Gram negative

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 synergized with PMB and SAEP-4 . LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic . LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb . SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum . Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells . Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.

Infect Immun, 2000 Mar, 68(3), 1235 - 42
Synergistic epithelial responses to endotoxin and a naturally occurring muramyl peptide; Flak TA et al.; We have investigated the synergistic interactions of a naturally occurring peptidoglycan fragment (muramyl peptide) and bacterial endotoxin in the induction of inflammatory processes within respiratory epithelial cells, at the levels of both signal transduction events and ultimate cellular metabolic effects . The source of the muramyl peptide is Bordetella pertussis, the causative agent of the respiratory disease pertussis . During log-phase growth, B . pertussis releases the muramyl peptide tracheal cytotoxin (TCT), which has the structure N - acetylglucosaminyl - 1,6 - anhydro - N - acetylmuramyl - (L) - alanyl - gamma - (D) - glutamyl - meso - diaminopimelyl - (D) - alanine, equivalent to a monomeric subunit of gram-negative bacterial peptidoglycan . When applied to hamster trachea epithelial (HTE) cells, TCT and endotoxin were found to be highly synergistic in the induction of interleukin-1alpha (IL-1alpha), type II (inducible) nitric oxide synthase (iNOS), nitric oxide production, and inhibition of DNA synthesis . Neither molecule alone significantly triggered these responses . The serine/threonine protein kinase inhibitor H7 blocked induction of both IL-1alpha and iNOS . More selective inhibitors of protein kinase C, cyclic AMP-dependent protein kinase, and cyclic GMP-dependent protein kinase were not capable of blocking the effects of TCT and endotoxin, suggesting that the H7-inhibited component in this pathway is not among the commonly described kinase targets of H7 . Treatment of HTE cells with exogenous IL-1 reproduced the induction of iNOS and DNA synthesis inhibition caused by TCT and endotoxin . H7 was not capable of interfering with effects caused by exogenous IL-1, implying that the H7-sensitive step in the pathway is upstream of IL-1 protein production . Similar assays with the phorbol ester phorbol myristate acetate indicate that it could effectively synergize with endotoxin but not with TCT, suggesting that TCT and endotoxin induce different signal transduction events that combine synergistically . The synergy observed with TCT and endotoxin in epithelial cells is significantly different from their interaction with other cell types, revealing a unique inflammatory response by epithelial cells to these natural bacterial products.

J Clin Invest, 2000 Feb, 105(4), 497 - 504
Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide; Lien E et al.; Lipopolysaccharide (LPS) is the main inducer of shock and death in Gram-negative sepsis . Recent evidence suggests that LPS-induced signal transduction begins with CD14-mediated activation of 1 or more Toll-like receptors (TLRs) . The lipid A analogues lipid IVa and Rhodobacter sphaeroides lipid A (RSLA) exhibit an uncommon species-specific pharmacology . Both compounds inhibit the effects of LPS in human cells but display LPS-mimetic activity in hamster cells . We transfected human TLR4 or human TLR2 into hamster fibroblasts to determine if either of these LPS signal transducers is responsible for the species-specific pharmacology . RSLA and lipid IVa strongly induced NF-kappaB activity and IL-6 release in Chinese hamster ovary fibroblasts expressing CD14 (CHO/CD14), but these compounds antagonized LPS antagonists in CHO/CD14 fibroblasts that overexpressed human TLR4 . No such antagonism occurred in cells overexpressing human TLR2 . We cloned TLR4 from hamster macrophages and found that human THP-1 cells expressing the hamster TLR4 responded to lipid IVa as an LPS mimetic, as if they were hamster in origin . Hence, cells heterologously overexpressing TLR4 from different species acquired a pharmacological phenotype with respect to recognition of lipid A substructures that corresponded to the species from which the TLR4 transgene originated . These data suggest that TLR4 is the central lipid A-recognition protein in the LPS receptor complex.

Anal Biochem, 2000 Mar 1, 279(1), 18 - 22
Separation of lipooligosaccharides by linear gradient gel electrophoresis; Noda K et al.; Lipooligosaccharides (LOSs) are one of the major antigenic and immunogenic components on the outer membrane of mucosal Gram-negative bacteria . These glycolipid antigens are in the M(r) range of 3-7 kDa, and SDS/PAGE has been used as an analytical tool . Although we are able to separate relatively higher M(r) LOS components by mini-PAGE, we encounter difficulties in resolving LOS components below 3.6 kDa present in heterogeneous LOS preparations . In the present study, we selected PID2 LOS consisting of six LOS components of 3.0-5.1 kDa as a model LOS and examined mini-PAGE conditions not only to resolve smaller M(r) LOS components but also to retain resolving capability of higher LOS components . We found that mini-PAGE with stepwise and linear gradient gels (glycine-SDS) resolved smaller M(r) LOS components . Mini-PAGE with linear gradient gels gave the best resolution, and LOS components of 3.0-5.1 kDa were separated as tight and even bands . Because of the resolution, LOS components were stained chemically and immunochemically much better than those on continuous or stepwise gradient gels . Our study also showed that preformed tricine-SDS (TSDS) minigels such as 16.5 and 10-20% (linear gradient) did not resolve PID2 LOS, which indicated that heterogeneous LOS preparations may not be fully analyzed by using these TSDS minigels . By using glycine-SDS linear gradient mini-PAGE, we should be able not only to screen expression of LOSs but also to characterize smaller M(r) LOS components present in heterogeneous LOS preparations whose identities may have been neglected in the past .

Adv Perit Dial, 1999, 15, 197 - 200
Peritoneal dialysis--associated peritonitis caused by gram-negative bacteria: characteristics similar to spontaneous bacterial peritonitis?
Suzuki K, Horigome I, Shishido Y, Chiba S, Miyazaki M.
The aim of the study was to investigate the characteristics of PD-related peritonitis caused by gram-negative bacteria (GNP) . We retrospectively studied the medical records of 164 patients (114 males, 50 females; mean age 46 +/- 15 years) who continued PD beyond 5 months between 1984 and 1998 . The average observation time was 40 +/- 28 months (total of 6609 patient-months) . A total of 166 episodes of peritonitis occurred during that time (mean incidence: 1 episode/40 patient-months) . Of these, 35 were GNPs, and GNP incidence stayed almost constant over time . Most GNP patients (63%) recovered without complication with an average of 14 days' antibiotic treatment . In only 4 cases was PD abandoned . Clinical features of GNP were similar to those of spontaneous bacterial peritonitis (SBP) . The unchanged incidence of GNP over time with advanced connection devices suggests that there are important mechanisms promoting micro-organisms of endogenous origin into the peritoneal cavity in PD patients.

Hunan Yi Ke Da Xue Xue Bao, 1998, 23(2), 187 - 8
{Clinical of 50 cases of aged patients with pneumonia caused by gram-negative bacteria}; Xiao Z; The diagnosis and treatment of 50 cases of aged patients with pneumonia caused by gram-negative G-bacteria (from Feb . 1993 to Aug . 1997) were analyzed . Before the results of drug sensitive tests were reported, aminoglycosides plus broad-spectrum semisynthetic penicillin or first generation cephalosporin could improve the curative effect in patients with good renal function . Bacterial culture and drug sensitive tests were crucial for choosing the proper antibiotics.

Curr Opin Microbiol, 2000 Feb, 3(1), 23 - 8
Endotoxin, toll-like receptor 4, and the afferent limb of innate immunity; Beutler B; Positional cloning work and subsequent biochemical analyses have revealed that Toll-like receptor 4 (Tlr4) transduces the lipopolysaccharide (LPS) signal, alerting the host to infection by Gram-negative bacteria . Moreover, it appears that the LPS sensing pathway is a solitary one: disruption of Tlr4 causes complete unresponsiveness to LPS . As several Tlr family members exist in vertebrates, it appears likely that the innate immune system defends the host by recognizing a small number of structurally conserved molecules that distinguish the microbial world from tissues of the host.

Curr Opin Microbiol, 2000 Feb, 3(1), 65 - 72
Bacterial pili: molecular mechanisms of pathogenesis; Sauer FG et al.; Gram-negative bacteria produce a diverse array of pili that mediate microbe-microbe and host-pathogen interactions important in the development of disease . The structural and functional characterization of these organelles, particularly their role in triggering signals in both the bacterium and the host upon attachment, has begun to reveal the molecular mechanisms of bacterial diseases.

Antimicrob Agents Chemother, 2000 Mar, 44(3), 522 - 7
Mechanism of uptake of a cationic water-soluble pyridinium zinc phthalocyanine across the outer membrane of Escherichia coli; Minnock A et al.; Previous studies have shown that a cationic water-soluble pyridinium zinc phthalocyanine (PPC) is a powerful photosensitizer that is able to inactivate Escherichia coli . In the current work incubation of E . coli cells with PPC in the dark caused alterations in the outer membrane permeability barrier of the cells, rendering the bacteria much more sensitive to hydrophobic compounds, with little effect seen with hydrophilic compounds . Addition of Mg(2+) to the medium prior to incubation of the cells with PPC prevented these alterations in the outer membrane permeability barrier . The presence of Mg(2+) in the medium also prevented the photoinactivation of E . coli cells with PPC . These results are consistent with the hypothesis that PPC gains access across the outer membrane of E . coli cells via the self-promoted uptake pathway, a mechanism of uptake postulated for the uptake of other cationic compounds across the outer membranes of gram-negative bacteria.

Alcohol, 2000 Jan, 20(1), 37 - 43
Lipopolysaccharide-stimulated nitric oxide production and inhibition of cell proliferation is antagonized by ethanol in a clonal macrophage cell line; Chang CY et al.; Both chronic and acute ethanol exposure have been shown to be cytotoxic and also to disrupt normal cell function or responses in a variety of cell types . Macrophage function has specifically been shown to be disrupted by chronic ethanol exposure by mechanisms that have not been elucidated . It is known that exposure of macrophages to lipopolysaccharide (LPS) from gram-negative bacteria will decrease the number of cells . Since increased exposure to endotoxin is often associated with chronic alcoholism, this may be one mechanism to account for loss of macrophages in alcoholic patients . The loss of macrophages, as a consequence of endotoxin treatment, appears to be linked to cell activation and, in particular, LPS-stimulated synthesis of nitric oxide which has been suggested to cause an increase in apoptosis . Ethanol also increases apoptosis in some cell types but, in general, ethanol inhibits activation of macrophages . Thus, the overall effect on cell numbers and cell proliferation elicited by treating macrophages concomitantly with ethanol and LPS depends on the balance between inhibiting LPS-mediated activation and the actions of ethanol . The interaction between ethanol and LPS was investigated in a macrophage cell line (RAW 264.7 cells) by measuring nitric oxide production and cell proliferation . A 24-h exposure to ethanol (100 mM) decreased {3H}-thymidine incorporation significantly . LPS treatment elicited a concentration-dependent decrease in {3H}-thymidine incorporation at LPS concentrations of 0.1 ng/ml to 1000 ng/ml and stimulated nitric oxide production at concentrations above 1 ng/ml . LPS-stimulated nitric oxide production was inhibited by ethanol (20 to 100 mM) and the nitric oxide synthesis inhibitor, N(G)Nitro-L-arginine methyl L-NAME) ester (100 and 500 microM) . However, LPS-inhibited {3H}-thymidine incorporation was not be totally reversed by ethanol- or L-NAME-treatment . A direct correlation between nitric oxide production and inhibition of cell proliferation could not be demonstrated . However, it appears that ethanol and LPS do affect some common mechanism(s) in this cell line.

J Biol Chem, 2000 Feb 18, 275(7), 4995 - 5002
A pattern-recognition protein for beta-1,3-glucan . The binding domain and the cDNA cloning of beta-1,3-glucan recognition protein from the silkworm, Bombyx mori; Ochiai M et al.; The beta-1,3-glucan recognition protein (betaGRP) has strong specific affinity for beta-1,3-glucan, a component of the fungal cell wall . Its interaction with beta-1,3-glucan initiates the activation of the prophenoloxidase cascade, which is an important defense system in invertebrates of many species . We cloned the cDNA of the betaGRP of the silkworm Bombyx mori . The betaGRP mRNA transcript was constitutively expressed in the hemocytes, fat body, and epithelial cells of the naive silkworm . At the same time, a bacterial or yeast challenge was indicated to intensify the transcription . Comparison of the deduced amino acid sequence with known sequences revealed that the betaGRP contained a region (Thr(264) to Pro(386)) displaying significant similarity to the catalytic regions of bacterial beta-1,3-glucanases and much higher similarity to the glucanase-like regions of Gram-negative bacteria-binding proteins found in the silkworm B . mori and the mosquito Anopheles gambiae . The region (Thr(264) to Pro(386)) of the betaGRP, however, was demonstrated not to have appreciable affinity for beta-1,3-glucan . A recombinant peptide corresponding to an N-terminal region (Tyr(1) to Ala(102)) of the betaGRP bound strongly to beta-1,3-glucan . These results indicate that the binding domain of the betaGRP for beta-1,3-glucan is located in the N-terminal region . Glucanases and the current pattern-recognition proteins that contain a glucanase-like region seem to have a common origin in their molecular evolution.

J Intern Med, 2000 Jan, 247(1), 153 - 5
Fatal hepatitis and renal failure during treatment with nimesulide; Schattner A et al.; A healthy 70-year-old woman who took nimesulide for 5 days, presented 2 weeks later with jaundice for which no other cause was found . Laboratory evidence of coagulopathy, hypoalbuminaemia and hypoglycaemia were present on admission, and liver biopsy showed massive necrosis of hepatocytes and severe inflammatory infiltrate . Despite supportive and corticosteroid treatment, her jaundice deepened and progressive acute renal failure developed, characterized by a 'prerenal' profile changing into irreversible acute tubular necrosis pattern, coma, occult Gram-negative sepsis and death . Although rare, nimesulide-associated hepatotoxicity and nephrotoxicity may occur and should be recognized as early as possible, to ensure immediate drug withdrawal and treatment.

Shock, 2000 Feb, 13(2), 100 - 9
Peritonitis in the baboon: a primate model which stimulates human sepsis; Kinasewitz GT et al.; The physiological, hemostatic, and immunological responses of 12 chronically instrumented conscious baboons with sepsis due to Escherichia coli peritonitis were compared with that of similarly instrumented controls . Chronic indwelling cannulae were placed in the aorta and pulmonary artery to monitor pressure, cardiac output, and obtain blood samples . At t = 0 a sterile or E . coli-laden fibrin clot containing 1.9-6.7 x 10(11) CFU/kg was introduced into the peritoneal cavity . The control animals were group 1 (n = 3) . The animals with peritonitis were divided into three groups depending on their clinical response . Group 2 animals (n = 3) were clinically well at the time of sacrifice (day 14), group 3 (n = 4) survived but were obviously sick on day 14, and group 4 (n = 5) died of sepsis . Implantation of a sterile fibrin clot was well tolerated with little hemodynamic change and a transient minimal inflammatory response in group 1 . Implantation of an E . coli-containing clot elicited a hyperdynamic cardiovascular response and evoked a marked inflammatory reaction and a disseminated intravascular coagulopathy . Five of 12 (42%) E . coli animals died from sepsis . In general, the physiological, hemostatic, and immunological disturbances tended to be greatest in these animals . Autopsy revealed residual peritoneal inflammation and varying degrees of inflammation in the lungs, adrenal, spleen, liver, and kidneys in all the animals that received E . coli with the inflammatory infiltrate increasing in severity from group 2 through group 4 . Tissue necrosis was observed only in the latter group . We conclude that the cardiovascular, hemostatic, and immunological responses of baboons with sepsis due to E . coli peritonitis exhibit a variable course that resembles the clinical manifestations of gram-negative sepsis in humans.

Biochim Biophys Acta, 2000 Feb 9, 1476(2), 350 - 62
Conformational studies of Actinobacillus actinomycetemcomitans leukotoxin: partial denaturation enhances toxicity; Lear JD et al.; A 114 kDa, water-soluble, cytotoxin secreted by the Gram-negative bacterium Actinobacillus actinomycetemcomitans (Aa) is similar in sequence to Escherichia coli alpha-hemolysin, but is non-hemolytic, killing leukocytes of select species, including humans . In this work, we investigated aspects of the water-soluble conformation of Aa toxin which relate to its biological, pore-forming activity . The toxin has five native tryptophans and fluorescence spectra were monitored in aqueous solutions in the presence of varying denaturants . Significant changes in the fluorescence spectra, without significant wavelength shifts, were induced by small additions of denaturants and changes in the temperature or pH . The fluorescence changes suggested that small perturbations in the aqueous environment resulted in structural changes in the toxin related not to a large unfolding but to more subtle conformational changes . Analytical ultracentrifugation showed the toxin to be a globular monomer in dilute aqueous solution . Circular dichroism spectroscopy showed about 25% alpha-helical structure which is largely maintained up to a temperature (65 degrees C) known to deactivate toxin activity . Changes in the cytotoxic properties of the toxin were monitored with flow cytometric analysis following preincubation of the toxin under mild conditions similar to those used in the fluorescence studies . These experiments showed that the pretreated toxin exhibited enhanced cell-killing potency on toxin-sensitive cells . The correlation of cytotoxicity with the changes in Trp fluorescence is consistent with the idea that partial unfolding of Aa toxin is an early, obligate step in toxin-induced cell kill.

J Infect Dis, 2000 Feb, 181(2), 621 - 5
Prognostic value of cytokine concentrations (tumor necrosis factor-alpha, interleukin-6, and interleukin-10) and clinical parameters in severe melioidosis; Simpson AJ et al.; Raised serum concentrations of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, or IL-10 are associated with mortality in patients with sepsis, but it is not known whether elevated cytokine levels are independently predictive of mortality . Cytokine assays (TNF-alpha, IL-6, and IL-10) were performed on admission plasma samples from 172 adult Thai patients with severe melioidosis . Mortality was 31.4% . APACHE II score; septicemia; plasma lactate; TNF-alpha, IL-6, and IL-10 concentrations; and IL-10/TNF-alpha and IL-6/IL-10 ratios were each associated with outcome (P</=.001 for all variables) . Only the APACHE II score and either IL-6 or IL-10 concentration were independent predictors of mortality, as determined by use of multiple logistic regression (with cytokine concentrations and ratios entered separately) . In a multivariate analysis, including both IL-6 and IL-10, the IL-10 concentration was no longer predictive . Therefore, APACHE II scores and either IL-6 or IL-10 concentration may be the most reliable parameters for stratification of patients in future studies of severe gram-negative sepsis.

Thromb Haemost, 2000 Jan, 83(1), 114 - 8
Induction of tissue factor procoagulant activity in myelomonocytic cells inoculated by the agent of human granulocytic ehrlichiosis; Behl R et al.; Human granulocytic ehrlichiosis (HGE) is a recently recognized rickettsial tick-borne febrile illness that may occasionally be complicated by coagulopathy . The agent of HGE (aHGE) is an obligate intracellular pathogen, which replicates in endosomes within neutrophils and their precursors . We hypothesized that aHGE might cause DIC via induction of monocyte tissue factor procoagulant activity (TF PCA) . Peripheral blood mononuclear cells (PBMNC) and HL-60 cells were used to model the effect of aHGE infection on monocytes/macrophages . Mononuclear cells inoculated with aHGE in vitro demonstrated approximately a 12-15-fold increase in TF PCA, with peak activity occurring at 8-12 h . HL-60 cells inoculated with aHGE also manifested a 4-6 fold induction of TF PCA, with maximal activity occurring at about 8 h . By comparison, E . Coli lipopolysaccharide (LPS) also induced an increase in TF PCA of an equivalent magnitude, and with a similar time course . Induction of TF did not require inoculation of HL-60 cells with live organism, since heat-inactivated aHGE still stimulated TF PCA expression in the target cells . Furthermore, filtered supernatants from heat-inactivated organisms induced TF PCA suggesting that the effect is due to a soluble mediator produced by the organism . Although aHGE is a gram negative organism, the soluble mediator did not appear to be classic endotoxin in that the supernatants tested negative for endotoxin by the Limulus Amoebocyte assay, and polymixin had no inhibitory effect on aHGE supernatants . We conclude that aHGE induces cells of the myelo-monocytic lineage to synthesize TF, which may contribute to the clinical coagulopathy that can be observed in this condition . An atypical soluble mediator or cellular component of the organism appears to be critically important in TF induction by aHGE.

Inflamm Res, 1999 Dec, 48(12), 613 - 20
The Lps locus: genetic regulation of host responses to bacterial lipopolysaccharide; Qureshi ST et al.; Lipopolysaccharide (LPS), an abundant glycolipid of the outer membrane of gram-negative bacteria, is able to provoke a generalized proinflammatory response in the infected host . Genetic regulation of this trait has been localized to the Lps locus on mouse chromosome 4 . Several inbred mouse strains, including C3H/HeJ, C57BL/10ScNCr and C57BL/10ScCr, bear mutations at the Lps locus (Lps(d)) that confer hyporesponsiveness to the immunostimulatory properties of LPS and susceptibility to overwhelming gram-negative bacterial infection . The phenotypic expression of Lps(d) is pleiotropic, affecting several cell types crucial to host defense, including the macrophage . By positional cloning, Toll-like receptor 4 (Tlr4), a transmembrane protein with a cytoplasmic domain that bears homology to the Interleukin-1 receptor, has been identified as the gene encoded by Lps . Tlr4 is a member of a novel gene family that participates in host defense against microbial infection in plants, invertebrates and mammals . Discovery of the molecular basis of the Lps mutation represents a significant advance in defining the fundamental mechanisms of cellular activation by LPS.

Blood, 2000 Feb 15, 95(4), 1124 - 9
Tissue factor pathway inhibitor dose-dependently inhibits coagulation activation without influencing the fibrinolytic and cytokine response during human endotoxemia; de Jonge E et al.; Inhibition of the tissue factor pathway has been shown to attenuate the activation of coagulation and to prevent death in a gram-negative bacteremia primate model of sepsis . It has been suggested that tissue factor influences inflammatory cascades other than the coagulation system . The authors sought to determine the effects of 2 different doses of recombinant tissue factor pathway inhibitor (TFPI) on endotoxin-induced coagulant, fibrinolytic, and cytokine responses in healthy humans . Two groups, each consisting of 8 healthy men, were studied in a double-blind, randomized, placebo-controlled crossover study . Subjects were studied on 2 different occasions . They received a bolus intravenous injection of 4 ng/kg endotoxin, which was followed by a 6-hour continuous infusion of TFPI or placebo . Eight subjects received 0.05 mg/kg per hour TFPI after a bolus of 0.0125 mg/kg (low-dose group), and 8 subjects received 0.2 mg/kg per hour after a bolus of 0.05 mg/kg (high-dose group) . Endotoxin injection induced the activation of coagulation, the activation and subsequent inhibition of fibrinolysis, and the release of proinflammatory and antiinflammatory cytokines . TFPI infusion induced a dose-dependent attenuation of thrombin generation, as measured by plasma F1 + 2 and thrombin-antithrombin complexes, with a complete blockade of coagulation activation after high-dose TFPI . Endotoxin-induced changes in the fibrinolytic system and cytokine levels were not altered by either low-dose or high-dose TFPI . The authors concluded that TFPI effectively and dose-dependently attenuates the endotoxin-induced coagulation activation in humans without influencing the fibrinolytic and cytokine response . (Blood . 2000;95:1124-1129)

Immunopharmacology, 2000 Jan, 46(1), 89 - 101
Ascorbic acid modulates in vitro the function of macrophages from mice with endotoxic shock; Victor VV et al.; The toxic effects of oxygen radicals produced by immune cells can be controlled to certain degree by endogenous antioxidants because of their scavenger action . This control is specially important in a type of immune cell, i.e., the phagocyte, which produces oxygen-free radicals and uses antioxidants in order to support its functions . Antioxidants, such as ascorbic acid (AA), are free radical scavengers and improve the immune response . In the pathogenesis of endotoxic shock, a disease with high mortality caused by gram-negative bacterial endotoxin, the reactive oxygen species (ROS) produced by phagocytes have been implicated . In a previous study, we observed in peritoneal macrophages from BALB/c mice suffering lethal endotoxic shock caused by intraperitoneal (i.p.) injection of Escherichia coli lipopolysaccharide (LPS; 100 mg/kg) a high production of superoxide anion . Therefore, in the present work, we have studied the in vitro effect of AA, at different concentrations (0.001, 0.01, 0.1, 1 and 2.5 mM), on the various steps of the phagocytic process, i.e., adherence to substrate, chemotaxis, ingestion of particles and superoxide anion production of murine peritoneal macrophages obtained from BALB/c mice with that of endotoxic shock, at 2, 4, 12 and 24 h after LPS injection . The increased adherence, ingestion and superoxide anion production by macrophages from animals with endotoxic shock were lower in the presence of AA, reaching similar values to those of the control animals . The most effective AA concentration in cells from mice with endotoxic shock was 0.01 mM . These data suggest that AA can regulate the phagocytic process in endotoxic shock, principally decreasing free radical production and thus it could reduce endotoxic shock severity.

Vet Microbiol, 2000 Jan, 71(1-2), 113 - 23
Extensive diversity in New Zealand Dichelobacter nodosus strains from infected sheep and goats; Zhou H et al.; Footrot is a contagious bacterial disease of ruminants spread by the Gram-negative, anaerobic organism, Dichelobacter nodosus . It is endemic in New Zealand and throughout sheep and goat farming regions of the world . Using the polymerase chain reaction (PCR) to amplify fragments of the fimbrial gene (fimA), D . nodosus was detected in 14 hoof scrapings, sampled from six farming regions within New Zealand . DNA sequencing revealed 15 strains covering eight serogroups on the New Zealand farms . The predominant serogroup was B which contained six strains, followed by serogroups F, H and G . No strains from serogroups D and I were detected in this investigation . Eleven out of the 15 D . nodosus strains had fimbriae sequences different to those previously reported and the presence of multiple strains on a single hoof was common (86% samples) . Individual sheep from the same farm, or the same paddock, were often infected by a different range of strains, which suggests a host role in mediating footrot infection.

Intensive Care Med, 1999 Dec, 25(12), 1437 - 9
Reversible tetraplegia due to polyneuropathy in a diabetic patient with hyperosmolar non-ketotic coma; Kennedy DD et al.; Critical illness polyneuromypathy has not previously been reported as a complication of diabetic coma . We describe a patient with hyperosmolar non-ketotic coma (HONK) complicating gram-negative sepsis in whom persistent coma and profound tetraplegia caused considerable concern . Although, initially, it was feared that the patient had suffered a central neurological complication such as stroke or cerebral oedema, a diagnosis of critical illness motor syndrome (CIMS) was subsequently confirmed neurophysiologically . Profound limb weakness associated with HONK is not necessarily due to a catastrophic cerebral event, rather it may be a result of CIMS, which has an excellent prognosis for full neurological recovery.

Mol Plant Microbe Interact, 2000 Feb, 13(2), 232 - 7
Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria; Heeb S et al.; The minimal replicon of the Pseudomonas plasmid pVS1 was genetically defined and combined with the Escherichia coli p15A replicon, to provide a series of new, oligocopy cloning vectors (5.3 to 8.3 kb) . Recombinant plasmids derived from these vectors were stable in growing and nongrowing cells of root-colonizing P . fluorescens strains incubated under different environmental conditions for more than 1 month.

Atherosclerosis, 2000 Feb, 148(2), 413 - 9
Hyperlipoproteinemia affects cytokine production in whole blood samples ex vivo . The influence of lipid-lowering therapy; Mohrschladt MF et al.; Low-density lipoprotein (LDL)-receptor deficient mice, thus hypercholesterolemic, combine protection against infection with an ex vivo two- to threefold higher pro-inflammatory cytokine production in macrophages . A pro-inflammatory cytokine profile ex-vivo is also associated with survival of gram-negative sepsis in man . We hypothesized that high lipoprotein levels would be associated with a pro-inflammatory cytokine production and could explain the resistance to fatal infection . We treated 10 patients with familial hypercholesterolemia (FH) with HMG-CoA reductase inhibitors, and 13 patients with endogenous hypertriglyceridemia (HTG) with fibrates . Blood samples were stimulated ex vivo with lipopolysaccharide (LPS), to assess the cytokine production capacity . FH patients had significantly lower tumor necrosis factor-alpha (TNF-alpha) production, compared to normolipidemic controls (P=0 . 001) . Lipid lowering treatment in FH patients did not affect TNF-alpha production . HTG patients showed significantly higher TNF-alpha production at baseline than matched normolipidemic controls (P<0.001), while lowering of serum triglycerides in these patients resulted in a significant decrease in TNF-alpha production (P=0.019) . The IL-10 production was not affected . These data refute our hypothesis that high LDL-cholesterol levels are associated with a pro-inflammatory cytokine production capacity . In contrast, the study suggests that very-low-density lipoprotein (VLDL) in hypertriglyceridemic patients augments TNF-alpha production.

J Biotechnol, 2000 Jan 21, 76(2-3), 97 - 119
Endotoxin removal from protein solutions; Petsch D et al.; Endotoxins liberated by gram-negative bacteria are frequent contaminations of protein solutions derived from bioprocesses . Because of their high toxicity in vivo and in vitro, their removal is essential for a safe parenteral administration . A general method for the removal of endotoxins from protein solutions is not available . Methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions . Various techniques described in the patent literature are not broadly applicable, as they are tailored to meet specific product requirements . Besides ion-exchangers and two-phase extraction, affinity techniques are applied with varying success . Also, taylor-made endotoxin-selective adsorber matrices for the prevention of endotoxin contamination and endotoxin removal are discussed for this purpose . After giving an overview of the properties of endotoxins and the significance of endotoxin contamination, this review intends to provide an overall picture of the various methods employed for their removal . Avenues are pointed out how to optimise a method with regard to the specific properties of endotoxins in aqueous solution.

Immunol Lett, 1999 Dec 1, 70(3), 185 - 9
Treatment of H . pylori infected mice with antioxidant astaxanthin reduces gastric inflammation, bacterial load and modulates cytokine release by splenocytes; Bennedsen M et al.; Helicobacter pylori is a gram-negative bacterium affecting about half of the world population, causing chronic gastritis type B dominated by activated phagocytes . In some patients the disease evolves into gastric ulcer, duodenal ulcer, gastric cancer or MALT lymphoma . The pathogenesis is in part caused by the immunological response . In mouse models and in human disease, the mucosal immune response is characterized by activated phagocytes . Mucosal T-lymphocytes are producing IFN-gamma thus increasing mucosal inflammation and mucosal damage . A low dietary intake of antioxidants such as carotenoids and vitamin C may be an important factor for acquisition of H . pylori by humans . Dietary antioxidants may also affect both acquisition of the infection and the bacterial load of H . pylori infected mice . Antioxidants, including carotenoids, have anti-inflammatory effects . The aim of the present study was to investigate whether dietary antoxidant induced modulation of H . pylori in mice affected the cytokines produced by H . pylori specific T-cells . We found that treatment of H . pylori infected mice with an algal cell extract containing the antioxidant astaxanthin reduces bacterial load and gastric inflammation . These changes are associated with a shift of the T-lymphocyte response from a predominant Th1-response dominated by IFN-gamma to a Th1/Th2-response with IFN-gamma and IL-4 . To our knowledge, a switch from a Th1-response to a mixed Th1/Th2-response during an ongoing infection has not been reported previously.

Microbiol Res, 1999 Dec, 154(3), 241 - 6
Comamonas acidovorans strain MC1: a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the herbicides 2,4-D and MCPA; Muller RH et al.; A gram-negative prototrophic bacterial species, strain MC1, was isolated from the vicinity of herbicide-contaminated building rubble and identified by 16S rDNA sequence analysis, its physiological properties, GC content, and fatty acid composition as Comamonas acidovorans . This strain displays activity for the productive degradation of the two enantiomers of dichlorprop {(RS)-2-(2,4-dichlorophenoxy-)propionate; (RS)-2,4-DP} and mecoprop {(RS)-2-(4-chloro-2-methyl-) phenoxypropionate; (RS)-MCPP} in addition phenoxyacetate herbicides, i.e . 2,4-dichlorophenoxyacetate (2,4-D) and 4-chloro-2-methylphenoxyacetate (MCPA), and various chlorophenols were utilized . Rates amounted to 1.2 mmoles/h g dry mass (2,4-D) and 2.7 mmoles/h g dry mass {(RS)-2,4-DP} . Degradation of (RS)-2,4-DP was not inhibited up to concentrations of 500 mg/l, nor of 2,4-D up to 200 mg/l . The optimum pH value of (RS)-2,4-DP degradation was around 8 . The application of respective primers for PCR amplification revealed the presence of tfdB and tfdC genes.

Eur J Clin Invest, 2000 Feb, 30(2), 167 - 79
Evaluation of CD14 in host defence; Antal-Szalmas P; An extensive search for the cell membrane targets for lipopolysaccharide (LPS), the major causative agent of Gram-negative septic shock, resulted in the identification of CD14 as the major endotoxin 'receptor' . Besides recognition of LPS, several new aspects of its biological functions have been described recently . In this review the different CD14 forms, their most important biological and biochemical features, signalling properties, cellular and subcellular distribution and association with different diseases are discussed in detail, showing that these molecules posses several unique biological functions and further proving their central role in innate immunity.

J Leukoc Biol, 2000 Jan, 67(1), 18 - 25
Synergistic action of pro-inflammatory agents: cellular and molecular aspects; Paludan SR; Generation of an inflammatory response is a complex process involving multiple factors acting in parallel and in concert . Viruses, parasites, and bacteria, particularly lipopolysaccharide (LPS), a component of the cell wall of gram-negative bacteria, act cooperatively with the cytokine interferon (IFN)-gamma to induce many of the genes involved in inflammation . In addition, these components synergistically induce secretion of tumor necrosis factor alpha (TNF-alpha), which also synergizes strongly with IFN-gamma . The molecular mechanisms underlying the synergistic gene induction discussed in this review involve cooperative activation of transcription factors . IFN-gamma-activated signal transducer and activator of transcription 1 and interferon regulatory factor-1 function synergistically with nuclear factor kappaB activated by LPS and TNF-alpha . In addition, cross-talk between the signal transduction pathways upstream of the activation of the transcription factors contributes to generation of the synergistic action . Cooperative activity of proinflammatory agents profoundly influences the immune response to infections and the efficiency of cellular clearance mechanisms.

J Surg Res, 2000 Feb, 88(2), 173 - 80
Platelet-activating factor and bacteremia-induced pulmonary hypertension; Clavijo LC et al.; BACKGROUND: Acute lung injury is a common complication of gram-negative sepsis . Pulmonary hypertension and increased lung vascular permeability are central features of lung injury following experimental bacteremia . Platelet-activating factor is a prominent proinflammatory mediator during bacterial sepsis . Our previous studies have demonstrated that exogenous administration of platelet-activating factor (PAF) induces pulmonary edema without causing pulmonary hypertension . Interestingly, inhibition of PAF activity during Escherichia coli bacteremia prevents the development of both pulmonary hypertension and pulmonary edema . These data suggest that PAF contributes to pulmonary hypertension during sepsis, but that this is unlikely to be a direct vascular effect of PAF . The goal of the present study was to investigate the mechanism by which acute E . coli bacteremia induces pulmonary injury and to define the role that PAF plays in this injury . We hypothesized that the effects of PAF on pulmonary hypertension during bacteremia are due to the effects of PAF on other vascular mediators . Several studies suggest that PAF induces the expression of endothelin-1 (ET), a potent peptide vasoconstrictor . Further, our previous studies have implicated ET as a central mediator of systemic vasoconstriction during bacteremia . We therefore sought to assess whether ET is modulated by PAF . E . coli has also been demonstrated to increase endothelial production of nitric oxide (NO), which contributes to maintenance of basal vascular tone in the pulmonary circulation . We hypothesized that PAF might increase pulmonary vascular resistance during bacteremia by activating neutrophils, increasing expression of ET, and decreasing the tonic release of NO . Furthermore, we hypothesized that hypoxic vasoconstriction did not contribute to pulmonary vasoconstriction during the first 120 min of E . coli bacteremia . METHODS: Pulmonary artery pressure (PAP), blood pressure (BP), heart rate (HR), and arterial blood gases (ABG) were measured in anesthetized spontaneously breathing adult male Sprague-Dawley rats . E . coli (10(9) CFU/100 g body wt) was injected at t = 0, and hemodynamic data were obtained at 10-min intervals and ABG data at 30-min intervals for a total of 120 min . Sham animals were treated equally but received normal saline in place of E . coli . In treatment groups, a 2.5 mg/kg dose of WEB 2086, a PAF receptor antagonist, was administered intravenously 15 min prior to the onset of sepsis or sham sepsis . The groups were (1) intravenous E . coli (n = 5); (2) intravenous WEB 2086 pretreatment + intravenous E . coli (n = 5); (3) intravenous WEB 2086 alone (n = 5); and (4) intravenous normal saline (n = 6) . Nitric oxide metabolites (NOx) and ET concentrations were assayed from arterial serum samples obtained at the end of the protocol . Lung tissue was harvested for measurement of myeloperoxidase (MPO) activity and pulmonary histology . RESULTS: E . coli bacteremia increased HR, PAP, and respiratory rate early during sepsis (within 20 min), while hypoxemia, hypotension, and hemoconcentration were not manifest until the second hour . Pretreatment with WEB 2086 completely abrogated all of these changes . E . coli bacteremia increased the activity of serum ET, lung MPO, and neutrophil sequestration in the lung parenchyma via a PAF-dependent mechanism . However, the mechanism of increased production of NO appears to be PAF independent . CONCLUSIONS: These data support the hypothesis that E . coli bacteremia rapidly induces pulmonary hypertension stimulated by PAF and mediated at least in part by endothelin-1 and neutrophil activation and sequestration in the lung . Microvascular injury with leak is also mediated by PAF during E . coli bacteremia, but the time course of resultant hypoxemia and hemoconcentration is slower than that of pulmonary hypertension . The contribution of hypoxic vasoconstriction in exacerbating pulmonary hypertension in gram-negative sepsis is probably a late

FEMS Microbiol Rev, 2000 Jan, 24(1), 21 - 44
Secretion and assembly of regular surface structures in Gram-negative bacteria; Fernandez LA et al.; Bacteria synthesize large-sized surface structures through the ordered polymerization of protein subunits . This results in planar or tubular regular structures that have evolved to accomplish specific functions related to the particular environment in which these bacteria are found . Tubular assemblies known as flagella are the most complex structures known in bacteria and consist of a helical rigid filament, a torsion adapter or hook and a proton-fueled rotator known as the basal body . Pili or fimbriae are less complicated helical filaments, which consist of a major subunit and 3-5 minor subunits or pilins, whose main function is the attachment to specific surfaces . Planar structures known as S-layers are the simplest of these regular assemblies and are generally made up of a single subunit packed as a bidimensional crystal around the whole cell surface . Most of the components of these structures have to be secreted through the inner membrane (IM), the periplasm and the outer membrane (OM) before reaching their final destination . The so called general secretory pathway (GSP), or type II secretion system, appears to be implicated in this process to varying degrees, depending on the structure considered . A few S-layers and pili require GSP components but also need specific terminal branches, such as the well known chaperone-usher pathway . On the other hand, only two of the nearly 40 proteins involved in flagellar assembly are dependent on the GSP, while the external components are secreted through a specific pathway similar to the type III systems identified in some pathogens . Moreover, secretion of subunits of S-layers using dedicated type I machinery, without the involvement of any GSP component, has also been observed.

Vet J, 2000 Jan, 159(1), 18 - 36
Actinobacillus species and their role in animal disease; Rycroft AN et al.; Actinobacillus species are Gram-negative bacteria responsible for several quite distinct disease conditions of animals . The natural habitat of the organisms is primarily the upper respiratory tract and oral cavity . A . lignieresii is the cause of actinomycosis (wooden tongue) in cattle: a sporadic, insidiously-developing granulomatous infection . In sharp contrast is A . pleuropneumoniae which is responsible for a rapidly spreading often fatal pneumonia, common among intensively reared pigs . Detailed investigation of this organism has provided a much clearer picture of the bacterial factors involved in causing disease . A . equuli similarly causes a potent septicaemia in the neonatal foal; growing apparently unrestricted once infection occurs . Other members of the genus induce characteristic pathogenesis in their preferred host, with one, A . actinomycetemcomitans, being a cause of human periodontal disease . This article reviews recent understanding of the taxonomy and bacteriology of the organisms, and the aetiology, pathogenicity, diagnosis and control of animal disease caused by Actinobacillus species .

Physiol Res, 1999, 48(4), 323 - 6
The common cDNA and amino acid sequences of the CD14 (myeloid cell-specific leucine-rich glycoprotein) receptor; Hubacek JA et al.; The CD14 receptor is a myeloid cell specific receptor, which plays a role in the recognition of lipopolysaccharides (endotoxins of gram-negative bacteria) and cell stimulation . To date, several sequences of the cDNA of the CD14 receptor have been described . We sought to establish whether the substitutions C(230)-->G, and G(560)-->A are polymorphic or if they result from a PCR or sequencing error . Using two mismatched PCRs, we confirmed (on 75 unrelated probands) that the substitutions are not due to common polymorphisms . The common cDNA sequence has the C in position 230 and G in position 560 . This corresponds to the amino acids Ala and Cys in positions 77 and 187, respectively.

Ann Thorac Cardiovasc Surg, 1999 Dec, 5(6), 408 - 10
Purulent pericarditis presenting as an extracardiac mass in a patient with untreated diabetes; Sugita T et al.; A 50-year-old man with symptoms of bi-ventricular heart failure was transferred to our hospital with a diagnosis of extracardiac tumor . He had a 10 year history of untreated diabetes . Chest computed tomography (CT) revealed an extracardiac mass in the right atrio-ventricular groove . Cardiac catheterization revealed an elevated mean right atrial pressure of 18 mmHg, mean pulmonary wedge pressure of 16 mmHg, and the right ventricular pressure curve demonstrated typical dips and plateaus . At surgery, there was severe adhesion between the pericardium and epicardium, and the pericardium was severely thickened and contained turbid pus . In the left thoracic cavity, there was large amount of pleural effusion and pus . Therefore, the patient was diagnosed with purulent pericarditis caused by left empyema . The thickened pericardium at the anterior portion of the heart was resected, however resection of the remaining portion was abandoned because the adhesion was so tight . After surgery, the patient underwent irrigation of the heart and left thoracic cavity by 1% povidone iodine solution and 0.5 mg/ml of imipenem for 7 days . Bacteriologic culture of the pus from the pericardium revealed anaerobic gram negative bacteria . After 4 months of antibiotics infusion, his C reactive protein became negative and the patient was subsequently discharged from our hospital.

J Neuroimmunol, 2000 Jan 24, 102(2), 172 - 81
Induction of inflammatory cytokines in the brain following respiratory infection with Bordetella pertussis; Loscher CE et al.; Parenteral injection of endotoxin has been used as a model to examine the role of pro-inflammatory cytokines in the centrally controlled responses to Gram-negative bacterial infection . However, the events that occur following mucosal exposure to live bacteria have received little attention . In this study, we have used a murine model to demonstrate that respiratory infection with Bordetella pertussis, which is associated with a number of systemic complications including fever, seizure and encephalopathy in children, resulted in persistent expression of mRNA transcripts for IL-1beta and TNFalpha and transient expression of IL-6 in the hippocampus and hypothalamus . These changes correlated with elevated levels of cytokine protein in the same brain areas . The results demonstrate that infection at a mucosal surface can result in the induction of pro-inflammatory cytokine production in the brain and suggest that these locally synthesized mediators may contribute to the centrally controlled clinical manifestations of B . pertussis infection.

Acta Otorhinolaryngol Belg, 1999, 53(3), 237 - 40
Pharyngeal flora in patients undergoing head and neck oncologic surgery; Barry B; In order to specify the correlation between pharyngeal flora and the onset of surgical wound infection, we conducted two prospective studies on patients undergoing oncologic surgical procedures with expected contamination by pharyngeal secretions . In the first study, an oropharyngeal swab and a specific swab of the tumour were collected the day before, or on the day of surgery . As potential pathogens were always isolated in the oropharyngeal swab, it was considered that the tumour is not infected but is colonised by the oropharyngeal flora . A second pharyngeal swab was collected at day 5-7 in the second study . Preliminary results in the second study showed that 50% (11/22) of patients were orpharyngeal carriers of pathogens before surgery . This rate is 70% (15/22) in the post-operative period with a higher rate of gram negative rods . WSI occurred in 7/22 patients (32%), mainly with isolated rods similar to those observed in the oropharyngeal post-operative flora and potential pathogens in 5/7 patients . More patients are necessary to establish a link between pre-operative ropharyngeal pathogens and the occurrence of SWI.

Haemophilia, 2000 Jan, 6(1), 41 - 3
Iliopsoas haemophiliac pseudotumours with bowel fistulation; Heaton DC et al.; Two cases of iliopsoas haemophilic pseudotumours are presented . In one patient a fistula developed between a pseudotumour and the large bowel . This resulted in an abscess involving the pseudotumour and adjacent tissues . It resolved after 5 years of therapy involving percutaneous drainage and closure of the fistula . The second patient had a massive pseudotumour that had obstructed both ureters . Later he suffered a fatal mixed Gram negative septicaemia probably related to erosion into the colon.

Clin Exp Immunol, 2000 Feb, 119(2), 376 - 81
Increased levels of circulating soluble CD14 in Kawasaki disease; Takeshita S et al.; The CD14 molecule, which is known to be a receptor for endotoxin, is expressed on monocytes and neutrophils . It is found as a soluble CD14 (sCD14) in circulation, and the plasma level has been shown to be increased in some infectious diseases, including sepsis . To investigate the potential significance of circulating sCD14 in Kawasaki disease (KD), the plasma level of sCD14 was measured using ELISA in patients with KD, patients with a Gram-negative bacterial infection (GNBI) including sepsis, patients with viral infection (VI), and healthy controls . We also analysed CD14 receptor expression in monocytes and neutrophils using flow cytometry and a semiquantitative reverse transcription-polymerase chain reaction . Although KD patients had significantly lower counts of peripheral neutrophils and monocytes than GNBI patients, KD patients had significantly higher levels of sCD14 than GNBI . No significant correlations were observed between sCD14 levels and clinical laboratory values or the cytokine (interferon-gamma, tumour necrosis factor-alpha) levels in the acute phase . The mean intensity of CD14 receptor expression on neutrophils markedly increased in the acute phases of KD and GNBI compared with that in their convalescent phases, while that on monocytes decreased . The expression of CD14 mRNA in neutrophils increased in the acute phases of KD and GNBI, while that in monocytes did not decrease but instead remained quite abundant . The present findings suggest that the elevated level of circulating sCD14 appears to be an important parameter for KD and that sCD14 shedding is accompanied by different kinetics regarding the expression of CD14 antigen and CD14 gene between monocytes and neutrophils.

J Periodontol, 1999 Dec, 70(12), 1429 - 34
Association between periodontitis and hyperlipidemia: cause or effect?
Cutler CW, Shinedling EA, Nunn M, Jotwani R, Kim BO, Nares S, Iacopino AM.
BACKGROUND: Epidemiological studies suggest a relationship between periodontitis and coronary artery disease, but the mechanism has not been established . Recent studies in animals indicate that low dose endotoxin, as in a gram-negative infection, can induce hyperlipidemia and myeloid cell hyperactivity . The association between periodontitis, systemic exposure to Porphyromonas gingivalis, lipopolysaccharides (LPS), and hyperlipidemia has not been examined in humans . METHODS: Sera were obtained from 26 adult periodontitis patients and 25 healthy control (C) subjects selected from patients and staff . Serum antibodies against Porphyromonas gingivalis and its LPS were analyzed by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively . Serum triglycerides (TG) and cholesterol (CHOL) were assayed by a commercial laboratory . The associations between AP and blood levels of TG, CHOL, and anti-P . gingivalis whole cells and LPS were examined by logistic regression analysis . Peripheral blood polymorphonuclear leukocytes (PMNs) from 6 healthy fasted donors were incubated with purified TG (0.1 mg/ml) for 2 hours at 37 degrees C, stimulated with 100 ng/ml P . gingivalis LPS, and the release of IL-1beta measured by ELISA . RESULTS: The presence of periodontitis was significantly associated with age (odds ratio = 3.5, P = 0.04), elevated TG levels (odds ratio = 8.6, P = 0.0009), elevated CHOL levels (odds ratio = 7, P = 0.004), elevated ELISA titer (odds ratio = 35, P = 0.003) and reactivity with P . gingivalis LPS (odds ratio = 41, P = 0.001) . PMNs from all 6 healthy patients released modest levels of IL-1beta (10 to 60 pg/ml) when stimulated with 100 ng/ml P . gingivalis LPS . Addition of TG resulted in a significant increase (P <0.05) in IL- 1beta secreted that ranged from 7 to 150% over LPS alone . No IL-1beta was elicited by TG or vehicle alone . CONCLUSIONS: The results of this study indicate the presence of a significant relationship between periodontitis, hyperlipidemia, and serum antibodies against P . gingivalis LPS that warrants further examination in a larger patient population . Furthermore, these studies indicate that elevated triglycerides are able to modulate IL-1beta production by PMNs stimulated with P . gingivalis LPS.

Microbiology, 1999 Dec, 145 ( Pt 12), 3523 - 8
Absence in Helicobacter pylori of an uptake sequence for enhancing uptake of homospecific DNA during transformation; Saunders NJ et al.; Uptake sequences are abundant sequence motifs, often located downstream of ORFs, that are used to facilitate the within-species horizontal transfer of DNA . A frequent word analysis of the complete genome sequence of Helicobacter pylori strain 26685 was performed to search for and determine the identity of an uptake sequence in this species . The results demonstrated that Hel . pylori does not possess an uptake sequence . This is the first naturally transformable Gram-negative species shown to lack such a transformation-targeting system.

J Biol Chem, 2000 Jan 14, 275(2), 1337 - 43
A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus . Purification, characterization, and cDNA cloning; Lee SY et al.; A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish Pacifastacus leniusculus . The LGBP was purified by chromatography on Blue-Sepharose and phenyl-Sepharose, followed by Sephacryl S-200 . The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions, respectively . The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80 . LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP . Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bacteria-binding proteins and beta-1, 3-glucanases . Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia foetida . To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the beta-1,3-glucan laminarin in the hemocyte lysate of crayfish . The PO activity was blocked completely by the anti-LGBP antibody . Moreover, the PO activity could be recovered by the addition of purified LGBP . These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.

EMBO J, 2000 Jan 4, 19(1), 57 - 66
Translocation of group 1 capsular polysaccharide to the surface of Escherichia coli requires a multimeric complex in the outer membrane; Drummelsmith J et al.; Surface expression of the group 1 K30 capsular polysaccharide of Escherichia coli strain E69 (O9a:K30) requires Wza(K30), a member of the outer membrane auxiliary (OMA) protein family . A mutation in wza(K30) severely restricts the formation of the K30 capsular structure on the cell surface, but does not interfere with the biosynthesis or polymerization of the K30 repeat unit . Here we show that Wza(K30) is a surface-exposed outer membrane lipoprotein . Wza(K30) multimers form ring-like structures in the outer membrane that are reminiscent of the secretins of type II and III protein translocation systems . We propose that Wza(K30) forms an outer membrane pore through which the K30-capsular antigen is translocated . This is the first evidence of a potential mechanism for translocation of high molecular weight polysaccharide across the outer membrane . The broad distribution of the OMA protein family suggests a similar process for polysaccharide export in diverse Gram-negative bacteria.

Clin Diagn Lab Immunol, 2000 Jan, 7(1), 21 - 4
Species-specific monoclonal antibodies for rapid identification of Bartonella quintana; Liang Z et al.; Seven species-specific monoclonal antibodies (MAbs) to Bartonella quintana were produced and characterized . The MAbs were of the immunoglobulin G class and reacted only with 13 B . quintana strains in indirect microimmunofluorescence and Western immunoblotting assays . They did not react with eight other Bartonella spp., including Bartonella henselae, the most closely related species, and a selected MAb did also not react with nine other strains of gram-negative bacteria . The MAbs reacted mainly with a 34-kDa protein epitope of B . quintana which was shown to be species specific by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Four of five body lice experimentally infected with B . quintana were found to be positive for the organism in microimmunofluorescence assays with one MAb . These MAbs may provide a specific, simple, rapid, and low-cost tool for the identification of B . quintana and the diagnosis of infections due to the microorganism.

J Clin Microbiol, 2000 Jan, 38(1), 456 - 7
Peritonitis due to Roseomonas fauriae in a patient undergoing continuous ambulatory peritoneal dialysis; Bibashi E et al.; Roseomonas is a newly described genus of pink-pigmented, nonfermentative, gram-negative bacteria that have been recognized as a cause of human infections . Roseomonas fauriae is a species rarely isolated from clinical specimens . We report the first known case of peritonitis caused by R . fauriae in a patient receiving continuous ambulatory peritoneal dialysis.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15178 - 83
Effect of cellular filamentation on adventurous and social gliding motility of Myxococcus xanthus; Sun H et al.; Filamentous bacterial cells often provide biological information that is not readily evident in normal-size cells . In this study, the effect of cellular filamentation on gliding motility of Myxococcus xanthus, a Gram-negative social bacterium, was investigated . Elongation of the cell body had different effects on adventurous and social motility of M . xanthus . The rate of A-motility was insensitive to cell-body elongation whereas the rate of S-motility was reduced dramatically as the cell body got longer, indicating that these two motility systems work in different ways . The study also showed that filamentous wild-type cells glide smoothly with relatively straight, long cell bodies . However, filamentous cells of certain social motility mutants showed zigzag, tangled cell bodies on a solid surface, apparently a result of a lack of coordination between different fragments within the filaments . Further genetic and biochemical analyses indicated that the uncoordinated movements of these mutant filaments were correlated with the absence of cell surface fibril materials, indicating a possible new function for fibrils.

J Biol Chem, 1999 Dec 31, 274(53), 37731 - 5
Channel formation by FhaC, the outer membrane protein involved in the secretion of the Bordetella pertussis filamentous hemagglutinin; Jacob-Dubuisson F et al.; Many virulence factors of pathogenic microorganisms are presented at the cell surface . However, protein secretion across the outer membrane of Gram-negative bacteria remains poorly understood . Here we used the extremely efficient secretion of the Bordetella pertussis filamentous hemagglutinin (FHA) to decipher this process . FHA secretion requires a single specific accessory protein, FhaC, the prototype of a family of proteins necessary for the extracellular localization of various virulence proteins in Gram-negative bacteria . We show that FhaC is heat-modifiable and localized in the outer membrane . Circular dichroism spectra indicated that FhaC is rich in beta-strands, in agreement with structural predictions for this protein . We further demonstrated that FhaC forms pores in artificial membranes, as evidenced by single-channel conductance measurements through planar lipid bilayers, as well as by liposome swelling assays and patch-clamp experiments using proteoliposomes . Single-channel conductance appeared to fluctuate very fast, suggesting that the FhaC channels frequently assume a closed conformation . We thus propose that FhaC forms a specific beta-barrel channel in the outer membrane for the outward translocation of FHA.

Pediatr Emerg Care, 1999 Dec, 15(6), 410 - 1
Surfactant treatment in a pediatric burn patient with respiratory failure; Tortorolo L et al.; This report describes surfactant treatment in a burned infant with severe respiratory failure . In this patient the instillation of surfactant rapidly improved compliance, oxygen index (OI), and alveolar-capillary oxygen gradient (AaDO2), while the need for oxygen supplementation and peak positive pressure drastically decreased . The treatment was repeated after 12 hours . Although the baby had severe clinical course complications as a Gram-negative sepsis and a subglottic stenosis, she was weaned from oxygen therapy and mechanical ventilation in few weeks . Surfactant dysfunctions seem to play a central role in the respiratory insufficiency of burned patients, and its exogenous replacements could improve their outcome.

Zentralbl Bakteriol, 1999 Oct, 289(4), 415 - 28
An effective, rapid and simple method for isolation of Shiga toxin-producing Escherichia coli O26, O111 and O157 from faeces and food samples; Fukushima H et al.; An effective, rapid and simple method was developed for isolating Shiga toxin-producing Escherichia coli (STEC) O26:H11, O111:H- and O157:H7 from faeces and food in less than 24 h . The procedure involves enrichment of these samples in Trypticase soy broth (TSB) at 42 degrees C for 6 h . The enrichment culture is exposed to 1/8N HCl +0.5% NaCl solution (1 + 1) for 30 sec, then plated onto MacConkey agar containing sorbitol, tellurite and cefixime (CT-SMAC) following culture at 37 degrees C for 18 h . The findings were compared with conventional enrichment methods for efficiency in recovering STEC from bovine faeces . The new method increased the sensitivity for isolation of < 10(2) STEC O26:H11, O111:HUT and O157:H7 CFU/g of bovine faeces and decreased the growth of other gram-negative microorganisms . This procedure is readily implemented for use in laboratories doing routine microbiological testing.

Zentralbl Bakteriol, 1999 Oct, 289(4), 389 - 97
Intracellular bacteria of Acanthamoebae resembling Legionella spp . turned out to be Cytophaga sp; Muller KD et al.; Acanthamoeba sp . isolated from the drinking water system of a hospital harboured gram-negative bacteria multiplying inside phagosomes and within the cytoplasm of their host cells . According to their morphology demonstrated by electron microscopy they resembled Llaps (Legionella-like amoebal pathogens) but turned out to be Cytophaga sp . as shown by a typical profile of cellular fatty acids obtained by means of gas-liquid chromatography.

Clin Exp Immunol, 2000 Jan, 119(1), 77 - 83
Pretreatment of mice with lipopolysaccharide (LPS) or IL-1beta exerts dose-dependent opposite effects on Shiga toxin-2 lethality; Palermo M et al.; Haemolytic uraemic syndrome (HUS) has been closely associated with infection with a group of Shiga toxin-producing enterohaemorrhagic Eschericchia coli in young children . Shiga toxins (Stx) have been implicated as pathogenic agents of HUS by binding to the surface receptor of endothelial cells . LPS is a central product of the Gram-negative bacteria and several reports have documented that both LPS and Stx are important for disease development . In this study the reciprocal interactions between LPS and Stx2 are analysed in a mouse model . The results demonstrated that LPS was able to reduce or enhance Stx2 toxicity, depending on the dose and the timing of the injection . The involvement of the main early cytokines induced by LPS, tumour necrosis factor alpha (TNF-alpha) and IL-1beta, in those LPS opposite effects on Stx2 toxicity was evaluated . Stx2 toxicity was enhanced by in vivo injection of murine TNF-alpha and low doses of murine IL-1beta . However, at higher doses of IL-1beta which induced corticosteroid increase in serum, Stx2 lethality was decreased . Considering that dexamethasone and IL-1beta reproduce the LPS protective effects, it is suggested that endogenous corticosteroids secondary to the inflammatory response induced by LPS, mediate the protection against Stx2 . It can be concluded that the fine equilibrium between proinflammatory and anti-inflammatory activities strongly influences Stx2 toxicity.

J Immunol, 2000 Jan 1, 164(1), 43 - 8
Human intestinal mast cells are capable of producing different cytokine profiles: role of IgE receptor cross-linking and IL-4; Lorentz A et al.; Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines . So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF) . We observed that these cells express the proinflammatory cytokines TNF-alpha, IL-1 beta, IL-6, IL-8, IL-16, and IL-18 without further stimulation . Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-alpha . Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking . If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone . By contrast, IL-6 expression was completely blocked in response to culture with IL-4 . In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines . IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.

Br J Cancer, 1999 Dec, 81(8), 1311 - 7
The role of endotoxin/lipopolysaccharide in surgically induced tumour growth in a murine model of metastatic disease; Pidgeon GP et al.; Surgical removal of a primary tumour is often followed by rapid growth of previously dormant metastases . Endotoxin or lipopolysaccharide, a cell wall constituent of Gram-negative bacteria, is ubiquitously present in air and may be introduced during surgery . BALB/c mice received a tail vein injection of 10(5) 4T1 mouse mammary carcinoma cells . Two weeks later, animals were subjected to surgical trauma or an intraperitoneal injection of endotoxin (10 microg per animal) . Five days later, animals which underwent open surgery, laparoscopy with air sufflation or received an endotoxin injection displayed increased lung metastasis compared to anaesthetic controls . These increases in metastatic tumour growth were reflected in increased tumour cell proliferation and decreased apoptosis within lung metastases . Circulating levels of the angiogenic cytokine, vascular endothelial growth factor (VEGF), were also elevated in these groups and correlated with increased plasma levels of endotoxin . Endotoxin treatment for 18 h (>10 ng ml(-1)) directly up-regulated VEGF production by the 4T1 tumour cells in vitro . Metastatic tumour growth in mice undergoing carbon dioxide laparoscopy, where air is excluded, was similar to anaesthetic controls . These data indicate that endotoxin introduced during surgery is associated with the enhanced growth of metastases following surgical trauma, by altering the critical balances governing cellular growth and angiogenesis.

Infect Immun, 2000 Jan, 68(1), 342 - 51
In vitro Brucella suis infection prevents the programmed cell death of human monocytic cells; Gross A et al.; During the complex interaction between an infectious agent and a host organism, the pathogen can interfere with the host cell's programmed death to its own benefit . Induction or prevention of host cell apoptosis appears to be a critical step for determining the infection outcome . Members of the gram-negative bacterial genus Brucella are intracellular pathogens which preferentially invade monocytic cells and develop within these cells . We investigated the effect of Brucella suis infection on apoptosis of human monocytic phagocytes . The present study provides evidence that Brucella infection inhibited spontaneously occurring apoptosis in human monocytes . Prevention of monocyte apoptosis was not mediated by Brucella lipopolysaccharide and required bacterial survival within infected cells . Both invaded and noninvaded cells were protected, indicating that soluble mediators released during infection were involved in the phenomenon . Analysis of Brucella-infected monocytes revealed specific overexpression of the A1 gene, a member of the bcl-2 family implicated in the survival of hematopoietic cells . Brucella infection also rendered macrophage-like cells resistant to Fas ligand- or gamma interferon-induced apoptosis, suggesting that Brucella infection protected host cells from several cytotoxic processes occurring at different steps of the immune response . The present data clearly show that Brucella suis modulated the monocyte/macrophage's apoptotic response to the advantage of the pathogen, thus preventing host cell elimination . This might represent a strategy for Brucella development in infected hosts.

Eur J Immunol, 2000 Jan, 30(1), 211 - 6
Binding of lipopolysaccharide (LPS) to CHO cells does not correlate with LPS-induced NF-kappaB activation; Hamann L et al.; Activation of myeloid cells by lipopolysaccharide (LPS) is a key event in the development of gram-negative sepsis . One crucial step within this process is the binding of LPS to CD14 . CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein requiring at least one additional membrane-spanning molecule for signal transduction . It is not clear whether the function of CD14 is to merely catalyze LPS binding, followed by the interaction of LPS with the signal transducer, or whether CD14 has a more specific function and may be a part of the signaling complex . To address this question we generated Chinese hamster ovary (CHO) cells expressing a human GPI-anchored form of LPS-binding protein (mLBP) to substitute for CD14 as LPS acceptor molecule . By comparison of CHO / mLBP with CHO / vector and CHO / CD14 cells we found that expression of GPI-linked LBP results in an enhanced binding of LPS but not in an increase in cell activation as determined by translocation of NF-kappaB . Furthermore, excess of recombinant soluble LBP resulted also in increased LPS binding without affecting NF-kappaB translocation . These data show that LPS binding alone is not sufficient to induce signaling . We conclude that CD14 is more than a catalyst for LPS binding: it seems to be directly involved in LPS signaling and thus appears to be an essential part of the signaling complex.

Neuroimmunomodulation, 2000, 7(1), 6 - 15
Inhibitory effects of endotoxin on LH secretion in the ovariectomized monkey are prevented by naloxone but not by an interleukin-1 receptor antagonist; Xiao E et al.; Endotoxin (lipopolysaccharides, LPS), the pathogenic moiety of gram-negative bacteria, is a well-known trigger for the central release of cytokines . The objective of this study is to evaluate the effects of systemic endotoxin administration on LH and cortisol secretion in a non-human primate model and to investigate whether these endocrine effects are mediated by centrally released interleukin-1 (IL-1) using the receptor antagonist to IL-1 (IL-1ra) . An additional objective is to investigate whether endogenous opioid peptides mediate these endocrine effects of LPS, using the opiate antagonist naloxone . The experiments were performed in long-term-ovariectomized rhesus monkeys . Blood samples for hormone determination were obtained at 15-min intervals for a period of 8 h, which included a 3-hour baseline period . Since the effective central dose of IL-1ra in the monkey was unknown, in the first experiment we tested the potency of several doses of this antagonist in preventing the effects of centrally administered IL-1alpha, a cytokine which is known to inhibit LH and stimulate cortisol release . Rhesus monkeys received a 30-min intracerebroventricular infusion of IL-1alpha (4.2 microg/30 min) alone or together with various doses of IL-1ra (30-180 microg/h i.c.v.) . IL-1ra infusion was initiated 1 h before IL-1 and extended over the experimental period . As previously reported, IL-1alpha induced a significant inhibition of LH, to 36.5 +/- 3.3% (mean +/- SE) by 5 h as a percentage from the 3-hour baseline . This inhibitory effect was reversed by cotreatment with the 180 microg/h dose of IL-1ra (to 82.5 +/- 3.8% by 5 h; NS vs . saline) but not with the lower doses . IL-1 stimulated cortisol release to 165.9 +/- 7.7%, but this increase was prevented by IL-1ra (66.6 +/- 8.9%; p < 0.05 vs . IL-1, NS vs . saline) . In the second experiment, LPS (50 microg) was administered intravenously, alone or in combination with intracerebroventricular IL-1ra infusion . LPS induced a significant decrease in LH secretion (to 57.1 +/- 5.2%) . These effects were not reversed by intracerebroventricular administration of IL-1ra (52.5 +/- 9.6%) . Cortisol secretion increased in response to LPS, but this stimulatory effect was not affected by IL-1ra (178.3 +/- 13.4 vs . 166.9 +/- 5.7%) . There were no effects of IL-1ra alone . In experiment 3, we investigated whether the opiate antagonist naloxone reverses the endocrine effects of endotoxin . Both LPS (50 microg) and naloxone (5-mg bolus + 5 mg/h) were infused intravenously . Naloxone was effective in preventing the inhibitory effect of LPS on LH (to 124.6 +/- 22.1%, NS vs . saline) but not the increase in cortisol (to 166.7 +/- 16.7%; p < 0.05 vs . saline, NS vs . LPS) . Naloxone alone has no significant effect on LH or cortisol secretion . These data demonstrate that, in the ovariectomized monkey, a systemic inflammatory/immune- like stress challenge acutely inhibits tonic LH secretion while concomitantly stimulating cortisol release . Although endotoxin is known to affect central cytokine release, these endocrine effects do not require a mediatory role of central IL-1 in the primate . In contrast, endogenous opioid pathways appear to be involved in this process .

Immunopharmacology, 1999 Sep, 43(2-3), 225 - 33
Correlation of kinin generating activity with Helicobacter pylori-associated gastric infection; Naidoo S et al.; The kallikrein-kinin system involves a biologically complex set of interactive proteases that signal the first-line onset of inflammation and associated cellular processes . The basic enzymatic cleavage of kininogen substrate by the serine protease tissue kallikrein to liberate kinins is regulated by a number of factors . These may include the recently discovered bacterial involvement in the causation of gastritis . The gram-negative Helicobacter pylori organism, colonises the human gastric epithelium and initiates ulcerogenesis and may induce, in the longer term, tumour formation . The aim of this study was to investigate the role of kinins in H . pylori-induced gastric dyspepsia . During endoscopic examination, lavage aspirates of 23 patients were collected, and the tissue kallikrein content measured by a kinin-generating assay and an enzyme-linked immunosorbent assay . Gastric antral and pyloric biopsy tissue was histologically examined for degrees of inflammation and H . pylori infection, and then immunolabelled for tissue kallikrein and kinin receptors . Results show that labelled tissue kallikrein in the fundic glands and parietal cells of the diseased antrum was elevated with increasing severity of gastritis . Further, kinin-generating potential of the lavage fluid appeared to be greater with increasing evidence of infection . Tissue kallikrein immunosorbent assay levels were significantly raised in patients showing mild to moderate H . pylori infection . One outcome of this study may be the inclusion of kinin antagonists in management of gastric dyspepsia.

Microbiol Immunol, 1999, 43(9), 853 - 61
The gene encoding the prepilin peptidase involved in biosynthesis of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli; Taniguchi T et al.; The assembly of pilus colonization factor antigen III (CFA/III) of human enterotoxigenic Escherichia coli requires the processing of CFA/III major pilin (CofA) by a peptidase, likely another type IV pilus formation system . Western blot analysis of CofA reveals that CofA is produced initially as a 26.5-kDa preform pilin (prepilin) and then processed to 20.5-kDa mature pilin by a prepilin peptidase . This processing is essential for exportation of the CofA from the cytoplasm to the periplasm . In this experiment, the structural gene, cofP, encoding CFA/III prepilin peptidase which cleavages at the Gly-30-Met-31 junction of CofA was identified, and the nucleotide sequence of the gene was determined . CofP consists of 819 bp encoding a 273-amino acid protein with a relative molecular mass of 30,533 Da . CofP is predicted to be localized in the inner membrane based on its hydropathy index . The amino acid sequence of CofP shows a high degree of homology with other prepilin peptidases which play a role in the assembly of type IV pili in several gram-negative bacteria.

J Biol Chem, 1999 Dec 17, 274(51), 36693 - 9
Activation of Helicobacter pylori VacA toxin by alkaline or acid conditions increases its binding to a 250-kDa receptor protein-tyrosine phosphatase beta; Yahiro K et al.; Helicobacter pylori, a Gram-negative gastric bacterium, secretes VacA, a cytotoxin that causes vacuolar degeneration of susceptible cells . Velocity sedimentation analysis showed that treatment of VacA at alkaline pH led to disassembly of VacA oligomers, an observation reported previously for acid-treated VacA . Exposure of VacA to acid or alkali increased its binding to AZ-521 cells, as shown by indirect immunofluorescence and flow cytometry . Moreover, immunoprecipitates with polyclonal antibodies against VacA from AZ-521 cells previously exposed to acid- or alkali-treated VacA had a 250-kDa glycoprotein containing galactose-beta(1-3)-N-acetylgalactosamine and galactose-beta(1-4)-N-acetylglucosamine . p250, purified by chromatography on peanut agglutinin affinity and Superose 6 columns, contained N-terminal and internal amino acid sequences of YRQQRKLVEEIGWSYT and LIIQDHILEATQDDY, respectively . These sequences are identical to those of a receptor protein-tyrosine phosphatase (RPTPbeta/PTPzeta); in agreement, p250 reacted with anti-human RPTPbeta monoclonal antibody . Immunoprecipitation with anti-human RPTPbeta antibody of solubilized membrane preparations previously incubated with VacA or heat-inactivated VacA demonstrated that RPTPbeta bound native, but not denatured, VacA . Acidic and alkaline treatments were associated with activation of VacA and increased binding to the cell surface RPTPbeta.

J Biol Chem, 1999 Dec 17, 274(51), 36579 - 84
Deacylation of lipopolysaccharide in whole Escherichia coli during destruction by cellular and extracellular components of a rabbit peritoneal inflammatory exudate; Katz SS et al.; Deacylation of purified lipopolysaccharides (LPS) markedly reduces its toxicity toward mammals . However, the biological significance of LPS deacylation during infection of the mammalian host is uncertain, particularly because the ability of acyloxyacyl hydrolase, the leukocyte enzyme that deacylates purified LPS, to attack LPS residing in the bacterial cell envelope has not been established . We recently showed that the cellular and extracellular components of a rabbit sterile inflammatory exudate are capable of extensive and selective removal of secondary acyl chains from purified LPS . We now report that LPS as a constituent of the bacterial envelope is also subject to deacylation in the same inflammatory setting . Using Escherichia coli LCD25, a strain that exclusively incorporates radiolabeled acetate into fatty acids, we quantitated LPS deacylation as the loss of radiolabeled secondary (laurate and myristate) and primary fatty acids (3-hydroxymyristate) from the LPS backbone . Isolated mononuclear cells and neutrophils removed 50% and 20-30%, respectively, of the secondary acyl chains of the LPS of ingested whole bacteria . When bacteria were killed extracellularly during incubation with ascitic fluid, no LPS deacylation occurred . In this setting, the addition of neutrophils had no effect, but addition of mononuclear cells resulted in removal of >40% of the secondary acyl chains by 20 h . Deacylation of LPS was always restricted to the secondary acyl chains . Thus, in an inflammatory exudate, primarily in mononuclear phagocytes, the LPS in whole bacteria undergoes substantial and selective acyloxyacyl hydrolase-like deacylation, both after phagocytosis of intact bacteria and after uptake of LPS shed from extracellularly killed bacteria . This study demonstrates for the first time that the destruction of Gram-negative bacteria by a mammalian host is not restricted to degradation of phospholipids, protein, and RNA, but also includes extensive deacylation of the envelope LPS.

J Appl Microbiol, 1999 Sep, 87(3), 438 - 46
Nisin, temperature and pH effects on growth and viability of pectinatus frisingensis, a gram-negative, strictly anaerobic beer-spoilage bacterium
Chihib N, Monnerat L, Membre JM, Tholozan J.
Pectinatus frisingensis, a Gram-negative and strictly anaerobic beer spoilage bacterium is sensitive to nisin . An increase in nisin concentration (0 to 1100 IU ml-1) added to the culture medium prolonged the lag phase, and decreased the growth rate of the bacterium . In addition, late exponential cells of P . frisingensis exposed to low concentrations of nisin lost immediately a part of their intracellular K+ . Presence of Mg2+ up to 15 mmol l-1 did not protect P . frisingensis from nisin-induced loss of viability and K+ efflux . Potassium leaks were also measured in P . frisingensis late exponential phase cells exposed to combined effects of nisin addition (100-500 IU ml-1), 10 min mild heat-treatment (50 degrees C) or rapid cooling (2 degrees C), and pH (4.0 and 6.2) . Net K+ efflux from both starving and glucose-metabolizing cells, was more important at pH 6.2, whatever the temperature treatment and nisin addition . Reincubation at 30 degrees C of P . frisingensis glucose-metabolizing cells exposed to a preliminary combination of nisin addition and mild heat or cooling down treatment, showed that cells exposed to rapid cooling reaccumulated more K+ than heat-treated cells, whatever the pH conditions . A combination of nisin and mild heat-treatment could thus be of interest to prevent P . frisingensis growth in beers.

Arch Surg, 1999 Dec, 134(12), 1293 - 8; discussion 1298-9
Analysis of aminoglycosides in the treatment of gram-negative infections in surgical patients; Crabtree TD et al.; HYPOTHESIS: Antibiotic regimens containing aminoglycosides result in a similar outcome compared with non-aminoglycoside regimens in the treatment of gram-negative infections in surgical patients . DESIGN: An inception cohort study of hospitalized surgical patients from December 1, 1996, through September 30, 1998 . Patients were observed from the time of diagnosis of infection to discharge . SETTING: University hospital . PATIENTS: Two hundred fifty-eight consecutive gram-negative infections occurring in general surgical and trauma patients and patients undergoing transplantation . Sixty-six patients received aminoglycosides as a component of their treatment regimen, whereas 192 received other agents . RESULTS: Patients treated with aminoglycosides were younger (mean +/- SEM age, 48+/-2 vs 53+/-1 years; P = .04 by univariate analysis) and had a similar APACHE II (Acute Physiology and Chronic Health Evaluation II) score (mean +/- SEM, 17+/-1 vs 15+/-1; P = .10), yet had a significantly higher mortality vs patients treated with other agents (29% vs 14%; P = .02) . A larger proportion of patients requiring hemodialysis were treated with aminoglycosides (33% vs 13%; P = .001) . Although there was no difference in the sites of infection between groups, surgical patients with gram-negative pneumonia had a higher mortality when treated with aminoglycosides (37% vs 18%; P = .04), despite similar APACHE II scores (mean +/- SEM, 20+/-1 vs 18+/-1; P = .40) . CONCLUSIONS: Despite a younger age and similar severity of illness, patients with gram-negative infections treated with aminoglycosides were associated with a higher mortality rate, although this may be related to selection bias in the use of aminoglycoside agents . The mortality rate associated with gram-negative pneumonia was also higher in patients treated with aminoglycosides, despite a similar severity of illness . Future randomized studies are necessary to reanalyze the role of aminoglycosides in treating surgical patients with gram-negative infections, particularly pneumonia.

Science, 1999 Dec 10, 286(5447), 2144 - 7
Microorganisms in the accreted ice of Lake Vostok, Antarctica; Karl DM et al.; Analysis of a portion of Vostok ice core number 5G, which is thought to contain frozen water derived from Lake Vostok, Antarctica (a body of liquid water located beneath about 4 kilometers of glacial ice), revealed between 2 x 10(2) and 3 x 10(2) bacterial cells per milliliter and low concentrations of potential growth nutrients . Lipopolysaccharide (a Gram-negative bacterial cell biomarker) was also detected at concentrations consistent with the cell enumeration data, which suggests a predominance of Gram-negative bacteria . At least a portion of the microbial assemblage was viable, as determined by the respiration of carbon-14-labeled acetate and glucose substrates during incubations at 3 degrees C and 1 atmosphere . These accreted ice data suggest that Lake Vostok may contain viable microorganisms.

Microbiol Immunol, 1999, 43(10), 937 - 46
Characterization of an outer membrane protein gene, pgmA, and its gene product from Porphyromonas gingivalis; Hongo H et al.; A gene upstream from fimA, the gene encoding fimbrilin, on the chromosome of Porphyromonas gingivalis was sequenced and shown to be the gene encoding an outer membrane protein in this organism based on homology and biochemical analyses . Therefore, the gene (formerly ORF5) was designated pgmA, the P . gingivalis outer membrane protein A gene . The gene product, PgmA, was sensitive to protease, and was detected as a 60-kDa protein from wild-type strains with trichloroacetic acid treatment, which was carried out to destroy intrinsic proteases, and from protease-deficient mutants without this treatment prior to electrophoresis . PgmA was indeed present in the membrane fraction . Its nature was determined to be that of outer membrane proteins in gram-negative bacteria based on attempts at differential extraction of inner membrane proteins with detergents . No evidence has been found thus far from functional analyses that this protein is related to fimbrial morphogenesis and functions or to serum resistance of this organism.

Eur J Clin Microbiol Infect Dis, 1999 Oct, 18(10), 723 - 5
Outbreak of bloodstream infections associated with dialysis machine waste ports in a hemodialysis facility; Block C et al.; Eight cases of gram-negative bloodstream infection without a clinically evident source occurred at a hemodialysis unit in Jerusalem between February and September 1997 . All infections could be traced to three of the 13 dialysis machines in use . Epidemiological investigation, including pulsed-field gel electrophoretic characterization of organisms isolated from the patients and dialysis machines, implicated the Waste Handling Option system of the machines as the source of the infections . Discontinuation of the Waste Handling Option system use was associated with prompt cessation of the outbreak.

Urol Clin North Am, 1999 Nov, 26(4), 687 - 99
Gram-negative bacterial sepsis and the sepsis syndrome; Lazaron V et al.; Gram-negative sepsis syndrome is an increasingly common complication in medical and surgical patients . The molecular and cellular mechanisms underlying this dreaded complication are yielding to investigation . These studies have led to a multiplicity of targets for novel therapies . Despite highly promising results in many animal studies, clinical studies have been disappointing.

Br J Surg, 1999 Nov, 86(11), 1410 - 4
Kupffer cell blockade, tumour necrosis factor secretion and survival following endotoxin challenge in experimental biliary obstruction; Kennedy JA et al.; BACKGROUND: Gram-negative sepsis and its sequelae frequently complicate invasive procedures in patients with obstructive jaundice . In response to endotoxin, Kupffer cells secrete tumour necrosis factor (TNF), a pivotal early mediator of sepsis . An investigation was carried out into the specific role of Kupffer cell TNF secretion following endotoxin challenge in obstructive jaundice . METHODS: Survival following intraperitoneal administration of endotoxin (2.0, 0.02 and 0.0002 mg per 100 g) was determined in rats following bile duct ligation (BDL) or sham operation . Plasma TNF concentration was quantified following endotoxin administration (0.0002 mg per 100 g) at 1, 2 and 6 h . Subsequently, the effect of Kupffer cell blockade by gadolinium chloride on survival and plasma TNF concentration was assessed . RESULTS: Jaundiced animals showed a significantly increased mortality rate following intraperitoneal injection of endotoxin 2.0 mg per 100 g (BDL 100 per cent versus sham 0 per cent) and 0.02 mg per 100 g (BDL 70 per cent versus sham 0 per cent; P = 0 . 002, Fisher's exact test) . Median plasma TNF concentration was significantly greater in jaundiced animals 1 h after endotoxin administration (BDL 943 (interquartile range (i.q.r.) 211-3900) pg/ml versus sham 64 (i.q.r . 47-127) pg/ml; P = 0.002, Mann-Whitney U test) . Kupffer cell blockade with gadolinium chloride increased the survival rate following endotoxin administration in BDL animals (BDL-GdCl3 100 per cent versus BDL-saline 40 per cent; P = 0.0003, Fisher's exact test) and decreased median plasma levels of TNF (BDL-GdCl3 88 (i.q.r . 0-1065) pg/ml versus BDL-saline 16 550 (1255-29 360) pg/ml; P = 0.002, Mann-Whitney U test) . CONCLUSION: Kupffer cell blockade improved survival and suppressed systemic TNF activity after endotoxin challenge . In obstructive jaundice, hypersecretion of TNF by Kupffer cells may supplement systemic cytokine production and be responsible for significant complications.

FEMS Immunol Med Microbiol, 1999 Dec, 26(3-4), 197 - 202
New insights into the role of serum amyloid P component, a novel lipopolysaccharide-binding protein; de Haas CJ; Serum amyloid P component (SAP) is a highly preserved plasma protein named for its ubiquitous presence in amyloid deposits . Although SAP is described to bind many ligands, no clear biological function has been ascribed to it as yet . This review summarizes the current knowledge about the protein SAP, its ligands and functional properties . Finally, the author focuses on the recent finding of the binding of SAP to lipopolysaccharide (LPS) and Gram-negative bacteria and the possible functional consequences of these interactions.

Eur J Surg, 1999 Oct, 165(10), 979 - 85
Pulmonary dynamics of radiolabelled erythrocytes and leucocytes in early gram-negative sepsis in pigs; Walther S et al.; OBJECTIVE: To study the pulmonary dynamics of erythrocytes and leucocytes in vivo in early experimental sepsis . DESIGN: Open, experimental study . SETTING: Academic research laboratory, Sweden . MATERIAL: 10 adolescent domestic pigs . INTERVENTIONS: Technetium (99mTc) labelling of erythrocytes (n = 5), and indium (111In) labelling of autologous leucocytes (n = 10) . Sepsis was induced by endotoxin (n = 4) or live Escherichia coli (n = 3), given intravenously . MAJOR OUTCOME MEASURES: Regional pulmonary scintigraphy, central haemodynamics, and gas exchange followed for 180 minutes . RESULTS: Septic animals developed arterial hypoxia, pulmonary hypertension, and systemic hypotension . They also had an early increase in mean (SD) regional pulmonary erythrocyte and leucocyte counts {+10.3 (7.7)% and +12.0 (3.5)%, respectively} with a simultaneous maximum 27-32 minutes after the start of the septic insult . CONCLUSIONS: The immediate sepsis-induced pulmonary accumulation of leucocytes as detected by external scintigraphy can be ascribed at least in part to a simultaneous sepsis-induced increase in pulmonary blood volume.

Vet Clin North Am Food Anim Pract, 1999 Nov, 15(3), 473 - 86, v
Metabolic acidosis in calves; Kasari TR; In neonatal calves metabolic acidosis is a common sequela to diarrhea-induced dehydration and endotoxemia in the aftermath of gram-negative bacterial infections . Without treatment, metabolic acidosis is a prime factor in the death of many of these calves . This article begins with a general discussion about the causes and recognition of metabolic acidosis . The remaining sections detail the subjective and objective methods available to assess the severity of acidosis and treatment options for this metabolic condition.

Biochim Biophys Acta, 1999 Oct 18, 1472(1-2), 13 - 24
Purification and characterization of a natural agglutinin from the serum of the hermit crab Diogenes affinis; Murali S et al.; A natural agglutinin from the serum of the hermit crab Diogenes affinis was purified to homogeneity by a single-step affinity chromatography using N-acetylglucosamine-coupled Sepharose 6B . The purified serum agglutinin (PSA) showed a strong affinity for rat RBC, and its hemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA . PSA in active form has a molecular mass estimate of 185 kDa and is composed of four non-identical subunits (51, 49, 42 and 39 kDa) cross-linked by interchain disulfide bonds . The homogeneity of PSA was corroborated by immunodiffusion and immunoelectrophoretic analyses using rabbit antiserum raised against the agglutinin . The antibodies in this antiserum appear to be specific for RBC-binding sites of the agglutinin molecules as revealed by the ability of the antiserum to neutralize HA activities of both whole serum and PSA of D . affinis . In HA-inhibition assays performed with several carbohydrates and glycoproteins, PSA showed a distinct and unique specificity for acetyl group in carbohydrates independently of the presence of this group on C-2 or C-5 and its stereochemical arrangement in the axial or equatorial orientation . Besides, this agglutinin appears to recognize the terminal N- and O- acetyl groups in the oligosaccharide chain of glycoconjugates . The HA activity of D . affinis agglutinin was also susceptible to inhibition by lipopolysaccharides from diverse gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections.

Pathol Int, 1999 Oct, 49(10), 849 - 52
Infectious etiology of xanthogranulomatous cholecystitis: immunohistochemical identification of bacterial antigens in the xanthogranulomatous lesions; Mori M et al.; The clinicopathologic features of 31 surgical patients with xanthogranulomatous cholecystitis were analysed in relation to the immunohistochemical demonstration of bacterial antigens using a polyclonal anti-E . coli antibody . The time period after the initial clinical manifestation was critical for identifying the bacterial antigens in the cytoplasm of foamy macrophages . Of 12 lesions removed within 4 weeks of the onset (subacute group), 7 lesions showed positive staining . No positivity was seen in 19 gall-bladder specimens with a clinical course of more than 4 weeks (chronic group) . Abscess formation was seen in 7 cases in the subacute group, 5 of which were positive for the bacterial antigens . Gram-negative bacteria were cultured from the bile in four in the subacute group and three in the chronic group . All the four culture-positive subacute lesions were immunoreactive for the bacterial antigens . Accumulation of ceroid pigment in the cytoplasm of foamy macrophages was characteristically seen in 16 of 20 chronic lesions . Three subacute lesions with ceroid pigmentation was negative for the bacterial antigens . In conclusion, xanthogranulomatous cholecystitis can be divided into two forms: subacute and chronic, and the subacute form is closely related to bacterial infection.

Kidney Int, 1999 Nov, 56(5), 1893 - 8
Nanobacteria: an infectious cause for kidney stone formation; Ciftcioglu N et al.; BACKGROUND: Nanobacteria are cytotoxic, sterile-filterable, gram-negative, atypical bacteria detected in bovine and human blood . Nanobacteria produce carbonate apatite on their cell walls . Data on Randall's plaques suggest that apatite may initiate kidney stone formation . We assessed nanobacteria in 72 consecutively collected kidney stones from Finnish patients . METHODS: Nanobacteria and kidney stone units were compared using scanning electron microscopy (SEM) . Demineralized kidney stones were screened for nanobacteria using a double-staining method and a specific culture method . Isolated nanobacteria were analyzed for mineral formation in vitro with Ca and 85Sr incorporation tests . RESULTS: SEM highlighted the resemblance in size and morphology of nanobacteria and the smallest apatite units in the kidney stones . Nanobacterial antigens could be detected after the demineralization of the stones in 1 N HCl . Nanobacteria were surprisingly resistant to this treatment, and cultures could be established from 93.1% of the stones . Only struvite stones had common bacteria, in addition to the nanobacteria . When the results of all of the assays were combined, 70 of the 72 stones (that is, 97.2%) were nanobacteria positive . Although apatite stones indicated highest nanobacteria antigen signals, the overall nanobacteria positivity did not depend on the stone type . The isolated nanobacteria produced apatite stones in vitro, measured by Ca and 85Sr incorporation . CONCLUSIONS: We propose that kidney stone formation is a nanobacterial disease analogous to Helicobacter pylori infection and peptic ulcer disease . Both diseases are initiated by bacterial infection and subsequently endogenous and dietary factors influence their progression.

Eur J Biochem, 1999 Dec, 266(2), 477 - 83
Effects of ligand binding on the internal dynamics of maltose-binding protein; Doring K et al.; Ligand binding to proteins often causes large conformational changes . A typical example is maltose-binding protein (MBP), a member of the family of periplasmic binding proteins of Gram-negative bacteria . Upon binding of maltose, MBP undergoes a large structural change that closes the binding cleft, i.e . the distance between its two domains decreases . In contrast, binding of the larger, nonphysiological ligand beta-cyclodextrin does not result in closure of the binding cleft . We have investigated the dynamic properties of MBP in its different states using time-resolved tryptophan fluorescence anisotropy . We found that the 'empty' protein exhibits strong internal fluctuations that almost vanish upon ligand binding . The measured relaxation times corresponding to internal fluctuations can be interpreted as originating from two types of motion: wobbling of tryptophan side-chains relative to the protein backbone, and orientational fluctuations of entire domains . After binding of a ligand, domain motions are no longer detectable and the fluctuations of some of the tryptophan side-chains become rather restricted . This transformation into a more rigid state is observed upon binding of both ligands, maltose and the larger beta-cyclodextrin . The fluctuations of tryptophan side-chains in direct contact with the ligand, however, are affected in a slightly different way by the two ligands.

Dev Biol Stand, 1999, 101, 131 - 9
Detection of endotoxins and other pyrogens using human whole blood; Fennrich S et al.; When cells of the immune system, i.e . primarily blood monocytes and macrophages, come into contact with pyrogens (fever-inducing contaminations) they release mediators transmitting the fever reaction through the organism to the thermoregulatory centres of the brain . The new test discussed here exploits this reaction for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample . If there is pyrogen contamination, the endogenous pyrogen interleukin-1 is released, which is then determined by ELISA . According to the pharmacopoeia, the rabbit pyrogen test determines the fever reaction following injection of a test sample . In comparison, the new whole blood assay is more sensitive, less expensive and determines the reaction of the targeted species . Compared to the well established in vitro alternative, i.e . the limulus amebocyte lysate assay (LAL), the new blood assay is not restricted to endotoxins of gram-negative bacteria, it is not affected by endotoxin-binding blood proteins and it reflects the potency of different endotoxin preparations in mammals . Here, interim results of the ongoing optimization and pre-validation are reported and the present state of the evaluation for biological and pharmaceutical drugs are presented.

J Clin Microbiol, 1999 Dec, 37(12), 4161 - 2
Rahnella aquatilis sepsis in an immunocompetent adult; Chang CL et al.; Rahnella aquatilis, a rare enteric gram-negative rod which is infrequently isolated in immunocompromised patients, was isolated as a causative organism of sepsis in a 26-year-old immunocompetent male patient . The contaminated intravenous fluid was confirmed to be the source of the organism.

J Clin Microbiol, 1999 Dec, 37(12), 3888 - 95
Western and dot blotting analyses of Ehrlichia chaffeensis indirect fluorescent-antibody assay-positive and -negative human sera by using native and recombinant E . chaffeensis and E . canis antigens; Unver A et al.; Human monocytic ehrlichiosis is an emerging infectious disease caused by Ehrlichia chaffeensis, a gram-negative obligatory intracellular bacterium closely related to E . canis . The immunoreactive recombinant fusion proteins rP28 and rP30 have become available after cloning and expressing of the 28- and 30-kDa major outer membrane protein genes of E . chaffeensis and E . canis, respectively . Western immunoblotting was performed to analyze the antibody responses of the 37 E . chaffeensis indirect fluorescent-antibody assay (IFA)-positive and 20 IFA-negative serum specimens with purified whole organisms, rP28, and rP30 . All IFA-negative sera were negative with purified whole organisms, rP28, or rP30 by Western immunoblot analysis (100% relative diagnostic specificity) . Of 37 IFA-positive sera, 34 sera reacted with any native proteins of E . chaffeensis ranging from 44 to 110 kDa, and 30 sera reacted with 44- to 110-kDa native E . canis antigens . The 28-kDa E . chaffeensis and 30-kDa E . canis native proteins were recognized by 25 IFA-positive sera . Fifteen IFA-positive sera reacted with rP28 by Western blot analysis, whereas 34 IFA-positive sera reacted with rP30 (92% relative diagnostic specificity), indicating that rP30 is more sensitive than rP28 for detecting the antibodies in IFA-positive sera . These 34 IFA-positive sera were positive by the dot blot assay with rP30, distinguishing them from IFA-negative sera . Except for three rP30-negative but IFA-positive specimens that instead showed an E . ewingii infection-like profile by Western immunoblotting, the results of Western and dot blot assays with rP30 matched 100% with the IFA test results . Densitometric analysis of dot blot reactions showed a positive correlation between the dot density and the IFA titer . These results suggest that rP30 antigen would provide a simple, consistent, and rapid serodiagnosis for human monocytic ehrlichiosis.

J Clin Microbiol, 1999 Dec, 37(12), 3851 - 5
Novel bacterium isolated from a lung transplant patient with cystic fibrosis; Pitulle C et al.; The major clinical problem for patients with cystic fibrosis (CF) is progressive loss of pulmonary function, usually due to chronic bacterial infections . A patient with CF and a lung transplant was severely infected with a previously unidentified gram-negative bacterium . We isolated this organism (strain DS15158) from the patient and characterized it by phylogenetic analysis of the small-subunit rRNA and biochemically by the BIOLOG GN MicroPlate assay, fatty acid analysis, and various standard laboratory tests . No close match to any other organism could be found . Isolate DS15158 represents a new genus-level divergence within the bacterial subdivision alpha-Proteobacteria on the basis of the 16S rRNA gene analysis.

Shock, 1999 Nov, 12(5), 350 - 4
The src family-selective tyrosine kinase inhibitor PP1 blocks LPS and IFN-gamma-mediated TNF and iNOS production in murine macrophages; Orlicek SL et al.; Tyrosine phosphorylation pathways are essential components of the process of macrophage activation and the resultant production of inflammatory mediators such as tumor necrosis factor (TNF) and nitric oxide (NO) . Several lines of evidence suggest that members of the src family of protein tyrosine kinases play important roles in macrophage activation by gram-negative bacterial lipopolysaccharide (LPS) or the cytokine interferon-gamma (IFN-gamma), but targeted disruption of three members of the src family (hck, fgr, and lyn) in mice failed to demonstrate a requirement for these particular kinases in macrophage activation . We report that the pyrazolopyrimidine PP1, a src family-selective tyrosine kinase inhibitor, potently inhibits the production of TNF and inducible nitric oxide synthase (iNOS) in RAW 264.7 murine macrophages stimulated with LPS, rlFN-gamma, or LPS + rIFN-gamma . Furthermore, the tested concentrations of PP1 inhibit LPS- and rlFN-gamma-mediated tyrosine phosphorylation of the hck tyrosine kinase and its putative substrate, vav, but fail to block rlFN-gamma-mediated JAK2 tyrosine phosphorylation . These findings provide additional support for a model of macrophage activation involving one or more src-related kinases . Selective inhibitors of this signaling pathway should be studied in animal models of sepsis.

Kansenshogaku Zasshi, 1999 Oct, 73(10), 1032 - 7
{Effect of preventive measures against central venous catheter-related infection: retrospective study in our division for recent 10 years}; Hanatani Y et al.; A study was made of 2202 central venous catheters (CVC), which were inserted for recent 10 years, to know the effect of preventive measures against CVC-related infection . We divided 10 years in 3 periods: 1987-1990 (the first period), 1991-1993 (the second period), and 1994-1996 (the third period) . Preventive measures such as thorough antiseptic precaution, shortening of CVC dwelling time, and prohibition of injection from three-way stopcocks were taken after the second period . In the third period, semiclosed infusion system (I-system) was introduced to our division . A febrile catheterized patient (higher than 38 degrees C) was diagnosed as CVC-related infection when the fever dropped immediately (within 72 hours) after removal of CVC, or when the tip of the CVC was positive for culture . The rate of CVC-related infection in the second (9.9%) or the third (7.3%) period was significantly lower than that (14.0%) of the first period (p < 0.05 and p < 0.001, respectively) . The mean dwelling time of CVC was 31.5 days for the first period, 27.0 days for the second period, and 24.8 days for the third period . The rate of long-term dwelling catheters (more than 29 days) in the second (34.5%) or the third (28.7%) period was significantly lower than that (40.5%) in the first period (p < 0.01 and p < 0.001, respectively) . The index of CVC-related infection (incidence of infection per 1,000 days) was 4.8 for the first period, 3.7 for the second period, and 2.9 for the third period . The rate of infection of short-term dwelling CVC (less than 28 days) in the second (9.5%) or the third (6.3%) period was significantly lower than that (16.0%) of the first period (p < 0.01 and p < 0.001, respectively) . As to cultures of CVC and/or blood sample, the isolation rate of fungi decreased significantly (p < 0.001), and that of gram-negative rods showed a tendency to increase after the second period . It was concluded that shortening of CVC dwelling time and application of semiclosed infusion system were effective to reduce the rate of CVC-related infection.

Am J Physiol, 1999 Nov, 277(5 Pt 2), H1857 - 62
Effect of tumor necrosis factor-alpha, IL-1beta, and IL-6 on interstitial fluid pressure in rat skin; Nedrebo T et al.; Interstitial fluid pressure (P(if)) decreases in several experimental models of acute inflammation, enhancing edema formation . The present study was designed to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6, and IL-1beta as well as lipopolysaccharides (LPS) on P(if) in a model of gram-negative sepsis . P(if) was measured in the paw skin of anesthetized rats (pentobarbital sodium, 50 mg/kg ip) using micropipettes (3-7 micrometer) and servo-controlled counterpressure technique . Test substances were injected intra-arterially (ia), intravenously (iv), or subdermally (sd) . After intra-arterial or intravenous administration, the test substances were circulated for 1 min before circulatory arrest was induced with an intravenous injection of KCl while the rats were under pentobarbital anesthesia . Circulatory arrest was induced to avoid edema formation, which would raise interstitial fluid volume to cause a more positive P(if) . Administration of 0.5 ml of LPS (5 mg/ml ia) lowered P(if) significantly from control values of -0.2 +/- 0.3 to -2.0 +/- 0.3 mmHg (P < 0.05) within 1 h . Corresponding values for TNF-alpha (500 ng/ml iv) were -0.4 +/- 0.2 to -2.3 +/- 0.1 mmHg (P < 0.05) . Administration of 5 microliter (5 mg/ml sd) of LPS did not affect P(if) significantly (P > 0.05), but TNF-alpha, IL-1beta, and IL-6 had a significant effect on P(if) when given subdermally . IL-6 (50 ng/ml) caused a decrease in P(if) from control values of -1.2 +/- 0.3 to -2 . 8 +/- 0.5 mmHg (P < 0.05) within 1 h . The experiments demonstrate that LPS, TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given intravenously or intra-arterially, whereas only TNF-alpha, IL-1beta, and IL-6 induce lowering of P(if) when given subdermally . We therefore suggest that the lowering of P(if) in this experimental model of sepsis is related to the release of and a local effect in skin of TNF-alpha, IL-1beta, and IL-6.

Arch Mal Coeur Vaiss, 1999 Oct, 92(10), 1389 - 92
{Stenotrophomonas maltophilia endocarditis following mitral valve prosthesis implantation . Report of a case}; Meimoun P et al.; The authors report the first case of early postoperative endocarditis after mitral valvuloplasty due to Stenotrophomonas Maltophilia, a Gram negative organism, in a 37 year old man with no special risk factors . Pyrexia and mitral valve vegetations were the main features, and, in the absence of complications or of embolism, the patient was treated initially with triple antibiotherapy (ceftazidime, amikacine, ciprofloxacine) . Relapse two weeks after withdrawal of treatment due to two variants of Stenotrophomas Maltophilia, one of which was resistant to ciprofloxacine, and the presence of a large vegetation, required repeat mitral valvuloplasty and a change in antibiotic therapy (ticarcilline with clavulanic acid, trimethoprim sulphamethoxazole, colistine) . This time, the outcome was good . The little known Stenotrophomonas Maltophilia infectious endocarditis is a serious complication and, in the absence of standardised management, the authors suggest that, in view of the multi-resistant character of the organism and in the light of this case, surgery should be considered in association with prolonged antibiotic therapy.

J Bacteriol, 1999 Nov, 181(22), 6948 - 57
Characterization of a periplasmic ATP-binding cassette iron import system of Brachyspira (Serpulina) hyodysenteriae; Dugourd D et al.; The nucleotide sequence of the pathogenic spirochete Brachyspira hyodysenteriae bit (for "Brachyspira iron transport") genomic region has been determined . The bit region is likely to encode an iron ATP-binding cassette transport system with some homology to those encountered in gram-negative bacteria . Six open reading frames oriented in the same direction and physically linked have been identified . This system possesses a protein containing ATP-binding motifs (BitD), two hydrophobic cytoplasmic membrane permeases (BitE and BitF), and at least three lipoproteins (BitA, BitB, and BitC) with homology to iron periplasmic binding proteins . These periplasmic binding proteins exhibit lipoprotein features . They are labeled by {(3)H}palmitate when tested in recombinant Escherichia coli, and their signal peptides are typical for substrates of the type II secretory peptidase . The FURTA system and Congo red assay indicate that BitB and BitC are involved in iron binding . The Bit system is detected only in B . hyodysenteriae and is absent from B . innocens and B . pilosicoli.

Transfus Med, 1999 Sep, 9(3), 177 - 88
Direct detection of bacteria in cellular blood products using bacterial ribosomal RNA-directed probes coupled to electrochemiluminescence; Chaney R et al.; Various techniques for detecting bacteria in blood products exist; however, none has been widely accepted . Our method permits direct bacterial detection in both platelet concentrates (PC) and packed red blood cells (RBC) . This novel procedure targets bacterial ribosomal RNA (rRNA) but does not utilize culture or nucleic acid amplification . The assay comprises five steps: (1) release of bacterial rRNA by cell lysis with a combination of detergents and high heat; (2) hybridization of bacterial rRNA using a biotin- and a ruthenium (ORIGEN)-labelled oligonucleotide probe pair; (3) capture of labelled rRNA with streptavidin-coated magnetic beads; (4) concentration of labelled rRNA/bead complexes out of solution and onto an electrode surface with a magnet; (5) detection of ruthenium-labelled bacterial rRNA by application of voltage and consequent generation of the electrochemiluminescent (ECL) signal . Results using PC and RBC samples, spiked with clinically relevant gram-negative and -positive bacterial species, consistently demonstrated a linear relationship between ECL signal (equates to rRNA level) and colony forming units (CFU) mL(-1) . Signals were generated in the range of 1400-80000 and 3500-500000 ECL units for unwashed and washed samples, respectively . This is equivalent to 10(5)-10(8)CFU mL(-1) . These data demonstrate that therapeutic blood products significantly contaminated with bacteria may be identified prior to issue.

Lab Anim Sci, 1999 Oct, 49(5), 545 - 50
Evaluation of surrogate markers of impending death in the galactosamine-sensitized murine model of bacterial endotoxemia; Krarup A et al.; BACKGROUND AND PURPOSE: When evaluating vaccines for efficacy against gram-negative endotoxemia, the challenge has historically required death of a large percentage of test subjects . We attempted to identify surrogate markers of impending death to allow for early euthanasia without interfering with experimental data collection . METHODS: Galactosamine-sensitized mice (n = 140) were inoculated intraperitoneally with various dosages of endotoxin, and development of clinical signs of disease--body temperature, body weight, hunched posture, ruffled coat, inability to ambulate, and loss of consciousness--was evaluated . RESULTS: Wide fluctuations in body temperature (+/- 4 degrees C) were observed in survivors and nonsurvivors . Posture, coat, and body weight were not accurate predictors of death . Only inability to ambulate, with a positive predictive value of 100% (11 of 11), accurately predicted death in the experimental mice of this study . CONCLUSION: Using this surrogate marker, loss of ability to ambulate, 11 of 13 mice that developed this sign could have been euthanized early, preventing anywhere from 2 to 22 h of potential distress prior to death.

Exp Eye Res, 1999 Nov, 69(5), 483 - 90
Ocular surface epithelia express mRNA for human beta defensin-2; McNAMARA NA et al.; Human skin, lung and trachea produce human beta defensin-2 (hBD-2), an inducible, transcriptionally regulated antibiotic peptide with activity against gram negative bacteria, which may explain the unusual resistance of these tissues to infection . Since an intact corneal epithelium is also highly resistant to infection, we examined whether human ocular surface epithelia might produce hBD-2 . Conjunctival epithelial cells were obtained from a human cadaver eye, while corneal epithelial cells were obtained from both a cadaver eye and the eye of a living human patient . Using reverse transcription-polymerase chain reaction and custom primers for hBD-2, a 257 bp sequence was amplified from both human corneal and conjunctival epithelial cell cDNA, and the amino acid sequence of this DNA band was computer-matched with the known gene sequence of hBD-2 available through GenBank (Z71389) . To determine whether bacterial by-products upregulate hBD-2 mRNA expression, we stimulated confluent SV 40-immortalized human corneal epithelial cells with bacterial culture supernatant prepared from either wild-type P . aeruginosa strain PAO1 or two different lipopolysaccharide (LPS) mutants of PAO1 . Both of these mutants, strains AK1012 and PAO1 algC::tet, are deficient in phosphomannomutase activity which is required for the synthesis of both a complete polysaccharide core and the O side chain structures of the LPS molecule . Neither of these mutations affects the lipid A portion of LPS . Cells treated with P . aeruginosa wild-type PAO1 bacterial culture supernatant demonstrated strong upregulation of hBD-2 mRNA expression, whereas cells stimulated with culture supernatant produced by either of the LPS mutants showed little or no change in hBD-2 gene expression . LPS extracted from the bacterial culture supernatant was used to demonstrate that upregulation of hBD-2 is caused by LPS . Genistein blocked this upregulation suggesting that protein tyrosine kinase activity is involved . Thus, both human corneal and conjunctival epithelium express mRNA for hBD-2, and this expression is upregulated by bacterial LPS . Data obtained from LPS mutants suggest that lipid A, which is responsible for initiating a number of the pathophysiological manifestations induced by endotoxin in mammals, is not required . Stimulation of endogenous hBD-2 production via the active portion of LPS might have therapeutic potential .

Semin Thromb Hemost, 1999, 25(4), 429 - 33
Dithiocarbamates ameliorate the effects of endotoxin in a rabbit model of disseminated intravascular coagulation; Drollinger AG et al.; Induction of tissue factor (TF) activity by endotoxin and cytokines is an important mechanism for initiation of disseminated intravascular coagulation (DIC) seen in patients with gram-negative sepsis . Based on data from an in vitro study in which dithiocarbamates abrogated endothelial cell TF activity by inhibition of the NF-kappaB pathway, we investigated whether dithiocarbamates had in vivo activity in an animal model of DIC . Dithiocarbamates ameliorated the adverse clinical and histological effects of endotoxin-induced DIC, including morbidity, hypofibrinogenemia, and target organ damage, especially in the liver and kidney, even when given up to 1 hour after administration of endotoxin . This pilot study confirms the key role of the nuclear factor-kappa beta (NF-kappaB) pathway in induction of TF activity in initiating sepsis-associated DIC and suggests that dithiocarbamates may be useful in treatment of DIC associated with excessive TF expression because of gram-negative sepsis . Additional studies of dithiocarbamates in DIC models are warranted.

Annu Rev Microbiol, 1999, 53, 353 - 87
Contributions of genome sequencing to understanding the biology of Helicobacter pylori; Ge Z et al.; About half of the world's population carries Helicobacter pylori, a gram-negative, spiral bacterium that colonizes the human stomach . The link between H . pylori and, ulceration as well as its association with the development of both gastric cancer and mucosa-associated lymphoid tissue lymphoma in humans is a serious public health concern . The publication of the genome sequences of two stains of H . pylori gives rise to direct evidence on the genetic diversity reported previously with respect to gene organization and nucleotide variability from strain to strain . The genome size of H . pylori strain 26695 is 1,6697,867 bp and is 1,643,831 bp for strain J99 . Approximately 89% of the predicted open reading frames are common to both of the strains, confirming H . pylori as a single species . A region containing approximately 45% of H . pylori strain-specific open reading frames, termed the plasticity zone, is present on the chromosomes, verifying that some strain variability exists . Frequent alteration of nucleotides in the third position of the triplet codons and various copies of insertion elements on the individual chromosomes appear to contribute to distinct polymorphic fingerprints among strains analyzed by restriction fragment length polymorphisms, random amplified polymorphic DNA method, and repetitive element-polymerase chain reaction . Disordered chromosomal locations of some genes seen by pulsed-field gel electrophoresis are likely caused by rearrangement or inversion of certain segments in the genomes . Cloning and functional characterization of the genes involved in acidic survival, vacuolating toxin, cag-pathogenicity island, motility, attachment to epithelial cells, natural transformation, and the biosynthesis of lipopolysaccharides have considerably increased our understanding of the molecular genetic basis for the pathogenesis of H . pylori . The homopolymeric nucleotide tracts and dinucleotide repeats, which potentially regulate the on- and off-status of the target genes by the strand-slipped mispairing mechanism, are often found in the genes encoding the outer-membrane proteins, in enzymes for lipopolysaccharide synthesis, and within DNA modification/restriction systems . Therefore, these genes may be involved in the H . pylori-host interaction.

AIDS, 1999 Oct 22, 13(15), 2013 - 21
HIV-1 exposed dendritic cells show increased pro-inflammatory cytokine production but reduced IL-1ra following lipopolysaccharide stimulation; Lore K et al.; OBJECTIVES: Dendritic cells (DC) are potential first target cells in sexually transmitted HIV-1 infection . They are also considered to be central in the activation of naive T cells, which thereupon can become permissive for HIV-1 . In addition, activated DC express effector molecules, which likely contribute to the direction of T helper (Th1/Th2)-specific immune responses . METHODS: The capacity of cytokine and chemokine production in in vitro DC infected and uninfected with HIV-1 was assessed by enzyme-linked immunosorbent assay (ELISA) and by in situ immunocytochemical detection at the single cell level . Fluorescent in situ 5'-nuclease assay (FISNA) was used for quantitative evaluation of HIV-1 gag-positive cells . RESULTS: Macrophage-tropic HIV-1 effectively infected 20-40% of in vitro cultured DC . However, this activity alone did not induce detectable cytokine or chemokine protein expression in DC . In contrast, lipopolysaccharide (LPS) stimulation of these HIV-1-infected DC resulted in a significantly increased level of cells producing tumour necrosis factor alpha (TNF-alpha) and interleukin (IL) 1beta but reduced frequencies of cells producing IL-1 receptor antagonist (IL-1ra) compared with the LPS-stimulated but uninfected DC cultures (P < 0.05) . Furthermore, an extensive production of the beta-chemokines {RANTES, macrophage inflammatory proteins (MIP) 1alpha and 1beta} was detected in DC in response to both LPS and HIV-1 plus LPS . CONCLUSIONS: These findings indicate that HIV-1 infected DC may have an increased proinflammatory activity . Elevated production of cytokines such as TNF-alpha and IL-1beta and reduced IL-1ra may contribute to enhanced replication of HIV-1 in bystander T cells . Gram-negative bacterial infection and gut-associated bacterial translocation in HIV-1-infected individuals may also result in endotoxin-mediated reactivation of HIV-1 in bystander CD4 CD45RO T cells caused by the increased production of proinflammatory cytokines in DC.

J Bacteriol, 1999 Nov, 181(21), 6642 - 9
Purification, characterization, and gene analysis of a chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001; Park JK et al.; The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified . The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C . The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives . Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+) . Internal amino acid sequences of the purified chitosanase were obtained . A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase . Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein . The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases . The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli . Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

Med Clin (Barc), 1999 Sep 11, 113(7), 241 - 5
{Development of Escherichia coli strands resistant to quinolones in stools of patients with liver cirrhosis submitted to selective bowel decontamination}; Aparicio JR et al.; BACKGROUND: Selective intestinal decontamination (SID) with norfloxacin in patients with cirrhosis may promote the development of quinolone-resistant (QR) gram-negative bacteria in stools . It is not known wether this fact may become a predisposing factor for the development of infections due to these bacteria . MATERIAL AND METHODS: We designed a prospective study to evaluate the incidence of Escherichia coli in stools at admission in patients with cirrhosis that had previously received norfloxacin as primary or secondary prophylaxis of spontaneous bacterial peritonitis (SBP) (group I, n = 28) vs those who did not (group II, n = 55) . RESULTS: QR strains of E . coli were observed in 37.5 and in 1.47% of patients from groups I and II, respectively (p < 0.001) . During admission, 36 patients underwent norfloxacin prophylaxis (group III), and 40 did not (group IV) . Eleven patients from group III and one patient from group IV showed QR E . coli in stools . We observed 5 bacterial infections in group III and 14 in group IV (p = 0.0039) . No patient with QR E . coli in stools developed infections due to this bacteria . CONCLUSION: The incidence of QR E . coli in stools of patients with cirrhosis is significantly increased in patients previously treated with prophylactic norfloxacin . However, this fact seems not to be associated with an increment in the prevalence of QR E . coli infections.

Int Arch Occup Environ Health, 1999 Oct, 72(7), 443 - 50
Endotoxins and IgG antibodies as indicators of occupational exposure to the microbial contaminants of metal-working fluids; Laitinen S et al.; OBJECTIVES: The aim of this study was to evaluate workers' exposure to microbes and bacterial endotoxins during the use of metal-working fluids (MWF) . METHODS: Air and bulk sampling with biomonitoring of workers' serum IgG antibodies were used to estimate the exposure to biological agents at 18 workplaces . The types of emulsified MWF used were synthetic fluid, mineral oil or rape seed oil, in grinding, turning and drilling work . RESULTS: The endotoxin concentrations in the air ranged from 0.04 to 600 ng/m(3) when the endotoxin levels in MWF were 0.03-25,000 ng/ml . A high correlation was found between the endotoxin levels and the bacterial counts from MWF, as well as between the total culturable bacteria and the gram-negative bacteria concentrations in the air . Comamonas testosteroni and C . acidovorans were the most common strains in the samples but also colonies of Ochrobactrum anthropi, Pantoea agglomerans and Stenotrophomonas maltophilia were isolated from the workplaces . Fungi like Aspergillus, Cladosporium and Penicillium species were identified in the air but only rarely in the MWF . Positive IgG antibodies were found in the sera of 22 of the 25 MWF workers examined . Antibodies against S . maltophilia, P . agglomerans and C . acidovorans were the most common, appearing in 72%, 64% and 64%, respectively, of the cases . The MWF workers showed significantly higher IgG antibody responses to bacterial antigens than did the controls . CONCLUSIONS: The results clearly proved that in occupational hygiene measurements, endotoxins serve as excellent indicators of exposure to the microbial contaminants of MWF . IgG antibodies against antigens identified from workplace samples could be a practical tool for occupational health physicians.

J Gastroenterol Hepatol, 1999 Sep, 14(9), 844 - 50
Helicobacter pylori and extra-digestive diseases; Tsang KW et al.; Helicobacter pylori is a recently rediscovered gram-negative bacteria that causes peptic ulcer disease, gastric lymphoma and gastric carcinoma . Helicobacter pylori achieves its pathogenetic role by triggering an intense leucocyte infiltration of the gastric submucosa which is mediated by proinflammatory cytokines . This pathogenetic mechanism is common to many other diseases and, therefore, Helicobacter pylori seroprevalence has also been investigated in other diseases . It is now known that H . pylori seropositivity is associated with an increasing number of cardiovascular, respiratory, extra-gastroduodenal digestive, neurological, skin, autoimmune, growth and miscellaneous disorders . Although the precise role for H . pylori is unknown in these diseases, it is of tremendous interest to most clinicians and scientists as H . pylori is amenable to eradication therapy using simple and reliable drug regimens . The conditions associated with H . pylori seropositivity are highlighted in this concise article.

Lab Invest, 1999 Oct, 79(10), 1181 - 99
Direct effects of endotoxin on the endothelium: barrier function and injury; Bannerman DD et al.; LPS directly disrupts EC barrier function in vitro and in vivo . This barrier dysfunction has been reported to occur in EC derived from both the macro- and microvasculature of varying species, including humans . Unlike other EC responses, LPS-induced loss of endothelial barrier function is protein-synthesis independent . In fact, protein synthesis inhibition enhances the LPS effect . The lipid A moiety is responsible for LPS-induced activation of the non-CD14-bearing EC, and agents that bind to and neutralize this highly conserved portion of the LPS molecule can crossprotect against EC barrier dysfunction elicited by LPS derived from diverse species of Gram-negative bacteria . Although the presentation of LPS to CD14-bearing cells such as macrophages and monocytes has been well characterized, far less is known about the interactions of LPS with the non-CD14-bearing EC . An EC receptor involved in LPS binding and cellular activation has yet to be identified . The presence of the accessory molecules, LBP and sCD14, are prerequisite to LPS-induced activation of EC at clinically relevant LPS concentrations . As with monocytes and macrophages, the CD14 dependence of LPS-induced endothelial barrier dysfunction can be overcome with high concentrations of LPS . In the absence of LBP and sCD14, a 200,000-fold increase in LPS concentration is required to elicit the same increments in EC monolayer permeability relative to when these accessory molecules are present . Within 30 minutes after LPS exposure, PTK activation is observed . PTK inhibition blocks LPS-induced EC actin depolymerization and endothelial barrier dysfunction which are seen only after a > or = 2-hour stimulus-to-response lag time . Furthermore this LPS-induced actin depolymerization is a prerequisite to opening up the paracellular pathway and loss of monolayer integrity . Interestingly LPS-induced increments in transendothelial 14C-BSA flux and EC detachment parallel caspase-mediated cleavage of ZA and FA proteins that participate in cell-cell and cell-matrix adhesion . The cleavage of the ZA components, beta- and gamma-catenin, does not affect their ability to bind the transmembrane protein, cadherin, or the actin-binding protein, alpha-catenin, suggesting that the linkage of the ZA to the actin cytoskeleton remains intact . LPS-induced cleavage of the FA protein, FAK, leads to dissociation of its catalytic domain from paxillin substrate and decreased paxillin phosphotyrosine content . Caspase inhibition protects against LPS-provoked apoptosis, cleavage of adherens junction proteins, paxillin dephosphorylation, cell-shape changes, and EC detachment . In contrast it fails to block LPS-induced increments in transendothelial 14C-BSA flux . PTK inhibition, which does protect against increased transendothelial 14C-BSA flux, does not block LPS-induced proteolytic cleavage events and only partially inhibits EC detachment . These findings suggest that the EC detachment and endothelial barrier dysfunction elicited by LPS are mediated through distinct pathways (Fig . 6) . Much of the work to date has focused on LPS interactions with mCD14-bearing cells, such as monocytes and macrophages, which are central to the inflammatory response elicited by endotoxin . EC, which line the vasculature, are one of the first host tissue barriers to encounter circulating LPS . Because damage to the endothelium is known to contribute to the development of multiorgan failure, including ARDS, understanding LPS-induced EC dysfunction in the setting of Gram-negative septicemia has clear pathophysiologic implications . (ABSTRACT TRUNCATED)

Eur J Pediatr Surg, 1999 Aug, 9(4), 236 - 41
Severe short-bowel syndrome in children . Clinical experience; de Agustin JC et al.; INTRODUCTION: Innovative surgical and pharmacological therapeutic measures in short-bowel syndrome (SBS) are constantly changing the prognosis of this devastating condition . The aim of this paper is to present our most recent experience in the treatment of this disease, with particular emphasis on the impact of home parenteral nutrition (HPN) and the use of growth hormone (GH) . METHODS: A group of 8 patients with severe SBS have been studied for the past 4 years . Intestinal length of less than 25% normal at the time of bowel resection was the criterion for inclusion in this study . RESULTS: Mean age at the time of diagnosis was 2 years (ranging from 1 day to 9 years) . The etiology of the SBS was Hirschsprung's disease (n = 3), midgut volvulus (n = 2), gastroschisis (n = 1), omphalocele with ileal atresia and necrotizing enterocolitis (n = 1) and Crohn's disease (n = 1) . Length of the residual bowel was 8 and 50 cm with ileocecal valve (ICV) preservation and 23, 27, 30, 50, 70, 100 cm without ICV . Sixty percent of the patients survived . Two patients died due to fulminant gram-negative sepsis and one due to cardiac malformation . Two patients are still on parenteral nutrition (PN) providing 30 and 60% of total calories . Human GH (0.3 U/kg/day) was used in two patients over a period of 28 days . In these patients, an increased tolerance to enteral feeding was observed . HPN was provided in 5 cases, allowing regular school attendance in 3 patients . In 3 cases, discontinuation of the PN was achieved at 24, 25 and 35 months respectively . CONCLUSIONS: Human GH can improve tolerance of enteral feeding . HPN has a beneficial effect on child behaviour . Intestinal transplantation must be considered when no other surgical or medical measures are available.

Infect Immun, 1999 Nov, 67(11), 5775 - 83
Outer membrane protein A-promoted actin condensation of brain microvascular endothelial cells is required for Escherichia coli invasion; Prasadarao NV et al.; Escherichia coli is the most common gram-negative bacterium that causes meningitis during the neonatal period . We have previously shown that the entry of circulating E . coli organisms into the central nervous system is due to their ability to invade the blood-brain barrier, which is composed of a layer of brain microvascular endothelial cells (BMEC) . In this report, we show by transmission electron microscopy that E . coli transmigrates through BMEC in an enclosed vacuole without intracellular multiplication . The microfilament-disrupting agents cytochalasin D and latrunculin A completely blocked E . coli invasion of BMEC . Cells treated with the microtubule inhibitors nocodazole, colchicine, vincristin, and vinblastine and the microtubule-stabilizing agent taxol also exhibited 50 to 60% inhibition of E . coli invasion . Confocal laser scanning fluorescence microscopy showed F-actin condensation associated with the invasive E . coli but no alterations in microtubule distribution . These results suggest that E . coli uses a microfilament-dependent phagocytosis-like endocytic mechanism for invasion of BMEC . Previously we showed that OmpA expression significantly enhances the E . coli invasion of BMEC . We therefore examined whether OmpA expression is related to the recruitment of F-actin . OmpA(+) E . coli induced the accumulation of actin in BMEC to a level similar to that induced by the parental strain, whereas OmpA(-) E . coli did not . Despite the presence of OmpA, a noninvasive E . coli isolate, however, did not show F-actin condensation . OmpA(+)-E . coli-associated condensation of F-actin was blocked by synthetic peptides corresponding to the N-terminal extracellular domains of OmpA as well as BMEC receptor analogues for OmpA, chitooligomers (GlcNAcbeta1-4GlcNAc oligomers) . These findings suggest that OmpA interaction is critical for the expression or modulation of other bacterial proteins that will subsequently cause actin accumulation for the uptake of bacteria.

Can J Microbiol, 1999 Sep, 45(9), 779 - 85
Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus; Ziola B et al.; Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells . All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding . Mab reactivity with purified PG confirmed this . Epitope mapping revealed the Mabs in total recognize four binding sites on the PG . Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells . No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria . These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria . These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.

FEMS Microbiol Ecol, 1999 Nov 1, 30(3), 273 - 284
Characterization of mercury resistance mechanisms in marine sediment microbial communities; Reyes NS et al.; While estuarine sediments are often severely polluted with mercury, few studies have focused on the mechanisms of adaptation to mercury contamination in marine sediment microbial communities . In this study, we report a high frequency of Gram-negative bacterial isolates that are resistant to the heavy metal mercury obtained from the aerobic culturable marine microbial community . We detected a low frequency of genes homologous to mer(Tn21) in isolates from three out of four different estuarine environments . Other mercury resistant culturable bacterial isolates lacking homology to the known mer genes were able to reduce Hg(II) to its volatile Hg(0) form, indicating the presence of divergent mer genes . In addition, a number of mercury resistant isolates, obtained from three of the four marine sites investigated, exhibited decreased resistance to mercury in the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone . Representative mercury resistant bacterial isolates were identified by phylogenetic analysis as belonging to the alpha and gamma subclasses of the class Proteobacteria.

Biochim Biophys Acta, 1999 Sep 22, 1440(2-3), 163 - 75
A novel glycosphingolipid from gram-negative aquatic bacteria; Batrakov SG et al.; The chloroform-methanol extractable lipids of the Gram-negative fresh-water bacteria Arcocella aquatica NO-502 and Flectobacillus major FM were found to contain an unusual ninhydrin-positive glycolipid . It was purified by two-stage silica gel-column chromatography . By the use of IR and (1)H-NMR spectroscopy, mass spectrometry and chemical-degradation experiment, the lipid was established to be 1-O-monoglycosyl ceramide, the carbohydrate moiety of which was the alpha-pyranose-ring form of 7-desoxy-7-amino-D-manno-heptulosonic acid, or 1-hydroxycarbonyl-6-deoxy-6-amino-alpha-D-mannopyranose . The ceramide portion consisted mainly (by 95% in the A . aquatica glycolipid and 80% in the F . major glycolipid) of 2-N-(2'-D-hydroxy-13'-methyltetradecanoyl)-15-methyl-4(E)-hexad ecasph ingenine . The minor molecular species differed from the major one only in fatty acid structure . The glycolipid accounted for 8 and 11% of the total lipids extracted from A . aquatica NO-502 and F . major FM cells, respectively.

Arterioscler Thromb Vasc Biol, 1999 Oct, 19(10), 2517 - 23
Endotoxin-induced activation of the coagulation cascade in humans: effect of acetylsalicylic acid and acetaminophen; Pernerstorfer T et al.; During Gram-negative septic shock, lipopolysaccharide (LPS, endotoxin) induces tissue factor (TF) expression . TF expression is mediated by nuclear factor kappaB and amplified by activated platelets . TF forms a highly procoagulant complex with activated coagulation factor VII (FVIIa) . Hence, we hypothesized that aspirin, which inhibits LPS-induced, nuclear factor kappaB-dependent TF expression in vitro and platelet activation in vivo, may suppress LPS-induced coagulation in humans . Therefore, we studied the effects of aspirin on systemic coagulation activation in the established and controlled setting of the human LPS model . Thirty healthy volunteers were challenged with LPS (4 ng/kg IV) after intake of either placebo or aspirin (1000 mg) . Acetaminophen (1000 mg) was given to a third group to control for potential effects of antipyresis . Neither aspirin nor acetaminophen inhibited LPS-induced coagulation . However, LPS increased the percentage of circulating TF(+) monocytes by 2-fold . This increase was associated with a decrease in FVIIa levels, which reached a minimum of 50% 24 hours after LPS infusion . Furthermore, LPS-induced thrombin generation increased plasma levels of circulating polymerized, but not cross-linked, fibrin (ie, thrombus precursor protein), whereas levels of soluble fibrin were unaffected . In summary, a single 1000-mg dose of aspirin did not decrease LPS-induced coagulation . However, our study showed, for the first time, that LPS increases TF(+) monocytes, substantially decreases FVIIa levels, and enhances plasma levels of thrombus precursor protein, which may be a useful marker of fibrin formation in humans.

Arch Neurol, 1999 Oct, 56(10), 1201 - 8
Molecular pathogenesis of Friedreich ataxia; Pandolfo M; Friedreich ataxia, the most common type of inherited ataxia, is itself caused in most cases by a large expansion of an intronic GAA repeat, resulting in decreased expression of the target frataxin gene . The autosomal recessive inheritance of the disease gives this triplet repeat mutation some unique features of natural history and evolution . Frataxin is a mitochondrial protein that has homologues in yeast and even in gram-negative bacteria . Yeast organisms deficient in the frataxin homologue accumulate iron in mitochondria and show increased sensitivity to oxidative stress . This suggests that Friedreich ataxia is caused by mitochondrial dysfunction and free radical toxicity.

Ther Drug Monit, 1999 Oct, 21(5), 540 - 3
Operator error: a critical determinant of false amikacin and tobramycin concentrations using fluorescence polarization immunoassay kits and TDX analyzer; Banerjee SK et al.; Amikacin and tobramycin are aminoglycosides used to treat severe Gram-negative infections . Therapeutic monitoring of both drugs is essential to avoid drug toxicity . Fluorescence polarization immunoassay kits for both amikacin and tobramycin are available from Abbott Laboratories, and assays can be run using the TDx analyzer . The test code for amikacin is 3, whereas the test code for tobramycin is 2 . Here the authors report that when the operator mistakenly programmed test code 3 (amikacin code) while using the tobramycin kit, the tobramycin values were falsely elevated . The instrument software does not have any mechanism to detect this error . Similarly, when the amikacin kit was used and the operator mistakenly programmed test code 2 (tobramycin code), the amikacin values were falsely lowered . For example, in a serum sample taken from a patient the true amikacin concentration was 20.9 microg/mL . When the amikacin kit was used correctly but test code 2 was programmed, the observed concentration was falsely lowered to 6.1 microg/mL . Similarly, in another specimen the true tobramycin concentration was 3.1 microg/mL . However, when the tobramycin kit was used correctly but test code 3 was used, the observed concentration was falsely elevated to 14.3 microg/mL . The authors also observed a similar effect with controls and spiked specimens containing either amikacin or tobramycin . They conclude that correct programming of the test code is vital to obtaining useful results in amikacin and tobramycin analysis using the TDx analyzer.

J Vet Dent, 1998 Dec, 15(4), 165 - 8
Oral malodor and its relevance to periodontal disease in the dog; Culham N et al.; Oral malodor has been studied extensively in humans but very little work has been done in dogs where it constitutes a significant problem . In this article we review its causes, methods of detection, and strategies for preventing it . Oral malodor arises from microbial metabolism of exogenous and endogenous proteinaceous substrates in the oral cavity and is exacerbated by periodontal disease and poor oral hygiene . Gram negative bacteria found in plaque, in periodontal pockets, and on the dorsum of the tongue are primarily responsible for odor production . The volatile sulfur compounds (VSCs) hydrogen sulfide and methyl mercaptan, which are produced by these bacteria, are not only primarily responsible for the objectionable odor but have been implicated in the pathogenesis of periodontal disease . Assessment of malodor by portable sulfide monitors correlates well with organoleptic measurements . Reduction of microbial load in the oral cavity due to good oral hygiene practices (such as tooth-brushing) or by the use of appropriate diets or chews may reduce malodor formation.

Med Pregl, 1999 Jun-Aug, 52(6-8), 275 - 7
{Helicobacter heilmanni (Gastrospirillum hominis) as a new cause of gastritis--case report}; Fenjvesi A et al.; INTRODUCTION: During the course of research into Helicobacter pylori, further microorganisms colonizing the gastric mucosa have been detected . The large spiral-shaped bacteria deviating from Helicobacter pylori was originally described by Dent . The bacteria was initially named Gastrospirillum hominis, but after sequencing of 16S rRNA it was classified as Helicobacter and named Helicobacter Heilmannii in honour of the pathologist Prof . Dr . Konrad Heilmann . We report two additional cases . CASE REPORTS: In 1997 at the Department of Pathology of General Hospital in Senta 268 gastric biopsies (151 male, 117 female), average age of 50.4 were analyzed . Helicobacter pylori was identified in 176 biopsies (65.7%) . Helicobacter Heilmannii was found in two cases (0.74%) . The clinical details were as follows: Case 1 . A 32-year-old male complained of dyspepsia . Esophagogastroduodenoscopy disclosed hyperemia and scattered erosions in the lower third of stomach . Case 2 . a 55-year-old female was admitted at the hospital with lumbar pain, loss of appetite and body weight . Esophagogastroduodenoscopy findings were normal . The patient lived in rural household in contact with various domestic animals . In the biopsy specimens of both cases in the antral gastric mucosa large bacteria resembling a corkscrew were found, 6-10 microns length, consisting of 5-9 regular tight spiral . The bacteria were midly eosinophilic and Gram negative . The bacteria stained strongly with the Warthin-Starry silver stain, and with Giemsa stain . The distribution of Helicobacter Heilmannii was patchy in the central zones of the gastric pits, deep or more superficial including the foveolar opening on the mucosal surface . A mild degree of chronic gastritis characterized both cases, with, focally active inflammation and lymphoid aggregates . None of biopsies had atrophy, intestinal metaplasia or epithelial damage . There was a symptomatic improvement after a 4-week course with triple therapy (H2 antagonist & metronidazole & amoxycillin) . After treatment rebiopsy specimens displayed mild chronic gastritis and no spiral organisms . CONCLUSION: Our reported cases are the first described cases of gastritis caused by Helicobacter Heilmannii in our region with successful eradication after therapy . Morphologically similar bacteria have been found in the stomachs of domestic animals . It is a general opinion that Helicobacter Heilmannii is characteristic for animals but it is occasionally transmitted to humans.

Am J Physiol, 1999 Oct, 277(4 Pt 2), R1188 - 95
Feeding status and bacterial LPS-induced cytokine and neuropeptide gene expression in hypothalamus; Gayle D et al.; This study determined the effects of feeding status on basal and lipopolysaccharide (LPS)-stimulated cytokine and neuropeptide gene expression in the hypothalamus . With the use of RNase protection assays, we measured mRNA levels of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1RA), IL-1 receptor type I (IL-1RI), IL-1R accessory proteins (AcP I and II), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1), glycoprotein 130 (Gp 130), leptin receptor (OB-R), neuropeptide Y (NPY), preprodynorphin, and proopiomelanocortin (POMC) . Analyses were done in ad libitum-fed, fasted, and fasted and refed rats treated with the intracerebroventricular administration of physiological saline or LPS . The data show that food deprivation increases the basal mRNA expression of IL-1beta, IL-1RA, TNF-alpha, IL-1RI, and IL-1R AcP I, whereas mRNA levels of POMC showed a decrease . Five hours of refeeding returned cytokine levels to those observed in the ad libitum-fed group . LPS administration induced a robust upregulation of IL-1beta, TNF-alpha, and IL-1RI during all three feeding conditions . Acute food deprivation did not modulate LPS-induced changes in hypothalamic cytokine mRNA profiles . These findings show that 1) cytokine modulation occurs as an adaptive response to the stress of acute fasting and 2) acute fasting does not affect LPS-induced cytokine mRNA modulation in the hypothalamus . The data have implications to gram-negative infections associated with acute anorexia.

J Bacteriol, 1999 Oct, 181(20), 6319 - 31
Induction of beta-lactamase influences the course of development in Myxococcus xanthus; O'Connor KA et al.; Myxococcus xanthus is a gram-negative bacterium that develops in response to starvation on a solid surface . The cells assemble into multicellular aggregates in which they differentiate from rod-shaped cells into spherical, environmentally resistant spores . Previously, we have shown that the induction of beta-lactamase is associated with starvation-independent sporulation in liquid culture (K . A . O'Connor and D . R . Zusman, Mol . Microbiol . 24:839-850, 1997) . In this paper, we show that the chromosomally encoded beta-lactamase of M . xanthus is autogenously induced during development . The specific activity of the enzyme begins to increase during aggregation, before spores are detectable . The addition of inducers of beta-lactamase in M . xanthus, such as ampicillin, D-cycloserine, and phosphomycin, accelerates the onset of aggregation and sporulation in developing populations of cells . In addition, the exogenous induction of beta-lactamase allows M . xanthus to fruit on media containing concentrations of nutrients that are normally too high to support development . We propose that the induction of beta-lactamase is an integral step in the development of M . xanthus and that this induction is likely to play a role in aggregation and in the restructuring of peptidoglycan which occurs during the differentiation of spores . In support of this hypothesis, we show that exogenous induction of beta-lactamase can rescue aggregation and sporulation of certain mutants . Fruiting body spores from a rescued mutant are indistinguishable from wild-type fruiting body spores when examined by transmission electron microscopy . These results show that the signal transduction pathway leading to the induction of beta-lactamase plays an important role in aggregation and sporulation in M . xanthus.

Clin Microbiol Rev, 1999 Oct, 12(4), 518 - 53
Q fever; Maurin M et al.; Q fever is a zoonosis with a worldwide distribution with the exception of New Zealand . The disease is caused by Coxiella burnetii, a strictly intracellular, gram-negative bacterium . Many species of mammals, birds, and ticks are reservoirs of C . burnetii in nature . C . burnetii infection is most often latent in animals, with persistent shedding of bacteria into the environment . However, in females intermittent high-level shedding occurs at the time of parturition, with millions of bacteria being released per gram of placenta . Humans are usually infected by contaminated aerosols from domestic animals, particularly after contact with parturient females and their birth products . Although often asymptomatic, Q fever may manifest in humans as an acute disease (mainly as a self-limited febrile illness, pneumonia, or hepatitis) or as a chronic disease (mainly endocarditis), especially in patients with previous valvulopathy and to a lesser extent in immunocompromised hosts and in pregnant women . Specific diagnosis of Q fever remains based upon serology . Immunoglobulin M (IgM) and IgG antiphase II antibodies are detected 2 to 3 weeks after infection with C . burnetii, whereas the presence of IgG antiphase I C . burnetii antibodies at titers of >/=1:800 by microimmunofluorescence is indicative of chronic Q fever . The tetracyclines are still considered the mainstay of antibiotic therapy of acute Q fever, whereas antibiotic combinations administered over prolonged periods are necessary to prevent relapses in Q fever endocarditis patients . Although the protective role of Q fever vaccination with whole-cell extracts has been established, the population which should be primarily vaccinated remains to be clearly identified . Vaccination should probably be considered in the population at high risk for Q fever endocarditis.

J Biol Chem, 1999 Oct 15, 274(42), 29805 - 11
Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia; Knodler LA et al.; Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum . The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions . We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia . Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site . The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria . The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa) . All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities . Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity . This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking . This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Appl Occup Environ Hyg, 1999 Sep, 14(9), 598 - 608
Exposure to biohazards in wood dust: bacteria, fungi, endotoxins, and (1-->3)-beta-D-glucans; Alwis KU et al.; Personal exposure to fungi, bacteria, endotoxin, and (1-->3)-beta-D-glucan was determined at different woodworking sites--logging sites, sawmills, woodchipping sites, and joineries . Exposure levels to fungi at logging sites and sawmills were in the range of 10(3)-10(4) cfu/m3, at the woodchipping mill, 10(3)-10(5) cfu/m3, and at joineries, 10(2)-10(4) cfu/m3 . Although mean endotoxin levels were lower than the suggested threshold value of 20 ng/m3, some personal exposures at sawmills and a joinery exceeded the standard . The geometric mean personal (1-->3)-beta-D-glucan exposure level at the woodchipping mill was 2.32 ng/m3, at sawmills, 1.37 ng/m3, at logging sites, 2.02 ng/m3, and at joineries, 0.43 ng/m3 . Highly significant associations were found between mean personal inhalable endotoxin exposures and Gram-negative bacteria levels (p < 0.0001), and mean personal inhalable (1-->3)-beta-D-glucan exposures and fungi levels (p = 0.0003) . The prevalence of cough, phlegm, chronic bronchitis, nasal symptoms, frequent headaches, and eye and throat irritations was significantly higher among woodworkers than controls . Dose-response relationships were found between personal exposures and work-related symptoms among joinery workers and sawmill and chip mill workers.

Bioorg Med Chem Lett, 1999 Sep 20, 9(18), 2741 - 6
Structure-activity relationships of biphenyl tetrazoles as metallo-beta-lactamase inhibitors; Toney JH et al.; Resistance to carbapenem antibiotics in gram-negative bacteria is due, in part, to expression of a wide spectrum metallo-beta-lactamase, which renders the drug inactive . Biphenyl tetrazoles containing 3-n-butyl-1-phenylpyrazole-5-carboxylates or the corresponding 5-ethyl esters were found to inhibit metallo-beta-lactamases as well as renal dehydropeptidase I to a lesser extent.

Biopolymers, 1999 Nov, 50(6), 579 - 88
Biopolymer excreted by pseudoalteromonas antarctica NF(3), as a coating and protective agent of liposomes against dodecyl maltoside
de La Maza A, Codech L, Lopez O, Parra JL, Sabes M, Guinea J.
The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative species Pseudoalteromonas antarctica NF(3), to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant dodecyl maltoside was investigated . Transmission electron microscopy (TEM) micrographs of freeze fractured liposome/GP aggregates reveal that the addition of the glycoprotein to liposomes led to the formation of a film (polymer adsorbed onto the bilayers) that tightly coated PC bilayers . The complete coating was already achieved at a PC : GP weight ratio of about 9:1 . Image analysis profiles of digitalized TEM micrographs (PC : GP weight ratio 8:2) show that this film was formed by a multilayer structure . The periods of the average distance of the pattern ordering in layer structures (9-10 layers) were of about 2-3 nm and the thickness of the complete film was of about 25 nm . Higher amounts of glycoprotein resulted in a growth of this film, which exhibited at the highest proportion of this compound (50% in weight) a multifilm structure . An increasing resistance of liposomes to be affected by dodecyl maltoside both at subsolubilizing and solubilizing levels occurred as the proportion of the glycoprotein in the system rose, although this protective effect was more effective at low proportions of this compound (PC : GP weight ratios from 9:1 to 8:2) . Thus, although a direct dependence was found between the growth of the enveloping structure and the resistance of the coated liposomes to be affected by the surfactant, the more effective protection occurred when this structure was a thin film formed by the assembly of various layers of GP of about 2-3 nm .

Curr Opin Microbiol, 1999 Oct, 2(5), 483 - 8
Resistance gene capture; Rowe-Magnus DA et al.; Integrons are the primary mechanism for antibiotic-resistance gene capture and dissemination among Gram-negative bacteria . The recent finding of super-integron structures in the genomes of several bacterial species has expanded their role in genome evolution and suggests that they are the source of mobile multi-resistant integrons.

Antimicrob Agents Chemother, 1999 Oct, 43(10), 2400 - 3
Characterization of mutations in the rpoB gene in naturally rifampin-resistant Rickettsia species; Drancourt M et al.; Rickettsiae are gram-negative, obligately intracellular bacteria responsible for arthropod-borne spotted fevers and typhus . Experimental studies have delineated a cluster of naturally rifampin-resistant spotted fever group species . We sequenced the 4, 122- to 4,125-bp RNA polymerase beta-subunit-encoding gene (rpoB) from typhus and spotted fever group representatives and obtained partial sequences for all naturally rifampin-resistant species . A single point mutation resulting in a phenylalanine-to-leucine change at position 973 of the Rickettsia conorii rpoB sequence and present in all the rifampin-resistant species was absent in all the rifampin-susceptible species . rpoB-based phylogenetic relationships among these rickettsial species yielded topologies which were in accordance with previously published phylogenies.

J Am Optom Assoc, 1999 Aug, 70(8), 525 - 30
Cat-scratch neuroretinitis; Lombardo J; BACKGROUND: Cat-scratch disease is a subacute regional lymphadenitis, usually preceded by a history of a cat scratch or exposure to kittens . The disease is caused by Bartonella henselae, and possibly Bartonella quintana, pleomorphic gram-negative rods formerly known as Rochalimaea henselae and Rochalimaea quintana . Ocular involvement is rare and typically manifests as either Parinaud's oculoglandular syndrome or neuroretinitis . Patients with neuroretinitis resulting from cat-scratch disease may be asymptomatic or experience mild-to-severe vision loss . The clinical features, angiographic appearance, differential diagnosis, and management of cat-scratch neuroretinitis are discussed . CASE REPORT: A 30-year-old white woman reported to the eye clinic with painless, decreased vision in the right eye . A diagnosis of cat scratch neuroretinitis was made on the basis of the history of cat scratch, clinical appearance, and angiographic findings . Treatment with oral ciprofloxacin restored vision to normal in 4 weeks . CONCLUSION: Painless vision loss associated with optic nerve swelling and macular star exudate should alert suspicion of systemic disease . Additional findings--including positive history of a cat scratch, lymphadenopathy, and flu-like symptoms--may indicate Bartonella henselae or Bartonella quintana infection . While treatment remains controversial, appropriate serology testing may aid in the diagnosis and management of the underlying infection.

Vaccine, 1999 Oct 1, 17 Suppl 2, S13 - 21
Immunotherapy of sepsis: flawed concept or faulty implementation?
Cross AS, Opal SM, Bhattacharjee AK, Donta ST, Peduzzi PN, Furer E, Que JU, Cryz SJ.
Gram-negative bacillary sepsis is a leading cause of death among patients hospitalized in intensive care units . While initial clinical studies with the passive administration of anti-endotoxin core-glycolipid (CGL) antibodies for the treatment and prophylaxis of sepsis showed promising results, subsequent studies failed to show a consistent benefit . There appears to be a good correlation between anti-CGL antibody levels at the onset of sepsis and maintenance of antibody levels during sepsis with outcome . Previous clinical studies may have failed because insufficient amounts of antibody were administered early in the course of sepsis . Unlike the case with anti-CGL antibodies, polyvalent, hyperimmune type-specific antibody preparations may prevent the development of infections; however, these antibodies also must be provided in adequate amounts and in close proximity to infection in order to provide a beneficial effect . These pharmacokinetic requirements may limit the utility of passive immunotherapy for the prophylaxis of sepsis . Active immunization of acutely traumatized patients or of rats subsequently rendered neutropenic with cyclophosphamide induced high antibody levels for extended periods of time . Since trauma and other conditions are associated with a Th(2) response, these conditions may favor antibody formation following active immunization . Active immunization with both anti-CGL and/or polyvalent-specific vaccines for the prophylaxis of sepsis with passive supplementation at the onset of sepsis is an approach that merits further investigation.

Eur J Biochem, 1999 Oct, 265(2), 524 - 9
The lipopolysaccharide of moraxella catarrhalis structural relationships and antigenic properties; Holme T et al.; Moraxella catarrhalis has recently been shown to be both widespread and pathogenic, in contrast to previous reports . Several factors have been suggested as virulence factors, lipopolysaccharide (LPS) being one . Recent studies have shown the LPS to be without the O-chain, i.e . the polysaccharide part, and to have specific structural features corresponding to each of the three serogroups, A, B and C . The structures resemble in many respects those present in other Gram-negative nonenteric bacteria, with a galabiosyl element as a prominent common denominator . The presence of such common structures suggests that the LPS of these bacteria might be a part of a mechanism of survival for bacteria colonizing the human host.

Microb Pathog, 1999 Oct, 27(4), 187 - 96
The form variation of the capsular polysaccharide K1 is not a critical virulence factor of Escherichia coli in a neonatal mouse model of infection; Colino J et al.; Escherichia coli K1 is a prevalent cause of Gram-negative neonatal bacteraemia and meningitis in humans . Its capsular polysaccharide K1 (CpsK1) has been identified as an important virulence factor . Nevertheless, the biological and pathogenic implications of its O-acetylated and non-O-acetylated forms are poorly understood . In an attempt to address this, we monitored the expression of both CpsK1 form variants in a neonatal mouse infection model . In the absence of anti-CpsK1 antibodies, no CpsK1 form variant selection was observed during the course of infection . The administration of monoclonal antibodies specific for CpsK1 provided a high level of protection . The monoclonal antibodies that recognized both CpsK1 forms (MGB12) provided protection from up to 850 LD(50) . By contrast, the administration of the monoclonal antibodies (MGB15) specific for non-O-acetylated CpsK1 cleared only bacteria expressing this CpsK1 form; a few mouse pups remained bacteraemic, and the bacteria in the blood had O-acetylated CpsK1 . In those pups, the infection progressed in a similar fashion to that in mice not treated with monoclonal antibody . Moreover, when the number of bacteria expressing the O-acetylated CpsK1 in the inoculated dose is considered independently, the LD(50)was similar to that for the original strain in pups that had not been treated with monoclonal antibodies (35 CFU) . These results suggest that whereas variation in acetylation form per se does not reinforce virulence, it could enable E . coli to avoid immune defenses . This highlights the importance of using highly specific monoclonal antibodies in immunotherapeutic approaches to E . coli K1 neonatal meningitis .

Semin Respir Infect, 1999 Sep, 14(3), 209 - 17
Pathogenesis of pneumococcal pneumonia; Novak R et al.; Pneumococcal pneumonia is the most severe of the common community-acquired pulmonary infections . The recent release of the complete DNA sequence reveals the entire capability of the bacteria and the current challenge is to map gene products to the mechanics of disease . This process will reveal antibiotic targets and protein vaccine candidates crossing serotype boundaries . Choline is a major constituent of surfactant and it is also a required nutrient for the pneumococcus . It appears that choline incorporated into the cell wall can bind the bacteria to the receptor for platelet activating factor, the gateway to invasion . The choline also serves as an antenna to which multifunctional proteins dock, thereby decorating the bacterial surface . This set of 12 choline binding proteins is subject to phase variation of expression resulting in display of different combinations of proteins that adapt the bacteria to survival on the mucosa versus the blood stream . These changes affect protective antigens, adhesions, and lytic proteins tying together the major elements of pneumococcal physiology: natural DNA transformation, adherence and invasion of host cells, and autolysis . Taking these components and building an understanding of disease is challenging . Clearly, the toxin pneumolysin is a major mediator of cell damage in the lung . Inflammation is also incited by cell wall components . The signal transduction pathways that explain pneumococcal inflammation are more complex than those for gram-negative endotoxin.

Proc Natl Acad Sci U S A, 1999 Sep 28, 96(20), 11584 - 8
Titanospirillum velox: a huge, speedy, sulfur-storing spirillum from Ebro Delta microbial mats; Guerrero R et al.; A long (20-30 micrometer), wide (3-5 micrometer) microbial-mat bacterium from the Ebro Delta (Tarragona, Spain) was grown in mixed culture and videographed live . Intracellular elemental sulfur globules and unique cell termini were observed in scanning-electron-microprobe and transmission-electron micrographs . A polar organelle underlies bundles of greater than 60 flagella at each indented terminus . These Gram-negative bacteria bend, flex, and swim in a spiral fashion; they translate at speeds greater than 10 body lengths per second . The large size of the spirillum permits direct observation of cell motility in single individual bacteria . After desiccation (i.e., absence of standing water for at least 24 h), large populations developed in mat samples remoistened with sea water . Ultrastructural observations reveal abundant large sulfur globules irregularly distributed in the cytoplasm . A multilayered cell wall, pliable and elastic yet rigid, distends around the sulfur globules . Details of the wall, multiflagellated termini, and large cytoplasmic sulfur globules indicate that these fast-moving spirilla are distinctive enough to warrant a genus and species designation: Titanospirillum velox genus nov., sp . nov . The same collection techniques at a similar habitat in the United States (Plum Island, northeast Essex County, Massachusetts) also yielded large populations of the bacterium among purple phototrophic and other inhabitants of sulfurous microbial-mat muds . The months-long survival of T . velox from Spain and from the United States in closed jars filled with mud taken from both localities leads us to infer that this large spirillum has a cosmopolitan distribution.

J Bacteriol, 1999 Oct, 181(19), 6152 - 9
Identification of a regulator that controls stationary-phase expression of catalase-peroxidase in Caulobacter crescentus; Rava PS et al.; Expression of the catalase-peroxidase of Caulobacter crescentus, a gram-negative member of the alpha subdivision of the Proteobacteria, is 50-fold higher in stationary-phase cultures than in exponential cultures . To identify regulators of the starvation response, Tn5 insertion mutants were isolated with reduced expression of a katG::lacZ fusion on glucose starvation . One insertion interrupted an open reading frame encoding a protein with significant amino acid sequence identity to TipA, a helix-turn-helix transcriptional activator in the response of Streptomyces lividans to the peptide antibiotic thiostrepton, and lesser sequence similarity to other helix-turn-helix regulators in the MerR family . The C . crescentus orthologue of tipA was named skgA (stationary-phase regulation of katG) . Stationary-phase expression of katG was reduced by 70% in the skgA::Tn5 mutant, and stationary-phase resistance to hydrogen peroxide decreased by a factor of 10 . Like the wild type, the skgA mutant exhibited starvation-induced cross-resistance to heat and acid shock, entered into the helical morphology that occurs after 9 to 12 days in stationary phase, and during exponential growth induced katG in response to hydrogen peroxide challenge . Expression of skgA increased 5- to 10-fold in late exponential phase . skgA is the first regulator of a starvation-induced stress response identified in C . crescentus . SkgA is not a global regulator of the stationary-phase stress response; its action encompasses the oxidative stress-hydrogen peroxide response but not acid or heat responses . Moreover, SkgA is not an alternative sigma factor, like RpoS, which controls multiple aspects of starvation-induced cross-resistance to stress in enteric bacteria . These observations raise the possibility that regulation of stationary-phase gene expression in this member of the alpha subdivision of the Proteobacteria is different from that in Escherichia coli and other members of the gamma subdivision.

Int Rev Cytol, 2000, 194, 133 - 96
Mitochondrial proteins at unexpected cellular locations: export of proteins from mitochondria from an evolutionary perspective; Soltys BJ et al.; Researchers in a wide variety of unrelated areas studying functions of different proteins are unexpectedly finding that their proteins of interest are actually mitochondrial proteins, although functions would appear to be extramitochondrial . We review the leading current examples of mitochondrial macromolecules indicated to be also present outside of mitochondria that apparently exit from mitochondria to arrive at their destinations . Mitochondrial chaperones, which have been implicated in growth and development, autoimmune diseases, cell mortality, antigen presentation, apoptosis, and resistance to antimitotic drugs, provide some of the best studied examples pointing to roles for mitochondria and mitochondrial proteins in diverse cellular phenomena . To explain the observations, we propose that specific export mechanisms exist by which certain proteins exit mitochondria, allowing these proteins to have additional functions at specific extramitochondrial sites . Several possible mechanisms by which mitochondrial proteins could be exported are discussed . Gram-negative proteobacteria, from which mitochondria evolved, contain a number of different mechanisms for protein export . It is likely that mitochondria either retained or evolved export mechanisms for certain specific proteins.

J Anim Sci, 1999 Sep, 77(9), 2523 - 32
Effect of a prolonged low-dose lipopolysaccharide infusion on feed intake and metabolism in heifers; Steiger M et al.; Prolonged infusions of bacterial lipopolysaccharides (LPS) are known to model gram-negative bacterial infections, but the basic mechanisms of the LPS effects on feed intake and metabolism and their potential interdependence are largely unknown . The aim of the present study was to distinguish and to better characterize the feeding suppressive and metabolic effects of LPS . Six heifers were infused intravenously for 100 min with either 1) LPS (2 microg/kg BW) with free access to feed, 2) saline with free access to feed, or 3) saline with feeding restricted to the amount of feed consumed after LPS infusion . Feed intake, body temperature, plasma concentrations of various metabolites and hormones, and the respiratory quotient and heat production were measured . The LPS reduced feed intake and induced pronounced changes in metabolic energy turnover and fat and carbohydrate metabolism that were largely independent of the concomitant feed intake reduction . Some of the metabolic changes were biphasic; the first phase resembled a stress response with increases in plasma glucose and cortisol, and the second phase reflected a beginning energy deficit with low plasma glucose and enhanced lipolysis . The coincidence of a short-term surge of plasma insulin with marked transient decreases in plasma FFA, glycerol, and beta-hydroxybutyrate as well as with the transition from hyper- to hypoglycemia indicates that insulin plays a role in some of the metabolic responses to LPS . The failure of LPS to clearly increase energy expenditure despite the increase in body temperature suggests that anaerobic mechanisms of heat production and, perhaps, a reduced peripheral blood flow contributed to the fever . Many of the initial metabolic responses occurred before and, therefore, independent of, an increase in circulating tumor necrosis factor-alpha.

J Biotechnol, 1999 Aug 20, 73(2-3), 261 - 73
Extended recombinant bacterial ghost system; Lubitz W et al.; Controlled expression of cloned PhiX174 gene E in Gram-negative bacteria results in lysis of the bacteria by formation of an E-specific transmembrane tunnel structure built through the cell envelope complex . Bacterial ghosts from a variety of bacteria are used as non-living candidate vaccines . In the recombinant ghost system, foreign proteins are attached on the inside of the inner membrane as fusions with specific anchor sequences . Ghosts have a sealed periplasmic space and the export of proteins into this space vastly extends the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens . In addition, S-layer proteins forming shell-like self assembly structures can be expressed in candidate vaccine strains prior to E-mediated lysis . Such recombinant S-layer proteins carrying foreign epitopes further extend the possibilities of ghosts as carriers of foreign epitopes . As ghosts have inherent adjuvant properties, they can be used as adjuvants in combination with subunit vaccines . Subunits or other ligands can also be coupled to matrixes like dextran which are used to fill the internal lumen of ghosts . Oral, aerogenic or parenteral immunization of experimental animals with recombinant ghosts induced specific humoral and cellular immune responses against bacterial and target components including protective mucosal immunity . The most relevant advantage of recombinant bacterial ghosts as immunogens is that no inactivation procedures that denature relevant immunogenic determinants are employed in this production . This fact explains the superior quality of ghosts when compared to other inactivated vaccines . The endotoxic component of the outer membrane does not limit the use of ghosts as vaccine candidates but triggers the release of several potent immunoregulatory cytokines . As carriers, there is no limitation in the size of foreign antigens that can be inserted in the membrane and the capacity of all spaces including the membranes, peri-plasma and internal lumen of the ghosts can be fully utilized . This extended recombinant ghost system represents a new strategy for adjuvant free combination vaccines.

J Comp Pathol, 1999 Oct, 121(3), 301 - 6
Systemic zygomycosis in farmed tilapia fish; Wolf JC et al.; Decreased feed intake and persistent low-level mortality in a production tank of hybrid tilapia (Oreochromis niloticus x Oreochromis mossambicus x Oreochromis aureus) prompted the submission of three affected fish for diagnosis . Consistent macrosopical findings included multifocal dermal haemorrhage, excess abdominal fluid and an enlarged friable liver . On microscopical examination, broad non-septate fungal hyphae and chlamydospores were identified within numerous internal organs, often within and adjacent to blood vessels . The fungal hyphae were readily seen by silver staining (GMS) and the chlamydospores were stained deep magenta by the periodic acid-Schiff reaction . In addition to several species of Gram-negative bacteria, moderate growths of woolly white fungal colonies were obtained from the posterior part of the kidney in two of the three tilapia . These colonies were identified as a Rhizomucor sp . on the basis of the morphological characteristics of the sporulating fungi in culture . This represents the first reported episode of zygomycosis in fish .

Endocrine, 1999 Jun, 10(3), 201 - 17
Role of cytokines in testicular function; Hales DB et al.; Inflammatory disease has been established to affect male reproductive function and fertility . Relevant inflammatory diseases include general and chronic infectious diseases as well as localized acute or chronic infections of the male genitourinary tract . Male accessory gland infections account for almost 15% of all cases of male infertility seen in infertility clinics while fertility usually is not a clinical objective among patients with acute systemic infections such as Gram-negative sepsis . Infections of the male accessory glands frequently are associated with increased counts of white blood cells in semen and elevated levels of proinflammatory cytokines in semen and the testis . There is a mounting body of evidence that demonstrates the importance of cytokines and chemokines in the regulation of testicular and glandular function during pathophysiological states as well as under normal physiological conditions when cytokines act as growth and differentiation factors . The purpose of this review is to examine the role of cytokines in the regulation of steroidogenesis and spermatogenesis in the testis under physiological and pathophysiological conditions and considers clinical investigations that help to improve the evaluation and treatment of male infertility.

J Bacteriol, 1999 Sep, 181(18), 5644 - 51
A nonessential signal peptidase II (Lsp) of Myxococcus xanthus might be involved in biosynthesis of the polyketide antibiotic TA; Paitan Y et al.; Myxococcus xanthus is a gram-negative soil bacterium that produces the polyketide antibiotic TA . In this study, we describe the analysis of an M . xanthus gene which encodes a homologue of the prolipoprotein signal peptidase II (SPase II; lsp) . Overexpression of the M . xanthus SPase II in Escherichia coli confers high levels of globomycin resistance, confirming its function as an SPase II . The M . xanthus gene encoding the lsp homologue is nonessential for growth, as determined by specific gene disruption . It has been mapped to the antibiotic TA gene cluster, and the disrupted mutants do not produce the antibiotic, indicating a probable involvement in TA production . These results suggest the existence of more than one SPase II protein in M . xanthus, where one is a system-specific SPase II (for TA biosynthesis).

Trends Cell Biol, 1999 Oct, 9(10), 402 - 8
Bacterial type II protein export and pilus biogenesis: more than just homologies?
Nunn D.
Protein export by Gram-negative bacteria requires devoted machineries to allow for the passage of hydrolytic enzymes and toxins through the cell envelope . The Type II export machinery has a number of distinct characteristics, which include its role as an extension of Sec-dependent secretion, its ability to recognize and export fully folded substrates efficiently and, perhaps most significantly, the relationship between a subset of its gene products with the Type IV pilus-biogenesis apparatus . An important question is whether we can extrapolate our knowledge, albeit limited, of Type IV pilus biogenesis to understand the structure and function of the Type II export apparatus . This and other questions relating to the energetics of assembly and specificity of the apparatus are addressed in this article.

Blood, 1999 Sep 1, 94(5), 1711 - 6
IkappaB kinase complex is an intracellular target for endotoxic lipopolysaccharide in human monocytic cells; Hawiger J et al.; Endotoxic lipopolysaccharide (LPS) is a proinflammatory agonist produced by gram-negative bacteria and a contributor to the majority of the 400,000 septic shock cases recorded annually in US hospitals . The primary target cells for LPS are monocytes and macrophages . Their response consists of massive production of proinflammatory cytokines, reactive oxygen- and nitrogen-intermediates, procoagulants, and cell adhesion molecules . In turn, expression of these LPS-responsive factors contributes to collapse of the circulatory system, to disseminated intravascular coagulation, and to a 30% mortality rate . A common intracellular mechanism responsible for the expression of septic shock genes in monocytes and macrophages involves the activation of NF-kappaB . This transcription factor is regulated by a family of structurally related inhibitors including IkappaBalpha, IkappaBbeta, and IkappaBepsilon, which trap NF-kappaB in the cytoplasm . In this report, the investigators show that LPS derived from different gram-negative bacteria activates cytokine-responsive IkappaB kinases containing catalytic subunits termed IKKalpha (IKK1) and IKKbeta (IKK2) . The kinetics of IKKalpha and IKKbeta activation in LPS-stimulated human monocytic cells differ from that recorded on their stimulation with tumor necrosis factor-alpha, thereby implying a distinct activation mechanism . LPS-activated IKK complexes phosphorylate all 3 inhibitors of NF-kappaB: IkappaBalpha, IkappaBbeta, and IkappaBepsilon . Moreover, LPS activates IKKbeta preferentially, relative to IKKalpha . Thus, IKK complex constitutes the main intracellular target for LPS-induced NF-kappaB signaling to the nucleus in human monocytic cells to activate genes responsible for septic shock.

Microbiol Mol Biol Rev, 1999 Sep, 63(3), 507 - 22
Transposon Tn21, flagship of the floating genome; Liebert CA et al.; The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria . The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes . The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon . We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains . Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon . Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron . The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium . A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon . Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon . The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents . The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.

FEMS Microbiol Lett, 1999 Aug 15, 177(2), 263 - 70
Detection of genes for periplasmic nitrate reductase in nitrate respiring bacteria and in community DNA; Flanagan DA et al.; A nested PCR primed by four degenerate oligonucleotides was developed for the specific amplification of sequences from the napA gene encoding the periplasmic nitrate reductase . This approach was used to amplify fragments of the napA gene from 10 Pseudomonas species and one Moraxella sp., previously shown to be able to express the periplasmic nitrate reductase activity, from Rhodobacter capsulatus and from community DNA extracted from a fresh-water sediment . Amino acid sequences encoded by the napA fragments were compared to one another and to the corresponding regions of related enzymes . This comparison indicates that the amplification protocol is specific for its intended target . The napA sequences amplified from community DNA were tightly clustered, which may indicate a degree of homogeneity in the sediment community . All tested Gram-negative strains capable of aerobic nitrate respiration were found to have periplasmic nitrate reductase genes . However, some strains which have and express the genes are incapable of aerobic nitrate respiration . The PCR primers and amplification protocols described will be useful in future studies of nitrate respiring populations.

Bioessays, 1999 Jul, 21(7), 590 - 5
Fibrils as extracellular appendages of bacteria: their role in contact-mediated cell-cell interactions in Myxococcus xanthus; Dworkin M; Social behavior in the myxobacterium Myxococcus xanthus involves epicellular, peritrichous appendages called fibrils . These are polysaccharide organelles containing a set of tightly adhering proteins . It is proposed that cell-cell contact is perceived by the fibrils and is mediated by the action of a fibrillar ADP-ribosyl transferase . Fibrils or fibril-like organelles have also been found on a variety of other gram-negative bacteria and at least one archaeon, and may mediate cell-cell contact between the bacteria themselves or between the bacteria and their eukaryotic host cells.

Trends Microbiol, 1999 Sep, 7(9), 356 - 61
The interaction between RTX toxins and target cells; Lally ET et al.; RTX toxins are important virulence factors produced by a wide range of Gram-negative bacteria . They fall into two categories: the hemolysins, which affect a variety of cell types, and the leukotoxins, which are cell-type- and species-specific . These toxins offer interesting models for targeting, insertion and translocation of aqueous proteins into lipid membranes.

Acta Microbiol Pol, 1999, 48(1), 39 - 51
Methylobacterium extorquens strain P14, a new methylotrophic bacteria producing poly-beta-hydroxybutyrate (PHB); Ostafin M et al.; Strain P14 of facultative methylotrophic bacteria that synthetisizes poly-beta-hydroxybutyrate has been isolated . The cells are gram-negative motile rods with a polar flagellum . They do not form spores or capsules, but do have a caretenoid pigment . Predominant in the fatty acid composition of the cells is cis-vaccenic acid (cis 18:1: omega 7)--72% . In the phospholipid composition phosphatidylcholine predominates (45%), along with phosphatidylenthanoloamine (27%) and phosphatidylglycerol (17%) . The main biquinone is Q-10; other ubiquinones (Q-8, Q-9, Q-11) are present in minor quantities . The cells accomplish the icl-variant of serine pathway . The GC content of DNA (Tm) is 65 mole% . A high level DNA-DNA homology with representatives of the genus Methylobacterium was observed . The strain has been identified as Methylobacterium extorquens strain P14.

J Chemother, 1999 Aug, 11(4), 248 - 54
Endotoxin release due to ciprofloxacin measured by three different methods; Trautmann M et al.; Antibiotics are known to induce the release of bioactive endotoxin (LPS) from gram-negative bacterial cells . Because varying data have been published on the influence of quinolone antibiotics on LPS liberation, we studied the effect of ciprofloxacin on a culture of Escherichia coli by determining bacterial killing and free LPS concentrations in comparison with imipenem and ceftazidime . LPS levels were measured by three different methods, namely (1) the Limulus amebocyte lysate test, (2) an ELISA method based on capture of LPS by monoclonal antibodies, and (3) indirect determination by measuring the ability of antibiotic-induced LPS to trigger TNFalpha release from a monocytic cell line . With both the Limulus and ELISA tests, a low endotoxin-releasing activity of ciprofloxacin was confirmed . In contrast to previous studies, this LPS also had low bioactivity in terms of TNFalpha induction . Limulus LPS determinations correlated more precisely with LPS bioactivity than did ELISA values, an observation which underlines the crucial role of LPS determination methods in studies of antibiotic-induced LPS release.

Am Ind Hyg Assoc J, 1999 Jul-Aug, 60(4), 480 - 5
A pilot study for monitoring changes in the microbiological component of metalworking fluids as a function of time and use in the system; Lonon MK et al.; This article describes the results of a pilot study to examine changes in the biological component of metalworking fluids (MWF) as a function of use . Fluid samples were taken from two newly charged systems, designated BT-7415 and BT-7707, at 1-week intervals for 8 weeks and characterized with respect to the kinds and numbers of bacteria present and presence of soluble protein in cell-free supernatants . In addition, lipid extracts of pelleted cells from fluids in BT-7415 were examined by gas chromatography/mass spectroscopy for the kinds and relative amounts of phospholipid fatty acids (PLFA) present . A total of 19 different bacterial species was cultured and identified, more than half (12/19) of which were gram-negative . Total colony-forming units (CFU) reached levels of 2.2 x 10(3)/mL in BT-7415 and 2.4 x 10(5)/mL in BT-7707 . The most common genus isolated was Pseudomonas . Estimations of cell numbers based on total biomass from PLFA in samples from BT-7415 indicated 1.1 x 10(7)/mL after 8 weeks of use . Both the numbers of PLFA identified and the amounts of each detected in BT-7415 increased as the fluids were used . The chromatograms were dominated by two fatty acids, the amounts of which increased with time . These fatty acids, 18:2 omega 6 and 18:1 omega 9c, are not commonly associated with pseudomonads . This suggests that there is an important component of the biological consortium in MWF is not being detected by currently used culture techniques . There was no soluble protein detected in any of the samples from either system.

J Antimicrob Chemother, 1999 Jul, 44(1), 11 - 8
Identification and characterization of class 1 integrons in bacteria from an aquatic environment; Rosser SJ et al.; In a survey of 3000 Gram-negative bacteria isolated from an estuarine environment over a 2 month period, the incidence of class 1 integrons was determined to be 3.6% . Of 85 integrons studied further, 11 lacked both the qacEdelta1 and sull genes usually present in the 3' conserved segment of the integron . The qacEdelta1 and sull genes were identified in the 3' conserved segment of 36 integrons . The remaining 38 integrons lacked a sull gene but contained a qacE gene . The variable region of 74 integrons was characterized by PCR and sequence analysis . Forty of the integrons were found to lack integrated gene cassettes, although 21 of these 'empty' integrons were shown to contain inserted DNA which has been tentatively identified as a novel insertion sequence (IS) element . Of the 34 integrons which contained inserted gene cassettes, the aadA1a gene was found to be the most prevalent (74%) . Nineteen integrons contained additional or other gene cassettes in their variable region, including those encoding resistance to trimethoprim (dfr1a, dfrIIc, dfrV, dfrVII, dfrXII), chloramphenicol (catB3, catB5), aminoglycosides (aadA2, aacA4, aacC1), beta-lactamases (oxa2) and erythromycin (ereA) . This study confirms the occurrence of integrons in bacteria from a natural habitat and suggests that in the absence of continued antibiotic selective pressures, integrons which persist appear to preferentially exist without integrated antibiotic resistance gene cassettes.

Infect Immun, 1999 Sep, 67(9), 4668 - 72
Lactoferrin-lipid A-lipopolysaccharide interaction: inhibition by anti-human lactoferrin monoclonal antibody AGM 10.14; Caccavo D et al.; Lactoferrin (LF) is a glycoprotein that exerts both bacteriostatic and bactericidal activities . The interaction of LF with lipopolysaccharide (LPS) of gram-negative bacteria seems to play a crucial role in the bactericidal effect . In this study, we evaluated, by means of an enzyme-linked immunosorbent assay, the binding of biotinylated LF to the S (smooth) and R (rough) (Ra, Rb, Rc, Rd1, Rd2, and Re) forms of LPS and different lipid A preparations . In addition, the effects of two monoclonal antibodies (AGM 10.14, an immunoglobulin G1 {IgG1} antibody, and AGM 2.29, an IgG2b antibody), directed against spatially distant epitopes of human LF, on the LF-lipid A or LF-LPS interaction were evaluated . The results showed that biotinylated LF specifically binds to solid-phase lipid A, as this interaction was prevented in a dose-dependent fashion by either soluble uncoupled LF or lipid A . The binding of LF to S-form LPS was markedly weaker than that to lipid A . Moreover, the rate of LF binding to R-form LPS was inversely related to core length . The results suggest that the polysaccharide O chain as well as oligosaccharide core structures may interfere with the LF-lipid A interaction . In addition, we found that soluble lipid A also inhibited LF binding to immobilized LPS, demonstrating that, in the whole LPS structure, the lipid A region contains the major determinant recognized by LF . AGM 10.14 inhibited LF binding to lipid A and LPS in a dose-dependent fashion, indicating that this monoclonal antibody recognizes an epitope involved in the binding of LF to lipid A or some epitope in its close vicinity . In contrast, AGM 2.29, even in a molar excess, did not prevent the binding of LF to lipid A or LPS . Therefore, AGM 10.14 may represent a useful tool for neutralizing selectively the binding of LF to lipid A . In addition, the use of such a monoclonal antibody could allow better elucidation of the consequences of the LF-lipid A interaction.

Acta Anaesthesiol Scand, 1999 Aug, 43(7), 726 - 30
Factors affecting gentamicin pharmacokinetics in septic patients; Tang GJ et al.; BACKGROUND: This prospective, comparative study was designed to estimate the volume of distribution (Vd) and elimination rate constant (K(e)) of gentamicin and to determine the clinical factors affecting the pharmacokinetics of gentamicin in different stages of sepsis . METHOD: Seventy-seven critically ill patients treated with gentamicin for gram-negative sepsis were included . These septic patients were divided into hyperdynamic septic and hypodynamic septic groups according to cardiac index . Twenty-seven patients who received postoperative prophylactic gentamicin were recruited as controls . RESULTS: Fifty-two patients in the hyperdynamic septic group had a significantly larger Vd than those in the hypodynamic septic and control groups . The Vd was correlated significantly with both Acute Physiological Score (APS) (r=0.340, P<0.01) and cardiac index (r=0.394, P<0.01) . The K(e) of gentamicin correlated significantly with both blood urea nitrogen (BUN) (r= 0.565, P<0.01) and serum creatinine level (r=0.563, P<0.01) . CONCLUSION: The increased Vd in the septic patients was related to the severity of illness and magnitude of cardiac output . The K(e) of gentamicin was correlated with the serum creatinine level.

J Biol Chem, 1999 Aug 27, 274(35), 24567 - 74
Skp, a molecular chaperone of gram-negative bacteria, is required for the formation of soluble periplasmic intermediates of outer membrane proteins; Schafer U et al.; Using a cross-linking approach, we have analyzed the function of Skp, a presumed molecular chaperone of the periplasmic space of Escherichia coli, during the biogenesis of an outer membrane protein (OmpA) . Following its transmembrane translocation, OmpA interacts with Skp in close vicinity to the plasma membrane . In vitro, Skp was also found to bind strongly and specifically to pOmpA nascent chains after their release from the ribosome suggesting the ability of Skp to recognize early folding intermediates of outer membrane proteins . Pulse labeling of OmpA in spheroplasts prepared from an skp null mutant revealed a specific requirement of Skp for the release of newly translocated outer membrane proteins from the plasma membrane . Deltaskp mutant cells are viable and show only slight changes in the physiology of their outer membranes . In contrast, double mutants deficient both in Skp and the periplasmic protease DegP (HtrA) do not grow at 37 degrees C in rich medium . We show that in the absence of an active DegP, a lack of Skp leads to the accumulation of protein aggregates in the periplasm . Collectively, our data demonstrate that Skp is a molecular chaperone involved in generating and maintaining the solubility of early folding intermediates of outer membrane proteins in the periplasmic space of Gram-negative bacteria.

Shock, 1999 Jun, 11(6), 436 - 42
Enhanced prostanoid-mediated vasorelaxation in pulmonary arteries isolated during experimental endotoxemia; Myers TP et al.; Endotoxemia secondary to gram-negative sepsis has been shown to inhibit endothelium-dependent vasomotion in numerous vascular beds, including guinea pig aortae and coronary arteries . We tested the hypothesis that in vivo endotoxin impairs endothelium-dependent nitric oxide-mediated relaxation responses of pulmonary arteries isolated from guinea pigs given intraperitoneal injections of Escherichia coli endotoxin lipopolysaccharide (LPS) or saline (control) 16 h before sacrifice . Pulmonary rings from the main artery and primary branches were isolated and studied in vitro using conventional isometric techniques . Interestingly, endotoxemia resulted in enhanced pulmonary artery relaxation in response to the endothelium-dependent receptor agonists acetylcholine (10(-10) -10(-5) M) and adenosine diphosphate (ADP; 10(-9) -10(-5) M), as compared with control responses (p < .05) . Nitric oxide synthase inhibitors N-monomethyl-L-arginine (300 microM) and N-nitro-L-arginine methyl ester (100 microm) reduced acetylcholine- and adenosine diphosphate-mediated relaxation in both groups (p < .05); however, vasodilation responses in arteries from LPS animals remained enhanced relative to those of control arteries . In contrast to nitric oxide synthase inhibitors, the cyclooxygenase inhibitor indomethacin markedly inhibited acetylcholine- and adenosine diphosphate-mediated relaxation responses of pulmonary arteries isolated from LPS-treated animals (p < .05) but not control arteries; indomethacin effectively reversed LPS-induced enhanced vasodilation of pulmonary arteries . Relaxation responses to the receptor-independent calcium ionophore (A23187) and to the direct smooth muscle vasodilator sodium nitroprusside (+ N-nitro-L-arginine methyl ester) were not significantly altered by LPS treatment (p > .05) . These data suggest that in pulmonary arteries, unlike aortae and coronary arteries isolated from the same model, in vivo LPS enhances agonist-mediated endothelium-dependent vasodilation responses to acetylcholine and adenosine diphosphate . Underlying mechanisms appear to involve increased dependency upon vasodilator prostanoids and decreased dependency on nitric oxide synthesis/release for LPS-induced alterations in pulmonary relaxation responses.

J Hepatol, 1999 Aug, 31(2), 277 - 83
Development of quinolone-resistant strains of Escherichia coli in stools of patients with cirrhosis undergoing norfloxacin prophylaxis: clinical consequences; Aparicio JR et al.; BACKGROUND/AIM: Norfloxacin prophylaxis decreases the incidence of bacterial infections in high-risk cirrhotic patients, but may promote the development of quinolone-resistant gram-negative bacteria in stools, and eventually lead to infections due to these bacteria . The aim of the study was to evaluate the prevalence of quinolone-resistant strains of E . coli in stools on admission, and the characteristics of any nosocomial infections . METHODS: Eighty-three consecutively hospitalized cirrhotic patients were included in this prospective study . The presence of quinolone-resistant strains of E . coli in stools on admission, and the characteristics of any nosocomial infections were recorded . RESULTS: Fourteen out of 83 patients (16.8%) showed quinolone-resistant E . coli in stools (Group I), and 69 did not (Group II) . Thirteen out of 14 from Group I (92.8%) and 17/69 (24.6) from Group II had received primary or secondary prophylaxis with norfloxacin (p<0.001) . During hospitalization, 12/12 (100%) of patients from Group I and 25/66 (37.8%) of patients from Group II underwent norfloxacin prophylaxis . Three bacterial infections in patients from Group I, 3 from Group II patients receiving norfloxacin and 16 from Group II patients not receiving norfloxacin were recorded (p<0.05) . No infections due to quinolone-resistant E . coli were observed in patients colonized with these bacteria . Treatment with norfloxacin induced the development of quinolone-resistant E . coli in 6/14 (42.8%) patients in a mean time of 18.5+/-9.8 days . CONCLUSIONS: The development of quinolone-resistant strains of E . coli is significantly associated with previous administration of norfloxacin prophylaxis . However, in our series this fact is not associated with an increased incidence of quinolone-resistant E . coli or other gram-negative infections.

Clin Infect Dis, 1999 May, 28(5), 1095 - 9
Bordetella bronchiseptica infection in human immunodeficiency virus-infected patients; Dworkin MS et al.; Bordetella bronchiseptica is a pleomorphic gram-negative coccobacillus that commonly causes respiratory tract infections in dogs . We identified nine human immunodeficiency virus (HIV)-infected persons with culture-confirmed B . bronchiseptica infections (eight respiratory tract and one disseminated infection) . The respiratory illnesses ranged in severity from mild upper respiratory tract infection to pneumonia . All nine patients had had at least one AIDS-defining condition before the B . bronchiseptica infection . Two patients had household contact with dogs before their illnesses, and one had household contact with cats . Infection due to B . bronchiseptica is uncommon in HIV-infected persons . Additional data are needed to fully define the spectrum of disease due to B . bronchiseptica infections and to evaluate the possibility that this infection may be acquired from pets . Treatment of B . bronchiseptica infection should be tailored to the patient and should be based on the results of susceptibility testing.

Biochemistry, 1999 Aug 17, 38(33), 10758 - 67
Lipopolysaccharide bilayer structure: effect of chemotype, core mutations, divalent cations, and temperature; Snyder S et al.; Lipopolysaccharide (LPS), the primary lipid on the surface of Gram-negative bacteria, is thought to act as a protective and permeability barrier . X-ray diffraction analysis of osmotically stressed LPS multilayers was used to determine the structure and interactive properties of LPSs from strains containing the minimum number of sugars necessary for bacterial survival (Re chemotype) to the maximum number of sugars found in rough bacteria (Ra chemotype) . At 20 degrees C in the absence of divalent cations, LPS suspensions gave a sharp wide-angle reflection at 4.23 A and a broad low-angle band centered at 50-68 A depending on the chemotype, indicating the presence of gel phase bilayers separated by large fluid spaces . As osmotic pressure was applied, the apposing bilayers were squeezed together and lamellar diffraction at 6 A resolution was obtained . At low applied pressures (<10(6) dyn/cm2), the total repulsive pressure between bilayers could be explained by electrostatic double layer theory . At higher applied pressures, there was a sharp upward break in each pressure-distance relation, indicating the presence of a hydrophilic steric barrier whose range depended strongly on the LPS chemotype . The positions of these upward breaks, along with electron density profiles, showed that the sugar core width systematically increased from 10 A for the Re chemotype to 27 A for the Ra chemotype . In excess buffer, the addition of divalent cations brought the bilayers into steric contact . Electron density profiles were used to determine the locations of cation binding sites and polar substituents on the LPS oligosaccharide core . The area per hydrocarbon chain was approximately 26 A2 in liquid-crystalline LPS bilayers, an indication of an acyl chain packing that is much tighter than that found in bilayers composed of typical membrane lipids . This unusually tight packing could be a critical factor in the permeability barrier provided by LPS.

Immunology, 1999 Jul, 97(3), 429 - 37
Lipopolysaccharide enhances FcgammaR-dependent functions in vivo through CD11b/CD18 up-regulation; Rubel C et al.; Fc receptors for immunoglobulin G (IgG) (FcgammaR) mediate several defence mechanisms in the course of inflammatory and infectious diseases . In Gram-negative infections, cellular wall lipopolysaccharides (LPS) modulate different immune responses . We have recently demonstrated that murine LPS in vivo treatment significantly increases FcgammaR-dependent clearance of immune complexes (IC) . In addition, we and others have reported the induction of adhesion molecules on macrophages and neutrophils by LPS in vivo and by tumour necrosis factor-alpha (TNF-alpha) in vitro . The aim of this paper was to investigate CD11b/CD18 participation in LPS enhancing effects on Fcgamma-dependent functionality of tissue macrophages . Our results have demonstrated that LPS can enhance antibody-dependent cellular cytotoxicity (ADCC) and IC-triggered cytotoxicity (IC-Ctx), two reactions which involve the Fcgamma-receptor but different lytic mechanisms . In vitro incubation of splenocytes from LPS-treated mice with anti-CD11b/CD18 abrogated ADCC and IC-Ctx enhancement, without affecting FcgammaR expression . Similar results were obtained with physiological concentrations of fibrinogen . In this way cytotoxic values of LPS-splenocytes decreased to the basal levels of control mice . Time and temperature requirements for such inhibition strongly suggested that anti-CD11b/CD18 could modulate intracellular signals leading to downregulation of FcgammaR functionality . Data presented herein support the hypothesis that functional and/or physical associations between integrins and FcgammaR could be critical for the modulation of effector functions during an inflammatory response.

Immunology, 1999 May, 97(1), 45 - 55
Increased inducible apoptosis in CD4+ T lymphocytes during polymicrobial sepsis is mediated by Fas ligand and not endotoxin; Ayala A et al.; Recent studies suggest that increased lymphocyte apoptosis (Ao) detected in peripheral blood T cells from burn patients appears to contribute to decreased lymphocyte immunoresponsiveness . However, while it is known that sepsis induces a marked depression in the splenocyte immune response (i.e . decreased interleukin-2, interferon-gamma production and proliferation) in response to the T-cell mitogen concanavalin A (Con A), it is unknown whether this depression is associated with an increase in inducible Ao and if so, which mediators control this process . To assess this, splenocytes were harvested from mice at 24 hr (a period associated with decreased Con A response) after the onset of polymicrobial sepsis {caecal ligation and puncture (CLP)} or sham-CLP (Sham) and then stimulated with 2.5 microg Con A/ml (24 hr) . Septic mouse splenocytes stimulated with Con A, while not showing a change in their phenotypic make-up, did exhibit a marked increase in the percentage of splenocyte that were Ao+ which was associated with altered cytokine release . This appears to be due to an increase in the percentage of Ao+ cells in the CD4+ CD8- population and was associated with enhanced Fas antigen expression as well as an increase in mRNA for the Fas-FasL gene family . To determine if the changes in Ao are due to either endotoxin (a product of Gram-negative bacteria seen in CLP mice) or the expression of Fas ligand (FasL; a mediator of activation-induced lymphocyte Ao), a second set of studies examining Con A-inducible Ao was performed with splenocytes harvested from septic endotoxin-tolerant C3H/HeJ and the FasL-deficient C3H/HeJ-Fasl gld mice . The results show that increased splenocyte Ao detected following CLP is due to a FasL-mediated process and not to endotoxin . Thus the inadvertent up-regulation of FasL-mediated splenocyte Ao may contribute to the depression of splenocyte immune responses seen during polymicrobial sepsis.

Scand J Infect Dis, 1999, 31(2), 185 - 9
Diagnostic value of procalcitonin serum levels in neutropenic patients with fever: comparison with interleukin-8; Engel A et al.; We assessed the predictive value of procalcitonin (PCT) serum levels in neutropenic patients with fever and various types of infection, using a prospective 3 times weekly blood sampling protocol during 103 patient episodes . Compared with pre-fever levels, median PCT levels increased after fever onset from 0.16 ng/ml (day -1) to 0.34 ng/ml (day +1) . In samples obtained within 32 h after fever onset, PCT levels differed significantly between (clinically or microbiologically) documented infection and unexplained fever (median 0.51 vs . 0.26 ng/ml), between bacteraemia and non-bacteraemic infection (median 0.8 vs . 0.27 ng/ml) and between Gram-negative bacteraemia and all other episodes (median 1.28 vs . 0.31 ng/ml) . Receiver-operating-characteristic (ROC) curves indicated that the discriminatory power of PCT was best for predicting bacteraemia vs . non-bacteraemic infection (sensitivity 73%; specificity 86%; area under the ROC curve 0.795; cut-off value 0.5 ng/ml) . Compared with interleukin-8 (IL-8) serum levels, test characteristics were similar in the prediction of bacteraemia vs . non-bacteraemic infection and in the prediction of documented infection vs . unexplained fever, while IL-8 was better than PCT in the prediction of Gram-negative bacteraemia (area under the ROC curve 0.965 vs . 0.758).

J Hand Surg {Am}, 1999 Jul, 24(4), 682 - 6
Upper extremity infections in patients with diabetes mellitus; Gonzalez MH et al.; Forty-five consecutive diabetic patients with 46 upper extremity infections who underwent surgical debridement were retrospectively reviewed . The infections involved the skin or subcutaneous tissue in 19 patients and the fascia, tendon, muscle, or bone in 27 . Twenty-three infections (50%) required a single operation and 23 required more than 1 . Eighteen infections (39%) required an amputation and there were 3 deaths directly related to an infection . Six of 7 infections in which anaerobic organisms were cultured culminated in amputation . Four patients were diagnosed with necrotizing fasciitis . Twenty-one cultures (46%) were polymicrobial . An increased rate of amputation was associated with deep infections below the subcutaneous tissue, renal failure, and infections with gram-negative, anaerobic, or polymicrobial cultures . An increased rate of repeat surgery and a prolonged hospitalization were associated with deep infection and polymicrobial infections.

Science, 1999 Aug 13, 285(5430), 1058 - 61
Structural basis of chaperone function and pilus biogenesis; Sauer FG et al.; Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway . Immunoglobulin-like periplasmic chaperones escort pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane . The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G(1) beta strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation . The structure of the PapD-PapK complex also suggests that during pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.

J Photochem Photobiol B, 1999 May, 50(1), 8 - 17
delta-Aminolaevulinic acid-induced porphyrin synthesis and photodynamic inactivation of Escherichia coli B; Szocs K et al.; The possibility and conditions for the induction of porphyrin synthesis by exogenous delta-aminolaevulinic acid (ALA) and its applicability for the inactivation of Gram-negative bacteria Escherichia coli B . by photodynamic therapy (PDT) have been studied . The bacteria are supplemented with ALA in the log phase of growth, and are grown in a synthetic medium at 37 degrees C in the dark . The efficiency of porphyrin synthesis is detected by fluorescence spectroscopy performed on the isolated bacterial cells and the medium, respectively, and compared with results of high-performance liquid chromatography (HPLC) analysis . ALA stimulates the synthesis of protoporphyrin in the bacteria by a factor of five to six, and an increased amount of the more hydrophilic derivatives with a significant contribution of mesoporphyrin by a factor of two to three is observed in the culturing medium . The optimal conditions of ALA treatment with respect to PDT are 10-15 min of incubation of a bacterial culture of 2 x 10(7) cells ml-1 with (5-9) x 10(-3) mol l-1 ALA . The ALA-treated cells are irradiated by white light of 80 mW cm-2 under growth conditions and a decrease to 0.6% of the number of colony-forming units (CFUs ml-1) is observed after 90 min of irradiation.

Curr Microbiol, 1999 Sep, 39(3), 146 - 52
Levansucrase from Acetobacter diazotrophicus SRT4 is secreted via periplasm by a signal-peptide-dependent pathway; Hernandez L et al.; Acetobacter diazotrophicus SRT4 secretes a constitutive levansucrase (LsdA) (EC 2.4.1.10) that is responsible for sucrose utilization . Immunogold electron microscopical studies revealed that LsdA accumulates in the periplasm before secretion . The periplasmic and extracellular forms of the enzyme were purified to homogeneity . Both proteins exhibited similar physical and biochemical characteristics indicating that LsdA adopts its final conformation in the periplasm . The N-terminal sequence of mature LsdA was pGlu-Gly-Asn-Phe-Ser-Arg as determined by PSD-MALDI-TOFMS (post-source decay-matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry) . Comparison of this sequence with the predicted precursor protein revealed the cleavage of a 30-residue typical signal peptide followed by the formation of the pyroglutamic acid (pGlu) residue . Thus, in contrast with other Gram-negative bacteria, A . diazotrophicus secretes levansucrase by a signal-peptide-dependent mechanism.

Pediatr Infect Dis J, 1999 Jul, 18(7), 591 - 5
Increasing incidence of gram-negative rod bacteremia in a newborn intensive care unit; Shah SS et al.; OBJECTIVES: To determine whether there has been an increase in the incidence or a change in the epidemiology of gram-negative rod (GNR) bacteremia in patients in a newborn special care unit . METHODS: Retrospective review of GNR bacteremia in patients hospitalized in the NBSCU at Yale-New Haven Hospital during a 10-year period . RESULTS: There were 120 isolates from 113 episodes of GNR bacteremia during the study period . The incidence of GNR bacteremia increased from a mean of 10.2 to 25.5 (P = 0.017) episodes of GNR bacteremia per 1000 admissions per year between the time periods 1988 to 1994 and 1995 to 1997, respectively, paralleling an increase in the overall incidence of bacteremia . The increase in GNR bacteremia in these two groups was not related to changes in the patient population, the number of admissions or duration of hospitalization . Stepwise multivariate analysis identified two independent variables associated with infants who had GNR bacteremia during the period 1995 to 1997 as compared with 1988 to 1994, maternal intrapartum antibiotics (odds ratio, 4.9; 95% confidence interval, 1.9 to 12.6) and the presence of a percutaneous central venous catheter (odds ratio, 4.6; 95% confidence interval, 1.8 to 11.8) . CONCLUSIONS: We observed changes in clinical obstetric and neonatal care that paralleled the increase in GNR bacteremia at our institution . A prospective study is needed to elucidate the impact of these changes on the incidence of GNR bacteremia in this population.

J Mol Biol, 1999 Aug 13, 291(2), 393 - 415
Crystallographic and calorimetric analysis of peptide binding to OppA protein; Sleigh SH et al.; Isothermal titration calorimetry has been used to study the binding of 20 different peptides to the peptide binding protein OppA, and the crystal structures of the ligand complexes have been refined . This periplasmic binding protein, part of the oligopeptide permease system of Gram negative bacteria, has evolved to bind and enclose small peptides of widely varying sequences . The peptides used in this study have the sequence Lys-X-Lys, where X is any of the 20 commonly occurring amino acids . The various side-chains found at position 2 on the ligand fit into a hydrated pocket . The majority of side-chains are restrained to particular conformations within the pocket . Water molecules act as flexible adapters, matching the hydrogen-bonding requirements of the protein and ligand and shielding charges on the buried ligand . This use of water by OppA to broaden the repertoire of its binding site is not unique, but contrasts sharply with other proteins which use water to help bind ligands highly selectively . Predicting the thermodynamics of binding from the structure of the complexes is highly complicated by the influence of water on the system .

J Tradit Chin Med, 1997 Mar, 17(1), 3 - 9
A study of Helicobacterium pylori and prevention and treatment of chronic atrophic gastritis; Zhang L et al.; The rate of detection of helicobacterium pylori (HP) in chronic atrophic gastritis (CAG) is 67%, HP being a slightly aerobic Gram-negative bacterium . Some drugs of traditional Chinese medicine (TCM) for clearing away heat and for removing blood stasis have significant effect on inhibition of HP . The drugs in both the recipe for nourishing yin, clearing away heat and invigorating blood circulation and the recipe for clearing away heat and invigorating blood circulation have inhibitory action on HP in vitro . All patients with CAG have symptoms of damp-heat and blood stasis . Radix et Rhizoma Rhei and Rhizoma Coptidis Decoction for Purging Stomach-Fire ({symbol: see text}) as the basic recipe and "Huo Wei" ({symbol: see text}) No . 2 recipe as the supplementary have good inhibitory effect on HP in inhibition tests . The results show several forms of the medicine all resulted in significant good therapeutic effect.

Antimicrob Agents Chemother, 1999 Aug, 43(8), 2084 - 6
Role of permeability in the activities of beta-lactams against gram-negative bacteria which produce a group 3 beta-lactamase; Matsumura N et al.; The production of a group 3 beta-lactamase permitted Escherichia coli to raise the MICs of ceftazidime, cefpirome, and meropenem greatly but those of imipenem and piperacillin only slightly . The ratios of maximum rate of hydrolysis to K(m) of ceftazidime, cefpirome, and piperacillin were lower than those of meropenem and imipenem for the group 3 beta-lactamase . The permeability coefficients for piperacillin and meropenem were higher than those for ceftazidime and cefpirome . Imipenem had the highest permeability coefficient.

Ital J Gastroenterol Hepatol, 1999 May, 31(4), 313 - 25
Immunological disorders in inflammatory bowel disease and immunotherapeutic implications; Amati L et al.; Ulcerative colitis and Crohn's disease, also called inflammatory bowel diseases, are characterised by altered mucosal and systemic immune responses . An increase in T helper (h) 1 cytokines, such as interleukin-2 and interferon-gamma, has been found in mucosa from patients affected by Crohn's disease . On the contrary, in patients with ulcerative colitis, mucosal cytokines seem to belong to the Th2 type with an increased release of interleukin-4, and -10 . B lymphocytes isolated from lamina propria of patients with ulcerative colitis produce perinuclear anti-neutrophil cytoplasmic antibodies, thus suggesting a status of hyperactivation of these cells in inflammatory bowel diseases, which may lead to autoimmune phenomena . Polymorphonuclear cells and monocytes/macrophages heavily infiltrate the intestinal mucosa and release proinflammatory cytokines, such as interleukin-1, -6, -8 and tumour necrosis factor-alpha . Endotoxins or lipopolysaccharides, major constituents of the gram-negative bacterial cell wall, are present in the circulation of patients with inflammatory bowel diseases and may account for the release of both cytokines and free radicals . Finally, besides immunosuppressive drugs (e.g . cyclosporin A), immunotherapy with neutralising monoclonal antibodies against tumour necrosis factor-alpha has been experimented in Crohn's disease with encouraging results . In addition, novel promising therapeutic approaches in these diseases include the administration of recombinant interleukin-10 or interleukin-11.

J Physiol Pharmacol, 1999 Jun, 50(2), 321 - 6
Effect of endotoxin on protein degradation and lipid peroxidation of erythrocytes; Bhattacharyya J et al.; Sepsis has often been associated with infection due to endotoxin (LPS) produced from gram-negative bacteria . Microcirculatory failure is one of the ultimate causes of septic shock . We studied the effect of endotoxin on the protein breakdown and lipid peroxidation of erythrocyte . In vivo (20 ug LPS/100 g) studies in rats showed increased tyrosine production from erythrocyte, as an index of protein degradation in erythrocyte . In vitro studies using 25 microg to 250 microg LPS per ml also showed similar type of increased effect of endotoxin in protein degradation . Washed erythrocyte devoid of plasma and leucocytes did not show any increased effect after endotoxin treatment . Lipid peroxidation was also increased after endotoxin treatment . However, protein degradation was more prominent than lipid peroxidation . We concluded therefore that the protein degradation and lipid peroxidation of erythrocytes caused by endotoxin are probably related to the production of septic shock.

Anesthesiology, 1999 Jul, 91(1), 215 - 21
Inhibition of nitric oxide synthase prevents hyporesponsiveness to inhaled nitric oxide in lungs from endotoxin-challenged rats; Holzmann A et al.; BACKGROUND: Inhalation of nitric oxide (NO) selectively dilates the pulmonary circulation and improves arterial oxygenation in patients with adult respiratory distress syndrome (ARDS) . In approximately 60% of patients with septic ARDS, minimal or no response to inhaled NO is observed . Because sepsis is associated with increased NO production by inducible NO synthase (NOS2), the authors investigated whether NOS inhibition alters NO responsiveness in rats exposed to gram-negative lipopolysaccharide (LPS) . METHODS: Sprague-Dawley rats were treated with 0.4 mg/kg Escherichia coli O111:B4 LPS with or without dexamethasone (inhibits NOS2 gene expression; 5 mg/kg), L-NAME (a nonselective NOS inhibitor; 7 mg/kg), or aminoguanidine (selective NOS2 inhibitor; 30 mg/kg) . Sixteen hours after LPS treatment, lungs were isolated-perfused; a thromboxane-analog U46619 was added to increase pulmonary artery pressure (PAP) by 5 mmHg, and the pulmonary vasodilator response to inhaled NO was measured . RESULTS: Ventilation with 0.4, 4, and 40 ppm NO decreased the PAP less than in lungs of LPS-treated rats (0.75+/-0.25, 1.25+/-0.25, 1.75+/-0.25 mmHg) than in lungs of control rats (3+/-0.5, 4.25+/-0.25, 4.5+/-0.25 mmHg; P < 0.01) . Dexamethasone treatment preserved pulmonary vascular responsiveness to NO in LPS-treated rats (3.75+/-0.25, 4.5+/-0.25, 4.5+/-0.5 mmHg, respectively; P < 0.01 vs . LPS, alone) . Responsiveness to NO in LPS-challenged rats was also preserved by treatment with L-NAME (3.0+/-1.0, 4.0+/-1.0, 4.0+/-0.75 mmHg, respectively; P < 0.05 vs . LPS, alone) or aminoguanidine (1.75+/-0.25, 2.25+/-0.5, 2.75+/-0.5 mmHg, respectively; P < 0.05 vs . LPS, alone) . In control rats, treatment with dexamethasone, L-NAME, and aminoguanidine had no effect on inhaled NO responsiveness . CONCLUSION: These observations demonstrate that LPS-mediated increases in pulmonary NOS2 are involved in decreasing responsiveness to inhaled NO.

Antonie Van Leeuwenhoek, 1999 Jan-Feb, 75(1-2), 109 - 24
Biosynthesis and molecular genetics of cephamycins . Cephamycins produced by actinomycetes; Liras P; Cephamycin C is produced in a nine steps pathway by the actinomycetes S . clavuligerus and N . lactamdurans . The genes encoding the biosynthesis enzymes are clustered in both microorganisms as well as in the cephabacin producer Lysobacter lactamgenus, a Gram negative bacterium . The clusters of genes include genes encoding enzymes common to the biosynthesis of penicillin and cephalosporin C by the eukaryotic producers Penicillium chrysogenum and Cephalosporium acremonium and genes for steps specific for the formation of the precursor alpha-aminoadipic acid as well as for the enzymes involved in the late modification of the cephalosporin intermediates of the pathway . Present are also genes for proteins involved in the export and/or resistance to cephamycin C . In S . clavuligerus a gene encoding a regulatory protein controlling the formation of cephamycin C and clavulanic acid is also present in the cluster.

Carbohydr Res, 1999 Mar 31, 316(1-4), 161 - 72
Solubilization of yeast cell-wall beta-(1-->3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction; Ohno N et al.; The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection . To diagnose fungal infections, a beta-(1-->3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically . We are concentrating our main efforts on developing a better standard to improve the precision of this method . To this end, we have successfully developed a protocol to obtain a soluble Candida spp . beta-(1-->3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations . The beta-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH . The product is composed mainly of beta-(1-->3)- and beta-(1-->6)-D-glucosidic linkages . The specific activity of the beta-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

J Bacteriol, 1999 Aug, 181(15), 4517 - 25
Citromicrobium bathyomarinum, a novel aerobic bacterium isolated from deep-sea hydrothermal vent plume waters that contains photosynthetic pigment-protein complexes; Yurkov VV et al.; We have taxonomically and phylogenetically characterized a new aerobic bacterial strain (JF-1) that contains photosynthetic pigment-protein complexes and which was recently isolated from black smoker plume waters of the Juan de Fuca Ridge . Strain JF-1 is a gram-negative, yellow-pigmented, motile bacterium that is salt-, pH-, and thermotolerant . These properties are consistent with an oligotrophic adaptation to varied environmental conditions thought to exist around deep-sea hydrothermal vents . The analysis of 16S rDNA sequences revealed that strain JF-1 forms a separate phylogenetic branch between the genus Erythromonas and the Erythromicrobium-Porphyrobacter-Erythrobacter cluster within the alpha subclass of the Proteobacteria . The taxonomic name Citromicrobium bathyomarinum (gen . nov., sp . nov.) is proposed for strain JF-1.

Vet Microbiol, 1999 Jun 30, 67(3), 213 - 9
Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR; Huang B et al.; Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry . A total of 35 R . anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence . This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures . Rep-PCR discriminated the R . anatipestifer isolates into 19 electrophoretic types . DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns . Substantial variation was seen among the rep-PCR fingerprints of different serotypes . Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype . These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R . anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype . Rep-PCR may serve as a useful molecular tool for subtyping R . anatipestifer isolates for epidemiologic investigations . The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 73 - 8
Determination of the genome size of Ehrlichia spp., using pulsed field gel electrophoresis; Rydkina E et al.; Ehrlichiae are obligatory intracellular, Gram-negative bacteria which belong to the alpha subclass of the phylum Proteobacteria and are responsible for infectious diseases of humans . Little is known about genetics and genomic organization of Ehrlichia spp . The genome sizes of four representatives of the genus Ehrlichia were determined for the first time by pulsed field gel electrophoresis . The sizes for E . sennetsu, E . risticii, E . chaffeensis (strain Arkansas and strain 91HE17), and the HGE agent were 878.5 kb, 880.3 kb, 1225.8 kb, 1262.3 kb and 1494 kb respectively.

Mol Microbiol, 1999 Aug, 33(3), 490 - 8
Host-dependent requirement for specific DnaA boxes for plasmid RK2 replication; Doran KS et al.; The replication origin of the broad-host-range plasmid RK2, oriV, contains four DnaA boxes, which bind the DnaA protein isolated from Escherichia coli . Using a transformation assay, mutational analysis of these boxes showed a differential requirement for replication in different Gram-negative bacteria . DnaA boxes 3 and 4 were required in E . coli and Pseudomonas putidabut not as strictly in Azotobacter vinelandii and not at all in P . aeruginosa . In vitro replication results using an extract prepared from E . coli demonstrated that the activity of origin derivatives containing mutations in boxes 3 or 4 or a deletion of all four DnaA boxes could be restored by the addition of increasing amounts of purified DnaA protein . High levels of DnaA protein in the presence of the TrfA protein also resulted in the stimulation of open complex formation and DnaB helicase loading on oriV, even in the absence of the four DnaA boxes . These observations at least raise the possibility that an alternative mechanism of initiation of oriV is being used in the absence of the four DnaA boxes and that this mechanism may be similar to that used in P . aeruginosa, which does not require these four DnaA boxes for replication.

Infect Immun, 1999 Aug, 67(8), 3824 - 9
Lipopolysaccharide-induced tumor necrosis factor alpha production by human monocytes involves the raf-1/MEK1-MEK2/ERK1-ERK2 pathway; van der Bruggen T et al.; During gram-negative sepsis, human monocytes are triggered to produce large quantities of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) in response to endotoxin (lipopolysaccharide {LPS}) . Several studies have identified signal transduction pathways that are activated by LPS, including activation of nuclear factor-kappaB (NF-kappaB) and activation of mitogen-activated protein kinases (MAPKs), including ERK1 and ERK2, c-Jun N-terminal kinase, and p38 . In this study, the relevance of ERK1 and ERK2 activation for LPS-induced TNF-alpha production by primary human monocytes has been addressed with PD-098059, which specifically blocks activation of MAPK kinase (MEK) by Raf-1 . TNF-alpha levels in the monocyte culture supernatant, induced by 10 ng of LPS/ml, were reduced by PD-098059 (50 microM) . In addition, PD-098059 also reduced TNF-alpha mRNA expression when cells were stimulated for 1 h with LPS . On the other hand, LPS-induced interleukin-10 (IL-10) levels in the monocyte supernatant were only slightly inhibited by PD-098059 . Ro 09-2210, a recently identified MEK inhibitor, completely abrogated TNF-alpha levels at nanomolar concentrations . IL-10 levels also were strongly reduced . To show the efficacy of PD-098059 and Ro 09-2210, ERK1 and -2 activation was monitored by Western blotting with an antiserum that recognizes the phosphorylated (i.e., activated) forms of ERK1 and ERK2 . Addition of LPS to human monocytes resulted in activation of both ERK1 and ERK2 in a time- and concentration (50% effective concentration between 1 and 10 ng of LPS/ml)-dependent manner . Activation of ERK2 was blocked by PD-098059 (50 microM), whereas ERK1 seemed to be less affected . Ro 09-2210 completely prevented LPS-induced ERK1 and ERK2 activation . LPS-induced p38 activation also was prevented by Ro 09-2210 . These data further support the view that the ERK signal transduction pathway is causally involved in the synthesis of TNF-alpha by human monocytes stimulated with LPS.

Ren Fail, 1999 May-Jul, 21(3-4), 433 - 42
Oxidant mechanisms in gentamicin nephrotoxicity; Walker PD et al.; Acute renal failure is a major complication of aminoglycoside antibiotics, which are widely used in the treatment of gram-negative infections . Sequential reduction of oxygen along the univalent pathway leads to the generation of superoxide anion, hydrogen peroxide, hydroxyl radical, and water . A large body of in vitro and in vivo evidence indicates that these partially reduced oxygen metabolites are important mediators of gentamicin nephrotoxicity . Gentamicin has been shown to enhance the generation of superoxide anion and hydrogen peroxide by renal cortical mitochondria . The interaction between superoxide anion and hydrogen peroxide in the presence of metal catalyst can lead to the generation of hydroxyl radical . Gentamicin has been shown to lead to release of iron from renal cortical mitochondria and to enhance generation of hydroxyl radical . These in vitro observations have been supported by in vivo studies in which scavengers of reactive oxygen metabolites and iron chelators have shown to be protective in gentamicin induced acute renal failure . There is evidence to suggest that studies may have broader implication in being relevant to other aminoglycosides including streptomycin and being applicable to other major toxicity of aminoglycoside such as ototoxicity.

J Immunol, 1999 Aug 1, 163(3), 1537 - 44
Regulation of macrophage chemokine expression by lipopolysaccharide in vitro and in vivo; Kopydlowski KM et al.; The host response to Gram-negative LPS is characterized by an influx of inflammatory cells into host tissues, which is mediated, in part, by localized production of chemokines . The expression and function of chemokines in vivo appears to be highly selective, though the molecular mechanisms responsible are not well understood . All CXC (IFN-gamma-inducible protein (IP-10), macrophage inflammatory protein (MIP)-2, and KC) and CC (JE/monocyte chemoattractant protein (MCP)-1, MCP-5, MIP-1alpha, MIP-1beta, and RANTES) chemokine genes evaluated were sensitive to stimulation by LPS in vitro and in vivo . While IL-10 suppressed the expression of all LPS-induced chemokine genes evaluated in vitro, treatment with IFN-gamma selectively induced IP-10 and MCP-5 mRNAs, but inhibited LPS-induced MIP-2, KC, JE/MCP-1, MIP-1alpha, and MIP-1beta mRNA and/or protein . Like the response to IFN-gamma, LPS-mediated induction of IP-10 and MCP-5 was Stat1 dependent . Interestingly, only the IFN-gamma-mediated suppression of LPS-induced KC gene expression was IFN regulatory factor-2 dependent . Treatment of mice with LPS in vivo also induced high levels of chemokine mRNA in the liver and lung, with a concomitant increase in circulating protein . Hepatic expression of MIP-1alpha, MIP-1beta, RANTES, and MCP-5 mRNAs were dramatically reduced in Kupffer cell-depleted mice, while IP-10, KC, MIP-2, and MCP-1 were unaffected or enhanced . These findings indicate that selective regulation of chemokine expression in vivo may result from differential response of macrophages to pro- and antiinflammatory stimuli and to cell type-specific patterns of stimulus sensitivity . Moreover, the data suggest that individual chemokine genes are differentially regulated in response to LPS, suggesting unique roles during the sepsis cascade.

Biochemistry, 1999 Jul 20, 38(29), 9541 - 8
HlyC, the internal protein acyltransferase that activates hemolysin toxin: roles of various conserved residues in enzymatic activity as probed by site-directed mutagenesis; Trent MS et al.; Hemolysin, a toxic protein produced by pathogenic Escherichia coli, is one of a family of homologous toxins and toxin-processing proteins produced by Gram-negative bacteria . HlyC, an internal protein acyltransferase, converts it from nontoxic prohemolysin to toxic hemolysin . Acyl-acyl carrier protein is the essential acyl donor . The acyltransferase reaction progresses through formation of a binary complex between acyl-ACP and HlyC to a reactive acyl-HlyC intermediate {Trent, M . S., Worsham, L . M., and Ernst-Fonberg, M . L . (1998) Biochemistry 37, 4644-4655} . The homologous acyltransferases of the family have a number of conserved amino acid residues that may be catalytically important . Experiments to illuminate the reaction mechanism were done . The formation of an acyl-enzyme intermediate suggested that the reaction likely proceeded through two partial reactions . The reversibility of the first partial reaction was shown by using separately subcloned, purified, and expressed substrates and enzyme . The effects of single site-directed mutations of conserved residues of HlyC on different portions of reaction progress (binary complex formation, acyl-enzyme formation, and enzyme activity, including kinetic parameters) were determined . Mutations of His23, the only residue essential for activity, formed normal binary complexes but were unable to form acyl-HlyC . The same was seen with S20A, a mutant with greatly impaired activity . Mutation of two conserved tyrosines separately to glycines results in greatly impaired binary complex and acyl-HlyC formation, but mutation of those residues to phenylalanines restored behavior to wild-type.

Eur J Pediatr, 1999 Jul, 158(7), 541 - 6
Gastritis and peptic ulcer disease in childhood; Blecker U et al.; Inflammation of the gastric and duodenal mucosa is the end result of an imbalance between mucosal defensive and aggressive factors . The degree of inflammation and imbalance between defensive and aggressive factors can then result in varying degrees of gastritis and/or frank mucosal ulceration . Gastritis and ulcers of the duodenum or stomach can be classified either as primary or secondary . The majority of children with chronic active or chronic gastritis and ulcers in the stomach or duodenum have secondary inflammation or mucosal ulceration . These ulcers generally occur due to a systemic condition like head trauma or overwhelming sepsis, or, as sequelae to drug ingestion (i.e., non-steroidal anti-inflammatory agents), but secondary gastroduodenal ulcers can also occur in specific disease conditions such as Zollinger-Ellison syndrome or Crohn's disease . The different causes of gastritis and peptic ulcer disease will be discussed in this paper . CONCLUSION: In almost all children presenting to their treating pediatric gastroenterologist with duodenal or gastric ulcers of these patients, mucosal inflammation and less frequently, ulceration is caused by a spiral shaped, gram-negative, microaerobic rod, properly named Helicobacter pylori . Recent epidemiological evidence has linked chronic H . pylori infection with the development of gastric carcinomas.

Cell Mol Life Sci, 1999 Jun, 55(6-7), 961 - 76
Enteropathogenic Escherichia coli: a pathogen that inserts its own receptor into host cells; DeVinney R et al.; Enteropathogenic Escherichia coli (EPEC) is a major cause of infant diarrhea, killing hundreds of thousands of children per year worldwide . Intimate attachment to the host cell leading to the formation of actin-rich pedestals beneath the adhering bacteria is an essential feature of EPEC pathogenesis . EPEC attaches to host cells via the outer membrane adhesin, intimin . It was recently shown that EPEC inserts its own receptor for intimate adherence, Tir (translocated intimin receptor) into the host cell membrane . The focus of this review is on the discovery and characterization of this novel receptor, and our current understanding of its role in pedestal formation . Gram-negative bacterial secretion systems, including type III secretion systems, are reviewed and discussed in the context of Tir delivery into the host cell membrane . The relationship and relevance of in vitro models compared to the actual in vivo situation is essential to understanding disease . We have critically reviewed the use of animal models in studying EPEC infection . Elucidating the function of Tir will contribute to our understanding of how EPEC mediates disease.

Proc Natl Acad Sci U S A, 1999 Jul 20, 96(15), 8432 - 7
A role for coenzyme M (2-mercaptoethanesulfonic acid) in a bacterial pathway of aliphatic epoxide carboxylation; Allen JR et al.; The bacterial metabolism of short-chain aliphatic alkenes occurs via oxidation to epoxyalkanes followed by carboxylation to beta-ketoacids . Epoxyalkane carboxylation requires four enzymes (components I-IV), NADPH, NAD(+), and a previously unidentified nucleophilic thiol . In the present work, coenzyme M (2-mercaptoethanesulfonic acid), a compound previously found only in the methanogenic Archaea where it serves as a methyl group carrier and activator, has been identified as the thiol and central cofactor of aliphatic epoxide carboxylation in the Gram-negative bacterium Xanthobacter strain Py2 . Component I catalyzed the addition of coenzyme M to epoxypropane to form a beta-hydroxythioether, 2-(2-hydroxypropylthio)ethanesulfonate . Components III and IV catalyzed the NAD(+)-dependent stereoselective dehydrogenation of R- and S-enantiomers of 2-(2-hydroxypropylthio)ethanesulfonate to form 2-(2-ketopropylthio)ethanesulfonate . Component II catalyzed the NADPH-dependent cleavage and carboxylation of the beta-ketothioether to form acetoacetate and coenzyme M . These findings evince a newfound versatility for coenzyme M as a carrier and activator of alkyl groups longer in chain-length than methane, a function for coenzyme M in a catabolic pathway of hydrocarbon oxidation, and the presence of coenzyme M in the bacterial domain of the phylogenetic tree . These results serve to unify bacterial and Archaeal metabolism further and showcase diverse biological functions for an elegantly simple organic molecule.

Mol Microbiol, 1999 Jul, 33(2), 296 - 306
The Per regulon of enteropathogenic Escherichia coli : identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler); Mellies JL et al.; Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram-negative bacteria that cause attaching and effacing (AE) intestinal lesions . All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE) . The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton . To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points . We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3 . The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4 . Using reporter gene fusion assays, we found that the previously described plasmid-encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC . Using single-copy lacZ fusions in K-12-derived strains, we determined that Per only directly activated the LEE1:lacZ fusion, and did not directly activate the other operons . Orf1 of the LEE1 operon activated the expression of single-copy LEE2:lacZ and LEE3:lacZ fusions in trans and modestly increased the expression of LEE4:lacZ in K-12 strains . Orf1 was therefore designated Ler, for LEE-encoded regulator . Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

Microbiology, 1999 Jun, 145 ( Pt 6), 1307 - 16
Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria; Reeve WG et al.; Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene . The nptII (encoding kanamycin resistance) or aacCI (encoding gentamicin resistance) genes were equipped with the tac promoter (Ptac) and the trpA terminator (TtrpA) and then cloned between NotI sites to construct the CAS-Nm (Ptac-nptII-TtrpA) and CAS-Gm (Ptac/PaacCI-aacCI-TtrpA) cassettes . The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-Ptac-nptII-TtrpA) or CAS-GGm (gusA-Ptac/PaacCI-aacCI-TtrpA) cassettes . Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional activity . The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid . This construct will enable transcriptional activity to be monitored in different genetic backgrounds . Each cassette was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons . The mTn5-Nm (containing Ptac-nptII-TtrpA) and mTn5-Gm (containing Ptac/PaacCI-aacCI-TtrpA) minitransposons have been constructed specifically for insertional inactivation studies . The minitransposons mTn5-GNm (containing gusA-Ptac-nptII-TtrpA) and mTn5-GGm (containing gusA-Ptac/PaacCI-aacCI-TtrpA) can be used for transcription signal localization or insertional inactivation . The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm . The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, mTn5-GNm and mTn5-GGm . The specific application of these constructs to generate acid- or nodule-inducible fusions is presented . The new constructs provide useful tools for insertional mutagenesis, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other gram-negative bacteria.

J Appl Physiol, 1999 Jul, 87(1), 299 - 307
ICAM-1 and CD11b inhibition worsen outcome in rats with E . coli pneumonia; Zeni F et al.; We investigated whether inhibiting an endothelial adhesion molecule {intracellular adhesion molecule 1 (ICAM-1)} would alter outcome and lung injury in a similar fashion to inhibition of a leukocyte adhesion molecule (integrin CD11b) in a rat model of gram-negative pneumonia . Inhibition of ICAM-1 with monoclonal antibody (MAb) 1A29 (1 mg/kg sc or 0.2 or 2 mg/kg iv, q 12 h x 3) or of CD11b with MAb 1B6 (1 mg/kg sc, q 12 h x 3) were compared against similarly administered placebo proteins in rats challenged with intrabronchial Escherichia coli . After challenge, all animals were treated with antibiotics . ICAM-1 MAb (6 mg/kg, iv, total dose) increased mortality vs . control (P = 0.03) . CD11b MAb (3 mg/kg, sc, total dose) did not significantly (P = 0.16) increase mortality rates, but this was not in a range of probability to exclude a harmful effect . All other doses of MAb had no significant effect on survival rates . ICAM-1 and CD11b MAbs had significantly different effects on the time course of lung injury, circulating white cells and lymphocytes, and lung lavage white cells and neutrophils (P = 0.04-0.003) . CD11b MAb decreased, whereas ICAM-1 MAb increased these measures compared with control from 6 to 12 h after E . coli . However, from 144 to 168 h after E . coli both MAbs increased these measures compared with control rats but to a greater level with CD11b MAb . Thus both ICAM-1 and CD11b appear to be necessary for survival during E . coli pneumonia . Although these adhesion molecules may participate differently in early lung injury, with CD11b increasing and ICAM-1 decreasing inflammation and injury, both are important for the resolution of later injury . During gram-negative pneumonia the protective roles of ICAM-1 and CD11b may make their therapeutic inhibition difficult.

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