ALTEX, 1994, 11(5), 24 - 29 {Investigation of the endotoxin content of veterinary vaccines}; Cussler K et al.; Vaccines composed of gram-negative bacteria contain endotoxin in considerable amounts . This may result in adverse effects after vaccination of sensitive animals . For reasons of safety and animal welfare the endotoxin level of veterinary vaccines should be limited . The limulus amebocyte lysate test is suitable to check the endotoxin content in most vaccines . Safety tests in animals should only be performed if the results of the limulus test are satisfactory. Curr Infect Dis Rep, 2001 Feb, 3(1), 13 - 19 What's New in beta-lactamases? Bradford PA. beta-lactamases continue to be the leading cause of resistance to beta-lactam antibiotics, among gram-negative bacteria . In recent years, both the incidence and the prevalence of extended-spectrum beta-lactamases, inhibitor-resistant beta-lactamases, AmpC-type enzymes, and both metallo- carbapenemases and nonmetallo-carbapenemases have increased . These beta-lactamases provide resistance to oximino-cephalosporins, beta-lactam/beta-lactamase inhibitor combinations, cephamycins, and carbapenems, respectively . Strains expressing these beta-lactamases will generate a host of therapeutic challenges as we begin the 21st century. Bull Exp Biol Med, 2000 Oct, 130(10), 997 - 1000 A single endotoxin aggression causes dose-dependent reversible activation of rat liver Ito cells without their transdifferentiation into myofibroblasts; Salakhov IM et al.; The effect of Gram-negative bacterial lipopolysaccharide on rat hepatocytes and sinusoidal cells was studied . The damage and regeneration potential of the liver were evaluated by activation of perisinusoidal Ito cells and proliferative activity of liver cells . Compensatory and repair reactions in the liver induced by lipopolysaccharide manifested by proliferation of liver cells and reversible activation of Ito cells without their transdifferentiation into myofibroblasts. Ann Med Interne (Paris), 2000 Dec, 151(8), 624 - 8 {Main complications of diabetes mellitus in Africa}; Sidibe EH; In Africa, a rise in complications of diabetes mellitus has gone in hand with the growing disease prevalence, clearly demonstrating the importance of assessing complications . Diabetes mellitus constitutes a major financial burden in developing countries in Africa with relatively limited resources . Ketoacidosis is observed in 24% of juvenile diabetes and is the inaugural sign in 76% of all cases, progressing to coma in 34% . Even in type 2 diabetes, acidoketosis occurs in 34% of the cases . Infection is particularly frequent and is often fatal in tropical Africa because of the involvement of Staphyococcus and Gram-negative microorganisms . Hyperleukocytosis and anemia are correlated with ineffective antibiotic therapy . Pulmonary tuberculosis is the ninth most frequent complication of diabetes . Overall mortality is 14.9 per 1000 person-years of diabetes . Mean age at death is 51.6 years for women and 57.6 years for men after a mean 12.5 year disease duration . Thirty percent of all deaths result from acute metabolic complications, infections and stroke . More than half of the patients with insulin-dependent-diabetes have retinopathy . Differences observed in patients with different ethnic origins is linked basically to unfavorable social and economic conditions that worsen the risk of poor blood glucose control . Retinopathy accounts for 32% of all ocular complications, similar to other African data and more generally in ophthalmology centers . The rate of neuropathy is high, reaching 70% in patients with microangiopathy . Impotence concerns 48.7% of the diabetic population with a mean age of 41.4+/-15.5 years . Coronary artery disease had a recognized influence on hemoglobin diseases, particularly when the coronarography is normal . Lower limb arteriopathy is observed in 18% of the diabetic patients. Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 323 - 5 Crystallization and preliminary crystallographic study of the periplasmic domain of the Escherichia coli TolR protein; Abergel C et al.; The TolR protein from Escherichia coli is part of the Tol-Pal multiprotein complex used by group A colicins to penetrate and kill cells . All genes of the Tol-Pal system are conserved in Gram-negative bacteria and this system is thought to play a role in the maintenance of the bacterial envelope integrity, although its exact function is not known . The TolR protein comprises 142 amino acids . The periplasmic domain of the TolR protein has been expressed, purified and crystallized . The crystals belong to the tetragonal space group P4(1)22, with unit-cell parameters a = 46.3, c = 178.0 A . There are one or two molecules in the asymmetric unit . Frozen crystals diffract to at least 3.2 A resolution using synchrotron radiation . Selenomethionine-substituted periplasmic TolR protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing. Acta Crystallogr D Biol Crystallogr, 2001 Feb, 57(Pt 2), 317 - 9 Crystallization and preliminary crystallographic study of the peptidoglycan-associated lipoprotein from Escherichia coli; Abergel C et al.; The peptidoglycan-associated lipoprotein (Pal) from Escherichia coli is part of the Tol--Pal multiprotein complex used by group A colicins to penetrate and kill cells . Pal homologues are found in many Gram-negative bacteria and the Tol--Pal system is thought to play a role in bacterial envelope integrity . The Pal protein comprises 152 amino acids . Crystals of the C-terminal 109-amino-acid fragment of the Pal protein have been produced . The crystals belong to the tetragonal space group I4(1), with unit-cell parameters a = b = 89.3, c = 67.2 A . There are two molecules in the asymmetric unit . Frozen crystals diffract to at least 2.8 A resolution using synchrotron radiation . Selenomethionine-substituted truncated Pal protein is currently being produced in order to use multiwavelength anomalous dispersion (MAD) for phasing. Clin Infect Dis, 2001 Jan 15, 32(2), 220 - 7 Epub 2000 Dec 22. Molecular typing demonstrating transmission of gram-negative rods in a neonatal intensive care unit in the absence of a recognized epidemic; Almuneef MA et al.; Molecular typing techniques have been used in outbreak investigations . In this study, molecular typing techniques were used to track the spread of gram-negative rods (GNRs) in a neonatal intensive care unit (NICU) in the absence of an outbreak . Stool or rectal swab cultures for GNRs were obtained from all infants on admission, weekly, and on discharge . GNRs were tested for gentamicin susceptibility and were typed by contour-clamped homogeneous electric field electrophoresis . Transmission of identical strains of GNRs among infants was noted . Shared strains were more gentamicin resistant compared with unique strains (53% vs . 10%; P=.0001) . Infants first colonized when they were >1 week of age had more total days of antibiotic treatment and had a higher rate of acquiring a shared and gentamicin-resistant strain, compared with infants colonized earlier . Antibiotic use increases colonization of infants in the NICU with resistant and shared strains of GNRs. Br J Haematol, 2000 Dec, 111(4), 1093 - 102 Procalcitonin in paediatric cancer patients: its diagnostic relevance is superior to that of C-reactive protein, interleukin 6, interleukin 8, soluble interleukin 2 receptor and soluble tumour necrosis factor receptor II; Fleischhack G et al.; Sensitive parameters of inflammation are rare in neutropenic cancer patients . In this study, procalcitonin (PCT), C-reactive protein (CRP), interleukin 6 (IL-6), IL-8, the soluble IL-2 receptor (sIL-2R) and the soluble tumour necrosis factor receptor II (sTNFRII) were evaluated for their diagnostic relevance in febrile episodes of cancer patients . Plasma or serum levels of these parameters were determined in neutropenic children with febrile episodes (n = 122) classified according to both the kind of infection {60 cases of fever of unknown origin (FUO), 28 cases of localized infection, 13 cases of pneumonia, 20 cases of bacteraemia, one case of fungaemia} and the World Health Organization (WHO) score of chemotherapy-induced mucositis . At baseline and during the febrile episodes, the highest levels of all parameters were observed in cases of gram-negative bacteraemia . However, in FUO and localized infections, low or only slightly elevated median levels of all parameters were documented . The degree of chemotherapy-induced mucositis did not influence the value of any parameter . In comparison with the other inflammatory parameters, PCT (optimum cut-off level 0.5 microg/l) was a more sensitive and more specific parameter in the diagnosis of high-risk (gram-negative bacteraemia) and low-risk (FUO) episodes, as well as in the sequential assessment of all febrile neutropenic episodes. Anaesthesia, 2001 Feb, 56(2), 130 - 44 The haemodynamics of human septic shock; MacKenzie IM; In the time it has taken medicine to develop the techniques to describe the circulatory changes of severe infections, both pattern and process have been profoundly influenced by the use of intravenous fluids, vasopressors, antibiotics, steroids, mechanical ventilation and haemoflltration . Constant features of severe sepsis include a reduction in peripheral vascular tone on both the arterial and venous sides of the circulation, a defect in oxygen utilisation resulting in lactic acidosis, and varying degrees of myocardial dysfunction . These events have a temporal progression, the precise pattern observed depending on the tempo of the infection, the influence of therapeutic manoeuvres, the age and comorbidities of the patient, and the time the observations are made in the course of events . Early sepsis is accompanied by a decrease in systemic vascular resistance and a metabolic acidosis . The clinical picture includes fever, tachycardia, tachypnoea, respiratory alkalosis and an increased cardiac output with warm, dry peripheries and a bounding pulse . Advanced sepsis involves varying degrees of venous and myocardial contractile failure, and is characterised by progressive acidaemia, respiratory failure and marked sympathetic adrenergic activation . In the absence of vigorous fluid resuscitation, the cardiac output is decreased and the patients are cold, clammy peripherally shut down, and frequently confused, obtunded or comatose . In infections with a silent primary focus (predominantly involving Gram-negative organisms), this stage is frequently the first to attract the attention of attending staff . Late sepsis is characterised by profound acidaemia, vascular hypo-responsiveness, multiple organ failure and death. Insect Biochem Mol Biol, 2001 Mar 1, 31(3), 219 - 29 Innate immune response of Aedes aegypti; Lowenberger C; Insects are able to protect themselves from invasion by pathogens by a rapid and potent arsenal of inducible immune peptides . This fast, extremely effective response is part of the innate immunity exhibited by all insects and many invertebrates, and shows striking similarities with the innate immune response of vertebrates . In Aedes aegypti invasion of the hemocoel by bacteria elicits the production of defensins, cecropins, a peptide active only against Gram-negative bacteria, and several other peptides that we are now characterizing . However, not all insects utilize the same peptides in the same concentrations, which may reflect the pathogens to which they may have been exposed through evolutionary time . These protective measures we see in mosquitoes are the current state of the evolution of a rapid immune response that has contributed to the success of insects in inhabiting essentially every niche on earth . The molecules involved in the response of Aedes aegypti to pathogens, and the potential role of these peptides against eukaryotic parasites ingested and transmitted by mosquitoes are discussed. J Insect Physiol, 2001 Apr, 47(4-5), 475 - 83 Suppression of pederin biosynthesis through antibiotic elimination of endosymbionts in Paederus sabaeus; Kellner RL; Biosynthesis of the unique defensive compound pederin is confined to female rove beetles of the genus Paederus (Coleoptera: Staphylinidae) . These (+)-females endow their eggs with toxin whereas (-)-females, which occur both naturally and in laboratory reared specimens, do not . The latter are aposymbionts lacking biosynthetic capabilities because of endosymbiotic deficiency . They can, however, be induced to accumulate pederin if fed with (+)-eggs during larval development . The endosymbionts can thus be transmitted by ingestion of (+)-Eggs . (+)-eggs treated with benzylpenicillin, erythromycin, oxytetracycline or streptomycin show that the induction of pederin accumulation depends on the antibiotic's spectrum of efficaciousness, its dosage and duration of the treatment . Certain bacteria, probably belonging to the gram-negative type, must be transmitted to produce (+)-females. Neuroscience, 2001, 102(2), 273 - 80 The effects of the bacterial endotoxin lipopolysaccharide on synaptic transmission and plasticity in the CA1-subiculum pathway in vivo; Commins S et al.; Lipopolysaccharide is derived from the cell wall of gram-negative bacteria and is a potent endotoxin which causes the release of cytokines in the CNS . We examined the effect of lipopolysaccharide on synaptic transmission and synaptic plasticity in the hippocampal area CA1-subicular pathway in vivo . We found that lipopolysaccharide did not affect baseline synaptic transmission in this pathway; it did, however, reduce the magnitude of paired-pulse facilitation, a form of short-term plasticity thought to be primarily presynaptic in origin . We then examined the interaction between lipopolysaccharide and two common models for the biological basis of memory: high-frequency stimulation induced long-term potentiation and low-frequency stimulation induced long-term depression of synaptic transmission . We found that lipopolysaccharide blocked long-term potentiation following high-frequency stimulation and also induced potentiation of synaptic transmission after low-frequency stimulation . Lipolysaccharide blocked paired-pulse facilitation selectively at short rather than longer interstimulus intervals . Thus, lipopolysaccharide has different effects on synaptic transmission in this pathway depending on the frequency and length of stimulation.These results provide new insights into the action of lipopolysaccharide on various forms of plasticity in the hippocampus, an area known to play a vital role in learning and memory. Trends Biochem Sci, 2001 Jan, 26(1), 3 - 6 Type I secretion and multidrug efflux: transport through the TolC channel-tunnel; Buchanan SK; The crystal structure of TolC from Escherichia coli was recently determined to 2.1-A resolution and shows a unique type of channel architecture: a 12-stranded beta-barrel spans the outer membrane and is attached to a long alpha-helical channel that penetrates far into the periplasm . The structure suggests a mechanism for its role in secretion of proteins and in efflux of toxic small molecules . The TolC export pathway is compared with several import pathways of gram-negative bacteria where the outer membrane protein structures are also known. Mech Ageing Dev, 2000 Dec 20, 121(1-3), 89 - 100 Chemokine production by peripheral blood mononuclear cells in elderly subjects; Pulsatelli L et al.; The function of chemokines in promoting and modulating leukocyte migration is essential for a prompt and efficacious inflammatory response and in host defence against infections . In order to investigate whether this important aspect of immunological response is influenced by ageing, we evaluated the basal levels as well as the ability of peripheral blood mononuclear cells from young and healthy elderly subjects to produce chemokines (IL-8, MCP-1, MIP-Ialpha, RANTES) in response to stimulation with anti-CD3 monoclonal antibody and lipopolysaccharide (LPS), a gram negative bacterial endotoxin . Our main findings are a spontaneous chemokine production; a 20% decrease of proliferative response to anti-CD3 monoclonal antibody accompanied by an age related increase of MIP-Ialpha and RANTES production and by a general increase of all chemokine production compared to unstimulated conditions; a proliferative defect of monocytes to LPS challenge associated with an increase of chemokine production compared to basal conditions with a progressive age-related increase of MIP-lalpha . In conclusion, this study suggests that chemokines could have a compensatory role in balancing the impaired mechanisms involved in 'specific' immune response during ageing . The successful activation of this strategy could contribute to the good performance of immune system so maintaining healthy status in elderly. Nucleic Acids Res, 2001 Feb 15, 29(4), 863 - 71 Polymorphism in the 3'-untranslated region of TNFalpha mRNA impairs binding of the post-transcriptional regulatory protein HuR to TNFalpha mRNA; Di Marco S et al.; Tumor necrosis factor alpha (TNFalpha) acts as a beneficial mediator in the process of host defence . In recent years major interest has focused on the AU-rich elements (AREs) present in the 3'-untranslated region (3'-UTR) of TNFalpha mRNA as this region plays a pivotal role in post-transcriptional control of TNFalpha production . Certain stimuli, such as lipopolysaccharides, a component of the Gram-negative bacterial cell wall, have the ability to relinquish the translational suppression of TNFalpha mRNA imposed by these AREs in macrophages, thereby enabling the efficient production of the TNFalpha . In this study we show that the polymorphism (GAU trinucleotide insertional mutation) present in the regulatory 3'-UTR of TNFalpha mRNA of NZW mice results in the hindered binding of RNA-binding proteins, thereby leading to a significantly reduced production of TNFalpha protein . We also show that the binding of macrophage proteins to the main ARE is also decreased by another trinucleotide (CAU) insertion in the TNFalpha 3'-UTR . One of the proteins affected by the GAU trinucleotide insertional mutation was identified as HuR, a nucleo-cytoplasmic shuttling protein previously shown to play a prominent role in the stability and translatability of mRNA containing AREs . Since binding of this protein most likely modulates the stability, translational efficiency and transport of TNFalpha mRNA, these results suggest that mutations in the ARE of TNFalpha mRNA decrease the production of TNFalpha protein in macrophages by hindering the binding of HuR to the ARE. Microbiology, 2001 Jan, 147(Pt 1), 11 - 9 Identification of a new class of biopolymer: bacterial synthesis of a sulfur-containing polymer with thioester linkages; Lutke-Eversloh T et al.; This is the first report on the biosynthesis of a hitherto unknown, sulfur-containing polyester and also the first report on a bacterial polymer containing sulfur in the backbone . The Gram-negative polyhydroxyalkanoate (PHA)-accumulating bacterium Ralstonia eutropha synthesized a copolymer of 3-hydroxybutyrate and 3-mercaptopropionate, poly(3HB-co-3MP), when 3-mercaptopropionic acid or 3,3'-thiodipropionic acid was provided as carbon source in addition to fructose or gluconic acid under nitrogen-limited growth conditions . The peculiarity of this polymer was the occurrence of thioester linkages derived from the thiol groups of 3MP and the carboxyl groups of 3MP or 3HB, respectively, which occurred in addition to the common oxoester bonds of PHAs . Depending on the cultivation conditions and the feeding regime, poly(3HB-co-3MP) contributed up to 19% of the cellular dry weight, with a molar fraction of 3MP of up to 43% . The chemical structure of poly(3HB-co-3MP) was confirmed by GC/MS, IR spectroscopy, (1)H- and (13)C-NMR spectroscopy, and elemental sulfur analysis . The identification of this novel biopolymer reveals a new quality regarding the substrate range of PHA synthases and their capability for the synthesis of technically interesting polymers. Antimicrob Agents Chemother, 2001 Feb, 45(2), 413 - 9 Plasmid location and molecular heterogeneity of the L1 and L2 beta-lactamase genes of Stenotrophomonas maltophilia; Avison MB et al.; An approximately 200-kb plasmid has been purified from clinical isolates of Stenotrophomonas maltophilia . This plasmid was found in all of the 10 isolates examined and contains both the L1 and the L2 beta-lactamase genes . The location of L1 and L2 on a plasmid makes it more likely that they could spread to other gram-negative bacteria, potentially causing clinical problems . Sequence analysis of the 10 L1 genes revealed three novel genes, L1c, L1d, and L1e, with 8, 12, and 20% divergence from the published strain IID 1275 L1 (L1a), respectively . The most unusual L1 enzyme (L1e) displayed markedly different kinetic properties, with respect to hydrolysis of nitrocefin and imipenem, compared to those of L1a (250- and 100-fold lower k(cat)/K(m) ratios respectively) . L1c and L1d, in contrast, displayed levels of hydrolysis very similar to that of L1a . Several nonconservative amino acid differences with respect to L1a, L1b, L1c, and L1d were observed in the substrate binding-catalytic regions of L1e, and this could explain the kinetic differences . Three novel L2 genes (L2b, L2c, and L2d) were sequenced from the same isolates, and their sequences diverge from the published sequence of strain IID 1275 L2 (L2a) by 4, 9, and 25%, respectively . Differences in L1 and L2 gene sequences were not accompanied by similar divergences in 16S rRNA gene sequences, for which differences of <1% were found . It is therefore apparent that the L1 and L2 genes have evolved relatively quickly, perhaps because of their presence on a plasmid. JAMA, 2000 Apr 5, 283(13), 1723 - 30 E5 murine monoclonal antiendotoxin antibody in gram-negative sepsis: a randomized controlled trial . E5 Study Investigators; Angus DC et al.; CONTEXT: Knowledge and understanding of gram-negative sepsis have grown over the past 20 years, but the ability to treat severe sepsis successfully has not . OBJECTIVE: To assess the efficacy and safety of E5 in the treatment of patients with severe gram-negative sepsis . DESIGN: A multicenter, double-blind, randomized, placebo-controlled trial conducted at 136 US medical centers from April 1993 to April 1997, designed with 90% power to detect a 25% relative risk reduction, incorporating 2 planned interim analyses . SETTING: Intensive care units at university medical centers, Veterans Affairs medical centers, and community hospitals . PATIENTS: Adults aged 18 years or older, with signs and symptoms consistent with severe sepsis and documented or probable gram-negative infection . INTERVENTION: Patients were assigned to receive 2 doses of either E5, a murine monoclonal antibody directed against endotoxin (n = 550; 2 mg/kg per day by intravenous infusion 24 hours apart) or placebo (n = 552) . MAIN OUTCOME MEASURES: The primary end point was mortality at day 14; secondary end points were mortality at day 28, adverse event rates, and 14-day and 28-day mortality in the subgroup without shock at presentation . RESULTS: The trial was stopped after the second interim analysis . A total of 1090 patients received study medication and 915 had gram-negative infection confirmed by culture . There were no statistically significant differences in mortality between the E5 and placebo groups at either day 14 (29.7% vs 31.1%; P = .67) or day 28 (38.5% vs 40.3%; P = .56) . Patients presenting without shock had a slightly lower mortality when treated with E5 but the difference was not significant (28.9% vs 33.0% for the E5 and placebo groups, respectively, at day 28; P = .32) . There was a similar profile of adverse event rates between E5 and placebo . CONCLUSIONS: Despite adequate sample size and high enrollment of patients with confirmed gram-negative sepsis, E5 did not improve short-term survival . Current study rationale and designs should be carefully reviewed before further large-scale studies of patients with sepsis are conducted. J Immunol, 2000 Apr 15, 164(8), 4301 - 6 IL-1 receptor-associated kinase modulates host responsiveness to endotoxin; Swantek JL et al.; Endotoxin triggers many of the inflammatory, hemodynamic, and hematological derangements of Gram-negative septic shock . Recent genetic studies in mice have identified the Toll-like receptor 4 as the transmembrane endotoxin signal transducer . The IL-1 intracellular signaling pathway has been implicated in Toll-like receptor signal transduction . LPS-induced activation of the IL-1 receptor-associated kinase (IRAK), and the influence of IRAK on intracellular signaling and cellular responses to endotoxin has not been explored in relevant innate immune cells . We demonstrate that LPS activates IRAK in murine macrophages . IRAK-deficient macrophages, in contrast, are resistant to LPS . Deletion of IRAK disrupts several endotoxin-triggered signaling cascades . Furthermore, macrophages lacking IRAK exhibit impaired LPS-stimulated TNF-alpha production, and IRAK-deficient mice withstand the lethal effects of LPS . These findings, coupled with the critical role for IRAK in IL-1 and IL-18 signal transduction, demonstrate the importance of this kinase and the IL-1/Toll signaling cassette in sensing and responding to Gram-negative infection. APMIS, 2000 Mar, 108(3), 201 - 8 The bactericidal/permeability-increasing protein (BPI) is membrane-associated in azurophil granules of human neutrophils, and relocation occurs upon cellular activation; Calafat J et al.; Neutrophilic granulocytes contain the 55 kDa bactericidal/permeability-increasing protein (BPI) . BPI binds to lipopolysaccharides (LPS), and exerts bacteriostatic and bactericidal effects against a wide variety of Gram-negative bacterial species . We have investigated the subcellular location of BPI in immature and mature neutrophils using cryotechnique for immunoelectron microscopy . BPI was found to colocate with myeloperoxidase (MPO), a marker for azurophil granules, and it also showed the same pattern of distribution as CD63, a transmembrane-anchored protein . This suggests that BPI is membrane-associated in the azurophil granules in neutrophils . Its presence in azurophil granules was further confirmed by the finding of BPI in the azurophil granules of neutrophil promyelocytes of the bone marrow . Induction of selective release of azurophilic granules by the Na-ionophore monensin resulted in fusion of endosomes with azurophil granules, leading to the formation of large vacuoles containing MPO, CD63, and BPI . After phagocytosis of serum-treated zymosan (STZ), BPI was detected in phagosomes, both in association with membranes as well as in the lumen, suggesting the release of BPI into activated compartments . The results show that BPI is present in azurophil granules, is probably primarily membrane-associated, and is relocated after activation, following the same route as MPO and CD63. J Biol Chem, 2001 Mar 23, 276(12), 8820 - 4 Epub 2001 Jan 02. Apolipoprotein E protects against bacterial lipopolysaccharide-induced lethality . A new therapeutic approach to treat gram-negative sepsis; Van Oosten M et al.; Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present . Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines . Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo . We now report that intravenously administered LPS strongly increases the serum levels of apoE . In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents . Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice . These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia. Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2151 - 6 Roseigium denhamense gen . nov., sp . nov . and Roseibium hemelinense sp . nov., aerobic bacteriochlorophyll-containing bacteria isolated from the east and west coasts of Australia; Suzuki T et al.; Phenotypic and phylogenetic studies were performed with 10 strains of bacteriochlorophyll-containing bacteria isolated from a variety of marine environments (surface of Rhodophyta, sand and algal sand mat) on the east and west coasts of Australia . The strains were aerobic, chemoheterotrophic, Gram-negative, motile rods with peritrichous flagella . Bacteriochlorophyll a was synthesized under aerobic conditions . Catalase, nitrate reductase, oxidase and phosphatase were produced . ONPG reaction was positive . The strains have been divided into genotype group 1 (seven strains) and genotype group 2 (three strains) according to previously described DNA-DNA hybridization data . Strains OCh 254T and OCh 368T have been included in genotype groups 1 and 2, respectively . The results of 165 rRNA gene sequence comparisons revealed that strains OCh 254T and OCh 368T formed a new cluster within the alpha-2 group of the alpha subclass of the Proteobacteria . The similarity value of the 16S rRNA gene sequences between strain OCh 254T and the most closely related species, Stappia aggregata, was 95.6 % . The sequence similarity value between strains OCh 254T and OCh 368T was 97.1% . It was concluded that these two strains should be placed into a new genus, Roseibium gen . nov., as Roseibium denhamense sp . nov . and Roseibium hamelinense sp . nov . The type species of the genus is Roseibium denhamense . The type strains of Roseibium denhamense and Roseibium hamelinense are OCh 254T (= JCM 10543T) and OCh 368T (= JCM 10544T), respectively. Minerva Med, 2000 Jul-Aug, 91(7-8), 161 - 7 {Infections and cardiovascular diseases on an ischemic basis . The case of Helicobacter pylori}; Pellicano R et al.; Cardiac and cerebrovascular diseases are an important cause of mortality in industrialized countries . "Classical" risk factors cannot fully explain epidemiological variations of these diseases . From several years infections have been linked to ischemic vascular events and recent publications pointed to the role of Helicobacter pylori, a Gram negative bacterium, involved in the pathogenesis of gastritis and peptic ulcer . Results on the association between this bacterium and acute myocardial infarction or stroke are controversial, due to the degree of studies heterogeneity . There is the need for extensive prospectic studies to evaluate the incidence of these diseases in relation to the presence of Helicobacter pylori infection . Interventional randomized studies employing an antibiotic treatment for patients affected by ischemic vascular diseases will rapidly answer the question of wheather Helicobacter pylori has a causal role in the pathogenesis of acute myocardial infarction and ischemic stroke. Toxicology, 2000 Nov 30, 155(1-3), 37 - 44 Pivotal role of nitric oxide in delayed pharmacological preconditioning against myocardial infarction; Xi L et al.; The phenomenon of 'ischemic preconditioning' (IP) has been vigorously investigated during the past 15 years . As our knowledge on the possible protective mechanisms of IP has been increasingly expanded, novel approaches based on preconditioning with pharmacological agents have recently emerged . Two drugs have been used to induce delayed preconditioning against myocardial infarction caused by ischemia/reperfusion . One of the drugs was monophosphoryl lipid A (MLA)--a detoxified derivative of lipopolysaccharide from gram-negative strains; and another drug was RC552--a novel synthetic glycolipid that mimics the chemical structure of MLA . We have shown that pretreatment of adult mice with MLA or RC552 (350 microg/kg) 24 h prior to the global ischemia and reperfusion in the isolated perfused heart attenuated myocardial injury . Infarct size was significantly reduced in MLA or RC552-treated groups as compared with the vehicle-treated group . The delayed cardioprotection was associated with a moderate but significant increase of nitric oxide level in the ischemic myocardium . Treatment with S-methylisothiourea (3 mg/kg), a selective inhibitor of inducible nitric oxide synthase (iNOS) abolished MLA or RC552-induced delayed protection . In addition, neither MLA nor RC552 reduced infarct size in iNOS knockout mice . Our findings suggest that both MLA and RC552 are able to induce delayed myocardial preconditioning via iNOS-dependent pathway. Oral Microbiol Immunol, 2000 Dec, 15(6), 383 - 7 Identification of an antigenic protein Pga30 from Porphyromonas gingivalis W50; Hendtlass A et al.; Porphyromonas gingivalis is a black-pigmented, gram-negative bacterium that has been implicated as a major pathogen in the development of adult periodontitis . In an approach to identify a P . gingivalis antigen uniquely seroreactive with healthy subjects, we produced a surface and periplasmic extract of P . gingivalis, separated that extract into 36 fractions using anion-exchange chromatography and screened each fraction for reactivity in an enzyme-linked immunosorbent assay (ELISA) using sera from eight periodontitis patients and eight age- and sex-matched healthy controls . All of the diseased subjects harboured subgingival P . gingivalis by DNA probe analysis and exhibited similar seroreactivity profiles to the anion exchange fractions . However, only two of the healthy subjects (C10 and C15) were seroreactive with the fractions . The highest reactivity for all the seropositive subjects was with the same anion-exchange fractions 13-15 . The anion exchange fraction (14) with the highest seroreactivity was subjected to gel filtration chromatography, and fraction 22 from this chromatography exhibited the highest reactivity with all the seropositive subjects . However, fraction 27 was found to be uniquely seroreactive with healthy subject C10, as it was not recognized by sera from any of the diseased or the other healthy subjects . This fraction was further purified by reversed-phase high-pressure liquid chromatography and shown to contain a 30-kDa protein as determined by SDS-PAGE . Control subject C10 had no pocket depths greater than 3 mm and no sites that bled on gentle probing; however, P . gingivalis was detected in subgingival plaque samples at a level of 10(5)-10(6) cells per site in two of the ten sites sampled . This subject was also unusual in that he exhibited a seroreactivity profile similar to that of diseased subjects, which was not characteristic of the healthy control subjects . The unique reactivity of the 30-kDa antigen, designated Pga30, with subject C10 serum was confirmed in a Western blot with the purified antigen . N-Terminal sequence analysis of Pga30 produced a single, unambiguous protein sequence confirming the purity of the protein . A search of the database using the N-terminal sequence obtained did not reveal any significant sequence similarity . In conclusion, we have identified a P . gingivalis antigen that was uniquely reactive in an ELISA and Western blot with serum from a subject with no clinical signs of periodontitis who harbored P . gingivalis in subgingival plaque. Oral Microbiol Immunol, 2000 Dec, 15(6), 378 - 82 Histatin 5 inhibits inflammatory cytokine induction from human gingival fibroblasts by Porphyromonas gingivalis; Imatani T et al.; Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease . It has been demonstrated that outer-membrane proteins as well as lipopolysaccharides from P . gingivalis ATCC 53977 can induce interleukin 6 (IL-6) and IL-8 from the cells of the periodontium in vitro . But, they cannot induce IL-1 and tumor necrosis factor-alpha from the cells . In the present study, we studied the effects of salivary protein on cytokine induction from human gingival fibroblasts by P . gingivalis outer-membrane protein . Histatin 5 suppressed the IL-6 and IL-8 induction by P . gingivalis outer-membrane protein . This activity was more effective when outer-membrane protein was incubated with histatin 5 before addition to the cell culture . The present study indicates that histatin 5 restrains induction of inflammatory cytokines by periodontal pathogens and that histatin is one of the salivary proteins responsible for this activity. Oral Microbiol Immunol, 2000 Aug, 15(4), 226 - 31 Sensitivity of Actinobacillus actinomycetemcomitans and Capnocytophaga spp . to the bactericidal action of LL-37: a cathelicidin found in human leukocytes and epithelium; Tanaka D et al.; The bactericidal activity of synthetic LL-37, a cathelicidin, was assessed against Actinobacillus actinomycetemcomitans (three strains) and Capnocytophaga spp . (three strains) . All strains were sensitive to LL-37, and exhibited 99% effective dose of 7.5-to-11.6 micrograms/ml . An amidated form of LL-37, pentamide-37, killed with about the same efficacy as LL-37 . Partial inhibition of killing was noted at physiologic concentrations of NaCl, and complete inhibition was observed at 400 mM NaCl . At approximately the 99% effective dose--i.e., 10 micrograms/ml--LL-37 also lost activity against A . actinomycetemcomitans in the presence of native or heat-inactivated 10-15% normal human AB serum . Pentamide-37 was less sensitive to serum inhibition than LL-37 . In conclusion, certain oral, gram-negative bacteria are sensitive to the bactericidal activity of LL-37 at low concentrations of serum and salt, a condition likely to be found within the membrane-delimited phagolysosome . Modified forms of LL-37, such as pentamide-37, may be more suitable for future therapeutic application in the presence of serum. FASEB J, 2001 Jan, 15(1), 155 - 163 Toll-like receptor 4: the missing link of the cerebral innate immune response triggered by circulating gram-negative bacterial cell wall components; Laflamme N et al.; The recent characterization of human homologues of Toll may be the missing link for the transduction events leading to NF-kappaB activity and proinflammatory gene transcription during innate immune response . Indeed, CD14 is not thought to participate directly in the cell signaling, but rather one or more of the mammalian Toll-like receptors (TLRs) acts in concert with the lipopolysaccharide (LPS) receptor to discriminate between microbial pathogens or their products and initiate transmembrane signaling . Mammalian cells may express as many as 10 distinct TLRs, although the importance of TLR4 in response to gram-negative bacteria and LPS is now supported by the fact that TLR4-mutated mice are LPS resistant . We investigated the expression of TLR4 across the rat brain under basal conditions and in response to systemic LPS and IL-1beta injection . We first cloned the rat TLR4 cDNA via RNA isolation and polymerase chain reaction (PCR) amplification with a proofreading polymerase . Total RNA was isolated from the rat liver tissue using Tri-Reagent and reverse transcribed into cDNA using Superscript II reverse transcriptase and an oligonucleotide primer with a degenerate 3' end of sequence 5'-T12(GAC)N-3' . Positive hybridization signal was found in the leptomeninges, choroid plexus (chp), subfornical organ, organum vasculosum of the lamina terminalis, median eminence, and area postrema . Scattered small cells also displayed a convincing hybridization signal within the brain parenchyma . Few well-defined nuclei exhibited positive TLR4 transcript: the supramamillary nucleus, cochlear nucleus, and the lateral reticular nucleus . The circumventricular organs, the leptomeninges, and chp also exhibited constitutive expression of the LPS receptor mCD14 . In contrast to the strong up-regulation of the gene encoding mCD14 during endotoxemia, neither LPS nor IL-1beta caused a convincing increase in the TLR4 mRNA levels across the CNS . A down-regulation of the gene encoding TLR4 was found in the cerebral tissue of immune-challenged animals . The constitutive expression of both mCD14 and TLR4 may explain the innate immune response in the brain, which originates from the structures devoid of blood-brain barrier in presence of circulating LPS. Hepatogastroenterology, 2000 Nov-Dec, 47(36), 1555 - 9 Application of peroral cholangioscopy in an endemic area with high prevalence of hepatocellular carcinoma and choledocholithiasis; Wang HP et al.; BACKGROUND/AIMS: Peroral cholangioscopy with a mother-baby scope system has been introduced for two decades . The paper presents the experience of peroral cholangioscopy at a university hospital in Taiwan where the prevalence of hepatocellular carcinoma and choledocholithiasis was high . METHODOLOGY: A total of 27 sessions of peroral cholangioscopy were performed in 26 patients during a period of 4 years . Of them, 20 patients were for diagnosis and the rest 6 for removing the retained biliary stones . RESULTS: The overall successful rate was 96.3% . The post-procedure complication rate was 11.5% with 2 cholangitis and 1 gram-negative septicemia . There were a total of 19 successful diagnostic sessions . These resulted in definite histological diagnosis in 5 patients and more precise diagnoses subsequently confirmed by surgery in 5 patients . In the remaining 9 patients with tentative diagnoses, 5 confirmed their diagnoses but 4 patients changed their diagnoses after peroral cholangioscopy . By this procedure, hepatocellular carcinoma and choledocholithiasis can be well identified and differentiated . CONCLUSIONS: Peroral cholangioscopy is a safe and valuable modality in diagnosing and treating difficult biliary tract disease when handled with care . It is particularly useful in an endemic area with high prevalence of hepatocellular carcinoma and choledocholithiasis. Biochemistry, 2001 Jan 16, 40(2), 514 - 23 Site-directed mutagenesis of the bacterial metalloamidase UDP-(3-O-acyl)-N-acetylglucosamine deacetylase (LpxC) . Identification of the zinc binding site; Jackman JE et al.; UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A in Gram-negative bacteria . Compounds targeting this enzyme are proposed to chelate the single, essential zinc ion bound to LpxC and have been demonstrated to stop the growth of Escherichia coli . A comparison of LpxC sequences from diverse bacteria identified 10 conserved His, Asp, and Glu residues that might play catalytic roles . Each amino acid was altered in both E . coli and Aquifex aeolicus LpxC and the catalytic activities of the variants were determined . Three His and one Asp residues (H79, H238, D246, and H265) are essential for catalysis based on the low activities (<0.1% of wild-type LpxC) of mutants with alanine substitutions at these positions . H79 and H238 likely coordinate zinc; the Zn(2+) content of the purified variant proteins is low and the specific activity is enhanced by the addition of Zn(2+) . The third side chain to coordinate zinc is likely either H265 or D246 and a fourth ligand is likely a water molecule, as indicated by the hydroxamate inhibition, suggesting a His(3)H(2)O or His(2)AspH(2)O Zn(2+)-polyhedron in LpxC . The decreased zinc inhibition of LpxC mutants at E78 suggests that this side chain may coordinate a second, inhibitory Zn(2+) ion . Given the absence of any known Zn(2+) binding motifs, the active site of LpxC may have evolved differently than other well-studied zinc metalloamidases, a feature that should aid in the design of safe antibiotics. Arterioscler Thromb Vasc Biol, 2001 Jan, 21(1), E1 - 8 Chlamydia pneumoniae proteins induce secretion of the 92-kDa gelatinase by human monocyte- derived macrophages; Vehmaan-Kreula P et al.; Chlamydia pneumoniae, an intracellular Gram-negative respiratory bacterium, and macrophages are present in inflammatory tissue sites such as atherosclerotic lesions, where abnormal degradation of the extracellular matrix takes place . To evaluate the potential of C pneumoniae for participation in matrix destruction, we studied the effect of this bacterium on the production of 3 matrix-degrading metalloproteinases, 92-kDa gelatinase, interstitial collagenase-1, and stromelysin-1, and their natural inhibitor TIMP-1 (tissue inhibitor of metalloproteinases-1) by human monocyte-derived macrophages differentiated in vitro . Spontaneous production of collagenase and stromelysin by these cells was minimal and was not influenced by C pneumoniae . In contrast, the cells secreted substantial basal quantities of 92-kDa gelatinase, the secretion of which was stimulated (on average, 2.5-fold) by C pneumoniae . C pneumoniae regulated the expression of 92-kDa gelatinase by macrophages at the pretranslational level . Macrophages secreted only small quantities of TIMP-1 . The chlamydial proteins Omp2, MOMP, and HSP60 were also found to participate in the induction of 92-kDa gelatinase by C pneumoniae . Denaturation of chlamydial proteins by boiling reduced 92-kDa gelatinase secretion only partially (by 35%), suggesting that the heat-stabile lipopolysaccharide molecules also stimulate secretion of the enzyme . The results show that production of 92-kDa gelatinase by human macrophages is selectively upregulated by C pneumoniae, which suggests that these bacteria, when present in a macrophage-containing inflammatory environment, actively participate in the destruction of the extracellular matrix. J Immunol, 2001 Jan 15, 166(2), 1075 - 8 Induction of a novel mechanism of accelerated bacterial clearance by lipopolysaccharide in CD14-deficient and Toll-like receptor 4-deficient mice; Haziot A et al.; Despite the lack of a proinflammatory response to LPS, CD14-deficient mice clear Gram-negative bacteria (Escherichia coli 0111) at least 10 times more efficiently than normal mice . In this study, we show that this is due to an early and intense recruitment of neutrophils following the injection of Gram-negative bacteria or LPS in CD14-deficient mice; in contrast, neutrophil infiltration is delayed by 24 h in normal mice . Similar results of early LPS-induced PMN infiltration and enhanced clearance of E . coli were seen in Toll-like receptor (TLR) 4-deficient mice . Furthermore, the lipid A moiety of LPS induced early neutrophil infiltration not only in CD14-deficient and TLR-4-deficient mice, but also in normal mice . In conclusion, the lipid A component of LPS stimulates a unique and critical pathway of innate immune responses that is independent of CD14 and TLR4 and results in early neutrophil infiltration and enhanced bacterial clearance. J Immunol, 2001 Jan 15, 166(2), 982 - 8 Viral infection causes rapid sensitization to lipopolysaccharide: central role of IFN-alpha beta; Nansen A et al.; LPS is the major active agent in the pathogenesis of Gram-negative septic shock . In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock . We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection . Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect . This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo . The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice . Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice . Thus hyperproduction of TNF-alpha was completely abrogated in IFN-alphabetaR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-alphabeta-mediated priming of mice for an augmented production of TNF-alpha in response to LPS . This conclusion was further supported by the finding that pretreatment of mice with rIFN-alphabeta mimicked the effect of viral infection . In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-alphabeta and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection. Genetica, 2000, 108(1), 19 - 24 Transposome insertional mutagenesis and direct sequencing of microbial genomes; Hoffman LM et al.; Preformed transposase-transposon complexes called 'Transposomes' have been electroporated into bacterial cells . The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo . The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0 x 10(4) and 1.0 x 10(7) kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species . Transposon integration sites were examined by direct genome sequencing of chromosomal DNA . Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon . The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences . Mutant phenotypes were rapidly correlated with genomic insertions sites. Clin, Eye Vis . Care . 2000 Dec, 12(3-4), 155 - 159 Neuroretinitis: a clinical syndrome of cat-scratch disease; Rost Monahan S; Cat-scratch disease is usually a benign self-limited illness, characterized by regional lymphadenopathy lasting between 3 and 6 weeks . The causative organism is Bartonella henselae, a small gram-negative rod . Between 1 and 2% of patients who contract the illness experience blurred vision, metamorphopsia and scotomas as a result of neuroretinitis, an associated clinical syndrome . The classical clinical findings in cat-scratch neuroretinitis include disc edema and a stellate pattern of exudates in the macula . However, a myriad of other signs has been documented, suggesting a much wider spectrum of intra-ocular disease . The following case report presents a young patient with neuroretinitis, and a history of lymphadenopathy secondary to cat-scratch disease. Br J Surg, 2001 Jan, 88(1), 22 - 30 Experimental models of gram-negative sepsis; Parker SJ et al.; BACKGROUND: The mortality rate from sepsis has improved little over the past two decades . One reason for this has been the use of flawed or inappropriate experimental models in preclinical sepsis studies . METHODS: A literature review of animal models of sepsis was performed following a Medline search based on the following medical subject headings: disease models, endotoxin, inflammation, peritonitis and sepsis . Additional references were identified from the papers identified in the search . RESULTS AND CONCLUSION: Many animal models of sepsis have been described but none has proved to be superior . Extrapolation of results from endotoxicosis or bacterial infusion models should be regarded with caution . Peritonitis models should be accepted as the 'gold standard' but the use of appropriate virulent bacterial species needs to be ensured . A standardized panel of animal models for the preclinical assessment of immunomodulatory agents should be established, including at least one immuno- suppressed model to simulate the immunocompromised patient with sepsis . A uniform and valid definition of sepsis applicable to both small and large animal species is required. Mol Cell Biol, 2001 Jan, 21(2), 438 - 48 Lipopolysaccharide-induced activation of beta2-integrin function in macrophages requires Irak kinase activity, p38 mitogen- activated protein kinase, and the Rap1 GTPase; Schmidt A et al.; Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, is a potent activator of macrophages . Besides inducing many transcriptional pathways, LPS also elicits rapid morphological changes such as cell spreading . Here we have investigated the signaling pathway that controls macrophage beta2-integrin-dependent spreading in response to LPS . We show that inhibition of the adapter protein MyD88, the interleukin-1 receptor-associated kinase Irak, the p38 mitogen-activated protein kinase, or the Ras-like GTPase Rap1 blocks LPS-induced spreading . In addition, Irak activates p38 and stimulates p38-dependent spreading . The activation of p38 by Irak requires Irak's kinase activity . We find that p38 controls spreading independently of its role in transcription but rather through activation of Rap1 . Together, our results suggest that beta2-integrin-dependent spreading of macrophages in response to LPS is controlled by a linear signaling pathway via MyD88, Irak, p38, and Rap1. J Bacteriol, 2001 Jan, 183(2), 725 - 35 Temporal regulation of genes encoding the flagellar proximal rod in Caulobacter crescentus; Boyd CH et al.; The gram-negative bacterium Caulobacter crescentus has a life cycle that includes two distinct and separable developmental stages, a motile swarmer phase and a sessile stalked phase . The cell cycle-controlled biogenesis of the single polar flagellum of the swarmer cell is the best-studied aspect of this developmental program . The flagellar regulon is arranged into a rigid trans-acting hierarchy of gene expression in which successful expression of early genes is required for the expression of genes that are later in the hierarchy and in which the order of gene expression mirrors the order of assembly of gene products into the completed flagellum . The flgBC-fliE genes were identified as a result of the C . crescentus genome sequencing project and encode the homologues of two flagellar proximal rod proteins, FlgB and FlgC, and one conserved protein, FliE, that is of unknown function . Footprint assays on a DNA fragment containing the operon promoter as well as in vivo mutant suppressor analysis of promoter mutations indicate that this operon is controlled by the cell cycle response regulator CtrA, which with sigma(70) is responsible for regulating transcription of other early flagellar genes in C . crescentus . Promoter analysis, timing of expression, and epistasis experiments place these genes outside of the flagellar regulatory hierarchy; they are expressed in class II mutants, and flgB deletions do not prevent class III gene expression . This operon is also unusual in that it is expressed from a promoter that is divergent from the class II operon containing fliP, which encodes a member of the flagellum-specific protein export apparatus. J Bacteriol, 2001 Jan, 183(2), 557 - 69 ihfA gene of the bacterium Myxococcus xanthus and its role in activation of carotenoid genes by blue light; Moreno AJ et al.; Myxococcus xanthus responds to blue light by producing carotenoids . Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes . We had already reported the isolation of a carotenoid-less, Tn5-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes . Here, we show that OmegaMR508::Tn5 affects all known light-inducible promoters in different ways . It blocks the activation of two of them by light but makes the activity of a third one light independent . The OmegaMR508 locus has been cloned and sequenced . The mutation had occurred at the promoter of a gene we propose is the M . xanthus ortholog of ihfA . This encodes the alpha subunit of the histone-like integration host factor protein . An in-frame deletion within ihfA causes the same effects as the OmegaMR508::Tn5 insertion . Like other IhfA proteins, the deduced amino acid sequence of M . xanthus IhfA shows much similarity to HU, another histone-like protein . Sequence comparison data, however, and the finding that the M . xanthus gene is preceded by gene pheT, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship . The M . xanthus ihfA gene shows some unusual features, both from structural and physiological points of view . In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of M . xanthus. J Appl Physiol, 2001 Jan, 90(1), 261 - 8 PAF increases vascular permeability without increasing pulmonary arterial pressure in the rat; Clavijo LC et al.; In vivo pulmonary arterial catheterization was used to determine the mechanism by which platelet-activating factor (PAF) produces pulmonary edema in rats . PAF induces pulmonary edema by increasing pulmonary microvascular permeability (PMP) without changing the pulmonary pressure gradient . Rats were cannulated for measurement of pulmonary arterial pressure (Ppa) and mean arterial pressure . PMP was determined by using either in vivo fluorescent videomicroscopy or the ex vivo Evans blue dye technique . WEB 2086 was administered intravenously (IV) to antagonize specific PAF effects . Three experiments were performed: 1) IV PAF, 2) topical PAF, and 3) Escherichia coli bacteremia . IV PAF induced systemic hypotension with a decrease in Ppa . PMP increased after IV PAF in a dose-related manner . Topical PAF increased PMP but decreased Ppa only at high doses . Both PMP (88 +/- 5%) and Ppa (50 +/- 3%) increased during E . coli bacteremia . PAF-receptor blockade prevents changes in Ppa and PMP after both topical PAF and E . coli bacteremia . PAF, which has been shown to mediate pulmonary edema in prior studies, appears to act in the lung by primarily increasing microvascular permeability . The presence of PAF might be prerequisite for pulmonary vascular constriction during gram-negative bacteremia. Appl Environ Microbiol, 2001 Jan, 67(1), 155 - 61 Exposure of workers to airborne microorganisms in open-air swine houses; Chang CW et al.; This study quantified the levels of airborne microorganisms in six swine farms with more than 10,000 pigs in subtropical Taiwan . We evaluated breeding, growing, and finishing stalls, which were primarily open-air buildings, as well as partially enclosed farrowing and nursery piggeries . Airborne culturable bacteria, gram-negative bacteria, and fungi were placed on appropriate media by using an all-glass impinger or single-stage Andersen microbial sampler . Results showed that mean concentrations of culturable bacteria and gram-negative bacteria were 3.3 x 10(5) and 143.7 CFU/m(3), respectively . The concentration of airborne culturable fungi was about 10(3) CFU/m(3), with Cladosporium the predominant genus . The highest airborne levels of culturable bacteria and gram-negative bacteria were identified in the finishing units . The air of the nursery stalls was the least contaminated with culturable and gram-negative bacteria . Irregular and infrequent cleaning, high pig density, no separation of wastes from pen floors, and accumulation of water as a result of the processes for cleaning and reducing pig temperature possibly compromise the benefits of the open characteristic of the finishing units with respect to airborne bacterial concentration. Appl Environ Microbiol, 2001 Jan, 67(1), 89 - 99 Direct cloning from enrichment cultures, a reliable strategy for isolation of complete operons and genes from microbial consortia; Entcheva P et al.; Enrichment cultures of microbial consortia enable the diverse metabolic and catabolic activities of these populations to be studied on a molecular level and to be explored as potential sources for biotechnology processes . We have used a combined approach of enrichment culture and direct cloning to construct cosmid libraries with large (>30-kb) inserts from microbial consortia . Enrichment cultures were inoculated with samples from five environments, and high amounts of avidin were added to the cultures to favor growth of biotin-producing microbes . DNA was extracted from three of these enrichment cultures and used to construct cosmid libraries; each library consisted of between 6,000 and 35,000 clones, with an average insert size of 30 to 40 kb . The inserts contained a diverse population of genomic DNA fragments isolated from the consortia organisms . These three libraries were used to complement the Escherichia coli biotin auxotrophic strain ATCC 33767 Delta(bio-uvrB) . Initial screens resulted in the isolation of seven different complementing cosmid clones, carrying biotin biosynthesis operons . Biotin biosynthesis capabilities and growth under defined conditions of four of these clones were studied . Biotin measured in the different culture supernatants ranged from 42 to 3,800 pg/ml/optical density unit . Sequencing the identified biotin synthesis genes revealed high similarities to bio operons from gram-negative bacteria . In addition, random sequencing identified other interesting open reading frames, as well as two operons, the histidine utilization operon (hut), and the cluster of genes involved in biosynthesis of molybdopterin cofactors in bacteria (moaABCDE). Schweiz Med Wochenschr, 2000 Dec 2, 130(48), 1837 - 44 {Evaluation of antibiotic prophylaxis in neutropenic patients with hematologic malignancies}; Delarive P et al.; The benefits of oral prophylaxis for neutropenia have remained controversial up to now . We evaluated retrospectively the effect of antibiotic prophylaxis with ciprofloxacin and penicillin on the prevention of bacterial infections in 112 cases of prolonged neutropenia in adult patients treated for haematological malignancies . 41 patients received prophylaxis between December 1993 and November 1994 while 71 patients did not receive prophylaxis between December 1994 and November 1995 . There were no significant differences between groups in age, sex, type or stage of haemopathy, type of chemotherapy and duration of neutropenia . The antibiotic prophylaxis reduced the number of overall infections (p = 0.05) and the number of gram-negative bacteraemias (p = 0.02) . The median time to the onset of fever, the duration of fever, the duration of antibiotic treatment, the duration of hospitalization or admission to the intensive care unit, the number of serious complications or death were not influenced by antibiotic prophylaxis . The prophylaxis did not reduce the overall incidence of bacteraemia, of clinically documented infections or of fever of unknown origin . This retrospective study confirms that oral prophylaxis with ciprofloxacin and penicillin decreases the incidence of infections and, in particular, of gram-negative bacteraemia, but does not modify the overall morbidity and mortality in our patients . In view of the risk of emergence of bacterial resistance, these data do not support the routine use of oral antibiotic prophylaxis in neutropenic patients with haematological malignancies. J Pediatr Hematol Oncol, 2000 Nov-Dec, 22(6), 552 - 7 Clinical manifestation of beta-thalassemia/hemoglobin E disease; Fucharoen S et al.; PURPOSE: To review the clinical manifestation and changes in hematologic parameters of patients with beta-thalassemia/hemoglobin (Hb) E . MATERIALS AND METHODS: Retrospective analysis of the clinical manifestation of 378 patients with beta-thalassemia/Hb E attending the hematology clinic at Siriraj Hospital between 1957 and 1982 . RESULTS: A wide spectrum of clinical phenotypes has been observed . Most patients show clinical symptoms by 10 years of age . The majority of patients survive with or without occasional transfusion . Splenectomy was performed in 26.5% of patients . Patients come to the hospital because of anemia, fever, abdominal mass, and jaundice . Gastrointestinal tract disturbances are the most common presenting symptoms (34.6%), especially abdominal pain (10%) and cholecystitis (5.1%) . Respiratory tract infections were found in 21.8% of patients and cardiovascular complications, including congestive heart failure, occurred in 11.9% . Other less common symptoms and complications included bone pain, chronic leg ulcers, paraplegia and hypertension-associated convulsions, and cerebral hemorrhage after multiple blood transfusion . Patients usually die between 20 to 40 years of age (67%), mainly from congestive heart failure and septicemia . Septicemia was often caused by Gram-negative bacteria . CONCLUSION: These clinical features observed in patients with beta-thalassemia/Hb E are probably the results of chronic anemia and iron overload . The study of the life history and clinical courses of patients with beta-thalassemia/Hb E should provide important information for the better management of these patients. Shock, 2000 Dec, 14(6), 599 - 604 Biliary obstruction exacerbates the hepatic microvascular inflammatory response to endotoxin; Ito Y et al.; Gram-negative sepsis is a serious complication for patients with obstructive jaundice . The present study was conducted to elucidate the response of hepatic microcirculation to endotoxin 2 weeks after bile duct ligation (BDL) or sham-operation in rats . Two hours after lipopolysaccharide (LPS) injection (1, 10, or 100 microg/kg, iv.), the hepatic microvasculature was examined using in vivo microscopy . BDL elicited increases in leukocytes adhering to the sinusoidal wall, swelling of sinusoidal endothelial cells as well as phagocytic activity of hepatic macrophages and a decrease in the numbers of perfused sinusoids . LPS (1, 10, 100 microg/kg) further increased leukocyte adhesion and reduced the numbers of perfused sinusoids in a dose-dependent manner . Leukocyte adhesion in response to LPS (1, 10, 100 microg/kg) in BDL rats was increased 6.1-fold, 5.9-fold, and 3.3-fold, respectively when compared with sham-operated rats . The numbers of perfused sinusoids in response to LPS (1, 10, 100 microg/kg) in BDL rats were decreased by 42%, 36%, and 45% . While 1 and 10 microg/kg LPS also elicited an increase in phagocytic activity in BDL rats when compared with sham-operated rats, the response to 100 microg/kg LPS was suppressed . LPS did not affect the numbers of swollen endothelial cell in BDL rats . The present study demonstrated that chronic biliary obstruction enhanced the hepatic microvascular response to low doses of endotoxin . This observation suggests that exaggerated hepatic microcirculatory dysfunction during sepsis contributes to the development of liver injury and a high incidence of morbidity and mortality in biliary obstruction. Biosci Biotechnol Biochem, 2000 Oct, 64(10), 2059 - 67 Isolation and some properties of a mesophilic and mixotrophic iron-oxidizing bacterium, OKM-9; Inoue T et al.; An iron-oxidizing bacterium strain, OKM-9, isolated from mud obtained from the bottom of a pond, Minamikata Ohike, in Okayama prefecture, Japan, grew well in an FeSO4 x 7H2O (3%)-medium (pH 2.5) with 0.03% yeast extract . However, the strain could not grow either in an FeSO4 x 7H2O (3%)-medium without yeast extract or in a yeast extract (0.03%)-medium (pH 2.5) without Fe2+ . The strain did not use elemental sulfur as an energy source and did not have the activity to fix carbon dioxide . Strain OKM-9 could grow in an FeSO4 x 7H2O (3%)-medium with twenty different L-amino acids instead of yeast extract . Incorporation of {U-14C} glutamic acid into the cells was dependent on the energy produced by the oxidation of Fe2+ . Strain OKM-9 did not grow heterotrophically using amino acids and hexoses as a sole energy and carbon source . The results that strain OKM-9 absolutely required ferrous iron (Fe2+) as a sole energy source and yeast extract or L-amino acids as a carbon source for growth strongly suggest that the strain is a mixotrophic iron-oxidizing bacterium . Strain OKM-9 was a gram-negative and rod-shaped bacterium (0.4-0.6 x 1.6-2.2 microm) and the mean G + C content of the DNA of the bacterium was 59.6 mol% . The optimum temperature and pH for growth were 30 degrees C and 2.1, respectively . However, the strain could not grow at temperatures above 45 degrees C . Iron-oxidizing activities of strain OKM-9 measured with intact cells and the plasma membrane were 14.3 and 5.7 microl O2 uptake/mg protein/min, respectively . The pyridine ferrohemochromes prepared from the plasma membrane of this strain showed absorption peaks characteristic of alpha-bands of heme a and b, but not heme c, at 587 and 557 nm, respectively . The results suggest that the cytochromes composing an iron-oxidation system of strain OKM-9 are different from those of the well-known mesophilic iron-oxidizing bacteria Thiobacillus ferrooxidans and Leptospirillum ferrooxidans. Eur Cytokine Netw, 2000 Dec, 11(4), 597 - 601 Differential response of a(2)-macroglobulin-deficient mice in models of lethal TNF-induced inflammation; Hochepied T et al.; Tumor necrosis factor (TNF) is an essential mediator in the pathogenesis of Gram-negative septic shock . Injection of TNF into normal mice leads to systemic, lethal inflammation, which is indistinguishable from lipopolysaccharide (LPS)-induced lethal inflammation . alpha(2)-macroglobulin (A2M) is a major positive acute phase protein with broad-spectrum protease-inhibitory activity . Mouse A2M-deficient (MAM-/-) mice were significantly protected against lethal systemic inflammation induced by TNF . The protection is not due to faster clearance of the injected TNF . The induction of tolerance to TNF-induced lethality by repetitive administration of small doses of human TNF for five consecutive days was equally efficient in both mutant mice compared to wild-type mice . In D-(+)-galactosamine (GalN)-sensitized mice, TNF induces lethal inflammatory hepatitis . MAM(-/-) mice are equally sensitive to the lethal combination of TNF/GalN . Furthermore, interleukin-1-induced desensitization to TNF/GalN was not impaired in MAM(-/-) mice . We conclude that MAM plays a mediating role in TNF-induced lethal shock and that MAM deficiency does not reduce changes in efficiency of tolerance and desensitization to TNF and TNF/GalN-induced lethality, respectively. Mol Microbiol, 2001 Jan, 39(1), 1 - 11 Emerging strategies in microbial haem capture; Genco CA et al.; Gram-negative pathogenic bacteria have evolved novel strategies to obtain iron from host haem-sequestering proteins . These include the production of specific outer membrane receptors that bind directly to host haem-sequestering proteins, secreted haem-binding proteins (haemophores) that bind haem/haemoglobin/haemopexin and deliver the complex to a bacterial cell surface receptor and bacterial proteases that degrade haem-sequestering proteins . Once removed from haem-sequestering proteins, haem may be transported via the bacterial outer membrane receptor into the cell . Recent studies have begun to define the steps by which haem is removed from bacterial haem proteins and transported into the cell . This review describes recent work on the discovery and characterization of these systems . Reference is also made to the transport of haem in serum (via haemoglobin, haemoglobin/haptoglobin, haemopexin, albumin and lipoproteins) and to mechanisms of iron removal from the haem itself (probably via a haem oxygenase pathway in which the protoporphyrin ring is degraded) . Haem protein-receptor interactions are discussed in terms of the criteria that govern protein-protein interactions in general, and connections between haem transport and the emerging field of metal transport via metallochaperones are outlined. J Pharmacol Exp Ther, 2001 Jan, 296(1), 71 - 6 A metabolic fragment of bradykinin, Arg-Pro-Pro-Gly-Phe, protects against the deleterious effects of lipopolysaccharide in rats; Morinelli TA et al.; Extensive research has provided few therapeutic agents for the treatment of septicemia . Bradykinin, an endogenous vasodepressor hormone, is a key mediator in the hypotension seen with septicemia . The present investigation shows that a stable metabolic fragment of bradykinin, arginine-proline-proline-glycine-phenylalanine (RPPGF), prevents the deleterious effects of endotoxin {lipopolysaccharide (LPS); a component of the membrane of Gram negative bacteria}, the signaling agent responsible for the effects of septicemia, in both anesthetized rats and in isolated rat aortic segments . Survival time of rats treated with LPS (12 mg/kg) was significantly (p < 0.05) prolonged by pretreatment with RPPGF {140.3 +/- 16 min (n = 10)} compared with rats receiving saline and LPS {93.2 +/- 8 min (n = 39)} . Prolongation of survival was not seen when rats were pretreated with either bradykinin or with PRGFP (proline-arginine-glycine-phenylalanine-proline) . Isolated aortic segments treated with LPS (30 microg/ml) showed a significantly reduced ability to contract in response to phenylephrine compared with control segments not receiving LPS . Pretreatment of the segments with RPPGF significantly reversed the LPS-induced reduction in contractile response of the segments . Removal of the endothelial layer did not alter the protection provided by RPPGF . These results demonstrate the ability of a stable metabolic fragment of bradykinin, RPPGF, to protect against the deleterious effects produced by LPS . The findings presented here may provide the basis for a new developmental area for novel therapeutic agents in the treatment of septicemia. J Immunol, 2001 Jan 1, 166(1), 574 - 81 CD11b/CD18 acts in concert with CD14 and Toll-like receptor (TLR) 4 to elicit full lipopolysaccharide and taxol-inducible gene expression; Perera PY et al.; Overproduction of inflammatory mediators by macrophages in response to Gram-negative LPS has been implicated in septic shock . Recent reports indicate that three membrane-associated proteins, CD14, CD11b/CD18, and Toll-like receptor (TLR) 4, may serve as LPS recognition and/or signaling receptors in murine macrophages . Therefore, the relative contribution of these proteins in the induction of cyclooxygenase 2 (COX-2), IL-12 p35, IL-12 p40, TNF-alpha, IFN-inducible protein (IP)-10, and IFN consensus sequence binding protein (ICSBP) genes in response to LPS or the LPS-mimetic, Taxol, was examined using macrophages derived from mice deficient for these membrane-associated proteins . The panel of genes selected reflects diverse macrophage effector functions that contribute to the pathogenesis of septic shock . Induction of the entire panel of genes in response to low concentrations of LPS or Taxol requires the participation of both CD14 and TLR4, whereas high concentrations of LPS or Taxol elicit the expression of a subset of LPS-inducible genes in the absence of CD14 . In contrast, for optimal induction of COX-2, IL-12 p35, and IL-12 p40 genes by low concentrations of LPS or by all concentrations of Taxol, CD11b/CD18 was also required . Mitigated induction of COX-2, IL-12 p35, and IL-12 p40 gene expression by CD11b/CD18-deficient macrophages correlated with a marked inhibition of NF-kappa B nuclear translocation and mitogen-activated protein kinase (MAPK) activation in response to Taxol and of NF-kappa B nuclear translocation in response to LPS . These findings suggest that for expression of a full repertoire of LPS-/Taxol-inducible genes, CD14, TLR4, and CD11b/CD18 must be coordinately engaged to deliver optimal signaling to the macrophage. Curr Rheumatol Rep, 2000 Aug, 2(4), 297 - 305 T-cell studies in the spondyloarthropathies; Marker-Hermann E et al.; A growing body of evidence suggests that T lymphocytes play an important role in initiating and maintaining the inflammatory process characteristic of the human leukocyte antigen (HLA)-B27-associated spondyloarthropathies . T cells seem to be involved in the primary defense reaction against arthritis-triggering gram-negative bacteria at the site of extra-articular infection, in determining the systemic cytokine pattern, in the recirculation process between gut mucosa and the joint, and in mediating secondary autoimmune joint inflammation . The factors involved in disease chronicity (namely in ankylosing spondylitis and psoriatic arthritis) are still unknown . Autoreactive T cells may contribute to this process by recognition of cross-reactive self-epitopes (ie, molecular mimicry between bacterial and self-antigens) . Autoreactive T cells may as well be inappropriately upregulated by bacterial superantigens, or by local inflammatory reactions leading to the uncovering of former cryptic self-epitopes . In this paper, we review recent studies on peripheral blood and synovial T cells in patients with reactive arthritis, enteropathic spondyloarthropathy, psoriatic arthritis, and ankylosing spondylitis. Infect Immun, 2001 Jan, 69(1), 543 - 6 Carboxy-terminal proteolytic processing of Helicobacter pylori vacuolating toxin; Nguyen VQ et al.; The vacA gene of Helicobacter pylori strain 60190 encodes a 1, 287-amino-acid protoxin, which undergoes cleavage of a 33-amino-acid amino-terminal signal sequence and carboxy-terminal proteolytic processing to yield a mature secreted toxin . Several features of VacA suggest that it belongs to the autotransporter family of gram-negative bacterial secreted proteins . Based on matrix-assisted laser desorption ionization-time of flight mass spectrometric analysis, we calculate that the mature toxin has a mass of 88.2+/-0.2 kDa and consists of approximately 821 amino acids. Infect Immun, 2001 Jan, 69(1), 486 - 93 Brucella suis-impaired specific recognition of phagosomes by lysosomes due to phagosomal membrane modifications; Naroeni A et al.; Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals . These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells . Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells . However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown . We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes . The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells . We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell . By in vitro reconstitution experiments, we observed a specific association between killed B . suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature . This association was observed with killed bacteria but not with live bacteria . Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. Infect Immun, 2001 Jan, 69(1), 479 - 85 Synthesis and surface expression of CD14 by human endothelial cells; Jersmann HP et al.; Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14) . By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface . Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes . Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages . Evidence for synthesis of CD14 by HUVEC was provided by the finding that L-{(35)S}methionine was incorporated into CD14 . In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis . Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14) . Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions . This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria. Clin Sci (Lond), 2001 Jan, 100(1), 101 - 10 Protein and energy metabolism in chronic bacterial infection: studies in melioidosis; Paton NI et al.; Chronic infection is often accompanied by a wasting process, the metabolic basis of which is not fully understood . The aims of the present study were to measure protein and energy metabolism in patients with melioidosis (a serious and antibiotic-refractory Gram-negative bacterial infection which is endemic in South-East Asia) in order to define the metabolic abnormalities that might contribute to wasting . Whole-body protein turnover was measured using the {(13)C}leucine technique, both in the fasted state and while consuming a high-energy meal . Resting energy expenditure was measured by indirect calorimetry, and total energy expenditure by the bicarbonate/urea method . Results were normalized for fat-free mass, as estimated from skinfold thickness . Protein turnover was increased in melioidosis patients compared with healthy controls during fasting (170.9 compared with 124.1 micromol x kg(-1) x h(-1); P=0.04), but the net rate of catabolism (22.2 compared with 20.5 micromol x kg(-1) x h(-1); P=0.77) and the anabolic response to feeding were similar in the two groups . Resting energy expenditure was higher in melioidosis patients compared with controls (191.4 and 157.3 kJ x kg(-1) x day(-1) respectively; P=0.04), but total energy expenditure (measured in a separate group of eight patients with melioidosis) was low (192.1 kJ x kg(-1) x day(-1)) . In conclusion, this study found no evidence of metabolic causative factors, such as accelerated net protein catabolism during fasting, a blunted anabolic response to feeding or increased daily energy expenditure, and therefore suggests that reduced energy intake is the prime cause of wasting . The observed normal response to feeding should encourage nutritional approaches to prevent wasting. Mol Microbiol, 2000 Nov, 38(4), 805 - 16 SdiA, an Escherichia coli homologue of quorum-sensing regulators, controls the expression of virulence factors in enterohaemorrhagic Escherichia coli O157:H7; Kanamaru K et al.; The quorum-sensing system in bacteria is a well-known regulatory system that controls gene expression in a cell density-dependent manner . A transcriptional regulator (LuxR homologue), signal synthase (LuxI homologue) and autoinducer (acyl homoserine lactone) are indispensable for this system in most Gram-negative bacteria . In this study, we found that SdiA, an Escherichia coli LuxR homologue, is a negative regulator of the expression of virulence factors EspD and intimin in enterohaemorrhagic E . coli (EHEC) O157:H7 . The expression of EspD and intimin was inhibited at the RNA level upon SdiA overexpression . SdiA has a DNA-binding motif in its C-terminal part and can bind to the promoter regions of the esp and eae genes in vitro . Extracellular factors, which accumulate in culture supernatants of O157:H7 at the stationary phase of growth and inhibit EspD and intimin synthesis, bind to the N-terminal part of SdiA in vivo and in vitro . O157:H7 overproducing the N-terminal part of SdiA exhibited hypertranscription of EspD and intimin, suggesting that the overproduced N-terminal part had inhibited the activity of intact SdiA through titration of the extracellular factors . These results indicate that a quorum-sensing system including the SdiA protein controls colonization by O157:H7. Proc Natl Acad Sci U S A, 2001 Jan 2, 98(1), 283 - 8 A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation; Sideraki V et al.; In Gram-negative bacteria, TEM-1 beta-lactamase provides the major mechanism of plasmid-mediated beta-lactam resistance . Natural variants of TEM-1 with increased antibiotic resistance have appeared in response to the use of extended-spectrum beta-lactam antibiotics (e.g., ceftazidime) and beta-lactamase inhibitors (e.g., clavulanic acid) . Some of the variant enzymes are more efficient at catalyzing beta-lactam hydrolysis, whereas others are more resistant to inhibitors . M182T is a substitution observed in both types of variant TEM-1 beta-lactamases . This mutation is found only in combination with other amino acid substitutions, suggesting that it may correct defects introduced by other mutations that alter the specificity . An engineered core mutation, L76N, which diminishes the periplasmic beta-lactamase activity by 100-fold, was used as a model to understand the mechanism of suppression of the M182T mutation . Biochemical studies of the L76N enzyme alone and in combination with the M182T mutation indicate that the M182T substitution acts at the level of folding but does not affect the thermodynamic stability of TEM-1 beta-lactamase . Thus, the M182T substitution is an example of a naturally occurring mutation that has evolved to alter the folding pathway of a protein and confer a selective advantage during the evolution of drug resistance. Eur J Intern Med, 2000 Dec 20, 11(6), 343 - 344 A rare manifestation of Kingella kingae infection; Reekmans A et al.; Kingella kingae is an aerobic gram-negative coccobacillus that has been associated predominantly with bone and joint infection but also with septicemia and endocarditis . Until now, only four cases of proven K . kingae meningitis have been reported . We describe a case of a K . kingae meningitis in a male adolescent who presented with a history of fever of unclear origin. Int J Med Microbiol, 2000 Oct, 290(4-5), 301 - 5 Genomics of Bordetella pertussis toxins; Antoine R et al.; Bordetella pertussis, the etiologic agent of whooping cough, produces numerous toxins including pertussis toxin (PTX), adenylate cyclase toxin (AC), dermonecrotic toxin (DNT) and tracheal cytotoxin (TCT) . PTX is composed of five different subunits organised in a typical A-B type structure of which the A part possesses an enzymatic ADP-ribosyltransferase activity and the B moiety expresses receptor-binding activity . The secretion of this toxin requires nine other genes (ptl) organised in an operon together with the five structural genes of PTX . To further characterise the genetic locus of this major virulence factor, we analysed the ptx/ptl upstream and downstream sequences . Comparison of these regions between three species of Bordetella (B . pertussis, Bordetella parapertussis and Bordetella bronchiseptica) revealed differences in the upstream region . Analysis of two strains of B . bronchiseptica naturally lacking the ptx genes showed that only the ptx/ptl genes were deleted in these strains, and that the upstream and downstream regions were conserved . Upstream of the PTX structural genes and the promoter, an open reading frame (bugT) was identified, the product of which is homologous with putative proteins from several other Gram-negative organisms . Detailed analysis of the genome of B . pertussis which is currently sequenced at the Sanger Centre revealed the presence of 90 genes coding for proteins homologous to BugT, which qualifies the bug gene family as the most populated one of Bordetella . These bug genes are located in various genetic environments, including the proximities of genes coding for other toxins, such as DNT and AC . The Bug proteins are highly conserved in terms of size and periodicity of predicted secondary structure elements, but have also a high variability in their amino acid composition reflected in their wide range of isoelectric points . The function of these genes which is currently unknown is under investigation . To characterise the expression and regulation of these genes, as well as of novel putative B . pertussis virulence factors, we designed a transcriptional fusion vector to be inserted in precise locations of the B . pertussis chromosome by homologous recombination . The reporter gene present in this vector allowed us to show that at least some of the bug genes are expressed. J Air Waste Manag Assoc, 2000 Nov, 50(11), 1957 - 67 Investigation of the concentration of bacteria and their cell envelope components in indoor air in two elementary schools; Liu LJ et al.; Bacterial cell envelope components are widely distributed in airborne dust, where they act as inflammatory agents causing respiratory symptoms . Measurements of these agents and other environmental factors are assessed in two elementary schools in a southeastern city in the United States . Muramic acid (MA) was used as a marker for bacterial peptidoglycan (PG), and 3-hydroxy fatty acids (3-OH FAs) were used as markers for Gram-negative bacterial lipopolysaccharide (LPS) . Culturable bacteria were collected using an Andersen sampler with three different culture media . In addition, temperature (T), relative humidity (RH), and CO2 were continuously monitored . Concentrations of airborne MA and 3-OH FAs were correlated with total suspended particulate (TSP) levels . Outdoor MA (mean = 0.78-1.15 ng/m3) and 3-OH FA levels (mean = 2.19-2.18 ng/m3) were similar at the two schools . Indoor concentrations of airborne MA and 3-OH FAs differed significantly between schools (MA: 1.44 vs . 2.84 ng/m3; 3-OH FAs: 2.96 vs . 4.57 ng/m3) . Although indoor MA levels were low, they were significantly related to teachers' perception of the severity of indoor air quality (IAQ) problems in their classrooms . Concentrations of CO2 correlated significantly with all bacteria measurements . Because CO2 levels were related to the number of occupants and the ventilation rates, these findings are consistent with the hypothesis that the children and teachers are sources of bacterial contamination . Many culturable bacteria present in indoor air are opportunistic organisms that can be infectious for compromised individuals, while both culturable and nonculturable bacterial remnants act as environmental toxins for both healthy and compromised individuals . Measuring the "total bacteria load" would be most accurate in assessing the biotoxicity of indoor air . Chemical analysis of MA and 3-OH FAs, when coupled with the conventional culture method, provides complementary information for assessing biocontamination of indoor air. Int Microbiol, 1999 Mar, 2(1), 29 - 31 Construction of plasmid vectors bearing a NotI-expression cassette based on the lac promoter; Jaenecke S et al.; We have constructed two plasmid vectors for cloning and expression of DNA fragments controlled by the lac promoter as a NotI-expression cassette . Whereas plasmid pSJ33 allows mobilization of the expression cassette into a wide variety of Gram-negative bacteria by RP4-mediated conjugation, the low-copy-number plasmid pSJP18Not facilitates cloning and expression in Escherichia coli when high gene dosage may be detrimental . In addition to their suitable cloning features (e.g . multiple cloning site, lacZ alpha fragment, compatible with ColE1-derived vectors), these plasmids are particularly useful as auxiliary vectors for cloning of the expression cassettes at the NotI site of mini-transposon elements {1, 2} and their eventual stable insertion into the host chromosome. Int Microbiol, 1998 Mar, 1(1), 19 - 26 Structure and properties of the outer membranes of Brucella abortus and Brucella melitensis; Moriyon I et al.; The brucellae are Gram-negative bacteria characteristically able to multiply facultatively within phagocytic cells and which cause a zoonosis of world-wide importance . This article reviews the structure and topology of the main components (lipopolysaccharide, native hapten polysaccharide, free lipids and proteins) of the outer membranes of Brucella abortus and B . melitensis, as well as some distinctive properties (permeability and interactions with cationic peptides) of these membranes . On these data, an outer membrane model is proposed in which, as compared to other Gram-negatives, there is a stronger hydrophobic anchorage for the lipopolysaccharide, free lipids, porin proteins and lipoproteins, and a reduced surface density of anionic groups, which could be partially or totally neutralized by ornithine lipids . This model accounts for the permeability of Brucella to hydrophobic permeants and for its resistance to the bactericidal oxygen-independent systems of phagocytes. J Electron Microsc (Tokyo), 2000, 49(3), 459 - 62 Deformation of the envelope of a spherical gram-negative bacterium during the atomic force microscopic measurements; Boulbitch A; A theoretical approach for the description of the deformation of the envelope of a spherical Gram-negative bacteria is presented . It is shown that the force-displacement relation taken on top of bacteria is accurately approximated by a linear dependence . The bacterial rigidity is shown to be controlled mainly by its turgor pressure, while the lateral rigidity of the bacterial wall determines the distance from the tip at which the displacement vanishes. ALTEX, 1999, 16(3), 146 - 149 New Applications of the Human Whole Blood Pyrogen Assay (PyroCheck); Fennrich S et al.; The absence of pyrogens in injectable drugs is an indispensable safety control because contaminants causing fever pose a life-threatening risk to the patient resulting in the worst case in death by shock . When fever- inducing agents, i.e.pyrogens, come into contact with the immunocompetent cells in blood, these cells release mediators which transmit the fever signal to the thermoregulatory centre of the brain . The Phamocopoeia lists currently two test systems for pyrogenicity: 1 . The in vivo rabbit pyrogen test which measures the fever reaction following injection of the sample to the animals . 2 . The in vitro Limulus Amebocyte Lysate assay (LAL) which measures the coagulation in a lysate prepared from the blood of the horseshoe crab specifically initiated by endotoxins, i.e . cell wall components from Gram-negative bacteria . The new test presented here (PyroCheck) exploits the reaction of monocytes/macrophages for the detection of pyrogens: human whole blood taken from healthy volunteers is incubated in the presence of the test sample in any form, be it a solution, a powder or even solid material . Pyrogenic contaminations initiate the release of the "endogenous pyrogen" Interleukin-1beta determined by ELISA after a fixed incubation time . The technology presently listed in the Pharmacopoeia is limited to parenteralia (rabbit test: biologicals and pharmaceuticals, LAL: predominantly pharmaceuticals) . In the EU Medical Devices Directive from 1995 the rabbit pyrogen test for medical products is in some cases requested . (in some cases LAL of an eluate from the device) . However, pyrogen-testing needs to cover also innovative high-tech products such as medical devices (implants, medical plastic materials, dialysis machines), cellular therapies and species-specific agents (e.g . recombinant proteins) . Here we report that the human blood test PyroCheck is suitable for testing filters in air quality control as well as for assessing medical devices and biocompatibility (dialysate fluid), i.e . that it can be extended to a wide spectrum of applications. Am J Respir Cell Mol Biol, 2000 Dec, 23(6), 780 - 7 CD14(+) cells are necessary for increased survival of eosinophils in response to lipopolysaccharide; Meerschaert J et al.; There has been considerable interest in the effect that gram-negative bacterial endotoxin (lipopolysaccharide {LPS}) can have in asthma, given that inhalation of LPS has been shown to cause bronchial hyperresponsiveness . Further, there is evidence that the endotoxin-binding protein CD14 may be a marker for asthma . Inhaled LPS has been shown to cause an influx of eosinophils into the nasal airway and to increase the survival of CD16-negatively selected eosinophils in vitro . In this study, we compared survival of eosinophils isolated via CD16-negative selection with eosinophils that were isolated using both CD16- and CD14-negative selection criteria . Survival of CD16-negatively selected eosinophils was enhanced by LPS in a dose-dependent manner and was inhibited by the endotoxin antagonists polymyxin B or lipid X . In contrast, depletion of CD14(+) cells within the eosinophil preparations (CD14/CD16-negatively selected eosinophils) decreased the effect of LPS on survival . Preincubation of CD16-negatively selected eosinophils with antibody 60bd, which blocks LPS binding to CD14, prevented the survival-enhancing effect of LPS . However, CD14 was not detected on eosinophils by flow cytometry, even after incubation with LPS for up to 24 h . These results suggest that the survival-enhancing effect of LPS on eosinophils requires the presence of CD14(+) cells in the population . It is our hypothesis that enhanced eosinophil survival with LPS involves the contribution of another cell type. J Surg Res, 2000 Dec, 94(2), 159 - 66 Lipopolysaccharide-binding protein accelerates and augments Escherichia coli phagocytosis by alveolar macrophages; Klein RD et al.; BACKGROUND: The first step in bacterial clearance by leukocytes is attachment and phagocytosis . Although lipopolysaccharide-binding protein (LBP) is best known for potentiating LPS-induced cytokine production through a CD14-dependent pathway, recent studies suggest that LBP plays a critical role in clearance of gram-negative bacteria and is essential for survival after bacterial challenge . We therefore sought to examine LBP's effect on Escherichia coli phagocytosis by alveolar macrophages (AMs) and to determine if this effect is mediated through CD14 . MATERIALS AND METHODS: Phosphatidylinositol-specific phospholipase C (PIPLC)-treated and untreated rat AMs were incubated in the presence of increasing doses of recombinant LBP or negative control protein (choramphenicol acetyltransferase) prior to E . coli-FITC (Ec-F) BioParticle challenge . Phagocytosed bacteria were assayed by fluorescence measurement . A time course study was also performed . RESULTS: LBP potentiated phagocytosis of Ec-F BioParticles by AMs in a dose-dependent fashion . Kinetic studies showed that LBP augmented Ec-F phagocytosis by 76% at 30 min . Treatment of AMs with PIPLC to remove CD14 resulted in only a partial decrease in LBP-mediated enhancement of phagocytosis . CONCLUSION: These results clearly demonstrate that LBP plays an important role in enhancing Ec-F binding and phagocytosis in a time- and dose-dependent manner . This observed increase may not require the presence of CD14 as significant potentiation of phagocytosis still occurred after PIPLC treatment . We postulate that the LBP-mediated increase in Ec-F phagocytosis can occur in the absence of CD14 through the presence of another receptor . Braz J Infect Dis, 1997 Aug, 1(4), 182 - 185 Central Venous Catheter-Related Infections in Intensive Care Units; Arruda E et al.; Objective: To determine the rates of colonization and infection related to central venous catheter (CVC), the causative microorganisms, and the influence of various factors . Methods: From June to August 1993, all CVC in 4 Intensive Care Units were evaluated from their insertion to removaL Data were collected by 3 nurses . Blood and catheter tips were cultured . Results: Of 84 catheters, 29.8% were colonized, 9.5% of patients showed evidence of local infection, and 4.8% had primary bloodstream infections . The internal jugular vein was the most common site for catheter insertion (81%) . Causes of removal were: end of need (48.3%), suspected infection (23.3%), malfunction (20%), routine change (8.3%) . Among removals because of suspected infection, 50% presented evidence of local infection, 43% were colonized(>15cfu), but there were no bloodstream infections . The average time of catheter use for those which became colonized was longer than for catheters that did not become colonized (p=0.008) . The average time of catheter use associated with removal for infection (local and bloodstream) was longer than for removal for other reasons (p=0.042) . Among colonized catheters, 16% developed bloodstream infection and 20% local infection . Immunosupressive drugs, cancer, diabetes mellitus, HIV-infection, and neutropenia were not associated with infection or colonization . The most common microorganisms were gram-negative rods and S.aureus . Conclusions: The duration of venous catheter use increased the risk of colonization and infection . This observation suggests that physicians must strive for the shortest time of use of venous catheters, but it does not indicate a need for routine central venous catheter removal. Curr Opin Chem Biol, 2000 Dec, 4(6), 653 - 60 Bacterial adhesins: structural studies reveal chaperone function and pilus biogenesis; Knight SD et al.; During the past year, remarkable progress has been made in understanding how periplasmic chaperones fold and protect protein modules that are destined for assembly into adhesive pili in Gram-negative bacteria . The first two three-dimensional structures of complexes of periplasmic chaperones with substrate pilus subunits have revealed much about the structural basis for chaperone-mediated folding and aggregation prevention, and have provided insight into the structure of adhesive pili. FEMS Microbiol Ecol, 2000 Dec 1, 34(2), 129 - 137 Isolation of new fastidious alpha Proteobacteria and Afipia felis from hospital water supplies by direct plating and amoebal co-culture procedures; La Scola B et al.; As water is a source of nosocomial infections in hospitals, the presence of fastidious Gram-negative bacteria in water samples taken in a university hospital was investigated . Water samples were inoculated onto agar plates and into amoebal microplates for co-culture . Sixty-eight alpha proteobacteria isolates were obtained and characterized using phenotypic methods and 16S rRNA gene sequence comparison . The latter approach divided the strains into seven clusters . Of these, one corresponded to previously recognized Afipia felis and it is likely that six were closely related new species . As these bacteria are fastidious and can not be cultivated on standard microbiological media, their possible role in hospital-acquired human infections should be investigated. Microbiology, 2000 Dec, 146 Pt 12, 3251 - 7 A functional water channel protein in the pathogenic bacterium Brucella abortus; Rodriguez MC et al.; The gene for a new bacterial aquaporin, AqpX, was cloned from the pathogenic Gram-negative bacterium BRUCELLA: abortus . The gene was mapped on the large chromosome of B . abortus . It is flanked by one upstream and two downstream copies of the BRUCELLA: repeated sequence Bru-RS . Prediction from the nucleotide sequence indicated that the protein is a member of the MIP family, which comprises channels for water and/or solute transport . Expression in XENOPUS: oocytes and cryoelectron microscopy of Escherichia coli cells transformed with the aqpX gene confirmed that the protein is an efficient water channel . Glycerol uptake experiments in E . coli also showed that the protein is not able to transport glycerol. J Am Soc Nephrol, 2000 Dec, 11(12), 2199 - 211 Basic fibroblast growth factor selectively enhances TNF-alpha-induced apoptotic cell death in glomerular endothelial cells: effects on apoptotic signaling pathways; Messmer UK et al.; Endothelial cell damage of glomeruli and kidney arterioles seems to play a pivotal role in several pathologic situations, such as Gram-negative sepsis, glomerulonephritis, and acute renal failure . Bacterial lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) have been identified as potent inducers of apoptotic cell death in bovine glomerular endothelial cells . Both agents elicited apoptotic DNA laddering within 12 to 24 h . Basic fibroblast growth factor (bFGF) was generally described as a protective factor for endothelial cells against radiation-, TNF-alpha-, and UV-light-induced programmed cell death . Therefore, whether bFGF also affects apoptosis of microvascular endothelial cells was questioned . Surprising was that simultaneous treatment of glomerular endothelial cells with bFGF and either LPS or TNF-alpha left LPS-induced death unaffected, whereas TNF-alpha-induced death induction was potentiated, amounting to 48.9+/-6.3% versus 22.4+/-4.3% DNA degradation with TNF-alpha alone . Comparably, acidic FGF also selectively potentiated TNF-alpha-induced apoptosis . In mechanistic terms, bFGF synergistically increased TNF-alpha-induced mitochondrial permeability transition, the release of cytochrome c from mitochondria to the cytosol, and upregulation of the proapoptotic protein Bak and significantly enhanced activation of caspase-8 protease activity . In contrast, stress-activated protein kinase and nuclear factor kappaB activation, which represent primary signals of TNF/TNF receptor interaction, downregulation of the antiapoptotic protein Bcl-x(L), and caspase-3-like protease activation, were unaffected . As bFGF did not affect LPS-induced apoptotic cell death, bFGF also left LPS-induced Bak upregulation and Bcl-x(L) downregulation unaffected . The results point to a selective bFGF-mediated enhancement of distinct proapoptotic pathways induced by TNF-alpha in glomerular endothelial cells. FEMS Microbiol Lett, 2000 Dec 1, 193(1), 25 - 30 The Bvg accessory factor (Baf) enhances pertussis toxin expression in Escherichia coli and is essential for Bordetella pertussis viability; Wood GE et al.; Pertussis toxin expression in the Gram-negative respiratory pathogen, Bordetella pertussis, is regulated by the BvgAS two-component system . Previous studies suggested that an additional gene encoding a Bvg accessory factor (Baf) was required, along with BvgAS, for expression of a ptx-lacZ fusion in Escherichia coli grown in rich medium . However, other studies showed that BvgAS is sufficient for ptx-lacZ expression in minimal medium . Here we show that Baf acts with BvgAS to further increase ptx-lacZ expression in E . coli grown in minimal media and this is concomitant with a two-fold increase in BvgA protein levels . Gene replacement experiments show that baf is essential for viability of B . pertussis, suggesting that Baf affects the expression of other genes in addition to ptx. J Biol Chem, 2001 Feb 23, 276(8), 5577 - 83 Epub 2000 Nov 28. Identification of two GDP-6-deoxy-D-lyxo-4-hexulose reductases synthesizing GDP-D-rhamnose in Aneurinibacillus thermoaerophilus L420-91T; Kneidinger B et al.; The glycan repeats of the surface layer glycoprotein of Aneurinibacillus thermoaerophilus L420-91T contain d-rhamnose and 3-acetamido-3,6-dideoxy-d-galactose, both of which are also constituents of lipopolysaccharides of Gram-negative plant and human pathogenic bacteria . The two genes required for biosynthesis of the nucleotide-activated precursor GDP-d-rhamnose, gmd and rmd, were cloned, sequenced, and overexpressed in Escherichia coli . The corresponding enzymes Gmd and Rmd were purified to homogeneity, and functional studies were performed . GDP-d-mannose dehydratase (Gmd) converted GDP-d-mannose to GDP-6-deoxy-d-lyxo-4-hexulose, with NADP+ as cofactor . The reductase Rmd catalyzed the second step in the pathway, namely the reduction of the keto-intermediate to the final product GDP-d-rhamnose using both NADH and NADPH as hydride donor . The elution behavior of the intermediate and end product was analyzed by high performance liquid chromatography . Nuclear magnetic resonance spectroscopy was used to identify the structure of the final product of the reaction sequence as GDP-alpha-d-rhamnose . This is the first characterization of a GDP-6-deoxy-d-lyxo-4-hexulose reductase . In addition, Gmd has been shown to be a bifunctional enzyme with both dehydratase and reductase activities . So far, no enzyme catalyzing these two types of reactions has been identified . Both Gmd and Rmd are members of the SDR (short chain dehydrogenase/reductase) protein family. Curr Infect Dis Rep, 2000 Apr, 2(2), 168 - 173 Lemierre's Syndrome; Armstrong AW et al.; Lemierre's syndrome is characterized by an oropharyngeal infection followed by internal jugular vein septic thrombophlebitis and metastatic emboli, most often to the lungs and joints . The syndrome is most commonly associated with the anaerobic gram-negative rod Fusobacterium necrophorum . Diagnosis is established with evidence of metastatic infection and internal jugular vein thrombophlebitis . CT is considered the diagnostic procedure of choice . Treatment should include an extended course of a beta-lactamase-resistant antibiotic and surgical drainage of any purulent fluid collection . Anticoagulation remains controversial, and ligation of the internal jugular vein is reserved for patients with persistent sepsis and recurrent emboli . With appropriate therapy, mortality is 4% to 12%; but mortality is increased when therapy is delayed. Curr Infect Dis Rep, 2000 Apr, 2(2), 141 - 146 Recent Advances in Diagnosis and Treatment of Cat Scratch Disease; Margileth AM; The cause of cat scratch disease (CSD), first described in France in 1950 and in the United States in 1951, was unknown until 1983 when the bacterium in lymph nodes was detected using a Warthin-Starry silver stain . Afipia felis has been an infrequent cause of CSD since1988, when this gram-negative bacterium was first isolated from 10 patients with CSD . In 1992 Bartonella organisms were isolated from immunocompetent and immunocompromised patients . An indirect fluorescent antibody test to detect bartonella-specific serum immunoglobulins was developed in 1992 . Since then multiple studies have shown that three Bartonella species may produce either CSD in humans, usually Bartonella henselae or Bartonella clarridgeiae, or bacteremia in healthy cats . Also, these two bacteria and Bartonella quintana cause bacillary angiomatosis, bacillary peliosis, or relapsing bacteremia in humans . Cats are healthy carriers of Bartonella organisms and may be bacteremic for months to years . Cat-to-cat transmission of Bartonella organisms involves the cat flea in absence of direct contact transmission . CSD is the most common cause of regional lymphadenitis in children and adolescents . Present knowledge on the etiology, clinical features, epidemiology, pathogenesis, diagnosis, and management of CSD are presented . Also, brief comments about the etiology, clinical presentation, and treatment of bacillary angiomatosis and bacillary peliosis are provided. Toxicology, 2000 Nov 2, 152(1-3), 37 - 45 Induction of proliferation and cytokine production in human T lymphocytes by lipopolysaccharide (LPS); Ulmer AJ et al.; Lipopolysaccharide (LPS), also known as endotoxin, is a compound of the cell wall of Gram-negative bacteria, which has been demonstrated to induce inflammatory reactions in vitro as well as in vivo, including lethal shock . A great number of different cells have been documented to be reactive to LPS, e.g . monocytes/macrophages, vascular cells, polymorphonuclear cells, and even B lymphocytes . We have now established that T lymphocytes could also contribute to an inflammatory reaction to LPS . LPS is a potent inducer of human T-lymphocyte proliferation and cytokine production . The activation of T lymphocytes by LPS requires direct cell-to-cell contact with viable accessory monocytes . This interaction was found to be MHC-unrestricted, but strongly dependent on costimulatory signals provided by B7/CD28 interactions . The frequency of responding T lymphocytes is less than 1:1000 . A very exciting finding was that not only monocytes, but also CD34+ hematopoietic stem cells, which circulate in peripheral blood in very low frequency, exert essential accessory cell activity during stimulation of T lymphocytes by LPS . In contrast, the response of T lymphocytes to conventional recall antigens is not controlled by blood stem cells . These conclusions are based on the observation that depletion of CD34-positive blood stem cells resulted in a complete loss of LPS-induced T-lymphocyte stimulation . Addition of CD34-enriched blood stem cells led to a recovery of reactivity of T lymphocyte to LPS . The characteristics of T-lymphocyte activation indicate that LPS is neither active as a mitogen, or as a superantigen, or as a classical antigen, but may activate T lymphocyte through a new, so far undescribed, mechanism . Furthermore, the involvement of hematopoietic blood stem cells in the activation of T lymphocytes by LPS demonstrates a role of these cells in inflammatory and immunological events. Intensive Care Med, 2000 Sep, 26(9), 1248 - 51 Bactericidal/permeability-increasing protein (BPI) in sepsis correlates with the severity of sepsis and the outcome; Rintala E et al.; OBJECTIVE: To compare the prognostic significance of bactericidal/permeability-increasing protein (BPI), group II phospholipase A2 (PLA2-II), C-reactive protein (CRP), tumour necrosis factor-alpha (TNF), interleukin-8 (IL-8) and interferon-gamma (IFN) in terms of predicting severity of sepsis and outcome . DESIGN: A prospective study . SETTING: Medical intensive care unit (ICU) of a university hospital . PATIENTS: Thirty-four patients with severe sepsis requiring ICU treatment . MEASUREMENTS AND RESULTS: The levels of BPI, PLA2-II, CRP, TNF, IL-8 and IFN were measured in these 34 patients . High levels of BPI were associated particularly with Gram-negative sepsis . BPI and BPI/neutrophil ratios correlated positively with PLA2-II, CRP, TNF and IL-8 and negatively with blood pressure . At 24 h, BPI/neutrophil ratios, IL-8 and Simplified Acute Physiology Scores II (SAPS II) scores were higher in non-survivors than in survivors . No such associations were noted in the levels of CRP, PLA2-II, TNF or IFN . The areas under the curve (AUC(ROC)s) of SAPS II scores and IL-8 were higher than AUC(ROC) of BPI/neutrophil ratio . CONCLUSION: The BPI and BPI/neutrophil ratios may serve as adjunctive tools to illustrate the severity of sepsis . However, their predictive power for sepsis-related death was not comparable to that of SAPS II scores and IL-8. J Biol Chem, 2001 Mar 16, 276(11), 8111 - 7 Epub 2000 Nov 21. Conserved residues Ser(16) and His(20) and their relative positioning are essential for TonB activity, cross-linking of TonB with ExbB, and the ability of TonB to respond to proton motive force; Larsen RA et al.; The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria . The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process . The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31) . Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation . In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished . Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the in