Lett Appl Microbiol, 2000 Apr, 30(4), 336 - 40 Alizarin-beta-D-galactoside: a new substrate for the detection of bacterial beta-galactosidase; James AL et al.; We describe the synthesis of a new substrate for the detection of bacterial beta-galactosidase . This substrate, alizarin-beta-D-galactoside, is readily hydrolysed to release alizarin which complexes with various metal ions to form brightly coloured chelates . A total of 367 strains of Gram-negative bacteria were examined for their ability to hydrolyse three chromogenic substrates: alizarin-beta-D-galactoside (Aliz-gal), cyclohexenoesculetin-beta-D-galactoside (CHE-gal) and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal) . A total of 182 strains (49.6%) were found to hydrolyse at least one of the three substrates . All of these 182 strains (100%) hydrolysed Aliz-gal whereas only 170 (93.4%) and 173 (95.1%) hydrolysed CHE-gal and X-gal, respectively . We conclude that alizarin-beta-D-galactoside is a highly sensitive substrate for the demonstration of beta-galactosidase. Int J Food Microbiol, 2000 Apr 10, 55(1-3), 53 - 61 Impact of thermal variations on biochemical and physiological traits in Pectinatus sp; Flahaut S et al.; The influence of temperature on cellular fatty acid composition and on heat stress tolerance was studied in the two species of Pectinatus, an anaerobic gram-negative bacterium . Cellular fatty acid (FA) patterns were determined for Pectinatus species cultivated in MRS medium at various defined conditions of temperature and pH . Our study shows that fluctuations of growth temperature and pH induced important changes in the ratio of unsaturated FAs (UFAs) to saturated FAs (SFAs) . The major differences in the FA composition as a function of growth temperature concerned C15:0 and C17:0 for the SFAs and C15:1 and C17:1 for the UFAs . The most significant adaptation of lipid composition to lower growth temperatures was the strong increase of UFAs, particularly for C15:1 and C17:1 concomitantly with a decrease of SFAs (C15:0 and C17:0) . When the pH of the culture medium was lowered from 6.2 to 4.0, a notable drop in the synthesis of the UFAs C15:1 and C17:1 was observed together with an important increase of C18-cyclopropane (C18-cyc) and high carbon number SFAs . Thermal modifications also provoked changes in Pectinatus behaviour . We observed that P . cerevisiiphilus was more heat sensitive than P . frisingensis . Mild exponential phase cells were treated for 1 h, at 40 degrees C for P . cerevisiiphilus or at 41 degrees C for P . frisingensis . This thermal adaptation induced tolerance against heat challenge (49 and 50 degrees C for P . cerevisiiphilus and P . frisingensis, respectively) . Survival of P . cerevisiiphilus and P . frisingensis adapted cells was, respectively, 3400- and 790-fold higher than control . Interestingly, adapted cells of P . cerevisiiphilus were more thermotolerant than P . frisingensis pretreated cells. Intensive Care Med, 2000, 26 Suppl 1, S51 - 6 The detection and interpretation of endotoxaemia; Cohen J; A considerable body of evidence has accumulated that implicates endotoxin in the pathogenesis of the sepsis syndrome . This has raised interest in the possibility of measuring endotoxin as a surrogate marker of Gram negative infection, particularly since conventional microbiological tests have an inevitable delay . The Limulus amoebocyte lysate (LAL) assay has been used most widely to measure endotoxin in clinical samples . However, there are several important limitations that need to be borne in mind . These include the dangers of contamination, lack of precision and accuracy, and both false positive and false negative results . Endotoxaemia is present in the blood of about 30% of patients with bacteraemia, but endotoxaemia does not predict either Gram negative bacteraemia or Gram negative infection, nor does it predict survival from sepsis . There is some correlation with severity of sepsis, but the level of precision is poor . At the present time, there is no place for routine endotoxin testing in clinical practice . In particular, the positive predictive value of the test is insufficiently high to be of clinical use . It may be that the LAL assay, or one of the newer developments, may be more useful in excluding Gram negative infection, but that remains to be shown. Nat Cell Biol, 2000 Apr, 2(4), 212 - 8 A new ABC transporter mediating the detachment of lipid-modified proteins from membranes; Yakushi T et al.; Lipoproteins in Escherichia coli are anchored to the periplasmic side of either the inner or the outer membrane by a lipid moiety that is covalently attached to the amino-terminal cysteine residue . Membrane specificity depends on a sorting signal at position 2 of the lipoprotein . Lipoproteins directed to the outer membrane are released from the inner membrane in an ATP-dependent manner through the formation of a complex with LolA, a periplasmic chaperone . However, the ATPase involved in this reaction has not been identified . Here we show, using reconstituted proteoliposomes, that a new complex, LolCDE, belonging to the ATP-binding cassette (ABC) transporter family, catalyses the release of lipoproteins in LolA- and sorting-signal-dependent manners . The LolCDE complex differs mechanistically from all other ABC transporters as it is not involved in the transmembrane transport of substrates . This new mechanism is evolutionarily conserved in other gram-negative bacteria. FEBS Lett, 2000 Apr 21, 472(1), 78 - 82 MerF is a mercury transport protein: different structures but a common mechanism for mercuric ion transporters? Wilson JR, Leang C, Morby AP, Hobman JL, Brown NL. Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present . We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase . We have confirmed that MerC of Tn21 is also a mercuric ion transporter . We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC . Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively . In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue . The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ. Vet Microbiol, 2000 May 11, 73(4), 301 - 10 Identification of the copper-zinc superoxide dismutase activity in Mycoplasma hyopneumoniae; Chen JR et al.; Copper-zinc superoxide dismutase (Cu/ZnSOD), a key enzyme in defense against toxic oxygen-free radicals, is widespread in eukaryotes and several species of gram-negative bacteria . The presence of this enzyme in Mycoplasma hyopneumoniae (M . hyopneumoniae), the primary pathogen of mycoplasmal pneumonia in pigs, was examined since the polyclonal antibody against bovine Cu/ZnSOD was dominantly cross-reactive with the M . hyopneumoniae Cu/ZnSOD from whole cellular proteins . In situ activity staining on SDS-PAGE showed that the molecular mass of M . hyopneumoniae Cu/ZnSOD in reducing form was approximately 17kDa . The presence of Cu and Zn ions at the active site of the enzyme was confirmed on the basis of inhibition by KCN and by H(2)O(2) . The activity of M . hyopneumoniae Cu/ZnSOD on both SDS- and native-polyacrylamide gels was completely inhibited by 2mM KCN and the gels showed no iron-containing SOD (FeSOD) or manganese-containing SOD (MnSOD) in the crude extracts . The activity of M . hyopneumoniae Cu/ZnSOD in crude extract was 70units/mg protein and was 55% inhibited by 5mM KCN and 56% inactivated by 40mM H(2)O(2) . This enzyme was growth-stage dependent and evidenced markedly higher production during the early log phase . Different expression levels of Cu/ZnSOD activity in field isolates were also detected . Taken together, the presence of Cu/ZnSOD in M . hyopneumoniae was identified for the first time. J Surg Res, 2000 May 1, 90(1), 51 - 7 Inhibition of alveolar neutrophil immigration in endotoxemia is macrophage inflammatory protein 2 independent; Duffy AJ et al.; BACKGROUND: Altered transendothelial migration and delayed apoptosis of neutrophils (PMN) have been implicated as contributing to infection in patients with gram-negative sepsis . Macrophage inflammatory protein 2 (MIP-2) signals PMN immigration and may alter other PMN functions . We tested the hypothesis that sequential endotoxin challenge in vivo alters PMN apoptosis and chemotactic responses . MATERIALS AND METHODS: Endotoxemia was induced in male Wistar rats (250 g) via intraperitoneal (IP) administration of LPS (4 mg/kg) . After 18 h, intratracheal (IT) injection of LPS (400 microg/kg) was performed . Control animals received saline injections . Four hours after IT-LPS, circulating and bronchoalveolar lavage (BAL) PMN were isolated . PMN yields were calculated, and apoptosis was quantified after 18 h in culture by annexin V-fluorescein isothiocyanate FACS analysis . BAL MIP-2 concentrations were determined by ELISA . PMN chemotaxis to MIP-2 and IL-8 was determined using a fluorescent in vitro migration assay . RESULTS: Endotoxemia (IP-LPS) significantly decreases BAL PMN yield in response to an in vivo IT-LPS challenge . IT-LPS inhibits BAL PMN apoptosis to the same extent as sequential IP/IT-LPS . Alveolar MIP-2 concentrations are similar in the two groups . In vitro migration to IL-8 and MIP-2 was inhibited in PMN from endotoxemic versus control animals . CONCLUSIONS: These data demonstrate that endotoxemia inhibits PMN migration despite similar MIP-2 concentrations in the alveolus . Sequential insults do not affect the inhibition of apoptosis . In vitro, PMN from endotoxemic animals display impaired chemotaxis to MIP-2 and interleukin-8 . This may result in an inadequate host defense that contributes to increased ICU-acquired pneumonia in septic patients . Eur Respir J, 2000 Apr, 15(4), 764 - 70 Tumour necrosis factor-alpha production in human alveolar macrophages: modulation by inhaled corticosteroid; Marshall BG et al.; Using an ex vivo alveolar macrophage model, the hypothesis that inhaled preparations of corticosteroids might have important anti-inflammatory effects on cells of the peripheral airway was tested . The tumour necrosis factor (TNF)-alpha-inducing potential of three glycolipid preparations from nonpathogenic (arabinofuranasyl lipoarabinomannan (LAM (Ara-LAM)) and virulent (mannase LAM (ManLAM)) mycobacteria and Gram-negative bacteria (lipopolysaccharide (LPS)), in primary alveolar macrophage preparations was investigated . A novel inhaled chlorofluorocarbon (CFC)-free preparation of beclomethasone dipropionate (hydrofluoroalkane 134a (HFA)-BDP) with increased peripheral lung deposition was investigated for its ability to modulate glycolipidinduced TNF-alpha production by human alveolar macrophages, in comparison with a CFC-containing preparation and placebo . Compared to the basal TNF-alpha bioactivity of 0.72 ng x mL-1 (geometric mean), the TNF-alpha bioactivity in the macrophage preparation increased following incubation with LPS (138 ng x mL-1, p<0.001), AraLAM (12.6 ng-mL-1, p<0.001) and ManLAM (1.42 ng x mL-1, p=0.02) . HFA-BDP, administered in vivo, significantly reduced LPS- and ManLAM-induced TNF-alpha production by alveolar macrophages cultured ex vivo . No change in glycolipid-induced TNF-alpha production was observed following in vivo administration of CFC-BDP or HFA-placebo . This is the first demonstration of an immunomodulatory effect on alveolar cells of corticosteroid delivered via metered dose inhaler . The present findings suggest that alveolar deposition of beclomethasone dipropionate is capable of modulating the inflammatory potential of the alveolar macrophage population. FEMS Microbiol Lett, 2000 May 1, 186(1), 11 - 9 Osmoregulated periplasmic glucans in Proteobacteria; Bohin JP; Large amounts of osmoregulated periplasmic glucans (OPGs) are found in the periplasmic space of Proteobacteria . Four families of OPGs are described on the basis of structural features of the polyglucose backbone . Depending on the species considered, OPGs can be modified to various extent by a variety of substituents . Genes governing the backbone synthesis are identified in a limited number of species . They belong to three unrelated families . OPG synthesis is subject to osmoregulation and feedback control . Osmoregulation can occur at the level of gene expression and/or at the level of enzyme activity . Mutants defective in OPG synthesis have a highly pleiotropic phenotype, indicative of an overall alteration of their envelope properties . Mutants of this kind were obtained as attenuated or avirulent derivatives of plant or animals pathogen . Thus, OPGs appear to be important intrinsic components of the Gram-negative bacterial envelope, which can be essential in extreme conditions found in nature, and especially when bacteria must interact with an eukaryotic host. Proc Natl Acad Sci U S A, 2000 May 9, 97(10), 5645 - 50 Early-life exposure to endotoxin alters hypothalamic-pituitary-adrenal function and predisposition to inflammation; Shanks N et al.; We have investigated whether exposure to Gram-negative bacterial endotoxin in early neonatal life can alter neuroendocrine and immune regulation in adult animals . Exposure of neonatal rats to a low dose of endotoxin resulted in long-term changes in hypothalamic-pituitary-adrenal (HPA) axis activity, with elevated mean plasma corticosterone concentrations that resulted from increased corticosterone pulse frequency and pulse amplitude . In addition to this marked effect on the development of the HPA axis, neonatal endotoxin exposure had long-lasting effects on immune regulation, including increased sensitivity of lymphocytes to stress-induced suppression of proliferation and a remarkable protection from adjuvant-induced arthritis . These findings demonstrate a potent and long-term effect of neonatal exposure to inflammatory stimuli that can program major changes in the development of both neuroendocrine and immunological regulatory mechanisms. J Biol Chem, 2000 Apr 28, 275(17), 12489 - 96 Enterotoxigenic Escherichia coli secretes active heat-labile enterotoxin via outer membrane vesicles; Horstman AL et al.; Escherichia coli and other Gram-negative bacteria produce outer membrane vesicles during normal growth . Vesicles may contribute to bacterial pathogenicity by serving as vehicles for toxins to encounter host cells . Enterotoxigenic E . coli (ETEC) vesicles were isolated from culture supernatants and purified on velocity gradients, thereby removing any soluble proteins and contaminants from the crude preparation . Vesicle protein profiles were similar but not identical to outer membranes and differed between strains . Most vesicle proteins were resistant to dissociation, suggesting they were integral or internal . Thin layer chromatography revealed that major outer membrane lipid components are present in vesicles . Cytoplasmic membranes and cytosol were absent in vesicles; however, alkaline phosphatase and AcrA, periplasmic residents, were localized to vesicles . In addition, physiologically active heat-labile enterotoxin (LT) was associated with ETEC vesicles . LT activity correlated directly with the gradient peak of vesicles, suggesting specific association, but could be removed from vesicles under dissociating conditions . Further analysis revealed that LT is enriched in vesicles and is located both inside and on the exterior of vesicles . The distinct protein composition of ETEC vesicles and their ability to carry toxin may contribute to the pathogenicity of ETEC strains. Acta Crystallogr D Biol Crystallogr, 2000 May, 56 ( Pt 5), 659 - 61 Crystallization and preliminary X-ray studies of membrane-associated Escherichia coli dihydroorotate dehydrogenase; Rowland P et al.; Dihydroorotate dehydrogenases (DHODs) are flavin-containing enzymes which catalyse the conversion of (S)-dihydroorotate to orotate, the fourth step in the de novo biosynthesis of pyrimidine nucleotides . Two major families of DHODs have now been identified based on their amino-acid sequence similarities . The two families differ in their reaction mechanisms, but structures are only known of enzymes belonging to family 1 . DHOD from Escherichia coli is a typical member of family 2, which contains the membrane-associated enzymes from Gram-negative bacteria and eukaryotes . Yellow crystals grown of this enzyme belong to the space group P4(1)2(1)2 or P4(3)2(1)2 . The unit-cell parameters are a = b = 119.2, c = 294.3 A . Owing to the rather large c axis, the currently available resolution of data is 2.2 A. J Exp Med, 2000 Apr 17, 191(8), 1437 - 42 Infectious agents are not necessary for murine atherogenesis; Wright SD et al.; Recent work has revealed correlations between bacterial or viral infections and atherosclerotic disease . One particular bacterium, Chlamydia pneumoniae, has been observed at high frequency in human atherosclerotic lesions, prompting the hypothesis that infectious agents may be necessary for the initiation or progression of atherosclerosis . To determine if responses to gram-negative bacteria are necessary for atherogenesis, we first bred atherosclerosis-prone apolipoprotein (apo) E(-/)- (deficient) mice with animals incapable of responding to bacterial lipopolysaccharide . Atherogenesis was unaffected in doubly deficient animals . We further tested the role of infectious agents by creating a colony of germ-free apo E(-/)- mice . These animals are free of all microbial agents (bacterial, viral, and fungal) . Atherosclerosis in germ-free animals was not measurably different from that in animals raised with ambient levels of microbial challenge . These studies show that infection is not necessary for murine atherosclerosis and that, unlike peptic ulcer, Koch's postulates cannot be fulfilled for any infectious agent in atherosclerosis. Infect Immun, 2000 May, 68(5), 2566 - 72 Outer membrane protein A, peptidoglycan-associated lipoprotein, and murein lipoprotein are released by Escherichia coli bacteria into serum; Hellman J et al.; Complexes containing lipopolysaccharide (LPS) and three outer membrane proteins (OMPs) are released by gram-negative bacteria incubated in human serum and into the circulation in an experimental model of sepsis . The same OMPs are bound by immunoglobulin G (IgG) in the cross-protective antiserum raised to Escherichia coli J5 (anti-J5 IgG) . This study was performed to identify the three OMPs . The 35-kDa OMP was identified as outer membrane protein A (OmpA) by immunoblotting studies using OmpA-deficient bacteria and recombinant OmpA protein . The 18-kDa OMP was identified as peptidoglycan-associated lipoprotein (PAL) based on peptide sequences from the purified protein and immunoblotting studies using PAL-deficient bacteria . The 5- to 9-kDa OMP was identified as murein lipoprotein (MLP) based on immunoblotting studies using MLP-deficient bacteria . The studies identify the OMPs released into human serum and into the circulation in an experimental model of sepsis as OmpA, PAL, and MLP. Infect Immun, 2000 May, 68(5), 2535 - 45 Identification of murine B-cell and T-cell epitopes of Escherichia coli outer membrane protein F with synthetic polypeptides; Williams KM et al.; The major pore-forming outer membrane proteins (Omps) of gram-negative bacteria demonstrate numerous immunomodulating properties and are involved in the virulence of pathogenic strains . Because Escherichia coli OmpF is the best-characterized porin in terms of structural and functional characteristics, in vitro B-cell and T-cell responses to this porin in six different strains of mice were analyzed . Mice were immunized with purified OmpF trimers or overlapping synthetic polypeptides (20-mers) spanning the entire 340-amino-acid sequence of the OmpF monomer . T-cell proliferative responses and immunoglobulin G antibody responses to native OmpF and the peptide analogues were determined . For each strain, patterns of T-cell proliferation were similar regardless of whether native OmpF or synthetic peptides were inoculated, although all strains recognized one or more cryptic determinants . Mice exhibited several haplotype-specific responses, but genetically permissive epitopes were also identified . Four peptides (75-94, 265-284, 295-314, and 305-324) elicited strong T-cell proliferative responses from all strains of mice when mice were presensitized with native OmpF or a homologous peptide . In general, 10 or fewer peptides were recognized by sera from mice immunized with native OmpF or synthetic peptides, and most sera from peptide-immunized mice reacted poorly with the native protein . Four peptides spanning amino acids 45 to 64, 95 to 114, 115 to 134, and 275 to 294 were recognized by sera from all strains immunized with native OmpF but not by sera from peptide-immunized mice . Peptides 245-264 and 305-324 were universally recognized by sera from peptide-immunized mice, but these sera reacted weakly or were negative when tested against the native protein . Based on the pattern of cytokine secretion by proliferating T cells, immunization with native OmpF polarizes T helper cells toward development of a TH1 response . T-cell and B-cell responses have been investigated based on the assumption that differences in epitope specificity could influence protective or pathologic host reactions . Because of the high level of structural homology of OmpF to porins isolated from other enteric pathogens, the identification of T- and B-cell-stimulatory determinants of E . coli OmpF may have broader application. Infect Immun, 2000 May, 68(5), 2418 - 23 Transforming growth factor-beta inhibits lipopolysaccharide-stimulated expression of inflammatory cytokines in mouse macrophages through downregulation of activation protein 1 and CD14 receptor expression; Imai K et al.; The septic shock that occurs in gram-negative infections is caused by a cascade of inflammatory cytokines . Several studies showed that transforming growth factor-beta1 (TGF-beta1) inhibits this septic shock through suppression of expression of the lipopolysaccharide (LPS)-induced inflammatory cytokines . In this study, we investigated whether TGF-beta1 inhibition of LPS-induced expression of inflammatory cytokines in the septic shock results from downregulation of LPS-stimulated expression of CD14, an LPS receptor . TGF-beta1 markedly inhibited LPS stimulation of CD14 mRNA and protein levels in mouse macrophages . LPS-stimulated expression of CD14 was dramatically inhibited by addition of antisense, but not sense, c-fos and c-jun oligonucleotides . Since TGF-beta1 pretreatment inhibited LPS-stimulated expression of c-fos and c-jun genes and also the binding of nuclear proteins to the consensus sequence of the binding site for activation protein 1 (AP-1), a heterodimer of c-Fos and c-Jun, in the cells, TGF-beta1 inhibition of CD14 expression may be a consequence of downregulation of AP-1 . LPS-stimulated expression of interleukin-1beta and tumor necrosis factor alpha genes in the cells was inhibited by addition of CD14 antisense oligonucleotide . Also, TGF-beta1 inhibited the LPS-stimulated production of both inflammatory cytokines by the macrophages . In addition, TGF-beta1 inhibited expression of the two cytokines in several organs of mice receiving LPS . Thus, our results suggest that TGF-beta1 inhibition of LPS-stimulated inflammatory responses resulted from downregulation of CD14 and also may be a possible mechanism of TGF-beta1 inhibition of LPS-induced septic shock. J Biol Chem, 2000 Apr 21, 275(16), 11929 - 33 Induction of endothelial nitric-oxide synthase in rat brain astrocytes by systemic lipopolysaccharide treatment; Iwase K et al.; In the brain, three isoforms of nitric oxide (NO) synthase (NOS), namely neuronal NOS (nNOS, NOS1), inducible NOS (iNOS, NOS2), and endothelial NOS (eNOS, NOS3), have been implicated in biological roles such as neurotransmission, neurotoxicity, immune function, and blood vessel regulation, each isoform exhibiting in part overlapping roles . Previous studies showed that iNOS is induced in the brain by systemic treatment with lipopolysaccharide (LPS), a Gram-negative bacteria-derived stimulant of the innate immune system . Here we found that eNOS mRNA is induced in the rat brain by intraperitoneal injection of LPS of a smaller amount than that required for induction of iNOS mRNA . The induction of eNOS mRNA was followed by an increase in eNOS protein . Immunohistochemical analysis revealed that eNOS is located in astrocytes of both gray and white matters as well as in blood vessels . Induction of eNOS in response to a low dose of LPS, together with its localization in major components of the blood-brain barrier, suggests that brain eNOS is involved in early pathophysiologic response against systemic infection before iNOS is induced with progression of the infection. Biosci Rep, 1999 Oct, 19(5), 421 - 32 Bacteria-host cell interaction mediated by cellular cholesterol/glycolipid-enriched microdomains; Shin JS et al.; Gram negative bacterial infection is a leading cause of fatality and is attributed, at least in part, to the bacteria's capacity to persist in the host in spite of appropriate antibiotic therapy . It has been suggested that bacteria evade antibiotics by hiding within host cells . We sought to investigate this important aspect of infections in mast cells, which are inflammatory cells found in close proximity to the host-environment interface and which have recently been reported to play a crucial role in the early innate immune response to bacteria . We examined mast cell interactions with FimH-expressing E . coli, one of the major opportunistic pathogens of humans . We determined that in serum free conditions, these bacteria were able to trigger mast cell uptake without loss of bacterial viability . CD48, a mannose containing GPI (glycosylphosphatidylinositol)-linked molecule was found to be the receptor of FimH-expressing E . coli in mouse mast cells . We found that the internalization via CD48 was blocked by filipin, a cholesterol binding drug known to disrupt cholesterol/glycolipid-enriched microdomains and the bacteria-encasing vacuoles were rich in cholesterol inside cells . Interestingly, we found that mast cells subsequently expelled majority of the intracellular bacteria in 24 hours . This expulsion process was blocked by lovastatin/cyclodextrin treatment, which is known to inhibit cellular trafficking of cholesterol/glycolipid-enriched microdomains . Thus, the bacterial entry into and expulsion from mast cells were critically dependent on cholesterol/glycolipid-enriched microdomains, which represents a novel mode of tussle between the pathogen and the mast cell occurring in opsonin deficient sites in the body or even at other sites in naive or immunocompromised hosts which have low systemic levels of E . coli specific antibody. Biochim Biophys Acta, 2000 Apr 12, 1484(2-3), 225 - 40 The polar-lipid composition of the sphingolipid-producing bacterium Flectobacillus major; Batrakov SG et al.; Polar lipids comprise about 90% of the total chloroform-methanol extractable lipids of the Gram-negative, fresh-water, ring-forming bacterium Flectobacillus major FM and consist of at least 10 constituents . These are aminophosphosphingolipids, 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenyl-1-phosphoethanolamine (36.8% of the total polar lipids) and its 2'-deoxy derivative (3.7%); sulfonic-acid analogues of ceramide, 2-D-(2'-D-hydroxy-13'-methyltetradecanoyl)amino-3-D-hydroxy-15-met hyl hexadecane-1-sulfonic acid (18.1%) and its 2'-deoxy derivative (3 . 5%); a lipoamino acid, N-{3-D-(15'-methylhexadecanoyloxy)-15-methylhexadecanoyl}-gl ycine (3 . 7%); a lipodipeptide, N- inverted question markN'-{3"-D-(15"'-methylhexadecanoyloxy)-15"-methylhexadecanoyl }glycy l inverted question mark-L-serine (7.8%); 1,2-diacyl-sn-glycero-3-phosphoethanolamine (7 . 7%), 1,2-diacyl-3-alpha-D-galactopyranosyl-sn-glycerol (2.9%); ceramide phospho-myo-inositol (4.9%), and a previously described unusual glycosphingolipid, 7-deoxy-7-amino-D-manno-heptulosonopyranosyl (1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranosyl) ceramide (10.9%); the last two lipids contain only 15-methyl-4(E)-hexadecasphingenine as a long-chain base . The sole structural type of amide-bound fatty acids in the sphingolipids, including the sulfonic-acid analogues, is iso-15:0, either non-hydroxylated or hydroxylated at 2-C, whereas 15-methylhexadecanoic acid is the major ester-bound fatty acid in the remaining lipids. Kidney Int, 2000 Apr, 57(4), 1736 - 42 Increased protein loss during peritonitis associated with peritoneal dialysis is neutrophil dependent; Luo Q et al.; BACKGROUND: Peritonitis in peritoneal dialysis patients is accompanied by an enhanced migration of neutrophils (PMNs) and increased protein loss into the peritoneal cavity; however, the role of PMNs in governing increased protein loss during peritonitis associated with peritoneal dialysis is unknown . METHODS: We determined the importance of PMNs in governing changes in peritoneal permeability to protein in New Zealand White rabbits in which acute peritonitis was induced by adding 4 x 106 colony-forming units of Escherichia coli to 35 mL/kg of 0.9% saline dialysate . The total leukocyte and PMN migration into the peritoneal cavity was assessed by differential cell counts in the dialysate, and peritoneal permeability to protein was evaluated by calculating the dialysate to plasma concentration ratio for total protein as a function of time during a six- or eight-hour dwell . In series 1 experiments, leukocytes were depleted from the rabbit circulation by an intravenous injection of mustine (1.2 mg/kg) three days before the experiment; in series 2 experiments, integrin-dependent PMN migration into the peritoneal cavity was inhibited by an intravenous injection of monoclonal antibody (mAb) 60.3 (2 mg/kg) directed against the integrin CD18 on leukocytes five minutes before the experiment . RESULTS: In series 1 experiments, mustine decreased circulating leukocytes by 82 +/- 5% (mean +/- SEM) and circulating PMNs by 93 +/- 3% . Total leukocyte and PMN migration into the peritoneal cavity and peritoneal permeability to protein were decreased in mustine-treated rabbits after exposure to E . coli in the dialysate to levels similar to those found in rabbits without bacterial peritonitis . In series 2 experiments, an intravenous injection of anti-CD18 antibody also abrogated both the enhanced PMN migration into the peritoneal cavity and the increased peritoneal permeability to protein after exposure to E . coli in the dialysate . CONCLUSIONS: PMN migration into the peritoneal cavity is integrin dependent . Increased protein loss during acute, gram-negative bacterial peritonitis in a rabbit model of peritoneal dialysis is PMN dependent. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 901 - 7 Idiomarina gen . nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp . nov . and Idiomarina zobellii sp . nov; Ivanova EP et al.; Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy . Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%) . The temperature for growth was 4-30 degrees C . Both strains were rod-shaped, with a single flagellum . However, strain KMM 231T revealed a single long fimbrium . Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca . 70%) . Also present were saturated and monounsaturated straight-chain fatty acids . Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria . The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization . The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles . Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen . nov., as Idiomarina abyssalis sp . nov . (type strain is KMM 227T) and Idiomarina zobellii sp . nov . (type strain is KMM 231T). Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 847 - 55 'Candidatus Xenohaliotis californiensis', a newly described pathogen of abalone, Haliotis spp., along the west coast of North America; Friedman CS et al.; Withering syndrome is a fatal disease of wild and cultured abalone, Haliotis spp., that inhabit the west coast of North America . The aetiological agent of withering syndrome has recently been identified as a member of the family Rickettsiaceae in the order Rickettsiales . Using a combination of morphological, serological, life history and genomic (16S rDNA) characterization, we have identified this bacterium as a unique taxon and propose the provisional status of 'Candidatus Xenohaliotis californiensis' . The Gram-negative, obligate intracellular pleomorphic bacterium is found within membrane-bound vacuoles in the cytoplasm of abalone gastrointestinal epithelial cells . The bacterium is not cultivable on synthetic media or in fish cell lines (e.g . CHSE-214) and may be controlled by tetracyclines (oxytetracycline) but not by chloramphenicol, clarithromycin or sarafloxicin . Phylogenetic analysis based on the 16S rDNA of 'Candidatus Xenohaliotis californiensis' places it in the alpha-subclass of the class Proteobacteria but not to the four recognized subtaxa of the alpha-Proteobacteria (alpha-1, alpha-2, alpha-3 and alpha-4) . The bacterium can be detected in tissue squashes stained with propidium iodide, microscopic examination of stained tissue sections, PCR or in situ hybridization . 'Candidatus Xenohaliotis californiensis' can be differentiated from other closely related alpha-Proteobacteria by its unique 16S rDNA sequence. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 831 - 4 Phylogenetic characterization of marine bacterium strain 2-40, a degrader of complex polysaccharides; Gonzalez JM et al.; The marine bacterium strain 2-40 was isolated from the salt marsh cord grass, Spartina alterniflora, in the Chesapeake Bay watershed, VA, USA . It is Gram-negative, requires sea salts and is a strict aerobe . It degrades numerous complex polysaccharides and synthesizes eumelanin . By 16S rDNA analysis, the isolate was shown to be a member of the gamma-subclass of the Proteobacteria, related to Microbulbifer hydrolyticus and to a cellulolytic nitrogen-fixing bacterium. Int J Syst Evol Microbiol, 2000 Mar, 50 Pt 2, 551 - 8 Description of strain 3CB-1, a genomovar of Thauera aromatica, capable of degrading 3-chlorobenzoate coupled to nitrate reduction; Song B et al.; A Gram-negative bacterium, strain 3CB-1, isolated from a 3-chlorobenzoate enrichment culture inoculated with a sediment sample is capable of degrading various aromatic compounds and halogenated derivatives with nitrate as electron acceptor . Compounds capable of serving as carbon and energy sources include 3-chlorobenzoate, 3-bromobenzoate, 2-fluorobenzoate, 4-fluorobenzoate, benzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 3-aminobenzoate, protocatechuate, m-cresol and p-cresol . Oxygen, nitrate and nitrite were used as electron acceptors for growth . Cells are Gram-negative short rods with peritrichous flagellation . The predominant fatty acids are cis-9-hexadecenoic acid (16:1 omega 7c), hexadecanoic acid (16:0), octadecanoic acid (18:0), octadecenoic acid (18:1), 3-hydroxydecanoic acid (10:0 3OH) and dodecanoic acid (12:0) . The sequence of the 16S rRNA gene, as well as the fatty acid composition, indicate that the strain is a member of the genus Thauera in the beta-subclass of the Proteobacteria and very close to Thauera aromatica . DNA-DNA hybridization and nutrient screening indicate that strain 3CB-1 is a genomovar of Thauera aromatica with the proposed name Thauera aromatica genomovar chlorobenzoica. Microbes Infect, 2000 Mar, 2(3), 295 - 304 CD14, new aspects of ligand and signal diversity; Landmann R et al.; The glycosyl-phosphatidylinositol-linked glycoprotein CD14 is expressed in myeloid cells and serum . It binds Gram-negative and -positive bacterial cell wall components and endogenous phospholipids . Toll-like receptors, NF-kappaB and MAP kinases participate in CD14 signaling of inflammation . Alterations of CD14 in inflammatory diseases support a pathogenic role for this microbial receptor. Rev Med Chir Soc Med Nat Iasi, 1998 Jul-Dec, 102(3-4), 65 - 8 {An update on human ehrlichiosis}; Corcaci DC; Human ehrlichiosis is a potentially lethal infections due to a new gram-negative microorganism . The first human case was reported in 1953 in Japan . Two of the animal agents are involved in human disease: Ehrlichia chaffeensis, Ehrlichia sennetsu . A third Ehrlichia type agent causes the disease in human beings only--Ehrlichia granulocytophila . The tick is natural vector; the highest frequency was observed in May-July, in the United States; the most important clinical signs are: fever, anorexia, rush, dyspnea etc.; the specific treatment consists in tetracycline administration (doxycycline minocycline) for up to seven days. Am J Physiol Regul Integr Comp Physiol, 2000 Apr, 278(4), R855 - 62 Glucocorticoid sensitivity of interleukin-1 agonist and antagonist secretion: the effects of age and gender; Daun JM et al.; Interleukin-1 (IL-1) is a primary mediator of inflammation that is regulated, in part, by the hypothalamic-pituitary-adrenal axis . The purpose of this study was to determine if gender- or age-related differences exist in the sensitivity of IL-1-producing cells to hydrocortisone . Peripheral blood mononuclear cells (PBMC) isolated from men and women (21-77 yr old) were incubated with hydrocortisone (0, 50, 100, 500, or 1,000 ng/ml) with or without lipopolysaccharide (LPS) . Secretion of IL-1beta and IL-1 receptor antagonist was inhibited in a dose-dependent manner (P = 0.001) without age- or gender-related differences . Hydrocortisone decreased soluble IL-1 receptor type II (sIL-1RII) secretion by unstimulated cells (P = 0 . 0001), but it increased secretion by LPS-stimulated cells (P = 0 . 0001) in all groups . Unstimulated cell supernatants from men contained greater concentrations of sIL-1RII than the supernatants from women (P = 0.011) . Compared with men, PBMCs from women were less responsive to hydrocortisone inhibition of sIL-1RII secretion, regardless of age (P = 0.001), and compared with the follicular phase, sIL-1RII secretion was lower in the luteal phase of the menstrual cycle (P < 0.05) . These data indicate that basal secretion and glucocorticoid modulation of sIL-1RII secretion by cultured PBMCs are gender dependent . Moreover, glucocorticoid influences on sIL-1RII secretion depend on the presence or absence of gram-negative bacterial toxins. J Biol Chem, 2000 Jun 9, 275(23), 17556 - 60 Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein; Hink MA et al.; Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry . We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP) . The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen . The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy . However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein . This result can only be explained by assuming a fast hinge motion between the two fused proteins . A modeled structure of scFv-GFP supports this observation. J Biol Chem, 2000 May 12, 275(19), 14009 - 12 Cell vacuolation induced by the VacA cytotoxin of Helicobacter pylori is regulated by the Rac1 GTPase; Hotchin NA et al.; Chronic gastric infection with the Gram-negative bacterium Helicobacter pylori is a major contributing factor in the development of duodenal ulcers and is believed to be a significant risk factor in the development of gastric tumors . The VacA cytotoxin of H . pylori is a 90-kDa secreted protein that forms trans-membrane ion channels . In epithelial cells, VacA activity is associated with the rapid formation of acidic vacuoles enriched for late endosomal and lysosomal markers . Rac1 is a member of the Rho family of small GTP-binding proteins that regulate reorganization of the actin cytoskeleton and intracellular signal transduction and are being shown increasingly to play a role in membrane trafficking events . In this study we report that: (i) green fluorescent-tagged Rac1 localizes around the perimeter of the vacuoles induced by VacA; (ii) expression of dominant negative Rac1 in epithelial cells inhibits vacuole formation; (iii) expression of constitutively active Rac1 potentiates the activity of VacA . Taken together, these data demonstrate a role for Rac1 in the regulation of VacA activity. Curr Opin Microbiol, 2000 Apr, 3(2), 215 - 20 Bacterial heme sources: the role of heme, hemoprotein receptors and hemophores; Wandersman C et al.; The major mechanisms by which Gram-negative bacteria acquire heme from host heme-carrier proteins involve either direct binding to specific outer membrane receptors or release of bacterial hemophores that take up heme from host heme carriers and shuttle it back to specific receptors . The ability to interact with and remove heme from carrier proteins distinguishes heme from conceptually similar siderophore and vitamin B12 receptors . Recent genetic, biochemical and crystallization studies have started to unravel the mechanism and molecular interactions between heme-carrier proteins and components of bacterial heme assimilation systems. Curr Opin Microbiol, 2000 Apr, 3(2), 154 - 8 Post-transcriptional control by global regulators of gene expression in bacteria; Nogueira T et al.; Several authentic or potential global regulators have recently been shown to act at the post-transcriptional level . This is the case for Hfq (HF-1), which is involved in the regulation of an increasing number of genes in Escherichia coli, and CsrA (RsmA) responsible for controlling the expression of genes for extracellular enzymes and secondary metabolism in Gram-negative bacteria . The cold-shock proteins of the CspA family are able to destabilise mRNA secondary structures at low temperature and, therefore, also seem to act post-transcriptionally . These findings illustrate a more general aspect of post-transcriptional control which, in the past, was generally restricted to regulators acting at a single target . The expression of several global transcriptional regulators, such as the stationary phase and heat-shock sigma factors and H-NS, have also recently been shown to be themselves under post-transcriptional control . These examples underline the importance of this type of control in bacterial gene regulation. Curr Opin Pulm Med, 2000 Mar, 6(2), 122 - 6 Management of acute exacerbations in chronic obstructive pulmonary disease; Fein A et al.; An acute exacerbation of chronic obstructive pulmonary disease (COPD) is characterized by an acute worsening of symptoms accompanied by lung infection . In severe cases, an acute exacerbation may cause respiratory failure and death . Successful management of acute exacerbation of COPD in either the inpatient or outpatient setting requires attention to a number of key issues . In this review, issues regarding the management of acute exacerbations of COPD are discussed . An inhaled beta-2 agonist along with the inhaled anticholinergic bronchodilator are recommended . Antibiotic therapy has been demonstrated to improve clinical recovery and physical outcomes . It should be directed against the most commonly occurring pathogens and, in more severe cases, coverage against Gram-negative bacteria is considered . Short course of systemic steroids does provide benefit in hospitalized patients . Supplemental oxygen is appropriate for all patients with hypoxemia . Ventilatory support treatment may be necessary, noninvasive ventilatory assistance being preferable early in the course of the acute episode . In a high number of cases, endotracheal intubation may be avoided . Promoting smoking cessation and the use of influenzae and pneumococcal vaccination may help decrease frequency of episodes of these exacerbations. Acta Crystallogr D Biol Crystallogr, 2000 Apr, 56 ( Pt 4), 516 - 9 Preliminary X-ray analysis of a new crystal form of the Escherichia coli KDO8P synthase; Radaev S et al.; 3-Deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the biosynthesis of an essential component of the lipopolysaccharide of all Gram-negative bacteria . The structure and mechanism of KDO8P synthase are being actively studied as this enzyme represents an important target for antibiotic therapy . The structure of the Escherichia coli KDO8P synthase in cubic crystals (space group I23) has recently been determined and the enzyme shown to be a tetramer of identical subunits . However, this information is challenged by biochemical studies, which suggest that the enzyme behaves in solution as a homotrimer . Here, the preparation and preliminary X-ray analysis of monoclinic crystals of KDO8P synthase are reported . The crystals belong to space group P2(1), with unit-cell parameters a approximately 50, b approximately 140, c approximately 74 A, beta approximately 105 degrees . The structure of KDO8P synthase in the monoclinic crystal form was determined by molecular replacement, using as a search model one of the subunits of the enzyme in the cubic crystals . A tetramer of KDO8P synthase with 222 local symmetry is also present in the asymmetric unit of the P2(1) crystals, with a solvent content of 43% . The observation that the same quaternary structure of KDO8P synthase is observed in two different crystal forms belonging to distinct crystal systems (monoclinic and cubic) suggests that a tetramer is the native form of the enzyme. Biochemistry (Mosc), 2000 Mar, 65(3), 332 - 40 Recent developments in the biochemistry and ecology of enhanced biological phosphorus removal; Kortstee GJ et al.; Most of the genes encoding the enzymes involved in polyP synthesis and degradation and in phosphate transport have been studied in various Gram-negative bacteria . Progress has also been made in studying the biochemical mechanisms underlying the process of enhanced biological phosphorus removal (EBPR), in particular in lab-scale systems fed with acetate or acetate plus glucose as the sole carbon and energy sources . By applying 13C-NMR, previous models concerning anaerobic carbon metabolism have been advanced and the role of glycogen in providing reducing equivalents in EBPR is definitely demonstrated . The role of the citric acid cycle in supplying reducing equivalents for the conversion of acetyl-CoA into poly-beta-hydroxybutyrate and poly-beta-hydroxyvalerate has been discussed . An incomplete citric acid cycle has been proposed to provide a small part of the reducing equivalents . Polyphosphate:AMP phosphotransferase and polyphosphatase were readily detectable in EBPR sludge fed with acetate plus glucose, but polyphosphate kinase remained undetected . In a lab-scale EBPR system, fed for several months with only acetate as carbon source, a Rhodocyclus-like bacterium (R6) was highly enriched and is therefore probably responsible for EBPR in systems fed with acetate only . This R6-type bacterium was however also present in other EBPR sludges (but to a lesser extent), and may therefore play an important role in EBPR in general . This organism accumulates polyhydroxyalkanoates anaerobically and polyP under aerobic conditions . Unlike members of the genus Rhodocyclus, bacterium R6 cannot grow phototrophically . Therefore a provisional new genus Candidatus and species Accumulibacter phosphatis was proposed. J Microbiol Methods, 2000 Mar, 40(1), 47 - 55 Extraction of violacein from Chromobacterium violaceum provides a new quantitative bioassay for N-acyl homoserine lactone autoinducers; Blosser RS et al.; Fatty acyl homoserine lactones (AHLs) are used as extracellular quorum sensing signals by a variety of gram-negative bacteria . By activating proteins belonging to the LuxR family of transcriptional regulators, these signal metabolites allow population density-dependent gene regulation within a species, as well as interspecies communication among different bacteria . The experimental detection of AHLs is important in the identification of quorum sensing capabilities in bacteria . Chromobacterium violaceum is a gram-negative bacterium that produces the purple pigment violacein in response to the presence of the AHL N-hexanoyl homoserine lactone (C6HSL) . The mini-Tn5 mutant strain C . violaceum CV0blu is deficient in the production of this signal molecule but retains the ability to synthesize violacein in response to the presence of C6HSL and a variety of other short-chain AHLs . We have developed a quantitative bioassay that measures the amount of violacein produced by this strain in response to the presence of different concentrations of various AHL molecules . This new assay provides a means of quantifying the amount of a given AHL present in a bacterial culture and can be used to measure differences in AHL production among different strains or different batch cultures of a given species. J Urol, 2000 Apr, 163(4), 1076 - 84 Gentamicin for the practicing urologist: review of efficacy, single daily dosing and "switch" therapy; Santucci RA et al.; PURPOSE: We review the literature on gentamicin, including single daily dosing and "switch" therapy . MATERIALS AND METHODS: We used MEDLINE to search the literature from 1966 to June 1997, and then manually searched bibliographies to identify studies that our initial search might have missed . RESULTS: Gentamicin has attractive characteristics, including wide spectrum, infrequent resistance, economy and familiarity . Although limited by well known toxicities, gentamicin remains a drug of choice for serious Gram-negative infections . Dosing strategies, such as single daily dosing and switch therapy, have renewed enthusiasm for this time-honored drug . CONCLUSIONS: Gentamicin remains a valuable drug in urology . Once daily dosing and switch therapy offer the potential to increase effectiveness and convenience while decreasing toxicity and costs. Semin Cell Dev Biol, 2000 Feb, 11(1), 27 - 34 PapD-like chaperones and pilus biogenesis; Sauer FG et al.; The assembly of adhesive pili from individual subunits by periplasmic PapD-like chaperones in Gram-negative bacteria offers insight into the complex process of organelle biogenesis . PapD-like chaperones bind, stabilize, and cap interactive surfaces of subunits until they are assembled into the pilus . Subunits lack the seventh *gb-strand necessary to complete their immunoglobulin-like folds; the chaperone supplies this missing strand . Indeed, the chaperone may act as a template, providing steric information to facilitate subunit folding . In the mature pilus, each subunit is thought to supply the missing strand to complete the fold of its neighbor . Thus, one general function of chaperones in organelle biogenesis may be to cap highly interactive surfaces of subunits until they reach the proper assembly site . J Biol Chem, 2000 Mar 31, 275(13), 9773 - 81 PU.1 and interferon consensus sequence-binding protein regulate the myeloid expression of the human Toll-like receptor 4 gene; Rehli M et al.; The protein product of the Toll-like receptor (TLR) 4 gene has been implicated in the signal transduction events induced by lipopolysaccharide (LPS) . In mice, destructive mutations of Tlr4 impede the normal response to LPS and cause a high susceptibility to Gram-negative infection . Expression of TLR4 mRNA in humans is restricted to a small number of cell types, including LPS-responsive myeloid cells, B-cells, and endothelial cells . To investigate the molecular basis for TLR4 expression in cells of myeloid origin, we cloned the human TLR4 gene and analyzed its putative 5'-proximal promoter . In transient transfections a region of only 75 base pairs upstream of the major transcription initiation site was sufficient to induce maximal luciferase activity in THP-1 cells . The sequence of this region is similar in human and mouse TLR4 genes and lacks a TATA box, typical Sp1-sites or CCAAT box sequences . Instead, it contains consensus-binding sites for Ets family transcription factors, octamer-binding factors, and a composite interferon response factor/Ets motif . The activity of the promoter in macrophages was strictly dependent on the integrity of both half sites of the composite interferon response factor/Ets motif, which was constitutively bound by the myeloid and B-cell-specific transcription factor PU.1 and interferon consensus sequence-binding protein . These results indicate that the two tissue-restricted transcription factors PU.1 and interferon consensus sequence-binding protein participate in the basal regulation of human TLR4 in myeloid cells . Cloning of the human TLR4 gene provides a basis for further investigation of the possible impact of genetic variations on the susceptibility to infection and sepsis. J Biol Chem, 2000 Mar 31, 275(13), 9476 - 84 Structure and mechanism of 3-deoxy-D-manno-octulosonate 8-phosphate synthase; Radaev S et al.; 3-deoxy-D-manno-octulosonate 8-phosphate (KDO8P) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with arabinose 5-phosphate (A5P) to form KDO8P and inorganic phosphate . KDO8P is the phosphorylated precursor of 3-deoxy-D-manno-octulosonate, an essential sugar of the lipopolysaccharide of Gram-negative bacteria . The crystal structure of the Escherichia coli KDO8P synthase has been determined by multiple wavelength anomalous diffraction and the model has been refined to 2.4 A (R-factor, 19.9%; R-free, 23.9%) . KDO8P synthase is a homotetramer in which each monomer has the fold of a (beta/alpha)(8) barrel . On the basis of the features of the active site, PEP and A5P are predicted to bind with their phosphate moieties 13 A apart such that KDO8P synthesis would proceed via a linear intermediate . A reaction similar to KDO8P synthesis, the condensation of phosphoenolpyruvate, and erythrose 4-phosphate to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), is catalyzed by DAH7P synthase . In the active site of DAH7P synthase the two substrates PEP and erythrose 4-phosphate appear to bind in a configuration similar to that proposed for PEP and A5P in the active site of KDO8P synthase . This observation suggests that KDO8P synthase and DAH7P synthase evolved from a common ancestor and that they adopt the same catalytic strategy. Hepatology, 2000 Apr, 31(4), 858 - 63 Effect of cisapride on intestinal bacterial overgrowth and bacterial translocation in cirrhosis; Pardo A et al.; Deranged intestinal motility, which occurs in cirrhosis, may facilitate the development of intestinal bacterial overgrowth (IBO), which can lead to bacterial translocation (BT) . To assess the effect of cisapride on IBO and BT in cirrhosis, cirrhotic rats received cisapride or a placebo for 7 days, and measurements of jejunal bacterial content and BT studies were performed . In addition, jejunal fluid from 46 cirrhotic patients was obtained for quantitative bacterial culture . Those patients in whom gram-negative IBO was detected were randomized to receive or not to receive cisapride (20 mg twice per day) for 1 week . Cisapride significantly reduced IBO in cirrhotic rats . In addition, no BT was documented in treated animals, whereas it occurred in 40% in nontreated cirrhotic rats . Total IBO was documented in 23 of 46 cirrhotic patients, which was caused by gram-negative organisms in 10 cases . Orocecal transit time (OCT) significantly decreased after cisapride therapy, and was associated with the abolishment of bacterial overgrowth caused by gram-negative organisms in 4 out of 5 treated patients, whereas it persisted in nontreated cases . Cisapride administration to cirrhotic rats resulted in a reduction of the IBO, which is associated with a marked decrease in BT . On the other hand, cisapride facilitates the abolition of IBO caused by gram-negative organisms in cirrhotic patients. Vet Microbiol, 2000 Apr 4, 73(1), 51 - 60 Selective media for isolation of Brucella abortus strain RB51; Hornsby RL et al.; Brucella abortus strain RB51 (SRB51) is the standard vaccine used to protect cattle against brucellosis and is currently being used to vaccinate bison in the United States (US) . Currently available media for culture of Brucella have not been evaluated for their ability to support growth of SRB51 . In this study, five selective media for isolating brucellae, four commercially available media for gram-negative bacteria, and tryptose agar with 5% bovine serum (TSA) were compared to two SRB51 selective media developed in this study (rifampin brucellae medium (RBM), and malachite green brucellae medium (MGB)), for their ability to support growth and enhance recovery of SRB51 . Four of the five media currently used for isolation of brucellae and two of the four media used for other Gram-negative bacteria did not support growth of SRB51 . Modified Kuzdas and Morse (MKM), Brilliant Green, Skirrow's, RBM, and MGB supported growth of SRB51 in a manner similar to TSA . Recovery of SRB51 from tissues of SRB51-vaccinated bison was attempted on TSA, MKM, RBM, and MGB . From a total of 436 samples, SRB51 was isolated from 9.6, 4.3, 5.5, and 9.0% on TSA, MKM, RBM, or MGB media, respectively . Strain RB51 was recovered on only one medium (nine on TSA; three on RBM; and 9 on MGB) from 21 samples . Overgrowth of contaminating bacteria prevented potential detection of SRB51 from 9 . 4, 5.5, 0.07, and 5.9% of samples on TSA, MKM, RBM, or MGB, respectively . These data suggest that the use of RBM and MGB, in combination with TSA, enhances the ability to recover SRB51 from tissue samples. J Med Assoc Thai, 1999 Nov, 82 Suppl 1, S63 - 8 Disseminated intravascular coagulation findings in 100 patients; Chuansumrit A et al.; A retrospective study of 100 patients with disseminated intravascular coagulation from 1993 to 1997 is reported . Forty-five patients were neonates with a mean age of 12.6 days and 55 patients were infants, children and adolescents with a mean age of 6 years and 3 months . Most of them (91.5%) had complicated underlying conditions which included congenital anomalies, prematurity, malignancy, hematological and various diseases . Additionally, every patient had triggering conditions commonly identified as gram-negative septicemia . Bleeding and thromboembolic manifestations were found in 59.4 per cent and 19.8 per cent, respectively . The laboratory findings revealed red blood cell fragmentation, 89.6 per cent and thrombocytopenia, 85.8 per cent . Natural anticoagulants were studied in a few cases and revealed low levels of antithrombin III and protein C . The prompt effective management included treatment of underlying diseases, identification and relief of triggering conditions, correction of thrombocytopenia and coagulopathy, and fully supportive care . The overall case-fatality rate was 41.6 per cent which was not correlated with age, underlying diseases, triggering conditions, manifestation of bleeding, thromboembolism or shock, and exchange transfusion . However, a significant lower case-fatality rate was found in patients with positive culture (25%) as compared to those with sepsis and negative culture (51.7%) (p = 0.044) . In addition, the febrile neutropenic patients, who showed good response to the administrated granulocyte-colony stimulating factor (G-CSF), survived from the DIC. Int J Immunopharmacol, 2000 Jun, 22(6), 403 - 10 Differential impact of nicotine on cellular proliferation and cytokine production by LPS-stimulated murine splenocytes; Hakki A et al.; The immunoregulatory effects of nicotine have not been fully clarified and the reported data are often conflicting . The present study investigated the role of nicotine as an immunomodulator of murine splenocytes stimulated by lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria . BALB/c female mice of two different ages, young (2-3 months) and old (18-22 months), were used . The cells were incubated with nicotine at two different time points, 3 h pre-incubation and concurrent incubation relevant to LPS stimulation, before further incubation for 48 or 72 h . Treatment of murine splenocytes with nicotine showed an impact on cellular proliferation as well as on the production of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) . The results indicated that nicotine significantly inhibited cellular proliferation of murine splenocytes in a concentration-related manner (32, 64 and 128 microg/ml) . Timing of nicotine exposure prior to LPS stimulation was critical in terms of immunological impact on cytokine production . TNF-alpha and IL-6 production were significantly enhanced by 1 microg/ml of nicotine when cells were pre-incubated with nicotine for 3 h compared to concurrent incubation relative to LPS stimulation . The alteration in cytokine production varied with the age of the mouse . TNF-alpha production was significantly inhibited by nicotine in young mice, while IL-6 production was significantly inhibited by nicotine in old mice . Since any immunomodulation that alters the profile of these cytokines may cause an imbalance in the immune system impinging on health status, these findings may be important when dealing with the concept of nicotine as a therapeutic agent. Biol Reprod, 2000 Apr, 62(4), 928 - 38 Trout ovulatory proteins: site of synthesis, regulation, and possible biological function; Coffman MA et al.; The mRNA transcripts for trout ovulatory proteins (TOPs) are dramatically up-regulated at the time of ovulation . Previous studies indicated that TOPs were produced by the ovaries and were also present in the coelomic fluid that bathes ovulated eggs . In the present study, Western analysis indicated that TOPs were not present in the coelomic fluid prior to ovulation and therefore must be secreted into the coelomic fluid in large quantities during and after ovulation . Using in situ hybridization and immunocytochemistry, TOP mRNA and proteins were localized to the granulosa cell layer of the postovulatory follicle . A whole-follicle in vitro incubation system was used to look at the effects of various mediators on TOP mRNA and protein levels . Results of several different secondary messenger agonists suggest that TOPs are regulated through a G protein-mediated pathway that does not involve cAMP but may involve the activation of protein kinase C . Other agonists that had significant effects on TOP RNA and/or protein included transforming growth factor alpha (TGF-alpha), serine proteases, corticosteroids, bacterial lipopolysaccharide, and the nitric oxide generator SNAP ({+/-}-S-nitroso-N-acetylpenicillamine) . Overall, while several compounds caused significant effects, none were able to reproduce the increase in TOP RNA and protein that occurs in vivo, suggesting that the natural mediator of TOPs may still be untested, or that a combination of mediators may be involved . Finally, coelomic fluid inhibited the growth of the Gram negative bacterium, P . aeruginosa, and this inhibition was lost following immunoprecipitation of TOPs . This suggests that one function of TOPs may be to protect ovulated eggs from bacterial infection. J Biol Chem, 2000 Mar 24, 275(12), 8515 - 22 Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400; Field SJ et al.; Two membranous c-type cytochromes from the Fe(III)-respiring bacterium Shewanella frigidimarina NCIMB400, CymA and OmcA, have been purified and characterized by UV-visible, magnetic circular dichroism, and electron paramagnetic resonance spectroscopies . The 20-kDa CymA is a member of the NapC/NirT family of multiheme cytochromes, which are invariably anchored to the cytoplasmic membrane of Gram-negative bacteria, and are postulated to mediate electron flow between quinols and periplasmic redox proteins . CymA was found to contain four low-spin c-hemes, each with bis-His axial ligation, and midpoint reduction potentials of +10, -108, -136, and -229 mV . The 85-kDa OmcA is located at the outer membrane of S . frigidimarina NCIMB400, and as such might function as a terminal reductase via interaction with insoluble Fe(III) substrates . This putative role is supported by the finding that the protein was released into solution upon incubation of harvested intact cells at 25 degrees C, suggesting an attachment to the exterior face of the outer membrane . OmcA was revealed by magneto-optical spectrocopies to contain 10 low-spin bis-His ligated c-hemes, with the redox titer indicating two sets of near iso-potential components centered at -243 and -324 mV. Infect Immun, 2000 Apr, 68(4), 1919 - 27 Bordetella pertussis TonB, a Bvg-independent virulence determinant; Pradel E et al.; In gram-negative bacteria, high-affinity iron uptake requires the TonB/ExbB/ExbD envelope complex to release iron chelates from their specific outer membrane receptors into the periplasm . Based on sequence similarities, the Bordetella pertussis tonB exbB exbD locus was identified on a cloned DNA fragment . The tight organization of the three genes suggests that they are cotranscribed . A putative Fur-binding sequence located upstream from tonB was detected in a Fur titration assay, indicating that the tonB exbB exbD operon may be Fur-repressed in high-iron growth conditions . Putative structural genes of the beta-subunit of the histone-like protein HU and of a new two-component regulatory system were identified upstream from tonB and downstream from exbD, respectively . A B . pertussis DeltatonB exbB::Km(r) mutant was constructed by allelic exchange and characterized . The mutant was impaired for growth in low-iron medium in vitro and could not use ferrichrome, desferal, or hemin as iron sources . Levels of production of the major bacterial toxins and adhesins were similar in the TonB(+)/TonB(-) pair . The DeltatonB exbB mutant was still responsive to chemical modulators of virulence; thus, the BvgA/BvgS two-component system is not TonB dependent . Nevertheless, in vivo in the mouse respiratory infection model, the colonization ability of the mutant was reduced compared to the parental strain. Infect Immun, 2000 Apr, 68(4), 1899 - 904 Comparable endotoxic properties of lipopolysaccharides are manifest in diverse clinical isolates of gram-negative bacteria; Luchi M et al.; In general there is a poor correlation between serum lipopolysaccharide (LPS; the biologically active constituent of endotoxin) levels and mortality in septic patients . The objective of this study was to determine if chemical, structural, or biological differences among LPS from different clinical isolates of gram-negative bacteria might explain this discrepancy . LPS preparations were made using the hot phenol-water extraction method from eight clinical isolates of gram-negative bacteria . As a percentage of the total weight of the LPS, the phosphate content ranged from 3.0 to 13.8% (average, 6.7 +/- 3.6%), and the 2-keto-3-deoxyoctonate content ranged from 1.9 to 27.4% (average, 8.9 +/- 8.5%) . These values were not dissimilar to those obtained for a reference endotoxin . In a standard measure of LPS activity, the Limulus amoebocyte lysate assay, there was approximately a twofold difference between the least and most active preparations . The two preparations with the greatest difference in their ability to elicit the secretion of tumor necrosis factor alpha from a mouse peritoneal macrophage cell line were similar in lethality when administered to mice sensitized to the effects of LPS by D(+)-galactosamine . These relatively minor differences in LPS activity seem unlikely to explain the generally observed discrepancy between serum endotoxin levels and mortality in patients with gram-negative sepsis. Infect Immun, 2000 Apr, 68(4), 1893 - 8 Induction of apoptotic cell death in peripheral blood mononuclear and polymorphonuclear cells by an oral bacterium, Fusobacterium nucleatum; Jewett A et al.; It is largely unknown why a variety of bacteria present in the oral cavity are capable of establishing themselves in the periodontal pockets of nonimmunocompromised individuals in the presence of competent immune effector cells . In this paper we present evidence for the immunosuppressive role of Fusobacterium nucleatum, a gram-negative oral bacterium which plays an important role in the generation of periodontal disease . Our studies indicate that the immunosuppressive role of F . nucleatum is largely due to the ability of this organism to induce apoptotic cell death in peripheral blood mononuclear cells (PBMCs) and in polymorphonuclear cells (PMNs) . F . nucleatum treatment induced apoptosis of PBMCs and PMNs as assessed by an increase in subdiploid DNA content determined by DNA fragmentation and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assays . The ability of F . nucleatum to induce apoptosis was abolished by either heat treatment or proteinase digestion but was retained after formaldehyde treatment, suggesting that a heat-labile surface protein component is responsible for bacterium-mediated cell apoptosis . The data also indicated that F . nucleatum-induced cell apoptosis requires activation of caspases and is protected by NF-kappaB . Possible mechanisms of F . nucleatum's role in the pathogenesis of periodontal disease are discussed. J Infect Dis, 2000 Mar, 181(3), 1044 - 8 Unique gonococcal phenotype associated with asymptomatic infection in men and with erroneous diagnosis of nongonococcal urethritis; Whittington WL et al.; The percentage of gonococcal isolates in King County, Washington, requiring citrulline and uracil (CU auxotype) increased from 1.6% in 1986 to 16.5% in 1997 . Among men, urethral infection with the CU auxotype (n=93), in comparison with infection by other auxotypes (n=1211), was associated with coexisting chlamydial infection, younger age, heterosexual contact, and fewer new recent partners (P< . 05) . Among heterosexual men, urethral infection with the CU auxotype, compared with infection with other auxotypes, less often produced symptoms of urethral discharge (75% vs . 92%) or dysuria (47% vs . 74%) or signs of moderate or profuse urethral discharge (57% vs . 89%, P<.05 for each comparison), produced symptoms of longer duration (7 . 0 vs . 4.5 days, P<.01), less often resulted in urethral smears showing gram-negative intracellular diplococci (67% vs . 95%, P<.01), and thus more often was erroneously diagnosed as nongonococcal urethritis . Several mechanisms could explain reduced inflammatory response to the CU auxotype and its recent spread. J Infect Dis, 2000 Mar, 181(3), 1034 - 43 Release of gram-negative outer-membrane proteins into human serum and septic rat blood and their interactions with immunoglobulin in antiserum to Escherichia coli J5; Hellman J et al.; Prior studies indicate that 3 bacterial outer-membrane proteins (OMPs) are released into serum associated with lipopolysaccharide (LPS) and are bound by IgG in antiserum to Escherichia coli J5 (anti-J5 IgG) . The present studies analyzed the interaction of the OMPs with anti-J5 IgG and evaluated their release in an infected burn model of gram-negative sepsis . Affinity purification studies were performed on filtrates of bacteria incubated in human serum and plasma from rats with sepsis by use of O chain-specific anti-LPS IgG and anti-J5 IgG . All 3 OMPs were captured from septic rat blood by anti-LPS IgG . Release of OMPs into serum was highest for immature bacterial cultures and was increased by antibiotics in vitro and in vivo . Anti-J5 IgG selectively captured an 18-kDa OMP released into serum and into plasma from septic rats . The results raise the possibility that anti-J5 IgG may, in part, protect via anti-OMP antibodies. Ann Allergy Asthma Immunol, 2000 Feb, 84(2), 249 - 54 Study of the effects of vacuuming on the concentration of dust mite antigen and endotoxin; Bellanti JA et al.; BACKGROUND: Dust mite antigens are major sources of allergens in house dust and together with endotoxin, a proinflammatory component of gram negative bacteria also found in house dust, are important causes of tissue injury involved in the pathogenesis and severity of allergic diseases, eg, asthma . OBJECTIVE: To determine the effectiveness of vacuuming in reducing the quantity of dust mite antigens, Dermatophagoides pteronyssinus (Der p and Der p2) and endotoxin using a quantitative ELISA assay and to correlate results with those obtained using a qualitative rapid dipstick method for Der p2 . METHODS: Four specimens of house dust were collected using a Kirby Model G5 vacuum cleaner with a Micron Magic Filtration system from an approximately 54" x 18" standardized area of rug from each of 20 homes at 4 time intervals over a 6-week period, ie, a baseline specimen #1 at 0 week; specimen #2 at 1 week; specimen #3 at 5 weeks (1 month after specimen #2); specimen #4 at 6 weeks (1 week after specimen #3) . Three intervals were compared, ie, period 1-2 (1 week), period 2-3 (1 month), and period 3 to 4 (1 week) . The concentrations of Der p1 and Der p2 were determined in dust samples using a standard ELISA assay and the concentration of endotoxin was detected using a limulus amebocyte lysate assay . Concentrations of Der p2, determined by the standard ELISA assay, were compared to those in the same samples determined by a rapid dipstick method . RESULTS: A wide range of values for total weight of unprocessed dust (0.3 to 59 g, X = 8.7) and finely sieved dust (0.1 to 19 g, X = 3) from all specimens were found . In finely sieved dust specimens the mean concentrations of Der p1, Der p2 and endotoxin were 775, 1310, and 3836 ng/g of dust, respectively . Following weekly vacuuming there was an increase in concentration of Der p1, Der p2, and endotoxin in 20%, 35%, and 63% of the houses, respectively, compared to in monthly vacuuming in which increases were seen in 65%, 50%, and 63% of the houses, respectively . In contrast, there was a decrease in concentration of Der p1, Der p2 and endotoxin with weekly vacuuming in 43%, 60%, and 37% of the houses respectively versus in monthly vacuuming in 15%, 35%, and 37% of the houses respectively . A correlation coefficient of 0.7 was found for the concentration of Der p2 in 37/40 samples tested detected using the ELISA method compared with rapid dipstick assay . CONCLUSION: The results of this study support the effectiveness of vacuuming on the reduction of dust mite antigens (Der p1 and Der p2) ie, Der p2 > Der p1 . This reduction was more pronounced with weekly compared with monthly vacuuming . No reduction in the concentration of endotoxin was found . A good correlation was found between results obtained by ELISA and rapid dipstick assay for Der p2. Immunol Rev, 2000 Feb, 173, 52 - 65 Collectins and pulmonary innate immunity; Crouch E et al.; The surfactant-associated proteins SP-A and SP-D are members of a family of host defense lectins, designated collectins . There is increasing evidence that these pulmonary, epithelial-derived proteins are important components of the innate immune response to microbial challenge and participate in other aspects of immune and inflammatory regulation within the lung . Both proteins bind to glycoconjugates and/or lipid moieties expressed by a wide variety of microorganisms, and to certain organic particles, such as pollens . SP-A and SP-D have the capacity to modulate leukocyte function and, in some circumstances; to opsonize and enhance the killing of microorganisms . The biologic activity of cell wall components, such as Gram-negative bacterial polysaccharides, or viral glycoproteins, such as the hemagglutinin of influenza viruses, may be altered by interactions with collectins . In addition, complementary or cooperative interactions between SP-A, SP-D and other host defense lectins could contribute to the efficiency of this defense system . Collectins could play particularly important roles in settings of inadequate or impaired specific immunity, and acquired alterations in the levels of active collectins within the airspaces and distal airways may increase susceptibility to infection. FEMS Microbiol Ecol, 2000 Mar 1, 31(3), 217 - 224 Francisella tularensis does not manifest virulence in viable but non-culturable state; Forsman M et al.; Francisella tularensis is a small Gram-negative bacterium that causes tularemia in animals and man . The disease can be transmitted by handling of infected animals, by contaminated dust, by insect vectors, or by drinking contaminated water . In the present study cells of F . tularensis were subjected to extended storage in cold water devoid of carbon sources . Total cell counts remained constant throughout a 70-day period and beyond, while plate counts decreased to an undetectable level after 70 days . Attempts to resuscitate the cells were unsuccessful . Quantitative PCR targeting the 16S rDNA of F . tularensis showed an increase in variability after 25 days and the signal was lost after 45 days . Metabolic activity, measured by accumulation of rhodamine 123, declined to approximately 35% after a 140-day period . Analyses of substrate responsiveness of cells stored for 140 days in cold water showed that approximately 30% of the population increased in size after incubation in rich medium in the presence of nalidixic acid . Approximately 10(5) of these cells were injected intraperitoneally into mice . No signs or symptoms of tularemia were observed during 3 weeks . In addition, there was no evidence of stimulation of lymphocytes with F . tularensis as recall antigen . In conclusion, viable but non-culturable cells of F . tularensis are avirulent in mice, giving new insight into the ecological niche of this bacterium. Inhal Toxicol, 2000 Mar, 12(3), 225 - 43 Induction of adaptation to inhaled lipopolysaccharide in young and old rats and mice; Elder AC et al.; Lipopolysaccharide (LPS) is a component of the gram-negative bacterial cell wall that is known to activate inflammatory cells and enhance the production of inflammatory mediators in the lung . As it is a ubiquitous compound, inhalation exposure is highly likely in the human environment . Adaptation is a phenomenon by which a previous exposure results in improved survival or reduced injury as compared to a single exposure alone . We hypothesized that the basic proinflammatory effects of LPS in the lung could result in the development of adaptation in animals . Based on evidence of age- and species-related differences in lung injury, we used an acute lung injury model with inhaled LPS to compare the development of adaptation in young and old Fisher 344 rats and C57Bl/6J mice . Animals were exposed to low-dose (predicted lung deposition approximately 20 ng in rats and approximately 5 ng in mice) LPS aerosols for 10 min on 3 consecutive days; on day 4, a high dose (rats approximately 200 ng; mice approximately 25 ng) was delivered . Another group of animals received only the high LPS dose on day 4, whereas controls were unexposed . Twenty-four hours after the last exposure, cellular and inflammatory parameters in bronchoalveolar lavage (BAL) were determined . An adaptive response was found in both rats and mice . Adapted animals showed significantly fewer BAL neutrophils compared to nonadapted ones; there was also a significantly lower release of oxidants from phorbol methyl ester-stimulated BAL cells from adapted compared to nonadapted animals, which, in turn, showed a greater response than controls . Furthermore, studies in old animals (21 mo of age) showed that adaptation also occurs in this age group . The adaptive response is clear in old mice; in rats, there is greater variability in the response, but an adaptive trend is apparent . Therefore, we have demonstrated that inhaled low-dose LPS can induce adaptation to subsequent higher doses, much as has been shown for other toxicants that induce oxidative lung injury. Bone Marrow Transplant, 2000 Mar, 25(5), 567 - 9 Central nervous system (CNS) tuberculosis following allogeneic stem cell transplantation; Campos A et al.; Tuberculosis is an uncommon infectious complication after stem cell transplantation . We report a patient who presented with a brain mass, 3 months after pulmonary tuberculosis had been diagnosed and while he was receiving triple antituberculous therapy . He had extensive chronic GVHD . The diagnosis was made after biopsy of the lesion . The cerebral mass was excised, antituberculous treatment was maintained and the patient made a complete neurologic recovery . Six months later, he died of gram-negative septic shock . Mycobacterial infections should be considered in allograft recipients with chronic GVHD and solid lesions in the brain . Bone Marrow Transplantation (2000) 25, 567-569. Biochem Biophys Res Commun, 2000 Mar 16, 269(2), 570 - 3 Effects of CTx and 8-bromo-cAMP on LPS-induced gene expression of cytokines in murine peritoneal macrophages; Feng W et al.; LPS, an endotoxin isolated from gram-negative bacterial, has been shown to be a potent cytokine initiator in murine peritoneal macrophages . CAMP-dependent pathway is generally considered to play a suppressive role in immune response . This study investigated the effect of cAMP on LPS-induced gene expression of cytokines in murine macrophages . Our data clearly demonstrated that in LPS-treated macrophages, cAMP elevator (CTx and 8-bromo-cAMP) could increase IL-1Ra and IL-10 gene expression, while mRNAs of IL-1alpha, IL-12, IL-6, and MIF were decreased and other cytokines like IL-1beta, and IFN-gamma did not give a definite tendency . This is the first report that CTx and 8-bromo-cAMP positively regulate IL-1Ra gene expression in LPS-stimulated macrophages . Our data also suggest that a cAMP-dependent pathway may play a regulatory role in Toll-receptor system . Pediatr Neurosurg, 1999 Sep, 31(3), 155 - 8 Multiple recurrent gram-negative cerebrospinal fluid shunt infections associated with a patient with a retained ventricular foreign body; Shingadia D et al.; A 3-year-old boy experienced 10 recurrent gram-negative ventriculoperitoneal shunt (VPS) infections with identical strains over a 17-month period . Multiple cranial MRI and CT scans to identify a retained foreign body were negative . CT myelography and pressure infusion radionuclide cisternography were also unhelpful . Ventriculoscopy revealed a small retained foreign body which was successfully removed, and cultures subsequently yielded gram-negative organisms identical to the previously identified bacteria by pulsed field gel electrophoresis . No further infections were noted after removal of the retained fragment . Exploratory ventriculostomy should be considered in patients with recurrent VPS infections where other techniques fail to reveal a cause. Mikrobiol Z, 1999 Nov-Dec, 61(6), 29 - 35 {The toxicity and interferon-inducing activity of the lipopolysaccharides of Cytophaga sp . (strains 81 and 92)}; Varbanets LD et al.; The experiments on mice and cell culture of a kidney of green monkey Vero have shown that lipopolysaccharides (LPS) of the studied strains 81 and 92 of Cytophaga sp . were nontoxic . They were less active as compared to LPS of other Gram-negative bacteria as to interferon-inducing activity . A comparative study of initial and dephosphorylated LPS has shown that phosphate groups were not responsible for the interferon-inducing activity of LPS of Cytophaga genus representatives. J Immunol, 2000 Mar 15, 164(6), 3255 - 63 CD14 employs hydrophilic regions to "capture" lipopolysaccharides; Cunningham MD et al.; CD14 participates in the host innate inflammatory response to bacterial LPS obtained from Escherichia coli and other Gram-negative bacteria . Evidence from several laboratories suggests that different regions of the amino-terminal portion of the molecule may be involved in LPS binding . In this report a series of single-residue serine replacement and charge reversal mutations were generated to further elucidate the mechanism by which this protein may bind a multitude of different LPS ligands . Single-residue CD14 mutation proteins were examined for their ability to bind LPS obtained from E . coli, Porphyromonas gingivalis, and Helicobacter pylori and facilitate the activation of E-selectin from human endothelial cells . In addition, the single-residue CD14 mutation proteins were employed to perform monoclonal epitope-mapping studies with three LPS-blocking Abs that bound tertiary epitopes . Evidence that several different hydrophilic regions of the amino-terminal region of CD14 are involved in LPS binding was obtained . Epitope-mapping studies revealed that these hydrophilic regions are located on one side of the protein surface . These studies suggest that CD14 employs a charged surface in a manor similar to the macrophage scavenger receptor to "capture" LPS ligands and "present" them to other components of the innate host defense system. Eur J Oral Sci, 2000 Feb, 108(1), 22 - 8 Water characteristics associated with the occurrence of Legionella pneumophila in dental units; Zanetti F et al.; This study evaluated the incidence of Legionella pneumophila in dental unit water samples and investigated how the occurrence of these bacteria may be related to some physical, chemical and bacteriological characteristics of the water . The samples were taken from the incoming tap water, oral rinsing cup, air-water syringe, ultrasonic scaler, and the turbine of 23 dental units of private and public institutions . Apart from L . pneumophila (serogroup 1 and 3) isolated in 22 out of the 101 (21.8%) water samples tested, two other species were found: L . bozemanii and L . dumoffii . The highest densities and frequency of L . pneumophila were observed in the water coming into the units and in the dental units of public institutions . A negative association between L . pneumophila and 36 degrees C and 22 degrees C heterotrophic total plate counts and other gram-negative bacteria was found . An inverse association between the concentration of L . pneumophila and water temperature was also observed . The values of pH and total hardness did not show any significant difference in the L . pneumophila-positive and -negative dental unit waters . Finally, the chemical oxygen demand (COD) and residual chlorine were found to correlate positively with L . pneumophila. Biochem Pharmacol, 2000 May 1, 59(9), 1155 - 61 Prophylaxis against lipopolysaccharide-induced lung injuries by liposome-entrapped dexamethasone in rats; Suntres ZE et al.; Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, stimulates phagocytes to generate metabolites that play an important role in the pathogenesis of acute lung injury . In this study, the prophylactic effect of liposome-entrapped dexamethasone (L-DEX) was evaluated in an animal acute lung injury model . Rats were pretreated intratracheally with L-DEX or dexamethasone phosphate (DEX) at a dose of 800 microg dexamethasone/kg body weight; 1 hr later, pretreated animals were challenged i.v . with LPS (Escherichia coli 0111:B4, 1 mg/kg body weight) and killed 24 hr later . Challenge of saline-pretreated animals with LPS resulted in lung injury, as evidenced by increases in wet lung weight and decreases in lung angiotensin-converting enzyme and alkaline phosphatase activities, injury markers of pulmonary capillary endothelial and alveolar type II epithelial cells, respectively . Also, LPS injection resulted in significant increases in plasma phospholipase A(2), thromboxane B(2), and leukotriene B(4) concentrations . The LPS challenge also increased pulmonary myeloperoxidase and elastase activities as well as chloramine concentrations, suggestive of neutrophil infiltration and activation of the inflammatory response . Pretreatment of animals with L-DEX was significantly more effective than pretreatment with the free drug in reducing lung inflammation and other lung injuries, as indicated by the appropriate injury markers used in this study . Our results suggested that the pulmonary delivery of liposome-entrapped anti-inflammatory drugs such as dexamethasone improves prophylactic efficacy in counteracting LPS-induced lung injury. Inflammation, 2000 Feb, 24(1), 33 - 44 Lactoferrin protects gut mucosal integrity during endotoxemia induced by lipopolysaccharide in mice; Kruzel ML et al.; The hypothesis that lactoferrin protects mice against lethal effects of bacterial lipopolysaccharide (LPS) is the subject of experimental investigations described in this article . Lipopolysaccharide is a powerful toxin produced by gram negative bacteria that when injected into humans or experimental animals reproduce many of the pathophysiologic and immune responses caused by live bacteria . Lactoferrin administered intraperitoneally 1 hr prior to injection of LPS significantly enhanced the survival of mice, reducing LPS-induced mortality from 83.3% to 16.7% . Changes in locomotor and other behavioral activities resulting from LPS injection were not present in mice treated with lactoferrin . Also, histological examination of intestine revealed remarkable resistance to injury produced by LPS if mice were pretreated with lactoferrin . Severe villus atrophy, edema and epithelial vacuolation were observed in LPS-treated animals but not in lactoferrin-treated counterparts . Electrophysiological parameters were used to assess secretory and absorptive functions in the small intestine . In mice treated with LPS, transmural electrical resistance was reduced and absorption of glucose was increased . Lactoferrin treatment had no significant influence on basal electrophysiological correlates of net ion secretion or glucose absorption nor on changes induced by LPS . Collectively, these results suggest that lactoferrin attenuates the lethal effect of LPS and modulates behavioral and histopathological sequela of endotoxemia. Clin Diagn Lab Immunol, 2000 Mar, 7(2), 168 - 74 Characterization of a predominant immunogenic outer membrane protein of Riemerella anatipestifer; Subramaniam S et al.; The ompA gene, encoding the 42-kDa major antigenic outer membrane protein OmpA of Riemerella anatipestifer, the etiololgical agent of septicemia anserum exsudativa, was cloned and expressed in Escherichia coli . Recombinant OmpA displayed a molecular mass similar to that predicted from the nucleotide sequence of the ompA gene but lower than that observed in total cell lysates of R . anatipestifer . The ompA gene showed a conserved C-terminal region comprising the OmpA-like domain and a variable N-terminal region . This structure is similar to those of the analogous outer membrane proteins of several gram-negative bacteria . However, OmpA of R . anatipestifer contains six EF-hand calcium-binding domains and two PEST regions, which distinguish it from other outer membrane proteins . The occurrence of these motifs in OmpA suggests a possible role in virulence for this protein . The ompA gene is present in the R . anatipestifer type strain and in all serotype reference strains . However, it exhibits some minor genetic heterogeneity among different serotypes, which seems not to affect the strong antigenic characteristics of the protein . OmpA is a conserved and strong antigenic determinant of R . anatipestifer and hence is suggested to be a valuable protein for the serodetection of R . anatipestifer infections, independent of their serotype. J Clin Microbiol, 2000 Mar, 38(3), 1258 - 62 Molecular characterization of Brucella strains isolated from marine mammals; Bricker BJ et al.; Recently, gram-negative bacteria isolated from a variety of marine mammals have been identified as Brucella species by conventional phenotypic analysis . This study found the 16S rRNA gene from one representative isolate was identical to the homologous sequences of Brucella abortus, B . melitensis, B . canis, and B . suis . IS711-based DNA fingerprinting of 23 isolates from marine mammals showed all the isolates differed from the classical Brucella species . In general, fingerprint patterns grouped by host species . The data suggest that the marine mammal isolates are distinct types of Brucella and not one of the classical species or biovars invading new host species . In keeping with historical precedent, the designation of several new Brucella species may be appropriate. J Physiol Pharmacol, 1999 Dec, 50(5), 787 - 805 Helicobacter pylori lipopolysaccharide-mediated gastric and extragastric pathology; Moran AP; Lipopolysaccharides (LPS) are a family of toxic phosphorylated glycolipids in the outer membrane of Gram-negative bacteria, including Helicobacter pylori, and are composed of a lipid moiety (termed lipid A), a core oligosaccharide, and a polymeric O-specific polysaccharide chain . Compared with LPS of other bacteria, H . pylori LPS and lipid A induce low immunological activities in a range of test systems . Nevertheless, these reduced levels of LPS-induced cytokines and toxic oxygen radicals can contribute, with those induced by bacterial proteins, to the H . pylori-associated inflammatory response . Whether the ability of H . pylori LPS to induce low production of both procoagulant activity and plasminogen activator inhibitor type 2 by human mononuclear cells contributes to localized inflammatory responses alone and, in addition, play a role in extragastric pathology remains an open question . The core oligosaccharide of H . pylori LPS, in part with a 25 kDa protein adhesin, mediates the binding of the bacterium to the host glycoprotein laminin, and hence interferes with gastric cell receptor-laminin interaction in the basement membrane . Also affecting mucosal integrity, the core sugars of certain H . pylori strains, particularly those associated with gastric ulceration, have been implicated in pepsinogen induction, but this is a strain-dependent phenomenon . Of particular interest, the O-chains of a large proportion of H . pylori strains mimic Lewis (Le) antigens . Although investigations have focussed on the role of these antigens in H . pylori-associated autoimmunity, which remains to be unequivocally established, other pathogenic consequences of Lewis mimicry are becoming apparent . Expression of Lewis antigens may be crucial for H . pylori colonization and adherence and, by aiding bacterial interaction with the gastric mucosa, thereby aid delivery of secreted products, and hence influence the inflammatory response. J Endocrinol, 2000 Mar, 164(3), 361 - 9 IGF-I/IGFBPs system response to endotoxin challenge in sheep; Briard N et al.; Endotoxin (LPS), a membrane component of gram-negative bacteria produces multiple endocrine and metabolic effects that mimic those seen in acute sepsis . It induces species-dependent alterations of the growth hormone (GH) axis that may participate in the shift of the metabolism towards catabolic events . Humans and sheep show increased GH secretion in response to LPS, as opposed to rats, which have been the most studied . The purpose of our work was to evaluate the effects in intact rams of an acute intravenous administration of a high dose of LPS on the insulin-like growth factor (IGF)-I/IGF-binding proteins (IGFBPs) system and to analyse the temporal relationship of GH axis changes with those of several hormonal and metabolic parameters such as somatostatin, cortisol, insulin, and glucose . LPS induced a late moderate decrease of total IGF-I plasma levels following a 5-h steady-state period (-26.6+/-4 . 2%, P<0.05, 9 h after LPS), despite a biphasic and sustained increase of GH secretion in the same animals (2.48+/-0.39 ng/ml 2 h after LPS and 2.7+/-0.37 ng/ml 5 h after LPS vs 0.77+/-0.10 before LPS; Briard et al . 1998a) . Western ligand blot analysis in IGFBPs showed an early short-lasting increase in IGFBP-1 (188.8+/-39% P<0 . 05, 3 h after LPS) . No significant change was seen for either IGFBP-2, -3 or -4 . We observed a marked and sustained increase in cortisol (128.18+/-7.21 ng/ml 3 h after LPS, vs 21.17+/-4.22 before LPS) . Insulin also increased (27.69+/-3.90 microU/ml 3 h after LPS, vs 13.48+/-1.69 before LPS) and its burst coincided with that of IGFBP-1 . Moderately decreased IGF-I and increased IGFBP-1 plasma levels contrasted with the sustained increase in GH secretion that we recently described, thereby suggesting that endotoxin causes a state of resistance to GH . This may be exacerbated by reduced IGF-I bioavailability and/or action, and which may participate in the pathophysiology of the catabolic state seen in sepsis . The temporal analysis of hormone responses suggests that endotoxin-induced alterations of the IGF-I/IGFBPs system may involve the prolonged and substantial somatostatin rise that we recently demonstrated, together with an increase in glucocorticoid and cytokine as more generally assumed. Br J Pharmacol, 2000 Jan, 129(2), 307 - 14 Lipopolysaccharide activates nuclear factor kappaB in rat intestine: role of endogenous platelet-activating factor and tumour necrosis factor; De Plaen IG et al.; 1 . We examined the effect of lipopolysaccharide (LPS), a cell wall constituent of Gram negative bacteria, on nuclear factor kappaB (NF-kappaB) activation in the intestine and the roles of endogenous platelet-activating factor (PAF), tumour necrosis factor-alpha (TNF) and neutrophils . We also compared the time course of NF-kappaB activation in response to PAF and LPS . 2 . Ileal nuclear extracts from LPS (8 mg kg(-1), IV)-injected rats were assayed for NF-kappaB-DNA-binding activity and identification of the subunits . Some rats were pretreated with WEB2170 (a PAF receptor antagonist), anti-TNF antibody, or anti-neutrophil antiserum . NF-kappaB p65 was localized by immunohistochemistry . An additional group was challenged with PAF (2 microg kg(-1), IV) for comparison . 3 . LPS activates intestinal NF-kappaB, both as p50-p50 and p50-p65 dimers within 15 min, and the effect peaks at 2 h . The effect is slower and more sustained than that of PAF, which peaks at 30 min . Activated NF-kappaB was immunolocalized within epithelial and lamina propria cells . LPS effect was reduced by 41, 37 and 44%, respectively, in animals pretreated with WEB2170, anti-TNF antibody, or anti-neutrophil antiserum (P<0.05) . 4 . LPS activates intestinal NF-kappaB in vivo and neutrophil activation is involved in its action . The LPS effect is mediated by both endogenous PAF and TNF. Int J Tuberc Lung Dis, 2000 Feb, 4(2), 97 - 107 A review of the diagnosis and treatment of smear-negative pulmonary tuberculosis; Colebunders R et al.; Recommendations on the management of smear-negative pulmonary tuberculosis (TB) are still based on the behaviour of this disease in populations unaffected by the human immunodeficiency virus (HIV) . Studies prior to the HIV epidemic estimated that there were 1.22 cases of smear-negative and extra-pulmonary TB for each smear-positive case . Patients with smear-negative pulmonary TB were found to be less infectious and to have a lower mortality, but a significant proportion (50%-71%) progressed to active disease justifying treatment . Moreover, a wide variety of regimens also proved effective in the treatment of smear-negative disease in HIV-negative patients . The advent of HIV has changed many of these parameters . Countries affected by both HIV and TB have experienced a disproportionate increase in smear-negative disease . While apparently remaining less infectious than smear-positive cases, HIV-positive patients with smear-negative pulmonary TB are generally more immunocompromised, have more adverse drug reactions, and suffer higher mortality rates on treatment . Clinical decision-making has also been complicated because HIV co-infection broadens the differential diagnoses of smear-negative pulmonary TB to include diseases such as Pneumocystis carinii pneumonia (PCP), pulmonary Kaposi's sarcoma, and Gram-negative bacteraemia . Our approach to smear-negative pulmonary TB must therefore adapt to these changed parameters . Management algorithms based on several features (clinical symptoms, response to antibiotic trials, smear investigations, and chest radiography) have been developed to improve case detection . These algorithms must be validated in each locale because their performance will vary depending on numerous local factors such as the regional prevalence of PCP . Alternative methods of specimen collection, such as sputum induction, and processing must be evaluated . National tuberculosis programmes should also consider extending the use of rifampicin-based short-course chemotherapy (SCC) to new patients with smear-negative disease . This latter intervention, and the much-needed establishment of additional microscopy and culture facilities, will depend on increased financial and technical support from the international community. Int J Clin Pract . 1999 Oct-Nov;53(7):565. Oral gastrografin in neonates: a note of caution; Tuladhar R et al.; Hyperosmolar feeds are known to increase gastrointestinal permeability, predisposing to absorption of toxins . They are also associated with necrotising enterocolitis (NEC) in neonates . A case of a neonate with suspected NEC who died following Gram-negative septicaemia possibly related to oral gastrografin is reported . Hyperosmolarity of gastrografin may have caused complete loss of mucosal integrity in the compromised bowel leading to Gram-negative septicaemia. J Bacteriol, 2000 Mar, 182(6), 1757 - 60 Isolation of an inner membrane-derived subfraction that supports in vitro replication of a mini-RK2 plasmid in Escherichia coli; Kim PD et al.; Previous results have demonstrated that the inner, but not the outer, membrane fraction of Escherichia coli is the site of membrane-associated DNA replication of plasmid RK2, a broad-host-range plasmid capable of replication in a wide variety of gram-negative hosts (K . Michaels, J . Mei, and W . Firshein, Plasmid 32:19-31, 1994) . To resolve the inner membrane replication site further, the procedure of Ishidate et al . (K . Ishidate, E . S . Creeger, J . Zrike, S . Deb, G . Glauner, T . J . MacAlister, and L . I . Rothfield, J . Biol . Chem . 261:428-443, 1986) was used to separate the inner membrane into a number of subfractions, of which only one, a small subfraction containing only 10% of the entire membrane, was found to synthesize DNA inhibited by antibody prepared against the plasmid-encoded initiation protein TrfA . This is the same subfraction that was also found to bind oriV and TrfA to the greatest extent in filter binding assays (J . Mei, S . Benashski, and W . Firshein, J . Bacteriol . 177:6766-6772, 1995). J Immunol, 2000 Mar 1, 164(5), 2592 - 601 Lipopolysaccharide inhibits HIV-1 infection of monocyte- derived macrophages through direct and sustained down-regulation of CC chemokine receptor 5; Franchin G et al.; It is now well established that HIV-1 requires interactions with both CD4 and a chemokine receptor on the host cell surface for efficient infection . The expression of the CCR5 chemokine receptor in human macrophages facilitates HIV-1 entry into these cells, which are considered important in HIV pathogenesis not only as viral reservoirs but also as modulators of altered inflammatory function in HIV disease and AIDS . LPS, a principal constituent of Gram-negative bacterial cell walls, is a potent stimulator of macrophages and has been shown to inhibit HIV infection in this population . We now present evidence that one mechanism by which LPS mediates its inhibitory effect on HIV-1 infection is through a direct and unusually sustained down-regulation of cell-surface CCR5 expression . This LPS-mediated down-regulation of CCR5 expression was independent of de novo protein synthesis and differed from the rapid turnover of these chemokine receptors observed in response to two natural ligands, macrophage-inflammatory protein-1alpha and -1beta . LPS did not act by down-regulating CCR5 mRNA (mRNA levels actually increased slightly after LPS treatment) or by enhancing the degradation of internalized receptor . Rather, the observed failure of LPS-treated macrophages to rapidly restore CCR5 expression at the cell-surface appeared to result from altered recycling of chemokine receptors . Taken together, our results suggest a novel pathway of CCR5 recycling in LPS-stimulated human macrophages that might be targeted to control HIV-1 infection. Mol Microbiol, 2000 Feb, 35(4), 711 - 7 Outer-membrane phospholipase A: known structure, unknown biological function; Dekker N; Outer-membrane phospholipase A (OMPLA) is one of the few enzymes present in the outer membrane of Gram-negative bacteria . The enzymatic activity of OMPLA is strictly regulated to prevent uncontrolled breakdown of the surrounding phospholipids . The activity of OMPLA can be induced by membrane perturbation and concurs with dimerization of the enzyme . The recently elucidated crystal structures of the inactive, monomeric and an inhibited dimeric form of the enzyme provide detailed structural insight into the functional properties of the enzyme . OMPLA is a serine hydrolase with a unique Asn-156-His-142-Ser-144 catalytic triad . Only in the dimeric state, complete substrate binding pockets and functional oxyanion holes are formed . A model is proposed for the activation of OMPLA in which membrane perturbation causes the formation of non-bilayer structures, resulting in the presentation of phospholipids to the active site of OMPLA and leading to the formation of the active dimeric species . Possible roles for OMPLA in maintaining the cell envelope integrity and in pathogenicity are discussed. Mol Microbiol, 2000 Feb, 35(4), 699 - 710 Families of transmembrane sugar transport proteins; Saier MH Jr; We describe here 20 families of secondary (pmf-driven) carriers which, in addition to nine families within the ATP-dependent ABC superfamily, and seven families of Gram-negative bacterial outer membrane porins, largely account for the stereospecific transport of sugars and their derivatives into and out of all living cells on earth . Family characteristics as well as struc-tural and functional properties of the family constituents are described . By reference to our website , phylogenetic relationships, detailed substrate specificity information and both primary and secondary references are also available . This review provides a comprehensive guide to the diversity of carriers that mediate the transport of sugar-containing molecules across cell and organellar membranes. Immunology, 2000 Feb, 99(2), 215 - 20 Repeat bacterial challenge in a subcutaneous chamber model results in augmented tumour necrosis factor-alpha and interferon-gamma response, and suppression of interleukin-10; Houri-Haddad Y et al.; The present study compared the effect of a single or a repeat challenge with the Gram-negative pathogen Porphyromonas gingivalis on the local inflammatory response within subcutaneous chamber model in mice . Subcutaneous chambers were implanted 2 weeks prior to the final challenge . The repeat-challenge (REP) group received two intrachamber bacterial injections 14 days apart, while the single-injection group (SIN) received only a single bacterial challenge . Injection of saline was used as the control . The cellular contents of the chamber exudates were used for differential cell counts, and the supernatants were analysed for tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and interleukin (IL)-10 levels . Immunoglobulin G1 (IgG1) and IgG2a levels to P . gingivalis in the exudates were also determined . The results showed that the leucocyte counts increased significantly post-challenge, and the REP group showed the highest number of lymphocytes and neutrophils . Both P . gingivalis-challenged groups exhibited significant increase in TNF-alpha and IL-10 levels at day 1 post-challenge . TNF-alpha levels in the chamber exudate were threefold higher in the REP group compared with the SIN group on day 1 post-challenge (P < 0.05) . In contrast, IL-10 levels were significantly lower in the REP group 1 day post-challenge compared with the SIN group . The REP group had significantly higher levels of IFN-gamma at baseline, and this difference remained significant 1 day post-challenge . Analysis of antibody levels to P . gingivalis showed that while the control and the SIN groups had no anti-P . gingivalis IgG in the chamber exudate during the 7-day study period, the REP group showed high anti-P . gingivalis IgG levels . In addition, the titres of IgG2a were fivefold higher than the IgG1 titres . The results showed that a repeat local challenge with P . gingivalis augmented the proinflammatory cytokines TNF-alpha and IFN-gamma, while inhibiting the accumulation of the anti-inflammatory cytokine IL-10 . This shift towards a T helper 1 (Th1)-dominant response was reflected in the relatively high anti-P . gingivalis IgG2a titres in the local inflammatory environment 7 days post-challenge. J Agric Food Chem, 2000 Feb, 48(2), 265 - 9 Lipophilization of lysozyme by short and middle chain fatty acids; Liu ST et al.; Hen egg white lysozyme was lipophilized with short and middle chain saturated fatty acids (caproic, capric, or myristic acid) . The yield, bactericidal properties, and structural properties of lipophilized lysozymes were investigated . The yield of lipophilization of lysozyme greatly increased with the decrease in the chain length of fatty acid . Lipophilization broadened the bactericidal action of lysozyme to Gram-negative bacteria with little loss of enzymatic activity . The bactericidal activity increased in proportion to the number of bound short chain fatty acids . The thermal stability of lipophilized lysozyme decreased in proportion to the chain length and number of bound fatty acids. Bull Soc Pathol Exot, 1999 Dec, 92(5), 288 - 91 {Bacterial meningitis in the adult . Study of 85 cases observed in the infectious disease unit of the Fondation Jeanne Ebori (F.J.E.), Libreville, Gabon}; Okome-Nkoumou M et al.; We conducted a retrospective review to specify the frequency, identify the aetiological factors of bacterial meningitis in adults (BMA) and to evaluate the therapeutic protocol used . This study was conducted on 85 (BMA) cases of hospitalised patients between January 1991 and December 1995 (5 years) on our service . The BMA represented 3% of all admissions for infectious diseases at the Foundation Jeanne Ebori in Libreville . It occurred in an endemosporadic fashion . All patients were Black Africans with an average age of 33 years (range: 16-60 years) . Males predominated by a ratio of 2.4 . Tha patients were seen late in the evolution of the disease, as shown by the folloxing clinical signs: neuropsychic problems (100%), 25 patients (29%) were in a profound coma, 5 (6%) had a hemiplegia, 2 (2%) an hypoacousie and 1 (1%) seizure . Aetiological factors were found in 17 cases (20%) to be in the ORL sphere (sinusitis: n = 8, ear infection: n = 4), pneumopathies (n = 4) and one case of breach dure-mere . The predominant germ was pneumocoque, isolated in 55 cases (65%), 15 cases had a LCR clear (18%) . Bacteria gram negative (6%) were identified in the immunocompromised HIV . Third generation cephems had an efficiency higher than beta lactamines: 83% against 73% . The mortality was 18%; 3% of the remaining patients had neurological deafness . The seriousness of the results of this survey calls for the urgent implementation of a surveillance programme. Science, 2000 Feb 25, 287(5457), 1497 - 500 Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion; Odenbreit S et al.; The Gram-negative bacterium Helicobacter pylori is a causative agent of gastritis and peptic ulcer disease in humans . Strains producing the CagA antigen (cagA(+)) induce strong gastric inflammation and are strongly associated with gastric adenocarcinoma and MALT lymphoma . We show here that such strains translocate the bacterial protein CagA into gastric epithelial cells by a type IV secretion system, encoded by the cag pathogenicity island . CagA is tyrosine-phosphorylated and induces changes in the tyrosine phosphorylation state of distinct cellular proteins . Modulation of host cells by bacterial protein translocation adds a new dimension to the chronic Helicobacter infection with yet unknown consequences. Infect Immun, 2000 Mar, 68(3), 1655 - 63 Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14; Iwagaki A et al.; Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock . Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety . Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential . The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays . PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells . The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergiz