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Moon H Nahm

Publications and source records attributed to Moon H Nahm.

At least 19 recordsLinked to original sources
PubMed2006

Lipoteichoic acid-induced nitric oxide production depends on the activation of platelet-activating factor receptor and Jak2.

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-alpha and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-alpha. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-alpha. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-beta expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-alpha production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-beta, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.

Animals↗
PubMed2006

Development and validation of a fourfold multiplexed opsonization assay (MOPA4) for pneumococcal antibodies.

Opsonophagocytic killing assays (OPAs) are essential for developing and improving pneumococcal vaccines. There is a need for a high-throughput, reliable, standardized, and fully characterized OPA for pneumococcal antibodies. To meet the need, we have developed and characterized a fourfold multiplexed OPA (MOPA4) against 13 serotypes (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F) of pneumococci. Thirteen target bacteria were made resistant to only one of the following antibiotics: optochin, streptomycin, spectinomycin, and trimethoprim. Following optimization of assay conditions, accuracy of MOPA4 was determined by testing 30 sera from old adults in the MOPA4 and the single-serotype assays. The opsonization titers obtained with both assays agreed well (r(2) > 0.95). Although 22 (out of 390; approximately 6%) results differed more than twofold, the differences were not reproducible. The assay was specific: preabsorbing test sera with homologous polysaccharide (PS) completely abrogated opsonic activity, but a pool of unrelated PS (5 mug/ml of each) had no effect. Intra- and interassay coefficients of variation were 10 and 22%, respectively. MOPA4 results were unaffected by having different target pneumococcal serotypes in each assay group. Also, HL60 cell-to-bacteria ratios could be varied twofold without affecting the results. We conclude that MOPA4 is sensitive, accurate, specific, precise, and robust enough for large-scale clinical studies. Furthermore, MOPA4 should allow evaluation of multivalent pneumococcal vaccines with the limited volume of serum typically available from young children.

Adult↗
PubMed2006

Platelet-activating factor-acetylhydrolase can monodeacylate and inactivate lipoteichoic acid.

Bacterial lipoteichoic acid (LTA) shares a structural motif with platelet-activating factor (PAF). Both molecules are strong inflammatory agents and have a glycerol backbone with two lipid chains at the sn-1 and sn-2 positions. PAF is normally inactivated by PAF-acetylhydrolase (PAF-AH), a phospholipase A2 (PLA2), which removes a short acyl group at the sn-2 position. To investigate whether PAF-AH can similarly degrade LTA, we studied the effects of porcine PLA2, bee venom PLA2, and recombinant human PAF-AH on pneumococcal LTA (PnLTA) and staphylococcal LTA (StLTA). After incubation with a porcine or bee venom PLA2, a large fraction of PnLTA lost 264 Da, which corresponds to the mass of the oleic acid group at the sn-2 position. After incubation with recombinant human PAF-AH, PnLTA lost 264 Da; the reduction did not occur when PAF-AH was exposed to Pefabloc SC, an irreversible inhibitor of the PAF-AH active site. Following PAF-AH treatment, PnLTA and StLTA were not able to stimulate mouse RAW 264.7 cells to produce tumor necrosis factor alpha but could stimulate CHO cells expressing human TLR2. This stimulation pattern has been observed with monoacyl PnLTA prepared by mild alkali hydrolysis (22). Taking these data together, we conclude that PAF-AH can remove one acyl chain at the sn-2 position of LTA and produce a monoacyl-LTA that is inactive against mouse cells.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed2006

Validation of a multiplex pneumococcal serotyping assay with clinical samples.

We have recently developed a rapid pneumococcal serotyping method called "multibead assay" (J. Yu et al., J. Clin. Microbiol. 43:156-162, 2005) based on a multiplexed immunoassay for capsular polysaccharides in lysates of pneumococcal cultures. The multibead assay can identify 36 serotypes (1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/33C, 11A/11D/11F, 12A/12B/12F, 14, 15B/5C, 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F). More than 90% of the U.S. isolates express one of these serotypes (J. B. Robbins et al., J. Infect. Dis. 148:1136-1159, 1983). To validate the new assay, we examined 495 clinical isolates of pneumococci obtained in Brazil, Denmark, and Mexico. Pneumococci were serotyped by the Neufeld test in their countries of origin, and lysates of each strain were coded and mailed to the United States for the multibead assay at ambient temperature without any thermal protection. After breaking the code, 54 discrepancies (11% of samples) were noted, but 46 were due to nonreproducible technical problems or insufficient growth of the pneumococci. All of the isolates grew well for a second test, and therefore, the culture medium used for the multibead assay is adequate. The discrepancies persisted for eight isolates, involving the 6A, 11A, and 18C serotypes. Additional studies of the eight isolates showed that the discrepancies were due to differences in the reagents used in the multibead or Neufeld tests for these three serotypes. For instance, five isolates were typed as 6A with the Neufeld test but as nontypeable by the multibead assay. Selection of another new monoclonal antibody (Hyp6AG1) for the multibead assay resulted in all five discrepant isolates typing as 6A. This finding indicates the validity of the multibead assay and emphasizes the need to validate any new pneumococcal serotyping assay with a large number of clinical isolates from different locations. It also suggests the presence of serological subtypes among isolates expressing the 6A serotype.

Bacterial Capsules↗
PubMed2005

Safety of varying dosages of 7-valent pneumococcal protein conjugate vaccine in seniors previously vaccinated with 23-valent pneumococcal polysaccharide vaccine.

In a phase I/II dose escalation study, varying volumes (0.1 ml, 0.5 ml, 1.0 ml and 2.0 ml) of 7-valent pneumococcal conjugate vaccine (PCV) (Prevnar or 0.5 ml of 23-valent pneumococcal polysaccharide vaccine (PPV) were administered to 220 adults 70 through 79 years of age previously vaccinated with 0.5 ml PPV at age 65 years or above and at least 5 years previously. Fever was uncommon and did not vary by study group. The rate of local reactions increased with higher volumes of PCV and the rate following 2.0 ml of PCV was comparable to that following 0.5 ml PPV.

Aged↗
PubMed2005

Simplified method to automatically count bacterial colony forming unit.

Bacterial colony counting is a significant technical hurdle for vaccine studies as well as various microbiological studies. We now show that an automated colony counter can process images obtained with a digital camera or document scanner and that any laboratory can efficiently have bacterial colonies enumerated by sending the images to a laboratory with a colony counter via internet.

Colony Count, Microbial↗
PubMed2005

Anti-idiotypic antibody as a potential candidate vaccine for Neisseria meningitidis serogroup B.

Sepsis and meningitis caused by Neisseria meningitidis serogroup B (NMGB) are serious diseases in infants and young adults, but no effective vaccine is available. The capsular polysaccharide (PS) of NMGB has poor immunogenicity and a structural similarity to polysialic acid (PSA) on neuronal tissue that may elicit autoantibodies. Using HmenB3, a protective and nonautoreactive monoclonal antibody (MAb) to NMGB capsular PS, we produced an anti-idiotypic MAb, Naid60, which mimics the capsular PS of NMGB. We produced an anti-anti-idiotypic MAb, MoB34, by using the immunogenic site on Naid60 responsible for inducing the anti-NMGB PS antibody response. MoB34 elicited the complement-mediated killing of representative strains of serogroup B meningococci. MoB34 did not bind to CHP-134, a neuroblastoma cell line expressing alpha(2-8) PSA, or to mouse brain cryosections at a high concentration. Naid60-keyhole limpet hemocyanin immunization inhibited the growth of live NMGB in intraperitoneally challenged mice; in contrast, three of five control mice developed bacteremia. Thus, Naid60 has an immunogenic site that elicits antibodies with bactericidal activity against NMGB and no autoimmunity to PSA. We suggest that the immunogenic region of Naid60 is a candidate for the development of a new vaccine against NMGB.

Animals↗
PubMed2005

Monoacyl lipoteichoic acid from pneumococci stimulates human cells but not mouse cells.

We have developed a method for obtaining pneumococcal lipoteichoic acid (LTA) with none, one, or two acyl chains. Anion-exchange chromatography at pH 9.5 yields pneumococcal LTA (labeled LTA-9.5) that has a mass spectrum identical to that of pre-ion-exchange LTA and loses 500 mass units after deacylation by alkali hydrolysis. Anion exchange at pH 10.5 produces LTA (labeled LTA-10.5) with mass peaks that are 264 mass units lower than those of pre-ion-exchange LTA, and deacylation of LTA-10.5 by alkali hydrolysis reduces the mass by only 239 mass units. This result indicates that LTA-10.5 has lost one of the two acyl chains, whereas LTA-9.5 has both acyl chains. When the biological properties of LTA-9.5 and LTA-10.5 are examined with mouse cells, only LTA-9.5 (and not LTA-10.5) is able to stimulate mouse cells to produce tumor necrosis factor alpha, interleukin-1beta, and nitric oxide. In contrast, both LTA-9.5 and LTA-10.5 can stimulate human cells. LTA became inactive when both acyl chains were removed. Thus, acyl chains are critical for LTA function, and small variations in acyl chains can alter biological properties of LTA.

Acylation↗
PubMed2005

Rapid multiplex assay for serotyping pneumococci with monoclonal and polyclonal antibodies.

We have developed and characterized a rapid semiautomated pneumococcal serotyping system incorporating a pneumococcal lysate preparation protocol and a multiplex serotyping assay. The lysate preparation incorporates a bile solubility test to confirm pneumococcal identification that also enhances assay specificity. The multiplex serotyping assay consists of 24 assays specific for 36 serotypes: serotypes 1, 2, 3, 4, 5, 6A, 6B, 7A/7F, 8, 9L/9N, 9V, 10A/10B/39/(33C), 11A/11D/11F, 12A/12B/12F, 14, 15B/(15C), 17F, 18C, 19A, 19F, 20, 22A/22F, 23F, and 33A/33F. The multiplex assay requires a flow cytometer, two sets of latex particles coated with pneumococcal polysaccharides, and serotype-specific antibodies. Fourteen newly developed monoclonal antibodies specific for common serotypes and a pool of polyclonal rabbit sera for some of the less-common serotypes are used. The two monoclonal antibodies specific for serotypes 18C and 23F recognize serotype-specific epitopes that have not been previously described. These monoclonal antibodies make the identification of the 14 common serotypes invariant. The specificity of the serotyping assay is fully characterized with pneumococci of all known (i.e., 90) serotypes. The assay is sensitive enough to use bacterial lysates diluted 20 fold. Our serotyping system can identify not only all the serotypes in pneumococcal vaccines but also most (>90%) of clinical isolates. This system should be very useful in serotyping clinical isolates for evaluating pneumococcal vaccine efficacy.

Animals↗
PubMed2004

Development of experimental carbohydrate-conjugate vaccines composed of Streptococcus pneumoniae capsular polysaccharides and the universal helper T-lymphocyte epitope (PADRE).

Experimental carbohydrate-conjugate vaccines composed of the 13 amino acid universal Pan HLA-DR Epitope (PADRE) and Streptococcus pneumoniae capsular polysaccharides from serotypes 14, 6B and 9V were produced. Simple carbodiimide-mediated condensation chemistry was used to conjugate the PADRE synthetic peptide to the three chemically different capsular polysaccharides in a 1:1 molar ratio. The immunogenicity of the PADRE peptide component of the conjugate vaccines was confirmed by the induction of PADRE-specific CD4+ helper T cell (HTL) responses following immunization of C57BL/6 mice. High titer antibody responses specific for polysaccharides of S. pneumoniae serotypes 14, 6B and 9V were induced using Complete Freund's Adjuvant (CFA) and alhydrogel Al(OH)3 formulations. The HTL, or carrier, effect of the PADRE synthetic peptide was only evident using the PADRE-polysaccharide conjugates; simple mixtures of the PADRE peptide and polysaccharides were essentially nonimmunogenic. The functional or potential protective value of the polysaccharide-specific antibodies was measured as a function of opsonophagocytic activity for the 6B serotype. High titers of opsonophagocytic activity were measured in sera from mice immunized with formulations containing both adjuvants. These data demonstrate that the PADRE synthetic peptide can induce the HTL responses needed to support the development of antibodies specific for bacterial carbohydrates used in conjugate vaccines.

Adjuvants, Immunologic↗
PubMed2004

A new murine model of humoral immuno-deficiency specifically affects class switching to T-independent antigens.

Immunoglobulin (Ig) isotype deficiencies are among the most common and least characterized humoral immunodeficiencies. A thorough understanding of their immunological and genetic features has been hampered by their extreme heterogeneity and the paucity of suitable animal models. Here, we report the initial characterization of a new mouse model with selective Ig deficiency. SENCARA mice display low serum IgG3 levels as well as severely deficient IgG3 responses to T cell-independent (TI) type 1 and 2 antigens. However, despite the significant block in class switching, expression of activation-induced deaminase and gamma3 germ-line transcription after TI antigen immunization are normal. IgG3 production in response to in vitro LPS stimulation was also normal, ruling out a specific defect in the Cgamma3 switch machinery. A decrease in the number of peritoneal B1a cells and enlarged splenic marginal zones were observed. The immunodeficiency is inherited as an autosomal, semi-dominant, essentially monogenic trait in SENCARA x C57BL/6 crosses. The SENCARA humoral immunodeficiency constitutes a novel immune phenotype, resembling human conditions such as IgG2 deficiency. This new mouse model will be of interest for the understanding of mechanisms involved in TI immune responses and may provide new insights into the molecular basis of human Ig deficiencies.

Animals↗
PubMed2003

The influence of maternal immunization on light chain response to Haemophilus influenzae type b vaccine.

The repertoire of human antibodies to Haemophilus influenzae type b polysaccharide (Hib PS) has been extensively characterized. In response to Hib PS vaccines, both IgG1 and IgG2 antibodies are produced. Based on the Vkappa gene used for the antibodies, heamophilus antibodies can be divided into A2 and non-A2 antibodies. A2 antibodies account for the majority of heamophilus antibodies, are generally more functional, and are specific for heamophilus polysaccharide. In contrast, non-A2 antibodies cross-react with Escherichia coli K100. Studies of placental transfer showed that IgG1 antibodies cross the placenta better than IgG2 antibodies and the A2 antibodies better than non-A2 antibodies. Babies born to mothers who were immunized with a heamophilus vaccine had a high level of antibodies to Hib PS but the antibody level decreased during the first 6 months. The children also responded to a heamophilus conjugate vaccine.

Adult↗
PubMed2003

Central role of complement in passive protection by human IgG1 and IgG2 anti-pneumococcal antibodies in mice.

Streptococcus pneumoniae is an important cause of morbitity and mortality worldwide. Capsule-specific IgG1 and IgG2 Abs are induced upon vaccination with polysaccharide-based vaccines that mediate host protection. We compared the protective capacity of human recombinant serogroup 6-specific IgG1 and IgG2 Abs in mice deficient for either leukocyte FcR or complement factors. Human IgG1 was found to interact with mouse leukocyte FcR in vitro, whereas human IgG2 did not. Both subclasses induced complement activation, resulting in C3c deposition on pneumococcal surfaces. Passive immunization of C57BL/6 mice with either subclass before intranasal challenge with serotype 6A induced similar degrees of protection. FcgammaRI- and III-deficient mice, as well as the combined FcgammaRI, II, and III knockout mice, were protected by passive immunization, indicating FcR not to be essential for protection. C1q or C2/factor B knockout mice, however, were not protected by passive immunization. Passively immunized C2/factor B(-/-) mice displayed higher bacteremic load than C1q(-/-) mice, supporting an important protective role of the alternative complement pathway. Spleens from wild-type and C1q(-/-) mice showed hyperemia and thrombotic vessel occlusion, as a result of septicemic shock. Notably, thrombus formation was absent in spleens of C2/factor B(-/-) mice, suggesting that the alternative complement pathway contributes to shock-induced intravascular coagulation. These studies demonstrate complement to play a central role in Ab-mediated protection against pneumococcal infection in vivo, as well as in bacteremia-associated thrombotic complications.

Animals↗
PubMed2003

Immunogenic protein contaminants in pneumococcal vaccines.

Currently available pneumococcal vaccines were examined for contamination by pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA). PspA could be detected in some (but not all) lots of 23-valent polysaccharide vaccine. Many lots of pneumococcal vaccines, including the heptavalent conjugate vaccine, were found to elicit small (but variable) antibody responses to PspA, PsaA, or both. The degree of contamination was highly variable, and this should be considered in pneumococcal vaccine evaluations or when capsular polysaccharide is used for pneumococcal antibody assays.

Adhesins, Bacterial↗
PubMed2003

Efficiency of a pneumococcal opsonophagocytic killing assay improved by multiplexing and by coloring colonies.

For evaluating pneumococcal vaccines, the opsonophagocytic killing assay (OPKA) is useful as a supplement to the pneumococcal antibody enzyme-linked immunosorbent assay (ELISA). However, evaluations of pneumococcal vaccines require the determination of antibody responses to 7 to 11 serotypes, and the OPKA is tedious to perform and requires more serum than the ELISA. Consequently, the OPKA is infrequently used for evaluating pneumococcal vaccines. To overcome these limitations, we have developed a simple multiplexed (double-serotype) OPKA by using antibiotic-resistant pneumococci for nine serotypes. Serotype 6B, 9V, 19A, and 23F strains were made streptomycin resistant, and serotype 4, 6A, 14, 18C, and 19F strains were made optochin resistant. The multiplexed OPKA was the same as the single-serotype OPKA except for two changes. First, the target bacteria were a mixture of one streptomycin-resistant strain and one optochin-resistant strain. Second, the surviving bacteria of each serotype were enumerated by plating on Todd-Hewitt agar plates with yeast extract and an agar overlay containing the appropriate antibiotics and 2,3,5-triphenyl tetrazolium chloride. The performance of the multiplexed OPKA was evaluated by analyzing 28 serum samples from adults immunized with a 23-valent polysaccharide vaccine by using the single-serotype OPKA and the multiplexed OPKA. The multiplexed OPKA was specific for the desired serotypes. The multiplexed and conventional OPKAs had comparable assay sensitivities and produced results that were highly correlated (r(2) values ranging from 0.92 to 0.98) for all nine serotypes. A simple modification of the conventional OPKA produces a multiplexed assay that greatly reduces effort, reagents, and the necessary amount of serum.

Adult↗
PubMed2003

Multilaboratory evaluation of a viability assay for measurement of opsonophagocytic antibodies specific to the capsular polysaccharides of Streptococcus pneumoniae.

Opsonophagocytosis is a correlate of protection that measures the functional activity of vaccine-induced antibodies. A standardized opsonophagocytosis assay (OPA) should be used as part of the evaluation of current and future pneumococcal (Pnc) polysaccharide (Ps)-based vaccines. We enrolled five laboratories to evaluate a previously standardized viability OPA. Each laboratory was provided with a detailed OPA protocol, seven target Pnc strains (serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F), two quality control sera and 12 paired sera (blinded) from adult donors who received one dose of the 23-valent Pnc Ps vaccine. Laboratories sent their results to the Centers for Disease Control and Prevention for analysis. Sera were tested in duplicate (single run), and the results were averaged to yield a single OPA titer (> or = 50% killing) for each serum sample. The percentage of sera within one or two dilutions of the calculated median OPA titer was determined for each laboratory and for each serotype. In general, laboratories were capable of detecting OPA titers within one or two dilutions of the median for at least 75 and 88%, respectively, of the sera tested. The level of agreement with the median OPA titers varied depending on the participating laboratory (overall agreement = 0.8 [99% confidence interval = 0.75 to 0.85]). All OPA median titers reported for quality control sera were within one dilution of the expected titer. We conclude that this OPA can be done in multiple laboratories with a high degree of interlaboratory reproducibility.

Adult↗