Eur J Immunol, 2000 Apr, 30(4), 1053 - 9 Substantial in vivo proliferation of CD4(+) and CD8(+) T lymphocytes during secondary Listeria monocytogenes infection; Mittrucker HW et al.; In mice Listeria monocytogenes infection induces a strong T cell response . In an attempt to quantitatively analyze the magnitude and kinetics of the CD4(+) and CD8(+) T cell response during L . monocytogenes infection in vivo we used a T cell transfer system that is independent of in vitro cell culture techniques and information about the identity of immunogenic T cell epitopes . Our results demonstrate substantial expansion of the in vivo primed and transferred T cell populations in response to L . monocytogenes . At the peak of response, transferred T cells represented more than one third of the total CD4(+) and CD8(+) T cell populations in blood and spleen of recipient mice . After stimulation in vitro, 40 % of these CD4(+) T cells responded to heat-killed listeriae with the production of IFN-gamma . Thus, our results reveal that in addition to the large CD8(+) T cell population an almost equally large population of Listeria-reactive CD4(+) T cells is generated in response to L . monocytogenes infection. J Immunol Methods, 2000 Apr 21, 238(1-2), 107 - 17 Intracellular staining for TNF and IFN-gamma detects different frequencies of antigen-specific CD8(+) T cells; Badovinac VP et al.; CD8(+) T lymphocytes are important mediators of adaptive immunity against certain viral, protozoan and bacterial pathogens . Activated CD8(+) T cells are able to induce cytolysis of infected cells (perforin and CD95-CD95L mediated pathways) and also elaborate cytokines, including IFN-gamma and TNF after appropriate MHC class I-peptide recognition . New technologies for the detection of antigen-specific CD8(+) T cells, including tetrameric MHC class I-peptide complexes, intracellular IFN-gamma staining and IFN-gamma ELISPOT analysis have revised our understanding of the magnitude of the CD8(+) T cell response to infection . Here, using intracellular cytokine staining, we compare detection of IFN-gamma and TNF in the analysis of pathogen-specific CD8(+) T cell lines and CD8(+) T cells after primary viral infection (LCMV) or secondary bacterial infection (Listeria monocytogenes) . Under multiple conditions and with multiple epitopes, we find that staining for intracellular IFN-gamma consistently detects a higher frequency of antigen-specific CD8(+) T cells than detection of intracellular TNF . However, (a) intracellular staining for TNF can be used to detect antigen-specific CD8(+) T cell responses and (b) intracellular staining for cytokines is a useful approach for in vitro characterization of antigen-specific CD8(+) T cell lines. Acta Microbiol Pol, 1999, 48(4), 331 - 40 Effect of benzylpenicillin on murein biosynthesis, turnover and structure in Listeria monocytogenes cells; Siwinska M et al.; The action of beta-lactam antibiotics such as ampicillin and benzylpenicillin on cells of Listeria monocytogenes appears to be bacteriostatic . However, after approximately two hours in the presence of 10 x MIC of benzylpenicillin the cells begin to rapidly lose viability without undergoing lysis . In this report we present the results of studies on the biosynthesis of murein in L . monocytogenes cells during the first 120 min of their exposure to benzylpenicillin as measured by the continuous and pulse incorporation of the precursor N-acetyl-D-{1-3H}glucosamine . The turnover of the murein sacculus in the presence of penicillin as well as the lack of discernible changes in the molecular structure of the murein synthesised in the presence of benzylpenicillin is also discussed. Acta Microbiol Pol, 1999, 48(4), 317 - 29 Intracellular growth of Listeria monocytogenes insertional mutant deprived of protein p60; Wisniewski JM et al.; The study of the mutant strain described here demonstrates that several characteristics contribute to maximal virulence of pathogenic strains of L . monocytogenes . The invasion levels of L . monocytogenes JB1115, a p60-deficient strain, were the same as for the parent strain L . monocytogenes 1043S in J774 macrophage-like cells . The invasion level of Listeria strains in Int407 cells was 100 times lower than in J774 cells . In epithelial Int407 cells, the time of division of p60- strain L . monocytogenes JB1115 was 43% slower than for the parent strain . In this study, two lisosomotrophic agents, ammonium chloride and chlorquinoline were tested in experimental L . monocytogenes 1043S and p60-deprived mutant JB1115 infection in both cell lines . The presence of ammonium chloride increased the level of infection (calculated as number of gentamicin-resistant cells) of both Listeria strains, but in the case of infection by p60 mutant, the increased amount of ammonium chloride showed only a minimal effect on the number of isolated bacteria . In both cell lines treated with chlorquinoline we observed a decrease in the number of viable intracellular bacteria isolated from infected monolayers . Our observation of parental and mutated strains of Listeria showed that phospholipase activity also depends on the presence of p60 protein . Mutated strain showed 31.46% reduction of PI-PLC activity measured in normal growth conditions . Protein p60 plays a role not only in listeriolysin O mediated haemolytic activity but full phospholipase C activity is also dependent on the presence of the Iap protein. J Zoo Wildl Med, 1999 Dec, 30(4), 545 - 9 Listeria monocytogenes subtypes associated with mortality among fallow deer (Dama dama); Tham W et al.; Different subtypes of Listeria monocytogenes were isolated from various animal and environmental samples during an episode of increased mortality on a fallow deer (Dama dama) farm . During a 4-wk period, six fallow deer died, including four does, one fawn, and one adult buck . Prior to death, one of the does had exhibited central nervous system signs characteristic of listeriosis . Postmortem examination of the six deer showed no histologic changes typical of listeriosis, although inflammatory changes were present in several organs . Different subtypes of L . monocytogenes were isolated from brain samples from six deer, from fodder and soil from the deer feeding area, and from faces of some healthy animals on the farm . Listeria monocytogenes, which was frequently isolated in the environment of the farm, was considered the probable major cause of mortality in these fallow deer. Lett Appl Microbiol, 2000 Mar, 30(3), 228 - 32 Variations in virulence between different electrophoretic types of Listeria monocytogenes; Norrung B et al.; A total of 245 strains of Listeria monocytogenes, representing 33 different electrophoretic types (ETs), were examined quantitatively for haemolytic activity . No significant difference was observed in the mean haemolytic activity between different ETs . Eighty four out of 91 strains examined were found to be virulent for chick embryos . Strains belonging to ET 2 and ET 4 were found to be less virulent than strains of other ETs (P = 0.0447) . Furthermore, strains from clinical cases were found to be more virulent (P = 0.0002) than strains from foods (the MTD among clinical strains was 2.46 in mean compared with 3.64 among food isolates) . The explanation for this may be that more virulent strains are more prone to cause human infection . It is, however, also possible that strains of L . monocytogenes may become more virulent while multiplying in a living organism compared with multiplying in foods. EMBO J, 2000 Apr 3, 19(7), 1458 - 66 gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes; Braun L et al.; InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase . Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q . Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry . Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads . Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB . Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1 . After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L . monocytogenes into mammalian cells. Int J Food Microbiol, 2000 Mar 10, 54(1-2), 109 - 15 Inhibitory combinations of nisin, sodium chloride, and pH on Listeria monocytogenes ATCC 15313 in broth by an experimental design approach; Bouttefroy A et al.; The influence of pH (5.0-8.2), NaCl concentrations (0-6% w/v), and incubation time (0-24 h) on the inhibitory activity of nisin (0-100 I.U./ml) against Listeria monocytogenes (10(3) cfu/ml) was studied using the Doehlert experimental design and was confirmed by kinetic experiments . Predicted values were in agreement with experimental values . Experiments were carried out at 22 degrees C in reconstituted TSB-YE1 broth with or without NaCl . Nisin had an immediate pH-dependent bactericidal effect, which increased with decreasing pH values . In modified TSB-YE1 broth without NaCl, the bactericidal efficacy of nisin (50 I.U./ml) was maximum at pH 6.6, with no L . monocytogenes survivors until 120 h at 22 degrees C . Nisin (50 I.U./ml) action decreased in the presence of NaCl, with a minimal inhibitory effect between 2 and 4% . This partially protective effect was cancelled at higher levels of nisin. Int J Food Microbiol, 2000 Mar 10, 54(1-2), 91 - 8 Pulsed electric fields inactivation of attached and free-living Escherichia coli and Listeria innocua under several conditions; Dutreux N et al.; The use of pulsed electric fields (PEF) is considered as a mild process in the inactivation of microorganisms present in liquid food products . PEF treatments of Escherichia coli and Listeria innocua suspended in milk and phosphate buffer, with same pH and same conductivities, yielded to similar inactivation . Reduction rates obtained in distilled water indicated that conductivity of the food product is a main parameter in bacterial inactivation . Bacteria attached to polystyrene beads were inactivated by PEF at a greater (E . coli) or equal rate (L . innocua) than free-living bacteria . Base on the use of selective and non-selective enumeration media, no clear indications were obtained for sublethal damage of microorganisms surviving the PEF treatment . E . coli cells subjected to 60 pulses at 41 kV/cm were examined by transmission and scanning electron microscopy . Changes in the cytoplasm were observed and the cell surface appeared rough . The cells outer membranes were partially destroyed allowing leaking of cell cytoplasm. Am J Respir Cell Mol Biol, 2000 Apr, 22(4), 481 - 90 Local activation of nonspecific defense against a respiratory model infection by application of interferon-gamma: comparison between rat alveolar and interstitial lung macrophages; Steinmuller C et al.; Pulmonary macrophages play a crucial role in the defense of inhaled pathogens . We characterized functional properties of alveolar (AM) and interstitial (IM) macrophages from rats . AM exhibited a pronounced microbicidal capacity as shown by an elevated production of reactive oxygen intermediates (ROI), nitric oxide (NO), tumor necrosis factor (TNF)-alpha, and tumor cytotoxicity when compared with IM . In contrast, IM were superior to AM regarding mechanisms mainly involved in the induction and maintenance of specific immune reactions (major histocompatibility complex {MHC} class II expression, interleukin {IL}-1 and IL-6) . In this line, we were interested in whether the microbicidal potential of AM could be augmented by treating Lewis rats with rat recombinant interferon (IFN)-gamma (5 x 10(2) to 1 x 10(5) U/animal) intratracheally, avoiding infection of interstitial lung macrophages or other organ-associated macrophages . The pulmonary cytokine application resulted in an activation of AM when macrophages from IFN-treated animals were compared with control macrophages from saline-treated rats 18 h after the treatment: (1) mediator release (ROI, NO, TNF-alpha, IL-6), (2) tumoricidal activity; (3) dose-dependent increase of MHC class II expression . The local immunomodulation enhanced the resistance of normal and immunosuppressed rats against respiratory infections with Listeria monocytogenes . Taken together, local activation of lung macrophages is a feasible therapeutic strategy against pulmonary infections. Appl Environ Microbiol, 2000 Apr, 66(4), 1706 - 10 Significance of inoculum size in the lag time of Listeria monocytogenes; Augustin JC et al.; The lag time of Listeria monocytogenes growing under suboptimal conditions (low nutrient concentrations, pH 6, and 6.5 degrees C) was extended when the inoculum was severely stressed by starvation and the inoculum size was very small . Predictive microbiology should deal with bacterial stress and stochastic approaches to improve its value for the agro-food industry. Appl Environ Microbiol, 2000 Apr, 66(4), 1405 - 9 Influence of catalase and superoxide dismutase on ozone inactivation of Listeria monocytogenes; Fisher CW et al.; The effects of ozone at 0.25, 0.40, and 1.00 ppm on Listeria monocytogenes were evaluated in distilled water and phosphate-buffered saline . Differences in sensitivity to ozone were found to exist among the six strains examined . Greater cell death was found following exposure at lower temperatures . Early stationary-phase cells were less sensitive to ozone than mid-exponential- and late stationary-phase cells . Ozonation at 1.00 ppm of cabbage inoculated with L . monocytogenes effectively inactivated all cells after 5 min . The abilities of in vivo catalase and superoxide dismutase to protect the cells from ozone were also examined . Three listerial test strains were inactivated rapidly upon exposure to ozone . Both catalase and superoxide dismutase were found to protect listerial cells from ozone attack, with superoxide dismutase being more important than catalase in this protection. Immunol Res, 1999, 20(3), 219 - 35 The interaction of intestinal epithelial cells and intraepithelial lymphocytes in host defense; Yoshikai Y; Intestinal intraepithelial lymphocytes (i-IEL) are located at the basolateral surfaces of intestinal epithelial cells (i-EC) and play important roles in the homeostasis of intestinal microenvironment . i-IEL comprise unique T cell populations including CD4-CD8alphaalpha+ T cells expressing T cell receptor (TCR)alphabeta or TCRgammadelta and CD4+ CD8alphaalpha+ T cells expressing TCR alphabeta . We show here that CD4+ CD8alphaalpha+ i-IEL belongs to Th1 type T cells capable of responding to self-MHC class I on i-EC and that a significant fraction of i-IEL expressed Fas ligand (Fas-L) and induced apoptosis in the i-EC via Fas-dependent pathway . i-IEL may recognize and eliminate the effete i-EC for homeostatic regulation of intestinal epithelia . The interaction of i-EC and i-IEL through E-cadherin/alphaEbeta7 integrin is important for homing and maintenance of i-IEL in intestine . Listeria monocytogenes are also known to interact with E-cadherin on i-EC and invade into the epithelial cells . Invasion of L . monocytogenes into i-EC activated NFkappa-B and subsequently up-regulated the expression of IL-15 gene, which has a NFkappa-B binding site at the promoter region . i-IEL, especially gammadelta T cells, were significantly activated to produce Th1 type cytokines at the early stage after oral infection with L . monocytogenes in mice and rats . The activation of i-IEL coincided with a peak response of IL-15 production by i-EC after infection . Taken together, mutual interaction of i-IEL and i-EC may be important not only for homeostatic regulation but also host defense against microbial infection in intestine. Theriogenology, 1999 Oct 15, 52(6), 1095 - 104 Pregnancy-associated glycoprotein levels in pregnant goats inoculated with Toxoplasma gondii or Listeria monocytogenes: a retrospective study; Zarrouk A et al.; The pregnancy-associated glycoprotein (PAG) concentration profiles of goats that had been experimentally inoculated with either Toxoplasma gondii or Listeria monocytogenes are described . All goats were examined regularly by ultrasonography . In T . gondii-infected females (n = 5), a slow decrease of PAG was observed throughout a period of 55 to 74 d after inoculation . Afterwards, the goats either aborted (n = 4) or kidded 1 dead and 1 weak fetus (n = 1) . In L . monocytogenes-infected females (n = 8), a marked decrease of PAG was observed from the day of inoculation . Abortion occurred within 9 to 11 d post inoculation (n = 7) . Only 1 goat kidded a healthy fetus. Int J Food Microbiol, 1999 Nov 15, 52(3), 163 - 8 Efficiency of four secondary enrichment protocols in differentiation and isolation of Listeria spp . and Listeria monocytogenes from smoked fish processing chains; Duarte G et al.; Four secondary enrichment protocols (conventional methods: UVM II, Fraser 24 h and Fraser 48 h: Impedimetric method: Listeria electrical detection medium) were studied for their ability to isolate Listeria spp . and Listeria monocytogenes from fish and environmental samples collected along the processing chain of cold-smoked fish . From all methods, Listeria spp . and L . monocytogenes were respectively present in 56 and 34 of 315 samples analysed . Fraser broth incubated for 48 h gave the fewest false negative Listeria spp . results {4/56; (7.1%)}, but concurrently only 15/34 (44.1%) samples were correctly identified as containing L . monocytogenes, Listeria electrical detection (LED) medium detected only 36/56 (64.3%) Listeria spp . positive samples . Despite this lower isolation rate, LED identified 20/34 (58.8%) L . monocytogenes positive samples correctly and gave fewer false positive results . The overall conclusion was that more than one isolation method is needed to accurately estimate L . monocytogenes contamination rates. J Vet Diagn Invest, 2000 Mar, 12(2), 173 - 6 Listeria monocytogenes septicemia in a Thoroughbred foal; Wilkins PA et al.; Listeria monocytogenes septicemia was diagnosed in a 6-day-old Thoroughbred foal . Primary clinical signs included fever, depression, diarrhea, and respiratory distress . Hematologic abnormalities included leukopenia, neutropenia, degenerative left shift, and hyperfibrinogenemia . Clinical chemistry and blood gas abnormalities included metabolic acidosis, hypoxemia, hypocapnia, hypoglycemia, and hyponatremia . Despite aggressive therapeutic intervention and intensive care, the foal died within 12 hours of admission . A postmortem examination was performed, and the primary gross lesion was bilaterally severe, focally extensive bronchopneumonia . Histopathology revealed severe subacute multifocal suppurative bronchopneumonia with necrotizing vasculitis and intralesional coccobacilli . Cultures of blood collected at admission and immediately prior to death were positive for L . monocytogenes, as were cultures obtained from lung and liver at necropsy . Immunohistochemical examination of formalin-fixed tissues revealed abundant intra- and extracellular L . monocytogenes antigen within the lung and intravascularly in multiple organs. Int J Food Microbiol, 1999 Feb 2, 46(2), 167 - 71 Behaviour of Listeria spp . in naturally contaminated chorizo (Spanish fermented sausage); Encinas JP et al.; Counts of Listeria spp . were determined during the manufacture and drying of 21 lots of five chorizo varieties produced by three different manufacturers . Presumptive Listeria were not isolated from any of the batches produced in a large factory (F3) using starter, sorbate and controlled ripening at high temperatures . Initial levels in factory 1 (F1), with no starter added, but controlled ripening at low temperatures, were ca 3.5 log10 cfu/g while those in factory 2 (F2), with no starter added and ripening under natural climatic conditions, were 1.17 log10 cfu/g . Numbers of listeriae in batches obtained from F1 remained almost constant before decreasing by ca 0.5 log units/g in the finished product (32 days), while the levels in F2 increased by 1.47 log units/g after 11 days of ripening and declined further to levels above the original amount . Manufacturing procedure and smoking significantly affected presumptive listeriae counts . Thirteen strains recovered from F1 batches were identified as: Listeria monocytogenes (three strains of serovar 1/2c), Listeria innocua (eight strains of serovar 6b) and Listeria welshimeri (two strains of serovar 6b) . Listeria strains from F2 were assigned to L . innocua and L . welshimeri. Int J Food Microbiol, 1999 Feb 2, 46(2), 151 - 7 The effect of homogenization of whole milk, skim milk and milk fat on nisin activity against Listeria innocua; Zapico P et al.; Whole milk, skim milk and an emulsion of milk fat in water, inoculated with approx . 10(5) cfu/ml of Listeria innocua, were treated at 30 degrees C with 100 IU/ml of nisin, homogenization at 200 bar or both procedures . Nisin activity and survival of L . innocua after treatments were determined . Recovery of nisin activity from non-homogenized whole milk treated with 100 IU/ml of nisin was complete, whereas a loss of 18 to 28% of activity was detected in non-homogenized fat-in-water emulsion . Loss in nisin activity due to homogenization represented up to 64% in whole milk and 62% in fat-in-water emulsion . Nisin addition by itself achieved a reduction in L . innocua counts of 3.7-3.8 log units in whole milk and 3.6 log units in fat-in-water emulsion compared to numbers in untreated samples . When nisin-containing whole milk and fat-in-water emulsion were homogenized, L . innocua counts were only reduced by 2.6-2.9 log units and 2.5 log units, respectively, compared to numbers in untreated samples . Homogenization of nisin-containing skim milk resulted in a loss of nisin activity of 20% but achieved a reduction of 3.0 log units in L . innocua counts. Int J Food Microbiol, 1999 Feb 2, 46(2), 135 - 49 Validation of predictive models describing the growth of Listeria monocytogenes; te Giffel MC et al.; In this study, predictions for growth rate of Listeria on food products were evaluated by both general applicable models and specific growth models . Literature values, obtained from a large number of publications, for growth rates in/on a variety of foods were compared by graphical and mathematical analysis with predictions given by various models . Apart for the great advantage of being generally applicable, the general models performed best . However, only small differences between the various models were observed . Model predictions were accurate within a factor of about two to four, depending on the type of product . The predictions should therefore not be considered as absolute; it is important to understand the limitations of the performance of models . All results and all assumptions should be criticised, but in many cases the accuracy will be sufficient to use these types of models as a tool in management decisions. Lett Appl Microbiol, 2000 Jan, 30(1), 23 - 7 Osmoprotectants and cryoprotectants for Listeria monocytogenes; Bayles DO et al.; Listeria monocytogenes is a foodborne pathogen that can grow in high osmotic strength environments and at refrigeration temperatures . Glycine betaine, proline betaine, acetylcarnitine, carnitine, gamma-butyrobetaine and 3-dimethylsulphoniopropionate all acted as osmoprotectants, as evidenced by an increase in growth rate of L . monocytogenes 10403S and Scott A when provided with these compounds, while being stressed in defined medium containing 0.7 M NaCl . These same compounds exhibited cryoprotective activity, as evidenced by increasing the growth rate of L . monocytogenes at 5 degrees C . Ectoine, hydroxy ectoine, pipecolic acid and proline were ineffective as osmoprotectants or cryoprotectants under these conditions . The presence of osmoprotectants and cryoprotectants in foods may provide compounds assisting L . monocytogenes to overcome the barriers of high osmotic strength and low temperature that otherwise control microbial growth. FEMS Immunol Med Microbiol, 2000 Apr, 27(4), 299 - 304 Novel bacterial systems for the delivery of recombinant protein or DNA; Spreng S et al.; On the basis of attenuated intracellular bacteria, we have developed two delivery systems for either heterologous proteins or DNA vaccine vectors . The first system utilizes attenuated strains of Gram-negative bacteria which are engineered to secrete heterologous antigens via the alpha-hemolysin secretion system of Escherichia coli . The second system is based on attenuated suicide strains of Listeria monocytogenes, which are used for the direct delivery of eukaryotic antigen expression vectors into professional antigen presenting cells (APC) like macrophages in vitro as well as in vivo. J Cell Sci, 2000 Apr, 113 ( Pt 8), 1415 - 26 Accumulation of profilin II at the surface of Listeria is concomitant with the onset of motility and correlates with bacterial speed; Geese M et al.; The spatial and temporal activity of the actin cytoskeleton is precisely regulated during cell motility by several microfilament-associated proteins of which profilin plays an essential role . We have analysed the distribution of green fluorescent protein (GFP)-tagged profilins in cultured and in Listeria-infected cells . Among the different GFP-profilin fusion proteins studied, only the construct in which the GFP moiety was fused to the carboxy terminus of profilin II (profilin II-GFP) was recruited by intracellular Listeria . The in vitro ligand-binding properties of this construct, e.g . the binding to monomeric actin, poly-L-proline and phosphatidylinositol 4,5-bisphosphate (PIP2), were unaffected by GFP . Profilin II-GFP co-localised with vinculin and Mena to the focal adhesions in REF-52 fibroblasts and was distributed as a thin line at the front of protruding lamellipodia in B16-F1 mouse melanoma cells . In Listeria-infected cells, profilin II-GFP was recruited, in an asymmetric fashion, to the surface of Listeria at the onset of motility whereas it was not detectable on non-motile bacteria . In contrast to the vasodilator-stimulated phosphoprotein (VASP), profilin II-GFP localised at the bacterial surface only on motile Listeria . Moreover, the fluorescence intensity of profilin II-GFP directly correlated with the speed of the bacteria . Thus, the use of GFP-tagged profilin II provides new insights into the role of profilins in cellular motility. Infect Immun, 2000 Apr, 68(4), 2196 - 204 CD8(+) T-cell priming against a nonsecreted Listeria monocytogenes antigen is independent of the antimicrobial activities of gamma interferon; Tvinnereim AR et al.; Sublethal infection of mice with recombinant Listeria monocytogenes expressing a model epitope in either secreted or nonsecreted form results in similar CD8(+) T-cell priming . Since nonsecreted bacterial proteins have no obvious access to the endogenous major histocompatibility complex (MHC) class I presentation pathway, presentation of these antigens requires destruction of the bacterium to reveal the nonsecreted molecules to an exogenous MHC class I presentation pathway . Gamma interferon (IFN-gamma), a cytokine made by multiple cell types in response to L . monocytogenes infection, could be required for exogenous presentation of nonsecreted bacterial antigens via its capacity to upregulate the expression of molecules involved in antigen presentation, its capacity to activate macrophages to kill bacteria to expose nonsecreted molecules or both . IFN-gamma knockout (KO) mice were used to address the requirement for IFN-gamma in CD8(+) T-cell priming against (i) a model exogenous antigen and (ii) secreted and nonsecreted L . monocytogenes antigens . We demonstrate that IFN-gamma KO mice are capable of cross-presenting the model exogenous antigen ovalbumin to prime CD8(+) T-cell responses that are only slightly weaker than that in wild-type (WT) mice . Despite their extreme susceptibility to primary L . monocytogenes infection, previously immunized and naive IFN-gamma KO mice were able to generate CD8(+) T-cell responses against both secreted and nonsecreted L . monocytogenes antigens which were similar to responses of WT mice . Interestingly, IFN-gamma KO mice were as capable as WT mice in mediating the characteristic drop in bacterial load in the liver at 4 h postinfection, although the IFN-gamma KO mice have exacerbated bacterial loads as early as 24 h postinfection . These results demonstrate that the regulatory functions of IFN-gamma are not required for priming of CD8(+) T cells by cross-presentation of a model exogenous antigen or in response to a nonsecreted L . monocytogenes antigen . In addition, the capacity of IFN-gamma to activate the microbicidal activities of macrophages is not required for the very early innate immune response to L . monocytogenes or priming of CD8(+) T cells against a nonsecreted bacterial antigen. Infect Immun, 2000 Apr, 68(4), 1988 - 96 Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon; Dreisbach VC et al.; Previous results have demonstrated that nonspecific protective immunity against lethal Francisella tularensis live vaccine strain (LVS) or Listeria monocytogenes infection can be stimulated either by sublethal infection with bacteria or by treatment with bacterial DNA given 3 days before lethal challenge . Here we characterize the ability of purified lipopolysaccharide (LPS) from F . tularensis LVS to stimulate similar early protective immunity . Treatment of mice with surprisingly small amounts of LVS LPS resulted in very strong and long-lived protection against lethal LVS challenge within 2 to 3 days . Despite this strong protective response, LPS purified from F . tularensis LVS did not activate murine B cells for proliferation or polyclonal immunoglobulin secretion, nor did it activate murine splenocytes for secretion of interleukin-4 (IL-4), IL-6, IL-12, or gamma interferon (IFN-gamma) . Immunization of mice with purified LVS LPS induced a weak specific anti-LPS immunoglobulin M (IgM) response and very little IgG; however, infection of mice with LVS bacteria resulted in vigorous IgM and IgG, particularly IgG2a, anti-LPS antibody responses . Studies using various immunodeficient mouse strains, including LPS-hyporesponsive C3H/HeJ mice, muMT(-) (B-cell-deficient) knockout mice, and IFN-gamma-deficient mice, demonstrated that the mechanism of protection does not involve recognition through the Lps(n) gene product; nonetheless, protection was dependent on B cells as well as IFN-gamma. J Food Prot, 2000 Mar, 63(3), 354 - 63 Twenty-four-hour direct presumptive enumeration of Listeria monocytogenes in food and environmental samples using the ISO-GRID method with LM-137 agar; Entis P et al.; A new culture medium, LM-137 agar, was developed for use with the ISO-GRID hydrophobic grid membrane filter system for direct presumptive enumeration of Listeria monocytogenes in 24 h . The method was validated against three-replicate, three-dilution most probable number procedures based on enrichment methods specified by the U.S . Department of Agriculture, the Association of Official Analytical Chemists International and the U.S . Food and Drug Administration . The study encompassed meats, dairy products, egg, produce, seafood, and environmental samples . The ISO-GRID filter method produced significantly higher recovery of L . monocytogenes from fermented sausage, hot dogs, pasteurized and raw milk, raw shrimp, and environmental swab samples (P < 0.05) . The reference methods yielded significantly higher counts from frozen raw pork and cole slaw (P < 0.05) . Confirmation rates of presumptive positive isolates from the filter method ranged from a low of 92% (frozen raw pork) to 100% (most other products) . Neither the recovery efficiency nor the confirmation rate were affected by the presence of competing aerobic flora. J Food Prot, 2000 Mar, 63(3), 347 - 53 Development and evaluation of a 24-hour method for the detection and quantification of Listeria monocytogenes in meat products; Carroll SA et al.; A 24-h filter monitor-based test, Listeria-SELeCT, has been developed to quantify Listeria monocytogenes organisms in meat samples with a sensitivity of < or = 1.0 CFU/g . The technique comprises a filter monitor-based system and a colony lift immunoassay to identify and enumerate the target organism . Meat homogenates were centrifuged and the eluate was filtered to trap and immobilize the microorganisms on the filter . Fraser broth was then added to the filter apparatus to allow the organisms to become established overnight and to inhibit contaminants, after which the filters were transferred onto Modified Oxford medium agar, a selective medium for L . monocytogenes . After 10 to 12 h, a colony lift immunoassay was used to confirm and enumerate suspect colonies on the filter . A correlation study between the Listeria-SELeCT method and the most probable number technique showed the Listeria-SELeCT to be considerably more accurate than the most probable number for quantitatively determining the number of viable organisms in meat samples . Because of ease and speed of testing, the Listeria-SELeCT system also provided major advantages over the most probable number technology. J Food Prot, 2000 Mar, 63(3), 343 - 6 Application of the BAX for screening/genus Listeria polymerase chain reaction system for monitoring Listeria species in cold-smoked fish and in the smoked fish processing environment; Norton DM et al.; The cold-smoked fish industry was used as a model for the development of a system for monitoring Listeria spp . in foods and in the food processing environment . A total of 214 samples including raw fish, fish during the cold-smoking process, finished product, and environmental samples were collected from three processing facilities over two visits to each facility . Samples were screened for Listeria spp . using the BAX for Screening/genus Listeria polymerase chain reaction system (PCR) and by culture . Listeria spp., confirmed by the API Listeria test strip or by a PCR assay targeting the L . monocytogenes hlyA gene, were isolated from a total of 89 (41.6%) samples . Of these, 80 samples also tested positive for Listeria spp . using the BAX system . Specifically, 42 (55.3%) environmental samples (n = 76), 11 (25.6%) raw materials samples (n = 43), 20 (35.1%) samples from fish in various stages of processing (n = 57), and 7 (18.4%) finished product samples (n = 38) tested positive for Listeria spp . using the BAX system . Five (4.0%) of the 125 culture-negative samples yielded BAX system-positive results . Listeria isolates from each of nine culture-positive/BAX system-negative samples yielded a positive reaction when tested in pure culture by the BAX system, suggesting that our false-negative results were likely due to the presence of low Listeria numbers in the initial enrichment as opposed to nonreacting isolates . The employment of alternative enrichment protocols, such as the two-step enrichment recommended by the manufacturer, may increase the sensitivity of the assay. J Food Prot, 2000 Mar, 63(3), 337 - 42 Rapid polymerase chain reaction/DNA probe membrane-based assay for the detection of Listeria and Listeria monocytogenes in food; O'Connor L et al.; We describe the development of polymerase chain reaction (PCR)/DNA probe membrane-based colorimetric assays for the detection and identification of Listeria and L . monocytogenes . PCR primers designed from the 16S to 23S rRNA intergenic spacer region amplified products that were reverse hybridized to membrane-bound oligonucleotide probes specific for Listeria and L . monocytogenes with a detection limit of 1 to 10 CFU/25 ml in inoculated raw and pasteurized milk samples . These qualitative assays have the potential to be integrated into testing laboratories for monitoring the microbiological quality of foods. J Food Prot, 2000 Mar, 63(3), 332 - 6 Single-strand conformation polymorphisms in the hly gene and polymerase chain reaction analysis of a repeat region in the iap gene to identify and type Listeria monocytogenes; Wagner M et al.; Two novel methods that allow the powerful identification of Listeria monocytogenes by polymerase chain reaction (PCR) and simultaneous differentiation by special electrophoresis formats are described . The first method involves a PCR-driven single-strand conformation polymorphism (SSCP-PCR) assay using a portion of the noncoding region of the hlv gene . The assay was evaluated with 120 genetically distinct L . monocytogenes strains of either foodborne or clinical origin . Distribution of listerial strains to at least 14 SSCP types was observed . In respect to the panel of strains, 39.7% were assigned to SSCP type 3, and 19% showed SSCP type 5 . Further, SSCP type 1 was found in 7.5% of all strains, SSCP type 10 in 6.7%, and 5.8% each for SSCP types 6 and 7 . The SSCP types 4, 9, and 11 were infrequently described in 2.55%, 3.3%, and 4.2%, respectively, of all isolates . At least 0.85% represented each of the SSCP types 2, 13, and 14, and 1.7% displayed SSCP types 8 and 12 . In the second method, the internal threonine-asparagine repeat portion of the L . monocytogenes p60 protein was used for setting up a PCR-based identification and parallel differentiation assay . Ten different repeat types (RTs), according to different sizes of PCR products, were observed . Of 163 strains tested, 35.58% of samples were assigned to RT 1, 39.26% to RT 2, 3.68% to RT 3, 6.13% to RT 4, 4.29% to RT 5, 2.45% to RT 6, 5.52% to RT 7, 0.61% to RT 8, 0.61% to RT 9, and 1.83% to RT 10 . The data suggest that both methods allow the simple identification and differentiation of L . monocytogenes isolates . Therefore, both the SSCP-PCR and the PCR-based identification and parallel differentiation assay could represent single-strand pretyping assays before laborious reference typing methods are applied. Cell Motil Cytoskeleton . 2000 Mar;45(3):247. Erratum: volume 45, number 1, january 2000 {Changes in the virulence factor expression level in Listeria monocytogenes under various environmental conditions} Ermolaeva SA, Belyi IuF, Tartakovskii IS. Effects of chelators Chelex-100 and activated charcoal on the production of proteins responsible for virulence of Listeria monocytogenes, facultative intracellular parasite were studied . Bivalent cation chelator Chelex-100 stimulates the production of only thiol-dependent hemolysin listeriolysin O . The presence of activated charcoal, a nonspecific chelator, in culture medium stimulated the expression of listeriolysin O and other main virulence factors by increasing the level of their transcription. Cell Stress Chaperones, 2000 Jan, 5(1), 21 - 9 Cloning, sequencing, and transcriptional analysis of the dnaK heat shock operon of Listeria monocytogenes; Hanawa T et al.; The complete dnaK operon of Listeria monocytogenes was isolated by chromosome walking using the previously cloned dnaK gene as a probe . Molecular analysis of the locus identified 6 genes in the order hrcA, grpE, dnaK, dnaJ, orf35, and orf29 . Primer extension analysis revealed 3 transcription start sites-S1, S2, and S3-upstream of the hrcA, grpE, and dnaJ, respectively . The transcription from S1 was heat inducible . Analysis of the sequences revealed the consensus promoter sequences of gram-positive bacteria, P1 and P2 upstream of the hrcA and dnaJ, respectively . The hrcA gene and a regulatory sequence, designated CIRCE (controlling inverted repeat of chaperone expression), play a role in the regulation of expression of the dnaK locus in response to heat shock in several gram-positive bacteria . Their presence upstream of the dnaK locus in L . monocytogenes suggested a similar regulatory mechanism for the transcription initiated at the promoter, P1 . Northern blot analysis led to the detection of 4 mRNA species of 4.9 kb, 3.6 kb, 3.6 kb, and 1.2 kb; the first 2 species were heat inducible . The current results indicate that 4 distinct transcripts directed by 3 promoters are involved in the expression of the dnaK operon of L . monocytogenes. J Microbiol Methods, 2000 Apr, 40(2), 147 - 51 Evaluation of mini-VIDAS rapid test for detection of Listeria monocytogenes from production lines of fresh to cold-smoked fish; Vaz-Velho M et al.; This study was conducted to evaluate the efficacy of the mini-VIDAS Listeria monocytogenes (LMO) system (BioMerieux Vitek, Inc., Missouri, USA) for detection of L . monocytogenes in environmental and fish samples from three Portuguese cold-smoking plants and from their fresh fish suppliers . Mini-VIDAS-LMO is a fully automated system that uses fluorescent ELFA (Enzyme Linked Fluorescent Assay) technology for detection of Listeria monocytogenes antigens in food . It can be a rapid screening method alternative to time consuming classical isolation and identification . Two hundred and ninety five samples were tested in mini-VIDAS-LMO and in parallel by the ISO 11290-1 traditional protocol . The mini-VIDAS-LMO detected 8 of the 11 confirmed positive samples and presented 11 false positive results . The specificity of the mini-VIDAS-LMO found in this experiment was 0.96 and the sensitivity 0.73. Adv Drug Deliv Rev, 2000 Mar 30, 41(2), 209 - 21 Bacterial pore-forming hemolysins and their use in the cytosolic delivery of macromolecules; Provoda CJ et al.; Advances in our understanding of fundamental cell biological processes have facilitated an expansion of therapeutic approaches to altering cellular physiology and phenotype . As many of these methods involve macromolecular agents that act on targets within the nucleus or cytoplasm, achieving their full potential ultimately requires the efficient delivery of these agents across the cell membrane barrier into the cytosol . Various strategies have been employed to enhance cytosolic delivery . These include either directly penetrating the plasma membrane, or avoiding degradation within the hydrolytic environment of the endosomal/lysosomal pathway after endocytic uptake . Some of the more promising methods in this regard have exploited the mechanisms utilized by certain viruses and bacteria for escaping into their host cell's cytosol . In this review, we will discuss some of these methods with an emphasis on the use of pore-forming proteins from bacteria . Particular attention will be drawn to the pH-sensitive endosomolytic bacterial hemolysins, such as listeriolysin O, and the potentiol for their use in cytosolic drug delivery systems. J Food Prot, 2000 Feb, 63(2), 186 - 9 Incidence and characterization of Listeria monocytogenes from domestic and imported foods in Korea; Baek SY et al.; A total of 1,537 domestic and imported food products were examined for the incidence of Listeria monocytogenes between 1993 and 1997 in Korea . L . monocytogenes was detected using the U.S . Department of Agriculture isolation method . Isolated L . monocytogenes was confirmed by polymerase chain reaction with hly1 and hly2 primers designed from the listeriolysin O . Overall, 122 samples (7.9%) contained L . monocytogenes . The rate of isolation was 4.3% for beef, 19.1% for pork, 30.2% for chicken, 1.2% for shellfish, 4.4% for raw milk, 4.4% for frozen smoked mussels, and 6.1% for ice cream . No L . monocytogenes was found in pasteurized milk, pasteurized processed cheese, saltwater fish, dried seafoods, or ham . The overall incidence was lower than that reported in previous studies from other countries . Most isolates were serotype 1/2b except for chicken, in which serotype 1/2a was predominant . The serotyping results might imply the presence of food or geography-specific L . monocytogenes strains. J Food Prot, 2000 Feb, 63(2), 179 - 85 Ribotype analysis of strain distribution in Listeria monocytogenes; Gendel SM et al.; Changes in the temporal and spatial patterns of strain distribution for the foodborne pathogen Listeria monocytogenes were studied by ribotyping using the Qualicon Riboprinter system . Ribotype patterns were obtained by using the restriction enzymes EcoRI and PvuII for 72 isolates of L . monocytogenes recovered from smoked salmon samples over a period of 3 years . Each pattern was classified both by comparison to a pattern library and by comparison among the 72 isolate patterns . Eleven EcoRI-based ribogroups and 16 PvuII groups were identified . Eight of the 11 EcoRI ribogroups were found in isolates obtained over a period of >12 months, and 75% of the EcoRI ribogroups that were found in more than one food sample were distributed nationally . Within the set of isolates, there were 26 instances where more than one isolate was obtained from a single food sample . In 35% of these instances, the co-isolates produced different ribotype patterns, indicating that multiple strains of L . monocytogenes commonly coexist in the same environment . Overall, these data indicate that the population of L . monocytogenes consists of a number of widely dispersed strains with little geographic or temporal stratification. Stomatologiia (Mosk), 2000, 79(1), 31 - 5 {The clinico-microbiological evaluation of the efficacy of using new drug forms of chlorhexidine--Corsodyl and Eludril--for the prevention of infectious complications in operations for endosseous implantation}; Ivanov SIu et al.; Prevention of infectious inflammatory complications of intraosseous implantation is a pressing problem . Chlorohexidine dosage forms Corsodyl and Eludril were used for this purpose . In the control group, furacillin was used . Microbiological studies showed that instillations of Corsodyl and Eludril solutions during the postoperative period did not modify normal oral microflora and led to disappearance of the most aggressive anaerobic bacteria in 1-3 days . These drugs are more effective that Listerine, and they are recommended for prevention of infectious inflammatory complications after intraosseous implantation. J AOAC Int, 2000 Jan-Feb, 83(1), 86 - 8 Rapid determination of Listeria monocytogenes by automated enzyme-linked immunoassay and nonradioactive DNA probe; Kerdahi KF et al.; A rapid and reliable analytical method was developed to detect and confirm the presence of Listeria monocytogenes in raw and partially processed foods . Forty-nine food samples (25 mixed cut vegetable salad, 12 smoked salmon, and 12 sterile smoked salmon) were individually inoculated with high levels {10-100 colony forming units (cfu)/25 g sample} and low levels (1-10 cfu/25 g sample) of L . monocytogenes, and were screened using the Vitek Immuno Diagnostic Assay (VIDAS) Listeria monocytogenes (VIDAS LMO)} . Positive test results were confirmed as L . monocytogenes by nonradioactive DNA probe . All samples inoculated with high levels of L . monocytogenes were detected by VIDAS and 96% were confirmed as L . monocytogenes by DNA probe . VIDAS LMO detected 89% of samples inoculated with low levels of L . monocytogenes, and 87% of these were confirmed as positive by DNA probe . In addition, 12 other samples (4 from each of mixed cut vegetable salad, smoked salmon, and sterile smoked salmon) were inoculated with high levels of L . ivanovii, L . seeligeri, L . welshimeri, L . innocua, L . grayi, and L . murrayi . Samples were assayed by the same protocol and all gave negative results . Compared with the cultural method, the VIDAS LMO nonradioactive DNA probe combination is highly specific, discriminates between L . monocytogenes and all other Listeria species, and reduces analytical time. Biophys J, 2000 Mar, 78(3), 1643 - 54 Growing an actin gel on spherical surfaces; Noireaux V et al.; Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement . On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced . We show experimentally that the stationary thickness of the gel depends on the radius of the beads . Moreover, the actin gel is not formed if the ActA surface density is too low . To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel . Our model also takes into account treadmilling of actin . We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN . Finally, we estimate from our theoretical model possible conditions for developing actin comet tails. Med Microbiol Immunol (Berl), 1999 Aug, 188(1), 15 - 21 Rat dorsal root ganglia neurons as a model for Listeria monocytogenes infections in culture; Dons L et al.; Neurotropism of Listeria monocytogenes was studied in rat dorsal root ganglia (DRG) and hippocampal neurons in culture . Using a system in which the DRG neurons can grow relatively free from other cells, it was observed that such DRG neurons, in contrast to hippocampal neurons, can be effectively infected by L . monocytogenes . The bacteria aligned along DRG axons, but not along hippocampal neurites . A mutant deficient in internalin, a protein required for entry into E-cadherin-expressing cells, did not interact with DRG neurons . Axonal migration of bacteria was studied in the DRG neurons grown in a double-chamber system, where either the neurites or the nerve cell bodies were exposed to the bacteria . The data suggest that L . monocytogenes can infect both axons and DRG nerve cell bodies, and that the bacteria can migrate in a retrograde as well as anterograde direction . These results support the notion that L . monocytogenes can spread via primary sensory neurons to the central nervous system . Infection of DRG primary sensory neurons, as employed in the present study, provides a model for analysis of bacterial and neuronal factors of importance for neurovirulence of L . monocytogenes. Hepatogastroenterology, 2000 Jan-Feb, 47(31), 29 - 43 Etiology and pathophysiology of inflammatory bowel disease--environmental factors; Andus T et al.; Environmental factors play an important role in the pathophysiology of inflammatory bowel disease . There is a strong and consistent association between smoking and Crohn's disease, and between nonsmoking and ulcerative colitis . Despite extensive research, the exact pathophysiological mechanisms for these associations remain unclear . In spite of this, some clinical trials with nicotine-patches showed beneficial effects for the treatment of ulcerative colitis . Associations of Crohn's disease and ulcerative colitis with other environmental factors are weaker like the association with use of oral contraceptives or those less well investigated such as the association with childhood hygiene . Most studies suggesting a potential pathogenetic role of Mycobacterium paratuberculosis or an effect of tuberculostatic therapy in Crohn's disease could not be reproduced by others . Perinatal or childhood infections by viruses like measles are heavily debated, but not proven to be causal for inflammatory bowel disease . Coagulation disorders have been described as protecting from inflammatory bowel disease, suggesting hypercoagulability to be a pathogenetic factor . Some studies described that appendectomy may prevent the onset of ulcerative colitis in man and mice . Other environmental factors such as hydrogen sulfide, tonsillectomy, diet, blood transfusions, and Listeria also require confirmation . There are, however, convincing data from genetic animal models and twin studies that environmental factors as the intestinal bacterial flora interact with susceptible hosts to cause inflammatory bowel disease . Inflammatory bowel diseases have multifactorial etiologies, which require a differentiated approach for treatment and prevention. Rinsho Shinkeigaku, 1999 Nov, 39(11), 1164 - 7 {Direct Gram staining of blood culture sample enabled the early diagnosis of brain abscess due to Listeria monocytogenes}; Takano A et al.; A 58 year old woman had a long history of immunocompromised state . Since age 28 she had multiple endocrine neoplasm type 2A: her thyroid gland and bilateral adrenal glands were removed because of pheochromocytoma and thyroid medullary carcinoma . Corticosteroid and levothyroxine were supplemented . At age 57 she was afflicted with systemic lupus erythematodes and nephrotic syndrome . Prednisolone therapy was started . Two months later she developed fever, lethergy, headache and left hemiparesis . MRI revealed multiple ring-enhancing lesions in the right cerebrum . CSF was negative for microorganisms . Blood culture hemolysed after 24 hours . Direct gram staining of the blood culture sample revealed gram-positive short rods without spore, suggested listeria . This enabled prompt initiation of high dose penicillin therapy before the official report of listseria infection . Neurological abnormality including left hemiparesis disappeared completely within one month . Enhancement of abscess wall decreased every month, but it persisted for five months despite continuous intravenous penicillin therapy . Listeria monocytogenes is well-recognized as an opportunistic pathogen . It requires prolonged therapy with antibiotics, since it is the intracellular organism . Monitoring of the brain abscess wall by the enhanced MRI is useful to determine the completion of therapy . Since listerial contamination is common among raw meat and unpasteurized milk, immunocompromised patients should be alarmed not to eat uncooked food products. Infect Immun, 2000 Mar, 68(3), 1498 - 506 Listeria monocytogenes as a short-lived delivery system for the induction of type 1 cell-mediated immunity against the p36/LACK antigen of Leishmania major; Soussi N et al.; Listeria monocytogenes has been used as an experimental live vector for the induction of CD8-mediated immune responses in various viral and tumoral experimental models . Susceptibility of BALB/c mice to Leishmania major infection has been correlated to the preferential development of Th2 CD4 T cells through an early production of interleukin 4 (IL-4) by a restricted population of CD4 T cells which react to a single parasite antigen, LACK (stands for Leishmania homologue of receptors for activated C kinase) . Experimental vaccination with LACK can redirect the differentiation of CD4(+) T cells towards the Th1 pathway if LACK is coadministrated with IL-12 . As IL-12 is known to be induced by L . monocytogenes, we have tested the ability of a recombinant attenuated actA mutant L . monocytogenes strain expressing LACK to induce the development of LACK-specific Th1 cells in both B10.D2 and BALB/c mice, which are resistant and susceptible to L . major, respectively . After a single injection of LACK-expressing L . monocytogenes, IL-12/p40 transcripts showed a rapid burst, and peaks of gamma interferon (IFN-gamma)-secreting LACK-specific Th1 cells were detected around day 5 in the spleens and livers of mice of both strains . These primed IFN-gamma-secreting LACK-reactive T cells were not detected ex vivo after day 7 of immunization but could be recruited and detected 15 days later in the draining lymph node after an L . major footpad challenge . Although immunization of BALB/c mice with LACK-expressing L . monocytogenes did not change the course of the infection with L . major, immunized B10.D2 mice exhibited significantly smaller lesions than nonimmunized controls . Thus, our results demonstrate that, in addition of its recognized use for the induction of effector CD8 T cells, L . monocytogenes can also be used as a live recombinant vector to favor the development of potentially protective IFN-gamma-secreting Th1 CD4 T lymphocytes. Curr Opin Microbiol, 2000 Feb, 3(1), 49 - 53 Bacterial replication in the host cell cytosol; Goebel W et al.; Intracellular bacteria in mammalian host cells can either live in a membrane-bound vacuole modified to support bacterial growth, or escape from the primary phagosome into the host cell cytoplasm . Phagosomal escape is best studied in Listeria monocytogenes in which a pore-forming cytolysin and two phospholipases are involved in the lysis of the phagosomal membrane . The mechanisms of and requirements for cytoplasmic growth are less clear but there is growing evidence that proficient replication of bacteria in the cytoplasmic compartment requires specific bacterial and cellular preconditions. Curr Opin Cell Biol, 2000 Feb, 12(1), 97 - 103 Control of actin assembly and disassembly at filament ends; Cooper JA et al.; The most important discovery in the field is that the Arp2/3 complex nucleates assembly of actin filaments with free barbed ends . Arp2/3 also binds the sides of actin filaments to create a branched network . Arp2/3's nucleation activity is stimulated by WASP family proteins, some of which mediate signaling from small G-proteins . Listeria movement caused by actin polymerization can be reconstituted in vitro using purified proteins: Arp2/3 complex, capping protein, actin depolymerizing factor/cofilin, and actin . actin depolymerizing factor/cofilin increases the rate at which actin subunits leave pointed ends, and capping protein caps barbed ends. Am J Respir Crit Care Med, 2000 Feb, 161(2 Pt 1), 535 - 42 Accumulation of macrophages with dendritic cell characteristics in the pulmonary response to Listeria; Kradin RL et al.; Pulmonary immunity reflects a balance between proinflammatory and immunosuppressive factors in the lung . To determine the immune activities of exudate macrophages in the pulmonary immune response, Lewis rats were injected intratracheally with heat-killed Listeria (HKL), labeled ex vivo with the lipophilic dye PKH-26 . At 24 h, macrophages from bronchoalveolar lavage fluid were purified on the basis of their surface membrane expression of RMA, a macrophage-specific antigen, which is brightly expressed by resident alveolar macrophages but dimly expressed by monocytes . Pulmonary macrophages were analyzed for uptake of PKH-26-HKL, and RMA(bright/dim) macrophages sorted by FACS were compared for cytokine expression, nitric oxide (NO) release, and APC activities . RMA(bright) macrophages were OX-62(-), B7(-), and factor XIIIa(-); they were the dominant mediators of phagocytosis when low doses of HKL were administered intratracheally but did not support the proliferation of T lymphocytes . RMA(dim) exudate macrophages were OX-62(+), B7(+), and factor XIIIa(+) . They expressed more IL-1 and TNF, but less nitric oxide, than did RMA(bright) macrophages; they were excellent APCs for T cell responses . We conclude that a subset of RMA(dim) exudate macrophages shows phenotypic and functional evidence of dendritic cell differentiation. Rev Inst Med Trop Sao Paulo, 1999 Nov-Dec, 41(6), 375 - 7 Listeria monocytogenes in renal transplant recipients; Hofer CB et al.; Five cases of Listeria monocytogenes bacteremia were observed from April to December 1985, among renal transplant recipients from the same hospital in Sao Paulo, Brazil . The patients were adults (mean age: 40.6 years), and the basic complaint was fever, with no report of meningeal syndrome . Laboratory tests revealed the presence of two serovars, (1/2)a and 4b, which were classified into three lysotypes . The four strains of serovar 4b showed the same antibiotype, with resistance to cefoxitin, clindamycin, oxacillin and penicillin. J Infect Dis, 2000 Feb, 181(2), 754 - 6 Recombinant human interleukin-11 has anti-inflammatory actions yet does not exacerbate systemic Listeria infection; Opal SM et al.; To determine whether recombinant human (rh) interleukin (IL)-11 disrupts the clearance of microbial pathogens, mice were challenged with Listeria monocytogenes after receiving high-dose rhIL-11, anti-tumor necrosis factor (TNF) monoclonal antibody (MAb), anti-IL-11 MAb, or saline control . The LD50 was not affected by rhIL-11 but was 10-fold lower in the anti-TNF MAb group (P<.001) . Plasma IL-6, IL-1beta, and TNF-alpha levels were not different between rhIL-11-treated animals and the control group; however, interferon-gamma levels were significantly reduced by IL-11 treatment (2477 vs . 0 pg/mL, P<.01) . Compared with the control group, the quantitative level of L . monocytogenes in hepatic and splenic tissue was unchanged by rhIL-11 but was significantly increased by TNF or IL-11 inhibition . The results indicate that IL-11 down-regulates cytokine production but does not exacerbate systemic infection in the murine Listeria infection model. Lett Appl Microbiol, 1999 Dec, 29(6), 416 - 20 Rapid detection of Listeria monocytogenes by PCR-ELISA; Scheu P et al.; A rapid detection system specific for Listeria monocytogenes based upon the polymerase chain reaction was developed . The specificity of the primers and the probe annealing to the coding region of the mpl gene proved positive with the DNA from a total of 103 L . monocytogenes strains, while DNA from another 73 Listeria and non-Listeria strains tested negative . To facilitate detection with large numbers of samples, a microtitre plate assay was established with biotinylated probes . Use of a standard DNA prevented false-negative results when used as an internal amplification control in the PCR-ELISA . As the described method required approximately 5-6 h to be completed it may prove useful in the detection of L . monocytogenes in food. Lett Appl Microbiol, 1999 Nov, 29(5), 350 - 3 Simple method to observe the adaptive response of Listeria monocytogenes in food; Bolton LF et al.; A simple, novel method for determining stress-adaptive response of Listeria monocytogenes in food systems is presented . The method involves plating samples on Listeria-selective agar (LSA) acidified to pH 5.25 with incubation at 36 degrees C for 60 h to detect acid adaptation and plating on LSA with 70 gl-1 NaCl and incubation at 7 degrees C for 7 d to detect cold-osmotic adaptation . Adapted cells produced larger colonies (> 1 mm) under these conditions than unadapted cells . Scot A (97%) and Brie-1 (100%) cells incubated in milk at pH 5 for 3 h manifested the acid-adapted colony type compared with 6% and 21% of viable cells in the unstressed control population . After a 5-d adaptation period at 4 degrees C in milk with 80 gl-1 salt, 29% of Scot A and 91% of Brie-1 viable cells exhibited the adapted colony type compared with < 1% of the unstressed control population . Stress-adapted L . monocytogenes were isolated from soft cheese held for 42 d at 10 C. J Appl Microbiol, 1999 Dec, 87(6), 915 - 22 Physical and metabolic causes of sub-lethal damage in Listeria monocytogenes after long-term chilled storage at 4 degrees C; Dykes GA; Cells of Listeria monocytogenes display sub-lethal injury when subjected to long-term chill-storage in a nutrient-poor environment . The physical and metabolic causes of sub-lethal injury to two meat (L61 and L62) and two clinical (L98 and L99) L . monocytogenes strains chill-stored (4 degrees C) for 4 weeks in phosphate-buffered saline at pH 7.0 and pH 5.5, and pH 5.5 in the presence of 0.3% potassium sorbate, were characterized . Causes of sub-lethal injury were determined by examining changes in the cell structure, leakage of nucleic acids and proteins from the cells, and cell recovery from injury in the presence of the metabolic inhibitors rifampicin, D-cycloserine, carbonyl cyanide m-chlorophenylhydrazone and chloramphenicol . Visible shrinkage of the cytoplasm and slight cell wall damage were apparent over the 4 week storage period by electron microscopy for all four strains and three storage conditions . By contrast, over the same storage period, only three of the strains (L62, L98 and L99) displayed slight leakage of cellular content in all three storage media, while one strain (L61) displayed greater leakage . The three strains also displayed similar storage media-dependent metabolic damage . For these strains, phosphate-buffered saline pH 5.5 caused the least damage and potassium sorbate, the most . Recovery experiments also indicated that at pH 5.5, the energy transduction system of these three strains remained undamaged, and that injury to the cell transcription machinery was greatest at pH 7.0 . The fourth strain, L61, displayed less damage than the others but this was attributed to the death of the injured cell sub-population in this strain . In this study, damage to sub-lethally injured chill-stored L . monocytogenes was different from that caused by other agents, such as heat . Therefore, cells injured by chill-storage under starvation conditions may require novel protocols to assure their effective recovery. Mol Gen Genet, 2000 Jan, 262(6), 1060 - 9 Expression and use of the green fluorescent protein as a reporter system in Legionella pneumophila; Kohler R et al.; The gene encoding the green fluorescent protein (GFP) was used as a reporter gene in Legionella pneumophila . To analyze GFP expression in Legionella, transcriptional fusions of gfp with the Legionella-specific mip (Macrophage Infectivity Potentiator) promoter (P(mip)) and the sod (SuperOxide Dismutase) promoter (P(sod)) derived from Listeria monocytogenes were constructed . Following transformation into the virulent L . pneumophila strain JR 32, strong GFP-mediated fluorescence was detected with both plasmids, although the sod promoter was associated with a 1ten-fold higher intensity . No fluorescence was observed in L . pneumophila transformed with the promoterless gfp gene . Comparison of fluorescence yields between various L . pneumophila strains that differ in their virulence characteristics and were transformed with the P(mip)-gfp carrying plasmid revealed no differences in GFP expression . Infection studies using Acanthamoeba castellanii as host and recombinant L . pneumophila strains carrying the P(mip)-gfp and P(sod)-gfp fusions indicated that the mip promoter was expressed when the bacteria replicated intracellularly . GFP expression was also used to monitor, in infected A . castellanii cells, the intracellular survival of, and incidence of host-cell killing by . L . pneumophila strains that vary in their virulence properties . As quantified by flow cytometry the highly virulent L . pneumophila strain Corby was twice as infectious to A . castellanii as the Philadelphia strain JR 32 . Using the avirulent Philadelphia derivative 25D invasion but no intracellular multiplication was observed . In addition, we examined by flow cytometry the influence of cytochalasin D, cycloheximide, and methylamine on the uptake of Legionella by A . castellanii . In conclusion, gfp appears to be a convenient reporter gene whose expression in Legionella can be followed in real time and allows analysis of promoter activities in Legionella and monitoring of the infection process. Science, 2000 Feb 4, 287(5454), 860 - 4 Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity; Ashkar S et al.; Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction . Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection {herpes simplex virus-type 1 (KOS strain)} and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas . Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased . A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression . These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression. Biochim Biophys Acta, 2000 Feb 2, 1495(2), 140 - 9 Reconstitution of Listeria motility: implications for the mechanism of force transduction; Olbris DJ et al.; Listeria monocytogenes and some other infectious bacteria polymerize their host cell's actin into tails that propel the bacteria through the cytoplasm . Here we show that reconstitution of this behavior in simpler media resolves two aspects of the mechanism of force transduction . First, since dilute reconstitution media have no cytoskeleton, we consider what keeps the tail from being pushed backward rather than the bacterium being propelled forward . The dependence of the partitioning of motion on the friction coefficient of the tail is derived . Consistent with experiments, we find that the resistance of the tail to motion is sensitive to its length . That even small tails are stationary in intact cells is attributed to anchoring to the cytoskeleton . Second, the comparatively low viscosity of some reconstitution media magnifies the effects of diffusion, such that a large gap will develop between the bacterium and its tail if they are unattached . At the viscosities of diluted platelet extracts, steady-state gaps of several bacterium lengths are predicted . Since such gaps are not observed, we conclude that Listeria must be attached to their tails . We consider what purposes such attachments might serve under physiological conditions . The implications for related pathogens and amoeboid locomotion are also discussed. Appl Environ Microbiol, 2000 Feb, 66(2), 860 - 3 Effect of flagella on initial attachment of Listeria monocytogenes to stainless steel; Vatanyoopaisarn S et al.; At 22 degrees C a flagellin mutant of Listeria monocytogenes was found to attach to stainless steel at levels 10-fold lower than wild-type cells, even under conditions preventing active motility . At 37 degrees C, when flagella are not produced, attachment of both strains was identical . Therefore, flagella per se facilitate the early stage of attachment. Appl Environ Microbiol, 2000 Feb, 66(2), 769 - 74 Carbon dioxide and nisin act synergistically on Listeria monocytogenes; Nilsson L et al.; This paper examines the synergistic action of carbon dioxide and nisin on Listeria monocytogenes Scott A wild-type and nisin-resistant (Nis(r)) cells grown in broth at 4 degrees C . Carbon dioxide extended the lag phase and decreased the specific growth rate of both strains, but to a greater degree in the Nis(r) cells . Wild-type cells grown in 100% CO(2) were two to five times longer than cells grown in air . Nisin (2.5 microg/ml) did not decrease the viability of Nis(r) cells but for wild-type cells caused an immediate 2-log reduction of viability when they were grown in air and a 4-log reduction when they were grown in 100% CO(2) . There was a quantifiable synergistic action between nisin and CO(2) in the wild-type strain . The MIC of nisin for the wild-type strain grown in the presence of 2.5 microg of nisin per ml increased from 3.1 to 12.5 microg/ml over 35 days, but this increase was markedly delayed for cultures in CO(2) . This synergism between nisin and CO(2) was examined mechanistically by following the leakage of carboxyfluorescein (CF) from listerial liposomes . Carbon dioxide enhanced nisin-induced CF leakage, indicating that the synergistic action of CO(2) and nisin occurs at the cytoplasmic membrane . Liposomes made from cells grown in a CO(2) atmosphere were even more sensitive to nisin action . Liposomes made from cells grown at 4 degrees C were dramatically more nisin sensitive than were liposomes derived from cells grown at 30 degrees C . Cells grown in the presence of 100% CO(2) and those grown at 4 degrees C had a greater proportion of short-chain fatty acids . The synergistic action of nisin and CO(2) is consistent with a model where membrane fluidity plays a role in the efficiency of nisin action. Mol Microbiol, 2000 Jan, 35(2), 312 - 23 Generalized transduction of serotype 1/2 and serotype 4b strains of Listeria monocytogenes; Hodgson DA; This is the first report of generalized transduction in the gram-positive, food-borne pathogen Listeria monocytogenes . Bacteriophages were isolated from the environment and from lysogens, or were obtained from other laboratories . Of the 59 bacteriophages tested, 34 proved to be capable of transduction . We exploited the ability of L . monocytogenes to grow at room temperature and isolated bacteriophages that were incapable of growth at 37 degrees C . Transductions at this temperature therefore eliminated transductant killing and lysogeny, as did inclusion of citrate and the use of a low multiplicity of infection . Transducing bacteriophages were found for each of the well-characterized L . monocytogenes strains: EGD, 10403, Mack (serotype1/2a), L028 (serotype 1/2c), Scott A (serotype 4b) and strains from the Jalisco and Halifax, Nova Scotia outbreaks (serotype 4b) . P35 (phiLMUP35) is a particularly useful generalized transducing bacteriophage with a wide host range (75% of all serotype 1/2 strains tested) . Its disadvantages are that it is small and transduction is relatively infrequent . U153(phiCU-SI153/95) is larger than P35 and transduction frequency increased 100-fold, but it has a very narrow host range . We demonstrated interstrain transduction and used transduction to test linkage between transposon insertions and mutant phenotypes in a variety of strains. Mol Microbiol, 2000 Jan, 35(2), 289 - 98 pH-regulated activation and release of a bacteria-associated phospholipase C during intracellular infection by Listeria monocytogenes; Marquis H et al.; Listeria monocytogenes grows in the cytosol of mammalian cells and spreads from cell to cell without exiting the intracellular milieu . During cell-cell spread, bacteria become transiently entrapped in double-membrane vacuoles . Escape from these vacuoles is mediated in part by a bacterial phospholipase C (PC-PLC), whose activation requires cleavage of an N-terminal peptide . PC-PLC activation occurs in the acidified vacuolar environment . In this study, the pH-dependent mechanism of PC-PLC activation was investigated by manipulating the intracellular pH of the host . PC-PLC secreted into infected cells was immunoprecipitated, and both forms of the protein were identified by SDS-PAGE fluorography . PC-PLC activation occurred at pH 7.0 and lower, but not at pH 7.3 . Total amounts of PC-PLC secreted into infected cells increased several-fold over controls within 5 min of a decrease in intracellular pH, and the active form of PC-PLC was the most abundant species detected . Bacterial release of active PC-PLC was dependent on Mpl, a bacterial metalloprotease that processes the proform (proPC-PLC), and did not require de novo protein synthesis . The amount of proPC-PLC released in response to a decrease in pH was the same in wild-type and Mpl-minus-infected cells . Immunofluorescence detection of PC-PLC in infected cells was performed . When fixed and permeabilized infected cells were treated with a bacterial cell wall hydrolase, over 97% of wild-type and Mpl-minus bacteria stained positively for PC-PLC, in contrast to less than 5% in untreated cells . These results indicate that intracellular bacteria carry pools of proPC-PLC . Upon cell-cell spread, a decrease in vacuolar pH triggers Mpl activation of proPC-PLC, resulting in bacterial release of active PC-PLC. Tuber Lung Dis, 1998, 79(2), 119 - 26 Suppression of lymphoproliferation by alveolar macrophages in the guinea pig; Zhang X et al.; SETTING: The relationship between alveolar macrophages (AM) and lymphocytes may be important in the early establishment of infection with Mycobacterium tuberculosis . AM in several species have been shown to suppress lymphoproliferation by producing inhibitors that include nitric oxide (NO) . OBJECTIVE: To study this phenomenon in the guinea pig, the mitogen-induced proliferation of splenic lymphocytes was quantified under various conditions of co-culture with resident AM . RESULTS: Guinea pig AM consistently and profoundly suppressed proliferation in the co-cultures at AM:lymphocyte ratios of 1:4 or greater . The inclusion of a NO synthesis inhibitor, N-monomethyl-L arginine (NMMA), in the co-culture medium did not influence the suppression of Con A-induced lymphoproliferation by resident guinea pig AM . No nitrite could be detected in supernatant fluids of co-cultured AM and splenocytes . Attempts to stimulate guinea pig AM with LPS in combination with recombinant murine and human IFN-gamma, infection with live Listeria monocytogenes, or incubation with the supernatants from ConA-activated guinea pig lymphocytes failed to generate NO metabolites . The addition of catalase or indomethacin to the Con A-induced AM-splenocyte co-cultures, to inhibit hydrogen peroxide (H2O2) or prostaglandin E2 (PGE2), respectively, did not counteract the suppression mediated by AM . Cell contact was necessary for the co-cultures to generate their inhibitory effects on lymphoproliferation, however, the suppression was actually mediated, at least in part, by soluble factors produced in the co-cultures . CONCLUSION: These results suggest that resident alveolar macrophages suppress lymphocyte proliferation in the guinea pig, but that the effect is not mediated by NO, PGE2 or H2O2 . The failure to demonstrate NO synthesis under a variety of stimulatory conditions, which resulted in macrophage activation, suggests that the guinea pig is similar to the human in that regard. J Food Prot, 2000 Jan, 63(1), 131 - 6 Survival of Listeria monocytogenes Scott A on vacuum-packaged raw beef treated with polylactic acid, lactic acid, and nisin; Ariyapitipun T et al.; Low-molecular-weight polylactic acid (LMW-PLA) and lactic acid (LA) were used to inhibit growth of Listeria monocytogenes Scott A on vacuum-packaged beef . Nisin was also used simultaneously as an additional hurdle to the growth of this pathogen . Inoculated beef cubes were immersed in a solution of 2% LMW-PLA, 2% LA, 400 IU/ml of nisin, or combinations of each acid and nisin for 5 min and drip-dried for 15 min . The cubes were then vacuum-packaged and stored at 4 degrees C for up to 42 days . Surface pH values of beef cubes treated with 2% LMW-PLA, the combination of 400 IU/ml of nisin and 2% LMW-PLA (2% NPLA), or 400 IU/ml of nisin alone were significantly reduced from 5.59 to 5.18, 5.01, and 5.19, respectively, whereas those decontaminated with 2% LA or 400 IU/ml of nisin and 2% LA (2% NLA) were significantly decreased from 5.59 to 4.92 and 4.83, respectively, at day 0 (P < or = 0.05) . The 2% LMW-PLA, 2% LA, 2% NPLA, 2% NLA, and 400 IU/ml of nisin showed immediate bactericidal effects on L . monocytogenes Scott A (1.22-, 1.56-, 1.57-, 1.94-, and 1.64-log10 reduction, respectively) compared with the initial number of 5.33 log10 CFU/cm2 of the untreated control at day 0 (P < or = 0.05) . These treatments, combined with vacuum-packaging and refrigeration temperature, succeeded to inhibit growth of L . monocytogenes during storage up to 42 days . At the end of 42 days, the numbers of L . monocytogenes Scott A remaining viable on these samples were 1.21, 0.36, 2.21, 0.84, and 0.89 log10 CFU/cm2, respectively. FEMS Immunol Med Microbiol, 2000 Feb, 27(2), 127 - 33 Determination of natural resistance of mice fed dietary lipids to experimental infection induced by Listeria monocytogenes; de Pablo MA et al.; Current understanding based on the effect of dietary lipid manipulation upon immune system function indicates that fatty acids are involved in the modulation of the immune response through different and complex pathways . Reduction of several immune parameters by fatty acid action may be applied in the treatment of diseases characterised by an overactivation of the immune system . As a consequence, a reduction of host resistance against infectious agents has been reported in animals fed dietary lipids . The present study confirms the action of dietary lipids on the survival of mice infected with the pathogenic bacterium Listeria monocytogenes . A significant increase in peritoneal cells from mice fed a hydrogenated coconut oil diet was found, while a significant reduction of bacterial recovery from spleens of these mice was observed in this group . In addition, both eicosanoid and phospholipase inhibitors did not promote any modification of lymphocyte proliferation from mice fed olive oil or fish oil. Infect Immun, 2000 Feb, 68(2), 999 - 1003 Role of listeriolysin O in cell-to-cell spread of Listeria monocytogenes; Gedde MM et al.; Listeria monocytogenes is a facultative intracellular bacterial pathogen that escapes from a host vacuolar compartment and grows rapidly in the cytosol . Listeriolysin O (LLO) is a secreted pore-forming protein essential for the escape of L . monocytogenes from the vacuole formed upon initial internalization . However, its role in intracellular growth and cell-to-cell spread events has not been testable by a genetic approach . In this study, purified six-His-tagged LLO (HisLLO) was noncovalently coupled to the surface of nickel-treated LLO-negative mutants . Bound LLO mediated vacuolar escape in approximately 2% of the mutants . After 5.5 h of growth, cytosolic bacteria were indistinguishable from wild-type bacteria with regard to formation of pseudopod-like extensions, here termed listeriopods, and spread to adjacent cells . However, bacteria in adjacent cells failed to multiply and were found in double-membrane vacuoles . Addition of bound LLO to mutants lacking LLO and two distinct phospholipases C (PLCs) also resulted in spread to adjacent cells, but these triple mutants became trapped in multiple-membrane vacuoles that are reminiscent of autophagocytic vacuoles . These studies show that neither LLO nor the PLCs are necessary for listeriopod formation and uptake of bacteria into neighboring cells but that LLO is required for the escape of L . monocytogenes from the double-membrane vacuole that forms upon cell-to-cell spread. Infect Immun, 2000 Feb, 68(2), 912 - 4 Mutants of Listeria monocytogenes defective in In vitro invasion and cell-to-cell spreading still invade and proliferate in hepatocytes of neutropenic mice; Appelberg R et al.; Listeria monocytogenes mutants defective in the actA gene, the plcB gene, and the inlA and inlB genes were less virulent when injected intravenously into BALB/c mice . The growth of these strains as well as of the virulent wild-type strains was increased by treating mice with a neutrophil-specific depleting monoclonal antibody, RB6-8C5 . Histologic examination of the livers of the treated animals showed intrahepatocytic proliferation of the listeriae in all cases . Our data show that more than one pathway exists that allows L . monocytogenes to invade parenchymal cells . One pathway most likely involves the actA and plcB gene products, and a second one probably involves the internalins. Proc Natl Acad Sci U S A, 2000 Jan 18, 97(2), 751 - 5 Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP; Taylor GA et al.; Interferon-gamma (IFN-gamma) is critical for defense against pathogens, but the molecules that mediate its antimicrobial responses are largely unknown . IGTP is the prototype for a family of IFN-gamma-regulated genes that encode 48-kDa GTP-binding proteins that localize to the endoplasmic reticulum . We have generated IGTP-deficient mice and found that, despite normal immune cell development and normal clearance of Listeria monocytogenes and cytomegalovirus infections, the mice displayed a profound loss of host resistance to acute infections of the protozoan parasite Toxoplasma gondii . By contrast, IFN-gamma receptor-deficient mice have increased susceptibility to all three pathogens . Thus, IGTP defines an IFN-gamma-regulated pathway with a specialized role in antimicrobial resistance. Int J Food Microbiol, 1999 Dec 15, 53(2-3), 195 - 203 Incidence and control of Listeria monocytogenes in foods in Denmark; Norrung B et al.; The Danish regulatory policy on Listeria monocytogenes in foods is based on the principles of HACCP and was developed using a health risk assessment approach . The Danish policy focuses examinations and criteria for L . monocytogenes in ready-to-eat foods and is based on a combination of inspection and product-testing . Based on current epidemiological information from several countries, a concentration of L . monocytogenes not exceeding 100 cfu/g of food at the time of consumption, seems to be of low risk to the consumers . In Denmark, ready-to-eat foods have been placed into six categories where absence of L . monocytogenes in 25 g is required in foods heat treated in the final package and in heat-treated as well as preserved, non heat-treated foods which can support growth within the shelf life . This level is necessary in foods capable of supporting growth, in order not to exceed 100 L . monocytogenes per g at the point of consumption . In heat-treated and preserved foods, which are not supportive of growth within the shelf-life and for raw, ready to eat foods, a level below 10 L . monocytogenes per g is regarded acceptable . A level between 10 and 100 L . monocytogenes per g is not satisfactory and a level above 100/g is not acceptable . Data on the qualitative and quantitative occurrence of L . monocytogenes in foods in Denmark are presented and discussed . In 1997 and 1998, greater than 15,000 samples from different categories of food were examined (semi-quantitatively) for the presence of L . monocytogenes . A significant difference could be seen in the number of samples containing more than 100 L . monocytogenes per g, between different categories of foods (1997, P = 0.001; 1998, P = 0.016) . In 1997, preserved meat products and preserved fish products and to a lesser extent vegetables and meat or vegetable mayonnaise were more likely to contain high numbers (i.e . above 100 cfu/g) of L . monocytogenes than other food categories . In 1998, preserved meat products, but also heat-treated meat products, vegetables and meat or vegetable mayonnaise had the highest frequency of samples with > 100 L . monocytogenes per g . In a survey performed in 1994 and 1995, 1.3% of ready-to-eat food samples (heat-treated meat products, preserved meat and fish products) were found to be contaminated with L . monocytogenes at a level above 100 cfu/g . The samples included in this survey were primarily products produced by authorized companies and were comprised mainly of vacuum packed products or products packed in modified atmosphere and with long shelf lives, typically above several weeks . The corresponding percentages of positive samples primarily processed in the retail outlets (heat-treated meat products, preserved meat and fish products) in 1997 and 1998 were 0.3% and 0.6%, respectively . The results suggest that ready-to-eat meat and fish products with extended shelf-lives produced by authorized companies are more likely to contain high numbers (> 100 cfu/g) of L . monocytogenes than products processed in the retail sector which often have a shorter shelf life. Int J Food Microbiol, 1999 Dec 15, 53(2-3), 127 - 40 Listeria monocytogenes in pork slaughtering and cutting plants . Use of RAPD, PFGE and PCR-REA for tracing and molecular epidemiology; Giovannacci I et al.; In order to determine the origin of pork cuts contamination by Listeria monocytogenes, 287 isolates, collected from five French pork slaughtering and cutting plants, from live pigs to pork cuts, were characterised using three molecular typing methods: random amplification of polymorphic DNA (RAPD) carried out with five different primers, genomic macrorestriction using ApaI with pulsed-field gel electrophoresis (PFGE) and a PCR-restriction enzyme analysis (PCR-REA) based on the polymorphism existing within the inlA and inlB genes . Results obtained from RAPD and PFGE were closely related and distinguished respectively 17 RAPD types (r1-r17) and 17 PFGE types (a1-a17) among the 287 isolates, whereas the PCR-REA analysis only yielded two profiles (p1 and p2) . Considering the combined results obtained with the three molecular typing methods, 19 Listeria monocytogenes genotypes (1-19) were distinguished . Serotyping led at least four serotypes being distinguished: 1/2a, 3a, 1/2c and 3c . The application of genotyping identified the predominance of a Listeria monocytogenes strain of type (1) and other very closely related ones (5, 9, 10, 12, 13, 14, 16 and 19) which were present on pork as well as in the environment within the five investigated plants . This study also pointed out the presence of these closely related Listeria monocytogenes strains over a 1-year period in the environments of two plants, even after cleaning and disinfection procedures . This highlights the possibility for some Listeria monocytogenes strains to persist in pork processing environments and raises the problem of the efficiency of cleaning and disinfection procedures used in pork slaughterhouses, chilling and cutting rooms. Nat Struct Biol, 2000 Jan, 7(1), 38 - 43 The dodecameric ferritin from Listeria innocua contains a novel intersubunit iron-binding site; Ilari A et al.; Ferritin is characterized by a highly conserved architecture that comprises 24 subunits assembled into a spherical cage with 432 symmetry . The only known exception is the dodecameric ferritin from Listeria innocua . The structure of Listeria ferritin has been determined to a resolution of 2.35 A by molecular replacement, using as a search model the structure of Dps from Escherichia coli . The Listeria 12-mer is endowed with 23 symmetry and displays the functionally relevant structural features of the ferritin 24-mer, namely the negatively charged channels along the three-fold symmetry axes that serve for iron entry into the cavity and a negatively charged internal cavity for iron deposition . The electron density map shows 12 iron ions on the inner surface of the hollow core, at the interface between monomers related by two-fold axes . Analysis of the nature and stereochemistry of the iron-binding ligands reveals strong similarities with known ferroxidase sites . The L . innocua ferritin site, however, is the first described so far that has ligands belonging to two different subunits and is not contained within a four-helix bundle. J Bacteriol, 2000 Feb, 182(3), 837 - 41 Sequence variations within PrfA DNA binding sites and effects on Listeria monocytogenes virulence gene expression; Williams JR et al.; Reporter gene fusions were used to investigate the contributions of PrfA DNA binding sites to Listeria monocytogenes virulence gene expression . Our results suggest that the DNA sequence of PrfA binding sites determines the levels of expression of certain virulence genes, such as hly and mpl . Other virulence genes, such as actA and plcB, may depend upon additional factors for full regulation of gene expression. Immunol Rev, 1999 Dec, 172, 163 - 9 Processing of Listeria monocytogenes antigens and the in vivo T-cell response to bacterial infection; Busch DH et al.; Presentation of antigens to T lymphocytes is a critical step in the clearance of pathogens from their hosts and in the establishment of protective immunity . Several animal models have been developed to study this process, but few have been as informative as the murine immune response to Listeria monocytogenes infection . Herein we review the presentation of L . monocytogenes proteins by the MHC class I antigen-processing pathway and the in vivo T-cell response to these bacterial antigens . These studies demonstrate the following: 1) The size of a peptide-specific T-cell response does not correlate with the amount of epitope presented by infected cells; 2) T cells specific for dominant epitopes do not, in the case of L . monocytogenes infection, inhibit responses to subdominant epitopes; 3) T cells responding to different epitopes presented by MHC class Ia molecules expand, contract and enter the memory pool synchronously; 4) Repeated in vivo expansion of antigen-specific T-cell populations results in a narrowing of their T-cell receptor repertoire and in an increase in their affinity for antigen; and 5) T cells restricted by H2-M3 MHC class Ib molecules constitute a major part of the primary response to bacterial infection, but appear to play a relatively smaller role in memory responses . These studies have provided a novel glimpse of the relationship between antigen processing and in vivo T-cell responses to infection, and provide a foundation for more detailed analyses of T-cell mediated adaptive immunity. Immunobiology, 1999 Dec, 201(2), 272 - 82 The need for a novel generation of vaccines; Kaufmann SH et al.; Although empirical vaccine development was highly successful, it has now reached its limits . Vaccines are only efficacious against those pathogens which are primarily controlled by antibodies . Protection against many infectious agents, however, strongly depends on T lymphocytes . Thus, novel vaccines have to stimulate the combination of T lymphocytes that is required for an optimum protective immune response . Although identification of antigens remains crucial, novel vaccine design also needs to consider the best way of introducing these antigens to the immune system . Intracellular antigen compartmentalisation, the early cytokine milieu and the appropriate surface expression of co-stimulatory molecules are of major relevance for understanding how novel vaccines could induce a protective immune response mediated by T lymphocytes . Intracellular bacteria are controlled by T lymphocytes and efficacious vaccines against these pathogens are not available yet . In this treatise, two experimental vaccination strategies will be described in more detail . These encompass recombinant vaccine carriers expressing, and naked DNA constructs encoding, heterologous antigens . Both vaccination strategies proved to be protective in the model of experimental listeriosis of mice. Immunobiology, 1999 Dec, 201(2), 205 - 9 MHC class Jb-restricted cell responses to Listeria monocytogenes infection; Kerksiek KM et al.; Murine infection with Listeria monocytogenes induces CD8+ T cell responses specific for bacterial peptides that are presented on the infected cell surface by MHC class Ia and MHC class Ib molecules . We have used MHC tetramers to demonstrate that CD8+ T cells restricted by the H2-M3 MHC class Ib molecules constitute a substantial portion of the T cell response to L . monocytogenes infection . The in vivo size and kinetics of MHC class Ib-restricted T cell populations suggests that they play a prominent role in bacterial clearance following primary L . monocytogenes infection. Immunobiology, 1999 Dec, 201(2), 196 - 204 A knockout approach to understanding CD8+ cell effector mechanisms in adaptive immunity to Listeria monocytogenes; Harty JT et al.; In the described experimental approach, we use an attenuated LM strain to evoke LM specific CD8+ T cell responses . In this fashion, we can immunize immunocompromised gene knockout mice, that would succumb to low level infection with virulent LM . We then generate antigen matched, LM-specific CD8+ T cell lines from wild-type and gene knockout mice, and compare their capacity to provide immunity to LM infection in vivo . To date, our results demonstrate that CD8+ T cell-derived IFN-gamma and TNF are not required effector functions . Perforin deficiency has an impact on CD8+ T cell immunity but our studies provide strong evidence for the existence of perforin independent pathways of CD8+ T cell immunity to LM . To assess the potential for redundancy in effector mechanisms, we have generated mice deficient in both perforin and IFN-gamma and are developing mice deficient in perforin and TNF . By removing the major CD8+ T cell effector mechanisms, singly and in combination, we will eventually determine whether immunity to LM can be provided by redundant effector pathways or if novel effector mechanisms exist beyond our current knowledge . The generation of MHC matched, single and double knockout mice, will also aid in continuing studies to analyze the role of these molecules in resistance to in vivo infection. Immunobiology, 1999 Dec, 201(2), 188 - 95 Immune reactions to Listeria monocytogenes in the brain; Schluter D et al.; Listeria monocytogenes (LM) is a common pathogen of cerebral infections . Experimental studies in mice have revealed that epithelial cells of the choroid plexus, ependymal cells, macrophages/microglia, and neurons are the target cells of LM . For the intracerebral pathogenesis of LM cell-to-cell spread via phospholipase C was particularly important . However, phospholipase C-deficient LM were not completely attenuated and, therefore, other virulence factors may also contribute to the intracerebral spread of LM . In general, all mice suffering from cerebral listeriosis rapidly succumbed to the disease . Active systemic immunization prior to intracerebral infection reduced the mortality rate to 40% . The favorable prognosis of immunized mice correlated with a reduced intracerebral bacterial load, an increased recruitment of protective CD4+ and CD8+ T cells as well as an upregulated mRNA production of protective cytokines. Immunobiology, 1999 Dec, 201(2), 178 - 87 Early host-pathogen interactions in the liver and spleen during systemic murine listeriosis: an overview; Conlan JW; Systemic listeriosis initiated by parenteral inoculation of mice with Listeria monocytogenes has been used extensively as a model infection for studying mammalian host defense against intracellular bacterial pathogens in general . Most effort has been expended on trying to understand the requirement for specific T cell-mediated immunity for combatting infection with this pathogen . By contrast, non-specific defenses have received much less attention . However, it is now obvious that these early innate defenses are critically important for the well-being of the host . If these early defenses fail to act, the murine host is rendered exquisitely susceptible to L . monocytogenes, and rapidly succumbs to overwhelming infection before T cell-mediated immunity can be generated and expressed . The most critical of these early defenses is mediated by neutrophils that rapidly accumulate in large numbers at foci of Listeria infection in the liver and spleen . These neutrophils act to curtail the growth of L . monocytogenes to levels that subsequently can be dealt with by specific defenses that are recruited into infectious foci later . In the absence of this neutrophil-mediated defense, an otherwise sublethal inoculum of L . monocytogenes rapidly grows to lethal numbers . An overview of this early aspect of murine listeriosis is presented below. Immunobiology, 1999 Dec, 201(2), 164 - 77 The mucosal phase of Listeria infection; Havell EA et al.; Listeria monocytogenes is an enteroinvasive bacterial pathogen of man and animals . Listeriae have been shown capable of infecting the host by translocating from the intestinal lumen through Peyer's Patches (PP), however, results of experiments now indicate that these facultative intracellular parasites may also translocate through PP-independent routes . With regards to this, on occasion we observed that listeriae were absent from the PP of mice inoculated intragastrically with L . monocytogenes, but were present in the mesenteric lymph nodes of these same mice . These observations suggested that PP were not necessary for listerial translocation from the intestinal lumen . Two experimental approaches were used to determine whether luminal listeriae could indeed infect the host through PP-independent routes . First, since it is known that: 1) following the intragastric inoculation of L . monocytogenes, listeriae rapidly transit the length of the gastrointestinal tract and reside in the colonic lumen for up to a week, 2) the colon lacks PP, and 3) the descending colon and rectum are drained exclusively by the caudal lymph node (CLN), it was determined whether colonic listeriae could access the CLN . Inoculation of listeriae into the rectum of mice resulted in the infection of the CLN which indicated that PP were not required for listerial translocation . Second, since germfree SCID mice lack PP, it was determined whether listeriae could translocate from the intestinal lumen and infect these immunoincompetent mice . Shortly after the intragastric inoculation of L . monocytogenes into germfree SCID mice, listeriae were found in the mesenteries, livers and spleens . These results also indicate that PP are not required for listerial translocation from the intestinal lumen . One possible route of translocation from the intestinal lumen might occur by listeriae entering enterocytes . Results were obtained showing that listeriae were capable of entering cultured mouse small intestine enterocytes . Internalized listeriae were observed to multiply and spread intracellularly between enterocytes. Immunobiology, 1999 Dec, 201(2), 155 - 63 Molecular and cell biological aspects of infection by Listeria monocytogenes; Chakraborty T; Bacterial pathogens have developed many subtle mechanisms to overcome and exploit cellular processes within the infected eukaryotic host cell . Listeria monocytogenes, a facultative intracellular non-spore forming gram-positive pathogen, uses a number of strategies to ursurp and harness host cell processes to invade, proliferate, move intracellularly and effect cell-to-cell spread during the course of infection . In this review progress in elucidating mechanisms by which the bacteria recruit and use components of the host actin-based cytoskeleton to generate intracellular motility is presented . Analysis of this fascinating property is giving us unexpected glimpses into the molecular mechanisms of complex cellular functions, here in particular, of actin-based cellular motility . Apart from an understanding of the fundamental biology of living processes these studies provide us with novel strategies to combat and halt infections by intracellular bacteria. Biochim Biophys Acta, 2000 Jan 15, 1463(1), 31 - 42 Electron paramagnetic resonance studies of the membrane fluidity of the foodborne pathogenic psychrotroph Listeria monocytogenes; Edgcomb MR et al.; Listeria monocytogenes is a foodborne psychrotrophic pathogen that grows at refrigeration temperatures . Previous studies of fatty acid profiles of wild-type and cold-sensitive, branched-chain fatty acid deficient mutants of L . monocytogenes suggest that the fatty acid 12-methyltetradecanoic (anteiso-C(15:0)) plays a critical role in low-temperature growth of L . monocytogenes, presumably by maintaining membrane fluidity . The fluidity of isolated cytoplasmic membranes of wild-type (SLCC53 and 10403S), and a cold-sensitive mutant (cld-1) of L . monocytogenes, grown with and without the supplementation of 2-methylbutyric acid, has been studied using a panel of hydrocarbon-based nitroxides (2N10, 3N10, 4N10, and 5N10) and spectral deconvolution and simulation methods to obtain directly the Lorentzian line widths and hence rotational correlation times (tau(c)) and motional anisotropies of the nitroxides in the fast motional region . tau(c) values over the temperature range of -7 degrees C to 50 degrees C were similar for the membranes of strains SLCC53 and 10403S grown at 10 degrees C and 30 degrees C, and for strain cld-1 grown with 2-methylbutyric acid supplementation (which restores branched-chain fatty acids) at 30 degrees C . However, strain cld-1 exhibited a threefold higher tau(c) when grown without 2-methylbutyric acid supplementation (deficient in branched-chain fatty acids) compared to strains SLCC53, 10403S, and supplemented cld-1 . No evidence was seen for a clear lipid phase transition in any sample . We conclude that the fatty acid anteiso-C(15:0) imparts an essential fluidity to the L . monocytogenes membrane that permits growth at refrigeration temperatures. J Med Microbiol, 2000 Jan, 49(1), 73 - 80 Molecular grouping of Listeria monocytogenes based on the sequence of the inIB gene; Ericsson H et al.; The major part of the gene inlB was sequenced in 24 strains of Listeria monocytogenes belonging to serovars 1/2a, 1/2b, 1/2c, 3b and 4b . A phylogenetic analysis based on the inlB nucleotide sequences showed that strains of serovars 1/2a and 1/2c were closely related, as well as those of serovars 1/2b and 3b . Strains sharing serovar 4b could be divided into two distinct groups . There were differences in amino-acid sequence between all serovars except between serovars 1/2b and 3b . Differences in amino-acid sequence were also seen within each of the serovars 1/2a and 4b . The data presented indicate that the inlB gene may be useful for typing purposes as an alternative or complement to serotyping. J Clin Microbiol, 2000 Jan, 38(1), 345 - 50 Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR; Al-Soud WA et al.; A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized . Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes . All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase AmpliTaq Gold . PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step . The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide . When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth . The inhibitory effect was reduced when PIgG was heated at 95 degrees C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture . However, heating of PIgG together with target DNA at 95 degrees C was found to block the amplification . Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested . The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR . The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads. Cell Motil Cytoskeleton, 2000 Jan, 45(1), 58 - 66 Listeria monocytogenes ActA protein interacts with phosphatidylinositol 4,5-bisphosphate in vitro; Steffen P et al.; The N-terminal region of the Listeria monocytogenes ActA protein, in conjunction with host cell factors, is sufficient for actin polymerization at the bacterial surface . Previous data suggested that ActA could protect barbed ends from capping proteins . We tested this hypothesis by actin polymerization experiments in the presence of the ActA N-terminal fragment and capping protein . ActA does not protect barbed ends from capping protein . In contrast, this polypeptide prevents PIP(2) from inhibiting the capping activity of capping protein . Gel filtration and tryptophan fluorescence experiments showed that the purified ActA N-terminal fragment binds to PIP(2) and PIP, defining phosphoinositides as novels ligands for this functional domain of ActA . Phosphoinositide binding to the N-terminal region of ActA may induce conformational changes in ActA and/or facilitate binding of other cell components, important for ActA-induced actin polymerization . J Leukoc Biol, 1999 Dec, 66(6), 961 - 7 Surface interleukin-10 inhibits listericidal activity by primary macrophages; Fleming SD et al.; Interleukin-10 (IL-10) down-regulates multiple functions of monocytes and macrophages, including the ability of macrophages to kill many intracellular microorganisms . The experiments presented here test the hypothesis that IL-10 expressed on the cell surface inhibits the ability of primary mouse macrophages to kill the facultative, intracellular bacterium Listeria monocytogenes . We show that, in contrast to macrophages from normal mice, both bone marrow-derived macrophages (BMDM) and thioglycollate-elicited macrophages obtained from IL-10-/- mice can kill L . monocytogenes . Treatment with anti-IL-10 monoclonal antibody (mAb) enables BMDM from normal mice and thioglycollate-elicited macrophages from RAG-2-/- mice (which lack T or B cell-derived IL-10) to kill L . monocytogenes, and concurrently down-regulates the expression of surface IL-10 . Surface IL-10 on paraformaldehyde-fixed cells can inhibit nitric oxide (NO) production by interferon-gamma (IFN-gamma)-stimulated macrophages from IL-10-/- mice, thus directly showing functional activ