ABSTRACT
To investigate the role of IL-13 during a severe systemic Candida albicans infection, BALB/c control and IL-13-/- mice were examined for colony forming units (CFU) in the kidneys and survival days after intravenous infection. Proinflammatory mediators and cell recruitment into the tissue were measured by quantitative real-time PCR, a multiple ELISA system, and morphological cell differentiation. The IL-13-/- group exhibited a lower CFU number in the kidneys at 4 days and survived longer than the control mice, which was accompanied by significantly higher expression of C-X-C motif ligand 2 (CXCL2), IFN-γ, and polymorphonuclear neutrophils (PMNs) in the infected kidneys. By contrast, the expression of transforming growth factor ß (TGF-ß) and IL-17 A on day 10 were significantly higher in the control mice than in the IL-13-/- group. When using an intratracheal infection model, the IL-13-/- group recruited a greater number of PMNs in 6 h, with rapidly increased CXCL2 in the alveolar space. In vitro testing with cultured bone-marrow-derived cells demonstrated rapid CXCL2 mRNA upregulation at 3 h after contact with C. albicans, which decreased with recombinant IL-13 pretreatment, whereas rIL-13 retained TGF-ß upregulation. In a murine model of Candida systemic infection, preexistent IL-13 limits both the rapid CXCL2 elevation and PMN aggregation in the target organ to suppress inflammatory mediators, which also attenuates local pathogen clearance within four days.
Subject(s)
Candida albicans/physiology , Candidiasis/immunology , Interleukin-13/metabolism , Kidney/immunology , Neutrophils/immunology , Animals , Cells, Cultured , Chemokine CXCL2/genetics , Chemokine CXCL2/metabolism , Disease Models, Animal , Disease Progression , Humans , Interferon-gamma/metabolism , Interleukin-13/genetics , Kidney/microbiology , Mice , Mice, Inbred BALB C , Mice, Knockout , Neutrophil Infiltration , Up-RegulationABSTRACT
BACKGROUND: With the improvements in immunosuppressive agents and graft survival, infections such as mycoses have become major complications after solid organ transplantation (SOT). METHODS: Our group has continuously updated an epidemiological database of visceral mycoses (VM) among autopsy cases in Japan since 1989. Data on infectious agents and clinical information were complied using similar procedures. RESULTS: Among the all autopsied cases studied, 356 undergone SOT. Of these, 214 (60.1%) suffered from one or more types of infections, including 51 (14.3%) with VM. Thus, the incidence of VM was higher in SOT recipients than in non-transplanted cases (P < 0.0001). Aspergillus spp. (Asp) was the most predominant agent and Candida spp. was second. Specifically, among SOT recipients, Asp was the most predominant in liver and lung transplant recipients. Among the 217 autopsied liver transplants cases, the incidence of VM was highest in those with fulminant hepatitis (FH, P = 0.01). The incidence of cytomegalovirus infection tended to be higher in cases with mycosis (P = 0.06). Multivariate logistic regression analysis identified FH (odds ratio, 3.61, 95% confidence interval 1.34-9.75; P = 0.03) as an independent risk factor for mycosis in liver transplant recipients. CONCLUSION: This epidemiological analysis of autopsied cases provides a strong incentive to intensify efforts to diagnose and treat post-SOT infectious diseases.
Subject(s)
Mycoses/epidemiology , Mycoses/microbiology , Transplant Recipients/statistics & numerical data , Adolescent , Adult , Autopsy , Bacterial Infections/epidemiology , Female , Humans , Japan/epidemiology , Male , Middle Aged , Retrospective Studies , Risk Factors , Virus Diseases/epidemiology , Young AdultABSTRACT
A series of molecular seesaw balances 1-5 have been developed to measure the relative strength of pyridinium-π (cation-π) interactions. The cycloaddition of 1-azaanthracene and o-quinodimethane under microwave irradiation afforded the efficient synthesis of 1 and 5. Introduction of substituents to the pyridine ring of balance 1 was achieved to produce 2-4 in good yields. Anion exchange of 1·MeI afforded 1·MeX with a variety of counteranions (X = Cl, Br, I, BF4, PF6, OAc). These balances adopt two distinct conformers, A and B, which are stabilized by a cation-π interaction and a π-π interaction, respectively. The conformer ratio was determined on the basis of the observed averaged 3J coupling constants for H1-C-C-H2 by comparison with the boundary JA and JB values, which were estimated by applying the Carplus-Altona equation to the dihedral angles of the optimized conformers A and B. The effects of the solvent, substituent and counteranion on the ΔG values were elucidated using these molecular balances. Thermodynamic parameters obtained from a van't Hoff plot as well as the electrostatic potential maps for both conformers A and B of the molecular balances helped us to better understand the obtained results.
ABSTRACT
We describe a protective early acquired immune response to pneumococcal pneumonia that is mediated by a subset of B1a cells. Mice deficient in B1 cells (xid), or activation-induced cytidine deaminase (AID(-/-) ), or invariant natural killer T (iNKT) cells (Jα18(-/-) ), or interleukin-13 (IL-13(-/-) ) had impaired early clearance of pneumococci in the lung, compared with wild-type mice. In contrast, AID(-/-) mice adoptively transferred with AID(+/+) B1a cells, significantly cleared bacteria from the lungs as early as 3 days post infection. We show that this early bacterial clearance corresponds to an allergic contact sensitivity-like cutaneous response, probably due to a subpopulation of initiating B1a cells. In the pneumonia model, these B1a cells were found to secrete higher affinity antigen-specific IgM. In addition, as in contact sensitivity, iNKT cells were required for the anti-pneumococcal B1a cell initiating response, probably through early production of IL-13, given that IL-13(-/-) mice also failed to clear infection. Our study is the first to demonstrate the importance of AID in generating an appropriate B1a cell response to pathogenic bacteria. Given the antibody affinity and pneumonia resistance data, natural IgM produced by conventional B1a cells are not responsible for pneumonia clearance compared with the AID-dependent subset.
Subject(s)
Adaptive Immunity , B-Lymphocytes/enzymology , Cytidine Deaminase/metabolism , Lung/enzymology , Phagocytosis , Pneumonia, Pneumococcal/enzymology , Streptococcus pneumoniae/immunology , Adoptive Transfer , Agammaglobulinaemia Tyrosine Kinase , Animals , Antigens, Bacterial/immunology , B-Lymphocytes/immunology , B-Lymphocytes/microbiology , B-Lymphocytes/transplantation , Complement Activation , Cytidine Deaminase/deficiency , Cytidine Deaminase/genetics , Cytidine Deaminase/immunology , Cytokines/immunology , Cytokines/metabolism , Dermatitis, Contact/enzymology , Dermatitis, Contact/immunology , Dermatitis, Contact/microbiology , Disease Models, Animal , Genotype , Immunoglobulin M/immunology , Immunoglobulin M/metabolism , Interleukin-13/deficiency , Interleukin-13/genetics , Lung/immunology , Lung/microbiology , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Knockout , Natural Killer T-Cells/immunology , Natural Killer T-Cells/microbiology , Phenotype , Pneumonia, Pneumococcal/immunology , Pneumonia, Pneumococcal/microbiology , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Spleen/enzymology , Spleen/immunology , Spleen/microbiology , Streptococcus pneumoniae/pathogenicity , Time FactorsABSTRACT
We propose that there is a special B-1a B cell subset ("sB-1a" cells) that mediates linked processes very early after immunization to initiate cutaneous contact sensitivity (CS), delayed-type hypersensitivity (DTH), and immune resistance to pneumococcal pneumonia. Our published data indicate that in CS and DTH, these initiating processes are required for elicitation of the delayed onset and late-occurring classical T cell-mediated responses. sB-1a cells resemble memory B2 cells, as they are stimulated within 1 h of immunization and depend on T helper cytokines-uniquely IL-4 from hepatic iNKT cells--for activation and rapid migration from the peritoneal cavity to the spleen to secrete IgM antibody (Ab) and Ab-derived free light chains (FLCs) by only 1 day after immunization. Unlike conventional B-1a (cB-1a) cell-produced IgM natural Ab, IgM Ab produced by sB-1a cells has high Ag affinity owing to immunoglobulin V-region mutations induced by activation-induced cytidine deaminase (AID). The dominant cB-1a cells are increased in immunized AID-deficient mice but do not mediate initiation, CS, or pneumonia resistance because natural Ab has relatively low Ag affinity because of unmutated germ-line V regions. In CS and DTH, sB-1a IgM Ag affinity is sufficiently high to mediate complement activation for generation of C5a that, together with vasoactive mediators such as TNF-α released by FLC-sensitized mast cells, activate local endothelium for extravascular recruitment of effector T cells. We conclude by discussing the possibility of functional sB-1 cells in humans.
Subject(s)
B-Lymphocyte Subsets/immunology , Cytidine Deaminase/immunology , Dermatitis, Contact/immunology , Pneumonia, Pneumococcal/immunology , Animals , B-Lymphocyte Subsets/metabolism , Cytidine Deaminase/deficiency , Dermatitis, Contact/metabolism , Humans , Hypersensitivity , Immunization , Mast Cells/immunology , Mast Cells/metabolism , Mice , Pneumonia, Pneumococcal/prevention & controlABSTRACT
A molecular seesaw balance 1·MeI has been developed to measure pyridinium-π interactions. This balance adopts two distinct conformers, A and B, which are stabilized by a cation-π interaction and a π-π interaction, respectively. The conformer ratio was determined on the basis of the averaged (3)J coupling constants for H1-C-C-H2 and the corresponding boundary J values for conformers A and B. The effects of the solvent and the counteranion on the ΔG values were investigated using this molecular balance. Thermodynamic parameters obtained from a van't Hoff plot helped us to better understand the solvent and counteranion effects.
ABSTRACT
BACKGROUND: Epicutaneous (EC) immunization is a potential new strategy of a needle-free and self-administrable immunization by using a topically applied patch to deliver vaccine. We have previously shown that EC immunization with various protein antigens inhibits both Th1- and Tc1-mediated contact hypersensitivity (CHS) in mice. Our further work showed that maneuver of EC immunization with an antigen and Toll-like receptor (TLR) ligands prior to hapten sensitization reverses skin-induced suppression. METHODS: Animal model of pneumococcal pneumonia was used to study efficacy of EC induced immunopotentiation. RESULTS: Current work showed that EC immunization with phosphorylcholine conjugated to bovine serum albumin (PC-BSA) and CpG prior to Streptococcus pneumoniae infection results in smaller decrease of body weight when compared to PBS treated mice. Consistent with the behavioral observations and body weight, smaller numbers of bacteria were quantitated in lung homogenates of mice patched with PC-BSA and CpG prior inoculation with S. pneumonia when compared to mice patched with PBS alone. In vitro experiments showed that lymph node cells and spleen cells from mice EC immunized with PC-BSA plus CpG produced high levels of IFN-γ and IL-17A when compared to PBS or PC-BSA or CpG treated mice. CONCLUSION: This work shows that EC immunization with PC-BSA plus TLR9 ligand CpG may be a potential tool to boost immunity to S. pneumoniae.
Subject(s)
CpG Islands/immunology , Phosphorylcholine/analogs & derivatives , Pneumococcal Vaccines/immunology , Pneumonia, Pneumococcal/prevention & control , Serum Albumin, Bovine/immunology , Toll-Like Receptor 9/immunology , Administration, Cutaneous , Animals , Disease Models, Animal , Interferon-gamma/biosynthesis , Interferon-gamma/immunology , Interleukin-17/biosynthesis , Interleukin-17/immunology , Ligands , Male , Mice, Inbred CBA , Phosphorylcholine/immunology , Pneumonia, Pneumococcal/immunology , Skin/immunology , Streptococcus pneumoniae/pathogenicityABSTRACT
The efficacy of 1% chlorhexidine-gluconate ethanol and 10% povidone-iodine for skin antisepsis of central venous catheter (CVC) sites were compared among hematology patients. The CVC site colonization rates of those groups were 11.9% and 29.2%, respectively, and the catheter-associated blood stream infections were 0.75 and 3.62 per 1,000 catheter-days, respectively. One percent chlorhexidine-gluconate ethanol was superior to povidone-iodine to reduce skin colonizers at CVC sites even when catheters were used for long duration.
Subject(s)
Central Venous Catheters/microbiology , Chlorhexidine/analogs & derivatives , Disinfectants/pharmacology , Disinfection/methods , Povidone-Iodine/pharmacology , Sepsis/prevention & control , Skin Diseases, Infectious/prevention & control , Adolescent , Adult , Aged , Aged, 80 and over , Chlorhexidine/pharmacology , Ethanol/pharmacology , Female , Hematology , Hospital Departments , Humans , Male , Middle Aged , Prospective Studies , Sepsis/epidemiology , Skin Diseases, Infectious/epidemiology , Young AdultABSTRACT
Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.
Subject(s)
CARD Signaling Adaptor Proteins/physiology , Interferon-gamma/biosynthesis , Lung Diseases, Fungal/microbiology , T-Lymphocytes/immunology , Animals , Chemokines/biosynthesis , Cryptococcus neoformans/genetics , Disease Models, Animal , Immunity, Innate/physiology , Lung Diseases, Fungal/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Th1 Cells/cytology , Th17 Cells/cytologyABSTRACT
Invariant NK T (iNKT) cells are known to play a critical role in the regulation of inflammatory responses in various clinical settings. In the present study, we assessed the contribution of iNKT cells to the development of acute lung injury (ALI), which was caused by intra-tracheal administration of LPS. Jα18 gene-disrupted mice lacking these cells underwent neutrophilic inflammatory responses in lungs at an equivalent level as control mice. Next, mice were sensitized intra-tracheally with α-galactosylceramide, an activator of iNKT cells, followed by challenge with LPS. In this model, mice showed severe lung injury, and all mice were killed within 72 h after LPS injection. IFN-γ and tumor necrosis factor (TNF)-α were strikingly elevated in the lungs of these mice. Administration of neutralizing mAb against IFN-γ and TNF-α attenuated lung injury in a histopathological analysis and improved their survival rate. Flow cytometric analysis revealed that IFN-γ was expressed in NK cells, iNKT cells and also Gr-1(dull+)Ly-6C(+) monocytes and TNF-α was detected mainly in Gr-1(bright+)Ly-6G(+) neutrophils and Gr-1(dull+)Ly-6C(+) monocytes. Otherwise, in mice treated with LPS alone, IFN-γ was not detected in the lungs and Gr-1(bright+)Ly-6G(+) neutrophil was a main cellular source of TNF-α production. Anti-Gr-1 mAb resulted in the attenuation of ALI and decrease in the level of these cytokines. These results indicated that activation of iNKT cells led to striking exacerbation of ALI caused by LPS and that Gr-1(+) monocytes were recruited in the lungs with expressing IFN-γ and TNF-α and played an important role in the development of these responses.
Subject(s)
Acute Lung Injury/physiopathology , Interferon-gamma/immunology , Lipopolysaccharides/pharmacology , Monocytes/immunology , Natural Killer T-Cells/immunology , Receptors, Chemokine/immunology , Tumor Necrosis Factor-alpha/immunology , Acute Lung Injury/chemically induced , Acute Lung Injury/immunology , Animals , Lung/drug effects , Lung/immunology , Lymphocyte Activation , Mice , Mice, Knockout , Mice, TransgenicABSTRACT
OBJECTIVES: Streptococcus pyogenes causes various diseases in humans. While the prevalence of fluoroquinolone-resistant S. pyogenes isolates has been increasing since 2000 in the USA and Europe, it has remained very low in Japan. We isolated a fluoroquinolone-resistant S. pyogenes strain and analysed its genetics. METHODS: TU-296, a strain of S. pyogenes resistant to levofloxacin (MIC 16 mg/L), was isolated from the throat of a patient in their thirties with pharyngitis in autumn 2007. We carried out susceptibility tests for various antimicrobial agents and PCR analysis of the genes gyrA, gyrB, parC and parE in the quinolone resistance-determining region, followed by sequencing of the PCR products to find mutation(s) and the resulting amino acid substitution(s). We then sequenced the PCR product of the emm gene and determined the emm genotype. RESULTS: S. pyogenes TU-296 was found to have the following mutations and amino acid substitutions: adenine 476 to cytosine in gyrA and cytosine 367 to thymine in parC, resulting in Glu-85âAla in GyrA and Ser-79âPhe in ParC. The genotype of the isolate was emm11. CONCLUSIONS: Amino acid substitutions in fluoroquinolone-resistant S. pyogenes have already been reported from Europe and the USA, including Ser-81âPhe or Tyr and Met-99âLeu in GyrA, as well as Ser-79âPhe, Tyr or Ala and others in ParC. Numerous point mutations were found in parC and parE of S. pyogenes TU-296. In addition, a new amino acid substitution was detected (Glu-85âAla in GyrA). To our knowledge, there have been no previous reports of this substitution in a clinical isolate of S. pyogenes.
Subject(s)
Amino Acid Substitution/genetics , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Fluoroquinolones/pharmacology , Point Mutation , Streptococcus pyogenes/drug effects , Streptococcus pyogenes/genetics , Adult , Antigens, Bacterial/genetics , Bacterial Outer Membrane Proteins/genetics , Carrier Proteins/genetics , DNA Gyrase/genetics , DNA Topoisomerase IV/genetics , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Humans , Japan , Microbial Sensitivity Tests , Pharyngitis/microbiology , Pharynx/microbiology , Polymerase Chain Reaction , Sequence Analysis, DNA , Streptococcal Infections/microbiology , Streptococcus pyogenes/isolation & purificationABSTRACT
Methicillin resistant Staphylococcus aureus (MRSA) is exceptionally critical to infection treatment and control in the health-care setting. MRSA has been detected at high levels in Japan, and the frequency of MRSA infection must be ascertained to provide a baseline with which to assess various infection control efforts. We studied MRSA infection rate at a general hospital in Japan in all 65,135 inpatients of Sendai Kousei Hospital from January 2004 to December 2008. MRSA's prevalence among strains of S. aureus and the rate of MRSA detection were studied. Identification of MRSA infection is according to the laboratory-based ward liaison surveillance. The minimal inhibitory concentrations (MICs) of vancomycin, teicoplanin, and arbekacin for the various isolates were determined. During the period studied, there were 621 MRSA-positive patients. MRSA prevalence among strains of S. aureus was 45.5% (621/1,365). The rate of MRSA detection in inpatients was 0.953/100 inpatients. Of the 621 patients from whom MRSA was isolated, 51 (8.2%) had an MRSA infection (MRSA infection rate 0.078/100 inpatients). MRSA was often detected from the respiratory tract, but this seldom led to infection, since many of those affected were merely carriers. MICs against MRSA was 0.5-4 µg/ml for vancomycin, 0.5-16 µg/ml for teicoplanin, and 0.5 to >16 µg/ml for arbekacin, with no tendency for tolerance observed for these drugs. Findings suggest that whereas MRSA remains prevalent, there is a low incidence of infection in a general hospital in Japan.
Subject(s)
Carrier State/epidemiology , Hospitals, Community/statistics & numerical data , Methicillin-Resistant Staphylococcus aureus/isolation & purification , Staphylococcal Infections/epidemiology , Aged , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Carrier State/microbiology , Humans , Japan/epidemiology , Methicillin/pharmacology , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Population Surveillance/methods , Prevalence , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effectsABSTRACT
Dectin-2 (gene symbol Clec4n) is a C-type lectin expressed by dendritic cells (DCs) and macrophages. However, its functional roles and signaling mechanisms remain to be elucidated. Here, we generated Clec4n(-/-) mice and showed that this molecule is important for host defense against Candida albicans (C. albicans). Clec4n(-/-) DCs had virtually no fungal alpha-mannan-induced cytokine production. Dectin-2 signaling induced cytokines through an FcRgamma chain and Syk-CARD9-NF-kappaB-dependent signaling pathway without involvement of MAP kinases. The yeast form of C. albicans induced interleukin-1beta (IL-1beta) and IL-23 secretion in a Dectin-2-dependent manner. In contrast, cytokine production induced by the hyphal form was only partially dependent on this lectin. Both yeast and hyphae induced Th17 cell differentiation, in which Dectin-2, but not Dectin-1, was mainly involved. Because IL-17A-deficient mice were highly susceptible to systemic candida infection, this study suggests that Dectin-2 is important in host defense against C. albicans by inducing Th17 cell differentiation.
Subject(s)
Candida albicans/immunology , Candidiasis/immunology , Cell Differentiation , Interleukin-17/metabolism , Lectins, C-Type/immunology , Mannans/immunology , T-Lymphocytes, Helper-Inducer , Animals , Cells, Cultured , Immunoassay , Interleukin-1beta/immunology , Interleukin-23/immunology , Lectins, C-Type/genetics , Male , Mice , Mice, Knockout , Signal Transduction , T-Lymphocytes, Helper-Inducer/cytology , T-Lymphocytes, Helper-Inducer/immunologyABSTRACT
The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-alpha and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-kappaB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.
Subject(s)
Cordyceps , DNA, Fungal/pharmacology , DNA/pharmacology , Dendritic Cells/drug effects , Myeloid Cells/drug effects , Toll-Like Receptor 9/immunology , Adjuvants, Immunologic/pharmacology , Animals , Cells, Cultured , Cloning, Molecular , Cordyceps/genetics , Dendritic Cells/cytology , Dendritic Cells/immunology , Female , Flow Cytometry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/cytology , Myeloid Cells/immunology , Signal Transduction/drug effects , Toll-Like Receptor 9/genetics , Up-Regulation , beta-Glucans/immunologyABSTRACT
Pseudomonas (P.) aeruginosa is a major opportunistic pathogen especially in immunocompromised patients. To evaluate the invasiveness of respiratory pathogens, we developed monolayer culture systems and examined the degree of invasion by P. aeruginosa and invasive Salmonella (S.) typhimurium strains using human respiratory cell lines: A549 (derived from lung cancer), BEAS-2B (normal bronchial epithelium), and Calu-3 (pleural effusion of a patient with adenocarcinoma of the lung). Cells were seeded into filter units containing 0.33 cm(2) filter membranes with 3.0 microm pores, and were incubated at 37 degrees C under 5% CO(2) for 4-10 days. By monitoring the trans-monolayer electrical resistance (TER), we judged that BEAS-2B cells (TER values: 436.2 +/- 16.8 to 628.8 +/- 66.3 Omega cm(2)) and Calu-3 cells (TER values: 490.5 +/- 25.2 to 547.8 +/- 21.6 Omega cm(2)) formed monolayers with tight junctions, but not A549 cells. On day 8 of culture, monolayer cultures were infected with bacteria, and the number of microorganisms penetrating into the basolateral medium was counted. Wild-type P. aeruginosa PAO1 (PAO1 WT) and S. typhimurium SL1344 were detected in the basolateral medium of BEAS-2B monolayer system by 3 h after inoculation, while only P. aeruginosa PAO1 WT was detected in the basolateral medium of Calu-3 monolayer, indicating poor invasiveness of S. typhimurium SL1344 in the Calu-3 system. These findings suggest that BEAS-2B or Calu-3 monolayer system could be useful for evaluating the invasiveness of respiratory pathogens. Because of the difference in bacterial invasiveness, we may need to choose a suitable cell system for each target pathogen.
Subject(s)
Bacteria/pathogenicity , Cell Culture Techniques/methods , Epithelial Cells/cytology , Epithelial Cells/microbiology , Respiratory System/cytology , Respiratory System/microbiology , Animals , Cell Line , Cell Separation , Dogs , Electric Impedance , HumansABSTRACT
Hybrisep is an in situ hybridization (ISH) method to detect phagocytosed bacteria in peripheral blood neutrophils and macrophages. We report 10 actual clinical cases tested using Hybrisep with new DNA probes, and the data were compared to the actual blood culture results. A normal Hybrisep strategy employs 5 DNA probes to detect the following bacterial DNA: "SA" probe for S. aureus, "SE" for S. epidermidis, "PA" for P. aeruginosa, "EF" for E. faecalis, and "EK" for E. coli, E. cloacae, and K. pneumoniae. Six newly designed DNA probes were used in this study: "GB" probe for 49 common bacteria, "SP" for S. pneumoniae, "BF" for B. fragilis, "HI" for H. influenzae, "GC" for Candida species, and "CA" for C. albicans. Three cases were positive on ISH, but all their blood cultures were negative. One case showed a positive blood culture, but was negative on ISH. In another 6 cases, both were negative. We postulated that empirical therapy of antibiotics resulted in only positive ISH. Cases only showing a positive outcome on blood culture might be due to a diminished phagocytic function during patients' severe disease conditions. In conclusion, ISH with Hybrisep has clinical advantages such as being able to defect causative pathogens even after the use of antibiotics, and facilitates more rapid identification than routinely performed bacterial cultures only.
Subject(s)
In Situ Hybridization/methods , Sepsis/microbiology , Adult , Aged , Bacteria/genetics , Bacteria/isolation & purification , DNA Probes , DNA, Bacterial/analysis , Female , Humans , Macrophages/microbiology , Male , Middle Aged , Neutrophils/microbiology , Phagocytosis , Sepsis/diagnosisABSTRACT
We analyzed 4 cases that were not determined as incidents and another 7 cases determined as incidents, found at the department of clinical laboratory of us from April 2009 to March 2010. The former cases were, excess values of LD, and Cl, and glucose leveled as 0 mg/dl incorrectly, and misdirected blood samples at the ER. Our routine equipment and sample flow did not detect these false values. Resetting for auto dilution system and secondary check by every worker were reconsidered for these measurements. Antiseptic drug usage was notified by clinicians, and actually affected to the excessive Cl value. Real incidents were, two unprocessed samples, leakage of a sample, missing processes that caused delay of clinical practice, mixed up sample labels, a lost narcotic patch during cardiac ultrasonography. A lack of checking, carelessness, and accidental mistakes were reevaluated and reminded for workers on the duties. Also inadequate pharmaceutical knowledge and responsibilities of this section might severely affect on these lessons. Efforts were taken so that all workers shared the accurate information. Code blue cases are defined here as those of life-threatening events and sudden vital changes occurred in highest emergency, involve all health care workers, patients, and families. It is very important here to keep regular trainings for workers to cope with such events as well as preemptive assessments on environment and underlying risks in our laboratory. In line with the continuous advances in clinical medicine, medical safety managements are growing issues. To achieve safer environment and minimize various type of risks in the hospital, these incidents are to be assessed and reported regularly.
Subject(s)
Laboratories, Hospital , Risk Management , Cardiopulmonary Resuscitation , Humans , Risk , Specimen HandlingABSTRACT
In this study, we elucidated the role of tumor necrosis factor (TNF)-alpha in the host defense to pulmonary infection with Streptococcus pneumoniae and defined the cellular source of this cytokine at an early stage of infection. Administration of anti-TNF-alpha monoclonal antibody (mAb) resulted in the reduced accumulation of neutrophils in bronchoalveolar lavage fluids (BALFs) and severe exacerbation of this infection. In a flow cytometric analysis, the intracellular expression of TNF-alpha was detected in Gr-1(bright+) and Gr-1(dull+) cells during the time intervals postinfection, and F4/80(+) cells expressed intracellular TNF-alpha before Gr-1(dull+) cells appeared. The Gr-1(bright+) and Gr-1(dull+) cells sorted from BALF cells at 24 h were identified as neutrophils and macrophage-like cells, respectively, and the Gr-1(dull+) cells expressing CD11c, partially CD11b and a marginal level of F4/80 secreted TNF-alpha in in vitro cultures. Finally, deletion of Gr-1(+) cells by administration of the specific mAb significantly reduced the concentrations of this cytokine in BALF at 6 and 12 h postinfection, but not the expression of TNF-alpha in F4/80(+) cells. Thus, these results demonstrated that neutrophils, F4/80(+) macrophages and Gr-1(dull+) CD11c(+) macrophage-like cells played an important role in the production of TNF-alpha in lungs at an early stage of infection with S. pneumoniae.