Gram negative

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 genogroup, and since its first description in Slovenia in 1997, details of 15 patients have been published . Diagnosis requires careful consideration of all circumstances and symptoms (history of tick bite and the presence of a flu-like syndrome with variable degrees of anemia, thrombocytopenia, and leukopenia, and elevated liver enzymes) . Some differences can be seen between US and European HGE patients . European HGE cases have a less severe course, and the presence of morulae is uncommon . In Europe, verification of HGE has been based on PCR and immunofluorescence antibody tests, because no isolation from humans has been reported.

J Clin Microbiol, 2003 Jan, 41(1), 192 - 6
Characteristics of Massilia timonae and Massilia timonae-like isolates from human patients, with an emended description of the species; Lindquist D et al.; The description of Massilia timonae, a nonfermentative aerobic gram-negative rod, was based on a single strain . A subsequent report of a second isolate has been recently published . Phenotypic descriptions of these two strains were based primarily on commercial test kit results . We have identified three additional strains as M . timonae by 16S rRNA sequence analysis and have characterized them phenotypically in parallel with the type strain of M . timonae, CIP 105350, by conventional test methods . A fourth strain, designated M . timonae-like, was also characterized . All four strains were isolated from human patients: two were blood isolates, one was isolated from cerebrospinal fluid, and one was isolated from bone . The four strains and the type strain were quite similar phenotypically . However, in contrast to the original description, the strains were found to be oxidase positive and arginine dihydrolase negative and to have lateral flagella as well as a single polar flagellum . Additionally the strains produced acid oxidatively from some carbohydrates . Other phenotypic characteristics, including cellular fatty acids, agreed with the original description . Based on our emended description, M . timonae and M . timonae-like strains can be differentiated from other aerobic nonfermentative gram-negative rods by conventional biochemical tests combined with cellular fatty acid analysis.

Zhonghua Shao Shang Za Zhi, 2002 Apr, 18(2), 92 - 4
{An experimental study of the LPS release from gram-negative bacteria induced by antibiotics (Part two)}; Xu N et al.; OBJECTIVE: To explore the effects of different beta-lactam antibiotics on the inducing of LPS release from gram-negative bacteria and on the protection of infected animals . METHODS: Wistar rats were employed as the model and were inflicted by 30% TBSA III degree scalding and sepsis caused by PA103 . The rats were randomly divided into 3 groups, i.e . simple antibiotic treatment group (A), treatment after sensitization with galactosamine (GalN) group (G) and treatment after blocking with carrageenan (CGN) group (C) . The rats were injected intra-peritoneally with imipenem (IMP, 5 mg) and ceftazidime (CTZ, 10 mg) for single time, respectively . Same amount of aseptic normal saline was injected in the control group, and GalN (50 mg) was added in G and CGN (1 mg) in C groups . The blood bacterial concentration and plasma LPS levels were determined at different time points after the treatment by antibiotics . The mortality was observed in G and C groups at 10 days after treatment . RESULTS: The blood bacterial amount could be decreased by both IMP and CTZ evidently . Large amounts of LPS released from PA103 could be induced by IMP and CTZ during their bactericidal process . But the plasma LPS level in rats treated by CTZ was markedly higher than that by IMP (P < 0.05 approximately 0.01) . The mortality in G group treated by CTZ was much higher than that by IMP (P < 0.05) . Nevertheless, the mortality in C groups was the same no matter CTZ or IMP was applied (P < 0.05) . CONCLUSION: There was no difference of the bactericidal power between IMP and CTZ . But CTZ was more powerful in inducing LPS release from bacteria than IMP . It was implied by the difference between these two antibiotics that IMP might be better choice in clinical application for burn infection due to its lower potential of inducing LPS release from the bacteria.

Appl Environ Microbiol, 2003 Jan, 69(1), 468 - 74
Online monitoring of Escherichia coli ghost production; Haidinger W et al.; Controlled expression of cloned phi X174 gene E in gram-negative bacteria results in lysis of the bacteria by the formation of a transmembrane tunnel structure built through the cell envelope complex . Production of bacterial ghosts is routinely monitored by classical microbiological procedures . These include determination of the turbidity of the culture and the total number of cells and the number of reproductive cells present during the time course of growth and lysis . Although conceptually simple, these methods are labor intensive and time consuming, providing a complete set of results after the determination of viable cell counts . To avoid culturing methods for bacterial growth, an alternative flow cytometric procedure is presented for the quantification of ghosts and polarized, as well as depolarized, nonlysed cells within a culture . For this method, which is based on the discriminatory power of the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol, a staining protocol was developed and optimized for the maximum discrepancy in fluorescence between bacterial ghosts and viable cells . The total quantitative analysis procedure takes less than 2 min . The results derived from classical or cytometric analyses correlate with respect to the total cell numbers and the viability of the culture.

BMC Cell Biol . 2003 Jan 06;4(1):1.
Lipopolysaccharide enhances the cytotoxicity of 2-chloroethyl ethyl sulfide; Stone WL et al.; BACKGROUND: The bacterial endotoxin, lipopolysaccharide (LPS), is a well-characterized inflammatory factor found in the cell wall of Gram-negative bacteria . In this investigation, we studied the cytotoxic interaction between 2-chloroethyl ethyl sulfide (CEES or ClCH2CH2SCH2CH3) and LPS using murine RAW264.7 macrophages . CEES is a sulfur vesicating agent and is an analog of 2,2'-dichlorodiethyl sulfide (sulfur mustard) . LPS is a ubiquitous natural agent found in the environment . The ability of LPS and other inflammatory agents (such as TNF-alpha and IL-1beta) to modulate the toxicity of CEES is likely to be an important factor in the design of effective treatments . RESULTS: RAW 264.7 macrophages stimulated with LPS were found to be more susceptible to the cytotoxic effect of CEES than unstimulated macrophages . Very low levels of LPS (20 ng/ml) dramatically enhanced the toxicity of CEES at concentrations greater than 400 microM . The cytotoxic interaction between LPS and CEES reached a maximum 12 hours after exposure . In addition, we found that tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1-beta) as well as phorbol myristate acetate (PMA) also enhanced the cytotoxic effects of CEES but to a lesser extent than LPS . CONCLUSION: Our in vitro results suggest the possibility that LPS and inflammatory cytokines could enhance the toxicity of sulfur mustard . Since LPS is a ubiquitous agent in the natural environment, its presence is likely to be an important variable influencing the cytotoxicity of sulfur mustard toxicity . We have initiated further experiments to determine the molecular mechanism whereby the inflammatory process influences sulfur mustard cytotoxicity.

Chembiochem, 2003 Jan 3, 4(1), 96 - 100
Evaluation of lipopolysaccharide aggregation by light scattering spectroscopy; Santos NC et al.; Lipopolysaccharides (LPS) are cell wall components of Gram-negative bacteria . These molecules behave as bacterial endotoxins and their release into the bloodstream is a determinant of the development of a wide range of pathologies . These amphipathic molecules can self-aggregate into supramolecular structures with different shapes and sizes . The formation of these structures occurs when the LPS concentration is higher than the apparent critical micelle concentration (CMC(a)) . Light scattering spectroscopy (both static and dynamic) was used to directly characterize the aggregation process of LPS from Escherichia coli serotype 026:B6 . The results point to a CMC(a) value of 14 microg mL(-1) and the existence of premicelle LPS oligomers below this concentration . Both structures were characterized in terms of molecular weight (5.5 x 10(6) and 16 x 10(6) g mol(-1) below and above the CMC(a), respectively), interaction with the aqueous environment, gyration radius (56 and 105 nm), hydrodynamic radius, (60 and 95 nm) and geometry of the supramolecular structures (nearly spherical) . Our data indicates that future in vitro experiments should be carried out both below and above the CMC(a) . The search for drugs that interact with the aggregates, and thus change the CMC(a) and condition LPS interactions in the bloodstream, could be a new way to prevent certain bacterial-endotoxin-related pathologies.

Thorax, 2003 Jan, 58(1), 63 - 7
Reduced interferon-gamma release in patients recovered from Legionnaires' disease; Lettinga KD et al.; BACKGROUND: Legionella pneumophila, a Gram negative intracellular pathogen, causes Legionnaires' disease (LD) . Interferon (IFN)-gamma is important for host defence against L pneumophila so reduced IFN-gamma production capacity and/or responsiveness might render humans more susceptible to infection with L pneumophila . METHODS: Seventy seven patients who suffered from LD after a point source outbreak one year earlier participated in the study . Whole blood was incubated with non-specific stimuli (lipopolysaccharide (LPS) or interleukin (IL)-12) or specific stimuli (viable or heat killed L pneumophila) to evaluate IFN-gamma production, and with IFN-gamma to evaluate IFN-gamma responsiveness . Expression of complement receptor 3 on monocytes was determined by flow cytometry . Thirty seven companions who were also exposed but had not developed LD served as controls . RESULTS: Patients released less IFN-gamma than controls in response to stimulation with LPS (mean (SE) 393 (58) pg/ml v 914 (178) pg/ml; p=0.001) and IL-12 (96 (14) pg/ml v 177 (41) pg/ml; p=0.058) . IFN-gamma responsiveness, measured by release of IFN-gamma inducible protein (IP)-10, tumour necrosis factor alpha, IL-12 production capacity, and monocyte expression of complement receptor 3, did not differ between patients and controls . IFN-gamma release after stimulation with LPS and IP-10 release after stimulation with IFN-gamma were weakly associated with severity of LD in the former patient group (rho=-0.3, p=0.011 and rho=-0.3, p=0.037, respectively) . CONCLUSION: These results suggest that impaired IFN-gamma production may contribute to susceptibility to L pneumophila infection.

J Bacteriol, 2003 Jan, 185(2), 489 - 95
Identification of secretion determinants of the Bordetella pertussis BrkA autotransporter; Oliver DC et al.; The autotransporters comprise a functionally diverse family of gram-negative proteins that mediate their own export across the bacterial outer membrane . They consist of an amino-terminal passenger region called the "alpha-domain" and the structural hallmark of the autotransporter family, a carboxy-terminal transporter region usually referred to as the "beta-domain." The passenger region can be quite diverse and constitutes the effector functions of these proteins, whereas the C-terminal region is conserved and is responsible for translocating the passenger moiety across the outer membrane . BrkA is the 103-kDa autotransporter protein in Bordetella pertussis that is cleaved to yield a 73-kDa N-terminal alpha-domain and a 30-kDa C-terminal beta-domain . We have previously shown that a recombinant form of the beta-domain of BrkA is capable of forming channels in artificial membranes . Here, we define two additional secretion determinants of BrkA . N-terminal sequencing of the 73-kDa BrkA passenger from B . pertussis and Escherichia coli revealed that BrkA has a 42-amino-acid signal peptide . In addition, deletion analysis of BrkA identified a 31- to 39-amino-acid region found immediately upstream of the beta-domain that was essential for surface expression . This 31- to 39-amino-acid linker region, together with the beta-domain, defines the minimal BrkA translocation unit . The linker region may also serve to anchor the BrkA passenger to the bacterial surface.

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2261 - 9
Teredinibacter turnerae gen . nov., sp . nov., a dinitrogen-fixing, cellulolytic, endosymbiotic gamma-proteobacterium isolated from the gills of wood-boring molluscs (Bivalvia: Teredinidae); Distel DL et al.; A cellulolytic, dinitrogen-fixing bacterium isolated from the gill tissue of a wood-boring mollusc (shipworm) Lyrodus pedicellatus of the bivalve family Teredinidae and 58 additional strains with similar properties, isolated from gills of 24 bivalve species representing 9 of 14 genera of Teredinidae, are described . The cells are Gram-negative, rigid, rods (0.4-0.6 x 3-6 microm) that bear a single polar flagellum . All isolates are capable of chemoheterotrophic growth in a simple mineral medium supplemented with cellulose as a sole source of carbon and energy . Xylan, pectin, carboxymethylcellulose, cellobiose and a variety of sugars and organic acids also support growth . Growth requires addition of combined nitrogen when cultures are vigorously aerated, but all isolates fix dinitrogen under microaerobic conditions . The pH, temperature and salinity optima for growth were determined for six isolates and are approximately 8.5, 30-35 degrees C and 0.3 M NaCl respectively . The isolates are marine . In addition to NaCl, growth requires elevated concentrations of Ca2+ and Mg2+ that reflect the chemistry of seawater . The DNA G+C content ranged from 49 to 51 mol% . Four isolates were identical with respect to small-subunit rRNA sequence over 891 positions compared and fall within a unique clade in the gamma-subclass of the Proteobacteria . Based on morphological, physiological and phylogenetic characteristics and specific symbiotic association with teredinid bivalves, a new genus and species, Teredinibacter turnerae gen . nov., sp . nov., is proposed . The type strain is T7902(T) (= ATCC 39867(T) = DSM 15152(T)).

Int J Syst Evol Microbiol, 2002 Nov, 52(Pt 6), 2203 - 9
Pseudomonas thermotolerans sp . nov., a thermotolerant species of the genus Pseudomonas sensu stricto; Manaia CM et al.; A bacterium, strain CM3(T), which has an optimal growth temperature of approximately 47 degrees C and a maximal growth temperature of 55 degrees C, was isolated from a hexadecane enrichment culture, obtained from a sample of the industrial cooking water of a cork-processing plant . 16S rRNA gene sequence analysis demonstrated that this Gram-negative, aerobic, rod-shaped bacterium is related to species of the genus Pseudomonas . Phosphatidylethanolamine and phosphatidylglycerol were the major polar lipids of this isolate and the total fatty acid methyl ester profile revealed predominantly C16:1 and C18:1 at a growth temperature of 25 degrees C, whereas, at a growth temperature of 50 degrees C, C16:0 was the dominant fatty acid . This is the first report of a species of Pseudomonas sensu stricto that may be considered to be truly thermotolerant . Isolate CM3(T) (= DSM 14292(T) = LMG 21284(T)) represents the type strain of a novel species of the genus Pseudomonas, named Pseudomonas thermotolerans sp . nov.

Comput Methods Programs Biomed, 2003 Feb, 70(2), 99 - 105
Use of correspondence discriminant analysis to predict the subcellular location of bacterial proteins; Perriere G et al.; Correspondence discriminant analysis (CDA) is a multivariate statistical method derived from discriminant analysis which can be used on contingency tables . We have used CDA to separate Gram negative bacteria proteins according to their subcellular location . The high resolution of the discrimination obtained makes this method a good tool to predict subcellular location when this information is not known . The main advantage of this technique is its simplicity . Indeed, by computing two linear formulae on amino acid composition, it is possible to classify a protein into one of the three classes of subcellular location we have defined . The CDA itself can be computed with the ADE-4 software package that can be downloaded, as well as the data set used in this study, from the Pole Bio-Informatique Lyonnais (PBIL) server at http://pbil.univ-lyon1.fr.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 80 - 5
{Serine proteases of Gram-negative bacteria: structure, mechanisms of secretion, biological activity}; Bondarenko VM et al.; Current data on the characterization of bacterial serine proteases, forming the family of proteins with specific structural and functional features and secreted by type V (autotransport), are presented . The expression of many of these proteases is believed to be linked with pathogenicity of Gram negative bacteria, which necessitates their further study with a view to obtain more profound concepts . These enzymes have been shown to facilitate the bacterial colonization of the skin and mucous membranes . They are believed to be linked with the resistance of microorganisms to lysosomal proteolysis by phagocytes and their ensuing dissemination in the course of the infectious process . Serine proteases split coagulating factor V and enhance the permeability of blood vessels, thus inducing the hemorrhagic syndrome . The detailed study of serine proteases is closely linked with the prospects of the development of protease inhibiting preparations aimed at the suppression of the pathogenetic activity of proteases by their blocking or by affecting the mechanisms of their secretion.

J Gen Appl Microbiol, 1998 Feb, 44(1), 75 - 84
Isolation and phylogenetic analysis of aerobic copiotrophic ultramicrobacteria from urban soil; Iizuka T et al.; Free-living, aerobic, copiotrophic ultramicrobacteria (UMB) that passed through a 0.45 &mgr;m membrane filter and had a cell volume of less than 0.3 &mgr;m(3) were isolated from polluted urban soil by using both the direct plating method and the membrane-filter enrichment technique . The efficiency of recovering UMB from the soil was much higher in the latter method than in the former . All of the UMB isolates grew well with a doubling time of less than 6 h either in a complex nutrient medium or a chemically defined medium . The average cell volumes of the UMB isolates, as measured by scanning electron microscopy and epifluorescent microscopy with an image analysis, ranged from 0.07 to 0.22 &mgr;m(3) . The cell size was larger at the exponential phase of growth than at the stationary growth stage in general . Ultrathin-section electron microscopy of representatives of the UMB isolates showed that they had complete cell wall structures like typical Gram-negative or -positive bacteria . Phenotypic studies and phylogenetic analyses on the basis of 16S rDNA sequences showed that the UMB isolates were classified into three major groups, the beta and gamma subdivisions of the Proteobacteria and the Actinobacteria (the high G+C DNA group of Gram-positives) . However, none of these isolates were assigned to any previously known species . These results demonstrate that free-living, relatively fast-growing, copiotrophic UMB strains undescribed so far are widely distributed in terrestrial environments, including urban soil.

J Biol Chem, 2003 Mar 7, 278(10), 8678 - 85 Epub 2002 Dec 25.
The core of the tetrameric mycobacterial porin MspA is an extremely stable beta-sheet domain; Heinz C et al.; MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length . Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel beta-strands organized in a coherent domain . Heating to 92 and 112 degrees C was required to dissociate the MspA tetramer and to unfold the beta-sheet domain in the monomer, respectively . The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14 . These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming beta-barrel as a core structure of MspA . Surprisingly, the channel activity of MspA in 2% SDS and 7.6 m urea at 50 degrees C was reduced 13- and 30-fold, respectively, although the MspA tetramer and the beta-sheet domain were stable under those conditions . Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations . This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are beta-sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far . Thus, MspA may be of special interest for biotechnological applications.

J Oral Implantol, 2001, 27(4), 174 - 9
Effect of lipopolysaccharide contamination on the attachment of osteoblast-like cells to titanium and titanium alloy in vitro; Nouneh RA et al.; Failing implants with loss of alveolar bone are associated with gram-negative bacteria that carry lipopolysaccharide (LPS) in the bacterial cell wall . Bony regeneration around these implants is still an unpredictable procedure due to the many clinical factors involved . One important factor is the presence of contaminants such as LPS on the implant surface . The effect of implant-associated LPS on the attachment of bone cells to the implant surface is unknown . This project investigated the effect of LPS on the attachment of osteoblast-like cells (MC3T3-E1) to titanium and titanium alloy surfaces in vitro . We hypothesized that LPS would inhibit bone cell attachment either through loss of cellular attachment sites or alteration of cellular function . Three experimental approaches were used . First, alloy surfaces were exposed to LPS (100 microgram/mL) before the cells were allowed to attach . In the second approach, the cells were exposed to the LPS before they were allowed to attach . Last, the cells were allowed to attach before exposure to LPS . Cellular attachment to implant materials was measured by using a histochemical stain (MTT) . The results indicated that LPS presence did not significantly (P > .05) alter osteoblast attachment to titanium or titanium alloy surfaces whether the exposure occurred before or after cellular adherence . It was concluded that LPS did not directly effect the attachment of the MC3T3-E1 osteoblasts to these implant surfaces in vitro . Further research is needed to define the clinical liabilities of LPS during implant placement and maintenance.

Presse Med, 2002 Nov 30, 31(38), 1810 - 2
{Peptide-deformylase inhibitors, a new class of antibiotics}; Dubreuil L; PEPTIDE-DEFORMYLASE: During protein synthesis in bacteria, a transformylase coding the fmt gene provides a formyl group on methionine before binding to the ARNm-ARNt complex . This tormylated methionine initiates the protein synthesis . The adjunction of an amino acid to the peptide chain leads to a peptide associated with a formylated methionine . The final stage requires a metallo-enzyme, peptide deformylase, which releases the peptide and regenerates the methionin . PEPTIDE-DEFORMYLASE INHIBITORS (PDF): Often rejected by the efflux pumps of Gram negative bacteria, PDF inhibitors are administered in the form of pro-drugs, capable of acting even in the bacteria that have lost their transformylase gene . TWO PRODUCTS: These are VCR 4887 developed by Versicor and Novartis and BB 83698 developed by British Biotechnology Genesoft . They are presently in the process of clinical predevelopment . They represent an important innovation and widen the range of new antibiotic classes.

Ann Surg, 2003 Jan, 237(1), 44 - 51
Effect of prolonged hyperdynamic endotoxemia on jejunal motility in fasted and enterally fed pigs; Bruins MJ et al.; OBJECTIVE: To determine the effects of hyperdynamic endotoxemia on the motility of the small intestine . SUMMARY BACKGROUND DATA: Motility disorders of the gastrointestinal tract are a common complication of sepsis . It has been suggested that gram-negative endotoxin plays a role in the pathogenesis of the accompanying diarrhea frequently observed . METHODS: Pigs were infused with lipopolysaccharide for a 24-hour period . During this fasting period jejunal motility was measured using ambulatory manometry . One and 4 days after cessation of endotoxin, pigs were enterally fed, and again motility was recorded . RESULTS: Hyperdynamic endotoxemia was achieved in this model . Manometric pressure recordings revealed that endotoxin infusion accelerated the migrating motor complex (MMC) migration along the jejunum . Also, a simultaneous increase in MMC cycling frequency was observed in the endotoxin-treated group . Elevated MMC migration velocity and cycling frequency were maintained the following day after endotoxin during feeding and returned to basal values 4 days after endotoxin . CONCLUSIONS: A small dose of continuously infused endotoxin significantly provokes jejunal motility disturbances that may contribute to diarrhea.

Infect Immun, 2003 Jan, 71(1), 109 - 16
Generation of Helicobacter pylori ghosts by PhiX protein E-mediated inactivation and their evaluation as vaccine candidates; Panthel K et al.; Bacterial ghosts are empty cell envelopes, which may be generated by the controlled expression of the PhiX174 lysis gene E in gram-negative bacteria to obtain vaccine candidates . We describe here the application of this technology to Helicobacter pylori . The lysis gene cassette was cloned into an Escherichia coli-Helicobacter pylori shuttle vector and introduced into an H . pylori recipient strain by bacterial conjugation . Temperature induction of the lysis gene cassette revealed a quantitative killing of the H . pylori culture without induction of lysis-resistant bacteria . Biochemical and transmission electron microscopic studies identified structurally intact H . pylori . Prophylactic oral vaccination experiments using these H . pylori ghosts in the BALB/c mouse model showed a significant reduction of the bacterial load in the ghost group, as measured by a quantitative bacterial reisolation procedure . Ten of 10 and 5 of 10 mice were protected, respectively, without the use of a mucosal adjuvant . Coadministration of ghosts with cholera toxin as mucosal adjuvant resulted in a complete protection of 10 of 10 and 8 of 8 mice against H . pylori challenge, with three animals showing a sterile immunity.

Infect Immun, 2003 Jan, 71(1), 86 - 94
Role of Bordetella O antigen in respiratory tract infection; Burns VC et al.; Lipopolysaccharide (LPS), as the major surface molecule of gram-negative bacteria, interacts with the host in complex ways, both inducing and protecting against aspects of inflammatory and adaptive immunity . The membrane-distal repeated carbohydrate structure of LPS, the O antigen, can prevent antibody functions and may vary as a mechanism of immune evasion . Genes of the wbm locus are required for the assembly of O antigen on the animal pathogen Bordetella bronchiseptica and the human pathogen B . parapertussis . However, the important human pathogen B . pertussis lacks these genes and a number of in vitro and in vivo characteristics associated with O antigen in other organisms . To determine the specific functions of O antigen in these closely related Bordetella subspecies, we compared wbm deletion (Deltawbm) mutants of B . bronchiseptica and B . parapertussis in a variety of assays relevant to natural respiratory tract infection . Complement was not activated or depleted by wild-type bordetellae expressing O antigen, but both Deltawbm mutants activated complement and were highly sensitive to complement-mediated killing in vitro . Although the O-antigen structures appear to be substantially similar, the two mutants differed strikingly in their defects within the respiratory tract . The B . parapertussis Deltawbm mutant was severely defective in colonization of the tracheas and lungs of mice, while the B . bronchiseptica Deltawbm mutant showed almost no defect . While in vitro characteristics such as serum resistance may be attributable to O antigen directly, the role of O antigen during infection appears to be more complex, possibly involving factors differing among the closely related bordetellae or different interactions between each one and its host.

AIHA J (Fairfax, Va), 2002 Jul-Aug, 63(4), 430 - 8
Comparison of endotoxin assays using agricultural dusts; Reynolds SJ et al.; Endotoxins from gram-negative bacteria pose a significant respiratory hazard . Establishing dose-response relationships is problematic because there are no standard procedures for sampling and analysis . The goal of this study was to compare endotoxin analyses in six laboratories using Limulus-based assays for analysis of organic dusts from three agricultural environments: chicken barns, swine barns, and corn processing facilities . For each dust generation experiment 14 side-by-side air samples were collected on 37-mm glass fiber filters at flows of 1.8 L/min . Each laboratory was randomly allocated two filters from each of seven experiments per dust type . Three laboratories used the QCL-1000 endpoint assay, and three used the kinetic-QCL method . To eliminate variability among different lots, a single lot of Limulus amebocyte lysate for endpoint assays and one similar lot for kinetic assays was provided . Precision of assays performed within labs was very good, with pooled coefficients of variation for replicate samples ranging from 1 to 11% over all labs and all dust types . There were significant differences between laboratories for all three dust types (p < 0.01) . The pattern of differences between labs varied by dust type . For chicken dust, labs using the endpoint method reported higher results than those using kinetic methods . For swine and corn dusts, labs using the kinetic method reported the highest endotoxin values . For chicken dust, results from all labs except A and B were highly correlated (r = 0.86-1.00) . For swine dust, only labs B and E, and C and D were correlated . For corn, A, B, and D were significantly correlated with most other labs . In conclusion, statistical differences in performance between laboratories were apparent and may be related to the extraction and analytical methods . The results of this study will be useful for standardization of sampling and analysis of airborne endotoxin in agriculture.

Proteins, 2003 Feb 1, 50(2), 213 - 21
Interaction of E . coli outer-membrane protein A with sugars on the receptors of the brain microvascular endothelial cells; Datta D et al.; Esherichia coli, the most common gram-negative bacteria, can penetrate the brain microvascular endothelial cells (BMECs) during the neonatal period to cause meningitis with significant morbidity and mortality . Experimental studies have shown that outer-membrane protein A (OmpA) of E . coli plays a key role in the initial steps of the invasion process by binding to specific sugar moieties present on the glycoproteins of BMEC . These experiments also show that polymers of chitobiose (GlcNAcbeta1-4GlcNAc) block the invasion, while epitopes substituted with the L-fucosyl group do not . We used HierDock computational technique that consists of a hierarchy of coarse grain docking method with molecular dynamics (MD) to predict the binding sites and energies of interactions of GlcNAcbeta1-4GlcNAc and other sugars with OmpA . The results suggest two important binding sites for the interaction of carbohydrate epitopes of BMEC glycoproteins to OmpA . We identify one site as the binding pocket for chitobiose (GlcNAcbeta1-4GlcNAc) in OmpA, while the second region (including loops 1 and 2) may be important for recognition of specific sugars . We find that the site involving loops 1 and 2 has relative binding energies that correlate well with experimental observations . This theoretical study elucidates the interaction sites of chitobiose with OmpA and the binding site predictions made in this article are testable either by mutation studies or invasion assays . These results can be further extended in suggesting possible peptide antagonists and drug design for therapeutic strategies .

Ann N Y Acad Sci, 2002 Nov, 973, 477 - 80
Cyclooxygenase-2 expression by transcription factors in Helicobacter pylori-infected gastric epithelial cells: comparison between HP 99 and NCTC 11637; Seo JH et al.; Helicobacter pylori, a gram-negative spiral bacterium, has been associated with chronic gastritis and gastric cancer . HP 99, isolated from the Korean patients, and NCTC 11637, obtained from ATCC, have different genotypes . The present study aims to investigate whether these H . pylori strains show the discrepancy for activating transcription factors (NF-kappaB, AP-1, C/EBP) and induction of cyclooxygenase-2 (COX-2) gene in gastric epithelial AGS cells . After treatment of H . pylori to AGS cells at the ratio of 300:1, the activation of transcription factors was assessed by electrophoretic mobility shift assay . COX-2 protein was determined by Western blot analysis . The level of 6-keto-prostaglandin F(1alpha) (6-keto-PGF(1alpha)), a COX-2 product, was measured in the medium by enzyme-linked immunosorbent assay . As a result, both H . pylori strains similarly induced COX-2 expression via activation of NF-kappaB, not C/EBP, and increased the level of 6-keto-PGF(1alpha) . HP 99 showed much higher activation of AP-1 compared to NCTC 11637 . NF-kappaB might play an important role in COX-2 expression in H . pylori-induced gastric inflammation as compared to other transcription factors.

J Biol Chem, 2003 Apr 11, 278(15), 12903 - 12 Epub 2002 Dec 13.
Identification of oligomerization and drug-binding domains of the membrane fusion protein EmrA; Borges-Walmsley MI et al.; Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance . These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein . However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer . The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single alpha-helix, which contains a leucine zipper dimerization domain . Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a beta-sheet subdomain and an alpha-helical coiled-coil . We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC . The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices . The beta-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP.

J Control Release, 2002 Dec 13, 85(1-3), 17 - 25
Bacterial ghosts as vaccine candidates for veterinary applications; Jalava K et al.; The application of new strategies to develop effective vaccines is essential in modern veterinary medicine . The bacterial ghost system is a novel vaccine delivery system endowed with intrinsic adjuvant properties . Bacterial ghosts are nonliving Gram-negative bacterial cell envelopes devoid of cytoplasmic contents while maintaining their cellular morphology and native surface antigenic structures including bioadhesive properties . They are produced by PhiX174 protein E-mediated lysis of Gram-negative bacteria . The intrinsic adjuvant properties of bacterial ghost preparations enhance immune responses against envelope bound antigens, including T-cell activation and mucosal immunity . Since native and foreign antigens can be expressed in the envelope complex of ghosts before E-mediated lysis, multiple antigens of various origins can be presented to the immune system simultaneously . The advantages of bacterial ghosts include the simplicity of the production method, safety, independence from the cold chain, and versatility as a combination vaccine.

Curr Drug Targets Immune Endocr Metabol Disord, 2001 Aug, 1(2), 131 - 7
Metabolic control through L calcium channel, PKC and opioid receptors modulation by an association of naloxone and calcium salts; Minoia P et al.; beta-endorphins (beta-ends) are released from the anterior pituitary and from lymphocytes directly into inflamed tissue in response to stress and pain . At the site of inflammation and trauma, the link of beta- ends to opioid receptors hyperpolarizes nerve terminal, by blocking L-calcium gated channels, induces modifications of receptor stereoisomerism and alters the bond-energy . Opioids increase potassium and decrease calcium and sodium currents through interactions with G-protein . In some pathologies, it has been found a loss of desensitization and down regulation of opioid receptors by means of Ca++ blocking that, in turn, inhibits PKC-activation . The physiopathological mechanism dependent on the high concentration of linked opioids affects cellular level of Ca++, ATP and NADH . This biochemical reaction exerts deep influence on energetic cell status and metabolism . In gram negative bacteria, expression of mu-receptors on cell surface has been observed, with a possibility to interfere with host cell metabolism . There are many human and veterinary pathologies in which the reported mechanisms are well known: polycystic ovary syndrome, gross cystic breast disease, milk fever, ruminal tympanites, pyometra, equine colic syndrome, ovarian follicular cyst in dairy cows, calcium deficit in post-partum cows, uterine involution in cows . Also incoming pathologies such as Electro-Magnetic-Field exposure may induce alteration of calcium channel activity through the same mechanism . On clinical bases, it has been pointed out that the therapeutic administration of an association of calcium salts and naloxone controls calcium turnover, pain and functional activity of endocrine glands, via down regulation/desensitization of opioid receptors, PKC stimulation and energy restoration.

Biochemistry, 2002 Dec 17, 41(50), 14659 - 68
Structure of bacterial 3beta/17beta-hydroxysteroid dehydrogenase at 1.2 A resolution: a model for multiple steroid recognition; Benach J et al.; The enzyme 3beta/17beta-hydroxysteroid dehydrogenase (3beta/17beta-HSD) is a steroid-inducible component of the Gram-negative bacterium Comamonas testosteroni . It catalyzes the reversible reduction/dehydrogenation of the oxo/beta-hydroxy groups at positions 3 and 17 of steroid compounds, including hormones and isobile acids . Crystallographic analysis at 1.2 A resolution reveals the enzyme to have nearly identical subunits that form a tetramer with 222 symmetry . This is one of the largest oligomeric structures refined at this resolution . The subunit consists of a monomer with a single-domain structure built around a seven-stranded beta-sheet flanked by six alpha-helices . The active site contains a Ser-Tyr-Lys triad, typical for short-chain dehydrogenases/reductases (SDR) . Despite their highly diverse substrate specificities, SDR members show a close to identical folding pattern architectures and a common catalytic mechanism . In contrast to other SDR apostructures determined, the substrate binding loop is well-defined . Analysis of structure-activity relationships of catalytic cleft residues, docking analysis of substrates and inhibitors, and accessible surface analysis explains how 3beta/17beta-HSD accommodates steroid substrates of different conformations.

Analyst, 2002 Nov, 127(11), 1450 - 6
Reconstructed protein arrays from 3D HPLC/tandem mass spectrometry and 2D gels: complementary approaches to Porphyromonas gingivalis protein expression; Wang T et al.; We compare typical qualitative protein identification data from two-dimensional (2D) polyacrylamide gel electrophoresis and reconstructed protein arrays, in the context of measuring protein expression by the Gram-negative periodontal pathogen Porphyromonas gingivalis . The arrays were assembled computationally from genome annotations and tandem mass spectrometry data from an off-line HPLC fractionation combined with 2D capillary HPLC analysis of whole proteome enzymatic digests . The 2D separation was carried out with a standard binary gradient HPLC system, modified only slightly with readily available components . Compared to 2D gels, the number of annotated open reading frames identified using the 3D HPLC approach was typically larger by at least a factor of 30 . However, the newer technology is currently limited in its ability to reflect the many protein variants derived from posttranscriptional and posttranslational processing.

Ren Fail, 2002 Nov, 24(6), 687 - 90
Effects of gentamicin, lipopolysaccharide, and contrast media on immortalized proximal tubular cells; Cunha MA et al.; Aminoglycosides are widely used in the treatment of gram-negative bacterial infections . Gentamicin (GE) acts mainly in proximal tubular cells, where it is uptake via organic anion transport system and it induces a high incidence of nephrotoxicity, which is characterized by tubular necrosis {5} leading to acute renal failure in 10 to 50% of patients . Gram-negative bacteria has lipopolysaccharide (LPS) which is an endotoxin that cause renal damage . {1} Moreover, many patients are undergone exams using radiologic contrast, which is a risk factor to induce a hemodynamic change in the kidney and to develop acute renal failure . {6} Intracellular calcium {Ca2+}i is involved in renal cellular injury {7,3} and maybe mediate the effects provoked by these drugs . This study was performed to evaluate necrosis, apoptosis, and intracellular calcium levels ({Ca2+}i) in LLC-PK1 (epithelial cell line from pig kidney) induced by GE associated with LPS and a low-osmolality media, Hexabrix (HE).

Curr Top Microbiol Immunol, 2002, 270, 47 - 61
Signal transduction pathways activated by the IL-1 receptor/toll-like receptor superfamily; O'Neill LA; Toll-like receptors (TLRs) are an important point of first contact between host and microbe, and once activated generate signals which culminate in the induction of genes important for host defence . TLRs respond to different microbial products, and the signalling pathways activated are very similar to that generated by the pro-inflammatory cytokine interleukin-1 (IL-1) . This is because the Type I IL-1 receptor and TLRs are highly homologous in their cytosolic portions, possessing a Toll/IL-1 receptor (TIR) domain . Signals triggered include the important transcription factor NF-kappa B and two MAP kinases, p38 and Jun N-terminal kinase . Receptor-proximal proteins involved include the adapter MyD88, IRAK, IRAK-2, Tollip, TRAF6 and TAK-1 . These latter two proteins need to be ubiquitinated in order to be active . Differences between signals generated by TLRs are emerging, with TLR-4 signalling requiring an additional adapter termed MyD88-adapter-like (Mal), which may regulate the expression of genes specific for the response required to eliminate infection by Gram-negative bacteria . Future studies on TLR signalling may reveal hitherto unsuspected specificities in the innate immune response to infection.

Ann Otolaryngol Chir Cervicofac, 2002 Nov, 119(5), 264 - 70
{Acute Mastoiditis in Children: a series of 38 Cases}; Urwald O et al.; OBJECTIVE: We reviewed retrospectively the clinical, radiological and therapeutic findings in 38 infants and children with acute mastoiditis . MATERIAL AND METHODS: From 1988 to 2001, 38 children (mean age 36 months) were treated for acute mastoiditis and periostitis or retroauricular abscess . RESULTS: Diagnosis was usually made on the basis of typical retroauricular signs (60% of the cases), but was sometimes delayed owing to inappropriate antibiotics in 16 cases (42%) or atypical presentation in 3 (8%) . Twenty-four children (70%) had no otolaryngological history . Pneumococci were isolated in most of the cases (n=15, 57%) with 13% having penicillin-resistant pneumococci . Lateral sinus thrombophlebitis was the most frequent complication, observed in 3 children (8%) . Mastoiditis revealed congenital or acquired cholesteatoma in 4 children, who were all four infected with Gram-negative bacteria . Intravenous antibiotic therapy was associated with antroatticotomy in 36 children . CONCLUSION: Despite emergence of new antibiotic resistance, the annual rate of mastoiditis has remained unchanged, remaining a serious complication of middle ear acute otitis . Cholesteatoma should be suspected in case of Gram-negative bacteria or in older children.

J Mammary Gland Biol Neoplasia, 2002 Apr, 7(2), 109 - 21
Defense of the bovine mammary gland by polymorphonuclear neutrophil leukocytes; Paape M et al.; The primary phagocytic cells of the bovine mammary gland, polymorphonuclear neutrophil leukocytes (PMN), and macrophages, comprise the first line of defense against invading pathogens . In the normal healthy mammary gland, macrophages predominate and act as sentinels to invading mastitis-causing pathogens . Once invaders are detected, macrophages, and possibly mammary epithelial cells, release chemoattractants that direct migration of PMN into the area . In the mammary gland, protection is only effective if rapid influx of PMN from the circulation and subsequent phagocytosis and killing of bacteria occur . The second line of defense against infection consists of a network of memory cells and immunoglobulins that interact with the first line of defense . To minimize mammary tissue damage caused by bacterial toxins and oxidative products released by PMN, elimination of invading bacteria must proceed quickly . Therefore, the inflammatory response needs to be regulated . Hormones, metabolites, and acute phase proteins act to influence the outcome of mastitis, especially around parturition . The number of circulating PMN in cows during early lactation is highly heritable and closely related to susceptibility to clinical mastitis at this time . Advances in molecular biology are making available the tools, techniques, and products to study and modulate host-pathogen interactions . For example, the cloning and expression of proteins such as recombinant bovine soluble (rbos) CD (cluster of differentiation) 14 antigens, may provide ways of minimizing damaging effects of endotoxin during acute coliform mastitis . Soluble CD14 binds and neutralizes lipopolysacharide (LPS) and causes local recruitment of PMN after binding of CD14-LPS complexes to mammary epithelial cells . Development of transgenic animals that express rbosCD14 in their milk could prevent infection by Gram-negative pathogens.

Shock, 2002 Dec, 18(6), 580 - 4
A novel water-soluble vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol, protects against acute lung injury and mortality in endotoxemic rats; Ochiai J et al.; Satisfactory therapy for acute lung injury related to endotoxemia remains to be established . However, in vivo antioxidant treatment with N-acetylcysteine reportedly suppresses acute lung injury and proinflammatory cytokine production induced by endotoxin (lipopolysaccharide, LPS) . In addition, intrinsic vitamin E is protective against LPS-induced insults . We determined the effects of a novel water-soluble vitamin E derivative, 2-(alpha-D-glucopyranosyl)methyl-2,5,7,8-tetramethylchroman-6-ol (TMG), on acute lung injury and mortality induced by LPS in rats . Intravenous injection of TMG (4 or 40 mg/kg) effectively decreased mortality and prevented the increased alveolar permeability and pulmonary edema that were caused by intravenous injection of LPS (20 mg/kg) . Treatment with TMG decreased the enhanced lung expression of TNF-alpha caused by LPS . TMG also suppressed the sequestration of neutrophils in the lung induced by LPS . These results indicate that TMG is a possible therapeutic agent for acute lung injury and mortality, especially that caused by gram-negative bacteria . The therapeutic effects could be mediated at least partly through suppression of the increased expression of TNF-alpha and neutrophil sequestration in the lung that are caused by LPS.

Int J Occup Med Environ Health, 2002, 15(3), 289 - 301
Occupational exposure to organic dust associated with municipal waste collection and management; Krajewski JA et al.; The aim of the study was to assess the occupational exposure of workers employed in the municipal waste collection and management industry . Air samples were collected in the workers' breathing zone; two samples were collected parallely . The assessment concerned exposure to organic dust, endotoxins, bacteria and fungi . The questionnaire data showed that workers found themselves in good or very good health . They also regarded noise, dustiness, odor, physical effort and changeable atmospheric conditions as the most strenuous factors . The highest dust concentrations were observed on the sites of waste collection (mean, 7.7 mg/m3) and composting (mean, 4.6 mg/m3) . Samples collected on the composting site contained the largest amount of endotoxins (mean concentration, 76 ng/m3) . This was followed by sorting facility (61 ng/m3) and waste collection area (36 ng/n3) . Gram-negative rods (primarily intestinal) were found in all samples collected at workposts, which justified the adoption of the air concentrations of endotoxins as a criterion for assessing their hygienic conditions . Taking MAC for total suspended dust (4.0 mg/m3) and the concentration of 10 ng/m3 for endotoxin as the criteria for the exposure evaluation, it must be considered that waste collectors and composting site workers are working in poor hygienic conditions . Workers employed at the most hazardous workposts did not use personal protective equipment (glasses, antirespirators), thus infringing a fundamental condition for limiting health hazards (Directive 2000/54/EC).

Gene, 2002 Oct 16, 299(1-2), 251 - 61
A partial copy of msDNA from a new retron element is likely a retrotransposed DNA found in the myxobacterium Nannocystis exedens; Lampson BC et al.; Retrons are reverse transcriptase (RT) encoding genetic elements usually located on the chromosome of a wide variety of mostly Gram-negative bacteria . Here we describe a new retron, designated Ne144, found in the chromosome of the myxobacterium Nannocystis exedens . This element codes for a 515-amino-acid RT that is most closely related to those found in other myxobacterial retrons . The RT is responsible for the production of a small satellite DNA called msDNA . This msDNA is composed of a 144 base, single-stranded DNA that is linked to a 72 base single-stranded RNA . The RNA strand is joined to the 5' end of the DNA chain via a 2'-5' linkage that occurs from the 2' position of an internal guanosine residue in the RNA . In addition to the retron element, the chromosome of N . exedens also contains several partial copies of the msDNA sequence as revealed by DNA hybridization experiments using msDNA as a probe . One of these partial copies was characterized from a chromosome restriction fragment and found to contain a sequence that matches the last 82 bases of the DNA strand and five bases of the RNA strand in msDNA-Ne144 . This partial copy of msDNA is very likely a retrotransposed sequence that was generated by reverse transcription using an RNA (the primer-template RNA for msDNA) as a template and the 3' end of a nick in the chromosome as a primer, followed by incorporation into an open reading frame . The presence of this truncated copy of msDNA is strong evidence of retrotransposition in N . exedens causing an alteration in the bacterial genome.

Expert Opin Investig Drugs, 2002 Dec, 11(12), 1795 - 812
Novel therapies for sepsis: antiendotoxin therapies; Manocha S et al.; Severe sepsis and septic shock is a common problem encountered in the critical care unit with an estimated incidence in the US of 750,000 cases/year and a mortality rate of 30-50% . Sepsis involves a complex interaction between bacterial factors and the host immune system producing a systemic inflammatory state that may progress to multiple organ failure and death . Endotoxin (a lipopolysaccharide) released from Gram-negative bacteria has been implicated as a potent, prototypical stimulus of the immune response to bacterial infection . Current antiendotoxin strategies utilise various approaches ranging from the prevention of binding to endotoxin receptors with antibodies (monoclonal or polyclonal) against endotoxin or endotoxin receptor/carrier molecules (antiCD14 or antilipopolysaccharide-binding protein antibodies), enhancing clearance or neutralisation (haemoperfusion, lipoproteins, lipopolysaccharide-neutralising proteins) or impairing cellular signalling (lipid A analogues, tyrosine kinase inhibitors) . In the future, innovative therapies involving Toll-like receptors and their downstream signalling elements will be developed . This review discusses current knowledge regarding endotoxin signalling, antiendotoxin therapies currently under development, and future areas for research.

Microbiol Mol Biol Rev, 2002 Dec, 66(4), 617 - 29, table of contents
Helicobacter pylori interactions with host serum and extracellular matrix proteins: potential role in the infectious process; Dubreuil JD et al.; Helicobacter pylori, a gram-negative spiral-shaped bacterium, specifically colonizes the stomachs of humans . Once established in this harsh ecological niche, it remains there virtually for the entire life of the host . To date, numerous virulence factors responsible for gastric colonization, survival, and tissue damage have been described for this bacterium . Nevertheless, a critical feature of H . pylori is its ability to establish a long-lasting infection . In fact, although good humoral (against many bacterial proteins) and cellular responses are observed, most infected persons are unable to eradicate the infection . A large body of evidence has shown that the interaction between H . pylori and the host is very complex . In addition to the effect of virulence factors on colonization and persistence, binding of specialized bacterial proteins, known as receptins, to certain host molecules (ligands) could explain the success of H . pylori as a chronically persisting pathogen . Some of the reported interactions are of high affinity, as revealed by their calculated dissociation constant . This review examines the binding of host proteins (serum and extracellular matrix proteins) to H . pylori and considers the significance of these interactions in the infectious process . A more thorough understanding of the kinetics of these receptin interactions could provide a new approach to preventing deeper tissue invasion in H . pylori infections and could represent an alternative to antibiotic treatment.

Arch Immunol Ther Exp (Warsz), 2002, 50(5), 317 - 24
Immunotherapy in the management of sepsis; Sikora JP; This work presents the role of Gram-negative bacteria endotoxins, pro- and anti-inflammatory cytokines and reactive oxygen species (ROS) in the complex and not fully explained pathogenesis of sepsis . The so-called "respiratory burst" of neutrophils and the antioxidant mechanisms of the host are also discussed . The work focuses on possible approaches to the management of sepsis connected with immunotherapy . Neutralization of endotoxin lipopolysaccharide (LPS), anti-tumor necrosis factor alpha (TNF-alpha) therapy with monoclonal antibodies or pentoxifylline (PTXF), as well as soluble recombinant cytokine agonists and antagonists used in clinical trials are taken into consideration . In addition, cytokine manipulation therapy, anti-adhesion techniques, glucocorticoides and antioxidant barrier interference are also described . So far there has been no immunotherapy of sepsis in children of proven clinical efficacy, which prompts an aggressive examination of the immune system aimed at affecting its function.

J Clin Microbiol, 2002 Dec, 40(12), 4536 - 43
Helicobacter cetorum sp . nov., a urease-positive Helicobacter species isolated from dolphins and whales; Harper CG et al.; A novel helicobacter with the proposed name Helicobacter cetorum, sp . nov . (type strain MIT 99-5656; GenBank accession number AF 292378), was cultured from the main stomach of two wild, stranded Atlantic white-sided dolphins (Lagenorhynchus acutus) and from the feces of three captive cetaceans (a Pacific white-sided dolphin {Lagenorhynchus obliquidens}; an Atlantic bottlenose dolphin {Tursiops truncatus}; and a beluga whale {Delphinapterus leucas}) . The infected captive cetaceans were either subclinical, or clinical signs included intermittent regurgitation, inappetance, weight loss, and lethargy . Ulcers were observed in the esophagus and forestomach during endoscopic examination in two of the three captive animals . In the third animal, esophageal linear erosions were visualized endoscopically, and histopathological evaluation of the main stomach revealed multifocal lymphoplasmacytic gastritis with silver-stained spiral-shaped bacteria . Helicobacter cetorum is a fusiform gram-negative bacterium with a single bipolar flagellum . The isolates grow under microaerobic conditions at 37 and 42 degrees C but not at 25 degrees C . H . cetorum is urease, catalase, and oxidase positive, and it is sensitive to cephalothin . The isolates from the wild, stranded dolphins were sensitive to nalidixic acid, whereas the isolates from the collection animals were resistant . By 16S rRNA sequencing it was determined that H . cetorum represented a distinct taxon that clusters most closely with H . pylori . Further studies are necessary to determine the role of H . cetorum in the development of gastric ulcers and gastritis of cetaceans . This is the first description and formal naming of a novel Helicobacter species from a marine mammal.

J Biol Chem, 2003 Feb 7, 278(6), 4353 - 7 Epub 2002 Nov 25.
Endotoxin enhances liver alcohol dehydrogenase by action through upstream stimulatory factor but not by nuclear factor-kappa B; Potter JJ et al.; Liver alcohol dehydrogenase (ADH) is increased by physiological stress and by chronic administration of growth hormone (GH) . Endotoxin plays a role in the pathogenesis of alcoholic liver disease . The effect of lipopolysaccharide (LPS), the endotoxin component of Gram-negative bacteria, was determined on liver ADH . LPS given daily to rats for 3 days increased ADH mRNA, ADH protein, and ADH activity . Nuclear factor-kappaB (NF-kappaB) in the liver nuclear extracts bound to an oligonucleotide specifying region -226 to -194 of the ADH promoter, whereas upstream stimulatory factor (USF) was shown previously to bind to a more proximal site . LPS increased NF-kappaB and USF binding to the ADH promoter . The NF-kappaB (p65) and NF-kappaB (p50) expression vectors inhibited the transfected ADH promoter activity, which contrasts with the previously demonstrated stimulation by an USF expression vector . The binding activities of STAT5b and of C/EBPbeta, which mediate the effect of GH on ADH, were not changed or decreased, respectively, by LPS, indicating that GH plays no intermediary role in the effect of LPS . This study shows that LPS increases ADH and that this effect is mediated by increased binding of USF to the ADH promoter and not by NF-kappaB, which has an inhibitory action.

Int J Dermatol, 2002 Nov, 41(11), 768 - 70
The seroprevalence of Helicobacter pylori and nitric oxide in acne rosacea; Gurer MA et al.; BACKGROUND: Acne rosacea is a dermatosis with unknown etiology . Some studies have reported a high prevalence of Helicobacter pylori infection in acne rosacea . Other studies have reported a decrease in the severity of the lesions of acne rosacea after eradication of H . pylori . H . pylori is a Gram-negative bacterium which colonizes the gastric mucosa and increases the synthesis of oxygen radicals, such as superoxide and proinflammatory cytokines . These cytokines have been demonstrated to stimulate the synthesis of the inflammatory species nitric oxide (NO) . In this study, we examined the role of NO in the possible effect of H . pylori in acne rosacea . METHODS: Thirty-three acne rosacea patients were included in the study and the control group comprised 20 healthy individuals . The levels of immunoglobulin G antibodies against H . pylori in the serum samples were measured using the enzyme-linked immunoabsorbent assay method . Measurement of nitrate was performed using chemiluminescence in accordance with the method described by Braman and Hendrix (Braman RS, Hendrix SA . Nanogram nitrite and nitrate determination in environmental and biological materials by vanadium(iii) reduction with chemiluminescence detection . Anal Chem 1989; 61: 2715-2718) . For statistical analysis, the t-test was used . RESULTS: The seropositivity of H . pylori in acne rosacea patients was found to be high; however, the serum nitrate levels were found to be normal . CONCLUSIONS: The results of the study indicate that the inflammatory species NO, which has been hypothesized to be associated with H . pylori, has no role in the inflammatory mechanism of acne rosacea.

Appl Environ Microbiol, 2002 Dec, 68(12), 6292 - 9
Coexistence of multiple proteobacterial endosymbionts in the gills of the wood-boring Bivalve Lyrodus pedicellatus (Bivalvia: Teredinidae); Distel DL et al.; Wood-boring bivalves of the family Teredinidae (commonly called shipworms) are known to harbor dense populations of gram-negative bacteria within specialized cells (bacteriocytes) in their gills . These symbionts are thought to provide enzymes, e.g., cellulase and dinitrogenase, which assist the host in utilizing wood as a primary food source . A cellulolytic, dinitrogen-fixing bacterium, Teredinibacter turnerae, has been isolated from the gill tissues of numerous teredinid bivalves and has been proposed to constitute the sole or predominant symbiont of this bivalve family . Here we demonstrate that one teredinid species, Lyrodus pedicellatus, contains at least four distinct bacterial 16S rRNA types within its gill bacteriocytes, one of which is identical to that of T . turnerae . Phylogenetic analyses indicate that the three newly detected ribotypes are derived from gamma proteobacteria that are related to but distinct (>6.5% sequence divergence) from T . turnerae . In situ hybridizations with 16S rRNA-directed probes demonstrated that the pattern of occurrence of symbiont ribotypes within bacteriocytes was predictable and specific, with some bacteriocytes containing two symbiont ribotypes . However, only two of the six possible pairwise combinations of the four ribotypes were observed to cooccur within the same host cells . The results presented here are consistent with the existence of a complex multiple symbiosis in this shipworm species.

J Chromatogr B Analyt Technol Biomed Life Sci, 2002 Dec 5, 781(1-2), 419 - 32
Chromatographic removal of endotoxin from protein solutions by polymer particles; Hirayama C et al.; Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products . Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions . Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions . To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective . In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents . We also provide the design of novel endotoxin-specific polymer adsorbents.

Arch Oral Biol, 2002 Dec, 47(12), 859 - 66
Effects of lipopolysaccharide extracted from Prevotella intermedia on bone formation and on the release of osteolytic mediators by fetal mouse osteoblasts in vitro; Pelt P et al.; Prevotella intermedia, a Gram-negative obligate anaerobic black-pigmented oral bacterium, belongs to a small group of microorganisms that is closely associated with the initiation of periodontal diseases . Lipopolysaccharide (LPS), an outer membrane component, is one of the main virulence factors of this bacterium . The aim of this study was to examine the effects of Prev . intermedia lipopolysaccharide, extracted by the hot-phenol-water method, on differentiation (alkaline phosphatase activity) and mineralisation (calcium incorporation) of fetal mouse calvarial cells in vitro and to determine the release of the important osteolytic factors nitric oxide, interleukin-6 (IL-6) and matrix metalloproteinases by these cells after treatment with different concentrations of Prev . intermedia lipopolysaccharide (0.2-25 microg/ml) . By gelatin zymography, we also characterized the matrix metalloproteinases released by these osteoblasts . Treatment with Prev . intermedia lipopolysaccharide dose-dependently inhibited bone formation by reducing alkaline phosphatase activity and calcium incorporation and induced the release of nitric oxide, IL-6 and the latent proforms of MMP-2 and MMP-9 by fetal mouse osteoblasts in organoid culture . These results indicate that the lipopolysaccharide from Prev . intermedia not only participates in periodontal tissue destruction and alveolar bone resorption, but also inhibits bone formation .

Biotechniques, 2002 Nov, 33(5), 1038 - 40, 1042-3
Broad-host-range mobilizable suicide vectors for promoter trapping in gram-negative bacteria; Izallalen M et al.; Here we report the construction of three different vectors for the identification of bacterial genes induced in vitro and/or in vivo . These plasmids contain kanamycin, gentamicin, or tetracycline resistance genes as selectable markers . A promoterless cat and an improved GFP (mut3-gfp) can be used to follow the induction of gene expression by measuring chloramphenicol resistance and fluorescence, respectively.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 33 - 42
Sensing and adapting to acid stress; Boot IR et al.; Bacteria and archaea occupy a considerable diversity of niches that vary with respect to the physical conditions . Survival and colonisation requires the capacity to sense, and adapt to, environmental change . In this short review we consider the issues of adaptation to acidic conditions, in particular the mechanisms that might be employed by different bacteria to respond to the specific challenges of their niche . We lay particular emphasis on the protection of the cytoplasm during alterations of the cytoplasmic pH and, in the Gram negative bacteria, on recent work that suggests that protection of the periplasm is critical for survival of exposure to extreme acid . Finally, we discuss potential mechanisms by which pH might be sensed and consider the insights gained from proteins that sense and respond specifically to changes in pH.

Water Res, 2002 Nov, 36(19), 4745 - 52
Efficiency of chitosans applied for flocculation of different bacteria; Strand SP et al.; Three types of well-characterized chitosans of different composition were applied to flocculate 8 different bacterial species . The aim of this study was to relate chitosan structure and flocculation characteristic to general bacterial characteristics such as the cell surface charge and hydrophobicity . Large differences in the flocculation efficiency of chitosan were found between different bacterial suspensions, both regarding the effective chitosan concentrations and the optimal type of chitosan . However, no correlation was observed between general surface characteristics of bacteria and flocculation by chitosan of different composition . It may be concluded that purely electrostatic interactions did not play a dominant role in flocculation of Gram-negative bacteria in this study . The presence of GlcNAc residues had clearly beneficial effects on flocculation in such cases.

Proc Natl Acad Sci U S A, 2002 Dec 10, 99(25), 15926 - 31 Epub 2002 Nov 21.
Combining computational and experimental screening for rapid optimization of protein properties; Hayes RJ et al.; We present a combined computational and experimental method for the rapid optimization of proteins . Using beta-lactamase as a test case, we redesigned the active site region using our Protein Design Automation technology as a computational screen to search the entire sequence space . By eliminating sequences incompatible with the protein fold, Protein Design Automation rapidly reduced the number of sequences to a size amenable to experimental screening, resulting in a library of approximately equal 200,000 mutants . These were then constructed and experimentally screened to select for variants with improved resistance to the antibiotic cefotaxime . In a single round, we obtained variants exhibiting a 1,280-fold increase in resistance . To our knowledge, all of the mutations were novel, i.e., they have not been identified as beneficial by random mutagenesis or DNA shuffling or seen in any of the naturally occurring TEM beta-lactamases, the most prevalent type of Gram-negative beta-lactamases . This combined approach allows for the rapid improvement of any property that can be screened experimentally and provides a powerful broadly applicable tool for protein engineering.

Biochem Biophys Res Commun, 2002 Dec 6, 299(3), 404 - 9
Chemotaxis of a Ralstonia sp . SJ98 toward co-metabolizable nitroaromatic compounds; Pandey G et al.; We have earlier reported chemotaxis of a Gram-negative, motile Ralstonia sp . SJ98 towards p-nitrophenol (PNP), 4-nitrocatechol (NC), o-nitrobenzoate (ONB), p-nitrobenzoate (PNB), and 3-methyl-4-nitrophenol (MNP) that also served as sole source of carbon and energy to the strain {S.K . Samanta, B . Bhushan, A . Chauhan, R.K . Jain, Biochem . Biophy . Res . Commun . 269 (2000) 117; B . Bhushan, S.K . Samanta, A . Chauhan, A.K . Chakraborti, R.K . Jain, Biochem . Biophy . Res . Commun . 275 (2000) 129} . In this paper, we report chemotaxis of a Ralstonia sp . SJ98 toward seven different nitroaromatic compounds (NACs) by drop assay, swarm plate assay, and capillary assay . These NACs do not serve as sole carbon and energy source to strain SJ98 but are partially transformed in the presence of an alternate carbon source such as succinate . This is the first report showing chemotaxis of a bacterial strain toward co-metabolizable NACs.

Paediatr Perinat Epidemiol, 2002 Oct, 16(4), 342 - 9
Epidemiology of neonatal necrotising enterocolitis: a population-based study; Llanos AR et al.; We examined the birthweight-, gender- and race-specific incidence as well as the biodemographic and clinical correlates of necrotising enterocolitis (NEC) in a well-defined six-county perinatal region in upstate New York . We conducted a retrospective, 8-year population-based survey to identify all cases of proven NEC (modified Bell stage II and above) in the area's regional neonatal intensive care unit (NICU) . The denominator used to calculate the incidence was obtained from the Statewide Planning Research Cooperative System . Incidence was expressed as cases per 1000 live births . A total of 85 documented cases of proven NEC was identified in a six-county perinatal region that experienced 117 892 live births during the 8-year period . The average annual incidence was 0.72 cases per 1000 live births {95% CI 0.57, 0.87 per 1000 live births} . The highest incidence of NEC occurred among infants weighing 750-1000 g at birth and declined with increasing birthweight . The urban county had a 1.53 times higher risk of NEC than rural counties {95% CI 0.9, 2.6} . The overall incidence of NEC for non-Hispanic blacks was significantly greater than that for non- Hispanic whites (2.2 vs . 0.5 cases per 1000 live births, P = 0.00) . The differences remained statistically significant even after correction for birthweight . Most cases (93%) in this series were preterm (gestational age <37 weeks) . Only two patients were never fed before the diagnosis of NEC was confirmed . Positive blood cultures were documented in 27% of the cases with a predominance of Gram-negative enteric micro-organisms . NEC remains an important health problem especially for preterm infants and the non-Hispanic black population.

Eur J Surg, 2002, 168(4), 204 - 14
Role of the neutrophil in septic shock and the adult respiratory distress syndrome; Aldridge AJ; The adult respiratory distress syndrome (ARDS) is a serious complication of many medical and surgical conditions, most of which do not involve direct pulmonary injury . In surgical practice, septic shock has long been recognised as an important cause of ARDS and it presents many management challenges . Endotoxin released from dead and dying Gram-negative bacteria induces a generalised inflammatory response that results in multiple organ dysfunction, the lung being just one target of this injurious process . In recent years, with the discovery of several key inflammatory mediators, many aspects of this complex condition have been elucidated . The neutrophil has emerged as the central effector cell and possesses a formidable armamentarium of cytokines, enzymes, and oxygen radicals that are capable of inflicting damage to cells . In this review I examine the mechanisms underlying the recruitment and activation of neutrophils in ARDS.

Biochem Cell Biol, 2002, 80(5), 517 - 23
Molecular basis of voltage gating of OmpF porin; Robertson KM et al.; OmpF and PhoE from Escherichia coli and related homologous proteins from other Gram-negative bacteria allow the passive transport of small polar molecules across the bacterial outer membrane . In vitro, they exhibit voltage gating depending on the experimental conditions . We review current hypotheses on the underlying molecular mechanism of voltage gating of OmpF porin and show how computer simulations can be used to examine each of the proposed mechanisms.

Infect Immun, 2002 Dec, 70(12), 7165 - 8
Rough lipopolysaccharide from Brucella abortus and Escherichia coli differentially activates the same mitogen-activated protein kinase signaling pathways for tumor necrosis factor alpha in RAW 264.7 macrophage-like cells; Jarvis BW et al.; The intracellular, gram-negative pathogen Brucella abortus establishes chronic infections in host macrophages while downregulating cytokines such as tumor necrosis factor alpha (TNF-alpha) . When producing TNF-alpha, Brucella abortus rough lipopolysaccharide (LPS) activates the same mitogen-activated protein kinase signaling pathways (ERK and JNK) as Escherichia coli LPS, but Brucella LPS is a much less potent agonist.

Infect Immun, 2002 Dec, 70(12), 6741 - 50
Cloning and porin activity of the major outer membrane protein P1 from Coxiella burnetii; Varghees S et al.; Coxiella burnetii, the etiological agent of Q fever, is a gram-negative obligate intracellular bacterium . Two striking characteristics of this microorganism are its ability to thrive within a phagolysosome and its ability to persist in the environment outside a host cell . These abilities have been attributed to the existence of C . burnetii developmental cycle variants: large-cell variants (LCV), small-cell variants (SCV), and small dense cells (SDC) . Variants differ in protein profiles, including differential expression of a major outer membrane protein (MOMP) of C . burnetii, designated P1 . The approximately 29-kDa MOMP is highly expressed in LCV, down-regulated in SCV, and not apparent in SDC . We sought to characterize P1 through purification of native protein for N-terminal analysis, cloning, and functional studies . Highly purified P1, extracted from C . burnetii membranes by using the zwitterionic detergent Empigen, allowed the determination of N-terminal and internal peptide sequences . The entire P1 coding locus was cloned by PCR amplification based upon these peptide sequences, followed by inverse PCR . Comparison of the predicted P1 amino acid sequences among the C . burnetii isolates Nine Mile, Koka, Scurry, and Kerns indicated a high degree of conservation . Structural prediction suggests that the peptide has a predominantly beta-sheet conformation, consistent with bacterial porins . Typical porin characteristics were observed for native P1, including detergent solubilization properties, heat modification of purified protein, and channel formation in a planar lipid bilayer . Characterization of differentially expressed P1 as a porin increases our understanding of the function of morphological variants and their role in pathogenesis.

Infect Immun, 2002 Dec, 70(12), 6541 - 8
Induction of keratinocyte growth factor 1 Expression by lipopolysaccharide is regulated by CD-14 and toll-like receptors 2 and 4; Putnins EE et al.; Periodontal disease is a chronic inflammatory condition that is associated with increased concentrations of gram-negative pathogenic bacteria and epithelial cell proliferation . Regulation of this proliferation is poorly understood but is most likely controlled by locally expressed growth factors . Keratinocyte growth factor 1, an epithelium-specific growth factor, is expressed by gingival fibroblasts, and its expression is regulated in a concentration-dependent manner by lipopolysaccharide . In this study, induction of keratinocyte growth factor 1 protein expression was dependent on gingival fibroblast expression of membrane CD14 (mCD14) and Toll-like receptors 2 and 4 . Lipopolysaccharides from Escherichia coli and Porphyromonas gingivalis induced membrane expression of CD14 at 1, 3, and 24 h . Specifically, lipopolysaccharide induced low mCD14 expression gingival fibroblasts to express mCD14 at a level consistent with that of high mCD14 expression cells . Functional studies with specific blocking antibodies for CD14 and Toll-like receptors 2 and 4 implicated all of these molecules in signal transduction . The rapid decrease in cell membrane expression of Toll-like receptors 2 and 4 after treatment with lipopolysaccharide was consistent with receptor internalization, and blocking of either of these receptors completely inhibited keratinocyte growth factor 1 protein expression . The transcription factors AP-1 and NF-kappaB were involved in lipopolysaccharide induction of keratinocyte growth factor 1 mRNA and protein expression . These results suggest that lipopolysaccharide may induce proliferation of periodontal epithelial cells by upregulating keratinocyte growth factor 1 expression via the CD14 and Toll-like receptor signaling pathway.

Semin Dial, 2002 Nov-Dec, 15(6), 430 - 3
APD in the elderly; Kadambi P et al.; Few elderly patients with end-stage renal disease (ESRD) are treated with peritoneal dialysis (PD) . Among dialysis patients >or=65 years of age reported by the U.S . Renal Data System (USRDS), the percentage treated with chronic peritoneal dialysis (CPD) is lower than in any other age group . To evaluate the effectiveness of automated peritoneal dialysis (APD) in the elderly, we compared several outcome measures in cohorts of different ages maintained on APD . The outcome measures we examined included mortality and technique failure rates, peritonitis rates, outcomes of peritonitis, and quality of life measures . We found that while patients >or=65 years of age have a higher mortality rate than younger patients, technique failure rates, overall peritonitis rates, and most quality of life measures are not different than for younger patients . However, patients >or=65 years of age have higher gram-negative peritonitis rates and lower scores on the physical component score of the SF-36 than younger patients . The present study suggests that APD is a reasonable treatment modality for elderly patients with ESRD and that elderly patients with progressive renal failure should be considered as candidates for APD.

Ophthalmol Clin North Am, 2002 Sep, 15(3), 297 - 307
HLA-B27--associated uveitis; Smith JR; HLA-B27--associated acute anterior uveitis, a relatively common form of uveal inflammation, has a sufficiently characteristic presentation that the diagnosis is often suggested by clinical assessment, although testing for HLA-B27 status provides strong support for the diagnosis . This condition usually responds readily to topical corticosteroid therapy, and the prognosis is generally good . Atypical cases may be chronic, complicated by visually disabling complications or posterior eye involvement . Chronic or progressive forms of the disorder may require systemic immunosuppression . A diagnosis of HLA-B27--associated uveitis may bring an associated systemic condition to medical attention for the first time . Ongoing research promises to elucidate the intriguing relationship between the HLA-B27 molecule, gram-negative bacterial infection, and inflammatory disease.

Int Immunopharmacol, 2002 Oct, 2(11), 1585 - 97
The effect of soluble beta-1,3-glucan and lipopolysaccharide on cytokine production and coagulation activation in whole blood; Engstad CS et al.; Soluble beta-1,3-glucan has been demonstrated to protect against infection and shock in rats and mice, and clinical studies suggest that administration of soluble glucans to trauma/surgical patients decreases septic complications and improves survival . However, little is known about the precise mechanisms by which glucans influence the state of activation of blood cells, which are responsible for the fulminant cytokine production and the activation of the coagulation system observed in serious gram-negative infection . We studied therefore the effect of an underivatized, soluble yeast beta-1,3-glucan and lipopolysaccharide (LPS), either alone or in combination, on tumor necrosis factor-alpha (TNFalpha), interleukin-6 (IL-6), IL-8 and IL-10 secretion and monocyte tissue factor (TF) expression in human whole blood . As expected, LPS induced the secretion of substantial amounts of all measured parameters, whereas only minor amounts of TNFalpha, IL-6, and IL-10 were induced by beta-glucan itself . However, beta-glucan itself induced the production of significant amounts of IL-8 and TF . Soluble beta-1,3-glucan had a strong synergistic effect on the LPS-induced secretion of IL-8, IL-10, and on monocyte TF activity, but not on TNFalpha and 1L-6 production . On the other hand, soluble beta-glucan strongly primed LPS stimulation of all parameters, including TNFalpha and IL-6 . beta-Glucan also induced detectable neutrophil degranulation within 15 min, whereas a response to LPS was first detected after 90 min . In conclusion, soluble beta-1,3-glucan upregulated leukocyte activity, both on its own and in concert with LPS.

Structure (Camb), 2002 Nov, 10(11), 1489 - 98
Crystallographic structure of SurA, a molecular chaperone that facilitates folding of outer membrane porins; Bitto E et al.; The SurA protein facilitates correct folding of outer membrane proteins in gram-negative bacteria . The sequence of Escherichia coli SurA presents four segments, two of which are peptidyl-prolyl isomerases (PPIases); the crystal structure reveals an asymmetric dumbbell, in which the amino-terminal, carboxy-terminal, and first PPIase segments of the sequence form a core structural module, and the second PPIase segment is a satellite domain tethered approximately 30 A from this module . The core module, which is implicated in membrane protein folding, has a novel fold that includes an extended crevice . Crystal contacts show that peptides bind within the crevice, suggesting a model for chaperone activity whereby segments of polypeptide may be repetitively sequestered and released during the membrane protein-folding process.

Structure (Camb), 2002 Nov, 10(11), 1469 - 71
Periplasmic chaperones--new structural and functional insights; Behrens S; Although chaperones exist in the periplasmic compartment of Gram-negative bacterial cells, how they function is not well understood . New intriguing functional insights are provided by the solved crystal structure of the periplasmic chaperone SurA.

Microbiology, 2002 Nov, 148(Pt 11), 3671 - 80
Identification of strain-specific genes located outside the plasticity zone in nine clinical isolates of Helicobacter pylori; Chanto G et al.; Helicobacter pylori is a Gram-negative bacterium that is associated with the development of peptic ulcers and gastric carcinoma in humans . This species appears to be one of the most genetically variable bacteria described to date . The overall level of heterogeneity within strains of this organism was determined by comparing the genome sequences of two reference strains, J99 and 26695 . The aim of this study was to measure the genetic diversity within strains of H . pylori by looking for strain-specific genes in nine H . pylori strains isolated from patients suffering from chronic gastritis (n=3), duodenal ulcers (n=3) or gastric cancer (n=3) . Seven loci that contained strain-specific genes in strains J99 and 26695 were studied . These regions were subsequently amplified from most of the clinical isolates studied and their sequences were determined . ORFs were predicted from the sequence data and were compared to sequences within the databases . The results showed that the genes flanking the ORFs specific to either strain J99 or strain 26695 were also present in a similar configuration in the genomes of the nine clinical isolates . Moreover, in most regions, ORFs homologous to those found in the corresponding loci in the two reference strains were detected . However, in 10 regions, genes similar to those located at another locus in the genome of J99 or 26695 were found . Finally, six strain-specific genes were identified in three regions of three of the H . pylori strains isolated from patients with duodenal ulcers (n=2) and gastric cancer (n=1) . Of these six genes, five were putative genes and one was an orthologue of a gene encoding a transposase in Thermotoga maritima . However, no association with disease was found for these genes.

Microbiology, 2002 Nov, 148(Pt 11), 3569 - 82
Tn5041-like transposons: molecular diversity, evolutionary relationships and distribution of distinct variants in environmental bacteria; Kholodii G et al.; A detailed study on the geographic distribution, molecular diversity and evolutionary relationships of 24 closely related variants of the Tn5041 transposon found among 182 mercury resistant environmental Gram-negative strains from the IMG-Hg Reference Collection is reported here . RFLP analysis, followed by the determination of partial DNA sequences, identified 14 distinct types of these transposons, which differed from each other by 1-7 single-event DNA polymorphisms . No polymorphisms were detected at the right arm of the transposons except an insertion of a new mobile DNA element carrying a mer operon (named the mer2 cassette) within the Tn5041 mer operon . According to the model presented here, the insertion occurred via homologous recombination with a circular form of the mer2 cassette . A total of 8 point mutations, 1 internal deletion, 2 end-involving deletions, 3 mosaic regions and 2 insertions were detected at the left arm of the transposons . The insertions were a transposon closely related to Tn21 but lacking the integron and a new group II intron (named INT5041C) . Inspection of the geographic distribution of the Tn5041 variants suggested that at least three long-distance waves of dissemination of these variants had occurred, accompanied by homologous recombination between different Tn5041 lineages . Movements of circular DNAs by homologous recombination as a source of mosaic genes and new mer genes, and formation of unusual mosaics ending or beginning at the Tn5041 att site are discussed.

Curr Rheumatol Rep, 2002 Dec, 4(6), 518 - 24
The role of infection in the pathogenesis of spondyloarthropathies with special reference to human leukocyte antigen-B27; Penttinen MA et al.; Spondyloarthropathies consist of many inflammatory diseases that are closely associated with human leukocyte antigen (HLA)-B27 . One of these diseases is reactive arthritis (ReA), which is a joint inflammation that occurs after infections that are caused by certain gram-negative bacteria . The importance of these infections as causative agents of ReA has been clearly established . It is not clear, however, whether these infections contribute to the development of other forms of spondyloarthropathies . The exact mechanism by which HLA-B27 influences disease susceptibility in spondyloarthropathies remains to be determined . The role of HLA-B27 as an antigen-presenting molecule is certainly important in the pathogenesis of these diseases; however, recent data indicate that this molecule may exhibit other functions unrelated to antigen presentation, which may be important in the pathogenesis of ReA . In this paper, the authors summarize the current knowledge of the role of infection in the spondyloarthropathies.

Biochemistry, 2002 Nov 19, 41(46), 13587 - 94
Solution structure and characterization of the heme chaperone CcmE; Arnesano F et al.; The covalent attachment of the heme cofactor in c-type cytochromes is a surprisingly complex process, which in bacteria involves a number of different proteins . Among the latter, the ccmE gene product is known to perform a key role in the heme delivery pathway in Gram-negative bacteria . The solution structure of the soluble domain of apo-CcmE from Shewanella putrefaciens was determined through NMR spectroscopy on a 13C,15N-labeled sample . The structure is characterized by a compact core with large regions of beta structure, while the N-terminal and C-terminal regions are essentially unstructured . The overall folding is similar to that of the so-called oligo-binding proteins (OB fold) . Solvent-exposed aromatic residues, conserved in all CcmE homologues, have been found in the proximity of His131, the putative heme-binding residue, that could have a role in the interaction with heme . No interaction between CcmE and heme, as well as between CcmE and holocytochrome c, could be detected in vitro by electronic spectroscopy or by NMR . The data available suggest that the heme transfer process is likely to involve a heterooligomeric protein complex and occur under a tight enzymatic control.

Biochimie, 2002 May-Jun, 84(5-6), 489 - 97
Use of colicin-based genetic tools for studying bacterial protein transport; Filloux A et al.; Transport of proteins across the envelope of Gram-negative bacteria is a very challenging domain of investigation, which involves membrane-embedded proteinaceous complexes at which specific targeting occurs . These transporters (translocon or secreton) have been studied both with genetics and biochemistry . In this review we report recent developments that should help to identify novel interactions that exist within these complexes, and to decipher the signals that specifically direct transported proteins to the cognate system . These developments are exclusively based on the re-routing of colicins to these molecular machineries . The re-routing induces a lethal situation in the case of efficient or inefficient transport, depending on the system, thus creating a genetic tool for selection of mutations that correct or generate a transport default.

Biochimie, 2002 May-Jun, 84(5-6), 391 - 7
The Tol proteins of Escherichia coli and their involvement in the translocation of group A colicins; Lazzaroni JC et al.; The Tol proteins are involved in outer membrane stability of Gram-negative bacteria . The TolQRA proteins form a complex in the inner membrane while TolB and Pal interact near the outer membrane . These two complexes are transiently connected by an energy-dependent interaction between Pal and TolA . The Tol proteins have been parasitized by group A colicins for their translocation through the cell envelope . Recent advances in the structure and energetics of the Tol system, as well as the interactions between the N-terminal translocation domain of colicins and the Tol proteins are presented.

Am J Rhinol, 2002 Sep-Oct, 16(5), 249 - 53
Bacteriology of chronic maxillary sinusitis in relation to sinoscopic appearance; Jiang RS et al.; BACKGROUND: The bacteriology of chronic maxillary sinusitis in relation to different sinoscopic appearances is reported in this study . METHODS: When transantral sinoscopy via an anterior wall puncture was used to examine the maxillary antra in patients with chronic maxillary sinusitis, the sinoscopic appearances were divided into five types . Maxillary antra in which ostia were recognized under the endoscope and no secretion existed were classified as type 1 . Maxillary antra in which ostia were not recognized and no secretion existed belonged to type 2 . Maxillary antra in which seromucoid secretion existed belonged to type 3 . Maxillary antra in which mucopurulent discharge existed belonged to type 4 . Maxillary antra in which fungal balls existed belonged to type 5 . Swab specimens of these antra were sent for aerobic and anaerobic cultures . RESULTS: Between 1988 and 1998, 493 sinoscopic appearances of maxillary antra were classified . Among them, 72 were classified as type 1, 59 were type 2, 75 as type3, 227 were type 4, and 60 were type 5 . The culture rate of swab specimens from those antra with type 1 sinoscopic appearance was 56.9% . The culture rates were 59.3% for type 2, 48% for type 3, 64.8% for type 4, and 88.3% for type 5 . Common cultured bacteria were similar among antra with different types of sinoscopic appearances, except type 5 antra, but less gram-negative aerobes grew from type 1 antra . CONCLUSIONS: This study shows that the bacteriology of noninvasive fungal sinusitis was different from that of ordinary chronic maxillary sinusitis . However, among ordinary chronic maxillary sinusitis, the bacteriology was similar between maxillary antra with different sinoscopic appearances except with mild disease.

Mol Microbiol, 2002 Nov, 46(4), 1069 - 80
Overexpression of ccl1-2 can bypass the need for the putative apocytochrome chaperone CycH during the biogenesis of c-type cytochromes; Deshmukh M et al.; In Gram-negative bacteria, including Rhodobacter capsulatus, the membrane protein CycH acts as a putative apocytochrome chaperone during the biogenesis of c-type cytochromes . CycH-null mutants are unable to produce various c-type cytochromes and sustain photosynthetic (Ps) growth that requires the cytochromes c1 and c2 or cy . However, Ps+ revertants are readily obtained only on minimal, but not on enriched, medium . To obtain further information about the biogenesis of c-type cytochromes, these suppressor mutants were studied . Complementation of a CycH-null mutant for Ps+ growth by a genomic library constructed using DNA from a Ps+ suppressor yielded a plasmid carrying the ccl1-2 operon, the products of which, Ccl1 and Ccl2, are also involved in the biogenesis of c-type cytochromes . DNA sequence analysis revealed that the complementing activity resulted from a single point mutation, G488A, located upstream of the coding region of ccl1-2 . This mutation changed the -35 region of the ccl1-2 promoter from TTGGCC to TTGACC, improving its similarity to the consensus sequence of Escherichia colisigma 70-dependent promoters . That the G488A mutation indeed enhanced transcription of ccl1-2 was demonstrated by the use of reporter gene fusions . An appropriate ccl1-2::lacZ transcriptional-translational fusion carrying the G488A mutation produced in R . capsulatus over 30-fold higher beta-galactosidase activity than a wild-type construct . Immunoblot analyses confirmed that Ccl1 and Ccl2 were overproduced in the Ps+ suppressors . Deletion of either ccl1 or ccl2, from the ccl1-2 cluster carrying the G488A mutation abolished the complementing ability, indicating that overexpression of both ccl1 and ccl2 was required to confer the Ps+ phenotype on a CycH-null mutant . These findings therefore demonstrate that, during R . capsulatus growth on minimal medium, the requirement for CycH in c-type cytochrome biogenesis could be bypassed by overexpressing the ccl1-2 operon.

Food Chem Toxicol, 2002 Dec, 40(12), 1843 - 7
The effect of calcium load and the calcium channel blocker verapamil on gentamicin nephrotoxicity in rats; Ali BH et al.; Gentamicin (GM) is used against serious and life-threatening Gram negative infections . However its use is limited by the occurrence of nephrotoxicity . Reports on the interaction between GM nephrotoxicity and calcium (Ca(2+)) or Ca blockers are conflicting . Therefore, in the present work we assessed the effect of treatment of rats with graded doses of calcium carbonate, CaCO(3) (0.25, 0.5 or 1.0 g/kg) orally, or the Ca(2+) channel blocker verapamil (1.75, 3.5 or 7.0 mg/ kg) intramuscularly (i.m.), on the nephrotoxicity induced by concomitant i.m . treatment with GM (80 mg /kg/day for 6 days) . Nephrotoxicity was evaluated histopathologically by light microscopy and biochemically by measuring the concentrations of urea and creatinine in plasma, reduced glutathione (GSH), lipid peroxidation and superoxide dismutase (SOD) activity in kidney cortex . The results indicated that the administration of CaCO(3) produced a dose-dependent amelioration in the biochemical indices of nephrotoxicity in plasma and renal cortex, which was significant at the two higher doses used . The histological picture of the renal proximal tubules followed a similar pattern . Treatment with verapamil induced a dose-dependent potentiation in the biochemical parameters of nephrotoxicity that was significant only at the highest dose used (7 mg/kg) . This dose also exacerbated the GM-induced histological necrosis . The above interactions may be clinically relevant in patients treated concurrently with these agents.

Mol Immunol, 2002 Nov, 39(7-8), 453 - 8
Role of the human C8 subunits in complement-mediated bacterial killing: evidence that C8 gamma is not essential; Parker CL et al.; Human C8 is one of five complement components (C5b, C6, C7, C8 and C9) that interact to form the cytolytic membrane attack complex (MAC) on bacterial cell membranes . It is an oligomeric protein composed of a disulfide-linked C8 alpha-gamma heterodimer and a non-covalently associated C8 beta chain . Previous studies revealed that C8 alpha and C8 beta have distinct roles in the formation of the MAC on simple cells such as erythrocytes and that both subunits are essential for cell lysis . These studies also determined that C8 gamma is not required for expression of MAC hemolytic activity . To determine if these conclusions are applicable to more biologically relevant systems, the C8 subunits were examined for their ability to support complement-mediated killing of Gram-negative bacteria . Results indicate: (1) C8 alpha-gamma, C8 alpha, C8 beta and C8 gamma have no independent bactericidal activity; (2) bacterial killing requires C8 beta and either C8 alpha-gamma or C8 alpha; (3) C8 alpha is an effective substitute for C8 alpha-gamma in bacterial killing; and (4) C8 gamma enhances, but is not required for C8 bactericidal activity . Together, these data suggest that C8 alpha and C8 beta have correspondingly similar roles in MAC-mediated lysis of erythrocytes and bacterial killing . Furthermore, they provide the first direct evidence that C8 gamma is not required for complement-mediated killing of Gram-negative bacteria.

J Clin Microbiol, 2002 Nov, 40(11), 4235 - 43
Serological diagnosis of ovine enzootic abortion by enzyme-linked immunosorbent assay with a recombinant protein fragment of the polymorphic outer membrane protein POMP90 of Chlamydophila abortus; Longbottom D et al.; Ovine enzootic abortion (OEA) resulting from infection of sheep and goats with Chlamydophila abortus is of major economic importance worldwide . Over the last 50 years the serological diagnosis of infection has been based mainly on the complement fixation test (CFT), which lacks both sensitivity and specificity because of cross-reactive antibodies to other gram-negative bacteria, including another common chlamydial pathogen of sheep, Chlamydophila pecorum . In the present study, a series of overlapping recombinant antigens representing the polymorphic outer membrane protein POMP90 of C . abortus was assessed by enzyme-linked immunosorbent assay (ELISA) with a panel of 143 serum samples from sheep experimentally infected with C . abortus, from sheep clinically free of OEA, and from specific-pathogen-free lambs experimentally infected with different subtypes of C . pecorum . The results were compared to those obtained by CFT and another recently described test, an indirect ELISA (iELISA) with the recombinant OMP91B (rOMP91B) fragment (rOMP91B iELISA) (D . Longbottom, E . Psarrou, M . Livingstone, and E . Vretou, FEMS Microbiol . Lett . 195:157-161, 2001) . The rOMP90-3 and rOMP90-4 ELISAs were identified as being more sensitive and specific than CFT . Assays with both fragments were evaluated further with a panel of 294 field serum samples from flocks with documented histories of abortion, from flocks with no clinical histories of abortion but which had a high proportion of samples seropositive by CFT, and from animals with no histories of abortion but from which various C . pecorum subtypes had been isolated . ELISAs with both POMP90 fragments outperformed CFT with serum samples from C . pecorum-infected animals, producing no false-positive results . However, the ELISA with the rOMP90-4 fragment appeared to be more sensitive than the one with rOMP90-3, as it identified more of the OEA-positive samples . The ELISA with the rOMP90-4 fragment was also able to identify apparently healthy animals that were infected with an enteric strain of C . abortus in flocks that were probably infected with both enteric C . abortus and C . pecorum strains . The identification of animals infected with enteric C . abortus is extremely important in controlling the spread of OEA . Overall, the new rOMP90-4 ELISA was found to be a more sensitive and specific test than CFT for differentiating animals infected with C . abortus from those infected with C . pecorum.

Biochim Biophys Acta, 2002 Oct 11, 1565(2), 318 - 32
TonB-dependent receptors-structural perspectives; Ferguson AD et al.; Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms . In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors . A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane . The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined . These outer membrane transporters show a novel fold consisting of two domains . A 22-stranded antiparallel beta-barrel traverses the outer membrane and adjacent beta-strands are connected by extracellular loops and periplasmic turns . Located inside the beta-barrel is the plug domain, composed primarily of a mixed four-stranded beta-sheet and a series of interspersed alpha-helices . Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.

Res Microbiol, 2002 Sep, 153(7), 417 - 24
Mechanism of maltodextrin transport through LamB; Klebba PE; The Gram-negative bacterial outer membrane contains several independent, biochemically distinct transport systems for the acquisition of solutes from the environment . Three or more different classes of membrane proteins exist within the porin superfamily, that facilitate the uptake of sugars, amino acids, nucleotides, vitamins and metals . In spite of crystallographic descriptions of these protein transporters over the past decade, the mechanisms by which porins catalyze solute internalization are controversial, and in some cases still obscure . For many years the research of Maurice Hofnung endeavored to explain the transport of maltose and maltodextrins by LamB, also known as maltoporin . In the shadow of recent crystal structures, his work helped outline a different picture of outer membrane transport physiology, that is a tribute to the powerful genetic approaches Maurice pioneered . These data suggest that the principal determinant of maltodextrin recognition by maltoporin derives from the configuration of aromatic amino acids in its surface loops.

J Infect Dis, 2002 Nov 15, 186(10), 1522 - 5 Epub 2002 Oct 29.
Human toll-like receptor 4 mutations but not CD14 polymorphisms are associated with an increased risk of gram-negative infections; Agnese DM et al.; Human toll-like receptor 4 (hTLR4) and CD14 are known to be components of the lipopolysaccharide receptor complex . Our study investigated the association between TLR4 mutations (Asp299Gly and Thr399Ile) and CD14 polymorphism(s) with outcome in an intensive care unit (ICU) population at risk for sepsis . By use of a polymerase chain reaction-based restriction fragment-length polymorphism analysis technique, the hTLR4 gene was altered in 14 (18%) of 77 ICU patients (all positive for systemic inflammatory response syndrome) and in 5 (13%) of 39 volunteers . There was a significantly higher incidence of gram-negative infection among patients with the mutations (11 {79%} of 14), compared with that in the wild-type population (11 {17%} of 63; P=.004) . No association between CD14 polymorphism(s) and the incidence of infection or outcome was observed . These findings indicate that hTLR4 mutations are associated with an increased incidence of gram-negative infections in critically ill patients in a surgical setting.

J Biol Chem, 2002 Dec 27, 277(52), 50867 - 75 Epub 2002 Oct 24.
Mutations of bacterial RNA polymerase leading to resistance to microcin j25; Yuzenkova J et al.; A mutation in the conserved segment of the rpoC gene, which codes for the largest RNA polymerase (RNAP) subunit, beta', was found to make Escherichia coli cells resistant to microcin J25 (MccJ25), a bactericidal 21-amino acid peptide active against Gram-negative bacteria (Delgado, M . A., Rintoul, M . R., Farias, R . N., and Salomon, R . A . (2001) J . Bacteriol . 183, 4543-4550) . Here, we report that mutant RNAP prepared from MccJ25-resistant cells, but not the wild-type RNAP, is resistant to MccJ25 in vitro, thus establishing that RNAP is a true cellular target of MccJ25 . We also report the isolation of additional rpoC mutations that lead to MccJ25 resistance in vivo and in vitro . The new mutations affect beta' amino acids in evolutionarily conserved segments G, G', and F and are exposed into the RNAP secondary channel, a narrow opening that connects the enzyme surface with the catalytic center . We also report that previously known rpoB (RNAP beta subunit) mutations that lead to streptolydigin resistance cause resistance to MccJ25 . We hypothesize that MccJ25 inhibits transcription by binding in RNAP secondary channel and blocking substrate access to the catalytic center.

J Bacteriol, 2002 Nov, 184(22), 6260 - 9
Bacterial outer membrane ushers contain distinct targeting and assembly domains for pilus biogenesis; Thanassi DG et al.; Biogenesis of a superfamily of surface structures by gram-negative bacteria requires the chaperone/usher pathway, a terminal branch of the general secretory pathway . In this pathway a periplasmic chaperone works together with an outer membrane usher to direct substrate folding, assembly, and secretion to the cell surface . We analyzed the structure and function of the PapC usher required for P pilus biogenesis by uropathogenic Escherichia coli . Structural analysis indicated PapC folds as a beta-barrel with short extracellular loops and extensive periplasmic domains . Several periplasmic regions were localized, including two domains containing conserved cysteine pairs . Functional analysis of deletion mutants revealed that the PapC C terminus was not required for insertion of the usher into the outer membrane or for proper folding . The usher C terminus was not necessary for interaction with chaperone-subunit complexes in vitro but was required for pilus biogenesis in vivo . Interestingly, coexpression of PapC C-terminal truncation mutants with the chromosomal fim gene cluster coding for type 1 pili allowed P pilus biogenesis in vivo . These studies suggest that chaperone-subunit complexes target an N-terminal domain of the usher and that subunit assembly into pili depends on a subsequent function provided by the usher C terminus.

Mol Hum Reprod, 2002 Nov, 8(11), 1031 - 4
The CARD15 2936insC mutation and TLR4 896 A>G polymorphism in African Americans and risk of preterm premature rupture of membranes (PPROM); Ferrand PE et al.; Infection is believed to be a leading cause of preterm premature rupture of membranes (PPROM) . The bacterial cell wall component, lipopolysaccharide (LPS), is thought to initiate tissue responses leading to PPROM in the setting of Gram negative infection . LPS is recognized by the innate immune system, including the proteins encoded by the CARD15 and TLR4 genes . A recently described mutation (2936insC) in CARD15 and a polymorphism in TLR4 896 A>G impair responses to LPS . The objective of this study was to determine if African Americans, who have a higher incidence of PPROM than Caucasians, have different frequencies of the mutant CARD15 allele and the TLR4 hyporesponsive variant, and if risk of PPROM is influenced by fetal carriage of these alleles . The allele frequencies for the CARD15 mutation and the TLR4 896G variant in African Americans were similar to those reported for Caucasians . There was no association between the TLR4 alleles examined and PPROM . However, the CARD15 mutation was only detected in controls and not in PPROM cases . We conclude that the CARD15 mutation and hyporesponsive TLR4 allele do not contribute to ethnic variation in the incidence of PPROM.

Am J Respir Cell Mol Biol, 2002 Nov, 27(5), 593 - 602
Interleukin-13 mediates a fundamental pathway for airway epithelial mucus induced by CD4 T cells and interleukin-9; Whittaker L et al.; Mucus hyperproduction in asthma results from Th2-induced airway inflammation . Controversy exists about the precise mechanism of this Th2 effect . Although we showed that mucus can be induced by Th2 cells in the absence of interleukin (IL)-4, IL-5, eosinophils, and mast cells, but not without IL-4Ralpha signaling, others demonstrated that IL-4 and IL-9 can directly stimulate airway epithelial mucus . Using a system in which in vitro-generated T cell receptor transgenic Th2 cells are transferred into recipient mice and activated in the respiratory tract with inhaled antigen, we now show that CD4 Th cells can stimulate mucus only through a common, IL-13-mediated pathway . All Th cytokines depend on IL-13 for this effect and IL-13 acts, not through intermediate inflammatory cells, but on structural cells within the lung, likely the airway epithelium itself . The potency of IL-13 is shown, requiring its complete blockade for a significant reduction in mucus production . We show that mucus induction by Th2 cells does not require nuclear factor-kappaB, unlike mucins induced by gram-negative infection . These studies define in vivo pathways that lead to mucus induction and indicate that, whereas IL-13 mediates a dominant pathway for CD4 Th induced inflammation, other inflammatory stimuli activate the epithelium to produce mucus by different pathways.

Hepatology, 2002 Nov, 36(5), 1070 - 8
Terlipressin inhibits in vivo aortic iNOS expression induced by lipopolysaccharide in rats with biliary cirrhosis; Moreau R et al.; In cirrhosis, lipopolysaccharide (LPS, a product of Gram-negative bacteria) in the blood may cause septic shock . LPS-elicited induction of arterial inducible nitric oxide synthase (iNOS) results in nitric oxide (NO)-induced vasodilation, which causes arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors . In vitro studies have suggested that vasopressin inhibits iNOS expression in cultured vascular smooth muscle cells exposed to LPS . Thus, the aim of this study was to investigate the effects of terlipressin administration (a vasopressin analog) on in vivo LPS-induced aortic iNOS in rats with cirrhosis . LPS (1 mg/kg, intravenously) was administered followed by the intravenous administration of terlipressin (0.05 mg/kg, intravenously) or placebo 1 hour later . Arterial pressure was measured, and contractions to phenylephrine (an alpha(1)-adrenoceptor agonist), iNOS activity, and iNOS expressions (mRNA and protein) were investigated in isolated aortas . LPS-induced arterial hypotension and aortic hyporeactivity to phenylephrine were abolished in rats that received terlipressin . LPS-induced aortic iNOS activity and expression were suppressed in terlipressin-treated rats . In conclusion, in LPS-challenged rats with cirrhosis, terlipressin administration inhibits in vivo LPS-induced aortic iNOS expression . Terlipressin administration may be a novel approach for the treatment of arterial hypotension and hyporeactivity to alpha(1)-adrenergic constrictors in patients with cirrhosis and septic shock.

Anaesthesist, 2002 Oct, 51(10), 843 - 52
{Immunonutritive enteral feeding in the critically ill}; Steinau F et al.; Since imbalances in the immune system of the critically ill patient have been demonstrated, the role of the gastrointestinal tract for the pathogenesis of multiple organ failure has been a focus of research in intensive care medicine . Particularly, the integrity of the intestinal barrier function has been studied experimentally and clinically . The enormous number of gram-negative bacteria up to 10(11)/ml intestinal liquid inducing the release of significant amounts of endotoxin, is considered to be a vital threat to the intensive care unit (ICU) patient . Acute failure of the intestinal barrier following various types of severe shock or following parenteral nutrition inducing atrophy of intestinal mucosa may lead to multiple organ dysfunction . Maintenance of hemodynamic stability is a mainstay of therapy of the critically ill . In addition, the intestinal integrity can be preserved by the early onset of enteral nutrition . Moreover, recent concepts of enteral nutrition using immunomodulating nutrients like omega-3-fatty acids, glutamine, arginine, and nucleotides are under clinical evaluation.

Curr Opin Hematol, 2002 Nov, 9(6), 479 - 84
Cytokines, viruses, and graft-versus-host disease; Holler E; Proinflammatory cytokines released by host tissues during conditioning treatment and interferon gamma released from donor T cells play a major role in acute graft-versus-host disease (GVHD) . In the past year the interaction of cytokines has been elucidated further . Host antigen-presenting cells play a key role in the induction of allogeneic recognition . Their activity is modulated by cytokines such as flt3-ligand, viruses, and donor T cells . Expansion of donor T cells is crucial for the pathogenesis of acute GVHD . Cytokines of the T helper 1 response-IFN-gamma, IL-12, and IL-18-regulate the expansion of donor and host cells via the induction of Fas and FasL and subsequent apoptosis . However TNF-alpha, FasL, and IL-1 also cause damage to target cells . Cytokine and receptor gene polymorphism has an impact on the activity of both host and donor cell activation . Genetic factors, conditioning treatment, lipopolysaccharides (LPS) from gram-negative microorganisms, viral infections, and donor T cells determine the activity level of host antigen-presenting cells and macrophages, which have an impact on acute GVHD and other complications of allogeneic stem cell transplantation.

Proc Natl Acad Sci U S A, 2002 Oct 29, 99(22), 14446 - 51 Epub 2002 Oct 22.
Bradyoxetin, a unique chemical signal involved in symbiotic gene regulation; Loh J et al.; Bradyrhizobium japonicum is a symbiotic bacterium that nodulates soybean . Critical for the infection and establishment of this symbiosis are the bacterial nodulation genes (nod, nol, noe), which are induced in the presence of plant produced isoflavones . Transcription of the nodulation genes is also controlled in a population density-dependent fashion . Expression of the nod genes is maximal at low population densities, and decreases significantly at higher culture densities . Population density control of the nodulation genes involves NolA and NodD2, both of which function in tandem to repress nod gene expression . An extracellular secreted factor (CDF) is known to mediate this repression . Here, we report that CDF is a novel signaling molecule, designated bradyoxetin, different from other Gram-negative quorum signals . The proposed structure of bradyoxetin is 2-{4-{{4-(3-aminooxetan-2-yl)phenyl}(imino)methyl}phenyl}oxetan-3-ylamine . Interestingly, expression of bradyoxetin is iron-regulated, and is maximally produced under iron-starved conditions . Consistent with this, expression of the nodulation genes occurred in an iron-dependent fashion . Addition of iron to B . japonicum cultures at high optical densities resulted in decreased bradyoxetin production, and a concomitant reduction in nolA expression . A corresponding increase in nodY-lacZ expression was observed with iron treatment.

Lung, 2002, 180(4), 215 - 20
Lipopolysaccharide (LPS) enhancement of outward current in lung pericytes; Herskovic JJ et al.; Patch clamp methods were used to study the effect of lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, on voltage-dependent outward current of lung pericytes . Pericytes are located in capillary walls and may mediate pathological changes in microvascular hemodynamics and permeability that accompany endotoxin-mediated pulmonary edema . Previous studies have shown that LPS reduces lung pericyte contractility . Lung pericytes exhibited a voltage-dependent outward current, presumed to be K+ current, and this current increased in magnitude in response to LPS . Cells incubated for 48 hr without LPS (control) had an average peak current at 50 mV of 101 pA (n = 5 cells), whereas cells incubated with 100 mg/ml LPS had an average peak current of 927 pA (n = 9 cells, P<0.01 compared to control) . When held at 50 mV for 50 msec, net outward current decreased in control cells by 10.7% and in LPS-treated cells by 2.6% (P<0.05) . The increased activation of outward current in LPS-treated cells may be due to a previously inactive potassium channel and may mediate LPS-induced relaxation of the lung pericyte.

Am J Trop Med Hyg, 2002 Aug, 67(2), 166 - 9
Polymerase chain reaction-based diagnosis of Mediterranean spotted fever in serum and tissue samples; Leitner M et al.; A nested polymerase chain reaction (PCR) assay has been developed and used in the diagnosis of fatal and benign cases of Mediterranean spotted fever (MSF) . The test was based on specific primers derived from a Rickettsia conorii 17-kD protein gene . A positive signal was obtained from spotted fever group (SFG) and typhus group (TG) rickettsiae . Discrimination between SFG and TG rickettsiae was based on a restriction fragment length polymorphism test . Other gram-negative bacterial species tested did not generate a signal, attesting for the specificity of the assay . The SFG-specific DNA fragment was detected in four of 29 acute-phase sera from serologically confirmed patients with MSF, while acute-phase sera from 25 patients without MSF were PCR negative . Acute-phase sera samples (five of five) and tissue autopsies (six of seven) from fatal suspected cases of MSF were PCR positive . The results demonstrate that sera and tissue samples are suitable specimens for the nested PCR tests, especially in fatal cases.

Clin Pharmacol Ther, 2002 Oct, 72(4), 403 - 10
Pharmacodynamics of active site-inhibited factor VIIa in endotoxin-induced coagulation in humans; Jilma B et al.; BACKGROUND: Inhibition of the tissue factor-factor VIIa pathway attenuated the activation of coagulation and prevented death in a gram-negative bacteremia primate model of sepsis . This lethal animal model suggested that tissue factor also influences inflammatory cascades . METHODS: This trial examined the pharmacodynamic effects of active site-inhibited factor VIIa (FFR-recombinant factor VIIa {rFVIIa}; ASIS) on endotoxin-induced procoagulant, fibrinolytic, and inflammatory responses in healthy humans . A double-blind, randomized, placebo-controlled, parallel-group study was conducted in 12 healthy male volunteers . Subjects received a bolus infusion of 2-ng/kg endotoxin, followed by a bolus infusion of ASIS (400 microg/kg) or placebo 10 minutes later . RESULTS: Endotoxin injection induced inflammation, activation of coagulation, and activation and subsequent inhibition of fibrinolysis . ASIS infusion completely blocked thrombin and fibrin generation, as measured by plasma levels of prothrombin fragment (no increase in the ASIS group, as compared with a 13-fold increase in the placebo group at 4 hours; P <.01), soluble fibrin, and fibrin split product D-dimer . ASIS did not alter endotoxin-induced changes in the fibrinolytic system, cytokine levels, or markers of endothelial (E-selectin, thrombomodulin) or platelet (P-selectin) activation . CONCLUSIONS: In summary, ASIS effectively and selectively attenuates tissue factor-induced thrombin generation . Because ASIS was well tolerated, this study provides seminal data to further characterize its anticoagulant and putative anti-inflammatory effects in critically ill patients.

J Laryngol Otol, 2002 Jun, 116(6), 443 - 6
Correlation between bacteriology of the middle meatus and ethmoid sinus in chronic sinusitis; Jiang RS et al.; In this study we report on the correlation between the bacteriology of the middle meatus and ethmoid sinus in chronic sinusitis . When patients with chronic sinusitis underwent functional endoscopic sinus surgery (FESS), swab specimens were taken from the middle meati before surgery . After removing the ethmoid bullae, swab specimens were taken from the ethmoid sinuses . Between November 1998 and February 2001, 186 pairs of middle meatal and ethmoid sinus specimens were collected from 186 patients . No bacteria were cultured from either specimen in 32 patients . The same bacteria were cultured from both specimens in another 60 patients . The culture results differed between the middle meatal and ethmoid sinus specimens in the remaining 94 patients . There was a significant difference between culture rates of 63.4 per cent for middle meatal specimens and 76.3 per cent for the ethmoid sinus specimens . In analysing bacterial species, significantly more aerobic gram negative bacteria were cultured from the ethmoid sinus specimens than from the middle meatal specimens . This study shows that the bacteriology of the middle meatus was different from that of the ethmoid sinus . Therefore, the bacteriological findings in the middle meati may not reflect the real bacteriology in chronic sinusitis.

Gene, 2002 Sep 4, 297(1-2), 123 - 7
Sequence of the Escherichia coli O26 O antigen gene cluster and identification of O26 specific genes; D'Souza JM et al.; Escherichia coli associated with outbreaks of gastroenteritis and hemolytic uremic syndrome include clones with O antigens O157 and O111 . However, O26 has emerged as an O antigen present in pathogenic strains, particularly those implicated in cases of infantile gastroenteritis worldwide . The O26 O antigen gene cluster was sequenced . It was found to contain the genes expected for biosynthesis of nucleotide sugars L-rhamnose, N-acetyl-L-fucosamine and N-acetyl-glucosamine, as well genes for O unit flippase, O antigen polymerase and potential transferase genes . By polymerase chain reaction testing against representative strains for the 166 Escherichia coli O serogroups and some randomly selected Gram-negative bacteria, we identified three O antigen genes that are highly specific to O26 . This work provides the basis for a sensitive test for the rapid detection of pathogenic clones with the O26 antigen, which has implications for public health, especially in the control of food-borne outbreaks.

Am J Ophthalmol, 2002 Oct, 134(4), 613 - 4
Chronic postoperative endophthalmitis due to pseudomonas oryzihabitans; Yu EN et al.; PURPOSE: To report a case of chronic postoperative endophthalmitis caused by the gram-negative bacterium Pseudomonas oryzihabitans.DESIGN: Interventional case report.METHODS: A 77-year-old man was referred to our service for nonpainful uveitis in the right eye accompanied by increased intraocular pressure of 2 years' duration with onset 4 months after uncomplicated extracapsular cataract extraction and posterior chamber intraocular lens implantation . The uveitis partially responded to topical corticosteroid therapy but was recurrent with tapering of steroids . An anterior chamber tap and vitreous biopsies sent for cultures were negative . Owing to the persistence of inflammation despite intraocular vancomycin injection, the lens implant and capsule were removed and culture of the latter revealed the bacterium P . oryzihabitans.RESULTS: There was no recurrence of inflammation after removal of the lens implant and capsule.CONCLUSIONS: Unlike other gram-negative organisms, which are associated with poor outcomes, P . oryzihabitans may masquerade as chronic uveitis because of its low virulence.

Ren Fail, 2002 Sep, 24(5), 655 - 8
Effects of gentamicin, lipopolysaccharide, and contrast media on immortalized proximal tubular cells; Cunha MA et al.; Aminoglycosides are widely used in the treatment of gram-negative bacterial infections . Gentamicin (GE) acts mainly in proximal tubular cells, where it is uptake via organic anion transport system and it induces a high incidence of nephrotoxicity, which is characterized by tubular necrosis leading to acute renal failure in 10 to 50% of patients . Gram-negative bacteria have lipopolysaccharide (LPS) which is an endotoxin that causes renal damage . Moreover, many patients are undergone exams using radiologic contrast, which is a risk factor to induce a hemodynamic change in the kidney and to develop acute renal failure . Intracellular calcium {Ca2+}i is involved in renal cellular injury and maybe mediate the effects provoked by these drugs . This study was performed to evaluate necrosis, apoptosis and intracellular calcium levels ({Ca2+}i) in LLC-PK1 (epithelial cell line from pig kidney) induced by GE associated with LPS and a low-osmolality media, Hexabrix (HE).

Infect Immun, 2002 Nov, 70(11), 6273 - 83
Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary-phase forms; Garduno RA et al.; When Legionella pneumophila grows in HeLa cells, it alternates between a replicative form and a morphologically distinct "cyst-like" form termed MIF (mature intracellular form) . MIFs are also formed in natural amoebic hosts and to a lesser extent in macrophages, but they do not develop in vitro . Since MIFs accumulate at the end of each growth cycle, we investigated the possibility that they are in vivo equivalents of stationary-phase (SP) bacteria, which are enriched for virulence traits . By electron microscopy, MIFs appeared as short, stubby rods with an electron-dense, laminar outer membrane layer and a cytoplasm largely occupied by inclusions of poly-beta-hydroxybutyrate and laminations of internal membranes originating from the cytoplasmic membrane . These features may be responsible for the bright red appearance of MIFs by light microscopy following staining with the phenolic Gimenez stain . In contrast, SP bacteria appeared as dull red rods after Gimenez staining and displayed a typical gram-negative cell wall ultrastructure . Outer membranes from MIFs and SP bacteria were equivalent in terms of the content of the peptidoglycan-bound and disulfide bond cross-linked OmpS porin, although additional proteins, including Hsp60 (which acts as an invasin for HeLa cells), were detected only in preparations from MIFs . Proteomic analysis revealed differences between MIFs and SP forms; in particular, MIFs were enriched for an approximately 20-kDa protein, a potential marker of development . Compared with SP bacteria, MIFs were 10-fold more infectious by plaque assay, displayed increased resistance to rifampin (3- to 5-fold) and gentamicin (10- to 1,000-fold), resisted detergent-mediated lysis, and tolerated high pH . Finally, MIFs had a very low respiration rate, consistent with a decreased metabolic activity . Collectively, these results suggest that intracellular L . pneumophila differentiates into a cyst-like, environmentally resilient, highly infectious, post-SP form that is distinct from in vitro SP bacteria . Therefore, MIFs may represent the transmissible environmental forms associated with Legionnaires' disease.

Cell Calcium, 2002 Oct, 32(4), 183 - 92
Direct measurement of free Ca(2+) shows different regulation of Ca(2+) between the periplasm and the cytosol of Escherichia coli; Jones HE et al.; As in eukaryotes, bacterial free Ca(2+) can play an important role as an intracellular signal . However, because free Ca(2+) is difficult to measure in live bacteria, most of the evidence for such a role is indirect . Gram-negative bacteria also have an outer membrane separating the external fluid from the periplasm as well as the cytosol where most bacterial metabolism takes place . Here we report, for the first time, direct measurement of free Ca(2+) in the periplasmic space of living Escherichia coli . Periplasmic free Ca(2+) was measured by targeting the Ca(2+)-activated photoprotein aequorin to this compartment using the N-terminal OmpT signal sequence . Cytosolic free Ca(2+) was determined using aequorin alone . We show that, under certain conditions, the periplasm can concentrate free Ca(2+), resulting in the inner membrane being exposed to free Ca(2+) concentrations several fold higher than in the bulk external fluid . Manipulation of periplasmic membrane-derived oligosaccharides (MDOs) altered the free Ca(2+) as predicted by the Donnan potential . With micromolar concentrations of external free Ca(2+), the periplasm concentrated free Ca (2+) some three to sixfold with respect to the external medium . A Ca(2+) gradient also existed between the periplasm and the cytosol under these conditions, the periplasmic free Ca(2+) being some one to threefold higher . At millimolar levels of external free Ca(2+), a similar concentration was detected in the periplasm, but the bacteria still maintained tight control of cytosolic free Ca(2+) in the micromolar range . We propose that the highly anionic MDOs in the periplasmic space generate a Donnan potential, capable of concentrating Ca(2+) in this compartment, where it may constitute a sink for regulation of Ca(2+)-dependent processes in the cytoplasm.

Chest, 2002 Oct, 122(4), 1271 - 9
Cost-effectiveness of IV-to-oral switch therapy: azithromycin vs cefuroxime with or without erythromycin for the treatment of community-acquired pneumonia; Paladino JA et al.; STUDY OBJECTIVE: To conduct a cost-effectiveness analysis of IV-to-oral regimens of azithromycin vs cefuroxime with or without erythromycin in the treatment of patients hospitalized with community-acquired pneumonia (CAP) . PATIENTS: Of the 268 evaluable patients enrolled into a randomized, multicenter clinical trial of adults, 266 patients had sufficient data to be included in this cost-effectiveness analysis . One hundred thirty-six patients received azithromycin, and 130 patients received cefuroxime with or without erythromycin . METHODS: A pharmacoeconomic analysis from the hospital provider perspective was conducted . Health-care resource utilization was extracted from the clinical database and converted to national reference costs . Decision analysis was used to structure and characterize outcomes . Sensitivity analyses were performed, and statistics were applied to the cost-effectiveness ratios . RESULTS: The clinical success and adverse event rates and antibiotic-related length of stay were 78%, 11.8%, and 5.8 days for the azithromycin group and 75%, 20.7%, and 6.4 days for the group receiving cefuroxime with or without erythromycin, respectively . Geometric mean treatment costs were 4,104 US dollars (95% confidence interval {CI}, 3,874 to 4,334 US dollars) for the azithromycin group, and 4,578 US dollars (95% CI, 4,319 to 4,837 US dollars) for the group receiving cefuroxime with or without erythromycin (p = 0.06) . The cost-effectiveness ratios were 5,265 US dollars per expected cure for the azithromycin group, and 6,145 US dollars per expected cure for group receiving cefuroxime with or without erythromycin (p = 0.05) . CONCLUSIONS: Despite a higher per-dose purchase price, overall costs with azithromycin tended to be lower due to decreased duration of therapy, lower preparation and administration costs, and reduced hospital length of stay . As empiric therapy, azithromycin monotherapy was cost-effective compared to cefuroxime with or without erythromycin for patients hospitalized with CAP who have no underlying cardiopulmonary disease, and no risk factors for either drug-resistant pneumococci or enteric Gram-negative pathogens.

Am J Physiol Regul Integr Comp Physiol, 2002 Nov, 283(5), R1263 - 74
Effect of hemorrhagic shock on gut barrier function and expression of stress-related genes in normal and gnotobiotic mice; Yang R et al.; We sought to determine whether gut-derived microbial factors influence the hepatic or intestinal inflammatory response to hemorrhagic shock and resuscitation (HS/R) . Conventional and gnotobiotic mice contaminated with a defined microbiota without gram-negative bacteria were subjected to either a sham procedure or HS/R . Tissue samples were obtained 4 h later for assessing ileal mucosal permeability to FITC dextran and hepatic and ileal mucosal steady-state IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, and TNF mRNA levels . Whereas HS/R significantly increased ileal mucosal permeability in conventional mice, this effect was not apparent in gnotobiotic animals . HS/R markedly increased hepatic mRNA levels for several proinflammatory genes in both conventional and gnotobiotic mice . HS/R increased ileal mucosal IL-6 and COX-2 mRNA expression in conventional but not gnotobiotic mice . If gnotobiotic mice were contaminated with Escherichia coli C25, HS/R increased ileal mucosal permeability and upregulated expression of IL-6 and COX-2 . These data support the view that the hepatic inflammatory response to HS/R is largely independent of the presence of potentially pathogenic gram-negative bacteria colonizing the gut, whereas the local mucosal response to HS/R is profoundly influenced by the microbial ecology within the lumen during and shortly after the period of hemorrhage.

Biochem Biophys Res Commun, 2002 Oct 11, 297(5), 1149 - 53
Porphyromonas gingivalis lipopolysaccharide interferes with salivary mucin synthesis through inducible nitric oxide synthase activation by ERK and p38 kinase; Slomiany BL et al.; Porphyromonas gingivalis is a Gram-negative periodontopathic bacterium colonizing the oral cavity and its lipopolysaccharide (LPS) is a key factor in the development of periodontitis . We investigated the effect of P . gingivalis LPS on the cellular responses associated with mucin synthesis in sublingual salivary gland acinar cells . Exposure of the acinar cells to the LPS led to a dose-dependent decrease in mucin synthesis and was accompanied by a massive induction in inducible nitric oxide synthase (NOS-2) activity and the increase in NO production, caspase-3 activity and apoptosis . Inhibition of extracellular signal-regulated kinase (ERK) with PD98059 accelerated the LPS-induced decrease in the glycoprotein synthesis and caused further increase in apoptosis and NOS-2 activity, while the blockade of p38 mitogen-activated kinase (MAPK) with SB203580 countered the LPS-induced reduction in the glycoprotein synthesis and obviated the induced increases in NOS-2 and apoptosis . Introduction of NOS-2 inhibitor, L-NAME, not only countered the LPS-induced increase in NO generation, caspase-3 activity and apoptosis, but caused the impedance of the LPS inhibition on mucin synthesis . The findings point to the upregulation in NOS-2 expression by P . gingivalis LPS as a key detrimental culprit affecting salivary mucin synthesis.

Mini Rev Med Chem, 2002 Aug, 2(4), 343 - 51
The biochemical and physiological characteristics of surface receptors of gram negative bacteria; Letellier L et al.; This review focuses on the properties of ferric iron surface receptors of Gram negative bacteria . We discuss the different strategies to acquire iron, and the fundamental role of these receptors in pathogenicity . The structure of some of these receptors, iron transport and regulation mechanisms are presented here.

J Virol, 2002 Nov, 76(21), 10579 - 87
Transient induction of cyclin T1 during human macrophage differentiation regulates human immunodeficiency virus type 1 Tat transactivation function; Liou LY et al.; The human immunodeficiency virus type 1 (HIV-1) Tat protein is essential for viral replication and stimulates transcription of the integrated provirus by recruiting the kinase complex TAK/P-TEFb, composed of cyclin T1 (CycT1) and Cdk9, to the viral TAR RNA element . TAK/P-TEFb phosphorylates the RNA polymerase II complex and stimulates transcriptional elongation . In this report, we investigated the regulation of TAK/P-TEFb in primary human macrophages, a major target cell of HIV infection . While Cdk9 levels remained constant, CycT1 protein expression in freshly isolated monocytes was very low, increased early during macrophage differentiation, and, unexpectedly, decreased to very low levels after about 1 week in culture . The kinase activity of TAK/P-TEFb paralleled the changes in CycT1 protein expression . RNA analysis indicated that the transient induction of CycT1 protein expression involves a posttranscriptional mechanism . In transient transfection assays, the ability of Tat to transactivate the HIV long terminal repeat (LTR) in the late differentiated macrophages was greatly diminished relative to its ability to transactivate the HIV LTR in early differentiated cells, strongly suggesting that CycT1 is limiting for Tat function in late differentiated macrophages . Interestingly, lipopolysaccharide, a component of the cell wall of gram-negative bacteria, reinduced CycT1 expression late in macrophage differentiation . These results raise the possibility that regulation of CycT1 expression may be involved in establishing latent infection in macrophages and that opportunistic infection may reactivate the virus by inducing CycT1 expression.

Mol Microbiol, 2002 Oct, 46(1), 1 - 11
Chaperones of the type III secretion pathway: jacks of all trades; Page AL et al.; The type III secretion (TTS) pathway is used by many Gram-negative bacteria to inject virulence proteins into cells of their host . The activity of the TTS apparatus is controlled by external signals and, in certain conditions, production and secretion are not coupled . Storage of some proteins before secretion involves their association with specific chaperones . Three classes of TTS chaperones have been distinguished according to whether they associate with: (i) one; (ii) several effector proteins; or (iii) the two translocators that allow passage of effectors across the membrane of eukaryotic cells . These chaperones are required for stabilization of their substrate(s) and prevention of their premature interactions with other partners during storage . They also play a role in secretion of their substrate(s) . Some chaperones are also involved in transcriptional regulation of certain genes in response to the activity of secretion . The flagellar export apparatus is closely related to the TTS apparatus and some proteins of the flagellar export system have also been proposed to be chaperones that prevent premature interactions between the flagellum subunits.

Acta Anaesthesiol Scand, 2002 Oct, 46(9), 1111 - 8
Bactericidal/permeability-increasing protein inhibits endotoxin-induced vascular nitric oxide synthesis; Ciornei CD et al.; BACKGROUND: Endotoxin (lipopolysaccharide, LPS) up-regulates inducible nitric oxide synthase (iNOS) in blood vessels during septic shock . This promotes the production of nitric oxide (NO), leading to dilation of the vessels . The aim of the study was to investigate the effects of the LPS-binding endogenous antibiotic bactericidal/permeability-increasing protein (BPI) on the action of LPS on the blood vessels wall and to identify possible influence on underlying NO-related mechanisms . METHODS: Isolated segments of rat thoracic aorta and cultured primary smooth muscle cells were incubated for 5-48 h in the presence of the following combinations of compounds: (a) LPS; (b) interleukin-1beta (IL-1beta); (c) BPI; (d) BPI + LPS; (e) BPI + IL-1beta or (f) neither BPI, LPS nor IL-1beta (control) . After incubation of intact segments, we measured smooth muscle contraction in response to phenylephrine and accumulation of the NO end products nitrate and nitrite in surrounding medium . Western blot was used to assess the levels of inducible nitric oxide synthase (iNOS) in cultured cells . RESULTS: Both LPS and IL-1beta decreased contractility and increased NO production, as well as iNOS . Co-incubation with BPI attenuated all the effects of LPS but only the effects of prolonged exposure to IL-1beta in cultured cells . CONCLUSION: We conclude that BPI attenuates the LPS-induced changes in vascular reactivity by inhibiting the expression of iNOS resulting in decreased NO formation and restored responsiveness to vasoconstrictors . The data suggest that BPI can prevent circulatory disturbances during Gram-negative sepsis.

Thromb Haemost, 2002 Oct, 88(4), 627 - 31
Recombinant nematode anticoagulant protein c2, a novel inhibitor of tissue factor-factor VIIa activity, abrogates endotoxin-induced coagulation in chimpanzees; Moons AH et al.; Systemic activation of coagulation leading to disseminated intra-vascular coagulation (DIC) is an important feature in patients with severe sepsis . Tissue factor has been shown to play a primary role in this pathological response, as revealed by the use of specific inhibitors and antagonists of the tissue factor/factor VIIa pathway . This class of agents has been demonstrated to attenuate the coagulation response in human volunteers with induced low-grade endotoxemia and to reduce mortality in primate models of Gram-negative sepsis . The efficacy of these agents in attenuating the activation of coagulation and formation of microvascular thrombosis in sepsis may depend on the mechanism of inhibition . Here we demonstrate the efficacy of recombinant nematode anticoagulant protein c2 (rNAPc2) that specifically inhibits the tissue factor/factor VIIa complex by a novel mechanism, in a model of endotoxin-induced coagulation activation in chimpanzees . Administration of a low dose of Gram-negative endotoxin induced marked increases of thrombin generation as measured by plasma levels of prothrombin activation fragment F(1+2) and thrombin-antithrombin complexes, which were completely blocked by rNAPc2 . In chimpanzees receiving rNAPc2 alone, there was a significant reduction in the activation of factor X but not factor IX, compared to animals receiving placebo . In contrast to the effect of rNAPc2 on thrombin generation, there was no effect of this inhibitor on the well known enhanced systemic fibrinolytic response induced by endotoxin . In conclusion, the recombinant peptide rNAPc2 is an effective inhibitor of tissue factor-driven thrombin generation during low grade endotoxemia . These results suggest that rNAPc2 may be a promising therapeutic option to inhibit coagulation activation in patients with sepsis.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1799 - 806
Methanocalculus taiwanensis sp . nov., isolated from an estuarine environment; Lai MC et al.; Two novel hydrogenotrophic methanogens, designated strains P2F9704aT and P2F9705, were isolated from an estuary in Eriln Shi, Taiwan . The cells of strain P2F9704aT were non-motile, irregular cocci 0.9-1.4 microm in diameter . They stained gram-negative . The cells catabolized formate and H2+CO2 to produce methane, but did not utilize acetate, methanol, trimethylamine, ethanol or secondary alcohols as methanogenic substrates . The optimal growth parameters for strain P2F9704aT were pH 6.7, 37 degrees C and 0.5% NaCl . Acetate was required for cell growth even though it was not a substrate for methanogenesis . The trace element tungsten was not required but slightly stimulated the growth of strain P2F9704aT . However, tungsten extended the growth ranges relating to temperature, pH and salt . The sequences of the 16S rRNA genes of strains P2F9704aT and P2F9705 were nearly identical and possessed 99.1 and 98.5% similarity to the genes of Methanocalculus pumilus and Methanocalculus halotolerans, respectively . In addition, strain P2F9704aT possessed 14 and 12% DNA relatedness with respect to Methanocalculus pumilus and Methanocalculus halotolerans, respectively . In addition, the optimal salt concentrations, the cellular protein profiles and the molecular masses of surface-layer protein subunits of strain P2F9704aT were different from those of the other two known Methanocalculus species . On the basis of these observations, it is proposed that these two organisms should be placed in a new species, namely Methanocalculus taiwanensis . The type strain is P2F9704aT (= OCM 671T = CCRC 16182T = DSM 14663T).

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1759 - 66
Pseudoalteromonas translucida sp . nov . and Pseudoalteromonas paragorgicola sp . nov., and emended description of the genus; Ivanova EP et al.; On the basis of phenotypic and genotypic characteristics and analysis of 16S rRNA sequences, two novel species belonging to the genus Pseudoalteromonas are described . A pale-orange-pigmented strain, KMM 3548T, isolated from a sponge and a non-pigmented strain, KMM 520T, isolated from sea water are marine, gram-negative, aerobic, rod-shaped organisms . One of the strains, KMM 520T, had bipolar flagella . Both strains had the ability to degrade gelatin, DNA and Tween 80 but not chitin or agar . Strain KMM 520T decomposed elastin and grew at NaCl concentrations of 1-8%, while strain KMM 3548T grew at 1-6% NaCl . The temperature range for both strains was 4-30 degrees C . The DNA G+C contents were 46.3 (KMM 520T) and 41.1 mol% (KMM 3548T) . The level of DNA relatedness between the two strains was 20% . DNA from strain KMM 520T showed 8-34% genetic relatedness and that of KMM 3548T showed 17-53% relatedness to the DNA of other type strains of the genus Pseudoalteromonas . 16S rRNA analysis indicated a clear affiliation of these novel bacteria with the genus Pseudoalteromonas . The type strains of the novel species are Pseudoalteromonas translucida sp . nov . KMM 520T (= LMG 19696T = ATCC BAA-3157T) and Pseudoalteromonas paragorgicola sp . nov . KMM 3548T (= LMG 19694T = ATCC BAA-322T).

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1655 - 61
Erythrobacter citreus sp . nov., a yellow-pigmented bacterium that lacks bacteriochlorophyll a, isolated from the western Mediterranean Sea; Denner EB et al.; Two facultatively oligotrophic, intensely yellow-pigmented bacterial strains, RE35F/1T and RE10F/45, have been previously isolated from the western Mediterranean Sea (Bay of Calvi, Corsica, France) by 0.2 microm membrane filtration . The organisms were gram-negative, catalase- and oxidase-positive, strictly aerobic, rod-shaped and non-motile . Their respiratory lipoquinone profiles consisted exclusively of ubiquinone-10 (Q-10) and the G+C contents of their DNAs were 62.0 and 62.4 mol%, respectively . Among the cellular fatty acids, octadecenoic acid (18:1omega7c) was the major component . Both isolates also contained hydroxy fatty acids (14:0 2-OH, 18:1 2-OH and 16:0 iso 3-OH) and branched fatty acids (15:0 anteiso, 16:0 anteiso and 17:0 anteiso) . Polar lipid fingerprints were characterized by the presence of a sphingoglycolipid . Comparative analyses of their 16S rRNA gene sequences indicated that both isolates were phylogenetically closely related (sequence similarity of 99.9%) and formed a coherent cluster with aerobic bacteriochlorophyll a-containing species of the Erythrobacter/Porphyrobacter/Erythromicrobium cluster within the family Sphingomonadaceae . The closest relative was Erythrobacter litoralis DSM 8509T (97.4 and 97.5% 16S rRNA gene sequence similarity between this strain and RE35F/1T and RE10F/45, respectively) . DNA-DNA reassociation studies confirmed that strains RE35F/1T and RE10F/45 represent a single species (79.6% DNA homology), but also demonstrated that they do not belong to the species Erythrobacter litoralis (25.2 and 34.2% DNA homology, respectively) . Notably, both RE35F/1T and RE10F/45 lacked bacteriochlorophyll a . Based upon phenotypic and molecular evidence, a novel species of the genus Erythrobacter, Erythrobacter citreus sp . nov., is proposed . Strain RE35F/1T (= CIP 107092T = DSM 14432T) is the type strain.

Int J Syst Evol Microbiol, 2002 Sep, 52(Pt 5), 1485 - 96
Emendation of the genus Sphingomonas Yabuuchi et al . 1990 and junior objective synonymy of the species of three genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastomonas ursincola; Yabuuchi E et al.; The 16S rDNA sequence similarities between the type strains of Sphingomonas paucimobilis and 32 other Sphingomonas species range from 90.2 to 99.6% . It might be possible to divide the genus into several new genera according to a dendrogram drawn from 16S rDNA sequence similarity . However, the phenotypic and biochemical information needed to define clusters of strains representing distinct genera within this group of organisms was not previously available . Although the cellular lipids of type strains of all 28 Sphingomonas species tested contained glucuronosyl-(1 --> 1)-ceramide together with 2-hydroxymyristic acid, other molecular species of sphingoglycolipids were distributed randomly . Sphingomonas natatoria and Sphingomonas ursincola, bacteriochlorophyll a-containing, gram-negative facultative phototrophs, belong to the cluster of the genus Sphingomonas . Other phototrophic Porphyrobacter and Erythrobacter species in the Sphingomonadaceae were classified into a cluster different from the genus Sphingomonas, as reported previously . None of the physiological and biochemical characteristics considered, including cellular lipids and fatty acid composition, provided evidence for the division of the current genus Sphingomonas . It is therefore concluded that the genus Sphingomonas should remain undivided at this time . The species of three recently proposed genera, Sphingobium, Novosphingobium and Sphingopyxis, in conjunction with Blastobacter ursincola, are junior objective synonyms of species of the genus Sphingomonas.

Bull Exp Biol Med, 2002 Mar, 133(3), 281 - 4
Possible participation of endotoxin of gram-negative bacteria in pathogenesis of liver damage during viral hepatitis; Sozinov AS; Possible role of gram-negative bacteria in the genesis of hepatic damage during chronic viral hepatitis B and C was studied . Histological activity, hepatocyte proliferative activity, and expression of alpha-smooth muscle actin were compared with the severity of endotoxemia in patients with chronic hepatitis B and C . The study revealed a correlation between the degree of intralobular necroses, hepatocyte proliferation, and myofibroblast transdifferentiation of Ito cells with endotoxin concentration in the blood.

FEBS Lett, 2002 Oct 2, 529(1), 78 - 85
Iron transport and signaling in Escherichia coli; Braun V et al.; Bacteria solve the iron supply problem caused by the insolubility of Fe(3+) by synthesizing iron-complexing compounds, called siderophores, and by using iron sources of their hosts, such as heme and iron bound to transferrin and lactoferrin . Escherichia coli, as an example of Gram-negative bacteria, forms sophisticated Fe(3+)-siderophore and heme transport systems across the outer membrane . The crystal structures of three outer membrane transport proteins now allow insights into energy-coupled transport mechanisms . These involve large long-range structural transitions in the transport proteins in response to substrate binding, including substrate gating . Energy is provided by the proton motive force of the cytoplasmic membrane through the activity of a protein complex that is inserted in the cytoplasmic membrane and that contacts the outer membrane transporters . Certain transport proteins also function in siderophore-mediated signaling cascades that start at the cell surface and flow to the cytoplasm to initiate transcription of genes encoding proteins for transport and siderophore biosynthesis.

Syst Appl Microbiol, 2002 Aug, 25(2), 275 - 83
Characterization of Pseudoalteromonas citrea and P . nigrifaciens isolated from different ecological habitats based on REP-PCR genomic fingerprints; Ivanova EP et al.; DNA primers corresponding to conserved repetitive interspersed genomic motifs and PCR were used to show that REP, ERIC and BOX-like DNA sequences are present in marine, oxidative, gram-negative Pseudoalteromonas strains . REP, ERIC and BOX-PCR were used for rapid molecular characterization of both the type species of the genus and environmental strains isolated from samples collected in different geographical areas . PCR-generated genomic fingerprint patterns were found to be both complex and strain specific . Analysis of the genotypic structure of phenotypically diverse P . citrea revealed a geographic clustering of Far Eastern brown-pigmented, agar-digesting strains of this species . Marine isolates of P . nigrifaciens with 67-70% DNA relatedness generated genomic patterns different from those of the type strain and formed a separate cluster . It is concluded that REP, ERIC and BOX-PCR are effective in generating strain specific patterns that can be used to elucidate geographic distribution, with these genomic patterns providing a valuable biogeographic criterion.

Syst Appl Microbiol, 2002 Aug, 25(2), 194 - 7
Dysgonomonas mossii sp . nov., from human sources; Lawson PA et al.; Phenotypic and phylogenetic studies were performed on seven unidentified gram-negative, facultatively anaerobic, coccobacillus-shaped organisms isolated from human clinical specimens . Comparative 16S rRNA gene sequencing demonstrated that four of the strains corresponded to Dysgonomonas capnocytophagoides whereas the remaining three isolates represent a new sub-line within the genus Dysgonomonas, displaying greater than 5% sequence divergence with Dysgonomonas capnocytophagoides and Dysgonomonas gadei . The three novel isolates were readily distinguished from D.capnocytophagoides and D . gadei by biochemical tests . The DNA base composition of the novel species was consistent with its assignment to the genus Dysgonomonas . Based on phylogenetic and phenotypic evidence it is proposed that the unknown species, be classified as Dysgonomonas mossii sp . nov . The type strain of Dysgonomonas mossii is CCUG 43457T (= CIP 107079T).

Int Immunopharmacol, 2002 Jul, 2(8), 1173 - 81
Inhibition of TNF-alpha, IL-1beta, and IL-6 productions and NF-kappa B activation in lipopolysaccharide-activated RAW 264.7 macrophages by catalposide, an iridoid glycoside isolated from Catalpa ovata G . Don (Bignoniaceae); An SJ et al.; Catalposide, the major iridoid glycoside isolated from the stem bark of Catalpa ovata G . Don (Bignoniaceae), was found to inhibit the productions of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), and the activation of nuclear factor kappaB (NF-kappaB) in RAW 264.7 macrophages activated with lipopolysaccharide (LPS) . Catalposide also inhibited the expressions of TNF-alpha, IL-1beta, and IL-6 genes and the nuclear translocation of p65 subunit of NF-kappaB in LPS-activated RAW 264.7 cells . Flow cytometric analysis revealed that catalposide suppressed the binding of FITC-conjugated LPS to CD14 on the surface of cells, probably resulting in the inhibitory effects on TNF-alpha, IL-1beta, and IL-6 productions and NF-kappaB activation . These findings suggest that catalposide could be an attractive candidate for adjunctive therapy in gram-negative bacterial infections.

AIDS Asia, 1996 May-Aug, 3(3-4), 21 - 3
The prevalence of HIV infection in children with extensive tuberculosis and chronic diarrhoea; Merchant RH et al.; PIP: A prospective study was conducted to determine the seroprevalence of HIV in children with extensive tuberculosis (TB) and with chronic diarrhea, and the most likely mode of infection in the seropositive population . 100 pediatric patients, 50 with extensive TB and 50 with chronic diarrhea, were tested for HIV seropositivity . Nine of the children with extensive TB and 12 of the children with chronic diarrhea were HIV positive . The largest number of children with extensive TB were 1-3 years old, while 50% of the children with chronic diarrhea were less than 1 year old . 94% of HIV transmission appeared to be perinatal, while 6% could have acquired HIV through the transfusion of unscreened blood . AIDS encephalopathy was confirmed in the two cases with central nervous system infection . Among the children with chronic diarrhea, there were eight cases of gram negative bacteria infection, 10 cases of fungal infection, four cases of giardia, and three cases of cryptosporidium . During the 2-year follow-up period, three children with extensive TB and three with chronic diarrhea died .

Toxicology, 2002 Oct 30, 180(1), 45 - 63
Fighting the stranger-antioxidant protection against endotoxin toxicity; Cadenas S et al.; Septic shock is a serious problem in critically ill and surgical patients throughout the world . It is a systemic inflammatory response caused by excessive secretion of proinflammatory mediators, such as tumor necrosis factor-alpha, mainly induced by endotoxin, a major component of the Gram-negative bacterial outer membrane . Experimental evidence suggests that reactive oxygen species (ROS) may be important mediators of cellular injury during endotoxemia, either as a result of macromolecular damage or by interfering with extracellular and intracellular regulatory processes . In addition, nitric oxide is thought to play a key role in the pathogenesis of sepsis . This review begins with a brief overview of the toxic effects of endotoxin at organism level, paying particular attention to cardiovascular damage . It continues by analysing the mechanism by which endotoxin is recognized by specific cells of the immune system, which then respond to bacterial infection and the pathway leading to nuclear factor-kappaB activation and proinflammatory gene transcription . With regard to this process, the review focuses on the involvement of reactive oxygen and nitrogen species . Lastly, the protective role of antioxidants against endotoxin toxicity and their potential clinical use is discussed.

Respir Res . 2002;3(1):23 . Epub 2002 Sep 05.
Activation of macrophage nuclear factor-kappaB and induction of inducible nitric oxide synthase by LPS; Li YH et al.; BACKGROUND: Chronic lung disease (CLD) of prematurity is a major problem of neonatal care . Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease . In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-kappaB (NF-kappaB) in vitro . In addition, we investigated the impact of dexamethasone and budesonide on these processes . METHODS: Griess reaction was used to measure the nitrite level . Western blot and a semi-quantitative RT-PCR were performed to detect iNOS expression . Electrophoretic mobility shift assay (EMSA) was performed to analyze the activation of NF-kappaB . RESULTS: We found that LPS stimulated the rat alveolar macrophages to produce NO in a dose (>or=10 ng/ml) and time dependent manner (p < 0.05) . This effect was further enhanced by IFN-gamma (>or=10 IU/ml, p < 0.05), but was attenuated by budesonide (10(-4)-10(-10) M) and dexamethasone (10(-4)-10(-6) M) (p < 0.05) . The mRNA and protein levels of iNOS were also induced in response to LPS and attenuated by steroids . LPS triggered NF-kappaB activation, a mechanism responsible for the iNOS expression . CONCLUSION: Our findings imply that Gram-negative bacterial infection and the inflammatory responses are important factors in the development of CLD . The down-regulatory effect of steroids on iNOS expression and NO production might explain the beneficial effect of steroids in neonates with CLD.

Rev Biol Trop, 2002 Mar, 50(1), 45 - 8
{Bacterial present in the xylem of coffee (Rubiaceae: coffea arabica) with "Crespera" disease}; Vargas Cartagena L et al.; "Crespera" is an infectious disease of coffee plants that affects both the coffee production and the economy of the coffee producer countries . This disease affects morphologically the plant: long and narrow leaves with wavy borders and marginal necrosis; strong chlorosis results in drying of the leave, and leads to bad conditions of the plant . The internodes are short, producing the appearance of multiple sprouts in the axial sprout, the flowers can turn greenish, and the plant can present branches with severe symptoms, and branches without apparent symptoms at the same time . As a result, the coffee bean production decreases strikingly . The aim of this work was to determine the occurrence of the possible causative agent in the coffee plants using transmission electron microscopy . Normal and infected plants were compared looking at the leaves, central vein, lateral veins and petiole . It was determined that xylematic vessels show the presence of gram negative bacilliform bacteria (of thick-wavy walls), with dimensions of 0.3-0.5 micron diameter and 1-4 microns, length . The control plants did not show bacteria in the xylem.

FEBS Lett, 2002 Sep 25, 528(1-3), 53 - 7
Orientation and interactions of dipolar molecules during transport through OmpF porin; Robertson KM et al.; The outer membrane of Gram-negative bacteria contains porins, large sieve-like proteins allowing small molecules to diffuse in and out of the periplasm . We have simulated transport of the dipolar molecules alanine and methylglucose through the OmpF porin from Escherichia coli using non-equilibrium steered molecular dynamics simulations in a realistic bilayer environment . Structural perturbation of the protein is minimal . During the permeation process, both alanine and methylglucose align strongly in the electric field in the eyelet region, where the adhesion force on the permeating molecule has a maximum . Binding of the permeating dipolar molecules in the eyelet region is not observed.

Nutrition, 2002 Sep, 18(9), 751 - 4
Effects of three intravenous lipid emulsions on the survival and mononuclear phagocyte function of septic rats; Garnacho-Montero J et al.; The immunosuppressive effects of intravenous lipid emulsions are a matter of great concern and debate . In a rat model of gram-negative bacteremia, we assessed whether the use of three intravenous lipid emulsions with different triacylglycerol compositions could influence mortality, bacterial clearance, and prostaglandin E(2) (PGE(2)) levels and compared these groups with groups of orally fed rats and rats that received a small amount of calories in form of glucose without enteral feeding (starvation).RATS WERE ASSIGNED TO ONE OF FIVE GROUPS: group 1 (control, n = 15) received rodent chow ad libitum and saline infusion; group 2 (starvation group, n = 12) had no access to chow and received an infusion of 5% glucose; group 3 (n = 17) received total parenteral nutrition (TPN) with long-chain triacylglycerols; group 4 (n = 12) received TPN with medium- and long-chain triacylglycerols; and group 5 (n = 15) received TPN with its emulsion based on olive oil . Animals received isonitrogenous and isocaloric TPN . After 2 d of TPN, a dose of 10(8) colony-forming units of Escherichia coli was introduced via the venous catheter; 2 d later the animals were killed . Blood, spleen, liver, and lungs were cultured . Circulating levels of PGE(2) were measured.Bacterial growth in the liver and lungs were significantly higher in groups 3 and 4 than in group 1, with no differences among the other groups . Rates of bacteremia were significantly higher in groups 3 and 4 than in group 1, with no differences among the other groups . Plasma levels of PGE(2) did not differ, and mortality was unaffected.Bacterial clearance clearly was preserved in orally fed, control rats when compared with rats on TPN with long-chain triacylglycerols or medium- plus long-chain triacylglycerols . However, the use of a lipid emulsion enriched intravenously with oleic acid was a valid way of reducing this disturbance, although plasma levels of PGE(2) and survival were not modified.

Microbes Infect, 2002 Jul, 4(8), 837 - 51
Structure and function of lipopolysaccharides; Erridge C et al.; The lipopolysaccharides of Gram-negative bacteria have a profound effect on the mammalian immune system and are of great significance in the pathophysiology of many disease processes . Consideration is given in this review to the relationship between structure and function of these lipopolysaccharides.

Mar Pollut Bull, 2002 Aug, 44(8), 726 - 32
Mercury and lead tolerance in hypersaline sulfate-reducing bacteria; Harithsa S et al.; Sulfate-reducing bacteria (SRB) HSR1, HSR4, and HSR14 isolated from the salt pans of Goa grew best at 90-100/1000 salinity on substrates like formate, acetate, lactate, butyrate, ethanol and benzoate . They were gram negative, non-sporulating, non-motile rods lacking in desulfoviridin and cytochromes . Examination of these isolates for heavy metal tolerance and response studies in terms of growth and sulfate-reducing activity (SRA) were carried out using HgCl2 and Pb(NO3)2 at final concentration of 50, 100, and 200 and 100, 200 and 500 microg ml(-1) respectively . With Hg, HSR1 showed approximately 80% of the control's growth at 100 and 200 microg ml(-1) but SRA reached only 60% of the control values at the end of 14 days . HSR14 could reach >100% of the control's growth at 200 microg ml(-1) but the SRA reached only up to 60% of the control without metal at 100 microg ml(-1) . Though the concentration of Pb was double that of Hg, HSR4 could grow and respire better than the control, the growth being stimulated by 160% and respiration by 170% in the presence of 500 microg ml(-1) of Pb(NO3)2 . It is probable that some hypersaline SRB are more tolerant to heavy metals than the mesohaline counterparts and could be more effectively used for precipitating these metals in bioremediatory measures . Further examination of their responses to varied concentration of metals under different salinities would indicate their range of applicability.

Mol Cells, 2002 Aug 31, 14(1), 35 - 42
TAK1 mediates lipopolysaccharide-induced RANTES promoter activation in BV-2 microglial cells; Jang SB et al.; Lipopolysaccharide (LPS), a part of the outer membrane of gram-negative bacteria activates the expression of the regulated upon activation, normal T cell expressed and secreted (RANTES), which plays an important role in the chemo-attraction of leukocytes during the inflammatory response . Recently, we found that LPS-induced RANTES production is mediated by the activation of NF-kappaB, but, the upstream regulatory mechanism involved in mediating this NF-kappaB activation was unclear . In this study, we investigated signal transducing molecules that mediate LPS-induced RANTES promoter activation and found the followings . First, LPS activates the RANTES gene promoter through NF-kappaB binding sites . Second, the expression of dominant negative mutants of TGF-beta-activated kinasel (TAK1) and NF-kappaB-inducing kinase (NIK), blocked the LPS-induced transcriptional activation of RANTES promoter . Moreover, the overexpression of TAK1 along with TAK1-binding protein 1 (TAB1), or NIK stimulated the transcriptional activation of RANTES in the absence of external stimuli . Third, we showed that endogenous TAK1 is phosphorylated by LPS stimulation, and that the association between TAK1 and tumour necrosis factor receptor-associated factor 6 (TRAF6) is constitutive and not induced by LPS treatment . These results indicate that NF-kappaB mediates LPS-triggered RANTES induction and that TAK1 as well as NIK, as NF-kappaB activators participates in LPS-triggered RANTES induction.

J Pathol, 2002 Oct, 198(2), 270 - 5
Heterogeneous expression of cell adhesion molecules by endothelial cells in ARDS; Muller AM et al.; ARDS (acute respiratory distress syndrome) can be associated with septic shock and multiple organ failure caused by an uncontrolled systemic inflammatory response to Gram-negative bacterial infection . While in animal models the key role of the endothelial adhesion molecules ICAM-1, E-selectin, and VCAM in ARDS has been extensively studied, there are scarcely any corresponding pathomorphological studies of human lung tissue . Hence, little is known about whether there is a comparable, or even heterogeneous, expression pattern of these molecules in the human pulmonary vasculature . This study was therefore undertaken to investigate the immunohistochemical expression of the constitutively expressed PECAM (CD31) and the inducible molecules ICAM-1, E-selectin, and VCAM in ARDS lungs from patients who had died in septic shock induced by Gram-negative bacteria . While in all specimens (ARDS and normal lungs) there was homogeneous strong expression of PECAM in all vessels, ICAM-1 was clearly up-regulated in ARDS lungs . E-selectin and VCAM were not expressed by endothelial cells (ECs) in normal lungs, but in ARDS lungs there was strong expression of both molecules in larger vessels, while in the capillaries there was only mosaic-like weak expression of a few ECs . This immunohistochemical investigation demonstrates the induction and up-regulation of adhesion molecules in human ARDS lungs, comparable to that described in animal models . There is also markedly heterogeneous expression of E-selectin and VCAM, indicating toporegional differences in the function of pulmonary ECs .

Plant Cell, 1997 Aug, 9(8), 1425 - 1433
Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves; Ji C et al.; Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D . The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene . A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained . The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating . A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s) . Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity . However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves . Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene . Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

Infection, 2002 Aug, 30(4), 240 - 2
Eikenella corrodens brain abscess after repeated periodontal manipulations cured with imipenem and neurosurgery; Asensi V et al.; Eikenella corrodens is a facultatively anaerobic gram-negative rod that colonizes the oral cavity and very rarely produces central nervous system (CNS) infections . Frontal lobe abscesses are occasionally associated with a dental source of infection . We report a case of an adult man with overzealous dental cleaning habits who developed a right frontal brain abscess caused by E . corrodens . He underwent neurosurgical drainage of the pus and was successfully treated with imipenem 4 g/i.v./day for 4 weeks with no complications . Repeated periodontal trauma could explain the Eikenella brain abscess in this case.

Am J Physiol Heart Circ Physiol, 2002 Oct, 283(4), H1645 - 55 Epub 2002 Jun 13.
TLR4 inactivation and rBPI(21) block burn-induced myocardial contractile dysfunction; Thomas JA et al.; Both large burns and severe gram-negative sepsis are associated with acute myocardial contractile dysfunction . Because others have reported that burn injury may be followed by transient endotoxemia, we hypothesized that bacterial endotoxin induces contractile impairment after burn trauma . We tested this hypothesis in two rodent models . In each model, postburn myocardial contractility was assessed using Langendorff preparations of excised hearts . In the first model, mice expressing either a mutant form of or no Toll-like receptor 4 (TLR4), a critical element of the mammalian endotoxin receptor, were resistant to postburn myocardial contractile dysfunction . In the second model, starting 30 min or 4 h after burn injury, rats were infused with recombinant bactericidal/permeability-increasing protein (rBPI(21)), a protein that binds and neutralizes endotoxin . Hearts from rBPI(21)-treated animals were completely protected from postburn contractile impairment . Because burn-induced contractile dysfunction can be prevented either by blocking signaling through the endotoxin receptor or by neutralizing circulating LPS, bacterial endotoxin may contribute to impaired myocardial contractility after burn injury.

Onderstepoort J Vet Res, 2002 Jun, 69(2), 123 - 7
An overview of the eradication of Brucella melitensis from KwaZulu-Natal; Emslie FR et al.; Brucella melitensis is a Gram-negative bacterium whose primary hosts are goats and sheep . Like the other BrucelIa spp., with the exception of Brucella ovis, it is not particularly host specific as it is pathogenic for a variety of other mammal species including humans . In humans the disease caused by it is rated as one of the most important zoonoses . Three outbreaks have been recorded in goats and sheep in South Africa; the first outbreak occurred in sheep in 1965 in the Mpumalanga and Northern Provinces (then both part of the Transvaal Province), the second occurred in sheep in 1989 near Pretoria, Gauteng Province, and the third and current outbreak was diagnosed in a flock of goats in northern KwaZulu-Natal in September 1994 . Following the initial diagnosis of B . melitensis in north-eastern KwaZulu-Natal, a serological survey was conducted in order to identify foci of infection in the goat and sheep populations . Six positive foci were identified . In March 1996 a test-and-slaughter eradication campaign was initiated in these areas . Initial test results revealed a prevalence of between 1.23% and 4.02 % . All positive animals were identified and slaughtered . Eradication programmes were repeated between March 1996 and June 2000, in the populations at risk, and the disease prevalence was reduced in all the affected populations.

Infect Immun, 2002 Oct, 70(10), 5390 - 403
BhuR, a virulence-associated outer membrane protein of Bordetella avium, is required for the acquisition of iron from heme and hemoproteins; Murphy ER et al.; Iron (Fe) is an essential element for most organisms which must be obtained from the local environment . In the case of pathogenic bacteria, this fundamental element must be acquired from the fluids and tissues of the infected host . A variety of systems have evolved in bacteria for efficient acquisition of host-bound Fe . The gram-negative bacterium Bordetella avium, upon colonization of the avian upper respiratory tract, produces a disease in birds that has striking similarity to whooping cough, a disease caused by the obligate human pathogen Bordetella pertussis . We describe a B . avium Fe utilization locus comprised of bhuR and six accessory genes (rhuIR and bhuSTUV) . Genetic manipulations of B . avium confirmed that bhuR, which encodes a putative outer membrane heme receptor, mediates efficient acquisition of Fe from hemin and hemoproteins (hemoglobin, myoglobin, and catalase) . BhuR contains motifs which are common to bacterial heme receptors, including a consensus FRAP domain, an NPNL domain, and two TonB boxes . An N-terminal 32-amino-acid segment, putatively required for rhuIR-dependent regulated expression of bhuR, is present in BhuR but not in other bacterial heme receptors . Two forms of BhuR were observed in the outer membrane of B . avium: a 91-kDa polypeptide consistent in size with the predicted mature protein and a smaller 82-kDa polypeptide which lacks the 104 amino acids found at the N terminus of the 91-kDa form . A mutation in hemA was engineered in B . avium to demonstrate that the bacterium transports heme into the cytoplasm in a BhuR-dependent manner . The role of BhuR in virulence was established in turkey poults by use of a competitive-infection model.

Crit Care, 2002 Aug, 6(4), 342 - 8 Epub 2002 May 02.
Measurement of endotoxin activity in critically ill patients using whole blood neutrophil dependent chemiluminescence; Marshall JC et al.; BACKGROUND: Lipopolysaccharide (endotoxin) from the cell wall of Gram-negative bacteria is a potent trigger for the release of host-derived inflammatory mediators . The relationship between endotoxaemia, Gram-negative infection and the clinical syndrome of sepsis has been difficult to establish, in part because of the limitations of available endotoxin assays . METHODS: We performed an observational cohort study in critically ill patients in the medical/surgical intensive care unit (ICU) of a tertiary care hospital . Whole blood endotoxin levels on the day of ICU admission were measured using a novel chemiluminescent assay--the endotoxin activity assay (EAA)--and the chromogenic modification of the limulus amoebocyte lysate (LAL) assay . RESULTS: We studied 74 consecutive admissions . Endotoxin levels were higher in patients with a diagnosis of sepsis (470 +/- 57 pg/ml) than in patients admitted with a diagnosis other than sepsis (157 +/- 140 pg/ml; P < 0.001) . Endotoxaemia was significantly associated with Gram-negative infection (P < 0.05); no patient with a Gram-negative infection had an endotoxin level below 50 pg/ml . White blood cell counts of patients with EAA-detected endotoxaemia were significantly higher (15.7 +/- 9.1 x 10(9) cells/l for endotoxaemic patients versus 10.8 +/- 6.2 x 10(9) cells/l for patients without endotoxaemia; P < 0.05) . CONCLUSION: Endotoxaemia is associated with Gram-negative infection from any source, and with a diagnosis of sepsis and leukocytosis . These correlations were not apparent using the LAL method . The EAA may be a useful diagnostic tool for the investigation of invasive Gram-negative infection and incipient sepsis.

Ann Hematol, 2002 Aug, 81(8), 462 - 6 Epub 2002 Aug 27.
Liposomal daunorubicin (DaunoXome) for treatment of poor-risk acute leukemia; Russo D et al.; Toxicity limits the use of anthracyclines in elderly sick patients and in heavily pretreated patients . Since the liposomal preparation of daunorubicin (DNR) (DaunoXome, or DNX) is expected to be less toxic than conventional DNR, we tested DNX combined with high-dose arabinosyl cytosine (HDAC) in 42 adult poor-risk acute leukemia patients . Thirty-one patients had acute non-lymphocytic leukemia (ANLL) . Of these, 12 patients were newly diagnosed but were not eligible for standard induction treatment, 13 were in first relapse, and 6 were in second or subsequent relapse . Eleven patients had acute lymphocytic leukemia (ALL), in first (eight cases) or second (three cases) relapse . DNX was given i.v . in three doses of 80 or 100 mg/m(2) each (days 1-3) by a 60-min infusion in glucose 5%, followed by a 4-h infusion of HDAC 2 g/m(2) (days 1-5) . Among 31 ANLL patients there were 16 (51%) complete remissions (CR), 5 deaths during induction, and 10 failures . Among 11 ALL patients there were 10 CRs and 1 failure . The response rate was not affected by the overexpression of MDR-related proteins (PgP, MRP-1, and LRP) . Non-hemopoietic toxicity was negligible, with no intestinal toxicity and only one case of gram-negative bacteremia . We conclude that DNX, in combination with HDAC, is an effective treatment for poor-risk adult AL . Because of the low non-hematologic toxicity, it can be used to reinduce remission in poor-risk patients who are candidates for allogeneic bone marrow transplantation . The high CR rate observed in ALL requires confirmation.

Eur J Immunol, 2002 Sep, 32(9), 2543 - 50
Bacterial fimbriae activate human peripheral blood monocytes utilizing TLR2, CD14 and CD11a/CD18 as cellular receptors; Ogawa T et al.; Bacterial fimbriae are associated with a specific adherence factor, adhesin, in their microbial etiology . Porphyromonas gingivalis, as an anaerobic Gram-negative periodontopathogenic organism, is known to possess fimbriae on its cell surfaces . In this study, we demonstrated that P . gingivalis fimbriae and an active synthetic peptide composed of residues 69 - 73 of thefimbrial subunit protein, ALTTE, induced IL-6 mRNA expression and cytokine production, p38 mitogen-activated protein (MAP) kinase phosphorylation, and NF-kappaB activation in human peripheral blood monocytes . P . gingivalis fimbriae and ALTTE also induced IL-6 production via human Toll-like receptor (TLR) 2, CD14, and CD11a/CD18 (LFA-1) molecules on human monocytes . These results suggest that P . gingivalis fimbriae and these degraded peptides may play an important role in the inflamed gingival and periodontal tissues seen in the development and progression of periodontal diseases.

Mycopathologia, 2002, 154(4), 181 - 4
Candida parapsilosis peritonitis in patients on CAPD; Kaitwatcharachai C; Twenty-four episodes of C . parapsilosis peritonitis in 23 patients on continuous ambulatory peritoneal dialysis (CAPD) over 6 years were reviewed . Clinical manifestations and laboratory findings were similar to those of other pathogens . All started treatment with intravenous amphotericin B . In six cases it was attempted to maintain a peritoneal catheter in situ, but removal became essential to relieve fungal peritonitis . Of the patients who developed peritonitis, 15 episodes (62.5%) continued the CAPD program . Nine cases could not resume CAPD because of death in 4, patient preference in 2, and abdominal adhesion in 3 . Antifungal treatment alone was ineffective in most cases . It was found that peritonitis developing after gram negative bacterial peritonitis and the use of fluconazole after catheter removal were associated with CAPD discontinuation . It was suggested that C . parapsilosis peritonitis in CAPD patients should be treated with rapid catheter removal, particularly those with fungal peritonitis who had prior gram negative peritonitis.

Int J Cancer, 2002 Oct 10, 101(5), 415 - 22
Lipopolysaccharide-induced metastatic growth is associated with increased angiogenesis, vascular permeability and tumor cell invasion; Harmey JH et al.; Endotoxin/lipopolysaccharide (LPS), a cell wall component of Gram-negative bacteria, is a potent inflammatory stimulus . We previously reported that LPS increased the growth of experimental metastases in a murine tumor model . Here, we examined the effect of LPS exposure on key determinants of metastasis-angiogenesis, tumor cell invasion, vascular permeability, nitric oxide synthase (NOS) and matrix metalloproteinase 2 (MMP2) expression . BALB/c mice bearing 4T1 lung metastases were given an intraperitoneal (i.p.) injection of 10 microg LPS or saline . LPS exposure resulted in increased lung weight and incidence of pleural lesions . LPS increased angiogenesis both in vivo and in vitro . Vascular permeability in lung tissue was increased 18 hr after LPS injection . LPS increased inducible nitric oxide synthase (iNOS) and MMP2 expression in lung tumor nodules . 4T1 cells transfected with green fluorescent protein (4T1-GFP) were injected via lateral tail vein . LPS exposure resulted in increased numbers of 4T1-GFP cells in mouse lung tissue compared to saline controls, an effect blocked by the competitive NOS inhibitor, N(G) methyl-L-arginine (NMA) . LPS-induced growth and metastasis of 4T1 experimental lung metastases is associated with increased angiogenesis, vascular permeability and tumor cell invasion/migration with iNOS expression implicated in LPS-induced metastasis .

Br J Pharmacol, 2002 Sep, 137(2), 146 - 52
Accumulation of platelets in the lung and liver and their degranulation following antigen-challenge in sensitized mice; Yoshida A et al.; 1 . Mast cells and basophils are believed to trigger allergic reactions and anaphylaxis . They rapidly release histamine (H), a typical mediator of inflammation, in response to antigens . In the mouse, platelets contain much 5-hydroxytryptamine (5HT), an additional inflammatory mediator, while human platelets contain both H and 5HT . Here, we examined the response of platelets in sensitized mice to antigen challenge . 2 . Platelets accumulated in the lung and liver almost immediately after intravenous injection of ovalbumin (OVA), in mice sensitized to it, and platelet degranulation occurred during these reactions . 3 . These responses of platelets preceded H release from mast cells and/or basophils, occurred at doses of OVA lower than those inducing H release, and contributed to the signs of shock . 4 . We reported previously that intravenous injection into mice of LPS (a membrane constituent of gram-negative bacteria) induces a similar platelet response (accumulation of platelets in the lung and liver) and shock . 5 . Blood that has passed through the body (other than the digestive tract) passes first to the lungs before being recirculated by the heart, and blood that has passed through the digestive tract passes next to the liver . Thus, our findings suggest that in addition to their role in haemostasis, platelets, tiny anuclear cytoplasts, may be important in both innate and acquired immunity, and that the lung and liver may be the fronts at which platelets wage war on pathogens.

Mol Microbiol, 2002 Sep, 45(5), 1289 - 302
Imp/OstA is required for cell envelope biogenesis in Escherichia coli; Braun M et al.; In Gram-negative bacteria, all components of the outer membrane are synthesized in the cytoplasm or the cytoplasmic leaflet of the inner membrane and must thus traverse the inner membrane and the periplasm on the way to their final destination . In this study, we show Imp/OstA to have characteristics typical for proteins involved in envelope biogenesis . Imp is essential and forms a high-molecular-weight disulphide-bonded complex in the outer membrane . Upon depletion of Imp, lipids and outer membrane proteins appear in a novel membrane fraction with higher density than the outer membrane . We propose Imp to be part of a targeting/usher system for components of the outer membrane.

J Clin Microbiol, 2002 Sep, 40(9), 3346 - 9
Leptotrichia amnionii sp . nov., a novel bacterium isolated from the amniotic fluid of a woman after intrauterine fetal demise; Shukla SK et al.; A novel bacterium was isolated and characterized from the amniotic fluid of a woman who experienced intrauterine fetal demise in the second trimester of pregnancy . The bacterium was a slow-growing, gram-negative anaerobic coccobacillus belonging to the genus LEPTOTRICHIA: Unlike Leptotrichia sanguinegens, the isolate did not grow in chopped-meat glucose broth or on sheep blood agar upon subculturing . The isolate was characterized by sequencing and analyzing its 16S rRNA gene . The 1,493-bp 16S ribosomal DNA sequence had only 96% homology with L . sanguinegens . Several phylogenetic analyses indicated that L . amnionii is a distinct species and most closely related to L . sanguiegens.

Int J Med Microbiol, 2002 Jul, 292(2), 95 - 105
Molecular analysis of efflux pump-based antibiotic resistance; Zgurskaya HI; Multidrug efflux transporters are normal constituents of bacterial cells . These transporters are major contributors to intrinsic resistance of bacteria to many anti-microbial agents . In clinical settings, exposure to antibiotics promotes the mutational overexpression of active or silent multidrug transporters, leading to increased antibiotic resistance without acquisition of multiple, specific resistance determinants . The paradoxical ability of multidrug transporters to recognize and efficiently expel from cells scores of dissimilar organic compounds has been in the focus of extensive research for many years . Several independent studies implied that the mechanistic basis of such ability lies in a distinctive locus of the transporter-substrate interaction: the multidrug transporters select and bind their substrates within the phospholipid bilayer . The recently reported high-resolution structure of a complete MsbA transporter of Escherichia coli provides a solid structural basis for these studies . Although the majority of multidrug transporters function as single-component pumps, major transporters of Gram-negative bacteria are organized as three-component structures . Special outer membrane channels and periplasmic proteins belonging to the membrane fusion protein family enable drug efflux across a Gram-negative two-membrane envelope, directly into the external medium . This minireview focuses on the current status of research in the field of multidrug efflux mechanisms.

Int J Med Microbiol, 2002 Jul, 292(2), 115 - 25
The role of integrons in antibiotic resistance gene capture; Rowe-Magnus DA et al.; Although recently discovered, integrons have played a primordial role in the evolution of bacterial genomes . They are best known as the genetic agents responsible for the capture and spread of antibiotic resistance determinants among diverse Gram-negative clinical isolates, and this activity is at the root of the antibiotic resistance phenomenon that has evolved over the last 60 years . The discovery of the ancestral chromosomal super-integrons, novel integron classes, and the multitude of gene cassettes they propagate solidify the crucial role of this system in adaptive bacterial evolution . Recent evidence suggests that evolutionarily old genetic recombination mechanisms for gene transfer have been adapted to the new antibiotic environment due to the heavy selective pressure of liberal antibiotic use in human medicine and animal husbandry.

J Infect Dis, 2002 Aug 15, 186(4), 570 - 3 Epub 2002 Jul 24.
Toll-like receptor 4 is not involved in host defense against pulmonary Legionella pneumophila infection in a mouse model; Lettinga KD et al.; Legionella pneumophila is a gram-negative microorganism that causes a severe pneumonia known as "legionnaires disease." Toll-like receptor 4 (TLR4) transduces the lipopolysaccharide signal and is therefore considered to play a role in host defense against gram-negative bacterial infection . To determine the role of TLR4 in L . pneumophila pneumonia, C3H/HeJ mice, which display a nonfunctional gene encoding TLR4 (TLR4), and wild-type (wt) C3H/HeN mice were intranasally inoculated with L . pneumophila serogroup 1 . Infection proceeded in an identical way in TLR4 mutant and wt mice, as reflected by similar bacterial outgrowth in the lungs . In addition, the inflammatory responses to L . pneumophila infection-as assessed by histopathologic analysis, cell influx in bronchoalveolar lavage fluid, myeloperoxidase activity in lungs, and lung cytokine concentrations-were indistinguishable in TLR4 mutant and wt mice . These data suggest that, in this mouse model, TLR4 does not play a role in resistance to L . pneumophila.

Pediatr Res, 2002 Sep, 52(3), 373 - 6
Association between the Asp299Gly polymorphisms in the Toll-like receptor 4 and premature births in the Finnish population; Lorenz E et al.; Premature birth causes significant health risks of the neonate and increases the cost for neonatal care . Urogenital infection, often caused by Gram-negative bacteria, is a known risk factor . Toll-like receptor-4 (TLR4) is the major endotoxin-signaling receptor and as such is crucial for the initiation of the innate immune response against Gram-negative bacteria . Recently, a variant in the human TLR4 gene was shown to be associated with impaired receptor function and an increased likelihood of Gram-negative sepsis . In the present study, we determined whether the same polymorphism in TLR4 gene is associated with an increased risk for premature birth . We analyzed genotypes for a Finnish study population consisting of a total of 351 term infants and 440 premature infants (gestational age <35 wk; 282 singletons, 158 multiples) and 94 mothers for the presence of the TLR4 polymorphisms Asp299Gly and Thr399Ile . These polymorphisms were in linkage disequilibrium . The 299Gly allele frequencies were 10.6% (93 of 880) in premature infants and 8.3% (58 of 72) in term infants . Excluding multiple pregnancies that often result in premature births, 23.8% (67 of 282) of premature infants and 24.2% (15 of 62) of the mothers of premature infants compared with 15.9% (55 of 345) of term infants and 15.0% (3 of 20) of the mothers delivering at term were carriers of the TLR4 variant . The frequencies of 299Gly allele and Asp/Gly or Gly/Gly genotype carrier status in premature singleton infants were higher than in term singleton infants (p = 0.024, p = 0.028, respectively) or in premature multiples (p = 0.036, p = 0.044, respectively) . According to the present results an allelic variation in the TLR4 receptor was associated with increased risk of premature birth.

Scand J Immunol, 2002 Sep, 56(3), 294 - 302
Differences in LPS-induced activation of bronchial epithelial cells (BEAS-2B) and type II-like pneumocytes (A-549); Schulz C et al.; Lipopolysaccharide (LPS) as a major component of the outer membrane of gram-negative bacteria stimulates various cells to initiate a signalling cascade which ultimately leads to cell activation and expression of immunoregulatory or inflammatory cytokines . The human respiratory epithelium is an important environmental interface, but differences in LPS-induced cell activation between bronchial and alveolar epithelial cells have not yet been investigated in detail . First, the expression of Toll-like receptors (TLRs), as pattern-recognition receptors, was investigated for the bronchial epithelial cells and type II-like pneumocytes, demonstrating that they fulfil the prerequisites for LPS signalling . Thereafter, the effects of LPS, soluble CD14 (sCD14) and LPS-binding protein (LBP) on the release of interleukin-6 (IL-6) and IL-8 were studied . In the presence of LPS, sCD14 induced a significant and concentration-dependent cytokine release in type II-like pneumocytes, whereas the response of bronchial epithelial cells to sCD14 stimulation was low, implicating sCD14-independent activation mechanisms . Furthermore, LBP revealed inhibitory effects on the activation of alveolar epithelial cells, which may represent a novel local defence mechanism during gram-negative infection . We conclude that distinct pathways exist for LPS-induced activation of bronchial and alveolar epithelial cells.

Neurosurgery, 2002 Sep, 51(3), 807 - 9; discussion 809-10
Rare Actinobacillus infection of the cavernous sinus causing painful ophthalmoplegia: case report; Tobias S et al.; OBJECTIVE AND IMPORTANCE: Actinobacillus actinomycetemcomitans is a gram-negative coccobacillus that is known to cause a wide array of clinical infections in debilitated patients, including periodontal disease, soft tissue abscess, pleural empyema, endocarditis, cerebral abscess, and meningitis . We report a rare A . actinomycetemcomitans cavernous sinus abscess that caused painful ophthalmoplegia in a healthy man . CLINICAL PRESENTATION: A 34-year-old man presented with a 3-month history of acute onset of left-sided retro-orbital pain that progressed to a complete left Cranial Nerve VI palsy and Cranial Nerve V1 and V2 hypesthesia . Magnetic resonance imaging revealed the presence of an enhancing lesion in the left cavernous sinus, which encased and narrowed the ipsilateral intracavernous segment of the internal carotid artery . Routine blood and cerebrospinal fluid analyses were normal . Tolosa-Hunt syndrome was suspected, and the patient was treated with high-dose corticosteroids . An open biopsy was performed after failed prolonged corticosteroid therapy . INTERVENTION: A left frontotemporal craniotomy and cavernous sinus exploration through an interdural approach were performed . A soft reddish mass was found in the cavernous sinus around Cranial Nerve V1 and V2 . Multiple biopsies were obtained . Pathological analysis revealed a purulent infection containing multiple gram-negative coccobacilli . The patient's pain improved immediately, and cranial neuropathy resolved during the next several weeks . After cultures demonstrated growth of A . actinomycetemcomitans, a regimen of orally administered amoxicillin and metronidazole was initiated . Eight months after surgery, the patient was free of symptoms and a repeat magnetic resonance imaging scan was normal . CONCLUSION: We present a rare bacterial abscess of the cavernous sinus causing painful ophthalmoplegia . In patients who present with presumed Tolosa-Hunt syndrome and do not improve with prolonged high-dose corticosteroid therapy, an open biopsy is recommended to exclude a bacterial infection.

Int Rev Immunol, 2002 Jan-Feb, 21(1), 27 - 31
Immune activation as effect modifier of atherogenesis in chronic infection; Ricevuti G et al.; Although no study has proven that infections cause atherosclerotic disease, findings suggest that exposure to infections such as Chlamydia pneumoniae, Helicobacter pylori, and bacterial infections related to periodontal disease or smoking might influence the development of heart, carotid, and peripheral vascular disease . Epidemiological data convincingly demonstrate risk for atherosclerotic disease associated with bacterial infection and endotoxemia; however, the independent contributions to disease and pathogenic mechanisms of endotoxin remained elusive . Investigation into this relation tested the correlation between endotoxin and neopterin . Data indicated that neopterin, which reflects immune activation of monocytes/macrophages, functions as atherogenic effect modifying factor on the effects of endotoxin . We hypothesize that immune activation via induction of endotoxin hyperresponsiveness determines the atherogenic potential of Gram-negative infections.

Am J Respir Crit Care Med, 2002 Aug 15, 166(4), 514 - 7
Prevention of gram-negative translocation reduces the severity of hepatopulmonary syndrome; Rabiller A et al.; Hepatopulmonary syndrome (HPS) is characterized by intrapulmonary vascular dilatations and an increased alveoloarterial oxygen difference (AaPO(2)) . These abnormalities are related to augmented pulmonary nitric oxide (NO) production, dependent primarily on increases in the expression and activity of inducible NO-synthase (iNOS) within pulmonary intravascular macrophages and, to a lesser extent, of endothelial NOS (eNOS) . Production of iNOS by pulmonary intravascular macrophages might be related to translocated gut bacteria present in the pulmonary circulation . To test this hypothesis, we determined whether macrophage sequestration, lung iNOS expression and activity, and HPS severity were decreased after norfloxacin was given for 5 weeks to prevent Gram-negative bacterial translocation in rats with common bile duct ligation-induced cirrhosis . Norfloxacin decreased the incidence of Gram-negative translocation from 70 to 0% and the percentage of pulmonary microvessels containing more than 10 macrophages from 52 +/- 7 to 21 +/- 8% (p < 0.01) . AaPO(2) and cerebral uptake of intravenous (99m)Tc-labeled albumin macroaggregates (reflecting intrapulmonary vascular dilatations) were intermediate to those of untreated cirrhotic and sham-operated rats . The activity and expression of lung iNOS, but not eNOS, were reduced to normal . Norfloxacin may reduce HPS severity by inhibiting Gram-negative bacterial translocation, thereby decreasing NO production by pulmonary intravascular macrophages . Bacterial translocation may be the key to the pathogenesis of HPS.

J Biol Chem, 2002 Oct 25, 277(43), 40456 - 61 Epub 2002 Aug 15.
Two nonadjacent regions in enteroaggregative Escherichia coli flagellin are required for activation of toll-like receptor 5; Donnelly MA et al.; Flagellin is the major structural protein of the flagella of Gram-negative bacteria . Recent work has demonstrated that flagellin is a potent trigger of innate immune responses in a number of eukaryotic cells and organisms, including both mammals and plants . In several different human epithelial cell lines, this innate immune response involves toll-like receptor 5 (TLR5) . The mechanisms by which flagellin activates TLR5 and the importance of this interaction in other model systems of flagellin-induced inflammation remain unknown . In this work, random and site-directed mutagenesis of the inflammatory flagellin from enteroaggregative Escherichia coli identified two regions in the conserved D1 domain that are required for interleukin-8 release and TLR5 activation . In contrast, large regions of the variable domain could be excised without reducing the inflammatory activity . In addition, regions of the protein analogous to epitopes that trigger innate immune responses in plants are not involved in Caco-2 flagellin responses . These results highlight the complexity of the interaction between bacterial flagellin and its eukaryotic recognition partners and provide the basis for further studies to characterize the innate immune response to flagellin.

Przegl Lek, 2002, 59(3), 142 - 6
{Chlamydia pneumoniae infections--diagnostic methods}; Stepien E et al.; Gram-negative bacteria Chlamydia pneumonia was found in 1989 to cause acute and chronic respiratory tract infections . This agent has been as well associated with other disease: atherogenesis and coronary heart disease . This study is aimed both at making an introduction to the issues related to C . pneumoniae diagnosis and presenting contemporary laboratory methods . Given the limitations of traditional diagnostics methods, serodiagnosis (EIA) and nucleic acids amplification (PCR, hybridisation) provide the most convincing evidence of C . pneumoniae infections . Culture and direct fluorescence antibody (DFA) may be useful in confirming these results . A variety of methods applied can provide an opportunity to detect bacteria in different clinical samples--incl . sputum, nasopharyngeal and throat swabs, bronchoalveolar lavage (BAL) and tissues from biopsy and autopsy.

Genes Dev, 2002 Aug 15, 16(16), 2156 - 68
DegS and YaeL participate sequentially in the cleavage of RseA to activate the sigma(E)-dependent extracytoplasmic stress response; Alba BM et al.; All cells have stress response pathways that maintain homeostasis in each cellular compartment . In the Gram-negative bacterium Escherichia coli, the sigma(E) pathway responds to protein misfolding in the envelope . The stress signal is transduced across the inner membrane to the cytoplasm via the inner membrane protein RseA, the anti-sigma factor that inhibits the transcriptional activity of sigma(E) . Stress-induced activation of the pathway requires the regulated proteolysis of RseA . In this report we show that RseA is degraded by sequential proteolytic events controlled by the inner membrane-anchored protease DegS and the membrane-embedded metalloprotease YaeL, an ortholog of mammalian Site-2 protease (S2P) . This is consistent with the mechanism of activation of ATF6, the mammalian unfolded protein response transcription factor by Site-1 protease and S2P . Thus, mammalian and bacterial cells employ a conserved proteolytic mechanism to activate membrane-associated transcription factors that initiate intercompartmental cellular stress responses.

Protein Expr Purif, 2002 Aug, 25(3), 465 - 71
Purification of secreted leukotoxin (LtxA) from Actinobacillus actinomycetemcomitans; Kachlany SC et al.; The RTX (repeats in toxin) family of toxins is important in the pathogenesis of many Gram-negative bacteria . The oral and systemic human pathogen Actinobacillus actinomycetemcomitans produces a member of this family known as leukotoxin (LtxA) . Previously, we found that LtxA is secreted into culture supernatants of A . actinomycetemcomitans and that this protein is abundant and relatively pure . Here, we report a large-scale method for the isolation and purification of LtxA from culture supernatants of A . actinomycetemcomitans strain JP2 . The purification scheme involves ammonium sulfate precipitation of culture supernatants, dialysis, and ultrafiltration to concentrate LtxA to approximately 10mg/ml . We found that LtxA remained soluble in buffer that contained at least 250mM NaCl . Purified LtxA was >98% pure and the final preparations were active against HL-60 cells . The entire purification protocol can be completed within 2 days . The ability to readily obtain a large amount of purified leukotoxin should accelerate investigations into the structure and biology of this important virulence factor.

Ter Arkh, 2002, 74(7), 56 - 61
{Tumor necrosis factor, interleukin-6, endotoxin and procalcitonin in septic shock in patients with hematologic neoplasms}; Galstian GM et al.; AIM: To study changes in proinflammatory markers and mediators in septic shock in patients with hematologic malignancy (HM) . MATERIAL AND METHODS: The examination of 33 patients with HM and septic shock included measurement of plasma concentrations of tumor necrosis factor (TNF), interleukine-6 (IL-6), endotoxin, procalcitonin (PCT) 12-24 hours before and each 12 hours after shock; registration of central hemodynamics parameters, the condition severity by APACHE II . RESULTS: Out of 33 patients 18 died of refractory shock, 15 survived the shock . Within the first shock hour TNF fell from 571.2 +/- 195 to 115.8 +/- 71.1 pg/ml (p < 0.02), later being stable . In those who died and survived TNF was the same . IL-6 fall was seen 36 hours after shock and was observed in the survivors; in those who died IL-6 was unchanged . Endotoxin in the blood was detected in 21 of 33 patients . In the survivors endotoxinemia declined after 2 days of treatment . 72 hours after beginning of the shock the survivors had no endotoxin . In shock APACH II severity of the patient's condition was graver in patients with endotoxinemia than without it (31.6 +/- 1.6 and 28.1 +/- 1.6 scores, p < 0.05) . Blood endotoxin levels and APACHE II scores correlated (r = 0.24, p < 0.05) positively and negatively with deficiency of buffer bases (r = -0.29, p < 0.05) and blood pH) r = -0.3, p < 0.05), left ventricular contractility index (r = -0.46, p < 0.01) and right ventricle (r = -0.52, p < 0.01), mean AP (r = -0.22, p < 0.03) . PCT concentration was lower before shock than on its hour 1 (4.2 +/- 2.9 and 6.9 +/- 1.1 ng/ml, p < 0.05) . No significant changes in PCT were found later . CONCLUSION: PCT is a specific marker of a severe infection . Rapid elimination from the blood of TNF and IL-6 makes them inadequate in sepsis diagnosis . Endotoxinemia aggravates the patients condition . Positive LAL-test results were obtained in gram-negative and fungal infections.

J Membr Biol, 2002 Aug 1, 188(3), 237 - 48
The phytotoxic lipodepsipeptide syringopeptin 25A from Pseudomonas syringae pv syringae forms ion channels in sugar beet vacuoles; Carpaneto A et al.; Syringopeptin 25A (SP(25)A) belongs to a family of cyclic lipodepsipeptides (LDPs) produced by the gram-negative bacterium Pseudomonas syringae, a phytopathogenic organism that affects several plants of agronomic interest . LDPs increase the permeability of plasma and, possibly, intracellular membranes in plant cells . Consistently, SP(25)A forms ion channels in planar lipid bilayers and other model membranes . Here we used sugar beet tonoplasts as a new biological model system to study toxin action . When applied to the vacuoles by a fast perfusion procedure, SP(25)A increases membrane permeability by forming discrete ion channels even at low applied potentials . The SP(25)A channel displays anion selectivity (with a Cl-/K+ permeability ratio of 6.7 +/- 1.3) and has intrinsic rectification properties that derive from a different channel conductance at negative and positive voltages, presumably owing to an asymmetric distribution of fixed charges on the pore . Substitution of chloride with different anions reveals the following selectivity sequence NO3- approximately Cl-> F- > gluconate-, suggesting that the permeation pore is filled with water . The properties of the SP(25)A channels in vacuolar membranes are similar to those observed in planar lipid membranes prepared with asolectin . This work provides a direct demonstration of toxin effects on a native plant membrane, extending to a biological system previous results obtained on artificial planar lipid membranes.

Mol Microbiol, 2002 Aug, 45(4), 1029 - 41
Proteomic analysis of the Caulobacter crescentus stalk indicates competence for nutrient uptake; Ireland MM et al.; Caulobacter crescentus, a Gram-negative alpha-purple proteobacterium, is an oligotroph that lives in aquatic environments dilute in nutrients . This bacterium divides asymmetrically . Part of this asymmetric cell division involves the formation of a prosthecum at one pole, referred to as the stalk, which replaces the flagellum of the motile swarmer cell . Little is known about the synthesis or function of the stalk . The stalk is an extension of the cell membranes and peptidoglycan layer, and stalk elongation is stimulated by phosphate starvation . In this study, we have taken advantage of two-dimensional gel (2D gel) electro-phoresis as well as the fully sequenced genome of Caulobacter to study the proteome of the stalk . We modified a stalk-shedding mutant strain of Caulobacter crescentus to increase the yield of stalk material shed and performed 2D gel electrophoresis of purified stalks and cellular fractions . Comparison of the stalk 2D gel with the 2D gels of cell membrane and soluble fractions showed that the stalk is mostly free of cytoplasmic proteins and has a profile very similar to that of the cell membrane . Of the 172 proteins on a stalk 2D gel, we report the identification of 64 spots, corresponding to 39 different proteins present in the stalk of Caulobacter . The identifications include several TonB-dependent receptors, two OmpA family proteins, a dipeptidase, GlpQ, two alkaline phosphatases, 3-phytase, a putative TolC protein and 11 proteins of unknown function . These identifications are consistent with the hypothesis that the stalk plays a role in nutrient uptake.

Mol Microbiol, 2002 Aug, 45(4), 905 - 16
Coiled-coil proteins associated with type III secretion systems: a versatile domain revisited; Delahay RM et al.; The pathogenic potential of many Gram-negative bacteria is indicated by the possession of a specialized type III secretion system that is used to deliver virulence effector proteins directly into the cellular environment of the eukaryotic host . Extracellular assemblies of secreted proteins contrive a physical link between the pathogen and host cytosol and enable the translocated effectors to bypass the bacterial and host membranes in a single step . Subsequent interactions of some effector proteins with host cytoskeletal and signalling proteins result in modulation of the cytoskeletal architecture of the aggressed cell and facilitate entry, survival and dissemination of the pathogen . Although the secreted components of type III secretion systems are diverse, many are predicted to share a common coiled-coil structural feature . Coiled-coils are ubiquitous and highly versatile assembly motifs found in a wide range of structural and regulatory proteins . The prevalence of these domains in secreted virulence effector proteins suggests a fundamental contribution to multiple aspects of their function, and evidence accumulating from functional studies suggests an intrinsic involvement of coiled-coils in subunit assembly, translocation and flexible interactions with multiple bacterial and host proteins . The known functional flexibility that coiled-coil domains confer upon proteins provides insights into some of the pathogenic mechanisms used during interaction with the host.

J Biol Chem, 2002 Nov 1, 277(44), 42003 - 10 Epub 2002 Aug 10.
Crystallographic and biochemical studies of DivK reveal novel features of an essential response regulator in Caulobacter crescentus; Guillet V et al.; DivK is an essential response regulator in the Gram-negative bacterium Caulobacter crescentus and functions in a complex phosphorelay system that precisely controls the sequence of developmental events during the cell division cycle . Structure determinations of this single domain response regulator at different pH values demonstrated that the five-stranded alpha/beta fold of the DivK protein is fully defined only at acidic pH . The crystal structures of the apoprotein and of metal-bound DivK complexes at higher pH values revealed a synergistic pH- and cation binding-induced flexibility of the beta4-alpha4 loop and of the alpha4 helix . This motion increases the solvent accessibility of the single cysteine residue in the protein . Solution state studies demonstrated a 200-fold pH-dependent increase in the affinity of manganese for the protein between pH 6.0 and 8.5 that seems to involve deprotonation of an acido-basic couple . Taken together, these results suggest that flexibility of critical regions of the protein, ionization of the cysteine 99 residue and improved K(D) values for the catalytic metal ion are coupled events . We propose that the molecular events observed in the isolated protein may be required for DivK activation and that they may be achieved in vivo through the specific protein-protein interactions between the response regulator and its cognate kinases.

Ann Clin Lab Sci, 2002 Summer, 32(3), 292 - 8
Ketamine reduces NFkappaB activation and TNFalpha production in rat mononuclear cells induced by lipopolysaccharide in vitro; Yu Y et al.; Ketamine may be advantageous for anesthesia of patients with sepsis caused by gram-negative bacteria, because ketamine may suppress LPS-induced production of proinflammatory cytokines, such as TNFalpha and IL-6 . NFkappaB is an important transcription factor that is involved in the post-transcriptional regulation of mRNA expression for several immunoinflammatory mediators in response to endotoxemia . This study examined the effect of ketamine on NFkappaB activation and TNFalpha production in rat peripheral blood mononuclear cells (PBMC) . The PBMC were incubated in the presence or absence of LPS and with graded concentrations of ketamine . The culture supernatants and cells were collected for each group and duration of incubation . Activation of NFkappaB was determined by electrophoretic mobility shift assay (EMSA), and the expression of IkappaBalpha, its inhibitor, in PBMC was analysed by Western blotting . TNFalpha levels in the supernatants were measured using a specific enzyme-linked immunosorbent assay (ELISA) . LPS stimulation of rat PBMC increased TNFalpha production and NFkappaB activation, with corresponding loss of IkappaBalpha . Ketamine significantly reduced the LPS-induced NFkappaB activation and inhibited TNFalpha production in a dose-dependent manner . These in vitro findings suggest that ketamine is a potent inhibitor of NFkappaB activation and cytokine production in rat PBMC.

Harefuah, 2002 May, 141 Spec No, 78 - 83, 120
{Tularemia as a biological weapon}; Katz L et al.; Tularemia is a zoonosis, caused by the Gram-negative bacterium, Francisella tularensis . The organism penetrates the human body through interrupted skin or mucous membranes, via animal contact or bites from ticks, deer-flies and mosquitoes . Contaminated aerosol and water are alternative modes of transmitting the germ through the respiratory and alimentary tracks . In light of its high infectivity in aerosol and its offensive occupation in the past, tularemia may appear in a biological warfare context . After an incubation period of 3-5 days, the disease begins with systemic symptoms, which abate, leaving a clinical picture, dominated by one of the listed patterns: ulceroglandular, typhoidal, glandular, occuloglandular, pharyngeal or pneumonic . Diagnosis and identification of the bacterium is difficult and even hazardous . Most diagnoses are established by serology which is positive in 50-70% of the patients after 2 weeks of illness, and in most of them by 4-8 weeks . The treatment of choice is streptomycin intra-muscular, or gentamicin intra-venous for 10-14 days . Prophylaxis may be achieved by tetracycline treatment, beginning up to 24 hours from exposure, for 2 weeks, or by a live attenuated, investigational vaccine . Nevertheless, post-exposure, or even better so, pre-exposure intervention is the most effective way of preventing the devastating results of the attack.

Am J Physiol Lung Cell Mol Physiol, 2002 Sep, 283(3), L655 - 63
Total extracellular surfactant is increased but abnormal in a rat model of gram-negative bacterial pneumonia; Russo TA et al.; An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL) . Total extracellular surfactant was increased in infected rats compared with controls . BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels (R(2) = 0.908, P < 0.0001) . Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E . coli . SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls . The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein . There was a significant correlation (R(2) = 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein . Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction . In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo.

Shock, 2002 Aug, 18(2), 182 - 8
Chylomicron-bound endotoxin selectively inhibits NF-kappaB activation in rat hepatocytes; Kumwenda ZL et al.; Triglyceride-rich lipoproteins (chylomicrons and very low-density lipoproteins) bind endotoxin (lipopolysaccharide {LPS}), forming lipoprotein-LPS complexes, and protect against endotoxic shock and death in rodent models of gram-negative sepsis . Hepatocytes play a central role in the protective process, as demonstrated by the increased uptake of chylomicron (CM)-bound LPS by these cells . We have previously reported that CM-LPS complexes inhibit nitric oxide (NO) production by hepatocytes as compared with LPS or CM alone . Herein, we report that CM-LPS selectively inhibits NF-kappaB in hepatocytes . Pretreating cultured primary hepatocyte spheroids with CM-bound LPS inhibited cytokine-induced NF-kappaB activation by approximately 60% vs . untreated control cells (P < 0.03) . The lipoprotein-mediated inhibition of NF-kappaB was non-toxic, selective, and associated with inhibition of IkappaB degredation . These data indicate that the mechanism by which CM protect against LPS involves inhibition of the hepatocellular response to proinflammatory stimulation and also support a role for triglyceride-rich lipoproteins as components of the innate host immune response to infection.

Shock, 2002 Aug, 18(2), 138 - 41
Different serotypes of endotoxin (lipopolysaccharide) cause different increases in albumin extravasation in rats; Nedrebo T et al.; Endotoxin (lipopolysaccharide {LPS} from cell membranes of gram-negative bacteria) is frequently used in experimental models of septic shock that are characterised by hypotension, peripheral vasodilation, and edema formation, as well as greatly enhanced flux of macromolecules and fluid from plasma to tissues . The edema formation and increased albumin extravasation (Ealb) could be caused by increased permeability and/or increased capillary net filtration pressure . We have measured interstitial fluid pressure (Pif) and Ealb after i.v . injection of two different serotypes of LPS in female Wistar Moller rats (200-250 g) in pentobarbital anaesthesia . Two experimental groups and one control group were studied (n = 8 in each group) . Group 1, serotype 0111:B4, received 3 mg/kg LPS, Group 2, serotype 0127:B8, received 1.5 mg/kg LPS, and controls received saline vehicle (0.4 mL) . Five minutes after injection of LPS or saline vehicle, human serum albumin labelled with 125I (125I-HAS; 0.05 MBq) was injected i.v . and was followed 55 min later by 131I-HSA (0.05 MBq) . Five minutes thereafter the rats were killed and tissue samples were obtained from skin, muscle, and small intestine . Ealb was estimated as the difference between the plasma equivalent distribution volumes of 125I-HSA and 131I-HSA . The pattern of extravasation between the groups was the same in all the tissues studied . Group 1 serotype (0111:B4) and controls had much lower Ealb than Group 2 serotype (0127:B8; P < 0.05) . Ealb differed among the tissues both in relative and absolute numbers, being largest in the intestine and smallest in skeletal muscle . We previously demonstrated a lowering of Pif after LPS injection using serotype (0127:B8) . The present results demonstrate that the same serotype of LPS also causes a significant increase of Ealb, and is therefore likely caused by the lowering of Pif.

Eur Respir J, 2002 Jul, 20(1), 210 - 22
Activation of second messenger pathways in alveolar macrophages by endotoxin; Monick MM et al.; The alveolar macrophage plays an important role in immune surveillance of the lung . Early responses to infectious agents by macrophages can decrease tissue injury and promote recovery of the host . Macrophage responses to pathogens are the cornerstone of the innate or nonspecific immune system . In particular, the response of macrophages to endotoxin from gram negative bacteria has been the focus of many recent studies . The recent discovery of the endotoxin receptor has accelerated the study of signalling in macrophages . This review focuses on the downstream events that occur following exposure of the alveolar macrophage to endotoxin.

J Immunol, 2002 Aug 15, 169(4), 1665 - 8
Cutting edge: a murine, IL-12-independent pathway of IFN-gamma induction by gram-negative bacteria based on STAT4 activation by Type I IFN and IL-18 signaling; Freudenberg MA et al.; IFN-alphabeta is a potent immunoregulatory cytokine involved in the defense against viral and bacterial infections . In this study, we describe an as yet undefined IFN-alphabeta-dependent pathway of IFN-gamma induction in mice . This pathway is based on a synergism of IFN-alphabeta and IL-18, and is independent of IL-12 signaling yet dependent on STAT4 . In contradiction to current dogma, we show further that IFN-alphabeta alone induces tyrosine phosphorylation of STAT4 in murine splenocytes of different mouse strains . This pathway participates in the induction of IFN-gamma by Gram-negative bacteria and is therefore expected to play a role whenever IFN-alpha or IFN-beta and IL-18 are produced concomitantly during bacterial, viral, or other infections.

Scand J Infect Dis, 2002, 34(6), 474 - 7
Rat-bite fever in children: case report and review; Ojukwu IC et al.; We report 2 cases of rat-bite fever (RBF), a multisystem zoonosis, in children and review the literature . RBF is caused by I of 2 Gram-negative organisms: Streptobacillus moniliformis or, less commonly, Spirillum minus . Both of our cases developed in school-aged girls with a history of rat exposure who presented with a multisystem illness consisting of fever, petechial and purpuric rash, arthralgia and polyarthritis . Both responded promptly to antibiotic treatment . An additional 10 cases from a MEDLINE review (1960-2000) are reviewed . RBF must be included in the differential diagnosis of febrile patients with rashes and a history of exposure to rats.

Leuk Lymphoma, 2002 Jun, 43(6), 1329 - 32
Augmentation of mast cell bactericidal activity by the anti-leukemic drug, 4-(3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline; Malaviya R et al.; Mast cells play a pivotal role in host innate immune defense against gram negative bacterial infections by killing gram negative bacteria and recruiting neutrophils to the sites of active infection through the release of TNFalpha and leukotrienes . Here, we report that the anti-leukemic compound 4-(3'bromo-4'-hydroxylphenyl)-amino-6,7-dimethoxyquinazoline, designated as MASTPROM, augments the bactericidal activity of mast cells by increasing the binding of bacteria to and their phagocytosis by mast cells . MASTPROM also promoted the bacterial clearance in a mouse model of bacterial peritonitis . MASTPROM may provide the basis for novel supportive care regimens aimed at augmenting the bactericidal activity of mast cells and thereby potentiating the innate immune response against gram negative organisms.

Orthopade, 2002 Jun, 31(6), 591 - 3
{Eikenella corrodens-induced spondylitis . Detection with 16s-RNA polymerase chain reaction}; Zeifang F et al.; Isolation of the relevant organism in patients with spondylitis even after an open biopsy is successful only in 75-90% . The rare case of an Eikenella corrodens-induced spondylitis is presented, which could only be identified using 16S ribosomal DNA polymerase chain reaction following unsuccessful microbiological cultivation . Eikenella corrodens is a facultative anaerobic gram-negative organism, which is mostly found in the oropharynx of healthy patients.

J Pediatr Surg, 2002 Aug, 37(8), 1177 - 82
Prospective study of the incidence and complications of bacterial contamination of enteral feeding in neonates; Mehall JR et al.; PURPOSE: The goal of this study was to determine incidence of, and complications resulting from, bacterial contamination of enteral feedings in neonates . METHODS: A prospective study of 50 tube-fed neonates was conducted . Infants were bolus fed via an open gravity drained system; demographic and clinical data were gathered . The lumen of the tube was cultured quantitatively after 7 days . All organisms were isolated, identified, frozen, and stored to correlate with clinical cultures . RESULTS: The 50 neonates were fed for a mean of 17.6 days each and represent 125 patient weeks (1 tube per patient per week) . A total of 71 of 125 tubes were "contaminated" (>1,000 colony forming units {CFU}/mL), with a mean 908,173 CFU and 3 different bacteria types . Among formula-fed infants, feeding intolerance occurred in 24 of 32 weeks with contaminated tubes versus 0 of 44 weeks with noncontaminated tubes (P <.05) . Contamination occurred in 41 of 48 weeks in patients on H2 antagonists versus 32 of 66 weeks in patients with normal gastric acidity (P <.05) . Necrotizing enterocolitis developed in 7 patients; all were fed formula contaminated with greater than 100,000 CFU/mL of Gram-negative bacteria . Four required operation; intraoperative cultures found the same organism as cultured previously in the tube in all 4 infants . CONCLUSION: Bacterial contamination of enteral feeding occurs frequently, causes significant feeding intolerance, and may contribute to NEC .

Appl Environ Microbiol, 2002 Aug, 68(8), 3943 - 7
Endotoxin activity of Moraxella osloensis against the grey garden slug, Deroceras reticulatum; Tan L et al.; Moraxella osloensis is a gram-negative bacterium associated with Phasmarhabditis hermaphrodita, a slug-parasitic nematode that has prospects for biological control of mollusk pests, especially the grey garden slug, Deroceras reticulatum . This bacterium-feeding nematode acts as a vector that transports M . osloensis into the shell cavity of the slug, and the bacterium is the killing agent in the nematode-bacterium complex . We discovered that M . osloensis produces an endotoxin(s), which is tolerant to heat and protease treatments and kills the slug after injection into the shell cavity . Washed or broken cells treated with penicillin and streptomycin from 3-day M . osloensis cultures were more pathogenic than similar cells from 2-day M . osloensis cultures . However, heat and protease treatments and 2 days of storage at 22 degrees C increased the endotoxin activity of the young broken cells but not the endotoxin activity of the young washed cells treated with the antibiotics . This suggests that there may be a proteinaceous substance(s) that is structurally associated with the endotoxin(s) and masks its toxicity in the young bacterial cells . Moreover, 2 days of storage of the young washed bacterial cells at 22 degrees C enhanced their endotoxin activity if they were not treated with the antibiotics . Furthermore, purified lipopolysaccharide (LPS) from the 3-day M . osloensis cultures was toxic to slugs, with an estimated 50% lethal dose of 48 microg per slug, thus demonstrating that the LPS of M . osloensis is an endotoxin that is active against D . reticulatum . This appears to be the first report of a biological toxin that is active against mollusks.

Arch Biochem Biophys, 2002 Aug 15, 404(2), 317 - 25
Mutational analysis of tetracycline resistance protein transmembrane segment insertion; Lewis GS et al.; The tetracycline resistance proteins (TetA) of gram-negative bacteria are secondary active transport proteins that contain buried charged amino acids that are important for tetracycline transport . Earlier studies have shown that insertion of TetA proteins into the cytoplasmic membrane is mediated by helical hairpin pairs of transmembrane (TM) segments . However, whether helical hairpins direct spontaneous insertion of TetA or are required instead for its interaction with the cellular secretion (Sec) machinery is unknown . To gain insight into how TetA proteins are inserted into the membrane, we have investigated how tolerant the class C TetA protein encoded by plasmid pBR322 is to placement of charged residues in TM segments . The results show that the great majority of charge substitutions do not interfere with insertion even when placed at locations that cannot be shielded internally within helical hairpins . The only mutations that frequently block insertion are proline substitutions, which may interfere with helical hairpin folding . The ability of TetA to broadly tolerate charge substitutions indicates that the Sec machinery assists in its insertion into the membrane . The results also demonstrate that it is feasible to engineer charged residues into the interior of TetA proteins for the purpose of structure-function analysis .

Bioresour Technol, 2002 Oct, 85(1), 103 - 5
Cadmium biosorption by Sphingomonas paucimobilis biomass; Tangaromsuk J et al.; Among microorganisms isolated in Bangkok, the gram-negative bacterium Sphingomonas paucimobilis exhibited the greatest cadmium tolerance . It was able to survive in the medium containing cadmium as high as 200 mg/l . However, concentrations of cadmium at 25-200 mg/l inhibited its growth . The biosorption properties for cadmium of this bacterial biomass and the effects of environmental factors (i.e., biosorbent type, initial pH and biosorbent concentration) on the cadmium biosorption were explored . The results showed that the cadmium removal capacity of living cells was markedly higher than that of nonliving cells . Cadmium biosorption by S . paucimobilis biomass was also affected by the initial pH and biosorbent concentration.

J Bacteriol, 2002 Aug, 184(16), 4620 - 5
Mutational analysis of the TolA C-terminal domain of Escherichia coli and genetic evidence for an interaction between TolA and TolB; Dubuisson JF et al.; The Tol proteins are involved in the outer membrane stability of gram-negative bacteria . The C-terminal domain of TolA was mutagenized to identify residues important for its functions . The isolation of suppressor mutants of tolA mutations in the tolB gene confirmed an interaction between TolAIII and the N-terminal domain of TolB.

Int J Med Microbiol, 2002 Jun, 292(1), 7 - 15
Molecular genetics and role in infection of environmentally regulated lipopolysaccharide expression; Preston A et al.; Lipopolysaccharides (LPSs) from different Gram-negative bacteria are structurally distinct . Even closely related serovars of single bacterial species may possess different LPS molecules . Further variability may then be superimposed on this ground-state structural diversity as a result of variable expression of other LPS structures . This variable expression is due in some cases to high-frequency, reversible, random "on-off" switching of genes required for biosynthesis of particular LPS structures . In other cases differential expression of LPS biosynthesis genes may be part of a programmed response to environmental stimuli, which may occur as adaptations by pathogenic bacteria to changing environments within the host during the course of infection.

Mikrobiologiia, 2002 May-Jun, 71(3), 308 - 15
{Low-molecular-weight autoregulatory factors in bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum}; Loiko NG et al.; The haloalkaliphilic, lithoautotrophic, sulfur-oxidizing gram-negative bacteria Thioalkalivibrio versutus and Thioalkalimicrobium aerophilum were found to possess a special system for the autoregulation of their growth . The system includes the extracellular autoinducers of anabiosis (the d1 factor) and autolysis (the d2 factor) . The principal components of the d1 factor are alkylhydroxybenzenes . The principal components of the d2 factor are free unsaturated fatty acids dominated by oleic acid isomers . Like the respective autoregulators of neutrophilic bacteria, the d1 factor of haloalkaliphilic bacteria presumably controls their growth and transition to a anabiotic state, while the d2 factor controls autolytic processes . Alkylhydroxybenzenes of both microbial and chemical origin were found to influence bacterial respiration . The low-molecular-weight osmoprotectant glycine betaine enhanced the thermostability of trypsin . This suggests that glycine betaine, like the d1 factor, serves as a molecular chaperone.

Clin Chim Acta, 2002 Sep, 323(1-2), 59 - 72
Initial responses to endotoxins and Gram-negative bacteria; Heumann D et al.; The innate immune system initiates host defence against invasive microbial pathogens using specific recognition mechanisms . Here we review the current concepts and the molecular basis of innate immune responses to bacterial infections, focusing our attention on the actors involved in the response to Gram-negative bacteria.Lipopolysaccharide (LPS) is the major virulence factor of Gram-negative bacteria . During the past decade, enormous progress has been obtained in the elucidation of LPS recognition and signalling in mammalian phagocytes . According to the current model, recognition of LPS is initialized by the cooperative interplay between the LPS-binding protein (LBP), the membrane-bound or soluble forms of CD14 and the recently identified Toll-like receptor 4 (TLR4)-MD-2 complex . Recognition of LPS leads to the rapid activation of an intracellular signalling pathway, highly homologous to the signalling pathway of interleukin-1, which results in the release of pro-inflammatory mediators.In vivo models in which animals are challenged with LPS or Gram-negative bacteria have highlighted opposite roles for LBP, CD14 and TLRs . Regarding LPS challenge, there is a large body of evidence in favour of a detrimental role played by LBP, CD14 and TLRs . These molecules sensitize the host to a LPS-induced uncontrolled acute inflammatory response that results in animal death . However, when the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted . Under these conditions, LBP, CD14 and TLRs are required to trigger a pro-inflammatory response which is crucial for keeping infection under control . Therefore, caution should be the rule about the development of therapeutic approaches aimed at blocking the pro-inflammatory response during Gram-negative infections.

Transplantation, 2002 Jul 15, 74(1), 7 - 13
Activation of the lipopolysaccharide signaling pathway in hepatic transplantation preservation injury; Tsoulfas G et al.; BACKGROUND: Endotoxin or lipopolysaccharide (LPS) initiates a cascade of complications of septic shock and multiple organ failure seen in Gram-negative bacterial infections . The first step of this pathway, which leads to activated nuclear factor (NF)-kappaB, activating protein (AP)-1, and other transcription factors, is the formation of the LPS receptor complex by LPS, LPS-binding protein (LBP), CD14, and toll-like receptor (TLR) 2 or 4 . We examined whether the LPS signaling pathway is activated by hepatic ischemia/reperfusion injury in the transplant setting . METHODS: Orthotopic syngeneic rat liver transplantation was performed with 0 to 18 hr of cold preservation in University of Wisconsin solution . Animals were killed 1 to 48 hr after reperfusion . Northern blot analysis for CD14, LBP, and TLR2 mRNA, immunohistochemistry for LBP, liver enzyme analysis, and gel shift assay for NF-kappaB and AP-1 were performed . RESULTS: LPS levels were elevated early after reperfusion . Aspartate aminotransferase and alanine aminotransferase maximally increased 12 hr after transplantation . LBP mRNA and protein and CD14 mRNA were significantly up-regulated peaking at 6 to 12 hr after reperfusion . TLR2 mRNA was also increased . NF-kappaB activity showed a biphasic peak at 1 to 3 hr and 12 hr after reperfusion, whereas AP-1 activity showed a peak at 3 to 6 hr . The induction of CD14 mRNA correlated with the length of cold ischemia time . CONCLUSIONS: These data indicate that multiple components of the LPS signaling pathway are activated during ischemia/reperfusion injury after liver transplantation.

J Virol, 2002 Aug, 76(16), 8169 - 78
Bacteriophage PM2 has a protein capsid surrounding a spherical proteinaceous lipid core; Kivela HM et al.; The marine double-stranded DNA (dsDNA) bacteriophage PM2, studied since 1968, is the type organism of the family Corticoviridae, infecting two gram-negative Pseudoalteromonas species . The virion contains a membrane underneath an icosahedral protein capsid composed of two structural proteins . The purified major capsid protein, P2, appears as a trimer, and the receptor binding protein, P1, appears as a monomer . The C-terminal part of P1 is distal and is responsible for receptor binding activity . The rest of the structural proteins are associated with the internal phospholipid membrane enclosing the viral genome . This internal particle is designated the lipid core . The overall structural organization of phage PM2 resembles that of dsDNA bacteriophage PRD1, the type organism of the family TECTIVIRIDAE:

Cornea, 2002 Aug, 21(6), 625 - 7
Positive polymerase chain reaction and histology with borderline serology in Parinaud's oculoglandular syndrome; Starck T et al.; PURPOSE: To report a case of Parinaud's oculoglandular syndrome (POS) in which, despite a borderline serology, polymerase chain reaction (PCR) testing for a conjunctival biopsy was positive for Bartonella henselae, a source of cat-scratch disease . A Steiner silver stain demonstrated the organism . METHODS: Case Report . RESULTS: A 65-year-old man was seen for a foreign body in his left eye (OS) associated with chemosis and a preauricular node . CONCLUSION: B . henselae is a known cause of POS . This gram-negative pleomorphic rod has been more frequently discovered in connection with this syndrome due to improved diagnostic testing such as indirect immunofluorescence antibody and PCR testing . Frequently, serology is positive if the organism is present . This report describes a patient with clinical findings of POS who, despite borderline serology, had pleomorphic rods on Steiner silver stain and positive PCR testing compatible with Bartonella henselae.

J Vet Med Sci, 2002 Jun, 64(6), 523 - 6
Immunohistochemical and ultrastructural identification of Fusobacterium necrophorum subsp . necrophorum in bovine fatal necrotizing glossitis; Shibahara T et al.; A 37-day-old male Japanese black calf showing marked salivation and leucocytosis died and was examined the tissues histologically . Histological lesions were characterized by severe focal necrotic glossitis on the ventral side of the root of the tongue . Immunohistochemically, Fusobacterium necrophorum subsp . necrophorum antigen was detected in the necrotic tissues and its distribution corresponded to that of the gram-negative, nonsporeforming, long filamentous organisms . Ultrastructural similarities between the organism and F . necrophorum subsp . necrophorum, but not subsp . funduliforme were observed . These findings clearly demonstrated that the fatal necrotic glossitis was caused by F . necrophorum subsp . necrophorum . This is the first report of bovine fatal necrotizing glossitis with leucocytosis caused by F . necrophorum subsp . necrophorum infection, and this organism may be an important fatal pathogen in calves with glossal lesions.

J Biol Chem, 2002 Oct 4, 277(40), 37567 - 72 Epub 2002 Jul 18.
A tetrameric porin limits the cell wall permeability of Mycobacterium smegmatis; Engelhardt H et al.; Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant against many antibiotics . Pore proteins mediate the diffusion of hydrophilic nutrients across this membrane . Electron microscopy revealed that the outer membrane of Mycobacterium smegmatis contained about 1000 protein pores per microm(2), which are about 50-fold fewer pores per microm(2) than in Gram-negative bacteria . The projection structure of the major porin MspA of M . smegmatis was determined at 17 A resolution . MspA forms a cone-like tetrameric complex of 10 nm in length with a single central pore . Thus, MspA is drastically different from the trimeric porins of Gram-negative bacteria and represents a new class of channel proteins . The formation of MspA micelles indicated that the ends of MspA have different hydrophobicities . Oriented insertion of MspA into membranes was demonstrated in lipid bilayer experiments, which revealed a strongly asymmetrical voltage gating of MspA channels at -30 mV . The length of MspA is sufficient to span the outer membrane and contributes in combination with the tapering end of the pore and the low number of pores to the low permeability of the cell wall of M . smegmatis for hydrophilic compounds.

N Engl J Med, 2002 Jul 18, 347(3), 185 - 92
Toll-like receptor 4 polymorphisms and atherogenesis; Kiechl S et al.; BACKGROUND: The ability to mount a prominent inflammatory response to bacterial pathogens confers an advantage in innate immune defense but may signal an increased risk of atherosclerosis . We determined whether recently discovered genetic variants of toll-like receptor 4 (TLR4) that confer differences in the inflammatory response elicited by bacterial lipopolysaccharide are related to the development of atherosclerosis . METHODS: As part of the five-year follow-up in the Bruneck (Italy) Study, we screened 810 persons in the study cohort for the TLR4 polymorphisms Asp299Gly and Thr399Ile . The extent and progression of carotid atherosclerosis were assessed by high-resolution duplex ultrasonography . RESULTS: As compared with subjects with wild-type TLR4, the 55 subjects with the Asp299Gly TLR4 allele had lower levels of certain proinflammatory cytokines, acute-phase reactants, and soluble adhesion molecules, such as interleukin-6 and fibrinogen . Although these subjects were found to be more susceptible to severe bacterial infections, they had a lower risk of carotid atherosclerosis (odds ratio, 0.54; 95 percent confidence interval, 0.32 to 0.98; P=0.05) and a smaller intima-media thickness in the common carotid artery (regression coefficient, -0.07; 95 percent confidence interval, -0.12 to -0.02; P=0.01) . CONCLUSIONS: The Asp299Gly TLR4 polymorphism, which attenuates receptor signaling and diminishes the inflammatory response to gram-negative pathogens, is associated with a decreased risk of atherosclerosis . This finding is consistent with the hypothesis that innate immunity may play a part in atherogenesis .

Am J Physiol Heart Circ Physiol, 2002 Aug, 283(2), H461 - 7
Lipopolysaccharide induces apoptosis in adult rat ventricular myocytes via cardiac AT(1) receptors; Li HL et al.; Lipopolysaccharide (LPS) from gram-negative bacteria circulates in acute, subacute, and chronic conditions . It was hypothesized that LPS directly induces cardiac apoptosis . In adult rat ventricular myocytes (isolated with depyrogenated digestive enzymes to minimize tolerance), LPS (10 ng/ml) decreased the ratio of Bcl-2 to Bax at 12 h; increased caspase-3 activity at 16 h; and increased annexin V, propidium iodide, and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining at 24 h . Apoptosis was blocked by the caspase inhibitor benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone (Z-VAD-fmk), captopril, and angiotensin II type 1 receptor (AT(1)) inhibitor (losartan), but not by inhibitors of AT(2) receptors (PD-123319), tumor necrosis factor-alpha (TNFRII:Fc), or nitric oxide (N(G)-monomethyl-L-arginine) . Angiotensin II (100 nmol/l) induced apoptosis similar to LPS without additive effects . LPS in vivo (1 mg/kg iv) increased apoptosis in left ventricular myocytes for 1-3 days, which dissipated after 1-2 wk . Losartan (23 mg . kg(-1) . day(-1) in drinking water for 3 days) blocked LPS-induced in vivo apoptosis . In conclusion, low levels of LPS induce cardiac apoptosis in vitro and in vivo by activating AT(1) receptors in myocytes.

Transpl Int, 2002 Jul, 15(7), 329 - 34 Epub 2002 Jun 13.
Selective bowel decontamination in elective liver transplantation: no improvement in endotoxaemia, initial graft function and post-operative morbidity; Maring JK et al.; Peri-operative endotoxaemia during liver transplantation has been linked to compromised graft function and infection . Selective decontamination of the digestive tract (SDD) could prevent endotoxaemia by eradicating Gram-negative bacteria from the intestine . In a randomized placebo controlled study we investigated the effects of endotoxaemia and the efficacy of SDD to prevent its occurrence . Thirty-one patients undergoing elective orthotopic liver transplantation received either SDD ( n=15) or placebo ( n=16), which was started at least 7 days before transplantation . Endotoxin levels were measured in blood peroperatively . Patients were scored daily for signs of liver dysfunction and infection . Endotoxaemia was neither associated with initial poor function nor any routine liver function test . Infections were more prominent in patients without endotoxaemia . SDD did not prevent endotoxaemia . Endotoxaemia does not affect post-operative graft function or the incidence of post-operative infections . SDD cannot prevent peri-operative endotoxaemia . Translocation of endotoxin may not be relevant in liver transplantation.

J Biol Chem, 2002 Oct 4, 277(40), 37573 - 81 Epub 2002 Jul 16.
Molecular mimicry of an HLA-B27-derived ligand of arthritis-linked subtypes with chlamydial proteins; Ramos M et al.; HLA-B27 is strongly associated with spondyloarthropathies, including ankylosing spondylitis and reactive arthritis . The latter disease is triggered by various Gram-negative bacteria . A dodecamer derived from the intracytoplasmic tail of HLA-B27 was a natural ligand of three disease-associated subtypes (B*2702, B*2704, and B*2705) but not of two (B*2706 and B*2709), weakly or not associated to spondyloarthropathy . This peptide was strikingly homologous to protein sequences from arthritogenic bacteria, particularly to a region of the DNA primase from Chlamydia trachomatis . A synthetic peptide with this bacterial sequence bound in vitro disease-associated subtypes equally as the natural B27-derived ligand . The chlamydial peptide was generated by the 20 S proteasome from a synthetic 28-mer with the sequence of the corresponding region of the bacterial DNA primase . Molecular modeling suggested that the B27-derived and chlamydial peptides adopt very similar conformations in complex with B*2705 . The results demonstrate that an HLA-B27-derived peptide mimicking arthritogenic bacterial sequences is a natural ligand of disease-associated HLA-B27 subtypes and suggest that the homologous chlamydial peptide might be presented by HLA-B27 on Chlamydia-infected cells.

Jpn J Pharmacol, 2002 Jun, 89(2), 176 - 83
Effect of anti-CD14 antibody on experimental periodontitis induced by Porphyromonas gingivalis lipopolysaccharide; Wang PL et al.; The lipopolysaccharide (LPS) released by Porphyromonas gingivalis, a Gram-negative bacterium found in the periodontal pockets of patients with periodontitis, induces bone resorbing activity in vivo . We previously showed that a receptor for LPS on human gingival fibroblasts and gingival epithelial cells is CD14 . In this study, we established a mouse model of experimental periodontitis by applying a P . gingivalis LPS solution to the buccal region of mice . P . gingivalis LPS-induced bone resorption and interleukin-6 production in the gingival tissues were significantly inhibited by pretreatment with anti-CD14 antibody for 5 weeks prior to LPS treatment . This result suggests that anti-CD14 antibody may be usable as a prototype for the development of drugs for the treatment of periodontal disease.

Nephron, 2002 Jul, 91(3), 406 - 15
Inflammatory cytokines and lipopolysaccharide induce Fas-mediated apoptosis in renal tubular cells; Jo SK et al.; BACKGROUND/AIMS: Increased susceptibility of the kidney to acute renal failure (ARF) in the setting of sepsis even in the absence of systemic hypotension is well known . In the hypothesis that the proinflammatory cytokines and lipopolysaccharide (LPS) in gram-negative sepsis can directly cause renal tubular cell apoptosis via Fas- and caspase-mediated pathways, we examined apoptosis and Fas, Fas ligand, FADD expression, as well as PARP cleavage in cultured human proximal tubular cells under the cytokine and LPS-stimulated conditions . METHODS: HK-2 cell, immortalized human proximal tubular cell lines, were treated with 5 and 30 ng/ml of tumor necrosis factor-alpha (TNF-alpha), 5 and 20 ng/ml of interleukin-1beta (IL-1beta) and 30 ng/ml LPS for 24 h . Fas expression was examined by RT-PCR and Fas ligand, Fas-associated protein with death domain (FADD) and poly ADP ribose polymerase (PARP) cleavage were examined by Western blot analysis . Apoptosis was assessed by flow cytometer using Annexin V-FITC and propidium iodide (PI) staining and also by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods . RESULTS: Fas mRNA expression (ratio of Fas/L-19) increased in the TNF-alpha 5, 30 ng/ml and LPS treated group (p < 0.01, p < 0.01, p = 0.02), but there was no difference between the low- and high-dose TNF-alpha groups . Fas ligand protein expression did not increase in the low-dose TNF-alpha treated group, but it increased significantly in the high-dose TNF-alpha treated group (p < 0.01), IL-1beta- and LPS-treated groups (p < 0.01, p = 0.01, p < 0.01, p = 0.02) . The intracellular adaptor protein, FADD expression also increased significantly in the high-dose TNF-alpha- and IL-beta-treated groups (p = 0.04, p = 0.04), but in the low-dose TNF-alpha and IL-beta treated group, it did not show statistically significant differences . In the LPS group, FADD expression also showed an increased tendency, but it was not statistically significant (p = 0.09) . Western blot for PARP, a DNA repair enzyme mainly cleaved by caspase 3, showed increased 89- and 24-kD PARP cleavage products in TNF-alpha, IL-1beta and LPS treated cells . The degree of apoptosis examined by DNA fragmentation and translocation of membrane phosphatidyl serine significantly increased in cytokines and LPS treated groups . CONCLUSION: These results suggest that Fas- and caspase-mediated apoptosis of tubular cells by inflammatory cytokines and LPS can be one of the possible mechanisms of renal dysfunction in endotoxemia .

Proc Natl Acad Sci U S A, 2002 Jul 23, 99(15), 9703 - 8 Epub 2002 Jul 15.
The CcmE protein of the c-type cytochrome biogenesis system: unusual in vitro heme incorporation into apo-CcmE and transfer from holo-CcmE to apocytochrome; Daltrop O et al.; Three key steps of cytochrome c biogenesis in many Gram-negative bacteria, the uptake of heme by the heme chaperone CcmE, the covalent attachment of heme to CcmE, and its subsequent release from CcmE to an apocytochrome c, have been achieved in vitro . apo-CcmE from Escherichia coli preferentially bound to ferric, with high affinity (K(d), 200 nM), rather than ferrous heme . The preference for ferric heme was confirmed by competition with 8-anilino-1-naphthalenesulfonate, which bound to a hydrophobic pocket in apo-CcmE . Reduction under certain conditions of the ferric heme-CcmE complex, which has characteristics of a b-type cytochrome, resulted in covalent attachment of heme to the protein . The resulting in vitro-produced holo-CcmE was identical to the in vivo-produced holo-CcmE, proving that unmodified Fe-protoporphyrin IX is incorporated into CcmE . Only noncovalent binding of mesoheme to CcmE was observed, thus implicating at least one vinyl group in covalent binding of heme to CcmE . Heme transferred in vitro from holo-CcmE to apocytochrome c, provided the heme was reduced . The necessity for reduced holo-CcmE might explain the role of the heme chaperone, i.e., prevention of reaction of ferric heme with apocytochrome and thus avoidance of incorrect side products . In addition, an AXXAH mutant of the CXXCH binding motif in the apocytochrome c was unable to accept heme from holo-CcmE . These in vitro results mimic, and thus have implications for, the molecular pathway of heme transfer during c-type cytochrome maturation in many species of bacteria in vivo.

Eur J Immunol, 2002 Jul, 32(7), 1958 - 68
Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments; Ahmad-Nejad P et al.; Recognition by innate immune cells of the pathogen associated molecular patterns (PAMP) lipopolysaccharide (LPS) from Gram-negative bacteria and bacterial CpG-DNA depends on Toll-like receptor4 (TLR4) and TLR9, respectively . To define differences in the response to these distinct PAMP we compared a key intracellular event, namely recruitment of myeloid differentiation marker 88 (MyD88) to the respective PAMP-initiated TLR signaling . Using MyD88-GFP fusion protein expressing macrophages we demonstrate that LPS and CpG-DNA trigger signaling from two different cellular locations: theformer at the cell membrane and the latter at the lysosomal compartment . While LPS does not require endocytosis to functionally associate with the membrane expressed TLR4/MD2 complex, internalization and endosomal maturation is conditional for CpG-DNA to activate TLR9 . In support of these data TLR9 is not localized at the cell surface, but intracellularily . These data stress the need to characterize individual TLR at the very beginning of signal initiation in order to understand their diverse biological functions.

Clin Infect Dis, 2002 Aug 1, 35(3), 228 - 35 Epub 2002 Jul 02.
Risk factors for late-onset nosocomial pneumonia caused by Stenotrophomonas maltophilia in critically ill trauma patients; Hanes SD et al.; Patients with nosocomial pneumonia caused by Stenotrophomonas maltophilia often receive inadequate empiric antibiotic therapy, potentially increasing mortality . Knowledge of the risk factors associated with S . maltophilia pneumonia may better guide the selection of empiric antibiotic therapy . Potential risk factors for S . maltophilia pneumonia were retrospectively analyzed for critically ill trauma patients with late-onset gram-negative pneumonia . The effects of S . maltophilia infection and inadequate empiric antibiotic therapy on patient outcomes were also assessed . By multivariate analysis, S . maltophilia pneumonia was found to be associated with cefepime exposure and tracheostomy in patients with a single pneumonia episode and with higher Injury Severity Score and pulmonary contusion in patients with multiple pneumonia episodes . S . maltophilia pneumonia was associated with increased patient morbidity; only inadequate empiric antibiotic therapy was associated with a higher mortality rate . In critically ill trauma patients with late-onset ventilator-associated pneumonia and these risk factors, empiric antibiotic therapy should include agents active against S . maltophilia.

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