J Biotechnol, 1999 Jun 11, 72(1-2), 85 - 93 A novel gene encoding a sulfur-regulated outer membrane protein in Thiobacillus ferrooxidans; Buonfiglio V et al.; Thiobacillus ferrooxidans is a Gram-negative chemolithotrophic bacterium able to oxidize ferrous iron, elemental sulfur and inorganic sulfur compounds . The oxidation of sulfur by T . ferrooxidans resulted in an expression of some outer membrane proteins (OMPs) at a level higher than that observed during ferrous iron oxidation . Among these OMPs, a protein with a molecular mass of 54 kDa was purified and 18 amino acids of the N-terminal sequence determined . Using a 54 bp PCR generated DNA product as a probe for the protein, we isolated a 4.5 kb Pst I DNA chromosomal fragment containing the corresponding gene . Sequencing 2169 bp of this fragment revealed the open reading frame codifying for the protein, consisting of 467 amino acids and a molecular mass of 49,674 Da . The mature protein was produced by the removal of a 32 amino acid signal peptide-like sequence from the N-terminus of a 499 amino acid peptide . Although no significant homology with any known protein has been found and its physiological role remains unclear, its high expression on sulfur substrates suggests a role in sulfide mineral oxidation. Biometals, 1999 Jun, 12(2), 141 - 9 Oxidation of arsenite to arsenate by a bacterium isolated from an aquatic environment; Weeger W et al.; Arsenic is ubiquitous in the biosphere and frequently reported to be an environmental pollutant . Global cycling of arsenic is affected by microorganisms . This paper describes a new bacterial strain which is able to efficiently oxidize arsenite (As{III}) into arsenate (As{V}) in liquid medium . The rate of the transformation depends on the cell density . Arsenic species were separated by high performance liquid chromatography (HPLC) and quantified by inductively coupled plasma-atomic emission spectrometry (ICP-AES) . The strain also exhibits high minimum inhibitory concentrations (MICs) for As{III} (6.65 mM (500 mg L-1)) and other heavy metals, such as cadmium (1.42 mM (160 mg L-1)) or lead (1.20 mM (250 mg L-1)) . Partial identification of the strain revealed a chemoorganotrophic, Gram-negative and motile rod . The results presented here demonstrate that this strain could represent a good candidate for arsenic remediation in heavily polluted sites. J Clin Microbiol, 1999 Aug, 37(8), 2568 - 75 Comparison of Ehrlichia chaffeensis recombinant proteins for serologic diagnosis of human monocytotropic ehrlichiosis; Yu XJ et al.; Diagnosis of human monocytotropic ehrlichiosis (HME) generally depends on serology that detects the antibody response to immunodominant proteins of Ehrlichia chaffeensis . Protein immunoblotting was used to evaluate the reaction of the antibodies in patients' sera with the recombinant E . chaffeensis 120- and 28-kDa proteins as well as the 106- and the 37-kDa proteins . The cloning of the genes encoding the latter two proteins is described in this report . Immunoelectron microscopy demonstrated that the 106-kDa protein is located at the surfaces of ehrlichiae and on the intramorular fibrillar structures associated with E . chaffeensis . The 37-kDa protein is homologous to the iron-binding protein of gram-negative bacteria . Forty-two serum samples from patients who were suspected to have HME were tested by immunofluorescence (IFA) using E . chaffeensis antigen and by protein immunoblotting using recombinant E . chaffeensis proteins expressed in Escherichia coli . Thirty-two serum samples contained IFA antibodies at a titer of 1:64 or greater . The correlation of IFA and recombinant protein immunoblotting was 100% for the 120-kDa protein, 41% for the 28-kDa protein, 9.4% for the 106-kDa protein, and 0% for the 37-kDa protein . None of the recombinant antigens yielded false-positive results . All the sera reactive with the recombinant 28- or the 106-kDa proteins also reacted with the recombinant 120-kDa protein. J Bacteriol, 1999 Jul, 181(14), 4223 - 36 The genes encoding formamidopyrimidine and MutY DNA glycosylases in Escherichia coli are transcribed as part of complex operons; Gifford CM et al.; Escherichia coli formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine . The genes encoding these proteins, fpg and mutY, are both cotranscribed as part of complex operons . fpg is the terminal gene in an operon with the gene order radC, rpmB, rpmG, and fpg . This operon has transcription initiation sites upstream of radC, in the radC coding region, and immediately upstream of fpg . There is a strong attenuator in the rpmG-fpg intergenic region and three transcription termination sites downstream of fpg . There is an additional site, in the radC-rpmB intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site . mutY is the first gene in an operon with the gene order mutY, yggX, mltC, and nupG . This operon has transcription initiation sites upstream of mutY, in the mutY coding region, and immediately upstream of nupG . There also appear to be attenuators in the yggX-mltC and mltC-nupG intergenic regions . The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms. Curr Microbiol, 1999 Aug, 39(2), 79 - 84 Characterization of Methanosarcina mazei N2M9705 isolated from an aquaculture fishpond; Lai MC et al.; A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan . This strain grew on trimethylamine and methanol, but it did not catabolize H2-CO2, acetate, or formate . The cells were stained Gram-negative, nonmotile, irregular coccus 0.6-0.8 micrometer in diameter . Gas vacuoles were observed and cell aggregated to form various sizes of granules . Cells grew optimally at 32 degrees -37 degrees C with 1% NaCl . The pH range of growth was 6.2-7.4, and higher pH inhibited the cell growth . The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone . A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei . This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Science, 1999 Jul 9, 285(5425), 248 - 51 HMG-1 as a late mediator of endotoxin lethality in mice; Wang H et al.; Endotoxin, a constituent of Gram-negative bacteria, stimulates macrophages to release large quantities of tumor necrosis factor (TNF) and interleukin-1 (IL-1), which can precipitate tissue injury and lethal shock (endotoxemia) . Antagonists of TNF and IL-1 have shown limited efficacy in clinical trials, possibly because these cytokines are early mediators in pathogenesis . Here a potential late mediator of lethality is identified and characterized in a mouse model . High mobility group-1 (HMG-1) protein was found to be released by cultured macrophages more than 8 hours after stimulation with endotoxin, TNF, or IL-1 . Mice showed increased serum levels of HMG-1 from 8 to 32 hours after endotoxin exposure . Delayed administration of antibodies to HMG-1 attenuated endotoxin lethality in mice, and administration of HMG-1 itself was lethal . Septic patients who succumbed to infection had increased serum HMG-1 levels, suggesting that this protein warrants investigation as a therapeutic target. J Pathol, 1999 Jun, 188(2), 220 - 6 Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles . Uptake of released toxin and vesicles by gastric epithelium; Fiocca R et al.; The mechanisms by which Helicobacter pylori releases its virulence factors are poorly known . Active secretion has been proposed for some products, including a vacuolating toxin (VacA) . Outer membrane vesicles represent another mechanism by which some Gram-negative bacteria may release virulence factors . This study sought to localize VacA by immunocytochemistry in H . pylori cells, to determine whether H . pylori produces outer membrane vesicles, and to investigate whether such vesicles might constitute a vehicle for the delivery of bacterial virulence factors to the gastric mucosa . Small (50-300 nm) membrane vesicles were found in H . pylori culture media from both H . pylori strain 60190 and strain CCUG 17874 . These vesicles appeared to originate from blebs arising on the bacterial outer membrane . VacA was immunolocalized in the periplasm and outer membrane of intact bacteria and also in outer membrane blebs and vesicles . Both soluble secreted VacA and VacA-containing vesicles bound to, and were internalized by, MKN28 cells and were detectable in the gastric mucosa from H . pylori-infected humans . The release of outer membrane vesicles by H . pylori may represent a mechanism, additional to secretory pathways, for the delivery of bacterial toxins and antigens to the gastric mucosa . J Biomed Mater Res, 1999 Sep 5, 46(3), 434 - 7 Decontaminating particles exposed to bacterial endotoxin (LPS); Hitchins VM et al.; Lipopolysaccharide (LPS), which comes from the cell wall of gram-negative bacteria, can stimulate murine macrophage cells to produce nitric oxide (NO), cytokines, such as tumor necrosis factor-alpha, and interleukins, such as IL-6 . When examining the biological effects of particles on macrophages, it is important to have no contaminating LPS associated with the particles and none with any cell culture media or supplies since even very low levels of LPS are stimulatory . The presence or absence of LPS was observed in two ways: (1) the amount of NO produced by RAW 264.7 murine macrophage cells, and (2) the Limulus amebocyte lysate (LAL) test . Treating particles with 70% ethanol at room temperature for 48 h, followed by washing the polymethylmethacrylate (PMMA) particles with endotoxin-free phosphate-buffered saline three times, decontaminated LPS and LPS-treated PMMA particles . When given LPS that had been treated with 70% ethanol for 48 h at room temperature or at 37 degrees C, cells did not produce NO above control levels . Negative LAL tests indicated the presence of extremely low levels or the complete absence of LPS in 70% ethanol-treated LPS . J Biol Chem, 1999 Jul 9, 274(28), 19601 - 5 Chaperone activity of DsbC; Chen J et al.; DsbC, a periplasmic disulfide isomerase of Gram-negative bacteria, displays about 30% of the activities of eukaryotic protein disulfide isomerase (PDI) as isomerase and as thiol-protein oxidoreductase . However, DsbC shows more pronounced chaperone activity than does PDI in promoting the in vitro reactivation and suppressing aggregation of denatured D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during refolding . Carboxymethylation of DsbC at Cys98 decreases its intrinsic fluorescence, deprives of its enzyme activities, but lowers only partly its chaperone activity in assisting GAPDH reactivation . Simultaneous presence of DsbC and PDI in the refolding buffer shows an additive effect on the reactivation of GAPDH . The assisted reactivation of GAPDH and the protein disulfide oxidoreductase activity of DsbC can both be inhibited by scrambled and S-carboxymethylated RNases, but not by shorter peptides, including synthetic 10- and 14-mer peptides and S-carboxymethylated insulin A chain . In contrast, all the three peptides and the two nonnative RNases inhibit PDI-assisted GAPDH reactivation and the reductase activity of PDI . DsbC assists refolding of denatured and reduced lysozyme to a higher level than does PDI in phosphate buffer and does not show anti-chaperone activity in HEPES buffer . Like PDI, DsbC is also a disulfide isomerase with chaperone activity but may recognize different folding intermediates as does PDI. Antimicrob Agents Chemother, 1999 Jul, 43(7), 1549 - 55 Prospective evaluation of the effect of an aminoglycoside dosing regimen on rates of observed nephrotoxicity and ototoxicity; Rybak MJ et al.; The nephrotoxicity and ototoxicity associated with once-daily versus twice-daily administration of aminoglycosides was assessed in patients with suspected or proven gram-negative bacterial infections in a randomized, double-blind clinical trial . Patients who received therapy for >/=72 h were evaluated for toxicity . Patients also received concomitant antibiotics as deemed necessary for treatment of their infection . Plasma aminoglycoside concentrations, prospective aminoglycoside dosage adjustment, and serial audiologic and renal status evaluations were performed . The probability of occurrence of a nephrotoxic event and its relationship to doses and daily aminoglycoside exposure served as the main outcome measurement . One hundred twenty-three patients were enrolled in the study, with 83 patients receiving therapy for at least 72 h . For 74 patients plasma aminoglycoside concentrations were available for analysis, and the patients formed the group evaluable for toxicity . The primary infectious diagnosis for the patients who were enrolled in the study were bacteremia or sepsis, respiratory infections, skin and soft tissue infections, or urosepsis or pyelonephritis . Of the 74 patients evaluable for toxicity, 39 received doses twice daily and 35 received doses once daily and a placebo 12 h later . Nephrotoxicity occurred in 6 of 39 (15.4%) patients who received aminoglycosides twice daily and 0 of 35 patients who received aminoglycosides once daily . The schedule of aminoglycoside administration, concomitant use of vancomycin, and daily area under the plasma concentration-time curve (AUC) for the aminoglycosides were found to be significant predictors of nephrotoxicity by multivariate logistic regression analysis (P </= 0.001) . The time to a nephrotoxic event was significantly influenced by vancomycin use and the schedule of administration, as assessed by Cox proportional hazards modeling (P </= 0.002) . The results of the multivariate logistic regression analysis and the Cox proportional hazards modeling demonstrate that both the probability of occurrence and the time to occurrence of aminoglycoside nephrotoxicity are influenced by the schedule on which the aminoglycoside is administered as well as by the concomitant use of vancomycin . Furthermore, this risk of occurrence is modulated by the daily AUC for aminoglycoside exposure . These data suggest that once-daily administration of aminoglycosides has a predictably lower probability of causing nephrotoxicity than twice-daily administration. Appl Environ Microbiol, 1999 Jul, 65(7), 2969 - 76 Isolation and characterization of a sulfate-reducing bacterium that anaerobically degrades alkanes; So CM et al.; An alkane-degrading, sulfate-reducing bacterial strain, AK-01, was isolated from an estuarine sediment with a history of chronic petroleum contamination . The bacterium is a short, nonmotile, non-spore-forming, gram-negative rod . It is mesophilic and grows optimally at pH 6.9 to 7.0 and at an NaCl concentration of 1% . Formate, fatty acids (C4 to C16) and hydrogen were readily utilized as electron donors . Sulfate, sulfite, and thiosulfate were used as electron acceptors, but sulfur, nitrite, and nitrate were not . Phenotypic characterization and phylogenetic analysis based on 16S rRNA gene sequence indicate that AK-01 is most closely related to the genera Desulfosarcina, Desulfonema, and Desulfococcus in the delta subdivision of the class Proteobacteria . It is phenotypically and phylogenetically different from strains Hxd3 and TD3, two previously reported isolates of alkane-degrading, sulfate-reducing bacteria . The alkanes tested to support growth of AK-01 had chain lengths of C13 to C18 . 1-Alkenes (C15 and C16) and 1-alkanols (C15 and C16) also supported growth . The doubling time for growth on hexadecane was 3 days, about four times longer than that for growth on hexadecanoate . Mineralization of hexadecane was indicated by the recovery of 14CO2 from cultures grown on {1-14C}hexadecane . Degradation of hexadecane was dependent on sulfate reduction . The stoichiometric ratio (as moles of sulfate reduced per mole of hexadecane degraded) was 10.6, which is very close to the theoretical ratio of 12.25, assuming a complete oxidation to CO2 . Anaerobic alkane degradation by sulfate reducers may be a more widespread phenomenon than was previously thought. Biochemistry, 1999 Jun 29, 38(26), 8582 - 9 Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli: chemical modification and mass spectrometry analysis; Gonzalez de Peredo A et al.; Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein . Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli . In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines . Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein . The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry . Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom . Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups . Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands . Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site . Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue. Circulation, 1999 Jun 29, 99(25), 3218 - 20 C(-260)-->T polymorphism in the promoter of the CD14 monocyte receptor gene as a risk factor for myocardial infarction; Hubacek JA et al.; BACKGROUND: The CD14 receptor of monocytes is an important mediator for the activation of monocytes/macrophages by endotoxins from the envelope of Gram-negative bacteria (lipopolysaccharides) . We identified a polymorphism in the CD14 receptor and examined whether this genetic marker influenced the expression of the CD14 receptor on monocytes and affected the predisposition to myocardial infarction . METHODS AND RESULTS: We identified a C(-260)-->T nucleotide change, creating a HaeIII polymorphism in the promoter of the CD14 gene . The polymorphism was determined in 178 male patients <65 years old (cases; average age, 55.9+/-6.3 years) at the time of their first myocardial infarction and in 135 representative selected male control subjects (controls; average age, 55.2+/-11.5 years) . The frequency of the T allele (absence of the cutting site) was 0.49 in cases and 0.35 in controls (P=0.0005; OR, 1.781; 95% CI, 1.286 to 2.465) . Subsequently, we measured the expression of monocyte CD14 by flow cytometry in 18 volunteers with different CD14 genotypes . A significantly higher density of the CD14 receptor was shown in the T/T homozygotes than in the others (P=0.0028) . CONCLUSIONS: A higher frequency of allele T(-260) in the promoter of the CD14 receptor gene was found in myocardial infarction survivors than in controls . At the same time, this variation was associated with a higher density of CD14 receptors in healthy volunteers . Therefore, we can conclude that in addition to the well-established risk factors, a genetically determined reaction of monocytes/macrophages to infectious stimuli could play an important role in the process of atherosclerosis. Parasitol Res, 1999 Jul, 85(7), 601 - 3 Isolation and identification by partial sequencing of the 18S ribosomal gene of free-living amoebae from necrotic tissue of Basilliscus plumifrons (Sauria: Iguanidae); Walochnik J et al.; A 3-year-old Basiliscus plumifrons developed a necrotic lesion on the tail resulting from nodules of unknown etiology . Investigation of necrotic tissue revealed several gram-negative bacteria as well as three different species of free-living amoebae . The amoebae were identified by morphological characters as belonging to the genera Acanthamoeba, Echinamoeba, and Naegleria, respectively . Partial sequencing of the 18S ribosomal gene was performed for reliable systematic determination . Two of the isolates showed thermotolerance . No isolate was growable in conventional liquid media, but the Acanthamoeba strain readily grew on a human cell line (HEp2) . It remains unclear whether the amoebae fed on the coexisting bacteria or on host tissue. Pediatr Radiol, 1999 May, 29(5), 327 - 30 Respiratory foreign bodies and Eikenella corrodens brain abscess in two children; Sane SM et al.; We report the coexistence of aspirated foreign bodies and brain abscess in two boys . One child had aspirated a metallic needle, and in the other boy partially embedded sunflower seeds were found in the bronchial wall . Both patients had growth of Eikenella corrodens (oral gram-negative flora) from the abscess . Aspirated foreign body in the respiratory tract should be one of the diagnostic considerations if any of the normal oropharyngeal organisms such as E . corrodens is the causative organism of brain abscess. J Leukoc Biol, 1999 Jun, 65(6), 815 - 21 Inhibition of microglial cell RANTES production by IL-10 and TGF-beta; Hu S et al.; Using human fetal microglial cell cultures, we found that the gram-negative bacterial cell wall component lipopolysaccharide (LPS) stimulated RANTES (regulated upon activation of normal T cell expressed and secreted) production through the protein kinase C signaling pathway and that activation of transcription nuclear factor (NF)-kappaB was required for this effect . Similarly, the proinflammatory cytokines interleukin (IL)-1beta and tumor necrosis factor-alpha dose-dependently stimulated microglial cell RANTES production via NF-kappaB activation . Anti-inflammatory cytokines, IL-10, and transforming growth factor (TGF)-beta sequentially inhibited LPS- and cytokine-induced microglial cell NF-kappaB activation, RANTES mRNA expression, and protein release . Proinflammatory cytokines but not LPS also stimulated RANTES production by human astrocytes . These findings demonstrate that human microglia synthesize RANTES in response to proinflammatory stimuli, and that the anti-inflammatory cytokines IL-10 and TGF-beta down-regulate the production of this beta-chemokine . These results may have important therapeutic implications for inflammatory diseases of the brain. Biospectroscopy, 1999, 5(3), 189 - 98 Circular dichroism and molecular modeling of the E . coli TolA periplasmic domains; Derouiche R et al.; Colicins are killer proteins that use envelope proteins from the outer and the inner membranes to reach their cellular target in susceptible cells of Escherichia coli . Each group A colicin uses a combination of Tol proteins to cross the outer membrane of gram-negative bacteria and to exert their killing activity . The TolA protein, necessary for the import of all the group A colicins, is a 421-amino acid residue protein composed of three domains (TolAI, TolAII, and TolAIII) . TolAIII interacts with the N-terminal domain of colicin A (AT1) . Analytical ultracentrifugation reveals that TolAII and TolAIII are monomer structures, TolAII has an elongated structure, and TolAIII is rather globular . Circular dichroism (CD) spectra were done with TolAII-III, TolAII, TolAIII, AT1, and the AT1-TolAII-III complex . TolA CD spectra reveal the presence of alpha-helix structure in aqueous solution and the intensity of the a-helix signal is the highest with TolAII . Few structural changes are observed with the complex AT1-TolAII-III . Molecular modeling was done for TolAII-III, taking into account CD and ultracentrifugation data and show that domain II can adopt a barrel structure made of three twisted alpha-helices similar to coiled coil helices while domain III can adopt a globular structure. Infection, 1999 May-Jun, 27(3), 183 - 6 Effect of polyclonal immunoglobulins on neutrophil phagocytic capacity and reactive oxygen production in patients with gram-negative septicemia; Wenisch C et al.; The effect of immunoglobulin (Ig) preparations on neutrophil phagocytic ability and oxidative burst in response to Escherichia coli stimulation was analyzed in 14 patients with gram-negative septicemia by an ex vivo whole blood assay using flow cytometry . In patients, neutrophils exhibited a decreased capacity to phagocytize E . coli and generate reactive oxygen products compared to healthy controls (median -68%, P < 0.01) . The addition of both 7S-Ig and 19S-Ig enriched preparations in vitro resulted in a dose-dependent increase in neutrophil reactive oxygen production at concentrations of 10 g/l (median +153% and +211%, P < 0.01, respectively) and 20 g/l (median +205% and +282%, P < 0.01, respectively) . A decreased neutrophil phagocytic ability was seen in patients with septicemia (median -58%) compared to healthy controls (P < 0.01) . Again, the addition of 7S and 19S-Igs enhanced the phagocytic ability in a dose-dependent manner (10 g/l: median +56 and +126%; 20 g/l: median +126% and +165%, P < 0.01 for all) . It can be concluded that both polyclonal Igs can increase depressed neutrophil reactive oxygen production and neutrophil phagocytosis in patients with gram-negative septicemia. Infect Immun, 1999 Jul, 67(7), 3662 - 6 Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease; Hales LM et al.; We report the identification of a set of Legionella pneumophila genes that encode products with homology to proteins of the type II general secretion pathway of gram-negative bacteria . A strain containing a deletion-substitution mutation of two of these genes was unable to secrete the Msp protease . This strain was unable to multiply within the free-living amoeba Acanthamoeba castellanii yet was able to kill HL-60-derived macrophages . Because Msp is not required for growth in amoebae, other proteins which are important for growth in amoebae are likely secreted by this pathway. Infect Immun, 1999 Jul, 67(7), 3566 - 70 The capsule supports survival but not traversal of Escherichia coli K1 across the blood-brain barrier; Hoffman JA et al.; The vast majority of cases of gram-negative meningitis in neonates are caused by K1-encapsulated Escherichia coli . The role of the K1 capsule in the pathogenesis of E . coli meningitis was examined with an in vivo model of experimental hematogenous E . coli K1 meningitis and an in vitro model of the blood-brain barrier . Bacteremia was induced in neonatal rats with the E . coli K1 strain C5 (O18:K1) or its K1(-) derivative, C5ME . Subsequently, blood and cerebrospinal fluid (CSF) were obtained for culture . Viable bacteria were recovered from the CSF of animals infected with E . coli K1 strains only; none of the animals infected with K1(-) strains had positive CSF cultures . However, despite the fact that their cultures were sterile, the presence of O18 E . coli was demonstrated immunocytochemically in the brains of animals infected with K1(-) strains and was seen by staining of CSF samples . In vitro, brain microvascular endothelial cells (BMEC) were incubated with K1(+) and K1(-) E . coli strains . The recovery of viable intracellular organisms of the K1(+) strain was significantly higher than that for the K1(-) strain (P = 0.0005) . The recovery of viable intracellular K1(-) E . coli bacteria was increased by cycloheximide treatment of BMEC (P = 0.0059) but was not affected by nitric oxide synthase inhibitors or oxygen radical scavengers . We conclude that the K1 capsule is not necessary for the invasion of bacteria into brain endothelial cells but is responsible for helping to maintain bacterial viability during invasion of the blood-brain barrier. Anim Genet, 1999 Apr, 30(2), 157 - 60 Associations of the bovine major histocompatibility complex DRB3 (BoLA-DRB3) with production traits in Canadian dairy cattle; Sharif S et al.; Associations of two alleles of the bovine major histocompatibility complex DRB3 gene (BoLA-DRB3) with lowered somatic cell score (SCS) and occurrence of disease (BoLA-DRB3.2* 16 and *23, respectively) have previously been documented . The objective of this study was to evaluate potential relationships between BoLA-DRB3 alleles with production traits, namely 305-day milk, milk fat and milk protein yield, in a population of Canadian dairy cattle (Holstein, n = 835 and Jersey, n = 66) over the course of two lactations . No significant associations were detected between BoLA alleles and production traits in Jerseys . In Holsteins, alleles *16 and *23 also did not show associations with production traits but allele *8 was significantly associated with increased 305-day milk, fat and protein yields in the previous lactation (the lactation prior to immunization with a gram negative core antigen vaccine), and with increased protein production in the subsequent (with reference to the time of immunization) lactation . Allele *22 was associated with decreased milk and protein yield in both previous and subsequent lactations . Therefore, it can be concluded that increasing or decreasing the frequency of BoLA alleles *16 and *23 to reduce SCS or increase resistance to mastitis in this population would not have adverse effects on production in this population, and that certain BoLA alleles (*8 and *22) are associated with altered production traits in Canadian Holsteins. Swed Dent J, 1999, 23(1), 11 - 5 Activation of human B-lymphocytes by Prevotella intermedia; Homayounfar A et al.; Black pigmented Gram negative Prevotella intermedia is a potential pathogenic bacterium in periodontal disease . Lipopolysaccharides (LPS) from Gram negative bacteria are the main antigens that stimulate antibody production and cytokine synthesis . Many reports indicate a strong correlation of specific antibodies of the immunoglobulin G (IgG) class with antigens of periopathogenic bacteria in periodontal disease . The aim of this study was to measure lymphocyte proliferation and immunoglobulin (IgA, IgG and IgM) production from B-lymphocytes in response to P . intermedia stimulation . P . intermedia was originally isolated from human periodontal pockets by means of a gingival crevice lavage method . The periodontal pocket was washed with salt solution and the solution was then aspirated into a cannula for further culturing in the laboratory . The identification of P . intermedia was made on Brucella agar . Lymphocytes were isolated from human peripheral blood . P . intermedia was added to the lymphocytes in concentrations from 0.005% to 0.1% . The immunoglobulin production was measured by enzyme-linked immunosorbent assay (ELISA) . The results showed that IgA and IgG were produced in response to stimulation with P . intermedia . The IgG response was dose-dependent . No stimulation of IgM production was obtained . It is concluded from the present observations that P . intermedia can stimulate proliferation of lymphocytes and production of IgA and IgG. J Bacteriol, 1999 Jun, 181(12), 3842 - 4 Identification of two new proteins in spermidine nucleoids isolated from Escherichia coli; Murphy LD et al.; The Escherichia coli nucleoid contains DNA in a condensed but functional form . Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type E . coli . Partial amino-terminal sequencing has identified them as the products of rdgC and yejK . These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles. J Bacteriol, 1999 Jun, 181(12), 3784 - 91 Porphyrin-mediated binding to hemoglobin by the HA2 domain of cysteine proteinases (gingipains) and hemagglutinins from the periodontal pathogen Porphyromonas gingivalis; DeCarlo AA et al.; Heme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogen Porphyromonas gingivalis . We therefore examined the potential role of the dominant P . gingivalis cysteine proteinases (gingipains) in the acquisition of heme from the environment . A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd = 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki = 2.5 microM) . Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd = 2.1 nM) was also inhibited by protoporphyrin IX (Ki = 10 microM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding . Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins . A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released by P . gingivalis cells in a batch culture, in parallel with proteinase activity . Cysteine proteinases from P . gingivalis appear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here . Detailed understanding of the biochemical pathways for heme acquisition in P . gingivalis may allow precise targeting of this critical metabolic aspect for periodontal disease prevention. Circulation, 1999 Jun 15, 99(23), 3056 - 62 Reduction in lipopolysaccharide-induced thrombocytopenia by triflavin in a rat model of septicemia; Sheu JR et al.; BACKGROUND: Thrombocytopenia frequently occurs early in the course of Gram-negative bacterial infections . Triflavin, an Arg-Gly-Asp-containing disintegrin, has been suggested to interfere with the interaction of fibrinogen with the glycoprotein IIb/IIIa complex . The present study was undertaken to determine whether triflavin could prevent thrombocytopenia in lipopolysaccharide (LPS)-treated rats . METHODS AND RESULTS: In this study, 51Cr-labeled platelets were used to assess blood and tissue platelet accumulation after LPS challenge . The administration of LPS (4 mg/kg IV bolus) for 4 hours induced a reduction in radiolabeled platelets in blood and an obvious accumulation of platelets in liver . Triflavin (500 microg/kg) but not GRGDS (20 mg/kg) significantly prevented the alteration of radiolabeled platelet distribution in blood and liver when induced by LPS . Furthermore, triflavin but not GRGDS markedly suppressed the elevation in plasma thromboxane B2 concentration within the 4-hour period of LPS administration . In LPS-treated rats, the 5-hydroxytryptamine level was lower in the blood and higher in the liver compared with levels in normal saline-treated rats . Pretreatment with triflavin (500 microg/kg) significantly reversed the 5-hydroxytryptamine concentration in blood and liver of LPS-treated rats . In histological examinations and platelet adhesion assay, triflavin markedly inhibited the adhesion of platelets to subendothelial matrixes in vivo and in vitro . CONCLUSIONS: The results indicate that triflavin effectively prevents thrombocytopenia, possibly through the following 2 mechanisms: (1) Triflavin markedly inhibits platelet aggregation, resulting in decreased thromboxane A2 formation . (2) It inhibits the adhesion of platelets to subendothelial matrixes, thereby leading to a reversal in the distribution of platelets in blood and liver in LPS-treated rats. J Periodontol, 1999 May, 70(5), 504 - 9 Persistence of oral colonization by the same Actinobacillus actinomycetemcomitans strain(s); Saarela MH et al.; BACKGROUND: The Gram-negative facultatively anaerobic coccobacillus Actinobacillus actinomycetemcomitans is the major pathogen in localized juvenile periodontitis (LJP) and some forms of adult periodontitis (AP) . A . actinomycetemcomitans can be grouped into 5 serotypes (a through e) based on differences in the carbohydrate moiety of cell surface lipopolysaccharide . The A . actinomycetemcomitans population is genetically heterogeneous . Since the studies on A . actinomycetemcomitans colonization have mostly applied only culture techniques, the clonality of the follow-up isolates has not been established . Thus, it is possible that, although A . actinomycetemcomitans could be repeatedly isolated from an individual, the initial colonizing strain was replaced by another strain . The aim of the study was to determine whether oral A . actinomycetemcomitans strains change spontaneously over time or after periodontal treatment . METHODS: A total of 922 A . actinomycetemcomitans isolates were recovered from 115 subjects . From each subject A . actinomycetemcomitans isolates were obtained from 2 to 9 follow-up samples 0.5 to 11.5 years apart . After the first sampling occasion, 99 subjects were treated for either LJP or AP, whereas the 16 non-periodontitis subjects received no treatment . All A . actinomycetemcomitans isolates were serotyped and 235 isolates from 52 subjects genotyped with AP-PCR and/or with ribotyping . RESULTS: Isolates of only one serotype, or non-serotypeable isolates alone, were repeatedly found in 104 subjects; serotype a occurred in 25%, b in 33%, c in 23%, d in 5%, e in 7%, and non-serotypeable isolates in 8% of these subjects . Two serotypes (or serotypeable isolates together with non-serotypeable isolates) occurred simultaneously in 9 subjects and in each of these subjects at least one of the serotypes was detected at each sampling occasion . In one subject the initial serotype reappeared although a different serotype was once seen alone, whereas in another subject the initial serotype could not be recovered later . Identical genotypes of A . actinomycetemcomitans were repeatedly detected in each of 52 subjects with follow-up isolates of the same serotype . CONCLUSIONS: The results showed that spontaneous or treatment-induced change in the oral A . actinomycetemcomitans strain(s) is extremely rare and that colonization with the same strain(s) seems to be remarkably persistent. Vet Res, 1999 Mar-Jun, 30(2-3), 181 - 202 Secretion of virulence factors by Escherichia coli; China B et al.; In order to interact with their host, pathogenic strains of E . coli need to secrete some virulence factors which can modify the metabolism of host cells, contributing to disease . Since E . coli is a Gram-negative bacteria, this secretion process involves the crossing of both the inner and the outer membranes . E . coli uses mainly four secretion mechanisms called type I, type II, type III and type IV secretion systems . In the type I secretion system, the secretion machinery is composed of three proteins forming a channel through the inner and outer membranes . It is a one-step mechanism . The secretion signal is present in the carboxyterminal region of the secreted protein but without proteolytic cleavage . In E . coli, the best studied type I secreted protein is haemolysin . In type II and type IV secretion systems, the crossing of the inner membrane involves the sec machinery with the cleavage of an aminoterminal signal sequence . The crossing of the outer membrane involves the formation of a pore either by other proteins (type II) or by the carboxyterminal region of the protein (type IV) . The A-B toxins, such as heat labile enterotoxin, are secreted by the first mechanism and members of the IgA proteases are secreted by the second . The type III secretion system involves at least 20 proteins including cytoplasmic, inner membrane and outer membrane proteins . The originality of this system is the ability to inject secreted bacteria into the cytosol of the host cells . Such a system is found in attaching and effacing E . coli and in diffusely adhering E . coli. Indian J Physiol Pharmacol, 1999 Apr, 43(2), 247 - 50 Gentamicin induced inhibition of steroidogenic enzymes in rat testis; Ghosh S et al.; Gentamicin is an aminoglycoside antibiotic, widely used for treating many gram negative bacterial infections . Though nephrotoxicity is the most highlighted side effect, it has also been found to cause an alteration in the phosphatase activities of testes and accessory sex organs and a decline in the sperm count . This study was designed to assess the effects of gentamicin on testicular steroidogenesis and to ascertain whether such alterations are reversible . Laboratory inbred adult, male, 'Wistar' strain rats were chosen as the experimental animal . A significant dose-dependant reduction in the activities of the two steroidogenic enzymes, accompanied with a significant decrease in ascorbic acid and elevation of level of cholesterol was observed . The effects were maximum at a dose of 100 mg/kg, b.wt . After 15 days of withdrawal of the drug therapy the biochemical parameters namely ascorbic acid and cholesterol returned to normal levels whereas the activities of the two dehydrogenases showed a compensatory increase . This indicates that gentamicin affects the steroidogenic enzymes, causing an alteration in the formation of testosterone, which was manifested in the elevated cholesterol in the adult rat testes . However, these alterations were reversible. J Hosp Infect, 1999 May, 42(1), 21 - 6 Endemic nosocomial gram-negative bacteraemias resulting from contamination of intravenous heparin infusions; Playford EG et al.; Following several cases of Gram-negative bacteraemia secondary to intravenous heparin infusion contamination, we retrospectively reviewed nosocomial bacteraemias associated with heparin infusions at our institution . Thirty-one episodes of heparin-infusion related bacteraemia occurred in 30 patients over a 23-month period affecting 2% patients receiving heparin infusions for more than 48 h . Gram-negative bacteria were responsible for all bacteraemias . The care of infusions during clinical use was prospectively surveyed, revealing that approximately 20% of lines and cannulae were left for more than 72 h before replacement, and significant discordance occurred between line replacement and syringe and cannula exchange . We concluded that contamination of the infusions was probably extrinsic and secondary to manipulations of the system during use . Prolonged usage and discordant exchange of infusion components were likely important factors in initial contamination and subsequent bacterial proliferation . The problem resolved following the introduction of a policy for routine and simultaneous replacement of lines and syringes at 24-h intervals and upon cannula exchange. Am J Orthod Dentofacial Orthop, 1999 Jun, 115(6), 634 - 9 Endotoxin affinity for orthodontic brackets; Knoernschild KL et al.; Endotoxin, cell envelope lipopolysaccharide produced by gram-negative bacteria can activate an immune response through a variety of pathways . In addition, it can stimulate bone resorption and reduce the periodontal tissue's healing capacity . Previous studies have documented the affinity of lipopolysaccharide for restorative materials . This study evaluated the affinity of lipopolysaccharide for commercially available orthodontic brackets . Stainless steel, ceramic, plastic, and "gold" brackets were exposed to 10 EU/mm2radiolabeled Porphyromonas gingivalis or Escherichia coli lipolpoysaccharide in water and incubated for 24 hours at 37 degrees C . Brackets were then transferred to fresh lipopolysaccharide-free water and incubated for 24 hours at 37 degrees C to evaluate elution . This elution transfer was continued up to 96 hours total incubation . Lipopolysaccharide adherence and elution levels were calculated after treatment, and elution solutions were evaluated through liquid scintillation spectrometry . Mean initial lipopolysaccharide adherence ranged from 2.42 +/- 0.26 EU/mm2(E . coli, plastic) to 6.75 +/- 0.34 EU/mm2 (P . gingivalis, stainless steel) . P . gingivalis lipopolysaccharide adherence was significantly greater than E . coli lipopolysaccharide adherence for all bracket types . Moreover, for each lipopolysaccharide type, stainless steel brackets exhibited significantly greater lipopolysaccharide adherence . Regarding elution, only the P . gingivalis lipopolysaccharide-exposed ceramic and plastic brackets at 24 hours and the stainless steel and ceramic brackets at 48 hours eluted measurable lipopolysaccharide . Results from this study demonstrate that P . gingivalis and E . coli LPS exhibit a high affinity for orthodontic brackets . In vivo, this affinity could affect the concentration of LPS in the gingival sulcus, thereby contributing to inflammation in tissues adjacent to the brackets. J Immunol, 1999 Jun 15, 162(12), 7454 - 60 Monoclonal antibodies to murine lipopolysaccharide (LPS)-binding protein (LBP) protect mice from lethal endotoxemia by blocking either the binding of LPS to LBP or the presentation of LPS/LBP complexes to CD14; Le Roy D et al.; Cellular responses to LPS, the major lipid component of the outer membrane of Gram-negative bacteria, are enhanced markedly by the LPS-binding protein (LBP), a plasma protein that transfers LPS to the cell surface CD14 present on cells of the myeloid lineage . LBP has been shown previously to potentiate the host response to LPS . However, experiments performed in mice with a disruption of the LBP gene have yielded discordant results . Whereas one study showed that LBP knockout mice were resistant to endotoxemia, another study did not confirm an important role for LBP in the response of mice challenged in vivo with low doses of LPS . Consequently, we generated rat mAbs to murine LBP to investigate further the contribution of LBP in experimental endotoxemia . Three classes of mAbs were obtained . Class 1 mAbs blocked the binding of LPS to LBP; class 2 mAbs blocked the binding of LPS/LBP complexes to CD14; class 3 mAbs bound LBP but did not suppress LBP activity . In vivo, class 1 and class 2 mAbs suppressed LPS-induced TNF production and protected mice from lethal endotoxemia . These results show that the neutralization of LBP accomplished by blocking either the binding of LPS to LBP or the binding of LPS/LBP complexes to CD14 protects the host from LPS-induced toxicity, confirming that LBP is a critical component of innate immunity. J Biomater Sci Polym Ed, 1999, 10(5), 557 - 72 Ability of the exopolymer excreted by Pseudoalteromonas antarctica NF3, to coat liposomes and to protect these structures against octyl glucoside; de la Maza A et al.; The ability of an exopolymer of glycoproteic character (GP) excreted by a new gram-negative specie Pseudoalteromonas antarctica NF3, to coat phosphatidylcholine (PC) liposomes and to protect these bilayers against the action of the nonionic surfactant octyl glucoside (OG) has been investigated . TEM micrographs of freeze-fractured liposome/GP aggregates reveal that the addition of GP to liposomes led to the formation of a covering structure (polymer adsorbed onto the bilayers) that tightly coated PC bilayers . The complete coating was already achieved when the proportion of GP assembled with liposomes was approximately 10% (wt% vs total PC) . Higher GP amounts resulted in a growth of this coating structure which exhibited at the highest GP proportion in the system (31% of assembled GP) a multilayered structure . An increasing resistance of PC liposomes to be affected by OG both at sublytic and lytic levels occurred as the proportion of GP in the system rose; this protective effect being more effective when the proportion of assembled GP was 10-20% in weight . 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 OG, the best protection occurred when the proportion of assembled GP was about 10 wt%. Kidney Int, 1999 Jun, 55(6), 2322 - 37 Tumor necrosis factor-alpha and lipopolysaccharide induce apoptotic cell death in bovine glomerular endothelial cells; Messmer UK et al.; BACKGROUND: The glomerular endothelial cell is a specialized microvascular cell type involved in the regulation of glomerular ultrafiltration . During gram-negative sepsis, glomerulonephritis, and acute renal failure, bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) may cause severe cell damage . Our aim was to study and compare the direct effects of TNF-alpha and LPS on the induction of apoptosis in bovine glomerular endothelial cells . METHODS: Primary bovine glomerular endothelial cells were stimulated with TNF-alpha or LPS, and apoptotic cell death was investigated by DNA fragmentation analysis, morphological studies, measurement of cytochrome c efflux and mitochondrial permeability transition, Bak, Bad, Bax, Bcl-2, Bcl-xL protein expression, and caspase-3-like protease activity . RESULTS: TNF-alpha, as well as LPS, elicited apoptotic cell death both time and concentration dependently . Along with DNA ladder formation, we detected the formation of 50 kbp high molecular weight DNA fragments, nuclear condensation, and mitochondrial permeability transition . Concerning all parameters, LPS signaling proved to be more rapid than TNF-alpha . Mechanistically, TNF-alpha-induced cell death was preceded by an efflux of mitochondrial cytochrome c into the cytosol and, subsequently, by a marked increase in the proapoptotic protein Bak and a decrease in the anti-apoptotic Bcl-xL protein content . Comparable but more pronounced effects were seen with LPS . Later, caspase-3-like protease activity was first detectable after 10 hours and was continuously increased up to 24 hours in both TNF-alpha- and LPS-stimulated cells . Correspondingly, we detected an extended cleavage of the nuclear enzyme poly(ADP-ribose) polymerase . Caspase inhibitors Z-Asp-CH2-DCB and Z-VAD-fmk blocked both TNF-alpha- and LPS-induced apoptosis in a comparable manner . Only Z-Asp-CH2-DCB was able to block apoptotic cell death completely . CONCLUSION: Both bacterial LPS and TNF-alpha potently induced apoptotic cell death in glomerular endothelial cells . Direct endotoxin-induced apoptosis may therefore be relevant in the progression of acute renal failure, which is a frequent complication of gram-negative sepsis. Clin Chem Lab Med, 1999 Mar, 37(3), 373 - 9 Endotoxin adsorbent based on immobilized human serum albumin; Zimmermann M et al.; Extracorporeal apheresis of endotoxins and pro-inflammatory cytokines is still a therapeutic option in the early hyper-inflammatory phase of gram-negative sepsis . There is therefore ongoing interest in adsorber materials suitable for that kind of clinical application . Here we describe lipopolysaccharide (LPS) and cytokine adsorption characteristics of a new adsorbent based on purified human serum albumin (HSA) covalently linked to macroporous polymer beads (iHSA) . Multipoint attachment of HSA to acrylic beads via carboxyl groups of the protein resulted in an increased affinity to LPS . In adsorption experiments (adsorbent/ plasma ratio 1:3) a 70-80% reduction of limulus amoebocyte lysate (LAL) activity from 8.59+/-2.07 EU/ml (mean+/-SD) to 1.82+/-0.77 EU/ml (S . abortus equi; n=40) (p < 0.001) and from 115.13+/-53.76 EU/ml to 17.70+/-11.68 EU/ml (E . coli F583; n=6) (p < 0.01) was achieved . iHSA-purified plasma samples showed a decreased capability of inducing cytokine release from peripheral monocytes . Direct haemoperfusion of LPS pre-stimulated whole blood over iHSA resulted in decreased tumour necrosis factor alpha (TNFalpha) concentrations (30-40% reduction) whereas induced levels of interleukin (IL)-1beta and IL-6 were not affected . Depending on the means of immobilization, iHSA shows higher affinity for LPS than native albumin present in plasma . We demonstrated an efficient removal of LPS from plasma in vitro . Adsorption over immobilized HSA appears to be a simple and effective means of removing LPS and perhaps pro-inflammatory cytokines from the circulation. Pediatr Nephrol, 1999 Apr, 13(3), 245 - 8 Survival and complications of cuffed catheters in children on chronic hemodialysis; Sharma A et al.; Central venous catheters are being increasingly used as hemodialysis vascular access . We evaluated catheter survival, outcome predictors, and complications in a total of 36 catheters used in 13 children and young adults undergoing chronic maintenance hemodialysis through catheter for a duration of 10.4+/-5.6 months . Reasons for catheter failure were: thrombosis 12 of 36 (33%), infection 6 of 36 (17%), and extrusion 2 of 36 (5.4%) . Catheters were lost to infection and thrombosis at 1.1 and 2.2 episodes per 1,000 catheter days, respectively . Symptomatic infections, Gram-negative and polymicrobial sepsis increased the risk of catheter failure . Most of the thrombotic episodes occurred in patients with inherent thrombotic tendency . The survival of the 36 catheters was 62% at 1 year . The survival of 13 randomly chosen catheters, 1 from each patient, was 85% at 1 year . The time from insertion to first complication correlated significantly with the outcome (P<0.03) . We conclude that central venous catheters are still associated with a high rate of failure and may be a regular access choice only in a selected patient population with no inherent thrombotic tendency and no other option available for long-term hemodialysis. Am J Respir Cell Mol Biol, 1999 Jun, 20(6), 1155 - 64 Early-onset inflammatory responses in vivo to adenoviral vectors in the presence or absence of lipopolysaccharide-induced inflammation; Thorne PS et al.; Adenoviral vectors (Ad) have potential for use in pulmonary gene transfer for treating cystic fibrosis (CF) . However, Ad may induce inflammation even in the absence of gene expression . Endotoxin from gram-negative bacteria in the airways of CF patients may also induce inflammation, and may further inhibit vector delivery and gene transfer . We used a mouse model to study the time course of Ad-induced lung inflammation and to assess additivity with lipopolysaccharide (LPS)-induced inflammatory responses . C3H/HeJ endotoxin-resistant (RES) mice hyporesponsive to inflammatory stimuli and normoresponsive C3HeB/FeJ endotoxin-sensitive (SEN) mice were studied to characterize inflammatory responses that follow intratracheal instillation of inactivated Ad, with or without simultaneous inhalation exposure to LPS . Instillation of 10(10) Ad particles dramatically increased bronchoalveolar lavage fluid (BALF) concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 at 3 to 6 h and induced profound neutrophilia, maximal at 12 to 24 h . SEN mice had tenfold greater responses than did RES mice at 6, 12, and 24 h . Mice exposed to Ad alone, LPS alone, or Ad + LPS had significant inflammation at the 3-h time point as demonstrated by BALF neutrophils, TNF-alpha, and IL-6 . With all three treatments, SEN mice had a five- to 300-fold greater response than did RES mice . Importantly, Ad + LPS yielded no greater inflammatory response than LPS without Ad . These data demonstrate that replication-deficient Ad induce early inflammation and LPS-induced inflammation is not augmented by concurrent treatment with Ad. Infect Dis Clin North Am, 1999 Jun, 13(2), 371 - 86, ix Antiendotoxin strategies; Hellman J et al.; Endotoxin is a potent stimulator of the inflammatory response and is believed to initiate the pathology in Gram-negative sepsis . Agents are being developed that bind and neutralize or block the effects of endotoxin, with the goal of improving outcome in the treatment of sepsis . Strategies discussed in this article include anti-LPS antibodies, LPS binding proteins and lipoproteins, polymyxin B conjugates, lipid A analogues, and extracorporeal techniques for endotoxin removal. Infect Dis Clin North Am, 1999 Jun, 13(2), 355 - 69, vii Vaccines and antibodies in the prevention and treatment of sepsis; Bhattacharjee AK et al.; Antibodies to various core glycolipid antigens have been shown to correlate with survival from Gram-negative sepsis . Recent preclinical data also support efficacy of the anti-core glycolipid antibodies in the treatment of sepsis . Failure of some of the previous clinical trials with anti-core glycolipid antibody was probably due to inadequate levels of antibody in those preparations . Future clinical trials must ensure that sufficient amounts of anti-core glycolipid antibodies are present in the circulation of patients with sepsis. Infect Dis Clin North Am, 1999 Jun, 13(2), 341 - 53, vii Lipopolysaccharide recognition, CD14, and lipopolysaccharide receptors; Ingalls RR et al.; The ability of a host to sense invasion by a pathogenic organism, and to respond appropriately to control infection, is paramount to survival . To that end, an array of receptors and binding proteins has evolved as part of the innate immune system to detect Gram-negative bacteria . This article reviews the role of CD14, other LPS binding proteins, and the Toll family of receptors in the innate recognition of bacterial lipopolysaccharide. Infect Dis Clin North Am, 1999 Jun, 13(2), 313 - 40 Structure-function relationships of bacterial endotoxins . Contribution to microbial sepsis; Morrison DC et al.; A substantial body of knowledge has emerged over the past several decades concerning the primary and tertiary, and quaternary structure of endotoxic LPS and their contribution to the pathogenesis of gram-negative sepsis; however, important questions remain . Among them are the precise three-dimensional configuration of the LPS macromolecule and the contribution of the quaternary structure to the ability of these potent microbial factors to interact with host humoral and cellular inflammatory mediator systems . Also remaining to be sufficiently addressed is the relative contribution of endotoxin interactions with the host to the overall manifestation of disease and conditions under which such contributions serve as the pivotal event in determining outcome . The answers to these questions can be expected to provide valuable insights into potential novel therapeutic intervention strategies and approaches that will ultimately reduce both morbidity and mortality in infection from gram-negative microbes. Infect Immun, 1999 Jun, 67(6), 2790 - 6 Lipopolysaccharide (LPS)-binding synthetic peptides derived from serum amyloid P component neutralize LPS; de Haas CJ et al.; Lipopolysaccharide (LPS) is the major mediator of gram-negative septic shock . Molecules that bind LPS and neutralize its toxic effects could have important clinical applications . We showed that serum amyloid P component (SAP) neutralizes LPS . A SAP-derived peptide, consisting of amino acids 27 to 39, inhibited LPS-mediated effects in the presence of human blood . In this study, we used a pepscan of overlapping 15-mer peptides and distinguished two additional LPS-binding regions within the SAP molecule, identified in the regions spanning amino acids 61 to 75 and 186 to 200 . The corresponding SAP-derived peptides, pep61-75 and pep186-200, inhibited the binding of fluorescein isothiocyanate-labeled LPS to monocytes as efficiently as a bactericidal/permeability-increasing protein (BPI)-derived 15-mer peptide comprising amino acids 85 to 99 . The same SAP-derived peptides very potently inhibited LPS-induced priming of phagocytes in human blood . Also, SAP-derived pep186-200 caused a prolonged survival of actinomycin D-sensitized mice treated with LPS to induce septic shock, indicating a potential use of this peptide in the defense against serious gram-negative sepsis in humans. J Trauma, 1999 May, 46(5), 800 - 8; discussion 808-10 Pulmonary capillary sieving of hetastarch is not altered by LPS-induced sepsis; Conhaim RL et al.; BACKGROUND: Gram-negative lipopolysaccharide (LPS) has been demonstrated to increase pulmonary capillary permeability as judged by the increased flow of protein-rich lymph from the lungs of sheep infused with LPS . This finding suggests that LPS-injured pulmonary capillaries might be less restrictive than uninjured capillaries to the filtration of large hetastarch molecules . Hetastarch has a broad molecular mass spectrum (35-1,500 kilodaltons (kDa)), and one way to test the restrictiveness of pulmonary capillaries is to measure the size of the largest hetastarch molecules that cross the microvascular barrier and enter the lymph . To evaluate the effects of LPS, we compared hetastarch molecular distributions in the lung lymph of normal and LPS-injured sheep . METHODS: Adult sheep (38.2 +/- 0.8 kg) were surgically prepared for the collection of lung lymph, with study initiation after a 5- to 7-day recovery period . Hetastarch (6%) was infused (10 mL/kg) 24 hours before study to allow for stabilization of the hetastarch molecular distribution . On the day of study, LPS (Escherichia coli lipopolysaccharide, 2 microg/kg; n = 6) was infused, and plasma and lymph samples were collected for 12 hours . An additional group of animals not infused with LPS (n = 6) served as controls . Hetastarch molecular distributions in plasma and lymph were measured by using high performance size exclusion chromatography . RESULTS: In control sheep, the largest hetastarch molecules in lymph averaged 861 +/- 18 kDa (mean +/- SEM) (plasma, 1,065 +/- 18 kDa) . In LPS-treated sheep, the largest hetastarch molecules in lymph averaged 845 +/- 19 kDa (not significant vs . normal) (plasma, 1,025 +/- 14 kDa) . Hetastarch concentrations in plasma and lung lymph of normal sheep, respectively, were 0.61 +/- 0.05% and 0.34 +/- 0.07% . In LPS-treated sheep, hetastarch concentrations in plasma and lymph were 0.56 +/- 0.08 (not significant vs . normal) and 0.29 +/- 0.07, respectively (p < or = 0.05) . Lymph concentrations were lower after LPS because of increased lymph flows (19.9 +/- 5.4 mL/30 min, compared with 3.6 +/- 0.8 mL/30 min in normal sheep) . CONCLUSION: Our results suggest that LPS does not alter the diameter of the largest pores perforating the walls of pulmonary capillaries . Rather, the number of these pores in the capillary wall appears to be increased . This increase would explain why lymph flows rise after LPS with little change in the lymph protein concentration . Our results are also consistent with a filtration model in which capillaries are assumed to be perforated by small pores (protein reflection coefficient = 1) as well as large pores (protein reflection coefficient = 0). Science, 1999 May 21, 284(5418), 1322 - 8 Type III secretion machines: bacterial devices for protein delivery into host cells; Galan JE et al.; Several Gram-negative pathogenic bacteria have evolved a complex protein secretion system termed type III to deliver bacterial effector proteins into host cells that then modulate host cellular functions . These bacterial devices are present in both plant and animal pathogenic bacteria and are evolutionarily related to the flagellar apparatus . Although type III secretion systems are substantially conserved, the effector molecules they deliver are unique for each bacterial species . Understanding the biology of these devices may allow the development of novel prevention and therapeutic approaches for several infectious diseases. Arch Biochem Biophys, 1999 May 15, 365(2), 299 - 306 Energetics underlying the process of long-chain fatty acid transport; Azizan A et al.; The gram negative bacterium Escherichia coli has evolved a highly specific system for the transport of exogenous long-chain fatty acids (C12-C18) across the cell envelope that requires the outer membrane protein FadL and the inner membrane associated fatty acyl CoA synthetase . The transport of oleate (C18:1) across the cell envelop responds to metabolic energy . In order to define the source of metabolic energy which drives this process, oleate transport was measured in wild-type and ATP synthase-defective (Deltaatp) strains which were (i) subjected to osmotic shock and (ii) starved and energized with glucose or d-lactate in the presence of different metabolic inhibitors . Osmotic shock did not eliminate transport but rather reduced the rate to 33-55% of wild-type levels . These results suggested a periplasmic protein may participate in this process or that osmotic shock disrupts the energized state of the cell which in turn reduces the rate of oleate transport . Transport systems which are osmotically sensitive also require ATP . The process of long-chain fatty acid transport requires ATP generated either by substrate-level or oxidative phosphorylation . Following starvation, the basal rate of transport for wild-type cells was 340.4 pmol/min/mg protein compared to 172.0 pmol/min/mg protein for the Deltaatp cells . When cells are energized with glucose, the rates of transport were increased and comparable (1242.6 and 1293.8 pmol/min/mg protein, respectively) . This was in contrast to cells energized with d-lactate in which only the wild-type cells were responsive . The role of ATP is likely due to the ATP requirement of fatty acyl CoA synthetase for catalytic activity . The process of oleate transport is also influenced by the energized state of the inner membrane . In the presence of carbonyl cyanide-m-chlorophenylhydrazone oleate transport is depressed to 30-50% of wild-type levels in wild-type and Deltaatp strains under starvation conditions . These results are mirrored in cells energized with glucose and d-lactate, indicating that an energized membrane is required for optimal levels of oleate transport . These data support the hypothesis that the fatty acid transport system of E . coli responds to both intracellular pools of ATP and an energized membrane for maximal proficiency . Cutis, 1999 Jan, 63(1), 49 - 51 Tularemia: a case transmitted from a sheep; Senol M et al.; Tularemia is an arthropod-borne infectious disease caused by Francisella tularensis, a gram-negative microorganism that normally resides in a wide range of wild and domestic animals . The disease is characterized by a sudden onset with high fever, headache, malaise, chills, myalgia, and arthralgia . A short time after exposure, an inflamed and ulcerated lesion rapidly appears at the site of entry . A regional lymphadenopathy follows the cutaneous presentation.Cultures from the lesions or blood generally give negative results . Histopathologic examination reveals either a nonspecific inflammatory infiltrate or an infectious granuloma . The most useful laboratory procedure in the diagnosis of tularemia is serologic tests . Streptomycin, gentamicin, and tetracycline are the drugs of choice in the treatment . Quinolones are also effective . Tularemia is fairly rare in Turkey . We present a typical case of ulceroglandular tularemia transmitted from a sheep to a young man. Zentralbl Chir, 1999, 124(3), 206 - 9 {Production of chemokines by the human peritoneum}; Riese J et al.; AIM OF THE STUDY: Peritonitis is characterised by a continued infiltration of the peritoneal cavity with leukocytes, attracted by chemotactic mediators . The aim of this study was to demonstrate the capacity of the human peritoneum to secrete chemokines and to show a therapeutic option by impairing the proinflammatory function of the peritoneum . METHODS: Peritoneum was obtained from 12 consenting patients undergoing abdominal surgery for noninflammatory diseases . After opening the peritoneal cavity a piece of the parietal peritoneum was excised and subsequently incubated with lipopolysaccharide (LPS, 50 ng/ml) +/- interleukin-10 (IL-10, 100 U/ml) for five hours in vitro . The culture supernatants were assayed for concentrations of interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and monocyte inflammatory protein-1 alpha (MIP-1 alpha) by using the ELISA . RESULTS: The cultured peritoneum secreted MCP-1 (mean (SEM): 3416 (659) pg/ml) and IL-8 (2946 (894) pg/ml) . The presence of LPS resulted in a fourfold enhancement of this secretion (MCP-1: 13563 (1613), IL-8: 9854 (1305) pg/ml) and led to the production of MIP-1 alpha (1476 (240) pg/ml) . The LPS-stimulated production of all of these chemokines was significantly diminished by the presence of IL-10 . CONCLUSION: The reaction of the peritoneum to LPS indicates its proinflammatory function in the context of peritonitis caused by gram-negative bacteria . This inflammatory reaction might be diminished by application of IL-10. J Clin Microbiol, 1999 Jun, 37(6), 2068 - 70 Two cases of Chromobacterium violaceum infection after injury in a subtropical region; Lee J et al.; Chromobacterium violaceum is a gram-negative rod and is isolated from soil and water in tropical and subtropical regions . The species have pigmented and nonpigmented colony types . Infections caused by nonpigmented strains are rare . We report on two cases of infection caused by both pigmented and nonpigmented strains of C . violaceum . Two 24-year-old Korea Airline stewardesses were admitted to Inha University Hospital, Inchon, South Korea, on 9 August 1997, 3 days after an airplane accident in Guam . Both had multiple lacerations on exposed parts of their bodies . There was swelling, tenderness, and pus discharge . The wounds contained many small fragments of stones and weeds . A pigmented strain was isolated from the left hand and a nonpigmented strain was isolated from the left knee of one patient . For the other patient only a nonpigmented strain was isolated from a foot wound . The nonpigmented colonies from the left-knee and the left-foot wounds did not produce any pigment even after an extended period of incubation . The biochemical characteristics were the same for each strain except for oxidase and indole reactions . The pigmented strain was oxidase negative and indole positive, whereas the nonpigmented strains were oxidase positive and indole negative . The patients were successfully treated by debridement and with appropriate antibiotics. J Clin Microbiol, 1999 Jun, 37(6), 1899 - 905 Culture of Bartonella quintana and Bartonella henselae from human samples: a 5-year experience (1993 to 1998); La Scola B et al.; Bartonella quintana and Bartonella henselae are fastidious gram-negative bacteria responsible for bacillary angiomatosis, trench fever, cat scratch disease, and endocarditis . During a 5-year period, we received 2,043 samples for culture of Bartonella sp . We found Bartonella sp . to be the etiologic agent in 38 cases of endocarditis, 78 cases of cat scratch disease, 16 cases of bacteremia in homeless people, and 7 cases of bacillary angiomatosis . We correlated the results of positive cultures with the clinical form of the disease, type of sample, culture procedure, PCR-based genomic detection, and antibody determination . Seventy-two isolates of B . quintana and nine isolates of B . henselae from 43 patients were obtained . Sixty-three of the B . quintana isolates and two of the B . henselae isolates, obtained from patients with no prior antibiotic therapy, were stably subcultured . The sensitivity of culture was low when compared with that of PCR-based detection methods in valves of patients with endocarditis (44 and 81%, respectively), skin biopsy samples of patients with bacillary angiomatosis (43 and 100%, respectively), and lymph nodes of cat scratch disease (13 and 30%, respectively) . Serological diagnosis was also more sensitive in cases of endocarditis (97%) and cat scratch disease (90%) . Among endocarditis patients, the sensitivity of the shell vial culture assay was 28% when inoculated with blood samples and 44% when inoculated with valvular biopsy samples, and the sensitivity of both was significantly higher than that of culture on agar (5% for blood {P = 0.045} and 4% for valve biopsy samples {P < 0.0005}) . The most efficient culture procedure was the subculture of blood culture broth into shell vials (sensitivity, 71%) . For patients with endocarditis, previous antibiotic therapy significantly affected results of blood culture; no patient who had been administered antibiotics yielded a positive blood culture, whereas 80% of patients with no previous antibiotic therapy yielded positive blood cultures (P = 0.0006) . Previous antibiotic therapy did not, however, prevent isolation of Bartonella sp . from cardiac valves but did prevent the establishment of strains, as none of the 15 isolates from treated patients could be successfully subcultured . For the diagnosis of B . quintana bacteremia in homeless people, the efficiency of systematic subculture of blood culture broth onto agar was higher than that of direct blood plating (respective sensitivities, 98 and 10% {P < 10(-7)}) . Nevertheless, both procedures are complementary, since when used together their sensitivity reached 100% . All homeless people with positive blood cultures had negative serology . The isolation rate of B . henselae from PCR-positive lymph nodes, in patients with cat scratch disease, was significantly lower than that from valves of endocarditis patients and skin biopsy samples from bacillary angiomatosis patients (13 and 33%, respectively {P = 0.084}) . In cases of bacillary angiomatosis for which an agent was identified to species level, the isolation rate of B . henselae was lower than the isolation rate of B . quintana (28 and 64%, respectively {P = 0.003}) . If culture is to be considered an efficient tool for the diagnosis of several Bartonella-related diseases, methodologies need to be improved, notably for the recovery of B . henselae from lymph nodes of patients with cat scratch disease. Drug Discov Today, 1999 May, 4(5), 209 - 221 Strategies for the control of LPS-mediated pathophysiological disorders; Chaby R; Lipopolysaccharides released from Gram-negative bacteria after infection initiate an alarm response in the host, which has supposedly evolved to protect it . However, an exaggerated response leads to a cascade of pathophysiological events termed sepsis . In the USA alone, the annual number of deaths caused by sepsis ( approximately 70,000) is comparable with that caused by AIDS . The author describes the major advances of knowledge in this field and the attempts to convert this into successful therapeutics . Anti-endotoxin and anti-inflammatory agents have been disappointing, but new strategies might result in effective treatments in the forthcoming years. Crit Care Med, 1999 Apr, 27(4), 807 - 14 Effect of the immunomodulating agent, pentoxifylline, in the treatment of sepsis in prematurely delivered infants: a placebo-controlled, double-blind trial; Lauterbach R et al.; OBJECTIVE: To evaluate the influence of the methylxanthine derivative, pentoxifylline, on plasma levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1, and IL-6 in prematurely delivered infants with generalized bacterial infections and to assess the effect of this immunomodulating drug on the clinical outcome in newborns with sepsis . DESIGN: A prospective, randomized, double-blind trial . SETTING: The neonatal intensive therapy units in university teaching hospitals . PATIENTS: One hundred patients with sepsis admitted during a 1.5-yr period . INTERVENTIONS: Patients were randomly assigned to receive pentoxifylline (pentoxifylline group) in a dose of 5 mg/kg/hr for 6 hrs on 6 successive days or an identically presented placebo (placebo group) . MEASUREMENTS AND MAIN RESULTS: Only infants with sepsis confirmed by positive blood culture were recruited into the study . There were no significant differences at randomization between the pentoxifylline and placebo groups with regard to the birth weight, gestational age, gender, Apgar score, hypotension, neutropenia, thrombocytopenia, metabolic acidosis, plasma levels of cytokines, and occurrence of shock . Plasma levels of TNF, IL-1, and IL-6 were evaluated before and after the drug or placebo administration on the first, third, and sixth days of therapy . Cytokines were determined by immunoenzymetric test EASIA (TNF) and Endogen Interleukin-Elisa (IL-1, IL-6) . The frequency of gram-negative sepsis was similar in both groups (37.5% and 36.8%) . Pentoxifylline significantly diminished plasma TNF levels (p = .009) but had no effect on plasma IL-1 levels . Mean plasma IL-6 levels, which were measured in the pentoxifylline group on the 6th day of the study, were significantly lower compared with respective data obtained in the placebo group . Only 1 of 40 infants with sepsis in the pentoxifylline group died, whereas 6 of 38 infants in the placebo group did not survive (p = .046) . An increased incidence of disordered peripheral circulation and metabolic acidosis (p = .048), anuria or oliguria (p = .03), disseminated intravascular coagulation (p = .043), and the occurrence of clinical symptoms of necrotizing enterocolitis (p = .025) was observed in the course of sepsis in infants in the placebo group . CONCLUSION: Pentoxifylline significantly affects the synthesis of TNF and IL-6 as well as reduces the mortality rate in premature infants with sepsis . The dosage and schedule of drug administration in this study attenuated the severity of the clinical course of sepsis in this group of patients. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 779 - 82 Phylogenetic analysis of Formivibrio citricus, Propionivibrio dicarboxylicus, Anaerobiospirillum thomasii, Succinimonas amylolytica and Succinivibrio dextrinosolvens and proposal of Succinivibrionaceae fam . nov; Hippe H et al.; The phylogenetic position of Gram-negative, strictly anaerobic, non-spore-forming bacteria, representing four different genera, was determined by analysis of their 16S rDNA sequences . Formivibrio citricus and Propionivibrio dicarboxylicus are members of the beta-subclass of the class Proteobacteria . While Formivibrio citricus stands phylogenetically isolated, Propionivibrio dicarboxylicus is moderately related to members of the genus Rhodocyclus . Succinimonas amylolytica and Succinivibrio dextrinosolvens are members of the gamma-subclass of the class Proteobacteria in which they, together with members of the genus Anaerobiospirillum and Ruminobacter amylophilus, form a separate line of descent . This phylogenetic group is described as Succinivibrionaceae fam . nov. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 629 - 34 Roseivivax halodurans gen . nov., sp . nov . and Roseivivax halotolerans sp . nov., aerobic bacteriochlorophyll-containing bacteria isolated from a saline lake; Suzuki T et al.; Phenotypic and phylogenetic studies were performed with two strains (OCh 239T and OCh 210T, T = type strain) of aerobic bacteriochlorophyll-containing bacteria isolated from the charophytes and the epiphytes on the stromatolites, respectively, of a saline lake located on the west coast of Australia . Both strains were chemoheterotrophic, Gram-negative and motile rods with subpolar flagella . Catalase and oxidase were produced . ONPG reaction was positive . Cells utilized D-glucose, acetate, butyrate, citrate, DL-lactate, DL-malate, pyruvate, succinate, L-aspartate and L-glutamate . Acids were produced from D-fructose and D-glucose . Bacteriochlorophyll a was synthesized under aerobic conditions . Strain OCh 239T had nitrate reductase and phosphatase . Acids were produced from L-arabinose, D-galactose, lactose, maltose, D-ribose and sucrose . The strain could grow in 0-20.0% (w/v) NaCl . Strain OCh 210T had urease . Hydrolysis of gelatin was positive . Acids were produced from D-xylose . The strain could grow in 0.5-20.0% (w/v) NaCl . The results of 16S rRNA sequence comparisons revealed that strains OCh 239T and OCh 210T formed a new cluster within the alpha-3 group of the alpha subclass of the class Proteobacteria . The similarity value of the 16S rRNA sequences between strains OCh 239T and OCh 210T was 95.8% . Therefore, it was concluded that these two strains should be placed in a new genus, Roseivivax gen . nov., as the new species Roseivivax halodurans sp . nov . and Roseivivax halotolerans sp . nov . The type species of the genus is Roseivivax halodurans . The type strains of Roseivivax halodurans and Roseivivax halotolerans are OCh 239T (= JCM 10272T) and OCh 210T (= JCM 10271T), respectively. Int J Syst Bacteriol, 1999 Apr, 49 Pt 2, 345 - 50 Desulfobacca acetoxidans gen . nov., sp . nov., a novel acetate-degrading sulfate reducer isolated from sulfidogenic granular sludge; Oude Elferink SJ et al.; A mesophilic sulfate reducer, strain ASRB2T, was isolated with acetate as sole carbon and energy source from granular sludge of a laboratory-scale upflow anaerobic sludge bed reactor fed with acetate and sulfate . The bacterium was oval-shaped, 1.3 x 1.9-2.2 microns, non-motile and Gram-negative . Optimum growth with acetate occurred around 37 degrees C in freshwater medium (doubling time: 1.7-2.2 d) . Enzyme studies indicated that acetate was oxidized via the carbon monoxide dehydrogenase pathway . Growth was not supported by other organic acids, such as propionate, butyrate or lactate, alcohols such as ethanol or propanol, and hydrogen or formate . Sulfite and thiosulfate were also used as electron acceptors, but sulfur and nitrate were not reduced . Phylogenetically, strain ASRB2T clustered with the delta subclass of the Proteobacteria . Its closest relatives were Desulfosarcina variabilis, Desulfacinum infernum and Syntrophus buswellii . Strain ASRB2T is described as the type strain of Desulfobacca acetoxidans gen . nov., sp . nov. J Biol Chem, 1999 May 14, 274(20), 13993 - 8 Membrane expression of soluble endotoxin-binding proteins permits lipopolysaccharide signaling in Chinese hamster ovary fibroblasts independently of CD14; Ingalls RR et al.; The activation of phagocytes by lipopolysaccharide (LPS) has been implicated in the pathogenesis of Gram-negative sepsis . Although the interaction between CD14 and LPS is a key event in the signaling cascade, the molecular mechanism by which cellular activation occurs remains obscure . We hypothesized that the main function of CD14 was to bind LPS and transfer it to a second receptor, which then initiates the subsequent signal for cellular activation . Thus, surface binding of LPS to the cell membrane would be the critical step that CD14 carries out . To test this hypothesis, we examined the activity of two other proteins known to bind LPS, lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein . We found that when these normally soluble proteins were expressed in Chinese hamster ovary-K1 fibroblasts as glycosylphosphatidylinositol-anchored proteins, both could substitute for CD14 in initiating LPS signaling . Pharmacological studies with synthetic lipid A analogues demonstrated that these surface expressed LPS-binding proteins had characteristics that were qualitatively identical to membrane CD14 . These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer and suggest that surface binding of LPS to the cell membrane is the crucial first step for initiating downstream signaling events. Indian J Physiol Pharmacol, 1997 Oct, 41(4), 344 - 52 Alterations in intracellular calcium during sepsis; Bhattacharyya J et al.; Tissue injury and/or infection produce significant alterations in intracellular calcium ion regulation . These alterations in cellular calcium has recently been studied following both short term and long term septic model which uses two types of gram-negative bacteria frequently encountered human abdominal sepsis . Changes in calcium flux as well as functional disturbances has been observed in the major organs, specially in skeletal muscle . The changes in calcium flux in different organs were studied using 45Ca exchange, 19F NMR study or by using calcium-fluorescence probes . Calcium-channel blockers attenuate the increased effects of calcium flux . Further anti-cytokines may be useful to prevent septic injury in tissues. FEMS Microbiol Lett, 1999 May 1, 174(1), 179 - 84 Mutations in dsbA and dsbB, but not dsbC, lead to an enhanced sensitivity of Escherichia coli to Hg2+ and Cd2+; Stafford SJ et al.; The Dsb proteins are involved in disulfide bond formation, reduction and isomerisation in a number of Gram-negative bacteria . Mutations in dsbA or dsbB, but not dsbC, increase the proportion of proteins with free thiols in the periplasm compared to wild-type . We investigated the effects of mutations in these genes on the bacterial resistance to mercuric and cadmium salts . Mutations in genes involved primarily in disulfide formation (dsbA and dsbB) generally enhanced the sensitivity to Hg2+ and Cd2+ while a mutation of the dsbC gene (primarily an isomerase of disulfide bonds) had no effect . Mutations of the dsb genes had no effect on the expression of the mercury-resistance determinants of the transposon Tn501. Schweiz Med Wochenschr, 1999 Mar 27, 129(12), 475 - 83 {Endotoxins in the workplace and in the environment}; Danuser B et al.; Endotoxins, cell-wall fragments of gram-negative bacteria, are found in various work environments and first measurements have been made in general indoor environments . Endotoxins cause an inflammatory response of the respiratory tract . The response is mediated by the proinflammatory cytokines IL-1, IL-6, IL-8, and TNF-alpha and gives rise to general symptoms (fever, headache, malaise), respiratory symptoms (tightness of chest, dry cough), and lung function decrements . In the work environment endotoxins have been identified in all environments which produce similar symptoms . The qualitative results of experimental and epidemiological studies agree well . The related question whether endotoxins are the biologically active component of organic dust cannot yet be answered because of the gap between the concentration of lipopolysaccharides and of endotoxins necessary to induce the same quantitative effect . Different possible explanations are discussed . Endotoxins are also found in the general environment, especially indoors . Their health relevance needs to be assessed in more detail, especially in subjects with bronchial hyperreactivity. Rapid Commun Mass Spectrom, 1999, 13(7), 604 - 6 Corona plasma discharge for rapid analysis of microorganisms by mass spectrometry; Birmingham J et al.; Corona plasma discharge provides a rapid and reliable tool for release of biomarkers from gram negative and positive bacteria, spores and viruses for characterization by mass spectrometry. Intensive Care Med, 1999 Mar, 25(3), 279 - 87 Mixed agonistic-antagonistic cytokine response in whole blood from patients undergoing abdominal aortic aneurysm repair; Ziegenfuss T et al.; OBJECTIVE: To characterize the impact of abdominal aortic aneurysm repair (AAAR) on spontaneous as well as lipopolysaccharide (LPS)-induced gene expression of pro- and anti-inflammatory cytokines . DESIGN: Prospective, controlled in vivo/ex vivo study . SETTING: University hospital . PATIENTS AND INTERVENTIONS: Whole blood from 14 consecutive patients undergoing AAAR withdrawn prior to surgery (T1), at the end of ischemia (T2), 90 min after declamping (T3) and on the first postoperative day (T4) was cultured in the absence or presence of LPS . Five patients undergoing elective inguinal hernia repair served as controls . MEASUREMENTS AND RESULTS: While tumor necrosis factor (TNF), Interleukin (IL)-1 and IL-10 plasma concentrations did not increase significantly, IL-6 was elevated at each time point, as compared with T1 . Despite the spontaneous release of trace amounts of IL-6, the ability of cultured whole blood to mount a cytokine response in vitro to LPS was impaired for all cytokines studied at T2 (TNF-62%, IL-1-51%, IL-6 -20%, IL-10-51%) . The stimulated IL-6 response was restored early after declamping (T3: +56 %) and enhanced 1 day after operation (T4: +144%) . In contrast, stimulated TNF and IL-1 responses remained depressed at T3 (TNF -48%, IL-1-64%) and T4 (TNF-40%, IL-1-24%) . A biphasic pattern was observed for IL-10 with initial depression at T3 (-51%) and restoration at T4 (+40%) . Among the different cytokines monitored, only impaired TNF responsiveness at early reperfusion (T3) correlated with the postoperative course, as reflected by APACHE II . Cytokine response to LPS was maintained or even increased during and after surgery in the whole blood from patients undergoing hernia repair . CONCLUSIONS: Despite consistent development of clinical signs of systemic inflammatory response syndrome (SIRS) and spontaneous release of IL-6 abdominal aortic aneurysm repair produces a state of impaired pro-inflammatory cytokine response upon a subsequent in vitro Gram-negative stimulus . This early impairment of TNF responsiveness seems to correlate with an unfavorable postoperative course. FEMS Microbiol Lett, 1999 Apr 15, 173(2), 347 - 52 Altered temperature induction sensitivity of the lambda pR/cI857 system for controlled gene E expression in Escherichia coli; Jechlinger W et al.; Cell lysis of Gram-negative bacteria can be efficiently achieved by expression of the cloned lysis gene E of bacteriophage PhiX174 . Gene E expression is tightly controlled by the rightward lambda pR promoter and the temperature-sensitive repressor cI857 on lysis plasmid pAW12 . The resulting empty bacterial cell envelopes, called bacterial ghosts, are currently under investigation as candidate vaccines . Expression of gene E is stringently repressed at temperatures up to 30 degrees C, whereas gene E expression, and thus cell lysis, is induced at temperatures higher than 30 degrees C due to thermal inactivation of the cI857 repressor . As a consequence, the production of ghosts requires that bacteria have to be grown at 28 degrees C before the lysis process is induced . In order to reflect the growth temperature of pathogenic bacteria in vivo, it seemed favorable to extend the heat stability of the lambda pR promoter/cI857 repressor system, allowing pathogens to grow at 37 degrees C before induction of lysis . In this study we describe a mutation in the lambda pR promoter, which allows stringent repression of gene E expression at temperatures up to 36 degrees C, but still permits induction of cell lysis at 42 degrees C. Infect Immun, 1999 May, 67(5), 2103 - 9 Identification and characterization of an Escherichia coli invasion gene locus, ibeB, required for penetration of brain microvascular endothelial cells; Huang SH et al.; Escherichia coli K1 is the most common gram-negative organism causing neonatal meningitis, but the mechanism by which E . coli K1 crosses the blood-brain barrier is incompletely understood . We have previously described the cloning and molecular characterization of a determinant, ibeA (also called ibe10), from the chromosome of an invasive cerebrospinal fluid isolate of E . coli K1 strain RS218 (O18:K1:H7) . Here we report the identification of another chromosomal locus, ibeB, which allows RS218 to invade brain microvascular endothelial cells (BMEC) . The noninvasive TnphoA mutant 7A-33 exhibited <1% the invasive ability of the parent strain in vitro in BMEC and was significantly less invasive in the central nervous system in the newborn rat model of hematogenous E . coli meningitis than the parent strain . The TnphoA insert with flanking sequences was cloned and sequenced . A 1,383-nucleotide open reading frame (ORF) encoding a 50-kDa protein was identified and termed ibeB . This ORF was found to be 97% identical to a gene encoding a 50-kDa hypothetical protein (p77211) and located in the 13-min region of the E . coli K-12 genome . However, no homology was observed between ibeB and other known invasion genes when DNA and protein databases in GenBank were searched . Like the TnphoA insertion mutant 7A-33, an isogenic ibeB deletion mutant (IB7D5) was unable to invade BMEC . A 7 . 0-kb locus containing ibeB was isolated from a LambdaGEM-12 genomic library of E . coli RS218 and subcloned into a pBluescript KS vector (pKS7-7B) . pKS7-7B was capable of completely restoring the BMEC invasion of the noninvasive TnphoA mutant 7A-33 and the ibeB deletion mutant IB7D5 to the level of the parent strain . More importantly, the ibeB deletion mutant IB7D5 was fully complemented by pFN476 carrying the ibeB ORF (pFN7C), indicating that ibeB is required for E . coli K1 invasion of BMEC . Taken together, these findings indicate that several E . coli determinants, including ibeA and ibeB, contribute to crossing of the blood-brain barrier. Scand J Infect Dis, 1998, 30(6), 579 - 83 In vitro expression of beta-lactam-induced response by clinical gram-negative bacteria with the potential for inducible beta-lactamase production; Bongaerts G et al.; The aim of this study was to investigate the in vitro beta-lactam-induced response of clinical Gram-negative bacteria with the potential for inducible beta-lactamase production, in order to be able to discriminate diagnostically between inducible and constitutive beta-lactamase production . A total of 242 clinical Gram-negative isolates of species with inducible chromosomal beta-lactamase were subjected to a disc diffusion test involving agar dilution of the inducers . Cefoxitin (FOX) and imipenem (IPM) were used as inducers and the antibiotics ceftazidime, cefuroxim, cefazolin, amoxycillin and piperacillin as indicators . beta-lactamase induction was observed at concentrations as low as 0.06 mg/l FOX or 0.008 mg/l IPM . In our test, 2 types of antibiotic phenomenon often interfered with inductive effects . Firstly a minor antibiotic effect was seen as an increase in inhibition zones at increasing inducer concentration and, secondly, absence of growth was caused by too high antibiotic activity of the inducer . The induced decrease in zone diameters varied strongly (up to 22 mm) . Expressions of resistance were combined in inducibility profiles . Compilation of these profiles allowed an explanation to be proposed for the multi(-beta-lactam)-resistance, i.e . that most isolates combine inducible beta-lactamase synthesis with one or more resistance-potentiating factors . Only a few isolates demonstrated non-inducible resistance, which was probably due to mutation-mediated de-repression of beta-lactamase synthesis . The test presented here may be well-suited to studying inducibility of beta-lactamase production in the diagnostic laboratory. Appl Environ Microbiol, 1999 May, 65(5), 1843 - 8 Export of virulence genes and Shiga toxin by membrane vesicles of Escherichia coli O157:H7; Kolling GL et al.; Membrane vesicles released by Escherichia coli O157:H7 into culture medium were purified and analyzed for protein and DNA content . Electron micrographs revealed vesicles that are spherical, range in size from 20 to 100 nm, and have a complete bilayer . Analysis of vesicle protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates vesicles that contain many proteins with molecular sizes similar to outer membrane proteins and a number of cellular proteins . Immunoblot (Western) analysis of vesicles suggests the presence of cell antigens . Treatment of vesicles with exogenous DNase hydrolyzed surface-associated DNA; PCR demonstrated that vesicles contain DNA encoding the virulence genes eae, stx1 and stx2, and uidA, which encodes for beta-galactosidase . Immunoblot analysis of intact and lysed, proteinase K-treated vesicles demonstrate that Shiga toxins 1 and 2 are contained within vesicles . These results suggest that vesicles contain toxic material and transfer experiments demonstrate that vesicles can deliver genetic material to other gram-negative organisms. Vet Microbiol, 1999 Mar 31, 66(1), 67 - 80 Serological characterization of Actinobacillus pleuropneumoniae serotype 2 strains by using polyclonal and monoclonal antibodies; Bouh KC et al.; The antigenic differences between strains of serotype 2 of both biotypes I and II of Actinobacillus pleuropneumoniae were studied by using several serological techniques . Monoclonal antibodies (MAbs) against A . pleuropneumoniae biotype I serotype 2 were produced by fusion of spleen cells of BALb/c mice immunized with whole-cell (WC) suspension with SP2/O-Ag14 murine myeloma cells . Desirable MAbs were selected by enzyme-linked immunosorbent assay (ELISA) using WC as antigen . MAbs MK-7 and MK-10 identified multiple bands of lipopolysaccharide in Western-blot . Treatment of WC with proteinase K and sodium periodate indicated that both MAb binding sites were carbohydrates in nature . In both ELISA and Western-blot, MAbs MK-7 and MK-10 recognized only biotype I serotype 2 isolates . Neither MAb MK-7 nor MK-10 reacted with reference strains of remaining serotypes of A . pleuropneumoniae and other Gram-negative bacteria tested . The results obtained with various serological tests showed that strains of serotype 2 biotype I shared antigenic determinants with strain N-282 of serotype 2 biotype II, but not with strain N-273 of serotype 1 biotype II . It is suggested that data obtained from this study may be helpful in the development of specific serotyping and serodiagnostic reagents of A . pleuropneumoniae strains. Arch Biochem Biophys, 1999 May 1, 365(1), 71 - 4 Phenyl-N-tert-butylnitrone demonstrates broad-spectrum inhibition of apoptosis-associated gene expression in endotoxin-treated rats; Stewart CA et al.; Systemic exposure to gram-negative bacterial substances such as lipopolysaccharide (LPS, or endotoxin) induces an uncontrolled, massive inflammatory reaction which culminates in multiple system organ failure and death . Septic shock often does not respond to corticosteroids; however, certain low-molecular-weight antioxidant compounds have been discovered to possess potent anti-inflammatory action, and some of these novel compounds can rescue animals from experimentally induced septic shock . Phenyl-N-tert-butylnitrone (PBN) is the archetype of the nitrone class of antioxidants which we have previously shown to suppress LPS-induced cytokine biosynthesis in vivo . Using a multiprobe ribonuclease protection assay, we now demonstrate the ability of PBN to suppress proapoptotic gene expression in the LPS-induced model of endotoxic shock . The broad-spectrum gene-suppressive affects of PBN are discussed in the context of inflammatory signal transduction and models are proposed to explain why certain antioxidants may also possess anti-inflammatory and antiapoptotic properties . Int J Antimicrob Agents, 1999 Feb, 11(2), 115 - 9 Impact of cefuroxime administration on endotoxin (LPS) and tumour necrosis factor-alpha (TNFalpha) blood levels in patients suffering from acute pyelonephritis: a preliminary report; Giamarellou-Bourboulis EJ et al.; It has been suggested that treatment of systemic infections caused by Gram-negative bacteria with beta-lactam agents might add to the inflammatory process by resulting in the release of endotoxins (LPS) upon death of the Gram-negative bacteria . To evaluate that hypothesis, 25 patients with acute pyelonephritis of Gram-negative aetiology were given intravenous cefuroxime 1.5 g tid . Blood samples were collected at various time intervals for blood culture and for the determination of LPS, tumour necrosis factor-alpha (TNFalpha) and cefuroxime levels . LPS remained elevated at levels equal to those before the administration of cefuroxime over the first 24 h of therapy . A positive correlation was detected between LPS and drug levels 6 h after the initiation of therapy . Fever persisted in 50, 37.5 and 16.7% of patients 48, 72 and 96 h after the start of treatment, respectively, followed by a rise of LPS at levels above the baseline . Blood cultures taken at the same time were sterile . A wide range of TNFalpha levels were found at similar times of sampling, indicating that LPS triggers considerable TNFalpha production in the serum of some patients but not in others . It is concluded that antibiotic-induced endotoxaemia is a phenomenon that might be observed in patients receiving cefuroxime and that might be responsible for the persistence of fever despite negative blood cultures. Ned Tijdschr Geneeskd, 1999 Feb 6, 143(6), 288 - 90 {Chlamydia pneumoniae as a causative agent in atherosclerosis}; van Deventer SJ; There are indications that inflammatory mechanisms play a part in the development of atherosclerosis, and infections by various micro-organisms have been related to the development of atherosclerotic lesions of vascular walls . Epidemiological observations, immunohistochemical examination of samples of vascular wall and studies in laboratory animals have yielded strong indications that infections with Chlamydia pneumoniae, a Gram-negative bacterium, are related to the development of atherosclerosis and occurrence of myocardial infarction . Complications after a myocardial infarction are also connected with seropositivity for C . pneumoniae . These findings suggest a connection between (airway) infections caused by C . pneumoniae and the development of atherosclerosis . A possible pathogenic factor is that endotoxins are moved by lipoproteins and after incorporation of the lipoproteins into the vascular wall may cause a local inflammation. FEMS Immunol Med Microbiol, 1999 Mar, 23(3), 259 - 69 Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55); el-Samalouti VT et al.; The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis . Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist . In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells . Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14 . Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques . Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes . However, the involvement of CD55 in LPS-induced signaling remains to be elucidated. Mol Med Today, 1999 Mar, 5(3), 123 - 32 The molecular pathogenesis of endotoxic shock and organ failure; Karima R et al.; Sepsis is still associated with a high mortality rate . Septic shock and sequential multiple organ failure have a strong correlation with poor outcome . Lipopolysaccharide (LPS) plays a pivotal role in the initiation of host responses to Gram-negative infection . A number of mediators, such as cytokines, nitric oxide and eicosanoids, are responsible for most of the manifestations caused by LPS, and circulatory failure, leukocyte-induced tissue injury and coagulation disorder appear to be critical determinants in the development of sequential organ failure . Although several anti-LPS or anti-cytokine clinical trials have been attempted, none of them has so far been successful. Electrophoresis, 1999 Mar, 20(3), 462 - 5 Use of a bilayer stacking gel to improve resolution of lipopolysaccharides and lipooligosaccharides in polyacrylamide gels; Inzana TJ et al.; Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are important antigenic and integral components of the outer membrane of Gram-negative bacteria . Alteration or heterogeneity of LPS/LOS structure is most often assessed by alteration of electrophoretic band profiles using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . In order to discern minor differences in the electrophoretic profile of closely spaced bands, particularly the low molecular weight bands of LOS, optimum resolution is required . Unfortunately, many publications of LPS/LOS in polyacrylamide gels show a diffuse, smeared pattern without discernible bands . We report here a formulation for polyacrylamide gels that reproducibly yields LPS/LOS bands with sharp resolution . A key feature of this formulation is the use of a separate comb gel containing electrode buffer layered on top of the conventional stacking gel. Am J Ind Med, 1999 May, 35(5), 481 - 90 Work-related symptoms and dose-response relationships for personal exposures and pulmonary function among woodworkers; Mandryk J et al.; BACKGROUND: Four sawmills, a wood chipping mill, and five joineries in New South Wales, Australia, were studied for the effects of personal exposure to wood dust, endotoxins . (1-->3)-beta-D-glucans, Gram-negative bacteria, and fungi on lung function among woodworkers . METHODS: Personal inhalable and respirable dust sampling was carried out . The lung function tests of workers were conducted before and after a workshift . RESULTS: The mean percentage cross-shift decrease in lung function was markedly high for woodworkers compared with the controls . Dose-response relationships among personal exposures and percentage cross-shift decrease in lung function and percentage predicted lung function were more pronounced among joinery workers compared with sawmill and chip mill workers . Woodworkers had markedly high prevalence of regular cough, phlegm, and chronic bronchitis compared with controls . Significant associations were found between percentage cross-shift decrease in FVC and regular phlegm and blocked nose among sawmill and chip mill workers . Both joinery workers and sawmill and chip mill workers showed significant relationships between percentage predicted lung function (FVC, FEV1, FEV1/FVC, FEF25-75%) and respiratory symptoms . CONCLUSIONS: Wood dust and biohazards associated with wood dust are potential health hazards and should be controlled. Lett Appl Microbiol, 1999 Apr, 28(4), 275 - 9 An investigation of the presence of ultramicrocells in natural mineral water; Jones CR et al.; The presence of 'ultramicrocells' in natural mineral water, capable of passing through a 0.2 micron filter, has been demonstrated . Filters allowing the greatest proportion of viable (culturable) cells to pass ranked in the order, 0.4 micron polycarbonate (5.02%) > 0.2 micron polycarbonate (0.02%) > or = 0.45 micron cellulose nitrate (0.02%) > 0.2 micron cellulose acetate (< 0.002%) . Following incubation for 4 d at 22 degrees C, viable counts in filtered mineral water increased from < 2-8.7 x 10(2) cfu ml-1(-2).8 x 10(4)-1.9 x 10(6) cfu ml-1 . Successive filtration/incubation cycles of mineral water increased the proportion of cells passing through a 0.2 micron cellulose acetate filter from < 0.003% to 0.11% and 0.69%, suggesting selection for 'ultramicrocells' . Cells isolated from this process and grown on liquid R2A medium were thin, Gram-negative rods, of 0.15-0.40 micron wide and 0.50-6.20 microns long . Membrane filtration techniques used for pathogen detection in mineral waters will not retain all the cells present . If pathogens are able to form ultramicrocells, these may go undetected. J Biol Chem, 1999 Apr 23, 274(17), 11727 - 35 A hemocyte-like cell line established from the malaria vector Anopheles gambiae expresses six prophenoloxidase genes; Muller HM et al.; Cell lines from the malaria vector Anopheles gambiae have been established as a tool for the study of the mosquito innate immune system in vitro . Here, we describe the first continuous insect cell line that produces prophenoloxidase (PPO) . This cell line (4a-3B) expresses constitutively six PPO genes, three of which are novel (PPO4, PPO5, and PPO6) . The PPO genes show distinct temporal expression profiles in the intact mosquito, spanning stages from the embryo to the adult in an overlapping manner . Transient induction of larva-specific PPO genes in blood-fed adult females suggests that the developmental hormone 20-hydroxyecdysone may be involved in PPO gene regulation . Indeed, exposure of 4a-3B cells to 20-hydroxyecdysone in culture results in induction of those PPO genes that are mainly expressed in early developmental stages, and repression of PPO5, which is preferentially expressed at the adult stage . The cell line shows bacteria-induced immune transcripts that encode defensin and Gram-negative bacteria-binding protein, but no induction of PPO transcripts . This cell line most likely derives from a hemocyte lineage, and represents an appropriate in vitro model for the study of the humoral and cellular immune defenses of A . gambiae. Ann Surg, 1999 Apr, 229(4), 542 - 50 Heparin and enoxaparin enhance endotoxin-induced tumor necrosis factor-alpha production in human monocytes; Heinzelmann M et al.; OBJECTIVE: To determine whether heparin or the low-molecular-weight heparin enoxaparin alter lipopolysaccharide (LPS)-induced monocyte activation . SUMMARY BACKGROUND DATA: Heparin is widely used in clinical practice to inhibit the coagulation cascade . However, heparin also is a naturally occurring glucosaminoglycan and a pleiotropic immunomodulator that binds to a variety of proteins . LPS is a component of gram-negative bacteria and is thought to be responsible for many of the deleterious effects seen in sepsis . The binding of LPS to CD14 induces a signaling cascade that results in the release of many inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha) . METHODS: Monocytes from healthy volunteers were isolated and cultured in the presence of saline, LPS (10 ng/ml), heparin (0.1 to 1000 microg/ml), or enoxaparin (0.1 to 1000 microg/ml) . In blocking experiments, cells were pretreated for 60 minutes with the monoclonal anti-CD14 antibody MY4 (10 microg/ml) or with isotype-matched control IgG2 (10 microg/ml) . TNF-alpha values were measured with enzyme-linked immunosorbent assay . Significance was assessed with analysis of variance . RESULTS: Heparin (10 to 1000 microg/ml) and enoxaparin (1000 microg/ml) significantly enhanced LPS-induced TNF-alpha release . Heparin (1000 microg/ml) or enoxaparin (1000 microg/ml) did not produce TNF-alpha in the absence of LPS . Blockade of CD14 abrogated both LPS-induced TNF-alpha release and the effect of heparin or enoxaparin to enhance LPS-induced TNF-alpha release . CONCLUSIONS: The effect of heparin to enhance LPS-induced TNF-alpha release is a biologic phenomenon that reveals a novel and potentially important host defense mechanism during endotoxemia and sepsis . Binding of LPS to CD14 is necessary to induce this phenomenon, suggesting that both heparin and enoxaparin induce signaling mechanisms that are downstream from the initial binding of LPS on CD14. Mol Microbiol, 1999 Mar, 31(5), 1307 - 19 Structure, assembly and regulation of expression of capsules in Escherichia coli; Whitfield C et al.; Many Escherichia coli strains are covered in a layer of surface-associated polysaccharide called the capsule . Capsular polysaccharides represent a major surface antigen, the K antigen, and more than 80 distinct K serotypes result from structural diversity in these polymers . However, not all capsules consist of K antigen . Some are due to production of an extensive layer of a polymer structurally identical to a lipopolysaccharide O antigen, but distinguished from lipopolysaccharide by the absence of terminal lipid A-core . Recent research has provided insight into the manner in which capsules are organized on the Gram-negative cell surface, the pathways used for their assembly, and the regulatory processes used to control their expression . A limited repertoire of capsule expression systems are available, despite the fact that the producing bacteria occupy a variety of ecological niches and possess diverse physiologies . All of the known capsule assembly systems seen in Gram-negative bacteria are represented in E . coli, as are the majority of the regulatory strategies . Escherichia coli therefore provides a variety of working models on which studies in other bacteria are (or can be) based . In this review, we present an overview of the current molecular and biochemical models for capsule expression in E . coli . By taking into account the organization of capsule gene clusters, details of the assembly pathway, and regulatory features that dictate capsule expression, we provide a new classification system that separates the known capsules of E . coli into four distinct groups. Arterioscler Thromb Vasc Biol, 1999 Apr, 19(4), 932 - 8 A new promoter polymorphism in the gene of lipopolysaccharide receptor CD14 is associated with expired myocardial infarction in patients with low atherosclerotic risk profile; Unkelbach K et al.; Recent findings suggest that inflammation plays a role in atherosclerosis and its acute complications . Cellular response in infections with Gram-negative bacteria is mediated by bacterial lipopolysaccharide (LPS), which activates monocytes to expression of cytokines, growth factors, and procoagulatory factors via LPS receptor CD14 . Endothelial cells and smooth muscle cells are stimulated by a complex of LPS and soluble CD14 . In this study, LPS receptor CD14 was analyzed to find genetic variants and check them for an association with coronary artery disease or myocardial infarction (MI) . When screening the CD14 gene by single-strand conformation polymorphism analysis, a promoter polymorphism was detected and confirmed as a T-to-C exchange at position -159 . We determined the genotypes of 2228 men who had undergone coronary angiography for diagnostic purposes . Within the total study group there was no significant association of either genotype with MI or coronary artery disease . However, in a subgroup with low coronary risk (normotensive nonsmokers), a relative risk for MI in probands homozygous for the T allele could be evaluated (OR, 1.6; 95% CI, 1.0 to 2.4; P<0.05) . The association was even stronger in low-risk patients older than 62 years (OR, 3.8; 95% CI, 1.6 to 9.0; P<0.01) . In conclusion, we describe a new CD14 promoter polymorphism that is associated with MI, especially in older patients with a low atherosclerotic risk profile. Vaccine, 1999 Mar 26, 17(13-14), 1643 - 9 New strategies for combination vaccines based on the extended recombinant bacterial ghost system; Eko FO 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 have been produced from a great variety of bacteria and 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 extents the capacity of ghosts or recombinant ghosts to function as carriers of foreign antigens, immunomodulators or other substances . In addition, S-layer proteins forming shell-like self assembly structures can be expressed in bacterial candidate vaccine strains prior to E-mediated lysis . Such recombinant S-layer proteins carrying inserts of foreign epitopes of up to 600 amino acids within the flexible surface loop areas of the S-layer further extend the possibilities of ghosts as carriers of foreign epitopes . As ghosts do not need the addition of adjuvants to induce immunity in experimental animal