Infect Immun, 2003 Aug, 71(8), 4398 - 404 Oral pretreatment of mice with CpG DNA reduces susceptibility to oral or intraperitoneal challenge with virulent Listeria monocytogenes; Ray NB et al.; Listeria monocytogenes is an enteroinvasive intracellular bacterial pathogen that infects humans and other animals, including mice, sometimes resulting in severe systemic infections . Previous studies showed that intraperitoneal (i.p.) pretreatment of susceptible BALB/c mice with immune-stimulatory CpG DNA 48 to 96 h prior to i.p . challenge with virulent L . monocytogenes reduces bacterial numbers in livers by greater than 100-fold, correlating with recovery from infection . Here we show that oral pretreatment of BALB/c mice with CpG DNA results in decreased susceptibility to either oral or i.p . challenge with L . monocytogenes . A single dose of 200 microg of CpG DNA administered to BALB/c mice orally by gavage 48 h or 7 days before oral challenge with virulent L . monocytogenes reduces bacterial numbers approximately 10- to 100-fold in livers and spleens . Lymphotoxin alpha knockout mice lacking Peyer's patches (PPs) and pretreated orally with CpG DNA 48 h prior to oral challenge with L . monocytogenes also have reduced susceptibility to infection, suggesting that PPs are required neither for oral infection nor for CpG-induced resistance against oral infection with L . monocytogenes . Surprisingly, 48-h oral pretreatment of BALB/c mice with 100 to 200 microg of CpG DNA results in approximately 100-fold-decreased bacterial numbers in livers following i.p . challenge with L . monocytogenes, suggesting, along with other data in this report, that orally delivered CpG DNA induces systemic resistance to infection . These results indicate that oral administration of CpG DNA induces systemic innate immune defenses against either oral or systemic infection with virulent L . monocytogenes. J Immunol, 2003 Aug 1, 171(3), 1148 - 55 Macrophages of the splenic marginal zone are essential for trapping of blood-borne particulate antigen but dispensable for induction of specific T cell responses; Aichele P et al.; Rapid removal of pathogens from the circulation by secondary lymphoid organs is prerequisite for successful control of infection . Blood-borne Ags are trapped mainly in the splenic marginal zone . To identify the cell populations responsible for Ag trapping in the marginal zone, mice were selectively depleted of marginal zone macrophages and marginal metallophilic macrophages . In the absence of these cells, trapping of microspheres and Listeria monocytogenes organisms was lost, and early control of infection was impaired . Depletion of marginal zone macrophages and marginal metallophilic macrophages, however, did not limit Ag presentation because Listeria-specific protective T cell immunity was induced . Therefore, marginal zone macrophages and marginal metallophilic macrophages are crucial for trapping of particulate Ag but dispensable for Ag presentation. Immunity, 2003 Jul, 19(1), 59 - 70 TNF/iNOS-producing dendritic cells mediate innate immune defense against bacterial infection; Serbina NV et al.; Dendritic cells (DCs) present microbial antigens to T cells and provide inflammatory signals that modulate T cell differentiation . While the role of DCs in adaptive immunity is well established, their involvement in innate immune defenses is less well defined . We have identified a TNF/iNOS-producing (Tip)-DC subset in spleens of Listeria monocytogenes-infected mice that is absent from CCR2-deficient mice . The absence of Tip-DCs results in profound TNF and iNOS deficiencies and an inability to clear primary bacterial infection . CD8 and CD4 T cell responses to L . monocytogenes antigens are preserved in CCR2-deficient mice, indicating that Tip-DCs are not essential for T cell priming . Tip-DCs, as the predominant source of TNF and iNOS during L . monocytogenes infection, orchestrate and mediate innate immune defense against this intracellular bacterial pathogen. Immunity, 2003 Jul, 19(1), 2 - 4 Monocyte heterogeneity and innate immunity; Taylor PR et al.; Peripheral monocyte heterogeneity is widely acknowledged in humans but until now comparable heterogeneity has not been characterized in mice . In this issue, Geissmann et al . use chemokine receptors to define two monocyte subsets and Serbina et al . highlight the importance of selective monocyte recruitment in the innate immune response to Listeria. J Food Prot, 2003 Jul, 66(7), 1283 - 7 Evaluation of antibodies for immunomagnetic separation combined with flow cytometry detection of Listeria monocytogenes; Jung YS et al.; Four polyclonal anti-Listeria antibodies were evaluated for the detection of Listeria monocytogenes in direct and indirect assays using immunomagnetic separation with flow cytometry . The efficiency of immunocapturing using magnetic beads was also determined . None of the tested antibodies exhibited sufficient specificity or avidity to allow sufficient separation and detection of L . monocytogenes for a useful test that differentiated between negative (without cell) and positive (with cell) samples . Plating results confirmed that cells were captured with Dynabeads anti-Listeria and magnetic beads coated with goat anti-Listeria antibody with recovery ranging from 7 to 23% . Fluorescent-labeled polyclonal antibodies used in this study were not sufficiently specific to allow the detection of L . monocytogenes cells captured by the beads. J Food Prot, 2003 Jul, 66(7), 1216 - 21 A predictive model to determine the effects of temperature, sodium pyrophosphate, and sodium chloride on thermal inactivation of starved Listeria monocytogenes in pork slurry; Lihono MA et al.; The effects and interactions of 27 combinations of heating temperature (57.5 to 62.5 degrees C), sodium pyrophosphate (SPP) level (0 to 0.5%, wt/vol), and salt (NaCl) level (0 to 6%, wt/vol) on the thermal inactivation of starved Listeria monocytogenes ATCC 19116 in pork slurry were investigated . A split-split plot experimental design was used to compare all 27 combinations . L . monocytogenes survivors were enumerated on tryptic soy agar supplemented with 0.6% yeast extract . The natural logarithm (loge) of the means of decimal reduction times (D-values) were modeled as a function of temperature, SPP level, and NaCl level . Increasing concentrations of SPP or NaCl protected starved L . monocytogenes from the destructive effect of heat . For example, D-values for the pathogen at 57.5 degrees C in pork slurry with 0, 3, and 6% NaCl were 2.79, 7.75, and 14.59 min, respectively . All three variables interacted to affect the thermal inactivation of L . monocytogenes . A mathematical model describing the combined effect of temperature, SPP level, and NaCl level on the thermal inactivation of starved L . monocytogenes was developed . There was strong correlation (R2 = 0.97) between loge D-values predicted by the model and those observed experimentally . The model can predict D-values for any combination of variables that falls within the range of those tested . This predictive model can be used to assist food processors in designing thermal processes that include an adequate margin of safety for the control of L . monocytogenes in processed meats. J Food Prot, 2003 Jul, 66(7), 1208 - 15 Growth kinetics and cell morphology of Listeria monocytogenes Scott A as affected by temperature, NaCl, and EDTA; Zaika LL et al.; Growth kinetics and morphological characteristics of Listeria monocytogenes Scott A grown under stress conditions induced by increasing levels of NaCl and EDTA were studied as a function of temperature . L . monocytogenes Scott A was inoculated into brain heart infusion broth (pH 6) at 19, 28, 37, and 42 degrees C . Test cultures contained NaCl (at concentrations of 4.5, 6.0, and 7.5%) or EDTA (at concentrations of 0.1, 0.2, and 0.3 mM); control cultures contained 0.5% NaCl . Growth curves were fitted from plate count data by the Gompertz equation, and growth kinetics parameters were derived . Stationary-phase cells were examined by scanning and transmission electron microscopy . Generation times (GTs) and lag phase duration times (LPDs) increased as additive levels were increased . The bacterium grew at all NaCl levels . At 37 and 42 degrees C, growth was slow in media containing 7.5% NaCl, and no growth occurred in media containing 0.3 mM EDTA . Temperature was a major factor in certain stress conditions that led to cell elongation and loss of flagella . Cells in control media at 28 degrees C grew as short rods (0.5 by 1.0 to 2.0 microm), while at 42 degrees C most cells were 4 to 10 times as long . Higher levels of NaCl at higher temperatures resulted in longer and thicker cells . At 28 degrees C, 0.1 mM EDTA had little effect on growth kinetics and morphology; however, 0.3 mM EDTA caused a sixfold increase in GT and LPD and loss of flagellae, with most cells being two to six times as long as normal . Cell length did not correlate with growth kinetics . The results of this study suggest that the effect of altered morphological characteristics of L . monocytogenes cells grown under stress on the virulence and subsequent survival of these cells should be investigated. Syst Appl Microbiol, 2003 Jun, 26(2), 236 - 44 An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates; Keto-Timonen RO et al.; Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles . AFLP also distinguished between L . monocytogenes, L . innocua, L . ivanovii, L . seeligeri, L . welshimeri and L . grayi species . All Listeria species showed species-specific clusters, with less than 33% similarity between different species . A total of 34 L . monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE) . The results of AFLP analysis of L . monocytogenes strains were in concordance with those obtained by PFGE . Both methods identified 29 different genotypes of L . monocytogenes and had a high discrimination index (> 0.999) . By combining the results of AFLP and PFGE, subtype discrimination was further improved . Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L . monocytogenes strains . AFLP was found to be faster and less labour-intensive than PFGE . We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification. Indian J Med Res, 2003 Jan, 117, 19 - 24 Typing of Listeria monocytogenes isolates by random amplification of polymorphic DNA; Dhanashree B et al.; BACKGROUND & OBJECTIVES: Listeria monocytogenes is an important food-borne pathogen causing meningitis and septicaemia in newborns and immunocompromised persons, abortion and preterm labour in pregnant women . Though various methods are available for typing L . monocytogenes, RAPD analysis has been used for epidemiological purposes in developed countries due to its greater discriminating ability . However, as there are no published reports from India on the typing of L . monocytogenes by RAPD technique the present study was undertaken to type isolates of L . monocytogenes from clinical, food and veterinary samples . METHODS: Isolates of L . monocytogenes were subjected to RAPD using four decamer random primers R1, R2, R3 and R4 . Amplified products were analysed by agarose gel electrophoresis . RESULTS: Eight strains of L . monocytogenes on RAPD analysis generated 4 distinct profiles each with R1 and R4 primers and 3 different profiles with R2 and R3 primers . The isolates from fish, clinical and veterinary samples showed different profiles with respect to each other . Isolate from flat fish (serovar 4) showed a different profile from that of clams (serovar 1) . Two isolates from placenta (serovar 1) showed similar profiles and all the isolates from veterinary samples generated similar profiles . INTERPRETATION & CONCLUSION: RAPD analysis in the present study allowed discrimination of isolates among the same serotype but from different sources . Since RAPD is a rapid technique and offers greater discrimination of strains, this method may be used for typing L . monocytogenes in India. Immunol Res, 2003, 27(2-3), 451 - 62 Rational approaches to immune regulation; Paterson Y; Our laboratory is interested in the properties of proteins that render them immunogenic, and how such immunogenicity may be modulated in vivo . We are attempting to enhance the immune response in the design of more effective vaccines against viral diseases, such as HIV, and against tumor antigens expressed on breast, ovarian, and cervical cancer and B cell lymphomas . Our main approach is to use a facultative intracellular bacterium, Listeria monocytogenes, which has the unusual ability to live and grow in the cytoplasm of the cell and is thus an excellent vector for targeting passenger antigens to the major histocompatibility complex (MHC) class I pathway of antigen processing with the generation of authentic cytotoxic T lymphocytes (CTL) epitopes . In the field of tumor immunotherapy, we are also developing nonliving vaccine vectors for tumor antigens. J Biol Chem, 2003 Sep 19, 278(38), 36810 - 8 Epub 2003 Jul 10. Design of N-substituted peptomer ligands for EVH1 domains; Zimmermann J et al.; Ena/VASP proteins are implicated in cytoskeletal reorganization during actin-dependent motility processes . Recruitment to subcellular sites of actin polymerization is mediated by the highly conserved N-terminal EVH1 domain, which interacts with target proteins containing proline-rich motifs . The VASP EVH1 domain specifically binds peptides with the consensus motif FPPPP present in all its binding partners, including the Listerial ActA protein . Previous studies have shown that the Phe and first and final Pro residues are highly conserved and cannot be substituted with any other natural amino acid without significant loss of binding affinity . We have incorporated peptoid building blocks (sarcosine derived, non-natural amino acids) into the peptide SFEFPPPPTEDEL from the Listerial ActA protein and were able to substitute the most highly conserved residues of this motif while maintaining binding to the VASP EVH1 domain with affinities in the range of 45-180 microm . We then used NMR chemical shift perturbations to locate specific domain residues involved in particular interactions . These studies may open up the way for designing selective modulators of VASP function for biological studies and for the development of novel therapeutics for diseases involving pathologically altered cell adhesion or cell motility. J Immunol, 2003 Jul 15, 171(2), 533 - 7 Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88; Way SS et al.; In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response . The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM) . In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM . Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM . These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88. Acta Paediatr Taiwan, 2003 Mar-Apr, 44(2), 106 - 8 Early-onset listeriosis in prematurity; Chen JM et al.; Listeria monocytogenes has been recognized as a human pathogen for more than 70 years . It causes illness mainly in pregnant women, newborns, elderly, and immunocompromised persons . Although L . Monocytogenes is a relatively uncommon pathogen in neonates, it can cause considerable morbidity and mortality in this age group, especially in the early-onset form of the disease . In Taiwan, neonatal listeriosis is rarely reported . We report one case of a premature newborn with early-onset listeria sepsis and meningitis. J Dairy Sci, 2003 Jun, 86(6), 1865 - 75 ADSA Foundation Scholar Award--An integrated science-based approach to dairy food safety: Listeria monocytogenes as a model system; Wiedmann M; ADSA Foundation; Transmission of food- and milkborne pathogens often involves complex interactions among the pathogen, the environment, and one or multiple host species . A complete understanding of these interactions is critical to allow the development of science-based, effective intervention strategies for foodborne infectious diseases . This article summarizes our studies on the transmission, ecology, pathogenesis and population genetics of Listeria monocytogenes, which we have used as model for a food- and milkborne pathogen that infects multiple hosts and also has considerable ability to survive and multiply in nonhost environments . Application of molecular subtyping tools in conjunction with phenotypic characterization of selected strains has allowed us to define distinct L . monocytogenes subtypes and clonal groups that appear to differ in relevant phenotypic characteristics that may affect their abilities to be transmitted through food systems . For example, a genetic group designated as lineage I has been shown to be not only more common among human listeriosis cases than among animal cases, but lineage I strains also appear to show an increased in vitro ability to spread intracellularly from host cell to host cell . These findings are consistent with the fact that while genetically diverse strains may be classified to one bacterial species, these strains often differ from one another in important genetic and phenotypic characteristics . I thus propose that evolutionary- and molecular subtyping-based definitions of bacterial subtypes and clonal groups will provide critical insight into the microbial ecology of dairy food systems, including not only foodborne pathogens, but also organisms important for dairy fermentation and spoilage. J Biol Chem, 2003 Sep 12, 278(37), 35102 - 8 Epub 2003 Jun 27. Tumor cell killing enabled by listeriolysin O-liposome-mediated delivery of the protein toxin gelonin; Provoda CJ et al.; Gelonin is a type I plant toxin that has potential as an effective anti-tumor agent by virtue of its enzymatic capacity to inactivate ribosomes and arrest protein synthesis, thereby effectively limiting the growth of cancer cells . Being a hydrophilic macromolecule, however, gelonin has limited access to its target subcellular compartment, the cytosol; it is effectively plasma membrane-impermeant and subject to rapid degradation within endosomes and lysosomes upon cellular uptake as it lacks the membrane-translocating capability that is typically provided by a disulfide-linked B polypeptide found in the type II toxins (e.g . ricin) . These inherent characteristics generate the need for the development of a specialized cytosolic delivery strategy for gelonin as an effective anti-tumor therapeutic agent . Here we describe an efficient means of delivering gelonin to the cytosol of B16 melanoma cells . Gelonin was co-encapsulated inside pH-sensitive liposomes with listeriolysin O, the pore-forming protein that mediates escape of the intracellular pathogen Listeria monocytogenes from the endosome into the cytosol . In in vitro experiments, co-encapsulated listeriolysin O enabled liposomal gelonin-mediated B16 cell killing with a gelonin IC50 of approximately 0.1 nM with an extreme efficiency requiring an incubation time of only 1 h . By contrast, cells treated with equivalent concentrations of unencapsulated gelonin or gelonin encapsulated alone in pH-sensitive liposomes exhibited no detectable cytotoxicity . Moreover, treatment by direct intratumor injection into subcutaneous solid tumors of B16 melanoma in a mouse model showed that pH-sensitive liposomes containing both listeriolysin O and gelonin were more effective than control formulations in curtailing tumor growth rates. FEMS Microbiol Lett, 2003 Jun 27, 223(2), 205 - 10 Identification of Listeria innocua by PCR targeting a putative transcriptional regulator gene; Liu D et al.; Listeria innocua is a common, non-pathogenic bacterial species that shares morphological, biochemical and molecular characteristics with the pathogenic species L . monocytogenes . The presence of L . innocua may cause difficulty or confusion in the laboratory identification of L . monocytogenes or other Listeria spp . In this report, through examining the recently published genome sequence of L . innocua strain CLIP 11262 (serovar 6a), we identified a L . innocua-specific gene (lin0464) encoding a putative transcriptional regulator and evaluated its efficacy for species-specific detection by polymerase chain reaction (PCR) . The specificity of the oligonucleotide primers (lin0464F and lin0464R) derived from this gene was confirmed with the formation of a 749-bp fragment in PCR from genomic DNA of L . innocua strains only . We expect that this assay will be useful in confirming identification of L . innocua or in studies where rapid detection of L . innocua is necessary. Cell Mol Life Sci, 2003 May, 60(5), 904 - 18 Pathogen, host and environmental factors contributing to the pathogenesis of listeriosis; Roberts AJ et al.; Listeriosis is a severe human and animal disease caused by two species of pathogenic bacteria from the genus Listeria, L . monocytogenes and L . ivanovii . In humans, listeriosis is overwhelmingly a foodborne disease, yet much remains to be learned regarding the transmission dynamics of pathogenic Listeria from the environment, through food, to humans . Similarly, our understanding of the various host, pathogen and environmental factors that impact the pathogenesis of listeriosis at the cellular and molecular level is incomplete . This review will summarize what is currently known about animal and human listeriosis, detail the pathogen, host and environmental factors that contribute to pathogenesis and, finally, examine the interactions among those factors that influence the occurrence of human infection. Transplant Proc, 2003 Jun, 35(4), 1485 - 7 Infection with Listeria monocytogenes following orthotopic liver transplantation: case report and review of the literature; Rettally CA et al.; Infection with Listeria monocytogenes is rare with a reported annual incidence of 4.4 cases/million individuals . Epidemiological data have identified certain groups to be higher risk of developing listeriosis, including neonates, pregnant women, adults older than 60 years of age, individuals afflicted with hematologic malignancies, acquired immunodeficicency syndrome, cirrhosis, and those receiving corticosteroid therapy and organ transplants . Within this last group, multiple cases have been described following bone marrow and renal transplantation, but only a few following liver transplantation . We report a case of a 66-year-old woman presenting with Listeria monocytogenes bacteremia at 32 months following orthotopic liver transplantation. Jpn J Infect Dis, 2003 Apr, 56(2), 60 - 1 Listeriosis in second trimester of pregnancy: case report from India; Gupta V et al.; Although Listeria monocytogenes infection occurs in sporadic and epidemic forms throughout the world, there are certain countries (especially Asian countries) that have reported only a few cases or failed to report even a single case . During her third visit at 17(+5) weeks of gestation, a 22-year-old primigravida presented with the complaint of an acute painful abdomen, leaking per vaginum and low-grade fever for the 2 preceding days . On ultrasonography, a single live fetus with no amniotic fluid was seen and the pregnancy was therefore terminated . L . monocytogenes was isolated from a high vaginal swab. J Immunol, 2003 Jul 1, 171(1), 291 - 8 Class Ia MHC-deficient BALB/c mice generate CD8+ T cell-mediated protective immunity against Listeria monocytogenes infection; D'Orazio SE et al.; CD8(+) T cells are required for protective immunity against intracellular pathogens such as Listeria monocytogenes . In this study, we used class Ia MHC-deficient mice, which have a severe reduction in circulating CD8(+) T cells, to determine the protective capacity of class Ib MHC-restricted T cells during L . monocytogenes infection . The K(b-/-)D(b-/-) mutation was backcrossed onto a C.B10 (BALB/c congenic at H-2 locus with C57BL/10) background, because BALB/c mice are more susceptible to Listeria infection than other commonly studied mouse strains such as C57BL/6 . C.B10 K(b-/-)D(b-/-) mice immunized with a sublethal dose of L . monocytogenes were fully protected against a subsequent lethal infection . Adoptive transfer of Listeria-immune splenocyte subsets into naive K(b-/-)D(b-/-) mice indicated that CD8(+) T cells were the major component of this protective immune response . A CD8(+) T cell line isolated from the spleen of a Listeria-infected class Ia MHC-deficient mouse was shown to specifically recognize Listeria-infected cells in vitro, as determined by IFN-gamma secretion and cytotoxicity assays . Adoptive transfer of this T cell line alone resulted in significant protection against L . monocytogenes challenge . These results suggest that even a limited number of class Ib MHC-restricted T cells are sufficient to generate the rapid recall response required for protection against secondary infection with L . monocytogenes. J Immunol, 2003 Jul 1, 171(1), 37 - 46 The uterine NK cell population requires IL-15 but these cells are not required for pregnancy nor the resolution of a Listeria monocytogenes infection; Barber EM et al.; During pregnancy in mice, uterine natural killer (uNK) cells abundantly accumulate on the mesometrial side of the placenta . In this study, we show that the presence of both mature and immature uNK cells requires IL-15 . Bone marrow transplantation of NK cell-negative mice due to null mutations in the recombination-activating gene (Rag) 2/common cytokine receptor gamma-chain (Rag2(-/-)gamma(c)(-/-)) genes indicated that uNK cells originate from the bone marrow and require IL-15 to develop . NK cells are thought to be central players in the immune response to intracellular pathogens such as Listeria monocytogenes, a bacterium that also has a predilection for replication in the placenta . However, IL-15(-/-), NK cell-deficient mice were relatively protected from this infection compared with wild-type mice, and during pregnancy the absence of NK cells did not compromise the immune response at this site . The loss of uNK cells results in decidual abnormalities, including thickening of the arterial walls with luminal narrowing and a hypocellular decidua basalis . These defects were rescued by bone marrow transplantation of the Rag2(-/-)gamma(c)(-/-) mice that restored the uNK cell population . The decidual abnormalities in the IL-15(-/-) mice however did not result in infertility as gestation times and litter sizes were comparable to those of wild-type mice . Fetal weights were mildly compromised, consistent with the arterial pathologies . These results show that uNK cells are not required for successful pregnancy and that NK cells are not essential for an adequate immune response to L . monocytogenes in either pregnant or nonpregnant mice. Cell Microbiol, 2003 Jul, 5(7), 455 - 68 Cytoplasmic bacteria can be targets for autophagy; Rich KA et al.; Autophagy is an important constitutive cellular process involved in size regulation, protein turnover and the removal of malformed or superfluous subcellular components . The process involves the sequestration of cytoplasm and organelles into double-membrane autophagic vacuoles for subsequent breakdown within lysosomes . In this work, we demonstrate that the intracellular pathogen Listeria monocytogenes can also be a target for autophagy . If infected macrophages are treated with chloramphenicol after phagosome lysis, the bacteria are internalized from the cell cytoplasm into autophagic vacuoles . The autophagic vacuoles appear to form by fusion of small cytoplasmic vesicles around the bacteria . These vesicular structures immunolabel with antibodies to protein disulphide isomerase, a marker for the rough ER . Internalization of metabolically arrested cytoplasmic L . monocytogenes represents an autophagic process as the vacuoles have double membranes and the process can be inhibited by the autophagy inhibitors 3-methyladenine and wortmannin . Additionally, the rate of internalization can be accelerated under starvation conditions and the vacuoles fuse with the endocytic pathway . Metabolic inhibition of cytoplasmic bacteria prevents them from adapting to the intracellular niche and reveals a host mechanism utilizing the autophagic pathway as a defence against invading pathogens by providing a route for their removal from the cytoplasm and subsequent delivery to the endocytic pathway for degradation. Int J Food Microbiol, 2003 Aug 1, 84(3), 285 - 97 Genetic variability among isolates of Listeria monocytogenes from food products, clinical samples and processing environments, estimated by RAPD typing; Martinez I et al.; RAPD analysis with four primers was used to examine the genetic relationship among 432 strains of Listeria monocytogenes isolated from clinical and veterinarian cases of listeriosis, dairy, vegetable, meat- and fish-based food items, environmental samples and samples collected from one transport terminal, one poultry-processing company and four Atlantic salmon-processing plants . The purpose of the study was to determine whether clinical isolates belonged to a specific genetic group, whether links could be made between food groups and clinical cases and whether specific genetic groups were associated with specific food products or processing units . There was great genetic variability among the isolates, which produced a total of 141 RAPD composites based on the RAPD analysis with four primers . The RAPD composites divided in two major clusters and clinical isolates were evenly distributed in both of them . None of the isolates from food products had the same RAPD composite as isolates from human patients, thus, no particular food commodity could be linked to clinical cases . Each food-processing environment was contaminated with more than one RAPD composite and the genetic variability found within each company was, in most cases, of approximately the same magnitude as the variability found when considering all the samples . In each plant, one or a few types persisted over time, indicating the presence of an established in-house flora . Our results indicate that most of the analysed cases of listeriosis were sporadic and, further, that these cases cannot be traced to a few specific food sources . We also found that no particular RAPD composite was better suited for survival in specific food types or food-processing environments, indicating that although differences may be found in virulence properties of individual strains, all L . monocytogenes must be treated as potentially harmful. Gene Ther, 2003 Jul, 10(13), 1105 - 15 Cytosolic delivery of antisense oligonucleotides by listeriolysin O-containing liposomes; Mathew E et al.; Antisense oligodeoxynucleotides (ODNs) possess great potential as sequence-specific therapeutic agents . Sufficient concentrations of intact ODN must bypass membrane barriers and access the cytosol and nucleus, for ODNs to be therapeutically effective . A cytosolic delivery strategy was designed to improve the efficiency of ODN delivery in bone-marrow-derived macrophages . This liposome-based formulation utilizes listeriolysin O (LLO), the endosomolytic hemolysin from Listeria monocytogenes, to mediate the escape of ODN from endocytic compartments into the cytosol . To monitor the cytosolic delivery of ODN, subcellular trafficking of fluorescently labeled ODNs was visualized using epifluorescence microscopy . The expression of target protein and mRNA after delivery was measured using flow cytometry and Northern blot analysis, respectively . ODN specific for murine intercellular adhesion molecule-1 (ICAM-1) encapsulated in LLO-liposomes was released to the cytosol and trafficked to the nucleus, efficiently and specifically suppressing activation-induced expression of ICAM-1 at both protein and mRNA levels . Delivery without LLO resulted in sequestration of ODN in vesicular compartments leading to little inhibition of ICAM-1 expression, which supports the requirement of LLO for efficient cytosolic delivery using this system . The data clearly demonstrate that LLO-mediated escape of ODN from intracellular vesicles is an effective approach to achieve full therapeutic antisense activity in cultured macrophages. Immunology, 2003 Jul, 109(3), 450 - 60 Anti-human immunodeficiency virus-gag CD8+ memory T cells generated in vitro from Listeria-immunized mice; Rayevskaya M et al.; The goal of vaccination is the generation of immune memory, an immune state that permits rapid and intense recall responses to a pathogen . Considerable effort is being made to understand the nature of memory T cells . We report here that by extending the length of in vitro culture following a single restimulation with specific peptide, preparations of highly enriched, highly active antigen-specific CD8+ memory T cells could be obtained . These cultures were begun with splenocytes from mice primed by infection either with an attenuated strain of Listeria monocytogenes or vaccinia virus, both expressing the human immunodeficiency virus-1-gag gene . In the cultures, antigen-specific cytotoxic T lymphocyte (CTL) activity reached a maximum at about 9 days and thereafter fell to negligible values . Concomitant with the fall of CTL activity, however, we observed enrichment for a subset of CD11ahigh antigen-specific gag-tetramerpos CD8+ T cells . The cells showed little or no 4-hr CTL activity, but had high delayed (18-hr) CTL activity, and very high cytolytic activity after restimulation . They rapidly expressed interferon-gamma production . Their growth and survival after sorting was completely dependent on interleukin-2 or -15 . As few as 5000 of the fluorescence-activated cell sorting-purified cells protected recipients against challenge 3 months after transfer . In response to the challenge, the cells repopulated lymphoid and non-lymphoid organs and showed a sizeable increase in number . The cells therefore demonstrate high protective activity for long periods of time . These cultured cells are thus a potential source of enriched natural memory T cells for reperfusion studies and in which the mechanisms that underlie the generation, differentiation and persistence of memory can be examined. Immunology, 2003 Jul, 109(3), 407 - 14 Induction of interleukin-12 production in mouse macrophages by berberine, a benzodioxoloquinolizine alkaloid, deviates CD4+ T cells from a Th2 to a Th1 response; Kim TS et al.; In this study we investigated whether berberine-mediated induction of interleukin-12 (IL-12) production in antigen-presenting cells could regulate a cytokine profile of antigen-primed CD4+ T helper (Th) cells . Pretreatment with berberine induced IL-12 production in both macrophages and dendritic cells, and significantly increased the levels of IL-12 production in lipopolysaccharide-stimulated macrophages and in CD40 ligand-stimulated dendritic cells . Importantly, berberine pretreatment of macrophages increased their ability to induce interferon-gamma (IFN-gamma) and reduced their ability to induce IL-4 in antigen-primed CD4+ T cells . Berberine did not influence the macrophage cell surface expression of the class II major histocompatibility complex molecule, the co-stimulatory molecules CD80 and CD86, and intracellular adhesion molecule-1 . Addition of neutralizing anti-IL-12p40 monoclonal antibody to cultures of berberine-pretreated macrophages and CD4+ T cells restored IL-4 production in antigen-primed CD4+ T cells . The in vivo administration of berberine resulted in the enhanced induction of IL-12 production by macrophages when stimulated in vitro with lipopolysaccharide or heat-killed Listeria monocytogenes, leading to the inhibition of the Th type 2 cytokine profile (decreased IL-4 and increased IFN-gamma production) in antigen-primed CD4+ T cells . These findings may point to a possible therapeutic use of berberine or medicinal plants containing berberine in the Th type 2 cell-mediated immune diseases such as allergic diseases. AIDS Res Hum Retroviruses, 2003 May, 19(5), 409 - 20 Enhancement of immune responses to an HIV env DNA vaccine by a C-terminal segment of listeriolysin O; Bu Z et al.; An effective vaccine against AIDS should induce both cellular and humoral immune responses . Here we report that immunization of mice with a DNA plasmid encoding a chimeric protein consisting of HIV89.6 Env gp140 and the listeriolysin O (LLO) C-terminal segment (59 amino acids) significantly enhanced both humoral and cellular immune responses against the HIV89.6 Env protein . Plasmid DNA expression vectors with genes codon-optimized for mammalian expression were synthesized for HIV89.6 gp140 as well as for chimeric protein gp140-LLO, in which the coding sequence for the C-terminal 59 amino acids of LLO were fused in frame to the 3' end of the codon-optimized gene for gp140 . All plasmid vectors produced high levels of protein expression, and the gp140-LLO chimeric protein was cleaved and secreted as efficiently as gp140 . Analysis of humoral immune responses by ELISA showed that the chimeric gp140-LLO construct induced higher antibody responses than the gp140 construct in immunized mice, more notably in the IgG2a antibody subtype . Intracellular cytokine staining and flow cytometry analysis showed that the gp140-LLO construct induced significantly higher levels of cytotoxic T lymphocyte immune responses against the HIV 89.6 Env protein than those observed with the gp140 construct . Our results thus demonstrate that the C-terminal segment of LLO can be effectively employed to enhance both cellular and humoral immune responses against the HIV89.6 Env antigen in the context of a DNA vaccine. J Food Prot, 2003 Jun, 66(6), 993 - 8 Ionizing radiation sensitivity of Listeria monocytogenes ATCC 49594 and Listeria innocua ATCC 51742 inoculated on endive (Cichorium endiva); Niemira BA et al.; Ionizing radiation inactivates the pathogenic bacteria that can contaminate leafy green vegetables . Leaf pieces and leaf homogenate of endive (Cichorium endiva) were inoculated with the pathogen Listeria monocytogenes (ATCC 49594) or Listeria innocua (ATCC 51742), a nonpathogenic surrogate bacterium . The radiation sensitivity of the two strains was similar, although L . innocua was more sensitive to the type of suspending leaf preparation . During refrigerated storage after irradiation, the population of L . monocytogenes on inoculated endive was briefly suppressed by 0.42 kilogray (kGy), a dose calibrated to achieve a 99% reduction . However, the pathogen regrew after 5 days until it exceeded the bacterial levels on the control after 19 days in storage . Treatment with 0.84 kGy, equivalent to a 99.99% reduction, suppressed L . monocytogenes throughout refrigerated storage . Doses up to 1.0 kGy had no significant effect on the color of endive leaf material, regardless of whether taken from the leaf edge or the leaf midrib . The texture of leaf edge material was unaffected by doses up to 1.0 kGy, whereas the maximum dose tolerated by leaf midrib material was 0.8 kGy . These results show that endive leaves may be treated with doses sufficient to achieve at least a 99.99% reduction of L . monocytogenes with little or no impact on the product's texture or color. J Food Prot, 2003 Jun, 66(6), 985 - 92 Acid adaptation does not promote survival or growth of Listeria monocytogenes on fresh beef following acid and nonacid decontamination treatments; Ikeda JS et al.; The objective of this study was to evaluate the survival and growth of acid-adapted and nonadapted Listeria monocytogenes inoculated onto fresh beef subsequently treated with acid or nonacid solutions . Beef slices (2.5 by 5 by 1 cm) from top rounds were inoculated with acid-adapted or nonadapted L . monocytogenes (4.6 to 5.0 log CFU/cm2) and either left untreated (control) or dipped for 30 s in water at 55 degrees C, water at 75 degrees C, 2% lactic acid at 55 degrees C, or 2% acetic acid at 55 degrees C . The beef slices were vacuum packaged and stored at 4 or 10 degrees C and were analyzed after 0, 7, 14, 21, and 28 days of storage . Dipping in 75 degrees C water, lactic acid, and acetic acid resulted in immediate pathogen reductions of 1.4 to 2.0, 1.8 to 2.6, and 1.4 to 2.4 log CFU/cm2, respectively . After storage at 10 degrees C for 28 days, populations of L . monocytogenes on meat treated with 55 degrees C water increased by ca . 1.6 to 1.8 log CFU/cm2 . The pathogen remained at low population levels (1.6 to 2.8 log CFU/cm2) on acid-treated meat, whereas populations on meat treated with 75 degrees C water increased rapidly, reaching levels of 3.6 to 4.6 log CFU/cm2 by day 14 . During storage at 4 degrees C, there was no growth of the pathogen for at least 21 days in samples treated with 55 and 75 degrees C water, and periods of no growth were longer for acid-treated samples . There were no differences between acid-adapted and nonadapted organisms across treatments with respect to survival or growth . In conclusion, the dipping of meat inoculated with L . monocytogenes into acid solutions reduced and then inhibited the growth of the pathogen during storage at 4 and 10 degrees C, while dipping in hot water allowed growth despite initial reductions in pathogen contamination . The results of this study indicate a residual activity of acid-based decontamination treatments compared with water-based treatments for refrigerated (4 degrees C) or temperature-abused (10 degrees C) lean beef tissue in vacuum packages, and these results also indicate that this activity may not be counteracted by prior acid adaptation of L . monocytogenes. J Food Prot, 2003 Jun, 66(6), 962 - 9 Survival and recovery of Listeria monocytogenes on ready-to-eat meats inoculated with a desiccated and nutritionally depleted dustlike vector; De Roin MA et al.; Dust from construction was theorized to serve as a vector for L . monocytogenes transmission to ready-to-eat (RTE) meats after heat processing but before packaging . A five-strain Listeria monocytogenes culture including serotype 4b was continually stressed on a sand vector under four sets of nutritionally depleted and dry conditions to simulate postprocessing contamination by dustlike particulates . The stresses included that associated with sand stored at different temperatures (10 and 22 degrees C) and levels of humidity (40% relative humidity {RH}, 88% RH, or complete desiccation) . Irradiated RTE meats, including frankfurters, bologna, chopped ham, and deli-style roast beef, were inoculated with the L . monocytogenes-contaminated sand every 2 to 3 days over a period of 1 1/2 months . After inoculation, the RTE meats were vacuum packed and stored at 4 degrees C for 24 h . Populations of L . monocytogenes were enumerated by surface plating on nonselective and selective media to recover cells on the basis of the different stresses presented (osmotic or antibiotic) . L . monocytogenes was demonstrated to be capable of surviving on the sand vector for > 151 days at 10 degrees C and 88% RH, 136 days at 10 degrees C and 0% RH, 73 days at 22 degrees C and 40% RH, and 82 days at 22 degrees C and 0% RH . These results show that under the most conservative scenario, the 73-day-old L . monocytogenes-contaminated sand was able to attach to and be recovered from the RTE meats . This study illustrated that dust contaminated with L . monocytogenes, once in contact with meat surfaces, can survive and grow, posing a health hazard to consumers. Biotechnol Bioeng, 2003 Aug 20, 83(4), 416 - 27 Micro-assembly of functionalized particulate monolayer on C18-derivatized SiO2 surfaces; Huang TT et al.; This work describes a simple approach to immobilize functionalized colloidal microstructures onto a C(18)-coated SiO(2) substrate via specific or non-specific bio-mediated interactions . Biotinylated bovine serum albumin pre-adsorbed onto a C(18) surface was used to mediate the surface assembly of streptavidin-coated microbeads (2.8 microm), while a bare C(18) surface was used to immobilize anti-Listeria antibody-coated microbeads (2.8 microm) through hydrophobic interactions . For a C(18) surface pre-adsorbed with bovine serum albumin, hydrophobic polystyrene microbeads (0.8 microm) and positively charged dimethylamino microbeads (0.8 microm) were allowed to self-assemble onto the surface . A monolayer with high surface coverage was observed for both polystyrene and dimethylamino microbeads . The adsorption characteristics of Escherichia coli and Listeria monocytogenes on these microbead-based surfaces were studied using fluorescence microscopy . Both streptavidin microbeads pre-adsorbed with biotinylated anti-Listeria antibody and anti-Listeria antibody-coated microbeads showed specific capture of L . monocytogenes, while polystyrene and dimethylamino microbeads captured both E . coli and L . monocytogenes non-specifically . The preparation of microbead-based surfaces for the construction of microfluidic devices for separation, detection, or analysis of specific biological species is discussed . J Dent, 2003 Jul, 31(5), 313 - 9 The erosive potential of commercially available mouthrinses on enamel as measured by Quantitative Light-induced Fluorescence (QLF); Pretty IA et al.; DESIGN: Longitudinal in vitro . METHODS: Previously extracted, caries free, human premolars were selected and prepared by gentle pumicing and coating in an acid-resistant nail-varnish save for an exposed enamel window on the buccal surface . Each was assigned to one of eight groups (six per group, 10 in positive control); positive control (citric acid, pH 2.7, F(-) 0 ppm), negative control (pH 7.0, F(-) 0 ppm) Listerine (pH 3.87, F(-) 0.021 ppm), Tesco Value (pH 6.05, F(-) 289.00 ppm), Tesco Total Care (pH 6.20, F(-) 313.84 ppm), Sainsbury's (pH 6.15, F(-) 365.75 ppm), Sensodyne (pH 6.12, F(-) 285.30 ppm) and Corsodyl (pH 5.65, F(-) 0 ppm) . The titratable acid values (TAV) for each rinse were established using volume (ml) of 0.1 M NaOH to achieve pH 7 . Fluoride values were obtained by ion selective electrode . The solutions were kept at 37 degrees C and gently agitated . Teeth were removed at hourly intervals for 15 h, air-dried and subjected to Quantitative Light-induced Fluorescence (QLF) examination by a blinded examiner and DeltaQ values recorded . At the conclusion of the study each of the positive control teeth and one from each other group were sectioned through the eroded lesion, ground and polished to 100 micrometers and subjected to transverse microradiography and DeltaZ recorded for validation . RESULTS: TAVs were: Listerine 2.45 L > Sainsbury's 0.35 ml >Tesco Total Care 0.14 ml > Tesco Value 0.08 ml > Corsodyl 0.10 ml >Sensodyne 0.9 ml . DeltaQ increased over time for the positive control, (0 h 0.2, 10 h 95.2, 15 h 152.3) . Negative controls remained stable . The increase in DeltaQ for each rinse after 15 h was Listerine (9.3(+/-7.2)), Corsodyl (1.5(+/-1.2)), Tesco Value (1.8(+/-1.2)), Tesco Total Care (1.4(+/-1.1)), Sainsbury's (3.4(+/-2.2)), Sensodyne (0.9(+/-1.6)) . TMR confirmed the presence/absence of erosive lesions . CONCLUSIONS: QLF effectively monitored erosion in the positive controls and lack of erosion in the NC . Only one mouthrinse (Listerine) caused any erosion compared to the negative control, but this was only significant after 14 h of continuous use. Cell Immunol, 2003 Mar, 222(1), 1 - 14 IL-12-assisted immunization generates CD4+ T cell-mediated immunity to Listeria monocytogenes; Miller MA et al.; Mice infected with virulent Listeria monocytogenes develop long-lived acquired immunity . We previously reported that acquired immunity to Listeria could also be elicited by immunizing mice with non-viable Listeria or listerial proteins/peptides in combination with IL-12 . Here we show that this IL-12-assisted immunization strategy was effective in class I but not in class II MHC-deficient mice, suggesting that antigen-specific CD4(+) T cells are selectively generated using this adjuvant system . We have also evaluated the importance of endogenous production of IFN-gamma and IL-12 for the efficacy of IL-12-assisted immunization . IFN-gamma-deficient mice immunized with HKLM and IL-12 failed to produce effective Listeria-specific responses . In contrast, IL-12-deficient mice were able to generate protective antigen-specific T cell responses in response to immunization with HKLM and IL-12, indicating that exogenous IL-12 is sufficient to initiate a cytokine cascade that results in a potent T(H)1 response . IL-12-assisted immunization provides a model in which both the generation and effector mechanisms of anti-bacterial antigen-specific CD4(+) effector cells can be analyzed. Int J Immunopathol Pharmacol, 2003 May-Aug, 16(2), 119 - 27 Heterogeneity of virulence-related properties in Listeria monocytogenes strains isolated from patients with haematological malignancies; Longhi C et al.; Listeria monocytogenes is an intracellular foodborne pathogen of humans and animals for which there are indications of virulence differences among strains . Various virulence properties related to different phases of infection process were investigated in L . monocytogenes strains isolated from patients affected by haematological malignancies . In these isolates, besides to the clinical history, we analysed the haemolysin production, the survival to acidic pH, the ability to enter and proliferate in human intestinal-like and human macrophagic-like cells, as well as the allelic polymorphism of the actA gene involved intracellular movement . A general heterogeneity in the virulence properties was detected which did not appear correlated with the clinical outcome of listeriosis but more probably was influenced by the status of the immune defence of the host. Mol Microbiol, 2003 Jun, 48(6), 1537 - 51 Isolation of Listeria monocytogenes mutants with high-level in vitro expression of host cytosol-induced gene products; Shetron-Rama LM et al.; The facultative intracellular bacterial pathogen Listeria monocytogenes dramatically increases the expression of several key virulence factors upon entry into the host cell cytosol . actA, the protein product of which is required for cell-to-cell spread of the bacterium, is expressed at low to undetectable levels in vitro and increases in expression more than 200-fold after L . monocytogenes escape from the phagosome . To identify bacterial factors that participate in the intracellular induction of actA expression, L . monocytogenes mutants expressing high levels of actA during in vitro growth were selected after chemical mutagenesis . The resulting mutant isolates displayed a wide range of actA expression levels, and many were less sensitive to environmental signals that normally mediate repression of virulence gene expression . Several isolates contained mutations affecting actA gene expression that mapped at least 40 kb outside the PrfA regulon, supporting the existence of additional regulatory factors that contribute to virulence gene expression . Two actA in vitro expression mutants contained novel mutations within PrfA, a key regulator of L . monocytogenes virulence gene expression . PrfA E77K and PrfA G155S mutations resulted in high-level expression of PrfA-dependent genes, increased bacterial invasion of epithelial cells and increased virulence in mice . Both prfA mutant strains were significantly less motile than wild-type L . monocytogenes . These results suggest that, although constitutive activation of PrfA and PrfA-dependent gene expression may enhance L . monocytogenes virulence, it may conversely hamper the bacterium's ability to compete in environments outside host cells. Mol Microbiol, 2003 Jun, 48(6), 1525 - 36 Aromatic amino acids at the surface of InlB are essential for host cell invasion by Listeria monocytogenes; Machner MP et al.; The surface protein InlB of the pathogen Listeria monocytogenes promotes invasion of this bacterium into host cells by binding to and activating the receptor tyrosine kinase Met . The curved leucine-rich repeat (LRR) domain of InlB, which is essential for this process, contains a string of five surface-exposed aromatic amino acid residues positioned along its concave face . Here, we show that the replacement of four of these residues (F104, W124, Y170 or Y214) by serine leads to a complete loss of uptake of latex beads coated with InlB', a truncated functional variant of InlB . The mutants correspondingly display severely reduced binding to Met . To abrogate fully invasion of bacteria expressing full-length InlB, exchange of at least four aromatic amino acids is required . We conclude that InlB binds to Met through its concave surface of the LRR domain, and that aromatic amino acids are critical for binding and signalling before invasion. Appl Environ Microbiol, 2003 Jun, 69(6), 3640 - 5 Adhesion, invasion, and translocation characteristics of Listeria monocytogenes serotypes in Caco-2 cell and mouse models; Jaradat ZW et al.; Adhesion is a crucial first step in Listeria monocytogenes pathogenesis . In this study, we examined how the adhesion properties of serotypes correlate with their invasion efficiencies in a cell culture model (Caco-2) and in a mouse model . Adhesion characteristics of all 13 serotypes of L . monocytogenes (25 strains) were analyzed, which yielded three distinct groups (P < 0.05) with high-, medium-, and low-level-adhesion profiles . The efficiency of these strains in invading the Caco-2 cell line was analyzed, which produced two groups; however, the overall correlation (R(2)) was only 0.1236 . In the mouse bioassay, all selected strains, irrespective of their adhesion profiles, translocated to the liver and the spleen with almost equal frequencies that did not show any clear relationship with adhesion profiles . However, the serotypes with increased adhesion showed a slightly increased translocation to the brain (R(2) = 0.3371) . Collectively, these results indicate that an in vitro adhesion profile might not be an accurate assessment of a strain's ability to invade a cultured cell line or organs or tissues in a mouse model. Appl Environ Microbiol, 2003 Jun, 69(6), 3368 - 76 Development of a Listeria monocytogenes EGDe partial proteome reference map and comparison with the protein profiles of food isolates; Ramnath M et al.; A partially annotated proteome reference map of the food pathogen Listeria monocytogenes was developed for exponentially growing cells under standardized, optimal conditions by using the sequenced strain EGDe (serotype 1/2a) as a model organism . The map was developed by using a reproducible total protein extraction and two-dimensional (2-D) polyacrylamide gel electrophoresis analysis procedure, and it contained 33 identified proteins representing the four main protein functional classes . In order to facilitate analysis of membrane proteins, a protein compartmentalization procedure was assessed . The method used provided partial fractionation of membrane and cytosolic proteins . The total protein 2-D profiles of three serotype 1/2a strains and one serotype 1/2b strain isolated from food were compared to the L . monocytogenes EGDe proteome . An average of 13% of the major protein spots in the food strain proteomes were not matched in the strain EGDe proteome . The variation was greater for the less intense spots, and on average 28% of these spots were not matched . Two of the proteins identified in L . monocytogenes EGDe were missing in one or more of the food isolates . These two proteins were proteins involved in the main glycolytic pathway and in metabolism of coenzymes and prosthetic groups . The two corresponding genes were found by PCR amplification to be present in the four food isolates . Our results show that the L . monocytogenes EGDe reference map is a valuable starting point for analyses of strains having various origins and could be useful for analyzing the proteomes of different isolates of this pathogen. Appl Environ Microbiol, 2003 Jun, 69(6), 3137 - 43 Identification of Listeria monocytogenes genes involved in salt and alkaline-pH tolerance; Gardan R et al.; The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation . We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance . We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses . The position of the insertion of the transposon was located in 15 of these mutants . In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes . Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters. J Clin Periodontol, 2003, 30 Suppl 5, 13 - 6 Evidence-based control of plaque and gingivitis; Santos A; Most adults brush and floss inadequately, and constant education and/or reinforcement is often required . Bacteria are usually left behind with mechanical oral health routines, and chemotherapeutic agents may have a key role as adjuncts to daily home-care . To date, two antiseptic mouthwashes have received the ADA Seal of Acceptance: Peridex (Zila Pharmaceuticals, Phoenix, AZ, USA; CHX, chlorhexidine) and Listerine (Pfizer Consumer Healthcare, Morris Plains, NJ, USA; essential oil (EO) mouthwash) . CHX has a strong affinity for tooth and tissue surfaces, but can cause brown staining on the teeth and tongue . Patients must also wait until all traces of toothpaste are removed before rinsing with CHX . Long-term use of an EO mouthwash is microbiologically safe, with no changes observed in the bacterial composition of supragingival plaque, and no evidence of antimicrobial resistance . A number of trials have demonstrated the long-term plaque- and gingivitis-reducing properties of both CHX and EO mouthwashes . These studies clearly demonstrate that these agents have lasting efficacy, and can access hard-to-reach areas. Int J Immunopathol Pharmacol, 1999 Sep, 12(3), 149 - 155 The anti-invasive effect of bovine lactoferrin requires an interaction with surface proteins of Listeria Monocytogenes; Conte MP et al.; The anti-invasive effect of bovine lactoferrin (BLf) and of bovine transferrin (BTf) towards L . monocytogenes, an intracellular facultative food-borne pathogen, was assayed in the enterocyte-like cell line Caco-2 . When 0.5 mg/ml BLf were added during the infection time or preincubated with bacteria the number of internalized bacteria was noticeably decreased whereas BLf was ineffective when preincubated with the enterocytes or added post infection . BTf was deprived of any effect . Results from direct binding and Western blotting assays provided evidence that two L . monocytogenes surface proteins, of approximately 80 and 60 kDa, specifically reacted with BLf . These findings strongly support the hypothesis that the antiinvasive mechanism of BLf is due to its interaction with bacterial surfaces, but not to its binding with eukaryotic cells. Int J Parasitol, 2003 May, 33(5-6), 495 - 505 Haemolysin A and listeriolysin--two vaccine delivery tools for the induction of cell-mediated immunity; Dietrich G et al.; Haemolysin A of Escherichia coli and listeriolysin of Listeria monocytogenes represent important bacterial virulence factors . While such cytolysins are usually the reason for morbidity and even mortality, vaccine researchers have turned haemolysin A and listeriolysin into tools for vaccine delivery . Both cytolysins have found widespread application in vaccine research and are highly suitable for the elicitation of cell-mediated immunity . In this paper, we will review vaccine delivery mediated by the haemolysin A secretion system and listeriolysin and will highlight their use in vaccination approaches against protozoan parasites. Int J Food Microbiol, 2003 Jul 15, 84(1), 79 - 85 Rapid enumeration of Listeria monocytogenes in milk using competitive PCR; Choi WS et al.; Competitive polymerase chain reaction (cPCR) was used to develop a direct enumeration method of Listeria monocytogenes in milk . Sterile milk was artificially inoculated with L . monocytogenes and DNA was extracted using guanidine thiocyanate/phenol/chloroform, followed by PCR . Several primers for L . monocytogenes hlyA gene were tested for specific detection and DG69/DG74 primer set was selected . The primer set produced a 636-bp band from L . monocytogenes, but no band appeared from the other six Listeria spp . tested . A detection limit was as few as 10(3) colony-forming unit (cfu) per 0.5 ml of milk with this primer set . When the samples were cultured at 25 degrees C for 15 h in a TSBY medium, even a single bacterium could be detected with this primer set by PCR . For the cPCR, hlyA gene segment was cloned in pGem-4Z vector and was modified to produce competitor DNA . The competitor DNA has the same primer binding sites and sequences as the target DNA except EcoRI site . Known amount of competitor DNA was coamplified with L . monocytogenes total DNA isolated from artificially inoculated milk . The target DNA and competitor DNA were distinguished by EcoRI digestion after cPCR . The cell number determined by cPCR was approximately equal to the colony-forming unit from conventional plate counting method . For the whole procedure, it took only 5 h. Int J Food Microbiol, 2003 Jul 25, 84(2), 237 - 44 Effects of a bacteriocin-like inhibitory substance from Carnobacterium piscicola against human and salmon isolates of Listeria monocytogenes; Schobitz R et al.; The aim of this study was to characterize the antagonism of a bacteriocin-like inhibitory substance (BLIS) produced by Carnobacterium piscicola L103 against Listeria monocytogenes strains isolated from salmon and human samples . The inhibitory effect of the BLIS was evaluated in Tryptic soy agar (TSA) during different growth phases of L . monocytogenes at 5 degrees C, using the well diffusion method . Also, the type of inhibition, either bacteriostatic or bactericidal of the BLIS in Tryptic soy broth (TSB), was studied and the development of resistant cells investigated.Results showed an antagonistic effect of the BLIS on all the strains of L . monocytogenes . Four selected strains presented a higher sensitivity to the BLIS in the exponential growth phase and were more resistant in the stationary phase . In TSB, the inhibitory substance showed a partially bactericidal effect on L . monocytogenes . After inactivation of the BLIS with a protease, however, a regrowth of L . monocytogenes was found . The isolate most affected by the action of the BLIS was one of salmon origin . From the 86 isolated colonies that grew in the presence of the BLIS, 93% showed total resistance and 7% partial resistance, which was maintained through five consecutive culture cycles in the absence of the BLIS. Int J Food Microbiol, 2003 Jul 25, 84(2), 207 - 16 Stress response of Listeria monocytogenes isolated from cheese and other foods; Faleiro ML et al.; The responses to pH and sodium chloride of four strains of Listeria monocytogenes isolated from Portuguese cheese, with a sodium chloride concentration of about 2% (w/v) and a pH value from 5.1 to 6.2, were studied . Two isolates from meat and two clinical isolates related to food-borne listeriosis, in which the implicated food product had about 2-3.5% (w/v) sodium chloride, also were studied . The effect of temperature on pH and sodium chloride sensitivity was also determined . The results show that natural isolates vary in response to these stresses and the data were often at variance with previously published data . Strains varied in sensitivity to low pH and to high sodium chloride concentration but the cheese isolates tended to be more resistant . A lower temperature was associated with a decrease in resistance to low pH and to sodium chloride . All strains showed an acid tolerance response induction when grown at pH 5.5 and although the time required for maximum induction of the response varied between strains, 2 h of acid adaptation, at least, was necessary which is longer than previously reported . Some strains showed an osmotolerance response after incubation in 3.5% (w/v) sodium chloride . Osmoadaptation, in addition to inducing an osmotolerance response, also induced cross-protection against acid shock conditions (pH 3.5) . The acid tolerance response also induced a cross-protection against osmotic shock conditions (20% (w/v) sodium chloride) . In some cases there was a relationship between the degree of resistance and adaptation, but usually the behaviour of a particular strain was independent of the conditions from which it was isolated. Int J Food Microbiol, 2003 Jul 25, 84(2), 133 - 43 The effect of growth atmosphere on the ability of Listeria monocytogenes to survive exposure to acid, proteolytic enzymes and bile salts; King T et al.; Four isolates of Listeria monocytogenes from food, human and environmental sources were grown separately in broth (pH 6.0 at 8 degrees C) under atmospheres of air, 100% N(2), 40% CO(2):60% N(2) or 100% CO(2) . Exponential and stationary phase cells were harvested to determine if growth atmosphere and growth phase influenced this pathogen's ability to survive exposure to an acid environment coupled with proteolytic enzymes, and the activity of bile salts . In general, isolates were more resistant to the acid environment than the bile salts environment and stationary phase cells were significantly more resistant to both environments than exponential phase cells . Irrespective of prior growth atmosphere, none of the isolates when in exponential phase remained detectable following full exposure to the acid environment (110 min at 37 degrees C) or the bile environment (3 h at 37 degrees C) . With the exception of one isolate grown under the atmosphere of 40% CO(2):60% N(2), all isolates when in stationary phase were detectable following full exposure to the acid environment but death rates varied significantly . Stationary phase cells of all isolates grown under 40% CO(2):60% N(2) and 100% CO(2) were highly susceptible to the bile salts environment: cells were not detectable after a 2-min exposure whereas stationary phase cells grown under air or 100% N(2) were recovered following full exposure to the bile environment . Survival curves were characterised by a population decline of at least 3 log(10)/ml (from an initial level of 7 log(10) CFU/ml) in the first 15 min; thereafter a constant population number of approximately 4 log(10)/ml was maintained over the remaining exposure period . No survival was observed when stationary phase cells of L . monocytogenes FRRB 2538 grown in air and 100% N(2) were subjected to the acid environment followed by immediate exposure to the bile salts environment . The results showed that growth atmosphere and growth phase could influence survival of this pathogen against conditions that imitate the extremes of the most important nonspecific defence mechanisms against microbial infection: the acid environment of the stomach coupled with the activity of proteolytic enzymes, and the activity of bile salts in the small intestine. Int Immunopharmacol, 2003 Jun, 3(6), 889 - 900 Protective effects of Chlorella vulgaris in lead-exposed mice infected with Listeria monocytogenes; Queiroz ML et al.; Chlorella vulgaris extract (CVE) was examined for its chelating effects on the myelosuppression induced by lead in Listeria monocytogenes-infected mice . The reduction in the number of bone marrow granulocyte-macrophage progenitors (CFU-GM) observed after the infection was more severe in the groups previously exposed to lead . Extramedullar hematopoiesis, which was drastically increased after the infection, was not altered by the presence of lead . Treatment with CVE, given simultaneously or following lead exposure, restored to control values the myelosuppression observed in infected/lead-exposed mice and produced a significant increase in serum colony-stimulating activity . The benefits of the CVE treatment were also evident in the recovery of thymus weight, since the reduction produced by the infection was further potentiated by lead exposure . The efficacy of CVE was evident when infected and infected/lead-exposed mice were challenged with a lethal dose of L . monocytogenes after a 10-day treatment with 50 mg/kg CVE/day, given simultaneously to the exposure to 1300 ppm lead acetate in drinking water . Survival rates of 30% for the infected group and of 20% for the infected/lead-exposed groups were observed . Evidence that these protective effects of CVE are partly due to its chelating effect was given by the changes observed in blood lead levels . We have observed in the group receiving the CVE/lead simultaneous exposure a dramatic reduction of 66.03% in blood lead levels, when compared to lead-exposed nontreated control . On the other hand, CVE treatment following lead exposure produced a much less effective chelating effect . CVE treatments for 3 or 10 days, starting 24 h following lead exposure, produced a reduction in blood lead levels of 13.5% and 17%, respectively, compared to lead-exposed nontreated controls . The significantly better response observed with the simultaneous CVE/lead administration indicates that the immunomodulation effect of CVE plays an important role in the ability of this algae to reduce blood lead levels . In this regard, additional experiments with gene knockout C57BL/6 mice lacking a functional IFN-gamma gene demonstrated that this cytokine is of paramount importance in the protection afforded by CVE . The antibacterial evaluation measured by the rate of survival demonstrated that, in face of a 100% survival in the control group composed of normal C57BL/6 mice, which are resistant to L . monocytogenes, we observed no protection whatsoever in the IFN-gamma knockout C57BL/6 mice treated with CVE and inoculated with L . monocytogenes. Emerg Infect Dis, 2003 Jun, 9(6), 672 - 80 Molecular subtyping to detect human listeriosis clusters; Sauders BD et al.; We analyzed the diversity (Simpson's Index, D) and distribution of Listeria monocytogenes in human listeriosis cases in New York State (excluding New York City) from November 1996 to June 2000 by using automated ribotyping and pulsed-field gel electrophoresis (PFGE) . We applied a scan statistic (p<or=0.05) to detect listeriosis clusters caused by a specific Listeria monocytogenes subtype . Among 131 human isolates, 34 (D=0.923) ribotypes and 74 (D=0.975) PFGE types were found . Nine (31% of cases) clusters were identified by ribotype or PFGE; five (18% of cases) clusters were identified by using both methods . Two of the nine clusters (13% of cases) corresponded with investigated multistate listeriosis outbreaks . While most human listeriosis cases are considered sporadic, highly discriminatory molecular subtyping approaches thus indicated that 13% to 31% of cases reported in New York State may represent single-source clusters . Listeriosis control and reduction efforts should include broad-based subtyping of human isolates and consider that a large number of cases may represent outbreaks. EMBO Rep, 2003 May, 4(5), 523 - 9 A crucial role for profilin-actin in the intracellular motility of Listeria monocytogenes; Grenklo S et al.; We have examined the effect of covalently crosslinked profilin-actin (PxA), which closely matches the biochemical properties of ordinary profilin-actin and interferes with actin polymerization in vitro and in vivo, on Listeria monocytogenes motility . PxA caused a marked reduction in bacterial motility, which was accompanied by the detachment of bacterial tails . The effect of PxA was dependent on its binding to proline-rich sequences, as shown by the inability of PH133SxA, which cannot interact with such sequences, to impair Listeria motility . PxA did not alter the motility of a Listeria mutant that is unable to recruit Ena (Enabled)/VASP (vasodilator-stimulated phosphoprotein) proteins and profilin to its surface . Finally, PxA did not block the initiation of actin-tail formation, indicating that profilin-actin is only required for the elongation of actin filaments at the bacterial surface . Our findings provide further evidence that profilin-actin is important for actin-based processes, and show that it has a key function in Listeria motility. Toxicol Sci, 2003 Aug, 74(2), 325 - 34 Epub 2003 May 28. Immune changes during acute cold/restraint stress-induced inhibition of host resistance to Listeria; Cao L et al.; Experiments were conducted to delineate the cellular changes modulated by acute cold/restraint stress (ACRS), a physical and psychological stressor, in response to a Listeria monocytogenes(LM) infection . In addition to wild type (WT) BALB/c mice, CD4-deficient (CD4-/-) BALB/c mice, which have no effective adaptive immunity, were used to determine the involvement of adaptive versus innate immunity . ACRS-induced suppression of host resistance to LM was not observed in CD4-/- mice, suggesting the involvement of CD4+T cells in the acute cold/restraint stress (ACRS)-induced inhibition . The in vivo splenic leukocyte phenotypes and activities of WT BALB/c mice after infection and in vitro lymphocyte responses to heat-killed LM (HKLM) also were examined . There were no significant differences in the numbers of splenic T and B lymphocytes, natural killer cells, macrophages, or neutrophils between nonstressed and ACRS-treated WT mice . However, higher levels of activated T cells and non-T lymphocytes were observed in the ACRS-treated mice; beta-adrenergic receptor (beta-ADR) antagonists (propranolol and atenolol) eliminated these elevated levels of activation, as well as the ACRS-induced suppression of host resistance . ACRS and control mice also had equivalent activation of macrophages . With in vitro HKLM stimulation, splenocytes from ACRS-treated mice produced significantly higher levels of IFNgamma and slightly higher levels of IL-6 in comparison with the nonstressed mice, although equivalent levels of lymphocyte proliferation were obtained . Additionally, ACRS-treated mice showed comparable elevation of serum nitric oxide after infection, indicating macrophage bactericidal activity similar to nonstressed mice . Thus, it appears that ACRS inhibits host resistance through regulatory CD4+ T cells and/or effector cell functions downstream of CD4+ T cell activation, as well as through beta-ADR signaling, in that blockage of these receptors appears to aid host defenses by means other than elevation of helper T cell activity . Because CD4 T cell deficiency and beta-ADR blockage produced equivalent effects, beta-ADR+ CD4+ T cells may have a negative role on host defenses after ACRS. Int J Cancer, 2003 Jul 20, 105(6), 811 - 9 Recombinant E . coli efficiently delivers antigen and maturation signals to human dendritic cells: presentation of MART1 to CD8+ T cells; Radford KJ et al.; The generation of tumour-specific cytotoxic T-lymphocyte (CTL) responses is the primary focus in the design of immunotherapeutic cancer vaccines . We have recently demonstrated generation of ovalbumin (OVA)-specific CTLs and tumour-protection in a murine tumour model using vaccination with dendritic cells (DCs) pulsed with E . coli expressing listeriolysin O (LLO) and OVA as a model antigen . In this system paraformaldehyde fixation of E . coli/LLO provided an additional safety feature without compromising vaccine efficacy . We therefore reasoned that paraformaldehyde-fixed recombinant E . coli expressing LLO would be an efficient vehicle for the delivery of human tumour antigens to human DCs . In the present study, we demonstrate that fixed E . coli expressing LLO are taken up efficiently by human monocyte-derived DCs (MoDCs) with minimal toxicity . As a consequence of the interaction with bacteria, human DCs undergo marked phenotypic and functional maturation . Furthermore, we show that fixed E . coli/LLO expressing the well-characterised human melanoma antigen, MART1, efficiently deliver the HLA-A2-restricted MART1(27-35) epitope for processing and presentation on human MoDCs, suggesting the potential of this system as a novel strategy for human tumour immunotherapy . Apoptosis, 2003 Mar, 8(2), 179 - 90 Potential role of the EPEC translocated intimin receptor (Tir) in host apoptotic events; Malish HR et al.; Apoptosis, or programmed cell death, is a well-ordered process that allows damaged or diseased cells to be removed from an organism without severe inflammatory reactions . Multiple factors, including microbial infection, can induce programmed death and trigger reactions in both host and microbial cellular pathways . Whereas an ultimate outcome is host cell death, these apoptotic triggering mechanisms may also facilitate microbial spread and prolong infection . To gain a better understanding of the complex events of host cell response to microbial infection, we investigated the molecular role of the microorganism Enteropathogenic Escherichia coli (EPEC) in programmed cell death . We report that wild type strain of EPEC, E2348/69, induced apoptosis in cultured PtK2 and Caco-2 cells, and in contrast, infections by the intracellularly localized Listeria monocytogenes did not . Fractionation and concentration of EPEC-secreted proteins demonstrated that soluble protein factors expressed by the bacteria were capable of inducing the apoptotic events in the absence of organism attachment, suggesting adherence is not required to induce host cell death . Among the known EPEC proteins secreted via the Type III secretion (TTS) system, we identified the translocated intimin receptor (Tir) in the apoptosis-inducing protein sample . In addition, host cell ectopic expression of an EPEC GFP-Tir showed mitochondrial localization of the protein and produced apoptotic effects in transfected cells . Taken together, these results suggest a potential EPEC Tir-mediated role in the apoptotic signaling cascade of infected host cells. Clin Nutr, 2003 Jun, 22(3), 313 - 9 Anti-oxidant properties of N-acetyl-L-cysteine do not improve the immune resistance of mice fed dietary lipids to Listeria monocytogenes infection; Puertollano MA et al.; BACKGROUND & AIMS: Current knowledge of the potential effects that several dietary lipids exert on immune functions indicates that these substances participate actively in the modulation of immune system by which they contribute to the improvement of the conditions of patients suffering from inflammatory disorders . However, long-chain n-3 polyunsaturated fatty acids induce an immunosuppressive status that leads to a reduction of the host natural resistance to infectious agents as well as to an enhancement of oxidative damage . Hence, the present study has been designed to evaluate the effects on the immune system of the antioxidant N-acetyl-L-cysteine (NAC) in mice fed dietary lipids and infected with Listeria monocytogenes . METHODS: Balb/c mice were fed for 4 weeks with diets containing either olive oil (OO, 20% by weight), fish oil (FO, 20% by weight) or hydrogenated coconut oil (HCO, 20% by weight) . After dietary lipid administration mice were experimentally infected with L . monocytogenes or treated with NAC (25mg/ml intraperitoneally) . RESULTS: NAC at a concentration of 1mM promoted a loss of cell viability, although no differences were observed among the four groups . After injection of NAC in combination with L . monocytogenes, 25% of mice fed a low-fat (LF) diet survived . However, in the groups fed dietary lipids no effect on survival of mice was found . NAC participated in the reduction of superoxide anion generation measured with nitroblue tetrazolium (NBT) in the group fed a FO diet . Finally, NAC reduced the recovery of L . monocytogenes from spleen of mice fed diets containing LF or HCO . CONCLUSIONS: On the basis of these results, we can confirm that the administration of NAC improves survival in mice fed LF diet, whereas a reduction in the generation of superoxide radicals was measured in mice fed a FO diet and infected with L . monocytogenes . Similarly, bacterial recovery was diminished in mice fed diets containing LF or HCO . Hence, these data reveal a beneficial effect of NAC in mice fed LF or HCO and a detrimental action of this antioxidant in mice fed diets containing FO or OO. Vaccine, 2003 Jun 1, 21 Suppl 2, S102 - 9 Induction of immune responses by attenuated isogenic mutant strains of Listeria monocytogenes; Darji A et al.; We have generated isogenic Listeria monocytogenes mutant strains to study the induction of protective immunity in mice . These strains harbored either a specific deletion within the actin nucleator (actA) and/or have multiple deletions within the actA and phospholipase B (plcB) genes . In comparison to the wild type parental L . monocytogenes EGDe strains, the mutant strains were extremely low in virulence and were rapidly eliminated by the host during the first days of infection . Nevertheless, a single immunization with both mutant strains (EGDe DeltaactA2 and DeltaactADeltaplcB) efficiently induced and maintained effector memory (CD8(+)) T cells and has provided animals with a state of long-lasting protective immunity against wild type L . monocytogenes . These mutant strains can be used as live vaccines against the corresponding virulent pathogen and as carriers for introducing heterologous protective antigens into animals and humans. Infect Immun, 2003 Jun, 71(6), 3614 - 8 Listeriolysin O-mediated calcium influx potentiates entry of Listeria monocytogenes into the human Hep-2 epithelial cell line; Dramsi S et al.; To investigate factors which modulate the entry of Listeria monocytogenes into mammalian cells, we have analyzed the role of Ca(2+) . We show that L . monocytogenes induced Ca(2+) transients into the human Hep-2 epithelial cell line . The nonpathogenic species L . innocua or a L . monocytogenes mutant strain defective in listeriolysin O (LLO) production was unable to induce these calcium fluxes . Addition of plasma membrane calcium channel antagonists or chelation of extracellular calcium markedly reduced L . monocytogenes entry . In contrast, chelation of host cytosolic Ca(2+) or blockade of Ca(2+) release from intracellular stores did not affect invasion . These results indicate that L . monocytogenes-induced mobilization of extracellular Ca(2+) by LLO and activation of downstream Ca(2+)-dependent signaling are required for efficient cell invasion. Infect Immun, 2003 Jun, 71(6), 3473 - 84 Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and loss of actin-based motility; Pilgrim S et al.; Protein p60 encoded by the iap gene is regarded as an essential gene product of Listeria monocytogenes . Here we report, however, the successful construction of a viable iap deletion mutant of L . monocytogenes EGD . The mutant, which produces no p60, shows abnormal septum formation and tends to form short filaments and hooked forms during logarithmic growth . These abnormal bacterial cells break into almost normal sized single bacteria in the late-stationary-growth phase . The iap mutant is strongly attenuated in a mouse model after intravenous injection, demonstrating the importance of p60 during infection, and the invasiveness of the Deltaiap mutant for 3T6 fibroblasts and Caco-2 epithelial cells is slightly reduced . Upon uptake by epithelial cells and macrophages, the iap mutant escapes from the phagosome into the cytosol with the same efficiency as the wild-type strain, and the mutant bacteria also grow intracellularly at a rate similar to that of the wild-type strain . Intracellular movement and cell-to-cell spread are drastically reduced in various cell lines, since the iap-negative bacteria fail to induce the formation of actin tails . However, the bacteria are covered with actin filaments . Most intracellular bacteria show a nonpolar and uneven distribution of ActA around the cell, in contrast to that for the wild-type strain, where ActA is concentrated at the old pole . In an iap(+) revertant strain that produces wild-type levels of p60, intracellular movement, cell-to-cell spread, and polar distribution of ActA are fully restored . In vitro analysis of ActA distribution on the filaments of the Deltaiap strain shows that the loss of bacterial septum formation leads to ActA accumulation at the presumed division sites . In the light of data presented here and elswhere, we propose to rename iap (invasion-associated protein) cwhA (cell wall hydrolase A). Infect Immun, 2003 Jun, 71(6), 3429 - 36 Experimental validation of low virulence in field strains of Listeria monocytogenes; Roche SM et al.; Several reports have described Listeria monocytogenes strains which were nonpathogenic or weakly pathogenic, but little is known about these low-virulence strains . We found that 9 field L . monocytogenes strains were hypovirulent and 17 were avirulent, based on the number of mice contaminated and the colonization of their spleens after subcutaneous inoculation . All these strains possessed the known virulence genes . We have now assessed the low virulence of these strains in other assays before determining how they differ from virulent strains . We have shown that the low-virulence strains exhibited a phenotypic stability and were not a mixture of virulent and avirulent bacteria . They did not recover virulence after many passages in mice and colonized the spleens of mice more poorly than virulent strains after i.v . inoculation . Their lethal capacities, determined by 50% lethal dose (LD(50)), were lower than those of virulent strains . Like Listeria innocua, 14 of 17 avirulent strains had no LD(50) and were eliminated by the lymph nodes after subcutaneous inoculation . The virulent, hypovirulent, and avirulent strains were always significantly different, whatever the tests of virulence used, confirming the importance of these low-virulence field strains in identifying the proteins involved in virulence. Curr Infect Dis Rep, 2003 Jun, 5(3), 220 - 226 Microbiology and Treatment of Halitosis; Loesche WJ; The many thousands of individuals who experience oral malodor that stems from the overgrowth of proteolytic, anaerobic bacteria on their tongue surfaces can be successfully treated by a regimen that includes tongue brushing and tooth brushing, often in combination with a mouthrinse containing an antibacterial agent . Several candidate mouthrinses containing essential oils (Listerine; Warner-Lambert, Morris Plains, NJ), ZnCl(2), chlorine dioxide, or an oil:water-cetylpyridium chloride mouthrinse have reduced the organoleptic scores of individuals with moderate levels of oral malodor in the absence of tongue brushing . Very little long-term data beyond 6 weeks of use are available. C R Biol, 2003 Feb, 326(2), 161 - 70 Actin-based motility as a self-organized system: mechanism and reconstitution in vitro; Carlier MF et al.; Site-directed actin polymerisation in response to signalling is responsible for the formation of cell protrusions . These elementary 'actin-based motility processes' are involved in cell locomotion, cell metastasis, organ morphogenesis and microbial pathogenesis . We have reconstituted actin-based propulsive movement of particles of various sizes and geometries (rods, microspheres) in a minimum motility medium containing five pure proteins . The ATP-supported treadmilling of actin filaments, regulated by Actin Depolymerizing Factor (ADF/cofilin), profilin and capping proteins provides the thermodynamic basis for sustained actin-based movement . Local activation of Arp2/3 complex at the surface of the particle promotes autocatalytic barbed end branching of filaments, generating a polarized arborescent array . Barbed end growth of branched filaments against the surface generates a propulsive force and is eventually arrested by capping proteins . Understanding the mechanism of actin-based movement requires elucidation of the biochemical properties and mode of action of Arp2/3 complex in filament branching, in particular the role of ATP binding and hydrolysis in Arp2/3, and a physical analysis of the movement of functionalised particles . Because the functionalisation of the particle by an activator of Arp2/3 complex (N-WASP or the Listeria protein ActA) and the concentrations of effectors in the medium are controlled, the reconstituted motility assay allows an analysis of the mechanism of force production at the mesoscopic and molecular levels. Neurol Sci, 2003 Apr, 24(1), 40 - 3 Syringomyelia following Listeria meningoencephalitis: report of a case; Nardone R et al.; A case of symptomatic syringomyelia which appeared six years after Listeria meningoencephalitis is described . Chronic spinal arachnoiditis, as shown by standard MRI and dynamic phase contrast (PC) cine-MRI, may occur after spinal infection and is likely the cause of syringomyelia . To our knowledge, there are no previous reports of delayed spinal complications following Listeria monocytogenes infection . The possibility of developing syringomyelia should be always considered in any patient with a history of central nervous system infection. Surg Neurol, 2003 Apr, 59(4), 320 - 8 Multiple cerebral abscesses because of Listeria monocytogenes: three case reports and a literature review of supratentorial listerial brain abscess(es); Cone LA et al.; BACKGROUND: Central nervous system involvement often follows bacteremia because of Listeria monocytogenes . Meningitis is clinically the most common manifestation, while brain abscess occurs in about 1% of patients . Brain abscess is usually solitary but in recent years, probably in part because of the availability of computerized tomography and magnetic resonance imaging, several reports have described two or more separate supratentorial abscesses . METHODS: We have described three patients with listerial brain abscesses and reviewed the North American and European literature of brain abscess(es) because of L . monocytogenes through December 2001 . We have evaluated the role of underlying diseases and therapeutic immunosuppression on the development of solitary or greater than one brain abscess . RESULTS: In contrast to meningitis, where immunosuppression does not predispose either to disease incidence or to higher mortality, patients with solitary and particularly those with more than one supratentorial abscess usually are immunosuppressed either by disease or by therapy . Corticosteroids in particular are significant predisposing factors, especially in those patients with two or more brain abscesses . Mortality resulting from listerial brain abscess, whether solitary or multiple, is nearly three times higher than nonlisterial brain abscess, probably in part because of both underlying diseases and immunosuppressive therapy . CONCLUSIONS: Therapy with high-dose ampicillin in combination with gentamicin appear to be the drugs of choice, followed by trimethoprim/sufamethoxazole and vancomycin . In general, antimicrobial therapy appears to be satisfactory treatment without surgical intervention. J Food Prot, 2003 May, 66(5), 819 - 24 Gamma irradiation of fine-emulsion sausage containing sodium diacetate; Sommers C et al.; Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a frequent postprocess contaminant of ready-to-eat (RTE) meat products, including frankfurters and bologna . Ionizing radiation can eliminate L . monocytogenes from RTE meats . Sodium diacetate (SDA) incorporated into fine-emulsion sausages inhibits the growth of L . monocytogenes . Irradiation of L . monocytogenes suspended in SDA solutions resulted in synergistic reductions of the microorganism . L . monocytogenes populations were reduced by > 9 log10 units at a radiation dose of 1.5 kGy when suspended in 0.125% SDA solution . In contrast, the D10-values (the ionizing radiation doses required to reduce the population by 90%) were 0.58, 0.59, 0.57, and 0.53 kGy for L . monocytogenes populations suspended in emulsions containing 0, 0.125, 0.25, and 0.5% SDA, respectively . The D10-values for L . monocytogenes surface inoculated onto frankfurters dipped in 0, 0.125, 0.25, and 0.5% SDA solutions were 0.58, 0.53, 0.54, and 0.52 kGy, respectively . Postirradiation growth of L . monocytogenes suspended in beef bologna emulsion at 9 degrees C was dependent on SDA concentration and ionizing radiation dose . Very small, but statistically significant, changes in bologna redness, lipid oxidation, and shear force were observed for the beef bologna emulsion with the highest SDA concentration (0.5%) and irradiation dose (3.0 kGy) . SDA can inhibit the proliferation of L . monocytogenes surviving the irradiation process with minimal impact on fine-emulsion sausage color, lipid oxidation, and firmness when used within regulatory limits. J Food Prot, 2003 May, 66(5), 812 - 8 Reducing levels of Listeria monocytogenes contamination on raw salmon with acidified sodium chlorite; Su YC et al.; The antimicrobial activity of acidified sodium chlorite (ASC) against Listeria monocytogenes in salmon was studied . Raw salmon (whole fish and fillets) inoculated with L . monocytogenes (10(3) CFU/cm2 or 10(4) CFU/g) were washed with ASC solution (50 ppm) for 1 min and stored at -18 degrees C for 1 month (whole salmon) or in ice for 7 days (fillets) . L . monocytogenes populations were determined for whole salmon after frozen storage and for fillets on days 1, 3, 5, and 7 of storage . A wash with ASC solution followed by ASC glazing did not reduce L . monocytogenes on the skin of whole salmon during frozen storage . However, the wash resulted in an L . monocytogenes reduction of 0.5 log CFU/g for salmon fillets . The populations of L . monocytogenes in fillets increased slowly during ice storage, but the growth of these populations was retarded by ASC ice . By day 7, the populations were 0.25 log units smaller in fillets stored in ASC ice and 0.62 log units smaller in fillets that had been washed with ASC solution and stored in ASC ice than in control fillets . Treatment with ASC also reduced total plate counts (TPCs) by 0.43 log CFU/cm2 on the skin of whole salmon and by 0.31 log CFU/g in fillets . The TPCs for skin decreased during frozen storage but increased gradually for fillets stored at 5 degrees C or in ice . However, TPCs of ASC-treated samples were lower than those for controls at any point during the study . Washing with ASC solution significantly (P < 0.05) reduced TPCs on the skin of whole salmon and in fillets, as well as L . monocytogenes in fillets . The antimicrobial activity of ASC was enhanced when salmon was washed with ASC solution and stored in ASC ice. J Food Prot, 2003 May, 66(5), 804 - 11 Predictive model for the combined effect of temperature, sodium lactate, and sodium diacetate on the heat resistance of Listeria monocytogenes in beef; Juneja VK; The effects of heating temperature (60 to 73.9 degrees C), sodium lactate (NaL; 0.0 to 4.8% {wt/wt}), and/or sodium diacetate (SDA; 0.0 to 0.25% {wt/wt}) and of the interactions of these factors on the heat resistance of a five-strain mixture of Listeria monocytogenes in 75% lean ground beef were examined . Thermal death times for L . monocytogenes in filtered stomacher bags in a circulating water bath were determined . The recovery medium was tryptic soy agar supplemented with 0.6% yeast extract and 1% sodium pyruvate . Decimal reduction times (D-values) were calculated by fitting a survival model to the data with a curve-fitting program . The D-values were analyzed by second-order response surface regression for temperature, NaL level, and SDA level . The D-values observed for beef with no NaL or SDA at 60, 65, 71.1, and 73.9 degrees C were 4.67, 0.72, 0.17, and 0.04 min, respectively . The addition of 4.8% NaL to beef increased heat resistance at all temperatures, with D-values ranging from 14.3 min at 60 degrees C to 0.13 min at 73.9 degrees C . Sodium diacetate interacted with NaL, thereby reducing the protective effect of NaL and rendering L . monocytogenes in beef less resistant to heat . A mathematical model describing the combined effect of temperature, NaL level, and SDA level on the thermal inactivation of L . monocytogenes was developed . This model can predict D-values for any combination of temperature, NaL level, and SDA level that is within the range of those tested . This predictive model will have substantial practical importance to processors of cooked meat, allowing them to vary their thermal treatments of ready-to-eat meat products in a safe manner. Int J Food Microbiol, 2003 Jun 25, 83(3), 325 - 30 Susceptibility of Listeria monocytogenes isolated from food in Italy to antibiotics; Aureli P et al.; The susceptibility of 148 strains of Listeria monocytogenes isolated from food to antibiotics currently used in veterinary and human therapy was determined by standard agar dilution and disk diffusion methods . The antibiotics included amikacin, amoxicillin, cefazolin, chloramphenicol, erythromycin, flumequine, fosfomycin, gentamicin, kanamycin, lincomycin, oxytetracycline, rifampicin, spiramycin, streptomycin, tetracycline, tobramycin and vancomycin . Soussy's breakpoints and MIC(50)-MIC(90) values were used to classify the strains into sensitive, moderately sensitive and resistant groups.This work is part of a wider surveillance program on listeriosis started in Italy in 1995. Proc Natl Acad Sci U S A, 2003 May 27, 100(11), 6493 - 8 Epub 2003 May 08. Compression forces generated by actin comet tails on lipid vesicles; Giardini PA et al.; Polymerizing networks of actin filaments generate force for a variety of movements in living cells, including protrusion of filopodia and lamellipodia, intra- and intercellular motility of certain bacterial and viral pathogens, and motility of endocytic vesicles and other membrane-bound organelles . During actin-based motility, coexisting populations of actin filaments exert both pushing and retarding forces on the moving cargo . To examine the distribution and magnitude of forces generated by actin, we have developed a model system where large artificial lipid vesicles coated with the protein ActA from the bacterial pathogen Listeria monocytogenes are propelled by actin polymerization in cytoplasmic extract . We find that motile vesicles associated with actin comet tails are significantly deformed due to an inward compression force exerted by actin polymerization orthogonal to the direction of motion, which is >10-fold greater in magnitude than the component of the force exerted in the direction of motion . Furthermore, there is a spatial segregation of the pushing and retarding forces, such that pushing predominates along the sides of the vesicle, although retarding forces predominate at the rear . We estimate that the total net (pushing minus retarding) force generated by the actin comet tail is approximately 0.4-4 nN . In addition, actin comet tail formation is associated with polarization of the ActA protein on the fluid vesicle surface, which may reinforce the persistence of unidirectional motion by helping to maintain a persistent asymmetry of actin filament density. Med Microbiol Immunol (Berl), 2003 May, 192(2), 85 - 91 Epub 2002 Oct 19. A Listeria adhesion protein-deficient Listeria monocytogenes strain shows reduced adhesion primarily to intestinal cell lines; Jaradat ZW et al.; Listeria monocytogenes adheres and penetrates intestinal cell linings for systemic infection . A 104-kDa Listeria adhesion protein (LAP) from L . monocytogenes was previously demonstrated to be responsible for adhesion to intestinal enterocyte-like Caco-2 cells . We investigated the adhesion and invasion characteristics of a LAP-deficient mutant L . monocytogenes strain (A572) to various human intestinal and non-intestinal cell lines to assess the possible target host cells . Among the intestinal cell lines, A572 showed significantly reduced adhesion than the wild type (WT) strain to the cells of ileum-cecum (HCT-8) and colon (Caco-2 and HT-29), whereas A572 and WT did not show any significant differences in adhesion to other intestinal cell lines from duodenum (HuTu-80) or jejunum (Int-407) . Differences in adhesion between A572 and WT were little or none in non-intestinal cell lines from liver, kidney, bladder, ovary, cervix, breast, larynx, or skin . Invasion data showed that A572 was invasive but the invasion efficiency was proportional to its adhesion characteristics to respective cell lines . In mouse bioassay, A572 was not found in liver following oral administration, suggesting that LAP mutant was possibly unable to pass through intestinal cell linings . Immuno-electron microscopy revealed that the LAP is localized in the bacterial surface as well as the cytoplasm . In summary, this study indicated that the LAP-mediated adhesion is associated with the intestinal cells originating from the lower part of small intestine and from the upper part of large intestine, and possibly plays an important role during the intestinal phase of infection. J Immunol, 2003 May 15, 170(10), 5228 - 34 Neutrophilia in LFA-1-deficient mice confers resistance to listeriosis: possible contribution of granulocyte-colony-stimulating factor and IL-17; Miyamoto M et al.; LFA-1 (CD11a/CD18) plays a crucial role in various inflammatory responses . In this study, we show that LFA-1(-/-) mice are far more resistant to Listeria monocytogenes infection than LFA-1(+/-) mice . Consistent with this, we found the following: 1) the numbers of granulocytes infiltrating the liver were markedly higher in LFA-1(-/-) mice than in LFA-1(+/-) mice, 2) increased antilisterial resistance in LFA-1(-/-) mice was abrogated by depletion of granulocytes, and 3) the numbers of granulocytes in peripheral blood, and the serum levels of both G-CSF and IL-17 were higher in LFA-1(-/-) mice than in LFA-1(+/-) mice . Neither spontaneous apoptosis nor survival of granulocytes from LFA-1(-/-) mice were affected by physiological concentrations of G-CSF . Our data suggest regulatory effects of LFA-1 on G-CSF and IL-17 secretion, and as a corollary on neutrophilia . Consequently, we conclude that increased resistance of LFA-1(-/-) mice to listeriosis is due to neutrophilia facilitating liver infiltration by granulocytes promptly after L . monocytogenes infection, although it is LFA-1 independent. J Immunol, 2003 May 15, 170(10), 5210 - 8 The lymphotoxin beta receptor is critically involved in controlling infections with the intracellular pathogens Mycobacterium tuberculosis and Listeria monocytogenes; Ehlers S et al.; Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-gamma and TNF . To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT beta R pathway . When mice deficient in LT alpha or LT beta were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT alpha beta heterotrimers in resistance to infection . Indeed, mice deficient in the receptor for LT alpha(1)beta(2) heterotrimers (LT beta R-knockout (KO) mice) also had significantly higher numbers of M . tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection . Furthermore, LT beta R-KO mice were dramatically more susceptible than wild-type mice to i.p . infection with Listeria monocytogenes . Compared with wild-type mice, LT beta R-KO mice had similar transcript levels of TNF and IFN-gamma and recruited similar numbers of CD3(+) T cells inside granulomatous lesions in M . tuberculosis-infected lungs . Flow cytometry revealed that the LT beta R is expressed on pulmonary macrophages obtained after digestion of M . tuberculosis-infected lungs . LT beta R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections . Since LIGHT-KO mice proved to be equally resistant to M . tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT alpha(1)beta(2) heterotrimers via the LT beta R is an essential prerequisite for containment of intracellular pathogens. J Immunol, 2003 May 15, 170(10), 5176 - 87 The induction of HIV Gag-specific CD8+ T cells in the spleen and gut-associated lymphoid tissue by parenteral or mucosal immunization with recombinant Listeria monocytogenes HIV Gag; Peters C et al.; The induction of mucosal immunity is crucial in controlling viral replication during HIV infection . In this study we compare the ability of a recombinant Listeria monocytogenes that expresses and secretes the HIV Ag Gag to induce CD8(+) T cells against this Ag in the spleen, mesenteric lymph nodes, and Peyer's patches and the ability to provide effector Gag-specific CD8(+) T cells to the lamina propria after i.v., oral, or rectal administration of the vaccine . The levels of Ag-specific CD8(+)-activated T cells were measured ex vivo using intracellular cytokine staining for IFN-gamma and H-2K(d) Gag peptide tetramer staining . We found that all routes of immunization induced Gag-specific CD8(+) T cells in the spleen . After secondary infection, we observed substantial increases in splenic levels of CD8(+) T cells, and levels of Gag-specific cells were similar to those against listeriolysin O, the immunodominant Ag of L . monocytogenes . Both primary and secondary oral immunization resulted in abundant Gag-specific CD8(+)-activated T cells in the lamina propria that constituted approximately 35% of the CD8 compartment . However, significant levels of Gag and listeriolysin O-specific CD8(+) T cells were observed in mucosal lymphoid tissue only after two immunizations, perhaps because they had already entered the lamina propria compartment after a single immunization . In the context of HIV, a mucosally administered vaccine seems best calculated to prompt an immune response that is capable of preventing infection . The data presented in this report demonstrate that mucosally administered Listeria can prompt such a response and that booster doses can maintain this response. J Immunol, 2003 May 15, 170(10), 4933 - 42 Regulation of CD8+ T cells undergoing primary and secondary responses to infection in the same host; Badovinac VP et al.; Naive Ag-specific CD8(+) T cells expand, contract, and become memory cells after infection and/or vaccination . Memory CD8(+) T cells provide faster, more effective secondary responses against repeated exposure to the same pathogen . Using an adoptive transfer system with low numbers of trackable nontransgenic memory CD8(+) T cells, we showed that secondary responses can be comprised of both primary (naive) and secondary (memory) CD8(+) T cells after bacterial (Listeria monocytogenes) and/or viral (lymphocytic choriomeningitis virus) infections . The level of memory CD8(+) T cells present at the time of infection inversely correlated with the magnitude of primary CD8(+) T cell responses against the same epitope but directly correlated with the level of protection against infection . However, similar numbers of Ag-specific CD8(+) T cells were found 8 days postinfection no matter how many memory cells were present at the time of infection . Rapid contraction of primary CD8(+) T cell responses was not influenced by the presence of memory CD8(+) T cells . However, contraction of secondary CD8(+) T cell responses was markedly prolonged compared with primary responses in the same host mice . This situation occurred in response to lymphocytic choriomeningitis virus or L . monocytogenes infection and for CD8(+) T cell responses against multiple epitopes . The delayed contraction of secondary CD8(+) T cells was also observed after immunization with peptide-coated dendritic cells . Together, the results show that the level of memory CD8(+) T cells influences protective immunity and activation of naive precursors specific for the same epitope but has little impact on the magnitude or program of the CD8(+) T cell response. Curr Microbiol, 2003 Jun, 46(6), 461 - 6 The role of the sigB gene in the general stress response of Listeria monocytogenes varies between a strain of serotype 1/2a and a strain of serotype 4c; Moorhead SM et al.; The ability of Listeria monocytogenes to resist many adverse environmental conditions has been attributed in part to activation of the alternative sigma factor sigma(B), encoded by the sigB gene . The ability of this pathogen to survive and grow under stress conditions varies between strains within the species . The current study was undertaken to determine whether the role played by the sigB gene in the stress response varies among strains of different serotypes . Null mutations were generated in the sigB genes of L . monocytogenes L61 (serotype 1/2a) and L99 (serotype 4c), and the survival of the resulting mutants was compared with that of the wild-type strains under osmotic, oxidative, and carbon starvation stress conditions and on exposure to bacteriocins, ethanol, acid, and heat . Except in a few cases, strain L61 displayed greater dependence on the sigB products for survival of adverse conditions than did strain L99 . The results of this study indicated that the relative importance of the s