacin during the period from 1995 to 1996 were 0.063 and 1 micro g/ml, and they increased to 4 and 8 micro g/ml, respectively, in the present study. All 19 cases of gonoccocal urethritis in the present study were cured with a single intramuscular injection of 2 g spectinomycin.