Mol Microbiol, 1999 Mar, 31(6), 1709 - 22 Listeriolysin O-dependent activation of endothelial cells during infection with Listeria monocytogenes: activation of NF-kappa B and upregulation of adhesion molecules and chemokines; Kayal S et al.; The facultative intracellular bacterium Listeria monocytogenes is an invasive pathogen that crosses the vascular endothelium and disseminates to the placenta and the central nervous system . Its interaction with endothelial cells is crucial for the pathogenesis of listeriosis . By infecting in vitro human umbilical vein endothelial cells (HUVEC) with L . monocytogenes, we found that wild-type bacteria induced the expression of the adhesion molecules (ICAM-1 and E-selectin), chemokine secretion (IL-8 and monocyte chemotactic protein-1) and NF-kappa B nuclear translocation . The activation of HUVEC required viable bacteria and was abolished in prfA-deficient mutants of L . monocytogenes, suggesting that virulence genes are associated with endothelial cell activation . Using a genetic approach with mutants of virulence genes, we found that listeriolysin O (LLO)-deficient mutants inactivated in the hly gene did not induce HUVEC activation, as opposed to mutants inactivated in the other virulence genes . Adhesion molecule expression, chemokine secretion and NF-kappa B activation were fully restored by a strain of Listeria innocua transformed with the hly gene encoding LLO . The relevance in vivo of endothelial cell activation for listerial pathogenesis was investigated in transgenic mice carrying an NF-kappa B-responsive lacZ reporter gene . NF-kappa B activation was visualized by a strong lacZ expression in endothelial cells of capillaries of mice infected with a virulent haemolytic strain, but was not seen in those infected with a non-haemolytic isogenic mutant . Direct evidence that LLO is involved in NF-kappa B activation in transgenic mice was provided by injecting intravenously purified LLO, thus inducing stimulation of NF-kappa B in endothelial cells of blood capillaries . Our results demonstrate that functional listeriolysin O secreted by bacteria contributes as a potent inflammatory stimulus to inducing endothelial cell activation during the infectious process. Mol Microbiol, 1999 Mar, 31(6), 1631 - 41 Delivery of protein to the cytosol of macrophages using Escherichia coli K-12; Higgins DE et al.; Listeriolysin O (LLO) is an essential determinant of pathogenicity whose natural biological role is to mediate lysis of Listeria monocytogenes containing phagosomes . In this study, we report that Escherichia coli expressing cytoplasmic recombinant LLO can efficiently deliver co-expressed proteins to the cytosol of macrophages . We propose a model in which subsequent or concomitant to phagocytosis the E . coli are killed and degraded within phagosomes causing the release of LLO and target proteins from the bacteria . LLO acts by forming large pores in the phagosomal membrane, thus releasing the target protein into the cytosol . Delivery was shown to be rapid, within minutes after phagocytosis . Using this method, a large enzymatically active protein was delivered to the cytosol . Furthermore, we demonstrated that the E . coli/LLO system is very efficient for delivery of ovalbumin (OVA) to the major histocompatibility (MHC) class I pathway for antigen processing and presentation, greater than 4 logs compared with E . coli expressing OVA alone . Moreover, the time required for processing and presentation of an OVA-derived peptide was similar to that previously reported when purified OVA was introduced directly into the cytosol by other methods . Using this system, potentially large amounts of any protein that can be expressed in E . coli can be delivered to the cytosol without protein purification . The potential use of this system for the delivery of antigenic protein in vivo and the delivery of DNA are discussed. J Biol Chem, 1999 Apr 23, 274(17), 11459 - 62 Increased expression of Rab5a correlates directly with accelerated maturation of Listeria monocytogenes phagosomes; Alvarez-Dominguez C et al.; Previous studies have shown that Listeria monocytogenes (LM) modulates phagocytic membrane traffic . Here we explore whether Rab5a, a GTPase associated with phagosome-endosome fusion, is related to phagosome maturation and to the intracellular survival of LM . Stable transfection of Rab5a cDNA into macrophages accelerates intracellular degradation of LM . Morphological studies confirmed that phagosome maturation and phagosome-lysosome fusion is enhanced by overexpression of Rab5a . Down-regulation experiments using antisense oligonucleotides targeted to the Rab5a mRNA efficiently reduced Rab5a synthesis, reduced phagosome-endosome traffic, blocked phagosome-lysosome fusion, and extended intraphagosomal survival of LM . Down-regulation of Rab5a had no effect on LM internalization . Down-regulation of Rab5c had no effect on phagosome maturation and phagosome-lysosome fusion . The results indicate that Rab5a controls early phagosome-endosome interactions and governs the maturation of the early phagosome leading to phagosome-lysosome fusion. J Immunol, 1999 Apr 15, 162(8), 4781 - 9 Enhancement of the Listeria monocytogenes p60-specific CD4 and CD8 T cell memory by nonpathogenic Listeria innocua; Geginat G et al.; The contact of T cells to cross-reactive antigenic determinants expressed by nonpathogenic environmental micro-organisms may contribute to the induction or maintenance of T cell memory . This hypothesis was evaluated in the model of murine Listeria monocytogenes infection . The influence of nonpathogenic L . innocua on the L . monocytogenes p60-specific T cell response was analyzed . We show that some CD4 T cell clones raised against purified p60 from L . monocytogenes cross-react with p60 purified from L . innocua . The L . monocytogenes p60-specific CD4 T cell clone 1A recognized the corresponding L . innocua p60 peptide QAAKPAPAPSTN, which differs only in the first amino acid residue . In vitro experiments revealed that after L . monocytogenes infection of APCs, MHC class I-restricted presentation of p60 occurs, while MHC class II-restricted p60 presentation is inhibited . L . innocua-infected cells presented p60 more weakly but equally well in the context of both MHC class I and MHC class II . In contrast to these in vitro experiments the infection of mice with L . monocytogenes induced a strong p60-specific CD4 and CD8 T cell response, while L . innocua infection failed to induce p60-specific T cells . L . innocua booster infection, however, expanded p60-specific memory T cells induced by previous L . monocytogenes infection . In conclusion, these findings suggest that infection with a frequently occurring environmental bacterium such as L . innocua, which is nonpathogenic and not adapted to intracellular replication, can contribute to the maintenance of memory T cells specific for a related intracellular pathogen. Lett Appl Microbiol, 1999 Mar, 28(3), 216 - 20 Behaviour of Listeria monocytogenes under combined chilling processes; Membre JM et al.; The behaviour of Listeria monocytogenes under chilling processes was investigated . Growth kinetics were measured at 7 degrees C in TSBYE culture medium as a function of pH (7.2 and 6.2), pre-incubation temperatures (4 or 7 degrees C), cooling (0.05 or 0.1 degree C min-1) and freezing (0 and -5 degrees C) treatments . Growth curves generated were fitted by Gompertz and Baranyi functions . The Baranyi function gave better parameter estimation values than the Gompertz equation which over-estimated the specific growth rate values . Listeria monocytogenes grew at 7 degrees C without a lag phase, except when the sub-culture was performed at 37 degrees C, whereas the specific growth rate was affected by the chilling processes . In fact, L . monocytogenes grew slightly faster at 7 degrees C when a 4 degrees C pre-incubation treatment was applied than with a 7 degrees C pre-incubation treatment . These results suggest that to mimic the processes of contamination in industry, predictive microbiology studies with L . monocytogenes should be performed with organisms cultured at low temperatures. J Appl Microbiol, 1999 Mar, 86(3), 544 - 56 Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy; Arroyo G et al.; Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min . Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa . The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa . Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa . Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment . The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L . monocytogenes, than in the other test micro-organisms . Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive . The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables . Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior . Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs. J Appl Microbiol, 1999 Mar, 86(3), 469 - 76 Effect of osmotic, alkaline, acid or thermal stresses on the growth and inhibition of Listeria monocytogenes; Vasseur C et al.; Five strains of Listeria monocytogenes (a, b, c, d and e) isolated from industrial plants have been subjected to different osmotic, alkaline, acid or thermal stresses . The effects of these treatments on lag-phase (L) and growth rate (mu) of cells in mid-log phase have been followed using an automated optical density monitoring system . Increasing the osmotic pressure by the addition of different amounts of NaCl increased the lag phase and decreased the growth rate . The same phenomena were observed after decreasing the pH of the medium to 5.8, 5.6 or 5.4 by addition of acetic, lactic or hydrochloric acids . The inhibitory effect was: acetic acid > lactic acid > hydrochloric acid . The addition of NaOH to attain pH values of 9.5, 10.0, 10.5 or 11.0 in the medium produced a dramatic increase of the lag phase at pH 10.5 and 11 . Growth rates were also decreased while the maximal population increased with high pH values . These effects varied according to strains . Strains d and e were the most resistant to acidic and alkaline stresses, and e was the most affected by the addition of NaCl . A cold shock of 30 min at 0 degree C had limited effects on growth parameters . On the other hand, hyperthermal shocks (55 or 63 degrees C, 30 min) led to similar increased lag phases and to significant increases of the maximal population in all five strains. J Virol, 1999 May, 73(5), 4120 - 6 CpG-containing oligonucleotides are efficient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines; Oxenius A et al.; Synthetic nonmethylated oligonucleotides containing CpG dinucleotides (CpG-ODNs) have been shown to exhibit immunostimulatory activity . CpG-ODNs have the capacity to directly activate B cells, macrophages, and dendritic cells, and we show here that this is reflected by cell surface binding of oligonucleotides to these cell subsets . However, T cells are not directly activated by CpG-ODNs, which correlates with the failure to bind to the T-cell surface . Efficient competition for CpG-induced B-cell activation by non-CpG-containing oligonucleotides suggests that oligonucleotides might bind to an as yet undefined sequence-nonspecific receptor prior to cellular activation . Induction of protective T-cell responses against challenge infection with lymphocytic choriomeningitis virus (LCMV) or with recombinant vaccinia virus expressing the LCMV glycoprotein was achieved by immunizing mice with the immunodominant major histocompatibility complex class I-binding LCMV glycoprotein-derived peptide gp33 together with CpG-ODNs . In these experiments, B cells, potentially serving as CpG-ODN-activated antigen-presenting cells (APCs), were not required for induction of protective immunity since CpG-ODN-gp33-immunized B-cell-deficient mice were equally protected against challenge infection with both viruses . This finding suggested that macrophages and/or dendritic cells were sufficiently activated in vivo by CpG-ODNs to serve as potent APCs for the induction of naive T cells . Furthermore, treatment with CpG-ODN alone induced protection against infection with Listeria monocytogenes via antigen-independent activation of macrophages . These data suggest that CpG activation of macrophages and dendritic cells may provide a critical step in CpG-ODN adjuvant activity. Mol Gen Genet, 1999 Mar, 261(2), 323 - 36 Differential expression of Listeria monocytogenes virulence genes in mammalian host cells; Bubert A et al.; We have used RT-PCR and GFP-mediated fluorescence to analyse the regulation of PrfA-dependent virulence genes of Listeria monocytogenes during proliferation in mammalian host cells . Our data show that most of the PrfA-regulated virulence genes are more efficiently expressed, as measured by transcript levels, when L . monocytogenes is grown in macrophages and macrophage-like cells rather than in epithelial cells, hepatocytes or endothelial cells . The promoters for hly and plcA are predominantly activated within the phagosomal compartment, while those for actA and inlC are predominantly activated in the host cell cytosol . Expression of actA and plcB precedes that of inlC after infection of epithelial cells and macrophages . Little transcription of inlA or inlB is observed in epithelial cells and there is only slightly more in macrophages . In both cell types the level of transcription of the inlAB operon is lower than is seen under extracellular growth conditions in rich media, which is compatible with the assumption that InlA and InlB are not required during intracellular growth of the bacteria . Activation of the PrfA-independent iap promoter is also low during intracellular growth, although the gene product (p60) is required for cell viability . The levels of the PrfA-dependent virulence gene transcripts do not correlate with the amount of prfA transcript present, which is low under all intracellular conditions analysed, suggesting that the prfA transcript is either highly unstable in bacteria that are growing intracellularly, or that the small amount of PrfA produced is highly activated by additional component(s). Int J Food Microbiol, 1999 Feb 18, 46(3), 251 - 61 A model describing the relationship between regrowth lag time and mild temperature increase for Listeria monocytogenes; Breand S et al.; In order to comply with the consumer demand for ready-to-eat and look 'fresh' products, mild heat treatment will be used more and more in the agrofood industry . Nonetheless there is no tool to define the most appropriate mild heat treatment . In order to build this tool, it is necessary to study and describe the response of a bacterial population to a mild increase in temperature, from the dynamic point of view . The response to a mild increase in temperature, defined by stress duration and temperature, consisted in a mortality phase followed by the lag time of the survivors and their exponential growth . The effect of the mild increase in temperature on the mortality phase was described in a previous paper (Breand et al., Int . J . Food Microbiol., in press) . The effect of the stress duration on the lag was presented in a previous paper (Breand et al., Int . J . Food Microbiol . 38 (1997) 157-167) . In particular, the biphasic relationship between the lag and the stress duration was observed and modelled with a four parameter nonlinear model: the primary model (Breand et al., Int . J . Food Microbiol . 38 (1997) 157-167) . The study presented in this paper deals with the effect of the stress temperature on the biphasic relationship between the lag time and the stress duration . The secondary models describing the effect of the stress temperature on this biphasic relationship, were empirically built from our experimental data concerning Listeria monocytogenes . This work pointed out that the higher the stress temperature, the narrower the range of stress duration for which the lag time increased . Since the primary and the secondary models of the lag time were available, the global model describing the effect of the mild increase duration and temperature directly on the lag was fitted . This model allowed an improvement of the parameter estimator precision . The potential contribution in mild heat treatment optimization of this global model and the one built for the mortality phase (Breand et al., Int . J . Food Microbiol., in press) is discussed. Int J Food Microbiol, 1999 Feb 18, 46(3), 187 - 92 Characterization of Listeria monocytogenes from an ice cream plant by serotyping and pulsed-field gel electrophoresis; Miettinen MK et al.; One dominating strain of serotype 1/2b was found when serotyping and pulsed-field gel electrophoresis (PFGE) patterns were used for the characterization of 41 Listeria monocytogenes isolates originating from an ice cream plant . Samples were taken from the production environment, equipment and ice cream during the years 1990-1997 . Serotyping divided the isolates into two serovars, 1/2b and 4b . Three rare-cutting enzymes (ApaI, AscI and SmaI) were used in the creation of PFGE patterns . AscI resulted in the best restriction enzyme digestion patterns (REDPs) for visual comparison . Eight different AscI REDPs were obtained, whereas ApaI produced six and SmaI seven banding patterns . When one-band differences are taken into account, 12 different PFGE types were distinguished based on information obtained with all three enzymes . The dominant PFGE type was found to have persisted in the ice cream plant for seven years . Improved and precisely targeted cleaning and disinfection practices combined with structural changes making for easier cleaning of the packaging machine, resulted in eradication of L . monocytogenes from this plant. J Clin Periodontol, 1999 Mar, 26(3), 164 - 8 Comparative antiplaque effectiveness of an essential oil and an amine fluoride/stannous fluoride mouthrinse; Riep BG et al.; The adjunctive use of antimicrobial mouthrinses to help control supragingival plaque and gingivitis has been shown to contribute significantly to patients' daily oral hygiene regimens . This controlled clinical study used an observer-blind, randomized, cross-over design in a 4-day plaque regrowth model to determine the relative efficacies of an essential oil-containing mouthrinse (Listerine Antiseptic) and an amine fluoride/stannous fluoride-containing mouthrinse (Meridol) in inhibiting the development of supragingival plaque . A 0.1% chlorhexidine mouthrinse (Chlorhexamed-Fluid) was used as a positive control, and a 5% hydroalcohol solution was used as a negative control . Dosing for each of the test mouthrinses was based on the manufacturers' label directions . Because the volume and rinse time for each of the test mouthrinses were different, each test mouthrinse had its own negative control group . On day 1 of each test period, subjects received an oral soft and hard tissue examination and a dental prophylaxis to remove all plaque, calculus, and extrinsic stain . Starting the same day, subjects refrained from all mechanical oral hygiene procedures for the next 4 days and rinsed 2x daily under supervision with their randomly-assigned mouthrinse . On day 5, each subject received a plaque assessment as well as an oral examination to assess side effects . Each test period was separated by a 2-week washout period . 23 volunteers with a median age of 26 years completed the study . Compared to the respective placebos, the median percent plaque reductions at 5 days were 23.0%, 12.2%, and 38.2% for the essential oil, amine/stannous fluoride, and chlorhexidine rinses, respectively . The plaque reductions seen in the essential oil and chlorhexidine rinse groups were statistically significant (p < 0.001), while the plaque reduction in the amine/stannous fluoride rinse group was not statistically significant (p > 0.05) . Additionally, the essential oil rinse was significantly more effective (p < 0.001) than the amine/stannous fluoride rinse in inhibiting plaque accumulation in this clinical model. Zentralbl Bakteriol, 1999 Feb, 289(1), 31 - 6 Detection of Listeria monocytogenes in milk by the polymerase chain reaction; Salmanzadeh Ahrabi S et al.; A on the polymerase chain reaction (PCR) method based was developed for detection of Listeria monocytogenes in milk samples after enrichment culture . It consists of culturing samples in Listeria enrichment broth, followed by DNA extraction and detection of the organism using PCR . Dilutions of L . monocytogenes in milk were subjected to PCR amplification after enrichment culture . When determining the sensitivity of the method, it was found to be possible to detect 37 CFU (colony forming unit gl/ml) of the bacterium in milk . The method was assessed as a sensitive, specific, times-saving and practical way of detecting L . monocytogenes in milk samples. Acta Crystallogr D Biol Crystallogr, 1999 Feb, 55 ( Pt 2), 552 - 3 Crystallization and preliminary X-ray crystallographic analysis of the unusual ferritin from Listeria innocua; Ilari A et al.; Single crystals of ferritin extracted from Listeria innocua have been obtained by the vapour-diffusion method using PEG 1000 as precipitant . The crystals are orthorhombic, space group P212121, with unit-cell dimensions a = 87.7, b = 137.5, c = 173.1 A . The crystals diffract to 2.9 A resolution on a rotating-anode X-ray source and to 2.35 A resolution on a synchrotron X-ray source . The asymmetric unit contains one molecule formed by 12 subunits, corresponding to a packing density of 2.41 A3 Da-1 J Leukoc Biol, 1999 Feb, 65(2), 265 - 9 LPS down-regulates the expression of chemokine receptor CCR2 in mice and abolishes macrophage infiltration in acute inflammation; Zhou Y et al.; Interactions between chemokines and their specific receptors are important for leukocyte trafficking . The CC-chemokine monocyte chemoattractant protein-1 (MCP-1) and its specific receptor CCR2 are essential in monocytic infiltration and have been associated with several inflammatory diseases . It has been reported that several endotoxin and proinflammatory cytokines inhibit CCR2 expression in vitro in human monocytes . We report here that lipopolysaccharides (LPS) down-regulated CCR2 expression both in vitro and in vivo . Injection of LPS into mice dramatically reduced the expression of CCR2 on the surface of peripheral blood cells and completely blocked macrophage infiltration into the peritoneal cavity in response to thioglycollate elicitation . In addition, treatment of mice with LPS reduced their efficiency to clear Listeria monocytogenes infection . These results suggest that down-regulation of CCR2 and blockage of monocyte infiltration may contribute to the inhibition of macrophage function in vivo by a low dose of LPS. J Leukoc Biol, 1999 Feb, 65(2), 256 - 64 Functional deficiencies of peritoneal cells from gene-targeted mice lacking G-CSF or GM-CSF; Zhan Y et al.; Gene-targeted mice lacking the hemopoietic growth factors, granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage (GM)-CSF, show increased susceptibility to infection with the facultative intracellular bacterium, Listeria monocytogenes . The resident peritoneal cell populations from G-CSF(-/-) and GM-CSF(-/-) mice showed reduced production of the bactericidal molecule nitric oxide . Macrophage-mediated tumoricidal activity and phagocytosis of Listeria were reduced in G-CSF(-/-), but not in GM-CSF(-/-), mice . In G-CSF(-/-) mice, there was an unexpected expansion (from 18% in WT to 38%) of a population of cells with morphology intermediate between typical macrophages and typical lymphocytes . These cells had some of the features of poorly differentiated macrophages, being adherent to plastic but poorly phagocytic, nonspecific esterase positive but myeloperoxidase negative . They were largely negative for the macrophage marker F4/80 and for Thy1, B220, and Gr1 . Their disproportionate presence, and the corresponding deficiency in typical macrophages, possibly accounts for some of the functional deficiencies observed in G-CSF(-/-) mice. J Cell Biol, 1999 Mar 22, 144(6), 1245 - 58 Role of proteins of the Ena/VASP family in actin-based motility of Listeria monocytogenes; Laurent V et al.; Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization . We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility . Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force . The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA . VASP also interacts with F-actin via its COOH-terminal EVH2 domain . Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement . The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase . Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly . We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament. Infect Immun, 1999 Apr, 67(4), 1901 - 9 Effects of interleukin-1 receptor antagonist overexpression on infection by Listeria monocytogenes; Irikura VM et al.; Interleukin-1 receptor antagonist (IL-1ra) is a naturally occurring cytokine whose only known function is the inhibition of interleukin-1 (IL-1) . Using a reverse genetic approach in mice, we previously showed that increasing IL-1ra gene dosage leads to reduced survival of a primary listerial infection . In this study, we characterize further the role of endogenously produced IL-1ra and, by inference, IL-1 in murine listeriosis . IL-1ra overexpression inhibits, but does not eliminate, primary immune responses, reducing survival and increasing bacterial loads in the target organs . We demonstrate that IL-1ra functions in the innate immune response to regulate the peak leukocyte levels in the blood, the accumulation of leukocytes at sites of infection, and the activation of macrophages during a primary infection . Reduced macrophage class II major histocompatibility complex expression was observed despite increased gamma interferon (IFN-gamma) levels, suggesting that IL-1 activity is essential along with IFN-gamma for macrophage activation in vivo . We also show that IL-1ra plays a more limited role during secondary listeriosis, blunting the strength of the delayed-type hypersensitivity response to listerial antigen while not significantly altering cellular immunity to a second infectious challenge . When these results are compared to those for other mutant mice, IL-1ra appears to be unique among the cytokines studied to date in its regulation of leukocyte migration during primary listeriosis. Infect Immun, 1999 Apr, 67(4), 1844 - 52 Examination of Listeria monocytogenes intracellular gene expression by using the green fluorescent protein of Aequorea victoria; Freitag NE et al.; The ActA protein of Listeria monocytogenes is an essential virulence factor and is required for intracellular bacterial motility and cell-to-cell spread . plcB, cotranscribed with actA, encodes a broad-specificity phospholipase C that contributes to lysis of host cell vacuoles and cell-to-cell spread . Construction of a transcriptional fusion between actA-plcB and the green fluorescent protein gene of Aequorea victoria has facilitated the detailed examination of patterns of actA/plcB expression within infected tissue culture cells . actA/plcB expression began approximately 30 min postinfection and was dependent upon entry of L . monocytogenes into the host cytosol . L . monocytogenes Deltahly mutants, which are unable to escape from host cell vacuoles, did not express actA/plcB at detectable levels within infected tissue culture cells; however, complementation of the hly defect allowed entry of the bacteria into the host cytoplasm and subsequent actA/plcB expression . These results emphasize the ability of L . monocytogenes to sense the different host cell compartment environments encountered during the course of infection and to regulate virulence gene expression in response. Infect Immun, 1999 Apr, 67(4), 1770 - 8 Listeria monocytogenes phospholipase C-dependent calcium signaling modulates bacterial entry into J774 macrophage-like cells; Wadsworth SJ et al.; Listeria monocytogenes secretes several proteins that have been shown to contribute to virulence . Among these is listeriolysin O (LLO), a pore-forming hemolysin that is absolutely required for virulence . Two other virulence factors are phospholipases: a phosphatidylinositol-specific phospholipase C (PI-PLC {plcA}) and a broad-range PLC (plcB) . Although mutations in plcA or plcB resulted in small increases in mouse 50% lethal dose (LD50), deletions in both genes resulted in a 500-fold increase in LD50 . We have examined the role of these secreted proteins in host intracellular signaling in the J774 macrophage-like cell line . Measurements of cytosolic free calcium ({Ca2+}i) have revealed a rapid spike upon exposure of these cells to wild-type L . monocytogenes . This is followed by a second peak at 5 min and a third prolonged peak with a maximal {Ca2+}i of 800 to 1,000 nM . The pattern of calcium changes was greatly altered by deletion of any of the three virulence factors . An LLO mutant produced none of these elevations in {Ca2+}i; however, a transient elevation was observed whenever these bacteria entered the cell . A PI-PLC mutant produced a diminished single elevation in {Ca2+}i at 15 to 30 min . A broad-range PLC mutant produced only the first calcium spike . Studies with inhibitors suggested that the first elevation arises from influx of calcium from the extracellular medium through plasma membrane channels and that the second and third elevations come from release of Ca2+ from intracellular stores . We observed that internalization of wild-type bacteria and the broad-range PLC mutant was delayed for 5 to 10 min, but the LLO and PI-PLC mutants were internalized rapidly upon infection . Inhibitors that affected calcium signaling changed the kinetics of association of wild-type bacteria with J774 cells, the kinetics of entry, and the efficiency of escape from the primary phagosome. Immunobiology, 1999 Feb, 200(1), 120 - 7 Effect of fetal calf serum on cytokine release by bone marrow-derived macrophages during infection with intracellular bacteria; Flesch IE et al.; Bone marrow-derived macrophages (BMM) comprise a population of quiescent cells which can be activated by defined signals . Here, we directly compare the release of chemokines and monokines by BMM raised either in serum-supplemented or in serum-free medium in response to Listeria monocytogenes EGD or Mycobacterium bovis BCG infection . We focused on this issue because there have been several controversial reports on the production of cytokines by BMM due to different in vitro culture conditions . Culture in serum-supplemented medium primed BMM for release of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-6, and IL-12, but had no effect on macrophage inflammatory protein (MIP)-1alpha and tumor necrosis factor (TNF)-alpha production in response to L . monocytogenes infection . After challenge infection with M . bovis, BMM raised and stimulated in serum-supplemented medium secreted higher levels of MCP-1, MIP-1alpha, IL-6, and TNF-alpha but not of IL-12 as compared to BMM cultured and infected in a serum-free medium . The effects of serum could be partially mimicked by interferon-gamma . Because the serum components responsible for BMM priming are undefined, BMM cultured under serum-free conditions provide an appropriate cell population for defining macrophage activating signals. Immunopharmacol Immunotoxicol, 1999 Feb, 21(1), 109 - 24 Petiveria alliacea L . extract protects mice against Listeria monocytogenes infection--effects on bone marrow progenitor cells; Quadros MR et al.; In this study we have investigated the effects of Petiveria alliacea on the hematopoietic response of mice infected with Listeria monocytogenes . Our results demonstrate a protective effect of the crude extract of P . alliacea since the survival of the treated/infected was higher than that in the infected group . Moreover, the number of granulocyte/macrophage colonies (CFU-GM) and the serum colony stimulating activity levels were increased in the treated/infected mice in relation to the infected group . These results suggest an immunomodulation of Petiveria alliacea extract on hematopoiesis, which may be responsible, at least in part, for the increased resistance of mice to Listeria monocytogenes infection. FEMS Microbiol Lett, 1999 Feb 15, 171(2), 209 - 14 Evaluation of the API test, phosphatidylinositol-specific phospholipase C activity and PCR method in identification of Listeria monocytogenes in meat foods; Paziak-Domanska B et al.; The aim of this work was to compare the possibility of identifying Listeria monocytogenes strains isolated from meat and sausage on the basis of the API-Listeria test, production of phosphatidylinositol-specific phospholipase C (PI-PLC) and polymerase chain reaction (PCR) for a DNA fragment of the hlyA gene encoding listeriolysin O . Forty-six strains were isolated and examined . The lethality of some Listeria isolates for BALB/c mice was also determined . In this study, all isolates identified as L . monocytogenes in the API test gave a positive signal in the PCR . Listeriae identified as L . innocua or L . welshimeri in the API test were negative in the PCR conducted with the primers for listeriolysin O . All strains identified as L . monocytogenes on the basis of the API test and the PCR produced PI-PLC . However, this activity was not limited to the bacteria of this species . Four out of 17 L . innocua and three out of 10 L . welshimeri isolates were PI-PLC-positive . None of the L . innocua or L . welshimeri isolates (neither PI-PLC+ or PI-PLC-) showed lethality for BALB/c mice . In contrast, two L . monocytogenes isolates as well as a reference L . monocytogenes strain killed all mice used for the experiment. Tidsskr Nor Laegeforen, 1999 Jan 30, 119(3), 375 - 9 {Listeria monocytogenes--the perfect parasite?}; Wyller VB et al.; The pathogen Listeria monocytogenes has an extraordinary intracellular life cycle, based on adaption to and exploitation of normal cellular mechanisms . Extracellular organisms induce their own phagocytosis, followed by destruction of the phagosomal membrane . Cytoplasmatic bacteria organize the intracellular protein actin into "comet tails", and thus gain motility . In contact with the plasma membrane, motile bacteria induce a pseudopodium, corresponding to an invagination of the plasma membrane of the neighbouring cell . Eventually, the pseudopodium is engulfed by the neighbouring cell, creating a double membrane vacuole . L . monocytogenes destroys the double membrane, and escapes into the cytoplasm . This article reviews the molecular biology of Listeria infection, and how research in this field has yielded increased insight into normal cellular processes . Finally, we propose that the neuroinvasive properties of L . monocytogenes is caused by actin-dependent transport within axons from the periphery to the central nervous system. Tidsskr Nor Laegeforen, 1999 Jan 30, 119(3), 373 - 4 {Brain stem infection caused by Listeria monocytogenes}; Antal EA et al.; Rhombencephalitis due to Listeria monocytogenes is a serious form of brainstem inflammation . It is difficult to diagnose on the basis of clinical and laboratory findings alone . We describe a fatal case of Listeria monocytogenes rhombencephalitis in a previously healthy female teenager . Clinical and pathogenetic aspects of this condition are discussed. J Vasc Surg, 1999 Mar, 29(3), 554 - 6 Infrarenal endoluminal bifurcated stent graft infected with Listeria monocytogenes; Heikkinen L et al.; Prosthetic graft infection as a result of Listeria monocytogenes is an extremely rare event that recently occurred in a 77-year-old man who underwent endoluminal stent grafting for infrarenal abdominal aortic aneurysm . The infected aortic endoluminal prosthesis was removed by means of en bloc resection of the aneurysm and contained endograft with in situ aortoiliac reconstruction . At the 10-month follow-up examination, the patient was well and had no signs of infection. Vet Res Commun, 1998 Dec, 22(8), 505 - 16 Kinetics of interferon-gamma production and its comparison with anti-listeriolysin O detection in experimental bovine listeriosis; Barbuddhe SB et al.; The kinetics of the production of interferon gamma (IFN-gamma) in whole blood culture and its comparison with anti-listeriolysin O (ALLO) detection by ELISA were studied during oral infection of calves with Listeria monocytogenes . Culture filtrate antigen (CFA), listeriolysin O (LLO), and sonicated antigen (SA) were used to prime the peripheral blood mononuclear cells (PBMCs) and the plasma from orally infected calves . IFN-gamma and ALLO appeared as early as day 7 of an oral infection . IFN-gamma was detected earlier with LLO than with SA . The Max50 interleukin (IL-2) activity and IFN-gamma estimated in the culture supernatant from PBMCs primed in vitro with different antigens of L . monocytogenes revealed high induction of IL-2 and IFN-gamma by CFA, LLO and live antigen . IFN-gamma assay and ALLO detection were used for testing cases of repeat breeding in dairy cattle . It appeared that detection of IFN-gamma employing LLO can be used to diagnose listerial infections. Immunol Lett, 1999 Feb, 65(3), 167 - 73 Dietary omega-3 polyunsaturated fatty acids from fish oil reduce interleukin-12 and interferon-gamma production in mice; Fritsche KL et al.; The objective of this study was to investigate the impact of feeding mice a diet rich in n-3 polyunsaturated fatty acids (PUFA) from fish oil on the interleukin-12 (IL-12) and interferon-gamma (IFNgamma) production during the early stage of an infectious challenge with Listeria monocytogenes . Weanling female C3H/HeN mice were fed AIN-93G experimental diets containing 20%, by weight one of three fat sources: lard (low PUFA), soybean oil (n-6 PUFA) or a mixture (9:1) of menhaden fish oil and corn oil (n-3 PUFA) . After 4 weeks, mice were injected intraperitoneally with 10(5) Listeria monocytogenes and the concentration of IL-12(p70) and IFNgamma in serum was determined 24 h post-infection by ELISA . IL-12p35, IL-12p40 mRNA, and IFNgamma mRNA in the spleen were quantified by RNase protection assay . The number of IFNgamma-producing cells in the spleen was determined by flow cytometry using an intracellular staining procedure . We found that n-3 PUFA-fed mice had lower levels of circulating IL-12 at 24 h post-infection than n-6 PUFA- or low PUFA-fed mice (9.7+/-3.4 pg/ml vs . 61.6+/-10.6, and 44.4+/-12.5 pg/ml, respectively; P=0.002, n = 10/trt) . The level of IL-12 p35 mRNA did not significantly differ among dietary treatment groups . However, IL-12p40 mRNA was significantly lower in n-3 PUFA- and n-6 PUFA-fed mice compared to low-PUFA-fed mice . Further, the n-3 PUFA group also had the lowest circulating IFNgamma (4.4+/-1.8 ng/ml vs . 9.1+/-1.0, and 9.7+/-2.1 ng/ml, respectively; P = 0.007 . n = 8-10/trt) . The n-3 PUFA-fed mice had significantly lower IFNgamma mRNA in their spleens compared to the mice fed the other fat sources . In agreement with having lower circulating IFNgamma and lower splenic IFNgamma mRNA, n-3 PUFA-fed mice had a significantly lower percentage of IFNgamma-producing cells in their spleens compared with the n-6 PUFA-fed group (2.1+/-0.6 vs . 4.2+/-0.7%; P = 0.037, n = 10/trt) . In summary, feeding mice a diet rich in n-3 PUFA from fish oil significantly lowered the production of both IL-12 and IFNgamma during the early phase of a Listeria infection. Immunol Lett, 1999 Jan, 65(1-2), 93 - 8 T lymphocyte dynamics during Listeria monocytogenes infection; Busch DH et al.; Infection of mice with Listeria monocytogenes results in a robust T lymphocyte response that clears the pathogen and provides long term immunity from reinfection . The number of splenic CD4+ and CD8+ T cells and natural killer cells increases during primary and recall infection with L . monocytogenes, however the proportional increase is greatest for CD8+ T cells . The proportion of CD8 T cells expressing low levels of CD62L, a sign of activation, was increased among immune splenocytes, suggesting a substantial expansion of L . monocytogenes specific CTL . Analysis of CTL specific for the immunodominant LLO 91-99 epitope showed that essentially all were CD62Llo during the primary response, but that many upregulated expression of CD62L during the memory phase . Interestingly, the antigen specificity of nearly all additional CD62Llo CTL detected in spleens during recall L . monocytogenes infection can be accounted for with MHC class I tetramers complexed with four different epitopes . These studies demonstrate the complex T lymphocyte dynamics during infection with intracellular pathogens. Eur J Immunol, 1999 Feb, 29(2), 581 - 91 The resistance against Listeria monocytogenes and the formation of germinal centers depend on a functional death domain of the 55 kDa tumor necrosis factor receptor; Plitz T et al.; The biological functions mediated by the death domain of the 55-kDa tumor necrosis factor receptor (TNFRp55) in vivo are still elusive . TNFRp55 mutants lacking a functional death domain were expressed in TNFRp55-/- and in TNFRp55+/- mice as transgenes under control of the H-2Kb promoter . Analysis of these animals revealed that signals originating from the TNFRp55 death domain are indispensable for the protection against Listeria monocytogenes, the expression of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in the spleen and the development of splenic germinal centers . Furthermore, the transgenic coexpression of the TNFRp55 mutants in TNFRp55+/- mice exerts a dominant negative effect on the signaling of the endogenous receptor chains in vivo. Vet Microbiol, 1999 Feb 12, 64(4), 333 - 41 Cytotoxic T-cell, delayed type hypersensitive and listeriolysin O responses in experimental bovine listeriosis; Barbuddhe SB et al.; Five different antigens of Listeria monocytogenes NCTC 11994 were assayed for their lymphoproliferative, cytotoxic, humoral and delayed type hypersensitivity (DTH) responses in experimentally infected calves . The antigens used were culture filtrate antigen (CFA), listeriolysin O (LLO), sonicated antigen (SA), live antigen (LA) and heat killed antigen (HKA) . CFA was most lymphoproliferative when assayed in vitro . In an autologous monocyte cytotoxicity assay, soluble protein as well as bacterial cells elicited cytolytic responses, however, LA and LLO primed monocytes showed a higher cytotoxic effect . The expression of cell surface markers of lymphocyte subpopulations was almost identical in experimentally infected as well as non-infected calves . SA elicited the highest humoral and DTH responses . LLO being a major virulence factor with appreciable humoral as well as cellular responses may be used as a candidate antigen to develop a reliable diagnostic immunoassay. Can Vet J, 1998 Feb, 39(2), 100 - 2 Listeria monocytogenes and Escherichia coli septicemia and meningoencephalitis in a 7-day-old llama; Frank N et al.; Listeria monocytogenes and Escherichia coli were isolated from blood collected on presentation and tissues samples taken postmortem . Listeria monocytogenes was isolated from cerebrospinal fluid collected antemortem . The importance of passive transfer of immunity, the subtlety of neurologic signs in early meningitis, and considering blood-CSF penetration in antimicrobial selection are discussed. Proc Natl Acad Sci U S A, 1999 Mar 2, 96(5), 2274 - 8 Dopamine beta-hydroxylase deficiency impairs cellular immunity; Alaniz RC et al.; Norepinephrine, released from sympathetic neurons, and epinephrine, released from the adrenal medulla, participate in a number of physiological processes including those that facilitate adaptation to stressful conditions . The thymus, spleen, and lymph nodes are richly innervated by the sympathetic nervous system, and catecholamines are thought to modulate the immune response . However, the importance of this modulatory role in vivo remains uncertain . We addressed this question genetically by using mice that lack dopamine beta-hydroxylase (dbh-/- mice) . dbh-/- mice cannot produce norepinephrine or epinephrine, but produce dopamine instead . When housed in specific pathogen-free conditions, dbh-/- mice had normal numbers of blood leukocytes, and normal T and B cell development and in vitro function . However, when challenged in vivo by infection with the intracellular pathogens Listeria monocytogenes or Mycobacterium tuberculosis, dbh-/- mice were more susceptible to infection, exhibited extreme thymic involution, and had impaired T cell function, including Th1 cytokine production . When immunized with trinitrophenyl-keyhole limpet hemocyanin, dbh-/- mice produced less Th1 cytokine-dependent-IgG2a antitrinitrophenyl antibody . These results indicate that physiological catecholamine production is not required for normal development of the immune system, but plays an important role in the modulation of T cell-mediated immunity to infection and immunization. Protein Expr Purif, 1999 Mar, 15(2), 243 - 5 A method for purification of listeriolysin O from a hypersecretor strain of Listeria monocytogenes; Walton CM et al.; A simple and convenient method for the purification of the hemolytic toxin listeriolysin O (LLO) from Listeria monocytogenes is described . Supernatants from bacteria cultures were purified by application to a CH2 spiral cartridge concentrator (Amicon) and ion exchange chromatography . A critical step is removal of contaminating RNA . The purified proteins had characteristics described for bacterial thiol-activated hemolysins: activation by a reducing agent (DTT) and inactivation by cholesterol . In addition, the molecular weight of 58, 000 and pH-dependent hemolytic activity of this purified protein are consistent with the previously published characteristics of LLO . Curr Opin Cell Biol, 1999 Feb, 11(1), 117 - 21 The Arp2/3 complex: a multifunctional actin organizer; Machesky LM et al.; The actin-related proteins (Arps) constitute a recently characterized family of proteins, many of which function as members of multiprotein complexes . The discovery that two family members, Arp2 and Arp3, act as multifunctional organizers of actin filaments in all eukaryotes has generated much excitement . Over the past two years, newly discovered properties of the Arp2/3 complex have suggested a central role in the control of actin polymerization . First, it promotes actin assembly on the surface of the motile intracellular pathogen Listeria monocytogenes . Second, it can nucleate and cross-link actin filaments in vitro . Third, it localizes with dynamic actin-rich spots of mammalian cells suggesting a role in protrusion; it is found in cortical actin patches in the budding and fission yeasts where it may control patch movement and cortical actin function . Clearly, the complex has a central role in actin cytoskeletal function and will be the subject of much research in the coming years. J Food Prot, 1999 Feb, 62(2), 170 - 6 Development and validation of a dynamic growth model for Listeria monocytogenes in fluid whole milk; Alavi SH et al.; Listeria monocytogenes, a psychrotrophic microorganism, has been the cause of several food-borne illness outbreaks, including those traced back to pasteurized fluid milk and milk products . This microorganism is especially important because it can grow at storage temperatures recommended for milk (< or =7 degrees C) . Growth of L . monocytogenes in fluid milk depends to a large extent on the varying temperatures it is exposed to in the postpasteurization phase, i.e., during in-plant storage, transportation, and storage at retail stores . Growth data for L . monocytogenes in sterilized whole milk were collected at 4, 6, 8, 10, 15, 20, 25, 30, and 35 degrees C . Specific growth rate and maximum population density were calculated at each temperature using these data . The data for growth rates versus temperature were fitted to the Zwietering square root model . This equation was used to develop a dynamic growth model (i.e., the Baranyi dynamic growth model or BDGM) for L . monocytogenes based on a system of equations which had an intrinsic parameter for simulating the lag phase . Results from validation of the BDGM for a rapidly fluctuating temperature profile showed that although the exponential growth phase of the culture under dynamic temperature conditions was modeled accurately, the lag phase duration was overestimated . For an alpha0 (initial physiological state parameter) value of 0.137, which corresponded to the mean temperature of 15 degrees C, the population densities were underpredicted, although the experimental data fell within the narrow band calculated for extreme values of alpha0 . The maximum relative error between the experimental data and the curve based on an average alpha0 value was 10.42%, and the root mean square error was 0.28 log CFU/ml. Lett Appl Microbiol, 1999 Jan, 28(1), 71 - 5 Resistance of heat-shocked cells of Listeria monocytogenes to mano-sonication and mano-thermo-sonication; Pagan R et al.; Heat shocks did not increase the resistance of Listeria monocytogenes to an ultrasonication treatment under pressure (Mano-Sonication; MS) . While heat-shocked cells (180 min, 45 degrees C) became sixfold more heat resistant than native cells (D62 = 1.8 min vs D62 = 0.24 min), the resistance of native and heat-shocked cells to MS (200 kPa, 117 microns) was the same (DMS = 1.6 min) . The inactivation rate of non-heat-shocked cells of L monocytogenes by a combined heat/ultrasonication treatment under pressure (Mano-Thermo-Sonication; MTS) was an additive effect . On the contrary, on heat-shocked cells, the inactivation rate of MTS was greater than that of heat added to the inactivation rate of MS (synergistic effect) in the range 62-68 degrees C. Lett Appl Microbiol, 1999 Jan, 28(1), 45 - 8 Sub-lethal damage of Listeria monocytogenes after long-term chilled storage at 4 degrees C; Dykes GA et al.; The possibility that long term in vitro chilled storage may result in sub-lethal damage to Listeria monocytogenes cells was investigated by comparing growth of chill-stored (starvation at 4 degrees C) and fresh cultures on selective and non-selective media . Growth of freshly grown cells was minimally (3-8%) affected by selective LSAMM agar compared with non-selective Brain Heart Infusion agar . In contrast, numbers of chill-stored strains were reduced by greater than 99% after direct plating on the same selective and non-selective media . Furthermore, chill-stored strains were able to grow in standard selective broth (Listeria Selective broth and Fraser broth) only if undiluted inocula (approximately 10(5)-10(6) cfu ml-1) were used, whereas they were capable of growth in Brain Heart Infusion broth even when the lowest dilutions were used (approximately 10(1) cfu ml-1) . The potential public health consequences of this finding for the isolation of Listeria monocytogenes from foods is considered. Infect Immun, 1999 Mar, 67(3), 1303 - 9 Noncompetitive expansion of cytotoxic T lymphocytes specific for different antigens during bacterial infection; Vijh S et al.; Listeria monocytogenes is an intracellular bacterium that elicits complex cytotoxic T-lymphocyte (CTL) responses in infected mice . The responses of CTL populations that differ in antigen specificity range in magnitude from large, dominant responses to small, subdominant responses . To test the hypothesis that dominant T-cell responses inhibit subdominant responses, we eliminated the two dominant epitopes of L . monocytogenes by anchor residue mutagenesis and measured the T-cell responses to the remaining subdominant epitopes . Surprisingly, the loss of dominant T-cell responses did not enhance subdominant responses . While mice immunized with bacteria lacking dominant epitopes developed L . monocytogenes-specific immunity, their ability to respond to dominant epitopes upon rechallenge with wild-type bacteria was markedly diminished . Recall responses in mice immunized with wild-type or epitope-deficient L . monocytogenes showed that antigen presentation during recall infection is sufficient for activating memory cells yet insufficient for optimal priming of naive T lymphocytes . Our findings suggest that T-cell priming to different epitopes during L . monocytogenes infection is not competitive . Rather, T-cell populations specific for different antigens but the same pathogen expand independently. Infect Immun, 1999 Mar, 67(3), 1125 - 30 Role of Listeria monocytogenes exotoxins listeriolysin and phosphatidylinositol-specific phospholipase C in activation of human neutrophils; Sibelius U et al.; Polymorphonuclear leukocytes (PMN) are essential for resolution of infections with Listeria monocytogenes . The present study investigated the role of the listerial exotoxins listeriolysin (LLO) and phosphatidylinositol-specific phospholipase C (PlcA) in human neutrophil activation . Different Listeria strains, mutated in individual virulence genes, as well as purified LLO were used . Coincubation of human neutrophils with wild-type L . monocytogenes provoked PMN activation, occurring independently of phagocytosis events, with concomitant elastase secretion, leukotriene generation, platelet-activating factor (PAF) synthesis, respiratory burst, and enhanced phosphoinositide hydrolysis . Degranulation and leukotriene formation were noted to be solely dependent on LLO expression, as these features were absent when the LLO-defective mutant EGD- and the avirulent strain L . innocua were used . These effects were fully reproduced by a recombinant L . innocua strain expressing LLO (INN+) and by the purified LLO molecule . LLO secretion was also required for PAF synthesis . However, wild-type L . monocytogenes was more potent in eliciting PAF formation than mutants expressing LLO, suggesting the involvement of additional virulence factors . This was even more obvious for phosphoinositide hydrolysis and respiratory burst: these events were provoked not only by INN+ but also by the LLO-defective mutant EGD- and by a recombinant L . innocua strain producing listerial PlcA . We conclude that human neutrophils react to extracellularly provided listerial exotoxins by rapid cell activation . Listeriolysin is centrally involved in triggering degranulation and lipid mediator generation, and further virulence factors such as PlcA apparently contribute to trigger neutrophil phosphoinositide hydrolysis and respiratory burst . In this way, listerial exotoxins may influence the host defense against infections with L . monocytogenes. Immunity, 1999 Jan, 10(1), 29 - 38 Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase; Shiloh MU et al.; The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2) . Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics . Neither parental strain showed such infections . Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully . Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria . Their killing of S . typhimurium, E . coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2. J Exp Med, 1999 Feb 15, 189(4), 657 - 62 Impaired antibacterial host defense in mice lacking the N-formylpeptide receptor; Gao JL et al.; N-formylpeptides derive from bacterial and mitochondrial proteins, and bind to specific receptors on mammalian phagocytes . Since binding induces chemotaxis and activation of phagocytes in vitro, it has been postulated that N-formylpeptide receptor signaling in vivo may be important in antimicrobial host defense, although direct proof has been lacking . Here we test this hypothesis in mice lacking the high affinity N-formylpeptide receptor (FPR), created by targeted gene disruption . FPR-/- mice developed normally, but had increased susceptibility to challenge with Listeria monocytogenes, as measured by increased mortality compared with wild-type littermates . FPR-/- mice also had increased bacterial load in spleen and liver 2 d after infection, which is before development of a specific cellular immune response, suggesting a defect in innate immunity . Consistent with this, neutrophil chemotaxis in vitro and neutrophil mobilization into peripheral blood in vivo in response to the prototype N-formylpeptide fMLF (formyl-methionyl-leucyl-phenylalanine) were both absent in FPR-/- mice . These results indicate that FPR functions in antibacterial host defense in vivo. J Med Microbiol, 1999 Feb, 48(2), 117 - 24 Surface protein p104 is involved in adhesion of Listeria monocytogenes to human intestinal cell line, Caco-2; Pandiripally VK et al.; Adhesion of Listeria monocytogenes to intestinal endothelial cells is an important initial event in the pathogenesis of infection which is not well understood . The suggestion has been made that some proteins, including internalin and actin polymerisation protein (ActA), and carbohydrate molecules mediate, at least in part, the adhesion of listeria to certain cultured mammalian cells . This study investigated the role of a L . monocytogenes cell-surface protein of 104 kDa (p104) in adhesion to human intestinal enterocyte-like Caco-2 cell lines by transposon (Tn916) mutagenesis and a p104-specific monoclonal antibody (MAb-H7) . Genotypic and phenotypic characteristics of Tn916-transformed L . monocytogenes strains, AAMU530 and AAMU572, revealed that these strains did not express p104, and the transposon had been inserted at a single locus in the structural gene . Strains AAMU530 and AAMU572 yielded only 10 and 6.3% adhesion to Caco-2 cells . Coating of L . monocytogenes and L . innocua wild-type strains with MAb-H7 reduced adhesion to Caco-2 cells from 100% to 50 and 45%, respectively, whereas on isotype control MAb EM-7G1 had no effect . Western blot analysis with MAb-H7 indicated that p104 is present in all Listeria spp . except in L . grayi . Furthermore, p104 is also present in internalin (BUG8) and ActA (LUT12) deficient strains, suggesting that p104 is indeed different from internalin or ActA proteins . Cytotoxicity analysis of strains AAMU530 and AAMU572 demonstrated that these strains, although haemolytic and phospholipase-positive, were avirulent when tested with a hybridoma B-lymphocyte cell line . Loss of virulence could be attributed to the interruption of adhesion of mutant strains to the hybridoma cell line . These results strongly suggest that p104 is an adhesion factor in L . monocytogenes and possibly in other Listeria species and is involved in adhesion to intestinal cells. Mol Gen Mikrobiol Virusol, 1998, (4), 18 - 22 {Ultrastructural and immunocytochemical study of Listeria monocytogenes with varying levels of pathogenicity factor production}; Didenko LV et al.; Wild and mutant strains of Listeria monocytogenes are examined by electron and immunoelectron microscopy . The mutant strain was characterized as a strain with a high level of production of pathogenic factors . No essential morphological criteria permitting the differentiation between wild and mutant Listeria strains were detected . Addition of activated charcoal to nutrient medium resulted in similar morphological changes in both strains . Enlargement of cells, a thicker cell wall, and changes in the cytoplasm structure are objective morphological signs of functional activity of bacteria with a higher level of pathogenic factor production . Indirect immunocytochemical method demonstrated the localization of specific phosphatidyl inosityl phospholipase C. J Immunol, 1999 Feb 15, 162(4), 2368 - 74 Immunostimulatory CpG-oligodeoxynucleotides cause extramedullary murine hemopoiesis; Sparwasser T et al.; Bacterial DNA and the synthetic CpG-oligodeoxynucleotides (ODNs) derived thereof have attracted attention because they activate cells of the adaptive immune system (lymphocytes) and the innate immune system (APCs) in a sequence-dependent manner . Here, we addressed whether CpG-ODNs affect hemopoiesis . Challenging mice with immunostimulatory CpG-ODN sequences led to transient splenomegaly, with a maximum increase of spleen weight at day 6 . The induction of splenomegaly by CpG-ODNs was sequence-specific, dose-dependent, and associated with an increase in splenic cell count, in numbers of granulocyte-macrophage CFUs (GM-CFUs), and early erythroid progenitors (burst-forming units-erythroid) . The transfer of spleen cells from CpG-ODN-pretreated animals into lethally irradiated syngeneic mice yielded an increase of spleen CFUs . Furthermore, the challenge of sublethally irradiated mice with CpG-ODNs caused radioprotective effects, in that recovery of GM-CFUs and cytotoxic T cell function was enhanced . The increase in GM-CFU and CTL function correlated with an enhanced resistance to Listeria infection in irradiated mice . We conclude from these data that CpG-ODNs trigger extramedullary hemopoiesis, and that this finding could be of therapeutic relevance in myelosuppression. J Immunol, 1999 Feb 15, 162(4), 2291 - 8 Bacterial DNA containing CpG motifs stimulates lymphocyte-dependent protection of mice against lethal infection with intracellular bacteria; Elkins KL et al.; Bacterial DNA containing unmethylated CpG motifs activates mammalian lymphocytes and macrophages to produce cytokines and polyclonal Ig . These include IFN-gamma, IL-12, TNF-alpha, and IL-6, which are important in the control of intracellular bacterial infection . Here, we show that bacterial DNA, as well as synthetic oligonucleotides containing CpG motifs, induce protection against large lethal doses of Francisella tularensis live vaccine strain (LVS) and Listeria monocytogenes . Methylation of DNA at CpG dinucleotides or inversion of the motif abolished this protection . Surprisingly, DNA-mediated protection was highly dependent on lymphocytes, particularly B cells, as well as the production of IFN-gamma . Optimal protection was elicited 2-3 days after inoculation with DNA and persisted for up to 2 wk . Further, animals surviving lethal challenge developed pathogen-specific secondary immunity . These findings indicate that host innate immune responses to bacterial DNA may contribute to the induction of protective immunity to bacteria and the subsequent development of memory. J Immunol, 1999 Feb 1, 162(3), 1723 - 9 A transgenic model to analyze the immunoregulatory role of IL-10 secreted by antigen-presenting cells; Groux H et al.; IL-10 is a cytokine secreted by a wide variety of cells type that has pleiotropic stimulatory and suppressive activities on both lymphoid and myeloid cells in vitro . To analyze the consequences of high IL-10 secretion by APCs in immune responses, we produced transgenic mice expressing human IL-10 directed by the MHC class II Ea promoter . Despite alterations in the development of T and B cells, no gross abnormalities were detected in peripheral lymphocyte populations or serum Ig levels . However, when immunized using conditions that give either a Th2-type or a Th1-type response, IL-10 transgenic mice failed to mount a significant T or B cell immune response to OVA . IL-10 transgenic mice were also highly susceptible to infection with intracellular pathogens like Listeria monocytogenes or Leishmania major, in contrast to IL-10 transgenic mice, where the transgene was express in T cells . Finally, the recently described stimulatory effect of IL-10 on CD8+ T cells was confirmed by the ability of IL-10 transgenic mice to limit the growth of immunogenic tumors by a CTL-mediated mechanism . These results demonstrate, that, depending on the type of immune response, IL-10 can mediate immunosuppressive or immunostimulatory activities in vivo. J Toxicol Environ Health A, 1999 Feb 12, 56(3), 205 - 19 Xenobiotic-metabolizing enzymes in carp (Cyprinus carpio) liver, spleen, and head kidney following experimental Listeria monocytogenes infection; Chambras C et al.; Infection of carp with Listeria monocytogenes 4b resulted in decreased liver, spleen, and head kidney enzyme activities, involved in the metabolism of xenobiotics . After infection, cytochrome P-450 levels and ethoxyresorufin O-deethylase (EROD) activity were decreased while conjugation enzymes remained unaffected . The maximum decrease for phase I enzymes occurred on d 3 . This loss of monooxygenase levels and activity could not be directly correlated with an increase in the number of organisms, as consistently high bacterial counts were observed in all three organs during infection . The effect of L . monocytogenes infection was also measured in carp exposed to 3-methylcholanthrene (MCA) . Cytochrome P-450 levels and EROD activity were significantly reduced, especially on d 3 . A significant decreased activity of conjugation enzymes such as glutathione S-transferase (GST) and UDP-glucuronosyltransferase (UDPGT) was also observed for all days studied . Listeria infection inhibited MCA-induced increases in xenobiotic-metabolizing enzyme activities . These results indicate that infection may have deleterious effects on basal cytochrome P-450 monooxygenase levels . Furthermore, MCA treatment aggravates the insult to xenobiotic biotransformation enzymes by L . monocytogenes infection, by impairing a number of detoxification enzymes . These findings could result in significant changes in the susceptibility of fish to pollutants. FEMS Microbiol Lett, 1999 Jan 15, 170(2), 349 - 53 Characterisation of Listeria ivanovii isolates from the UK using pulsed-field gel electrophoresis; Ramage CP et al.; Forty-three Listeria ivanovii isolates were collected in the UK between 1991 and 1997 from: 35 animal infections; two human infections; five foods; and one environmental source . A further two type strains of L . ivanovii (subsp . ivanovii and subsp . londoniensis) were obtained from a culture collection . These bacteria were characterised by conventional phenotypic methods and by pulsed-field gel electrophoresis (PFGE) using ApaI and SmaI . Forty-two of the isolates from the UK were identified as L . ivanovii subsp . ivanovii and the remaining culture as L . ivanovii subsp . londoniensis . Six and four PFGE profiles were obtained using ApaI and SmaI digestion respectively; six composite profiles were obtained combining the results for both enzymes . The PFGE profile of the UK L . ivanovii subsp . londoniensis (isolated from processed shrimps) was similar to the type strain of this subspecies and differed from all of the L . ivanovii subsp . ivanovii tested . The majority of isolates (38 out of 45) belonged to one profile showing that the UK population of this bacterium is much less genetically diverse than similar studies have shown for Listeria monocytogenes. Biochem J, 1999 Feb 15, 338 ( Pt 1), 71 - 5 Incorporation of iron by the unusual dodecameric ferritin from Listeria innocua; Stefanini S et al.; The polypeptide chain that assembles into the unusual dodecameric shell of Listeria innocua apoferritin lacks the ferroxidase centre characteristic of H-type mammalian chains, but is able to catalyse both Fe(II) oxidation and nucleation of the iron core . A cluster of five carboxylate residues, which correspond in part to the site of iron core nucleation typical of L-type mammalian ferritins, has been proposed to be involved in both functions . The features of the iron uptake kinetics and of Fe(II) autoxidation in the presence of citrate followed spectrophotometrically confirm this assignment . In Listeria the kinetics of iron uptake is hyperbolic at low Fe(II)-to-dodecamer ratios and becomes sigmoidal when iron exceeds 150 Fe(II) atoms per dodecamer, namely when a fast crystal growth phase follows a slow initial nucleation step . Iron autoxidation in the presence of citrate displays a similar behaviour . Thus the time course is sigmoidal at low citrate-to-Fe ratios at which Fe(III) polymerization is predominant, but is hyperbolic at ligand concentrations high enough to prevent polymerization . The marked inhibitory effect of Tb(III) on the kinetics of iron incorporation confirms that carboxylates provide the iron ligands in L . innocua apoferritin . Iron uptake followed in steady-state fluorescence experiments allows one to distinguish Fe(II) binding and oxidation from the subsequent movement of Fe(III) into the apoferritin cavity as in mammalian ferritins despite the different localization of the tryptophan residues. Gastroenterol Hepatol, 1998 Dec, 21(10), 489 - 91 {Spontaneous bacterial peritonitis caused by Listeria monocytogenes}; Jorquera Plaza F et al.; We present a 68 year old male with alcoholic cirrhosis that was admitted with abdominal pain and fever . Hepatocarcinoma and spontaneous bacterial peritonitis by Listeria monocytogenes was diagnosed . The patient was treated with ampicillin and tobramycin during 25 days following a favorable course although ascitic fluid remained abnormal during 21 days . It is noted the rarity of Listeria as a cause of bacterial peritonitis in cirrhotic patients although they are immunodeficient . It is also important to establish the etiological origin because standard treatment of spontaneous bacterial peritonitis is cefotaxime and Listeria is resistant to this antibiotic . The 66% of spontaneous bacterial peritonitis secondary to Listeria monocytogenes infection in cirrhotic patients has been reported in Spain and this might be due to a higher incidence of human listeriosis in this country. J Food Prot, 1999 Jan, 62(1), 46 - 50 Combined effect of nisin and moderate heat on destruction of Listeria monocytogenes in cold-pack lobster meat; Budu-Amoako E et al.; The combined effect of nisin and moderate heat to increase the killing of Listeria monocytogenes in cans of "cold-pack" lobster was investigated . Adding nisin at a level of 25 mg/kg of can contents to the brine surrounding the lobster, in combination with a heat process giving internal can temperatures of 60 degrees C for 5 min and 65 degrees C for 2 min, resulted in decimal reductions of inoculated L . monocytogenes of 3 to 5 logs, whereas heat or nisin alone resulted in decimal reductions of 1 to 3 logs . Such a reduced heat process to that currently commercially used (65.5 degrees C for 13 to 18 min, depending on the can size) results in significant reduction in drained weight loss, thus allowing considerable cost savings to the lobster-processing industry. J Food Prot, 1999 Jan, 62(1), 40 - 5 High-pressure destruction kinetics of Listeria monocytogenes on pork; Mussa DM et al.; Packaged fresh pork chops (30-g samples) containing an indigenous bacterial population of approximately 10(7) CFU/g were inoculated with 10(7) CFU of Listeria monocytogenes Scott A per g, heat sealed, and subjected to high-pressure processing at 200 to 400 MPa for up to 90 min . Total counts and the number of surviving L . monocytogenes cells were determined by a spread plate technique on tryptic soy agar and modified Oxford medium, respectively . The pressure destruction was characterized by a dual-behavior, consisting of a step change in the number of survivors (Pk0) with the application of a pressure pulse and a first-order rate drop in the number of survivors during the pressure hold period . Higher pressures resulted in higher rates of microbial inactivation, as indicated by their associated lower D values (and higher k values) . The pressure sensitivities of the kinetic parameters were evaluated on the basis of Arrhenius and pressure death time (PDT)-type models . The results suggested that L . monocytogenes was more resistant to pressure inactivation than the indigenous microflora (the volume change of activation, deltaV* {Arrhenius model}), and Zp values (PDT model) were -4.17 x 10(-5) m3 mole(-1) and 134 MPa for indigenous microflora and -3.43 x 10(-5) m3 mole(-1) and 163 MPa for L . monocytogenes respectively. MMWR Morb Mortal Wkly Rep, 1999 Jan 8, 47(51-52), 1117 - 8 Update: multistate outbreak of listeriosis--United States, 1998-1999; Restraint stress-induced immunosuppression by inhibiting leukocyte migration and Th1 cytokine expression during the intraperitoneal infection of Listeria monocytogenes; Department of Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, JapanIn this study, a murine model of Listeria monocytogenes infection was used to investigate effects of restraint stress (RST) on host defense . We observed that the L . monocytogenes infection as well as RST induced an elevation of endogenous corticosterone (CORT) levels and RST synergistically enhanced endogenous CORT levels during the listerial infection . RST suppressed the migration of leukocytes including macrophages, neutrophils, NK cells and lymphocytes into the peritoneal cavities after the intraperitoneal inoculation of L . monocytogenes . RST also suppressed the increase of the surface MHC class II antigen expression in both peritoneal macrophages and B cells during the listerial infection . Interestingly, gene expression of iNOS, MCP-1 (JE) and Th1-type cytokines including IFN-gamma and IL-12 was down-regulated but Th2-type cytokine (IL-4 and IL-6) gene expression in the PEC was rather up-regulated on day 7 after infection, indicating that Th2-type immune response is more resistant to the elevated endogenous CORT levels than Th1-type response . Treatment of mice with RU486, a glucocorticoid receptor antagonist, restored the immune responses suppressed by RST to their normal levels in the infected mice, suggesting that the RST-induced elevation of endogenous corticosterone levels is mainly responsible for the induction of the immunosuppressive events during L . monocytogenes infection. J Immunol, 1999 Jan 15, 162(2), 980 - 8 Perforin-deficient CD8+ T cells: in vivo priming and antigen-specific immunity against Listeria monocytogenes; White DW et al.; CD8+ T cells require perforin to mediate immunity against some, but not all, intracellular pathogens . Previous studies with H-2b MHC perforin gene knockout (PO) mice revealed both perforin-dependent and perforin-independent pathways of CD8+ T cell-mediated immunity to Listeria monocytogenes (LM) . In this study, we address two previously unresolved issues regarding the requirement for perforin in antilisterial immunity: 1) Is CD8+ T cell-mediated, perforin-independent immunity specific for a single Ag or generalizable to multiple Ags? 2) Is there a deficiency in the priming of the CD8+ T cell compartment of PO mice following an immunizing challenge with LM? We used H-2d MHC PO mice to generate CD8+ T cell lines individually specific for three known Ags expressed by a recombinant strain of virulent LM . Adoptive transfer experiments into BALB/c host mice revealed that immunity can be mediated by PO CD8+ T cells specific for all Ags examined, indicating that perforin-independent immunity is not limited to CD8+ T cells that recognize listeriolysin O . Analysis of epitope-specific CD8+ T cell expansion by MHC class I tetramer staining and ELISPOT revealed no deficiency in either the primary or secondary response to LM infection in PO mice . These results demonstrate that the perforin-independent pathway of antilisterial resistance mediated by CD8+ T cells is generalizable to multiple epitopes . Furthermore, the results show that reduced antilisterial resistance observed with polyclonal PO CD8+ T cells is a consequence of a deficiency in effector function and not a result of suboptimal CD8+ T cell priming. J Immunol, 1999 Jan 15, 162(2), 965 - 73 IL-12 is not required for induction of type 1 cytokine responses in viral infections; Oxenius A et al.; To investigate the physiological role of IL-12 in viral infections in terms of T cell cytokine responses involved in virus-specific Ig isotype induction and in antiviral protection, immune responses elicited upon infection of IL-12-deficient mice with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV) were studied . Infection of IL-12-deficient mice with LCMV induced a virus-specific type 1 cytokine response as determined by in vitro cytokine secretion patterns as well as by in vivo intracellular cytokine staining of LCMV-specific CD4+ TCR transgenic T cells that had clonally expanded in LCMV-infected IL-12-deficient recipient mice . In addition, LCMV- and VSV-specific IgG responses exhibited normal serum IgG2a/IgG1 ratios, demonstrating again virus-specific CD4+ T cell induction of type 1 phenotype in IL-12-deficient mice upon viral infection . LCMV and VSV immune mice were found to be protected against challenge immunization with recombinant vaccinia viruses expressing either the LCMV- or the VSV-derived glycoprotein, respectively . This protection is known to be mediated by T cell-secreted type 1 cytokines IFN-gamma and TNF-alpha . In contrast, IL-12-deficient mice showed impaired abilities to control infection with the facultative intracellular bacterium Listeria monocytogenes at early time points after infection . However, at later time points of infection, IL-12-deficient mice were able to clear infection . These findings may indicate that viruses are able to induce type 1 T cell responses in the absence of IL-12 as opposed to some bacterial or parasitical infections that are crucially dependent on the presence of IL-12 for the induction of type 1 immune responses. Infect Immun, 1999 Feb, 67(2), 568 - 75 Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O; Tanabe Y et al.; Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice . Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma . In this study, mice were immunized with L . monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome) . LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma . Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection . In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner . The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells . These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria. MMWR Morb Mortal Wkly Rep, 1998 Dec 25, 47(50), 1085 - 6 Multistate outbreak of listeriosis--United States, 1998; Intramedullary brain stem cyst and trapped IV ventricle after infection with Listeria monocytogenes; Neurochirurgische Klinik im Neurozentrum, Albert-Ludwigs-Universitat, Freiburg i.Br., GermanyWe present the rare case of a girl surviving intrauterine listeria brain stem meningoencephalitis, who subsequently developed hydrocephalus, a trapped IV ventricle and an intramedullary cyst . Such cases have been reported only infrequently, and in earlier cases modern imaging studies were not available . Magnetic resonance imaging has been helpful in our patient to delineate the lesions and plan further treatment. Prev Vet Med, 1998 Dec 1, 37(1-4), 129 - 45 Quantitative risk assessment of human listeriosis from consumption of soft cheese made from raw milk; Bemrah N et al.; Microbial hazards have been identified in soft cheese made from raw milk . Quantification of the resulting risk for public health was attempted within the frame of the Codex Alimentarius Commission, 1995 approach to quantitative risk assessment, using Monte Carlo simulation software . Quantitative data could only be found for Listeria monocytogenes . The complete process of cheese making was modeled, from milking to consumption . Using data published on the different sources of milk contamination (environment and mastitis) and bacterial growth, distributions were assumed for parameters of the model . Equations of Farber, J.M., Ross, W.H., Harwing, J . (1996) for general and at-risk populations were used to link the ingested dose of L . monocytogenes to the occurrence of listeriosis . The probability of milk contamination was estimated to be 67% with concentration ranging from 0 to 33 CFU ml-1 . The percentage of cheese with a predicted concentration of L . monocytogenes greater than 100 CFU g-1 was low (1.4%) . The probability of consuming a contaminated cheese serving was 65.3% . Individual annual cumulative risk of listeriosis, in a population each consuming 50 servings of 31 g, ranged from 1.97 x 10(-9) to 6.4 x 10(-8) in a low-risk sub-population and 1.04 10(-6) to 7.19 10(-5) in a high-risk sub-population . The average number of expected cases of listeriosis per year was 57 for a high-risk sub-population and one for a low-risk healthy sub-population . When the frequency of environmental milk contamination was reduced in the model and L . monocytogenes mastitis was eliminated, the expected incidence of listeriosis decreased substantially; the average number of expected cases was reduced by a factor of 5 . Thus the usefulness of simulation to demonstrate the efficiency of various management options could be demonstrated, even if results should be interpreted with care (as many assumptions had to be made on data and their distributions. J Vet Med Sci, 1998 Dec, 60(12), 1341 - 3 Detection of Listeria monocytogenes in humans, animals and foods; Iida T et al.; The Listeria monocytogenes-carrying rates were 100% for listeriosis patients and 1.3% for healthy humans . The L . monocytogenes contamination rates for retail sliced beef (34.2%) and pork (36.4%) were significantly higher (p < 0.05) than those for cattle (2.0%) and pigs (0.8%) and for cattle (4.9%) and swine (7.4%) carcasses . The percentages of serotypes 1/2a, 1/2b and 4b which are most dominant in human patients were high in isolates from fresh (90.0%) and processed (100%) fish and shellfish and imported natural cheese (96.7%). Physiol Behav, 1998 Dec 1, 65(3), 535 - 43 2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice; Miller ES et al.; Exposure to different forms of psychological and physiological stress can elicit a host stress response, which alters normal parameters of neuroendocrine homeostasis . The present study evaluated the influence of the metabolic stressor 2-deoxy-D-glucose (2-DG; a glucose analog, which when administered to rodents, induces acute periods of metabolic stress) on the capacity of mice to resist infection with the facultative intracellular bacterial pathogen Listeria monocytogenes . Female BDF1 mice were injected with 2-DG (500 mg/kg b . wt.) once every 48 h prior to, concurrent with, or after the onset of a sublethal dose of virulent L . monocytogenes . Kinetics of bacterial growth in mice were not altered if 2-DG was applied concurrently or after the start of the infection . In contrast, mice exposed to 2-DG prior to infection demonstrated an enhanced resistance to the listeria challenge . The enhanced bacterial clearance in vivo could not be explained by 2-DG exerting a toxic effect on the listeria, based on the results of two experiments . First, 2-DG did not inhibit listeria replication in trypticase soy broth . Second, replication of L . monocytogenes was not inhibited in bone marrow-derived macrophage cultures exposed to 2-DG . Production of neopterin and lysozyme, indicators of macrophage activation, were enhanced following exposure to 2-DG, which correlated with the increased resistance to L . monocytogenes . These results support the contention that the host response to 2-DG-induced metabolic stress can influence the capacity of the immune system to resist infection by certain classes of microbial pathogens. Appl Environ Microbiol, 1999 Jan, 65(1), 150 - 5 Sources of Listeria monocytogenes contamination in a cold-smoked rainbow trout processing plant detected by pulsed-field gel electrophoresis typing; Autio T et al.; Sites of Listeria monocytogenes contamination in a cold-smoked rainbow trout (Oncorhynchus mykiss) processing plant were detected by sampling the production line, environment, and fish at different production stages . Two lots were monitored . The frequency of raw fish samples containing L . monocytogenes was low . During processing, the frequency of fish contaminated with L . monocytogenes clearly rose after brining, and the most contaminated sites of the processing plant were the brining and postbrining areas . A total of 303 isolates from the raw fish, product, and the environment were characterized by pulsed-field gel electrophoresis (PFGE) . PFGE yielded nine pulsotypes, which formed four clusters . The predominating L . monocytogenes pulsotypes of the final product were associated with brining and slicing, whereas contaminants of raw fish were not detected in the final product . Air-mediated contamination in the plant could not be proved . In accordance with these results, an L . monocytogenes eradication program was planned . The use of hot steam, hot air, and hot water seemed to be useful in eliminating L . monocytogenes . None of the control samples taken in the 5 months after the eradication program was implemented contained L . monocytogenes. Lett Appl Microbiol, 1998 Dec, 27(6), 362 - 8 Natural milk fatty acids affect survival and invasiveness of Listeria monocytogenes; Petrone G et al.; The effects of 12 fatty acids, naturally occurring in milk from several mammalian species, on the survival and invasion ability of Listeria monocytogenes, a food-borne pathogen, were determined . The survival was tested in the presence of 200 micrograms ml-1 fatty acids suspended in brain hearth infusion broth or in storage conditioning solution (NaCl 1%) of Mozzarella cheese, an Italian soft unripened cheese, at pH 7.0 or 5.0 . Lauric (C12:0), linoleic (C18:2) and linolenic (C18:3) acids exerted the strongest bactericidal activity . The invasive efficiency of L . monocytogenes, determined in the Caco-2 enterocyte-like cell line, was strongly decreased in the presence of the fatty acids tested (from about 20 to 500-fold) . This research suggests that naturally occurring fatty acids of milk, supplemented in milk derivatives, could affect both bacterial growth and invasiveness and consequently, could serve as barriers towards L . monocytogenes infection. Kekkaku, 1998 Nov, 73(11), 639 - 44 {Mechanisms of induction and expression of anti-tuberculous immunity}; Mitsuyama M; The induction of anti-tuberculous immunity highly depends on the cytokines produced endogenously at the initial stage of immunization . Among several cytokines, IFN-gamma appears to be the most important to generate antigen-specific Th1 type of protective T cells in mice . IL-12 and IL-18, which are produced by macrophages in response to virulent mycobacteria, are responsible for stimulating NK cells to produce IFN-gamma . Once antigen-specific Th1 cells are generated, Th1-dependent macrophage activation was effective in the elimination of infected bacteria through enhanced production of reactive oxygen intermediates and reactive nitrogen intermediates . In Listeria monocytogenes, one of the intracellular bacteria, listeriolysin O (LLO) appeared to be responsible for the induction of endogenous IFN-gamma from NK cells . The possible mechanisms operating in the induction and expression of anti-tuberculous immunity are discussed with special reference to cytokine responses . An application of LLO to the induction of protective immunity is also discussed. Clin Ter, 1998 Jul-Aug, 149(4), 307 - 11 {A case of maternal and neonatal infection due to Listeria monocytogenes}; Tridente V et al.; A Listeria monocytogenes infection may develop during pregnancy by eating sausages, fresh meats and milk products derived from infected animals . According to the period in which the infection starts, the pregnancy outcome can be abortion or pre-term or at term delivery . The infection can pass from mother to fetus and can cause a serious neonatal sepsis . Listeriosis in pregnant women can be asymptomatic or may present as an influenza-like syndrome . This case report, along with other published cases, demonstrates how hard is to make a correct diagnosis of listeriosis during pregnancy . Since this is mainly related to the aspecificity of symptoms, it is very important to have a high suspicion and to take a careful patient history. Infect Immun, 1999 Jan, 67(1), 253 - 8 Existing antilisterial immunity does not inhibit the development of a Listeria monocytogenes-specific primary cytotoxic T-lymphocyte response; Bouwer HG et al.; Infection of BALB/c mice with Listeria monocytogenes stimulates an antilisterial immune response evident by the appearance of H2-Kd-restricted CD8(+) cytotoxic T lymphocytes (CTLs) specific for the nanomer peptides amino acids (aa) 91 to 99 of listeriolysin O (LLO 91-99) and aa 217 to 225 of the p60 molecule (p60 217-225) . We have introduced point mutations at anchor residues within LLO 91-99 (92F) or p60 217-225 (218F), and BALB/c mice infected with L . monocytogenes strains containing these point mutations do not develop CTLs specific for LLO 91-99 or p60 217-225, respectively . We have used these strains to test whether primary CTL responses against L . monocytogenes-derived determinants can be stimulated within an environment of existing antilisterial immunity . We found that the development of a primary L . monocytogenes-specific CTL response is not altered by existing immunity to L . monocytogenes . For example, primary immunization with the p60 218F strain of L . monocytogenes followed by a secondary immunization with wild-type L . monocytogenes results in stimulation of p60 217-225-specific CTLs at primary response levels and LLO 91-99-specific effectors at levels consistent with a memory CTL response . Similarly, primary immunization with the 92F strain of L . monocytogenes followed by a secondary immunization with wild-type L . monocytogenes results in stimulation of LLO 91-99-specific CTLs at primary response levels and p60 217-225-specific effectors at levels consistent with a memory CTL response . These results provide additional support for the use of L . monocytogenes as a recombinant vaccine vector and show that antivector immunity does not inhibit the development of a primary CTL response when the epitope is delivered by L . monocytogenes as the vaccine strain. Infect Immun, 1999 Jan, 67(1), 182 - 6 Mutagenesis of active-site histidines of Listeria monocytogenes phosphatidylinositol-specific phospholipase C: effects on enzyme activity and biological function; Bannam T et al.; Listeria monocytogenes, a gram-positive facultative intracellular pathogen, produces two distinct phospholipases C . PC-PLC, encoded by plcB, is a broad-range phospholipase, whereas PI-PLC, encoded by plcA, is specific for phosphatidylinositol . It was previously shown that PI-PLC plays a role in efficient escape of L . monocytogenes from the primary phagosome . To further understand the function of PI-PLC in intracellular growth, site-directed mutagenesis of plcA was performed . Two potential active-site histidine residues were mutated independently to alanine, serine, and phenylalanine . With the exception of the activity of the enzyme containing H38F, which was unstable, the PI-PLC enzyme activities of culture supernatants containing each mutant enzyme were <1% of wild-type activity . In addition, the levels of expression of the mutant PI-PLC proteins were equivalent to wild-type expression . Derivatives of L . monocytogenes containing these specific plcA mutations were found to have phenotypes similar to that of the plcA deletion strain in an assay for escape from the primary vacuole, in intracellular growth in a murine macrophage cell line, and in a plaquing assay for cell-to-cell spread . Thus, catalytic activity of PI-PLC is required for all its intracellular functions. J Immunol, 1998 Dec 15, 161(12), 6715 - 23 Two families of GTPases dominate the complex cellular response to IFN-gamma; Boehm U et al.; IFN-gamma induces a number of cellular programs functional in innate and adaptive resistance to infectious pathogens . It has recently become clear that the complete cellular response to IFN-gamma is extraordinarily complex, with >500 genes (i.e., approximately 0.5% of the genome) activated . We made suppression-subtractive hybridization differential libraries from IFN-gamma-stimulated primary mouse embryonic fibroblasts and from a mouse macrophage cell line, ANA-1, in each case with reference to unstimulated cells . Of approximately 250 clones sequenced at random from the two libraries, >35% were representatives of one or the other of two small unrelated families of GTPases, the 65-kDa and 47-kDa families . These families dominate the IFN-gamma-induced response in both cell types . We report here the full-length sequences of one new 65-kDa and two new 47-kDa family members . The 65-kDa family members are under transcriptional control of IRF-1, whereas the 47-kDa family members are inducible in embryonic fibroblasts from IRF-1(-/-) mice . Members of both GTPase families are strongly up-regulated in livers of wild-type mice infected with the pathogenic bacterium, Listeria monocytogenes, but not in IFN-gammaR(0/0) mice . These GTPases appear to be dedicated to the IFN-gamma response, since resting levels are negligible and since neither family shows any significant relationship to any other described family of GTPases . Understanding the role of these GTPases in IFN-gamma-mediated resistance against pathogens is the task for the future. Mol Gen Genet, 1998 Nov, 260(2-3), 144 - 58 The gene cluster inlC2DE of Listeria monocytogenes contains additional new internalin genes and is important for virulence in mice; Raffelsbauer D et al.; In this work we identified and characterized a gene cluster containing three internalin genes of Listeria monocytogenes EGD . These genes, termed inlG, inlH and inlE, encode proteins of 490, 548 and 499 amino acids, respectively, which belong to the family of large, cell wall-bound internalins . The inlGHE gene cluster is flanked by two listerial house-keeping genes encoding proteins homologous to the 6-phospho-beta-glucosidase and the succinyl-diaminopimelate desuccinylase of E . coli . A similar internalin gene cluster, inlC2DE, localised to the same position on the L . monocytogenes EGD chromosome was recently described in a different isolate (Dramsi S, Dehoux P, Lebrun M, Goossens PL, Cossart P (1997) Infect Immun 65: 1615-1625) . Sequence comparison of the two inl gene clusters indicates that inlG is a new internalin gene, while inlH was generated by a site-specific recombination, leading to an in-frame deletion which removed the 3'-terminal end of inlC2 and the 5'-terminal part of inlD . The third gene of the inlGHE cluster, inlE, is almost identical to the previously reported inlE gene . Our data show that the inlGHE gene cluster is probably transcribed from a major PrfA-independent promoter located upstream of inlG . PCR analysis revealed the presence of the newly identified inl genes inlG and inlH in most L . monocytogenes isolates tested . A mutant which has lost inlG, inlH and inlE by an in-frame deletion exhibited, after oral infection of mice, a significant loss in virulence and shows drastically reduced numbers of viable bacteria in both liver and spleen when compared to the wild-type strain. J Bacteriol, 1998 Dec, 180(24), 6655 - 60 Functional similarities between the Listeria monocytogenes virulence regulator PrfA and cyclic AMP receptor protein: the PrfA* (Gly145Ser) mutation increases binding affinity for target DNA; Vega Y et al.; Most Listeria monocytogenes virulence genes are positively regulated by the PrfA protein, a transcription factor sharing sequence similarities with cyclic AMP (cAMP) receptor protein (CRP) . Its coding gene, prfA, is regulated by PrfA itself via an autoregulatory loop mediated by the upstream PrfA-dependent plcA promoter . We have recently characterized prfA* mutants from L . monocytogenes which, as a result of a single amino acid substitution in PrfA, Gly145Ser, constitutively overexpress prfA and the genes of the PrfA virulence regulon . Here, we show that about 10 times more PrfA protein is produced in a prfA* strain than in the wild type . Thus, the phenotype of prfA* mutants is presumably due to the synthesis of a PrfA protein with higher promoter-activating activity (PrfA*), which keeps its intracellular levels constantly elevated by positive feedback . We investigated the interaction of PrfA and PrfA* (Gly145Ser) with target DNA . Gel retardation assays performed with a DNA fragment carrying the PrfA binding site of the plcA promoter demonstrated that the PrfA* mutant form is much more efficient than wild-type PrfA at forming specific DNA-protein complexes . In footprinting experiments, the two purified PrfA forms interacted with the same nucleotides at the target site, although the minimum amount required for protection was 6 to 7 times lower with PrfA* . These results show that the primary functional consequence of the Gly145Ser mutation is an increase in the affinity of PrfA for its target sequence . Interestingly, similar mutations at the equivalent position in CRP result in a transcriptionally active, CRP* mutant form which binds with high affinity to target DNA in the absence of the activating cofactor, cAMP . Our observations suggest that the structural similarities between PrfA and CRP are also functionally relevant and support a model in which the PrfA protein, like CRP, shifts from transcriptionally inactive to active conformations by interaction with a cofactor. Int J Food Microbiol, 1998 Oct 20, 44(1-2), 141 - 4 Initial numbers, serovars and phagevars of Listeria monocytogenes isolated in prepared foods in the city of Barcelona (Spain); de Simon M et al.; A total of 1100 samples of prepared foods purchased in restaurants and delicatessen shops in the city of Barcelona was examined for the presence of Listerio . L . monocytogenes was more frequently isolated from foods intended to be consumed without further cooking (9.3%) than from foods intended to receive further cooking prior to consumption (2.9%) . A quantitative study, carried out with 773 samples, yielded 1% of samples with numbers of L . monocytogenes higher than 100 CFU/g . Strains of L . monocytogenes belonged to serovar 4b (36.3%), 1/2a (29.5%), 1/2b (22.7%) and 1/2c (11.3%) . L . monocytogenes serovar 4b phagevar 3274: 2671 (6 of 11) was prevalent among the strains studied. Int J Food Microbiol, 1998 Oct 20, 44(1-2), 83 - 92 The effect of the growth environment on the lag phase of Listeria monocytogenes; Robinson TP et al.; The duration of lag in Listeria monocytogenes was examined in relation to the physico-chemical properties of the growth environment . It was supposed that lag would be determined by two hypothetical quantities, the amount of work that a cell has to perform to adapt to new conditions and the rate at which it can perform that work . If the rate at which the cell can perform the necessary work is a function of the maximum specific growth rate in the new environment, the hypothesis predicts that lag time should be related in some way to growth rate, provided cells are initially in approximately the same physiological state . Literature data suggest this is true for many organisms when temperature is the sole growth limiting factor . However, lag times of L . monocytogenes displayed an unusual response to temperature in which lag times of cells precultured at 37 degrees C were shorter at 15 degrees C than at 20 degrees C or 25 degrees C . Analysis of data from the Food Micromodel in which growth of L . monocytogenes was controlled by combinations of pH, NaCl concentration and temperature, showed that there was a linear relationship between lag time and mean generation time although there was much scatter in the data . When the effects of pH, solute type and concentration were investigated individually in this work the correlation between lag time and mean generation time was often poor . It would thus appear that the relationship between growth environment and lag time is more complex than the corresponding relationship between growth environment and maximum specific growth rate. Dig Surg, 1998, 15(4), 364 - 8 Listeria monocytogenes causing solitary liver abscess . Case report and review of the literature; Bronnimann S et al.; The authors report on a case of a solitary liver abscess due to Listeria monocytogenes in a 53-year-old diabetic white male and review all published cases of solitary listerial abscesses of the liver . L . monocytogenes is a rare cause of solitary liver abscess which occurs in elderly patients with diabetes mellitus . The clinical signs are variable and often mimic malignancy, with epigastric pain, night sweats and weight loss . Prevalent features are poor control of glycemia, temperature up to 38.5 degrees C and elevated alkaline phosphatase . Optimal treatment includes percutaneous drainage of the hepatic abscess and antibiotic therapy with an aminopenicillin or trimethoprim/sulfamethoxazole . Outcome of the reviewed patients was favourable with no mortality and no relapse of the disease. Eur J Cell Biol, 1998 Oct, 77(2), 81 - 90 The suitability and application of a GFP-actin fusion protein for long-term imaging of the organization and dynamics of the cytoskeleton in mammalian cells; Choidas A et al.; The product of a GFP-actin gene fusion, permanently or transiently transfected in diverse mammalian cell lines, was shown to be a suitable, intrinsic probe of both the organization and dynamics of the actin cytoskeleton . In live Swiss 3T3 and NIH 3T3 cells, the fusion protein was found to accumulate in lamellipodia, filopodia, focal contacts and stress fibers . Furthermore, comparisons of fluorescence images of GFP-actin and Cy3.5-phalloidin, an independent marker of F-actin, in permeabilized cells showed a complete overlap of the two fluorescence signals . In GFP-actin-transfected Hela cells that had been infected with Listeria monocytogenes, the fluorescence of the fusion protein was shown to dynamically associate in the F-actin rich comet tail that formed behind a motile bacterium . In stable transfectants of PC12 cells, GFP-actin constituted on the average 5% of the total actin - these cells exhibited normal growth behavior and responded to treatment with nerve growth factor by extending neurite-like extensions, the filopodia-like tips of which were densely packed with filamentous GFP-actin . Finally, the photobleaching decay time of GFP-actin in live cells of 63 seconds was much longer than that of fluorescein-labeled actin conjugates and little or no damage to the cytoskeleton was found during the photobleaching of GFP-actin . Having shown the suitability of GFP-actin as a probe of the cytoskeleton, its fluorescence was used in long-term imaging studies aimed at documenting changes in the cytoskeleton of rat bladder NBT-II carcinoma cells during the 24-hour growth factor-mediated epithelia to mesenchyme transformation . The intrinsic fluorescent probe was also used to investigate the organization of the actin cytoskeleton and behavior of individual mesenchyme NBT-II cells slowly migrating through a colony of epithelia cells. Vet Immunol Immunopathol, 1998 Oct 23, 65(2-4), 125 - 38 Effect of feline immunodeficiency virus on cytokine response to Listeria monocytogenes in vivo; Dean GA et al.; Feline immunodeficiency virus (FIV) is a lentivirus that induces an acquired immunodeficiency in domestic cats . The objective of this study was to compare the immune response of chronically FIV-infected cats and specific pathogen free (SPF) cats to Listeria monocytogenes, a facultative intracellular bacterium . Regional lymph nodes were removed at various times after subcutaneous inoculation with L . monocytogenes and evaluated . Lymph nodes of chronically FIV-infected cats enlarged more slowly and to a lesser degree than SPF cats . This was due to delayed and blunted lymphoid follicle formation and markedly diminished histiocyte influx . The cellular response correlated with a marked upregulation in IL10 transcription and delayed increase in TNF-alpha upregulation in FIV-infected cats . Transcriptional upregulation of IFN-gamma, IL4, and the p40 chain of IL12 was similar in lymph nodes of FIV-infected and SPF cats . Clinically, FIV-infected cats had a more severe response at the site of L . monocytogenes injection and showed signs of systemic bacterial dissemination while SPF cats remained clinically normal . FIV-infected cats generated a delayed hypersensitivity response similar to SPF cats but also had a significantly greater antibody response . Taken together, these data suggest excessive IL10 production may be responsible for the deficiency observed in the innate immune response of chronically FIV-infected cats challenged with L . monocytogenes. Appl Environ Microbiol, 1998 Dec, 64(12), 4883 - 90 Purification and genetic characterization of enterocin I from Enterococcus faecium 6T1a, a novel antilisterial plasmid-encoded bacteriocin which does not belong to the pediocin family of bacteriocins; Floriano B et al.; Enterocin I (ENTI) is a novel bacteriocin produced by Enterococcus faecium 6T1a, a strain originally isolated from a Spanish-style green olive fermentation . The bacteriocin is active against many olive spoilage and food-borne gram-positive pathogenic bacteria, including clostridia, propionibacteria, and Listeria monocytogenes . ENTI was purified to homogeneity by ammonium sulfate precipitation, binding to an SP-Sepharose fast-flow column, and phenyl-Sepharose CL-4B and C2/C18 reverse-phase chromatography . The purification procedure resulted in a final yield of 954% and a 170,000-fold increase in specific activity . The primary structure of ENTI was determined by amino acid and nucleotide sequencing . ENTI consists of 44 amino acids and does not show significant sequence similarity with any other previously described bacteriocin . Sequencing of the entI structural gene, which is located on the 23-kb plasmid pEF1 of E . faecium 6T1a, revealed the absence of a leader peptide at the N-terminal region of the gene product . A second open reading frame, ORF2, located downstream of entI, encodes a putative protein that is 72.7% identical to ENTI . entI and ORF2 appear to be cotranscribed, yielding an mRNA of ca . 0.35 kb . A gene encoding immunity to ENTI was not identified . However, curing experiments demonstrated that both enterocin production and immunity are conferred by pEF1. Rev Med Chil, 1998 Jul, 126(7), 828 - 32 {Listeria monocytogenes rhomboencephalitis . Clinical case}; Bravo M et al.; We report a previously healthy 44 years old female, that presented with mild clouding of consciousness, a left cerebellar syndrome, involvem