Appl Environ Microbiol, 2004 Apr, 70(4), 2289 - 95 Pulsed electric field alters molecular chaperone expression and sensitizes Listeria monocytogenes to heat; Lado BH et al.; Pulsed electric field (PEF)-resistant and PEF-sensitive Listeria monocytogenes strains were sublethally treated with electric pulses at 15 kV/cm for 29 micro s and held at 25 degrees C for 5 to 30 min prior to protein extraction . The levels of the molecular chaperones GroEL, GroES, and DnaJ were determined by immunoblotting . After 10 to 20 min after sublethal PEF treatment, a transient decrease in molecular chaperone expression was observed in the PEF-sensitive strain (Scott A) . The levels of GroEL and DnaJ increased back to the basal expression level within 30 min . A substantial decrease in GroES expression persisted for at least 30 min after PEF treatment . Chaperone expression was suppressed after PEF treatment to a smaller extent in the PEF-resistant (OSY-8578) than in the PEF-sensitive strain, and no clear expression pattern was identified in OSY-8578 . Inactivation of Scott A and OSY-8578 in phosphate buffer was compared when lethal PEF (27.5 kV/cm, 144 micro s) and heat (55 degrees C, 10 min) were applied in sequence . When PEF and heat treatments were applied separately, the populations of L . monocytogenes Scott A and OSY-8578 decreased 0.5 to 0.6 log CFU/ml . Cells treated first with PEF and incubated at 25 degrees C for 10 min showed substantial sensitivity to subsequent heat treatment; the decrease in counts for Scott A and OSY-8578 was 6.1 and 2.8 log CFU/ml, respectively . The sequence and time lapse between the two treatments were crucial for achieving high inactivation rates . It is concluded that PEF sensitized L . monocytogenes to heat and that maximum heat sensitization occurred when chaperone expression was at a minimum level. Appl Environ Microbiol, 2004 Apr, 70(4), 2193 - 203 Multilocus sequence typing of Listeria monocytogenes by use of hypervariable genes reveals clonal and recombination histories of three lineages; Meinersmann RJ et al.; In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L . monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L . monocytogenes ATCC 19115 (serotype 4b) . Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80% . Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L . monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant) . Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested . Population genetics analyses revealed three lineages of L . monocytogenes that differed in their history of apparent recombination . Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination . Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I . Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes . Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage . In the L . monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent . While it appears that different lineages of L . monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer. Appl Environ Microbiol, 2004 Apr, 70(4), 2180 - 5 Use of PCR-restriction fragment length polymorphism of inlA for rapid screening of Listeria monocytogenes strains deficient in the ability to invade Caco-2 cells; Rousseaux S et al.; A PCR-restriction fragment length polymorphism (RFLP) method was developed in order to screen a large number of strains for impaired adhesion to epithelial cells due to expression of truncated InlA . inlA polymorphism was analyzed by PCR-RFLP in order to correlate inlA PCR-RFLP profiles and production of truncated InlA . Thirty-seven Listeria monocytogenes strains isolated from various sources, including five noninvasive and two invasive reference strains, were screened . Two endonucleases (AluI and Tsp509I) were used, and they generated five composite profiles . Thirteen L . monocytogenes isolates were characterized by two specific PCR-RFLP profiles similar to PCR-RFLP profiles of noninvasive reference strains previously described as strains that produce truncated InlA . Ten of the 13 isolates showed low abilities to invade human epithelial Caco-2 cells . However, 4 of the 13 isolates were able to invade Caco-2 cells like reference strains containing complete InlA . Sequencing of inlA and Western blot analysis confirmed that truncated InlA was expressed in the 10 L . monocytogenes strains which were isolated from food . This PCR-RFLP method allowed us to identify 10 new strains expressing a truncated internalin . Based on the results obtained in this study, the PCR-RFLP method seems to be an interesting method for rapidly screening L . monocytogenes strains deficient in the ability to invade Caco-2 cells when a sizeable number of strains are studied. Appl Environ Microbiol, 2004 Apr, 70(4), 1935 - 43 The htrA (degP) gene of Listeria monocytogenes 10403S is essential for optimal growth under stress conditions; Wonderling LD et al.; This report describes a mutant of Listeria monocytogenes strain 10403S (serotype 1/2a) with a defective response to conditions of high osmolarity, an environment that L . monocytogenes encounters in some ready-to-eat foods . A library of L . monocytogenes clones mutagenized with Tn917 was generated and scored for sensitivity to 4% NaCl in order to identify genes responsible for growth or survival in elevated-NaCl environments . One of the L . monocytogenes Tn917 mutants, designated strain OSM1, was selected, and the gene interrupted by the transposon was sequenced . A BLAST search with the putative translated amino acid sequence indicated that the interrupted gene product was a homolog of htrA (degP), a gene coding for a serine protease identified as a stress response protein in several gram-positive and gram-negative bacteria . An htrA deletion strain, strain LDW1, was constructed, and the salt-sensitive phenotype of this strain was complemented by introduction of a plasmid carrying the wild-type htrA gene, demonstrating that htrA is necessary for optimal growth under conditions of osmotic stress . Additionally, strain LDW1 was tested for its response to temperature and H(2)O(2) stresses . The results of these growth assays indicated that strain LDW1 grew at a lower rate than the wild-type strain at 44 degrees C but at a rate similar to that of the wild-type strain when incubated at 4 degrees C . In addition, strain LDW1 was significantly more sensitive to a 52 degrees C heat shock than the wild-type strain . Strain LDW1 was also defective in its response to H(2)O(2) challenge at 37 degrees C, since 100 or 150 micro g of H(2)O(2) was more inhibitory for the growth of strain LDW1 than for that of the parent strain . The stress response phenotype observed for strain LDW1 is similar to that observed for other HtrA(-) organisms, which suggests that L . monocytogenes HtrA may play a role in degrading misfolded proteins that accumulate under stress conditions. Mol Microbiol, 2004 Apr, 52(2), 601 - 11 Negative control of Listeria monocytogenes virulence genes by a diffusible autorepressor; Ermolaeva S et al.; Virulence genes from the facultative intracellular pathogen Listeria monocytogenes are controlled by the transcriptional regulator PrfA . Although PrfA synthesis is activated at 37 degrees C, PrfA-dependent expression remains low in rich medium . However, a strong induction of the PrfA regulon is observed when L . monocytogenes is cultured in the presence of activated charcoal . Here, we show that the 'charcoal effect' results from the adsorption of a diffusible autorepressor substance released by L . monocytogenes during exponential growth . Analyses using an L . monocytogenes strain in which the prfA gene is expressed constitutively at 37 degrees C from a plasmid indicate that the autoregulatory substance represses PrfA-dependent expression by inhibiting PrfA activity . PrfA presumably functions via an allosteric activation mechanism . The inhibitory effect is bypassed by a PrfA* mutation that locks PrfA in fully active conformation, suggesting that the autorepressor interferes with the allosteric shift of PrfA . Our data indicate that the listerial autorepressor is a low-molecular-weight hydrophobic substance . We suggest that this diffusible substance mediates a quorum-sensing mechanism by which L . monocytogenes restricts the expression of its PrfA virulence regulon . This autoregulatory pathway could serve L . monocytogenes to ensure the silencing of virulence genes during extracellular growth at 37 degrees C . It may also play a role during intracellular infection, by limiting the damage to the host cell caused by an excess production of cytotoxic PrfA-dependent virulence factors in the PrfA-activating cytosolic compartment. J Biotechnol, 2004 Apr 8, 109(1-2), 13 - 20 Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications; Giammarini C et al.; The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol . This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria . A protocol for large-scale expression and purification of recombinant LLO was previously optimized . By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l(-1) of cell culture) in a hemolytically active form (1.2 x 10(6) HU mg(-1) of protein) . This procedure can solve the problem of LLO isolation from L . monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media . Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis. FEMS Microbiol Lett, 2004 Apr 15, 233(2), 257 - 67 Simultaneous quantitative detection of Listeria spp . and Listeria monocytogenes using a duplex real-time PCR-based assay; Rodriguez-Lazaro D et al.; We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp . and the food-borne pathogen Listeria monocytogenes . The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp . and L . monocytogenes, respectively . Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L . monocytogenes and 120 Listeria spp . strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions . Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases . Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L . monocytogenes. J Microbiol Methods, 2004 May, 57(2), 251 - 8 Atypical colonial morphology and low recoveries of Listeria monocytogenes strains on Oxford, PALCAM, Rapid'L.mono and ALOA solid media; Leclercq A; The performance of four commercial media, polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM), Oxford, Rapid'L.mono (Bio-Rad, Marne la Coquette, France) and Agar Listeria according to Ottaviani and Agosti (ALOA: AES Laboratoire, Combourg, France; Biolife, Milan, Italy), used to detect and enumerate 176 Belgian strains of Listeria monocytogenes of human and food origin, was evaluated . Four strains showed a low recovery and/or atypical colonies on one or more media . These results showed that a combination of these media, especially alternative media (Rapid'L.mono and/or ALOA) with esculin-containing media (PALCAM and/or Oxford), should therefore be recommended to detect or enumerate atypical strains of L . monocytogenes . In outbreak case investigation for example, incubation of plates should be extended to at least 96 h if no colonies are typical or growth does not appear after 48 h . This is a cost/benefit calculation that should be done in the context of recent listeriosis risk assessments. C R Biol, 2004 Feb, 327(2), 115 - 23 Exploitation of host cell cytoskeleton and signalling during Listeria monocytogenes entry into mammalian cells; Pizarro-Cerda J et al.; Deciphering how Listeria monocytogenes exploits the host cell machinery to invade mammalian cells during infection is a key issue for the understanding how this food-borne pathogen causes a pleiotropic disease ranging from gastro-enteritis to meningitis and abortions . Using multidisciplinary approaches, essentially combining bacterial genetics and cell biology, we have identified two bacterial proteins critical for entry into target cells, InlA and InlB . Their cellular ligands have been also identified: InlA interacts with the adhesion molecule E-cadherin, while InlB interacts with the receptor for the globular head of the complement factor C1q (gC1q-R), with the hepatocyte growth factor receptor (c-Met) and with glycosaminoglycans (including heparan sulphate) . The dynamic interaction between these cellular receptors and the actin cytoskeleton is currently under investigation . Several intracellular molecules have been recognized as key effectors for Listeria entry into target cells, including catenins (implicated in the connection of E-cadherin to actin) and the actin depolymerising factor/cofilin (involved in the rearrangement of the cytoskeleton in the InlB-dependent internalisation pathway) . At the organism level, species specificity has been discovered concerning the interaction between InlA and E-cadherin, leading to the generation of transgenic mice expressing the human E-cadherin, in which the critical role of InlA in the crossing of the intestinal barrier has been clearly determined . Listeria appears as an instrumental model for addressing critical questions concerning both the complex process of bacterial pathogenesis and also fundamental molecular processes, such as phagocytosis. Curr Microbiol, 2004 May, 48(5), 373 - 8 Lack of apoptosis in Listeria monocytogenes-infected thymocytes from mice fed with dietary lipids; Puertollano MA et al.; The potential action of certain fatty acids has been studied since the early 1970s . Numerous effects on immune system functions have been related to dietary lipid administration; therefore, several of them have been applied in the treatment of inflammatory disorders . Nevertheless, n-3 polyunsaturated fatty acids may affect host resistance to infectious diseases . In addition, several studies have demonstrated that certain fatty acids are involved in apoptosis induction . Here, we have examined the action of different dietary lipids on the promotion of apoptosis in thymocytes from mice fed with dietary lipids and infected with Listeria monocytogenes . Thus, L . monocytogenes promoted an important cytotoxic effect in all of the groups, but it did not increase the percentage of DNA fragmentation . Similarly, an important increase of caspase-3 activity was demonstrated in OO and FO groups, but infection with L . monocytogenes exerted an inhibitory effect . Finally, L . monocytogenes did not modify proteasome activity among groups fed with dietary lipids . On the basis of this preliminary study, we can state that the infection of thymocytes from mice fed with dietary lipids does not promote a synergistic effect in the induction of apoptosis . Hence, these results may partially serve to elucidate the immune mechanisms involved in cells from mice fed with dietary lipids in an infectious process. Pediatr Emerg Care, 2004 Apr, 20(4), 233 - 7 Fulminant Listeria monocytogenes meningitis complicated with acute hydrocephalus in healthy children beyond the newborn period; Ulloa-Gutierrez R et al.; We describe 3 previously healthy Costa Rican children who had Listeria monocytogenes meningitis, an uncommon cause of bacterial meningitis beyond the newborn period in normal subjects . Two of them had initial normal brain computed tomography, but all 3 developed acute hydrocephalus at days 7, 3, and 5, respectively . All required immediate ventriculostomy placement and only 1 of 3 survived . L . monocytogenes should be considered among the etiologies of bacterial meningitis in children who do not respond initially to conventional antimicrobial treatment or who deteriorate rapidly. J Antimicrob Chemother, 2004 May, 53(5), 863 - 6 Epub 2004 Mar 31. Effect of listeriolysin O-loaded erythrocytes on Mycobacterium avium replication within macrophages; Rossi L et al.; OBJECTIVE: To evaluate the efficacy of erythrocytes loaded with the haemolytic toxin listeriolysin O against Mycobacterium avium replication within human macrophages . METHODS: Recombinant listeriolysin O was loaded in human erythrocytes by a procedure of hypotonic dialysis and isotonic resealing . Loaded erythrocytes were modified to allow them to be recognized and taken up by human macrophages infected with M . avium . The antimycobacterial activity of the erythrocytes loaded with listeriolysin O was evaluated by supernatant and intracellular cfu counts on days 4 and 7 post-erythrocyte administration . RESULTS: Recombinant listeriolysin O was encapsulated in human erythrocytes to reach final concentrations ranging from 1 to 4 ng/mL of erythrocytes . Erythrocytes loaded with increasing quantities of recombinant protein were able to reduce (at most by 50%) M . avium replication in a dose-dependent fashion when administered to infected macrophages . CONCLUSIONS: Erythrocytes loaded with listeriolysin O are effective against M . avium replication within macrophages . We are confident that the strategy presented could be useful against mycobacteria other than M . avium (such as Mycobacterium tuberculosis and Mycobacterium leprae) by itself or as part of an antimycobacterial treatment. Roum Arch Microbiol Immunol, 2002 Oct-Dec, 61(4), 275 - 83 Listeria monocytogenes--host interaction; Zlei M et al.; Both innate and adaptive immunity against Listeria monocytogenes are essential in circumventing or fighting the disease, although there is no clear delineation of the precise role of either in listeriosis . A consistent amount of in vivo experiments have generated a complex picture over the impact of listerial infection on the hosts, and they are shortly reviewed in this paper. Mol Microbiol, 2004 Apr, 52(1), 257 - 71 GW domains of the Listeria monocytogenes invasion protein InlB are required for potentiation of Met activation; Banerjee M et al.; The Listeria monocytogenes protein InlB promotes intracellular invasion by activating the receptor tyrosine kinase Met . Earlier studies have indicated that the LRR fragment of InlB is sufficient for Met activation, but we show that this is not the case unless the LRR fragment is artificially dimerized through a disulphide bond . In contrast, activation of Met proceeds through monomers of intact InlB and, at physiologically relevant concentrations, requires coordinated action in cis of both InlB N-terminal LRR region and C-terminal GW domains . The GW domains are shown to be crucial for potentiating Met activation and inducing intracellular invasion, with these effects depending on association between GW domains and glycosaminoglycans . Glycosaminoglycans do not alter the monomeric state of InlB, and are likely to enhance Met activation through a receptor-mediated mode, as opposed to the ligand-mediated mode observed for the LRR fragment . Surprisingly, we find that gC1q-R, a host protein implicated in InlB-mediated invasion, specifically antagonizes rather than enhances InlB signalling, and that interaction between InlB and gC1q-R is unnecessary for bacterial invasion . Lastly, we demonstrate that HGF, the endogenous ligand of Met, substitutes for InlB in promoting intracellular invasion, suggesting that no special properties are required of InlB in invasion besides its hormone-like mimicry of HGF. Mol Microbiol, 2004 Apr, 52(1), 39 - 52 In vitro transcription of the Listeria monocytogenes virulence genes inlC and mpl reveals overlapping PrfA-dependent and -independent promoters that are differentially activated by GTP; Luo Q et al.; Most known virulence genes of Listeria monocytogenes are regulated by the transcriptional factor PrfA . Using our recently established in vitro transcription system, we have studied the PrfA-dependent promoter (PinlC) regulating the expression of the small, secreted internalin C . PrfA-dependent and PrfA-independent transcription is observed starting from PinlC in vitro and in vivo, suggesting the presence of two apparently overlapping promoters both of which use the same -10 box . Although the PrfA-dependent transcription requires, as expected, the PrfA-box, PrfA-independent transcription depends on a -35 box located directly downstream of the PrfA-box . PrfA-independent transcription starts at A, 7 bp downstream of the common -10 box (A7), and is strongly inhibited by PrfA because of the close proximity of the PrfA binding site to the -35 box . PrfA-dependent transcription starts preferentially at G5 but, in the absence of this start nucleotide, alternative start sites at A positions 7 or 8 bp downstream of the -10 box can also be used . The -35 box of the PrfA-independent promoter can be functionally inactivated without affecting PrfA-dependent transcription as long as the distance between the PrfA-box and the -10 box remains fixed to 22 (or 23) bp . Vice versa, the PrfA-box can be deleted without affecting PrfA-independent transcription from PinlC, which is no longer inhibited by PrfA . The PrfA-dependent transcription initiation needs, in contrast to the PrfA-independent one, the presence of a high concentration of GTP (and ATP) but not of CTP and UTP . Overlapping PrfA-dependent and PrfA-independent promoter activity was also demonstrated for the mpl promoter (Pmpl) . Again, PrfA-dependent transcription starting at Pmpl is dominant at high GTP concentration and PrfA-independent transcription at low GTP . Here too, the PrfA-dependent and the PrfA-independent promoters share the same -10 box characteristic of SigA-loaded RNA polymerase . High GTP concentration also appears to be necessary for transcription initiation at other PrfA-dependent promoters (Phly, PactA) but not at the PrfA-independent promoter PinlC-m8. Eur J Immunol, 2004 Apr, 34(4), 913 - 20 Mini-review: Presentation of pathogen-derived antigens in vivo; Lauvau G et al.; Most intracellular pathogens induce robust T cell responses upon infection of mammalian hosts . In most cases, these T cell responses are protective and result in pathogen clearance . It is therefore important to determine how T cells are primed and how they differentiate into cytokine-secreting and/or cytotoxic effector cells . In contrast to B cells, which recognize soluble Ag, CD8(+) and CD4(+) T cells react to Ag-derived peptides bound to MHC I or MHC II molecules, respectively . Therefore, elucidating the mechanisms by which pathogen-derived Ag become available for presentation is necessary to understand how pathogens trigger T cell responses in vivo . Although many excellent reviews have focused on the mechanisms involved in Ag processing, very few have pointed to the specificity of host-pathogen interactions . In this respect, it should be noticed that these interactions are very different from one pathogen to another, and may result in the involvement of different cells and molecules . Because of space limitations, we have decided to focus this review on two intracellular pathogens--vaccinia virus and Listeria monocytogenes . We have chosen these two pathogens because they both induce a strong CD8(+) T cell response and because they have been extensively studied by both microbiologists and immunologists. Biosens Bioelectron, 2004 May 15, 19(10), 1331 - 5 A generic approach for the detection of whole Listeria monocytogenes cells in contaminated samples using surface plasmon resonance; Leonard P et al.; The opportunistic food pathogen Listeria monocytogenes is of great concern to the food industry and its rapid detection is of major importance . This paper describes the detection of L . monocytogenes with a polyclonal antibody by means of a new subtractive inhibition assay using a BIAcore 3000 biosensor . Incubating L . monocytogenes cells and antibody for a short period of time, followed by subsequent separation of free unbound antibody with a stepwise centrifugation process, allowed the detection of 1 x 10(5) L . monocytogenes cells/ml in less than 30 min . Free antibody was passed over an anti-Fab ligand-coated sensor chip surface with the generated response being inversely proportional to the inhibiting cell concentration . The method was simple, rapid and needed minimum sample preparation . This assay format has the potential for the quick and sensitive detection of pathogens with limited sample handling and preparation. FEMS Microbiol Lett, 2004 Apr 1, 233(1), 159 - 64 Listeria monocytogenes: comparative interpretation of mouse virulence assay; Liu D; Being an opportunistic bacterial pathogen, Listeria monocytogenes demonstrates significant strain variations in virulence and pathogenicity . The availability of laboratory procedures to ascertain the pathogenic potential of L . monocytogenes bacteria would greatly enhance the control and prevention of listerial infections . As a method that measures all virulent determinants, mouse virulence assay has been frequently used for assessing L . monocytogenes virulence . The pathogenic potential of a given L . monocytogenes strain as determined by mouse virulence assay is often calculated from mouse mortality data in combination with colony forming units (CFUs) derived from plate counts, and expressed by medium lethal dose (LD(50)) . In this report, we describe an alternative method {i.e., relative virulence (%)} that does not involve CFU estimation, and is comparable to LD(50) for interpretation of mouse virulence assay for L . monocytogenes . The relative virulence (%) is obtained by dividing the number of dead mice with the total number of mice tested for a particular strain using a known virulent strain (e.g., L . monocytogenes EGD) as reference . Besides providing a more direct interpretation in comparison with LD(50) values for mouse virulence assay, this method requires fewer dosage groups per L . monocytogenes strain, and eliminates CFU estimation that is step subject to variations between runs and also between laboratories. Nat Rev Immunol, 2004 Feb, 4(2), 100 - 9 p47 GTPases: regulators of immunity to intracellular pathogens; Taylor GA et al.; Activation of the innate immune system by interferon-gamma (IFN-gamma is crucial for host resistance to infection . IFN-gamma induces the expression of a wide range of mediators that undermine the ability of pathogens to survive in host cells, including a newly discovered family of 47-kDa GTPases . Elimination of different p47 GTPases in mice by gene targeting severely cripples IFN-gamma-regulated defence against Toxoplasma gondii, Listeria monocytogenes, Mycobacterium spp . and other pathogens . In this article, we review our understanding of the role of p47 GTPases in resistance to intracellular infection and discuss the present evidence concerning their mode of action. Int Immunol, 2004 Apr, 16(4), 597 - 605 DNA vaccination with gp96-peptide fusion proteins induces protection against an intracellular bacterial pathogen; Rapp UK et al.; Effective vaccination using in vitro peptide-loaded heat-shock proteins (HSP), tumor-derived HSP and HSP fusion proteins has been shown in viral, parasite and tumor model systems . We demonstrate protective DNA vaccination using gp96-peptide fusion proteins against the intracellular bacterial pathogen Listeria monocytogenes in a mouse model . In contrast to previous studies using pathogen-derived HSP as vaccine vehicles, we used recombinant endogenous (Mus musculus) gp96 (GRP94) as a carrier for immunodominant listerial peptides . Analyses of the cellular immune response revealed profound epitope-specific IFN-gamma and cytotoxic T cell responses . Our findings suggest a predominantly MHC I-restricted T cell response to DNA vaccination with gp96 fusion proteins in the model employed . Most importantly, DNA vaccination induced protection against an otherwise lethal dose of L . monocytogenes. Infect Immun, 2004 Apr, 72(4), 2131 - 9 Toll-like receptor 2 is required for optimal control of Listeria monocytogenes infection; Torres D et al.; The control of Listeria monocytogenes infection depends on the rapid activation of the innate immune system, likely through Toll-like receptors (TLR), since mice deficient for the common adapter protein of TLR signaling, myeloid differentiation factor 88 (MyD88), succumb to Listeria infection . In order to test whether TLR2 is involved in the control of infections, we compared the host response in TLR2-deficient mice with that in wild-type mice . Here we show that TLR2-deficient mice are more susceptible to systemic infection by Listeria than are wild-type mice, with a reduced survival rate, increased bacterial burden in the liver, and abundant and larger hepatic microabscesses containing increased numbers of neutrophils . The production of tumor necrosis factor, interleukin-12, and nitric oxide and the expression of the costimulatory molecules CD40 and CD86, which are necessary for the control of infection, were reduced in TLR2-deficient macrophages and dendritic cells stimulated by Listeria and were almost abolished in the absence of MyD88, coincident with the high susceptibility of MyD88-deficient mice to in vivo infection . Therefore, the present data demonstrate a role for TLR2 in the control of Listeria infection, but other MyD88-dependent signals may contribute to host resistance. Infect Immun, 2004 Apr, 72(4), 2014 - 21 Induction of protective cellular immunity against Mycobacterium tuberculosis by recombinant attenuated self-destructing Listeria monocytogenes strains harboring eukaryotic expression plasmids for antigen 85 complex and MPB/MPT51; Miki K et al.; We report here the induction of specific protective cellular immunity against Mycobacterium tuberculosis by the employment of vaccination with recombinant attenuated Listeria monocytogenes strains . We constructed self-destructing attenuated L . monocytogenes Delta 2 strains carrying eukaryotic expression plasmids for the antigen 85 complex (Ag85A and Ag85B) and for MPB/MPT51 (mycobacterial protein secreted by M . bovis BCG/mycobacterial protein secreted by M . tuberculosis) molecules . Infection of these recombinant bacteria allowed expression of the genes in the J774A.1 murine macrophage cell line . Intraperitoneal vaccination of C57BL/6 mice with these recombinant bacteria was capable of inducing purified protein derivative-specific cellular immune responses, such as foot pad reactions, proliferative responses of splenocytes, and gamma interferon production from splenocytes, suggesting the efficacy of vaccination against mycobacterial infection by use of these recombinant L . monocytogenes strains . Furthermore, intravenous vaccination with recombinant bacteria carrying expression plasmids for Ag85A, Ag85B, or MPB/MPT51 in BALB/c mice elicited significant protective responses, comparable to those evoked by a live Mycobacterium bovis BCG vaccine . Notably, this is the first report to show that MPB/MPT51 is a major protective antigen in addition to Ag85A and Ag85B, which have been reported to be major mycobacterial protective antigens. Curr Opin Microbiol, 2004 Feb, 7(1), 45 - 50 T cell responses to Listeria monocytogenes; Lara-Tejero M et al.; Owing to its unique intracellular biology that allows it to gain access to the host cell cytosol, Listeria monocytogenes induces potent, protective CD8 responses . The study of these responses has served as a paradigm to understand cell-mediated immunity to microbial pathogens . The availability of mutants specifically defective in unique aspects of the intracellular biology of this pathogen has greatly aided these studies . During the past few years, progress has been made to understand the contribution of the innate immune system and CD4 T cells in the generation of robust, long lasting CD8 responses to L . monocytogenes. Biochemistry, 2004 Mar 30, 43(12), 3688 - 95 Membrane fusion induced by the catalytic activity of a phospholipase C/sphingomyelinase from Listeria monocytogenes; Montes LR et al.; Listeria monocytogenes is a bacterium responsible for localized and generalized infections in humans and animals . It has the ability to spread from the cytoplasm of an infected cell to neighboring cells without becoming exposed to the extracellular space . The bacterium secretes a phospholipase C (PLC(LM)) that is active on glycerophospholipids, e.g., phosphatidylcholine, and on sphingomyelin; thus, PLC(LM) should be described more appropriately as a phospholipase C/sphingomyelinase . We have obtained PLC(LM) free from a frequent contaminant, listeriolysin O, using an improved purification procedure . PLC(LM) has been assayed on large unilamellar liposomes of defined lipid composition . The enzyme is activated by K(+) and Mg(2+), and readily degrades phospholipids in bilayer form, in the absence of detergents . Enzyme activity is accompanied by important changes in the structure of the phospholipid vesicles, namely, vesicle aggregation, intervesicular mixing of lipids, and mixing of aqueous contents, with very low leakage of vesicular contents . The data are interpreted as indicative of PLC(LM)-induced vesicle fusion . This is confirmed by the demonstration of intervesicular mixing of inner monolayer lipids, using a novel procedure . The observation of PLC(LM)-induced membrane fusion suggests a mechanism for the cell-to-cell propagation of the bacterium, which requires disruption of a double-membrane vacuole. J Food Prot, 2004 Mar, 67(3), 480 - 5 Inhibition of Listeria monocytogenes on the surface of individually packaged hot dogs with a packaging film coating containing nisin; Franklin NB et al.; The objective of this study was to determine the effectiveness of packaging films coated with a methylcellulose/hydroxypropyl methylcellulose-based solution containing 10,000, 7,500, 2,500, or 156.3 IU/ml nisin for controlling Listeria monocytogenes on the surfaces of vacuum-packaged hot dogs . Barrier film coated with a methylcellulose/hydroxypropyl methylcellulose-based solution containing nisin or no nisin (control) was heat sealed to form individual pouches . Hot dogs were placed in control and nisin-containing pouches and inoculated with a five-strain L . monocytogenes cocktail (approximately 5 log CFU per package), vacuum sealed, and stored for intervals of 2 h and 7, 15, 21, 28, and 60 d at 4 degrees C . After storage, hot dogs and packages were rinsed with 0.1% peptone water . Diluent was spiral plated on modified oxford agar and tryptic soy agar and incubated to obtain counts (CFU per package) . L . monocytogenes counts on hot dogs packaged in films coated with 156.3 IU/ml nisin decreased slightly (approximately 0.5-log reduction) through day 15 of refrigerated storage but was statistically the same (P > 0.05) as hot dogs packaged in films without nisin after 60 d of storage . Packaging films coated with a cellulose-based solution containing 10,000 and 7,500 IU/ml nisin significantly decreased (P < 0.05) L . monocytogenes populations on the surface of hot dogs by greater than 2 log CFU per package throughout the 60-d study . Similar results were observed for hot dogs packaged in films coated with 2,500 IU/ml nisin; however, L . monocytogenes populations were observed to be approximately 4 log CFU per package after 60 d of refrigerated storage from plate counts on tryptic soy and modified oxford agars. J Food Prot, 2004 Mar, 67(3), 475 - 9 Development and characterization of an antimicrobial packaging film coating containing nisin for inhibition of Listeria monocytogenes; Grower JL et al.; The purpose of this study was to develop and characterize a packaging film coating containing nisin . A spot-on-lawn assay was used to determine the effect of acid type (ascorbic, acetic, hydrochloric, lactic) and nisin level (equal increments from 10,000 IU to 9 IU) to be used in the formulation of the film coating . Zones of inhibition were measured after incubation on tryptic soy agar (37 degrees C, 48 h) . Low-density polyethylene films coated with differing levels of nisin were characterized by field emission scanning electron microscopy, tensile strength, elongation, and water vapor transmission rate . The MIC of nisin in solution was 157 mg/ml . All acids were equally inhibitory (P > 0.05), but acetic acid produced the largest zone of inhibition (21 mm) . Field emission scanning electron microscopy confirmed that the cloudy appearance of the films was due to sodium chloride found in the commercially prepared nisin . Tensile strength increased as nisin concentration increased, which also corresponded to increasing film thickness . The nisin coatings (10,000 and 2,500 IU/ml) did not have a significant effect (P > 0.05) on the water vapor transmission rate of the low-density polyethylene film. J Food Prot, 2004 Mar, 67(3), 470 - 4 Radiation resistance and virulence of Listeria monocytogenes Scott A following starvation in physiological saline; Mendonca AF et al.; The influence of starvation on the resistance of Listeria monocytogenes Scott A to electron beam irradiation in 0.85% (wt/vol) NaCl (saline) and in ground pork was investigated . Exponential- or stationary-phase cells (control) were grown at 35 degrees C in tryptic soy broth supplemented with 0.6% yeast extract . Washed cells were starved for 12 days in saline, and virulence of the pathogen was evaluated at 0, 8, and 12 days during starvation . Samples of saline and irradiation-sterilized ground pork, inoculated with control or starved cells, were irradiated at doses ranging from 0.0 to 2.5 kGy . L . monocytogenes survivors were determined by plating diluted samples of saline or pork on tryptic soy agar supplemented with 0.6% yeast extract and counting bacterial colonies following incubation (35 degrees C, 48 h) . Virulence of starved cells and control was not significantly different (P > 0.05) . Cells exhibited the highest radiation resistance at 8 days of starvation . Irradiation (0.5 kGy) in saline resulted in approximately 7.14, 5.55, and 2.38 log reduction in exponential, stationary, and starved cells, respectively . Irradiation of ground pork at 2.5 kGy reduced controls by approximately 6.0 log, whereas starved cells were reduced by only 3.8 log . Starved cells consistently exhibited higher irradiation D10-values than controls (P < 0.05) . D10-values for exponential, stationary, and starved cells were 0.07, 0.09, and 0.21 kGy and 0.35, 0.42, and 0.66 kGy in saline and ground pork, respectively . These results indicate that starvation cross-protects L . monocytogenes Scott A against radiation inactivation and should be considered when determining this pathogen's irradiation D-value. J Food Prot, 2004 Mar, 67(3), 463 - 9 Temperature effect on Listeria monocytogenes growth in the event of contamination of cooked pork products; Membre JM et al.; The aim of this study was to describe the effect of temperature on the growth of Listeria monocytogenes in the event of postprocess contamination of packaged pork meats . This study was carried out in two steps . In the first step, the effect of temperature on L . monocytogenes growth rates was determined in duplicates at 13 temperatures between 2 and 43 degrees C by turbidimetric methods and adjusted by a quantitative secondary model . Then, seven sets of growth kinetics were collected by challenge testing in white pudding and roulade, both cooked pork products prepared according to an industrial process and stored at suboptimal temperatures ranging from 2 to 20 degrees C . In the second step, objectives were to (i) collect direct information on the temperature effect of L . monocytogenes on the two pork products, (ii) compare the two products regarding L . monocytogenes exposure, and (iii) compare results given by modeling (step i) with results obtained independently and then evaluate the model application domain . Each kinetic was built with at least 10 experimental data and two replicates . Comparison between L . monocytogenes behavior at 4 degrees C on white pudding and roulade indicated that both meat products were affected by food safety problems . Indeed, after contamination and storage for 10 days at 4 degrees C, the bacterial population increased by 2 log CFU/g in both products . Comparison between growth kinetic simulations and experimental data obtained separately gave satisfactory conclusions; the difference between observed and predicted bacterial population values was always less than 1 log CFU/g and a bias factor of 1.18 when growth rates were compared . These results applied to L . monocytogenes contamination of white pudding or roulade can now be used either in the management of optimal process and distribution networks or in risk assessment (exposure assessment). J Food Prot, 2004 Mar, 67(3), 456 - 62 Attachment of Listeria monocytogenes on ready-to-eat meats; Foong SC et al.; Five individual strains of Listeria monocytogenes and a mixed cocktail of all five were studied for attachment on frankfurters, ham, bologna, and roast beef relative to their cell surface characteristics . The ratio of strongly attached (sessile) L . monocytogenes cells compared with total (sessile and planktonic) attached cells on ready-to-eat meats was also determined . Because bacterial cell surfaces were characterized by net negative charge and hydrophobicity, electrostatic interaction chromatography and cationized ferritin methods were chosen to study net negative charge distribution on the bacterial cell surface, whereas hydrophobic interaction chromatography and contact angle measurement were used to examine the cell surface hydrophobicity . No differences (P > 0.05) were observed in cell surface charge or cell surface hydrophobicity among strains . Approximately 84 to 87% L . monocytogenes were found to attach strongly to ready-to-eat meats within 5 min . No differences (P > 0.05) were found among strains or among meats . Micrographs observed from scanning electron microscopy showed no differences among the strains but showed a difference in age of cells (mixed culture) in terms of surface negative charge distribution . More surface negatively charged sites were observed at 0 and 7 days and much fewer at 3 days during storage of washed, harvested cells in buffer at 4 degrees C (aged cells under cold and nutrient deprivation), indicating a possible change in cell surface properties . Because no difference in strains was observed, the contact angle measurement study was carried out with the five-strain mixed culture . The surface hydrophobicity increased in frankfurters, decreased in roast beef, and was unchanged in ham and bologna as a result of inoculation. J Immunol, 2004 Apr 1, 172(7), 4418 - 24 The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice; Drevets DA et al.; Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host . In the bloodstream these cells are generically referred to as monocytes . However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known . Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria . More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells . Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell . Interestingly, systemic infection with wild-type or DeltaactA mutants of L . monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset . In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly . Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes . Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection . This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression . These data indicate that the Ly-6C(high) monocyte subset transports L . monocytogenes into the brain and establish their role as Trojan horses in vivo. J Immunol, 2004 Apr 1, 172(7), 4410 - 7 Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response; Sunderkotter C et al.; Blood monocytes are well-characterized precursors for macrophages and dendritic cells . Subsets of human monocytes with differential representation in various disease states are well known . In contrast, mouse monocyte subsets have been characterized minimally . In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L . The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation . By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets . Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation . These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes . Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation . Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes . In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes . Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites. Int J Food Microbiol, 2004 Apr 1, 92(1), 95 - 103 Effects of several environmental factors on the anti-Listeria monocytogenes activity of an essential oil of Picea excelsa; Canillac N et al.; The effects of several environmental factors on the anti-Listeria monocytogenes activity of an essential oil of Picea excelsa were explored by determination of active concentrations using two methods and with survival curves . In trial conditions, the serovars 1/2c and 4b behaved similarly . A dose of 0.2-0.3% (v/v) of essential oil was bactericidal for 10(5)-10(7) cells contained in 1 ml of Tryptone Soy Broth Yeast Extract at pH 6 and 7 incubated at 13 and 37 degrees C and of medium supplemented with levan . Introduction of sodium caseinate, agar or fat into the test medium and use of a cheese medium decreased the bactericidal effects of the essential oil . Basic pH, addition of NaCl or use of Tryptone Soy Broth and saline solution increased its antilisterial activity . Serovar 1/2c survival curves exhibited an exponential death rate followed by a tailing effect in the presence of the minimal bactericidal concentration of the essential oil . A three log10 reduction of cell viability was obtained within 100 min in Tryptone Soy Broth Yeast Extract, within longer exposure in media supplemented with NaCl or at basic pH. Int J Food Microbiol, 2004 Apr 1, 92(1), 89 - 94 Growth of Listeria monocytogenes in melon, watermelon and papaya pulps; Penteado AL et al.; Growth of Listeria monocytogenes in low-acid fruits (melon, watermelon and papaya) at different times of incubation and at temperatures of 10, 20 and 30 degrees C was studied . Fruit pulp portions with an average pH of 5.87, 5.50 and 4.87 for melon, watermelon and papaya, respectively, were obtained aseptically, homogenized, weighed and inoculated with suspensions (approximately 10(2) CFU/g) of L . monocytogenes . Generation times of 7.12, 13.03 and 15.05 h at 10 degrees C, 1.74, 2.17 and 6.42 h at 20 degrees C and 0.84, 1.00 and 1.16 h at 30 degrees C were obtained, respectively, for melon, watermelon and papaya . The results showed that L . monocytogenes grew in low-acid fruits at all tested temperatures, although growth was diminished, but not inhibited at 10 degrees C. Int J Food Microbiol, 2004 Apr 1, 92(1), 15 - 33 Listeria monocytogenes and listeriosis: a review of hazard characterisation for use in microbiological risk assessment of foods; McLauchlin J et al.; Considerable effort has been put into the application of quantitative microbiological risk assessment for Listeria monocytogenes, and data are available for England and Wales (probably more so than most other countries) on the adverse health effects, together with incidence data on different age and risk groups for human L . monocytogenes infections . This paper reviews aspects of Listeria and human listeriosis, especially from a public health perspective and provide hazard characterisation data, i.e . the qualitative and/or quantitative evaluation of the adverse health effect associated with the hazard, which is the relationship between exposure levels (dose) and frequency of illness . The majority of cases of human listeriosis are food-borne; however, the disease process is complex with multiple routes of infection . The dose-response relationship is poorly understood, and data from human volunteer studies are not available and would be unethical to produce . Data are available from a range of different animal and in vitro models, although these poorly mimic the natural disease process in route of infection, end point, host and history of prior exposure to the bacterium . Epidemiological data provide some information on infective doses and dose responses, but because of the characteristics of the disease (the hugely variable and potentially very long incubation periods, the low attack rates and the rarity of identification of specific food vehicles), this also provides limited data for calculation of dose responses . There is some, albeit limited, evidence for strain variation, but this is an area of considerable uncertainty despite great advances in the genetic basis of the virulence of this bacterium, and almost all strains seem capable of causing serious disease . A variety of mathematical approaches have been used to model dose responses . The review is written to provide a clinical and epidemiological background to the mathematically oriented, as well as to outline the mathematical approaches to those interested in food-borne infection. Curr Drug Targets Inflamm Allergy, 2004 Mar, 3(1), 97 - 104 Estrogen, a double-edged sword: modulation of TH1- and TH2-mediated inflammations by differential regulation of TH1/TH2 cytokine production; Salem ML; Estrogen appears to play a central role in the immune response and immune-mediated diseases . Estrogen receptors are expressed in a variety of immunocompetent cells, including CD4(+) and CD8(+) T cells and macrophages . Clinical observations indicate that some autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis, frequently remit during pregnancy but exacerbate, or have their onset during the postpartum period . Pharmacological levels of estrogen also appear to ameliorate certain autoimmune diseases . In addition, estrogen is known to suppress certain infectious diseases, as well as T cell-mediated responses toward oxazolone, keyhol lympet hemocyanin, Listeria soluble protein and purified protein derivatives . The immune basis for these phenomena is poorly understood . Based on a distinctive profile of cytokine production, data accumulated thus far have revealed modulatory effects for estrogen on the TH1-type and TH2-type cells, which represent two polarized forms of the effector specific immune response . Recent evidence indicates that estrogens inhibit the production of TH1 proinflammatory cytokines, such as IL-12, TNF-alpha and IFN-gamma, whereas they stimulate the production of TH2 anti-inflammatory cytokines, such as IL-10, IL-4, and TGF-beta . This can explain why estrogen suppresses and potentiates TH1- and TH2-mediated diseases, respectively . We hypothesize that exacerbation or suppression of inflammatory diseases by estrogen is mediated by skewing TH1-type to TH2-type response . This view represents a novel mechanism for the modulatory effect of estrogen on certain inflammatory diseases that can lead to beneficial or detrimental impacts depending on the type of immune involved . Such a concept is valuable when considering the application of combination therapies that include estrogen. FDA Consum, 2004 Jan-Feb, 38(1), 10 - 1 Preventing Listeria contamination in foods; Rados C; Keeping ready-to-eat foods cold is key to reducing listeriosis, a serious infection in humans . That's one of the conclusions of a recent Food and Drug Administration risk assessment on the relationships between foodborne listeriosis and human health. Biochem Biophys Res Commun, 2004 Apr 2, 316(2), 379 - 86 Characterization of the calcium-binding sites of Listeria monocytogenes InlB; Marino M et al.; The Listeria monocytogenes protein InlB promotes invasion of mammalian cells through activation of the receptor tyrosine kinase Met . The InlB N-cap, a approximately 40 residue part of the domain that binds Met, was previously observed to bind two calcium ions in a novel and unusually exposed manner . Because subsequent work raised questions about the existence of these calcium-binding sites, we assayed calcium binding in solution to the InlB N-cap . We show that calcium ions are bound with dissociation constants in the low micromolar range at the two identified sites, and that the sites interact with one another . We demonstrate that the calcium ions are not required for structure, and also find that they have no appreciable effect on Met activation or intracellular invasion . Therefore, our results indicate that the sites are fortuitous in InlB, but also suggest that the simple architecture of the sites may be adaptable for protein engineering purposes. Am J Ophthalmol, 2004 Mar, 137(3), 579 - 81 Listeria monocytogenes-induced endogenous endophthalmitis: bioultrasonic findings; Mendez-Hernandez C et al.; PURPOSE: To report bioultrasonic findings in Listeria monocytogenes-induced endophthalmitis (LMIE) that have not been described previously . DESIGN: Interventional case report . METHODS: To rule out intraocular tumor, ultrasound biomicroscopy was performed in a patient referred for a 2-day history of uveitis with elevated intraocular pressure, dark hypopyon, and pigment dispersion in the anterior chamber . RESULTS: Ultrasound biomicroscopy examination showed increased iris thickness with rarefaction of tissue and irregular echogenicity as well as iris pigment epithelial detachment . A small choroidal detachment was also detected . The anterior chamber and vitreous sample confirmed the LMIE diagnosis . CONCLUSIONS: The detection of both pigment epithelial detachment and changes in the iris tissue could explain why black hypopyon frequently develops in LMIE with significant pigment dispersion in some cases. FEBS Lett, 2004 Mar 12, 561(1-3), 99 - 104 The interplay between classical and alternative isoprenoid biosynthesis controls gammadelta T cell bioactivity of Listeria monocytogenes; Begley M et al.; Isoprenoids are synthesised either through the classical, mevalonate pathway, or the alternative, non-mevalonate, 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway . The latter is found in many microbial pathogens and proceeds via (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), a potent activator of human Vgamma9/Vdelta2 T cells . Listeria monocytogenes is the only pathogenic bacterium known to contain both pathways concurrently . Strategic gene knockouts demonstrate that either pathway is functional but dispensable for viability . Yet, disrupting the mevalonate pathway results in a complementary upregulation of the MEP pathway . Vgamma9/Vdelta2 T cell bioactivity is increased in DeltalytB mutants where HMB-PP accumulation is expected, and lost in DeltagcpE mutants which fail to produce HMB-PP. Mol Microbiol, 2004 Mar, 51(6), 1601 - 14 Auto, a surface associated autolysin of Listeria monocytogenes required for entry into eukaryotic cells and virulence; Cabanes D et al.; Listeria monocytogenes is an opportunistic food-borne human and animal pathogen . Several surface proteins expressed by this intracellular pathogen are critical for the infectious process . By in silico analysis we compared the surface protein repertories of L . monocytogenes and of the non-pathogenic species Listeria innocua and identified a gene encoding a surface protein of L . monocytogenes absent in L . innocua . This gene that we named aut encodes a protein (Auto) of 572 amino acids containing a signal sequence, a N-terminal autolysin domain and a C-terminal cell wall-anchoring domain made up of four GW modules . We show here that the aut gene is expressed independently of the virulence gene regulator PrfA and encodes a surface protein with an autolytic activity . We provide evidence that Auto is required for entry of L . monocytogenes into cultured non-phagocytic eukaryotic cells . The low invasiveness of an aut deletion mutant correlates with its reduced virulence following intravenous inoculation of mice and oral infection of guinea pigs . During infection, the autolytic activity of Auto may also be critical . Auto appears thus as a novel type of L . monocytogenes virulence factor. J Obstet Gynaecol Res, 2004 Apr, 30(2), 117 - 9 Maternal listeriosis in pregnancy associated with measles virus infection; Naruse K et al.; A case of maternal listeriosis associated with measles infection is described . Maternal listeriosis without fetal or neonatal involvement is relatively rare . This case was diagnosed early and treated appropriately, and the baby showed no symptoms or signs of feto-maternal infection. Appl Environ Microbiol, 2004 Mar, 70(3), 1669 - 79 pbp2229-mediated nisin resistance mechanism in Listeria monocytogenes confers cross-protection to class IIa bacteriocins and affects virulence gene expression; Gravesen A et al.; It was previously shown that enhanced nisin resistance in some mutants was associated with increased expression of three genes, pbp2229, hpk1021, and lmo2487, encoding a penicillin-binding protein, a histidine kinase, and a protein of unknown function, respectively . In the present work, we determined the direct role of the three genes in nisin resistance . Interruption of pbp2229 and hpk1021 eliminated the nisin resistance phenotype . Interruption of hpk1021 additionally abolished the increase in pbp2229 expression . The results indicate that this nisin resistance mechanism is caused directly by the increase in pbp2229 expression, which in turn is brought about by the increase in hpk1021 expression . We also found a degree of cross-protection between nisin and class IIa bacteriocins and investigated possible mechanisms . The expression of virulence genes in one nisin-resistant mutant and two class IIa bacteriocin-resistant mutants of the same wild-type strain was analyzed, and each mutant consistently showed either an increase or a decrease in the expression of virulence genes (prfA-regulated as well as prfA-independent genes) . Although the changes mostly were moderate, the consistency indicates that a mutant-specific change in virulence may occur concomitantly with bacteriocin resistance development. Appl Environ Microbiol, 2004 Mar, 70(3), 1366 - 77 Quantitative detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology; Rodriguez-Lazaro D et al.; We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L . innocua . The target genes were hly and iap for L . monocytogenes and lin02483 for L . innocua . The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions . Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R(2) values of >0.996 . There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%) . The hly-based assay accurately quantified L . monocytogenes in all of the samples tested . The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability . The combination of the two assays enabled us to classify L . monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II . We also assessed the new AmpliFluor technology for the quantitative detection of L . monocytogenes by RTi-PCR . The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules). Mol Biol Cell, 2004 May, 15(5), 2164 - 75 Epub 2004 Mar 05. Listeria monocytogenes actin-based motility varies depending on subcellular location: a kinematic probe for cytoarchitecture; Lacayo CI et al.; Intracellular Listeria monocytogenes actin-based motility is characterized by significant individual variability, which can be influenced by cytoarchitecture . L . monocytogenes was used as a probe to transmit information about structural variation among subcellular domains defined by mitochondrial density . By analyzing the movement of a large population of L . monocytogenes in PtK2 cells, we found that mean speed and trajectory curvature were significantly larger for bacteria moving in mitochondria-containing domains (generally perinuclear) than for bacteria moving in mitochondria-free domains (generally peripheral) . Analysis of bacteria that traversed both mitochondria-containing and mitochondria-free domains revealed that these motile differences were not intrinsic to bacteria themselves . Disruption of mitochondrial respiration did not affect bacterial mean speed, speed persistence, or trajectory curvature . In contrast, microtubule depolymerization lead to decreased mean speed per bacterium and increased mean speed persistence of L . monocytogenes moving in mitochondria-free domains compared with untreated cells . L . monocytogenes were also observed to physically collide with mitochondria and push them away from the bacterial path of motion, causing bacteria to slow down before rapidly resuming their speed . Our results show that subcellular domains along with microtubule depolymerization may influence the actin cytoskeleton to affect L . monocytogenes speed, speed persistence, and trajectory curvature. Mol Biol Cell, 2004 May, 15(5), 2312 - 23 Epub 2004 Mar 05. Biophysical parameters influence actin-based movement, trajectory, and initiation in a cell-free system; Cameron LA et al.; Using a biochemically complex cytoplasmic extract to reconstitute actin-based motility of Listeria monocytogenes and polystyrene beads coated with the bacterial protein ActA, we have systematically varied a series of biophysical parameters and examined their effects on initiation of motility, particle speed, speed variability, and path trajectory . Bead size had a profound effect on all aspects of motility, with increasing size causing slower, straighter movement and inhibiting symmetry-breaking . Speed also was reduced by extract dilution, by addition of methylcellulose, and paradoxically by addition of excess skeletal muscle actin, but it was enhanced by addition of nonmuscle (platelet) actin . Large, persistent individual variations in speed were observed for all conditions and their relative magnitude increased with extract dilution, indicating that persistent alterations in particle surface properties may be responsible for intrinsic speed variations . Trajectory curvature was increased for smaller beads and also for particles moving in the presence of methylcellulose or excess skeletal muscle actin . Symmetry breaking and movement initiation occurred by two distinct modes: either stochastic amplification of local variation for small beads in concentrated extracts, or gradual accumulation of strain in the actin gel for large beads in dilute extracts . Neither mode was sufficient to enable spherical particles to break symmetry in the cytoplasm of living cells. J Immunol, 2004 Mar 15, 172(6), 3725 - 35 Increased dendritic cell numbers impair protective immunity to intracellular bacteria despite augmenting antigen-specific CD8+ T lymphocyte responses; Alaniz RC et al.; Dendritic cells (DCs) reside in tissues, where they function as sentinels, providing an essential link between innate and adaptive immunity . Increasing the numbers of DCs in vivo augments T cell responses, and can cause dramatic CTL-dependent tumor regression . To determine whether greater DC numbers promoted T cell-mediated protection in the context of host defense against intracellular bacteria, we treated mice with Flt3 ligand (Flt3-L) to increase DCs in vivo and challenged them with Listeria monocytogenes . Unexpectedly, after primary challenge with Listeria, the overall control of Listeria infection was impaired in Flt3-L-treated mice, which had greater bacterial burden and mortality than controls . Similar results were obtained when DC numbers were increased by treatment with polyethylene glycol-conjugated GM-CSF rather than Flt3-L and in mice infected with Mycobacterium tuberculosis . Impaired protection was not due to dysfunctional T cell responses, as Flt3-L-treated mice had a greater frequency and absolute number of Ag-specific CD8+ T cells, which produced IFN-gamma, exhibited cytolytic activity, and transferred protection . The increased Listeria burden in Flt3-L-treated mice was preferentially associated with DCs, which were unable to kill Listeria and more resistant to CTL lysis compared with macrophages in vitro . Although we cannot exclude the possibility that other potential effects, in addition to increased numbers of DCs, are shared by Flt3-L and polyethylene glycol-conjugated GM-CSF and contributed to the increase in susceptibility observed in treated mice, these results support the notion that DC numbers must be properly controlled within physiological limits to optimize host defense to intracellular bacterial pathogens. J Immunol, 2004 Mar 15, 172(6), 3491 - 500 Prolonged antigen presentation, APC-, and CD8+ T cell turnover during mycobacterial infection: comparison with Listeria monocytogenes; van Faassen H et al.; We expressed the CTL epitope of OVA (OVA(257-264)) in an acute (Listeria monocytogenes (LM)-OVA) and a chronic intracellular pathogen (Mycobacterium bovis (BCG)-OVA), to evaluate the kinetics of Ag presentation . LM-OVA proliferated rapidly in vivo, resulting in profound LM-OVA expansion within the first 24 h of infection, culminating in the generation of a potent CD8+ T cell response, which peaked on day 7 but underwent a rapid attrition subsequently . In contrast, BCG-OVA exhibited reduced growth in vivo, resulting in a delayed CD8+ T cell response that increased progressively with time . Relative to LM-OVA, BCG-OVA induced persistently increased numbers of apoptotic (annexin V+) CD8+ T cells . Ag presentation in vivo was evaluated by transferring Thy1.2+ carboxyfluorescein-labeled OT1 transgenic CD8+ T cells into infected Thy1.1+ congeneic recipient mice . LM-OVA induced rapid Ag presentation that was profound in magnitude, with most of the transferred cells getting activated within 4 days and resulting in a massive accumulation of activated donor CD8+ T cells . In contrast, Ag presentation induced by BCG-OVA was delayed, weaker in magnitude, which peaked around the second week of infection and declined to a low level subsequently . Increasing the dose of BCG-OVA while enhancing the magnitude of Ag presentation did not change the kinetics . Furthermore, a higher dose of BCG-OVA also accelerated the attrition of OVA(257-264)-specific CD8+ T cells . Relative to LM-OVA, the dendritic cells in BCG-OVA-infected mice were apoptotic for prolonged periods, suggesting that the rapid death of APCs may limit the magnitude of Ag presentation during chronic stages of mycobacterial infection. J Immunol, 2004 Mar 15, 172(6), 3385 - 9 Cutting edge: long-lived CD8 memory and protective immunity in the absence of CD40 expression on CD8 T cells; Sun JC et al.; CD8 T cells need CD4 T cells to develop into long-lived, functional memory cells that provide protection against pathogen rechallenge . We investigated whether signaling via CD40 expressed on the CD8 cells themselves is involved in this cooperation . In murine responses to Listeria monocytogenes and lymphocytic choriomeningitis virus, we found no evidence of any requirement for CD40-CD40 ligand interaction at this level . No differences were observed between CD40(-/-) and CD40(+/+) CD8 T cells that had matured in the same environment when comparing their expansion in a primary or secondary response, their contribution to memory, and their ability to enter nonlymphoid tissues such as the liver . Thus, we find no evidence that CD40 ligand-expressing CD4 T cells are required to activate CD40 on CD8 T cells directly for the full differentiation of the cytotoxic T cell response. J Comp Pathol, 2004 Feb-Apr, 130(2-3), 130 - 6 Suppurative gastritis in BALB/c mice infected with Listeria monocytogenes via the intragastric route; Park JH et al.; Suppurative gastritis was demonstrated in BALB/c mice 3 days after intragastric inoculation with 10(9) organisms of Listeria monocytogenes strain ATCC19113 (serotype 3) . Also tested were four other strains of mice (C3H, C57BL/6, FVB and ICR) and three other strains of L . monocytogenes (HPB 3 {serotype 4b}, HPB 410 {serotype 1/2a} and HPB 503 {serotype 1/2b}) . After inoculation with ATCC19113 the numbers of bacteria found in the stomach wall were greater in C57BL/6 and ICR mice than in C3H and FVB mice; moreover, the gastritis produced in BALB/c and C57BL/6 mice was more severe than that produced in the other mouse strains . The gastritis produced in BALB/c mice with L . monocytogenes HPB 3, HPB 410 and HPB 503 was much more severe than that produced by ATCC19113 . The inflammatory response occurred in the lamina muscularis and mucosa of the fundus . Massive necrosis of the gastric epithelium was observed, and there was oedema in a large part of the mucosal layer of the fundus . In addition, the submucosal layer was apparently expanded due to oedema, and in the cardia, the mucosal layer had become thin and flattened . Immunohistochemically, a polyclonal antibody against Listeria spp . produced labelling in areas of the gastric mucosa in which there was an inflammatory response and gastric epithelial necrosis. J Mol Biol, 2004 Mar 19, 337(2), 453 - 61 Folding and stability of the leucine-rich repeat domain of internalin B from Listeri monocytogenes; Freiberg A et al.; Internalin B (InlB), a surface protein of the human pathogen Listeria monocytogenes, promotes invasion into various host cell types by inducing phagocytosis of the entire bacterium . The N-terminal half of InlB (residues 36-321, InlB321), which is sufficient for this process, contains a central leucine-rich repeat (LRR) domain that is flanked by a small alpha-helical cap and an immunoglobulin (Ig)-like domain . Here we investigated the spectroscopic properties, stability and folding of InlB321 and of a shorter variant lacking the Ig-like domain (InlB248) . The circular dichroism spectra of both protein variants in the far ultraviolet region are very similar, with a characteristic minimum found at approximately 200 nm, possibly resulting from the high 3(10)-helical content in the LRR domain . Upon addition of chemical denaturants, both variants unfold in single transitions with unusually high cooperativity that are fully reversible and best described by two-state equilibria . The free energies of GdmCl-induced unfolding determined from transitions at 20 degrees C are 9.9(+/-0.8)kcal/mol for InlB321 and 5.4(+/-0.4)kcal/mol for InlB248 . InlB321 is also more stable against thermal denaturation, as observed by scanning calorimetry . This suggests, that the Ig-like domain, which presumably does not directly interact with the host cell receptor during bacterial invasion, plays a critical role for the in vivo stability of InlB. DNA Cell Biol, 2004 Feb, 23(2), 93 - 106 Cytotoxic T-lymphocyte-, and helper T-lymphocyte-oriented DNA vaccination; Nagata T et al.; DNA vaccines have advantages over other types of vaccines in that they can induce strong cellular immune responses, namely cytotoxic T lymphocytes (CTL) and helper T lymphocytes (Th) . DNA vaccines are therefore considered a promising alternative to attenuated live vaccines in the field of infectious diseases . So far, various DNA vaccines have been generated and tried to induce a particular cellular immune response by virtue of recombinant DNA technology . DNA vaccines have been designed for efficient transcription and translation of target genes by a variety of strategies . Also, various DNA vaccine strategies for induction of specific CTL and Th have been reported by taking into consideration antigen presentation pathways and the strategies have been shown to be effective to elicit particular T-cell responses . In this paper, we have reviewed these strategies, including our study on epitope-specific T-cell induction by DNA vaccination against Listeria monocytogenes infection . From this review, it has been surmised that, to induce strong immune responses by DNA vaccines, the immunization route and the immunization regimen, such as heterologous "prime-boost" regimen, should also be considered. Int J Food Microbiol, 2004 Mar 1, 91(2), 167 - 74 Incidence of Listeria monocytogenes in two milk processing environments, and assessment of Listeria monocytogenes blood agar for isolation; Kells J et al.; A year-long survey of two Northern Ireland milk processing plants for Listeria monocytogenes was carried out . Sample sites included the milk processing environment (walls, floors, drains, and steps), processing equipment, raw and pasteurised milk . The FDA listeria-selective enrichment procedure was used to process samples and an additional agar medium, L . monocytogenes Blood Agar (LMBA), was utilized as part of the isolation procedure in order to compare its performance to that of the recommended Oxford and Palcam agars . LMBA proved to be a very useful tool and was able to detect L . monocytogenes from 94.1% of sites compared to the 76.5% and 79.4% detection rate displayed by Oxford and Palcam agars, respectively . The overall incidence of listeria on equipment was 18.8% (6.3% L . monocytogenes), in the environment was 54.7% (40.6% L . monocytogenes) and in raw milk 44.4% (22.2% L . monocytogenes) . On one occasion, L . welshimeri was isolated from pasteurised milk, probably demonstrating post-pasteurisation contamination of product . The main environmental sources of L . monocytogenes were considered to be a floor drain and stainless steel steps. Int J Food Microbiol, 2004 Mar 1, 91(2), 119 - 27 A contribution to the improvement of Listeria monocytogenes enumeration in cold-smoked salmon; Besse NG et al.; For the enumeration of Listeria monocytogenes in food, a sensitive enumeration method based on membrane filtration followed by transfer of the filter to a selective medium has been developed . This study was carried out with cold-smoked salmon, a product likely to be contaminated with L . monocytogenes . The operating protocol utilizes three filtration runs in parallel (5, 15 and 30 ml) of a 1 in 10 dilution of the salmon suspension through 0.45-microm pore-size cellulose ester membranes, and then culture of the filters on Aloa agar (AES Laboratoires, Combourg, France) . The results obtained with the technique were compared with those from the reference EN ISO 11290-2 method and found to provide more precise results in the enumeration of L . monocytogenes from both artificially and naturally contaminated cold-smoked salmon . Moreover, for several samples contaminated at low levels, L . monocytogenes could be recovered only by the filtration method . The examination of increasing volumes of salmon suspension enabled readable results to be obtained for all levels of L . monocytogenes and competitive microflora investigated . In most cases, the optimised operating protocol enabled 5.1 g of salmon to be examined, instead of 0.01-0.1 g with the reference EN ISO 11290-2 method, thus improving the sensitivity of the method. Int J Food Microbiol, 2004 Mar 15, 91(3), 297 - 304 Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes; Liu D et al.; Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide . In this study, through comparative analysis of L . innocua and L . monocytogenes genomic sequences, we selected a L . monocytogenes specific gene (lmo0733) that has the potential for specific detection of L . monocytogenes . Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L . monocytogenes strains . PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay . Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L . monocytogenes. Cell Motil Cytoskeleton, 2004 May, 58(1), 17 - 29 Palladin is a novel binding partner for Ena/VASP family members; Boukhelifa M et al.; Palladin is an actin-associated protein that contains proline-rich motifs within its amino-terminal sequence that are similar to motifs found in zyxin, vinculin, and the Listeria protein ActA . These motifs are known to be potential binding sites for the Vasodilator-Stimulated Phosphoprotein (VASP) . Here, we demonstrate that palladin is an additional direct binding partner for VASP, by using co-immunoprecipitation and blot overlay techniques with both endogenous palladin and recombinant myc-tagged palladin . These results show that VASP binds to full-length palladin and also to the amino-terminal half of palladin, where the polyproline motifs are located . Using a synthetic peptide array, two discrete binding sites for VASP were identified within palladin's proline-rich amino-terminal domain . Using double-label immunofluorescence staining of fully-spread and actively-spreading fibroblasts, the extent of co-localization of palladin and VASP was explored . These proteins were found to strongly co-localize along stress fibers, and partially co-localize in focal adhesions, lamellipodia, and focal complexes . These results suggest that the recently described actin-associated protein palladin may play an important role in recruiting VASP to sites of actin filament growth, anchorage, and crosslinking . Heart Lung, 2004 Jan-Feb, 33(1), 61 - 4 Listeria monocytogenes encephalitis mimicking West Nile encephalitis; Cunha BA et al.; We present a case of a 50-year-old man who presented to Winthrop-University Hospital in the midst of the 2002 West Nile encephalitis (WNE) outbreak with the cardinal clinical findings of WNE, ie, fever, encephalopathy, weakness, and muscle tremors . During the summer of 2002, several cases of aseptic meningitis/viral encephalitis were admitted to our emergency room weekly . In addition, cases of WNE were being admitted at the same time . During this period we had 3 cases of WNE . Our patient presented with the clinical findings of WNE . However, laboratory and radiologic findings suggested the possibility of Listeria monocytogenes encephalitis . The cerebrospinal fluid findings included red blood cells, which, in the absence of a traumatic tap or HSV encephalitis, argue against the diagnosis of WNE but are consistent with L . monocytogenes encephalitis . Computed tomography scan showed communicating hydrocephalus, which also suggests the possibility of L . monocytogenes and argued against the diagnosis of WNE . Clinicians should be vigilant for the mimics of WNE in geographical areas where WNE outbreaks are occurring. Mol Microbiol, 2004 Mar, 51(5), 1483 - 92 Listeria monocytogenes virulence proteins induce surface expression of Fas ligand on T lymphocytes; Zenewicz LA et al.; Virulence factors secreted by Listeria monocytogenes are known to interfere with host cellular signalling pathways . We investigated whether L . monocytogenes modulates T-cell receptor signalling by examining surface expression of proteins known to be upregulated on activated T cells . In vitro culture of murine splenocytes with L . monocytogenes resulted in a specific and dose-dependent upregulation of Fas ligand (FasL) . Induction of FasL expression was also observed for pathogenic Listeria ivanovii but not for non-pathogenic Listeria innocua, indicating involvement of Listeria virulence protein(s) . Examination of L . monocytogenes strains deficient in different virulence genes demonstrated that FasL upregulation was dependent on the expression of two secreted proteins: listeriolysin O (LLO) and phosphatidylcholine-preferring phospholipase C (PC-PLC) . Treatment of cells with purified proteins demonstrated that LLO was sufficient for inducing FasL, while PC-PLC synergized with LLO for the induction of FasL expression . FasL-expressing cells induced by L . monocytogenes were capable of killing Fas-expressing target cells . Furthermore, L . monocytogenes infection results in upregulation of FasL on T cells in mice . These results describe a novel function for LLO and PC-PLC and suggest that L . monocytogenes may use these virulence factors to modulate the host immune response. J Immunol, 2004 Mar 1, 172(5), 3167 - 72 Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis; Kursar M et al.; Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L . monocytogenes infection . We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses . In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L . monocytogenes . During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production . In summary, we demonstrate that combining nonviable L . monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells. Int Immunol, 2004 Mar, 16(3), 415 - 21 Protective T cell response against intracellular pathogens in the absence of Toll-like receptor signaling via myeloid differentiation factor 88; Kursar M et al.; Toll-like receptors (TLR) have been indicated as germline-encoded receptors for sensing a variety of pathogens . Although the role of TLR in innate immunity is beyond question, their function in acquired immunity, in particular in T cell immunity, is less clear . Here, we used experimental Listeria monocytogenes infection of mice to analyze requirements for TLR2, TLR4 and the central TLR adaptor protein myeloid differentiation factor 88 (MyD88) in the generation of specific T cell responses . We demonstrate that following L . monocytogenes infection, mice deficient in TLR2, TLR4 and MyD88 can generate Listeria-specific CD8+ and CD4+ Th1 responses . These T cell responses are sufficient to control secondary infection with a high dose of L . monocytogenes even in the absence of TLR signaling via MyD88 . Thus, TLR2-, TLR4- and MyD88-dependent signals are not essential for the generation of CD4+ Th1 and CD8+ T cells, and T cells can protect mice against infection in the absence of these signals. Front Biosci, 2004 May 01, 9, 1294 - 310 ENA/VASP proteins: multifunctional regulators of actin cytoskeleton dynamics; Sechi AS et al.; The spatial and temporal regulation of the actin cytoskeleton is fundamental to several cellular processes as diverse as cell motility and immune responses . At the molecular level, the remodelling of the actin cytoskeleton depends on two key events: actin filament nucleation and elongation . Seminal studies on the actin-based intracellular motility of the bacterial pathogen Listeria monocytogenes have been instrumental for the characterisation of a class of actin filament elongating factors, the proteins of the Ena/VASP family . Ena/VASP proteins enhance actin filament elongation via the recruitment of profilin:actin complexes to sites of active actin remodelling such as the tips of spreading lamellipodia and the surface of intracellular Listeria . Moreover, Ena/VASP proteins not only enhance actin filament elongation but also influence the activity of the Arp2/3 complex and counteract the inhibition of actin polymerisation by capping proteins . These findings, taken together with the observation that Ena/VASP proteins can influence actin filament architecture by affecting the actin filament branching activity of the Arp2/3 complex, define Ena/VASP proteins as multifunctional organisers of the actin cytoskeleton. Rev Argent Microbiol, 2003 Oct-Dec, 35(4), 224 - 7 {Monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for Listeria monocytogenes control}; Mengoni GB et al.; The monitoring of a HACCP (Hazard Analysis Critical Control Point) plan for the Listeria monocytogenes control in the cooked and frozen meat section of a thermo-processing meat plant was evaluated . Seventy "non-product-contact" surface samples and fourteen finished product samples were examined . Thirty eight positive sites for the presence of Listeria sp . were obtained . Twenty-two isolates were identified as L . monocytogenes, two as L . seeligeri and fourteen as L . innocua . Non isolates were obtained from finished product samples . The detection of L . monocytogenes in cooked and frozen meat section environment showed the need for the HACCP plan to eliminate or prevent product contamination in the post-thermal step. An Med Interna, 2004 Feb, 21(2), 75 - 8 {Listeriosis in the adult . Revision of 10 cases}; Arias Miranda IM et al.; Listeria monocytogenes is still a very rare opportunist infection in immunosuppressive patients . The clinical-epidemiological and therapeutic characteristics in 10 patients with infection produced by LM are reported--four of them had primary bacteriemia, three patients had a meningeal involvement, there were two patients with spontaneous bacterial peritonitis and one suffered from abdominal access . All of the patients had underlying disorders favouring the infection . Sepsis and meningeal syndrome were the most common presenting forms . Ampicillin was the most used antibiotic . The overall mortality was 40%. Mol Cell Biochem, 2004 Jan, 255(1-2), 257 - 65 Metal composition and solubility determine lung toxicity induced by residual oil fly ash collected from different sites within a power plant; Antonini JM et al.; Residual oil fly ash (ROFA) is a particulate pollutant comprised of soluble and insoluble metals and is produced by the combustion of fossil fuels . The objective was to examine the pulmonary responses to chemically distinct ROFA samples collected from either a precipitator or air heater within the same power plant . The collected ROFA samples were suspended in saline (total sample), incubated for 24 h at 37 degrees C, centrifuged, separated into soluble and insoluble fractions, and the metal composition was determined . In addition, electron spin resonance (ESR) was used to detect short-lived free radical intermediates produced by the ROFA samples and the different fractions . On day 0, Male Sprague-Dawley rats were intratracheally instilled with saline (vehicle control) or the ROFA samples (1 mg/100 g body wt) . At day 1, bronchoalveolar lavage was performed, and lung inflammation was assessed . On day 3, additional rats that had been treated with ROFA were intratracheally inoculated with 5 x 10(5) Listeria monocytogenes, and pulmonary bacterial clearance was measured at days 6, 8, and 10 . The precipitator ROFA was found to be more soluble and acidic with a significantly greater mass of each metal compared with the air heater ROFA . A prominent hydroxyl radical signal was measured for the total and soluble precipitator ROFA after the addition of H2O2, whereas the air heater ROFA and its fractions did not produce a signal . Precipitator ROFA induced a greater inflammatory response than air heater ROFA illustrated by a significant elevation in lung neutrophils . In addition, pulmonary clearance of L . monocytogenes was greatly diminished in the rats treated with the soluble and total precipitator ROFA samples . None of the air heater ROFA samples had an effect on lung bacterial clearance . In conclusion, precipitator ROFA, particularly the soluble fraction, generated a metal-dependent hydroxyl radical as measured by ESR and was shown to cause more inflammation and result in reduced lung defense against infection compared with air heater ROFA . These results are most likely due to differences in metal composition and solubility of the ROFA samples. Ann Ig, 2003 Sep-Oct, 15(5), 575 - 81 {Lysteria monocytogenes in samples of swines sent to the human alimentary chain}; Adorisio E et al.; Animal food-stuffs are known to be potential vehicles of Listeria monocytogenes . The contamination can be caused from processing or enviromental sources and from infected animals . This hypothesis has been checked in the present work . The authors found that 13.2% of 189 swines were carriers of Listeria monocytogenes, the microrganism was isolated from salivary glands, mesenterial gangles and tonsils . The authors suggest some preventive intervention to reduce both the environmental circulation of Listeria monocytogenes and the human risk of infection. J Food Prot, 2004 Feb, 67(2), 383 - 6 Thermal inactivation of Listeria innocua in salmon (Oncorhynchus keta) caviar using conventional glass and novel aluminum thermal-death-time tubes; Al-Holy M et al.; Differences in the come-up times and thermal inactivation parameters of Listeria innocua in salmon (Oncorhynchus keta) caviar containing 2.5% salt using conventional thermal-death-time (TDT) glass tubes and a novel aluminum tube were tested and compared . Generally, the come-up times and decimal reduction times (D-values) were shorter and the change in temperature required to change the D-value (z-value) was longer in the aluminum than in the glass tubes . The D-values at 60, 63, and 65 degrees C for the aluminum TDT tubes were 2.97, 0.77, and 0.40 min, respectively, and for the glass TDT tubes, these values were 3.55, 0.84, and 0.41 min . The z-values were 5.7 degrees C in the aluminum and 5.3 degrees C in the glass . Because of the shorter come-up time, the aluminum TDT tubes may provide a more precise measurement of microbial thermal inactivation than the glass TDT tubes, particularly for viscous materials, solid foods, and foods containing particulate matter. J Food Prot, 2004 Feb, 67(2), 378 - 82 Survival of Listeria monocytogenes in vanilla-flavored soy and dairy products stored at 8 degrees C; Tipparaju S et al.; The survival of Listeria monocytogenes V37 in vanilla-flavored yogurt (low-fat and nonfat) and soy milk (low-fat and Plus) stored at 8 degrees C for 31 days was investigated . Commercial samples of yogurt and soy milk were used . These samples were inoculated with either 10(4) or 10(7) CFU of L . monocytogenes per ml . Sampling was carried out every 3 to 4 days initially and was then carried out weekly, for a total storage time of 31 days . Each time a sample was collected, the pH of the sample was measured . After 31 days, low-fat plain, low-fat vanilla, and nonfat plain yogurt samples inoculated with 10(4) CFU/ml showed 2.5-log reductions in viable cell populations, and nonfat vanilla yogurt showed a 3.5-log reduction . For yogurt inoculated with 10(7) CFU/ml, reductions of 2.5 log CFU/ml were observed for plain low-fat and nonfat yogurts, and reductions of 5 log CFU/ml were observed for vanilla-flavored low-fat and nonfat yogurts . In vanilla-flavored and plain low-fat and Plus soy milk samples, cell counts increased from 10(4) and 10(7) CFU/ml to 10(9) CFU/ml at 7 and 3 days of storage, respectively, at 8 degrees C . Coagulation in soy milk samples was observed when the cell population reached 10(9) CFU/ml . In soy milk, the L . monocytogenes population did not change for up to 31 days . Vanillin had an inhibitory effect on L . monocytogenes in yogurt but not in soy milk. J Food Prot, 2004 Feb, 67(2), 328 - 41 Tracking of Listeria monocytogenes in smoked fish processing plants; Thimothe J et al.; Four smoked fish processing plants were used as a model system to characterize Listeria monocytogenes contamination patterns in ready-to-eat food production environments . Each of the four plants was sampled monthly for approximately 1 year . At each sampling, four to six raw fish and four to six finished product samples were collected from corresponding lots . In addition, 12 to 14 environmental sponge samples were collected several hours after the start of production at sites selected as being likely contamination sources . A total of 234 raw fish, 233 finished products, and 553 environmental samples were tested . Presumptive Listeria spp . were isolated from 16.7% of the raw fish samples, 9.0% of the finished product samples, and 27.3% of the environmental samples . L . monocytogenes was isolated from 3.8% of the raw fish samples (0 to 10%, depending on the plant), 1.3% of the finished product samples (0 to 3.3%), and 12.8% of the environmental samples (0 to 29.8%) . Among the environmental samples, L . monocytogenes was found in 23.7% of the samples taken from drains, 4.8% of the samples taken from food contact surfaces, 10.4% of the samples taken from employee contact surfaces (aprons, hands, and door handles), and 12.3% of the samples taken from other nonfood contact surfaces . Listeria spp . were isolated from environmental samples in each of the four plants, whereas L . monocytogenes was not found in any of the environmental samples from one plant . Overall, the L . monocytogenes prevalence in the plant environment showed a statistically significant (P < 0.0001) positive relationship with the prevalence of this organism in finished product samples . Automated EcoRI ribotyping differentiated 15 ribotypes among the 83 L . monocytogenes isolates . For each of the three plants with L . monocytogenes-positive environmental samples, one or two ribotypes seemed to persist in the plant environment during the study period . In one plant, a specific L . monocytogenes ribotype represented 44% of the L . monocytogenes-positive environmental samples and was also responsible for one of the two finished product positives found in this plant . In another plant, a specific L . monocytogenes ribotype persisted in the raw fish handling area . However, this ribotype was never isolated from the finished product area in this plant, indicating that this operation has achieved effective separation of raw and finished product areas . Molecular subtyping methods can help identify plant-specific L . monocytogenes contamination routes and thus provide the knowledge needed to implement improved L . monocytogenes control strategies. J Food Prot, 2004 Feb, 67(2), 316 - 21 Changes in heat resistance resulting from pH and nutritional shifts of acid-adapted and non-acid-adapted Listeria monocytogenes Scott A; Bayles DO; Stationary-phase Listeria monocytogenes cells that were either pH dependent acid adapted or not acid adapted were heat challenged at 60 degrees C in a two-level full factorial design for three variables . The three variables and the levels consisted of tryptic soy broth (TSB) and sterile cell-free culture supernatant (sterile TSB), the presence and absence of 1% added glucose, and pH 4.8 and pH 7 . Non-acid-adapted cells were most heat resistant when challenged in TSB (mean decimal reduction times at 60 degrees C: D60 = 1.16 min) . In the absence of added glucose, non-acid-adapted cells had similar D60-values for inactivations at pH 4.8 and pH 7; however, the presence of glucose caused non-acid-adapted cells challenged at pH 4.8 to be more heat sensitive (D60 = 0.65 min) than those inactivated at pH 7 (D60 = 1.03 min), indicating an interaction between glucose and pH . Overall, the significantly decreased heat resistance of the acid-adapted cells was due to the presence of glucose (D60 = 0.78 min without glucose, D60 = 0.59 min with glucose) . Acid-adapted cells heat challenged in TSB had similar D60-values for inactivations at pH 4.8 and pH 7; however, acid-adapted cells in sterile TSB challenged at pH 4.8 (D60 = 0.52 min) had significantly lower heat resistance than did cells challenged at pH 7 (D60 = 0.76 min), indicating an interaction between the medium and pH . The L . monocytogenes survivor data were modeled to extract information on the frequency distribution of heat resistance within heat-challenged populations, and the frequency distribution characteristics of mean, mode, and variance were compared among treatment conditions . Significant differences in the frequency distribution data were compared with the D60-values . These data indicated that the presence and level of cross-protection is highly dependent on the physiological state of the cells and nutrient availability at the time of heat challenge . Such conditions should be considered to ensure that stressed pathogens in foods are destroyed or inactivated. Int J Food Microbiol, 2004 Feb 15, 91(1), 63 - 72 Influence of the sigB gene on the cold stress survival and subsequent recovery of two Listeria monocytogenes serotypes; Moorhead SM et al.; The influence of serotype and the role of the sigB gene of Listeria monocytogenes during the survival and recovery on different substrates were determined . Wild-type and sigB mutants of two serotypes of L . monocytogenes were inoculated into buffer and onto beef steaks, and incubated at 4 degrees C for 8 weeks during which samples were removed and Listeria numbers determined . Growth kinetics of stationary phase wild-type and sigB mutant cells were compared, without prechilling and after prechilling at 4 degrees C . The two serotypes had similar survival capabilities under the conditions examined, and the sigB gene was influential in survival of chill stress, but was dependent upon additional nutritional factors . Prechilling cells prior to growth extended the lag phase of both strains, and this lag phase extension was compounded by the absence of a functional sigB gene . In conclusion, the sigB gene is involved in the survival and recovery from chill stress by the two serotypes tested . Additional factors such as previous growth conditions, nutritional requirements and serotype susceptibility are also contributory . This study adds relevant information regarding the influence of the sigB gene, in conjunction with the historical growth conditions and serotype differences . Understanding the significance of these factors may be useful in creating improved recovery systems for the detection of L . monocytogenes from at-risk foods. Int J Food Microbiol, 2004 Feb 15, 91(1), 13 - 8 Effects of chlorine and pH on efficacy of electrolyzed water for inactivating Escherichia coli O157:H7 and Listeria monocytogenes; Park H et al.; The effects of chlorine and pH on the bactericidal activity of electrolyzed (EO) water were examined against Escherichia coli O157:H7 and Listeria monocytogenes . The residual chlorine concentration of EO water ranged from 0.1 to 5.0 mg/l, and the pH effect was examined at pH 3.0, 5.0, and 7.0 . The bactericidal activity of EO water increased with residual chlorine concentration for both pathogens, and complete inactivation was achieved at residual chlorine levels equal to or higher than 1.0 mg/l . The results showed that both pathogens are very sensitive to chlorine, and residual chlorine level of EO water should be maintained at 1.0 mg/l or higher for practical applications . For each residual chlorine level, bactericidal activity of EO water increased with decreasing pH for both pathogens . However, with sufficient residual chlorine (greater than 2 mg/l), EO water can be applied in a pH range between 2.6 (original pH of EO water) and 7.0 while still achieving complete inactivation of E . coli O157:H7 and L . monocytogenes. Biochemistry, 2004 Feb 24, 43(7), 1939 - 49 A ratiometric expressible FRET sensor for phosphoinositides displays a signal change in highly dynamic membrane structures in fibroblasts; Cicchetti G et al.; Phosphoinositides are important signal transduction intermediates in cell growth, survival, and motility . We have invented a fluorescence sensor for polyphosphorylated phosphoinositides based on a peptide derived from the Listeria protein ActA that undergoes a random coil to helix transition upon lipid binding . The sensor, termed CAY, is a fusion protein of cyan and yellow fluorescent proteins flanking the peptide at its N- and C-termini, respectively . CAY displays fluorescence resonance energy transfer in vitro in the absence of phosphorylated phosphoinositides, and this energy transfer is lost upon interaction with these phospholipids . These results demonstrate that a short peptide undergoing a coil to helix transition can be sufficient for the engineering of a FRET-based biosensor . CAY is predominantly localized to the cytoplasm in fibroblasts expressing the sensor but shows loss of fluorescence resonance energy transfer in regions of active actin dynamics such as ruffles that have previously been demonstrated to contain high levels of phosphoinositides. Infez Med, 1997, 5(2), 118 - 24 {A case report of Listeria monocytogenes infection in a patient with AIDS . Efficacy of treatment with cotrimoxazole associated with rHuG-CSF (filgrastim)}; Manfredi R et al.; Listeriosis is an emerging opportunistic infection in the immunocompromised host . A case of sepsis due to Listeria monocytogenes in a patient with advanced HIV infection and severe neutropenia, treated for an underlying non-Hodgkin's lymphoma, is described . Therapy with cotrimoxazole associated with rHuG-CSF (filgrastim) led to a rapidly favourable clinical and microbiological outcome, and to the correction of concurrent neutropenia . The case report is discussed according to a literature review of all cases of listeriosis reported until now in the setting of HIV infection and AIDS . In particular, the role of both cotrimoxazole and rHuG-CSF adjunct in the treatment of listeriosis in the immunocompromised patient is focused. Lett Appl Microbiol, 2004, 38(3), 181 - 4 A comparison of the traditional method of counting viable cells and a quick microplate method for monitoring the growth characteristics of Listeria monocytogenes; Horakova K et al.; AIMS: To determine: (i) the growth parameters (specific growth rate, lag time, asymptotic amount of growth, generation time and time for maximum growth rate) of Listeria monocytogenes in different broths by standard cultivation methods and (ii) whether a microplate method in conjunction with a standard nondedicated plate reader could be adapted to routine assay . METHODS AND RESULTS: Growth curves were determined from cell numbers in a standard tube method at 2 h intervals by serial dilution and plating, and in a microplate method by absorbance measurements . Growth curves were fitted with a modified Gompertz function . CONCLUSIONS: The microplate method was similar to the standard cultivation methods in accuracy, required less chemical reagents, and considerably reduced the time required for analyses . This work also illustrates that growth characteristics of bacteria are not necessarily constant, and depend on the methodology used . SIGNIFICANCE AND IMPACT OF THE STUDY: It is not the intended purpose of this paper to present all the data for the media tested but instead to illustrate the success of the microplate method for studying growth kinetics compared to a standard cultivation method and system precision . The method will be of considerable benefit to laboratories unable to afford dedicated workstations. Microbiology, 2004 Feb, 150(Pt 2), 335 - 40 Physiological implications of class IIa bacteriocin resistance in Listeria monocytogenes strains; Vadyvaloo V et al.; High-level resistance to class IIa bacteriocins has been directly associated with the absent EIIAB(Man) (MptA) subunit of the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (EIIt(MAN)) in Listeria monocytogenes strains . Class IIa bacteriocin-resistant strains used in this study were a spontaneous resistant, L . monocytogenes B73-MR1, and a defined mutant, L . monocytogenes EGDe-mptA . Both strains were previously reported to have the EIIAB(Man) PTS component missing . This study shows that these class IIa bacteriocin-resistant strains have significantly decreased specific growth and glucose consumption rates, but they also have a significantly higher growth yield than their corresponding wild-type strains, L . monocytogenes B73 and L . monocytogenes EGDe, respectively . In the presence of glucose, the strains showed a shift from a predominantly lactic-acid to a mixed-acid fermentation . It is here proposed that elimination of the EIIAB(Man) in the resistant strains has caused a reduced glucose consumption rate and a reduced specific growth rate . The lower glucose consumption rate can be correlated to a shift in metabolism to a more efficient pathway with respect to ATP production per glucose, leading to a higher biomass yield . Thus, the cost involved in obtaining bacteriocin resistance, i.e . losing substrate transport capacity leading to a lower growth rate, is compensated for by a higher biomass yield. Microbiology, 2004 Feb, 150(Pt 2), 321 - 33 Deregulation of Listeria monocytogenes virulence gene expression by two distinct and semi-independent pathways; Milenbachs Lukowiak A et al.; Expression of the major virulence cluster in Listeria monocytogenes is positively regulated by the transcription factor PrfA and is influenced by several environmental factors, including the presence of readily metabolized carbohydrates such as cellobiose and glucose . Although little is understood about the mechanisms through which environmental factors influence expression of the PrfA regulon, evidence for structural and functional similarities of PrfA to the CRP-FNR family of regulatory proteins suggests the possibility that PrfA activity could be modulated by a small molecule ligand . The identity of components of the PrfA-associated regulatory pathway was sought through the isolation of mutants that exhibit high levels of PrfA-controlled gene expression in the presence of cellobiose or glucose . Here are described the properties and preliminary genetic analysis in two different genetic loci, gcr and csr, both unlinked by general transduction to the major virulence cluster . A mutation in gcr deregulates the expression of PrfA-controlled genes in the presence of several repressing sugars and other environmental conditions, a phenotype similar to that of a G145S substitution in PrfA itself . A mutation in the csr locus, within csrA, results in a cellobiose-specific defect in virulence gene regulation . Gene products encoded by the csr locus