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1.  Simultaneous Quantification of Multiple Food- and Waterborne Pathogens by Use of Microfluidic Quantitative PCR 
The direct quantification of multiple pathogens has been desired for diagnostic and public health purposes for a long time. In this study, we applied microfluidic quantitative PCR (qPCR) technology to the simultaneous detection and quantification of multiple food- and waterborne pathogens. In this system, multiple singleplex qPCR assays were run under identical detection conditions in nanoliter-volume chambers that are present in high densities on a chip. First, we developed 18 TaqMan qPCR assays that could be run in the same PCR conditions by using prevalidated TaqMan probes. Specific and sensitive quantification was achieved by using these qPCR assays. With the addition of two previously validated TaqMan qPCR assays, we used 20 qPCR assays targeting 10 enteric pathogens, a fecal indicator bacterium (general Escherichia coli), and a process control strain in the microfluidic qPCR system. We preamplified the template DNA to increase the sensitivity of the qPCR assays. Our results suggested that preamplification was effective for quantifying small amounts of the template DNA without any major impact on the sensitivity, efficiency, and quantitative performance of qPCR. This microfluidic qPCR system allowed us to detect and quantify multiple pathogens from fecal samples and environmental water samples spiked with pathogens at levels as low as 100 cells/liter. These results suggest that the routine monitoring of multiple pathogens in food and water samples is now technically feasible. This method may provide more reliable information for risk assessment than the current fecal contamination indicator approach.
PMCID: PMC3623133  PMID: 23435884
2.  Identification and isolation of active N2O reducers in rice paddy soil 
The ISME Journal  2011;5(12):1936-1945.
Dissolved N2O is occasionally detected in surface and ground water in rice paddy fields, whereas little or no N2O is emitted to the atmosphere above these fields. This indicates the occurrence of N2O reduction in rice paddy fields; however, identity of the N2O reducers is largely unknown. In this study, we employed both culture-dependent and culture-independent approaches to identify N2O reducers in rice paddy soil. In a soil microcosm, N2O and succinate were added as the electron acceptor and donor, respectively, for N2O reduction. For the stable isotope probing (SIP) experiment, 13C-labeled succinate was used to identify succinate-assimilating microbes under N2O-reducing conditions. DNA was extracted 24 h after incubation, and heavy and light DNA fractions were separated by density gradient ultracentrifugation. Denaturing gradient gel electrophoresis and clone library analysis targeting the 16S rRNA and the N2O reductase gene were performed. For culture-dependent analysis, the microbes that elongated under N2O-reducing conditions in the presence of cell-division inhibitors were individually captured by a micromanipulator and transferred to a low-nutrient medium. The N2O-reducing ability of these strains was examined by gas chromatography/mass spectrometry. Results of the SIP analysis suggested that Burkholderiales and Rhodospirillales bacteria dominated the population under N2O-reducing conditions, in contrast to the control sample (soil incubated with only 13C-succinate). Results of the single-cell isolation technique also indicated that the majority of the N2O-reducing strains belonged to the genera Herbaspirillum (Burkholderiales) and Azospirillum (Rhodospirillales). In addition, Herbaspirillum strains reduced N2O faster than Azospirillum strains. These results suggest that Herbaspirillum spp. may have an important role in N2O reduction in rice paddy soils.
PMCID: PMC3223309  PMID: 21677691
denitrification; Herbaspirillum; nitrous oxide; rice paddy soil; single-cell isolation; stable isotope probing
3.  Advantages of functional single-cell isolation method over standard agar plate dilution method as a tool for studying denitrifying bacteria in rice paddy soil 
AMB Express  2012;2:50.
We recently established a method for isolating functional single cells from environmental samples using a micromanipulator (Functional single-cell (FSC) isolation), and applied it to the study of denitrifying bacteria in rice paddy soil (Ashida et al. 2010. Appl Microbiol Biotechnol 85:1211–1217). To further examine the advantages and possible disadvantages of the FSC method, we isolated denitrifying bacteria from the same rice paddy soil sample using both FSC and standard agar plate dilution (APD) methods and compared in this study. The proportion of denitrifying bacteria in the total isolates was more than 6-fold larger with FSC isolation (57.1%) compared with the APD method (9.2%). Denitrifying bacteria belonging to Alphaproteobacteria and Bacilli were commonly isolated using both methods, whereas those belonging to Betaproteobacteria, which had been found to be active in the denitrification-inductive paddy soil, were isolated only with the FSC method. On the other hand, Actinobacteria were only isolated using the APD method. The mean potential denitrification activity of the FSC isolates was higher than that of the APD isolates. Overall, FSC isolation was confirmed to be an excellent method for studying denitrifying bacteria compared with the standard agar plate dilution method.
PMCID: PMC3488030  PMID: 22985609
16S rRNA gene; Denitrifying bacteria; Functional single-cell isolation; Phylogenetic analysis; Rice paddy soil
4.  Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Azoarcus sp. Strain KH32C 
Journal of Bacteriology  2012;194(5):1255.
We report the finished and annotated genome sequence of a denitrifying and N2O-reducing betaproteobacterium, Azoarcus sp. strain KH32C. The genome is composed of one chromosome and one megaplasmid and contains genes for plant-microbe interactions and the gene clusters for aromatic-compound degradations.
PMCID: PMC3294784  PMID: 22328754
5.  Complete Genome Sequence of the Denitrifying and N2O-Reducing Bacterium Pseudogulbenkiania sp. Strain NH8B 
Journal of Bacteriology  2011;193(22):6395-6396.
Pseudogulbenkiania sp. strain NH8B is a Neisseriales bacterium isolated from an agricultural field. This strain has strong denitrification and N2O reduction activities. Here, we report the finished and annotated genome sequence of this organism.
PMCID: PMC3209224  PMID: 22038961
6.  Increased globotriaosylceramide levels in a transgenic mouse expressing human α1,4-galactosyltransferase and a mouse model for treating Fabry disease 
Journal of Biochemistry  2010;149(2):161-170.
Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/−)M(+/−)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/−)M(+/−)/KO mouse was 1.4 µg/mg protein, higher than in the TgM(+/−)/KO (<0.1 µg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/−) M(+/−)/KO mouse organs. These data indicated that the TgG3S(+/−) M(+/−)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.
PMCID: PMC3031308  PMID: 20961863
active-site-specific chaperone therapy; Fabry disease; globotriaosylceramide; mouse model
7.  Pharmacological chaperone therapy for Fabry disease 
Fabry disease is an inherited lysosomal storage disorder caused by deficient α-galactosidase A activity. Many missense mutations in Fabry disease often cause misfolded gene products, which leads to their retention in the endoplasmic reticulum by the quality control system; they are then removed by endoplasmic reticulum-associated degradation. We discovered that a potent α-galactosidase A inhibitor, 1-deoxygalactonojirimycin, acts as a pharmacological chaperone to facilitate the proper folding of the mutant enzyme by binding to its active site, thereby improving its stability and trafficking to the lysosomes in mammalian cells. The oral administration of 1-deoxygalactonojirimycin to transgenic mice expressing human mutant α-galactosidase A resulted in significant increases in α-galactosidase A activity in various organs, with concomitant reductions in globotriaosylceramide, which contributes to the pathology of Fabry disease. Seventy-eight missense mutations were found to be responsive to 1-deoxygalactonojirimycin. These data indicate that many patients with Fabry disease could potentially benefit from pharmacological chaperone therapy.
PMCID: PMC3278969  PMID: 22241068
pharmacological chaperone; Fabry disease; α-galactosidase A; therapy
8.  Large Scale Analysis of Virulence Genes in Escherichia coli Strains Isolated from Avalon Bay, CA 
Water research  2010;44(18):5463-5473.
Contamination of recreational waters with E. coli and Enterococcus sp. is a widespread problem resulting in beach closures and loss of recreational activity. While E. coli is frequently used as an indicator of fecal contamination, and has been extensively measured in waterways, few studies have examined the presence of potentially pathogenic E. coli strains in beach waters. In this study, a combination of high-throughput, robot-assisted colony hybridization and PCR-based analyses were used to determine the genomic composition and frequency of virulence genes present in E. coli isolated from beach water in Avalon Bay, Santa Catalina Island, CA. A total of 24,493 E. coli isolates were collected from two sites at a popular swimming beach between August through September 2007 and from July through August 2008. All isolates were examined for the presence of shiga-like toxins (stx1/stx2), intimin (eaeA), and enterotoxins (ST/LT). Of the 24,493 isolates examined, 3.6% contained the eaeA gene, indicating that these isolates were potential EPEC strains. On five dates, however, greater than 10% of the strains were potential EPEC, suggesting that incidence of virulence genes at this beach has a strong temporal component. No STEC or ETEC isolates were detected, and only eight (<1.0%) of the potential EPEC isolates were found to carry the EAF plasmid. The potential EPEC isolates mainly belonged to E. coli phylogenetic groups B1 or B2, and carried the beta intimin subtype. DNA fingerprint analyses of the potential EPEC strains indicated that the isolates belonged to several genetically diverse groups, although clonal isolates were frequently detected. While the presence of virulence genes alone cannot be used to determine the pathogenicity of strains, results from this study show that potential EPEC strains can be found in marine beach water and their presence needs to be considered as one of the factors used in decisions concerning beach closures.
PMCID: PMC2952692  PMID: 20643468
Bacteria; E. coli; Beach water; virulence genes; phylogenetic groups; DNA fingerprinting
9.  Isolation of Oligotrophic Denitrifiers Carrying Previously Uncharacterized Functional Gene Sequences▿ †  
Oligotrophic denitrifying bacteria, including those belonging to the genera Herbaspirillum, Azospirillum, and Bradyrhizobium, were obtained using a single-cell isolation technique. The taxonomic composition of the denitrifier population was similar to those assessed by previous culture-independent studies. The sequencing of nitrite reductase and N2O reductase genes of these strains revealed previously unknown links between 16S rRNA and the denitrification-functional gene phylogenies. In particular, we identified Bradyrhizobium strains that harbor nirS sequences previously detected only in culture-independent studies.
PMCID: PMC3019733  PMID: 21075882
11.  Role of PAF Receptor in Proinflammatory Cytokine Expression in the Dorsal Root Ganglion and Tactile Allodynia in a Rodent Model of Neuropathic Pain 
PLoS ONE  2010;5(5):e10467.
Neuropathic pain is a highly debilitating chronic pain following damage to peripheral sensory neurons and is often resistant to all treatments currently available, including opioids. We have previously shown that peripheral nerve injury induces activation of cytosolic phospholipase A2 (cPLA2) in injured dorsal root ganglion (DRG) neurons that contribute to tactile allodynia, a hallmark of neuropathic pain. However, lipid mediators downstream of cPLA2 activation to produce tactile allodynia remain to be determined.
Principal Findings
Here we provide evidence that platelet-activating factor (PAF) is a potential candidate. Pharmacological blockade of PAF receptors (PAFRs) reduced the development and expression of tactile allodynia following nerve injury. The expression of PAFR mRNA was increased in the DRG ipsilateral to nerve injury, which was seen mainly in macrophages. Furthermore, mice lacking PAFRs showed a reduction of nerve injury-induced tactile allodynia and, interestingly, a marked suppression of upregulation of tumor necrosis factor α (TNFα) and interleukin-1β (IL-1β) expression in the injured DRG, crucial proinflammatory cytokines involved in pain hypersensitivity. Conversely, a single injection of PAF near the DRG of naïve rats caused a decrease in the paw withdrawal threshold to mechanical stimulation in a dose-dependent manner and an increase in the expression of mRNAs for TNFα and IL-1β, both of which were inhibited by pretreatment with a PAFR antagonist.
Our results indicate that the PAF/PAFR system has an important role in production of TNFα and IL-1β in the DRG and tactile allodynia following peripheral nerve injury and suggest that blocking PAFRs may be a viable therapeutic strategy for treating neuropathic pain.
PMCID: PMC2862737  PMID: 20454616
12.  Microbial Populations Responsive to Denitrification-Inducing Conditions in Rice Paddy Soil, as Revealed by Comparative 16S rRNA Gene Analysis▿ †  
Applied and Environmental Microbiology  2009;75(22):7070-7078.
Rice paddy soil has been shown to have strong denitrifying activity. However, the microbial populations responsible for nitrate respiration and denitrification have not been well characterized. In this study, we performed a clone library analysis of >1,000 clones of the nearly full-length 16S rRNA gene to characterize bacterial community structure in rice paddy soil. We also identified potential key players in nitrate respiration and denitrification by comparing the community structures of soils with strong denitrifying activity to those of soils without denitrifying activity. Clone library analysis showed that bacteria belonging to the phylum Firmicutes, including a unique Symbiobacterium clade, dominated the clones obtained in this study. Using the template match method, several operational taxonomic units (OTUs), most belonging to the orders Burkholderiales and Rhodocyclales, were identified as OTUs that were specifically enriched in the sample with strong denitrifying activity. Almost one-half of these OTUs were classified in the genus Herbaspirillum and appeared >10-fold more frequently in the soils with strong denitrifying activity than in the soils without denitrifying activity. Therefore, OTUs related to Herbaspirillum are potential key players in nitrate respiration and denitrification under the conditions used.
PMCID: PMC2786546  PMID: 19767468
13.  Opposing Effects of Platelet-activating Factor and Lyso-Platelet-activating Factor on Neutrophil and Platelet Activation 
Molecular pharmacology  2008;75(1):227-234.
Platelet-activating factor (PAF) is a potent, bioactive phospholipid that acts on multiple cells and tissues through its G protein-coupled receptor (GPCR). PAF is not stored, but rapidly generated via enzymatic acetylation of the precursor, lysoPAF. The bioactivity of PAF is effectively and tightly regulated by PAF acetylhydrolases, which convert PAF back to lysoPAF. Previous studies report that lysoPAF is an inactive precursor and metabolite of PAF. However, lysoPAF has not been carefully studied in its own context. Here we report that lysoPAF has an opposing effect of PAF in the activation of neutrophils and platelets. Whereas PAF potentiates neutrophil NADPH oxidase activation, lysoPAF dose-dependently inhibits this function. Inhibition by lysoPAF is not affected by the use of a PAF receptor antagonist or genetic deletion of the PAF receptor gene. The mechanism of lysoPAF-mediated inhibition of neutrophils involves an elevation in the intracellular cAMP level, and pharmacological blockade of adenylyl cyclase completely reverses the inhibitory effect of lysoPAF. In addition, lysoPAF increases intracellular cAMP levels in platelets and inhibits thrombin-induced platelet aggregation, which can be reversed by inhibition of PKA. These findings identify lysoPAF as a bioactive lipid with opposing functions of PAF, and suggest a novel and intrinsic regulatory mechanism for balance of the potent activity of PAF.
PMCID: PMC2646610  PMID: 18931035
14.  Escherichia coli Populations in Great Lakes Waterfowl Exhibit Spatial Stability and Temporal Shifting▿ † 
Populations of Escherichia coli from juvenile and adult ring-billed gulls, juvenile common terns, and adult Canada geese were sampled over 6 years at five locations on Lake Superior (Duluth, MN, and Wisconsin) and Lake Michigan (Wisconsin, Illinois, and Indiana) to determine the extent of spatial and temporal variability in E. coli strains. Strain identity was determined using horizontal fluorophore-enhanced repetitive element palindromic DNA fingerprinting. Multivariate statistics were used to determine if spatial or temporal changes in E. coli populations occurred in waterfowl species. Pairwise multivariate analyses of variance revealed that E. coli populations of adult gulls from three regions of Lake Michigan and the Wisconsin shore of Lake Superior were similar to one another but different from an E. coli population of gulls from the Duluth region of Lake Superior. Juvenile and adult gulls from the Duluth area harbored different E. coli populations. The E. coli strains from juvenile gulls, however, were similar to those found in juvenile terns obtained from the same island rookery. Temporal changes in E. coli populations from several waterfowl species were also demonstrated for this site. Although portions of source tracking databases might be successfully used in other geographic regions, it is clear that juvenile birds should not be the sole source of E. coli strains used for source tracking databases, and multiple-year libraries should be constructed in order to identify the potential sources of E. coli in the environment.
PMCID: PMC2655468  PMID: 19139226
15.  Relationship between Phylogenetic Groups, Genotypic Clusters, and Virulence Gene Profiles of Escherichia coli Strains from Diverse Human and Animal Sources▿ †  
Applied and Environmental Microbiology  2007;73(18):5703-5710.
Escherichia coli strains in water may originate from various sources, including humans, farm and wild animals, waterfowl, and pets. However, potential human health hazards associated with E. coli strains present in various animal hosts are not well known. In this study, E. coli strains from diverse human and animal sources in Minnesota and western Wisconsin were analyzed for the presence of genes coding for virulence factors by using multiplex PCR and biochemical reactions. Of the 1,531 isolates examined, 31 (2%) were found to be Shiga toxin-producing E. coli (STEC) strains. The majority of these strains, which were initially isolated from the ruminants sheep, goats, and deer, carried the stx1c and/or stx2d, ehxA, and saa genes and belonged to E. coli phylogenetic group B1, indicating that they most likely do not cause severe human diseases. All the STEC strains, however, lacked eae. In contrast, 26 (1.7%) of the E. coli isolates examined were found to be potential enteropathogenic E. coli (EPEC) strains and consisted of several intimin subtypes that were distributed among various human and animal hosts. The EPEC strains belonged to all four phylogenetic groups examined, suggesting that EPEC strains were relatively widespread in terms of host animals and genetic background. Atypical EPEC strains, which carried an EPEC adherence factor plasmid, were identified among E. coli strains from humans and deer. DNA fingerprint analyses, done using the horizontal, fluorophore-enhanced repetitive-element, palindromic PCR technique, indicated that the STEC, potential EPEC, and non-STEC ehxA-positive E. coli strains were genotypically distinct and clustered independently. However, some of the potential EPEC isolates were genotypically indistinguishable from nonpathogenic E. coli strains. Our results revealed that potential human health hazards associated with pathogenic E. coli strains varied among the animal hosts that we examined and that some animal species may harbor a greater number of potential pathogenic strains than other animal species.
PMCID: PMC2074926  PMID: 17644637
16.  Presence and Sources of Fecal Coliform Bacteria in Epilithic Periphyton Communities of Lake Superior▿  
Applied and Environmental Microbiology  2007;73(12):3771-3778.
Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and Escherichia coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 105 CFU cm−2 by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (≥92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.
PMCID: PMC1932738  PMID: 17468280
17.  Cladophora (Chlorophyta) spp. Harbor Human Bacterial Pathogens in Nearshore Water of Lake Michigan†  
Cladophora glomerata, a macrophytic green alga, is commonly found in the Great Lakes, and significant accumulations occur along shorelines during the summer months. Recently, Cladophora has been shown to harbor high densities of the fecal indicator bacteria Escherichia coli and enterococci. Cladophora may also harbor human pathogens; however, until now, no studies to address this question have been performed. In the present study, we determined whether attached Cladophora, obtained from the Lake Michigan and Burns Ditch (Little Calumet River, Indiana) sides of a breakwater during the summers of 2004 and 2005, harbored the bacterial pathogens Shiga toxin-producing Escherichia coli (STEC), Salmonella, Shigella, and Campylobacter. The presence of potential pathogens and numbers of organisms were determined by using cultural methods and by using conventional PCR, most-probable-number PCR (MPN-PCR), and quantitative PCR (QPCR) performed with genus- and toxin-specific primers and probes. While Shigella and STEC were detected in 100% and 25%, respectively, of the algal samples obtained near Burns Ditch in 2004, the same pathogens were not detected in samples collected in 2005. MPN-PCR and QPCR allowed enumeration of Salmonella in 40 to 80% of the ditch- and lakeside samples, respectively, and the densities were up to 1.6 × 103 cells per g Cladophora. Similarly, these PCR methods allowed enumeration of up to 5.4 × 102 Campylobacter cells/g Cladophora in 60 to 100% of lake- and ditchside samples. The Campylobacter densities were significantly higher (P < 0.05) in the lakeside Cladophora samples than in the ditchside Cladophora samples. DNA fingerprint analyses indicated that genotypically identical Salmonella isolates were associated with geographically and temporally distinct Cladophora samples. However, Campylobacter isolates were genetically diverse. Since animal hosts are thought to be the primary habitat for Campylobacter and Salmonella species, our results suggest that Cladophora is a likely secondary habitat for pathogenic bacteria in Lake Michigan and that the association of these bacteria with Cladophora warrants additional studies to assess the potential health impact on beach users.
PMCID: PMC1489363  PMID: 16820442
18.  Presence and Growth of Naturalized Escherichia coli in Temperate Soils from Lake Superior Watersheds 
The presence of Escherichia coli in water is used as an indicator of fecal contamination, but recent reports indicate that soil populations can also be detected in tropical, subtropical, and some temperate environments. In this study, we report that viable E. coli populations were repeatedly isolated from northern temperate soils in three Lake Superior watersheds from October 2003 to October 2004. Seasonal variation in the population density of soilborne E. coli was observed; the greatest cell densities, up to 3 × 103 CFU/g soil, were found in the summer to fall (June to October), and the lowest numbers, ≤1 CFU/g soil, occurred during the winter to spring months (February to May). Horizontal, fluorophore-enhanced repetitive extragenic palindromic PCR (HFERP) DNA fingerprint analyses indicated that identical soilborne E. coli genotypes, those with ≥92% similarity values, overwintered in frozen soil and were present over time. Soilborne E. coli strains had HFERP DNA fingerprints that were unique to specific soils and locations, suggesting that these E. coli strains became naturalized, autochthonous members of the soil microbial community. In laboratory studies, naturalized E. coli strains had the ability to grow and replicate to high cell densities, up to 4.2 × 105 CFU/g soil, in nonsterile soils when incubated at 30 or 37°C and survived longer than 1 month when soil temperatures were ≤25°C. To our knowledge, this is the first report of the growth of naturalized E. coli in nonsterile, nonamended soils. The presence of significant populations of naturalized populations of E. coli in temperate soils may confound the use of this bacterium as an indicator of fecal contamination.
PMCID: PMC1352292  PMID: 16391098
19.  Dual phase regulation of experimental allergic encephalomyelitis by platelet-activating factor 
Experimental allergic encephalomyelitis (EAE) serves as a model for multiple sclerosis and is considered to be a CD4+ Th1 cell–mediated autoimmune disease. To investigate the role of platelet-activating factor (PAF) in this disease, PAF receptor (PAFR) KO (PAFR-KO) and wild-type (WT) mice, on a C57BL/6 genetic background, were immunized with myelin oligodendrocyte glycoprotein 35–55. The levels of PAF production and PAFR mRNA expression in the spinal cord (SC) correlated with the EAE symptoms. PAFR-KO mice showed lower incidence and less severe symptoms in the chronic phase of EAE than WT mice. However, no difference was observed in T cell proliferation, Th1-cytokine production, or titer of IgG2a between both genotypes. Before onset, as revealed by microarray analysis, mRNAs of inflammatory mediators and their receptors—including IL-6 and CC chemokine receptor 2—were down-regulated in the SC of PAFR-KO mice compared with WT mice. Moreover, in the chronic phase, the severity of inflammation and demyelination in the SC was substantially reduced in PAFR-KO mice. PAFR-KO macrophages reduced phagocytic activity and subsequent production of TNF-α. These results suggest that PAF plays a dual role in EAE pathology in the induction and chronic phases through the T cell–independent pathways.
PMCID: PMC2212945  PMID: 16172262
20.  Staphylococcal lipoteichoic acid inhibits delayed-type hypersensitivity reactions via the platelet-activating factor receptor 
Journal of Clinical Investigation  2005;115(10):2855-2861.
Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor–positive and –negative (PAF-R–positive and –negative) cells and PAF-R–deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R–positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R–deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R–expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.
PMCID: PMC1224300  PMID: 16184199
21.  Absence of platelet-activating factor receptor protects mice from osteoporosis following ovariectomy 
While platelet-activating factor (PAF) is produced in various diseases associated with bone resorption, its functions in bone metabolism remain unknown. Using PAF receptor–deficient mice, we evaluated the role of PAF in the development of bone resorption following ovariectomy, a model of postmenopausal osteoporosis. Through observations of bone mineral density and histomorphometric parameters, it was found that bone resorption was markedly attenuated in PAF receptor–deficient mice, indicating that PAF links estrogen depletion and osteoporosis in vivo. Osteoclasts expressed higher amounts of the enzymes required for PAF biosynthesis than osteoblasts. TNF-α and IL-1β increased the acetyl-coenzyme A:lyso-PAF acetyltransferase activity in osteoclasts. Osteoclasts, but not osteoblasts, expressed the functional PAF receptor. PAF receptor stimulation prolonged the survival of osteoclasts in vitro. Furthermore, osteoclasts treated with a PAF receptor antagonist, and also those from PAF receptor–deficient mice, showed reductions in survival rate and Ca resorption activity. Consistently, in organ cultures, bone resorption was significantly suppressed by a PAF receptor antagonist treatment or genetic PAF receptor deficiency. Thus, these results suggest that, through the inflammatory cytokines, estrogen depletion enhances PAF production as a unique autocrine factor for osteoclast functions. Inhibition of PAF function might pave the way for a new strategy to prevent postmenopausal bone loss without disturbing osteoblast functions.
PMCID: PMC437965  PMID: 15232615
22.  Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice 
Journal of Clinical Investigation  1999;104(8):1071-1076.
Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration–induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration–induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.
PMCID: PMC481051  PMID: 10525045
23.  Impaired Anaphylactic Responses with Intact Sensitivity to Endotoxin in Mice Lacking a Platelet-activating Factor Receptor  
The Journal of Experimental Medicine  1998;187(11):1779-1788.
Platelet-activating factor (PAF) is a potent phospholipid mediator with diverse biological activities in addition to its well-known ability to stimulate platelet aggregation. Pharmacologic studies had suggested a role for PAF in pregnancy, neuronal cell migration, anaphylaxis, and endotoxic shock. Here we show that disruption of the PAF receptor gene in mice caused a marked reduction in systemic anaphylactic symptoms. Unexpectedly, however, the PAF receptor–deficient mice developed normally, were fertile, and remained sensitive to bacterial endotoxin. These mutant mice clearly show that PAF plays a dominant role in eliciting anaphylaxis, but that it is not essential for reproduction, brain development, or endotoxic shock.
PMCID: PMC2212308  PMID: 9607919
platelet-activating factor; platelet-activating factor receptor; anaphylaxis; endotoxic shock; gene targeting

Results 1-23 (23)