Gram negative

Am J Respir Crit Care Med, 1996 Aug, 154(2 Pt 1), 460 - 8
Synergism of alveolar endotoxin "priming" and intravascular exotoxin challenge in lung injury; Walmrath D et al.; Both endotoxin (lipopolysaccharides of gram-negative bacteria; LPS) and bacterial exotoxins may induce pulmonary microcirculatory disturbances when infused into the lung vasculature, and synergism between these types of microbial challenge has recently been noted . We now asked whether a bronchoalveolar LPS load in perfused rabbit lungs alters the responsiveness to a subsequent intravascular challenge with Escherichia coli hemolysin (ECH) . In control lungs (sham aerosolization) and lungs undergoing LPS nebulization (alveolar deposition of approximately 22 micrograms), normal pulmonary artery pressure (PAP), lung weight, and ventilation/perfusion (V/Q) matching were observed . Intravascular ECH (0.013 hemolytic units/ml buffer fluid) increased PAP by approximately 10 mm Hg and lung weight by approximately 4 g within 10 min, paralleled by V/Q mismatch and a shunt flow of approximately 15% . In lungs "primed" for 3 h by a preceding bronchoalveolar LPS deposition, the same ECH dose provoked a dramatic increase in PAP to 40 to 50 mm Hg, a weight gain of approximately 10 g, and shunt flow of 60% . Both vasoconstrictor response and V/Q mismatch were completely suppressed by preadministration and "rescue" application of the thromboxane receptor antagonist BM13.505 . We conclude that a bronchoalveolar endotoxin load, though effecting no changes in pulmonary function by itself and showing no spillover into the vascular compartment, primes the lungs for a manifold increased vascular response to a subsequently infused exotoxin . Enhanced thromboxane-mediated vasoconstriction, largely redistributing perfusate flow from normally ventilated to shunt areas, is suggested as the predominant underlying event.

Am J Respir Crit Care Med, 1996 Aug, 154(2 Pt 1), 394 - 9
Intermittent enteral feeding: the influence on respiratory and digestive tract colonization in mechanically ventilated intensive-care-unit patients; Bonten MJ et al.; Continuous enteral feeding (CEF) has been associated with decreased gastric acidity, thereby stimulating gastric colonization and ventilator-associated pneumonia (VAP) . Intermittent enteral feeding (IEF) could induce a temporary increase in gastric acidity and decrease the risk of VAP . We studied the influence of IEF (18 h/d) and CEF (24 h/d) on gastric and oropharyngeal colonization . Sixty patients were randomized to receive either IEF or CEF, and continuous intragastric pH monitoring was performed in 50 patients . Median intragastric pH levels were similar before enteral feeding was instituted (pH 2.5 for CEF and pH 2.4 for IEF), and median pH values increased slightly after institution of nutrition (NS) . In patients receiving IEF, median pH decreased from 3.5 to 2.2 (p = 0.0002) when enteral feeding was discontinued . However, despite this, 80% of the patients in both study groups were colonized in the stomach after 7 d in study . In addition, colonization rates of the oropharynx and trachea, the incidence of VAP, and mortality were similar in both study groups . IEF was less well tolerated than CEF . We conclude that almost all patients receiving enteral feeding are colonized in the stomach with gram-negative bacteria . IEF resulted in a slight decrease in intragastric pH without influencing rates of colonization and infection of the respiratory tract.

J Bacteriol, 1996 Aug, 178(15), 4438 - 44
Starvation-induced expression of retron-Ec107 and the role of ppGpp in multicopy single-stranded DNA production; Herzer PJ; Multicopy single-stranded DNA is found as a small single-stranded RNA-DNA complex in certain wild-type strains of Escherichia coli as well as in other gram-negative bacteria . Using the promoter region of the previously characterized retron-Ec107 from E . coli ECOR70, I constructed a chromosomally located lacZ operon fusion . Examination of expression from the PEc107 promoter showed that activity increased sharply when cells entered stationary phase in rich medium or when they were starved for phosphate . The nucleotide guanosine-3',5'-bispyrophosphate was found to be a positive regulator of retron-Ec107 expression . Its presence is required for starvation-induced transcription of retron-Ec107 and multicopy single-stranded DNA production . It was also found that expression from the retron promoter is independent of the sigma factor sigmaS.

Crit Care Med, 1996 Aug, 24(8), 1293 - 301
Perioperative endotoxemia and bacterial translocation during major abdominal surgery: evidence for the protective effect of endogenous prostacyclin?
Brinkmann A, Wolf CF, Berger D, Kneitinger E, Neumeister B, Buchler M, Radermacher P, Seeling W, Georgieff M.
OBJECTIVE: To investigate the potential role of endogenous prostacyclin (PGI2) released after mesenteric traction during major abdominal surgery on perioperative endotoxemia and bacterial translocation . DESIGN: Prospective, randomized, double-blind clinical study . SETTING: Operating room and surgical intensive care unit in a university hospital . PATIENTS: Fifty consecutive patients scheduled for major abdominal surgery (pancreas resection, abdominal aortic surgery) . INTERVENTIONS: Fifteen minutes before skin incision, either 400 mg of ibuprofen or a placebo equivalent were administered intravenously . Immediately after peritoneal incision, eventration and action of the small bowel was intentionally performed in a uniform fashion . MEASUREMENTS AND MAIN RESULTS: Baseline values were obtained before induction of anesthesia . Additional measurements, along with assessments of hemodynamics and gas exchange, were performed before incision of the peritoneum and at 5, 30, and 45 mins and 3, 6, and 24 hrs after mesenteric traction . Arterial plasma concentrations of 6-keto-prostaglandin F1 alpha and thromboxane B2 (stable metabolites of PGI2 and thromboxane A2) were determined by radioimmunoassay . Endotoxin was measured by limulus amebocyte lysate test . Mesenteric lymph nodes were sampled in 31 patients (ibuprofen n = 14, placebo n = 17) and sent for culture under sterile conditions . Transient hypotension and a marked increase of plasma 6-keto-prostaglandin F1 alpha concentrations occurred up to 6 hrs after mesenteric traction in untreated patients with median peak concentrations (2243 vs . 72 ng/L {p < .0001, placebo vs . ibuprofen}, observed 5 mins after mesenteric traction) . Endotoxemia occurred in both study groups . However, after mesenteric traction, plasma endotoxin concentrations were significantly higher in the ibuprofen group . Median peak concentrations (0.12 vs . 0.27 EU/mL {p < .001, placebo vs . ibuprofen}) were observed 3 hrs after mesenteric traction . Gram-negative bacteria in mesenteric lymph nodes were detected exclusively in the ibuprofen group (n = 5, p < .01) . CONCLUSIONS: In ibuprofen-pretreated patients, significantly higher endotoxin concentrations as well as bacterial translocation to mesenteric lymph nodes occurred, despite the absence of a transient decrease in mean arterial pressure that had been associated with PGI2 release . Therefore, we hypothesized that during major abdominal surgery, endogenous PGI2 released in response to mesenteric traction may play a crucial role in maintaining splanchnic microcirculation and thus preserving gut mucosal barrier function.

Appl Environ Microbiol, 1996 Aug, 62(8), 2961 - 5
Distribution of class II transposase and resolvase genes in soil bacteria and their association with mer genes; Pearson AJ et al.; Southern hybridization was performed on 30 gram-negative, mercury-resistant soil bacteria isolated from three terrestrail sites in Great Britain; two of these sites were mercury polluted (SO and SE), and one was pristine (SB) . Most of the isolates (20 of 30) hybridized to probes encoding regions of the transposase (tnpA) and resolvase (tnpR) genes from Tn501 and Tn21 . Isolates SE9 and SB3 hybridized to the Tn21 but not the Tn501 tnpA probe; however, they differed in that SB3 hybridized to both Tn501 and Tn21 tnpR probes while SE9 did not hybridize to either tnpR probe . The remaining isolates (7 of 30) did not hybridize to any of the transposon gene probes under the conditions used . tnpA and tnpR regions were PCR amplified from most of the hybridizing isolates and from Tn501 and Tn21, and variation was assessed by restriction fragment length polymorphism analysis . On the basis of these data, tnpA regions were divided into eight restriction fragment length polymorphism classes and tnpR regions were divided into five classes . Similarity coefficients were calculated between classes and used to construct dendrograms showing percent similarity . A compilation of the data from this study on tnpA and tnpR regions and a previous study on merRT delta P regions (A . M . Osborn, K . D . Bruce, P . Strike, and D . A . Ritchie, Appl . Environ . Microbiol . 59:4024-4030, 1993) indicates the presence of hybrid transposons and provides evidence for extensive recombination, both between transposon genes and between transposon and mer genes, within these natural populations of bacteria.

Laryngoscope, 1996 Aug, 106(8), 1028 - 33
Reduction of endotoxin-induced inflammation of the middle ear by polymyxin B; Darrow DH et al.; Endotoxin (ET) is an aggregate of lipo-oligosaccharide and protein found in the cell wall of gram-negative bacteria . A potent mediator of inflammatory responses, ET has been detected in middle ear effusions from patients with otitis media with effusion and chronic suppurative otitis media and used to induce inflammation of the middle ear mucosa and disruption of mucociliary transport in experimental animals . Polymyxin B, a polypeptide antibiotic, has been shown to bond to and inactivate the ET molecule . This study investigated the efficacy of polymyxin B as a modulator of the inflammatory response to endotoxin in the middle ear . In a guinea pig model, cellular infiltrate, effusion volume, and mucosal edema in response to ET were reduced in the presence of polymyxin B . These results suggest a potential role for the use of polymyxin B in the management of middle ear effusion.

J Am Acad Dermatol, 1996 Aug, 35(2 Pt 2), 285 - 7
Widespread cutaneous bacillary angiomatosis and a large fungating mass in an HIV-positive man; Fagan WA et al.; Bacillary angiomatosis (BA), an infection caused by a gram-negative rod, can be a multiorgan disease . The usual causative organism, Bartonella (formerly Rochalimaea) hensalae, has only recently been identified . Bartonella quintana has also been shown to cause some cases of cutaneous BA . We describe a patient with widespread cutaneous BA with probable bone involvement and a large fungating mass.

Anesth Analg, 1996 Aug, 83(2), 242 - 6
Plasma potentiates the priming effects of endotoxin on platelet activating factor-induced pulmonary hypertension in the rabbit lung; Goldsmith JA et al.; During Gram-negative sepsis, endotoxin lipopolysaccharide (LPS) may activate host inflammatory responses, resulting in the systemic inflammatory response syndrome and the adult respiratory distress syndrome . In cell culture systems, LPS activation of cellular responses may be potentiated by plasma proteins . In the isolated perfused rabbit lung, LPS administration markedly increases the pulmonary hypertensive response to subsequent administration of platelet activating factor (PAF) . We examined whether plasma would potentiate the priming effects of LPS in this model . Male New Zealand White rabbits were used in a standard, isolated buffer-perfused rabbit lung preparation, and the pulmonary hypertensive response to 5 nM PAF was measured after 2 h of perfusion with different LPS doses (0, 1, and 10 ng/mL), with and without plasma (10% by volume) . In the absence of plasma, 10 ng/mL LPS, but not 1 ng/mL LPS, increased the pulmonary hypertensive response to subsequent administration of 5 nM PAF . However, in the presence of plasma, 1 ng/mL LPS significantly increased the hypertensive response to subsequent administration of 5 nM PAF . We conclude that components of plasma--possibly LPS binding protein and soluble CD14--potentiate the priming effect of endotoxin, resulting in an augmented pulmonary hypertensive response to PAF . Thus, plasma proteins decrease the threshold at which endotoxin primes the lung and may have a critical role in the pathogenesis of endotoxin-induced acute lung injury.

EMBO J, 1996 Aug 1, 15(15), 3792 - 805
Molecular basis of two subfamilies of immunoglobulin-like chaperones; Hung DL et al.; The initial encounter of a microbial pathogen with the host often involves the recognition of host receptors by different kinds of bacterial adhesive organelles called pili, fimbriae, fibrillae or afimbrial adhesins . The development of over 26 of these architecturally diverse adhesive organelles in various Gram-negative pathogens depends on periplasmic chaperones that are comprised of two immunoglobulin-like domains . All of the chaperones possess a highly conserved sheet in domain 1 and a conserved interdomain hydrogen-bonding network . Chaperone-subunit complex formation depends on the anchoring of the carboxylate group of the subunit into the conserved crevice of the chaperone cleft and the subsequent positioning of the COOH terminus of subunits along the exposed edge of the conserved sheet of the chaperone . We discovered that the chaperones can be divided into two distinct subfamilies based upon conserved structural differences that occur in the conserved sheet . Interestingly, a subdivision of the chaperones based upon whether they assemble rod-like pili or non-pilus organelles that have an atypical morphology defines the same two subgroups . The molecular dissection of the two chaperone subfamilies and the adhesive fibers that they assemble has advanced our understanding of the development of virulence-associated organelles in pathogenic bacteria.

Curr Microbiol, 1996 Aug, 33(2), 89 - 93
Flexibacter japonensis sp . nov., a new species that produces a novel inhibitor of human leukocyte elastase isolated from soil; Fujita T et al.; This strain 758 of a new member of Flexibacter species isolated from a soil is a Gram-negative, nonflagellated, gliding, long rod or filamentous . The GC contents of the deoxyribonucleic acid of this strain is 49.8 mol %GC . The isolate can be distinguished from other Flexibacter species on the basis of physiological and biochemical properties and DNA relatedness data . Therefore, we propose a new species, Flexibacter japonensis, for this strain . The strain 758 is deposited to the Japan Collection of Microorganisms as JCM 9735.

Ann N Y Acad Sci, 1996 Jul 23, 791, 378 - 401
Pore-forming activity of Coxiella burnetii outer membrane protein oligomer comprised of 29.5- and 31-kDa polypeptides . Inhibition of porin activity by monoclonal antibodies 4E8 and 4D6; Banerjee-Bhatnagar N et al.; Envelopes of large-cell variant Coxiella burnetii, the agent of Q fever, were the starting material for purification of an outer membrane protein (OMP) oligomer with aggregate molecular mass of approximately 2 x 10(4) kDa . The oligomer was resistant to trypsin and dissociation by SDS at 100 degrees C . Reducing agents dissociated the oligomer into monomers of 29.5 and 31 kDa, which migrated as a doublet during SDS-polyacrylamide gel electrophoresis . Both monomers were reactive in an immunoblot assay with monoclonal antibodies (mAbs) 4E8 and 4D6, which were previously selected for their reactivity with purified and SDS-denatured 29.5 kDa protein . Proteoliposomes were functional in an equilibrium assay at pH 7 and a swelling assay at pH 7 and 4.5 . The pores in proteoliposomes allowed the passage of arabinose, glucose, and sucrose, but restricted stachyose . Polyclonal antibodies to C . burnetii cells and the mAbs were able to bind C . burnetii at pH 7 and 4.5 . The uptake of 14C-glucose at pH 4.5 was inhibited by polyclonal antibodies and mAbs after binding to cells at pH 7 . The mAbs did not inhibit 14C-glucose uptake at pH 4.5 after binding to cells at pH 4.5 . Although the mAbs bind C . burnetii porin epitopes before and after acid activation, the mAbs bound under acidic conditions were unable to inhibit porin function . The inhibition of porin channel function by mAbs confirms the role of porin as a permeability barrier for the subsequent active transport of glucose by C . burnetii . In another study, we showed that the 29.5 kDa OMP antigen induced active immunity against virulent challenge . This information, combined with the recent confirmation that porins are important antigens in the induction of specific protective immune responses against infection by gram-negative bacteria, suggests that humoral immunity directed against C . burnetii porins might play an important role in immunity against Q fever (human infection) and coxiellosis (animal infection), global enzootic diseases.

Proc Natl Acad Sci U S A, 1996 Jul 23, 93(15), 7888 - 93
Purification and molecular cloning of an inducible gram-negative bacteria-binding protein from the silkworm, Bombyx mori; Lee WJ et al.; A 50-kDa hemolymph protein, having strong affinity to the cell wall of Gram(-) bacteria, was purified from the hemolymph of the silkworm, Bombyx mori . The cDNA encoding this Gram(-) bacteria-binding protein (GNBP) was isolated from an immunized silkworm fat body cDNA library and sequenced . Comparison of the deduced amino acid sequence with known sequences revealed that GNBP contained a region displaying significant homology to the putative catalytic region of a group of bacterial beta-1,3 glucanases and beta-1,3-1,4 glucanases . Silkworm GNBP was also shown to have amino acid sequence similarity to the vertebrate lipopolysaccharide receptor CD14 and was recognized specifically by a polygonal anti-CD14 antibody . Northern blot analysis showed that GNBP was constitutively expressed in fat body, as well as in cuticular epithelial cells of naive silkworms . Intense transcription was, however, rapidly induced following a cuticular or hemoceolien bacterial challenge . An mRNA that hybridized with GNBP cDNA was also found in the l(2)mbn immunocompetent Drosophila cell line . These observations suggest that GNBP is an inducible acute phase protein implicated in the immune response of the silkworm and perhaps other insects.

Acta Med Port, 1996 Jul-Sep, 9(7-9), 187 - 95
{Individualized monitoring of the therapy with gentamycin using pharmacokinetic methods . Which method to choose?}; Carvalho A et al.; Gentamicin has an excellent cost/efficacy ratio for gram negative infections treatment . Its use is often limited in clinical practice by its narrow safety margins and a high incidence of toxicity . Gentamicin related nephrotoxicity is a major adverse effect, mostly in patients with other concomitant potential risk factors . As many other Authors we have found in our Internal Medicine Service during 1992 a gentamicin related nephrotoxicity incidence of 22.5% . Various empiric methods and nomograms have shown a significant incidence of error in predicting individualized gentamicin dosage regimens . Pharmacokinetics methods have demonstrated much better results regarding efficacy and toxicity . The aim of this prospective study carried out during 1993-1994 was to individualize by pharmacokinetics methods dosage regimens of gentamicin in patients with one or more concomitant risk factors of nephrotoxicity . The purpose of pharmacokinetics dosage regimens has been to achieve trough serum concentrations of gentamicin in therapeutics range-0.5 to 2 micrograms/ml-on the first 24 to 48 hours of treatment, and the maintenance in this range during all the treatment, avoiding both toxic and under therapeutic levels . The incidence of gentamicin related nephrotoxicity has been evaluated in this population . Twenty patients were studied: 18 males and 2 females aged 59.6 years (19 to 85) . All had one or more potential risk factors for nephrotoxicity-65 years or more: 13, previous renal failure: 6, other nephrotoxic drugs: 10, diuretics: 4, dehydration: 5, congestive heart failure: 5, diabetes: 3, hypertension: 3 . For the first 10 patients gentamicin dosage regimens have been determined by Sawchuk-Zaske pharmacokinetics method and for the subsequent 10 patients by Bayesian method . The two subpopulations had no significant differences regarding mean age, sex and potential risk factors for nephrotoxicity . Results of Sawchuk-Zaske method: 53 trough gentamicin serum concentration were obtained; 86.8% were within the therapeutic range, 7.5% were toxic and 5.7% were under therapeutic . Results of Bayesian method: 44 determinations of gentamicin through concentrations were obtained; 86.3% within therapeutic range, 2.4% were toxic and 11.3% were under therapeutic . A great variability in pharmacokinetic patient's profile has been found and explains the great variability of individualized dosage regimens of gentamicin (30 to 320 mg/day) . No patients had gentamicin related nephrotoxicity . Both pharmacokinetics methods lead to a efficient and save employment of gentamicin in patients with previous renal failure and other potential risk factors for nephrotoxicity.

Appl Microbiol Biotechnol, 1996 Jul, 45(6), 778 - 84
Isolation and characterization of a bacterial growth-stimulating peptide from a peptic bovine hemoglobin hydrolysate; Zhao QY et al.; A peptide with a bacterial-growth-stimulating activity was isolated from a bovine hemoglobin hydrolysate by reversed-phase high-performance liquid chromatography . Its primary structure and molecular mass, determined by amino acid analysis and fast-atom bombardment mass spectrometry, were identical to those of fragment 48-52 (Ser-Thr-Ala-Asp-Ala) of the beta chain of bovine hemoglobin . The microbiological tests in solid media demonstrated that this peptide exhibited a growth-stimulating activity on gram-negative bacteria.

Laryngorhinootologie, 1996 Jul, 75(7), 403 - 7
{Cat scratch disease . An overview for the ENT physician}; Dreher A et al.; BACKGROUND: Cat scratch disease (CSD) is a relatively common cause of chronic lymphadenopathy in the USA . In the present paper the authors describe recent advances in the understanding of this disorder focusing on etiology, clinical aspects, diagnostic management, and therapy . ETIOLOGY: Rochalimaea henselae and Afipia felis, two gram-negative bacteria, have recently been isolated from lymph node tissue of patients suffering from CSD . The current literature reveals that Rochalimaea henselae seems to be the most probable agent responsible for CSD . Serum samples from CSD patients' cats have shown titers of 1:64 or higher for antibodies to Rochallmaea henselae . EPIDEMIOLOGY: The incidence of CSD in the USA is between 0.77 and 9.3 per 100,000 per year . The incidence of CSD in Europe is unknown, but the prevalence of antibodies to Rochalimaea henselae among cats is comparable . CLINICAL MANIFESTATIONS: Most common symptoms of CSD are regional lymphadenopathy, fever, and malaise . Other manifestations occur in about 5% of patients and include encephalitis, granulomatous hepatitis, and Parinaud's oculoglandular syndrome . Fatal complications and irreversible sequelae have not been reported . DIAGNOSIS: To establish the diagnosis of CSD, the presence of regional lymphadenopathy, cat contact, and papula are required . Additional procedures include indirect fluorescent antibody assays and PCR . THERAPY: Up to now there is no standard therapy for CSD . Antibiotic treatment, however, might be considered for CSD patients with severe symptoms . Rifampicin, ciprofloxacin, trimethoprim sulfamethoxazole, and gentamicin are known to be effective antibiotic agents.

Trans R Soc Trop Med Hyg, 1996 Jul-Aug, 90(4), 406 - 8
Infections in haematological malignancies: an autopsy study of 72 cases; Srivastava VM et al.; Autopsy material from 72 patients with haematological malignancies treated in India was reviewed . Thirty-seven patients (51%) had documented infections; 20 (27%) had bacterial infections, 14 of which were Gram-negative organisms (Pseudomonas species in 10); tuberculosis was present in 2 patients (2.7%) . Twenty-one patients (29%) had systemic fungal infections; invasive pulmonary aspergillosis and gastrointestinal candidiasis were present in 10 patients each . Only 3 patients (4%) had viral infection, all of which were due to cytomegalovirus . Eleven patients (15%) had polymicrobial infections . No patient had any parasitic infection . Systemic fungal infections due to Aspergillus and Candida predominated, while Gram-negative bacterial infections were also common.

Gut, 1996 Jul, 39(1), 136 - 40
Intestinal immunisation with Escherichia coli protects rats against Escherichia coli induced cholangitis; Aagaard BD et al.; BACKGROUND: Cholangitis, an infection of the biliary tract, is most commonly caused by Gram negative bacteria, particularly Escherichia coli . Factors governing the severity of cholangitis, including the role of biliary IgA, are poorly understood . AIMS: The aim of this work was to find out if biliary IgA directed against E coli protects rats against hepatobiliary infection with E coli . SUBJECTS: Male Sprague-Dawley rats weighing 270-350 grams were used in all of the experiments . METHODS: At laparotomy, rats were immunised by injecting killed E coli or normal saline (controls) into Peyer's patches . With or without subsequent antigenic boosting (by oral administration of killed E coli), bile was collected at a second laparotomy, and rats were infected by introducing viable E coli into the bile duct . Production of IgA anti-E coli antibody was measured by enzyme linked immunosorbent assay of bile, and the presence of hepatobiliary infection was determined by quantitative culture of liver homogenates . RESULTS: Systemic infection was present in six of 12 control rats and in one of 24 immunised rats (p = 0.005) after death . There was an inverse correlation between immunisation and E coli colony counts in cultured liver homogenates (p = 0.024) . CONCLUSION: The findings suggest that biliary IgA directed against E coli protected rats against hepatobiliary E coli infection and systemic sepsis.

Eur J Clin Invest, 1996 Jul, 26(7), 596 - 601
Detection of gram-negative bacteraemia in early sepsis by a quantitative chromogenic and kinetic endotoxin assay . The Study Group; Massignon D et al.; A kinetic chromogenic limulus test was carried out in order to investigate the possibility of a sensitive and specific detection of circulating endotoxin during the first 24 h of septic shock or severe sepsis in 76 patients . Two commercial kits, Whittaeker (W) and Chromogenix (C), were used . Blood culture was taken as a reference . At 1:10 plasma dilution (a currently used dilution in the end point limulus test) abnormal reaction kinetics were found in 13% and 41% of tests, for C and W respectively (P = 0.0008), resulting in unreliable results . Retesting plasma at a greater dilution, until the reaction kinetic was identical to calibration curve control values, gave similar results between the two kits and a better accuracy . Beyond a 0.5 EU mL-1 endotoxin level, the probability of Gram-negative bacteraemia was high (sensitivity = 0.53 and 0.47; specificity = 0.95 and 0.93 for C and W respectively) . This kinetic limulus amoebocyte lysate (LAL) test may be useful in therapeutic decisions for treatment of endotoxaemia.

Mol Microbiol, 1996 Jul, 21(2), 221 - 31
Coating the surface: a model for expression of capsular polysialic acid in Escherichia coli K1; Bliss JM et al.; Capsules are well-studied components of the bacterial surface that modulate interactions between the cell and its environment . Generally composed of polysaccharide, they are key virulence determinants in invasive infections in humans and other animals . Genetic determinants involved in capsule expression have been isolated from a number of organisms, but perhaps the best characterized is the kps cluster of Escherichia coli K1 . In this review, the current understanding of the functions of the kps gene products is summarized . Further, a proposed mechanistic model for capsule expression is presented and discussed . The model is based on the premise that the numerous components of the kps cluster form a hetero-oligomeric complex responsible for synthesis and concurrent translocation of the capsular polysialic acid through sites of inner and outer membrane fusion . We view the ATP-binding cassette (ABC) transporter, KpsMT, to be central to the functioning of the complex, interacting with the biosynthetic apparatus as well as the extracytoplasmic components of the cluster to co-ordinate synthesis and translocation . The model provides the basis for additional experimentation and reflects emerging similarities among systems responsible for macromolecular export in Gram-negative bacteria.

J Formos Med Assoc, 1996 Jul, 95(7), 551 - 4
Pulmonary hyperinfection with Strongyloides stercoralis; Huang MS et al.; Strongyloides stercoralis is an intestinal parasite in humans . Infected patients may be asymptomatic or have mild to moderate abdominal symptoms . It may spread to the lungs and, finally, disseminate in the immunocompromised patient . S . stercoralis is an important cause of severe pulmonary infection and death in many areas of the world . Here we describe an 87-year-old man with S . stercoralis pulmonary hyperinfection . He had respiratory failure with severe abdominal distention . Chest x-ray showed infiltration over the right upper lung field . Papanicolaou stain of sputum demonstrated the rabditiform larvae of S . stercoralis . Stool examination revealed S . stercoralis larvae and eggs . He received two courses of albendazole treatment, but died 5 weeks after admission from Gram-negative bacteremia . This case is a classic presentation of the S . stercoralis pulmonary hyperinfection syndrome.

Antonie Van Leeuwenhoek, 1996 Jul, 70(1), 79 - 87
Interaction of the salivary low-molecular-weight mucin (MG2) with Actinobacillus actinomycetemcomitans; Groenink J et al.; Periodontitis is associated with the presence of certain Gram-negative bacteria in the oral cavity, among these Actinobacillus actinomycetemcomitans . In order to determine which types of salivary components interact with A . actinomycetemcomitans two strains (HG 1175 and FDC Y4) were incubated with whole saliva and individual glandular secretions, viz . parotid, submandibular, and sublingual saliva . Immunochemical analysis by immunoblotting of bacteria-bound salivary proteins showed that IgA, the low-molecular mucin MG2, parotid agglutinin, and a 300 kDa sublingual and submandibular glycoprotein, were bound to the bacterial strains tested . In addition, adherence of A . actinomycetemcomitans to salivary proteins in a solid-phase was studied . After electrophoresis and transfer of salivary proteins to nitrocellulose membranes A . actinomycetemcomitans adhered only to MG2 . In this assay periodate treatment, mild acid hydrolysis or neuraminidase digestion of the saliva glycoproteins abolished binding of two clinical isolates (HG 1175 and NY 664), suggesting that sialic acid residues on MG2 are involved in the binding . In contrast, adherence of the smooth laboratory strain Y4 was not affected by removal of sialic acid residues or even periodate treatment of MG2.

ASAIO J, 1996 Jul-Aug, 42(4), 321 - 3
Management of acute airway obstruction using extracorporeal membrane oxygenation; Morneault L et al.; The authors describe, to their knowledge, the first management of acute airway obstruction in a newborn infant using Extracorporeal Membrane Oxygenation (ECMO) . The infant had a primary diagnosis of gram negative sepsis complicated by pulmonary hemorrhage resulting in a left main stem bronchus obstruction . Despite full ventilatory support, the infant could not be adequately oxygenated . The infant was placed on venovenous ECMO . Airway management also included vigorous physiotherapy, suctioning, and bronchoscopy . The infant was successfully weaned from ECMO after 208 hours . The authors suggest that ECMO could be useful to manage life threatening airway obstruction in the neonate.

Microvasc Res, 1996 Jul, 52(1), 84 - 100
Lung microvessel injury from peritoneal abscesses and gram-negative bacteremia; Jones R et al.; To analyze the effect of an extrathoracic focus of infection on lung vessel wall structure, we produced peritoneal abscesses in the rat, over a period of 3 to 7 weeks, by implanting capsules containing live gram-negative bacteria (3.0 x 10(7) Escherichia coli and 5.0 x 10(7) Bactercides fragilis) with an adjutant . We document here by arteriography, morphometric analysis, and high resolution microscopy, microvessel cell injury, and wall remodeling . In the sepsis-injured lung, both dilated and thick-walled microvessels are present . The walls of dilated vessels are disrupted by extensive endothelial and precursor smooth muscle cell injury . In thick-walled vessels these cells are hypertrophied, and the precursor smooth muscle cells express filaments, demonstrating a shift toward a contractile phenotype . Infiltrating monocytic cells focally consolidate alveolar regions . In the residual nonconsolidated regions, alveolar-capillary membrane cells are attenuated and the capillaries dilated . Vessel changes after 7 weeks are similar to those after 3 weeks but alveolar-capillary membrane injury is more extensive . These structural changes, including the development of precursor smooth muscle cells, may contribute to the known increase in reactivity of these lung vessels to challenge by vasoactive agents.

Zentralbl Veterinarmed A, 1996 Jul, 43(5), 271 - 9
Prevalence of antibodies to lipid A in Danish cattle; Andersen PH et al.; A cross-sectional study was performed on the occurrence of IgG antibodies to lipid A of the Gram-negative bacterial lipopolysaccharide (LPS, endotoxin) on serum of 2272 cattle distributed on 19 Danish dairy herds . The relationship between the concentration of antibodies to lipid A (ALI) and age, herd, pregnancy rate and occurrence of mastitis, bovine virus diarrhoea (BVD), reproductive and digestive disorders, diarrhoea, pneumonia, foot disorders, various infections and traumatic udder lesions was investigated . ALI generally was low in calves and increased during their first 1.5 years of life to a steady state, which could be altered by the occurrence of disease . There were significant differences in the mean ALI among the herds (P < 0.001) . High ALI was associated with a low herd pregnancy rate, to preceding occurrence of mastitis (P < 0.048), BVD (P < 0.01), reproduction diseases (P < 0.041) and digestion disorders (P < 0.064) in animals older than 2 years . The calf mortality rate was not associated to ALI and there was no correlation between the ALI in calves and their dams . The occurrence of high ALI levels on a herd basis may be an indication of increased challenge or enhanced immunological defense to Gram-negative bacteria or endotoxin.

Can J Microbiol, 1996 Jul, 42(7), 672 - 84
The extreme N-terminus of the Caulobacter crescentus surface-layer protein directs export of passenger proteins from the cytoplasm but is not required for secretion of the native protein; Bingle WH et al.; The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane . Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface . To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5'-region of the rsaA gene . This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein . Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (delta CenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9% . No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell . RsaA:PhoA hybrid proteins were also exported from the E . coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA . Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.

J Pharmacol Exp Ther, 1996 Jul, 278(1), 378 - 83
The thrombin inhibitor, hirudin, attenuates lipopolysaccharide-induced liver injury in the rat; Pearson JM et al.; The administration of gram-negative bacterial lipopolysaccharide (LPS) to rats results in hepatic parenchymal cell injury within 6 hr . The coagulation system is critical to the pathogenesis, but previously reported results suggested that its critical role is independent of insoluble clot formation and that thrombin may be a key mediator of liver injury . To test the hypothesis that thrombin is involved in LPS-induced liver injury, animals were treated with the selective thrombin inhibitor, hirudin . The hirudin treatment regimen effectively inhibited thrombin, as evidenced by prolonged activated partial thromboplastin time and by maintenance of plasma fibrinogen concentrations in LPS-treated rats . Treatment with hirudin prevented LPS-induced liver injury, assessed by plasma alanine aminotransferase activity and histological evidence of hepatocellular necrosis . Previous studies have shown that LPS exposure results in the accumulation of neutrophils and platelets within the liver and that both of these cell types are critical for the development of LPS-induced liver injury . Hirudin attenuated in part the decrease in blood platelet concentration that accompanied LPS administration, but did not alter hepatic platelet or neutrophil accumulation . These results support the hypothesis that thrombin is required for hepatic injury from LPS exposure, but that it does not act by promoting the accumulation of platelets or neutrophils within the liver.

J Bacteriol, 1996 Jul, 178(14), 4323 - 6
Identification and properties of a novel clt locus in the Streptomyces phaeochromogenes plasmid pJV1; Servin-Gonzalez L; A novel clt locus required for efficient transfer of the Streptomyces phaeochromogenes plasmid pJV1 was identified and mapped . The clt region was functional in both orientations, and its absence caused a severe reduction in plasmid transfer . Chromosome mobilization, on the other hand, was not affected by absence of the clt locus . The clt region showed structural, but not sequence, similarity to transfer origins of gram-negative plasmids.

Clin Exp Immunol, 1996 Jul, 105(1), 74 - 8
Elevated levels of soluble CD14 in serum of patients with acute Plasmodium falciparum malaria; Wenisch C et al.; Serum sCD14, tumour necrosis factor-alpha (TNF-alpha), IL-6, and endotoxin were analysed in 45 patients with complicated malaria, in 14 patients with Gram-negative septicaemia and in 24 healthy subjects by ELISA . Malaria patients with renal failure (n = 16) had higher levels than patients without renal failure (n = 29) (8116 + 1440 micrograms/l versus 9453 + 1017 micrograms/l; P < 0.05) and both had higher levels than patients with septicaemia (6155 + 1635 micrograms/l) and normal subjects (2776 + 747 micrograms/l) . A significant correlation between sCD14 and IL-6 (r = 0.756) and TNF (r = 0.822) existed . However, no relation between sCD14 and serum endotoxin or indices of clinical disease severity (parasitaemia, fever, parasite or fever clearance time) was seen . Although the role of sCD14 in malaria remains to be determined, elevated levels may participate in the inflammatory response in complicated malaria.

Br J Cancer, 1996 Jul, 74(2), 312 - 7
The reduction of radiation mucositis by selective decontamination antibiotic pastilles: a placebo-controlled double-blind trial; Symonds RP et al.; The aim of this study was to see if antibiotic pastilles could reduce radiation mucositis, pain, dysphagia and weight loss in patients undergoing radical radiotherapy for head and neck cancer . A total of 275 patients with T1-T4 tumours entered the study; 136 were allocated to suck four times daily a pastille containing amphotericin, polymyxin and tobramycin . The remaining 139 patients received an identical placebo . In all, 54 patients were unevaluable (24 active, 30 placebo) . Bacteriological monitoring was carried out before and twice weekly during treatment . Both arms of the study were well balanced for T and N stage, age, sex and radiation dose (60 Gy) . There was a slight imbalance in the site of disease which had no substantive effect on the results . The primary study end point was the percentage of patients who developed intermediate or thick pseudomembranes . No statistically significant difference was found in this end point, with 36% of patients in the active arm developing this type of membrane compared with 48% in the placebo arm (P = 0.118) . The estimated odds ratio (placebo/active) of developing an intermediate or thick pseudomembrane was 1.59 (95% CI 0.89-2.82) . However a more sensitive test comparing the worst recorded mucositis grade between the two arms was statistically significant (P = 0.009) . This indicated that the active pastilles had a beneficial effect, but the magnitude was probably smaller than the trial was designed to detect . There was a reduction in mucositis distribution (P = 0.002), mucositis area (P = 0.028), dysphagia (P = 0.006) and weight loss (P = 0.009) in the active arm . There was a clear tendency for patients with positive cultures for aerobic Gram-negative bacteria (AGNB) (P = 0.003) and yeasts (P = 0.026) during treatment to have more severe mucositis . The active pastilles reduced the percentage of patients with yeast cultures (P = 0.003) but had less effect on AGNB . The benefit derived from the pastilles should materially increase patient tolerance to radical radiotherapy for head and neck cancer.

J Bacteriol, 1996 Jul, 178(13), 3715 - 21
Polyamines decrease Escherichia coli outer membrane permeability; Dela Vega AL et al.; The permeability of the outer membranes of gram-negative bacteria to hydrophilic compounds is mostly due to the presence of porin channels . We tested the effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on two processes known to depend on intact porin function: fluxes of beta-lactam antibiotics in live cells and chemotaxis . In both cases, inhibition was observed . Measurements of the rate of permeation of cephaloridine and of chemotaxis in swarm plates and capillary assays were used to determine the concentration dependence of this modulation . The effective concentration ranges depended on the nature of the polyamine and varied from submillimolar for spermine to tens of millimolar for cadaverine . Both OmpC and OmpF porins were inhibited, although the effects on OmpC appeared to be milder . These results are in agreement with our observations that polyamines inhibit porin-mediated ion fluxes in electrophysiological experiments, and they suggest that a low-affinity polyamine binding site might exist in these porins . These results reveal the potential use of porins as targets for blocking agents and suggest that polyamines may act as endogenous modulators of outer membrane permeability.

Chest, 1996 Jul, 110(1), 243 - 8
Intermittent enteral feeding in mechanically ventilated patients . The effect on gastric pH and gastric cultures; Spilker CA et al.; OBJECTIVE: To evaluate the effect of intermittent (16 h/d) enteral feeding (IEF) on gastric pH and gastric microbial growth in mechanically ventilated patients . DESIGN: Prospective, case-controlled study . SETTING: Medical ICU and infectious disease research laboratory in a university hospital . PATIENT POPULATION: Thirteen mechanically ventilated patients receiving continuous enteral feeding (CEF) . METHODS: Gastric pH and quantitative gastric cultures were obtained while patients received CEF . Each patient's feeding schedule was changed to IEF . Daily gastric pH and quantitative gastric cultures were obtained for 5 consecutive days . RESULTS: Gastric microbial growth was found in 85% (11/13) of patients receiving CEF . Implementation of IEF did not clear gastric microbial growth, as only one patient subsequently reverted to negative culture . Similar gastric microbial growth continued in 90% (10/11) of patients after institution of IEF . Gastric pH did not decrease with the administration of IEF (gastric pH with IEF, 3.8 +/- 0.6 vs 4.7 +/- 0.5 with CEF (not significant {NS}) . The amount of microbial growth was also unchanged with IEF (total growth with IEF, 7.8 x 10(5) +/- 5.2 x 10(5) cfu/mL vs 8.7 x 10(5) +/- 4.6 x 10(5) cfu/mL with CEF) (NS) . Thirty-eight percent (5/13) of patients developed new Gram-negative rod growth in gastric cultures while receiving IEF . Gram-negative rod isolates increased from 25% of total isolates (CEF) to 40% (IEF) . CONCLUSION: Our preliminary data suggest gastric pH was not lowered and existing microbial growth was not cleared in ventilated patients receiving IEF after previously receiving CEF . Further controlled study in a larger group of patients is necessary to determine whether IEF is of benefit in decreasing gastric colonization and nosocomial pneumonia.

J Virol, 1996 Jul, 70(7), 4394 - 410
Disruption of M-T5, a novel myxoma virus gene member of poxvirus host range superfamily, results in dramatic attenuation of myxomatosis in infected European rabbits; Mossman K et al.; Myxoma virus is a pathogenic poxvirus that induces a lethal myxomatosis disease profile in European rabbits, which is characterized by fulminating lesions at the primary site of inoculation, rapid dissemination to secondary internal organs and peripheral external sites, and supervening gram-negative bacterial infection . Here we describe the role of a novel myxoma virus protein encoded by the M-T5 open reading frame during pathogenesis . The myxoma virus M-T5 protein possesses no significant sequence homology to nonviral proteins but is a member of a larger poxviral superfamily designated host range proteins . An M-T5- mutant virus was constructed by disruption of both copies of the M-T5 gene followed by insertion of the selectable marker p7.5Ecogpt . Although the M-T5- deletion mutant replicated with wild-type kinetics in rabbit fibroblasts, infection of a rabbit CD4+ T-cell line (RL5) with the myxoma virus M-T5- mutant virus resulted in the rapid and complete cessation of both host and viral protein synthesis, accompanied by the manifestation of all the classical features of programmed cell death . Infection of primary rabbit peripheral mononuclear cells with the myxoma virus M-T5-mutant virus resulted in the apoptotic death of nonadherent lymphocytes but not adherent monocytes . Within the European rabbit, disruption of the M-T5 open reading frame caused a dramatic attenuation of the rapidly lethal myxomatosis infection, and none of the infected rabbits displayed any of the characteristic features of myxomatosis . The two most significant histological observations in rabbits infected with the M-T5-mutant virus were (i) the lack of progression of the infection past the primary site of inoculation, coupled with the establishment of a rapid and effective inflammatory reaction, and (ii) the inability of the virus to initiate a cellular reaction within secondary immune organs . We conclude that M-T5 functions as a critical virulence factor by allowing productive infection of immune cells such as peripheral lymphocytes, thus facilitating virus dissemination to secondary tissue sites via the lymphatic channels.

J Trauma, 1996 Jul, 41(1), 10 - 4
Fluconazole increases bactericidal activity of neutrophils; Zervos EE et al.; BACKGROUND: Candida infections after injury are associated with significant mortality . Death often results from gram negative sepsis . Because antifungal therapy lowers mortality seen with Candida infections, this study was undertaken to determine whether fluconazole (FCZ) augments host response against bacteria, specifically, the bactericidal activity of polymorphonuclear neutrophils (PMN) . METHODS: PMN from 23 volunteers were incubated with phosphate-buffered saline or FCZ (20 micrograms/mL or 40 micrograms/mL) . Bactericidal activity of these PMN, as well as FCZ alone, was determined using Escherichia coli and a colony forming unit protocol . RESULTS: PMN possess potent bactericidal activity while FCZ possesses minimal inherent bactericidal activity . PMN bactericidal activity is significantly augmented by preincubation with FCZ . This enhanced bactericidal state is greater than the combined individual bactericidal properties of either FCZ concentration and PMN . CONCLUSIONS: FCZ augments PMN bactericidal activity . This augmentation may help to explain the improved survival of critically injured patients receiving FCZ.

Crit Care Med, 1996 Jul, 24(7), 1203 - 7
High-dose recombinant endotoxin neutralizing protein improves survival in rabbits, with Escherichia coli sepsis; Saladino RA et al.; OBJECTIVE: To assess the benefit of a recombinant endotoxin neutralizing protein from Limulus polyphemus in treating Gram-negative bacterial sepsis in rabbits . DESIGN: Prospective, blinded, controlled, laboratory trial . SETTING: Animal research laboratory . SUBJECTS: New Zealand White rabbits . INTERVENTIONS: We established a rabbit model of Escherichia coli peritonitis and bacteremia, with high mortality rate, despite treatment with gentamicin and ceftriaxone . Twenty-five pairs of male New Zealand White rabbits were challenged intraperitoneally with E . coli O18ac K1 in 5% porcine mucin (mean 7 x 10(1) colony-forming units) . All animals were treated with intravenous gentamicin (2.5 mg/kg) and ceftriaxone (100 mg/kg), and with either intravenous endotoxin neutralizing protein (50 mg/kg) or saline 1 hr after E . coli challenge . MEASUREMENTS AND MAIN RESULTS: All animals were bacteremic 1 hr after challenge (mean 3.6 x 10(5) colony-forming units/mL) . Animals in both groups developed tachycardia, hypotension, and acidosis (NS) . Geometric mean serum endotoxin and tumor necrosis factor (TNF) concentrations were significantly ( p < .001) higher 1 hr after challenge compared with baseline prechallenge concentrations in both groups . From 1 to 2 hrs after challenge, endotoxin concentrations increased 2.5-fold in control animals (95% confidence interval = 13.1 to 32.9 endotoxin units/mL, p = .024), whereas endotoxin concentrations increased only 1.2-fold in endotoxin neutralizing protein-treated animals (95% confidence interval = 20.4 to 23.6 endotoxin units/mL, NS) . TNF concentrations increased significantly (p < .001) in both groups from 1 to 2 hrs after challenge . Eighteen (72%) of 25 endotoxin neutralizing protein-treated animals vs . 11 (44%) of 25 controls survived 24 hrs (p = .032) . CONCLUSIONS: Treatment with endotoxin neutralizing protein had the following effects: a) the increase in serum endotoxin was blunted, but not TNF concentrations measured 1 hr after antibiotic treatment; and b) survival in rabbits with E . Coli sepsis was improved.

J Infect Dis, 1996 Jul, 174(1), 69 - 74
Cytomegalovirus inhibits CD14 expression on human alveolar macrophages; Hopkins HA et al.; Cytomegalovirus (CMV) infection in transplant patients is associated with an increased incidence of gram-negative pneumonia; the mechanism for this is unknown . Human alveolar macrophages (HAM) are an important part of the response of the lung to gram-negative bacteria . They interact with lipopolysaccharide (LPS) via the surface receptor CD14 . The effect of CMV on CD14 expression by HAM was examined . HAM were obtained from normal volunteers by bronchoalveolar lavage, and some were exposed to CMV . CD14 expression was assessed by immunofluorescent microscopy and flow cytometry . CMV inhibited the surface expression of CD14 on HAM . Release of soluble CD14 was also reduced from infected cells, and Northern blot analysis revealed that CD14 mRNA was reduced in CMV-exposed cells . These findings were specific for CD14 expression . These results demonstrate that CMV inhibits the ability of HAM to express CD14.

J Biotechnol, 1996 Jun 27, 47(2-3), 89 - 97
The SIGNAL experiment in BIORACK: Escherichia coli in microgravity; Thevenet D et al.; Microgravity affects certain physical properties of fluids, such as convection movement and surface tension . As a consequence, cells and living organisms may exhibit different behaviour in space, which may result from differences in the immediate environment of the cell or changes in the structure of the membrane in microgravity . Two experiments to examine the effects of microgravity on cell microenvironment and signal transduction through membranes were performed using a well-characterized system with different strains of the non-pathogenic Gram-negative bacterium Escherichia coli . Our results indicate that (i) microgravity appears to reduce the lag period of a non-motile culture of E . coli, and (ii) the ompC gene, regulated by the two-component system EnvZ-OmpR, is induced as well or better in microgravity than in ground controls.

FEBS Lett, 1996 Jun 17, 388(2-3), 169 - 72
A dnaA box can functionally substitute for the priming signals in the oriV of the broad host-range plasmid RSF1010; Taguchi Y et al.; The initiation of replication from oriV RSF1010, the replication origin of the broad host-range plasmid RSF1010, depends on RepA (helicase), RepB' (primase), and RepC (initiator protein), encoded by RSF1010 itself, while this initiation event in E . coli is independent of dnaA, dnaB, dnaC, and dnaG {Scherzinger et al . (1984) Proc . Natl . Acad . Sci . USA 81, 654-658; Scholz et al . (1985) in: Plasmids in Bacteria, pp . 243-259, Plenum, New York; Haring and Scherzinger (1989) in: Promiscuous Plasmids of Gram-negative Bacteria, pp . 95-124, Academic Press, London; Scherzinger et al . (1991) Nucl . Acids Res . 19, 1203-1211} . We showed in this work that a newly constructed origin consisting of an oriV RSF1010 and a DnaA protein binding site, the dnaA box, inserted near oriV RSF1010 (oriV RSF1010-dnaA box) could function without RepB' primase, but required RepA and RepC . This oriV RsF1010-dnaA box could not replicate in a dnaA46 strain in which only RepA and RepC were supplied, even at a permissive temperature . These results indicate that an inserted dnaA box can functionally substitute for the RSF1010-specific ssi signals, the RepB' dependent priming signals in oriV RSF1010, and can direct a priming pathway different from the RSF1010-specific one, but related to DnaA protein.

J Immunol, 1996 Jun 15, 156(12), 4969 - 73
Release of bactericidal/permeability-increasing protein in experimental endotoxemia and clinical sepsis . Role of tumor necrosis factor; von der Mohlen MA et al.; Bactericidal/permeability-increasing protein (BP}) is contained within the azurophilic granules of neutrophils and is able to neutralize endotoxin and kill Gram-negative bacteria . TNF has been implicated as a mediator of endotoxin-induced neutrophil degranulation . To assess the role of TNF in the elevated BPI levels during sepsis, the following studies were performed . 1) In 31 consecutive patients with sepsis syndrome, plasma BPI levels were markedly elevated compared with those in healthy controls, but showed no correlation with simultaneously measured TNF concentrations . 2) In four healthy men, i.v . injection of recombinant human TNF (50 microg/m2) induced a rapid rise in plasma BPI levels . 3) In eight normal subjects, i.v . administration of Escherichia coli endotoxin (4 ng/kg) elicited subsequent increases in the plasma concentrations of TNF and BPI . 4) Eight healthy chimpanzees were investigated after i.v . injection of endotoxin (4 ng/kg); four animals received endotoxin only, and four animals received an anti-TNF mAb simultaneously . Although anti-TNF completely prevented the endotoxin-induced appearance of TNF activity, the rise in BPI levels remained unaltered . These results suggest that TNF is not critical for the release of BPI from neutrophils during experimental endotoxemia or clinical sepsis.

Gut, 1996 Jun, 38(6), 925 - 31
Biliary decompression promotes Kupffer cell recovery in obstructive jaundice; Clements WD et al.; BACKGROUND: Jaundiced patients undergoing surgical procedures have an increased risk of Gram negative sepsis with potential morbidity and mortality . Depressed Kupffer cell clearance capacity (KCCC) predisposes jaundiced patients to endotoxaemia and its sequelae . Biliary decompression remains the main therapeutic strategy in obstructive jaundice . AIMS: This study investigates the efficacy of internal (ID) and external biliary drainage (ED) on KCCC in an experimental model of extrahepatic biliary obstruction . METHODS: Adult male Wistar rats (250-300 g) were assigned to one of six groups: sham operated, where the bile duct was mobilised but not divided; bile duct ligation (BDL) for three weeks, and sham operated or BDL for three weeks followed by a second laparotomy and further 21 days of ID or ED, by way of choledochoduodenostomy or choledochovesical fistula respectively . KCCC was measured using an isolated hepatic perfusion technique with FITC labelled latex particles (0.75 mu) as the test probe . Plasma was assayed for bilirubin, endotoxin, and anticore glycolipid antibody (ACGA) concentrations . RESULTS: Jaundiced rats had reduced KCCC (p < 0.001), increased concentrations of ACGA (p < 0.001), and endotoxin (p < 0.001) compared with controls . Biliary drainage for three weeks produced a recovery in KCCC and normalisation of endotoxin and ACGA concentrations, however, external drainage was less effective than ID (p < 0.01) . CONCLUSIONS: These data support the hypothesis that endotoxaemia and its mediated effects are integral in the pathophysiology of jaundice . Furthermore, a short period of internal biliary drainage is a useful therapeutic strategy in restoring Kupffer cell function and negating systemic endotoxaemia and consequent complications in biliary obstruction.

Immunopharmacology, 1996 Jun, 33(1-3), 374 - 6
Does resistance to endotoxin in primates correlate with levels of plasma prekallikrein?
Veloso D.
Increased release of BK by HK hydrolysis has been correlated with the severity of hypotension in septic patients and animals challenged with gram-negative bacteria or endotoxin (ETX) . Since HK hydrolysis in vivo is attributed mainly to the catalytic reaction of kallikrein (KAL) formed by activation of plasma PK, I tested whether the known resistance to ETX-rhesus and baboon > man and chimp-correlated with PK levels . Immunoblots and amidolytic assays showed that PK levels did not correlate . They were in rhesus and man > baboon and chimp . Also, PK did not correlate with levels of other modulators of free KAL levels in plasma-factor XII, HK and KAL inhibitors . Nonetheless, the distribution of PK and its activation products in plasmas activated with kaolin at 37 degrees C is analogous in the 4 primates, suggesting a similar mechanism for KAL inhibition in vitro . The results suggest that factors not yet known must contribute to the differential resistance of primates to ETX . Knowledge of these factors will help in prophylaxis and therapy of septicemia.

Hybridoma, 1996 Jun, 15(3), 225 - 32
Production and characterization of strain-specific monoclonal antibodies against outer membrane components of Azospirillum brasilense Sp245; Schloter M et al.; Several hybridoma cell lines producing murine monoclonal antibodies (MAbs) directed against outer membrane components of the Gram-negative rhizosphere bacterium Azospirillum brasilense Sp245 have been established and characterized . Whole bacterial cells were used as immunogens . Among the clones obtained, 14 hybridoma cell lines were selected for further characterization . Eight MAbs were strain-specific and 6 MAbs showed cross-reactivity with a closely related strain Azospirillum brasilense Sp246 . According to the biochemical characterization of the antigenic determinants, MAbs were classified into four groups . The corresponding antigens were lipopolysaccharides (class 1) and an outer membrane protein (class 4), which is common to Azospirillum brasilense Sp245 and Azospirillum brasilense Sp246 as well as two outer membrane proteins (class 2 and class 3) that are characteristic for Azospirillum brasilense Sp245 . The number of antigens per cell varied from 4000 (class 1) to 100 (class 4) . In each class high affinity MAbs were identified, which made a sensitive direct quantification of Azospirillum brasilense Sp245 possible.

Infect Agents Dis, 1996 Jun, 5(3), 127 - 43
The biology of rickettsiae; Hackstadt T; Rickettsiae are bacterial obligate intracellular parasites ranging from harmless endosymbionts to the etiologic agents of some of the most devastating diseases known to mankind . Rickettsiae are primarily associated with arthropod vectors in which they may exist commensally and, in most cases, only accidentally infect humans . These fascinating microbes are the prototypical obligate intracellular parasites . Other than being extremely fastidious in their growth requirements, however, rickettsiae are typical gram-negative bacteria . Only a few intracellular parasites multiply within the cytoplasm of eukaryotic cells . In this environment, rickettsiae are provided with a rich source of biosynthetic precursors not normally encountered by free-living bacteria and have evolved a number of unique mechanisms to transport such metabolites as nucleotides and nucleotide sugars . The physiologic basis for their obligate parasitism, however, has remained elusive for > 90 years . Other than the obvious property of replicating inside eukaryotic cells, the molecular mechanisms of cellular damage are ill defined . The typhus-group rickettsiae multiply within host cells to great numbers without profound damage until lysis occurs . In contrast, the spotted fever-group rickettsiae spread rapidly from cell to cell by an actin-based motility . This property, in itself, is not sufficient to cause cell death, because avirulent spotted fever-group rickettsiae also spread by actin-based movement but do not cause lysis of the host cell . Despite the obvious limitations imposed by their obligate intracellular lifestyle and the current lack of methods for genetic manipulation, there are enough interesting biological properties of rickettsiae to offer an attractive area for research.

Comp Immunol Microbiol Infect Dis, 1996 Jun, 19(3), 213 - 7
Prevalence of Helicobacter-like organisms in porcine gastric mucosa: a study of swine slaughtered in Italy; Grasso GM et al.; Recent reports described some cases of gastritis in man caused by an uncultured gram-negative spiral bacterium morphologically identical to organisms observed in the stomachs of mammalians (e.g . cats, dogs, pigs) . The aim of the present study is to confirm the presence of these bacteria in Italian swine . Tightly spiralled organisms (Gastrospirillum suis) were found in the stomach of eight (9.4%) out of 85 pigs examined . The bacteria were always associated with macroscopic lesions indicative of gastritis . Attempts to culture H . pylori or Helicobacter-like organisms were unsuccessful . The possibility that Gastrospirillum may be a zoonotic pathogen, with transmission occurring from pigs to humans, is discussed.

Alcohol Clin Exp Res, 1996 Jun, 20(4), 607 - 14
Hepatic sinusoidal endothelial cell in alcoholemia and endotoxemia; Deaciuc IV et al.; The experimental data reviewed in this study tend to indicate that the hepatic sinusoidal endothelial cell (SEC) is, chronologically, the first hepatic cell that undergoes pathologic changes in alcoholemia . Due to its strategic position in the liver sinusoid, SEC dysfunction and structural alterations have far-reaching repercussions for the whole liver . The authors gather experimental evidence suggesting that alcohol-induced SEC alterations are mostly due to Kupffer cell activation induced by alcohol rather than to a direct action of alcohol on SEC . Once activated, the Kupffer cell secretes a spectrum of mediators that affect both function and structure of SEC . Kupffer cell activation is regarded as a result of both direct and indirect actions of alcohol on the cell . The indirect action of alcohol is ascribed to alcohol-induced elevated plasma levels of Gram-negative bacterial lipopolysaccharide (LPS), a strong activator of Kupffer cell . However, a comparison of alcohol and LPS effects on SEC functions and structure reveals that these two agents may have, under many circumstances, different actions on the SEC, at least in laboratory animals . However, this issue continues to be a matter of debate . Also the review presents justification for the necessity to extend research on mechanisms underlying alcoholic liver disease to the effects of alcohol on the SEC . Finally, several future research directions are suggested in this review to better understand the mechanisms underlying alcohol-induced liver dysfunction.

Ann Pharmacother, 1996 Jun, 30(6), 596 - 602
Sequential parenteral and oral ciprofloxacin regimen versus parenteral therapy for bacteremia: a pharmacoeconomic analysis; Amodio-Groton M et al.; OBJECTIVE: To compare, in patients with gram-negative bacteremia, a course of parenteral antibiotic therapy alone with initial parenteral therapy followed by oral ciprofloxacin in terms of the length of hospitalization, clinical effectiveness, toxicity, and cost . DESIGN: A prospective, controlled, randomized, open trial in select hospitalized patients . SETTING: Large metropolitan teaching hospital . PATIENTS: Fifty hospitalized patients with proven gram-negative bacteremia were randomized to receive either oral ciprofloxacin (group 1) following a 72-hour initial intravenous antibiotic regimen or to continue parenteral therapy alone (group 2) . To compare the length of hospitalization, an additional group of 50 hospitalized patients with bacteremia (not enrolled in the study, group 3) were analyzed . INTERVENTION: Parenteral antibiotics for 72 hours followed by continuation of a parenteral regimen or oral ciprofloxacin 750 mg bid . MAIN OUTCOME MEASURES: Clinical response, toxicity, and length of hospitalization . RESULTS: Clinical resolution was comparable in the 24 group 1 patients receiving intravenous antibiotics followed by oral ciprofloxacin (83%), the 26 group 2 patients receiving parenteral therapy alone (77%), and the 50 comparison patients (76%) . There was little toxicity noted in any group, and the initial parenteral antibiotic regimens were similar . The mean numbers of hospital days on antibiotics were 9.1, 11.2, and 10.6 days in groups 1,2, and 3, respectively (p < 0.05 for group 1 vs . group 2 or 3), and the lengths of hospitalization were 9.8, 15.7, and 12.1 days, respectively (p < 0.05 for group 1 vs . group 2 or 3) . Shortening the length of hospitalization and days of antibiotic therapy was associated with a cost savings of up to $78 000 for group 1 patients . CONCLUSIONS: Parenteral therapy for 72 hours followed by oral ciprofloxacin significantly shortened both the number of hospital days taking antibiotics and the length of stay compared with parenteral therapy alone . Both regimens were equally effective and safe in the therapy of gram-negative bacteremia, and initial parenteral therapy followed by oral ciprofloxacin was cost-effective.

Clin Chest Med, 1996 Jun, 17(2), 183 - 97
The pathogenesis of sepsis . Factors that modulate the response to gram-negative bacterial infection; Marsh CB et al.; Gram-negative bacteria gain access to the bloodstream by evading host defenses . Once in circulation, lipopolysaccharide interacts with the host receptor CD14 and initiates the host's immune response . Lipolysaccharide stimulates the host to produce a cascade of mediators that activate and target leukocytes, opsonize the bacteria, and induce fever to defend against the invading bacteria . Unregulated release of these mediators, however, leads to the production of vasoactive substances, activation of the clotting cascade, and diminution of cardiac performance, which leads to the sepsis syndrome . This article discusses the pathogenic events that lead to sepsis syndrome and reviews critical steps in regulating these inflammatory mediators to allow the host to recover from gram-negative bacteremia.

Chest, 1996 Jun, 109(6), 1556 - 61
Chlorhexidine gluconate 0.12% oral rinse reduces the incidence of total nosocomial respiratory infection and nonprophylactic systemic antibiotic use in patients undergoing heart surgery; DeRiso AJ 2nd et al.; STUDY OFJECTIVE: The purpose of this study was to test the effectiveness of oropharyngeal decontamination on nosocomial infections in a comparatively homogeneous population of patients undergoing heart surgery . DESIGN: This was a prospective, randomized, double-blind, placebo-controlled clinical trial . Experimental and control groups were selected for similar infection risk parameters . SEETTING: Cardiovascular ICU of a tertiary care hospital . PATIENTS: Three hundred fifty-three consecutive patients undergoing coronary artery bypass grafting, valve, or other open heart surgical procedures were randomized to an experimental (n=173) or control (n=180) group . Heart and lung transplantations were excluded . INTERVENTIONS: The experimental drug chosen was 0.12% chlorhexidine gluconate (CHX) oral rinse . MEASUREMENTS AND RESULTS: The overall nosocomial infection rate was decreased in the CHX-treated patients by 65% (24/180 vs 8/173; p<0.01) . We also noted a 69% reduction in the incidence of total respiratory tract infections in the CHX-treated group (17/180 vs 5/173; p<0.05) . Gram-negative organisms were involved in significantly less (p<0.05) of the nosocomial infections and total respiratory tract infections by 59% and 67%, respectively . No change in bacterial antibiotic resistance patterns in either group was observed . The use of nonprophylactic IV antibiotics was lowered by 43% (42/180 vs 23/173; p<0.05) . A reduction in mortality in the CHX-treated group was also noted (1.16% vs 5.56%) . CONCLUSIONS: Inexpensive and easily applied oropharyngeal decontamination with CHX oral rinse reduces the total nosocomial respiratory infection rate and the use of nonprophylactic systemic antibiotics in patients undergoing heart surgery . This results in significant cost savings for those patients who avoid additional antibiotic treatment.

Microbiologia, 1996 Jun, 12(2), 171 - 84
Virulence factors of the swine pathogen Actinobacillus pleuropneumoniae; Tascon RI et al.; The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia, a highly contagious respiratory infection with great economic implications . In recent years, considerable efforts have been invested in the study of its virulence mechanisms . Here we review the current knowledge on the determinants of A . pleuropneumoniae pathogenicity, paying particular attention to the capsule, the lypopolysaccharide, the outer membrane proteins, and the RTX exotoxins . The contribution of other factors is also discussed.

Biol Reprod, 1996 Jun, 54(6), 1390 - 6
Lipopolysaccharide inhibits in vitro luteinizing hormone-stimulated rat ovarian granulosa cell estradiol but not progesterone secretion; Taylor CC et al.; Endotoxin, known as lipopolysaccharide (LPS), is a component of gram-negative bacterial cell walls and is a potent immunostimulator, inducing the release of several cytokines, such as tumor necrosis factor alpha (TNF alpha) and interleukins (IL) 1, 6, and 8 . A previous study with immature rats revealed that exogenous administration of LPS inhibits ovarian estradiol secretion in response to eCG . The present study was undertaken in order to determine whether LPS could directly inhibit rat granulosa cell (GC) steroid secretion . GC were collected and purified from 26-day-old hypophysectomized female rats (hypophysectomy on Day 23) . Purified GC were highly responsive to FSH (1-100 ng/ml), leading to increased estradiol, progesterone, and cAMP accumulation in culture media . GC were also capable of binding 125I-labeled hCG and were responsive to LH stimulation . Treatment of GC with LPS (1-100 ng/ml) led to a significant (p < 0.01) dose-dependent decrease in LH-stimulated estradiol accumulation in culture media (maximum 75% inhibition) . However, treatment of GC with LPS had no significant effect on FSH-stimulated progesterone or estradiol, or LH-stimulated progesterone accumulation in culture media . GC stimulated with 8-bromo cAMP were also insensitive to the effects of LPS . LPS had no significant effect on 125I-labeled hCG binding to GC homogenates, nor did it have any significant effect on FSH or LH-stimulated cAMP accumulation . Treatment of both FSH and LH-stimulated GC with LPS was associated with an increase in IL-6 bioactivity in culture media . This effect could be blocked with the nonreceptor tyrosine kinase inhibitor herbimycin A . TNF alpha bioactivity was undetectable with or without LPS challenge . Direct challenge of GC with recombinant murine IL-6 had no effect on either FSH or LH-stimulated estradiol whereas TNF alpha inhibited FSH-stimulated estradiol secretion . Collectively, these results suggest that the inhibitory effects of LPS were not mediated by either IL-6 or TNF alpha . Treatment of GC with the epidermal growth factor receptor tyrosine kinase inhibitor, tyrphostin A46, blocked the inhibitory effects of LPS on steroid secretion and was associated with an increased cAMP accumulation in culture media . The results indicate that LPS inhibits in vitro GC estradiol secretion . This effect appears to be restricted to the LH-stimulated aromatization of androgens to estrogen and may involve a tyrosine kinase signaling pathway.

Infect Immun, 1996 Jun, 64(6), 2041 - 6
Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins; Ancuta P et al.; We studied the ability of the lipopolysaccharide (LPS) extracted from a vaccine strain of Francisella tularensis (LPS-Ft) to mimic LPSs from other gram-negative bacteria for activation of various murine cell types or to antagonize the effects of other LPSs . We found that activation of macrophages for the production of tumor necrosis factor alpha and NO, of pre-B lymphocytes for the expression of surface immunoglobulins, and of bone marrow cells for the expression of LPS-binding sites was either undetectable with LPS-Ft or required concentrations 100 to 1,000 times higher than for standard LPSs . Preexposure of macrophages to LPS-Ft also failed to trigger down-regulation of tumor necrosis factor alpha (desensitization) or up-regulation of NO responses to an endotoxin challenge . In contrast to other atypical LPSs, LPS-Ft was also unable to antagonize any of the endotoxin-induced cellular responses mentioned above, suggesting that this LPS does not interact with LPS receptors.

Curr Microbiol, 1996 Jun, 32(6), 320 - 6
Natroniella acetigena gen . nov . sp . nov., an Extremely Haloalkaliphilic, Homoacetic Bacterium: A New Member of Haloanaerobiales
Zhilina TN, Zavarzin GA, Detkova EN, Rainey FA.
A new extremely haloalkaliphilic, chemoorganotrophic, homoacetogenic bacterium strain Z-7937(T)(T-type strain) was isolated from the bottom mud of the soda-depositing Lake Magadi, Kenya . It is an obligately anaerobic, motile, Gram-negative, spore-forming rod growing in the pH range pH 8.1 to 10.7 and optimally in the range pH 9.7 to 10.0 under conditions of high alkalinity caused by saturation with trona . It has an obligate requirement for sodium carbonate and chloride ions . The optimum salt concentration for growth is in the range 12-15% wt/vol, and growth occurs within the range from 10% to 26% . Strain Z-7937(T) is a mesophile with an optimal temperature for growth of 37°C, and a maximum of 42°C . The G + C content of strain Z-7937(T) is 31.9 mol% . A limited number of compounds are utilized, including lactate, ethanol, pyruvate, glutamate, and propanol . Acetate is the main end product . 16S rDNA sequence analysis shows strain Z-7937(T) to be a member of the order Haloanaerobiales and to represent a new branch within the family Halobacteroidaceae . On the basis of its novel physiology and phylogenetic position, we propose strain Z-7937 as a new species of a new genus, Natroniella acetigena gen . nov . sp . nov . The type strain is Z-7937(T) (= DSM 9952).

J Surg Res, 1996 Jun, 63(1), 44 - 8
A novel endotoxin antagonist attenuates tumor necrosis factor-alpha secretion; Dahlberg PS et al.; Twenty-seven amino acid peptides with sequences corresponding to a proposed endotoxin binding region of bactericidal permeability increasing protein (BPI):1) inhibit lipopolysaccharide induced macrophage tumor necrosis factor-alpha (TNF-alpha) secretion, 2) have bactericidal activity against gram-negative bacteria, and 3) protect mice from a lethal lipopolysaccharide (LPS) challenge . Unfortunately, peptides have a short halflife in vivo . Therefore, we have chemically conjugated the BPI based peptide, BG38, to a larger carrier protein, keyhole limpet hemocyanin (KLH), and characterized its ability: 1) to inhibit LPS induced macrophage TNF-alpha secretion and 2) to decrease plasma endotoxin and TNF-alpha levels following an i.v . injection of E . coli 0111:B4 LPS . BG38-KLH inhibited cultured macrophage TNF-alpha secretion in response to LPS derived from four pathogenic strains of gram-negative bacteria in a dose dependent manner (>90% inhibition at 50 microgram/ml, P < 0.05 Student's t test) . BG38-KLH also decreased serum endotoxin (>90%, P < 0.05 Student's t test) and peak TNF-alpha levels (>30% inhibition, P < 0.05 Student's t test) following E . coli LPS challenge in a murine gram-negative bacterial sepsis model . Novel endotoxin antagonists based upon a small domain of BPI represent promising reagents for the treatment of serious gram-negative bacterial infections.

J Immunol, 1996 May 15, 156(10), 3986 - 92
Neutrophils undergo apoptosis following ingestion of Escherichia coli; Watson RW et al.; Apoptosis is a distinct mechanism by which eukaryotic cells die . Neutrophils (PMN) play a fundamental role in the systemic inflammatory response syndrome . Clearance of PMN during resolution of the acute inflammatory process occurs by apoptosis, but factors inducing this process are unknown . The aims of this study were to determine whether PMN ingestion of Escherichia coli would result in PMN apoptosis and whether the mechanism was related to the respiratory burst . PMN from 10 healthy volunteers were cultured with different ratios of PMN:E . coli (1:0 to 1:25) for 12 h . Apoptosis was then assessed by propidium iodide DNA staining, morphology, gel electrophoresis, and Fc gamma RIII expression . There was a significant induction of PMN apoptosis on incubation with E . coli at a ratio of 1:10 and 1:25 PMN:E . coli as well as decreases in Fc gamma RIII . This correlated with increased ingestion of FITC-labeled E . coli and intracellular reactive oxygen intermediates after a 2-h coculture . To clarify the role of reactive oxygen intermediates in E . coli-induced PMN apoptosis, we assessed the effects of the antioxidants catalase, DMSO, glutathione, and N-acetylcysteine . There was a significant decrease in E . coli-induced PMN apoptosis on incubation with DMSO (1.0%), glutathione (25 mM), and N-acetylcysteine (25 mM) compared with control PMN:E . coli . This study demonstrates for the first time that E . coli induces PMN apoptosis through an oxygen-dependent mechanism . The removal of effete PMN by the process of apoptosis rather than necrosis may be teleologically beneficial during Gram-negative septicemia.

Biochemistry, 1996 May 7, 35(18), 5647 - 54
An NMR spectroscopy and molecular mechanics study of the molecular basis for the supramolecular structure of lipopolysaccharides; Wang Y et al.; Lipopolysaccharides from Gram-negative bacteria interact with the mammalian immune system to trigger a cascade of physiological events leading to a shock syndrome which results in the death in over 70% of cases of severe shock . It is known that the supramolecular structures of lipopolysaccharide aggregates are critical contributors to their biological activities . Despite this, the molecular basis for the formation if the regular hexagonal plates and arrays observed in lipopolysaccharide films and suspensions is unknown . Since these structures are two dimensional, it is unlikely that X-ray crystallographic methods will shed much light on their detailed structure . Knowing this structure is important since it is becoming increasingly likely that the insertion of the lipopolysaccharide hydrocarbon chains in the target host cell membrane may be involved in triggering host responses . This work describes the three-dimensional structure of the lipopolysaccharide lipid A moiety . The structure was obtained by a combination of molecular mechanics calculations and nuclear magnetic resonance spectroscopy . This involved calculation of the dihedral angle between the two glucosamine residues of the lipid A molecule from coupling constants and measuring critical interresidue NOE values . The study also takes into account information from X-ray powder diffraction and electron microscopy studies.

Zhonghua Yi Xue Za Zhi, 1996 May, 76(5), 355 - 8
{Changes of circulating Lps and cytokines in burned patients after anti-endotoxin therapy}; Fu W et al.; OBJECTIVE: Endotoxin as the inciting agent of cytokines and other mediators, whose high level expression correlates with the septic shock and MOF, has been the one of leading causes of death in ICU . METHODS: For treating sepsis and MOF caused by endotoxin, the anti-lipid A of LPS antibody was used, 19 burned patients whose TBSA varied from 50% to 100% were divided into anti-LPS treatment group and nontreated group . RESULTS: The levels of serum endotoxin, IL-6, IL-8, TNF and soluble IL-2R were lower obviously in patients of anti-LPS group than those of nontreated group (P < 0.05) . CONCLUSION: Clinical study surggests that anti-lipid A of LPS antibody can act as an therapeutic agent against gram-negative bacterin infection in burned patients.

Zhonghua Zheng Xing Shao Shang Wai Ke Za Zhi, 1996 May, 12(3), 167 - 70
{Protective effect of lipid A monoclonal antibody against burn sepsis in rats}; Xiao H et al.; A lipid A monoclonal antibody(mAb) was prepared and it was used to study its protective effect against burn sepsis . Wistar rats were inflicted with 30% TBSA third degress burn, and they were given LPS to mimic early sepsis after burn . The rats were divided randomly into burn with LPS, monoclonal antibody treatment, and control groups . The levels of endotoxin, tumor necrosis factor, light and electron microscopic studies of the morphological changes in the liver were studied . The results showed that the anti-lipid A monoclonal antibody demonstrated capacity to cross-react with several Gram-negative bacteria and their endotoxins . The mAb improved the survival rate of rats and decreased the levels of endotoxin and TNF as well as the liver damage significantly.

Shock, 1996 May, 5(5), 378 - 84
Ultrastructural changes in skeletal muscle mitochondria in gram-negative sepsis; Welty-Wolf KE et al.; Energy metabolism during sepsis is incompletely understood, but alterations in mitochondrial structure and function appear important . We measured time-dependent changes in mitochondrial structure during sepsis using serial skeletal muscle biopsies in anesthetized baboons injected with 10(10) CFU/kg of live Escherichia coli (LD(100)) . Skeletal muscle biopsies were taken before bacterial challenge (0 h controls) and at 12 h, 24 h, and death . By qualitative electron microscopy, the organelles became enlarged with distorted cristae and developed electron lucent areas within the matrix . With advanced injury the inner membrane became fragmented . Quantitative morphometric analysis showed a 50% increase in mean cristal membrane surface density by 24 h (p < .05) accompanied by a 100% increase in intermembrane space (p < .01) . Matrix volume density decreased progressively (p < .01) . These changes in mitochondrial ultrastructure occur within 12 h after the onset of the bacterial insult . This damage, including destruction or reorganization of both membrane and matrix proteins, is severe enough to compromise oxidative metabolism in muscle in Gram-negative sepsis.

Anesteziol Reanimatol, 1996 May-Jun, (3), 4 - 9
{The therapy of gram-negative septicotoxic diseases with pentaglobin, an immunoglobulin with an elevated IgM content (a prospective, randomized clinical study)}; Schedel I et al.; The aim of this study was to assess the efficacy of pentaglobin, a polyclonal polyvalent immunoglobulin containing IgG, IgM, and IgA, in therapy of septicotoxic diseases . Fifty-five patients with sepsis were divided into 2 perspective randomized groups . Group 1 (27 patients) were infused pentaglobin containing specific antibodies to bacterial endotoxin determinant . Immunoglobulin therapy was carried out during the first 3 days after the group was selected for study . In the other group (n = 28) no immunoglobulin therapy was carried out . During 6 weeks from the beginning of the study one patient out of 27 in group 1 (4%) died because of sepsis, whereas in group 2 nine patients died out of 28 (32%) (p < 0.01) . A reliably higher titer of circulating endotoxins and a lower titer of antibodies to endotoxin determinant were revealed during the first 48 hours of experiment in the serum or plasma of patients who died in the course of the follow-up period, in comparison with the survivors.

J Invertebr Pathol, 1996 May, 67(3), 279 - 88
Intracytoplasmic Bacteria in Male Germ Cells of Philudoria potatoria L . (Lasiocampidae, Lepidoptera, Insecta)
Wolf KW, Glatzel S.
The fine structure of bacteria, detected in the cytoplasm of spermatogonia, spermatocytes, and spermatids of a Lepidoptera species, Philudoria potatoria L . (Lasiocampidae), was described using transmission electron microscopy of ultrathin sections through testes . The effects of the bacteria on the germ cells were studied in semithin sections using light microscopy and in ultrathin sections using transmission electron microscopy . Larvae of the animal were collected in the field at the same location in two subsequent summers and the picture was very similar in both preparations . We examined a total of five animals and all were infected . The intracytoplasmic bacteria were usually rod-shaped, up to 1.7 mum long, and about 0.5 mum thick . They lay in intracellular cavities of the germ cells and were separated from the host cell by a membrane . The envelope of the bacteria consisted of two thin electron-dense layers interspersed by a transparent zone which represent the plasma membrane and the cell wall, respectively . Thus, the bacteria are most probably gram-negative . When the cells contained a small number of intracytoplasmic bacteria, pachytene spermatocytes and the development and structure of the meiotic spindles appeared regular . A prominent layer of perispindle membranes that is typical of male meiosis in the Lepidoptera (Wolf, 1994) was, however, missing in the infected animals . A low number of bacteria apparently does not interfere with the onset of spermiogenesis and it is possible that the microorganisms are transmitted into the next generation via the male germ line . In contrast, high numbers of intracytoplasmic bacteria result in cell degeneration in late prophase I of meiosis . Under these circumstances, electron-dense bodies formed within the nuclei and the nucleoplasm became transparent . The most prominent event in the cytoplasm was the aggregation of the mitochondria into large clusters . Finally, the nuclear envelope dissolved and the germ cells degenerated . The intracytoplasmic bacteria of P . potatoria are interpreted as parasites.

Mol Med, 1996 May, 2(3), 373 - 83
Cyclooxygenase-2-dependent bronchoconstriction in perfused rat lungs exposed to endotoxin; Uhlig S et al.; BACKGROUND: Lipopolysaccharides (LPS), widely used to study the mechanisms of gram-negative sepsis, increase airway resistance by constriction of terminal bronchioles . The role of the cyclooxygenase (COX) isoenzymes and their prostanoid metabolites in this process was studied . MATERIALS AND METHODS: Pulmonary resistance, the release of thromboxane (TX) and the expression of COX-2 mRNA were measured in isolated blood-free perfused rat lungs exposed to LPS . RESULTS: LPS induced the release of TX and caused increased airway resistance after about 30 min . Both TX formation and LPS-induced bronchoconstriction were prevented by treatment with the unspecific COX inhibitor acetyl salicylic acid, the specific COX-2 inhibitor CGP-28238, dexamethasone, actinomycin D, or cycloheximide . LPS-induced bronchoconstriction was also inhibited by the TX receptor antagonist BM-13177 . The TX-mimetic compound, U-46619, increased airway resistance predominantly by constricting terminal bronchioles . COX-2-specific mRNA in lung tissue was elevated after LPS exposure, and this increase was attenuated by addition of dexamethasone or of actinomycin D . In contrast to LPS, platelet-activating factor (PAF) induced immediate TX release and bronchoconstriction that was prevented by acetyl salicylic acid, but not by CGP-28238 . CONCLUSIONS: LPS elicits the following biochemical and functional changes in rat lungs: (i) induction of COX-2; (ii) formation of prostaglandins and TX; (iii) activation of the TX receptor on airway smooth muscle cells; (iv) constriction of terminal bronchioles; and (v) increased airway resistance . In contrast to LPS, the PAF-induced TX release is likely to depend on COX-1.

New Horiz, 1996 May, 4(2), 265 - 75
Platelet activating factor and its role in trauma, shock, and sepsis; Ayala A et al.; Platelet activating factor (PAF) is a phospholipid mediator released upon stimulation of cells, such as mast cells, basophils, neutrophils, and macrophages, by opsonized agents . This mediator produces a variety of biological effects and acts via specific binding sites present on various cell types . This article briefly reviews the nature of PAF, as well as what is understood about its role in the inflammatory response associated with trauma, shock, and sepsis . Much of what is known of PAF biology and experimental pathophysiology has come from the discovery and subsequent use of selective PAF antagonists . In this respect, several of the PAF antagonists have been examined experimentally and some have been tested clinically in patients with sepsis and septic shock . Experimental and clinical studies suggest that PAF antagonists appear to be effective in cases of severe Gram-negative septic shock . Nonetheless, this mediator may not be a major component involved in the systemic inflammatory response syndrome.

Cent Afr J Med, 1996 May, 42(5), 144 - 7
The outcome of surgical treatment of pressure sores at Mpilo Central Hospital, Zimbabwe; Muguti GI et al.; OBJECTIVE: To analyze the results of surgical management of pressure sores at Mpilo Central Hospital and also to identify the characteristics of patients who develop pressure sores in our community . DESIGN: A retrospective study of 25 patients who were treated surgically for pressure sores at Mpilo Central Hospital between January 1991 and December 1994 . SETTING: Mpilo Central Hospital, Bulawayo, Zimbabwe . SUBJECTS: Twenty five patients who under-went surgical treatment for pressure sores . MAIN OUTCOME MEASURES: Site of pressure sore, underlying cause of pressure sore, investigations, surgical procedures, complications, recurrence of pressure sores and length of hospital stay . RESULTS: The mean age of the patients was 35 years (standard deviation, SD 17) . There were more patients with pressure sores from rural areas (n = 15) than from urban areas (n = 10) . Most pressure sores occurred in the trochanteric region, 36pc (12/33), ischial region, 33pc (11/33) and in the sacral region, 21pc (7/33) . The commonest condition predisposing to pressure sores was spinal cord trauma, 48pc (12/25) . Most of the patients underwent excision and flap repair (58pc) and excision and direct closure of the pressure sore (35pc) . The main post operative complication was wound infection which occurred in about half the cases (n = 12) . The only death in this series occurred in one patient who developed a gram negative septicaemia . The mean hospital stay was 66 days (SD37) . CONCLUSION: This study indicates that spinal cord trauma, 48pc (912/25), is the commonest condition predisposing to pressure sores in our community . The incidence of such injuries can be reduced by strict enforcement of Road Traffic Regulations and improving safety precautions at the work place . As shown in this study the use of suitable flaps reduces the recurrence rate of pressure sores.

Mol Immunol, 1996 May-Jun, 33(7-8), 725 - 33
Horse complement protein C9: primary structure and cytotoxic activity; Esser AF et al.; Lack of hemolytic activity of horse serum is an inherent property of horse C9 . To understand the molecular reasons for this deficiency we have cloned C9 cDNA from a horse liver cDNA library and have sequenced the cDNA yielding the complete coding sequence for horse C9 . Purification of C9 from horse plasma and microsequencing established the N-terminus of the mature protein and verified that the correct horse C9 cDNA clone had been isolated . The deduced amino acid sequence corresponds to a mature protein of 526 amino acids that is 77% identical to human C9 . It has the same domain structure as human C9 and contains 22 cysteines and four invariant tryptophans . The few differences include the N-terminus, which is an unblocked glycine in horse C9 but pyroglutamine in human C9, and three potential N-glycosylation sites compared to two in human C9 . The N-terminal difference is unimportant since microsequencing of bovine C9, which is strongly hemolytic, established that it also has an unblocked glycine identical to horse C9 . There are no obvious structural differences apparent that could resolve the differences in hemolytic potency between the two molecules . Aside from a few conservative replacements, both C9 sequences are identical between positions 250 and 360 . This region includes the membrane interaction domain in C9 and the postulated transmembrane segment that is thought to constitute the wall of a putative transmembrane pore and, therefore, should be required for cytotoxicity . In agreement with this prediction we have observed that, in contrast to the marked decrease in hemolytic activity, horse C9 is very efficient in killing a variety of Gram-negative bacteria . These results demonstrate that horse C9 is a structurally competent molecule with efficient cytotoxic activity . Its inability to lyse erythrocytes may be related to the action of control proteins on target cell membranes.

Pathology, 1996 May, 28(2), 173 - 7
Pitfalls in the use of random amplified polymorphic DNA (RAPD) for fingerprinting of gram negative organisms; Gao Z et al.; Two arbitrary PCR primers for random amplified polymorphic DNA (RAPD) bacterial fingerprinting were used to test factors which may affect RAPD PCR results . These primers have been used in previously published RAPD fingerprinting studies . As expected, the MgCl2 concentration and template concentration in the reaction mixture may affect the RAPD banding patterns . The results obtained were not comparable between runs when using the Hybaid thermal cycler when all other conditions were kept constant . Addition of DMSO, gelatin and repeated subculturing did not appear to affect the banding patterns . A second set of primers directed against known repetitive sequences in Gram negative bacteria (REP1/REP2 and ERIC2) were examined to compare with RAPD as a means of fingerprinting organisms . The reproducibility was excellent . The results suggest RAPD primers can provide some useful comparative information on suspected related strains when tested on the same day and under the same conditions . PCR using REP1/REP2 and ERIC2 primers may provide a more reliable and reproducible alternative method for fingerprinting Gram negative bacteria.

Int J Clin Pharmacol Ther, 1996 May, 34(5), 212 - 8
Activation of protein tyrosine kinase: a possible requirement for fixed-bacteria and lipopolysaccharide-induced increase in human natural killer cell activity; Puente J et al.; Preincubation of peripheral blood lymphocytes (PBL) from drug-free, healthy volunteers with either the protein tyrosine kinase inhibitor genistein (GNT, n = 10, final concentration 200 microM) or the protein kinase A activator dybutiryl-cyclic-AMP (cAMP, n = 11, final concentration 10 microM), resulted in a significant inhibition of natural killer cell activity (NKCA, expressed as percentage of specific chromium release) . With the exception of 4 out of the 11 cAMP-treated samples, individual values for NKCA in the drug preincubated specimens were at least 20% below the same subject baseline activity; furthermore, NKC lytic function was non-detectable in 4 out of the 10 and in 1 out of the 11 samples pretreated with either GNT or cAMP, respectively . PBL preincubation with glutaraldehyde-fixed Gram-negative bacteria (GNB, n = 13, final GNB-to-effector cell ratio of 50 : 1) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 21.6 +/- 16.4 and bacteria treated samples of 41.5 +/- 24.6, respectively, Student's paired t-test p < 0.05) . At least a 20% increase in NKC lytic function over its own baseline value was recorded for 11 out of the 13 samples tested (Table 1) . Preincubation with GNB and GNT (5 samples) not only blocked the immunostimulant effects of GNB (Student's paired t-test p < 0.05), but in most cases individual values for NKCA were similar to those recorded for GNT-only treated samples . Use of cAMP instead of GNT also blocked, but to a smaller extent, the GNB-produced increases in NKC lytic function (paired Student's t-test < 0.05) . PBL preincubation with lipopolysaccharide (LPS, n = 11, final concentration 50 micrograms/ml) resulted in a statistically significant increase in NKCA (baseline (x +/- SD) of 20.7 +/- 14.1 and LPS treated samples of 39.2 +/- 18.5, respectively, Student's paired t-test < 0.05) . At least a 20% increase in NKCA over its own baseline value was observed for each and everyone of the 11 samples studied (Table 2) . Addition of LPS and GNT to the incubation mixture resulted not only in inhibition of the NKCA upmodulating LPS effects (Student's paired t-test p < 0.05), but each and everyone of the individual samples' NKCA were, in fact, significantly lower than their corresponding control baseline values and similar to those recorded for GNT-only treated samples . However, the use of LPS and cAMP (Table 2) produced less dramatic results, significant inhibition of LPS effect were recorded in only 2 samples (Nos 8 and 10), and individual NKCA in the remaining 3 specimens was significantly higher than the corresponding baseline value . Whereas experimental results obtained with GNT support the involvement of PTK-dependent pathways in the stimulation of human NKCA produced by GNB and LPS, cAMP experiments suggest modulation of PKA-dependent pathways as responsible for the decrease in NK lytic function produced by a number of chemicals involved in the pathophysiology associated with certain forms of stress, including septic shock . Further research in this area could help in the rational design of pharmacological approaches for the treatment of these conditions.

Eur Heart J, 1996 May, 17(5), 682 - 8
Association of Chlamydia pneumoniae and acute coronary heart disease events in non-insulin dependent diabetic and non-diabetic subjects in Finland; Miettinen H et al.; OBJECTIVE: It has been suggested that Chlamydia pneumoniae, a Gram-negative intracellular bacterium, is a risk factor for both myocardial infarction and chronic coronary heart disease . Previous studies have been done predominantly in non-diabetic subjects and thus the effect of diabetes on the association between C . pneumoniae antibodies and coronary heart disease has not been analysed . The aim of our study was to investigate the association between prior chlamydial infection and the risk of serious coronary heart disease events (myocardial infarction or coronary death) in a 7-year prospective study of cohorts of diabetic and non-diabetic subjects in two areas of Finland . RESULTS: It was found that the prevalence of elevated chlamydial antibodies at baseline was higher in nondiabetic subjects who had serious coronary heart disease events during the follow-up than in subjects without coronary heart disease events (32 vs 15%, relative risk 2.56, P = 0.013) in East Finland . In non-diabetic subjects living in West Finland we did not find this association . The association between the C . pneumoniae antibodies and coronary heart disease events did not markedly change after controlling for other risk factors for coronary heart disease (OR 2.44, P = 0.055) in non-diabetic subjects living in eastern Finland . In diabetic patients we did not find any association between chlamydial antibodies and coronary heart disease events . CONCLUSION: We found an association between C . pneumoniae antibodies and coronary heart disease events in non-diabetics living in eastern Finland . This association remained strong even after controlling for the other risk factors for coronary heart disease . In diabetic patients with high risk for coronary heart disease, C . pneumoniae was not a risk factor for coronary heart disease.

Eur J Immunol, 1996 May, 26(5), 1103 - 9
Inhibition of interleukin-6 synthesis in an animal model of septic shock by anti-C5a monoclonal antibodies; Hopken U et al.; The complement activation fragment C5a was recently shown to induce interleukin (IL)-6 synthesis by peripheral blood mononuclear cells . To understand better the role of C5a in cytokine regulation in vivo, we investigated the effects of complement depletion by cobra venom factor (CVF) or of anti-C5a monoclonal antibodies (mAb) on IL-6 generation in an animal model of septic shock . Complement-depleted pigs which were subsequently challenged with Escherichia coli generated significantly (p < 0.05) less IL-6 during the 6-h observation period than complement-sufficient controls . To address specifically the role of C5a in IL-6 regulation, we produced a C5a(57-74) peptide-specific mAb (T13/9) which neutralizes the bioactivity of porcine C5a . The mAb T13/9 does not cross-react with the precursor protein C5 . The pretreatment of pigs with anti-C5a mAb T13/9 prior to the induction of sepsis resulted in a decrease of over 75% in serum IL-6 bioactivity compared to control animals (p < 0.0001) . These results indicate a role for C5a in the modulation of IL-6 synthesis in Gram-negative bacteremia.

J Clin Invest, 1996 May 1, 97(9), 2152 - 7
Endotoxin and cytokines induce expression of leptin, the ob gene product, in hamsters; Grunfeld C et al.; The expression of leptin, the ob gene product, is increased in adipose tissue in response to feeding and energy repletion, while leptin decreases food intake . Because adipose tissue gene expression is regulated by cytokines induced during infection and because infection is associated with anorexia, we tested whether induction of leptin might occur during the host response to infection . Administration of endotoxin (LPS), a model for gram negative infections, induces profound anorexia and weight loss in hamsters . In fasted adipose tissue to levels similar to fed control animals . There is a strong inverse correlation between mRNA levels of leptin and subsequent food intake . TNF and IL-1, mediators of the host response to LPS, also induced anorexia and increased levels of leptin in mRNA in adipose tissue . As assessed by immuknoprecipitation and Western blotting, circulating leptin protein is regulated by LPS and cytokines in parallel to regulation of adipose tissue leptin mRNA . Induction of leptin during the host response to infection may contribute to the anorexia of infection.

J Immunol, 1996 May 1, 156(9), 3501 - 8
Modulation of endogenous IL-1 beta and IL-1 receptor antagonist results in opposing effects on HIV expression in chronically infected monocytic cells; Goletti D et al.; A proportion of HIV-infected individuals experience episodes of localized or systemic bacterial infections caused by Gram-negative bacteria . Many of the clinical side effects of these infections are associated with the production of proinflammatory cytokines, which are induced primarily by LPS, a constituent of the bacterial cell wall of Gram-negative bacteria . The present study examines the mechanisms involved in LPS-mediated induction of HIV expression in U1 cells, a promonocytic cell line chronically infected with HIV . Stimulation of U1 cells by LPS alone induced minimal levels of HIV expression, which was significantly enhanced by granulocyte-macrophage colony-stimulating factor (GM-CSF) . Costimulation of U1 cells with LPS plus GM-CSF resulted in the accumulation of steady-state levels of HIV RNA; however, only a weak induction of HIV long terminal repeat-driven transcription, which was not associated with the activation of the cellular transcription factor nuclear factor-kappa B, was noted . Costimulation of cells with LPS plus GM-CSF induced the production of proinflammatory cytokines, IL-8, IL-1 beta and IL-6, but not TNF-alpha . IL-1 receptor antagonist (ra) inhibited LPS enhancement of HIV expression in GM-CSF-stimulated cells, suggesting that endogenous IL-1 was involved in LPS-mediated viral production . In this regard, anti-inflammatory cytokines inhibited LPS plus GM-CSF-stimulated HIV expression, and this effect closely correlated with inhibition of IL-1 beta release and, in particular, with up-regulation of endogenous IL-1ra production . Thus, the balance between an endogenously produced viral inducer (IL-1 beta ) and an inhibitor (IL-1ra) may represent an important pathway leading to modulation of HIV expression from monocytic cells.

Am Surg, 1996 May, 62(5), 350 - 5
Effects of reconstituted high-density lipoprotein in persistent gram-negative bacteremia; Casas AT et al.; Reconstituted high-density lipoproteins (rHDL) have been shown bind bacterial LPS and reduce its toxic effects . Since the effect of rHDL on LPS in vitro cannot be directly extrapolated to the in-vivo picture of Gram-negative septic shock, we have investigated the effects of rHDL in a rabbit model of Gram-negative bacteremia . Rabbits were anesthetized, ventilated, and invasively monitored for 6 hours . Escherichia coli (4 x 10(9) CFU/kg) were infused over 2 hours in rabbits given rHDL (75 mg/kg) before the bacterial challenge . Antibiotics were not used in this model . The bacterial infusion resulted in a bacteremia that persisted until the end of the study . The sepsis-induced TNF peak was significantly lowered by rHDL treatment (10 +/- 3 ng/mL in rHDL treated versus 33 +/- 5 in controls, P = 0.001) . Blood pressure, although not statistically significant, tended to be higher in the rHDL group . Acidosis was significantly attenuated up to 3 hours after the beginning of the bacterial challenge (7.39 +/- 0.05 versus 7.27 +/- 0.05 in controls, P = 0.041) . rHDL treatment produced some transient beneficial effects in this model of persistent Gram-negative bacteremia . Additional studies, investigating the effects of rHDL in combination with antibiotics, are warranted.

Infect Immun, 1996 May, 64(5), 1679 - 84
De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells; Susa M et al.; Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells . The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival . Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L . pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies . We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L . pneumophila . The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection . A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells . The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics . Synthesis of this antigen was not dependent on protein synthesis of the host cell . Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L . pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene . We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L . pneumophila.

Infect Immun, 1996 May, 64(5), 1510 - 5
Adenovirus-mediated transfer of a gene encoding acyloxyacyl hydrolase (AOAH) into mice increases tissue and plasma AOAH activity; Coulthard MG et al.; Although the host response to gram-negative bacterial infection follows largely from the interactions of bacterial lipopolysaccharides (LPS or endotoxin) with host cells, little information is available concerning the mechanisms by which the host eliminates or detoxifies LPS . Acyloxyacyl hydrolase (AOAH) is an enzyme, found in phagocytic cells, that catalyzes the enzymatic deacylation of the lipid A moiety of LPS . Enzymatically deacylated LPS is much less potent than LPS at inducing responses in human cells, and it can antagonize the ability of LPS to activate human macrophages, neutrophils, and endothelial cells . Despite these observations, the physiologic role of LPS deacylation remains undefined . To investigate the ability of AOAH to carry out LPS deacylation in vivo, we produced a recombinant adenovirus carrying a gene encoding (AOAH) (Ad.CMV-AOAH) and employed this vector to elicit transient overexpression of AOAH in mice . Mice infected with Ad.CMV-AOAH expressed high levels of the enzyme in plasma, liver, spleen, and kidney . Although adenovirus-induced hepatitis reduced hepatic uptake of intravenously injected {3H}LPS, animals expressing the transgene deacylated a larger fraction of the {3H}LPS taken up by their livers than did mice infected with a control adenovirus . These studies indicate that AOAH can catalyze the deacylation of LPS in vivo, and they provide evidence that the rates of hepatic LPS uptake and deacylation are not closely linked.

Blood, 1996 May 1, 87(9), 3919 - 28
Induction of the chemotactic S100 protein, CP-10, in monocyte/macrophages by lipopolysaccharide; Hu SP et al.; The murine S100 protein CP-10 is a potent chemotactic factor for murine and human myeloid cells in vivo and in vitro . This is the first report describing regulations of the CP-10 gene by a proinflammatory stimulus, lipopolysaccharide (LPS), in cells of the monocyte/macrophage lineage . Murine monocyte/macrophage-like WEHI 265 and RAW 264.7 cells preexposed to 5 to 50 ng/mL LPS expressed significant levels of CP-10 mRNA 4 hours, and maximal at 20 hours, after a secondary LPS challenge . This was accompanied by increasing levels of cell-associated and released CP-10 protein . In contrast, a single dose of LPS upregulated CP-10 mRNA in elicited peritoneal macrophages, whereas mRNA and protein levels decreased following LPS challenge . The state of macrophage differentiation may control responsiveness as LPS had no effect on CP-10 basal levels in bone marrow derived macrophages . LPS-induced CP-10 expression was controlled at the transcriptional level and nuclear run-on and protein synthesis inhibition assays indicated that LPS priming and challenge of RAW cells occurred via distinct pathways . MRP14, another S100 protein generally coordinately expressed with human MRP8, was not induced by LPS under the same conditions . We propose that CP-10 may play a key role in recruitment of leukocytes into tissues in response to gram-negative bacterial infection.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4142 - 6
Cell density regulates cellular reversal frequency in Myxococcus xanthus; Shi W et al.; Myxococcus xanthus is a Gram-negative bacterium that aggregates to form fruiting bodies when nutrients are limiting . Previous studies showed that the frz mutants that are defective in chemotaxis exhibited irregular and infrequent patterns of cellular reversal . In contrast, wild-type cells, when examined individually, reverse relatively frequently, about once every 6 min . It is not known how the change of reversal frequency effects cellular aggregation during fruiting body formation in M . xanthus . In this study, we stained cells with a tetrazolium dye so that we could track the reversal frequencies of single cells and cells in groups . We found that developmental cells in large groups reverse much less than cells in small groups or as single cells . This reduced cellular reversal frequency is related to the frz signal transduction system and correlated with the methylation of FrzCD (a methyl-accepting chemotaxis protein) . Cells containing a mutation in the frz genes or in the genes required for social motility do not respond in this way . The reduction in cellular reversals as developmental cells accumulate in groups suggests a simple hypothesis for the aggregation of cells into discrete mounds during fruiting body formation . We also found that M . xanthus cells glide with equal frequency in the forward or reverse directions, indicating that cells do not contain a "head" or "tail."

Ugeskr Laeger, 1996 Apr 22, 158(17), 2397 - 8
{Capnocytophaga canimorsus bacteremia}; Uldbjerg IB; A case of bacteraemia in a 54-year-old previously healthy man caused by the gram-negative rod Capnocytophaga canimorsus is presented . The patient developed severe ischaemia in both feet, thrombocytopenic purpura and renal failure . The bacteraemia was apparently caused by saliva from the patient's dog who had licked ulcers on the patient's legs . The patient regained full health but had a minor inferior myocardial infarction seven weeks after start of the infection.

J Immunol Methods, 1996 Apr 19, 190(2), 185 - 8
Technical problems arising from the use of the immunoblot for determination of the reactivity of natural antibodies with different lipopolysaccharides (LPS); Schoenherr G et al.; Natural polyreactive antibodies (NPAB) appear to play an important role in the first-line defence against invading bacteria . The major constituent of the outer membrane of Gram-negative bacteria is the lipopolysaccharide (LPS) . Therefore, reactivity against this structure could be of importance in protecting the organism from the harmful effects of LPS . Immunoblotting has become a common method to verify the specificity of antigen antibody interactions . Various immunoblot techniques for testing the reactivity of monoclonal antibodies with LPS have been published using nitrocellulose and detergent-free blocking buffer systems . These methods are not suitable for the investigation of NPABs due to the broad reactivity and a high background staining which gives rise to interpretational difficulties . In the present study we demonstrate an immunoblot technique using polyvinylidene difluoride (PVDF) membranes and a detergent-containing buffer system which permits to detect LPS reactivity of NPABs . The polyreactive monoclonal human antibody CB03 used was screened for lipid A/LPS reactivity in ELISA experiments . The binding was confirmed in the described blot system and depends on the membranes and blocking agents used . The use of nitrocellulose versus PVDF was also tested for monospecific anti-LPS antibodies and the latter can be recommended due to the production of stronger reaction patterns without any background staining.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3559 - 64
Copy-up mutants of the plasmid RK2 replication initiation protein are defective in coupling RK2 replication origins; Blasina A et al.; The broad host range plasmid RK2 replicates and regulates its copy number in a wide range of Gram-negative bacteria . The plasmid-encoded trans-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-negative bacteria tested . The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication . A replication control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than normal copy number . Therefore, according to this model, the coupling activity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to form coupled complexes . To test this model for plasmid RK2, two previously characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TFrfA protein TrfA-254D/267L was characterized . Despite initiating runaway (uncontrolled) replication in vivo, the copy-up double-mutant TrfA protein exhibited replication kinetics similar to the wild-type protein in vitro . Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay . It was found that both single and double TrfA mutant proteins exhibited substantially reduced (single mutants) or barely detectable (double mutant) levels of coupling activity while not being diminished in their capacity to bind to the origin of replication . These observations provide direct evidence in support of the coupling model of replication control.

Biochem J, 1996 Apr 15, 315 ( Pt 2), 679 - 86
Titration calorimetric studies to elucidate the specificity of the interactions of polymyxin B with lipopolysaccharides and lipid A; Srimal S et al.; Lipopolysaccharide (LPS), the major cell wall constituent of Gram-negative bacteria, evokes a multitude of biological effects in mammals including pyrogenicity and toxic shock syndrome . Polymyxin B (PmB), a polycationic cyclic peptide, is known to neutralize most of its activities . The nature of the interaction of PmB with LPS and lipid A was investigated by isothermal titration calorimetry . PmB binds to LPS as well as lipid A stoichiometrically and non-co-operatively with micromolar affinity . These interactions are driven primarily by a favourable change in entropy (delta S) and are endothermic in nature . These positive changes in enthalpies decrease with increasing temperature, yielding a heat capacity change, delta Cp, of -2385 J.mol-1.degree-1 for PmB-LPS interactions while the binding of PmB to lipid A displays a delta Cp of -2259 J.mol-1.degree-1 . The negative heat capacity changes provide strong evidence for the role of hydrophobic interactions as the driving force for the association of PmB with LPS and lipid A . A correlation of the energetics of these interactions with analyses of the molecular models of PmB suggests that a cluster of solvent-exposed non-polar amino acid side-chains that line one surface of the molecule, together with a ring of positively charged residues on its other surface, are responsible for its strong and stoichiometric binding to LPS.

Presse Med, 1996 Apr 13, 25(13), 628 - 30
{Brucella pancarditis with fatal outcome}; Chevalier P et al.; Brucella endocarditis was diagnosed in a 21-year-old itinerant farm worker hospitalized for acute pulmonary edema . History taking revealed cough, fever and sweating one month earlier which had been treated with antibiotics . At admission, echography showed lesions on the aortic valve and hemocultures identified Brucella meltensis . On day 7 of specific treatment with doxycycline (200 mg/day) and rifamycine (1200 mg/day), and despite digitalics and diuretics, left ventricular failure rapidly worsened, leading to cardiac arrest and death before emergency surgery could be performed . Autopsy showed occlusive vegetations on the aortic valves facing the right coronary ostium, deep ulceration of the valsava sinus with abscess formation and fibrino-hemorragic pericarditis involving both the anterior and posterior walls of the epicardium . Gram negative germs were evidenced in the abscess alone . This case emphasizes the potentially rapid destructive effect of Brucella melitensis and confirms that surgery is the safest therapeutic alternative for aortic valve localizations . Surgery should be performed without delay.

Arch Microbiol, 1996 Apr, 165(4), 272 - 8
Sulfide pulsing as the controlling factor of spinae production in Chlorobium limicola strain UdG 6038
Pibernat IV IV, Abella CA.
Chlorobium limicola UdG 6038, a green sulfur bacterium, was isolated from anoxic sediments . Cells were gram-negative, non-motile, ovoid shaped, and contained chlorobactene and bacteriochlorophyll c as the main photosynthetic pigments . The DNA G+C content was 56.4 mol% . Ultrastructural studies revealed the presence of abundant spinae (45-110 spinae per cell) attached to the cell wall . India-ink-stained cells observed under the optical microscope were surrounded by a large capsule (5-11 &mgr;m total diameter) . The presence of this capsule was coincident with the presence of a large number of spinae (> 30 spinae per cell) . The mucilaginous capsule was attached to the spinae without penetrating it . In batch culture, the synthesis of spinae in strain UdG 6038 was not affected by changes in temperature, pH, salt concentration, or illumination at physiological ranges and hence, the cells remained spined . The control of spinae production was experimentally confirmed using a semicontinuous batch culture refed by sulfide pulsing . The culture remained at a low spination level (> 30 spinae per cell) only when the duration of sulfide starvation between pulses was less than 5 h . After longer sulfide starvation periods, the cells remained spined (more than 38 ± 6.3 spinae per cell) . This observation supports the idea that the duration of sulfide limitation in the culture plays a key role in controlling the spination process in strain C . limicola UdG 6038 . Chlorobium spinae may play an eco-physiological role in buoyancy capacity and adhesion of sulfur globules to the cells in natural environments where sulfide concentrations are expected to be highly variable.

J Pediatr Surg, 1996 Apr, 31(4), 486 - 9
The microenvironment influences the pattern of bacterial translocation in formula-fed neonates; Ford HR et al.; The authors previously demonstrated that neonatal rabbits fed conventional formula have a significantly greater incident of bacterial translocation than do neonatal rabbits fed breast milk . They hypothesized that exogenous bacteria in the formula and/or the microenvironment of the neonatal rabbit may contribute to the higher incidence of bacterial translocation . In the present study, the authors examined the incidence of bacterial translocation in neonatal rabbits fed pasteurized formula, unsterile formula, or breast milk while being housed in a clean or unsterile environment . The rabbits were divided into five groups . Groups I and II were fed pasteurized formula; groups III and IV were fed unsterile formula . In addition, groups I and III were housed in a clean environment, and groups II and IV were kept in an unsterile environment . The neonates in group V were fed breast milk and were kept in an unsterile environment . On the seventh day, the animals were killed, and the mesenteric lymph nodes, liver, and spleen were cultured for the presence of bacteria . Bacterial translocation occurred in 100% of group IV neonates . A clean environment (groups I and III) eliminated gram-negative bacterial translocation . A reduction (50%) in the overall incidence of bacterial translocation was obtained by pasteurizing the formula (group I v group III) . Group II had significantly less gram-negative bacterial translocation than did group IV . None of the neonates in group V had translocation . The data show that a clean environment abrogates gram-negative bacterial translocation . Pasteurizing the formula significantly reduces the incidence of gram-negative bacterial translocation, and further reduces overall bacterial translocation in a clean environment . The authors hypothesize that control of the microenvironment can significantly influence the pattern of bacterial translocation in formula-fed neonates, and thus potentially reduce the incidence of gut-origin sepsis . Factors present in breast milk inhibit bacterial translocation, regardless of the microenvironment.

Aliment Pharmacol Ther, 1996 Apr, 10 Suppl 1, 39 - 50
The role of lipopolysaccharide in Helicobacter pylori pathogenesis; Moran AP; The present review describes the structure, attributes and properties of Helicobacter pylori lipopolysaccharides (LPS), and their potential role in pathogenesis . Although possessing certain attributes similar to those of LPS of other Gram-negative bacteria, H . pylori LPS possess unique biological properties . H . pylori LPS has, in general, low immunological activity and this property may aid the persistence of infection . The O-specific chain of the LPS mimics Lewis blood group antigens in structure . As these antigens are present in the gastric mucosa, the expression of Lewis antigens on the bacterial surface may camouflage the bacterium and aid survival of H . pylori . Alternatively, since autoantibodies against human antral gastric mucosa have been observed in H . pylori-positive patients, the relevance of LPS in the development of autoimmunity in H . pylori-associated disease requires further investigation . H . pylori LPS in part mediates the binding of the bacterium to laminin, and interferes with gastric cell receptor-laminin interaction, thereby potentially contributing to the loss of mucosal integrity . In vitro observations of inhibition of sulphated mucin synthesis and stimulation of pepsinogen secretion by LPS suggest new mechanisms for H . pylori-induced mucosal damage . Nevertheless, further in vivo studies are required to support their pathogenic role.

Biochem J, 1996 Apr 1, 315 ( Pt 1), 257 - 64
Cloning, sequencing and expression of the transferrin-binding protein 1 gene from Actinobacillus pleuropneumoniae; Daban M et al.; Two outer-membrane proteins are involved in the uptake of iron from transferrin by certain Gram-negative bacteria, transferrin-binding proteins 1 and 2 . The gene encoding transferrin-binding protein 1 from a serotype 1 isolate of the Gram-negative pathogen Actinobacillus pleuropneumoniae was cloned, and a fragment encoding 700 amino acids of Tbp1 was expressed in Escherichia coli . We also report here sequencing of the tbpl gene and a comparison of the deduced amino acid sequence with Tbpls from related species . The predicted polypeptide product of tbpl is a 106 kDa protein with a 22-residue signal peptide.

Hepatology, 1996 Apr, 23(4), 788 - 96
Kupffer cell inactivation prevents lipopolysaccharide-induced structural changes in the rat liver sinusoid: an electron-microscopic study; Sarphie TG et al.; Scanning and transmission electron-microscopic examination of the rat liver sinusoid was performed in this study after in vivo treatment of rats with gram-negative bacterial lipopolysaccharide (LPS, 1 mg/Kg(-1) body weight), with or without pretreatment with gadolinium chloride (GdCl3 10 mg(Kg(-1) body weight) . Twenty-seven and 48 hours after GdCl3 administration, to inactivate/eliminate part of the Kupffer cell population, a decrease in the number of visualized Kupffer cells was observed, without evident effects on the sinusoidal endothelial cell or on the hepatocyte . Three and 24 hours after its administration, LPS produced ultrastructural changes in the sinusoid characterized by morphological evidence of Kupffer cell activation (i.e., swelling and expanded philopodia anchoring the Kupffer cell to the luminal surface of the sinusoidal wall), and a marked decrease in the population of endothelial cell fenestration . The reduction in the number of fenestrae was associated with a change in the diameter of fenestrae and can be interpreted as a component of the "capillarization" process of the hepatic sinusoid . Such ultrastructural changes were prevented by the administration of GdCl3 24 hours before LPS injection . Hence, these findings suggest that LPS-induced structural changes in the liver sinusoid are mediated by an LPS-induced Kupffer cell activation . Coupled with previous experimental data, showing similar effects of GdCl3 on one of the hepatic sinusoidal endothelial cell (SEC) functions, i.e., hyaluronan scavenging, the data presented in this study strongly support the view that Kupffer cells modulate both the hepatic SEC's functional as well as ultrastructural properties.

Crit Care Med, 1996 Apr, 24(4), 566 - 74
Pyruvate dehydrogenase inactivity is not responsible for sepsis-induced insulin resistance; Shangraw RE et al.; OBJECTIVE: To determine whether activation of pyruvate dehydrogenase with dichloroacetate can reverse sepsis-induced insulin resistance in humans or rats . DESIGN: Prospective, controlled study . SETTING: Intensive care unit (ICU) and laboratory at a university medical center . SUBJECTS: Nine patients were admitted to the ICU with Gram-negative sepsis, confirmed by cultures . In addition, chronically instrumented, Sprague-Dawley rats, either controls or with live Escherichia coli-induced sepsis . INTERVENTIONS: Hyperinsulinemic euglycemic clamp, with or without coadministration of dichloroacetate . MEASUREMENTS AND MAIN RESULTS: In humans, a primed, constant infusion of {6,6-2H2}glucose was used to determine endogenous glucose production and whole-body glucose disposal . Septic humans exhibited impaired maximal insulin-stimulated glucose utilization (39.5 +/- 2.7 mumol/kg/min), despite complete suppression of endogenous glucose production . In rats, a primed, constant infusion of {3-3H}glucose was used to determine endogenous glucose production and whole-body glucose disposal . Tissue glucose uptake in vivo was determined by {14C}-2-deoxyglucose uptake . Maximal, whole-body, insulin-stimulated glucose utilization was 205 +/- 11 and 146 +/- 9 mumol/kg/min in control and septic rats, respectively . The defect was specific to skeletal muscle and heart . Stimulation of pyruvate dehydrogenase with dichloroacetate caused a 50% decrease in plasma lactate concentration but failed to improve whole-body insulin-stimulated glucose utilization in either the septic human or rat . Dichloroacetate reversed the impairment of insulin-stimulated myocardial glucose uptake in septic rats, but did not influence skeletal muscle glucose uptake either under basal conditions or during insulin stimulation . CONCLUSIONS: Activation of pyruvate dehydrogenase with dichloroacetate does not ameliorate the impairment of whole-body, insulin-stimulated glucose uptake in septic humans or rats, or reverse the specific defect in insulin-mediated skeletal muscle glucose uptake by septic rats . Therefore, the decreased pyruvate dehydrogenase activity associated with sepsis does not appear to mediate sepsis-induced insulin resistance during insulin-stimulated glucose uptake at either the whole-body or tissue level.

Immunity, 1996 Apr, 4(4), 407 - 14
Resistance to endotoxin shock and reduced dissemination of gram-negative bacteria in CD14-deficient mice; Haziot A et al.; Endotoxin shock is the result of activation of the immune system by endotoxin/LPS, a component of Gram-negative bacteria . CD14, a GPI-anchored glycoprotein expressed strongly by monocyte/macrophages, is one of several receptors for endotoxin/LPS . The role of CD14 in bacterial-induced and LPS-induced shock was tested in CD14-deficient mice produced by gene targeting in embryonic stem cells . CD14-deficient mice were found to be highly resistant to shock induced by either live Gram-negative bacteria or LPS; however, at very high concentrations of LPS or bacteria, responses through non-CD14 receptors could be detected . Surprisingly, CD14-deficient mice also showed dramatically reduced levels of bacteremia, suggesting an unexpected role for CD14 in the dissemination of Gram-negative bacteria.

J Bacteriol, 1996 Apr, 178(7), 2102 - 7
An O-antigen processing function for Wzx (RfbX): a promising candidate for O-unit flippase; Liu D et al.; O antigen is the major cell surface antigen of gram-negative bacteria, and the genes responsible for its synthesis are located in a single gene cluster . The wzx (rbfX) gene, which is characteristic of the major class of O-antigen gene clusters, encodes a hydrophobic protein with 12 potential transmembrane segments . We demonstrate that a wzx mutant accumulates undecaprenol pyrophosphate-linked O units which appear to be on the cytoplasmic side of the cytoplasmic membrane, suggesting that the wzx gene encodes a flippase for O-unit translocation across that membrane.

J Infect Dis, 1996 Apr, 173(4), 927 - 33
Late administration of a lipophilic tyrosine kinase inhibitor prevents lipopolysaccharide and Escherichia coli-induced lethal toxicity; Vanichkin A et al.; Septic shock induced by gram-negative bacteria results primarily from excessive stimulation by lipopolysaccharide (LPS) of macrophages to produce tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 . The cellular effects of LPS, TNF-alpha, and IL-1 are mediated via tyrosine phosphorylation pathways . A recent report indicated that selective inhibitors of tyrosine kinases, tyrphostins of the AG126 family, protect mice against LPS-induced lethal toxicity in mice . Protection was most effective when the tyrphostin was injected before the LPS . In the present study, tyrphostin AG556, which is more lipophilic than those of the AG126 family, was effective in preventing LPS-induced lethal toxicity when administered 2 h after LPS . AG556 also prevented viable Escherichia coli-induced lethal toxicity when given 2 h before and, to a lesser extent, 2 h after the bacterial inoculation . AG556 may block a critical step downstream of the signaling pathway induced by LPS after TNF-alpha production.

Biochem Biophys Res Commun, 1996 Mar 27, 220(3), 979 - 82
Pit structure on bacterial cell surface; Hisano T et al.; The yellow-pigmented bacterium isolated from a ditch was a gram negative rod with a G+C content of 63 mol%, and was classified in the genus Sphingomonas . Electron microscopy revealed that the bacterial cell surface was covered with many large plaits . When grown in a medium containing a polysaccharide as an essential nutrient, a pit of 0.02-0.1 micrometers in diameter was formed on the cell surface, and a thin section showed the rearrangement of the plaits and the presence of a region where the cell membrane sinks into the cytosol . The dependence of the pit formation on the presence of macromolecule may predict the existence of a direct uptake mechanism for macromolecules through a mouth-like pit, possibly in endocytosis fashion . The confirmation of the pit structure is the first such finding in the history of microbiology and may provide a new insight into the cell morphology and biochemistry of macromolecule transport in microbial cell system.

Eur J Biochem, 1996 Mar 15, 236(3), 871 - 6
Surface-associated material from the bacterium Actinobacillus actinomycetemcomitans contains a peptide which, in contrast to lipopolysaccharide, directly stimulates fibroblast interleukin-6 gene transcription; Reddi K et al.; The oral commensal Gram-negative bacterium Actinobacillus actinomycetemcomitans is believed to be the causative organism of localized juvenile periodontitis, a disease in which there is rapid loss of alveolar bone supporting the teeth . Previously, we have reported that gentle saline extraction of this bacterium removed a loosely adherent proteinaceous fraction from the cell surface of the bacterium, which we have termed surface-associated material . This material contained potent bone-resorbing activity . We now report that surface-associated material is also a potent stimulator of cytokines, and in particular, interleukin-6 (IL-6) synthesis, while the lipopolysaccharide from this bacterium is only a weak stimulator of IL-6 synthesis by fibroblasts and monocytes . In contrast to enteric lipopolysaccharide (LPS), which induces fibroblast IL-1, IL-6 and tumour necrosis factor (TNF) alpha synthesis, surface-associated material stimulated gingival fibroblasts to synthesize only IL-6, with no induction of IL-1 or TNF (the normal inducers of IL-6 synthesis) . Reverse transcriptase PCR also failed to detect mRNA for IL-1 or TNF in surface-associated-material-stimulated fibroblasts, although both mRNAs were present in Escherichia coli LPS-stimulated cells . Neutralizing antibodies to IL-1 and/or TNF or the natural IL-1 receptor antagonist (IL-1ra) inhibited enteric LPS-induced IL-6 synthesis, but did not inhibit surface-associated-material-induced synthesis . In addition, dexamethasone, which completely suppressed LPS-induced IL-6 synthesis, only inhibited surface-associated-material-induced IL-6 synthesis by 50% . This suggests that the active constituent in the surface-associated material stimulates IL-6 gene transcription by a transcriptional control mechanism distinct to that of E . coli LPS . The IL-6 stimulating activity of the surface-associated material is inhibited by both heat and trypsin, suggesting that it is proteinaceous . The activity has been isolated using anion-exchange, reverse-phase and size-exclusion HPLC . The active moiety is a peptide of molecular mass 2kDa which may be the product of a bacterial short open reading frame.

Appl Biochem Biotechnol, 1996 Mar, 56(3), 277 - 88
Characterization of a lactate oxidase from a strain of gram negative bacterium from soil; Xu P et al.; A lactate oxidase was purified about 36-fold from a newly screened strain KY6 of gram negative bacterium from soil to yield a homogeneous protein . The native enzyme had a molecular mass of 204 kDa measured by Sephadex G-200 and that of subunit on the SDS-PAGE was found to be 45 kDa . The enzyme was optimally active at pH 7.7 and showed stability at pH range of 5.7 to 9.5 for 24 h at 4 degrees C . The optimum temperature was 70 degrees C and the enzyme activity was stable for 10 min up to 45 degrees C . The half-life of the enzyme activity was about 10 min at 55 degrees C . The best substrate of the enzyme was D-lactate and Km value for D-lactate was 0.14 mM . The Km value for DL-lactate was 0.20 mM . Substrate inhibition of the enzyme was observed at higher concentrations than 20 mM of DL-lactate and 10 mM of D-lactate.

PDA J Pharm Sci Technol, 1996 Mar-Apr, 50(2), 129 - 35
Effect of steam-heat treatment with/without divalent cations on the inactivation of lipopolysaccharides from several bacterial species; Bamba T et al.; The inactivation of endotoxin from six species of smooth gram-negative bacteria (S-form) by steam-heat treatment was investigated using the Limulus amebocyte lysate (LAL) assay . Biphasic decreases of endotoxins from four species of bacteria were observed upon steam-heat treatment of 1 microgram/ml endotoxin solution at 121 degrees C in a steam sterilizer . A lag time, however, was observed in the inactivation profiles of V . cholerae and P . aeruginosa . Distinct differences in heat resistance were observed among the bacterial species . The decrease rate was found to be concentration-dependent, and endotoxins at low concentrations (less than 10 ng/ml) were inactivated by the treatment to below the detection limit of the LAL assay . The time-course of the decrease of endotoxin from rough strains (R-form) resembled that of the respective S-form . The inactivation of R-form, especially Rc mutant, endotoxin was markedly affected by divalent cations such as Mg2+ and Ca2+, which appear to promote reaggregation of the endotoxin.

Eur J Clin Invest, 1996 Mar, 26(3), 237 - 9
Increased serum concentrations of the carboxy-terminal cross-linked telopeptide of collagen type I in patients with Gram-negative septicaemia; Wenisch C et al.; The authors determined serum levels of the carboxy-terminal cross-linked telopeptide and the carboxy-terminal propeptide of type I collagen (ICTP and PICP) in 18 patients with Gramnegative septicaemia before (day 0) and 28 days after therapy and in 18 age- and sex-matched controls by radioimmunoassay . Elevated levels of ICTP were observed in septicaemic patients {median (range): 15 (7-49) mu g L-1 before therapy and 14 (6-45) mu g L-1 28 days after therapy vs . 2 center dot 1 (1 center dot 4-4 center dot 3) mu g L-1 in normal subjects; P < 0 center dot 01 for both}, whereas PICP levels were not different between patients and controls {median (range): 119 (52-275) mu g L-1 (day 0) and 133 (79-288) mu g L-1 (day 28) vs . 91 (54-213) mu g L-1 in normal subjects, P > 0 center dot 05 for all} . The findings suggest an increased production or release of ICTP in Gram-negative septicaemia, presumably owing to an alteration of extracellular matrix during septicaemia-related vascular inflammation.

Biosci Biotechnol Biochem, 1996 Mar, 60(3), 377 - 82
DNA replication of IncQ broad-host-range plasmids in gram-negative bacteria; Sakai H et al.; Bacterial plasmids of Escherichia coli incompatibility group Q (IncQ) are broad-host-range plasmids that are able to proliferate in almost all Gram-negative bacteria . They are small, nonconjugative, and multicopy plasmids . They can be mobilized into many species of Gram-negative bacteria by coresident conjugative plasmids . Plasmids RSF1010, R1162, and R300B have DNAs of a size of 8.7 kb, and are best studied among IncQ plasmids . These plasmids encode by themselves three major proteins essential for the initiation of DNA replication . This makes the plasmid DNA replication less dependent on the DNA replication apparatus of host cells, and leads to promiscuity or a broad host range . Considering the biological features of these plasmids, they are potent DNA cloning vehicles . Moreover, their characteristic DNA replication mechanism that makes IncQ plasmids promiscuous is elaborate, and is an interesting object of scientific studies.

Semin Respir Infect, 1996 Mar, 11(1), 13 - 23
New etiopathogenic concepts of ventilator-associated pneumonia; Cassiere HA et al.; Ventilator-associated pneumonia develops as a consequence of impaired host defenses and exposure to large numbers of potential pathogens . Both of these processes interact and allow colonization of the airways with enteric gram-negative bacteria to occur through the molecular mechanism of bacterial adherence . Bacterial adherence is a cell-cell interaction mediated by the following variables: (1) host receptors, (2) bacterial adhesins, and (3) the proper microenvironment on the respiratory mucosa . For gram-negative bacteria, an important adhesin may be the pili that project from the cell surface, and important epithelial receptors for adhesins may be glycoproteins contained in respiratory mucus . The proper microenvironment for adherence to take place is a mixture of exposed cell receptors, prolonged bacterial-epithelial cell contact, impaired airway defenses, presence of proteases, and an optimal pH . In some way, all of the risk factors discussed impact either directly or indirectly with this basic molecular process . Once adherence is accomplished, potential pathogens may begin to colonize the respiratory tract . Once colonization occurs, bacteria may proliferate on the respiratory mucosa . At this point, the general status of host defenses determines whether colonization progresses to overt parenchymal lung infection.

Pediatr Cardiol, 1996 Mar-Apr, 17(2), 108 - 11
Infective endocarditis caused by an unusual gram-negative rod, Rahnella aquatilis; Matsukura H et al.; An 11-month-old girl with congenital heart disease developed infective endocarditis . Blood cultures revealed an unusual gram-negative rod, Rahnella aquatilis . The patient was successfully treated with a combination of netilmicin and ceftazidime . This is the first case report of infective endocarditis caused by this organism . R . aquatilis should be recognized as a clinical pathogen capable of causing life-threatening infection in children and adults.

Proteins, 1996 Mar, 24(3), 407 - 8
Crystallization and preliminary crystallographic studies of 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Escherichia coli; Tolbert WD et al.; 3-Deoxy-D-manno-octulosonate-8-phosphate (KDOP) synthase catalyzes the production of KDOP from phosphoenolpyruvate (PEP) and arabinose-5-phosphate (A5P) . In gram-negative bacteria KDOP is subsequently dephosphorylated, cytidylylated, and linked to lipid A and is required for lipid A incorporation into the outer membrane (Raetz, Annu . Rev . Biochem . 59:129-170, 1990) . We have crystallized two forms of KDOP synthase belonging to space groups I23 or I2(1)3, one with a = b = c = 118.0 A and the other with a = b = c = 233 A.

ORL J Otorhinolaryngol Relat Spec, 1996 Mar-Apr, 58(2), 68 - 73
Attenuation of aminoglycoside ototoxicity by glutathione; Nishida I et al.; Aminoglycoside antibiotics are commonly used for the treatment of serious gram-negative infections despite a high incidence of associated ototoxicity . Attempts to elucidate the mechanisms of toxicity or prevent the adverse effects have previously been unsuccessful . Recently, the damaging effects of aminoglycosides on the inner ear have been shown to be caused by a metabolite of the drug, implying an enzymatic conversion of the parent compound . Glutathione has been suggested to be closely related to the detoxification mechanisms of this metabolite . This study revealed the possible attenuation of aminoglycoside ototoxicity by glutathione . Guinea pigs were given amikacin alone or amikacin with prior intramuscular injection of glutathione . The pretreatment of glutathione significantly reduced the damage of outer hair cells of the organ of Corti . This may indicate that glutathione reduces the aminoglycoside ototoxicity and can be favorably applied in clinical use.

Arch Oral Biol, 1996 Mar, 41(3), 307 - 9
The oral prevalence of aerobic and facultatively anaerobic gram-negative rods and yeasts in semi-recluse human vegetarians; Sedgley CM et al.; Limited data exist on the oral ecology of vegetarians . Hence the dental and periodontal status, and the oral prevalence of aerobic and facultatively anaerobic Gram-negative rods (AGNR) and yeasts, were studied in 36 semi-recluse, vegetarian, Buddhist monks and nuns in Hong Kong . The oral prevalence of AGNR and yeasts was 61.1% and 33.3% . There was no correlation between the prevalence of AGNR and/or yeasts and the incidence of carious or filled teeth and the health status of the periodontium . Rather, the results of this study combined with those of previous studies suggest that increasing age and the consumption of food prepared in communal kitchens might be more important contributory factors in the oral prevalence of AGNR than the nature of the diet itself or the health of the dentition and periodontium.

Zh Mikrobiol Epidemiol Immunobiol, 1996 Mar-Apr, (2), 76 - 9
{The Fc-dependent binding of gram-negative bacterial endotoxins by human blood polymorphonuclear leukocytes}; Likhoded VG et al.; The treatment of thin blood smears with antibodies to glycolipid of chemotype Re, conjugated with horseradish peroxidase, revealed that under physiological conditions about 3.5% of leukocytes bound endotoxin of gram-negative bacteria by means of the Fc-dependent mechanism . In addition, about 4.9% of leukocytes may bind endotoxin as the result of the treatment of blood smears with the preparation of glycolipid of chemotype Re . At the acute period of bacterial cerebrospinal meningitis leukocytes capable of binding endotoxin in the body or during the treatment of blood smears are practically absent . The conclusion was made that the binding of endotoxins by leukocytes had a protective character.

Can Vet J, 1996 Mar, 37(3), 157 - 60
Tissue and serum concentrations of amikacin after intramuscular and intrauterine administration to mares in estrus; Orsini JA et al.; Concentrations of amikacin in endometrial tissue and plasma were studied in mares in estrus after intrauterine infusion of 1.0 or 2.0 g once a day for 3 consecutive d, and after 9.7 or 14.5 mg/kg body weight (BW) had been injected intramuscularly once a day for 3 consecutive d to determine concentrations of amikacin sulfate in plasma and endometrial tissues, and whether parenteral administration provides any advantages over intramuscular infusion . No amikacin was detected in serum at the 1.0 g dose . At the infusion dose of 2.0 g once a day, very low levels of serum amikacin were detected at 1 and 4 h postinfusion on the 1st treatment day . Amikacin was found to penetrate the endometrium after intramuscular injection; however, the levels attained were not as high as those achieved following intrauterine infusion . Based on the tissue and serum concentrations of amikacin, an intrauterine infusion at a dose of 4.4 mg/kg BW/d would appear to be an appropriate therapeutic regimen for the treatment of gram-negative endometritis.

Radiats Biol Radioecol, 1996 Mar-Apr, 36(2), 195 - 208
{Radiation-protective action of microbial substances}; Andrushchenko VN et al.; The referenced and our data on the radioprotective effect of microbial substances have been analysed . It was shown that Gram-negative microbe polysaccharides produce the maximum effect . The radioprotective mechanism of polysaccharides is considered in connection with their effect on the immune and hemopoietic systems.

Infect Immun, 1996 Mar, 64(3), 905 - 12
Surface localization of Helicobacter pylori urease and a heat shock protein homolog requires bacterial autolysis; Phadnis SH et al.; Helicobacter pylori is a gram-negative bacterium which causes chronic gastritis and is associated with peptic ulcer disease, gastric carcinoma, and gastric lymphoma . The bacterium is characterized by potent urease activity, thought to be located on the outer membrane, which is essential for survival at low pH . The purpose of the present study was to investigate mechanisms whereby urease and HspB, a GroEL homolog, become surface associated in vitro . Urease, HspB, and catalase were located almost exclusively within the cytoplasm in fresh log-phase cultures assessed by cryo- immunoelectron microscopy . In contrast, significant amounts of surface-associated antigen were observed in older or subcultured preparations concomitantly with the appearance of significant amounts of extracellular antigen, amorphous debris, and membrane fragments . By use of a variety of biochemical methods, a significant fraction of urease and HspB was associated with the outer membrane in subcultured preparations of H . pylori . Taken together, these results strongly suggest that H . pylori cells undergo spontaneous autolysis during culture and that urease and HspB become surface associated only concomitant with bacterial autolysis . By comparing enzyme sensitivity to flurofamide (a potent, poorly diffusible urease inhibitor) in whole cells with that in deliberately lysed cells, we show that both extracellular and intracellular urease molecules are active enzymatically . Autolysis of H . pylori is an important phenomenon to recognize since it likely exerts significant effects on the behavior of H . pylori . Furthermore, the surface properties of H . pylori must be unique in promoting adsorption of cytoplasmic proteins.

Infect Immun, 1996 Mar, 64(3), 825 - 8
Protection against endotoxic shock and lipopolysaccharide-induced local inflammation by tetracycline: correlation with inhibition of cytokine secretion; Shapira L et al.; Septic shock results from excessive stimulation of host immune cells, particularly monocytes and macrophages, by lipopolysaccharide (LPS) released from gram-negative bacteria . Macrophage-derived cytokines, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-1beta (IL-1 beta), have been identified as central mediators in the pathogenesis of septic shock and the resultant mortality . Therefore, these cytokines were targets for experimental therapy for septic shock . Because of tetracycline's ability to intervene in cellular mechanisms involved in cytokine secretion, we tested the effect of tetracycline on LPS-induced septic shock and inflammatory lesions in mice . Tetracycline was found to protect mice against LPS-induced lethality and to abolish clinical signs of LPS-induced inflammatory lesions . This protection correlates with tetracycline's ability to reduce LPS-induced TNF-alpha levels in serum . Furthermore, tetracycline was found to inhibit LPS-induced TNF-alpha and IL-1 beta secretion, but not cytokine mRNA accumulation, in human monocytes in vitro . The results presented here suggest that tetracycline is a potent drug for LPS-induced pathology and that its mechanism of action involves blockage of posttranscriptional events of cytokine production.

Gastroenterology, 1996 Mar, 110(3), 733 - 9
Inflammatory bowel disease is associated with increased mucosal levels of bactericidal/permeability-increasing protein; Monajemi H et al.; BACKGROUND & AIMS: Clinical sepsis seldom accompanies inflammatory bowel disease . The aim of this study was to measure colonic mucosal levels of the neutrophil product bactericidal/permeability-increasing protein (BPI), which kills gram-negative bacteria in addition to inactivating endotoxin . METHODS: Enzyme-linked immunosorbent assay and immunohistochemistry for BPI were performed on homogenates and tissue secretions of biopsy specimens from patients with ulcerative colitis (n=11) and Crohn's disease (n=5) and from normal controls (n=5) . RESULTS: Mucosal neutrophil content (144 +/- 23 vs . 35 +/- 9 neutrophils/mg protein; P<0.007) and BPI content (2.07 +/- 0.75 vs . 0.12 +/- 0.02 ng/mg protein; P<0.002) were greater in the colitis groups and correlated closely (r=0.68; P<0.001) . This relationship held for both ulcerative colitis (P<0.002) and Crohn's disease (P<0.01) with a trend towards greater levels in Crohn's disease . There was a trend towards higher BPI levels with an increasing endoscopic inflammation score (grade I, 1.32 +/- 0.6 ng/mg protein; grade II, 2.82 +/- 1.4 ng/mg protein) . Immunohistochemistry and the biopsy culture showed BPI to be both intracellular and extracellular, to be present in the crypt lumen, and to be released into incubating medium . CONCLUSIONS: Mucosal levels of BPI are increased in colitis . Such localization may ameliorate mucosal responses to gram-negative bacteria and their products.

J Surg Res, 1996 Feb 15, 61(1), 206 - 14
Effects of urinastatin and free radical scavengers on hepatic lipid peroxidation in endotoxemia; Htwe T et al.; In gram-negative septicemia, endotoxin-induced free radicals probably damage the liver cells by membrane lipid peroxidation . Phosphotidylcholine hydroperoxide (PCOOH), a primary lipid peroxidation product can be applied as a parameter to measure the extent of liver damage . The protective effects of urinastatin and free radical scavengers, superoxide dismutase (SOD), and catalase against hepatic lipid peroxidation and tissue energy reserves in the liver during endotoxemia were evaluated in rats with gram-negative septicemia induced by cecal ligation and puncture (CLP) . One hundred and sixty-five rats were divided into three groups . The first two groups consisted of 45 rats each . Group (1) was used for blood endotoxin level and liver function tests, group (2) for hepatic energy charge and PCOOH measurement, and group (3) (n = 75) for survival study . In each group, control animals received saline injection only . Urinastatin was injected twice intravenously through tail veins using 50,000 u/kg at 0 and 12 hr after CLP . SOD 90,000 u/kg and catalase 50,000 u/kg were given subcutaneously just before CLP and every 3 hr thereafter up to 24 hr . Liver and blood specimens were taken at time points 0, 12, and 24 hr after CLP . Increased concentration of PCOOH in liver denotes that endotoxemia can damage the liver by hepatocellular lipid peroxidation . Attenuation of lipid peroxidation, which correlated with liver enzyme leakage, was noted by finding significant decreased concentrations of PCOOH (P < 0.001), improvement in energy charge (P < 0.05), and survivability (P < 0.05) was seen in urinastatin or radical scavenger-treated groups . These results suggested that urinastatin has protective effect against free radical-induced lipid peroxidation probably by inhibiting proteases especially elastase, from polymorphonuclear leucocytes . SOD and catalase, which scavenged oxygen free radicals, also suppressed free radical-induced lipid peroxidation . Improvement in survivability was also seen in treated groups.

J Immunol, 1996 Feb 15, 156(4), 1525 - 30
Age-associated differences in TNF-alpha and nitric oxide production in endotoxic mice; Chorinchath BB et al.; Gram-negative bacterial infection is a common cause of septic shock in the older population in the U.S . We employed an experimental model of sepsis to study the cause of increased lethality due to LPS in older animals . Three ages of male B6JC3J/Nia mice, young (2 mo old), mature (12 mo old), and senescent (24 mo old), were treated with bacterial LPS, and the older mice were found to be 10 times more sensitive to LPS lethality . Increased sensitivity to LPS in senescent mice correlated with significantly elevated plasma TNF-alpha and nitric oxide levels . Abs to TNF-alpha afforded aged animals passive protection against a supralethal dose of LPS, establishing a central role for TNF in the increased sensitivity to LPS seen in the aged animals . Other cytokines, such as IL-1 and IFN-gamma, appeared secondary to TNF and nitric oxide in the age-associated sensitivity to LPS . Plasma corticosterone levels were increased by LPS at a time when maximal levels of plasma TNF-alpha were observed in both age groups, although the kinetics of hormone production and the magnitude of TNF-alpha release varied among the age groups . Exogenously administered dexamethasone protected senescent animals given a high dose of LPS, by decreasing cytokine levels . The increased sensitivity to LPS observed in aged animals, therefore, seems to be due to excessive TNF and nitric oxide production, resulting from perturbed endogenous hormonal control of cytokine production.

JAMA, 1996 Feb 14, 275(6), 470 - 3
The spectrum of septic encephalopathy . Definitions, etiologies, and mortalities; Eidelman LA et al.; OBJECTIVE--To determine whether the severity of septic encephalopathy is correlated with gram-negative bacteremia and mortality and whether there exists a single or combination of metabolic derangements(s) that cause septic encephalopathy . DESIGN AND SETTING--Prospective case series in an academic medical center . PATIENTS--Fifty patients selected according to clinical and laboratory criteria for severe sepsis . The criteria included temperature, heart rate, respiratory rate, and hypotension and/or signs of systemic hypoperfusion . MAIN OUTCOME MEASURES--A single or combination of metabolic and laboratory derangements and organ failures, three different methods to grade the severity of septic encephalopathy, Acute Physiology and Chronic Health Evaluation II (APACHE II) scores, gram-negative bacteremia and infection, and mortality . RESULTS--Encephalopathy was associated with an increase in mortality when graded by the Glasgow Coma Score; a score of 15 had 16% mortality, 13 to 14 had 20%, 9 to 12 had 50%, and 3 to 8 had 63% mortality (P < .05) . Bacteremia was associated with encephalopathy; 13% of septic patients without encephalopathy vs 59% of patients with encephalopathy had bacteremia (P < .001) when graded by altered mental status . Septic encephalopathic patients had elevated serum urea nitrogen and bilirubin levels, increased APACHE II scores, and a higher incidence of renal failure . CONCLUSIONS--The severity of septic encephalopathy correlated with mortality, bacteremia, and renal and hepatic dysfunction . The Glasgow Coma Score is a useful tool for characterizing septic encephalopathy . Considerable variations can be found according to different criteria used to classify septic encephalopathy.

J Aerosol Med, 1995 Summer, 8(2), 177 - 86
Technique for measurement of oropharyngeal clearance in the elderly; Diot P et al.; In elderly patients, gram negative bacterial colonization often preceeds nosocomial pneumonia . As we propose that a critical factor influencing this change from normal to gram negative predominance is an alteration in oral clearance, we designed this study to validate a technique for measurement of oropharyngeal clearance in a large number of nursing home residents . We modified a protocol of La Force et al who utilized an atomizer to radiolabel oropharyngeal secretions . We determined the output per spray of a DeVILBISS model 152 atomizer and found that 3 sprays of 5 mCi of 99mTc-HSA in 4 ml saline delivered 263 microCi in 0.21 ml . To measure clearance, we designed a portable, collimated ratemeter . It has a lead lined tapered aluminium frame 15 cm high, originating from a 7.5 cm rectangular base which is fitted to the scintillator . On the bench we demonstrated that this collimator, used to confine detection to the face, did not alter sensitivity and linearity of the ratemeter in our specific experimental conditions . When the ratemeter was collimated and its window off, its sensitivity was 5 times greater than the gamma camera with no loss of linearity . However, distance had a significant effect on the ratemeter's sensitivity whereas it had little effect on the gamma camera . Finally, in thirteen patients we assessed the ratemeter's accuracy in measurement of oropharyngeal clearance by comparing curves obtained simultaneously from the ratemeter and gamma camera . While each curve had its own characteristics, both devices provided remarkably similar data and there were no significant differences (r = 0.967, p < 0.0001) . We conclude that oropharyngeal clearance can be conveniently and accurately studied in elderly patients at the bedside with a collimated ratemeter . The high sensitivity provides a measure of clearance with low levels of radioactivity exposure, allowing repeated studies over time.

Mol Ecol, 1996 Feb, 5(1), 133 - 43
Dissemination of the strA-strB streptomycin-resistance genes among commensal and pathogenic bacteria from humans, animals, and plants; Sundin GW et al.; Gene transfer within bacterial communities has been recognized as a major contributor in the recent evolution of antibiotic resistance on a global scale . The linked strA-strB genes, which encode streptomycin-inactivating enzymes, are distributed worldwide and confer streptomycin resistance in at least 17 genera of gram-negative bacteria . Nucleotide sequence analyses suggest that strA-strB have been recently disseminated . In bacterial isolates from humans and animals, strA-strB are often linked with the suIII sulfonamide-resistance gene and are encoded on broad-host-range nonconjugative plasmids . In bacterial isolates from plants, strA-strB are encoded on the Tn3-type transposon Tn5393 which is generally borne on conjugative plasmids . The wide distribution of the strA-strB genes in the environment suggests that gene transfer events between human, animal, and plant-associated bacteria have occurred . Although the usage of streptomycin in clinical medicine and animal husbandry has diminished, the persistence of strA-strB in bacterial populations implies that factors other than direct antibiotic selection are involved in maintenance of these genes.

Protein Eng, 1996 Feb, 9(2), 239 - 47
Display of beta-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-beta-lactamase fusions; Georgiou G et al.; Bacterial cell-surface exposure of foreign peptides and soluble proteins has been achieved recently by employing a fusion protein methodology . An Lpp'-OmpA(46-159)-Bla fusion protein has been shown previously to display the normally periplasmic enzyme beta-lactamase (Bla) on the cell surface of the Gram-negative bacterium Escherichia coli . Here, we have investigated the role of the OmpA domain of the tripartite fusion protein in the surface display of the passenger domain (Bla) and have characterized the effects of the fusion proteins on the integrity and permeability of the outer membrane . We show that in addition to OmpA(46-159), a second OmpA segment, consisting of amino acids 46-66, can also mediate the display of Bla on the cell surface . Other OmpA domains of various lengths (amino acids 46-84, 46-109, 46-128, 46-141 and 46-145) either anchored the Bla domain on the periplasmic face of the outer membrane or caused a major disruption of the outer membrane, allowing the penetration of antibodies into the cell . Detergent and antibiotic sensitivity and periplasmic leakage assays showed that changes in the permeability of the outer membrane are an unavoidable consequence of displaying a large periplasmic protein on the surface of E . coli . This is the first systematic report on the effects that cell surface engineering may have on the integrity and permeability properties of bacterial outer membranes.

Microbiology, 1996 Feb, 142 ( Pt 2), 337 - 45
The mercury resistance operon of the IncJ plasmid pMERPH exhibits structural and regulatory divergence from other Gram-negative mer operons; Osborn AM et al.; The bacterial mercury resistance determinant carried on the IncJ plasmid pMERPH has been characterized further by DNA sequence analysis . From the sequence of a 4097 bp Bg/II fragment which confers mercury resistance, it is predicted that the determinant consists of the genes merT, merP, merC and merA . The level of DNA sequence similarity between these genes and those of the mer determinant of Tn21 was between 56 center dot 4 and 62 center dot 4% . A neighbour-joining phylogenetic tree of merA gene sequences was constructed which suggested that pMERPH bears the most divergent Gram-negative mer determinant characterized to date . Although the determinant from pMERPH has been shown to be inducible, no regulatory genes have been found within the Bg/II fragment and it is suggested that a regulatory gene may be located elsewhere on the plasmid . The cloned determinant has been shown to express mercury resistance constitutively . Analysis of the pMERPH mer operator/promoter (O/P) region in vivo has shown constitutive expression from the mer PTCPA promoter, which could be partially repressed by the presence of a trans-acting MerR protein from a Tn21-like mer determinant . This incomplete repression of mer PTCPA promoter activity may be due to the presence of an extra base between the -35 and -10 sequences of the promoter and/or to variation in the MerR binding sites in the O/P region . Expression from the partially repressed mer PTCPA promoter could be restored by the addition of inducing levels of Hg2+ ions . Using the polymerase chain reaction with primers designed to amplify regions in the merP and merA genes, 1 center dot 37 kb pMERPH-like sequences have been amplified from the IncJ plasmid R391, the environmental isolate SE2 and from DNA isolated directly from non-cultivated bacteria in River Mersey sediment . This suggests that pMERPH-like sequences, although rare, are nevertheless persistent in natural environments.

Genomics, 1996 Feb 1, 31(3), 283 - 94
A high-resolution map in the chromosomal region surrounding the Lps locus; Qureshi ST et al.; The Lps locus on mouse chromosome 4 controls host responsiveness to lipopolysaccharide, a major component of the outer membrane of Gram-negative bacteria . The C3H/HeJ inbred mouse strain is characterized by a mutant Lps allel (Lpsd) that renders it hyporesponsive to LPS and naturally tolerant of its lethal effects . To identify the Lps gene by a positional cloning strategy, we have generated a high-resolution linkage map of the chromosomal region surrounding this locus . We have analyzed a total of 1604 backcross mice from a preexisting interspecific backcross panel of 259 (Mus spretus x C57BL/6J)F1 x C57BL/6J and two novel panels of 597 (DBA/2J x C3H/HeJ)F1 x C3H/HeJ and 748 (C57BL/6J x C3H/HeJ)F1 x C3H/HeJ segregating at Lps . A total of 50 DNA markers have been mapped in a 11.8-cM span overlapping the Lps locus . This positions the Lps locus within a 1.1-cM interval, flanked proximally by a large cluster of markers, including three know genes (Cd301, Hxb, and Ambp), and distally by two microsatellite markers (D4Mit7/D4Mit178) . The localization of the Lps locus is several centimorgans proximal to that previously assigned.

J Chemother, 1996 Feb, 8(1), 17 - 24
Amikacin as first-choice aminoglycoside in a medical intensive care unit: a one-year bacteriological surveillance study; Peetermans WE et al.; The aim of this study was to determine the baseline pattern of resistance to aminoglycosides in a medical intensive care unit and to evaluate the influence on resistance rates of the use of amikacin as first-line aminoglycoside . A prospective bacteriological surveillance study was done during a 3-month baseline period with all aminoglycosides freely available, followed by a 9-month study period with predominant amikacin use . All patients admitted for more than 24 hours who were colonized or infected were included . Susceptibility rates for all isolates during the baseline period were 69% for amikacin, 32% for gentamicin, 35% for tobramycin and 46% for netilmicin . During the amikacin study period susceptibility rates rose to 75%, 43%, 44% and 51%, respectively . The increase in susceptibility to gentamicin and tobramycin, considering all isolates, was statistically significant . There was also a significant increase in susceptibility to netilmicin for gram-negative bacteria . There was no significant change in susceptibility to amikacin . We conclude that the use of amikacin as a first-choice aminoglycoside in a medical intensive care unit is warranted because of the better susceptibility rates and does not lead to a rapid rise in amikacin resistance . The predominant use of amikacin may have a favorable influence on susceptibility to the other aminoglycosides . To answer the question of whether these conclusions will remain valid over a longer period of predominant amikacin use, follow-up surveillance studies are mandatory.

Antimicrob Agents Chemother, 1996 Feb, 40(2), 500 - 2
Unique susceptibility of Helicobacter pylori to simethicone emulsifiers in alimentary therapeutic agents; Kane AV et al.; Helicobacter pylori is killed in vitro by polyoxyethylene acyl esters and ethers similar to simethicone emulsifiers in therapeutic antifoams . The MBC of these compounds for Helicobacter pylori was less than 20 micrograms/ml, while other gram-negative bacteria were unaffected by much higher concentrations of up to 50 mg/ml.

Mol Microbiol, 1996 Feb, 19(4), 705 - 13
Suppression of transcription polarity in the Escherichia coli haemolysin operon by a short upstream element shared by polysaccharide and DNA transfer determinants; Nieto JM et al.; Expression of the Escherichia coli hlyCABD operon encoding synthesis, maturation and export of haemolysin toxin was strongly dependent upon a 35 bp DNA sequence, spanning the element GGCGGTAG, located 2 kbp upstream . When the hly operon was placed under the control of the inducible tac promoter, expression remained dependent upon this element, when transcribed in its native orientation 3' of the promoter . The increase in ptac-directed transcription was strongest for the distal, export genes of the hly operon, and was particularly striking when ptac and the element were placed far upstream . The element did not influence transcript stability, and we suggest that it is a key component of a novel regulatory mechanism may suppresses transcription polarity within operons . The mechanism that be of widespread importance in bacterial gene expression because the 8 bp element is present in many Gram-negative species as an upstream component of operons encoding the production of toxins and the surface assembly of polysaccharides and components required for the conjugal transfer of DNA . We name it the ops element for operon polarity suppressor.

J Clin Microbiol, 1996 Feb, 34(2), 352 - 4
Rejection criteria for endotracheal aspirates from pediatric patients; Zaidi AK et al.; Endotracheal aspirates (ETAs) from mechanically ventilated pediatric patients frequently are cultured as part of an evaluation for suspected sepsis . There are now well-defined criteria for rejecting low-yield ETAs from adults, but it is uncertain whether the same criteria can be applied to ETAs from children . Therefore, we compared the Gram stain and culture results for 361 consecutive ETA specimens collected from pediatric patients over a 1-year period . Results for patients for whom a blood culture was performed within 48 h of the time that a culture of ETA was performed were also reviewed . Gram stains were examined under x100 magnification to quantitate the number of polymorphonuclear neutrophils and squamous epithelial cells (SECs) per low-power field and under x1,000 magnification for the presence of organisms . No organisms were seen by Gram staining in 225 (62%) of the ETAs . Culture of these specimens rarely yielded useful information: 52% were sterile, 32% grew rare to 1+ quantities of expected respiratory flora only, 12% grew rare to 1+ quantities of gram-negative rods mixed with expected respiratory flora, and only 10 (4%) yielded a pure or predominant growth of a potential respiratory pathogen . Unlike adult patients, we did not find the number of SECs to be a useful screening criterion . Only 17 (5%) of the ETAs had greater than 10 SECs per low-power field, and 5 (29%) of these yielded pure growth of a gram-negative rod . When blood culture results were positive, they correlated with ETA culture results in only 6 of 10 cases . On the basis of our findings, the absence of organisms on Gram staining is a useful criterion for rejecting ETAs from pediatric patients for culture and would have excluded 62% of the specimens from further processing.

J Chemother, 1996 Feb, 8 Suppl 2, 37 - 47
Bacterial resistance to cephalosporins as a function of outer membrane permeability and access to their target; James PA et al.; Resistance of Gram-negative bacteria to cephalosporins, as with other beta-lactam antibiotics, is a function of a combination of outer-membrane permeability, affinity and stability to beta-lactamases, and their activity against target sites (penicillin-binding proteins) . Permeation through the outer-membrane is largely governed by the presence and properties of porins, which are water-filled channels facilitating the movement of hydrophilic molecules across the membrane . The properties of porins vary considerably between wild-type bacterial species, and their numbers (and hence the ability of a bacterial cell to exclude the antibiotic) may be reduced in strains with acquired resistance . In the case of cephalosporins, ability to cross the outer-membrane is related to physico-chemical properties such as molecular size, hydrophobicity and the number and charge of ionised groups . Thus, for example, dianionic compounds have in general lower permeability rates than dipolar cephalosporins . These relationships are discussed in detail . The phenotypically expressed susceptibility of a particular bacterial strain to a cephalosporin is brought about by a dynamic combination of permeation, the ability of the agent to resist degradation or binding to the beta-lactamases in the periplasmic space which act upon the relatively low concentration of cephalosporin present there, and target affinity . The interplay of these factors is discussed.

J Chemother, 1996 Feb, 8 Suppl 2, 23 - 30
Comparative affinities for penicillin-binding proteins of multipolar ionic amphoteric cephalosporins in gram-negative bacteria; Fontana R et al.; Chemical modification of the highly reactive 7-aminocephalosporanic acid has yielded many compounds with improved activities and expanded clinical spectra . Introduction of an alpha-oxyimino group in C-7 position has given rise to improved activity against Gram-negative bacteria as a consequence of the high affinity of compounds carrying this substituent for the essential penicillin-binding protein (PBP) 3 . The spectrum of activity of oxyimino cephalosporins has been further expanded by introduction of substituents with a quaternary nitrogen in C-3 position . These compounds have maintained their high affinity for the essential PBP 3, typical of the third generation cephalosporins and have acquired an improved ability to cross the outer membranes of Gram-negative bacteria . Among these compounds cefepime also exhibits high affinity for PBP 2, a very unusual property among cephalosporins.

Shock, 1996 Feb, 5(2), 116 - 21
A selective inhibitor of inducible in nitric oxide synthase prolongs survival in a rat model of bacterial peritonitis: comparison with two nonselective strategies; Aranow JS et al.; We evaluated the effects on survival of three different strategies for blocking the actions of nitric oxide (NO) during Gram-negative sepsis in rats . Male Sprague-Dawley rats underwent placement of a jugular vein catheter and i.p . implantation of a gelatin capsule containing a paste (.11 +/- 0.1 g final weight) consisting of sterile rat feces mixed with a suspension (.2 mL) of viable Escherichia coli (strain sm 18; 5.7 x 10(5) colony-forming units) in saline . Beginning at T = 6h, all animals received i.v . ampicillin (85 mg/kg every 12 h) until death or the administration of five doses . At the same time points, pairs of animals received an i.v . dose of either an experimental treatment agent or an appropriate control substance . The following experimental regimens were tested: 5 mg/kg per dose of S-methylisothiourea sulfate (SMT), a selective inhibitor of the inducible isoform of nitric oxide synthase (NOS); 10 mg/kg per dose or 25 mg/kg per dose of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of the inducible and constitutive isoforms of NOS; 200 mg/kg per dose of cross-linked human hemoglobin (HGB), an NO scavenger . SMT significantly prolonged survival in septic rats, although cumulative survival at T = 168 h was approximately equivalent in SMT- or saline-treated animals . In contrast, HGB and the higher dose of L-NAME significantly shortened survival times . At T = 20 h, arterial PO2 was significantly lower in rats treated with HGB as compared to time-matched controls . We conclude that SMT, a compound with reported activity as a selective inhibitor of the inducible isoform of NOS, prolongs survival in a rat model of antibiotic-treated Gram-negative sepsis.

Infect Immun, 1996 Feb, 64(2), 406 - 12
Role of endotoxemia in cardiovascular dysfunction and lethality: virulent and nonvirulent Escherichia coli challenges in a canine model of septic shock; Hoffman WD et al.; We investigated whether the severity of septic shock is determined by virulence factors associated with or the levels of endotoxemia produced by two Escherichia coli strains . Canines were challenged intraperitoneally with an E . coli strain (O6:H1:K2) that has virulence factors associated with human disease or with an equal dose of a nonvirulent strain (O86:H8) that lacks these factors . Both strains were administered in viable, heat-killed, and purified endotoxin forms . Median survival times with the virulent strain compared with the nonvirulent strain were shorter with viable bacteria (5 x 10(10) CFU/kg) (144 h versus > 672 h; Wilcoxon, P = 0.03), longer with heat-killed bacteria (5 x 10(9) CFU/kg) ( > 676 h versus 26 h; P = 0.03), and similar with purified endotoxin (15 mg/kg) (28 h versus 48 h; P = 0.71) . However, whether the challenge contained viable bacteria, heat-killed bacteria, or purified endotoxin, the virulent strain produced less endotoxemia (P = 0.001) . Hence, the changing outcomes with differing forms of the two strains cannot be attributed solely to endotoxin levels . The viable virulent strain caused less endotoxemia but more harm, and this does not appear to be explained by a more potent endotoxin or other heat-stable component . This study suggests that circulating endotoxin levels per se are less important in the outcome of septic shock than virulence factors associated with E . coli strains . Furthermore, the data call into question the significance of the endotoxin concentration in the blood in predicting the severity of shock and the lethality of gram-negative infections.

Dtsch Med Wochenschr, 1996 Jan 26, 121(4), 94 - 8
{Lemierre's post-tonsillitis sepsis with meningitis and intravascular consumption coagulopathy as complication of infectious mononucleosis with pansinusitis}; Busch N et al.; HISTORY AND CLINICAL FINDINGS: 24 days after the onset of infectious mononucleosis, clinically and serologically confirmed, an otherwise healthy 18-year-old schoolboy developed a fulminant septicaemia with acute meningitis and loss of consciousness, consumptive coagulopathy and acute renal failure . INVESTIGATIONS: Computed tomography demonstrated pansinusitis . Lumbar puncture produced purulent cerebrospinal fluid with 11,500 cells/microliters, predominantly granulocytes, protein 205 mg/dl, glucose 19 mg/dl, indicating bacterial meningitis . The suspected diagnosis of posttonsillitis septicaemia (Lemierre's syndrome) was confirmed by repeated demonstration of fusiform gram-negative bacteria in anaerobic blood cultures, identified as Fusobacterium necrophorum . Anaerobic CSF culture grew Prevotella bivia of the Bacteroidaceae family . TREATMENT AND COURSE: Both the consumptive coagulopathy and the renal failure were successfully treated and the patient's condition stabilized . The sinuses were surgically drained under high doses of piperacillin/sulbactam and chloramphenicol . Despite the sensitivity of the cultured bacteria to the administered antibiotics the septic temperature continued, but disappeared within 4 days of metronidazole having been added . After 5 weeks of antibiotic treatment, three of them in an intensive care unit, the patient was discharged in good general condition . CONCLUSION: This case illustrates that severe septicaemia caused by rare bacteria may follow an attack of infectious mononucleosis which had taken an uncomplicated course.

Biochemistry, 1996 Jan 16, 35(2), 579 - 85
Acetyltransfer precedes uridylyltransfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU protein of Escherichia coli; Gehring AM et al.; The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine {Mengin-Lecreulx, D., & van Heijenoort, J . (1994) J . Bacteriol . 176, 5788-5795}, a fundamental precursor in bacterial peptidoglycan biosynthesis and the source of activated N-acetylglucosamine in lipopolysaccharide biosynthesis in Gram-negative bacteria . In the work described here, the GlmU protein and truncation variants of GlmU (N- and C-terminal) were purified and kinetically characterized for substrate specificity and reaction order . It was determined that the GlmU protein first catalyzed acetyltransfer followed by uridylyltransfer . The N-terminal portion of the enzyme was capable of only uridylyltransfer, and the C-terminus catalyzed only acetyltransfer . GlmU demonstrated a 12-fold kinetic preference (kcat/Km, 3.1 x 10(5) versus 2.5 x 10(4) L.mol-1.s-1) for acetyltransfer from acetyl-CoA to glucosamine-1-P as compared to UDP-glucosamine . No detectable uridylyltransfer from UTP to glucosamine-1-P was observed in the presence of GlmU; however, the enzyme was competent in catalyzing the formation of UDP-N-acetylglucosamine from UTP and N-acetylglucosamine-1-P (kcat/Km 1.2 x 10(6) L.mol-1.s-1) . A two active site model for the GlmU protein was indicated both by domain dissection experiments and by assay of the bifunctional reaction . Kinetic studies demonstrated that a pre-steady-state lag in the production of UDP-N-acetylglucosamine from acetyl-CoA, UTP, and glucosamine-1-P was due to the release and accumulation of steady-state levels of the intermediate N-acetylglucosamine-1-P.

Harefuah, 1996 Jan 15, 130(2), 90 - 2, 143
{Strongyloides stercoralis hyperinfection}; Gelber M et al.; Strongyloides stercoralis (SS) is an intestinal nematode which infects a large proportion of tropical and subtropical populations . The endemic areas are southeast Asia, South America and sub-Saharan Africa . Infection is acquired when the worms penetrate the skin, pass via the blood stream to the lungs, ascend the respiratory tract, are swallowed and grow into adult worms in the mucosa of the small intestine . The eggs laid by the female release larvae which pass down the lumen and reach the soil via the feces . While passing down the intestinal tract, a few of the rhabditiform larvae may be transformed into infective filariform larvae which can penetrate the distal intestinal and anal mucosa, returning to the blood stream . This "autoinfection" cycle is unique to SS, and explains why the infection can be perpetuated without further exposure to exogenous, infective larvae and can persist decades after departure from endemic areas . A substantial proportion of those affected are asymptomatic, but overwhelming infection may occur in immunosuppressed patients . This "hyperinfection" state is characterized by severe gastrointestinal and respiratory tract involvement, along with skin rash, Gram-negative bacteremia and cerebral signs . While frequently fatal, it is curable when diagnosed and treated promptly . We present a 79-year-old woman with idiopathic polymyositis, who was immunosuppressed by prolonged corticosteroid treatment . Her illness was complicated by SS hyperinfection . Diagnosis was made from stool and bronchial smears . Treatment with thiabendazole was started early and within a week there was complete cure . This is the fourth reported case of SS hyperinfection treated in Israel.

Biochemistry, 1996 Jan 9, 35(1), 299 - 308
Intermembrane molecular contacts by polymyxin B mediate exchange of phospholipids; Cajal Y et al.; Direct intermembrane exchange of dimyristoylphosphatidylmethanol is mediated by polymyxin B (PxB), a cationic amphipathic cyclic decapeptide . The possibility that the phospholipid exchange is mediated by solubilization of phospholipids or by fusion of vesicles is ruled out . By kinetic and spectroscopic methods it is shown that the exchange occurs directly through vesicle-vesicle contacts formed by a few PxB molecules . The contact is stable on the time scale of several minutes such that neither PxB nor the vesicles in the pair forming a contact exchange with excess vesicles . Several contacts may be formed on a vesicle, which leads to the formation of a cluster of vesicles, and the lipid molecules on the outer monolayers of vesicles exchange throughout the cluster . Kinetics of substrate replenishment during processive interfacial catalysis suggests that the exchange of anionic lipids over the contact occurs at a rate considerably faster than 300 s-1 . The exchange through the contact is specific for certain lipids, and phospholipids with a modified head group or phospholipase A2 bound to a vesicle are not transferred to the other vesicle in contact . Since this phenomenon has not been described before, possible implications of direct vesicle-vesicle exchange of phospholipids through peptide-mediated molecular contacts are discussed . Such a mechanism for intermembrane transfer of phospholipids could be responsible for intracellular trafficking and sorting of phospholipids; it could be a necessary first step for the sequence of events leading to budding, vesiculation, and secretion; and PxB-mediated transfer between the inner and outer membranes of Gram-negative bacteria could also account for its antibiotic action.

Mol Membr Biol, 1996 Jan-Mar, 13(1), 41 - 48
Combinatorial mutagenesis analysis of residues in the channel constriction loop L3 and neighbouring beta-strands in the LamB glycoporin of Escherichia coli; Chan WC et al.; Members of the LamB family of sugar-selective porins (glycoporins) are beta-barrel proteins in the outer membrane of Gram-negative bacteria . To study the determinants of structure and sugar selectivity, 68 non-identical single amino acid substitutions were introduced into the stretch of sequence consisting of residues 106 through 125 in Escherichia coli LamB . This region includes all bar one residue of the channel constriction loop L3 and extends into the transmembrane beta 6 strand in the LamB structure . Mutants were assayed for dextrin utilization, starch binding, A binding, monoclonal antibody binding and for qualitative changes in protein expression . The importance of the L3 amino acids was emphasized by the observation that only four residues permitted a majority of neutral substitutions . Changes to the channel constriction zone strongly affected sugar binding yet no single amino acid change of residues exposed to the channel lumen caused a complete defect in maltodextrin utilization (i.e . were still Dex+) . Substitutions in the L3 loop did not affect phage lambda binding, except one change at residue 122, nor changed recognition by anti-LamB antibodies specific for surface epitopes, consistent with the lack of a role of L3 residues in surface receptor function . In marked contrast, four substitutions in transmembrane strand beta 5 resulted in a Dex- phenotype and gross changes in protein properties, indicating the significance of beta 5 in the architecture of LamB.

Scand J Infect Dis Suppl, 1996, 101, 3 - 8
Evidence for antibiotic-mediated endotoxin release as a contributing factor to lethality in experimental gram-negative sepsis; Morrison DC et al.; Endotoxic lipopolysaccharide (LPS) is a major constituent of the outer membrane of the Gram-negative microbe . Following its release from the bacterium, LPS serves as a potent proinflammatory stimulus by interacting with humoral and cellular mediator systems to stimulate production of an array of inflammatory molecules . Cell-wall active antibiotics are known to promote endotoxin release . To assess the contribution of antibiotic-induced endotoxin release in the pathogenesis of Gram-negative sepsis, we have developed several experimental models in which mice have been pretreated with various agents to make them sensitive to Gram-negative (E . coli, pseudomonas) infection and/or the lethal effects of endotoxin . For the former, both cyclophosphamide (which renders mice neutropenic) and the reversible hepatotoxin D-galactosamine (D-gal) have been used . D-gal also sensitized mice to the lethal effects of LPS . Infected mice treated with cell-wall active antibiotics are protected approximately five- to 10-fold (as assessed by increases in LD50) if they are sensitive to LPS lethality (D-gal treatment) but 500-fold if they are resistant to LPS lethality . Importantly, different antibiotics that have been documented to cause different amounts of endotoxin release in vitro also differ in their protective efficacy in vivo . Thus, imipenem, which causes relatively low endotoxin release, is significantly more protective (8-fold) than ceftazidime or meropenem (3-fold, P < 0.005) under conditions of equivalent MICs . Lethality data correlate well with circulating levels of interleukin-6 (Il-6) in vivo and with induction of Il-6 in ex vivo studies in which anticoagulated mouse blood is incubated with bacteria and antibiotics . Finally, antiendotoxin agents manifest additional levels of protection in vivo under conditions in which antibiotics alone are not protective . Collectively, these results strongly implicate antibiotic-induced endotoxin release as a significant contributing factor in experimental Gram-negative sepsis.

Injury, 1996, 27 Suppl 3, SC9 - 15
Relevance, pathogenicity and virulence of microorganisms in implant related infections; Printzen G; It is impossible to imagine modern medicine today without indwelling devices of various kinds . The time that these implants or prostheses remain in the patient's body can vary from a few hours, e.g . intravenous catheter, to his entire life, e.g . hip prosthesis, heart valve . Besides the indisputable use and advantages of this type of medical intervention for the patient, e.g . saving his life or improving its quality, the associated complications should not be overlooked . One of the most frequent and significant complications of implant surgery is the manifestation of infection in the tissue around the implant . That infection occurs is not surprising since the indwelling devices predispose to bacterial and mycotic infection on the one hand and impede its eradication on the other . The consequences of infection for the patient may mean the loss of regained mobility and independence, hospitalization for sepsis, or even death . Microbes per se are not necessarily pathogenic, however, there are numerous virulence factors which affect the degree of pathogenicity of the microorganisms . These include, for example, various enzymes, (e.g . catalase, hyaluronidase, collagenase and other proteases), and specific surface structures, e.g . the polysaccharide capsules of pneumococci or the lipopolysaccharides of Gram negative bacteria, and the production of bacterial toxins, e.g . leucozidin, streptolysine . The strategies which the pathogenic bacteria employ in their efforts to occupy the host include adherence, penetration and multiplication, antiphagocytosis and serum resistance, the formation of siderophores, antiimmunity, and cell and tissue damage . An attempt will be made here to present an overview of this multifactorial event in which the host obviously plays an important role.

Postepy Hig Med Dosw, 1996, 50(4), 333 - 50
{Neutrophil activation by bacterial endotoxins}; Klink M et al.; The endotoxin (lipopolysaccharide, LPS), major component of Gram-negative bacteria cell wall, activate numerous types of cells, neutrophils included . The chemical compositions of polysaccharide (O-specific polysaccharide and core oligosaccharide) and hydrophobic lipid A parts of LPS are presented . The bindings of LPSs to neutrophils resulted in signal transduction and neutrophils activation . Neutrophils, under LPS stimulation, generate oxygen radicals, nitric oxide and other components of inflammatory processes.

Khirurgiia (Sofiia), 1996, 49(3), 41 - 4
{The diagnostic-therapeutic problems in acute suppurative destructive pleuropulmonary diseases in childhood}; MIkhailova V; Over a ten-year period (1985 through 1994), 183 children with acute purulent-destructive pleuropulmonary disease undergo treatment in the pediatric chest surgery department of the Medical Faculty-Sofia . Their distribution by age is as follows: 0-1 y . -28 children (15.2 per cent), 1-3 y.-67 (36.4 per cent), and 3-15 y . -88 (48.4 per cent) . To assess the lung condition, and specify the form of disease and therapeutic approach, conventional and contrast roentgenography, thoracoscopy and CAT are performed . St . aureus is the etiological contributory cause in 28.9 per cent of patients, gram-negative flora-in 32.4 per cent, mixed microbial flora-in 33.2 per cent, and in the remainder no causing agent is isolated (15.5 per cent) . All patients are subjected to complex treatment . In 23 children (12.5 per cent) the pleural complication is treated by pleural puncture, and in 138 (75.5 per cent)-by drainage of the pleural cavity . In 22 children (12.0 per cent) thoracotomy is done with varying in size pulmonary resection . In six children (3.2 per cent) the outcome is fatal.

DNA Seq, 1996, 6(6), 319 - 30
Identification of genes encoding for peptide synthetases in the gram-negative bacterium Lysobacter sp . ATCC 53042 and the fungus Cylindrotrichum oligospermum; Bernhard F et al.; Genes encoding for the multifunctional peptide synthetases lysobactin synthetase and peptolide SDZ 214-103 synthetase were identified by hybridization of genomic libraries with oligonucleotides derived from consensus motifs of various genes encoding for delta-(L-alpha amino-adipoyl)-L-cysteinyl-D-valine (ACV) synthetases and gramicidin S synthetase . The sequence of subcloned gene fragments revealed core motifs and a modular structure typical for the family of peptide synthetase genes . A fragment of 4.6 kb of the lysobactin synthetase gene was sequenced and one amino acid activating module was localized . The cloning of lysobactin synthetase was verified by marker-exchange mutagenesis and the lysobactin minus phenotype of the mutant . The sequenced 3.1 kb fragment of peptolide SDZ 214-103 synthetase contained parts of two modules and was highly homologous to corresponding regions of module 6 and 7 of cyclosporin synthetase . Therefore, the localized modules may activate the amino acids threonine and glycine.

Vestn Ross Akad Med Nauk, 1996, (8), 26 - 31
{Prospects for designing immunoglobulins to prevent and treat purulent-inflammatory diseases}; Levina LA et al.; The paper gives the results of prophylactic and therapeutic evaluations made in gram-negative opportunistic bacteria-induced pyoinflammatory diseases during retrospective and controlled studies of blood plasma preparations and immunoglobulins that contain antibodies to lipopolysaccharides . The data available in the literature and the authors' own findings confirm the obligatory presence of antibodies to tumor necrosis factor and to other interleukins, as well as the importance of IgM antibodies to various lipopolysaccharide determinants . The paper also presents the results the results of the authors' own investigations to design and evaluate the activity of intravenous immunoglobulin that contains antibodies to IgG and IgM lipopolysaccharides, which is termed panglobulin . Prospects for designing a combined preparation containing IgM and IgG antibodies with antiendotoxic activity and antibodies to tumor necrosis factor and other interleukins.

Biochimie, 1996, 78(6), 390 - 8
Bacterial poly(A) polymerase: an enzyme that modulates RNA stability; Raynal LC et al.; We have constructed a strain that overexpresses E coli poly(A) polymerase (PAP I) . The recombinant protein was soluble, and a partially purified extract had high levels of poly(A) polymerising activity . An antiserum raised against the overexpressed PAP I has permitted two types of analysis: the identification of other E coli proteins that may interact with PAP I, and the search for PAP I-like proteins in other bacteria . Immunoprecipitation experiments suggest that PAP I is associated with a 48-kDa protein . This protein remains to be identified . Western blotting using the antiserum against E coli PAP I revealed related proteins in a variety of Gram-negative bacteria and in B subtilis . A comparison of the E coli protein with putative poly(A) polymerases recently identified in H influenza and B subtilis showed highly conserved sequences in the amino terminal and central portions of the proteins that may be important for enzyme activity.

Vox Sang, 1996, 71(3), 155 - 64
Production and characterization of a reconstituted high density lipoprotein for therapeutic applications; Lerch PG et al.; A method is described for the large scale preparation of reconstituted high density lipoproteins (rHDL) suitable for therapeutic use . Apolipoprotein A-I (apoA-I was isolated from precipitates obtained by cold ethanol fractionation of human plasma . This process includes several steps for virus removal and virus inactivation, among them pasteurization . Reconstitution of lipoprotein particles was performed by cholate dialysis using soybean phosphatidylcholine as the lipid source . An apoA-I:lipid ratio of 1:150 (mol:mol) was obtained . Redissolved rHDLs were disc-shaped particles resembling nascent HDL, as assessed by electron microscopy . The method was optimized for low content of free apoA-I protein as well as the low concentration of free lipid . The product was stabilized by lyophilization in the presence of sucrose . In vitro studies show potential effects it the prevention of gram-negative septic shock and in the inhibition of atherosclerosis.

Crit Rev Oral Biol Med, 1996, 7(3), 278 - 91
Periodontitis vs . peri-implantitis: the same disease? The same treatment?
Meffert RM.
The microbial flora in the natural dentition sulcus/pocket and the implant crevice/pocket is very similar in both health and disease . In health, coccal forms predominate, and in disease, large numbers of Gram-negative pathogens are associated with both tooth and implant . It has also been demonstrated that the bacteria in the partially edentulous implant case may be more pathogenic (especially Gram-negative rods and spirochetes) than in the fully edentulous case, indicating a possible seeding mechanism from tooth pocket to implant crevice . Detoxification procedures involving the use of tetracycline and citric acid prior to regenerative procedures with the use of barrier membranes and grafting materials are necessary, and the same problems attendant to premature exposure of the barrier membrane(s) in the natural dentition situation apply to the implant case . It is apparent that periodontitis = peri-implantitis in etiology and therapy.

Emerg Infect Dis, 1996 Jan-Mar, 2(1), 18 - 29
Emergence of the ehrlichioses as human health problems; Walker DH et al.; Ehrlichiae are small, gram-negative, obligately intracellular bacteria that reside within a phagosome . The first human ehrlichial infection was recognized in the United States in 1987 . It was later shown to be caused by a new species, Ehrlichia chaffeensis . In 1994, an ehrlichial pathogen within neutrophils that is closely related to the known veterinary pathogens E . equi and E . phagocytophila was found to infect humans . Molecular methods were required to detect, characterize, and identify these fastidious and uncultivated bacteria . Subsequently, E . chaffeensis infection was documented in more than 400 patients in 30 states, Europe, and Africa . Likewise, approximately 170 cases of human granulocytic ehrlichiosis have been diagnosed, most since 1994, predominantly in the upper midwestern and northeastern states, but also in northern California . The disease caused by ehrlichiae is generally undifferentiated but is often associated with leukopenia, thrombocytopenia, and elevated serum hepatic transaminase levels in tick-exposed patients . Infection ranges from subclinical to fatal; tetracycline appears to be an effective therapy . The emergence of these two newly recognized tickborne infections as threats to human health is probably due to increased clinical cognizance, but as in other emerging tickborne infections, it is likely that the rapid increase in identified cases signals a true emergence of disease associated with a changing vector-host ecology.

Int Orthop, 1996, 20(1), 12 - 4
One stage revision arthroplasty of the hip for deep gram negative infection; Raut VV et al.; We describe the use of one stage revision arthroplasty of the hip for deep gram negative infections . We have followed up 15 patients for an average of 8 years following operation . Two procedures failed, one at two years due to aseptic loosening and another because of persistent infection . We attribute the low reinfection rate (1/15) to meticulous surgical technique, preoperative parenteral antibiotics and antibiotic loaded cement . Both failures occurred in the two operations performed without antibiotic loaded cement . We do not recommend the use of plain cement for exchange hip arthroplasty in the presence of gram negative infection.

Z Rheumatol, 1996 Jan-Feb, 55(1), 4 - 18
{New aspects in the pathogenesis of Bechterew disease}; Marker-Hermann E et al.; The association of HLA-B27 with ankylosing spondylitis (AS), first described more than 20 years ago, triggered intensive research all over the world . AS is a disease model to study the interplay between genetic, immunologic, and environmental factors in the induction of rheumatic disease . Over the past years, substantial advances have taken place in the area of the molecular and cellular immunology of the HLA-B27 molecule, HLA-B27 subtype polymorphism, peptide binding and presentation to cytotoxic T cells, and their relevance to disease . New insights into the pathogenesis of the spondylarthropathies come from the development of animal models, namely HLA-B27/human beta 2-microglobulin transgenic rats, and HLA-B27 transgenic, beta 2-microglobulin knock-out mice . The role of gram-negative bacteria and gut inflammation in the development of ankylosing spondylitis continues to be the focus of interest in many studies . In this review, recent hypotheses of the pathogenesis of AS and its relationship to HLA-B27 are discussed.

Scand J Infect Dis, 1996, 28(3), 297 - 303
Trimethoprim-sulfamethoxazole plus amikacin versus ceftazidime monotherapy as empirical treatment in patients with neutropenia and fever; Engervall P et al.; In a prospective randomized comparison, 217 episodes of fever (oral temperature > 38.5 degrees C on 1, or 38.0 degrees C on 2 occasions with a minimum interval of 4 h between recordings) during neutropenia (neutrophil count < 0.5 x 10(9)/I), patients were empirically treated with trimethoprim-sulfamethoxazole plus amikacin (TMP/SMZ plus AMI) or ceftazidime . Successful antibiotic treatment was defined as eradication of all signs, symptoms and microbiological evidence of infection on the primary therapy alone . The overall success rate did not differ between the 2 treatment groups: 31/102 (30%; 21-39%, 95% confidence interval, CI) for TMP/SMZ plus AMI and 41/115 (36%; 27-44%) for ceftazidime (difference 0.06 +/- 0.13, 95% CI) . The corresponding numbers for documented infections were 12/50 (24%; 12-36%) and 14/60 (23%; 12-35%), respectively (difference 0.01 +/- 0.16) . One patient in the TMP/SMZ plus AMI group and 2 patients in the ceftazidime group died from Gram-negative bacteraemias within 72 h . No other early deaths were observed . Antibiotics were changed due to adverse events in 2 episodes of each treatment group . In conclusion, this study demonstrates that TMP/SMZ plus AMI combination is comparable (i.e . a difference of < 20%) to ceftazidime monotherapy with regard to efficacy and safety in haematological patients with severe neutropenia . Both regimens require frequent modifications, particularly in bacteraemic fever episodes . However, in centres with a low frequency of isolation of Pseudomonas and especially of multi-resistent Pseudomonas strains, TMP/SMZ plus AMI offers an inexpensive alternative for the empirical treatment of febrile neutropenia.

Acta Neurochir (Wien), 1996, 138(6), 714 - 9
Multiloculated hydrocephalus related to cerebrospinal fluid shunt infection; Jamjoom AB et al.; This study is an attempt to establish that CSF shunt infection has a role in the aetiology of multiloculated hydrocephalus . The authors carried out a review of 12 cases of multiloculated hydrocephalus who were treated at King Khalid University Hospital between 1988-1994 . The multiloculation appears to have developed following the shunt infection in all cases . The hydrocephalus was related to an intraventricular haemorrhage (IVH) in 9 patients and was congenital in 2 patients and post-meningitic in 1 patient . The shunt infection was caused by a gram-negative organism in 8 patients and duration of external ventricular drainage ranged from 9-24 (median 13) days . The diagnosis of multiloculated hydrocephalus was made on average 2 months after the shunt infection . In three patients endoscopic fenestration of intraventricular septations was attempted but was effective in only one case . The other patients were managed by two shunts (9 patients) and three shunts (2 patients) . At a mean follow-up of 15 months, the shunt revision rate of the patients was 0.4/year . One patient died of multiple brain abscesses and 6 patients remain severely disabled . The poor outcome may also be related to the original IVH as well as the multiloculated hydrocephalus . The study also shows that patients with post-haemorrhagic hydrocephalus, who develop a shunt infection due to gram-negative organisms and in whom the CSF fails to be cleared of the infection following 12 days of external drainage appear to be at risk of developing multiloculated hydrocephalus.

Shock, 1996, 6 Suppl 1, S57 - 64
Treatment of LPS-induced tissue injury: role of liposomal antioxidants; Suntres ZE et al.; Tissue injury is a common occurrence in multiple organ failure, a possible clinical complication of Gram-negative bacterial sepsis . Gram-negative bacteria, in part through lipopolysaccharide (LPS), tumor necrosis factor, and other cytokines, activate neutrophils to increase oxygen consumption and produce reactive oxygen species (ROS) . ROS have been suggested to play a critical role in the pathogenesis of multiple organ failure . Accordingly, we hypothesized that the susceptibility of tissues to ROS can be reduced by augmenting the antioxidant status of the affected tissues . Rats were challenged intravenously with LPS (Escherichia coli: 0111:B4) at a dose of 1 mg/kg body weight, and 0, 2, 4, or 6 h later were treated intravenously with plain liposomes or alpha-tocopherol liposomes (20 mg alpha-tocopherol/kg body weight); treated rats were then killed 24 h after LPS challenge . Animals challenged with LPS were extensively damaged in the liver, as evidenced by an increase in plasma alanine aminotransferase and aspartate aminotransferase activities, and also in the lung, as indicated by a decrease in pulmonary angiotensin-converting enzyme and alkaline phosphatase activities . The injection of LPS also resulted in increased myeloperoxidase activities in the two organs, suggestive of activation of the inflammatory response . Within the pulmonary and hepatic organs of LPS-challenged animals, the involvement of oxidative stress mechanisms was evident, because a significant decrease in reduced glutathione and an increase in lipid peroxidation were observed . In contrast, the administration of alpha-tocopherol liposomes in the post-LPS-challenge period resulted in a significant alleviation of both lung and liver injuries, evidenced by a general reversal of the altered biochemical indices toward normal among treated animals . The therapeutic effect was found to be greater when liposomal alpha-tocopherol treatment was given earlier during the development of injury . Plain liposomes administered immediately after LPS injection also protected hepatic and pulmonary tissues from injuries . However, unlike alpha-tocopherol liposomes, plain liposomes did not confer any beneficial effect when administered at later timepoints post-LPS injection . These data suggest that alpha-tocopherol, administered in a liposomal form, may serve as a potentially effective pharmacological agent in the treatment of LPS-induced tissue injuries.

Shock, 1996, 6 Suppl 1, S50 - 6
The systemic inflammatory response: perspective of human endotoxemia; Santos AA et al.; Improvements in detection of cytokines and other intermediary substances has allowed a new wave of investigations to determine the role that endotoxin plays in initiating these mediators . We have reviewed all studies of endotoxin administration (Escherichia coli, Lot EC-5, 4 ng/kg) to healthy humans in an effort to collate the currently available data that describes mediator elaboration and therapy directed against them . More than 60 studies included descriptions of the effects of administering endotoxin alone and with pretreatments, such as antiendotoxin molecules (n = 8), mediator receptor antagonist (n = 9), antimediator therapy (n = 5), and anti-inflammatory agents (n = 49), which were given in an attempt to modify the inflammatory response . Endpoints that were monitored included vital signs and symptoms, leukocyte and platelet patterns, alterations in coagulation, hormonal secretion, plasma enzyme activities, plasma cytokine and anticytokine concentrations, plasma metabolic substrates, and ex vivo mononuclear cell responses . Analysis of investigations of human endotoxemia with and without pretreatments suggests that such trials a) are safe, b) are unable to achieve the success of small animal studies using pretreatments of endotoxemia, and c) have results similar to antimediator therapy administered recently to patients with Gram-negative bacteremia . Establishment of endotoxemia in humans may provide a valuable screening method to determine which antimediator treatments should be submitted to carefully constructed clinical trials.

Shock, 1996, 6 Suppl 1, S43 - 9
Liposomal cefoxitin in a porcine model of intra-abdominal sepsis: hemodynamic changes; Soltes S et al.; The effects of free versus liposomal cefoxitin on various physiological parameters in a porcine model of Gram-negative intra-abdominal sepsis were evaluated . Four different doses of Escherichia coli inoculum mixed with sterile pig feces were used (10(8), 10(9), 10(10), and 10(11) cfu/animal), and the most consistent hemodynamic changes were observed with an inoculum of approximately 10(11) bacteria/20 kg animal . Two treatment groups were established as follows: free cefoxitin (n = 9) and liposomal cefoxitin (n = 9) . All animals were maintained under anesthesia for the duration of the study, and then euthanized 24 h following intra-abdominal inoculation . The inoculated and nontreated animals showed increases in heart rate, mean pulmonary arterial pressure, systemic and pulmonary vascular resistance, and decreases in mean systemic arterial pressure and cardiac index . These changes were significant (p < .05) compared with a control group injected with normal saline . Liposomal cefoxitin-treated animals showed significantly lower decreases in mean systemic arterial pressure and increases in heart rate (p < .05) compared with both the inoculated nontreated and free cefoxitin-treated groups . Both liposomal and free cefoxitin significantly modulated the mean pulmonary arterial pressure compared with the inoculated nontreated animals (p < .05) . Acidosis that developed during intra-abdominal infection diminished 6 h following the first dose of liposomal cefoxitin (p < .05) . The results of these experiments demonstrate that liposomal cefoxitin exerts a beneficial modulation of some of the hemodynamic disturbances during intra-abdominal Gram-negative sepsis.

Clin Infect Dis, 1996 Jan, 22(1), 152 - 6
Genotyping may provide rapid identification of Escherichia coli K1 organisms that cause neonatal meningitis; Bingen E et al.; Escherichia coli K1 is the most common cause of gram-negative neonatal bacterial meningitis and septicemia . In an attempt to identify genetic markers in E . coli K1 that are associated with the capacity of the organism to cause neonatal meningitis, we used rRNA gene restriction patterns . E . coli strains isolated from the CSF of neonates with meningitis (n = 43) on two continents were compared to strains isolated from the blood of neonates with bacteremia who did not have meningitis (n = 29) and to isolates from the vaginas of asymptomatic pregnant women whose neonates remained without infection (n = 39) . E . coli strains from CSF are genetically less heterogeneous than isolates from blood and the vagina: 44.2% of the CSF isolates belonged to only two types, whereas no more than two blood vaginal strains were of the same type . After HindIII digestion, a 14.9-kb rDNA-containing fragment was found in 81.3% of the strains from CSF vs . 28.0% of the isolates from blood and only 12.8% of the vaginal isolates (P = .001) . Thus, genotyping might provide markers to identify organisms in the maternal vaginal flora that are highly likely to cause neonatal meningitis . This observation may have very practical implications for the early identification of these organisms in pregnant women and thus for the selective establishment of preventive measures per partum or for the early treatment of colonized neonates.

Free Radic Biol Med, 1996, 21(1), 117 - 21
Purification and characterization of the Cu,Zn SOD from Escherichia coli; Benov LT et al.; The periplasmic Cu,Zn superoxide dismutase has been purified to homogeneity by a procedure, which depended upon osmotic shock followed by two chromatographic columns . Its subunit weight, determined by electrospray ionization mass spectrometry, was found to be 15,737 +/- 1.6 . The second derivative ultraviolet spectrum indicated a lack of tryptophan . The amino acid composition as well as a partial N-terminal amino acid sequence is reported . The specific activity was 3700 U/mg and the corresponding copper content was 0.77 atoms Cu/subunit . The enzyme was quite unstable and overnight dialysis against EDTA or even prolonged dialysis against neutral phosphate buffer caused partial loss of activity and of copper and visible precipitation . It is likely that some losses occurred during the isolation procedure, and if these could have been prevented the copper content would have been 1.0 Cu/subunit and the specific activity would have been 4800 U/mg . It now appears likely that gram negative bacteria will commonly be found to contain a periplasmic Cu,Zn SOD.

Ann Chir Gynaecol Suppl, 1996, 211, 1 - 53
The effect of tumor necrosis factor-alpha on wound healing . An experimental study; Rapala K; The inflammatory phase in wound healing is considered to be a preparatory process for the formation of new tissue . A monocyte-derived cytokine, tumor necrosis factor-alpha (TNF-alpha), is a highly conserved molecule known to play a major role in the pathogenesis of gram-negative shock . Besides this, previous experimental studies show that TNF-alpha may have either a beneficial or detrimental role in wound healing . The purpose of the present study was to examine the effects of TNF-alpha on developing granulation tissue in rats as well as on rat and human granulation tissue cells in culture . Subcutaneously implanted cylindrical hollow sponges were used for studying the effects of locally applied TNF-alpha on granulation tissue in rats . These implants were treated either on the day of implantation or for the first 4 or 7 days after implantation with a solution containing various amounts of TNF-alpha while the control implants were treated correspondingly with the carrier solution only . The analyses of the granulation tissue were carried out 4, 7, 14 and 21 days after implantation . In the histological specimen these sponges were cut into small pieces and stained with Weigert van Gieson to visualize collagen . The amount of granulation tissue grown into the sponge was calculated from the cross section of every sponge . For the cell culture studies fibroblasts were released from human and rat granulation tissue which was cut into small pieces and digested by collagenase and DNase in Hank's balanced salt solution . The cells were exposed to 1, 10, or 100 ng/ml of TNF-alpha and the rate of collagen synthesis was measured as synthesis of protein-bound 3H-hydroxyproline . The number of cells in the culture dishes was counted with Burger's hemocytometer after detaching the cells with trypsin treatment . As interleukin-1 (IL-1) and TNF-alpha overlap in many of their functions, the effects of lipopolysaccharide (LPS), human interleukin 1 beta (IL-1) and prostaglandin E2 (PGE2) on experimental granulation tissue in rats as well as on rat granulation tissue cells in culture were studied with the same method . After a single application of TNF-alpha into the sponge, no essential differences between the groups were detected . However, after daily applications of TNF-alpha for 4 days, an inhibitory effect on tissue repair was observed after 4 and 7 days . Collagen formation, indicated by the hydroxyproline content of the sponge, was significantly lower in the group treated with TNF-alpha than in the controls . This effect was not observed after 14 or 21 days . These findings were confirmed in the histological samples . In the cultures of rat granulation tissue fibroblasts TNF-alpha decreased 3H-hydroxyproline production to about 75% of that in the controls and it had also a decreasing effect on pro alpha 1(I) and pro alpha 1(III) collagen mRNA levels maximally by 67% and 77% of the control level, respectively . In the cultures of human granulation tissue fibroblasts a similar inhibiting effect on the production of collagen was seen . TNF-alpha decreased the production of 3H-hydroxyproline to 56% of the control value with a dose of 100 ng/ml . Similarly, IL-1 beta decreased hydroxyproline content of granulation tissue seven days postoperatively and PGE2 decreased nonsignificantly the amounts of hydroxyproline but the steady-state levels of pro alpha 1(I) and pro alpha 1(III) collagen chain mRNAs were slightly elevated . In the IL-1 beta-treated fibroblast cultures collagen production decreased by 15% compared with that of the controls . PGE2 decreased collagen production by 34% of that in the controls . This effect could be abolished with indomethacin . Indomethacin alone stimulated collagen production by 40% . In vivo IL-1 decreases the formation of normal granulation tissue . This effect may be partly due to IL-1 stimulated secretion of PGE2.

Infect Agents Dis, 1996 Jan, 5(1), 21 - 8
Acellular pertussis vaccines: a turning point in infant and adolescent vaccination; Rappuoli R; Whooping cough, an infectious disease caused by the gram-negative bacterium Bordetella pertussis, is a life-threatening disease that cannot be controlled by antibiotic treatment or other procedures of modern medicine . Immunization, using a vaccine made of heat-killed bacteria, has been the only way to prevent the disease and keep the infection under control . However, the high reactogenicity of the whole-cell vaccine available so far has made vaccination very controversial, and vaccine use has been restricted to the minimum doses strictly necessary to protect infants during the first few years of life, when the disease is most dangerous . This policy left unsolved the problem of controlling the circulation of the pathogens that are still spreading undisturbed in the population, even after decades of vaccine use . Today, the introduction of acellular vaccines that are efficacious and virtually free of side effects suggests that the new vaccines can be used safely to immunize not only infants, toddlers, and preschool children, but also adolescents and adults, making possible the complete control of the disease and infection, so that policies addressing the eradication of the disease become feasible . The absence of constraints for the use of pertussis vaccine will allow the rational design of the optimal combinations of vaccines for each age.

Antimicrob Agents Chemother, 1996 Jan, 40(1), 65 - 9
Synergistic effect of a recombinant N-terminal fragment of bactericidal/permeability-increasing protein and cefamandole in treatment of rabbit gram-negative sepsis; Lin Y et al.; As a consequence of their bactericidal actions, many antibiotics cause the release of endotoxin, a primary mediator of gram-negative sepsis . Bactericidal/permeability-increasing protein (BPI) has bactericidal activity and neutralizes endotoxin in vitro and in vivo . We sought to examine the effect of a recombinant N-terminal fragment of BPI (rBPI21) in conjunction with cefamandole, a cephalosporin antibiotic, in the treatment of Escherichia coli bacteremia and septic shock in rabbits . Cefamandole (100 mg/kg of body weight) was injected intravenously . This was followed by simultaneous 10-min infusions of E . coli O7:K1 (9 x 10(9) CFU/kg) and rBPI21 (10 mg/kg) . rBPI21 was continuously infused for an additional 110 min at 10 mg/kg/h . The administration of rBPI21 in conjunction with the administration of cefamandole prevented the cefamandole-induced increase of free endotoxin in plasma, accelerated bacterial clearance, ameliorated cardiopulmonary dysfunction, and thereby, prevented death, whereas neither agent alone was protective in this animal model . The efficacy of the combined treatment with rBPI21 and cefamandole suggests a synergistic interaction between the two agents . The data indicate that rBPI21 may be useful in conjunction with traditional antibiotic therapy.

Am J Physiol, 1996 Jan, 270(1 Pt 2), R289 - 97
Bidirectional effects of hepatic ischemia/reperfusion on E . coli-induced TNF-alpha gene expression; Epperly NA et al.; We tested the hypothesis that gram-negative bacteremia (GNB) and brief (30 min) reductions in the hepatic O2 supply by low-flow ischemia differentially modulate tumor necrosis factor-alpha (TNF-alpha) gene expression owing to sequence-specific activation of cyclooxygenase vs . complement (C) pathways . Buffer-perfused Sprague-Dawley rat livers (n = 82) were studied over 180 min after intraportal 10(9) live E . coli serotype 055:B5 (EC) or 0.9% NaCl (NS) at t = 0 . Compared with EC and NS controls receiving constant-flow perfusion, sequential GNB and ischemia/reperfusion (I/R) were studied in EC + 30 I/R and NS + 30 I/R livers, in which 30 min of ischemia (I) beginning 0.5 h after EC or NS was followed by 120 min of reperfusion (R) . This sequence was reversed in 30 I/R + EC and 30 I/R + NS groups . Bacterial clearance, bioactive and antigenic TNF-alpha, prostaglandin E2 (PGE2), and hepatic O2 uptake and performance were serially assessed . Venous TNF-alpha increased in EC controls to peak at 155 +/- 29 U/ml after 180 min (P < 0.001 vs . NS controls) as did hepatic TNF-alpha mRNA . Both TNF-alpha transcripts and protein levels were markedly attenuated in EC + 30 I/R (P < 0.001 vs . EC) despite equivalent EC clearance by Kupffer cells . Indomethacin (10(-5) M) decreased I/R-induced PGE2 secretion and restored TNF-alpha to control levels . In contrast, TNF-alpha levels in 30 I/R + EC perfusates exceeded those of EC + 30 I/R livers (P < 0.05) and were indistinguishable from EC controls . Allopurinol pretreatment but not heat inactivation of C or infusion of soluble human complement receptor type 1 inhibited TNF-alpha production in 30 I/R + EC organs . These results identify a novel sequence-dependent interaction whereby hepatic O2 deprivation after GNB downregulates TNF-alpha via generation of cyclooxygenase metabolites, whereas ischemia preceding GNB increases cytokine expression via reactive O2 species but not C activation.

Respiration, 1996, 63(3), 170 - 3
The effect of diabetes mellitus on the outcome of patients with chronic obstructive pulmonary disease exacerbated due to respiratory infections; Loukides S et al.; During the last 2 years, 597 cases of chronic obstructive pulmonary disease (COPD) exacerbated due to respiratory infections were treated in our department . Eighty-eight (14.7%) of them suffered from diabetes mellitus (DM) . Thirty-four had insulin-dependent DM and 54 non-insulin-dependent DM . In these groups, we studied the duration of hospitalization, their clinical features, chest X-rays, blood tests (white blood cells, erythrocyte sedimentation rate), blood gases and the sputum culture for bacteria . The paired t test was used for statistical analysis . Our results showed that the mean duration of hospitalization in patients with DM was 10.76 +/- 2.7 days (X +/- SD), whereas for patients without DM it was 8.53 +/- 1.9 days . Patients with insulin-dependent DM had a mean hospitalization of 15.63 +/- 3.6 days, which was statistically significant in relation to the group without DM (p < 0.0001) . As for the chest X-rays, clinical features, blood tests and blood gases, no statistically significant differences were found among the groups . The sputum culture for bacteria showed that in patients with insulin-dependent DM the isolation of gram-negative bacteria was 51.6%, which was statistically significant in relation to the group without DM, where it was 27.3% (p < 0.0001) . In conclusion, a significant percentage of patients with COPD suffers from DM . Patients with insulin-dependent DM and COPD with respiratory infections were found to have longer periods of hospitalization and a larger percentage of gram-negative bacteria in the sputum culture.

Eur Surg Res, 1996, 28(3), 179 - 89
Intraperitoneal cholelithiasis after laparoscopic cholecystectomy--behavior of 'lost' concrements and their role in abscess formation; Hornof R et al.; In two experimental studies we sought preliminary information about the behavior of concrements lost in the peritoneal cavity during laparoscopic cholecystectomy . Materials and Methods: In study 1, human gallstones were analyzed using X-ray diffraction, classified in three groups and examined with an ultramicroscope; then they were implanted in the peritoneal cavity of rats . After 8 weeks or 6 months, the animals were sacrificed and the concrements analyzed again as before . The tissues surrounding the calculi were also examined histologically . In study 2, human gallstones were examined with regard to bacterial contamination on the surface or in the middle of the calculi . The cholesterol content was analyzed, and the stones were divided into three groups and implantated in the rats as in the first study . After 8 weeks, the animals were sacrificed and areas with identifiable tissue reactions were examined histologically and microbiologically . Results: The concrements lost their crystalline formation without any relation to their former cholesterol content, as shown by X-ray diffraction as well as ultramicroscopy . Mineralogically, these changes are a certain sign of structural dissolution . Cholesterol stones only caused abscess formations in association with gram-negative bowel germs . Sterile pigment concrements often led to a mesenchymal reaction such as granulomas . Contaminated pigment stones also resulted in extensive abscess formations.

Perit Dial Int, 1996, 16 Suppl 1, S215 - 9
Peritoneal dialysis in liver disorders; Selgas R et al.; The purposes of this paper is to review the specific role of peritoneal dialysis (PD) in patients with liver disorders . We will pay attention to the confluence of liver diseases and situations for which chronic dialysis treatment is required . Hemodialysis (HD) and peritoneal membranes are safe barriers against the passage of the hepatitis C virus; consequently, while peritoneal effluent or HD ultrafiltrate drained from hepatitis B patients/carriers is infective, that from hepatitis C patients does not appear to present this risk . An important issue is horizontal transmission, which appears to occur with both viruses in HD units, and which is absent in peritoneal dialysis units . The incidence of hepatitis C among continuous ambulatory peritoneal dialysis (CAPD) patients is quite low, while it may reach almost 50%-60% of HD patients in some units . While hepatitis C transmission mechanisms are not completely understood and a vaccine is not available, PD provides some degree of protection when compared with HD, for and-stage renal disease patients . In summary, our experience and that of others, with a total of 19 PD-treated chronic liver disease patients, supports CAPD as the treatment of choice for cirrhotic patients with ascites who require chronic dialysis . Data on peritoneal diffusion of low molecular weight substances revealed a marked increase in most patients . The ultrafiltration capacity was clearly augmented with respect to noncirrhotic patients, making the use of hypertonic bags unnecessary . Hemodynamic tolerance was excellent . Complications and death were mainly related to liver disease complications . Spontaneous bacterial peritonitis (SBP), caused by gram-negative germs, is the most important complication directly related to ascites and may have some points in common with PD-related peritonitis . However, and in contrast to most PD peritonitis, two pathogenetic mechanisms have been suggested for SBP: (1) translocation of bacteria from the gut to the mesenteric lymph nodes, and (2) bacteremia in these patients is secondary to the general abnormal host defense mechanisms . Local factors such as intrahepatic shunting and the impairment of bactericidal activity in ascitic fluid favor the bacteria ascites . The hypothesis of a direct transmural contamination from bowel to ascitic fluid has been relegated to secondary bacterial peritonitis . Would cirrhotic patients with temporal or permanent renal function compromise benefit from peritoneal catheter placement and other PD practices to perform repetitive small ascitic drainages at home? Perhaps the time has arrived when hepatologists and PD nephrologists begin to work shoulder to shoulder in this particular field, as we have a common problem, the peritoneal cavity filled with fluid.

Pathology, 1996 Jan, 28(1), 80 - 3
Bartonella (Rochalimaea) quintana causing fever and bacteremia in an immunocompromised patient with non-Hodgkin's lymphoma; Rathbone P et al.; A 48-yr-old man with stage IV non-Hodgkin's lymphoma, became neutropenic following chemotherapy and developed a fever . His blood cultures were processed to enhance the yield of fastidious bacteria . A slow-growing, capnophilic Gram-negative rod was isolated . The febrile episode was treated with cefotaxime, imipenem and vancomycin and resolved . The bacterial isolate was identified as Bartonella (Rochalimaea) quintana by 16S-rDNA gene sequencing . The isolate showed 99.8% sequence homology with the type strain . This is the first isolation of Bartonella (Rochalimaea) quintana from a bacteremic patient in Australia . This bacterium is a fastidious Gram-negative rod requiring prolonged culture for its isolation . Patients with culture-negative pyrexia, especially immunocompromised patients, may need to be investigated for infection with this agent.

Avian Dis, 1996 Jan-Mar, 40(1), 181 - 5
Runting of ducklings associated with Cochlosoma anatis infection; Bollinger TK et al.; Ducklings, especially common goldeneye (Bucephala clangula), experiencing poor weight gain and delayed development were reported from a waterfowl park during June and July of 1990 . Runting was first noticed between days 5 and 10 post-hatch in the "brooder" building, and although ducklings appeared active and were feeding, they developed at a slower rate than other members of their clutch . Many ultimately died of emaciation and gram-negative bacterial septicemia . Necropsies of affected ducklings revealed large numbers of the intestinal flagellate Cochlosoma anatis, in both the small and the large intestine; however, autolysis impaired histological interpretation in many cases . Inoculation of 1-day-old Muscovy ducklings with feces containing large numbers of C . anatis resulted in poor weight gain and delayed tail-feather development compared with uninoculated control ducklings . Weight gain improved after treatment with metronidazole . Cochlosoma anatis is associated with the runting syndrome observed in ducklings at the waterfowl park.

Med Pregl, 1996, 49(5-6), 221 - 4
{Postoperative infection as an indication for hysterectomy after cesarean section}; Milasinovic L et al.; Even today infection is the most important cause of morbidity and mortality after cesarean section . The aim of this paper is to point to the significance of hysterectomy in treatment of puerperal infection in the contemporary obstetrics . During a 15- year period (1981 - 1995) 85231 deliveries were performed at the Clinic for Gynecology and Obstetrics in Novi Sad (Yugoslavia) . There were 8609 (10.10%) cesarean sections . Hysterectomy was performed in 16 cases (0.186%) of women undergoing cesarean section due to dehiscence of uterine suture, endometritis, diffuse peritonitis or sepsis . Infection during delivery was diagnosed in 3 (18.75%) cases, the delivery itself began by amnion rupture in 5 (31.25%) cases, it lasted more than 12 hours in 6 (37.50%) cases . Elective cesarean section was performed in 3 (18.75%) cases, cesarean section during delivery in 13 (81.25%) and iterative cesarean section in 5 (31.25%) cases . Total hysterectomy was performed in 9 (56.25%) cases and subtotal in 7 (43.75%) cases . Postoperative treatment by antibiotics was carried out in all cases . There were postoperative complications after hysterectomy in 7 (43.75%) cases . According to results of microbiologic analyses in 7 (43.75%) cases one cause was determined, while in 9 (56.25%) cases two or more causes were determined . Gram-negative-bacteria were the most common cause (68.75%).

Eur J Clin Invest, 1996 Jan, 26(1), 45 - 8
Immunosuppressive effects of endotoxins and bile acids in vivo in the rat; Aouad K et al.; Cell-mediated immunity is impaired during cholestasis . The aim of this study was to evaluate in vivo the effects on this immune defect of high serum levels of endotoxin and bile acids . Heterotopic cardiac allotransplantations were performed in the DA/Lewis rat combination . Cholestasis, induced by ligation/section of the common bile duct, was responsible for a significant delay in the rejection time (16 +/- 0.5 vs . 7.1 +/- 0.4 days in controls, P < 0.01) . Elimination of Gram-negative intestinal bacteria from cholestatic rats by a vancocin/colimycin/tobramycin (VCT) mixture induced a significant reduction in endotoxin levels and a reduction in rejection times (9.5 +/- 1.0 days, P < 0.01) that remained, however, significantly longer than those of controls (P < 0.05) . Oral administration of chenodeoxycholic acid in non-cholestatic rats significantly enhanced the serum concentration of total bile acids (60.6 +/- 15.3 mumol L(-1) vs . 17.4 +/- 1.9 mol L(-1) in controls, P < 0.01) and postponed allograft rejection (10.7 +/- 0.6 days, P < 0.01 vs . controls) . These data suggest that increased endotoxin level and serum bile acid concentration may play a role in the immunosuppressive effect of cholestasis.

Pediatr Radiol, 1996, 26(2), 141 - 4
Jugular thrombophlebitis complicating bacterial pharyngitis (Lemierre's syndrome); De Sena S et al.; Lemierre s syndrome is a rare syndrome caused by Fusobacterium necrophorum, a gram-negative anaerobic organism that normally inhabits the oropharynx . The syndrome follows primary oropharyngeal infection and affects previously healthy adolescents in a characteristic manner with fatal results if left untreated . The authors present two cases seen at their institution and discuss the clinical and radiologic features of the syndrome, along with considerations for patient management.

Biometals, 1996 Jan, 9(1), 78 - 83
Characterization of a novel Spirillum-like bacterium that degrades ferrioxamine-type siderophores; Winkelmann G et al.; A novel Gram-negative Spirillum-like bacterium (ASP-1) was isolated from lake water by enrichment culture on desferrioxamine B as sole source of carbon and energy . ASP-1 was able to degrade the siderophores desferrioxamine B and E . The property of siderophore degradation was inducible in the presence of desferrioxamine B . The ferric complexes, however, were not measurably degraded but served as an iron source . Degradation of desferrioxamines in culture was followed by measuring the residual ferrioxamines colorimetrically at 430 nm after addition of iron . Degradation in cell-free assays was followed quantitatively by HPLC on a reversed-phase column measuring the time-dependent disappearance of the desferrioxamines B and E . Cell-free assays also revealed that degradation of the cyclic desferrioxamine E was rapid and complete, whereas degradation of the linear desferrioxamine B yielded two intermediate iron-binding metabolites of shorter chain length . Preparative isolation by HPLC and mass spectrometric analysis of the metabolites revealed masses at 361 and 419 a.m.u., respectively, suggesting a splitting at the two amide bonds . ASP-1 is a nitrogen fixing Spirillum bacterium which could also use ammonium and glucose or several organic acids as a carbon source but grew poorly with amino acids . Physiological comparisons with Aquaspirillum and Azospirillum failed to assign ASP-1 to any of the presently known Spirillum species . Based on 16S rDNA sequence analysis the strain could be placed within the radiation of the Azospirillum/Rhodocista group . The closest relative was Azospirillum irakense, showing 98.8% similarity.

Int J Syst Bacteriol, 1996 Jan, 46(1), 16 - 22
Transfer of "Pseudomonas riboflavina" (Foster 1944), a gram-negative, motile rod with long-chain 3-hydroxy fatty acids, to Devosia riboflavina gen . nov., sp . nov., nom . rev; Nakagawa Y et al.; The taxonomic position of "Pseudomonas riboflavina" was studied by 16S rRNA gene sequencing and chemotaxonomic methods . This organism is a gram-negative, strictly aerobic rod and has a DNA guanine-plus-cytosine content of 61.4 mol%; the major isoprenoid quinone is ubiquinone 10, and the unusual cellular fatty acids 3-hydroxytetracosenoic acid (3-OH 24:1) and 3-hydroxyhexacosenoic acid (3-OH 26:1) are the major 3-hydroxy cellular fatty acids . A phylogenetic analysis based on 16S rRNA sequences revealed that "P . riboflavina" IFO 13584T (T = type strain) occupies an independent position in the alpha subclass of the Proteobacteria . On the basis of our data, we propose that "P . riboflavina" IFO 13584T should be transferred to the genus Devosia gen . nov . as Devosia riboflavina sp . nov., nom . rev.

Am J Hematol, 1996 Jan, 51(1), 26 - 31
Selective discharge of patients with acute myeloid leukemia during chemotherapy-induced neutropenia; Gillis S et al.; PURPOSE . It is common practice for patients with acute myeloid leukemia (AML) to be observed in hospital during the entire nadir after intensive chemotherapy . In an attempt to lessen the likelihood of developing infections with hospital acquired pathogens, we usually discharge patients upon completion of chemotherapy and follow them as outpatients . They are readmitted if fever develops . We evaluated the feasibility and safety of this practice . PATIENTS AND METHODS . We studied 29 patients with AML (median age 40 years, range 16-63) who were treated with intensive remission-induction and consolidation chemotherapy . Afebrile patients not receiving antibiotics were discharged immediately following chemotherapy and were followed every 3-4 days at the day care unit . Patients were instructed to return immediately if fever rose to 38.2 degrees C or a fever of 38 degrees C persisted for 2 hr . The 29 patients received a total of 86 courses . Following 50 courses, patients were discharged . These 50 ambulatory nadir periods (ANPs) were monitored . RESULTS . Median WBC and platelet counts on discharge were 2,900 per cubic millimeter (range 300-8,300) and 137,000 per cubic millimeter (range 17,000-618,000), respectively . Mean traveling time from the hospital by car was 1.6 hr (range 15 min-3 hr) . In three of the 50 ANPs (6%), patients were not readmitted during their entire nadir . During 47 of the ANPs, patients returned to the hospital (because of fever in 44 cases), a mean of 7.2 days (range 1.0-12.7 days) after discharge . In 45 ANPs, patients were readmitted in good general condition . Four patients had life-threatening complications . Two patients were admitted in septic shock due to delay in seeking admission, but rapidly recovered . Two other patients died, one of cardiogenic shock within 24 hr of readmission and one 24 days later . Only one of the 11 gram negative bacteria cultured was resistant to mezlocillin and gentamicin . After 45 ANPs, patients were discharged a mean of 12.2 days (range 5-42 days) following readmission . We estimate that approximately 383 hospital days were saved by this policy, a mean of 7.6 days per patient, representing 16% of total inpatient hospital days . CONCLUSIONS . For AML patients who are reliable and without complicating medical conditions, selected discharge following chemotherapy is a low-risk practice and may reduce the incidence of infection with resistant hospital-acquired pathogens.

Lab Invest, 1996 Jan, 74(1), 241 - 52
Coexpression of heat-shock protein 60 and intercellular-adhesion molecule-1 is related to increased adhesion of monocytes and T cells to aortic endothelium of rats in response to endotoxin; Seitz CS et al.; Bacterial cell-wall lipopolysaccharide (LPS) is the main endotoxin contributing to local inflammation and systemic toxicity during Gram-negative infections and induces aortic endothelial injury with or without cell death and replication followed by increased leukocyte adhesion . Heat-shock protein (hsp) 60 is under study in our laboratory as a potential antigen inducing immunologic attack to endothelial cells in atherogenesis . To investigate the mechanism of LPS-induced endothelial injury and the phenotypes of adhering cells, Lewis rats were treated in vivo or, in aortic organ cultures, with LPS to determine the expression of intercellular-adhesion molecule-1 (ICAM-1) and hsp60 on aortic endothelium and to characterize phenotypes of adhering leukocytes . Increased ICAM-1 expression by aortic endothelium was observed as early as 3 hr after LPS injection and persisted up to 72 hr, whereas elevated levels of hsp60 were found between 6 and 48 hr . In vitro application of various types of stress, such as LPS, H2O2, and high temperature, not only stimulated endothelial expression of hsp60 but, concomitantly, that of ICAM-1 . The number of adhering leukocytes was significantly increased on aortic endothelium 6 hr after LPS administration, and the predominant leukocytes adhering to stressed endothelium were monocytes (80%) and T lymphocytes (8 to 20%) . In organ cultures of rat aortic intimal, LPS, and H2O2 evoked increased leukocyte adhesion, which proved to be selective, because adherent leukocytes were mostly Ia+ monocytes and T cells, i.e., activated . Adhering T cells were gamma/delta antigen-receptor positive in 8 to 16% after LPS stress, whereas these cells amount to only 2 to 4% of peripheral blood T cells . Blocking of adhesion molecules ICAM-1, LFA-1 alpha, and/or LFA-1 beta reduced adhesion up to 34% . Increased coordinated LPS-dependent expression of hsp60 and ICAM-1 correlates with monocyte and T-cell adhesion to aortic endothelium . These observations may be significant for elucidating the mechanism of the initiating events in the development of atherosclerosis.

Infect Immun, 1996 Jan, 64(1), 146 - 53
Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells; Prasadarao NV et al.; Escherichia coli is the most common gram-negative bacteria causing meningitis during the neonatal period, but is unclear what microbial factors mediate traversal of E . coli across the blood-brain barrier . Outer membrane protein A (OmpA), a highly conserved 35-kDa protein, was examined for its role in E . coli K1 invasion of brain microvascular endothelial cells (BMEC) . The invasive capability of the OmpA+ strains was 25- to 50-fold greater than that of OmpA- strains, and the invasive capability of OmpA- strains was restored to the level of the OmpA+ strain by complementation with the OmpA+ E . coli into BMEC . Two short synthetic peptides (a hexamer, Asn-27-Glu-32, and a pentamer, Gly-65-Asn-69) generated from the N-terminal amino acid sequence of OmpA exhibited significant inhibition of OmpA+ E . coli invasion, suggesting that these two sequences represent the OmpA domains involved in E . coli invasion of BMEC . These findings suggest that OmpA is the first microbial structure identified to enhance E . coli invasion of BMEC, an important event in the pathogenesis of E . coli meningitis.

Arch Surg, 1996 Jan, 131(1), 51 - 6
Interleukin-10 attenuates the release of proinflammatory cytokines but depresses splenocyte functions in murine endotoxemia; Ertel W et al.; OBJECTIVE: To determine whether interleukin (IL)-10, besides its potent anti-inflammatory properties, causes depression of splenocyte functions in a murine model of gram-negative endotoxemia . DESIGN: Mice (strain C3H/HeN) were injected intravenously with 1 mg of Escherichia coli lipopolysaccharide at 15 minutes after intravenous injection of either 200 U of recombinant murine IL-10 or saline solution . Serum levels of tumor necrosis factor alpha, IL-6, and IL-1 alpha were determined at 90 minutes and 12 hours after lipopolysaccharide challenge . In addition, splenocyte proliferation and lymphokine release (IL-2, IL-6, and interferon gamma) were measured . RESULTS: Pretreatment with IL-10 markedly reduced (P < .05) serum levels of tumor necrosis factor alpha (-79%), IL-6 (-94%), and IL-1 alpha (-69%), but it significantly inhibited splenocyte proliferation (-32%) and IL-2 (-40%), IL-6 (-49%), and interferon gamma (-54%) release of splenocytes . CONCLUSIONS: Interleukin-10 prevents E coli lipopolysaccharide-induced cytokinemia but dampens antigen-driven cellular immune responses . Although IL-10 protects against the detrimental effects of proinflammatory cytokines by deactivation of macrophages, its immunosuppressive effect may augment susceptibility to repeated or continuous invasion of microorganisms, as it is observed during clinical sepsis.

Radiol Clin North Am, 1996 Jan, 34(1), 47 - 58
Nosocomial pneumonia; Lipchik RJ et al.; Nosocomial pneumonia continues to be a major problem plaguing hospitalized patients, especially those on ventilators . Gram-negative bacteria and S . aureus are the most common causitive organisms . Alteration of the normal oropharyngeal flora and contamination of the respiratory tract from the pharynx and stomach are now recognized to be important factors in its development . As there is no definitive diagnostic test, nosocomial pneumonia remains a clinical diagnosis; however bronchoscopy with protected specimen brush cultures and BAL are diagnostic methods under study . Noninvasive radiologic examinations and clinical criteria have poor specificity in diagnosis.

Orthop Clin North Am, 1996 Jan, 27(1), 37 - 46
Spinal infections in the immunocompromised host; Broner FA et al.; There is an increasing population of immunocompromised patients with HIV, IV drug abuse, organ transplantation, and long-term steroid treatment developing spinal infections . Delayed diagnosis because of blunted host immune response and lack of outward signs and symptoms places the treating physician at a disadvantage in the treatment of this type of disease, which presents at a later stage of development . Immunocompromised patients are infected by a different group of pathogens than their healthier cohorts (e.g., Pseudomonas, gram-negative bacteria and fungal infections) because their host defenses are diminished . Osteomyelitis with or with out pyomyositis and epidural abscess may occur . The overriding symptom is back pain . Radiculopathy, myelopathy, and sensory loss may accompany local pain and tenderness . Plain film radiography, CT scan, MR image, and bone scan is invaluable in the diagnosis of these infections . The cornerstone of treatment is identification of the responsible pathogen, appropriate medical therapy, immobilization of the affected segment of the spine, and physical therapy to combat physical deconditioning . Psoas abscesses may require surgical debridement if they cannot be adequately drained by CT-guided percutaneous catheterization . Epidural abscesses with neurologic compromise require surgical drainage . Impingement of the spinal cord or cauda equina by collapsed osteomyelitic vertebral bodies requires surgical debridement by anterior vertebrectomy, with an autologous tricortical iliac crest strut and immobilization of the spine using external bracing or posterior instrumentation as dictated by the disease.

J Crit Care, 1995 Dec, 10(4), 198 - 212
Applied molecular biology of sepsis; Remick DG; The effective treatment of sepsis and septic shock has remained elusive despite intense research efforts . The tools of molecular biology have been applied to the problem of sepsis in an attempt to design more rational, directed therapy . Cellular interactions with invading microorganisms begin a series of stimulation events within the cell . One of the important interactions is the binding of lipopolysaccharide (LPS) from gram-negative bacteria to the LPS binding protein, and then this complex binding to CD14 on monocytes . Cell stimulation occurs through activation of signal transduction pathways within the cell, many of which have been defined . These include the kinases that phosphorylate proteins, and phosphatases that dephosphorylate proteins . The next step after activation of the signal transduction pathways is stimulation of nuclear regulatory factors . One of the best characterized of these is nuclear regulatory factor kappa B (NF-kappa B), which is a trans activating element that binds to specific DNA nucleotide sequences to allow transcription of downstream elements . Many inflammatory mediators are located downstream of NF-kappa B so that activation of NF-kappa B causes upregulation of the inflammatory mediators . The cytokines have been identified as a group of mediators important in the pathogenesis of sepsis, because several studies have shown that higher levels are correlated with a worse outcome in patients . Additionally, in experimental animal models, inhibition of cytokines improves survival, and administration of exogenous, recombinant cytokines reproduces many of the pathophysiologic alterations observed in sepsis . Molecular biology has played a critical role in the understanding of sepsis by providing the tools to make the recombinant cytokines of sufficient purity and quantity for infusion into experimental animals . The cellular response for the production of cytokines occurs through classic protein chemistry, with the signal transduction inducing messenger RNA (mRNA) coding for the cytokines, which are then translated and secreted . The relative contribution of local versus systemic cytokine production is beginning to be appreciated, with several diseases showing substantially higher local cytokine levels . The cytokines exert their activity on other cells by binding to their specific cytokine receptors . These receptors are part of the immune response and may be shed from the cell surface . These soluble receptors bind to and inactivate the cytokines . Inhibition of cytokine activity has been hypothesized as a potential therapy for sepsis . This inhibition has been done with antibodies directed against either the cytokines themselves or their receptors . Naturally occurring cytokine inhibitors have been cloned and expressed by molecular biologists and used for treatment of sepsis and other diseases . Using molecular biology techniques, the murine antibodies have been "humanized" to reduce their immunogenicity . The measurement of cytokines is critically important to our understanding of their role in health and disease . Cytokines may be measured by either immunologic methods or biological assays . Molecular biology has made important contributions to our understanding of sepsis by precisely identifying some of the mediators and providing reagents for therapeutic use.

J Crit Care, 1995 Dec, 10(4), 154 - 64
The influence of clinical study design on cost-effectiveness projections for the treatment of gram-negative sepsis with human anti-endotoxin antibody; Linden PK et al.; PURPOSE: This study was performed to compare the effect of entry criteria, patient population, and study design on outcome and projected cost-effectiveness of human anti-endotoxin antibody (HA-1A) . MATERIALS AND METHODS: Patients with suspected or documented gram-negative bacteremia (GNB) with sepsis syndrome or shock received HA-1A during an open-label protocol . The patient characteristics and outcome measures of this series were compared with those of a placebo-controlled randomized clinical trial (RCT) of HA-1A . Both data sets were subjected to three published cost-effectiveness models of anti-endotoxin therapy, which were derived from RCT data . RESULTS: One hundred thirty-one patients (43 with gram-negative bacteremia) received HA-1A during a 19-month open-label protocol . Comparison with the RCT results demonstrated greater severity of illness and higher 28-day mortality in the open-label protocol . When projected for open-label recipients, HA-1A was considerably less cost-effective than in the original projections based on RCT-derived data . This reduction in cost-effectiveness was consistent across all three models and their respective sensitivity analyses . CONCLUSIONS: Extrapolating cost-effectiveness from RCT-derived analyses to open-label usage may yield widely inaccurate projections because of only small differences in patient population and the drug administration protocol.

FEMS Microbiol Rev, 1995 Dec, 17(4), 381 - 99
Bacteriocin release proteins: mode of action, structure, and biotechnological application; van der Wal FJ et al.; The mechanism by which Gram-negative bacteria like Escherichia coli secrete bacteriocins into the culture medium is unique and quite different from the mechanism by which other proteins are translocated across the two bacterial membranes, namely through the known branches of the general secretory pathway . The release of bacteriocins requires the expression and activity of a so-called bacteriocin release protein and the presence of the detergent-resistant phospholipase A in the outer membrane . The bacteriocin release proteins are highly expressed small lipoproteins which are synthesized with a signal peptide that remains stable and which accumulates in the cytoplasmic membrane after cleavage . The combined action of these stable, accumulated signal peptides, the lipid-modified mature bacteriocin release proteins (BRPs) and phospholipase A cause the release of bacteriocins . The structure and mode of action of these BRPs as well as their application in the release of heterologous proteins by E . coli is described in this review.

Nephrol Dial Transplant, 1995 Dec, 10(12), 2325 - 7
Cuff-shaving procedure . A rescue treatment for exit-site infection unresponsive to medical therapy; Scalamogna A et al.; We performed 41 cuff-shaving procedures in 38 patients on continuous ambulatory peritoneal dialysis (CAPD) with exit-site infection unresponsive to medical treatment . Cuff shaving was performed on three patients with two catheters each . This procedure was effective in eliminating 50% of S . aureus exit-site infection and all S . epidermidis exit-site infection, but was ineffective in Gram-negative exit-site infection . After cuff-shaving procedure, 20 catheters (49%) were removed; 11 for persistent tunnel infection and nine because of development of secondary peritonitis . The probability of catheter survival at 1 year was 50% and remained stable thereafter . Cuff-shaving procedure may be a valuable mode of therapy for treating patients with S . aureus and/or S . epidermidis exit-site infection unresponsive to medical treatment.

Trends Microbiol, 1995 Dec, 3(12), 480 - 3
Biological effects of RTX toxins: the possible role of lipopolysaccharide; Czuprynski CJ et al.; RTX toxins are a family of related exotoxins with hemolytic, leukotoxi c and leukocyte-stimulating activities that are produced by a diverse array of Gram-negative bacteria . Lipopolysaccharide might be required for the maximal production of some RTX toxins and might be a cofactor in some of the biological effects of RTX toxins.

Compr Ther, 1995 Dec, 21(12), 737 - 40
Sepsis-related alterations in non-immune cell-signaling; Ahmad S; Traditionally, sepsis is defined as a systemic inflammatory reaction of the organism to Gram-negative bacterial leading to septic shock--characterized by hemodynamic derangements--and eventually to septic multi-organ malfunction . Sepsis syndrome is diagnosed when fever and other abnormalities of vital signs are present along with abnormalities of one or more organ systems that are not the site of infection and trauma (but with an identifiable locus of infection), and is associated with a range of 30% to 50% mortality . In the United States, one of the most frequent and serious problems confronting clinicians is the management of a serious infection and the systemic response to the infection, such as sepsis . Endotoxins are responsible for initiation of septic shock, which increases the number of fatalities in Gram-negative bacteremia among patients . Inflammation is meant to preserve health, but it is a double-edged sword because of its potential to cause irreversible tissue damage . Like other physiologic systems, the inflammatory response must be turned on and off as required . At present, our knowledge of the pathophysiologic changes at the initiation of the inflammatory process is in infancy, and the mechanism(s) of these signals are relatively less understood . Sepsis gives rise to pronounced metabolic alterations in various organs and tissues, particularly with increased muscle protein breakdown and stimulated hepatic protein synthesis . Although it leads to muscle wasting and increased nitrogen secretion, protein metabolic alteration also serves as an adaptive response in early sepsis as it provides amino acids for hepatic acute phase protein synthesis and gluconeogenesis . Calcium plays vital roles in the intracellular regulation of a variety of cellular responses (for example, contraction, secretion, cell-cell communication, cell proliferation) under physiologic conditions in various cell-types . Alterations in intracellular Ca2+ regulation leading to elevated cytosolic Ca2+ concentration could not only interfere with the cellular responses but also activate lytic enzymes such as proteinases and phospholipases . The objective of this article is to discuss the experimental findings that indicates relationship between alterations in cellular signaling and protein metabolic derangements in non-immune cells (skeletal muscle or liver) during sepsis and inflammation.

Mod Pathol, 1995 Dec, 8(9), 924 - 9
Demonstration of Chlamydia trachomatis in inguinal lymphadenitis of lymphogranuloma venereum: a light microscopy, electron microscopy and polymerase chain reaction study; Hadfield TL et al.; Intravacuolar organisms in vacuolated macrophages were associated with areas of necrosis and suppuration in 12 patients with suppurative inguinal lymphadenitis . The intravacuolar organisms measured 0.2 to 2.0 micrometers in diameter, stained Gram negative with the Brown-Hopp's tissue Gram stain, faintly blue with hematoxylin and eosin stain, and black with the Warthin-Starry silver impregnation stain . The organisms lined vacuolar membranes and/or clumped in centers of vacuoles . Electron microscopy revealed elementary and reticulate bodies and intermediate forms characteristic of the genus Chlamydia . Cultures of three lymph nodes in McCoy cells grew Chlamydia trachomatis, lymphogranuloma venereum (LGV) serovars . Polymerase chain reaction using primers for chlamydial 16S ribosomal DNA confirmed the organisms as Chlamydia in lymph nodes from nine patients . Recognition of chlamydial organisms by light microscopy in tissue sections of lymph nodes allows a definitive diagnosis of lymphogranuloma venereum.

Pediatr Emerg Care, 1995 Dec, 11(6), 379 - 80
Necrobacillosis: an unusual cause of purulent otitis media and sepsis; Cron RQ et al.; Necrobacillosis is a rare systemic illness caused by the anaerobic gram-negative bacterium, Fusobacterium necrophorum . We describe a case of necrobacillosis in a previously healthy child who developed purulent otitis media with sepsis caused by F . necrophorum . This case demonstrates that not all cases of purulent ear drainage are caused by the usual otopathic organisms and emphasizes the importance of culturing ear discharge in a child with an unusual presentation.

Wei Sheng Wu Xue Bao, 1995 Dec, 35(6), 450 - 4
{Isolation and reassociation of acetogen and methanogen in a syntrophobic coculture degrading butyrate anaerobically}; Cheng G et al.; Anaerobic coculture BF2 which degraded butyrate into acetate and produced methane was isolated from granular methanogenic sludge . The coculture is associated syntrophically the Syntrophomonas subsp . saponavida strain CF2 with Methanobacterium formicicum strain MF2 and appeared to degraded C4 approximately C18 fatty acids including isobutyrate . The optimal temperature and pH for growth was 37 degrees C and 7.7 respectively . The strain CF2 was obtained in pure culture with crotonate as substrate and produces acetate and butyrate . The doubling time of strain CF2 in crotonate media was about 20 hours . Strain CF2 is Gram negative, slightly curved 0.2 approximately 0.3 x 2.0 approximately 3.0 micron with round ends, motile by lateral flagellation at the concave side, non sporeforming . With a hydrogen scavenging organism, such as Methanospillum hungatei JF1, Methanobacterium formicicum 1535, Methanobrevibacterium bryantii 1125 and Desulfovibrio sp . B11, the strain CF2 paired up and the defined coculture degraded butyrate to acetate . When the strain CF2 associated with the original accompanist, Methanobacterium formicius strain MF2, the reassociated couculture degraded butyrate to acetate and produced methane again.

Pediatr Infect Dis J, 1995 Dec, 14(12), 1087 - 91
Plasma bactericidal/permeability-increasing protein concentrations in critically ill children with the sepsis syndrome; Wong HR et al.; Bactericidal/permeability-increasing protein (BPI) is a neutrophil azurophilic granule component that is bactericidal towards Gram-negative bacteria and inhibits lipopolysaccharide-mediated inflammatory responses . We conducted a prospective study to measure plasma BPI concentrations in 36 critically ill children with and without the sepsis syndrome . Plasma BPI concentrations ranged from 0.5 to 452 ng/ml . Patients with the sepsis syndrome had higher median plasma BPI concentrations than critically ill controls (5.1 vs . 1.8 ng/ml, P = 0.006) . Patients with organ system failure had higher median plasma BPI concentrations than those with no organ system failure (4.5 vs . 1.3 ng/ml, P = 0.001) . Plasma BPI concentrations were positively associated with pediatric risk of mortality score (P = 0.03, rs = 0.4) . These data provide the first clinical insights regarding the role of endogenous BPI production in critically ill children and suggest that BPI may play an important role in host defenses.

Eur J Clin Microbiol Infect Dis, 1995 Dec, 14(12), 1039 - 45
Plasma endotoxin and cytokine levels in neutropenic and non-neutropenic bacteremic patients; Hynninen M et al.; Plasma endotoxin, tumor necrosis factor-alpha (TNF-alpha), interleukin 1 beta (IL-1 beta), interleukin 1 receptor antagonist (IL-1ra), and interleukin 6 (IL-6) concentrations in 69 bacteremic patients were compared with those in 54 nonbacteremic patients suffering from suspected bacterial infections . Only three (11%) of the 27 patients with gram-negative bacteremia showed detectable levels of endotoxin . TNF-alpha was detected in 6% of the bacteremic patients and in none of the nonbacteremic patients . Median IL-6 levels were significantly higher in bacteremic than in nonbacteremic patients (55 vs . 0 pg/ml, p = 0.0008) . IL-6 concentrations were similar in neutropenic and non-neutropenic bacteremic patients (median 55 vs . 74 pg/ml) . In contrast, neutropenic bacteremic patients had significantly lower concentrations of IL-1ra than non-neutropenic bacteremic patients (250 vs . 1,950 pg/ml, p < 0.0001) . Patients with fatal bacteremia had significantly higher concentrations of IL-6 and IL-1ra than the survivors (median, 450 vs . 40, p = 0.012 and 7,600 vs . 420 pg/ml, p = 0.0075, respectively) . Determinations of endotoxin or TNF-alpha in patients with suspected bacteremia failed to offer clinically relevant data on the prognosis of these patients . IL-6 levels correlated with both the presence of bacteremia and the risk of death . Granulocytopenic patients with bacteremia had lower levels of circulating IL-1ra than patients with normal granulocyte counts, and these levels correlated with poor outcome.

Infect Agents Dis, 1995 Dec, 4(4), 254 - 72
Battling against host phagocytes: the wherefore of the RTX family of toxins?
Welch RA, Bauer ME, Kent AD, Leeds JA, Moayeri M, Regassa LB, Swenson DL.
The RTX family of bacterial exotoxins is a group of related cytolytic proteins produced by a wide variety of gram-negative human and animal pathogens . While diverse in their associated diseases and in their target cell specificities, there remain several themes common to RTX toxins, including genetic organization, structural and functional features, and effects on target cells . In this review, we summarize and discuss the genetics, regulation, epidemiology, structure/function relationships, and in vivo and in vitro activities of the best characterized RTX toxins, and speculate on their roles in pathogenesis and their use in immunotherapy.

Am J Vet Res, 1995 Dec, 56(12), 1592 - 8
Serum sensitivity of field isolates and laboratory strains of Brucella abortus; Eisenschenk FC et al.; OBJECTIVE--To evaluate the ability of bovine complement to kill a variety of field isolates and laboratory strains of Brucella abortus . DESIGN--The experimental approach was to determine the sensitivity of B abortus isolates to killing by bovine serum, and to document the role of complement in brucellacidal activity . SAMPLE POPULATION--Six laboratory isolates and 12 field isolates of B abortus were tested . PROCEDURE--The ability of B abortus to survive exposure to undiluted bovine serum for 2 hours at 37 C was assessed . The role of complement in killing was determined by examining the ability of heat (56 C for 60 minutes) and cobra venom factor to obliterate the activity in serum, and by detecting binding of the ninth component of bovine complement to serum-sensitive target cells . RESULTS--Isolates of B abortus that were resistant to the bactericidal activity of normal bovine serum were revealed . These included field isolates and laboratory strains . Furthermore, the study confirmed earlier reports that bovine serum-mediated killing of B abortus is caused by the complement cascade . CONCLUSIONS--Some isolates of B abortus, like other gram-negative bacteria, were resistant to complement-mediating killing . Resistance was associated with smooth colony morphology . Isolates lacking detectable O antigen were serum sensitive.

Inflammation, 1995 Dec, 19(6), 637 - 50
Regulation of superoxide anion generation in bovine alveolar macrophages by bacterial lipopolysaccharide, serum proteins, and modulators of signal transduction; Jian ZJ et al.; The respiratory burst of phagocytes in an important leukocyte function which results in generation of oxygen species that are both microbicidal and potentially damaging to host tissues . We investigated regulation of the respiratory burst of alveolar macrophages in response to lipopolysaccharide (LPS) derived from gram-negative bacteria, serum proteins, and several modulators of signal transduction . When employed as a single stimulus, LPS (E . coli 055:B5, 10 ng/ml-1 microgram/ml) was a weak stimulus for generation of superoxide anion (O2-) as compared to the potent effect of the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA; 500 ng/ml) . However, when LPS was combined with fetal bovine serum (FBS; 0.4-1.0% vol/vol, equivalent to 128-320 micrograms protein/ml), O2- generation was enhanced approximately two-fold over LPS alone . A chromatographically-derived bovine serum fraction which contained bovine lipopolysaccharide-binding protein (bLBP; 0.25-1.0 microgram/ml) was an effective substitute for FBS at a much lower protein concentration than whole FBS, and a similar synergistic effect with LPS on O2- generation was observed . Stimulation of macrophages for generation of O2- either with LPS alone or with LPS plus serum/serum fraction was suppressed by the protein tyrosine kinase inhibitor heribimycin A (0.2 ng/ml), and the calcium chelator BAPTA (12 microM), but not by modulators of G-proteins, including pertussis toxin (10 ng/ml) and cholera toxin (5 micrograms/ml protein) . Essentially complete inhibition of O2- synthesis by herbimycin A and BAPTA occurred in the presence of LPS and the bLBP-containing serum fraction (1 microgram/ml protein), but only partial inhibition (46.7% and 64.1%, respectively) was observed in the presence of LPS plus FBS (256 micrograms/ml protein) . These results indicate that when LPS is used as a sole stimulus it induces modest respiratory burst activity . However, when LPS is combined with appropriate serum components, it stimulates alveolar macrophages to generate larger amounts of O2- . Cellular signaling pathways important in stimulation of macrophages by LPS and serum components are protein tyrosine kinase- and Ca(++)-dependent, but do not relay on G-protein-mediated signaling.

Arch Microbiol, 1995 Dec, 164(6), 406 - 13
Desulfuromonas palmitatis sp . nov., a marine dissimilatory Fe(III) reducer that can oxidize long-chain fatty acids; Coates JD et al.; Studies on the microorganisms living in hydrocarbon-contaminated sediments in San Diego Bay, California led to the isolation of a novel Fe(III)-reducing microorganism . This organism, designated strain SDBY1, was an obligately anaerobic, non-motile, non-flagellated, gram-negative rod . Strain SDBY1 conserves energy to support growth from the oxidation of acetate, lactate, succinate, fumarate, laurate, palmitate, or stearate . H2 was also oxidized with the reduction of Fe(III), but growth with H2 as the sole electron donor was not observed . In addition to various forms of soluble and insoluble Fe(III), strain SDBY1 also coupled growth to the reduction of fumarate, Mn(IV), or S0 . Air-oxidized minus dithionite-reduced difference spectra exhibited peaks at 552.8, 523.6, and 422.8 nm, indicative of c-type cytochrome(s) . Strain SDBY1 shares physiological characteristics with organisms in the genera Geobacter, Pelobacter, and Desulfuromonas . Detailed analysis of the 16S rRNA sequence indicated that strain SDBY1 should be placed in the genus Desulfuromonas . The new species name Desulfuromonas palmitatis is proposed . D . palmitatis is only the second marine organism found (after D . acetoxidans) to oxidize multicarbon organic compounds completely to carbon dioxide with Fe(III) as an electron acceptor and provides the first pure culture model for the oxidation of long-chain fatty acids coupled to Fe(III) reduction.

Arch Microbiol, 1995 Dec, 164(6), 383 - 9
Molecular mechanisms of endotoxin activity; Schletter J et al.; Endotoxin (lipopolysaccharide, LPS), a constituent of the outer membrane of the cell wall of gram-negative bacteria, exerts a wide variety of biological effects in humans . This review focuses on the molecular mechanisms underlying these activities and discusses structure-function relationships of the endotoxin molecule, its interaction with humoral and cellular receptors involved in cell activation, and transmembrane and intracellular signal transduction pathways.

Microbiology, 1995 Dec, 141 ( Pt 12), 3161 - 70
Phase and electron microscopic observations of osmotically induced wrinkling and the role of endocytotic vesicles in the plasmolysis of the Gram-negative cell wall; Schwarz H et al.; When a Gram-negative bacterium is challenged with a sufficient concentration of a non-penetrating solute such as sucrose, water is sucked out of the cell . Plasmolysis spaces may form if the cell's cytoplasmic membrane (CM) separates from the murein wall (M) and the outer membrane (OM) . However, we suggest that first wrinkling of the wall envelope, forced by dehydration of the cytoplasm, occurs . The cryofixation, freeze-substitution electron microscope studies used here are much too slow to study the kinetics of shrinkage, wrinkling and plasmolysis . However, they are consistent with faster phase microscope studies and previous stopflow experiments . For the electron microscopy studies reported here, only sucrose was used as the osmotic agent and under conditions that do not cause extreme plasmolysis . Plasmolysis spaces were associated with the formation of small membrane-bound vesicles in the nearby cytoplasm . Such vesicles formed by osmotic challenge are called 'endocytotic' in plant cell systems . They had been recorded in earlier plasmolysis studies in bacteria, but not interpreted as a concomitant part of plasmolysis space formation in certain locations of the cell . We suggest that the endocytotic vesicles form because the phospholipid membranes are capable of very little contraction so extra membrane must be disposed of when plasmolysis spaces form . In the case of plasmolysis spaces forming at poles and constriction sites, for geometric reasons the surface area of the CM may be conserved without disposition of excess membrane . We suggest that it is this biophysical property of lipid membranes that leads to the frequent formation of plasmolysis spaces at a pole and at the site of future division . We also observed a novel structure, this is seen only under mild osmotic up-shock, and consists of very thin, straight, uniform and long plasmolysis spaces which were called 'lamellar spaces'; these commonly formed inside the sidewalls and were usually associated with the formation of endocytotic vesicles . Since lipoprotein links the M to the OM layers and thus could affect plasmolysis, we examined both wild-type and deficient strains . Some effects were observed, but they were minimal . The volume of the periplasmic space of growing unshocked cells was determined to be about 7%.

Scand J Immunol, 1995 Dec, 42(6), 701 - 4
Lipopolysaccharide effectively up-regulates B7-1 (CD80) expression and costimulatory function of human monocytes; Schmittel A et al.; The influence of lipopolysaccharide (LPS) and various cytokines on the expression of the costimulatory molecule B7-1 and intercellular adhesion molecule-1 (ICAM-1), lymphocyte function associated antigen-3 (LFA-3) and human histocompatibility leucocyte antigen-DR (HLA-DR) on human monocytes and their effect on the costimulatory function was investigated . Freshly isolated human monocytes constitutively express ICAM-1, LFA-3 and HLA-DR, but no B7-1 . B7-1 expression was up-regulated by LPS and, to a lesser extent, by interferon-gamma (IFN-gamma) . The other stimuli tested, including IFN-alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor-alpha (TNF-alpha) and GM-CSF+TNF-alpha, did not influence expression of B7-1 on monocytes . ICAM-1 and HLA-DR were up-regulated by IFN-gamma and LPS; LFA-3 expression was not influenced . LPS also effectively enhanced costimulatory function of monocytes as determined in the tetanustoxoid (TT) assay . Blocking of B7 by CTLA-4Ig inhibited the LPS-induced enhancement of costimulatory function almost completely . Our results indicate that the LPS-mediated up-regulation of the costimulatory function of human monocytes is mediated by B7 . This mechanism may be important for host defence against Gram-negative bacteria.

J Bacteriol, 1995 Dec, 177(24), 7125 - 30
Biosynthetic origin of mycobacterial cell wall arabinosyl residues; Scherman M et al.; Designing new drugs that inhibit the biosynthesis of the D-arabinan moiety of the mycobacterial cell wall arabinogalactan is one important basic approach for treatment of mycobacterial diseases . However, the biosynthetic origin of the D-arabinosyl monosaccharide residues themselves is not known . To obtain information on this issue, mycobacteria growing in culture were fed glucose labeled with 14C or 3H in specific positions . The resulting radiolabeled cell walls were isolated and hydrolyzed, the arabinose and galactose were separated by high-pressure liquid chromatography, and the radioactivity in each sugar was determined . {U-14C}glucose, {6-3H}glucose, {6-14C}glucose, and {1-14C}glucose were all converted to cell wall arabinosyl residues with equal retention of radioactivity . The positions of the labeled atoms in the arabinose made from {1-14C}glucose and {6-3H}glucose were shown to be C-1 and H-5, respectively . These results demonstrated that the arabinose carbon skeleton is formed via the nonoxidative pentose shunt and not via hexose decarboxylation or via triose condensations . Since the pentose shunt product, ribulose-5-phosphate, is converted to arabinose-5-phosphate as the first step in 3-keto-D-manno-octulosonic acid biosynthesis by gram-negative bacteria, such a conversion was then searched for in mycobacteria . However, cell-free enzymatic analysis using both phosphorous nuclear magnetic resonance spectrometry and colorimetric methods failed to detect the conversion . Thus, the conversion of the pentose shunt intermediates to the D-arabino stereochemistry is not via the expected isomerase but rather must occur via novel metabolic transformations.

Infect Immun, 1995 Dec, 63(12), 4686 - 94
Porphyromonas gingivalis lipopolysaccharide is poorly recognized by molecular components of innate host defense in a mouse model of early inflammation; Reife RA et al.; Porphyromonas gingivalis is a gram-negative bacterium that is associated with periodontitis . It has been hypothesized that destruction of bone and periodontal connective tissue is associated with colonization of the subgingival crevicular space by P . gingivalis, although how these bacteria overcome innate host defenses is largely unknown . To examine the early cellular and molecular events of P . gingivalis interaction with host tissues, we compared lipopolysaccharide (LPS) isolated from this bacterium with Escherichia coli LPS, a potent inflammatory mediator, in a mouse model of acute inflammation . In these studies, mice were given intramuscular injections of either P . gingivalis LPS or E . coli LPS and then sacrificed after 4 h . Reverse transcriptase-PCR analysis showed that expression of mRNAs for E- and P-selectins was higher in E . coli LPS-injected muscles than in P . gingivalis LPS-injected or control phosphate-buffered-saline-injected muscles . Similarly, monocyte chemotactic protein 1 and fibroblast-induced cytokine mRNAs were expressed in E . coli LPS-injected muscles whereas their expression was reduced or absent in P . gingivalis LPS-injected samples . These results were confirmed by in situ hybridization whereby stronger hybridization for selectin mRNAs was observed in the endothelium of capillaries from E . coli LPS-injected samples than in that from P . gingivalis LPS-injected muscles . In addition, many monocytes expressing monocyte chemotactic protein 1 mRNA and polymorphonuclear leukocytes expressing fibroblast-induced cytokine mRNA were observed in E . coli LPS-injected muscles whereas only a few cells were identified in P . gingivalis LPS-injected muscles . These results demonstrate that compared with E . coli, P . gingivalis has a low biologically reactive LPS as measured by its weak activation of inflammation . This may allow P . gingivalis to evade innate host defense mechanisms, resulting in colonization and chronic disease.

Endocrinology, 1995 Dec, 136(12), 5527 - 32
Lipopolysaccharide inhibits rat ovarian thecal-interstitial cell steroid secretion in vitro; Taylor CC et al.; Lipopolysaccharide (LPS), a major component of gram-negative cell walls, is a potent immunostimulator . Treatment of monocytes/macrophages in vitro with LPS induces the secretion of cytokines such as tumor necrosis factor-alpha and interleukin-1 alpha, -1 beta, and -6 . LPS is thought to require LPS-binding protein or CD14 to act at low concentrations (< 100 ng LPS/ml) . In the present study, rat ovarian thecal-interstitial cells (TIC) were cultured in a serum-free culture system (in the absence of LPS-binding protein or soluble CD14) and challenged with LPS . Treatment with LPS led to a dose-dependent (1-100 ng LPS/ml) decrease in LH-stimulated progesterone and androstenedione secretion . LPS had no effect on radiolabeled hCG binding to TIC homogenates or cAMP accumulation in culture medium . LPS treatment was associated with an increase in interleukin-6 bioactivity in the medium of thecal-interstitial cell cultures; however, tumor necrosis factor-alpha bioactivity was undetectable . Herbimycin A, an src tyrosine kinase inhibitor, blocked the actions of LPS and was associated with an increase in cAMP accumulation in TIC culture medium . The results suggest that LPS can act directly on ovarian thecal-interstitial cells and that this can occur in a LPS-binding protein/CD14-independent manner . The actions of LPS appear to be specific and require a nonreceptor tyrosine kinase.

FEBS Lett, 1995 Nov 27, 376(1-2), 6 - 10
A recombinant polypeptide, composed of the alpha-helical neck region and the carbohydrate recognition domain of conglutinin, self-associates to give a functionally intact homotrimer; Wang JY et al.; A recombinant polypeptide composed of the alpha-helical neck region and carbohydrate recognition domain (CRD) of bovine conglutinin was expressed in Escherichia coli . The recombinant protein formed inclusion bodies but could be solubilised using a denaturation-renaturation cycle based on urea and then purified by affinity chromatography on a TSK-N-acetylglucosamide column . The purified product behaved as a homotrimer in non-dissociating conditions, with three CRDs held together by the alpha-helical neck regions . The trimer, although lacking the N-terminal and collagen regions of the native conglutinin, showed the same binding carbohydrate specificities as the native molecule, for the complement fragment C3b and for lipopolysaccharides derived from Gram-negative bacteria.

Science, 1995 Nov 17, 270(5239), 1170 - 6
Crystal structure of the xanthine oxidase-related aldehyde oxido-reductase from D . gigas; Romao MJ et al.; The crystal structure of the aldehyde oxido-reductase (Mop) from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas has been determined at 2.25 A resolution by multiple isomorphous replacement and refined . The protein, a homodimer of 907 amino acid residues subunits, is a member of the xanthine oxidase family . The protein contains a molybdopterin cofactor (Mo-co) and two different {2Fe-2S} centers . It is folded into four domains of which the first two bind the iron sulfur centers and the last two are involved in Mo-co binding . Mo-co is a molybdenum molybdopterin cytosine dinucleotide . Molybdopterin forms a tricyclic system with the pterin bicycle annealed to a pyran ring . The molybdopterin dinucleotide is deeply buried in the protein . The cis-dithiolene group of the pyran ring binds the molybdenum, which is coordinated by three more (oxygen) ligands.

Med Clin (Barc), 1995 Nov 11, 105(16), 619 - 21
{Intense neutropenia of 14 years duration as the only manifestation of a myelodysplastic syndrome}; Las Heras G et al.; Myelodysplastic syndromes (MDS) are a group of acquired hemopathies characterized by peripheral cytopenias due to ineffective hematopoiesis and a high risk of transformation into acute non lymphoblastic leukemia (ANLL) which, in most cases, usually occurs from 6 months to 4 years after diagnosis . A patient with extreme neutropenia with intense dysgranulopoiesis as the only manifestations of MDS is described . The patient was controlled over 14 years and presented multiple infectious episodes, in various locations, throughout the evolution, some being very severe and generally caused by gram-negative germs . Likewise, during this time the patient received different treatments (oxymetholone, prednisone and lithium carbonate) with no hematologic response being observed . The leukocyte count remained around 3 x 10(9)/L with a mean proportion of neutrophils of 12% with no variations being found in the bone marrow aspirates carried out throughout the evolution (total of 9) . At 14 years the diagnosis of MDS evolved to ANLL . The patient died shortly after the acute transformation due to respiratory failure secondary to bilateral pneumonia . In this case three peculiar features are of note: the almost exclusive involvement of the granulopoietic series without either anemia or thrombocytopenia, the long evolution of AREB, with acute transformation 14 years after diagnosis and the severity of the infections, among which recurrent lingual granulopenic ulcers were of note.

FEBS Lett, 1995 Nov 6, 374(3), 351 - 5
Molecular characterization of an Arabidopsis thaliana cDNA encoding a novel putative adenylate translocator of higher plants; Kampfenkel K et al.; We have isolated an Arabidopsis thaliana cDNA encoding a highly hydrophobic membrane protein of 589 amino acids which contains 12 potential transmembrane helices and shows a high degree of similarity (43.5% identity, 66.2% similarity) to the ATP/ADP translocase of the Gram-negative bacterium Rickettsia prowazekii, an obligate intracellular parasite responsible for the epidemic typhus . This rickettsial translocator resides in the cytoplasmic membrane and allows the bacterium to exploit the host cytoplasmic ATP pool . We hypothesize that the A . thaliana homolog of the R . prowazekii ATP/ADP translocase is the functional eukaryotic equivalent and resides in the plastid inner envelope membrane where it functions as an ATP importer.

J Biol Chem, 1995 Nov 3, 270(44), 26178 - 83
A bacterial thioredoxin-like protein that is exposed to the periplasm has redox properties comparable with those of cytoplasmic thioredoxins; Loferer H et al.; The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-negative soil bacterium Bradyrhizobium japonicum was initially discovered due to its essential role in the maturation of cytochrome aa3 . A soluble form of TlpA lacking the N-terminal membrane anchor acts as a protein thiol:disulfide oxidoreductase . TlpA possesses an active-site disulfide bond common to all members of the thiol:disulfide oxidoreductase family . In addition, it contains two non-active-site cysteines that form a structural disulfide bond (Loferer, H., Bott, M., and Hennecke, H . (1993) EMBO J . 12, 3373-3383; Loferer, H., and Hennecke, H . (1994) Eur . J . Biochem . 223, 339-344) . Here, we compare the far- and near-UV CD spectra of TlpA before and after reduction of both disulfides by dithiothreitol and show that the non-active-site disulfide bond is not required for the integrity of TlpA's native conformation . In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the active-site disulfide and leaves the non-active-site disulfide bond intact, even at high molar excess over TlpA . The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA at 355 nm, which may be interpreted as a quenching of tryptophan fluorescence by the active-site disulfide bond . Using the specific fluorescence of TlpA as a measure of its redox state, a value of 1.9 +/- 0.2 M was determined for the TlpA:glutathione equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant, like cytoplasmic thioredoxins . The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia coli, is not capable of oxidizing the active-site cysteines of TlpA . This suggests that TlpA's primary role in vivo is keeping the thiols of certain proteins reduced and that TlpA's active, reduced state may be maintained owing to its kinetically restricted oxidation by other periplasmic disulfide oxidoreductases such as DsbA.

Cent Afr J Med, 1995 Nov, 41(11), 355 - 7
A case of gram negative pericarditis in a patient with the Acquired Immunodeficiency Syndrome; Manyemba JE et al.; A diagnosis of pericarditis due to Escherichia coli was made in a 38 year old male patient with AIDS . The source of infection in this patient was not established . This case highlights the need to carry out a diagnostic pericardiocentesis in immunocompromised patients presenting with pericardial effusions and the importance of adequate antibiotic cover when treating such patients prior to microbiological confirmation of the causative organism.

Pathol Biol (Paris), 1995 Nov, 43(9), 779 - 87
{Is it still possible to reduce the incidence of nephrotoxicity of aminoglycosides?}; Beauchamp D et al.; Aminoglycosides are still widely used alone or in combination with a beta-lactam antibiotic for the treatment of severe Gram negative infection . Oto- and nephrotoxicity are the major side effects associated with the use of these drugs . Although several risk factors associated with aminoglycoside nephrotoxicity have been identified, only few therapeutic approaches were suggested to reduce the incidence of their toxicity in patients . The single daily injection is the only approach actually used to reduce the renal toxicity of aminoglycosides in patients . However, the relationship between the nephrotoxicity of aminoglycosides and the time of the day these drugs should be given has never been explored in patients . Data obtained in laboratory animals indicated that temporal variations can be detected in the renal toxicity of aminoglycosides: the nephrotoxicity was observed during the rest period of the animals while no toxicity was found at other times of the day . Other studies suggested also that food intake can modulate the temporal variations in the nephrotoxicity of aminoglycosides . A better knowledge of the risk factors associated with the renal toxicity of aminoglycosides, a reduction in the number of daily injections of aminoglycosides, administration of aminoglycosides at the time of the lowest toxicity of the drug in patients submitted to an appropriate diet are the most interesting approaches to reduce the incidence of the renal toxicity of these agents.

Gastroenterol Clin Biol, 1995 Nov, 19(11), 871 - 5
{Treatment of diversion colitis with short-chain fatty acids . Bacteriological study}; Neut C et al.; OBJECTIVES: Bacterial imbalance may be involved in the pathogenesis of diversion colitis, via diminished production of short chain fatty acids . The aim of the study was to evaluate the effectiveness of short chain fatty acids on microbial flora of patients with diversion colitis and to compare this flora to the microbial flora of controls . METHODS: We prospectively evaluated the effectiveness of short chain fatty acids irrigation on bacterial flora of the excluded colon in 13 patients (8 males, 5 females; mean age: 43.7 years) . The causes of diversion were inflammatory bowel disease (n = 4) colonic cancer (n = 2) sigmoid diverticulitis with perforation (n = 3) ischio-rectal abscess (n = 2) and miscellaneous (n = 2) . Patients were given, twice a day for 14 days in a double blind manner, a 60 mL enema containing either short chain fatty acids (acetate: 60 mmol/L; propionate: 30 mmol/L; and n-butyrate: 40 mmol/L) (group 1; n = 7) or isotonic NaCl (group 2; n = 6) . Bacteriological studies were carried on before starting the trial (D1) and 14 days later (D14) . RESULTS: Before and after treatment, there was no difference between group 1 and group 2 concerning bacterial counts and species . Bacterial flora of patients with diversion colitis was characterized by: a) an increase of the count of aerobic bacteria; b) an increase of aerobic and aeroanaerobic species; c) the presence of black pigmented Gram negative anaerobic rods such as Prevotella intermedia and Porphyromonas asaccharolytica which were not found in rectal flora of the control group (16 volunteers, mean age: 27 years) . CONCLUSIONS: These data suggest that: a) enema with short chain fatty acids does not induce significant changes in the composition of the microbial flora in patients with diversion colitis; b) bacterial dysbiosis may be involved in pathogenesis of diversion colitis without involving the action of short chain fatty acids.

Shock, 1995 Nov, 4(5), 373 - 8
Liposomal cefoxitin in a porcine model of intra-abdominal sepsis: bactericidal efficacy; Soltes S et al.; The bactericidal effect of free versus liposomal cefoxitin was evaluated in the major reticuloendothelial organs in a porcine model of intra-abdominal sepsis . Yorkshire Landrace pigs were inoculated with 3.2 x 10(10) (n = 5) or 1.4 x 10(11) (n = 7) cfu of Escherichia coli mixed in sterile feces/animal . Two treatment groups inoculated with 1.4 x 10(11) cfu were established: free cefoxitin (n = 9) and liposomal cefoxitin (n = 9) . All animals were maintained under anesthesia and euthanized after 24 h . The number of E . coli recovered in the liver, lungs, and spleen was significantly affected by inoculum size (p < .05) . The liver had significantly higher numbers of bacteria (p < .05) compared with the other organs, regardless of the inoculum size . The liver and the lung of the liposomal cefoxitin-treated group showed significantly lower numbers of E . coli (5.0 x 10(4) and 6.3 x 10(2), respectively) compared with the untreated (liver, 6.3 x 10(7); lung, 2.0 x 10(6)) and free cefoxitin (liver, 5.0 x 10(6); lung, 7.9 x 10(4))-treated groups (p < .05) . At 2 h following the injection of free and liposomal cefoxitin, the decrease of E . coli in peritoneal fluid compared with the nontreated septic group was significant (p < .05) . No growth was observed from blood cultures taken 24 h after sepsis induction . All control experiments yielded negative cultures . The results of these experiments demonstrated that liposomal cefoxitin exerts an enhanced bactericidal effect in liver and lungs during Gram-negative sepsis.

Clin Infect Dis, 1995 Nov, 21(5), 1300 - 2
Discontinuation of intravenous antibiotic therapy during persistent neutropenia in patients receiving prophylaxis with oral ciprofloxacin; Cornelissen JJ et al.; To evaluate the mortality and morbidity associated with early discontinuation of intravenously administered antibiotics, we prospectively examined the incidence and cause of recurrent fever in patients with persistent neutropenia who responded to a short course of intravenous antibiotic therapy . Preventive measures included the use of oral ciprofloxacin as prophylaxis for infection by gram-negative bacteria during the entire neutropenic episode . The rate of response to either initial or modified intravenous antibiotic therapy was 96% (149 of 156 episodes of fever) . Eighty-five patients had an episode of persistent neutropenia (median duration, 7 days; range, 1-36 days) after they responded to treatment . Seven of these patients had recurrent fever, including 2 with bacteriologically documented infections, 4 with probable fungal pneumonia, and 1 with documented pneumonia due to Aspergillus fumigatus . Two patients with probable fungal pneumonia died, while the other infectious episodes resolved completely . These results do not support the continuation of intravenous antibiotic therapy for febrile patients with persistent neutropenia who have responded to the antibiotic regimen while receiving prophylaxis with oral ciprofloxacin.

New Horiz, 1995 Nov, 3(4), 680 - 7
Biocompatible intermittent hemodialysis; Lang S et al.; Since intermittent hemodialysis was first used systemically during the Korean war, the mortality of acute renal failure (ARF) in critically ill patients has remained high ( > or 50%) . The lack of improvement may be a result of better resuscitation techniques and intensive care management that allow more severely ill patients to survive long enough to develop ARF . The concept that those patients with ARF die with, but not of, renal failure was challenged recently by the results of three prospective randomized trials . Each tested the hypothesis that the course of ARF and the fate of critically ill patients may be affected adversely by bioincompatibility reactions due to the dialysis membrane used (activation of complement and neutrophils) . Schiffl and colleagues were the first to publish a full report on the results of their investigation comparing bioincompatible cuprophane (CUP) and biocompatible acrylonitrile AN 69 (Hospal, Lyon, France) membranes in 52 patients with ARF following cardiovascular surgery . The AN 69 group had a lower death rate (38% vs . 65%, p = 0.052), a lower proportion of patients dying from Gram-negative sepsis (40% vs . 71%, p = 0.0162), and an improved recovery of renal function . A similar trial comparing the use of CUP with biocompatible polymethyl-methacrylate (PMMA) was performed in 72 patients with medical categories of ARF . Again, the use of a biocompatible membrane resulted in an improved survival rate (57% vs . 37%, p = 0.11) and better recovery of renal function (62% vs . 37%, p = 0.04) . Of the 20 patients in each group who initially had nonoliguric ARF, the survival rates were 80% with PMMA and 40% with CUP (p = 0.01) . The preliminary results of another multicenter study including 121 patients dialyzed with either bioincompatible cellulosic membranes or PMMA or polysulfone membranes seem to confirm these findings . The management of critically ill patients is sophisticated and expensive . The use of biocompatible membranes adds little to the overall costs and appears to be justified.

J Dent Res, 1995 Nov, 74(11), 1802 - 11
LPS responsiveness in periodontal ligament cells is regulated by tumor necrosis factor-alpha; Quintero JC et al.; Gingival fibroblasts function as accessory immune cells and are capable of synthesizing cytokines in response to lipopolysaccharides (LPS) from Gram-negative microbes . Recently, we have isolated, cloned, and characterized two cell lines which exhibit characteristics of periodontal ligament (PDL) cells . In this report, we demonstrate that PDL cells showing osteoblast-like phenotype are not LPS-responsive cells . However, treatment of PDL cells with tumor necrosis factor-alpha (TNF-alpha) inhibits the expression of their osteoblast-like characteristics . As a consequence of this TNF-alpha-induced phenotypic change, PDL cells become LPS-responsive, i.e., synthesize several pro-inflammatory cytokines in response to LPS . These phenotypic changes occur at concentrations of TNF-alpha that are frequently observed in tissue exudates during periodontal inflammation, suggesting a physiological significance for these in vitro observations . It is of interest that TNF-alpha-induced phenotypic changes in PDL cells are transient, since removal of rhTNF-alpha from the supernatants of PDL cell cultures results in re-acquisition of the osteoblast-like characteristics and lack of LPS responsiveness of PDL cells . These results suggest that TNF-alpha, by regulating the PDL cell functions, may allow these cells to participate in the disease process as accessory immune cells at the expense of their structural properties.

J Immunol, 1995 Nov 1, 155(9), 4497 - 503
Treatment with bacterial LPS renders genetically resistant C57BL/6 mice susceptible to Theiler's virus-induced demyelinating disease; Pullen LC et al.; Theiler's murine encephalomyelitis virus (TMEV) induces a demyelinating disease in susceptible strains, which clinically and histopathologically resembles human multiple sclerosis . Since bacterial LPS produced by Gram-negative bacteria is known to potentiate an immune response and trigger resident central nervous system cells to produce various inflammatory cytokines, we examined the ability of LPS to affect resistance to TMEV-induced demyelinating disease (TMEV-IDD) . Intraperitoneal injection of LPS, concomitant with intracerebral of genetically resistant C57BL/6 mice with TMEV, resulted in clinical symptoms in approximately 50% of the group . The increase in susceptibility following LPS treatment correlated with the enhanced levels of TMEV-specific delayed-type hypersensitivity and T cell proliferative responses . Similar treatment with LPS, however, did not accelerate the clinical course of susceptible (SJL/J) or intermediately susceptible (C3H) mice . The LPS-treated C57BL/6 mice displayed an increased viral persistence in the central nervous system when compared with nontreated control mice . Intraperitoneal administration of IL-1 beta could mimic the LPS effect in C57BL/6 mice, suggesting that the increase in susceptibility to TMEV-IDD may function via IL-1 produced following LPS stimulation.

Chest, 1995 Nov, 108(5), 1420 - 4
Pulmonary melioidosis; Ip M et al.; Melioidosis is the name given to all diseases caused by the bacterium Pseudomonas pseudomallei . Melioidosis is a tropical disease and prevails in parts of Southeast Asia, northern Australia, and Central and South America . However, in recent years, cases of melioidosis have been reported in the United States and other areas . The organism can infect any organ system, although the lung is the most common organ affected . Pulmonary melioidosis presents either as an acute fulminant pneumonia or as an indolent cavitary disease . In northeastern Thailand, the incidence of P pseudomallei infection is extremely high with significant mortality . One of the key problems with treating melioidosis is its recalcitrance to therapy and high relapse rate . In addition, this Gram-negative rod is resistant to aminoglycosides . In nonendemic regions, patients with melioidosis more typically present with reactivation disease occurring months to years after initial exposure to the organism . The pulmonary disease is mainly in the apices and resembles tuberculosis . With the increasing mobility of people throughout the world and the influx of immigrants from endemic to nonendemic areas, it is important that clinicians be aware of this disease . This article will review the epidemiology, clinical presentations, diagnosis, and treatment of pulmonary melioidosis.

Can J Microbiol, 1995 Nov, 41(11), 1053 - 5
New mini-Tn5 derivatives for insertion mutagenesis and genetic engineering in gram-negative bacteria; Alexeyev MF et al.; Five mini-Tn5 derivatives encoding resistance to Km, Cm, Gm, Tc, and Sm, coupled with the polylinker of the pBluescriptII plasmid, were constructed . These derivatives are carried by an ampicillin-resistant plasmid that has a conditional origin of replication from plasmid R6K and origin of conjugal transfer from the broad host range plasmid RP4 . The new vectors are smaller than those previously described and possess numerous unique restriction sites inside the minitransposons for gene cloning in addition to SfiI and NotI sites found in their predecessors.

Biochemistry, 1995 Oct 31, 34(43), 14230 - 6
Denaturant unfolding of the ferric enterobactin receptor and ligand-induced stabilization studied by site-directed spin labeling; Klug CS et al.; FepA is an integral outer membrane protein that is the specific receptor for the siderophore, ferric enterobactin, and is thus primarily responsible for iron uptake in many Gram-negative bacteria . A site-specific mutant of FepA, containing a single introduced cysteine in the ligand-binding domain, was spin labeled and used to examine the denaturant-induced unfolding of this receptor with guanidine hydrochloride (Gdn-HCl) and urea . Electron spin resonance (ESR) spectra showed conversion of the spin label from a motionally-restricted, immobilized environment to a freely-accessible, rotationally-mobile state upon denaturation . Unfolding was also followed by nondenaturing polyacrylamide gel electrophoresis (PAGE), which is sensitive to loss of the putative transmembrane beta-structure, and displayed a similar concentration dependence . Unfolding occurred over relatively narrow ranges of denaturant concentration, indicating a high degree of cooperativity . Unfolding was fully reversible under the conditions employed . Rapid, spontaneous refolding occurred in the presence of Triton X-100 and did not require exogenous lipids . Refolding could be induced by either dialysis, dilution to low denaturant concentration, or ethanol precipitation . At ambient temperature the free energy of unfolding extrapolated to zero denaturant concentration (delta GU zero) was 6.24 +/- 0.63 kcal/mol . Values of delta GU zero obtained with Gdn-HCl and urea were in good agreement, as were values obtained from linear extrapolation and nonlinear regression fitting to a two-state equilibrium . This is the first report of a quantitative evaluation of the free energy of unfolding for an integral membrane protein.

Dtsch Med Wochenschr, 1995 Oct 27, 120(43), 1468 - 72
{Neurological symptoms after an infection by the sandfly fever virus}; Pauli C et al.; CASE 1: A few days after returning from a holiday in Italy a 50-year-old man developed acute gastrointestinal symptoms, followed by headache, fever and joint pains . After transitory remission he had a relapse with fever, headache and meningitis 2 weeks later . Cerebrospinal fluid contained gram-negative diplococci, but no bacteria grew on culture . Under the suspected diagnosis of meningitis he was treated with penicillin . Ten days later he suddenly developed deafness in his right ear . The various signs and symptoms gradually disappeared and the patient was discharged after 24 days in hospital . Retrospectively serological tests indicated sandfly fever (SF) virus infection with serotype Toscana . This is thought to be the first reported case of deafness associated with this disease . CASE 2: One week after returning from a holiday in Tunisia a 34-year-old man fell ill with fever, headache, rigor, nausea, joint pains and a maculopapular rash for which he was treated as an outpatient . The symptoms improved after 7 days, except for the headache which persisted another 5 days . Serology demonstrated an acute infection by SF virus, serotype Sicilian . CONCLUSION: Sandfly fever should be included in the differential diagnosis of headache, fever and signs of meningitis in persons who have recently been to mediterranean countries.

JAMA, 1995 Oct 18, 274(15), 1221 - 5
The role of polyneuropathy in motor convalescence after prolonged mechanical ventilation; Leijten FS et al.; OBJECTIVE--To test the hypothesis that prolonged motor recovery after long-term ventilation may be due to polyneuropathy and can be foreseen at an early stage by electromyography (EMG) . DESIGN--Cohort study with an entry period of 18 months . Polyneuropathy was identified by EMG studies in the intensive care unit (ICU) . During a 1-year follow-up, amount of time was recorded to reach a rehabilitation end point . SETTING--The general ICU of a community hospital . PATIENTS--Fifty patients younger than 75 years who were receiving mechanical ventilation for more than 7 days . MAIN OUTCOME MEASURES--A rehabilitation end point was defined as return of normal muscle strength and ability to walk 50 m independently . RESULTS--In 29 of 50 patients, an EMG diagnosis of polyneuropathy was made in the ICU . Patients with polyneuropathy had a higher mortality in the ICU (14 vs 4; P = .03), probably related to multiple organ failure (22 vs 11; P = .08) or aminoglycoside treatment of suspected gram-negative sepsis (17 vs 4; P = .05) . Rehabilitation was more prolonged in 12 patients with polyneuropathy than in 12 without polyneuropathy (P = .001) . Of nine patients with delays beyond 4 weeks, eight had polyneuropathy, five of whom had persistent motor handicap after 1 year . In particular, axonal polyneuropathy with conduction slowing on EMG indicated a poor prognosis . CONCLUSIONS--Polyneuropathy in the critically ill is related to multiple organ failure and gram-negative sepsis, is associated with higher mortality, and causes important rehabilitation problems . EMG recordings in the ICU can identify patients at risk.

J Immunol, 1995 Oct 15, 155(8), 3994 - 4003
Lipopolysaccharide stimulates the tyrosine phosphorylation of mitogen-activated protein kinases p44, p42, and p41 in vascular endothelial cells in a soluble CD14-dependent manner . Role of protein tyrosine phosphorylation in lipopolysaccharide-induced stimulation of endothelial cells; Arditi M et al.; Vascular endothelial cell (EC) injury or activation by LPS plays a critical role in the pathogenesis of Gram-negative meningitis and endotoxic shock . EC do not express membrane CD14, but respond to LPS in a soluble CD14-dependent manner . The signal transduction mechanisms involved in LPS-induced EC responses are largely unknown . We used bovine and human brain microvessel EC (BBMEC, and HBMEC) to study LPS-induced protein tyrosine phosphorylation . LPS rapidly induced the tyrosine phosphorylation of several proteins in BBMEC and HBMEC, which was detectable by 5 to 15 min, reached a maximum by 30 min, and declined by 60 to 90 min . The increase in tyrosine phosphorylation was apparent following stimulation with LPS at 0.1 ng/ml and was dose dependent up to 100 ng/ml . Similar changes in tyrosine phosphorylation were induced by smooth and rough LPS as well as lipid A, but not by the inactive lipid A analogue, Rhodopseudomonas sphaeroides diphosphoryl lipid A . Pretreatment of EC with the tyrosine kinase inhibitor, herbimycin A, inhibited LPS-stimulated protein tyrosine phosphorylation and LPS-mediated lactic dehydrogenase release from BBMEC and IL-6 release from HBMEC in a dose-dependent manner . Three proteins with apparent m.w . of 44, 42, and 41 kDa were predominant among the LPS-induced tyrosine phosphoproteins, and they were identified as mitogen-activated protein kinase isoforms ERK1, ERK2, and p38, respectively . LPS-induced protein tyrosine phosphorylation in HBMEC and BBMEC was soluble CD14 dependent, since pretreatment of these cells with anti-hCD14 mAb inhibited the LPS-induced tyrosine phosphorylation of p44, p42, and p41 . Additionally, LPS induced a mobility shift in p44 and p42 mitogen-activated protein kinase isozymes, which was inhibited by herbimycin A pretreatment of the EC . These findings demonstrate for the first time that increased protein tyrosine phosphorylation and activation of mitogen-activated protein kinases occur rapidly after LPS stimulation of EC in the presence of soluble CD14 . Our data also suggest that a herbimycin-sensitive step, presumably a tyrosine kinase, is involved in mediating LPS-induced human EC activation and IL-6 secretion.

J Immunol Methods, 1995 Oct 12, 186(1), 1 - 15
Antibodies to lipopolysaccharide; Poxton IR; Lipopolysaccharides (LPS) are indispensable structural components of the Gram-negative bacterial outer membrane and are major determinants of virulence in pathogenic species . In the infected host LPS is better known as endotoxin where it acts as a potent stimulator of the inflammatory response . This article reviews the methods for the production and measurement of anti-LPS antibodies, and then describes the uses to which these methods have been employed . Antibodies to LPS (either monoclonal or polyclonal) may be used directly as immunotherapeutic agents for the treatment of Gram-negative sepsis or endotoxaemia, or as probes for the diagnosis and epidemiological investigation of Gram-negative bacterial infections . Antibodies are useful tools for investigation of the chemical structure of LPS, its expression on bacteria and to study the role of LPS in pathogenic mechanisms . The detection and quantitation of anti-LPS antibodies has formed the basis of classical and more recent serological studies of major bacterial infections.

Clin Infect Dis, 1995 Oct, 21 Suppl 2, S190 - 5
Knowledge of cellular receptors for bacterial endotoxin--1995; Viriyakosol S et al.; Septic shock due to infections with gram-negative bacteria remains a major clinical problem for infectious disease specialists, although our understanding of the pathophysiology of this syndrome has improved greatly over the past 5 years . The discovery of lipopolysaccharide (LPS) binding protein, a serum protein that catalyses the transfer of LPS to cellular receptors for LPS, was a major breakthrough . The finding that CD14, a glycophosphatidylinositol-linked membrane protein expressed by macrophages, is a receptor for LPS has made a major difference in our understanding of cellular activation by LPS . We will review studies dealing with LPS's binding to these proteins and its activation of cells . A better understanding of septic shock at the molecular level should lead to the development of new treatments for this lethal disease.

Clin Infect Dis, 1995 Oct, 21 Suppl 2, S186 - 9
Anti-endotoxin human monoclonal antibody A6H4C5 (HA-1A) utilizes the VH4.21 gene; Bieber MM et al.; Human IgM monoclonal antibody A6H4C5 was manufactured by Centocor (Malvern, PA) and used in clinical trials as HA-1A (Centoxin) . In vitro, A6H4C6 binds to lipid A and rough-strain, gram-negative bacteria endotoxin . Further analysis of A6H4C5 has shown that it is a polyreactive, cold agglutinin that utilizes the VH4.21 gene segment in germline configuration . It is also a human antibody that binds to human B cells . We have characterized several other independently derived VH4.21 human monoclonal antibodies with the same characteristics as A6H4C5 . This group of antibodies may represent a conserved host immune response.

J Clin Periodontol, 1995 Oct, 22(10), 780 - 7
Clinical and microbiological changes associated with an altered subgingival environment induced by periodontal pocket reduction; Mombelli A et al.; The purpose of the present investigation was to study the effect of an altered subgingival environment, induced by changing the local soft tissue morphology, i.e., pocket depth reduction, on the subgingival microbiota and the clinical conditions . 7 patients aged 30-60 years with generalized marginal periodontitis were selected . Patients were instructed in proper oral hygiene and all teeth were cleaned supragingivally . Mucoperiosteal flaps were raised and the bone re-contoured to eliminate angular bony defects . While the control teeth were carefully debrided and thoroughly root planed, no root instrumentation was performed on the test teeth . Calculus deposits visible to the naked eye were only chipped-off with the tip of a scaler . The flaps were apically repositioned and sutured at the level of the bone crest . Clinical parameters showed a similar pattern of response in the test and control sites over a one year observation period post therapy . Probing depths and probing attachment levels were significantly reduced one month after surgery and remained at a lower level . A significant decrease was also noted for total anaerobic viable bacterial counts . The proportion of the Gram-negative anaerobic rods decreased significantly in both groups . P . gingivalis, Fusobacterium sp., C rectus were detected significantly less often after treatment in both groups . Capnocytophaga and A . odontolyticus, on the other hand, were more frequently isolated after therapy . These findings corroborate the concept that the reduction of selected subgingival microorganisms is the key element for the success of periodontal therapy, rather than the removal of tooth substance and mineralized deposits by root instrumentation.

Clin Microbiol Rev, 1995 Oct, 8(4), 557 - 84
beta-Lactamases in laboratory and clinical resistance; Livermore DM; beta-Lactamases are the commonest single cause of bacterial resistance to beta-lactam antibiotics . Numerous chromosomal and plasmid-mediated types are known and may be classified by their sequences or phenotypic properties . The ability of a beta-lactamase to cause resistance varies with its activity, quantity, and cellular location and, for gram-negative organisms, the permeability of the producer strain . beta-Lactamases sometimes cause obvious resistance to substrate drugs in routine tests; often, however, these enzymes reduce susceptibility without causing resistance at current, pharmacologically chosen breakpoints . This review considers the ability of the prevalent beta-lactamases to cause resistance to widely used beta-lactams, whether resistance is accurately reflected in routine tests, and the extent to which the antibiogram for an organism can be used to predict the type of beta-lactamase that it produces.

Biochem Mol Biol Int, 1995 Oct, 37(3), 439 - 45
Cycloheximide induces nitric oxide synthase mRNA in vascular smooth muscle cells by prolonging mRNA lifetime; Hattori Y et al.; Bacterial lipopolysaccharide (LPS) and other immunostimulants induce an isoform of NO synthase (iNOS) in vascular smooth muscle (VSM) which produces large quantities of NO and profound vasodilation; this process has been implicated as the cause of gram-negative septic shock . Although regulation of iNOS has been considered to occur at the level of transcription, it is unclear whether post-transcriptional events also contribute to changes in iNOS mRNA expression . We show that cycloheximide (CH), an inhibitor of protein synthesis, induces iNOS mRNA in VSM and potentiates the induction of iNOS mRNA caused by LPS . For many early-response genes, protein-synthesis inhibitors enhance mRNA levels by increasing mRNA stability . Since iNOS mRNA contains multiple copies of a consensus sequence (AUUUA) in common with these eary-response genes and responsible for mRNA destabilization, we tested whether CH induces iNOS mRNA by prolonging mRNA lifetime . In the absence of CH, iNOS mRNA decayed with biphagic kinetics and a half-life of 2 h . In the presence of CH, half-life was prolonged to approximately 7 h . Our observations indicate that in VSM the stability of iNOS mRNA may be under the control of a labile protein factor which awaits identification and characterization . Regulation of mRNA lifetime represents a novel site for control of iNOS gene expression.

J Clin Pharm Ther, 1995 Oct, 20(5), 253 - 8
Expanded gentamicin volume of distribution in critically ill adult patients receiving total parenteral nutrition; Ronchera-Oms CL et al.; Aminoglycoside antibiotics distribute into the extracellular fluid compartment and are eliminated by the kidney via glomerular filtration . Malnutrition and total parenteral nutrition influence the fluid and electrolyte status of the patient, and cause organ changes . The purpose of this clinical study was to characterize the kinetic behaviour of gentamicin in the parenterally fed critically ill adult patient . Eighty-six critically ill adult patients treated with gentamicin for severe Gram-negative infections were enrolled in the study (mean +/- SD): age, 60 +/- 14 years; weight, 69.4 +/- 10.2 kg; height, 163 +/- 10 cm; 22 females and 64 males . Four study groups were defined (2 x 2): total parenteral nutrition vs . fluid therapy, and acute renal failure vs . normal renal function . The drug was administered by intermittent intravenous infusion . Blood samples were drawn at steady-state, 5 min before the next dose ('trough') and 30 min after the termination of the infusion ('peak') . Gentamicin serum concentration was determined by fluorescence polarization immunoassay . Gentamicin pharmacokinetic parameters were estimated by non-linear regression analysis, assuming a one-compartment model and first-order elimination from the central compartment . Treatment of malnutrition with total parenteral nutrition increased gentamicin volume of distribution (P < 0.001), but did not affect total body clearance (P = 0.75) . This change tended to produce lower peak concentrations (< 4 micrograms/ml, P = 0.07), thus potentially compromising therapeutic effectiveness . There was no significant influence on trough concentrations (P = 0.56) . Patients receiving fluid therapy had a volume of distribution of 0.34 +/- 0.08 litre/kg, while those fed by the intravenous route showed larger values (0.43 +/- 0.12 litre/kg), irrespective of their renal function . This may be explained by the extracellular water expansion caused by stress, malnutrition, and parenteral refeeding . Gentamicin dosage regimens in critically ill adult patients on total parenteral nutrition should be formulated on the basis of larger volumes of distribution and to attain therapeutic serum concentrations higher doses may be required.

Trends Microbiol, 1995 Oct, 3(10), 381 - 6
Role of O-antigen variation in the immune response; Reeves P; The O antigen is an extremely variable surface polysaccharide of Gram-negative bacteria . This variation is thought to allow the various clones of a species each to present a surface that offers a selective advantage in the niche occupied by that clone . The interactions between O antigen and the immune system are central to determining the selective advantage of each clone.

J Bacteriol, 1995 Oct, 177(20), 5884 - 90
Cloning, sequencing, and characterization of a membrane-associated Prevotella ruminicola B(1)4 beta-glucosidase with cellodextrinase and cyanoglycosidase activities; Wulff-Strobel CR et al.; Prevotella ruminicola B(1)4 is a gram-negative, anaerobic gastrointestinal bacterium . A 2.4-kbp chromosomal fragment from P . ruminicola encoding an 87-kDa aryl-glucosidase (CdxA) with cellodextrinase activity was cloned into Escherichia coli DH5 alpha and sequenced . CdxA activity was found predominantly in the membrane fraction of both P . ruminicola and E . coli, but P . ruminicola localized the protein extracellularly while E . coli did not . The hydrolase had the highest activity on cellodextrins (3.43 to 4.13 mumol of glucose released min-1 mg of protein-1) and p-nitrophenyl-beta-D-glucoside (3.54 mumol min-1 mg of protein-1) . Significant activity (70% of p-nitrophenyl-beta-D-glucoside activity) was also detected on arbutin and prunasin . Less activity was obtained with cellobiose, amygdalin, or gentiobiose . CdxA attacks cellodextrins from the nonreducing end, releasing glucose units, and appears to be an exo-1,4-beta-glucosidase (EC 3.2.1.74) which also is able to attack beta-1,6 linkages . Comparison of the deduced amino acid sequence with other glycosyl-hydrolases suggests that this enzyme belongs to family 3 (B . Henrissat, Biochem . J . 280:309-316, 1991) . On the basis of this sequence alignment, the catalytic residues are believed to be Asp-275 and Glu-265 . This is the first report of a cloned ruminal bacterial enzyme which can cleave cyanogenic plant compounds and which may therefore contribute to cyanide toxicity in ruminants.

Crit Care Med, 1995 Oct, 23(10), 1694 - 702
Autonomic modulation of heart rate variability during endotoxin shock in rabbits; Goldstein B et al.; OBJECTIVE: Gram-negative septic shock is associated with severe hypotension and autonomic cardiovascular dysfunction . We hypothesized that in an anesthetized rabbit model of endotoxin shock, autonomic modulation of cardiac activity, as measured by power spectral analysis of heart rate (HR) variability, would be decreased compared with the anesthetized control rabbits . DESIGN: Experimental, comparative study . SETTING: Laboratory of a university hospital . SUBJECTS: Fourteen adult male New Zealand white rabbits (2.7 to 3.1 kg body weight) were studied under anesthesia . INTERVENTIONS: None . MEASUREMENTS AND MAIN RESULTS: We studied the absolute and temporal changes in HR power spectra and plasma catecholamine concentrations in eight experimental and six control New Zealand white rabbits during Escherichia coli endotoxin-induced shock . HR, respirations, arterial blood pressure (BP), HR power spectra, and plasma catecholamine concentrations were measured at 5- to 10-min intervals for 60 mins in control rabbits or until the mean arterial pressure (MAP) decreased by > or = 20 mm Hg in experimental rabbits . There were no differences in basal HR, respiratory rate, BP, HR power spectra, or catecholamine concentrations between groups . After endotoxin administration, MAP decreased (82 +/- 7 vs . 62 +/- 5 mm Hg; p < .05) as did log low-frequency HR power (-2.14 +/- 2.46 vs . -2.20 +/- 2.48 beats/min2; p < .05) . Low-frequency HR power and MAP remained unchanged in control animals . Log high-frequency HR power decreased in control and experimental rabbits (-1.02 +/- 1.34 vs . -1.69 +/- 2.12 {control}, p < .05; -1.53 +/- 2.19 vs . -2.19 +/- 2.85 beats/min2 {experimental}, p < .05) . While there was an inverse relationship between low- and high-frequency HR power and MAP, the direction of change was opposite in six of six rabbits in the control group and in six of eight rabbits in the experimental group . Plasma catecholamine concentrations did not change during the experiment in either group . CONCLUSIONS: Sympathetic modulation of cardiac activity decreased, while the sympathomedullary response remained unchanged during endotoxin shock . We speculate that a concomitant decrease in low-frequency HR power as MAP decreases may prove to be an early marker for impending shock.

Mol Ecol, 1995 Oct, 4(5), 605 - 12
Genetic diversity within mer genes directly amplified from communities of noncultivated soil and sediment bacteria; Bruce KD et al.; Individual merRT delta P regions were amplified from DNA directly isolated from soil and sediment samples using consensus primers derived from the conserved mer sequences of Tn501, Tn21 and pMER419 . Soil and sediment samples were taken from four sites in the British Isles; one 'pristine' (SB) and three polluted (SO, SE, T2) with respect to mercury . The sizes of the PCR products amplified (approximately 1 kb) were consistent with their generation from mer determinants related to the archetypal elements found in Gram negative bacteria . Forty-five individual clones of sequences obtained from these four sites were isolated which hybridized (> 70% homology) to a merRT delta P probe from Tn501 . The diversity of these amplified mer genes was analysed using Restriction Fragment Length Polymorphism (RFLP) profiling . Fourteen RFLP classes were distinguished, 12 of which proved to be novel and only two of which had been identified in an earlier study of 40 Gram negative mercury resistant bacteria cultured from the same four sites . UPGMA analysis was used to examine the relationships between the 22 classes of determinant identified . The T2 site, which has the longest history of mercury exposure, was found to have the greatest level of diversity in terms of numbers of classes of determinant, while the SO site, which had the highest mercury levels showed relatively low variation . Variation of mer genes within and between the sequences from cultivated bacteria and from total bacterial DNA shows clearly that analysing only sequences from cultivated organisms results in a gross underestimation of genetic variation.

Microbiology, 1995 Oct, 141 ( Pt 10), 2719 - 27
High frequency of conjugation versus plasmid segregation of RP1 in epiphytic Pseudomonas syringae populations; Bjorklof K et al.; The maintenance and transfer of the broad host-range plasmid RP1 in epiphytically growing populations of Pseudomonas syringae was monitored in the phyllosphere of bush bean (Phaseolus vulgaris) . When foliage was inoculated with plasmid-containing bacteria, the plasmid was lost from the majority of the cells within 2 d but was stably maintained in 0.8% of the population . A high frequency of conjugation between added donors and recipients was observed under high humidity conditions . In 1 d, the number of transconjugants rose to 10(-1) of the donors and the proportional level of transconjugants continued to increase until 3 d after inoculation . Under these conditions the proportion of plasmid-containing bacteria stabilized at about 0.8% of the total population . The conjugation rate appeared to be in equilibrium with plasmid loss and the slower growth of the plasmid-carrying cells . A factor that influenced the high conjugation frequency observed was the available nutrients provided by the leaf and also, to a lesser extent, the leaf surface itself . Transfer of the plasmid from added donors to indigenous bacteria was also studied, using a donor-specific bacteriophage for counterselection of the donor . Transfer was observed to 10 different species of Gram-negative epiphytically growing bacteria . The bean leaf surface appears to function as a hotspot at least for intraspecific transfer of plasmids in high humidity . The frequency of transfer was higher than in soil or in rhizosphere habitats . This is likely to be the result of an environment that is nutritionally rich in combination with a limited colonizable surface area which permits close contact between the bacterial cells.

J Surg Res, 1995 Oct, 59(4), 428 - 32
Pharmacokinetics of endotoxin in a rhesus macaque septic shock model; Premaratne S et al.; Using a prospective, randomized, controlled study, we tested the hypothesis that the initial administered dose of endotoxin determines its pharmacokinetics in a rhesus macaque septic shock model . Twelve adult male rhesus macaques, weighing 6 to 10 kg, were equally divided into two groups . The first group received a 20 mg/kg intravenous bolus of the gram-negative endotoxin . The second group received a bolus comparable to the concentration of endotoxin found in the plasma of the first group, 12 hr postendotoxin injection . Both groups were monitored for 12 hr and sacrificed . Plasma endotoxin concentrations were measured using the limulus amebocyte lysate assay and a pharmacokinetic model was applied to the concentration curves . Results of the pharmacokinetic evaluation revealed differences in half-life, clearance, and total apparent volume of distribution between the two groups of animals, suggesting that the changes in these parameters may have a biphasic pattern and may be related to the initial dose of endotoxin injected.

J Infect Dis, 1995 Oct, 172(4), 1137 - 40
Thalidomide inhibits tumor necrosis factor-alpha production by lipopolysaccharide- and lipoarabinomannan-stimulated human microglial cells; Peterson PK et al.; Tumor necrosis factor-alpha (TNF-alpha) is a pathogenic factor in bacterial meningitis . The effect of thalidomide on TNF-alpha production by microglia, the resident macrophages of the brain, was evaluated . In primary human fetal microglial cell cultures stimulated with lipopolysaccharide or lipoarabinomannan, thalidomide inhibited TNF-alpha release in a dose-dependent manner . The inhibitory effect of thalidomide was similar to that of dexamethasone, although expression of TNF-alpha mRNA in microglial cells was reduced only by thalidomide . The results of this in vitro study suggest that thalidomide could have therapeutic potential in gram-negative bacterial and tuberculous meningitis.

Infect Immun, 1995 Oct, 63(10), 3914 - 9
Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection; Kato S et al.; The gram-negative bacterium Actinobacillus actinomycetemcomitans is considered an important etiological agent in periodontal diseases . In this study, we show that A . actinomycetemcomitans strains are cytotoxic for the murine macrophage cell line J774.1 . On the other hand, Porphyromonas gingivalis strains, other gram-negative oral species implicated in adult periodontitis, showed weak cytotoxic effects . For this to occur, A . actinomycetemcomitans had to gain entry into the macrophages, since cytotoxicity was prevented by cytochalasin D . We demonstrate that cell death induced by A . actinomycetemcomitans Y4 occurs through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis . We further sought to determine whether the cytotoxicity induced by A . actinomycetemcomitans Y4 could be modulated by the protein kinase inhibitors H7 and HA1004 . Apoptotic cell death induced by A . actinomycetemcomitans Y4 was suppressed by H7 but was relatively unaffected by HA1004 . These findings suggest that the signals of protein kinases may regulate apoptosis induced by A . actinomycetemcomitans Y4 . The ability of A . actinomycetemcomitans to promote the apoptosis of macrophages may be important for the initiation of infection and the development of periodontal diseases.

Can J Surg, 1995 Oct, 38(5), 459 - 63
Spontaneous rupture of a pancreatic pseudocyst into the portal vein; Skarsgard ED et al.; An unusual complication of pancreatic pseudocyst is reported . A 47-year-old woman with chronic liver disease and a history of recurrent pancreatitis died of fulminant, gram-negative septic shock . Acute, bilateral thigh cellulitis with tissue culture positive for Escherichia coli was the only potential infectious source identified . Autopsy revealed a pseudocyst in the head of the pancreas that had eroded into the portal vein, with embolization of mucoid cyst material into intrahepatic portal vein branches . Fibrous organization and recanalization of some of the occluded portal vein branches indicated that this process had been present for weeks to months and was therefore not the direct cause of death . The literature on this unusual complication of pancreatic pseudocyst is also reviewed.

Am J Respir Cell Mol Biol, 1995 Oct, 13(4), 387 - 98
Induction of heme oxygenase-1 gene expression by lipopolysaccharide is mediated by AP-1 activation; Camhi SL et al.; Gram-negative sepsis is the most common cause of the adult respiratory distress syndrome (ARDS) . Lipopolysaccharide (LPS) when administered in vivo produces pathophysiologic changes similar to those seen in ARDS . The pathogenesis of these changes is mediated in part by oxidative stress . We demonstrate that LPS induces high mRNA levels of the stress-inducible gene heme oxygenase-1 (HO-1) in the rat lung . Increased HO-1 mRNA levels correlate with increased HO-1 protein and enzyme activity . Immunohistochemical analyses of lung tissues from rats treated with LPS reveal abundant HO-1 expression in inflammatory and bronchoalveolar epithelial cells . We further examined the molecular regulation of HO-1 gene expression after exposure of RAW 264.7 macrophage cells to LPS in vitro . These cells respond to LPS with increased HO-1 mRNA expression and HO-1 gene transcription . Transcriptional activation of the mouse HO-1 gene by LPS is mediated by a 5' distal enhancer fragment located approximately 4 kbp upstream from the transcription site . Electrophoretic mobility shift assays show increased activator protein-1 (AP-1) binding activity in RAW 264.7 cells after LPS treatment . Mutation of the AP-1 binding site in this enhancer fragment completely abolishes HO-1 gene activation while mutation of CCAAT/enhancer-binding protein (C/EBP) binding site exerts negligible effect, suggesting that the AP-1 family of transcription factors plays a critical role in regulating HO-1 gene activation following LPS treatment . Furthermore, upstream phosphorylation events modulate this AP-1-dependent expression of the HO-1 gene after LPS treatment.

J Antimicrob Chemother, 1995 Sep, 36(3), 561 - 75
Prospective audit of costs and outcome of aminoglycoside treatment and of therapy for gram-negative bacteraemia; Davey PG et al.; The aims of this study were to audit the monitoring of aminoglycoside treatment to develop a method for measurement of the cost and outcome of treatment, and to assess the accuracy of a previously published sepsis score to predict cost and outcome . Measurements of costs and outcomes were also made for all patients (n = 85) with Gram-negative bacteraemia . Of these, 40 received an aminoglycoside and an additional 215 patients received aminoglycosides for other indications (total aminoglycoside patients 255; total patients 300) . There were no interpretable assays for 82 (32%) of the aminoglycoside patients and only 33/173 (19%) patients assayed had first peak serum concentrations within the recommended range of 8-10 mg/L . Median costs of aminoglycoside treatment were 599 pounds in neutropenic patients, 471 pounds in ICU patients and 185 pounds in other patients . In the bacteraemic patients, median costs of aminoglycoside regimens (278 pounds) were higher than for non-aminoglycoside regimens (97 pounds) . Death in hospital was twice as common in bacteraemic patients (20% versus 10%) and there was a stepwise increase in rate of mortality with sepsis scores . Treatment costs were markedly higher in patients who failed to respond to initial treatment, the mean difference in cost was 418 pounds per patient (95% CI 89 pounds - 747) . Sepsis scores only explained 2.6% of the variance in treatment costs, and 22 patients with zero sepsis scores received prolonged courses of i.v . antibiotic treatment at an average cost of 209 pounds per patient . In conclusion, aminoglycoside regimens rarely conformed to accepted standards of care and treatment failure was associated with markedly increased treatment costs . Three readily measurable indicators of adverse outcome were identified (death in hospital, change of i.v . treatment and readmission within 2 weeks of discharge) and all were related to initial severity of illness as measured by sepsis score . The sepsis score may prove useful for assessment of individual risk but would benefit from further analysis to validate and possibly reduce the number of items in the score.

Pediatr Pol, 1995 Sep, 70(9), 733 - 7
{Septicemia in children treated with exchange transfusions because of hemolytic disease of the newborn}; Kordyasz E; Clinical signs and laboratory test results were analyzed in 70 neonates (42 boys and 28 girls) hospitalized because of neonatal haemolytic disease who were treated with exchange transfusion and later developed septicaemia . Serological Rh-D incompatibility was diagnosed in 11 children, ABO incompatibility in 59 . Signs of infection appeared between days 1 and 7 after transfusion . Pneumonic signs and diarrhoea dominated clinically in 45 newborns, skin abscesses were observed in 10, osteomyelitis in 4 . Septic shock occurred in 7 . Gram-negative bacteria predominated (52.85%) . A significant diagnostic value of the following was found (chi2): granulocytic band forms (expressed as percentages > or = 0.10, a neutrophil index > 0.2 and toxic granulations in neutrophils . These results were obtained in the early, asymptomatic stage of infection, i.e . before the exchange transfusion was performed . The importance of the presence of risk factors, not exchange transfusion per se, is stressed.

Shock, 1995 Sep, 4(3), 178 - 86
Platelet participation in liver injury from gram-negative bacterial lipopolysaccharide in the rat; Pearson JM et al.; Intravenous administration of lipopolysaccharide (LPS) to rats results in multifocal, primarily midzonal hepatic necrosis . The hepatic injury is associated with inflammation and is dependent on neutrophils and the coagulation system . After LPS injection into rats, plasma fibrinogen concentration and numbers of blood platelets and leukocytes decrease . Results of our studies, using immunocytochemistry for the detection of neutrophils and 111indium-labeling to identify platelets, indicate that both neutrophils and platelets accumulate within the liver early after administration of LPS to rats . The accumulation of platelets in the liver before the onset of injury suggested that platelets contribute to the manifestation of LPS-induced hepatotoxicity . To test this hypothesis, the number of circulating blood platelets was decreased by the administration of an anti-rat platelet serum (APS) before LPS administration . The consequent thrombocytopenia by APS administration was associated with an attenuation of both LPS-induced liver injury and the activation of the coagulation system . However, the APS treatment did not prevent the hepatic neutrophil accumulation . These results suggest that platelets contribute to the pathogenesis of liver injury after LPS administration, perhaps through their integral role in coagulation and/or interaction with neutrophils, but they do not appear to contribute to hepatic neutrophil accumulation.

Bone Marrow Transplant, 1995 Sep, 16(3), 381 - 5
Mismatched bone marrow transplantation for Omenn syndrome: a variant of severe combined immunodeficiency; Loechelt BJ et al.; Omenn syndrome is a variant of SCID, inherited as an autosomal recessive disorder, and characterized by severe eczematoid dermatitis, eosinophilia, elevated serum IgE and a distinctive histology in enlarged lymph nodes . The etiology of Omenn syndrome is unknown, however, unlike other forms of SCID; patients with Omenn syndrome have activated T lymphocytes in their circulation capable of non-MHC restricted cytotoxic function . Recently, it has been observed that the use of immunosuppressive therapy, particularly cyclosporine, can modify the clinical manifestations of the disorder . Prior to the use of bone marrow transplantation this disease was universally fatal . Death typically occurred in infancy as the result of opportunistic infections and/or malignancies, most notably lymphomas . While bone marrow transplantation has become quite successful for many phenotypes of SCID, even with the use of alternative donors other than histocompatible siblings, in Omenn syndrome it remains a challenge . In our experience, patients with Omenn syndrome exhibit a higher incidence of Gram negative sepsis, before and during transplantation, and carry a significant risk of post-transplant rejection when compared with patients with other phenotypes of SCID . We report the results of six patients treated with bone marrow transplantation from alternative donors, three had unrelated donors (URD) and three had haplo-identical parental donors . Five of the six patients achieved complete and/or durable donor cell engraftment and only one patient experienced acute GVHD . Three patients died of transplant-related complications (infection or EBV-associated B cell lymphoma) between day +22 and day +95 post-transplant . Three patients survived more than 1 year post-transplant.(ABSTRACT TRUNCATED AT 250 WORDS)

Childs Nerv Syst, 1995 Sep, 11(9), 511 - 6
Morphological modifications of the choroid plexus in a rodent model of acute ventriculitis induced by gram-negative liquoral sepsis . Possible implications in the pathophysiology of hypersecretory hydrocephalus; Cardia E et al.; Gram-negative bacterial infections of the central nervous system are generally associated with high morbidity and mortality rates . In patients with ventriculitis induced by gram-negative liquoral sepsis, a reduction in cerebrospinal fluid formation has been reported, suggesting that gram-negative ventriculitis is able per se to alter the normal functioning of the choroid plexus . The aim of the present study was to analyse, for the first time in the rat, the effects of acute ventriculitis on the ultrastructure of the choroid plexus . A simple and inexpensive experimental model of acute ventriculitis was developed: we injected into the cisterna magna of rats 10(3) CFU of live Escherichia coli, inducing septic ventriculitis without major neurological deficits . Histological examinations of rodent choroid plexus 24 h after the injection revealed patches of altered epithelium, with swollen and vacuolated ependymal cells associated with leukocyte infiltration . Electron microscopy demonstrated a reduced number of microvilli and flattening of the epithelial surface . These results (a) indicate that gram-negative septic ventriculitis is able to induce visible ultrastructural alterations of the choroid plexus which (b) are consistent with a picture of marked reduction of the functioning epithelial choroid plexus surface, and (c) highlight the potential usefulness of our rodent acute ventriculitis model for developing treatment modalities.

J Infect Dis, 1995 Sep, 172(3), 794 - 804
Lipopolysaccharide (LPS)-specific monoclonal antibodies regulate LPS uptake and LPS-induced tumor necrosis factor-alpha responses by human monocytes; Pollack M et al.; Lipopolysaccharide (LPS)-monocyte/macrophage interactions are central to the infected host's inflammatory response to gram-negative bacteria . Flow cytometry was used to analyze the regulation by LPS-specific monoclonal antibodies (MAbs) of fluorescein isothiocyanate-conjugated LPS uptake by human peripheral blood monocytes . The uptake of LPS was stimulated by fresh or heat-inactivated serum (NHS or delta NHS) or by LPS-binding protein and inhibited by alpha-LPS or alpha-CD14 (LPS receptor) MAbs . The inhibition of alpha-LPS uptake was offset in the presence of NHS by a simultaneous MAb-mediated increase in LPS uptake that was blocked by alpha-complement receptor 1 . Monocyte tumor necrosis factor-alpha responses to LPS were augmented by NHS and delta NHS and inhibited by alpha-LPS MAbs . Thus, alpha-LPS MAbs down-regulate the proinflammatory uptake of LPS by human monocytes via membrane-bound CD14 while promoting complement-mediated opsonic uptake through membrane-associated CR1.

Infect Immun, 1995 Sep, 63(9), 3693 - 6
Purification and characterization of Eikenella corrodens type IV pilin; Hood BL et al.; Eikenella corrodens is a gram-negative human pathogen associated with periodontal diseases and soft-tissue infections . Pilin was purified by association-dissociation and fast protein liquid chromatography; it had an apparent molecular mass of about 14.8 kDa and an N-terminal amino acid sequence reflective of type IV pilins . Antibodies to the purified protein reacted with pili on whole cells . This is the first report of purification of type IV pili/pilin from this organism . Other type IV pili are important virulence factors; we are currently investigating the biological role of pili in E . corrodens.

Infect Immun, 1995 Sep, 63(9), 3484 - 90
Intracellular multiplication and toxic destruction of cultured macrophages by Capnocytophaga canimorsus; Fischer LJ et al.; Capnocytophaga canimorsus is a gram-negative rod that causes opportunistic infections resulting in bacteremia, septicemia, meningitis, and death in immunocompromised, splenectomized, and alcoholic individuals . Infections caused by a related species, Capnocytophaga cynodegmi, remain localized at the site of the wound where the organism is introduced . Both organisms are part of the normal canine oral flora and are introduced through puncture wounds via dog bites . We found that both C . canimorsus and C . cynodegmi attach, are phagocytized, and multiply intracellularly in J774 mouse macrophage cells . After 48 h of infection by C . canimorsus, large sections of the macrophage cell layer were observed to detach and lyse, while the monolayer infected with C . cynodegmi demonstrated no cytotoxic effects . Tissue culture supernatants from the C . canimorsus-infected J774 cells filtered through a 0.22-micron-pore membrane produced a similar effect on fresh monolayers, while filtrates from C . cynodegmi and uninfected controls produced no effect . No endotoxin release was observed in these supernatants . We conclude that the cytotoxic phenotype of C . canimorsus is the likely result of a toxin produced by this organism.

J Clin Microbiol, 1995 Sep, 33(9), 2358 - 65
Characterization of a 17-kilodalton antigen of Bartonella henselae reactive with sera from patients with cat scratch disease; Anderson B et al.; A library of Bartonella (Rochalimaea) henselae DNA was constructed in the cloning vector lambda ZAPII and screened for expression of antigenic proteins by using a pool of sera from patients who had been diagnosed with cat scratch disease (CSD) and had antibodies to Bartonella spp., as determined by indirect fluorescent-antibody (IFA) assay . Ten immunoreactive phages were subcloned as recombinant plasmids by in vivo excision . All 10 recombinants expressed a protein of approximately 17 kDa when they were examined by immunoblot with the pool of human sera . Restriction endonuclease digestion of each recombinant plasmid indicated seven profiles, suggesting that cloning bias was not the reason for repeated isolation of clones expressing the 17-kDa antigen . The gene coding for the 17-kDa antigen was sequenced and shown to code for an open reading frame of 148 amino acids with a predicted molecular mass of 16,893 Da . The amino terminus of the deduced amino acid sequence was hydrophobic in nature and similar in size and composition to signal peptides found in gram-negative bacteria . The remainder of the deduced amino acid sequence was more hydrophilic and may represent surface-exposed epitopes . Further subcloning of the 17-kDa antigen as a biotinylated fusion protein in the expression vector PinPoint Xa-2 resulted in a 30-kDa protein that was highly reactive on immunoblots with individual serum samples from patients with CSD . The agreement between reactivity with the 30-kDa fusion protein on immunoblot analysis and the results obtained by IFA assay was 92% for IFA-positive sera and 88% for IFA-negative sera . The recombinant-expressed 17-kDa protein should be of value as an antigen for serologic diagnosis of CSD and Bartonella infections and warrants further study in attempts to develop a subunit vaccine to prevent long-term Bartonella infection in cats and the potential for further spread of these organisms to humans.

Biochim Biophys Acta, 1995 Aug 23, 1238(1), 34 - 41
Actinobacillus actinomycetemcomitans leukotoxin forms large conductance, voltage-gated ion channels when incorporated into planar lipid bilayers; Lear JD et al.; Actinobacillus actinomycetemcomitans leukotoxin is a member of the bacterial RTX (repeats in toxin) toxin family, produced by a diverse group of Gram-negative pathogens . Members of this group of toxins, although similar in sequence, differ in target cell specificity with Actinobacillus actinomycetemcomitans leukotoxin demonstrating a unique species- and cell-type specificity . Purified A . actinomycetemcomitans leukotoxin added to pre-formed POPE/POPS lipid bilayers showed no spontaneous incorporation (to concentrations of 250 ng/ml) . Reproducible channel activity was seen when the bilayer was reformed from lipid monolayers in the presence of toxin (50 ng/ml) in one of the aqueous chambers . Control experiments with heat-inactivated toxin did not display channel activity under the same experimental conditions . The channel behavior showed a complex pattern of multiple conductance levels of 118, 262 and 406 pS in solutions containing 0.140 M NaCl . The first two states showed voltage-dependent channel gating with approximately equal but opposite apparent gating charges of 1.4 electrons . A model accounting for the multiple conducting states and gating properties is presented.

J Immunol, 1995 Aug 15, 155(4), 2005 - 12
Differences in the shedding of soluble TNF receptors between endotoxin-sensitive and endotoxin-resistant mice in response to lipopolysaccharide or live bacterial challenge; Carpenter A et al.; TNF-alpha plays a pivotal role in the pathogenesis of septic shock . It exerts its effects by binding two cell surface receptors, designated TNF-R I and II, also referred to as the p55 and p75 receptors, respectively . TNF-Rs are transmembrane proteins, which on cleavage of their extracellular domains, result in the release of soluble fragments (sTNF-R) . sTNF-R levels increase markedly during infection, and may serve to modulate TNF-alpha bioactivity . The mechanisms regulating this process are uncertain . To investigate this, we measured sTNF-R release in endotoxin-sensitive C3H/HeN and endotoxin-resistant C3H/HeJ mice given LPS or live Gram-negative bacteria . In C3H/HeN mice, there was a rapid early response during the first 4 h, and a second peak at 8 h, particularly noticeable in the case of the p75 receptor . Prior administration of neutralizing Abs to TNF-alpha or IFN-gamma had no effect on receptor shedding . Surprisingly, C3H/HeJ mice also responded to both bacterial challenge and to LPS by shedding sTNF-R; the magnitude and duration of the early response was not substantially different from C3H/HeN mice, although the second peak was absent . Peritoneal macrophages from C3H/HeN mice responded promptly (5 h) when stimulated with LPS in vitro, and by 22 h levels had increased five- to 10-fold . In contrast, cells from C3H/HeJ mice demonstrated only a very modest response at 22 h following maximal stimulation . The data suggest that there may be at least two separately regulated pathways that control sTNF-R shedding in these mice.

Singapore Med J, 1995 Aug, 36(4), 367 - 70
Adult idiopathic thrombocytopenic purpura (ITP)--a prospective tracking of its natural history; Kueh YK; Thirty-seven Asian patients (30 women, 7 men) with chronic idiopathic thrombocytopenic purpura (ITP) followed prospectively for 4 to 15 years showed a highly variable clinical course . The women as a group had a much lower initial platelet count than the men (28 x 10(9)/l versus 54 x 10(9)/l) . All the women but only 2 men required treatment for symptomatic thrombocytopenia . Six women developed secondary autoimmune disorders (4 systemic lupus erythematosus and 2 Evan's syndrome) after 14 to 33 months of clinical follow up . Although their responses to corticosteroid therapy were suboptimal when initiated for ITP, these 6 patients uniformly demonstrated a complete platelet response when corticosteroid treatment was re-introduced following the evolution of secondary autoimmune disorders . Four of the 5 untreated men were over 55 years of age . Their mild to moderate thrombocytopenia was discovered incidentally and they remained symptom-free after a follow up of at least 5 years . The overall response rates of this cohort of Asian patients to corticosteroid therapy and splenectomy are compared with those reported from the West . Three deaths are recorded in this study, one from intracranial haemorrhage and 2 gram negative septicaemia in steroid-dependent postsplenectomy patients . The variable behaviour of this cohort of ITP patients emphasises the need for individualised management . Asymptomatic thrombocytopenia can be observed without treatment . Two fatalities from gram negative septicaemia in asplenic, steroid-dependent patients caution against the hasty recommendation of splenectomy for refractory ITP.

Clin Exp Immunol, 1995 Aug, 101(2), 357 - 61
Increased lipopolysaccharide-induced tumour necrosis factor levels and death in hypercholesterolaemic rabbits; Brito BE et al.; Nutritional-induced hypercholesterolaemia in New Zealand rabbits causes increased susceptibility to experimental infections . Rabbits fed cholesterol (0.5 g%) for 8 weeks were injected intravenously with varying doses of Escherichia coli 0127: B8 lipopolysaccharide (LPS; 3-100 micrograms/kg) . The levels of cholesterol, triglycerides, tumour necrosis factor (TNF), and the survival rates of treated rabbits were then measured . Rabbits fed either normal chow or chow impregnated with sesame oil were used as controls . LPS induced higher serum TNF levels in hypercholesterolaemic rabbits than in normal rabbits or rabbits fed with chow containing sesame oil . TNF levels rose faster in hypercholesterolaemic rabbits than in normal rabbits, reaching maximum levels at 60 min and 120 min, respectively, after LPS injection . The survival rate of hypercholesterolaemic rabbits (1/11) was lower than in normal rabbits (6/7) or rabbits fed with the sesame oil chow (4/4) at the higher LPS doses . No death occurred at lower doses . One possible interpretation of these results, also supported by neutralization experiments, is that increased TNF secretion in hypercholesterolaemic rabbits raises the host's susceptibility to experimental endotoxaemia and possibly to Gram-negative infection.

J Bacteriol, 1995 Aug, 177(16), 4742 - 7
Characterization of the exbBD operon of Escherichia coli and the role of ExbB and ExbD in TonB function and stability; Ahmer BM et al.; TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria . ExbB protein has been identified as an auxiliary protein in this process . In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB . The phenotypes of exbB exbD+ strains were essentially indistinguishable from the phenotypes of exbB+ exbD strains . Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80 . Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained . In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min . These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex . While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition . Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB+ strain under iron-limiting conditions . Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.

Ann Surg, 1995 Aug, 222(2), 179 - 85
Pathogenesis and prevention of early pancreatic infection in experimental acute necrotizing pancreatitis; Foitzik T et al.; OBJECTIVE: The authors test antibiotic strategies aimed at either mitigating bacterial translocation from the gut or delivering antibiotics specifically concentrated by the pancreas for prevention of early secondary infection after acute necrotizing pancreatitis . BACKGROUND: Infection currently is the principal cause of death after severe pancreatitis . The authors have shown that the risk of bacterial infection correlates directly with the degree of tissue injury in a rodent model of pancreatitis . Bacteria most likely arrive by translocation from the colon . METHODS: Severe acute necrotizing pancreatitis was induced in rats by a combination of low-dose controlled intraductal infusion of glycodeoxycholic acid superimposed on intravenous cerulein hyperstimulation . At 6 hours, animals were randomly allocated to five treatment groups: controls, selective gut decontamination (oral antibiotics and cefotaxime), oral antibiotics alone, cefotaxime alone, or imipenem . At 96 hours, surviving animals were killed for quantitative bacterial study of the cecum, pancreas, and kidney . RESULTS: The 96-hour mortality (35%) was unaffected by any treatment regimen . Cecal gram-negative bacteria were significantly reduced only by the oral antibiotics . Pancreatic infection was significantly reduced by full-gut decontamination and by imipenem, but not by oral antibiotics or by cefotaxime alone . Renal infection was reduced by both intravenous antibiotics . CONCLUSIONS: Early pancreatic infection after acute necrotizing pancreatitis can be reduced with a full-gut decontamination regimen or with an antibiotic concentrated by the pancreas (imipenem) but not by unconcentrated antibiotics of similar spectrum (cefotaxime) or by oral antibiotics alone . These findings suggest that 1) both direct bacterial translocation from the gut and hematogenous seeding interplay in pancreatic infection while hematogenous seeding is dominant at extrapancreatic sites and 2) imipenem may be useful in clinical pancreatitis.

Cell Immunol, 1995 Aug, 164(1), 36 - 46
Induction of human T cells that coexpress CD4 and CD8 by an immunomodulatory protein produced by Actinobacillus actinomycetemcomitans; Shenker BJ et al.; Actinobacillus actinomycetemcomitans, a gram-negative, capnophilic bacterium, is associated with several human diseases and is the suspected etiologic agent in certain forms of periodontal disease . We have previously shown that this organism produces an immunosuppressive factor (ISF) which is capable of inhibiting both T- and B-cell activation . Furthermore, these effects appear to be associated with the activation of a population of suppressor cells . We now report that the ISF induces a unique population of CD4+CD8+ dual-positive T-cells . By utilizing multiparameter flow cytometric analysis, we were able to detect the presence of dual-positive cells in cultures of human T-cells treated with PHA and ISF . The cells appeared within 48 hr and their induction was dependent upon the presence of both CD4 and CD8 cells in the culture . Dual expression of CD4 and CD8 was stable in that the cells continued to express both surface proteins after being sorted and cultured for an additional 24 hr . Phenotypic analysis indicates that these cells are also CD3+, CD2+, CD5+, TCR alpha beta+, CD45RA+ (and RO+), and CD29+ . The dual-positive cells express surface markers associated with T-cell activation: CD25+, CD69+, CD71+, and HLA-DR+ . In contrast, the cells were negative for CD34, CD57, CD56, and CD16 . Cell cycle analysis indicates that > 80% of the dual-positive cells were in the S phase . Finally, functional analysis of these cells indicates that they are capable of suppressing the proliferative response of autologous T-cells to PHA.

J Infect Dis, 1995 Aug, 172(2), 481 - 9
Opsonic activities of surfactant proteins A and D in phagocytosis of gram-negative bacteria by alveolar macrophages; Pikaar JC et al.; Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages . The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated . In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages . Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing . Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A . In contrast, SP-D did not mediate phagocytosis . SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

Zhonghua Liu Xing Bing Xue Za Zhi, 1995 Aug, 16(4), 231 - 3
{Analysis of nosocomial infection in hospitalized critical and serious patients}; Liu MD et al.; An investigation was made on 298 hospitalized critical and serious patients in Chongqing . the results showed that the nosocomial infection rate was 43.3% (129/298) . The higher infection rate was found in the departments of brain surgery and hematopathy . The lower respiratory tract was found to be the most commonly seen infection sites (65.1%) . Of 46 strains of the pathogenic organisms causing the nosocomial infection, gram-negative becilli were accounted for 52.2% and fungi 28.3% . The case fatality rate of infected patients (37.9%) was significantly higher than that of non-infected patient's (10.7%).

J Ind Microbiol, 1995 Aug, 15(2), 67 - 70
Sequence of the gene encoding the 16S rRNA of the beer spoilage organism Megasphaera cerevisiae; Doyle LM et al.; The 16S ribosomal RNA gene from the beer-spoilage organism, Megasphaera cerevisiae was polymerase chain reaction (PCR)-amplified and sequenced . Analysis confirmed the phylogenetic position of M . cerevisiae as a sister taxon of Megasphaera elsdenii, within the obligately anaerobic, Gram-negative cocci . The sequence obtained should facilitate the development of DNA probes for early detection of this spoilage organism.

Appl Microbiol Biotechnol, 1995 Aug-Sep, 43(4), 656 - 66
Cloning, characterization and phenotypic expression in Escherichia coli of catF, which encodes the catalytic subunit of catalase isozyme CatF of Pseudomonas syringae; Klotz MG et al.; The phytophathogenic, gram-negative bacterium Pseudomonas syringae pv . syringae 61 contains three isozymes of catalase (EC 1.11.1.6), which have been proposed to play a role in the bacterium's responses to various environmental stresses . To study the role of individual isozymes, the gene coding for the catalytic subunit of one catalase isozyme was cloned from a cosmid library hosted in Escherichia coli DH5 alpha by using a designed catalase-specific DNA probe for the screening . One out of four clones with a catalase-positive genotype was subcloned and a pUC19-based 2.7 x 10(3)-base (2.7-kb) insert subclone, pMK3E5, was used to transform catalase-deficient E . coli strain UM255 (HPI-, HPII-) . The transformants contained a single isozyme of catalase that had electrophoretic and enzymic properties similar to catalase isozyme CatF from P . syringae pv . syringae 61 . Analysis of the sequenced 2.7-kb insert DNA revealed six putative open-reading frames (ORF) . The 1542-base-pair DNA sequence of ORF2, called catF, encodes a peptide of 513 amino acid residues with a calculated molecular mass of 66.6 kDa . The amino acid sequence deduced from catF had homology to the primary structure of true catalases from mammals, plants, yeasts and bacteria . The activity of the recombinant catalase was inhibited by 3-amino-1,2,4-triazole and azide and stimulated by chloramphenicol . The N terminus contained a signal sequence of 26 amino acids necessary for secretion into the periplasm, a so-far unique property of Pseudomonas catalases.

J Bacteriol, 1995 Aug, 177(15), 4474 - 80
Transcriptional analysis of rolling circle replicating plasmid pVT736-1: evidence for replication control by antisense RNA; Galli DM et al.; Several plasmids have been described in Actinobacillus actinomycetemcomitans, a gram-negative coccobacillus . Recently, the nucleotide sequence of pVT736-1, a cryptic plasmid of A . actinomycetemcomitans VT736, was determined . This plasmid possesses all the features necessary for rolling circle replication . The present study involved a transcriptional analysis of pVT736-1 . Results of Northern (RNA) blot analyses and primer extension studies indicated that the two open reading frames identified in pVT736-1 are each preceded by at least one promoter . Expression of these promoters varied with growth phase . In addition, an antisense RNA (Cop RNA) appeared to control the synthesis of the putative replication protein . To our knowledge, this is the first rolling circle replicating plasmid isolated from a gram-negative organism that has been subjected to such detailed analysis.

Infect Immun, 1995 Aug, 63(8), 2995 - 9
Leptospira icterohemorrhagiae and leptospire peptidolgycans induce endothelial cell adhesiveness for polymorphonuclear leukocytes; Dobrina A et al.; We have examined the effect of the virulent Leptospira interrogans strain Teramo, serotype icterohemorrhagiae, on the adherence of human neutrophilic polymorphonuclear leukocytes (PMN) to cultured human umbilical vein endothelial cells (HEC) . Selective pretreatment of HEC with intact or sonicated leptospires caused a dose- and time-dependent increase of HEC-PMN adhesion (13.2% +/- 2.5% adherence to untreated HEC versus 46.3% +/- 5.6% adherence to HEC pretreated for 4 h with 10(8) intact leptospires per ml {mean +/- standard error of six experiments; P < 0.001}) . In contrast, selective leptospire pretreatment of PMN or the addition of leptospires during the adherence assay did not alter HEC-PMN adherence . Leptospire induction of endothelial-cell adhesiveness occurred without detectable HEC damage and was prevented by RNA and protein synthesis inhibitors and by monoclonal antibodies to the CD11/CD18 adhesion complex of neutrophils and to the endothelial-leukocyte adhesion molecule 1 (ELAM-1) of endothelial cells . Similar results were obtained with pretreatment of HEC with interleukin-1 or with the lipopolysaccharide (LPS) of the gram-negative bacterium Escherichia coli . The possibility that contamination by the LPS of gram-negative bacteria could be involved in the induction of HEC adhesiveness was ruled out by the observation that the LPS inhibitor polymyxin B, which abolished the proadhesive effect of E . coli LPS, was ineffective in inhibiting leptospire- as well as interleukin-1-induced adherence . Similarly, leptospire LPSs seemed to have no role in the increase of endothelial-cell adhesiveness, since pretreatment of HEC with a leptospire LPS extract (phenol-water method) or with a leptospire total lipid extract failed to induce the proadhesive phenotype for neutrophils . Instead, peptidoglycans extracted from our leptospires actively stimulated the endothelial proadhesive activity for neutrophils (16.5% +/- 2.1% adherence to untreated HEC versus 51.2% +/- 2.9% adherence to HEC pretreated for 4 h with 1 microgram of peptidoglycan per ml; {mean +/- standard error of four experiments; P < 0.001}) . This peptidoglycan-induced activity was inhibited by monoclonal antibodies to the CD11/CD18 adhesion complex and to ELAM-1 but not by polymyxin B . We conclude that peptidoglycans from pathogenic leptospires are among the molecules that can directly activate vascular endothelial cells to increase their adhesiveness for neutrophilic granulocytes . These observations may contribute to a better understanding of the mechanisms whereby non-gram-negative bacteria modulate the local and systemic inflammatory reaction.

Infect Immun, 1995 Aug, 63(8), 2899 - 905
Glycosphingolipids from Sphingomonas paucimobilis induce monokine production in human mononuclear cells; Krziwon C et al.; Glycosphingolipids (GSL) isolated from the gram-negative lipopolysaccharide (LPS)-free bacterium Sphingomonas paucimobilis have remarkable structural similarities with LPS and its hydrophobic part, termed lipid A . Like LPS, but in contrast to the structurally related ceramides and cerebrosides, GSL contain an alpha-linked, negatively charged pyranosidic glycosyl component adjacent to the lipid portion and are capable of forming membranes . Because of these similarities, it was of interest to investigate whether these GSL are also able to induce monokine production in human mononuclear cells (MNC) . Our results show that a GSL containing four sugar residues (GSL-4A) induced the release of tumor necrosis factor, interleukin-6, and interleukin-1 in MNC, whereas GSL-1, containing only one glycosyl residue, was inactive . A minimal concentration of 1 microgram of GSL-4A per ml was necessary to induce monokine production in MNC, whereas LPS was as active at a 10,000-fold-lower concentration (0.1 ng/ml) . Both GSL-4A-induced monokine production and LPS-induced monokine production were reduced by the bactericidal/permeability-increasing protein and GSL-1 . In contrast to LPS, GSL-4A-induced monokine release could be inhibited neither by an anti-CD14 monoclonal antibody nor by lipid A partial structures . We therefore conclude that at the receptor level, different mechanisms are involved in the LPS- and GSL-4A-induced monokine release.

Microcirculation, 1995 Aug, 2(2), 151 - 63
Activation-dependent isolation and culture of murine pulmonary microvascular endothelium; Gerritsen ME et al.; OBJECTIVE: Establish a reproducible method for the isolation and cultivation of murine pulmonary microvascular endothelium . To this end, we exploited the localized pattern of microvascular endothelial activation induced in vivo by inflammatory stimuli to isolate a subpopulation of endothelium for in vitro study . METHODS: Immunohistochemical analyses of the pulmonary vasculature of mice treated systemically with gram-negative bacterial endotoxin (LPS) demonstrated selective expression of VCAM-1 (CD106) in the endothelial lining of small collecting veins, venules, septal capillaries, and, infrequently, small arteries, which was not observed in control mice . Single cell suspensions prepared by enzymatic dissociation of peripheral lobular tissues dissected from the lungs of LPS-stimulated mice were incubated with a phycoerythrin-conjugated antimouse VCAM-1 monoclonal antibody (MK 1.91) . Cells expressing this antigen were isolated by sterile fluorescence-activated cell sorting (FACS) . Positive cell populations were collected and cultured for 1-2 weeks . When confluent, these primary cultures were further FACS enriched for endothelium, positively selecting for cells incorporating a fluorescent derivative of acetylated low density lipoprotein (Di-I-Ac-LDL) . RESULTS: The resulting population of cells (mouse lung endothelial cells, MLEC) were uniformly positive for the endothelial markers von Willebrand factor, thrombomodulin, and Dil-Ac-LDL uptake . MLEC readily formed tube-like structures when cultured on Matrigel and spontaneously demonstrated a sprouting phenotype on fibronectin or collagen matrices . MLEC retained responsiveness to cytokines (IL-1 alpha, IL-1 beta, TNF alpha, IFN gamma) up to at least eight passages from primary culture and demonstrated upregulation of E-selectin (CD62E) and P-selectin (CD62P) mRNA as early as 2 hr after LPS stimulation . Characteristic temporal expression patterns of cell surface E-selectin (maximal at 4 hr and declining toward baseline by 24 hr), VCAM-1 (maximal at 6-8 hr and remaining elevated for 24-48 hr), and ICAM-1 (maximal at 6-8 hr and maintained at 24 hr) were observed when cultured MLEC were treated with recombinant murine TNF alpha or recombinant human (rh) IL-1 alpha or rhIL-1 beta . The rolling, adhesion, and transmigration of human polymorphonuclear leukocytes was markedly increased on cytokine-activated MLEC monolayers under defined flow conditions . CONCLUSION: The strategy of activation-dependent isolation allows for the reproducible selection of a specific subset of microvascular endothelial cells for in vitro study . This experimental approach should further facilitate study of the functional heterogeneity of endothelium and its pathophysiologic dysfunction.

Shock, 1995 Aug, 4(2), 139 - 42
Effects of endotoxin on the guinea pig heart response to ischemia reperfusion injury; McDonough KH et al.; Ischemia causes significant damage to the heart as manifested by decreases in ventricular performance . Several different methods have been shown to protect the heart from ischemic injury--one is operative over a short period (an hour) and the other over longer periods (a day) . The latter form of protection has been demonstrated in rats after induction of Gram-negative sepsis or administration of endotoxin or cytokines . In the present study we determined whether guinea pigs would also show induction of cardiac protection subsequent to a dose of endotoxin . Male guinea pigs were injected with 1 mg of endotoxin and studied the following day . Hearts were perfused at a constant perfusion pressure and studied in an isovolumic mode . Left ventricular developed pressure was significantly lower in the endotoxin-treated group than in the control group . After 35 min of total ischemia and 25 min of reperfusion, recovery of left ventricular developed pressure was complete in the endotoxin group but significantly decreased in the control group such that after ischemia and reperfusion, there was no significant difference in left ventricular performance between the two groups . Coronary flow was significantly greater in the endotoxin group than in the control group both prior to and after ischemia . Hearts from endotoxin-treated guinea pigs resumed spontaneous contractile activity sooner and released less lactate upon reperfusion than did the control group . Thus prior treatment of guinea pigs with endotoxin resulted in depression of the isolated heart but also resulted in protection of the isolated heart from further damage due to ischemia/reperfusion injury.

Alcohol Clin Exp Res, 1995 Aug, 19(4), 834 - 9
1-week withdrawal from 8 weeks alcohol consumption protects the heart from sepsis-induced dysfunction; McDonough KH; Gram-negative sepsis causes a depression of the myocardium such that ventricular function curves generated on isolated perfused hearts removed from septic rats are displaced downward and to the right of control . Alcohol consumption can also cause a depression of the myocardium, especially if the period of alcohol feeding is prolonged . However, even before overt changes in the myocardium can be measured as a result of alcohol consumption, chronic alcoholism can result in a potentiation of sepsis-induced cardiac depression (Am . J . Physiol . 250:H1857-H1863, 1991) . The purpose of the present study was to determine if 1 week of withdrawal of alcohol from the diet after 8 weeks of alcohol consumption would reverse the potentiation by alcohol of sepsis-induced cardiac depression . Animals were fed an ethanol-containing diet in which ethanol contributed 36% of the total calories . Rats were fed this diet or a control liquid diet for 8 weeks, and then some animals were taken off the alcohol diet and placed on the control diet for 1 week . Sepsis was induced in control-fed, alcohol-fed or withdrawal animals by the administration of Escherichia coli into the dorsal subcutaneous space . Nonseptic animals received sterile saline in this space . The following day animals were anesthetized, and the hearts were removed and studied as isolated working hearts . Hearts removed from septic and alcohol septic animals showed severe depression of cardiac contractile performance . Hearts from the withdrawal group, however, were less compromised by sepsis and showed only a few signs of cardiac dysfunction . Withdrawal from alcohol for 1 week thus resulted in protection of the heart from sepsis-induced cardiac depression.

J Biol Chem, 1995 Jul 28, 270(30), 17934 - 8
Lipopolysaccharide (LPS) neutralizing peptides reveal a lipid A binding site of LPS binding protein; Taylor AH et al.; Endotoxic shock follows a cascade of events initiated by release of lipopolysaccharide during infection with Gram-negative organisms . Two overlapping 15-mer peptides were identified, corresponding to residues 91-108 of human lipopolysaccharide binding protein that specifically bound the lipid A moiety of lipopolysaccharide with high affinity . The peptides inhibited binding of lipopolysaccharide to lipopolysaccharide binding protein, inhibited the chromogenic Limulus amebocyte lysate reaction, and blocked release of tumor necrosis factor alpha following lipopolysaccharide challenge both in vitro and in vivo . These results suggest lipopolysaccharide binding protein residues 91-108 form at least part of the lipopolysaccharide binding site . Moreover, derivatives of lipopolysaccharide binding protein residues 91-108 might modulate lipopolysaccharide toxicity in the clinical setting.

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