Listeriosis

Infect Immun, 1998 May, 66(5), 2368 - 73
Roles for tumor necrosis factor and gamma interferon in resistance to enteric listeriosis; Beretich GR Jr et al.; Listeria monocytogenes normally infects the host by translocating from the intestinal lumen . Experiments were carried out to determine if, when, and where tumor necrosis factor (TNF) and gamma interferon (IFN-gamma) function in antibacterial resistance during enteric listeriosis . Groups of normal mice and severe combined immunodeficient (SCID) mice were injected with neutralizing monoclonal antibodies (MAb) specific for each cytokine and then inoculated intragastrically with L . monocytogenes . The course of infection was monitored by enumerating listeriae in gut-associated lymphoid tissues, livers, and spleens . By the third day of infection, bacterial numbers in infected tissues and organs were greatly exacerbated in all mice treated with anti-TNF MAb, whereas bacterial numbers in the organs of mice treated with anti-IFN-gamma MAb did not differ from those present in the respective organs of control mice . However, by the fifth day of infection, bacterial numbers in the organs of anti-IFN-gamma MAb-treated normal mice and SCID mice were much greater than in the corresponding organs of control mice . Experiments with Listeria-immune mice revealed that TNF and IFN-gamma are involved in the expression of anti-Listeria memory immunity; however, it was also found that the anti-IFN-gamma MAb was relatively ineffective in inhibiting the expression of anti-Listeria immunity, whereas a polyclonal anti-IFN-gamma was quite effective.

J Clin Microbiol, 1998 May, 36(5), 1318 - 23
Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages; Gerner-Smidt P et al.; Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared . The two systems use different approaches to compare patterns from different gels . In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern . In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel . The molecular size estimates obtained with the two systems for 12 restriction fragments of phage lambda were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments . At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of HhaI-restricted DNA of Listeria monocytogenes . Both systems require the user to make critical decisions in the analysis . It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing . It is also important to verify results obtained in the pattern matching or clustering portions of the analysis.

J Immunol, 1998 May 1, 160(9), 4441 - 8
MHC class I/peptide stability: implications for immunodominance, in vitro proliferation, and diversity of responding CTL; Busch DH et al.; Infection of BALB/c mice with Listeria monocytogenes primes CD8+ cytotoxic T cells specific for four different H2-Kd-restricted peptides . In vitro restimulation of L . monocytogenes immune splenocytes with each of these peptides resulted in larger T cell responses to p60 217-225 and mpl 84-92 than to LLO 91-99 and p60 449-457 . Direct frequency analyses of immune splenocytes, however, revealed that LLO 91-99 and p60 217-225 elicit dominant T cell responses, while p60 449-457 and mpl 84-92 elicit minor, subdominant responses . Restimulation of immune splenocytes with a range of peptide concentrations revealed that T cells with dominant specificities respond optimally to low peptide concentrations, while T cells specific for subdominant epitopes expand maximally to high peptide concentrations . This disparity correlates with the stability of H2-Kd/epitope complexes: the two dominant epitopes form stable complexes, while the subdominant epitopes form less stable complexes with H2-Kd . Interestingly, T cells specific for LLO 91-99 and p60 217-225 express more complex TCR-Vbeta repertoires than p60 449-457- and mpl 84-92-specific T cells . Thus, in our system, dominant T cell responses have relatively diverse TCR repertoires and are specific for peptides that form stable complexes with MHC class I molecules . Determining the precise roles of epitope/MHC class I stability and TCR repertoire in the generation of dominant T cell responses will require further investigation.

Infect Immun, 1998 May, 66(5), 2284 - 9
Disruption of the cellular inflammatory response to Listeria monocytogenes infection in mice with disruptions in targeted genes; DiTirro J et al.; The results of this study to dissect the nature of the acquired immune response to infection with Listeria monocytogenes in mice with targetted gene disruptions show that successful resolution of disease requires the essential presence of alphabeta T cells and the capacity to elaborate gamma interferon . In the absence of either of these entities, mice experience increasingly severe hepatitis and tissue necrosis and die within a few days . The data from this study support the hypothesis that the protective process is the efficient replacement of neutrophils in lesions by longer-lived mononuclear phagocytes; alphabeta-T-cell-knockout mice died from progressive infection before neutrophil replacement could occur, whereas in gammadelta-T-cell-knockout mice this replacement process in the liver has previously been shown to be much slower . In the present study we attribute this delay to reduced production of the macrophage-attracting chemokine MCP-1 in the gammadelta-T-cell-knockout animals . These data further support the hypothesis that gammadelta T cells are important in controlling the inflammatory process rather than being essential to the expression of protection.

Infect Immun, 1998 May, 66(5), 2200 - 6
Production of proinflammatory cytokines and inflammatory mediators in human intestinal epithelial cells after invasion by Trichinella spiralis; Li CK et al.; Epithelial cells are the first point of host contact for invasive intestinal pathogens and may initiate mucosal inflammatory responses via production of proinflammatory cytokines and mediators . The aim of the present study was to investigate in vitro the initial invasion of a parasitic nematode (Trichinella spiralis), to measure the early production of specific epithelial cytokines and inflammatory mediators after invasion, and to compare these responses with those to invasive bacteria . Monolayers of human colonic epithelial cell lines (HT29, T84, and Caco-2) were infected by T . spiralis or Listeria monocytogenes . Bile-activated infective larvae of T . spiralis invaded and migrated into the epithelial cell monolayers, leaving trails of dead cells . Transmission electron microscopy studies of damaged cells along the trail showed a progressive increase in size, disruption of cell membranes, loss or dilution of cytoplasmic proteins, and swelling of mitochondria and nuclei . However, no nuclear fragmentation was observed . With reverse transcription-PCR and an enzyme-linked oligonucleotide chemiluminescent assay, mRNA transcripts of interleukin-1beta (IL-1beta), IL-8, and epithelial neutrophil-activating peptide 78 were shown to increase in epithelial cells invaded by T . spiralis or L . monocytogenes, but only L . monocytogenes elicited increased inducible nitric oxide synthase (iNOS) mRNA . No increase in tumor necrosis factor alpha or transforming growth factor beta mRNA was seen after T . spiralis invasion . Increased levels of IL-8 were also released from the basolateral surfaces of infected monolayers as detected by sandwich enzyme-linked immunosorbent assay . Induction and secretion of proinflammatory cytokines in epithelial cells after nematode or bacterial invasion may initiate the acute inflammatory response of the small intestine . The upregulation of iNOS in bacterial infections may contribute to mucosal defense and may also be associated with subsequent cell death, whereas different mechanisms appear to operate after nematode invasion.

Microbiol Immunol, 1998, 42(2), 129 - 32
Sequence analysis of the actA gene of Listeria monocytogenes isolated from human; Moriishi K et al.; The region encoding proline-rich units of actA genes was amplified from 24 strains of Listeria monocytogenes using polymerase chain reaction (PCR) . PCR products of 13 strains showed the expected size of 623 bp, whereas those of 11 strains showed a short size of 518 bp . The shortening of these PCR products resulted from the deletion of one proline-rich unit . These results indicate that ActA proteins are divided into at least two different types which are unrelated to bacterial serotypes.

Anal Biochem, 1998 May 1, 258(2), 230 - 5
Evaluation of five imidazopyrazinone-type chemiluminescent superoxide probes and their application to the measurement of superoxide anion generated by Listeria monocytogenes; Shimomura O et al.; Superoxide-triggered chemiluminescence of five new imidazopyrazinone derivatives was investigated using the hypoxanthine-xanthine oxidase system as the source of superoxide anion . The results showed that they are highly sensitive and have favorable properties in measuring superoxide anion . With those new probes, the generation of superoxide anion from the bacteria Listeria monocytogenes was examined . The results confirmed the previous report that L . monocytogenes is an unusual organism that extracellularly and continuously generates a high level of superoxide anion in the presence of acetaldehyde . The data indicated that two of the probes, 3,7-dihydro-2-methyl-6-phenylethynylimidazo{1,2-a}pyrazin-3- one (4) and its methoxy derivative (5), are highly sensitive and useful in the measurements of superoxide anion and are clearly superior to 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazo{1,2-a}pyrazin-3-on e (MCLA), which-has been generally considered the most sensitive superoxide probe in the past . When tested at a probe concentration of 3.3 microM, the luminescence response and the signal-background ratio of compound 4 were 1.5 and 2.5 times those of MCLA, respectively, and the signal-background ratio of compound 5 was almost 15 times that of MCLA, though the luminescence response of this compound was slightly lower than that of MCLA . The low probe concentration used enhances the usefulness of probes in the measurements of superoxide in functioning biological systems.

Mol Microbiol, 1998 Mar, 27(6), 1235 - 45
The ClpC ATPase of Listeria monocytogenes is a general stress protein required for virulence and promoting early bacterial escape from the phagosome of macrophages; Rouquette C et al.; Under stress conditions, the facultative intracellular pathogen Listeria monocytogenes produces a ClpC ATPase, which is a general stress protein encoded by clpC and belonging to the HSP-100/Clp family . A ClpC-deficient mutant was obtained by gene disruption in strain LO28, which became highly susceptible to stress conditions in vitro . Intracellular growth of this mutant was restricted within macrophages, one of the major target cells of L . monocytogenes, during the infectious process . A quantitative electron microscope study showed that, contrary to wild-type bacteria that rapidly gain access to the cytoplasm of macrophages, mutant bacteria remained confined to membrane-bound phagosomes . Only a few mutant bacteria disrupted the phagosome membrane after 4h of incubation, then polymerized actin filaments and multiplied within the cytoplasm . The ClpC ATPase, therefore, promotes early bacterial escape from the phagosome of macrophages, thus enhancing intracellular survival . The ClpC ATPase was produced in vivo during experimental infection by wild-type bacteria . The virulence of the ClpC-deficient mutant was severely attenuated in mice, with a three-log decrease in its 50% lethal dose compared with wild-type bacteria . Bacterial growth of mutant bacteria was strongly restricted in organs, presumably because of an impairment of intracellular survival in host tissues . Our results provide evidence that a general stress protein is required for the virulence of L . monocytogenes, which behaves as a virulence factor promoting intracellular survival of this pathogen.

Microbiol Immunol, 1998, 42(3), 203 - 9
Protective effect of administration of skim milk on exogenous and endogenous infection in mice; Kobayashi T et al.; In order to minimize the denaturation of proteins in milk, normal cow's milk was pasteurized at 61 C for 20 min . The protective effects of the thus prepared skim milk (low-heat skim milk) on exogenous and endogenous infection were examined as compared with conventional skim milk which was pasteurized at 121 C for 2 sec . The antibody titers to Listeria monocytogenes and Escherichia coli of low-heat skim milk were almost equal to that of raw milk, while no antibody was detected in the conventional skim milk . When mice were given low-heat skim milk or conventional skim milk, the incidence of the translocation of orally inoculated Listeria monocytogenes to the spleen was lower in the low-heat skim milk group than that in the conventional skim milk group . The life span of 7 Gy X-ray irradiated mice given low-heat skim milk was significantly prolonged in comparison to that of mice given conventional skim milk . However, there were no differences in the number of bacteria in the feces or IgA production by Peyer's patch cells between the two groups . These results suggest that antibodies in low-heat skim milk, which still have reactivity to exogenous or indigenous bacteria, may contribute to the protective effects against bacterial infection.

Int J Syst Bacteriol, 1998 Jan, 48 Pt 1, 127 - 39
Comparison of PCR-based DNA fingerprinting techniques for the identification of Listeria species and their use for atypical Listeria isolates; Vaneechoutte M et al.; Four PCR-based DNA fingerprinting techniques were compared for their ability to identify at the species level a heterogeneous collection of isolates belonging to the six valid Listeria species . 16S rDNA-RFLP analysis identified all species and 16S rDNA-SSCP analysis identified almost all species . Also, isolates with unusual biochemical characteristics and/or unusual antigenic composition could be identified correctly . rRNA-intracistronic length polymorphism analysis suffered from high intraspecific variability, a limited number of fragments per profile, and small length differences between the spacers of different species . tRNA-intergenic length polymorphism analysis resulted in identification of all isolates but one, when fluorescent DNA capillary electrophoresis was used such that fragment length differences of 1 bp could be resolved . The four techniques yielded comparable results relevant to the taxonomy of Listeria . They all indicate a high degree of genetic relatedness between L . innocua and L . welshimeri, homogeneity of L . grayi, distinct but clear relatedness of L . grayi to the other Listeria species, a clear distinction between the two subspecies of L . ivanovii, and a clear distinction between Listeria isolates and isolates from closely related taxa or from species which are phenotypically difficult to distinguish from Listeria . New sequence determination of the 16S rRNA gene was necessary to obtain sequences in accordance with the findings of 16S rDNA-RFLP analysis.

Immunobiology, 1998 Feb, 198(4), 343 - 60
Activation and suppression of natural cellular immune functions by Pneumocystis carinii; Warschkau H et al.; The regulatory role of soluble cytokines in innate cellular immune responses induced by Pneumocystis carinii was assessed in vitro in direct comparison to induction by Listeria monocytogenes . This report shows that P . carinii organisms, as well as L . monocytogenes, stimulated in whole spleen cell cultures of SCID mice the release of IFN-gamma, TNF-alpha/beta, IL-10, IL-12, and iNO . This response was independent of functional T cells . Both macrophages (M phi) and natural killer (NK) cells were necessary for either microorganism to induce release of these cytokines . Cocultures of purified M phi--including alveolar M phi--and purified NK cells indicated that no other cell population was necessarily involved . Microbial induction of NK cell-derived IFN-gamma has been reported to be mediated by the combined effects of TNF-alpha and IL-12 released by M phi upon adequate microbial stimulation . Interestingly, only L . monocytogenes, but not P . carinii organisms could directly induce detectable amounts of TNF-alpha/beta, IL-12, or iNO in purified M phi cultures . In dose-response experiments, release of IFN-gamma, TNF-alpha/beta, and iNO was reduced at high relative concentrations of either microorganism . This high-dose suppression was at least partially controlled by M phi-produced IL-10 . Our data show that, P . carinii potently induces activating and inhibitory innate cellular immune response mechanisms and indicate that the initial step of macrophage-mediated NK cell activation might also involve other pathways than those described to date.

J Vet Med Sci, 1998 Mar, 60(3), 311 - 4
Prevalence of Listeria species in raw milk from farm bulk tanks in Nagano prefecture; Yoshida T et al.; Raw milk samples from bulk tanks of a total of 943 farms, which corresponded to approximately 60% of all dairy farms in Nagano Prefecture were examined for Listeria species between December 1990 and April 1991 . Listeria spp . were isolated from 29 (3.1%) of 943 milk specimens . In the southern, central, eastern and northern areas of the prefecture, Listeria spp . were isolated from 6.1% (22/362), 1.5% (4/272), 1.4% (2/143) and 0.6% (1/166) of samples, respectively . Listeria monocytogenes was isolated from three (0.3%) bulk tanks in the southern area: two of the strains isolated from two different farm bulk tanks were serovar 4b, and the other one was 1/2a . Besides, between February 1991 and January 1992, 504 samples of raw milk from farm bulk tanks were collected nine times from 56 farms in the southern area, where the prevalence of Listeria spp . was the highest, and examined for the seasonal variation in the presence of Listeria spp . The prevalence of Listeria spp . was higher in spring (14.3%) than in autumn (4.8%) . The 56 farms were divided into three groups according to the prevalence of Listeria spp., namely, three farms in Group 1 gave a high contamination rate (50% < or =), 14 farms in Group 2 a low contamination rate and the remaining 39 farms in Group 3 no recovering of Listeria spp . Sixteen strains of L . monocytogenes serovar 4b were isolated from four farms.

Jpn J Med Sci Biol, 1997 Apr, 50(2), 63 - 71
Discrimination of Listeria monocytogenes strains of serotype 4b by restriction enzyme analysis of chromosomal DNA; Saito A et al.; Epidemiologically related cheese and environmental strains and epidemiologically unrelated strains of Listeria monocytogenes serotype 4b were examined by restriction enzyme analysis of chromosomal DNA with a total of 10 restriction enzymes . The DNA fingerprint patterns generated from each restriction enzyme digest of total DNA of all strains were classified . The restriction enzyme patterns of seven strains recovered from cheese and environmental samples in the same plant were identical to each other, but differed from those of seven epidemiologically unrelated strains . Two, originating from sporadic human patients, of eight epidemiologically unrelated strains exhibited the identical restriction enzyme patterns . Excepting these two strains, restriction enzyme analysis of the chromosomal DNA of L . monocytogenes serotype 4b can discriminate serologically indistinguishable strains.

J Immunol, 1998 Apr 15, 160(8), 4018 - 25
Impaired macrophage function and enhanced T cell-dependent immune response in mice lacking CCR5, the mouse homologue of the major HIV-1 coreceptor; Zhou Y et al.; The CC-chemokine receptor CCR5 has been shown to be the major coreceptor for HIV-1 entry into cells, and humans with homozygous mutation in the ccr5 gene are highly resistant to HIV-1 infection, despite the existence of many other HIV-1 coreceptors . To investigate the physiologic function of CCR5 and to understand the cellular mechanisms of these clinical observations, we generated a CCR5-deficient mouse model (ccr5{-/-}) by targeted deletion of the ccr5 gene . We found that although developed normally in a pathogen-free environment, CCR5-deficient mice showed reduced efficiency in clearance of Listeria infection and exert a protective effect against LPS-induced endotoxemia, reflecting a partial defect in macrophage function . In addition, CCR5-deficient mice had an enhanced delayed-type hypersensitivity reaction and increased humoral responses to T cell-dependent antigenic challenge, indicating a novel role of CCR5 in down-modulating T cell-dependent immune response.

J Immunol, 1998 Apr 15, 160(8), 3971 - 7
Effect of antigen-processing efficiency on in vivo T cell response magnitudes; Vijh S et al.; T lymphocytes eradicate and provide long-term immunity to infections caused by intracellular pathogens . The mechanisms that determine in vivo T cell response sizes are poorly understood . Although it is speculated that the relative processing efficiency of different epitopes determines the hierarchy of T cell responses following immunization, this hypothesis has not been rigorously tested . We therefore mutagenized the secreted p60 Ag of Listeria monocytogenes to alter the efficiency of T cell epitope generation . Ag-processing efficiencies in cells infected with the different L . monocytogenes mutants ranged from one H2-Kd-associated p60 217-225 epitope generated per 15 intracellularly degraded p60 molecules (1/15) to one epitope per 350 degraded p60 molecules (1/350), i.e., a spectrum encompassing a 20-fold range of efficiencies . Mice infected with L . monocytogenes secreting inefficiently processed p60 (1/350) did not mount p60 217-225-specific T cell responses . However, increasing the efficiency of Ag processing by a factor of 5 to 1/70 restored the T cell response size to normal, while further increases in the efficiency of p60 217-225 generation to 1/50, 1/35, and 1/17 did not further augment specific T cell responses . Our studies demonstrate an Ag-processing threshold for in vivo T cell activation . Surprisingly, once this threshold is achieved, further enhancement of Ag-processing efficiency does not enhance the size of T cell responses.

Eur J Epidemiol, 1998 Feb, 14(2), 205 - 10
Genetic typing of human and food isolates of Listeria monocytogenes from episodes of listeriosis; Franciosa G et al.; Ten clinical and food Listeria monocytogenes strains isolated during the epidemiological investigations of episodes of listeriosis (one outbreak and two sporadic cases) that occurred in northern Italy during 1993-1995 have been examined by DNA macrorestriction pattern analysis obtained by PFGE and RAPD typing, in order to confirm the food vehicle of infections . The same DNA profiles within the isolates from the three episodes were obtained by both techniques . The Apal and Smal PFGE profiles and RAPD patterns with primer OPM-01 confirmed the close relationship between strains from two distinct episodes . However, RAPD analysis with primer UBC-127 distinguished between these L . monocytogenes isolates.

Int J Food Microbiol, 1998 Feb 17, 39(3), 231 - 6
Characterization of plasmids from Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses; Margolles A et al.; The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from short-ripened cheeses was analysed . The isolates of L . monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2 kbp), whereas the serogroup 4 isolates did not contain plasmids . One group of L . innocua strains harboured the plasmid pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp) . These plasmid groups were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of Listeria isolates . Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp . Plasmid pLI56 did not encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different characteristics to the other three plasmids . It was also the only one cured from the parent strain and the sole plasmid not digested by the restriction enzyme PstI . In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the possibility of a different origin for plasmid pLI56.

Int J Food Microbiol, 1998 Feb 17, 39(3), 167 - 73
Predictive modelling of inactivation of Listeria spp . in bovine milk during high-temperature short-time pasteurization; Piyasena P et al.; A linear model was derived to describe the thermal inactivation of Listeria innocua in bovine whole milk in a high-temperature short-time pilot scale pasteurizer . Integrated lethal effect, or pasteurization effect (PE), was obtained by converting times at different temperatures in the various sections of the pasteurizer to the equivalent time at the reference temperature (72 degrees C) . PE was then related by a simple linear function to the log10 of the % viable counts with a power transformation of the PE values to improve the linear fit . R2 values for the five L . innocua trials varied from 0.728 to 0.974 . Validation of this model with Listeria monocytogenes confirmed that L . monocytogenes was more heat sensitive . Inter-trial variation was incorporated into the model using the @RISK simulation software . Output from simulations confirmed that pasteurization at the IDF standard conditions of 72 degrees C for 15 sec can ensure at least an 11-log reduction of L . monocytogenes . The results showed that L . innocua may be used as a model microorganism to assess the thermal inactivation of L . monocytogenes, since its heat resistance is at least equal to or greater than that of the pathogenic species.

J Immunol, 1998 Jan 1, 160(1), 376 - 84
Chronic Listeria infection in SCID mice: requirements for the carrier state and the dual role of T cells in transferring protection or suppression; Bhardwaj V et al.; Listeriosis in mice with the SCID mutation results in a chronic infection . The chronic infection is characterized by abundant granulomas and neutrophil infiltrates . Both lesions were particularly noticeable in the liver . In the liver, about 95% are granulomas with 5% microabscesses involving intrahepatic infection . The majority of Listeria resided in membrane-bound vacuolar structures of the macrophages and not in the cytosol . Three manipulations resulted in alterations in the equilibrium between granulomas and liver microabscesses, with massive transfer of the infection to the hepatocyte and dissolution of the granulomas: depletion of neutrophils and neutralization of IFN-gamma and TNF-alpha . We did not find a role for IL-12, IL-10, or nitric oxide . Adoptive transfer studies showed a decisive role for both CD4+ and CD8+ T cells for an effective immune response, i.e., clearance of bacteria, granuloma formation with lymphocytes, and disappearance of microabscess . Clearance of Listeria was induced by transfer of CD8+ T cells from mice with targeted disruption of the IFN-gamma structural gene (IfgTM1KO), even in the presence of neutralizing mAb to IFN-gamma . In marked contrast, transfer of CD4+ T cells from IfgTM1KO mice exacerbated the infection in the chronically infected SCID mice, resulting in increased mortality with dissolution of the granulomas and severe hepatic infection with neutrophil infiltration . Thus, these data indicate that both IFN-gamma-dependent and -independent mechanisms are operative in the context of a chronic listerial infection.

J Immunol, 1998 Jan 15, 160(2), 898 - 905
Perforin-deficient CD8+ T cells provide immunity to Listeria monocytogenes by a mechanism that is independent of CD95 and IFN-gamma but requires TNF-alpha; White DW et al.; CD8+ T cells are effective mediators of immunity against Listeria monocytogenes, but the mechanisms by which they provide antilisterial immunity are poorly understood . CD8+ T cells efficiently lyse target cells in vitro by at least two independent pathways . To test the hypothesis that CD8+ T cell-mediated immunity to L . monocytogenes is dependent on perforin or CD95 (Fas, Apo-1), we used C57BI/6 (B6) and perforin-deficient (PO) mice to generate CD8+ T cell lines specific for the L . mono cytogenes-encoded Ag listeriolysin O (LLO) . Both lines specifically produce IFN-gamma and TNF-alpha, and mediate target cell lysis in vitro . Cytolysis mediated by the PO-derived CD8+ T cell line is delayed relative to the B6-derived line and is completely inhibited by anti-CD95 Abs . In vivo, PO-derived CD8+ T cells provide specific antilisterial immunity in B6 hosts, CD95-deficient hosts, and IFN-gamma-depleted hosts . However, PO-derived CD8+ T cells fail to provide antilisterial immunity in hosts depleted of TNF-alpha . These results indicate that single Ag-specific CD8+ T cells derived from PO mice can mediate antilisterial immunity by a mechanism that is independent of CD95 or IFN-gamma, but requires TNF-alpha.

Dtsch Med Wochenschr, 1998 Mar 20, 123(12), 347 - 52
{Polyposis of the gastrointestinal tract as a manifestation of diffuse follicular lymphatic hyperplasia}; Storr M et al.; HISTORY AND ADMISSION FINDINGS: A 21-year-old previously healthy Turkish man who had been living in Germany for 15 years was admitted because of worsening cramp-like abdominal pain with nausea, vomiting and watery diarrhoea . Palpation elicited diffuse muscular guarding over the entire abdomen and a mass of about 8 cm in the right lower abdomen . INVESTIGATIONS: Abnormal laboratory results were erythrocyte sedimentation rate (55 mm), C-reactive protein (6.2 mg/dl), total bilirubin (2.1 mg/dl), creatine kinase (137 U/l) and thymidine kinase (5.5 U/l) . There was a slight leucocytosis (13,700/microliter) and mild anaemia (haemoglobin 13.4 g/dl) with a normal differential count . Listeria ivanovii was repeatedly cultured from stool . Ultrasonography and computed tomography of the abdomen demonstrated a 6 cm mass in the right lower abdomen, splenomegaly (15.5 x 5 cm) and several lymphomas, up to 1.8 cm in diameter . Endoscopy revealed dense, in part grass-like, polyps, 3 to 6 mm deep, in the mucosa from the terminal ileum to the rectum, and to a lesser extent also in the duodenum . Histological examination of the polyps demonstrated diffuse follicular hyperplasia without evidence of malignancy . TREATMENT AND COURSE: On antibiotic treatment with ofloxacin (2 x 400 mg intravenously) the symptoms quickly regressed, but the endoscopic findings remained unchanged . CONCLUSION: Diffuse follicular lymphatic hyperplasia manifested itself in this patient as diffuse gastrointestinal polyposis . Listeria ivanovii cannot be ruled out as a causative factor.

J Immunol, 1997 Dec 15, 159(12), 5787 - 94
Evidence that the same gamma delta T cells respond during infection-induced and autoimmune inflammation; Mukasa A et al.; Inflammatory responses are induced in both testes of a mouse following injection of Listeria monocytogenes into one testis . Although the uninjected testis contains no detectable bacteria, it undergoes an autoimmune attack . Normally, the testis lacks lymphocytes, but in the infected and autoimmune state, both gamma delta and alpha beta T cells are found as infiltrates . Here, we have examined the repertoire of the infiltrating gamma delta T cells, using two different methods, and found a high frequency of V gamma 6/V delta 1 gamma delta T cells in both infected and autoimmune testes . All of these expressed the invariant V gamma 6/V delta 1 TCR previously reported . However, secondary gamma and delta transcripts present within V gamma 6/V delta 1 hybridomas indicated nonclonality . Interestingly, some of these secondary transcripts were derived from gamma gene rearrangements not previously found in this gamma delta T cell subset, implying a difference in its origin . The increase in V gamma 6/V delta 1 cells observed here in both infected and autoimmune testes, together with our previous finding of a preferential response by the same subset in Listeria-infected liver, indicates that their response is triggered by the inflammation rather than by the infectious agent or because they are already resident in the tissue . We and others have previously reported that the presence of gamma delta T cells during certain inflammatory conditions correlates with less host tissue damage . This result, together with the evidence presented here, further implies that a response by the V gamma 6/V delta 1 subset in some way exerts a controlling influence on the host inflammatory response.

Vet Microbiol, 1998 Jan 16, 59(2-3), 193 - 202
The effects of inoculation of Listeria monocytogenes into the ovine mammary gland; Tzora A et al.; In each of two experiments, the effects of inoculation of Listeria monocytogenes into the ovine mammary gland were studied . In the first experiment, ewes were challenged with one or other of five different Listeria spp . isolates to study differences in their pathogenicity . In the second, ewes were challenged with L . monocytogenes serotype 1/2a to study the sequential features of the infection . The reaction of the mammary glands was assessed by bacteriological, cytological and histological methods . No distinct variation in the pathogenicity of L . monocytogenes isolates was evident: all produced subclinical mastitis, independently of their origin or serotype; a L . innocua isolate caused only a transient increase of milk somatic cell counts . After challenge, L . monocytogenes was isolated for 88 days from the milk of inoculated glands, whose milk somatic cell counts were greater than 1.0 x 10(6) cells ml-1 . The organism was also isolated from the mammary lymph nodes, but not from any internal organ of any inoculated ewe . In early stages of the infection neutrophilic infiltration was the predominant histological feature, but hyperaemia, and degeneration of alveolar epithelial cells were also recorded . Later, chronic inflammatory features predominated, with lymphocytes as the principal cell types, destruction of alveoli and fibrous tissue proliferation . In the final stage of the experiment, fibrosis was the salient finding . It is concluded that L . monocytogenes can cause subclinical mastitis after intramammary inoculation into ewes.

FEMS Immunol Med Microbiol, 1998 Feb, 20(2), 159 - 64
Neuropeptides in the livers of mice during bacterial infections; Nakane A et al.; Neuropeptides such as substance P (SP) and vasoactive intestinal peptide (VIP) are known to act as immunomodulators . We investigated the induction of SP and VIP in the livers of mice infected with Listeria monocytogenes or injected with Tsukamurella paurometabolum . VIP was detected in the livers of mice after L . monocytogenes infection by an immunohistochemical technique and preproVIP mRNA, which was detected by reverse transcription-polymerase chain reaction (PCR), was induced post infection . However, no SP was detected . In contrast, SP, but not VIP was detected within granulomas in the livers of T . paurometabolum-injected mice, suggesting VIP and SP might be selectively induced in the liver by different bacterial infections.

Immunology, 1998 Jan, 93(1), 73 - 9
Effect of granulocyte-macrophage colony-stimulating factor on the number of leucocytes and course of Listeria monocytogenes infection in naive and leucocytopenic mice; Buisman AM et al.; This study concerns the effect of recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) on the number of circulating leucocytes, activation of peritoneal macrophages and proliferation of Listeria monocytogenes in various organs of naive and leucocytopenic mice . Mice were rendered leucocytopenic by sublethal total body irradiation or cyclophosphamide treatment . GM-CSF treatment enhanced the number of granulocytes and monocytes in peripheral blood during L . monocytogenes infection in naive mice, but not in irradiated or cyclophosphamide-treated mice . In naive mice, irradiated and cyclophosphamide-treated mice, GM-CSF did not affect the course of L . monocytogenes infection in thigh muscle, spleen and liver . However, GM-CSF treatment significantly increased the number of macrophages in the peritoneal cavity of naive mice during infection; these macrophages were more enlarged and showed a higher frequency of binucleated and multinucleated cells relative to non-GM-CSF-treated mice . Together, these results demonstrated that GM-CSF increased the number of circulating granulocytes and monocytes, and the number of peritoneal macrophages during infection with L . monocytogenes in naive mice, but did not affect the course of the infection in thigh muscle, spleen or liver of these mice . In leucocytopenic mice, however, GM-CSF did not affect the number of circulating phagocytes, which explains that this factor had no effect on the proliferation of the bacteria in the various organs.

Mol Microbiol, 1998 Mar, 27(5), 1077 - 87
The InIB protein of Listeria monocytogenes is sufficient to promote entry into mammalian cells; Braun L et al.; InIB is one of the two Listeria monocytogenes invasion proteins required for bacterial entry into mammalian cells . Entry into human epithelial cells such as Caco-2 requires InIA, whereas InIB is needed for entry into cultured hepatocytes and some epithelial or fibroblast cell lines such as Vero, HEp-2 and HeLa cells . InIB-mediated entry requires tyrosine phosphorylation, cytoskeletal rearrangements and activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor . In this study, we demonstrate for the first time that InIB is sufficient to promote internalization . Indeed, coating of normally non-invasive bacteria or inert latex beads with InIB leads to internalization into mammalian cells . In addition, a soluble form of InIB also appears to promote uptake of non-invasive bacteria, albeit at a very low level . Similar to entry of L . monocytogenes, uptake of InIB-coated beads required tyrosine phosphorylation in the host cell, PI 3-kinase activity and cytoskeletal reorganization . Taken together, these data indicate that InIB is sufficient for entry of L . monocytogenes into host cells and suggest that this protein is an effector of host cell signalling pathways.

Mol Microbiol, 1998 Mar, 27(5), 915 - 28
Differential interaction of the transcription factor PrfA and the PrfA-activating factor (Paf) of Listeria monocytogenes with target sequences; Dickneite C et al.; The interaction of the purified PrfA transcription factor with the regulatory sequences located upstream of the PrfA-dependent listeriolysin (hly) and internalin (inlA) genes was studied in the presence and in the absence of Paf (PrfA-activating factor)-containing extracts . It is shown that PrfA protein is able to bind, independently of additional factors, to a 109bp DNA fragment including the entire hly promoter sequence with the anticipated PrfA binding site ('PrfA-box') . PrfA alone, but not in combination with Paf, can also bind to a shorter target sequence of 28 bp comprising essentially the PrfA-box of the hly promoter . The addition of a Paf-containing extract does not lead to significant protein binding to these two hly target sequences in the absence of PrfA but converts the complex (CIII) consisting of PrfA and the 109 bp hly DNA fragment to a slower migrating PrfA-Paf-DNA complex (CI) . Incubation of cell-free extracts of wild-type Listeria monocytogenes with the 109 bp DNA fragment leads to the formation of CI . The addition of polyclonal PrfA antibodies causes a supershift of CIII . Purified PrfA and PrfA-Paf also bind to a DNA fragment containing the PrfA-dependent promoter P2 of inlA, albeit at a lower rate when compared with the corresponding hly sequence . In contrast to the hly target DNA, the inlA promoter sequence efficiently binds Paf alone, and this Paf binding reduces that of PrfA and PrfA-Paf to the inlA target DNA . DNase I footprint experiments show that purified PrfA protects sequences of dyad symmetry previously proposed as PrfA binding sites in the hly and in the inlA promoter regions.

Microbiology, 1998 Mar, 144 ( Pt 3), 807 - 14
Determination of a 15437 bp nucleotide sequence around the inhA gene of Mycobacterium avium and similarity analysis of the products of putative ORFs; Labo M et al.; A 15437 bp region encompassing the inhA locus from the Mycobacterium avium chromosome was cloned and sequenced . From the sequencing data generated and the results of homology searches, the primary structure of this region was determined . This region contains four known genes (acnA, fabG, inhA and hemH) and two genes, invA and invB, whose products display homology with p60 invasion protein of Listeria monocytogenes . Six proteins encoded by putative ORFs contained an RGD motif (often involved in binding to macrophage integrins), while ORF1 and MoxR are probably transcriptional regulators . The rest of the putative products encoded by ORFs in the sequenced region showed little homology with the proteins contained in the databases and were considered to be unknown proteins.

Int Immunol, 1998 Feb, 10(2), 117 - 30
The regulatory role of heat shock protein 70-reactive CD4+ T cells during rat listeriosis; Kimura Y et al.; Protection against infection with Listeria monocytogenes depends primarily on Listeria-specific T cells . We show here that CD4+ TCR alphabeta+ T cells are capable of recognizing the mycobacterial heat shock protein (HSP) 70, that appears in the peritoneal cavity of F344 rats infected i.p . with L . monocytogenes . The HSP70-reactive CD4+ T cells recognized a peptide comprising 234-252 residues as present in the 70 kDa HSP of Mycobacterium tuberculosis in the context of RT1.B MHC class II molecules . Analysis of TCR Vbeta gene expression with RT-PCR revealed that the HSP70-reactive CD4+ T cells predominantly used the Vbeta16 gene segment, whereas the heat-killed Listeria (HKL)-specific T cells expressed a diverse set of Vbeta gene segments . In contrast to the HKL-specific T cells producing IFN-gamma, the HSP70-reactive CD4+ T cells produced TGF-beta1 and IL-10 but neither Th1- or Th2-type cytokines . Adoptive transfer with HSP70-reactive T cells rendered rats susceptible to listerial infection . Collectively, these results proposed that the HSP70-reactive CD4+ T cells appearing during rat listeriosis may be involved in termination of Th1 cell-mediated excessive inflammation after the battle against L . monocytogenes has been won.

Vet Clin North Am Food Anim Pract, 1998 Mar, 14(1), 113 - 25
Listeriosis; Cooper J et al.; Listeria monocytogenes is ubiquitous in nature and is part of the normal flora of the distal portion of the intestinal tract of numerous animal species . Listeriosis is an emerging food borne disease that is responsible for approximately 1,700 cases of human illness each year and 650 deaths . Listeria is the cause of three main disease entities in animals and humans: neural, visceral, and reproductive . Clinical signs associated with the three forms are discussed along with diagnosis, therapy, prevention, and control.

Immunity, 1998 Mar, 8(3), 353 - 62
Coordinate regulation of complex T cell populations responding to bacterial infection; Busch DH et al.; Bacterial infections activate complex T cell populations that differ in size and antigen specificity . We used tetramerized MHC class I molecules complexed with Listeria monocytogenes-derived epitopes to characterize four distinct CD8+ T lymphocyte populations during bacterial infection . Surprisingly, T cell populations differing in antigen specificity expand, contract, and enter the T cell memory compartment synchronously . Because the four L . monocytogenes epitopes are presented with different efficiencies and have distinct stabilities in infected cells, our findings suggest that these factors do not determine in vivo T cell dynamics . While T cell activation requires antigen presentation, the timing and extent of T cell expansion appear to be regulated in a coordinated fashion independent of antigen quantity and stability.

Microb Pathog, 1998 Jan, 24(1), 17 - 23
Mice lacking the murine interleukin-8 receptor homologue demonstrate paradoxical responses to acute and chronic experimental infection with Listeria monocytogenes; Czuprynski CJ et al.; In this study we demonstrate that mice which lack the murine interleukin-8 receptor homologue exhibit enhanced resistance during the early stage of infection (1-4 days after i.v . challenge with Listeria monocytogenes) . This result is surprising in that interleukin-8 and other CXC chemokines are key players in the accumulation of inflammatory neutrophils, which is thought to be critical for resistance to listeriosis . Paradoxically, some of the interleukin-8 receptor knockout mice that survived acute infection with L . monocytogenes demonstrated evidence of chronic infection with L . monocytogenes .

J Med Microbiol, 1998 Mar, 47(3), 211 - 5
Killing mechanism of Listeria monocytogenes in activated macrophages as determined by an improved assay system; Ohya S et al.; Exposure of Listeria monocytogenes to gentamicin 5 mg/L for 4 h resulted in the killing of most extracellular bacteria, but had no effect on the survival of bacteria inside macrophages . Higher concentrations of gentamicin caused a reduction in the number of intracellular bacteria . This effect was associated with cellular uptake of gentamicin, but was unaffected by activation of macrophages by interferon-gamma and lipopolysaccharide . In experiments in which exposure to gentamicin 5 mg/L for 4 h was used to kill extracellular bacteria, killing by activated macrophages was impaired when O2- production was inhibited by superoxide dismutase, but not when nitric oxide production was blocked by NG-monomethyl-L-arginine . These data suggest that the reactive oxygen intermediates are more important than nitric oxide in the killing of L . monocytogenes, at least in macrophages activated in vitro.

J Med Microbiol, 1997 Aug, 46(8), 681 - 92
Cell proliferation enhances entry of Listeria monocytogenes into intestinal epithelial cells by two proliferation-dependent entry pathways; Velge P et al.; Bacterial entry into intestinal host cells is the result of a fairly sophisticated manipulation of host cell machinery by the pathogens . To study further the potential cell target of Listeria spp., the in-vitro entry of L . monocytogenes strains into intestinal cells was examined in relation to the metabolism, proliferation and differentiation of the cells by the alamarBlue assay, {3H} thymidine incorporation, and brush border-associated enzyme activities, respectively . The study showed that cell metabolism was not involved in the entry of L . monocytogenes in three cell models (two human and one porcine) . On the other hand, entry was closely related to the proliferation process and poorly related to the differentiation state of the cells . The use of L . monocytogenes mutants lacking invasion proteins showed that InlA and InlB acted in synergy to mediate the entry of L . monocytogenes into proliferative cells, whereas InlA alone seemed to be involved in the entry into non-proliferative cells . These two entry pathways could correspond to the two cellular processes used by L . monocytogenes to enter proliferative and non-proliferative cells, as suggested by the use of cytochalasin D, nocodazole, chloroquine and monodansylcadaverine . Taken together, we propose a hypothesis in which the entry of L . monocytogenes is mediated by interaction between randomly distributed E-cadherin on the surface of proliferative cells . In contrast, the entry into non-proliferative cells may involve pp60c-src, a proto-oncogenic tyrosine kinase signal that modifies E-cadherin localisation . In conclusion, these results suggest that L . monocytogenes may preferentially enter crypt cells in vivo by a microfilament-dependent process, whereas the few bacteria that infect villus cells enter by an E-cadherin-internalin interaction that mediates microtubule-dependent endocytosis.

Microb Pathog, 1997 Nov, 23(5), 255 - 63
Penetration of Listeria monocytogenes in mice infected by the oral route; Marco AJ et al.; In this study, it is suggested that the Peyer's patches are the most important point of entry of Listeria monocytogenes in the host after subclinical infection by the oral route . Microbiological, histopathological and ultrastructural evidence of infection was obtained in mice inoculated with a sublethal dose of 10(9) cfu . No mortality was observed . L . monocytogenes was isolated from the mesenteric lymph nodes from 6 hours post infection (hpi) through day 7 p.i . and from the liver and spleen from 24 h p.i . until days 5 and 7 p.i . respectively . Lesions were mainly restricted to the dome area of Peyer's patches and consisted of a purulent to pyogranulomatous inflammatory reaction . Scarce and minor lesions were also observed in the mesenteric lymph nodes and liver . L . monocytogenes was detected by immunohistochemistry in the Peyer's patches from 12 h p . i . to day 6 p.i . Ultrastructural study of Peyer's patches showed that the majority of Listeria cells were free within the cytoplasm of neutrophils and macrophages, not surrounded by a phagosomal membrane, and some of them were dividing .

Int J Food Microbiol, 1997 Sep 16, 38(2-3), 217 - 27
Inhibition of Listeria monocytogenes on cold-smoked salmon by nisin and carbon dioxide atmosphere; Nilsson L et al.; The bacteriostatic and bacteriocidal effect of nisin in combination with carbon dioxide, NaCl and low temperature on the survival of Listeria monocytogenes was investigated in in vitro model studies and in trials with cold-smoked salmon . Addition of nisin caused various degrees of inhibition and sometimes death of L . monocytogenes in model experiments performed at 10 degrees C . The antilisterial effect of nisin was improved in the presence of 100% CO2 and increasing NaCl concentrations (0.5 to 5.0% w/v) . Minimal bactericidal concentrations (MBC) of nisin varied from 30 to more than 500 IU/ml . The most pronounced effect of nisin was found when 10(2) cfu/ml was grown in media with 5.0% NaCl and incubated in CO2 atmosphere (MBC = 30 IU/ml) . The bactericidal effect of nisin was reduced in air and vacuum, and did not increase systematically with increasing NaCl concentrations . In general, nisin concentration < or = 50 IU/ml resulted in the survival and growth of L . monocytogenes in all combinations with other preservatives (NaCl, CO2) . Addition of nisin (500 or 1000 IU/g) to cold-smoked salmon inoculated with L . monocytogenes and stored at 5 degrees C delayed, but did not prevent growth of L . monocytogenes in vacuum-packs . Numbers of L . monocytogenes increased to 10(8) cfu/g in vacuum packed cold-smoked salmon in 8 days, whereas CO2 packing of cold-smoked salmon resulted in an 8-day lag phase of L . monocytogenes, with numbers eventually reaching 10(6) cfu/g in 27 days . Addition of nisin to CO2 packed cold-smoked salmon resulted in a 1 to 2 log reduction of L . monocytogenes followed by a lag phase of 8 and 20 days in salmon with 500 and 1000 IU nisin/g, respectively . The levels of L . monocytogenes remained below 10(3) cfu/g during 27 days of storage at both concentrations of nisin.

Int J Food Microbiol, 1997 Sep 16, 38(2-3), 157 - 67
A model describing the relationship between lag time and mild temperature increase duration; Breand S et al.; When a bacterial population undergoes an unfavourable increase in temperature for a given duration, called stress duration, a death phase followed by a lag and a growth phase are observed . The lag phase is actually of great interest in regard to foodstuff safety in choosing a suitable protocol for the detection of microorganisms which have undergone a mild heat treatment . The extension of lag time with the severity of the increase in temperature has been highlighted by previous papers . Our experimental results concerning Listeria monocytogenes and Escherichia coli revealed that a two phase relationship between lag time and stress duration is observed for a specific increase in temperature . The first phase consists of an increase in lag time up to a peak; the second one consists of a decrease from this peak to a steady threshold . The mathematical model presented, describing the relationship between lag time and stress duration was empirically built from our experimental data concerning L . monocytogenes and E . coli . The fit evaluation carried out led us to consider this model as a good description of the relationship studied . The potential contribution of our model in heat treatment optimization is discussed.

Mol Cell Probes, 1997 Dec, 11(6), 459 - 62
Single-strand conformation polymorphism (SSCP) analysis of Listeria monocytogenes iap gene as tool to detect different serogroups; Manzano M et al.; PCR-single-strand conformation polymorphism (PCR-SSCP) analysis is a convenient technique for the detection of mutations . As the mobility of single-stranded DNA is sequence-dependent it could therefore be used to determine serotype-related sequence variations in Listeria monocytogenes . Sero-specific patterns were observed in different L . monocytogenes serogroups.

Mol Cell Probes, 1997 Dec, 11(6), 453 - 5
A PCR-microplate capture hybridization method to detect Listeria monocytogenes in blood; Cocolin L et al.; In order to improve the diagnosis of Listeria monocytogenes infection, we have developed a polymerase chain reaction (PCR)-based assay combined with microplate capture hybridization technique . The system is based on selective amplification of L . monocytogenes with two specific primers based on the iap gene . The amplicon produced, with digoxigenin 11-dUTP incorporated during PCR, is hybridized in streptavidin-coated microtitre plates prepared with biotinylated specific DNA probe . The method involved requires approximately 6-8 h, and its high sensitivity, rapidity and simplicity should make it valuable for diagnosis and for epidemiological studies of listeriosis.

Int J Food Microbiol, 1997 Aug 19, 38(1), 77 - 81
The identification of Listeria species; McLauchlin J; The purpose of this study was to compare methods for the identification of Listeria species . Three hundred and fifty cultures representing the six species of Listeria were tested using conventional sugar fermentation and haemolytic reactions, as well as the hydrolysis of the DL-alanine beta-naphthylamide (DLABN), and the API Listeria identification test kit . Using conventional tests, 99% of cultures were correctly identified: four L . monocytogenes were misidentified as L . innocua . The DLABN hydrolysis test distinguished L . monocytogenes from the remainder of the genus for 98% of the cultures: 6 out of 14 L . ivanovii isolates gave atypical results . There was correct identification for 97% of the cultures using the API Listeria test kit and no misidentifications were obtained: nine cultures (six L . monocytogenes and three L . innocua) gave equivocal profiles which were not ascribed to any species.

Int J Food Microbiol, 1997 Aug 19, 38(1), 71 - 6
Addition of 2.5% lactate and 0.25% acetate controls growth of Listeria monocytogenes in vacuum-packed, sensory-acceptable servelat sausage and cooked ham stored at 4 degrees C; Blom H et al.; A study of the inhibitory effects of propylparaben and of a combination of lactate and acetate against growth of Listeria monocytogenes in inoculated liquid medium, sliced servelat sausage and cooked ham, were performed using rifampicin resistant Listeria strains in inoculation experiments . A consumer acceptance test of products produced with and without the compounds was also performed . Propylparaben was found to be effective in a model liquid non-fat medium, but was without effect in the actual products . This illustrates the potential pitfalls in translating results from studies in liquid media to fat-containing food products . The combined inhibitory and sensory results showed that a mixture of 2.5% lactate and 0.25% acetate (w/w, calculated on the water phase), could be used to increase the margins of safety for sliced and spreadable vacuum-packed ready-to-eat cooked meat products stored for 4-6 weeks . In addition, strict control of temperature during production and storage is very important.

New Microbiol, 1998 Jan, 21(1), 87 - 92
Classification of Listeria monocytogenes by PCR-restriction enzyme analysis in the two genes of hlyA and iap; Saito A et al.; PCR-restriction enzyme analysis in the two virulence-associated genes was performed . The hlyA gene cording for listeriolysin O and the iap gene cording for an invasion associated factor were amplified with primers SH2 or SI3 . The PCR products obtained were cleaved with 32 restriction enzymes, and restriction profiles from 12 strains, 6 each of serotypes 1/2a and 4b, were compared . We obtained two profiles for the hlyA using 4 restriction enzymes and eight profiles for the iap by using AluI, and the results of the profiles did not correlate with the serotypes . The polymorphism in the iap region was of a higher degree than that in the hlyA region, and the PCR-restriction enzyme analysis of the iap gene with primers SI3 and AluI was confirmed as one of the useful epidemiological analysis methods for listerosis outbreaks.

Indian J Dent Res, 1993 Jul-Dec, 4(3-4), 103 - 11
A comparative, qualitative and quantitative antimicrobial efficacies of mouthrinses containing chlorhexidine gluconate and essential oils; Shah HM et al.; A project was launched to evaluate and compare antimicrobial efficacy of Hexidine and Listerine over placebo in 10 day human experimental gingivitis study . A rigid study schedule for subject compliance spanning well over 3 months was sorted out and volunteers recruited for study rinsed all the 3 assigned mouthrinses containing (a) chlorhexidine gluconate, (b) "essential oils" and (c) flavoured distilled water one after the other at certain prefixed intervals . After 10 days of assigned mouthrinse regimen, where the assigned mouthrinse was the only method practiced by the volunteers for oral hygiene, Supragingival Plaque was quantitatively and qualitatively assayed . Qualitatively supragingival plaque was assayed by Gram staining (Direct smear and Thioglycollate Broth) and growth characteristics i.e; Aerobic, Microaerophilic and Anaerobic growth was noted . Quantitatively plaque was assayed by calculating total microbial load per tooth by preparing Mafarland Nephelometer standard and Spectrophotometric analysis . It is concluded that Hexidine and Listerine exert similar antimicrobial efficacy in 10 days experimental gingivitis study . Hexidine and Listerine exert their antimicrobial influence through reduction of total Aerobes and Anaerobes reducing total microbial load per tooth by 58% and 53% respectively as compared to placebo.

FEMS Microbiol Lett, 1998 Mar 1, 160(1), 159 - 68
In situ detection of a virulence factor mRNA and 16S rRNA in Listeria monocytogenes; Wagner M et al.; Simultaneous in situ analysis of the structure and function of bacterial cells present within complex communities is a key for improving our understanding of microbial ecology . A protocol for the in situ identification of Listeria spp . using fluorescently tagged, rRNA-targeted oligonucleotide probes was developed . Ethanol fixation and enzymatic pretreatment with lysozyme and proteinase K were used to optimize whole cell hybridization of exponential phase and stationary phase Listeria spp . cells . In parallel, transcript probes carrying multiple digoxigenin molecules were combined with anti-digoxigenin Fab antibody fragments labeled with horseradish peroxidase to detect, via the catalytic deposition of fluorescein-tyramide, the iap-mRNA in single Listeria monocytogenes cells . The iap gene encodes the associated virulence factor p60 . Application of the new signal amplification technique resulted in strong signals comparable in intensity to those obtained with fluorescently labeled rRNA-targeted oligonucleotide probes.

FEMS Microbiol Lett, 1998 Mar 1, 160(1), 87 - 90
The microtubule depolymerizing drugs nocodazole and colchicine inhibit the uptake of Listeria monocytogenes by P388D1 macrophages; Kuhn M; Uptake of Listeria monocytogenes by different mammalian cells like macrophages and epithelial cells is dependent on functional actin filaments and hence susceptible to inhibition by cytochalasin . Here we show that phagocytic uptake of L . monocytogenes by P388D1 macrophages is also highly sensitive to treatment with the microtubule depolymerizing drugs nocodazole and colchicine . This sensitivity is cell type specific and much less pronounced in bone marrow-derived macrophages and Caco-2 epithelial cells . In contrast to nocodazole and colchicine, the microtubule stabilizing drug taxol has no significant effect on the uptake of L . monocytogenes by all three cell types tested.

Cell, 1998 Feb 20, 92(4), 535 - 45
Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity; Shen H et al.; Bacterial pathogens synthesize numerous proteins that are either secreted or localized within bacterial cells . To address the impact of antigen compartmentalization on T cell immunity, we constructed recombinant Listeria monocytogenes that express a model CD8T cell epitope as a secreted or nonsecreted fusion protein . Both forms of the antigen, either secreted into the host cell cytoplasm or retained within bacterial cells, efficiently prime CD8 T cell responses . However, epitope-specific CD8 T cells confer protection only against bacteria secreting the antigen but not against the bacteria expressing the nonsecreted form of the same antigen . This dichotomy as a result of antigen compartmentalization suggests that bacterial antigens are presented by multiple MHC class I pathways to prime CD8 T cells, but only the endogenous pathway provides target antigens for CD8 T cell-mediated protective immunity.

Mol Gen Genet, 1998 Jan, 257(2), 186 - 97
Sequence comparison of the chromosomal regions encompassing the internalin C genes (inlC) of Listeria monocytogenes and L . ivanovii; Engelbrecht F et al.; We have recently cloned and characterized the inlC gene of Listeria monocytogenes which belongs to the listerial internalin multigene family and codes for a 30-kDa secreted protein containing five consecutive leucine-rich repeats . Here, we show that in L . monocytogenes inlC is located between the rplS gene (encoding the 50S ribosomal protein L19), and the infC gene (encoding the translation initiation factor 3) . By direct and inverse polymerase chain reactions (PCR), we cloned a 5.4-kb region containing a homologous gene (termed i-inlC) from L . ivanovii, the other pathogenic member of the genus Listeria . In this microorganism, the i-inlC gene is preceded by another internalin gene, i-inlD, which seems to be specific for L . ivanovii, as this gene could not be detected in L . monocytogenes by Southern hybridization with an i-inlD gene probe . The i-inlD gene also encodes a small secretory internalin (i-InlD), which shares extended homology with (i-)InlC . Upstream of i-inlD are genes for 23S rRNA and 5S rRNA, and two tRNA genes {Asn-tDNA (GTT) and Thr-tDNA(GTT)} . The 3' terminus of the Thr-tRNA gene appears to be the site of an insertion of a genetic element including i-inlC and i-inlD . A putative transcriptional regulator gene, the product of which contains the TetR family signature, is located downstream of i-inlC . This chromosomal position of the two inlC genes on their respective chromosomes may be due to horizontal transfer of this gene . Transcription of i-inlC and i-inlD is strictly dependent on the transcriptional activator PrfA, which regulates transcription of most of the known virulence genes (including inlC) of L . monocytogenes and of L . ivanovii.

Lett Appl Microbiol, 1998 Jan, 26(1), 5 - 8
Fitness costs associated with class IIa bacteriocin resistance in Listeria monocytogenes B73; Dykes GA et al.; In order to assess the potential for the spread of class IIa bacteriocin resistance in natural populations of Listeria monocytogenes, the fitness costs associated with resistance to leucocins A, B and E and sakacin A in L . monocytogenes B73 in the absence of bacteriocin were examined . The resistant phenotype had a lower growth rate (and thus relative fitness) than the sensitive phenotype in monoculture experiments . Furthermore, resistant phenotypes were unable to invade populations of the sensitive strain, even at frequencies of 10(-1) or higher, when grown in co-culture . These results held true for resistant strains that had been exposed to bacteriocin for 25 successive growth cycles . It was concluded that the class IIa bacteriocin-resistant phenotype of L . monocytogenes B73 is unlikely to become stable in natural populations based on this evidence . Due to the possibility of variations in the frequencies of spontaneous mutation and fitness among Listeria strains, however, the extrapolation of these results to the species as a whole should not be made.

Infect Immun, 1998 Mar, 66(3), 1106 - 12
Listeria monocytogenes invasion of epithelial cells requires the MEK-1/ERK-2 mitogen-activated protein kinase pathway; Tang P et al.; PD98059, a specific inhibitor of MEK-1 mitogen-activated protein (MAP) kinase kinase, blocked Listeria monocytogenes invasion into HeLa epithelial cells . The effects of PD98059 were reversible, as adherent extracellular bacteria were internalized upon removal of the drug . Previously, we reported that L . monocytogenes could activate ERK-1 and ERK-2 MAP kinases through the action of listeriolysin O (LLO) on the host cell (P . Tang, I . Rosenshine, P . Cossart, and B . B . Finlay, Infect . Immun . 64:2359-2361, 1996) . We have now found that two other MAP kinase pathways, those of p38 MAP kinase and c-Jun N-terminal kinase, are also activated by wild-type L . monocytogenes . Mutants lacking functional LLO (hly mutants) were still invasive but only activated ERK-2 and only activated it at later (90-min) postinfection times . Two inhibitors of L . monocytogenes invasion, cytochalasin D, which disrupts actin polymerization, and wortmannin, which blocks phosphatidylinositol (PI) 3-kinase activity, did not block ERK-2 activation by wild-type L . monocytogenes and hly mutants . However, genistein, an inhibitor of tyrosine kinases, and PD98059 both blocked invasion and decreased ERK-2 activation . These results suggest that MEK-1 and ERK-2 activities are essential for L . monocytogenes invasion into host epithelial cells . This is the first report to show that a MAP kinase pathway is required for bacterial invasion.

Nat Biotechnol, 1998 Feb, 16(2), 181 - 5
Delivery of antigen-encoding plasmid DNA into the cytosol of macrophages by attenuated suicide Listeria monocytogenes; Dietrich G et al.; Eukaryotic expression vectors can be delivered to macrophages using attenuated self-destructing Listeria monocytogenes . L . monocytogenes cells are preferentially lysed in the host cell macrophage cytosol by the production of a PactA-dependent Listeria-specific phage lysin . Efficient expression of the cloned reporter genes by the macrophages and subsequent antigen presentation were achieved after the delivery of eukaryotic expression vectors by the attenuated suicide L . monocytogenes strain . After delivery by L . monocytogenes plasmid DNAs were found to integrate into the macrophage cell's genome at a frequency of about 10(-7).

Curr Opin Cell Biol, 1998 Feb, 10(1), 45 - 51
Control of actin dynamics; Carlier MF; Actin-based motility processes are tightly linked to the rapid turnover of actin filaments . Factors that control the steady state of actin assembly, such as capping proteins and actin-depolymerizing factor/cofilin, directly affect motility . Actin-depolymerizing factor increases the treadmilling of actin filaments in vitro and in vivo . Cellular factors that are involved in linking initiation of barbed end assembly to cell signaling are being identified using Listeria monocytogenes and Saccharomyces cerevisiae as model systems.

Trends Microbiol, 1998 Jan, 6(1), 11 - 5
Host cell signalling during Listeria monocytogenes infection; Kuhn M et al.; Macrophages and other mammalian cells respond to infection with Listeria monocytogenes by the transient or persistent activation of host cell signal transduction pathways . In addition, L . monocytogenes infection influences expression of various host cell genes, some of which may hinder or favour bacterial replication . The observed host cell responses vary with the different subcellular locations inhabited by L . monocytogenes during its intracellular life cycle.

Schweiz Arch Tierheilkd, 1997, 139(11), 490 - 4
{A case of acute disseminated Mucor encephalitis in a heifer}; Schonmann M et al.; The case of a 2 1/2 year old Swiss Braunvieh heifer suffering from an acute disseminated mycotic encephalitis caused by a Mucorales spp . infection is presented . Clinical signs and analysis of cerebrospinal fluid (increased protein concentration and pleocytosis) were typical for an acute encephalitis, probably due to a listeriosis . The histological examination of the brain revealed an acute disseminated thrombo-embolic encephalomyelitis due to a fungi infection, morphologically consistent with Mucorales spp . The occurrence of bovine cerebral mucormycosis is rare and therefore the veterinarian should become aware of a case which was clinically not distinguishable from a listeriosis.

Immunol Res, 1998, 17(1-2), 13 - 22
Role of gamma delta T cells in immunity to infectious diseases and the regulation of hematolymphoid cell development; Carding SR; My research interests are twofold . The first is to define the biochemical and molecular mechanisms that regulate hematopoietic cell development . In particular, the role that the cytokine interleukin-2 (IL2) plays in regulating the development and selection of lymphocyte progenitor cells, and in myelopoiesis are primary areas of research . The second is to understand the role that gamma delta T cells play in pathogen-induced immune responses and autoimmunity . Their involvement in the immune response to the intracellular bacteria Listeria monocytogenes in mice and Mycobacteria tuberculosis (Mtb) in humans, in T cell-mediated inflammatory bowel disease in humans, and the nature of the antigens they recognize during these responses are major areas of interest . Research material includes patient-derived tissues as well as both conventional and genetically engineered (transgenic) strains of mice.

Biophys Chem, 1997 Oct, 68(1-3), 73 - 82
The role of actin binding proteins in epithelial morphogenesis: models based upon Listeria movement; Golsteyn RM et al.; We summarize recent findings on the organization of the protein actin in eucaryotic cells . In particular we focus on how actin can be used to generate a vectorial force that is required for cell movement . These forces arise from protein molecules that recruit actin to the plasma membrane in such a manner that actin filaments extend outward from the cell body . This type of actin dependent force generation has been described in a nucleation-release model, which is one of several models currently being tested to explain actin dependent cell movement . Data in support of this model has arisen unexpectedly from studies of an intracellular bacteria, Listeria monocytogenes . This bacteria uses actin to propel itself during infection of eucaryotic cells . By studying Listeria movement, the roles of several eucaryotic actin interacting proteins have been identified . One of these is zyxin, a human protein that shares important structural and possibly functional properties with ActA, an actin dependent force generating protein of Listeria . We intend to test the function of these and other actin interacting proteins in a simplified system that should facilitate precise measurement of their properties of force generation in vitro.

Microbiology, 1998 Jan, 144 ( Pt 1), 109 - 18
Lectin reactivity and virulence among strains of Listeria monocytogenes determined in vitro using the enterocyte-like cell line Caco-2; Facinelli B et al.; Forty-six cultures of Listeria monocytogenes (including clinical, food and collection strains) were serotyped, characterized for motility, haemolysis and phospholipase activities and tested for lectin agglutination using a four-lectin set . Lectin reactivity (i.e . agglutination by one or more of the four lectins) was observed in all 12 clinical isolates, 16 of the 23 food isolates and eight of the 11 collection strains . Virulence was evaluated in vitro based on strains' ability to invade the human enterocyte-like cell line Caco-2 . In gentamicin survival experiments, recovery of viable intracellular bacteria among lectin-unreactive strains was usually 100-1000-fold lower than among lectin-reactive haemolytic strains, and lower than among nonhaemolytic strains . Considerable cytopathogenic effects were produced by lectin-reactive haemolytic strains in trypan-blue-stained cell monolayers, whereas lectin-unreactive and nonhaemolytic strains produced no detectable cytopathogenic effect . Among lectin-reactive strains, the number of listerial cells associated with Caco-2 monolayers was more than tenfold greater than among lectin-unreactive strains . Cell invasion was inhibited by pretreatment of Caco-2 cells with sugars recognized by the lectins or of listeriae with enzymes which removed the same sugars from the bacterial surface . The results suggest that the study of lectin interactions could be helpful in understanding the pathogenicity potential of isolates of food and environmental origin.

Zentralbl Veterinarmed B, 1997 Dec, 44(10), 617 - 24
{Presence of Listeria monocytogenes and Listeria sp . in fresh and cured sausages . Use of different conditions of time and temperature for incubation}; Rota C et al.; Two different temperatures for enrichment of Listeria monocytogenes and related species have been studied (1) cold enrichment at 4 degrees C (2) enrichment at 30 degrees C (FDA method) . Also, two selective media for isolation were tested: Acriflavine-Ceftazidime agar (A.C.) and Palcam agar . We have studied 72 samples of dry-cured sausage (called 'longaniza') at different stages of maturation: fresh, semi-cured and cured samples . The most efficient method was cold enrichment at 4 degrees C during 5 days followed by isolation in Palcam agar, but results were only significant for fresh sausages (P < 0.05).

Appl Environ Microbiol, 1998 Feb, 64(2), 800 - 3
Purification and characterization of anti-Listeria compounds produced by Geotrichum candidum; Dieuleveux V et al.; Geotrichum candidum can produce and excrete compounds that inhibit Listeria monocytogenes . These were purified by ultrafiltration, centrifugal partition chromatography, thin-layer chromatography, gel filtration, and high-pressure liquid chromatography, and analyzed by liquid chromatography-mass spectrometry, infrared spectrometry, nuclear magnetic resonance spectrometry, and optical rotation . Two inhibitors were identified: D-3-phenyllactic acid and D-3-indollactic acid.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Nov-Dec, (6), 68 - 70
{The effect of Listeria monocytogenes on the blood system}; Iurkina OA et al.; The influence of L . monocytogenes on the hemopoietic system was studied in mouse experiments . All elements of hemopoiesis were damaged . The most pronounced changes developed in erythropoiesis . This was testified by a decrease in erythrocyte count and hemoglobin content in peripheral blood, by the hypoplasia of the hemopoietic erythroid germ in the marrow, as well as by the decrease of yield in culture CFU-E . These changes in the hemopoietic tissue supposedly caused by the direct action of L . monocytogenes and its toxins.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Sep-Oct, (5), 78 - 82
{The ecological aspects of listeriosis in the Maritime Territory}; Somov GP et al.; In the process of batch cultivation the strains under study are capable of prolonged growth at low temperature in rich and poor nutrient media (with the term of observation equal to 4 months), while at a temperature of 37 degrees C microbial populations quickly die (in 8-35 days) . In the absence of compounds containing carbon, hydrogen and nitrogen in the nutrient medium, Listeria can proliferate under such conditions . As established with the use of gas chromatography and the radioisotopic method, they can uptake carbon dioxide, hydrogen and nitrogen from the air gas mixture, using carbon of the first gas for the synthesis of the main biopolymers (proteins, lipids, carbohydrates, DNA and RNA) and the second one as the source of energy . During the cultivation of Listeria at low temperature in poor nutrient media (soil microecosystems, synthetic mineral media) they are capable of preserving and under favorable conditions also increasing their virulence . Its increase is facilitated by capsule formation, mobility, chemotaxis, adhesion and invasion enhancing under such conditions.

J Gen Virol, 1997 Oct, 78 ( Pt 10), 2633 - 7
Characterization of the vaccinia virus F8L protein; Higley S et al.; Vaccinia virus infection dramatically affects the host actin cytoskeleton by inducing disassembly of actin stress fibres and formation of actin tails which propel the virus intra- and intercellularly . The viral factors responsible for these actin rearrangements remain unknown . Sequence analysis reveals significant homology between the vaccinia F8L ORF and the proline repeats of iActA, the protein which initiates actin tail assembly and motility in the bacterial pathogen Listeria ivanovii . We characterized the F8L gene product to examine its possible role in vaccinia rearrangements of the host actin cytoskeleton . F8L is a approximately 8 kDa protein expressed early during infection and is found throughout the cytoplasm, with no discernible association with viral or cellular structures . Furthermore, the F8L deletion strain, WR deltaF8L, forms particles and actin tails indistinguishable from WR . Our observations demonstrate that F8L is not required for vaccinia virus morphogenesis or the actin rearrangements observed during infection.

Ocul Immunol Inflamm, 1997 Dec, 5(4), 245 - 57
Immune privilege in the anterior chamber of the eye is not extended to intraocular Listeria monocytogenes; Li XY et al.; Resistance to many pathogens, such as Listeria monocytogenes, is correlated with the host's capacity to generate a ThI cell-mediated immune response in which delayed-type hypersensitivity (DTH) is activated . A wide variety of antigens induce down-regulation of DTH when introduced into the anterior chamber of the eye . This immunoregulatory phenomenon has been termed anterior chamber-associated immune deviation (ACAID) and is believed to be a primary mechanism for the immune privilege of the anterior chamber . Suppression of DTH, as a result of anterior chamber priming, could carry significant risk to the host's well-being as the resistance to many pathogens relies heavily on DTH-dependent ThI responses . Studies were performed to determine if a bacterial pathogen, L . monocytogenes, introduced into the anterior chamber of the eye would induce a down-regulation of systemic DTH . Intracameral inoculation of infectious L . monocytogenes into genetically susceptible C3H and BALB/c mice did not induce suppression of DTH, but instead resulted in a significant footpad swelling response to bacterial antigens . Likewise, intracameral inoculation of L . monocytogenes into genetically resistant C57BL/6 mice also induced vibrant bacterial-specific DTH . Using an in-vitro model of ACAID, we showed that macrophage suspensions that were simultaneously exposed to L . monocytogenes and bovine serum albumin (BSA) antigens, in the presence of aqueous humor (AH), induced listerial-specific DTH responses, yet simultaneously induced suppression of BSA-specific DTH . Collectively, the results indicate that immune privilege is not extended to all foreign antigens that enter the anterior chamber of the eye, and as a result, some intraocular antigens can provoke strong systemic DTH . However, non-ACAID-inducing antigens do not prejudice the down-regulation of DTH by other antigens which normally induce ACAID.

Infect Immun, 1998 Feb, 66(2), 747 - 55
Comprehensive study of the intestinal stage of listeriosis in a rat ligated ileal loop system; Pron B et al.; The intestinal stage of listeriosis was studied in a rat ligated ileal loop system . Listeria monocytogenes translocated to deep organs with similar efficiencies after inoculation of loops with or without Peyer's patches . Bacterial seeding of deep organs was demonstrated as early as 15 min after inoculation . It was dose dependent and nonspecific, as the delta inlAB, the delta hly, and the delta actA L . monocytogenes mutants and the nonpathogenic species, Listeria innocua, translocated similarly to wild-type L . monocytogenes strains . The levels of uptake of listeriae by Peyer's patches and villous intestine were similar and low, 50 to 250 CFU per cm2 of tissue . No listeria cells crossing the epithelial sheet of Peyer's patches and villous intestine were observed by transmission electron microscopy . The lack of significant interaction of listeriae and the follicle-associated epithelium of Peyer's patches was confirmed by scanning electron microscopy . The follicular tissue of Peyer's patches was a preferential site of Listeria replication . With all doses tested, the rate of bacterial growth was 10 to 20 times higher in Peyer's patches than in villous intestine . At early stages of Peyer's patch infection, listeriae were observed inside mononuclear cells of the dome area . Listeriae then disseminated throughout the follicular tissue except for the germinal center . The virulence determinants hly and, to a lesser extent, actA, but not inlAB, were required for the completion of this process . This study suggests that Peyer's patches are preferential sites for replication rather than for entry of L . monocytogenes, due to the presence of highly permissive mononuclear cells whose nature remains to be defined.

Infect Immun, 1998 Feb, 66(2), 620 - 6
The role of sialic acid in opsonin-dependent and opsonin-independent adhesion of Listeria monocytogenes to murine peritoneal macrophages; Maganti S et al.; The adhesion of listeriae to host cells employs mechanisms which are complex and not well understood . Listeria monocytogenes is a facultative intracellular pathogen responsible for meningoencephalitis, septicemia, and abortion in susceptible and immunocompromised individuals . Subsequent to colonization and penetration of the gut epithelium, the organism attaches to resident macrophages and replicates intracellularly, thus evading the humoral immune system of the infected host . The focus of these studies was to investigate the attachment of the organism to murine peritoneal macrophages in an opsonin-dependent and opsonin-independent fashion . Assessment of competitive binding experiments by immunofluorescence and enzyme-linked immunosorbent assays showed that adhesion of the organism to macrophages in the presence or absence of opsonins was inhibited (90%) by N-acetylneuraminic acid (NAcNeu) . In addition, the lectin from Maackia amurensis, with affinity for NAcNeu-alpha(2,3)galactose, blocked binding of L . monocytogenes to host cells . Oxidation of the surface carbohydrates on the organism by using sodium metaperiodate resulted in a dose-dependent reduction (up to 98%) in adherence to macrophages . Monoclonal antibody to complement receptor 3 did not prevent listeriae from binding to mouse macrophages or from replicating within the infected cells whether or not normal mouse serum was present . Based on our results, we propose the involvement of NAcNeu, a member of the sialic acid group, in the attachment of L . monocytogenes to permissive murine macrophages.

FEMS Microbiol Lett, 1998 Jan 1, 158(1), 45 - 50
Evidence for expressional crosstalk between the central virulence regulator PrfA and the stress response mediator ClpC in Listeria monocytogenes; Ripio MT et al.; Virulence is a multifactorial trait which depends on the coordinated expression of many bacterial products, hence it is to be expected that the regulatory circuits that control the relevant genetic determinants are somehow interconnected . Two pleiotropic regulatory elements acting at different levels, the transcription factor PrfA which controls virulence gene expression and the potential chaperone ClpC which is involved in tolerance to environmental stress, are required for Listeria monocytogenes survival within the host . We analyzed the influence of PrfA on clpC expression in L . monocytogenes . clpC transcription is maximal under heat-shock conditions, i.e . at 42 degrees C, and is very weak or undetectable at 37 degrees C . In a prfA* mutant which constitutively overexpresses PrfA and PrfA-dependent virulence genes, clpC transcription dropped to basal levels during exponential growth at 42 degrees C . This repression was not observed during stationary phase, indicating growth phase-dependent regulation of clpC . Culture in charcoal-treated medium, which triggers in wild-type strains the transcriptional activation of the PrfA regulon, also caused a strong downregulation of clpC . Moreover, in a prfA deletion mutant, clpC transcription during exponential growth at 37 degrees C was clearly enhanced, reaching the same high levels of the wild-type at 42 degrees C . Overall, our results indicate that clpC expression is negatively controlled at the transcriptional level, directly or indirectly, by the central virulence regulator PrfA.

Eur J Clin Microbiol Infect Dis, 1997 Nov, 16(11), 827 - 33
In vitro extracellular and intracellular activity of two newer and two earlier fluoroquinolones against Listeria monocytogenes; Facinelli B et al.; Two new fluoroquinolones (trovafloxacin and sparfloxacin) with enhanced activity against gram-positive pathogens and two earlier compounds (ciprofloxacin and ofloxacin) were tested for their in vitro inhibitory and bactericidal activity against 80 strains of Listeria monocytogenes . All strains were uniformly highly susceptible to trovafloxacin, the MIC90 being 0.25 mg/l . Resistance to sparfloxacin was not detected, however the MIC90 of sparfloxacin was eight times that of trovafloxacin . A few strains were resistant to ciprofloxacin and ofloxacin (MIC90 4 mg/l for both drugs) . MBCs usually exceeded MICs by 2 to 4 times . The MBC90 of trovafloxacin (1 mg/l) was lower than that of the other three drugs (8 mg/l) . After checking their ability to enter and grow within human enterocyte-like Caco-2 cells, four strains were used to study the intracellular activity and eradicating power of the four quinolones . Trovafloxacin was more active than sparfloxacin and the earlier fluoroquinolones in terms of both intracellular killing and inhibition of a cytopathogenic effect . The uniform high-level activity of trovafloxacin against Listeria monocytogenes isolates in conventional in vitro assays and its extracellular and intracellular killing of invasive strains suggest that this and maybe other new fluoroquinolones should be further investigated as possible anti-listerial agents.

Blood, 1998 Feb 1, 91(3), 863 - 9
Essential roles for granulocyte-macrophage colony-stimulating factor (GM-CSF) and G-CSF in the sustained hematopoietic response of Listeria monocytogenes-infected mice; Zhan Y et al.; The in vivo roles of granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte (G)-CSF were studied in factor-deficient gene-targeted knockout mice infected with the facultative intracellular bacterium Listeria monocytogenes . Previous results showed that G-CSF-/- mice had an underlying selective deficiency in granulopoiesis, but GM-CSF-/- mice had little disturbance in resting hematopoiesis . Nevertheless, in this study it is revealed that 3 days after intraperitoneal infection with 2 x 10(5) Listeria, GM-CSF-/- mice harbored 50-fold more organisms in their spleen and liver than similarly infected wild-type mice . This was accompanied by a severe depletion of bone marrow hematopoietic cells and a deficient inflammatory response in their peritoneal cavity . Thus, GM-CSF is essential for emergency, but not resting, hematopoiesis . In contrast, G-CSF-/- mice were markedly susceptible to low doses (2 x 10(4)) of Listeria intraperitoneally . After infection, the acute (1 day) granulocyte infiltration to the peritoneal cavity was normal compared with wild type, but the more prolonged monocyte response was deficient, reflecting a continued decrease in bone marrow cellularity and hematopoiesis over 3 days, which was not observed in infected wild-type mice . It is thus apparent that G-CSF deficiency affects monocytopoiesis as well as granulopoiesis during infection.

Microbiol Immunol, 1997, 41(11), 847 - 53
The host response to Listeria monocytogenes mutants defective in genes encoding phospholipases C (plcA, plcB) and actin assembly (actA); Rudnicka W et al.; Several genes involved in the determination of Listeria monocytogenes pathogenesis have been identified . Among them, plcA gene encodes phosphatidylinositol-specific phospholipase C (PI-PLC), plcB gene encodes a broad-range phospholipase C (PC-PLC), and actA encodes a protein contributing to actin assembly in infected cells . The interaction of L . monocytogenes wild type (LO 28) strain and two derivative mutants, plcA- (BUG 206) and actA-/plcB- (LUT 12), with macrophages and T lymphocytes was investigated in a mouse model of listeriosis . Both mutants showed evidence of attenuation . The plcA- mutant, but not the plcB- mutant, expressed an increase in susceptibility to the anti-listerial activity of macrophages . Both mutants showed a decreased ability to induce IL-12 production by bone marrow macrophages when co-stimulated with E . coli LPS or IFN-gamma . In vivo, L . monocytogenes plcA- mutant was found to be a more effective stimulator of T cells than the wild LO 28 strain.

Annu Rev Genet, 1997, 31, 113 - 38
Host-pathogen interactions during entry and actin-based movement of Listeria monocytogenes; Ireton K et al.; Listeria monocytogenes is a pathogenic bacterium that induces its own uptake into mammalian cells, and spreads from one cell to another by an actin-based motility process . Entry into host cells involves the bacterial surface proteins InlA (internalin) and InlB . The receptor for InlA is the cell adhesion molecule E-cadherin . InlB-mediated entry requires activation of the host protein phosphoinositide (PI) 3-kinase, probably in response to engagement of a receptor . Actin-based movement of L . monocytogenes is mediated by the bacterial surface protein ActA . The N-terminal region of this protein is necessary and sufficient for polymerization of host cell actin . Other host proteins involved in bacterial motility include profilin, Vasodilator-Stimulated Phosphoprotein (VASP), the Arp2/Arp3 complex, and cofilin . Studies of entry and intracellular movement of L . monocytogenes could lead to a better understanding of receptor-ligand signaling and dynamics of actin polymerization in mammalian cells.

Toxicol Appl Pharmacol, 1997 Dec, 147(2), 180 - 9
Interleukin-12 promotes enhanced resistance to Listeria monocytogenes infection of lead-exposed mice; Kishikawa H et al.; The heavy metal lead (Pb) has been shown to downregulate various parameters of cell-mediated immune (CMI) responses . This inhibition of CMI responses by Pb is exemplified by a higher mortality rate upon infections with sublethal doses of a variety of pathogens . Unlike Pb, which lowers host resistance, interleukin-12 (IL-12) exerts a substantial stimulatory influence on the host response to intracellular bacteria such as Listeria monocytogenes . To explore the influence of IL-12 in mice rendered susceptible to Listerial infection by oral exposure to Pb, we determined bacterial burdens and production of interferon gamma (IFN-gamma) . As expected, Pb-exposed mice had increased morbidity due to higher Listerial titers as compared to control mice . However, administration of exogenous IL-12 reversed the Pb-induced inhibition of host defense and boosted the resistance of the non-Pb-treated mice . The enhanced CMI responses observed in both IL-12-treated groups were accompanied with elevations of IFN-gamma in the sera and spleens . Significant reduction in the number of viable Listeria in Pb-exposed mice upon IL-12 administration suggests that the processes downstream of IL-12 production were intact in the Pb-exposed mice and that the inhibition by Pb was due to the lack of functional IL-12 . Alternatively, the exogenous IL-12 may have overcome a downstream effect by enhancing an secondary pathway . Support for the former hypothesis is based on the observation that Pb induced elevated levels of p40 splenic messenger RNA since increased p40 expression would result from lack of IL-12 formation . Contrary to the IFN-gamma levels, significantly higher levels of IL-6 and corticosterone were observed in the sera and spleens of Pb-exposed mice upon infection, suggesting heightened stress in the absence of IL-12 . Overall, the results suggest that an environmental pollutant such as Pb can enhance the stress response, which naturally occurs during an infection, and can further compromise health by lowering host resistance by altering cytokine levels.

Poult Sci, 1997 Dec, 76(12), 1661 - 4
Effects of gamma irradiation on the survival of Listeria monocytogenes and on its growth at refrigeration temperature in poultry and red meat; Gursel B et al.; Gamma irradiation sensitivity of a strain of Listeria monocytogenes was determined in trypticase soy broth supplemented with yeast extract (TSB-YE), in a slurry of chicken breast meat and in raw ground beef . D10 values in these different media were 0.364, 0.599, and 0.699 kGy, respectively . This organism appeared most sensitive in TSB-YE, more resistant in minced fresh chicken breast meat, and most resistant in fresh minced beef . It was found that irradiation at 2.5 kGy prior to refrigeration is an efficient way for the preservation of meat products contaminated at 10(3) to 10(4) per gram initial load of L . monocytogenes for about 7 d . However, with this initial load, the injured cells might repair themselves and cause a health hazard during storage at 4 C in the presence of air after 7 d.

J Dairy Sci, 1997 Dec, 80(12), 3445 - 8
Mycobacterium paratuberculosis: a potential food-borne pathogen?
Collins MT.
Mycobacterium paratuberculosis commonly infects dairy cattle, leading to Johne's disease, which is also known as paratuberculosis . The infection is chronic progressive, and incurable . As the infection progresses, excretion of M . paratuberculosis in feces and milk occurs, and the bacterium spreads through the blood to multiple internal organs . Consequently, raw products originating from cattle may harbor M . paratuberculosis . Thermal treatments, such as pasteurization, are commonly relied on to kill food-borne bacterial pathogens that can infect humans . The small number of studies conducted to determine the thermal resistance of M . paratuberculosis suggest that it is less susceptible to destruction by heat killing than are milkborne zoonotic bacterial pathogens such as Listeria spp . or Mycobacterium bovis . Published reports concerning the thermal resistance of M . paratuberculosis in milk are reviewed herein, and key issues concerning the efficacy of pasteurization for elimination of M . paratuberculosis from milk are summarized.

Environ Health Perspect, 1998 Feb, 106(2), 71 - 7
Risk assessment for the harmful effects of UVB radiation on the immunological resistance to infectious diseases; Goettsch W et al.; Risk assessment comprises four steps: hazard identification, dose-response assessment, exposure assessment, and risk characterization . In this study, the effects of increased ultraviolet B(UVB, 280-315 nm) radiation on immune functions and the immunological resistance to infectious diseases in rats were analyzed according to this strategy . In a parallelogram approach, nonthreshold mathematical methods were used to estimate the risk for the human population after increased exposure to UVB radiation . These data demonstrate, using a worst-case strategy (sensitive individuals, no adaptation), that exposure for approximately 90 min (local noon) at 40 degrees N in July might lead to 50% suppression of specific T-cell mediated responses to Listeria monocytogenes in humans who were not preexposed to UVB (i.e., not adapted) . Additionally, a 5% decrease in the thickness of the ozone layer might shorten this exposure time by approximately 2.5% . These data demonstrate that UVB radiation, at doses relevant to outdoor exposure, may affect the specific cellular immune response to Listeria bacteria in humans . Whether this will also lead to a lowered resistance (i.e.,increased pathogenic load) in humans is not known, although it was demonstrated that UVB-induced immunosuppression in rats was sufficient to increase the pathogenic load . Epidemiology studies are needed to validate and improve estimates for the potential effects of increased UVB exposure on infectious diseases in humans.

Appl Environ Microbiol, 1998 Jan, 64(1), 231 - 7
Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype; Crandall AD et al.; Nisin resistance in Listeria monocytogenes ATCC 700302 is a complex phenotype involving alterations in both the cytoplasmic membrane and the cell wall and a requirement for divalent cations . In addition to a lower ratio of C15 to C17 fatty acids than in the wild-type strain (A . S . Mazzotta and T.J . Montville, J . Appl . Microbiol . 82: 32-38, 1997), this nisin-resistant (Nisr) strain contained significantly more zwitterionic phosphatidylethanolamine and less anionic phosphatidylglycerol and cardiolipin . The extraction of cardiolipin was enhanced by a penicillin-lysozyme step to disrupt the cell wall . This study is the first to quantify the phosphatidylethanolamine component of the L . monocytogenes cytoplasmic membrane . While these cytoplasmic membrane changes were induced by nisin, the Nisr strain also showed altered sensitivities to cell wall-acting compounds, even when grown in the absence of nisin, suggesting a constitutive alteration in the strain's cell wall . A model which integrates the roles of the cell membrane, cell wall, and divalent cations is presented . Finally, nisin resistance in L . monocytogenes ATCC 700302 conferred cross-resistance to the class IIa bacteriocin pediocin PA-1 and the class IV leuconocin S.

Respirology, 1996 Jun, 1(2), 127 - 32
Effect of antibacterial antibody on bactericidal activities of superoxide and lysosomal enzyme from alveolar macrophages in rabbits; Suga M et al.; Alveolar macrophages (AM) have many Fc receptors for IgG, but are less reactive to lymphokines . They have a well-developed oxidative metabolism and contain large amounts of lysosomal enzyme . This suggests that the antibacterial antibody plays an important role in early resistance by AM to intracellular bacterial infection and that a bactericidal agent, dependent on oxygen and lysosomal enzyme, participates in the effects of the antibacterial antibody on bactericidal activities of superoxide (O2-) and lysosomal enzyme from rabbit AM . The number of Listeria monocytogenes in AM increased after pretreatment with saline or normal IgG but decreased by 60% after pretreatment with anti-Listeria and 120 min incubation . Alveolar macrophage-phagocytized Listeria monocytogenes and Bacille Calmette Guerin (BCG) bound with antibacterial antibody enhanced release of O2-, and nitroblue tetrazolium (NBT) formazan reduced by O2- was observed around the bacteria in the phagosomes of AM . We also confirmed that Listeria and BCG were killed extracellularly by O2-released by a superoxide-generating system in vitro and/or by lysosomal concluded that the antibacterial antibody of the IgG class enhances the antibacterial activity of AM thereby increasing the production of 02- and lysosomal enzyme in the phagosome . This finding may be important in the early resistance to intracellular bacteria infection by AM in the alveolar spaces.

Arch Latinoam Nutr, 1996 Dec, 46(4), 292 - 4
{Presence of various pathogenic microorganisms in fresh vegetables in Costa Rica}; Monge R et al.; This study reports the occurrence of some pathogenic microorganisms in vegetable consumed on a daily basis by Costa Ricans . Cryptosporidium sp . oocysts were found in 5.2% (4/80) of cilantro leaves, in 8.7% (7/80) of cilantro roots and 2.5% of lettuce samples . A 1.2% (1/80) incidence was found in other vegetables samples (carrot, cucumber, radish and tomatoe) . Oocysts of this parasite were absent in cabbage . Giardia intestinalis was only detected in 5.2% (4/80) of cilantro leaves and in 2.5% (2/ 80) of cilantro roots . Entamoeba histolytica cysts were found in 6.2% (5/80) of cilantro leaves, in 2.5% (2/80) cilantro roots, in 3.8% (3/80) lettuce and in 2.5% (2/80) radish samples . At least a 2% incidence of this amoeba was found in other vegetable samples (carrot, cucumber, cabbage and tomatoe) . Listeria monocytogenes was isolated in 20% (10/50) of the samples of cabbage salad . Hepatitis A virus and Rotavirus were evidenciated in three of the lettuce pooles, suggesting that at least three of the samples were contaminated with these viruses.

Rev Argent Microbiol, 1997 Apr-Jun, 29(2), 63 - 7
{Effect of different dairy products on the growth of Listeria monocytogenes in selective media}; Santillan MA et al.; We have studied the influence of the incorporation of milk cream and skim milk on the growth kinetic of Listeria monocytogenes in listeria enrichment broth with 15 mg/l or 7.5 mg/l of acriflavine . Acriflavine was responsible, at least partially, for delayed growth of Listeria monocytogenes in the enrichment broths . A longer lag phase of the growth was produced by the addition of milk cream or skim milk to the enrichment broth containing 7.5 mg/l of acriflavine . However, the maximum population obtained at 48 h did not show significant differences . In the presence of 15 mg/l of acriflavine, we observed a decrease of the viable counts during the early phase of the growth cycle, which was enhanced by the addition of milk cream or skim milk . Moreover, the maximum growth rate was reduced by the addition of skim milk and maximum population was not reached at 48 h . These results suggest the need to validate the methodology of recuperation of Listeria monocytogenes from each dairy product, since its efficiency may be affected by product composition, specially when the sample biocharge is low.

Rev Argent Microbiol, 1997 Apr-Jun, 29(2), 109 - 13
{Listeria monocytogenes and Listeria sp in heat-processed products}; Tobia MB et al.; Presence of Listeria monocytogenes in thermoprocessed food which is vacuum packaged, refrigerated stored and eaten uncooked or minimally heated was investigated . Thirty samples including sausage, large sausage with ham, spring large sausage, liverwurst and bologna were examined . Listeria monocytogenes was isolated and identified according to USDA-FSIS method for meat products, simultaneously with McBride agar . Seven out of 30 samples were found to contain listeriae . Five isolates were identified as Listeria monocytogenes through Gram coloration, culture appearance, biochemical test and serotyping . This product results potentially risky for the susceptible population . The presence of this microorganism in this kind of product suggests environmental post-process contamination or insufficient thermal process.

Infect Immun, 1998 Jan, 66(1), 232 - 8
Listeria monocytogenes virulence factors that stimulate endothelial cells; Drevets DA; Listeria monocytogenes infection of endothelial cells upregulates surface expression of adhesion molecules and stimulates neutrophil adhesion to infected cell monolayers . The experiments presented here tested the roles of specific bacterial virulence factors as triggers for this inflammatory phenotype and function . Human umbilical vein endothelial cell (HUVEC) monolayers were infected with wild-type L . monocytogenes or L . monocytogenes mutants; then surface expression of E-selectin and neutrophil adhesion were measured . The results showed that delta hly and prfA mutants were the most crippled, requiring 100-fold more mutant bacteria than wild-type bacteria for analogous stimulation . By comparison, L . monocytogenes mutants with deletions of actA, inlA, inlB, inlAB, plcA, and plcB resembled their parent strains, and a delta plcA delta plcB mutant displayed decreased intracellular growth rate but only a minor decrease in stimulation of E-selectin or neutrophil adhesion . Other experiments showed that cytochalasin D-treated HUVEC monolayers bound bacteria, but internalization and increased surface E-selectin and intercellular adhesion molecule-1 expression were profoundly inhibited . However, cytochalasin D had no effect on the HUVEC response to stimulation with lipopolysaccharide or tumor necrosis factor alpha . These data suggest that listeriolysin O production by infecting L . monocytogenes contributes to increased expression of surface E-selectin and intercellular adhesion molecule-1, but neither it nor intracellular replication are directly responsible for this event . Nonetheless it is possible that listeriolysin O potentiates the effect(s) of an other molecule(s) that directly triggers this response . Additionally, cellular invasion by L . monocytogenes appears to be critical for initiating the HUVEC response, potentially by providing a signal which results in upregulation of the necessary bacterial genes.

J AOAC Int, 1997 Nov-Dec, 80(6), 1208 - 14
Comparison of antibody-direct epifluorescent filter technique with the most probable number procedure for rapid enumeration of Listeria in fresh vegetables; Tortorello ML et al.; The antibody-direct epifluorescent filter (Ab-DEFT) technique was evaluated as a rapid alternative to the most probable number (MPN) method for enumeration of artificially inoculated Listeria monocytogenes in ready-to-eat packaged salads and other fresh vegetables . Ab-DEFT was performed by homogenization of food in mesh-lined Stomacher bags, followed by prefiltration of homogenate through a 5 microns pore nylon filter, and passage of filtrate through a 0.4 micron pore black polycarbonate filter to collect and concentrate Listeria cells . After cells were stained with a fluorochrome-labeled polyclonal antibody to Listeria, the filter surface was examined by epifluorescence microscopy, and fluorescent cells were counted . A 3-tube MPN procedure was performed by successive enrichments of homogenized foods in Listeria enrichment and Fraser broths, followed by selective plating . Ab-DEFT provided quantitative determinations of Listeria cells that correlated with plate counts and MPN estimates in a linear response over a range of cell concentrations from 10 to 10(7) colony forming units (CFU)/mL . Microbial backgrounds as high as 10(8) CFU/mL did not affect performance of Ab-DEFT . In contrast to the MPN method, which required 5 days to perform, quantitation by Ab-DEFT could be completed in less than 1 h . Despite cross-reactivities demonstrated by the polyclonal fluorescent antibody, the potential of Ab-DEFT as a rapid alternative to MPN for microbial cell enumeration was evident.

J Infect Dis, 1998 Jan, 177(1), 155 - 60
Listeriosis outbreak associated with the consumption of rillettes in France in 1993; Goulet V et al.; An outbreak of listeriosis involving 38 patients occurred in France between 18 June and 5 October 1993 . The epidemic clone was characterized by serovar 4b, phagovar 2671:108:312, and DNA macrorestriction patterns 12 and 13 . Thirty-one case-patients were materno-neonatal patients and 7 patients were nonpregnant adults . Preliminary analysis of a case-control study implicated a pork product, rillettes, of a particular brand (odds ratio, 18; 95% confidence interval, 2.2-208) as the vehicle of infection . Rillettes is a ready-to-eat food prepared with ham meat cooked with grease . The implicated lots of rillettes were recalled in mid-August, and the French authorities issued a warning to the general public . Microbiologic analysis of unopened plastic cans of rillettes confirmed the results of the case-control study 3 weeks after the recall . Final analysis showed that the rillettes was the major vehicle of the outbreak but suggested that other brand A meat products could also have been involved.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 163 - 70
Listeria monocytogenes-infected human umbilical vein endothelial cells: internalin-independent invasion, intracellular growth, movement, and host cell responses; Greiffenberg L et al.; The interaction of Listeria monocytogenes with human umbilical vein endothelial cells was studied . We show that L . monocytogenes invades human umbilical vein endothelial cells independently of internalin A, internalin B, internalin C, and ActA . L . monocytogenes replicates efficiently inside the cells and moves intracellularly by the induction of actin polymerization . We further show that L . monocytogenes-infection of human umbilical vein endothelial cells induces interleukin-6 and interleukin-8 expression during the first 6 h of infection . The expression of MCP-1 and the adhesion molecules VCAM-1 and ICAM-1 was not altered under the experimental conditions used here.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 131 - 6
Involvement of MAP-kinases and -phosphatases in uptake and intracellular replication of Listeria monocytogenes in J774 macrophage cells; Kugler S et al.; In this study we show that protein tyrosine kinases and also protein tyrosine phosphatases are involved in the uptake of Listeria monocytogenes by J774 macrophages to a different extent than in the uptake of inert latex beads . In addition, protein tyrosine kinases are necessary for the intracellular growth and survival of L . monocytogenes . The expression of the MAP kinase phosphatase MKP-1, a protein tyrosine phosphatase, is induced upon infection, and phagocytosis of L . monocytogenes by J774 cells overexpressing the MKP-1 protein is reduced compared to control cells . The decreased phagocytosis of L . monocytogenes as a result of the MKP-1 overexpression in J774 macrophages suggests that the activation of the MAP kinase(s) ERK-1 and/or ERK-2 is an essential requirement for the uptake of L . monocytogenes by J774 macrophages.

Lett Appl Microbiol, 1997 Nov, 25(5), 367 - 70
The use of ethidium bromide to assess a novel injury/recovery phenomenon in Listeria monocytogenes in inhibitory NaCl conditions; Robinson TP et al.; An injury and recovery phenomenon was observed in Listeria monocytogenes inoculated into a medium containing 2.2 mmol l -1 NaCl, a concentration that was inhibitory to growth . The apparent loss then recovery of viability, as determined by plate counts, was compared with the uptake of ethidium bromide by the cells and found to be inversely related . Injury was caused not only by the initial osmotic up-shock but also by the subsequent down-shock involved in the spread plate protocol.

Immunology, 1997 Oct, 92(2), 274 - 83
Macrophages activated by Listeria monocytogenes induce organ-specific autoimmunity; Sonoda KH et al.; We have previously reported an experimental autoimmune model induced by the local infection of Listeria monocytogenes . The unilateral inoculation of virulent Listeria into a testis of a normal mouse induced a delayed-type hypersensitivity response against testicular antigen and caused autoimmune orchitis in the contralateral testis . The orchitis was transferred to naive mice by T cells from the intratesticularly infected mice . In this paper, we demonstrated that avirulent Listeria, which lacks the expression of listeriolysin O, failed to induce any anti-testicular responses or contralateral orchitis even when it was inoculated at a high dose into the testis . Furthermore, the intraperitoneal inoculation of virulent Listeria with testicular antigen induced the anti-testicular responses and orchitis although intraperitoneal inoculation of testicular antigen with avirulent Listeria failed to induce them . The difference between virulent and avirulent Listeria in the induction of anti-testicular responses was supposed to be dependent on the difference in macrophage activation by the two bacterial strains because, first, the anti-testicular responses were elicited in normal mice when macrophages from virulent Listeria-infected mice were intraperitoneally transferred with testicular antigen although no viable bacteria were detected from the macrophages, and secondly, in contrast, the intraperitoneal co-inoculation of macrophages from avirulent Listeria-infected mice and testicular antigen failed to elicit any anti-testicular responses . Finally, we found that the virulent Listeria-induced macrophages expressed a higher level of CD80 (B7-1) and CD86 (B7-2) molecules than did the avirulent Listeria-induced macrophages and naive peritoneal macrophages . These results thus suggest that virulent Listeria activates macrophages to induce autoreactive T cells while avirulent Listeria does not . The up-regulation of B7 molecules by virulent Listeria infection is a candidate of the mechanism for the activation of autoreactive T cells.

Cell, 1997 Dec 12, 91(6), 765 - 75
Oral somatic transgene vaccination using attenuated S . typhimurium; Darji A et al.; An attenuated strain of S . typhimurium has been used as a vehicle for oral genetic immunization . Eukaryotic expression vectors containing truncated genes of ActA and listeriolysin--two virulence factors of Listeria monocytogenes--have been used to transform S . typhimurium aroA . Multiple or even single oral immunizations with such transformants induced excellent cellular and humoral responses . In addition, protective immunity was induced with listeriolysin transformants . The quality of the responses suggested a transfer of plasmid DNA from the bacterial carrier to the host . Such transfer was unequivocally shown in vitro with primary peritoneal macrophages . We describe a highly versatile system for antigen delivery, identification of protective antigens for vaccination, and efficient generation of antibodies against the product of open reading frames present on virtually any DNA segment.

Medicine (Baltimore), 1997 Nov, 76(6), 415 - 22
Pneumocystis carinii infection in heart transplant recipients . Efficacy of a weekend prophylaxis schedule; Munoz P et al.; Most series of heart transplant patients report incidences of Pneumocystis carinii pneumonia (PCP) below 5% but do not individually describe the cases . From August 1988 to March 1994, 138 patients received 1 or more heart transplants at our institution . No anti-PCP chemoprophylaxis was provided, and 5 (3.6%) patients developed PCP . Incidence for listeriosis was 0.7% and for nocardiosis, 3.6% . We found descriptions of 14 more heart transplant patients with PCP in the medical literature . Data from the 19 patients follow . Mean age was 52 years, and PCP was diagnosed a median of 75 days after heart transplant (range, 37-781 d) . Clinical presentation was acute (less than 48 h) with fever (89%), shortness of breath (84%), dry cough (74%), and hypoxia (63%) . Cytomegalovirus was isolated from lung or blood in 74% of patients . Chest X-ray usually showed interstitial pneumonia (84%) . Three patients required ventilatory support . All patients were treated with trimethoprim-sulfamethoxazole (TMP/SMX) (4 also with corticosteroids and 5 with ganciclovir) . Mortality was 26% . Older age was the only significant poor prognostic factor (61 versus 49 years; p < 0.03) . From March 1994, 50 heart transplant patients were given TMP/SMX prophylaxis at our institution (1 double-strength tablet, 160/800 mg, every 12 hours on Saturdays and Sundays), and no new cases of PCP, Listeria or Nocardia have been detected since then . Tolerance has been excellent . Heart transplant recipients are at a substantial risk of PCP pneumonia, which presents with an abrupt onset and a high mortality . Weekend TMP/SMX chemoprophylaxis was very effective at our institution.

J Exp Med, 1997 Nov 17, 186(10), 1757 - 62
Defects in macrophage recruitment and host defense in mice lacking the CCR2 chemokine receptor; Kurihara T et al.; Chemokines are a structurally related family of cytokines that are important for leukocyte trafficking . The C-C chemokine monocyte chemoattractant protein-1 (MCP-1) is a potent monocyte activator in vitro and has been associated with monocytic infiltration in several inflammatory diseases . One C-C chemokine receptor, CCR2, has been identified that mediates in vitro responses to MCP-1 and its close structural homologues . CCR2 has also recently been demonstrated to be a fusion cofactor for several HIV isolates . To investigate the normal physiological function of CCR2, we generated mice with a targeted disruption of the ccr2 gene . Mice deficient for CCR2 developed normally and had no hematopoietic abnormalities . However, ccr2(-/-) mice failed to recruit macrophages in an experimental peritoneal inflammation model . In addition, these mice were unable to clear infection by the intracellular bacteria, Listeria monocytogenes . These results suggest that CCR2 has a nonredundant role as a major mediator of macrophage recruitment and host defense against bacterial pathogens and that MCP-1 and other CCR2 ligands are effectors of those functions.

Pediatr Dent, 1997 Sep-Oct, 19(6), 404 - 8
Acute ethanol toxicity from ingesting mouthwash in children younger than 6-years of age; Shulman JD et al.; The purpose of our study was to analyze reports of the American Association of Poison Control Centers (AAPCC) of suspected overingestion of ethanol from mouthrinses by children younger than 6 years of age between 1989 and 1994 . Annual incidence rates of reported ethanol exposures attributed to mouthrinses were calculated . Lethal and toxic amounts of several mouthrinses were calculated using peak blood ethanol concentrations of 500 and 50 mg per 100 mL, respectively . In 1994, there were 2937 calls reported by poison control centers related to ethanol-containing mouthrinses, an estimated incidence of 168 reported exposures per 100,000 children younger than 6 years of age . A 15-kg child who ingests 212 mL (7.2 oz.) of Listerine (26.9% ethanol) ingests 57 mL (1.9 oz.) of ethanol, which is potentially lethal . Approximately one-tenth that amount of ethanol can produce a toxic reaction . Physicians, dentists, and other health care providers should inform parents of the dangers associated with accidental ingestion of mouthrinse and encourage them to keep mouthrinse out of the reach of children . The Food and Drug Administration (FDA) should require readily visible warning labels and child-resistant caps for containers with potentially toxic volumes of ethanol . The American Dental Association (ADA) should re-evaluate its acceptance criteria for advertising cosmetic mouthrinses in its publications and consider including child-resistant caps and warning labeling.

Appl Environ Microbiol, 1997 Dec, 63(12), 4945 - 7
A mutant of Listeria monocytogenes LO28 unable to induce an acid tolerance response displays diminished virulence in a murine model; Marron L et al.; Exposing Listeria monocytogenes LO28 to sublethal pH induces protection against normally lethal pH conditions, a phenomenon known as the acid tolerance response . We identified a mutant, L . monocytogenes ATR1, which is incapable of inducing such tolerance, either against low pH or against any other stress tested . The virulence of this mutant was considerably decreased, suggesting that the acid tolerance response contributes to in vivo survival of L . monocytogenes.

Appl Environ Microbiol, 1997 Dec, 63(12), 4770 - 7
Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles; Chen Y et al.; The purpose of this study was to characterize in detail the binding of pediocin PA-1 and its fragments to target membranes by using tryptophan fluorescence as a probe . Based on a three-dimensional model (Y . Chen, R . Shapira, M . Eisenstein, and T . J . Montville, Appl . Environ . Microbiol . 63:524-531, 1997), four synthetic N-terminal pediocin fragments were selected to study the mechanism of the initial step by which the bacteriocin associates with membranes . Binding of pediocin PA-1 to vesicles of phosphatidylglycerol, the major component of Listeria membranes, caused an increase in the intrinsic tryptophan fluorescence intensity with a blue shift of the emission maximum . The Stern-Volmer constants for acrylamide quenching of the fluorescence of pediocin PA-1 in buffer and in the lipid vesicles were 8.83 +/- 0.42 and 3.53 +/- 0.67 M-1, respectively, suggesting that the tryptophan residues inserted into the hydrophobic core of the lipid bilayer . The synthetic pediocin fragments bound strongly to the lipid vesicles when a patch of positively charged amino acid residues (K-11 and H-12) was present but bound weakly when this patch was mutated out . Quantitative comparison of changes in tryptophan fluorescence parameters, as well as the dissociation constants for pediocin PA-1 and its fragments, revealed that the relative affinity to the lipid vesicles paralleled the net positive charge in the peptide . The relative affinity for the fragment containing the YGNGV consensus motif was 10-fold lower than that for the fragment containing the positive patch . Furthermore, changing the pH from 6.0 to 8.0 decreased binding of the fragments containing the positive patch, probably due to deprotonation of His residues . These results demonstrate that electrostatic interactions, but not the YGNGV motif, govern pediocin binding to the target membrane.

Eur J Clin Microbiol Infect Dis, 1997 Oct, 16(10), 756 - 60
Pseudo-outbreak of listeriosis elucidated by pulsed-field gel electrophoresis; La Scola B et al.; Listeria monocytogenes was isolated from three patients hospitalized in two departments of the same hospital over a two-week period . A nosocomial outbreak of listeriosis was suspected . Patients presented with tenosynovitis, central venous catheter infection, and bacteremia . The first patient had a community-acquired infection, the second a nosocomial infection, and the source of the third patient's illness was uncertain . Epidemiological investigations failed to identify a common source of contamination within the hospital . The three strains were nontypeable by phage typing, but Smal macrorestriction analysis and pulsed-field gel electrophoresis yielded three distinct profiles . Therefore, the three cases seemed to represent a cluster of sporadic cases as opposed to an outbreak of listeriosis . Rapid typing of isolates is essential in the early investigation of potential outbreaks of listeriosis and may prevent the initiation of expensive and time-consuming epidemiological investigations.

Bioconjug Chem, 1997 Nov-Dec, 8(6), 781 - 4
Listeriolysin O potentiates immunotoxin and bleomycin cytotoxicity; Kerr DE et al.; Antitumor immunotoxins were formed by covalently attaching the ribosome-inactivating protein ricin A chain (RA) to the antitumor antibodies BR96 and L6 . In vitro cytotoxicity assays established that BR96-RA was cytotoxic to H2987 human lung adenocarcinoma cells (IC50 = 6 nM), while L6-RA exhibited very low levels of cytotoxic activity (18% cell kill at 67 nM) . The virulence factor from the intracellular pathogen Listeria monocytogenes, listeriolysin O (LLO), was able to potentiate the cytotoxicity of BR96-RA and L6-RA by 120- and > 1340-fold, respectively, resulting in IC50 values of approximately 50 pM . LLO also potentiated the cytotoxicity of the peptide anticancer drug bleomycin by a factor of > 2500 but had no effect on the cytotoxic activities of the anticancer drugs cytarabine and etoposide phosphate . In addition, LLO did not potentiate the cytotoxic activity of unconjugated ricin A chain or L6-RA on H2987 cells that were saturated with L6 prior to conjugate treatment . These results are attributed to LLO-induced alteration of the intracellular trafficking of molecules that are incorporated into acidic vesicles.

J Leukoc Biol, 1997 Dec, 62(6), 795 - 804
Effector molecules in expression of the antimicrobial activity of macrophages against Mycobacterium avium complex: roles of reactive nitrogen intermediates, reactive oxygen intermediates, and free fatty acids; Akaki T et al.; We studied microbicidal activities of reactive nitrogen intermediates (RNI), free fatty acids (FFA), and reactive oxygen intermediates (ROI) against Mycobacterium avium complex (MAC) and the mode of macrophage (mphi) production of these effectors . (1) Intracellular growth of MAC in murine peritoneal mphis was accelerated by scavengers for ROI or RNI and inhibitors of nitric oxide synthase or phospholipase A2, indicating roles of ROI, RNI, and FFA in mphi anti-MAC functions . (2) Acidified NaNO2-derived RNI, FFA (linolenic and arachidonic acids), and the H2O2-mediated halogenation system exhibited a significant anti-MAC bactericidal activity . The combination of RNI with FFA showed a synergistic effect . However, the H2O2-halogenation system in combination with either RNI or FFA showed an antagonism . When Listeria monocytogenes (Lm) was used as a target organism, the combinations of RNI + FFA and RNI + H2O2-halogenation gave a synergistic effect, whereas FFA + H2O2-halogenation showed an antagonism in exerting bactericidal activity . In addition, when ROI generated by the xanthine oxidase-acetaldehyde system was combined with RNI, anti-Lm but not anti-MAC activity was potentiated . (3) ROI production by murine peritoneal mphis was observed immediately after contact with MAC organisms (MAC stimulation) and ceased within 2 h . FFA release was seen 1-24 h after MAC stimulation . RNI production was initiated from 3 h and increased during the first 36 h and continued at least for 4 days . These findings suggest that RNI and FFA rather than ROI are important effectors of anti-MAC functions of mphis, and the collaborating action of RNI with FFA temporarily participates in mphi-mediated killing of MAC in the relatively early phase after MAC stimulation.

J Leukoc Biol, 1997 Dec, 62(6), 726 - 32
Innate resistance to Listeria monocytogenes in tumor-bearing mice; Gahan CG et al.; We have previously described the isolation, cloning, and characterization of a tumorigenic murine fibrosarcoma, designated JBS . Growth of JBS tumors in syngeneic mice initiates an anti-tumor immune response that initially manifests as progressive splenic hyperplasia and an increased proliferative ability in cultured splenocytes . In animals with tumors progressing beyond the 2 cm stage there is a reduction in spleen size and a gradual decrease in splenocyte proliferative abilities, leading to anergy at heavy tumor burdens (>3.5 cm) . During the phase of immune hyperresponsiveness in tumor-bearing mice clearance of Listeria monocytogenes by components of the innate immune system is increased . This heightened resistance to infection is most likely macrophage-mediated because these mice demonstrate an increased ability to recruit macrophages to the peritoneal cavity during Listeria infection . In addition, these macrophages are highly activated in vivo as evidenced by an elevated capacity to express class II MHC (Ia) molecules . This increase in macrophage activation status is coincident with an increased capacity of splenocytes from tumor-bearing mice to secrete IFN-gamma . In mice with much heavier tumor burdens (>3.5 cm), down-regulation of the immune response leads to a reduction in peritoneal macrophage numbers, decreased macrophage Ia expression, and diminished splenic clearance of L . monocytogenes . Our data demonstrate that activation of macrophages distal to the tumor site occurs as an initial consequence of tumor growth . It is only in mice with very heavy tumor burdens that functionality of macrophages is sufficiently suppressed to allow increased splenic growth of L . monocytogenes.

Infect Immun, 1997 Dec, 65(12), 5326 - 9
A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytolytic T lymphocytes; Busch DH et al.; Listeria monocytogenes is an intracellular bacterium that secretes proteins into the cytosol of infected macrophages . Major histocompatibility complex (MHC) class I molecules bind peptides that are generated by the degradation of bacterial proteins and present them to cytolytic T lymphocytes (CTL) . In this study we have investigated CTL responses in L . monocytogenes-immunized mice to peptides that (i) derive from the L . monocytogenes proteins phosphatidylinositol-specific phospholipase C, lecithinase (most active on phosphatidylcholine), metalloprotease (Mpl), PrfA, and the ORF-A product and (ii) conform to the binding motif of the H2-Kd MHC class I molecule . We identified a nonamer peptide, Mpl 84-92, that is presented to L . monocytogenes-specific CTL by H2-Kd MHC class I molecules . Unlike other motif-conforming peptides derived from the secreted Mpl of L . monocytogenes, Mpl 84-92 is bound with high affinity by H2-Kd . Mpl 84-92 is the fourth L . monocytogenes-derived peptide found to be presented to CTL by the H2-Kd molecule during infection and demonstrates the importance of high-affinity interactions between antigenic peptides and MHC class I molecules for CTL priming.

Infect Immun, 1997 Dec, 65(12), 5279 - 88
Comparison of inducible nitric oxide synthase expression in the brains of Listeria monocytogenes-infected cattle, sheep, and goats and in macrophages stimulated in vitro; Jungi TW et al.; The expression of inducible nitric oxide synthase (iNOS) was studied in the brains of cattle, sheep, and goat that succumbed to a natural infection with Listeria monocytogenes . The lesions in infected brains are characterized by microabscesses, perivascular cuffs, gliosis, glial nodules, and large areas of malacia . Using immunocytochemistry, we detected bacteria in microabscesses, particularly in sheep and goats, and in areas without signs of inflammation, but not in perivascular infiltrates . iNOS was expressed by macrophage (Mphi)-type cells of microabscesses and glial nodules but rarely by Mphi in areas of malacia, as determined by immunohistochemistry with iNOS-specific antibodies . iNOS was not detected in perivascular cuffs . Major histocompatibility complex class II molecules (MHC-II), another marker of cell activation, showed a different pattern of distribution . Perivascular cuffs contained high numbers of MHC-II-positive cells, including some with Mphi characteristics . Microabscesses in sheep and goats showed low expression of MHC-II, particularly in iNOS-expressing cells . In cattle, the expression of markers for activated or recruited phagocytes, the calcium-binding proteins S100A8 and S100A9 (formerly called MRP-8 and MRP-14, respectively), was largely restricted to cells showing weak or undetectable iNOS expression; iNOS-positive Mphi showed a low expression of S100A8 and S100A9 . Thus, iNOS is expressed by a restricted subset of Mphi in listeric encephalitis . In cultured sheep and goat Mphi, a low proportion of cells expressed iNOS upon activation by L . monocytogenes and gamma interferon, resulting in nitrite generation at least 1 order of magnitude lower than that in similarly treated cattle Mphi . Since these species differences were much less obvious in vivo, it appears that the well-known species variation in iNOS expression by Mphi could reflect an in vitro phenomenon.

Infect Immun, 1997 Dec, 65(12), 5137 - 41
Internalin B promotes the replication of Listeria monocytogenes in mouse hepatocytes; Gregory SH et al.; The uptake of Listeria monocytogenes by a variety of cell types in vitro is facilitated by the protein products of the inlAB (internalin) operon expressed by the organism . In the case of mouse hepatocytes, the extent to which inlAB expression influenced the uptake of Listeria in vitro was markedly dependent upon the ratio of bacteria to cells . At a ratio of 100:1, greater than 40-fold fewer transposon-induced inl4B mutant listeriae entered hepatocytes compared to the isogenic wild-type control; the difference was only fourfold, however, in cultures inoculated at a 1:1 ratio . Similarly, the uptake of in-frame inlB or inlAB deletion mutants differed only fourfold from the uptake of wild-type or inlA mutant Listeria at a 1:1 multiplicity of infection . Mutations affecting inlB or inlAB, on the other hand, resulted in a marked decrease in the capacity of Listeria to proliferate within mouse hepatocytes in vivo and in vitro . Electron micrographs of Listeria-infected hepatocytes demonstrated the impaired capacity of inlB mutants to escape from endocytic vacuoles and to enter the cytoplasm where proliferation occurs . These findings indicate that the protein product of inlB exerts a significant effect on the intracellular replication of Listeria.

Infect Immun, 1997 Dec, 65(12), 5003 - 9
Transient control of interleukin-4-producing natural killer T cells in the livers of Listeria monocytogenes-infected mice by interleukin-12; Emoto Y et al.; Unconstrained development of gamma interferon (IFN-gamma)-secreting natural killer (NK) cells and T helper (Th) 1 cells is central to protection against Listeria monocytogenes . In contrast, interleukin 4 (IL-4) is considered harmful . IL-12 produced by infected macrophages promotes, and IL-4 interferes with, protective antilisterial immunity . The liver NK T lymphocytes, which are a potent source of IL-4, are downregulated at an intermediate stage of listeriosis . Here we demonstrate that endogenous IL-12 participates in the control of IL-4-producing liver NK T lymphocytes during listeriosis . The effects of L . monocytogenes infection on IL-4-producing liver NK T lymphocytes were reversed by antibody neutralization of IL-12 but not of IFN-gamma or tumor necrosis factor alpha (TNF-alpha) . IL-4 production by liver NK T lymphocytes was virtually unaffected by heat-killed L . monocytogenes (HKL) . Viable L . monocytogenes markedly increased the numbers of IL-12 producers in livers in parallel with an increase in macrophage numbers, whereas HKL failed to do so with similar efficiency . These results indicate that in the liver endogenous IL-12 improves protective immunity against listeriosis by downregulating IL-4-producing NK T lymphocytes . Moreover, our findings that HKL have a low level of IL-12-inducing activity and fail to control IL-4-producing NK T lymphocytes in the liver are consistent with the lesser protective capacity of HKL compared to that of live listeriae.

J Bacteriol, 1997 Nov, 179(22), 7174 - 80
Glucose-1-phosphate utilization by Listeria monocytogenes is PrfA dependent and coordinately expressed with virulence factors; Ripio MT et al.; Virulence genes of the facultative intracellular pathogen Listeria monocytogenes are coordinately regulated by the activator protein PrfA, encoded by prfA, a member of the cyclic AMP receptor protein family of bacterial transcription factors . We found that prfA* mutants that constitutively overexpress the virulence regulon due to a Gly145Ser substitution in PrfA (M.-T . Ripio, G . Dominguez-Bernal, M . Lara, M . Suarez, and J.-A . Vazquez-Boland, J . Bacteriol . 179:1533-1540, 1997) rapidly utilized glucose-1-phosphate (G-1-P) as a carbon source for growth, in contrast to wild-type strains, which characteristically do not . Wild-type strains acquired the capacity for readily metabolizing G-1-P upon exposure to environmental conditions that activate the expression of prfA and PrfA-dependent virulence genes (i.e., culture at 37 degrees C in charcoal-treated medium) . In these strains, G-1-P utilization followed an expressional pattern identical to that of virulence genes controlled by PrfA, with repression at 20 degrees C . Tn917 insertions in L . monocytogenes mutants selected for G-1-P utilization deficiency mapped to the plcA-prfA operon, a deltaprfA strain was totally unable to utilize G-1-P, and trans complementation with prfA constructs restored the ability to efficiently metabolize and grow on G-1-P to these mutants . Thus, G-1-P utilization by L . monocytogenes is under the tight positive control of the central virulence regulator, PrfA, and is coexpressed with PrfA-dependent pathogenicity determinants . It was recently reported that readily utilized carbohydrates, such as glucose or cellobiose, repress virulence genes in L . monocytogenes . We confirmed this but, interestingly, found that G-1-P does not inhibit expression of the PrfA regulon, indicating that this sugar follows a catabolic pathway that bypasses the repressor mechanism triggered by other readily metabolized carbon sources . PrfA dependence and coexpression with virulence genes suggest that utilization of exogenous G-1-P may be relevant to Listeria pathogenesis . G-1-P is the precursor metabolite and primary degradation product of glycogen and is therefore available within the mammalian cell . Based on our results, we hypothesize that G-1-P could play an important role as a growth substrate for intracellular Listeria.

J Bacteriol, 1997 Nov, 179(22), 6979 - 85
Betaine and L-carnitine transport by Listeria monocytogenes Scott A in response to osmotic signals; Verheul A et al.; The naturally occurring compatible solutes betaine and L-carnitine allow the food-borne pathogen Listeria monocytogenes to adjust to environments of high osmotic strength . Previously, it was demonstrated that L . monocytogenes possesses an ATP-dependent L-carnitine transporter (A . Verheul, F . M . Rombouts, R . R . Beumer, and T . Abee, J . Bacteriol . 177:3205-3212, 1995) . The present study reveals that betaine and L-carnitine are taken up by separate highly specific transport systems and support a secondary transport mechanism for betaine uptake in L . monocytogenes . The initial uptake rates of betaine and L-carnitine are not influenced by an osmotic upshock, but the duration of transport of both osmolytes is directly related to the osmotic strength of the medium . Regulation of uptake of both betaine and L-carnitine is subject to inhibition by preaccumulated solute . Internal betaine inhibits not only transport of external betaine but also that of L-carnitine and, similarly, internal L-carnitine inhibits transport of both betaine and L-carnitine . The inhibition is alleviated upon osmotic upshock, which suggests that alterations in membrane structure are transmitted to the allosteric binding sites for betaine and L-carnitine of both transporters at the inner surface of the membrane . Upon osmotic downshock, betaine and L-carnitine are rapidly released by L . monocytogenes as a consequence of activation of a channel-like activity . The osmolyte-sensing mechanism described is new and is consistent with various unexplained observations of osmoregulation in other bacteria.

Eur J Immunol, 1997 Oct, 27(10), 2549 - 56
Normal macrophage functions, but impaired induction of gamma delta T cells, at the site of bacterial infection in CD45 exon 6-deficient mice; Fujise S et al.; We investigated the protective functions of macrophages and gamma delta T cells in adult CD45 exon 6-deficient (CD45 -/-) mice against an intraperitoneal (i.p.) infection with Listeria monocytogenes . gamma delta T cells are preferentially localized in the spleen, liver, and intraperitoneal cavity of the adult CD45-/- mice . Increased numbers of gamma delta T cells were observed after i.p . infection with L . monocytogenes in the peritoneal cavity of C57BL/6 (CD45 +/+) mice but not in CD45 -/- mice . The gamma delta T cells showed predominant usage of V delta 5 and V delta 6 rearranged to J delta 1 in the infected CD45 -/- mice which are the same as those used by resident gamma delta T cells of noninfected CD45 +/+ and CD45 -/- mice . Furthermore, we analyzed the protective abilities of the CD45 -/-, CD45 +/+, and gamma delta T cell-depleted mice at the early stage of the listerial infection . The numbers of bacteria in the spleens and livers of the CD45 -/- mice 5 days after the listerial infection were almost ten times larger than those in the CD45 -/- and gamma delta T cell-depleted CD45 +/+ mice . Macrophages showed normal antigen presentation, nitric oxide production and bactericidal activity for L . monocytogenes despite their lacking CD45 surface expression, suggesting that CD45-negative macrophages have a minimal influence on the increased bacterial multiplication in the CD45-/- mice . These results suggest that the gamma delta T cells are induced by the bacterial infection in a CD45-dependent manner, and that unresponsiveness of the gamma delta T cells results in only weak protection against L . monocytogenes in CD45 -/- mice.

J Leukoc Biol, 1997 Nov, 62(5), 577 - 80
In vivo properties of monocyte chemoattractant protein-1; Gu L et al.; Monocyte chemoattractant protein-1 (MCP-1) attracts monocytes, memory T lymphocytes, and natural killer (NK) cells in vitro . Its expression has been documented in disorders characterized by mononuclear cell infiltrates, suggesting that it may contribute to the inflammatory component of such diseases as atherosclerosis, multiple sclerosis, or rheumatoid arthritis . To prove a causal association, the in vivo properties of MCP-1 must be understood . Several lines of transgenic mice have been constructed to address this question . A transgenic line in which MCP-1 expression is controlled by the MMTV-LTR expressed high levels of MCP-1 in multiple organs but showed no evidence for monocyte infiltration . Instead, these mice were more susceptible to infection by the intracellular pathogens, Listeria monocytogenes and Mycobacterium tuberculosis . These mice had high serum levels of MCP-1, suggesting that their circulating monocytes may have been desensitized or that MCP-1 stimulated a Th2-dominant response . In contrast, another model in which MCP-1 expression was controlled by the insulin promoter demonstrated a monocytic infiltrate in pancreatic islets . These results indicate that MCP-1 expression at low levels in an anatomically confined area results in monocyte infiltration, suggesting that when properly expressed, MCP-1's in vitro properties are reproduced in vivo . This justifies the examination of MCP-1-deficient mice in disease models in order to explore MCP-1's role in pathogenesis.

Appl Environ Microbiol, 1997 Nov, 63(11), 4441 - 8
Sensitive detection of viable Listeria monocytogenes by reverse transcription-PCR; Klein PG et al.; Detection of pathogens in contaminated food products by PCR can result in false-positive data due to the amplification of DNA from nonviable cells . A new method based on reverse transcription-PCR (RT-PCR) amplification of mRNA for the specific detection of viable Listeria monocytogenes was developed . The expression of three L . monocytogenes genes, iap, hly, and prfA, was examined to determine a suitable target for amplification of RT-PCR . Total RNA from L . monocytogenes was isolated, and following DNase treatment, the RNA was amplified by both RT-PCR and PCR with primers specific for the three genes . Amplicon detection was accomplished by Southern hybridization to digoxigenin-labeled gene probes . The levels of expression of these three genes differed markedly, and the results indicated that the iap gene would provide a good target for development of a specific method for detection of viable L . monocytogenes based on RT-PCR amplification . After a 1-h enrichment, the 371-bp iap-specific product was detected with a sensitivity of ca . 10 to 15 CFU/ml from pure culture . Detection of the 713-bp hly-specific amplicon was ca . 4,000 times less sensitive after 1 h, whereas detection of the 508-bp prfA product showed the lowest level of sensitivity, with detection not observed until after a 5-h enrichment period . The amplification of the iap mRNA was specific for L . monocytogenes . Overall, the assay could be completed in ca . 54 h . The use of RT-PCR amplification for the detection of viable L . monocytogenes was validated in artificially contaminated cooked ground beef . Following a 2-h enrichment incubation, the iap-specific amplification product could be detected in a cooked meat sample that was originally inoculated with ca . 3 CFU/g . These results support the usefulness of RT-PCR amplification of mRNA as a sensitive method for the specific detection of viable L . monocytogenes and indicate that this method may prove useful in the detection of this pathogen in ready-to-eat, refrigerated meat products.

Ann Ig, 1997 Jul-Aug, 9(4), 281 - 8
Identification of Listeria monocytogenes by colony hybridization test using the virulence-associated hly and inlA genes as probes; Petrone G et al.; We developed a method of identification of Listeria monocytogenes based on colony hybridization with nonradioactively labeled DNA probes, represented by the hly and inlA virulence-associated genes . The procedure described in this paper results simple, rapid, specific and reproducible . Since it can be performed in a short time, the above technique can be applied to detect L . monocytogenes from different source and constitutes a noteworthy and alternative tool to identify this gram-positive pathogenic bacterium.

J Infect, 1997 Sep, 35(2), 195 - 7
Sporadic case of listeriosis associated with the consumption of a Listeria monocytogenes-contaminated 'Camembert' cheese; Gilot P et al.; Listeria monocytogenes is an intracellular gram-positive organism responsible for severe infections in both humans and animals . Whereas the food-borne transmission of listeriosis was demonstrated in several outbreaks, most cases of listeriosis occur sporadically and are rarely linked with consumption of contaminated foods . In this paper a case of septicaemia with L . monocytogenes in a 73-year-old immunocompromised man is described . Evidence for the association of this case of listeriosis with the consumption of a contaminated 'Camembert' cheese is provided by serotyping, esterase typing, DNA macrorestriction patterns analysis and level of virulence of the isolated strains for mice.

Zentralbl Hyg Umweltmed, 1996 Nov, 199(1), 60 - 8
Prevalence of Listeria monocytogenes and other listerias in Italian-made soft cheeses; Pinto B et al.; The frequency of L . monocytogenes and other listerias was determined in different types of Italian-made soft cheeses purchased from retail outlets (shops and supermarkets) located in different areas and different towns of central Italy . Of the 164 examined samples, eight proved to be positive for L . monocytogenes (4.9%), seven strains belonged to serotype 1, and one strain to serotype 4 . Thirty-six samples were positive for the presence of other listeria species (22%); of these, L . innocua was prevalent (72% of positive samples) . The cheeses bought in supermarkets displayed a higher and statistically significant (Fisher's exact test: 0.0016) positivity for Listeria spp . than those sold by the shops, independently of the type of cheese . One particular type of cheese proved to be frequently contaminated (Fis . ex . test: 0.0013) . Technical, analytical and epidemiological aspects are discussed.

Zentralbl Hyg Umweltmed, 1996 Nov, 199(1), 51 - 9
Occurrence and detection of viable Listeria in food scrap compost; Droffner ML et al.; Listeria species (L . innocua, L . ivanovii, L . seeligeri, and L . grayi) were readily detected in food scraps by Nucleic Acid Hybridization (NAH) probes using a standard Listeria selective medium (UVM-1) at ambient temperature . Various food scrap compost recipes artificially contaminated with Listeria at 10(7) cells per gram wet weight were composted in thermally insulated bench scale reactor vessels . These Listeria were not detected when the compost temperature became elevated . Different isolation methods for the Listeria showed this result to be a false negative occurring apparently because the heat stressed Listeria were unable to survive in the selective medium (UVM-1) . Once incubated at 37 degrees C in Universal Listeria medium (ULM), the Listeria were detectable for a short period in compost at temperatures as high as 64 degrees C.

Zentralbl Hyg Umweltmed, 1995 Dec, 198(2), 124 - 37
Comparison of different selective methods for detection of Listeria species in surface water; Bernagozzi M et al.; Tests were carried out to evaluate the efficiency of various combinations of selective enrichment and plating techniques using pure cultures of Listeria monocytogenes, Listeria seeligeri and Listeria innocua in suspension and samples of surface water . The best yields for the various Listeria spp . were obtained after a single passage in LEB (Oxoid) or LEB Buffered (Oxoid) and after using Palcam and Oxford agar (Oxoid) . Palcam agar was, however, shown to be the most efficient means of detecting the Listeria spp . in the natural water samples.

Lett Appl Microbiol, 1997 Oct, 25(4), 257 - 60
Significance of temperature and preincubation temperature on survival of Listeria monocytogenes at pH 4.8; Gay M et al.; Listeria monocytogenes is a food-borne pathogenic bacterium that can be found in soft cheese . At the beginning of cheese ripening, the pH is about 4.85-4.90 . The aim of this work was to study the influence of temperature, preincubation temperature (temperature at which the inoculum was cultivated) and initial bacterial concentration on the survival of L . monocytogenes (strain Scott A) at pH 4.8 . It was demonstrated in an earlier study that these factors did influence growth kinetics . Survival studies of L . monocytogenes were done in a laboratory broth simulating cheese composition . Four test temperatures (2, 6, 10 and 14 degrees C) and two preincubation temperatures were studied (30 degrees C or the test temperature) . Listeria monocytogenes (strain Scott A) was unable to grow at pH 4.8 under all conditions tested . The time for 10% survival was about 11 and 2 d, at 2 degrees C with preincubation at 2 degrees C and 30 degrees C, respectively; 9 d at 6 degrees C with preincubation at 6 degrees C; 4 d at 6 degrees C with preincubation at 30 degrees C; and 1 d at 14 degrees C with preincubation at 14 degrees C or at 30 degrees C . The results show that survival of L . monocytogenes (strain Scott A) at pH 4.8 is not dependent on initial bacterial concentration but on both the test and preincubation temperatures.

J Appl Microbiol, 1997 Sep, 83(3), 381 - 8
Subtyping of Listeria monocytogenes on the basis of plasmid profiles and arsenic and cadmium susceptibility; McLauchlin J et al.; The susceptibilities to arsenic and cadmium together with the detection of plasmid DNA were evaluated for use as epidemiological markers for the subtyping of Listeria monocytogenes . Plasmid DNA was detected in 34% of 322 apparently unrelated isolates of L . monocytogenes . The resistance to cadmium and arsenic differentiated 565 apparently unrelated cultures into four groups, the smallest being 5% of cultures resistant to both agents, and the largest (53%) being sensitive to cadmium and resistant to arsenic . The resistance patterns to these agents and the presence of plasmid DNA varied markedly between the serotypes of the cultures . The detection of plasmid DNA was strongly associated with cadmium resistance in serogroup 1/2 cultures, but not within those of serogroup 4 . Arsenic resistance was not associated with plasmid DNA . All methods were sufficiently stable to be useful for epidemiology investigations . The techniques described here offer simple methods which can be easily utilized in laboratories without a specialized expertise for this bacterium.

J Clin Microbiol, 1997 Nov, 35(11), 2904 - 7
An outbreak of listeriosis suspected to have been caused by rainbow trout; Ericsson H et al.; An outbreak of listeriosis in Sweden, consisting of nine cases, was investigated by means of molecular typing of strains from patients and strains isolated from suspected foodstuffs, together with interviews of the patients . Listeria monocytogenes was isolated from six of the patients, and all isolates were of the same clonal type . This clonal type was also isolated from a "gravad" rainbow trout, made by producer Y, found in the refrigerator of one of the patients . Unopened packages obtained from producer Y were also found to contain the same clonal type of L . monocytogenes . Based on the interview results and the bacteriological typing, we suspect that at least six of the nine cases were caused by gravad or cold-smoked rainbow trout made by producer Y . To our knowledge, this is the first rainbow trout-borne outbreak of listeriosis ever reported.

Am J Pathol, 1997 Oct, 151(4), 897 - 904
Evidence for intrathecal synthesis of alternative pathway complement activation proteins in experimental meningitis; Stahel PF et al.; Complement has been shown to contribute to intrathecal inflammation in bacterial meningitis . However, the cellular source of complement in the infected central nervous system has not been determined . In this study, we analyzed protein and mRNA expression of two alternative pathway complement activation proteins, C3 and factor B, in the brains of mice with Listeria monocytogenes meningitis . Complement protein levels were found elevated in the cerebrospinal fluid of infected mice, compared with mock-infected animals . In the course of the disease, enhanced C3 and factor B mRNA expression was detected on pyramidal neurons and Purkinje cells within 6 hours, peaking at 12 hours and then gradually decreasing by 72 hours after infection . In addition, leukocytes infiltrating the subarachnoid space, within 12 to 24 hours, expressed mRNA for C3 and factor B . The cellular infiltration increased dramatically up to 72 hours . Intraperitoneal injection of tumor necrosis factor (TNF)-alpha up-regulated C3 and factor B mRNA expression on neurons in normal mice, suggesting that TNF-alpha may represent one cytokine regulating complement expression in this model of bacterial meningitis . However, additional mediators may be involved in regulation of intrathecal complement expression, as infected mice deficient of TNF/lymphotoxin-alpha genes did not demonstrate attenuated complement expression in the brain.

Appl Environ Microbiol, 1997 Oct, 63(10), 3887 - 94
Critical role of anteiso-C15:0 fatty acid in the growth of Listeria monocytogenes at low temperatures; Annous BA et al.; Listeria monocytogenes is a food-borne pathogen capable of growth at refrigeration temperatures . Membrane lipid fatty acids are major determinants of a sufficiently fluid membrane state to allow growth at low temperatures . L . monocytogenes was characterized by a fatty acid profile dominated to an unusual extent (> 95%) by branched-chain fatty acids, with the major fatty acids being anteiso-C15:0, anteiso-C17:0, and iso-C15:0 in cultures grown in complex or defined media at 37 degrees C . Determination of the fatty acid composition of L . monocytogenes 10403S and SLCC 53 grown over the temperature range 45 to 5 degrees C revealed two modes of adaptation of fatty acid composition to lower growth temperatures: (i) shortening of fatty acid chain length and (ii) alteration of branching from iso to anteiso . Two transposon Tn917-induced cold-sensitive mutants incapable of growth at low temperatures had dramatically altered fatty acid compositions with low levels of i-C15:0, a-C15:0, and a-C17:0 and high levels of i-C14:0, C14:0, i-C16:0, and C16:0 . The levels of a-C15:0 and a-C17:0 and the ability to grow at low temperatures were restored by supplementing media with 2-methylbutyric acid, presumably because it acted as a precursor of methylbutyryl coenzyme A, the primer for synthesis of anteiso odd-numbered fatty acids . When mid-exponential-phase 10403S cells grown at 37 degrees C were temperature down-shocked to 5 degrees C they were able, for the most part, to reinitiate growth before the membrane fatty acid composition had reset to a composition more typical for low-temperature growth . No obvious evidence was found for a role for fatty acid unsaturation in adaptation of L . monocytogenes to cold temperature . The switch to a fatty acid profile dominated by a-C15:0 at low temperatures and the association of cold sensitivity with deficiency of a-C15:0 focus attention on the critical role of this fatty acid in growth of L . monocytogenes in the cold, presumably through its physical properties and their effects, in maintaining a fluid, liquid-crystalline state of the membrane lipids.

J AOAC Int, 1997 Sep-Oct, 80(5), 1139 - 42
Comparative study of colorimetric and fully automated enzyme-linked immunoassay system for rapid screening of Listeria spp . in foods; Kerdahi KF et al.; Two enzyme-linked immunoassay (ELISA) systems for rapid screening of Listeria spp . were compared for their use in analysis of spiked foods regulated by the U.S . Food and Drug Administration . The Tecra Listeria kit is a 48 h visual ELISA that detects Listeria spp . through colorimetry . It has been approved for first action by AOAC INTERNATIONAL . The Vitek immunodiagnostic assay system for Listeria (VIDAS LIS) is a fully automated 48 h ELISA that detects Listeria spp . by immunofluorescence . Fifty-two food samples were artificially contaminated with high (11-42 colony-forming units {cfu}/25 g food) and low (2-8 cfu/25 g food) levels of L . monocytogenes and screened by the 2 protocols . Unspiked samples were also assayed as negative controls . Six unspiked samples were found positive for Listeria spp . by both methods: 3 were identified as L . monocytogenes and 3 as L . innocua by official methods . Both ELISA methods detected all spiked samples . One unspiked sample was assayed positive by Tecra and negative by VIDAS LIS . No Listeria spp . were recovered when the sample was tested by the conventional method . No interference due to background fluorescence of food matrixes was observed in the VIDAS LIS method . Results suggest a modified VIDAS LIS preenrichment medium may be used in place of the VIDAS standard medium in the protocol.

Fundam Appl Toxicol, 1997 Sep, 39(1), 53 - 9
Effects of fumonisin B1 on the immune system of sprague-dawley rats following a 14-day oral (gavage) exposure; Tryphonas H et al.; The effects of fumonisin B1 (FB1) on the immune system of Sprague-Dawley rats were investigated . Groups of male and female rats (10 rats/group) were gavaged daily for 14 days with doses of 0, 5, 15, and 25 mg/kg body wt/day and the primary (IgM) response to sheep red blood cells expressed as plaque-forming cell numbers/10(6) spleen mononuclear leukocytes (PFC/10(6) splenocytes) and PFC/spleen was determined . There was a significant dose-related linear trend toward decreased PFC/10(6) splenocytes (p = 0.003) and PFC/spleen cells (p = 0.001) in the male rats . Body weights, expressed as a percentage of the control, were significantly reduced (p = 0.002) in the male rats administered 15 and 25 mg/kg doses . The PFC numbers in female rats were not affected significantly by treatment (p > 0.05) . For the remaining immunotoxicity studies, groups of male rats (10 rats/group) were gavaged with FB1 doses of 0, 1, 5, and 15 mg/kg body wt/day for 14 days . There was a weakly significant dose-related trend toward increased numbers of serum immunoglobulin class G (p = 0.04) . Also a significant dose-related increase (p = 0.013) in Listeria monocytogenes numbers was observed in the spleen at 24 hr postinfection . Treatment did not have a significant effect on organ weights, hematology, mitogen-induced lymphocyte transformation, calcium mobilization, the numbers of leukocytes and T-lymphocyte subsets, the natural killer cell activity, and phagocytosis (p >/= 0 . 05) . These observations suggested that FB1 may have indirect consequences for human health and warrant further investigations .

Immunity, 1997 Sep, 7(3), 419 - 32
Listeriosis in p47(phox-/-) and TRp55-/- mice: protection despite absence of ROI and susceptibility despite presence of RNI; Endres R et al.; The significance of host defense mechanisms in primary listeriosis in vivo is incompletely understood . Here, we show that tumor necrosis factor receptor p55-/- (TRp55-/-) mice are susceptible to Listeria monocytogenes infection in the presence of leukocyte recruitment, inflammatory cytokine production (including IFNgamma), nitric oxide synthesis, and oxidative burst formation . Mice deficient for oxidative burst (p47{phox-/-} mice) are relatively resistant to listeriosis . Despite activation of these antibacterial effector systems, TRp55-/- phagocytes in vivo are incapable of confining and eradicating L . monocytogenes inside phagolysosomes . Bone marrow chimeras reveal that for eradication of L . monocytogenes, TRp55 is crucially required only on cells from hematopoietic origin . Unexpectedly, prior to death, exocrine pancreatic cells undergo apoptosis in TRp55-/- mice . Collectively, these data demonstrate that in vivo, TRp55 initiates a protective, listericidal mechanism in phagocytes that differs from nitric oxide production and oxidative burst formation and that uncontrolled listeriosis results in necrotizing pancreatitis in TRp55-/- mice.

J Virol, 1997 Nov, 71(11), 8467 - 74
Recombinant Listeria monocytogenes vaccination eliminates papillomavirus-induced tumors and prevents papilloma formation from viral DNA; Jensen ER et al.; Listeria monocytogenes is a gram-positive, facultative intracellular bacterium that enters the cytoplasm of infected cells and spreads directly into neighboring cells without encountering the extracellular environment . Cytoplasmic L . monocytogenes efficiently presents secreted proteins to the major histocompatibility complex class I pathway which can stimulate protective T-cell-mediated immune responses . We have used a cottontail rabbit papillomavirus (CRPV) rabbit model to test the ability of recombinant L . monocytogenes strains secreting the viral E1 protein (E1-rLm) to protect outbred rabbits against CRPV- and CRPV DNA-induced tumors . CRPV infection of outbred rabbits serves as a model for oncogenic papillomaviruses since CRPV-induced papillomas progress with high frequency to malignant carcinoma . Rabbits were vaccinated with wild-type L . monocytogenes or E1-rLm and then challenged with CRPV or viral DNA . In contrast to 0% papilloma regression in control animals, 77% of E1-rLm-vaccinated rabbits generated protective immunity that controlled and induced complete regression of tumors induced by CRPV . Latent viral DNA was not detected at 71% of the papilloma regression sites examined 4.5 months postregression . E1-rLm responder rabbits were completely resistant to papilloma formation from viral DNA . In contrast to controls, peripheral blood mononuclear cells from E1-rLm responder rabbits were able to proliferate in response to in vitro E1 stimulation . These results indicate that E1-rLm immunization generated a systemic anti-CRPV E1 cell-mediated immune response which protected outbred rabbits from tumors induced by CRPV or CRPV DNA challenge.

Mol Gen Genet, 1997 Sep, 256(1), 54 - 62
The Listeria monocytogenes iap gene as an indicator gene for the study of PrfA-dependent regulation; Bubert A et al.; The iap gene of Listeria monocytogenes encodes the extracellular protein p60, which possesses a murein hydrolase activity necessary for septum separation . We constructed L . monocytogenes EGD strains harbouring plasmids that carry the iap gene under the control of the PrfA-regulated promoters of the L . monocytogenes genes hly, mpl, and actA . After insertional inactivation of the chromosomal iap gene in L . monocytogenes EGD, p60 synthesis was strictly dependent on PrfA . Elevated temperature (40 degrees C) enhanced synthesis of p60 in L . monocytogenes when the iap gene was under the control of the hly promoter; this appeared to be associated with increased synthesis of PrfA at this temperature . Synthesis of p60 in L . monocytogenes was significantly lower when the iap gene was placed under the control of the actA or the mpl promoter . Transcription of the iap gene was repressed in L . monocytogenes in the presence of PrfA when iap expression was under the control of the prfA promoter P2 . Under the control of the hly promoter the gene produced low levels of secreted p60 in the presence of low amounts of PrfA, and this in turn led to the generation of long listerial cell filaments consisting of bacteria that had failed to separate . Overexpression of p60 in the presence of high levels of PrfA caused formation of single cells, which showed reduced viability depending on the level of secreted p60 . These data suggest that the iap gene may be a valuable tool for monitoring virulence gene regulation by PrfA under in vivo conditions, without disturbing the integrity of the infected host cells.

Immunology, 1997 Aug, 91(4), 520 - 8
The characterization of testicular cell (TC)-specific T-cell clones induced by intratesticular Listeria monocytogenes infection: TC-specific T cells with atypical cytokine profile transfer orchitis; Matsuzaki G et al.; A unilateral infection of Listeria monocytogenes into the testis of mice induces not only Listeria-specific T cells but also autoreactive T cells that can transfer experimental autoimmune orchitis (EAO) into naive mice . To investigate the characteristics of the autoreactive T cells, we established six testicular cell (TC)-specific T-cell clones from the spleen of the intratesticularly infected mice . All the clones expressed CD4 and T-cell receptor (TCR) alpha beta, and four of the six clones expressed V beta 8 . They showed proliferative response to TC in the presence of syngeneic spleen antigen-presenting cells, but did not cross-react to Listeria antigen (Ag) . They produced interferon-gamma (IFN-gamma) when stimulated with TC, but interleukin-2 (IL-2), IL-4 and IL-10 were undetectable . IL-2 production was not detected even when they were restimulated with TC after a 10-day resting culture without Ag and IL-2, although they proliferated in the restimulation culture . Even in the presence of anti-IL-2 mAb, the TC-specific T-cell clones showed proliferative response against TC . The observations indicate that the TC-specific IFN-gamma-producing T cells proliferate in the absence of autocrine . Both intravenous and intratesticular injection of these clones transferred EAO in syngeneic naive mice . These results suggest that L . monocytogenes infection in the testis induces autoreactive orchitogenic CD4+ T cells without cross-reactivity to bacterial Ag . Furthermore, these data demonstrate that CD4+ T cells with an atypical cytokine profile can efficiently cause EAO.

Immunology, 1997 Aug, 91(4), 511 - 9
TCR alpha beta+ CD4- CD8- T cells differentiate extrathymically in an lck-independent manner and participate in early response against Listeria monocytogenes infection through interferon-gamma production; Kadena T et al.; T-cell receptor (TCR) alpha beta+ CD4- CD8- (double-negative; DN) T cells appear in the peritoneal cavity at an early stage of intraperitoneal (i.p.) infection with the intracellular pathogen Listeria monocytogenes . In the present report, we analysed the developmental pathway and functions of the TCR alpha beta+ DN T cells using the L . monocytogenes infection system . The TCR alpha beta+ DN T cells appeared in the peritoneal cavity after L . monocytogenes i.p . infection in adult-thymectomized lethally irradiated bone marrow chimeras and p56lck-deficient mice . The results demonstrated that the TCR alpha beta+ DN T cells can develop extrathymically in a p56lck-independent manner . Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the TCR alpha beta+ DN T cells expressed genes for interferon-gamma (IFN-gamma), the macrophage chemotactic factors MCP-1 and Eta-1, and granulocyte-macrophage colony-stimulating factor (GM-CSF) but lacked expression of genes for interleukin-2 (IL-2), IL-4 and IL-10 . As expected from the RT-PCR analysis, the TCR alpha beta+ DN T cells produced IFN-gamma in response to anti-TCR beta monoclonal antibody (mAb), anti-CD3 mAb and L . monocytogenes-infected macrophages but IL-4 was undetectable after the stimulation . Furthermore, the intracellular cytokine staining analysis demonstrated that approximately half of the TCR alpha beta+ DN T cells detectable at the early stage of L . monocytogenes infection were IFN-gamma-producing cells . All of the results suggest that the TCR alpha beta+ DN T cells develop through a unique extrathymic p56lck-independent pathway and participate in early protection against bacterial infection through activation and accumulation of macrophages.

Thorax, 1997 Aug, 52(8), 745 - 6
Listeria monocytogenes empyema in an HIV infected patient; Marron A et al.; Listeriosis in HIV infected patients is uncommon and usually presents as meningitis or bacteraemia . Pleural fluid infections caused by this organism are extremely rare . A case is described of empyema caused by Listeria monocytogenes in an HIV infected patient that was successfully treated with medical treatment only.

J Immunol, 1997 Oct 15, 159(8), 3675 - 9
A synthetic peptide administered with IL-12 elicits immunity to Listeria monocytogenes; Miller MA et al.; IL-12 is a pivotal cytokine signal for the development of Th1-type cellular responses that are required for control of intracellular pathogens . We previously demonstrated that coinjection of IL-12 with heat-killed Listeria monocytogenes, which was not immunogenic when injected alone, elicited intense Ag-specific T cell responses that conferred protection against subsequent challenge with Listeria . Herein we describe the remarkable finding that a nonimmunogenic synthetic peptide corresponding to a dominant MHC class II (H-2k)-restricted listerial determinant, when coinjected i.p . with murine IL-12, elicited potent Ag-specific immune responses that conferred protective immunity against Listeria.

J Immunol, 1997 Oct 1, 159(7), 3364 - 71
Phenotypic and functional characterization of mice that lack the type I receptor for IL-1; Glaccum MB et al.; IL-1 alpha and IL-1 beta bind to receptors termed the type I and type II IL-1 receptors . The type I IL-1 receptor is responsible for specific signaling, while the type II IL-1 receptor functions as a nonsignaling decoy receptor . To determine the effect of a defect in IL-1-mediated signaling, mice have been produced with a genetically disrupted type I IL-1 receptor gene . Mice lacking type I IL-1 receptors are of normal vigor and exhibit no overt phenotype . B cells from type I IL-1R-/- mice activated in vitro with anti-IgM do not proliferate in response to IL-1, but do so in response to IL-4 . Injection of murine IL-1 alpha does not induce detectable serum IL-6 levels in type I IL-1R-/- mice, but equivalent levels are produced in response to LPS . Type I IL-1R-/- mice have normal serum Ig levels and generate equivalent primary and secondary Ab responses as wild-type mice . In response to LPS, acute phase protein mRNA induction are equivalent in type I IL-1R-/- and wild-type mice . Type I IL-1R-/- mice do not differ from control mice in susceptibility to either a lethal challenge with D-galactosamine plus LPS or high dose LPS . Interestingly, ICE-/-/type I IL-1R-/- double mutant mice are resistant to high dose LPS . Type I IL-1R-/- mice backcrossed to the C57BL/6 background were as equally resistant as wild-type mice to Listeria monocytogenes.

Infect Immun, 1997 Oct, 65(10), 4267 - 72
In vivo and in vitro activation and expansion of gammadelta T cells during Listeria monocytogenes infection in humans; Jouen-Beades F et al.; Serial flow cytometry analyses of peripheral blood mononuclear cells obtained from 8 patients infected with Listeria monocytogenes showed a higher percentage (P < 0.01) of gammadelta T cells (median, 11.7; range, 3.7 to 35.3) than did 16 age-matched uninfected controls (1.7, 0.4 to 13) . Most in vivo-expanded gammadelta T cells expressed the Vgamma9 and Vdelta2 gene products and displayed a memory phenotype (CD45RO{high}), and patients' gammadelta T cells expressed significantly more (P < 0.01) activation marker HLA-DR than did controls (19.8% {median} and 0.9 to 87.6% {range} versus 2.3% and 0 to 4.7%, respectively) . When peripheral blood mononuclear cells from normal donors were cultured in vitro with heat-killed Listeria cells, analysis of CD25 and HLA-DR expression on gammadelta and alphabeta T cells indicated that a high percentage of gammadelta T cells was activated early compared to alphabeta T cells . In addition, depletion of gammadelta T cells before culture abrogated the early lymphocyte proliferative response induced by the pathogen . Taken together, these results argue for the involvement of gammadelta T cells during L . monocytogenes infection in humans.

J Exp Med, 1997 Oct 6, 186(7), 1159 - 63
pH-dependent perforation of macrophage phagosomes by listeriolysin O from Listeria monocytogenes; Beauregard KE et al.; The pore-forming toxin listeriolysin O (LLO) is a major virulence factor implicated in escape of Listeria monocytogenes from phagocytic vacuoles . Here we describe the pH-dependence of vacuolar perforation by LLO, using the membrane-impermeant fluorophore 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) to monitor the pH and integrity of vacuoles in mouse bone marrow-derived macrophages . Perforation was observed when acidic vacuoles containing wild-type L . monocytogenes displayed sudden increases in pH and release of HPTS into the cytosol . These changes were not seen with LLO-deficient mutants . Perforation occurred at acidic vacuolar pH (4.9-6.7) and was reduced in frequency or prevented completely when macrophages were treated with the lysosomotropic agents ammonium chloride or bafilomycin A1 . We conclude that acidic pH facilitates LLO activity in vivo.

Immunol Rev, 1997 Aug, 158, 159 - 69
Listeria monocytogenes: a potent vaccine vector for neoplastic and infectious disease; Weiskirch LM et al.; Listeria monocytogenes (L . monocytogenes) is a promising candidate vaccine vector that naturally infects antigen-presenting cells, and targets antigen delivery to both the class I MHC pathway of endogenous antigen presentation and the class II pathway of exogenous antigen presentation . At the same time, L . monocytogenes stimulates the innate immune response to produce cytokines that enhance antigen-presenting function and induce a Th1-type cytokine profile associated with cell-mediated immune responses . Immune responses with these features are considered to be particularly important for clearance of viruses, tumors, and intracellular infections . In this review, we describe the development of methods to transform L . monocytogenes to express and secrete foreign antigens and the studies that have demonstrated that genetically engineered L . monocytogenes mutants are highly effective vectors for the induction of potent immune responses against viral antigens and tumor cells . In addition, we discuss the strengths and weaknesses of L . monocytogenes as a vaccine vector.

Immunol Rev, 1997 Aug, 158, 147 - 57
Recombinant Listeria monocytogenes as a live vaccine vehicle and a probe for studying cell-mediated immunity; Jensen ER et al.; The ability of Listeria monocytogenes (L . monocytogenes) to enter the cytosol of host cells allows secreted proteins to efficiently enter the endogenous antigen-processing pathway leading to presentation by MHC class I molecules . L . monocytogenes has recently been exploited as a live vaccine vehicle for the induction of immunological memory against heterologous antigens . We have established a genetic system for site-specific integration of antigen expression cassettes into the Listeria genome which allows regulated expression and secretion of heterologous proteins . The ability of recombinant strains to stimulate long-term immunological memory and CD8+ T-cell-mediated protective immunity was investigated using the lymphocytic choriomeningitis virus (LCMV) murine infection model . Vaccination of mice with recombinant Listeria strains expressing LCMV antigens induced LCMV-specific CD8+ T cells which protected mice against LCMV challenge . We have also used a cottontail rabbit papillomavirus model to test the ability of recombinant Listeria strains to stimulate protective antitumor immunity in domestic rabbits . These studies have demonstrated the protective efficacy of recombinant L . monocytogenes vaccines and have established an experimental system for systematic analysis of cytotoxic T-cell induction by an intracellular bacterium.

Immunol Rev, 1997 Aug, 158, 137 - 46
Acquired immunity to an intracellular pathogen: immunologic recognition of L . monocytogenes-infected cells; Bouwer HG et al.; Listeria monocytogenes (L . monocytogenes) is a pathogenic bacterium, and subclinical infection in mice is utilized as a prototypic model to investigate the development and expression of acquired resistance to facultative intracellular organisms . A key virulence factor of L . monocytogenes is the hemolysin listeriolysin O (LLO), and BALB/c mice immunized with hemolysin-secreting strains of L . monocytogenes develop specific acquired resistance, while mice immunized with hemolysin-negative strains or non-viable preparations of L . monocytogenes do not develop a protective immune response . Adoptive transfer studies show that L . monocytogenes-immune CD8+ T cells mediate acquired resistance . The L . monocytogenes-immune CD8+ population is cytotoxic, and target cells infected with hemolysin-secreting strains of L . monocytogenes are lysed, while target cells infected with hemolysin-negative strains or non-viable preparations of L . monocytogenes are not lysed . MHC class Ia and Ib molecules present L . monocytogenes-derived peptides, and we have identified Qa-Ib, a T-region-encoded MHC class Ib molecule, as a restriction element for L . monocytogenes-specific CD8+ CTL . MHC class Ib-restricted CTL are stimulated following infection with L . monocytogenes and are a significant component of the total MHC class I-restricted CTL population . These findings support the observation that cytoplasmic L . monocytogenes-derived antigens are endogenously processed and presented in association with MHC class Ia and Ib molecules to CD8+ effector cells, and that both populations of effector cells contribute to the immune response to this intracellular pathogen.

Immunol Rev, 1997 Aug, 158, 129 - 36
MHC class I antigen processing of Listeria monocytogenes proteins: implications for dominant and subdominant CTL responses; Pamer EG et al.; Listeria monocytogenes (L . monocytogenes) secretes proteins associated with its virulence into the cytosol of infected cells . These secreted proteins are degraded by host cell proteasomes and processed into peptides that are bound by MHC class I molecules in the endoplasmic reticulum . We have found that the MHC class I antigen-processing pathway is very efficient at generating the epitopes that are presented to cytolytic T lymphocytes (CTL) . Depending on which antigen is investigated, from 3 to 30% of degraded antigens are processed into nonamer peptides that are bound by MHC class I molecules . Surprisingly, neither the efficiency of epitope generation nor the absolute number of epitopes per infected cell determines the magnitude of the in vivo CTL response . One of the least prevalent epitopes, derived from an antigen that is virtually undetectable in infected cells, primes the immunodominant CTL response in L . monocytogenes-infected mice . Our studies suggest that immunodominant and subdominant T-cell responses cannot be predicted by the prevalence of antigens or epitopes alone, and that additional factors, yet to be determined, are involved.

Immunol Rev, 1997 Aug, 158, 123 - 8
Characterization of the murine H2-M3wt-restricted CD8 response against a hydrophobic, protease-resistant, phospholipid-associated antigen from Listeria monocytogenes; Kurlander R et al.; Mice infected with Listeria monocytogenes (LM) generate protective CD8 cells of varying specificity . One subset, unlike conventional LM-immune CD8 cells, can respond to antigen-presenting cells (APC) treated with heat-killed LM (HKLM) . These cells proved to have surprisingly uniform specificity, recognizing a product we designated HKLM-associated antigen (HAA) presented by the non-classical class Ib product H2-M3wt . HAA proved to be extremely hydrophobic and the bioactive portion of the molecule was highly protease-resistant, leading us initially to speculate that it might be a non-peptide . Recent studies, however, identify HAA as a complex containing lemA, a listerial protein bearing the immunogenic amino terminal peptide sequence fMIGWII, tightly associated with bacterial cardiolipin . A variety of cell types can process and present exogenous HAA/lemA, and the phospholipid component appears essential for this processing . Endosomal acidification and proteolysis are required for processing, but the site where antigen binds to H2-M3wt within APC remains uncertain . HAA/lemA-immune effectors are unusually cross-reactive . We could readily detect H2-M3wt-restricted responses to APC incubated with unrelated N-formylated peptides, and bacteria . HAA-like products represent an intriguing new set of bacterial antigens recognizable by immune CD8 cells.

Immunol Rev, 1997 Aug, 158, 115 - 21
CTL responses to H2-M3-restricted Listeria epitopes; Lenz LL et al.; Cytotoxic T cells (CTL) play a critical role in the murine immune response to Listeria monocytogenes (Listeria) . Bacterial antigens are presented to Listeria-specific CTL by products of both conventional, polymorphic MHC class Ia and non-polymorphic MHC class Ib alleles . The H2-M3 class Ib gene product, M3, preferentially presents formylmethionine-initiating (fMet) peptides derived from the N termini of bacterial and mitochondrial proteins . Thus, M3 signals the presence of bacterial invaders to CTL effectors . Listeria-encoded fMet peptide epitopes for H2-M3-restricted CTL have recently been identified . These and other identified fMet peptides are predominantly comprised of hydrophobic residues and appear to be cleaved from membrane-bound proteins . The subcellular location and membrane topology of such proteins may be significant factors in their selection as target antigens for H2-M3-restricted CTL . Such rules may prove useful for prediction of candidate fMet peptide epitopes from other bacterial proteins and species . Studies using synthetic fMet peptides to stimulate CTL ex vivo are also discussed . These latter studies indicate that Listeria infection boosts H2-M3-restricted CTL responses . However, in contrast to MHC class Ia-restricted CTL responses, fMet peptide-specific CTL are observed in a large proportion of cultures from non-immunized, conventionally housed (non-SPF) mice . The CTL activity in these latter cultures may reflect priming in vivo on cross-reactive antigens, or may indicate that requirements for priming of H2-M3-restricted CTL are less stringent than for class Ia-restricted responses.

Immunol Rev, 1997 Aug, 158, 107 - 14
The cytokine stew and innate resistance to L . monocytogenes; Mocci S et al.; The Listeria monocytogenes (L . monocytogenes) infection model has been a useful system to evaluate the cellular interactions leading to host immunity . The initiation of the innate immune response in naive animals and subsequent progression to acquired immunity represent an integrated system with numerous layers of complexity . Coincident with experimental infection is the induction of cytokines . Cytokines, which are soluble mediators of cell growth, maintenance and function, from a network of pleiotropic stimuli that serve as one of the main driving forces for the progressive development of cellular responses . A variety of in vivo approaches, such as injection of the recombinant cytokines themselves or antibodies to neutralize their activity, have been used to define these stimuli . Perhaps one of the most useful tools is that of germline-manipulated animals . One of the many cytokines implicated in resistance to L . monocytogenes infection is interleukin (IL)-6, a molecule associated with diverse infectious and pathophysiological disease states . This review concentrates on various cytokines (IL-1, TNF alpha, IFN-gamma, IL-12, IL-10 and the colony-stimulating factors (CSF)) thought to play a role during the innate host response to L . monocytogenes infection, with a special emphasis on studies using IL-6-deficient mice . Additionally, we show unpublished data obtained when the concepts learned from L . monocytogenes infection in IL-6-deficient mice were applied to other infection models.

Immunol Rev, 1997 Aug, 158, 95 - 105
Interleukin-4 and listeriosis; Kaufmann SH et al.; Experimental infection of mice with Listeria monocytogenes (L . monocytogenes) has served as an appropriate model for analyzing Th1-cell-driven immune responses . Generally, Th2 responses are absent and IL-4 is not detectable . Here, we describe experimental settings under which IL-4 is detectable in listeriosis . Our data suggest that IL-4 is rapidly produced after infection . This prompt IL-4 burst seems to stimulate chemokine responses and, therefore, may participate in the regulation of the early antilisterial host response . Soon thereafter, IL-4 production wanes . At least partially this seems to be caused by downregulation of IL-4-producing CD4+ NK1+ TCR alpha beta int lymphocytes by IL-12 . In the absence of IFN-gamma responsiveness, IL-4 production is demonstrable during acquired immunity against L monocytogenes, and this elevated IL-4 production apparently contributes to disease exacerbation . In conclusion, the data are consistent with a detrimental role of IL-4 in listeriosis and active control of IL-4 synthesis by the antilisterial immune response . The rapid, but transient, IL-4 burst in listeriosis probably contributes to host defense without impairing development of the acquired T-cell response because of its shortness.

Immunol Rev, 1997 Aug, 158, 79 - 93
Cytokines in the induction and expression of T-cell-mediated granuloma formation and protection in the murine model of listeriosis; Mielke ME et al.; Lymphocyte-mediated inflammation is a hallmark of autoimmune diseases, such as multiple sclerosis . Crohn's disease, rheumatoid arthritis and sarcoidosis . However, this type of inflammation probably developed under evolutionary pressure from pathogenic microorganisms, such as mycobacteria and other intracellular infective agents . One such pathogen, the gram-positive bacterium Listeria monocytogenes (L . monocytogenes), induces a cascade of tissue alterations that ultimately results in the eradication of the bacteria associated with a granulomatous response . Consequently, murine listeriosis has been established as a model to analyze not only T-cell-dependent antibacterial protection but also T-cell-mediated mononuclear inflammation in parenchymal organs . Extensive studies of the molecular basis of the latter phenomenon led to the conclusion that the most decisive step from non-specific microabscess formation to granulomatous inflammation is the activation of non-specifically invading CD4+ T cells, which results in high local concentrations of TNF-alpha and IFN-gamma in the presence of IL-2 . This in turn induces CD11b-independent mechanisms of intraparenchymal monocyte accumulation . Because any attempt to neutralize the effects of TNF-alpha and IFN-gamma to modulate T-cell-mediated inflammation will also dramatically decrease host resistance, other anti-inflammatory strategies based on the modulation of TNF-alpha and IFN-gamma-induced mechanisms of monocyte accumulation must be developed . Recalling the classical work by Dienes & Schoenheit on the induction of bacterial allergies (1), the cytokine phenotype of granuloma formation also has implications as regards the most potent adjuvant environment for the development of a T-cell response . The murine listeriosis model is the basis for all conclusions in this article on the role of cytokines in the induction and expression of T-cell-mediated inflammation and, as we will show, promises to yield still more insights into the rational design of vaccines.

Immunol Rev, 1997 Aug, 158, 69 - 77
Some macrophages kill Listeria monocytogenes while others do not; Fleming SD et al.; It is not known why some macrophages can kill certain microbes, such as the facultative intracellular bacterium Listeria monocytogenes (L . monocytogenes), while other macrophages cannot . Perhaps listericidal activity is a property of macrophages at specific stages of differentiation; may be the ability to kill this bacterium is regulated by the microenvironment of the cell: or it is possible that other regulatory forces are important . We describe here three characteristics that distinguish macrophages which can kill L . monocytogenes from those which cannot . First, listericidal macrophages must have neither too much nor too little intracellular iron-they must have an intermediate amount . Second, the receptor a macrophage uses to phagocytose L . monocytogenes seems to influence the intracellular fate of this bacterium . And third, macrophages which have cell-surface interleukin-10 (IL-10), a known downregulator of macrophage function, cannot kill L . monocytogenes . These traits of macrophages and their effects on listericidal activity are reviewed here, and the possibility that these properties might interact to control macrophage bactericidal activity is discussed.

Immunol Rev, 1997 Aug, 158, 57 - 67
Responses by murine macrophages infected with Listeria monocytogenes crucial for the development of immunity to this pathogen; Kuhn M et al.; Macrophages and other mammalian cells respond to infection with Listeria monocytogenes (L . monocytogenes) by the transient or persistent activation of host cell signal transduction pathways . In addition, L . monocytogenes infection influences expression of various host cell genes, such as stress genes, genes from the MHC I and II complex, cytokine genes, and cytokine receptor genes . The possible influences of the different host cell responses on the outcome of an L . monocytogenes infection in vitro as well as for the development of immunity are discussed.

Immunol Rev, 1997 Aug, 158, 47 - 56
Non-specific resistance mechanisms to listeriosis: implications for experimental and naturally occurring infection; Czuprynski CJ et al.; Use of murine listeriosis as an experimental model has greatly increased our understanding of the complex interplay of cells and mediators in non-specific antibacterial resistance (innate immunity) . Important contributions made with this experimental model include demonstrating the ability of inflammatory cytokines (i.e . IFN-gamma, IL-1 alpha, TNF-alpha) to protect against bacterial infection, and illustrating the rapidity of the host cytokine response (detectable within 1 h of challenge) during bacterial infection . Most experimental studies of host defense against Listeria monocytogenes (L . monocytogenes) have used a parenteral challenge (i.v . or i.p.) . This ignores the pathogenesis of naturally occurring listeriosis, which usually results from ingestion of Listeria-contaminated food products . In this review, we will include consideration of the host-pathogen interactions that occur when L . monocytogenes invades through its natural portal of entry, the gastrointestinal tract . Although resistance to facultative intracellular pathogens, such as L . monocytogenes, was formerly thought to revolve exclusively around the T helper cell/macrophage axis, more recent evidence indicates that neutrophils are able to ingest and kill L . monocytogenes and prevent the unrestricted multiplication of listeriae in parenchymal cells . Exploring the mechanisms involved in this process will provide new insights into the communication between leukocytes and tissue cells in host defense.

Immunol Rev, 1997 Aug, 158, 37 - 46
Listeria monocytogenes in laboratory mice: a model of short-term infectious and pathogenic processes controllable by regulated protective immune responses; Milon G; The mammalian immune system is an integrated network of tissue, leukocytes and effector and regulatory molecules . All these components operate i) to maintain the proper structure of and processes expressed by each tissue . and ii) to protect the hosts from those microorganisms that generally invade them as part of their life cycle . Among the invading microorganisms . Listeria monocytogenes (L . monocytogenes) can persist as a live organism independent of the host, and is, thus, able to drive short-term infectious and pathogenic processes that are controlled by integrated innate and adaptive protective immune responses driven by CD8 and CD4 type 1 T lymphocytes acting on non-T non-B leukocytes . Although the effector functions and the fine specificity of T lymphocytes have been more and more characterized, an understanding of the precise regulation of both leukocyte traffic and T-lymphocyte migration depends on knowledge of the early tissue distribution of L . monocytogenes, points that are addressed in this review.

Immunol Rev, 1997 Aug, 158, 27 - 36
Murine listeriosis as a model of antimicrobial defense; North RJ et al.; Murine listeriosis was introduced 35 years ago as a model with which to analyze mechanisms of antibacterial defense that are independent of antibodies . Listeria monocytogenes was shown to be an intramacrophage pathogen with capacity to induce the generation of a state of specific immunity in the form of DTH and a macrophage system with enhanced non-specific bactericidal activity . The demonstration that anti-Listeria immunity and DTH can be passively transferred with T cells was taken to indicate that the T cells responsible for DTH function upregulate the listericidal function of macrophages . This interpretation is contradicted by recent research showing that CD8 T cells, rather than CD4 T cells, are responsible for mediating adoptive immunity . However, T-cell depletion studies show that primary infection can eventually be resolved in the absence of either CD8 or CD4 T cells . On the other hand, infection becomes lethal in the absence of neutrophils or NK cells . It is apparent, therefore, that the most important defense against primary listeriosis resides with the functions of neutrophils and NK cells that are mobilized early in infection . Antigen-specific T cells function at a later time to resolve infection more efficiently . It is apparent that T cells are much more important in defense against secondary infection.

Immunol Rev, 1997 Aug, 158, 11 - 25
Studies in listeriosis show the strong symbiosis between the innate cellular system and the T-cell response; Unanue ER; Resistance to infection with Listeria monocytogenes involves a series of cellular interactions, many of which are carried out by cytokines . Macrophages, NK cells and neutrophils participate in early stages of Listeria resistance . The neutrophil is specially important for clearance of the liver phase of listeriosis . Macrophages and NK cells interact by way of IL-12 and TNF, which induce the NK cell to produce IFN-gamma . IFN-gamma is the major macrophage-activating cytokine . The CB-17 SCID mouse shows these cellular interactions restricting the growth of Listeria, without its elimination . CD4 and/or CD8 T cells bring about sterilizing immunity . Macrophages influence the lymphocyte response by way of antigen presentation and also by promoting Th 1 differentiation . Thus, elimination of Listeria requires a symbiosis between innate immunity and the T-cell system.

Tierarztl Prax, 1997 Jul, 25(4), 336 - 43
{Cerebral listeriosis in cattle: literature review and retrospective analysis of individual cases}; Hirsbrunner G et al.; Between 1990 and 1994, 89 cattle with signs of affection of the central nervous system were referred to the Clinic for Food Animals and Horses, University of Bern; in 17 cases of which, cerebral listeriosis was diagnosed . History, clinical, hematologic findings and cerebrospinal fluid analysis at admission were evaluated retrospectively . Four cattle were slaughtered after cerebral listeriosis had been diagnosed clinically because of economic reasons . Therapy, consisting of administration of penicillin (i.m./i.v.), intravenous fluids (including bicarbonate replacement), and oral fluids and rumen juice was initiated in the remaining 13 cases . Five of the 13 cattle were refractory to treatment and had to be euthanatized, one to two days after initiation of therapy . Clinical diagnosis of cerebral listeriosis was confirmed by histological examination of brain stem tissue in all nine cattle that were slaughtered or euthanatized; however, Listeria monocytogenes was isolated by standard culturing of brain stem tissue in two of these cases only . Eight to 62 months after discharge from the clinic, telephone interview with the owners of the surviving animals revealed that recovery had been uneventful and satisfactory in all of the eight cases.

EMBO J, 1997 Sep 1, 16(17), 5433 - 44
A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP family; Niebuhr K et al.; The ActA protein of the intracellular pathogen Listeria monocytogenes induces a dramatic reorganization of the actin-based cytoskeleton . Two profilin binding proteins, VASP and Mena, are the only cellular proteins known so far to bind directly to ActA . This interaction is mediated by a conserved module, the EVH1 domain . We identify E/DFPPPPXD/E, a motif repeated 4-fold within the primary sequence of ActA, as the core of the consensus ligand for EVH1 domains . This motif is also present and functional in at least two cellular proteins, zyxin and vinculin, which are in this respect major eukaryotic analogs of ActA . The functional importance of the novel protein-protein interaction was examined in the Listeria system . Removal of EVH1 binding sites on ActA reduces bacterial motility and strongly attenuates Listeria virulence . Taken together we demonstrate that ActA-EVH1 binding is a paradigm for a novel class of eukaryotic protein-protein interactions involving a proline-rich ligand that is clearly different from those described for SH3 and WW/WWP domains . This class of interactions appears to be of general importance for processes dependent on rapid actin remodeling.

Int J Food Microbiol, 1997 Jul 22, 37(2-3), 215 - 9
Risk factors for contamination of smoked salmon with Listeria monocytogenes during processing; Rorvik LM et al.; Forty smoked salmon processing plants were examined for the occurrence of Listeria monocytogenes and other Listeria spp . in the smoked salmon and the drains . L . monocytogenes was detected in smoked salmon from 13 (33%) and in the drains samples from 25 (63%) of the plants . Other Listeria spp . were found in smoked salmon samples from 16 (40%) and in the drains of 30 (75%) of the plants . Multivariate analyses of data on hygiene, management, production facilities of the plants and bacteriological results showed that job rotation was the strongest expressed risk factor for isolation of L . monocytogenes from the smoked salmon (hazard ratio, HR = 11.0, p = 0.002) . Well-maintained facilities (HR = 0.31, p = 0.064) and use of vats for salting of the fillets (HR = 0.33, p = 0.109), showed a preventive effect . L . monocytogenes in the drains was found to be a sensitive predictor for the presence of L . monocytogenes in the smoked salmon . In general, detection of other Listeria spp . in the smoked salmon or the drains could not be demonstrated to have any association with detection of L . monocytogenes.

Int J Food Microbiol, 1997 Jul 22, 37(2-3), 209 - 14
Distribution and behavior of Listeria monocytogenes in three lots of naturally-contaminated vacuum-packed smoked salmon stored at 2 and 10 degrees C; Cortesi ML et al.; The incidence, the number and the behavior of L . monocytogenes in three lots of naturally-contaminated vacuum-packed sliced smoked salmon, processed in different plants, were investigated during storage at 2 and 10 degrees C . L . monocytogenes was isolated from 20 of the 100 packages stored at 2 degrees C (16 from lot 1, 1 from lot 2 and 3 from lot 3) and from 12 of the 65 packages stored at 10 degrees C (all from lot 1) . The levels detected were 15, 20, 290, 1100 and > 1100 cfu/g in 5 packages, all belonging to lot 1, and < 10 cfu/g in the remaining samples . The high incidence of L . monocytogenes in lot 1 was assumed to be due mainly to the use of causal workers for the slicing and packing operations.

Chemotherapy, 1997 Sep-Oct, 43(5), 303 - 10
In vitro susceptibility of Listeria monocytogenes: comparison of the E test with the agar dilution test; Heger W et al.; In vitro susceptibility testing was performed on 66 strains of Listeria monocytogenes . Recently obtained clinical isolates from Austria, France, Norway and Switzerland and 17 reference strains from three type culture collections were tested against ampicillin, gentamicin, trimethoprim-sulfamethoxazole, ampicillin-sulbactam, meropenem, and first-, second-, third- and fourth-generation cephalosporins . All isolates were susceptible to ampicillin, gentamicin, and trimethoprim-sulfamethoxazole, ampicillin-sulbactam, and meropenem and more than 86% of the minimal inhibitory concentration E test values were within plus/minus one dilution step of the agar dilution test . In vitro susceptibility of L . monocytogenes has not changed markedly during the last decades; the level of susceptibility of old reference strains did not differ from that of recently encountered clinical isolates . The E test seems to be a suitable method for Listeria.

Zh Mikrobiol Epidemiol Immunobiol, 1997 May-Jun, (3), 3 - 6
{Pathogenic Listeria in the soil and in association with algae: a reversible transition into a nonculturable state}; Pushkareva VI et al.; The analysis of the dynamics of the amount of L . monocytogenes in soil extract in association with green algae and in their absence, carried out in parallel by the bacteriological method and in polymerase chain reaction (PCR), revealed the mass transition of L . monocytogenes into the nonculturable state . The proportion of vegetative (bacteriologically detected) forms in the bacterial population rapidly decreased, while its total amount remained unchanged due to the formation of inactive (nonculturable) L . monocytogenes forms . Algae or their metabolites considerable accelerated this process: by day 26 L . monocytogenes could not be isolated, but in PCR they could be registered at their initial concentration: 10(6) microbial cells/ml . Inactive L . monocytogenes forms were shown to revert into the vegetative (culturable) state under the action of fetal serum, live and killed infusoria, auxin . The concentration of revertants grown on a solid culture medium was high: 10(4)-10(5) colony-forming units/ml.

J Immunol, 1997 Sep 15, 159(6), 2795 - 801
MHC class Ib-restricted cells contribute to antilisterial immunity: evidence for Qa-1b as a key restricting element for Listeria-specific CTLs; Bouwer HG et al.; Subclinical infection of BALB/c mice with the intracellular pathogen Listeria monocytogenes results in the development of MHC class Ia- and Ib-restricted CTLs . L . monocytogenes-infected TAP-/- bone marrow macrophage targets are not lysed by MHC class Ia- or Ib-restricted CTLs, showing a requirement for transport of peptides into the endoplasmic reticulum for development of the MHC class Ib-peptide target . L . monocytogenes-infected B6.Tla(a)-derived bone marrow macrophages (Kb Qa-1a) are not lysed by BALB/c (Kd Qa-1b)-derived antilisterial CTLs, confirming an earlier finding that the Ib-restricting element is T region encoded . We have further determined that Qa-1b is a restricting element for antilisterial CTLs using L . monocytogenes-infected Qa-1b-transformed mouse L cells as well as human-derived HeLa cells as target populations . These L . monocytogenes-infected Qa-1b-transformed cell lines are lysed by BALB/c (Qa-1b)- or C57BL/6 (Qa-1b)-derived antilisterial CTLs, but are not lysed by B6.AKM (Qa-1a)-derived antilisterial CTLs . Using L . monocytogenes-infected targets, we found that MHC class Ia- and Ib-restricted CTLs are evident within 4 days following infection, peak on day 5 following infection, and although Ib-restricted CTLs disappear by day 6 postinfection, la-restricted antilisterial CTL activity can still be detected . These results demonstrate that Qa-1b is a restricting element for antilisterial CTLs, and expression of the MHC class Ib-presented target at the cell surface is TAP dependent . In addition, these results show that following L . monocytogenes infection, MHC class Ib-restricted CTLs are evident in vivo.

Electrophoresis, 1997 Aug, 18(8), 1464 - 71
Stress proteins in Listeria monocytogenes; Phan-Thanh L et al.; The proteins induced by the different stress conditions in Listeria monocytogenes were analyzed by two-dimensional (2-D) electrophoresis with the aid of a computerized 2-D gel analysis system . The stress conditions imposed were pH 4, pH 10, 0.015% sodium, dodecyl sulfate (SDS), 0.03% sodium deoxycholate and 4% ethanol . As previously seen for heat shock and cold shock, more than half of the proteins normally synthesized by Listeria cells were repressed under these stress conditions . Conversely, the synthesis of a great number of proteins was increased and novel proteins appeared upon stress . Each stress factor induced a specific set of proteins . These stress proteins were characterized by their apparent molecular mass and isoelectric point . No universal stress proteins were found to be common to all the stresses studied, while some proteins were commonly induced by two or three stress conditions . The degree of dissimilarity in stress responses was best illustrated by the induction of only two proteins common to exposure to the two detergents SDS and sodium deoxycholate . This work together with that on heat and cold shock, constitutes the basic step for the identification of stress proteins in Listeria.

Schweiz Arch Tierheilkd, 1997, 139(8), 354 - 62
{Frequency of neurologic diseases in cattle}; Heim D et al.; The cases of neurological diseases at the Institute of Animal Neurology, University of Berne, from 1985-1994 were assessed . During this period 532 cattle with neurological symptoms were examined . After 1980 differential diagnostic investigation of rabies negative brains were not pursued anymore and the number of examined cattle brains had declined to 25-30 per year . With the occurrence of bovine spongiform encephalopathy (BSE) in 1990 in Switzerland the number of cattle brains examined has increased to 75-80 yearly . The most frequently diagnosed neurological diseases found are BSE, followed by listeriosis and viral encephalitides.

J Cell Sci, 1997 Aug, 110 ( Pt 16), 1893 - 906
Structural and functional similarities between the human cytoskeletal protein zyxin and the ActA protein of Listeria monocytogenes; Golsteyn RM et al.; The intracellular bacterial parasite Listeria monocytogenes produces ActA protein at its surface to facilitate the localized assembly of actin-filled comets that are required for movement . The organization of actin in Listeria comets shows striking similarity to the organization of actin at the plasma membrane of mammalian cells . Therefore we examined the possibility that an ActA-like protein is present in mammalian cells . By using antibodies directed against ActA, we identified zyxin as an ActA related protein in a number of cell types . We compared the functions of ActA and zyxin by transient expression of variants tagged with an inner plasma membrane localization sequence (a CAAX box) . Targeting of the proline rich domain of zyxin to the plasma membrane disrupts the actin cytoskeleton and cell shape in a manner similar to that which occurs with membrane-targeted ActA sequences . A chimeric protein composed of the N-terminal domain of ActA fused to the N-terminal and central domains of zyxin induced a full ActA response in cells . Furthermore, zyxin and ActA exhibit common protein partners in vitro . On the basis of the shared properties of zyxin and ActA, we propose that zyxin enhances actin organizing activity in mammalian cells.

Res Microbiol, 1996 Nov-Dec, 147(9), 677 - 85
Esculetin antagonizes iron-chelating agents and increases the virulence of Listeria monocytogenes; Coulanges V et al.; Iron is an essential compound for the growth and virulence of Listeria monocytogenes . In extracellular environments, iron often requires a siderophore to be acquired by microorganisms . Although it does not produce siderophores, L . monocytogenes can use some exogenous bacterial or fungal siderophores as well as a number of animal or plant o-diphenol compounds to overcome growth inhibition by the iron-chelating agents tropolone and 8-hydroxyquinoline . Esculin, a plant glycoside, can be hydrolysed by L . monocytogenes to the o-diphenol aglucon, esculetin . The latter neutralized in vitro growth inhibition induced by the iron-chelating agents . Furthermore, when injected into infected mice, esculetin enhanced mortality in a dose-dependent manner and increased bacterial counts in spleen induced by sublethal doses of L . monocytogenes . Esculetin apparently functioned as a siderophore for L . monocytogenes in murine tissues.

Proc Natl Acad Sci U S A, 1997 Sep 16, 94(19), 10034 - 9
ActA is a dimer; Mourrain P et al.; ActA, a surface protein of Listeria monocytogenes, is able to induce continuous actin polymerization at the rear of the bacterium, in the cytosol of the infected cells . Its N-terminal domain is sufficient to induce actin tail formation and movement . Here, we demonstrate, using the yeast two-hybrid system, that the N-terminal domain of ActA may form homodimers . By using chemical cross-linking to explore the possibility that ActA could be a multimer on the surface of the bacteria, we show that ActA is a dimer . Cross-linking experiments on various L . monocytogenes strains expressing different ActA variants demonstrated that the region spanning amino acids 97-126, and previously identified as critical for actin tail formation, is also critical for dimer formation . A model of actin polymerization by L . monocytogenes, involving the ActA dimer, is presented.

Appl Environ Microbiol, 1997 Sep, 63(9), 3695 - 7
Effects of pH on distribution of Listeria ribotypes in corn, hay, and grass silage; Ryser ET et al.; Listeria app, isolated from 13 of 129 (10%) corn silage samples, 21 of 76 (28%) hay silage samples, and 3 of 5 (60%) grass silage samples during a previous Vermont survey were subjected to automated ribotype (RT) analysis . The 13 positive corn silage samples contained 3 Listeria monocytogenes isolated (three RTs, including one known clinical RT) and 10 L . innocua isolates (four RTs) . Similarly, 2 L . monocytogenes isolates (two RTs) and 19 L . innocua isolates (three RTs) were identified in the 21 positive hay silage samples . The three positive grass silage samples contained two L . innocua isolates (two RTs) and one isolate of L . welshimeri . One hundred seven of 129 (83%) high-quality (pH < 4.0) corn silage samples accounted for 8 of 13 Listeria isolates from corn silage, including isolates belonging to one L . monocytogenes clinical RT . In contrast, low-quality hay silage (70 of 76 {92%} samples having a pH of > or = 4.0) harbored 20 of 21 isolates, including isolates belonging to two nonclinical L . monocytogenes RTs . Poor-quality silage is readily discernible by appearance; however, these findings raise new concerns regarding the safety of high-quality (pH < 4.0) corn silage, which can contain Listeria spp., including L . monocytogenes strains belonging to RTs of clinical importance in cases of food-borne listeriosis.

Appl Environ Microbiol, 1997 Sep, 63(9), 3480 - 7
Construction of the temperature-sensitive vectors pLUCH80 and pLUCH88 for delivery of Tn917::NotI/SmaI and use of these vectors to derive a circular map of Listeria monocytogenes Scott A, a serotype 4b isolate; He W et al.; A physical map of Listeria monocytogenes Scott A was generated by the pulsed-field technique of contour-clamped-homogeneous-electric-field (CHEF) electrophoresis . The circular genome of this serotype 4b strain contains 12 AscI fragments (38 to 790 kb), 5 NotI fragments (55 to 1,400 kb), 3 SrfI fragments (110, 1,110, and 2,000 kb), and 2 SfiI fragments (1,320 and 1,920 kb) . Summation of individually sized fragments derived by digestion of Scott A genomic DNA with each of these four enzymes provided an average estimated genome length of 3,210 +/- 60 kb . Efforts to assemble the macrorestriction map benefited greatly from the construction and use of pLUCH80 and pLUCH88, temperature-sensitive vectors for delivering transposon Tn917::NotI/SmaI to the chromosome of Scott A . As another component of this study, the positions of four known virulence genes (inlA, mpl, hly, and prf) and three L . monocytogenes-specific sequences (lisM44, lisM51, and lisM52) were localized on the physical map of Scott A by hybridization . Probes prepared from lisM44, lisM51, and the four virulence genes hybridized within a cluster on a 150-kb fragment of the Scott A genome that overlaps part of the NotI-B and AscI-D fragments . The lisM52 probe hybridized with the AscI-F2 (120-kb) fragment of Scott A, which is separated from the NotI-B-AscI-D region by about 300 kb . These results established the first physical and genetic map of a serotype 4b strain of L . monocytogenes and provided further insight on this important food-borne pathogen at the genome level.

Appl Environ Microbiol, 1997 Sep, 63(9), 3451 - 7
Modifications of membrane phospholipid composition in nisin-resistant Listeria monocytogenes Scott A; Verheul A et al.; A nisin-resistant (NISr) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth . The NISr strain was about 12 times more resistant to nisin than was the wild-type (WT) strain . Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NISr strain than in the WT strain . Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NISr derivative revealed no significant differences . From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NISr strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does . Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NISr strain, reflecting qualitative differences in nisin sensitivity . Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NISr strains showed a comparable sensitivity to lysozyme . Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol (R.A . Demel, T . Peelen, R.J . Siezen, B . de Kruijff, and O.P . Kuipers, Eur . J.Biochem . 235:267-274, 1996) . Collectively, the mechanism of nisin resistance in this L . monocytogenes NISr strain is attributed to a reduction in the DPG content of the cytoplasmic membrane.

Appl Environ Microbiol, 1997 Sep, 63(9), 3374 - 7
Evaluation of seven experimental phages for inclusion in the international phage set for the epidemiological typing of Listeria monocytogenes; van der Mee-Marquet N et al.; The purpose of our study was to evaluate the inclusion of seven experimental phages into the international phage set for subtyping Listeria monocytogenes . The seven additional phages included the broad-host-range virulent Myoviridae phage A511 (M . J . Loessner, Appl . Environ . Microbiol . 57:1912-1918, 1991), three temperate phages from the Danish subsystem for typing serotype 1/2 strains (12682, 6223, and 5775) (P . Gerner-Smidt, V.T . Rosdahl, and W . Frederiksen, APMIS 101:160-167, 1993), and three temperate phages isolated by this laboratory in France (9425, 1313, and 197) . A panel of 395 Listeria monocytogenes isolates (including 180 that were non-phage typeable by the international set) were used in the study for a comparison of the lytic spectra of the various bacteriophages . These results showed that the inclusion of five of the experimental phages contributed greatly to the overall typeability and discriminatory power of the system, especially for strains within serogroup 1/2.

Am J Pathol, 1997 Sep, 151(3), 785 - 92
Lymphocyte apoptosis during early phase of Listeria infection in mice; Merrick JC et al.; During the acute phase of growth of Listeria monocytogenes in spleen and lymph nodes, the infective foci consist of macrophages and neutrophils accompanied by extensive death of lymphocytes . Many of the lymphocytes die by apoptosis . The lesions are found by 48 hours after infection and can regress with time . Depending on the dose, the infected foci can be restricted to the thymus-dependent areas or can occupy the entire lymphoid tissue . The Listeria in the lesions are primarily found inside macrophages, but a few are extracellular amid cellular debris . Lymphocyte death appears to be an obligatory step in primary Listeria infection, the extent of which is controlled by the early restriction of Listeria growth by the innate cellular system.

Infect Immun, 1997 Sep, 65(9), 3976 - 80
Listeriolysin and IrpA are major protein targets of the human humoral response against Listeria monocytogenes; Grenningloh R et al.; We have examined the human humoral immune response directed against proteins of Listeria monocytogenes in both healthy individuals and listeriosis patients . Two major targets for an antibody response were found in individuals that did not suffer from listeriosis: listeriolysin (Hly) and the recently described internalin-related protein (IrpA) . In contrast, the humoral response in listeriosis patients appears to be more heterogeneous and included Hly, IrpA, InlB, and ActA as major targets.

J Immunol, 1997 Sep 1, 159(5), 2452 - 61
Absence of IL-1 signaling and reduced inflammatory response in IL-1 type I receptor-deficient mice; Labow M et al.; IL-1alpha and IL-1beta are potent inflammatory cytokines that contribute to a number of normal physiologic processes and to the development of a number of inflammatory diseases . Two IL-1R, the type I and type II receptors, have been identified . This work describes the derivation and characterization of mice deficient in expression of the type I IL-1R (IL-1RI) . IL-1RI-deficient mice were viable and fertile, but failed to respond to IL-1 in a variety of assays, including IL-1-induced IL-6 and E-selectin expression and IL-1-induced fever . Similar to IL-1beta-deficient mice, IL-1RI-deficient mice had a reduced acute phase response to turpentine . In contrast, IL-1RI-deficient mice had a reduced delayed-type hypersensitivity response and were highly susceptible to infection by Listeria monocytogenes . These data demonstrate that the IL-1RI is essential for all IL-1-mediated signaling events examined, and that both IL-1alpha and IL-1beta are critical to the animals' response to injury and infection . These data also demonstrate that IL-1 function is not required for normal development or homeostasis.

J Cell Biol, 1997 Aug 25, 138(4), 783 - 97
Actin filament cables in Drosophila nurse cells are composed of modules that slide passively past one another during dumping; Guild GM et al.; At a late stage in Drosophila oogenesis, nurse cells rapidly expel their cytoplasm into the oocyte via intracellular bridges by a process called nurse cell dumping . Before dumping, numerous cables composed of actin filaments appear in the cytoplasm and extend inward from the plasma membrane toward the nucleus . This actin cage prevents the nucleus, which becomes highly lobed, from physically blocking the intracellular bridges during dumping . Each cable is composed of a linear series of modules composed of approximately 25 cross-linked actin filaments . Adjacent modules overlap in the cable like the units of an extension ladder . During cable formation, individual modules are nucleated from the cell surface as microvilli, released, and then cross-linked to an adjacent forming module . The filaments in all the modules in a cable are unidirectionally polarized . During dumping as the volume of the cytoplasm decreases, the nucleus to plasma membrane distance decreases, compressing the actin cables that shorten as adjacent modules slide passively past one another just as the elements of an extension ladder slide past one another for storage . In Drosophila, the modular construction of actin cytoskeletons seems to be a generalized strategy . The behavior of modular actin cytoskeletons has implications for other actin-based cytoskeletal systems, e.g., those involved in Listeria movement, in cell spreading, and in retrograde flow in growth cones and fibroblasts.

Proc Natl Acad Sci U S A, 1997 Aug 19, 94(17), 9394 - 9
Listeria monocytogenes infection of P388D1 macrophages results in a biphasic NF-kappaB (RelA/p50) activation induced by lipoteichoic acid and bacterial phospholipases and mediated by IkappaBalpha and IkappaBbeta degradation; Hauf N et al.; As previously reported, Listeria monocytogenes infection of P388D1 macrophages results in a rapid induction of NF-kappaB DNA-binding activity . Here we show that this induction of NF-kappaB activity occurs in a biphasic mode: first, a transient, IkappaBalpha degradation-dependent phase of activity, also induced by the nonvirulent species Listeria innocua, which is mediated by binding of the bacteria to the macrophage, or by adding Listeria-derived lipoteichoic acid to the macrophage; the second persistent phase of activation is only markedly induced when the bacteria enter the cytoplasm of the host cell and express the virulence genes plcA and plcB, encoding two phospholipases . We suggest that products of the enzymatic activity of phospholipases directly interfere with host cell signal transduction pathways, thus leading to persistent NF-kappaB activation via persistent IkappaBbeta degradation.

J Immunol, 1997 Aug 15, 159(4), 1970 - 6
Listeria monocytogenes potently induces up-regulation of endothelial adhesion molecules and neutrophil adhesion to cultured human endothelial cells; Krull M et al.; Infection of endothelial cells by Listeria monocytogenes is an essential step in the pathogenesis of listeriosis . Listeriolysin (Hly) is one of its major virulence factors . In the early phase of the disease polymorphonuclear leukocytes (PMN) substantially contribute to the nonspecific anti-listerial resistance . We characterized the effects of L . monocytogenes on the expression of endothelial adhesion molecules and on subsequent PMN adhesion to cultured HUVEC . P-selectin, E-selectin, intracellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) were up-regulated in HUVEC after cell incubation with L . monocytogenes (wild type), but not with the nonpathogenic Listeria innocua strain . P-selectin expression peaked after 30 min and could be mimicked with similar kinetics by exposure to L . innocua engineered to produce large amounts of Hly or by addition of purified Hly . Listeriolysin production, however, was not necessary for an up-regulation of E-selectin after 6 h or of ICAM-1 and VCAM-1 after 18 to 24 h in HUVEC, because L . monocytogenes defective for Hly synthesis was almost as effective as the wild type . Listeria-induced up-regulation of endothelial adhesion molecules was accompanied by an increased binding of PMN to infected HUVEC . PMN adhesion was significantly reduced in the presence of anti-beta2 integrin, anti-E-selectin, and anti-ICAM-1, but not anti-VCAM-1 Abs . Our data indicate that infection of endothelial cells with L . monocytogenes induced up-regulation of adhesion molecules by two different mechanisms: a Hly-dependent up-regulation of P-selectin and a Hly-independent expression of E-selectin, ICAM-1, and VCAM-1 . The ability of L . monocytogenes to stimulate PMN adhesion to endothelial cells may be an important mechanism in the pathogenesis of severe listeriosis.

J Appl Microbiol, 1997 Aug, 83(2), 181 - 8
The effect of high hydrostatic pressure on Listeria monocytogenes in phosphate-buffered saline and model food systems; Simpson RK et al.; Three strains of Listeria monocytogenes (NCTC 1194, a poultry isolate and the Scott A strain) were exposed to a range of pressures (300, 350, 375, 400 and 450 MPa) in 10 mmol 1(-1) phosphate-buffered saline (PBS) at pH 7.0 for up to 30 min at ambient temperature . Generally, increasing the magnitude and duration of compression resulted in increasing levels of inactivation, although the inactivation kinetics varied depending on the strain and pressure applied . The three strains also exhibited a wide variation in their resistance to high pressure . The resistance of the three strains to high pressure (375 MPa) was also assessed in a series of model food systems containing one of each of the three main food constituents: protein (1, 2, 5 and 8% w/v bovine serum albumin in PBS), carbohydrate (1, 2, 5 and 10% w/v glucose in PBS) and lipid (olive oil (30% v/v) in PBS emulsion) . Overall, increasing the concentrations of bovine serum albumin (BSA) and glucose in the suspending medium resulted in decreasing levels of inactivation of all three strains; however, the minimum concentration of BSA and glucose required to increase survival to a level greater than that observed in PBS alone varied depending on the strain and on the duration of the treatment . The survival of all three strains was greater in the olive oil/PBS emulsion than in PBS alone at all treatment times.

Br J Obstet Gynaecol, 1997 Aug, 104(8), 942 - 5
Zoonotic and viral infection in fetal loss after 12 weeks; Sanghi A et al.; One hundred and thirty-six women from an urban, rural and farming community were recruited to a study of infectious causes of midtrimester miscarriage (n = 85), stillbirth (n = 32), or termination for developmental (n = 17) or chromosomal (n = 2) abnormalities . No woman had evidence of acute infection with toxoplasma, listeria, leptospira or Chlamydia psittaci (ovine enzootic abortion) . One woman had midtrimester miscarriage associated with primary cytomegolovirus infection and five women had evidence of parvovirus B19 infection, although fetal infection was not proven . Zoonoses were not identified as a cause of fetal loss or malformation in this at-risk population, but parvovirus B19 was a significant cause of midtrimester fetal loss.

Appl Environ Microbiol, 1997 Aug, 63(8), 3225 - 32
Effects of several factors on the heat-shock-induced thermotolerance of Listeria monocytogenes; Pagan R et al.; The influence of the temperature at which Listeria monocytogenes had been grown (4 or 37 degrees C) on the response to heat shocks of different durations at different temperatures was investigated . For cells grown at 4 degrees C, the effect of storage, prior to and after heat shock, on the induced thermotolerance was also studied . Death kinetics of heat-shocked cells is also discussed . For L . monocytogenes grown at 37 degrees C, the greatest response to heat shock was a fourfold increase in thermotolerance . For L . monocytogenes grown at 4 degrees C, the greatest response to heat shock was a sevenfold increase in thermotolerance . The only survival curves of cells to have shoulders were those for cells that had been heat shocked . A 3% concentration of sodium chloride added to the recovery medium made these shoulders disappear and decreased decimal reduction times . The percentage of cells for which thermotolerance increased after a heat shock was smaller the milder the heat shock and the longer the prior storage.

Appl Environ Microbiol, 1997 Aug, 63(8), 3085 - 9
Host-mediated modification of Sau3AI restriction in Listeria monocytogenes: prevalence in epidemic-associated strains; Zheng W et al.; Most major food-related outbreaks of listeriosis have been traced to a cluster of genetically related strains of serovar 4b (epidemic clone) . In spite of numerous searches, distinct bacteriologic or virulence-related features unique to these strains have eluded identification, although a restriction fragment length polymorphism (RFLP) characteristic of the epidemic clone has previously been described (W . Zheng and S . Kathariou, Appl . Environ . Microbiol . 61:4310-4314, 1995) . We found that DNAs from 75 strains which were derived from three separate outbreaks and which had the epidemic clone-specific RFLP were also invariably resistant to digestion by Sau3AI and other restriction endonucleases sensitive to cytosine methylation at 5' GATC 3' sites . This modification of Sau3AI restriction was host mediated, as it did not persist when DNA was cloned and propagated in Escherichia coli, and was uncommon among other Listeria strains . Epidemic-associated strains with this modification were resistant to infection by phage propagated in a serotype 4b strain which was not known to be involved in an epidemic and which lacked the epidemic clone-specific RFLP . Screening for susceptibility to MboI digestion revealed that these epidemic strains lacked methylation of adenines at GATC sites . This type of modification was rare among Listeria strains and was found in only three (of eight screened) strains of serovar 1/2b, possibly representing one clonal lineage.

Appl Environ Microbiol, 1997 Aug, 63(8), 2961 - 5
Evaluation of luciferase reporter bacteriophage A511::luxAB for detection of Listeria monocytogenes in contaminated foods; Loessner MJ et al.; A511::luxAB is a recombinant derivative of a broad-host-range bacteriophage specific for the genus Listeria, transducing bacterial bioluminescence into infected cells . In this study, we have evaluated its use for rapid and easy testing of contaminated foods and environmental samples for the presence of viable Listeria cells, in comparison to the standard plating procedure . With a short preenrichment step of 20 h, the system was capable of detecting very low initial contamination rates in several foods artificially contaminated with Listeria monocytogenes Scott A cells . In ricotta cheese, chocolate pudding, and cabbage, less than one cell per g of food could be clearly identified by comparing the light emission of phage-infected samples to that of controls without lux phage . In foods having a large and complex microbial background flora, such as minced meat and soft cheese, at least 10 cells per g were necessary to produce a positive bioluminescence signal . Of 348 potentially contaminated natural food and environmental samples, 55 were found to be Listeria positive by the lux phage method . The standard plating procedure detected 57 positive samples . Some differences were observed with respect to the individual samples, i.e., the lux phage procedure detected more positive samples among the dairy products and environmental samples, whereas the plating procedure revealed more contaminated meat and poultry samples . Overall, both methods performed similarly, i.e., were equally sensitive . However, the minimum time required for detection of Listeria with the luciferase phage assay was 24 h, which is much shorter than the 4 days needed by the standard plating method . Furthermore, a most probable number technique with three parallels, based on the use of A511::luxAB for differentiation of positive and negative tubes, is described . The method enables rapid enumeration of low levels of Listeria cells in several foods tested, against the background of a competing microflora.

Microb Pathog, 1997 Aug, 23(2), 101 - 6
CD8alphaalpha T cells in lesions of Listeria monocytogenes-infected beta2m-deficient mice; Thoma-Uszynski S et al.; The beta2-microglobulin (beta2m)-deficient mutant mice lack alphabeta TCR CD8alphabeta T cells . We found markedly impaired granuloma formation in Listeria monocytogenes-infected beta2m-/- mice . Abundant CD8alphaalpha T cells were identified in loosely structured infiltrative liver lesions . Microfluorescence analysis disclosed that these CD8alphaalpha T cells expressed mostly the gammadelta TCR . CD8alphaalpha T cells were also found in the spleen of Listeria-infected beta2m-/- mice . These data provide first evidence for CD8alphaalpha T cells in listerial lesions of beta2m-/- mice.

J Biol Chem, 1997 Aug 1, 272(31), 19261 - 8
The Listeria monocytogenes-secreted p60 protein is an N-end rule substrate in the cytosol of infected cells . Implications for major histocompatibility complex class I antigen processing of bacterial proteins; Sijts AJ et al.; Cytosolic antigen degradation is an initial step in the generation of major histocompatibility complex (MHC) class I-associated cytolytic T lymphocyte epitopes . Intracellular Listeria monocytogenes secretes p60, a murein hydrolase, into the host cell cytosol, where it is degraded by proteasomes . Roughly 3% of degraded p60 gives rise to p60 217-225, a nonamer peptide that is bound by H-2Kd MHC class I molecules . Herein, we introduce targeted deletions throughout the p60 gene to identify potential proteolytic signals within p60 . Degradation of mutant forms of p60 was investigated in macrophages infected with recombinant L . monocytogenes . We found that deletions within the amino-terminal two-thirds of p60 enhanced cytosolic degradation . In contrast, truncation of the C terminus resulted in modest stabilization of p60 in the host cell cytosol . Because a protein's N-terminal amino acid can determine its rate of degradation, we mutagenized this residue in p60 into known stabilizing and destabilizing residues . Valine substitution dramatically stabilized cytosolic p60 molecules, while substitution with aspartic acid resulted in rapid degradation . The number of p60 217-225 epitopes isolated from infected cells directly correlated with the rates of p60 degradation . Our data, therefore, indicate that the N-terminal amino acid and multiple internal regions of p60 influence its stability in the cytosol of infected cells . Antigen degradation and epitope generation are linked, and different degradation signals can channel bacterial proteins into the MHC class I antigen processing pathway.

Mol Cell Biol, 1997 Aug, 17(8), 4572 - 88
Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins; Plevy SE et al.; Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is critical for T-helper 1 responses . The gene for the IL-12 p40 subunit is expressed in macrophages following induction by bacterial products, and its expression is augmented by gamma interferon . In this study, we performed a functional analysis of the murine and human p40 promoters in the murine macrophage cell line RAW 264.7 . Transcription from the murine p40 promoter was strongly induced by lipopolysaccharide and heat-killed Listeria monocytogenes (HKLM), but promoter activity was not enhanced by gamma interferon . Multiple cis-acting elements involved in activated transcription were identified through an extensive mutant analysis . The most critical element, whose activity is conserved in mice and humans, is located between positions -96 and -88 relative to the murine transcription start site . This element exhibits functional synergy with a previously described NF-kappaB half-site which interacts with Rel proteins . DNase I footprinting and electrophoretic mobility shift assays demonstrated that C/EBP proteins interact with the critical element, but in nuclear extracts, cooperative binding of C/EBP and Rel proteins to their respective sites was not observed . Interestingly, promoter activity was induced by HKLM in the presence of cycloheximide, consistent with induction by posttranslational mechanisms . The results suggest that C/EBP and Rel proteins play important roles in the activation of IL-12 p40 transcription by bacteria . However, many complex interactions will need to be clarified to fully understand p40 regulation.

J Clin Microbiol, 1997 Aug, 35(8), 2155 - 6
Simple color tests based on an alanyl peptidase reaction which differentiate Listeria monocytogenes from other Listeria species; Clark AG et al.; The hydrolysis of DL-alanine-beta-naphthylamide and D-alanine-p-nitroanilide for identification of Listeria spp . has been studied with 227 cultures . All species of Listeria, except L . monocytogenes, hydrolyzed these substrates . The reactions were detected by simple chromogenic reactions and could substitute for the CAMP test.

J Cell Biol, 1997 Jul 28, 138(2), 375 - 84
The human Arp2/3 complex is composed of evolutionarily conserved subunits and is localized to cellular regions of dynamic actin filament assembly; Welch MD et al.; The Arp2/3 protein complex has been implicated in the control of actin polymerization in cells . The human complex consists of seven subunits which include the actin related proteins Arp2 and Arp3, and five others referred to as p41-Arc, p34-Arc, p21-Arc, p20-Arc, and p16-Arc (p omplex) . We have determined the predicted amino acid sequence of all seven subunits . Each has homologues in diverse eukaryotes, implying that the structure and function of the complex has been conserved through evolution . Human Arp2 and Arp3 are very similar to family members from other species . p41-Arc is a new member of the Sop2 family of WD (tryptophan and aspartate) repeat-containing proteins and may be posttranslationally modified, suggesting that it may be involved in regulating the activity and/or localization of the complex . p34-Arc, p21-Arc, p20-Arc, and p16-Arc define novel protein families . We sought to evaluate the function of the Arp2/3 complex in cells by determining its intracellular distribution . Arp3, p34-Arc, and p21-Arc were localized to the lamellipodia of stationary and locomoting fibroblasts, as well to Listeria monocytogenes assembled actin tails . They were not detected in cellular bundles of actin filaments . Taken together with the ability of the Arp2/3 complex to induce actin polymerization, these observations suggest that the complex promotes actin assembly in lamellipodia and may participate in lamellipodial protrusion.

J Immunol, 1997 Jul 15, 159(2), 861 - 9
TNF-alpha-mediated expression of the receptor for anaphylatoxin C5a on neurons in experimental Listeria meningoencephalitis; Stahel PF et al.; The anaphylatoxin C5a has been implicated in the pathogenesis of bacterial meningitis as a potent mediator of inflammation in the subarachnoid space . We investigated the expression of the receptor for C5a (C5aR) in brains of mice with experimental Listeria monocytogenes (LM) meningoencephalitis . In the course of the disease, infiltrating cells in the meninges and the ventricles were found to express C5aR mRNA and protein . In the brain parenchyma, very low constitutive C5aR expression was seen on pyramidal neurons and Purkinje cells . However, in LM-infected mice, a dramatic increase in C5aR expression occurred on neurons starting 6 h after infection and was maximal between 24 and 36 h . TNF-alpha was identified as an essential mediator of neuronal C5aR expression, since mice lacking the genes for TNF and lymphotoxin-alpha (TNF/lymphotoxin-alpha -/- mice) showed significantly attenuated C5aR expression after LM infection . Furthermore, i.p . injection of recombinant TNF-alpha induced enhanced C5aR expression in the brains of TNF/lymphotoxin-alpha -/- mice and in normal animals even in the absence of a bacterial infection . We also assessed the levels of anaphylatoxin C5a in the cerebrospinal fluid of patients with infectious meningitis . C5a was detected in all patients with bacterial meningitis (n = 9), in 6 of 18 patients with aseptic meningitis, and in 1 of 66 control patients . The finding of TNF-alpha-mediated C5aR expression on neurons in experimental Listeria meningitis and the detection of the ligand, C5a, in the cerebrospinal fluid of human patients with infectious meningitis present new directions in the investigation of the pathophysiologic sequelae leading to secondary brain damage.

Biochemistry, 1997 Jul 8, 36(27), 8384 - 92
Profilin interacts with the Gly-Pro-Pro-Pro-Pro-Pro sequences of vasodilator-stimulated phosphoprotein (VASP): implications for actin-based Listeria motility; Kang F et al.; Intracellular actin-based motility of Listeria monocytogenes requires protein-protein interactions involving two different proline-rich sequences: first, the tightly bound bacterial surface protein ActA uses its multiple oligoproline registers {consensus sequence = FE(D)FPPPPTD(E)E(D)} to tether vasodilator-stimulated phosphoprotein (VASP) to the bacterial surface; and second, VASP then deploys its own multiple GPPPPP (or GP5) registers to localize the actin-regulatory protein profilin to promote actin polymerization . We now report that fluorescence titration showed that GP5GP5GP5 peptide binds to profilin (KD of 84 microM), and the peptide weakly inhibits exchange of actin-bound nucleotide in the absence or presence of profilin . Microinjection of synthetic GPPPPP triplet into Listeria-infected PtK2 cells promptly arrested motility at an intracellular concentration of 10 microM . This inhibition was completely neutralized when equimolar concentrations of profilin and GP5GP5GP5 were simultaneously microinjected . Fluorescence studies with {His-133-Ser}-profilin, a site-directed mutant previously shown to be defective in binding poly-l-proline {Bjorkegren, C., Rozycki, M., Schutt, C . E., Lindberg, U., & Karlsson, R . (1993) FEBS Lett . 333, 123-126}, exhibits little or no evidence of saturable GP5GP5GP5 binding . When an equimolar concentration of this {His-133-Ser}-profilin mutant was co-injected with GP5GP5GP5, the peptide's inhibitory action remained completely unaffected, indicating that GP5GP5GP5 binding to wild-type profilin represents a key step in actin-based pathogen motility . We also present a model that shows how the focal binding of VASP with its GPPPPP registers can greatly increase the local concentration of profilin and/or profilin-actin-ATP complex at the bacteria/rocket-tail interface.

Mol Microbiol, 1997 Jul, 25(2), 285 - 94
InlB: an invasion protein of Listeria monocytogenes with a novel type of surface association; Braun L et al.; Listeria monocytogenes is an intracellular bacterial pathogen that expresses several surface proteins critical for the infectious process . Such proteins include InlA (internalin) and InlB, involved in bacterial entry into the host cell, and ActA, required for bacterially induced actin-based motility . Although the molecular mechanisms of attachment of InlA and ActA have been characterized, essentially nothing is known about how InlB is anchored to the bacterial surface . Using a genetic approach, we demonstrate that the last 232 amino acids of InlB are both necessary and sufficient for anchoring this protein to the bacterial surface . An InlB mutant protein deleted for the last 232 amino acids was secreted and not detected at the cell surface . A 'domain-swapping' strategy in which these 232 amino acids were used to replace the normal cell wall-anchoring domain of InlA resulted in a chimeric protein that was anchored to the cell surface and able to confer entry . Interestingly, surface association of InlB also occurred when InlB was added externally to bacteria, suggesting that association may be able to occur after secretion . This association was productive for invasion, as it conferred bacterial entry into host cells . The C-terminal anchoring region in InlB contains 80-amino-acid repeats beginning with the sequence GW that is also present in a newly identified surface-associated bacteriolysin of L . monocytogenes, called Ami . Addition of GW repeats to the C-terminal of InlB improves anchoring of the protein to the cell surface . These and other data suggest that such 'GW' repeats may constitute a novel motif for cell-surface anchoring in Listeria and other Gram-positive bacteria . This motif may have important consequences for the release of surface proteins involved in interactions with eukaryotic cells.

Microb Pathog, 1997 Jul, 23(1), 39 - 48
A gerbil model for rhombencephalitis due to Listeria monocytogenes; Blanot S et al.; Rhombencephalitis due to Listeria monocytogenes is a frequent complication of human listeriosis, inducing a high mortality and severe neurological sequelae despite antibiotic therapy . However, there is no animal model which consistently reproduces clinical rhombencephalitis . Here, we present a model of Listeria rhombencephalitis in gerbils . Animals were inoculated in the middle ears with a low infective dose of L . monocytogenes, thus creating prolonged otitis media with persistent bacteremia . Gerbils developed a severe rhombencephalitis with circling syndrome, paresia, ataxia, rolling movements . The invasion of the central nervous system was visualized on living animals by resonance magnetic imaging and characterized by bacterial growth in the brain, reaching about 10(7) bacteria in the rhombencephalum by day 12 of infection . The histological lesions were mainly located in the brainstem, and consisted in coalescent, necrotic abscesses with perivascular sheaths, mimicking those observed in human rhombencephalitis . Bacteria were detected by electronmicroscopy inside infectious foci, either free in necrotic material or inside inflammatory cells, mainly polymorphonuclear cells . This gerbil model of Listeria rhombencephalitis will be useful to study the molecular mechanisms allowing bacteria to cross the blood-brain barrier, and to evaluate the intracerebral efficacy of antibiotics.

Lett Appl Microbiol, 1997 Jul, 25(1), 48 - 53
The effect of high hydrostatic pressure on the activity of intracellular enzymes of Listeria monocytogenes; Simpson RK et al.; The effect of high hydrostatic pressure (100-550 MPa, 15 min, ambient temperature) on the activity of 13 metabolic enzymes produced by all three strains of Listeria monocytogenes (NCTC 11994, a poultry isolate and Scott A) was examined using gel electrophoresis . The enzymes assayed exhibited a wide variation in barotolerance . The pressure resistance of each particular enzyme was not dependent on the strain from which it was derived . This would seem to indicate that these enzymes were not a determining factor in relation to previously observed differences in the overall pressure resistance of the three strains.

Eur J Immunol, 1997 Jul, 27(7), 1696 - 703
Antigen-specific T cell receptor antagonism by antigen-presenting cells treated with the hemolysin of Listeria monocytogenes: a novel type of immune escape; Darji A et al.; We have examined the influence of listeriolysin O (LLO), the hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, on major histocompatibility complex class II-dependent T cell activation . Stimulation of T cells by native antigens but not by peptides is inhibited upon pretreatment of antigen-presenting cells (APC) with LLO . Experiments presented here reveal that this inhibition is not due to a lack in processing of antigen by APC but is the result of an irreversible inactivation of T cells that recognize antigen on LLO-treated APC . Incubation of mixtures of two different T cells where only one antigen was presented on LLO-treated APC suggested that T cell inactivation is antigen specific . The inactivation was dominant and could be observed even in the presence of amounts of synthetic peptides that normally lead to T cell responses . This condition is reminiscent of the T cell inhibition observed when antagonistic and stimulatory peptides are added to APC at the same time . Our results thus reveal a novel type of interference by pathogens with antigen presentation and T cell stimulation that could give the pathogen a decisive advantage in dissemination and disease.

J Appl Microbiol, 1997 Jul, 83(1), 31 - 5
Physiological and biochemical studies on psychrotolerance in Listeria monocytogenes; Jones CE et al.; Studies were undertaken to explain the ability of Listeria monocytogenes to grow at low temperatures in a chemostat . It was found that when grown in continuous culture at a dilution rate of 0.02 h-1 L . monocytogenes had a lower proportion of anteiso-17:0, and a higher proportion of anteiso-15:0, and smaller chain fatty acids when grown at 10 degrees C compared to 30 degrees C . A previously unreported glycolipid was only seen after growth at low temperature . Growth temperature had no effect on the rate of glucose uptake.

J AOAC Int, 1997 Jul-Aug, 80(4), 913 - 9
Predictive microbiology of dairy products: influence of biological factors affecting growth of Listeria monocytogenes; Brouillaud-Delattre A et al.; The growth potential of Listeria monocytogenes was evaluated at low temperature in sterilized milk and raw dairy products . Sterilized and raw milk were inoculated with different strains of L . monocytogenes in 2 physiological states and at various contamination levels . Raw cheese was naturally contaminated with Listeria spp . The results suggest that some biological factors influence the growth capacity of L monocytogenes in dairy products . Significant strain effect was observed at low temperature whatever the growth medium . By contrast, no inoculum effect was observed in the 3 dairy products . In raw matrixes, growth of L . monocytogenes was influenced greatly by bacterial interactions and physiological state of inoculum cells.

J AOAC Int, 1997 Jul-Aug, 80(4), 791 - 805
Visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria species detection in selected foods: collaborative study; Feldsine PT et al.; Six foods representing a variety of food products were analyzed by the Visual Immunoprecipitate Assay (VIP) and either the Bacteriological Analytical Manual (BAM) or the U.S . Department of Agriculture culture methods for detection of Listeria monocytogenes and related Listeria spp . Samples of each food type at each inoculation level were simultaneously analyzed by both methods . A total of 23 laboratories representing federal agencies and private industry in the United States and Canada participated in this collaborative study . Foods were inoculated with Listeria species including L . monocytogenes, with the exception of 3 lots of green beans that were naturally contaminated . During this study, 1509 samples and controls were analyzed and confirmed, of which 370 were positive and 921 were negative by both methods . One hundred and fifteen samples were positive by culture methods but negative by VIP . One hundred and thirty-two were negative by culture methods but positive by the VIP . Twenty-nine samples were negative by VIP and by culture methods but confirmed positive when VIP selective enrichment broths were subcultured to selective agars . The VIP method for detection of L . monocytogenes and related Listeria spp . in foods has been adopted first action by AOAC INTERNATIONAL.

J AOAC Int, 1997 Jul-Aug, 80(4), 775 - 90
Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria species in selected foods: collaborative study; Feldsine PT et al.; Six foods representing a variety of food products were analyzed by the Assurance Listeria polyclonal enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S . Department of Agriculture culture method for detecting Listeria monocytogenes and related Listeria species . Samples of each food type, at each inoculation level, were analyzed simultaneously by both methods . A total of 19 laboratories representing federal government agencies and private industry in the United States and Canada participated . Food types were inoculated with Listeria species including L . monocytogenes, with the exception of 3 lots of green beans, which were naturally contaminated . During this study, 1764 samples and controls were analyzed and confirmed, of which 492 were positive and 947 were negative by both methods . There were 159 samples that were positive by culture method but negative by the EIA and 188 that were negative by culture method but positive by EIA . Twenty-two samples were negative by EIA and by culture method but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar . The Assurance polyclonal EIA for detecting L . monocytogenes and related Listeria species in foods has been adopted first action by AOAC INTERNATIONAL.

Trends Microbiol, 1997 Jul, 5(7), 272 - 6
How the Listeria monocytogenes ActA protein converts actin polymerization into a motile force; Smith GA et al.; The ActA protein is an essential determinant of pathogenicity that is responsible for the actin-based motility of Listeria monocytogenes in mammalian cells and cell-free extracts . ActA appears to control at least four functions that collectively lead to actin-based motility: (1) initiation of actin polymerization, (2) polarization of ActA function, (3) transformation of actin polymerization into a motile force and (4) acceleration of movement mediated by the host protein profilin.

Am J Respir Crit Care Med, 1997 Jul, 156(1), 223 - 8
Expression of inducible nitric oxide synthase by macrophages in rat lung; Liu HW et al.; Nitric oxide (NO) is a short-lived free radical that is secreted by pulmonary macrophages (Mo) . An inducible isoform of NO synthase (iNOS) catalyses the production of NO and is activated by lipopolysaccharide and certain T-helper(h) 1 cytokines, including interferon-gamma and TNF-alpha . In the present study, iNOS+ interstitial cells were demonstrated in the alveolar wall of normal Lewis rat lung . Enzymatic digests of normal lung showed that approximately one third of pulmonary ED1+ interstitial Mo (IM) were iNOS+ and secreted modest amounts of NO without ex vivo stimulation, whereas normal alveolar macrophages (AM) were iNOS- and showed no basal NO secretion . When incubated with heat-killed Listeria monocytogenes (HKL) in vitro, AM secreted larger amounts of NO than did IM . Recombinant murine GM-CSF stimulated production of NO by AM but not by IM . However, when IM were costimulated with GM-CSF and IFN-gamma, they expressed a marked increase in NO production . Intratracheal challenge with HKL yielded decreased NO production by IM . We conclude that iNOS+ IM are present in normal rat lung, where they regulate the pulmonary cell-mediated immune response to antigen.

FEMS Microbiol Lett, 1997 Jul 1, 152(1), 149 - 54
Characterisation of the peptidoglycan hydrolases of Listeria monocytogenes EGD; McLaughlan AM et al.; The peptidoglycan hydrolase profile of Listeria monocytogenes EGD has been characterised under a variety of environmental and physiological conditions, using renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The profiles show activities ranging from 29 to 186 kDa . The 186-kDa enzyme was only observable under specific medium and aeration conditions . The enzyme activities show differential substrate specificity and sensitivity to incubation conditions . The peptidoglycan hydrolase profile of several different Listeria strains was also compared.

Int J Syst Bacteriol, 1997 Jul, 47(3), 863 - 9
Inter- and intraspecies comparison of the 16S-23S rRNA operon intergenic spacer regions of six Listeria spp; Graham TA et al.; The 16S-23S rRNA intergenic spacer (IGS) regions found in six Listeria species were characterized . PCR amplification of the 16S-23S IGS with a "generic primer" set generated products of about 340 bp (small) and 550 to 590 bp (large) with DNA from all Listeria strains tested . Seven Listeria monocytogenes serotype 4b strains and one L . monocytogenes serotype 4d strain also had an additional PCR product of ca . 360 bp . The 360-bp PCR product from one of these L . monocytogenes serotype 4b strains was identical in nucleotide sequence to the small 340-bp IGS, except that it contained an 18-bp tandem repeat . The small rRNA IGSs of L . innocua, L . ivanovii, L . seeligeri, L . welshimeri, and L . grayi were 83 to 99% homologous to that of L . monocytogenes . The large rRNA IGS of L . monocytogenes was 81 to 96% homologous to those of the other Listeria species and agreed with current taxonomic division among these species . The nucleotide sequences of the central 274 bp of the large rRNA IGS of strains from seven different L . monocytogenes serotypes were highly homologous; however, serotype-specific differences were noted, and four groups were identified within L . monocytogenes based on this analysis.

Am J Vet Res, 1997 Jul, 58(7), 733 - 7
Investigation of a listeriosis epizootic in sheep in New York state; Wiedmann M et al.; OBJECTIVE: To investigate potential sources of an epizootic of listerial encephalitis, using molecular diagnostic and typing methods . SAMPLE POPULATION: A flock of about 655 sheep . PROCEDURE: An epizootiologic investigation was performed . Clinical, feed, and environmental samples were tested for Listeria monocytogenes, using polymerase chain reaction and culture methods; recovered isolates were "fingerprinted," using an automated ribotyping system . RESULTS: Listeria monocytogenes was recovered from brain specimens of 7 sheep with clinical signs of listerial encephalitis . All clinical isolates had fingerprints identical to those of isolates from farm equipment used to transport silage . Corn silage, which was not fed to the sheep, also contained L monocytogenes of the same pattern type as defined by ribotyping . Listeria monocytogenes was not isolated from the stored haylage designated for feeding the sheep (the cut-off point for isolation being < 10(2) colony-forming units/g) . CONCLUSIONS: Corn silage was implicated as the source of a listeriosis epizootic . It appears to have cross-contaminated the haylage destined for the sheep during handling with a front-end loader . Suspension of silage feeding coincided with cessation of listeriosis cases . CLINICAL RELEVANCE: Use of advanced molecular techniques can help to identify the sources and restrict the scope of an epizootic . In epizootics, a single L monocytogenes strain can lead to infection of multiple animals, with rapid progression of the disease.

Curr Biol, 1997 Jul 1, 7(7), 519 - 29
The complex containing actin-related proteins Arp2 and Arp3 is required for the motility and integrity of yeast actin patches; Winter D et al.; BACKGROUND: Structural modeling and biochemical experiments in vitro have implicated a multi-protein complex containing two actin-related proteins, Arp2 and Arp3, as a potential actin-filament nucleation factor . This 'Arp2/3 complex' has been identified in Acanthamoeba and human cells and has been shown to localize to regions involved in actin-based motility, such as the leading edge of moving cells and the 'tail' of actin that forms behind the intracellular pathogen Listeria . The function of this complex in vivo has not been characterized, however, and the sequences of the non-actin-related subunits remain to be determined . RESULTS: An Arp3 homologue from the budding yeast Saccharomyces cerevisiae was found to localize to cortical actin patches, highly motile structures that concentrate at sites of polarized growth during the yeast cell cycle . A conditional arp3 mutant allele inhibited cortical actin motility at the restrictive temperature and eventually disrupted actin patches . Most Arp3 protein is found in a multi-protein complex; we purified this complex and determined the sequences of each of the protein subunits using a high-accuracy mass peptide-mapping technique . The proteins found in the complex are similar to those in the Acanthamoeba and human Arp2/3 complexes except that the yeast complex lacks a 40 kDa subunit, which is therefore not required for the structural integrity of the complex . CONCLUSIONS: The Arp2/3 protein complex is conserved from yeast to man, and in yeast the complex is required in vivo for the motility and integrity of cortical actin patches . We hypothesize that these patches may move by a Listeria-like mechanism driven by actin polymerization.

J Immunol, 1997 Jul 1, 159(1), 7 - 10
IL-4 secretion by CD4+ NK1+ T cells induces monocyte chemoattractant protein-1 in early listeriosis; Flesch IE et al.; IL-4 is a major promotor of Th2 differentiation and an antagonist of IFN-gamma production . Although experimental listeriosis is characterized by a Th1 response, IL-4-producing cells were detected in spleens of mice promptly after Listeria monocytogenes infection . We identified this early IL-4 as inducer of the chemokine, monocyte chemoattractant protein-1 (MCP-1), which mainly attracts monocytes/macrophages, but not neutrophils . MCP-1-secreting cells were demonstrable in spleens of mice infected with L . monocytogenes, and IL-4 neutralization with anti-IL-4 mAb 11B11 markedly diminished frequencies of MCP-1-producing cells . Cell depletion experiments and studies with gene disruption mutant mice lacking distinct T cell subsets and surface MHC molecules point to CD4+ NK1+ T cells as a cellular source of early IL-4 . Since monocyte infiltration to infective foci contributes to early control of listeriosis, our results suggest that IL-4-producing CD4+ NK1+ T cells participate in the innate immune response against L . monocytogenes through MCP-1 induction.

Infect Immun, 1997 Jul, 65(7), 2778 - 85
Utilization of iron-catecholamine complexes involving ferric reductase activity in Listeria monocytogenes; Coulanges V et al.; Listeria monocytogenes is a ubiquitous potentially pathogenic organism requiring iron for growth and virulence . Although it does not produce siderophores, L . monocytogenes is able to obtain iron by using either exogenous siderophores produced by various microorganisms or natural catechol compounds widespread in the environment . In the presence of tropolone, an iron-chelating agent, growth of L . monocytogenes is completely inhibited . However, the growth inhibition can be relieved by the addition of dopamine or norepinephrine under their different isomeric forms, while the catecholamine derivatives 4-hydroxy-3-methoxyphenylglycol and normetanephrine did not relieve the inhibitory effect of tropolone . Preincubation of L . monocytogenes with chlorpromazine and yohimbine did not antagonize the growth-promoting effect of catecholamines in iron-complexed medium . In addition, norepinephrine stimulated the growth-promoting effect induced by human transferrin in iron-limited medium . Furthermore, dopamine and norepinephrine allowed 55Fe uptake by iron-deprived bacterial cells . The uptake of iron was energy dependent, as indicated by inhibition of 55Fe uptake at 0 degrees C as well as by preincubating the bacteria with KCN . Inhibition of 55Fe uptake by L . monocytogenes was also observed in the presence of Pt(II) . Moreover, when assessed by a whole-cell ferric reductase assay, reductase activity of L . monocytogenes was inhibited by Pt(II) . These data demonstrate that dopamine and norepinephrine can function as siderophore-like compounds in L . monocytogenes owing to their ortho-diphenol function and that catecholamine-mediated iron acquisition does not involve specific catecholamine receptors but acts through a cell-bound ferrireductase activity.

Infect Immun, 1997 Jul, 65(7), 2707 - 16
Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential; Wiedmann M et al.; A total of 133 Listeria monocytogenes isolates were characterized by ribotyping and allelic analysis of the virulence genes hly, actA, and inlA to uncover linkages between independent phylogenetic and specific virulence markers . PCR-restriction fragment length polymorphisms revealed 8 hly, 11 inl4, and 2 actA alleles . The combination of these virulence gene alleles and ribotype patterns separated L . monocytogenes into three distinct lineages . While distinct hly and inlA alleles were generally found to cluster into these three lineages, actA alleles segregated independently . These three phylogenetic lineages were confirmed when 22 partial actA DNA sequences were analyzed . The clinical history of the L . monocytogenes strains showed evidence for differences in pathogenic potential among the three lineages . Lineage I contains all strains isolated during epidemic outbreaks of listeriosis, while no human isolates were found in lineage III . Animal isolates were found in all three lineages . We found evidence that isolates from lineages I and III have a higher plaquing efficiency than lineage II strains in a cell culture assay . Strains from lineage III also seem to form larger plaques than strains from lineage II . A distinctive ribotype fragment and unique 16S rRNA gene sequences furthermore suggest that lineage III might represent a L . monocytogenes subspecies . None of the 20 human isolates available but 11% of our animal isolates were grouped in this lineage, indicating that strains in this lineage might have reduced virulence for humans.

Int J Food Microbiol, 1997 Jun 17, 37(1), 37 - 45
Predictive model of the effect of CO2, pH, temperature and NaCl on the growth of Listeria monocytogenes; Fernandez PS et al.; The growth responses of L . monocytogenes as affected by CO2 concentration (0-100% v/v, balance nitrogen), NaCl concentration (0.5-8.0% w/v), pH (4.5-7.0) and temperature (4-20 degrees C) were studied in laboratory medium . Growth curves were fitted using the model of Baranyi and Roberts, and specific growth rates derived from the curve fit were modelled . Predictions for specific growth rate, doubling time and time to a 1000-fold increase could be made for any combination of conditions within the matrix . Predictions of growth from the model were compared with published data and this showed the model to be suitable for predicting growth of L . monocytogenes in a range of foods packaged under a modified atmosphere.

J Cell Biol, 1997 Jun 16, 137(6), 1381 - 92
Proteolytic pathways of activation and degradation of a bacterial phospholipase C during intracellular infection by Listeria monocytogenes; Marquis H et al.; Listeria monocytogenes is a facultative intracellular bacterial pathogen that spreads cell to cell without exposure to the extracellular environment . Bacterial cell-to-cell spread is mediated in part by two secreted bacterial phospholipases C (PLC), a broad spectrum PLC (PC-PLC) and a phosphatidylinositolspecific PLC (PI-PLC) . PI-PLC is secreted in an active state, whereas PC-PLC is secreted as an inactive proenzyme (proPC-PLC) whose activation is mediated in vitro by an L . monocytogenes metalloprotease (Mpl) . Analysis of PI-PLC, PC-PLC, and Mpl single and double mutants revealed that Mpl also plays a role in the spread of an infection, but suggested that proPC-PLC has an Mpl-independent activation pathway . Using biochemical and microscopic approaches, we describe three intracellular proteolytic pathways regulating PCPLC activity . Initially, proPC-PLC secreted in the cytosol of infected cells was rapidly degraded in a proteasome-dependent manner . Later during infection, PCPLC colocalized with bacteria in lysosome-associated membrane protein 1-positive vacuoles . Activation of proPC-PLC in vacuoles was mediated by Mpl and an Mpl-independent pathway, the latter being sensitive to inhibitors of cysteine proteases . Lastly, proPC-PLC activation by either pathway was sensitive to bafilomycin A1, a specific inhibitor of vacuolar ATPase, suggesting that activation was dependent on acidification of the vacuolar compartment . These results are consistent with a model in which proPC-PLC activation is compartment specific and controlled by a combination of bacterial and host factors.

J Immunol, 1997 Jun 15, 158(12), 5581 - 3
Mice lacking reduced nicotinamide adenine dinucleotide phosphate oxidase activity show increased susceptibility to early infection with Listeria monocytogenes; Dinauer MC et al.; Mice lacking the gp91 protein of reduced nicotinamide adenine dinucleotide phosphate oxidase showed heightened susceptibility to Listeria infection . The enhanced susceptibility was noted 2 days after infection, the usual peak time of the neutrophil-dependent lesion in the liver . Infected gp91phox -/- mice had an increase of approximately 30-fold in Listeria in the liver and an increase in the number of microabscesses . The study of the gp91phox mice underscores the early role of the neutrophil and of an oxidative burst in this infection with an intracellular pathogen . These results contrast with others showing that the late macrophage-dependent stage of the infection is dependent on nitric oxide.

Leuk Lymphoma, 1997 Jun, 26(1-2), 83 - 8
Antimicrobial prophylaxis to prevent opportunistic infections in patients with chronic lymphocytic leukemia after allogeneic blood or marrow transplantation; Mehta J et al.; Opportunistic infections have been a problem after BMT in CLL . We have allografted seven patients with B-CLL (n = 6) or B-prolymphocytic leukemia (n = 1) from matched siblings (n = 6) or a mismatched unrelated donor (n = 1) . Amongst the first six, we saw two cases of recurrent or prolonged cytomegaloviremia and CMV disease, one listeria meningitis, and one fatal toxoplasma encephalitis . The latter two developed in the setting of steroid therapy of GVHD with extensive prior fludarabine therapy . Prophylaxis for opportunistic infections was developed on an ongoing basis as new infectious complications were seen . The current drug prophylaxis, which has been successful for eight months in the last patient despite pretreatment with fludarabine and steroid therapy for GVHD, is directed against pneumocystis, toxoplasma, fungi, and pneumococci . It includes immunoglobulin (for 3 1/2 months), pyrimethamine-sulfadiazine (for 4 months and during steroids), fluconazole (for 2 1/2 months), cotrimoxazole or pentamidine (for 2 years) and penicillin (lifelong) . Dietary precautions are followed for 4 months and during steroids to prevent listeriosis . Four patients are alive in remission with no active infectious problems 8-44 months (median 29) after BMT . We recommend adoption of these or similar prophylactic measures for BMT in CLL as a baseline which can be modified if new infections are identified and according to individual needs.

Acta Paediatr Jpn, 1997 Jun, 39(3), 382 - 4
A case of congenital Listeria septicemia associated with high levels of inflammatory cytokines; Suda H et al.; A case of congenital Listeria septicemia is reported . A 2256 g male infant suffering from respiratory and circulatory failure with shock-like symptoms and high levels of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1 beta, -6, and -8), was admitted to the Morioka Red Cross Hospital . Listeria monocytogenes was cultured from cord blood, contents from the external ear canal, rectum and stomach . The infant was treated with surfactant replacement as well as conventional therapy . The high levels of interleukin-1 beta decreased with the improvement of the circulatory function, which might have been the major cause of the poor clinical state.

Zentralbl Bakteriol, 1997 Jun, 286(1), 46 - 55
Cell wall-deficient forms (L-forms) of Listeria monocytogenes in experimentally infected rats; Markova N et al.; Experimental infections were induced with different bacterial forms of Listeria monocytogenes: parental (S-forms), protoplastic (L-forms) and combined inoculum of both forms by i.p . injection of rats . The parental bacterial forms (S-forms) were isolated up to 7 days after challenge from the peritoneal cavity and the liver, while the L-forms were isolated up to 60 days from the peritoneal cavity . Continuous adhesion of L-forms on the peritoneal macrophage surface was found by scanning-electron microscopy . Erythrocyte and leucocyte count as well as some clinical chemistry parameters were measured during infections . They showed different dynamics in the three experimental groups . Histomorphological changes in the liver (microabscesses and mononuclear cellular granulomas) of infected animals were observed . They were less intensive and appeared later in rats infected with L-forms . The experiments demonstrated that infections caused by parental bacterial forms and by combined inoculum took an acute course, while the infection caused by L-forms could be distinguished as a prolonged and persistent one.

Zentralbl Bakteriol, 1997 Jun, 286(1), 33 - 40
Typing of Austrian Listeria monocytogenes isolates by automated laser fluorescence analysis of randomly amplified polymorphic DNA; Allerberger F et al.; We used automated laser fluorescence analysis of randomly amplified polymorphic DNA (RAPD-Alfa) to study the epidemiology of listeriosis in western Austria . There were no discrepancies between RAPD-Alfa patterns and serotypes found in 18 food isolates and 18 clinical isolates . The results of our study suggest that the food isolates typed were not at the origin of the human cases in western Austria . Using RAPD-Alfa, it was possible to link 9 out of 16 "sporadic" Listeria infections (mother-child cases counted as one) to the occurrence of other cases . Our results underline the necessity of epidemiological clarification of listeriosis cases as a prerequisite for specific preventive measures by public health services (e.g . confiscation of contaminated food products, issue of public warnings) . To establish the chain of infection, more is needed than just speciation of bacteria in incriminated food products . Automated laser fluorescence analysis of randomly amplified polymorphic DNA seems a suitable, easy and rapid method for the typing of Listeria monocytogenes strains.

Immunol Lett, 1997 Jun 1, 57(1-3), 33 - 7
T-cell anergy induced by antigen presenting cells treated with the hemolysin of Listeria monocytogenes; Darji A et al.; Antigen presenting cells (APC) that are infected with listeriolysin (LLO) secreting Listeria lack the ability to stimulate MHC class II restricted T-cells by conventional antigens . Similarly, T-cell activation by native proteins but not by peptides was inhibited upon pretreatment of APC with purified listeriolysin . The inhibition is due to an irreversible inactivation of T-cells that recognize antigen on infected or LLO treated APC . Inhibition was found to dominate over stimulation by peptides . This condition is reminiscent of T-cells inactivation by antagonistic peptides and represents a novel type of immune escape.

Zentralbl Veterinarmed B, 1997 Jun, 44(4), 253 - 6
A fatal case of Listeria endocarditis in a man following his tending of goats suggests an epidemiological link which is not supported by the results; Danielsson-Tham ML et al.; A man died in endocarditis due to listeriosis in the late autumn . He had been looking after two goats during the summer . Listeria monocytogenas was isolated from a rectal swab from one of the goats . The goat faeces isolate and the human blood isolate were of identical serovar . The two isolates, however, were shown to be different by multilocus electrophoretic enzyme analysis and ribotyping, as well as by biotyping . Thus, these results do not support the hypothesis that the man was infected by the goat.

FEMS Immunol Med Microbiol, 1997 Jun, 18(2), 99 - 104
A RE-PCR method to distinguish Listeria monocytogenes serovars; Comi G et al.; Strains (107) of L . monocytogenes were tested with a PCR-restriction enzyme analysis with two new original primers . A segment of 1395 bp containing the entire iap gene in L . monocytogenes was amplified by the PCR technique . The PCR product was cleaved with the restriction enzymes HindIII and RsaI, and the fragments generated were separated by gel electrophoresis . Two groups of serovars were obtained: one group contained serovars 1/2a and 1/2c, the other group contained serovars 1/2b, 3b and 4b . The PCR-restriction enzyme analysis method described in this paper could be a useful tool for the unambiguous division of L . monocytogenes into two serovar groups, and it could be used to study the evolution of different serotypes and groups of serotypes in foods produced in the same processing plant and processed during the same month . The RE-PCR method used can give a rapid confirm at the subgroup level in the laboratory of an epidemiological association between human disease and suspected sources of contaminated food.

J Cell Sci, 1997 Jun, 110 ( Pt 12), 1361 - 71
Ligand recruitment by vinculin domains in transfected cells; Bubeck P et al.; Vinculin, a prominent protein component of microfilament-membrane attachment sites, consists of three major domains: an N-terminal, compact head and a C-terminal rod-like tail that are connected by a flexible, proline-rich hinge . In vitro, the protein has been shown to interact with numerous ligands, including other components of the microfilament system . To characterize the ligand recruitment ability of the different vinculin domains in a cellular environment, we used a novel approach of comprising chimeric proteins of either the vinculin head, hinge or tail regions, fused to the membrane anchor sequence of ActA, a surface protein of the intracellular bacterial pathogen Listeria monocytogenes . When PtK2 cells were transfected with the corresponding constructs, the ActA membrane anchor directed the chimeric polypeptides to mitochondrial membranes . In this position, they accumulated microfilament proteins, as seen by immunofluorescence analysis . A chimera comprising the full length vinculin clone recruited a substantial amount of the cellular F-actin, the vasodilator stimulated phosphoprotein (VASP) and paxillin, but little alpha-actinin and talin . The presence of only the vinculin head directed some of the fusion protein to focal contacts, and alpha-actinin recruitment was still ineffective . Prominent recruitment of F-actin and of VASP required the presence of the tail and proline-rich hinge, respectively . Reducing the vinculin tail to short pieces harboring only one of the two F-actin binding sequences, which were defined by in vitro experiments, resulted in loss of activity, possibly by incorrect polypeptide folding . The proline-rich hinge domain could be exchanged for the analogous region of the ActA protein, and the number of such proline-clusters, containing an FPPPP motif, correlated with the extent of VASP recruitment . The results show that this system can be used to analyze in vivo the activity of vinculin domains responsible for the assembly of various cytoskeletal ligands.

Eur J Immunol, 1997 Jun, 27(6), 1570 - 5
The defined attenuated Listeria monocytogenes delta mp12 mutant is an effective oral vaccine carrier to trigger a long-lasting immune response against a mouse fibrosarcoma; Paglia P et al.; Listeria monocytogenes has been proposed as a carrier to elicit major histocompatibility complex class-I restricted immune responses able to protect against tumor challenge . In this study the properties of the attenuated L . monocytogenes delta mp12 mutant has been evaluated in vivo against a highly aggressive mouse fibrosarcoma which expresses beta-galactosidase (beta-gal) as a tumor-associated antigen (TAA) . Immunization with the vaccine prototypes resulted in both elicitation of specific antibodies and generation of cytotoxic lymphocytes (CTL) . Oral vaccination protected 55-64% of the immunized animals from tumor take (p < 0.01) and strongly reduced the average size of the tumor in the other 34-45% (p < 0.01) . Vaccinated mice developed a long-lasting response, which resulted in 100% protection from a subsequent tumor challenge . Substitution of the whole TAA by its CTL-defined immunodominant epitope resulted in 43% protection, suggesting a contribution of the humoral response to the observed antitumor effect . No statistically significant differences were observed in the antitumor response when mice were immunized with strains expressing the immunodominant TAA epitope in the context of carrier proteins which were either exported or restricted to the bacterial cytoplasm . This suggests that the topology of the recombinant antigen does not play a major role in the outcome of the protective response.

Eur J Immunol, 1997 Jun, 27(6), 1353 - 9
TAP-dependent major histocompatibility complex class I presentation of soluble proteins using listeriolysin; Darji A et al.; Immunization of mice with mixtures of listeriolysin, a pore-forming hemolysin secreted by the pathogenic bacterium Listeria monocytogenes, together with soluble ovalbumin, nucleoprotein of influenza virus, or beta-galactosidase of Escherichia coli, resulted in strong cytotoxic CD8 T cell responses to each of the respective passenger proteins in vivo . Also, the concomitant addition of either protein with listeriolysin to target cells elicited efficient sensitization of these cells which could be attributed to the pore-forming activity of listeriolysin . This response was dependent upon a functional TAP transporter and was inhibitable by brefeldin A, indicating the transfer of the soluble proteins into the cytosol and the classical major histocompatibility (MHC) class I presentation pathway . The treatment of target cells with listeriolysin under our experimental conditions did not affect cell viability and the pores generated by listeriolysin treatment were repaired within 60 min . Introduction of soluble proteins into the MHC class I presentation pathway by listeriolysin provides a powerful system to study the cytotoxic response towards intracellular pathogens and would allow for rapid screening of potential antigens in vaccine formulations.

Lett Appl Microbiol, 1997 Jun, 24(6), 445 - 50
A comparison of immunomagnetic and surface adhesion immunofluorescent techniques for the rapid detection of Listeria monocytogenes and Listeria innocua in meat; Duffy G et al.; An immunomagnetic immunofluorescent method was investigated for the rapid detection of Listeria monocytogenes and Listeria innouca . This technique involved enrichment of the suspect sample at 30 degrees C overnight . Listeria monocytogenes cells were isolated from the enriched sample using immunomagnetic separation and Listeria were subsequently visualized using an immunofluorescent microscopy technique . This technique was used in the detection of Listeria cells from pure culture, inoculated beef mince samples and naturally contaminated retail beef mince samples . A detection level of approximately 1 x 10(3) cfu ml-1 was achieved . When compared with traditional detection methods no false negatives or positives were recorded for L . monocytogenes or L . innocua . The immunomagnetic immunofluorescent technique had a detection level similar to a previously described surface adhesion immunofluorescent technique . Isolation of the Listeria cells by surface adhesion involved dipping a membrane attached to a microscope slide into the enriched sample for 10 min . This was quicker and simpler to perform than the immunomagnetic separation technique which took 2 h to carry out.

J Appl Microbiol, 1997 Jun, 82(6), 780 - 2
Research note: in vitro inhibition of Listeria monocytogenes growth by veillonellae cultures grown on tartrate media; Hinton A Jr et al.; Veillonellae cultures were grown on agar media supplemented with tartrate and examined for inhibitory effects on the growth of Listeria monocytogenes . Veillonellae cultures grown on media supplemented with 0 or 50 mmol l-1 of tartrate did not inhibit the growth of L . monocytogenes; however, veillonellae grown on media supplemented with 100, 150 or 200 mmol l-1 of tartrate did inhibit the growth of L . monocytogenes . The inhibition of the growth of L . monocytogenes by the veillonellae was correlated with the increased production of acetate and propionate from tartrate by veillonellae and with the reduction of the pH of the media by L . monocytogenes.

J Appl Microbiol, 1997 Jun, 82(6), 759 - 62
Bioactivity of selected plant essential oils against Listeria monocytogenes; Lis-Balchin M et al.; Ninety-three different commercial essential oils were screened for activity against 20 Listeria monocytogenes strains in vitro and the results correlated against the actual chemical composition of each oil . There was a substantial difference in the activity between different essential oils as expected, but there was also a difference in activity between different samples of the same essential oil . Strong anti-Listeria activity was often correlated with essential oils containing a high percentage of monoterpenes, eugenol, cinnamaldehyde, thymol, and sometimes with citronellol, limonene and geraniol . However, as there was often no correlation between the anti-Listeria activity and the main chemical components, it is possible that either there is a more complex relationship with the chemical composition (which includes the minor components) or that substantial adulteration had occurred in some essential oil samples.

J Neuroimmunol, 1997 Jun, 76(1-2), 10 - 4
Inhibition of gamma interferon synthesis by catecholamines; Andrade-Mena CE; In vitro incubation of Listeria monocytogenes immune spleen cells in the presence of the catecholamines epinephrine or norepinephrine inhibited the gamma interferon (IFN-gamma) synthesis induced by the mitogen PHA, in a manner that appeared to be concentration dependent . Moreover, the inhibitory effect of both catecholamines epinephrine and norepinephrine on the synthesis of IFN-gamma was prevented by incubating immune spleen cells in the presence of propranolol, a beta adrenergic antagonist agent.

Clin Exp Immunol, 1997 Jun, 108(3), 456 - 62
Persistent production of interferon-gamma (IFN-gamma) and IL-12 is essential for the generation of protective immunity against Listeria monocytogenes; Xiong H et al.; IFN-gamma and IL-12 are believed to be important in the host defence against Listeria infection in mice . However, the relationship between these two cytokines and generation of protective immunity remains poorly understood . In the present study, it was found that at least 4 days of immunizing infection were required for the generation of protective immunity against L . monocytogenes . Protective immunity was generated only by immunizing infection with virulent strain . Even repeated injections of avirulent strain failed to induce protective immunity . When the immunizing infection was terminated with antibiotics, generation of protective immunity and IFN-gamma-producing ability was impaired, while expression of IFN-gamma and IL-12 was also impaired . The mutual relationship between IFN-gamma and IL-12 in L . monocytogenes infection was analysed in vitro . After neutralization of IL-12, IFN-gamma production was completely blocked and IFN-gamma expression was also inhibited . In contrast, there was no change of IL-12 expression after neutralization of IFN-gamma . Taking all facts into consideration, it may be concluded that persistent production of IFN-gamma induced by persistent production of IL-12 during immunizing infection is essential for the generation of protective immunity against L . monocytogenes.

J Immunol, 1997 Jun 1, 158(11), 5305 - 13
Listeria monocytogenes infection of cultured endothelial cells stimulates neutrophil adhesion and adhesion molecule expression; Drevets DA; Microbial infection of the endothelium with the resultant up-regulation of adhesion molecule expression and stimulated leukocyte adhesion to endothelial cells can promote an inflammatory response . Previous work demonstrated that Listeria monocytogenes can replicate within cultured endothelial cells; thus, we tested whether L . monocytogenes infection of HUVEC stimulated an inflammatory phenotype on these cells . Infection with 10(4) CFU of bacteria increased neutrophil adhesion to HUVEC 40-fold and up-regulated E-selectin, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1 expression . Approximately 80% of neutrophil adhesion to infected HUVEC was blocked by anti-E-selectin mAb, 35% was blocked by anti-CD18 mAb, and anti-vascular cell adhesion molecule-1 mAb was without effect . Microscopy of infected HUVEC monolayers showed that neutrophils bound to infected and uninfected cells and that infected and uninfected HUVEC expressed E-selectin . Interestingly, uninfected HUVEC that bound neutrophils or expressed E-selectin typically were adjacent to infected cells . However, infected monolayers did not produce soluble factors that stimulated E-selectin expression on uninfected cells . Nuclear translocation of the p65 subunit of the transcription factor NF-kappaB accompanied the HUVEC response, and hemolysin secretion appeared critical for stimulating HUVEC . These studies show that L . monocytogenes infection stimulates up-regulation of adhesion molecule expression on endothelial cells, resulting in neutrophil adhesion to them . This response includes induction of an inflammatory phenotype on uninfected cells and may be triggered by listeriolysin O-mediated activation of host response mechanisms . Additionally, cell-to-cell spread of L . monocytogenes throughout the monolayer, without stimulating secondarily infected endothelial cells for neutrophil adhesion, is a possible means of immune avoidance.

J Immunol, 1997 Jun 1, 158(11), 5297 - 304
Impaired macrophage listericidal and cytokine activities are responsible for the rapid death of Listeria monocytogenes-infected IFN-gamma receptor-deficient mice; Dai WJ et al.; IFN-gamma receptor-deficient (IFN-gammaR -/-) mice were used to study the innate immune responses during infection with Listeria monocytogenes . Mutant mice were unable to limit bacterial growth and died of sepsis even with an infection dose of 70 Listeria . At day 2, they showed an exacerbated listeriosis and mice succumbed to infection before the onset of an effective specific immunity, demonstrating a defective innate immunity . Recruitment and extravasation of phagocytic cells to infected organs was present and dominated by neutrophils . However, during the early course of infection, mutant mice responded by an elevated inflammatory type 1 cytokine response, as determined by IL-12, IFN-gamma, TNF-alpha, and IL-1alpha-specific RNA expression . Induction of inducible nitric oxide synthase was present and also increased in mutant mice . Interestingly, IFN-gammaR -/- neutrophils expressed substantial TNF-alpha- and IL-1alpha-specific RNA, suggesting a substantial contribution in the overall inflammatory cytokine response . In contrast, IFN-gammaR -/- macrophages showed reduced MHC class II surface expression levels and impaired TNF-alpha and IL-1alpha but normal IL-6 production after restimulation with heat-killed L . monocytogenes . Moreover, IFN-gammaR -/- macrophages showed defective listericidal activities . In contrast to normal macrophages, Listeria escaped rapidly from the phagosome in IFN-gammaR -/- macrophages to the cytoplasm, where they productively survived . In conclusion, these data suggest that IFN-gammaR signaling activates yet unknown functions in macrophages, preventing Listeria-induced escape from the phagosome and consequent killing of the invader . Together with the impaired cytokine responses, these macrophage defects seem to be responsible for the dramatic susceptibility during innate immunity, whereas predominant neutrophil responses mediate limited protective role in mutant mice.

J Immunol, 1997 Jun 1, 158(11), 5211 - 8
Multifocal defects in immune responses in RelB-deficient mice; Weih F et al.; Mice with a targeted disruption of the Rel/nuclear factor-kappaB family member RelB develop a complex inflammatory phenotype, myeloid hyperplasia, and splenomegaly due to extramedullary hemopoiesis . In this work, we report that RelB-deficient mice, in addition to the pathologic changes, were highly susceptible to infection by the facultative intracellular bacterium Listeria monocytogenes . RelB binds transcriptionally active kappaB motifs in the TNF-alpha promoter in normal cells, and in vitro studies with macrophages isolated from RelB-deficient animals revealed impaired production of TNF-alpha in response to LPS and IFN-gamma . RelB-deficient mice also were unable to mount a protective immune response against lymphocytic choriomeningitis virus . These results indicate a defective T cell-macrophage interaction and cytotoxic T cell response, respectively, in mice lacking RelB . Analysis of resting and specific Ab production demonstrated that while RelB is not required for the secretion of Ig isotypes that result from heavy chain class switching, it is necessary for normal production of Ag-specific IgG in response to T cell-dependent and -independent stimuli . Thus, RelB is not only essential for a normal hemopoietic system in the unchallenged animal, but also involved in various specific and nonspecific immune responses.

J Clin Microbiol, 1997 Jun, 35(6), 1606 - 8
Serological diagnosis of bovine, caprine, and ovine mastitis caused by Listeria monocytogenes by using an enzyme-linked immunosorbent assay; Bourry A et al.; An enzyme-linked immunosorbent assay for detecting Listeria monocytogenes antibodies in bovine (n = 35), caprine (n = 27), and ovine (n = 30) milk samples was evaluated by comparison with bacteriological examination . Microtiter plates were coated with proteins obtained from culture supernatant, and antibodies were revealed with a monoclonal antibody able to react with the immunoglobulins belonging to the three animal species . The arithmetic mean optical density (OD) of milk samples infected with L . monocytogenes was above that of uninfected milk samples or milk samples infected with pathogens others than L . monocytogenes . With an OD threshold of 0.2 for goat and ewe milk samples, the sensitivity and specificity of the test were 100 and 88%, respectively . The choice of a different OD threshold (0.5) for cows allowed the discrimination of all of the infected cows and yielded no false positives, and both sensitivity and specificity were 100%.

Int J Food Microbiol, 1997 May 20, 36(2-3), 179 - 86
A heterogeneous population model for the analysis of bacterial growth kinetics; McKellar RC; A two-compartment, heterogeneous population model (HPM) was derived using the simulation software SB ModelMaker to describe the growth of Listeria monocytogenes in bacteriological media at 5-35 degrees C . The model assumed that, at time t = 0, the inoculum was distributed between two distinct compartments, Non-Growing and Growing, and that growth could be described by four parameters: initial total cell population (N0), final maximum cell population (Nmax), maximum specific growth rate (mu(max)), and initial cell population in the Growing compartment (G0) . The model was fitted to the data by optimizing the four parameters, and lag phase duration (lambda) was calculated . The resulting values of mu(max) and lambda were similar to those determined using the modified Gompertz equation . A new parameter, w0, was defined which relates to the proportion of the initial cell population capable of growth, and is a measure of the initial physiological state of the cells . A modified model in which mu(max) was replaced with a temperature function, and w0 replaced G0, was used to predict the effect of temperature on the growth of L . monocytogenes . The results of this study raise questions concerning the current definition of the lag phase.

Int J Food Microbiol, 1997 May 20, 36(2-3), 155 - 61
Antilisterial activity of three bacteriocins used at sub minimal inhibitory concentrations and cross-resistance of the survivors; Song HJ et al.; The kinetics of inhibition of Listeria innocua strain Lin11 in a nutrient broth containing either nisin, pediocin AcH, or enterococcin EFS2 at concentrations ensuring a 2-3 log reduction of the population were first investigated . The rate of inhibition differed considerably between the bacteriocins . At the time that maximum viability loss occurred in the cultures containing either nisin (12 AU ml(-1)) or pediocin AcH (50 AU ml(-1)) the survivors resumed growth, although the medium retained most of its initial inhibitory activity . The survivors displayed increased resistance not only toward the bacteriocin they were in contact with, but toward the two other bacteriocins under study.

FEMS Microbiol Lett, 1997 May 15, 150(2), 189 - 95
Isolation and characterization of a Listeria monocytogenes mutant strain hyperproducing virulence factors; Ermolaeva S et al.; A mutant strain characterized by hyperproduction of a number of cell wall and supernatant proteins was isolated after N'methyl-N'-nitro-N-nitrosoguanidine treatment of Listeria monocytogenes strain NCTC10527 . Several of these proteins were identified as virulence factors . When a wild-type strain was grown in the presence of activated charcoal it was shown to exhibit the same protein pattern as the isolated mutant.

J Interferon Cytokine Res, 1997 May, 17(5), 271 - 7
Dietary fish oil enhances circulating interferon-gamma in mice during listeriosis without altering in vitro production of this cytokine; Fritsche KL et al.; The objective of this study was to investigate the impact of feeding mice a diet rich in n-3 polyunsaturated fatty acids (PUFA) from fish oil on the interferon-gamma (IFN-gamma) response during an active infection with Listeria monocytogenes . Weanling female C3H/Hen mice were fed experimental diets containing 20% by weight one of the following fats: soybean oil, lard, or a mixture of menhaden fish oil and corn oil (17:3, w/w) . After 4 weeks, mice were injected with 10(5) live L . monocytogenes, and the concentration of IFN-gamma in serum and spleen was determined 0, 2, 4, and 7 days postinfection by enzyme-linked immunosorbent assay (ELISA) . Fish oil-fed mice showed significantly higher IFN-gamma in their blood at 2 and 4 days postchallenge compared with mice fed the soybean oil-containing or lard-containing diets (p < 0.001) . A higher concentration of IFN-gamma was also found in the spleen homogenate of fish oil-fed mice on day 4 postchallenge (p < 0.005) . To examine in vitro IFN-gamma production, splenocytes were isolated from fish oil-fed and soybean oil-fed mice on day 4 postchallenge and cultured with concanavalin A (1 microgram/ml and 10 micrograms/ml) for 24 and 48 h . There were no significant differences in the IFN-gamma concentration in cell culture supernatants between these diet treatments . This study demonstrated that the elevation in the concentration of IFN-gamma in blood and spleen during murine listeriosis is accentuated and prolonged by dietary n-3 PUFA, and these effects may not be due to changes in IFN-gamma production.

Infection, 1997 May-Jun, 25(3), 135 - 9
Nosocomial infections by Listeria monocytogenes: analysis of a cluster of septicemias in immunocompromised patients; Elsner HA et al.; From December 1994 to November 1995 an unusual accumulation of Listeria infections occurred at the University Hospital Hamburg-Eppendorf, Germany . Eleven immunosuppressed patients from different departments developed septicemia due to Listeria monocytogenes during hospitalization . In a retrospective study, serotyping and pulsed-field gel electrophoresis revealed that six isolates were identical or genetically related . Four of them had been isolated from renal transplant recipients . Listeria monocytogenes was neither detected in food samples of the renal transplantation ward, nor in stool specimens obtained from the ward staff . There had been no close contacts among the infected patients . Before transplantation, the renal transplant recipients had been dialysed in different dialysis centers . Nosocomial foodborne transmission could not be proven but seems likely.

Lett Appl Microbiol, 1997 May, 24(5), 421 - 5
Optimization of haemolysis in enhanced haemolysis agar (EHA)--a selective medium for the isolation of Listeria monocytogenes; Beumer RR et al.; The presence of Listeria monocytogenes in enrichment media can be masked by faster growth of other Listeria spp . Therefore, enhanced haemolysis agar (EHA) is a good alternative for another isolation media, because the presence of a few L . monocytogenes colonies can be detected in a majority of colonies of other listeriae on the basis of haemolysis . In this study the haemolysis reaction in EHA was optimized . In a collaborative study using reference samples, no significant differences in counts on EHA, Palcam and Oxford agar were shown.

Lett Appl Microbiol, 1997 May, 24(5), 343 - 6
The use of the bacteriocin, nisin, as a preservative in ricotta-type cheeses to control the food-borne pathogen Listeria monocytogenes; Davies EA et al.; The efficacy of nisin to control the food-borne pathogen Listeria monocytogenes in ricotta-type cheeses over long storage (70 d) at 6-8 degrees C was determined . Cheeses were prepared from unpasteurized milk by direct acidification with acetic acid (final pH 5.9) and/or calcium chloride addition during heat treatment . Nisin was added in the commercial form of Nisaplin pre-production to the milk . Each batch of cheese was inoculated with 10(2)-10(3) cfu g-1 of a five-strain cocktail of L . monocytogenes before storage . Shelf-life analysis demonstrated that incorporation of nisin at a level of 2.5 mg l-1 could effectively inhibit the growth of L . monocytogenes for a period of 8 weeks or more (dependent on cheese type) . Cheese made without the addition of nisin contained unsafe levels of the organism within 1-2 weeks of incubation . Measurement of initial and residual nisin indicated a high level of retention over the 10-week incubation period at 6-8 degrees C, with only 10-32% nisin loss.

J Appl Microbiol, 1997 May, 82(5), 641 - 7
Prevalence of Listeria sp . in droppings from urban rooks (Corvus frugilegus); Bouttefroy A et al.; Droppings from 112 urban rooks (Corvus frugilegus) were cultured for the presence of Listeria sp . Overall, 46% of rooks sampled harboured one or more Listeria species . Of all birds examined, 33%, 24% and 8%, respectively, were infected with Listeria monocytogenes, Listeria innocua and Listeria seeligeri . Differentiation of L . monocytogenes and L . seeligeri carried out by several phenotypic typing methods proved the diversity of strains and the major role of rooks which widely contribute to spreading this bacteria in our environment . The results also suggest that the ability to recover specific Listeria strains from the same sample is at least partially dependent on the methodology . These findings reinforce the need for strain-specific typing of multiple L . monocytogenes isolates from the same sample.

J Appl Microbiol, 1997 May, 82(5), 567 - 77
Expanded models for the non-thermal inactivation of Listeria monocytogenes; Buchanan RL et al.; Previously developed four-variable response surface models for describing the effects of temperature, pH/lactic acid, sodium chloride and sodium nitrite on the time to achieve a 4-log, non-thermal inactivation (t4D) of Listeria monocytogenes in aerobic, acidic environments were expanded to five-variable models that distinguish the effects of pH and acidulant concentration . A total of 18 new variable combinations were evaluated and the inactivation kinetics data appended onto a consolidation of two data sets from earlier studies . The consolidated data set, which included 315 inactivation curves representing 209 unique combinations of the five variables, was analysed by response surface analysis . The quadratic model without backward elimination regression was selected for further evaluation . Three additional quadratic models were generated using the concentrations of undissociated lactic and/or nitrous acids as variables in place of percentage lactic acid and sodium nitrite concentration . Comparison of predicted t4D values against literature values for various food systems indicated that the models provide reasonable initial estimates of the inactivation of L . monocytogenes . The models based on the concentration of undissociated lactic and nitrous acids support the hypothesis that antimicrobial activity is associated with this form of the compounds . Evaluation of several examples suggests that these models may be useful for predicting the equivalent of the compounds' "minimal inhibitory concentrations' for accelerating inactivation under various conditions.

Infect Immun, 1997 May, 65(5), 1883 - 91
CD95 (Fas) may control the expansion of activated T cells after elimination of bacteria in murine listeriosis; Fuse Y et al.; CD95 (Fas) is known to mediate activation-induced T-cell death by apoptosis . To understand the role of CD95 during the course of bacterial infection, we examined the kinetics of alphabeta and gammadelta T cells in the peritoneal cavities and livers of 5-week-old CD95-defective MRL/lpr mice after an intraperitoneal infection with Listeria monocytogenes . The number of bacteria in the spleen decreased to an undetectable level by day 10 after infection with 7 x 10(3) Listeria cells similar to the number in MRL/+/+ mice . The number of alphabeta T cells expressing CD44 and CD95 reached a maximum in the peritoneal cavity on day 6 after listerial infection and thereafter decreased gradually in MRL/+/+ mice, whereas CD44+ alphabeta T cells without CD95 expression continued to increase throughout the course of listerial infection in MRL/lpr mice . Freshly isolated T cells from MRL/+/+ mice infected with L . monocytogenes 10 days previously showed DNA fragmentation with apoptosis, whereas such fragmentation was not prominent in T cells from infected MRL/lpr mice . In correlation with the increased number of CD44+ alphabeta T cells, Listeria-specific T-cell proliferation of peritoneal exudate cells was significantly greater in MRL/lpr mice than in MRL/+/+ mice on day 10 after listerial infection . In contrast to alphabeta T cells, gammadelta T cells increased in number only transiently in the peritoneal cavity and liver after listerial infection in both MRL/lpr mice and MRL/+/+ mice . These results suggest that CD95-mediated cell death with apoptosis may be involved in termination of the alphabeta-T-cell-mediated immune response after the battle against L . monocytogenes has been won, whereas gammadelta T cells may undergo apoptosis independently of CD95 during the course of listerial infection.

Infect Immun, 1997 May, 65(5), 1615 - 25
Identification of four new members of the internalin multigene family of Listeria monocytogenes EGD; Dramsi S et al.; Listeria monocytogenes is a bacterial pathogen that is able to invade nonphagocytic cells . Two surface proteins, internalin, the inlA gene product, and InlB, play important roles in the entry into cultured mammalian cells . These proteins also have extensive sequence similarities . Previously, Southern hybridization predicted the existence of an internalin multigene family . Recently, InlC, a secreted protein of 30 kDa homologous to InlA and InlB, was identified . In this work, we identified and characterized four new members of the internalin multigene family, inlC2, inlD, inlE, and inlF which encode proteins of 548, 567, 499, and 821 amino acids respectively . inlC2, inlD, and inlE are contiguous on the chromosome of L . monocytogenes EGD, whereas inlF is located in a different chromosomal region . These four inl gene products display the principal features of internalin, namely, a signal sequence, two regions of repeats (or LRR and B repeats), and a putative cell wall anchor sequence containing the sorting motif LPXTG . The four inl genes were maximally expressed albeit at a low level during early exponential growth in bacterial medium at 37 degrees C . The role of these inl genes in L . monocytogenes invasion was assessed by constructing isogenic chromosomal deletion mutants and testing them for entry into various nonphagocytic cells . Unexpectedly, the inlC2, inlD, inlE, and inlF null mutants were not affected for entry into any of the cell lines tested, raising the possibility that these genes are needed for an aspect of pathogenicity other than invasion . The identity of such an aspect remains to be determined.

Ugeskr Laeger . 1997 Apr 28;159(18):2716.
{Listeriosis in the third trimester of pregnancy}; Lorentzen U et al.; A case of fatal intrauterine listeriosis in the third trimester of pregnancy is described . The patient presented with preterm labour and was delivered by emergency caesarean section on suspicion of foetal distress . The child was stillborn . The diagnosis was based on specific histopathological findings in the foetus and the placenta.

J Exp Med, 1997 Apr 21, 185(8), 1403 - 11
Enhanced intracellular dissociation of major histocompatibility complex class I-associated peptides: a mechanism for optimizing the spectrum of cell surface-presented cytotoxic T lymphocyte epitopes; Sijts AJ et al.; Association of antigenic peptides with newly synthesized major histocompatibility complex (MHC) class I molecules occurs in the endoplasmic reticulum and is a critical early step for the initiation of cytotoxic T lymphocyte (CTL)-mediated immune defenses . Pathogen-derived peptides compete with a plethora of endogenous peptides for MHC class I grooves . We find that two H2-K(d)-restricted peptides, which derive from the Listeria monocytogenes p60 antigen, accumulate in infected cells with different kinetics . Although competition assays suggest that both epitopes are bound with equivalent affinity, they dissociate from MHC class I molecules at markedly different rates . p60 217-225 forms complexes with H2-K(d) with a half-life >6 h, while p60 449-457 dissociates from H2-K(d) with a half-life of approximately 1 h . We find that p60 449-457-H2-K(d) complexes retained intracellularly with brefeldin A have a half-life of 30 min, and thus are less stable than surface complexes . While peptide dissociation from retained MHC class I molecules is enhanced, retained H2-K(d) molecules maintain a remarkable capacity to bind new T cell epitopes . We find that intracellular H2-K(d) molecules can bind new CTL epitopes for up to 3 h after their synthesis . Our studies provide a glimpse of peptide interaction with MHC class I molecules in the endoplasmic reticulum/proximal Golgi complex of intact, infected cells . We propose that the increased intracellular lability of peptide-MHC class I complexes may function to optimize the spectrum of peptides presented to T lymphocytes during cellular infection.

Int J Food Microbiol, 1997 Apr 15, 35(3), 281 - 5
PCR detection of Listeria monocytogenes in 'gravad' rainbow trout; Ericsson H et al.; 'Gravad' rainbow trout artificially contaminated with Listeria monocytogenes was analyzed by use of a 4 h enrichment period followed by extraction of DNA and PCR amplification . This procedure made it possible to detect 10-100 cfu L . monocytogenes per gram 'gravad' rainbow trout, within 12 h . After a prolonged enrichment period of 24 h, numbers as low as 1-10 cfu L . monocytogenes per gram could be detected . The method described may be a useful tool for screening samples of 'gravad' rainbow trout for the presence of L . monocytogenes, since it is sensitive, rapid and simple.

Int J Food Microbiol, 1997 Apr 15, 35(3), 275 - 80
Sample preparation and DNA extraction procedures for polymerase chain reaction identification of Listeria monocytogenes in seafoods; Agersborg A et al.; Five grams of seafood products were inoculated with one to 500 viable or 10(9) heat-killed cells of Listeria monocytogenes . The presence of the pathogen was detected by the polymerase chain reaction (PCR) with primers specific for fragments of the listeriolysin O (hly) gene (two sets) and for the invasion-associated protein (iap) gene (one set) . For DNA preparation, boiling, either alone or in combination with lysozyme and proteinase K treatment, was not always sufficient to lyse L . monocytogenes, while treatment with Triton X-100 produced consistently good DNA suitable for amplification . To avoid false-negative and false-positive results, 48 h incubations were necessary and a subculturing step after an initial 24 h incubation greatly improved the results . The primers that amplified regions of the listeriolysin O gene gave clearer and stronger products than primers for the invasion-associated protein gene . Using this method we were able to detect one to five L . monocytogenes cells in 5 g of product in a total of 55 h.

J Cell Biol, 1997 Apr 7, 137(1), 155 - 67
The isolated comet tail pseudopodium of Listeria monocytogenes: a tail of two actin filament populations, long and axial and short and random; Sechi AS et al.; Listeria monocytogenes is driven through infected host cytoplasm by a comet tail of actin filaments that serves to project the bacterium out of the cell surface, in pseudopodia, to invade neighboring cells . The characteristics of pseudopodia differ according to the infected cell type . In PtK2 cells, they reach a maximum length of approximately 15 microm and can gyrate actively for several minutes before reentering the same or an adjacent cell . In contrast, the pseudopodia of the macrophage cell line DMBM5 can extend to >100 microm in length, with the bacteria at their tips moving at the same speed as when at the head of comet tails in bulk cytoplasm . We have now isolated the pseudopodia from PtK2 cells and macrophages and determined the organization of actin filaments within them . It is shown that they possess a major component of long actin filaments that are more or less splayed out in the region proximal to the bacterium and form a bundle along the remainder of the tail . This axial component of filaments is traversed by variable numbers of short, randomly arranged filaments whose number decays along the length of the pseudopodium . The tapering of the tail is attributed to a grading in length of the long, axial filaments . The exit of a comet tail from bulk cytoplasm into a pseudopodium is associated with a reduction in total F-actin, as judged by phalloidin staining, the shedding of alpha-actinin, and the accumulation of ezrin . We propose that this transition reflects the loss of a major complement of short, random filaments from the comet, and that these filaments are mainly required to maintain the bundled form of the tail when its borders are not restrained by an enveloping pseudopodium membrane . A simple model is put forward to explain the origin of the axial and randomly oriented filaments in the comet tail.

J Exp Med, 1997 Apr 7, 185(7), 1241 - 51
Bystander activation of cytotoxic T cells: studies on the mechanism and evaluation of in vivo significance in a transgenic mouse model; Ehl S et al.; Bystander activation, i.e., activation of T cells specific for an antigen X during an immune response against antigen Y may occur during viral infections . However, the low frequency of bystander-activated T cells has rendered it difficult to define the mechanisms and possible in vivo relevance of this nonspecific activation . This study uses transgenic mice expressing a major histocompatibility complex class I-restricted TCR specific for glycoprotein peptide 33-41 of lymphocytic choriomeningitis virus (LCMV) to overcome this limitation . CD8+ T cells from specific pathogen-free maintained, unimmunized "naive" TCR transgenic mice can differentiate into LCMV-specific cytolytic effector CTL during infections with vaccinia virus or Listeria monocytogenes in vivo or mixed lymphocyte culture in vitro . We show that in these model situations (a) nonspecifically activated CTL are able to confer antiviral protection in vivo, (b) bystander activation is largely independent of the expression of a second T cell receptor of different specificity, (c) bystander activation is not mediated by a broadly cross-reactive TCR, but rather by cytokines, (d) bystander activation can be mediated by cytokines such as IL-2, but not alpha/beta-IFN in vitro; (e) bystander activation is, overall, a rare event, occuring in vivo in roughly 1 in 200 of the LCMV-specific CTL during infection of TCR transgenic mice with vaccinia virus; (f) bystander activation does not have a significant functional impact on nontransgenic CTL memory under the conditions tested; and (g) even in the TCR transgenic situation, where unphysiologically high numbers of T cells of a single specificity are present, bystander activation is not sufficient to cause clinically manifest autoimmune disease in a transgenic mouse model of diabetes . We conclude that although bystander activation via cytokines may generate cytolytically active CTL from naive precursors, quantitative considerations suggest that this is usually not of major biological consequence.

Leukemia, 1997 Apr, 11 Suppl 2, S29 - 34
Present status of purine analogs in the therapy of chronic lymphocytic leukemias; Bergmann L; Chronic lymphocytic leukemia (CLL) is considered an incurable disease and therefore the management is palliative and more disease-related symptoms directed . Recently, the high activity of nucleoside analogs as fludarabine (FAMP), 2-chlorodeoxyadenosine (2-CDA) and 2-deoxycoformycin (DCF) in low-grade NHLs has caused a new reawakening interest in CLL concerning new treatment strategies, the biology and prognostic factors of this disease . Predominantly FAMP has widely been studied in CLL with impressive remission rates of 30-70%, including some complete remission (CR) in refractory or relapsed CLL . In previously untreated patients, the remission rate is about 80% with a CR rate of up to 60% . These results open new treatment strategies, even with a curative intention such as high-dose chemotherapy combined with autologous stem cell support or allogeneic stem cell transplantation . The clinical experience with 2-CDA in CLL is limited, but the preliminary results suggest a similar efficacy as FAMP, whereas DCF seems to be less effective . The major treatment-related morbidity is due to myelo- and immunosuppression by long-lasting T cell depletion, which may facilitate a greater susceptibility of infections including those with opportunistic organisms as herpes simplex or herpes zoster, cytomegalovirus, Pneumocystis carinii, mycobacteria, listeriosis, candida and aspergillus in pretreated patients . However, in previously untreated patients no increased incidence of infections has been reported compared with other schedules . Whether FAMP treated patients have any advantage for overall or progression-free survival has to be answered by ongoing randomized trials . Presently, the position of FAMP and 2-CDA as two extremely active single agents in CLL is that of second-line therapy . Their appropriate indication in the first-line strategy of CLL has, however, still to be defined by clinical studies in progress.

Hematol Cell Ther, 1997 Apr, 39(2), 89 - 91
Listeriosis after fludarabine treatment for chronic lymphocytic leukemia; Hequet O et al.; The authors report a case of Listeria monocytogenes septicemia in a patient with advanced CLL after a single course of fludarabine, without any other immunosuppressive therapy e.g . corticosteroids . The immunosuppressive action of fludarabine in patients who are already severely immunosuppressed must be considered from a diagnostic and therapeutic point of view . Listeriosis and other opportunistic infections, like pneumocystis carinii pneumonia, have been reported during and after treatment with purine analogues . Prophylaxis with cotrimoxazole must therefore be discussed in patients with CLL treated with fludarabine.

Heart, 1997 Apr, 77(4), 380 - 3
Listeria endocarditis: current management and patient outcome--world literature review; Spyrou N et al.; This is believed to be the 58th reported case of Listeria monocytogenes infective endocarditis . Published reports worldwide were reviewed as to treatment, outcome, and prognostic features . There is controversy over whether all patients with this condition should have surgery . Moreover the best antibiotic treatment is not known, which accounts for the heterogeneity of regimens used . Listeria endocarditis has a high mortality rate (37%) . This was higher in men (41% v 32%) and in patients with valve prostheses (41% v 31%), though neither observation reached statistical significance . There was no significant difference in mortality between surgical and non-surgical treatment, but untreated listeria endocarditis proved universally fatal . From the data, treatment with ampicillin is recommended, with resort to surgery in cases where the infection cannot be eradicated or where haemodynamic compromise has occurred.

J Dairy Sci, 1997 Apr, 80(4), 667 - 74
Antibacterial peptides of bovine lactoferrin: purification and characterization; Dionysius DA et al.; Three peptides with antibacterial activity toward enterotoxigenic Escherichia coli have been purified from a pepsin digest of bovine lactoferrin . All peptides were cationic and originated from the N-terminus of the molecule in a region where a bactericidal peptide, lactoferricin B, had been previously identified . The most potent peptide, peptide I, was almost identical to lactoferricin B; the sequence corresponded to residues 17 to 42, and the molecular mass was 3195 as determined by mass spectrometry . A second, less active peptide, peptide II, consisted of two sequences, residues 1 to 16 and 43 to 48 (molecular mass of 2673), linked by a single disulfide bond . The third peptide, peptide III, also a disulfide-linked heterodimer, corresponded to residues 1 to 48 (molecular mass of 5851), cleaved between residues 42 and 43 . Peptides I and II displayed antibacterial activity toward a number of pathogenic and food spoilage microorganisms, and peptide I inhibited the growth of Listeria monocytogenes at concentrations as low as 2 microM . Bacterial growth curves showed that bactericidal effects of peptides I and II were observable within 30 min of exposure . The results confirmed and extended those of earlier studies suggesting that the bactericidal domain of lactoferrin was localized in the N-terminus and did not involve iron-binding sites.

Zentralbl Bakteriol, 1997 Apr, 285(4), 491 - 500
Listeria monocytogenes infection in mice treated with pentoxifylline; Brzychcy M et al.; The course of L . monocytogenes infection was followed in mice treated with pentoxifylline (POF), a known inhibitor of endogenous tumor necrosis factor (TNF) formation . Administration of POF caused a delay in L . monocytogenes elimination which was probably related to a reduction the listericidal activity of macrophages and to an attenuation of delayed type hypersensitivity (DTH) to Listeria antigens . In spite of this, some POF-treated mice were protected from lethal effects of virulent L . monocytogenes bacteria.

Int Immunol, 1997 Apr, 9(4), 563 - 71
TCR-mediated target cell lysis by CD4+NK1+ liver T lymphocytes; Emoto M et al.; In the liver, an unusual T lymphocyte population exists with the intriguing phenotype CD4+NK1+ TCR alpha beta int . Thus far, functions of these lymphocytes remained elusive . Recently, however, CD4+NK1+ liver T lymphocytes have been shown to produce cytokines . Here we show that sorted CD4+NK1+ liver lymphocytes from naive mice lyse target cells after TCR alpha beta or CD3, but not TCR gamma delta, engagement . Liver lymphocytes from beta 2-microglobulin-deficient gene disruption mutant mice failed to express such cytolytic activities and in vivo treatment with anti-NK1.1 mAb or anti-CD4 mAb, but not anti-CD8 mAb, markedly reduced target cell lysis . In vivo administration or rIL-12 impaired TCR alpha beta-mediated target cell lysis by liver lymphocytes . A similar down-regulation of cytolytic activities was observed with liver lymphocytes from mice infected with Listeria monocytogenes or Mycobacterium bovis BCG, which are potent IL-12 inducers . We anticipate (i) that cytolytic CD4+NK1+ T lymphocytes contribute to immunosurveillance of inflammatory processes in the liver and (ii) that they are influenced by IL-12.

Int Immunol, 1997 Apr, 9(4), 467 - 74
Effects of IL-13 on murine listeriosis; Flesch IE et al.; Acquired resistance against Listeria monocytogenes is a typical T helper (Th) 1 dominated immune response, whereas Th2 cytokines are thought to worsen listeriosis . We investigated effects of recombinant IL-13 (rIL-13) on the host response to L . monocytogenes in mice . Although IL-13 has been described as a Th2 cytokine with deactivating anti-inflammatory activities, it was found to enhance antilisterial resistance . In vitro, rIL-13 increased IL-12 p40 and p70 production by bone marrow macrophages infected with L . monocytogenes . In vivo, numbers of viable bacteria in spleens and livers were decreased after treatment of mice with rIL-13 . In addition, granuloma formation was impaired and NK cell activity of spleen cells was enhanced . At the onset of infection, frequencies of IL-12-producing cells were increased and numbers of IL-4- and IFN-gamma-secreting cells were diminished in rIL-13-treated mice as compared to controls . In contrast, on day 6 after infection, IL-12, IL-4 and IFN-gamma levels in rIL-13-treated animals were equal to or even higher than those in controls . Although direct activation of host macrophages by IL-13 is possible, we consider it more likely that IL-13 acted indirectly through stimulation of IL-12 production and inhibition of IL-4 release early after infection . In contrast, our data argue against an apparent role of IFN-gamma in IL-13-induced antilisterial resistance.

EMBO J, 1997 Apr 1, 16(7), 1531 - 40
Identification of two regions in the N-terminal domain of ActA involved in the actin comet tail formation by Listeria monocytogenes; Lasa I et al.; The ActA protein of Listeria monocytogenes induces actin nucleation on the bacterial surface . The continuous process of actin filament elongation provides the driving force for bacterial propulsion in infected cells or cytoplasmic extracts . Here, by fusing the N-terminus of ActA (residues 1-234) to the omega fragment of beta-galactosidase, we present the first evidence that this domain contains all the necessary elements for actin tail formation . A detailed analysis of ActA variants, in which small fragments of the N-terminal region were deleted, allowed the identification of two critical regions . Both are required to initiate the actin polymerization process, but each has in addition a specific role to maintain the dynamics of the process . The first region (region T, amino acids 117-121) is critical for filament elongation, as shown by the absence of actin tail in a 117-121 deletion mutant or when motility assays are performed in the presence of anti-region T antibodies . The second region (region C, amino acids 21-97), is more specifically involved in maintenance of the continuity of the process, probably by F-actin binding or prevention of barbed end capping, as strongly suggested by both a deletion (21-97) leading to 'discontinuous' actin tail formation and in vitro experiments showing that a synthetic peptide covering residues 33-74 can interact with F-actin . Our results provide the first insights in the molecular dissection of the actin polymerization process induced by the N-terminal domain of ActA.

Eur J Immunol, 1997 Apr, 27(4), 1035 - 42
Mice heterozygous for a deletion of the tumor necrosis factor-alpha and lymphotoxin-alpha genes: biological importance of a nonlinear response of tumor necrosis factor-alpha to gene dosage; Amiot F et al.; The tumor necrosis factors (TNF-alpha and lymphotoxin, or LT-alpha) are important mediators of the immune and inflammatory responses, and it has been proposed that a positive feedback loop could boost the expression of the TNF to sufficiently high levels to fend off infections . To investigate this phenomenon and its biological consequences, we have generated LT-alpha/TNF-alpha knockout mice and compared mice having one or two functional LT-alpha/TNF-alpha alleles . In response to lipopolysaccharide (LPS) stimulation, TNF-alpha levels in the circulation or in the supernatant of macrophage cultures were 20- to 100-fold lower in heterozygous samples than in their wild-type counterparts . This differential increased with the intensity of stimulation and throughout the response, supporting the involvement of a positive feedback loop . Moreover, the heterozygous mice had an increased bacterial load following Listeria monocytogenes infection and exhibited a bimodal response to the association of D-galactosamine and LPS which was similar to that of wild-type mice at low doses of LPS and more like that of homozygous mutants at high doses . These results therefore establish the biological importance of the nonlinear response of TNF-alpha levels to gene dosage, and these mice provide a unique tool to study how the propensity to produce TNF can determine the immunological fitness of individuals.

Eur J Immunol, 1997 Apr, 27(4), 866 - 70
Interleukin-12 promotes a chronic intestinal nematode infection; Bancroft AJ et al.; Resistance and susceptibility to the intestinal parasite Trichuris muris has been shown to be due to a dominant T helper 2 (Th2) and a dominant Th1 response, respectively . The factors determining the initial polarization of the immune response remain largely unresolved, although the cytokine environment at the time of antigen presentation clearly plays an essential role . Interleukin (IL)-12, a cytokine produced mainly by macrophages, dendritic cells, and other monocytes has been shown to be important in driving a strong Th1 response by stimulating the production of interferon (IFN)-gamma from natural killer and Th0 cells and therefore forms a link between the innate and adaptive immune system . IL-12 has been shown to play an important role in resistance to a number of intracellular pathogens, including Listeria and Leishmania . It has also been proposed as an anti-tumor agent and for use in the treatment of HIV . Conversely, IL-12 has been shown to prolong the survival of Nippostrongylus brasiliensis and to accelerate autoimmunity . Our studies demonstrate that by driving a strong Th1 response, IL-12 promotes chronic T . muris infection when given to normally resistant BALB/K mice . Parasite-specific IgG2a, a Th1 parameter of infection, was greatly up-regulated, whereas some Th2 parameters of infection were down-regulated . IL-12 treatment could be delayed until 1 week after infection had started and still promote a strong Th1 response . The actions of IL-12 in promoting a chronic infection were IFN-gamma dependent as an anti-IFN-gamma mAb abrogated the effects of IL-12.

J Immunol, 1997 Apr 1, 158(7), 3366 - 71
Immunodominant and subdominant CTL responses to Listeria monocytogenes infection; Vijh S et al.; Protective immunity to infection by intracellular pathogens begins with expansion of Ag-specific, effector T lymphocytes and is followed by persistence of pathogen-specific memory T cells . Infection by Listeria monocytogenes, an intracellular bacterium, induces cytolytic T lymphocytes that mediate systemic sterilization and long term immunity . In cells infected with L . monocytogenes, H2-Kd class I molecules present three nonamer peptides, listeriolysin (LLO) 91-99, p60 217-225, and p60 449-457, to CTL . Herein we show that during the peak CTL response to L . monocytogenes infection, the ratio of T cells specific for LLO 91-99, p60 217-225, and p60 449-457 is approximately 20:10:1, respectively . While the number of Ag-specific T lymphocytes decreases in the weeks after infection, the proportion of T lymphocytes specific for the three epitopes is maintained . Repertoire analysis of a subset of L . monocytogenes-specific T cells, using alanine-substituted variants of p60 217-225, indicates that the range of T cell specificities is maintained by memory cells . These results indicate that the breadth and relative magnitude of T cell specificities initially elicited by an infection are transmitted to the memory compartment . Our results suggest that T lymphocytes with different gross and fine Ag specificities are equally likely to become memory T cells.

Infect Immun, 1997 Apr, 65(4), 1515 - 8
Evidence that PrfA, the pleiotropic activator of virulence genes in Listeria monocytogenes, can be present but inactive; Renzoni A et al.; All virulence genes of Listeria monocytogenes identified to date are positively regulated by PrfA, a transcriptional activator belonging to the Crp-Fnr family . Low temperature and cellobiose are two environmental signals known to repress expression of virulence genes in L . monocytogenes . In the present work, we analyzed the effect of temperature and cellobiose on the expression of the PrfA protein . At low temperature, PrfA was undetected, although prfA monocistronic transcripts are present . In contrast, PrfA was fully expressed in the presence of cellobiose . These results strongly suggest that virulence gene activation depends on both the presence of PrfA and additional regulatory pathways that either modify PrfA or act synergistically with PrfA.

J Bacteriol, 1997 Apr, 179(8), 2707 - 16
Identification, cloning, and characterization of the Ima operon, whose gene products are unique to Listeria monocytogenes; Schaferkordt S et al.; The lmaA gene of Listeria monocytogenes encodes a protein capable of inducing delayed-type hypersensitivity reactions in L . monocytogenes-immune mice (S . Gohmann, M . Leimeister-Wachter, E . Schiltz, W . Goebel, and T . Chakraborty, M . Microbiol . 4:1091-1099, 1990) . Here we show that it is the last gene of the lma operon, which now comprises four genes, lmaDCBA . Maxicell analysis of peptides encoded by the lma operon identified four polypeptides of 16.7, 16.4, 14.9, and 21 kDa which correspond to the gene products encoded by the lmaD, -C, -B, and -A genes, respectively . Northern blot analysis of the lma operon showed that lmaA is expressed by two transcripts: the longer lmaDCBA transcript of 2,100 nucleotides, which was observed at growth temperatures of 37 and 20 degrees C, and a shorter transcript consisting of lmaBA, which is detected only at low temperatures (20 degrees C) . Two promoters, one preceding the lmaD gene and another located upstream of the lmaB gene, were detected . An extended stem-loop structure resembling box elements found in other gram-positive pathogens was also present in the lmaC-lmaB intergenic region . By immunoblot analysis, we found that although LmaA was produced at both temperatures (20 and 37 degrees C), it was secreted into culture supernatants only at 20 degrees C . However, LmaA lacks a bona fide signal peptide sequence and could, like flagellin, be secreted by a type III transport system . DNA hybridization studies indicate that the lma operon is species specific and restricted to pathogenic strains of L . monocytogenes.

Appl Environ Microbiol, 1997 Apr, 63(4), 1338 - 43
Typing Listeria monocytogenes isolates from fish products and human listeriosis cases; Boerlin P et al.; Seventy-two Listeria monocytogenes isolates originating from 10 different fish products of 12 producers and 47 isolates from human listeriosis cases were typed by serotyping and multilocus enzyme electrophoresis . Seventy-five of these isolates were further subtyped by restriction analysis of genomic DNA with the enzyme XhoI and by pulsed-field gel electrophoresis using the enzymes ApaI and SmaI . The results show that several L . monocytogenes clones identified by multilocus enzyme electrophoresis are frequently found in fish products of different origins . One of these clones is the same as another previously shown to be frequently associated with meat and meat products . The epidemic-associated electrophoretic type 1 was only rarely found in fish products . No association was found between any type of fish product and a particular lineage of L . monocytogenes . Both long-term persistence of a strain and simultaneous presence of several clearly distinct strains in the products of single producers were observed . The comparison of L . monocytogenes isolates from human clinical listeriosis cases in Switzerland and those from imported fish products by use of multilocus enzyme electrophoresis showed that they do not form two clearly distinct lineages but nevertheless belong to two separate populations . None of the 48 subtypes distinguished by the combination of all four typing methods could be found in both populations of human origin and those of fish origin.

Appl Environ Microbiol, 1997 Apr, 63(4), 1252 - 5
Adaptation to sublethal environmental stresses protects Listeria monocytogenes against lethal preservation factors; Lou Y et al.; A sublethal dose of ethanol (5%, vol/vol), acid (HCl, pH 4.5 to 5.0), H2O2 (500 ppm), or NaCl (7%, wt/vol) was added to a Listeria monocytogenes culture at the exponential phase, and the cells were allowed to grow for 1 h . Exponential-phase cells also were heat shocked at 45 degrees C for 1 h . The stress-adapted cells were then subjected to the following factors at the indicated lethal levels--NaCl (25%, wt/vol), ethanol (17.5%, vol/vol), hydrogen peroxide (0.1%, wt/vol), acid (pH 3.5), and starvation on 0.1 M phosphate buffer at pH 7.0 (up to 300 h) . Viable counts of the pathogen, after the treatment, were determined on Trypticase soy agar-yeast extract, and survivor plots were constructed . The area (h.log10 CFU/ml) between the control and treatment curves was calculated to represent the protective effect resulting from adaptation to the sublethal stress factor . Adaptation to pH 4.5 to 5.0 or 5% ethanol significantly (P < 0.05) increased the resistance of L . monocytogenes to lethal doses of acid, ethanol, and H2O2 . Adaptation to ethanol significantly (P < 0.05) increased the resistance to 25% NaCl . When L . monocytogenes was adapted to 500 ppm of H2O2, 7% NaCl, or heat, resistance of the pathogen to 1% hydrogen peroxide increased significantly (P < 0.05) . Heat shock significantly (P < 0.05) increased the resistance to ethanol and NaCl . Therefore, the occurrence of stress protection after adaptation of L . monocytogenes to environmental stresses depends on the type of stress encountered and the lethal factor applied . This "stress hardening" should be considered when current food processing technologies are modified or new ones are developed.

J Cell Biol, 1997 Mar 24, 136(6), 1323 - 32
Xenopus actin depolymerizing factor/cofilin (XAC) is responsible for the turnover of actin filaments in Listeria monocytogenes tails; Rosenblatt J et al.; In contrast to the slow rate of depolymerization of pure actin in vitro, populations of actin filaments in vivo turn over rapidly . Therefore, the rate of actin depolymerization must be accelerated by one or more factors in the cell . Since the actin dynamics in Listeria monocytogenes tails bear many similarities to those in the lamellipodia of moving cells, we have used Listeria as a model system to isolate factors required for regulating the rapid actin filament turnover involved in cell migration . Using a cell-free Xenopus egg extract system to reproduce the Listeria movement seen in a cell, we depleted candidate depolymerizing proteins and analyzed the effect that their removal had on the morphology of Listeria tails . Immunodepletion of Xenopus actin depolymerizing factor (ADF)/cofilin (XAC) from Xenopus egg extracts resulted in Listeria tails that were approximately five times longer than the tails from undepleted extracts . Depletion of XAC did not affect the tail assembly rate, suggesting that the increased tail length was caused by an inhibition of actin filament depolymerization . Immunodepletion of Xenopus gelsolin had no effect on either tail length or assembly rate . Addition of recombinant wild-type XAC or chick ADF protein to XAC-depleted extracts restored the tail length to that of control extracts, while addition of mutant ADF S3E that mimics the phosphorylated, inactive form of ADF did not reduce the tail length . Addition of excess wild-type XAC to Xenopus egg extracts reduced the length of Listeria tails to a limited extent . These observations show that XAC but not gelsolin is essential for depolymerizing actin filaments that rapidly turn over in Xenopus extracts . We also show that while the depolymerizing activities of XAC and Xenopus extract are effective at depolymerizing normal filaments containing ADP, they are unable to completely depolymerize actin filaments containing AMPPNP, a slowly hydrolyzible ATP analog . This observation suggests that the substrate for XAC is the ADP-bound subunit of actin and that the lifetime of a filament is controlled by its nucleotide content.

J Cell Biol, 1997 Mar 24, 136(6), 1307 - 22
Actin depolymerizing factor (ADF/cofilin) enhances the rate of filament turnover: implication in actin-based motility; Carlier MF et al.; Actin-binding proteins of the actin depolymerizing factor (ADF)/cofilin family are thought to control actin-based motile processes . ADF1 from Arabidopsis thaliana appears to be a good model that is functionally similar to other members of the family . The function of ADF in actin dynamics has been examined using a combination of physical-chemical methods and actin-based motility assays, under physiological ionic conditions and at pH 7.8 . ADF binds the ADP-bound forms of G- or F-actin with an affinity two orders of magnitude higher than the ATP- or ADP-Pi-bound forms . A major property of ADF is its ability to enhance the in vitro turnover rate (treadmilling) of actin filaments to a value comparable to that observed in vivo in motile lamellipodia . ADF increases the rate of propulsion of Listeria monocytogenes in highly diluted, ADF-limited platelet extracts and shortens the actin tails . These effects are mediated by the participation of ADF in actin filament assembly, which results in a change in the kinetic parameters at the two ends of the actin filament . The kinetic effects of ADF are end specific and cannot be accounted for by filament severing . The main functionally relevant effect is a 25-fold increase in the rate of actin dissociation from the pointed ends, while the rate of dissociation from the barbed ends is unchanged . This large increase in the rate-limiting step of the monomer-polymer cycle at steady state is responsible for the increase in the rate of actin-based motile processes . In conclusion, the function of ADF is not to sequester G-actin . ADF uses ATP hydrolysis in actin assembly to enhance filament dynamics.

Nature, 1997 Mar 20, 386(6622), 292 - 6
A role for macrophage scavenger receptors in atherosclerosis and susceptibility to infection; Suzuki H et al.; Macrophage type-I and type-II class-A scavenger receptors (MSR-A) are implicated in the pathological deposition of cholesterol during atherogenesis as a result of receptor-mediated uptake of modified low-density lipoproteins (mLDL) . MSR-A can bind an extraordinarily wide range of ligands, including bacterial pathogens, and also mediates cation-independent macrophage adhesion in vitro . Here we show that targeted disruption of the MSR-A gene in mice results in a reduction in the size of atherosclerotic lesions in an animal deficient in apolipoprotein E . Macrophages from MSR-A-deficient mice show a marked decrease in mLDL uptake in vitro, whereas mLDL clearance from plasma occurs at a normal rate, indicating that there may be alternative mechanisms for removing mLDL from the circulation . In addition, MSR-A-knockout mice show an increased susceptibility to infection with Listeria monocytogenes or herpes simplex virus type-1, indicating that MSR-A may play a part in host defence against pathogens.

Int J Food Microbiol, 1997 Mar 18, 35(1), 91 - 5
A chemically defined minimal medium for the optimal culture of Listeria; Phan-Thanh L et al.; Unlike most other bacteria, many Listeria strains do not grow well in the minimal media described so far in the literature . Among the minimal media tested, a chemically defined medium modified from that of Premaratne and co-workers was found to support the best growth of Listeria spp . The promoting effect was due to the incorporation of several indispensable vitamins and growth factors.

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