Int J Food Microbiol, 2001 Oct 22, 70(1-2), 121 - 9 Validation of ISO method 11290 part 2 . Enumeration of Listeria monocytogenes in foods; Scotter SL et al.; The European and International Standard method for the enumeration of Listeria monocytogenes, described in EN ISO 11290 Part 2: 1998 {EN ISO 11290-2 Microbiology of Food and Animal Feedingstuffs-Horizontal Method for the Detection and Enumeration of L . monocytogenes: Part 2 . Enumeration; International Organisation for Standardisation, Geneva.} was validated by order of the European Commission (Standards, Measurement and Testing Fourth Framework Programme Project SMT4-CT96-2098) . The objective was to determine the precision of the method in terms of repeatability (r) and reproducibility (R) using three different food types inoculated with various levels of L . monocytogenes and a typical background flora . The results are intended for publication in the associated standards . Cheese, meat, dried egg powder and reference materials were examined by 21 laboratories in 16 countries in Europe . Each participant received eight test materials per food type: blind duplicates at four inoculum levels (0, 10(2), 10(3), 10(4) cfu/g) . In addition, two reference materials containing L . monocytogenes were included in the study . All test materials were subjected to stringent homogeneity and stability testing before being used in the collaborative trial . Participants were required to use only PALCAM agar for enumeration of L . monocytogenes, as prescribed by the reference method . Statistical analyses has been performed using a newly introduced approach for food microbiology (draft standard prEN ISO 16140 {prEN ISO 16140 Microbiology of Food and Animal Feedingstuffs-Protocol for the Validation of Alternative Methods, International Organisation for Standardisation, Geneva.}, the precision data being calculated using robust estimates . Overall values for repeatability (r) of EN ISO 11290-2 when used with food test materials were r = log10 0.58 (expressed as an absolute difference between log10-transformed test results) or r = 3.8 (expressed as an absolute ratio between test results on the normal scale) . For the reference materials (capsules containing approximately 5000 cfu), r = log10 0.34 (expressed as an absolute difference between log10-transformed test results) or r = 2.2 (expressed as an absolute ratio between test results on the normal scale) . Overall values for reproducibility (R) of EN ISO 11290-2 when used with food test materials were R = log10 0.81 (expressed as a difference between log10-transformed test results) or R = 6.5 (expressed as an absolute ratio between test results on the normal scale) . For the reference materials, R = log10 0.51 (expressed as a difference between log10-transformed test results) or R = 3.2 (expressed as an absolute ratio between test results on the normal scale) . Further studies have been initiated by ISO TC34/SC9 to try to enhance the isolation of L . monocytogenes from foods and improve the confirmation procedures. Proc Natl Acad Sci U S A, 2002 Jan 8, 99(1), 431 - 6 Epub 2001 Dec 26. Hpt, a bacterial homolog of the microsomal glucose- 6-phosphate translocase, mediates rapid intracellular proliferation in Listeria; Chico-Calero I et al.; Efficient replication in vivo is essential for a microparasite to colonize its host and the understanding of the molecular mechanisms by which microbial pathogens grow within host tissues can lead to the discovery of novel therapies to treat infection . Here we present evidence that the foodborne bacterial pathogen Listeria monocytogenes, a facultative intracellular parasite, exploits hexose phosphates (HP) from the host cell as a source of carbon and energy to fuel fast intracellular growth . HP uptake is mediated by Hpt, a bacterial homolog of the mammalian translocase that transports glucose-6-phosphate from the cytosol into the endoplasmic reticulum in the final step of gluconeogenesis and glycogenolysis . Expression of the Hpt permease is tightly controlled by the central virulence regulator PrfA, which upon entry into host cells induces a set of virulence factors required for listerial intracellular parasitism . Loss of Hpt resulted in impaired listerial intracytosolic proliferation and attenuated virulence in mice . Hpt is the first virulence factor to be identified as specifically involved in the replication phase of a facultative intracellular pathogen . It is also a clear example of how adaptation to intracellular parasitism by microbial pathogens involves mimicry of physiological mechanisms of their eukaryotic host cells. J Virol, 2002 Jan, 76(2), 918 - 22 Safety and immunogenicity in neonatal mice of a hyperattenuated Listeria vaccine directed against human immunodeficiency virus; Rayevskaya M et al.; CD8(+) T cells are a major component of the adaptive response of a host to infections by viruses and other intracellular pathogenic agents . However, because of the intrinsic immaturity of the immune system of neonatal animals, neonates are highly sensitive to a variety of pathogens and may be unable to respond in a protective manner . Here we explore whether a hyperattenuated strain of Listeria monocytogenes that can be used as a live vaccine vector in adults is safe and able to induce an effective response in neonates . We answer both questions affirmatively. J Immunol, 2002 Jan 1, 168(1), 308 - 15 Complementary adhesion molecules promote neutrophil-Kupffer cell interaction and the elimination of bacteria taken up by the liver; Gregory SH et al.; Most bacteria that enter the bloodstream are taken up by the liver . Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature . Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface . In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v . was reduced from approximately 60% of the inoculum in normal mice to approximately 15% in mice rendered Kupffer cell deficient . Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells . Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells . Complementary adhesion molecules promoted the interaction between these two cell populations . Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae . Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils. FEMS Microbiol Lett, 2001 Dec 18, 205(2), 179 - 83 Early translocation of acid-adapted Listeria monocytogenes during enteric infection in TNF/LTalpha-/- mice; Fontan E et al.; TNF/LTalpha deficient mice are devoid of Peyer's patches and lack mesenteric lymph nodes . Translocation, especially in the early steps after intragastric delivery of Listeria monocytogenes, has been explored in this study, and the role of TNFalpha has been addressed . We showed that L . monocytogenes translocation occurred at least as efficiently in TNF/LTalpha-/- mice as in TNF/LTalpha+/+ littermates . Even very low inocula (2.7x10(4) cfu) could initiate infection in the TNF/LTalpha deficient mice . Early kinetics of dissemination to the spleen and liver were similar, L . monocytogenes reaching these organs at 8 h post inoculation . However, a 10-fold higher bacterial load was observed at this early time point in the TNF/LTalpha deficient mice . rTNF pretreatment (4 h before intragastric inoculation) had no effect on the L . monocytogenes associated with the caecum-colon walls at 10 h after inoculation, although bacterial levels in the caecum-colon lumen and in spleen and liver were already controlled. FEMS Immunol Med Microbiol, 2001 Dec, 32(1), 47 - 52 Immunomodulatory effects of dietary lipids alter host natural resistance of mice to Listeria monocytogenes infection; Puertollano MA et al.; Over the past two decades, unsaturated fatty acids have received particular attention due to their ability to suppress immune functions . Nevertheless, suppression of immune functions also involves a reduction of host natural resistance to eliminate the infectious agents . We have analyzed the role of dietary lipids on immune functions in cells cultured with Listeria monocytogenes . Bactericidal efficiency of peritoneal cells from mice fed a fish oil diet against this bacterium was reduced and the incubation of peritoneal cells with polyunsaturated fatty acids led to similar results . The levels of superoxide radicals in the presence of L . monocytogenes increased in cells from mice fed olive oil or fish oil diets . Proteasome activity, a mechanism that participates in T cell activation, was inhibited in all of the dietary groups assayed in the presence of L . monocytogenes, but this inhibition was abolished in the presence of both MG132 (a proteasome inhibitor) and L . monocytogenes . Overall, these results underline the potential role of fatty acids in the modulation of many functions of the immune system. Infect Immun, 2002 Jan, 70(1), 416 - 8 STK receptor tyrosine kinase regulates susceptibility to infection with Listeria monocytogenes; Lutz MA et al.; We have previously identified the STK receptor tyrosine kinase as a key regulator of macrophage activation and cell-mediated immune responses . Here we demonstrate that, although MSP activation of STK inhibits NO production by macrophages in response to heat-killed Listeria monocytogenes, STK-deficient mice exhibit increased susceptibility to infection with Listeria. Infect Immun, 2002 Jan, 70(1), 153 - 62 CD8(+)-T-cell response to secreted and nonsecreted antigens delivered by recombinant Listeria monocytogenes during secondary infection; Tvinnereim AR et al.; Understanding how existing antivector immunity impacts live vaccine delivery systems is critical when the same vector system may be used to deliver different antigens . We addressed the impact of antivector immunity, elicited by immunization with attenuated actA-deficient Listeria monocytogenes, on the CD8(+)-T-cell response to a well-characterized lymphocytic choriomeningitis virus epitope, NP118-126, delivered by infection with recombinant L . monocytogenes . Challenges of immune mice with actA-deficient and with wild-type recombinant L . monocytogenes generated similar numbers of CD8(+) T cells specific for the NP118-126 epitope . High-dose immunization with actA-deficient L . monocytogenes resulted in substantial numbers of CD8(+) T cells specific for the L . monocytogenes LLO91-99 epitope in the effector and memory stages of the T-cell response . Challenge of these immune mice with recombinant L . monocytogenes resulted in rapid control of the infection and decreased CD8(+)-T-cell responses against both the secreted and nonsecreted form of the recombinant antigen compared to the response of naive mice . In contrast, mice immunized with a low dose of actA-deficient L . monocytogenes had approximately 10-fold fewer effector and memory T cells specific for LLO91-99 and a substantially higher CD8(+)-T-cell response against the recombinant antigen after challenge with recombinant L . monocytogenes . Although mice immunized with low-dose actA-deficient L . monocytogenes had a substantial recall response to LLO91-99, which reached the same levels by 5 to 7 days postchallenge as that in high-dose-immunized mice, they exhibited decreased ability to control L . monocytogenes replication . Thus, the level of antivector immunity impacts the control of infection and efficiency of priming responses against new antigens introduced with the same vector. Enferm Infecc Microbiol Clin, 2001 Aug-Sep, 19(7), 297 - 303 {Listeria monocytogenes infections in the adult . Clinical and microbiological issues of a changing disease}; Julian A et al.; Thirty-one cases of human listeriosis seen from 1971-1999 were reviewed . cases were grouped as follows: Group I composed of 14 patients were studied in the period 1971-1984; and group II composed of 17 cases studied in the period 1985-1999 . We tried to assess changes in the incidence, clinical findings and outcome in both periods . The incidence of listeriosis remained constant along the years, 1.2 cases/20,000 discharges.The mean age of the patients significantly increased along the years (55 11 years versus 68 12 years; p 0.002) . 77% of cases had one or more underlying diseases predisposing to listeriosis . We observed an increasing number of listeriosis in patients without chronic diseases in recent years . Listeriosis presented as meningitis or primary sepsis . Mortality was 61% and was strictly associated with the severity of the underlying disease . Patients with meningoencephalitis and seizures had a worse prognosis . We did not observe differences in mortality of patients who were treated with beta-lactam monotherapy in comparison with those who were treated with beta-lactam/aminoglucoside combination . Cotrimoxazole was uniformly successful treatment of human listeriosis in this series. Emerg Infect Dis, 2001 Nov-Dec, 7(6), 983 - 9 Effect of prevention measures on incidence of human listeriosis, France, 1987-1997; Goulet V et al.; To assess the impact of preventive measures by the food industry, we analyzed food monitoring data as well as trends in the incidence of listeriosis estimated through three independent sources: the National Reference Center of Listeriosis; a laboratory-based active surveillance network; and two consecutive nationwide surveys of public hospital laboratories . From 1987 to 1997, the incidence of listeriosis decreased by an estimated 68% . A substantial reduction in the proportion of Listeria monocytogenes-contaminated products was observed at the retail level . The temporal relationship between prevention measures by the food industry, reduction in L . monocytogenes-contaminated foodstuffs, and reduction in listeriosis incidence suggests a causal relationship and indicates that a substantial part of the reduction in illness is related to prevention efforts. Eur J Immunol, 2001 Dec, 31(12), 3714 - 25 PUMA-G, an IFN-gamma-inducible gene in macrophages is a novel member of the seven transmembrane spanning receptor superfamily; Schaub A et al.; IFN-gamma is a key immunoregulatory cytokine that plays a predominant role in innate immunity . By employing PCR-Select to search for genes differentially expressed in IFN-gamma/TNF-alpha stimulated macrophages, we identified a novel IFN-gamma-induced transcript designated PUMA-G (protein up-regulated in macrophages by IFN-gamma) . PUMA-G codes for a protein with seven transmembrane helices, a feature commonly shared with the G protein-coupled receptor superfamily (GPCR) . The PUMA-G protein is most similar to the human orphan GPCR HM74 (73 % identity) and GPR31 (30 % identity) . PUMA-G mRNA was readily induced in macrophages after stimulation with IFN-gamma, LPS, polyIC and CpG oligonucleotides . In vivo PUMA-G was up-regulated in mice suffering from microbial sepsis or from Listeria monocytogenes infection . Characterization of the genomic locus revealed an intronless PUMA-G open reading frame . Genomic Southern blot analysis indicates that PUMA-G is a single-copy gene . PUMA-G maps to mouse chromosome 5F . Confocal microscopy of transiently transfected 264.7 RAW macrophages and 293T cells with a PUMA-G-EGFP fusion construct showed predominant fluorescence at the cell surface, suggesting a localization at the cell membrane . Taken together, our data indicate that PUMA-G is a new inducible representative of GPCR, with potential importance in macrophage functions. J Immunol, 2001 Dec 15, 167(12), 6859 - 68 Immunoproteasomes largely replace constitutive proteasomes during an antiviral and antibacterial immune response in the liver; Khan S et al.; The proteasome is critically involved in the production of MHC class I-restricted T cell epitopes . Proteasome activity and epitope production are altered by IFN-gamma treatment, which leads to a gradual replacement of constitutive proteasomes by immunoproteasomes in vitro . However, a quantitative analysis of changes in the steady state subunit composition of proteasomes during an immune response against viruses or bacteria in vivo has not been reported . Here we show that the infection of mice with lymphocytic choriomeningitis virus or Listeria monocytogenes leads to an almost complete replacement of constitutive proteasomes by immunoproteasomes in the liver within 7 days . Proteasome replacements were markedly reduced in IFN-gamma(-/-) mice, but were only slightly affected in IFN-alphaR(-/-) and perforin(-/-) mice . The proteasome regulator PA28alpha/beta was up-regulated, whereas PA28gamma was reduced in the liver of lymphocytic choriomeningitis virus-infected mice . Proteasome replacements in the liver strongly altered proteasome activity and were unexpected to this extent, since an in vivo half-life of 12 days had been previously assigned to constitutive proteasomes in the liver . Our results suggest that during the peak phase of viral and bacterial elimination the antiviral cytotoxic T lymphocyte response is directed mainly to immunoproteasome-dependent T cell epitopes, which would be a novel parameter for the design of vaccines. Mol Microbiol, 2001 Nov, 42(4), 955 - 65 Synergy between the N- and C-terminal domains of InlB for efficient invasion of non-phagocytic cells by Listeria monocytogenes; Jonquieres R et al.; InlB is a Listeria monocytogenes protein promoting entry in non-phagocytic cells, and has been shown recently to activate the hepatocyte growth factor receptor (HGFR or Met) . The N-terminal domain of InlB (LRRs) binds and activates Met, whereas the C-terminal domain of InlB (GW modules) mediates loose attachment of InlB to the listerial surface . As HGF activation of Met is tightly controlled by glycosaminoglycans (GAGs), we tested if GAGs also modulate the Met-InlB interactions . We show that InlB-dependent invasion of non-phagocytic cells decreases up to 10 times in the absence of GAGs, and that soluble heparin releases InlB from the bacterial surface and promotes its clustering . Furthermore, we demonstrate that InlB binds cellular GAGs by its GW modules, and that this interaction is required for efficient InlB-mediated invasion . Therefore, GW modules have an unsuspected dual function: they attach InlB to the bacterial surface and enhance entry triggered by the LRRs domain . Our results thus provide the first evidence for a synergy between two host factor-binding domains of a bacterial invasion protein, and reinforce similarities between InlB and mammalian growth factors. Cell Microbiol, 2001 Dec, 3(12), 853 - 64 A role for ActA in epithelial cell invasion by Listeria monocytogenes; Suarez M et al.; We assessed the role of the actin-polymerizing protein, ActA, in host cell invasion by Listeria monocytogenes . An in frame DeltaactA mutant was constructed in a hyperinvasive strain of prfA* genotype, in which all genes of the PrfA-dependent virulence regulon, including actA, are highly expressed in vitro . Loss of ActA production in prfA* bacteria reduced entry into Caco-2, HeLa, MDCK and Vero epithelial cells to basal levels . Reintroduction of actA into the DeltaactA prfA* mutant fully restored invasiveness, demonstrating that ActA is involved in epithelial cell invasion . ActA did not contribute to internalization by COS-1 fibroblasts and Hepa 1-6 hepatocytes . Expression of actA in Listeria innocua was sufficient to promote entry of this non-invasive species into epithelial cell lines, but not into COS-1 and Hepa 1-6 cells, indicating that ActA directs an internalization pathway specific for epithelial cells . Scanning electron microscopy of infected Caco-2 human enterocytes suggested that this pathway involves microvilli . prfA* bacteria, but not wild-type bacteria (which express PrfA-dependent genes very weakly in vitro) or prfA* DeltaactA bacteria, efficiently invaded differentiated Caco-2 cells via their apical surface . Microvilli played an active role in the phagocytosis of the prfA* strain, and actA was required for their remodelling into pseudopods mediating bacterial uptake . Thus, ActA appears to be a multifunctional virulence factor involved in two important aspects of Listeria pathogenesis: actin-based motility and host cell tropism and invasion. Mol Cell Probes, 2001 Oct, 15(5), 275 - 80 Rapid detection of Listeria monocytogenes in foods, by a combination of PCR and DNA probe; Ingianni A et al.; Listeria monocytogenes is a frequent contaminant of water and foods . Its rapid detection is needed before some foods can be prepared for marketing . In this work L . monocytogenes has been searched for in foods, by a combination of polymerase chain reaction (PCR) and a DNA probe . Both PCR and the probe were prepared for recognizing a specific region of the internalin gene, which is responsible for the production of one of the most important pathogenic factors of Listeria . The combined use of PCR and the DNA probe was used for the detection of L . monocytogenes in over 180 environmental and food samples . Several detection methods were compared in this study, namely conventional culture methods; direct PCR; PCR after an enrichment step; a DNA probe alone; a DNA probe after enrichment and another commercially available gene-probe . Finally PCR and the DNA probe were used in series on all the samples collected . When the DNA probe was associated with the PCR, specific and accurate detection of listeria in the samples could be obtained in about a working-day . The present molecular method showed some advantages in terms of rapidity and specificity in comparison to the other aforementioned tests . In addition, it resulted as being easy to handle, even for non-specialized personnel in small diagnostic microbiology laboratories . Rev Sci Tech, 2001 Dec, 20(3), 777 - 83 First report of an outbreak of ovine septicaemic listeriosis in Saudi Arabia; al-Dughaym AM et al.; Although a case of human listeriosis has recently been reported in Saudi Arabia, the disease has not been reported in animals to date . The authors describe an outbreak of septicaemic listeriosis in sheep, which occurred during winter . Adult animals and pregnant ewes were principally affected, with a morbidity rate of 7.1% and a mortality rate of 2.4%; no abortions were recorded during the outbreak . Clinical signs included inappetence, weakness, fever, respiratory distress, keratoconjunctivitis and compulsive circling . Listeria monocytogenes was isolated in pure culture from affected sheep . Pathological findings indicated septicaemic listeriosis with encephalitis . Hygienic measures and antibiotics were successful in treating the rest of the flock . Aspects of the outbreak and public health implications are discussed. Commun Dis Public Health, 2001 Sep, 4(3), 188 - 93 Surveillance of listeriosis in England and Wales, 1995-1999; Smerdon WJ et al.; A total of 543 cases of listerIosis were ascertained by the Public Health Laboratory Service Communicable Disease Surveillance Centre (PHLS CDSC) in residents of England and Wales with onset of illness between 1st January 1995 and 31st December 1999 . Of 452 non-pregnancy associated cases, 326 (72%) had major medical conditions, 140 (31%) had received immuno-suppressive therapy and 97 (21%) were aged 60 years or over . Serovar 4b predominated in both pregnancy and non-pregnancy associated cases . Of ninety-one pregnancy associated cases, five had underlying medical conditions . Foetal death or early neonatal death was reported in 24 (26%) of the cases giving a mean perinatal mortality rate of 7.5 per million conceptions per year . Two episodes of neonatal cross infection occurred . Two non-pregnancy associated cases in 1999 were linked by a common food; all other cases were unlinked . No trend was observed from 1995 to 1999. J Food Prot, 2001 Nov, 64(11), 1739 - 43 Detection of heat injury in Listeria monocytogenes Scott A; Novak JS et al.; Methods of detecting live pathogens in foods that may be growth inhibited following heat treatment are essential to food safety . Among the techniques available, reverse transcription polymerase chain reaction (RT-PCR) amplification of messenger RNA from heat-injured Listeria monocytogenes Scott A is preferable to direct PCR in an attempt to avoid false positives from dead cells . The RT-PCR has a detection limit of 3 x 10(6) CFU/g, compared to 3 CFU/g for untreated controls, but may not be suitable for the identification of all viable cells . Physically apparent changes in cellular structures from heat injury in L . monocytogenes are expected to result . Ultrastructural analyses did depict notable heat damage as cytoplasmic clearing after 5 min at 60 degrees C . The heat-injured survivors can be readily distinguished from total viable cells using selective media . As a result, combinations of molecular and visual methods including selective media improve detectability of heat-injured, viable L . monocytogenes Scott A. J Food Prot, 2001 Nov, 64(11), 1730 - 8 Reduction of listeria monocytogenes on green peppers (Capsicum annuum L.) by gaseous and aqueous chlorine dioxide and water washing and its growth at 7 degrees C; Han Y et al.; Reduction of Listeria monocytogenes Scott A on uninjured and injured surfaces of green peppers after 0.3- and 3-mg/ liter gaseous and aqueous ClO2 treatment and water washing for 10 min at 20 degrees C was studied . Growth of the L . monocytogenes untreated or treated with 0.6 mg/liter ClO2 gas for 30 min at 20 degrees C on green peppers also was investigated . A membrane-surface-plating method was used for resuscitation and enumeration of L monocytogenes treated with ClO2 . The bacterial viability on pepper surfaces was visualized using confocal laser scanning microscopy (CLSM) . Live and dead cells of L . monocytogenes were labeled with a fluorescein isothiocyanate-labeled antibody and propidium iodide, respectively . More than 6 log CFU/5 g L . monocytogenes on uninjured surfaces and about 3.5 log CFU/5 g on injured surfaces were inactivated by both 3-mg/liter and 0.6-mg/liter ClO2 gas treatments . The 3-mg/liter aqueous ClO2 treatment achieved 3.7- and 0.4-log reductions on uninjured and injured surfaces, respectively; whereas, water washing alone showed 1.4- and 0.4-log reductions, respectively . ClO2 gas treatment was the most effective in reducing L . monocytogenes on both uninjured and injured green pepper surfaces, when compared with aqueous ClO2 treatment and water washing . The significant difference (P < 0.05) between log reductions on uninjured and injured surfaces and the results from CLSM analysis suggested that injured surfaces protected more bacteria from sanitation treatments than did uninjured surfaces . Not only could L . monocytogenes grow on green pepper surfaces at 7 degrees C, bacteria that survived the 0.6-mg/liter ClO2 gas treatment also could grow. J Food Prot, 2001 Nov, 64(11), 1722 - 9 Organic acids and their salts as dipping solutions to control listeria monocytogenes inoculated following processing of sliced pork bologna stored at 4 degrees C in vacuum packages; Samelis J et al.; Postprocessing contamination of cured meats with Listeria monocytogenes has become a major concern for the meat processing industry and an important food safety issue . This study evaluated aqueous dipping solutions of organic acids (2.5 or 5% lactic or acetic acid) or salts (2.5 or 5% sodium acetate or sodium diacetate, 5 or 10% sodium lactate, 5% potassium sorbate or potassium benzoate) to control L . monocytogenes on sliced, vacuum-packaged bologna stored at 4 degrees C for up to 120 days . Organic acids and salts were applied by immersing (1 min) in each solution inoculated (10(2) to 10(3) CFU/cm2) slices of bologna before vacuum packaging . Growth of L . monocytogenes (PALCAM agar) on inoculated bologna slices without treatment exceeded 7 log CFU/cm2 (P < 0.05) at 20 days of storage . No significant (P > 0.05) increase in L . monocytogenes populations occurred on bologna slices treated with 2.5 or 5% acetic acid, 5% sodium diacetate, or 5% potassium benzoate from day 0 to 120 . Products treated with 5% potassium sorbate and 5% lactic acid were stored for 50 and 90 days, respectively, before a significant (P < 0.05) increase in L . monocytogenes occurred . All other treatments permitted growth of the pathogen at earlier days of storage, with sodium lactate (5 or 10%) permitting growth within 20 to 35 days . Extent of bacterial growth on trypticase soy agar plus 0.6% yeast extract (TSAYE) was similar to that on PALCAM, indicating that the major part of total bacteria grown on TSAYE agar plates incubated at 30 degrees C was L . monocytogenes . Further studies are needed to evaluate organic acids and salts as dipping solutions at abusive temperatures of retail storage, to optimize their concentrations in terms of product sensory quality, and to evaluate their effects against various other types of microorganisms and on product shelf life . In addition, technologies for the commercial application of postprocessing antimicrobial solutions in meat plants need to be developed. Appl Environ Microbiol, 2001 Dec, 67(12), 5840 - 3 Molecular typing by pulsed-field gel electrophoresis of Spanish animal and human Listeria monocytogenes isolates; Vela AI et al.; A total of 153 strains of Listeria monocytogenes isolated from different sources (72 from sheep, 12 from cattle, 18 from feedstuffs, and 51 from humans) in Spain from 1989 to 2000 were characterized by pulsed-field gel electrophoresis . The strains of L . monocytogenes displayed 55 pulsotypes . The 84 animal, 51 human, and 18 feedstuff strains displayed 31, 29, and 7 different pulsotypes, respectively, indicating a great genetic diversity among the Spanish L . monocytogenes isolates studied . L . monocytogenes isolates from clinical samples and feedstuffs consumed by the diseased animals were analyzed in 21 flocks . In most cases, clinical strains from different animals of the same flock had identical pulsotypes, confirming the existence of a listeriosis outbreak . L . monocytogenes strains with pulsotypes identical to those of clinical strains were isolated from silage, potatoes, and maize stalks . This is the first study wherein potatoes and maize stalks are epidemiologically linked with clinical listeriosis. Vet Ophthalmol, 2001 Sep, 4(3), 217 - 9 Listeria keratitis in a horse; Sanchez S et al.; Listeria monocytogenes is ubiquitous in the environment but is rarely reported as a cause of keratitis in animals . In this case, a mare was presented with epiphora and evidence of pain in the right eye . Listeria monocytogenes was isolated from a corneal lesion, and bacteria were also seen in the cytologic evaluation . This is the first reported case of ulcerative keratitis associated with L . monocytogenes in a horse. J Appl Microbiol, 2001 Nov, 91(5), 888 - 99 Molecular epidemiology of Listeria monocytogenes isolates collected from the environment, raw meat and raw products in two poultry- and pork-processing plants; Chasseignaux E et al.; AIMS: In order to study the transmission of Listeria monocytogenes in a poultry and a pork meat plant, we analysed the contamination by this pathogen over several months . METHODS AND RESULTS: Five hundred and two isolates of L . monocytogenes were collected and characterized by genotyping and serotyping . Thirty-seven genotypes were obtained by ApaI-restriction analysis-pulsed field gel electrophoresis (REA-PFGE) and 35 by SmaI-REA-PFGE and resulted in 50 combined genotypes . The tracing of the contamination in both plants showed that some clones were able to survive for several months . However, some other clones were found only during processing operations, were not detectable after cleaning and seemed to enter continuously into the plant . CONCLUSIONS: Some L . monocytogenes strains may persist for a long period in the plant environment . Different genotypes can be associated with poultry as well as pork meat . SIGNIFICANCE AND IMPACT OF THE STUDY: Listeria monocytogenes contamination can be due to contaminated raw materials, bacterial spread and also ineffective cleaning procedures. MLO Med Lab Obs, 2001 Nov, 33(11), 8 - 15; quiz 16-9 Serologic markers in inflammatory bowel disease (IBD); Nakamura RM et al.; Inflammatory bowel disease (IBD) is a generic term that refers to Crohn's disease and ulcerative colitis . Crohn's disease (CD) is a granulomatous enteritis which can involve the ileum, colon, and other parts of the intestinal tract . The serologic responses seen in Crohn's disease include antibodies to Saccharomyces cerevisiae, mycobacteria, bacteroides, listeria and E . coli . Many of these organisms may be involved in the pathogenesis of the Crohn's disease . Ulcerative colitis is characterized by inflammation of the mucosa and submucosa of the large intestine . The CD and UC are considered to be distinct forms of IBD; however, there is a subgroup of CD with a UC-like presentation . In recent years, several serologic markers have been found to be useful for the diagnosis and differentiation of CD and UC . These markers include the following antibodies (a) 2pANCA, (b) ASCA, (c) pancreatic antibody, and (d) OmpC antibody . The application of a panel of markers with the use of an algorithm can identify specific subtypes of IBD that have different clinical courses and progression of the diseases . The application of the serologic markers is useful for diagnosis and management of CD and UC patients. Science, 2001 Nov 23, 294(5547), 1735 - 9 Priming of memory but not effector CD8 T cells by a killed bacterial vaccine; Lauvau G et al.; Killed or inactivated vaccines targeting intracellular bacterial and protozoal pathogens are notoriously ineffective at generating protective immunity . For example, vaccination with heat-killed Listeria monocytogenes (HKLM) is not protective, although infection with live L . monocytogenes induces long-lived, CD8 T cell-mediated immunity . We demonstrate that HKLM immunization primes memory CD8 T lymphocyte populations that, although substantial in size, are ineffective at providing protection from subsequent L . monocytogenes infection . In contrast to live infection, which elicits large numbers of effector CD8 T cells, HKLM immunization primes T lymphocytes that do not acquire effector functions . Our studies show that it is possible to dissociate T cell-dependent protective immunity from memory T cell expansion, and that generation of effector T cells may be necessary for long-term protective immunity. Biophys J, 2001 Dec, 81(6), 3193 - 203 Effects of intermediate filaments on actin-based motility of Listeria monocytogenes; Giardini PA et al.; How does subcellular architecture influence the intracellular movements of large organelles and macromolecular assemblies? To investigate the effects of mechanical changes in cytoplasmic structure on intracellular motility, we have characterized the actin-based motility of the intracellular bacterial pathogen Listeria monocytogenes in normal mouse fibroblasts and in fibroblasts lacking intermediate filaments . The apparent diffusion coefficient of L . monocytogenes was two-fold greater in vimentin-null fibroblasts than in wild-type fibroblasts, indicating that intermediate filaments significantly restrict the Brownian motion of bacteria . However, the mean speed of L . monocytogenes actin-based motility was statistically identical in vimentin-null and wild-type cells . Thus, environmental drag is not rate limiting for bacterial motility . Analysis of the temporal variations in speed measurements indicated that bacteria in vimentin-null cells displayed larger fluctuations in speed than did trajectories in wild-type cells . Similarly, the presence of the vimentin meshwork influenced the turning behavior of the bacteria; in the vimentin-null cells, bacteria made sharper turns than they did in wild-type cells . Taken together, these results suggest that a network of intermediate filaments constrains bacterial movement and operates over distances of several microns to reduce fluctuations in motile behavior. FEMS Immunol Med Microbiol, 2001 Oct, 31(3), 219 - 25 Uptake and killing of Listeria monocytogenes by normal human peripheral blood granulocytes and monocytes as measured by flow cytometry and cell sorting; Raybourne RB et al.; Cellular components of innate immunity (NK cells, monocytes and granulocytes) play an important role in early resistance to Listeria monocytogenes in the mouse model . Minimally invasive methods of measuring the bacteriocidal capacity of these cells may be useful as a biomarker of susceptibility in humans . A technique was developed whereby the uptake and survival of L . monocytogenes could be measured in human granulocytes and monocytes using small volumes of peripheral blood . This method used flow cytometry to detect the presence of PKH-2-labeled bacteria within these cells . Survival of bacteria was determined by sorting of infected cells based on a combination of fluorescence and light scattering properties . Considerable variation in bacterial recovery was seen between normal volunteers . There was consistently greater survival of a fully virulent strain of L . monocytogenes within monocytes and granulocytes compared with an isogenic strain lacking the hemolysin, listeriolysin O, when measured at baseline . There was no evidence of longer-term bacterial survival or growth at 2 or 24 h . This technique may be useful for assessment of both host resistance and pathogen virulence. Acta Microbiol Pol, 2001, 50(2), 155 - 60 Cloning and characterization of M.LmoA118I, a novel DNA:m4C methyltransferase from the Listeria monocytogenes phage A118, a close homolog of M.NgoMXV; Bujnicki JM et al.; A homolog of M.NgoMXV DNA:m4C methyltransferase has been identified among the open reading frames deduced from the genomic sequence of Listeria monocytogenes phage A118 {Loessner et al., 2000} . The gene coding for this putative protein has been cloned in Escherichia coli and its enzymatic activity in vivo in this host have been analyzed . Remarkably, despite M.NgoMXV and M.LmoA118I exhibit high sequence similarity (58% identical and 19% conservatively substituted residues), their target preferences differ: both proteins exhibit "relaxed" sequence specificity, but while M.LmoA118I more efficiently methylates GGCC sites, it seems to target only a subset of CCWGG sites methylated by M.NgoMXV. New Microbiol, 2001 Oct, 24(4), 333 - 9 Detection of Listeria monocytogenes by polymerase chain reaction oriented to inlB gene; Pangallo D et al.; Primers were designed for the detection of Listeria monocytogenes by the polymerase chain reaction oriented to specific sequences of the inlB gene encoding an internalin . At optimized reaction conditions, 100% sensitivity (on a panel of 33 strains of L . monocytogenes) and 100% specificity (on panels of 15 strains of other Listeria spp . and 41 other bacteria), were determined for the inlB-L/R primers . The detection limit of PCR with these primers was 10(4) cfu/ml and was not affected by up to 10(8) cfu/ml of L . innocua. J Immunol, 2001 Dec 1, 167(11), 6480 - 6 Immunization with gp96 from Listeria monocytogenes-infected mice is due to N-formylated listerial peptides; Sponaas AM et al.; N-Formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2-M3-restricted CD8 T cell response in C57BL/6 mice . N-f-met peptide-specific CTL were generated in vitro when mice previously immunized with gp96 isolated from donor mice infected with L . monocytogenes were stimulated with these peptides . No significant peptide-specific CTL activity was observed in mice immunized with gp96 from uninfected animals . Masses corresponding to one N-f-met peptide were found by matrix-assisted laser desorption/ionization-mass spectrometry on gp96 isolated from C57BL/6 mice infected with L . monocytogenes, but not on gp96 from noninfected mice . Therefore, bacterial N-f-met peptides from intracellular bacteria can bind to gp96 in the infected host, and gp96 loaded with these peptides can generate N-f-met-peptide-specific CTL . We assume a unique role of gp96 in Ag processing through the H2-M3 pathway. J Immunol, 2001 Dec 1, 167(11), 6471 - 9 Two Listeria monocytogenes vaccine vectors that express different molecular forms of human papilloma virus-16 (HPV-16) E7 induce qualitatively different T cell immunity that correlates with their ability to induce regression of established tumors immortalized by HPV-16; Gunn GR et al.; Two recombinant Listeria monocytogenes (rLm) strains were produced that secrete the human papilloma virus-16 (HPV-16) E7 protein expressed in HPV-16-associated cervical cancer cells . One, Lm-E7, expresses and secretes E7 protein, whereas a second, Lm-LLO-E7, secretes E7 as a fusion protein joined to a nonhemolytic listeriolysin O (LLO) . Lm-LLO-E7, but not Lm-E7, induces the regression of the E7-expressing tumor, TC-1, established in syngeneic C57BL/6 mice . Both recombinant E7-expressing rLm vaccines induce measurable anti-E7 CTL responses that stain positively for H-2D(b) E7 tetramers . Depletion of the CD8+ T cell subset before treatment abrogates the ability of Lm-LLO-E7 to impact on tumor growth . In addition, the rLm strains induce markedly different CD4+ T cell subsets . Depletion of the CD4+ T cell subset considerably reduces the ability of Lm-LLO-E7 to eliminate established TC-1 tumors . Surprisingly, the reverse is the case for Lm-E7, which becomes an effective anti-tumor immunotherapeutic in mice lacking this T cell subset . Ab-mediated depletion of TGF-beta and CD25+ cells improves the effectiveness of Lm-E7 treatment, suggesting that TGF-beta and CD25+ cells are in part responsible for this suppressive response . CD4+ T cells from mice immunized with Lm-E7 are capable of suppressing the ability of Lm-LLO-E7 to induce the regression of TC-1 when transferred to tumor-bearing mice . These studies demonstrate the complexity of L . monocytogenes-mediated tumor immunotherapy targeting the human tumor Ag, HPV-16 E7. J Immunol, 2001 Dec 1, 167(11), 6180 - 7 Tolerance or immunity to a tumor antigen expressed in somatic cells can be determined by systemic proinflammatory signals at the time of first antigen exposure; Frazer IH et al.; Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells . Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors . Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin . Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L . monocytogenes exposure, whereas recipients of an E7 graft given without L . monocytogenes do not reject a second graft, even if given with L . monocytogenes . Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag . Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor . These observations have implications for immunotherapy for epithelial cancers. Infect Immun, 2001 Dec, 69(12), 7234 - 41 Effect of 6-hydroxydopamine on host resistance against Listeria monocytogenes infection; Miura T et al.; Recent studies have shown that immunocompetent cells bear receptors of neuropeptides and neurotransmitters and that these ligands play roles in the immune response . In this study, the role of the sympathetic nervous system in host resistance against Listeria monocytogenes infection was investigated in mice pretreated with 6-hydroxydopamine (6-OHDA), which destroys sympathetic nerve termini . The norepinephrine contents of the plasma and spleens were significantly lower in 6-OHDA-treated mice than in vehicle-treated mice . The 50% lethal dose of L . monocytogenes was about 20 times higher for 6-OHDA-treated mice than for vehicle-treated mice . Chemical sympathectomy by 6-OHDA upregulated interleukin-12 (IL-12) and tumor necrosis factor-alpha (TNF-alpha) production in enriched dendritic cell cultures and gamma interferon (IFN-gamma) and TNF-alpha production in spleen cell cultures, whereas chemical sympathectomy had no apparent effect on phagocytic activities, listericidal activities, and nitric oxide production in peritoneal exudate cells and splenic macrophages . Augmentation of host resistance against L . monocytogenes infection by 6-OHDA was abrogated in IFN-gamma(-/-) or TNF-alpha(-/-) mice, suggesting that upregulation of IFN-gamma, IL-12, and TNF-alpha production may be involved in 6-OHDA-mediated augmentation of antilisterial resistance . Furthermore, adoptive transfer of spleen cells immune to L . monocytogenes from 6-OHDA-treated mice resulted in untreated naive recipients that had a high level of resistance against L . monocytogenes infection . These results suggest that the sympathetic nervous system may modulate host resistance against L . monocytogenes infection through regulation of production of IFN-gamma, IL-12, and TNF-alpha, which are critical in antilisterial resistance. Infect Immun, 2001 Dec, 69(12), 7213 - 23 Exaggerated proinflammatory and Th1 responses in the absence of gamma/delta T cells after infection with Listeria monocytogenes; Skeen MJ et al.; While gamma/delta T cells are involved in host defense and immunopathology in a variety of infectious diseases, their precise role is not yet clearly defined . In the absence of gamma/delta T cells, mice die after infection with a dose of Listeria monocytogenes that is not lethal in immunologically intact animals . Morbidity might result from insufficient levels of cytokines normally produced by gamma/delta T cells or conversely from an excess of cytokines due to a lack of down-regulation of the inflammatory response in the absence of gamma/delta T cells . Consistent with a regulatory role, we found that systemic levels of proinflammatory cytokines (interleukin-6 {IL-6}, IL-12, and gamma interferon {IFN-gamma}) were significantly higher in the absence of gamma/delta T cells during the innate phase of the response . Using combinations of genetically altered and immunodepleted mice, we found evidence for gamma/delta T-cell-mediated regulation of IFN-gamma production by multiple cell types of both lymphoid and myeloid lineages . The antigen-specific alpha/beta T-cell response that followed the exaggerated innate response was also increased in gamma/delta T-cell-deficient mice . These findings are consistent with an emerging picture from a variety of immune response models of a critical role for gamma/delta T cells in down-modulation of the immune response. Biochemistry, 2001 Nov 20, 40(46), 14037 - 46 Conformational changes in pediocin AcH upon vesicle binding and approximation of the membrane-bound structure in detergent micelles; Watson RM et al.; Pediocin AcH is a 44-residue antimicrobial peptide with bactericidal potency against Gram-positive bacteria such as Listeria . It belongs to a family of bacteriocins that, when membrane-associated, is predicted to contain beta-sheet and alpha-helical regions . All bacteriocins in this family have a conserved N-terminal disulfide bond . An additional C-terminal disulfide bond in pediocin AcH is thought to confer enhanced potency and broader specificity range against sensitive bacteria . The C-terminal disulfide bond may also affect the conformation of the C-terminus . The secondary structures of pediocin AcH in aqueous solution and vesicles from susceptible cells, as well as the ability of trifluoroethanol (TFE) and detergent systems to induce secondary structures like those induced in vesicles, were studied by circular dichroism (CD) spectroscopy . Like related peptides, pediocin AcH was highly unordered in aqueous solution, 56% . However, it also contained 20% beta-strand and 15% beta-turn structures . Upon complete binding to vesicles, 32% alpha-helical structure formed, the unordered structure decreased to 32%, and the beta-strand and beta-turn structures remained largely unchanged . Thus, a betaalpha domain structure formed in vesicles . The helical structure likely forces the C-terminal tail to loop back on the helix so that the C24-C44 disulfide bond can form . Detergent micelles were superior to TFE in their ability to induce secondary structural fractions in pediocin AcH comparable to those observed in vesicles . This demonstrates the importance of a hydrocarbon-water interface to pediocin AcH structure induction and suggests that it is preferable to use detergent micelles as solvents in NMR studies of pediocin AcH structure. Am J Epidemiol, 2001 Nov 15, 154(10), 944 - 50 Two consecutive nationwide outbreaks of Listeriosis in France, October 1999-February 2000; de Valk H et al.; In France, listeriosis surveillance is based on mandatory notification of all culture-confirmed cases, with systematic typing of isolates and routine collection of the patient's food history . From October 1999 to March 2000, two outbreaks of listeriosis were detected through this enhanced surveillance system . In outbreak 1, analysis of the food histories of cases suggested brand X "rillettes," a pate-like meat product, as the vehicle of infection, and the outbreak strain of Listeria monocytogenes was subsequently isolated from the incriminated rillettes . In outbreak 2, a case-control study showed that consumption of jellied pork tongue was strongly associated with infection with the outbreak strain (odds ratio = 75.5, 95% confidence interval: 4.7, 1,216.0) . However, trace-back results did not permit incrimination of any particular manufacturer of jellied pork tongue, and the outbreak strain was not isolated from the incriminated food or from any production sites . Consumption of jellied pork tongue was discouraged on epidemiologic evidence alone . The consecutive occurrence of these two outbreaks confirms the epidemic potential of listeriosis, even in a context of decreasing incidence, and underlines the importance of timely case-reporting and systematic typing of human L . monocytogenes strains to allow early detection and separate investigation of different clusters. J Immunol, 2001 Nov 15, 167(10), 5620 - 7 Role of CD28 for the generation and expansion of antigen-specific CD8(+) T lymphocytes during infection with Listeria monocytogenes; Mittrucker HW et al.; Infection of mice with the intracellular bacterium Listeria monocytogenes results in a strong CD8(+) T cell response that is critical for efficient control of infection . We used CD28-deficient mice to characterize the function of CD28 during Listeria infection, with a main emphasis on Listeria-specific CD8(+) T cells . Frequencies and effector functions of these T cells were determined using MHC class I tetramers, single cell IFN-gamma production and Listeria-specific cytotoxicity . During primary Listeria infection of CD28(-/-) mice we observed significantly reduced numbers of Listeria-specific CD8(+) T cells and only marginal levels of specific IFN-gamma production and cytotoxicity . Although frequencies were also reduced in CD28(-/-) mice during secondary response, we detected a considerable population of Listeria-specific CD8(+) T cells in these mice . In parallel, IFN-gamma production and cytotoxicity were observed, revealing that Listeria-specific CD8(+) T cells in CD28(-/-) mice expressed normal effector functions . Consistent with their impaired CD8(+) T cell activation, CD28(-/-) mice suffered from exacerbated listeriosis both after primary and secondary infection . These results demonstrate participation of CD28 signaling in the generation and expansion of Ag-specific CD8(+) T cells in listeriosis . However, Ag-specific CD8(+) T cells generated in the absence of CD28 differentiated into normal effector and memory T cells. J Immunol, 2001 Nov 15, 167(10), 5603 - 9 Listeria monocytogenes infection overcomes the requirement for CD40 ligand in exogenous antigen presentation to CD8(+) T cells; Hamilton SE et al.; In vivo priming of CD8(+) T lymphocytes against exogenously processed model Ags requires CD4(+) T cell help, specifically interactions between CD40 ligand (CD40L) expressed by activated CD4(+) T cells and CD40, which is present on professional APC such as dendritic cells (DCs) . To address this issue in the context of bacterial infection, we examined CD40L-CD40 interactions in CD8(+) T cell priming against an exogenously processed, nonsecreted bacterial Ag . CD40L interactions were blocked by in vivo treatment with anti-CD40L mAb MR-1, which inhibited germinal center formation and CD8(+) T cell cross-priming against an exogenous model Ag, OVA . In contrast, MR-1 treatment did not interfere with CD8(+) T cell priming against a nonsecreted or secreted recombinant Ag expressed by Listeria monocytogenes . Memory and secondary responses of CD8(+) T cells against nonsecreted and secreted bacterial Ags were also largely unimpaired by transient MR-1 treatment . When MR-1-treated mice were concurrently immunized with L . monocytogenes and OVA-loaded splenocytes, cross-priming of OVA-specific naive CD8(+) T cells occurred . No significant decline in cross-priming against OVA was measured when either TNF or IFN-gamma was neutralized in L . monocytogenes-infected animals, demonstrating that multiple signals exist to overcome CD40L blockade of CD8(+) T cell cross-priming during bacterial infection . These data support a model in which DCs can be stimulated in vivo through signals other than CD40, becoming APC that can effectively stimulate CD8(+) T cell responses against exogenous Ags during infection. Lett Appl Microbiol, 2001 Nov, 33(5), 357 - 61 The effect of inoculum size and sublethal injury on the ability of Listeria monocytogenes to initiate growth under suboptimal conditions; Pascual C et al.; AIMS: To investigate the effect of inoculum size and physiological state on the ability of Listeria monocytogenes cells to initiate growth under suboptimal conditions of salt concentration and pH . METHODS AND RESULTS: Cell suspensions were serially diluted in media of different salt concentration or pH and replicate inocula distributed into 96-well microplates . The proportion of wells showing growth at each dilution level was determined after incubation for 6 weeks for each set of conditions . Growth occurred from single cells up to a concentration of 1.2 mol l-1 NaCl; above this threshold, the inoculum size needed to initiate growth became progressively larger . A similar effect was seen with decreasing pH but only very close to the growth/no growth boundary . The threshold for inoculum-dependent growth was lower in exponential phase cells than in stationary phase ones and sublethal injury greatly decreased the probability of growth from small inocula . CONCLUSIONS: The growth/no growth boundary for L . monocytogenes is not an absolute cut-off point but represents a region where the probability of growth rapidly decreases as conditions become more extreme . We interpret the requirement for a critical inoculum size for growth as being due to death of a proportion of cells in the inoculum rather than to co-operative population effects . SIGNIFICANCE AND IMPACT OF THE STUDY: Physiological heterogeneity within the cell population and inoculum size will affect the risk of L . monocytogenes growing in food. Immunopharmacol Immunotoxicol, 2001 Aug, 23(3), 367 - 82 Evaluation of Caesalpinia ferrea extract on bone marrow hematopoiesis in the murine models of listeriosis and Ehrlich ascites tumor; Queiroz ML et al.; The capacity of hematopoietic tissues to produce and mobilize phagocytes to the site of infection and tumor growth is of central importance to mediate the early immunological response . In this perspective, studies from our laboratory have defined Listeria monocytogenes infection and the Ehrlich ascites tumor (EAT) as useful models to investigate the effects of natural compounds on the growth and differentiation of granulocyte-macrophage progenitor cells (CFU-GM) . As expected, a significant reduction in the number of bone marrow CFU-GM was observed in the initial stages of infection with a sublethal dose of Listeria . Similarly, the bone marrow CFU-GM decreased sharply 4 days after the EAT transplantation . Treatment of infected and tumor-bearing mice with 500 and 1,000 mg/kg of Caesalpinia ferrea aqueous extract, given 3 times orally, significantly stimulated myelopoiesis, whereas no effects were observed with the 250 mg/kg dose . Similar results were obtained in normal mice . The administration of the two higher doses of the extract also protected 15-20% of mice from a lethal dose of Listeria and significantly prolonged survival of EAT-bearing mice . In summary, these results demonstrate that C . ferrea extract acts as a positive regulator of myelopoiesis, and suggest that the therapeutic effect of C . ferrea may be partially mediated by this action. Infection, 2001 Oct, 29(5), 278 - 9 Rapidly growing tumor-like brain lesion; Ackermann G et al.; Listeria monocytogenes accounts for 8-11% of the cases of bacterial meningitis which is associated with high mortality in patients with serious underlying diseases or those receiving immunosuppressive treatment . Brain abscess due to L . monocytogenes is a very rare occurrence . The case reported here concerns a 54-year-old female patient with a rapidly growing tumor-like brain lesion . L . monocytogenes type 4b could be cultured from blood and brain biopsy . Despite antimicrobial therapy with ampicillin and gentamicin, the patient died 11 days after admission to the hospital . The growing numbers of elderly and immunocompromised patients will increasingly confront physicians with patients with listeriosis . Delayed therapy in patients treated with corticosteroids may result in a fatal outcome. Clin Microbiol Infect, 2001, 7 Suppl 4, 43 - 6 Antimicrobial therapy of infections with aerobic Gram-positive rods; von Graevenitz A; This review presents data on in vitro susceptibilities of aerobically growing Gram-positive rods and in vivo activities of antibiotics used against Gram-positive rods . While in some instances susceptibility and efficacy are predictable (e.g . penicillin vs . Listeria and microaerophilic coryneforms, or metronidazole vs . Gardnerella) susceptibility testing by dilution techniques seems necessary for many Gram-positive rods as long as they are deemed clinically relevant. Can J Microbiol, 2001 Sep, 47(9), 883 - 7 RAPD analysis, serotyping, and esterase typing indicate that the population of Listeria monocytogenes strains recovered from cheese and from patients with listeriosis in Belgium are different; Malak M et al.; Populations of Listeria monocytogenes strains isolated in Belgium from cheese and from patients with listeriosis were characterised by randomly amplified polymorphic DNA (RAPD) analysis using two 10-mers primers (OPA-04 and OPA-13) . High discrimination levels were obtained with each of these primers alone (discrimination indices (DI) of 0.899 and 0.935 for OPA13 and OPA04, respectively) or in combination (DI of 0.960) . The clustering of strains obtained by RAPD was compared with a clustering previously made using serotyping and esterase typing . RAPD allowed the subdivision of each serovar cluster and of most of the clusters determined by the polymorphism of the bacterial esterases . Our analysis indicates that the population of strains of L . monocytogenes found in cheese differs from the one isolated from patients with listeriosis during the same period. Curr Microbiol, 2001 Oct, 43(4), 271 - 7 Characterization of the prfA virulence gene cluster insertion site in non-hemolytic Listeria spp.: probing the evolution of the Listeria virulence gene island; Cai S et al.; The prfA virulence gene cluster is present between prs and ldh in the pathogenic L . monocytogenes and L . ivanovii, but absent from the non-pathogenic L . innocua and L . welshimeri . To probe the evolution of this virulence gene cluster, we sequenced the prs-ldh intergenic region in L . welshimeri and L . innocua . Two ORFs (ORFA and ORFB) were found in both species as well as in L . monocytogenes . Another ORF of unknown function (ORFZ) was found in L . monocytogenes and L . innocua, while two unique ORFs were present in L . welshimeri . ORFA and ORFB showed significant functional constraint, suggesting that further investigations in the functions of these genes, including possible roles in horizontal gene transfer or sequence deletion, are warranted . DNA sequences homologous to Tn1545 integration consensus sequences were found downstream of prs and ORFB, thus defining the likely junctions of the virulence gene island and indicating that the prs-ldh intergenic region may represent a Tn insertion hot spot . Our results are consistent with the hypothesis that a combination of horizontal gene transfer and deletion events mayhave been involved in the evolution of the prfA virulence gene cluster in Listeria. FEMS Microbiol Lett, 2001 Oct 16, 204(1), 111 - 5 Listeria monocytogenes response regulators important for stress tolerance and pathogenesis; Kallipolitis BH et al.; Environmental sensing by two-component signal transduction systems is likely to play a role for growth and survival of Listeria monocytogenes both during transmission in food products and within a host organism . Two-component systems typically consist of a membrane-associated sensor histidine kinase and a gene regulatory protein, the response regulator (RR) . We have identified seven putative RR genes in L . monocytogenes LO28 by PCR using degenerate oligonucleotide primers . By insertional inactivation we obtained data suggesting that three of the putative RRs contribute to the pathogenicity of L . monocytogenes in mice . Strikingly, the mutants that were attenuated in virulence also had a decreased ability to grow in the presence of various stress conditions potentially encountered in an infection process . Thus, our data point to a connection between the ability of the putative two-component systems to sense and respond to certain environmental stimuli, and the virulence of L . monocytogenes. Science, 2001 Oct 26, 294(5543), 849 - 52 Comparative genomics of Listeria species; Glaser P et al.; Listeria monocytogenes is a food-borne pathogen with a high mortality rate that has also emerged as a paradigm for intracellular parasitism . We present and compare the genome sequences of L . monocytogenes (2,944,528 base pairs) and a nonpathogenic species, L . innocua (3,011,209 base pairs) . We found a large number of predicted genes encoding surface and secreted proteins, transporters, and transcriptional regulators, consistent with the ability of both species to adapt to diverse environments . The presence of 270 L . monocytogenes and 149 L . innocua strain-specific genes (clustered in 100 and 63 islets, respectively) suggests that virulence in Listeria results from multiple gene acquisition and deletion events. Appl Environ Microbiol, 2001 Nov, 67(11), 5339 - 42 Pyrosequencing as a method for grouping of Listeria monocytogenes strains on the basis of single-nucleotide polymorphisms in the inlB gene; Unnerstad H et al.; By using pyrosequencing (i.e., sequencing by synthesis) 106 strains of different serovars of Listeria monocytogenes were rapidly grouped into four categories based on nucleotide variations at positions 1575 and 1578 of the inlB gene . Strains of serovars 1/2a and 1/2c constituted one group, and strains of serovars 1/2b and 3b constituted another group, whereas serovar 4b strains were separated into two groups. Mol Microbiol, 2001 Oct, 42(1), 111 - 20 In vitro transcription of PrfA-dependent and -independent genes of Listeria monocytogenes; Lalic-Multhaler M et al.; In vitro transcription starting from the promoters of the Listeria monocytogenes genes hly, plcA, actA, mpl, prfA and iap has been studied . Whereas transcription from Phly, PplcA and PactA is strictly PrfA-dependent, that from Piap, PprfA1/2 and, unexpectedly, also from Pmpl is independent . Initiation of in vitro transcription at all tested promoters except PprfA requires high concentrations of ATP but not GTP . The nucleotides required in higher concentrations for efficient in vitro transcription are always included in the first three nucleotides of the corresponding transcript . RNA polymerase prepared from L . monocytogenes cultured either in rich culture medium (RNAP(BHI)), exposed to heat shock conditions (RNAP48) or conditioned in minimal essential medium (RNAP(MEM)) shows significant differences in the transcription efficiencies when transcription is initiated at these promoters . Transcription starting from the PrfA-dependent promoters PactA and Phly is enhanced with RNAP48 and RNAP(MEM) (in relation to Piap-mediated transcription), while transcription from the other promoters is reduced when compared with RNAP(BHI) . These data suggest that in vivo transcription of the genes actA and hly may not function optimally with RNA polymerase loaded with the vegetative sigma factor 43, but may require a modified RNA polymerase, possibly loaded with an alternative sigma factor. Ann Hematol, 2001 Sep, 80(9), 549 - 52 Fulminant hepatitis B virus reactivation with concomitant listeriosis after fludarabine and rituximab therapy: case report; Ng HJ et al.; Chronic hepatitis B infection poses a problem in patients with malignancies undergoing chemotherapy . Not uncommonly, hepatitis B virus (HBV) reactivates during or soon after chemotherapy . We report a case of a woman with acute prolymphocytic leukemia who received chemotherapy containing fludarabine and cyclophosphamide, followed by the CD 20 monoclonal antibody, rituximab . She developed fatal reactivation of hepatitis B with fulminant liver failure 3 months after completing treatment . Listeria monocytogenes was concomitantly isolated . This case indicates the importance of identifying all hepatitis B carriers and the need to closely monitor and detect reactivation early . Newer chemotherapeutic agents with potentially long-lasting effects on T cells, such as purine analogues, require extended vigilance for reactivation of HBV as well as opportunistic infections . Rituximab is increasingly used alone or in combination with other chemotherapy agents . Its possible contributory role in hepatitis B reactivation needs to be defined. Scand J Infect Dis, 2001, 33(9), 714 - 6 Epidural abscess and vertebral osteomyelitis caused by Listeria monocytogenes: case report and literature review; Khan KM et al.; A 69-y-old male with an epidural abscess and concomitant vertebral osteomyelitis caused by Listeria monocytogenes is presented, together with a brief review of the literature . To our knowledge, no cases of epidural abscess and only 3 cases of osteomyelitis have previously been reported in association with this organism. J Commun Dis, 2000 Dec, 32(4), 295 - 9 Seropositivity for intracellular bacterial infections among abattoir associated personnels; Barbuddhe SB et al.; Studies were carried out to detect the antibodies against zoonotic intracellular bacterial infections viz., brucellosis, listeriosis and tuberculosis in abattoir associated personnel employing dot-ELISA . Out of 165 serum samples tested 25.5, 40.0 and 10.9 per cent were detected as positive for brucellosis, listeriosis and tuberculosis, respectively . Immunodetection of these occupationally exposed persons for IgM as well as IgG antibodies revealed positivity for IgM, IgG and IgM + IgG in 8.5, 17.6 and 13.9 per cent for listeriosis and 5.5, 2.4 and 3.0 for tuberculosis respectively . Antibodies against brucellosis and listeriosis both were detected in 10.9 per cent persons, while 6.7 per cent persons were positive for both listeriosis and tuberculosis . No person was found positive with both brucellosis and tuberculosis . All the three infections were detected in 3.6 per cent persons. J Ind Microbiol Biotechnol, 2001 Aug, 27(2), 111 - 6 Effects of vegetable type, package atmosphere and storage temperature on growth and survival of Escherichia coli O157:H7 and Listeria monocytogenes; Francis GA et al.; The survival and growth of Escherichia coli O157:H7 (ATCC 43888 and NCTC 12900) and Listeria monocytogenes (ATCC 19114 and NCTC 11994) during storage (4 and 8 degrees C) on ready-to-use (RTU) packaged vegetables (lettuce, swedes (rutabaga), dry coleslaw mix, soybean sprouts) were studied . The vegetables were sealed within oriented polypropylene packaging film, and modified atmospheres developed in packs during storage due to produce respiration . Survival and growth patterns were dependent on vegetable type, package atmosphere, storage temperature and bacterial strain . Populations of L . monocytogenes and E . coli O157:H7 increased (P<0.05, by 1.5 to 2.5 log cycles, depending on strain) during a 12-day storage period on shredded lettuce (8 degrees C) . L . monocytogenes populations also increased (by approximately 1 log cycle) on packaged swedes, did not change significantly (P>0.05) in packages of soybean sprouts and decreased by approximately 1.5 log cycles (P<0.05) on coleslaw mix (8 degrees C) . E . coli O157:H7 populations on packaged coleslaw and soybean sprouts increased (by 1.5 to 2.5 log cycles) up to day 5, but declined during subsequent storage (8 degrees C) . On packaged swedes (8 degrees C), populations of E . coli O157:H7 strain ATCC 43888 increased (by approximately 1 log cycle) during storage, whereas populations of strain 12900 increased between days 2 and 5, and declined during subsequent storage . Reducing the storage temperature from 8 to 4 degrees C reduced the growth of L . monocytogenes and E . coli O157:H7 on packaged RTU vegetables . However, viable populations remained at the end of the storage period at 4 degrees C. Pediatr Cerrahi Derg, 1990, 4, 136 - 38 G . Buchanan on inguinal hernia treatment: a review 111-years after; Etker S; The Scottish surgeon G . Buchanan (1827-1905) has contributed to the development of pediatric inguinal herniorrhaphy in line with Listerian principles, and is the author of the first separate publication on the subject to appear as early as 1879 . His largely forgotten work is being reassessed. Hist Cienc Saude Manguinhos, 1994 Nov-1995 Feb, 1(2), 20 - 38 {From miasmata to germs . The impact of bacteriology on medical practice in Canadian territory from 1870 to 1930}; Goulet D; This article analyzes some aspects concerning the introduction of bacteriological theory and practice into Canadian territory from 1870 to 1930 . The author begins by presenting the principal explanatory models characterizing the transition period from pre-bacteriological discourse--referring to infectious diseases and surgical infections--to new bacteriological paradigms . He goes on to analyze the different ways in which the germ theory and then bacteriology spread into Canadian territory . The transition periods, where old and new coexisted, reveal a kind of syncretism by physicians in relation to both aetiology of infectious diseases and surgical procedures . The author then shows that there were two distinct phases in the overall context of the history of antiseptic surgery from 1868 to 1890 . He demonstrates that the use of antiseptics did not necessarily mean either adherence to Pasteur's postulates on fermentation or strict observance of Listerian methods . Finally, the article expounds on the role played by European institutions in importing bacteriological knowledge and institutionalizing this new discipline in Canadian territory. Int J Food Microbiol, 2001 Sep 28, 69(3), 227 - 35 Random amplification of polymorphic DNA typing of Listeria monocytogenes isolated from meat; Byun SK et al.; To investigate the epidemiological characteristics of Listeria monocytogenes isolated from imported or domestic meats, L . monocytogenes was isolated and identified through biochemical and serological tests, and epidemiological analysis of the isolates was carried out through the random amplification of polymorphic DNA (RAPD) method . Fifty-four isolates were identified as L . monocytogenes through biochemical tests, of which 36 (67%) were confirmed as serotype 1, and 18 (33%) were serotype 4, through the microagglutination test . In the molecular epidemiological analysis using RAPD method, the isolates could be classified into 10, 6 and 6 types using three random primers, PB1, PB4, and HLWL74, respectively . Forty composite profiles were identified by a combination of the three primers . RAPD analysis demonstrated the relationships between the isolates from beef from Korea and the USA, pork from Korea and Denmark . These results suggested that RAPD could be a useful typing tool for the epidemiological study of L . monocytogenes and other bacteria. FEBS Lett, 2001 Oct 12, 506(3), 249 - 52 A possible regulatory role for the metal-binding domain of CadA, the Listeria monocytogenes Cd2+-ATPase; Bal N et al.; Using the baculovirus/Sf9 expression system, we produced CadA and DeltaMBD, a metal-binding domain, truncated CadA . Both proteins had the expected properties of P-type ATPases: ATP-induced Cd2+ accumulation, Cd2+-sensitive ATP and Pi phosphorylation and ATPase activity . DeltaMBD displayed lower initial transport velocity as well as lower maximal ATPase activity than CadA . MBD truncation flattened the Cd2+ dependence of the ATPase activity and increased apparent Cd2+ affinity, suggesting a positive cooperativity between MBD and membranous transport sites . We propose that occupancy of MBD by Cd2+ modulates CadA activity. J Food Prot, 2001 Oct, 64(10), 1627 - 30 Efficacy of trisodium phosphate solutions in reducing Listeria monocytogenes populations on chicken skin during refrigerated storage; Capita R et al.; Chicken skin inoculated with l0(8) CFU/ml of Listeria monocytogenes was dipped for 15 min in sterile water (control) and in 8, 10, or 12% trisodium phosphate (TSP) solutions . Skin samples were stored at 2 degrees C for 5 days, with microbial monitoring on days 0, 1, 3, and 5 after treatment . Compared to the water dip, all TSP treatments significantly (P < 0.05) reduced L monocytogenes populations on chicken skin . The concentration of the TSP was a significant factor in reducing the populations of the bacteria at days 0, 1, 3, and 5 of refrigerated storage . For all sampling times, the best outcomes were attained with the highest TSP concentration studied (12%) . Bacterial reductions in counts during the first day of storage were between 1.52 and 2.70 log10 cycles for 8 and 12% TSP-treated samples, respectively . Significantly greater reductions were observed from the third day of refrigerated storage onward . This occurred largely because populations of L . monocytogenes on control samples increased somewhat, but on TSP-treated samples the pathogen remained practically constant . Differences between L monocytogenes counts in skin samples immersed in water and those treated with TSP ranged from 2.10 (8% TSP-treated samples) and 3.63 (12% TSP-treated samples) log10 cycles on day 5 of storage . These results indicated that TSP is effective against L . monocytogenes in chicken meat, especially after several days of refrigerated storage. J Food Prot, 2001 Oct, 64(10), 1521 - 6 Comparative evaluation of culture- and BAX polymerase chain reaction-based detection methods for Listeria spp . and Listeria monocytogenes in environmental and raw fish samples; Hoffman AD et al.; Two commercial polymerase chain reaction (PCR)-based Listeria detection systems, the BAX for Screening/Listeria monocytogenes and the BAX for Screening/Genus Listeria, and a culture-based detection system, the Biosynth L . monocytogenes Detection System (LMDS), were evaluated for their ability to detect L . monocytogenes and Listeria spp . in raw ingredients and the processing environment . For detection of L . monocytogenes from raw fish, enrichment was performed in Listeria enrichment broth (LEB), followed by plating on both Oxford agar and LMDS L . monocytogenes plating medium (LMPM) . Detection of Listeria and L . monocytogenes from environmental samples was performed using LMDS enrichment medium, followed by plating on both Oxford agar and LMPM . A total of 512 environmental samples and 315 raw fish were taken from two smoked fish processing facilities and screened using these molecular and cultural Listeria detection methods . The BAX for Screening/L monocytogenes was used to screen raw fish and was 84.8% sensitive and 100% specific . The BAX for Screening/Genus Listeria was evaluated on environmental samples and had 94.7% sensitivity and 97.4% specificity . In conjunction with enrichment in LEB, LMPM had a sensitivity and specificity for detection of L . monocytogenes from raw fish of 97.8 and 100%, respectively . Use of LMDS enrichment medium followed by plating on LMPM allowed for sensitivity and specificity rates of 94.8 and 100%, respectively, for detection of L . monocytogenes from environmental samples . We conclude that both the BAX systems and the use of LMPM allow for reliable and rapid detection of Listeria spp . and L . monocytogenes . While the BAX systems provide screening results in about 3 days, the use of LMPM allows for L . monocytogenes isolation in 4 to 5 days. EMBO J, 2001 Oct 15, 20(20), 5603 - 14 A WASp-VASP complex regulates actin polymerization at the plasma membrane; Castellano F et al.; Proteins of the Wiskott-Aldrich syndrome and Ena/VASP families both play essential functions in the regulation of actin dynamics at the cell leading edge . However, possibilities of functional interplay between members of these two families have not been addressed . Here we show that, in hemopoietic cells, recruitment of the C-terminal VCA (Verprolin homology, Cofilin homology, Acidic) domain of WASp at the plasma membrane by a ligand technique using rapamycin as an intermediate is not sufficient to elicit efficient Arp2/3 complex-mediated actin polymerization . Other domains of WASp, in particular the proline-rich domain, are required for the formation of actin-rich structures . An in vitro analysis demonstrates that the proline-rich domain of WASp binds VASP with an affinity of approximately 10(6) M(-1) . In addition, WASp and VASP both accumulate in actin-rich phagocytic cups . Finally, in a reconstituted motility medium, VASP enhances actin-based propulsion of WASp-coated beads in a fashion reminiscent of its effect on Listeria movement . We propose that VASP and WASp cooperation is essential in stimulating actin assembly and membrane protrusion at the leading edge. Crit Care Med, 2001 Oct, 29(10), 1943 - 9 Intraoperative prostaglandin E1 improves antimicrobial and inflammatory responses in alveolar immune cells; Kotani N et al.; OBJECTIVE: Anesthesia and surgery decrease antimicrobial and increase proinflammatory functions of alveolar immune cells . Thus, anti-inflammatory agents that do not further suppress antimicrobial functions are required . We tested the hypothesis that intraoperative prostaglandin E1 (PGE1) suppresses proinflammatory responses and prevents the reduction in antimicrobial responses of alveolar immune cells . DESIGN: Prospective, randomized, controlled, double-blind study . SETTING: University hospital . PATIENTS: A total of 40 patients undergoing elective orthopedic surgery under propofol/fentanyl anesthesia . INTERVENTION: In double-blind fashion, the patients received PGE1 from the beginning to the end of surgery (PGE1 group, n = 20) or nothing (control group, n = 20) . METHODS AND MAIN RESULTS: Alveolar immune cells were harvested by bronchoalveolar lavage immediately after induction of anesthesia; 2, 4, and 6 hrs after induction of anesthesia; and at the end of surgery . We measured opsonized and nonopsonized phagocytosis . Microbicidal activity was evaluated to directly kill Listeria monocytogenes in alveolar macrophages . Finally, we determined the expression of proinflammatory cytokines including interleukin (IL)-1beta, IL-8, interferon-gamma, and tumor necrosis factor-alpha, and that of anti-inflammatory cytokines (IL-4 and IL-10) by semiquantitative polymerase chain reaction . Nonopsonized and opsonized phagocytosis and microbicidal activity of alveolar macrophages decreased and the expression of genes for all pro- and anti-inflammatory cytokines increased significantly over time in both groups . Starting 2-4 hrs after induction of anesthesia, the increases in gene expression of proinflammatory cytokines were 1.5-3 times smaller in the PGE1 than in the control group . Starting 6 hrs after anesthesia, the increase in gene expression of IL-10 was 1.5-3 times greater in the PGE1 than in the control group . Intraoperative decreases in phagocytic and microbial activities were the same in the two groups . CONCLUSION: Intraoperative PGE1 not only suppressed proinflammatory responses, but also protected antimicrobial functions of alveolar macrophages, possibly because PGE1 is mostly inactivated in the pulmonary intravascular space . Our results suggest that intraoperative PGE1 protects the pulmonary immune defense in alveolar immune cells. Br J Pharmacol, 2001 Oct, 134(3), 571 - 8 Inhibition of interleukin-12 production by auranofin, an anti-rheumatic gold compound, deviates CD4(+) T cells from the Th1 to the Th2 pathway; Kim TS et al.; 1 . Interleukin-12 (IL-12) may play a central role in the development and progression of rheumatoid arthritis by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN-gamma and low IL-4 production . In this study we investigated the effect of auranofin (AF), an anti-rheumatic gold compound, on IL-12 production in mouse macrophages and dendritic cells, and studied whether AF-mediated inhibition of IL-12 production could regulate a cytokine profile of antigen (Ag)-primed CD4(+) Th cells . 2 . Treatment with AF significantly inhibited IL-12 production in lipopolysaccharide (LPS)-stimulated macrophages and also in CD40L-stimulated dendritic cells . AF-pretreated macrophages reduced their ability to induce IFN-gamma and increased the ability to induce IL-4 in Ag-primed CD4(+) T cells . AF did not influence the cell surface expression of the class II MHC molecule and the costimulatory molecules CD80 and CD86 . 3 . Addition of recombinant IL-12 to cultures of AF-pretreated macrophages and CD4(+) T cells restored IFN-gamma production in Ag-primed CD4(+) T cells . 4 . The in vivo administration of AF resulted in the inhibition of IL-12 production by macrophages stimulated in vitro with LPS or heat-killed Listeria monocytogenes (HKL), leading to the inhibition of Th1 cytokine profile (decreased IFN-gamma and increased IL-4 production) in Ag-primed CD4(+) T cells . 5 . These findings may explain some known effects of AF including anti-rheumatic effects and the inhibition of encephalitogenicity, and point to a possible therapeutic use of AF in the Th1-mediated immune diseases such as autoimmune diseases. FEMS Microbiol Lett, 2001 Sep 25, 203(2), 185 - 9 Difference in cholesterol-binding and cytolytic activities between listeriolysin O and seeligeriolysin O: a possible role of alanine residue in tryptophan-rich undecapeptide; Ito Y et al.; We have constructed recombinant listeriolysin O (rLLO) and seeligeriolysin O (rLSO) from Listeria monocytogenes and Listeria seeligeri, respectively . In hemolysis and cholesterol-binding assays, the specific activity of recombinant toxin was lower for LSO as compared to LLO . To understand the molecular basis of this difference, in particular with respect to the conserved Trp-rich undecapeptide, a naturally occurring Ala to Phe substitution in LSO was introduced into rLLO . The rLLO:A488F hemolysin exhibited a reduced activity in both hemolysis and cholesterol-binding . The reverse mutation, inserted into rLSO, also increased the hemolytic activity of this mutant LSO . These results suggested that the natural replacement of Ala to Phe is involved in the weak cytolytic activity of LSO. J Cell Biol, 2001 Oct 1, 155(1), 89 - 100 Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motility; Skoble J et al.; The Listeria monocytogenes ActA protein mediates actin-based motility by recruiting and stimulating the Arp2/3 complex . In vitro, the actin monomer-binding region of ActA is critical for stimulating Arp2/3-dependent actin nucleation; however, this region is dispensable for actin-based motility in cells . Here, we provide genetic and biochemical evidence that vasodilator-stimulated phosphoprotein (VASP) recruitment by ActA can bypass defects in actin monomer-binding . Furthermore, purified VASP enhances the actin-nucleating activity of wild-type ActA and the Arp2/3 complex while also reducing the frequency of actin branch formation . These data suggest that ActA stimulates the Arp2/3 complex by both VASP-dependent and -independent mechanisms that generate distinct populations of actin filaments in the comet tails of L . monocytogenes . The ability of VASP to contribute to actin filament nucleation and to regulate actin filament architecture highlights the central role of VASP in actin-based motility. Microbiology, 2001 Oct, 147(Pt 10), 2689 - 96 Relationship between nucleic acid ratios and growth in Listeria monocytogenes; Milner MG et al.; Listeria monocytogenes is a pathogen whose distribution in a range of foodstuffs requires the development of methods for sensitive and rapid detection . Molecular biological methods usually rely on specific detection of L . monocytogenes rDNA directly amplified by the application of PCR to DNA extracts . Information on the metabolic status of L . monocytogenes populations would be valuable and can, in theory, be provided by quantitative detection of rRNA itself . Both fluorometry and oligonucleotide probe assays were applied to L . monocytogenes cultures to quantify RNA and DNA and produced more meaningful data than previous estimates for bacteria based on eukaryotic nucleic acid standards . In batch culture, the RNA-DNA ratio was found to be greatest at the end of exponential growth, after which RNA became degraded in accordance with the rapid decrease in viability . When the pH of the medium was controlled at neutrality, culture viability was dramatically extended and although RNA was degraded, intact DNA was maintained for the duration of the experiment . Ribosome numbers per cell were estimated to decrease from about 25000 observed during mid-exponential growth to about 600 during stationary phase, under pH-controlled conditions . Like Escherichia coli, therefore, L . monocytogenes loses viability and rRNA rapidly once exponential growth has ceased in batch culture . However, much improved survival of a culturable L . monocytogenes population when pH is controlled has clear implications for the persistence of this species in buffered environments such as dairy products. J Mol Biol, 2001 Sep 28, 312(4), 783 - 94 Internalins from the human pathogen Listeria monocytogenes combine three distinct folds into a contiguous internalin domain; Schubert WD et al.; Listeria monocytogenes is an opportunistic, food-borne human and animal pathogen . Host cell invasion requires the action of the internalins A (InlA) and B (InlB), which are members of a family of listerial cell-surface proteins . Common to these proteins are three distinctive N-terminal domains that have been shown to direct host cell-specific invasion for InlA and InlB . Here, we present the high-resolution crystal structures of these domains present in InlB and InlH, and show that they constitute a single "internalin domain" . In this internalin domain, a central LRR region is flanked contiguously by a truncated EF-hand-like cap and an immunoglobulin (Ig)-like fold . The extended beta-sheet, resulting from the distinctive fusion of the LRR and the Ig-like folds, constitutes an adaptable concave interaction surface, which we propose is responsible for the specific recognition of the host cellular binding partners during infection . J Cell Biol, 2001 Oct 1, 155(1), 101 - 12 Epub 2001 Sep 24. A role for cofilin and LIM kinase in Listeria-induced phagocytosis; Bierne H et al.; The pathogenic bacterium Listeria monocytogenes is able to invade nonphagocytic cells, an essential feature for its pathogenicity . This induced phagocytosis process requires tightly regulated steps of actin polymerization and depolymerization . Here, we investigated how interactions of the invasion protein InlB with mammalian cells control the cytoskeleton during Listeria internalization . By fluorescence microscopy and transfection experiments, we show that the actin-nucleating Arp2/3 complex, the GTPase Rac, LIM kinase (LIMK), and cofilin are key proteins in InlB-induced phagocytosis . Overexpression of LIMK1, which has been shown to phosphorylate and inactivate cofilin, induces accumulation of F-actin beneath entering particles and inhibits internalization . Conversely, inhibition of LIMK's activity by expressing a dominant negative construct, LIMK1(-), or expression of the constitutively active S3A cofilin mutant induces loss of actin filaments at the phagocytic cup and also inhibits phagocytosis . Interestingly, those constructs similarly affect other actin-based phenomenons, such as InlB-induced membrane ruffling or Listeria comet tail formations . Thus, our data provide evidence for a control of phagocytosis by both activation and deactivation of cofilin . We propose a model in which cofilin is involved in the formation and disruption of the phagocytic cup as a result of its local progressive enrichment. J Biol Chem, 2001 Nov 23, 276(47), 43597 - 603 Epub 2001 Sep 24. Internalin B activates nuclear factor-kappa B via Ras, phosphoinositide 3-kinase, and Akt; Mansell A et al.; Internalin B (InlB), a 630-amino acid protein loosely attached to the surface of Listeria monocytogenes, participates in the entry of the bacterium into mammalian cells . This process requires the activation of phosphoinositide (PI) 3-kinase by InlB . Previously, we demonstrated that InlB activates the transcription factor Nuclear Factor-kappaB in murine J774 macrophage-like cells, an event that also requires PI 3-kinase . Here we have further investigated this phenomenon . InlB activated the small G-protein Ras in J774 cells . Inhibition of Ras with the farnesyltransferase inhibitor manumycin A inhibited NF-kappaB activation and the recruitment of the p85 subunit of PI 3-kinase, implying that Ras is required for PI 3-kinase activation . InlB also activated the PI 3-kinase downstream effector, Akt, as assessed by increased phosphorylation of Akt on serine 473 . Transfection of Hep2 cells with dominant negative Ras N17 or dominant negative Akt inhibited the induction of a reporter gene linked to the interleukin-8 promoter by InlB . Furthermore, the Ras inhibitor manumycin A, the PI 3-kinase inhibitor LY294002, and an Akt inhibitor all blocked the induction of interleukin-8 by InlB . Our study is the first report of a bacterial product activating a pathway involving Ras, PI 3-kinase, and Akt, which leads to NF-kappaB activation . This process could be involved in host defense or the inhibition of apoptosis during infection. Appl Environ Microbiol, 2001 Oct, 67(10), 4560 - 5 Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection; Sleator RD et al.; The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene . Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E . coli (DeltaproBA) background . While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L . monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E . coli . Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis . However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria. Appl Environ Microbiol, 2001 Oct, 67(10), 4454 - 7 Role of sigma(B) in heat, ethanol, acid, and oxidative stress resistance and during carbon starvation in Listeria monocytogenes; Ferreira A et al.; To determine the contribution of sigma B (sigma(B)) to survival of stationary-phase Listeria monocytogenes cells following exposure to environmental stresses, we compared the viability of strain 10403S with that of an isogenic nonpolar sigB null mutant strain after exposure to heat (50 degrees C), ethanol (16.5%), or acid (pH 2.5) . Strain viabilities were also determined under the same conditions in cultures that had been previously exposed to sublethal levels of the same stresses (45 degrees C, 5% ethanol, or pH 4.5) . The DeltasigB and wild-type strains had similar viabilities following exposure to ethanol and heat, but the DeltasigB strain was almost 10,000-fold more susceptible to lethal acid stress than its parent strain . However, a 1-h preexposure to pH 4.5 yielded a 1,000-fold improvement in viability for the DeltasigB strain . These results suggest the existence in L . monocytogenes of both a sigma(B)-dependent mechanism and a pH-dependent mechanism for acid resistance in the stationary phase . sigma(B) contributed to resistance to both oxidative stress and carbon starvation in L . monocytogenes . The DeltasigB strain was 100-fold more sensitive to 13.8 mM cumene hydroperoxide than the wild-type strain . Following glucose depletion, the DeltasigB strain lost viability more rapidly than the parent strain . sigma(B) contributions to viability during carbon starvation and to acid resistance and oxidative stress resistance support the hypothesis that sigma(B) plays a role in protecting L . monocytogenes against environmental adversities. Vet Rec, 2001 Sep 8, 149(10), 289 - 93 Prevalence, incidence, signs and treatment of clinical listeriosis in dairy cattle in England; Erdogan HM et al.; The prevalence, incidence and clinical signs of listeriosis in dairy cattle in England were investigated by means of a postal questionnaire survey of 1500 dairy farmers . The response rate was 64.1 per cent . Overall the farm prevalence of listeriosis was 11.7 per cent, 9.3 per cent for milking cows, 5.0 per cent for replacement heifers and 1.4 per cent for dairy calves . The within-herd incidence rate per thousand animal-years was 51.4 for all cases, 39.7 for milking cows, 86.6 for replacement heifers and 73.7 for dairy calves . Most cases of clinical listeriosis were reported between December and May, and the most common signs were silage eye, followed by nervous signs . The results of the questionnaire were validated internally by re-estimating the farm prevalence by including only those cases diagnosed by a veterinarian or veterinary investigation centre; the prevalence did not change significantly . The proportion of cases which were culled or died of encephalitic listeriosis was compared with the proportion diagnosed during statutory BSE reporting . The fact that the two proportions were similar provided external validation for the results of the questionnaire. Mil Med, 2001 Sep, 166(9), 833 - 5 Cardiac asystole after mouthwash ingestion: a case report and review of the contents; Westermeyer RR et al.; In the search for intoxication, alcoholic patients often ingest toxic alcohols or other products containing ethanol . We report a patient who presented with intoxication from Listerine and rapidly progressed to cardiac asystole . Several mouthwash products have a high concentration of ethanol and are easily obtained . We review the contents of this product and their possible toxicologic effects. J Food Prot, 2001 Sep, 64(9), 1362 - 8 Role of the glutamate decarboxylase acid resistance system in the survival of Listeria monocytogenes LO28 in low pH foods; Cotter PD et al.; The glutamate decarboxylase (GAD) acid resistance system of the foodborne pathogen Listeria monocytogenes plays a major role in its survival at low pH . It was found that survival of the wild-type strain . LO28, in acidified reconstituted skim milk, diluted to reduce free glutamate levels . improves in response to supplementation with monosodium glutamate . A mutant, in which the two listerial GAD homologs have been deleted (and in which there is no discernible GAD activity), did not respond to glutamate supplementation and displayed greatly enhanced sensitivity in a number of low pH foods, even when levels of free glutamate were as low as 0.22 mM . We thus show that the GAD system plays a major role in the survival of L . monocytogenes in acidic foods even when levels of free glutamate are low. Int Immunopharmacol, 2001 Sep, 1(9-10), 1669 - 77 Protective effect of a traditional Japanese medicine Hochu-ekki-to (Chinese name: Bu-zhong-yi-qi-tang), on the susceptibility against Listeria monocytogenes in infant mice; Yamaoka Y et al.; In this study, the effect of traditional Japanese (Chinese) medicine, Hochu-ekki-to, HOT (Chinese name: Bu-zhong-yi-qi-tang), on the susceptibility against Listeria monocytogenes in postneonatal infant mice was examined . Numbers of bacteria in infant mice (infected at 4 weeks of age) were significantly higher than those in adult mice (infected at 8 weeks of age) on day 3 (non-specific resistance phase) and day 5 (specific resistance phase) after infection . Oral administration of 1,000 mg/kg of HOT for 7 days to infant mice reduced bacterial numbers in the liver and spleen at 5 days after the infection . The amount of IFN-gamma and the number of IFN-gamma-producing CD4+ T cells were lower in infant mice than adult mice but those in infant mice enhanced by HOT treatment . HOT also enhanced the antigen-presenting function along with the expression of MHC class II in infant macrophages induced by heat-killed L . monocytogenes . Further, HOT enhanced the IFN-gamma production from infant CD4+ T cells independent of the deficiency in the antigen-presenting function . These findings suggest that HOT induced simultaneously functional maturation of both infant antigen-presenting cells and T cells, and consequently developed an anti-listerial Th1 response. J Virol, 2001 Oct, 75(20), 9654 - 64 Regression of established human papillomavirus type 16 (HPV-16) immortalized tumors in vivo by vaccinia viruses expressing different forms of HPV-16 E7 correlates with enhanced CD8(+) T-cell responses that home to the tumor site; Lamikanra A et al.; Using vaccinia virus as a live vector, we show that the expression of human papillomavirus type 16 (HPV-16) E7 fused to a nonhemolytic portion of the Listeria monocytogenes virulence factor, listeriolysin O (LLO), induces an immune response that causes the regression of established HPV-16 immortalized tumors in C57BL/6 mice . The vaccinia virus construct expressing LLO fused to E7 (VacLLOE7) was compared with two previously described vaccinia virus constructs: one that expresses unmodified E7 (VacE7) and another that expresses E7 in a form designed to direct it to intracellular lysosomal compartments and improve major histocompatibility complex class II-restricted responses (VacSigE7LAMP-1) . C57BL/6 mice bearing established HPV-16 immortalized tumors of 5 or 8 mm were treated with each of these vaccines . Fifty percent of the mice treated with VacLLOE7 remained tumor free 2 months after tumor inoculation, whereas 12 to 25% of the mice were tumor free after treatment with VacSigE7LAMP-1 (depending on the size of the tumor) . No mice were tumor free in the group given VacE7 . Compared to VacE7, VacSigE7LAMP-1 and VacLLOE7 resulted in increased numbers of H2-D(b)-specific tetramer-positive CD8(+) T cells in mouse spleens that produced gamma interferon and tumor necrosis factor alpha upon stimulation with RAHYNIVTF peptide . In addition, the highest frequency of tetramer-positive T cells was seen in the tumor sites of mice treated with VacLLOE7 . An increased efficiency of E7-specific lysis by splenocytes from mice immunized with VacLLOE7 was also observed . These results indicate that the fusion of E7 with LLO not only enhances antitumor therapy by improving the tumoricidal function of E7-specific CD8(+) T cells but may also increase the number of antigen-specific CD8(+) T cells in the tumor, the principle site of antigen expression. J Appl Microbiol, 2001 Sep, 91(3), 556 - 62 Characterization of Listeria monocytogenes isolated from production lines of fresh and cold-smoked fish; Vaz-Velho M et al.; AIMS: The aims of this study were to characterize strains of Listeria monocytogenes isolated from cold-smoking fish plants to establish possible routes of contamination through the processing chain . METHODS AND RESULTS: Listeria monocytogenes from fresh fish suppliers, raw materials, factory sites and finished products isolated in Portugal (162 isolates) and England (28 isolates) were characterized by serotyping, phage typing, tetracycline, cadmium and arsenic resistance, and plasmid profiling . On the basis of serotyping and phage typing, the isolates were categorized into eight groups . Although cultures within some of the groups could be further differentiated on the basis of plasmid profiling and cadmium and arsenite typing, consideration of all typing data predominantly clustered together isolates from a single location . L . monocytogenes strains: from fresh salmon suppliers were not found in the processing lines; from fresh salmon from different locations differed; and from the water where salmon trout were farmed differed from those isolated from the fish samples . SIGNIFICANCE AND IMPACT OF THE STUDY: No clear source or route of contamination in the cold-smoked processing chain could be established; however, these results highlight the complexity in tracking this bacterium through food chains. J Appl Microbiol, 2001 Sep, 91(3), 506 - 13 Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems; Himelbloom B et al.; AIMS: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b . METHODS AND RESULTS: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7.2), with or without glucose . No bacteriocin was produced in APT broth when a low inoculum level (0.001%) was used . In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose . Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture . Increasing concentrations of NaCl (2-7%) reduced bacteriocin production and maximum cell density of C . piscicola A9b when grown in cooked fish juice at 4 degrees C . CONCLUSION: Media composition, inoculum level and sodium chloride concentration affected production . SIGNIFICANCE AND IMPACT OF THE STUDY: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C . piscicola A9b against Listeria monocytogenes in salty foods. J Appl Microbiol, 2001 Sep, 91(3), 463 - 9 Inactivation and injury of pressure-resistant strains of Escherichia coli O157 and Listeria monocytogenes in fruit juices; Jordan SL et al.; AIMS: To investigate methods for inactivating a pressure-resistant strain of Escherichia coli O157 in fruit juices . METHODS AND RESULTS: Cells of a pressure-resistant strain of E . coli O157 (C9490) were exposed to pressures of between, 0.1 and 500 MPa for 5 min in orange, apple or tomato juice . Treatment at 500 MPa achieved an immediate reduction of 5 log units in apple juice (pH 3.5) and tomato juice (pH 4.1), but only about a 1-2 log10 reduction in orange juice (pH 3.8) . The greater level of inactivation in tomato juice than in orange juice of lower pH was due to the presence of low levels (0.7%) of salt in the tomato juice . With the type-strain of E . coli (ATCC 11775) and Listeria monocytogenes NCTC 11994, similar levels of inactivation were achieved at pressures 200 MPa lower . Following storage of pressure-treated orange juice at 4 degrees C for 24 h or 25 degrees C for 3 h, the level of inactivation of E . coli O157 strain C9490 increased to 4.4 or > 7 log10 units, respectively . CONCLUSION: Treatment at 500 MPa may be insufficient to achieve a '5D' reduction in counts of pressure-resistant strains of E . coli, but subsequent death during storage substantially increases process lethality . SIGNIFICANCE AND IMPACT OF THE STUDY: Commercially-practicable pressure processes can be used to inactivate even the most pressure-and acid-resistant strains of E . coli O157, provided that processing and subsequent storage conditions are carefully optimized. Brain Pathol, 2001 Oct, 11(4), 432 - 8 Evidence for intraaxonal spread of Listeria monocytogenes from the periphery to the central nervous system; Antal EA et al.; Rhombencephalitis due to Listeria monocytogenes is characterized by progressive cranial nerve palsies and subacute inflammation in the brain stem . In this paper, we report observations made on mice infected with L . monocytogenes . Unilateral inoculation of bacteria into facial muscle, or peripheral parts of a cranial nerve, induced clinical and histological signs of mainly ipsilateral rhombencephalitis . Similarly, unilateral inoculation of bacteria into lower leg muscle or peripheral parts of sciatic nerve was followed by lumbar myelitis . In these animals, intraaxonal bacteria were seen in the sciatic nerve and its corresponding nerve roots ipsilateral to the bacterial application site . Development of myelitis was prevented by transsection of the sciatic nerve proximally to the hindleg inoculation site . Altogether, our results support the hypothesis that Listeria rhombencephalitis is caused by intraaxonal bacterial spread from peripheral sites to the central nervous system. Southeast Asian J Trop Med Public Health, 2001 Jun, 32(2), 402 - 7 Prevalence of Listeria spp and Listeria monocytogenes in meat and fermented fish in Malaysia; Hassan Z et al.; Fermented fish and meat samples were purchased from supermarket and wet market for microbiological analysis of Listeria species and Listeria monocytogenes contamination . Listeria species were isolated from 17 (73.9%) of 23 samples of imported frozen beef, 10 (43.5%) of the 23 samples of local beef and 14 (56%) of the 25 samples of fermented fish from wet market . Listeria monocytogenes occurred in 15 (75%) of the frozen beef samples, 6 (30.4%) of the 23 samples of local meat and 3 (12%) of the 25 samples from fermented fish . Listeria species was not isolated from any of the 23 samples of imported frozen beef from supermarket and from the 5 samples of buffalo meat examined . This highlights the possibility of Listeria spp or L . monocytogenes to persist in meat and fermented fish in wet market and raises the problem of illness due to the handling and consumption of Listeria-contaminated meat or fermented fish are likely as evidence by the high contamination rates of samples sold at the wet market. Cell Microbiol, 2001 Sep, 3(9), 599 - 609 Eukaryotic expression plasmid transfer from the intracellular bacterium Listeria monocytogenes to host cells; Hense M et al.; The facultative intracellular, Gram-positive bacterium Listeria monocytogenes invades phagocytic and non-phagocytic cells from the tissues and organs of a wide variety of animals and humans . Here, we report the use of these bacteria as vehicles for gene transfer . Eukaryotic expression plasmids were introduced into the nucleus of host cells following lysis of the intracytosolic, plasmid-carrying bac