Nat Struct Biol, 2003 May, 10(5), 394 - 401 Substrate-induced transmembrane signaling in the cobalamin transporter BtuB; Chimento DP et al.; The outer membranes of Gram-negative bacteria possess transport proteins essential for uptake of scarce nutrients . In TonB-dependent transporters, a conserved sequence of seven residues, the Ton box, faces the periplasm and interacts with the inner membrane TonB protein to energize an active transport cycle . A critical mechanistic step is the structural change in the Ton box of the transporter upon substrate binding; this essential transmembrane signaling event increases the affinity of the transporter for TonB and enables active transport to proceed . We have solved crystal structures of BtuB, the outer membrane cobalamin transporter from Escherichia coli, in the absence and presence of cyanocobalamin (vitamin B(12)) . In these structures, the Ton box is ordered and undergoes a conformational change in the presence of bound substrate . Calcium has been implicated as a necessary factor for the high-affinity binding (K(d) approximately 0.3 nM) of cyanocobalamin to BtuB . We observe two bound calcium ions that order three extracellular loops of BtuB, thus providing a direct (and unusual) structural role for calcium. Proc Natl Acad Sci U S A, 2003 Apr 1, 100(7), 3814 - 9 Epub 2003 Mar 21. A redox switch in CopC: an intriguing copper trafficking protein that binds copper(I) and copper(II) at different sites; Arnesano F et al.; The protein CopC from Pseudomonas syringae has been found capable of binding copper(I) and copper(II) at two different sites, occupied either one at a time or simultaneously . The protein, consisting of 102 amino acids, is known to bind copper(II) in a position that is now found consistent with a coordination arrangement including His-1, Glu-27, Asp-89, and His-91 . A full solution structure analysis is reported here for Cu(I)-CopC . The copper(I) site is constituted by His-48 and three of the four Met residues (40, 43, 46, 51), which are clustered in a Met-rich region . Both copper binding sites have been characterized through extended x-ray absorption fine structure studies . They represent novel coordination environments for copper in proteins . The two sites are approximately 30 A far apart and have little affinity for the ion in the other oxidation state . Oxidation of Cu(I)-CopC or reduction of Cu(II)-CopC causes migration of copper from one site to the other . This behavior is observed both in NMR and EXAFS studies and indicates that CopC can exchange copper between two sites activated by a redox switch . CopC resides in the periplasm of Gram-negative bacteria where there is a multicopper oxidase, CopA, which may modulate the redox state of copper . CopC and CopA are coded in the same operon, responsible for copper resistance . These peculiar and novel properties of CopC are discussed with respect to their relevance for copper homeostasis. Eur J Clin Microbiol Infect Dis, 2003 Mar, 22(3), 137 - 43 Epub 2003 Mar 05. Prospective evaluation of the epidemiology, microbiology, and outcome of bloodstream infections in hematologic patients in a single cancer center; Velasco E et al.; Bloodstream infections (BSIs) have an important impact on the outcome of cancer patients . A prospective cohort study was undertaken at a referral cancer center in order to describe the clinical and microbiological characteristics of patients with hematologic malignancies and BSIs and to identify independent predictors associated with mortality . The study enrolled 110 consecutive BSI episodes during an 18-month period . Patients were monitored for 30 days after the last positive blood culture . There were 10.24 BSI episodes per 1,000 patient-days . The median age of the patients was 25 years . Most patients had acute leukemia ( n=72) . The origin of the BSI was unknown in 43.6% of the episodes and was associated with known sites in 32.7% . There were 58 concomitant infectious sites (lungs, 43%, and soft tissue, 22.4%) and 195 noninfectious comorbid factors (poor performance status, 30.2%; undernourishment, 14.3%) . The median neutrophil count was 215 cells/mm(3) . Indwelling catheters were present in 70% of the episodes . The majority of isolates obtained within the first 48 h of the BSI episode (61%) were gram-negative rods . Overall mortality was 24.5% . Multivariate analysis using logistic regression showed relapsed leukemia, poor performance status, recent weight loss, and ventilatory failure requiring ventilatory support as independent predictors of mortality . Hematologic cancer patients with BSIs should be regarded as a distinct group of patients at high risk of death . The knowledge of variables amenable to intervention would help diminish or prevent serious medical complications. Protein Sci, 2003 Apr, 12(4), 748 - 59 Prediction of the plant beta-barrel proteome: a case study of the chloroplast outer envelope; Schleiff E et al.; In the postgenomic era, the transformation of genetic information into biochemical meaning is required . We have analyzed the proteome of the chloroplast outer envelope membrane by an in silico and a proteomic approach . Based on its evolutionary relation to the outer membrane of Gram-negative bacteria, the outer envelope membrane should contain a large number of beta-barrel proteins . We therefore calculated the probability for the existence of beta-sheet, beta-barrel, and hairpin structures among all proteins of the Arabidopsis thaliana genome . According to the existence of these structures, a number of candidates were selected . This protein pool was analyzed by TargetP to discard sequences with signals that would direct the protein to other organelles different from chloroplasts . In addition, the pool was manually controlled for the presence of proteins known to function outside of the chloroplast envelope . The approach developed here can be used to predict the topology of beta-barrel proteins . For the proteomic approach, proteins of highly purified outer envelope membranes of chloroplasts from Pisum sativum were analyzed by ESI-MS/MS mass spectrometry . In addition to the known components, four new proteins of the outer envelope membranes were identified in this study. Arch Oral Biol, 2003 Feb, 48(2), 117 - 23 Oral colonisation by aerobic and facultatively anaerobic Gram-negative rods and yeast in Tibetans living in Lhasa; Leung WK et al.; Sample groups of children (n=50) and adults (n=38) were selected from pools of 207 children, (11-13-year olds from two primary schools) and 94 adults (25-44-year olds from four governmental agencies) who were the subjects of an oral health survey among Tibetans living in Lhasa, Tibet Autonomous Region . Mean ages of the study groups of children (38% females) and adults (61% females) were 11.6+/-0.9 and 37.1+/-6.1 years, respectively . All had lived in Tibet since birth . Oral rinse samples were selective cultured to isolate, quantify and speciate aerobic and facultatively anaerobic Gram-negative rods (using the API 20E kit) and yeasts (using API 20C AUX and API ZYM kits) . For children, the isolation rates for oral coliform bacteria and yeasts were 84 and 14%, respectively, for adults, the respective rates were 26 and 40% . The corresponding quantities of coliforms/yeasts for children and adults were 0.4+/-1.6 x 10(3)c.f.u./15.8+/-72.3 and 0.2+/-0.6 x 10(3)c.f.u./57.2+/-137.5c.f.u . per millilitre oral rinse, respectively . Aerobic and facultatively anaerobic Gram-negative rods and Stenotrophomonas maltophilia, a free-living saprophytic and ubiquitous bacterial species of wide geographic distribution, were significantly more frequently recovered from the children's oral rinses . The isolation rates of facultatively anaerobic Gram-negative rods in adults and yeasts in both groups were similar to those found in similar cohorts from southern China in earlier studies . Randomly amplified polymeric DNA analysis showed that the S . maltophilia spp . isolated from children were of several different clonal types and were school specific . This study shows that the colonisation rate of facultatively anaerobic Gram-negative rods in adults and yeasts in both groups are similar to those in populations living at lower altitudes, the native young, urban Tibetans appear to exhibit a high oral carriage rate of S . maltophilia spp. Lett Appl Microbiol, 2003, 36(4), 197 - 202 Development of a miniaturized four-culture method for the rapid enumeration of four bacterial groups in ground beef; Kang DH et al.; AIMS: A simplified and rapid method was developed to identify and enumerate total mesophilic microbial load, gram-negative bacteria, coliforms, and Escherichia coli in ground beef, using a single module . METHODS AND RESULTS: A 96-well microtiter plate was divided into four sections of two lanes (24 wells), with each section dedicated to each of the four critical bacterial groups . Double strength of four different selective broths (100 microl) was added to each well and an aliquot of sample (100 microl) from the first well was transferred to the next to achieve a twofold serial dilution in each series (24 wells) . Initial bacterial load was calculated using the final consecutive positive reaction exhibiting color change or fluorescence . To validate the method, ground beef (n = 32), inoculated with a three-strain mixture of E . coli, together with 30 samples of commercial ground beef, were assayed by the four-culture method and conventional plating . For all media tested, the coefficients of determinations (r2) between the results derived from the four-culture method and those of conventional plating method ranged from 0.83 to 0.92 . CONCLUSIONS: The high degree of association between the two methods indicates that the four-culture method could be easily applied to the enumeration of four critical bacterial groups in ground beef . SIGNIFICANCE AND IMPACT OF THE STUDY: This method will facilitate sanitation monitoring at meat processing plants by shortening time required for results, reducing consumable material costs, simplifying procedures, reducing the space required, and cutting the cost of start up equipment. Med Sci Monit, 2003 Mar, 9(3), BR120 - 6 A human whole blood model of LPS-mediated suppression of T cell activation; Yaqub S et al.; BACKGROUND: Lipopolysaccharide (LPS) is the main initiator of the early signaling events leading to sepsis caused by Gram-negative bacteria . Late stages of sepsis are associated with impairments of T lymphocyte function, a condition associated with nosocomial infection and poor outcome . The molecular basis for septic immunosuppression is not fully understood . MATERIAL/METHODS: Human whole blood was incubated ex vivo with purified LPS . Cytokine responses and T cell proliferation were assessed, and the role of cyclic 3',5'-adenosine monophosphate (cAMP) in T cell suppression by LPS was studied using a cAMP-antagonist (Rp-8-Br-cAMPS) . RESULTS: Adding LPS (0.01 to 10 microg/ml) to human blood ex vivo caused a release of prostaglandin E2 (PGE2) in a concentration- and time-dependent manner, with maximal levels of PGE2 obtained with 10 microg LPS per ml blood after 10 hours of incubation . Adding PGE2-concentrations ranging from 0.03 to 10 microM to purified T cells completely abrogated T cell activation and proliferative response, which was largely reversed by adding Rp-8-Br-cAMPS . Peripheral blood mononuclear cells (PBMC) and T cells harvested from whole blood cultured in the presence of LPS ex vivo showed attenuated proliferative response (30-70%) (purified T cells and PBMC) and reduced IL-2 production (85%) upon T cell receptor (TCR)/CD3 activation with anti-CD3 . The proliferation in T cells and PBMC was in part restored by Rp-8-Br-cAMPS . CONCLUSIONS: The human whole blood model of LPS-mediated T lymphocyte suppression described in this paper is time and cost efficient, as well as easy to use. Water Sci Technol, 2003, 47(3), 103 - 7 PCR as a test for the presence or absence of Legionella in (cooling) water; Declerck P et al.; Legionella pneumophila, a Gram-negative bacterium, is the causative agent of legionellosis . Traditionally, culture methods are normally used to detect Legionella species in different types of water (e.g . surface or tap water, circulating systems, air conditioners and their cooling devices) . In this study the PCR conditions to detect Legionella were optimised based on the EnviroAmp Legionella kit (Perkin-Elmer) which is no longer commercially available . The PCR is very sensitive and specific in indicating the presence or absence (no quantification with classical PCR) of Legionella spp in general and more specifically L . pneumophila . To identify L . pneumophila . DNA sequences from the mip (macrophage infectivity potentiator) gene were amplified . The mip gene is conserved and specific for L . pneumophila although mip-like genes are also present in other Legionella spp . The PCR techniques were able to detect small amounts of Legionella in tap water samples . Cooling water, however, often contained PCR-inhibiting substances that could result in false negative PCR results for Legionella. Rev Med Chir Soc Med Nat Iasi, 2002 Apr-Jun, 107(2), 411 - 3 {Osteomyelitis, a cause of prolonged fever in adults}; Georgescu N et al.; A 35 years old male patient with an unexplained prolonged fever, after serial unsuccessful investigations in 3 different services, suffers a fracture of the lower third of the right femur produced by a minor trauma . This raises the suspicion of a pathological fracture . The surgical intervention and the microbiological exam confirms the existence of an osseous infection with Gram-negative germs, and the clinical course is uneventful after stabilization with an external fixator and appropriate antibiotic therapy . Osteomyelitis, although rare in adults, should be included in the differential diagnosis of any prolonged fever of unknown origin in adults. Am J Med, 2003 Feb 15, 114(3), 194 - 8 Pharmacokinetic dosing of aminoglycosides: a controlled trial; Bartal C et al.; PURPOSE: To evaluate whether individualized pharmacokinetic dosing of aminoglycosides can reduce nephrotoxicity and improve the outcome of patients with gram-negative sepsis . METHODS: We conducted a prospective controlled trial at a tertiary care university hospital . Eighty-one patients with suspected or documented gram-negative infections were enrolled . All were treated with either gentamicin or amikacin, according to clinical judgement . Patients were allocated to one of two groups based on the last digit (odd/even) of their identification number . In the study group (pharmacokinetic dosing) of 43 patients, plasma aminoglycoside levels were determined 1 hour after initiation of drug infusion and 8 to 16 hours later to estimate the elimination half-life and volume of distribution, from which the subsequent dosage schedule was calculated . Target peak plasma levels were 20 microg/mL for gentamicin and 60 microg/mL for amikacin . Target trough levels were <1 microg/mL for both drugs . The control group (fixed once-daily dosing) consisted of 38 patients who were prescribed single daily doses of gentamicin or amikacin . The primary endpoints were renal toxicity (> or = 25% increase in serum creatinine level or a serum creatinine level > or = 1.4 mg/dL) and 28-day mortality . RESULTS: The two study groups were similar in age, sex, indications for therapy, Acute Physiology and Chronic Health Evaluation (APACHE) II score, and clinical assessment at baseline . Although the pharmacokinetic group received significantly greater doses of aminoglycosides than did the once-daily group, the incidence of nephrotoxicity was significantly lower in the pharmacokinetic group (5% {2/43} vs . 21% {8/38}, P = 0.03) . There was no statistically significant difference in 28-day mortality (27% {12/43} vs . 22% {8/38}, P = 0.3) . CONCLUSION: These results suggest that individualized pharmacokinetic dosing of aminoglycosides reduces the incidence of nephrotoxicity and allows the use of greater doses of aminoglycosides. Environ Sci Technol, 2003 Feb 15, 37(4), 701 - 6 Cd bioaccumulation by a freshwater bacterium, Rhodospirillum rubrum; Smiejan A et al.; Cd bioaccumulation by Rhodospirillum rubrum, a Gram-negative freshwater bacterium, was studied in a synthetic medium . The free ion (Cd2+) was the best predictor of the Cd internalization fluxes . Representation of the short-term uptake fluxes as a function of {Cd2+} in the medium demonstrated a linear relationship, as would be expected for a rate-limiting, first-order internalization with a single transporter . Nonetheless, several different accumulation profiles were observed, depending on the Cd concentration . Cd uptake was regulated differently for concentrations above and below 10(-6) M (or was regulated only above {Cd2+} = 10(-6) M) . Short-and long-term studies revealed that regulation was rapidly initiated for the highest Cd concentrations examined, effectively decreasing both adsorbed and internalized Cd . Anodic stripping voltammetry demonstrated that a Cd complexing ligand was produced within minutes upon exposure to 5 x 10(-6) M Cd2+ and that an extracellular sequestration of Cd was one mechanism regulating Cd uptake . Competition studies with other cations revealed a competitive inhibition of Cd uptake by Zn and an uptake enhancement in the presence of Mn and Cu. Microbiol Immunol, 2003, 47(1), 27 - 35 The reduction of Fusobacterium nucleatum in mice is irrelevant to the nitric oxide induced by iNOS; Kato C et al.; Previously we reported that mice infected recurrently with live Fusobacterim nucleatum (Fn) synthesize a significant amount of NO between 12 hr and 24 hr after the Fn injection . We now investigated whether the NO has the capability of killing Fn, a gram-negative rod periodontal pathogen . The mice were divided into three groups: treated with live bacteria (LB), treated with heat-killed bacteria (HKB) and untreated: normal (N) . The Fn reduction, NO production and cell number after Fn injection were then compared in these mice . In the LB group, no Fn was detected at 6 hr, whereas it was still detected in the HKB and N groups at 24 hr as assessed by both colony counts and PCR assays . A significant amount of NO was synthesized in the LB group at 24 hr after the Fn injection . Fn is not killed by SNAP-generated NO in vitro . An increase in the total cell number was accompanied by an increase of the neutrophil numbers in the LB group . Intracellular O2(-) generation (including ONOO(-)) was visualized using dihydrorhodamine (DHR)-123 . The peak of O2(-) generation by PEC was shown to be at 3 hr in all 3 groups . The number of O2(-) positive cells in the LB group at 3 hr was remarkably high, and most of them were likely to be neutrophils . The Fn reduction would be performed cooperatively via oxygen dependent and oxygen independent mechanisms . Thus reactive oxygen species (ROS) included in the oxygen dependent mechanism appear to be important for Fn reduction . However the significant amounts of NO derived from the iNOS synthesized in the LB group between 12 hr and 24 hr after injection of LFn were not involved in the Fn reduction. Schweiz Rundsch Med Prax, 2003 Jan 22, 92(4), 127 - 33 {Ototoxicity of aminoglycoside antibiotics}; Reiss M et al.; In gram-negative infections aminoglycosides still remain to present a first-line antibiotic . Their use is limited by the high risk for side effects and especially nephrotoxicity and ototoxicity . The more recently developed members of this group, although purporting to be less ototoxic, are also capable of producing inner ear damage in sufficiently high doses . This paper reviews the morphological, histochemical, and electrophysiological evidence concerning the mode of action of aminoglycosides on the auditory and vestibular systems and discusses the available methods for the detection of possible clinical ototoxicity . With recent advances in aminoglycoside monitoring techniques, the risk of toxicity has been greatly reduced . Guidelines are proposed about the prevention of ototoxicity and the management of patients with established auditory or vestibular dysfunction. Shock, 2003 Mar, 19(3), 215 - 22 Sympathetic blockade in a canine model of gram-negative bacterial peritonitis; Solomon SB et al.; We investigated, in a well-established canine model of human sepsis, the effects of two different techniques of sympathetic blockade during bacterial peritonitis on pain relief, hemodynamics, and survival rate . Twenty-two purpose-bred beagles (12-28 months old, weighing 10-12 kg) were studied . Fourteen animals received an epidural infusion of bupivicaine and morphine, and the other eight received either a celiac plexus block (n = 4) or a sham block (n = 4) . Eighteen of the 22 animals received an intraperitoneal challenge of Escherichia coli (1-10 x 10(9) CFU kg(-1) body weight) . At comparable doses of intraperitoneal-implanted E . coli (2.5-5 x 10(9) CFU kg(-1) body weight), the addition of sympathetic blockade produced a synergistic decrease in survival times (P = 0.002) and mean left ventricular ejection fraction (P = 0.008), and increase in creatinine levels (P = 0.02) . There was also a significant increase in tumor necrosis factor (TNF) levels (P = 0.004) and decrease in blood endotoxin clearance (P = 0.006) associated with sympathetic blockade during sepsis . The celiac plexus-blocked animals had no improvement in pain scores, and subjectively looked clinically worse than animals with sepsis without a celiac plexus block . In contrast, the epidural block was effective in blocking the pain and discomfort associated with low lethality doses of intraperitoneal bacteria reflected by no increase in pain scores compared with animals not receiving bacterial challenge . This study shows that during severe bacterial peritonitis, maintenance of sympathetic tone irrespective of pain relief provided is necessary for clearance of bacterial toxins, control of proinflammatory mediator release, hemodynamic stability, and survival. Chest, 2003 Mar, 123(3), 772 - 7 Efficacy and safety of azithromycin vs levofloxacin in the outpatient treatment of acute bacterial exacerbations of chronic bronchitis; Amsden GW et al.; STUDY OBJECTIVES: To compare the safety and efficacy of oral azithromycin and levofloxacin in the treatment of outpatients with acute bacterial exacerbations of chronic bronchitis (ABECB) . DESIGN: Randomized, double-blinded, double-dummy, multicenter trial with 1:1 treatment allocation . SETTING: Outpatient treatment setting . PATIENTS: Two hundred thirty-five male or female outpatients between the ages of 35 and 75 years who had received a clinical diagnosis of ABECB . INTERVENTIONS: Blinded treatment with either oral azithromycin, 500 mg on day 1 and 250 mg per day for days 2 to 5, or, oral levofloxacin, 500 mg q24h for 7 days . RESULTS: Both treatments were well-tolerated, with the majority of adverse events being GI in nature . Favorable clinical outcomes in clinically evaluable patients were demonstrated in 89% of patients receiving azithromycin and in 92% of patients receiving levofloxacin by day 4 of therapy . At day 24, the posttherapy visit, favorable responses were approximately 82% and 86%, respectively, for patients in the two treatment groups . The bacterial eradication rates of respiratory pathogens were 96% for azithromycin and 85% for levofloxacin . CONCLUSIONS: Despite increasing concerns over macrolide resistance and a higher incidence of Gram-negative pathogens, a standard 5-day course of oral azithromycin was clinically and bacteriologically equivalent to a 7-day course of oral levofloxacin in the treatment of patients with ABECB. An Pediatr (Barc), 2003 Mar, 58(3), 228 - 31 {Once-daily gentamicin dosing versus thrice-daily dosing in infants with acute pyelonephritis}; Calvo Rey C et al.; BACKGROUND: Once-daily dosing (ODD) of gentamicin is advocated as an effective and safe treatment of Gram-negative bacterial infections in adults . There are insufficient data in the literature to justify its use in infants . OBJECTIVES: To compare the efficacy of ODD of gentamicin with that of classical thrice-daily (t.i.d.) administration in infants with acute pyelonephritis . METHODS: We performed a quasi-experimental study comparing 33 infants who received ODD of gentamicin with a historical control group of 25 infants treated with gentamicin t.i.d . Leukocytosis, C-reactive protein, creatinine, gentamicin dose, peak and trough values, time required for disappearance of fever, and outcome were analyzed . RESULTS: The mean doses of gentamicin (mg/kg/day) were higher in the t.i.d . group (6.4 1.14) than in the ODD group (5.06 0.22; p < 0.001) . Peak serum gentamicin concentrations (micro g/ml) were significantly higher in the ODD group (9.32 1.4) than in the t.i.d . group (5.09 1.15; p < 0.001) . Mean trough gentamicin concentrations (micro g/ml) were lower in the ODD group than in the t.i.d . group (0.23 0.26 vs 0.78 0.45; p 0.001) . There were no significant differences in the duration of fever between the groups (30.64 32 hours in the t.i.d . group vs . 28.57 32 hours in the ODD group) . Serum creatinine levels were normal during treatment in both groups . In all patients outcome was good and no adverse effects were noted . CONCLUSIONS: Treatment with ODD of gentamicin in our population of infants with acute pyelonephritis was as effective as traditional administration t.i.d . and possibly was equally safe or safer. Crit Care Med, 2003 Mar, 31(3), 924 - 8 Endotoxin binding to erythrocyte membrane and erythrocyte deformability in human sepsis and in vitro; Poschl JM et al.; OBJECTIVE: Several studies have shown that lipopolysaccharide and lipid A impair red blood cell deformability and . However, it is unclear whether impaired red blood cell deformability is associated with binding of lipopolysaccharide to the red blood cell membrane . DESIGN: Analysis of hydroxymyristic acid content in red blood cell membranes and red blood cell deformation in patients with Gram-negative septicemia and after incubation of red blood cells from healthy adults with 100 microg of lipid A or 1 mg of lipopolysaccharide per milliliter of red blood cell in buffer solution and in whole blood . Hydroxymyristic acid is a fatty acid of the lipid A part of lipopolysaccharide in most Gram-negative bacteria . SETTING: University research laboratories . SUBJECTS: Ten healthy adults and four patients with clinical and laboratory signs of septicemia . INTERVENTIONS: Blood sampling . MEASUREMENTS AND MAIN RESULTS: Red blood cell deformation was measured with a laser-diffraction shearing device (Rheodyn) and a computerized micropore filtration system (CTA) . Lipopolysaccharide and lipid A binding to red blood cell membranes was studied by measuring the amide-linked hydroxymyristic acid by gas chromatography . The detection rates of hydroxymyristic acid were 82% for lipopolysaccharide and 79% for lipid A in buffer solution . In membranes of washed red blood cell, the detection rates of lipopolysaccharide and lipid A were 0.26 +/- 0.03% (2.6 +/- 0.3 microg/mL) and 1.3 +/- 0.5% (1.3 +/- 0.5 microg/mL), and in red blood cell membranes of whole blood the detection rates were 2.6% (25.5 microg/mL) and 4.1% (4.1 microg/mL), respectively . The lipopolysaccharide content in red blood cell membranes of septic patients ranged from 47 to 103 microg/mL of red blood cell . Red blood cell deformation in the Rheodyn and in the CTA were not influenced by lipopolysaccharide incubated with washed red blood cells . In the Rheodyn, red blood cell deformation was significantly decreased by 18% after lipid A incubation in washed red blood cells, by 26% after lipopolysaccharide incubation in whole blood, and by 31% in septic patients . Similar effects were observed when we used the CTA . CONCLUSIONS: Red blood cell deformation is decreased in septic patients, after incubation of washed red blood cells with lipid A and of whole blood with lipopolysaccharide . Lipopolysaccharide did not influence red blood cell deformation after incubation with washed red blood cells . The decrease of red blood cell deformation was related to the amount of hydroxymyristic acid measured in red blood cell membranes, suggesting that endotoxin binding directly affects mechanical properties of red blood cells. Crit Care Med, 2003 Mar, 31(3), 781 - 6 Inadequate salivary flow and poor oral mucosal status in intubated intensive care unit patients; Dennesen P et al.; OBJECTIVE: To investigate salivary flow and frequency of oral mucositis in intensive care unit patients compared with patients admitted because of elective coronary artery bypass graft (CABG) surgery . In addition, the pattern of oropharyngeal colonization was investigated in these patients . DESIGN: Prospective study . SETTING: Mixed intensive care unit and cardiosurgical ward . PATIENTS: In this study, 24 ventilated intensive care unit patients and 20 CABG patients were included . MEASUREMENTS AND MAIN RESULTS: Two dental hygienists examined intensive care unit patients for the presence of periodontal disease and mucositis at admission and subsequently every week during their stay in the intensive care unit . At the same time, stimulated salivary flow and salivary total immunoglobulin A output were measured . Oropharyngeal cultures were obtained as well . CABG patients were examined the day before the operation, 1 day, 1 wk, and 2 wks after the operation . The following results were obtained: a) temporarily reduced postoperative stimulated salivary flow and total salivary immunoglobulin A output in CABG patients and nearly absent stimulated salivary flow in intensive care unit patients; b) oropharyngeal colonization with potentially pathogenic microorganisms in intensive care unit and not in CABG patients; and c) the increase in mucositis index in intensive care unit patients paralleled the increase in potentially pathogenic microorganism oropharyngeal colonization, especially and . CONCLUSIONS: Absence of adequate salivary flow in intubated intensive care unit patients causes severe xerostomia, which may contribute to the development of mucositis and oropharyngeal colonization with Gram-negative bacteria. Glycobiology, 2003 Apr, 13(4), 295 - 303 Epub 2003 Jan 03. Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans; Maki M et al.; Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients . The serotype a-specific polysaccharide antigen of A . actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification . This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s) . In the synthesis of GDP-6- deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto-6-deoxy-D-mannose and then reduced to GDP-6-deoxy-D-talose by GDP-6-deoxy-D-talose synthetase (GTS) . In this study, we cloned and overexpressed in Escherichia coli the A . actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose . The recombinant A . actinomycetemcomitans GTS enzyme expressed in E . coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose . The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDI-TOF MS, and NMR spectroscopy . The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases. Physiol Res, 2003, 52(1), 101 - 10 Changes in the superoxide production and other macrophage functions could be related to the mortality of mice with endotoxin-induced oxidative stress; Victor VM et al.; Free radicals and proinflammatory cytokines from phagocytes have been implicated in the pathogenesis of endotoxic shock, a disease with high mortality caused by Gram-negative bacterial endotoxin . In the present study, male BALB/c and Swiss mice received intraperitoneally lipopolysaccharide (LPS) at 100 mg/kg and 150 mg/kg, respectively, that led to a lethal endotoxic shock (100 % of mortality before 30 h) . Swiss mice injected with 100 mg/kg, that did not show lethal endotoxic shock, were also studied . Peritoneal macrophages were obtained from animals at 2, 4, 12 or 24 h after injection of LPS or saline (control) solutions . Superoxide anion and tumor necrosis factor (TNFalpha) production were determined in these cells as well as other functions such as adherence capacity, chemotaxis and phagocytosis . The increase in superoxide anion production after endotoxin injection was higher in cells from mice with lethal shock than in those with non-lethal shock . However, the enhancement of TNFalpha production was similar in all cases, although in Swiss mice the highest levels of TNFalpha were observed at 1.5 h after endotoxin injection, while in BALB/c mice they occurred at 2 h after LPS injection . This oxidative stress was also revealed by the other functions analyzed, since adherence to substrate and phagocytosis were stimulated and chemotaxis was decreased after endotoxin injection as compared to controls, the differences being even more significant in animals with lethal shock . These data suggest that these changes, mainly the increased production of free radicals even more than the TNFalpha release, could be involved in mouse mortality caused by LPS. Zhonghua Bing Li Xue Za Zhi, 2002 Dec, 31(6), 534 - 7 {The effect of transmembrane- and secreted-tumor necrosis factor-alpha on shock liver}; Yin B et al.; OBJECTIVE: To observe the dynamic expression of transmembrane (TM)-tumor necrosis factor (TNF)-alpha and secreted (S)-TNF-alpha in the development of endotoxic shock and explore the actions and mechanism of TM-TNF-alpha in liver of the rat with endotoxic shock . METHODS: Endotoxic shock in rats was induced by intravenous injection of dead gram negative bacteria E . Coli; the kinetics of TM-TNF-alpha on peritoneal macrophages and S-TNF-alpha in serum of these rats were determined . Pretreatment with TNF alpha converting enzyme antisense oligonucleotide (5 mg/kg) 30 minutes before rats were administrated dead bacteria inhibited enzymatic cleavage of TM-TNF-alpha into S-TNF-alpha . Six hours after bacteria injection, TM-TNF-alpha and S-TNF-alpha were also detected respectively . The pathological injury in the livers of rats with endotoxin shock was examined, and artery pressure was constantly measured . RESULTS: The kinetics of TM-TNF-alpha expression in the development of endotoxic shock was different from that of S-TNF-alpha expression in serum . The expression of TM-TNF-alpha began to increase on the surface of peritoneal macrophages and liver within 30 min, after bacteria challenge and peaking within a period of 4.5 hours followed by a gradual decrease to a relatively high level which was maintained for at least 24 hours . The TACE antisense oligonucleotide pretreated rats showed remarkable increase in TM-TNF-alpha expression by peritoneal macrophages and liver (P < 0.001), and their arterial blood pressure were maintained within normal levels and there were no detectable pathological changes in their livers . CONCLUSIONS: These findings suggested that TM-TNF-alpha may be a potent endogenous regulator involved in anti-inflammatory responses to maintain normal arterial pressure and protect liver tissue from pathological injury in during endotoxin shock . This study confirmed the important role of TNF-alpha in endotoxic shock which is not only of important theoretical significance, but also of practical interest in providing experimental basis for clinical treatment of endotoxin shock. Genetics, 2003 Feb, 163(2), 457 - 66 An analysis of why highly similar enzymes evolve differently; Majiduddin FK et al.; The TEM-1 and SHV-1 beta-lactamases are important contributors to resistance to beta-lactam antibiotics in gram-negative bacteria . These enzymes share 68% amino acid sequence identity and their atomic structures are nearly superimposable . Extended-spectrum cephalosporins were introduced to avoid the action of these beta-lactamases . The widespread use of antibiotics has led to the evolution of variant TEM and SHV enzymes that can hydrolyze extended-spectrum antibiotics . Despite being highly similar in structure, the TEM and SHV enzymes have evolved differently in response to the selective pressure of antibiotic therapy . Examples of this are at residues Arg164 and Asp179 . Among TEM variants, substitutions are found only at position 164, while among SHV variants, substitutions are found only at position 179 . To explain this observation, the effects of substitutions at position 164 in both TEM-1 and SHV-1 on antibiotic resistance and on enzyme catalytic efficiency were examined . Competition experiments were performed between mutants to understand why certain substitutions preferentially evolve in response to the selective pressure of antibiotic therapy . The data presented here indicate that substitutions at position Asp179 in SHV-1 and Arg164 in TEM-1 are more beneficial to bacteria because they provide increased fitness relative to either wild type or other mutants. Sex Transm Dis, 2003 Mar, 30(3), 257 - 61 Glycosidase and proteinase activity of anaerobic gram-negative bacteria isolated from women with bacterial vaginosis; Olmsted SS et al.; BACKGROUND: It is known that glycosidases and proteases are produced by the anaerobic gram-negative bacteria associated with bacterial vaginosis (BV) . We hypothesized that these enzymes enzymatically degrade mucins, thereby destroying the mucus gel that otherwise helps protect against sexually transmitted pathogens, including HIV . GOAL: The goal was to determine glycosidase and protease production by vaginal bacteria associated with BV and to compare these with symptoms and signs of abnormal discharge and to test vaginal fluid viscosity . STUDY DESIGN: The anaerobic gram-negative rods recovered from the vaginas of 153 women with normal flora, intermediate flora, or BV were tested for production of sialidase, fucosidase, galactosidase, glucosaminidase, and glycine and arginine aminopeptidases . RESULTS: Women with BV had higher frequencies and concentrations of bacteria producing mucin-degrading enzymes than did women with intermediate and normal flora (P < 0.001) . Women with higher concentrations of bacteria producing mucin-degrading enzymes were more likely to have a thin discharge associated with BV (P < 0.001) . The viscosity of diluted vaginal fluid samples from women with BV was significantly lower than those from women with normal flora (P = 0.001) . CONCLUSION: These data support the hypothesis that BV organisms degrade the protective mucus gel. Curr Opin Microbiol, 2003 Feb, 6(1), 7 - 14 The various and varying roles of specific chaperones in type III secretion systems; Parsot C et al.; The type III secretion pathway is used by numerous Gram-negative pathogenic bacteria to deliver proteins within the membrane or the cytoplasm of eukaryotic cells with which these bacteria interact . Secretion is regulated by external signals . This requires that, before being secreted, proteins are stored in the cytoplasm where they need to be stabilised, separated from other interaction partners, and maintained in a secretion-competent state . Specialised, energy-independent chaperones play various roles in these functions by associating in the cytoplasm with proteins before their secretion . Some chaperones are also directly involved in modulating transcription in response to secretion. J Biol Chem, 2003 May 23, 278(21), 19453 - 62 Epub 2003 Feb 27. Activation of p38 plays a pivotal role in the inhibitory effect of lipopolysaccharide and interleukin-1 beta on long term potentiation in rat dentate gyrus; Kelly A et al.; Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, has been shown to induce profound changes both peripherally and centrally . It has recently been reported that intraperitoneal injection of LPS inhibited long term potentiation (LTP) in perforant path-granule cell synapses and that this effect was coupled with an increase in the concentration of the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) . The LPS-induced effects were abrogated by inhibition of caspase-1, suggesting that IL-1 beta may mediate the effects of LPS . Here we report that the inhibition of LTP induced by LPS and IL-1 beta was coupled with stimulation of the stress-activated protein kinase p38 in hippocampus and entorhinal cortex and that this effect was abrogated by the p38 inhibitor SB203580, while the effect of LPS was markedly attenuated in C57BL/6 IL-1RI-/- mice . The data also indicate that activation of the transcription factor, nuclear factor kappa B (NF kappa B), may play a role, since the inhibitory effect of LPS and IL-1 beta on LTP was attenuated by the NF kappa B inhibitor, SN50; consistently, LPS and IL-1 beta led to activation of NF kappa B in entorhinal cortex . We suggest that one consequence of these LPS and IL-1 beta-induced changes is a compromise in glutamate release in dentate gyrus, which was coupled with the inhibition of LTP . The evidence is consistent with the idea that the LPS-induced impairment in LTP is mediated by IL-1 beta and is a consequence of activation of p38. J Matern Fetal Neonatal Med, 2002 Nov, 12(5), 313 - 21 Lipopolysaccharide-binding protein in microbial invasion of the amniotic cavity and human parturition; Espinoza J et al.; OBJECTIVE: Lipopolysaccharide-binding protein (LBP) is an acute-phase protein of predominantly hepatic origin, capable of binding the lipid A fraction of bacterial lipopolysaccharide (LPS) . The complex LBP-LPS binds to CD14, and has been implicated in the host response to gram-negative infection . The purpose of this study was to determine whether microbial invasion of the amniotic cavity (MIAC) and parturition (term and preterm) are associated with changes in the amniotic fluid concentration of LBP . STUDY DESIGN: Amniotic fluid was retrieved by amniocentesis from 343 patients in the following groups: (1) those in mid-trimester with a subsequent normal pregnancy outcome (n = 84); (2) those in mid-trimester with a fetal loss after the procedure (n = 10); (3) those with preterm labor and intact membranes without MIAC who delivered at term (n = 36) or prematurely (n = 52), and those with preterm labor with MIAC (n = 26); (4) those with preterm premature rupture of membranes (PROM) with (n = 26) and without (n = 26) MIAC; and (5) those delivering at term with intact membranes in the absence of MIAC, in labor (n = 52) and not in labor (n = 31) . The concentration of LBP in amniotic fluid was determined with a specific and sensitive immunoassay . Non-parametric statistics were used . A p value of < 0.05 was considered significant . RESULTS: LBP was detected in 98% (335/343) of the amniotic fluid samples . MIAC was associated with a significant increase in amniotic fluid concentration of LBP in women with preterm labor and intact membranes, but not in preterm PROM . Spontaneous preterm parturition was associated with a significant increase in amniotic fluid concentration of LBP . Patients who had a spontaneous fetal loss after a mid-trimester amniocentesis had a significantly higher median amniotic fluid LBP concentration than those who had a mid-trimester amniocentesis and a normal perinatal outcome . CONCLUSION: Preterm labor with MIAC and preterm parturition are associated with higher amniotic fluid concentrations of LBP than those with sterile amniotic fluid. Pancreas, 2003 Mar, 26(2), 122 - 9 Role of the gut in the course of severe acute pancreatitis; Ammori BJ; INTRODUCTION: The pathogenesis of acute pancreatitis remains elusive . Sepsis and multiple organ failure continue to cause death (overall mortality rate, approximately 10%) despite immense improvements in supportive, radiologic, and surgical therapy . The gut appears to play a key role in the development of these complications . AIM: To critically review the evidence implicating the gut in the pathogenesis of acute pancreatitis . METHODS: Relevant English-language literature or abstracts cited in the MEDLINE database were reviewed . RESULTS AND CONCLUSION: Gram-negative enteric organisms account for most infections of pancreatic necrosis and subsequent sepsis, which suggests the gut as a source . Intestinal permeability is increased early in patients with severe acute pancreatitis and correlates with endotoxemia, which suggests translocation as a possible mechanism . The pathogenesis of the deranged function of the gut mucosal barrier and the possible sites of increase in intestinal permeability are discussed . The gut also plays a role in priming neutrophils and the release of inflammatory cytokines, which initiate and propagate nearly all the detrimental consequences of severe inflammation and sepsis . Future research avenues and potential therapeutic measures that may restore and preserve gut barrier function are explored. Mol Microbiol, 2003 Mar, 47(5), 1367 - 83 A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain; Oliver DC et al.; Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface . BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells . BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain . The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter . In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain . In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger . This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600 . However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E . coli strains expressing OmpT suggesting that the region is required to maintain a stable structure . The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane. J Am Chem Soc, 2003 Mar 5, 125(9), 2426 - 35 Synthesis of lipid A derivatives and their interactions with polymyxin B and polymyxin B nonapeptide; Yin N et al.; Lipid A is the causative agent of Gram-negative sepsis, a leading cause of mortality among hospitalized patients . Compounds that bind lipid A can limit its detrimental effects . Polymyxin B, a cationic peptide antibiotic, is one of the simplest molecules capable of selectively binding lipid A and may serve as a model for further development of lipid A binding agents . However, association of polymyxin B with lipid A is not fully understood, primarily due to the low solubility of lipid A in water and inhomogeneity of lipid A preparations . To better understand lipid A-polymyxin B interaction, pure lipid A derivatives were prepared with incrementally varied lipid chain lengths . These compounds proved to be more soluble in water than lipid A, with higher aggregation concentrations . Isothermal titration calorimetric studies of these lipid A derivatives with polymyxin B and polymyxin B nonapeptide indicate that binding stoichiometries (peptide to lipid A derivative) are less than 1 and that affinities of these binding partners correlate with the aggregation states of the lipid A derivatives . These studies also suggest that cooperative ionic interactions dominate association of polymyxin B and polymyxin B nonapeptide with lipid A. Electrophoresis, 2003 Feb, 24(4), 757 - 61 Multiple polypeptide forms observed in two-dimensional gels of Methylococcus capsulatus (Bath) polypeptides are generated during the separation procedure; Berven FS et al.; We have examined two-dimensional electrophoresis (2-DE) gel maps of polypeptides from the Gram-negative bacterium Methylococcus capsulatus (Bath) and found the same widespread trains of spots as often reported in 2-DE gels of polypeptides of other Gram-negative bacteria . Some of the trains of polypeptides, both from the outer membrane and soluble protein fraction, were shown to be generated during the separation procedure of 2-DE, and not by covalent post-translational modifications . The trains were found to be regenerated when rerunning individual polypeptide spots . The polypeptides analysed giving this type of trains were all found to be classified as stable polypeptides according to the instability index of Guruprasad et al . (Protein Eng . 1990, 4, 155-161) . The phenomenon most likely reflects conformational equilibria of polypeptides arising from the experimental conditions used, and is a clear drawback of the standard 2-DE procedure, making the gel picture unnecessarily complex to analyse. Am J Clin Nutr, 2003 Mar, 77(3), 744 - 50 Membrane peroxidation by lipopolysaccharide and iron-ascorbate adversely affects Caco-2 cell function: beneficial role of butyric acid; Courtois F et al.; BACKGROUND: Membrane lipid peroxidation may play a role in immune-mediated bowel diseases . OBJECTIVE: We examined the effects of lipopolysaccharide (LPS), a ubiquitous endotoxin mediator of gram-negative bacteria, alone and in combination with iron-ascorbate, on enterocyte function . Furthermore, we assessed the antioxidant capacity of butylated hydroxytoluene (BHT) and butyric acid, which are known to play a significant role in the welfare of intestinal mucosa . DESIGN: Differentiated intestinal Caco-2 cells were used to study the induction of membrane peroxidation by LPS (100 micro g/mL) and iron-ascorbate (0.2 and 2 mmol/L, respectively) and to examine the beneficial effects of BHT and butyric acid . RESULTS: A significant dose-dependent increase in malondialdehyde, accompanied by lower apical membrane fluidity and significantly decreased sucrase activity, was observed when Caco-2 cells were incubated with LPS . LPS also augmented paracellular permeability ({(14)C}polyethylene glycol flux), prostaglandin E(2) production, and cyclooxygenase-2 (EC 1.14.99.1) expression . These abnormalities were exacerbated by the coadministration of iron-ascorbate, but most of them were suppressed by butyric acid and BHT . CONCLUSION: Bacterial endotoxin and prooxidants may overwhelm antioxidant defenses and become deleterious to enterocyte function, whereas butyric acid and BHT may provide antioxidant protection. J Gastrointest Surg, 2003 Feb, 7(2), 191 - 8; discussion 198-9 Cholangitis: bacterial virulence factors that facilitate cholangiovenous reflux and tumor necrosis factor-alpha production; Stewart L et al.; In previous studies we noted that biliary bacteria produce slime and possess P1-fimbriae . The presence of gram-negative bacteria killed by complement correlated with serious biliary infections and induced more tumor necrosis factor-alpha (TNF-alpha) production in sera, suggesting a role for cytokine production and complement activation in biliary sepsis . This study examined bacterial virulence factors that facilitate cholangiovenous reflux (CVR) and TNF-alpha production in a rat model . Twenty-one biliary bacteria and two stool isolates were tested for slime production, sensitivity to complement killing, and hemolysin production . 10(7) Bacterial colony-forming units/ml (or saline control) were injected retrograde into the common bile ducts of Sprague-Dawley rats at a pressure of 30 cm H(2)O . Blood was obtained at 5 and 60 minutes after infusion for bacterial culture and TNF-alpha assay, respectively . The magnitude of slime production correlated inversely with the magnitude of bacterial CVR . Average bacterial colony-forming units were 1.4 x 10(5), 6.8 x 10(4), or 2.1 x 10(3) for bacteria with slime production 0 to 10, 11 to 99, or more than 100, respectively (P < 0.0001, analysis of variance) . CVR was greater for serum-resistant bacteria (1.2 x 10(5) vs . 5.5 x 10(4) {P = 0.007, resistant vs . sensitive}), but TNF-alpha production was greater in serum-sensitive bacteria . TNF-alpha production as a function of bacterial reflux followed a logarithmic curve (R(2) = 0.75) for serum-sensitive bacteria but was linear (R(2) = 0.60) for serum-resistant bacteria . These data show how specific virulence factors explain why some bacterial species colonize without causing illness, whereas others colonize and cause sepsis . Although slime production was necessary for colonization, too much slime inhibited CVR . Although complement killing cleared bacteria from the circulation, it was also associated with increased TNF-alpha production, which can lead to septic manifestations . The most virulent bacterial species (from patients with sepsis) were killed by complement, but they still had significant CVR and were associated with increased TNF-alpha production. Rev Esc Enferm USP, 2002 Jun, 36(2), 177 - 83 {Nosocomial infection and mortality}; Turrini RN; A retrospective study with 69 deaths occurred at a pediatric hospital in 1993 was undertaken to identify the relationship of nosocomial infection with death . Pneumonia and bloodstream infection were the main site of infection . A higher prevalence of gram-negative bacteria was also observed . The hospital infection was causally related to death in 30.4% and contributed to death in 50.8% of children . The nosocomial infection was causally related to death more frequently in patients classified as having a non fatal disease at admission. Infect Immun, 2003 Mar, 71(3), 1481 - 90 Role of the Brucella suis lipopolysaccharide O antigen in phagosomal genesis and in inhibition of phagosome-lysosome fusion in murine macrophages; Porte F et al.; Brucella species are gram-negative, facultative intracellular bacteria that infect humans and animals . These organisms can survive and replicate within a membrane-bound compartment inside professional and nonprofessional phagocytic cells . Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both cell types . However, the molecular mechanisms and the microbial factors involved are poorly understood . Smooth lipopolysaccharide (LPS) of Brucella has been reported to be an important virulence factor, although its precise role in pathogenesis is not yet clear . In this study, we show that the LPS O side chain is involved in inhibition of the early fusion between Brucella suis-containing phagosomes and lysosomes in murine macrophages . In contrast, the phagosomes containing rough mutants, which fail to express the O antigen, rapidly fuse with lysosomes . In addition, we show that rough mutants do not enter host cells by using lipid rafts, contrary to smooth strains . Thus, we propose that the LPS O chain might be a major factor that governs the early behavior of bacteria inside macrophages. Surg Infect (Larchmt), 2000 Autumn, 1(3), 227 - 37 Prevention and treatment of multiple organ dysfunction syndrome: lessons learned and future prospects; Dunn DL; Gram-negative bacteria commonly cause serious infections in hospitalized patients, and those that lead to bacteremic episodes and sepsis syndrome are associated with the highest mortality rate . Sepsis syndrome frequently progresses to multisystem organ dysfunction and failure, with as many as 400,000 cases occurring annually . Unfortunately, the associated mortality rate remains about 40% . Lipopolysaccharide (LPS, endotoxin), an integral component of the gram-negative bacterial outer membrane, plays a critical role in the pathophysiology of this lethal disease process . It is capable of interacting with host macrophages, a process that leads to the secretion of an increasingly well-characterized array of macrophage cytokines . Several different classes of compounds that bind directly to LPS and thereby neutralize its effects are being examined . These consist of anti-LPS monoclonal antibodies (mAbs), naturally occurring proteins and their derivatives (e.g., bactericidal/permeability-increasing protein {BPI}, Limulus anti-LPS factor {LALF}), and certain antibiotics (polymyxin B, taurolidine) . The molecular biology of BPI, LALF, and LPS binding protein (LBP, which augments the host response to LPS) is of considerable interest, as each demonstrates considerable genetic sequence homology . Although two anti-LPS monoclonal antibodies (HA-1A, E5) did not demonstrate efficacy during sepsis syndrome, information obtained from these clinical trials provided investigators with the ability to better understand this disease process . However, a detailed understanding of the biology of endotoxin antagonism is beginning to emerge, and the application of this knowledge in the clinical setting provides hope that it may be possible to reduce the mortality of sepsis syndrome caused by these microorganisms to a statistic well below the current 40%. Surg Infect (Larchmt), 2001, 2 Suppl 1, S3 - 11 Basic aspects of and general problems in surgical infections; Fry DE; Antibiotics are important in the prophylaxis and treatment of surgical infections as well as in the management of nosocomial infections acquired postoperatively in surgical patients . Surgeons encounter a range of infectious conditions, including established single-pathogen infections of soft tissues, polymicrobial intra-abdominal infections, and resistant gram-negative nosocomial infections such as ventilator-associated and aspiration pneumonia . Preoperative antibiotic administration has been shown to reduce the risk of surgical site infections and is now an accepted part of the standard care for most surgical patients . In patients with established single-pathogen or polymicrobial infections requiring surgery, studies have shown appropriate empiric antibiotic therapy to be an important adjunct to surgical intervention and general supportive measures in improving patient outcome . Antibiotics are also essential for those who develop postoperative nosocomial infections . Empiric coverage of the most likely causative organisms, especially in synergistic polymicrobial mixed infections, is one of the keys to successful prophylaxis and treatment of surgical infections. FEMS Microbiol Lett, 2003 Feb 14, 219(1), 33 - 8 Envelope ultrastructure of uncultured naturally occurring magnetotactic cocci; Freitas F et al.; Magnetotactic bacteria are microorganisms that respond to magnetic fields . We studied the surface ultrastructure of uncultured magnetotactic cocci collected from a marine environment by transmission electron microscopy using freeze-fracture and freeze-etching . All bacteria revealed a Gram-negative cell wall . Many bacteria possessed extensive capsular material and a S-layer formed by particles arranged with hexagonal symmetry . No indication of a metal precipitation on the surface of these microorganisms was observed . Numerous membrane vesicles were observed on the surface of the bacteria . Flagella were organized in bundles originated in a depression on the surface of the cells . Occasionally, a close association of the flagella with the magnetosomes that remained attached to the replica was observed . Capsules and S-layers are common structures in magnetotactic cocci from natural sediments and may be involved in inhibition of metal precipitation on the cell surface or indirectly influence magnetotaxis. J Soc Gynecol Investig, 2003 Feb, 10(2), 110 - 7 Lipopolysaccharide induces interleukin-8 production by human cervical smooth muscle cells; Watari M et al.; OBJECTIVE: We examined the effect of lipopolysaccharide (LPS), a component of the outer wall of gram-negative bacteria, on expression of the neutrophil chemoattractant interleukin-8 (IL-8) and the effects of IL-8 treatment on release of matrix metabolizing enzymes in human cervical smooth muscle cells (CSMCs) . METHODS: Human CSMCs were exposed to Escherichia coli LPS, and the expression of IL-8 mRNA was analyzed by Northern blotting . The IL-8 promoter activity was examined by dual luciferase assay, and the IL-8 concentration was assessed by enzyme-linked immunosorbent assay . We also treated the CSMCs with human IL-8 and examined the expression of matrix-degrading enzymes . RESULTS: E coli LPS (100 ng/mL) increased the expression of IL-8 mRNA 12.8-fold after 3 hours . This up-regulation was maintained for up to 24 hours . Lipopolysaccharide treatment produced a fivefold increase in IL-8 promoter activity in CSMCs transfected with an IL-8 promoter-reporter construct . IL-8 concentrations in conditioned medium of CSMC cultures treated with E coli LPS increased approximately 18-fold compared with the control cultures . Northern blot analysis and zymography revealed that exogenous human IL-8 had no significant effect on the expression of matrix metalloproteinase (MMP)-1, -3, and tissue inhibitor of metalloproteinase (TIMP)-1 mRNAs, and on the secretion MMP-2 and -9 in CSMCs . CONCLUSION: We conclude that CSMCs respond to LPS with increased expression of IL-8 mRNA and secreted IL-8, and that expression of matrix metabolizing enzymes in CSMCs is not directly affected by IL-8 . IL-8 produced by CSMCs in response to gram-negative infection may promote neutrophil invasion, and release of neutrophil matrix-degrading enzymes may participate in the matrix remodeling associated with parturition. Lijec Vjesn, 2002 Sep, 124 Suppl 1, 10 - 3 {Helicobacter pylori--bacterial characteristics}; Kalenic S et al.; Helicobacter pylori lives in the gastric mucosa of about half of world population . H . pylori is a gram-negative, microaerophilic, facultatively acidophilic rod, very sensitive to drying and usual disinfectants . H . pylori strains show great genetic variability, the main mechanism of this phenomenon being in vivo genetic recombination . The principal virulence factors are: vacuolating cytotoxin, enzyme urease, motility, adhesive molecules on the cell surface, catalase and superoxide-dysmutase, receptor for human lactoferin and factors which promote proinflammatory cytokine secretion . Strains of H . pylori are sensitive to ampicillin and tetracycline; resistance to macrolide antibiotics is 7-15% in different parts of the world, and to metronidazole is 7-50% or even more . Because in vitro sensitivity of H . pylori is the most important factor for successful therapy, it is necessary to monitor this sensitivity continuously in a particular area. Biol Bull, 2003 Feb, 204(1), 21 - 7 Amebocyte production begins at stage 18 during embryogenesis in Limulus polyphemus, the American horseshoe crab; Coursey Y et al.; Limulus polyphemus, the American horseshoe crab, has a single type of circulating blood cell, the granular amebocyte, which is the horseshoe crab's primary cellular defense against microbial infection . On exposure to gram-negative bacteria or their endotoxins, the amebocytes degranulate, releasing the clotting protein coagulogen and a number of proteases . The protease cascade converts the soluble coagulogen to insoluble coagulin, which forms fibrous clots that seal off the site of infection . The first description of this clotting reaction in the 1950s initiated development of Limulus amebocyte lysate and spurred an intensive study of the amebocytes . However, the site or sites and timing of amebocyte production have yet to be determined . We report here that during embryonic development in Limulus polyphemus, amebocyte production begins at stage 18 . The first amebocytes detected are found in developing hemocoel cavities, and the cells may derive from previously undifferentiated yolk nuclei. FEMS Microbiol Lett, 2003 Jan 21, 218(1), 65 - 70 Mutations in sit B and sit D genes affect manganese-growth requirements in Sinorhizobium meliloti; Platero RA et al.; Two transposon-induced mutants of Sinorhizobium meliloti 242 were isolated based on their inability to grow on rich medium supplemented with the metal chelator ethylenediamine di-o-hydroxyphenylacetic acid (EDDHA) and either heme-compounds or siderophores as iron sources . Tagged loci of these mutants were identified as sit B and sit D genes . These genes encode components of an ABC (ATP-binding cassette) metal-type permease in several Gram-negative bacteria . In this work, the phenotypes of these two mutants were compared with those of two siderophore-mediated iron transport mutants . The results strongly implicate a role of the sit genes in manganese acquisition when this metal is limiting in S . meliloti. Shock, 2003 Feb, 19(2), 131 - 7 Flagellin from gram-negative bacteria is a potent mediator of acute pulmonary inflammation in sepsis; Liaudet L et al.; Flagellin is a recently identified bacterial product that elicits immune response via toll-like receptor 5 . Here, we demonstrate that flagellin is an extraordinarily potent proinflammatory stimulus in the lung during sepsis . In vitro, flagellin triggers the production of interleukin (IL)-8 by human lung epithelial (A549) cells, with 50% of the maximal response obtained at a concentration of 2 x 10(-14) M . Flagellin also induces the expression of ICAM-1 in vitro . Intravenous administration of flagellin to mice elicited a severe acute lung inflammation that was significantly more pronounced than following lipopolysaccharide (LPS) administration . Flagellin induced a local release of proinflammatory cytokines, the accumulation of inflammatory cells, and the development of pulmonary hyperpermeability . These effects were associated with the nuclear translocation of the transcription NF-kappaB in the lung . Flagellin remained active in inducing pulmonary inflammation at doses as low as 10 ng/mouse . In the plasma of patients with sepsis, flagellin levels amounted to 7.1 +/- 0.1 ng/mL . Plasma flagellin levels showed a significant positive correlation with the lung injury score, with the alveolar-arterial oxygen difference as well as with the duration of the sepsis . Flagellin emerges as a potent trigger of acute respiratory complications in gram-negative bacterial sepsis. Crit Care Med, 2003 Feb, 31(2), 367 - 73 Multicenter, double-blind, placebo-controlled study of the use of filgrastim in patients hospitalized with pneumonia and severe sepsis; Root RK et al.; OBJECTIVE: To determine the safety and efficacy of filgrastim (r-metHuG-CSF) in combination with intravenous antibiotics to reduce the rate of mortality in patients with pneumonia and sepsis . DESIGN: This study was multicenter, double-blind, and randomized . SETTING: Intensive care units PATIENTS Adult patients with bacterial pneumonia, either acquired or nosocomial, as confirmed by chest radiograph and positive culture or Gram-negative stain, and severe sepsis, defined as sepsis-induced hypotension or organ dysfunction . INTERVENTIONS: Standard antibiotic therapy with or without filgrastim (300 microg/day) or placebo administered as a 30-min intravenous infusion . The study drug was started within 24 hrs of enrollment and was continued for 5 days or until the white blood cell count reached >75.0 x 10(9) cells/L . MEASUREMENTS AND MAIN RESULTS: The primary end point was the occurrence of mortality through day 29; secondary end points included occurrence of subsequent organ dysfunction, time to discharge from intensive care unit, number of days on mechanical ventilatory support, and time to death . Study-related observations were recorded through day 10 and included vital signs, onset of organ dysfunction, clinical laboratory variables, and adverse events . Filgrastim increased the white blood cell count to a median peak of 31.7 x 10(9) cells/L from a baseline of 12.3 x 10(9) cells/L . The two groups were well matched and did not differ significantly with regard to severe adverse events, time to death, occurrence of end-organ dysfunction, days of intensive care unit hospitalization, or days on mechanical ventilatory support . Mortality was low in both treatment groups; the mortality rate in patients with adult respiratory distress syndrome was similar between the two groups . CONCLUSIONS: The addition of filgrastim to the antibiotic and supportive care treatment of patients with pneumonia complicated by severe sepsis appeared to be safe, but not efficacious in reducing mortality rates or complications from this infection. Microbiology, 2003 Jan, 149(Pt 1), 177 - 84 A novel ColV plasmid encoding type IV pili; Gophna U et al.; Many septicaemic Escherichia coli strains harbour ColV virulence plasmids . This paper describes pO78V, a conjugative ColV plasmid from an avian pathogenic E . coli strain that encodes type IV pili in addition to other virulence-related genes and tetracycline resistance . Plasmid location of type IV pili genes was demonstrated using Southern hybridization and expression of the pili was demonstrated using RT-PCR and phage sensitivity assays . This is a first report of a ColV plasmid encoding type IV pili . Plasmid pO78V is a mosaic plasmid containing replicons and other genes typical to both IncI1 and IncFII groups . As type IV pili of Gram-negative bacteria are involved in several stages of infection, their presence on a ColV virulence plasmid could expand the repertoire of pathogenesis-related genes. Mol Biol Evol, 1998 Nov, 15(11), 1548 - 61 Evolution of genes, evolution of species: the case of aminoacyl-tRNA synthetases; Diaz-Lazcoz Y et al.; All of the aminoacyl-tRNA synthetase (aaRS) sequences currently available in the data banks have been subjected to a systematic analysis aimed at finding gene duplications, genetic recombinations, and horizontal transfers . Evidence is provided for the occurrence (or probable occurrence) of such phenomena within this class of enzymes . In particular, it is suggested that the monomeric PheRS from the yeast mitochondrion is a chimera of the alpha and beta chains of the standard tetrameric protein . In addition, it is proposed that the dimeric and tetrameric forms of GlyRS are the result of a double and independent acquisition of the same specificity within two different subclasses of aaRS . The phylogenetic reconstructions of the evolutionary histories of the genes encoding aaRS are shown to be extremely diverse . While large segments of the population are consistent with the broad grouping into the three Woesian domains, some phylogenetic reconstructions do not place the Archae and the Eucarya as sister groups but, rather, show a gram-negative bacteria/eukaryote clustering . In addition, many individual genes pose difficulties that preclude any simple evolutionary scheme . Thus, aaRS's are clearly a paradigm of F . Jacob's "odd jobs of evolution" but, on the whole, do not call into question the evolutionary scenario originally proposed by Woese and subsequently refined by others. Jpn J Antibiot, 2000 Jun, 53 Suppl B, 60 - 71 {Transport to an infected site of azithromycin by phagocyte cells}; Enogaki K; Azithromycin is a new macrolide antibiotic developed by Pfizer . This compound has a 15 ring structure formed by adding a methyl-nitrogen to the 14-member lactone ring of erythromycin . Azithromycin has acid stability and significant improved activity against gram negative bacteria compared to other macrolides . Further, sustained high tissue levels of azithromycin have been demonstrated clinically and in basic research . There has been particular interest in the phagocyte delivery system of azithromycin to the site of infection . The mechanism is characterized by the intake of azithromycin by phagocytic cells which release the antibiotic at the site of infection . This report describes the mechanism of sustained high tissue levels by summarizing the data of Japanese and western clinical trials and research. Curr Drug Targets Infect Disord, 2003 Mar, 3(1), 9 - 23 Beta-lactamases: a survey of protein diversity; Helfand MS et al.; Bacterial resistance to beta-lactam antibiotics and beta-lactamase inhibitors is an ever increasing problem that threatens the clinical utility of drugs that form the cornerstone of the antibiotic armamentarium . Especially among Gram-negative pathogens, elaboration of structurally and mechanistically novel beta-lactamase enzymes is the most important means by which resistance occurs . An appreciation of the tremendous diversity of these drug-modifying enzymes will assist in understanding why so few generally effective inhibitory agents exist for these unique drug targets . This review will give a general background on the reaction mechanisms and classification schemes of the more than 340 beta-lactamase enzymes described to date . A discussion will follow highlighting the emerging Class A SHV and TEM-derived extended-spectrum (ESBLs), and inhibitor-resistant enzymes, non-TEM, non-SHV Class A ESBLs, and carbapenemases, Class B metallo-beta-lactamases and some of their novel inhibitors, plasmid and chromosomally encoded Class C enzymes, and finally, the OXA-type oxacillinases, ESBLs, and carbapenemases of Class D . The clinical importance of multiple resistance mechanisms in conjunction with the production of beta-lactamase enzymes is emphasized. AIHA J (Fairfax, Va), 2003 Jan-Feb, 64(1), 88 - 94 Bactericidal treatment of raw cotton as the method of byssinosis prevention; Hend IM et al.; In early studies, research to control byssinosis focused on methods to reduce the trash in the textile mill environment . Dust control has been effective in reducing the prevalence of byssinosis, but simple reduction in dust levels does not always assure its prevention . Also, bacteria and fungi present in cotton do not in themselves cause byssinosis, but the endotoxins-heat-stable lipopolysaccharide-protein complexes contained in the cell wall of Gram-negative bacteria-are responsible for the development of this respiratory disease of workers on cotton, flax, and some other fibers . Experimental work was carried out in cotton fields in different cotton growing countries . Opened cotton capsules were treated by spraying them with bactericidal water solutions of benzododecinium bromide to avoid the growth of bacteria by bacteriostatic effect during transportation and storage and thus to prevent the formation of endotoxins . To simulate transport conditions, treated and nontreated cotton samples were incubated under high air humidity . The endotoxin contents were determined by Limulus amebocyte lysate assay depending on the duration of incubation . In nontreated samples the endotoxin content grew to over 5,000 ng/mg . In comparison, in treated samples the endotoxin content grew extremely slowly . Thus, the bactericidal treating of raw cotton showed high efficiency as a potential method of byssinosis prevention . The irradiation by gamma-rays is also efficient, but it is not realistic in cotton growing areas of developing countries at the present time. Novartis Found Symp, 2002, 248, 181 - 97; discussion 197-200, 277-82 Non-allergic models of mucous cell metaplasia and mucus hypersecretion in rat nasal and pulmonary airways; Harkema JR et al.; Mucous cell proliferation and hypersecretion of airway mucus are important pathological features of human respiratory disorders such as asthma and chronic bronchitis . In addition to airborne allergens and infectious agents, inhaled chemical irritants such as ozone and cigarette smoke have been demonstrated to induce changes in airway mucus production . Cellular and molecular mechanisms involved in non-allergic, toxicant-induced mucous cell metaplasia (MCM; transformation of airway epithelium, normally devoid of mucous cells, to secretory epithelium containing numerous mucus-secreting cells) are still unclear . We have used two experimental models of toxicant-induced MCM in the airways of rats to study the epithelial and inflammatory factors involved in the pathogenesis of MCM . Mucin-specific gene expression and MCM are induced in the nasal transitional epithelium (NTE), but not in the bronchiolar epithelium of F344 rats acutely exposed to ozone, an important air pollutant of photochemical smog . Inhalation of endotoxin, a lipopolysaccharide-protein molecule of gram-negative bacteria, induces MCM in the bronchiolar epithelium, but not in the NTE, of rats . Both ozone- and endotoxin-induced MCM are dependent on neutrophilic inflammation . Interestingly, each toxicant enhances the MCM induced by the other toxicant . These synergistic effects elicited by coexposure to ozone and endotoxin are also mediated, in part, by neutrophils. Nat Rev Mol Cell Biol, 2003 Feb, 4(2), 105 - 16 Acquisition of siderophores in gram-negative bacteria; Faraldo-Gomez JD et al.; The outer membrane of Gram-negative bacteria constitutes a permeability barrier that protects the cell from exterior hazards, but also complicates the uptake of nutrients . In the case of iron, the challenge is even greater, because of the scarcity of this indispensable element in the cell's surroundings . To solve this dilemma, bacteria have evolved sophisticated mechanisms whereby the concerted actions of receptor, transporter and energy-transducing proteins ensure that there is a sufficient supply of iron-containing compounds, such as siderophores. J Dent Res, 2003 Feb, 82(2), 82 - 90 Resolution of inflammation: a new paradigm for the pathogenesis of periodontal diseases; Van Dyke TE et al.; The periodontal diseases are infectious diseases caused by predominantly Gram-negative bacteria . However, as our understanding of the pathogenesis of the periodontal diseases grows, it is becoming clear that most of the tissue damage that characterizes periodontal disease is caused by the host response to infection, not by the infectious agent directly . Investigation into the mechanism of action of host-mediated tissue injury has revealed that the neutrophil plays an important role in destruction of host tissues . In this paper, we review the biochemical pathways and molecular mediators that are responsible for regulation of the inflammatory response in diseases such as periodontitis, with a focus on lipid mediators of inflammation . Pro-inflammatory mediators, such as prostaglandins and leukotrienes, are balanced by counter-regulatory signals provided by a class of molecules called lipoxins . The role of lipoxins in the control and resolution of inflammation is discussed, as is the possibility of the development of new therapeutic strategies for the control and prevention of neutrophil-mediated tissue injury in inflammatory diseases like periodontitis. J Bacteriol, 2003 Feb, 185(4), 1218 - 28 In vitro and in vivo functional activity of Chlamydia MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase involved in peptidoglycan synthesis and fosfomycin resistance; McCoy AJ et al.; Organisms of Chlamydia spp . are obligate intracellular, gram-negative bacteria with a dimorphic developmental cycle that takes place entirely within a membrane-bound vacuole termed an inclusion . The chlamydial anomaly refers to the fact that cell wall-active antibiotics inhibit Chlamydia growth and peptidoglycan (PG) synthesis genes are present in the genome, yet there is no biochemical evidence for synthesis of PG . In this work, we undertook a genetics-based approach to reevaluate the chlamydial anomaly by characterizing MurA, a UDP-N-acetylglucosamine enolpyruvyl transferase that catalyzes the first committed step of PG synthesis . The murA gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible, glucose-repressible ara promoter and transformed into Escherichia coli . After transduction of a lethal DeltamurA mutation into the strain, viability of the E . coli strain became dependent upon expression of the C . trachomatis murA . DNA sequence analysis of murA from C . trachomatis predicted a cysteine-to-aspartate change in a key residue within the active site of MurA . In E . coli, the same mutation has previously been shown to cause resistance to fosfomycin, a potent antibiotic that specifically targets MurA . In vitro activity of the chlamydial MurA was resistant to high levels of fosfomycin . Growth of C . trachomatis was also resistant to fosfomycin . Moreover, fosfomycin resistance was imparted to the E . coli strain expressing the chlamydial murA . Conversion of C . trachomatis elementary bodies to reticulate bodies and cell division are correlated with expression of murA mRNA . mRNA from murB, the second enzymatic reaction in the PG pathway, was also detected during C . trachomatis infection . Our findings, as well as work from other groups, suggest that a functional PG pathway exists in Chlamydia spp . We propose that chlamydial PG is essential for progression through the developmental cycle as well as for cell division . Elucidating the existence of PG in Chlamydia spp . is of significance for the development of novel antibiotics targeting the chlamydial cell wall. Clin Exp Immunol, 2003 Feb, 131(2), 217 - 24 Endogenous glucocorticoids attenuate Shiga toxin-2-induced toxicity in a mouse model of haemolytic uraemic syndrome; Gomez SA et al.; The concept that during an immune challenge the release of glucocorticoids (GC) provides feedback inhibition on evolving immune responses has been drawn primarily from studies of autoimmune and/or inflammatory processes in animal models . The epidemic form of haemolytic uraemic syndrome (HUS) occurs secondary to infection with Gram-negative bacteria that produce Shiga toxin (Stx) . Although Stx binding to the specific receptors present on renal tissue is the primary pathogenic mechanism, inflammatory or immune interactions are necessary for the development of the complete form of HUS . The aim of this study was to investigate the influence of endogenous GC on Stx-toxicity in a mouse model . Stx2 was injected into GC-deprived mice and survival rate, renal damage and serum urea levels were evaluated . Plasma corticosterone and cytosolic GC receptor (GR) concentration were also determined at multiple intervals post-Stx2 treatment . Higher sensitivity to Stx2 was observed in mice lacking endogenous GC, evidenced by an increase in mortality rates, circulating urea levels and renal histological damage . Moreover, Stx2 injection was associated with a transient but significant rise in corticosterone secretion . Interestingly, 24 h after Stx inoculation significant increases in total GR were detected in circulating neutrophils . These results indicate that interactions between the neuroendocrine and immune systems can modulate the level of damage significantly during a bacterial infection. Clin Exp Dermatol, 2003 Jan, 28(1), 46 - 9 Giant condyloma of Buschke-Loewenstein in association with erythroderma; Antony FC et al.; The Buschke-Loewenstein tumour is regarded as a type of verrucous carcinoma occurring on anogenital mucosal surfaces . The tumour is locally invasive but displays a benign cytology and rarely metastasizes . It is associated with human papillomavirus types 6 and 11 . We describe a case of Buschke-Loewenstein tumour occurring in a 61-year-old man which behaved in a locally aggressive manner and was associated with human papillomavirus type 16 and erythroderma which proved resistant to treatment . The patient refused surgery and therapy with interferon alpha was ineffective . Chemotherapy with systemic cisplatin and 5-fluorouracil produced a partial response before the patient succumbed from gram-negative septicaemia. Environ Microbiol, 2003 Feb, 5(2), 127 - 32 A novel assay to monitor predator-prey interactions for Bdellovibrio bacteriovorus 109 J reveals a role for methyl-accepting chemotaxis proteins in predation; Lambert C et al.; Bdellovibrio bacteriovorus are Gram-negative bacteria that prey upon other Gram-negative bacteria, including some pathogens, in a wide variety of habitats including soil, sewage, marine and estuarine environments . In order to facilitate studies on predation by this organism, we have developed a method that assays killing of luminescent Escherichia coli by B . bacteriovorus . Moreover, we have used this assay to compare predation of cells by derivatives of B . bacteriovorus containing targeted mutations in genes we have identified . Two genes are described; one, mcp2, encoding a methyl-accepting chemotaxis protein (MCP) and the other, an mviN homologue . Bdellovibrio bacteriovorus mcp2::aphII were less efficient predators on luminescent E . coli than B . bacteriovorus containing a randomly inserted aphII gene via TnphoA transposition . These and other chemotaxis experiments implicated at least a minor role for chemotaxis in predation by B . bacteriovorus . They also open the way for further studies on Bdellovibrio ecology, genomics and predator-prey interactions . The results further confirm that Bdellovibrio uses a chemotaxis system in order to sense, and respond to, changes in its environment, including prey. Shock, 2003 Jan, 19(1), 91 - 6 Diferuloylmethane inhibits neutrophil infiltration and improves survival of mice in high-dose endotoxin shock; Madan B et al.; Gram-negative septic shock is a systemic inflammatory response of the body caused primarily by the cell wall component (lipopolysaccharide) of the gram-negative bacteria . During high-dose endotoxin shock, neutrophils infiltrate and accumulate in the liver, causing hepatocellular injury . Cell adhesion molecules, specifically intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), play an important role in the infiltration of neutrophils in the liver tissue . In this study, we demonstrate that diferuloylmethane exerts protective effect in high-dose endotoxin shock by improving survival and reducing the severity of endotoxin shock symptoms such as lethargy, diarrhea, and watery eyes following a challenge with lipopolysaccharide . We demonstrate here that diferuloylmethane inhibits the transmigration and infiltration of neutrophils from blood vessels to the underlying liver tissue and, hence, inhibits the damage to the tissue . Diferuloylmethane blocks the induced expression of ICAM-1 and VCAM-1 in liver and lungs . Diferuloylmethane, being a natural compound, may have few side effects and may be useful in attenuating multiple organ injury in pathological conditions arising due to excessive infiltration of neutrophils into the tissues. Expert Opin Investig Drugs, 2003 Feb, 12(2), 139 - 52 Recent developments in the treatment of sepsis; Sharma VK et al.; Despite advances in supportive care, septic shock remains a major cause of morbidity and mortality . With the identification of the systemic inflammatory response as a major component in the pathogenesis of the septic shock syndrome, much of the recent work has focused on modulating this response . This includes antiendotoxin therapies in patients with Gram-negative sepsis, and therapies to modulate the pro-inflammatory mediators produced in response to infection, such as TNF-alpha, platelet-activating factor and complement . High-flow haemofiltration has the potential advantage of clearing both endotoxin and pro-inflammatory mediators . Antithrombotic strategies have been investigated and have yielded the first major success in the treatment of sepsis with activated protein C . Nitric oxide produces the cardiovascular features of sepsis and investigators have looked at both reducing its production and mopping up the excess . Attempts to reduce apoptosis have been a new focus in the treatment of sepsis . There have also been recent developments in supportive care suggesting a role for vasopressin and replacement corticosteroid therapy. Equine Vet J, 2003 Jan, 35(1), 35 - 9 Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS; Dagleish MR et al.; REASONS FOR PERFORMING STUDY: Lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-like peptides are Gram-negative bacterial cell wall components which, when released into the peripheral circulation in endotoxaemia, have the potential to activate leucocytes . In vitro, equine neutrophils require priming with LPS in order to generate reactive oxygen intermediates (ROI) in response to fMLP . OBJECTIVES: The aim of this study was to examine whether the release of other neutrophil products is similarly dependent on prior priming with LPS . In particular, neutrophil elastase (NE), a potent proteolytic enzyme, and its major inhibitor, alpha-1 proteinase inhibitor, were investigated . METHODS: Neutrophils were isolated from equine peripheral blood (n = 5) by discontinuous Percoll gradient preparative centrifugation and primed with LPS prior to stimulation with fMLP . ROI were measured by lucigenin dependent chemiluminescence (LDCL) . Concentrations of NE and API were determined by ELISA on cell free supernatants taken at 0, 2, 10, 30, 60 and 90 mins post stimulus . Data was analysed by Kruskal-Wallis and Mann-Whitney Tests . RESULTS: Sequential exposure of Percoll purified equine blood neutrophils in vitro to LPS followed by fMLP resulted in the greatest release of NE from equine neutrophils and was required for ROI generation . However, LPS or fMLP stimulation alone resulted in an increase in NE release compared to unstimulated control cells . In contrast, significant API release was only induced by LPS stimulation or fMLP stimulation only after LPS priming, not fMLP on its own . CONCLUSIONS: These results suggest that different stimuli (fMLP or LPS) are capable of invoking similar responses from equine neutrophils with respect to NE release yet different ones with respect to API release . Potential relevance: In addition, demonstration of elastase release induced by LPS and/or fMLP suggests that monitoring serum elastase levels is a potential diagnostic tool for detecting the early onset of endotoxaemia in the horse. Environ Res, 2003 Jan, 91(1), 35 - 44 Reactions of macrophages exposed to particles <10 microm; Monn C et al.; This study describes experiments on cytotoxic effects and the production of oxidative radicals and the proinflammatory cytokine tumor growth factor alpha (TNFalpha) in a cell line of rat lung macrophages exposed to aqueous extracts from ambient air particles <10 microm (PM(10)) collected on Teflon filters . The particles were collected during the four seasons at two urban sites, one rural site, and one alpine site in Switzerland . Cytotoxic effects, determined as a reduction in the metabolic activity, were found in particle extracts from all sites and seasons . Taking together the data from all sites and seasons, a dose-response function was observed between the particle mass on the filter and toxicity (r(2)=0.633, linear regression) . The release of the pro-inflammatory cytokine TNFalpha as well as of oxidative radicals was most pronounced in particles collected in spring-summer and autumn . While at Montana (alpine), the stimulation of the cells was positively correlated with the particle mass on the filters, this correlation was negative at the urban sites Zurich and Lugano . It is interpreted that at high PM(10) levels, as in these cities, macrophages are inhibited by increasing air pollution due to toxic effects . Cytotoxic effects and the release of oxidative radicals could be inhibited when the extracts were treated with an endotoxin-neutralizing protein . This suggests that endotoxin, a cell-wall constituent of gram-negative bacteria, is one of the factors which modulates macrophage activity . All together, the experiments indicate that in the PM(10) fraction, water-soluble macrophage-toxic and macrophage-stimulating compounds are present . The data offer an explanation for at least some of the known harmful effects of PM(10), and confirm endotoxin as a possible reactant. Wei Sheng Wu Xue Bao, 1998 Oct, 38(5), 390 - 2 {Isolation and identification of Rhodopseudomonas acidophila from Fujian province, China}; Zeng H et al.; A strain P301 of Gram negative purple nonsulfur bacterium was isolated from the ditch's mud collected in Fuzhou, Fujian . The cells are rod-shaped . Multiplication occur by budding without stalk formation . Growth optimum pH is 5.0-5.7 . No growth factors required . The cells contain bacteriochlorophyll a . The photosynthetic membrane system consists of parallel lamellae . According to Bergery's Manual of Determinative Bacteriology, 8th ed . (1974) and 9th ed . (1994), the strain was identified to be Rhodopseudomonas acidophila . But on the side of enzyme activity, utilization of organic substrates and others the strain some differ from the type strain described by N Pfennig (1969) . Therefore the strain P301 was identified to be Rhodopseudomonas acidophila var . fujianensis n . var. Crit Care Med, 2003 Jan, 31(1), 237 - 45 Modulation of serum cytokine levels by a novel superoxide dismutase mimetic, M40401, in an Escherichia coli model of septic shock: correlation with preserved circulating catecholamines; Macarthur H et al.; OBJECTIVES: We have shown previously that inactivation of catecholamines by superoxide anions contributes to the loss of vascular reactivity to norepinephrine and the subsequent hypotension that develops in Gram-negative endotoxic shock . In addition to their vasopressor actions, catecholamines, via beta-adrenoceptor activation, are important regulators of cytokine production . Here we examined if maintenance of serum catecholamine levels by the superoxide dismutase mimetic, M40401, modulates serum cytokine levels and arterial hypotension in an Escherichia coli-infected conscious rat model of septic shock . DESIGN: Controlled laboratory animal study . SETTING: University animal research laboratory . SUBJECTS: Pathogen-free male Sprague-Dawley rats (n = 51) . INTERVENTIONS: Conscious, antibiotic-treated animals with chronic in-dwelling carotid arterial and jugular venous catheters were intravenously infected with 10(10) live E . coli bacteria (O55:B5, n = 51) over 30 mins, ending at time = 0 hrs . At 0.5 or 3 hrs, infected rats were administered an intravenous infusion of either M40401 (n = 33) or 0.9% saline (n = 18) for 6 hrs at a rate of 1 mL/h . In additional experiments, anesthetized animals with catheterized left femoral arteries and veins were administered a dose-range of norepinephrine (0.1-1 microg/kg) as bolus intravenous injections . Thereafter, E . coli lipopolysaccharide (4 mg/kg, n = 6) was administered as a 0.3-mL slow bolus intravenous injection . One hour later, the norepinephrine protocol was repeated, after which the rats were administered an intravenous infusion of either M40401 or 0.9% saline for 15 mins . At 2 hrs, the dose response to norepinephrine was repeated . MEASUREMENTS AND MAIN RESULTS: Rats infected with live E . coli exhibited a biphasic fall in mean arterial pressure, with mortality reaching 83% by 24 hrs . Rats treated with M40401 (0.25, 2.5, or 25 microg x kg-1 x hr-1 ) 3 hrs after bacteremic sepsis maintained a normal mean arterial pressure, and mortality was dose-dependently reduced to 44, 33, and 22%, respectively, at 24 hrs . Furthermore, serum catecholamine levels were diminished in E . coli-infected rats treated with saline compared with rats treated with M40401 . In separate experiments, E . coli-infected rats were administered M40401 (25 microg x kg-1 x hr-1 ) 0.5 hr after bacterial challenge . Blood samples taken at 0, 1.5, 3.5, and 6 hrs were analyzed for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, and IL-10 and for norepinephrine and epinephrine . Serum levels of tumor necrosis factor-alpha and IL-1 beta were significantly depressed in M40401-treated septic rats, whereas IL-10 was elevated . Moreover, serum catecholamine levels were greater in M40401-treated septic rats at the same time points . IL-6 levels were unaffected by M40401 treatment . Finally we examined whether treatment with M40401 could reverse the hyporeactivity to norepinephrine typifying early septic shock . Using the E . coli lipopolysaccharide (4 mg/kg) challenged anesthetized rat model of shock, we demonstrated that the vasoconstrictor ability of norepinephrine was indeed restored after M40401 treatment (25 microg/kg) . CONCLUSION: Postinfection treatment with the superoxide dismutase mimetic M40401 protects against hypotension, vascular hyporeactivity to catecholamines, and mortality associated with septic shock . Such beneficial effects correlate with both reduced oxidative inactivation of serum catecholamines and a reduction in canonical cytokine mediators of inflammation. Crit Care Med, 2003 Jan, 31(1 Suppl), S57 - 64 Endotoxin as a drug target; Opal SM et al.; OBJECTIVE: To review the preclinical and clinical evidence that antiendotoxin therapeutic strategies are potentially useful in the prevention and treatment of septic shock . STUDY DESIGN: A critical review of the literature over the past 30 yrs relating basic and clinical research on the therapeutic value of endotoxin as a target for the prevention and treatment of severe sepsis and septic shock . MAIN RESULTS: Bacterial endotoxin is a potent and predominant microbial mediator that induces an intense inflammatory and procoagulant response by elements of the innate immune response . This macromolecule is capable of inducing lethal septic shock in experimental animals, and a large number of preclinical studies consistently demonstrate the survival advantage of endotoxin inhibition in experimental models of sepsis . Clinical studies indicate that endotoxin may be found in the systemic circulation in the majority of humans with septic shock . Endotoxemia is largely independent of the nature of the infecting microorganism despite the fact that this molecule is specifically found in the outer membrane of Gram-negative bacteria only . Antiendotoxin strategies studied thus far have not provided reproducible survival benefits in clinical trials in septic patients . CONCLUSIONS: Despite compelling evidence of the critical importance of endotoxin in the pathogenesis of Gram-negative bacterial sepsis in preclinical investigations and numerous clinical interventional trials, the utility of antiendotoxin approaches to significantly reduce the mortality rate in human septic shock remains unproven . Ongoing clinical trials with specific endotoxin inhibitors should determine the potential value of this therapeutic approach to the management of septic shock. Infect Immun, 2003 Feb, 71(2), 794 - 800 Colonization of C57BL/6J and BALB/c wild-type and knockout mice with Helicobacter pylori: effect of vaccination and implications for innate and acquired immunity; Panthel K et al.; The gram-negative bacterial pathogen Helicobacter pylori is a major cause of peptic ulcer disease and a risk factor for gastric cancer in humans . Adapted H . pylori strains, such as strain SS1, are able to infect mice and are a useful model for gastric colonization and vaccination studies . In this study we used a streptomycin-resistant derivative of H . pylori SS1 to analyze the colonization behavior and the success of vaccination in wild-type (wt) and various knockout mice of the BALB/c and C57BL/6J genetic backgrounds . We here report that BALB/c interleukin-4 knockout (IL-4(-/-)) mice are weakly overcolonized compared to the wt strain but that the IL-12(-/-) knockout results in a strong overcolonization (500%) . Unexpectedly, in the C57BL/6J background the same knockouts behaved in diametrically opposed manners . The IL-4(-/-) mutation caused a 50% reduction and the IL-12(-/-) knockout caused a 95% reduction compared to the wt colonization rate . For C57BL/6J mice we further analyzed the IL-18(-/-) and Toll-like receptor 2 knockout mutations, which showed reductions to 66 and 57%, respectively, whereas mice with the IL-10(-/-) phenotype were hardly infected at all (5%) . In contrast, the tumor necrosis factor receptor knockout (p55(-/-) and p55/75(-/-)) mice showed an overcolonization compared to the C57BL/6J wt strain . With exception of the low-level infected C57BL/6J IL-10(-/-) and IL-12(-/-) knockout mice, all knockout mutants were accessible to a prophylactic vaccination and their vaccination behavior was comparable to that of the wt strains. J Pharmacol Exp Ther, 2003 Feb, 304(2), 624 - 33 Histamine inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production in an intercellular adhesion molecule-1- and B7.1-dependent manner; Morichika T et al.; Lipopolysaccharide (LPS) is recognized as a key molecule in the pathogenesis of Gram negative sepsis and septic shock . In the present study, we demonstrate that LPS (1-1000 pg/ml) concentration dependently up-regulated the expression of intercellular adhesion molecule (ICAM)-1, B7.1, and B7.2 on human monocytes using fluorescence-activated cell sorting analysis, and that tumor necrosis factor (TNF)-alpha production induced by LPS in peripheral blood mononuclear cells (PBMCs) was inhibited by the addition of antibodies against these adhesion molecules, suggesting the dependence of TNF-alpha production on cell-cell interaction through these adhesion molecules . Moreover, we found that histamine (10(-7)-10(-4) M) concentration dependently inhibited the expression of ICAM-1 and B7.1, but not B7.2 on monocytes induced by LPS . Histamine also inhibited the responses of TNF-alpha production induced by LPS . The modulatory effects of histamine on ICAM-1 and B7.1 expression and TNF-alpha production were all concentration dependently antagonized by famotidine but not by d-chlorpheniramine and thioperamide, and were mimicked by selective H2-receptor agonists but not by H1-, H3-, and H4-receptor agonists, indicating the involvement of H2-receptors in the histamine action . Dibutyryl cAMP down-regulated ICAM-1 and B7.1 expression on monocytes stimulated by LPS, suggesting the mediation by the cyclic adenosine monophosphate-protein kinase A pathway of H2-receptor activation . These results as a whole indicated that histamine via H2-receptor inhibited the LPS-induced TNF-alpha production through the regulation of ICAM-1 and B7.1 expression, leading to the reduction of innate immune response stimulated by LPS. J Am Soc Nephrol, 2003 Feb, 14(2), 454 - 61 Bioartificial kidney ameliorates gram-negative bacteria-induced septic shock in uremic animals; Fissell WH et al.; The bioartificial kidney (BAK) consists of a conventional hemofiltration cartridge in series with a renal tubule assist device (RAD) containing 10(9) porcine renal proximal tubule cells . BAK replaces filtration, transport, and metabolic and endocrinologic activities of a kidney . Previous work in an acutely uremic dog model demonstrated that BAK ameliorated endotoxin (lipopolysaccharide {LPS})-induced hypotension and altered plasma cytokine levels . To further assess the role of BAK in sepsis in acute renal failure, dogs were nephrectomized and 48 h later administered intraperitoneally with 30 x 10(10) bacteria/kg of E . coli . One hour after bacterial administration, animals were placed in a continuous venovenous hemofiltration circuit with either a sham RAD without cells (n = 6) or a RAD with cells (n = 6) . BP, cardiac output, heart rate, pulmonary capillary wedge pressure, and systemic vascular resistance were measured throughout the study . All animals tested were in renal failure, with blood urea nitrogen and serum creatinine concentrations greater than 60 and 6 mg/dl, respectively . RAD treatment maintained significantly better cardiovascular performance, as determined by arterial BP (P < 0.05) and cardiac output (P < 0.02), for longer periods than sham RAD therapy . Consistently, all sham RAD-treated animals, except one, expired within 2 to 9 h after bacterial administration, whereas all RAD-treated animals survived more than 10 h . Plasma levels of TNF-alpha, IL-10, and C-reactive protein (CRP) were measured during cell RAD and sham RAD treatment . IL-10 levels were significantly higher (P < 0.01) during the entire treatment interval in the RAD animals compared with sham controls . These data demonstrated in a pilot large animal experiment that the BAK with RAD altered plasma cytokine levels in acutely uremic animals with septic shock . This change was associated with improved cardiovascular performance and increased survival time . These results demonstrate that the addition of cell therapy to hemofiltration in an acutely uremic animal model with septic shock ameliorates cardiovascular dysfunction, alters systemic cytokine balance, and improves survival time. Br J Anaesth, 2003 Feb, 90(2), 221 - 32 Oxidative stress and gene expression in sepsis; Macdonald J et al.; Dysregulation of the immuno-inflammatory response, as seen in sepsis, may culminate in host cell and organ damage . Lipopolysaccharide from Gram-negative bacterial cell walls induces gene activation and subsequent inflammatory mediator expression . Gene activation is regulated by a number of transcription factors at the nuclear level, of which nuclear factor kappaB appears to have a central role . The redox (reduction-oxidation) cellular balance is important for normal cellular function, including transcription factor regulation . In sepsis, a state of severe oxidative stress is encountered, with host endogenous antioxidant defences overcome . This has implications for cellular function and the regulation of gene expression . This review gives an overview of the mechanisms by which transcription factor activation and inflammatory mediator overexpression occur in sepsis, together with the events surrounding the state of oxidative stress encountered and the effects on the host's antioxidant defences . The effect of oxidative stress on transcription factor regulation is considered, together with the role of antioxidant repletion in transcription factor activation and in sepsis in general . Other interventions that may modulate transcription factor activation are also highlighted. J Bacteriol, 2003 Feb, 185(3), 909 - 17 Heme-responsive transcriptional activation of Bordetella bhu genes; Vanderpool CK et al.; Bordetella pertussis and Bordetella bronchiseptica, gram-negative respiratory pathogens of mammals, possess a heme iron utilization system encoded by the bhuRSTUV genes . Preliminary evidence suggested that expression of the BhuR heme receptor was stimulated by the presence of heme under iron-limiting conditions . The hurIR (heme uptake regulator) genes were previously identified upstream of the bhuRSTUV gene cluster and are predicted to encode homologs of members of the iron starvation subfamily of extracytoplasmic function (ECF) regulators . In this study, B . pertussis and B . bronchiseptica DeltahurI mutants, predicted to lack an ECF sigma factor, were constructed and found to be deficient in the utilization of hemin and hemoglobin . Genetic complementation of DeltahurI strains with plasmid-borne hurI restored wild-type levels of heme utilization . B . bronchiseptica DeltahurI mutant BRM23 was defective in heme-responsive production of the BhuR heme receptor; hurI in trans restored heme-inducible BhuR expression to the mutant and resulted in BhuR overproduction . Transcriptional analyses with bhuR-lacZ fusion plasmids confirmed that bhuR transcription was activated in iron-starved cells in response to heme compounds . Heme-responsive bhuR transcription was not observed in mutant BRM23, indicating that hurI is required for positive regulation of bhu gene expression . Furthermore, bhuR was required for heme-inducible bhu gene activation, supporting the hypothesis that positive regulation of bhuRSTUV occurs by a surface signaling mechanism involving the heme-iron receptor BhuR. Chemistry, 2003 Jan 20, 9(2), 378 - 88 A process in need is a process indeed: scalable enantioselective synthesis of chiral compounds for the pharmaceutical industry; Ikunaka M; This report deals with enantioselective synthesis of viracept 1 (nelfinavir mesylate, AG 1343), a potent HIV protease inhibitor, and 3-hydroxytetradecanoic acid 3, a component of lipid A comprising lipopolysaccharide embedded in the cell surface of Gram-negative bacteria, from both strategic and practical perspectives . As regards the synthesis of 1, the synthetic approaches to its central intermediate 2 possessing the common structural motif of 1,4-differentially substituted-2-amino-3-hydroxylbutane are mainly discussed with emphasis on the molecular symmetry that has helped streamline the synthetic strategy . In the discussion of the synthetic strategies to access a single enantiomer of 3, the chiral methodologies that have been applied so far are assessed for industrial viability; the synthetic alternatives explored include resolution via diastereomeric salt formation, lipase-catalyzed kinetic resolution, asymmetric synthesis, and chiral pool approaches. Am J Gastroenterol, 2003 Jan, 98(1), 77 - 81 Surveillance cultures to monitor quality of gastrointestinal endoscope reprocessing; Moses FM et al.; OBJECTIVES: High-level disinfection of GI endoscopes can be reliably obtained under controlled conditions with approved reprocessing methods . However, there are scant data regarding the effectiveness of these methods in clinical practice and no published methods of verification . The purpose of this study is to review retrospectively the results of environmental cultures of flexible endoscopes and to analyze the pattern of results . METHODS: Cultures of selected GI endoscopes listed as ready to use were obtained by adding 5-15 ml of trypticase soy broth or saline or 30-50 ml of sterile water to the biopsy channel of an endoscope . This wash was collected in a sterile container, plated onto blood and MacConkey agar, incubated at 37 degrees C, and examined for growth at 24 and 48 h . Personnel trained in accordance with approved procedures performed endoscope reprocessing . RESULTS: A total of 312 surveillance cultures were performed between 1990 and 1999 . Initially, three of 17 water bottles were found to be contaminated with Pseudomonas species . The bottles were sterilized daily; only sterile water was used and subsequent cultures were negative . Between 1992 and 1994, 15/129 (11.6%) cultures were positive; 14 (93%) were from duodenoscopes . From 1995 to 1997, 18/124 (14.5%) cultures were positive, but only six (33%) were from duodenoscopes . However, 10 (55.6%) positive cultures were obtained from therapeutic upper endoscopes, attributed to faulty mechanical cleaning by nonnursing personnel after emergent procedures . The reprocessing procedure was altered, with improvement . One duodenoscope was persistently culture positive and was found to have a damaged biopsy channel . There were no recognized iatrogenic infections associated with endoscopic procedures . Organisms cultured were commonly gram-negative rods . CONCLUSIONS: The use of environmental endoscope culturing is a rapid, simple, inexpensive method to monitor effectiveness of standard reprocessing procedures . Disinfection is less effective with poor mechanical cleansing, and high-titer positivity is a marker for poor cleaning technique . Standard upper and lower scopes are commonly culture negative . Duodenoscopes, because of their inherent complexity, and other scopes used in emergent conditions require particular attention . Surveillance culture results can be used to identify patterns of poor technique, to reinforce proper procedure, and to modify clinical practice . No associated clinical illness was apparent during this study. Curr Biol, 2003 Jan 8, 13(1), 27 - 36 Naip5 affects host susceptibility to the intracellular pathogen Legionella pneumophila; Wright EK et al.; BACKGROUND: Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease . Legionella produces disease because it can replicate inside a specialized compartment of host macrophages . Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella . A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified . RESULTS: Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella . Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation . Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains . A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness . In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages . CONCLUSIONS: We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication . We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins. Clin Chem Lab Med, 2002 Nov, 40(11), 1097 - 100 Polymorphisms in the lipopolysaccharide-binding protein and bactericidal/permeability-increasing protein in patients with myocardial infarction; Hubacek JA et al.; Gram-negative bacterial infection, namely Chlamydia pneumoniae has been recently discussed as a risk factor for myocardial infarction . The lipopolysaccharide-binding protein (LBP) and the bactericidal/permeability-increasing protein (BPI) play a role in the processes leading to recognition and neutralisation of the Chlamydia pneumoniae and their endotoxins lipopolysaccharides (LPS) . LPS interact with plasma LBP, and LBP-LPS complex activates monocytes/macrophages, which can influence the atherosclerotic process . BPI is cytotoxic for Gram-negative bacteria and BPI-LPS complexes do not activate monocytes . We have analysed the polymorphisms in the LBP gene (Gly98-->Cys; Pro436-->Leu) and BPI gene (Lys216-->Glu; PstI polymorphism in intron-5; G545-->C) in 313 patients after myocardial infarction (MI) and in 302 control individuals . Genotype frequencies in the LBP gene and BPI gene did not differ between MI patients and control individuals . Our findings suggest that LBP and BPI polymorphisms do not influence the risk of MI. Clin Microbiol Infect, 2002 Dec, 8(12), 763 - 72 Human granulocytic ehrlichiosis in Europe; Blanco JR et al.; Ehrlichiosis comprises a group of emerging tick-borne infectious diseases caused by obligate intracellular Gram-negative bacteria that infect leukocytes . Infections caused by members of the genus Ehrlichia have been described in animals and humans, but to date there are no convincing reports of the presence of other types of human ehrlichiosis different from human granulocytic ehrlichiosis (HGE) in Europe . The European vector is the same as that of Lyme borreliosis, the hard tick Ixodes ricinus, and HGE has a similar epidemiology to that of Borrelia burgdorferi infection . Across Europe, I . ricinus is infected to a variable extent (0.4-66.7%) with the causative agent Ehrlichia (Anaplasma) phagocytophila